1,5-Diarylpyrazole and vanillin hybrids: Synthesis, biological activity and DFT studies

Article abstract of DOI:10.1016/j.ejmech.2015.06.010

Herein, we report the design and synthesis of 13 diarylpyrazole hybrids with vanillin constructed as dual compounds against oxidative stress and diabetes. Compounds were tested in two different antioxidant assays. It was found that all compounds showed an

Full text of DOI:10.1016/j.ejmech.2015.06.010

European Journal of Medicinal Chemistry 100 (2015) 106e118
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
1,5-Diarylpyrazole and vanillin hybrids: Synthesis, biological activity
and DFT studies
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Eduardo Hernandez-Vazquez , Romina Castaneda-Arriaga ,
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Juan Jose Ramírez-Espinosa , Omar Noel Medina-Campos , Francisco Hernandez-Luis ,
Jose Pedraza Chaverri , Samuel Estrada-Soto
Facultad de Química, Departamento de Farmacia, Universidad Nacional Autonoma de Mexico, Mexico, DF 04510, Mexico
Facultad de Química, Departamento de Física y Química Teorica, Universidad Nacional Autonoma de Mexico, Mexico, DF 04510, Mexico
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Facultad de Química, Departamento de Biología, Universidad Nacional Autonoma de Mexico, Mexico, DF 04510, Mexico
Facultad de Farmacia, Universidad Autonoma del Estado de Morelos, Cuernavaca, Morelos 62209, Mexico
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a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we report the design and synthesis of 13 diarylpyrazole hybrids with vanillin constructed as dual
compounds against oxidative stress and diabetes. Compounds were tested in two different antioxidant
assays. It was found that all compounds showed an important antioxidant activity in both DPPH and
ORAC models and the activity was even more remarkable than vanillin. In addition, the hypoglycemic
effect of compounds 1, 2, 4 and 12 was evaluated. Interestingly, compound 1 had the most potent hy-
poglycemic effect with a glycemia reduction of 71%, which was higher than rimonabant. Finally, a DFT
study to propose a reasonable antioxidant mechanism is detailed. Both thermodynamic and kinetic
studies indicated that the most feasible mechanism consists in the HAT abstraction of the phenolic
hydrogen due to the formation of an stable transition state through the most rapid and exergonic path,
while the SPLET mechanism is the most significant at higher pH values.
Received 23 February 2015
Received in revised form
2 June 2015
Accepted 3 June 2015
Available online 5 June 2015
Keywords:
Diarylpyrazole hybrids
Vanillin
Antioxidant capacity
DFT calculations
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
other glucose catabolic pathways become activated, such as hex-
osamine and polyol (sorbitol-aldose reductive pathway) metabolic
Oxidative stress is defined as a redox imbalance generated by
the overproduction of highly reactive species (oxygen, ROS; nitro-
gen, RNS), the deficiency of the antioxidant system (small mole-
cules, proteins and enzymes) or both [1]. Several chronic diseases
have been proposed to be either caused or enhanced by oxidative
stress, such as obesity, cancer, aging, inflammation, neurodegen-
erative disorders, hypertension, and cardiovascular diseases [2].
Furthermore, the participation of oxidative stress in the develop-
ment and progression of diabetes and its complications is widely
accepted [3,4]. ROS formation is a direct consequence of hyper-
glycemia (Fig. 1) [5], and several mechanisms contribute to its
formation. In mitochondria, high levels of glucose enhance super-
oxide production, which eventually activates the nuclear enzyme
poly (ADP-ribose) polymerase (PARP) and inhibits glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) [6,7]. As a result of that,
cascades. Furthermore, hexosamine pathway is a well-known
source of endogenous radicals, while the activation of polyol
route depletes nicotinamide adenine dinucleotide phosphate
(NADPH), a necessary cofactor to regenerate oxidized glutathione
[7,8]. Likewise, other mechanisms related to diabetic complications
are activated, like protein kinase C (PKC) and advanced glycation
end products (AGE) [9,10], which eventually lead to ROS produc-
tion. Besides the latter, transition-metal dependent autooxidation
and nonenzymatic glycation of glucose with amino groups of pro-
teins or nucleic acids, contribute to oxidative stress in hypergly-
cemia [11]. As a result of the activation of all those mechanisms,
oxidative stress participates in pathogenesis of diabetes due to their
ability to directly oxidize and damage lipids, DNA and proteins.
Some radicals can also act as signaling molecules to activate stress-
sensitive pathways [nuclear factor kappa B (NF-kB), p38 mitogen-
activated protein kinase (p38 MAPK), c-Jun N-terminal kinase
(JNK)/stress-activated protein kinase (SAPK)] [5,12], which finally
produce insulin resistance and
reasons, antioxidants could be employed in diabetes treatment and
b-cell damage [9,13,14]. For these
* Corresponding author.
E-mail address: eduardo.hervaz@gmail.com (E. Hernandez-Vazquez).
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http://dx.doi.org/10.1016/j.ejmech.2015.06.010
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

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107
Fig. 1. Relationship between oxidative stress and diabetes.
also in the prevention of its complications. However, only a limited
number of antioxidants are capable to reduce high blood glucose
2. Results and discussion
levels in diabetes such as melatonin, vitamins C and E (with
b
-
2.1. Chemistry
carotene) and lipoic acid [15e17].
On the other hand, it is well documented the participation of
cannabinoid receptor 1 (CB1) in metabolic control and food intake
balance [18]. In particular, the antagonism of CB1 triggers signal
pathways in peripheral organs (liver, adipose tissue and skeletal
muscle) that regulate metabolic functions like the reduction of
adipogenesis, hypoglycemia by glucose uptake, thermogenesis,
enhancement of lipogenesis and adiponectin up-regulation
[19e21]. In accordance to this, our group has reported a series of
1,5-diarylpyrazole with higher hypoglycemic activity than rimo-
nabant, a selective CB1 antagonist [22]. However, several trials have
shown that improving glycemic control does not necessarily reduce
the collateral oxidative stress [23]. Therefore, the main objective of
this work was the hybridization of 1,5-diarylpyrazole analogs and
vanillin, with the purpose of generating a new class of compounds
to offer an integral treatment to diabetes and metabolic syndrome
(Fig. 2). The design considered the formation of a hydrazone in
order to act as a bioisostere of guaiacol derivatives. The increase in
The synthetic pathway observed in Scheme 1 was followed for
the synthesis of hybrids 1e13. First, the 1,5-diaryl pyrazole-3-
carboylic acids were constructed. To achieve these intermediaries,
different substituted propiophenone derivatives were treated with
lithium bis(trimethylsilyl)amide (LiHMDS) in methylcyclohexane,
giving the corresponding lithium enolates. These enolates were left
to react with diethyl oxalate to obtain the expected ethyl 4-aryl-3-
methyl-2,4-dioxobutanoate (compounds 19e31), with yields
ranging from 60% to 97% (Scheme 1). The cyclocondensation of
previously obtained phenylhydrazines and the unpurified tri-
carbonylic compounds in a sulfuric acid/ethanol solution produced
the ethyl pyrazole-3-carboxylates. The esters were converted to the
corresponding carboxylic acids by treatment with potassium hy-
droxide at 50 ꢀC, resulting in an overall yield ranging from 50 to
87%.
After that, we focused on the synthesis of final products. Two
routes were considered to achieve compounds 1e13. In a first
attempt, the vanillin hydrazone was proposed as an intermediary;
however, the dimerization of the aldehyde was the major product
when treated vanillin with hydrazine hydrochloride; similar result
was observed when the N-Boc carbazate was deprotected. This
problem was solved by changing the order of the hydrazone for-
mation. As seen in Scheme 1, the pyrazole-3-carboxylic acids were
converted to the corresponding acyl chlorides by refluxing them
with thionyl chloride in toluene and later were stirred with tert-
butyl carbazate to obtain the desired carbohydrazides in good
yields. Without purification, Boc group was cleavage by treatment
with trifluoroacetic acid (TFA) and subsequently condensed with
vanillin to afford hydrazones 1e13. Table 1 resumes the physico-
chemical properties of compounds, which were obtained as solids
with a narrow melting point.
conjugation of
p-electrons would confer a higher degree of reso-
nance structures of the phenoxy radical, which has been proposed
to be the mechanism implicated in the antioxidant activity of these
derivatives [24,25], and thus would enhance the protective effect
against oxidative stress.
During the condensation of vanillin with 1,5-diarylpyrazole-3-
carbohydrazides, two possible configurations are expected: the Z
and the E hydrazones (Fig. 3); despite this possibility, only one
compound was observed. In the case of the E isomer, Ha (hydrogen
bonded to the nitrogen) can interact with the imine hydrogen Hb
and with the hydrogen at ortho position of phenol ring (Hc and Hꢀc),
while the Z isomer can only couple with Hc/Hꢀc. In order to verify the
Fig. 2. Design of pyrazole-based hybrids.

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E. Hernandez-Vazquez et al. / European Journal of Medicinal Chemistry 100 (2015) 106e118
108
Scheme 1. Synthesis of hybrids 1e13. Reagents and conditions: a) LiHMDS, MCH, 3 h, r.t. b) diethyl oxalate, r.t. c) corresponding arylhydrazine, H2SO4, EtOH, 80 ꢀC, ca. 8 h then KOH,
EtOH, 50 ꢀC, 12. d) SOCl2, PhMe, 110 ꢀC, ca. 2.5 h e) t-butyl carbazate, DIPEA, CHCl3, 0 ꢀC to r. t., 3 h f) TFA, then vanillin, AcOHcat, CHCl3, r.t.
Table 1
Physicochemical data of compounds 1e13.
Compound
R1
R2
Melting point (ꢀC)
Unoptimized yield (%)
1
2
3
4
5
6
7
8
2,4-Dichlorophenyl
2,4-Dichlorophenyl
Phenyl
Cl
H
H
Cl
Cl
Cl
Cl
F
F
F
Br
CF3
CF3
256e258
239e241
291e292
224
210e212
272e273
184e185
241e242
206
289e291
289e290
250e251
245e246
68
69
62
60
62
59
51
57
84
55
78
76
87
3,4-Dichlorophenyl
3-Chloro-4-fluorophenyl
7-Chloroquinolin-4-yl
4-(Trifluoromethyl)phenyl
2,4-Difluorophenyl
3-Chloro-4-fluorophenyl
7-Chloroquinolin-4-yl
4-(Trifluoromethyl)phenyl
2,4-Dichlorophenyl
2,4-Difluorophenyl
9
10
11
12
13
Fig. 3. Possible interactions throughout space in E and Z isomers of hybrids. Compound 1 is shown in the figure.
configuration of the series, the nuclear Overhauser effect spec-
troscopy (NOESY) spectrum of compound 2 was analyzed. Fig. 4
shows an expansion from 8 to 12 ppm of the spectrum of 2. In
this region, three signals can be observed: those corresponding to
the hydrogen of the hydrazone (Ha, 11.6 ppm), the imine (Hb,
8.4 ppm) and the phenol (Hd, 9.5 ppm), respectively. As discussed

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E. Hernandez-Vazquez et al. / European Journal of Medicinal Chemistry 100 (2015) 106e118
109
Fig. 4. NOESY spectrum of 2 (extension from 11.8 to 8.2 ppm). A correlation between Ha and Hb is clearly visible.
above, only the E isomer shows a correlation throughout space
between the hydrazine and imine hydrogens (Ha and Hb, respec-
tively), and this interaction is present in the NOESY spectrum of 2.
This interaction is due to the proximity of both atoms, which does
not occur in the Z isomer. Additionally, a density functional theory
(DFT) minimization with B3LYP 6-31Gþ in a PCM solvation model
was performed to further calculate the most stable isomer. Two
compounds were submitted to this calculation (2 and 13), and in
both cases, the E isomer was the most stable (Fig. 5). By analyzing
the optimized geometry of the isomers, it can be clearly notice that
the distance between Ha and Hb in E hydrazone is close enough to
generate a correlation in NOESY spectrum. In summary, NOESY
Fig. 5. DFT optimization of compounds 2 and 13. A) Compound 2. B) Compound 13. In both cases, the E isomer is the most stable.

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E. Hernandez-Vazquez et al. / European Journal of Medicinal Chemistry 100 (2015) 106e118
110
spectra and DFT calculations confirmed that E hydrazone is the
geometry observed in the series.
(Table 2) had an antioxidant capacity ranging from 401 to 1076
trolox equivalents and compound 2 was the most active. Unlike
DPPH results, vanillic acid showed a better antioxidant capacity,
being two-fold more active than 2. This result clearly indicates that
there is no correlation between the two methods employed,
although both assays are considered hydrogen atom transfer (HAT)
methodologies. Additionally, no structure-activity relationships
can be generated because the antioxidant effect is due to the
guaiacol moiety and the substitutions of phenyl ring slightly in-
terferes in the global effect. Both DPPH and ORAC assays demon-
strated the antioxidant properties of hybrids 1e13 and therefore
can be considered as new antioxidant compounds. However,
further scavenging studies comprising other ROS as well as in vivo
models are required and the publication of these results is
forthcoming.
2.2. Biology
2.2.1. In vitro antioxidant capacity
The design of compounds included an antioxidant portion, thus
we decided to evaluate their radical scavenging capability. Several
assays, such as trolox-equivalent antioxidant activity (TEAC), oxy-
gen radical absorbance capacity (ORAC) assays as well as 2,2-
diphenylpicrylhydrazyl (DPPH) scavenging, are commonly used to
study the radical-scavenging ability of phenolic compounds
[26,27], and provide a relative measure of antioxidant activity [28].
As a preliminary study, the DPPH-scavenging capacity was evalu-
ated. DPPH is a stable radical which has been used to test the
antioxidant capacity of foods and other compounds [29]. The EC50
of hybrids 1e13 is shown in Table 2. As it can be seen, all com-
pounds had a DPPH-scavenging effect under 7 mM and compound
7 was the most active (EC50 ¼ 2.34 mM). However, no significant
differences between compounds were found and all compounds
were less active than ascorbic acid. In order to corroborate if the
antioxidant activity is directly related to the presence of vanillin
moiety, the pirazole-3-carboxylic acid 19 was tested. As expected,
this compound did not show an important DPPH scavenging ac-
tivity (maximum scavenging of 17%), thus the antioxidant activity
may be explained by the phenolic hydrazone. In addition, vanillin
and vanillic acid were evaluated and, surprisingly, compounds
1e13 had a better DPPH scavenging capacity than those guaiacol
derivatives. These findings suggest that the hybridization of vanillin
with 1,5-diarylpyrazole derivatives not only maintained the anti-
oxidant properties of this phenolic compound but also indicated an
increase of activity due to the conjugation of both scaffolds.
Although interesting results were found with the DPPH-
scavenging assay, this radical has no biological importance
because it is not endogenous. For this reason, the ORAC assay was
carried out since this technique can evaluate the ability of com-
pounds to scavenge peroxyl radical (ROOꢁ), a reactive oxygen specie
with biological and clinical relevance [30]. ORAC method de-
termines the ability of antioxidants to protect the oxidative damage
in fluorescein caused by a source of peroxyl radicals through the
measurement of the decrease in its fluorescence. Hybrids tested
2.2.2. In vivo hypoglycemic evaluation
We have previously demonstrated that 1,5-diarylpyrazole de-
rivatives having electron-withdrawing groups show a remarkable
in vivo glucose reduction in diabetic rats [22], and in consequence,
hybrids 1e13 could exhibit this effect. To further corroborate this
statement, an in vivo diabetes mellitus type II (DMTII) test in pre-
viously induced diabetic rats was performed. Due to the poor sol-
ubility of compounds in water, we employed Tween 80 as vehicle.
Furthermore, glibenclamide was taken as reference drug while
vehicle was considered as control. The compounds tested showed a
significant glycemia reduction with respect to the control (Table 3).
Fig. 6 shows the glycemia reduction over the time of evaluation
(7 h). The most prominent effect was evident at 7 h after admin-
istration of analogues, in which compounds 1, 2, 4, and 12 showed a
plasma glucose reduction of 71, 32, 39 and 41%, respectively.
Compounds 4 and 12 had a similar response to that observed with
the administration of glibenclamide and, as it can be deduced from
Fig. 6, the effect would continue after 7 h. Even though this phar-
macologic effect is significant, compound 1 had the best antidia-
betic results, with an extraordinarily reduction of 71% at 7 h. It is
remarkable that compound 2, which 1,5-diarylpyrazole moiety
lacks of CB1 affinity [31], also displayed an important glycemia
reduction. Two assertions can be concluded from these data. First,
the hybrids showed an in vivo antidiabetic activity and thus, the
hybridization with vanillin did not eliminate the biological activity
of rimonabant derivatives but conserved the antioxidant capacity.
As result of that, it was confirmed that compounds 1e13 act as
hybrids, in which two components of diabetes were covered:
oxidative stress and hyperglycemia. Second, the hypoglycemic ac-
tivity of rimonabant was increased. In previously published works,
its percentage of glucose reduction in the same model was about
31% at 7 h [22]. Nonetheless, when rimonabant hybridizes with
vanillin hydrazone, the effect increases to 71%. The depletion could
be attributed to the antioxidant capacity, which could reduce the
oxidative stress that is involved in the development and progres-
sion of diabetes. Moreover, an increase in peripheral CB1 receptor
affinity is not discarded. This is a vital point in the design of CB1
antagonist/inverse agonist for the treatment of obesity and dia-
betes, because the main toxicity of these ligands is related to its
action in central nervous system [32].
Table 2
EC50 of DPPH-scavenging and trolox equivalents in ORAC for hybrids 1e13.
Compound
DPPH scavenging (EC50)
ORAC
(Mg/ml)
(mM)
Trolox equivalents/g
1
2
3
4
5
6
7
8
9
10
11
12
1.88
1.48
1.05
1.38
1.83
2.51
1.24
1.7
3.44
3.42
1.67
2.39
1.54
3.55
2.99
2.46
2.60
3.56
4.59
2.34
3.54
6.92
6.45
2.91
4.24
2.90
634 83
1076 224
611 52
432 32
882 63
ND
867 71
891 45
525 41
ND
ND
401 42
993 172
2979 262
ND
2.3. DFT calculations
13
Vanillic acid
Vanillin
19
11.06
65.83
Several antioxidant mechanisms have been attributed to
guaiacol derivatives and phenolic compounds such as HAT,
Sequential Proton-Loss Electron Transfer (SPLET) and Single Elec-
tron Transfer (SET) [33]. Taking this into account, a DFT study was
performed to propose a plausible mechanism of the antioxidant
action of hybrids and gain greater understanding of the
a
a
b
b
ND
Ascorbic acid
0.366 0.048
2.07 0.034
ND
ND: not determined.
a
Maximum scavenging of 7% at 20 g/mL (75% at 1 g/mL).
Maximum scavenging of 17% at 20 mg/mL.
b

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E. Hernandez-Vazquez et al. / European Journal of Medicinal Chemistry 100 (2015) 106e118
111
Table 3
In vivo hypoglycemic activity (at 50 mg/kg) of compounds 1, 2, 4 and 12.
Compound
Blood glucose-lowering profile
% of change of glucose
over control
at 7 h (mg/dL)a
1
2
4
12
ꢂ70.52 10.19***
ꢂ32.35 7.30
64.9
26.6
33.3
35.3
ꢂ39.00 6.50*
ꢂ41.00 7.10**
*p < 0.05; **p < 0.01, ***p < 0.001 as compared to untreated group. The negative
value (ꢂ) indicates a decrease in glycemia compared with time 0.
Fig. 7. Hydrogen atom numeration of virtual analog considered in the theoretical
study.
a
Values are represented as means SEM. (n ¼ 5).
afford the products. We considered the H1, H2, H3 and H5 abstrac-
tions mediated by some radicals with different electrophilicity. The
fully optimized geometries of the transition states (TS) are shown in
Fig. 8. All the transition states corresponding to HAT from the
phenolic OH (H1 abstraction) showed a delocalization of the radical
within the aromatic ring. This delocalization led to several canon-
ical structures and results in stabilization of the transition states.
phenomena. Several oxygen radicals with different electrophilic
nature were explored, named HOꢁ, HOOꢁ, CH3OOꢁ, CH2CHOOꢁ and
CCl3OOꢁ (in ascending electrophilic character); M06-2X functional
in conjunction with the 6e31þþG (d,p) basis set were used in the
study, since it has been recommended for kinetic calculations by its
developers [34].
Considering that the antioxidant effect is due to the phenolic
moiety and not to the 1,5-diarylpyrazole ring (in accordance to the
results found in the DPPH and ORAC tests), a virtual analog (Fig. 7)
was modeled. This compound lacks of aromatic rings at 1 and 5
positions, which were substituted by methyl groups; however this
reduction in atoms number was suitable for completing the study
in reasonable time and also contained the structural features
responsible of the DPPH and peroxyl radicals scavenging. Five
possible removable hydrogen atoms were considered for both
thermodynamic and kinetic determinations, labeled H1, H2, H3, H4
and H5 (Fig. 7). The Gibbs free energy of the studied reaction paths
involving hydrogen atom transfer are summarized in Table 4. As it
can be shown, only the H1 HAT pathway presented an exergonic
behavior independently of the reactive radical tested, ranging
from ꢂ2.88 to ꢂ35.43 kcal/mol. Other hydrogen atom transfer re-
actions that resulted in a viable path were the abstraction of the
amide hydrogen and the hydrogen at methyl group in the pyrazole
ring, which were found to be exergonic abstractions with either
nucleophilic (CH3Oꢁ) or electrophilic (CCl3OOꢁ) radicals.
Thermodynamic data showed above points that the abstraction
of the H1 trough a HAT pathway seems to be the main mechanism
implicated in the antioxidant activity of compounds 1e13. How-
ever, the kinetic of the reaction must also be considered, since a
high reaction rate would conduct to a rapid products formation and
thus could prevent cell damage caused by reactive radicals. The
endergonic paths were not included because, even if they take
place at a significant rate, the reversibility of the reaction will not
This hypothesis can be corroborated in the
states of the H1 (Table 5), which are lower than those calculated for
other hydrogen abstractions. For example, the
Gs for H1 path by
CH3Oꢁ was 11.50 kcal, whereas H2, H3 and H5 showed higher Gs
D
Gs of the transition
D
D
values (15.89, 18.52 and 13.60 kcal, respectively). Similar results
were found with the more electrophilic radical Cl3COOꢁ, in which
the D
Gs of H1 path was found to be 2 kcal lower than the calculated
for H5 (11.81 kcal for H1 versus 13.84 kcal for H5). Therefore, it is
expected that kinetics would favor the H1 HAT over other possible
and thermodynamically favored paths, such as H5.
In addition, the rate constants of each reaction path are reported
in Table 5. The calculated values of k range from 103 to 107 Mꢂ1sꢂ1
.
When comparing the possible mechanisms for HAT by CH3Oꢁ
radical, it is noteworthy that the H1 path has the higher rate con-
stant among them (8.42 ꢃ 107
M
ꢂ1sꢂ1), followed by the H5
abstraction with k of 8.54 ꢃ 106 Mꢂ1sꢂ1 and is ten-fold slower than
H1 entry. The same finding occurs with Cl3COOꢁ radical, where H1
abstraction was found to be more rapid than H5 path. According to
the latter, the most important mechanism that contributes to the
antioxidant properties of compounds 1e13 is the HAT of the
phenolic hydrogen (H1) through a reaction that involves the gen-
eration of a stabilized phenoxyl radical, followed by the H5 path, in
which the homolytic cleavage at this position conduct to the for-
mation of a benzyl-like radical. Notably, the rate constants obtained
in this work are higher than those calculated for guaiacol de-
rivatives, whose k values range from 102 to 104 in aqueous solution
[35]. This is logical since the TS in compounds 1e13 have more
resonance structures than guaiacol derivatives, in part, due to the
extended double bond of the hydrazone moiety.
Furthermore, the SET mechanism was also studied for several
radicals, albeit all the cases resulted in an endergonic reaction
(Table 4). Previous works have demonstrated that this mechanism
is not significant at physiological pH (pH ¼ 7.4) in guaiacol analogs,
in which almost all derivatives (such as eugenol, vanillic alcohol
Table 4
Gibbs free energies of reaction (kcal/mol) at 298.15 K for HAT and SET mechanisms.
Radical
HAT (kcal/mol)
H1 H2
SET (kcal/mol)
H3
H4
H5
HOꢁ
ꢂ35.43 ꢂ21.64 ꢂ15.31 ꢂ25.28 ꢂ28.35
8.12
3.79 30.74
HOOꢁ
ꢂ3.29
ꢂ20.16
ꢂ1.58
10.50
ꢂ6.37
12.21
10.91
2.80
16.83
6.86
CH3Oꢁ
ꢂ0.04 ꢂ10.01 ꢂ13.08 24.19
CH3OOꢁ
CH2CHOOꢁ
Cl3COOꢁ
18.54
17.24
9.12
8.57
7.27
ꢂ0.85
5.50 32.28
4.20 26.13
ꢂ3.92
ꢂ2.88
Fig. 6. Plasma glucose reduction after treatment with compounds 1, 2, 4 and 12
(50 mg/kg).
ꢂ11.00
9.78

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112
Fig. 8. Optimized geometries of the HAT transition states (the distances are expressed in Å).
and even guaiacol) are predominantly in its neutral state [36].
However, this mechanism becomes more important at basic pH
values, where the antioxidant species are the phenoxide anions due
to the known electro-donating capacity of this ion [35]. Accord-
ingly, this process actually corresponds to a sequential proton loss
electron transfer mechanisms (SPLET) that involves the electron
transfer between the anion of the antioxidant and the radical and
was first proposed by Litwinienko and Ingold [37e40]. Virtual
analog was also employed in these calculations and its pKa value
was estimated (pKa ¼ 11.14; molar fraction of the phenoxide anion
of 0.02 at physiologic pH). Considering the molar fraction, the
calculated rate constants ranged from 105 to 108 and are higher
than those obtained for HAT mechanism with except to the con-
stant for HOOꢁ, in which no significant difference was observed
(Table 5). In conclusion, even though the deprotonated fraction is
very low (i.e. 0.02), the rate constant of SPLET is faster than the HAT
occurring in the neutral molecule of the antioxidant. Thus, both
HAT at H1 position and SPLET mechanisms contribute to a signifi-
cant extent to the overall reactivity of compounds 1e13 toward
radicals.
3. Conclusions
Considering the relationship between diabetes mellitus, oxida-
tive stress and metabolic syndrome, thirteen hybrids of vanillin and
CB1 antagonists were designed and synthesized. The design
contemplated that the extension in conjugation of the
p system in
vanillin could increase the antioxidant activity of the guaiacol de-
rivative. In this sense, hybrids 1e13 showed a significant antioxi-
dant capacity in DPPH-scavenging and ORAC in vitro
determinations. In the case of DPPH test, all the compounds
exhibited a better scavenging effect than vanillin and vanillic acid,
in which compound 7 was the most active (EC50 ¼ 2.34 mM). On the
other hand, the ORAC assay corroborated the previously observed
results and indicated that hybrids had an antioxidant effect ranging
from 401 to 1076 trolox equivalents/g of compound. In this case,
compound 2 showed the best antioxidant capacity (1076 trolox
equivalents). Although the in vitro antioxidant capacity was
confirmed with these studies, structure-activity relationships could
not be established since the antioxidant capacity is due to the
guaiacol moiety.
Table 5
Gibbs free energies of activation and rate constants at 298.15 K.
Radical
CH3Oꢁ
SPLET
HAT
Rate constants (Mꢂ1sꢂ1
1.67 ꢃ 108
)
HAT pathway
D
Gs (kcal/mol)
Rate constants (Mꢂ1sꢂ1
)
H1
H2
H3
H5
H1
H5
H1
H1
H1
11.50
15.89
18.52
13.61
11.81
13.84
15.58
19.08
19.19
8.42 ꢃ 107
3.78 ꢃ 105
4.40 ꢃ 103
8.54 ꢃ 106
2.70 ꢃ 107
4.63 ꢃ 106
5.47 ꢃ 105
6.46 ꢃ 104
6.97 ꢃ 105
Cl3COOꢁ
1.53 ꢃ 108
CH2CHOOꢁ
CH3OOꢁ
HOOꢁ
1.45 ꢃ 108
4.04 ꢃ 107
2.89 ꢃ 105

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E. Hernandez-Vazquez et al. / European Journal of Medicinal Chemistry 100 (2015) 106e118
113
In addition, hybrids 1, 2, 4 and 12 were evaluated for its in vivo
4.1.2. General procedure for the synthesis of 1,5-diarylpyrazole-3-
carboxylic acids (19e31)
hypoglycemic effect in a DMTII model. Compounds presented a
significant plasma glucose reduction at 7 h after administration;
notably rimonabant hybrid 1 showed the higher effect with a
decrease of 70%. In closing, with the information described above,
the dual activity of compounds 1e13 was confirmed by presenting
hypoglycemic activity as well as antioxidant properties.
Finally, the DFT thermodynamic and kinetic studies corrobo-
rated that the most feasible and rapid mechanism that contributes
to the radical scavenging properties is the reaction of the phenolic
hydrogen through a HAT path. However, at physiologic pH, in which
there exists deprotonated species, the SPLET mechanism becomes
the most probable mechanism. Gratifyingly, compounds 1e13
consolidate as new pyrazole leads with both hypoglycemic and
antioxidant properties, which would further be used in the treat-
ment and prophylaxis of diabetes and metabolic syndrome.
A mixture of the lithium salt of the corresponding ethyl 3-
methyl-2,4-dioxo-4-phenylbutanoate (13.8 mmol) and substituted
phenylhydrazine (15.6 mmol) was suspended in 50 mL of EtOH and
H2SO4 was added dropwise in an ice bath. The mixture was heated
to reflux and stirred until the starting material was completely
consumed. The resulting solution was cooled to room temperature
and the excess of ethanol was removed in vacuo. 20 mL of water
were added and the suspension was neutralized with NaHCO3 and
then extracted with CHCl3 (3 ꢃ 20 mL). The organic layer was
successively dried over Na2SO4, filtered and concentrated in vacuo.
The residue containing the ester product was then dissolved in
20 mL of EtOH and KOH (23 mmol) was added. The mixture was
allowed to react 12 h at 50 ꢀC. The resulting suspension was
concentrated in vacuo and dissolved in 30 mL of water and later
washed with hexane (3 ꢃ 15 mL). The aqueous layer was acidified
with concentrated HCl until the pure carboxylic acids precipitated.
4. Experimental
4.1.2.1. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid (19). Beige solid in 87% yield, mp
208e210 ꢀC. FAB-MS m/z 381 [(Mþ1)þ], 383 [(Mþ3)þ], 385
4.1. Chemistry
Solvents and reagents were purchased from SigmaeAldrich and
were used without purification. Melting points were determined
on a Büchi melting point apparatus, model B-540 and are uncor-
rected. The progress of reactions and the purity of final products
were monitored by thin layer chromatography (TLC) whereas UV
and iodine oxidation were used as revealing agents. 1H and 13C
NMR spectra were obtained in DMSO-d6 and CDCl3 solutions at
400 MHz (for 1H) and 100 MHz (for 13C) on a Varian MR-400 (9.2 T)
instrument and on a Varian VNMRS-400 (9.2 T) instrument.
Chemical shifts (
signal.
Mass spectrometry spectra were obtained using a Thermo-
electron model DFS instrument. Glibenclamide, nicotinamide,
streptozotocin and Tween 80 were purchased from SigmaeAldrich
Co. (St. Louis, MO, USA).
[(Mþ5)þ], 363[(Mþ1)-18]. 1H NMR (400 MHz, DMSO-d6):
d 7.77 (d,
J ¼ 2.0 Hz, 1H, ArH), 7.69 (d, J ¼ 8.4 Hz, 1H, ArH), 7.56 (dd, J ¼ 8.8 y
2.4 Hz, 1H, ArH), 7.44 (d, J ¼ 8.4 Hz, 2H, ArH), 7.21 (d, J ¼ 8.4 Hz, 2H,
ArH), 2.23 (s, 3H, CH3).
4.1.2.2. 1-(2,4-Dichlorophenyl)-4-methyl-5-phenyl-1H-pyrazole-3-
carboxylic acid (20). Light brown solid in 68% yield, mp 189e191 ꢀC.
EI-MS m/z 346 [Mþ], 348 [(Mþ2)þ]. 1H NMR (300 MHz, DMSO-d6):
d) are reported in ppm relative to the solvent
d
12.95 (bs, 1H, ArCOOH), 7.77 (d, J ¼ 2.1 Hz, 1H, ArH), 7.69 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.56 (dd, J ¼ 8.6 and 2.1 Hz, 1H, ArH), 7.41e7.34
(m, 3H, ArH), 7.22e7.19 (m, 3H, ArH), 2.23 (s, 3H, CH3).
4.1.2.3. 4-Methyl-1,5-diphenyl-1H-pyrazole-3-carboxylic acid (21).
Yellowish solid in 74% yield, mp 256e258 ꢀC. FAB-MS m/z 279
[(Mþ1)þ], 261 [(Mþ1)-18], 233 [(Mþ1)-46]. 1H NMR (300 MHz,
4.1.1. General procedure for the synthesis of lithium salt of ethyl 3-
methyl-2,4-dioxo-4-phenylbutanoates (14e18)
DMSO-d6): d 7.44e7.26 (m, 6H, ArH), 7.23e7.18 (m, 4H, ArH), 2.20 (s,
3H, CH3).
A solution of the corresponding propiophenone (15.06 mmol) in
3 mL of anhydrous methylcyclohexane was added at room tem-
4.1.2.4. 5-(4-Chlorophenyl)-1-(3,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid (22). Light brown solid in 82% yield, mp
203e204 ꢀC. FAB-MS m/z 381 [(Mþ1)þ], 383 [(Mþ3)þ], 385
perature to
a magnetically stirred solution of lithium bis(-
trimethylsilyl)amide (16.1 mmol) in anhydrous methylcyclohexane
(3e5 mL). After 3 h of stirring, diethyl oxalate (16.6 mmol) was
added dropwise and then left to react for 16 h. The mixture was
filtered and washed with several portions of methylcyclohexane
and used in the next reaction without purification.
[(Mþ5)þ], 363[(Mþ1)-18]. 1H NMR (300 MHz, DMSO-d6):
d 7.85 (d,
J ¼ 2.4 Hz, 1H, ArH), 7.67 (d, J ¼ 8.7 Hz, 1H, ArH), 7.56 (d, J ¼ 8.4 Hz,
2H, ArH), 7.36 (d, J ¼ 8.4 Hz, 2H, ArH), 7.14 (dd, J ¼ 8.8 and 2.4 Hz,
1H, ArH), 2.21 (s, 3H, CH3).
4.1.1.1. Lithium salt of ethyl 3-methyl-2,4-dioxo-phenylbutanoate
(14). Yellow solid in 97% yield.
4.1.2.5. 1-(3-Chloro-4-fluorophenyl)-5-(4-chlorophenyl)-4-methyl-
1H-pyrazole-3- carboxylic acid (23). Light brown solid in 75% yield,
mp 169e171 ꢀC. FAB-MS m/z 365 [(Mþ1)þ], 367 [(Mþ3)þ], 347
[(Mþ1)-18]. 1H NMR (300 MHz, DMSO-d6):
d
7.6 (dd, J ¼ 6.6 and
4.1.1.2. Lithium salt of ethyl 4-(4-chlorophenyl)-3-methyl-2,4-
dioxobutanoate (15). Yellow solid in 89% yield.
2.7 Hz, 1H, ArH), 7.50 (d, J ¼ 8.4 Hz, 2H, ArH), 7.44 (t, J ¼ 9.0 Hz, 1H,
ArH), 7.28 (d, J ¼ 8.7 Hz, 2H, ArH), 7.19e7.14 (m,1H, ArH), 2.19 (s, 3H,
CH3).
4.1.1.3. Lithium salt of ethyl 4-(4-fluorophenyl)-3-methyl-2,4-
dioxobutanoate (16). Yellow solid in 93% yield.
4.1.2.6. 5-(4-Chlorophenyl)-1-(7-chloroquinolin-4-yl)-4-methyl-1H-
pyrazole-3- carboxylic acid (24). Yellow solid in 51% yield, mp
221e223 ꢀC. FAB-MS m/z 398 [(Mþ1)þ], 400 [(Mþ3)þ], 401
4.1.1.4. Lithium
salt
of
ethyl
3-methyl-2,4-dioxo-4-[4-(tri-
fluoromethyl)phenyl]butanoate (17). Yellow solid in 70% yield.
[(Mþ5)þ], 380 [(Mþ1)-18]. 1H NMR (300 MHz, DMSO-d6):
d 8.98 (d,
J ¼ 4.5 Hz, 1H, ArH), 8.78 (d, J ¼ 1.8 Hz, 1H, ArH), 7.71e7.62 (m 2H,
ArH), 7.53 (d, J ¼ 4.5 Hz, 1H, ArH), 7.36 (d, J ¼ 8.7 Hz, 2H, ArH), 7.20
(d, J ¼ 8.4 Hz, 2H, ArH), 2.27 (s, 3H, CH3).
4.1.1.5. Lithium salt of ethyl 4-(4-bromophenyl)-3-methyl-2,4-
dioxobutanoate (18). Yellow solid in 60% yield.

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114
4.1.2.7. 5-(4-Chlorophenyl)-4-methyl-1-[4-(trifluoromethyl)phenyl]-
1H-pyrazole-3-carboxylic acid (25). Light brown solid in 59% yield,
were filtered, dried over Na2SO4 and evaporated, which afforded
the desired N-Boc carbohydrazides in an acceptable purity.
mp 199e200 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
13.03(bs, 1H,
The unpurified N-Boc carbohydrazides were stirred with 1 mL of
TFA at room temperature for 2.5 h. Once the deprotection was
completed, the remnant TFA was co-distilled with 20 mL of CHCl3
(this procedure was repeated two more times). Finally, to the
resulting oil 20 mL of CHCl3 were added and 0.55 mmol of vanillin
and catalytic acetic acid were aggregated. The solution was stirred
for ca. 4 h at room temperature and then evaporated. The solid was
purified by column chromatography on silica gel (CHCl3 to
CHCl3eMeOH 9:1) to afford the desired carbohydrazydes 1e13.
ArCOOH), 7.79 (d, J ¼ 8.4 Hz, 2H, ArH), 7.52 (d, J ¼ 8.7 Hz, 2H, ArH),
7.46 (d, J ¼ 8.1 Hz, 2H, ArH), 7.30 (d, J ¼ 8.7 Hz, 2H, ArH), 2.20 (s, 3H,
CH3).
4.1.2.8. 1-(2,4-Difluorophenyl)-5-(4-fluorophenyl)-4-methyl-1H-pyr-
azole-3-carboxylic acid (26). White solid in 50% yield, mp
190e191 ꢀC. FAB-MS m/z 333 [(Mþ1)þ], 315 [(Mþ1)-18], 287
[(Mþ1)-46]. 1H NMR (300 MHz, DMSO-d6):
d 12.92 (bs, 1H,
ArCOOH), 7.73e7.65 (m,1H, ArH), 7.45e7.37 (m,1H, ArH), 7.30e7.20
(m, 5H, ArH), 2.21 (s, 3H, CH3).
4.1.3.1. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N0-[(Z)-(4-
hydroxy-3-methoxyphenyl)methylidene]-4-methyl-1H-pyrazole-3-
carbohydrazide (1). White solid in 68% yield; mp 256e258 ꢀC. 1H
4.1.2.9. 1-(3-Chloro-4-fluorophenyl)-5-(4-fluorophenyl)-4-methyl-
1H-pyrazole-3-carboxylic acid (27). Light brown solid in 68% yield,
mp 167e168 ꢀC. FAB-MS m/z 349 [(Mþ1)þ], 351 [(Mþ3)þ], 331
NMR (400 MHz, DMSO-d6):
d 2.29 (s, 3H, CH3), 3.83 (s, 3H, OCH3),
6.83 (d, J ¼ 8 Hz, 1H, ArH), 7.02 (dd, 1H, J ¼ 8 and 2 Hz, ArH), 7.27 (d,
J ¼ 8.4 Hz, 2H, ArH), 7.29 (d, J ¼ 2 Hz,1H, ArH), 7.47 (d, J ¼ 8.8 Hz, 2H,
ArH), 7.60 (dd, J ¼ 8.6 and 2.4 Hz, 1H, ArH), 7.78 (d, J ¼ 1.6 Hz, 1H,
ArH), 7.79 (d, J ¼ 8.8 Hz, 1H, ArH), 8.40 (s, 1H, NCH), 9.53 (s, 1H,
[(Mþ1)-18]. ]. 1H NMR (300 MHz, DMSO-d6):
d 12.98 (bs, 1H,
ArCOOH), 7.59 (dd, J ¼ 6.6 and 2.7 Hz, 1H, ArH), 7.45 (t, J ¼ 9.0 Hz,
1H, ArH), 7.35e7.25 (m, 4H, ArH), 7.22e7.16 (m,1H, ArH), 2.18 (s, 3H,
CH3).
ArOH), 11.60 (s, 1H, CO NH).13C NMR (100 MHz, DMSO- d6):
d 9.6,
56.0, 109.2, 115.8, 117.6, 122.6, 126.4, 127.6, 128.8, 129.2, 130.1, 131.8,
132.4, 132.6, 134.3, 135.6, 136.1, 143.1, 144.4, 148.5, 148.8, 149.4,
158.6. FTIR (cmꢂ1): 3536.96, 3166.88, 3075.88, 2984.14, 2941.87,
1645.54, 1602.45, 1508.51, 1387.98, 1266.22, 1116.45. FAB-MS (m/z):
529 [(Mþ1)]þ 531 [(Mþ2)]þ. HRFAB-MS m/z 528.0504 (calcd for
4.1.2.10. 1-(7-Chloroquinolin-4-yl)-5-(4-fluorophenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid (28). Light brown solid in 73% yield, mp
185e186 ꢀC. FAB-MS m/z 382 [(Mþ1)þ], 384 [(Mþ3)þ], 364
[(Mþ1)-18]. 1H NMR (300 MHz, DMSO-d6):
8.98 (d, J ¼ 4.5 Hz, 1H,
d
ArH), 8.20 (bs, 1H, ArH), 7.67 (d, J ¼ 1.2 Hz, 1H, ArH), 7.50 (d,
J ¼ 4.5 Hz, 1H, ArH), 7.34e7.23 (m, 3H, ArH), 7.18e7.12 (m, 2H, ArH),
2.29 (s, 3H, CH3).
C
25H19O3N345Cl3: 528.0517).
4.1.3.2. 1-(2,4-Dichlorophenyl)-N0-[(Z)-(4-hydroxy-3-
methoxyphenyl)methylidene]-4-methyl-5-phenyl-1H-pyrazole-3-
carbohydrazide (2). Beige solid in 69% yield; mp 239e241 ꢀC. 1H
4.1.2.11. 5-(4-Bromophenyl)-4-methyl-1-[4-(trifluoromethyl)
phenyl]-1H-pyrazole-3-carboxylic acid (29). Yellow solid in 60%
NMR (400 MHz, DMSO-d6):
d 2.29 (s, 3H, CH3), 3.83 (s, 3H, OCH3),
yield, mp 171e173 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d
13.03 (bs,
6.83 (d, J ¼ 8 Hz, 1H, ArH), 7.01 (dd, 1H, J ¼ 8.2 and 2 Hz, ArH),
7.25e7.22 (m, 2H, ArH), 7.28 (d, J ¼ 2 Hz, 1H, ArH), 7.41e7.34 (m, 3H,
ArH), 7.60 (dd, J ¼ 8.6 and 2.4 Hz, 1H, ArH), 7.76 (d, J ¼ 2.4 Hz, 1H,
ArH), 7.76 (d, J ¼ 8.4 Hz, 1H, ArH), 8.40 (s, 1H, NCH), 9.52 (s, 1H,
1H, ArCOOH), 7.80 (d, J ¼ 8.4 Hz, 2H, ArH), 7.52 (d, J ¼ 8.7 Hz, 2H,
ArH), 7.46 (d, J ¼ 8.4 Hz, 2H, ArH), 7.45 (d, J ¼ 8.4 Hz, 2H, ArH), 2.20
(s, 3H, CH3).
ArOH), 11.60 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6):
d 9.2,
4.1.2.12. 1-(2,4-Dichlorophenyl)-4-methyl-5-[4-(trifluoromethyl)
phenyl]-1H-pyrazole-3-carboxylic acid (30). Light orange solid in
81% yield, mp 156e158 ꢀC. FAB-MS m/z 415 [(Mþ1)þ], 417
55.5, 108.7, 115.4, 115.4, 122.1, 125.9, 128.19, 128.2, 128.6, 128.9,
129.51, 129.54, 132.0, 132.4, 135.0, 135.9,143.85, 143.89, 148.0, 148.2,
148.9, 158.3. FTIR (cmꢂ1): 3534.50, 3171.42, 3074.21, 1646.31,
1603.29, 1509.33, 1384.44, 1273.69, 1095.86. EI-MS m/z 494 [Mþ].
HREIꢂMS ¼ 494.0900 (calcd for C25H20O3N345Cl2: 494.0907).
[(Mþ3)þ], 397 [(Mþ1)-18]. 1H NMR (300 MHz, DMSO-d6):
d 7.82 (d,
J ¼ 2.4 Hz, 1H, ArH), 7.79 (d, J ¼ 8.1 Hz, 2H, ArH), 7.76 (d, J ¼ 8.7 Hz,
1H, ArH), 7.61 (dd, J ¼ 8.6 and 2.4 Hz, 1H, ArH), 7.46 (d, J ¼ 7.8 Hz,
2H, ArH), 2.29 (s, 3H, CH3).
4.1.3.3. N0-[(Z)-(4-hydroxy-3-methoxyphenyl)methylidene]-4-
methyl-1,5-diphenyl-1H-pyrazole-3-carbohydrazide (3). White solid
4.1.2.13. 1-(2,4-Difluorophenyl)-4-methyl-5-[4-(trifluoromethyl)
phenyl]-1H-pyrazole-3-carboxylic acid (31). Light brown solid in
in 62% yield; mp 291e292 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d 2.25
(s, 3H, CH3), 3.84 (s, 3H, OCH3), 6.84 (d, J ¼ 8 Hz, 1H, ArH), 7.04 (dd,
1H, J ¼ 8.2 and 2 Hz, ArH), 7.28e7.23 (m, 2H, ArH), 7.34e7.29 (m, 3H,
ArH), 7.45e7.36 (m, 6H, ArH), 8.40 (s, 1H, NCH), 9.52 (s, 1H, ArOH),
59% yield, mp 198e199 ꢀC. 1H NMR (300 MHz, DMSO-d6):
d 13.03
(bs, 1H, ArCOOH), 7.78 (d, J ¼ 8.4 Hz, 2H, ArH), 7.75e7.69 (m, 1H,
ArH), 7.46 (d, J ¼ 8.1 Hz, 2H, ArH), 7.44e7.40 (m, 1H, ArH), 7.30e7.23
(m, 1H, ArH), 2.25 (s, 3H, CH3).
11.52 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6):
d 9.1, 55.5,
108.8, 115.4, 117.7, 122.1, 125.3, 125.9, 128.0, 128.7, 128.7, 128.9, 129.1,
129.9, 139.2, 142.0, 143.3, 148.03, 148.05, 148.9, 158.5 FTIR (cmꢂ1):
3312.93, 3070.81, 3013.47, 2955.54, 2929.85, 1678.17, 1598.83,
1513.97, 1362.82, 1268.20, 1117.05. FAB-MS m/z 427
[(Mþ1)]þ.HRFAB-MS ¼ 427.1738 (calcd for C25H23O3N4: 427.1765).
4.1.3. General procedure for the synthesis of N0-[(Z)-(4-hydroxy-3-
methoxyphenyl)methylidene]-4-methyl-1H-pyrazole-3-
carbohydrazides (1e3)
To a stirred solution of 0.5 mmol of the corresponding 1,5-
diarylpyrazole-3-carboxylic acid (19e31) in 15 mL of toluene,
0.2 mL of SOCl2 was slowly added. The mixture was heated to reflux
for ca. 2.5 h. Then, the excess of solvent and SOCl2 was removed in
vacuo and the residue was dissolved in 10 mL of CHCl3. To this
stirred mixture at 0 ꢀC, a solution of 0.55 mmol of t-butyl carbazate
and 0.55 mmol of diisopropylethylamine (DIPEA) was added
dropwise. After the consumption of the acyl chloride, the chlor-
oformic solution was subsequently washed with citric acid 0.5 M
and saturated aqueous NaHCO3. The combined organic extracts
4.1.3.4. 5-(4-Chlorophenyl)-1-(3,4-dichlorophenyl)-N0-[(Z)-(4-
hydroxy-3-methoxyphenyl)methylidene]-4-methyl-1H-pyrazole-3-
carbohydrazide (4). White solid in 60% yield; mp 224e225 ꢀC. 1H
NMR (400 MHz, DMSO-d6):
d 2.23 (s, 3H, CH3), 3.84 (s, 3H, OCH3),
6.85 (d, J ¼ 8.4 Hz, 1H, ArH), 7.06 (dd, 1H, J ¼ 8.2 and 2.0 Hz, ArH),
7.14 (dd, J ¼ 8.8 and 2.4 Hz, 1H, ArH), 7.32 (d, J ¼ 2.4 Hz, 1H, ArH),
7.34 (d, J ¼ 8.4, 2H, ArH), 7.54 (d, J ¼ 8.4 Hz, 2H, ArH), 7.66 (d,
J ¼ 8.8 Hz, 1H, ArH), 7.81 (d, J ¼ 2.4 Hz, 1H, ArH), 8.40 (s, 1H, NCH),
9.54 (s, 1H, ArOH), 11.60 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-

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E. Hernandez-Vazquez et al. / European Journal of Medicinal Chemistry 100 (2015) 106e118
115
d6):
d
8.9, 55.5, 108.8, 115.4, 118.6, 122.2, 124,8, 125.8, 126.9, 127.4,
J ¼ 3.1 Hz), 125.9, 131.3 (d, J ¼ 10.6 Hz), 131.7 (d, J ¼ 8.6 Hz), 143.0,
144.1, 148.1, 148.2, 148.9, 156.4 (dd, J ¼ 251.7 and 13.4 Hz), 158.3,
162.2 (d, J ¼ 245.7 Hz),162.2(dd, J ¼ 248.7 and 11.5 Hz). FTIR (cmꢂ1):
3414.35, 3075.77, 3011.08, 2934.67, 1668.47, 1640.86, 1589.86,
1267.15, 1140.04. FAB-MS m/z 481 [(Mþ1)]þ. HRFAB-
MS ¼ 481.1468 (calcd for C25H20O3N4F3: 481.1482).
129.0, 130.5, 130.8, 131,6, 131.8, 134.0, 138.6, 140.8, 144.0, 148.1,
148.3, 149.0, 158.1. FTIR (cmꢂ1): 3538.62, 3249.38, 3098.37, 2925.57,
1655.38, 1599.43, 1509.20, 1363.81, 1268.71, 1127.16. FAB-MS m/z
529 [(Mþ1)]. HRFAB-MS ¼ 528.0464 (calcd for C25H19O3N345Cl3:
528.0517).
4.1.3.5. 1-(3-Chloro-4-fluorophenyl)-5-(4-chlorophenyl)-N0-[(Z)-(4-
hydroxy-3-methoxyphenyl)methylidene]-4-methyl-1H-pyrazole-3-
carbohydrazide (5). White solid in 62% yield; mp 210e212 ꢀC. 1H
4.1.3.9. 1-(3-Chloro-4-fluorophenyl)-5-(4-fluorophenyl)-N0-[(Z)-(4-
hydroxy-3-methoxyphenyl)methylidene]-4-methyl-1H-pyrazole-3-
carbohydrazide (9). Yellowish solid in 84% yield; mp 206e207 ꢀC.
NMR (400 MHz, DMSO-d6):
d
2.24 (s, 3H, CH3), 3.84 (s, 3H, OCH3),
1H NMR (400 MHz, DMSO-d6):
d 2.23 (s, 3H, CH3), 3.84 (s, 3H,
6.84 (d, J ¼ 8.0 Hz, 1H, ArH), 7.05 (dd, 1H, J ¼ 8.2 and 1.6 Hz, ArH),
7.21e7.17 (m, 1H, ArH), 7.34e7.31 (m, 3H, ArH), 7.46 (t, J ¼ 9.2 Hz,
1H, ArH), 7.53 (d, J ¼ 8.8 Hz, 2H, ArH), 7.78 (dd, J ¼ 6.6 and 2.8 Hz,
1H, ArH), 8.41 (s, 1H, NCH), 9.52 (s, 1H, ArOH), 11.56 (s, 1H,
OCH3), 6.84 (d, J ¼ 8 Hz, 1H, ArH), 7.05 (dd, J ¼ 8.2 and 2 Hz, 1H,
ArH), 7.21e7.17 (m, 1H, ArH), 7.37e7.28 (m, 5H, ArH), 7.44 (t, J ¼ 8.8.
Hz, ArH), 7.74 (dd, J ¼ 6.6 and 2.4 Hz, 1H, ArH), 8.41 (s, 1H, NCH),
9.55 (s, 1H, ArOH), 11.60 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-
CONH).13C NMR (100 MHz, DMSO-d6):
d
9.0, 55.5, 108.8, 115.4, 117.1
d6):
d
9.0, 55.6, 108.8, 115.4, 116.0 (d, J ¼ 21.6 Hz), 117.1 (d,
(d, J ¼ 22.4 Hz), 118.3, 119.9 (d, J ¼ 18.9 Hz), 122.2, 125.8, 125.9 (d,
J ¼ 7.6 Hz), 127.4, 127.7, 128.9, 131.9, 133.9,136.0 (d, J ¼ 3.5 Hz), 141.0,
143.7, 148.0, 148.2, 148.9, 156.5 (d, J ¼ 247.1 Hz), 158.2. FTIR (cmꢂ1):
3328.45, 3192.15, 3102.37, 2997.81, 1665.44, 1594.39, 1520.10,
J ¼ 22.3 Hz), 118.2, 119.9 (d, J ¼ 19.0 Hz), 122.2, 125.0 (d, J ¼ 3.0 Hz),
125.8 (d, J ¼ 8.2 Hz), 125.8, 127.6, 132.4 (d, J ¼ 8.5 Hz), 136.1 (d,
J ¼ 3.3 Hz), 141.3, 143.6, 148.1, 148.3, 149.0, 156.5 (d, J ¼ 247.3 Hz),
158.3, 162.3 (d, J ¼ 245.8 Hz). FTIR (cmꢂ1): 3326.20, 3224.58,
3104.14, 2997.21, 2933.401666.29, 1592.75, 1508.95, 1389.50,
1266.92, 1122.41. FAB-MS m/z 497 [(Mþ1)]þ. HRFAB-
MS ¼ 497.1168 (calcd for C25H20O3N345Cl1F2: 497.1187).
1390.39,
1267.18,
1123.19.
EI-MS
m/z
512(Mþ).
HREIꢂMS ¼ 512.0801 (calcd for C25H19O3N345Cl2F1: 512.0801).
4.1.3.6. 5-(4-Chlorophenyl)-1-(7-chloroquinolin-4-yl)-N'-[(Z)-(4-
hydroxy-3-methoxyphenyl)methylidene-4-methyl-1H-pyrazole-3-
carbohydrazide (6). Yellowish solid in 59% yield; mp 272e273 ꢀC.
4.1.3.10. 1-(7-Chloroquinolin-4-yl)-5-(4-fluorophenyl)-N0-[(Z)-(4-
hydroxy-3-methoxyphenyl)methylidene]-4-methyl-1H-pyrazole-3-
carbohydrazide (10). Light brown solid in 55% yield; mp
1H NMR (400 MHz, DMSO-d6):
d 2.35 s, 3H, CH3), 3.83 (s, 3H, OCH3),
6.83 (d, J ¼ 8 Hz, 1H, ArH), 7.01 (dd, J ¼ 8.2 and 2 Hz, 1H, ArH), 7.25
(d, J ¼ 8.8 Hz, 2H, ArH), 7.29 (d, J ¼ 2 Hz,1H, ArH), 7.39 (d, J ¼ 8.4, 2H,
ArH), 7.56 (d, J ¼ 4.8 Hz, 1H, ArH), 7.68 (dd, J ¼ 8.8 and 2.4 Hz, 1H,
ArH), 7.77 (d, J ¼ 8.8 Hz, 1H, ArH), 8.21 (d, J ¼ 2.4, 1H, ArH), 8.36 (s,
1H, NCH), 8.99 (d, J ¼ 4.8 Hz, 1H, ArH), 9.54 (s, 1H, ArOH), 11.61 (s,
289e291 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d 2.34 (s, 3H, CH3), 3.83
(S, 1H, OCH3), 6.83 (d, J ¼ 8 Hz, 1H, ArH), 7.02 (dd, J ¼ 8.2 and 2 Hz,
1H, ArH), 7.17 (t, J ¼ 8.8 Hz, 2H, ArH), 7.29e7.27 (m, 2H, ArH), 7.30
(d, J ¼ 2 Hz, 1H, ArH) 7.56 (d, J ¼ 4.4 Hz, 1H; ArH), 7.67 (dd, J ¼ 8.8
and 2.0 Hz, 1H, ArH), 7.75 (d, J ¼ 9.2 Hz, 1H, ArH), 8.19 (d, J ¼ 2.0 Hz,
1H, ArH), 8.36 (s, 1H, NCH), 8.99 (d, J ¼ 4.4 Hz, 1H, ArH), 9.54 (s, 1H;
1H, CONH). 13C NMR (100 MHz, DMSO-d6):
d 9.1, 55.5, 108.8, 115.4,
118.1, 120.5, 122.2, 122.6, 125.77, 125.79, 127.0, 127.9, 128.7, 128.4,
131.3, 133.9, 135.2, 142.9, 143.0, 144.8, 148.0, 148.5, 149.0, 149.2,
152.1, 158.1. FTIR (cmꢂ1): 3476.13, 3318.50, 3083.09, 2995.49,
2933.60, 1686.83, 1657.82, 1508.35, 1377.15, 1267.85 1123.72. EI-MS
m/z 545 (Mþ). HRꢂEIMS ¼ 545.0988 (calcd for C28H21O3N355Cl2:
545.1016).
ArOH), 11.60 (s, 1H, CONH). 13C NMR (100 MHz, DMSO-d6):
d 9.2,
55.6, 108.8, 115.4, 115.9 (d, J ¼ 21.7 Hz), 118.0, 120.5, 122.2, 122.7,
124.6 (d, J ¼ 3.2 Hz), 125.8, 128.0, 128.7, 131.9 (d, J ¼ 8.5 Hz), 135.2,
143.2, 143.3, 144.7, 148.1, 148.5, 149.0, 149.2, 152.1, 158.3, 162.2 (d,
J ¼ 245.8 Hz). FTIR (cmꢂ1): 3322.91, 3027.77, 2930.55, 2899.02,
1733.92, 1629.44, 1572.84, 1375.65, 1239.29, 1124.76. EI-MS m/z 529
(Mþ). HREꢂMS
¼
529.1300 (calcd para C
28H21O3N345Cl1F1:
4.1.3.7. 5-(4-Chlorophenyl)-N0-[(Z)-(4-hydroxy-3-methoxyphenyl)
methylidene]-4-methyl-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-
3-carbohydrazide (7). Yellowish solid in 51% yield; mp 184e185 ꢀC.
529.1300.
4.1.3.11. 5-(4-Bromophenyl)-N0-[(Z)-(4-hydroxy-3-methoxyphenyl)
methylidene]-4-methyl-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-
3-carbohydrazide (11). Brown solid in 78% yield; mp 289e290 ꢀC.
1H NMR (400 MHz, DMSO-d6):
d 2.24 (s, 3H, CH3), 3.83 (s, 3H,
OCH3), 6.84 (d, J ¼ 8 Hz, 1H, ArH), 7.05 (dd, J ¼ 8.2 and 2 Hz, 1H,
ArH), 7.34e7.31 (m, 3H, ArH), 7.55e7.52 (m, 4H, ArH), 7.82 (d,
J ¼ 8.4 Hz, 2H, ArH), 8.40 (s, 1H, NCH), 9.59 (s, 1H, ArOH), 11.62 (s,
1H NMR (400 MHz, DMSO-d6):
d 2.24 (s, 3H, CH3), 3.84 (s, 3H,
OCH3), 6.84 (d, J ¼ 7.8 Hz, 1H, ArH), 7.05 (dd, J ¼ 8.1 and 1.8 Hz, 1H,
ArH), 7.26 (d, J ¼ 8.7 Hz, 2H, ArH), 7.32 (d, J ¼ 1.8 Hz, 1H, ArH), 7.55
(d, J ¼ 8.4 Hz, 1H, ArH), 7.67 (d, J ¼ 8.4 Hz, 2H, ArH), 7.82 (d,
J ¼ 8.4 Hz, 1H, ArH), 8.40 (s, 1H, NCH), 9.59 (s, 1H, ArOH), 11.62 (s,
1H, CONH). 13C NMR (100 MHz, DMSO-d6): dd 9.0, 55.6, 108.9, 115.5,
118.8, 122.3, 122.7, 123.9 (q, J ¼ 270.9 Hz), 125.6, 125.8, 126.4 (q,
J ¼ 3.3 Hz), 128.0, 128.1 (q, J ¼ 32.1 Hz), 132.0, 132.1, 140.9, 142.1,
144.3, 148.1, 148.4, 149.0, 158.3. FTIR (cmꢂ1): 3543.35, 3211.82
3073.59, 2965.49, 2932.88, 1654.58, 1614.70, 1510.35, 1363.40,
1269.03, 1115.34. EI-MS m/z 572 (Mþ). HRꢂEMEI ¼ 572.0642 (calcd
for C26H20O3N749Br1F3: 572.0665).
1H, CONH). 13C NMR (100 MHz, DMSO-d6):
d 9.0, 55.6, 108.8, 115.5,
118.8,122.3,123.9 (q, J ¼ 270.9 Hz),125.6,125.9,126.4 (q, J ¼ 3.7 Hz),
127.6, 128.1 (q, J ¼ 32 Hz), 129.1, 131.9, 134.0, 140.9, 142.6, 144.3,
149.0, 148.4. FTIR (cmꢂ1): 3540.09, 3229.39, 3003.76, 2968.36,
2626.63, 1655.82, 1601.41, 1509.62, 1364.30, 1268.58, 1112.02. EI-MS
m/z 528 (Mþ). HREIꢂMS ¼ 528.1197 (calcd for C26H20O3N345Cl1F3:
528.1171).
4.1.3.8. 1-(2,4-Difluorophenyl)-5-(4-fluorophenyl)-N0-[(Z)-(4-
hydroxy-3-methoxyphenyl)methylidene]-4-methyl-1H-pyrazole-3-
carbohydrazide (8). Light pink solid in 57% yield; mp 241e242 ꢀC.
1H NMR (400 MHz, DMSO-d6):
d
2.27 (s, 3H, CH3), 3.83 (s, 3H,
4.1.3.12. 1-(2,4-Dichlorophenyl)-N0-[(Z)-(4-hydroxy-3-
methoxyphenyl)methylidene]-4-methyl-5-[4-(trifluoromethyl)
phenyl]-1H-pyrazole-3-carbohydrazide (12). Light pink solid in 76%
OCH3), 6.83 (d, J ¼ 8.4 Hz,1H, ArH), 7.02 (d, J ¼ 8.2 y 1.6 Hz, 1H, ArH),
7.33e7.23 (m, 6H, ArH), 7.44e7.39 (m, 1H, ArH), 7.80e7.74 (m, 1H,
ArH), 8.39 (s, 1H, NCH), 9.53 (s, 1H, ArOH), 11.60 (s, 1H, CONH). 13
C
yield; 250e251 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d 2.23 (s, 3H,
NMR (100 MHz, DMSO-d6):
d
9.1, 55.6, 105.05 (dd, J ¼ 28.6 and
CH3), 3.83 (s, 3H, OCH3), 6.83 (d, J ¼ 8.4 Hz,1H, ArH), 7.02 (dd, J ¼ 8.4
and 2 Hz, 1H, ArH), 7.30 (d, J ¼ 2 Hz, 1H, ArH), 7.49 (d, J ¼ 8.0 Hz, 2H,
ArH), 7.62 (dd, J ¼ 8.4 and 2.4 Hz, 1H, ArH), 7.78 (d, J ¼ 8.8 Hz, 2H,
23.7 Hz), 108.8, 112.3 (dd, J ¼ 21.4 and 1.8 Hz), 115.4, 115.8 (d,
J ¼ 21.5 Hz), 117.1, 122.2, 123.7 (dd J ¼ 12.3 and 3.6 Hz), 124.7 (d,

ꢀ ꢀ
E. Hernandez-Vazquez et al. / European Journal of Medicinal Chemistry 100 (2015) 106e118
116
ArH), 7.80 (d, J ¼ 2 Hz, 1H, ArH), 7.83 (d, J ¼ 8.4 Hz, 1H, ArH), 8.40 (s,
1H, NCH), 9.53 (s, 1H, ArOH), 11.6 (s, 1H, CONH).
13C NMR
(100 MHz, DMSO-d6): 9.1, 55.5, 108.8, 115.4, 117.7, 122.2, 123.9 (q,
J ¼ 270.7 Hz), 125.6 (q, J ¼ 3.8 Hz), 125.9, 128.4, 129.1 (q, J ¼ 31.9 Hz),
129.7, 130.4, 132.0, 132.1, 132.4, 135.3, 135.6, 142.4, 144.1, 148.0,
148.4, 148.9, 158.1. FTIR (cmꢂ1): 3240.65, 3097.74, 3044.12, 2969.86,
2936.80, 1640.40, 1583.88, 1516.88, 1376.32, 1263.22, 1124.64. FAB-
Regulations for Animal Experimentation and Care (NOM-062-ZOO-
1999, SAGARPA, Mexico) and approved by the Institutional Animal
Care and Use Committee based on the US National Institute for
Health (publication No. 85-23, revised 1985).
d.
d
4.5. Induction of diabetes
MS m/z 563(Mþ). HRFAB-MS
¼
563.0875 (calcd for
Diabetes was induced in overnight fasted rats by intraperitoneal
(i.p.) injection of 110 mg/kg of nicotinamide 15 min before an i.p.
injection of 65 mg/kg of streptozotocin dissolved in cold citrate
buffer (pH 4.5). Rats with hyperglycemia (i.e. those with blood
glucose between 200 and 350 mg/dL) were detected after 72 h
using a glucometer and these were used for antidiabetic screening.
C
26H20O3N345Cl2F3: 563.0859).
4.1.3.13. 1-(2,4-Difluorophenyl)-N0-[(Z)-(4-hydroxy-3-
methoxyphenyl)methylidene]-4-methyl-5-[4-(trifluoromethyl)
phenyl]-1H-pyrazole-3-carbohydrazide (13). White solid in 86%
yield; 245e246 ꢀC. 1H NMR (400 MHz, DMSO-d6):
d 2.31 (s, 3H,
CH3), 3.84 (s, 3H, OCH3), 6.84 (d, J ¼ 8 Hz, 1H, ArH), 7.03 (dd, J ¼ 8.2
and 2 Hz, 1H, ArH) 7.32e7.23 (m, 2H, ArH), 7.46e7.41 (m, 1H, ArH),
7.50 (d, J ¼ 8.0 Hz, 2H, ArH), 7.79 (d, J ¼ 8.0 Hz, 2H, ArH), 7.84e7.78
(m, 1H, ArH), 8.39 (s, 1H, NCH), 9.53 (s, 1H, ArOH), 11.64 (s, 1H,
4.6. In vivo assay for antidiabetic activity
All compounds (50 mg/kg) were dissolved in Tween 80 at 10% in
water (vehicle) and administered intragastrically to groups of five
diabetic rats; control groups of vehicle (1 mL/rat) and glibencla-
mide (5 mg/kg) were also administered for negative control and
drug reference. Blood samples were collected from the tail vein at 0,
3, 5 and 7 h after administration and glucose was measured using a
commercial glucometer. The percentage of glycemia variation was
calculated in relation to initial levels (0 h) using the formula: %
variation of glycemia ¼ [(Gx-G0)/G0] X 100, where G0 were the
initial glycemia values and Gx were the glycemia values at T1, T3, T5
and T7 h, respectively. All values are expressed as mean S.E.M. the
significance was estimated by an analysis of variance (ANOVA),
p < 0.05 implies significance [43].
CONH). 13C NMR (100 MHz, DMSO-d6):
d 9.0, 55.5, 105.1 (dd,
J ¼ 34.8 and 23.8 Hz), 108.8, 112.5 (dd, J ¼ 18.6 and 3.6 Hz), 115.4,
117.7, 122.2, 123.9 (q, J ¼ 270.8 Hz), 123.5 (dd, J ¼ 12.2 and
3.6 Hz),125.6, 125.9, 129.1 (q, J ¼ 32 Hz), 130.2, 131.2 (d, J ¼ 10.6 Hz),
132.5, 142.3, 144.3, 148.0, 148.3, 148.9, 156.3 (dd, J ¼ 251.6 and
13.1 Hz), 158.1, 162.3 (dd, J ¼ 248.5 and 11.4 Hz). FTIR (cmꢂ1):
3335.10, 3195.73, 3073.61, 2998.76, 1666.57, 1594.92, 1517.11,
1391.94,
1270.70,
1118.24.
EI-MS
m/z
530.
(Mþ).
HREIꢂMS ¼ 530.1333 (calcd for C26H19O3N4F5: 530.1372).
4.2. DPPH radical scavenging activity
DPPH radical scavenging activity was investigated according to
the method of Sreejayan and Rao [41]. Briefly, 2.95 ml of ethanolic
4.7. Computational details
solution of 100
mM DPPH and 0.05 mL of samples at different
All electronic calculations were performed with the Gaussian 09
package of programs [44] using a methodology previously reported
[45,46]. For the identification of the most stable hydrazone isomer,
geometry optimizations and frequency calculations of compounds
2 and 13 were carried out using the functional B3LYP and the 6-
31Gþ(d) basis set, in conjunction with the PCM [47] continuum
model using water as solvent. Since B3LYP has not be recom-
mended for kinetic studies, we employed an M06-2X [34] and the
6e31þþG(d,p) basis set, in conjunction with the SMD continuum
model using water as solvent [48] to carried out geometry opti-
mizations and frequency calculations. This methodology has been
previously used in other kinetic studies with high accuracy and
successfulness. Unrestricted calculations were used for open shell
systems. Local minima and transition states were identified by the
number of imaginary frequencies (0 and 1, respectively). Relative
energies are calculated with respect to the sum of the isolated re-
actants. Thermodynamic corrections at 298.15 K were included in
the calculation of relative energies, which correspond to 1 M
standard state. In addition the solvent cage effects have been
included according to the corrections proposed by Okuno [49],
taking into account the free volume theory [50]. The expression
used to correct the Gibbs free energy is:
concentrations were mixed. The control tube contains distilled
water instead of sample. After incubation for 20 min at room
temperature and in darkness the absorbance at 517 nm was
recorded. The antioxidant capacity is given as percent (%) DPPH
scavenging, calculated as [(optical density of control e optical
density of sample)/(optical density of control) x 100].
4.3. ORAC assay
The assays were performed in a Synergy™ HT Multi-Mode
Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA)
and were based on the method described by Huang et al. [42]. In
these assays 2,20-Azobis(2-methylpropionamidine) dihydro-
chloride (AAPH), a water-soluble azo compound, was used as a
peroxyl radical generator. Trolox,
analogue, was used as standard and fluorescein was used as fluo-
rescent probe. Briefly, 25 L of water, Trolox standards, diluted
samples and diluted vehicle were mixed with 25 L of 153 mM
AAPH and with 150
L of 50 nM fluorescein and incubated at 37 ꢀC.
a water-soluble vitamin E
m
m
m
The fluorescence was measured every min for a total of 90 min
using fluorescence filters for an excitation wavelength of 485 nm
and an emission wavelength of 520 nm. The ORAC values were
calculated using the net area under the curves and were expressed
n
h
i
o
FV
sol
DG ¼ DGs0ol ꢂ RT ln n10ð2nꢂ2Þ ꢂ ðn ꢂ 1Þ
(1)
as
mmol of Trolox equivalents (TE) per gram of samples.
4.4. Animals
where n represents the molecularity of the reaction. According to
expression (1), the entropy loss in solution causes
DG to decrease
Male Wistar rats with 200e250 g of body weight were housed at
by 2.54 kcal molꢂ1 for bimolecular reactions, at 298.15 K. This
correction is important because the cage effects of the solvent
reduce the entropy loss associated with any chemical reaction
whose molecularity is equal or larger than two.
standard laboratory conditions (22 3 ꢀC, 12 h day/night cycle), fed
with a rodent pellet diet and water ad libitum. Animals described as
‘fasted’ were deprived of food for 16 h, but had free access to water.
All animal procedures were conducted in accordance with Federal
The rate constants (k) were calculated using the Conventional

ꢀ
ꢀ
E. Hernandez-Vazquez et al. / European Journal of Medicinal Chemistry 100 (2015) 106e118
117
carotene on the antioxidative status in kidneys of rats with streptozotocin-
induced diabetes, Nahrung 39 (1995) 257e261.
Transition State Theory (TST) [51e53]:
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s
k ¼ sk
eꢂDG
(2)
kBT
h
RT
ꢀ
Hyiaman, S. Ohnuma, S.V. Ambudkar, J. Pickel, A. Makriyannis, G. Kunos, Pe-
ripheral CB1 cannabinoid receptor blockade improves cardiometabolic risk in
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where kB and h are the Boltzmann and Planck constants;
Gibbs free energy of activation;
generacy, accounting for the number of equivalent reaction paths;
and accounts for tunneling corrections. The latter, which are
defined as the Boltzmann average of the ratio of the quantum and
the classical probabilities, were calculated using the Zero Curvature
Tunneling corrections (ZCT) [54].
D
G
s is the
s
represents the reaction path de-
k
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F. Hernandez-Luis, Synthesis, hupoglycemic activity and molecular modeling
studies of pyrazole-3-carbohydrazides designed by a CoMFA model, Eur. J.
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A. Revol, Oxidative stress parameters in type I, type II and insulin-treated type
2 diabetes mellitus; insulin treatment efficiency, Clin. Chim. Acta 321 (2002)
89e96.
Financial support from the Instituto de Ciencia y Tecnología del
Distrito Federal (ICYTDF235/2010) is acknowledged. Eduardo
ꢀ
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ꢀ
Hernandez-Vazquez is granted to the CONACyT for his fellowship
(number 216082). We also thank to Rosa Isela del Villar, Georgina
ꢀ
ꢀ
Duarte, Margarita Guzman and Marisela Gutierrez for the analytical
support.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.06.010.
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