Novel pyrazole derivatives: Synthesis and evaluation of anti-angiogenic activity

Article abstract of DOI:10.1016/j.bmc.2010.04.076

The synthesis of a series of novel trisubstituted pyrazole derivatives and their PIFA-mediated conversion to molecules bearing the fused pyrazolo[4,3-c]quinoline ring system is reported. The anti-angiogenic activity of these compounds was evaluated by using in vitro assays for endothelial cell proliferation and migration, and in the chicken chorioallantoic membrane (CAM) assay. Compounds containing the fused pyrazolo[4,3-c]quinoline motifs emerged as potent anti-angiogenic compounds, which also had the ability to inhibit the growth of human breast (MCF-7) and cervical (Hela) carcinoma cells in vitro.

Full text of DOI:10.1016/j.bmc.2010.04.076

Bioorganic & Medicinal Chemistry 18 (2010) 4338–4350
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel pyrazole derivatives: Synthesis and evaluation of anti-angiogenic activity
Michael S. Christodoulou a, Sandra Liekens b, , Konstantinos M. Kasiotis a,c, Serkos A. Haroutounian a,
*
*
a Chemistry Laboratory, Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece
b Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium
c Benaki Phytopathological Institute, 8 St. Delta Street, Athens, Kifissia 14561, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of a series of novel trisubstituted pyrazole derivatives and their PIFA-mediated conversion
to molecules bearing the fused pyrazolo[4,3-c]quinoline ring system is reported. The anti-angiogenic
activity of these compounds was evaluated by using in vitro assays for endothelial cell proliferation
and migration, and in the chicken chorioallantoic membrane (CAM) assay. Compounds containing the
fused pyrazolo[4,3-c]quinoline motifs emerged as potent anti-angiogenic compounds, which also had
the ability to inhibit the growth of human breast (MCF-7) and cervical (Hela) carcinoma cells in vitro.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 26 October 2009
Revised 21 March 2010
Accepted 26 April 2010
Available online 20 May 2010
Keywords:
Pyrazole
Pyrazolo[4,3-c]quinoline
Angiogenesis
Endothelial cell proliferation and migration
CAM assay
Inhibition of tumor growth
1. Introduction
Various assays have been developed that recapitulate the differ-
ent steps of the angiogenesis process, including endothelial cell
Angiogenesis, the process of new blood vessel formation is piv-
otal for many physiological events including embryogenesis, organ
development, the female reproductive cycle and wound healing.1,2
Under these conditions, angiogenesis is a highly regulated process,
that is, turned on for brief periods and then completely inhibited.
On the contrary, unregulated angiogenesis has been associated
with the development of a diversity of disease states including can-
cer, retinopathy, rheumatoid arthritis, psoriasis, atherosclerosis
and haemangioma.3–5
During the last decade several molecules containing various
azaheterocyclic rings—including the pyrazole moiety6—in their
structural framework have been considered, designed, synthesized
and investigated as potent inhibitors of angiogenesis.7–9 Thus, in
the course of our investigations concerning the exploitation of no-
vel bioactive molecules containing the pyrazole residue,10,11 we
envisioned the synthesis of novel fused pyrazolo[4,3-c]quinoline
derivatives as potential anti-angiogenic agents. The rational to de-
sign these compounds was to combine in a single molecule the
known pharmacophore quinoline ring with a substituted pyrazole
ring backbone. This was achieved through the PIFA [phenylio-
dine(III) bis(trifluoroacetate)]12 mediated ring closure of the appro-
priate trisubstituted pyrazole derivatives 6 to form the fused
pyrazolo[4,3-c]quinoline ring system.
proliferation, migration and tube formation in vitro. To study angi-
ogenesis in vivo the most widely used assay is the chicken chorioa-
llantoic membrane assay (CAM).13 In the present study, all novel
compounds (n = 18) were evaluated for their capacity to inhibit
endothelial cell proliferation. Based on this preliminary screening,
two series of compounds were selected for further evaluation in an
in vitro wound healing assay and the CAM assay. Finally, in order to
determine whether the inhibitory activity of the compounds is spe-
cific for endothelial cells, we also investigated their potency to in-
hibit the growth of human cervical (Hela) and breast (MCF-7)
carcinoma cells in vitro. Our data indicate that these novel pyrazole
derivatives target both tumor and endothelial cells. In particular,
the hydroxypyrazoloquinolin-4-ones emerged as potent anti-
angiogenic compounds, which may be used as lead compounds
but also deserve further study in order to gain insight into their
mechanism of action.
2. Results
2.1. Chemistry
The synthesis of hydroxypyrazolocarboxaldehydes
4 was
accomplished in a three steps synthetic sequence as is outlined
in Scheme 1. Specifically, the acetophenone substrates (1a–e) were
condensed with 4-methoxyphenylhydrazine hydrochloride to
produce hydrazines 2, which by subsequent treatment with the
* Corresponding authors. Tel.: +30 2105294247; fax: +30 210 529 4265 (S.A.H.).
E-mail address: sehar@aua.gr (S.A. Haroutounian).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.076

M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
4339
R3
4'''
R3
R7
5''
R2
R6
3''
R2
R1
5''' R4
6'''
4''
R3
3'''
2'''
R4
R8
R2
R1
R4
R1
R5
2
2''
1'''
6''
O
1''
b
1''
c
a
3
O
H
2
N
N
N
2''
4
1
NH
1
1'
N
N
O
H
1'
2'
5
2'
4'
4'
O
O
HO
3'
4
1
2
3
3'
4a
4b
1a, 2a, 3a
R5= H, R6= H, R7= H, R8= H
R5= H, R6= OH, R7= H, R8= H
R1= H, R2= H, R3=
R1= H, R2= OCH3,
H, R 4= H
1b, 2b, 3b
4c R5= H, R6= H, R7= OH , R8= H
4d R5= H, R6= OH, R7= OH, R8= H
4e R5= OH, R6= H, R7= H, R8= OH
R3= H, R4= H
1c, 2c, 3c
R1= H, R2= H, R3=
OCH3 , R4= H
1d, 2d, 3d
R1= H, R2= OCH3,
R3= OCH3, R4= H
1e, 2e, 3e R1= OCH3, R2= H,
R3= H, R4= OCH3
Scheme 1. Reagents and conditions: (a) 4-methoxyphenylhydrazine hydrochloride, acetic acid, Et3N, EtOH; (b) TCT, DMF; (c) BBr3, CH2Cl2, ꢀ78 °C.
iminium salt—produced from the reaction of 2,4,6-trichloro-
[1,3,5]triazine (TCT, cyanuric chloride)—in DMF provided the core
pyrazole backbone (compounds 3). Then, the subsequent removal
of the methoxy protective groups with BBr3 at ꢀ78 °C afforded
the hydroxy pyrazolocarboxaldehydes 4.
Oxidation of the pyrazolocarboxaldehydes 3 with NaOCl2 at
0 °C, in the presence of sulfamic acid as scavenger, furnished quan-
titatively the corresponding acids 5, which were treated with
methoxylamine hydrochloride in the presence of the uronium-cou-
pling reagent TBTU to provide amides 6. The latter were cyclized by
a nucleophilic substitution reaction, which was promoted by PIFA
that served as the source of hypervalent iodine and trifluoroacetic
acid (TFA) as additive, to furnish the target pyrazoloquinolin-4-one
fused ring system 7. Finally, the removal of the methoxy protective
groups with BBr3 at ꢀ78 °C afforded the desired hydroxypyrazolo-
quinolin-4-ones 8 (Scheme 2).
R3
R2
R3
R3
R2
R2
R4
R4
R4
R1
R1
R1
O
N
O
O
H
a
b
N
N
N
N
N
NH
OH
MeO
O
O
O
5
6
3
c
R7
R6
R3
R2
7
8
6
9
R8
R4
R5
R1
5a
9a
d
1
1a
OH
N5
OMe
N
O
N
4
N
2N
3a
N
O
1'
2'
3
4'
HO
3'
O
8
7
3a, 5a, 6a, 7a R1= H, R2= H,
R3= H, R4= H
3b, 5b, 6b, 7b R1= H, R2=
OCH3, R3= H, R4= H
3c, 5c, 6c, 7c R1= H, R2= H,
R3= OCH3 , R4= H
8a R5= H, R6= H, R7= H, R8= H
8b R5= H, R6= OH, R7= H, R8= H
8c R5= H, R6= H, R7= OH , R8= H
8d R5= H, R6= OH, R7= OH, R8= H
8e R5= OH, R6= H, R7= H, R8= OH
3d, 5d, 6d, 7d R1= H, R2=
OCH3, R3= OCH3, R4= H
3e, 5e, 6e, 7e R1= OCH3, R2=
H, R3= H, R4= OCH3
Scheme 2. Reagents and conditions: (a) NaClO2, H2NSO3H, acetone/H2O, 0 °C; (b) Et3N, NH2OMeꢁHCl, TBTU, MeCN; (c) PIFA, TFA, CH2Cl2; (d) BBr3, CH2Cl2, ꢀ78 °C.

4340
M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
R7
4'''
R3
R3
R2
R2
R6
3'''
R4
R4
O
R8
2'''
5'''
6'''
R1
R1
R5
2' N
1'''
3'
O
b
a
O
N
3
N
1
OCH2CH3
OCH2CH3
N
1' N
4'
N
2
H
1''
5'
2''
4''
O
O
HO
3''
3
9
10
3a, 9a, R1= H, R2= H, R3 = H, R4= H
10a R 5= H, R6= H, R7= H, R8= H
10b R5= H, R6= OH, R7= H, R8= H
10c R5= H, R6= H, R7= OH , R8= H
10d R5= H, R6= OH, R7= OH, R8= H
3b, 9b, R1= H, R2= OCH3, R3= H, R4= H
3c, 9c, R1= H, R2= H, R3= OCH3 , R4= H
3d, 9d, R1= H, R2= OCH3, R3= OCH3, R4= H
3e, 9e, R1= OCH3, R2= H, R3= H, R4= OCH3
10e R5= OH, R6= H, R7 = H, R8= OH
Scheme 3. Reagents and conditions: (a) Ph3PCHCO2CH2CH3, MeCN, reflux; (b) BBr3, CH2Cl2, ꢀ78 °C.
Table 1
The synthesis of esters 9 was accomplished through the Wittig
Cytostatic activity of novel pyrazole derivatives in MAEC (mouse aortic endothelial
cells).
reaction of pyrazolocarboxaldehydes 3 with the stabilized ylide
Ph3PCHCO2CH2CH3 which formed in very good yields the desirable
E-isomer of the corresponding esters 9. Demethylation of esters 9
with BBr3 at ꢀ78 °C afforded the desired unprotected esters 10
(Scheme 3).
Finally, esters 9 were hydrolyzed almost quantitatively to acids
11, which were subsequently demethylated at ꢀ78 °C, in the pres-
ence of BBr3, to produce derivatives 12 (Scheme 4).
Aldehydes
4a
4b
4c
4d
4e
a
IC50
P100
42 3.4
44 7.2
12 2.9
43 15
Closed rings
IC50
8a
7.0 0.3
8b
25 1.4
8c
42 4.2
a
Esters
IC50
10a
29 15
10b
14 0.6
10c
25 1.1
10d
6.2 1.3
10e
12 2.8
a
Acids
IC50
12a
>100
12b
>100
12c
>100
12d
>100
12e
>100
a
2.2. Biological activity
a
Data are expressed as micromolar concentrations.
2.2.1. Inhibition of endothelial cell proliferation by pyrazole
derivatives
Comparison of compounds activities within each series indi-
In order to evaluate the anti-angiogenic activity of the pyrazole
derivatives, we first assessed their in vitro effect (Table 1) on the
proliferation of mouse aortic endothelial cells (MAEC).
Several compounds elicited a dose-dependent inhibition of
endothelial cell proliferation with 50% inhibitory concentrations
cated that the presence of two hydroxyl groups in meta and para
positions of the B-phenyl moiety (compounds d) increased their
anti-proliferative activities. On the other hand, the ring closure of
compounds 4 to produce the hydroxypyrazoloquinolin-4-ones 8
potentiated the cytostatic effect of the compounds, with 8a being
(IC50) ranging from 6 to 44
oxaldehydes, compound 4d proved to be the most potent exhibit-
ing an IC50 of 12 M, whereas 4a did not inhibit endothelial cell
proliferation (IC50 higher than 100 M). The replacement of their
lM. Among the hydroxypyrazolocarb-
the most active compound of this series (IC50 of 7 lM).
l
2.2.2. Inhibition of endothelial cell migration by pyrazole
derivatives
l
aldehyde group with an alkylated ester (10a–10e) resulted in a
slight improvement of their anti-proliferative activities, displaying
IC50 values between 6 and 29 lM. On the contrary, hydrolysis of
the esters and their subsequent demethylation to the correspond-
ing acids (12a–12e) rendered the compounds inactive (IC50
>100 lM).
Endothelial cell migration is an essential step in the formation
of new blood vessels. In order to further evaluate the in vitro
anti-angiogenic activity of the pyrazole derivatives, we focused
on compounds 4 and 8 (Fig. 1). The hydroxypyrazolocarboxaldehy-
des 4b and in particular 4d significantly inhibited wound closure of
R3
R3
R7
R6
R2
R2
R4
O
R4
O
R8
O
R1
R1
R5
b
a
N
N
N
OCH2CH3
OH
OH
N
N
N
O
O
HO
9
11
12
9a, 11a R1= H, R2= H, R3= H, R4= H
12a R5= H, R6= H, R7= H, R8= H
12b R5= H, R6= OH, R7= H, R8= H
12c R5= H, R6= H, R7= OH , R8= H
12d R5= H, R6= OH, R7= OH, R8= H
12e R5= OH, R6= H, R7= H, R8= OH
9b, 11b R1= H, R2= OCH3, R3= H, R4= H
9c, 11c R1= H, R2= H, R3= OCH3 , R4= H
9d, 11d R1= H, R2= OCH3, R3= OCH3, R4= H
9e, 11e R1= OCH3, R2= H, R3= H, R4= OCH3
Scheme 4. Reagents and conditions: (a) EtOH, DMSO, NaOH; (b) BBr3, CH2Cl2, ꢀ78 °C.

M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
4341
Figure. 2. Effects of hydroxypyrazolocarboxaldehydes (4) and hydroxypyrazolo-
quinolin-4-ones (8) on angiogenesis in the CAM assay. At day 9 of incubation, discs
containing either 50 nmol of the test compounds or SU5416 were applied onto the
CAM. A control disc containing an equal amount of solvent (DMSO) was positioned on
the CAM 1 cm away from the test compounds. At day 11, the percentage of inhibition
of blood vessel formation, compared to untreated controls, was determined (see
*
***
p <0.01
materials and methods). Results are expressed as mean SEM. p <0.05;
(A). (B) CAMs treated with a control disc are characterized by extensive neovascu-
larization, whereas a large avascular zone is visible in CAMs treated with 8b.
Figure 1. Effects of hydroxypyrazolocarboxaldehydes (4d) and hydroxypyrazolo-
quinolin-4-ones (8b) on endothelial wound repair. Confluent MAEC were wounded
with a 1.0 mm wide tip and incubated with two concentrations (10 and 50 lM) of
2.2.4. Inhibition of tumor cell proliferation by pyrazole
derivatives
the test compounds. After 8 h, the wounds were photographed and endothelial cells
invading the wound were quantified by computerized analysis of the digitalized
Next, we investigated if the inhibitory activity of the com-
pounds examined is specific for endothelial cells and whether
these compounds also affect tumor cell proliferation. Therefore,
the anti-proliferative activity of hydroxyl pyrazolocarboxaldehy-
des 4 and hydroxyyrazoloquinolin-4-ones 8 was compared in
MAEC (see also Table 1), bovine aortic endothelial cells (BAEC),
and the human cervical (Hela) and breast carcinoma (MCF-7) cells.
All compounds showed comparable activity in the endothelial and
tumor cell lines (Table 2).
*
***
p
pictures, Results are expressed as mean SEM.
p
0
<0.05,
and 8 h after wounding.
M.
<0.01 (A). (B)
Representative images of control monolayers at
Compounds 4d and 8b significantly delayed wound healing at 50
l
the endothelial cells at 50
of endothelial cell migration was hydroxypyrazoloquinolin-4-one
8b, which showed 76% inhibition at 50 M.
lM. However, the most potent inhibitor
l
2.2.3. Inhibition of angiogenesis in the CAM assay
Next, the anti-angiogenic activity of the compounds was evalu-
ated in the CAM assay, which represents the most widely used as-
say to study angiogenesis in vivo. In this regard we used SU5416
(Fig. 3), a well-established anti-angiogenic agent, which inhibits
vascular endothelial growth factor (VEGF), as a positive control.14
At 50 nmol/CAM, this compound elicited 54% inhibition of angio-
genesis (p <0.01). Compound 4a did not show a significant anti-
angiogenic activity at this concentration. However, 4b and 4c
inhibited blood vessel formation by respectively 50% and 48% (p
<0.05) (Fig. 2). Compounds 4d and 4e induced an inflammatory re-
sponse in the CAM, which made an evaluation of angiogenesis
impossible. All compounds of 8 series significantly inhibited blood
vessel formation in the CAM, with compound 8b being the most
potent one, eliciting 64% of inhibition (p <0.01, Fig. 2). It should
be noted however, that a 100% inhibition of angiogenesis cannot
be achieved by this method, since the pre-existing vessels—already
present at day 9 when the compounds are put on the CAM—are
also being counted when angiogenesis is assessed at day 11. Thus,
8b nearly completely inhibited the formation of new blood vessels,
as shown in Figure 2B. In fact, the assessed activity of this com-
pound is comparable with the reference anti-angiogenic molecule
SU5416.
3. Discussion
Several literature studies have provided evidences that pyrazole
derivatives inhibit angiogenesis by interacting with various molec-
ular targets. In addition, these compounds may combine anti-
angiogenic and direct anti-tumor activities. More specifically, pyr-
azole derivatives were found to inhibit the proliferation of endo-
thelial cells and angiogenesis in
a matrigel plug assay by
blocking the intermediate-conductance Ca2+-activated K+ channel
(IKCa1).15 It was hypothesized that an upregulation of the expres-
sion of IKCa1 by angiogenic factors, such as basic fibroblast growth
factor (FGF2) and vascular endothelial growth factor (VEGF) is re-
quired to stimulate endothelial cell proliferation and angiogenesis
in vivo. Thus, selective IKCa1 blockers might inhibit angiogenesis
by interfering with growth factor activity.
Pyrazole derivatives were also identified as antagonists of the
integrin
avb3, which is upregulated on endothelial cells during
angiogenesis.16 These integrin antagonists were shown to inhibit
angiogenesis in the mouse corneal pocket model.
Another class of pyrazoles (3,4-diaryl pyrazole resorcinol deriv-
atives) was identified to inhibit the heat-shock protein HSP90.
Thus, the reference compound CCT018159 (Fig. 3) was shown to

4342
M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
Figure 3. 3D chemical structures of compound 8b and reference compounds SU5416, CCT018159 and XRP44X.
Table 2
of novel fused pyrazole-quinolin-4-one derivatives. Figures 3 and 4
delineate the structural similarities of pyrazole 8b with the corre-
sponding lead molecules.
Cytostatic activity of compounds 4 and 8 in endothelial and tumor cell lines
a
Compound
IC50
(lM)
Of 18 novel pyrazole derivatives synthesized and studied, we
have identified several compounds that inhibit endothelial cell
proliferation. Two classes of compounds (hydroxypyrazolocarbox-
aldehydes and hydroxyl pyrazoloquinolin-4-ones) were selected
for further investigation. Compound 8b—bearing the fused pyraz-
olo[4,3-c]quinoline structural motif—emerged as a promising lead
compound, with inhibitory activity against endothelial and tumor
cell proliferation in vitro and angiogenesis in vivo.
MAEC
BAEC
Hela
MCF-7
4a
4b
4c
4d
4e
8a
8b
8c
P100
64 8.4
29 4.5
32 8.7
17 4.2
P100
4.6 0.9
38 9.1
52 11
79 8.8
37 4.9
37 9.5
39 1.4
P100
8.9 0.1
37 2.6
38 4.3
47 5.1
29 6.8
38 6.0
24 0.4
P100
5.8 1.8
26 2.2
25 8.1
42 3.4
44 7.2
12 2.9
43 15
7.0 0.25
25 1.4
42 4.2
Therefore, further studies should aim at determining the mech-
anism of action of this compound as well as its potential anti-tu-
mor activity in vivo.
a
IC50 = concentration of compound that reduces cell proliferation by 50%.
4. Experimental
4.1. General experimental conditions
All reactions were carried out in oven-dried glassware under ar-
gon atmosphere. All starting materials were purchased from Al-
drich, while the solvents used were purified by distillation prior
to use. Solvent mixtures employed in chromatography were re-
ported as volume to volume ratios. Thin layer chromatography
(TLC) was performed on Merck precoated aluminium sheets of Sil-
ica Gel 60 F254, visualization of products being accomplished by UV
absorbance at 254 nm and by an alcohol solution of anisaldehyde
with heating. Flash column chromatography was performed on a
SDS silica gel (35–70 lm). Melting points were determined on a
Figure 4. Superimposition of the 3D chemical structure of compound 8b and the
Stuart apparatus (SMP3) and are uncorrected. IR spectra were re-
corded on a Thermo electron corporation Nicolet 6700 FT-IR spec-
trometer using CH2Cl2 in ZnSe round windows. 1H NMR and 13C
NMR spectra were recorded at 400 and 50 MHz respectively on
Bruker DRX-400 and DRX-200 spectrometers in the indicated sol-
vents. Chemical shifts (d) for proton and carbon resonances are
quoted in parts per million (ppm) relative to tetramethylsilane
(TMS), which was used as an internal standard. MS spectra were
recorded using electrospray ionisation (ESI) technique on a Thermo
Accela LC TSQ Quantum Access MS–MS spectometer.
anti-angiogenic pyrazole derivative CCT018159.
inhibit cell proliferation by inducing G1 arrest and apoptosis.
CCT018159 also inhibited both endothelial and tumor cell func-
tions implicated in invasion and angiogenesis.17
Finally, pyrazole XRP44X (Fig. 3) has found to inhibit FGF2-in-
duced activation of the Ras-Net (Elk-3) signaling pathway.18
XRP44X also abrogated cell growth, cell cycle progression and
in vitro angiogenesis. In addition, this compound binds to the col-
chicine-binding site of tubulin, resulting in depolymerization of
the microtubules and affects the actin cytoskeleton. These data
indicate that XRP44X may combine anti-angiogenic and vascular-
targeting properties.
4.2. Synthesis
4.2.1. Typical procedure for the synthesis of hydrazines 2a–c
4.2.1.1. Synthesis of 1-(40-methoxyphenyl)-2-(1’’-phenylethyli-
dene) hydrazine (2a). To a stirred solution of acetophenone 1a
(2.4 mL, 20 mmol) in 35 mL of ethanol and acetic acid (1.5 mL), a
The herein reported compounds share several structural simi-
larities with the aforementioned lead compounds containing a
fused rigid backbone that combines in a single molecule the substi-
tuted pyrazole ring and the pharmacophore quinoline in the form

M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
4343
solution of 4-methoxyphenylhydrazine hydrochloride (4.3 g,
oil (95% yield). IR (film)
m
: 3343 cmꢀ1
;
1H NMR (DMSO): d = 8.89
24 mmol) and Et3N (4.2 mL, 30 mmol) in 40 mL of ethanol was
added. The reaction mixture was stirred overnight at room temper-
ature and the resulting precipitate was filtered and washed with
ethanol and diethylether to produce the hydrazine product 2a as
(1H, s, NH), 7.10 (2H, d, J 8.8 Hz, H-2’), 6.96 (1H, d, J 8.8 Hz, H-
3’’’), 6.92 (1H, d, J 3.2 Hz, H-6’’’), 6.87 (1H, dd, J 8.8 Hz J 3.2 Hz, H-
4’’’), 6.82 (2H, d, J 8.8 Hz, H-3’), 3.75 (3H, s, C2’’’–OCH3), 3.73 (3H,
s, C5’’’–OCH3), 3.68 (3H, s, C4’–OCH3), 2.16 (3H, s, H-2’’); 13C NMR
(DMSO): d = 153.4 (C-4’), 153.0 (C-5’’’), 151.6 (C-2’’’), 141.8 (C-1’’),
140.8 (C-1’), 124.4 (C-1’’’), 120.0 (C-2’), 114.8 (C-3’), 114.7 (C-4’’’),
114.3 (C-6’’’), 113.9 (C-3’’’), 55.96 (C4’–OCH3), 55.83 (C5’’’–OCH3),
55.67 (C2’’’–OCH3), 17.36 (C-2’’); MS: 301.33 (M+H+). Anal. Calcd
for C17H20N2O3: C, 67.98; H, 6.71; N, 9.33. Found: C, 67.84; H,
6.77; N 9.41.
white solid (95% yield). IR (film) m ;
: 3343 cmꢀ1 1H NMR (DMSO):
d = 9.07 (1H, s, NH), 7.78 (2H, d, J 7.6 Hz, H-2’’’ and H-6’’’), 7.38
(2H, t, J 7.2 Hz, H-3’’’ and H-5’’’), 7.28 (1H, t, J 7.2 Hz, H-4’’’), 7.19
(2H, d, J 8.4 Hz, H-2’), 6.86 (2H, d, J 8.8 Hz, H-3’), 3.70 (3H, s, OCH3),
2.23 (3H, s, H-2’’); 13C NMR (DMSO): d = 153.1 (C-4’), 140.6 (C-1’’),
139.8 (C-1’’’ and C-1’), 128.7 (C-3’’’ and C-5’’’), 127.7 (C-4’’’), 125.4
(C-2’’’ and C-6’’’), 114.8 (C-2’), 114.3 (C-3’), 55.68 (C-OCH3), 13.11
(C-2’’); MS: 241.29 (M+H+). Anal. Calcd for C15H16N2O: C, 74.97; H,
6.71; N, 11.66. Found: C, 74.78; H, 6.58; N 11.82.
4.2.3. General procedure for the synthesis of pyrazolo
carboxaldehydes 3a–e
4.2.3.1. Synthesis of 1-(4’-methoxyphenyl)-3-phenyl-1H-pyra-
zole-4-carbaldehyde (3a). 2,4,6-Trichloro-[1,3,5]-triazine (11 g,
60 mmol) was added to DMF (8.3 mL) and maintained at 25 °C.
After the formation of a white solid, the reaction was monitored
(TLC) until complete disappearance of TCT (20 min). Next, hydra-
zine 2a (3.0 g, 12 mmol) in DMF (32 mL) was added and the mix-
ture was stirred for 2 h. After completion, the reaction was
cooled at 0 °C and water was added. The organic phase was ex-
tracted twice with EtOAc. The combined organic extracts were
washed with saturated Na2CO3, brine, dried with Na2SO4, filtered
and evaporated. The product was obtained after purification by
flash column chromatography (Hex/EtOAc, 8:2) and evaporation
of the solvent as white solid (60% yield). mp 123–124 °C; IR (film)
4.2.1.2. 1-(4’-Methoxyphenyl)-2-(1”-(3’’’-methoxyphenyl) ethyl-
idene) hydrazine (2b). According to the typical procedure, hydra-
zine 2b was obtained from acetophenone 1b as white solid (95%
yield). IR (film) m ;
: 3345 cmꢀ1 1H NMR (DMSO): d = 9.09 (1H, s,
NH), 7.34–7.30 (2H, m, H-4’’’ and H-6’’’), 7.27 (1H, t, J 7.6 Hz,
H-5’’’), 7.18 (2H, d, J 8.8 Hz, H-2’), 6.86–6.82 (3H, m, H-2’’’ and
H-3’), 3.78 (3H, s, C3’’’–OCH3), 3.68 (3H, s, C4’–OCH3), 2.20 (3H, s,
H-2’’); 13C NMR (DMSO): d = 159.7 (C-3’’’), 153.1 (C-4’), 141.3 (C-
1’’), 140.6 (C-1’), 139.5 (C-1’’’), 129.7 (C-5’’’), 118.0 (C-6’’’), 114.8
(C-2’), 114.2 (C-3’), 113.1 (C-4’’’), 110.8 (C-2’’’), 55.6 (C4’-OCH3),
55.4 (C3’’’-OCH3), 13.31 (C-2’’); MS: 271.30 (M+H+). Anal. Calcd
for C16H18N2O2: C, 71.09; H, 6.71; N, 10.36. Found: C, 70.99; H,
6.60; N 10.52.
m
: 1670 cmꢀ1; 1H NMR (CDCl3): d = 10.0 (1H, s, CHO), 8.46 (1H, s, H-
5), 7.83 (2H, d, J 6.8 Hz, H-2’’ and H-6’’), 7.71 (2H, d, J 8.8 Hz, H-2’),
7.54–7.49 (3H, m, H-3’’, H-4’’ and H-5’’), 7.03 (2H, d, J 8.8 Hz, H-3’),
3.88 (3H, s, OCH3); 13C NMR (CDCl3): d = 185.2 (CHO), 159.3 (C-4’),
154.6 (C-3), 132.6 (C-1’), 131.4 (C-5), 130.7 (C-1’’), 129.2 (C-4’’),
129.0 (C-3’’ and C-5’’), 128.8 (C-2’’ and C-6’’), 122.2 (C-4), 121.4
(C-2’), 114.7 (C-3’), 55.64 (OCH3); MS: 279.10 (M+H+). Anal. Calcd
for C17H14N2O2: C, 73.37; H, 5.07; N, 10.07. Found: C, 73.20; H,
5.14; N 9.93.
4.2.1.3. 1-(4’-Methoxyphenyl)-2-(1’’-(4’’’-methoxyphenyl) ethyl-
idene) hydrazine (2c). According to the typical procedure, hydra-
zine 2c was obtained from acetophenone 1c as white solid (95%
yield). IR (film) m ;
: 3347 cmꢀ1 1H NMR (DMSO): d = 8.91 (1H, s,
NH), 7.70 (2H, d, J 16.8 Hz, H-2’’’ and H-6’’’), 7.15 (2H, d, J 16.8 Hz,
H-2’), 6.92 (2H, d, J 16.8 Hz, H-3’’’ and H-5’’’), 6.83 (2H, d, J 16.8 Hz,
H-3’) 3.76 (3H, s, C4’’’-OCH3), 3.68 (3H, s, C4’-OCH3), 2.18 (3H, s, H-
2’’); 13C NMR (DMSO): d = 159.4 (C-4’’’), 152.9 (C-4’), 140.9 (C-1’’),
140.0 (C-1’), 132.5 (C-1’’’), 126.8 (C-2’’’ and C-6’’’), 114.8 (C-2’),
114.1 (C-3’’’, C-5’’’ and C-3’), 55.65 (C4’-OCH3), 55.54 (C4’’’-OCH3),
13.20 (C-2’’); MS: 271.31 (M+H+). Anal. Calcd for C16H18N2O2: C,
71.09; H, 6.71; N, 10.36. Found: C, 71.19; H, 6.64; N 10.44.
4.2.3.2. 1-(4’-Methoxyphenyl)-3-(3’’-methoxyphenyl)-1H-pyra-
zole-4-carbaldehyde (3b). According to the general procedure,
pyrazolocarboxaldehyde 3b was obtained from hydrazine 2b after
purification by flash column chromatography (Hex/EtOAc, 1:1) and
evaporation of the solvent as a yellow solid (55% yield). mp 95–
4.2.2. Typical procedure for the synthesis of hydrazines 2d, e
4.2.2.1. Synthesis of 1-(4’-methoxyphenyl)-2-(1’’-(3’’’,4’’’-dime-
thoxyphenyl)ethylidene) hydrazine (2d). To a stirred solution of
3’,4’-dimethoxy acetophenone (2.30 g, 12.5 mmol) in 30 mL of eth-
anol and acetic acid (1.00 mL), a solution of 4-methoxyphenylhy-
drazine hydrochloride (3.12 g, 17.5 mmol) and Et3N (2.80 mL,
20.1 mmol) in 30 mL of ethanol was added. The reaction mixture
was stirred overnight at room temperature furnishing a precipitate
which was filtered off and washed with ethanol and diethylether.
Hydrazine 2d was obtained as a white solid (95% yield). IR (film)
96 °C; IR (film) m ;
: 1676 cmꢀ1 1H NMR (CDCl3): d = 10.0 (1H, s,
CHO), 8.46 (1H, s, H-5), 7.71 (2H, d, J 9.2 Hz, H-2’), 7.43–7.40 (3H,
m, H-4’’, H-5’’, H-6’’), 7.05 (1H, d, J 2.0 Hz, H-2’’), 7.02 (2H, d, J
8.8 Hz, H-3’), 3.90 (3H, s, C3’’–OCH3), 3.88 (3H, s, C4’–OCH3); 13C
NMR (CDCl3): d = 185.3 (CHO), 159.8 (C-3’’), 159.3 (C-4’), 154.5
(C-3), 132.6 (C-1’), 132.5 (C-1’’), 130.7 (C-5), 129.8 (C-5’’), 122.3
(C-4), 121.5 (C-6’’), 121.4 (C-2’), 115.2 (C-4’’), 114.7 (C-3’), 114.1
(C-2’’), 55.64 (C4’–OCH3), 55.42 (C3’’–OCH3); MS: 309.14 (M+H+).
Anal. Calcd for C18H16N2O3: C, 70.12; H, 5.23; N, 9.09. Found: C,
70.30; H, 5.14; N 9.19.
m
: 3341 cmꢀ1 1H NMR (DMSO): d = 8.99 (1H, s, NH), 7.42 (1H, s,
;
H-2’’’), 7.22 (1H, d, J 8.4 Hz, H-6’’’), 7.17 (2H, d, J 8.8 Hz, H-2’),
6.94 (1H, d, J 8.4 Hz, H-5’’’), 6.83 (2H, d, J 8.8 Hz, H-3’), 3.81 (3H,
s, C3’’’–OCH3), 3.77 (3H, s, C4’’’–OCH3), 3.69 (3H, s, C4’–OCH3),
2.21 (3Y, s, H-2’’); 13C NMR (DMSO): d = 152.9 (C-4’), 149.1 (C-
4’’’), 149.0 (C-3’’’), 140.9 (C-1’’), 140.1 (C-1’), 132.8 (C-1’’’), 118.3
(C-6’’’), 114.8 (C-2’), 114.1 (C-3’), 111.7 (C-5’’’), 108.8 (C-2’’’),
55.93 (C3’’’–OCH3), 55.80 (C4’’’–OCH3), 55.66 (C4’–OCH3), 13.23
(C-2’’); MS: 301.34 (M+H+). Anal. Calcd for C17H20N2O3: C, 67.98;
H, 6.71; N, 9.33. Found: C, 69.12; H, 6.60; N 9.42.
4.2.3.3. 1-(4’-Methoxyphenyl)-3-(4’’-methoxyphenyl)-1H-pyra-
zole-4-carbaldehyde (3c). According to the general procedure,
pyrazolocarboxaldehyde 3c was obtained from hydrazine 2c after
purification by flash column chromatography (Hex/EtOAc, 1:1)
and evaporation of the solvent as a yellow solid (55% yield). mp
135–136 °C; IR (film)
m ;
: 1679 cmꢀ1 1H NMR (CDCl3): d = 10.0
(1H, s, CHO), 8.43 (1H, s, H-5), 7.80 (2H, d, J 8.8 Hz, H-2’’ and H-
6’’), 7.70 (2H, d, J 8.8 Hz, H-2’), 7.17 (2H, d, J 8.8 Hz, H-3’’ and H-
5’’), 7.02 (2H, d, J 8.8 Hz, H-3’), 3.89 (3H, s, C4’’–OCH3), 3.87 (3H,
s, C4’–OCH3); 13C NMR (CDCl3): d = 185.2 (CHO), 160.4 (C-4’’),
159.2 (C-4’), 154.3 (C-3), 132.6 (C-1’), 131.0 (C-5), 130.2 (C-2’’
and C-6’’), 123.9 (C-1’’), 122.0 (C-4), 121.3 (C-2’), 114.7 (C-3’’ and
C-5’’), 114.2 (C-3’), 55.64 (C4’–OCH3), 55.40 (C-4’’–OCH3); MS:
4.2.2.2. 1-(4’-Methoxyphenyl)-2-(1’’-(2’’’,5’’’-dimethoxyphenyl)
ethylidene) hydrazine (2e). According to the typical procedure,
hydrazine 2e was obtained from acetophenone 1e as yellowish

4344
M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
309.13 (M+H+). Anal. Calcd for C18H16N2O3: C, 70.12; H, 5.23; N,
9.09. Found: C, 69.98; H, 5.16; N 9.19.
yellow solid (95% yield). Mp 224–225 °C; IR (film)
m: 3388,
1662 cmꢀ1 1H NMR (DMSO): d = 9.96 (1H, s, CHO), 9.88 (1H, br.
;
s, OH), 9.64 (1H, br s, OH), 9.09 (1H, s, H-5), 7.76 (2H, d, J 8.8 Hz,
H-2’), 7.31 (3H, m, H-2’’, H-4’’ and H-6’’), 6.92 (2H, d, J 8.8 Hz, H-
3’), 6.88 (1H, ddd, J 7.2 Hz J 2.4 Hz, H-5’’); 13C NMR (DMSO):
d = 185.1 (CHO), 157.9 (C-3’’), 157.5 (C-4’), 153.0 (C-3), 134.0 (C-
1’), 133.0 (C-5), 131.3 (C-5’’), 130.1 (C-1’’), 122.1 (C-4), 121.5 (C-
2’), 120.0 (C-2’’), 116.6 (C-4’’), 116.4 (C-3’), 115.8 (C-6’’); MS:
281.06 (M+H+). Anal. Calcd for C16H12N2O3: C, 68.56; H, 4.32; N,
9.99. Found: C, 68.74; H, 4.46; N, 10.20.
4.2.3.4. 1-(4’-Methoxyphenyl)-3-(3’’,4’’-dimethoxyphenyl)-1H-
pyrazole-4-carbaldehyde (3d). According to the general proce-
dure, pyrazolocarboxaldehyde 3d was obtained from hydrazine
2d after purification by flash column chromatography (Hex/EtOAc,
1:1) and evaporation of the solvent as a yellow solid (50% yield).
mp 113–114 °C; IR (film)
m: ;
1686 cmꢀ1 1H NMR (DMSO):
d = 9.95 (1H, s, CHO), 9.16 (1H, s, H-5), 7.88 (2H, d, J 8.8 Hz, H-2’),
7.54 (1H, s, H-2’’), 7.51 (1H, d, J 8.4 Hz, H-6’’), 7.10 (2H, d, J
8.8 Hz, H-3’), 7.06 (1H, d, J 8.4 Hz, H-5’’) 3.83 (3H, s, C3’’–OCH3),
3.81 (6H, s, C4’–OCH3 and C4’’–OCH3); 13C NMR (DMSO):
d = 185.1 (CHO), 159.0 (C-4’), 152.9 (C-3), 150.1 (C-4’’), 149.0 (C-
3’’), 134.9 (C-1’), 132.6 (C-5), 124.3 (C-1’’), 122.2 (C-4), 121.9 (C-
6’’), 121.3 (C-2’), 115.2 (C-3’), 112.4 (C-2’’), 112.0 (C-5’’), 55.98
(C3’’–OCH3, C4’’–OCH3 and C4’–OCH3); MS: 339.12 (M+H+). Anal.
Calcd for C19H18N2O4: C, 67.44; H, 5.36; N, 8.28. Found: C, 67.64;
H, 5.23; N, 8.19.
4.2.4.3. 1-(4’-Hydroxyphenyl)-3-(4’’-hydroxyphenyl)-1H-pyrazole-
4-carboxaldehyde (4c). According to the general procedure,
demethylated pyrazolocarboxaldehyde 4c was obtained from pyr-
azolocarboxaldehyde 3c after purification by flash column chroma-
tography (Hex/EtOAc 1:1) and evaporation of the solvent as a light
brown solid (95% yield). mp 216–217 °C; IR (film)
m: 3320,
1650 cmꢀ1 1H NMR (DMSO): d = 9.91 (1H, s, CHO), 9.82 (2H, br. s,
;
OH), 9.04 (1H, s, H-5), 7.74 (4H, d, J 6.8 Hz, H-2’’, H-6’’ and H-2’),
6.89 (2H, d, J 9.2 Hz, H-3’’ and H-5’’) 6.87 (2H, d, J 9.2 Hz, H-3’);
13C NMR (DMSO): d = 185.0 (CHO), 158.8 (C-4’’), 157.4 (C-4’),
153.0 (C-3), 134.2 (C-1’), 131.4 (C-5), 130.5 (C-2’’ and C-6’’), 122.6
(C-1’’), 121.8 (C-4), 121.4 (C-2’), 116.3 (C-3’’ and C-5’’), 115.8
(C-3’); MS: 281.06 (M+H+). Anal. Calcd for C16H12N2O3: C, 68.56;
H, 4.32; N, 9.99. Found: C, 68.41; H, 4.40; N, 10.12.
4.2.3.5. 1-(4’-Methoxyphenyl)-3-(2’’,5’’-dimethoxyphenyl)-1H-
pyrazole-4-carbaldehyde (3e). According to the general proce-
dure, pyrazolocarboxaldehyde 3e was obtained from hydrazine
2e after purification by flash column chromatography (Hex/EtOAc,
1:1) and evaporation of the solvent as a yellow solid (50% yield).
mp 120–121 °C; IR (film) m ;
: 1688 cmꢀ1 1H NMR (CDCl3): d = 9.79
(1H, s, CHO), 8.42 (1H, s, H-5), 7.69 (2H, d, J 8.8 Hz, H-2’), 7.19
(1H, d, J 3.2 Hz, H-6’’), 7.01 (2H, d, J 8.8 Hz, H-3’), 6.99 (2H, d, J
6.8 Hz, H-3’’ and H-4’’), 3.87 (3H, s, C2’’–OCH3), 3.84 (3H, s, C5’’–
OCH3), 3.79 (3H, s, C4’–OCH3); 13C NMR (CDCl3): d = 185.0 (CHO),
159.2 (C-4’), 153.8 (C-3), 151.6 (C-5’’), 151.0 (C-2’’), 132.6 (C-1’),
130.7 (C-5), 123.2 (C-1’’), 122.7 (C-4), 121.1(C-2’), 117.7 (C-6’’),
114.1 (C-3’), 113.0 (C-3’’), 110.8 (C-4’’), 57.52 (C4’–OCH3), 57.27
(C5’’–OCH3), 57.02 (C2’’–OCH3); MS: 339.15 (M+H+). Anal. Calcd
for C19H18N2O4: C, 67.44; H, 5.36; N, 8.28. Found: C, 67.29; H,
5.19; N, 8.16.
4.2.4.4. 1-(4’-Hydroxyphenyl)-3-(3’’,4’’-dihydroxyphenyl)-1H-pyr-
azole-4-carboxaldehyde (4d). According to the general proce-
dure, demethylated pyrazolocarboxaldehyde 4d was obtained
from pyrazolocarboxaldehyde 3d after purification by flash column
chromatography (Hex/EtOAc 7:3) and evaporation of the solvent as
foam (90% yield). IR (film) m ;
: 3438, 1641 cmꢀ1 1H NMR (DMSO):
d = 9.94 (1H, s, CHO), 9.85 (1H, s, OH), 9.28 (1H, s, OH), 9.18 (1H,
s, OH), 9.03 (1H, s, H-5), 7.75 (2H, d, J 9.2 Hz, H-2’), 7.33 (1H, d, J
2.0 Hz, H-2’’), 7.23 (1H, dd, J 8.0 J 2.0 Hz, H-6’’), 6.92 (2H, d, J 8.8,
H-3’), 6.85 (1H, d, J 8.0 Hz, H-5’’); 13C NMR (DMSO): d = 185.6
(CHO), 157.0 (C-4’), 153.3 (C-3), 146.6 (C-4’’), 145.2 (C-3’’), 134.3
(C-1’), 131.4 (C-5), 123.0 (C-1’’), 121.6 (C-2’ and C-4), 121.0 (C-
2’’), 116.4 (C-3’), 116.2 (C-5’’), 116.0 (C-6’’); MS: 297.06 (M+H+).
Anal. Calcd for C16H12N2O4: C, 64.86; H, 4.08; N, 9.46. Found: C,
64.78; H, 4.01; N, 9.38.
4.2.4. General procedure for the demethylation of pyrazolo
carboxaldehydes 3a–e
4.2.4.1. Synthesis of 1-(4’-hydroxyphenyl)-3-phenyl-1H-pyra-
zole-4-carboxaldehyde (4a). To a stirred solution of the pro-
tected pyrazolocarboxaldehyde 3a (0.20 g, 0.72 mmol) in CH2Cl2
(22 mL) at ꢀ78 °C BBr3 (3.6 mmol, 1.0 M in CH2Cl2, 5 equiv per
bond, 3.6 mL) dropwise was added. The reaction was kept at
ꢀ78 °C for 1 h and then for 16 h at room temperature. The solution
was cooled at 0 °C, neutralized with MeOH and then HCl 1 N was
added. The organic phase was extracted twice with EtOAc. The
combined organic extracts were washed with water, brine, dried
with Na2SO4, filtered and evaporated. The product was obtained
after flash column chromatography (Hex/EtOAc 1:1) and evapora-
tion of the solvent as brown solid (95% yield). Mp 162–163 °C; IR
4.2.4.5. 1-(4’-Hydroxyphenyl)-3-(2’’,5’’-dihydroxyphenyl)-1H-pyr-
azole-4-carboxaldehyde (4e). According to the general proce-
dure, demethylated pyrazolocarboxaldehyde 4e was obtained
from pyrazolocarboxaldehyde 3e after purification by flash column
chromatography (Hex/EtOAc 1:1) and evaporation of the solvent as
a brown solid (90% yield). mp 208–209 °C; IR (film)
m: 3289,
1669 cmꢀ1 1H NMR (DMSO): d = 9.84 (1H, br. s, OH), 9.78 (1H, s,
;
CHO), 9.22 (1H, br. s, OH), 8.94 (2H, s, H-5 and OH), 7.75 (2H, d, J
8.4 Hz, H-2’), 6.94 (1H, d, J 2.4 Hz, H-6’’), 6.56 (2H, d, J 8.8 Hz, H-
3’), 6.82 (1H, d, J 8.4, H-3’’), 6.74 (1H, d, J 8.0 Hz, H-4’’); 13C NMR
(DMSO): d = 184.2 (CHO), 157.4 (C-4’) 151.9 (C-3), 150.4 (C-5’’),
147.8 (C-2’’), 131.5 (C-1’), 130.4 (C-5), 122.7 (C-1’’), 121.4 (C-2’),
119.2 (C-4), 117.7 (C-3’’), 117.3 (C-4’’), 117.1 (C-6’’), 116.3 (C-3’);
MS: 297.02 (M+H+). Anal. Calcd for C16H12N2O4: C, 64.86; H,
4.08; N, 9.46. Found: C, 64.72; H, 3.99; N 9.35.
(film)
m ;
: 3415, 1658 cmꢀ1 1H NMR (DMSO): d = 9.93 (1H, s,
CHO), 9.13 (1H, s, H-5), 7.88 (2H, d, J 8.0 Hz, H-2’’ and H-6’’), 7.75
(2H, d, J 8.8 Hz, H-2’), 7.47 (3H, m, H-3’’, H-4’’ and H-5’’), 6.91
(2H, d, J 8.8 Hz, H-3’); 13C NMR (DMSO): d = 185.0 (CHO), 157.5
(C-4’), 152.7 (C-3), 134.6 (C-1’), 131.8 (C-5), 131.3 (C-1’’), 129.5
(C-4’’), 129.1 (C-3’’ and C-5’’), 129.0 (C-2’’ and C-6’’), 122.1 (C-4),
121.5 (C-2’), 116.4 (C-3’); MS: 265.05 (M+H+). Anal. Calcd for
C16H12N2O2: C, 72.72; H, 4.58; N, 10.60. Found: C, 72.94; H, 4.47;
N, 10.74.
4.2.5. General procedure for the synthesis of carboxylic acids
5a–e
4.2.5.1. Synthesis of 1-(4’-Methoxy-phenyl)-3-phenyl-1H-pyra-
zole-4-carboxylic acid (5a). To a cooled (0 °C) solution of pyraz-
olocarboxaldehyde 3a (0.40 g, 1.4 mmol) in acetone (19 mL) a cold
solution (0 °C) of NaClO2 (0.38 g, 3.4 mmol) and H2NSO3H (0.38 g,
3.9 mmol) in water (19 mL) was added. The subsequent mixture
is stirred for 30 min, until total consumption of the starting mate-
4.2.4.2. 1-(4’-Hydroxyphenyl)-3-(3’’-hydroxyphenyl)-1H-pyrazole-
4-carboxaldehyde (4b). According to the general procedure,
demethylated pyrazolocarboxaldehyde 4b was obtained from pyr-
azolocarboxaldehyde 3b after purification by flash column chro-
matography (Hex/EtOAc 1:1) and evaporation of the solvent as a

M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
4345
rial. The solution was extracted twice with EtOAc and the com-
bined organic extracts were washed twice with an aqueous solu-
tion of 10% Na2S2O3, water and brine. The organic layer was
dried with Na2SO4, filtered and evaporated, to provide carboxylic
C2’’-OCH3), 3.74 (3H, s, C5’’-OCH3), 3.66 (3H, s, C4’-OCH3); 13C NMR
(DMSO): d =164.1 (COOH), 158.6 (C-4’), 153.1 (C-5’’), 152.1 (C-2’’),
150.4 (C-3), 133.0 (C-5), 131.8 (C-1’), 123.5 (C-4), 120.9 (C-2’),
116.5 (C-1’’) 115.1 (C-3’ and C-4’’) 114.9 (C-3’’), 112.8 (C-6’’), 56.29
(C4’-OCH3), 55.96 (C5’’-OCH3) 55.94 (C2’’-OCH3); MS: 355.14
(M+H+). Anal. Calcd for C19H18N2O5: C, 64.40; H, 5.12; N, 7.91. Found:
C, 64.57; H, 5.02; N 7.81.
acid 5a as white solid (95% yield). Mp 203–204 °C; IR (film)
m:
1697 cmꢀ1 1H NMR (DMSO): d = 12.6 (1H, br. s, COOH), 8.97 (1H,
;
s, H-5), 7.89 (2H, d, J 9.2 Hz, H-2’), 7.84 (2H, d, J 7.6 Hz, H-2’’ and
H-6’’), 7.46–7.42 (3H, m, H-3’’, H-4’’ and H-5’’), 7.08 (2H, d, J
9.2 Hz, H-3’), 3.82 (3H, s, OCH3); 13C NMR (DMSO): d = 164.3
(COOH), 158.8 (C-4’), 152.9 (C-3), 133.8 (C-1’), 132.8 (C-5), 132.7
(C-1’’), 129.6 (C-3’’ and C-5’’), 128.8 (C-4’’), 128.2 (C-2’’ and C-6’’),
121.1 (C-2’), 115.1 (C-3’), 113.9 (C-4), 55.94 (OCH3); MS: 295.21
(M+H+). Anal. Calcd for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52.
Found: C, 69.62; H, 4.89; N, 9.40.
4.2.6. General procedure for the synthesis of amides 6a–e
4.2.6.1. Synthesis of N-methoxy-1-(4’-methoxyphenyl)-3-phe-
nyl-1H-pyrazole-4-carboxamide (6a). Et3N (0.17 mL, 1.2 mmol),
was added dropwise into a solution of carboxylic acid 5a (0.17 g,
0.58 mmol) and NH2OMeꢁHCl (0.074 g, 0.87 mmol) in MeCN
(7.0 mL). After stirring the mixture at room temperature for
30 min, TBTU (0.28 g, 0.87 mmol) was added and the stirring was
continued for 30 min until total consumption of the starting mate-
rial. Then brine was added and the solution was extracted twice
with EtOAc. The combined organic extracts were washed with
HCl 1 N, water, 5% aq NaHCO3 and brine. The organic layer was
dried with Na2SO4, filtered and evaporated. After drying in vacuum
over P2O5 carboxamide 6a was obtained as white solid (95% yield).
4.2.5.2. 1-(4’-Methoxyphenyl)-3-(3’’-mehoxyphenyl)-1H-pyra-
zole-4-carboxylic acid (5b). According to the general procedure,
carboxylic acid 5b was obtained from pyrazolocarboxaldehyde 3b
as white solid (95% yield). mp 217–218 °C; IR (film) m ;
: 1696 cmꢀ1
1H NMR (DMSO): d = 12.5 (1H, br. s, COOH), 8.96 (1H, s, H-5), 7.89
(2H, d, J 8.8 Hz, H-2’), 7.45–7.42 (2H, m, H-2’’ and H-6’’), 7.36 (1H,
t, J 8.0 Hz, H-5’’), 7.09 (2H, d, J 8.8 Hz, H-3’), 7.00 (1H, d, J 8.8 Hz,
H-4’’) 3.82 (3H, s, C3’’- OCH3), 3.81 (3H, s, C4’- OCH3); 13C NMR
(DMSO): d = 164.3 (COOH), 159.1 (C-3’’), 158.8 (C-4’), 152.6 (C-3),
133.9 (C-1’), 133.8 (C-5), 132.8 (C-1’’), 129.3 (C-5’’), 121.8 (C-6’’),
121.1 (C-2’), 115.2 (C-4’’), 115.1 (C-3’), 114.4 (C-2’’), 113.9 (C-4)
55.93 (C4’-OCH3), 55.51 (C3’’-OCH3); MS: 325.11 (M+H+). Anal.
Calcd for C18H16N2O4: C, 66.66; H, 4.97; N, 8.64. Found: C, 66.82;
H, 4.89; N, 8.75.
mp 157–158 °C; IR (film) m ;
: 1664 cmꢀ1 1H NMR (DMSO): d = 11.4
(1H, s, NH) 8.75 (1H, s, H-5), 7.84 – 7.80 (4H, m, H-2’, H-2’’ and H-
6’’), 7.47–7.38 (3H, m, H-3’’, H-4’’ and H-5’’), 7.11 (2H, d, J 9.2 Hz, H-
3’), 3.82 (3H, s, NH-OCH3), 3.71 (3H, s, C4’-OCH3); 13C NMR
(DMSO): d = 161.2 (CONH), 158.6 (C-4’), 150.9 (C-3), 133.0 (C-1’),
132.6 (C-1’’), 130.2 (C-5), 128.7 (C-4’’), 128.6 (C-3’’ and C-5’’),
128.4 (C-2’’ and C-6’’), 120.8 (C-2’), 115.2 (C-3’), 114.6 (C-4),
63.41 (NH-OCH3), 55.96 (C4’-OCH3); MS: 324.13 (M+H+). Anal.
Calcd for C18H17N3O3: C, 66.86; H, 5.30; N, 13.00. Found: C,
66.73; H, 5.22; N 13.13.
4.2.5.3. 1-(4’-Methoxyphenyl)-3-(4’’-mehoxyphenyl)-1H-pyra-
zole-4-carboxylic acid (5c). According to the general procedure,
carboxylic acid 5c was obtained from pyrazolocarboxaldehyde 3c
4.2.6.2. N-Methoxy-1-(4’-methoxyphenyl)-3-(3’’-methoxyphenyl)-
1H-pyrazole-4-carboxamide (6b). According to the general pro-
cedure, carboxamide 6b was obtained from carboxylic acid 5b as
as white solid (95% yield). mp 236–237 °C; IR (film) m ;
: 1691 cmꢀ1
1H NMR (DMSO): d = 12.5 (1H, br. s, COOH), 8.93 (1H, s, H-5), 7.85
(2H, d, J 8.4 Hz, H-2’’ and H-6’’), 7.82 (2H, d, J 8.4 Hz, H-2’), 7.08
(2H, d, J 8.4 Hz, H-3’’ and H-5’’), 7.01 (2H, d, J 8.4 Hz, H-3’), 3.82
(6H, s, OCH3); 13C NMR (DMSO): d = 164.4 (COOH), 159.9 (C-4’’),
158.7 (C-4’), 152.7 (C-3), 133.7 (C-1’), 132.8 (C-5), 130.8 (C-2’’ and
C-6’’), 125.0 (C-1’’), 121.0 (C-2’), 115.0 (C-3’’ and C-5’’), 113.7
(C-3’), 113.4 (C-4), 55.92 (C4’-OCH3), 55.60 (C4’’-OCH3); MS:
325.11 (M+H+). Anal. Calcd for C18H16N2O4: C, 66.66; H, 4.97; N,
8.64. Found: C, 66.78; H, 4.88; N, 8.56.
white solid (90% yield). mp 176–177 °C; IR (film) m ;
: 1643 cmꢀ1
1H NMR (DMSO): d = 11.5 (1H, s, NH), 8.74 (1H, s, H-5), 7.82 (2H,
d, J 7.6 Hz, H-2’), 7.45–7.42 (2H, m, H-2’’ and H-6’’), 7.37 (1H, t, J
7.2 Hz, H-5’’), 7.12 (2H, d, J 7.6 Hz, H-3’), 6.99 (1H, d, J 6.4 Hz, H-
4’’), 3.82 (3H, s, NH-OCH3), 3.81 (3H, s, C3’’-OCH3), 3.72 (3H, s,
C4’-OCH3); 13C NMR (DMSO): d = 161.4 (CONH), 159.5 (C-3’’),
158.7 (C-4’), 150.5 (C-3), 133.9 (C-1’), 133.0 (C-5), 130.3 (C-1’’),
129.8 (C-5’’), 120.8 (C-2’), 120.6 (C-6’’), 115.2 (C-3’), 114.7 (C-4),
114.3 (C-4’’), 113.9 (C-2’’), 63.71 (NH-OCH3), 55.95 (C4’-OCH3),
55.93 (C3’’-OCH3); MS: 354.15 (M+H+). Anal. Calcd for
C19H19N3O4: C, 64.58; H, 5.42; N, 11.89. Found: C, 64.71; H, 5.32;
N, 12.03.
4.2.5.4. 1-(4’-Methoxyphenyl)-3-(3’’,4’’-dimethoyphenyl)-1H-
pyrazole-4-carboxylic acid (5d). According to the general proce-
dure, carboxylic acid 5d was obtained from pyrazolocarboxalde-
hyde 3d as white solid (95% yield). mp 172–173 °C; IR (film)
m:
1697 cmꢀ1 1H NMR (acetone-d6): d = 8.76 (1H, s, H-5), 7.91 (2H,
;
4.2.6.3. N-Methoxy-1-(4’-methoxyphenyl)-3-(4’’-methoxyphenyl)-
1H-pyrazole-4-carboxamide (6c). According to the general proce-
dure, carboxamide 6c was obtained from carboxylic acid 5c as
d, J 8.8 Hz, H-2’), 7.71 (1H, d, J 2.0 Hz, H-2’’), 7.62 (1H, dd, J
8.4 Hz J 2.0 Hz, H-6’’), 7.11 (2H, d, J 8.8 Hz, H-3’), 7.01 (1H, d, J
8.4 Hz, H-5’’) 3.88 (3H, s, C3’’-OCH3), 3.87 (3H, s, C4’’-OCH3), 3.86
(3H, s, C4’-OCH3); 13C NMR (acetone d6): d = 165.4 (COOH), 159.3
(C-4’), 152.4 (C-3’’), 151.9 (C-4’’), 144.6 (C-3), 133.1 (C-1’), 133.0
(C-5), 121.9 (C-6’’), 120.7 (C-1’’ and C-2’), 114.5 (C-3’), 113.4 (C-
4), 111.0 (C-5’’), 110.0 (C-2’’), 55.18 (OCH3); MS: 355.14 (M+H+).
Anal. Calcd for C19H18N2O5: C, 64.40; H, 5.12; N, 7.91. Found: C,
64.62; H, 4.99; N 7.79.
white solid (90% yield). mp 187–188 °C; IR (film) m ;
: 1659 cmꢀ1
1H NMR (DMSO): d = 11.4 (1H, s, NH), 8.72 (1H, s, H-5), 7.82 (4H,
d, J 5.6 Hz, H-2’, H-2’’ and H-6’’), 7.12 (2H, d, J 6.0 Hz, H-3’’ and
H-5’’), 7.02 (2H, d, J 6.0 Hz, H-3’), 3.83 (9H, s, OCH3); 13C NMR
(DMSO): d = 161.4 (CONH), 159.8 (C-4’’), 158.5 (C-4’), 150.7 (C-3),
133.0 (C-1’), 130.1 (C-5), 129.7 (C-2’’ and C-6’’), 125.0 (C-1’’),
120.6 (C-2’), 115.2 (C-3’’ and C-5’’), 114.0 (C-4 and C-3’), 63.73
(NH-OCH3), 55.92 (C4’-OCH3), 55.62 (C4’’-OCH3); MS: 354.14
(M+H+). Anal. Calcd for C19H19N3O4: C, 64.58; H, 5.42; N, 11.89.
Found: C, 64.69; H, 5.35; N 12.00.
4.2.5.5. 1-(4’-Methoxyphenyl)-3-(2’’,5’’-dimethoyphenyl)-1H-pyr-
azole-4-carboxylic acid (5e). According to the general procedure,
carboxylic acid 5e was obtained from pyrazolocarboxaldehyde 3e
as white solid (95% yield). mp 221–222 °C; I.R. (film)
m
: 1696 cmꢀ1
;
4.2.6.4. N-Methoxy-1-(4’-methoxyphenyl)-3-(3’’,4’’-dimethoxy-
phenyl)-1H-pyrazole-4-carboxamide (6d). According to the
general procedure, carboxamide 6d was obtained from carboxylic
acid 5d as white solid (85% yield). mp 193–194 °C; IR (film)
1H NMR (DMSO): d = 8.86 (1H, s, H-5), 7.84 (2H, d, J 8.8 Hz, H-2’),
7.07 (2H, d, J 8.8 Hz, H-3’), 7.00 (1H, d, J 8.8 Hz, H-3’’), 6.96 (1H, dd,
J 8.8 Hz J 2.8 Hz, H-4’’), 6.91 (1H, d, J 2.8 Hz, H-6’’), 3.81 (3H, s,

4346
M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
m
: 1650 cmꢀ1; 1H NMR (DMSO): d = 11.5 (1H, s, NH), 8.74 (1H, s, H-
4.2.7.3. 5,7-Dimethoxy-2-(4’-methoxyphenyl)-2H-pyrazolo[4,3-
c]quinolin-4(5H)-one (7c). According to the general procedure,
pyrazoloquinolin-4-one 7c was obtained from carboxamide 6c
after purification by flash column chromatography (Hex/EtOAc,
3:7) and evaporation of the solvent as white solid (55% yield).
5), 7.83 (2H, d, J 8.8 Hz, H-2’), 7.55 (1H, s, H-2’’), 7.45 (1H, dd, J
8.4 Hz J 2.0 Hz, H-6’’), 7.12 (2H, d, J 8.8 Hz, H-3’), 7.04 (1H, d, J
8.4 Hz, H-5’’) 3.83 (9H, s, C3’’-OCH3, C4’’-OCH3, and NH-OCH3),
3.74 (3H, s, C4’-OCH3); 13C NMR (DMSO): d = 161.6 (CONH), 158.6
(C-4’), 150.7 (C-3’’), 149.5 (C-4’’), 148.8 (C-3), 133.0 (C-1’), 130.2
(C-5), 125.3 (C-1’’), 121.0 (C-6’’), 120.7 (C-2’), 115.2 (C-3’), 114.3
(C-4), 112.2 (C-5’’), 112.0 (C-2’’), 63.72 (NH-OCH3), 55.94 (OCH3);
MS: 384.18 (M+H+). Anal. Calcd for C20H21N3O5: C, 62.65; H,
5.52; N, 10.96. Found: C, 62.81; H, 5.39; N, 11.09.
mp 175–176 °C; IR (film) m ;
: 1647 cmꢀ1 1H NMR (CDCl3): d = 8.63
(1H, s, H-3), 8.24 (1H, d, J 8.8 Hz, H-9), 7.76 (2H, d, J 8.4 Hz, H-
2’), 7.14 (1H, s, H-6), 7.06 (2H, d, J 8.8 Hz, H-3’), 6.94 (1H, d, J
8.8 Hz, H-8), 4.14 (3H, s, N-OCH3), 3.97 (3H, s, C7-OCH3), 3.91
(3H, s, C4’-OCH3); 13C NMR (CDCl3): d = 161.5 (C-4), 159.4 (C-7),
155.5 (C-4’), 148.0 (C-1a), 138.4 (C-1’), 133.2 (C-5a), 127.4 (C-3),
124.7 (C-9), 121.9 (C-2’), 114.8 (C-3’), 113.0 (C-9a), 110.3 (C-8),
108.1 (C-3a), 97.70 (C-6) 62.88 (N-OCH3), 55.67 (OCH3); MS:
352.28 (M+H+). Anal. Calcd for C19H17N3O4: C, 64.95; H, 4.88; N,
11.96. Found: C, 64.81; H, 4.78; N 12.08.
4.2.6.5. N-Methoxy-1-(4’-methoxyphenyl)-3-(2’’,5’’-dimethoxy-
phenyl)-1H-pyrazole-4-carboxamide (6e). According to the
general procedure, carboxamide 6e was obtained from carboxylic
acid 5e as white solid (80% yield). mp 177–178 °C; IR (film)
m:
1651 cmꢀ1 1H NMR (DMSO): d = 11.1 (1H, s, NH), 8.62 (1H, s,
;
H-5), 7.77 (2H, d, J 8.8 Hz, H-2’), 7.09 (2H, d, J 8.8 Hz, H-3’), 7.01
(1H, d, J 2.8 Hz, H-6’’), 6.98 – 6.94 (2H, m, H-3’’ and H-4’’), 3.81
(3H, s, NH-OCH3), 3.75 (3H, s, C2’’-OCH3), 3.65 (6H, s, C4’-OCH3
and C5’’-OCH3); 13C NMR (DMSO): d = 161.5 (CONH), 158.5 (C-4’),
153.3 (C-5’’), 151.6 (C-2’’), 149.0 (C-3), 133.2 (C-1’), 128.8 (C-5),
122.9 (C-1’’), 120.8 (C-2’), 116.8 (C-4), 116.3 (C-4’’), 115.2 (C-3’),
115.0 (C-3’’), 112.7 (C-6’’), 63.62 (NH-OCH3), 56.12 (C4’-OCH3),
55.97 (C5’’-OCH3), 55.94 (C2’’-OCH3); MS (M+H+): 384.17. Anal.
Calcd for C20H21N3O5: C, 62.65; H, 5.52; N, 10.96. Found: C,
62.85; H, 5.62; N 10.87.
4.2.7.4. 5,7,8-Trimethoxy-2-(4’-methoxyphenyl)-2H-pyrazolo-
[4,3-c]quinolin-4(5H)-one (7d). According to the general proce-
dure, pyrazoloquinolin-4-one 7d was obtained from carboxamide
6d after purification by flash column chromatography (EtOAc)
and evaporation of the solvent as yellowish oil (50% yield). IR (film)
m
: 1655 cmꢀ1 1H NMR (CDCl3): d = 8.65 (1H, s, H-3), 7.77 (2H, d, J
;
8.8 Hz, H-2’), 7.75 (1H, s, H-9), 7.16 (1H, s, H-6), 7.07 (2H, d, J
8.8 Hz, H-3’), 4.15 (3H, s, N-OCH3), 4.06 (3H, s, C8-OCH3), 4.05
(3H, s, C7-OCH3), 3.91 (3H, s, C4’-OCH3); 13C NMR (CDCl3):
d = 159.5 (C-4), 155.0 (C-4’), 151.4 (C-8), 148.0 (C-1a), 145.9
(C-7), 133.2 (C-1’), 131.9 (C-3), 127.5 (C-5a), 122.0 (C-2’), 114.8
(C-3’), 113.5 (C-9a), 107.1 (C-3a), 104.9 (C-9), 96.58 (C-6), 62.92
(N-OCH3), 56.42 (C7-OCH3), 56.29 (C8-OCH3), 55.66 (C4’-OCH3);
MS: 382.31 (M+H+). Anal. Calcd for C20H19N3O5: C, 62.99; H,
5.02; N, 11.02. Found: C, 63.10; H, 4.94; N 10.98.
4.2.7. Typical procedure for the cyclization of amides 6a–e with
PIFA
4.2.7.1 . Synthesis of 5-Methoxy-2-(4’-methoxyphenyl)-2H-pyr-
azolo[4,3-c]quinolin-4(5H)-one (7a). A solution of PIFA (0.21 g,
0.46 mmol) and TFA (0.070 mL, 0.93 mmol) in CH2Cl2 (6.6 mL)
was added into a cold (ꢀ20 °C) solution of amide 6a (0.10 g,
0.31 mmol) in CH2Cl2 (3.1 mL). The mixture was stirred at the same
temperature during 1 h until total consumption of the starting
material and then quenched with 10% aq Na2CO3 and extracted
twice with CH2Cl2. The combined organic extracts were washed
with brine, dried with Na2SO4, filtered and evaporated. The product
was obtained after flash column chromatography (Hex/EtOAc, 3:7)
and evaporation of the solvent as white solid (57% yield). mp 200–
4.2.7.5. 5,6,9-Trimethoxy-2-(4’-methoxyphenyl)-2H-pyrazolo-
[4,3-c]quinolin-4(5H)-one (7e). According to the general proce-
dure, pyrazoloquinolin-4-one 7e was obtained from carboxamide
6e after purification by flash column chromatography (EtOAc)
and evaporation of the solvent as yellowish oil (50% yield). IR (film)
m
: 1656 cmꢀ1 1H NMR (CDCl3): d = 8.67 (1H, s, H-3), 7.89 (2H, d, J
;
9.2 Hz, H-2’), 7.76 (1H, d, J 8.8 Hz, H-7), 7.18 (1H, d, J 8.8 Hz, H-8),
7.03 (2H, d, J 9.2 Hz, H-3’), 4.17 (3H, s, N-OCH3), 3.96 (3H, s, C6-
OCH3), 3.91 (3H, s, C9-OCH3), 3.91 (3H, s, C4’-OCH3); 13C NMR
(CDCl3): d = 160.1 (C-4), 159.7 (C-4’), 159.6 (C-6), 139.4 (C-1a),
139.3 (C-9), 133.3 (C-1’), 133.0 (C-3), 127.0 (C-5a), 122.4 (C-2’),
115.1 (C-3’), 114.8 (C-7), 114.6 (C-3a), 111.9 (C-9a), 106.4 (C-8),
63.12 (N-OCH3), 55.87 (C9-OCH3), 55.66 (C6-OCH3), 52.22 (C4’-
OCH3); MS: 382.33 (M+H+). Anal. Calcd for C20H19N3O5: C, 62.99;
H, 5.02; N, 11.02. Found: C, 63.14; H, 4.90; N 10.89.
201 °C; IR (film) m
: 1646 cmꢀ1; 1H NMR (CDCl3): d = 8.68 (1H, s, H-
3), 8.36 (1H, d, J 7.2 Hz, H-9), 7.78 (2H, d, J 7.6 Hz, H-2’), 7.66 (1H, d,
J 7.2 Hz, H-6), 7.61 (1H, t, J 6.4 Hz, H-7), 7.36 (1H, t, J 6.4 Hz, H-8),
7.07 (2H, d, J 7.6 Hz, H-3’), 4.15 (3H, s, N-OCH3), 3.90 (3H, s, C4’-
OCH3); 13C NMR (CDCl3): d = 159.5 (C-4), 155.1 (C-4’), 148.0 (C-
1a), 136.9 (C-1’), 133.2 (C-5a), 130.0 (C-3), 127.4 (C-7), 123.3 (C-
9) 123.2 (C-8), 121.9 (C-2’), 114.8 (C-3’ and C-9a), 113.0 (C-6 and
C-3a) 62.95 (N-OCH3) 55.67 (C4’-OCH3); MS: 322.22 (M+H+). Anal.
Calcd for C18H15N3O3: C, 67.28; H, 4.71; N, 13.08. Found: C, 67.41;
H, 4.59; N 13.21.
4.2.8. General procedure for the demethylation of pyrazoloquin-
olin-4-ones 7a–e
4.2.8.1. Synthesis of 5-Hydroxy-2-(4’-hydroxyphenyl)-2H-pyraz-
olo[4,3-c]quinolin-4(5H)-one (8a). To a stirred solution of the
protected pyrazoloquinolin-4-one 7a (0.050 g, 0.16 mmol) in
CH2Cl2 (5.0 mL) at ꢀ78 °C BBr3 (1.6 mmol, 1.0 M in CH2Cl2, 5 equiv
per bond, 1.6 mL) was added dropwise. The reaction was kept at -
78 °C for 1 h and then for 16 h at room temperature. The solution
was cooled at 0 °C, neutralized with MeOH and then HCl 1 N was
added. The organic phase was extracted twice with EtOAc. The
combined organic extracts were washed with water, brine, dried
with Na2SO4, filtered and evaporated. The product was obtained
after flash column chromatography (EtOAc) and evaporation of
the solvent as brown solid (95% yield). mp 269–270 °C; IR (film)
4.2.7.2. 5,8-Dimethoxy-2-(4’-methoxyphenyl)-2H-pyrazolo[4,3-
c]quinolin-4(5H)-one (7b). According to the general procedure,
pyrazoloquinolin-4-one 7b was obtained from carboxamide 6b after
purification by flash column chromatography (Hex/EtOAc, 4:6) and
evaporation of the solvent as white solid (55% yield). mp 166–
167 °C; IR (film) m
: 1663 cmꢀ1; 1H NMR (CDCl3): d = 8.66 (1H, s, H-
3), 7.80 (1H, d, J 2.8 Hz, H-9), 7.78 (2H, d, J 8.8 Hz, H-2’), 7.57 (1H,
d, J 9.2 Hz, H-6), 7.19 (1H, dd, J 9.2 Hz J 2.8 Hz, H-7), 7.07 (2H, d, J
8.8 Hz, H-3’), 4.13 (3H, s, N-OCH3), 3.96 (3H, s, C8-OCH3), 3.90 (3H,
s, C4’-OCH3); 13C NMR (CDCl3): d = 159.5 (C-4), 155.8 (C-8), 154.7
(C-4’), 147.8 (C-1a), 133.1 (C-1’), 131.1 (C-5a), 127.6 (C-3), 122.0
(C-2’), 118.4 (C-7), 115.5 (C-9a), 114.8 (C-3’), 114.6 (C-6), 114.3 (C-
3a), 105.5 (C-9), 62.97 (N-OCH3), 55.90 (C4’-OCH3), 55.69 (C8-
OCH3); MS: 352.29 (M+H+). Anal. Calcd for C19H17N3O4: C, 64.95;
H, 4.88; N, 11.96. Found: C, 65.10; H, 4.74; N 11.82.
m
: 3390, 1642 cmꢀ1 1H NMR (DMSO): d = 11.0 (1H, s, N-OH),
;
9.90 (1H, s, C4’-OH) 9.30 (1H, s, H-3), 8.19 (1H, d, J 7.2 Hz, H-9),
7.89 (2H, d, J 8.0 Hz, H-2’), 7.73 (1H, d, J 8.0 Hz, H-6), 7.62 (1H,
t, J 6.8 Hz, H-7), 7.34 (1H, t, J 6.8 Hz, H-8), 6.95 (2H, d, J 8.0 Hz,

M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
4347
H-3’); 13C NMR (DMSO): d = 157.7 (C-4), 155.1 (C-4’), 147.0
(C-1a), 138.8 (C-1’), 131.9 (C-5a), 130.3 (C-3), 128.4 (C-7), 123.0
(C-9) 122.7 (C-8), 122.0 (C-2’), 116.4 (C-3’) 114.1 (C-9a) 113.9
4.2.9. General procedure for the synthesis of esters 9a-e
4.2.9.1. Synthesis of (E)-ethyl 3-(1’-(4’’-methoxyphenyl)-3’-phe-
nyl-1’H-pyrazol-4’-yl) acrylate (9a). To a stirred solution of the
protected aldehyde 3a (1.2 g, 4.3 mmol) in MeCN (14 mL),
Ph3PCHCO2CH2CH3 (1.6 g, 4.7 mmol) was added and the reaction
mixture was refluxed overnight. After the completion of the reac-
tion EtOAc was added and the organic layer was washed with
water, brine, dried with Na2SO4, filtered and evaporated. The prod-
uct was obtained after flash column chromatography (Hex/EtOAc,
8:2) and evaporation of the solvent as white solid (90% yield).
(C-6) 113.8 (C-3a); MS: 294.17 (M+H+). Anal. Calcd for C16H11
-
N3O3: C, 65.53; H, 3.78; N, 14.33. Found: C, 65.39; H, 3.86; N
14.46.
4.2.8.2. 5,8-Dihydroxy-2-(4’-hydroxyphenyl)-2H-pyrazolo[4,3-c]
quinolin-4-one (8b). According to the general procedure,
demethylated pyrazoloquinolin-4-one 8b was obtained from pyraz-
oloquinolin-4-one 7b after purification by flash column chromatog-
raphy (EtOAc) and evaporation of the solvent as yellowish oil (95%
mp 162–163 °C; IR (film) m ;
: 1703 cmꢀ1 1H NMR (CDCl3): d = 8.16
(1Y, s, H-5’), 7.76 (1H, d, J 16 Hz, H-3), 7.69 (2H, d, J 7.2 Hz, H-2’’’
and H-6’’’), 7.68 (2H, d, J 8.8 Hz, H-2’’), 7.51 (2H, t, J 7.2 Hz, H-3’’’
and H-5’’’), 7.45 (1H, t, J 7.2 Hz, H-4’’’), 7.02 (2H, d, J 8.8 Hz, H-
3’’), 6.28 (1H, d, J 16 Hz, H-2), 4.26 (2H, q, J 7.2 Hz, OCH2CH3),
3.88 (3H, s, OCH3), 1.34 (3H, t, J 7.2 Hz, OCH2CH3); 13C NMR
(CDCl3): 167.1 (C-1), 158.8 (C-4’’), 153.0 (C-3’), 135.2 (C-4’), 133.2
(C-1’’), 132.4 (C-1’’’), 128.7 (C-3, C-3’’’ and C-5’’’), 128.6 (C-2’’’ and
C-6’’’), 128.5 (C-4’’’), 126.4 (C-5’), 121.0 (C-2’’), 117.3 (C-2), 114.6
(C-3’’), 60.32 (OCH2CH3), 55.60 (OCH3), 14.35 (OCH2CH3); MS:
349.28 (M+H+). Anal. Calcd for C21H20N2O3: C, 72.40; H, 5.79; N,
8.04. Found: C, 72.19; H, 5.69; N 8.20.
yield). IR (film) m ;
: 3416, 1652 cmꢀ1 1H NMR (DMSO): d = 10.94
(1H, s, N-OH), 9.94 (1H, s, OH), 9.70 (1H, s, OH), 9.23 (1H, s, H-3),
7.87 (2H, d, J 7.2 Hz, H-2’), 7.56 (1H, d, J 8.0 Hz, H-6), 7.07 (1H, d, J
8.0 Hz, H-7), 6.95 (2H, d, J 7.2 Hz, H-3’), 6.58 (1H, s, H-9); 13C NMR
(DMSO): d = 157.8 (C-4), 157.7 (C-8), 153.4 (C-4’), 146.9 (C-1a),
132.1 (C-1’), 132.0 (C-5a), 128.3 (C-3), 122.0 (C-2’), 118.5 (C-9a),
116.4 (C-3’), 115.7 (C-7), 114.8 (C-6), 114.2 (C-3a), 107.4 (C-9);
MS: 310.39 (M+H+). Anal. Calcd for C16H11N3O4: C, 62.14; H, 3.58;
N, 13.59. Found: C, 62.27; H, 3.67; N 13.48.
4.2.8.3. 5,7-Dihydroxy-2-(4’-hydroxyphenyl)-2H-pyrazolo[4,3-c]-
quinolin-4(5H)-one (8c). According to the general procedure,
demethylated pyrazoloquinolin-4-one 8c was obtained from pyr-
azoloquinolin-4-one 7c after purification by flash column chroma-
tography (EtOAc) and evaporation of the solvent as yellowish oil
4.2.9.2. (E)-ethyl 3-(1’-(4’’-methoxyphenyl)-3’-(3’’’-methoxy-
phenyl)-1’H-pyrazol-4’-yl) acrylate (9b). According to the gen-
eral procedure, ester 9b was obtained from aldehyde 3b after
purification by flash column chromatography (Hex/EtOAc, 7:3)
and evaporation of the solvent as white solid (80% yield). mp
(95% yield). IR (film) m ;
: 3381, 1635 cmꢀ1 1H NMR (acetone-d6):
119–120 °C; IR (film)
m ;
: 1703 cmꢀ1 1H NMR (CDCl3): d = 8.15
d = 9.10 (1H, s, N-OH), 8.94 (1H, s, OH), 8.92 (1H, s, H-3), 8.86
(1H, s, OH), 8.12 (1H, d, J 8.4 Hz, H-9), 7.90 (2H, d, J 8.4 Hz, H-2’),
7.31 (1H, s, H-6), 7.04 (2H, d, J 8.0 Hz, H-3’), 6.90 (1H, d, J 8.4 Hz,
H-8); 13C NMR (acetone-d6): d = 159.2 (C-4), 157.2 (C-7), 154.6
(C-4’), 147.5 (C-1a), 138.6 (C-1’), 132.5 (C-5a), 126.7 (C-3), 124.1
(C-9), 121.6 (C-2’), 115.9 (C-3’), 111.7 (C-9a), 111.1 (C-8), 106.5
(C-3a), 99.39 (C-6); MS: 310.37 (M+H+). Anal. Calcd for
C16H11N3O4: C, 62.14; H, 3.58; N, 13.59. Found: C, 61.96; H, 3.69;
N 13.44.
(1Y, s, H-5’), 7.76 (1H, d, J 16 Hz, H-3), 7.68 (2H, d, J 8.8 Hz, H-
2’’), 7.41 (1H, t, J 8.0 Hz, H-5’’’), 7.25 (2H, d, J 6.4 Hz, H-2’’’ and
H-6’’’), 7.00 (3H, d, J 8.8 Hz, H-3’’ and H-4’’’), 6.28 (1H, d, J 16 Hz,
H-2), 4.26 (2H, q, J 7.2 Hz, OCH2CH3), 3.89 (3H, s, C3’’’-OCH3),
3.86 (3H, s, C4’’-OCH3), 1.33 (3H, t, J 7.2 Hz, OCH2CH3); 13C NMR
(CDCl3): 167.0 (C-1), 159.8 (C-3’’’), 158.8 (C-4’’), 152.8 (C-3’),
135.2 (C-4’), 133.6 (C-1’’), 133.2 (C-1’’’), 129.7 (C-3), 126.4 (C-5’’’),
121.2 (C-5’), 121.0 (C-2’’), 117.3 (C-4’’’ and C-6’’’), 114.6 (C-3’’
and C-2’’’), 113.7 (C-2), 60.31 (OCH2CH3), 55.60 (C4’’-OCH3), 55.36
(C3’’’-OCH3), 14.36 (OCH2CH3); MS: 379.14 (M+H+). Anal. Calcd
for C22H22N2O4: C, 69.83; H, 5.86; N, 7.40. Found: C, 70.01; H,
5.73; N 7.53.
4.2.8.4. 5,7,8-Trihydroxy-2-(4’-hydroxyphenyl)-2H-pyrazolo[4,3-
c]quinolin-4(5H)-one (8d). According to the general procedure,
demethylated pyrazoloquinolin-4-one 8d was obtained from pyr-
azoloquinolin-4-one 7d after purification by flash column chroma-
tography (EtOAc) and evaporation of the solvent as yellowish oil
4.2.9.3. (E)-ethyl 3-(1’-(4’’-methoxyphenyl)-3’-(4’’’-methoxy-
phenyl)-1’H-pyrazol-4’-yl) acrylate (9c). According to the gen-
eral procedure, ester 9c was obtained from aldehyde 3c after
purification by flash column chromatography (Hex/EtOAc, 7:3)
(90% yield). IR (film)
m ;
: 3398, 1650 cmꢀ1 1H NMR (DMSO):
d = 9.14 (1H, s, H-3), 7.84 (2H, d, J 8.8 Hz, H-2’), 7.48 (1H, s, H-9),
7.13 (1H, s, H-6), 6.92 (2H, d, J 8.8 Hz, H-3’); 13C NMR (DMSO):
d = 157.4 (C-4), 154.9 (C-4’), 152.0 (C-8), 148.6 (C-1a), 142.2 (C-
7), 133.0 (C-1’), 132.0 (C-3), 127.9 (C-5a), 121.8 (C-2’), 116.4 (C-3’
and C-9a), 108.2 (C-3a), 105.6 (C-9), 101.2 (C-6); MS: 326.28
(M+H+). Anal. Calcd for C16H11N3O5: C, 59.08; H, 3.41; N, 12.92.
Found: C, 59.23; H, 3.49; N 13.07.
and evaporation of the solvent as foam (80% yield). IR (film)
m:
1703 cmꢀ1 1H NMR (CDCl3): d = 8.13 (1Y, s, H-5’), 7.74 (1H, d, J
;
16 Hz, H-3), 7.67 (2H, d, J 9.2 Hz, H-2’’’ and H-6’’’), 7.62 (2H, d, J
8.8 Hz, H-2’’), 7.03 (2H, d, J 9.2 Hz, H-3’’’ and H-5’’’),7.01(2H, d, J
8.8 Hz, H-3’’), 6.27 (1H, d, J 16 Hz, H-2), 4.26 (2H, q, J 7.2 Hz,
OCH2CH3), 3.89 (3H, s, C4’’’-OCH3), 3.88 (3H, s, C4’’-OCH3), 1.34
(3H, t, J 7.2 Hz, OCH2CH3); 13C NMR (CDCl3): 167.2 (C-1), 160.0
(C-4’’’), 158.7 (C-4’’), 152.8 (C-3’), 135.4 (C-4’), 133.2 (C-1’’), 129.9
(C-2’’’ and C-6’’’), 126.3 (C-3), 124.9 (C-1’’’), 121.0 (C-2’’), 117.1
(C-5’), 117.0 (C-2), 114.6 (C-3’’’ and C-5’’), 114.2 (C-3’’), 60.29
(OCH2CH3), 55.60 (C4’’-OCH3), 55.35 (C4’’’-OCH3), 14.34
(OCH2CH3); MS: 379.20 (M+H+). Anal. Calcd for C22H22N2O4: C,
69.83; H, 5.86; N, 7.40. Found: C, 69.99; H, 5.99; N 7.55.
4.2.8.5. 5,6,9-Trihydroxy-2-(4’-hydroxyphenyl)-2H-pyrazolo[4,3-
c]quinolin-4(5H)-one (8e). According to the general procedure,
demethylated pyrazoloquinolin-4-one 8e was obtained from pyr-
azoloquinolin-4-one 7e after purification by flash column chroma-
tography (EtOAc) and evaporation of the solvent as yellowish oil
(90% yield). IR (film)
m ;
: 3412, 1652 cmꢀ1 1H NMR (DMSO):
d = 9.24 (1H, s, H-3), 7.90 (2H, d, J 8.8 Hz, H-2’), 7.63 (1H, d, J
8.4 Hz, H-7), 7.13 (1H, d, J 8.4 Hz, H-8), 6.95 (2H, d, J 8.8 Hz, H-
3’); 13C NMR (DMSO): d = 157.2 (C-4), 155.3 (C-4’), 153.4 (C-6),
142.4 (C-1a), 139.3 (C-9), 133.1 (C-1’), 132.8 (C-3), 127.3 (C-5a),
122.1 (C-2’), 115.3 (C-3’), 115.0 (C-7), 114.6 (C-3a), 111.9 (C-9a),
106.4 (C-8); MS: 326.29 (M+H+). Anal. Calcd for C16H11N3O5: C,
59.08; H, 3.41; N, 12.92. Found: C, 58.89; H, 3.53; N 12.79.
4.2.9.4. (E)-ethyl 3-(1’-(4’’-methoxyphenyl)-3’-(3’’’,4’’’-dime-
thoxyphenyl)- 1’H-pyrazol-4’-yl) acrylate (9d). According to
the general procedure, ester 9d was obtained from aldehyde 3d
after purification by flash column chromatography (Hex/EtOAc,
1:1) and evaporation of the solvent as white solid (80% yield).
mp 122–123 °C; IR (film) m ;
: 1704 cmꢀ1 1H NMR (CDCl3): d = 8.13

4348
M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
(1Y, s, H-5’), 7.76 (1H, d, J 16 Hz, H-3), 7.67 (2H, d, J 9.2 Hz, H-2’’),
7.24 (1H, d, J 2.0 Hz, H-2’’’), 7.20 (1H, dd, J 8.2 Hz J 2.0 Hz, H-6’’’),
7.00 (2H, d, J 9.2 Hz, H-3’’), 6.99 (1H, d, J 8.2 Hz, H-5’’’), 6.27 (1H,
d, J 16 Hz, H-2), 4.25 (2H, q, J 7.2 Hz, OCH2CH3), 3.97 (3H, s, C3’’’-
OCH3), 3.96 (3H, s, C4’’’-OCH3), 3.87 (3H, s, C4’’-OCH3), 1.33 (3H, t, J
7.2 Hz, OCH2CH3); 13C NMR (CDCl3): 167.1 (C-1), 158.8 (C-4’’),
152.8 (C-3’), 149.5 (C-3’’’), 149.2 (C-4’’’), 135.4 (C-4’), 133.2 (C-1’’),
126.4 (C-1’’’), 125.1 (C-3), 121.4 (C-5’), 121.0 (C-2’’), 120.9 (C-6’’’)
117.1 (C-5’’’), 114.6 (C-3’’), 111.6 (C-2’’’), 111.3 (C-2), 60.29
(OCH2CH3), 55.98 (C3’’’-OCH3 and C4’’’-OCH3), 55.59 (C4’’-OCH3),
14.35 (OCH2CH3); MS: 409.34 (M+H+). Anal. Calcd for C23H24N2O5:
C, 67.63; H, 5.92; N, 6.86. Found: C, 67.71; H, 5.83; N 6.94.
(1H, dd, J 8.0 Hz J 1.6 Hz, H-6’’’), 6.44 (1H, d, J 16 Hz, H-2), 4.19
(2H, q, J 7.2 Hz, OCH2CH3), 1.27 (3H, t, J 7.2 Hz, OCH2CH3); 13C
NMR (acetone-d6): 166.5 (C-1), 157.7 (C-3’’’), 156.7 (C-4’’), 152.1
(C-3’), 136.2 (C-4’), 135.0 (C-1’’ and C-1’’’), 129.7 (C-3), 127.2 (C-
5’’’), 120.6 (C-5’and C-2’’), 119.7 (C-6’’’), 116.9 (C-4’’’), 115.9 (C-
3’’), 115.4 (C-2’’’), 115.4 (C-2), 59.71 (OCH2CH3), 13.78 (OCH2CH3);
MS: 351.02 (M+H+). Anal. Calcd for C20H18N2O4: C, 68.56; H, 5.18;
N, 8.00. Found: C, 68.75; H, 5.32; N 8.18.
4.2.10.3. (E)-ethyl 3-(1’-(4’’-hydroxyphenyl)-3’-(4’’’-hydroxy-
phenyl)-1’H-pyrazol-4’-yl) acrylate (10c). According to the gen-
eral procedure, deprotected ester 10c was obtained from ester 9c
after purification by flash column chromatography (Hex/EtOAc,
1:1) and evaporation of the solvent as white solid (85% yield). mp
4.2.9.5. (E)-ethyl 3–1’-(4’’-methoxyphenyl)-3’-(2’’’,5’’’-dimethoxy-
phenyl)-1’H-pyrazol-4’-yl) acrylate (9e). According to the general
procedure, ester 9e was obtained from aldehyde 3e after purification
by flash column chromatography (Hex/EtOAc, 7:3) and evaporation
248–249 °C; IR (film) m ;
: 1693 cmꢀ1 1H NMR (DMSO): d = 9.00 (1Y,
s, H-5’), 7.67 (2H, d, J 8.4 Hz, H-2’’’ and H-6’’’), 7.54 (1H, d, J 16 Hz,
H-3), 7.41 (2H, d, J 8.0 Hz, H-2’’), 6.93–6.90 (4H, m, H-3’’ H-3’’’ and
H-5’’’), 6.46 (1H, d, J 16 Hz, H-2), 4.16 (2H, q, J 6.8 Hz, OCH2CH3),
1.24 (3H, t, J 6.8 Hz, OCH2CH3); 13C NMR (DMSO): 167.0 (C-1), 158.3
(C-4’’’), 156.9 (C-4’’), 152.5 (C-3’), 135.6 (C-4’), 131.8 (C-1’’), 130.1
(C-2’’’ and C-6’’’), 128.4 (C-3), 123.9 (C-1’’’), 120.8 (C-2’’), 116.6 (C-
5’), 116.4(C-2), 116.3(C-3’’’ and C-5’’), 116.1(C-3’’), 60.26(OCH2CH3),
14.68 (OCH2CH3); MS: 351.10 (M+H+). Anal. Calcd for C20H18N2O4: C,
68.56; H, 5.18; N, 8.00. Found: C, 68.40; H, 5.31; N 7.88.
of the solvent as white solid (80% yield). mp 116–117 °C; IR (film)
m:
1704 cmꢀ1 1H NMR (DMSO): d = 8.14 (1Y, s, H-5’), 7.69 (2H, d, J
;
9.2 Hz, H-2’’), 7.68 (1H, d, J 16 Hz, H-3), 7.08 (1H, d, J 1.6 Hz,
H-6’’’), 7.00 (2H, d, J 9.2 Hz, H-3’’), 6.97 (2H, d, J 8.8 Hz, H-3’’’ and
H-4’’’), 6.12 (1H, d, J 16 Hz, H-2), 4.22 (2H, q, J 7.2 Hz, OCH2CH3),
3.87 (3H, s, C5’’’-OCH3), 3.83 (3H, s, C2’’’-OCH3), 3.80 (3H, s,
C4’’-OCH3),1.21 (3H, t, J 7.2 Hz, OCH2CH3); 13C NMR (DMSO):
167.3 (C-1), 158.7 (C-4’’), 153.7 (C-5’’’), 151.4 (C-2’’’), 150.6 (C-3’),
136.0 (C-4’), 133.6 (C-1’’), 133.3 (C-3), 126.0 (C-5’) 121.0 (C-2’’),
118.7 (C-1’’’) 116.7 (C-4’’’), 116.0 (C-3’’’), 115.8 (C-6’’’), 114.6
(C-3’’), 112.6 (C-2) 60.12 (OCH2CH3), 56.11 (C4’’-OCH3) 55.86
(C5’’’-OCH3) 55.58 (C2’’’-OCH3), ,14.35 (OCH2CH3); MS: 409.35
(M+H+). Anal. Calcd for C23H24N2O5: C, 67.63; H, 5.92; N, 6.86.
Found: C, 67.85; H, 5.78; N 7.01.
4.2.10.4. (E)-ethyl 3-(1’-(4’’-hydroxyphenyl)-3’-(3’’’,4’’’-dihydroxy-
phenyl)- 1’H-pyrazol-4’-yl) acrylate (10d). According to the gen-
eral procedure, deprotected ester 10d was obtained from ester 9d
after purification by flash column chromatography (EtOAc) and
evaporation of the solvent as brown solid (85% yield). mp 203–
204 °C; IR (film)
m ;
: 1698 cmꢀ1 1H NMR (acetone-d6): d = 8.78
(1Y, s, H-5’), 7.76 (2H, d, J 9.2 Hz, H-2’’),7.73 (1H, d, J 16 Hz, H-3),
7.21 (1H, d, J 2.0 Hz, H-2’’’), 7.04 (1H, dd, J 8.0 Hz J 2.0 Hz, H-6’’’),
6.99 (2H, d, J 9.2 Hz, H-3’’), 6.98 (1H, d, J 8.2 Hz, H-5’’’), 6.41 (1H, d,
J 16 Hz, H-2), 4.19 (2H, q, J 7.2 Hz, OCH2CH3), 1.27 (3H, t, J 7.2 Hz,
OCH2CH3); 13C NMR (acetone-d6): 166.5 (C-1), 156.4 (C-4’’), 152.4
(C-3’), 145.6 (C-3’’’), 145.1 (C-4’’’), 135.3 (C-4’), 132.5 (C-1’’), 127.0
(C-3), 124.7 (C-5’), 120.5 (C-2’’ C-1’’’ and C-6’’’), 116.5 (C-5’’’), 115.8
(C-3’’), 115.5 (C-2’’’), 115.3 (C-2), 59.63 (OCH2CH3), 13.75
(OCH2CH3); MS: 367.27 (M+H+). Anal. Calcd for C20H18N2O5: C,
65.57; H, 4.95; N, 7.65. Found: C, 65.75; H, 5.12; N 7.78.
4.2.10. General procedure for the demethylation of esters 9a-e
4.2.10.1. Synthesis of (E)-ethyl 3-(1’-(4’’-hydroxyphenyl)-3’-phe-
nyl-1’H-pyrazol-4’-yl) acrylate (10a). To a stirred solution of the
protected ester 9a (0.20 g, 0.57 mmol) in CH2Cl2 (18 mL) at -78 °C
BBr3 (2.8 mmol, 1.0 M in CH2Cl2, 5 equiv per bond, 2.8 mL) was
added dropwise. The reaction was kept at -78 °C for 1 h and then
for 16 h at room temperature. The solution was cooled at 0 °C, neu-
tralized with MeOH and then HCl 1 N was added. The organic
phase was extracted twice with EtOAc. The combined organic ex-
tracts were washed with water, brine, dried with Na2SO4, filtered
and evaporated. The product was obtained after flash column chro-
matography (Hex/EtOAc, 1:1) and evaporation of the solvent as
4.2.10.5. (E)-ethyl 3–1’-(4’’- hydroxyphenyl)-3’-(2’’’,5’’’- dihydroxy-
phenyl)-1’H-pyrazol-4’-yl) acrylate (10e). According to the gen-
eral procedure, deprotected ester 10e was obtained from ester 9e
after purification by flash column chromatography (Hex/EtOAc,
1:1) and evaporation of the solvent as yellow solid (85% yield).
white solid (85% yield). mp 158–159 °C; IR (film) m ;
: 1689 cmꢀ1
1H NMR (acetone-d6): d = 8.84 (1Y, s, H-5’), 7.79 (2H, d, J 8.8 Hz,
H-2’’), 7.71 (1H, d, J 16 Hz, H-3), 7.70 (2H, d, J 7.2 Hz, H-2’’’ and
H-6’’’), 7.55 (2H, t, J 7.2 Hz, H-3’’’ and H-5’’’), 7.48 (1H, t, J 7.2 Hz,
H-4’’’), 7.01 (2H, d, J 8.8 Hz, H-3’’), 6.44 (1H, d, J 16 Hz, H-2), 4.19
(2H, q, J 7.2 Hz, OCH2CH3), 1.27 (3H, t, J 7.2 Hz, OCH2CH3); 13C
NMR (acetone-d6): 166.4 (C-1), 156.7 (C-4’’), 152.2 (C-3’), 134.8
(C-4’), 132.9 (C-1’’), 132.4 (C-1’’’), 128.6 (C-3’’’ and C-5’’’), 128.5
(C-2’’’ and C-6’’’), 128.3 (C-4’’’), 127.3 (C-3), 120.6 (C-2’’), 117.1
(C-5’), 116.8 (C-2), 115.9 (C-3’’), 59.68 (OCH2CH3), 13.74(OCH2-
CH3); MS: 335.22 (M+H+). Anal. Calcd for C20H18N2O3: C, 71.84;
H, 5.43; N, 8.38. Found: C, 71.99; H, 5.52; N 8.21.
mp 236–237 °C; IR (film) m ;
: 1696 cmꢀ1 1H NMR (DMSO): d = 8.96
(1Y, s, H-5’), 7.66 (2H, d, J 9.2 Hz, H-2’’), 7.47 (1H, d, J 16 Hz, H-3),
6.91 (2H, d, J 8.8 Hz, H-3’’), 6.80 (1H, d, J 8.4 Hz, H-3’’’), 6.72 (1H,
dd, J 8.4 Hz J 2.8 Hz, H-4’’’), 6.71 (1H, d, J 2.8 Hz, H-6’’’), 6.32 (1H, d,
J 16 Hz, H-2), 4.13 (2H, q, J 7.2 Hz, OCH2CH3), 1.22 (3H, t, J 7.2 Hz,
OCH2CH3); 13C NMR (DMSO): 167.1 (C-1), 156.9 (C-4’’), 150.8
(C-3’), 150.2 (C-5’’’), 148.0 (C-2’’’), 136.9 (C-4’), 131.9 (C-1’’), 127.9
(C-3), 120.7 (C-2’’), 119.9 (C-5’), 118.2 (C-4’’’), 117.4 (C-3’’’) 117.2
(C-6’’’), 117.1 (C-1’’’), 116.3 (C-3’’), 115.7 (C-2), 60.10 (OCH2CH3),
14.67 (OCH2CH3); MS: 367.29 (M+H+). Anal. Calcd for C20H18N2O5:
C, 65.57; H, 4.95; N, 7.65. Found: C, 65.77; H, 5.10; N 7.78.
4.2.10.2. (E)-ethyl 3-(1’-(4’’-hydroxyphenyl)-3’-(3’’’-hydroxy-
phenyl)-1’H-pyrazol-4’-yl) acrylate (10b). According to the gen-
eral procedure, deprotected ester 10b was obtained from ester 9b
after purification by flash column chromatography (Hex/EtOAc,
1:1) and evaporation of the solvent as yellow solid (85% yield).
4.2.11. General procedure for the synthesis of acids 11a-e
4.2.11.1. Synthesis of (E)-3-(1’-(4’’-methoxyphenyl)-3’-phenyl-
1’H-pyrazol-4’-yl) acrylic acid (11a). A solution of ester 9a
(0.90 g, 2.6 mmol) and NaOH (1.8 mL, 5 N) in 1.3:1 EtOH-DMSO
(46 mL), was stirred overnight at room temperature. After comple-
tion of the reaction, DEE was added and the water layer was
extracted twice with H2O. Next, the solution was acidified with
mp 190–191 °C; IR (film)
m ;
: 1692 cmꢀ1 1H NMR (acetone-d6):
d = 8.82 (1Y, s, H-5’), 7.78 (2H, d, J 8.8 Hz, H-2’’), 7.73 (1H, d, J
16 Hz, H-3), 7.36 (1H, t, J 8.0 Hz, H-5’’’), 7.20 (1H, d, J 1.6 Hz, H-
2’’’), 7.16 (1H, d, J 8.0 Hz, H-4’’’), 7.00 (2H, d, J 8.8 Hz, H-3’’), 6.95

M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
4349
HCl (1 N) to pH = 2 to form a precipitate that was filtered off,
washed with water and dried over P2O5 atmosphere to provide
acid 11a as white solid (90% yield). mp 205–206 °C; IR (film) m:
d, J 2.8 Hz, H-6’’’), 6.20 (1H, d, J 16 Hz, H-2), 3.82 (3H, s, C5’’’-
OCH3), 3.76 (3H, s, C2’’’-OCH3), 3.70 (3H, s, C4’’-OCH3); 13C NMR
(DMSO): 168.3 (C-1), 158.5 (C-4’’), 153.6 (C-3’), 151.4 (C-5’’’),
150.2 (C-2’’’), 135.7 (C-4’), 133.2 (C-1’’), 127.9 (C-3), 122.4 (C-5’),
120.6 (C-2’’), 118.6 (C-1’’’), 117.0 (C-3’’’ and C-4’’’), 115.7 (C-6’’’),
115.1 (C-3’’), 113.5 (C-2), 56.40 (C4’’-OCH3), 55.86 (C2’’’-OCH3
and C5’’’-OCH3); MS: 381.30 (M+H+). Anal. Calcd for C21H20N2O5:
C, 66.31; H, 5.30; N, 7.36. Found: C, 66.44; H, 5.20; N 7.49.
1693 cmꢀ1 1H NMR (DMSO): d = 9.11 (1Y, s, H-5’), 7.84 (2H, d, J
7.2 Hz, H-2’’’ and H-6’’’), 7.62–7.51 (6H, m, H-3, H-2’’, H-3’’’, H-4’’’
and H-5’’’), 7.12 (2H, d, J 7.6 Hz, H-3’’), 6.44 (1H, d, J 16 Hz, H-2),
3.82 (3H, s, OCH3); 13C NMR (DMSO): 168.2 (C-1), 158.7 (C-4’’),
152.2 (C-3’), 134.7 (C-3), 133.1 (C-4’), 132.6 (C-1’’), 129.3 (C-2’’’
and C-6’’’), 129.0 (C-1’’’), 128.8 (C-3’’’ and C-5’’’), 128.6 (C-4’’’),
120.7 (C-2’’), 118.6 (C-5’), 117.1 (C-2), 115.2 (C-3’’), 55.95 (OCH3);
MS: 321.23 (M+H+). Anal. Calcd for C19H16N2O3: C, 71.24; H,
5.03; N, 8.74. Found: C, 71.07; H, 5.12; N 8.88.
4.2.12. General procedure for the demethylation of acids 11a-e
4.2.12.1. Synthesis of (E)-3-(1’-(4’’-hydroxyphenyl)-3’-phenyl-
1’H-pyrazol-4’-yl) acrylic acid (12a). To a stirred solution of the
protected acid 11a (0.20 g, 0.62 mmol) in CH2Cl2 (19 mL) at -78 °C,
BBr3 (3.1 mmol, 1.0 M in CH2Cl2, 5 equiv per bond, 3.1 mL) was
added dropwise. The reaction was kept at -78 °C for 1 h and then
for 16 h at room temperature. The solution was cooled at 0 °C, neu-
tralized with MeOH and then HCl 1 N was added. The organic phase
was extracted twice with EtOAc. The combined organic extracts
were washed with water, brine, dried with Na2SO4, filtered and
evaporated. The formatting precipitate was filtered off, washed with
DEE and dried over P2O5 atmosphere to provide the deprotected
acid 12a as white solid (85% yield). mp 190–191 °C; IR (film)
4.2.11.2. (E)-3-(1’-(4’’-methoxyphenyl)-3’-(3’’’-methoxyphenyl)-
1’H-pyrazol-4’-yl) acrylic acid (11b). According to the general
procedure, acid 11b was obtained from ester 9b as white solid
(90% yield). mp 168–169 °C; IR (film)
m ;
: 1685 cmꢀ1 1H NMR
(CDCl3): d = 8.20 (1Y, s, H-5’), 7.87 (1H, d, J 16 Hz, H-3), 7.70 (2H,
d, J 8.8 Hz, H-2’’), 7.43 (1H, t, J 8.0 Hz, H-5’’’), 7.29 (2H, m, H-2’’’
and H-6’’’), 7.02 (3H, m, H-3’’ and H-4’’’), 6.28 (1H, d, J 16 Hz, H-
2), 3.90 (3H, s, C3’’’-OCH3), 3.88 (3H, s, C4’’-OCH3); 13C NMR
(CDCl3): 168.1 (C-1), 159.9 (C-3’’’), 158.6 (C-4’’), 152.2 (C-3’),
134.8 (C-4’), 133.5 (C-1’’), 133.1 (C-1’’’), 131.7 (C-3), 127.1 (C-5’’’),
122.3 (C-5’), 120.5 (C-2’’), 117.8 (C-4’’’ and C-6’’’), 115.0 (C-3’’ and
C-2’’’), 114.0 (C-2), 55.93 (C4’’-OCH3), 55.66 (C3’’’-OCH3); MS:
351.02 (M+H+). Anal. Calcd for C20H18N2O4: C, 68.56; H, 5.18; N,
8.00. Found: C, 68.72; H, 5.11; N 8.08.
m
: 1693 cmꢀ1 1H NMR (DMSO): d = 9.03 (1Y, s, H-5’), 7.71 (2H,
;
d, J 8.8 Hz, H-2’’), 7.61 (2H, d, J 7.2 Hz, H-2’’’ and H-6’’’), 7.54 (2H, t,
J 7.2 Hz, H-3’’’ and H-5’’’), 7.52 (1H, d, J 16 Hz, H-3), 7.47 (1H, 7,
J 7.2 Hz, H-4’’’), 6.92 (2H, d, J 8.8 Hz, H-3’’), 6.41 (1H, d, J 16 Hz,
H-2); 13C NMR (DMSO): 168.2 (C-1), 157.0 (C-4’’), 152.0 (C-3’),
134.8 (C-3), 132.7 (C-4’), 131.9 (C-1’’), 129.3 (C-2’’’ and C-6’’’),
128.9 (C-1’’’), 128.8 (C-3’’’ and C-5’’’), 128.5 (C-4’’’), 121.0 (C-2’’),
118.4 (C-5’), 116.8 (C-2), 116.4 (C-3’’); MS: 307.23 (M+H+). Anal.
Calcd for C18H14N2O3: C, 70.58; H, 4.61; N, 9.15. Found: C, 70.44;
H, 4.50; N 9.29.
4.2.11.3. (E)-3-(1’-(4’’-methoxyphenyl)-3’-(4’’’-methoxyphenyl)-
1’H-pyrazol-4’-yl) acrylic acid (11c). According to the general
procedure, acid 11c was obtained from ester 9c as white solid
(90% yield). mp 210–211 °C; IR (film)
m ;
: 1693 cmꢀ1 1H NMR
(DMSO): d = 9.02 (1Y, s, H-5’), 7.78 (2H, d, J 8.8 Hz, H-2’’’ and H-
6’’’), 7.50 (2H, d, J 8.4 Hz, H-2’’), 7.47 (1H, d, J 16 Hz, H-3), 7.06
(4H, m, H-3’’, H-3’’’ and H-5’’’), 6.36 (1H, d, J 16 Hz, H-2), 3.78
(3H, s, C4’’’-OCH3), 3.77 (3H, s, C4’’-OCH3); 13C NMR (DMSO):
168.3 (C-1), 160.0 (C-4’’’), 158.7 (C-4’’), 152.1 (C-3’), 134.9 (C-4’),
133.1 (C-1’’), 130.1 (C-2’’’ and C-6’’’), 128.4 (C-3), 124.9 (C-1’’’),
120.6 (C-2’’), 118.1 (C-5’), 117.0 (C-2), 115.1 (C-3’’’ and C-5’’’),
114.7 (C-3’’), 55.92 (C4’’-OCH3), 55.68 (C4’’’-OCH3); MS: 351.15
(M+H+). Anal. Calcd for C20H18N2O4: C, 68.56; H, 5.18; N, 8.00.
Found: C, 68.44; H, 5.09; N 7.89.
4.2.12.2. (E)-3-(1’-(4’’-hydroxyphenyl)-3’-(3’’’-hydroxyphenyl)-
1’H-pyrazol-4’-yl) acrylic acid (12b). According to the general
procedure, deprotected acid 12b was obtained from acid 11b as
white solid (85% yield). mp 246–247 °C; IR (film) m ;
: 1682 cmꢀ1
1H NMR (acetone-d6): d = 8.83 (1Y, s, H-5’), 7.79 (2H, d, J 8.8 Hz,
H-2’’), 7.76 (1H, d, J 16 Hz, H-3), 7.37 (1H, t, J 7.6 Hz, H-5’’’), 7.21
(1H, s, H-2’’’), 7.17 (1H, d, J 7.6 Hz, H-4’’’), 7.01 (2H, d, J 8.8 Hz, H-
3’’), 6.95 (1H, dd, J 7.6 Hz J 1.6 Hz, H-6’’’), 6.43 (1H, d, J 16 Hz, H-
2); 13C NMR (acetone-d6): 167.2 (C-1), 157.6 (C-3’’’), 156.6 (C-
4’’), 152.1 (C-3’), 135.4 (C-4’), 134.1 (C-1’’), 132.5 (C-1’’’), 129.7
(C-3), 127.2 (C-5’’’), 120.6 (C-2’’ and C-6’’’), 119.8 (C-2), 117.0 (C-
5’), 116.8 (C-4’’’), 115.9 (C-3’’), 115.4 (C-2’’’); MS: 323.96 (M+H+).
Anal. Calcd for C18H14N2O4: C, 67.07; H, 4.38; N, 8.69. Found: C,
66.91; H, 4.50; N 8.55.
4.2.11.4.
(E)-3-(1’-(4’’-methoxyphenyl)-3’-(3’’’,4’’’-dimethoxy-
phenyl)-1’H-pyrazol-4’-yl) acrylic acid (11d). According to the
general procedure, acid 11d was obtained from ester 9d as white
solid (90% yield). mp 173–174 °C; IR (film)
m ;
: 1694 cmꢀ1 1H
NMR (CDCl3): d = 8.18 (1Y, s, H-5’), 7.85 (1H, d, J 16 Hz, H-3),
7.69 (2H, d, J 8.0 Hz, H-2’’), 7.25 (1H, s, H-2’’’), 7.20 (1H, d, J
8.0 Hz, H-6’’’), 7.02–6.99 (3H, m, H-3’’ and H-5’’’), 6.27 (1H, d, J
16 Hz, H-2), 3.97 (3H, s, C3’’’-OCH3), 3.96 (3H, s, C4’’’-OCH3), 3.88
(3H, s, C4’’-OCH3); 13C NMR (CDCl3): 167.4 (C-1), 158.6 (C-4’’),
152.7 (C-3’), 149.7 (C-3’’’), 149.3 (C-4’’’), 135.0 (C-4’), 132.8 (C-
1’’), 126.1 (C-1’’’), 122.8 (C-3), 121.1 (C-5’), 121.0 (C-2’’), 120.9
(C-6’’’), 118.4 (C-5’’’), 115.0 (C-3’’), 112.8 (C-2’’’), 112.1 (C-2),
56.21 (C3’’’-OCH3 and C4’’’-OCH3), 55.69 (C4’’-OCH3); MS: 381.31
(M+H+). Anal. Calcd for C21H20N2O5: C, 66.31; H, 5.30; N, 7.36.
Found: C, 66.39; H, 5.23; N 7.42.
4.2.12.3. (E)-3-(1’-(4’’-hydroxyphenyl)-3’-(4’’’-hydroxyphenyl)-
1’H-pyrazol-4’-yl) acrylic acid (12c). According to the general
procedure, deprotected acid 12c was obtained from acid 11c as
white solid (85% yield). mp 202–203 °C; IR (film) m ;
: 1682 cmꢀ1
1H NMR (acetone-d6): d = 8.78 (1Y, s, H-5’), 7.78 (2H, d, J 8.8 Hz,
H-2’’’ and H-6’’’), 7.72 (1H, d, J 16 Hz, H-3), 7.55 (2H, d, J 8.4 Hz,
H-2’’), 7.01 (2H, d, J 8.4 Hz, H-3’’), 7.00 (2H, d, J 8.8 Hz, H-3’’’ and
H-5’’’), 6.40 (1H, d, J 16 Hz, H-2); 13C NMR (acetone-d6): 167.2
(C-1), 157.9 (C-4’’’), 156.5 (C-4’’), 152.4 (C-3’), 135.6 (C-4’), 132.5
(C-1’’), 129.9 (C-2’’’ and C-6’’’), 127.0 (C-3), 124.1 (C-1’’’), 120.5
(C-2’’), 116.6 (C-5’), 116.5 (C-2), 115.8 (C-3’’’ and C-5’’’), 115.5 (C-
3’’); MS: 323.14 (M+H+). Anal. Calcd for C18H14N2O4: C, 67.07; H,
4.38; N, 8.69. Found: C, 66.93; H, 4.31; N 8.60.
4.2.11.5. (E)-3–1’-(4’’-methoxyphenyl)-3’-(2’’’,5’’’-dimethoxy-
phenyl)-1’H-pyrazol-4’-yl) acrylic acid (11e). According to the
general procedure, acid 11e was obtained from ester 9e as white
solid (90% yield). mp 187–188 °C; IR (film)
m
: 1685 cmꢀ1
;
1H
4.2.12.4.
(E)-3-(1’-(4’’-hydroxyphenyl)-3’-(3’’’,4’’’-dihydroxy-
NMR (DMSO): d = 9.01 (1Y, s, H-5’), 7.80 (2H, d, J 8.8 Hz, H-2’’),
7.23 (1H, d, J 16 Hz, H-3), 7.12 (1H, d, J 8.8 Hz, H-3’’’), 7.10 (2H,
d, J 8.8 Hz, H-3’’), 7.05 (1H, dd, J 8.8 Hz J 2.8 Hz, H-4’’’), 6.94 (1H,
phenyl)-1’H-pyrazol-4’-yl) acrylic acid (12d). According to the
general procedure, deprotected acid 12d was obtained from acid
11d as white solid (85% yield). mp 237–238 °C; IR (film)
m:

4350
M. S. Christodoulou et al. / Bioorg. Med. Chem. 18 (2010) 4338–4350
1710 cmꢀ1
;
1H NMR (acetone-d6): d = 8.78 (1Y, s, H-5’), 7.77 (2H,
lophane tape and the eggs were returned to the incubator until day
9 when the compounds were applied. The compounds were placed
on sterile plastic discs (Ø 8 mm), which were allowed to dry under
d, J 8.8 Hz, H-2’’), 7.75 (1H, d, J 16 Hz, H-3), 7.22 (1H, d, J 2.0 Hz,
H-2’’’), 7.05 (1H, dd, J 8.0 Hz J 2.0 Hz, H-6’’’), 7.00 (2H, d, J 8.8 Hz,
H-3’’), 6.98 (1H, d, J 8.0 Hz, H-5’’’), 6.40 (1H, d, J 16 Hz, H-2); 13C
NMR (acetone-d6): 167.2 (C-1), 156.5 (C-4’’), 152.4 (C-3’), 145.7
(C-3’’’), 145.1 (C-4’’’), 135.7 (C-4’), 132.5 (C-1’’), 127.0 (C-1’’’),
124.7 (C-3), 120.4 (C-5’ and C-2’’), 116.6 (C-6’’’), 116.5 (C-2),
115.8 (C-3’’), 115.6 (C-2’’’), 115.4 (C-5’’’); MS: 339.23 (M+H+). Anal.
Calcd for C18H14N2O5: C, 63.90; H, 4.17; N, 8.28. Found: C, 64.03; H,
4.25; N 8.40.
sterile conditions. A solution of cortisone acetate (100 lg/disc, Sig-
ma, St. Louis, MO) was added to all discs in order to prevent an
inflammatory response. A loaded and dried control disc was placed
on the CAM approximately 1 cm away from the disc containing the
test compound(s). Next, the windows were covered and the eggs
further incubated until day 11 when the area around the discs
was cut-off and photographed. Next 2 concentric circles were posi-
tioned on the digitalized pictures and all vessels intersecting these
circles were counted. A two-tailed paired Student’s t-test was used
to assess the significance of the obtained results.
4.2.12.5. (E)-3–1’-(4’’-hydroxyphenyl)-3’-(2’’’,5’’’-dihydroxy-
phenyl)-1’H-pyrazol-4’-yl) acrylic acid (12e). According to the
general procedure, deprotected acid 12e was obtained from acid
Acknowledgments
11e as white solid (85% yield). mp 258–259 °C; IR (film)
m:
1698 cmꢀ1 1H NMR (DMSO): d = 8.93 (1Y, s, H-5’), 7.66 (2H, d, J
;
The authors wish to thank Mrs. Eef Meyen for dedicated techni-
cal help. This research was supported by grants from the Flemish
FWO (Krediet nr. G. 0486.08), the Concerted Actions (GOA nr.
05/19), the Centers of Excellence (Krediet nr. 05/15) (to S.L.) and
COST action CM 602 (Angiokem, to S.L. and S.H.).
8.8 Hz, H-2’’), 7.42 (1H, d, J 16 Hz, H-3), 6.75 (2H, d, J 8.8 Hz, H-
3’’), 6.80 (1H, d, J 8.4 Hz, H-3’’’), 6.72 (1H, dd, J 8.4 Hz J 3.2 Hz, H-
4’’’), 6.71 (1H, d, J 3.2 Hz, H-6’’’), 6.24 (1H, d, J 16 Hz, H-2); 13C
NMR (DMSO): 168.5 (C-1), 156.8 (C-4’’), 150.7 (C-3’), 150.2 (C-
5’’’), 148.0 (C-2’’’), 136.4 (C-4’), 132.0 (C-1’’), 127.6 (C-3), 120.7
(C-2’’), 119.9 (C-5’), 118.3 (C-2), 117.4 (C-6’’’), 117.1 (C-3’’’ and C-
4’’’), 116.9 (C-1’’’), 116.3 (C-3’’); MS: 339.22 (M+H+). Anal. Calcd
for C18H14N2O5: C, 63.90; H, 4.17; N, 8.28. Found: C, 64.11; H,
4.27; N 8.44.
References and notes
1. Folkman, J. N. Eng. J. Med. 1971, 285, 1182.
2. Liekens, S.; De Clercq, E.; Neyts Biochem. Pharmacol. 2001, 61, 253.
3. Carmeliet, P. Nature 2005, 438, 932.
4. Folkman, J. Nat. Rev. Drug Disc. 2007, 6, 273.
5. Folkman, J. Nat. Med. 1995, 1, 27.
4.3. Biological assays
6. Zheng, L. W.; Wu, L. L.; Zhao, B. X.; Dong, W. L.; Miao, J. Y. Bioorg. Med. Chem.
2008, 17, 1957.
4.3.1. Cell cultures
7. (a) Hennequin, L. F.; Thomas, A. P.; Johnstone, C.; Stokes, E. S. E.; Plé, P. A.;
Lohmann, J. J. M.; Ogilvie, D. J.; Dukes, M.; Wedge, S. R.; Curwen, J. O.; Kendrew,
J.; Lambert-van der Brempt, C. J. Med. Chem. 1999, 42, 5369; (b) Kallander, L. S.;
Lu, Q.; Chen, W.; Tomaszek, T.; Yang, G.; Tew, D.; Meek, T. D.; Hofmann, G. A.;
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Johanson, K. O.; Kirkpatrick, R. B.; Ho, T. F.; Fisher, P. W.; Mattern, M. R.;
Johnson, R. K.; Hansbury, M. J.; Winkler, J. D.; Ward, K. W.; Veber, D. F.;
Thompson, S. K. J. Med. Chem. 2005, 48, 5644; (c) Carraro, F.; Naldini, A.; Pucci,
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Mouse aortic endothelial cells (MAEC) and bovine aortic endo-
thelial cells (BAEC) were kindly provided by Prof. M. Presta (Bre-
scia, Italy). Human cervical carcinoma (Hela) and human breast
carcinoma (MCF-7) cells were obtained from the American Type
Culture Collection (ATCC, Middlesex, UK). The cells were grown
in Dulbecco’s modified minimum essential medium (DMEM, Life
Technologies, Inc., Rockville, MD) supplemented with 10 mM
Hepes (Life Technologies, Inc., Rockville, MD) and 10% fetal bovine
serum (FBS, Harlan Sera-Lab Ltd., Loughborough, UK).
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Cells (MAEC, BAEC, Hela or MCF-7) were seeded in 48-well
plates at 10,000 cells per cm2. After 16 h, the cells were incubated
in fresh medium in the presence of the test compounds, as indi-
cated in the Results section. On day 5, cells were trypsinized and
counted by a Coulter counter (Analis, Belgium).
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4.3.3. Cell migration assay
Wounds were created in confluent MAE cell monolayers with a
1.0-mm wide micropipette tip. Then, cells were incubated in fresh
medium containing 10% FCS in the presence of the test compounds.
After 8 h, the wounds were photographed, and endothelial cells
invading the wound were quantified by computerized analysis of
the digitalized images.
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4.3.4. Chorioallantoic membrane (CAM) assay in fertilized
chicken eggs
The in vivo CAM angiogenesis model was performed as de-
scribed with slight modifications.19 Fertilized eggs were incubated
for 3 days at 37 °C when 3 ml of albumen was removed (to detach
the shell from the developing CAM) and a window was opened on
the eggshell exposing the CAM. The window was covered with cel-
19. Liekens, S.; Hernández, A. I.; Ribatti, D.; De Clercq, E.; Camarasa, M. J.; Pérez-
Pérez, M. J.; Balzarini, J. J. Biol. Chem. 2004, 279, 29598.