Novel pyrazole and indazole derivatives: Synthesis and evaluation of their anti-proliferative and anti-angiogenic activities

Article abstract of DOI:10.1002/ardp.201200057

The synthesis of several new pyrazole and indazole derivatives from acetophenone and tetralone substrates is reported. The bioactivities of the new compounds were evaluated through in vitro assays for endothelial cell proliferation and tube formation. Res

Full text of DOI:10.1002/ardp.201200057

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
1
Full Paper
Novel Pyrazole and Indazole Derivatives: Synthesis and
Evaluation of Their Anti-Proliferative and Anti-Angiogenic
Activities
Evangelia Tzanetou1, Sandra Liekens2, Konstantinos M. Kasiotis3, Nikolas Fokialakis4,
and Serkos A. Haroutounian1
1 Chemistry Laboratory, Agricultural University of Athens, Athens, Greece
2 Rega Institute for Medical Research, Leuven, Belgium
3 Laboratory of Pesticides Toxicology, Benaki Phytopathological Institute, Athens, Kifissia, Greece
4 Faculty of Pharmacy, Department of Pharmacognosy and Natural Products Chemistry, University of
Athens, Zografou, Athens, Greece
The synthesis of several new pyrazole and indazole derivatives from acetophenone and tetralone
substrates is reported. The bioactivities of the new compounds were evaluated through in vitro
assays for endothelial cell proliferation and tube formation. Results herein indicate that the easily
prepared compounds containing the indazole structural framework exhibit potent cytostatic
properties against all cell lines tested, with compounds 13 and 14 being the most active
displaying IC50 values of 1.5 ꢀ 0.4 mM and 5.6 ꢀ 2.5 mM, respectively, against MCF-7 cells. In
addition, the indazole derivative 16 was assessed as a competent inhibitor of endothelial tube
formation at 30 mM.
Keywords: Angiogenesis inhibition / Antiproliferative activity / Indazoles / Pyrazoles / Tube formation
Received: February 15, 2012; Revised: May 9, 2012; Accepted: May 11, 2012
DOI 10.1002/ardp.201200057
Introduction
diverse azaheterocyclic rings in their structural framework
have been considered, designed, synthesized, and investi-
gated as potent inhibitors of angiogenesis. These compounds
act on different targets, such as the vascular endothelial
growth factor (VEGF) and platelet-derived growth factor
(PDGF), integrins and heat shock proteins (HSP) [6–8].
Active heterocyclic derivatives containing the pyrazole
moiety have also been reported to serve as scaffolds for the
synthesis of apoptosis-inducing agents [9].
Angiogenesis (the formation of new blood vessels) is a multi-
step process that involves a complex interplay between a
multitude of soluble factors, cell surface receptors, and extra-
cellular matrix components [1–3]. Though angiogenesis is
related to various physiological functions of organisms,
such as embryogenesis, wound healing, organ development,
menstrual cycle, an excessive or insufficient angiogenesis is
connected with the appearance of diverse human diseases
including, retinopathy, rheumatoid arthritis, psoriasis, ath-
erosclerosis, haemangioma, and cancer [4, 5]. Thus, the dis-
covery of specific anti-angiogenesis agents constitutes an
attractive therapeutic approach for the treatment of these
diseases, leading to the development of new highly active
small organic molecules acting as potent inhibitors of angio-
genesis. During the last decade several molecules containing
In the course of our investigations concerning the develop-
ment of novel bioactive molecules containing the pyrazole
residue, we have conceived and implemented the synthesis of
several pyrazole derivatives as potential anti-angiogenic
agents. The rationale of their design was to delineate the effect
of the substitution pattern of the pharmacophore pyrazole
on their anti-angiogenic activities. In this respect, we have
found that compounds containing the fused pyrazole[4,3-c]-
quinoline framework are promising lead compounds display-
ing inhibitory activity against endothelial and tumor cell
proliferation in vitro and angiogenesis in vivo (exemplified by
compound 8b depicted in Fig. 2) [10]. As a continuation, we
present herein the outcome of our research concerning the
Correspondence: Serkos A. Haroutounian, Chemistry Laboratory,
Agricultural University of Athens, Iera odos 75, Athens 11855, Greece.
E-mail: sehar@aua.gr
Fax: þ30 210 529-4265
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
synthesis of novel molecules which contain the pyrazole
structural backbone either as trisubstiuted flexible molecule
or as cornerstone of the rigid indazole skeleton. The outcome
of their bioactivity evaluation indicated that the synthesized
rigid pyrazole derivatives inhibit efficiently the in vitro endo-
thelial and tumor cell proliferation as well as the tube for-
mation process (angiogenesis).
tion yield. We have also investigated the reduction of the
aromatic nitro group of pyrazole 3, considering a variety of
procedures. Best yield was obtained using SnCl2 as reducing
agent and the employment of ultrasonic irradiation.
A similar strategy/reaction sequence was also used for the
efficient condensation of 6-methoxy-1-tetralone 12 substrate
with hydrazine and 4-methoxy-phenylhydrazine (Scheme 3).
Thus, a variety of novel indazole derivatives (molecules con-
taining the fused tetralone–pyrazole backbone) were
obtained in good overall yields (63–70%).
Results
Chemistry
The condensation of hydrazines with various 1,3-diketones
constitutes the most common synthetic procedure for the
construction of the pyrazole backbone, since initial methods
not involving the 1,3-diketones require the adaptation of
additional synthetic steps [11–13]. Pyrazole derivatives can
also be prepared by the palladium-catalyzed four-component
coupling of a terminal alkyne, hydrazine, carbon monoxide
under ambient pressure and an aryl iodide as was recently
reported by Ahmed et al. [14]. Since the mechanism involved
in hydrazine condensation proceeds through their nucleo-
philic attack on carbonyl functions, the use of unsymmet-
rical diketones as substrates leads to the possibility of
forming tautomeric products. However the proton migration
between the nitrogen atoms of the pyrazole ring with sub-
stitutes possessing similar electronic properties is so fast that
usually individual tautomers cannot be seen in the spectra
(Scheme 1) [15]. This was also our case where the pyrazole
proton could not be detected in standard 1D or 2D NMR
experiments.
Bioactivity evaluation
Inhibition of cell proliferation
Several in vitro and in vivo assays have been developed to study
different steps of the angiogenesis process, including endo-
thelial cell proliferation and tube formation [18, 19]. We first
investigated the anti-proliferative activity of the pyrazoles in
two endothelial cell lines [human microvascular endothelial
cells (HMEC-1) and mouse brain endothelial cells (MBEC)] and
two tumor cell lines [human cervical carcinoma cells (HeLa)
and human breast carcinoma cells (MCF-7)]. The biological
results are summarized in Table 1, indicating that all flexible
pyrazole derivatives synthesized herein (compounds 7, 9, 10,
and 11) failed to inhibit the cell proliferation at concen-
trations of up to 100 mM. On the contrary, the investigated
indazoles (1,2,3,4-tetrahydronaphthalene-fused pyrazoles
13–16) were determined as cytostatic to all cell lines tested,
displaying IC50 values between 11 and 40 mM. Compounds 13
and 14 showed more pronounced activity against MCF-7 cells,
with IC50 values of 1.5 ꢀ 0.4 and 5.6 ꢀ 2.5 mM, respectively.
Overall, compound 13 proved to be two- to fivefold more
active than the other compounds, with IC50 values ranging
from 1.5 ꢀ 0.4 mM (in MCF-7 cells) to 13 ꢀ 3.2 mM (in
HMEC-1 cells).
Acetophenones 1, 2, and 8 were used as substrates for the
synthesis of flexible pyrazole derivatives (Scheme 2). More
specifically, all acetophenones in the presence of lithium
hexamethyl disilazane (LHMDS) base and anhydrous con-
ditions provided the corresponding anion which was sub-
sequently condensed with 4-nitrobenzoylchloride to yield
the 1,3-diketone derivative [16]. The in situ condensation
of the latter with hydrazine gave in one step the desired
pyrazole derivatives 3, 4, and 9 (45–50% yield).
Inhibition of endothelial tube formation
When endothelial cells are seeded on top of matrigel, a
protein mixture is secreted by Engelbreth–Holm–Swarm
(EHS) mouse sarcoma cells [19], forming tube-like structures
that are comparable to blood vessels. All novel compounds
inhibited the tube formation at 100 mM (Fig. 1), but the
flexible pyrazole derivatives (7, 9, 10, and 11) and the rigid
pyrazoles (14 and 15) lost most of their activity at lower
concentrations. On the contrary, pyrazole 16, and to a lesser
Phenol 5 was derivatized with 1,2-dibromoethane as alky-
lating agent, in the presence of TBAF [17] and microwave
(MW) irradiation. Thus, we were able to shorten substantially
the reaction time compared with the refluxing procedure
(from 24 h to 30 min) and simultaneously improve the reac-
Scheme 1. The tautomeric forms of pyrazol derivatives.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Biologically Active Pyrazolic and Indazolic Compounds
3
Scheme 2. Pyrazole derivatives from acetophenone substrates.
extent pyrazole 13, retained their activity at 30 mM doses,
indicating that they may be considered new leads for the
development of novel, potent anti-angiogenic agents.
matrigel [6]. Pyrazole XRP44X was found to inhibit the acti-
vation of the transcription factor Net, resulting in G2–M cell
cycle arrest, suppression of growth of multiple cell types
(IC50 ꢁ 2 nM) and inhibition of angiogenesis. This compound
also acts as a microtubule depolymerizing agent in vitro, and
may thus be endowed with both anti-angiogenic and vascu-
lar-targeting activity [7]. Another pyrazole derivative with
pronounced activity is TAK-593, a potent inhibitor of VEGF
receptor 2 (VEGFR2) and platelet-derived growth factor recep-
tor b (PDGFR b), by a two-step binding mechanism [20]. The
Discussion and conclusion
Several pyrazole-containing compounds are endowed with
anti-angiogenic and/or anti-tumor activity (Fig. 2). In particu-
lar, CCT018159, a specific inhibitor of Hsp90, was shown to
abrogate endothelial cell proliferation and tube formation in
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Scheme 3. Synthesis of novel indazole derivatives.
development of multi-target compounds presents a promis-
ing strategy to inhibit tumor angiogenesis. TAK-593 shows a
prominent effect at low doses, its inhibitory activity against
VEGFR2 being similar to that of axitinib, a highly potent
inhibitor of VEGFR2, which is in clinical study for various
solid tumors. Also pazopanib is a potent and selective multi-
targeted receptor tyrosine kinase inhibitor of VEGFR-1,
VEGFR-2, VEGFR-3, PDGFR-a/b, and c-kit that blocks tumor
growth and inhibits angiogenesis [21]. An additional pyrazole
compound with pronounced biological action is celecoxib
(Fig. 2) which is used clinically for the therapy of osteoar-
thritis and rheumatoid arthritis [22]. Currently a recent
report implicated it in the potentiation of the chemotherapic
effect of vinorelbine in the medullary cancer TT cell line [23].
We recently reported the anti-proliferative and anti-
angiogenic activity of pyrazolo[4,3-c]quinolines [10]. Here,
we demonstrated that tetralone-fused pyrazoles inhibit endo-
thelial and tumor cell proliferation and the formation of
Table 1. Cytostatic activities of pyrazoles against endothelial and
tumor cell lines.
Compound
a)IC50 (mM)
HMEC-1
MBEC
HeLa
MCF-7
7
9
ꢂ100
ꢂ100
>100
>100
>100
ꢂ100
ꢂ100
>100
>100
10
11
13
14
15
16
ꢂ100
ꢂ100
77 ꢀ 19
>100
>100
>100
>100
13 ꢀ 3.2
21 ꢀ 9.2
36 ꢀ 18
17 ꢀ 6.1
11 ꢀ 2.8
13 ꢀ 2.5
39 ꢀ 4.6
25 ꢀ 4.8
13 ꢀ 1.9
21 ꢀ 3.1
40 ꢀ 4.2
40 ꢀ 1.4
1.5 ꢀ 0.4
5.6 ꢀ 2.5
34 ꢀ 2.2
11 ꢀ 1.5
HeLa, Human cervical carcinoma cells; HMEC-1, human micro-
vascular endothelial cells; MBEC, mouse brain endothelial cells;
MCF-7, human breast carcinoma cells.
a)
IC50 ¼ concentration of compound that reduces cell prolifer-
ation by 50%.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Biologically Active Pyrazolic and Indazolic Compounds
5
indicated solvents. The coupling constants are recorded in Hertz
(Hz) and the chemical shifts are reported in parts per million
(d, ppm), downfield from tetramethylsilane (TMS) that was used
as an internal standard.
Infrared spectra were obtained on a Nicolet Magna 750, series
II spectrometer. HPLC separations were performed using an
Agilent 1100 series instrument with a variable wavelength UV
detector and coupled to HP Chem-Station utilizing the manu-
facturer’s 5.01 software package.
5-(4-Methoxyphenyl)-3-(4-nitrophenyl)-1H-pyrazole (3)
To an ice-cold (08C) stirred solution of 4-methoxyacetophenone
(0.5 g, 3.3 mmol) in 9 mL of anhydrous toluene, under argon
atmosphere, 3.5 mL of LiHMDS (1.0 M in THF, 3.5 mmol) were
added in one pot via syringe. After the anion formation, 0.31 g of
4-nitrobenzoylchloride (1.65 mmol) dissolved in 3 mL of DMF
were added in one portion under stirring. The ice-bath was
removed, the solution was allowed to stand for 1 min and then
were added successively 4 mL of AcOH, 17 mL EtOH, and 4 mL
THF. To the homogeneous mixture formed, 0.7 mL of hydrazine
monohydrate (13.2 mmol) was added. The mixture was refluxed
for 15 min and TLC indicated that the diketone was consumed.
The reaction mixture was quenched with 1 M NaOH extracted
with EtOH (3 ꢃ 10 mL). The combined organic fractions were
washed with brine, dried over Na2SO4 and evaporated under
reduced pressure. The resulting residue was purified by flash
column chromatography using a mixture of n-hexane/EtOAc
(7.5:2.5) as eluent to provide compound 3 (0.44 g, 45%) as yellow
crystalline needles (Mp 111–1138C). 1H NMR (acetone-d6):
d ¼ 3.86 (3H, s, OCH3), 7.06 (2H, d, J 8.5 Hz, H-30, H-50), 7.23
Figure 1. Tube formation in matrigel. HMEC-1 cells were seeded on
top of matrigel in the absence (control) or presence of the test
compounds. (A) Angiogenesis was evaluated after 6 h of incubation
by scoring the tubular network (0: no tubes, i.e., complete inhibition –
score 3: comparable to untreated control culture, no inhibition). (B)
Representative pictures showing inhibition of tube formation by
compound 16.
(1H, s, H-4), 7.80 (2H, d,
J
8.5 Hz, H-20, H-60), 8.17 (2H,
d, J 9.0 Hz, H-200, H-600), 8.30 (2H, d, J 8.5 Hz, H-300, H-500).
Analysis for C16H13N3O3: Theoretical: C, 65.08; H, 4.44; N, 14.23;
Found C, 65.08; H, 4.44; N, 14.23.
5-(3,4-Dimethoxyphenyl)-3-(4-nitrophenyl)-1H-pyrazole
(4)
tube-like structures in vitro. Based on the above, these
compounds may serve as scaffolds which with slight modi-
fications on their structure – based on molecular modeling –
can prove useful in the development of anti-angiogenic
agents. A parallel research on the apoptotic pathway will
also vastly contribute to their better evaluation as anti-
angiogenic compounds.
This compound was synthesized as previously described, using as
substrates the molecules of 2,5-dimethoxyacetophenone (0.5 g,
2.8 mmol) and 4-nitrobenzoylchloride (0.26 g, 1.4 mmol).
The resulting residue was purified by recrystallization from
Et2O yielding compound 4 (0.43 g, 47%) as a yellow crystalline
solid (Mp 121–1228C). 1H NMR (acetone-d6): d ¼ 3.87 (3H, s,
OCH3), 3.91 (3H, s, OCH3), 7.07 (1H, d, J 8.5 Hz, H-30), 7.27
(1H, s, H-4), 7.42 (1H, dd, J 2.0, 8.5 Hz, H-20), 7.49 (1H, d,
J 2.0 Hz, H-60), 8.17 (2H, d, J 9.0 Hz, H-200, H-600), 8.31 (2H, d,
J 9.0 Hz, H-300, H-500). 13C NMR (acetone-d6): d ¼ 55.3 (2 OCH3),
100.1 (C-4), 109.3 (C-60), 112.1 (C-30), 117.9 (C-20), 123.9 (C-300and
C-500), 125.8 (C-200 and C-600), 127.8 (C-10), 139.8 (C-100), 145.9 (C-5),
146.9 (C-400), 148.6 (C-3), 149.7 (C-40), 150.1 (C-50). Analysis
for C17H15N3O4: Theoretical: C, 62.76; H, 4.65; N, 12.92. Found:
C, 62.70; H, 4.60; N, 12.82.
Experimental
Chemistry-general procedures
All anhydrous reactions were carried out under argon atmos-
phere. Solvents were dried by distillation prior to use. Solvent
mixtures used in chromatographic separations are reported as
volume to volume ratios. Starting materials were purchased
from Aldrich as analytical reagent grades and used without
further purification. Analytical thin-layer chromatography
(TLC) was conducted on Merck glass plates coated with silica
gel 60 F254 and spots were visualized with UV light or/and an
alcohol solution of anisaldehyde. Flash column chromatography
was performed using Merck silica gel 60 (230–400 mesh ASTM).
Melting points (Mp) were determined on a Bu¨chi melting point
apparatus and are uncorrected. 1H and 15N NMR spectra were
recorded at 400 MHz on a Bruker DRX-400 spectrometer in the
4-[3-(4-Nitrophenyl)-1H-pyrazol-5-yl]phenol (5)
To a stirred solution of the protected pyrazole 3 (0.1 g,
0.34 mmol) in CH2Cl2 (15 mL) at ꢄ788C, 2.38 mL BBr3 was added
(1.0 M in CH2Cl2, 2.38 mmol) dropwise. The reaction was run for
1 h at ꢄ788C and for 18 h at room temperature. Then, the
solution was cooled at 08C, quenched with MeOH and 15 mL
of water acidified with 1 N HCl was added. The mixture
was extracted twice with EtOAc (2 ꢃ 20 mL) and the combined
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Figure 2. Potent anti-angiogenic pyrazoles.
organic extracts were washed successively with brine, dried over
Na2SO4, filtered, and evaporated. The desired deprotected com-
pound 5 was obtained by recrystallization from Et2O (0.89 g,
93%) as a pale green crystalline solid (Mp 109–1118C). 1H NMR
(acetone-d6): d ¼ 6.97 (2H, d, J 8.5 Hz, H-20, H-60), 7.19 (1H, s, H-4),
7.7 (2H, d, J 8.5 Hz, H-30, H-50), 8.17 (2H, d, J 8.5 Hz, H-200, H-600), 8.31
(2H, d, J 8.5 Hz, H-300, H-500). 13C NMR (acetone-d6): d ¼ 100.0 (C-4),
115.3 (C-30 and C-50), 120.0 (C-10), 123.5 (C-300and C-500), 126.2 (C-200
and C-600), 126.7 (C-20 and C-60), 137.9 (C-100), 146.2 (C-5), 147.4 (C-400),
148.5 (C-3), 157.9 (C-40). Analysis for C15H11N3O3: Theoretical: C,
64.05; H, 3.94; N, 14.94; Found: C, 64.00; H, 3.99; N, 14.79.
(2 mL) was stirred for 20 min at 308C. Then, 1,2-dibromoethane
(0.1 mL, 1.08 mmol) was added dropwise, the stirring was con-
tinued for an additional 1 h and the solution was refluxed for
24 h (or MW-irradiated for 30 min). The reaction mixture was
cooled, the solvent was evaporated under reduced pressure and
extracted with EtOAc (10 mL). The organic layer was washed with
brine, dried over Na2SO4 and evaporated under reduced pressure.
The residue was purified by silica gel flash column chromatog-
raphy, using a mixture of n-hexane/EtOAc (7:3) as eluent.
Recrystallization from Et2O afforded compound 7 (30 mg, 22%
for reflux and 42 mg 30% for MW procedure) as yellow crystalline
needles (Mp 135–1368C). 1H NMR (acetone-d6): d ¼ 3.82 (2H, t,
J 5.5 Hz, –CH2Br), 4.45 (2H, t, J 5.5 Hz, –OCH2–), 7.11 (2H, d,
J 8.5 Hz, H-30, H-50), 7.26 (1H, s, H-4), 7.84 (2H, d, J 8.5 Hz, H-20,
H-50), 8.19 (2H, d, J 8.5 Hz, H-200, H-600), 8.32 (2H, d, J 9.0 Hz, H-300,
H-500). Analysis for C16H12BrN3O3: Theoretical: C, 51.36; H, 3.23;
N, 11.23; Found: C, 51.31; H, 3.13; N, 11.20.
4-(3-(4-Nitrophenyl)-1H-pyrazol-5-yl)benzene-1,2-diol (6)
This compound was obtained from pyrazole 4 as yellow crystalline
solid, in accordance with the previously described procedure (yield,
90%, Mp 114–1158C). 1H NMR (DMSO-d6): d ¼ 6.93 (1H, d, J 8.5 Hz,
H-30), 7.15 (1H, s, H-4), 7.10(1H, dd, J 8.5, 2.0 Hz, H-20), 7.25(1H, d,
J 2.0 Hz H-60), 8.16 (2H, d, J 8.5 Hz, H-200 and H-600), 8.33 (2H, d,
J 8.5 Hz, H-300 and C-500). Analysis for C15H11N3O4: Theoretical: C,
60.61; H, 3.73; N, 14.14. Found: C, 60.55; H, 3.76; N, 14.11.
5-(4-Methoxyphenyl)-4-methyl-3-(4-nitrophenyl)-1H-
pyrazole (9)
Compound 9 was synthesized in respect to the above-mentioned
procedure for the synthesis of pyrazoles using 4-methoxypropio-
phenone (0.5 g, 3.0 mmol) and 4-nitrobenzoylchloride (0.28 g,
1.5 mmol) as substrates. The resulting product was purified by
recrystallization from Et2O to provide compound 9 (0.46 g, 50%)
as a white crystalline solid (Mp 140–1418C). 1H NMR (acetone-d6):
5-[4-(2-Bromoethoxy)phenyl]-3-(4-nitrophenyl)-1H-
pyrazole (7)
A mixture of phenol 5, (0.1 g, 0.36 mmol), NaOH (0.17 g,
0.43 mmol), TBAF (0.02 mL, 1 M in THF, 0.02 mmol) and EtOH
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
Biologically Active Pyrazolic and Indazolic Compounds
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d ¼ 2.39 (3H, s, –CH3), 3.87 (3H, s, OCH3), 7.09 (2H, d, J 8.5 Hz, H-30,
H-50), 7.59 (2H, d, J 8.5 Hz, H-20, H-60), 8.05 (2H, d, J 8.5 Hz, H-200, H-
600), 8.34 (2H, d, J 9.0 Hz, H-300, H-500). 13C NMR (acetone-d6): d ¼ 9.4
(–CH3), 54.6 (–O CH3), 11.0 (C-4), 114.2 (C-30 and C-50), 128.4 (C-10),
123.5 (C-300 and C-500), 127.7 (C-200 and C-600), 129.0 (C-20 and C-60),
140.9 (C-100), 142.6 (C-5), 146.9 (C-400), 147.1 (C-3), 159.9 (C-40).
Analysis for C17H15N3O3: Theoretical: C, 66.01; H, 4.89;
N, 13.58; Found: C, 66.12; H, 4.81; N, 13.47.
3-(4-Fluorophenyl)-7-methoxy-4,5-dihydro-1H-
benzo[g]indazole (13)
To an ice-cold (08C) stirred solution of 6-methoxy-1-tetralone
(0.5 g, 2.8 mmol) in 9 mL of anhydrous toluene under argon
atmosphere, 3.0 mL of LiHMDS (1.0 M in THF, 3.0 mmol) was
added via syringe in one pot. After the anion formation, 4-fluo-
robenzoyl chloride (0.17 mL, 1.4 mmol) was added in one pot,
the ice-bath was removed and the solution was allowed to pro-
ceed at room temperature. Then were added successively 4 mL
of AcOH, 17 mL of EtOH and 4 mL THF to form a homogeneous
mixture, to which an excess of hydrazine hydrochloride (0.5 mL,
10 mmol) was added. The reaction mixture was allowed to auto-
reflux for 15 min, when TLC indicated that the added amount of
diketone had reacted. The reaction mixture was quenched with
the addition of NaOH (1 M) and extracted with EtOAc (30 mL).
The organic layer was separated, washed with brine, dried over
Na2SO4, and evaporated under reduced pressure. The remaining
residue was purified by flash column chromatography with a
mixture of n-hexane/EtOAc (3:7) providing compound 13 (0.55 g,
67%) as pale yellow crystals (Mp 150–1528C). 1H NMR (DMSO-d6):
d ¼ 2.85 (2H, m, H-5), 2.87(2H, m H-6), 3.78 (3H, –OCH3), 6.45 (1H,
dd, J 8.5, 2.4 Hz, H-10), 6.52 (1H, d, J 8.5 Hz, H-11), 6.76 (1H, d, J 2.4
H-8), 7.00 (2H, m, H-30 and H-50), 7.87 (2H, m, H-20 and H-60).
Analysis for C18H15FN2O: Theoretical: C, 73.45; H, 5.14N, 9.52;
Found C, 73.25; H, 5.24; N, 9.46.
4-[4-Methyl-3-(4-nitrophenyl)-1H-pyrazol-5-yl]phenol (10)
Obtained as previously described by the deprotection of com-
pound 9 as a pale yellow solid (yield 50%, Mp 146–1478C). 1H NMR
(MeOH-d4) d ¼ 2.34 (3H, s, –CH3), 6.97 (2H, d, J 8.5 Hz, H-30, H-50),
7.50 (2H, d, J 8.5 Hz, H-20, H-60), 7.91 (2H, d, J 8.5 Hz, H-200, H-600),
8.40 (2H, d, J 8.5 Hz, H-300, H-500). Analysis for C16H13N3O3:
Theoretical: C, 65.08; H, 4.44; N, 14.23; Found: C, 65.23;
H, 4.40; N, 14.30.
4-[5-(4-Methoxyphenyl)-4-methyl-1H-pyrazol-3-yl]aniline
(11)
Procedure A
To a suspension of 3 (0.1 g, 0.34 mmol) in a mixture of glacial
AcOH (1 mL), EtOH (1 mL), and H2O (0.5 mL), 0.095 g of reduced
Fe (17 mmol) was added. The resulting suspension was exposed
to ultrasonic irradiation for 1 h at 308C and the reaction course
was monitored with TLC, which revealed its completion. Then,
the reaction mixture was filtered to remove the iron residue,
which was washed with EtOAc (10 mL). The filtrate was parti-
tioned with 2 M and extracted with tAc (3 ꢃ 15 mL). The organic
extracts were washed with brine, dried over Na2SO4, and con-
centrated under reduced pressure. The crude residue was puri-
fied by recrystallization from Et2O, yielding compound 11
(31.6 mg, 35%) as an orange colored crystalline solid (Mp 129–
1318C).
3-(4-Fluorophenyl)-4,5-dihydro-1H-benzo[g]indazol-7-ol
(14)
The deprotection was perfomed according to the above-described
procedure to yield compound 14 as a pale yellow solid (Yield,
1
77%, Mp 148–1498C). H NMR (DMSO-d6): d ¼ 2.85 (2H, m, H-5),
2.87 (2H, m H-6), 6.43 (1H, dd, J 8.5, 2.4 Hz, H-10), 6.51 (1H, d, J
8.5 Hz, H-11), 6.73 (1H, d, J 2.4 H-8), 7.26 (2H, m, H-30 and H-50),
7.70 (2H, m, H-20 and H-60). Analysis for C17H13FN2O: Theoretical:
C, 72.85; H, 4.67; N, 9.99; Found 72.66; H, 4.58; N, 9.99.
Procedure B
3-(4-Fluorophenyl)-7-methoxy-1-(4-methoxyphenyl)-4,5-
dihydro-1H-benzo[g]indazole (15)
To a solution of 3 (0.1 g, 0.34 mmol) in EtOH (2 mL), 0.64 g of
SnCl2 (3.4 mmol) was added. The reaction mixture was exposed
to ultrasonic irradiation for 2 h at 308C. After the reaction
completion (revealed by TLC), the solvent was evaporated under
reduced pressure and the remaining residue was partitioned
with 2 M and extracted with tAc (3 ꢃ 15 mL). The organic
extracts were washed with brine, dried over Na2SO4, and con-
centrated under reduced pressure. The crude residue was puri-
fied by recrystallization from Et2O yielding compound 11
(36 mg, 40%) as orange crystals.
This compound was synthesized in respect to the previously
described procedure using as substrates the 6-methoxy-1-tetra-
lone (0.5 g, 2.8 mmol), 4-fluorobenzoyl chloride (0.17 mL,
1.4 mmol), and 4-methoxyphenylhydrazine hydrochloride
(1.74 g, 10 mmol). The resulting residue was purified by flash
column chromatography with a mixture of n-hexane/EtOAc (8:2)
as eluent, providing 0.7 g of the title compound (62%) as a white
crystalline solid (Mp 168–1708C). 1H NMR (CHCl3-d6): d ¼ 2.96
(2H, m, H-5), 3.03 (2H, m H-6), 3.81 (3H, –OCH3), 3.91 (3H, –OCH3),
6.59 (1H, dd, J 8.5, 2.4 Hz, H-10), 6.77 (1H, d, J 8.5 Hz, H-11), 6.89
(1H, d, J 2.4 H-8), 7.03 (2H, d, J 8.5 Hz, H-300 and H-500), 7.16 (2H, m,
H-30 and H-50), 7.49 (2H, d, J 8.5 Hz, H-200 and H-600), 7.78 (2H, m,
H-20 and H-60). Analysis for C25H21FN2O2: Theoretical: C, 74.98;
H, 5.29; N, 7.00. Found: C, 75.12; H, 5.35; N, 6.82.
Procedure C
A solution of 3 (0.1 g, 0.34 mmol) and 10 mg of Pd/C (10%) in
anhydrous THF (30 mL) was hydrogenated (1 bar pressure) for
24 h. The reaction mixture was filtered from Celite1 and
extracted with tAc (3 ꢃ 15 mL). The combined organic extracts
were washed with brine, dried over Na2SO4, and concentrated
under reduced pressure. The resulting residue was purified by
recrystallization from Et2O providing compound 11 (34 mg, 38%)
3-(4-Fluorophenyl)-1-(4-hydroxyphenyl)-4,5-dihydro-1H-
benzo[g]indazol-7-ol (16)
Following the general deprotection procedure, compound 16
was obtained as a pale yellow solid (yield, 81%, Mp 161–
1628C). 1H NMR (DMSO-d6): d ¼ 2.86 (2H, m, H-5), 2.88 (2H, m
H-6), 6.44 (1H, dd, J 8.5, 2.4 Hz, H-10), 6.53 (1H, d, J 8.5 Hz, H-11),
6.76 (1H, d, J 2.4 H-8), 6.89 (2H, d, J 8.5 Hz, H-300, H-500), 7.27 (2H, d,
1
as orange crystals. H NMR (acetone-d6): d ¼ 3.86 (3H, s, OCH3),
7.08 (2H, d, J 8.5 Hz, H-30, H-50), 7.23 (1H, s, H-4), 7.80 (2H, d,
J 8.5 Hz, H-20, H-60), 8.17 (2H, d, J 9.0 Hz, H-200, H-600), 8.30 (1H, d,
J 8.5 Hz, H-300, H-500). Analysis for C16H15N3O: Theoretical C, 72.43;
H, 5.70; N, 15.84; Found: C, 72.23; H, 5.79; N, 15.95.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
E. Tzanetou et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–8
J 8.5 Hz, H-200 and H-600), 7.28 (2H, m, H-30 and H-50), 7.72 (2H, m,
H-20 and H-60). 13C NMR (DMSO-d6): d ¼ 20.5 (C-5), 30.8 (C-6), 113.4
(C-10), 115.1 (C-4), 115.9 (C200 and C-600), 116.0 (C-8), 116.2 (C-300 and
C-500), 118.0 (C12), 124.0 (C-11), 127.8 (C-30and C-50), 129.1 (C-20 and
C-60), 130.4 (C-10), 132.4 (C-100), 139.4 (C-7), 146.2 (C-3), 149.0 (C-13),
157.4 (C-9), 158.0 (C-400), 162.0 (C-40). Analysis for C23H17FN2O2:
Theoretical: C, 74.18; H, 4.60; N, 7.52; Found C, 74.31; H, 4.68; N,
7.42.
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Mouse brain endothelial cells (MBEC) were kindly provided by
Prof. M. Presta (Brescia, Italy). Human cervical carcinoma (HeLa)
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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
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The authors have declared no conflict of interest.
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ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com