Aryl azoles with neuroprotective activity-Parallel synthesis and attempts at target identification

Article abstract of DOI:10.1016/j.bmc.2007.10.090

A parallel synthesis of aryl azoles with neuroprotective activity is described. All compounds obtained were evaluated in an in vitro assay using a NMDA toxicity paradigm showing a neuroprotective activity between 15% and 40%. The potential biological target of the active compounds was investigated by extensive literature searches based around similar scaffolds with reported neuroprotective activity. The most interesting molecules active in the NMDA toxicity assay (3a and 2g) showed moderate but significant activity in the inhibition of the Site 2 Sodium Channel binding assay at 10 μM. To confirm our hypothesis compounds 3a, c, f and 2g were tested in the Veratridine assay which is one of the excitotoxicity assays of revelance to NaV channels. The compounds tested showed an activity between 40% and 70%. The identification of neuroprotective small molecules and the identification of NaV channels as the potential site of action were the most important goals of this work.

Full text of DOI:10.1016/j.bmc.2007.10.090

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2043–2052
Aryl azoles with neuroprotective activity—Parallel synthesis
and attempts at target identification
a,
a
a
Giuseppe Cocconcelli,a,* Enrica Diodato, Andrea Caricasole, Giovanni Gaviraghi,
Eva Genesio,a Chiara Ghiron,a Letizia Magnoni,a Elena Pecchioli,a
Pier Vincenzo Plazzib and Georg C. Terstappena
*
aSiena Biotech S.p.A., via Fiorentina 1, 53100 Siena, Italy
`
Dipartimento Farmaceutico, Universita degli Studi di Parma, V.le G.P. Usberti, 27/A, I-43100 Parma, Italy
b
Received 27 July 2007; revised 23 October 2007; accepted 30 October 2007
Available online 4 November 2007
Abstract—A parallel synthesis of aryl azoles with neuroprotective activity is described. All compounds obtained were evaluated in
an in vitro assay using a NMDA toxicity paradigm showing a neuroprotective activity between 15% and 40%. The potential bio-
logical target of the active compounds was investigated by extensive literature searches based around similar scaffolds with reported
neuroprotective activity. The most interesting molecules active in the NMDA toxicity assay (3a and 2g) showed moderate but sig-
nificant activity in the inhibition of the Site 2 Sodium Channel binding assay at 10 lM. To confirm our hypothesis compounds 3a, c,
f and 2g were tested in the Veratridine assay which is one of the excitotoxicity assays of revelance to NaV channels. The compounds
tested showed an activity between 40% and 70%. The identification of neuroprotective small molecules and the identification of NaV
channels as the potential site of action were the most important goals of this work.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
mostly limited to symptomatic treatments. In Parkin-
son’s disease for example, where the neurochemical
deficit produced by the disease is comparatively well-de-
fined, symptomatic treatment is in general relatively
successful.3,4 Carbidopa/levodopa, bromocriptine (a
dopamine receptor agonist), entacapone (a caethcol-O-
methyl-transferase inhibitor), selegiline (a MAO-B
inhibitor) and amantidine are examples of commonly
used medications for the treatment of PD. Drugs ap-
proved by US Food and Drug Administration for the
treatment of AD are galantamine, rivastigmine, donepe-
zil, tacrine (cholinesterase inhibitors) and memantine
(an NMDA antagonist). The pharmacological treatment
of Huntington’s disease is currently limited to the ame-
lioration of movement and psychiatric abnormalities
associated with the illness and includes antipsychotic
agents and benzodiazepines. Likewise, current therapy
for ALS is symptomatic and includes GABA-B agonists
and benzodiazepines. These symptomatic treatments do
not alter the course of these disorders. The lack of dis-
ease-modifying therapies for most neurodegenerative
disorders commands significant efforts towards the
development of novel neuroprotective strategies. These
efforts have resulted in the identification of general
mechanisms such as excitotoxicity (determined by an
Neurodegenerative disorders are characterized by a pro-
gressive dysfunction and death of neurons from specific
regions of the brain. These disorders include debilitating
diseases as Parkinson’s disease (PD), Alzheimer’s dis-
ease (AD), Huntington’s disease (HD) and amyotrophic
lateral sclerosis (ALS). Although clinical and neuro-
pathological aspects of these syndromes are distinct,
their unifying feature is that each one has a characteris-
tic pattern of neuronal degeneration in anatomically de-
fined regions.1
Neuroprotection may be defined as the interference with
the cellular and functional outcomes of neurotoxic in-
sults, with the aim to preserve neuronal survival and
function.2 The result of a neuroprotective therapy may
be the salvage, recovery or regeneration of the nervous
system, its cells, structure and functions. Presently phar-
macological therapy of neurodegenerative disorders is
Keywords: Aryl azoles; Neuroprotection; Target identification; Site 2
Sodium Channel.
*
Corresponding authors. Tel.: +39 0577 381416; fax: +39 0577 381410
(G.C.); e-mail: gcocconcelli@sienabiotech.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.090

2044
G. Cocconcelli et al. / Bioorg. Med. Chem. 16 (2008) 2043–2052
is the reaction of 1,3-dicarbonyl compounds with hydra-
zine hydrate. However, when substituted hydrazines and
unsymmetrical ketones are used, the reaction often re-
sults in a mixture of regioisomeric pyrazoles.11
The 3,5-diarylpyrazoles (2a–h and 3a–f) described in this
paper (see Table 1) were obtained following the syn-
thetic routes represented in Scheme 1. Since the desired
pyrazoles are substituted in position 1 and the aryl
groups in position 3 and 5 of the heterocyclic ring are
different (2a–h), the development of a regioselective
synthesis was necessary. In order to obtain compounds
2a–h, we therefore decided to explore the reaction of
substituted hydrazines with a,b-unsaturated ketones;
this reaction has been reported to lead to the regioselec-
tive formation of 4,5-dihydro-1H-pyrazoles that can
then be oxidized to the corresponding pyrazoles.12,13
Figure 1. Erythropoietin sensitizer 1 and compounds 2a–h; 3a–f.
excess of glutamate in the brain5), aberrant neuronal
energy metabolism6 and the production of reactive com-
pounds, such as peroxides and radicals,7 as contributors
to the cell death observed in various neurodegenerative
diseases. In addition to generic toxicity responses which
contribute to many neurodegenerative conditions, dis-
ease-specific mechanisms have been identified through
the study of familial cases, resulting in the definition of
key proteins associated with individual pathologies such
as the amyloid precursor protein (APP) and the secreta-
ses (b- and c-secretase) in AD, Parkin in PD, Huntingtin
in HD and Superoxide Dismutase 1 (SOD1) in ALS.
Many of these proteins represent targets currently
exploited for pharmacological intervention in drug
discovery efforts, aimed at defining disease-modification
approaches. In addition, the mechanisms underlying the
neuroprotective properties of specific proteins (such as
erythropoietin,8 NGF and others9) are being studied to
identify tractable targets upon which neuroprotective
pharmacological strategies could be developed.
Compounds 6a, c, f are commercially available. Com-
pounds 6b, d, e, g were synthesized under basic condi-
tions, according to reported literature methods.14
Compound 6h was successfully synthesized under micro-
wave irradiation (lW) with acidic catalysis after the
observation that 1H-indole-3-carbaldehyde did not react
under basic conditions, possibly due to a tautomeric
equilibrium between the indole NH and the carbonyl.15
Although products 2a–h could be obtained directly from
compounds 6a–h,12 the intermediate 4,5-dihydro-1H-
pyrazoles 7a–h were also isolated and characterized in
order to test them in our biological assay. However,
these intermediates proved to be air-sensitive especially
in solution (spontaneous slow oxidative aromatization
was observed), and their biological evaluation was not
possible. Compounds 7a–d, 7h were synthesized from
the respective ketones under basic conditions, while
compounds 7e–g could only be obtained under neutral
conditions, as they showed a high degree of instability
when submitted to our standard procedure (see Scheme
1 and Table 1).
As part of a research project directed towards the devel-
opment of small neuroprotective molecules, we were ini-
tially interested in compounds which could act as
erythropoietin mimetics as these might also display neu-
roprotective properties.8 Compound 1 has been reported
to improve the erythropoietic process by sensitizing red
blood cells to the effect of erythropoietin and possesses a
chemically tractable chemical scaffold.8 We therefore
decided to consider 1 as starting point for our work
and test it in in vitro neuroprotection assays. As the
molecule did indeed show some level of neuroprotective
activity, we explored the chemical class further by syn-
thesizing a small array of compounds (Fig. 1) and inves-
tigating their neuroprotective properties. Finally, we
proceeded to attempt deconvoluting the cellular target
of this chemical class. As a result, classical erythropoie-
tin receptor-mediated responses were excluded and NaV
channels were identified as the likely targets for the neu-
roprotective action of these compounds.
3,5-Diaryl-4,5-dihydro-1H-pyrazoles 7a–f were then oxi-
dized under Pd-catalysis in acetic acid according to the
procedure described by Nakamichi et al.16 This oxida-
tive aromatization procedure in acidic medium led how-
ever in our hands to a high degree of decomposition for
the molecules bearing an indole moiety. To circumvent
this problem we resorted to a different method for 4,5-
dihydro-1H-pyrazoles 7g, h which were finally success-
fully oxidized using MnO2 in toluene. The regioselective
1
mechanism of this synthetic route was confirmed by H
NMR–NOESY (see Supplementary material). Since we
noted that compounds 7a–h underwent a spontaneous
oxidative aromatization, we planned to obtain the 3,5-
diarylpyrazoles 3a–f directly from compounds 6a–h by
treatment with hydrazine. However, using literature re-
ported conditions for this kind of reaction (ethanol at
room temperature),17 only low yield of the desired prod-
ucts was obtained. These were only deemed acceptable
for commercially available starting a,b-unsaturated
ketones 6a, c, f. Investigating what parameters would
affect the reaction profile, we tried to use acetic acid as
the solvent at refluxing temperature. Although we
2. Results and discussion
2.1. Chemistry
Numerous methods for the synthesis of 1,3,5-substituted
pyrazoles are known.10 One frequently reported method

G. Cocconcelli et al. / Bioorg. Med. Chem. 16 (2008) 2043–2052
Table 1. Chemical structure of final synthesized compounds 2a–h, 3a–f, 8b, d, e
2045
O
CH3
X
CH3
N
H
N
N
N
N
N
X
X
2
8
3
R
R
R
R
X
OCH3
H
Cl
H
NO2
H
H
H
*
*
*
*
*
*
*
*
HN
h
NO2
N
OMe
Cl
H
g
a
b
c
d
e
f
Scheme 1. Reagents and conditions: (a) NaOH, EtOH (6b, d, e, g); (b) MW, 65 W, 65 ꢁC, 4 · 10 min (6h); (c) methylhydrazine sulfate, KOH, EtOH
(7a–d, h); (d) methylhydrazine, EtOH (7e–g); (e) 10% Pd/C, CH3COOH (2a–f); (f) MnO2, toluene (2g, h); (g) hydrazine hydrate, EtOH (3a, c, f); (h)
hydrazine hydrate, CH3COOH (3b, d, e; 8b, d, e).
observed an increase in the yield of the desired pyra-
zoles, this was accompanied by the formation of side
products identified as the N-acetyl-4,5-dihydro-1H-pyr-
azoles 8b, d, e. As we did not observe further oxidation
to the corresponding N-acetylpyrazoles, we supposed
compounds 8b, d, e to be more stable than the corre-
sponding N-methyl and N-H 4,5-dihydro-1H-pyrazoles.
The N-acetyl-4,5-dihydro-1H-pyrazoles proved indeed
to be more stable and compounds 8b, d, e could be
isolated and tested in our biological assays. The struc-
tures of these N-acetyl-4,5-dihydro-1H-pyrazoles were
confirmed by 1H NMR–NOESY (see Supplementary
material). Some of the final compounds are described
in the literature.31–37
2.2. In vitro pharmacology
The neuroprotective properties of compounds 1, 2a–h,
3a–f, 8b, 8d, and 8e were first evaluated at the single dose
of 10 lM (co-treatment, see Sections 4.2.2 in 4.2) in the
NMDA toxicity paradigm in mixed rat cortical neurons.
The results for each compound are presented in Table 2

2046
G. Cocconcelli et al. / Bioorg. Med. Chem. 16 (2008) 2043–2052
Table 2. Results of neuroprotective effect of all compounds (10 lM,
co-treatment) tested in NMDA toxicity assay
Table 3. Results of neuroprotective effects of compounds 2g and 3a
(1 lM and 10 lM co- and pre-treatment) tested in NMDA toxicity
assay
N
Mean
SEM
R
E
N
1 lM
10 lM
Mean
R
E
Co-treatment (10 lM)
1
61.1*
70.4
5.29
7.01
6.49
5.38
5.67
5.67
4.79
3.26
5.05
3.28
3.34
3.41
3.47
3.91
3.21
3.42
3.72
3.28
3.05
4
4
4
4
4
4
4
8
4
8
8
8
8
8
8
8
8
8
8
3
2
3
3
3
3
3
2
3
4
4
4
4
4
4
4
3
4
2
Mean
SEM
SEM
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
3e
3f
8b
8d
8e
9
Pre-treatment
78.3
87.3
2g
3a
90.2
91.1
2.03
2.65
86.0
95.0
2.03
2.60
8
8
3
3
65.6
86.3
Co-treatment
2g
3a
80.3*
81.7*
2.65
2.32
72.0*
77.5*
2.65
2.22
8
8
3
3
85.5
67.1*
76.7*
67.2*
76.0*
66.1*
96.4
Insult: NMDA (60 lM); positive control: MK801 (10 lM). Normal-
ized data (expressed as percentage of LDH released by NMDA) are
displayed as means and SEM of three independent experiments with
eight technical replicates for each sample. R, replicates; E, experiments.
* p < 0.05 Tukey’s honestly significant difference criterion post ANO-
VA test.
89.0
75.6*
81.0*
86.4
Intrigued by the levels of neuroprotection shown by the
molecules synthesized and by the observation that eryth-
ropoietin, in contrast to the behaviour of our
compounds, can act as neuroprotectant agent in
pre-treatment assays,18 we reasoned that the observed
neuroprotection could possibly be related to activity
on targets other than those associated with classical
erythropoietin signalling. Furthermore, the structural
differences between our compounds and starting mole-
cule 1 prompted us to make a further attempt at the def-
inition of putative targets for our molecules through
literature searches based around generic azole scaffolds
with reported neuroprotectant activity. Structurally
related acetyl-4,5 dihydro-1H-pyrrazoles were recently
reported19 to display neuroprotectant activity, however
our attention was focused on structural analogies with
Sodium Channel Site 2 ligands and AMPA receptor
antagonists (i.e., compound 9—Fig. 2), suggesting these
proteins as possible targets for our compounds.20
85.5*
80.3*
Insult: NMDA (60 lM); positive control: MK801 (10 lM). Normal-
ized data (expressed as percentage of LDH released by NMDA) are
displayed as means and SEM of at least two independent experiments
with at least four technical replicates for each sample. R, replicates; E,
experiments.
* p < 0.05 Tukey’s honestly significant difference criterion post ANO-
VA test.
and show the percentage of cell damage due to the toxic
insult (NMDA) in presence of the tested compounds.
The neuroprotective effect is therefore deduced as differ-
ence between the maximum insult (100%) and the cell
damage in presence of test compound. All compounds
tested demonstrated neuroprotective activity (15–40%),
with compounds 3a, c, f; 2g, d and 1 demonstrating the
strongest neuroprotective effects.
We therefore decided to test 3a and 2g, the best perform-
ing amongst the synthesized compounds, in binding as-
says against the above-mentioned receptors.21–23 We
found evidence supporting our hypothesis, with 3a and
2g showing moderate activity in the inhibition in the Site
2 Sodium Channel binding assay at 10 lM as shown in
Table 4. Negligible activity was detected on the AMPA
and Kainate receptors. All compounds synthesized were
then tested on Site 2 Sodium Channel binding assay (Ta-
ble 4).
More detailed experiments were then carried out in or-
der to better profile the neuroprotective activity of
these compounds. In the NMDA toxicity assay in
mixed rat cortical cultures compounds 3a and 2g were
evaluated at the doses of 1 lM and 10 lM in both co-
treatment and pre-treatment paradigms. The results
are shown in Table 3. Compounds 3a and 2g displayed
significant protection from toxicity induced by NMDA
at both 1 lM and 10 lM, when tested in the co-treat-
ment paradigm. In addition, when tested at three con-
centrations (0.1 lM, 1 lM and 10 lM), compounds 3a
and 2g showed activity only at the higher concentra-
tions (i.e., were inactive at 0.1 lM; data not shown),
suggestive of a concentration-dependent neuroprotec-
tive activity. However, when tested in the pre-treat-
ment paradigm, no neuroprotective activity was
evident. This may be due to a number of reasons, for
example, the compounds may elicit a transient cellular
response whose neuroprotective effect is efficaceous
only when delivered simultaneously with the neuro-
toxic insult. For instance, this effect may be produced
as the result of desensitization of a receptor recogniz-
ing the small molecules.
Example 17 from WO99/54314 (compound 9) prepared
according to the patent experimental description was
also tested, and its activity as neuroprotectant was con-
firmed in our NMDA (co-treatment) toxicity assay as
Figure 2. Example 17 from WO99/54314.

G. Cocconcelli et al. / Bioorg. Med. Chem. 16 (2008) 2043–2052
2047
Table 4. Percent inhibition of Veratridine (V) specific binding in the
Na+ channel (site 2) assay as measured by displacement of 10 nM
[3H[batrachotoxinin by synthesized compounds 1, 2a–h, 3a–f, 8b, d, e
and 9
in pure versus mixed cortical cultures suggests a pre-
dominantly neuronal expression of the analyzed NaV
subunits, consistent with published data.24 One of the
excitotoxicity assays of relevance to NaV channels is
the Veratridine assay. Analogues of compound 1, show-
ing significant neuroprotection against NMDA, were
tested in the Veratridine toxicity assay.25 Compounds
3a, c, f and 2g were tested in both pre- and co-treatment
at the doses of 1 lM and 10 lM in both pure neuronal
and mixed cortical cultures. While no protective effects
were evident in the pre-treatment paradigm (data not
shown), the dose of 10 lM was neuroprotective (2g:
40%, 3a: 50%; 3c, f: 70%) respectively, against Veratri-
dine-induced neuronal toxicity in the co-treatment para-
digm for all tested compounds in mixed rat cortical
neurons (Table 5). At the lower dose (1 lM), only com-
pounds 3a, c and f demonstrated a low but significant
(ca. 20%) neuroprotective activity, while no activity
was observed at the concentration of 0.1 lM (data not
shown). These data are suggestive of a concentration-
dependent neuroprotective activity of the compounds.
In pure neuronal cultures, all tested compounds dis-
played significant neuroprotective activity (ca. 40–50%)
at the dose of 10 lM. Significant but limited (20–30%)
neuroprotective effects were observed at 1 lM for com-
pounds 3a and 3c, while compounds 3f and 2g were
inactive. In agreement to the binding data on Side 2 So-
dium Channel, compound 1 showed no significant activ-
ity in the Veratridine paradigm; this compound could be
acting as neuroprotectant agent exploiting another
mechanism. Thus, all compounds tested demonstrated
neuroprotective activity against excitotoxicity induced
by NMDA (15–40%) and in the Veratridine assay (40–
70%). As the observed toxicity (measured as LDH re-
lease) induced by NMDA and Veratridine in the mixed
cultures is comparable (data not shown), these data are
consistent with the compounds acting as NaV blockers,
as their neuroprotective activity increases in a more
N
% Inhibitiona
N
% Inhibitiona
V
100%
Inactive
3a
3b
3c
3d
3e
3f
8b
8d
8e
9
30
27
1
2a
2b
2c
2d
2e
2f
2g
2h
8
16
21
12
13
15
8
14
15
16
27
4
41
14
17
Inactive
a Concentration of positive control (Veratridine): 300 lM: concentra-
tion of test compounds: 10 lM.
shown in Table 2. Surprisingly, compound 9 did not
show activity in the Site 2 Sodium Channel binding as-
say (Table 4). Although these data are supported by
the inactivity on the Veratridine toxicity assay described
after, perhaps indicating that the compounds from our
study are more potent against Site 2 Sodium Channel,
the binding data in Table 4 should be evaluated in a
qualitative way and more experiments will be necessary
to allow for structure–activity-relationship analysis. We
next sought evidence for the expression of NaV genes in
the primary neuronal cultures employed in the neuro-
protection assays. To this end, levels of mRNA of
NaV genes known to be expressed in CNS neurons were
evaluated by real time PCR. At least four NaV a sub-
units (Ia, IIa, IIIa and VIIIa) are known to be expressed
in CNS neurons in the rat.24 In rat primary cortical cul-
tures, Scn1a, 2a, 3a and 8a are all expressed in both pure
cultured neurons and in mixed cortical cultures of neu-
rons and glia, with higher expression levels of Scn2a rel-
atively to other subunits (Fig. 3). The expression profile
Table 5. Neuroprotective effects of compounds 2g, 3a, 3c and 3f in
Veratridine (60 lM) toxicity assay
N
1 lM
10 lM
Mean
R
E
Mean
SEM
SEM
Mixed rat cortical neurons
3a
3c
3f
80.9*
74.1*
77.6*
87.9
5.58
4.82
4.93
3.10
49.2*
24.2*
26.6*
63.2*
6.37
5.85
3.94
2.90
8
8
8
8
3
3
3
3
2g
Pure rat cortical neurons
3a
3c
3f
70.4*
75.0*
80.9
5.81
3.25
9.17
8.12
64.9*
56.5*
51.2*
56.8*
2.87
6.10
9.45
7.41
8
8
8
8
3
3
3
3
2g
82.7
Compounds were tested at the doses of 1 lM and 10 lM in co-treat-
ment paradigm using mixed rat cortical neurons and pure rat cortical
neurons. Insult: Veratridine. Normalized data (expressed as percentage
of LDH or XTT released by Veratridine) are displayed as means and
SEM of three independent experiments with eight technical replicates
for each sample. R, replicates; E, experiments.
Figure 3. Messenger RNA expression levels of Scn1a, 2a, 3a and 8a in
pure and mixed rat cortical cultures. Data are normalized relatively to
b-actin levels, and are expressed as SEM SD. A representative
experiment is shown.
* p < 0.05 Tukey’s honestly significant difference criterion post ANO-
VA test.

2048
G. Cocconcelli et al. / Bioorg. Med. Chem. 16 (2008) 2043–2052
direct assay of NaV activation-mediated neurotoxicity.
In particular, analogues 3a, c, f and 2g showed the most
significant neuroprotective effects in our toxicity assay.
These data, together with the results demonstrating
moderate binding of our class of compounds to the
NaV channel, point to the inhibition of NaV channel
as an important component of the neuroprotective
properties of these compounds, consistent with the
known neuroprotective properties of published inhibi-
tors of voltage-gated sodium channels.26,27
4.1.1.3. 3-(3-Chlorophenyl)-1-phenylpropenone (6d).
The product was used without further purification in
the next reaction. Yield: 89%; mp 74–75 ꢁC (lit. 73–
74 ꢁC28). 1H NMR (CDCl3) d 7.32–7.40 (m, 2H) d
7.48–7.63 (m, 6H) d 7.73 (d, J = 15.6 Hz, 1H) d 8.01
(d, 2H).
4.1.1.4. 1-(4-Nitrophenyl)-3-phenylpropenone (6e). The
product was purified by crystallization from ethanol.
Yield: 82%; mp 115–116 ꢁC (lit. 146–148 ꢁC29). 1H
NMR (DMSO-d6) d 7.47–7.49 (m, 3H) d 7.77–7.98 (m,
4H) d 8.33–8.40 (m, 4H).
3. Conclusion
4.1.1.5. 3-(1H-Indol-5-yl)-1-phenylpropenone (6g).
The product was purified by chromatography using
CH2Cl2/EtOAc (20:1) as eluant. Yield: 80%; mp 168–
In summary, we have identified novel neuroprotective
small molecules active against excitotoxic insults, and
have identified the inhibition of NaV channels as a likely
site of action of their neuroprotective activity.
1
170 ꢁC. H NMR (DMSO-d6) d 6.52 (br s, 1H) d 7.42
(t, 1H) d 7.46 (d, 1H) d 7.57 (t, 2H) d 7.63–7.71 (m,
2H) d 7.82 (d, J = 15.6 Hz, 1H) d 7.89 (d, J = 15.0 Hz,
1H) d 8.06 (s, 1H) d 8.15 (d, 2H) d 11.36 (s, 1H).
4. Experimental
4.1. Chemistry
4.1.1.6. Synthesis of 3-(1H-indol-3-yl)-1-phenylprope-
none (6h). A mixture of 0.15 mol of ketone 5, 0.10 mol of
aldehyde 6 and 10 g Al2O3 [activated, acid (Brockman
I)] was irradiated in the microwave reactor (65 W,
65 ꢁC, 4 · 10 min). The mixture was then extracted with
CH3OH and the solvent was evaporated under reduced
pressure. The product was purified by chromatography
using CH2Cl2/CH3OH (10:1) as eluant. Yield: 76%; mp
Melting points are not corrected and were determined
with a Gallenkamp melting point apparatus. Purity of
final compounds 2a–f, 2h, 3a–c, 3f and 8b, d was deter-
mined by ThermoQuest (Italia) FlashEA 1112 Elemen-
tal Analyzer, for C, H, and N. The percentages found
were within 0.4% of the theoretical values. Character-
ization of final compounds 2g, 3e and 8e was determined
by HR-MS using a Thermo LTQ Orbitrap Mass Spec-
1
168–169 ꢁC (lit. 166–167 ꢁC30). H NMR (DMSO-d6) d
7.20–7.27 (m, 2H) d 7.48–7.67 (m, 5H) d 8.03–8.12 (m,
5H) d 11.91 (s, 1H).
1
trometer. H NMR spectra were recorded on a Bruker
Avance 300 Spectrometer (300 MHz); chemical shifts
4.1.2. Preparation of 3,5-diaryl-1-methyl-4,5-dihydro-1H-
pyrazoles (7a–h)
(d scale) are reported in parts per million (ppm) relative
to the central peak of the solvent. H NMR spectra are
1
4.1.2.1. General synthesis of compounds 7a–d, h. A
solution of 10.0 mmol of 1,3-diarylpropenones 6a–d, h,
37.5 mmol (5.4 g) of methylhydrazine sulfate and
94.5 mmol (5.3 g) of KOH in 100 mL of EtOH was re-
fluxed for 3 h under a nitrogen atmosphere. The solvent
was then evaporated under reduced pressure and the res-
idue was diluted with H2O and extracted with Et2O. The
organic phases were combined, dried over Na2SO4 and
evaporated under reduced pressure.
reported in the following order: multiplicity, approxi-
mate coupling constants (J value) in Hertz (Hz) and
number of protons. Reactions were monitored by
TLC, on Kieselgel 60 F 254 (DC-Alufolien, Merck).
Final compounds and intermediates were purified by
chromatography on preparative Gilson MPLC, using a
SiO2 column (LiChroprep, Si 60, 25-40 lm, Merck).
Microwave reactions (MW) were conducted using a
CEM Discover Synthesis Unit (CEM Corp., Matthews,
NC). Abbreviations for solvents are the following:
EtOAc: ethyl acetate, DMSO: dimethylsulfoxide.
4.1.2.2. 3-(4-Methoxyphenyl)-1-methyl-5-phenyl-4,5-
dihydro-1H-pyrazole (7a). The product was purified by
chromatography using CH2Cl2/EtOAc (20:1) as eluant
4.1.1. Preparation of 1,3-diarylpropenones (6a–h)
4.1.1.1. General synthesis of compounds 6b, d, e, g. To
a solution of 0.10 mol of ketone 4 and 0.10 mol of alde-
hyde 5, in a minimum amount of ethanol, 5 mL NaOH
(40% w/v) was added dropwise. The mixture was stirred
for 2 h, then the residue was filtered and washed with
cold EtOH, H2O and cold EtOH again.
1
obtaining an air-sensitive solid. Yield: 69%. H NMR
(CDCl3) d 2.81 (s, 3H) d 2.92–3.16 (m, 1H) d 3.43–
3.60 (m, 1H) d 3.83 (s, 3H) d 4.03–4.29 (m, 1H) d 6.90
(d, J = 8.7 Hz, 2H) d 7.32–7.48 (m, 5H) d 7.61 (d,
J = 8.7 Hz, 2H).
4.1.2.3. 5-(3-Methoxyphenyl)-1-methyl-3-phenyl-4,5-
dihydro-1H-pyrazole (7b). The product was purified by
chromatography using CH2Cl2/EtOAc (20:1) as eluant
4.1.1.2. 3-(3-Methoxyphenyl)-1-phenylpropenone (6b).
The product was purified by chromatography using
CH2Cl2 as eluant. Yield: 67%; mp 56–57 ꢁC (lit. 59–
1
obtaining an oil. Yield: 61%. H NMR (CDCl3) d 2.84
1
61 ꢁC28). H NMR (DMSO-d6) d 3.82 (s, 3H), d 7.03
(s, 3H) d 3.01–3.11 (m, 1H) d 3.52 (dd, 1H) d 3.82 (s,
3H) d 4.10–4.32 (m, 1H) d 6.88 (d, 1H) d 7.00–7.04
(m, 2H) d 7.29–7.41 (m, 4H) d 7.67 (d, 2H).
(d, 1H), d 7.34–7.75 (m, 7H) d 7.95 (d, J = 15.7 Hz,
1H) d 8.16 (d, 2H).

G. Cocconcelli et al. / Bioorg. Med. Chem. 16 (2008) 2043–2052
2049
4.1.2.4. 3-(4-Chlorophenyl)-1-methyl-5-phenyl-4,5-di-
hydro-1H-pyrazole (7c). The product was purified by
chromatography using CH2Cl2/EtOAc (20:1) as eluant
ture was then filtered on Hyflo Super Celꢂ medium,
carefully basified with an aqueous solution of NaOH
and extracted with EtOAc. The organic phases were col-
lected and evaporated under reduced pressure.
1
obtaining an oil. Yield: 69%. H NMR (CDCl3) d 2.86
(s, 3H) d 3.00 (dd, J = 16.1, 14.4 Hz, 1H) d 3.47 (dd,
J = 16.1, 10.0 Hz, 1H) d 4.18 (dd, J = 14.3, 10.1 Hz,
1H) d 7.33–7.45 (m, 5H) d 7.49 (d, 2H) d 7.60 (d, 2H).
4.1.3.2. 3-(4-Methoxyphenyl)-1-methyl-5-phenyl-1H-
pyrazole (2a). The product was purified by chromatogra-
phy using CH2Cl2/EtOAc (10:1) as eluant. Yield: 35%;
1
4.1.2.5. 5-(3-Chlorophenyl)-1-methyl-3-phenyl-4,5-di-
hydro-1H-pyrazole (7d). The product was purified by
chromatography using CH2Cl2/EtOAc (20:1) as eluant
mp 90–91 ꢁC (lit. 92–93 ꢁC31). H NMR (DMSO-d6) d
3.78 (s, 3H) d 3.87 (s, 3H) d 6.79 (s, 1H) d 6.79 (d,
J = 9.0 Hz, 2H) d 7.43–7.60 (m, 5H) d 7.75 (d,
J = 8.7 Hz, 2H). MS (ES+) 265 [M+1]+. Anal. Calcd
for C17H16N2O (264.32). C, 77.24; H, 6.10; N, 10.60.
Found: C, 77.19; H, 6.04; N, 10.30.
1
obtaining an oil. Yield: 67%. H NMR (CDCl3) d 2.86
(s, 3H) d 3.02 (dd, J = 16.1, 14.1 Hz, 1H) d 3.54 (dd,
J = 16.2, 10.0 Hz, 1H) d 4.17 (dd, J = 13.9, 10.5 Hz,
1H) d 7.32–7.43 (m, 6H) d 7.51 (s, 1H) d 7.67 (dd, 2H).
4.1.3.3. 5-(3-Methoxyphenyl)-1-methyl-3-phenyl-1H-
pyrazole (2b). The product was purified by chromatogra-
phy using CH2Cl2/EtOAc (10:1) as eluant obtaining an
4.1.2.6. 3-(2-Methyl-5-phenyl-3,4-dihydro-2H-pyrazol-
3-yl)-1H-indole (7h). The product was purified by chro-
matography using CH2Cl2/EtOAc (20:1) as eluant
1
oil. Yield: 52%. H NMR (DMSO-d6) d 3.83 (s, 3H) d
1
obtaining an oil. Yield: 50%. H NMR (CDCl3) d 2.90
3.90 (s, 3H) d 6.90 (s, 1H) d 7.03 (d, 1H) d 7.12–7.17
(m, 2H) d 7.30 (t, 1H) d 7.38–7.46, (m, 3H) d 7.83, (d,
2H). MS (ES+) 265 [M+1]+. Anal. Calcd for
C17H16N2O (264.32). C, 77.24; H, 6.10; N, 10.60.
Found: C, 76.97; H, 6.01; N, 10.55.
(s, 3H) d 3.25 (dd, J = 13.5, 16.2 Hz, 1H) d 3.53 (dd,
J = 10.2, 16.2 Hz, 1H) d 4.52–4.58 (m, 1H) d 7.10–7.23
(m, 2H) d 7.30–7.43 (m, 5H) d 7.70 (d, 3H) d 8.24 (s,
1H).
4.1.2.7. General synthesis of compounds 7e–g. A solu-
tion of 10.0 mmol of the 1,3-diarylpropenones 6e–g and
37.5 mmol (1.73 g) of methylhydrazine in 100 mL of eth-
anol was refluxed for 3 h under nitrogen. The solvent
was then evaporated under reduced pressure.
4.1.3.4. 3-(4-Chlorophenyl)-1-methyl-5-phenyl-1H-
pyrazole (2c). The product was purified by chromatogra-
phy using CH2Cl2/EtOAc (20:1) as eluant. Yield: 56%;
1
mp 127–129 ꢁC (lit. 127–128 ꢁC31). H NMR (DMSO-
d6) d 3.89 (s, 3H) d 6.92 (s, 1H) d 7.45–7.60 (m, 7H) d
7.85 (d, 2H). MS (ES+) 269 [M+1]+. Anal. Calcd for
C16H13ClN2 (268.74). C, 71.51; H, 4.87; N, 10.42.
Found: C, 71.34; H, 4.76; N, 10.27.
4.1.2.8. 1-Methyl-3-(4-nitrophenyl)-5-phenyl-4,5-dihy-
dro-1H-pyrazole (7e). The product was purified by crys-
tallization from ethanol. Yield: 56%; mp 115–116 ꢁC. 1H
NMR (DMSO-d6) d 2.79 (s, 3H) d 2.97 (dd, J = 14.4,
16.5 Hz, 1H) d 3.63 (dd, J = 10.5, 16.5 Hz, 1H) d 4.35
(dd, J = 10.5, 14.1 Hz, 1H) d 7.31–7.48 (m, 5H) d 7.83
(d, J = 8.7 Hz, 2H) d 8.23 (d, J = 8.9 Hz, 2H).
4.1.3.5. 5-(3-Chlorophenyl)-1-methyl-3-phenyl-1H-
pyrazole (2d). The product was purified by chromatogra-
phy using CH2Cl2/EtOAc (20:1) as eluant obtaining an
1
oil. Yield: 55%. H NMR (DMSO-d6) d 3.91 (s, 3H) d
6.98 (s, 1H) d 7.32 (d, 1H) d 7.39–7.44 (m, 2H) d 7.53–
7.59 (m, 3H) d 7.69 (s, 1H) d 7.83 (d, 2H). MS (ES+)
269 [M+1]+. Anal. Calcd for C16H13ClN2 (268.74). C,
71.51; H, 4.87; N, 10.42. Found: C, 71.80; H, 4.98; N,
9.98.
4.1.2.9. 1-Methyl-5-(3-nitrophenyl)-3-phenyl-4,5-dihy-
dro-1H-pyrazole (7f). The product was purified by chro-
matography using CH2Cl2/EtOAc (20:1) as eluant
1
obtaining an oil. Yield: 73%. H NMR (CDCl3) d 2.88
(s, 3H) d 3.02 (dd, J = 16.1, 14.1 Hz, 1H) d 3.61 (dd,
J = 16.2, 10.2 Hz, 1H) d 4.29 (dd, J = 14.0, 10.2 Hz,
1H) d 7.40 (d, 3H) d 7.60 (t, J = 7.9 Hz, 1H) d 7.67
(dd, J = 8.0, 1.9 Hz, 2H) d 7.88 (d, J = 7.8 Hz, 1H) d
8.22 (d, J = 8.18 Hz, 1H) 8.37 (s, 1H).
4.1.3.6. 1-Methyl-3-(4-nitrophenyl)-5-phenyl-1H-pyra-
zole (2e). The product was purified by chromatography
using CH2Cl2/EtOAc (40:1) as eluant. Yield: 43%; mp
1
138–139 ꢁC (lit. 141–142 ꢁC31). H NMR (DMSO-d6) d
3.94 (s, 3H) d 7.12 (s, 1H) d 7.46–7.63 (m, 5H) d 8.10
(d, J = 8.1 Hz, 2H) d 8.28 (d, J = 8.7 Hz, 2H). MS
(ES+) 280 [M+1]+. Anal. Calcd for C16H13N3O2
(279.29). C, 68.80; H, 4.69; N, 15.05. Found: C, 68.63;
H, 4.59; N, 15.03.
4.1.2.10. 5-(2-Methyl-5-phenyl-3,4-dihydro-2H-pyra-
zol-3-yl)-1H-indole (7g). The product was purified by
chromatography using CH2Cl2/EtOAc (20:1) as eluant
obtaining a air-sensitive solid. Yield: 50%. 1H NMR
(DMSO-d6) d 2.70 (s, 3H) d 2.89–299 (m, 1H) d 3.53
(dd, 1H) d 4.18 (dd, 1H) d 6.41 (br s, 1H) d 7.21 (dd,
1H) d 7.34–7.41 (m, 5H) d 7.60–7.66 (m, 3H) d 11.11
(s, 1H).
4.1.3.7. 1-Methyl-5-(3-nitrophenyl)-3-phenyl-1H-pyra-
zole (2f). The product was purified by chromatography
using CH2Cl2/EtOAc (20:1) as eluant. Yield: 33%; mp
125–127 ꢁC (lit. 98–95 ꢁC32). 1H NMR (DMSO-d6) d
3.96 (s, 3H) d 7.10 (s, 1H) d 7.32 (t, 1H) d 7.43 (t, 2H)
d 7.80–7.87 (m, 3H) d 8.09 (d, 1H) d 8.31 (d, 1H) d
8.40 (s, 1H). MS (ES+) 280 [M+1]+. Anal. Calcd for
C16H13N3O2 (279.29). C, 68.80; H, 4.69; N, 15.05.
Found: C, 68.54; H, 4.68; N, 14.80.
4.1.3. Preparation of 3,5-diaryl-1-methylpyrazoles
4.1.3.1. General synthesis of compounds 2a–f. A solu-
tion of 10.0 mmol of 3,5-diaryl-1-methyl-4,5-dihydro-
1H-pyrazole (7a–f) and 0.5 g 10% Pd/C in 45 mL of
CH3COOH was heated under stirring for 6 h. The mix-

2050
G. Cocconcelli et al. / Bioorg. Med. Chem. 16 (2008) 2043–2052
4.1.3.8. General synthesis of compounds 2g, h. A solu-
[M+1]+. Anal. Calcd for C15H11N3O2 (265.27). C,
67.92; H, 4.18; N, 15.84. Found: C, 67.71; H, 4.11; N,
15.24.
tion of 1.0 mmol of 3,5-diaryl-1-methyl-4,5-dihydro-1H-
pyrazole (7g, h) and 13.0 mmol (1.13 g) of MnO2 in
45 mL of toluene was refluxed for 2 h. The mixture
was then filtered on Hyflo Super Celꢂ medium and the
solvent was evaporated under reduced pressure.
4.1.4.5. General synthesis of compounds 3b, d, e; 8b, d,
e. A solution of 10.0 mmol of 1,3-diarylpropenone (6b,
d, e) and 20.0 mmol (1.0 g) of hydrazine hydrate in
150 mL of CH3COOH was refluxed for 6 h. The mixture
was carefully basified with an aqueous solution of
NaOH and extracted with EtOAc. The organic phases
were collected and evaporated under reduced pressure.
4.1.3.9. 5-(2-Methyl-5-phenyl-2H-pyrazol-3-yl)-1H-in-
dole (2g). The product was purified by chromatography
using CH2Cl2/EtOAc (20:1) as eluant. Yield: 36%; mp
161–162 ꢁC. 1H NMR (DMSO-d6) d 3.89 (s, 3H) d
6.52 (br s, 1H) d 6.81 (s, 1H) d 7.25–7.32 (m, 2H) d
7.38–7.46 (m, 3H) d 7.52 (d, 1H) d 7.74 (s, 1H) d 7.84
(d, 2H) d 11.31 (s, 1H). HR-MS m/z 274.13363
[M+1]+. Calcd mass for C18H16N3 273.13387 (deviation
0.9 ppm).
4.1.4.6. 5-(3-Methoxyphenyl)-3-phenyl-1H-pyrazole
(3b). The product was purified by chromatography using
CH2Cl2/EtOAc (5:1) as eluant. Yield: 20%; mp 100–
1
102 ꢁC. H NMR (DMSO-d6) d 3.82 (s, 3H) d 6.91 (s,
1H) d 7.22 (s, 1H) d 7.31–7.47 (m, 6H) d 7.80–7.89 (m,
2H) d 13.38 (s, 1H). MS (ES+) 251 [M+1]+. Anal. Calcd
for C16H14N2O (250.29). C, 76.78; H, 5.64; N, 11.19.
Found: C, 76.49; H, 5.52; N, 11.40.
4.1.3.10. 3-(2-Methyl-5-phenyl-2H-pyrazol-3-yl)-1H-
indole (2h). The product was purified by chromatogra-
phy using CH2Cl2/EtOAc (10:1) as eluant and then
crystallized from ethanol/water. Yield: 41%; mp 206–
1
207 ꢁC. H NMR (DMSO-d6) d 3.93 (s, 3H) d 6.91 (s,
4.1.4.7. 1-[5-(3-Methoxyphenyl)-3-phenyl-4,5-dihydro-
pyrazol-1-yl]-ethanone (8b). The product was purified by
chromatography using CH2Cl2/EtOAc (5:1) as eluant.
Yield: 40%; mp 112–113 ꢁC (lit. 115–117 ꢁC36). 1H
NMR (DMSO-d6) d 2.32 (s, 3H) d 3.12 (dd, J = 4.8,
18.0 Hz, 1H) d 3.72 (s, 3H) d 3.82 (dd, J = 12.0,
18.0 Hz, 1H) d 5.52 (dd, J = 5.1, 12.0 Hz, 1H) d 6.72–
6.75 (m, 2H) d 6.80–6.84 (m, 1H) d 7.24 (t, 1H) d
7.44–7.46 (m, 3H) d 7.76–7.79 (m, 2H). MS (ES+) 295
[M+1]+. Anal. Calcd for C18H18N2O2 (294.35). C,
73.45; H, 6.16; N, 9.52. Found: C, 73.21; H, 6.14; N,
9.40.
1H) d 7.10–7.23 (m, 2H) d 7.29 (t, 1H) d 7.41 (t, 2H) d
7.49 (d, 1H) d 7.69–7.73 (m, 2H) d 7.88 (d, 2H) d
11.58 (s, 1H). MS (ES+) 274 [M+1]+. Anal. Calcd for
C18H15N3 (273.33). C, 79.10; H, 5.53; N, 15.37. Found:
C, 78.97; H, 5.36; N, 15.26.
4.1.4. Preparation of 3,5-diarylpyrazoles (3a–f) and 1-
(3,5-diaryl)-4,5-dihydro-pyrazol-1-yl-ethanones (8b, d, e)
4.1.4.1. General synthesis of compounds 3a, c, f. A
solution of 10.0 mmol of 1,3-diaryl-propenone (6a, c, f)
and 20.0 mmol (1.0 g) of hydrazine hydrate in 100 mL
of ethanol was stirred for 48 h at room temperature.
The solvent was then evaporated under reduced pressure.
4.1.4.8. 5-(3-Chlorophenyl)-3-phenyl-1H-pyrazole (3d).
The product was purified by chromatography using
CH2Cl2/EtOAc (10:1) as eluant. Yield: 12%; mp 177–
178 ꢁC (lit. 168–169 ꢁC34). 1H NMR (DMSO-d6) d
7.30–7.51 (m, 6H) d 7.79–7.91 (m, 4H) d 13.47 (d, 1H).
MS (ES+) 255 [M+1]+. Anal. Calcd for C15H11ClN2
(254.71). C, 70.73; H, 4.35; N, 11.00. Found: C, 70.45;
H, 4.29; N, 11.26.
4.1.4.2. 3-(4-Methoxyphenyl)-5-phenyl-1H-pyrazole
(3a). The product was purified by chromatography using
CH2Cl2/EtOAc (20:1) as eluant and then crystallized
from CH2Cl2/petroleum ether. Yield: 8%; mp 158–
1
159 ꢁC (lit. 159–160 ꢁC33). H NMR (DMSO-d6) d 3.79
(s, 3H) d 6.96–7.07 (m, 2H) d 7.08 (s, 1H) d 7.26–7.52
(m, 3H) d 7.72–7.92 (m, 4H) d 13.20 (s, 1H). MS
(ES+) 251 [M+1]+. Anal. Calcd for C16H14N2O
(250.29). C, 76.78; H, 5.64; N, 11.19. Found: C, 76.53;
H, 5.50; N, 11.08.
4.1.4.9. 1-[5-(3-Chlorophenyl)-3-phenyl-4,5-dihydropy-
razol-1-yl]-ethanone (8d). The product was purified by
chromatography using CH2Cl2/EtOAc (10:1) as eluant.
Yield: 46%; mp 124–125 ꢁC. 1H NMR (DMSO-d6) d
2.31 (s, 3H) d 3.18 (dd, J = 4.5, 18.3 Hz, 1H) d 3.85
4.1.4.3. 3-(4-Chlorophenyl)-5-phenyl-1H-pyrazole (3c).
The product was purified by chromatography using
CH2Cl2/EtOAc (20:1) as eluant and then crystallized
from CH2Cl2/petroleum ether. Yield: 12%; mp 216–
(dd, J = 12.3, 18.0 Hz, 1H)
d 5.55 (dd, J = 5.1,
12.3 Hz, 1H) d 7.15 (d, 1H) d 7.26 (s, 1H) d 7.30–7.39
(m, 2H) d 7.46–7.48 (m, 3H) d 7.77–7.80 (m, 2H). MS
(ES+) 299 [M+1]+. Anal. Calcd for C17H15ClN2O
(298.77). C, 68.34; H, 5.06; N, 9.38. Found: C, 67.95;
H, 4.98; N, 9.25.
1
217 ꢁC (lit. 216–217 ꢁC34). H NMR (DMSO-d6) d 7.23
(s, 1H) d 7.31–7.57 (m, 5H) d 7.79–7.90 (m, 4H) d
13.44 (s, 1H). MS (ES+) 255 [M+1]+. Anal. Calcd for
C15H11ClN2 (254.71). C, 70.73; H, 4.35; N, 11.00.
Found: C, 70.60; H, 4.27; N, 11.10.
4.1.4.10. 3-(4-Nitrophenyl)-5-phenyl-1H-pyrazole (3e).
The product was purified by chromatography using
CH2Cl2/CH3OH (5:1) as eluant. Yield: 8%; mp 270 ꢁC
4.1.4.4. 3-3-Nitrophenyl)-5-phenyl-1H-pyrazole (3f).
The product was purified by chromatography using
CH2Cl2/EtOAc (5:1) as eluant. Yield: 9%; mp 198–
199 ꢁC (lit. 204–205 ꢁC35) 1H NMR (DMSO-d6) d
7.36–7.52 (m, 4H) d 7.71–7.91 (m, 3H) d 8.16–8.33 (m,
2H) d 8.68 (d, 1H) d 13.65 (d, 1H). MS (ES+) 266
1
dec. (lit. 278–279 ꢁC37). H NMR (DMSO-d6) d 7.39–
7.53 (m, 4H) d 7.81–7.90 (m, 2H) d 8.07–8.16 (m, 2H)
d 8.30–8.38 (m, 2H) d 13.73 (s, 1H). HR-MS m/z
266.09259 [M+1]+. Calcd mass for C15H12N3O2
266.09240 (deviation 0.7 ppm).

G. Cocconcelli et al. / Bioorg. Med. Chem. 16 (2008) 2043–2052
2051
4.1.4.11. 1-[3-(4-Nitrophenyl)-5-phenyl-4,5-dihydro-
pyrazol-1-yl]-ethanone (8e). The product was purified by
chromatography using CH2Cl2/CH3OH (5:1) as eluant.
Yield: 44%; mp 194–195 ꢁC. 1H NMR (DMSO-d6) d
2.34 (s, 3H) d 3.21 (dd, J = 5.4, 18.6 Hz, 1H) d 3.93 (dd,
J = 11.4, 17.4 Hz, 1H) d 5.60 (dd, J = 4.8, 12.0 Hz, 1H)
d 7.19–7.36 (m, 5H) d 8.03 (d, J = 9.6 Hz, 2H) d 8.30 (d,
J = 9.0 Hz, 2H). HR-MS m/z 310.11900 [M+1]+. Calcd
mass for C17H16N3O3 310.11862 (deviation 1.2 ppm).
Data have been background-subtracted (na¨ıve cells) and
expressed as percentages of LDH or XTT release by
NMDA or Veratridine (respectively) for each indepen-
dent experiment and have been analyzed with a blocked
ANOVA model (each experiment was at least replicated
twice) followed by Tukey’s honestly significant difference
criterion for identifying compounds significantly differ-
ent from NMDA (or Veratridine) toxicity (p < 0.05).
4.2.3. RT-PCR. Total RNA was extracted as described
previously.45 Total RNA was subjected to DnaseI treat-
ment (Roche, Hertforshire, UK) and 2 lg of total RNA/
sample was used for cDNA synthesis using Superscript
II (Invitrogen, San Diego, CA) and an oligo(dT) primer.
Each reverse transcriptase (RT) product was diluted to
100 lL with sterile, distilled water, and 1 lL of cDNA
was used in the subsequent PCR amplification. Real-
time PCR was performed using an I-Cycler and the iQ
SYBR Green Supermix (Bio-Rad, Hercules, CA)
according to manufacturer’s instructions. Primer se-
quences were designed relevant (neuronal expressed) to
rat NaV coding sequences and to the rat b-actin gene.
Primer sequences were as follows: Scn1a-FOR 50-GAG
GAACAGCCTGTCATGGAA-30; Scn1a-REV 50-CTG
CACACAGCCTTCAGTGAA-30; Scn2a1-FOR 50-TC
TTGCTGATGCCGCTGTAT-30; Scn2a1-REV 50-CG
ACGTATGGCTTCCCTGTT-30; Scn3a-FOR 50-CCC
GGGACCTGAGAGCTT-30; Scn3a-REV 50-GCAGC
ACGCTTTTCGATAGC-30; Scn8a1-FOR 50-TGCA
CCGTTCACTGTATTTGG-30; Scn8a1-REV 50-AGG
TGTACGAAAGAATCCTACACTGA-30; b-actin-FOR
4.2. Biology
4.2.1. Primary neuronal cultures. Pure neuronal and
mixed primary cortical cultures were prepared from
E16 embryos Sprague–Dawley rat neocortex by
mechanical dissociation38 and were grown in Neuroba-
sal medium supplemented with B27 (Invitrogen—pure
cortical neurons), or with Neurobasal medium supple-
mented with 5% of FBS (Fetal Bovine Serum—Hy-
clone-mixed
cortical
cultures).
Cultures
were
maintained in a humidified incubator with an atmo-
sphere of 95% air/ 5% CO2 at 37 ꢁC and the culture med-
ium was replaced every 3–4 days. All primary neuronal
cultures were grown in 24-well plates (500 lL medium/
well) and used after 12 days in vitro.
4.2.2. In vitro neuroprotection assay. Two different
in vitro models of excitotoxicity, N-methyl-D-aspartate
and Veratridine, were implemented in mixed and pure
cortical neurons, respectively. Primary neuronal cultures
from rodents challenged with different neurotoxic agents
(e.g., NMDA or other excitotoxins, Abpeptide) repre-
sent well-validated and characterized in vitro models
of neurodegeneration.39–42 They were therefore em-
ployed to analyze the capacity of compounds to protect
neurons from neuronal cell death induced by excitotox-
icity mediated by overactivation of NMDA receptors or
from activation of Sodium Channels. N-Methyl-D-
aspartate (NMDA) (Sigma) was added to culture med-
ium in presence or absence of 10 lM dizolcipine maleate
(MK-801) (Sigma). MK801 is a non-selective NMDA
channel blocker, and represents the positive control
for activity in the neuroprotection assay as it can reverse
the neuronal cell death induced by NMDA. Experimen-
tal compounds were added to culture medium 24 h (pre-
treatment) or 20 min (co-treatment) before addition of
either NMDA or Veratridine. In both experimental par-
adigms, neuronal viability was assessed 24 h after the
excitotoxic insult. Neuronal cell death was determined
according to manufacturer’s instructions using a Cyto-
toxicity Detection Kit (LDH) (Roche Applied Science,
IN, USA) or a Cell Proliferation kit II (XTT) (Roche
Applied Science, IN, USA) in mixed cortical cultures
or pure neuronal cultures, respectively. The XTT assay
measures mitochondrial activity, which is inversely pro-
portional to cell death. The LDH assay measures the
presence in the medium of an intracellular enzyme (lac-
tate dehydrogenase) whose release outside the cell is in-
versely proportional to cell membrane integrity, and is
therefore directly proportional to neuronal cell death.
This assay is typically preferred in mixed neuronal
cultures (comprising both a neuronal and a glial
component).43,44
50-CCCTGGCTCCTAGCACCAT-30;
50-GAGCCACCAATCCACACAGA-30.
b-actin-REV
Reaction
conditions were as follows: 95 ꢁC/50; 35 · (95 ꢁC/3000;
55 ꢁC/3000). Data were analyzed with the I-Cycler Opti-
cal System software version 3.0a. Results are repre-
sented as mean normalized expression levels SEM.
4.2.4. In vitro pharmacology. AMPA, Kainate and Site 2
Sodium Channel binding assays were performed by Cer-
ep-France (www.cerep.com).
Acknowledgments
Financial support from Siena Biotech S.p.A. is grate-
fully acknowledged. We thank Dr. Graeme M. Robert-
son. We are grateful to Dr. Stefano Gotta for HR-MS
`
analysis. We are also grateful to the Centro Interfacolta
Misure of the University of Parma for providing the
NMR instrumentation.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2007.10.090.
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