New styryl sulfones as anticancer agents

Article abstract of DOI:10.1016/S0223-5234(03)00144-2

New styryl sulfone compounds have been synthesized and evaluated for their anti-proliferative activity. Among the compounds synthesized, one compound (7k) has shown 51% tumor growth inhibition in mice implanted with HT-29 human carcinoma at 400 mg kg^-1 orally.

Full text of DOI:10.1016/S0223-5234(03)00144-2

European Journal of Medicinal Chemistry 38 (2003) 811Á824
/
www.elsevier.com/locate/ejmech
Original article
New styryl sulfones as anticancer agentsꢀ
Manohar Sharma Vedula a,*, Aravind Babu Pulipaka a, Chandrasekhar Venna a,
Vamsee Krishna Chintakunta a, Sreenu Jinnapally a, Venkata Adiseshu Kattuboina a,
Ravi Krishna Vallakati a, Vishnu Basetti a, Venkateswarlu Akella a, Sriram Rajgopal b,
Ajaya Kumar Reka b, Sravan Kumar Teepireddy b, Prem Kumar Mamnoor b,
Ramanujam Rajagopalan b, Gopalakrishnan Bulusu c, Akash Khandelwal c,
Vijay V. Upreti d, Srinivas Rao Mamidi d
a Discovery Chemistry, Discovery Research, Dr. Reddy’s Laboratories, Bollaram Road, Hyderabad 500 050, India
b Discovery Biology, Discovery Research, Dr. Reddy’s Laboratories, Bollaram Road, Hyderabad 500 050, India
c Molecular Modeling & Drug Design, Discovery Research, Dr. Reddy’s Laboratories, Bollaram Road, Hyderabad 500 050, India
d Drug Metabolism & Pharmacokinetics, Discovery Research, Dr. Reddy’s Laboratories, Bollaram Road, Hyderabad 500 050, India
Received 20 January 2003; received in revised form 11 July 2003; accepted 11 July 2003
Abstract
New styryl sulfone compounds have been synthesized and evaluated for their anti-proliferative activity. Among the compounds
synthesized, one compound (7k) has shown 51% tumor growth inhibition in mice implanted with HT-29 human carcinoma at 400
mg kgꢀ1 orally.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Substituted benzimidazole styryl sulfones; MAP kinase inhibitors; Anti-cancer activity
1. Introduction
upstream dual specifying kinases [5Á7]. The best under-
/
stood MAPK pathway involves extra cellular signal
regulated kinases, which constitute the Ras/Raf/MEK/
ERK kinase cascade [8]. Once this pathway is activated
by different stimuli, MAPK phosphorylates a variety of
proteins including several transcription factors, which
translocate into the nucleus and activate gene transcrip-
tion. Negative regulation of this pathway could arrest
the cascade of these events and will inhibit the prolif-
eration of cancer cells.
Recently, it was reported [9] that certain styryl sulfone
derivatives like FRI-20 inhibit the tumor cell growth
and viability by inhibiting the MAPK signal transduc-
tion pathway. The compounds regulate the ERK and
inhibit the proliferation of breast and prostate tumor
cells in a dose-dependent manner without affecting
normal cell growth. The cell growth inhibitory activity
of this compound is dictated by the nature and position
of the functional groups.
Extra cellular signals received at transmembrane
receptors and relayed into the cells by the signal
transduction pathways [1], have been implicated in a
wide array of physiological processes such as induction
of cell proliferation, differentiation or apoptosis [2]. The
mitogen activated protein kinase (MAPK) cascade is a
major signaling system by which cells transduce extra
cellular cues into intracellular responses [3,4]. In mam-
malian cells, the extra cellular signal-regulated kinases
(ERKs), ERK-1 and ERK-2 are the archetypal and best
studied members of the MAPK family and are activated
by phosphorylation on threonine/tyrosine residues by
ꢀ DRL Publication No. 282.
* Correspondence and reprints.
E-mail address: sarmavm@drreddys.com (M.S. Vedula).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00144-2

812
M.S. Vedula et al. / European Journal of Medicinal Chemistry 38 (2003) 811Á824
/
Fig. 1. Synthetic routes to compounds 7aÁk.
/
The objective of the present study is to synthesize
2. Chemistry
novel styryl sulfone compounds exhibiting wide spec-
trum of activity and increased potency on different types
of tumor cells both in-vitro and in-vivo. In order to
facilitate the design and predict the activity of this new
styryl sulfone compounds we performed a 3D QSAR
study using comparative molecular similarity indices
analysis (CoMSIA) [10,11]. We report here the synth-
esis, QSAR, in-vitro and in-vivo pharmacological
evaluation and pharmacokinetic data of these com-
pounds.
Synthesis of these compounds is outlined in Figs. 1
and 2. Commercially available O-phenylene diamines
were condensed with thioglycolic acid to get 1H-
benzo[d]imidazol-2-ylmethanethiol (2) which were
further reacted with substituted phenacyl chlorides in
presence of potassium carbonate in acetone to get 2-
[{1H-benzo[d]imidazol-2-yl methyl}sulfanyl]-1-phenyl-
1-ethanone (3aÁ/k). These were easily converted to 2-
[{1H-benzo[d]imidazol-2-ylmethyl}sulfanyl]-1-phenyl-1-

M.S. Vedula et al. / European Journal of Medicinal Chemistry 38 (2003) 811Á
/824
813
Fig. 2. Synthetic routes to compounds 12aÁh.
/
ethanol (4aÁ
Compounds 4aÁ
zo[d]imidazol-2-ylmethyl}sulfonyl]-1-phenyl-1-ethanol
(5aÁk). These compounds were subjected to dehydra-
tion with methane sulfonyl chloride to afford 1-{methyl-
sulfonyl}-2-{([(E)-2-phenyl-1-ethenyl] sulfonyl}6aÁk)
which in turn were demesylated to 1H-benzo[d]imida-
zol-2-ylmethyl[(E)-2-phenyl-1-ethenyl]sulfones (7aÁk)
with 33% HBr in AcOH.
/
k) by reduction with sodium borohydride.
(10aÁ
benzo[d]imidazol-2-ylsulfonyl}-1-phenyl-1-ethanol
(11aÁh). Dehydration with methane sulfonyl chloride
followed by demesylation with 33% HBr in AcOH
afforded 1H-benzo[d]imidazol-2-yl-[{E}-2-phenyl-1-
ethenyl]sulfone (12aÁh).
/
h), which subsequently were oxidized to 2-{1H-
/k were oxidized to get 2-[{1H-ben-
/
/
/
/
/
3. Results and discussion
In a similar manner, 1H-benzo[d]imidazol-2-ylhydro-
sulfides (8) was prepared by condensing commercial O-
phenylene diamine with carbon disulfide in the presence
of potassium hydroxide and ethanol. Compound 8 was
reacted with substituted phenacyl chlorides in potassium
carbonate in acetone to get 2-{1H-benzo[d]imidazol-2-
All the compounds were evaluated for their in-vitro
cytotoxic activity against different types of cancer cell
lines. The tested compounds showed moderate to good
cytotoxic activity and indeed, some of them were more
potent than FRI-20. The compound 1 of this series
showed weak anti-proliferative activity in the tested cell
lines. Compounds substituted with halogens on the
phenyl ring at the para position namely, 12d and 12e
ylsulfanyl}-1-phenyl-1-ethanones (9aÁh). These were
/
reduced with sodium borohydride to afford 2-{1H-
benzo[d]imidazol-2-ylsulfanyl}-1-phenyl-1-ethanol

Table 1
In-vitro anti-cancer activities (GI50 in mM) of compounds 12aÁ
/
12h
Compound
R
R1
R2
R3
Breast
CNS
Colon
Lung
H522
Melanoma
UACC62
Ovarian
Prostate
Renal
MCF7/ADR
MCF7
U251
SW620
HT29
SKOV3
PA1 DU145
PC3 A498
FRI-20
12a
12b
80
80
ND
70
9
70
19
15
6
70
16
18
12
1
ND
10
10
90
8
ND
20
50
20
ND
30
15
15
5
6
40
ND
80
9
90
33
10
3
H
H
Cl
F
H
H
H
H
H
H
H
H
CH3
CH3
H
28
29
13
16
18
15
5
16
ND
ND
15
20
28
ND
ND
10
12c
12d
H
Cl
Br
F
17
10
H
8
18
2
0.35
15
20
12
2
10
12
12e
12f
H
H
4.5
100
100
0.3
8
100
ND
ND
0.06
ND
100
35
12
100
100
2
ND
50
100
ND
12
5
5OMe
5-OMe
H
H
Br
Cl
100
100
0.35
50
100
6.5
40
30
100
20
5
100
ND
ND
100
25
12g
12h
CH3
C(CH3)3
20
7
OH C(CH3)3
3.5
15
10
ND, not done.

M.S. Vedula et al. / European Journal of Medicinal Chemistry 38 (2003) 811Á
/824
815
showed activity equal to the standard molecule FRI-20
(Table 1). It is interesting to note that 12d with bromo
substituent on the para position of the phenyl ring
showed activity with an average GI50 of 8.5 mM. The
compound showed significant activity particularly in
breast (MCF-7), renal (A-498), CNS (U-251) and
ovarian (PA1) cell lines. Compounds 12b and 12c were
prepared to test the tolerability of further substitution
ortho to the halo substituent. Unfortunately both the
compounds failed to show appreciable cytotoxic activ-
ity. Further investigations were carried out to improve
the activity of compounds in this series and it was found
that 3,5-di-tert-butyl-4-hydroxy phenyl compound (12h)
showed activity better than either unsubstituted or halo
substituted benzimidazole derivatives. Compound 12h
detrimental to biological activity. Blue colored regions
indicate areas where electropositive groups are predicted
to enhance biological activity, while red regions repre-
sent areas where electronegative groups are predicted to
favor activity. The hydrophobic, hydrogen bond donor
and hydrogen bond acceptor contours are displayed in
Figs. 5 and 6, respectively. The hydrophobic fields
(yellow, hydrophobic group favored; white, hydropho-
bic disfavored), H-bond donor (Cyan, favored; purple,
disfavored) and the H-bond acceptor (magenta, favored;
red, disfavored) fields indicate areas around the mole-
cules where changes increased or decreased activity.
The higher activities of 12h and 12a as compared to
other compounds may be due to the fact that the t-butyl
substituent and the phenyl ring of 12h and 12a occupy
green contours. The methyl group of compound 7k
occupies green contours and hence exhibits higher
activity. The phenyl ring of 6j, 6i, 6g, 6e, 6d and 6b
occupies yellow contours and hence exhibits lower
activity. The cyan contour (H-bond favor) is embedded
inside the purple contour (H-bond disfavored). The
SO2CH3 group of 6j, 6i, 6g, 6e and 6b touches cyan
contour and protrudes out of purple contour. A graph
of actual and predicted pGI50 values is shown in Fig. 7.
The predictive power of each of the 3D-QSAR models
was evaluated by predicting the pGI50 of 16 additional
ligands not included in the training set. In all the cases,
the predictive values fall close to the actual pGI50 values,
deviating by not more than one logarithmic unit.
showed a GI50 value of B5 mM in most of the cell lines
/
and also showed activity in adriamycin resistant breast
cancer cell line indicating that these substituents play an
important role in the cytotoxicity as predicted by QSAR
studies. Compounds like 12f and 12g were also prepared
with a methoxy group on the benzimidazole moiety.
These compounds did not show any activity.
In continuation of SAR studies, compounds 7a and
7h (Table 2) were synthesized by incorporating an
additional methylene between benzimidazole and sul-
fone moiety. Even though, 7h showed better activity
than 7a, it was not comparable with 12h. To check the
tolerability of additional substitution on the imidazole
ring, the free nitrogen in benzimidazole was substituted
with a methyl sulfonyl group. All the compounds in this
series 6aÁ
7k was found to be the most active in this series showing
50% growth inhibition in 4 cell lines at B1 mM
/
j failed to show activity (Table 3). Compound
5. Conclusions
/
concentration. The pharmacokinetic properties in mice
of 7k and FRI-20 were studied and are summarized in
Table 4. Compound 7k, which has relatively good
pharmaco kinetic properties, was selected for in-vivo
experiments using human colon adenocarcinoma (HT-
29) xenograft in nude mice. At 400 mg kgꢀ1 dose
compound 7k, when administered orally, showed 51%
tumor growth inhibition as shown in Fig. 3.
In conclusion, we have described the synthesis of a
novel class of styryl sulfone compounds, their 3D QSAR
studies using CoMSIA method and demonstrated that
these compounds have potent anti-proliferative activity.
Based on pharmacokinetic profiles we selected a com-
pound 7k for in-vivo studies. The in-vivo studies
revealed that the compound showed efficacy against
HT-29 human carcinoma inhibiting 51% tumor growth
in a QDx20 schedule.
4. COMSIA studies
6. Experimental
Partial least-square analysis (PLS) (Table 5) shows
results obtained from atom based alignment. The atom-
based alignment shows a cross-validated r2 of 0.921 with
five components, a non-cross-validated r2 of 0.977, and
standard error of estimate (s) of 0.057. The residual
values of the training and test set molecules are shown in
(Tables 6 and 7), respectively. The steric and electro-
static contour plots are shown in Fig. 4. The green
regions indicate areas where steric bulk is predicted to
enhance biological activity, whereas yellow contours
indicate regions where steric bulk is predicted to be
6.1. Chemistry
The reported molecules were synthesized in the
Discovery Research department of Dr. Reddy’s Labora-
tories. All other chemicals and reagents used in the
synthesis were of reagent grade. Melting points were
determined on an Electro thermal melting point appa-
ratus (Buchi 535) and were uncorrected. Pre-coated
silica gel plates (20ꢁ 20 cm, silica gel 60 F254, Merck)
/
1
were used for TLC. H-NMR spectra were recorded on

Table 2
In-vitro anti-cancer activities (GI50 in mM) of compounds 7aÁ
/
7k
Compound
R
R1
R2
R3
Breast
CNS
Colon
Lung
H522
Melanoma
UACC62
Ovarian
Prostate
Renal
MCF7/ADR
MCF7
U251
SW620
HT29
SKOV3
PA1 DU145
PC3 A498
FRI-20
7a
7b
80
20
30
40
30
15
25
20
45
15
100
25
ND
100
100
60
9
100
100
30
6
100
100
40
ND
100
100
40
10
100
100
35
ND
100
100
25
50
ND
ND
25
ND
100
10
6
40
30
18
52
3
ND
ND
ND
40
9
15
35
20
50
30
20
35
20
30
35
30
H
H
H
H
C(CH3)3
H
OH
OMe
Br
F
C(CH3)3
H
H
H
H
H
H
H
H
H
H
7c
7d
H
CH3
25
19
18
15
100
100
20
50
18
12
15
14
10
ND
ND
ND
ND
30
ND
ND
ND
ND
25
7e
6-Cl CH3
F
0.05
15
15
7f
7g
6-Cl
H
H
Cl
Cl
Cl
Cl
F
0.02
100
45
15
100
20
30
3
18
18
15
15
20
2
Cl
H
100
25
100
30
100
30
100
20
100
30
7h
7i
H
20
30
100
13
H
6-Cl
CH3
H
50
13
30
13
35
30
20
13
20
12
20
20
25
7j
7k
ND
ND
100
16
ND
ND
6-Cl CH3
Cl
0.03
0.01
10
15
0.15
0.3
ND, not done.

Table 3
In-vitro anti-cancer activities (GI50 in mM) of compounds 6aÁ
/
k
Compound
R
R1
R2
R3
Breast
CNS
Colon
Lung
H522
Melanoma
UACC62
Ovarian
Prostate
DU145
Renal
MCF7/ADR
MCF7
U251
SW620
HT29
SKOV3
PA1
ND
PC3 A498
FRI-20
6a
6b
80
4.5
60
ND
60
9
4.5
70
6
5
ND
5.5
100
40
10
30
ND
18
40
20
55
25
20
35
25
100
40
60
50
100
30
6
4.5
35
18
25
20
20
20
20
30
15
30
ND
30
9
60
H
H
H
H
C(CH3)3
H
OH
OMe
Br
F
C(CH3)3
3.5
H
H
H
H
H
H
H
H
H
H
30
30
100
45
70
30
20
20
20
20
20
15
20
30
20
20
100
40
90
18
6c
6d
H
CH3
20
25
50
20
100
100
35
100
90
100
50
100
60
100
40
100
15
100
60
100
15
6e
6-Cl CH3
F
30
20
6f
6g
6-Cl
H
H
Cl
Cl
Cl
Cl
F
65
25
25
25
40
18
25
20
20
30
Cl
H
100
30
100
40
25
25
100
80
100
45
100
60
6h
6i
H
25
30
30
70
H
6-Cl
CH3
H
100
80
100
65
100
35
100
100
100
100
90
100
90
4.5
35
100
35
6j
6k
12
100
6-Cl CH3
Cl
80
90
90
100
100
35
15
ND, not done.

818
M.S. Vedula et al. / European Journal of Medicinal Chemistry 38 (2003) 811Á824
/
Table 4
Pharmacokinetic parameters of 7k and FRI-20 in mice at 100 mg kgꢀ1
Table 6
Actual and predicted pGI50 values of training set molecules
FRI-20
7k
S. No. Code Actual pGI50
Predicted pGI50
Residuals
AUC(0 Á t ) (mg h mLꢀ1
)
1.27
1.95
0.35
2.00
0.21
3.34
10.57
11.80
9.90
0.50
0.27
2.57
1
6l
4.6383
4.5686
4.5528
4.2924
4.4815
4.1079
4.2366
4.3565
4.1024
4.2076
4.2007
4.1871
4.7696
4.7959
4.8239
4.7696
4.7447
4.6383
5.3010
5.0458
5.0398
4.6401
4.5866
4.5366
4.3472
4.6189
4.1405
4.1940
4.3208
4.1839
4.2229
4.1683
4.1700
4.6847
4.7921
4.8558
4.7583
4.7092
4.6049
5.3074
5.0359
5.0165
ꢀ
/
0.0018
0.0180
0.0162
0.0548
0.1374
0.0326
0.0426
0.0357
0.0815
0.0153
0.0324
0.0171
0.0849
0.0038
0.0319
0.0113
0.0355
0.0334
0.0064
0.0099
0.0233
AUC(0 Á ꢀ) (mg h mLꢀ1
)
2
7i
ꢀ
/
Cmax (mg mLꢀ1
Tmax (h)
Kel (hꢀ1
t1/2,b (h)
)
3
7h
6j
4
ꢀ
/
)
5
7c
6i
ꢀ
/
6
ꢀ/
7
6g
6e
8
9
6d
6b
6m
6n
12c
7f
ꢀ
/
10
11
12
13
14
15
16
17
18
19
20
21
ꢀ/
7e
ꢀ/
12i
12e
12j
12h
12a
12f
ꢀ/
Table 7
Actual and predicted pGI50 values of test set molecules
S. No. Code Actual pGI50
Predicted pGI50
Residuals
Fig. 3. Xenograft data of 7k.
1
7m
6o
6h
6f
4.0555
4.5229
4.4437
4.5850
4.5686
4.5686
4.1549
4.1739
4.0969
4.5686
4.4089
4.3768
4.7696
5.0000
4.2676
4.7447
4.2818
4.1812
4.3071
4.1527
4.1376
4.6368
4.7386
4.6894
4.5884
4.7263
4.8347
5.0525
4.8105
4.6621
ꢀ
/
0.6892
0.2411
0.2625
0.2779
0.4159
0.4310
0.4819
0.5647
0.5925
0.0198
0.3174
0.4579
0.2829
0.1895
0.3945
2
Table 5
Summary of PLS analysis
3
4
5
6c
CoMSIA
6
6a
7g
7b
7a
7l
r2cv
0.921
0.977
0.057
5
7
ꢀ
/
r2conv
SEE
N
8
ꢀ
/
9
ꢀ
/
10
11
12
13
14
15
ꢀ
/
SDEP
0.106
7d
7j
ꢀ
/
ꢀ
/
Field contributions (%)
Steric
12b
7k
12g
ꢀ/
13.7
24.1
29.5
23.6
9.1
Electrostatic
Hydrophobic
Donor
ꢀ/
Acceptor
SEE, standard error of estimate; r2cv, cross-validated correlation
coefficient; r2conv, non-cross-validated correlation coefficient; N, opti-
mum number of components; SDEP, standard error of prediction.
6.1.1. General procedure for the synthesis of 1H-
benzo[d]imidazol-2-yl methanethiol (2)
a Varian-200 MHz, Gemini-200-software spectrometer
(Varian USA). TMS was used as internal standard and
chemical shifts are given in ppm. IR Spectra were
recorded on a Perkin Elmer 1600 series FT IR
Commercially available O-phenylene diamines (1)
(0.04 mol) and thioglycolic acid (0.04 mol) were added
to 4 N HCl (120 mL) and contents were stirred at reflux
temperature for 5 h. The mixture was basified with aq.
sodium bi-carbonate solution till pH 10 and the solid
precipitated was filtered to get 1H-benzo[d]imidazol-2-yl
methanethiol as a solid.
(PerkinÁElmer, USA). Mass spectra were recorded on
/
a Hewlett Packard 5989-A mass spectrometer. Starting
materials were either commercially available or synthe-
sized according to known literature methods.

M.S. Vedula et al. / European Journal of Medicinal Chemistry 38 (2003) 811Á
/824
819
Fig. 4. CoMSIA stdev*coeff. Regions favoring bulkier substituents are represented by green contours while regions disfavors for bulky groups are
represented by yellow contours. Areas favoring positive charges are represented by blue contours while negative charges are represented by red
contours.
Fig. 5. CoMSIA stdev*coeff. Hydrophobic contour plots; yellow
contours indicate regions where hydrophobic groups increases activity,
whereas white contours indicate regions where hydrophobic group
decreases activity.
Fig. 7. Graph of actual vs. predicted pGI50 of training and test set
molecules for CoMSIA 3D QSAR model.
Fig. 6. CoMSIA stdev*coeff H-bond donor and acceptor plots: cyan contours indicate regions where H-bond donor group increases activity,
whereas purple contours indicate regions where H-bond donor group decreases activity. Magenta contours indicate regions where H-bond acceptor
group increases activity, whereas red contours indicate regions where H-bond acceptor group decreases activity.

820
M.S. Vedula et al. / European Journal of Medicinal Chemistry 38 (2003) 811Á824
/
6.1.2. General procedure for the synthesis of 2-[{1H-
benzo[d]imidazol-2-ylmethyl}sulfanyl]-1-phenyl-1-
6.1.6. General procedure for the synthesis of 1H-
benzo[d]imidazol-2-ylmethyl[(E)-2-phenyl-1-
ethanone (3aÁ
/
j)
ethenyl]sulfone (7aÁj)
/
NaH (60%, 21 mmol) was added to a solution of 1H-
benzo[d]imidazol-2-ylmethanethiol (2) (7 mmol) in 15
mL of DMF at 0 8C and the contents were stirred for 10
min. Appropriate phenacyl chloride (7 mmol) was added
to the reaction mixture and contents were stirred for 2 h
at room temperature (r.t.). Cold water (100 mL) was
added to the reaction mixture and solid precipitated was
filtered to give 2-[{1H-benzo[d]imidazol-2-ylmethyl}sul-
fanyl]-1-phenyl-1-ethanone.
Three milliliters of 33% HBr in AcOH was added
slowly to 1-{methylsulfonyl}-2-{([(E)-2-phenyl-1-ethe-
nyl]sulfonyl}methyl}-1H-benzo[d] imidazole (6) (2.3
mmol) at 0 8C and the contents were stirred at r.t. for
15 min. Ice was added to reaction mixture and the solid
formed was filtered to give crude product. This was
purified by column chromatography to give 1H-ben-
zo[d]imidazol-2-yl
fone.
methyl[(E)-2-phenyl-1-ethenyl]sul-
6.1.3. General procedure for the synthesis of 2-[{1H-
benzo[d]imidazol-2-ylmethyl}sulfanyl]-1-phenyl-1-
6.1.7. Synthesis of 2,6-di(tert-butyl)-4-[(E)-2-({[1-
(methylsulfonyl)-1H-benzo[d]imidazol-2-
yl]methyl}sulfonyl)-1-ethenyl] phenol (6a)
ethanol (4aÁj)
/
Sodium borohydride (8.1 mmol) was added slowly to
a solution of 2-[{1H-benzo[d] imidazol-2-yl methyl}sul-
fanyl]-1-phenyl-1-ethanone (3) (2.7 mmol) in 20 mL of
1:1 chloroform and methanol at 0 8C and the contents
were stirred for 30 min at r.t. Water (50 mL) was added
to reaction mixture and extracted with ethyl acetate. The
organic layer was evaporated to give 2-[{1H-benzo[d]i-
midazol-2-yl methyl}sulfanyl]-1-phenyl-1-ethanol.
Yield (42%); m.p. 197Á :
199 8C; IR (KBr) cmꢀ1
/
3554.5, 3006, 2961, 2916, 1616, 1594, 1537, 1453, 1420,
1373, 1323, 1293, 1259, 1226, 1178, 1147, 1100; 1H-
NMR (DMSO-d6), dH 7.2 (m, 7H, aromatic), 6.9 (d, 1H,
Jꢂ
/
15.4 Hz, Ar), 5.8 (s, 1H, Á
/
OH), 5.2 (s, 2H, ÁCH2),
/
3.8 (s, 3H, SO2CH3), 1.4 (s, 18H, di-tert-butyl); Mass
(m/z) (DIP): 505 (Mꢃ), 427 (100%), 363, 233.
6.1.4. General procedure for the synthesis of 2-[{1H-
benzo[d]imidazol-2-yl methyl}sulfonyl]-1-phenyl-1-
6.1.8. Synthesis of 2-({[(E)-2-(4-methoxyphenyl)-1-
ethenyl] sulfonyl} methyl)-1-(methylsulfonyl)-1H-
benzo[d]imidazole (6b)
ethanol (5aÁj)
/
Yield (45%); m.p. 169Á
171 8C; IR (KBr) cmꢀ1: 3024,
2929, 1602, 1511, 1451, 1366, 1290, 1254, 1172, 1122;
/
Potassium mono persulphate triple salt (5.5 mmol)
was added to a solution of 2-[{1H-benzo[d]imidazol-2-
ylmethyl}sulfanyl]-1-phenyl-1-ethanol (4) (2.7 mmol) in
1:1:1 dichloromethane:tert-butanol:water (30 mL) and
contents were stirred at r.t. for 7 h. Water (50 mL) was
added to reaction mixture and extracted with ethyl
acetate. Evaporation of the organic solvent gave crude
product. This crude was washed with acetone to give
pure 2-[{1H-benzo[d]imidazol-2-ylmethyl}sulfonyl]-1-
phenyl-1-ethanol.
1H-NMR (CDCl3), dH 7.2 (m, 7H, Ar), 6.8 (m, 3H, Ar),
5.2 (s, 2H, ÁCH2), 3.8 (s, 3H, SO2Me), 3.6 (s, 3H, CH3);
/
Mass (m/z) (DIP): 407 (Mꢃ), 329 (100%), 265, 211.
6.1.9. Synthesis of 2-({[(E)-2-(4-bromophenyl)-1-
ethenyl] sulfonyl}methyl)-1-(methyl sulfonyl)-1H-
benzo[d]imidazole (6c)
Yield (45%); m.p. 199Á
201 8C; IR (KBr) cmꢀ1: 3009,
2930, 1622, 1586, 1487, 1450, 1368, 1328, 1240, 1108,
/
6.1.5. General procedure for the synthesis of 1-
{methylsulfonyl}-2-{([(E)-2-phenyl-1-
1
1057; H-NMR (CDCl3), dH 7.2 (m, 9H, Ar), 7.0(d,1H,
Jꢂ
/
15.6 Hz, styryl), 5.2 (s, 2H, ÁCH2), 3.8 (s, 3H,
/
ethenyl]sulfonyl}methyl}-1H-benzo[d] imidazole (6aÁ
2-[{1H-Benzo[d]imidazol-2-ylmethyl}sulfonyl]-1-
/
j)
SO2CH3); Mass (m/z) (DIP): 457 (Mꢃ), 457, 379, 241,
211, 190 (100%).
phenyl-1-ethanol (5) (2.7 mmol) was taken in dichloro
methane (20 mL), triethyl amine (5.5 mmol) was added
and the contents were stirred for 15 min. Methane
sulfonyl chloride (5.5 mmol) was added slowly to the
reaction mixture at 0 8C and stirred for 45 min. Water
(100 mL) was added to the reaction mixture and
extracted with dichloro methane. Evaporation of the
organic layer and purification of the residue by column
6.1.10. Synthesis of 2-({[(E)-2-(4-fluoro-3-
methylphenyl)-1-ethenyl]sulfonyl}methyl)-1-
(methylsulfonyl)-1H-benzo[d]imidazole (6d)
Yield (43%); m.p. 179Á
1818C; IR (KBr) cmꢀ1: 3064,
3022, 2938, 1624, 1592, 1527, 1502, 1452, 1408, 1363,
/
1
1308, 1155, 1226; H-NMR (CDCl3), dH 6.9 (m, 9H,
chromatography over 100Á
/
200 mesh silica gel gave 1-
{methylsulfonyl}-2-{([(E)-2-phenyl-1-ethenyl]sulfonyl-
Ar), 5.2 (s, 2H, Á
/
CH2), 3.5 (s, 3H, SO2CH3) 2.2 (s, 3H,
CH3); Mass m/z (DIP): 409 (Mꢃ), 331 (100%), 190.
methyl}-1H-benzo[d] imidazole.

M.S. Vedula et al. / European Journal of Medicinal Chemistry 38 (2003) 811Á
/824
821
6.1.11. Synthesis of 6-chloro-2-({[(E)-2-(4-fluoro-3-
methyl phenyl)-1-ethenyl]sulfonyl}methyl)-1-(methyl
sulfonyl)-1H-benzo[d]imidazole (6e)
SO2Me); Mass (m/z) (DIP): 429 (Mꢃ), 391, 351
(100%), 287, 279, 254, 245, 231.
Yield (35%); m.p. 194Á
2932, 1607, 1501, 1368, 1302, 1234, 1168, 1125, 1048;
1H-NMR (CDCl3): dH 7.0 (m, 8H, Ar), 5.2 (s, 2H, Á
/
195 8C; IR (KBr) cmꢀ1: 3018,
6.1.17. Synthesis of4-{(E)-2-[(1H-benzo[d]imidazol-2-
yl methyl} sulfonyl]-1-ethenyl}-2,6-di{tert-butyl} phenol
(7a)
/
Yield (50%); m.p. 142Á
2963, 1713, 1674, 1616, 1548, 1439, 1362, 1310, 1225,
1201; 1H-NMR (CDCl3), dH 7.6 (m, 2H, Ar), 7Á
7.45 (m,
5H, Ar), 6.8 (d, 1H, Jꢂ15.6 Hz, styryl), 6.2 (s, 1H, OH),
/
144 8C; IR (KBr) cmꢀ1: 3610,
CH2), 3.8 (s, 3H, SO2Me), 2.2 (s, 3H,ÁCH3); Mass m/z
/
(DIP): 443 (Mꢃ), 365 (100%), 301, 245, 167.
/
/
6.1.12. Synthesis of 6-chloro-2-({[(E)-2-(4-
chlorophenyl)-1-ethenyl]sulfonyl}methyl)-1-
(methylsulfonyl)-1H-benzo[d]imidazole (6f)
4.6 (s, 2H, CH2), 1.4 (s, 18H, di-tert.butyl); Mass (m/z)
(DIP): 427 (, Mꢃ,100%), 362, 217, 132.
Yield (39%); m.p. 230Á
232 8C; IR (KBr) cmꢀ1: 3020,
2950, 1619, 1527, 1492, 1454, 1367, 1303, 1259, 1168,
/
6.1.18. Synthesis of 1-(1H-benzo[d]imidazol-2-yl
methyl[(E)-2-{4-methoxy phenyl}-1-ethenyl]sulfone
(7b)
1
1129, 1097; H-NMR (CDCl3): dH 7.2 (m, 9H, Ar), 7.0
(d, 1H, styryl, Jꢂ
3H, Á
SO2Me); Mass (m/z) (DIP): 406 (Mꢃ, 100%), 367,
303.
/
15.6 Hz), 5.2 (s,2H, Á/CH2), 3.8 (s,
Yield (53%); m.p. 190Á
191 8C; IR (KBr) cmꢀ1: 3060,
3002, 2940, 2835, 1599, 1511, 1433, 1404, 1308, 1291,
/
/
1
1257, 1173; H-NMR (CDCl3), dH 7.2 (m, 7H, Ar), 6.6
(m, 3H, Ar), 4.8 (s, 2H, CH2), 3.8 (s, 3H, OCH3); Mass
(m/z) (DIP): 329 (Mꢃ, 100%), 264, 249, 149, 132.
6.1.13. Synthesis of 2-({[(E)-2-(3,4-dichlorophenyl)-1-
ethenyl]sulfonyl}methyl)-1-(methylsulfonyl)-1H-
benzo[d]imidazole (6g)
Yield (44%); m.p. 194Á
2854, 1614, 1584, 1467, 1371, 1302, 1251, 1172, 1129;
1H-NMR (CDCl3), dH 7.0 (m, 9H, Ar), 5.2 (s, 2H, Á
/
196 8C; IR (KBr) cmꢀ1: 3026,
6.1.19. Synthesis of 1H-benzo[d]imidazol-2-yl
methyl[{E}-2-(4-bromophenyl)-1-ethenyl]sulfone (7c)
211 8C; IR (KBr) cmꢀ1: 3331,
/
Yield (49%); m.p. 209Á
3057, 1613.8, 1587, 1488, 1454, 1433, 1305;1H-NMR
(CDCl3), dH 7.2 (m, 9H, Ar), 6.8 (d, 1H, styryl, Jꢂ15.2
2), 381
/
CH2), 3.8(s, 3H, ÁSO2CH3); Mass (m/z) (DIP): 445
/
(Mꢃ), 367 (100%), 333, 303, 211.
/
Hz), 4.8 (s, 2H); Mass (m/z) (DIP): 379 (Mꢃꢃ
/
(Mꢃ), 315, 299, 183, 133, 113.
6.1.14. Synthesis of 2-({[(E)-2-(4-chlorophenyl)-1-
ethenyl]sulfonyl}methyl)-1-(methylsulfonyl)-1H-
benzo[d]imidazole (6h)
6.1.20. Synthesis of 1H-benzo[d]imidazol-2-yl
methyl[{E}-2-(4-fluoro-3-methyl phenyl)-1-
ethenyl]sulfone (7d)
Yield (45%); m.p. 189Á
1591, 1490, 1449, 1365, 1295, 1170, 1126, 1053, 1013;
1H-NMR (CDCl3), dH 7.7(dd, 2H, Jꢂ
9.2 Hz, Ar), 7.3
/
191 8C; IR (KBr) cmꢀ1: 3022,
79 8C; IR (KBr) cmꢀ1: 3282,
/
Yield (45%); m.p. 177Á
/
(m,7H, Ar), 7.0 (d,1H, Jꢂ
/
15.6 Hz, styryl), 5.2 (s, 2H, Á
/
3026, 2998, 1703, 1615, 1589, 1502, 1442, 1402, 1328,
CH2), 3.6 (s, 3H); Mass (m/z) (DIP): 411(Mꢃ), 333, 269,
211, 133, 104, 94.
1
1299, 1257, 1190, 1130; H-NMR (DMSO-d6ꢃ
dH 2.2 (s, 2H), 6.8 (d, 1H, Jꢂ8.4 Hz), 7.0 (t, 1H, Jꢂ
7.6 (m, 3H); Mass (m/z) (CI):
/
CDCl3),
/
/8
Hz), 7.1Á
/
7.4 (m, 4H), 7.4Á
/
330 (Mꢃ), 260, 132 (100%), 104, 91.
6.1.15. Synthesis of 2-({[(E)-2-(4-chloro-3-
methylphenyl)-1-ethenyl]sulfonyl}methyl)-1-
(methylsulfonyl)-1H-benzo[d]imidazole (6i)
6.1.21. Synthesis of 6-chloro-1H-benzo[d] imidazol-2-
yl)methyl[(E)-2-(4-fluoro-3-methyl phenyl)-1-
ethenyl]sulfone (7e)
Yield (45%); m.p. 204Á
206 8C; IR (KBr) cmꢀ1: 3423,
3058, 3022, 2939, 1620,1527, 1483, 1452, 1363, 1307,
/
1
1246, 1168; H-NMR (CDCl3), dH 7.7 (m, 2H, Ar), 7.2
Yield (37%); m.p. 111Á
/
113 8C; IR (KBr) cmꢀ1: 3421,
2928, 1604, 1501, 1431, 1311, 1256, 1147, 1110; 1H-
(m, 6H), 7.0 (d, 1H, Jꢂ15.2 Hz, styryl), 3.6 (s, 3H), 2.4
/
(s, 3H); Mass (m/z) (DIP): 425 (Mꢃ), 347, 283, 211, 195,
185, 165, 153, 133 (100%), 112.
NMR (CDCl3): 2.2 (s, 3H), 4.8 (s, 2H), 6.7Á7.8 (m, 8H);
/
Mass (m/z) (CI): 365 (Mꢃ), 300, 285, 165 (100%), 136,
109, 97.
6.1.16. Synthesis of 6-chloro-2-({[(E)-2-(4-
fluorophenyl)-1-ethenyl]sulfonyl}methyl)-1-
(methylsulfonyl)-1H-benzo[d]imidazole (6j)
6.1.22. Synthesis of 6-chloro-1H-benzo[d]imidazol-2-
yl)methyl[(E)-2-(4-chloro phenyl)-1-ethenyl sulfone
(7f)
Yield (44%); m.p. 196Á
98 8C; IR (KBr) cmꢀ1: 3443,
3020, 2928, 1601, 1510, 1455, 1367, 1302, 1238, 1167,
/
Yield (33%); m.p. 187Á
2928, 1616, 1591,1528, 1491, 1431, 1309, 1145; 1H-NMR
(CDCl3): 4.8 (s, 2H), 6.8 (d,1H, Jꢂ16 Hz), 7.2Á7.8 (m,
/
185 8C; IR (KBr) cmꢀ1: 3384,
1126; 1H-NMR (CDCl3) dH 7.1 (m, 8H, Ar), 7.0 (d, 1H,
styryl, Jꢂ
/
14.6Hz), 5.2 (s, 2H, Á/CH2), 3.6 (s, 3H,
/
/

822
M.S. Vedula et al. / European Journal of Medicinal Chemistry 38 (2003) 811Á824
/
8H); Mass (m/z) (CI): 366 (Mꢃ), 302, 165 (100%), 140,
138, 102, 90.
was filtered to give 1H-benzo[d]imidazole-2-yl hydro-
sulfide.
6.1.29. Synthesis of 1H-benzo[d]imidazol-2-yl[(E)-2-
(4-chloro phenyl)-1-ethenyl sulfone (12a)
6.1.23. Synthesis of 1H-benzo[d] imidazol-2-yl
methyl[(E)-2-(3,4-dichloro phenyl)-1-ethenyl] sulfone
(7g)
Yield (35%); m.p. 235Á
37 8C; IR (KBr) cmꢀ1: 3387,
3247, 3040, 3000, 2920, 2850, 2581, 2293, 1918, 1747,
/
Yield (45%); m.p. 201Á
/
202 8C; IR (KBr) cmꢀ1:1607,
1605, 1563, 1487, 1405, 1355, 1332, 1226; 1H-NMR
1584, 1528, 1469, 1437, 1366, 1292, 1200, 1128; 1H-
(DMSO-d6), dH 7.9 (t, 4H, Jꢂ8.2 Hz, Ar), 7.7 (m, 2H),
/
NMR (CDCl3), dH 7.0 (m, 9H, Ar), 4.8 (s, 2H, ÁCH2);
/
Mass (m/z) (DIP): 366 (Mꢃ, 10%), 302, 267, 131
(100%), 104.
7.5 (d, 2H, styryl, Jꢂ8.2 Hz), 7.4 (m, 2H); Mass (m/z)
/
(DIP): 319 (Mꢃ, 100%), 254, 219, 159, 139, 119.
6.1.30. Synthesis of 1H-benzo[d]imidazol-2-yl[(E)-2-
(4-fluoro-3-methyl phenyl)-1-ethenyl sulfone (12b)
6.1.24. Synthesis of 1H-benzo[d]imidazol-2-yl
methyl[(E)-2-(4-chloro phenyl)-1-ethenyl] sulfone (7h)
209 8C; IR (KBr) cmꢀ1: 3282,
Yield (50%); m.p. 208Á
210 8C; IR (KBr) cmꢀ1: 3056,
2999, 2360, 1615, 1491, 1453, 1433, 1408, 1306, 1245,
/
Yield (30%); m.p. 208Á
3026, 2998, 1703, 1615, 1589, 1502, 1442, 1402, 1328,
1299, 1257, 1190, 1130, 1003; 1HNMR (DMSO-d6ꢃ
/
1140; 1H-NMR (CDCl3), dH 7.2 (m, 9H, Ar), 6.7 (d, 1H,
/
CDCl3), dH 2.2 (s, 3H), 7Á8 (m, 9H), 13.4 (brs, 1H);
/
styryl, Jꢂ15.4 Hz), 4.7 (s, 2H, CH2); Mass (m/z) (DIP):
/
Mass (m/z) (DIP): 316 (Mꢃ), 251, 133, 118 (100%), 115,
91.
333 (Mꢃ), 268, 253, 131 (base peak).
6.1.25. Synthesis of 1H-benzo[d]imidazol-2-yl
methyl[(E)-2-(4-chloro-3-methyl phenyl)-1-ethenyl
sulfone (7i)
6.1.31. Synthesis of 1H-benzo[d]imidazol-2-yl-[(E)-2-
(4-chloro-3-methyl phenyl)-1-ethenyl sulfone (12c)
Yield (28%); m.p. 135Á
/
137 8C; IR (KBr) cmꢀ1: 2929,
1712, 1615, 1431, 1303, 1238, 1113; H-NMR (CDCl3),
Yield (48%); m.p. 122 8C; IR (KBr) cmꢀ1: 3280, 3015,
3000, 1703, 1615, 1589, 1502, 1442, 1402, 1328, 1299,
1257, 1190, 1130; 1H-NMR (CDCl3), dH 7.0 (m, 8H), 6.8
1
dH 2.4 (s, 3H), 4.9 (s, 2H), 6.9 (d, 1H), 7.2Á7.7 (m, 8H);
/
Mass (m/z) (DIP): 316 (Mꢃ), 281, 166 (100%), 152, 115,
91.
(d, 1H, styryl, Jꢂ15.2 Hz), 4.7 (s, 2H), 2.3 (s, 3H); Mass
/
(m/z) (DIP): 347 (Mꢃ, 100%), 327, 313, 283, 133.
6.1.32. Synthesis of 1H benzo[d]imidazol-2-yl[(E)-2-
(4-bromo phenyl)-1-ethenyl sulfone (12d)
6.1.26. Synthesis of 6-chloro-1H-benzo[d]imidazol-2-
yl)methyl[(E)-2-(4-fluoro phenyl)-1-ethenyl]sulfone
(7j)
Yield (25%); m.p. 228Á
/
230 8C; IR (KBr) cmꢀ1: 3389,
3286, 1609, 1586, 1441, 1401, 1329, 1137, 1071; 1H-
Yield (37%); m.p. 116Á
/
118 8C; IR (KBr) cmꢀ1: 2927,
NMR (CDCl3ꢃ
/
DMSO-d6), dH 7Á8 (m, 10H); Mass (m/
/
1599, 1510, 1449, 1328, 1233, 1158, 1040; 1H-NMR
z) (DIP): 364(Mꢃ), 299, 218, 118(100%), 102, 90.
(CDCl3): 4.8 (s, 2H), 6.7Á
/
6.8 (d, 1H, Jꢂ
/
15.8 Hz), 7.2Á
/
7.8 (m, 8H); Mass (m/z) (CI): 350 (Mꢃ), 286, 165
(100%), 138, 122, 101, 91.
6.1.33. Synthesis of 1H-benzo[d]imidazol-2-yl[(E)-2-
(4-fluoro phenyl)-1-ethenyl sulfone (12e)
Yield (30%); m.p. 217Á
/
218 8C; IR (KBr) cmꢀ1: 3265,
3007, 1616, 1599, 1510, 1443, 1329, 1233, 1194, 1162;
6.1.27. Synthesis of 6-chloro-1H-benzo[d]imidazol-2-
yl)methyl[(E)-2-(4-chloro-3-methyl phenyl)-1-
ethenyl]sulfone (7k)
1H-NMR (DMSO- d6), dH 7Á
8 (m, 10H), 14 (brs, 1H);
/
Mass (m/z) (DIP): 302 (Mꢃ), 237, 118 (100%), 101, 90.
Yield (40%); m.p. 166Á
3062, 2992, 2929, 2282, 1612, 1528, 1477, 1427; 1HNMR
(DMSO-d6) 12.8 (s, 1H), 7.2Á8.0 (m, 8H), 4.9 (s, 2H),
2.4 (s, 3H); Mass (m/z) (CI): 382 (Mꢃ2), 381 (Mꢃ1),
380 (Mꢃ), 316, 167, 152.
/
168 8C; IR(KBr) cmꢀ1: 3658,
6.1.34. Synthesis of (E)-2-(4-bromo phenyl)-1-ethenyl-
(5-methoxy-1H-benzo[d]imidazol-2-yl)sulfone (12f)
/
213 8C; IR (KBr) cmꢀ1: 3261,
3005, 2917, 2848, 1735, 1620, 1585, 1585, 1500, 1459,
/
/
Yield (27%); m.p. 211Á
/
1
1401, 1314; HNMR (DMSO-d6), dH 3.9 (s, 3H), 6.9Á
/
6.1.28. General procedure for the synthesis of 1H-
benzo[d]imidazole-2-yl hydrosulfide (8)
7.9 (m,9H); Mass (m/z)( DIP): 393 (Mꢃ), 149, 99
(100%).
Potassium hydroxide (1 mmol), carbon disulfide (1
mmol) were added to commercially available O-phen-
ylene diamine (1 mmol) in 10 mL of ethanol and the
contents were refluxed for 3 h. Norit was added to the
contents, refluxed for 15 min and filtered. Water (30
mL) was added to the filtrate and the solid precipitated
6.1.35. Synthesis of (E)-2-(4-chloro-3-methyl phenyl)-
1-ethenyl(5-methoxy-1H-benzo[d]imidazol-2-yl)sulfone
(12g)
Yield (25%); m.p. 205-207 8C; IR (KBr) cmꢀ1: 3256,
3000, 1611, 1510, 1477, 1327, 1200, 1160, 1133; 1H-

M.S. Vedula et al. / European Journal of Medicinal Chemistry 38 (2003) 811Á
/824
823
Percentage inhibitionꢂ((Tf ꢀTs)=(Cf ꢀCs))ꢁ100
NMR (DMSO-d6), dH 2.2 (s, 3H), 3.9 (s, 3H), 6.9Á
8H); Mass (m/z) (DIP): 363 (Mꢃ2), 297, 191, 163, 149
(100%).
/
8 (m,
/
where Tf is the relative tumor volume of treated animals
on final day, Ts is the relative tumor volume of treated
animals on starting day, Cf is the relative tumor volume
of control animals on final day and Cs is the relative
tumor volume of control animals on starting day.
6.1.36. Synthesis of 4-[(E)-2-(1H-benzo[d]imidazol-2yl
sulfonyl)-1-ethenyl]-2,6-di(tert-butyl)phenol (12h)
Yield (26%); m.p. 237Á
/
240 8C; IR (KBr) cmꢀ1: 3609,
3427, 3056, 2959, 1599, 1438 1422, 1332, 1273; 1H-NMR
6.2.3. Determination of maximum tolerated dose (MTD)
To determine the MTD mice were grouped based on
their body weights. The test compounds were formu-
lated as suspension using appropriate vehicle and
administered to the animals through oral route. The
animals were observed for clinical signs of toxicity, body
weight change for 14 days. Based on the mortality and
body weight loss the MTD was calculated.
(CDCl3),dH 1.2 (s, 18H), 7Á8 (m, 8H), 13.8 (s, 1H);
/
Mass (m/z) (CI): 413 (Mꢃ, 100%), 397, 357, 333, 311,
119.
6.2. Pharmacology
6.2.1. In vitro cell growth assay
Cells were seeded on a 96-well cell culture plates at a
concentration of 10,000 cells/well in a volume of 100 mL
of RPMI medium with 10% fetal bovine serum and were
incubated at 37 8C in a CO2 incubator. After 24 h cells
were treated with varied concentrations of compound in
100 mL of medium and the control wells received vehicle.
The plates were further incubated at 37 8C in a CO2
incubator for 48 h. The assay was terminated by
addition of 50% cold TCA to a final concentration of
10% to the cells, followed by incubation at 4 8C for 1 h.
At the time of compound addition a separate set of cells
having a 24 h growth were terminated for time zero
growth (T0). The TCA fixed plates were washed thrice
with distilled water, air dried and stained with 0.4% SRB
in 1% acetic acid for 30 min at room temperature. The
plates were washed with 1% acetic acid, dried and the
protein bound dye was dissolved in 100 mL of 10 mM
Tris buffer and read at 515 nm. The percentage growth
of treated cells is calculated compared to control with
reference to time zero optical densities [12].
6.3. Pharmaco kinetic studies
Single dose oral pharmacokinetic studies for 7k and
FRI-20 were conducted in male Swiss albino mice (nꢂ
/
12 animals for each compound, weight range: 20Á26 g)
/
at 100 mg kgꢀ1 given in 0.25% CMC suspension. Blood
samples were collected at predetermined time intervals
over 0Á24 h post-dose. Concentration of analyte was
/
determined by suitable HPLC bioanalytical methods.
Plasma concentration versus time profiles was generated
over the 0Á24 h time period and pharmacokinetic
/
parameters were calculated based on non-compartmen-
tal analysis [14].
6.4. Molecular modeling
Molecular modeling and three-dimensional structure
building were performed using SYBYL 6.8 [15] installed
on a Silicon Graphics workstation with IRIX 6.5
6.2.2. In vivo xenograft studies
CD-1 Nude mice were used for HT-29 (colon) human
tumor xenografts. HT-29 tumors were initiated by
implantation of tumor fragments (ca. 60 mm3) from
established tumors. Tumor fragments were implanted at
s.c. into the axillary region of the animal. When the
tumors reached a size of ca. 100 mm3 experimental
compounds were administered p.o. once daily for 20
days at 100, 200 and 400 mg kgꢀ1 doses. The s.c. tumors
were measured with calipers, and mice were weighed
every alternate day and the volumes were calculated
using the equation Vꢂ
/
(D ꢁ
d2)/2 where V (mg) is
/
tumor volume, D is longest diameter in mm, and d is
shortest diameter in mm [13]. Tumor volumes were
converted into relative tumor volumes using the for-
mula, Relative tumor volumeꢂAverage tumor volume
/
on each reading day/Average tumor volume on starting
day. From the relative tumor volumes the percentage
tumor growth inhibition of compound treated animals
compared to control was calculated using the formula,
Fig. 8. Aligned molecules.

824
M.S. Vedula et al. / European Journal of Medicinal Chemistry 38 (2003) 811Á824
/
operating system. Energy minimizations were performed
using Tripos force field with distance dependent di-
electric and the Powell conjugate gradient method with a
convergence criterion of 0.01 kcal molꢀ1. Partial atomic
charges were calculated using Gasteiger Huckel method.
The CoMSIA method was employed for deriving 3D
QSAR models for these compounds keeping activity
pGI50 as a dependent variable. The conformations of the
molecules used in the study were generated by systemic
search method. Atom based alignment (RMS fit) was
employed in this study. Compound 12h was taken as a
template and all the molecules were aligned on it using
atoms of benzimidazole ring. The aligned molecules are
shown in Fig. 8.
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Acknowledgements
[14] M. Gibaldi, D. Perrier, J. Swarbrick, Pharmacokinetics, vol. 4,
Marcel Dekker, New York, 1982, p. 149.
[15] SYBYL 6.8, Tripos Associates: St. Louis, MO, USA.
Spectral data recorded by the analytical group is
gratefully acknowledged.