Synthesis and molecular docking of N′-arylidene-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbohydrazides as novel hypoglycemic and antioxidant dual agents

Article abstract of DOI:10.1016/j.bmc.2016.04.007

Herein, the design and synthesis of 10 novel N′-arylidene pyrazole-3-carbohydrazides are described. Compounds were pretended to act as dual agents against diabetes and oxidative stress, two correlated pathologies involved in metabolic syndrome development and progression. The antioxidant capacity was evaluated by means of DPPH and FRAP in vitro assays. It was found that compounds bearing a hydroxyl group at 4-position of the hydrazone moiety are potent antioxidant entities, being compound 3g (a syringaldehyde derivative) the most active compound. In addition, the in vivo hypoglycemic effect of the analogues was determined. With regard to the above, the cinnamaldehyde derivatives showed a scarce biological activity, while the 4-hydroxy analogues showed the higher glycemia reduction at 7 h after administration. Interestingly, the most potent antioxidants 3b and 3g also were of the most active compounds in reducing the plasma glucose, reaching 80% of reduction in the case of 3g. Molecular docking binding poses conducted to a plausible interpretation of the biological outcomes and a possible interaction between a hydroxy group and Asn287 of CB1R was proposed as an important feature for enhancing the observed activity.

Full text of DOI:10.1016/j.bmc.2016.04.007

Accepted Manuscript
Synthesis and molecular docking of N ´-arylidene-5-(4-chlorophenyl)-1-(3,4-
dichlorophenyl)-4-methyl-1H-pyrazole-3-carbohydrazides as novel hypoglyce-
mic and antioxidant dual agents
Eduardo Hernández-Vázquez, Sandybel Salgado-Barrera, Juan José Ramírez-
Espinosa, Samuel Estrada-Soto, Francisco Hernández-Luis
PII:
S0968-0896(16)30240-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.04.007
BMC 12921
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 February 2016
23 March 2016
1 April 2016
Please cite this article as: Hernández-Vázquez, E., Salgado-Barrera, S., Ramírez-Espinosa, J.J., Estrada-Soto, S.,
Hernández-Luis, F., Synthesis and molecular docking of ´-arylidene-5-(4-chlorophenyl)-1-(3,4-
N
dichlorophenyl)-4-methyl-1H-pyrazole-3-carbohydrazides as novel hypoglycemic and antioxidant dual agents,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.04.007
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Synthesis and molecular docking of N´-arylidene-5-(4-chlorophenyl)-1-
(3,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carbohydrazides as novel
hypoglycemic and antioxidant dual agents
Eduardo Hernández-Vázquez,a* Sandybel Salgado-Barrera,a Juan José Ramírez-
Espinosa,b Samuel Estrada-Soto,b Francisco Hernández-Luisa
a)
México, México, DF 04510, México.
b) Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos
Facultad de Química, Departamento de Farmacia, Universidad Nacional Autónoma de
62209, México
Keywords: diarylpyrazole hybrids, cannabinoid receptor 1, antioxidant capacity,
antidiabetic effect, molecular docking
*Corresponding author. Tel: +52 55 56225287, e-mail: eduardo.hervaz@gmail.com

Abstract
Herein, the design and synthesis of ten novel N´-arylidene pyrazole-3-carbohydrazides are
described. Compounds were pretended to act as dual agents against diabetes and oxidative stress,
two correlated pathologies involved in metabolic syndrome development and progression. The
antioxidant capacity was evaluated by means of DPPH and FRAP in vitro assays. It was found that
compounds bearing a hydroxyl group at 4-position of the hydrazone moiety are potent antioxidant
entities, being compound 3g (a syringaldehyde derivative) the most active compound. In addition,
the in vivo hypoglycemic effect of the analogues was determined. With regard to the above, the
cinnamaldehyde derivatives showed a scarce biological activity, while the 4-hydroxy analogues
showed the higher glycemia reduction at 7 h after administration. Interestingly, the most potent
antioxidants 3b and 3g also were of the most active compounds in reducing the plasma glucose,
reaching 80 % of reduction in the case of 3g. Molecular docking binding poses conducted to a
plausible interpretation of the biological outcomes and a possible interaction between a hydroxy
group and Asn287 of CB1R was proposed as an important feature for enhancing the observed
activity.
1. Introduction
Cannabinoid receptor 1 (CB1R) antagonists/inverse agonists are some of the most studied leads
directed for the treatment of obesity and metabolic syndrome [1, 2]. Since rimonabant (1, Figure 1)
was released as a selective CB1R antagonist, there has been an increase in the number of
research groups whose efforts focus in the optimization of this kind of molecules [3]. In this sense,
1,5-diarylpyrazole derivatives, such as rimonabant, have a plethora of physiological effects related
to food intake control and weight balance [4]. Among the physiological responses mediated by the
CB1R blockade, it is noteworthy the metabolic effects in peripheral organs (such as adipose tissue,
liver and skeletal muscle), which includes the reduction of adipogenesis, hypoglycemia,
thermogenesis, enhancement of lipogenesis, and adiponectin up-regulation [5–8]. The latter effects

not only contribute to weight loss in an independent manner of food intake, but it is also implicated
in insulin sensitization and lipid homeostasis. Nonetheless, rimonabant was withdrawn in 2009 due
to severe side effects such as depression and suicide [9], though it is still considered as a lead for
metabolic disorders treatment.
On the other hand, it is well documented that oxidative stress is a condition implicated in the
development and progression of diabetes and its complications [10, 11]. Oxidative stress
participates in pathogenesis of diabetes due to their ability to oxidize and damage lipids, DNA and
proteins and also by acting as signaling molecules to activate stress-sensitive pathways (i. e. NF-
B, p38 MAPK, SAPK) [12, 13], events that finally produce insulin resistance and -cell damage
[14–16].
Figure 1. Structures of the pyrazole derivative rimonabant (1) and the lead 2
During our current research concerning the design and synthesis of novel antidiabetic compounds,
we found that rimonabant analogues possess an in vivo hypoglycemic effect due to its interactions
with peripheral CB1R [17]. The latter discovery led us to the synthesis of the 1,5-diarylpyrazole
derivative 2 (Figure 1) [18], a dual compound that was demonstrated to exhibit both in vivo
hypoglycemic and in vitro antioxidant activities (in DPPH and ORAC tests). Interestingly, compound
2 bears a vanillinic hydrazone moiety that is responsible for its radical scavenging properties and
can be replaced for any other antioxidant portions. Hence, the main goal of this work was the
design and preparation of hybrids of compound 2 with several benzaldehyde and cinnamaldehyde
derivatives with different oxygenated patterns. Those modifications were selected to verify the

effects in the observed biological effect (both antidiabetic and antioxidant properties) (Figure 2).
Additionally, molecular docking studies on a previously modeled CB1R [17] were performed as a
computational method to explain the results and also confirm a plausible mechanism of action.
Figure 2. Optimization of compound 2. R1 corresponds to different hydroxylated patterns.
2.
Results and discussion
2.1. Chemistry
The synthesis of compounds was achieved by following the route showed in Scheme 1. First, 4-
chloropropiophenone 4 was treated with LiHMDS in methylcyclohexane and then reacted with
diethyl oxalate to obtain the tricarbonylic compound 5. Afterwards, the condensation of 5 with 3,4-
dichlorophenylhydrazine in sulfuric acid and ethanol gave the desired pyrazole ring, which upon a
basic hydrolysis provided the pyrazole-3-carboxylic acid 6. Stirring of 6 in SOCl2/PhMe and the
subsequent reaction with tert-butyl carbazate, produced the corresponding N´-Boc carbohydrazide
7. Finally, the cleavage of the Boc group with trifluoroacetic acid and the successive condensation
with the corresponding aromatic aldehyde generated the hydrazones 3a–3j in good yields. The
structures and purity of all compounds was confirmed by spectroscopic (IR and NMR) and
spectrometric (mass spectrometry) techniques (See supporting information). Notably, as was
previously reported [18], an E geometry of the hydrazone double bond is observed and was further
corroborated by the NOESY spectrum of compound 3h and 3j (Figure S1 and S2 in supporting
information). The structure of the diversely decorated N´-arylidenehydrazones obtained in this work
is shown in Table 1. It is interesting to note that the theoretical pKi values of compounds 3a–3j
(ranging from 7.9 to 8.28) on a highly predictable CoMFA model [7] (Table 1) are in the same

magnitude than rimonabant (experimental pKi = 7.94 [19]), which demonstrates that compounds
would probably have affinity to CB1R and thus a potential hypoglycemic effect.
Scheme 1. Synthesis of compounds 3a–3j. Reactions and conditions: (a) LiHMDS, diethyl oxalate,
MCH, r.t., 17 h; (b) 3,4-dichlorophenyl hydrazine, EtOH, H2SO4, reflux, 8 h; (c) KOH, EtOH, 50 °C,
12 h; (d) SOCl2, PhMe, reflux, 2 h; (e) tert-butyl carbazate, DIPEA, CHCl3, 0 °C to r.t., 4 h; (f) TFA,
then vanillin, AcOHcat, CHCl3, r.t.

Table 1. Structures and DPPH scavenging of N’-arylidenehydrazones synthesized in this work
% DPPH scavenging
b
IC50
Predicted
Compound
X
R1
Yield (%)
pKia
(M)
1 M
2.76
9.85
3.57
1.15
2.25
0.72
11.19
1.58
0.51
1.45
3.06
2.65
-
10 M
2.71
58.60
12.20
2.46
3.73
0.53
76.24
16.80
12.57
29.39
14.52
23.29
-
100 M
6.43
92.70
22.15
3.44
2.81
1.85
94.30
90.33
85.33
91.37
78.73
90.61
-
3a
CH
4-OH
82
83
75
78
73
68
54
58
47
61
-
8.03
8.02
8.00
8.28
8.05
7.99
8.22
8.09
8.15
8.01
-
ND
3b
CH
3,4-diOH
10.82  1.55
ND
3c
CH
2,4-diOH
3d
CH
3-OH,4-OMe
ND
3e
CH
3,4-diOMe
ND
3f
CH
3,4-(OCH2)2
ND
3g
3h
CH
4-OH,3,5-diOMe
8.67±0.25
43.41±2.39
50.09  1.96
32.60  1.52
47.72  2.31
35.78  1.74
31.74±1.04
(E)-CH-CH=CH
H
3i
(E)-CH-CH=CH
4-OH,3-OMe
3j
(E)-CH-CH=CH
4-OH,3,5-diOMe
Ferulic acid
Sinapic acid
Quercetin
-Tocopherol
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10.89±0.47
a) Obtained by a CoMFa model [Reference 6]
b) IC are reported as mean ± standard error (SEM) of n observations (n = 3). ND: not determined.
50
2.2. Biological activity
Having in hand hydrazones 3a–3j, we proceeded with the biological evaluation of compounds
employing both in vitro and in vivo methods. Since compounds were prepared with the main goal of
acting as dual compounds, this section was divided in two principal parts, one concerning to the
antioxidant capacity of the analogues and the second related to their potential in vivo hypoglycemic
effect. The outcomes of these studies are described below.

2.2.1. In vitro antioxidant assays
As described previously, there is a narrow relationship between the metabolic syndrome/diabetes
and oxidative stress [10, 11]; thus, chemical entities with antioxidant properties are desired in such
chronic diseases. According to the latter, derivatives 3a–3j are decorated with different
hydroxylated substituents and could exhibit antioxidant effect. Since several antioxidant
mechanisms have been attributed to guaiacol derivatives and phenolic compounds such as
Hydrogen Atom Transfer (HAT), Sequential Proton-Loss Electron Transfer (SPLET) and Single
Electron Transfer (SET) [20], we tested compounds in the DPPH scavenging test, a commonly used
antioxidant assay [21]. This method determines the capacity of compounds for neutralize radicals,
regardless its biological importance; however, it is a preliminary test to identify novel and potential
antioxidant entities. As shown in Table 1, none of the analogues lacking a hydroxyl group in the
hydrazone ring had an observable response (compounds 3d–3f), while compound 3a, whose only
substituent is hydroxyl at para position, neither demonstrated a scavenging activity. The preceding
observation clearly indicates that this group is a necessary but not a sufficient requirement for
exhibiting and antioxidant effect. Surprisingly, however, the cinnamaldehyde derivative 3h had a
significant 90 % of DPPH scavenging, though it lacks of substituents at phenyl ring. The latter
finding could be explained by the participation of additional hydrogen abstractions in other positions,
such as the amidic hydrogen, which conducts to a stabilized radical intermediate [18]. On the other
hand, compounds bearing two hydroxyl groups (3b and 3c) or those derived from well-known
antioxidant motifs (syringaldehyde, 3g; coniferylaldehyde, 3i and sinapinaldehyde, 3j), also
scavenged DPPH radical, ranging from 22 to 94 % of inhibition. Notably, analogues owning oxygen
substituents at both 2 or 3 and 4 positions (3b, 3g and 3i–3j) were the most active and their IC50 are
shown in Table 1. It is noteworthy that those derivatives had a similar or higher activity than ferulic
and sinapinic acid as well as the potent flavonoid quercetin, while the antioxidant effect of 3b and
3g was on the same magnitude as vitamin E (Table 1).

Figure 3 shows some plausible mechanisms in which compounds would scavenge DPPH radical;
after hydrogen abstraction caused by DPPH by a HAT pathway, the phenoxy radical can be
stabilized through resonance within the phenyl ring and even with the hydrazone moiety. On the
other hand, the hydrazone could donate an electron to DPPH radical (SET pathway) generating a
cationic-radical species, which would eventually lose a proton to produce the identical radical
intermediary as the HAT path.
Figure 3. Schematization of several antioxidant mechanisms that might be attributed to N´-
acylhydrazones 3a–3j. The figure shows the most active compound 3g.
Although some of the compounds (i.e. those derived from reported antioxidant portions) seemed to
be excellent scavengers in DPPH test, such radical has no biological relevance. For this reason, we
also evaluated the capacity of pyrazole derivatives 3a–3j for reducing Fe3+ to Fe2+ by employing the
ferric reducing antioxidant power (FRAP) test [22]. FRAP evaluates the antioxidant capacity by
means of a SET or SPLET paths (Figure 3). Despite iron is essential for cellular metabolism, its
accumulation may be a source of reactive species and thus oxidative stress [23, 24]. Similar
findings were detected as in the case of the DPPH scavenging test: compounds lacking a hydroxyl

group at para position (3d–3h) had a minimum effect in reducing ferric to ferrous iron, expressed as
trolox equivalents (mmol Trolox/g of compound) (Figure 4). The same outcome was observed in
the case of the 4-hydroxy derivative (3a) and the dihydroxylated (3c). However, the 4-hydroxy-3-
methoxy derivatives 3g, 3i and 3j, as well as the dihydroxylated 3b, had the higher effect in
reducing ferric iron, ranging from 20,046 to 45,667 trolox equivalents. Once more, the
syringaldehyde derivative 3g was the most active compound, reaching more than 45,000 trolox
equivalents in FRAP test. It is important to highlight that we do not discard the possibility of
compound 3b to coordinate with iron, a well-documented antioxidant mechanism of catechol
derivatives and polyphenols [25, 26].
Figure 4. Antioxidant capacity of hydrazones 3a–3j in FRAP test. Results are expressed as trolox
equivalents (mol trolox/g compound)  SEM (n = 4)
2.2.2. Antidiabetic study
It is well-known that 3-carbonyl-1,5-diarylpyrazole compounds are selective CB1R antagonists,
whose interaction with that receptor triggers several physiological responses, including glycemia
reduction [27]. Moreover, the predicted pKi (see Table 1) suggests that derivatives 3a–3j are
potential CB1R blockers. In order to confirm this hypothesis, we tested the compounds for their in
vivo hypoglycemic capacity on an experimental noninsulin-dependent diabetes mellitus (NIDDM)
model [28]. Compounds were evaluated at a dose of 50 mg/kg, which has been proven by our
group to be suitable for discriminating among active and non-active candidates [29]. Figure 5 shows
the plasma glucose reduction at 1, 3, 5 and 7 h after administration (for better understanding, the

derivatives were separated in two groups). It is note to worth that almost all pyrazole-3-
carbohydrazides showed a biological response since 3 h after administration and was sustained
until 7 h of the study, though the dimethoxy compound 3e did not reduce the plasma glucose in any
time of the antidiabetic experiment. This outcome clearly demonstrated that analogues must have a
free hydroxyl group at 3 or 4 positions to exert a biological response. According to the later,
compounds bearing a 4-hydroxy group (3a, 3b and 3g) showed a glucose reduction higher than 50
% at 7 hours after administration activity (70, 67 and 83 % of reduction for 3a, 3b and 3g,
respectively) and were even more active that the lead 2. Gratifyingly, there exists a correlation
between the antioxidant capacity and the glycemia reduction, in which the best antioxidant
derivatives 3b and 3g also demonstrated the higher glucose reduction. The increasing in the
biological effect could be attributed to either the enhancement of the affinity to CB1R or to the
antioxidant capacity, which could reduce oxidative stress that is involved in the development and
progression of diabetes. Surprisingly, however, the benzodioxane derivative 3f was also active in
the in vivo model, though it lacks of hydroxyl groups in its structure. One hypothesis for this
unexpected phenomenon is that 3f could suffer and oxidative dealkylation to produce a
hydroxylated compound and thus enhance the affinity to CB1R; however, further experimentation
must be done to corroborate it. On the other hand, the cinnamaldehyde derivatives showed a lower
glycemia reduction than the lead 2 and therefore demonstrating that the vinylogation of the
hydrazone moiety in this kind of compounds negatively impacts the biological effect, probably due
to disfavored interactions with CB1R. In summary, compounds 3a, 3b, 3f and 3g are promising
candidates for further optimization in the search for more potent antidiabetic compounds, especially
those with an intrinsic antioxidant capacity (i.e. 3b and 3g) because of their potential to lessen the
complications of metabolic syndrome and diabetes associated with oxidative stress.

Figure 5. Effect of
a single dose of hydrazones 3a–3e (A) and 3f–3j (B) in a
streptozotocin/nicotinamide rat model of diabetes (50 mg/kg; intragastric, n = 5). *p < 0.05; **p <
0.01; ***p < 0.001.
2.3. Molecular docking study
In order to corroborate the possible mechanism of action of pyrazole derivatives and understand the
detailed ligand-receptor interactions, we carried out a molecular docking study in a previously
published CB1R [17]. Such G protein-coupled receptor (GPCR) was modeled by using the B chain
of 1-adrenergic receptor from Meleagris gallopavo (PDB: 4AMJ, resolution of 2.30) [30] in the
SWISSMODEL workspace [31]. Compounds were docked into the binding site of CB1 model using
Autodock 4.2 program. The binding pose of each compound was selected according the existence
of a hydrogen bond between the carbonyl moiety of hydrazones with Lys192, which is reported to
be a crucial interaction for inverse agonist response [32]. Accordingly, the binding modes of the top-
score docking poses are shown in Figure 5. With exception of compound 3j, all analogues were
satisfactorily docked into the binding site of CB1R with exothermic G values (Table 2). Satisfyingly,
compounds show a similar pose as rimonabant does [33], in which the carbonyl group interacts with
Lys192 through a hydrogen bond and the aromatic rings point to a hydrophobic domain comprising
Phe170, 174, 177 and 200, TRP356 and Ile277 (Figure 6). It is note to worth that a - stacking
interaction is detected between Phe170 and the aromatic rings at 1 and 5 positions of the pyrazole
ring. These similarities suggest that the derivatives described in this work could reduce the plasma

glucose by CB1R interaction in the peripheral tissues [34]. Nonetheless, there is no relationship
between the binding energies and the observed hypoglycemic effect (Table 2) as the inactive
compound 3e also had a negative G value (-10.2 kcal/mol) which was even higher than those
calculated for the most active compounds 3b and 3g (-9.62 and -9.79 kcal/mol, respectively).
Notwithstanding, several asseverations can be concluded from Figure 6; first the cinnamaldehyde
derivatives 3h and 3i had a different conformation in the binding pocket of CB1R while 3j could not
be docked satisfactorily. This finding can be a plausible explanation of the moderate hypoglycemic
activity of those analogues. Similarly, the bicyclic analogue 3f also showed the same pose and thus
a moderate activity is expected; contrarily, 3f was one of the most potent compounds so that the
hypothesis of the enhancement of activity through metabolic dealkylation is supported. On the other
hand, the most active analogues had an additional interaction with Asn187, which is more
significant in hydroxy than alkoxy substituents. The last observation could explain the increased
hypoglycemic effect of 3b and 3g when compared with the 4-methoxy derivatives 3d and 3e.
Although we proposed a plausible explanation to the observed biological effect in base of the
computational outcomes, experimental CB1R binding assay is required to confirm the mechanism
of action of derivatives 3a–3j; those studies are currently underway in our laboratory.
Table 2. Gbind of hydrazones 3a–3j and rimonabant.
Compound

Gbind (kcal/mol)
-9.99
3a
3b
3c
3d
3e
3f
-9.62
-9.42
-9.38
-10.2
-9.87
3g
3h
3i
-9.79
-10.35
-9.51
3j
Rimonabant
(2)
--
-9.81

Figure 6. Predicted poses of hydrazones 3a–3j inside the binding pocket of CB1R. Dashed lines
illustrate hydrogen bonds between the carbonyl group and Lys192
Finally, the two-dimensional complex diagrams for rimonabant (1) and the most active 3g obtained
from the Poseview server [35] are shown in Figure 7. Both pyrazole derivatives share the hydrogen
bond with Lys192 and the hydrophobic interactions with Phe170 (- stacking). However, it was
soon discovered that compound 3g, as described previously, possesses another hydrogen bond
with Asn187, interaction that would enhance the affinity to CB1R and thus increasing the
hypoglycemic effect. Summarizing, we demonstrated that compounds would act as dual entities by
scavenging radicals through HAT and SET mechanisms and also reducing the glycemia in diabetic
rats via blockade of CB1R and become excellent candidates for further optimization and
investigation.

Figure 7. Two-dimensional complex diagrams of the binding pose of 1 (A) and compound 3g (B)
inside the binding pocket of CB1R.
3. Conclusions
Considering the close relationship between diabetes mellitus and metabolic syndrome, as well as
their association with oxidative stress, ten hybrids of CB1R antagonists were designed and
synthesized. The analogues included different hydroxylated patterns as a key feature for antioxidant
capacity. Accordingly, hydrazones from well-known antioxidant portions among with the 3,4-
dihydroxylated 3b were demonstrated to be potent antioxidant compounds by scavenging DPPH
radical and reducing Fe3+ to Fe2+ through HAT and SET mechanisms. Surprisingly, the IC50 of 3g
has the same order of magnitude than the effective -tocopherol and was more potent than
quercetin. Regarding the experimental NIDDM model, cinnamaldehyde-containing analogues
showed lower activity than the lead 2 and the dimethoxylated 3e lacked of biological effect.
Contrarily, compounds with a 4-hydroxy moiety on the hydrazone ring exhibited a remarkable
glycemia reduction and was more prominent than rimonabant and the lead 2. The most active
compounds were the potent antioxidants 3b and 3g (70 and 83 % of glycemia reduction,
respectively), the phenolic 3a (70 %) and, surprisingly, the benzodioxane derivative 3f, with a 90 %
of plasma glucose reduction, probably due to a metabolic dealkylation. Molecular docking on a
previously modeled CB1R showed that compounds would interact with this GPCR in a similar way
as rimonabant does, revealing a hydrogen bond with Lys192 and the aromatic rings interacting with

a phenylalanine-rich domain. Additionally, cinnamaldehyde derivatives possess a different
conformation that could account for the reduction in biological response, while the hydroxylated
analogues could interact with Asn187 through a hydrogen bond, a new proposed feature to
enhance the affinity to CB1R and increase the hypoglycemic effect. The results presented in this
work clearly corroborate the importance of considering CB1R as a biological target for the
generation of more potent hypoglycemic compounds for the treatment of diabetes and metabolic
syndrome, and place hydrazones 3a–3j as interesting antioxidant and antidiabetic entities.
4. Experimental
4.1. Chemistry
Solvents and reagents were purchased from Sigma-Aldrich and were used without purification.
Melting points were determined on a Büchi melting point apparatus, model B-540 and are
uncorrected. The progress of reactions and the purity of final products were monitored by thin layer
1
chromatography (TLC) whereas UV and iodine oxidation were used as revealing agents. H and 13
C
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 () are reported in ppm relative to the solvent signal. Elemental analyses were
conducted on a PerkinElmer® 2400 Series II CHNS/O instrument. Mass spectrometry spectra were
obtained using a Thermo- electron model DFS instrument. Glibenclamide, nicotinamide,
streptozotocin and Tween 80 were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
4.1.1. Synthesis of lithium salt of ethyl 3-methyl-2,4-dioxo-4-(4-chlorophenyl)butanoate (5)
To a solution of 4-chloropropiophenone 4 (15.06 mmol) in 3 mL of anhydrous methylcyclohexane
was added, at room temperature, a solution of lithium bis(trimethylsilyl)amide (16.1 mmol) in
anhydrous methylcyclohexane (5 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.

4.1.2. Synthesis of 5-(4-Chlorophenyl)-1-(3,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic
acid (6)
A mixture of the lithium salt 5 (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 x 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 x 15 mL). The aqueous layer was acidified with concentrated HCl until the pure
1
carboxylic acids precipitated. Light brown solid in 82 % yield, mp 203–204 °C. H NMR (300 MHz,
DMSO-d6):  7.85 (d, J = 2.4 Hz, 1H, ArH), 7.67 (d, J = 8.7 Hz, 1H, ArH), 7.56 (d, J = 8.4 Hz, 2H,
ArH), 7.36 (d, J = 8.4 Hz, 2H, ArH), 7.14 (dd, J = 8.8 and 2.4 Hz, 1H, ArH), 2.21 (s, 3H, CH3). MS
(FAB) m/z 381 [(M+1)+], 383 [(M+3)+], 385 [(M+5)+], 363[(M+1)-18].
4.1.3. General synthesis of N´-arylidene-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-4-methyl-1H-
pyrazole-3-carbohydrazides (3a–3j)
To a stirred solution of the 1,5-diarylpyrazole-3-carboxylic acid 6 (0.5 mmol) in 15 mL of toluene,
SOCl2 (0.2 mL) was slowly added. The mixture was heated to reflux for ca. 2.5. h. Then, the excess
of solvent and SOCl2 were removed in vacuo and the residue was dissolved in 10 mL of CHCl3. To
this stirred mixture at 0°C, a solution of t-butylcarbazate (0.55 mmol) and diisopropylethylamine
(DIPEA, 0.55 mmol) in 10 mL of CHCl3 was added dropwise. After the consumption of the acyl
chloride, the chloroformic solution was subsequently washed with citric acid 0.5 M and saturated
aqueous NaHCO3. The combined organic extracts were filtered, dried over Na2SO4 and evaporated,
which afforded the desired N-Boc carbohydrazide 7 in an acceptable purity.

The unpurified N-Boc carbohydrazide 7 was stirred with TFA (1 mL) 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, CHCl3 (20 mL) were added
and the corresponding aldehyde (0.55 mmol) and catalytic acetic acid were aggregated. The
solution was stirred for ca. 4 h at room temperature and then evaporated. The resulting mixture was
purified by column chromatography on silica gel (CHCl3 to CHCl3–MeOH 9:1) to afford the desired
carbohydrazydes 3a–3j.
4.1.3.1.
(E)-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-N'-(4-hydroxybenzylidene)-4-methyl-1H-
pyrazole-3-carbohydrazide (3a). Off-White solid in 82 % yield; mp 285–286 °C. IR (cm-1): 3323,
1
3184, 3095, 2925, 1667, 1541, 1598, 1512, 1475, 1219.55; H-NMR (DMSO-d6, δ ppm, 400 MHz):
2.23 (s, 3H, CH3Ar), 6.85 (AA´BB´, 2H, ArH), 7.13 (dd, J = 8.8 and 2.4 Hz, 1H, ArH), 7.33 (AA´BB¨,
2H, ArH), 7.53–7.56 (comp, 4H, ArH), 7.65 (d, J = 8.8 Hz, 1H, ArH), 7.81 (d, J = 2.4 Hz, 1H, ArH),
8.43 (s, 1H, N=CH), 9.94 (s, 1H. ArOH), 11.56 (s, 1H, NH); 13C-NMR (DMSO-d6, δ ppm, 100 MHz):
9.0, 115.8, 118.7, 124.9, 125.4, 127.0, 127.4, 128.8, 129.0, 130.6, 130.8, 131.6, 131.9, 134.0,
138.6, 140.9, 144.0, 148.2, 158.1, 159.4; MS (EI) m/z: 498; Anal. calcd for C24H17Cl3N4O2 C,57.68;
H, 3.43; N, 11.21; found C, 58.17; H, 2.81; N, 11.43.
4.1.3.2. (E)-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-N'-(3,4-dihydroxybenzylidene)-4-methyl-1H-
pyrazole-3-carbohydrazide (3d). White solid in 83 % yield; mp 244–245 °C. IR (cm-1): 3561, 3542,
1
3131, 1666, 1514.93, 1590, 1475, 1288.01; H-NMR (DMSO-d6, δ ppm, 400 MHz): 2.23 (s, 3H,
CH3Ar), 6.79 (d, J = 8 Hz, 1H, ArH), 6.92 (dd, J = 8.4 and 2 Hz, 1H, ArH), 7.13 (dd, J = 8.6 and 2.4
Hz, 1H, ArH), 7.23 (d, J = 2 Hz, 1H, ArH), 7.34 (AA´BB´, 2H, ArH), 7.54 (AA´BB´, 2H, ArH), 7.66 (d,
J = 8.8 Hz, 1H, ArH), 7.81 (d, J = 2.4 Hz, 1H, ArH), 8.35 (s, 1H, N=CH), 9.30 (br s, 1H, ArOH), 9.34
(br s, 1H, ArOH); 11.51 (s, 1H, NH); 13C-NMR (DMSO-d6, δ ppm, 100 MHz): 9.0, 112.7, 115.6,
118.6, 120.5, 124.9, 125.8, 126.9, 127.4, 129.0, 130.6, 130.8, 131.6, 131.9, 134.0, 138.6, 140.8,
144.0, 145.7, 147.9, 148.4, 158.1; MS (EI) m/z: 514; Anal. calcd for C24H17Cl3N4O3 C,55.89; H,
3.32; N, 10.86; found C, 55.86; H, 2.76; N, 11.08.
4.1.3.3. (E)-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-N'-(2,4-dihydroxybenzylidene)-4-methyl-1H-
pyrazole-3-carbohydrazide (3c) Beige solid in 75 % yield; mp 275–275 °C. IR (cm-1): 3321, 3252,
1
3093, 2978, 1669, 1536, 1536, 1477, 1217; H-NMR (DMSO-d6, δ ppm, 400 MHz): 2.24 (s, 3H,

CH3Ar), 6.32 (d, J = 2.4 Hz, 1H, ArH), 6.37 (dd, J = 8.4 and 2.4 Hz, 1H, ArH), 7.14 (dd, J = 8.6 and
2.4 Hz, 1H, ArH), 7.25 (d, J = 8.4 Hz, 1H, ArH), 7.34 (AA´BB´, 2H, ArH), 7.54 (AA´BB´, 2H, ArH),
7.67 (d, J = 8.8 Hz, 1H, ArH), 7.82 (d, J = 2.4 Hz, 1H, ArH), 8.61 (s, 1H, N=CH), 9.96 (s, 1H, ArOH),
11.52 (s, 1H, ArOH), 11.90 (s, 1H, NH); 13C-NMR (DMSO-d6, δ ppm, 100 MHz): 8.9, 102.7, 107.7,
110.5, 118.7, 124.8, 127.0, 127.3, 129.0, 130.6, 130.8, 131.4, 131.5, 131.8, 138.6, 139.0, 140.9,
143.5, 149.5, 157.8, 159.5, 160.6; MS (EI) m/z: 514; Anal. calcd for C24H17Cl3N4O3 C,55.89; H,
3.32; N, 10.86; found C, 56.07; H, 2.51; N, 11.32.
4.1.3.4.
(E)-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-N'-(3-hydroxy-4-methoxybenzylidene)-4-
methyl-1H-pyrazole-3-carbohydrazide (3d). White solid in 78 % yield; m.p. 240242 °C. IR (cm-1)
3530, 3172, 3074, 1646, 1602, 1511, 1377, 1279, 1095; 1H-NMR (DMSO-d6, δ ppm, 400 MHz): 2.24
(s, 3H, CH3Ar), 3.81 (s, 3H, OCH3), 6.98 (d, J = 8.4 Hz, 1H, ArH), 7.04 (dd, J = 8.4 and 2 Hz, 1H,
ArH), 7.13 (dd, J = 2.4 and 8.6 Hz, 1H, ArH), 7.26 (d, J = 2 Hz, 1H, ArH), 7.34 (AA´BB´, 2H, ArH),
7.55 (AA´BB´2H, ArH), 7.67 (d, J = 8.8 Hz, 1H, ArH), 7.82 (d, J = 2.8 Hz, 1H, ArH), 8.37 (s, 1H,
N=CH), 9.33 (s,1H, ArOH), 11.59 (s, 1H, NH); 13C-NMR (DMSO-d6, δ ppm, 100 MHz): 9.0, 55.6,
111.9, 112.3, 118.6, 120.2, 124.9, 127.0, 127.2, 127.4, 129.0, 130.6, 130.8, 131.6, 131.9, 134.0,
138.6, 140.9, 143.9, 146.9, 148.0, 149.8 158.2; MS (EI) m/z: 528; Anal. calcd for C24H19Cl3N4O3
C,56.68; H, 3.61; N, 10.58; found C, 56.37; H, 3.31; N, 10.32.
4.1.3.5. (E)-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-N'-(3,4-dimethoxybenzylidene)-4-methyl-1H-
pyrazole-3-carbohydrazide (3e). Beige solid in 73 % yield; mp 233–234 °C. IR (cm-1): 3184, 3078,
1
3007, 2959, 2933, 2839, 1648, 1512, 1575, 1479, 1265, 1240; H-NMR (DMSO-d6, δ ppm, 400
MHz): 2.23 (s, 3H, CH3Ar), 3.81 (s, 3H, CH3O), 3.83 (s, 3H, CH3O), 7.03 (d, J = 8.4 Hz, 1H, ArH),
7.14 (dd, J= 8.6 and 2.4 Hz, 1H, ArH), 7.17 (dd, J = 8.2 and 2 Hz, 1H, ArH), 7.34 (AA´BB´, 2H,
ArH), 7.35 (m, 1H, ArH), 7.54 (AA´BB´, 2H, ArH), 7.66 (d, J = 8.8 Hz, 1H, ArH), 7.82 (d, J = 2.4 Hz,
1H, ArH), 8.46 (s, 1H, N=CH), 11.65 (s, 1H, NH); 13C-NMR (DMSO-d6, δ ppm, 100 MHz): 9.0, 55.5,
55.6, 108.0, 111.5, 118.9, 122.0, 124.9, 127.0, 127.1, 127.4, 129.0, 130.6, 130.8, 131.6, 131.9,
134.0, 138.6, 140.9, 143.9, 148.0, 149.1, 150.8, 158.2. MS (EI) m/z: 542; Anal. calcd for
C26H21Cl3N4O3 C,57.42; H, 3.89; N, 10.30; found C, 57.56; H, 3.89; N, 10.50.
4.1.3.6.
(E)-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-N'-((2,3-dihydrobenzo[b][1,4]dioxin-6-
yl)methylene)-4-methyl-1H-pyrazole-3-carbohydrazide (3f). Off-white solid in 68 % yield; mp 205–

1
206 °C. IR (cm-1): 3330, 1549, 3064), 2923, 2876, 1670, 1506, 1477, 1259, 1240; H-NMR (DMSO-
d6, δ ppm, 400 MHz): 2.23 (s, 3H, CH3Ar), 4.28 (br s, 4H, (CH2O)2), 6.94 (d, J = 8.4 Hz, 1H, ArH),
7.14 (dd, J = 8.8 and 2.8 Hz, 1H, ArH), 7.17–7.21 (comp, 2H, ArH), 7.34 (AA´BB´, 2H, ArH), 7.55
(AA´BB´, 2H, ArH), 7.66 (d, J = 8.8 Hz, 1H, ArH), 7.82 (d, J = 2.8 Hz, 1H, ArH), 8.42 (s, 1H, N=CH),
11.65 (s, 1H, NH); 13C-NMR (DMSO-d6, δ ppm, 100 MHz): 9.0, 64.0, 64.3, 115.2, 117.5, 118.7,
120.7, 124.8, 126.9, 127.4, 127.8, 129.0, 130.6, 130.8, 131.6, 131.9, 134.0, 138.6, 140.9, 143.6,
143.9, 145.2, 147.5, 158.2; MS (EI) m/z: 540; Anal. calcd for C26H19Cl3N4O3 C,57.64; H, 3.53; N,
10.34; found C, 57.89; H, 2.82; N, 10.46.
4.1.3.7. (E)-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-N'-(4-hydroxy-3,5-dimethoxybenzylidene)-4-
methyl-1H-pyrazole-3-carbohydrazide (3g). Yellowish solid in 54 % yield; mp 269–279 °C. IR (cm-1):
1
3252, 3087, 3059, 2955, 2933, 2836, 1667, 1554, 1512, 1477, 1209; H-NMR (DMSO-d6, δ ppm,
400 MHz): 2.23 (s, 3H, CH3Ar), 3.83 (s, 6H, OCH3), 6.97 (s, 2H, ArH), 7.13 (dd, J = 8.6 and 2.4 Hz,
ArH), 7.34 (AA´BB´, 2H, ArH), 7.65 (AA´BB´, 2H, ArH), 7.66 (d, J = 8.8 Hz, 1H, ArH), 7.82 (d, J =
2.4 Hz, 1H, ArH), 8.41 (s, 1H, N=CH), 8.91 (s, 1H, ArOH), 11.63 (s, 1H, NH); 13C-NMR (DMSO-d6, δ
ppm, 100 MHz): 9.0, 56.0, 104.5, 118.6, 124.6, 124.9, 127.0, 127.4, 129.1, 130.6, 130.8, 131.6,
131.9, 134.0, 137.9, 138.6, 140.9, 144.0, 148.1, 148.5, 158.2; MS (EI) m/z: 558; Anal. calcd for
C26H21Cl3N4O4 C,55.78; H, 3.78; N, 10.01; found C, 56.13; H, 3.19; N, 10.21.
4.1.3.8.
(E)-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-4-methyl-N'-((E)-3-phenylallylidene)-1H-
pyrazole-3-carbohydrazide (3h). White solid in 58 % yield; mp 163–164 °C. IR (cm-1): 3381, 3001,
1
3027, 3060, 2925, 1655, 1548, 1477; H-NMR (DMSO-d6, δ ppm, 400 MHz): 2.23 (s, 3H, CH3Ar),
7.00 (d, J = 16 Hz, 1H, ArCH=CH), 7.07 (dd, J = 16 and 8.4 Hz, 1H, ArCH=CH), 7.14 (dd, J = 8.6
and 2.4 Hz, 1H, ArH), 7.35–7.31 (comp, 3H, ArH), 7.42–7.38 (m, 2H, ArH), 7.54 (AA´BB´, 2H, ArH),
7.64–7.62 (m, 2H, ArH), 7.67 (d, J = 8.8 Hz, 1H, ArH), 7.81 (d, J = 2.4 Hz, 1H, ArH), 8.33 (d, J = 8
Hz, 1H, N=CH), 11.69 (s, 1H, NH); 13C-NMR (DMSO-d6, δ ppm, 100 MHz): 9.0, 118.7, 125.0, 125.8,
127.08, 127.1, 127.4, 128.8, 129.0, 130.7, 130.8, 131.6, 131.9, 134.0, 135.9, 138.6, 138.9, 140.9,
143.8, 149.8, 158.3; MS (EI) m/z: 508; Anal. calcd for C26H19Cl3N4O C,61.25; H, 3.76; N, 10.99;
found C, 61.74; H, 3.22; N, 10.10.
4.1.3.9.
(E)-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-N'-((E)-3-(4-hydroxy-3-
methoxyphenyl)allylidene)-4-methyl-1H-pyrazole-3-carbohydrazide (3i). Orange solid in 47 % yield;

1
mp 213–214 °C. IR (cm-1): 3536, 3252, 3093, 2966, 2940, 2842, 1668, 1528, 1593, 1475, 1208; H-
NMR (DMSO-d6, δ ppm, 400 MHz): 2.23 (s, 3H, CH3Ar); 3.82 (s, 3H, OCH3), 6.78 (d, J = 8 Hz, 1H,
ArH), 6.87 (d, J = 16.4 Hz, 1H, ArCH=CH), 6.96 (dd, J = 16 and 8 Hz, 1H, ArCH=CH); 7.03 (dd, J =
8.2 and 2 Hz, 1H, ArH), 7.15 (dd, J = 8.8 and 2.4Hz, 1H, ArH), 7.2 (d, J = 2 Hz, 1H, ArH), 7.33
(AA´BB, 2H, ArH), 7.54 (AA´BB´, 2H, ArH), 7.67 (d, J = 8.6 Hz, 1H, ArH), 7.81 (d, J = 2.4 Hz, 1H,
ArH), 8.28 (d, J = 8.4 Hz, 1H, N=CH), 9.36 (s, 1H, ArOH), 11.58 (s, 1H, NH); 13C-NMR (DMSO-d6, δ
ppm, 100 MHz): 9.0, 55.6, 110.2, 115.6, 118.6, 121.3, 122.8, 124.9, 127.0, 127.4, 127.6, 129.0,
130.6, 130.8, 131.6, 131.9, 134.0, 138.6, 139.5, 140.9, 143.9, 147.8, 147.9, 150.4, 158.1; MS (EI)
m/z: 554; Anal. calcd for C27H21Cl3N4O3 C,58.34; H, 3.81; N, 10.08; found C, 58.43; H, 3.99; N,
9.66.
4.1.3.10.
(E)-5-(4-chlorophenyl)-1-(3,4-dichlorophenyl)-N'-((E)-3-(4-hydroxy-3,5-
dimethoxyphenyl)allylidene)-4-methyl-1H-pyrazole-3-carbohydrazide (3j). Yellow solid in 61 % yield;
mp 249–250 °C; IR (cm-1):3276, 3072, 3030, 2989, 2960, 2937, 2837, 1666, 1540, 1594, 1515,
1252, 1201; 1H-NMR (DMSO-d6, δ ppm, 400 MHz): 2.23 (s, 3H, CH3Ar), 3.81 (s, 6H, CH3O), 6.88 (d,
J = 16 Hz, 1H, ArCH=CH), 6.92 (s, 2H, ArH), 7.01 (dd, J = 25.8 and 9.2Hz, 1H, ArCH=CH), 7.14
(dd, J = 8.6 and 2.4 Hz, 1H, ArH), 7.33 (AA´BB, 2H, ArH), 7.54 (AA´BB´, 2H, ArH), 7.66 (d, J = 8.4
Hz, 1H, ArH), 7.81 (d, J = 2.4 Hz, 1H, ArH), 8.29 (d, J = 8.8 Hz, 1H, N=CH), 8.73 (s, 1H, ArOH),
11.59 (s, 1H, NH); 13C-NMR (DMSO-d6, δ ppm, 100 MHz): 9.1, 56.0, 104.8, 118.7, 123.4, 125.0,
126.5, 127.1, 127.4, 129.0, 130.6, 130.8, 131.6, 131.9, 134.0, 136.8, 138.6, 139.7, 140.9, 143.9,
148.1, 150.3, 158.1; MS (EI) m/z: 584; Anal. calcd for C28H23Cl3N4O4 C,57.40; H, 3.96; N, 9.56;
found C, 57.91; H, 3.72; N, 9.28.
4.2. In vitro antioxidant assays
4.2.1. DPPH-scavenging test
DPPH radical scavenging activity was investigated according to the method of Sreejayan and Rao
[36]. Briefly, 2.95 mL of ethanolic solution of 100 μM DPPH and 0.05 mL of samples at different
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 – optical density of sample)/(optical density of control) x 100]. The
samples where measured by triplicate and the results are represented as mean± standard error
(SEM).
4.2.2. FRAP test
The FRAP assay was conducted in accordance with Benzie & Strain with some modifications [37].
Briefly FRAP reagent was prepared by mixing 2.5 mL of TPTZ in HCl (40 mM), 2.5 g of FeCl3.6H2O
(20 mM) and 25 mL of acetate buffer (300 mM, pH = 3.6). Afterwards, 30 L of samples at different
concentrations or Trolox standards/vehicle, 90 L of deionizated water and 900 L of FRAP solution
mixture were mixed and left at rest in the dark for 30 min at 37 ºC. The absorbance of the samples
was measured in comparison to a blank at a wavelength of 595 nm. The FRAP values were
calculated using the net area under the curves and were expressed as μmoles of Trolox equivalents
per gram of samples.
4.3. Animals
Male Wistar rats with 200–250 g of body weight were housed at 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 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).
4.4. Induction of diabetes

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.
4.5. Antidiabetic model
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 glibenclamide
(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 = [(Gx-G0) / G0] X 100, where G0 were the
initial glycemia values and Gx were the glycemia values at 1, 3, 5 and 7 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 [38].
Acknowledgments
Financial support from the Instituto de Ciencia y Tecnología del Distrito Federal (ICYTDF235/2010)
is acknowledged. Eduardo Hernández-Vázquez is granted to the CONACyT for his fellowship
(number 216082). We also thank to Rosa Isela del Villar, Georgina Duarte, Margarita Guzmán and
Marisela Gutiérrez for the analytical support.
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Legends
Table 1. Structures and DPPH scavenging of N’-arylidenehydrazones synthesized in this work
Table 2. Gbind of hydrazones 3a–3j and rimonabant.
Scheme and figures captions
Scheme 1. Synthesis of compounds 3a–3j. Reactions and conditions: (a) LiHMDS, diethyl oxalate,
MCH, r.t., 17 h; (b) 3,4-dichlorophenyl hydrazine, EtOH, H2SO4, reflux, 8 h; (c) KOH, EtOH, 50 °C,
12 h; (d) SOCl2, PhMe, reflux, 2 h; (e) tert-butyl carbazate, DIPEA, CHCl3, 0 °C to r.t., 4 h; (f) TFA,
then vanillin, AcOHcat, CHCl3, r.t.
Figure 1. Structures of the pyrazole derivative rimonabant (1) and the hybrid 2
Figure 2. Optimization of compound 2. R1 corresponds to different hydroxylated patterns.

Figure 3. Schematization of several antioxidant mechanisms that might be attributed to N´-
acylhydrazones 3a–3j. The figure shows the most active compound 3g.
Figure 4. Antioxidant capacity of hydrazones 3a–3j in FRAP test. Results are expressed as trolox
equivalents (mol trolox/g compound)  SEM (n = 4)
Figure 5. Effect of
a single dose of hydrazones 3a–3e (A) and 3f–3j (B) in a
streptozotocin/nicotinamide rat model of diabetes (50 mg/kg; intragastric, n = 5). *p < 0.05; **p <
0.01; ***p < 0.001.
Figure 6. Predicted poses of hydrazones 3a–3j inside the binding pocket of CB1R. Dashed lines
illustrate hydrogen bonds between the carbonyl group and Lys192
Figure 7. Two-dimensional complex diagrams of the binding pose of 1 (A) and compound 3g (B)
inside the binding pocket of CB1R.

Graphical Abstract

Highlights
Ten pyrazole-based hybrids were synthesized as hypoglycemic and antioxidant entities
Compounds bearing a 4-hydroxy group showed antioxidant activity in DPPH and FRAP assays
Analogues 3f and 3g had a glycemia reduction in an in vivo model of 83 and 90 %, respectively
Molecular docking demonstrated that compounds could reduce glucose by blockade of CB1R