Polysubstituted pyrazoles, part 6. Synthesis of some 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-3-carbonyl derivatives linked to nitrogenous heterocyclic ring systems as potential antitumor agents

Article abstract of DOI:10.1016/j.bmc.2010.02.006

The synthesis of two novel series of 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazoles linked to either polysubstituted 1H-pyrazole counterparts through a carbonyl bridge, or to some biologically-active nitrogenous heterocycles by an amide linker, is described. 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. The most active six compounds 2, 3, 6, 7, 13 and 14 revealed a significant broad spectrum of antitumor potential against most of the tested subpanel tumor cell lines at the GI₅₀ and TGI levels, together with a mild cytotoxic (LC₅₀) activity. The pyrazolinedione analog 7 displayed remarkable growth inhibition and cytostatic effects (GI₅₀ and TGI MG-MID values 0.67 and 53.8 μM, respectively). Compounds 13 (GI₅₀, TGI, and LC₅₀ MG-MID values 0.08, 30.9 and 93.3 μM) and 14 (GI₅₀, TGI, and LC₅₀ MG-MID values 0.36, 8.78 and 69.3 μM, respectively) proved to be the most active antitumor members identified in this study.

Full text of DOI:10.1016/j.bmc.2010.02.006

Bioorganic & Medicinal Chemistry 18 (2010) 2767–2776
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Polysubstituted pyrazoles, part 6. Synthesis of some 1-(4-chlorophenyl)-4-
hydroxy-1H-pyrazol-3-carbonyl derivatives linked to nitrogenous heterocyclic
ring systems as potential antitumor agents q,qq
*
Sherif A. F. Rostom
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdul-Aziz University, PO Box 80260, Jeddah 21589, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of two novel series of 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazoles linked to either poly-
substituted 1H-pyrazole counterparts through a carbonyl bridge, or to some biologically-active nitroge-
nous heterocycles by an amide linker, is described. Ten of the newly synthesized compounds were
selected by the National Cancer Institute (NCI) in vitro disease-oriented antitumor screening to be eval-
uated for their antitumor activity. The most active six compounds 2, 3, 6, 7, 13 and 14 revealed a signif-
icant broad spectrum of antitumor potential against most of the tested subpanel tumor cell lines at the
GI50 and TGI levels, together with a mild cytotoxic (LC50) activity. The pyrazolinedione analog 7 displayed
Received 24 December 2009
Revised 2 February 2010
Accepted 4 February 2010
Available online 10 February 2010
Keywords:
Antitumor activity
1H-Pyrazoles
Acid hydrazide derivatives
Indole
remarkable growth inhibition and cytostatic effects (GI50 and TGI MG-MID values 0.67 and 53.8
lM,
respectively). Compounds 13 (GI50, TGI, and LC50 MG-MID values 0.08, 30.9 and 93.3 M) and 14 (GI50
l
,
TGI, and LC50 MG-MID values 0.36, 8.78 and 69.3
mor members identified in this study.
l
M, respectively) proved to be the most active antitu-
Isoindole
Quinazoline
Pyrrole, b-Carboline
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
beside their capability to exert remarkable anticancer effects
through inhibiting different types of enzymes that play important
roles in cell division.12–14
Several lines of evidence support the view that chemotherapy
has become one of the most significant treatment modalities in
cancer management. Nevertheless, from early on in the develop-
ment of chemotherapy, it was realized that the window in which
the dose range is both efficacious and safe is small. Consequently,
the principal obstacles to the clinical efficacy of chemotherapy re-
main their possible toxicity to normal tissues of the body, beside
the development of cellular drug resistance especially to conven-
tional anticancer agents.1,2 As a result, the design and discovery
of non-traditional, efficient and safe chemical classes of agents
are prime targets in contemporary medicinal chemistry. Over the
past two decades, pyrazole-containing compounds have received
considerable attention owing to their diverse chemotherapeutic
potentials including versatile antineoplastic activities. Literature
survey revealed that some pyrazoles have been implemented as
antileukemic,3–5 antitumor6–9 and anti-proliferative10,11 agents,
As a part of an ongoing research program devoted to the synthe-
sis and characterization of different heterocyclic ring systems en-
dowed with potential chemotherapeutic activities,15–28 particular
attention has been given to the pronounced anticancer activity of
several new 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole-3-carbox-
ylic acid hydrazide derivatives having the general formula A
(Fig. 1).18,19 According to the protocol of the NCI’s in vitro
disease-oriented antitumor screen unit, Bethesda, Maryland,
USA,29–31 seven analogs exhibited potential antitumor activity, of-
ten associated with high or moderate selectivity for certain human
tumor cell lines. As a consequence, three compounds incorporating
the above-mentioned pyrazole counterpart together with either
1,3,4-oxadiazole or 1,2,4-triazole moieties were further selected
by the NCI’s biological committee for subsequent confirmatory
screening procedures in order to optimize the exposure time to
the agent and to define its possible mechanism of action,32 which
would be useful for determining the necessity of the subsequent
in vivo studies.
q
For part 5: see Ref. 18.
qq
Accepted for presentation in the 8th Saudi International Pharmaceutical
Prompted by the above-mentioned results, it was rationalized
to further optimize this chemical series (4-hydroxy-1H-pyrazoles)
by exploring additional modifications at position 3 of the pyrazole
Conference and Exhibition, Riyadh, Saudi Arabia, April 26–28/2010.
* Tel.: +966507654566; fax: +9662 6400000 22327.
E-mail address: sherifrostom@yahoo.com
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.006

2768
S. A. F. Rostom / Bioorg. Med. Chem. 18 (2010) 2767–2776
R
R
1
O
R
O
N
2
N-Heterocycle
X
N
N
HO
N
HO
N
HO
N
N
H
R
3
N
N
N
N
Cl
Cl
Cl
A
B
C
(X = O or N-R2)
Figure 1.
ring, keeping the same 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole
scaffold responsible for the anticancer profile unchanged. Structure
modifications suggested in the present investigation focussed
mainly on studying the effect of linking various polysubstituted
pyrazole counterparts to position 3 by a carbonyl bridge (Formula
B; Fig. 1). The substitution pattern of the target compounds include
various functionalities that would act as hydrogen-bond forming
centers, such as the carbonyl, amide, amino, cyano, hydroxy and
carbethoxy groups. In addition, variation in the nature and size
of other substituents was also attempted, as it would offer variable
electronic, lipophilic and steric environment that would influence
the targeted biological activity. It was considered also worthwhile
to extend structure variation by attaching some biologically-active
nitrogenous heterocycles such as indole, isoindole, quinazoline,
pyrrole, b-carboline at position 3 of the main pyrazole moiety
through an amide linker (Formula C; Fig. 1). Interest in such substi-
tution profile stemmed from the reported contribution of different
linear and semi-linear fused di- and tricyclic ring systems as a scaf-
fold for DNA intercalation and kinase inhibition.33–35 These combi-
nations were suggested in an attempt to investigate the possible
synergistic influence of such structure hybridizations on the antic-
ipated activity, hoping to discover a new lead structure that would
have a significant antitumor potential at very small concentrations.
lute ethanol, in the presence of anhydrous potassium carbonate
resulted in the formation of the corresponding 5-amino-4-cyano-
1H-pyrazol-1-yl 14 and the 5-amino-4-ethoxycarbonyl-1H-pyra-
zol-1-yl 15 derivatives, respectively. Analogously, when 1 was
reacted with diethyl ethoxymethylenemalonate in refluxing aceto-
nitrile, in the presence of anhydrous potassium carbonate, the tar-
geted 4-ethoxycarbonyl-5-hydroxy-1H-pyrazole analog 16 was
obtained.
On the other hand, Scheme 3 illustrates the preparation of the
target compounds 17–21 in which the 1-(4-chlorophenyl)-4-hy-
droxy-1H-pyrazole scaffold was linked to different nitrogenous
heterocyclic ring systems through a carboxamido functionality.
Thus, the starting acid hydrazide 1 was allowed to react with
maleic anhydride in glacial acetic acid in the presence of anhy-
drous sodium acetate to produce N-(2,4-dioxo-2,5-dihydropyrrol-
1-yl)-1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole-3-carboxamide
17. Whereas, heating 1 with phthalic anhydride in boiling xylene
afforded the N-(1,3-dioxo-1,3-dihydroisoindol-2-yl) analog 18.
Moreover, when 1 was reacted with acetanthranil37 or 3,4-dihy-
dropyrano[3,4-b]indol-1(9H)-one38 in glacial acetic acid, the
corresponding 1-(4-chlorophenyl)-4-hydroxy-N-(2-methyl-4-oxo-
quinazolin-3(4H)-yl)-1H-pyrazole-3-carboxamide 19 and the N-
(3,4-dihydro-1-oxo-1H-pyrido[3,4-b]indol-2(9H)-yl) analogs 20,
respectively, were obtained. Finally, condensing the acid hydrazide
1 with isatin in glacial acetic acid yielded the azomethine deriva-
tive 21.
2. Results and discussion
2.1. Chemistry
2.2. In vitro antitumor activity
The synthesis of the target compounds 2–21 is depicted in
Schemes 1–3. In Scheme 1, the starting compound 1-(4-chloro-
phenyl)-4-hydroxy-1H-pyrazole-3-carboxylic acid hydrazide 136
was allowed to react with ethyl acetoacetate in ethanol to produce
the pyrazolinone derivative 2. Condensing 1 with various substi-
tuted diketones in glacial acetic acid gave rise to the corresponding
3,5-disubstituted pyrazoles 3–6. Whereas, heating the same acid
hydrazide 1 with diethyl malonate at 200 °C afforded the targeted
pyrazolidine-3,5-dione 7. On the other hand, reacting 1 with vari-
ous substituted diethyl malonate in the presence of sodium meth-
oxide resulted in the formation of the 4-substituted-pyrazolidine-
3,5-diones 8–10. Condensing 1 with 2-acetylbutyrolactone in
refluxing bromobenzene afforded the 5-(2-hydroxyethyl)-4-
methyl-3-oxopyrazol-2-yl derivative 11. Analogously, compound
12 was synthesized by direct fusion of 1 with ethyl cyclopenta-
none-2-carboxylate. Whereas, reacting the same starting
compound 1 with 2-acetyl-1-tetralone afforded the targeted 3-
methyl-4,5-dihydronaphtho[1,2-c]pyrazol-2-yl analog 13.
2.2.1. Primary in vitro one-dose assay
Out of the newly synthesized compounds, ten derivatives 2–7,
13–15 and 18; were selected by the National Cancer Institute
(NCI) in vitro disease-oriented human cells screening panel assay
to be evaluated for their in vitro antitumor activity. Effective
one-dose assay has been added to the NCI 60 Cell screen in order
to increase compound throughput and reduce data turnaround
time to suppliers while maintaining efficient identification of ac-
tive compounds.29–31 All the selected compounds were tested ini-
tially at a single high dose (10 lM) in the full NCI 60 cell panel
including leukemia, non-small cell lung, colon, CNS, melanoma,
ovarian, renal, prostate and breast cancer cell lines. Only com-
pounds which satisfy pre-determined threshold inhibition criteria
would progress to the five-dose screen. The threshold inhibition
criteria for progression to the five-dose screen was designed to
efficiently capture compounds with anti-proliferative activity
and is based on careful analysis of historical Development Ther-
apeutic Program (DTP) screening data. The data are reported as a
mean-graph of the percent growth of treated cells, and presented
Referring to Scheme 2, condensation of 1 with ethoxymethyl-
enemalononitrile or ethyl ethoxymethylenecyanoacetate in abso-

S. A. F. Rostom / Bioorg. Med. Chem. 18 (2010) 2767–2776
2769
F3C
O
O
N
O
N
O
N
HO
N
HO
HO
N
N
N
N
R
R
O
N
H
N
N
N
Cl
Cl
Cl
5: R = CH3
6: R = 2-furyl
3: R = CH3
4: R = C6H5
7
iv
iii
ii
O
O
O
N
O
N
R
O
HO
HO
NH2
N
N
N
HO
N
O
N
N
i
H
v
H
N
N
N
Cl
Cl
Cl
2
8: R = C2H5
9: R = n-C4H9
10: R = C6H5
1
vi
viii
vii
O
O
O
O
N
O
HO
N
N
HO
HO
N
N
N
N
N
OH
H
N
N
H
N
N
Cl
Cl
Cl
11
13
12
Scheme 1. Reagents and reaction conditions: (i) CH3COCH2COOC2H5, abs ethanol, reflux 10 h; (ii) CH3COCH2COR, gl. acetic acid, reflux 8–12 h; (iii) CF3COCH2COR, gl. acetic
acid, reflux 8–12 h; (iv) CH2(COOC2H5)2, 200 °C, 2 h; (v) RCH(COOC2H5)2, sod. methoxide, methanol, reflux 18 h; (vi) 2-acetylbutyrolactone, bromobenzene, reflux 6 h; (vii)
ethyl cyclopentanone-2-carboxylate, 200 °C, 2 h; (viii) 2-acetyl-1-tetralone, gl. acetic acid, reflux 3 h.
as percentage growth inhibition (GI%) caused by the test com-
pounds. The obtained results showed that six compounds,
namely; 2, 3, 6, 7, 13 and 14, passed successfully this primary
anticancer assay and were consequently carried over to the
five-dose screen against a panel of about 60 different tumor cell
lines.
to create log concentration-% growth inhibition curves. Three re-
sponse 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 concen-
tration 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.
2.2.2. In vitro full panel (five-dose) 60-cell line assay
About 60 cell lines of nine tumor subpanels, including leuke-
mia, 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

2770
S. A. F. Rostom / Bioorg. Med. Chem. 18 (2010) 2767–2776
O
NH2
HO
N
H
N
N
Cl
1
iii
ii
i
O
HO
H2N
O
H2N
O
N
O
N
O
N
CN
O
HO
O
N
N
HO
N
N
HO
N
N
N
N
N
Cl
Cl
Cl
16
15
14
Scheme 2. Reagents and reaction conditions: (i) ethoxymethylenemalononitril, abs ethanol, potassium carbonate, reflux 6 h; (ii) ethyl ethoxymethylenecyanoacetate, abs
ethanol, potassium carbonate, reflux 8 h; (iii) diethyl ethoxymethylenemalonate, acetonitrile, potassium carbonate, reflux 12 h.
In the present study, the active six compounds; 2, 3, 6, 7, 13 and
14, exhibited potential antitumor activities against most of the
tested subpanel tumor cell lines (GI50, TGI and LC50 values
30.9 and 8.78 lM, respectively (Tables 2 and 3). In addition, with
the exception of compounds 2 and 3, compounds 6, 7, 13 and 14;
exhibited mild cytotoxic efficacy with LC50 (MG-MID) values of
<100
lM). These compounds showed a distinctive pattern of sensi-
97.7, 93.3, 93.3 and 69.3 lM, respectively (Table 3).
tivity against some individual cell lines (Table 1), as well as a broad
spectrum of antitumor activity (Tables 2 and 3).
Further interpretation of the obtained data revealed that, com-
pounds 13 (GI50, TGI, and LC50 MG-MID values 0.08, 30.9 and
Concerning the sensitivity against some individual cell lines
(Table 1), compound 2 displayed a distinguished sensitivity profile
93.3
l
M, respectively) and 14 (GI50, TGI, and LC50 MG-MID values
M, respectively) proved to be the most active
0.36, 8.78 and 69.3
l
towards leukemia cell lines with GI50 range of 0.42–1.87
addition to a remarkably high activity against non-small cell lung
cancer NCI-H460 and CNS SF-295 cell lines (GI50 0.37 and
l
M, in
members in this study with a significantly potential activity
against all the tested subpanel tumor cell lines at the nanomolar
concentration level. Moreover, both compounds revealed distinc-
tive effectiveness on the leukemia subpanel at both the GI50
0.23
inhibitory activity towards most of the tested subpanel tumor cell
lines with GI50 range of 1.27–18.5 M. Furthermore, compound 6
exhibited potential activity toward the renal RXF 393 cell line
(GI50 value of 0.03 M). Whereas, compound 7 was able to inhibit
the growth of about 50 out of the 60 tested cell lines at GI50 value
<1 M, with special high activity against the CNS SF-295 and renal
A498 cell lines (GI50 0.15 and 0.11 M, respectively). On the other
lM, respectively). The analog 3 showed appreciable growth
(0.042 and 0.27
lM, respectively) and the TGI levels (10.2 and
l
3.23 M, respectively) (Tables 2 and 3). Additionally, compound
l
14 was able to show a broad spectrum of cytostatic and cytotoxic
potentials (TGI and LC50 MG-MID ranges of 3.23–23.2 and 44.3–
88.5 lM, respectively) against most of the tested subpanel tumor
cell lines (Table 3). Compound 7 displayed a remarkable growth
l
l
l
inhibitory and cytostatic effects (GI50 and TGI MG-MID values
hand, compound 13 showed the ever highest growth inhibitory po-
tential against all the tested tumor cell lines, with an outstanding
sensitivity profile towards about 47 different cancer cell lines with
GI50 values lying in the nanomolar concentration range (GI50 range
0.67 and 53.8
On the other hand, compounds 2 (GI50 and TGI MG-MID values
1.55 and 63.1 M, respectively) and 6 (GI50 and TGI MG-MID val-
ues 2.65 and 77.0 M, respectively) showed an observable antitu-
lM, respectively) when compared with 13 and 14.
l
l
0.02–0.09
other cell lines with GI50 range of 0.12–5.54
pound 14 was able to display a remarkable activity against all the
tested tumor cell lines, with GI50 value <1 M.
l
M), in addition to the potential activity against the
mor activity towards most of the tested tumor cell lines with
particular high sensitivity towards the leukemia subpanel (GI50
and TGI MG-MID values 1.03, 1.76 and 52.9, 80.7 lM, respectively).
Finally, compound 3 was found to be relatively the least effective
lM. Additionally, com-
l
With regard to the broad spectrum antitumor activity, the re-
sults revealed that all of the six active compounds; 2, 3, 6, 7, 13
and 14; revealed effective growth inhibition GI50 (MG-MID) values
antitumor agent in the present investigation with GI50 and TGI
(MG-MID) values of 5.75 and 67.6
any cytotoxic effect (Tables 2 and 3).
The ratio obtained by dividing the full panel MG-MID (
the compounds by their individual subpanel MG-MID ( M) is con-
lM, respectively; and without
of 1.55, 5.75, 2.65, 0.67, 0.08 and 0.36
lM, respectively, beside a
lM) of
cytostatic activity TGI (MG-MID) values of 63.1, 67.6, 77.0, 53.8,
l

S. A. F. Rostom / Bioorg. Med. Chem. 18 (2010) 2767–2776
2771
O
O
N
O
N
NH2
O
N
N
HO
HO
N
N
H
O
HO
N
O
H
H
H
N
N
N
N
N
v
i
Cl
21
Cl
Cl
1
iv
17
ii
iii
H
N
N
O
O
O
N
O
O
O
N
N
HO
N
O
HO
HO
N
N
H
H
H
N
N
N
N
N
N
Cl
Cl
Cl
20
19
18
Scheme 3. Reagents and reaction conditions: (i) maleic anhydride, anhydrous sodium acetate, gl. acetic acid, reflux 10 h; (ii) phthalic anhydride, xylene, reflux 12 h; (iii)
acetanthranil, gl. acetic acid, reflux 14 h; (iv) 3,4-dihydropyrano[3,4-b]indol-1(9H)-one, acetic acid, reflux 18 h; (v) isatin, acetic acid, reflux 2 h.
sidered as a measure of compound selectivity. Ratios between 3
and 6 refer to moderate selectivity, ratios greater than six indicate
high selectivity towards the corresponding cell line, while com-
pounds not meeting either of these criteria are rated non-selec-
tive.39 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 between 0.3–2.0 and 0.14–1.2 at the GI50 and TGI
levels, respectively, except compounds 13 and 14 which showed
moderate selectivity towards the leukemia subpanel at the TGI le-
vel (selectivity ratios 3.0 and 3.5, respectively).
change in the antitumor activity. In this view, when compared with
the structure analog 3, compound 6 (R = 2-furyl) showed about
twofold improvement in the growth inhibitory activity (GI50 MG-
MID 2.65 vs 5.75
duced (TGI MG-MID 77.0 vs 67.6
was developed (LC50 MG-MID 97.7
l
M), while its cytostatic effect was slightly re-
M), and a weak cytotoxic activity
M). On the other hand, intro-
l
l
duction of another carbonyl group as in the pyrazolidin-3,5-dione
7 (as a possible H-bond forming center) in place of the methyl group
in 2, resulted in a noticeable enhancement (twofold) in the overall
growth inhibition activity (GI50 MG-MID 0.67 vs 1.55 lM) and 2–3-
fold improvement in the individual subpanel tumor cell lines (Table
Structurally, the active analogs 2, 3, 6, 7, 13 and 14 are bipyraz-
olyl derivatives linked together through a carbonyl bridge, compris-
ing essentially the 1-(4-chlorophenyl)-4-hydroxy-1H-pyrazol-3-
carbonyl counterpart, which was found to be an essential pharma-
cophore for potential anticancer activity,18,19 together with other
functionalities that could assist in H-bond formation. In this view,
the pyrazol-5-one derivative 2 showed remarkable growth inhibi-
tory and cytostatic activities (GI50 and TGI MG-MID values 1.55
2), meanwhile, its cytostatic activity was slightly enhanced (TGI
MG-MID 53.8 vs 63.1
about fivefold improvement in the cytostatic activity against the
leukemia subpanel (TGI MG-MID 10.8 vs 52.9 M). Here, it should
lM). However, the same compound showed
l
be pointed out that, substituting the second pyrazole moiety with
two adjacent highly active functionalities (cyano and amino) led
to a potentially active analog 14 which showed a distinctive broad
spectrum of antitumor activities at the GI50, TGI and LC50 MG-MID
levels. This compound showed a significant growth inhibitory po-
tential against all the tested subpanel tumor cell lines falling in
the sub-micromolar concentration level with (GI50 MG-MID range
and 63.1 lM, respectively), while it lacked cytotoxic efficacy.
Replacement of the carbonyl functionality at position 5 with a
methyl group (3; R = CH3), resulted in about threefold reduction
of the overall growth inhibitory activity (GI50 MG-MID 5.75
l
M),
0.27–0.61
3.23–19.6
l
l
M), in addition to reliable cytostatic (TGI MG-MID range
M) and cytotoxic (LC50 MG-MID range 44.3–88.5 M)
whereas its cytostatic and cytotoxic activities were almost not af-
fected. Enlargement of the size of the substituent by introducing
an aromatic ring as in compound 4 (R = C6H5), led to complete abol-
ishment of the activity. Furthermore, increasing compounds’ lipo-
philicity by introducing a trifluromethyl moiety together with
changing the nature of substituent at position 3 led to a significant
l
activities. Replacement of the cyano group in compound 14 with
a ethoxycarbonyl functionality as in the structure analog 15, led
to a complete abolishment of activity.
Finally, an outstanding profile of antitumor activity was ob-
tained when the substitution pattern was extended to involve

2772
S. A. F. Rostom / Bioorg. Med. Chem. 18 (2010) 2767–2776
Table 1
the incorporation of a pyrazole moiety in a tricyclic ring system as
in the naphthopyrazole 13 (GI50, TGI, and LC50 MG-MID values
0.08, 30.9 and 93.3 M, respectively). This structure modification
Growth inhibitory concentrations (GI50, l
M) of compounds 2, 3, 6, 7, 13 and 14.a
l
Cell lines
2
3
6
7
13
14
resulted in a significant potentiation in the growth inhibitory activ-
ity against all the tested subpanel tumor cell lines with GI50 MG-
MID values lying at the nanomolar concentration level (0.04–
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
0.42
1.17
0.65
1.87
1.66
0.42
3.66
0.69
2.42
1.72
2.55
1.42
0.42
0.77
0.29
0.41
0.38
0.26
0.057
0.051
0.030
0.043
0.038
0.032
0.28
0.33
0.25
0.29
0.21
0.28
3.96
NAb
0.22 lM), together with a broad spectrum of cytostatic and cyto-
NTc
toxic potentials. This finding substantiates the idea behind the pos-
sible role of different linear and semi-linear fused di- and tricyclic
ring systems in anticancer activity as DNA intercaletors and kinase
inhibitors.33–35
3.34
NA
NT
Non-small cell Lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322
NCI-H460
NCI-H522
0.82
2.21
1.33
3.67
1.40
2.90
0.85
0.37
2.84
3.72
7.23
0.36
0.85
0.51
0.50
0.73
0.49
0.53
0.31
0.30
0.034
0.071
0.044
0.052
0.045
0.041
0.054
0.028
0.055
0.20
0.38
0.34
0.40
0.37
0.37
0.44
0.19
0.21
4.87
5.21
4.52
4.66
3.31
6.17
2.38
7.37
1.99
1.98
4.90
2.71
5.59
0.83
0.78
2.67
3. Conclusion
In conclusion, the aim of the present investigation was to syn-
thesize novel 4-hydroxy-1H-pyrazole lead compounds possessing
potential antitumor activity, as an extension to such previously-re-
ported template of compounds. Such objective has been achieved
by the synthesis of two novel series of 1-(4-chlorophenyl)-4-hy-
droxy-1H-pyrazoles linked to either substituted 1H-pyrazole coun-
terparts through a carbonyl bridge, or to some biologically-active
nitrogenous heterocycles by an amide linker for synergistic pur-
pose. The most active six compounds 2, 3, 6, 7, 13 and 14 revealed
a significant broad spectrum of antitumor potential against most of
the tested subpanel tumor cell lines at the GI50 and TGI levels, to-
gether with a mild cytotoxic (LC50) activity. The pyrazolinedione
derivative 7 displayed a remarkable growth inhibition and cyto-
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
4.81
3.67
0.68
0.68
6.16
2.27
3.41
18.5
15.1
3.42
3.53
3.29
5.07
8.02
2.97
10.5
0.94
4.74
0.34
0.49
0.58
1.16
2.32
0.291
0.502
0.034
0.052
0.241
0.070
0.360
0.91
0.65
0.26
0.34
1.19
0.38
0.52
2.00
1.44
4.56
5.88
4.75
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
0.49
0.23
1.70
3.16
0.53
0.41
4.13
1.81
3.05
3.60
3.52
2.68
3.63
0.48
2.81
4.37
0.82
5.86
0.51
0.15
0.37
0.51
0.24
0.18
0.048
0.020
0.041
0.045
0.029
0.031
0.46
0.16
0.32
0.45
0.25
0.29
static effects (GI50 and TGI MG-MID values 0.67 and 53.8 lM,
respectively) together with a weak cytotoxic effect. The tricyclic
naphthopyrazole 13 (GI50, TGI, and LC50 MG-MID values 0.08,
30.9 and 93.3
lM) and the aminocyanopyrazole 14 (GI50, TGI,
Melanoma
LOX IMVI
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
and LC50 MG-MID values 0.36, 8.78 and 69.3
lM, respectively)
1.55
1.78
NT
NT
3.06
1.70
1.06
1.80
3.94
4.69
2.37
4.28
5.82
3.82
9.22
2.49
2.67
2.16
1.37
0.71
0.38
NA
5.26
1.92
1.86
0.39
0.075
0.096
0.046
5.544
0.092
0.370
0.210
0.051
0.44
0.48
0.33
0.91
0.63
0.48
0.56
0.30
could be classified as the most active antitumor members identi-
fied in this study as evidenced by their significantly potential
growth inhibitory activity at the nanomolar concentration level
and remarkable cytostatic and cytotoxic activities, regardless to
the subpanel being tested, with special high selectivity profile
against some individual cell lines.
NT
NT
6.28
6.76
16.6
3.46
It is worth-mentioning that, compounds 7, 13 and 14 were fur-
ther selected by the NCI’s biological committee for subsequent
confirmatory screening procedures in order to assess their exact
antitumor potentialities. These tests include the repetition of the
testing in the primary screen, referring to the Biological Evaluation
Committee and a final review by the Biological Evaluation Commit-
tee. Interestingly, those three potential compounds have passed
successfully all the above-mentioned in vitro stages and are re-
quired as candidates for the next in vivo hollow fiber assay.40
All these favorable features make such type of 4-hydroxypyraz-
ole derivatives the appropriate candidates for further testing and
derivatization in the hope of finding more selective and active anti-
cancer compounds in the nano- or even sub-nanomolar concentra-
tion levels.
Ovarian cancer
IGROV1
0.51
1.21
2.40
3.32
1.49
1.53
3.16
1.27
9.19
4.98
5.96
7.04
0.92
1.72
2.43
2.60
1.88
3.55
0.38
0.93
1.52
8.08
0.64
1.92
0.043
0.099
0.264
0.072
0.053
0.160
0.36
0.55
0.46
0.64
0.48
0.52
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786–0
A498
6.45
0.45
0.88
1.06
9.64
2.90
3.77
4.23
4.58
3.37
4.71
3.01
6.60
8.71
2.53
2.43
0.03
5.18
1.45
3.84
3.77
0.11
0.58
0.65
0.31
0.50
0.35
0.55
0.319
0.024
0.048
0.127
0.047
0.041
0.033
0.099
0.66
0.17
0.32
0.28
0.51
0.35
0.28
0.30
ACHN
CAKI-1
RXF 393
SN12C
TK-10
15.4
1.42
0.79
0.67
UO-31
Prostate cancer
PC-3
DU-145
NT
5.22
7.05
6.23
26.3
10.5
0.36
0.98
0.037
0.050
0.33
0.57
4. Experimental
4.1. Chemistry
Breast cancer
MCF7
NCI/ADR-RES
HS 578T
MDA-MB-435
BT-549
T-47D
3.61
1.89
0.28
1.20
0.65
2.66
4.57
4.52
4.42
3.21
3.94
2.19
3.66
8.90
3.51
1.56
0.94
0.19
0.33
0.37
0.45
0.72
0.121
0.046
0.021
0.037
0.029
0.059
0.262
0.36
0.37
0.19
0.29
0.29
0.30
0.95
1.60
0.64
1.97
1.14
4.16
4.66
Melting points were determined in open glass capillaries on a
Gallenkamp melting point apparatus and were uncorrected. The
infrared (IR) spectra were recorded on Perkin–Elmer 297 infrared
spectrophotometer using the NaCl plate technique. The 1H NMR
spectra were recorded on a Varian EM 360 spectrometer using tet-
ramethylsilane as the internal standard and DMSO-d6 as the sol-
vent (Chemical shifts in (d, ppm). Splitting patterns were
designated as follows: s: singlet; d: doublet; m: multiplet. Elemen-
MDA-MB-468
aData obtained from NCI’s in vitro disease-oriented human tumor cell screen.
b
NA: Not active (GI50 value >100
NT: Not tested.
lM).
c

S. A. F. Rostom / Bioorg. Med. Chem. 18 (2010) 2767–2776
2773
Table 2
Median growth inhibitory concentrations (GI50
,
l
M) of in vitro subpanel tumor cell linesa
Subpanel tumor cell linesb
Compd no.
MG-MIDc
I
II
III
IV
V
VI
VII
VIII
5.22
6.64
18.4
0.67
0.044
0.45
IX
2
3
6
7
13
14
1.03
3.65
1.76
0.42
0.042
0.27
1.82
4.69
3.19
0.51
0.047
0.32
3.09
8.13
4.59
1.51
0.221
0.61
1.09
3.13
2.99
0.33
0.036
0.32
1.82
4.95
4.14
1.60
0.140
0.46
1.74
5.27
2.18
1.08
0.115
0.50
1.67
4.52
3.85
0.85
0.092
0.36
2.02
4.96
3.07
0.82
0.082
0.43
1.55
5.75
2.65
0.67
0.08
0.36
a
b
c
Median values calculated according to the data obtained from NCI’s in vitro disease-oriented human tumor cell screen.
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.
GI50 M) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines towards the test agent.
(l
Table 3
Median total growth inhibitory concentrations (TGI,
l
M) and lethal concentration 50 (LC50, l
M) of in vitro subpanel tumor cell linesa
Compd no.
Subpanel tumor cell linesb
MG-MIDc
I
II
III
IV
V
VI
VII
VIII
IX
d
2
3
6
7
13
14
52.9
74.3
80.7
10.8
10.2 (90.7)
3.23 (44.3)
78.2
81.7
78.9
80.3
68.1
10.8 (69.4)
95.4
85.1
88.5
86.2
86.3
12.6 (64.3)
79.6
69.2
90.5
87.9
69.1
70.5 (93.6)e
71.4 (92.8)
63.7 (89.4)
50.1 (85.2)
52.1 (86.4)
83.8
87.8
94.5
84.0
83.6
19.6 (88.5)
77.0
71.3
75.6
71.8
67.7
—
90.1
90.7
—
79.6
91.7
14.4 (64.3)
63.1
67.6
77.0 (97.7)
53.8 (93.3)
30.9 (93.3)
8.78 (69.3)
—
—
—
—
13.4
23.1 (74.7)
11.9 (65.0)
8.99 (75.0)
a
b
c
Median values calculated according to the data obtained from NCI’s in vitro disease-oriented human tumor cell screen.
For subpanel tumor cell lines, see footnote (b) of Table 3.
TGI (
l
M) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines towards the test agent.
TGI (MG-MID) value >100 M.
LC50 M) full panel mean-graph mid point (MG-MID) = the average sensitivity of all cell lines towards the test agent.
d
e
l
(l
tal 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 reactions and checking the homogeneity of the compounds
were made by TLC on silica gel-protected aluminum sheets (Type
60 F254, Merck) and the spots were detected by exposure to UV-
lamp at k 254. The synthesis of the starting 1-(4-chlorophenyl)-
4-hydroxy-1H-pyrazole-3-carboxylic acid hydrazide 1,36 acetan-
thranil37 and 3,4-dihydropyrano[3,4-b]indol-1(9H)-one,38 was per-
formed according to reported literature procedures.
thoroughly washed with cold ethanol, dried and recrystallized
from ethanol.
4.1.2.1. 3-(3,5-Dimethyl-1H-pyrazol-1-yl)carbonyl-1-(4-chloro-
phenyl)-4-hydroxy-1H-pyrazole (3). (Using pentane-2,4-dione)
Yield: 67%, mp: 260–61 °C, IR (cmÀ1): 3280–3050 (OH), 1670
(C@O); 1H NMR (d, ppm): 2.19 (s, 3H, CH3), 2.38 (s, 3H, CH3),
6.21 (s, 1H, pyrazole-C4-H), 7.43–7.78 (m, 4H, Ar-H), 8.27 (s, 1H,
pyrazole-C5-H), 9.09 (s, 1H, OH). Anal. Calcd for C15H13ClN4O2
(316.74): C, 56.88; H, 4.14; N, 17.69. Found: C, 56.65; H, 4.47; N,
17.31.
4.1.1. 3-(4,5-Dihydro-3-methyl-5-oxo-1H-pyrazol-1-
4.1.2.2. 3-(5-Methyl-3-phenyl-1H-pyrazol-1-yl)carbonyl-1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazole (4). (Using 1-phenylbu-
tane-1,3-dione) Yield: 38%, mp: 382–84 °C, IR (cmÀ1): 3475–3120
(OH), 1665 (C@O); 1H NMR (d, ppm): 2.49 (s, 3H, CH3), 6.30 (s,
1H, pyrazole-C4-H), 7.31–7.89 (m, 9H, Ar-H), 8.31 (s, 1H, pyra-
zole-C5-H), 9.15 (s, 1H, OH). Anal. Calcd for C20H15ClN4O2
(378.81): C, 63.41; H, 3.99; N, 14.79. Found: C, 63.61; H, 3.78; N,
14.59.
yl)carbonyl-1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole (2)
Ethyl acetoacetate (0.39 g, 3 mmol) was added to a solution of
the acid hydrazide 1 (0.5 g, 2 mmol) in absolute ethanol (15 mL),
and the reaction mixture was heated under reflux for 10 h. Solvent
was removed under reduced pressure and the remaining residue
was recrystallized from aqueous ethanol. Yield: 70%, mp: 284–
86 °C, IR (cmÀ1): 3245–2980 (OH), 1740, 1675 (C@O); 1H NMR (d,
ppm): 2.09 (s, 3H, CH3), 4.24 (s, 2H, CH2), 7.38–7.71 (m, 4H, Ar-
H), 8.23 (s, 1H, pyrazole-C5-H), 9.12 (s, 1H, OH). Anal. Calcd for
C14H11ClN4O3 (318.72): C, 52.76; H, 3.48; N, 17.58. Found: C,
52.41; H, 3.56; N, 17.39.
4.1.2.3. 3-(3-Methyl-5-trifluoromethyl-1H-pyrazol-1-yl)carbonyl-
1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole (5). (Using 1,1,1-
trifluoropentane-2,4-dione) Yield: 63%, mp: 229–31 °C, IR (cmÀ1):
3380–2975 (OH), 1660 (C@O); 1H NMR (d, ppm): 2.32 (s, 3H,
CH3), 7.19–7.77 (m, 5H, 4 Ar-H and pyrazole-C4-H), 8.33 (s, 1H, pyr-
azole-C5-H), 9.17 (s, 1H, OH). Anal. Calcd for C15H10ClF3N4O2
(370.71): C, 48.60; H, 2.72; N, 15.11. Found: C, 48.92; H, 2.48; N,
15.27.
4.1.2. General method for the synthesis of 3-(5-methyl-3-
substituted-1H-pyrazol-1-yl)carbonyl-1-(4-chlorophenyl)-4-
hydroxy-1H-pyrazoles (3,4) and 3-(3-substituted-5-
trifluoromethyl-1H-pyrazol-1-yl)carbonyl-1-(4-chlorophenyl)-
4-hydroxy-1H-pyrazoles (5,6)
To a solution of 1 (0.5 g, 2 mmol) in glacial acetic acid (15 mL),
was added the appropriate diketone (2 mmol). The reaction mix-
ture was heated under reflux for 8–12 h, then allowed to cool to
room temperature. The solid product thus formed was filtered,
4.1.2.4. 3-(3(2-Furyl)-5-trifluoromethyl-1H-pyrazol-1-yl)carbonyl-
1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole (6). (Using 1-(2-fur-
yl)butane-1,3-dione) Yield: 54%, mp: 280–82 °C, IR (cmÀ1): 3240–
3120 (OH), 1665 (C@O); 1H NMR (d, ppm): 7.09–8.27 (m, 8H, 4

2774
S. A. F. Rostom / Bioorg. Med. Chem. 18 (2010) 2767–2776
Ar-H, 3 furyl-H and pyrazole-C4-H), 8.37 (s, 1H, pyrazole-C5-H), 9.16
(s, 1H, OH). Anal. Calcd for C18H10ClF3N4O3 (422.75): C, 51.14; H,
2.38; N, 13.25. Found: C, 50.89; H, 2.56; N, 13.38.
(cmÀ1): 3640–3020 (OH, NH), 1690, 1670 (C@O); 1H NMR (d,
ppm): 1.94 (s, 3H, CH3), 2.42 (t, 2H, CH2), 3.51 (t, 2H, –CH2–),
6.79 (s, 1H, –CH2–OH), 7.41–7.69 (m, 4H, Ar-H), 8.19 (s, 1H, pyra-
zole-C5-H), 9.08 (s, 1H, pyrazole-4-OH), 10.38 (s, 1H, NH). Anal.
Calcd for C16H15ClN4O4 (362.77): C, 52.97; H, 4.17; N, 15.44. Found:
C, 52.76; H, 4.35; N, 15.12.
4.1.3. 3-(3,5-Dioxopyrazolidine-1-yl)carbonyl-1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazole (7)
A mixture of the starting compound 1 (0.5 g, 2 mmol) and
diethyl malonate (0.46 g, 4 mmol) was heated at 200 °C in an oil
bath for 2 h. After being cooled to room temperature, the solidified
product was treated with cold diethyl ether, filtered, washed with
diethyl ether, dried and recrystallized from aqueous ethanol. Yield:
46%, mp: 282–84 °C, IR (cmÀ1): 3370–2755 (OH, NH), 1720, 1700,
1670 (C@O); 1H NMR (d, ppm): 3.51 (s, 2H, CH2), 7.41–7.79 (m,
4H, Ar-H), 8.25 (s, 1H, pyrazole-C5-H), 9.21 (s, 1H, OH), 10.35 (s,
1H, NH). Anal. Calcd for C13H9ClN4O4 (320.69): C, 48.69; H, 2.83;
N, 17.47. Found: C, 48.81; H, 2.64; N, 17.29.
4.1.6. 3-(3-Oxo-1,2,5,6-tetrahydrocyclopenta[c]pyrazol-2-
yl)carbonyl-1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole (12)
The acid hydrazide 1 (0.5 g, 2 mmol) and ethyl cyclopentanone-
2-carboxylate (0.47 g, 3 mmol) were heated at 200 °C in an oil bath
for 2 h. After being cooled to room temperature, the solidified
product was treated with cold methanol, filtered, washed with cold
methanol, dried and recrystallized from toluene/petroleum ether
(60/80). Yield: 65%, mp: 188–90 °C, IR (cmÀ1): 3350–2860 (OH,
NH), 1690, 1665 (C@O); 1H NMR (d, ppm): 1.76–1.79 (m, 2H,
CH2), 2.70 (t, 2H, CH2), 2.92 (t, 2H, CH2), 7.29–7.61 (m, 4H, Ar-H),
8.16 (s, 1H, pyrazole-C5-H), 8.99 (s, 1H, OH), 9.96 (s, 1H, NH). Anal.
Calcd for C16H13ClN4O3 (344.75): C, 55.74; H, 3.80; N, 16.25. Found:
C, 55.87; H, 3.55; N, 16.08.
4.1.4. General method for the synthesis of 3-(3,5-dioxo-4-
substituted pyrazolidine-1-yl)carbonyl-1-(4-chlorophenyl)-4-
hydroxy-1H-pyrazoles (8–10)
A mixture of 1 (0.5 g, 2 mmol) and the appropriate 2-substi-
tuted diethyl malonate (4 mmol) and sodium methoxide (0.22 g,
4 mmol) was refluxed in methanol (20 mL) for 18 h. Excess solvent
was removed under reduced pressure, the remaining residue was
dissolved in water (20 mL) and extracted with chloroform
(2 Â 15 mL) to get rid of any unreacted esters. The aqueous solu-
tion was acidified with 2 N hydrochloric acid to pH 3–4 to give a
creamy white precipitate which was filtered, washed with water,
dried and recrystallized from ethanol.
4.1.7. 3-(3-Methyl-4,5-dihydronaphtho[1,2-c]pyrazol-2-
yl)carbonyl-1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole (13)
To a solution of 1 (0.5 g, 2 mmol) in glacial acetic acid (10 mL),
was added 2-acetyl-1-tetralone (0.38 g, 2 mmol). The reaction mix-
ture was refluxed for 3 h, during which a heavy yellow solid was
crystallized out. After cooling to room temperature, the separated
product was filtered, washed with cold ethanol, dried and recrys-
tallized from ethanol. Yield: 54%, mp: 363–65 °C, IR (cmÀ1):
3230–2780 (OH), 1670 (C@O); 1H NMR (d, ppm): 2.39 (s, 3H,
CH3), 2.62–2.74 (m, 4H, 2 CH2), 7.38–8.09 (m, 9H, 8 Ar-H and pyr-
azole-C5-H), 8.87 (s, 1H, OH). Anal. Calcd for C22H17ClN4O2
(404.85): C, 65.27; H, 4.23; N, 13.84. Found: C, 65.04; H, 4.51; N,
13.69.
4.1.4.1. 3-(4-Ethyl-3,5-dioxopyrazolidine-1-yl)carbonyl-1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazole (8). (Using diethyl 2-eth-
ylmalonate) Yield: 27%, mp: 242–44 °C, IR (cmÀ1): 3420–2810 (OH,
NH), 1725, 1700, 1675 (C@O); 1H NMR (d, ppm): 1.09 (t, 3H, CH3),
2.11 (q, 2H, CH2), 3.38–3.44 (m, 1H, pyrazole-C4-H), 7.37–7.81 (m,
4H, Ar-H), 8.22 (s, 1H, pyrazole-C5-H), 9.24 (s, 1H, OH), 10.47 (s, 1H,
NH). Anal. Calcd for C15H13ClN4O4 (348.74): C, 51.66; H, 3.76; N,
16.07. Found: C, 51.48; H, 3.88; N, 15.93.
4.1.8. 3-(5-Amino-4-cyano-1H-pyrazol-1-yl)carbonyl-1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazole (14)
A mixture of 1 (0.5 g, 2 mmol), ethoxymethylenemalononitrile
(0.24 g, 2 mmol) and anhydrous potassium carbonate (0.3 g,
2 mmol) in absolute ethanol (15 mL) was heated under reflux for
6 h. After being cooled to room temperature, the yellowish solid
product was separated, suspended in water (10 mL) and acidified
with 2 N hydrochloric acid till pH 3–4. The resulting creamy white
precipitate was filtered, washed thoroughly with water, dried and
recrystallized from ethanol. Yield: 79%, mp: 288–90 °C, IR (cmÀ1):
3690–3050 (OH, NH), 2215 (CN), 1665 (C@O); 1H NMR (d, ppm):
6.73 (s, 2H, NH2), 7.46–7.98 (m, 5H, 4 Ar-H and pyrazole-C3-H),
8.36 (s, 1H, pyrazole-C5-H), 9.12 (s, 1H, OH). Anal. Calcd for
C14H9ClN6O2 (328.71): C, 51.15; H, 2.76; N, 25.57. Found: C,
51.34; H, 2.65; N, 25.34.
4.1.4.2. 3-(4-Butyl-3,5-dioxopyrazolidine-1-yl)carbonyl-1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazole (9). (Using diethyl 2-
butylmalonate) Yield: 32%, mp: 250–52 °C, IR (cmÀ1): 3460–2670
(OH, NH), 1720, 1710, 1665 (C@O); 1H NMR (d, ppm): 1.01–1.52
(m, 9H, n-butyl-9H), 3.32–3.37 (m, 1H, pyrazole-C4-H), 7.41–7.76
(m, 4H, Ar-H), 8.23 (s, 1H, pyrazole-C5-H), 9.19 (s, 1H, OH), 10.38
(s, 1H, NH). Anal. Calcd for C17H17ClN4O4 (376.79): C, 54.19; H,
4.55; N, 14.87. Found: C, 53.94; H, 4.67; N, 14.68.
4.1.4.3. 3-(3,5-Dioxo-4-phenylpyrazolidine-1-yl)carbonyl-1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazole (10). (Using diethyl 2-phe-
nylmalonate) Yield: 41%, mp: 357–59 °C, IR (cmÀ1): 3460–2670 (OH,
NH), 1715, 1700, 1670 (C@O); 1H NMR (d, ppm): 4.97 (s, 1H, pyra-
zole-C4-H), 7.29–7.78 (m, 9H, Ar-H), 8.21 (s, 1H, pyrazole-C5-H),
9.17 (s, 1H, OH), 10.45 (s, 1H, NH). Anal. Calcd for C19H13ClN4O4
(396.78): C, 57.51; H, 3.30; N, 14.12. Found: C, 57.64; H, 3.07; N,
14.26.
4.1.9. 3-(5-Amino-4-ethoxycarbonyl-1H-pyrazol-1-yl)carbonyl-
1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole (15)
The title compound was prepared as described for compound
14 by refluxing
a mixture of 1 (0.5 g, 2 mmol), ethyl eth-
oxymethylenecyanoacetate (0.35 g, 2 mmol) and anhydrous potas-
sium carbonate (0.3 g, 2 mmol) in absolute ethanol (15 mL) for 8 h.
Yield: 82%, mp: 267–69 °C, IR (cmÀ1): 3580–2940 (OH, NH), 1665,
1715 (C@O); 1H NMR (d, ppm): 1.29 (t, 3H, CH3), 3.47 (q, 2H, CH2);
6.61 (s, 2H, NH2), 7.59–8.07 (m, 5H, 4 Ar-H and pyrazole-C3-H),
8.31 (s, 1H, pyrazole-C5-H), 9.09 (s, 1H, OH). Anal. Calcd for
C16H14ClN5O4 (375.77): C, 51.14; H, 3.76; N, 18.64. Found: C,
50.93; H, 3.87; N, 18.55.
4.1.5. 3-(1,2-Dihydro-5-(2-hydroxyethyl)-4-methyl-3-
oxopyrazol-2-yl)carbonyl-1-(4-chlorophenyl)-4-hydroxy-1H-
pyrazole (11)
A solution of the acid hydrazide 1 (0.5 g, 2 mmol) and 2-acetyl-
butyrolactone (0.38 g, 3 mmol) in bromobenzene (10 mL), was re-
fluxed for 6 h. After cooling to room temperature, the separated
solid product was filtered, washed with cold ethanol, dried and
recrystallized from ethanol. Yield: 32%, mp: 179–81 °C, IR

S. A. F. Rostom / Bioorg. Med. Chem. 18 (2010) 2767–2776
2775
4.1.10. 3-(4-Ethoxycarbonyl-5-hydroxy-1H-pyrazol-1-
19. Yield: 35%, mp: 274–75 °C, IR (cmÀ1): 3450–2920 (OH, NH),
1670, 1645 (C@O); 1H NMR (d, ppm): 3.36 (t, 2H, CH2), 3.51 (t,
2H, CH2), 7.19–7.79 (m, 8H, Ar-H), 8.40 (s, 1H, pyrazole-C5-H),
8.91 (s, 2H, amide NH and indole-NH), 9.10 (s, 1H, OH). Anal. Calcd
for C21H16ClN5O3 (421.84): C, 59.79; H, 3.82; N, 16.60. Found: C,
59.63; H, 3.91; N, 16.44.
yl)carbonyl-1-(4-chlorophenyl)-4-hydroxy-1H-pyrazole (16)
An equimolar mixture of 1 (0.5 g, 2 mmol), diethyl ethoxymeth-
ylenemalonate (0.45 g, 2 mmol) and anhydrous potassium carbon-
ate (0.3 g, 2 mmol) in acetonitrile (15 mL) was refluxed for 12 h.
The reaction mixture was concentrated in vacuo, and allowed to
attain room temperature. The separated solid product was sus-
pended in water (10 mL) and acidified with 2 N hydrochloric acid
till pH 3–4. The resulting creamy white precipitate was filtered,
washed thoroughly with water, dried and recrystallized from eth-
anol. Yield: 91%, mp: 294–96 °C, IR (cmÀ1): 3470–2710 (OH), 1725,
1670 (C@O); 1H NMR (d, ppm): 1.34 (t, 3H, CH3), 4.25 (q, 2H, CH2);
7.67–8.12 (m, 5H, 4 Ar-H and pyrazole-C3-H), 8.39 (s, 1H, pyrazole-
C5-H), 8.92 (s, 1H, OH), 10.20 (s, 1H, OH). Anal. Calcd for
C16H13ClN4O5 (376.75): C, 51.01; H, 3.48; N, 14.87. Found: C,
50.86; H, 3.63; N, 14.64.
4.1.15. 1-(4-Chlorophenyl)-4-hydroxy-3-(2-oxoindolin-3-
ylidene)hydrazinocarbonyl)-1H-pyrazole (21)
A solution of the acid hydrazide 1 (0.5 g, 2 mmol) and isatin
(0.3 g, 2 mmol) in glacial acetic acid (10 mL) was heated under re-
flux for 2 h, during which a deep yellow solid was partially crystal-
lized out. The solid separated upon cooling was filtered, washed
with cold ethanol and recrystallized from ethanol. Yield: 86%,
mp: 346–48 °C, IR (cmÀ1): 3460–3040 (OH, NH), 1690, 1655
(C@O); 1H NMR (d, ppm): 7.08–7.69 (m, 8H, Ar-H), 8.35 (s, 1H, pyr-
azole-C5-H), 9.24 (s, 2H, amide NH and indole-NH), 9.55 (s, 1H, OH).
Anal. Calcd for C18H12ClN5O3 (381.77): C, 56.63; H, 3.17; N, 18.34.
Found: C, 56.47; H, 3.06; N, 18.12.
4.1.11. N-(2,4-Dioxo-2,5-dihydropyrrol-1-yl)-1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazole-3-carboxamide (17)
A mixture of the acid hydrazide 1 (0.5 g, 2 mmol), maleic anhy-
dride (0.2 g, 2 mmol) and anhydrous sodium acetate (0.2 g,
2.5 mmol) in glacial acetic acid (20 mL) was heated under reflux
for 10 h. The reaction mixture was concentrated in vacuo to half
its volume and allowed to cool. The precipitated solid product
was filtered, washed with cold ethanol, dried and recrystallized
from acetic acid. Yield: 37%, mp: 236–38 °C, IR (cmÀ1): 3490–
2820 (OH, NH), 1705, 1680, 1665 (C@O); 1H NMR (d, ppm): 6.44–
6.48 (m, 2H, olefinic 2H), 7.54–8.01 (m, 5H, 4 Ar-H and pyrazole-
C5-H), 8.45 (s, 1H, NH), 9.15 (s, 1H, OH). Anal. Calcd for
C14H9ClN4O4 (332.7): C, 50.54; H, 2.73; N, 16.84. Found: C, 50.38;
H, 2.81; N, 16.69.
4.2. In vitro antitumor activity
4.2.1. Preliminary in vitro one-dose antitumor screening
Out of the newly synthesized compounds, ten derivatives
namely; 2–7, 13–15 and 18; were selected by the National Cancer
Institute (NCI) in vitro disease-oriented human cells screening pa-
nel assay to be evaluated for their in vitro anticancer activity. Pri-
mary in vitro one-dose anticancer assay was performed using the
full NCI 60 cell panel in accordance with the current protocol of
the Drug Evaluation Branch, NCI, Bethesda.29–31 These cell lines
were incubated with one concentration (10 lM) for each tested
compound. A 48 h continuous drug exposure protocol was used,
and a sulphorhodamine B (SRB) protein assay was employed to
estimate cell viability or growth. Six compounds namely; 2, 3, 6,
7, 13 and 14, passed this primary anticancer assay and conse-
quently were carried over to the five-dose screen against a panel
of about 60 different tumor cell lines.
4.1.12. N-(1,3-Dioxo-1,3-dihydroisoindol-2-yl)-1-(4-
chlorophenyl)-4-hydroxy-1H-pyrazole-3-carboxamide (18)
A mixture of the acid hydrazide 1 (0.5 g, 2 mmol) and phthalic
anhydride (0.3 g, 2 mmol) in xylene (10 mL) was heated under re-
flux for 12 h. After cooling, the obtained solid product was filtered,
washed with cold ethanol, and recrystallized from acetic acid con-
taining few drops of water. Yield: 88%, mp: 325–27 °C, IR (cmÀ1):
3370–2730 (OH, NH), 1735, 1700, 1665 (C@O); 1H NMR (d, ppm):
7.33–7.96 (m, 8H, Ar-H), 8.43 (s, 1H, pyrazole-C5-H), 8.76 (s, 1H,
NH), 9.32 (s, 1H, OH). Anal. Calcd for C18H11ClN4O4 (382.76): C,
56.48; H, 2.90; N, 14.64. Found: C, 56.71; H, 2.69; N, 14.53.
4.2.2. Full in vitro five-dose antitumor assay
Compounds 2, 3, 6, 7, 13 and 14 were subjected to the NCI
in vitro disease-oriented human cells screening panel assay to
screen their antitumor activities. 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 bo-
4.1.13. 1-(4-Chlorophenyl)-4-hydroxy-N-(2-methyl-4-
oxoquinazolin-3(4H)-yl)-1H-pyrazole-3-carboxamide (19)
To a solution of the acid hydrazide 1 (0.5 g, 2 mmol) in glacial
acetic acid (10 mL) was added acetanthranil (0.32 g, 2 mmol) and
the reaction mixture was heated under reflux for 14 h. After being
cooled to room temperature, the mixture was poured onto crushed
ice and the separated solid product was filtered, thoroughly
washed with water and dried. It was recrystallized from dioxan
containing few drops of water. Yield: 48%, mp: 229–31 °C, IR
(cmÀ1): 3240–2810 (OH, NH), 1690, 1665 (C@O); 1H NMR (d,
ppm): 2.51 (s, 3H, CH3), 7.41–7.86 (m, 8H, Ar-H), 8.41 (s,1H, pyra-
zole-C5-H), 8.85 (s, 1H, NH), 9.07 (s, 1H, OH). Anal. Calcd for
C19H14ClN5O3 (395.8): C, 57.66; H, 3.57; N, 17.69. Found: C,
57.46; H, 3.66; N, 17.51.
vine serum and 2 mM L-glutamine. For a typical screening experi-
ment, 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 measurement of the cell pop-
ulation for each cell line at the time of drug addition. Experimental
drugs were solubilized in dimethyl sulphoxide at 400-fold the de-
sired final maximum 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 medium containing 50 mg mLÀ1 gen-
tamicin. 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. Fol-
lowing drug addition, the plates were incubated for an additional
4.1.14. 1-(4-Chlorophenyl)-N-(3,4-dihydro-1-oxo-1H-
pyrido[3,4-b]indol-2(9H)-yl)-4-hydroxy-1H-pyrazole-3-
carboxamide (20)
A mixture of the acid hydrazide 1 (0.5 g, 2 mmol) and 3,4-dihy-
dropyrano[3,4-b]indol-1(9H)-one (0.37 g, 2 mmol) in glacial acetic
acid (20 mL) was heated under reflux for 18 h. working up of the
reaction mixture was carried out as described under compound

2776
S. A. F. Rostom / Bioorg. Med. Chem. 18 (2010) 2767–2776
11. Daidone, G.; Raffa, D.; Maggio, B.; Raimondi, M. V.; Plescia, F.; Schillaci, D. Eur. J.
Med. Chem. 2004, 39, 219.
12. Warshakoon, N. C.; Wu, S.; Boyer, A.; Kawamoto, R.; Renock, S.; Xu, K.; Pokross,
M.; Evdokimov, A. G.; Zhou, S.; Winter, C.; Walter, R.; Mekel, M. Bioorg. Med.
Chem. Lett. 2006, 16, 5687.
13. Huang, S.; Lin, R.; Yu, Y.; Lu, Y.; Connolly, P. J.; Chiu, G.; Li, S.; Emanuel, S. L.;
Middleton, S. A. Bioorg. Med. Chem. Lett. 2007, 17, 1243.
14. Zhu, G.-D.; Gong, J.; Gandhi, V. B.; Woods, K.; Luo, Y.; Liu, X.; Guan, R.;
Klinghofer, V.; Johnson, E. F.; Stoll, V. S.; Mamo, M.; Li, Q.; Rosenberg, S. H.;
Giranda, V. L. Bioorg. Med. Chem. 2007, 15, 2441.
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. 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 tempera-
ture. 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 an automated plate reader at a wave-
length of 515 nm. For 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 each cell line.29–31
15. Fahmy, H. T. Y.; Rostom, S. A. F.; Bekhit, A. A. Arch. Pharm. Pharm. Med. Chem.
2002, 335, 213.
16. Fahmy, H. T. Y.; Rostom, S. A. F.; Saudi, M. N. S.; Zjawiony, J. K.; Robins, D. J.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 216.
17. Rostom, S. A. F.; Fahmy, H. T. Y.; Saudi, M. N. S. Sci. Pharm. 2003, 71, 57.
18. Rostom, S. A. F.; Shalaby, M. A.; El-Demellawy, M. A. Eur. J. Med. Chem. 2003, 38,
959.
19. Al-Saadi, M. S. M.; Rostom, S. A. F.; Faid Allah, H. M. Saudi Pharm. J. (SPJ) 2005,
13, 89.
20. Al-Saadi, M. S. M.; Rostom, S. A. F.; Faid Allah, H. M. Alex. J. Pharm. Sci. 2005, 19,
15.
21. Rostom, S. A. F. Bioorg. Med. Chem. 2006, 14, 6475.
22. Faid-Allah, H. M.; Al-Saadi, M. S.; Rostom, S. A. F.; Fahmy, H. T. Med. Chem. Res.
2007, 16, 300.
23. Al-Saadi, M. S. M.; Rostom, S. A. F.; Faid Allah, H. M. Arch. Pharm. Chem. Life Sci.
2008, 341, 181.
Acknowledgments
24. Faid-Allah, H. M.; Al-Saadi, M. S.; Rostom, S. A. F. Saudi Pharm. J. (SPJ) 2008, 16,
33.
25. Al-Saadi, M. S. M.; Faid Allah, H. M.; Rostom, S. A. F. Arch. Pharm. Chem. Life Sci.
2008, 341, 424.
26. Rostom, S. A. F.; Ashour, H. M. A.; Abd El Razik, H. A. Arch. Pharm. Chem. Life Sci.
2009, 342, 299.
27. Rostom, S. A. F.; Ashour, H. M. A.; Abd El Razik, H. A.; Abd El Fattah, A. H.; El-
Din, N. N. Bioorg. Med. Chem. 2009, 17, 2410.
The author is deeply thankful to the staff members of the
Department of Health and Human Services, National Cancer Insti-
tute (NCI), Bethesda, Maryland, USA for carrying out the anticancer
screening of the newly synthesized compounds.
28. Rostom, S. A. F.; Hassan, G. S.; El-Subbagh, H. I. Arch. Pharm. Chem. Life Sci. 2009,
342, 584.
References and notes
29. Grever, M. R.; Schepartz, S. A.; Chabner, B. A. Semin. Oncol. 1992, 19, 622.
30. Boyd, M. R.; Paull, K. D. Drug Rev. Res. 1995, 34, 91.
31. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose,
C.; Jangley, J.; Cronisie, P.; Viagro-Wolff, A.; Gray-Goodrich, M.; Campell, H.;
Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757.
32. Chu, E.; DeVita, V. T., Jr. In Cancer Principles and Practice of Oncology; DeVita, V.
T., Jr., Hellman, S., Rosenberg, S. A., Eds., 7th ed.; Lippincott Williams and
Wilkins: Philadelphia, USA, 2005; p 295.
33. Joseph, B.; Facompre, M.; Da Costa, H.; Routier, S.; Merour, J.-Y.; Colson, P.;
Houssier, C.; Bailly, C. Bioorg. Med. Chem. 2001, 9, 1533.
34. Lynch, M. A.; Duval, O.; Sukhanova, A.; Devy, J.; MacKay, S. P.; Waigh, R. D.;
Nabiev, I. Bioorg. Med. Chem. Lett. 2001, 11, 2643.
35. Antonini, I.; Polucci, P.; Magnano, A.; Gatto, B.; Palumbo, M.; Menta, E.; Pescalli,
N.; Martelli, S. J. Med. Chem. 2003, 46, 3109.
36. Farghaly, A. M.; Soliman, F. S. G.; El Semary, M. M.; Rostom, S. A. F. Pharmazie
2001, 56, 28.
37. Errede, L. A.; McBrady, J. J.; Oien, H. T. J. Org. Chem. 1976, 41, 1765.
38. Lehmann, J.; Knoch, F.; Jiang, N. Arch. Pharm. (Weinheim) 1993, 326, 947.
39. Acton, E. M.; Narayanan, V. L.; Risbood, P. A.; Shoemaker, R. H.; Vistica, D. T.;
Boyd, M. R. J. Med. Chem. 1994, 37, 2185.
1. Cozzi, P. Il Farmaco. 2003, 58, 213.
2. Johnston, S. R. D.; Ford, H.; Ross, P. In The Royal Marsden Hospital Hand Book of
Cancer Chemotherapy; Brighton, D., Wood, M., Eds.; Elsevier Churchill
Livingstone: London, New York, Oxford, 2005; pp 1–17.
3. Daidone, G.; Maggio, B.; Raffa, D.; Plescia, S.; Schillaci, D.; Raimondi, M. V. Il
Farmaco. 2004, 59, 413.
4. Chou, L.-C.; Huang, L.-J.; Yang, J.-S.; Lee, F.-Y.; Teng, C.-M.; Kuo, S.-C. Bioorg.
Med. Chem. 2007, 15, 1732.
5. Manetti, F.; Brullo, C.; Magnani, M.; Mosci, F.; Chelli, B.; Crespan, E.; Schenone,
S.; Naldini, A.; Bruno, O.; Trincavelli, M. L.; Maga, G.; Carraro, F.; Martini, C.;
Bondavalli, F.; Botta, M. J. Med. Chem. 2008, 51, 1252.
6. Li, J.; Zhao, Y. F.; Zhao, X. L.; Yuan, X. Y.; Gong, P. Arch. Pharm. Chem. Life Sci.
2006, 339, 593.
7. Xia, Y.; Dong, Z.-W.; Zhao, B.-X.; Ge, X.; Meng, N.; Shin, D.-S.; Miao, J.-Y. Bioorg.
Med. Chem. 2007, 15, 6893.
8. Xia, Y.; Fan, C.-D.; Zhao, B.-X.; Zhao, J.; Shin, D.-S.; Miao, J.-Y. Eur. J. Med. Chem.
2008, 43, 2347.
9. Farag, A. M.; Mayhoub, A. S.; Barakat, S. E.; Bayomi, A. H. Bioorg. Med. Chem.
2008, 16, 881.
10. Schenone, S.; Bruno, O.; Ranise, A.; Bondavalli, F.; Brullo, C.; Fossa, P.; Mosti, L.;
Menozzi, G.; Carraro, F.; Naldini, A.; Bernini, C.; Manettic, F.; Botta, M. Bioorg.
Med. Chem. Lett. 2004, 14, 2511.
40. Freije, M. P.; Lawrence, J. A.; Hollingshead, M. G.; De La Rosa, A.; Narayanan, V.
L.; Grever, M.; Sausville, E. A.; Paull, K.; Steeg, P. S. Nat. Med. 1997, 3, 395.