Polysubstituted pyrazoles, part 5.1. Synthesis of new 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole-3-carboxylic acid hydrazide analogs and some derived ring systems. A novel class of potential antitumor and anti-HCV agents

Article abstract of DOI:10.1016/j.ejmech.2003.08.003

A novel series of 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole-3-carboxylic acid hydrazide analogs and some derived 4-substituted-1,2,4-triazolin-3-thiones, 2-substituted-1,3,4-thiadiazole and 2-substituted-1,3,4-oxadiazoles has been synthesized. Ten of the newly synthesized compounds were selected by the National Cancer Institute (NCI)-in vitro-disease oriented antitumor screening to be evaluated for their antitumor activity. Seven compounds, namely 7a-c, 9, 11, 13 and 14, exhibited potential and broad spectrum antitumor activity against most of the tested subpanel tumour cell lines (GI₅₀<100 μM). Compounds 14 (GI₅₀, TGI, and LC₅₀ MG-MID values of 0.08, 15.8 and 64.6 μM, respectively) and 11 (GI₅₀, TGI, and LC₅₀ MG-MID values of 0.20, 11.7 and 87.1 μM, respectively) proved to be the most active members in this study with potential activity against all the tested subpanel tumour cell lines and particular effectiveness on the leukaemia subpanel at both the GI₅₀ (0.03 and 0.09 μM, respectively) and the TGI levels (35.2 and 28.1 μM, respectively). Moreover, compound 14 exhibited a super sensitivity profile towards about 26 different cancer cell lines with GI₅₀ values lying in the nanomolar concentration range (GI₅₀ values<0.01 μM). In addition, compounds 2-5, 6a-d, 7a, 8-11, 12a, 13, 14 were investigated for their in vitro effect on the replication of hepatitis-C virus (HCV) in HepG2 hepatocellular carcinoma cell line infected with the virus using the reverse transcription-polymerase chain reaction (RT-PCR) technique. The results revealed that compounds 2 and 5 were capable of inhibiting the replication of both the HCV RNA (+) and (-) strands at 10-100 μg mL^-1 concentration range.

Full text of DOI:10.1016/j.ejmech.2003.08.003

European Journal of Medicinal Chemistry 38 (2003) 959ꢀ974
/
www.elsevier.com/locate/ejmech
Original article
Polysubstituted pyrazoles, part 5.1 Synthesis of new 1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazole-3-carboxylic acid hydrazide
analogs and some derived ring systems. A novel class of potential
antitumor and anti-HCV agentsꢀ
Sherif A.F. Rostom a,*, Manal A. Shalaby b, Maha A. El-Demellawy b
a Department of Medicinal Chemistry, Faculty of Medicine and Allied Sciences, King Abdul-Aziz University, P.O. Box 80205,
Jeddah 21589, Saudi Arabia
b Genetic Engineering and Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research and Technology Applications,
Bourg El-Arab, Alexandria, Egypt
Received 14 March 2003; received in revised form 4 August 2003; accepted 4 August 2003
Abstract
A novel series of 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole-3-carboxylic acid hydrazide analogs and some derived 4-substituted-
1,2,4-triazolin-3-thiones, 2-substituted-1,3,4-thiadiazole and 2-substituted-1,3,4-oxadiazoles has been synthesized. Ten of the newly
synthesized compounds were selected by the National Cancer Institute (NCI)-in vitro-disease oriented antitumor screening to be
evaluated for their antitumor activity. Seven compounds, namely 7aꢀ/c, 9, 11, 13 and 14, exhibited potential and broad spectrum
antitumor activity against most of the tested subpanel tumour cell lines (GI50
B
/
100 mM). Compounds 14 (GI50, TGI, and LC50 MG-
MID values of 0.08, 15.8 and 64.6 mM, respectively) and 11 (GI50, TGI, and LC50 MG-MID values of 0.20, 11.7 and 87.1 mM,
respectively) proved to be the most active members in this study with potential activity against all the tested subpanel tumour cell
lines and particular effectiveness on the leukaemia subpanel at both the GI50 (0.03 and 0.09 mM, respectively) and the TGI levels
(35.2 and 28.1 mM, respectively). Moreover, compound 14 exhibited a super sensitivity profile towards about 26 different cancer cell
lines with GI50 values lying in the nanomolar concentration range (GI50 valuesB
8ꢀ11, 12a, 13, 14 were investigated for their in vitro effect on the replication of hepatitis-C virus (HCV) in HepG2 hepatocellular
carcinoma cell line infected with the virus using the reverse transcriptionꢀpolymerase chain reaction (RT-PCR) technique. The
) and (ꢂ) strands at
/
0.01 mM). In addition, compounds 2ꢀ
/
5, 6aꢀ
/
d, 7a,
/
/
results revealed that compounds 2 and 5 were capable of inhibiting the replication of both the HCV RNA (ꢁ
/
/
10ꢀ
/
100 mg mLꢂ1 concentration range.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 1H-pyrazoles; acid hydrazide derivatives; 1,2,4-triazolin-3-thiones; 1,3,4-thiadiazole; 1,3,4-oxadiazoles; antitumor activity; anti-HCV
activity; RT-PCR
1. Introduction
inal chemistry researchers. In this view, the discovery of
the natural 4-hydroxypyrazole C-glycoside antibiotic
Identification of novel structure leads that may be of
use in designing new, potent, selective and less toxic
anticancer agents remains a major challenge for medic-
Pyrazofurin; 4-hydroxy-3b-D-ribofuranosyl-1H-pyra-
zole-5-carboxamide (Fig. 1); has provided a basis for
more rationale design and synthesis of new pyrazoles as
potential antimicrobial [1ꢀ
ancer agents [7ꢀ9]. Pyrazofurin; isolated from Strepto-
myces candidus; bears a clear structure resemblance to
ribaverin; 1b- -ribofuranosyl-1H-1,2,4-triazole-3-car-
/
3], antiviral [4ꢀ
/
6] and antic-
/
ꢀ This work was partly presented at the 8th Ibn-Sina International
Conference on Pure and Applied Heterocyclic Chemistry; Luxor,
D
Egypt; February 16ꢀ19, 2002; Poster P-157.
/
boxamide (Fig. 1) and other five-membered nucleosides
but unlike those, it inhibits the de novo synthesis of
pyrimidines rather than the structurally related purines.
* Corresponding author.
E-mail address: sherifrostom@yahoo.com (Sh.A.F. Rostom).
1
For part 4: see Ref. [18].
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.08.003

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Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ974
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transformation and hepatocellular carcinoma [14].
Moreover, some clinical studies indicated a significant
correlation between HCV infection and some lympho-
proliferative disorders, especially the high prevalence of
auto-antibodies, mixed cryoglobulinemia and non-
Hodgkin lymphoma, suggesting that HCV not only
causes a liver disease but rather a multifaceted clinical
syndrome [15]. The inherent instability of this virus gave
rise to multiple types and subtypes which have seriously
hampered efforts to develop an efficient HCV vaccine
[16]. As yet, the only available therapy for chronic HCV
infection is the treatment with interferon-alpha (Ifn-a)
either alone or in combination with the nucleoside
analog ribaverin. Unfortunately, only 40% of treated
patients develop a sustained response that is defined by
the absence of viral RNA for more than 6 months after
cessation of the therapy. In spite of the beneficial effect
of rebaverin, the side effects are intensified with
combination therapy as it can cause severe haemolytic
anaemia in addition to its possible teratogenic effect
[15,17]. These complications clearly document the need
for more effective, less toxic anticancer and antiviral
therapies.
In view of the above-mentioned findings, and in
continuation of our interest in biologically active
Fig. 1.
pyrazoles [18ꢀ22], the aim of the present study was to
/
This antibiotic was believed to act as an antagonist of
uridine metabolism and the monophosphate inhibits the
OMP decarboxylase enzyme [10]. Pyrazofurin has
received considerable attention as a result of its various
biological effects including the potent antimicrobial and
broad-spectrum antiviral activities against many RNA
and DNA viruses. As has been also noted, pyrazofurin
has been evaluated against several tumour cell lines,
consequently, it has only been used clinically as an
anticancer agent. Although pyrazofurin and its analogs
will continue to be pursued for their chemotherapeutic
value, the critical hurdle still to be overcome is the
unavailability and inherent toxicity of the drug [11].
On the other hand, over the past decade, there has
been growing interest in acute and chronic liver diseases
that are caused by an infection with hepatitis-C virus
(HCV), such as hepatocellular carcinoma and liver
cirrhosis. It has been recently reported that about 3%
of the world population is infected with this harmful
pathogen that can cause a variety of complicated clinical
synthesize and investigate the in vitro anticancer activity
of some novel 4-hydroxy-1H-pyrazole-3-carboxylic acid
hydrazide analogs A carrying the vicinal hydroxyl and
amidic pharmacophores of pyrazofurin, in addition to
some derived ring systems having the general formula B
(Fig. 1). It was also considered of interest to confirm the
ability of the newly synthesized compounds to inhibit
the replication of the HCV RNA (as a possible
carcinogen) by a reliable and reproducible biotechnolo-
gical technique; the RT-PCR. Careful literature survey
for functional groups which could be considered as
pharmacophores for the anticancer and antiviral activ-
ities revealed that the amide function is common among
most of the natural antibiotic antitumor and antiviral
agents such as bleomycin, netropsin and pyrazofurin
[23]. Therefore, the target compounds were rationalized
so as to comprise the amidic pharmacophores that are
believed to be responsible for the biological significance
of some relevant chemotherapeutic agents, such as the
N-formyl, N-acetyl, semicarbazide, thiosemicarbazide
symptoms, 90ꢀ95% of which through blood transfusion
/
and azomethine functionalities [24ꢀ29]. The substitution
/
and haemodialysis [12]. HCV is a small-enveloped virus
belonging to the Flaviviridea with single-stranded RNA
genome that codes for a single polyprotein [13]. Re-
cently, HCV is believed to act as a carcinogen by virtue
of the increased risk of hepatocellular carcinoma among
persistently infected patients with chronic active hepa-
titis. This was attributed to the fact that HCV causes an
immune-mediated chronic inflammation in the liver with
the resulting cell damage eventually giving rise to
pattern of such amide derivatives was carefully selected
so as to confer different electronic environment to the
molecules. Based on the fact that substituted 1,2,4-
triazoles and 1,3,4-thiadiazoles have been contributed to
a variety of chemotherapeutic activities [30ꢀ33], it was
/
planned to synthesize hybrid compounds that comprise
both the 4-hydroxypyrazole and the aforementioned
heterocyclic ring systems. Such hybridization was de-
signed in an attempt to obtain synergistic chemother-

Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ
/974
961
apeutic activity with higher selectivity and less toxicity.
Moreover, owing to the increasing biological impor-
tance of 1,3,4-oxadiazoles, particularly in the field of
formation of the corresponding 4-phenyl-1-(1-(4-chlor-
ophenyl)-4-hydroxy-1H-pyrazole-3-carbonyl)semi-car-
bazide 4 and the 4-substituted thiosemicarbazides 6aꢀd,
/
chemotherapy [34ꢀ36], it was considered of interest to
/
respectively. On the other hand, heating 1 with ammo-
nium thiocyanate in the presence of dilute hydrochloric
acid afforded the 1-(1-(4-chlorophenyl)-4-hydroxy-1H-
pyrazole-3-carbonyl)thiosemicarbazide 5 in a moderate
yield. Alkaline cyclisation of the 4-substituted thiosemi-
link the 4-hydroxypyrazole backbone to some 1,3,4-
oxadiazoles and their 1,3,4-oxadiazoline-5-thione, 1,3,4-
oxadiazoline-5-one analogs in order to investigate the
effect of such structure variation on the anticipated
antitumor and/or antiviral activities.
carbazides 6aꢀ
corresponding
/
c using sodium hydroxide afforded the
5-(1-(4-chlorophenyl)-4-hydroxy-1H-
pyrazol-3-yl)-4-substituted-1,2,4-triazolin-3-thiones 7aꢀ
/
c in excellent yields. Furthermore, reacting the thiose-
micarbazide 6a with cold concentrated sulphuric acid
resulted in the formation of the corresponding 5-(1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazol-3-yl)-2-phenyla-
mino-1,3,4-thiadiazole 8. Cyclisation of 6a to 5-(1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazol-3-yl)-2-phenyla-
mino-1,3,4-oxadiazole 9 could be achieved by boiling of
the former with mercuric oxide in absolute ethanol (Fig.
2).
In Fig. 3, the preparation of the 5-(1-(4-chlorophe-
nyl)-4-hydroxy-1H-pyrazol-3-yl)-1,3,4-oxadiazolin-2-
thione 10 was achieved by adopting a simple one-pot
procedure that involves reacting 1 with carbon disulfide
under strong basic conditions followed by acidification
with dilute hydrochloric acid. The synthesis of the 5-(1-
2. Chemistry
The synthetic strategies adopted to obtain the target
compounds are depicted in Figs. 2 and 3. The key
intermediate in the present study is the 1-(4-chlorophe-
nyl)-4-hydroxy-1H-pyrazole-3-carboxylic acid hydra-
zide 1, which was prepared according to a previously
described procedure [18]. When 1 was refluxed with
formic acid, the target N-formyl-1-(4-chlorophenyl)-4-
hydroxy-1H-pyrazole-3-carboxylic acid hydrazide 2 was
obtained in a good yield. Warming 1 with acetic
anhydride afforded the N-acetyl-4-acetyloxy-1-(4-chlor-
ophenyl)-1H-pyrazole-3-carboxylic acid hydrazide 3.
Reacting the same starting compound 1 with phenyl
isocyanate or a variety of isothiocyanates resulted in the
Fig. 2. Synthesis of compounds 2ꢀ9.
/

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Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ974
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Fig. 3. Synthesis of compounds 10ꢀ14.
/
(4-chlorophenyl)-4-hydroxy-1H-pyrazol-3-yl)-1,3,4-oxa-
diazolin-2-one 11 was accomplished by treating the key
intermediate 1 with 1,1‘-carbonyldiimidazole (CDI) in
dry tetrahydrofuran (THF). Moreover, condensing the
acid hydrazide 1 with a variety of aldehydes or ketones
in acetic acid yielded the corresponding 3-(alkylidenehy-
drazinocarbonyl)-1-(4-chlorophenyl)-4-hydroxy-1H-
iented human cells screening panel assay [37ꢀ39] to
/
investigate their antitumor activities. About 60 cell lines
of nine tumour subpanels, including leukaemia, non-
small cell lung, colon, CNS, melanoma, ovarian, renal,
prostate and breast cancer cell lines, were incubated with
five concentrations (0.01ꢀ100 mM) for each compound
/
and were used to create log concentration -% 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%
decrease in net cell growth, the TGI value (cytostatic
activity) 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
midpoint 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 [37]. The NCI antitumor drug
discovery was designed to distinguish between broad
spectrum antitumor compounds and tumour or sub-
panel-selective agents.
pyrazoles 12aꢀe. Conversion of 12a into the 3-acetyl-5-
/
(4-acetyloxy-1-(4-chlorophenyl)-1H-pyrazol-3-yl)-2-
(2,4-difluorophenyl)-1,3,4-oxadiazoline 13 was effected
by treatment of the former compound with acetic
anhydride. Finally, a convenient method for the synth-
esis of 5-(1-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-3-
yl)-2-phenyl-1,3,4-oxadiazole 14 was deduced from
refluxing the acid hydrazide 1 with an equimolar
amount of benzoic acid in the presence of an excess of
phosphorus oxychloride (Fig. 3).
3. Results and discussion
3.1. In vitro antitumor screening
Out of newly synthesized pyrazole derivatives, com-
pounds 3, 7aꢀ
/
c, 8ꢀ
/
11, 13 and 14 were selected by the
National Cancer Institute (NCI) in vitro disease-or-
In the present study, only seven compounds, namely;
the 5-(1-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-3-yl)-

Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ
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963
4-substituted-1,2,4-triazolin-3-thiones 7aꢀ
/
c and the 5-
well as a broad spectrum of antitumor activity (Tables
2ꢀ4).
With regard to the sensitivity against some individual
cell lines, compounds 7c, 11 and 14 proved to be very
sensitive towards most of the tested subpanel tumour
(1-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-3-yl)-2-sub-
stituted-1,3,4-oxadiazoles 9, 11, 13 and 14, exhibited
potential antitumor activities against most of the tested
subpanel tumour cell lines (GI50, TGI and LC50
/
cell lines with GI50 values range of B
/
0.01ꢀ0.66 mM.
/
valuesB100 mM), whereas the other three compounds
/
Compound 14 showed a super sensitivity profile to-
wards about 26 different cancer cell lines with GI50
values lying in the nanomolar concentration range (GI50
3, 8 and 10 proved to be totally inactive. Those seven
compounds showed a distinctive potential pattern of
sensitivity against some individual cell lines (Table 1), as
Table 1
Growth inhibitory concentration (GI50, mM) of some selected in vitro tumour cell lines a
Cell lines
7a
7b
7c
9
10
13
14
Leukaemia
HL-60(TB)
K-562
4.13
3.95
62.7
NT
NT b
NT
NT
NT
NT
NT
0.03
0.05
0.05
0.12
NT
B
B/
/
0.01
0.01
0.02
0.09
0.01
12.6
B
/
0.01
0.63
0.25
0.01
30.0
MOLT-4
RPMI-8226
SR
14.7
22.2
10.0
18.5
20.5
35.8
5.45
2.97
B
/
NT
B/
Non-small cell lung cancer
A549/ATCC
HOP-62
14.1
13.2
22.7
26.6
11.9
0.93
0.47
20.4
17.7
8.39
0.18
33.2
28.8
B
/
0.01
0.01
0.01
0.01
9.92
6.22
0.20
0.27
0.04
B
/
NCI-H226
NCI-460
NT
0.24
NT
B
/
10.6
19.9
34.4
B/
Colon cancer
HCT-116
HCT-15
HT29
6.35
3.36
11.6
11.3
43.2
13.0
NT
0.15
39.1
0.39
16.1
10.4
0.09
0.30
2.02
0.12
33.4
32.2
40.1
25.4
B
B/
/
0.01
0.01
0.25
0.01
19.8
4.35
9.56
KM12
17.8
B/
CNS cancer
SF-268
SF-295
SF-539
U251
10.4
14.7
14.8
15.1
15.8
11.8
0.01
14.6
18.9
15.3
17.5
0.17
29.6
20.2
29.8
38.5
B
/
0.01
0.01
0.01
0.01
4.95
5.91
B
/
NT
B
/
NT
NT
0.03
0.02
B
/
10.8
B/
Melanoma
MALME-3M
SK-MEL-5
UACC-257
UACC-62
16.2
4.19
25.9
14.3
24.2
12.0
B
/
0.01
0.23
0.87
0.01
25.3
11.4
21.3
16.5
B/
0.01
0.36
0.43
0.28
14.8
18.4
27.2
28.8
8.34
0.01
0.51
0.23
B/
20.7
11.4
B/
Ovarian cancer
OVCAR-3
OVCAR-5
OVCAR-8
2.51
22.0
23.0
27.3
B/
0.01
2.17
0.29
31.9
88.8
20.9
NT
0.60
0.23
19.7
44.4
34.6
0.02
0.66
0.01
24.2
14.9
B/
Renal cancer
786-0
11.6
17.8
22.0
14.2
28.1
16.9
28.1
25.0
0.54
0.84
0.41
16.6
16.1
18.5
14.3
0.50
0.31
0.01
0.23
26.1
33.6
21.1
31.9
B
B/
/
0.01
0.01
0.29
0.01
SN12C
TK-10
UO-31
13.5
B/
Prostate cancer
PC-3
DU-145
27.5
22.3
14.8
22.0
10.0
NT
0.45
0.05
27.9
39.2
0.36
0.31
0.33
15.4
Breast cancer
MCF7
4.41
24.8
15.4
29.1
B
B/
/
0.01
0.01
0.3
16.9
14.9
24.1
16.8
0.35
0.18
0.08
0.01
30.0
42.9
31.8
27.9
B
/
0.01
0.03
0.01
0.01
NCI/ADR-RES
HS 578T
MDA-MB-435
3.79
7.64
1.94
B
/
4.93
B/
0.01
B/
a
Data obtained from NCI’s in vitro disease-oriented human tumour cell screen.
NT, not tested.
b

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Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ
/974
Table 2
Median growth inhibitory concentration (GI50, mM) of in vitro subpanel tumour cell lines
Cpd. No.
Subpanel tumour cell lines a
MG-MID b
I
II
III
IV
V
VI
VII
VIII
IX
7a
7b
7c
9
15.8
13.3
22.9
21.2
10.3
27.2
31.7
15.7
11.7
25.0
14.1
20.3
19.3
39.0
11.3
36.0
14.7
28.7
19.3
19.0
24.9
18.4
5.16
15.4
0.25
33.6
0.34
15.8
11.0
20.9
0.65
18.6
0.20
31.6
0.08
28.7
14.2
18.0
0.23
NT c
0.09
26.5
0.03
9.60
17.4
0.23
32.4
2.45
4.11
18.0
0.13
33.4
0.30
4.17
18.8
2.24
34.4
4.06
10
13
14
0.93
35.8
0.20
0.54
39.5
5.10
0.90
31.4
1.49
1.05
35.7
0.04
a
I, Leukaemia; 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.
b
GI50 (mM) full panel mean-graph mid point (MG-MID)ꢃthe average sensitivity of all cell lines towards the test agent.
/
c
Not tested.
values B
/
0.01 mM). Furthermore, compound 7a showed
exhibited cytotoxic activity with LC50 (MG-MID)
values of 85.1, 95.5, 79.4, 87.1 and 64.6 mM, respectively
(Table 4).
also a remarkable sensitivity against most of the tested
tumour cell lines with GI50 values ranging between 1.94
and 27.5 mM. Except for the leukaemia subpanel,
compounds 7b and 9 exhibited almost the same pattern
of sensitivity against most of the tested subpanel tumour
cell lines with special pronounced activity against breast
cancer MDA-MB-435 cell line (GI50 values 4.93 and
16.8 mM, respectively). Although compound 13 proved
to be less sensitive towards the nine subpanel tumour
cell lines when compared with the pre-mentioned
compounds, it showed an appreciable antitumor profile
with GI50 values ranging between 14.8 and 44.4 mM
(Table 1).
Further interpretation of the obtained data revealed
that, compounds 14 (GI50, TGI, and LC50 MG-MID
values of 0.08, 15.8 and 64.6 mM, respectively) and 11
(GI50, TGI, and LC50 MG-MID values of 0.20, 11.7 and
87.1 mM, respectively) proved to be the most active
members in this study with potential activity against all
the tested subpanel tumour cell lines and particular
effectiveness on the leukaemia subpanel at both the GI50
(0.03 and 0.09 mM, respectively) and the TGI levels (35.2
and 28.1 mM, respectively) (Tables 2ꢀ/4). Compound 7c
(GI50 and TGI MG-MID values of 0.65 and 41.6 mM,
respectively) displayed a remarkable growth inhibition
and cytostatic effects when compared with 11 and 14,
however, it lacks cytotoxic activity (Tables 2 and 4). On
the other hand, compounds 7a (GI50, TGI, and LC50
MG-MID values of 11.0, 44.7 and 85.1 mM, respec-
tively), 7b (GI50, TGI, and LC50 MG-MID values of
20.9, 75.9 and 95.5 mM, respectively) and 9 (GI50, TGI,
and LC50 MG-MID values of 18.6, 40.7 and 79.4 mM,
respectively) displayed a considerable antitumor activity
With regard to the broad spectrum antitumor activity,
the results revealed that all of the seven active com-
pounds; 7aꢀc, 9, 11, 13 and 14 showed effective growth
/
inhibition GI50 (MG-MID) values of 11.0, 20.9, 0.65,
18.6, 0.20, 31.6 and 0.08 mM, respectively, beside a
cytostatic activity TGI (MG-MID) values of 44.7, 75.9,
41.6, 40.7, 11.7, 87.1 and 15.8 mM, respectively (Tables 2
and 3). In addition, except for compounds 7c and 13, the
rest of the active compounds; 7a, 7b, 9, 11, 13 and 14
Table 3
Median total growth inhibitory concentration (TGI, mM) and mean LC50 (mM) values of in vitro subpanel tumour cell lines
Cpd. No.
Subpanel tumour cell lines a
MG-MID b
I
II
III
IV
V
VI
VII
VIII
IX
c
7a
7b
7c
9
99.0
50.4
70.0
91.7
40.2
31.8
88.9
22.2
30.7
84.0
73.6
34.8
57.8
91.3
11.6
56.3
74.5
46.8
38.9
80.0
90.3
28.4
54.2
66.2
84.7
36.9
51.2
84.9
19.5
43.9
87.3
52.1
70.9
57.3
90.8
92.7
49.1
40.0
84.8
23.3
ꢀ/
49.4
81.7
71.5
42.1
43.9
90.4
26.2
44.7
75.9
41.6
40.7
11.7
87.1
15.8
ꢀ/
ꢀ/
ꢀ/
86.2
NT d
28.1
42.1
10
13
14
ꢀ/
92.0
36.8
52.4
86.8
35.2
ꢀ/
61.3
a
For subpanel tumour cell lines see footnote (a) of Table 2.
b
c
TGI (mM) full panel mean-graph mid point (MG-MID)ꢃ
/
the average sensitivity of all cell lines towards the test agent.
Subpanel TGI valueꢀ100 mM.
/
d
Not tested.

Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ
/974
965
Table 4
Median lethal concentration 50 (LD50, mM) values of in vitro subpanel tumour cell lines
Cpd. No.
Subpanel tumour cell lines a
MG-MID b
I
II
III
IV
V
VI
VII
VIII
IX
c
7a
7b
7c
9
ꢀ/
85.3
96.6
78.2
92.7
86.0
96.1
79.9
94.5
83.3
95.0
95.4
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
85.0
85.1
95.5
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
76.6
85.5
ꢀ
/
ꢀ/
78.9
ꢀ/
74.1
95.6
ꢀ/
80.9
94.6
ꢀ/
72.5
94.1
ꢀ/
93.3
ꢀ/
79.4
87.1
NT d
86.0
10
13
14
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
45.3
ꢀ/
81.6
ꢀ/
57.9
ꢀ/
68.9
ꢀ/
64.6
69.4
80.3
72.0
82.4
a
For subpanel tumour cell lines see footnote (a) of Table 2.
b
c
TGI (mM) full panel mean-graph mid point (MG-MID)ꢃ
/
the average sensitivity of all cell lines towards the test agent.
Subpanel LC50 valueꢀ100 mM.
/
d
Not tested.
towards most of the tested tumour cell lines (Tables 2
and 4). Compound 13 was found to be the least effective
antitumor agent in the present investigation with GI50
and TGI MG-MID values of 31.6 and 87.1 mM,
MDA-MB-435 cancer cell lines at both the GI50 and
TGI levels.
Structurally, the tested compounds represent two
different main hybrids, namely the 5-(1-(4-chlorophe-
nyl)-4-hydroxy-1H-pyrazol-3-yl)-4-substituted-1,2,4-
respectively; without any cytotoxic effect (Tables 2ꢀ4).
/
triazolin-3-thiones 7aꢀc and the 5-(1-(4-chlorophenyl)-
/
The ratio obtained by dividing the full panel MG-
MID (mM) of the compounds by their individual
subpanel MG-MID (mM) is considered as a measure
of compound selectivity. 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 are rated
non-selective [40]. All the active compounds in the
present study proved to be non-selective with broad
spectrum antitumor activity against the nine tumour
subpanels tested with ratios ranging between 0.02 to
2.83 for the GI50 and 0.15 to 1.36 for the TGI. At the
GI50 level, compounds 7c and 14 revealed mild selectiv-
ity towards the leukaemia subpanel with selectivity
ratios near 3 (2.8 and 2.7, respectively). Regarding the
selectivity of the tested compounds against some of the
individual subpanel tumour cell lines, compound 7a was
found to exhibit moderate selectivity towards leukaemia
SR and breast MDA-MB-435 cancer cell lines at the
GI50 level with selectivity ratios of 3.7 and 5.7,
respectively. This selectivity was retained at the TGI
level for the latter cell line (selectivity ratio 8.9).
Compound 7c showed a significant high selectivity
towards the same subpanel tumour cell lines at the
GI50 level with selectivity ratios of 65.0 and 20.0,
respectively. Moreover, the same compound was proved
to be highly selective towards CNS SF-295, ovarian
OVCAR-3 and breast MCF7 cancer cell lines at the GI50
(selectivity ratio 65.0 for the three cell lines) and TGI
levels (selectivity ratios 102, 133 and 61, respectively).
On the other hand, compounds 11 and 14 revealed high
selectivity ratios ranging between 6.7 and 20.0 towards
CNS SF-539, CNS U251, renal TK-10 and breast
4-hydroxy-1H-pyrazol-3-yl)-2-substituted-1,3,4-oxadia-
zoles 9, 11, 13 and 14 (Figs. 2 and 3). Within the first
series (Fig. 2), the substituent at position 4 of the 1,2,4-
triazolin-3-thione moiety seems to modulate the anti-
tumor activity. The cyclohexyl group (7c, Rꢃcyclohex-
/
yl); was the most favourable substituent among this
series. Thus, 7c is considered to be the most active
member within this series with distinctive antitumor
activities against all the tested subpanel tumour cell lines
(GI50 and TGI MG-MID values of 0.65 and 41.6 mM,
respectively), however without any cytotoxic effect.
Replacement of the alicyclic group with an aromatic
moiety as in 7a (Rꢃ
/
C6H5), resulted in a 2ꢀ5-fold
/
reduction in the growth inhibitory activity against most
of the tested subpanel tumour cell lines, except for the
leukaemia subpanel which showed a dramatic reduction
(82-fold) in activity. The antitumor activities against the
ovarian, renal and breast cancer cell lines were margin-
ally affected (Table 2). However, this compound re-
vealed some cytotoxic activity with LC50 value of 85.1
mM (Table 4). Substituting this phenyl group with a
fluorine atom as in 7b (Rꢃ
full panel activity by 2-fold at both the GI50 and TGI
levels (Tables 2 and 3) and showed 2ꢀ3-fold reduction in
/4-F-C6H4), decreased the
/
activity against most of the tested subpanel tumour cell
lines except for the leukaemia, melanoma and prostate
cancer subpanels where the activity was retained nearly
within the same level (Table 2). The higher growth
inhibitory and cytostatic activities of 7c relative to 7a
and 7b are concordant with some previous reports [41ꢀ
/
43] which have discussed the biological significance of
the cyclohexyl moiety rather than other aromatic
substituents in enhancing the antitumor properties of

966
Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ974
/
such type of 1,2,4-triazolin-3-thiones attached to a
variety of heterocyclic ring systems.
3.2. In vitro effect on the replication of hepatitis-C virus
in HCV-infected HepG2 heptocellular carcinoma cell line
Regarding the 2-substituted-1,3,4-oxadiazoles struc-
ture variants 9, 11, 13 and 14; the antitumor activity of
these compounds appears to be closely related to the
nature of the substituent at position-2 of the 1,3,4-
oxadiazole counterpart. Compound 14; 5-(1-(4-chloro-
phenyl)-4-hydroxy-1H-pyrazol-3-yl)-2-phenyl-1,3,4-ox-
adiazole (Fig. 3); was proved to be the most active
member with a unique antitumor potency against all the
tested subpanel tumour cell lines (GI50, TGI, and LC50
MG-MID values of 0.08, 15.8 and 64.6 mM, respec-
Compounds 2ꢀ
/
5, 6aꢀ
/
d, 7a, 8ꢀ11, 12a, 13, and 14
/
were investigated for their in vitro effect on the
replication of HCV in HepG2 heptocellular carcinoma
cell line infected with the virus. In the last few years, a
number of cell culture systems have been developed that
support reliable and efficient progression of this virus.
Among several human hepatocyte cell lines analysed,
the hepatocellular carcinoma HepG2 cell line was found
to be most susceptible to the HCV infection [44]. On the
other hand, monitoring of the HCV viremia pre- and
tively) (Tables 2ꢀ4). Separation of the phenyl group at
/
post-antiviral therapy through the detection of viral (ꢁ
/)
position 2 from the 1,3,4-oxadiazole counterpart with an
NH linker as in 9 (Fig. 2), resulted in an obvious
reduction in activity (GI50, TGI, and LC50 MG-MID
values of 18.6, 40.7 and 79.4 mM, respectively), against
all the tested subpanel tumour cell lines except for
leukaemia, at both the GI50 and TGI levels (Tables 2
and 3). Here, it should be pointed out that replacing the
1,3,4-oxadiazole with its isosteric 1,3,4-thiadiazole ring
system as in 8 (Fig. 2), led to a total loss of the
antitumor activity. Furthermore, replacing the aromatic
moiety in 14 with an oxo function as in 11; 5-(1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazol-3-yl)-1,3,4-oxa-
diazol-2-one (Fig. 3); led to a highly active analogue
with GI50, TGI, and LC50 MG-MID values of 0.2, 11.7
and 87.1 mM, respectively (Tables 2 and 4). When
and/or (ꢂ) RNA strands by the use of qualitative
/
reverse transcription-polymerase chain reaction (RT-
PCR) has become the most frequently-used, reliable and
sensitive technique. Recently, it has been reported that
the detection of the (ꢂ) strand HCV RNA using the
/
RT-PCR is a very important tool for understanding the
life cycle of the HCV and provides a reliable marker for
the diagnosis of HCV and monitoring the viral response
to antiviral therapy [45].
Based on these facts, the adopted protocol in the
present study contributes to the simultaneous detection
of the (ꢁ
/
) and/or (ꢂ) HCV RNA strands in HepG2
/
hepatoma cells infected with HCV. Inhibition of viral
replication was detected by amplification of viral RNA
segments using the RT-PCR technique, both in the
uninfected cultivated cells (as a positive control) and in
the presence of variable concentrations (10, 25, 50 and
100 mg mLꢂ1) of the test compound at optimal
temperature. The test compound is considered to be
active when it is capable of inhibiting the viral replica-
tion inside the HCV-infected HepG2 cells, as evidenced
compared with 14, this analog showed 2ꢀ10-fold
/
improvement in the antitumor activity against most of
the tested subpanel tumour cell lines, except for the
leukaemia and the breast cancer subpanels which
showed 3- and 25-fold reduction in activity, respectively.
It is worth mentioning that, isosteric replacement of the
2-oxo with 2-thioxo function as in 10 (Fig. 3), led to
complete abolishment of the antitumor activity. On the
other hand, the 3-acetyl-5-(4-acetyloxy-1-(4-chlorophe-
nyl)-1H-pyrazol-3-yl)-2-(2,4-difluorophenyl)-1,3,4-oxa-
diazoline 13 (Fig. 3); proved to be the least active
member among the substituted 1H-pyrazolyl-1,3,4-ox-
adiazole analogs with GI50 and TGI MG-MID values of
31.6 and 87.1 mM, respectively and without any
by the disappearance of the (ꢁ
/
) and/or (ꢂ) strands
/
viral RNA-amplified products detected by the RT-PCR
(compared with the positive control).
Out of the compounds tested, only two derivatives
namely; the N-formyl-1-(4-chlorophenyl)-4-hydroxy-
1H-pyrazole-3-carboxylic acid hydrazide 2 and 1-(1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazole-3-carbonyl)-
thiosemicarbazide 5 proved to inhibit the viral replica-
tion inside HCV-infected hepatoma HepG2 cell line as
revealed from their corresponding gel pictures illu-
strated in Figs. 4 and 5. The N-formyl derivative 2
cytotoxic activity (Tables 2ꢀ4). Such reduction in the
/
biological activity might be attributed to the acetylation
of the 4-hydroxyl group of the 1H-pyrazole as well as
the 1,3,4-oxadiazoline ring which would block an active
hydrogen-bond forming centre in the molecule. This
suggestion was intensified by the fact that the diacetyl
pyrazole derivative 3 (Fig. 2) proved to be totally
inactive in vitro under the same experimental condi-
tions. Collectively, these results highlight the synergestic
role of the 4-hydroxy-1H-pyrazole nucleus together with
the substitution pattern of the 1,3,4-oxadiazole counter-
part in manipulating the enhanced biological activity of
such model of compounds.
was able to inhibit the replication of the (ꢁ) strand
/
HCV RNA at a dose of 100 mg mLꢂ1 as indicated by
the absence of the amplified PCR products at lane 5
(Fig. 4). On the other hand, the thiosemicarbazide 5
exhibited a distinctive HCV inhibitory profile as re-
vealed from Fig. 5, where no (ꢁ
/
) or (ꢂ) viral RNA
/
products were detected at lower doses (25 and 50 mg
mLꢂ1). At the 10 mg mLꢂ1 dose level, the compound
inhibited the replication of the (ꢁ
/
) strand HCV RNA
rather than the (ꢂ) strand. It is worth mentioning that
/

Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ
/974
967
the substituted thiosemicarbazides 6aꢀ
/
d and the N-
phenylsemicarbazide 4 failed to exhibit any antiviral
activity under the same test conditions and dose level
used.
4. Conclusion
In conclusion, the aim of the present research work
was to synthesize novel 1H-pyrazole lead compounds
possessing potential antitumor as well as anti-HCV
activities and are structurally related to the natural 4-
hydroxypyrazole; pyrazofurin. Our aim has been
achieved by the synthesis of two different groups of
structure hybrids comprising the 4-hydroxy-1H-prya-
zole moiety with either the 4-substituted-1,2,4-triazolin-
3-thione (compounds 7aꢀc) or the 2-substituted-1,3,4-
/
oxadiazole (compounds 9, 11, 13 and 14) counterparts in
one and the same entity for synergistic purpose. The
most active seven compounds at the GI50 level exhibited
TGI levels with nearly the same order of activity; 14ꢀ
/
11ꢀ7cꢀ7aꢀ7b:9ꢀ13 (Tables 2 and 3). Com-
/
/
/
/
/
pounds 11 and 14 proved to be the most active
derivatives identified in this study as evidenced by their
potential growth inhibitory, cytostatic and cytotoxic
activities, regardless to the subpanel being tested, with
special high selectivity profile against some individual
cell lines. Compounds 11 and 7c were selected by the
NCI Developmental Therapeutic Program for further in
vitro biological evaluation. Interestingly, compound 14
is currently under review by the NCI Biological Evalua-
tion Committee at the last stage of the in vitro screening.
On the other hand, compounds 2 and 5 revealed
potential inhibitory effect on the replication of the
Fig. 4. PCR amplification picture of HCV RNA (ꢁ
in the presence of compound 2. Lane 1 is the molecular weight marker
(MWM) 50bp ladder. Lane 2ꢃcontrol (C). Lanes 3, 5, 7, 9 are the
effect of compound 2 (10, 25, 50, 100 mg, respectively) on the HCV
RNA (ꢁ) strand. Lanes 4, 6, 8, 10 are the effect of compound 2 (10,
25, 50, 100 mg, respectively) on the HCV RNA (ꢂ) strand.
/
) and (ꢂ) strands
/
/
/
/
HCV RNA (ꢁ
/
) and (ꢂ) strands in HepG2 hepatocel-
/
lular carcinoma cell line infected with the HCV at 10ꢀ
/
100 mg mLꢂ1 concentration range in the RT-PCR
experimental protocol. All these favourable features
make such type of 4-hydroxypyrazole derivatives the
appropriate candidates for future testing and derivatisa-
tion in the hope of finding more selective and active
anticancer and antiviral compounds in the nanomolar
concentration level or less.
5. Experimental
5.1. Chemistry
Melting points were determined in open-glass capil-
laries on a Stuart melting point apparatus and were
uncorrected. The infrared (IR) spectra were recorded on
470-Shimadzu infrared spectrophotometer using the
KBr disc technique. The 1H-NMR (d-ppm) spectra
were recorded on a Bruker (500 MHz) spectrometer
using tetramethylsilane as the internal standard and
Fig. 5. PCR amplification picture of HCV RNA (ꢁ
in the presence of compound 5. Lane 1 is the molecular weight marker
(MWM) 50bp ladder. Lane 2ꢃcontrol (C). Lanes 3, 5, 7, 9 are the
effect of compound 5 (10, 25, 50, 100 mg, respectively) on the HCV
RNA (ꢁ) strand. Lanes 4, 6, 8, 10 are the effect of compound 5 (10,
25, 50, 100 mg, respectively) on the HCV RNA (ꢂ) strand.
/
) and (ꢂ) strands
/
/
/
/

968
Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ974
/
Table 5
Physical and analytical data of compounds 2ꢀ14
/

Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ
/
974
969

970
Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ974
/
DMSO-d6 as the solvent. Splitting patterns were desig-
nated as follows: s; singlet; d: doublet; m: multiplet.
Mass spectra were recorded on a Finnigan SSQ 7000
2.5 mmol). The reaction mixture was refluxed for 1 h,
then allowed to attain room temperature. The separated
solid was filtered, washed with cold ethanol and
recrystallised. Physical and analytical data are listed in
GCꢀMS, ionisation energy 70 eV. Elemental analyses
/
were performed at the Microanalytical Unit, Faculty of
Science, Cairo University, Cairo, Egypt, and the found
Table 5. IR (cmꢂ1): 3670ꢀ
1617 (CÄO), 1578, 1553, 1496 (CÄ
aromatics), 827 (CÃ
NH), 8.2 (s, 1H, pyrazole-C5-H), 8.0ꢀ
H).
/
2860 (OH, NH), 1729 (CÄ
C, CÄN, amide II,
Cl). H-NMR: d 9.3ꢀ9.2 (m, 3H,
7.26 (m, 9H, Ar-
/
O),
/
/
/
1
values were within 9
/
0.4% of the theoretical values.
/
/
Follow up of the reactions and checking the homo-
geneity of the compounds were made by TLC on silica
gel-protected aluminium sheets (Type 60 F254, Merck)
and the spots were detected by exposure to UV-lamp at
l 254 nm for a few seconds. Compound 1 was
synthesized as described in Ref. [18].
/
5.1.4. 1-(1-(4-Chlorophenyl)-4-hydroxy-1H-pyrazole-3-
carbonyl)-thiosemicarbazide (5)
A mixture of 1 (0.5 g, 2 mmol) and ammonium
thiocyanate (0.45 g, 6 mmol) in hydrochloric acid (20%,
20 mL) was heated under reflux for 3h. The reaction
mixture was concentrated, cooled and the separated
solid was filtered, washed with water, dried and
recrystallised. Physical and analytical data are listed in
5.1.1. N-Formyl-1-(4-chlorophenyl)-4-hydroxy-1H-
pyrazole-3-carboxylic acid hydrazide (2)
A solution of 1-(4-chlorophenyl)-4-hydroxy-1H-pyr-
azole-3-carboxylic acid hydrazide 1 (0.5 g, 2 mmol) in
formic acid (5 mL) was heated under reflux for 1 h,
during which a yellow solid was partially crystallized
out. After being cooled to room temperature, the
product was filtered, washed and recrystallised. Physical
and analytical data are recorded in Table 5. IR (cmꢂ1):
Table 5. IR (cmꢂ1): 3375ꢀ
1593, 1571, 1531, 1499, 1460 (CÄ
aromatics), 994 (NCS), 824 (CÃ
Cl). 1H-NMR: d 9.95 (s,
1H, CONH), 9.3 (s, 1H, CSNH), 8.1 (s, 1H, pyrazoleÃ
C5ÃH), 7.75ꢀ7.3 (m, 6H, ArÃH and CSNH2).
/
2655 (OH, NH), 1667 (CÄ
/
O),
/
C, CÄN, amide II,
/
/
/
/
/
/
3145ꢀ
O amide), 1593, 1496, 1377 (CÄ
aromatics), 829 (CÃ
Cl). 1H-NMR: d 10.12 (s, 1H, NH),
8.1 (s, 1H, pyrazoleÃC5ÃH), 8.0 (s, 1H, CHO), 7.91 (d,
H). MS, m/z (%): 282.0
/
2930 (OH, NH), 1667 (CÄ
/
O aldehyde), 1630 (CÄ
/
/
C, CÄN, amide II,
/
5.1.5. 4-Substituted-1-(1-(4-chlorophenyl)-4-hydroxy-
1H-pyrazole-3-carbonyl)thiosemicarbazides (6aꢀd)
To a solution of the acid hydrazide 1 (1 g, 4 mmol) in
/
/
/
/
2H, ArÃ
/
H), 7.55 (d, 2H, ArÃ
/
ethanol (20 mL) was added the appropriate isothiocya-
(5.5), 280.0 (16.9, Mꢁ), 254.0 (5.6), 252.0 (16.9), 223.0
(33.1), 221.0 (100), 140.0 (16.4), 137.9 (49.6), 112.9
(16.8), 111.0 (53.4), 77.0 (5.0), 75.0 (25.2).
nate (5 mmol). The reaction mixture was refluxed for 6ꢀ
/
8 h, then allowed to attain room temperature. The
separated solid was filtered, washed with cold ethanol
and recrystallised. Physical and analytical data are listed
5.1.2. N-Acetyl-4-acetyloxy-1-(4-chlorophenyl)-1H-
pyrazole-3-carboxylic acid hydrazide (3)
The acid hydrazide 1 (0.5 g, 2 mmol) was warmed
with acetic anhydride (5 mL) for 1 h, then the mixture
was allowed to attain room temperature. The deposited
in Table 5. IR (cmꢂ1): 3470ꢀ
O), 1593, 1555, 1531, 1494, 1460 (CÄ
aromatics), 982ꢀ958 (NCS), 850ꢀ829 (Cꢀ
of 6c: d 9.69ꢀ9.3 (m, 2H, NH), 8.1 (s, 1H, pyrazoleÃ
C5ÃH), 7.96 (d, 2H, ArÃH), 7.9 (s, 1H, NH), 7.65 (d,
2H, ArÃH), 4.1 (s, 1H, cyclohexylÃH), 1.79ꢀ1.03 (m,
10H, cyclohexylÃH).
/
2935 (OH, NH), 1665 (CÄ
C, CÄN, amide II,
Cl). 1H-NMR
/
/
/
/
/
/
/
/
/
/
yellow solid was filtered, washed with pet. ether (60ꢀ
80 8C) and recrystallised. Physical and analytical data
are listed in Table 5. IR (cmꢂ1): 3550ꢀ
3105 (NH), 1758
(CÄO acetyl), 1734 (CÄO acetyl), 1667 (CÄO amide),
1655, 1544, 1497, 1475, 1427 (CÄC, CÄN, amide II,
aromatics), 1252 (CÃOÃC), 831 (CÃ
8.8 (s, 1H, pyrazoleÃC5ÃH), 7.81 (d, 2H, ArÃ
(d, 2H, ArÃH), 2.56 (s, 3H, CH3), 2.34 (s, 3H, CH3).
/
/
/
/
/
/
/
/
/
5.1.6. 5-(1-(4-Chlorophenyl)-4-hydroxy-1H-pyrazol-3-
yl)-4-substituted-1,2,4-triazolin-3-thiones (7aꢀc)
The appropriate 4-substituted-1-(1-(4-chlorophenyl)-
/
/
/
1
/
/
/
Cl). H-NMR: d
H), 7.63
/
/
/
4-hydroxy-1H-pyrazole-3-carbonyl)-thiosemicarbazide
/
6aꢀc (1 mmol) in sodium hydroxide (2N, 10 mL) was
/
MS, m/z (%): 339.1 (6.6), 338.0 (15.9), 337.1 (20.8),
336.0 (41.1, Mꢁ), 296.0 (29.9), 294.0 (79.4), 281.0 (20.3),
279.0 (62.4), 265.0 (29.8), 263 (80.4), 254.0 (34.4), 252.0
(89.8), 223.0 (59.4), 220.9 (100), 139.9 (19.1), 137.9
(54.9), 127.0 (37.1), 125.0 (83.9), 112.0 (14.3), 110.0
(44.6), 101.0 (7.6), 99.0 (22.6), 77.0 (2.0), 75.0 (16.7).
heated under reflux for 4 h, cooled, neutralized to pH 6
using dilute hydrochloric acid. The separated solid
product was filtered, washed with water, dried and
recrystallised. Physical and analytical data are listed in
Table 5. IR (cmꢂ1): 3530ꢀ
1567, 1496 (CÄC, CÄN, aromatics), 1375ꢀ
834ꢀ824 (CÃCl). H-NMR of 7a: d 14.12 (s, 1H, OH),
9.31 (s, 1H, NH), 8.0 (s, 1H, pyrazole-C5-H), 7.92ꢀ7.33
/2800 (OH, NH), 1618, 1591,
/
/
/1317 (NCS),
1
/
/
5.1.3. 4-Phenyl-1-(1-(4-chlorophenyl)-4-hydroxy-1H-
pyrazole-3-carbonyl)semicarbazide (4)
To a solution of the acid hydrazide 1 (0.5 g, 2 mmol)
in ethanol (10 mL), was added phenyl isocyanate (0.3 g,
/
(m, 10H, NH and Ar-H). MS, m/z (%) of 7c: 377.2
(14.7), 375.2 (38.3, Mꢁ), 296.0 (11.2), 295.0 (38.8), 294.1
(30.7), 293.0 (100), 222.0 (2.3), 220.0 (6.3), 169.0 (5.9),

Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ
/974
971
167.0 (18.0), 141.0 (12.5), 140.0 (29.1), 139.0 (37.7),
138.0 (73.4), 132.0 (26.8), 129.0 (20.2), 128.0 (67.5),
127.0 (76.2), 112.9 (17.1), 111.0 (52.3), 83.1 (3.5), 81.1
(12.2), 77.0 (10.3), 75.0 (22.6).
5.1.9. 5-(1-(4-Chlorophenyl)-4-hydroxy-1H-pyrazol-3-
yl)-1,3,4-oxadiazolin-2-thione (10)
The acid hydrazide 1 (0.5 g, 2 mmol) was dissolved in
a solution of potassium hydroxide (0.11 g, 2 mmol) in
water (2 mL) and ethanol (20 mL). Carbon disulfide (2
mL) was then added while stirring and the reaction
mixture was heated under reflux for 8 h. The solvents
were removed under reduced pressure, the residue was
treated with water then filtered. The filtrate was cooled,
neutralized to pH 6 using dilute hydrochloric acid and
the separated product was filtered, washed with water,
dried and recrystallised. Physical and analytical data are
5.1.7. 5-(1-(4-Chlorophenyl)-4-hydroxy-1H-pyrazol-3-
yl)-2-phenylamino-1,3,4-thiadiazole (8)
To an ice-cold stirred solution of 1-(1-(4-chlorophe-
nyl)-4-hydroxy-1H-pyrazole-3-carbonyl)-4-phenylthio-
semicarbazide 6a (0.5 g, 1.3 mmol) in absolute ethanol
(10 mL), sulphuric acid (5 mL) was carefully added over
a period of 15 min. Stirring was maintained at room
temperature for 4 h then the reaction mixture was
poured carefully to an equal volume of ice water. The
precipitate formed was filtered, washed with water,
dried and recrystallised. Physical and analytical data
listed in Table 5. IR (cmꢂ1): 3555ꢀ
1628, 1545, 1495 (CÄC, CÄN, aromatics), 1374 (CÄ
Cl). H-NMR: d 10.4 (s, 1H, OH), 9.66 (s, 1H,
NH), 8.2 (s, 1H, pyrazoleÃC5ÃH), 7.76ꢀ7.23 (m, 4H,
ArÃ
H). MS, m/z (%): 296.2 (9.2), 294.1 (25.2, Mꢁ),
/3000 (OH, NH),
/
/
/S),
1
823 (CÃ
/
/
/
/
/
239.1 (4.3), 238.1 (23.8), 237.1 (9.8), 236.1 (81.2), 224.2
(2.5), 223.1 (14.5), 222.1 (5.4), 221.1 (42.6), 149.0 (13.7),
141.1 (4.5), 140.0 (16.8), 139.1 (10.3), 138.1 (47.5), 129.1
(19.7), 128.1 (19.0), 127.1 (58.2), 126.1 (40.6), 125.1
(100), 114.1 (4.1), 113.0 (25.8), 112.0 (11.4), 111.0 (77.8),
105.1 (28.9), 101.0 (20.8), 99.0 (59.5), 97.1 (30.1), 93.0
(6.8), 92.0 (22.7), 91.0 (18.3), 77.0 (33.8), 75.0 (57.6).
are listed in Table 5. IR (cmꢂ1): 3380ꢀ
1653, 1599, 1552, 1496 (CÄC, CÄN, aromatics), 831 (CÃ
Cl). H-NMR: d 10.13 (s, 1H, OH), 9.77 (s, 1H, NH),
8.12 (s, 1H, pyrazoleÃC5ÃH), 7.93 (d, 2H, ArÃH), 7.58
(d, 2H, ArÃH), 7.49ꢀ7.14 (m, 5H, ArÃH). MS, m/z
/
3145 (OH, NH),
/
/
/
1
/
/
/
/
/
/
(%): 369.1 (2.0, Mꢁ), 296.0 (2.1), 294.0 (5.7), 254.0
(19.1), 252.0 (56.5), 224.0 (4.5), 223.0 (40.5), 222.0
(14.2), 221.0 (100), 140.0 (12.5), 139.0 (37.7), 137.9
(73.8), 135.0 (79.6), 112.9 (12.5), 111.0 (38.9), 93.0
(14.6), 77.0 (47.0), 75.0 (20.5).
5.1.10. 5-(1-(4-Chlorophenyl)-4-hydroxy-1H-pyrazol-3-
yl)-1,3,4-oxadiazolin-2-one (11)
To an ice-cold stirred solution of the acid hydrazide 1
(0.5 g, 2 mmol) and triethylamine (0.5 mL) in tetra-
hydrofuran (30 mL) was added 1,1?-carbonyldiimidazole
(CDI) (0.48 g, 3 mmol). Stirring at 0 8C was continued
for 5 h then the reaction mixture was treated with
additional amounts of triethylamine (0.5 mL) and CDI
(0.48 g, 3 mmol), then stirring was maintained at room
temperature for an overnight. The solvent was evapo-
rated to dryness under reduced pressure, the residue was
treated with ethanol then filtered. The resulting pre-
cipitate was washed with ethanol, dried and recrystal-
lised. Physical and analytical data are listed in Table 5.
5.1.8. 5-(1-(4-Chlorophenyl)-4-hydroxy-1H-pyrazol-3-
yl)-2-phenylamino-1,3,4-oxadiazole (9)
Finely-powdered yellow mercuric oxide (0.4 g, 1.8
mmol) was, portionwise, added over a period of 30 min
to a boiling solution of 1-(1-(4-chlorophenyl)-4-hy-
droxy-1H-pyrazole-3-carbonyl)-4-phenylthiosemicarba-
zide 6a (0.5 g, 1.3 mmol) in absolute ethanol (20 mL).
The resulting suspension was heated under reflux for 2
h, then filtered while hot. The black precipitate was
washed three times with boiling ethanol (3ꢄ
/
10 mL) and
IR (cmꢂ1): 3650ꢀ
1631, 1590, 1495 (CÄ
/
2995 (OH, NH), 1778 (CÄ
C, CÄN, aromatics), 1250 (CÃ
Cl). H-NMR: d 10.3 (s, 1H, OH), 9.7 (s,
1H, NH), 8.1 (s, 1H, pyrazoleÃC5ÃH), 7.82ꢀ7.33 (m,
4H, ArÃ
H). MS, m/z (%): 280.1 (5.5), 278.0 (17.1, Mꢁ),
/
O), 1665,
the combined filtrate and washings were concentrated to
a small volume and set aside for an overnight at room
temperature. The separated product was filtered, dried
and recrystallised. Physical and analytical data are listed
/
/
/
OÃ
/
1
C), 823 (CÃ
/
/
/
/
/
in Table 5. IR (cmꢂ1): 3320ꢀ
1593, 1552 (CÄC, CÄN, aromatics), 830 (CÃ
NMR: d 10.0 (s, 1H, OH), 9.47 (s, 1H, NH), 8.1 (s, 1H,
H), 7.83ꢀ H). MS, m/z
/
3120 (OH, NH), 1630,
254.1 (3.8), 252.1 (12.2), 239.1 (2.1), 238.2 (5.9), 237.1
(6.8), 236.0 (14.4), 224.1 (3.2), 223.1 (25.1), 222.0 (12.6),
221.0 (84.3), 141.0 (11.5), 140.0 (40.0), 139.0 (27.2),
138.0 (97.9), 129.0 (6.6), 128.0 (6.5), 127.0 (18.0), 126.0
(18.3), 125.0 (34.6), 114.0 (5.5), 112.9 (40.7), 112.0
(18.5), 111.0 (100), 101.0 (9.0), 99.0 (16.6), 77.0 (14.3),
75.0 (55.0).
1
/
/
/
Cl). H-
pyrazoleÃ
/
C5Ã
/
/
7.11 (m, 9H, ArÃ
/
(%): 355.1 (34.1), 353.1 (100, Mꢁ), 254.0 (8.1), 252.0
(25.9), 224.0 (2.6), 223.0 (21.9), 222.0 (9.2), 221.0 (73.1),
220.0 (3.4), 219.0 (14.9), 188.0 (14.8), 187.0 (46.0), 169.0
(5.6), 166.9 (17.1), 141.0 (13.9), 140.0 (34.1), 138.9
(42.6), 138.0 (97.9), 134.9 (47.9), 132.0 (31.3), 118.0
(20.4), 117.0 (15.9), 112.9 (27.6), 112.0 (11.4), 110.9
(85.4), 93.0 (19.4), 92.0 (22.7), 91.0 (10.5), 77.0 (82.3),
75.0 (41.5).
5.1.11. 3-(Alkylidenehydrazinocarbonyl)-1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazoles (12aꢀe)
/
A solution of 1 (0.5 g, 2 mmol) and the appropriate
aldehyde or ketone (2 mmol) in gl. acetic acid (10 mL)

972
Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ974
/
was refluxed for 6ꢀ
/
8 h. The solid separated upon
(15.0), 172.1 (58.2), 169.1 (6.7), 167.1 (15.2), 141.1
cooling was filtered, washed with cold ethanol and
recrystallised. Physical and analytical data are listed in
(13.7), 140.1 (28.6), 139.1 (39.7), 138.1 (79.0), 132.1
(26.8), 114.0 (3.6), 113.0 (22.6), 112.0 (9.4), 111.0 (69.0),
105.1 (48.1), 103.1 (17.6), 78.1 (5.1), 77.1 (68.6), 76.1
(15.6), 75.1 (34.7).
Table 5. IR (cmꢂ1): 3540ꢀ
(CÄ
amide II, aromatics), 840ꢀ
d 9.99 (s, 1H, OH), 9.7 (s, 1H, NH), 8.11 (s, 1H,
pyrazole-C5-H), 7.9ꢀ755 (m, 9H, Ar-H), 3.29 (m, 4H,
CH2 Ã CH2), 1.9 (s, 3H, CH3). MS, m/z (%) of 12a:
/
2775 (OH, NH), 1689ꢀ
O), 1638, 1612, 1591, 1561, 1532, 1494, (CÄC, CÄ
821 (CÃ
Cl). 1H-NMR of 12e:
/
1649
/
/
/N,
/
/
5.2. In vitro antitumor screening
/
/
Compounds 3, 7aꢀ
/
c, 8ꢀ11, 13 and 14 were subjected
/
378.1 (12.1), 376.1 (30.0, Mꢁ), 239.0 (6.7), 237.0 (19.4),
224.0 (4.7), 223.0 (41.6), 222.0 (26.3), 221.0 (100), 141.0
(9.0), 140.0 (45.0), 139.0 (23.5), 138.0 (85.5), 128.0 (2.8),
127.0 (2.7), 126.0 (12.1), 125.0 (13.1), 114.0 (4.3), 112.9
(31.3), 111.9 (9.2), 110.9 (68.3), 101.0 (6.8), 100.0 (17.1),
77.0 (6.9), 75.0 (28.2).
to the NCI in vitro disease-oriented human cells screen-
ing panel assay to screen their antitumor activities.
About 60 cell lines of nine tumour subpanels, including
leukaemia, non-small cell lung, colon, CNS, melanoma,
ovarian, renal, prostate and breast cancer cell lines were
utilized. The human tumour cell lines of the cancer
screening panel were grown in RPMI 1640 medium
5.1.12. 3-Acetyl-5-(4-acetyloxy-1-(4-chlorophenyl)-1H-
pyrazol-3-yl)-2-(2,4-difluorophenyl)-1,3,4-oxadiazoline
(13)
containing 5% foetal 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 8C, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experi-
mental drugs. After 24 h, two plates of each cell line
were fixed in situ with TCA, to represent a measurement
of the cell population for each cell line at the time of
drug addition. Experimental drugs were solubilised in
dimethyl sulphoxide at 400-fold the desired final max-
imum test concentration and stored frozen 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 med-
ium containing 50 mg mLꢂ1 gentamicin. Additional
four, 10-fold or 1/2 log serial dilutions 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 appropriate microtiter wells already
containing 100 mL of medium, resulting in the required
final drug concentrations. Following drug addition, the
plates were incubated for an additional 48 h at 37 8C,
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 8C. The supernatant was discarded, and the plates
were washed five times with tap water and air dried.
Sulphorhodamine 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 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 solubilised with 10
mM trizma base, and the absorbance was read on an
automated plate reader at a wavelength of 515 nm. For
A mixture of 3-(2,4-difluorophenylidenehydrazino-
carbonyl)-1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole
12a (0.4 g, 1 mmol) and acetic anhydride (5 mL) was
heated under reflux for 1 h. After the reaction mixture
was attained room temperature, excess acetic anhydride
was decomposed by water and the mixture was stirred
for further 30 min. The separated product was filtered,
washed with water, dried and recrystallised. Physical
and analytical data are listed in Table 5. IR (cmꢂ1):
1765 (CÄ
aromatics), 1210 (CÃ
8.8 (s, 1H, pyrazoleÃC5Ã
7.1 (s, 1H, oxadiazoleÃC2Ã
/
O), 1660 (CÄ
OÃ
H), 7.87ꢀ
H), 2.3 (s, 3H, CH3), 2.21 (s,
/
O), 1616, 1499, 1439 (CÄ
/
C, CÄ
Cl). H-NMR: d
7.33 (m, 7H, ArÃH),
/
N,
1
/
/
C), 821 (CÃ
/
/
/
/
/
/
/
3H, CH3). MS, m/z (%): 462.1 (4.0), 460.1 (12.2, Mꢁ),
378.1 (7.3), 376.1 (21.2), 265.0 (11.0), 263.0 (33.6), 239.0
(6.9), 237.0 (21.2), 224.0 (3.6), 223.0 (33.0), 222.0 (18.7),
221.0 (100), 141.0 (6.2), 140.0 (21.4), 139.0 (9.8), 138.0
(46.0), 112.9 (12.6), 111.0 (32.1), 100.0 (10.6), 75.0
(10.9).
5.1.13. 5-(1-(4-Chlorophenyl)-4-hydroxy-1H-pyrazol-3-
yl)-2-phenyl-1,3,4-oxadiazole (14)
To a cooled mixture of equimolar amounts of the acid
hydrazide 1 (0.5 g, 2 mmol) and benzoic acid (0.24 g, 2
mmol) was added phosphorous oxychloride (5 mL)
while stirring. The reaction mixture was then heated
under reflux for 2 h, cooled, poured carefully to an equal
volume of ice water then neutralized with solid sodium
bicarbonate. The precipitate formed after standing for 1
h was filtered, washed with water, dried and recrystal-
lised. Physical and analytical data are listed in Table 5.
IR (cmꢂ1): 3450ꢀ
(CÄC, CÄN, aromatics), 1111 (CÃ
1H-NMR: d 9.8 (s, 1H, OH), 8.2 (s, 1H, pyrazoleÃ
/
3015 (OH), 1657, 1560, 1495, 1458
OÃC), 825 (CÃCl).
C5Ã
H). MS, m/z (%): 340.2
/
/
/
/
/
/
/
H), 7.79ꢀ
/
7.18 (m, 9H, ArÃ
/
(33.9), 338.2 (100, Mꢁ), 254.1 (12.9), 252.0 (26.6), 224.1
(2.7), 223.1 (21.6), 222.1 (10.6), 221.1 (64.1), 173.1

Sh.A.F. Rostom et al. / European Journal of Medicinal Chemistry 38 (2003) 959ꢀ
/974
973
suspension cells, the methodology was the same except
that the assay was terminated by fixing settled cells at
the bottom of the wells by gently adding 50 mL of 80%
TCA (final concentration, 16% TCA). Three response
parameters (GI50, TGI, and LC50) were calculated for
Go RT-PCR beads (Pharmacia Amersham Biotech,
USA), 10 mM from each of the RT downstream primer,
PCR forward primer and reverse primer P2. The
thermal cycling protocol was manipulated as follows:
30 min at 42 8C for cDNA synthesis followed by 5 min
at 95 8C and 30 cycles of 1 min at 94 8C, 1 min at 55 8C
and 1 min at 72 8C. The nested PCR amplification was
performed in 50 mL reaction mixture containing 0.2 mM
from each dNTP, 10 mM from each of the reverse nested
primer and the forward nested primer, two units of taq
DNA polymerase (Promega, USA), 10 mL from the RT-
PCR reaction in a 1X buffer supplied by Vendor. A
fragment of 174 bp length was identified in positive
samples.
each cell line [37ꢀ39].
/
5.3. In vitro effect on the replication of hepatitis-C virus
in HCV-infected HepG2 heptocellular carcinoma cell line
5.3.1. Cell culture
HepG2 cells were washed twice in EMEM (Bio
Whittaker, USA) media supplemented with 200 mM
L-
glutamine (Bio Whittaker), 100U Penicillin, 100 mg
streptomycin (Bio Whittaker), and 25 mM HEPES
buffer; N-[2-hydroxyethyl]piperazine-N?-[2-ethanesulfo-
nic acid] (Bio Whittaker). The cells were suspended at
Acknowledgements
2ꢄ
105 cells mLꢂ1 in EMEM Culture media (EMEM
/
supplemented media, 10% foetal bovine serum (FBS);
Bio Whittaker) then cells were left to adhere on
polystyrene 6-well plates for 24 h in 37 8C, 5% CO2,
95% humidity incubator. The cells were washed twice
from debris and dead cells using EMEM supplemented
media, then infected with 2% HCV-infected serum in
EMEM culture medium with 8% FBS. Each of the
tested compounds was added at concentrations of 10,
25, 50 and 100 mg mLꢂ1. Positive and negative control
cultures were included. After 96 h incubation in 37 8C,
5% CO2, 95% humidity incubator, another dose of the
test compound was added. The cells were further
incubated for another 96 h at 37 8C, 5% CO2, 95%
humidity, then the RNA was extracted. The positive
strand and its replicating form (negative strand) of HCV
were detected by RT-PCR using HCV specific primers
to the 5?-untranslated region of the virus.
The authors are very grateful to the staff members of
the Department of Health and Human Services, Na-
tional Cancer Institute (NCI), Bethesda, Maryland,
USA for carrying out the anticancer screening of the
newly synthesized compounds. Extendable thanks are
due to Dr. Sherif El-Kafrawy for his assistance in the
standardization of the anti-HCV assay.
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