Benzimidazole clubbed with triazolo-thiadiazoles and triazolo-thiadiazines: New anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.07.011

Two series of Benzimidazole clubbed with triazolo-thiadiazoles (5a-q, 5r, 5s and 5x-a¹) and triazolo-thiadiazines (5t-w) were synthesized with an aim to produce promising anticancer agents. In vitro anticancer activities of synthesized compound

Full text of DOI:10.1016/j.ejmech.2012.07.011

European Journal of Medicinal Chemistry xxx (2012) 1e14
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Benzimidazole clubbed with triazolo-thiadiazoles and triazolo-thiadiazines: New
anticancer agents
*
Asif Husain , Mohd Rashid, M. Shaharyar, Anees A. Siddiqui, Ravinesh Mishra
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India
a r t i c l e i n f o
a b s t r a c t
Two series of Benzimidazole clubbed with triazolo-thiadiazoles (5aeq, 5r, 5s and 5xea1) and triazolo-
thiadiazines (5tew) were synthesized with an aim to produce promising anticancer agents. In vitro
anticancer activities of synthesized compounds were investigated at the National Cancer Institute (NCI)
against NCI 60 cell line panel; results showed good to remarkable broad-spectrum anticancer activity.
Among them, the compound 5h (NCS: 760452, 1-(1H-benzo [d] imidazol-2-yl)-3-(6-(2,4-
dichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl) propan-1-one) exhibited significant growth
Article history:
Received 27 March 2012
Received in revised form
2 July 2012
Accepted 5 July 2012
Available online xxx
inhibition with GI50 values ranging from 0.20 to 2.58
cell lines and further screened at 10-fold dilutions of five different concentrations (0.01, 0.1, 1, 10 and
mM and found superior selectivity for the leukemia
Keywords:
Benzimidazole
Triazole
Thiadiazole
Thiadiazine
Anticancer
100
m
M). The 5h may possibly be used as lead compound for developing new anticancer agents.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
Benzimidazole attached with other heterocyclic moieties
including fused rings resulted in compounds (hybrid molecules)
Cancer, a disease of the cell cycle, is one of the major health
problems in the world from decades. The effectiveness of many
existing anticancer drugs is limited by their toxicity to normal
rapidly growing cells and may develop resistance to that drug.
Another drawback is that majority of the drugs currently in the
market are not specific. Different classes of heterocyclic and fused
heterocyclic compounds have been identified through molecular
biology, empirical screening and rational drug development in
search of anticancer agents during the recent times [1e3].
Among the heterocyclic compounds, benzimidazoles are of
great importance due to their important biological actions as well
as synthetic applications in medicinal chemistry. Benzimidazoles
have been reported to have potential antitumor/antiproliferative/
anticancer activity [4e12] along with antibacterial [13,14], anti-
fungal [14e17], antiviral including anti-HIV [11,17] and antioxidant
activities [16,18]. Similarly, triazolo-thiadiazoles and triazolo-
thiadiazines are a class of fused heterocyclic compounds, which
have attracted great interest in medicinal chemistry owing to their
wide range of pharmacological activities including antitumor/
antiproliferative/anticancer activities [19e28].
with improved pharmacological profile [10e12]. Some biologically
active antitumor agents having benzimidazole and other heterocy-
clic moiety, for example, Nocodazole (NSC-238189) (1) [29], FB642
(2)[30], A-620223 (3)[31], Hoechst-33258 (4)[32], ABT-888(5)[33],
Phortress (6) [34], SNS-032 (7) [35], Proxazole (8) [36], CYC116 (9)
[37], thiadiazole derivative (10) [38], Levamisole (11) [39], imidazo
[2,1-b] [1, 3,4]thiadiazole analogs (12) [40], triazolo[1,3,4]thiadia-
zole derivative (13) [41] and similarly synthetic target compounds
(5h) are presented in Fig. 1. Here described in detail some rationally
designed target compounds with marketed drug Treanda (bend-
amustine hydrochloride) as antitumor agents made up by Cephalon
Inc. USA and comprises by mechlorethamine group and benzimid-
azole heterocyclic ring with a butyric acid substituent chemically as,
1H-benzimidazole-2-butanoicacid-5-[bis(2-chloroethyl)amino]-1-
methyl monohydro chloride, this is a rationally designed purine
analog and alkylator hybrid of chlorambucil chemotherapeutic
agent. On March 20, 2008, it was approved by the FDA (U.S.) for the
treatment of chronic lymphocytic leukemia (CLL) and about 6
months later, on October 31, 2008 this also approved for patients
with indolent B-cell non-Hodgkin’s lymphoma (NHL) [42,43]. The
chemically drawn of newly prepared target compounds rationally
designed with bendamustine along with chlorambucil represented
in Fig. 2.
* Corresponding author. Tel.: þ91 11 26059681/688x5890, þ91 9891116086
(mobile); fax: þ91 11 16988874.
In view of these points, and in continuation of our work on
benzimidazole derivatives bearing oxadiazole moiety [44] which
E-mail addresses: drasifhusain@yahoo.com, ahusain@jamiahamdard.ac.in
(A. Husain), yarmsy@rediffmail.com (M. Shaharyar).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.011
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2
A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
Fig. 1. Structure of some biologically active antitumor agents containing the benzimidazole and other heterocyclic scaffold and rationally designed targeted compounds (5h).
showed significant anticancer activities, it was thought worthwhile
to study new benzimidazoles clubbed with fused heterocyclic ring
systems; triazolo-thiadiazoles and triazolo-thiadiazines moieties,
with the hope of getting promising anticancer agents. Therefore,
a number of hybrid molecules were synthesized and screened for
their in vitro anticancer activities at the Development Therapeutic
Program (DTP), National Cancer Institute (NCI), Chemotherapeutic
Research division, USA, against full NCI 60 cell line panel according
their applied protocol. Twenty seven selected compounds among
thirty two newly synthesized hybrid molecules were submitted to
NCI and granted NCS codes (Table 1).
propan-1-one), was obtained by reacting hydrazine hydrate with
compound 4 (Scheme 1). After that different acids were reacted with
compound 5 to obtain the desired product as 1-(1H-benzo[d]imida-
zol-2-yl)-3-(6-(substituted)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-
yl)propan-1-one (5aeq) and the compounds (5r and 5s) obtained by
reacting compound 5 with KOH/CS2 and chloro-isothiocyanate,
respectively. The Compound 5 treated with different types of
a-chloro containing carbonyl compounds to produce 1-(1H-benzo[d]
imidazol-2-yl)-3-(6-substituted-7H-[1,2,4]triazolo[3,4-b][1,3,4]thia-
diazin-3-yl)propan-1-one (5tew) and 1-(1H-benzo[d]imidazol-2-
yl)-3-(6-(substituted)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)pr-
opan-1-one (5xea1) (Scheme 2). The products were purified by
recrystallization with suitable solvent and found pure upon TLC
examination. Their structures were established on the basis of
modernanalyticaltechniques (IR,1HNMR,13-NMR and mass spectral
data). The elemental analysis results were within ꢀ0.4% of the theo-
retical values.
2. Chemistry
The title compounds were synthesized as outlined in Schemes 1
and 2 following reported methods with appropriate modifications
[27,44e46]. The starting material, 4-(1H-benzo[d]imidazol-2-yl)-4-
oxobutanoic acid (1) was synthesized by reacting o-phenylenedi-
amine and
a-ketoglutaric acid in presence of HCl. 4-(1H-benzo[d]
3. Pharmacology
imidazol-2-yl)-4-oxobutanehydrazide (3) was obtained by treating
the compound 1 with hydrazine hydrate through ethyl-ester inter-
mediate(2).Thecompound3wastreatedwithCS2/KOHusingethanol
as solvent toget1-(1H-benzo[d]imidazol-2-yl)-3-(5-mercapto-1,3,4-
oxadiazol-2-yl)propan-1-one (4), and compound 5, (3-(4-amino-5-
mercapto-4H-1,2,4-triazol-3-yl)-1-(1H-benzo[d]imidazol-2-yl)
3.1. In vitro anticancer activity
All the selected 25 compounds were submitted to National
Cancer Institute (NCI), USA for evaluation of their in vitro anticancer
activity at single dose (1 ꢁ 10ꢂ5 M) against full NCI 60 cell lines
Please cite this article in press as: A. Husain, et al., Benzimidazole clubbed with triazolo-thiadiazoles and triazolo-thiadiazines: New anticancer
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A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
3
untreated control cells [49,50]. The output from the single dose
screen is analyzed by the COMPARE program and compounds
which exhibit significant growth inhibition at a single dose of
10
mM are further evaluated against the 60 cell panel at five
concentration levels.
4. Results and discussion
4.1. Chemistry
Thirty-two new compounds were prepared (Schemes 1 and 2)
and their IR,1H NMR,13C NMR and Mass spectral data and elemental
analysis results found in full agreement with their assigned struc-
tures. In general, IR spectra of the compounds showed peaks at 3326
and 2557 cmꢂ1 for NH and SH, respectively. In 1H NMR spectra, there
appeared a singlet at around
d 11.9 indicative of ring HeN and
another singlet at 13.4 for SeH, both disappeared by addition of
d
D2O as confirmation for these groups. The appeared peak in IR
spectra at around 1728 and 1600 cmꢂ1 accounted for C]O and C]N.
Thechemical shift in 13C NMR spectra at
d 173.56 and 156.24could be
accounted for C]O and C]N. The characteristic peaks at around
1384 and 1273 cmꢂ1 for N]CeS and NeN]C as indicative the
formation of thiadiazole and triazole ring. All the synthetic
compounds showed two triplets at appropriate signals and chemical
shifts in 1H NMR spectra at around
d
2.8 (J ¼ 6.9 Hz) and 3.3
Fig. 2. Chemical structure of active chemotherapeutic anticancer agents (bendamus-
tine, chlorambucil) and rationally designed template for targeted compound (5h).
(J ¼ 7.2 Hz) and 13C NMR spectra showed at around
d 30.9 and 22.4
which could be accounted for two methylene groups (eCH2eCH2e)
panels representing on full nine human systems as leukemia,
melanoma and cancers of lung, colon, brain, breast, ovary, kidney
and prostate. The screening is a two-stage process, beginning with
the evaluation of all compounds against the 60 cell lines at a single
dose. The compounds added at a concentration (1 ꢁ 10ꢂ5 M) and
the culture incubated for 48 h. End point determinations made with
a protein binding dye, sulforhodamine B [47,48]. Results for each
compound were reported as a mean graph of the percent growth of
the treated cells. Results of each test agents are reported as
percentage growth of the treated cells when compared with
forming
a linker chain through which benzimidazole nuclei
attached with triazole/thiadiazole nuclei made up the complete
back bone of synthetic compounds. The presence of doublet, triplet
at around
imidazole hydrogens. The multiplets peaks shown at around
7.2e6.9 accounted for aromatic protons. The signals in 13C NMR
spectra which appeared at around 166.2 could be for thiadiazole
carbon ring and other signals at 162.7, 158.3 indicative of triazole
carbon ring. Other peaks were observed at appropriate values
supporting the structure. The mass spectra (ESI MS) showed the
d 7.7, 7.5 and 7.2 (7.8 Hz, 7.5 Hz, 7.5 Hz) indicated benz-
d
d
d
d
Table 1
Sensitivity, NSC: Code, growth percent, delta value, mean growth percent of NCI cancer cell lines treated with synthesized compounds (10
m
M).
Delta Range Activitya
Compd. NSC: Code The most sensitive cell line Growth % of the most sensitive cell line Range of growth, % Mean
4
759213/1
760446/1
760447/1
760448/1
760449/1
760450/1
760451/1
760452/1
760455/1
760453/1
760454/1
760456/1
760457/1
760458/1
760459/1
760460/1
760461/1
760462/1
760463/1
760464/1
760465/1
760466/1
760468/1
760469/1
761987/1
HOP-92(Non-Small Cell Lung Cancer)
MCF7(Breast Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
NCI-H522(Non-Small Cell Lung Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
IGROV1(Ovarian Cancer)
NCI-H322M(Non-Small Cell Lung Cancer) 73.88
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
UO-31(Renal Cancer)
T-47D(Breast Cancer)
UO-31(Renal Cancer)
HOP-62(Non-Small Cell Lung Cancer)
69.36
61.97
63.03
64.29
30.17
75.55
76.24
0.38
69.36e128.74
61.97e126.96
63.03e143.70
64.29e122.39
30.17e107.44
75.55e123.55
76.24e112.60
ꢂ94.66e15.24
73.88e133.80
54.88e125.77
51.29e139.62
79.38e130.64
69.58e144.62
67.68e134.34
83.90e132.53
73.78e129.94
76.61e126.21
64.18e114.65
80.40e159.00
79.85e126.44
80.35e153.14
78.94e143.32
84.18e125.78
80.06e123.52
28.66e116.98
104.14
86.81
93.15
98.45
70.41
101.57
103.05
34.78
24.84
30.12
34.16
40.24
26.02
26.81
59.38 Inactive
64.99 Active
80.67 Active
58.10 Active
77.27 Active
54.60 Inactive
58.58 Inactive
5a
5b
5c
5d
5e
5g
5h
5i
ꢂ47.05b 47.61 109.90 Active
96.31
89.16
83.74
22.43
34.28
32.45
23.98
30.46
33.80
20.18
30.20
25.79
28.55
22.10
24.53
23.16
24.81
19.75
24.10
54.66
59.92 Inactive
70.89 Active
88.33 Active
51.26 Inactive
75.04 Inactive
66.66 Active
48.63 Inactive
56.16 Inactive
50.58 Inactive
54.64 Active
78.60 Inactive
46.59 Inactive
72.79 Inactive
64.38 Inactive
41.60 Inactive
50.92 Inactive
91.72 Active
5j
54.88
51.29
79.38
69.58
67.68
83.90
73.78
76.61
64.18
80.40
79.85
80.35
78.94
84.18
80.06
28.66
5k
5m
5n
5o
5p
5q
5r
103.36
100.04
101.48
104.08
103.98
102.40
92.73
102.50
104.38
103.51
103.75
103.93
104.16
79.92
5s
5t
5u
5v
5w
5x
5z
5a1
a
Compounds active of that particular cell lines, which showed growth inhibition ꢃ32% cell growth reduction following 48-h incubation with test compounds.
b
Negative indicates the cell kill.
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A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
Scheme 1. Protocol for synthesis of compound 5.
presence of peak at definite m/z value in accordance tothe molecular
ion peak. In case of aryl groups having chloro-substituent (s) the
molecular ion peak appeared as cluster of peaks.
(Renal Cancer), Compound 5r, 76.61% against UO-31 cell lines
(Renal Cancer), Compound 5s, 64.18% against UO-31 cell lines
(Renal Cancer), Compound 5t, 80.40% against UO-31 cell lines
(Renal Cancer), Compound 5u, 79.85% against UO-31 cell lines
(Renal Cancer), Compound 5v, 80.35% against UO-31 cell lines
(Renal Cancer), Compound 5w, 78.94% against UO-31 cell lines
(Renal Cancer), Compound 5x, 84.18% against T-47D cell lines
(Breast Cancer) and Compound 5z, 80.06% against UO-31 cell lines
(Renal Cancer), Compound 5a1 exhibited 28.66% growth inhibition
against HOP-62 cell lines (Non-Small Cell Lung Cancer) (Table 1).
The compounds which reduced the growth of the cell lines to 32%
or less (negative number indicate kills) is considered in vitro active
[51,52]. Compound (5h, NCS: 760452) satisfied pre-determined
threshold growth inhibition criteria and further selected for NCI
full panel five dose assay at 10-fold dilutions of five different
4.2. In vitro anticancer activity
The synthesized compounds (25 in no.) were submitted to
National Cancer Institute (NCI), USA for evaluation of their in vitro
anticancer activity at single dose (1 ꢁ 10ꢂ5 M) against full NCI 60
cell lines panels representing on full nine human systems as
leukemia, melanoma and cancers of lung, colon, brain, breast,
ovary, kidney and prostate. The compounds added at a concentra-
tion (1 ꢁ 10ꢂ5 M) and the culture incubated for 48 h. End point
determinations made with a protein binding dye, sulforhodamine B
[47,48]. Results for each compound were reported as a mean graph
of the percent growth of the treated cells. Results of each test agents
are reported as percentage growth of the treated cells when
compared with untreated control cells [49,50]. Compound 4 shown
69.36% growth inhibition against HOP-92 cell line (Non-Small Cell
Lung Cancer), Compound 5a, 61.97% against MCF7 cell lines (Breast
Cancer), Compound 5b, 63.03% against UO-31 cell lines (Renal
Cancer), Compound 5c, 64.29% against UO-31 cell lines (Renal
Cancer), Compound 5d, 30.17% against NCI-H522 cell lines (Non-
Small Cell Lung Cancer), Compound 5e, 75.55% against UO-31 cell
lines (Renal Cancer), Compound 5g, 76.24% against UO-31 cell lines
(Renal Cancer), Compound 5h, 0.38% against IGROV1 cell lines
(Ovarian Cancer), Compound 5i, 73.88% against NCI-H322M cell
lines (Non-Small Cell Lung Cancer), Compound 5j, 54.88% against
UO-31 cell lines (Renal Cancer), Compound 5k, 51.29% against UO-
31 cell lines (Renal Cancer), Compound 5m, 79.38% against UO-31
cell lines (Renal Cancer), Compound 5n, 69.58% against UO-31
cell lines (Renal Cancer), Compound 5o, 67.68% against UO-31 cell
lines (Renal Cancer), Compound 5p, 83.90% against UO-31 cell lines
(Renal Cancer), Compound 5q, 73.78% against UO-31 cell lines
concentrations (0.01, 0.1, 1, 10 and 100 mM). The result of tested
compound is given by three response parameters (GI50, TGI and
LC50) for each cell line from log concentration vs % growth inhibi-
tion curves on nine cancer disease (Fig. 3). The GI50 value (growth
inhibitory activity) corresponds to the concentration of the
compound causing 50% decrease in net cell growth, the TGI value
(cytostatic activity) is the concentration of the compound resulting
in total growth inhibition and LC50 value (cytotoxic activity) is the
concentration of the compound causing net 50% loss of initial cells
at the end of the incubation period of 48 h. Furthermore, a mean
graph midpoint (MG-MID) is calculated giving an averaged activity
parameter overall cell lines.
The title compound under investigation (5h, NCS: 760452)
exhibited remarkable anticancer activity against all the tested cell
lines representing nine different subpanels with GI50 values
between 0.20 and 2.58 mM under sensitive range an outstanding
activity (Table 2). With regard to the sensitivity against some
individual cell lines the compound showed high activity against
Leukemia CCRF-CEM and MOLT-4 cell lines with GI50 0.26 and
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A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
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Scheme 2. Protocol for synthesis of title compounds.
0.28
profile towards non-small cell Lung cancer subpanel (GI50 value
ranging from 0.24 to 2.0 M), least for NCI-H226 and maximum for
A549/ATCC cell line. The compound proved to be sensitive towards
all the tested Colon cancer cell lines with not more than 1.63
concentrations. All the tested Leukemia cancer cell lines were
sensitive with not more than GI50 > 1 M concentrations of the
m
M respectively. Obtained data revealed an obvious sensitivity
their individual subpanel MIDb (the average sensitivity of all cell
lines of a particular subpanel towards the test agent). The ratios
between 3 and 6 refer to moderate selectivity; ratios greater than 6
indicate high selectivity towards the corresponding cell line, while
compounds not meeting either of these criteria rated non-selective
[53]. As per this criterion, compound 5h in the study was found to
be mild selective towards Leukemia cancer subpanel. Over all the
GI50 values of the screening process resulted in a sensitive range
m
mM
m
tested compound. The highest growth inhibitory activity was
observed against the Colon SW-620 cancer cell line with GI50 value
inferior of 2.58
m
M denoting an outstanding activity and the values
M (Table 2). The log molar
0.20
mM. The all remaining subpanel cell line showed maximum
of LC50 are in most of the cell lines >100 m
sensitive towards tested compound with not more than 2.58
m
M
concentration of the resulted screening as log GI50 ranged
from ꢂ6.58 to ꢂ5.66, log TGI ꢂ6.26 to ꢂ5.18 except MOLT-4, RPMI-
8226, SR and OVCAR-8 (>ꢂ4.00) and all Leukemia cell lines showed
log LC50 > ꢂ4.00. A mean graph midpoint (MG-MID) calculated for
concentrations (Table 2). The criterion for selectivity of a compound
depends upon the ratio obtained by dividing the full panel MIDa
(the average sensitivity of all cell lines towards the test agent) by
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A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
Fig. 3. Dose response curves (% growth verses sample concentration at NCI fixed protocol, mM) obtained from the NCI’s in vitro disease-oriented human tumor cells line of
compound (5h) on nine cancer disease. The different color and shape of NCI, subpanel cell lines indicative of growth percentage inhibition with concentration of sample. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
c. On the other hand the electron releasing groups like methyl
attached to phenyl ring showed decrease in the sensitivity
(84.18%) as shown by compound 5x, 1-(1H-benzo[d]imidazol-
2-yl)-3-(6-methyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)
propan-1-one.
each of parameters, giving as log GI50 (ꢂ6.07), log TGI (ꢂ5.51) and
log LC50 (ꢂ4.85) and insensitive cell lines are also included with the
highest concentration (Table 3).
4.2.1. Structural activity relationship (SAR)
d. Presence of diphenylmethane group (5d) or electron with
drawing group (s) significantly improved anticancer activity
against the human cancer cell lines of nine subpanels.
e. Compound 5h may serve as novel template for development of
potential and selective agents in the field of cancer
chemotherapy.
Following points could be drawn regarding the SAR of newly
synthesized compounds in relation to their anticancer activity:
a. Benzimidazoles clubbed with triazolo-thiadiazole were found
to have better anticancer activities than those of benzimid-
azoles clubbed with triazolo-thiadiazine.
b. The anticancer activity was influenced by the presence of elec-
tron with drawing group like chloro on ortho, meta or para
position on aromatic ring. Compound 5s having chloro group on
para position of phenyl ring, 1-(1H-benzo[d]imidazol-2-yl)-3-
(6-(4-chlorophenyl-amino)-[1,2,4]triazolo[3,4-b][1,3,4]thiadia-
zol-3-yl)propan-1-one, showed increased in sensitivity of cell
line (64.18%) and similarly the same group disubstituted on meta
and para position, showed synergistic action in the sensitivity
(ꢂ47.05%) as exhibited by the compound 5h, 1-(1H-benzo[d]
imidazol-2-yl)-3-(6-(2,4-dichlorophenyl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-3-yl) propan-1-one.
5. Conclusion
Two series of benzimidazole clubbed with triazolo-thiadiazole
and triazolo-thiadiazine rings, comprising of 32 new compounds
were successfully synthesized and among them 25 compounds were
evaluated for their in vitro anticancer activity at the NCI, USA.
Compounds showed good to remarkable and broad-spectrum anti-
cancer activity. One compound (5h), namely 1-(1H-benzo [d]imida-
zol-2-yl)-3-(6-(2,4-dichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thia-
diazol-3-yl)propan-1-one emerged as lead compound with broad
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A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
7
Fig. 3. (continued).
spectrum of anticancer activities against tumor cell lines (MG-
MID ꢂ6.07, ꢂ5.51 and ꢂ4.85 value of log10 GI50, log10 TGI and
log10 LC50 respectively). Based on these observations, it could be
concluded that the compound (5h) may be used as template for
development of novel anticancer agents.
HCl (4 N; 5 mL) was refluxed for 6 h and then left to cool to room
temperature. 10% NaOH solution was added slowly to neutralize
the reaction mixture, when a solid mass precipitated out, which
was filtered, washed with water and recrystallized with ethanol.
Yield: 87%, m.p. 261e262 ꢄC, Rf 0.71 [toluene:ethyl acetate:formic
acid (5:4:1)]; IR (KBr, cmꢂ1): 3394 (OeH), 3326 (NeH), 3114
(CeH, AreH), 2972 (CeH, CH2), 1728 (C]O), 1600 (C]N), 1562
(C]C); 1H NMR (DMSO-d6): 12.97 (s, 1H, OH, D2O exchangeable),
12.34 (s, 1H, NH, D2O exchangeable), 7.66 (d, 1H, J ¼ 7.5 Hz, H-4,
benzimidazole), 7.48 (t, 1H, J ¼ 7.2 Hz, H-7, benzimidazole), 7.27
(t, 2H, J ¼ 8.7 Hz, H-5,6, benzimidazole), 3.05 (t, 2H, J ¼ 6.9 Hz,
CH2), 2.76 (t, 2H, J ¼ 6.9 Hz, CH2); 13C NMR (DMSO-d6): 175.57
(C]O), 168.21 (C]O, COOH), 159.67 (C]N), 138.27, 132.21,
130.19, 128.57, 124.65, 123.64 (AreC), 34.23 (CH2, CH2CO), 31.36
(CH2, CH2COOH); ESI MS (m/z): 218 (Mþ); Anal. calcd. for
6. Experimental protocols
6.1. General materials and instrumentations
All chemicals (reagents and solvents) were procured from E.
Merck Ltd. and S.D. Fine Chemicals (India). Melting points were
taken on a liquid paraffin bath in open capillary tubes and are
uncorrected. Progress of the reactions was monitored by using TLC
plates (silica gel G), Toluene:Ethyl acetate:Formic acid (5:4:1, v/v/v)
and benzene: acetone (9:1, v/v) used as solvent systems. The spots
were located by exposure to iodine vapors or under UV-light.
Nuclear magnetic resonance (1H NMR and 13C NMR) spectra were
recorded on Bruker spectrospin DPX-300 MHz in DMSO-d6/CDCl3;
C
11H10N2O3: C, 60.55; H, 4.62; N, 12.84. Found: C, 60.63; H, 4.65;
N, 12.98.
6.2.2. Synthesis of ethyl-4-(1H-benzo[d]imidazol-2-yl)-4-oxobutanoate
(2)
chemical shift (d) values reported in parts per million (ppm) and
coupling constants (J) in Hz using tetramethylsilane as internal
reference. The splitting pattern abbreviations are as follows: s,
singlet; bs, broad singlet; d, doublet; dd, double doublet; t, triplet;
q, quadruplet; m, multiplet. The exchangeable protons (OH and NH)
confirmed by the addition of D2O. Mass spectra recorded on LCMS/
MS (PerkineElmer and LABINDIA, Applied Biosystems) model no.
API 3000, presented as m/z. IR spectra recorded on FT/IR (Jasco,
Japan), model no.410. Elemental analyses performed on a Per-
kineElmer 240 analyzer and found in the range of ꢀ0.4% for each
element analyzed (C, H and N).
A solution of compound (1) (0.01 mol) in absolute ethanol
(25 mL) was refluxed for 10 h in presence of conc. H2SO4, (0.1 mL).
After completion of the reaction, it was cooled to room temperature,
diluted with cold waterand then neutralized with NaHCO3 to pH 7. A
precipitate formed which was filtered and crystallized from ethanol.
Yield: 81%, m.p. 253e254 ꢄC, Rf ¼ 0.60, [toluene:ethyl acetate:formic
acid (5:4:1)]; IR (KBr, cmꢂ1): 3330 (NeH), 3047 (CeH, AreH), 2958
(CeH, CH2), 1718 (C]O), 1640 (C]N),1570 (C]C); 1H NMR (DMSO-
d6): 12.51 (bs,1H, NH, D2O exchangeable), 7.73 (d,1H, J ¼ 7.8 Hz, H-4,
benzimidazole), 7.54 (t, 1H, J ¼ 7.5 Hz, H-7, benzimidazole), 7.12
(t, 2H, J ¼ 7.5 Hz, H-5,6, benzimidazole), 3.82 (q, 2H, J ¼ 5.4 Hz, CH2),
3.03 (t, 2H, J ¼ 7.2 Hz, CH2), 2.71 (t, 2H, J ¼ 7.2 Hz, CH2), 1.15 (t, 3H,
J ¼ 1.2 Hz, CH3); 13C NMR (DMSO-d6):177.61 (C]O), 165.37 (C]O,
ester), 156.47 (C]N), 138.32, 137.93, 129.13, 128.17, 124.55, 123.74
(AreC), 30.81 (CH2, CH2CO), 28.36 (CH2, CH2COO), 69.07 (CH2, ester),
6.2. Synthesis
6.2.1. Synthesis of 4-(1H-benzo[d]imidazol-2-yl)-4-oxobutanoic acid (1)
A solution of o-phenylenediamine (0.01 mol),
a-ketoglutaric
acid (equimolar; 0.01 mol) in methanol/water mixture (1:1) and
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8
A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
Table 2
Calculated values of GI50, TGI, LC50 of the cell lines, NCI: full cell lines panel, MG-MID and selectivity ratio of the compound (5h, NCS: 760452).
Panel
Cell line
GI50(10ꢂ6 M)
TGI (10ꢂ6 M)
LC50 (10ꢂ6 M)c
Concentration per cell line
Subpanel concentration
2.094
Subpanel MIDb
0.418
Selectivity ratio
2.485
Leukemia
CCRF-CEM
HL-60(TB)
MOLT-4
RPMI-8226
SR
0.265
0.862
0.282
0.352
0.333
2.07
0.974
5.93
>100
1.76
>100
5.63
3.69
2.12
0.696
2.48
3.74
2.17
2.96
2.21
3.20
1.24
2.21
1.81
2.99
0.545
2.14
2.73
4.86
5.44
4.65
2.61
1.59
2.94
2.45
0.842
6.67
1.67
2.69
5.01
2.44
3.51
1.80
2.82
3.98
>100
5.80
3.68
3.06
2.32
1.35
3.44
1.99
3.10
3.55
2.02
3.90
2.46
1.50
2.42
e
>100
>100
>100
>100
>100
>100
Nt
6.16
2.59
Nt
7.94
5.21
6.82
5.50
6.29
5.06
Nt
>100
7.70
2.48
5.15
Nt
22.8
>100
17.1
5.34
Nt
Nt
Nt
Nt
>100
4.24
5.19
22.5
5.61
Nt
4.60
8.04
8.39
>100
>100
7.28
5.92
4.89
4.67
Nt
Non-Small Cell
Lung Cancer
A549/ATCC
EKVX
1.53
HOP-62
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
0.576
0.244
0.719
1.76
0.766
1.28
0.767
1.63
0.320
0.489
0.362
1.16
0.204
0.740
1.03
2.17
1.37
8.945
1.118
0.929
Colon Cancer
4.932
8.55
0.704
1.425
1.476
0.729
CNS Cancer
Melanoma
1.96
1.28
0.363
0.762
0.596
0.265
1.76
0.474
1.39
2.10
1.06
1.31
0.514
0.971
1.88
2.58
1.56
1.86
1.59
1.10
0.339
1.45
0.674
1.23
2.01
0.576
0.695
1.20
0.355
1.00
0.478
2.16
0.753
0.699
1.04
8.77
10.675
8.969
0.974
1.525
1.121
1.067
0.681
0.927
Ovarian Cancer
Renal Cancer
4.55
Nt
6.30
4.63
>100
5.02
5.97
5.87
>100
>100
7.06
5.41
Prostate Cancer
Breast Cancer
1.895
5.445
0.947
0.907
1.097
1.145
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
4.77
2.51
2.15
MIDa
a
Mean graph midpoint (arithmetical mean value of treated cancer cell lines) representing the average sensitivity of all cell lines (full panel) towards the test agent in mM.
b
c
Average sensitivity of all cell lines of a particular subpanel towards the test agent in
Nt indicates not tested.
mM.
20.31 (CH3); ESI MS (m/z): 246 (Mþ); Anal. calcd. for C13H14N2O3: C,
63.40; H, 5.73; N, 11.38. Found: C, 62.77; H, 5.81; N, 11.35.
was cooled, a solid precipitate separated out, which was filtered,
dried and recrystallized from methanol. Yield: 85%, m.p.
241e242 ꢄC, Rf ¼ 0.53 [toluene:ethyl acetate:formic acid (5:4:1)]; IR
(KBr, cmꢂ1): 3494 (NeH, NH2), 3359 (NeH, NH), 3151 (CeH, AreH),
2947 (CeH, CH2), 1691 (C]O), 1643 (C]N), 1600 (C]C); 1H NMR
(DMSO-d6): 11.51 (s, 1H, NH, benzimidazole, D2O exchangeable),
10.72 (s, 1H, NH, hydrazide, D2O exchangeable), 9.05 (bs, 2H, NH2,
6.2.3. Synthesis of 4-(1H-benzo[d]imidazol-2-yl)-4-oxobutane
hydrazide (3)
A solution of compound 2 (0.01 mol) and hydrazine hydrate
(0.015 mol) in ethanol was refluxed for 8 h. The reaction mixture
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A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
9
232 (Mþ); Anal. calcd. for C11H12N4O2: C, 56.89; H, 5.21; N, 24.12.
Found: C, 56.73; H, 5.27; N, 24.15.
Table 3
Values of the log molar concentration of response parameter (log10GI50, log10TGI
and log10LC50) of the title compound (5h, NCS: 760452).
a
Cancer disease
Leukemia
Used cell lines
log10 GI50
log10 TGI
log10 LC50
6.2.4. Synthesis of 1-(1H-benzo[d]imidazol-2-yl)-3-(5-mercapto-
1,3,4-oxadiazol-2-yl)propan-1-one (4)
CCRF-CEM
HL-60(TB)
MOLT-4
RPMI-8226
SR
ꢂ6.58
ꢂ6.06
ꢂ6.55
ꢂ6.45
ꢂ6.48
ꢂ5.68
ꢂ5.81
ꢂ6.24
ꢂ6.61
ꢂ6.14
ꢂ5.75
ꢂ6.12
ꢂ5.89
ꢂ6.12
ꢂ5.79
ꢂ6.50
ꢂ6.31
ꢂ6.44
ꢂ5.94
ꢂ6.69
ꢂ6.13
ꢂ5.99
ꢂ5.66
ꢂ5.86
ꢂ5.71
ꢂ5.89
ꢂ6.44
ꢂ6.12
ꢂ6.22
ꢂ6.58
ꢂ5.75
ꢂ6.32
ꢂ5.86
ꢂ5.68
ꢂ5.97
ꢂ5.88
ꢂ6.29
ꢂ6.01
ꢂ5.72
ꢂ5.59
ꢂ5.81
ꢂ5.73
ꢂ5.80
ꢂ5.96
ꢂ6.47
ꢂ5.84
ꢂ6.17
ꢂ5.91
ꢂ5.70
ꢂ6.24
ꢂ6.16
ꢂ5.92
ꢂ6.45
ꢂ6.00
ꢂ6.32
ꢂ5.67
ꢂ6.12
ꢂ6.16
ꢂ6.07
0.62
ꢂ6.01
5.23
>ꢂ4.00
>ꢂ4.00
>ꢂ4.00
>ꢂ4.00
>ꢂ4.00
>ꢂ4.00
Nt
ꢂ5.21
ꢂ5.59
Nt
ꢂ5.10
ꢂ5.28
ꢂ5.17
ꢂ5.26
ꢂ5.20
ꢂ5.30
Nt
>ꢂ4.00
ꢂ5.11
ꢂ5.60
ꢂ5.29
Nt
ꢂ4.64
>ꢂ4.00
ꢂ4.77
ꢂ5.27
Nt
Nt
Nt
Nt
>ꢂ4.00
ꢂ5.37
ꢂ5.28
ꢂ4.65
ꢂ5.25
Nt
ꢂ5.34
ꢂ5.09
ꢂ5.08
>ꢂ4.00
>ꢂ4.00
ꢂ5.14
ꢂ5.23
ꢂ5.31
ꢂ5.33
Nt
A mixture of compound 3 (0.01 mol) and CS2/KOH (0.01 mol) in
ethanol/water (1:1) refluxed for 6 h. After completion of reaction,
the reaction mixture was reduced to half of its original volume and
cold water (20 mL) added to it. The contents were then acidified
with conc. HCl. A solid precipitate obtained which was filtered,
washed with water and recrystallized from methanol. Yield: 75%;
m.p. 220e221 ꢄC; Rf ¼ 0.47 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3341 (NH), 3066 (CH), 2900 (CH2), 2599
(SH), 1685 (C]O), 1654 (C]N), 1627 (C]C), 1280 (NeN]C), 1161
(CeOeC, asymmetric), 1060 (CeOeC, symmetric); 1H NMR (CDCl3):
13.40 (s, 1H, SH, D2O exchangeable), 11.31 (s, 1H, NH, D2O
exchangeable), 7.81 (d, 1H, J ¼ 8.1 Hz, H-4, benzimidazole), 7.49 (t,
1H, J ¼ 7.5 Hz, H-7, benzimidazole), 7.32 (t, 2H, J ¼ 7.5 Hz, H-5,6,
benzimidazole), 3.31 (t, 2H, J ¼ 6.9 Hz, CH2), 2.92 (t, 2H, J ¼ 6.9 Hz,
CH2CO); 13C NMR (DMSO-d6): 179.14 (C]O), 162.77, 156.31
(C-oxadiazole), 159.84 (C]N), 133.93, 132.26, 129.62, 128.45,
122.96, 116.86 (AreC), 30.14 (CH2, CH2CO), 22.25 (CH2); ESI MS
(m/z): 274 (Mþ); Anal. calcd. for C12H10N4O2S: C, 52.54; H, 3.67; N,
20.43. Found: C, 52.61; H, 4.05; N, 20.51.
>ꢂ4.00
ꢂ5.75
>ꢂ4.00
ꢂ5.25
ꢂ5.43
ꢂ5.67
ꢂ6.16
ꢂ5.61
ꢂ5.43
ꢂ5.66
ꢂ5.53
ꢂ5.66
ꢂ5.50
ꢂ5.91
ꢂ5.65
ꢂ5.74
ꢂ5.52
ꢂ6.26
ꢂ5.67
ꢂ5.56
ꢂ5.31
ꢂ5.26
ꢂ5.33
ꢂ5.58
ꢂ5.80
ꢂ5.53
ꢂ5.61
ꢂ6.07
ꢂ5.18
ꢂ5.78
ꢂ5.57
ꢂ5.30
ꢂ5.61
ꢂ5.46
ꢂ5.74
ꢂ5.55
ꢂ5.40
> ꢂ4.00
ꢂ5.24
ꢂ5.43
ꢂ5.51
ꢂ5.63
ꢂ5.87
ꢂ5.46
ꢂ5.70
ꢂ5.51
ꢂ5.45
ꢂ5.69
ꢂ5.41
ꢂ5.61
ꢂ5.82
ꢂ5.62
e
Non-Small Cell
Lung Cancer
A549/ATCC
EKVX
HOP-62
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
Colon Cancer
CNS Cancer
Melanoma
6.2.5. Synthesis of 3-(4-amino-5-mercapto-4H-1,2,4-triazol-3-yl)-
1-(1H-benzo[d]imidazol-2-yl) propan-1-one (5)
To a solution of compounds 4 (0.01 mol) in n-butanol (40 mL),
hydrazine hydrate (0.03 mol) was added. The reaction mixture was
refluxed for 5 h and after completion of the reaction, KOH
(0.015 mol) was added to reaction media. A solid precipitate
separated out, which was acidified with conc. HCl, filtered, washed
with H2O and recrystallized from dichloromethane. Yield: 70%;
m.p. 231e232 ꢄC; Rf ¼ 0.45 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3429 (NH), 3105 (CH), 2904 (CH2), 2557
(SH), 1728 (C]O), 1670 (C]N), 1566 (C]C), 1303 (NeN]C); 1H
NMR (DMSO-d6): 13.43 (s, 1H, SH, D2O exchangeable), 12.40 (s, 1H,
NH, benzimidazole, D2O exchangeable), 7.73 (d, 1H, J ¼ 7.8 Hz, H-4-
benzimidazole), 7.51 (t, 1H, J ¼ 7.5 Hz, H-7-benzimidazole), 7.32
(t, 2H, J ¼ 7.8 Hz, H-5,6-benzimidazole), 3.53 (s, 2H, NH2), 3.20 (t,
2H, J ¼ 6.3 Hz, CH2), 2.76 (t, 2H, J ¼ 7.2 Hz, CH2CO); 13C NMR (DMSO-
d6): 166.16 (C]O),160.32,152.14 (C-triazole),155.06 (C]N),132.29,
131.94, 130.03, 128.59, 123.54, 115.80 (AreC), 29.24 (CH2, CH2CO),
21.41 (CH2); ESI MS (m/z): 288 (Mþ); Anal. calcd. for C12H12N6OS: C,
49.99; H, 4.19; N, 29.15. Found: C, 49.85; H, 4.25; N, 29.37.
Ovarian Cancer
Renal Cancer
ꢂ5.34
Nt
ꢂ5.20
ꢂ5.33
>ꢂ4.00
ꢂ5.30
ꢂ5.22
ꢂ5.23
>ꢂ4.00
>ꢂ4.00
ꢂ5.15
ꢂ5.27
ꢂ4.85
0.75
Prostate Cancer
Breast Cancer
6.2.6. General procedure for synthesis of 1-(1H-benzo[d]imidazol-
2-yl)-3-(6-(substituted)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)
propan-1-one (5aeq)
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
Compounds (5aeq) were prepared by reacting compound 5
(0.003 mol) and different types of aliphatic/aromatic acids (equi-
molar; 0.003 mol) in phosphorus oxychloride (5 mL) under reflux
for 5e6 h. After completion of reaction, the reaction mixture was
cooled to room temperature and then gradually poured on to
crushed ice with continuous stirring. The obtained solid precipitate
was treated with dilute sodium hydroxide solution and washed
thoroughly with cold water to remove the inorganic component.
The compound so obtained was filtered, dried and recrystallized
from ethanol.
ꢂ5.32
ꢂ5.60
ꢂ5.67
ꢂ5.51
0.75
MID
Delta
Range
1.1
2.26
1.6
a
Nt indicates compound not evaluated on that particular cell lines; showed
M.
values >100
m
D2O exchangeable), 7.68 (d,1H, J ¼ 7.8 Hz, H-4, benzimidazole), 7.50
(t, 1H, J ¼ 7.2 Hz, H-7, benzimidazole), 7.27 (t, 2H, J ¼ 7.5 Hz, H-5,6,
benzimidazole), 3.02 (t, 2H, J ¼ 7.5 Hz, CH2), 2.52 (t, 2H, J ¼ 7.8 Hz,
CH2CO); 13C NMR (DMSO-d6):179.15 (C]O), 162.72 (C]O, hydra-
zide), 156.32 (C]N), 133.99, 132.28, 129.59, 128.45, 122.92, 116.85
(AreC), 30.17 (CH2, CH2CO), 22.27 (CH2, hydrazide); ESI MS (m/z):
6.2.6.1. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(2-aminophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadia-zol-3-yl)propan-1-one (5a). Yield: 66%;
m.p. 215e216 ꢄC; Rf ¼ 0.53 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3357 (NH), 3063 (CH), 2955 (CH2), 1711
(C]O), 1657 (C]N), 1563 (C]C), 1385 (N]CeS), 1320 (NeN]C);
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10
A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
1H NMR (DMSO-d6): 12.37 (s, 1H, NH, D2O exchangeable), 7.71 (d,
1H, J ¼ 7.8 Hz, H-4, benzimidazole), 7.48 (t, 1H, J ¼ 7.5 Hz, H-7,
benzimidazole), 7.30 (t, 2H, J ¼ 7.5 Hz, H-5,6, benzimidazole),
7.27e6.95 (m, 4H, phenyl), 5.92 (s, 2H, NH2), 3.23 (t, 2H, J ¼ 6.9 Hz,
CH2), 2.87 (t, 2H, J ¼ 6.9 Hz, CH2CO); 13C NMR (DMSO-d6): 170.41
(C]O), 169.23 (C-thiadiazole), 161.23, 158.01 (C-triazole), 154.93
(C]N), 138.21, 133.05, 132.75, 131.45, 129.84, 129.31, 128.53, 127.47,
124.37, 123.92, 117.32, 115.03 (AreC), 33.73 (CH2, CH2CO), 24.56
(CH2); ESI MS (m/z): 389 (Mþ); Anal. calcd. for C19H15N7OS: C,
58.60; H, 3.88; N, 25.18. Found: C, 58.71; H, 3.97; N, 25.21.
(NH), 3076 (CH), 2966 (CH2), 2335 (CN), 1718 (C]O), 1664 (C]N),
1569 (C]C), 1384 (N]CeS), 1263 (NeN]C); 1H NMR (CDCl3):11.76
(s, 1H, NH, D2O exchangeable), 7.81 (d, 1H, J ¼ 8.7 Hz, H-4, benz-
imidazole), 7.52 (t, 1H, J ¼ 7.8 Hz, H-7, benzimidazole), 7.33 (t, 2H,
J ¼ 7.8 Hz, H-5,6, benzimidazole), 3.72 (s, 2H, CH2CN), 3.34 (t, 2H,
J ¼ 7.2 Hz, CH2), 2.95 (t, 2H, J ¼ 6.9 Hz, CH2CO); 13C NMR (DMSO-d6):
174.39 (C]O), 172.89 (C-thiadiazole), 160.73, 160.42 (C-triazole),
155.01 (C]N), 132.11, 131.83, 129.97, 128.50, 115.72 (AreC), 60.24
(CN), 30.01 (CH2, CH2CO), 28.07 (CH2), 14.60 (CH2, CH2CN); ESI MS
(m/z): 337 (Mþ); Anal. calcd. for C15H11N7OS: C, 53.40; H, 3.29; N,
29.06. Found: C, 53.65; H, 3.41; N, 29.11.
6.2.6.2. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(2-chlorobenzyl)-[1,2,4]
triazolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5b). Yield: 65%;
m.p. 212e213 ꢄC; Rf ¼ 0.56 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3373 (NH), 3042 (CH), 2930 (CH2), 1720
(C]O), 1625 (C]N), 1561 (C]C), 1383 (N]CeS), 1280 (NeN]C),
713 (CCl); 1H NMR (DMSO-d6): 11.97 (s, 1H, NH, D2O exchange-
able), 7.87 (d, 1H, J ¼ 8.1 Hz, H-4, benzimidazole), 7.64 (t, 1H,
J ¼ 8.1 Hz, H-7, benzimidazole), 7.36 (t, 2H, J ¼ 7.8 Hz, H-5,6,
benzimidazole), 7.30e7.01 (m, 4H, phenyl), 4.10 (s, 2H, CH2), 3.68 (t,
2H, J ¼ 6.9 Hz, CH2), 2.88 (t, 2H, J ¼ 6.9 Hz, CH2CO); 13C NMR
(DMSO-d6): 174.61 (C]O), 166.23 (C-thiadiazole), 160.42, 158.74
(C-triazole), 156.52 (C]N), 136.13, 135.98, 134.57, 132.45, 130.35,
129.17, 128.86, 127.45, 124.90, 124.21, 123.16, 115.38 (AreC), 34.81
(CH2, CH2CO), 30.51 (CH2), 22.14 (CH2); ESI MS (m/z): 422 (Mþ);
Anal. calcd. for C20H15ClN6OS: C, 56.80; H, 3.58; N, 19.87. Found: C,
57.15; H, 3.65; N, 19.95.
6.2.6.6. 2-(3-(3-(1H-Benzo[d]imidazol-2-yl)-3-oxopropyl)-[1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazol-6-yl)phenyl acetate (5f). Yield: 66%;
m.p. 195e196 ꢄC; Rf ¼ 0.61 [benzene:acetone (9:1)]; IR (KBr, cmꢂ1):
3327 (NH), 3053 (CH), 2941 (CH2), 1692 (C]O), 1628 (C]N), 1526
(C]C), 1384 (N]CeS), 1260 (NeN]C); 1H NMR (CDCl3): 12.05
(s, 1H, NH, D2O exchangeable), 8.13e7.78 (m, 4H, phenyl), 7.73 (d,
1H, J ¼ 7.5 Hz, H-4, benzimidazole), 7.51 (t, 1H, J ¼ 7.8 Hz, H-7,
benzimidazole), 7.35 (t, 2H, J ¼ 8.1 Hz, H-5,6, benzimidazole), 3.27
(t, 2H, J ¼ 6.9 Hz, CH2), 2.74 (t, 2H, J ¼ 6.9 Hz, CH2CO), 2.53 (s, 3H,
CH3); 13C NMR (CDCl3): 175.21 (C]O), 172.45 (CO, COCH3), 169.81
(C-thiadiazole), 162.52, 158.21 (C-triazole), 156.37 (C]N), 132.10,
129.83, 128.46, 126.87, 125.23, 123.41, 122.35, 121.89, 120.53, 118.76,
118.21, 116.71 (AreC), 32.13 (CH2, CH2CO), 24.17 (CH2), 18.61 (CH3);
ESI MS (m/z): 432 (Mþ); Anal. calcd. for C21H16N6O3S: C, 58.32; H,
3.73; N, 19.43. Found: C, 57.43; H, 3.83; N, 19.47.
6.2.6.3. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(phenoxymethyl)-[1,2,4]
triazolo[3,4-b][1,3,4] thiadiazol-3-yl) propan-1-one (5c). Yield: 67%;
m.p. 200e201 ꢄC; Rf ¼ 0.52 [benzene:acetone (9:1)]; IR (KBr, cmꢂ1):
3341 (NH), 3010 (CH), 2963 (CH2), 1710 (C]O), 1647 (C]N), 1573
(C]C), 1373 (N]CeS), 1314 (NeN]C), 1247 (CeOeC, asymmetric),
1040 (CeOeC, symmetric), 1193 (OCH2); 1H NMR (DMSO-d6): 12.27
(s, 1H, NH, D2O exchangeable), 7.81 (d, 1H, J ¼ 7.8 Hz, H-4, benz-
imidazole), 7.54 (t, 1H, J ¼ 7.8 Hz, H-7, benzimidazole), 7.41 (t, 2H,
J ¼ 7.5 Hz, H-5,6, benzimidazole), 7.31e6.95 (m, 5H, phenyl), 5.44
(s, 2H, CH2), 3.31 (t, 2H, J ¼ 7.2 Hz, CH2), 2.94 (t, 2H, J ¼ 6.9 Hz,
CH2CO); 13C NMR (DMSO-d6): 179.15 (C]O), 167.23 (C-thiadiazole),
163.42, 159.32 (C-triazole), 156.17 (C]N), 141.57, 134.12, 133.61,
132.41, 129.70, 128.23, 127.74, 124.75, 123.82, 122.34, 117.52, 112.93
(AreC), 51.35 (OCH2), 30.92 (CH2, CH2CO), 23.41 (CH2); ESI MS
(m/z): 404 (Mþ); Anal. calcd. for C20H16N6O2S: C, 59.39; H, 3.99; N,
20.78. Found: C, 59.45; H, 3.83; N, 20.89.
6.2.6.7. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-vinyl-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-3-yl)- propan-1-one (5g). Yield: 70%; m.p.
186e187 ꢄC; Rf ¼ 0.51 [benzene:acetone (9:1)]; IR (KBr, cmꢂ1): 3328
(NH), 3012 (CH), 2974 (HC]CH2), 2904 (CH2), 1728 (C]O), 1666
(C]N), 1569 (C]C), 1342 (N]CeS), 1230 (NeN]C); 1H NMR
(CDCl3):12.41 (s, 1H, NH, D2O exchangeable), 7.80 (d, 1H, J ¼ 7.8 Hz,
H-4, benzimidazole), 7.51 (t, 1H, J ¼ 7.2 Hz, H-7, benzimidazole),
7.33 (t, 2H, J ¼ 7.5 Hz, H-5,6, benzimidazole), 4.06 (t, 1H, J ¼ 7.2 Hz,
CH), 3.92 (d, 2H, J ¼ 6.9 Hz, CH2]CH), 3.29 (t, 2H, J ¼ 6.9 Hz, CH2),
2.90 (t, 2H, J ¼ 6.9 Hz, CH2CO); 13C NMR (CDCl3): 171.51 (C]O),
167.31 (C-thiadiazole), 160.41, 157.13 (C-triazole), 153.47 (C]N),
133.27, 128.91 (CH]CH2) 128.78, 128.13, 124.31, 123.82, 120.53,
118.46 (AreC), 34.73 (CH2, CH2CO), 22.53 (CH2); ESI MS (m/z): 324
(Mþ); Anal. calcd. for C15H12N6OS: C, 55.54; H, 3.73; N, 25.91.
Found: C, 55.68; H, 3.85; N, 25.97.
6.2.6.8. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(2,4-dichlorophenyl)-
[1,2,4]triazolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5h). Yield:
69%; m.p. 225e226 ꢄC; Rf ¼ 0.41 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3336 (NH), 3093 (CH), 2900 (CH2), 1685
(C]O), 1654 (C]N), 1627 (C]C), 1373 (N]CeS), 1234 (NeN]C);
1H NMR (CDCl3): 12.17 (s, 1H, NH, D2O exchangeable), 8.20 (s, 1H,
H-3-phenyl), 8.11 (d, 1H, J ¼ 7.8 Hz, H-5-phenyl), 7.70 (d, 1H,
J ¼ 7.5 Hz, H-4, benzimidazole), 7.51 (t, 1H, J ¼ 7.5 Hz, H-7, benz-
imidazole), 7.36 (t, 2H, J ¼ 7.8 Hz, H-5,6, benzimidazole), 3.29 (t, 2H,
J ¼ 6.9 Hz, CH2), 2.90 (t, 2H, J ¼ 6.9 Hz, CH2CO); 13C NMR (CDCl3):
173.63 (C]O), 173.01 (C-thiadiazole), 157.29, 156.42 (C-triazole),
154.21 (C]N), 138.21, 132.97, 129.25, 128.15, 126.91, 126.08, 125.95,
125.21, 124.01, 117.40, 116.63, 109.97 (AreC), 35.10 (CH2, CH2CO),
25.03 (CH2); ESI MS (m/z): 443 (Mþ); Anal. calcd. for
6.2.6.4. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-benzhydryl-[1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazol-3-yl)propan-1-one (5d). Yield: 65%;
m.p. 197e198 ꢄC; Rf ¼ 0.59 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3326 (NH), 3050 (CH), 2890 (CH2), 2873
(CH), 1724 (C]O), 1633 (C]N), 1537 (C]C), 1380 (N]CeS), 1306
(NeN]C); 1H NMR (DMSO-d6): 12.01 (s, 1H, NH, D2O exchange-
able), 8.05 (d, 1H, J ¼ 7.5 Hz, H-4, benzimidazole), 7.75 (t, 1H,
J ¼ 7.5 Hz, H-7, benzimidazole), 7.57 (t, 2H, J ¼ 7.5 Hz, H-5,6,
benzimidazole), 7.33e6.80 (m, 10H, phenyl), 5.61 (s, H, CH), 3.24 (t,
2H, J ¼ 7.2 Hz, CH2), 2.71 (t, 2H, J ¼ 7.2 Hz, CH2CO); 13C NMR (DMSO-
d6): 172.46 (C]O), 169.72 (C-thiadiazole), 162.72, 159.86 (C-tri-
azole), 156.39 (C]N), 140.23, 134.72, 134.01, 133.91, 132.31, 131.82,
130.51, 129.45, 128.41, 125.74, 124.86, 124.21, 123.37, 122.30, 120.15,
118.41, 111.32 (AreC), 47.24 (CH), 36.10 (CH2, CH2CO), 22.71 (CH2);
ESI MS (m/z): 464 (Mþ); Anal. calcd. for C26H20N6OS: C, 67.22; H,
4.34; N, 18.09. Found: C, 67.25; H, 4.45; N, 18.17.
C19H12Cl2N6OS: C, 51.48; H, 2.73; N, 18.96. Found: C, 51.49; H, 3.21;
N, 18.87.
6.2.6.9. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(2-mercaptophenyl)-
[1,2,4]triazolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5i). Yield:
60%; m.p. 189e190 ꢄC; Rf ¼ 0.63 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3356 (NH), 3080 (CH), 2925 (CH2), 2571
6.2.6.5. 2-(3-(3-(1H-Benzo[d]imidazol-2-yl)-3-oxopropyl)-[1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazol-6-yl)acetonitrile (5e). Yield: 63%; m.p.
217e218 ꢄC; Rf ¼ 0.53 [benzene:acetone (9:1)]; IR (KBr, cmꢂ1): 3344
Please cite this article in press as: A. Husain, et al., Benzimidazole clubbed with triazolo-thiadiazoles and triazolo-thiadiazines: New anticancer
agents, European Journal of Medicinal Chemistry (2012), http://dx.doi.org/10.1016/j.ejmech.2012.07.011

A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
11
(SH), 1703 (C]O), 1643 (C]N), 1534 (C]C), 1380 (N]CeS), 1290
(NeN]C); 1H NMR (CDCl3): 12.51 (s, 1H, NH, D2O exchangeable),
7.83 (d, 1H, J ¼ 7.8 Hz, H-4, benzimidazole), 7.73 (t, 1H, J ¼ 7.5 Hz,
H-7, benzimidazole), 7.61e7.39 (m, 4H, phenyl), 7.27 (t, 2H,
J ¼ 7.5 Hz, H-5,6, benzimidazole), 4.63 (s, H, SH), 3.04 (t, 2H,
J ¼ 6.9 Hz, CH2), 2.61 (t, 2H, J ¼ 7.2 Hz, CH2CO); 13C NMR (CDCl3):
172.60 (C]O), 169.71 (C-thiadiazole), 158.37, 157.52 (C-triazole),
155.43 (C]N), 136.73, 133.53, 130.23, 129.85, 126.57, 125.81, 124.35,
123.71, 122.13, 118.42, 111.60, 110.87 (AreC), 36.23 (CH2, CH2CO),
23.45 (CH2); ESI MS (m/z): 406 (Mþ); Anal. calcd. for C19H14N6OS2:
C, 56.14; H, 3.47; N, 20.67. Found: C, 56.17; H, 3.53; N, 20.85.
m.p. 204e205 ꢄC; Rf ¼ 0.55 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3321 (NH), 3097 (CH), 2896 (CH2), 2545
(SH), 1724 (C]O), 1612 (C]N), 1504 (C]C), 1384 (N]CeS), 1253
(NeN]C); 1H NMR (CDCl3): 12.05 (s, 1H, NH, D2O exchangeable),
7.78 (d, 1H, J ¼ 7.8 Hz, H-4, benzimidazole), 7.51 (t,1H, J ¼ 7.8 Hz, H-7,
benzimidazole), 7.33 (t, 2H, J ¼ 8.4 Hz, H-5,6, benzimidazole), 3.72 (s,
1H, CH2SH), 3.32 (t, 2H, J ¼ 6.9 Hz, CH2), 2.92 (t, 2H, J ¼ 7.2 Hz,
CH2CO), 1.73 (s, 1H, SH); 13C NMR (CDCl3): 172.60 (C]O), 170.14
(C-thiadiazole), 158.19, 157.07 (C-triazole), 155.42 (C]N), 131.64,
130.78, 129.84, 128.85, 124.18, 116.70 (AreC), 51.80 (CH2SH), 30.30
(CH2, CH2CO), 28.06 (CH2); ESI MS (m/z): 344 (Mþ); Anal. calcd. for
C14H12N6OS2: C, 48.82; H, 3.51; N, 24.40. Found: C, 48.93; H, 3.54; N,
6.2.6.10. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(prop-1-enyl)-[1,2,4]tri-
azolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5j). Yield: 64%;
m.p. 210e211 ꢄC;Rf ¼ 0.62[toluene:ethylacetate:formicacid(5:4:1)];
IR (KBr, cmꢂ1): 3321 (NH), 3109 (¼CH), 3004 (CH), 2908 (CH2), 2877
(CH3), 1658 (C]O), 1631 (C]N), 1566 (C]C), 1384 (N]CeS), 1145
(NeN]C); 1H NMR (CDCl3): 12.63 (s,1H, NH, D2O exchangeable), 8.11
(d, 1H, J ¼ 7.5 Hz, H-4, benzimidazole), 7.61 (t, 1H, J ¼ 7.2 Hz, H-7,
benzimidazole), 7.48 (t, 2H, J ¼ 7.2 Hz, H-5,6, benzimidazole), 4.54 (d,
1H, J ¼ 8.7 Hz, CH), 4.17 (m, 1H, CHCH3), 3.31 (t, 2H, J ¼ 6.6 Hz, CH2),
2.90 (t, 2H, J ¼ 6.9 Hz, CH2CO), 1.26 (d, 3H, J ¼ 6.9 Hz, CH3); 13C NMR
(CDCl3):174.15 (C]O), 170.53 (C-thiadiazole), 160.71, 158.43 (C-tri-
azole),154.71 (C]N),135.46,132.67 (CH]CH),130.21, 129.75,125.17,
124.83, 120.37, 119.71 (AreC), 32.75 (CH2, CH2CO), 25.31 (CH2), 14.67
(CH3); ESI MS (m/z): 338 (Mþ); Anal. calcd. for C16H14N6OS: C, 56.79;
H, 4.17; N, 24.84. Found: C, 56.81; H, 4.31; N, 24.73.
24.87.
6.2.6.14. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(4-methoxyphenyl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)propan-1-one (5n). Yield:
67%; m.p. 199 ꢄC; Rf ¼ 0.57 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3370 (NH), 3065 (CH), 2931 (CH2), 1705
(C]O), 1654 (C]N), 1573 (C]C), 1382 (N]CeS), 1281 (NeN]C);
1H NMR (DMSO-d6): 12.13 (s, 1H, NH, D2O exchangeable), 8.11 (d,
1H, J ¼ 7.8 Hz, H-4, benzimidazole), 7.61 (t, 1H, J ¼ 7.2 Hz, H-7,
benzimidazole), 7.50 (t, 2H, J ¼ 7.2 Hz, H-5,6, benzimidazole),
7.40e7.26 (m, 4H, phenyl), 3.71 (s, 3H, CH3), 3.29 (t, 2H, J ¼ 6.9 Hz,
CH2), 2.90 (t, 2H, J ¼ 6.9 Hz, CH2CO); 13C NMR (DMSO-d6): 175.32
(C]O), 164.23 (C-thiadiazole), 162.42, 157.53 (C-triazole), 155.13
(C]N), 133.62, 130.75, 130.45, 128.57, 127.83, 124.13, 122.34, 120.65,
119.37, 115.27, 113.41, 112.19 (AreC), 61.31 (OCH3), 34.17 (CH2,
CH2CO), 23.83 (CH2). ESI MS (m/z): 404 (Mþ); Anal. calcd. for
6.2.6.11. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(2-hydroxyphenyl)-
[1,2,4]triazolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5k). Yield:
66%; m.p. 198 ꢄC; Rf ¼ 0.62 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3391 (OH), 3317 (NH), 3025 (CH), 2913
(CH2), 1690 (C]O), 1636 (C]N), 1585 (C]C), 1380 (N]CeS), 1236
(NeN]C); 1H NMR (DMSO-d6): 12.53 (s, 1H, NH, D2O exchange-
able), 7.84 (d, 1H, J ¼ 7.8 Hz, H-4, benzimidazole), 7.63 (t, 1H,
J ¼ 7.5 Hz, H-7, benzimidazole), 7.54 (t, 2H, J ¼ 7.5 Hz, H-5,6,
benzimidazole), 7.45e7.23 (m, 4H, phenyl), 6.52 (s, 1H, OH), 3.21 (t,
2H, J ¼ 6.9 Hz, CH2), 2.87 (t, 2H, J ¼ 6.9 Hz, CH2CO); 13C NMR
(DMSO-d6): 170.87 (C]O), 170.10 (C-thiadiazole), 163.51, 157.85
(C-triazole), 155.23 (C]N), 138.53, 133.56, 130.63, 129.41, 127.45,
124.81, 124.15, 120.35, 118.71, 116.31, 115.67, 113.75 (AreC), 37.27
(CH2, CH2CO), 24.10 (CH2); ESI MS (m/z): 390 (Mþ); Anal. calcd. for
C20H16N6O2S: C, 59.39; H, 3.99; N, 20.78. Found: C, 59.43; H, 3.87; N,
20.91.
6.2.6.15. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(4-hydroxyphenyl)-
[1,2,4]triazolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5o). Yield:
72%; m.p. 207e208 ꢄC; Rf ¼ 0.56 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3483 (OH), 3321 (NH), 3027 (CH), 2890
(CH2), 1728 (C]O), 1671 (C]N), 1620 (C]C), 1370 (N]CeS), 1283
(NeN]C); 1H NMR (DMSO-d6): 12.32 (s, 1H, NH, D2O exchange-
able), 7.81 (d, 1H, J ¼ 7.8 Hz, H-4, benzimidazole), 7.63 (t, 1H,
J ¼ 7.8 Hz, H-7, benzimidazole), 7.54 (t, 2H, J ¼ 7.5 Hz, H-5,6,
benzimidazole), 7.31e6.92 (m, 4H, phenyl), 6.23 (s, 1H, OH), 3.32 (t,
2H, J ¼ 7.2 Hz, CH2), 2.94 (t, 2H, J ¼ 7.2 Hz, CH2CO); 13C NMR (DMSO-
d6): 172.15 (C]O), 168.43 (C-thiadiazole), 163.52, 157.75 (C-tri-
azole), 153.87 (C]N), 141.65, 135.73, 130.48, 128.25, 127.62, 123.17,
122.54, 120.19, 119.38, 113.27, 112.43, 111.15 (AreC), 38.05 (CH2,
CH2CO), 26.73 (CH2); ESI MS (m/z): 390 (Mþ); Anal. calcd. for
C
19H14N6O2S: C, 58.45; H, 3.61; N, 21.53. Found: C, 59.12; H, 3.67; N,
21.47.
6.2.6.12. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(5-amino-2-hydroxy-
C19H14N6O2S: C, 58.45; H, 3.61; N, 21.53. Found: C, 58.48; H, 3.75; N,
phenyl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-3-yl)propan-1-one
21.63.
(5l). Yield: 70%; m.p. 215 ꢄC; Rf ¼ 0.49 [toluene:ethyl acetate:for-
mic acid (5:4:1)]; IR (KBr, cmꢂ1): 3495 (OH), 3371 (NH), 3071 (CH),
2890 (CH2), 1711 (C]O), 1651 (C]N), 1582 (C]C), 1378 (N]CeS),
1280 (NeN]C); 1H NMR (CDCl3): 12.01 (s, 1H, NH, D2O
exchangeable), 8.04 (d, 1H, J ¼ 7.5 Hz, H-4, benzimidazole), 7.81 (t,
1H, J ¼ 7.8 Hz, H-7, benzimidazole), 7.54 (t, 2H, J ¼ 7.5 Hz, H-5,6,
benzimidazole), 7.42 (s,1H, H-6-phenyl), 7.34 (d,1H, J ¼ 7.8 Hz, H-3-
phenyl), 7.22 (d, 1H, J ¼ 7.5 Hz, H-4, phenyl), 6.83 (s, 1H, OH), 6.29 (s,
2H, NH2), 3.17 (t, 2H, J ¼ 6.9 Hz, CH2), 2.94 (t, 2H, J ¼ 6.9 Hz, CH2CO);
13C NMR (CDCl3): 172.56 (C]O), 169.43 (C-thiadiazole), 162.71,
156.93 (C-triazole), 154.75 (C]N), 139.50, 137.35, 131.26, 129.31,
128.57, 123.62, 123.51, 122.48, 120.73, 118.35, 116.63, 115.17 (AreC),
36.21 (CH2, CH2CO), 23.63 (CH2); ESI MS (m/z): 405 (Mþ); Anal.
calcd. for C19H15N7O2S: C, 56.29; H, 3.73; N, 24.18. Found: C, 57.15;
H, 3.77; N, 24.27.
6.2.6.16. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(chloromethyl)-[1,2,4]
triazolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5p). Yield: 68%;
m.p. 209 ꢄC; Rf ¼ 0.44 [toluene:ethyl acetate:formic acid (5:4:1)]; IR
(KBr, cmꢂ1): 3301 (NH), 3012 (CH), 2904 (CH2), 1728 (C]O), 1670
(C]N), 1608 (C]C), 1384 (N]CeS), 1180 (NeN]C), 759 (CCl); 1H
NMR (CDCl3): 11.94 (s, 1H, NH, D2O exchangeable), 7.81 (d, 1H,
J ¼ 8.4 Hz, H-4, benzimidazole), 7.49 (t, 1H, J ¼ 7.5 Hz, H-7, benz-
imidazole), 7.32 (t, 2H, J ¼ 7.5 Hz, H-5,6, benzimidazole), 3.72 (s, 2H,
CH2Cl), 3.34 (t, 2H, J ¼ 7.2 Hz, CH2), 2.92 (t, 2H, J ¼ 6.6 Hz, CH2CO);
13C NMR (CDCl3): 173.61 (C]O), 173.14 (C-thiadiazole), 159.19,
159.07 (C-triazole), 156.42 (C]N), 132.64, 130.84, 129.88, 128.87,
124.16,115.70 (AreC), 60.52 (CH2, CH2Cl), 30.30 (CH2, CH2CO), 28.02
(CH2); ESI MS (m/z): 346 (Mþ); Anal. calcd. for C14H11ClN6OS: C,
48.49; H, 3.20; N, 24.23. Found: C, 48.63; H, 3.35; N, 24.26.
6.2.6.13. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(mercaptomethyl)-[1,2,4]
6.2.6.17. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(3-aminopropyl)-[1,2,4]
triazolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5m). Yield: 63%;
triazolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5q). Yield: 65%;
Please cite this article in press as: A. Husain, et al., Benzimidazole clubbed with triazolo-thiadiazoles and triazolo-thiadiazines: New anticancer
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12
A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
m.p. 221e222 ꢄC; Rf ¼ 0.55 [toluene:ethyl acetate:formic acid (5:4:1)];
IR (KBr, cmꢂ1): 3398 (NH), 2974 (CH), 2850 (CH2), 1728 (C]O), 1653
(C]N), 1508 (C]C),1338 (N]CeS),1145 (NeN]C); 1H NMR (DMSO-
d6):12.77 (s, 1H, NH, D2O exchangeable), 7.79 (d, 1H, J ¼ 7.8 Hz, H-4,
benzimidazole), 7.28 (t,1H, J ¼ 7.8 Hz, H-7, benzimidazole), 7.07 (t, 2H,
J ¼ 7.5 Hz, H-5,6, benzimidazole), 6.46 (s, 2H, NH2), 3.32 (t, 2H,
J ¼ 7.2 Hz, CH2), 3.01 (t, 2H, J ¼ 6.9 Hz, CH2g, CH2gCH2CH2NH2), 2.87 (t,
2H, J ¼ 7.2 Hz, CH2CO), 2.74 (t, 2H, J ¼ 6.9 Hz, CH2aNH2), 2.30 (m, 2H,
CH2b, CH2bCH2NH2); 13C NMR (DMSO-d6): 176.25 (C]O), 170.53 (C-
thiadiazole), 164.46, 158.31 (C-triazole), 155.17 (C]N), 130.45, 129.52,
128.63, 122.73, 121.68, 117.21 (AreC), 50.13, 42.37, 27.56
(CH2CH2CH2NH2), 33.21 (CH2, CH2CO), 24.15 (CH2); ESI MS (m/z): 355
(Mþ); Anal. calcd. for C16H17N7OS: C, 54.07; H, 4.82; N, 27.59. Found: C,
54.13; H, 4.87; N, 27.68.
room temperature and then neutralized with ammonia solution. A
solid product obtained which was filtered, washed with water,
dried and recrystallized from dichloromethane.
6.2.9.1. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-amino-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazin-3-yl)propan-1-one (5t). Yield: 62%; m.p.
238 ꢄC; Rf ¼ 0.57 [benzene:acetone (9:1)]; IR (KBr, cmꢂ1): 3372
(NH), 3061 (CH), 2913 (CH2), 1690 (C]O), 1651 (C]N), 1583 (C]C),
1383 (N]CeS), 1287 (NeN]C); 1H NMR (CDCl3): 12.25 (s, 1H, NH,
D2O exchangeable), 7.36 (d, 1H, J ¼ 7.5 Hz, H-4, benzimidazole), 7.18
(d, 1H, J ¼ 7.2 Hz, H-7, benzimidazole), 7.02 (t, 2H, J ¼ 8.7 Hz, H-5,6,
benzimidazole), 6.91 (s, 2H, NH2), 3.78 (s, 2H, CH2, cyclic), 2.91 (t,
2H, J ¼ 7.2 Hz, CH2), 2.27 (t, 2H, J ¼ 6.9 Hz, CH2CO); 13C NMR
(CDCl3): 171.37 (C]O), 160.13, 157.74 (C-triazole), 156.27, 155.32
(C]N), 131.53, 130.15, 127.21, 126.77, 119.61, 118.37 (AreC), 50.32
(CH2, cyclic), 32.73 (CH2, CH2CO), 25.47 (CH2); ESI MS (m/z): 327
(Mþ); Anal. calcd. for C14H13N7OS: C, 51.36; H, 4.00; N, 29.95.
Found: C, 51.37; H, 4.10; N, 29.99.
6.2.7. Synthesis of 1-(1H-benzo[d]imidazol-2-yl)-3-(6-mercapto-
[1,2,4]triazolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5r)
A mixture of compound 5 (0.003 mol) and KOH/CS2 (0.003 mol)
in ethanol (15 mL) was refluxed for 4 h. After completion of the
reaction, excess solvent was removed under reduced pressure and
then cold water (20 mL) added into reaction media with stirring. A
solid separated out which was filtered off, washed with water, dried
and recrystallized from methanol. Yield: 63%; m.p. 232 ꢄC; Rf ¼ 0.61
[benzene:acetone (9:1)]; IR (KBr, cmꢂ1): 3398 (NH), 3097 (CH),
2835 (CH2), 2545 (SH), 1724 (C]O), 1612 (C]N), 1504 (C]C), 1334
(N]CeS), 1176 (NeN]C); 1H NMR (CDCl3): 12.12 (s, 1H, NH þ SH,
D2O exchangeable), 7.80 (d, 1H, J ¼ 8.1 Hz, H-4, benzimidazole), 7.51
(t, 1H, J ¼ 7.5 Hz, H-7, benzimidazole), 7.35 (t, 2H, J ¼ 7.8 Hz, H-5,6,
benzimidazole), 3.34 (t, 2H, J ¼ 7.2 Hz, CH2), 2.95 (t, 2H, J ¼ 7.2 Hz,
CH2CO); 13C NMR (CDCl3): 174.21 (C]O), 171.16 (C-thiadiazole),
163.61, 157.32 (C-triazole), 155.03 (C]N), 131.23, 129.58, 128.47,
120.37, 119.71, 115.13 (AreC), 31.64 (CH2, CH2CO), 22.57 (CH2); ESI
MS (m/z): 330 (Mþ); Anal. calcd. for C13H10N6OS2: C, 47.26; H, 3.05;
N, 25.44. Found: C, 48.31; H, 3.17; N, 25.51.
6.2.9.2. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-hydroxy-7H-[1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazin-3-yl)propan-1-one (5u). Yield: 66%;
m.p. 215 ꢄC; Rf ¼ 0.53 [benzene:acetone (9:1)]; IR (KBr, cmꢂ1): 3475
(OH), 3367 (NH), 3058 (CH), 2904 (CH2), 1677 (C]O), 1600 (C]N),
1504 (C]C),1384 (N]CeS),1180 (NeN]C); 1H NMR (CDCl3): 12.10
(s, 1H, NH, D2O exchangeable), 7.71 (d, 1H, J ¼ 8.1 Hz, H-4, benz-
imidazole), 7.38 (t, 1H, J ¼ 8.1 Hz, H-7, benzimidazole), 7.27 (dd, 2H,
J ¼ 6.3 Hz, J ¼ 7.2 Hz, H-5,6, benzimidazole), 3.69 (s, 2H, CH2, cyclic),
3.20 (t, 2H, J ¼ 7.2 Hz, CH2), 2.84 (t, 2H, J ¼ 6.9 Hz, CH2CO), 2.59 (s,
1H, OH); 13C NMR (CDCl3): 173.53 (C]O), 161.25, 158.75 (C-tri-
azole), 155.31, 154.85 (C]N), 130.41, 129.57, 124.32, 123.83, 120.13,
119.85 (AreC), 47.83 (CH2, cyclic), 33.82 (CH2, CH2CO), 24.81 (CH2);
ESI MS (m/z): 328 (Mþ); Anal. calcd. for C14H12N6O2S: C, 51.21; H,
3.68; N, 25.59. Found: C, 51.45; H, 3.71; N, 26.82.
6.2.9.3. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-ethoxy-7H-[1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazin-3-yl)propan-1-one (5v). Yield: 61%;
m.p. 226e227 ꢄC; Rf ¼ 0.55 [benzene:acetone (9:1)]; IR (KBr, cmꢂ1):
3321 (NH), 3070 (CH), 2974 (CH2), 1728 (C]O), 1643 (C]N), 1573
(C]C), 1330 (N]CeS), 1218 (NeN]C), 1149 (CeOeC, asymmetric),
1064 (CeOeC, symmetric); 1H NMR (CDCl3): 11.94 (s, 1H, NH, D2O
exchangeable), 7.78 (d, 1H, J ¼ 8.4 Hz, H-4, benzimidazole), 7.46 (t,
1H, J ¼ 7.5 Hz, H-7, benzimidazole), 7.32 (t, 2H, J ¼ 7.5 Hz, H-5,6,
benzimidazole), 4.14 (q, 2H, J ¼ 7.2 Hz, OCH2), 3.72 (s, 2H, CH2,
cyclic), 3.29 (t, 2H, J ¼ 6.9 Hz, CH2), 2.92 (t, 2H, J ¼ 6.6 Hz, CH2CO),
1.27 (t, 3H, J ¼ 7.2 Hz, CH3); 13C NMR (CDCl3): 175.61 (C]O), 161.25,
157.54 (C-triazole), 156.71, 156.37 (C]N), 131.23, 129.81, 123.43,
122.76, 122.30, 120.51 (AreC), 50.17 (CH2, cyclic), 42.51 (OCH2),
35.25 (CH2, CH2CO), 23.42 (CH2), 18.32 (CH3); ESI MS (m/z): 356
(Mþ); Anal. calcd. for C16H16N6O2S: C, 53.92; H, 4.52; N, 23.58.
Found: C, 53.97; H, 4.55; N, 23.73.
6.2.8. Synthesis of 1-(1H-benzo[d]imidazol-2-yl)-3-(6-(4-chloropheny
lthio)-[1,2,4]triazolo[3,4-b] [1,3,4]thiadiazol-3-yl)propan-1-one (5s)
Compound 5 (0.003 mol) and p-chlorophenyl isothiocyanate
(0.003 mol) were completely dissolved in dimethylformamide and
then refluxed for 10 h. After completion of the reaction, the reaction
mixture was cooled to room temperature and then gradually
poured on to crushed ice with stirring. A solid product separated
out which was filtered, washed thoroughly with water, dried and
crystallized from ethanol. Yield: 60%; m.p. 223e224 ꢄC; Rf ¼ 0.46
[benzene:acetone (9:1)]; IR (KBr, cmꢂ1): 3310 (NH), 3073 (CH),
2907 (CH2), 1711 (C]O), 1671 (C]N), 1543 (C]C), 1384 (N]CeS),
1171 (NeN]C), 751 (CCl); 1H NMR (CDCl3): 11.95 (s, 1H, NH,
benzimidazole, D2O exchangeable), 7.73 (d, 1H, J ¼ 7.8 Hz, H-4,
benzimidazole), 7.57 (t, 1H, J ¼ 7.5 Hz, H-7, benzimidazole), 7.48 (t,
2H, J ¼ 8.1 Hz, H-5,6, benzimidazole), 7.39e7.12 (m, 4H, phenyl),
5.18 (s, 1H, NH, D2O exchangeable), 3.27 (t, 2H, J ¼ 7.2 Hz, CH2), 2.81
(t, 2H, J ¼ 6.9 Hz, CH2CO); 13C NMR (CDCl3): 173.15 (C]O), 168.37
(C-thiadiazole), 165.25, 158.36 (C-triazole), 153.87 (C]N), 133.21,
132.51, 130.41, 129.92, 128.27, 124.71, 123.67, 120.56, 118.71, 115.32,
112.43, 111.64 (AreC), 36.47 (CH2, CH2CO), 23.51 (CH2); ESI MS
(m/z): 423 (Mþ); Anal. calcd. for C19H14ClN7OS: C, 53.84; H, 3.33; N,
23.13. Found: C, 53.89; H, 4.65; N, 23.27.
6.2.9.4. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-chloro-7H-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazin-3-yl)propan-1-one (5w). Yield: 63%, m.p.
228e229 ꢄC; Rf ¼ 0.47 [benzene:acetone (9:1)]; IR (KBr, cmꢂ1):
3336 (NH), 3012 (CH), 2904 (CH2), 1666 (C]O), 1608 (C]N), 1569
(C]C), 1342 (N]CeS), 1145 (NeN]C), 759 (CCl); 1H NMR (CDCl3):
12.34 (s, 1H, NH, D2O exchangeable), 7.68 (d, 1H, J ¼ 7.5 Hz, H-4,
benzimidazole), 7.50 (t, 1H, J ¼ 7.2 Hz, H-7, benzimidazole), 7.27 (t,
2H, J ¼ 8.7 Hz, H-5,6, benzimidazole), 3.60 (s, 2H, CH2, cyclic), 3.05
(t, 2H, J ¼ 6.9 Hz, CH2), 2.79 (t, 2H, J ¼ 6.9 Hz, CH2CO); 13C NMR
(CDCl3): 173.60 (C]O), 159.73, 159.13 (C-triazole), 156.25, 155.23
(C]N), 132.62, 130.81, 129.88, 128.90, 124.16, 115.60 (AreC), 51.79
(CH2, cyclic), 30.01 (CH2, CH2CO), 28.03 (CH2); ESI MS (m/z): 346
(Mþ); Anal. calcd. for C14H11ClN6OS: C, 48.49; H, 3.20; N, 24.23.
Found: C, 48.51; H, 3.27; N, 25.87.
6.2.9. General method for synthesis of 1-(1H-benzo[d]imidazol-2-yl)-
3-(6-substituted-7H-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazin-3-yl)
propan-1-one (5te5w)
A solution of compound 5 (0.003 mol) and different types of
a
-chloro containing carbonyl methyl compound (equimolar;
0.003 mol) in absolute ethanol (15 mL) was reflux for 3e4 h. After
completion of the reaction, the reaction solution was cooled to
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A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
13
6.2.10. General method for synthesis of 1-(1H-benzo[d]imidazol-2-
yl)-3-(6-(substituted)-[1,2,4] triazolo[3,4-b][1,3,4]thiadiazol-3-yl)
propan-1-one (5x-5a1)
J ¼ 8.1 Hz, H-4, benzimidazole), 7.51 (t, 1H, J ¼ 7.5 Hz, H-7, benz-
imidazole), 7.32 (t, 2H, J ¼ 7.5 Hz, H-5,6, benzimidazole), 3.97 (q, 2H,
J ¼ 7.2 Hz, OCH2), 3.32 (t, 2H, J ¼ 7.2 Hz, CH2), 2.81 (t, 2H, J ¼ 6.9 Hz,
CH2CO), 1.36 (t, 3H, J ¼ 6.9 Hz, CH3); 13C NMR (DMSO-d6): 173.61
(C]O), 165.27, 163.45 (C-triazole), 160.72 (C-thiadiazole), 156.17
(C]N), 131.35, 130.17, 127.97, 126.87, 120.72, 115.64 (AreC), 55.63
(OCH2), 35.41 (CH2, CH2CO), 25.61 (CH2), 16.23 (CH3); ESI MS (m/z):
360 (Mþ); Anal. calcd. for C15H14N6O2S: C, 52.62; H, 4.12; N, 24.55.
Found: C, 52.72; H, 4.26; N, 24.85.
A solution of compound 5 (0.003 M) and different types of
a
-chloro containing carbonyl compounds (0.003 M) in absolute
ethanol was allowed to reflux for 3e4 h. After completion the
reaction, cooled to room temperature and neutralized with
ammonia solution. The solid precipitate so obtained was filtered,
washed with water and recrystallized from dichloromethane.
6.2.10.1. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-methyl-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazol-3-yl) propan-1-one (5x). Yield: 66%; m.p.
190e191 ꢄC; Rf ¼ 0.52 [toluene:ethyl acetate:formic acid (5:4:1)];
IR (KBr, cmꢂ1): 3344 (NH), 3008 (CH), 2904 (CH2), 2854 (CH3), 1670
(C]O), 1612 (C]N), 1566 (C]C), 1385 (N]CeS), 1180 (NeN]C);
1H NMR (CDCl3): 11.91 (s, 1H, NH, D2O exchangeable), 7.78 (d, 1H,
J ¼ 8.1 Hz, H-4, benzimidazole), 7.46 (t, 1H, J ¼ 7.5 Hz, H-7, benz-
imidazole), 7.34 (t, 2H, J ¼ 7.2 Hz, H-5,6, benzimidazole), 3.29 (t, 2H,
6.3. Anticancer screening
The human tumor cell lines were grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM L-glutamine. After cell
inoculation, the microtitre plates incubated at 37 ꢄC, 5% CO2, 95% air
and 100% relative humidity for 24 h prior to addition of tested
compounds. After 24 h, two plates of each cell line fixed in situ with
TCA, to represent a measurement of the cell population for each cell
line at the time of sample addition (Tz). The sample solubilized in
DMSO at 400-fold the desired final maximum test concentration
and stored frozen prior to use. At the time of compounds addition,
an aliquot of frozen concentrate was thawed and diluted to twice,
the desired final maximum test concentration with complete
J ¼ 6.9 Hz, CH2), 2.92 (t, 2H, J ¼ 6.9 Hz, CH2CO), 1.82 (s, 3H, CH3); 13
C
NMR (CDCl3): 172.01 (C]O), 158.23, 156.71 (C-triazole), 155.03
(C]N), 148.10 (C-thiadiazole), 132.72, 132.63, 128.53, 128.01,
123.57,115.63 (AreC), 33.27 (CH2, CH2CO), 24.05 (CH2), 14.83 (CH3);
ESI MS (m/z): 312 (Mþ); Anal. calcd. for C14H12N6OS: C, 53.83; H,
3.87; N, 26.91. Found: C, 53.95; H, 3.82; N, 25.97.
medium containing 50
or ½ log serial dilutions are made to provide a total of five drug
concentrations plus control. Aliquots of 100 L of these different
sample dilutions added to the appropriate microtitre wells already
containing 100 L of medium, resulting in the required final sample
mg/mL gentamicin. Additional four, 10-fold
6.2.10.2. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(3-chloropropyl)-[1,2,4]
triazolo[3,4-b][1,3,4] thiadiazol-3-yl)propan-1-one (5y). Yield: 62%;
m.p. 234e235 ꢄC; Rf ¼ 0.46 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3325 (NH), 3040 (CH), 2932 (CH2), 1710
(C]O), 1657 (C]N), 1520 (C]C), 1380 (N]CeS), 1160 (NeN]C),
750 (CCl); 1H NMR (DMSO-d6): 12.67 (s, 1H, NH, D2O exchange-
able), 7.70 (d, 1H, J ¼ 7.8 Hz, H-4, benzimidazole), 7.51 (t, 1H,
J ¼ 7.8 Hz, H-7, benzimidazole), 7.38 (t, 2H, J ¼ 7.5 Hz, H-5,6,
benzimidazole), 3.74 (t, 2H, J ¼ 6.9 Hz, CH2Cl), 3.23 (t, 2H, J ¼ 7.2 Hz,
CH2), 2.90 (t, 2H, J ¼ 7.2 Hz, CH2CO), 2.51 (t, 2H, J ¼ 7.2 Hz, CH2,
CH2CH2CH2Cl), 1.73 (m, 2H, CH2, CH2CH2Cl); 13C NMR (DMSO-d6):
173.31 (C]O), 160.26, 157.21 (C-triazole), 156.81 (C-thiadiazole),
155.13 (C]N), 130.61, 130.01, 129.53, 128.96, 122.51, 118.13 (AreC),
47.32, 27.15, 26.42 (CH2CH2CH2Cl), 30.83 (CH2, CH2CO), 23.37
(CH2); ESI MS (m/z): 374 (Mþ); Anal. calcd. for C16H15ClN6OS: C,
51.27; H, 4.03; N, 22.42. Found: C, 51.43; H, 4.12; N, 22.45.
m
m
concentrations. The tested compounds addition, the plates was
incubated for an additional 48 h at 37 ꢄC, 5% CO2, 95% air, and 100%
relative humidity. Cells fixed in situ by the gentle addition of 50 mL
of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated
for 60 min at 4 ꢄC. The supernatant discarded and plates washed
five times with tap water and air dried. Sulforhodamine B (SRB)
solution (100 mL) at 0.4% (w/v) in 1% acetic acid added to each well
and plates incubated for 10 min at RT. Bound stain subsequently
solubilized with 10 mM trizma base and the absorbance was read
on an automated plate reader at a wavelength of 515 nm. Using the
seven absorbance measurements [time zero, (Tz), control growth
(C) and test growth in the presence of sample at the five concen-
tration levels (Ti)]. Percentage growth inhibition calculated as:
6.2.10.3. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-(2-chloroethyl)-[1,2,4]
triazolo[3,4-b][1,3,4] thiadiazole-3-yl)propan-1-one (5z). Yield: 58%;
m.p. 217e218 ꢄC; Rf ¼ 0.41 [toluene:ethyl acetate:formic acid
(5:4:1)]; IR (KBr, cmꢂ1): 3312 (NH), 3020 (CH), 2936 (CH2), 1704
(C]O), 1645 (C]N), 1563 (C]C), 1383 (N]CeS), 1174 (NeN]C),
770 (CCl); 1H NMR (DMSO-d6): 12.13 (s, 1H, NH, D2O exchange-
able), 7.80 (d, 1H, J ¼ 7.8 Hz, H-4, benzimidazole), 7.63 (t, 1H,
J ¼ 7.8 Hz, H-7, benzimidazole), 7.44 (t, 2H, J ¼ 7.8 Hz, H-5,6,
benzimidazole), 3.75 (t, 2H, J ¼ 6.9 Hz, CH2Cl), 3.26 (t, 2H, J ¼ 6.6 Hz,
CH2), 2.84 (t, 2H, J ¼ 6.9 Hz, CH2CO), 2.54 (t, 2H, J ¼ 7.2 Hz,
CH2CH2Cl); 13C NMR (DMSO-d6): 172.67 (C]O), 161.32, 158.41
(C-triazole), 157.92 (C-thiadiazole), 155.01 (C]N), 131.52, 130.23,
128.94, 128.12, 120.75, 119.38 (AreC), 50.43, 36.23 (CH2CH2Cl),
33.27 (CH2, CH2CO), 24.15 (CH2); ESI MS (m/z): 360 (Mþ); Anal.
calcd. for C15H13ClN6OS: C, 49.93; H, 3.63; N, 23.29. Found: C, 49.97;
H, 3.75; N, 23.37.
[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz
[(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz
Three dose response parameters (GI50, TGI and LC50) were
calculated for each experimental agent. Growth inhibition of 50%
(GI50) calculated from [(Ti ꢂ Tz)/(C ꢂ Tz)] ꢁ 100 ¼ 50 (which was
the drug concentration resulting in a 50% reduction in the net
protein increase), Total growth inhibition (TGI) calculated from
Ti ¼ Tz (concentration at which the total growth inhibition is 100%)
and LC50calculated from [(Ti ꢂ Tz)/Tz] ꢁ 100 ¼ ꢂ50 (concentration
of drug resulting in a 50% reduction in the measured protein at the
end of the drug treatment as compared to that at the beginning)
indicating a net loss of the cells. Values were calculated for each of
these three parameters if the level of activity was reached;
however, if the effect was not reached or was exceeded, the value
for that parameter was expressed as greater or less than the
maximum or minimum concentration tested [39e42]. The logs
molar concentration also calculated of individual GI50, TGI and
LC50and represented as log10 GI50, log10 TGI and log10 LC50 respec-
tively. The lowest values of response parameter were obtained with
the most sensitive cell lines.
6.2.10.4. 1-(1H-Benzo[d]imidazol-2-yl)-3-(6-ethoxy-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazol-3-yl) propan-1-one (5a1). Yield: 61%; m.p.
208e209 ꢄC; Rf ¼ 0.47 [toluene:ethyl acetate:formic acid (5:4:1)];
IR (KBr, cmꢂ1): 3327 (NH), 3010 (CH), 2950 (CH2), 1713 (C]O), 1657
(C]N), 1547 (C]C), 1387 (N]CeS), 1168 (NeN]C); 1H NMR
(DMSO-d6): 12.06 (s, 1H, NH, D2O exchangeable), 7.81 (d, 1H,
Please cite this article in press as: A. Husain, et al., Benzimidazole clubbed with triazolo-thiadiazoles and triazolo-thiadiazines: New anticancer
agents, European Journal of Medicinal Chemistry (2012), http://dx.doi.org/10.1016/j.ejmech.2012.07.011

14
A. Husain et al. / European Journal of Medicinal Chemistry xxx (2012) 1e14
[23] A.A.O. Sarhan, A. Al-Dhfyan, M.A. Al-Mozaini, C.N. Adra, T.A. Fadl, Eur. J. Med.
Acknowledgments
Chem. 45 (2010) 2689e2694.
[24] B.S. Holla, K.N. Poojary, B.S. Rao, M.K. Shivananda, Eur. J. Med. Chem. 37 (2002)
511e517.
[25] R. Lesyk, O. Vladzimirska, S. Holota, L. Zaprutko, A. Gzella, Eur. J. Med. Chem.
42 (2007) 641e648.
[26] G.L. Almajan, S.F. Barbuceanu, G. Bancescu, I. Saramet, G. Saramet, C. Draghici,
Eur. J. Med. Chem. 45 (2010) 6139e6146.
[27] B.S. Holla, B.S. Rao, B.K. Sarojini, P.M. Akberali, N.S. Kumari, Eur. J. Med. Chem.
41 (2006) 657e663.
We are thankful to Dr. Thelma Dizon (Project Manager), Devel-
opmental Therapeutics Program (DTP), National Cancer Institute
(NCI), Chemotherapeutic Agents Repository, Fisher Bio Services,
Rockville, MD, USA, for in vitro anticancer screening of the
compounds. Thanks are also to UGC, New Delhi, Government of
India, for financial assistance in the form of Major Research Project
[file No. 36-107/2008 (SR)].
[28] V. Padmavathi, S.G. Reddy, A. Padmaja, P. Kondaiah, A. Shazia, Eur. J. Med.
Chem. 44 (2009) 2106e2112.
[29] C. Duanmu, L.K. Shahrik, H.H. Holly, E. Hamel, Cancer Res. 49 (1989)
1344e1348.
[30] C.D. Britten, M. Delioukina, L. Boulos, L. Reiswig, N. Gicanov, J. Rizzo, D. Hao,
A. Tolcher, S. Weitman, T. Rugg, D. Von Hoff, J. Camden, L.S. Rosen, Proc. Am.
Soc. Clin. Oncol. 20 (2001) 2129.
[31] T.D. Penning, G.D. Zhu, V.B. Gandhi, J. Gong, S. Thomas, W. Lubisch, R. Grandel,
W. Wernet, C.H. Park, E.H. Fry, Y. Luo, X. Liu, Y. Shi, V. Klinghofer, E.F. Johnson,
D.J. Frost, C.K. Donawho, D.V. Bontcheva, J.J. Bouska, A.M. Olson, K.C. Marsh,
S.H. Rosenberg, V.L. Giranda, Bioorg. Med. Chem. 16 (2008) 6965e6975.
[32] B. Tolner, J.A. Hartley, D. Hochhauser, Mol. Pharmacol. 59 (2001) 699e708.
[33] J.M. Albert, C. Cao, K.W. Kim, C.D. Willey, L. Geng, D. Xiao, H. Wang, A. Sandler,
D.H. Johnson, A.D. Colevas, J. Low, M.L. Rothenberg, B. Lu, Clin. Cancer Res. 13
(2007) 3033e3042.
[34] T.D. Bradshaw, A.D. Westwell, Curr. Med. Chem. 11 (2004) 1241e1253.
[35] S.J. Mcclue, D. Blake, R. Clarke, A. Cowan, L. Cummings, P.M. Fisher,
M. Mackenzie, J. Melville, K. Stewart, S. Wang, N. Zhelev, D. Zheleva, D.P. Lane,
Int. J. Cancer 102 (2002) 463e478.
[36] K. Dalip, P. Gautam, K. Angela, Chavers, H.C. Kuei, S. Kavita, Eur. J. Med. Chem.
46 (2011) 3085e3092.
[37] G. Griffiths, F. Scaerou, C. Midgley, S. Mcclue, Proc. Am. Assoc. Cancer Res. 49
(2008), Abstract 5644.
[38] J. Matysiak, A. Opolski, Bioorg. Med. Chem. 14 (2006) 4483e4489.
[39] W.A. Remrs, A. Wilson, B. Gisvolds, Text Book of Organic, Medicinal and
Pharmaceuticals Chemistry, Lippincott Company, Philadeplphia, 1982.
[40] T.T. Azza, H.G. Hanan, E.S. Hussein, Eur. J. Med. Chem. 47 (2012) 445e451.
[41] D.A. Ibrahim, Eur. J. Med. Chem. 44 (2009) 2776e2781.
[42] T. Lissitchkov, G. Arnaudov, D. Peytchev, K. Merkle, J. Cancer Res. Clin. Oncol.
132 (2) (2006) 99e104.
References
[1] M. Kidwai, R. Venkataramanan, R. Mohan, P. Sapra, Curr. Med. Chem. 9 (2002)
1209e1228.
[2] J.F.L. Saez, C.D.L. Torre, J. Pincheira, G.G. Martin, Histol. Histopathol. 13 (1998)
1197e1214.
[3] L.H. Hartwell, M.B. Kastan, Science 266 (1994) 1821e1828.
[4] T.D. Penning, G.D. Zhu, V.B. Gandhi, J. Gong, X. Liu, Y. Shi, V. Klinghofer,
E.F. Johnson, C.K. Donawho, D.J. Frost, V.B. Diaz, J.J. Bouska, D.J. Osterling,
A.M. Olson, K.C. Marsh, Y. Luo, V.L. Giranda, J. Med. Chem. 52 (2009) 514e523.
[5] D. Hao, J.D. Rizzo, S. Stringer, R.V. Moore, J. Marty, D.L. Dexter, G.L. Mangold,
J.B. Camden, D.D. Von- Hoff, S.D. Weitman, Invest. New Drugs 20 (2002)
261e270.
[6] H.M. Refaat, Eur. J. Med. Chem. 45 (2010) 2949e2956.
[7] S.C. Lio, J. Johnson, A. Chatterjee, J.W. Ludwig, D. Millis, H. Banie, J.C. Sircar,
A. Sinha, M.L. Richards, Can. Chem. Pharmacol. 61 (2008) 1045e1058.
[8] R. Abonia, E. Cortes, B. Insuasty, J. Quiroga, M. Nogueras, J. Cobo, Eur. J. Med.
Chem. 46 (2011) 4062e4070.
[9] M.M. Ramla, M.A. Omar, A.M.M. El-Khamry, H.I. El-Diwani, Bioorg. Med. Chem.
14 (2006) 7324e7332.
[10] J. Styskala, L. Styskalova, J. Slouka, M. Hajduch, Eur. J. Med. Chem. 43 (2008)
449e455.
[11] S. Demirayak, A. Usama, A.C. Mohsen, K. Agri, Eur. J. Med. Chem. 37 (2002)
255e260.
[12] H.T.A. Mohsen, F.A.F. Ragab, M.M. Ramla, H.I.E. Diwani, Eur. J. Med. Chem. 45
(2010) 2336e2344.
[43] W.U. Knauf, T. Lissichkov, A. Aldaoud, J. Clin. Oncol. 27 (2009) 4378e4384.
[44] M. Rashid, A. Husain, R. Mishra, Eur. J. Med. Chem. xxx (2012) 1e12.
[45] D.J. Prasad, M. Ashok, P. Karegoudar, B. Poojary, B.S. Holla, N.S. Kumari, Eur. J.
Med. Chem. 44 (2009) 551e557.
[46] P. Karegoudar, D.J. Prasad, M. Ashok, M. Mahalinga, B. Poojary, B.S. Holla, Eur.
J. Med. Chem. 43 (2008) 808e815.
[13] B.V.S. Kumar, S.D. Vaidya, R.V. Kumar, S.B. Bhirud, R.B. Mane, Eur. J. Med.
Chem. 41 (2006) 599e604.
[14] O.O. Guven, T. Erdogan, H. Goker, S. Yildiz, Bioorg. Med. Chem. Lett. 17 (2007)
2233e2236.
[15] H. Goker, C. Kus, D.W. Boykin, S. Yildiz, N. Altanlar, Bioorg. Med. Chem. 10
(2002) 2589e2596.
[47] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. Mcmahon, D. Vistica,
J.R. Warren, H. Bokesch, S. Kenney, M.R. Boyd, J. Natl. Cancer Inst. 82 (1990)
1107e1112.
[48] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose,
J. Langley, P. Cronise, J. Natl. Cancer Inst. 83 (1991) 757e766.
[49] M.R. Grever, S.A. Schepartz, B.A. Chabner, Semin. Oncol. 19 (1992) 622e638.
[50] (a) M.R. Boyd, K.D. Paull, Drug Dev. Res. 34 (1995) 91e109.(b) , in: M.R. Boyd,
B.A. Teicher (Eds.), Can. Drug. Dis. Devel, vol. 2, 1997, pp. 23e43.
[51] N. Kode, L. Chen, D. Murthy, D. Adewumi, S. Phadtare, Eur. J. Med. Chem. 42
(2007) 327e333.
[16] I. Kerimov, G.A. Kilcigil, B.C. Eke, N. Altanlar, M. Iscan, J. Enzym. Inhib. Med.
Chem. 17 (2007) 696e701.
[17] D. Sharma, B. Narasimhan, P. Kumar, V. Judge, R. Narang, E.D. Clercq,
J. Balzarini, J. Enzym. Inhib. Med. Chem. 24 (2009) 1161e1168.
[18] G.A. Kilcgil, C. Kus, T. Coban, B.C. Eke, M. Iscan, J. Enzym. Inhib. Med. Chem. 19
(2004) 129e135.
[19] D. Kumar, N.M. Kumar, K.H. Chang, K. Shah, Eur. J. Med. Chem. 45 (2010)
4664e4668.
[20] R.E. Avanzo, C. Anesini, M.L. Fascio, M.I. Errea, N.B.D. Accorso, Eur. J. Med.
Chem. xxx (2011) 1e7.
[52] P. Corona, A. Carta, M. Loriga, G. Vitale, G. Paglietti, Eur. J. Med. Chem. 44
(2009) 1579e1591.
[53] S.A.F. Rostom, Bioorg. Med. Chem. 14 (2006) 6475e6485.
[21] H.B. El-Nassan, Eur. J. Med. Chem. 46 (2011) 2031e2036.
[22] N.S. El-Sayed, E.R. El-Bendary, S.M. El-Ashry, M.M. El-Kerdawy, Eur. J. Med.
Chem. 46 (2011) 3714e3720.
Please cite this article in press as: A. Husain, et al., Benzimidazole clubbed with triazolo-thiadiazoles and triazolo-thiadiazines: New anticancer
agents, European Journal of Medicinal Chemistry (2012), http://dx.doi.org/10.1016/j.ejmech.2012.07.011