Design and microwave-assisted synthesis of 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles: Novel agents with analgesic and anti-inflammatory properties

Article abstract of DOI:10.1016/j.ejmech.2007.07.018

In this work, we reported the synthesis and evaluation of the analgesic and anti-inflammatory properties of novel 3- or 4-substituted 5-trifluoromethyl-5-hydroxy-4,5-dihydro-1H-1-carboxyamidepyrazoles (where 3-/4-substituent = H/H, Me/H, Et/H, Pr/H, i-Pr/H, Bu/H, t-Bu/H, Ph/H, 4-Br-Ph/H and H/Me) designed in the exploration of the bioisosteric replacement of benzene present in salicylamide with a 5-trifluoromethyl-4,5-dihydro-1H-pyrazole scaffold. Target compounds were synthesized from the cyclocondensation of 4-alkoxy-1,1,1-trifluoromethyl-3-alken-2-ones with semicarbazide hydrochloride through a rapid one-pot reaction via microwave irradiation. In addition to spectroscopic data, the structure of the compounds was supported by X-ray diffraction. Subcutaneous administration of the 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles decreased pain-related behavior during neurogenic and inflammatory phases of the formalin test in mice. Moreover, the more active analgesic compounds (3-/4- = Et/H and H/Me) significantly decreased carrageenan-induced paw edema in mice. The data obtained in this work suggest that the synthesized compounds could be promising candidates for the future development of novel analgesic and anti-inflammatory agents.

Full text of DOI:10.1016/j.ejmech.2007.07.018

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European Journal of Medicinal Chemistry 43 (2008) 1237e1247
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Original article
Design and microwave-assisted synthesis of
5-trifluoromethyl-4,5-dihydro-1H-pyrazoles: Novel
agents with analgesic and anti-inflammatory properties
Patricia D. Sauzem b, Pablo Machado a, Maribel A. Rubin b,
Gabriela da S. Sant’Anna b, Henrique B. Faber b, Alessandra H. de Souza b,
Carlos F. Mello c, Paulo Beck a, Robert A. Burrow a, Helio G. Bonacorso a,
Nilo Zanatta a, Marcos A.P. Martins a,
*
a Nu´cleo de Qu´ımica de Heterociclos (NUQUIMHE), Departamento de Qu´ımica, Centro de Cieˆncias Naturais e Exatas,
Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil
b Laborato´rio de Neurotoxicidade e Psicofarmacologia, Departamento de Qu´ımica, Centro de Cieˆncias Naturais e Exatas,
Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil
c Departamento de Fisiologia e Farmacologia, Centro de Cieˆncias da Sau´de,
Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil
Received 23 March 2007; received in revised form 12 July 2007; accepted 20 July 2007
Available online 15 August 2007
Abstract
In this work, we reported the synthesis and evaluation of the analgesic and anti-inflammatory properties of novel 3- or 4-substituted 5-
trifluoromethyl-5-hydroxy-4,5-dihydro-1H-1-carboxyamidepyrazoles (where 3-/4-substituent ¼ H/H, Me/H, Et/H, Pr/H, i-Pr/H, Bu/H, t-Bu/H,
Ph/H, 4-Br-Ph/H and H/Me) designed in the exploration of the bioisosteric replacement of benzene present in salicylamide with a 5-trifluoro-
methyl-4,5-dihydro-1H-pyrazole scaffold. Target compounds were synthesized from the cyclocondensation of 4-alkoxy-1,1,1-trifluoromethyl-3-
alken-2-ones with semicarbazide hydrochloride through a rapid one-pot reaction via microwave irradiation. In addition to spectroscopic data, the
structure of the compounds was supported by X-ray diffraction. Subcutaneous administration of the 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles
decreased pain-related behavior during neurogenic and inflammatory phases of the formalin test in mice. Moreover, the more active analgesic
compounds (3-/4- ¼ Et/H and H/Me) significantly decreased carrageenan-induced paw edema in mice. The data obtained in this work suggest
that the synthesized compounds could be promising candidates for the future development of novel analgesic and anti-inflammatory agents.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Pyrazoles; Microwave-assisted synthesis; Analgesic; Anti-inflammatory; Pain
1. Introduction
Pyrazoles and their derivatives are widely known for their
excellent effectiveness as analgesics and antipyretics, dipyrone
being a well documented and commercially available example
[1]. This compound is a nonsteroidal anti-inflammatory drug
(NSAID) used with success in the treatment of fever and
pain showing a relatively weak anti-inflammatory effect
[1,2]. Other NSAIDs have been used in the treatment of
chronic arthropathies, as well as for fever and pain. However,
long-term use of these agents is limited by a high incidence of
unpleasant effects in the gastrointestinal tract since these
Although pain is the most common complaint in the med-
ical field, the arsenal of effective and safe analgesics is still
relatively small. Thus, the identification of compounds that
can effectively treat painful states without induction of side ef-
fects remains a major challenge in biomedical research [1].
* Corresponding author. Tel.: þ55 55 3220 8756; fax: þ55 55 3220 8031.
E-mail address: mmartins@base.ufsm.br (M.A.P. Martins).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.07.018

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P.D. Sauzem et al. / European Journal of Medicinal Chemistry 43 (2008) 1237e1247
complications are a major cause of morbidity and mortality
produced by NSAIDs [3]. Nonetheless, some data have shown
that the ulcerogenic activity of dipyrone in rats and humans is
substantially lower than the risk associated with other
NSAIDs, such as acetylsalicylic acid and diclofenac, com-
monly used for pain relief [4,5]. These findings suggest better
tolerability of dipyrone in relation to other NSAIDs.
for basic information about the structural requirements for
the analgesic properties of such compounds, we have synthe-
sized of a series of 3-alkyl(aryl)-5-hydroxy-5-trifluoromethyl-
4,5-dihydro-1H-1-carboxyamidepyrazoles using microwave
induced techniques (Scheme 1). Furthermore, we investigated
the analgesic and anti-inflammatory profile of the synthesized
compounds in mice. The effects of bioactive pyrazoles on
other behavioral measures, such as spontaneous and forced
locomotion were also evaluated.
In this context, our research group has reported the bio-
logical effect of novel pyrazole derivatives in animal models
of inflammation, fever and pain [6e11]. The lead-like com-
pound, 3-methyl-5-hydroxy-5-trichloromethyl-4,5-dihydro-1H-
1-carboxyamidepyrazole (MPCA) possessed analgesic, but
not anti-inflammatory properties against neurogenic, inflamma-
tory and visceral pain in rodents [6]. This effect seemed to be
related with the activation of spinal a2-adrenoceptors and
5-HT receptors, but not with opioid receptor stimulation [7].
In accordance with the increasing interest in the medicinal
chemistry community in technologies and concepts that facili-
tate a more rapid synthesis and, consequently, the screening of
novel chemical substances [12], we have demonstrated the use
of microwave irradiation in heterocyclic preparations [13,14].
This form of energy has been recently used to accelerate organic
reactions due to its high heating efficiency, providing a remark-
able rate of enhancement and a dramatic reduction in reaction
times. Reactions that require hours or even days by conventional
heating can often be accomplished in seconds or minutes by
microwave heating [15].
In this study, our initial efforts were focused on the design of
new pyrazole derivatives to be used as analgesics. For this pur-
pose, we have explored the hypothesis that benzene, which is
present in salicylamide, could be mimicked with an appropriate
3- or 4-substituted 5-trifluoromethyl-4,5-dihydro-1H-pyrazole
scaffold (Fig. 1). Salicylamide is a salicylate derivative en-
dowed with good analgesic and antipyretic activity [16,17]. In
addition, the presence of a trifluoromethyl group within cyclic
compounds, especially at a strategic position, has become an
important aspect to be studied in pharmaceutical research, due
to the unique physical and biological properties of fluorine
[18]. In many systems, the substitution of a methyl group with
a trifluoromethyl group, for example, has resulted in added
lipophilicity, which may lead to easier absorption and transpor-
tation of molecules within biological systems and thereby
improve the overall pharmacokinetic properties of drug candi-
dates [18,19].
2. Results and discussion
2.1. Chemistry
Over the last decade, our research group has reported the gen-
eral synthesis of 1,1,1-trihalo-4-alkoxy-3-alken-2-ones, which
are 1,3-dielectrophilic building blocks, and have demonstrated
their use in the preparations of a wide range of heterocyclic
compounds [20]. In this study, the 1,1,1-trifluoromethyl-4-alk-
oxy-3-alken-2-ones 1 were synthesized from the reaction of
the respective enol ether or acetal with trifluoroacetic anhydride
[21,22]. The 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles 2
were synthesized by the cyclocondensation reaction of the
enones 1 with semicarbazide hydrochloride (Scheme 1).
The mixture of enones 1 and semicarbazide hydrochloride
reacted in the presence of pyridine and methanol/water solu-
tion (3:1 v/v) as solvent. The solution was submitted to micro-
wave irradiation (100 W) for 4 min, at a temperature of 70 ꢀC
and at 2.2 bar of pressure, to produce 5-trifluoromethyl-4,5-di-
hydro-1H-pyrazoles 2 with yields of 82e96%. The new
method for forming 5-trifluoromethyl-4,5-dihydro-1H-pyra-
zoles under microwave irradiation offers several advantages
including faster reaction rates, fewer byproducts, and higher
yields, while the conventional method entails moderate yields
and a long process (ca. 24 h) [23].
1H and 13C NMR, mass spectral data and elemental analy-
sis of all compounds are in full agreement with the proposed
structure. In addition to these data, the structures of the two
most active compounds (2c and 2j) were supported by crystal-
lographic studies (Figs. 2 and 3). On the basis of these exper-
iments, it was observed that the presence of intramolecular
hydrogen bonds between the carbonyl and the hydroxyl groups
of tetrasubstituted pyrazole rings generated a six membered ring
˚
with O/H bond distances of 2.17 A [O(5)eH(5)/O(11)] and
˚
Thus, in an attempt to identify novel pyrazole derivatives
endowed with analgesic activity and as a part of our search
2.28 A [O(5)eH(5)/O(11)] in structures of 2c and 2j, respec-
tively [25].
2.2. Pharmacological studies
R2
R1
2.2.1. Formalin test
F3C
HO
All compounds 2aej were screened for analgesic activity
using the formalin test in mice. In Figs. 4 and 5 the effect of
these novel pyrazole derivatives on formalin-induced neuro-
genic and inflammatory pain is shown. The synthesized com-
pounds 2a, 2cee, 2g, 2i and 2j exhibited a significant
analgesic effect on neurogenic and inflammatory phases of
the formalin test. Compound 2b was ineffective on neurogenic
N
HO
Bioisosteric Replacement
N
NH2
O
O
NH2
Salicylamide
Fig. 1. Design of the new 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles based
on the bioisosteric replacement of the benzene scaffold of salicylamide.

P.D. Sauzem et al. / European Journal of Medicinal Chemistry 43 (2008) 1237e1247
1239
R2
R1
R2
R1
i
F3C
HO
N
OR
O
82-96%
N
CF3
O
NH2
1a-j
2a-j
Entry
R
R1
H
R2
H
H
H
H
H
Yield (%)
Entry
R
R1
Bu
R2
H
Yield (%)
a
b
c
d
e
Et
90
96
91
87
90
f
g
h
i
Me
Me
Me
Me
Et
89
82
85
91
94
Me
Me
Me
Me
Me
Et
t-Bu
Ph
H
H
Pr
4-Br-Ph
H
H
i-Pr
j
Me
Scheme 1. Reagents and conditions: (i) NH2NHCONH2$HCl, MeOH/H2O, Py, MW, 100 W, 70 ꢀC, 2.2 bar, 4 min.
pain, causing a significant analgesic effect only in the inflam-
matory phase of the test. On the other hand, compound 2f
showed a significant analgesic effect in the neurogenic phase
but was ineffective in the inflammatory phase (data not shown
in Fig. 5). Furthermore, the 5-trifluoromethyl-4,5-dihydro-1H-
pyrazole 2h proved to be ineffective against the pain response
induced by formalin (data not shown in Fig. 5). Interestingly,
compounds 2d and 2e presented an analgesic profile that was
not linear with the dose. In the inflammatory phase, 2d pre-
sented an effect at doses of 75 and 750 mmol/kg but not at
250 mmol/kg. On the other hand, 2e had an analgesic effect
at all doses tested in the inflammatory phase, but, surprisingly,
the greatest effect was achieved at the smallest dose (25 mmol/
kg). It would be possible to explain this dose-independent pro-
file through future research on the mechanism(s) of action for
the 5-trifluoromethyl-4,5-dihydro-1H-pyrazole derivatives.
Another interesting observation was the different profile
of the action of compound 2b in relation to its analogue
3-methyl-5-hydroxy-5-trichloromethyl-4,5-dihydro-1H-1-car-
boxyamidepyrazole (MPCA), which has been previously
reported [6]. Compound 2b was ineffective against formalin-
induced neurogenic pain, while MPCA caused analgesia at
a similar dose as that administered in the present study [6].
However, 2b was more potent than MPCA to induce analgesia
against inflammatory pain. Therefore, the replacement of the
trichloromethyl group at position 5 of the pyrazole ring by
a trifluoromethyl group seems to increase the analgesic po-
tency of the novel pyrazoles. Compound 2h is structurally
similar to 3-phenyl-5-hydroxy-5-trichloromethyl-4,5-dihydro-
1H-1-carboxyamidepyrazole (PPCA), a pyrazole derivative
also previously described [7,8]. Different from PPCA, 2h
Fig. 2. ORTEP [24] obtained from crystal structure of 3-ethyl-5-hydroxy-5-
trifluoromethyl-4,5-dihydro-1H-1-carboxyamidepyrazole (2c). Displacement
ellipsoids are drawn at the 50% probability level. The intramolecular hydrogen
bond is indicated with dashed lines.
Fig. 3. ORTEP [24] obtained from crystal structure of 5-hydroxy-4-methyl-5-
trifluoromethyl-4,5-dihydro-1H-1-carboxyamidepyrazole (2j). Displacement
ellipsoids are drawn at the 50% probability level. The intramolecular hydrogen
bond is indicated with dashed lines.

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P.D. Sauzem et al. / European Journal of Medicinal Chemistry 43 (2008) 1237e1247
did not exhibit an analgesic effect in mice. This discrepancy
may be explained by the different models of pain used to
evaluate the analgesic effect of PPCA and 2h. One signifi-
cant change caused by the replacement of trichloromethyl
with a trifluoromethyl group is related to their differences
in size. While the van der Waals radius of chlorine has
2.2.2. Hot-plate test
The subcutaneous administration of pyrazole compounds
2c and 2j (1000 mmol/kg) did not produce an analgesic effect
according to the thermal behavior model of pain, the hot-plate
test, maintained at 50 ꢀC (Fig. 7). On the other hand, dipyrone
(1500 mmol/kg), included in the experiment as an internal
standard, increased the latency response after subcutaneous
administration.
˚
been shown to be 1.81 A [26], the fluorine atom has shown
˚
a van der Waals radius of 1.47 A [27]. This greater size of
chlorine could cause steric effects at putative receptor
sites, thus changing the pharmacological properties of the
compounds.
There is controversy in the literature about the usefulness of
the thermal stimulus test for the evaluation of the analgesic ef-
ficacy of mild analgesics, such as NSAIDs. Some authors con-
sider that NSAIDs, as well as dipyrone, are not effective in
thermal stimulation tests [33,34]. In contrast, other groups
have found that dipyrone is effective against the nociception
produced by the thermal stimulus [35e38]. The discrepancies
in the literature are probably due to differences in the intensity
of the thermal stimulus applied [39e41]. When the intensity
of the thermal stimulus is around 50 ꢀC, the antinociceptive ef-
fect of mild analgesics has been detected [39,41]. In our work,
high doses of 2c and 2j were not evaluated due to their low
solubility in the vehicle used. The lack of analgesic effect
may, therefore, be attributed to the low doses. In summary,
our findings suggest that 2c and 2j have a different analgesic
profile in relation to dipyrone, perhaps because they present
a different mechanism of action or different analgesic potency.
In order to determine the most promising compounds for
the next tests, we calculated the analgesic efficacy of the
4,5-dihydro-1H-pyrazoles in both the neurogenic and inflam-
matory phases. As shown in Table 1, compounds 2c and 2j,
at a dose of 1000 mmol/kg, presented the best analgesic effi-
cacy against neurogenic and inflammatory pain. Therefore,
these compounds were selected for evaluation with hot-plate,
carrageenan-induced paw edema, rotarod and spontaneous lo-
comotor activity tests. Furthermore, the analgesic efficacy of
2c and 2j in the formalin test was compared with that of
dipyrone and salicylamide (Fig. 6). Compounds 2c, 2j and di-
pyrone, but not salicylamide, were effective against formalin-
induced neurogenic pain. Moreover, compounds 2c and 2j
were equally effective against inflammatory pain, but more ef-
fective than salicylamide and dipyrone. These data suggest
that the replacement of the benzene from salicylamide with
a
5-trifluoromethyl-4,5-dihydro-1H-pyrazole scaffold was
2.2.3. Evaluation of anti-inflammatory action
well tolerated leading to compounds with better analgesic ef-
ficacy than salicylamide. It is noteworthy that the crystallo-
graphic studies revealed that the 4,5-dihydro-1H-pyrazole
rings, similar to the benzene ring, showed a planar structure
where the sum of internal angles [N(1)eN(2)eC(3)eC(4)e
C(5)] was found to be 539.65ꢀ and 539.8ꢀ for compounds 2c
and 2j, respectively, deviating only slightly from the ideal
value of 540ꢀ. This fact may be associated with the retention
of analgesic activity of the pyrazole derivatives of salicyla-
mide as the structural planarity often guides the molecular
elements toward the correct binding pockets of the putative
target.
in carrageenan-induced paw edema test
The pyrazole compounds 2c and 2j (1000 mmol/kg) were
evaluated for anti-inflammatory activity by the carrageenan-
induced paw edema test, a suitable experimental animal
model, widely used for determining acute inflammation [42].
The inflammation induced by carrageenan is believed to be bi-
phasic [42]. The early phase (up to 1 h) involves the release of
histamine, serotonin and bradykinin and the late phase (over
1 h) is due to the release of prostaglandin-like substances
[43,44]. Based on this, a decrease in the second phase may
be, at last in part, explained by inhibition of cyclooxygenase
[44].
The analgesic profile of compounds 2aej also suggests that
the analgesic efficacy is greater when R1 is an ethyl or when
R2 is a methyl group such as in compounds 2c and 2j, respec-
tively. Furthermore, a change in the methyl group from R2 to
R1 seemed to decrease the analgesic effect (2j versus 2b
compound).
Another interesting finding is that most of the pyrazole de-
rivatives, similar to dipyrone and unlike most of the nonsteroi-
dal anti-inflammatory drugs (NSAIDs), were effective in
preventing the neurogenic pain response induced by formalin
[28e30]. The analgesic effect of such pyrazole compounds
during the neurogenic phase of the formalin test may suggest
that they have a different mechanism of action than classical
NSAIDs. Neurogenic pain generally responds poorly to con-
ventional analgesics and its treatment can be difficult
[31,32]. Therefore, to discover new drugs for treatment of
this kind of pain is clinically relevant.
Fig. 8 shows that the intraplantar injection of carrageenan
in mice produced an increase in paw volume, a signal of in-
flammation. However, carrageenan-induced paw edema was
significantly prevented in a time-dependent manner by previ-
ous treatment with compounds 2c and 2j at a dose of
1000 mmol/kg. In fact, when the paw volume was evaluated
30 min after the carrageenan injection, 2c and 2j did not pre-
vent the development of paw edema. In contrast, 2c and 2j pre-
sented an anti-edematogenic effect when evaluated 4 h after
the induction of inflammation. This inhibition of paw edema,
only in the late phase of inflammation, suggests that the 5-
trifluoromethyl-4,5-dihydro-1H-pyrazoles 2c and 2j act on
prostaglandin biosynthesis, which is involved in the late phase
of carrageenan-induced inflammation.
It is important to consider that 2c and 2j inhibited carra-
geenan-induced inflammation almost equally (43.7 ꢁ 8.1%
and 34.6 ꢁ 12.8%, respectively), suggesting that the R1 or R2

P.D. Sauzem et al. / European Journal of Medicinal Chemistry 43 (2008) 1237e1247
1241
Fig. 4. Effect of novel pyrazole derivatives 2aed on nociception during neurogenic and inflammatory phases of the formalin test in mice. Data are reported as
mean ꢁ SEM; *p < 0.05 compared to vehicle (SNK test).
substituent did not significantly affect the anti-inflammatory
effect.
In contrast with compounds 2c and 2j, the trichloromethy-
lated analogue, MPCA, was ineffective against carrageenan-
induced paw edema [6]. As the substitutions in R1 and R2
positions seem not to modify the anti-inflammatory efficacy
of these compounds, it is possible that such an effect is
due to the trifluoromethyl group attached at position 5 of

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P.D. Sauzem et al. / European Journal of Medicinal Chemistry 43 (2008) 1237e1247
Fig. 5. Effect of novel pyrazole derivatives 2e, 2g, 2i and 2j on nociception during neurogenic and inflammatory phases of the formalin test in mice. Data are
reported as mean ꢁ SEM; *p < 0.05 compared to vehicle (SNK test).
4,5-dihydro-1H-pyrazole ring. One plausible hypothesis for
this fact is that the presence of CF3 group provides compounds
metabolically more stable in comparison to their isostere CCl3
group [27]. Such an alteration is based on the premise
that fluorine forms a strong bond with carbon (bond energy
CeF ¼ 116 kcal/mol) [27], which has an increased oxidative
and thermal stability compared with the carbonechlorine
bond (CeCl ¼ 87 kcal/mol) [45]. Therefore, this greater sta-
bility can lead to better bioavailability of the trifluoromethy-
lated compounds in relation to their trichloromethylated

P.D. Sauzem et al. / European Journal of Medicinal Chemistry 43 (2008) 1237e1247
1243
Table 1
Analgesic efficacy of 4,5-dihydro-1H-pyrazoles 2aej (1000 mmol/kg) on neu-
rogenic and inflammatory phases of the formalin test in mice
behaves like benzene bioisosteres, supplying novel analgesic
and anti-inflammatory pyrazole derivatives. The synthesized
compounds 2aej were obtained with the use of microwave-
assisted synthesis in a significantly shorter time (4 min) and
with high yields (82e96%). Our results suggest that 4,5-dihy-
dro-1H-pyrazoles 2a, 2ceg, and 2iej, unlike most of the
NSAIDs, have an additional and interesting analgesic action,
in that these compounds were effective on neurogenic pain.
However, further investigations are necessary to elucidate their
mechanism of action and their effect on other models of pain,
including chronic and neuropathic pain models. Finally, the re-
sults showed that 5-hydroxy-5-trifluoromethyl-4,5-dihydro-
1H-1-carboxyamidepyrazoles are promising candidates for the
future development of novel drugs for the treatment of pain
and inflammatory diseases.
Entry
Analgesia (%)
N
Neurogenic phase
Inflammatory phase
Vehicle
Treated
2a
2b
2c
2d
2e
2f
32.8 ꢁ 6.2*
23.3 ꢁ 11.9
87.9 ꢁ 3.6*
48.2 ꢁ 4.7*
51.9 ꢁ 9.5*
46.0 ꢁ 7.2*
59.1 ꢁ 7.6*
10.0 ꢁ 11.8
33.3 ꢁ 5.9*
74.6 ꢁ 3.6*
70.8 ꢁ 9.6*
55.5 ꢁ 10.5*
98.2 ꢁ 1.3*
70.1 ꢁ 8.4*
67.0 ꢁ 8.8*
ꢂ22.3 ꢁ 15.1
72.9 ꢁ 11.9*
8.2 ꢁ 17.1
17
9
15
8
14
16
10
6
9
12
9
8
2g
2h
2i
12
8
11
8
47.3 ꢁ 9.8*
97.2 ꢁ 1.6*
17
10
18
10
2j
Values are reported as means ꢁ SEM; N is the number of animals in each
group.
* Statistically different from vehicle ( p < 0.05; SNK test).
4. Experimental protocols
analogues thus being able to demonstrate a significant effect in
the late phase of carrageenan-induced inflammation. However,
further investigation is necessary to clarify this point.
4.1. Chemistry
Unless otherwise indicated, all common reagents were used
as obtained from commercial suppliers without further purifica-
tion. The solvents were dried and purified according to recom-
mended procedures [46]. All melting points were measured
using a Reichert-Thermovar apparatus and are uncorrected.
Yields listed are of isolated compounds. 1H and 13C NMR spec-
tra were acquired on a Bruker DPX 200 or Bruker DPX 400
spectrometer (1H at 200.13 MHz or 400.13 MHz and 13C at
50.32 MHz or 100.63 MHz, respectively) at 300 K, 5 mm sam-
ple tubes, and with a digital resolution of ꢁ0.01 ppm. CDCl3
was used as solvent with TMS as internal standard. Mass spectra
were registered in a HP 5973 MSD connected to a HP 6890 GC
and interfaced by a Pentium PC. The GC was equipped with
a splitesplitless injector, autosampler cross-linked HP-5 capil-
lary column (30 m, 0.32 mm of internal diameter), and helium
was used as the carrier gas. The reactions were performed in
a multimode microwave ETHOS 1 (Milestone Inc) which pos-
sesses a twin magnetron with a maximum delivered power of
1300 W. The temperature and the pressure were measured
throughout with an ATC-400 CE and APC-55 detector, respec-
tively. The crystal data were recorded on a Bruker Kappa Apex
2.2.4. Rotarod test and spontaneous
locomotor activity evaluation
Tables 2 and 3 show that 2c and 2j did not alter rotarod
performance and spontaneous locomotor activity of mice, re-
spectively. However, salicylamide increased spontaneous loco-
motor activity. These results indicate that the observed decrease
in pain-related behavior in both the neurogenic and inflamma-
tory phases of the formalin test, after administration of 2c and
2j, was not related to gross motor impairment since locomotion
was not affected. Nevertheless, salicylamide increased the loco-
motor activity of mice, suggesting that it induces other effects
besides analgesia. In addition, the compounds did not cause
the death of the animals in a 72 h observation period, an indica-
tion of low toxicity, at least after acute administration.
3. Conclusion
In summary, we have demonstrated here, for the first time,
that the 5-trifluoromethyl-4,5-dihydro-1H-pyrazole scaffold
Fig. 6. Comparison between analgesic efficacy of 2c, 2j (1000 mmol/kg), salicylamide (1500 mmol/kg) (SALIC) and dipyrone (1000 mmol/kg) (DIP) in the formalin
test. Data are reported as mean ꢁ SEM for n ¼ 6e9, * denotes a significant difference with regards to salicylamide and dipyrone ( p < 0.05; SNK test).

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P.D. Sauzem et al. / European Journal of Medicinal Chemistry 43 (2008) 1237e1247
chloroform (2 ꢃ 20 mL) and ethyl acetate (2 ꢃ 20 mL). The
organic layers were washed with a solution of H2O/HCl
(10:1) (2 ꢃ 10 mL) and with distilled water (2 ꢃ 10 mL).
Finally, the organic layers were combined and dried with
magnesium sulfate, the solvent was removed by rotatory evap-
oration; the 5-hydroxy-5-trifluoromethyl-4,5-dihydro-1H-1-
carboxyamidepyrazoles 2aej were isolated. When necessary,
the products were recrystallized from hexane.
4.1.1.1. 5-Hydroxy-5-trifluoromethyl-4,5-dihydro-1H-1-carbox-
yamidepyrazole (2a). Yield 1.8 mmol (90%); mp 125e
1
127 ꢀC; H NMR (200 MHz, CDCl3) d (J, Hz) 3.18 (dd, 1H,
JH4aeH4b ¼ 19.6, H4a), 3.35 (dd, 1H, JH4beH4a ¼ 19.6, H4b),
6.89 (s, 1H, H3); 13C NMR (100 MHz, CDCl3) d (JCeF, Hz)
45.2 (C4), 89.4 (q, 2J ¼ 33.9, C5), 123.1 (q, 1J ¼ 286.1,
CF3), 143.1 (C3), 156.3 (CaO); MS-EI (70 eV): m/z
(%) ¼ 197 (Mþ, 1), 154 (9), 128 (4), 85 (100), 69 (14);
Anal. Calcd. for C5H6F3N3O2: C, 30.47%; H, 3.07%; N,
21.32%. Found: C, 30.69%; H, 2.86%; N, 21.12%.
Fig. 7. Effect of pyrazole derivatives 2c and 2j (1000 mmol/kg), dipyrone
(1500 mmol/kg) or vehicle on the latency to response from thermal stimulus
on the hot-plate test. Data are reported as mean ꢁ SEM for n ¼ 14e15 animals
per group; *p < 0.05 compared to vehicle (SNK test).
II CCD area detector with graphite monochromatized Mo Ka
˚
radiation (l ¼ 0.71073 A). The data were processed with
4.1.1.2. 5-Hydroxy-3-methyl-5-trifluoromethyl-4,5-dihydro-1H-
SAINT and SADABS. The structure was solved by direct
methods (SHELXS-97) and additional atoms were located in
the Fourier map and refined on F2 (SHELXL-97) using the
SHELXTL [47] and Wingx [48] packages. The CHN elemental
analyses were performed on a Perkin-Elmer 2400 CHN elemen-
1-carboxyamidepyrazole (2b). Yield 1.92 mmol (96%); mp
1
108e110 ꢀC; H NMR (200 MHz, CDCl3) d (J, Hz) 2.02 (s,
3H, Me), 3.11 (d, 1H, JH4aeH4b ¼ 19.0, H4a), 3.24 (d, 1H,
JH4beH4a ¼ 19.1, H4b), 5.6 (br, 2H, NH2); 13C NMR
(100 MHz, CDCl3) d (JCeF, Hz) 15.3 (CH3), 47.4 (C4), 90.6
(q, 2J ¼ 33.9, C5), 123.1 (q, 1J ¼ 286.1, CF3), 152.9 (C3),
156.7 (CaO); MS-EI (70 eV): m/z (%) ¼ 211 (Mþ, 6), 168
(26), 99 (100), 69 (14); Anal. Calcd. for C6H8F3N3O2: C,
34.13%; H, 3.82%; N, 19.90%. Found: C, 34.43%; H,
3.57%; N, 19.64%.
~
tal analyzer (Federal University of Sao Paulo, USP/Brazil).
4.1.1. General procedure for the synthesis
of 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles 2aej
A solution of 4-alkoxy-1,1,1-trifluoro-3-alken-2-ones 1
(2 mmol) and semicarbazide hydrochloride (0.268 g,
2.4 mmol) in methanol/water 3:1 v/v (6 mL) and pyridine
(2 mL) was stirred for a few minutes. The mixture was then
irradiated in a microwave ETHOS 1 at 100 W, 2.2 bar of pres-
sure for 4 min. The temperature was set to 70 ꢀC and the irra-
diation was automatically stopped at this temperature. After
cooling to room temperature, the solution was extracted with
4.1.1.3. 3-Ethyl-5-hydroxy-5-trifluoromethyl-4,5-dihydro-1H-
1-carboxyamidepyrazole (2c). Yield 1.82 mmol (91%); mp
1
83e85 ꢀC; H NMR (400 MHz, CDCl3) d (J, Hz) 1.15 (t,
3H, CH3), 2.34 (q, 2H, CH2), 3.11 (d, 1H, JH4aeH4b ¼ 19.0,
H4a), 3.24 (d, 1H, JH4aeH4b ¼ 19.0, H4b), 5.09 (br, 2H,
NH2); 13C NMR (100 MHz, CDCl3) d (JCeF, Hz) 9.7 (CH3),
Fig. 8. Effect of pyrazole derivatives 2c and 2j (1000 mmol/kg) or vehicle on the paw edema induced by carrageenan (20 ml, 1.5%, ipl) 30 min and 4 h after paw
edema-induction in mice. Data are reported as mean ꢁ SEM for n ¼ 14e16 animals per group; *p < 0.05 compared to vehicle (SNK test).

P.D. Sauzem et al. / European Journal of Medicinal Chemistry 43 (2008) 1237e1247
1245
59e61 ꢀC; 1H NMR (200 MHz, CDCl3) d (J, Hz) 0.93
(t, 3H, CH3), 1.36 (sex, 2H, CH2), 1.55 (quin, 2H, CH2),
2.33 (t, 2H, CH2), 3.09 (d, 1H, JH4aeH4b ¼ 18.8, H4a), 3.23
(d, 1H, JH4aeH4b ¼ 18.8, H4b), 5.06 (br, 2H, NH2); 13C
NMR (100 MHz, CDCl3) d (JCeF, Hz) 12.3 (CH3), 21.9
(CH2), 27.8 (CH2), 29.0 (CH2), 45.9 (C4), 90.0 (q,
Table 2
Effects of 4,5-dihydro-1H-pyrazoles 2c and 2j on rotarod performance
Group
Fall latency (s)
Number of falls
Vehicle
2c 1000 mmol/kg
2j 1000 mmol/kg
76.9 ꢁ 17.0
112.5 ꢁ 21.3
62.6 ꢁ 17.6
5.4 ꢁ 1.3
3.7 ꢁ 2.1
5.1 ꢁ 1.0
1
2J ¼ 33.9, C5), 123.1(q, J ¼ 286.1, CF3), 156.4 (C3), 156.8
Data are means ꢁ SEM for 14 to 15 animals per group.
(CaO); MS-EI (70 eV): m/z (%) ¼ 253 (Mþ, 9), 210 (25),
168 (71), 141 (100), 85 (43), 69 (16); Anal. Calcd. for
C9H14F3N3O2: C, 42.69%; H, 5.57%; N, 16.59%. Found: C,
42.83%; H, 5.19%; N, 16.21%.
22.7 (CH2), 45.7 (C4), 90.3 (q, 2J ¼ 33.9, C5), 123.1 (q,
1J ¼ 286.1, CF3), 156.8 (CaO), 157.3 (C3); MS-EI (70 eV):
m/z (%) ¼ 225 (Mþ, 5), 182 (17), 113 (100), 85 (18), 69 (7);
Anal. Calcd. for C7H10F3N3O2: C, 37.34%; H, 4.48%; N,
18.66%. Found: C, 37.15%; H, 4.13%; N, 18.29%.
4.1.1.7. 3-tert-Butyl-5-hydroxy-5-trifluoromethyl-4,5-dihydro-
1H-1-carboxyamidepyrazole (2g). Yield 1.64 mmol (82%);
mp 100e102 ꢀC; 1H NMR (200 MHz, CDCl3) d (J, Hz)
1.19 (s, 9H, C3H9), 3.13 (d, 1H, JH4aeH4b ¼ 18.8, H4a), 3.28
(d, 1H, JH4aeH4b ¼ 18.8, H4b), 4.99 (br, 2H, NH2); 13C
NMR (100 MHz, CDCl3) d (JCeF, Hz) 27.7 (C3H9), 34.0
(t-bu), 43.0 (C4), 90.8 (q, 2J ¼ 33.2, C5), 123.3 (q, 1J ¼
286.8, CF3), 156.7 (CaO), 163.2 (C3); MS-EI (70 eV): m/z
(%) ¼ 253 (Mþ, 7), 195 (63), 178 (7), 141 (100), 85 (53), 69
(22); Anal. Calcd. for C9H14F3N3O2: C, 42.69%; H, 5.57%;
N, 16.59%. Found: C, 43.90%; H, 5.69%; N, 16.80%.
4.1.1.4. 5-Hydroxy-3-propyl-5-trifluoromethyl-4,5-dihydro-1H-
1-carboxyamidepyrazole (2d). Yield 1.74 mmol (87%); mp
1
68e70 ꢀC; H NMR (200 MHz, CDCl3) d (J, Hz) 0.97 (t,
3H, CH3), 1.62 (sex, 2H, CH2), 2.31 (t, 2H, CH2), 3.09 (d,
1H, JH4aeH4b ¼ 18.9, H4a), 3.24 (d, 1H, JH4aeH4b ¼ 18.9,
H4b), 5.66 (br, 2H, NH2); 13C NMR (50 MHz, CDCl3)
d (JCeF, Hz) 13.4 (CH3), 19.4 (CH2), 31.5 (CH2), 46.0 (C4),
90.4 (q, 2J ¼ 33.9, C5), 123.2 (q, 1J ¼ 286.1, CF3), 156.4
(C3), 156.8 (CaO); MS-EI (70 eV): m/z (%) ¼ 239 (Mþ, 6),
196 (18), 168 (8), 127 (100), 85 (29), 69 (8); Anal. Calcd.
for C8H12F3N3O2: C, 40.17%; H, 5.06%; N, 17.57%. Found:
C, 40.40%; H, 4.85%; N, 17.28%.
4.1.1.8. 5-Hydroxy-3-phenyl-5-trifluoromethyl-4,5-dihydro-1H-
1-carboxyamidepyrazole (2h). Yield 1.7 mmol (85%); mp
1
129e131 ꢀC; H NMR (400 MHz, CDCl3) d (J, Hz) 3.56 (d,
4.1.1.5. 5-Hydroxy-3-isopropyl-5-trifluoromethyl-4,5-dihydro-
1H, JH4aeH4b ¼ 18.8, H4a), 3.70 (d, 1H, JH4aeH4b ¼ 18.8,
H4b), 5.68 (br, 2H, NH2), 7.43e7.69 (m, 5H, Ph); 13C NMR
(100 MHz, CDCl3) d (JCeF, Hz) 43.9 (C4), 91.0 (q,
1H-1-carboxyamidepyrazole (2e). Yield 1.8 mmol (90%);
1
mp 78e80 ꢀC; H NMR (200 MHz, CDCl3) d (J, Hz) 1.18
(d, 6H, C2H6), 2.63 (sep, 1H, CH), 3.10 (d, 1H, JH4aeH4b
¼
1
2J ¼ 33.9 Hz, C5), 123.2 (q, J ¼ 286.8, CF3), 126.4, 128.8,
18.8, H4a), 3.26 (d, 1H, JH4aeH4b ¼ 18.8, H4b), 5.35 (br,
129.8, 130.9 (Ph), 151.7 (C3), 156.4 (CaO); MS-EI (70 eV):
m/z (%) ¼ 273 (Mþ, 33), 230 (90), 161 (100), 118 (26), 103
(52), 77 (86); Anal. Calcd. for C11H10F3N3O2: C, 48.36%;
H, 3.69%; N, 15.38%. Found: C, 48.10%; H, 3.39%; N,
15.25%.
2H, NH2); 13C NMR (100 MHz, CDCl3) d (JCeF, Hz) 19.5
2
(C2H6), 29.4 (CH), 44.1 (C4), 90.5 (q, J ¼ 33.9, C5), 123.2
(q, 1J ¼ 286.1, CF3), 156.9 (CaO), 160.7 (C3); MS-EI
(70 eV): m/z (%) ¼ 239 (Mþ, 19), 196 (57), 181 (29), 127
(100), 85 (98), 69 (23); Anal. Calcd. for C8H12F3N3O2: C,
40.17%; H, 5.06%; N, 17.57%. Found: C, 40.30%; H,
4.90%; N, 17.52%.
4.1.1.9. 3-(4-Bromophenyl)-5-hydroxy-5-trifluoromethyl-4,5-
dihydro-1H-1-carboxyamidepyrazole (2i). Yield 1.82 mmol
(91%); mp 194e196 ꢀC; 1H NMR (200 MHz, CDCl3)
d (J, Hz) 3.52 (d, 1H, JH4aeH4b ¼ 18.8, H4a), 3.64 (d, 1H,
JH4aeH4b ¼ 18.8, H4b), 5.6 (br, 2H, NH2), 7.52 (d, 2H, Ph),
7.57 (d, 2H, Ph); 13C NMR (100 MHz, CDCl3) d (JCeF, Hz)
49.1 (C4), 91.4 (q, 2J ¼ 33.9, C5), 123.3 (q, 1J ¼ 286.8,
CF3), 130.9, 133.3, 134.3, 137.6 (Ph), 156.2 (CaO), 161.4
(C3); MS-EI (70 eV): m/z (%) ¼ 351 (Mþ, 15), 308 (47),
239 (100), 160 (47), 102 (55), 75 (26); Anal. Calcd. for
C11H9BrF3N3O2: C, 37.52%; H, 2.58%; N, 11.93%. Found:
C, 37.69%; H, 2.65%; N, 12.15%.
4.1.1.6. 3-Butyl-5-hydroxy-5-trifluoromethyl-4,5-dihydro-1H-
1-carboxyamidepyrazole (2f). Yield 1.78 mmol (89%); mp
Table 3
Effect of 4,5-dihydro-1H-pyrazoles 2c and 2j and salicylamide on spontaneous
locomotor activity
Group
Number of crossing
N
Vehicle
2c 1000 mmol/kg
146.1 ꢁ 24.7
165.6 ꢁ 30.5
10
8
Vehicle
2j 1000 mmol/kg
115.5 ꢁ 10.0
14
9
167.3 ꢁ 31.2
4.1.1.10. 5-Hydroxy-4-methyl-5-trifluoromethyl-4,5-dihydro-
1H-1-carboxyamidepyrazole (2j). Yield 1.88 mmol (94%);
mp 118e120 ꢀC; 1H NMR (400 MHz, CDCl3) d (J, Hz)
1.25 (d, 3H, JCH3eH4 ¼ 7.5, CH3), 3.43 (qd, 1H, JH4e
Vehicle
Salicylamide 1500 mmol/kg
45.6 ꢁ 7.2
9
7
102.1 ꢁ 26.4*
Vehicle
Dipyrone 1000 mmol/kg
114.3 ꢁ 22.8
6
7
87.7 ꢁ 29.2
¼ 7.5, JH4eH3 ¼ 1.5, H4), 5.64 (br, 2H, NH2), 6.79 (d,
CH3
1H, JH3eH4 ¼ 1.5 Hz, H3); 13C NMR (100 MHz, CDCl3)
Data are means ꢁ SEM; N, number of animals in each group. *p < 0.05 com-
2
pared to vehicle (SNK test).
d (JCeF, Hz) 10.0 (CH3), 48.2 (C4), 85.1 (q, J ¼ 33.2, C5),

1246
P.D. Sauzem et al. / European Journal of Medicinal Chemistry 43 (2008) 1237e1247
1
123.5 (q, J ¼ 286.8, CF3), 148.5 (C3), 156.8 (CaO); MS-EI
(70 eV): m/z (%) ¼ 211 (Mþ, 3), 168 (17), 142 (22), 99 (100),
69 (10); Anal. Calcd. for C6H8F3N3O2: C, 34.13%; H, 3.82%;
N, 19.90%. Found: C, 34.38%; H, 3.55%; N, 19.65%.
4.2.3. Hot-plate test
The hot-plate test was carried out to determine whether the
novel pyrazoles produced analgesia in a thermal model of
pain. The test was performed according to the method
described by Ferreira et al. [51]. In these experiments, the
hot-plate (Ugo Basile, model-DS 37) was maintained at
50 ꢁ 1 ꢀC. Animals were placed in a glass cylinder of 24 cm
diameter on the heated surface, and the time between place-
ment and shaking, lifting or licking of the hind paws, or jump-
ing, was recorded as the index of response latency. The cutoff
latency was set to 90 s to prevent tissue damage. Twenty-four
hours before the experiment, all mice were acclimated to the
experimental procedure in order to minimize novelty-induced
analgesia [52]. On the day of the experiment the animals were
injected with vehicle or pyrazole derivatives (1000 mmol/kg,
subcutaneously) and the latency time was determined 30 min
thereafter. Dipyrone (1500 mmol/kg, subcutaneously) was
used as an internal standard at effective doses previously deter-
mined by pilot experiments.
4.2. Pharmacological studies
4.2.1. Animals
Adult male albino mice (30e40 g) were housed in groups
of 20 per cage, at a controlled temperature (22 ꢁ 1 ꢀC), on
a 12 h light/12 h dark cycle and with standard lab chow and
water ad libitum. The animals were acclimated to the experi-
mental room for at least 2 h before the experiments. Each an-
imal was used only once. All experiments were performed in
accordance with the ethical guidelines established for investi-
gations of experimental pain in conscious animals [49] and
were approved by the Committee on the Use and Care of
Laboratory Animals of our University (register number
23081.018371/2006-94).
4.2.2. Formalin test
4.2.4. Carrageenan-induced paw edema
The analgesic activity was evaluated by the formalin test
carried out as previously described [50]. Animals were in-
jected subcutaneously with 20 ml of 1.5% formalin (v/v) into
the dorsal right hindpaw.
We employed the classical model of carrageenan-induced
paw edema to assess the anti-inflammatory effect of the novel
pyrazoles [42,53]. Briefly, carrageenan was diluted in physio-
logical saline at a concentration of 15 mg/ml. This solution
was boiled for 1e2 s and cooled at room temperature. The
mice received 20 ml of this solution in the right hindpaw, sub-
cutaneously, on the intraplantar side. Edema formation was
quantified by changes in paw volume measured before,
30 min and 4 h after carrageenan injection. The paw edema
evaluation was made by immersing the injected paw into a cu-
vette filled with a solution of 2.5% extran in water (v/v). The
cuvette was fixed on the plate of an electronic scale (precision of
0.01 g), and the careful immersion of the paw into cuvette’s so-
lution was accompanied by an increase in the weight displayed.
The weight in grams is related to the increase of the liquid
column in the cuvette, but not to the mass of the paw. Since
the cuvette’s solution density was 1 mg/ml, the value displayed
by the balance was promptly assumed as the paw volume. In
order to evaluate the effect of the pyrazole compounds, the ani-
mals received subcutaneously vehicle or pyrazole derivatives
(1000 mmol/kg) 30 min before intraplantar carrageenan
injection and the paw edema was calculated as to the following
equation: % edema ¼ [(Vinitial ꢂ Vfinal)/Vinitial] ꢃ 100; where
Vinitial is the volume of the paw before carrageenan injection
and Vfinal is the volume of the paw 30 min or 4 h after
carrageenan injection.
The time spent licking or biting the injected paw or leg was
recorded for 30 min at 5 min intervals. Subcutaneous adminis-
tration of formalin causes a typical biphasic response of lick-
ing and biting. During the first 5 min the animals lick or bite
the injected paw because formalin probably causes direct stim-
ulation of nerve terminals and this phase is termed ‘‘neuro-
genic’’. A second burst of licking or biting occurs between
15 and 30 min after the injection and seems to be related to
the inflammatory response elicited by formalin and this phase
is termed ‘‘inflammatory’’ [50].
Vehicle (saline plus 5% Tween 80) or the 4,5-dihydro-1H-
pyrazoles 2aej (25e1000 mmol/kg) were injected subcutane-
ously into the dorsal region 20 min before formalin injection
into the paw. Salicylamide and dipyrone were used as internal
standards, at effective doses previously reported [16,17] or de-
termined by pilot experiments. Immediately after formalin in-
jection, the mice were placed in individual glass cages
(20 ꢃ 20 ꢃ 20 cm3), which served as the observation chamber,
and were evaluated for 30 min.
Furthermore, the floor of the observation chamber was di-
vided into 12 equal areas and the number of areas that a mouse
crossed with all paws was recorded and was assessed simulta-
neously with the formalin-induced pain evaluation. The num-
ber of crossings was used as a spontaneous locomotor activity
index.
Initially, the compounds were administered at the maximal
possible dose in accordance with their solubility in vehicle (sa-
line plus 5% Tween 80). Therefore, 2aec, 2gej were injected
at a dose of 1000 mmol/kg, while 2def were injected at a dose
of 750 mmol/kg. When the compounds that produced analgesia
were identified, a doseeresponse curve was constructed for
each one.
4.2.5. Rotarod performance test
The rotarod test was used to assess whether the pyrazole
derivatives caused gross motor impairment in the animals.
Twenty-four hours before the experiment, all mice were
trained in the rotarod (3.7 cm in diameter, 8 rpm) until they
could remain in the apparatus for 60 s without falling. On
the day of the experiment, the animals were injected with ve-
hicle or the pyrazole derivatives (1000 mmol/kg) and subjected
to the rotarod 30 min thereafter. The latency to fall from the

P.D. Sauzem et al. / European Journal of Medicinal Chemistry 43 (2008) 1237e1247
1247
[17] C. Lazar, A. Kluczyk, T. Kiyota, Y. Konishi, J. Med. Chem. 47 (2004)
6973e6982.
apparatus was recorded with a stopwatch for up to 240 s, as
was the number of falls in a 4 min session [54].
[18] P. Lin, J. Jiang, Tetrahedron 56 (2000) 3635e3671.
[19] M.S. Karthikeyan, B.S. Holla, N.S. Kumari, Eur. J. Med. Chem. 42
(2007) 30e36.
4.2.6. Statistical analysis
[20] M.A.P. Martins, W. Cunico, C.M.P. Pereira, A.F.C. Flores,
H.G. Bonacorso, N. Zanatta, Curr. Org. Synth. 1 (2004) 391e403 and
references therein.
Data were analyzed by one-way ANOVA. Post hoc
StudenteNewmaneKeuls test (SNK) was carried out when
appropriate.
[21] A. Colla, M.A.P. Martins, G. Clar, S. Krimmer, P. Fischer, Synthesis 6
(1991) 483e486.
[22] M.A.P. Martins, G.P. Bastos, H.G. Bonacorso, N. Zanatta, A.F.C. Flores,
G.M. Siqueira, Tetrahedron Lett. 40 (1999) 4309e4312.
[23] H.G. Bonacorso, M.A.P. Martins, S.R.T. Bittencourt, R.V. Lourega,
N. Zanatta, A.F.C. Flores, J. Fluorine Chem. 99 (1999) 177e182.
[24] L.J. Farrugia, J. Appl. Crystallogr. 30 (1997) 565.
[25] Full crystallographic data for structures reported in this paper have been
deposited with the Cambridge Crystallographic Data Center (CCDC
638958 and CCDC 639836 for 2c and 2j, respectively). Copies of the
data can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (Fax: þ44-01223-336033) or via
www.cam.ac.uk/datarequest/cif.
Acknowledgements
The authors thank Dr. Erico M.D. Flores (Chemistry De-
partment, Federal University of Santa Maria, Brazil) for the
use of his laboratory and the multimode microwave ETHOS
1 (Milestone Inc) used in the synthesis of the compounds.
The authors also thank the Conselho Nacional de Desen-
´
´
volvimento Cientıfico e Tecnologico (CNPq/PRONEX), the
`
~
Fundac¸ao de Amparo a Pesquisa do Estado do Rio Grande
do Sul (FAPERGS) and the Financiadora de Estudos e Proje-
tos (FINEP) for financial support. The fellowships from CNPq,
CAPES and FAPERGS are also acknowledged.
[26] P.R. Watson, S.M. Mokler, J. Phys. Chem. 91 (1987) 5705e5709.
[27] B. Kevin Park, N.R. Kitteringham, P.M. O’Neill, Annu. Rev. Pharmacol.
Toxicol. 41 (2001) 443e470.
[28] Z.R. Vaz, V. Cechinel Filho, R.A. Yunes, J.B. Calixto, J. Pharmacol. Exp.
Ther. 278 (1996) 304e312.
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