Analogues of N,1-diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3- carboxamide and N,1-diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3- carboxamide-6,6-dioxide: Syntheses, characterization, antimicrobial, antituberculosis, and antitumor activity

Article abstract of DOI:10.1039/c3ra23124k

A series of N,1-diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3- carboxamides (11a-m) and N,1-diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c] pyrazole-3-carboxamide-6,6-dioxides (12a-m) were synthesized by varying the active part (carboxamide group) of the pyrazole and were characterized by IR, ¹H-NMR, ¹³C-NMR, mass spectral data, and elemental analyses. All compounds were evaluated for their antibacterial and antifungal activity. Compounds 11k and 12k showed higher activity than chloroamphenicol against Klebsiella pneumonia and Escherichia coli. Compounds 11b, 11c, 11l, 12b, 12c, and 12l displayed higher activity towards amikacin in inhibiting the growth of Escherichia coli (MIC 3.125 mg mL^-1). Compounds 11k and 12k were equipotent to clotrimazole in inhibiting the growth of Candida albicans (MIC 3.125 mg mL^-1). All compounds were screened for their cytotoxic activity against two tumor cell lines, namely the human colon tumor cell line (HCT116) and human cervical cancer cell line (HeLa). Most of the test compounds exhibited potent antitumor activity, especially compounds 11k and 12k, which displayed the highest activity among the test compounds with an IC₅₀ equal to 18 and 12 μM for HeLa cells, and 16 and 10 μM for HCT116 cells, respectively. All the synthesized compounds showed low to moderate inhibitory activities against M. tuberculosis (MTB) H₃₇Rv, whereas 11k and 12k were found to be more active against M. tuberculosis, with MIC values of 8.2 and 7.8 μM, compared to other analogues.

Full text of DOI:10.1039/c3ra23124k

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PAPER
Analogues of N,1-diphenyl-4,5-dihydro-1H-
[1]benzothiepino[5,4-c]pyrazole-3-carboxamide and
N,1-diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-
c]pyrazole-3-carboxamide-6,6-dioxide: syntheses,
characterization, antimicrobial, antituberculosis, and
antitumor activity3
Cite this: DOI: 10.1039/c3ra23124k
Pandaram Palanisamy and Sudalaiandi Kumaresan*
A series of N,1-diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3-carboxamides (11a–m) and
N,1-diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3-carboxamide-6,6-dioxides (12a–m) were
synthesized by varying the active part (carboxamide group) of the pyrazole and were characterized by
IR, 1H-NMR, 13C-NMR, mass spectral data, and elemental analyses. All compounds were evaluated for their
antibacterial and antifungal activity. Compounds 11k and 12k showed higher activity than chloroam-
phenicol against Klebsiella pneumonia and Escherichia coli. Compounds 11b, 11c, 11l, 12b, 12c, and 12l
displayed higher activity towards amikacin in inhibiting the growth of Escherichia coli (MIC 3.125 mg
mL21). Compounds 11k and 12k were equipotent to clotrimazole in inhibiting the growth of Candida
albicans (MIC 3.125 mg mL21). All compounds were screened for their cytotoxic activity against two tumor
cell lines, namely the human colon tumor cell line (HCT116) and human cervical cancer cell line (HeLa).
Most of the test compounds exhibited potent antitumor activity, especially compounds 11k and 12k,
which displayed the highest activity among the test compounds with an IC50 equal to 18 and 12 mM for
HeLa cells, and 16 and 10 mM for HCT116 cells, respectively. All the synthesized compounds showed low to
moderate inhibitory activities against M. tuberculosis (MTB) H37Rv, whereas 11k and 12k were found to be
more active against M. tuberculosis, with MIC values of 8.2 and 7.8 mM, compared to other analogues.
Received 30th November 2012,
Accepted 28th January 2013
DOI: 10.1039/c3ra23124k
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purine, and many derivatives of pyrazolo[3,4-d]pyrimidines
have been reported as antitumoragents14–23 and cannabinoid
1 Introduction
The pyrazole moiety is present in many natural products.1 Its
derivatives are reported to have a broad spectrum of biological
activities, such as antitumour,2 anticoagulant,3 antihypergly-
cemic, analgesic, antipyretic, antimicrobial, and hypoglycemic
activity.4–8 These derivatives have applications in drug devel-
opment.9 Arylpyrazoles are important in medicinal and
pesticidal chemistry.10,11 Some arylpyrazoles were reported to
have non-nucleoside HIV-1 reverse transcriptase inhibitory
activity.12 Li et al. have reported a series of N,1,3-triphenyl-
1H-pyrazole-4-carboxamide derivatives that exhibited potent
antiproliferative activities against HTC116, MCF-7 cells, and
Aurora-A kinase inhibitory activities.13 Pyrazolo[3,4-d]pyrimi-
dines are of considerable chemical and pharmacological
importance because of their structural similarities with
type-1 (CB1) receptor antagonists.24,25 Ding et al. reported a
series of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carboxamide
derivatives which inhibited the proliferation of A549 cells.26
The azole group of heterocyclic compounds possesses a
significant pharmacokinetic property, and lipophilicity that
influences the ability of a drug to reach the target by
transmembrane diffusion, and shows promising activity
against resistant TB by inhibiting the biosynthesis of
lipids.27,28 Ahsan et al. have reported the antimycobacterial
activity of 4-dihydro-3H-indeno[1,2-c]pyrazole-2-carboxamide
analogues.29–31
The synthesis and in vitro antitumour activity of several
benzo[b]thiophenesulfonamide-1,1-dioxide derivatives have
been reported.32 Benzo[b]thiophene-4-carboxamide-1,1-diox-
ide derivatives have been described in the literature as
preventives for various inflammatory and neoplastic diseases
caused by an abnormal production of interleukin-6 or
interleukin-12.33 Alonso34 and Sagardoy35 and co-workers
Department of Chemistry, Manonmaniam Sundaranar University, Tirunelveli-627
012, Tamilnadu, India. E-mail: skumarmsu@yahoo.com
Electronic supplementary information (ESI) available. See DOI: 10.1039/
3
c3ra23124k
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CH2 group), a quartet at d 4.2 ppm (–O–CH2 group) and a
multiplet in the region of d 6.8–7.5 ppm (aromatic protons).
The mass spectrum revealed a molecular ion peak at m/z =
350.11. The IR spectrum of compound 8 showed a carbonyl
absorption band at 1696 cm21 and sulfone absorption bands
synthesized a series of benzo[b]thiophene-6-carboxamide-1,1-
dioxide derivatives which exhibited growth inhibition of HTB-
54, CCRF-CEM, and HeLa tumour cells.
In view of the above-mentioned facts to identify new
candidates that may be valued as potent and less toxic
antimicrobial, antimycobacterial and antitumor agents, we
report herein the syntheses, characterization, and biological
evaluation of a series of pyrazole carboxamides.
1
at 1156 (sym) and 1308 (asym) cm21. The H-NMR spectrum
showed a triplet at d 1.1 ppm (–CH3 group), two more triplets
at d 3.1 and 3.8 ppm (–SO2–CH2–CH2 and –SO2–CH2–CH2
group), a quartet at d 4.3 ppm (–O–CH2 group) and a multiplet
in the region of d 6.9–7.6 ppm (aromatic protons). The mass
spectrum of 8 revealed a molecular ion peak at m/z = 382.08.
Alkaline hydrolysis of 7 and 8 afforded the corresponding
1,4-dihydro-1-phenylthiochromeno[4,3-c]pyrazole-3-carboxylic
acid (9) and 1-phenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-
c]pyrazole-3-carboxylic acid-6,6-dioxide (10), respectively. The
IR spectrum of compound 9 showed a strong band at 1688
cm21, which was due to the conjugated carbonyl. The 1H-NMR
spectrum showed two triplets at d 2.9 and 3.0 ppm (–S–CH2
and –CH2 group), and a multiplet in the region of 6.8–7.7 ppm
(9H, aromatic). The mass spectrum displayed a molecular ion
peak at m/z = 322.10. The IR spectrum of compound 10
displayed a strong band at 1668 cm21, which was due to the
conjugated carbonyl, and the sulfone absorption bands were at
1152 (sym) and 1303 cm21(asym). The 1H-NMR spectrum
showed two triplets at d 3.1 and 3.8 ppm (–SO2–CH2–CH2 and –
SO2–CH2–CH2 group), and a multiplet in the region of 6.9–7.9
ppm (9H, aromatic). The mass spectrum displayed a molecular
ion peak at m/z = 354.05.
2 Results and discussion
2.1 Chemistry
The strategies adopted for the synthesis of the intermediates
and target compounds are depicted in Scheme 1 and Table 1.
Compound 1 and 2 were prepared as per the reported
methods.36 Addition of 1 equiv. of the requisite thiochro-
man-4-one (1) and 3,4-dihydro-1-benzothiepin-5(2H)-one-1,1-
dioxide (2) to diethyl oxalate in ethanol at room temperature in
the presence of 2 equiv. of a base, afforded the Claisen
condensation products 3/5 and 4/6 respectively.37
Subsequent reaction of 1 equiv. of a mixture of 3/5 and 4/6
with 1.15 equiv. of the phenylhydrazine hydrochloride at reflux
in ethanol afforded the ethyl 1-phenyl-4,5-dihydro-1H-[1]ben-
zothiepino[5,4-c]pyrazole-3-carboxylate (7) and ethyl 1-phenyl-
4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3-carboxylate-
6,6-dioxide (8) respectively. The IR spectrum of 7 showed a
carbonyl absorption band at 1707 cm21
.
The 1H-NMR
The pyrazole acid
9 and 10 were converted to the
spectrum showed a triplet at d 1.2 ppm (–CH3 group), two
more triplets at d 2.9 and 3.0 ppm (–S–CH2–CH2 and –S–CH2–
corresponding acid chloride followed by treatment with an
Scheme 1 Synthetic route for the pyrazole carboxamide analogues (11a–m and 12a–m). Reagents and conditions: (a) ClCH2CH2CH2COOH/NaOH; PPA (b) Na, dry
EtOH, (COOEt)2 (c) C6H5NHNH2. HCl, EtOH (d) KOH, MeOH; D (e) i) C6H5CH3, SOCl2 ii) CH2Cl2, TEA, R-NH2
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Table 1 Pyrazole carboxamide analogues (11a–m and 12a–m from 1-phenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3-carboxylic acid and amines
11a–m
12a–m
No
1
R
Compound 11 and 12
Time (h)
M. p. (uC)
Yield (%)
71
Time (h)
M. p.(uC)
Yield (%)
51
a
3.0
3.5
238–241
3.5
3.5
268–271
2
b
246–249
75
275–278
52
3
4
c
3.5
4.0
245–248
257–260
68
62
4.0
4.0
276–279
286–289
54
49
d
5
6
e
f
4.0
2.5
258–261
247–250
60
68
4.0
3.0
286–289
246–249
46
56
7
8
g
2.5
2.5
246–249
259–262
64
66
3.0
3.0
245–248
287–290
54
52
h
9
i
j
3.0
3.0
253–256
259–262
70
72
3.0
3.0
282–286
287–290
54
56
10
11
k
2.5
264–267
72
3.0
292–292
56
12
13
l
3.0
3.5
262–265
266–269
68
64
3.0
4.0
290–293
293–296
52
54
m
excess of the appropriate amine (Table 1) to give 11a–m and
12a–m. The sulfone derivatives 12a–m were obtained in lower
yield compared to 11a–m. The nitro substituted compounds
12d and 12e were obtained in moderate yield (46–48%) and the
chloro- and 4-aminoantipyrine substituted compounds were
obtained in good yields (72–75%). The toludine- and 4-ami-
noantipyrine-substituted compounds readily reacted with the
corresponding acid chlorides.
characteristic absorption bands in the IR spectra around
3117–3196 and 1632–1657 cm21, which were due to the N–H
and CLO stretching, respectively. The 1H-NMR spectra of 11a–
m exhibited a triplet at d 2.9 and 3.0 ppm (–S–CH2–CH2 and –
S–CH2–CH2 group), and a broad singlet at d 9.5–10.5, which
was due to the CONH proton. The mass spectra of products
11a–m agree well with the structures, with molecular ion peaks
at m/z 397.51, 431.07, 431.10, 442.13, 442.10, 411.12, 411.16,
412.12, 336.12, 412.16, 507.15, 454.11, and 447.12, which
correspond to 11a, 11b, 11c, 11d, 11e, 11f, 11g, 11h, 11i, 11j,
11k, 11l, and 11m respectively. Elemental analyses were
The structures of compounds 11a–m and 12a–m have been
elucidated on the basis of their IR, 1H-NMR, 13C-NMR and
mass spectral data (ESI ). Compounds 11a–m displayed
3
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satisfactory, which confirmed the elemental compositions and
purities of the newly synthesized compounds 11a–m.
derivatives displayed higher activity than amikacin in inhibit-
ing the growth of E. coli (MIC 3.125 mg mL21).
The toludine- and phenylhydrazine-substituted pyrazoles
showed reasonably good growth inhibitory profiles against S.
aureus (MIC 12.5 mg mL21) compared to chloroamphenicol
and amikacin. The chloro- and toludine-pyrazole analogues
displayed relatively moderate growth inhibitory profiles
against S. pneumonia (MIC 12.5 mg mL21). Regarding the
activity of the pyrazole ester, pyrazole carboxylic acid, and
pyrazole carboxamides, against antifungal strains, the results
revealed that the chloro- and 2-aminobenzothiazole derivatives
Compounds 12a–m displayed characteristic absorption
bands in the IR spectra at around 3124–3211 and 1624–1661
cm21, due to N–H and CLO stretching, respectively, and the
sulfone absorption bands were found at 1150–1156 and 1300–
1308 cm21. The 1H-NMR spectra of 12a–m exhibited two
triplets at d 3.0–3.1 and 3.7–3.8 ppm (–SO2–CH2–CH2 and –
SO2–CH2–CH2 group), and a broad singlet at d 9.7–10.9, which
was due to the CONH proton. The mass spectra of products
12a–m agree well with the structures, with molecular ion peaks
at m/z 429.19, 463.06, 463.05, 474.08, 474.06, 443.12, 443.10,
444.10, 368.09, 444.12, 539.14, 486.06, and 479.10, which
correspond to 12a, 12b, 12c, 12d, 12e, 12f, 12g, 12h, 12i, 12j,
12k, 12l, and 12m respectively. Elemental analyses confirmed
the elemental compositions of 12a–m.
showed
a
comparable activity against clotrimazole.
4-Aminoantipyrine analogues are equipotent to clotrimazole
in inhibiting the growth of C. albicans (MIC 3.125 mg mL21).
3.2 In vitro antituberculosis activity
All the compounds were screened for their in vitro antituber-
culosis activity against MTB (H37Rv). The primary screening
was carried out via the agar dilution method using two fold
dilution techniques. Isoniazid (INH) was used as a standard
drug. The pyrazole carboxamides (11a–m and 12a–m) dis-
played better antituberculosis activity compared to their
predecessors, the pyrazole ester (7 and 8) and pyrazole acids
(9 and 10). The observed data on the antituberculosis activity
of the title compounds and the standard drug are given in
Table 3. Thirty compounds were found to be active with
minimum inhibitory concentrations of 7.8–26 mM. The chloro-,
2-aminobenzothiazole-, and 4-aminoantipyrine-substituted
compounds produced more inhibitory activity than the nitro-,
phenyl-, and naphthyl-substituted systems. 4-Aminoantipyrine
substituted pyrazole carboxamide (11k and 12k) showed good
inhibitory activity against MTB at MIC 8.2 and 7.8 mM, while
compounds 11b–c, 12b–c, 11l and 12l showed moderate
inhibitory activity against MTB at MIC 8.9–9.8 (Table 3).
3 Pharmacology
3.1 Antimicrobial evaluation
All 15 newly synthesized compounds were evaluated for their
in vitro antibacterial activity against Staphylococcus aureus, and
Streptococcus pneumonia, as examples of Gram-positive bac-
teria and Klebsiella pneumonia, Pseudomonas aeruginosa, and
Escherichia coli as examples of Gram-negative bacteria. They
were also evaluated for their in vitro antifungal potential
against Candida albicans, Aspergillus flavus, and Aspergillus
niger fungal strains. The agar-diffusion method was used for
the determination of the preliminary antibacterial and
antifungal activity. Amikacin, chloroamphenicol, and clotri-
mazole were used as reference drugs. The results were
recorded for each tested compound as the average diameter
of inhibition zones (IZ) of bacterial or fungal growth around
the discs in mm. The minimum inhibitory concentration
(MIC) measurement for these compounds showed significant
growth inhibition zones (.20 mm) by a two fold serial dilution
method.38 The MIC (mg mL21) and inhibition zone diameter
values are recorded in Table 2.
The results depicted in Table 2 revealed that most of the
tested compounds displayed variable inhibitory effects on the
growth of the tested Gram-positive and Gram-negative
bacterial strains, and also against the fungal strains. In
general, these compounds revealed better activity against the
Gram-negative rather than the Gram-positive bacteria. It was
found that the intermediates 7, 8, 9 and 10 exhibited better
antibacterial potentials than the pyrazole carboxamides 11a–m
and 12a–m. (Scheme 1 and Table 1)
3.3 In vitro antitumor activity
The newly synthesized compounds 11a–m and 12a–m were
initially screened at a single concentration of two fold dilution
using the colorimetric MTT to test their in vitro cytotoxicity
against HeLa (cervical cancer cells) and HCT116 (colon cancer
cells). Doxorubicin was used as the reference drug in this
study. The cytotoxicity of the tested compounds was estimated
in terms of percent growth inhibition compared to untreated
control cells. All the compounds effected .70% inhibition and
were re-tested by a two fold dilution from 6.25 to 100 mM. The
results are expressed as IC50 (inhibitory concentration 50%),
the concentration of the compound which inhibits the tumor
cell growth by 50% and the data are presented in Table 3 and
Fig. 1 and Fig. 2.
Cell growth inhibition was analyzed by the MTT assay and
the results show that the compounds 7, 8, 9, 10, 11a–m, and
12a–m exhibit an inhibitory effect on the proliferation of HeLa
and HCT116 cells in a dose-dependent manner (Table 3).
Compounds 11k and 12k were found to exhibit a higher
cytotoxic potency (18, 12 mM and 16, 10 mM) than that of
doxorubicin (21 mM and 19 mM) against HeLa cells and
HCT116 cells. Of note is that the 4-aminoantipyrinepyrazole-
carboxamide derivatives (11k and 12k) have a higher ability
than the rest towards both the cancer cells. The m- and
Regarding the structure activity relationship (SAR) of the
pyrazole carboxamide against Gram-negative bacteria, the
results revealed that the chloro-, 4-aminoantipyrine-, and
2-aminobenzothiazole substituted pyrazole compounds exhib-
ited a broad spectrum antibacterial profile against the tested
organisms. In this view, 4-aminoantipyrine-substituted pyr-
azole was found to exhibit higher activity (MIC 3.125 mg mL21
)
than that of chloroamphenicol (MIC 6.25 mg mL21) against K.
pneumonia and E. coli. The chloro- and 2-aminobenzothiazole
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Table 2 Minimal inhibitory concentrations (MIC, mg mL21) and inhibition zone (mm) of compounds.7, 8 9, 10, 11a–m, and 12a–m
MIC in mg mL21, and zone of inhibition/mm
Bacteria
Fungi
Gram-positive bacteria
Gram-negative bacteria
Pseudomonas Escherichia
Compound
no.
Staphylococcus Streptococcus Klebsiella
Candida
albicans
Aspergillus
flavus
Aspergillus
niger
aureus
pneumonia
pneumonia
aeruginosa
coli
a
7
9
.100 (26–29)
50 (25–28)
25 (29–32)
25 (39–42)
25 (38–41)
25 (30–33)
25(31–34)
12.5(37–40)
12.5 (38–41)
25 (28–31)
25 (30–33)
12.5 (28–31)
6.25 (40–43)
12.5 (39–42)
25(24–27)
.100 (22–25)
50(28–31)
25 (33–36)
25 (40–43)
25 (41–43)
25 (32–35)
25(33–36)
50 (25–28)
25 (27–30)
25 (28–31)
—
12.5 (23–26)
12.5 (28–31)
12.5 (32–35)
6.25 (33–36)
6.25 (34–37)
12.5 (28–31)
12.5 (27–30)
12.5 (37–40)
12.5 (36–39)
12.5 (26–29)
25 (27–30)
50 (27–30)
50 (29–32)
25 (31–34)
3.125 (37–39) 6.25 (31–34)
3.125 (37–40) 6.25 (32–35)
25 (30–33)
25 (29–32)
12.5 (34–37)
12.5 (35–38)
25 (29–32)
12.5 (33–36)
12.5 (26–29)
50 (25–28)
25 (28–31)
25 (32–35)
—
25(28–31)
25 (29–32)
12.5 (31–34)
12.5 (38–41) 6.25 (36–39)
12.5 (39–42) 6.25 (36–39)
12.5 (30–33)
12.5 (31–34)
12.5 (38–41) 25 (36–39)
50 (29–30)
25 (29–32)
6.25(36–39)
6.25 (36–39) 3.25 (29–32)
12.5 (28–31) 12.5 (32–35)
12.5 (29–32) 12.5 (33–36)
12.5 (32–35) 25 (36–39)
12.5 (33–36) 25 (34–37)
25 (27–30)
12.5 (31–34)
3.25 (29–32)
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
8
25 (28–31)
25 (29–32)
25 (32–35)
25 (31–33)
25 (29–32)
25 (30–33)
25 (30–33)
12.5 (25–28)
12.5 (27–30)
12.5(39–42)
25 (31–33)
25 (29–32)
25 (32–35)
25(35–38)
12.5 (29–32)
12.5 (28–31)
12.5 (27–30)
25 (27–30)
12.5 (28–31) 12.5 (27–30)
12.5 (28–31) 6.25 (31–34)
12.5 (28–31)
25 (26–29)
6.25 (39–42) 3.125 (34–37) 6.25 (38–41)
6.25 (35–38) 6.25 (33–36)
3.125 (40–43) 3.125 (33–36) 6.25 (34–37) 3.125 (33–36)
3.125 (33–36) 6.25 (31–34) 6.25 (33–36) 3.125 (29–32)
25 (24–27) 12.5 (26–29)
.100(24–27) 12.5(23–26)
6.25 (24–25)
12.5 (25–28)
25 (30–33)
50(26–29)
25 (31–33)
25 (32–35)
12.5 (27–30)
.100 (23–25) 12.5(28–31)
25(28–31)
12.5 (33–36)
12.5 (28–31)
25(26–29)
50(30–33)
25 (32–35)
25 (29–32)
25(28–31)
25 (29–31)
25 (33–36)
10
12.5 (28–31)
12.5 (33–36)
6.25 (34–37)
6.25 (38–41)
12.5 (29–32)
12.5 (29–32)
12.5 (40–43)
12.5 (36–39)
12.5 (29–32)
25 (30–33)
50 (30–33)
25 (30–33)
6.25(38–41)
6.25 (38–41) 3.25 (31–34)
12.5 (30–33) 12.5 (34–37)
12.5 (30–33) 12.5 (35–38)
12.5 (34–37) 25 (39–42)
12.5 (33–36) 25 (34–37)
25 (29–31)
12.5 (32–35)
3.25 (31–34)
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12.5 (39–42) 6.25 (38–41)
12.5 (41–44) 6.25 (39–42)
12.5 (32–35)
12.5 (33–37)
12.5 (40–43) 25 (39–42)
3.125 (37–39) 6.25 (33–37)
3.125 (39–42) 6.25 (35–38)
25 (30–33)
25 (31–34)
25 (32–35)
25 (33–35)
12.5 (37–40)
12.5 (35–38)
25 (30–33)
12.5 (37–39)
12.5 (26–29)
25 (33–36)
25 (33–35)
25 (31–34)
25 (30–33)
25 (33–36)
12.5 (27–30)
12.5 (27–30)
12.5(39–42)
12.5 (38–41)
25 (32–35)
25 (33–36)
12.5 (28–31)
6.25 (43–46)
12.5 (40–43)
25(25–28)
12.5(39–42)
25 (34–37)
25 (31–34)
25 (32–35)
25(35–38)
12.5 (32–35)
12.5 (31–34)
12.5 (27–30)
25 (30–33)
12.5 (30–33) 12.5 (29–32)
12.5 (28–31) 6.25 (31–34)
12.5 (29–32)
25 (26–29)
6.25 (40–43) 3.125 (36–40) 6.25 (39–42)
3.125 (43–46) 3.125 (34–37) 6.25 (35–38) 3.125 (35–38)
3.125 (36–39) 6.25 (35–38) 6.25 (37–40) 3.125 (32–35)
12.5 (32–35)
6.25 (34–37)
6.25 (36–39)
NT
6.25 (38–41) 6.25 (36–39)
25 (33–36) 12.5 (30–33)
6.25 (38–41) 6.25 (32–35)
6.25 (34–37) 3.125 (37–40) 6.25 (29–32)
NT NT NT
6.25 (26–29)
12.5 (28–31)
6.25 (36–39)
25 (32–35)
25 (27–30)
NT
NT
12.5 (29–32)
NT
NT
Chloramphe-nicol 3.125 (38–41)
Amikacin
Clotrimazole
NTb
6.25 (35–38)
NT
NT
6.25 (29–32)
6.25 (28–31) 3.125 (27–30)
a
b
(—): totally inactive (no inhibition zone). NT: Not tested.
p-chloro substituted compounds (11b–c and 12b–c), and
2-aminobenzothiazole substituted compounds (11l and 12l)
show comparable IC50 values than the other substituted
compounds on both the cells. In general, many of the IC50
values for the HCT116 cells are lower than those for the
corresponding HeLa cells.
Compounds 12a–m exhibited higher activity than the 11a–m
analogues.
5 Experimental
5.1 Analysis and instruments
Melting points were obtained on TECHNICO melting point
apparatus and are uncorrected. IR spectra were recorded as
thin films (for oils) or KBr (for solids) on NaCl plates with a
Jasco FT-IR spectrophotometer and are expressed in cm21. All
NMR spectra were taken on a Brucker Advance 400 FT-NMR
spectrometer with 1H and 13C being observed at 400 MHz.
Chemical shifts for 1H and 13C-NMR spectra were reported in d
or ppm downfield from TMS [(CH3)4Si]. Multiplicities are
recorded as s (singlet), br s (broad singlet), d (doublet), t
(triplet), q (quartet), dd (double doublets), and m (multiplet).
ESI mass spectra were obtained on an Agilent 1100 series LC/
MSD spectrometer. All reactions involving air or moisture-
sensitive compounds were performed under a nitrogen atmo-
4 Conclusion
The objective of the present study was to synthesize and
investigate the antimicrobial activities of some new N,1-
diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3-car-
boxamides (11a–m) and carboxamide-6,6-dioxides (12a–m)
with the hope of discovering their bioactivity. Results obtained
clearly revealed that the chloro-, 2-aminobenzothiazole-, and
4-aminoantipyrine-linkages exhibited better antimicrobial
activity than their counterparts. Similarly, compounds 11k
and 12k displayed higher antimicrobial, antituberculosis, and
antitumor activity compared to the other derivatives.
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(CH3), 23.34 (C5), 35.86 (CH3–CH2), 60.54 (C4), 119.42–149.64
(aromatic and olefinic carbon), 160.12 (CO). ESI-MS calcd for
m/z: 350.11. Anal. Calcd for C20H18N2O2S: C, 68.55; H, 5.18; N,
7.99; O, 9.13; S, 9.15%. Found: C, 68.25; H, 5.16; N, 7.77; S,
9.11%.
Table 3 Antimycobacterial activity and antitumor activity of the title com-
pounds 7, 8, 9, 10, 11a–m and 12a–ma
Antitumor activity IC50(mM)b
Antituberculosis
Compound
HeLa cell
HCT116
activity MIC (mM)
5.2.2 1-(6,6-Dioxido-1-phenyl-4,5-dihydro-1H-[1]benzothie-
pino[5,4-c]pyrazol-3-yl)butan-1-one (8). A similar procedure
for the preparation of 7 was followed for the preparation of
8, with refluxing for 4 h. Compound 8 (0.92 g, 51%) was
obtained as a yellow solid. Rf 0.64 (petroleum ether/EtOAc,
8 : 2); mp 186–189 uC (triturated with petroleum ether); FT-IR
7
9
.100
.100
68
24
23
60
64
58
48
56
58
62
18
22
64
.100
95
60
20
21
52
54
53
42
50
52
.100
.100
62
23
24
58
62
54
46
50
54
60
16
20
60
.100
90
57
20
21
54
56
50
40
45
50
64
54
26
9.8
9.6
38
34
28
26
30
28
24
8.2
9.4
34
60
50
20
9.0
8.9
30
28
26
24
26
22
16
7.8
9.0
30
8.6
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
8
10
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
INH
Doxorubicind
1698, 1571, 1502, 1358, 1214, 1156, 1308 cm21 1H-NMR
.
(CDCl3) d 1.1 (t, 3H, –CH3), 3.1 and 3.8 (t, 2H, –SO2–CH2–CH2
and –SO2–CH2–CH2), 4.3 (q, 2H, –O–CH2), 6.9–7.6 (m, 9H,
aromatic); 13C-NMR (CDCl3) d 11.36 (C5), 21.36 (CH3), 34.72 (–
CH3–CH2), 68.32 (C4), 119.36–149.76 (aromatic and olefinic
carbon), 160.35 (CO). ESI-MS calcd for m/z: 382.08. Anal. Calcd
for C20H18N2O4S: C, 62.81; H, 4.74; N, 7.33; O, 16.73; S, 8.38%.
Found: C, 62.57; H, 4.56; N, 7.17; S, 8.31%.
5.2.3 1-Phenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyra-
zole-3-carboxylic acid (9). To a mixture of ester 7 (1.0 equiv.,
5 mmol) in methanol (25 mL), was added a solution of
potassium hydroxide (2.0 equiv., 10mmol) in methanol (18
mL). The resulting mixture was heated under reflux for 3.5 h,
cooled overnight, poured into water, and acidified with 1 N
hydrochloric acid. The precipitate was filtered, washed with
water, and air-dried to yield the analytically pure product of
1-phenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3-car-
boxylic acid (9) (1.87g, 95.2%) as a colorless solid. Rf 0.54
(CHCl3/MeOH 9 : 1); mp 261–263 uC; FT-IR 1688 (conjugated
carbonyl), 1517, 1431, 1379, 1200 cm21. 1H-NMR (CDCl3) d 2.9
and 3.0 (t, 2H, –S–CH2–CH2 and –S–CH2–CH2), 6.8–7.5 (m, 9H,
aromatic); 13C-NMR (CDCl3) d 22.35 (C4), 35.68 (C5), 119.17–
149.55 (aromatic and olefinic carbon), 159.454 (CO). ESI-MS
calcd for m/z: 322.10. Anal. Calcd for C18H14N2O2S: C, 67.06; H,
4.38; N, 8.69; O, 9.93; S, 9.95%. Found: C, 66.95; H, 4.29; N,
8.57; S, 9.65%.
68
12
20
54
10
18
58
54
c
c
—
—
c
21
19
—
a
b
Negative control DMSO, no activity. The IC50 value was defined as
the concentration at which a 50% survival of cells was observed. The
c
d
results are listed in the table. Not Tested. Used as a positive
control.
sphere. The separation of compounds was carried out by
column chromatography using silica gel. Unless otherwise
specified, all materials, solvents, and reagents were obtained
commercially.
5.2.4 1-Phenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyra-
zole-3-carboxylic acid 6,6-dioxide (10). A similar procedure
for the preparation of 7 was followed for the preparation of 8,
with refluxing for 4 h. Compound 10 (1.56g, 78%) was
obtained as a colorless solid. Rf 0.54 (CHCl3/MeOH 9 : 1);
mp 291–293 uC; FT-IR 1688 (conjugated carbonyl), 1517, 1431,
1379, 1200, and sulfone absorption bands at 1152 and 1303
5.2 Synthesis
5.2.1 Ethyl 1-phenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-
c]pyrazole-3-carboxylate (7). A stirred mixture of the diketoe-
ster (3/5) (1.0 equiv., 4 mmol) and the phenylhydrazine
hydrochloride (1.15 equiv.) in EtOH (28 mL) was heated under
reflux for 3.5 h. The reaction was allowed to cool to room
temperature and the insoluble material was collected by
filtration and washed with a small volume of ice-cold ethanol.
Purification by column chromatography afforded the analyti-
cally pure product ethyl 1-phenyl-4,5-dihydro-1H-[1]benzothie-
pino[5,4-c]pyrazole-3-carboxylate (7) (1.12 g, 71%) as a yellow
solid. Rf 0.64 (petroleum ether (40–60 uC)/EtOAc, 8 : 2); mp
156–159 uC (triturated with petroleum ether); FT-IR 1707, 1591,
1508, 1369, 1211 cm21; 1H-NMR (CDCl3) d 1.2 (t, 3H, –CH3), 2.9
and 3.0 (t, 2H, –S–CH2–CH2 and –S–CH2–CH2), 4.2 (q, 2H, –O–
CH2), 6.8–7.5 (m, 9H, aromatic); 13C-NMR (CDCl3) d 14.25
cm21 1H-NMR (CDCl3) d 3.1 and 3.8 (t, 2H, –SO2–CH2–CH2
.
and –SO2–CH2–CH2), 7.1–7.5 (m, 9H, aromatic); 13C-NMR
(CDCl3) d 14.35 (C4), 60.35 (C5), 117.35–149.75 (aromatic and
olefinic carbon), 160.45 (CO). ESI-MS calcd for m/z: 354.05.
Anal. Calcd for C18H14N2O4S: C, 61.01; H, 3.98; N, 7.90; O,
18.06; S, 9.05%. Found: C, 60.95; H, 3.69; N, 7.57; S, 8.95%.
5.2.5 General syntheses of carboxamides (11 and 12). A
mixture of the appropriate 1-phenyl-4,5-dihydro-1H-[1]ben-
zothiepino[5,4-c]pyrazole-3-carboxylic acid (9) or 1-phenyl-4,5-
dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3-carboxylic acid-
6,6-dioxide (10) (1 equiv., 4.0 mmol) and thionyl chloride
(3.0 equiv.) in toluene (30 mL) was refluxed for 30 min. The
solvent and the excess SOCl2 were removed under reduced
pressure and the resulting dark solid dissolved in CH2Cl2 (15
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Fig. 1 Growth of inhibition of compounds 7–11a–m and 12a–m, based on the concentration in the HeLa cells
Fig. 2 Growth of inhibition of compounds 7–11a–m and 12a–m, based on the concentration in the HCT116 cells
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mL) was dropwise added to a solution of the corresponding
amine (1.5 equiv.) and Et3N (1.5 equiv.) in CH2Cl2 (15 mL) at 0
uC. The mixture was refluxed for 3–4 h (Table 1), taken in a
separatory funnel and washed with brine. The aqueous layer
was separated and extracted with CH2Cl2. The combined
organic layers were washed with water, dried over Na2SO4, and
concentrated under reduced pressure. The analytically pure
product was isolated by an appropriate method of purification
as indicated below.
5.2.5.5 N-(4-Nitrophenyl)-1-phenyl-4,5-dihydro-1H-[1]benzothie-
pino[5,4-c]pyrazole-3-carboxamide (11e). The mixture was sepa-
rated by column chromatography [petroleum ether/EtOAc
(1 : 1)] to afford 11e (1.35 g, 65%) as a colorless solid. IR
1655, 3194 cm21 1H-NMR (CDCl3) d 2.9 and 3.0 (t, 2H,
;
–S–CH2–CH2 and –S–CH2–CH2), 7.1–7.8 (m, 13H, aromatic),
9.6 (s, 1H, –CONH). 13C-NMR (CDCl3) d 22.98 (C5), 35.98 (C4),
117.36–150.12 (aromatic and olefinic carbon), 161.96 (CO).
ESI-MS calcd for m/z: 442.10. Anal. Calcd for C25H21N3OS: C,
65.14; H, 4.10; N, 12.66; O, 10.85; S, 7.25%. Found: C, 65.04; H,
4.02; N, 12.48; S, 7.10%.
5.2.5.1 N,1-Diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyra-
zole-3-carboxamide (11a). The mixture was separated by
column chromatography [petroleum ether/EtOAc, (1 : 1)] to
afford 11a (1.42 g, 71%) as a colorless solid. IR 1657, 3175
5.2.5.6 1-Phenyl-N-m-tolyl-4,5-dihydro-1H-[1]benzothiepino[5,4-
c]pyrazole-3-carboxamide (11f). The mixture was separated by
column chromatography [petroleum ether/EtOAc (1 : 1)] to
furnish 11f (1.33 g, 68%) as a colorless solid. IR 1653, 3185
1
cm21. H-NMR (CDCl3) d 2.9 and 3.0 (t, 2H, –S–CH2–CH2 and
–S–CH2–CH2), 6.7–7.4 (m, 14 H, aromatic), 9.9 (s, 1H, –CONH).
13C-NMR (CDCl3) d 22.34 (C4), 35.86 (C5), 117.25–149. 65
(aromatic and olefinic carbon), 160.25 (CO). ESI-MS calcd for
m/z: 397.51. Anal. Calcd for C24H19N3OS: C, 72.52; H, 4.82; N,
10.57; O, 4.03; S, 8.07%. Found: C, 72.48; H, 4.78; N, 10.55; S,
8.03%.
1
cm21; H-NMR (CDCl3) d 2.7 (s, 3H, CH3), 2.9 and 3.0 (t, 2H,
–S–CH2–CH2 and –S–CH2–CH2), 6.8–7.6 (m, 13H, aromatic),
9.6 (s, 1H, –CONH). 13C-NMR (CDCl3) d 21.16 (CH3), 25.12 (C5),
36.12 (C4), 117.36–150.65 (aromatic and olefinic carbon),
161.34 (CONH). ESI-MS calcd for m/z: 411.12. Anal. Calcd for
C
25H21N3OS: C, 72.97; H, 5.14; N, 10.21; O, 3.89; S, 7.79%.
Found: C, 72.36; H, 5.04; N, 10.12; S, 7.40%.
5.2.5.2 N-(3-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-[1]benzothie-
pino[5,4-c]pyrazole-3-carboxamide (11b). The mixture was sepa-
rated by column chromatography [petroleum ether/EtOAc,
(1 : 1)] to furnish 11b (1.64 g, 80%) as a colorless solid. IR
1654, 3180cm21. 1H-NMR (CDCl3) d 2.9 and 3.0 (t, 2H, –S–CH2–
CH2 and –S–CH2–CH2), 6.7–7.4 (m, 13H, aromatic), 9.9 (s,1H,
–CONH). 13C-NMR (CDCl3) d 23.46 (C5), 35.98 (C4), 116.86–
149.68 (aromatic and olefinic carbon), 160.89 (CO). ESI-MS
calcd for m/z: 431.07. Anal. Calcd for C24H18ClN3OS: C, 66.74;
H, 4.20; Cl, 8.21; N, 9.73; O, 3.70; S, 7.42%. Found: C, 66.70; H,
4.18; N, 9.68; S, 7.38%.
5.2.5.7 1-Phenyl-N-p-tolyl-14,5-dihydro-1H-[1]benzothiepino[5,4-
c]pyrazole-3-carboxamide (11g). The mixture was separated by
column chromatography [petroleum ether/EtOAc (1;1)] to
obtain 11g (1.58 g, 71%) as a colorless solid. IR 1650, 3180
1
cm21; H-NMR (CDCl3) d 2.4 (s, 3H, CH3), 2.9 and 3.0 (t, 2H,
–S–CH2–CH2 and –S–CH2–CH2), 6.8–7.7 (m, 13H, aromatic),
9.8 (s, 1H, –CONH). 13C-NMR (CDCl3) d 21.96 (CH3), 27.43 (C5),
36.25 (C4), 118.14–150.46 (aromatic and olefinic carbon),
162.12 (CO). ESI-MS calcd for m/z: 411.16. Anal. Calcd for
C
25H21N3OS: C, 72.97; H, 5.14; N, 10.21; O, 3.89; S, 7.79%.
Found: C, 72.46; H, 5.10; N, 10.08; S, 7.64%.
5.2.5.3 N-(4-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-[1]benzothie-
pino[5,4-c]pyrazole-3-carboxamide (11c). The mixture was sepa-
rated by column chromatography [petroleum ether/EtOAc
(1 : 1)] to afford 11c (1.43 g, 75%) as a colorless solid. IR
5.2.5.8 N-(2-Aminophenyl)-1-phenyl-4,5-dihydro-1H-[1]benzothie-
pino[5,4-c]pyrazole-3-carboxamide (11h). The mixture was sepa-
rated by column chromatography [petroleum ether/EtOAc
(1 : 1)] afforded 11h (1.38 g, 67%) as a yellow solid. IR 1658,
3194 cm21; 1H-NMR (CDCl3) d 2.9 and 3.0 (t, 2H, –S–CH2–CH2
and –S–CH2–CH2), 5.8 (s, 2H, NH2), 6.8–7.6 (m, 13H, aromatic),
10.02 (s, 1H, –CONH). 13C-NMR (CDCl3) d 22.34 (C5), 31.12
(C4), 116.42–149.87 (aromatic and olefinic carbon), 161.85
(CO). ESI-MS calcd for m/z: 412.12. Anal. Calcd for
1656, 3180 cm21 1H-NMR (CDCl3) d 2.9 and 3.0 (t, 2H, –S–
;
CH2–CH2 and –S–CH2–CH2), 6.7–7.5 (m, 13H, aromatic), 9.8
(s,1H, –CONH). 13C-NMR (CDCl3) d 23.26 (C5), 35.98 (C4),
117.89–150.75 (aromatic and olefinic carbon), 161.14 (CO).
ESI-MS calcd for m/z: 431.11. Anal. Calcd for C24H18ClN3OS: C,
66.74; H, 4.20; Cl, 8.21; N, 9.73; O, 3.70; S, 7.42%. Found: C,
66.70; H, 4.18; N, 9.68; S, 7.38%.
C
24H20N4OS: C, 69.88; H, 4.89; N, 13.58; O, 3.88; S, 7.77%.
Found: C, 69.58; H, 4.59; N, 13.33; S, 7.59%.
5.2.5.4 N-(3-Nitrophenyl)-1-phenyl-4,5-dihydro-1H-[1]benzothie-
pino[5,4-c]pyrazole-3-carboxamide (11d). The mixture was sepa-
rated by column chromatography [petroleum ether/EtOAc
(1 : 1)] to give 11d (1.34 g, 63%) as a colorless solid. IR 1651,
3180 cm21; 1H-NMR (CDCl3) d 2.9 and 3.0 (t, 2H, –S–CH2–CH2
and –S–CH2–CH2), 7.1–7.8 (m, 13H, aromatic), 10.31 (s, 1H,
–CONH). 13C-NMR (CDCl3) d 22.85 (C5), 36.01 (C4), 117.24–
150.11 (aromatic and olefinic carbon), 161.01 (CONH). ESI-MS
calcd for m/z: 442.10. Anal. Calcd for C24H18N4O3S: C, 65.14; H,
4.10; N, 12.66; O, 10.85; S, 7.25%. Found: C, 65.10; H, 4.06; N,
12.58; S, 7.19%.
5.2.5.9 1-Phenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-
3-carbohydrazide (11i). The mixture was separated by column
chromatography [petroleum ether/EtOAc (1 : 1)] to obtain 11i
(1.45 g, 71%) as a colorless solid. IR 1655, 3196 cm21; 1H-NMR
(CDCl3) d 2.9 and 3.0 (t, 2H, –S–CH2–CH2 and –S–CH2–CH2),
4.7 (s, 2H, NH2), 7.2–7.8 (m, 9H, aromatic), 9.5 (s, 1H, –CONH).
13C-NMR (CDCl3) d 21.45 (C5), 36.28 (C4), 117.34–149.84
(aromatic and olefinic carbon), 161.21 (CO). ESI-MS calcd for
m/z: 336.12. Anal. Calcd for C18H16N4OS: C, 64.26; H, 4.79; N,
16.65; O, 4.76; S, 9.53,%. Found: C, 64.11; H, 4.64; N, 16.48; S,
9.41%.
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5.2.5.10 N9,1-Diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-
c]pyrazole-3-carbohydrazide (11j). The mixture was separated
by column chromatography [petroleum ether/EtOAc (1 : 1)] to
obtain 11j (1.54 g, 71%) as a colorless solid. IR 1645, 3184
5.2.6.2 N-(3-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-[1]ben-
zothiepino[5,4-c]pyrazole-3-carboxamide 6,6-dioxide (12b).
The mixture was separated by column chromatography
[petroleum ether/EtOAc, (1 : 1)] to furnish 12b (0.98 g, 52%)
1
cm21; H-NMR (CDCl3) d 2.9 and 3.0 (t, 2H, –S–CH2–CH2 and
1
as a colorless solid. IR 1651, 3160, 1151, 1305 cm21. H-NMR
–S–CH2–CH2), 4.2 (s, 1H, NH), 7.2–7.8 (m, 13H, aromatic),
10.31 (s, 1H, –CONH). 13C-NMR (CDCl3) d 21.89 (C5), 36.01
(C4), 113.11–151.46 (aromatic and olefinic carbon), 160.75
(CO). ESI-MS calcd for m/z: 412.16. Anal. Calcd for
C24H20N4OS: C, 69.88; H, 4.89; N, 13.58; O, 3.88; S, 7.77%.
Found: C, 69.55; H, 4.60; N, 13.48; S, 7.61%.
(CDCl3) d 3.1–3.8 (t, 2H, t, 2H, –SO2–CH2–CH2 and –SO2–CH2–
CH2), 6.8–7.5 (m, 13H, aromatic), 9.9 (s, 1H, –CONH). 13C-NMR
(CDCl3) d 14.36 (C5), 58.63 (C4), 116.34–150.36 (aromatic and
olefinic carbon), 161.54 (CO). ESI-MS calcd for m/z: 463.0. Anal.
Calcd for C24H18ClN3O3S: C, 62.13; H, 3.91; Cl, 7.64; N, 9.06; O,
10.35; S, 6.91%. Found: C, 62.04; H, 3.85; N, 9.01; S, 3.81%.
5.2.6.3 N-(4-Chlorophenyl)-1-phenyl-4,5-dihydro-1H-[1]ben-
zothiepino[5,4-c]pyrazole-3-carboxamide 6,6-dioxide (12c).
The mixture was separated by column chromatography
[petroleum ether/EtOAc (1 : 1)] to afford 12c (0.93 g, 54%) as
5.2.5.11 N-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-
4-yl)-1-phenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyrazole-3-
carboxamide (11k). The mixture was separated by column
chromatography [petroleum ether/EtOAc (1 : 1)] to obtain 11k
a colorless solid. IR 1652, 3170, 1152, 1304 cm21 1H-NMR
.
1
(1.3 g, 60%) as a colorless solid. IR 1632, 3117 cm21; H-NMR
(CDCl3) d 3.1–3.8 (t, 2H, –SO2–CH2–CH2 and –SO2–CH2–CH2),
6.9–7.6 (m, 13H, aromatic), 9.9 (s,1H, –CONH). 13C-NMR
(CDCl3) d 13.96 (C5), 58.69 (C4), 117.52–150.34 (aromatic and
olefinic carbon), 162.45 (CO). ESI-MS calcd for m/z: 463.05.
Anal. Calcd for C24H18ClN3O3S: C, 62.13; H, 3.91; Cl, 7.64; N,
9.06; O, 10.35; S, 6.91%. Found: C, 62.03; H, 3.86; N, 9.01; S,
6.86%.
(CDCl3) d 2.9 and 3.0 (t, 2H, –S–CH2–CH2 and –S–CH2–CH2),
2.8 and 3.2 (s, 3H, CH3), 6.8–7.6 (m, 14H, aromatic), 9.4 (s, 1H,
–CONH). 13C-NMR (CDCl3) d12.46 (CH3), 22.49 (C5), 36.12 (C4),
39.79 (CH3), 103.32–149.56 (aromatic and olefinic carbon),
160.45 (CO), 161.32 (CO). ESI-MS calcd for m/z: 507.15. Anal.
Calcd for C29H25N5O2S: C, 68.62; H, 4.96; N, 13.80; O, 6.30; S,
6.32%. Found: C, 68.58; H, 4.90; N, 13.68; S, 6.24%.
5.2.6.4 N-(3-Nitrophenyl)-1-phenyl-4,5-dihydro-1H-[1]ben-
zothiepino[5,4-c]pyrazole-3-carboxamide 6,6-dioxide (12d).
The mixture was separated by column chromatography
[petroleum ether/EtOAc (1 : 1)] to give 12d (0.81 g, 49%) as a
5.2.5.12 N-(1,3-Benzothiazol-2-yl)-1-phenyl-4,5-dihydro-1H-
[1]benzothiepino[5,4-c]pyrazole-3-carboxamide (11l). The mix-
ture was separated by column chromatography [petroleum
ether/EtOAc (1 : 1)] to obtain 11l (1.30 g, 64%) as a pale yellow
colorless solid. IR 1656, 3175, 1152, 1304 cm21 1H-NMR
.
(CDCl3) d 3.1 and 3.8 (t, 2H, –SO2–CH2–CH2 and –SO2–CH2–
CH2), 7.1–7.9 (m, 13H, aromatic), 10.46 (s, 1H, –CONH). 13C-
NMR (CDCl3) d 14.36 (C5), 58.36 (C4), 117.24–151.32 (aromatic
and olefinic carbon), 163.52 (CONH). ESI-MS calcd for m/z:
474.08. Anal. Calcd for C24H18N4O5S: C, 60.75; H, 3.82; N,
11.81; O, 16.86; S, 6.76%. Found: C, 60.62; H, 3.68; N, 11.75; S,
6.67%.
1
solid. IR 1631, 3154 cm21; H-NMR (CDCl3) d 2.9 and 3.0 (t,
2H, –S–CH2–CH2 and –S–CH2–CH2), 6.8–7.5 (m, 13H, aro-
matic). 13C-NMR (CDCl3) d 21.86 (C5), 37.42 (C4), 117.48–
149.86 (aromatic and olefinic carbon), 162.51 (CO), 174.23
(CLN). ESI-MS calcd for m/z: 454.11. Anal. Calcd for
C25H18N4OS2: C, 66.06; H, 3.99; N, 12.33; O, 3.52; S, 14.11%.
Found: C, 65.98; H, 3.59; N, 12.03; S, 14.09%.
5.2.6.5 N-(4-Nitrophenyl)-1-phenyl-4,5-dihydro-1H-[1] ben-
zothiepino[5,4-c]pyrazole-3-carboxamide 6,6-dioxide (12e).
The mixture was separated by column chromatography
[petroleum ether/EtOAc (1 : 1)] to afford 12e (0.76 g, 45%) as
5.2.5.13 N-(Naphthalen-1-yl)-1-phenyl-4,5-dihydro-1H-[1]ben-
zothiepino[5,4-c]pyrazole-3-carboxamide (11m). The mixture
was separated by column chromatography [petroleum ether/
EtOAc (1 : 1)] to afford 11m (1.24 g, 68%) as a colorless solid.
a colorless solid. IR 1650, 3184, 1155, 1306 cm21 1H-NMR
.
1
IR 1635, 3134 cm21; H-NMR (CDCl3) d 2.9 and 3.0 (t, 2H, –S–
(CDCl3) d 3.1 and 3.8 (t, 2H, –SO2–CH2–CH2 and –SO2–CH2–
CH2), 7.3–7.9 (m, 13H, aromatic), 10.36 (s, 1H, –CONH). 13C-
NMR (CDCl3) d 14.52 (C5), 56.96 (C4), 117.52–150.63 (aromatic
and olefinic carbon), 162.58 (CO). ESI-MS calcd for m/z: 474.06.
Anal. Calcd for C24H18N4O5S: C, 60.75; H, 3.82; N, 11.81; O,
16.86; S, 6.76%. Found: C, 60.75; H, 3.75; N, 11.76; S, 6.65%.
CH2–CH2 and –S–CH2–CH2), 6.8–7.8 (m, 12H, aromatic); and d
10.43 (s, 1H, –CONH). 13C-NMR (CDCl3) d 22.34 (C5), 35.98
(C4), 117.46–151.24 (aromatic and olefinic carbon), 160.98
(CO). ESI-MS calcd for m/z: 447.12. Anal. Calcd for
C28H21N3OS: C, 75.14; H, 4.73; N, 9.39; O, 3.57; S, 7.16%.
Found: C, 75.02; H, 4.57; N, 9.22; S, 7.08%.
5.2.6.6 1-Phenyl-N-m-tolyl-4,5-dihydro-1H-[1]benzothie-
pino[5,4-c]pyrazole-3-carboxamide 6,6-dioxide (12f). The mix-
ture was separated by column chromatography [petroleum
ether/EtOAc (1 : 1)] to furnish 12g (0.92 g, 56%) as a colorless
5.2.6.1 N,1-Diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-
c]pyrazole-3-carboxamide 6,6-dioxide (12a). The mixture was
separated by column chromatography [petroleum ether/EtOAc,
(1 : 1)] to afford 12a (0.96 g, 51%) as a colorless solid. IR 1638,
1
solid. IR 1656, 3195, 1155, 1306 cm21. H-NMR (CDCl3) d 2.7
1
3155, 1148, 1298 cm21. H-NMR (CDCl3) d 3.1 and 3.8 (t,2H,
(s, 3H, CH3), 3.1 and 3.8 (t, 2H, –SO2–CH2–CH2 and –SO2–CH2–
CH2), 7.1–7.7 (m, 13H, aromatic), 9.8 (s, 1H, –CONH). 13C-NMR
(CDCl3) d 14.63(CH3), 25.24 (C5), 36.12 (CH3), 60.35 (C4),
117.25–151.32 (aromatic and olefinic carbon), 162.48 (CO).
ESI-MS calcd for m/z: 443.12. Anal. Calcd for C25H21N3O3S: C,
67.70; H, 4.77; N, 9.47; O, 10.82; S, 7.23%. Found: C, 67.66; H,
4.70; N, 9.36; S, 7.16%.
–SO2–CH2–CH2 and –SO2–CH2–CH2), 6.8–7.5 (m, 14H, aro-
matic), 10.13 (s, 1H, –CONH). 13C-NMR (CDCl3) d 13.46 (C5),
57.86 (C4), 117.25–149.45 (aromatic and olefinic carbon),
162.35 (CO). ESI-MS calcd for m/z: 429.19. Anal. Calcd for
C24H19N3O3S: C, 67.12; H, 4.46; N, 9.78; O, 11.18; S, 7.47%.
Found: C, 67.08; H, 4.40; N, 9.71; S, 7.40%.
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5.2.6.7 1-Phenyl-N-p-tolyl-14,5-dihydro-1H-[1]benzothie-
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C
29H25N5O4S: C, 64.55; H, 4.67; N, 12.98; O, 11.86; S, 5.94%.
Found: C, 64.48; H, 4.60; N, 12.78; S, 5.86%.
pino[5,4-c]pyrazole-3-carboxamide 6,6-dioxide (12g). The mix-
ture was separated by column chromatography [petroleum
ether/EtOAc (1;1)] to obtain 12g (0.96 g, 54%) as a colorless
5.2.6.12 N-(1,3-Benzothiazol-2-yl)-1-phenyl-4,5-dihydro-1H-
[1]benzothiepino[5,4-c]pyrazole-3-carboxamide 6,6-dioxide
(12l). The mixture was separated by column chromatography
[petroleum ether/EtOAc (1 : 1)] to furnish 12l (0.96 g, 52%) as a
1
solid. IR 1650, 3180, 1155, 1306 cm21. H-NMR (CDCl3) d 2.8
(s, 3H, CH3), 3.1 and 3.8 (t, 2H, –SO2–CH2–CH2 and –SO2–CH2–
CH2), 7.1–7.7 (m, 13H, aromatic), 9.8 (s, 1H, –CONH). 13C-NMR
(CDCl3) d 27.64 (CH3), 35.63 (CH3), 15.36 (C5), 63.75 (C4),
118.75–150.75 (aromatic and olefinic carbon), 162.75 (CO).
ESI-MS calcd for m/z: 443.10. Anal. Calcd for C25H21N3O3S: C,
67.70; H, 4.77; N, 9.47; O, 10.82; S, 7.23%. Found: C, 67.63; H,
4.68; N, 9.40; S, 7.14%.
pale yellow solid. IR 1636, 3164, 1154, 1308 cm21 1H-NMR
.
(CDCl3) d 3.1 and 3.8 (t, 2H, –SO2–CH2–CH2 and –SO2–CH2–
CH2), 6.9–7.6 (m, 13H, aromatic); 13C-NMR (CDCl3) d 14.35
(C5), 56.78 (C4), 117.36–150.42 (aromatic and olefinic carbon),
163.45 (CO), 175.63 (CLN). ESI-MS calcd for m/z: 486.06. Anal.
Calcd for C25H18N4O3S2: C, 61.71; H, 3.73; N, 11.51; O, 9.86; S,
13.18%. Found: C, 61.63; H, 3.62; N, 11.46; S, 13.02%.
5.2.6.8 N-(2-Aminophenyl)-1-phenyl-4,5-dihydro-1H-[1]ben-
zothiepino[5,4-c]pyrazole-3-carboxamide6,6-dioxide (12h). The
mixture was separated by column chromatography [petroleum
ether/EtOAc (1 : 1)] to afford 12h (0.86 g, 52%) as a yellow
5.2.6.13 N-(Naphthalen-1-yl)-1-phenyl-4,5-dihydro-1H-
[1]benzothiepino[5,4-c]pyrazole-3-carboxamide 6,6-dioxi-
de(12m). The mixture was separated by column chromato-
graphy [petroleum ether/EtOAc (1 : 1)] to afford 12m (0. 93 g,
54%) as a colorless solid. IR 1644, 3164, 1152, 1305 cm21. 1H-
NMR (CDCl3) d 3.1 and 3.8 (t, 2H, –SO2–CH2–CH2 and –SO2–
CH2–CH2), 6.9–7.8 (m, 12 H, aromatic), 10.43 (s, 1H, –CONH).
13C-NMR (CDCl3) d 15.12 (C5), 57.38 (C4), 117.28–152.36
(aromatic and olefinic carbon), 161.86 (CO). ESI-MS calcd for
m/z: 479.10. Anal. Calcd for C28H21N3O3S: C, 70.13; H, 4.41; N,
8.76; O, 10.01; S, 6.69%. Found: C, 70.06; H, 4.39; N, 8.69; S,
6.58%.
1
solid. IR 1651, 3186, 1152 and 1304 cm21. H-NMR (CDCl3) d
3.1 and 3.8 (t, 2H, –SO2–CH2–CH2 and –SO2–CH2–CH2), d 5.9
(s, 2H, NH2), 7.1–7.7 (m, 13H, aromatic), 10.20 (s, 1H, –CONH).
13C-NMR (CDCl3) d 14.63 (C5), 57.86 (C4), 116.35–149.36
(aromatic and olefinic carbon), 162.58 (CO). ESI-MS calcd for
m/z: 444.10. Anal. Calcd for C24H20N4OS: C, 69.88; C, 64.85; H,
4.54; N, 12.60; O, 10.80; S, 7.21%. Found: C, 64.69; H, 4.39; N,
12.56; S, 7.19%.
5.2.6.9 1-Phenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-c]pyra-
zole-3-carbohydrazide 6,6-dioxide (12i). The mixture was
separated by column chromatography [petroleum ether/
EtOAc (1 : 1)] to obtain 12i (0.84 g, 54%) as a colorless solid.
IR 1651, 3185, 1152, 1304 cm21. 1H-NMR (CDCl3) d 3.1 and 3.8
(t, 2H, –SO2–CH2–CH2 and –SO2–CH2–CH2), 4.8 (s, 2H, NH2),
7.1–7.8 (m, 9H, aromatic), 9.8 (s, 1H, –CONH). 13C-NMR
(CDCl3) d 14.36 (C5), 57.36 (C4), 117.45–149.46 (aromatic and
olefinic carbon), 162.45 (CO). ESI-MS calcd for m/z: 368.09.
Anal. Calcd for C18H16N4O3S: C, 58.68; H, 4.38; N, 15.21; O,
13.03; S, 8.70; S, 9.53%. Found: C, 58.59; H, 4.34; N, 15.18; S,
9.43%.
6 Antimicrobial evaluation
Standard sterilized filter paper discs (5 mm diameter)
impregnated with a solution of the test compound in DMSO
(1 mg mL21) was placed on an agar plate seeded with the
appropriate test organism in triplicate. The utilized test
organisms were S. aureus, and S. pneumonia, as examples of
Gram-positive bacteria and K. pneumonia, P. aeruginosa, and E.
coli as examples of Gram-negative bacteria. They were also
evaluated for their in vitro antifungal potential against C.
albicans, A. flavus, and A. niger strains. Amikacin and
chloroamphenicol were used as a standard antibacterial agent
and clotrimazole was used as a standard antifungal agent.
DMSO alone was used as the control at the above-mentioned
concentration. The plates were incubated at 37 uC for 24 h for
bacteria and 28 uC for 48 h for fungi. Compounds that showed
significant growth inhibition zones (.20 mm) using the two
fold serial dilution technique were further evaluated for their
minimal inhibitory concentrations (MICs).
5.2.6.10 N9,1-Diphenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-
c]pyrazole-3-carbohydrazide 6,6-dioxide (12j). The mixture was
separated by column chromatography [petroleum ether/EtOAc
(1 : 1)] to afford 12j (0.96 g, 56%) as a colorless solid. IR 1648,
1
3191, 1152, 1304 cm21. H-NMR (CDCl3) d 3.1 and 3.8 (t, 2H,
–SO2–CH2–CH2 and –SO2–CH2–CH2), 4.3 (s, 1H, NH), 6.9–7.6
(m, 13H, aromatic), 10.23 (s, 1H, –CONH). 13C-NMR (CDCl3), d
14.36 (C5), 57.36 (C4), 113.42–151.36 (aromatic and olefinic
carbon), 161.52 (CO). ESI-MS calcd for m/z: 444.10. Anal. Calcd
for C24H20N4O3S: C, 64.85; H, 4.54; N, 12.60; O, 10.80; S, 7.21%.
Found: C, 64.76; H, 4.46; N, 12.54; S, 7.11%.
5.2.6.11 N-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-
1H-pyrazol-4-yl)-1-phenyl-4,5-dihydro-1H-[1]benzothiepino[5,4-
c]pyrazole-3-carboxamide 6,6-dioxide (12k). The mixture was
separated by column chromatography [petroleum ether/EtOAc
(1 : 1)] to afford 12k (0.99 g, 56%) as a colorless solid. IR 1644,
6.1 Minimal inhibitory concentration (MIC) measurement
The broth dilution test was used to determine the Minimum
Inhibitory Concentration (MIC) of the above mentioned
samples.39,40 The micro dilution susceptibility test was used
for the determination of antibacterial and antifungal activity.
Stock solutions of the tested compounds, amikacin, chlor-
oamphenicol, and clotrimazole were prepared in DMSO at
concentrations of 1000 mg mL21, followed by two fold dilution
at concentrations of 500, 250, … 3.125 mg mL21. All the plates
were incubated at 37 uC for 24 h for bacteria and at 28 uC for 48
1
3124, 1154, 1308 cm21. H-NMR (CDCl3) d 2.7 and 3.7 (t, 2H,
–SO2–CH2–CH2 and –SO2–CH2–CH2), 2.1 and 2.9 (s, 3H, CH3),
7.1–7.7 (m, 14H, aromatic), 9.5 (s, 1H, –CONH). 13C-NMR
(CDCl3) d 22.49 (CH3), 36.12 (CH3), 11.32 (C5), 60.35 (C4),
103.74–149.36 (aromatic and olefinic carbon), 161.54 (CO),
162.54 (CO). ESI-MS calcd for m/z: 539.14. Anal. Calcd for
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h for fungi and the MICs were determined. Control experi-
ments were also carried out.
and 100% relative humidity. The medium without samples
served as a control and a triplicate was maintained for all
concentrations.
MTT is a yellow water soluble tetrazolium salt. A mitochon-
drial enzyme in living cells, succinate-dehydrogenase, cleaves
the tetrazolium ring, converting the MTT to an insoluble
purple formazan. Therefore, the amount of formazan pro-
duced is directly proportional to the number of viable cells.
After 48h of incubation, 15 mL of MTT (5 mg mL21) in
phosphate buffered saline (PBS) was added to each well and
incubated at 37 uC for 4h. The medium with MTT was then
flicked off and the formed formazan crystals were solubilized
in 100 mL of DMSO. The absorbance was measured at 570 nm
using a micro plate reader. The % cell inhibition was
determined using the following formula:
7 Antimycobacterial activity
All the compounds were screened for their in vitro antimyco-
bacterial activity against MTB. The antimicrobacterial activity
of the compounds was tested by the resazurin microplate assay
(REMA) following the method of Martin et al.41,42 MTB H37Rv
was grown in Middlebrook 7H11 broth medium supplemented
with 10% OADC (oleic acid, albumin, dextrose, and catalase, 1,
10, 100 mg L21). After incubation at 37 uC for 7 days, 15 mL of
0.01% resazurin (Sigma, St. Louis. MO, USA) solution in sterile
water was added to the first growth control wells and
incubated for 24 h. Once the first set of growth controls
turned pink, the dye solution was added to the second set of
growth controls and the test wells, and incubated for 24 h at 37
uC. A blue color in the wells containing the test compounds
would indicate inhibition of growth and pink would indicate
lack of inhibition of growth of M. tuberculosis. The MIC was
defined as the minimum concentration of the compound
required to achieve 99.9% inhibition of bacterial growth.
% cell inhibition = 100 2 Abs (sample)/Abs (control) 6 100
A nonlinear regression graph was plotted between the %
cell inhibition and the log10 concentration. The IC50 was
determined using Graph Pad Prism software.
Acknowledgements
We are thankful to NTS-India, Mysore, for financial support. P.
8 Anticancer activity
1
P thanks CDRI-Lucknow for the H-NMR, 13C-NMR and mass
The in vitro anticancer activity was analyzed by the MTT assay
method.43,44 The human cervical cancer cell line (HeLa) and
colon cancer cell line (HCT116) were obtained from National
Centre for Cell Science (NCCS), Pune, and grown in Eagles
Minimum Essential Medium (EMEM) containing 10% fetal
bovine serum (FBS). The cells were maintained at 37 uC, 5%
CO2, 95% air, and 100% relative humidity. Maintenance
cultures were passaged weekly, and the culture medium was
changed twice a week.
The monolayer cells were detached with trypsin-ethylene-
diaminetetraacetic acid (EDTA) to make single cell suspen-
sions and viable cells were counted using a hemocytometer
and diluted with a medium containing 5% FBS to give a final
density of 1 6 105 cells mL21. 100 mL per well of cell
suspension were seeded into 96-well plates at a plating density
of 10 000 cells per well and incubated to allow for cell
attachment at 37 uC, 5% CO2, 95% air, and 100% relative
humidity. After 24 h, the cells were treated with serial
concentrations of the test samples. They were initially
dissolved in neat dimethylsulfoxide (DMSO) to prepare the
stock (200 mM) and were stored frozen prior to use. At the time
of drug addition, an aliquot of frozen concentrate was thawed
and diluted twice to the desired final maximum test
concentration with serum free medium. An additional three,
two fold serial dilutions were made to provide a total of four
drug concentrations. Aliquots of 100 mL of these different drug
dilutions were added to the appropriate wells already contain-
ing 100 mL of medium, resulting in the required final drug
concentrations. Following the drug addition, the plates were
incubated for an additional 48 h at 37 uC, 5% CO2, 95% air,
spectral data and Malar Microbiological Laboratory,
Tirunelveli, Tamilnadu, for the antimicrobial study.
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