Synthesis and in vitro antitumor evaluation of some indeno[1,2-c]pyrazol(in)es substituted with sulfonamide, sulfonylurea(-thiourea) pharmacophores, and some derived thiazole ring systems

Article abstract of DOI:10.1016/j.bmc.2006.06.020

The synthesis of a series of 3-(4-chlorophenyl)-[1,2-c]pyrazol(in)es substituted with benzenesulfonamide, N¹,N³-disubstituted sulfonylurea, sulfonylthiourea pharmacophores, and some derived thiazolidinone and thiazoline ring systems

Full text of DOI:10.1016/j.bmc.2006.06.020

Bioorganic & Medicinal Chemistry 14 (2006) 6475–6485
Synthesis and in vitro antitumor evaluation of some indeno[1,2-c]-
pyrazol(in)es substituted with sulfonamide, sulfonylurea(-thiourea)
pharmacophores, and some derived thiazole ring systems
Sherif A. F. Rostom*
Department of Medicinal Chemistry, Faculty of Medicine, King Abdulaziz University, PO Box 80205, Jeddah 21589, Saudi Arabia
Received 28 March 2006; revised 1 June 2006; accepted 8 June 2006
Available online 27 June 2006
Abstract—The synthesis of a series of 3-(4-chlorophenyl)-[1,2-c]pyrazol(in)es substituted with benzenesulfonamide, N1,N3-disubsti-
tuted sulfonylurea, sulfonylthiourea pharmacophores, and some derived thiazolidinone and thiazoline ring systems is described. All
the newly synthesized target compounds were subjected to the NCI-in vitro disease-oriented antitumor screening to be evaluated for
their antitumor activity. Eight compounds namely; 2–4, 7, 8, 10, 13, and 16; showed promising broad spectrum antitumor activity
against most of the tested subpanel tumor cell lines (GI50 < 100 lM). Compound 3, 4-(3-(4-chlorophenyl)-4H-indeno[1,2-c]pyrazol-
2-yl)-benzenesulfonamide; although it did not show the highest growth inhibitory value (GI50 (MG-MID) 13.2 lM), it proved to be
the most active analog in this study with the highest cytostatic and cytotoxic potentials (TGI and LC50 (MG-MID) concentrations
of 33.1 and 66.1 lM, respectively). In general, the oxidized pyrazoles displayed better antitumor activity than their parent pyrazoline
analogs, whereas the benzenesulfonamides and the N1, N3-disubstituted sulfonylureas showed significant better antitumor spectrum
than the sulfonylthioureido and the derived thiazole analogs.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
cytotoxicity and, as a consequence, the antitumor prop-
erties of the diarylsulfonylurea is due to the uncoupling
of mitochondria4,5 but other mechanisms, such as inhi-
bition of the mitochondrial isozyme V of carbonic anhy-
drase (CA V), have also been hypothesized, since
hydrolysis of the cytotoxic agent, leading to the forma-
tion of unsubstituted sulfonamides as the principal
products, has been reported both in vivo and in vitro.10
It is well known that aromatic/heterocyclic sulfonamides
(formed after such a hydrolytic process) act as very
potent inhibitors of CAs,11,12 and these enzymes are
Cancer is one of the most formidable afflictions in the
world. Despite immense advances in the field of basic
and clinical research, which have resulted in higher cure
rates for a number of malignancies, cancer remains the
second leading cause of death after heart disorders in
developing as well as advanced countries.1,2 Although
major advances have been made in the chemotherapeu-
tic management of some patients, the continued com-
mitment to the arduous task of discovering new
anticancer agents remains critically important. Among
the wide range of compounds tested as potential anti-
cancer agents, derivatives comprising the sulfonamide,
N1,N3-diarylsulfonylurea and -thiourea functionalities
have attracted great attention,3–8 especially after the dis-
covery of sulofenur (LY 186641) A and its structure ana-
logs8 (LY 295501) B, C, and D (Fig. 1). Sulofenur is an
antineoplastic sulfonylurea that has been clinically eval-
uated in lung, breast, colon, ovarian, pancreatic, and
gastric cancer.9 It is generally assumed that the strong
involved in
a
multitude of crucial physiologic
processes.13 However, clinical trials of sulofenur have
yielded unsatisfactory results because of its high protein
binding and dosing being limited by the appearance of
anemia due to methemoglobinemia, a side effect likely
associated with its aniline-related metabolites.14 In con-
trast LY 295501 (B) known now as ILX-295501; is prin-
cipally metabolized by hydroxylation with negligible
formation of aniline metabolites at relevant doses in
experimental animals and demonstrated impressive
activity against a broad spectrum of human tumor
xenografts.15
Keywords: Antitumor activity; Sulofenur; Sulfonamides; Diarylsulfo-
nylureas; Pyrazoles; Thiazoles.
On the other hand, over the past few years, much inter-
est has been given to the chemotherapeutic activity of
*
Tel.: +966 507654566; e-mail: sherifrostom@yahoo.com
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.020

6476
S. A. F. Rostom / Bioorg. Med. Chem. 14 (2006) 6475–6485
H
N
X
R
H
H
Cl
N
N
X
H
N
SO2
N
N
z
Y
SO2
A
B
C
Sulofenur: X = O, Y = H, Z = CH2
LY295501: X = O, Y = Cl, Z = O
X = S, Y = H, Z = CH2
E
Cl
R
Cl
Cl
Y
N
N
N
S
SO2
N
N
N
H
N
OH
SO2
H3C
F
Cl
D
Figure 1. Structures of sulofenur, its analogs A–D, and the novel series of compounds E and F.
pyrazoles as antimicrobial,16–18 antiviral,19–21 and po-
tential anticancer agents.22–24 It has been also reported
that some pyrazoles have remarkable antiproliferative
effect on certain cell lines in addition to their pro-
nounced inhibitory effect on the cyclin-dependent kinase
(CDK) enzyme which plays a key role during cell divi-
sion.25 In addition, some fused pyrazoles proved to have
potent glycogen synthase-3 inhibitory activity.26,27
turally related to reported anticancer agent D (Fig. 1).
The objective of forming these hybrids is an attempt
to reach an active antitumor agent with potentiated
activity and selectivity toward cancerous cells.
2. Results and discussion
2.1. Chemistry
In view of these facts, and in continuation of an on-
going program aiming at finding new structure leads
with potential chemotherapeutic activities,28–33 it was
rationalized to synthesize and investigate the antitumor
activity of some new compounds comprising the sulfon-
amido and the derived N1,N3-disubstituted sulfonylurea
and thiourea pharmacophores having the general struc-
ture E that are structurally related to sulofenur A and its
congeners B and C (Fig. 1). The thrust of efforts in the
area of derivatization of such type of compounds con-
centrated mainly on the aryl moiety of the sulfonamide
portion of the diarylsulfonylureas. In the present study,
such N3-aryl moiety was substituted basically with 3-(4-
chlorophenyl)-indeno[1,2-c]pyrazol(in)e moieties be-
cause of the reported potential anticancer activity of
pyrazoles hoping to induce some biological synergism.
In addition, isosteric replacement of the sulfonylurea
with a sulfonylthiourea functionality was considered as
an interesting structure variation in order to study the
influence of such modification on the anticipated antitu-
mor activity. The substitution pattern of the N1 part of
the sulfonylurea, thiourea pharmacophores was selected
so as to confer different electronic environment that
would affect the lipophilicity, and hence the activity of
the target molecules. Owing to the well-documented che-
motherapeutic activities associated with thiazole deriva-
tives,34–36 it was considered of interest to investigate the
effect of increasing compounds’ rigidity by cyclizing the
sulfonylthioureido derivatives to the sulfonylimino thia-
zolidinone and thiazoline ring systems F that are struc-
The synthetic pathways adopted for the preparation
of the intermediate and target products are illustrated
in Scheme 1. The starting chalcone 1 was prepared by
condensing equimolar amounts of 1-indanone with 4-
chlorobenzaldehyde in an aqueous ethanolic solution
of sodium hydroxide according to a previously described
procedure.37 Cyclocondensation of 1 with 4-hydrazino-
benzenesulfonamide hydrochloride in boiling ethanol
afforded the indeno[1,2-c]-pyrazoline 2 in a good yield.
Its 1H NMR spectrum exhibited a doublet at d
2.25 ppm due to two aliphatic protons at C-4, a charac-
teristic doublet at d 5.75 ppm attributed to the C-3 pro-
ton, and multiplet at d 2.64 ppm due to the C-3a proton.
Mild oxidation of the substituted indeno[1,2-c]pyrazo-
line 2 with bromine water at room temperature fur-
nished the corresponding indeno[1,2-c]pyrazole 3. The
1H NMR of 3 lacked the characteristic doublet and mul-
tiplet of C-3 and C-3a protons of the parent pyrazoline
2. The substituted sulfonylureas 4–6 were successfully
obtained by condensing the key intermediate sulfon-
amide 2 with the appropriate isocyanate in the presence
of anhydrous potassium carbonate as a mild basic
catalyst. At this stage, the targeted substituted
indeno[1,2-c]pyrazole benzenesulfonylureas 7–9 could
be synthesized via either of the following procedures.
The first method contributes to the condensation of
the pyrazolylbenzenesulfonamide 3 with the appropriate
isocyanate, while the second involves mild oxidation of
the substituted indeno[1,2-c]pyrazoline benzenesulfo-

S. A. F. Rostom / Bioorg. Med. Chem. 14 (2006) 6475–6485
6477
H
N
R
O
N
H
NH2
SO2
SO2
N
N
CHO
Cl
N
N
O
iv
+
Cl
Cl
7-9
3
H
N
R
O
i
N
iii
NH2
SO2
H
NHNH
2
SO2
O
.
HCl
N
N
SO NH
ii
2
2
N
N
Cl
Cl
N
1
2
Cl
4-6
O
R1
H
N
R1
S
R1
N
N
v
N
H
S
N
S
SO2
SO2
SO2
N
N
N
N
N
vii
N
vi
Cl
Cl
Cl
13-15
10-12
16-18
CH3
R1
N
N
S
R = 4,7: Cyclohexyl, 5,8: Phenyl, 6,9: 1-Naphthyl
R1 = 10, 13, 16: Phenyl
SO2
viii
N
11, 14, 17: Benzyl
N
12, 15, 18: Benzoyl
Cl
19-21
Scheme 1. Reagents and conditions: (i) bromine water, r.t., 3 h; (ii) RNCO, K2CO3, reflux, 18 h; (iii) bromine water, r.t., 6 h; (iv) RNCO, K2CO3,
reflux, 18 h; (v) RNCS, K2CO3, reflux, 10 h; (vi): BrCH2COOC2H5, Na acetate, reflux, 2 h; (vii) C6H5COCH2Br, Na acetate, reflux, 3 h; (viii)
CH3COCH2Cl.
nylureas 4–6 with bromine water at room temperature
to give the same products. Here, it should be mentioned
that the yield of first procedure was slightly better than
that of the second one. The IR spectra of compounds 4–
9 were characterized by a urea carbonyl band at 1640–
1665 cmÀ1. On the other hand, condensing the key inter-
mediate 2 with different isothiocyanates in the presence
of anhydrous potassium carbonate gave rise to the
substituted benzenesulfonylthioureas 10–12.Their IR
spectra revealed a characteristic C@S band at 1115–
1155 cmÀ1. The latter compounds in their turn were fur-
ther utilized for the preparation of some thiazole ring
systems. Thus, cyclization of the sulfonylthioureido
derivatives 10–12 with ethyl bromoacetate in the pres-
ence of anhydrous sodium acetate afforded the corre-
sponding thiazolidin-4-ones 13–15. The IR of these
compounds was characterized by a carbonyl band at
1720–1735 cmÀ1, whereas, their 1H NMR spectra re-
vealed a new singlet at d 4.15–4.28 ppm attributed to
the oxothiazolidine C-5 two protons. Analogously,
reacting the same derivatives 10–12 with phenacyl bro-
mide under similar reaction conditions resulted in the
formation of the desired 1,3-thiazolines 16–18. Their
1H NMR spectra showed a new singlet at d 6.11–
6.19 ppm due to the thiazoline C-5 proton. Here it
should be pointed out that, reacting 10–12 with chloro-
acetone using various reaction conditions, basic cata-
lysts and different solvents failed to produce the
desired 1,3-thiazolines 19–21.
2.2. In vitro antitumor activity
2.2.1. Primary in vitro one dose 3-cell line assay. All the
synthesized compounds were selected by the National
Cancer Institute (NCI) in vitro disease-oriented human
cells screening panel assay to be evaluated for their in vi-
tro antitumor activity. Primary in vitro one dose anti-
cancer assay was performed using the 3-cell line panel
consisting of NCI-H460 (lung), MCF7 (breast), and
SF-268 (CNS) in accordance with the protocol of the
Drug Evaluation Branch, NCI, Bethesda.38–40 The com-
pounds were added at a single concentration (10À4 M)
and the culture was incubated for 48 h. End point deter-
minations were made with a protein binding dye, sulfo-
rhodamine B (SRB). Results for each compound were
reported as the percent of growth of the treated cells
when compared to the untreated control cells. All the
compounds which reduced the growth of any one of
the cell lines to 32% or less were passed on for evalua-
tion in the full panel of 60 human tumor cell lines.38–40
The results revealed that eight compounds namely 2,
3, 4, 7, 8, 10, 13, and 16; passed this primary anticancer

6478
S. A. F. Rostom / Bioorg. Med. Chem. 14 (2006) 6475–6485
assay and consequently were carried over to be tested
against a panel of 60 different tumor cell lines.
Concerning the broad spectrum antitumor activity, the
results revealed that all of the eight active compounds
2, 3, 4, 7, 8, 10, 13, and 16, showed effective growth inhi-
bition GI50 (MG-MID) values of 9.12, 13.2, 9.12, 10.2,
19.5, 25.7, 30.2, and 41.7 lM, respectively, beside cyto-
static activity TGI (MG-MID) values of 34.7, 33.1,
33.9, 34.7, 63.1, 61.6, 77.6, and 91.2 lM, respectively
(Tables 2 and 3). In addition, except for compounds
13 and 16, the rest of the active compounds 2, 3, 4, 7,
8, and 10, exhibited a variable degree of cytotoxic effica-
cy with LC50 (MG-MID) values of 83.2, 66.1, 77.6, 81.3,
97.7, and 95.5 lM, respectively (Table 3).
2.2.2. In vitro full panel 60-cell line assay. About 60 cell
lines of nine tumor subpanels, including leukemia, non-
small cell lung, colon, CNS, melanoma, ovarian, renal,
prostate and breast cancer cell lines, were incubated with
five concentrations (0.01–100 lM) for each compound
and were used to create log concentrationÀ% growth
inhibition curves. Three response parameters (GI50,
TGI, and LC50) were calculated for each cell line. The
GI50 value (growth inhibitory activity) corresponds to
the concentration of the compounds causing 50% de-
crease in net cell growth, the TGI value (cytostatic activ-
ity) is the concentration of the compounds resulting in
total growth inhibition, and the LC50 value (cytotoxic
activity) is the concentration of the compounds causing
net 50% loss of initial cells at the end of the incubation
period (48 h). Subpanel and full panel mean-graph mid-
point values (MG-MID) for certain agents are the average
of individual real and default GI50, TGI, or LC50 values of
all cell lines in the subpanel or the full panel, respectively.
The NCI antitumor drug discovery program was designed
to distinguish between broad spectrum antitumor com-
pounds and tumor or subpanel-selective agents.
Further interpretation of the obtained results revealed
that, compounds 2 (GI50, TGI, and LC50 (MG-MID)
values 9.12, 34.7, and 83.2 lM, respectively) and 4
(GI50, TGI, and LC50 (MG-MID) values 9.12, 33.9,
and 77.6 lM, respectively) are nearly equipotent with
almost the same range of activity against most of the
tested subpanel tumor cell lines (Tables 2 and 3).
Regarding compound 3, although it did not show the
highest growth inhibitory value (GI50 (MG-MID)
13.2 lM; Table 2), it proved to be the most active
member in this study with the highest cytostatic and
cytotoxic potentials with TGI and LC50 (MG-MID)
concentrations of 33.1 and 66.1 lM, respectively (Table
3). It revealed an obvious activity against most of the
tested subpanel tumor cell lines and relative effectiveness
on the leukemia subpanel at both the GI50 and TGI lev-
els (5.93 and 19.1 lM, respectively). Moreover, com-
pounds 2, 3, and 4 displayed almost the same level of
growth inhibition against the leukemia subpanel (GI50
(MG-MID) values 5.92, 5.93, and 5.73 lM, respective-
ly). This pattern was not maintained at the TGI level
where compound 2 showed weak cytostatic activity
(TGI (MG-MID) 73.7 lM) relative to 3 and 4. When
compared with the NCI-DTP data of the standard
diarylsulfonylurea analog sulofenur (A); the growth
inhibitory activity of compound 7 was almost the half
against the leukemia subpanel (GI50 (MG-MID) values
1.27 vs 2.50 lM, respectively); whereas the cytostatic
activity of the same compound was about 25% of that
of sulofenur (TGI (MG-MID) 7.51 vs 1.98, respectively)
(Tables 2 and 3). In addition, particular sensitivity has
been shown by compound 4 toward the prostate cancer
subpanel at the GI50, TGI, and LC50 levels (4.77, 20.0
and 50.7 lM, respectively). On the other hand, com-
pound 16 was found to be the least effective antitumor
agent in the present investigation with GI50 and TGI
(MG-MID) values of 41.7 and 91.2 lM, respectively;
and without any cytotoxic effect (Tables 2 and 3).
In the present study, the active eight compounds exhib-
ited remarkable antitumor activities against most of the
tested subpanel tumor cell lines (GI50, TGI, and LC50
values <100 lM). These compounds showed a distinc-
tive pattern of sensitivity against some individual cell
lines (Table 1), as well as a broad spectrum of antitumor
activity (Tables 2 and 3).
With regard to the sensitivity against some individual
cell lines (Table 1), compound 2 showed high activity
against leukemia SR cell line (GI50 2.19 lM). The ana-
log 3 also exhibited noticeable antitumor activity against
non-small cell lung NCS-H23 cell line (GI50 3.27 lM).
Furthermore, compound 4 showed the ever highest
growth inhibitory activity against the renal A498 cancer
cell line with GI50 value of <0.01 lM. It also revealed an
obvious sensitivity profile toward leukemia SR cell line
with GI50 value of 0.41 lM, in addition to a good activ-
ity against renal UO-31 and breast MCF7 cell lines with
GI50 values of 2.00 and 2.52 lM, respectively.
Except for the SR cell line, compound 7 proved to be
sensitive toward most of the tested leukemia subpanel
tumor cell lines with GI50 values range of 1.13–
4.28 lM. It also showed particular effectiveness against
the melanoma LOX IMVI cell line (GI50 1.96 lM). Fur-
thermore, compound 13 exhibited a significant activity
against the colon COLO 205 cell line with GI50 value
of 1.73 lM. On the other hand, the colon SW-620 cell
line proved to be sensitive toward compounds 2, 3 and
7 with GI50 values of 4.71, 1.88, and 2.46 lM, respec-
tively. Melanoma SK-MEL-5, ovarian IGROV1 and re-
nal A498 cancer cell lines showed high sensitivity toward
compounds 2–4, with GI50 values range of 1.36–
5.59 lM. Moreover, the highest sensitivity against the
prostate cancer cell lines was displayed by compounds
4 and 7, with GI50 values range of 4.05–5.50 and 6.28–
6.39 lM, respectively.
The ratio obtained by dividing the full panel MG-MID
(lM) of the compounds by their individual subpanel
MG-MID (lM) is considered as a measure of com-
pound selectivity. Ratios between 3 and 6 refer to mod-
erate selectivity, ratios greater than 6 indicate high
selectivity toward the corresponding cell line, while com-
pounds not meeting either of these criteria are rated
non-selective.41 In this context, the active compounds
in the present study were found to be non-selective with
broad spectrum antitumor activity against the nine
tumor subpanels tested with selectivity ratios ranging

S. A. F. Rostom / Bioorg. Med. Chem. 14 (2006) 6475–6485
6479
Table 1. Growth inhibitory concentration (GI50, lM) of compounds 2–4, 7, 8, 10, 13, and 16a
Cell lines
2
3
4
7
8
10
13
16
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
6.48
2.96
NT
4.98
5.51
10.4
5.82
3.85
15.8
6.71
1.13
4.28
2.15
2.77
2.11
3.21
13.4
4.66
7.05
16.5
2.86
19.7
21.0
16.4
15.9
19.6
14.7
13.7
23.1
18.7
17.2
26.4
11.4
18.6
29.6
20.0
18.5
21.5
17.3
NTb
10.6
7.58
2.73
2.19
MOLT-4
RPMI-8226
SR
4.04
3.57
0.41
NT
Non-small cell lung cancer
A549/ATCC
EKVX
10.8
14.1
21.1
10.3
10.9
11.4
5.80
19.7
8.44
37.8
17.9
5.38
15.0
16.6
14.2
35.8
24.5
20.9
27.4
41.3
18.1
41.4
NT
24.2
43.2
56.8
27.0
45.1
NT
31.4
61.1
93.9
38.6
94.6
21.0
25.2
NAc
22.7
17.1
30.4
11.6
16.8
15.8
72.0
17.1
15.5
NT
HOP-62
HOP-92
10.8
19.8
12.2
6.45
6.15
NT
NCI-H226
NCI-H23
3.27
NCI-H322
NCI-H460
NCI-H522
27.9
32.1
4.26
14.2
19.8
18.2
25.3
22.3
19.0
16.3
22.2
18.1
19.3
19.7
36.7
4.45
9.43
5.63
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
4.99
10.3
6.04
18.2
15.7
5.55
4.71
22.8
18.3
11.7
36.2
NT
23.5
NT
21.6
NT
1.73
54.9
NT
3.56
5.15
NT
4.06
4.18
7.79
34.8
10.6
2.46
22.5
18.5
29.8
16.0
23.0
21.6
22.4
41.6
19.4
29.7
38.7
34.8
NT
47.5
47.7
44.0
19.9
35.0
19.7
15.4
19.9
14.5
32.5
7.90
10.3
KM12
SW-620
22.0
37.4
1.88
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
22.1
16.3
9.07
17.3
2.97
20.2
22.4
29.8
17.5
31.7
14.6
19.5
6.78
20.1
11.3
11.2
43.7
27.6
11.7
14.8
18.2
18.8
61.5
48.6
22.3
20.7
35.5
24.3
21.8
39.7
19.7
22.7
35.8
60.2
23.3
NT
NA
40.7
62.6
78.8
NA
26.1
15.9
12.9
21.6
13.2
10.1
27.8
Melanoma
LOX IMVI
M14
18.0
10.1
8.23
10.0
2.30
11.4
5.97
19.0
13.1
13.6
18.3
10.3
10.8
24.4
29.7
1.96
15.3
34.5
27.4
33.7
22.0
37.1
12.2
19.5
36.4
23.4
29.4
20.4
29.5
18.3
23.4
59.4
NA
NT
21.5
NA
36.0
67.6
25.2
83.5
32.7
13.7
19.4
21.9
12.1
18.4
10.9
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
1.36
3.69
32.2
92.9
30.1
20.6
13.7
19.7
14.9
Ovarian cancer
IGROV1
3.48
17.3
9.76
15.9
11.8
12.4
2.58
5.98
18.6
13.7
28.8
NT
28.5
18.3
45.2
NT
20.2
46.3
42.2
NT
37.8
48.5
NT
25.7
44.3
63.8
38.1
36.1
76.2
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
17.0
15.0
24.2
21.6
77.1
18.4
38.4
25.5
19.6
24.5
NT
11.9
31.6
24.7
53.9
26.8
40.7
34.6
31.4
Renal cancer
786-0
A498
6.28
2.55
17.5
18.2
5.07
6.74
8.32
7.88
14.6
13.3
5.07
18.5
7.93
4.52
20.0
7.54
13.3
27.5
21.8
12.9
32.4
NT
46.6
25.6
27.7
35.8
29.4
37.3
29.7
29.1
32.7
79.8
40.1
46.4
42.0
50.8
29.6
45.4
4.70
0.01
ACHN
CAKI-1
RXF 393
SN12C
TK-10
19.8
17.5
17.0
13.4
12.8
23.2
16.8
25.8
26.1
43.1
24.5
30.2
19.0
4.37
22.2
23.2
18.5
24.5
5.49
19.5
9.57
4.50
2.00
UO-31
3.64
Prostate cancer
PC-3
DU-145
7.17
14.2
14.0
15.1
4.05
5.50
6.28
6.39
16.7
19.4
13.8
24.9
22.6
33.9
19.1
54.3
Breast cancer
MCF7
NCI/ADR-RES
25.0
10.3
22.9
19.6
2.52
15.3
NT
11.2
NT
23.5
NT
18.7
NT
NA
51.5
20.6
(continued on next page)

6480
S. A. F. Rostom / Bioorg. Med. Chem. 14 (2006) 6475–6485
Table 1 (continued)
Cell lines
2
3
4
7
8
10
13
16
MDA-MB-231/ATCC
HS 578T
2.89
4.61
14.8
12.1
17.8
NT
12.8
6.43
5.03
8.27
3.52
4.98
14.7
19.6
16.8
21.0
49.9
87.1
30.0
44.8
33.2
36.3
86.0
29.4
30.6
35.6
41.7
70.1
33.0
24.2
41.4
NA
4.61
18.5
7.01
22.6
MDA-MB-435
BT-549
T-47D
16.0
NT
32.9
91.0
a Data obtained from NCI’s in vitro disease-oriented human tumor cell screen.
b NT, Not Tested.
c NA, not active; GI50 value >100 lM.
Table 2. Median growth inhibitory concentrations (GI50, lM) of in vitro subpanel tumor cell linesa
Compound
Subpanel tumor cell linesb
MG-MIDc
I
II
III
9.35
IV
V
VI
VII
9.07
VIII
IX
2
5.92
5.93
5.73
2.50
7.94
12.5
20.3
11.0
16.4
26.4
25.7
34.0
54.3
14.6
22.6
13.4
20.9
30.7
27.0
33.9
68.0
9.43
11.8
26.2
22.1
20.9
34.1
35.2
38.1
47.4
10.7
14.5
15.3
29.7
12.7
9.12
13.2
9.12
10.2
19.5
25.7
30.2
41.7
1.19
3
14.6
12.2
16.0
22.2
26.0
26.9
41.5
16.8
16.2
14.0
26.0
25.3
56.3
52.3
13.5
10.7
10.1
19.4
28.7
32.6
45.8
4
4.77
6.33
18.0
7
7.25
8
22.2
42.4
40.3
60.0
NT
10
13
16
Sd
17.9
18.4
20.9
19.3
28.2
36.7
NTe
1.27
1.05
1.21
1.21
1.30
1.16
1.16
a Data obtained from NCI’s in vitro disease-oriented human tumor cell screen.
b I, Leukemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer; VII, renal cancer; VIII, prostate
cancer; IX, breast cancer.
c GI50 (lM) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines toward the test agent.
d Sulofenur (NSC 656667): NCI cancer screen; August 2004.
e NT, not tested.
Table 3. Median total growth inhibitory concentrations (TGI, lM) of in vitro subpanel tumor cell linesa
Compound
Subpanel tumor cell linesb
MG-MIDc
I
II
III
IV
V
VI
VII
VIII
IX
2
73.3
19.1
21.1
39.7
52.2
33.3
48.0
82.5
62.7
89.3
92.6
63.3
45.0
36.6
76.9
74.2
64.2
74.9
90.6
44.9
55.3
52.6
55.3
79.2
63.8
89.7
95.9
31.1
29.0
47.2
48.6
76.9
59.1
87.7
88.3
37.4
46.2
70.0
62.8
88.2
84.1
98.8
97.5
24.0
28.3
35.0
40.2
74.3
72.4
87.6
NA
29.6
30.5
20.0
22.3
47.4
39.7
79.3
NA
43.8
39.1
48.8
42.2
65.4
83.5
87.3
NA
NT
34.7 (83.2)d
33.1 (66.1)
33.9 (77.6)
34.7 (81.3)
63.1 (97.7)
61.6 (95.5)
77.6 (NA)e
91.2 (NA)
1.63 (1.99)
3
4
7
7.51
8
27.7
48.1
50.8
71.6
10
13
16
Sf
1.98
1.50
1.60
1.66
1.69
1.55
1.59
NTg
a Data obtained from NCI’s in vitro disease-oriented human tumor cell screen.
b For subpanel tumor cell lines, I, Leukemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer; VII,
renal cancer; VIII, prostate cancer; IX, breast cancer.
c TGI (lM) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines toward the test agent.
d LC50 (lM) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines toward the test agent.
e NA, not active, (TGI or LC50 (MG-MID) values >100 lM).
f Sulofenur (NSC 656667): NCI cancer screen; August 2004.
g NT, not tested.
between 0.54–2.30 and 0.60–2.00 at the GI50 and TGI
levels, respectively, except for compound 7 which
showed moderate selectivity toward the leukemia sub-
panel at both the GI50 and TGI levels (selectivity ratios
4.06 and 4.62, respectively). However, compound 8 re-
vealed mild selectivity toward the leukemia subpanel
with selectivity ratio near 3 (2.50 and 2.44, for the
GI50 and TGI, respectively).
A close examination of the structures of the active
compounds revealed that, the key intermediate
indeno[1,2-c]pyrazoline sulfonamide
2 exhibited a
broad spectrum antitumor activity as indicated from
its GI50, TGI, and LC50 (MG-MID) values (9.12,
34.7, and 83.2 lM, respectively). Oxidation of
resulted in the formation of the corresponding pyra-
2
zole sulfonamide 3, which proved to be the most

S. A. F. Rostom / Bioorg. Med. Chem. 14 (2006) 6475–6485
6481
active member in this series with the highst cytostatic
3. Conclusion
and cytotxic potentials (TGI and LC50 (MG-MID)
values of 33.1 and 66.1, respectively; Table 3), with
special effectiveness on the leukemia subpanel at the
GI50, TGI, and LC50 (MG-MID) levels (5.93, 19.1,
and 59.2 lM, respectively). These findings were
substantiated by the idea that the sulfonamido func-
tionality plays certain role in the antitumor activi-
ty.8,42,43 Condensing the parent compound 2 with
different isocyanates furnished the disubstituted sulf-
onylureido derivatives 4–6, of which only one analog
(4; R = cyclohexyl) exhibited remarkable antitumor
activity, whereas the other congeners 5 and 6 proved
to be totally inactive. Compound 4 was nearly equi-
potent with 2 at the GI50 and TGI (MG-MID) levels
(9.12 vs 9.12 and 33.9 vs 34.7 lM, respectively;
Tables 2 and 3), meanwhile, the cytotoxic activity
of the same compound was slightly improved (LC50
(MG-MID) 77.6 vs 83.2 lM; Table 3). The synthesis
of the oxidized pyrazole derivatives 7–9 gave rise to
two relatively active substituted sulfonylureidopyraz-
ole derivatives 7 and 8. The nature of substituent
in the sulfonylureido functionality seems to modulate
the antitumor activity of this type of compounds as
evidenced by their GI50, TGI, and LC50 (Tables 2
and 3). The cyclohexyl moiety in compound 7 proved
to be the most favorable substituent among this ser-
ies with GI50, TGI, and LC50 (MG-MID) values of
10.2, 34.7, and 81.3 lM, respectively. Shifting the
In conclusion, the objective of the present study was to
synthesize and investigate the antitumor activity of new
compounds incorporating the sulfonamido, N1,N3-di-
substituted sulfonylurea and thiourea pharmacophores
structurally related to a well-documented sulfonylurea
anticancer agent sulofenur A and its structure congeners
B–D (Fig. 1). This aim has been verified by the synthesis
of hybrid compounds comprising the above-mentioned
pharmacophores substituted essentially with a 3-(4-
chlorophenyl)-[1,2-c]pyrazol(in)e counterpart at the N3
aryl moiety having the general structures E and F
(Fig. 1), for synergistic purpose. The results revealed
that eight compounds namely; 2–4, 7, 8, 10, 13, and
16; showed promising broad spectrum antitumor activi-
ty. In general, the oxidized pyrazole derivatives ap-
peared to exhibit better antitumor activity than their
parent pyrazoline analogs, whereas the benzenesulfona-
mides and N1, N3-disubstituted sulfonylureas showed
significant better antitumor spectrum than that of the
sulfonylthioureido and derived thiazole analogs. The ac-
tive compounds displayed remarkable growth inhibitory
and cytostatic potencies nearly with the same order of
activity (Tables 2 and 3). In addition, except for com-
pounds 13 and 16, the other six active compounds
showed a wide range of cytotoxic activity (Table 3).
The sulfonylureido derivative 7 displayed almost half
the growth inhibitory and 25% of the cytostatic activities
of the diarylsulfonylurea analog sulofenur (A) against
the leukemia subpanel (Tables 2 and 3). Compound 3,
although did not show the highest growth inhibitory val-
ue (GI50 (MG-MID) 13.2 lM; Table 2), it could be con-
sidered the most active derivative identified in this study
with the highest cytostatic and cytotoxic potentials (TGI
and LC50 (MG-MID) concentrations of 33.1 and
66.1 lM, respectively; Table 3).
substitution toward the aromatic side as in
8
(R = phenyl), resulted in a marked reduction in the
antitumor activity to the half at the GI50 and TGI
levels (19.5 and 63.1 lM, respectively), whereas its
cytotoxic effect was obviously reduced (LC50 (MG-
MID) 97.7 lM). Increasing the size and aromaticity
of the substituent as in 9 (R = 1-naphthyl) led to a
complete abolishment of the antitumor activity. When
7 was compared with its parent pyrazoline 4, both
displayed almost the same antitumor profile except
for the activity against leukemia and breast cancer
subpanels which showed about 2-fold improvement
in activity at the GI50 level (2.50 vs 5.73 and 7.25
vs 12.7 lM, respectively). On the other hand, conver-
sion of the sulfonamide 2 to the substituted sulfonyl-
thioureido derivatives 10–12 gave only one active
compound (10; R = phenyl). Such derivatization led
to a significant reduction (2- to 3-fold) in the overall
antitumor activity of 10 when compared with the
parent sulfonamide 2 and the sulfonylureido isostere
7 (Tables 2 and 3). Incorporation of the thioureido
moiety partly in a rigid structure resulted in two ac-
tive compounds, namely, the 2-oxothiazolidine (13;
R = phenyl) and thiazoline (16; R = phenyl) deriva-
tives, with a significant reduction in the growth inhib-
itory, cytostatic potencies and a complete abolishment
of the cytotoxic activity (Tables 2 and 3). However,
the oxothiazolidine derivative 13 showed a relatively
better activity (GI50 and TGI (MG-MID) 30.2 and
77.6 lM, respectively) when compared with the thiaz-
oline analog 16 (GI50 and TGI (MG-MID) 41.7 and
91.2 lM, respectively) (Tables 2 and 3). Therefore,
compound 16 could be considered the least active
member in this investigation.
Finally, the broad spectrum antitumor activity displayed
by these compounds will be of interest for future deriv-
atization in the hope of finding more active and selective
antitumor agents.
4. Experimental
4.1. Chemistry
Melting points were determined in open glass capillaries
on a Gallenkamp melting point apparatus and are uncor-
rected. The infrared (IR) spectrawererecorded on Perkin-
Elmer 297 infrared spectrophotometer using the KBr
plate technique. The 1H NMR spectra were recorded on
a Varian EM 360 spectrometer using tetramethylsilane
as the internal standard and DMSO-d6 as the solvent
(Chemical shifts in d, ppm). Splitting patterns were desig-
nated as follows: s, singlet; d, doublet; m, multiplet. Ele-
mental analyses were performed at the Microanalytical
Unit, Faculty of Science, King Abdul-Aziz University,
Jeddah, Saudi Arabia, and the found values were within
0.4% of the theoretical values. Follow-up of the reac-
tions and checking the homogeneity of the compounds
were made by TLC on silica gel-protected aluminum

6482
S. A. F. Rostom / Bioorg. Med. Chem. 14 (2006) 6475–6485
sheets (Type 60 F254, Merck) and the spots were detected
by exposure to UV-lamp at k 254.
H), 10.24 (s, 1H, NH), 10.35 (s, 1H, NH). Anal. Calcd
for C29H23ClN4O3S (543.04): C, 64.14; H, 4.27; N,
10.32; S, 5.90. Found: C, 64.02; H, 4.40; N, 10.45; S,
5.85.
4.1.1.
4-(3-(4-Chlorophenyl)-3a,4-dihydro-3H-indeno
[1,2-c]pyrazol-2-yl)-benzenesulfonamide (2). A solution
of the chalcone 137 (5.1 g, 0.02 mol) in ethanol was re-
fluxed with 4-hydrazino-benzenesulfonamide hydro-
chloride (4.9 g, 0.022 mol) for 4 h. The volume of the
reaction mixture was concentrated to the half and the
separated product was filtered, washed with cold etha-
nol, and recrystallized from benzene/ethanol (1:1).
Yield: 85%, mp: 203–204 °C. IR (cmÀ1): 3350 and
3210 (NH2), 1335 and 1180 (SO2N); 1H NMR (d,
ppm): 2.25 (d, J = 9 Hz, 2H, C4–2H), 2.64 (m, 1H,
C3a–H), 5.75 (d, 1H, C3–H), 6.85–8.4 (m, 12H, Ar-H),
10.64 (s, 2H, NH2). Anal. Calcd for C22H18ClN3O2S
(423.92): C, 62.33; H, 4.28; N, 9.91; S, 7.56. Found: C,
61.95; H, 4.36; N, 9.72; S, 7.78.
4.1.3.3. N1-(1-Naphthyl) N3-[4-(3-(4-chlorophenyl)-
3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfo-
nyl]-urea (6). Yield: 80%, mp: 188–189 °C (ethanol). IR
1
(cmÀ1): 1320 and 1190 (SO2N), 1665 (C@O); H NMR
(d, ppm): 2.26 (d, J = 9 Hz, 2H, C4–2H), 2.66 (m, 1H,
C3a–H), 5.72 (d, 1H, C3–H), 6.85–8.40 (m, 19H, Ar-
H), 10.30 (s, 1H, NH), 10.55 (s, 1H, NH). Anal. Calcd
for C33H25ClN4O3S (593.10): C, 66.83; H, 4.25; N,
9.45; S, 5.41. Found: C, 67.05; H, 4.13; N, 9.70; S, 5.23.
4.1.4. N1-substituted N3-[4-(3-(4-chlorophenyl)-3H-inde-
no[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-ureas
(7–9).
Method A. A mixture of the sulfonamide 3 (4.2 g,
0.01 mol) and anhydrous potassium carbonate (2.8 g,
0.02 mol) in dry acetone (25 mL) was heated under re-
flux with stirring, with the appropriate isocyanate
(0.011 mol) for 24 h. Working up of the reaction mixture
was carried out as described under 4–6.
4.1.2. 4-(3-(4-Chlorophenyl)-4H-indeno[1,2-c]pyrazol-2-
yl)-benzenesulfonamide (3). To a stirred suspension of
the pyrazoline 2 (4.2 g, 0.01 mol) in water (10 mL), bro-
mine water (5%, 15 mL) was gradually added over a
period of 30 min at room temperature. After stirring
for further 3 h, the pyrazole derivative thus formed
was collected by filtration, washed with water, and
dried. It was recrystallized from ethanol. Yield: 82%,
mp: 184–185 °C. IR (cmÀ1): 3340 and 3250 (NH2),
1320 and 1185 (SO2N); 1H NMR (d, ppm): 4.12 (s,
2H, C4–2H), 7.47–8.01 (m, 12H, Ar-H). Anal. Calcd
for C22H16ClN3O2S (421.90): C, 62.63; H, 3.82; N,
9.96; S, 7.60. Found: C, 62.91; H, 3.71; N, 10.08; S, 7.75.
Method B. To a stirred suspension of the appropriate
sulfonylurea 4–6 (0.01 mol) in water (10 mL), bromine
water (5%, 15 mL) was gradually added over a period
of 30 min at room temperature. After further stirring
for 6 h, the oxidation product thus formed was treated
as described under 3.
4.1.4.1. N1-Cyclohexyl N3-[4-(3-(4-chlorophenyl)-3H-
indeno[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-urea
(7).
4.1.3. N1-substituted N3-[4-(3-(4-chlorophenyl)-3a,4-dihy-
dro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-ureas
(4–6). A mixture of the pyrazoline 2 (2.1 g, 0.005 mol)
and anhydrous potassium carbonate (1.4 g, 0.01 mol)
in dry acetone (20 mL) was heated under reflux with stir-
ring, with the appropriate isocyanate (0.0055 mol) for
18 h. The solvent was removed under reduced pressure
and the remaining solid residue was dissolved in water
(30 mL). After acidification of the resulting solution
with 2 N hydrochloric acid, the precipitated crude prod-
uct was filtered, washed with water, dried, and
recrystallized.
Yield: 62%, mp: 136–138 °C (ethanol). IR (cmÀ1): 1305
and 1160 (SO2N), 1650 (C@O); 1H NMR (d, ppm):
1.57–2.36 (m, 11H, cyclohexyl-H), 4.23 (s, 2H,
C4–2H), 7.08–8.05 (m, 12H, Ar-H), 9.80 (s, 1H, NH),
10.25 (s, 1H, NH). Anal. Calcd for C29H27ClN4O3S
(547.07): C, 63.67; H, 4.97; N, 10.24; S, 5.86. Found:
C, 63.55; H, 5.07; N, 10.33; S, 5.70.
4.1.4.2. N1-Phenyl N3-[4-(3-(4-chlorophenyl)-3H-inde-
no[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-urea (8). Yield:
68%, mp: 166–167 °C (ethanol). IR (cmÀ1): 1320 and
1
1170 (SO2N), 1660 (C@O); H NMR (d, ppm): 4.28 (s,
2H, C4–2H), 7.12–8.17 (m, 17H, Ar-H), 9.68 (s, 1H,
NH), 10.40 (s, 1H, NH). Anal. Calcd for
C29H21ClN4O3S (541.02): C, 64.38; H, 3.91; N, 10.36;
S, 5.93. Found: C, 64.55; H, 3.76; N, 10.44; S, 5.83.
4.1.3.1. N1-Cyclohexyl N3-[4-(3-(4-chlorophenyl)-3a,4-
dihydro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-
urea (4). Yield: 72%, mp: 185–187 °C (ethanol). IR
1
(cmÀ1): 1300 and 1165 (SO2N), 1640 (C@O); H NMR
(d, ppm): 1.57–2.16 (m, 11H, cyclohexyl-H), 2.20 (d,
J = 9 Hz, 2H, C4–2H), 2.81 (m, 1H, C3a–H), 5.60 (d,
1H, C3–H), 6.98–8.12 (m, 12H, Ar-H), 10.40 (s, 1H,
NH), 10.52 (s, 1H, NH). Anal. Calcd for
C29H29ClN4O3S (549.08): C, 63.43; H, 5.32; N, 10.20;
S, 5.84. Found: C, 63.49; H, 5.29; N, 9.98; S, 5.98.
4.1.4.3. N1-(1-Naphthyl) N3-[4-(3-(4-chlorophenyl)-
3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-urea (9).
Yield: 60%, mp: 168–169 °C (ethanol). IR (cmÀ1): 1330
and 1210 (SO2N), 1665 (C@O); 1H NMR (d, ppm):
4.31 (s, 2H, C4–2H), 6.95–8.35 (m, 19H, Ar-H), 10.10
(s, 1H, NH), 10.36 (s, 1H, NH). Anal. Calcd for
C33H23ClN4O3S (591.08): C, 67.06; H, 3.92; N, 9.48; S,
5.42. Found: C, 66.89; H, 4.06; N, 9.60; S, 5.39.
4.1.3.2. N1-Phenyl N3-[4-(3-(4-chlorophenyl)-3a,4-
dihydro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-
urea (5). Yield: 78%, mp: 192–193 °C (ethanol). IR
4.1.5. N1-substituted N3-[4-(3-(4-chlorophenyl)-3a,4-dihy-
dro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-thiou-
1
(cmÀ1): 1310 and 1175 (SO2N), 1650 (C@O); H NMR
(d, ppm): 2.18 (d, J = 9 Hz, 2H, C4–2H), 2.57 (m, 1H,
C3a–H), 5.68 (d, 1H, C3–H), 6.90–8.35 (m, 17H, Ar-
reas (10–12).
isothiocyanate (0.011 mol) in dry acetone (5 mL) was
A
solution of the appropriate

S. A. F. Rostom / Bioorg. Med. Chem. 14 (2006) 6475–6485
6483
added to a stirred mixture of the sulfonamide 2 (4.2 g,
0.01 mol) and anhydrous potassium carbonate (2.8 g,
0.02 mol) in dry acetone (25 mL), and the reaction mix-
ture was heated under reflux with stirring for 10 h. The
solvent was removed under reduced pressure and the
remaining solid residue was dissolved in water (30 mL)
and acidified with 2 N hydrochloric acid. The precipitat-
ed crude product was filtered, washed with water, dried,
and recrystallized.
4.1.6.2. 3-Benzyl-2-[4-(3-(4-chlorophenyl)-3a,4-dihy-
dro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonylimino]-
thiazolidin-4-one (14). Yield: 66%, mp: 154–156 °C
(ethanol/benzene; 1:1). IR (cmÀ1): 1720 (C@O); 1H
NMR (d, ppm): 2.26 (d, J = 9 Hz, 2H, C4–2H), 2.61
(m, 1H, C3a–H), 4.21 (s, 2H, thiazol-CH2), 4.67 (s, 2H,
benzyl-CH2), 5.44 (d, 1H, C3–H), 7.05–8.30 (m, 17H,
Ar-H). Anal. Calcd for C32H25ClN4O3S2 (613.15): C,
62.68; H, 4.11; N, 9.14; S, 10.46. Found: C, 62.44; H,
4.25; N, 9.02; S, 10.51.
4.1.5.1. N1-Phenyl N3-[4-(3-(4-chlorophenyl)-3a,4-
dihydro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-
thiourea (10). Yield: 85%, mp: 155–157 °C (ethanol). IR
4.1.6.3. 3-Benzoyl-2-[4-(3-(4-chlorophenyl)-3a,4-dihy-
dro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonylimino]-
thiazolidin-4-one (15). Yield: 60%, mp: 177–178 °C
(ethanol/benzene; 1:1). IR (cmÀ1): 1690 (C@O), 1735
1
(cmÀ1): 1310 and 1175 (SO2N), 1155 (C@S); H NMR
(d, ppm): 2.25 (d, J = 9 Hz, 2H, C4–2H), 2.54 (m, 1H,
C3a–H), 5.60 (d, 1H, C3–H), 6.85–8.23 (m, 17H, Ar-H),
9.82 (s, 1H, NH), 10.12 (s, 1H, NH). Anal. Calcd for
C29H23ClN4O2S2 (559.10): C, 62.30; H, 4.15; N, 10.02;
S, 11.47. Found: C, 62.10; H, 4.22; N, 9.86; S, 11.70.
1
(C@O); H NMR (d, ppm): 2.19 (d, J = 9 Hz, 2H, C4–
2H), 2.54 (m, 1H, C3a–H), 4.28 (s, 2H, thiazol-CH2),
5.50 (d, 1H, C3–H), 6.93–8.25 (m, 17H, Ar-H). Anal.
Calcd for C32H23ClN4O4S2 (627.19): C, 61.28; H, 3.69;
N, 8.93; S, 10.22. Found: C, 60.93; H, 3.95; N, 9.11; S,
10.07.
4.1.5.2. N1-Benzyl N3-[4-(3-(4-chlorophenyl)-3a,4-
dihydro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-
thiourea (11). Yield: 78%, mp: 136–138 °C (ethanol). IR
4.1.7.
3-Substituted-4-phenyl-2-[4-(3-(4-chlorophenyl)-
1
(cmÀ1): 1315 and 1190 (SO2N), 1150 (C@S); H NMR
3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfo-
nylimino]-1,3-thiazolines (16–18). A solution of the
appropriate sulfonyl-thioureido derivative 6–8
(0.005 mol) in absolute ethanol (20 mL) was refluxed
with phenacyl bromide (1.1 g, 0.0055 mol) and anhy-
drous sodium acetate (0.82 g, 0.01 mol) for 3 h during
which the solid product was partially crystallized out.
The mixture was left to attain room temperature then fil-
tered, washed with cold ethanol, dried, and
recrystallized.
(d, ppm): 2.29 (d, J = 9 Hz, 2H, C4–2H), 2.61 (m, 1H,
C3a–H), 4.73 (d, 2H, benzyl CH2), 5.54 (d, 1H, C3–H),
6.92–8.34 (m, 17H, Ar-H), 9.90 (s, 1H, NH), 10.22 (s,
1H, NH). Anal. Calcd for C30H25ClN4O2S2 (573.13):
C, 62.87; H, 4.40; N, 9.78; S, 11.19. Found: C, 62.56;
H, 4.58; N, 9.90; S, 11.35.
4.1.5.3. N1-Benzoyl N3-[4-(3-(4-chlorophenyl)-3a,4-
dihydro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonyl]-
thiourea (12). Yield: 71%, mp: 160–162 °C (ethanol). IR
(cmÀ1): 1115 (C@S), 1330 and 1220 (SO2N), 1660
4.1.7.1.
3,4-Diphenyl-2-[4-(3-(4-chlorophenyl)-3a,4-
1
(C@O); H NMR (d, ppm): 2.22 (d, J = 9 Hz, 2H, C4–
dihydro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonyli-
mino]-1,3-thiazoline (16). Yield: 72%, mp: 146–148 °C
2H), 2.51 (m, 1H, C3a–H), 5.49 (d, 1H, C3–H), 6.88–
8.29 (m, 17H, Ar-H), 10.05 (s, 1H, NH), 10.28 (s, 1H,
NH). Anal. Calcd for C30H23ClN4O3S2 (587.12): C,
61.36; H, 3.95; N, 9.54; S, 10.92. Found: C, 61.03; H,
4.11; N, 9.81; S, 11.05.
1
(ethanol). IR (cmÀ1): 1600 (C@N); H NMR (d, ppm):
2.18 (d, J = 9 Hz, 2H, C4–2H), 2.61 (m, 1H, C3a–H),
5.39 (d, 1H, C3–H), 6.11 (s, 1H, thiazol-H), 7.03–8.32
(m, 22H, Ar-H). Anal. Calcd for C37H27ClN4O2S2
(659.22): C, 67.41; H, 4.13; N, 8.50; S, 9.73. Found: C,
67.30; H, 4.25; N, 8.66; S, 9.48.
4.1.6. 3-Substituted-2-[4-(3-(4-chlorophenyl)-3a,4-dihy-
dro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonylimino]-
thiazolidin-4-ones (13–15). To a solution of the appropri-
ate sulfonylthioureido derivative 6–8 (0.005 mol) in
absolute ethanol (20 mL) were added ethyl bromoace-
tate (1 g, 0.0055 mol) and anhydrous sodium acetate
(0.82 g, 0.01 mol) and the reaction mixture was heated
under reflux for 2 h. The mixture was left to attain room
temperature then poured into an ice-cold water (30 mL),
and the solid product thus formed was filtered, washed
with water, dried, and recrystallized.
4.1.7.2.
3-Benzyl-4-phenyl-2-[4-(3-(4-chlorophenyl)-
3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfo-
nylimino]-1,3-thiazoline (17). Yield: 69%, mp: 154–156 °C
1
(ethanol). IR (cmÀ1): 1560 (C@N); H NMR (d, ppm):
2.24 (d, J = 9 Hz, 2H, C4–2H), 2.55 (m, 1H, C3a–H),
4.69 (s, 2H, benzyl-CH2), 5.48 (d, 1H, C3–H), 6.15 (s,
1H, thiazol-H), 6.89–8.18 (m, 22H, Ar-H). Anal. Calcd
for C38H29ClN4O2S2 (673.25): C, 67.79; H, 4.34; N,
8.32; S, 9.53. Found: C, 67.90; H, 4.30; N, 8.12; S, 9.69.
4.1.6.1. 3-Phenyl-2-[4-(3-(4-chlorophenyl)-3a,4-dihy-
dro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfonylimino]-
thiazolidin-4-one (13). Yield: 68%, mp: 184–186 °C
(ethanol/benzene; 1:1). IR (cmÀ1): 1730 (C@O); 1H
NMR (d, ppm): 2.26 (d, J = 9 Hz, 2H, C4–2H), 2.76
(m, 1H, C3a–H), 4.15 (s, 2H, thiazol-CH2), 5.39 (d,
1H, C3–H), 7.20–8.15 (m, 17H, Ar-H). Anal. Calcd for
C31H23ClN4O3S2 (599.12): C, 62.15; H, 3.87; N, 9.35;
S, 10.70. Found: C, 61.97; H, 4.00; N, 9.30; S, 10.82.
4.1.7.3. 3-Benzoyl-4-phenyl-2-[4-(3-(4-chlorophenyl)-
3a,4-dihydro-3H-indeno[1,2-c]pyrazol-2-yl)-benzenesulfo-
nylimino]-1,3-thiazoline (18). Yield: 55%, mp: 191–
193 °C (ethanol). IR (cmÀ1): 1740 (C@O); 1H NMR
(d, ppm): 2.20 (d, J = 9 Hz, 2H, C4–2H), 2.56 (m, 1H,
C3a–H), 5.55 (d, 1H, C3–H), 6.19 (s, 1H, thiazol-H),
7.01–8.24 (m, 22H, Ar-H). Anal. Calcd for
C38H27ClN4O3S2 (687.29): C, 66.41; H, 3.96; N, 8.15;
S, 9.33. Found: C, 66.12; H, 4.16; N, 8.36; S, 9.09.

6484
S. A. F. Rostom / Bioorg. Med. Chem. 14 (2006) 6475–6485
4.2. In vitro antitumor screening
Cancer Institute (NCI), Bethesda, Maryland, USA,
for carrying out the anticancer screening of the newly
synthesized compounds.
Compounds 2–4, 7, 8, 10, 13, and 16 were subjected
to the NCI in vitro disease-oriented human cells
screening panel assay to screen their antitumor activi-
ties. About 60 cell lines of nine tumor subpanels,
including leukemia, non-small cell lung, colon, CNS,
melanoma, ovarian, renal, prostate, and breast cancer
cell lines, were utilized. The human tumor cell lines of
the cancer screening panel were grown in RPMI 1640
medium containing 5% fetal bovine serum and 2 mM
L-glutamine. For a typical screening experiment, cells
were inoculated into 96-well microtiter plates in 100
mL at plating densities ranging from 5000 to 40,000
cells/well depending on the doubling time of individual
cell lines. After cell inoculation, the microtiter plates
were incubated at 37 °C, 5% CO2, 95% air, and
100% relative humidity for 24 h prior to addition of
experimental drugs. After 24 h, two plates of each cell
line were fixed in situ with TCA, to represent a mea-
surement of the cell population for each cell line at
the time of drug addition. Experimental drugs were
solubilized in dimethyl sulfoxide at 400-fold the de-
sired final maximum test concentration and stored fro-
zen prior to use. At the time of drug addition, an
aliquot of frozen concentrate was thawed and diluted
to twice the desired final maximum test concentration
with complete medium containing 50 mg mLÀ1 genta-
micin. Additional four 10-fold or 1/2 log serial dilu-
tions were made to provide a total of five drug
concentrations plus control. Aliquots of 100 mL of
these different drug dilutions were added to the appro-
priate microtiter wells already containing 100 mL of
medium, resulting in the required final drug concen-
trations. Following drug addition, the plates were
incubated for an additional 48 h at 37 °C, 5% CO2,
95% air, and 100% relative humidity. For adherent
cells, the assay was terminated by the addition of cold
TCA. Cells were 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 °C. The
supernatant was discarded, and the plates were
washed five times with tap water and air-dried. Sulfo-
rhodamine B (SRB) solution (100 mL) at 0.4% (w/v)
in 1% acetic acid was added to each well, and plates
were incubated for 10 min at room temperature. After
staining, unbound dye was removed by washing five
times with 1% acetic acid and the plates were air-
dried. Bound stain was subsequently solubilized with
10 mM trizma base, and the absorbance was read on
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