Synthesis and biological evaluation of novel pyrazole derivatives with anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2011.08.014

We synthesized thirty-six novel pyrazole derivatives and studied their antiproliferative activity in human ovarian adenocarcinoma A2780 cells, human lung carcinoma A549 cells, and murine P388 leukemia cells. Four of these substances were selected because of their higher antiproliferative activity and further analyses showed that they were all able to induce apoptosis, although to a different extent. The expression of p53 and p21^waf1, which induce apoptosis and cell cycle arrest, was evaluated by western blot analysis in cells treated with compound 12d. The analysis of the cell cycle showed that all the selected compounds cause a partial G2/M block and the formation of polyploid cells. Furthermore, the four selected compounds were tested for their interaction with the microtubular cytoskeletal system by docking analysis, tubulin polymerization assay and immunofluorescence staining, demonstrating that the compound 12d, unlike the other active derivatives, was able to significantly bind dimers of α- and β-tubulin, probably causing a molecular distortion resulting in the disassembly of microtubules.

Full text of DOI:10.1016/j.ejmech.2011.08.014

European Journal of Medicinal Chemistry 46 (2011) 5293e5309
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of novel pyrazole derivatives with anticancer
activity
,
a
a
b
a
b
Alessandro Balbia , Maria Anzaldi , Chiara Macciò , Cinzia Aiello , Mauro Mazzei , Rosaria Gangemi ,
*
Patrizio Castagnolac, Mariangela Mielea, Camillo Rosanod, Maurizio Vialeb
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Genova, V.le Benedetto XV, 3, 16132 Genova, Italy
b Istituto Nazionale per la Ricerca sul Cancro, S.C. Terapia Immunologica, L.go R. Benzi 10, 16132 Genova, Italy
c Istituto Nazionale per la Ricerca sul Cancro, S.S. Biofisica e Citometria, L.go R. Benzi 10, 16132 Genova, Italy
d Istituto Nazionale per la Ricerca sul Cancro, S.C. Nanobiotecnologie, L.go R. Benzi 10, 16132 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized thirty-six novel pyrazole derivatives and studied their antiproliferative activity in human
ovarian adenocarcinoma A2780 cells, human lung carcinoma A549 cells, and murine P388 leukemia cells.
Four of these substances were selected because of their higher antiproliferative activity and further
analyses showed that they were all able to induce apoptosis, although to a different extent. The
expression of p53 and p21waf1, which induce apoptosis and cell cycle arrest, was evaluated by western
blot analysis in cells treated with compound 12d.
Received 20 April 2011
Received in revised form
19 July 2011
Accepted 9 August 2011
Available online 31 August 2011
The analysis of the cell cycle showed that all the selected compounds cause a partial G2/M block and
the formation of polyploid cells. Furthermore, the four selected compounds were tested for their
interaction with the microtubular cytoskeletal system by docking analysis, tubulin polymerization assay
and immunofluorescence staining, demonstrating that the compound 12d, unlike the other active
Keywords:
Pyrazole derivatives
Antitumor activity
Apoptosis
derivatives, was able to significantly bind dimers of
distortion resulting in the disassembly of microtubules.
Ó 2011 Elsevier Masson SAS. All rights reserved.
a- and b-tubulin, probably causing a molecular
Tubulin polymerization inhibitors
1. Introduction
a cyclohexenyl group linked to a heterocyclic moiety by a short
ethenylic chain, have shown antimicrobial [19], anti-inflammatory
and histoprotective properties [20]. The heterocyclic moiety (pyr-
azole, isoxazole, pyrimidine) and the substituents present in them,
deeply affected the biological activities. Our researches demon-
strated that these compounds have antiproliferative and pro-
apoptotic activities. Our attention was focused on a class of ionone-
derived 1,5-pyrazoles 1a-f that exhibited promising anti-
proliferative properties in preliminary experiments on the HL-60
leukemia cell line (Chart 1) [21]. In particular 1a, 1d-f inhibited
HL-60 cell growth and induced apoptosis in dose dependent
manner, while 1b-c displayed antiproliferative activity but were
unable to induce apoptosis.
The pyrazole scaffold represents a common motif in many
pharmaceutical active and remarkable compounds demonstrating
a wide range of pharmacological activities; the most important
activities are the anti-inflammatory [1,2], the antibacterial-
antifungal [3,4], the hypoglycemic [5,6], the anti-hyperlipidemic
[7], the inhibition of cyclooxigenase-2 [8], p38 MAP kinase [9]
and CDK2/Cyclin A [10,11], and the antiangiogenic [12]. Heterocy-
clic rings and, in particular, the pyrazole ring, represent an advan-
tageous choice for the synthesis of pharmaceutical compounds
with different activities and good safety profiles [13]. Different
pyrazole derivatives have also been tested for their antiproliferative
activities in vitro and antitumor activity in vivo, often resulting in
promising lead compounds [14e18].
R
N
N
Our research started some time ago with the synthesis of
heterocyclic ionone-like derivatives, which have a similarity to the
so-called ‘short heteroretinoids’. These compounds, formed by
1a: R=C(S)N(CH3)2
1b: R=4-ClC6H4
1c: R=4-BrC6H4
1d: R=4-(COOEt)C6H4
1e: R=3-OHC6H4CH2
1f: R=2-pyridinyl
1a-f
* Corresponding author. Tel.: þ39 0103533040; fax: þ39 0103533009.
E-mail address: balbi@unige.it (A. Balbi).
Chart 1. Chemical structure of previous reported compounds 1.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.014

5294
A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
Prompted by the above-mentioned results, we further opti-
R
N
N
mized this chemical series by exploring additional modifications.
Firstly we introduced new substituents in the N position of the
pyrazole (compd. 4) also changing the linker moiety in one
example (4h). Then, keeping the same pyrazole scaffold that
showed the best results on HL-60 cells, we planned to synthesize
two new sets of molecules: in the first set we chose to remove the
cyclohexenyl ring maintaining the ethenylic chain (A), while in the
second set we also removed the ethenylic chain (B). Finally we
evaluated the importance of introducing a third substituent in the
pyrazole ring (C) (Fig. 1).
All the synthesized compounds were preliminarily evaluated for
their antiproliferative activity in A2780 (ovary, adenocarcinoma)
and A549 (lung, carcinoma) human cell lines, and in murine P388
(leukemia) cells by MTT assay. 12a, 12a1, 12d, 12d1, the most active
compounds in the preliminary screening, have been further eval-
uated for their ability to induce apoptosis in all the above-
mentioned cell lines. In addition, to verify the involvement of 12d
in apoptotic triggering we analyzed the expression of p53 and
p21waf1 proteins both involved in the induction of apoptosis and
cell cycle arrest. On the basis of the results of these studies, which
suggest the involvement of the microtubule cytoskeletal system in
the mechanism of the action of 12d, we performed a docking
R1
R1= H
NMe2
4a-g
4h
CHO
α-ionone
a
R1= CH3
b
R1
2: R1= H,
3: R1= CH3
α-isomethylionone
R
N
N
4a, 5a: R = 3-ClC6H4
4b, 5b: R = CH2COOC2H5
4d, 5d: R = 4-(OCH3)C6H4
4f, 5f: R = 3,4-(CH3)2C6H4
4c:
4e, 5e: R = PhCH2
4g, 5g: R = 2-pyridinyl 4h:
R = C(S)NH2
5a-b, 5d-g
R = 2-pyridinyl
Scheme 1. Reagents and conditions: (a) Vilsmeier reaction: POCl3 (50.0 mmol), N,N-
dimethylformamide (3.78 mL), ionone (25.0 mmol); (b) method 1: RNHNH2*HCl;
method 2: RNHNH2 þ HCl conc.
The synthetic route followed for the synthesis of the trisubsti-
tuted pyrazoles is outlined in Schemes 4 and 5. Pyrazoles 17 were
easily prepared from intermediates 16a and 16b which, in turn,
were obtained from the commercial 1-(4-methoxyphenyl)propan-
1-one 15a and from the 1-(4-methoxyphenyl)-2-phenylethanone
15b which was synthesized by Friedel-Crafts acylation on anisole
as already reported [33]. A little amount of the isomer 18 was also
isolated (Scheme 4).
analysis, using the dimer of a- and b-tubulin as the putative target
for our molecules, and a tubulin polymerization assay.
2. Chemistry
The intermediate 1,3-dicarbonilyc compounds 19 and 21 were
obtained by Claisen condensation between 40-methoxyacetophe-
none and methyl benzoate or ethyl acetate, respectively, under
nitrogen atmosphere using sodium hydride as a deprotonating
agent [34,35], whereas 22 was obtained using sodium amide
instead of sodium hydride. Starting from those 1,3-diketones 19, 21,
22 we performed the cyclisation with 2-hydrazinopyridine to pyr-
azoles 20, 23, 24, respectively, with the same procedure described
above (Scheme 5).
Compounds 4 were synthesized by cyclisation with suitable
hydrazines of the already reported [20] b-(dimethylamino)vinyl-
aldehydes 2 and 3 following our method which was further
modified [21]: when hydrazine derivatives were available as
hydrochlorides, they were used as such (method 1), while all the
other hydrazine derivatives were made to react with equimolar
quantities of hydrochloridric acid 37% (method 2).
The pyrazole derivatives have been obtained in good yields
(56e76%) and in a very short period of time (1h). Moreover, in
several cases minor amounts of the 1,3-pyrazole derivative 5 have
been isolated together with the predominant 1,5-pyrazoles 4
(Scheme 1). Compound 4g was already reported as 1f [21]. The
treatment of commercially available phenylbutenone derivatives 6
with N,N-dimethylformamide dimethyl acetal (DMFDMA) afforded
the dimethylaminopentadienone intermediates 7, as reported in
the literature [22]. They in turn easily reacted with the hydrazine
derivatives giving the pyrazoles 8 according to method 1 or 2. In
most cases the 1,3-substituted isomers 9 were also isolated
(Scheme 2).
3. Results
3.1. Inhibition of cell proliferation
The analyses of concentration-response curves obtained from
each cell line treated with our 36 compounds together with the
resulting calculation of mean IC50s displayed a quite broad range of
sensitivity (from 0.64 ꢀ 0.31 to more than 100
mM, Table 1). The
Using the same procedure on different arylmethylketones 10
and following the synthetic routes described in the literature
[23e31], we first obtained the dimethylaminopropenone inter-
mediates 11 which cyclized with the opportune hydrazines to give
the pyrazoles 12. The 1,3-substituted isomers 13 were also isolated
in seven cases. When the intermediates 11c, d, f have a hydroxy
group in ortho position, the reaction with 2-hydrazinopyridine led
to the chromones 14c, d, f probably via the intramolecular cycli-
zation of the intermediate itself (Scheme 3) [32].
O
O
a
Ar
N(CH3)2
Ar
7
6
N
N
b
N
N
N
N
7
(+)
Ar
Ar
R
N
R
N
R
8a-e
8b; 9b: Ar=2-furyl
9a-d
N
N
N
N
8c; 9c: Ar=4-ClC6H4
8a; 9a: Ar=Ph
Ar
Ar
Ar
R1
8d; 9d: Ar=3-ClC6H4 8e: Ar=3,4-(OCH3)2C6H4
A
B
C
Scheme 2. Reagents and conditions: (a) DMFDMA, xylene, reflux; (b) method 1:
RNHNH2*HCl; method 2: RNHNH2 þ HCl conc.
Fig. 1. General structure of new synthesized compounds.

A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
5295
O
R
O
a
N(CH3)2
R
H3CO
H3CO
15a: R=CH3
15b: R=Ph
16b: R=Ph
16a: R=CH3
N
N
N
N
N
R
N
b
+
R
H3CO
H3CO
17a: R=CH3
17b: R=Ph
18: R=CH3
Scheme 4. Reagents and conditions: (a) DMFDMA, xylene, reflux; (b) 2-
hydrazinopyridine dihydrochloride, ethanol, reflux.
exposure to equitoxic concentrations (IC50 and IC90) of the selected
compounds (Fig. 2).
On A2780 cells, 12d showed the highest level of activity with
a peak at 48 h. On A549, all molecules showed a significant level of
apoptotic activity and a good concentration-response with the
maximal apoptotic activity expressed at 48 h for 12d, 12a and 12d1.
All compounds and in particular 12a1, showed a good level of
apoptotic activity, similar or even better than that of taxol and
vincristine (Fig. 2), and concentration-response on P388 cells,
although with a different timing of maximal expression.
3.3. Cell cycle analysis
P388 were analyzed for the modifications of cell-cycle phases
after exposure to the IC30 and IC75 of selected active substances. As
shown in Table 2, our data display that in general all compounds
were able to cause a partial block of cells in the G2/M phase of the
cell cycle, with a concomitant decrease of cells in the S and/or G0/
G1 phases, more evident after 48 h exposure. In particular, at the
higher concentration and after 24 h, 12d1, 12d and 12a1 caused the
formation of octaploid and aneuploid cells that died in the
following 24e48 h.
3.4. Western blot analysis of p53, p21waf1 and
a-tubulin
To verify the involvement of 12d in apoptotic triggering we
analyzed the expression of p53 and p21waf1 proteins both impli-
cated in the induction of apoptosis and cell cycle arrest.
Scheme 3. Reagents and conditions: (a) DMFDMA, xylene, reflux; (b) method 2:
RNHNH2 þ HCl conc.; method 1: RNHNH2*HCl.
In both human cell lines 12d was able to upregulate both
molecular markers in
a concentration- and time-dependent
comparison of the activities of each compound on each cell line
allowed the selection of four molecules endowed with a higher
activity, these compounds are: 12a, 12a1, 12d and 12d1. All these
molecules were particularly active on human ovarian adenocarci-
noma A2780 (12d > 12d1 ¼ 12a > 12a1, for significant differences
p < 0.001) and murine leukemia P388 cells (12d > 12a >
12d1 > 12a1, for significant differences p < 0.01). No one of our
compounds showed a particularly significant activity (arbitrarily
manner. The maximal effect on p53 and p21waf1 accumulation,
calculated by densitometric analysis on the basis of the relative
untreated control, was observed after 24 h of treatment in A2780
cells (p53, þ71% and 85% at 0.1
m
M and 10
M and 10 M, respectively, Fig. 3A)
M and
M and 100 M,
mM, respectively;
p21waf1, þ42% and þ62% at 0.1
m
m
and after 72 h in A549 cells (p53, þ40% and 1320% at 1
m
100
m
M, respectively; p21waf1, þ293% and 62% at 1
m
m
respectively, Fig. 3B).
defined as ꢁ 30
mM) against human lung carcinoma A549 cells
It is also of note that the expression of a-tubulin was significantly
reduced after incubation with 12d, this effect being mostly evident in
(12a ¼ 12d ¼ 12a1 ¼ 12d1).
A549 cells (ꢂ37% and ꢂ94% after 72 h incubationwith 10 and 100
mM
3.2. Determination of apoptosis by nuclear morphological
examination of cells stained by DAPI
for A2780 and A549, respectively). This effect could be due to the
great triggering of the apoptotic activity of our compounds that
necessarily involves the disruption of tubulin. This effect was in fact
mostly evident after 72 h of incubation, that is when almost all the
few cells alive were apoptotic and even reduced to debris.
Cell lines were then tested for the induction of apoptosis by
means of DAPI staining and morphological analysis of nuclei after

5296
A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
O
O
N
Ph
b
N
N
Ph
H3CO
a
19
H3CO
20
O
O
O
O
O
c
CH2COOH
+
H3CO
H3CO
H3CO
22
21
4'-methoxyacetophenone
b
b
d
N
N
O
O
N
N
N
N
e
CH2COOH
H3CO
H3CO
H3CO
21
24
23
Scheme 5. Reagents and conditions: (a) PhCOOCH3, NaH, toluene, reflux, N2; (b) 2-hydrazinopyridine dihydrochloride, ethanol, reflux; (c) AcOEt, NaNH2, toluene, reflux; (d) AcOEt,
NaH, toluene, reflux, N2; (e) 2-hydrazinopyridine dihydrochloride, ethanol, reflux.
Table 1
3.5. Effect of 12d on cell microtubules
ICa as calculated by the MTT assay.
50
Compounds
Cell lines
A2780
As we observed the generation of large cells after treatment
with 12d, whose presence was also confirmed by the flow cytom-
etry analysis, and on the basis of findings of polyploid cells in the
literature concerning the effects of pyrazole compounds with
anticancer activity, we hypothesized a possible involvement of the
microtubule cytoskeletal system in the action mechanism of this
active substance.
Therefore, we decided to study the microtubule network in
A549 cells treated with 12d by immunofluorescence microscopy.
Our results are reported in Fig. 4. The compound 12d was able to
cause the condensation of microtubules into “dots” in a great
percentage of cells, depending on the compound concentration
applied. These “dots” were only sporadically present in control cells
treated with 100 nM taxol. While typical bundles were never
observed in cells treated with 12d, these “dots” are similar to
tubulin aggregates observed after treatment with colchicine and
vinblastine (Fig. 4E) [36,37].
A549
P388
4a
4b
4c
4d
4e
4f
4g
4h
8a
8b
8c
8d
52.9 ꢀ 3.0
73.3 ꢀ 8.1
50.3 ꢀ 1.5
51.2 ꢀ 12.6
48.1 ꢀ 3.0
50.0 ꢀ 1.6
45.8 ꢀ 2.6
84.0 ꢀ 6.4
>100
>100
>100
>100
>100
2.89 ꢀ 0.72
5.62 ꢀ 1.1
50.9 ꢀ 5.5
>100
52.8 ꢀ 3.2
1.22 ꢀ 0.33
2.35 ꢀ 0.62
>100
57.1 ꢀ 1.5
>100
49.2 ꢀ 5.7
47.3 ꢀ 13.1
50.7 ꢀ 4.2
45.2 ꢀ 13.7
47.3 ꢀ 3.6
50.2 ꢀ 3.4
48.5 ꢀ 1.5
48.8 ꢀ 3.8
57.9 ꢀ 7.9
74.0 ꢀ 14.9
80.9 ꢀ 19.23
72.8 ꢀ 5.62
95.8 ꢀ 17.0
5.38 ꢀ 0.34
24.2 ꢀ 7.3
51.7 ꢀ 2.5
>100
49.3 ꢀ 4.9
48.4 ꢀ 3.4
57.8 ꢀ 11.8
55.8 ꢀ 1.4
55.8 ꢀ 7.7
>100
>100
>100
>100
>100
8e
>100
12a
12a1
12b
12c
12c1
12d
12d1
12e
12f
12f1
12g
12h
12i
12l
13a
13c
13d
13f
13l
17a
17b
20
44.7 ꢀ 10.4
53.5 ꢀ 4.7
67.9 ꢀ 5.8
>100
>100
48.5 ꢀ 4.1
1.56 ꢀ 0.42
7.51 ꢀ 0.71
>100
52.5 ꢀ 18.0
56.7 ꢀ 15.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
87.1 ꢀ 8.1
84.5 ꢀ 5.6
>100
>100
>100
>100
>100
It is also of note that no other selected compound was able to
cause, even at the maximal applied dose of 100
of these microtubule structures (data not shown).
On the basis of the immunofluorescence results we performed
a docking analysis using the dimer of - and -tubulin as the
putative target for our molecules. Docking simulation results
indicated that 12d is more likely to bind the -tubulin in a cleft next
to the interface with the -monomer (Fig. 5), with a calculated Ki of
mM, the formation
>100
>100
62.9 ꢀ 3.7
58.7 ꢀ 7.9
>100
>100
a
b
>100
>100
85.0 ꢀ 1.8
65.0 ꢀ 7.6
b
>100
>100
a
>100
>100
63.8 ꢀ 10.4
69.1 ꢀ 10.6
>100
>100
>100
>100
>100
>100
45.5 ꢀ 6.0
69.1 ꢀ 10.6
>100
5.3 ꢃ 10ꢂ8 M. This compound is stabilized by hydrogen bonding to
the peptidic N atom of Arg 2, the carboxylic groups of Arg 48, Leu
242 and Asn 249 and to the side chain atoms of Arg 48, Asn 50 and
Asp 251 (Fig. 5A). These residues, together with Val 51 and Leu 242,
form a pocket that is separated by ca. 7.0 Å from the colchicine
binding site (Fig. 5B) and 10 Å from the GTP moiety bound to the
>100
>100
62.4 ꢀ 10.0
23
67.6 ꢀ 5.4
2.3 ꢀ 0.8
NDb
>100
a
-subunit, these latter sites having been determined by X-ray
Taxol
Vincristine
21.5 ꢀ 5
4.9 ꢀ 0.4
0.7 ꢀ 0.3
crystallography [38]. Besides changing the surface characteristic at
the interface of the two tubulin molecules, the presence of 12d can
interfere with residues Glu71 and Asp 98 thus altering the overall
conformation of the polymer by bending the dimer (Fig. 5D). We
97.8 ꢀ 20.4
a
Mean ꢀ SD of 3e9 experiments. Data are expressed in
mM except for taxol and
vincristine where they were expressed in nM.
b
Not detected.

A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
5297
Fig. 2. Histograms represent the mean ꢀ SD of cells treated with the IC50 and IC90 of 12a, 12a1, 12d, 12d1, taxol and vincristine and showing nuclear fragmentation, as evaluated
after DAPI staining and microscopic analysis.
suggest that this bending could lead the molecular assembly to an
asset similar to the one present in the tubulinestathmin complex
that does not participate in microtubule assembly. If further
confirmed by crystallography, the binding site identified by our
simulations would be a novel target site to use for structure-based
drug design of inhibitors of tubulin polymerization (Fig. 5C).
Finally, we also tested “in silico” the activity and binding
modes of 12a. This molecule binds to the
b-subunit in a very

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A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
Table 2
Percentage of P388 cells in the different cell-cycle phases after 24e72 hour treatment with active compounds as determined by the MTT assay.
24 h
IC75
48 h
IC75
72 h
IC30
IC30
IC75
IC30
12a
G0/G1 (2n)
S
G2/M (4n)
Aneuploid cells
8n cells
29.2 ꢀ 1.4
37.1 ꢀ 5.9
33.7 ꢀ 5.9
e
31.3 ꢀ 3.7
48.9 ꢀ 5.9
19.9 ꢀ 2.3
e
30.0 ꢀ 10.9
41.8 ꢀ 11.4
28.2 ꢀ 2.5
e
34.7 ꢀ 3.9
39.7 ꢀ 12.7
25.6 ꢀ 9.1
e
NDa
ND
ND
ND
32.9 ꢀ 0.6
49.3 ꢀ 3.6
17.8 ꢀ 3.2
e
e
e
e
e
ND
e
12a1
G0/G1
S
3.0 ꢀ 0.9
32.5 ꢀ 1.2
23.5 ꢀ 2.8
32.7 ꢀ 2.0
8.3 ꢀ 0.1
0.0
30.4 ꢀ 2.1
52.5 ꢀ 5.4
17.2 ꢀ 3.4
e
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
21.5 ꢀ 1.8
24.7 ꢀ 1.0
7.4 ꢀ 1
35.9 ꢀ 6.5
10.6 ꢀ 6.3
10.3 ꢀ 1.6
29.0 ꢀ 3.5
32.3 ꢀ 13.2
26.4 ꢀ 9.9
2.1 ꢀ 1.4
48 h
34.3 ꢀ 8.4
38.6 ꢀ 14.7
27.1 ꢀ 7.3
e
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
31.7 ꢀ 3.0
50.7 ꢀ 5.1
17.6 ꢀ 2.3
e
G2/M (4nþ2n)
Aneuploid ells
8n cells
G0/G1 (2n)
S
G2/M (4nþ2n)
Aneuploid cells
8n cells
G0/G1 (2n)
S
G2/M (4nþ2n)
Aneuploid cells
8n cells
G0/G1 (2n)
S
G2/M (4nþ2n)
Aneuploid cells
8n cells
G0/G1 (2n)
S
e
e
e
12d
34.5 ꢀ 0.7
47.0 ꢀ 2.7
18.5 ꢀ 2.0
e
37.0 ꢀ 8.6
40.8 ꢀ 15.5
22.3 ꢀ 7.9
e
44.2 ꢀ 1.8
42.3 ꢀ 0.4
13.4 ꢀ 1.8
e
0.0
5.0 ꢀ 2.0
50.9 ꢀ 13.5
44.2 ꢀ 13.9
0.0
e
e
e
12d1
31.0 ꢀ 2.6
50.1 ꢀ 2.9
18.8 ꢀ 0.9
e
34.9 ꢀ 6.0
43.0 ꢀ 11.9
22.1 ꢀ 6.3
e
36.4 ꢀ 3.3
46.3 ꢀ 6.3
17.3 ꢀ 3.2
e
0.0
9.2 ꢀ 4.1
56.6 ꢀ 14.9
34.2 ꢀ 16.9
8.2 ꢀ 0.6
24.3 ꢀ 6.6
67.7 ꢀ 5.9
e
e
e
e
Taxol
Vincristine
7.3 ꢀ 0.1
36.2 ꢀ 11.4
56.6 ꢀ 11.5
e
20.1 ꢀ 5.2
25.9 ꢀ 3.9
8.5 ꢀ 1.3
38.2 ꢀ 3.6
7.5 ꢀ 3.5
25.3 ꢀ 1.5
57.8 ꢀ 3.9
17.0 ꢀ 2.4
e
15.5 ꢀ 1.7
25.3 ꢀ 2.3
18.6 ꢀ 0.8
38.2 ꢀ 1.6
2.5 ꢀ 0.2
11.9 ꢀ 2.7
29.6 ꢀ 2.8
24.0 ꢀ 3.5
32.7 ꢀ 4.0
1.9 ꢀ 0.6
13.5 ꢀ 2.8
27.1 ꢀ 2.7
17.3 ꢀ 2.9
40.0 ꢀ 2.6
2.2 ꢀ 0.1
23.1 ꢀ 0.2
61.1 ꢀ 0.5
15.9 ꢀ 0.7
e
e
e
29.8 ꢀ 5.1
52.3 ꢀ 16.3
18.0 ꢀ 11.2
e
32.5 ꢀ 6.7
52.0 ꢀ 10.8
12.4 ꢀ 5.5
e
G2/M (4nþ2n)
Aneuploid cells
8n cells
e
e
e
e
24 h
72 h
CTR
G0/G1 (2n)
S
G2/M (4n)
33.2 ꢀ 3.1
8.6 ꢀ 3.2
18.2 ꢀ 2.0
31.6 ꢀ 7.6
52.0 ꢀ 9.5
16.4 ꢀ 2.8
36.6 ꢀ 8.5
49.9 ꢀ 9.3
13.6 ꢀ 2.2
a
ND, not detected due to the low number of evaluable alive cells. Mainly apoptotic cells were still present.
similar way to 12d but with a much weaker affinity constant (Ki
ca. 1.8 ꢃ 10ꢂ6).
3.6. Effect of 13d and 12d1 on microtubules
Docking simulations performed on the two moieties tested
led to similar results. Both molecules in fact showed a similar
fitness function and a similar predicted Ki (using GOLD and
Autodock respectively) with a very favorable clusterization of
the results. Structurally, the simulation results indicated that the
inactive isomer 13d is more likely to bind the
a cleft close to the interface between the two protomers but
without a direct interaction with the -subunit (see Fig. 5A). 13d
b-tubulin, within
a
is stabilized by hydrogen bonding to the carboxylic group of Asn
258 and to the side chain atoms of Asn 249, Lys 254 and Asn 258.
The hydrophobic residues Leu 248, Ala 250, Leu 252, Leu 255 and
Met 259 contribute to form the pocket containing the colchicine
binding site. However, the smaller 13d resulted inactive on
tubulin probably because colchicine is able to interact both with
the
b
-subunit and with residues belonging to the molecule,
-subunit. On the other
whilst 13d is located more deeply in the
b
side, 12d1 is perfectly superpositioned on the 12d as the docking
simulations showed. The presence of a chlorine in para-position
confers to 12d1 the ability to interact with b-tubulin, possibly
Fig. 3. Western blot of p53 and p21waf1 and
A2780 (A) and A549 (B) cells to 0.1 and 10 mM and to 1 and 100 m
a
-tubulin after exposure for 24e72 h of
M 12d, respectively. Note
both the upregulation with the maximal expression after 48 h of both apoptosis and cell
cycle arrest markers and the reduction of -tubulin bands after exposure to high
forming an alogen bond with the carboxylic group of Leu 255 of
this molecule, however, the same atom can also interact with the
hydrophobic residues since the interaction of 12d1 (predicted Ki
of 1.8 ꢃ 10ꢂ6 M) to the tubulin dimer is less favorable than that
of 12d.
a
concentrations of 12d, in particular in A549 cells. Beta-actin was used for the densito-
metric normalization. Prestained molecular markers were always included as reference.

A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
5299
Fig. 4. Microtubular effect of 12d in A549 cells. Panel A: Untreated control cells. Panel B and C: Cells treated with 10 and 100
microstructures, as indicated by the arrowheads. Panel D: A549 cells treated with 100 nM taxol and showing typical bundles, as indicated by the arrows. Panel E: A549 cells treated
with 25 nM vinblastine. Note the round microstructure in some cells interpreted as tubulin aggregates. Bar ¼ 20 m (AeD). Bar ¼ 31.5 m (E).
mM 12d, respectively. Note the formation of many round
m
m
3.7. Tubulin polymerization assay
examined the effect of 12a, 12a1, 12d, and 12d1 on microtubule
polymerization using an in vitro assay. An increase in the absor-
bance at 460 nm indicates an increase in tubulin polymerization
(Fig. 6A). As expected, taxol gave an increase in absorbance above
In order to investigate the possible involvement of microtubule
cytoskeleton in the action mechanism of the active compounds, we
Fig. 5. Panel A: The interface between
involved in ligand binding are drawn as sticks. Panel B: 12d predicted binding site is displaced by ca. 7 Å from the colchicine binding site, this latter as determined by X-ray
crystallography. However both the molecules interfere with residues in loop Leu 242eArg 250 of -tubulin. Panel C: Identification of at least four different binding sites in -tubulin
(salmon ribbons) three-dimensional structure: 12d (white) and colchicine (yellow) bind at the interface between the (dark green) and (salmon) subunits, taxol (light green)
binds on the -subunit surface, vinblastine (orange) on the dimeric interface. Panel D: In salmon the structural arrangement of dimer bound to 12d. In cyan the -subunit in
the native position (pdb code 1JFF). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
a (green) and b (salmon) subunits. The 12d moiety position, as determined by docking simulation, is reported in light green. Residues
b
b
a
b
b
b:
a
a
:
b
a

5300
A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
the other aryl substituents, in spite of the presence of the 2-pyr-
idinyl moiety, gave inactive compounds (see 12e-12l).
We thus selected the four active compounds (12d, 12d1, 12a and
12a1) and confirmed their pro-apoptotic activity along with their
significant antiproliferative activity, although the latter was
observed in the micromolar range of IC50 instead of the nanomolar
range, as occurs with more classical antimicrotubule agents such as
taxol and vincristine.
Apoptosis was induced at a different extent, with respect to the
time of exposure and the concentrations used for each compound
and it was confirmed for 12d in human cells by western blot
analysis of two markers of apoptosis and cell cycle arrest such as
p53 and p21waf1. In particular, in A549 cells the apoptotic activity of
our compounds appeared to be higher than expected on the basis of
the IC50 for the antiproliferative activity (see Table 1 and Fig. 2).
Nevertheless, these results are not in contrast since we treated our
cells at equitoxic conditions, i.e. with compound IC50s and IC90s.
Once disconnected from the applied absolute concentrations
applied, these results suggested that the triggering of apoptotic
activity was the prevalent killing mechanism of our active
Fig. 6. In vitro tubulin polymerization assay. Porcine brain tubulin polymerization was
measured in absence (Control, 4% DMSO) or in presence of 3
mM Taxol, 1.6 mM
Vincristine, and 12a, 12a1, 12d and 12d1 compounds at 2 M concentation.
m
compounds,
a fortiori if we consider the final lower anti-
proliferative activity on A549 cells. On the other hand both taxol
and vincristine, used here as controls, showed a similar apoptotic
the control: in contrast, 1.6
mM vincristine strongly inhibits
activity in spite of their much lower applied IC50 and IC90
.
microtubule polymerization. Tested compounds showed a trend
similar to vincristine, suggesting that they destabilize microtubules
(Fig. 6), although they did not show significant differences in terms
of their concentrations inhibiting 50% polymerization that
reasonably was much higher than that of vincristine in our test
The analysis of the cell cycle phases showed a block of cells in the
G2/M phase with a concomitant decrease in the S and/or G0/G1
phases. In particular, after 24 h exposure to the IC75s, cells in S or G0/
G1 phases were slightly lower (see 12a) or absent (12d1, 12d and
12a1) compared to controls, in this case this was due to the appear-
ance of abnormal cell cycles and the formation of polyploid/aneu-
ploid cells. In fact, after the first 24 h, P388 cells exposed to the IC75 of
12d1, 12d and 12a1 compounds became unable to proceed through
the normal phases of mitosis due to their polyploidy and died in the
following 24e48 h generating a great number of cell debris and
a culture containing mainlyapoptotic cells. After 48 h cells exposed to
the compound IC30s showed a more or less marked reduction of cells
in the S phase of the cell cycle along with a relative increase of G2/M
cells. If compared to classical antimicrotubule drugs, such as
vincristine and taxol, our compounds show a much greater propen-
sity to cause accumulation of aneuploid/polyploid cells.
The results of cell cycle analysis, the simple microscopic obser-
vation of quite large cells and, finally, the observation of others
[39e42] that antiproliferative pyrazole compounds were often able
to inhibit tubulin polymerization through a binding site interaction,
similar in part to that of colchicine, suggested the possibility of
a mechanism of action directly or indirectly involving the micro-
tubular system.
conditions (IC50s: 1.48
1.46 0.07 M for 12a, 12a1, 12d1 and 12d, respectively,
vincristine < 1.6 M).
ꢀ
0.01, 1.78
ꢀ
0.03, 1.64
ꢀ
0.03 and
ꢀ
m
m
4. Discussion and conclusions
In the last decades pharmacological research has been strongly
devoted to in the discovery of new conventional and target-based
anticancer drugs with the aim of finding drugs with new mecha-
nisms of action, possibly involving new molecular pathways, with
a spectrum of activity toward those tumors with lower respon-
siveness and high mortality, and with the ability to overcome the
most common mechanisms of resistance. On this basis we under-
took the present study analyzing 36 new pyrazole derivatives for
their ability to inhibit tumor cell growth.
The preliminary screening on human ovarian adenocarcinoma
A2780, murine leukemia P388 and human lung carcinoma A549
cell lines evidenced a great difference in activity strictly related to
the pyrazole substituents. The presence of the cyclohexenylvinyl
moiety yielded compounds (4) showing an antiproliferative
activity in all the cell lines tested only at concentrations greater
Therefore, we chose to perform a microscopic analysis of the
microtubular cytoskeletal system, a docking analysis, and a tubulin
polymerization assay to verify the ability of our compounds to
interact with microtubules.
than 50 mM. The substitution of the cyclohexenylvinyl with an aryl
moiety caused a decrease of activity (see compd. 8), while the
further removal of the vinyl linker greatly enhanced the anti-
proliferative activity. In fact, compounds 12 were the most active
out of all those synthesized. It is worth noting that all the 1,3-
substituted pyrazoles 5 [21] and 13 (see Table 1) were inactive
or showed a dramatic loss of potency. Further SAR considerations
can be made by observing which substituents gave the most active
compounds in the 12 series. Firstly, the best substituent in position
The immunofluorescence microscopy of the microtubule
network revealed that 12d caused a complete breakdown of
microtubules with the formation of “dots” similar to the tubulin
aggregates observed after treatment with microtubule dis-
aggregating agents such as colchicine and vinblastine [36,37]. The
microscopic observations were also in agreement with results
obtained by western blot analysis for 12d. In fact, a partial or
complete reduction of a-tubulin was clearly observed and corre-
1
is the 2-pyridinyl moiety, since all the 4-chlorophenyl
lated with 12d concentration and time of exposure. Furthermore,
12d did not cause the formation of bundles or other similar struc-
tures, which are observed after treatment with taxol, and none of
the other active compounds tested (12d1, 12a and 12a1) caused
microtubular structures similar to aggregates or bundles, also at
high concentrations.
substituted were less active or inactive (compare compd. 12d
and 12d1). Moreover, the phenyl substituents R1 and R3 played
a fundamental role in determining the potency of the activity. In
particular, when both OH and OCH3 groups were present or R3 was
OCH3, we obtained the most active compounds 12d and 12a. All

A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
5301
Due to its antiproliferative and pro-apoptotic activities and its
apparent peculiarity as microtubule-interfering agent,12d was then
docked against the a-b dimer of tubulin and compared to the inac-
tive 13d and to the active compounds 12d1 and 12a, in order to
verify a possible interaction with this target and its molecular
features. Our results indicated that 12d may interfere with the
regular microtubule formation because of its ability to form a bridge
spots were located in UV light or by vanillin in sulfuric acid. Melting
points were obtained with a Fisher-Johns apparatus and are
uncorrected. IR spectra were recorded with a PerkineElmer 398
spectrometer in film or KBr disks. 1H and 13C NMR spectra were
determined with a Varian Gemini 200 (200 MHz, 1H; 50 MHz, 13C)
or a Bruker DPX 300 (300 MHz) instrument, in CDCl3 or (D6)DMSO;
chemical shifts (d) are given in part per million from the peak of
between
a
and
b
-subunits. This probablygreatly disturbs the regular
tetramethylsilane as internal standard; coupling constants (J) in Hz.
Elemental analyses: Carlo Erba 1106 Elemental Analyser. Micro-
analysis of all synthesized compounds agreed within ꢀ 0.3% of
calculated values. CG-MS analyses: HP 6890e5973, GC parameters:
injector temperature 250 ꢄC; HP5 poly(methylphenilsyloxane)
curvature of the tubulin complex and thus the correct growth of the
microtubule during polymerization giving prevalence to the
phenomena of depolymerization. This aspect could also explain the
formation of the “dots” observed by microscope. Furthermore, on
the basis of the docking analysis we also can draw out that, similarly
to 12d, 12d1 is more likely to interfere with the regular microtubule
formation with respect to 13d, because of its ability to form a bridge
between the two subunits, also in this case disturbing the regular
curvature of the tubulin complex in this case as well. On the other
hand, this interaction had a predicted Ki 10 times higher than that of
12d, which in part may justify the absence of microtubule break-
down by this compound as revealed by immunofluorescence, also
after exposure to high concentrations. Similarly, and in spite of its
depolymerizating activity, which was similar tothat of 12d as shown
by our specific cell free polymerization assay, 12a did not show
a great activity in our docking system. This together with the absent
activity at the microscope analysis were the reasons why we
preferred to focus our efforts on 12d.
Although all our results obtained by docking analysis should be
confirmed by crystallography, the most important result of this in
silico study is that the binding site identified by our simulations
would be a novel target site to use for structure-based drug design of
inhibitors of tubulin polymerization and different from those
already described for other pyrazole microtubule interfering agents
and anticancer drugs such as taxol, colchicine, vincristine and other
related compounds. On the other hand our data do not completely
exclude the hypothesis of other targets for our compounds. In
particular, the presence of a high percentage of polyploid/aneuploid
cells makes our compounds similar in behavior to aurora inhibitors
[43,44]. These compounds, some of which are in clinical phases of
study [44], are able to inhibit the activity of aurora kinases blocking
the correct development of the various phases of mitosis such as the
centrosome separation, the mitotic entry, the spindle assembly, the
correct alignment of chromosomes and cytokinesis. Alternatively,
column 30 m, 0.25 mm, 0.25 mm; temperature profile: from 100 to
300 ꢄC; 10 ꢄC/min; MS parameters mode SCAN 40e600 amu.
5.1.1. 3-Dimethylamino-4-methyl-5-(2,6,6-trimethyl-1-cyclohexen-
1-yl)-2,4-pentadienal (3)
Phosphorous oxychloride (50.0 mmol; 4.57 mL) was added
dropwise, within 15 min at
0
ꢄC, to 3.87 mL of N,N-
dimethylformamide in a two-necked flask protected from atmo-
spheric moisture and efficiently stirred with a magnetic bar. A
solution of
a-isomethylionone (25.0 mmol) in 3 mL of dime-
thylformamide was dropped into the above Vilsmeier reagent,
cooled at ꢂ20 ꢄC left to stir while the temperature was left to rise to
0
ꢄC for a total of 45 min and finally poured onto crushed ice. The
aqueous layer was alkalinized with NaOH 2N, stirred overnight and
then extract with CHCl3. The organic layers were combined, washed
with water, dried over sodium sulfate, and evaporated at reduced
pressure to give the crude product. It was purified by silica gel
chromatography using AcOEt as eluant. The white solid was crys-
tallized from AcOEt, mp. 80e81 ꢄC. Yield 53%. IR (KBr):
1626, 1548, 1379, 1181 cm-1; 1H NMR (200 MHz, CDCl3):
n
2961, 2921,
0.81 (3H,
d
s, CH3), 0.93 (3H, s, CH3), 1.20e1.24 (1H, m, H-50), 1.41e1.45 (1H, m,
H-50), 1.61 (3H, s, CH3), 1.92 (3H, s, CH3), 1.97e1.99 (2H, m, H-40),
2.52e2.55 (1H, m,, H-10), 2.92 (6H, s, NCH3), 5.11 (1H, d, CH-2,
J ¼ 8.6 Hz), 5.30e5.36 (2H, m, H-40 þ CH-5), 9.16 (1H, d, CHO,
J ¼ 8.6 Hz); 13C NMR (50 MHz, CDCl3):
d 16.6 (CH3), 22.3 (CH3); 22.4
(CH2); 26.2 (CH3); 26.7 (CH3); 30.3 (CH2); 31.9 (C); 48.4 (2CH3);
100.4 (CH); 120.7 (CH), 128.1 (C), 132.4 (CH), 136.4 (C), 170.9 (C),
189.3 (C). Anal. calcd. for C16H25NO: C, 77.68; H, 10.19; N, 5.66;
found: C, 77.81; H, 10.15; N, 5.31.
our compounds could inhibit the activity of
g
tubulin, a further
5.1.2. General procedure for cyclization to pyrazole from enamines
The hydrazines (method 2) or hydrazine hydrochlorides
(2 mmol) (method 1) were added in one portion to a stirred solu-
tion of enammines (2 mmol) in acidified ethanol (10 mL containing
0.17 mL of HCl 37%) (method 2) or ethanol (10 mL) (method 1). The
resulting solution was stirred for different time at different
temperatures. After cooling, several compounds were collected
from the reaction mixture by filtration and then crystallized; other
compounds were obtained by silica gel chromatography of the
residue after removal of the solvent.
component of the centrosome and spindle pole bodies [45,46],
whose inactivation may stimulate polyploidization. For both targets
we applied docking simulations whose results showed a very low
probability of binding of our compounds to the possible target sites
of aurora kinase and
range of concentrations.
g tubulin, with Ki ranging in the micromolar
In conclusion, very important indications emerge from the
biological investigations on these new pyrazole derivatives. In fact,
in spite of their lower antiproliferative activity, compared to the
more classic antimicrotubule anticancer compounds such as taxol
or vincristine, our new pyrazole compounds are likely to bind to the
possible newly described target site on microtubules and represent
the rational for the future development of new small molecules
acting as tubulin polymerization inhibitors potentially useful as
anticancer drugs.
5.1.2.1. 1-(3-chlorophenyl)-5-(2,6,6-trimethyl-2-cyclohex-1-yl)
ethenyl-1H-pyrazole (4a); 1-(3-chlorophenyl)-3-(2,6,6-trimethyl-2-
cyclohex-1-yl)ethenyl-1H-pyrazole (5a). From 3-chlorophenyly-
drazine hydrochloride at 25 ꢄC for 1 h (method 1). After evaporation
to dryness, the residue was chromatographed on silica gel eluting
with toluene. The first eluate gave 5a as a red thick oil. Yield 16%.
The second eluted gave 4a as a red thick oil. Yield 64%. Data of 4a: IR
5. Experimental section
(film):
n ;
2957, 2915, 1595,1488, 1433 cm-1 1H NMR (200 MHz,
5.1. Chemistry
CDCl3): d
0.88 (3H, s, CH3), 0.91 (3H, s, CH3), 1.28e1.33 (1H, m, H-50),
1.41e1.48 (1H, m, H-50), 1.60 (3H, s, CH3), 2.00e2.04 (2H, m, H-40),
2.23 (1H, d, J ¼ 8.2, H-10), 5.04e5.03 (1H, m, H-30), 6.10 (1H, dd,
J ¼ 8.2; 15.6, ethene), 6.18 (1H, d, J ¼ 15.6, ethene), 6.45 (1H, d,
The purity of all the compounds was checked by thin-layer
chromatography on silica gel 60-F-254 pre-coated plates and the

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A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
J ¼ 1.8, H-4), 7.36e7.50 (4H, m, Ar), 7.59 (1H, J ¼ 1.8, H-3). Anal.
calcd. for C20H23ClN2: C, 73.49; H, 7.09; N, 8.57; Cl, 10.85; found: C,
73.52; H, 7.02; N, 8.50.
(3H, s, CH3), 1.98e2.02 (2H, m, H-40), 2.19 (1H, d, H-10, J ¼ 8.7 Hz),
5.39 (2H, s, CH2), 5.41e5.43 (1H, m, H-30), 6.00 (1H, dd, J ¼ 8.7, 15.2,
ethene), 6.18 (1H, d, J ¼ 15.2, ethene), 6.34 (1H, d, J ¼ 2.2, H-4),
7.07e7.29 (5H, m, Ar), 7.47 (1H, d, H-3, J ¼ 2.2 Hz). Anal. calcd. for
C21H26N2: C, 82.31; H, 9.14; N, 8.55; found: C, 82.25; H, 8.94; N, 8.65.
Data of 5a: IR (film):
(200 MHz, CDCl3): 0.89 (3H, s, CH3), 0.90 (3H, s, CH3), 1.18e1.23
n
2961, 2915, 1595,1488, 1447 cm-1; 1H NMR
d
(1H, m, H-50), 1.42e1.48 (1H, m, H-50), 1.63 (3H, s, CH3), 2.01e2.05
(2H, m, H-40), 2.27 (1H, d, J ¼ 9.4, H-10), 5.43e5.45 (1H, m, H-30),
6.14 (1H, dd, J ¼ 9.4, 16.0, ethene), 6.48 (1H, d, J ¼ 16.0, ethene), 6.54
(1H, d, J ¼ 2.6, H-4), 7.20e7.38 (2H, m, Ar), 7.52e7.53 (1H, m, Ar),
7.71 (1H, t, J ¼ 2.0, Ar), 7.82 (1H, J ¼ 2.6, H-5). Anal. calcd. for
C20H23ClN2: C, 73.49; H, 7.09; N, 8.57; Cl, 10.85; found: C, 73.59; H,
7.05; N, 8.42.
Data of 5e: IR (KBr):
(200 MHz, CDCl3):
n
2967, 2953, 2906, 1451, 1050 cm-1
;
1H NMR
d
0.87 (3H, s, CH3), 0.92 (3H, s, CH3), 1.17e1.22
(1H, m, H-50), 1.42e1.47 (1H, m, H-50), 1.62 (3H, s, CH3), 1.99e2.03
(2H, m, H-40), 2.24 (1H, d, H-10 J ¼ 9.5 Hz), 5.27 (2H, s, CH2),
5.41e5.43 (1H, m, H-30), 6.02 (1H, dd, ethene, J ¼ 9.5, 16.0 Hz), 6.33
(1H, d, H-4, J ¼ 2.4 Hz), 6.40 (1H, d, ethene, J ¼ 16.0 Hz), 7.21e7.34
(6H, m, Ar þ H-5). Anal. Calcd. for C21H26N2: C, 82.31; H, 9.14; N,
8.55; found: C, 82.29; H, 8.99; N, 8.60.
5.1.2.2. Ethyl 2-{5-[2-(2,6,6-trimethylcyclohexen-2-yl)vinyl]-1H-pyr-
azol-1-yl}ethanoate (4b), ethyl 2-{3-[2-(2,6,6-trimethylcyclohexen-2-
yl)vinyl]-1H-pyrazol-1-yl}ethanoate (5b). From ethyl hydrazine
hydrochloride at 50 ꢄC for 1 h (method 1 e see ref. [21]).
5.1.2.6. 1-(3,4-dimethylphenyl)-5-(2,6,6-trimethyl-2-cyclohex-1-yl)
ethenyl-1H-pyrazole (4f); 1-(3,4-dimethylphenyl)-3-(2,6,6-trimethyl-
2-cyclohex-1-yl)ethenyl-1H-pyrazole (5f). From 3,4-dimethylphe-
nylydrazine hydrochloride at 50 ꢄC for 1 h (method 1). After
evaporation to dryness, the residue was chromatographed on
silica gel eluting with toluene. The first eluate gave 5f as thick
yellow oil. Yield 15%. The second eluated gave 4f as a thick yellow
5.1.2.3. 5-(2,6,6-Trimethyl-2-cyclohexen-1-yl)ethenyl-1H-pyrazole-
1-thiocarboxamide (4c). From thiosemicarbazide at reflux for 1 h
(method 2). After evaporation to dryness, the residue was chro-
matographed on silica gel eluting with toluene. The eluate gave 4c
as an already pure yellow crystals, mp 80e82 ꢄC. Yield 76%. IR
oil. Yield 60%. Data of 4f: IR (film):
n
2957, 2917, 1670, 1613, 1507,
0.87 (3H, s, CH3),
1449, 1383 cm-1 1H NMR (200 MHz, CDCl3):
;
d
(film):
n
3252, 3150, 2958, 2915, 1590, 1495, 1247 cm-1
;
1H NMR
0.90 (3H, s, CH3), 1.18e1.23 (1H, m, H-50), 1.40e1.48 (1H, m, H-50),
1.59 (3H, s, CH3), 1.99e2.04 (2H, m, H-40), 2.20 (1H, d, J ¼ 8.8,
H-10), 2.30 (3H, s, CH3), 2.31 (3H, s, CH3), 5.40e5.43 (1H, m, H-30),
6.03 (1H, dd, J ¼ 8.8; 15.6, ethene), 6.19 (1H, d, J ¼ 15.6, ethene),
6.44 (1H, d, H-4, J ¼ 2.0 Hz), 7.19e7.25 (3H, m, Ar), 7.58 (1H, d,
J ¼ 2.0, H-3). Anal. calcd. for C22H28N2: C, 82.45; H, 8.81; N, 8.74;
(200 MHz, CDCl3):
d
0.86 (3H, s, CH3), 0.93 (3H, s, CH3), 1.19e1.23
(1H, m, H-50), 1.41e1.48 (1H, m, H-50), 1.61 (3H, s, CH3), 2.00e2.05
(2H, m, H-40), 2.26 (1H, d, J ¼ 8.5, H-10), 5.44e5.46 (1H, m, H-30),
6.22 (1H, dd, J ¼ 8.5; 15, ethene), 6.34 (1H, d, J ¼ 15, ethene), 6.49
(1H, d, J ¼ 2.8, H-4), 6.84 (1H, s, NH), 8.26 (1H, s, NH), 8.55 (1H, d,
J ¼ 2.8, H-3). Anal. calcd. for C15H21 N3S: C, 65.41; H, 7.69; N, 15.26;
found: C, 65.64; H, 7.91; N, 15.02.
found: C, 82.33; H, 8.97; N, 8.84. Data of 5f: IR (film):
2919, 1670, 1613, 1518, 1454, 1381 cm-1 1H NMR (200 MHz,
CDCl3): 0.89 (3H, s, CH3), 0.94 (3H, s, CH3), 1.9e1.24 (1H, m,
n 2959,
;
d
5.1.2.4. 1-(4-methoxyphenyl)-5-(2,6,6-trimethyl-2-cyclohex-1-yl)
ethenyl-1H-pyrazole (4d); 1-(4-methoxyphenyl)-3-(2,6,6-trimethyl-
2-cyclohex-1-yl)ethenyl-1H-pyrazole (5d). From 4-methoxyphenylhy-
drazine hydrochloride at 50 ꢄC for 1 h (method 1). After evaporation
to dryness, the residue was chromatographed on silica gel eluting
with toluene. The first eluate gave 5d as a yellow thick oil. Yield 17%.
The second eluted gave 4d as a yellow thick oil. Yield 67%. Data of 4d:
H-50), 1.44e1.53 (1H, m, H-50), 1.63 (3H, s, CH3), 2.01e2.06 (2H, m,
H-40), 2.26 (3H, s, CH3), 2.30 (3H, s, CH3), 2.35 (1H, d, J ¼ 9.4, H-
10), 5.44e5.45 (1H, m, H-30), 6.06 (1H, dd, J ¼ 9.4; 16.0, ethene),
6.49 (1H, d, J ¼ 16.0, ethene), 6.51 (1H, d, J ¼ 2.4, H-4), 7.16 (1H,
d, J ¼ 8.0, Ar), 7.33 (1H, dd, J ¼ 2.2, 8.0, Ar), 7.47 (1H, d, J ¼ 2.2,
Ar), 7.77 (1H, d, J ¼ 2.4, H-5). Anal. calcd. for C22H28N2: C, 82.45;
H, 8.81; N, 8.74; found: C, 82.39; H, 8.77; N, 8.71.
IR (film):
n
2958, 2913, 1515, 1250 cm-1; 1H NMR (200 MHz, CDCl3):
d
0.85 (3H, s, CH3), 0.89 (3H, s, CH3),1.17e1.21 (1H, m, H-50), 1.38e1.44
5.1.2.7. 2-{5-[2-(2,6,6-trimethylcyclohex-2-enyl)vinyl]pyrazol-1-yl}
pyridine (4g), 2-{3-[2-(2,6,6-trimethylcyclohex-2-enyl)vinyl]pyrazol-
1-yl}pyridine (5g). From 2-hydrazinopyridine dihydrochloride at
50 ꢄC for 1 h (method 1 e see ref. [21]).
(1H, m, H-50), 1.57 (3H, s, CH3), 1.99e2.03 (2H, m, H-40), 2.19 (1H, d, H-
10 J ¼ 8.3 Hz), 3.85 (3H, s, CH3), 5.41e5.43 (1H, m, H-30), 6.01 (1H, dd,
ethene, J ¼ 8.3; 15.8 Hz), 6.16 (1H, d, ethene, J ¼ 15.8 Hz), 6.44 (1H, d,
H-4, J ¼ 2.0 Hz), 6.97 (2H, d, J ¼ 8.6 Hz, Ar), 7.36 (2H, d, J ¼ 8.6 Hz, Ar),
7.57 (1H, d, H-3, J ¼ 2.0 Hz). Anal. calcd. for C21H26N2O: C, 78.22; H,
5.1.2.8. (E)-2-(5-(1-(2,6,6-trimethylcyclohex-2-enyl)prop-1-en-2-
yl)-1H-pyrazol-1-yl)pyridine (4h). From 3 (1.77 mmol) and phe-
nylydrazine hydrochloride (1.77 mmol) at room temperature for 1 h
(method 1). After evaporation to dryness, the residue was chro-
matographed on silica gel eluting with toluene. The second eluate
8.13; N, 8.69; found: C, 77.91; H, 8.31; N, 8.74. Data of 5d: IR (film):
n
2959, 2914,1524, 1256 cm-1; 1H NMR (200 MHz, CDCl3):
d 0.88 (3H, s,
CH3), 0.93 (3H, s, CH3), 1.17e1.22 (1H, m, H-50), 1.42e1.50 (1H, m, H-
50), 1.63 (3H, s, CH3), 2.01e2.04 (2H, m, H-40), 2.27 (1H, d, J ¼ 8.9,, H-
10), 3.83 (3H, s, CH3), 5.43e5.45 (1H, m, H-30), 6.12 (1H, dd, J ¼ 8.9;
16.6, ethene), 6.48 (1H, d, J ¼ 16.6, ethene), 6.50 (1H, d, J ¼ 2.4, H-4),
6.94 (2H, d, J ¼ 9.0, Ar), 7.56 (2H, d, J ¼ 9.0, Ar), 7.72 (1H, d, J ¼ 2.4, H-
5). Anal. calcd. for C21H26N2O: C, 78.22; H, 8.13; N, 8.69; found: C,
77.87; H, 8.44; N, 8.69.
gave 4h as a thick yellow oil. Yield 60%. IR (film):
n
2960, 2916, 1591,
0.81 (3H, s,
1579, 1473, 1436 cm-1 1H NMR (200 MHz, CDCl3):
;
d
CH3), 0.92 (3H, s, CH3), 1.15e1.20 (1H, m, H-50), 1.35e1.41 (1H, m, H-
50), 1.62 (3H, s, CH3), 1.87 (3H, s, CH3), 1.94e1.96 (2H, m, H-40), 2.54
(1H, d,, H-10 J ¼ 10.3 Hz), 5.34e5.36 (2H, m, H-40, ethene), 6.22 (1H,
d, H-4, J ¼ 1.4 Hz), 7.15e7.20 (1H, m, Ar), 7.59 (1H, d, H-3, J ¼ 1.4 Hz),
7.65e7.81 (2H, m, Ar), 8.35e8.40 (1H, m, Ar) Anal. calcd. for
C20H25N3: C, 78.14; H, 8.20; N, 13.67; found: C, 77.11; H, 8.52;
N, 13.84.
5.1.2.5. 1-Benzyl-5-(2,6,6-trimethyl-2-cyclohex-1-yl)ethenyl-1H-pyr-
azole (4e); 1-benzyl-3-(2,6,6-trimethyl-2-cyclohex-1-yl)ethenyl-1H-
pyrazole (5e). From benzylhydrazine dihydrochloride at reflux for
1 h (method 1). After evaporation to dryness, the residue was
chromatographed on silica gel eluting with toluene. The first eluate
gave 5e as a white solid, mp 73e74 ꢄC. Yield 28%. The second eluted
5.1.2.9. 2-{5-[2-(phenyl)vinyl]pyrazol-1-yl}pyridine (8a); 2-{3-[2-
(phenyl)vinyl]pyrazol-1-yl}pyridine (9a). From 2-hydrazinopyridine
dihydrochloride and 7 at 50 ꢄC for 1 h (method 1). The residue was
chromatographed on silica gel eluting with CH2Cl2/toluene 1:1,
toluene. The first eluate gave 9a as a white solid which was
gave 4e as a yellow oil. Yield 56%. Data of 4e: IR (film):
1455,1400 cm-1; 1H NMR (200 MHz, CDCl3):
0.76 (3H, s, CH3), 0.86
(3H, s, CH3), 1.14e1.19 (1H, m, H-50), 1.35e1.41 (1H, m, H-50), 1.47
n 2958, 2917,
d

A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
5303
crystallized from diethyl ether; mp 77e81 ꢄC. Yield 11%. The second
eluate gave 8a as white solid, crystallized from diethyl ether; mp
d, J ¼ 2.0, H-4), 7.02 (1H, d, J ¼ 16,4, ethene), 7.24e7.37 (4H, m, Ar),
7.47 (1H, s, Ar), 7.67 (1H, d, J ¼ 2.0, H-3), 7.85e7.88 (1H, m, Ar), 7.95
(1H, d, J ¼ 16,4, ethene), 8.53 (1H, m, Ar); 13C NMR (50 MHz, CDCl3):
80e81 ꢄC. Yield 48%. Data of 8a: IR (KBr):
n
1590, 1471, 1446, 1382,
783 cm-1; 1H NMR (200 MHz, CDCl3):
d
6.71 (1H, d, J ¼ 1.8, H-4), 7.12
d 104.8 (CH); 116.0 (CH); 118.3 (CH); 120.8 (CH); 124.0 (CH); 125.5
(1H, d, J ¼ 16.4, ethene), 7.22e7.39 (4H, m, Ar), 7.47e7.53 (2H, m,
Ar), 7.65e7.68 (1H, m, Ar), 7.85 (1H, d, J ¼ 1.8, H-3), 8.83e7.88 (1H,
m, Ar), 7.94 (1H, d, J ¼ 16.4, ethene), 8.46e8.54 (1H, m, Ar). Anal.
calcd. for C16H13N3: C, 77.71; H, 5.30; N, 16.99; found: C, 77.65; H,
(CH); 126.9 (2CH); 128.9 (2CH); 133.6 (C); 137.6 (C); 137.8 (CH);
140.0 (CH); 141.0 (C); 146.8 (C). Anal. Calcd. for C16H12ClN3: C,
68.21; H, 4.29; Cl, 12.58; N, 14.91. Found: C, 68.14; H, 4.34; N, 15.17.
Data of 9d: IR (KBr):
(200 MHz, CDCl3):
n ;
1594, 1517, 1475, 1449, 1377 cm-1 1H NMR
6.68 (1H, d,, H-4 J ¼ 2.6 Hz), 7.15e7.41 (6H, m,
5.63; N, 17.21. Data of 9a: IR (KBr):
NMR (200 MHz, CDCl3):
n
1593, 1472, 1452, 770 cm-1; 1H
6.69 (1H, d, J ¼ 2.8, H-4), 7.13e7.37 (5H,
d
d
Ar þ ethene þ H-5), 7.50e7.51 (1H, m, Ar), 7.78e7.87 (1H, m, Ar),
m, Ar þ ethene), 7.33 (1H, d, J ¼ 2.8, H-5); 7.49e7.53 (2H, m,
Ar þ ethene), 7.80e7.97 (2H, m, Ar), 8.36e8.41 (1H, m, Ar),
8.53e8.55 (1H, m, Ar). Anal. calcd. for C16H13N3: C, 77.71; H, 5.30; N,
16.99; found: C, 77.61; H, 5.38; N, 16.81.
7.97e8.01 (1H, m, Ar), 8.40e8.42 (1H, m, Ar), 8.56 (1H, m, Ar); 13C
NMR (50 MHz, CDCl3):
d 107.9 (CH); 114.8 (CH); 123.9 (CH); 124.4
(CH); 127.3 (CH); 129.1 (2CH); 130.3 (2CH); 130.7(CH), 132.50 (C);
137.2 (C); 141.3 (CH); 150.6 (C); 153.2 (C). Anal. Calcd. for
C16H12ClN3: C, 68.21; H, 4.29; Cl, 12.58; N, 14.91; found: C, 68.13; H,
4.26; N, 14.74.
5.1.2.10. 2-{5-[2-(furan-2-yl)vinyl]-1H-pyrazol-1-yl}pyridine (8b);
2-{3-[2-(furan-2-yl)vinyl]-1H-pyrazol-1-yl}pyridine (9b). From 2-
hydrazinopyridine dihydrochloride and
7
at 60 ꢄC for
1
h
5.1.2.13. 2-{5-[2-(3,4-dimethoxyphenyl)vinyl]pyrazol-1-yl}pyridine
(8e). From 2-hydrazinopyridine dihydrochloride and 7 at 60 ꢄC for
1 h (method 1). After cooling, the solid was collected by filtration
from the mixture and crystallized from ethanol, mp 126e128 ꢄC.
(method 1). The residue was chromatographed on silica gel eluting
firstly with CH2Cl2/toluene 1:1 and, secondly, with toluene. The
first eluate gave 9b as a red solid which was crystallized from
diethyl ether, mp 75e78 ꢄC. Yield 8%. The second eluate gave 8b as
red solid, crystallized from diethyl ether, mp 50e53 ꢄC. Yield 44%.
Yield 73%. IR (KBr):
(200 MHz, CDCl3):
n
1591, 1512, 1470, 1438, 1379 cm-1
;
1H NMR
d
3.90 (3H, s, OCH3), 3.92 (3H, s, OCH3), 6.68 (1H,
Data of 8b: IR (KBr):
(200 MHz, CDCl3):
n
1587, 1472, 1437, 1373 cm-1
;
1H NMR
d, H-4, J ¼ 2.0 Hz), 6.85 (1H, d, Ar), 7.06 (1H, d J ¼ 16,4, ethene),
7.03e7.04 (2H, m, Ar), 7.25 (1H, s, Ar), 7.68 (1H, d, J ¼ 2.0, H-3), 7.73
(1H, d, J ¼ 16,4, ethene), 7.85e7.89 (2H, m, Ar), 8.48e8.53 (1H, m,
Ar). Anal. calcd. for C18H17N3O2: C, 70.34; H, 5.58; N,13.67; found: C,
70.42; H, 5.85; N, 13.48.
d
6.39e6.42 (2H, m, Ar), 6.64 (1H, d, H-4,
J ¼ 2.0 Hz), 6.87 (1H, d, J ¼ 16.2, ethene), 7.23e7.27 (1H, m, Ar),
7.39e7.40 (1H, m, Ar), 7.69 (1H, d, J ¼ 16.2, ethene), 7.82 (1H, d,
J ¼ 2.0, H-3), 7.82e7.85 (2H, m, Ar), 8.52e8.55 (1H, m, Ar). Anal.
calcd. for C14H11N3O: C, 77.71; H, 5.30; N, 16.99; found: C, 77.65; H,
5.42; N, 17.05. Data of 9b: IR (KBr):
n
1588, 1472, 1439, 1370 cm-1; 1H
5.1.3. General procedure for the synthesis of compounds 11
A suspension or a solution of each methylketone (10 mmol) in
xylene (30 mmol) was treated with dimethylformamide dimethy-
lacetal (10 mmol). The reaction mixture was refluxed for different
hours. After cooling, compound 11a, 11e, 11g, 11i and 11l were
obtained by silica gel chromatography of the residue after removal
of the solvent. All the other compounds were collected by filtration
from the mixture and crystallized.
NMR (200 MHz, CDCl3): d 6.58e6.61 (2H, m, Ar), 6.78 (1H, d, H-4,
J ¼ 2.6 Hz), 7.16 (1H, d, ethene, J ¼ 16.4 Hz), 7.25e7.43 (2H, m,
Ar þ ethene), 7.59e7.62 (1H, m, Ar), 7.93e8.01 (1H, m, Ar),
8.13e8.17 (1H, m, Ar), 8.56e8.58 (1H, m, Ar), 8.68 (1H, d,H-5,
J ¼ 2.6 Hz). Anal. Calcd. for C14H11N3O: C, 77.71; H, 5.30; N, 16.99;
found: C, 77.68; H, 5.34; N, 17.04.
5.1.2.11. 2-{5-[2-(4-chlorophenyl)vinyl]pyrazol-1-yl}pyridine (8c); 2-
{3-[2-(4-chlorophenyl)vinyl]pyrazol-1-yl}pyridine (9c). From 2-
hydrazinopyridine dihydrochloride and
5.1.3.1. (2E)-3-(dimethylamino)-1-(4-methoxyphenyl)-2-propen-1-
one (11a). From 40-methoxyacetophenone, refluxed for 2 h. The
residue was chromatographed on silica gel eluting with toluene/
AcOEt 1:1 and, then, AcOEt. The first eluate gave the starting
product, the second eluate gave 11a as a yellow solid, mp 89e91 ꢄC.
7 1 h
at 60 ꢄC for
(method 1). The residue was chromatographed on silica gel eluting
with CH2Cl2/toluene 1:1 and then toluene. The first eluate gave 9c
as a white solid which was crystallized from diethyl ether, mp
118e121 ꢄC. Yield 8%. The second eluate gave 8c as yellow solid,
crystallized from diethyl ether, mp 64e66 ꢄC. Yield 40%. Data of 8c:
Yield 62%. IR (KBr):
(200 MHz, CDCl3):
n
1638, 1580, 1539, 1359, 1241 cm-1
;
1H NMR
d
3.03 (6H, s, NCH3), 3.86 (3H, s, OCH3), 5.73 (1H,
IR (KBr):
n
1590, 1475, 1432, 1378 cm-1; 1H NMR (200 MHz, CDCl3):
d, J ¼ 12.6, CH-2), 6.92 (2H, d, J ¼ 8.5, Ar), 7.45 (1H, d, J ¼ 12.6, CH-3),
7.92 (2H, d, J ¼ 8.5, Ar); Anal. calcd. for C12H15NO2: C, 70.22; H, 7.37;
N, 6.82; found: C, 70.26; H, 7.30; N, 6.74.
d
6.70 (1H, d, J ¼ 2.0, H-4), 7.03 (1H, d, J ¼ 16,2, ethene), 7.23e7.44
(5H, m, H-40 and ethene), 7.67 (1H, d, J ¼ 2.0, H-3), 7.84e7.88 (2H, m,
Ar), 8.51e8.53 (1H, m, Ar). Anal. Calcd. for C16H12ClN3: C, 68.21; H,
4.29; Cl, 12.58; N, 14.91; found: C, 68.16; H, 4.36; N, 14.94. Data of
5.1.3.2. (2E)-3-(dimethylamino)-1-(2-hydroxy-5-methylphenyl)-2-
propen-1-one (11b). From 20-hydroxy-50-methylacetophenone,
refluxed for 2 h. An orange solid was collected by filtration from the
mixture and crystallized from EtOH, mp 136e138 ꢄC. Yield 65%. IR
9c: IR (KBr):
CDCl3): 6.67 (1H, d,
Ar þ ethane þ H-5), 7.82e8.00 (2H, m, Ar), 8.35e8.56 (2H, m, Ar).
Anal. calcd. for C16H12ClN3: C, 68.21; H, 4.29; Cl, 12.58; N, 14.91;
found: C, 68.18; H, 4.33; N, 14.97.
n
1595, 1471, 1447, 1377, 774 cm-1; 1H NMR (200 MHz,
d
J
¼
2.6, H-4), 7.09e7.47 (7H, m,
(KBr):
CDCl3):
n ;
1634, 1532, 1498, 1341, 1291 cm-1 1H NMR (200 MHz,
d
2.33 (3H, s, CH3), 3.01 (3H, s, NCH3), 3.21 (3H, s, NCH3),
5.80 (1H, d, J ¼ 12.0, CH-2), 6.87 (2H, d, J ¼ 8.2, CH-2), 7.20 (2H, dd,
J ¼ 8.2, 1.6, CH-2), 7.50 (2H, d, J ¼ 8.5, Ar), 7.90 (1H, d,, J ¼ 12.0, CH-
3). Anal. calcd. for C12H15NO2: C, 70.22; H, 7.37; N, 6.82; found: C,
70.44; H, 7.71; N, 6.75.
5.1.2.12. 2-{5-[2-(3-chlorophenyl)vinyl]pyrazol-1-yl}pyridine (8d);
2-{3-[2-(3-chlorophenyl)vinyl]pyrazol-1-yl}pyridine (9d). From 2-
hydrazinopyridine dihydrochloride and
7 1 h
at 60 ꢄC for
(method 1). The residue was chromatographed on silica gel eluting
with CH2Cl2/toluene 1:1 and then toluene. The first eluate gave 9d
as a white solid, crystallized from diethyl ether, mp 128e130 ꢄC.
Yield 10%. The second eluate gave 8d as yellow solid, crystallized
5.1.3.3. (2E)-1-(5-chloro-2-hydroxyphenyl)-3-(dimethylamino)-2-
propen-1-one (11c). From 50-chloro-20-hydroxyacetophenone,
refluxed for 2 h. A yellow solid was collected by filtration from the
mixture and crystallized from cyclohexane, mp 141 ꢄC. Yield 70%. IR
from cyclohexane, mp 68e70 ꢄC. Yield 44%. Data of 8d: IR (KBr):
n
1592, 1473, 1435, 1379 cm-1; 1H NMR (200 MHz, CDCl3):
d
6.70 (1H,
(KBr): n ;
1633, 1540, 1477, 1286 cm-1 1H NMR (200 MHz, CDCl3):

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A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
d
3.02 (3H, s, NCH3), 3.24 (3H, s, NCH3), 5.70 (1H, d, J ¼ 12.1, CH-2),
starting product, the second eluate gave 11i as a dark yellow oil.
Yield 32%. IR (KBr):
1631, 1541, 1442, 1368, 1237 cm-1 1H NMR
(200 MHz, CDCl3): 2.97 (3H, s, NCH3), 3.15 (3H, s, NCH3), 3.72 (3H,
s, OCH3), 3.83 (3H, s, OCH3), 5.74 (1H, d, J ¼ 12.6, CH-2), 6.57e6.68
(2H, m, Ar), 7.61 (2H, d, J ¼ 8.9, Ar), 7.78 (1H, d, J ¼ 12.6, CH-3),
7.83e7.85 (1H, m, Ar). Anal. calcd. for C15H17NO3: C, 66.36; H,
7.28; N, 5.95; found: C, 66.30; H, 7.25; N, 6.01.
6.90 (2H, d, J ¼ 8.8, Ar), 7.31 (2H, dd, J ¼ 8.8, 2.6 Ar), 7.65 (2H, d,
J ¼ 8.8 Ar), 7.92 (1H, d, J ¼ 12.1, CH-3). Anal. calcd. for C11H12ClNO2:
C, 58.54; H, 5.36; N, 6.21; found: C, 58.74; H, 5.71; N, 6.21.
n
;
d
5.1.3.4. (2E)-3-(dimethylamino)-1-(2-hydroxy-4-methoxyphenyl)-2-
propen-1-one (11d). From 20-hydroxy-40-methoxyacetophenone,
refluxed for 2 h. A yellow solid was collected by filtration from the
mixture and crystallized from EtOH, mp 145e146 ꢄC. Yield 56%. IR
(KBr):
CDCl3):
5.71 (1H, d, J ¼ 12.6, CH-2), 6.37e6.43 (2H, m, Ar), 7.61 (2H, d,
J ¼ 8.9, Ar), 7.88 (1H, d, J ¼ 12.6, CH-3). Anal. calcd. for C12H15NO3: C,
65.14; H, 6.83; N, 6.33; found: C, 65.20; H, 7.05; N, 6.59.
5.1.3.10. (2E)-3-(dimethylamino)-1-(2-methoxyphenyl)-2-propen-1-
one (11l). From 20 methoxyacetophenone, refluxed overnight. The
residue was chromatographed on silica gel eluting with toluene/
AcOEt 9:1 and, then, AcOEt. The first eluate gave the starting
product, the second eluate gave 11l as a yellow oil. Yield 44%. IR
n ;
1625, 1543, 1443, 1369, 1237 cm-1 1H NMR (200 MHz,
d
2.97 (3H, s, NCH3), 3.17 (3H, s, NCH3), 3.83 (3H, s, OCH3),
(KBr):
CDCl3):
n ;
1631, 1538, 1447, 1362, 1237 cm-1 1H NMR (200 MHz,
d
3.05 (6H, s, NCH3), 3.78 (3H, s, OCH3), 5.76 (1H, d, J ¼ 12.6,
5.1.3.5. N-[4-[(2E)-3-(dimethylamino)-1-oxo-2-propen-1-yl]phenyl]-
acetamide (11e). From N-(4-acetylphenyl)-acetamide, refluxed for
2 h. The residue was chromatographed on silica gel eluting firstly
with toluene/AcOEt 1:1 and, secondly, with AcOEt/EtOH. The first
eluate gave the starting product, the second eluate gave 11e as
a yellow solid which was crystallized from cyclohexane, mp
CH-2), 7.02e7.08 (1H, m, Ar), 7.40e7.56 (2H, m, Ar), 7.68 (1H, d,
J ¼ 12.6, CH-3), 7.78e7.85 (1H, m, Ar); Anal. calcd. for C12H15NO2: C,
70.22; H, 7.37; N, 6.82; found: C, 70.25; H, 7.32; N, 6.76.
5.1.4. General procedure for the synthesis of compounds 12 and 13
The hydrazines (method 2) or hydrazine hydrochlorides
(method 1) (2 mmol) were added in one portion to a stirred solu-
tion of the appropriate compound 11 (2 mmol) in acidified EtOH
[10 mL containing 0.17 mL of HCl 37% (method 2)] or EtOH [10 mL
(method 1)]. The resulting solution was stirred for 2 h at 60 ꢄC.
183e185 ꢄC. Yield 36%. IR (KBr):
NMR (200 MHz, CDCl3): 2.07 (3H, s, C(O)CH3), 2.97 (6H, s, NCH3),
5.73 (1H, d, J ¼ 12.6, CH-2), 7.62 (2H, d, J ¼ 8.6, Ar), 7.78 (1H, d,
J ¼ 12.6, CH-3), 7.86 (2H, d, J ¼ 8.6, Ar), 8.91 (1H, s, NH). Anal.
(C13H16N2O2) C, H, N. Anal. calcd. for C13H16N2O2: C, 67.22; H, 6.94;
N, 12.06; found: C, 67.40; H, 7.20; N, 11.93.
n ;
1690, 1638, 1552, 1262 cm-1 1H
d
5.1.4.1. 2-[5-(4-methoxyphenyl)-1H-pyrazol-1-yl]pyridine (12a); 2-
[3-(4-methoxyphenyl)-1H-pyrazol-1-yl]pyridine (13a). From 2-
hydrazinopyridine dihydrochloride and 11a (method 1). The
residue was chromatographed on silica gel eluting with toluene
and, then, toluene/AcOEt 1:1. The first eluate gave 13a as a pale
yellow solid which was crystallized from diethyl ether, mp
75e77 ꢄC. Yield 6%. The second eluate gave 12a as yellow oil. Yield
5.1.3.6. (2E)-3-(dimethylamino)-1-(2-hydroxyphenyl)-2-propen-1-
one (11f). From 20-hydroxyacetophenone, refluxed for 2 h. A yellow
solid was collected by filtration from the mixture and crystallized
from EtOH, mp 130e132 ꢄC. Yield 82%. IR (KBr):
n
1629, 1489,
1287 cm-1 1H NMR (200 MHz, CDCl3):
; d 2.99 (3H, s, NCH3), 3.21
(3H, s, NCH3), 5.80 (1H, d, J ¼ 12.2, CH-2), 6.80e6.98 (2H, m, Ar),
7.34e7.41 (1H, m, Ar), 7.70e7.75 (1H, m, Ar), 7.61 (2H, d, J ¼ 8.9, Ar),
7.91 (1H, d, J ¼ 12.2, CH-3), 13.97 (1H, s, OH). Anal. calcd. for
C11H13NO2: C, 69.09; H, 6.85; N, 7.32; found: C, 69.18; H, 6.51;
N, 7.45.
43%. Data of 12a: IR (film):
n
2936, 2836, 1591, 1499, 1473, 1458,
3.96 (3H, s, OCH3), 6.09 (1H,
1378 cm-1; 1H NMR (200 MHz, CDCl3):
d
d, J ¼ 1.8, H-4), 6.98e7.02 (2H, m, Ar), 7.32e7.39 (3H, m, Ar),
7.55e7.61 (1H, m, Ar), 7.83e7.96 (1H, m, Ar), 7.90 (1H, d, J ¼ 1.8, H-
3), 8.54e8.61 (1H, m, Ar); 13C NMR (50 MHz, CDCl3):
d 54.2 (OCH3),
107.3 (CH), 112.7 (2CH), 118.0 (CH), 121.4 (CH), 122.3 (C), 129.0
(2CH), 137.1 (CH), 140.0 (CH), 142.4 (C), 147.5 (CH), 151.5 (C), 158.5
(C). Anal. calcd. for C15H13N3O: C, 71.70; H, 5.21; N, 16.72. Found: C,
5.1.3.7. (2E)-3-(dimethylamino)-1-(3-methoxyphenyl)-2-propen-1-
one (11g). From 30-methoxyacetophenone, refluxed for 2 h. The
residue was chromatographed on silica gel eluting with toluene/
AcOEt 8:2 and, then, with AcOEt. The first eluate gave the starting
product, the second eluate gave 11g as an orange oil. Yield 79%. IR
71.63; H, 5.13; N, 16.48. Data of 13a: IR (film):
n
2936, 2836, 1591,
4.03 (3H,
1499, 1473, 1458, 1378 cm-1; 1H NMR (200 MHz, CDCl3):
d
s, OCH3), 6.89 (1H, d, J ¼ 2.6, H-4), 7.14 (2H, d, Ar), 7.30e7.39 (1H, m,
Ar), 7.55e7.61 (1H, m, Ar), 7.98e8.05 (3H, m, Ar), 8.25e8.30 (1H, m,
Ar), 8.54e8.61 (1H, m, Ar); 8.76 (1H, d, J ¼ 2.6, H-5). Anal. calcd. for
C15H13N3O: C, 71.70; H, 5.21; N, 16.72; found: C, 71.68; H, 5.19;
N, 16.89.
(film):
n
1638, 1560, 1437, 1358, 1261 cm-1
;
1H NMR (200 MHz,
CDCl3):
d 2.96 (3H, s, NCH3), 3.13 (3H, s, NCH3), 3.87 (3H, s, OCH3),
5.71 (1H, d, J ¼ 12.4, CH-2), 6.99e7.05 (1H, m, Ar), 7.29e7.37 (1H, m,
Ar), 7.45e7.49 (2H, m, Ar), 7.83 (1H, d, J ¼ 12.4, CH-3). Anal. calcd.
for C15H16N2O2: C, 70.22; H, 7.37; N, 6.82; found: C, 70.22; H, 7.58;
N, 6.97.
5.1.4.2. 1-(4-chlorophenyl)-5-(4-methoxyphenyl)-1H-pyrazole (12a1);
1-(4-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazole (13a1). From
4-chlorophenylhydrazine hydrochloride and 11a (method 1). The
residue was chromatographed on silica gel eluting with dichloro-
methane. The first eluate gave 13a1 as a pale yellow solid which
was crystallized from diethyl ether, mp 75e77 ꢄC. Yield 6%. The
second eluate gave 12a1 as a red oil. Yield 53%. Data of 12a1: IR
5.1.3.8. (2E)-3-(dimethylamino)-1-(4-morpholinophenyl)-2-propen-
1-one (11h). From 40-morpholinoacetophenone, refluxed over-
night. A white solid was collected by filtration from the mixture and
crystallized from AcOEt, mp 205e207 ꢄC. Yield 33%. IR (KBr):
n
1634, 1599, 1571, 1528 cm-1; 1H NMR (200 MHz, CDCl3):
d 3.04 (6H,
s, NCH3), 3.28 (4H, t, NCH2), 3.89 (4H, t, O CH2), 5.75 (1H, d, J ¼ 12.4,
CH-2), 6.91 (2H, d, J ¼ 9.0, Ar), 7.81 (1H, d, J ¼ 12.4, CH-3), 7.92 (2H,
d, J ¼ 8.9, Ar). Anal. calcd. for C15H20N2O2: C, 69.20; H, 7.74; N,10.76;
found: C, 69.21; H, 7.97; N, 10.43.
(film):
CDCl3):
n
2836, 1613, 1497, 1452, 1383, 1251 cm-1; 1H NMR (200 MHz,
d
3.98 (3H, s, OCH3), 6.11 (1H, d, J ¼ 1.8, H-4), 6.99e7.04 (2H,
m, Ar), 7.29e7.50 (6H, m, Ar), 7.86 (1H, d, J ¼ 1.8, H-3); 13C NMR
(50 MHz, CDCl3):
d 54.2 (OCH3), 106.7 (CH), 113.0 (2CH), 121.6 (C),
125.2 (2CH), 128.0 (2CH), 129.0 (2CH), 131.9 (C), 137.7 (C), 139.5
(CH), 141.9 (C), 158.6 (C). Anal. calcd. for C16H13ClN2O: C, 67.49; H,
4.60; Cl, 12.45; N, 9.84; found: C, 67.30; H, 4.90; N, 10.07. Data of
5.1.3.9. (2E)-3-(dimethylamino)-1-(2,4-dimethoxyphenyl)-2-propen-
1-one (11i). From 20, 40-dimethoxyacetophenone, refluxed for 2
days. The residue was chromatographed on silica gel eluting with
toluene/AcOEt 9:1 and, then, AcOEt. The first eluate gave the
13a1: 1H NMR (200 MHz, CDCl3):
d
4.02 (3H, s, OCH3), 6.88 (1H, d,
J ¼ 2.6, H-4), 7.13 (2H, d, Ar), 7.59 (2H, d, Ar), 7.87 (2H, d, Ar), 8.00

A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
5305
(2H, d, Ar), 8.06 (1H, d, J ¼ 2.6, H-5). Anal. calcd. for C16H13ClN2O: C,
87e88 ꢄC. Yield 27%. The second eluate gave 14d as a white solid.
The third eluate gave 12d as white solid, crystallized from diethyl
67.49; H, 4.60; Cl, 12.45; N, 9.84; found: C, 67.39; H, 4.67; N, 10.02.
ether, mp 95e97 ꢄC. Yield 48%. Data of 12d: IR (KBr):
n
3009, 1618,
5.1.4.3. 2-[1-(4-chlorophenyl)-1H-pyrazol-5-yl]-4-methylphenol (12b1).
From 4-chlorophenylhydrazine hydrochloride and 11b (method 1).
The residue was prified on silica gel eluting with toluene/AcOEt 9:1.
The eluate gave a white solid which was crystallized from cyclo-
1601,1574, 1477, 1292, 1164 cm-1; 1H NMR (200 MHz, CDCl3):
d
3.97
(3H, s, OCH3), 6.55 (1H, d, J ¼ 1.6, H-4), 6.67 (1H, dd, Ar), 6.85 (1H, d,
Ar), 7.22 (1H, d, Ar), 7.43e7.52 (1H, m, Ar), 7.91e7.95 (2H, m, Ar þ H-
3), 8.04e8.09 (1H, m, Ar), 8.44e8.48 (1H, m, Ar); 13C NMR (50 MHz,
hexane, mp 158e160 ꢄC. Yield 46%. IR (KBr):
n
3154, 1494, 1411,
CDCl3): d 54.3 (OCH3), 104.0 (CH), 106.2 (CH), 110.9 (CH), 112.3 (C),
1270 cm-1; 1H NMR (200 MHz, CDCl3):
d
2.38 (3H, s, CH3), 5.30 (1H,
118.1 (CH), 121.6 (CH), 132.2 (CH), 139.0 (CH), 140.4 (C), 140.7 (CH),
145.2 (CH), 151.2 (C), 155.8 (C), 161.1 (C). Anal. calcd. for C15H13N3O2:
C, 67.40; H, 4.90; N, 15.51; found: C, 67.28; H, 5.06; N, 15.51. GS-MS
s, OH, D2O exchangeable), 6.69 (1H, d, J ¼ 1.8, H-4), 6.94e7.01 (2H,
m, Ar), 7.20e7.29 (1H, m, Ar), 7.42(4H, m, Ar), 7.94 (1H, d, J ¼ 1.8, H-
3); 13C NMR (50 MHz, CDCl3):
d
19.3 (CH3), 108.0 (CH), 115.0 (CH),
tr ¼ 21.2 min, m/z : 267 (Mþ). Data of 13d: IR (KBr):
n
1634, 1587,
115.7 (C), 124.0 (2CH), 127.9 (2CH), 129.0 (C), 130.0 (CH), 130.4 (CH),
131.8 (C), 137.2 (C), 137.5 (C), 139.7 (CH), 150.0 (C). Anal. calcd. for
C16H13ClN2O: C, 67.49; H, 4.60; Cl, 12.45; N, 9.84; found: C, 67.46; H,
4.56; N, 9.73.
1438, 1254, 1032 cm-1 1H NMR (200 MHz, CDCl3):
; d 4.01 (3H, s,
OCH3), 6.65e6.79 (2H, m, Ar), 6.94 (1H, d, J ¼ 2.8, H-4), 7.32e7.43
(1H, m, Ar), 7.67e7.72 (1H, m, Ar), 8.00e8.03 (2H, m, Ar þ H-5),
8.58e8.62 (1H, m, Ar), 8.77e8.79 (1H, m, Ar), 10.95 (1H, s, OH, D2O
exchangeable); 13C NMR (50 MHz, CDCl3):
d 54.3 (OCH3), 100.6
5.1.4.4. 4-Chloro-2-[1-(pyridin-2-yl)-1H-pyrazol-5-yl]phenol (12c);
4-chloro-2-[1-(pyridin-2-yl)-1H-pyrazol-3-yl]phenol (13c); 6-chloro-
4H-chromen-4-one (14c). From 2-hydrazinopyridine dihydro-
chloride and 11c (method 1). The residue was chromatographed on
silica gel eluting with hexane/AcOEt 8:2. The first eluate gave 13c as
a white solid which was crystallized from diethyl ether, mp
189e190 ꢄC. Yield 14%. The second eluate gave 14c as a white solid.
The third eluate gave 12c as white solid, crystallized from diethyl
(CH), 103.3 (CH), 105.6 (CH), 108.3 (C), 110.7 (CH), 120.4 (CH), 126.8
(CH),126.9 (CH),137.8 (CH),147.2 (CH),149.5 (C),152.8 (C),156.5 (C),
160.2 (C). Anal. calcd. for C15H13N3O2: C, 67.40; H, 4.90; N, 15.51;
found: C, 67.30; H, 4.86; N, 15.72. GS-MS tr ¼ 23.4 min, m/z : 267
(Mþ). Data of 14d: GS-MS tr ¼ 15.4 min, m/z : 176 (Mþ).
5.1.4.7. 2-[1-(4-chlorophenyl)-1H-pyrazol-5-yl]-5-methoxyphenol
(12d1). From 4-chlorophenylhydrazine hydrochloride and 11d
(method 1). A yellow solid was collected by filtration from the
mixture and crystallized from diethyl ether, mp 147e148 ꢄC. Yield
ether, mp 40e42 ꢄC. Yield 38%. Data of 12c: IR (KBr):
1478, 1437 cm-1; 1H NMR (200 MHz, CDCl3):
n
3067, 1591,
d
6.59 (1H, d, J ¼ 1.8, H-
4), 7.21 (1H, d, Ar), 7.30 (1H, m, Ar), 7.43e7.53 (2H, m, Ar), 7.96 (1H,
d, Ar), 8.08 (1H, d, H-3, J ¼ 1.8, H-3), 7.98e8.03 (1H, m, Ar),
8.48e8.50 (1H, m, Ar), 8.83 (1H, m, Ar), 10.58 (1H, s, OH, D2O
66%. IR (KBr):
CDCl3): 3.94 (3H, s, OCH3), 6.01 (1H, s, OH, D2O exchangeable),
n ;
3135, 1496, 1208, 829 cm-1 1H NMR (200 MHz,
d
6.59e6.63 (2H, m, Ar), 6.66 (1H, d, J ¼ 1.8, H-4), 7.01e7.10 (1H, m,
exchangeable); 13C NMR (50 MHz, CDCl3):
d 111.9 (CH), 118.6 (CH),
Ar), 7.39e7.43 (4H, m, Ar), 7.91 (1H, d, J ¼ 1.8, H-3); 13C NMR
121.2 (C), 122.3 (C), 122.5 (CH), 125.0 (C), 130.4 (CH), 131.5 (CH),
139.7 (C),140.0 (CH),141.5 (CH),145.9 (CH),151.6 (C),154.0 (C). Anal.
calcd. for C14H10N3ClO: C, 61.89; H, 3.71; Cl, 13.05; N, 15.47; found:
C, 61.87; H, 4.00; Cl, N, 15.59. GS-MS tr ¼ 20.4 min, m/z : 271 (Mþ).
(50 MHz, CDCl3): d 54.6 (OCH3), 101.0 (CH), 104.6 (CH), 108.6 (CH),
109.8 (C), 124.3 (2CH), 128.3 (2CH), 130.5 (CH), 131.3 (C), 139.4 (C),
139.5 (C), 139.6 (CH), 155.4 (C), 160.6 (C). Anal. calcd. for
C16H13N2ClO2: C, 63.90; H, 4.36; N, 9.31; found: C, 63.78; H, 4.30;
N, 9.38.
Data of 13c: IR (KBr):
(200 MHz, CDCl3):
n ;
3121, 1581, 1471, 1452 cm-1 1H NMR
7.00 (1H, d, J ¼ 2.6, H-4), 7.17 (1H, d, Ar),
d
7.35e7.44 (3H, m, Ar), 7.75 (1H, d, J ¼ 2.6, H-5), 8.01e8.04 (2H, m,
Ar), 8.60e8.63 (1H, m, Ar), 8.82e8.83 (1H, m, Ar), 10.85 (1H, s, OH,
5.1.4.8. N-{4-[(1-(pyridin-2-yl)-1H-pyrazol-5-yl)]phenyl}ethana-
mide (12e). From 2-hydrazinopyridine dihydrochloride and 11e
(method 1). The residue was chromatographed on silica gel eluting
with toluene and, then, toluene/AcOEt 1:1. The eluate gave 12e as
a white solid, crystallized from EtOH, mp 179e181 ꢄC. Yield 23%. IR
D2O exchangeable); 13C NMR (50 MHz, CDCl3):
d 104.6 (CH), 111.5
(CH), 117.0 (C), 118.2 (CH), 121.5 (CH), 123.8 (C), 126.0 (CH), 127.9
(CH), 129.3 (CH), 138.6 (CH), 148.0 (CH), 150.0 (C), 152.2 (C), 154.3
(C). Anal. calcd. for C14H10N3ClO: C, 61.89; H, 3.71; Cl, 13.05; N,
15.47; found: C, 61.85; H, 3.83; N, 15.65. GS-MS tr ¼ 22.8 min, m/z :
271 (Mþ). Data of 14c: GS-MS tr ¼ 14.3 min, m/z : 180 (Mþ).
(KBr):
CDCl3):
n ;
1687, 1595, 1504, 1477, 1436 cm-1 1H NMR (200 MHz,
d
2.11 (3H, s, CH3), 6.64 (1H, d, J ¼ 1.8, H-4), 7.25 (2H, d, Ar),
7.43 (1H, s, NH, D2O exchangeable), 7.77 (1H, m, Ar), 7.90 (1H, d,
J ¼ 1.8, H-3), 7.97e8.04 (1H, m, Ar), 8.31e8.37 (1H, m, Ar), 8.51e8.58
5.1.4.5. 4-Chloro-2-[1-(4-chlorophenyl)-1H-pyrazol-5-yl]phenol
(12c1). From 4-chlorophenylhydrazine hydrochloride and 11c
(method 1). A yellow solid was collected by filtration from the
mixture and crystallized from cyclohexane, mp 181e183 ꢄC. Yield
(1H, m, Ar); 13C NMR (50 MHz, CDCl3):
d 23.3 (CH3), 104.7 (CH),
112.2 (CH), 118.1 (2CH), 121.8 (CH), 125.0 (C), 128.2 (2CH), 137.3 (C),
137.7 (CH), 140.1 (CH), 141.2 (C), 147.2 (CH), 151.6 (C), 160.7 (C ¼ 0).
Anal. calcd. for C16H14N4O: C, 69.05; H, 5.07; N, 20.13; found: C,
69.18; H, 5.20; N, 20.27.
63%. IR (KBr):
(D6)DMSO):
6.71 (1H, d, H-4 J ¼ 1.8 Hz), 6.96 (1H, m, Ar), 7.41e7.63
(6H, m, Ar), 7.91 (1H, d, H-3, J ¼ 1.8 Hz), 10.08 (1H, s, OH, D2O
exchangeable); 13C NMR (50 MHz, CDCl3):
108.3 (CH), 118.9 (CH),
n ;
3099, 1493, 1406, 1276 cm-1 1H NMR (200 MHz,
d
5.1.4.9. 2-[1-(pyridin-2-yl)-1H-pyrazol-5-yl]phenol (12f); 2-[1-(pyr-
idin-2-yl)-1H-pyrazol-3-yl]phenol (13f); 4-oxo-chromene (14f).
From 2-hydrazinopyridine dihydrochloride and 11f (method 1). The
residue was chromatographed on silica gel eluting with hexane/
AcOEt 9:1 and, then, AcOEt. The first eluate gave 13f as a white
solid, crystallized from diethyl ether, mp 96e98 ꢄC. Yield 28%. The
second eluate gave 14f as a white solid. The third eluate gave 12f as
white solid, crystallized from diethyl ether, mp 170e173 ꢄC. Yield
d
119.1 (C), 124.0 (2CH), 128.0 (2CH), 128.7 (C), 129.3 (CH), 129.5 (CH),
132.0 (C), 136.5 (C), 137.4 (C), 139.6 (CH), 154.6 (C). Anal. calcd. for
C15H10N2Cl2O: C, 59.04; H, 3.30; Cl, 23.24; N, 9.18; found: C, 58.78;
H, 3.52; N, 9.01.
5.1.4.6. 5-Methoxy-2-[1-(pyridin-2-yl)-1H-pyrazol-5-yl]phenol
(12d); 5-methoxy-2-[1-(pyridin-2-yl)-1H-pyrazol-3-yl]phenol (13d);
7-methoxy-4H-chromen-4-one (14d). From 2-hydrazinopyridine
dihydrochloride and 11d (method 1). The residue was chromato-
graphed on silica gel eluting with hexane/AcOEt 8:2. The first eluate
gave 13d as a white solid, crystallized from diethyl ether, mp
44%. Data of 12f : IR (KBr):
(200 MHz, d6-DMSO):
Ar), 7.27e7.42 (2H, m, Ar), 7.27e7.42 (2H, m, Ar), 7.43e7.46 (1H, m,
Ar), 7.77e7.84 (1H, m, Ar), 7.90 (1H, m, J ¼ 1.8 , H-3), 8.04e8.08 (1H,
m, Ar), 8.31e8.34 (1H, m, Ar), 9.54 (1H, s, OH, D2O exchangeable);
n
3050, 1478, 1438, 1365 cm-1; 1H NMR
6.64 (1H, d, J ¼ 1.8, H-4), 6.87e7.00 (2H, m,
d

5306
A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
13C NMR (50 MHz, CDCl3):
d
111.0 (CH), 117.9 (CH), 119.1 (CH), 119.6
Ar), 7.72e7.78 (1H, m, Ar), 7.89 (1H, d, J ¼ 1.8, H-5), 8.09e8.20 (1H,
m, Ar), 8.49e8.56 (1H, m, Ar); 13C NMR (50 MHz, CDCl3):
47.3
(CH), 120.2 (C), 121.6 (CH), 130.0 (CH), 131.4 (CH), 139.0 (CH), 140.4
(C), 140.7 (CH), 145.3 (CH), 151.1 (C), 154.5 (C). Anal. calcd. for
C14H11N3O: C, 70.87; H, 4.67; N, 17.71; found: C, 70.91; H, 4.62; N,
d
(2CH2), 65.6 (2CH2), 107.1 (CH), 113.8 (2CH), 119.1 (CH), 120.4 (CH),
122.8 (CH), 128.6 (2CH), 138.6 (CH), 140.2 (C), 143.0 (CH), 147.8 (C),
150.1 (CH) 152.1 (C). Anal. Calcd. for C18H18N4O: C, 70.57; H, 5.92; N,
18.29. Found: C, 70.35; H, 6.02; N, 18.59. GS-MS: tr ¼ 24.8 min, m/z :
306 (Mþ).
17.71. GS-MS tr ¼ 18.8 min, m/z : 237 (Mþ). Data of 13f : IR (KBr):
n
3142, 1576, 1472, 1445, 1365 cm-1; 1H NMR (200 MHz, CDCl3):
d 7.04
(1H, d,, H-4 J ¼ 2.8 Hz), 7.09e7.50 (4H, m, Ar þ H-4), 7.79e7.83 (1H,
m, Ar), 8.02e8.06 (2H, m, J ¼ 2.6, H-5), 8.59e8.63 (1H, m, Ar),
8.14e8.24 (1H, m, Ar), 10.87 (1H, s, OH, D2O exchangeable); 13C
5.1.4.13. 2-[5-(2,4-dimethoxyphenyl)-1H-pyrazol-1-yl]pyridine (12i).
From 2-hydrazinopyridine dihydrochloride and 11i (method 1). The
residue was chromatographed on silica gel eluting with cyclo-
hexane and, then, cyclohexane/AcOEt 7:3. The eluate gave 12i as
white solid, crystallized from cyclohexane, mp 105e107 ꢄC. Yield
NMR (50 MHz, CDCl3): d 103.9 (CH), 110.9 (CH), 115.0 (C), 116.1 (CH),
118.5 (CH), 120.6 (CH), 125.9 (CH), 127.0 (CH), 128.9 (CH), 137.9 (CH),
147.2 (CH), 149.4 (C), 152.8 (C), 155.0 (C). Anal. calcd. for C14H11N3O:
C, 70.87; H, 4.67; N, 17.71; found: C, 70.82; H, 4.70; N, 17.99. GS-MS
tr ¼ 21.0 min, m/z : 237 (Mþ). Data of 14f: GS-MS tr ¼ 11.5 min, m/z :
146 (Mþ).
52%. IR (KBr):
CDCl3):
n ;
1612, 1583, 1474, 1434 cm-1 1H NMR (200 MHz,
3.33 (3H, s, OCH3), 3.87 (3H, s, OCH3), 6.38 (1H, d, J ¼ 2.4,
d
Ar), 6.46 (1H, d, J ¼ 1.6, H-4), 6.58 (1H, dd, J ¼ 2.4, 8.4, Ar), 7.17e7.23
(1H, m, Ar), 7.32 (1H, d, J ¼ 8.4 Hz, Ar), 7.52e7.60 (1H, m, Ar), 7.80
(1H, d, J ¼ 1.6, H-3), 7.83e7.85 (1H, m, Ar), 8.36e8.40 (1H, m, Ar);
5.1.4.10. 2-[1-(4-chlorophenyl)-1H-pyrazol-5-yl]phenol (12f1). From
4-chlorophenylhydrazine hydrochloride and 11f (method 1). A
white solid was collected by filtration from the mixture and crys-
13C NMR (50 MHz, CDCl3):
d 53.7 (OCH3), 54.4 (OCH3), 97.6 (CH),
tallized from cyclohexane, mp 184e187 ꢄC. Yield 55%. IR (KBr):
n
103.3 (CH), 108.1 (CH), 112.7 (C), 116.0 (CH), 120.6 (CH), 130.0 (CH),
136.7 (CH), 139.2 (C), 139.7 (CH), 146.9 (CH), 152.6 (C), 156.2 (C),
160.4 (C). Anal. calcd. for C15H13N3O2: C, 68.31; H, 5.37; N, 14.94;
found: C, 68.42; H, 5.63; N, 14.66.
3428, 2967, 1497, 834, 756 cm-1
6.63(1H, d, J ¼ 1.8, H-4), 6.96e7.03 (2H, m, Ar), 7.29e7.43 (4H, m,
Ar), 7.52e7.58 (2H, m, Ar), 7.89 (1H, d, J ¼ 1.8, H-3), 9.80 (1H, s, OH,
D2O exchangeable); 13C NMR (50 MHz, CDCl3):
108.7 (CH), 115.4
;
1H NMR (200 MHz, (D6)DMSO):
d
d
(CH), 117.1 (C), 118.7 (CH), 124.3 (2CH), 128.3 (2CH), 130.1 (C), 130.7
(CH), 139.3 (C), 139.6 (C), 139.7 (CH), 154.3 (C). Anal. calcd. for
C15H11N2ClO: C, 66.55; H, 4.10; N, 10.35; found: C, 66.58; H, 4.16; N,
10.32. GS-MS tr ¼ 19.9 min, m/z : 270 (Mþ).
5.1.4.14. 2-[5-(2-methoxyphenyl)-1H-pyrazol-1-yl]pyridine (12g); 2-
[3-(2-methoxyphenyl)-1H-pyrazol-1-yl]pyridine (13g). From 2-
hydrazinopyridine dihydrochloride and 11g (method 1). The
residue was chromatographed on silica gel eluting with cyclo-
hexane and, then, cyclohexane/AcOEt 7:3. The first eluate gave 13g
as a white solid, mp. Yield 10%. The second eluate gave 12g as white
solid, crystallized from cyclohexane, mp 88e90 ꢄC. Yield 49%. Data
5.1.4.11. 2-[5-(3-methoxyphenyl)-1H-pyrazol-1-yl]pyridine (12l); 2-
[3-(3-methoxyphenyl)-1H-pyrazol-1-yl]pyridine (13l). From
2-
hydr-azinopyridine dihydrochloride and 11l (method 1). The
residue was chromatographed on silica gel eluting with toluene
and, then, toluene/AcOEt 1:1. The first eluate gave 13l as a pale
yellow oil. Yield 11%. The second eluate gave 12l as yellow solid,
crystallized from diethyl ether, mp 61e63 ꢄC. Yield 50%. Data of
of 12g: IR (KBr):
(200 MHz, CDCl3):
6.80 (1H, d, J ¼ 8.2, Ar), 7.02e7.09 (1H, m, Ar), 7.15e7.22 (1H, m, Ar),
7.34 (1H, d, J ¼ 1.4, H-3), 7.37e7.43 (1H, m, Ar), 7.58 (1H, d, J ¼ 8.2,
Ar), 7.74e7.81 (2H, m, Ar), 8.26e8.33 (1H, m, Ar); 13C NMR (50 MHz,
n ;
1596, 1477, 1466, 1452, 1351 cm-1 1H NMR
d
3.35 (3H, s, OCH3), 6.51 (1H, d, J ¼ 1.4, H-4),
12l: IR (KBr):
(200 MHz, CDCl3):
4), 6.97e7.04 (3H, m, Ar), 7.36e7.43 (2H, m, Ar), 7.58e7.64 (1H,
m, Ar), 7.85e7.97 (1H, m, Ar), 7.92 (1H, d, 1.8, H-3),
8.53e8.60 (1H, m, Ar); 13C NMR (50 MHz, CDCl3):
54.1 (OCH3),
n
2938, 2835, 1590, 1472, 1434, 1378 cm-1
;
1H NMR
CDCl3): d 54.2 (OCH3), 109.2 (CH), 110.7 (CH), 116.4 (CH), 120.0 (CH),
d
3.88 (3H, s, OCH3), 6.69 (1H, d, J ¼ 1.8, H-
120.2 (C), 120.6 (CH), 121.9 (CH), 129.6 (CH), 129.7 (CH), 138.2 (CH),
139.6 (C), 140.0 (CH), 146.9 (CH), 152.8 (C), 155.3 (C). Anal. calcd. for
C15H13N3O: C, 71.70; H, 5.21; N, 16.72; found: C, 71.68; H, 5.16; N,
J
¼
d
16.81. Data of 13g: IR (KBr):
n
1595, 1475, 1466, 1456, 1351, 1278 cm-
d 3.96 (3H, s, OCH3), 7.02e7.31 (5H, m,
107.8 (CH), 112.9 (CH), 113.1 (CH), 118.0 (CH), 120.1 (CH), 121.5
(CH), 128.3 (CH), 131.1 (C), 137.1 (CH), 140.0 (CH), 142.4 (C), 147.5
(CH), 151.4 (C), 158.3 (C). Anal. calcd. for C15H13N3O: C, 71.70; H,
5.21; N, 16.72. found: C, 71.66; H, 5.43; N, 16.67. GS-MS
1; 1H NMR (200 MHz, CDCl3):
Ar), 7.81e7.90 (1H, m, Ar), 8.11e8.17 (2H, m, Ar), 8.42e8.50 (1H, m,
Ar), 7.43e7.52 (1H, m, Ar), 7.91e7.95 (2H, m, Ar þ H-3), 8.04e8.09
(1H, m, Ar), 8.64 (1H, d, J ¼ 2.4, H-5); 13C NMR (50 MHz, CDCl3):
tr ¼ 19.5 min, m/z : 251 (Mþ). Data of 13l: IR (film):
n
2937,
d 54.5 (OCH3), 108.5 (CH), 110.4 (CH), 111.5 (CH), 119.8 (CH), 120.0
2835, 1595, 1578, 1473, 1454, 1355 cm-1
;
1H NMR (200 MHz,
(CH), 120.9 (C), 126.2 (CH), 127.8 (CH), 128.4 (CH), 137.5 (CH), 146.9
(CH), 149.9 (C), 150.6 (C), 156.2 (C). Anal. calcd. for C15H13N3O: C,
71.70; H, 5.21; N, 16.72; found: C, 71.44; H, 5.61; N, 16.57.
CDCl3):
d
4.06 (3H, s, OCH3), 6.69 (1H, d, J ¼ 2.6, H-4), 7.05e7.11
(1H, m, Ar), 7.48e7.56 (1H, m, Ar), 7.58e7.64 (2H, m, Ar),
7.92e8.04 (1H, m, Ar), 8.25e8.30 (1H, m, Ar), 8.57e8.59 (1H, m,
Ar), 8.76 (1H, d, J ¼ 2.6, H-5); 13C NMR (50 MHz, CDCl3):
d
54.3
5.1.5. 1-(4-methoxyphenyl)-2-phenylethanone (15b)
(OCH3), 104.4 (CH), 110.0 (CH), 111.4 (CH), 113.1 (CH), 117.5 (CH),
120.2 (CH), 127.2 (CH), 128.6 (CH), 133.2 (C), 137.6 (CH), 146.9
(CH), 150.2 (C), 152.5 (C), 158.8 (C). Anal. calcd. for C15H13N3O: C,
71.70; H, 5.21; N, 16.72; found: C, 71.76; H, 5.19; N, 16.84. GS-MS
tr ¼ 21.4 min, m/z : 251 (Mþ).
From anisole. A white solid was collected by filtration from the
mixture and crystallized from cyclohexane, mp 66e67 ꢄC. IR (KBr):
n
d
1676,1601,1574,1335,1260,1172 cm-1; 1H NMR (200 MHz, CDCl3):
4.02 (3H, s, OCH3), 4.41 (2H, s, CH2), 7.09 (2H, d, J ¼ 8.8, Ar),
7.35e7.55 (5H, m, Ar), 8.17 (2H, d, J ¼ 8.8, Ar). Anal. calcd. for
C15H14O2: C, 79.62; H, 6.24; found: C, 79.89; H, 5.89.
5.1.4.12. 4-{4-[1-(pyridin-2-yl)-1H-pyrazol-5-yl]phenyl}morpholine
(12h). From 2-hydrazinopyridine dihydrochloride and 11h
(method 1). The residue was chromatographed on silica gel eluting
with toluene and, then, toluene/AcOEt 1:1. The first eluate gave 12h
as a pale yellow solid, crystallized EtOH, mp 136e137 ꢄC. Yield 28%.
5.1.6. 3-(dimethylamino)-1-(4-methoxyphenyl)-2-methyl-2-
propen-1-one (16a)
Dimethylformamide dimethylacetal (10 mmol) was added to
a suspension of 15a (10 mmol) in xylene (30 mmol) and the mixture
was refluxed for 4 h. After solvent evaporation, the residue was
chromatographed on silica gel eluting with cyclohexane/AcOEt 7:3
and, then, AcOEt. The first eluate gave the starting product, the
IR (KBr):
DMSO):
d, J ¼ 1.8, H-4), 7.01 (2H, d, Ar), 7.21 (2H, d, Ar), 7.52e7.61 (1H, m,
n
2853, 1591, 1473, 1436 cm-1
;
1H NMR (200 MHz, (D6)
d
3.24e3.29 (4H, m, CH2), 3.84e3.89 (4H, m, CH2), 6.72 (1H,

A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
5307
second eluate gave 16a as a yellow oil. Yield 69%. 1H NMR (200 MHz,
CDCl3): 2.28 (3H, s, CH3), 3.16 [6H, s, N(CH3)2], 3.98 (3H, s, OCH3),
7.02 (1H, d, J ¼ 9.0, Ar), 7.06 (1H, s, ¼ CH), 7.56 (2H, d, J ¼ 9.0, Ar);
Anal. calcd. for C13H17NO2: C, 71.21; H, 7.81; N, 6.39; found: C, 71.16;
H, 7.85; N, 6.33.
form: d 1.60 (3H, s, CH3),3.93 (3H, s, OCH3), 6.84 (1H, s, CH enolic
d
form), 7.03 (2H, d, J ¼ 8.8, Ar), 7.90 (2H, d, J ¼ 8.8, Ar), 7.99e8.04 (5H,
m, Ar). Anal. calcd. for C16H14O3: C, 75.57; H, 5.55; found: C, 75.42;
H, 5.68.
5.1.10.2. 1-(4-methoxyphenyl)butane-1,3-dione (21). From ethyl
5.1.7. 3-(dimethylamino)-1-(4-methoxyphenyl)-2-phenyl-2-
propen-1-one (16b)
acetate. Mp 53-4 ꢄC. Yield 51%. IR (KBr):
n
2924, 2853, 1607, 1464,
1260, 1176 cm-1; 1H NMR (200 MHz, CDCl3) enolic form:
d
2.20 (3H,
Dimethylformamide dimethylacetal (10 mmol) was added to
a suspension of 15b (10 mmol) in xylene (30 mmol) and the
mixture was refluxed for 2 h. After solvent evaporation, the residue
was chromatographed on silica gel eluting with cyclohexane/AcOEt
7:3 and, then, AcOEt. The first eluate gave the starting product, the
second eluate gave 16b as a yellow solid, crystallized from diethyl
s, CH3), 3.20 (3H, s, OCH3), 6.15 (1H, s, CH enolic form), 6.98 (2H, d,
J ¼ 9.0, Ar), 7.54 (2H, d, J ¼ 9.0, Ar). Anal. calcd. for C11H12O3: C,
68.74; H, 6.29; found: C, 68.49; H, 5.41.
5.1.11. 2-[5-(4-methoxyphenyl)-3-phenyl-1H-pyrazol-1-yl]pyridine
(20)
ether, mp 92e94 ꢄC. Yield 57%. 1H NMR (200 MHz, CDCl3):
d
2.90
From 2-hydrazinopyridine dihydrochloride and 19 (method 1)
for 2 h at reflux. A white solid was collected by filtration from the
mixture and crystallized from EtOH, mp 122e123 ꢄC. Yield 38%. IR
[6H, s, N(CH3)2],3.98 (3H, s, OCH3), 6.92 (2H, d, J ¼ 8.8, Ar),
7.29e7.50 (5H, m, Ar þ ¼CH), 7.55e7.61 (3H, m, Ar). Anal. calcd. for
C18H19NO2: C, 76.84; H, 6.81; N, 4.98; found: C, 76.86; H, 6.84;
N, 4.95.
(KBr):
CDCl3):
n ;
1589, 1471, 1449, 1435, 1245 cm-1 1H NMR (200 MHz,
d
4.02 (3H, s, OCH3), 7.00 (1H, s, H-4), 7.16 (2H, d, J ¼ 9.0, Ar),
7.44e7.61 (7H, m, Ar), 8.03e8.12 (1H, m, Ar), 8.18e8.27 (1H, m, Ar)
8.85e8.94 (1H, m, Ar). Anal. calcd. for C21H17N3O: C, 77.04; H, 5.23;
N, 12.84; found: C, 76.98; H, 5.19; N, 12.93.
5.1.8. 2-[5-(4-methoxyphenyl)-4-methyl-1H-pyrazol-1-yl]pyridine
(17a); 2-[3-(4-methoxyphenyl)-4-methyl-1H-pyrazol-1-yl]pyridine
(18)
From 2-hydrazinopyridine dihydrochloride and 16a (method 1)
for 2 h at reflux. The residue was chromatographed on silica gel
eluting with cyclohexane and, then, cyclohexane/AcOEt 8:2. The
first eluate gave 18 as a yellow solid, crystallized from diethyl ether,
mp 70e72 ꢄC. Yield 8%. The second eluate gave 17a as white solid,
crystallized from cyclohexane, mp 74e75 ꢄC. Yield 45%. Data of 17a:
5.1.12. 2-[5-(4-methoxyphenyl)-3-methyl-1H-pyrazol-1-yl]
pyridine (23)
From 2-hydrazinopyridine dihydrochloride and 21 (method 1)
for 2 h at reflux. The residue was purified by chromatography on
silica gel eluting with cyclohexane/AcOEt 7:3. Yield 52%. Mp
82e84 ꢄC. IR (KBr):
n ;
1615, 1587, 1509, 1473, 1250 cm-1 1H NMR
IR (KBr):
n
1591, 1472, 1433, 1247 cm-1; 1H NMR (200 MHz, CDCl3):
(200 MHz, CDCl3): d 2.44 (3H, s, CH3), 3.84 (3H, s, OCH3), 6.31
d
2.29 (3H, s, CH3), 4.02 (3H, s, OCH3), 7.07 (2H, d, J ¼ 9.0, Ar),
(1H, s, H-4), 6.86 (2H, d, J ¼ 8.8, Ar), 7.18e7.38 (4H, m, Ar),
7.67e7.77 (1H, m, Ar), 8.21e8.29 (1H, m, Ar). Anal. calcd. for
C16H15N3O: C, 72.43; H, 5.70; N, 15.84; found: C, 72.30; H, 5.69;
N, 15.76.
7.32e7.53 (4H, m, Ar), 7.76e7.89 (2H, m, H-3 þ Ar), 8. 49e8.58 (1H,
m, Ar). Anal. calcd. for C16H15N3O: C, 72.43; H, 5.70; N, 15.84; found:
C, 72.14; H, 5.74; N, 15.73. Data of 18: IR (KBr):
n 1590, 1472, 1435,
1245 cm-1; 1H NMR (200 MHz, CDCl3):
d
2.47 (3H, s, CH3), 4.03 (3H,
s, OCH3), 7.02e7.43 (4H, m, Ar), 7.80e7.98 (2H, m, Ar), 8.18e8.22
(1H, m, Ar), 8. 54e8.58 (1H, m, Ar þ H-5). Anal. calcd. for
C16H15N3O: C, 72.43; H, 5.70; N, 15.84; found: C, 72.36; H, 5.69;
N, 15.93.
5.1.13. 7-(4-methoxyphenyl)-3,5,7-trioxoheptanoic acid (22)
A solution of 40-methoxyacetophenone (10 mmol) in 15 mL of
dried toluene was added to a stirred suspension of sodium amide
(20 mmol) in dried toluene (40 mL). The mixture was stirred for
20 min at 50 ꢄC, then AcOEt (11 mmol) was added slowly. The
resulting mixture was heated at reflux overnight, cooled to room
temperature, and quenched with water (20 mL). The organic layer
was removed, and the aqueous acidified with HCI 37%, followed by
extraction with ether. The organic layers were combined, washed
with water (20 mL), dried over sodium sulfate, and evaporated at
reduced pressure to give the crude product as an oil. The residue
was chromatographed on silica gel eluting with toluene and, then,
toluene/AcOEt 1:1. The title compound was obtained as a white
5.1.9. 2-[5-(4-methoxyphenyl)-4-phenyl-1H-pyrazol-1-yl]pyridine
(17b)
From 2-hydrazinopyridine dihydrochloride and 16b (method 1)
for 2 h at reflux. A white solid was collected by filtration from the
mixture and crystallized from EtOH, mp 66e67 ꢄC. Yield 73%. IR
(KBr):
CDCl3):
n ;
1580, 1471, 1443, 1429, 1247 cm-1 1H NMR (200 MHz,
d
3.97 (3H, s, OCH3), 6.98 (2H, d, J ¼ 8.4, Ar), 7.26e7.50 (9H,
m, Ar), 8.80e8.89 (1H, m, Ar), 8.11(1H, s, H-3), 8.56e8.58 (1H, m,
Ar). Anal. calcd. for C21H17N3O: C, 77.04; H, 5.23; N, 12.84; found: C,
76.86; H, 5.41; N, 12.85.
solid from CHCl3, mp. 143e145 ꢄC. Yield 29%. IR (KBr):
n 3364, 1682,
1658, 1605 cm-1 1H NMR (200 MHz, CDCl3):
;
d
3.50 (4H, s, CH2),
4.00(3H, s, OCH3), 7.22 (2H, d, J ¼ 8.8, Ar), 7.87 (2H, d, J ¼ 8.8, Ar),
13.02 (1H, s, COOH). Anal. calcd. for C12H12O5: C, 61.01; H, 5.12;
found: C, 61.28; H.
5.1.10. Synthesis of compounds 19 and 21. General procedure:
To a stirred suspension of sodium hydryde 60% (22.5 mmol) in
dried toluene (10 mL) was slowly added a solution of 40-methox-
yacetophenone (20 mmol) and the desired ester (20 mmol) in
20 mL of dried toluene under nitrogen atmosphere. The mixture
was stirred for 4 h at reflux, cooled to room temperature and
quenched with a solution of HCl 10%. The organic layer was
removed, and the aqueous extracted with ether. The organic layers
were combined, washed with water, dried over sodium sulfate, and
evaporated at reduced pressure to give the crude product.
5.1.14. 2-[5-(4-methoxyphenyl)-1-(pyridin-2-yl)-1H-pyrazol-3-yl]
acetic acid (24)
From 2-hydrazinopyridine dihydrochloride and 22 (method 1) for
2 h at reflux. The residuewas purified bychromatographyon silica gel
eluting with toluene and, then, AcOEt. The second eluate gave 24 as
white solid, crystallized from cyclohexane, mp 169e172 ꢄC. Yield 18%.
IR (KBr):
(200 MHz, CDCl3):
n
3000 (br), 1601, 1511, 1483, 1438, 1253 cm-1; 1H NMR
d
2.39 (2H, s, CH2), 3.91 (3H, s, OCH3),
5.1.10.1. 1-(4-methoxyphenyl)-3-phenylpropane-1,3-dione
6.84e6.91(2H, m, Ar þ H-4), 6.95 (2H, d, J ¼ 8.8, Ar), 7.45e7.51 (1H, m,
Ar), 7.58 (2H, d, J ¼ 8.8, Ar). Anal. calcd. for C17H15N3O3: C, 66.01; H,
4.89; N, 13.58; found: C, 66.06; H, 5.01; N, 12.95.
(19). From methyl benzoate. Mp 120-2 ꢄC. Yield 68%. IR (KBr):
n
1607, 1516, 1487, 1261 cm-1 1H NMR (200 MHz, CDCl3) enolic
;

5308
A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
5.2. Biology
actin monoclonal antibody (TU-02,1:5,000, Santa Cruz, CA, USA)
was used for the densitometric normalization.
5.2.1. MTT assay
Human cell lines A2780 (ovary, adenocarcinoma) and A549
(lung, carcinoma), and murine P388 (leukemia) cells were plated
(P388: 900 cells/well; A549: 1600 cells/well; A2780: 2000 cells/
well) into flat-bottomed (for adherent cells) and U-bottomed (for
non-adherent cells) 96-well microtiter plates, treated for 72 h with
5 concentrations of our pyrazole derivatives (1:10 fold concentra-
tions) and prepared as described elsewhere [47,48].
5.2.5. Microtubule immunofluorescence
Ten to five A549 cells were cultured in chamber slides and 24 h
later treated with 1, 10 and 100 mM of selected active substances
and with 100 nM taxol as positive control. After 24-h incubation
A549 cells were washed with cold PBS and fixed at room temper-
ature for 5 min with 3.7% paraformaldehyde in PBS plus 2% sucrose
(PBS-S). Once washed again 3 times with cold PBS-S on ice, they
were incubated with methanol for other 5 min at ꢂ20 ꢄC. Further 3
additional washes with cold PBS-S ended the fixation procedure.
Thirty min incubation with cold 20% goat serum in PBS (GS-PBS) on
ice was used to saturate non-specific immunoglobulin binding
5.2.2. Morphological determination of apoptotic cells by 40-6-
diamidino-2-phenylindole (DAPI)
Apoptosis was evaluated in all cell lines for the four pyrazole
derivatives that demonstrated to be more active by the MTT assay.
Cells (from 5 ꢃ 103 to 11 ꢃ 103) were plated in 1 mL of culture
medium into 24-well plates and treated with the IC50 and IC90
equitoxic concentrations, as determined by the MTT assay. Floating
and adherent cells were harvested after 3 days culture, washed
with cold phosphate buffered saline (PBS) and resuspended in
sites. The monoclonal anti-a-tubulin (T5168, Sigma) diluted 2000
times in GS-PBS was added for 2 h on ice, then cells were washed 3
times with cold PBS-S on ice. The Alexa 594 goat anti-mouse IgG1
(2.5 mg/mL in GS-PBS) was used to reveal the anti-a-tubulin anti-
body after incubation on ice for 30 min. The samples were finally
washed 3 times with cold PBS-S on ice and the slide mounted with
a coverslip using GelMount (Biomeda Inc., CA). Images were
acquired with an Axiovert 200M microscope (Carl Zeiss Micro-
Imaging GmbH, Goettingen, Germany).
100
microscopic examination. For the morphological analysis of nuclei,
L of a solution of 10 mg/mL DAPI in water were added and the
m
L of 70% ethanol in PBS. Samples were maintained at 4 ꢄC until
5
m
percentage of apoptotic segmented nuclei/cells stained with the
fluorescent dye evaluated (n ¼ 3e7).
5.2.6. Tubulin polymerization assay
The effect of tested compounds on in vitro assembly of tubulin
microtubules was evaluated using a fluorescence-based microtu-
bule polymerization assay kit (Cytoskeleton, Denver, USA) [49,50],
following the recommended protocol. Briefly, porcine brain tubulin
was dissolved in 80 mM piperazine-N,N0-bis[2-ethanesulfonic acid]
sequisodium salt, 2 mM magnesium chloride, 0.5 mM ethylene
5.2.3. Analysis of the cell cycle
P388 cells were also analyzed for the effect of active compounds
on cell cycle phases. To this end cells (5 ꢃ103 in 3e4 mL of complete
medium) were treated for 24, 48 and 72 h with the IC30 and IC75 of
our molecules, harvested, washed twice with cold PBS, and fixed in
70% ethyl alcohol overnight at ꢂ20 ꢄC. The day after they were
centrifuged, washed with cold PBS and incubated at room
temperature for 20 min with propidium iodide (PI) staining solu-
glycol-bis(b
-amino-ethyl ether) N,N,N0,N0-tetra-acetic-acid, 60% v/v
glycerol, pH 6.9. The assay was carried out in a half area 96-well
plate (Corning Costar), and the reaction was initiated by the addi-
tion of tubulin. The plate was incubated at 37 ꢄC on a fluorescence
microplate reader (Mithras LB 940, Berthold Technologies), and
microtubule assembly was monitored by measuring fluorescence
tion (50 mg/mL PI, 0.05% Triton X-100, 0.2 mg/mL RNase A in PBS).
Cells were finally analyzed by flow cytometric analysis of DNA
content using FACSort flow cytometer (BD Bioscences, Mountain.
View, CA). Doublets, cell clumps, and debris were excluded by
plotting PI fluorescence pulse-width versus pulse-area measure-
ments using CellQuest software (BD Biosciences). Cell cycle analysis
on the gated PI distribution was performed using Modfit software.
(
l
ex, 355 nm;
polymerization assay was performed in the presence of 10, 2, 1 and
0.5 M concentrations of 12a, 12a1, 12d1 and 12d compounds and
results compared with untreated controls containing fetal calf
serum plus 4% DMSO. Positive controls were treated with 3
taxol and 1.6 M vincristine.
lem, 460 nm) by the minute for 60 min. A tubulin
m
m
M
5.2.4. Western blot analysis of p53 and p21waf1
m
In a single experiment human A2780 and A549 cells were treated
with 0.1,10, and 100
m
M 12d. After 24, 48 and 72 h culture cells were
5.2.7. Preparation of ligands and receptor molecules for docking
harvested, washed twice with cold PBS and lysed for 30 min at 4 ꢄC
with a buffer containing 1% Triton X-100, 0.15 M NaCl, 10 mM Tris
A molecular structure of the selected compound was built and
energy minimized with the programs Insight II and Discover3
(Biosym/MSI, San Diego, CA, USA). To analyze the receptoreligand
complexes, we used the atomic coordinates of the alpha-beta
tubulin dimer [38]. The receptor and the ligands were prepared
for docking using ADT, the AutoDock tool graphical interface [51].
For each receptor structure polar hydrogens were added, Kollman
charges were assigned and atomic solvatation parameters were
added. Polar hydrogen charges of the Gasteiger-type were assigned
and the non-polar hydrogens were merged with the carbons, the
internal degrees of freedom and torsions were set for all the
designed small molecules. The resulting atomic models were then
submitted to different cycles of molecular dynamics followed by
energy minimization using the programs Insight II and Discover3.
The resulting model was used as target for molecular docking
simulations using the program AutoDock 3.05 [52].
(pH 7.4), 0.1 mg/mL phenylmethylsulphonyl fluoride and 50 mg/mL
aprotinin. The protein concentration was determined by the Brad-
ford method (Sigma Chemical Co., St. Louis, MO, USA). Fifty mg of
proteinwere separated on a 12% polyacrylamide gel (SDS-PAGE) and
then transferred onto a nitrocellulose membrane (Hybond C-Extra,
Amersham Italia Srl, Milan, Italy). The protein loading was checked
by Ponceau S staining. Nonspecific binding was blocked overnight at
4 ꢄC with 2% bovine albumin (Sigma) in Tris-buffered saline-Tween
20 [0.15 M NaCl, 10 mM Tris (pH 8.0), 0.05% Tween 20]. Blots were
probed with anti-p53 DO-1 (1:2,000, Santa Cruz, CA, USA), anti-
p21waf1 1:1000 (Exbio, Praha, Czech Republic) and anti-
a-tubulin
(1:2,000, Santa Cruz) monoclonal antibodies. The membranes were
then incubated with horseradish peroxidase-conjugated anti-
mouse IgG and bands visualized by chemiluminescent detection
(ECLÔ Western blotting analysis system, Amersham Italia Srl)
following the supplier’s recommended procedures. Prestained
molecular weight markers (New England Biolabs, Beverly, MA, USA)
5.2.8. Docking simulation
To better address our search, we performed three different
simulations using, as targets, the alpha monomer, the beta
were used as reference. The expression signal obtained by an anti-b-

A. Balbi et al. / European Journal of Medicinal Chemistry 46 (2011) 5293e5309
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lation, in a first phase, 12d was docked into the active site using
the program AutoDock 3.05 with the macromolecule considered
as a rigid body and the ligand being flexible. The searching grid
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