Synthesis, cytotoxic properties and tubulin polymerization inhibitory activity of novel 2-pyrazoline derivatives

Article abstract of DOI:10.1002/ardp.201100471

A series of novel 1-(3',4',5'-trimethoxybenzoyl)-3,5-diarylpyrazoline derivatives were synthesized and evaluated for their cytotoxic properties on different cancer cell lines and tubulin polymerization inhibitory activity. Compounds 6d and 6e exhibited re

Full text of DOI:10.1002/ardp.201100471

Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
535
Full Paper
Synthesis, Cytotoxic Properties and Tubulin Polymerization
Inhibitory Activity of Novel 2-Pyrazoline Derivatives
Mohamed Abdel-Aziz1, Omar M. Aly1, Sabine S. Khan2, Kamalika Mukherjee3, and Susan Bane3
1 Faculty of Pharmacy, Medicinal Chemistry Department, Minia University, Minia, Egypt
2 SUNY Upstate Medical University, Syracuse, NY, USA
3 Biological and Biophysical Chemistry Department, Binghamton University, Binghamton, NY, USA
A series of novel 1-(30,40,50-trimethoxybenzoyl)-3,5-diarylpyrazoline derivatives were synthesized and
evaluated for their cytotoxic properties on different cancer cell lines and tubulin polymerization
inhibitory activity. Compounds 6d and 6e exhibited remarkable cytotoxic activity against different
cancer cell lines with good tubulin polymerization inhibitory activity. Compound 6d exhibited
moderate selectivity toward renal cancer and breast cancer subpanels with selectivity ratios of
3.06 and 5.11, respectively, at the cytostatic activity (TGI) level. Compounds 6e and 6d achieved
good tubulin polymerization inhibitory activity with IC50 values of 17 and 40 mM, respectively. The
photomicrographs made for compounds 6d and 6e on cellular microtubules indicated that the
cytotoxicity of these compounds can be attributed to their ability to interfere with microtubule
assembly. Molecular modeling studies involving compound 6e with the colchicine binding site of
a,b-tubulin revealed hydrogen-bonding and hydrophobic interactions with several amino acids in the
colchicine binding site of b-tubulin.
Keywords: 2-Pyrazoline / Combretastatin A4 / Cytotoxicity / Tubulin polymerization
Received: December 29, 2011; Revised: February 22, 2012; Accepted: February 24, 2012
DOI 10.1002/ardp.201100471
Introduction
lin by binding to the colchicine-binding site of the b-tubulin
subunit [4]. Tubulin therefore remains an important target
for the design of anticancer agents. Interference with tubu-
lin/microtubule polymerization dynamics has two key anti-
cancer effects: (i) inhibition of cancer cell proliferation
through disturbance of mitotic spindle function, which leads
to cell apoptosis [5], and (ii) disruption of cell signaling path-
ways involved in regulating and maintaining the cytoskele-
ton of endothelial cells in tumor vasculature. It must be
emphasized that the therapeutic effects of these agents such
as CA4 are derived from their vasculature targeting proper-
ties and not antimitotic properties. Because of its structural
simplicity, a wide number of CA4 analogues have been devel-
oped and assessed as potential anticancer agents, some of
which have recently been reviewed [6–16]. Further interest in
combretastatin derivatives was stimulated by the discovery
that CA4 is also able to elicit irreversible vascular shutdown
within solid tumors, leaving normal vasculature intact
[17, 18]. In this way tumors are starved of oxygen and nutrients
and their constituent cells die. Agents that act in this manner
have the potential to make a significant impact on the
clinical management of cancer [19]. A prodrug of combreta-
Cancer is a major cause of death around the world. The WHO
projected 12 million deaths by cancer with existing thera-
peutics by 2030. Out of the 27 types of characterized cancers,
only lung, stomach, liver, colon, and breast cancers are
mainly implicated in cancer mortality. Taking into consider-
ation the existing cancer therapies, chemotherapy has
turned out to be one of the most significant treatments in
cancer management [1]. Moreover, cancer chemotherapy has
entered a new era of molecularly targeted therapeutics,
which is highly selective and not associated with the serious
toxicities of conventional cytotoxic drugs [2]. The combreta-
statins are a group of antimitotic agents isolated from the
bark of the South African tree Combretum caffrum [3]. The most
active of these, combretastatin A4 (CA4) (I), is a potent cyto-
toxic agent that strongly inhibits the polymerization of tubu-
Correspondence: Mohamed Abdel-Aziz, Faculty of Pharmacy, Medicinal
Chemistry Department, Minia University, Minia 61519, Egypt.
E-mail: abulnil@hotmail.com
Fax: þ2086 2369075
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536
M. Abdel-Aziz et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
statin A4, the water-soluble phosphate derivative Zybrestat is
now in phase III clinical trials for the treatment of cancer [20].
Many analogs of CA4 have been designed to study the struc-
ture-activity relationship of the molecule in order to enhance
both the cytotoxic and selective vascular targeting activity
[21–32]. Among synthetic small molecule tubulin inhibitors,
replacement of the olefinic bridge of I with a carbonyl group
furnished a benzophenone-type CA4 analogue named phen-
statin (IIa). This compound demonstrated interesting efficacy
activity in a numerous inhibitors of tubulin polymerization,
such as colchicine, CA4, and podophyllotoxin [51–54]. The
prepared pyrazoline derivatives that carry three different
aryl groups were evaluated for both in vitro cytotoxic proper-
ties on different cancer cell lines and tubulin polymerization
inhibitory activity. Furthermore, molecular modeling studies
of the most active tubulin inhibitor with the colchicine bind-
ing site of a,b-tubulin was also performed.
in a variety of tumor models, while retaining the character- Results and discussion
istics of I [33]. The 2-aminobenzophenone derivative (IIb) also
strongly inhibited cancer cell growth and tubulin polymeri-
zation and caused mitotic arrest, the same as in compound
IIa [34]. A series of methoxy substituted 2-(30,40,50-trimethox-
ybenzoyl)benzo[b]furan [35], -benzo[b]thiophene (IIIa and
IIIb) [36–38] (Chart 1), and indole [39] derivatives were found
to inhibit the growth of different cancer cell lines and tubu-
lin polymerization by binding to the colchicine site of tubu-
lin and caused G2-M phase arrest of the cell cycle. In addition,
pyrazole derivatives are well established in the literature as
important biologically effective heterocyclic compounds.
These derivatives are the subject of many research studies
due to their widespread potential pharmacological activities
such as anti-inflammatory [40], antipyretic [41], antimicrobial
[42], antiviral [43], antitumor [44], anticonvulsant [45], anti-
histaminic [46], and antidepressant [47] activities. Promoted
with the above-mentioned studies and as a continuation of
our research interest in the synthesis and biological activities
of novel pyrazole derivatives [48–50], and in order to further
investigate structural determinants of cytotoxic activity of
this class of compounds, the present work involves a flexible,
concise, and highly convergent protocol for the synthesis of
novel series of 1-(30,40,50-trimethoxybenzoyl)-3,5-diaryl-2-pyr-
azoline derivatives having variously substituted 3- and 5-aryl
rings aimed to investigate the effect of introducing
additional aryl unit, keeping in mind the presence of
30,40,50-trimethoxyphenyl of the 2-benzoyl moiety unchanged
because it is the characteristic structural requirement for
Chemistry
Chalcones (3a–g, Scheme 1) were synthesized by a base cata-
lyzed Claisen-Schmidt condensation reaction of appropri-
ately substituted acetophenones (1a–b) and substituted
aromatic aldehydes (2a–f) in the presence of 10% NaOH in
ethanol. Heating at reflux chalcones (3a–g) with hydrazine
monohydrate 95% in absolute ethanol afforded the corre-
sponding pyrazolines (4a–g). 1H NMR spectra recorded for
the prepared compounds clearly supported the proposed
structures, protons of pyrazoline ring in compounds 4a,
4c, 4d, 4e, 4f, and 4g showed a prominent ABX system,
with protons Ha, Hb, and Hx seen as doublets of doublets
at d 2.96–3.30, 3.40–3.54, and 4.82–5.27 ppm (JHa–Hb ¼ 16.30–
16.86 Hz, JHa–Hx ¼ 7.33–9.34 Hz, and JHb–Hx ¼ 9.90–10.62 Hz),
respectively. On the other hand, pyrazoline 4b with substi-
tuted benzaldehyde carrying highly electronegative groups
(2,6-dichloro) made Ha and Hb that appeared to be equival-
ent, so the pattern became AX system. The CH2 protons
appeared as doublet at d 3.46 ppm while the CH proton
appeared as triplet at d 5.80 ppm (J ¼ 11.90 Hz). The protons
belonging to the aromatic system and phenyl substituents
were observed at the expected chemical shifts and integral
values. Coupling of pyrazolines (4a–g) with 3,4,5-trimethox-
ybenzoyl chloride 5 in CH2Cl2 in the presence of triethyl-
amine afforded the formation of the corresponding 1-(30,40,50-
trimethoxybenzoyl)-3,5-diarylpyrazoline derivatives 6a–g
(Scheme 1). The structure of the prepared compounds was
OMe
MeO
MeO
O
R1
A
MeO
MeO
MeO
R2
O
HO
R1
A
B
MeO
OMe
OMe
B
S
OMe
MeO
MeO
R
1 = H, IIIa
OMe
Combretastatin A4 (CA4), I
R1= OH, IIIb
R1 = H, R2 = OH, Phenstatin, IIa
R1 = NH2, R2 = H, IIb
Chart 1. Different inhibitors for tubulin polymerization.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
Novel 2-Pyrazolines as Tubulin Polymerization Inhibitors
537
OCH3
R
O
O
R
R
Ar
CH3
a
b
Ar-CHO
H3CO
+
H3CO
N
Ar
N
H
3a–g
2a–f
1a–b
4a–g
3a; R = OCH3, Ar = Furyl
3b; R = OCH3, Ar = 2,6-Di-Cl phenyl
4a; R = OCH3, Ar = Furyl
4b; R = OCH3, Ar = 2,6-Di-Cl phenyl
4c; R = OCH3, Ar = 2,4-Di-OCH3 phenyl
4d; R = OCH3, Ar = 4-OCH3 phenyl
4e; R = OCH3, Ar = 4-CN phenyl
4f; R = OCH3, Ar = 3-OH phenyl
4g; R = H, Ar = 3-OH phenyl
1a; R = OCH3
1b; R = H
3c; R = OCH3, Ar = 2,4-Di-OCH3 phenyl
3d; R = OCH3, Ar = 4-OCH3 phenyl
3e; R = OCH3, Ar = 4-CN phenyl
3f; R = OCH3, Ar = 3-OH phenyl
3g; R = H, Ar = 3-OH phenyl
OCH3
R
COCl
H3CO
OCH3
OCH3
5
N
Ar
N
OCH3
OCH3
O
c
OCH3
6a–g
6a; R = OCH3, Ar = Furyl
6b; R = OCH3, Ar = 2,6-Di-Cl phenyl
6c; R = OCH3, Ar = 2,4-Di-OCH3 phenyl
6d; R = OCH3, Ar = 4-OCH3 phenyl
6e; R = OCH3, Ar = 4-CN phenyl
6f; R = OCH3, Ar = 3-OH phenyl
6g; R = H, Ar = 3-OH phenyl
Reagents: (a) 10% NaOH, EtOH; (b) NH2NH2.H2O, EtOH; (c) TEA, CH2Cl2.
Scheme 1. Synthesis of novel pyrazoline derivatives 6a–g.
confirmed by the appearance of an additional strong absorp-
ꢀ1
The results in Table 2 indicate that the pyrazoline deriva-
tive 6e exhibited broad spectrum of cell growth inhibition
activity against most of the tested cell lines.
–
tion band of (C O) stretching at 1570–1585 cm and dis-
–
appearance of that of NH stretching in IR spectra. The 1H NMR
spectra showed additional peaks each for the CH3 protons in
addition to the protons of the phenyl moiety at the expected
aromatic region. The structure of the prepared compounds
was confirmed on the basis of their IR, 1H NMR, 13C NMR,
mass spectra, and elemental analysis.
Pyrazoline derivative 6b exhibited moderate cell growth
inhibition activity against leukemia SR, melanoma SK-MEL-5,
and renal cancer UO-31 cell lines. Compound 6c also exhib-
ited broad-spectrum cell growth inhibition activity against
melanoma MDA-MB-435, renal cancer CAKI-1, and breast
cancer MCF7 cell lines. Pyrazoline derivative 6c revealed
moderate cell growth inhibition against melanoma SK-
MEL-5 and renal cancer UO-31 cell lines.
Biological investigations
Screening of anticancer activity
Compounds 6b, 6c, 6d, and 6e were selected by the National
Cancer Institute (NCI) according to the protocol of the Drug
Evaluation Branch of the National Cancer Institute, Bethesda,
USA for in vitro anticancer screening. Primary in vitro one dose
anticancer assay was performed in full NCI 60 cell lines
derived from nine tumor subpanels, including leukemia,
melanoma, lung, colon, CNS, ovarian, renal, prostate, and
breast cancer cell lines. The selected compounds were added
at a single concentration (10ꢀ5 M) and the culture was incu-
bated for 48 h. End point determinations were made with a
protein binding dye sulforhodamine B (SRB). Results for each
compound were reported as a mean graph of the percent
growth of the treated cells when compared to the untreated
control cells.
The obtained results indicate that the pyrazoline deriva-
tives 6d and 6e exhibited the highest ability to inhibit the
proliferation of different cancer cell lines (Tables 1 and 2)
compared to pyrazoline derivatives 6b and 6c. A different
aldehyde substituent used in the position 5 of the synthe-
sized pyrazoline derivative might contribute to the activity
of the synthesized compounds; the presence of only one
OCH3 group is preferable over the presence of two OCH3
groups (pyrazoline 6d has better anticancer activity against
different cancer cell lines, better than pyrazoline 6c).
Also the presence of an electron withdrawing group (CN)
in pyrazoline 6e has better anticancer activity against differ-
ent cancer cell lines.
The pyrazoline derivative 6d achieved remarkable cell
growth inhibition activity against most of the tested cell
lines and the results were illustrated in Table 1.
In vitro five dose full NCI 60 cell panel assay
The pyrazoline derivatives 6d and 6e were selected for
advanced five dose testing against the full panel of 60 human
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Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
Table 1. One dose mean graph of nine different cancer cell types of compound 6d
Panel
Cell line
Growth percent
Panel
Cell line
Growth percent
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
A549/ATCC
EKVX
HOP-62
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
24.31
5.49
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
ACHN
CAKI-1
RXF-393
SN12C
TK-10
31.49
ꢀ45.13
58.83
66.53
34.16
12.99
30.31
26.74
48.71
12.09
ꢀ21.41
41.49
18.39
21.07
30.53
11.62
27.60
12.55
ꢀ14.04
27.57
36.39
ꢀ32.54
28.31
47.08
50.20
20.62
27.23
48.16
34.76
51.94
46.60
37.72
6.07
Non-small cell lung cancer
Renal cancer
ꢀ35.93
UO-31
PC-3
DU-145
MCF7
Colon cancer
8.03
Prostate cancer
Breast cancer
61.11
20.59
32.84
ꢀ1.10
16.15
20.86
54.74
4.28
ꢀ14.46
38.90
26.05
36.06
57.74
ꢀ11.78
ꢀ26.63
12.61
70.51
5.80
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
CNS cancer
Melanoma
SF-295
SF-539
SNB-19
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
66.30
41.88
tumor cell lines. All the 60 cell lines representing nine tumor
subpanels were incubated at five different concentrations
(0.01, 0.1, 1, 10, and 100 mM). The outcomes were used to
create log concentration versus % growth inhibition curves
and three response parameters (GI50, TGI, and LC50) were
calculated for each cell line. The GI50 value (growth inhibi-
tory activity) corresponds to the concentration of the com-
pound causing 50% decrease in net cell growth, the TGI value
(cytostatic activity) is the concentration of the compound
resulting in total growth inhibition (TGI) and LC50 value
(cytotoxic activity) is the concentration of the compound
causing net 50% loss of initial cells at the end of the incu-
bation period of 48 h. Compound 6d exhibited remarkable
anticancer activity against most of the tested cell lines
representing nine different subpanels. Compound 6d showed
high activity against most of the tested cell lines with GI50
ranging from 0.19 to >100 mM (Table 3). The criterion
for selectivity of a compound depends upon the ratio
obtained by dividing the full panel MID (the average sensi-
tivity of all cell lines toward the test agent) (mM) by
their individual subpanel MID (mM). Ratios between 3
and 6 refer to moderate selectivity; ratios >6 indicate high
selectivity toward the corresponding cell line, while
compounds not meeting either of these criteria rated non-
selective [55]. In this context, compound 6d was found to
have broad-spectrum antitumor activity against the nine
tumor subpanels tested with selectivity ratios ranging
between 0.52 and 5.11 at the GI50 level. Compound 6d
exhibited moderate selectivity toward the renal cancer
subpanel with selectivity ratio of 3.06 at GI50 level and toward
the breast cancer subpanel with selectivity ratio of 5.11
at GI50 level.
Compound 6e exhibited remarkable anticancer activity
against most of the tested cell lines representing nine differ-
ent subpanels with GI50 values between 0.19 and 12.7 mM
(Table 4).
The criterion for selectivity of compound 6e was found to
be broad-spectrum antitumor activity against the nine tumor
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Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
Novel 2-Pyrazolines as Tubulin Polymerization Inhibitors
539
Table 2. One dose mean graph of nine different cancer cell types of compound 6e
Panel
Cell line
Growth
percent
Panel
Cell line
Growth
percent
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
A549/ATCC
EKVX
HOP-62
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
29.95
4.24
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
ACHN
CAKI-1
RXF-393
SN12C
TK-10
13.42
ꢀ47.81
62.13
58.79
32.83
2.92
19.78
25.13
44.56
5.45
ꢀ17.21
53.55
31.92
18.21
31.38
5.75
26.96
18.22
ꢀ18.29
28.63
37.14
ꢀ25.76
20.64
49.49
60.79
28.45
22.03
33.40
36.09
52.41
43.19
36.95
9.81
ꢀ15.98
ꢀ9.28
35.19
13.56
26.67
ꢀ8.40
15.12
16.09
49.33
ꢀ3.96
ꢀ13.27
21.54
33.58
39.46
ꢀ6.86
ꢀ40.37
35.64
65.29
17.74
69.25
43.60
Non-small cell lung cancer
Renal cancer
UO-31
PC-3
DU-145
MCF7
Colon cancer
Prostate cancer
Breast cancer
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
CNS cancer
Melanoma
subpanels tested with selectivity ratios ranging between 0.70
and 1.69 at the GI50 level. Compound 6e exhibited no selec-
tivity toward the tested cell lines.
produces a net increase in the emission intensity of DAPI.
The use of the fluorescence method has the following
advantages: rapid, easy to perform, with good accuracy
and reproducibility, lower sample volumes, and concen-
trations can be used in fluorescence spectroscopy. The sol-
ubility of the tested compounds was first determined in the
assay conditions specified under experimental procedures. It
was found that each drug approached its solubility limit at
40 mM concentration in 2% DMSO. The drugs (40 mM) were
then screened for their potential ability to inhibit micro-
tubule formation. In the polymerization experiments; a fixed
concentration of protein (5 mM) was induced to polymerize
in the presence of a fixed concentration of drug (40 mM). If a
test drug is strongly cytotoxic and the effect is mediated
through microtubules, then we expect to see it affect tubulin
assembly under these conditions. If it does not, then a non-
microtubule mechanism for cytotoxicity is more likely.
Tubulin polymerization inhibitory assay
To investigate whether the cytotoxicity of the synthesized
pyrazoline derivatives were related to the interaction with
tubulin, pyrazolines 6c, 6d, 6e, and 6f were tested in vitro
for tubulin polymerization inhibitory activity (Fig. 1 and
Table 5). Inhibition of paclitaxel-induced tubulin polymeri-
zation was determined by monitoring DAPI fluorescence as
previously described [56, 57]. Bonne et al. [57] demonstrated
that the DNA-binding dye, 40,6-diamidino-2-phenylindole
(DAPI), binds to tubulin at a site distinct from the three major
drug-binding sites, resulting in enhanced DAPI fluorescence.
The affinity of DAPI for assembled tubulin is greater than
its affinity for unpolymerized tubulin, so tubulin assembly
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Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
Table 3. NCI in vitro testing results of compound 6d at five dose level in mM
Panel
Cell line
GI50
TGI
LC50
Conc. per
cell line
Subpanel
MIDb
Selectivity ratio
(MIDa/MIDb)
Conc. per
cell line
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
4.29
3.80
1.78
11.10
4.27
6.16
2.88
7.60
1.67
0.19
0.59
3.08
>100
3.71
0.46
2.50
1.85
2.48
1.32
3.11
2.18
4.11
5.61
0.68
1.15
3.82
1.61
2.48
2.55
83.40
2.40
0.45
3.05
72.2
0.32
9.92
0.75
4.76
0.98
5.66
9.27
3.62
1.04
1.64
3.95
0.54
1.06
0.66
0.34
5.90
4.87
1.03
2.86
4.01
5.23
1.34
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.93
>100
>100
>100
>100
2.29
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Non-small cell lung cancer
A549/ATCC
EKVX
13.35
0.52
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF-393
SN12C
TK-10
Colon cancer
2.51
2.79
7.86
4.77
>100
>100
>100
39.80
>100
>100
>100
4.71
>100
>100
>100
>100
>100
>100
2.30
>100
>100
3.21
>100
>100
>100
2.91
>100
>100
>100
7.85
>100
>100
nd
>100
>100
3.05
>100
>100
>100
>100
>100
CNS cancer
Melanoma
2.56
2.73
0.36
19.45
Ovarian cancer
3.85
2.29
1.82
3.06
Renal cancer
UO-31
PC-3
DU-145
Prostate cancer
2.43
2.88
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Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
Table 3. (continued )
Novel 2-Pyrazolines as Tubulin Polymerization Inhibitors
541
Panel
Cell line
GI50
TGI
LC50
Conc. per
cell line
Subpanel
MIDb
Selectivity ratio
(MIDa/MIDb)
Conc. per
cell line
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
0.63
2.84
1.55
2.28
0.61
0.33
1.37
5.11
>100
>100
>100
21.8
>100
4.66
>100
>100
>100
>100
>100
>100
BT-549
T-47D
MDA-MB-468
MIDa
7.00
MIDa ¼ Average sensitivity of all cell lines in mM.
MIDb ¼ Average sensitivity of all cell lines of a particular subpanel in mM.
nd, Not determined.
Table 4. NCI in vitro testing results of compound 6e at five dose level in mM.
Panel
Cell line
GI50
TGI
LC50
Conc. per
cell line
Subpanel
MIDb
Selectivity ratio
(MIDa/MIDb)
Conc. per
cell line
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
A549/ATCC
EKVX
HOP-62
3.53
1.89
1.30
4.43
3.53
2.28
2.32
4.57
1.53
0.19
0.75
2.93
7.97
2.34
0.18
1.47
2.16
1.76
1.10
2.13
1.03
1.44
2.48
0.40
0.58
1.75
0.55
1.21
2.37
12.7
2.03
0.34
0.71
1.80
0.39
0.70
0.40
2.83
0.70
>100
5.62
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4.81
>100
>100
>100
>100
>100
>100
36.90
0.99
30.6
54.8
85.3
>100
0.49
4.03
6.63
19.5
Non-small cell lung cancer
2.53
0.78
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
Colon cancer
1.58
1.25
>100
>100
>100
>100
95.20
>100
>100
>100
69.90
>100
>100
>100
78.90
>100
>100
>100
39.60
48.90
>100
>100
>100
>100
continued
42.1
10.3
7.44
>100
44.40
7.23
CNS cancer
Melanoma
1.16
2.38
1.69
0.83
SF-295
SF-539
SNB-19
SNB-75
U251
3.35
46.50
11.00
11.90
15.00
62.20
12.40
1.01
4.12
30.20
5.65
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
28.30
28.20
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542
M. Abdel-Aziz et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
Table 4. (continued )
Panel
Cell line
GI50
TGI
LC50
Conc. per
cell line
Subpanel
MIDb
Selectivity ratio
(MIDa/MIDb)
Conc. per
cell line
Ovarian cancer
IGROV1
1.87
1.87
1.05
23.40
>100
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF-393
SN12C
TK-10
0.42
2.09
4.21
2.87
0.75
0.89
2.62
0.37
0.65
0.43
0.42
3.96
3.32
0.46
1.89
3.53
0.48
2.27
0.99
2.09
2.21
0.30
1.70
42.40
>100
18.20
41.50
7.05
23.80
4.34
44.20
25.40
3.03
>100
20.30
17.80
27.40
15.20
79.00
20.80
6.74
7.03
>100
>100
>100
>100
>100
>100
>100
>100
>100
48.00
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Renal cancer
1.53
1.29
UO-31
PC-3
DU-145
MCF7
Prostate cancer
Breast cancer
2.71
1.39
0.73
1.42
MDA-MB-231/ATCC
HS 578T
BT-549
11.00
95.30
1.92
T-47D
MDA-MB-468
MIDa
1.97
MIDa ¼ Average sensitivity of all cell lines in mM.
MIDb ¼ Average sensitivity of all cell lines of a particular subpanel in mM.
Table 5. Inhibition of tubulin assembly induced by compounds 6c–f
(40 mM)
Compound
Percent inhibitiona)
6c
6d
6e
6f
11
15
40
11
a)
Decrease in assembly relative to a control without added drug.
Data were collected as described in Fig. 1.
Figure 1 shows a typical polymerization profile. The relative
potencies of each compound are expressed as the decrease in
the fluorescence signal at the plateau relative to the control
(Table 5).
Figure 1. Effect of compounds 6c–f on paclitaxel-induced tubulin
assembly. Bovine brain tubulin (5 mM) in PME buffer; and 0.1 mM
GTP was incubated with 40 mM of various tested compounds for
40 min at room temperature. Microtubule assembly was then
induced by paclitaxel (5 mM) at 378C. The final concentration of
DMSO in each sample was 2% v/v. Polymerization was monitored
by the change in fluorescence of microtubule-bound DAPI at excita-
tion and emission wavelengths of 360 and 450 nm, respectively. The
curves shown are the control with no ligand (a), and samples con-
taining 40 mM of the tested drug; 6c (c), 6d (b), 6e (e), and 6f (d).
These results indicate that there is a positive correlation
between tubulin polymerization and cytoxicity activity of the
tested pyrazoline derivatives. The results indicate that pyr-
azolines 6e and 6d which exhibited good cytotoxic activity
against different cancer cell lines achieved also good tubulin
polymerization inhibitory activity. Compound 6d exhibited
moderate selectivity toward renal cancer subpanel with selec-
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Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
Novel 2-Pyrazolines as Tubulin Polymerization Inhibitors
543
Table 6. IC50 values for inhibition of tubulin assembly for
compounds 6d and 6e compared to podophyllotoxin
IC50 determination
Pyrazolines 6d and 6e were selected for more detailed
analysis. Due to the low solubility of these molecules,
the assay conditions were modified as described under
experimental procedures to obtain the full dose–response
curve for inhibition of tubulin assembly. An IC50 value was
calculated for each molecule from these data. The IC50
Compound
IC50 (mM) ꢁ SEM
6d
6e
40 ꢁ 2
17 ꢁ 2
Podophyllotoxin
1.3 ꢁ 0.26
of podophyllotoxin,
a potent polymerization inhibitor
that binds to the colchicine site on tubulin [58] was also
determined under identical experimental conditions. The
results are summarized in Table 6.
tivity ratio of 3.06 at GI50 level and toward breast cancer
subpanel with selectivity ratio of 5.11 at GI50 level. Both
cytotoxicity results and tubulin polymerization inhibitory
activity support the fact that pyrazoline derivatives 6d and 6e
are promising anticancer agents and tubulin polymerization
inhibitory activity may be the expected mechanism of action
of these compounds.
Confocal microscopy
To establish the effect of compounds 6d and 6e on cellular
microtubules, PC3 cells were treated with or without ligand
for 24 h, then fixed and immunostained for microtubules
(red) and nucleus (blue) (Fig. 2). The cells with 1% DMSO (no
Figure 2. Confocal microscopy images of PC3 cells treated with 1% DMSO (A), 20 mM compound 6e in 1% DMSO (B), and 20 mM
compound 6d in 1% DMSO (C) for 24 h.
Figure 3. The structure of compound 6e (S-isomer) docked into the colchicine binding site of tubulin.
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544
M. Abdel-Aziz et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
ligand) were uninucleated and exhibited a dense network of
microtubules whereas the cells treated with 20 mM of com-
pound 6d or 6e were multinucleated (indicated by an arrow)
and showed a diffused microtubule network. The photo-
micrographs thus indicate that the cytotoxicity of these
drugs can be attributed to their ability to interfere with
microtubule assembly.
amino acids in the colchicine binding site of b-tubulin.
Finally, the novel synthesized pyrazoline derivatives 6d
and 6e are promising anticancer agents and tubulin
polymerization inhibitory activity may be the expected
mechanism of action of these compounds.
Experimental
Molecular modeling
Chemistry
Molecular docking studies of pyrazoline 6e to tubulin protein
were performed. The X-ray crystal structure of the DAMA–
colchicine–tubulin complex (PDB code 1SA0) was used as the
tubulin protein template. As shown in Fig. 3 (S-isomer of
compound 6e), the pyrazoline bridge moiety of compound
6e causes the ring-A and ring-B to lie in similar positions to
that of the colchicine. In the ring-A (trimethoxyphenyl)
of compound 6e, the –OCH3 substituted on it can form
hydrogen bonds with Cys241 in b-subunit (3.63, 4.16, or
Melting points were determined on Stuart electrothermal melt-
ing point apparatus and were uncorrected. IR spectra were
recorded as KBr disks on a Brukar Vector 22 IR spectropho-
tometer. NMR spectra were carried out on 300 MHz Mercury
300BB NMR spectrophotometer and
a
Bruker Advance
300 MHz NMR spectrometer, using TMS as internal reference.
Chemical shifts (d values are given in parts per million (ppm)
relative to CDCl3 (7.29 for proton and 76.9 for carbon) or DMSO-d6
(2.50 for proton and 39.50 for carbon) and coupling constants (J)
in Hertz. Splitting patterns are designated as follows: s, singlet; d,
doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multip-
let. Accurate masses were obtained on a Micromass LCT mass
spectrometer and on an Agilent Technologies 1100 LC/MSD ion
trap mass spectrometer equipped with an atmospheric pressure
electrospray. The progress of reactions and the purity of the
prepared compounds were monitored by thin-layer chromatog-
raphy (TLC) using Merck 9385 pre-coated aluminum plate silica
gel (Kieselgel 60) 5 cm ꢂ 20 cm plates with a layer thickness of
0.2 mm. The spots were detected by exposure to UV-lamp at
l ¼ 254 nm. Elemental analysis was performed on a Perkin
Elmer 2400 CHN elemental analyzer, and the results were within
ꢁ0.4% of the theoretical values.
˚
4.92 A). The ring-B of compound 6e (dimethoyphenyl) the
p–OCH3 on it can support hydrogen bond interactions with
the backbone at Val315 or Lys352 in b-subunit (distances 2.15
˚
or 2.89 A, respectively). The CN group can accept a hydrogen
˚
bond with the Glu183 (distance 3.97 A). Hydrophobic inter-
actions were also observed between different methyl groups
of compound 6e with different amino acids including
Val181, Leu248, and Leu255 in the colchicine binding site
of b-tubulin.
Chalcones (3a–g) were prepared according to reported pro-
cedure [59].
Conclusions
Compounds (4a–g) were prepared according to reported pro-
cedure [48].
A group of novel 2-pyrazoline derivatives was prepared as
combretastatin A4 analogues and characterized by different
spectroscopic and elemental analysis techniques. The pre-
pared pyrazoline derivatives were evaluated for both anti-
cancer activity on different cancer cells and tubulin
polymerization inhibitory activity. The results revealed
that pyrazoline derivatives 6d and 6e exhibited remarkable
cytotoxic activity against different cancer cell lines. The
results indicate that there is a positive correlation between
tubulin polymerization inhibitory activity and cytoxicity of
the tested pyrazolines 6d and 6e. Compound 6d exhibited
moderate selectivity toward the renal cancer subpanel with
selectivity ratio of 3.06 at GI50 level and toward the breast
cancer subpanel with selectivity ratio of 5.11 at GI50 level. The
IC50 value was calculated for compounds 6e and 6d and was
found to be 17 and 40 mM, respectively. The photomicro-
graphs made for compounds 6d and 6e on cellular micro-
tubules indicate that the cytotoxicity of these compounds is
attributed to their ability to interfere with microtubule
assembly. Molecular modeling studies involving compound
6e with the colchicine binding site of a,b-tubulin revealed
hydrogen-bonding and hydrophobic interactions with several
5-(3-Hydroxyphenyl)-4,5-dihydro-3-(4-methoxyphenyl)-
1H-pyrazole (4g)
Yellowish white crystals (ethanol) in (2.00 g, 74.63% yield), mp
179–1808C. IR (KBr) ymax (cmꢀ1): 3410 (OH), 3322 (NH), 1592
(C N), and 1575 (C–C). 1H NMR (300 MHz, CDCl3) d (ppm):
–
–
3.15 (dd, 1H, J ¼ 16.76 and 7.40 Hz, CH2 of pyrazoline), 3.44
(dd, 1H, J ¼ 16.76 and 10.30 Hz, CH2 of pyrazoline), 3.70 (s,
3H, OCH3), 4.82 (dd, 1H, J ¼ 7.40 and 10.30 Hz, CH of pyrazoline),
6.41–6.68 (m, 5H, Ar–H), 6.94 (dd, 2H, J ¼ 8.10 and 2.10 Hz,
Ar–H), 7.17 (s, 1H, NH), 7.25 (d, 1H, J ¼ 1.83 Hz, Ar–H). MS: m/z
(%) 269 (100) [Mþ1], 268 (100) [Mþ], 267 (68), 155 (60). Anal. Calcd.
for C16H16N2O2: C, 71.62; H, 6.01; N, 10.44. Found: C, 71.73; H, 6.08;
N, 10.44.
General procedure for preparation of compounds (6a–g)
To a stirred solution of pyrazoline derivatives 4a–f (1.0 mmol)
in 20 mL CH2Cl2 was added compound 5 (0.63 g; 2.0 mmol)
followed by triethylamine (0.22 g; 2.2 mmol). The mixture was
stirred for 1 h at room temperature; the reaction was quenched
by the addition of 20 mL saturated NaCl and 20 mL EtOAc.
The aqueous layer was extracted with (2 ꢂ 20 mL) EtOAc, the
combined EtOAc extracts were washed subsequently with 10 mL
1 N HCl (2 ꢂ 10 mL) distilled H2O, 10 mL NaHCO3 (2 ꢂ 10 mL)
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Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
Novel 2-Pyrazolines as Tubulin Polymerization Inhibitors
545
distilled H2O and 10 mL saturated NaCl, and dried over MgSO4.
After complete evaporation of the organic solvents using a
vacuum pump the crude product was purified by silica gel
column chromatography using ethyl acetate/n-hexane as a
mobile phase to give compounds 6a–g.
42.65 (CH2), 57.37 (CH3), 57.55 (CH3), 57.86 (CH3), 57.98 (CH3),
58.17 (2CH3), 59.29 (CH3), 62.83 (CH), 100.56 (CH), 105.69 (CH),
109.90 (CH), 112.06 (CH), 121.81 (CH), 123.05 (CH), 125.78 (CH),
127.93 (C), 130.50 (C), 141.41 (C), 150.01 (C), 151.97 (C), 153.11 (C),
156.41 (C), 157.97 (C), 161.07 (C), 165.36 (CO). MS (ESI): 537.3
(C29H33N2O8, [MþH]þ). Anal. Calcd. for C29H32N2O8: C, 64.91; H,
6.01; N, 5.22. Found: C, 64.66; H, 5.97; N, 5.12.
5-(2-Furyl)-4,5-dihydro-1-(3,4,5-trimethoxybenzoyl)-3-
(3,4-dimethoxyphenyl)-1H-pyrazole (6a)
1-(3,4,5-Trimethoxybenzoyl)-4,5-dihydro-5-(4-
The residue was chromatographed with ethyl acetate/n-hexane
(1:4) as eluent to give 6a as a white powder in (0.39 g, 83.69%
methoxyphenyl)-3-(3,4-dimethoxyphenyl)-1H-pyrazole (6d)
The residue was chromatographed with ethyl acetate/n-hexane
(1:3) as eluent to give 6d as a yellow powder in (0.41 g, 80.93%
yield), mp 1808C. IR (KBr) ymax (cmꢀ1): 1615 (C N), 1578 (C O).
–
–
–
–
1H NMR (300 MHz, CDCl3) d (ppm): 3.50 (dd, 1H, J ¼ 17.40 and
5.10 Hz, CH2 of pyrazoline), 3.65 (dd, 1H, J ¼ 17.40 and 11.40 Hz,
CH2 of pyrazoline), 3.92 (s, 3H, CH3), 3.94 (s, 9H, 3CH3), 3.96 (s, 3H,
CH3), 5.94 (dd, 1H, J ¼ 5.10 and 11.40 Hz, CH of pyrazoline), 6.35–
6.37 (m, 1H, Ar–H), 6.44 (d, 1H, J ¼ 3.30 Hz, Ar–H), 6.91 (d, 1H,
J ¼ 8.10 Hz, Ar–H), 7.22 (dd, 1H, J ¼ 8.10 and 1.80 Hz, Ar–H), 7.35
(s, 1H, Ar–H), 7.41 (d, 1H, J ¼ 1.80 Hz, Ar–H), 7.47 (s, 2H, Ar–H).
13C NMR (75 MHz, CDCl3) d (ppm): 39.53 (CH2), 57.03 (CH3), 57.86
(CH3), 58.01 (CH3), 58.18 (2CH3), 62.84 (CH), 109.24 (CH), 109.50
(CH), 109.90 (CH), 111.94 (CH), 112.09 (CH), 121.91 (CH), 125.32
(CH), 129.98 (C), 141.59 (C), 142.93 (C), 150.09 (C), 152.18 (C),
152.89 (C), 153.11 (C), 155.57 (C), 165.80 (CO). MS (ESI): 467.3
(C25H27N2O7, [MþH]þ). Anal. Calcd. for C25H26N2O7: C, 64.37; H,
5.62; N, 6.01. Found: C, 64.14; H, 5.66; N, 5.97.
yield), mp 93–948C. IR (KBr) ymax (cmꢀ1): 1612 (C N) and 1583 (C
–
–
–
–
O). 1H NMR (300 MHz, CDCl3) d (ppm): 3.23 (dd, 1H, J ¼ 17.40 and
5.20 Hz, CH2 of pyrazoline), 3.70–3.74 (m, 1H, CH2 of pyrazoline),
3.81 (s, 3H, CH3), 3.90 (s, 3H, CH3) 3.94 (s, 6H, 2CH3), 3.95 (s, 6H,
2CH3), 5.76–5.81 (m, 1H, CH of pyrazoline), 6.85–6.91 (m, 3H,
Ar–H), 7.17–7.22 (m, 1H, Ar–H), 7.21–7.28 (m, 2H, Ar–H), 7.44 (s,
1H, Ar–H), and 7.49 (s, 2H, Ar–H). 13C NMR (75 MHz, CDCl3) d
(ppm): 43.53 (CH2), 57.29 (CH3), 57.87 (CH3), 58.01 (CH3), 58.18
(CH3), 60.36 (CH3), 62.84 (CH3) 63.19 (CH), 109.52 (CH), 109.90
(CH), 112.10 (CH), 115.59 (CH), 121.91 (CH), 125.45 (CH), 128.16
(C), 130.21 (C), 135.19 (C), 141.51 (C), 150.01 (C), 152.15 (C), 153.11
(C), 155.74 (C), 159.80 (C), 165.53 (CO). MS (ESI): 507.3
(C28H31N2O7, [MþH]þ). Anal. Calcd. for C28H30N2O7: C, 66.39;
H, 5.97; N, 5.53. Found: C, 66.25; H, 6.23; N, 5.25.
5-(2,6-Dichlorophenyl)-4,5-dihydro-1-(3,4,5-trimethoxy-
5-(4-Cyanophenyl)-4,5-dihydro-1-(3,4,5-trimethoxy-
benzoyl)-3-(3,4-dimethoxyphenyl)-1H-pyrazole (6b)
The residue was chromatographed with ethyl acetate/n-hexane
(1:3) as eluent to give 6b as a white solid in (0.44 g, 80.73% yield),
benzoyl)-3-(3,4-dimethoxyphenyl)-1H-pyrazole (6e)
The residue was chromatographed with ethyl acetate/n-hexane
(1:3) as eluent to give 6e as a white powder in (0.41 g, 81.74%
mp 191–1928C. IR (KBr) ymax (cmꢀ1): 1608 (C N), 1580 (C O).
–
–
–
–
1H NMR (300 MHz, CDCl3) d (ppm): 3.41 (dd, 1H, J ¼ 17.40 and
8.70 Hz, CH2 of pyrazoline), 3.77 (dd, 1H, J ¼ 17.40 and 12.60 Hz,
CH2 of pyrazoline), 3.92 (s, 3H, CH3), 3.93 (s, 3H, CH3), 3.94 (s, 6H,
2CH3), 3.96 (s, 3H, CH3), 6.48 (dd, 1H, J ¼ 8.70 and 12.60 Hz, CH of
pyrazoline), 6.92 (d, 1H, J ¼ 8.40 Hz, Ar–H), 7.16 (d, 1H,
J ¼ 7.80 Hz, Ar–H), 7.21 (d, 1H, J ¼ 8.4 Hz, Ar–H), 7.29 (d, 1H,
J ¼ 7.80 Hz, Ar–H), 7.40 (s, 1H, Ar–H), 7.42 (d, 1H, J ¼ 1.80 Hz,
Ar–H), 7.46 (s, 2H, Ar–H). 13C NMR (75 MHz, CDCl3) d (ppm): 40.23
(CH2), 57.88 (CH3), 58.02 (CH3), 58.15 (2CH3), 60.03 (CH3) 62.83
(CH), 109.58 (CH), 109.93 (CH), 112.14 (CH), 121.73 (CH), 125.29
(CH), 129.69 (CH), 129.74 (CH), 130.19 (C), 131.07 (C), 134.79 (C),
135.97 (C), 137.23 (C), 141.62 (C), 150.12 (C), 152.12 (C), 153.10 (C),
155.15 (C), 165.84 (CO). MS (ESI): 545.2 (C27H27Cl2N2O6, [MþH]þ).
Anal. Calcd. for C27H26Cl2N2O6: C, 59.46; H, 4.80; N, 5.14. Found:
C, 59.43; H, 5.04; N, 5.15.
–
–
yield), mp 188–1898C. IR (KBr) ymax (cmꢀ1): 2240 (CN), 1610 (C N),
1578 (C O). 1H NMR (300 MHz, CDCl3) d (ppm): 3.18 (dd, 1H,
–
–
J ¼ 17.70 and 5.10 Hz, CH2 of pyrazoline), 3.82–3.89 (m, 1H, CH2
of pyrazoline), 3.94 (s, 3H, CH3), 3.95 (s, 6H, 2CH3), 3.96 (s, 6H,
2CH3), 5.85 (dd, 1H, J ¼ 5.10 and 11.70 Hz, CH of pyrazoline), 6.91
(d, 1H, J ¼ 8.10 Hz, Ar–H), 7.19 (dd, 1H, J ¼ 8.1, 1.8 Hz, Ar–H),
7.41–7.48 (m, 3H, Ar–H), 7.51 (s, 2H, Ar–H), 7.65–7.68 (m, 2H,
Ar–H). 13C NMR (75 MHz, CDCl3) d (ppm): 43.20 (CH2), 57.88 (CH3),
58.04 (CH3), 58.21 (2CH3), 62.87 (CH3), 63.43 (CH), 109.55 (CH),
109.87 (CH), 112.15 (CH), 112.96 (CH), 119.80 (CN), 122.02 (CH),
124.87 (CH), 127.73 (C), 129.42 (C), 133.95 (C), 141.89 (C),
147.95 (C), 150.20 (C), 152.45 (C), 153.21 (C), 155.52 (C), 165.59
(CO). MS (ESI): 502.3 (C28H28N3O6, [MþH]þ). Anal. Calcd.
for C28H27N3O6: C, 67.05; H, 5.43; N, 8.38. Found: C, 66.91; H,
5.59; N, 8.37.
1-(3,4,5-Trimethoxybenzoyl)-4,5-dihydro-5-(2,4-
5-(3-Hydroxyphenyl)-4,5-dihydro-1-(3,4,5-trimethoxy-
dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)-1H-pyrazole (6c)
The residue was chromatographed with ethyl acetate/n-hexane
(1:4) as eluent to give 6c as a yellow crystals in (0.43 g, 80.13%
benzoyl)-3-(3,4-dimethoxyphenyl)-1H-pyrazole (6f)
The residue was chromatographed with ethyl acetate/n-hexane
(1:2) as eluent to give 6f as a white powder in (0.40 g, 81.30%
yield), mp 1058C. IR (KBr) ymax (cmꢀ1): 1615 (C O) and 1585 (C
yield), mp 118–1198C. IR (KBr) ymax (cmꢀ1): 3250 (OH), 1609 (C N),
–
–
–
–
–
–
and 1570 (C O). H NMR (300 MHz, CDCl ) d (ppm): 3.14 (dd, 1H,
O). 1H NMR (300 MHz, CDCl3) d (ppm): 3.11 (dd, 1H, J ¼ 17.40 and
4.80 Hz, CH2 of pyrazoline), 3.68–3.74 (m, 1H, CH2 of pyrazoline),
3.79 (s, 3H, CH3), 3.80 (s, 3H, CH3), 3.88 (s, 3H, CH3), 3.93 (s, 3H,
CH3), 3.94 (s, 3H, CH3), 3.95 (s, 6H, 2CH3), 5.98–6.03 (m, 1H, CH of
pyrazoline), 6.45 (dd, 1H, J ¼ 8.40 and 2.40 Hz, Ar–H), 6.51 (s, 1H,
Ar–H), 6.89 (d, 1H, J ¼ 8.40 Hz, Ar–H), 7.10 (d, 1H, J ¼ 8.40 Hz,
Ar–H), 7.19 (dd, 1H, J ¼ 8.40 and 1.80 Hz, Ar–H), 7.41 (s, 1H,
Ar–H), 7.53 (s, 2H, Ar–H). 13C NMR (75 MHz, CDCl3) d (ppm):
1
–
–
3
J ¼ 17.40 and 4.50 Hz, CH2 of pyrazoline), 3.77–3.80 (m, 1H,
CH2 of pyrazoline), 3.81 (s, 3H, CH3), 3.82 (s, 3H, CH3), 3.84 (s,
3H, CH3), 3.87 (s, 6H, 2CH3), 5.66 (dd, 1H, J ¼ 4.50 and 11.70 Hz,
CH of pyrazoline), 6.63–6.71 (m, 3H, Ar–H), 6.93 (d, 1H,
J ¼ 8.40 Hz, Ar–H), 7.09 (t, 1H, J ¼ 7.8 Hz, Ar–H), 7.21 (dd, 1H,
J ¼ 7.8, 2.10 Hz, Ar–H), 7.32 (d, 1H, J ¼ 1.80 Hz, Ar–H), 7.41
(s, 2H, Ar–H), 8.04 (s, 1H, OH). 13C NMR (75 MHz, CDCl3)
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M. Abdel-Aziz et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
d (ppm): 43.51 (CH2), 62.42 (CH3), 62.67 (CH3), 62.93 (2 CH3), 67.21
(CH3), 68.06 (CH), 114.22 (CH), 115.18 (CH), 115.23 (CH), 117.56
(CH), 118.44 (CH), 120.63 (CH), 122.26 (CH), 126.80 (CH), 130.04
(C), 135.22 (C), 135.72 (C), 146.06 (C), 149.68 (C), 154.76 (C), 156.86
(C), 157.85 (C), 160.90 (C), 163.52 (C), 169.46 (CO). MS (ESI): 493.3
(C27H29N2O7, [MþH]þ). Anal. Calcd. for C27H28N2O7: C, 65.84; H,
5.73; N, 5.69. Found: C, 65.55; H, 5.82; N, 5.57.
treated cells (measured by SRB staining) as compared to the
net protein increase seen in the control cells. The drug concen-
tration resulting in TGI was calculated from Ti ¼ Tz. The LC50
(concentration of drug resulting in a 50% reduction in the
measured protein at the end of the drug treatment as compared
to that at the beginning) indicating a net loss of cells following
treatment was calculated from [(Ti ꢀ Tz)/Tz] ꢂ 100 ¼ ꢀ50. Values
were calculated for each of these three parameters if the level of
activity is reached; however, if the effect was not reached or was
exceeded, the value for that parameter was expressed as more or
less than the maximum or minimum concentration tested. The
log GI50, log TGI, and log LC50 were then determined, defined as
the mean of the logs of the individual GI50, TGI, and LC50 values.
The lowest values are obtained with the most sensitive
cell lines. Compound having logGI50 values ꢀ4 and <ꢀ4 was
declared to be active.
5-(3-Hydroxyphenyl)-4,5-dihydro-1-(3,4,5-trimethoxy-
benzoyl)-3-(4-methoxyphenyl)-1H-pyrazole (6g)
The residue was chromatographed with ethyl acetate/n-hexane
(1:2) as eluent to give 6g as a white powder in (0.37 g, 89% yield),
mp 189–1918C. IR (KBr) ymax (cmꢀ1): 3380 (OH), 1610 (C N), and
–
–
1580 (C O). 1H NMR (300 MHz, CDCl3) d (ppm): 3.14 (dd, 1H,
–
–
J ¼ 17.40 and 4.50 Hz, CH2 of pyrazoline), 3.71 (dd, 1H, J ¼ 17.40
and 11.40 Hz, CH2 of pyrazoline), 3.86 (s, 3H, CH3), 3.91 (s, 3H,
CH3), 3.95 (s, 6H, 2CH3), 5.70 (dd, 1H, J ¼ 4.50 and 11.40 Hz, CH of
pyrazoline), 6.66 (dd, 1H, J ¼ 7.80 and 1.50 Hz, Ar–H), 6.76–6.80
(m, 2H, Ar–H), 6.93 (d, 2H, J ¼ 8.7 Hz, Ar–H), 7.12 (t, 1H,
J ¼ 7.8 Hz, Ar–H), 7.48 (s, 2H, Ar–H), 7.67 (d, 2H, J ¼ 8.7 Hz,
Ar–H), 8.10 (s, 1H, OH). 13C NMR (75 MHz, CDCl3) d (ppm):
43.73 (CH2), 57.43 (CH3), 58.23 (CH3), 62.42 (CH3), 62.84 (CH3),
63.64 (CH), 109.56 (CH), 114.47 (CH), 115.55 (CH), 116.33 (CH),
117.54 (CH), 124.92 (CH), 129.51 (CH), 129.92 (C), 131.27 (C),
141.62 (C), 143.98 (C), 153.15 (C), 156.68 (C), 157.82 (C), 162.32
(C), 166.20 (CO). MS (ESI): 463.3 (C26H27N2O6, [MþH]þ). Anal.
Calcd. for C26H26N2O6: C, 67.52; H, 5.67; N, 6.06. Found: C,
67.42; H, 5.87; N, 6.01.
Assay of microtubule assembly
Solubility of the tested compounds
A stock solution of each compound was prepared in DMSO. Each
compound stock was diluted in PME buffer {100 mM PIPES,
1 mM MgSO4, 2 mM ethylene glycol bis[b-aminoethyl
ether]N,N0-tetraacetic acid (EGTA), pH 6.90 at 258C} to yield con-
centrations up to 80 mM and a final DMSO concentration of
2–8%. Samples of identical concentrations were also prepared
in 100% DMSO. Absorbance spectra of these samples were
obtained using a HP 8453 UV–Vis spectrophotometer. The
compound’s aqueous solubility was assessed by the overlaid
absorbance spectra of the 2–8% and 100% DMSO samples for
the same concentration. Significant changes in light scattering
of the 2–8% DMSO sample compared to the 100% DMSO sample
suggested insolubility of the compound under experimental
conditions.
Biolgical evaluation
Anticancer activity
The methodology of the NCI anticancer screening has been
described in detail elsewhere (http://www.dtp.nci.nih.gov).
Briefly, the primary anticancer assay was performed at approxi-
mately 60 human tumor cell lines panel derived from nine
neoplastic diseases, in accordance with the protocol of the
Drug Evaluation Branch, National Cancer Institute, Bethesda.
Tested compounds were added to the culture at a single concen-
tration (10ꢀ5 M) and the cultures were incubated for 48 h. End
point determinations were made with a protein binding dye,
SRB. Results for each tested compound were reported as the
percent of growth of the treated cells when compared to the
untreated control cells. The percentage growth was evaluated
spectrophotometrically versus controls not treated with test
agents. The cytotoxic and/or growth inhibitory effects of the
most active selected compound were tested in vitro against
the full panel of about 60 human tumor cell lines at 10-fold
dilutions of five concentrations ranging from 10ꢀ4 to 10ꢀ8 M. A
48-h continuous drug exposure protocol was followed and an
SRB protein assay was used to estimate cell viability or growth.
Using the seven absorbance measurements [time zero (Tz), con-
trol growth in the absence of drug (C), and test growth in the
presence of drug at the five concentration levels (Ti)], the per-
centage growth was calculated at each of the drug concen-
trations levels. Percentage growth inhibition was calculated
as: [(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 for concentrations for which
Ti > Tz, and [(Ti ꢀ Tz)/Tz] ꢂ 100 for concentrations for which
Ti < Tz. Three dose–response parameters were calculated for
each compound. Growth inhibition of 50% (GI50) was calculated
from [(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 ¼ 50, which is the drug concen-
tration resulting in a 50% lower net protein increase in the
Screening for tubulin polymerization inhibition [60]
DMSO or 40 mM of the ligand in DMSO was added to bovine brain
tubulin (final concentration of 5 mM) in PME buffer and incu-
bated for 40 min at room temperature in a 96-well plate. GTP and
DAPI were added to obtain final concentrations of 0.1 mM and
10 mM, respectively. The samples were equilibrated to 378C in
the SynergyMx microplate reader for 5 min and the fluorescence
was recorded to establish a baseline. The excitation and emission
wavelengths were 360 and 450 nm, respectively. Paclitaxel in
DMSO was added to a final concentration of 5 mM to induce
microtubule assembly. The total concentration of DMSO was
limited to 4% v/v; ligand and paclitaxel were each added in
2% DMSO of the final volume. The extent of polymerization
was monitored over time by an increase in fluorescence at
450 nm.
IC50 determination
Bovine brain tubulin (8 mM), GTP (1 mM), and DAPI (10 mM) in
PME buffer were equilibrated to 378C in the 96-well plates. The
excitation and emission wavelengths were set at 360 and
450 nm, respectively, and the fluorescence was recorded to
obtain a baseline. Varying concentration of the ligand in
DMSO was added and the increase in fluorescence was monitored
over time. The control contained tubulin, GTP, DAPI, and DMSO
but no ligand. The final concentration of DMSO was 8% v/v in all
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Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
Novel 2-Pyrazolines as Tubulin Polymerization Inhibitors
547
samples. The extent of polymerization versus the log of ligand
concentration was plotted, and the IC50 value was calculated
from the sigmoidal fit of the dose–response curve.
[6] K. Odlo, J. Hentzen, J. F. dit Chabert, S. Ducki, O. Gani, I.
Sylte, M. Skrede, V. A. Florenes, T. V. Hansen, Bioorg. Med.
Chem. 2008, 16, 4829–4838.
[7] D. Simoni, R. Romagnoli, R. Baruchello, R. Rondanin, G.
Grisolia, M. Eleopra, M. Rizzi, M. Tolomeo, G. Giannini, D.
Alloatti, M. Castorina, M. Marcellini, J. Med. Chem. 2008, 51,
6211–6215.
Confocal microscopy
PC3 cells were grown on Lab-Tek II chambered cover glass slips
for 24 h and then treated with 1% DMSO or 20 mM ligand in 1%
DMSO for 24 h. Cells were washed three times with PBS (10 mM
sodium phosphate, pH 7.2, 0.9% sodium chloride w/v) and then
treated with methanol/acetone (1:1 v/v) for 10 min at 48C. The
cells were washed again three times with PBS and incubated with
5% bovine serum albumin (BSA) in PBST (PBS with 0.1% Tween)
for 30 min. The cells were then incubated with mouse mono-
clonal anti-a tubulin antibody (Zymed Technologies) for 1 h and
with Alexafluor 647 goat anti-mouse IgG (Molecular probes) for
45 min at room temperature. The nucleus was stained with DAPI
(0.1 mg/mL) and Gel/Mount (Biomedia Corp.) was added before
imaging. In between each step, the cells were washed with PBS
thrice. Photomicrographs were obtained using a Zeiss LSM 510
META confocal scanning laser microscope.
[8] D. Simoni, R. Romagnoli, R. Baruchello, R. Rondanin, M.
Rizzi, M. G. Pavani, D. Alloatti, G. Giannini, M. Marcellini, T.
Riccioni, M. Castorina, M. B. Guglielmi, F. Bucci, P.
Carminati, C. Pisano, J. Med. Chem. 2006, 49, 3143–3152.
[9] G. C. Tron, T. Pirali, G. Sorba, F. Pagliai, S. Bussacca, A. A.
Genazzani, J. Med. Chem. 2006, 49, 3033–3044.
[10] Q. Zhang, Y. Peng, X. I. Wang, S. M. Keenan, S. Arora, W. J.
Welsh, J. Med. Chem. 2007, 50, 749–754.
[11] J. P. Liou, Y. L. Chang, F. M. Kuo, C. W. Chang, H. Y. Tseng,
C. C. Wang, Y. N. Yang, J. Y. Chang, S. J. Lee, H. P. Hsieh, J. Med.
Chem. 2004, 47, 4247–4257.
[12] G. De Martino, M. C. Edler, G. La Regina, A. Coluccia, M. C.
Barbera, D. Barrow, R. I. Nicholson, G. Chiosis, A. Brancale,
E. Hamel, M. Artico, R. Silvestri, J. Med. Chem. 2006, 49, 947–
954.
Molecular docking
All the molecular modeling studies were carried out on an Intel
CoreTM i3 processor, 3 GB memory with Windows 7 operating
system using Molecular Operating Environment (MOE 2008,
Chemical Computing Group, Canada) as the computational soft-
[13] J. X. Duan, X. Cai, F. Meng, L. Lan, C. Hart, M. Matteucci,
J. Med. Chem. 2007, 50, 1001–1006.
[14] N. H. Nam, Curr. Med. Chem. 2003, 10, 1697–1722.
[15] L. Sun, N. I. Vasilevich, J. A. Fuselier, S. J. Hocart, D. H. Coy,
Bioorg. Med. Chem. Lett. 2004, 14, 2041–2046.
ware. All the minimizations were performed with MOE until a
˚
RMSD gradient of 0.05 kcal molꢀ1 ꢀ1 with MMFF94X force-field
A
and the partial charges were automatically calculated.
The X-ray crystallographic structure of tublin complexed with
DAMA–colchicine (PDB ID: 1SA0) was obtained from the protein
data bank. The enzyme was prepared for docking studies where:
(i) Ligand molecule was removed from the enzyme active site. (ii)
Hydrogen atoms were added to the structure with their standard
geometry. (iii) MOE Alpha Site Finder was used for the active sites
search in the enzyme structure and dummy atoms were created
from the obtained alpha spheres. (iv) The obtained model was
then used in predicting the ligand–enzyme interactions at the
active site.
[16] H. P. Hsieh, J. P. Liou, N. Mahindroo, Curr. Pharm. Des. 2005,
11, 1655–1677.
[17] D. J. Chaplin, G. J. Dougherty, Br. J. Cancer 1999, 80, 57–64.
[18] G. M. Tozer, C. Kanthou, C. S. Parkins, S. A. Hill, Int. J. Exp.
Pathol. 2002, 83, 21–38.
[19] C. Kanthou, G. M. Tozer, Exp. Opin. Ther. Targets 2007, 11,
1443–1457.
[20] D. W. Siemann, D. J. Chaplin, P. A. Walicke, Exp. Opin. Invest.
Drugs 2009, 18, 189–197.
[21] R. LeBlanc, J. Dickson, T. Brown, M. Stewart, H. N. Pati, D.
VanDerveer, H. Arman, J. Harris, W. Pennington, H. L. Holt,
Jr M. Lee, Bioorg. Med. Chem. 2005, 13, 6025–6034.
Authors thank the Development Therapeutics Program of the National
Cancer Institute, Bethesda, MD, USA, for in vitro evaluation of anticancer
activity.
[22] C. M. Lin, S. B. Singh, P. S. Chu, R. O. Dempcy, J. M. Schmidt,
G. R. Pettit, E. Hamel, Mol. Pharmacol. 1988, 34, 200–208.
[23] K. Ohsumi, T. Hatanaka, K. Fujita, R. Nakagawa, Y. Fukuda,
Y. Nihei, Y. Suga, Y. Morinaga, Y. Akiyama, T. Tsuji, Bioorg.
Med. Chem. Lett. 1998, 8, 3153–3158.
The authors have declared no conflict of interest.
[24] L. Wang, K. W. Woods, Q. Li, K. J. Barr, R. W. McCroskey, S. M.
Hannick, L. Gherke, R. B. Credo, Y. H. Hui, K. Marsh, R.
Warner, J. Y. Lee, N. Zielinski-Mozng, D. Frost, S. H.
Rosenberg, H. L. Sham, J. Med. Chem. 2002, 45, 1697–1711.
References
[1] M. Harrison, K. Holen, G. Liu, Clin. Adv. Hematol. Oncol. 2009, 7,
54–64.
[25] G. R. Pettit, M. R. Rhodes, D. L. Herald, E. Hamel, J. M.
Schmidt, R. K. Pettit, J. Med. Chem. 2005, 48, 4087–4099.
[2] P. A. Gehrig, V. L. Bae-Jump, Gynecol. Oncol. 2010, 116, 187–
194.
[26] G. R. Pettit, M. P. Grealish, D. L. Herald, M. R. Boyd, E. Hamel,
R. K. Pettit, J. Med. Chem. 2000, 43, 2731–2737.
[3] G. R. Pettit, S. B. Singh, E. Hamel, C. M. Lin, D. S. Alberts, D.
Garcia-Kendall, Experientia 1989, 45, 209–211.
[27] M. Medarde, A. C. Ramos, E. Caballero, R. Pelaez-Lamamie de
Clairac, J. L. Lopez, D. G. Gravalos, A. S. Feliciano, Bioorg. Med.
Chem. Lett. 1999, 9, 2303–2308.
[4] J. A. Woods, J. A. Hadfield, G. R. Pettit, B. W. Fox, A. T.
McGown, Br. J. Cancer 1995, 71, 705–711.
[5] E. K. Rowinsky, R. C. Donehower, Pharmacol. Ther. 1991, 52,
35–84.
[28] N. H. Nam, Y. Kim, Y. J. You, D. H. Hong, H. M. Kim, B. Z. Ahn,
Bioorg. Med. Chem. Lett. 2001, 11, 1173–1176.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

548
M. Abdel-Aziz et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 535–548
[29] N. H. Nam, Y. Kim, Y. J. You, D. H. Hong, H. M. Kim, B. Z. Ahn,
Bioorg. Med. Chem. Lett. 2002, 12, 1955–1958.
[42] S. Bondock, W. Khalifa, A. A. Fadda, Eur. J. Med. Chem. 2011,
46, 2555–2561.
[30] C. C. Kuo, H. P. Hsieh, W. Y. Pan, C. P. Chen, J. P. Liou, S. J. Lee,
Y. L. Chang, L. T. Chen, C. T. Chen, J. Y. Chang, Cancer Res.
2004, 64, 4621–4628.
[43] S. L. Janus, A. Z. Magdif, B. P. Erik, N. Claus, Monatsh. Chem.
1999, 130, 1167–1174.
[44] H. J. Park, K. Lee, S. J. Park, B. Ahn, J. C. Lee, H. Cho, K. I. Lee,
Bioorg. Med. Chem. Lett. 2005, 15, 3307–3312.
[31] C. Borrel, S. Thoret, X. Cachet, D. Guenard, F. Tillequin, M.
Koch, S. Michel, Bioorg. Med. Chem. 2005, 13, 3853–3864.
[45] V. Michon, C. H. Du Penhoat, F. Tombret, J. M. Gillardin, F.
Lepage, L. Berthon, Eur. J. Med. Chem. 1995, 30, 147–155.
[32] X. Ouyang, E. L. Piatnitski, V. Pattaropong, X. Chen, H. Y. He,
A. S. Kiselyov, A. Velankar, J. Kawakami, M. Labelle, L. Smith,
II J. Lohman, S. P. Lee, A. Malikzay, J. Fleming, J. Gerlak, Y.
Wang, R. L. Rosler, K. Zhou, S. Mitelman, M. Camara, D.
Surguladze, J. F. Doody, M. C. Tuma, Bioorg. Med. Chem. Lett.
2006, 16, 1191–1196.
[46] I. Yildirim, N. Ozdemir, Y. Akc¸amur, M. Dinc¸er, O. Andac¸,
Acta Crystallogr. 2005, E61, o256–o258
[47] D. M. Bailey, P. E. Hansen, A. G. Hlavac, E. R. Baizman, J.
Pearl, A. F. DeFelice, M. E. Feigenson, J. Med. Chem. 1985, 28,
256–260.
[33] G. R. Pettit, B. Toki, D. L. Herald, P. Verdier-Pinard,
M. R. Boyd, E. Hamel, R. K. Pettit, J. Med. Chem. 1998, 41,
1688–1695.
[48] M. Abdel-Aziz, A. M. Gamal-Eldeen, Pharm. Biol. 2009, 47,
854–863.
[49] M. E. Shoman, M. Abdel-Aziz, O. M. Aly, H. H. Farag, M. A.
Morsy, Eur. J. Med. Chem. 2009, 44, 3068–3076.
[34] J. P. Liou, C. W. Chang, J. S. Song, Y. N. Yang, C. F. Yeh, H. Y.
Tseng, Y. K. Lo, Y. L. Chang, C. M. Chang, H. P. Hsieh, J. Med.
Chem. 2002, 45, 2556–2562.
[50] el-S. M. M. N. Abdel-Hafez, Gel-D. A. A. Abuo-Rahma, M.
Abdel-Aziz, M. F. Radwan, H. H. Farag, Bioorg. Med. Chem.
2009, 17, 3829–3837.
[35] R. Romagnoli, P. G. Baraldi, T. Sarkar, M. D. Carrion, O. Cruz-
Lopez, C. Lopez-Cara, M. Tolomeo, S. Grimaudo, A.
Di Cristina, M. R. Pipitone, J. Balzarini, R. Gambari, L.
Ilaria, R. Saletti, A. Brancale, E. Hamel, Bioorg. Med. Chem.
2008, 16, 8419–8426.
[51] N. Mahindroo, J. P. Liou, J. Y. Chang, H. P. Hsieh, Expert Opin.
Ther. Pat. 2006, 16, 647–691.
[52] M. A. Jordan, L. Wilson, Nat. Rev. Cancer 2004, 4, 253–265.
[36] K. G. Pinney, A. D. Bounds, K. M. Dingerman, V. P. Mocharla,
G. R. Pettit, R. Bai, E. Hamel, Bioorg. Med. Chem. Lett. 1999, 9,
1081–1086.
[53] A. Chaudhary, S. N. Pandeya, P. Kumar, P. P. Sharma, S.
Gupta, N. Soni, K. K. Verma, G. Bhardwaj, Mini-Rev. Med. Chem.
2007, 7, 1186–1205.
[37] R. Romagnoli, P. G. Baraldi, M. D. Carrion, C. L. Cara, O. Cruz-
Lopez, D. Preti, M. Tolomeo, S. Grimaudo, A. Di Cristina, N.
Zonta, J. Balzarini, A. Brancale, T. Sarkar, E. Hamel, Bioorg.
Med. Chem. 2008, 16, 5367–5376.
[54] H. P. Hsie, J. P. Liou, N. Mahindroo, Curr. Pharm. Des. 2005, 11,
1655–1677.
[55] E. M. Acton, V. L. Narayanan, P. A. Risbood, R. H. Shoemaker,
D. T. Vistica, M. R. Boyd, J. Med. Chem. 1994, 37, 2185–2189.
[38] R. Romagnoli, P. G. Baraldi, O. Cruz-Lopez, M. Tolomeo, A.
Di Cristina, R. M. Pipitone, S. Grimaudo, J. Balzarini, A.
Brancale, E. Hamel, Bioorg. Med. Chem. Lett. 2011, 21, 2746–
2751.
[56] C. E. Kung, J. K. Reed, Biochemistry 1989, 28, 6678–6686.
[57] D. Bonne, C. Heusele, C. Simon, D. Pantaloni, J. Biol. Chem.
1985, 260, 2819–2825.
`
[58] S. Desbene, S. Giorgi-Renault, Curr. Med. Chem. Anticancer
Agents 2002, 2, 71–90.
[39] R. Romagnoli, P. G. Baraldi, T. Sarkar, M. D. Carrion, C.
Lopez-Cara, O. Cruz-Lopez, D. Preti, M. A. Tabrizi, M.
Tolomeo, S. Grimaudo, A. Di Cristina, N. Zonta, J.
Balzarini, A. Brancale, H. P. Hsieh, E. Hamel, J. Med. Chem.
2008, 51, 1464–1468.
[59] B. A. Bhat, K. L. Dhar, S. C. Puri, A. K. Saxena, M.
Shanmugavel, G. N. Qazi, Bioorg. Med. Chem. Lett. 2005, 15,
3177–3180.
[40] A. K. Tewari, A. Mishra, Bioorg. Med. Chem. 2001, 9, 715–718.
[60] D. M. Barron, S. K. Chatterjee, R. Ravindra, R. Roof, E.
Baloglu, D. G. Kingston, S. Bane, Anal. Biochem. 2003, 315,
49–56.
[41] R. H. Wiley, P. Wiley, Pyrazolones, Pyrazolidones and Derivatives,
John Wiley and Sons, New York 1964.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com