Synthesis, hypoglycemic activity and molecular modeling studies of pyrazole-3-carbohydrazides designed by a CoMFA model

Article abstract of DOI:10.1016/j.ejmech.2013.07.054

Diabetes and obesity are two universal health problems that constitute a research objective of several groups due to the lack of efficient and safe drug treatment. In this sense, cannabinoid receptor 1 (CB1) has attracted interest because of its role in food intake and metabolic balance, two targets in the control of metabolic syndrome. In this work, novel 1,5-diaryl pyrazole derivatives were synthesized in accordance with the pK_i prediction of a previously reported CoMFA model by our group. To further investigate the biological activity of these compounds in metabolic disorders, their hypoglycemic activity in an in vivo model was tested. Interestingly, a high degree of correlation was observed between the predicted pK_i and hypoglycemic effect 7 h after administration. Compounds 4, 9 and 13 showed the most significant plasma glucose reduction with decreases of 60%, 64% and 60% respectively. This result not only surpasses the activity of the lead rimonabant, but also that of the reference drug glibenclamide. Moreover, PASS prediction and molecular docking in an excellent validated homology model of CB1 suggest that these compounds would probably act as CB1 antagonists/inverse agonists and therefore, anti-obesity agents. The ligand-receptor complexes demonstrate that 1,5-diaryl pyrazole derivatives bind to the proposed binding site where a hydrophobic moiety interacts with the phenyl rings in the pyrazole nucleus and Lys192 forms a hydrogen bond with the oxygen of the carbonyl group. Dynamics simulations were also carried out to study the stability of the ligand-receptor complexes where the most active compounds showed smaller ΔG values and more hydrogen bonds throughout the simulation. These compounds are considered useful leads for further optimization in the treatment of such metabolic illnesses.

Full text of DOI:10.1016/j.ejmech.2013.07.054

Accepted Manuscript
Synthesis, hypoglycemic activity and molecular modeling studies of pyrazole-3-
carbohydrazides designed by a CoMFA model
Eduardo Hernández-Vázquez, Rodrigo Aguayo-Ortiz, Juan José Ramírez-Espinosa,
Samuel Estrada-Soto, Francisco Hernández-Luis
PII:
S0223-5234(13)00515-1
10.1016/j.ejmech.2013.07.054
EJMECH 6354
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 June 2013
Revised Date: 28 July 2013
Accepted Date: 30 July 2013
Please cite this article as: E. Hernández-Vázquez, R. Aguayo-Ortiz, J.J. Ramírez-Espinosa, S.
Estrada-Soto, F. Hernández-Luis, Synthesis, hypoglycemic activity and molecular modeling studies of
pyrazole-3-carbohydrazides designed by a CoMFA model, European Journal of Medicinal Chemistry
(2013), doi: 10.1016/j.ejmech.2013.07.054.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
1
Synthesis, hypoglycemic activity and molecular modeling studies of
pyrazole-3-carbohydrazides designed by a CoMFA model
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
a
a
b
b
Eduardo Hernández-Vázquez, Rodrigo Aguayo-Ortiz, Juan José Ramírez-Espinosa, Samuel Estrada-Soto,
Francisco Hernández-Luis *
a
a) Facultad de Química, Departamento de Farmacia, Universidad Nacional Autónoma de México, México,
DF 04510, México
b) Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos 62209,
México
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
Keywords: 1,5-diaryl pyrazole, cannabinoid receptor 1 antagonism, hypoglycemic activity, homology
modeling, molecular docking, molecular dynamics.
*Corresponding author. Tel: +52 55 56225287, e-mail: franher@servidor.unam.mx

ACCEPTED MANUSCRIPT
5
5
6
7
Abstract
5
5
6
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Diabetes and obesity are two universal health problems that constitute a research objective of several groups
due to the lack of efficient and safe drug treatment. In this sense, cannabinoid receptor 1 (CB1) has attracted
interest because of its role in food intake and metabolic balance, two targets in the control of metabolic
syndrome. In this work, novel 1,5-diaryl pyrazole derivatives were synthesized in accordance with the pKi
prediction of a previously reported CoMFA model by our group. To further investigate the biological activity
of these compounds in metabolic disorders, their hypoglycemic activity in an in vivo model was tested.
Interestingly, a high degree of correlation was observed between the predicted pK and hypoglycemic effect 7
i
hours after administration. Compounds 4, 9 and 13 showed the most significant plasma glucose reduction
with decreases of 60 %, 64 % and 60 % respectively. This result not only surpasses the activity of the lead
rimonabant, but also that of the reference drug glibenclamide. Moreover, PASS prediction and molecular
docking in an excellent validated homology model of CB1 suggest that these compounds would probably act
as CB1 antagonists/inverse agonists and therefore, antio-besity agents. The ligand-receptor complexes
demonstrate that 1,5-diaryl pyrazole derivatives bind to the proposed binding site where a hydrophobic
moiety interacts with the phenyl rings in the pyrazole nucleus and Lys192 forms a hydrogen bond with the
oxygen of the carbonyl group. Dynamics simulations were also carried out to study the stability of the ligand-
receptor complexes where the most active compounds showed smaller ∆G values and more hydrogen bonds
throughout the simulation. These compounds are considered useful leads for further optimization in the
treatment of such metabolic illnesses.
7
7
6
7
1. Introduction
7
7
8
8
8
8
8
8
9
0
1
2
3
4
Chronic degenerative diseases have become a major worldwide problem with steady rises in
morbidity and mortality over the years. Among these illnesses, obesity and insulin resistance, two major
components of the Metabolic Syndrome (MS) [1], have attracted the interest and efforts of several medicinal
chemistry research groups. The World Health Organization reported 1.4 billion overweight adults in 2004,
and of these, 500 million were obese [2]. Apart from the health, social, and psychological repercussions
related to them, obesity and insulin resistance also constitute risk factors in the development of other chronic
diseases such as diabetes mellitus type II (DMTII) and several cardiovascular diseases [3].

ACCEPTED MANUSCRIPT
8
8
8
8
8
9
9
9
9
9
9
9
9
9
9
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
Recent works reveal that 17–25 % of people suffer from metabolic syndrome with and body mass
index contributing to an increased risk [4, 5]. Due to the morbidity rates of MS and the lack of therapeutic
options that can offer integral management of this illness, new agents focused on the treatment of its
components are required. In this sense, 1,5-diaryl pyrazole derivatives such as N-(piperidin-1-yl)-5-(4-
chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, also known as rimonabant
(SR141716A, compound 1), have demonstrated interesting anti-obesity activity in several preclinical and
clinical trials [6–8]. These compounds present cannabinoid receptor antagonism, specifically the selective
blockade of cannabinoid receptor 1 (CB1) which is mainly located in the central nervous system and regulates
energy balance and food intake [9]. The antagonism of CB1 in peripheral organs such as liver, adipose tissue
and skeletal muscle has demonstrated interesting results by modulating metabolic functions, including the
reduction of adipogenesis, hypoglycemia by glucose uptake, thermogenesis, enhancement of lipogenesis, and
adiponectin up-regulation [10–12]. This not only contributes to weight loss independent of food intake, but it
is also implicated in insulin sensitization and lipid homeostasis. As a result, cannabinoid blockade is now
considered a potential route to the management of metabolic syndrome.
In order to rationalize the design of potent CB1 antagonists, CoMSIA and COMFA validated 3D-
QSAR models of 1,5-diaryl pyrazole derivatives (CB1 antagonists) were developed [13]. Based on this study
and others previously reported [14], it was found that the para position on the phenyl ring attached to C5 of
the pyrazole is an important moiety for increasing affinity to CB1 where hydrophobic, bulky, and electron
withdrawing groups are required (Figure 1). Moreover, the carbonyl group at the C3 position on the pyrazole
is also important because of its capacity to form a hydrogen bond with lysine 192 (K3.28), an interaction that
establishes the inverse agonist property of 1,5-diaryl pyrazoles [15, 16]. The methyl group at C4 and the aryls
on N1 and C5 are also important in determining the antagonism of ligands because they establish hydrophobic
and aromatic stacking interactions in the binding pocket [17].
1
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
10
11
<Insert Figure 1 here>
Considering the SAR mentioned above, novel 1,5-diaryl pyrazole analogs were designed and their CB1
receptor affinity was calculated with the CoMFA and CoMSIA models. Herein, the synthesis of some
pyrazole derivatives designed by a 3D-QSAR study and their evaluation in an in vivo model of DMTII are

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
reported. Molecular docking studies were also carried out on a homology-modeled CB1 receptor in order to
corroborate the CB1 affinity and thus explain a possible mechanism of action.
2. Results and discussion
2.1 Chemistry
The synthesis of target compounds 1–13 was carried out using a methodology derived from
previously published works [18, 19]. The enolates of substituted propiophenone derivatives were formed in
methylcyclohexane (MHC) by treatment with LiHMDS and were then treated with diethyl oxalate to obtain
the tricarbonylic lithium salt, compounds 19–31, with yields ranging from 60% to 97% (Scheme 1). The
cyclocondensation of previously obtained phenylhydrazines and unpurified tricarbonylic compounds in a
sulfuric acid-ethanol solution produced the ethyl pyrazole-5-carboxylates. These carboxylates were converted
to the corresponding carboxylic acids by treatment with potassium hydroxide at 50 °C, resulting in an overall
yield ranging from 50 to 87 %. Finally, the target products were obtained by formation of the acyl chloride
derivatives from pyrazole 5-carboxylic acids and the subsequent reaction with 1-aminopiperidine and DIPEA
in chloroform.
< Insert Scheme 1 here>
1
1
1
27
28
29
2.2 Hypoglycemic assay
Compounds 1–13 were evaluated for in vivo hypoglycemic activity using a STZ–nicotinamide rat
model of diabetes [20]. The anti-diabetic activity of compounds 1–13 was determined at 50 mg/kg of an
intragastric single dose, using glibenclamide as the positive control. Table 1 resumes the glycemia variations
at 7 hours after administration of the compounds. Compound 1 demonstrated good glycemia reduction (25.4
1
1
1
1
1
1
1
1
1
1
1
30
31
32
33
34
35
36
37
38
39
40
%) while compound 2, previously reported as a poor CB1 antagonist (pK = 6.930), showed no variation in
i
glycemia at 7 hours. Both compounds were evaluated in order to have an internal positive control (1) and
negative control (2) in the test. Interestingly, a high degree of correlation between CB1 pK and glycemia
i
reduction was observed so it can be hypothesized that a greater decrease in glycemia is indicative of an
improved affinity to CB1. This can be explained if the peripheral CB1 receptors are considered, specifically
those present in adipose tissue, skeletal muscle and liver, where the blockade of CB1 triggers several
metabolic functions such as glucose uptake and insulin sensitization [10]. A significant plasma glucose
reduction was observed in the rest of the set except in the case of compound 11. Although compound 11 did

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
not reduce the plasma glucose, probably due to an extensive metabolism, it is an important molecule because
it can be used as starting material for the synthesis of novel compounds via cross-coupling reactions, adding
to the library of compounds with hydrophobic and steric hindrance at this position, which is a key feature for
CB1 affinity. Surprisingly, compound 3, which lacks halogenated substitutions on the phenyl rings, showed a
greater hypoglycemic activity than other 5-(4-clorophenyl) compounds such as 5, 6 and rimonabant (1).
Although QSAR studies showed that halogenated atoms in the aryl groups at N1 and C5 of the pyrazole
enhance CB1 affinity and its predicted pK value was very low (pK = 5.683), compound 3 showed good
i
i
hypoglycemic activity. An explanation can be offered for this behavior is based on the logP values of the
compounds (Table 2), where compound 3 shows the lower logP value (3.043). This enhances aqueous
solubility and in consequence results in better bioavailability. However, further studies are necessary to
corroborate this statement and another possible mechanism of action cannot be discarded. A quinolone ring
instead of a phenyl ring did not reduce hypoglycemic activity like in the case of compounds 6 and 10, which
had glucose reduction similar to rimonabant (1). Among the compounds, the rimonabant isomer 4, C5 4-
flurophenyl compounds (8 and 9) and 4-(trifluoromethyl)phenyl compounds (12 and 13) showed the best
hypoglycemic activity of the set, with a higher decrease in glycemia with respect to glibenclamide (Figure 2
and 3). Figure 2 shows the glycemia variation of compounds 4, 8 and 9 during the 7 hours of the experiment.
The reduction of glycemia was time-dependent and the most important reduction was achieved 7 hours after
treatment with a 60 %, 55 % and 64 % of reduction for compounds 4, 8 and 9 respectively, all with a
demonstrating a significant difference when compared with glibenclamide. We can also predict, as seen in
Figure 2, that the effect would be higher after seven hours of the assay. Remarkably, these compounds have
better activity than the lead 1, both theoretically and in vivo.
< Insert Table 1 here>
< Insert Table 2 here>
The trifluoromethylated compounds 12 and 13 also showed a significant glycemia reduction
compared with untreated animals. Notably, these results correlate with the CoMFA prediction of the CB1 pKi,
where compounds 12 and 13 have the higher values (9.105 and 8.378 respectively). This could be a result of
the high steric hindrance and the electronegative potential produced by trifluoromethyl, which according to
QSAR-3D studies, are essential in enhancing the affinity to the transmembranal protein [13]. This effect

ACCEPTED MANUSCRIPT
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
increased with respect to time after the administration of the compounds and the most evident plasmatic
glucose reduction was observed at 7h (50 % of reduction for 12 and 60 % for 13) and it is supposed that the
effect is sustained for a long time, an important factor in potential drugs developed for the treatment of
chronic diseases such as diabetes. Summarizing, the hypoglycemic activity increased in order of
electronegative potential of groups attached at the para position of the phenyl ring at C5 of the pyrazole (Cl <
F, CF ), while substitution of hydrogen or chlorine atoms with fluorine on the phenyl ring at N1 enhanced
3
hypoglycemic activity (compounds 5, 8 and 9).
<Insert Figure 2>
<Insert Figure 3>
Compounds 3–13 were designed as CB1 antagonists and thus a weight reduction due to the control
of food intake and metabolic changes were expected. For this reason, the anti-obesity activity of compounds
was predicted by using the PASS® (Prediction of Activity Spectra for Substances) program [21]. PASS® is a
software designed for the prediction of general biological potential of organic drug-like molecules based on
the comparison between the chemical structures of the compounds designed and the structures (or
substructures) of well-known biologically active drugs included in the database of PASS®. Two parameters
are derived from this program: the probability “to be active” (Pa) and the probability “to be inactive” (Pi). A
Pa > 0.5 indicates that the compounds are very likely to reveal a specific activity in experiments. In
accordance with this information, the compounds have a high Pa value (Pa > 0.96) of anti-obesity activity
because they exhibit similar structures to known active compounds (i.e. rimonabant). Similarly, the
antidiabetic (type II) activity of the set was predicted. Even though Pa values were not high (Pa < 0.5), they
were meaningful. We have also demonstrated experimentally that 1,5-diaryl pyrazole compounds such as
compounds 2–13 are molecules capable of reducing the glycemia in diabetic rats and can be considered as
new biologically active chemical entities [22].
As the data suggests, these compounds showed in vivo antidiabetic activity even higher than
glibenclamide and rimonabant, and a high probable anti-obesity activity predicted with the PASS program so
they can be considered as leads for the design of molecules for the treatment of diabetes, obesity and in
consequence, metabolic syndrome.

ACCEPTED MANUSCRIPT
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
2.3. Homology modeling
In the absence of experimental information about the 3D coordinates of CB1, homology models were
generated based on the sequence similarity of previously solved GPCR structures. Table 3 shows the
BLASTp results obtained during the alignment of the partial CB1 sequence against the PDB database. The
comparative BLAST homologous search indicates that the β1 and β2-adrenergic receptors, rhodopsin and
adenosine A2A receptor were the most similar GPCR structures to CB1. It is noteworthy that a lipid G
protein-coupled receptor was recently deposited in the PDB, which shows the highest identity with the CB1
receptor sequence. Nevertheless, homology modeling studies are underway to validate this structure as a
template of CB1 receptor.
The template proposals from the GPCR-SSFE database [23, 24] were also tacken into account during
template selection. Interestingly, the β1-adrenergic receptor shows the highest sequence similarity in three
transmembrane helices (TMH1, 3 and 6) and for H8. This information, together with the sequence alignment
results, supports the use of the β1-adrenergic receptor as good template for CB1.
< Insert Table 3>
Three homology models of the CB1 receptor were generated using the B chain of β1-adrenergic
receptor structure as a template, determined at a resolution of 2.30 Å (PDB: 4AMJ). Table 4 shows the
evaluation of the models obtained by MODELLER 9v11, SWISS-MODEL and I-TASSER programs. The
best results were obtained with the SWISS-MODEL server (Fig. S1 and S2 in the Supplementary data), which
present the highest QMEAN6 score (0.44) and the lowest Z-score value (–3.81). Moreover, the
Ramachandran plot shows the highest reliability (98.2%) with 89.6% of the residues in the most favored zone.
Also, the isolated TMH were compared with the TMH model from the GPCR-SSFE database, where I-
TASSER model showed the lowest RMSD value. Nonetheless, only three of the seven TMHs presented a
RMSD bellow of 1.0 Å (data not shown), while in the case of the model generated by SWISS-MODEL, four
TMHs showed a lower value. Figure 4 shows the superposition of the TMHs from the model obtained from
the SWISS-MODEL server with the GPCR-SSFE TMH model. The table beneath the figure shows the amino
acid length of the TMHs and the RMSD values for each TMH. Indeed the quality of the SWISS-MODEL
CB1 model makes it the appropriate choice for embedding into a lipid bilayer and molecular docking studies.

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
The disulfide bond between C257 and C264 was included in the final CB1 model in order to evaluate
the proximity of the bridge with the binding site and to stabilize the extracellular domain during the
simulation.
< Insert Table 4 and Figure 4 here >
2.4. Construction of the receptor-membrane complex
The final model was embedded into an explicit POPC lipid bilayer in order to optimize the three-
dimensional structure in a more realistic environment. Figure 5A shows the hydrophobic surface
representation of the CB1 model. It is noteworthy that the hydrophobic amino acids are distributed almost
homogeneously in the lipid face space, while the hydrophilic amino acid residues are exposed to the
extracellular and cytoplasmic spaces. This result allowed us to identify the surface area that must be covered
during the lipid bilayer construction in order to generate a stable model.
Figure 5B shows the CB1 model after energy minimization in the lipid bilayer. The CB1-POPC
model exhibits seven TMHs, a cytoplasmic helix (H8), an extracellular N-terminus region, a cytoplasmic C-
terminus domain and three extracellular and three intracellular loops. The inner positive side [25] confirms an
increased presence of Lys and Arg residues in the cytoplasmic space (22 amino acids) compared to the
extracellular region (7 amino acids), showing the reliability of the model and the arrangement of the loops.
Furthermore, the disulfide bridge (C257–C264) was located at the extracellular loop 2 (EL2) next to the
binding pocket. These results were consistent with the expected structural features of GPCRs in a lipid bilayer
system [26].
< Insert Figure 5 here >
2.5. Docking studies and molecular dynamics simulations
To support the QSAR-calculated profile exerted by the synthesized derivatives, molecular docking
studies were carried out to understand the detailed ligand-receptor interactions. The compounds were docked
into the binding site of the CB1 model using AutoDock 4.2 program. It is well established that the inverse
agonist activity of rimonabant (1) derivatives is due to a hydrogen bond interaction between the carbonyl
group of the compound with the Lys192 [27]. Thus, the binding modes were selected based on the

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
conformations that promote this interaction. Figure 6 shows the binding modes of the top-score docking poses
for each compound and using compound 1 binding mode as a representative 2D schematic diagram. It is not a
surprise that the thirteen compounds present highly similar binding modes in the CB1 binding pocket. The
1,5-substituents are pointed towards the central transmembrane region, located in proximity of the
hydrophobic residues of Phe170, Phe174, Phe177, Gly195, Phe200, Trp279 and Trp356. Interestingly, a
possible π–π stacking (T-shaped configuration) interaction between the 5-substituent and the Phe170 is
observed in the PoseView schematic representation [28] (Figure 6B).
< Insert Figure 6 here >
On the other hand, the 3-substituent is pointed towards the extracellular region inserted within
TMH2, TMH3, TMH4 and EL2. This conformation is stabilized by a hydrogen bond interaction between the
carbonyl group and the Lys192 (H-bond distance of 2.21 Å in the case of compound 1). A hydrogen-bonding
between the piperidine group and the Lys192 was observed in all cases (H-bond distance of 2.48 Å). This
interaction could lead to the hydrogen transference between both groups, resulting in the protonated form of
the piperidine. The described interactions are typical of selective inverse agonists of CB1, confirming the
correlation between in vivo activity and binding mode.
Table 5 shows the binding free energies of the top-score docking poses of each compound compared
with the binding free energies calculated in the MD studies (see next subsection) and the QSAR-calculated
(pk ) results. In the case of binding free energies calculated from docking, all compounds showed negative
i
energies with a ∆G value under -8 kcal/mol. Although these results indicate that all compounds have a high
affinity to CB1 receptor, no further conclusions can be obtained in terms of differences in potency. In order to
propose a plausible explanation of the biological results and predicted pk , a 2 ns molecular dynamics was
i
performed. First, the stability of the ligands into the binding pocket was assessed. The stability of the ligand-
protein complex was considered as Root-Mean-Square Deviation (RMSD, Figure 8 and Table 6). As it can be
seen in Figure 7, compound 3 presents the higher RMSD in almost all the simulation (after 1 ns), and this
result could explain why this molecule shows the smaller predicted pK (5.683). However, as previously
i
described, compound 3 has demonstrated an important blood-glucose reduction in the in vivo test, such that
another mechanism could be responsible of the hypoglycemic activity.
< Insert Table 5 and Figure 7 here>

ACCEPTED MANUSCRIPT
2
2
2
2
2
2
2
2
2
2
2
81
82
83
84
85
86
87
88
89
90
91
Alternatively, compounds 7 and 11 also have high RMSD values probably due to the high steric
hindrance on the para position in the phenyl ring at N1. In the case of compound 13, the mild stability of the
complex could explain the lack of hypoglycemic activity as well as the hypothesis discussed in section 2.2.
The remaining compounds show a notably stable complex, with mean RMSD values lower than 0.34.
Noteworthy, the most active compounds, such as 4, 12 and 13 exhibited mean RMSD values lower than 0.30.
Moreover, the stability of these complexes can be seen as number of hydrogen bonds (Table 6); compounds 3,
7 and 11 have the lower number of hydrogen bonds (mean equal to 0.28, 0.51 and 0.82 respectively) while
compound 12, one of the most active compounds, has the maximum number of hydrogen bonds in the set
(2.22). The molecular dynamics not only demonstrated that these compounds produce a stable complex with
CB1 but also were served as a useful tool in explaining the observed biological activity.
< Insert Table 6 here >
2
2
2
92
93
94
3. Conclusions
Eleven novel compounds were designed using a CoMFA model. These compounds, along with rimonabant,
were synthesized and fully characterized by spectroscopic techniques. In order to evaluate the potential
hypoglycemic activity, an in vivo antidiabetic assay was performed where compounds 4, 8, 9 11 and 12
showed blood-glucose reduction higher than rimonabant and glibenclamide and in general, the predicted
results correlate with the observed biological activity.
2
2
2
2
2
3
3
3
3
3
3
3
95
96
97
98
99
00
01
02
03
04
05
06
Furthermore, a validated homology model of CB1 was achieved by using a β1-adrenergic receptor as
template. Docking of compounds with the CB1 homology-model resulted in ligand conformations and poses
in accordance to that previously reported for rimonabant, where the hydrogen bond with lys192 and the
interactions with the aromatic domains are conserved. These suggest a possible anti-obesity activity, as
observed with the PASS prediction. Molecular dynamics simulations corroborated the stability and mode of
action of compounds and could also be used as a tool for the interpretation of the observed biological activity
of some compounds. Hence, as result of these findings, compounds 4, 12 and 13 can be considered good
candidates in further optimization.
3
3
3
07
08
09
4. Experimental

ACCEPTED MANUSCRIPT
3
3
3
10
11
12
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 chromatography (TLC)
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1
13
whereas UV and iodine oxidation were used as revealing agents. H and CNMR spectra were obtained in
1
13
DMSO-d6 and CDCl solutions at 400 MHz (for H) and 100 MHz (for C) on Varian MR-400 (9.2 T)
3
instrument and on a Varian VNMRS-400 (9.2 T) instrument. Chemical shifts (δ) are reported in ppm relative
to the solvent signal.
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), while test strips and a glucometer were acquired from Roche (AccuChek Performa,
Mexico City, Mexico).
4.1.1. General procedure for the synthesis of lithium salt of Ethyl 3-methyl-2,4-dioxo-4-phenylbutanoates
(14–18)
A solution of the corresponding propiophenone (15.06 mmol) in 3 mL of anhydrous
methylcyclohexane was added at room temperature to a magnetically stirred solution of lithium
bis(trimethylsilyl)amide (16.1 mmol) in anhydrous methylcyclohexane (3–5 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.
3
3
31
32
4.1.1.1.Lithium salt of Ethyl 3-methyl-2,4-dioxo-phenylbutanoate (14)
3
3
3
3
3
3
33
34
35
36
37
38
Obtained as a yellow solid with a yield of 97 % yield.
4.1.1.2.Lithium salt of Ethyl 4-(4-chlorophenyl)-3-methyl-2,4-dioxobutanoate (15)
Obtained as a yellow solid with a yield of 89 % yield.
4.1.1.3.Lithium salt of Ethyl 4-(4-fluorophenyl)-3-methyl-2,4-dioxobutanoate (16)

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
Obtained as a yellow solid with a yield of 93 % yield.
4.1.1.4.Lithium salt of Ethyl 3-methyl-2,4-dioxo-4-[4-(trifluoromethyl)phenyl]butanoate (17)
Obtained as a yellow solid with a yield of 70 % yield.
4.1.1.5.Lithium salt of Ethyl 4-(4-bromophenyl)-3-methyl-2,4-dioxobutanoate (18)
Obtained as a yellow solid with a yield of 60 % yield.
4.1.2. General procedure for the synthesis of 1,5-Diarylpyrazole-3-carboxilic acids (19–31)
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) were suspended in 50 mL of EtOH and H SO was added
2
4
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 was added and the suspension was neutralized with NaHCO and then
3
extracted with CHCl (3 x 20 mL). The organic layer was successively dried over Na SO , filtered and
3
2
4
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 carboxylic acids precipitated.
4.1.2.1. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxilic acid (19)
Beige solid with a yield of 87 % yield, mp 208–210 °C. FAB-MS m/z 381 [(M+1)+], 383 [(M+3)+], 385
1
[(M+5)+], 363[(M+1)-18]. H NMR (400 MHz, DMSO-d ): δ 7.77 (d, J = 2.0 Hz, 1H, ArH), 7.69 (d, J = 8.4
6
Hz, 1H, ArH), 7.56 (dd, J = 8.8 y 2.4 Hz, 1H, ArH), 7.44 (d, J = 8.4 Hz, 2H, ArH), 7.21 (d, J = 8.4 Hz, 2H,
ArH), 2.23 (s, 3H, CH3).

ACCEPTED MANUSCRIPT
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
4.1.2.2. 1-(2,4-Dichlorophenyl)-4-methyl-5-phenyl-1H-pyrazole-3-carboxilic acid (20)
1
Light brown solid with a yield of 68 % yield, mp 189–191 °C. EI-MS m/z 346 [M+], 348 [(M+2)+]. H NMR
(300 MHz, DMSO-d ): δ 12.95 (bs, 1H, ArCOOH), 7.77 (d, J = 2.1 Hz, 1H, ArH), 7.69 (d, J = 8.4 Hz, 1H,
6
ArH), 7.56 (dd, J = 8.6 and 2.1 Hz, 1H, ArH), 7.41–7.34 (m, 3H, ArH), 7.22–7.19 (m, 3H, ArH), 2.23 (s, 3H,
CH3).
4.1.2.3. 4-Methyl-1,5-diphenyl-1H-pyrazole-3-carboxilic acid (21)
Yellowish solid with a yield of 74 % yield, mp 256–258 °C. FAB-MS m/z 279 [(M+1)+], 261 [(M+1)-18],
1
233 [(M+1)-46]. H NMR (300 MHz, DMSO-d ): δ 7.44-7.26 (m, 6H, ArH), 7.23–7.18 (m, 4H, ArH), 2.20 (s,
6
3H, CH3).
4.1.2.4. 5-(4-Chlorophenyl)-1-(3,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxilic acid (22)
Light brown solid with a yield of 82 % yield, mp 203–204 °C. FAB-MS m/z 381 [(M+1)+], 383 [(M+3)+],
1
385 [(M+5)+], 363[(M+1)-18]. H NMR (300 MHz, DMSO-d ): δ 7.85 (d, J = 2.4 Hz, 1H, ArH), 7.67 (d, J =
6
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).
4.1.2.5. 1-(3-Chloro-4-fluorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3- carboxilic acid (23)
Light brown solid with a yield of 75 % yield, mp 169–171 °C. FAB-MS m/z 365 [(M+1)+], 367 [(M+3)+],
1
347 [(M+1)-18]. H NMR (300 MHz, DMSO-d ): δ 7.6 (dd, J = 6.6 and 2.7 Hz, 1H, ArH), 7.50 (d, J = 8.4
6
Hz, 2H, ArH), 7.44 (t, J = 9.0 Hz, 1H, ArH), 7.28 (d, J = 8.7 Hz, 2H, ArH), 7.19–7.14 (m, 1H, ArH), 2.19 (s,
3H, CH3).
4.1.2.6. 5-(4-Chlorophenyl)-1-(7-chloroquinolin-4-yl)-4-methyl-1H-pyrazole-3- carboxilic acid (24)
Yellow solid with a yield of 51 % yield, mp 221–223 °C. FAB-MS m/z 398 [(M+1)+], 400 [(M+3)+], 401
1
[(M+5)+], 380 [(M+1)-18]. H NMR (300 MHz, DMSO-d ): δ 8.98 (d, J = 4.5 Hz, 1H, ArH), 8.78 (d, J = 1.8
6
Hz, 1H, ArH), 7.71–7.62 (m 2H, ArH), 7.53 (d, J = 4.5 Hz, 1H, ArH), 7.36 (d, J = 8.7 Hz, 2H, ArH), 7.20 (d,
J = 8.4 Hz, 2H, ArH), 2.27 (s, 3H, CH3).

ACCEPTED MANUSCRIPT
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
4.1.2.7. 5-(4-Chlorophenyl)-4-methyl-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxilic acid (25)
1
Light brown solid with a yield of 59 % yield, mp 199200°C. H NMR (300 MHz, DMSO-d ): δ 13.03(bs, 1H,
6
ArCOOH), 7.79 (d, J = 8.4 Hz, 2H, ArH), 7.52 (d, J = 8.7 Hz, 2H, ArH), 7.46 (d, J = 8.1 Hz, 2H, ArH), 7.30
(d, J = 8.7 Hz, 2H, ArH), 2.20 (s, 3H, CH3).
4.1.2.8. 1-(2,4-Difluorophenyl)-5-(4-fluorophenyl)-4-methyl-1H-pyrazole-3-carboxilic acid (26)
White solid with a yield of 50 % yield, mp 190–191 °C. FAB-MS m/z 333 [(M+1)+], 315 [(M+1)-18], 287
1
[(M+1)-46]. H NMR (300 MHz, DMSO-d ): δ 12.92 (bs, 1H, ArCOOH), 7.73–7.65 (m, 1H, ArH), 7.45–7.37
6
(m, 1H, ArH), 7.30–7.20 (m, 5H, ArH), 2.21 (s, 3H, CH3).
4.1.2.9. 1-(3-Chloro-4-fluorophenyl)-5-(4-fluorophenyl)-4-methyl-1H-pyrazole-3-carboxilic acid (27)
Light brown solid with a yield of 68 % yield, mp 167–168°C. FAB-MS m/z 349 [(M+1)+], 351 [(M+3)+],
1
331 [(M+1)-18]. ]. H NMR (300 MHz, DMSO-d ): δ 12.98 (bs, 1H, ArCOOH), 7.59 (dd, J = 6.6 and 2.7 Hz,
6
1H, ArH), 7.45 (t, J = 9.0 Hz, 1H, ArH), 7.35–7.25 (m, 4H, ArH), 7.22–7.16 (m, 1H, ArH), 2.18 (s, 3H, CH3).
4.1.2.10. 1-(7-Chloroquinolin-4-yl)-5-(4-fluorophenyl)-4-methyl-1H-pyrazole-3-carboxilic acid (28)
Light brown solid with a yield of 73 % yield, mp 185–186 °C. FAB-MS m/z 382 [(M+1)+], 384 [(M+3)+],
1
364 [(M+1)-18]. H NMR (300 MHz, DMSO-d ): δ 8.98 (d, J = 4.5 Hz, 1H, ArH), 8.20 (bs, 1H, ArH), 7.67
6
(d, J = 1.2 Hz, 1H, ArH), 7.50 (d, J = 4.5 Hz, 1H, ArH), 7.34–7.23 (m, 3H, ArH), 7.18–7.12 (m, 2H, ArH),
2.29 (s, 3H, CH3).
4.1.2.11. 5-(4-Bromophenyl)-4-methyl-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxilic acid (29)
1
Yellow solid with a yield of 60 % yield, mp 171–173 °C. H NMR (300 MHz, DMSO-d ): δ 13.03 (bs, 1H,
6
ArCOOH), 7.80 (d, J = 8.4 Hz, 2H, ArH), 7.52 (d, J = 8.7 Hz, 2H, ArH), 7.46 (d, J = 8.4 Hz, 2H, ArH), 7.45
(d, J = 8.4 Hz, 2H, ArH), 2.20 (s, 3H, CH3).
4.1.2.12. 1-(2,4-Dichlorophenyl)-4-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxilic acid (30)

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
Light orange solid with a yield of 81 % yield, mp 156–158 °C. FAB-MS m/z 415 [(M+1)+], 417 [(M+3)+],
1
397 [(M+1)-18]. ]. H NMR (300 MHz, DMSO-d ): δ 7.82 (d, J = 2.4 Hz, 1H, ArH), 7.79 (d, J = 8.1 Hz, 2H,
6
ArH), 7.76 (d, J = 8.7 Hz, 1H, ArH), 7.61 (dd, J = 8.6 and 2.4 Hz, 1H, ArH), 7.46 (d, J = 7.8 Hz, 2H, ArH),
2.29 (s, 3H, CH3).
4.1.2.13. 1-(2,4-Difluorophenyl)-4-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxilic acid (31)
1
Light brown solid with a yield of 59 % yield, mp 198 – 199 °C. H NMR (300 MHz, DMSO-d ): δ 13.03 (bs,
6
1H, ArCOOH), 7.78 (d, J = 8.4 Hz, 2H, ArH), 7.75–7.69 (m, 1H, ArH), 7.46 (d, J = 8.1 Hz, 2H, ArH), 7.44–
7.40 (m, 1H, ArH), 7.30–7.23 (m, 1H, ArH), 2.25 (s, 3H, CH3).
4.1.3. General procedure for the synthesis of N-(1-Piperidinyl)-1,5-diarylpyrazol-3-carboxamides (1–13)
A solution of the crude 1,5-diarylpyrazole acid (0.7 mmol) was suspended in Toluene (25 mL) and SOCl (2.8
2
mmol) and it was heated to 110 °C for 2.5 h. The excess of SOCl was removed by co-destilation with PhMe
2
(3 x 25 mL) and the residue was dissolved in 10 mL of CHCl . A solution of 1-aminopiperidine (3.1 mmol)
3
and DIPEA (3.1 mmol) in 10 mL of CHCl was added dropwise at 0°C. The reaction was stirred with an
3
anhydrous trap from 0°C to room temperature for 4 h. The solution was successively washed with a solution
of citric acid 0.02 M (3 x 10 mL), NaHCO pH 10 (3 x 10 mL) and a saturated solution of NaCl (10 mL). The
3
organic layer was dried over Na SO , filtered and concentrated in vacuo to obtain the crude carbohydrazides.
2
4
Purification was carried out by column chromatography (CHCl to CHCl -MeOH, 95:5) which afforded the
3
3
carbohydrazides 1–13 as solids.
4.1.3.1. 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide
(1)
1
White solid with a yield of 63 % yield, mp 151–152. H NMR (400 MHz, DMSO-d ): δ 9.05 (s, 1H, NH),
6
7.74 (d, J = 8.4 Hz, 1H ArH), 7.74 (d, J = 2.4 Hz, 1H, ArH), 7.57 (dd, J = 8.6 and 2.4 Hz, 1H, ArH), 7.44 (d,
J = 8.8 Hz, 2H, ArH), 7.22 (d, J = 8.4 Hz, 2H, ArH), 2.77 (t, J = 5.6 Hz, 4H, NCH ), 2.21 (s, 3H, CH ), 1.57
2
3
(quint, J = 5.6 Hz, 4H, CH ), 1.38–1.28 (m, 2H, CH ).
2
2

ACCEPTED MANUSCRIPT
1
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
C NMR (100 MHz, DMSO-d ): δ 159.4, 144.5, 142.4, 135.8, 135.1, 133.8, 132.1, 132.0, 131.3 (2C), 129.6,
6
128.8 (2C), 128.3, 127.3, 116.6, 55.3, 25.4, 23.1, 9.1.
FAB-MS m/z 463 [(M+1)+], 465 [(M+3)+], 467 [(M+5)+], 363 [(M+1)-100]. HR-FAB-MS m/z 463.0803
1
3
(calcd for C H O N Cl : 463.0854).
2
2
22
1
4
3
4.1.3.2. 1-(2,4-Dichlorophenyl)-N-(1-piperidinyl)-4-methyl-5-phenyl-1H-pyrazole-3-carboxamide (2)
1
Yellowish-white solid with a yield of 85 % yield, mp 190 °C. H NMR (400 MHz, DMSO-d ): δ 9.02 (s, 1H,
6
NH), 7.720–7.70 (m, 2H, ArH), 7.54 (dd, J = 8.6 and 2.4 Hz, 1H, ArH), 7.38–7.31 (m, 3H, ArH), 7.20–7.17
(m, 2H, ArH), 2.762 (t, J = 5.2 Hz, 4H, NCH ), 2.21 (s, 3H, CH ), 1.563 (quin, J = 5.2 Hz, 4H, CH ), 1.37–
2
3
2
1.27 (m, 2H, CH2).
1
3
C NMR (100 MHz, DMSO-d ): δ 159.9, 144.8, 144.0, 136.5, 135.3, 132.7, 132.5, 129.9, 129.8 (2C), 129.2,
6
129.0 (2C), 128.8, 128.6, 116.6, 55.7, 25.8, 23.5, 9.6
FAB-MS m/z 429 [(M+1)+], 431 [(M+3)+], 433 [(M+5)+], 329 [(M+1)-100]. HR-FAB-MS m/z 429.1228
1
3
(calcd for C H O N Cl : 429.1243).
2
2
23
1
4
2
4.1.3.3. 4-Methyl-1,5-diphenyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide (3)
1
White solid with a yield of 80 % yield, mp 188–189°C. H NMR (400 MHz, DMSO-d ): δ 9.03 (s, 1H, NH),
6
7.43–7.31 (m, 6H, ArH), 7.29–7.24 (m, 2H, ArH), 7.21–7.16 (m, 2H, ArH), 2.79 (t, J = 7.2 Hz, 4H, NCH2),
2.18 (s, 3H, CH ), a 1.59 (quint, J = 6.4 Hz, 4H, CH ), 1.40–1.29 (m, 2H, CH ).
3
2
2
1
3
C NMR (100 MHz, DMSO-d ): δ 159.7, 143.8, 141.7, 139.3, 129.8 (2C), 129.2, 128.9 (2C), 128.63, 128.60
6
(2C), 127.8, 125.3 (2C), 117.1, 55.4, 25.4, 23.0, 9.0.
FAB-MS m/z 361 [(M+1)+], 261 [(M+1)-100]. HR-FAB-MS m/z 361.1988 (calcd for C H O N :
2
2
25
1
4
361.2023).
4.1.3.4. 5-(4-Chlorophenyl)-1-(3,4-dichlorophenyl)-4-methyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide
(4)
1
White solid with a yield of 50 % yield, mp 113–114 °C. H NMR (400 MHz, DMSO-d ): δ 9.14 (s, 1H, NH),
6
7.75 (d, J = 2.4 Hz, 1H, ArH), 7.63 (d, J = 8.4 Hz, 1H, ArH), 7.52 (d, J = 8.4 Hz, 2H, ArH), 7.29 (d, J = 8.8

ACCEPTED MANUSCRIPT
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
Hz, 2H, ArH), 7.10 (dd, J = 8.4 and 2.4 Hz, 1H, ArH), 2.81 (t, J = 5.6 Hz, 4H, NHCH ), 2.16 (2, 3H, CH ),
2
3
1.59 (quint, J = 1.59, 4H, CH ), 1.40–1.31 (m, 2H, CH ).
2
2
1
3
C NMR (100 MHz, DMSO-d ): δ 159.4, 144.5, 140.6, 138.7, 133.9, 131.8 (2C), 131.5, 130.8, 130.4, 129.0
6
(2C), 127.6, 126.9, 124.9, 118.1, 55.4, 25.4, 23.1, 9.0.
FAB-MS m/z 463 [(M+1)+], 465 [(M+3)+], 467 [(M+5)+], 363 [(M+1)-100]. HR-FAB-MS m/z 463.0826
1
3
(calcd for C H O N Cl : 463.0854).
2
2
22
1
4
3
4.1.3.5.
carboxamide (5)
1-(3-Chloro-4-fluorophenyl)-5-(4-chlorophenyl)-4-methyl-N-(1-piperidinyl)-1H-pyrazole-3-
1
Beige solid with a yield of 56 % yield, mp 150–151 °C. H NMR (400 MHz, DMSO-d ): δ 9.13 (s, 1H, NH),
6
7.73 (dd, J = 6.6 and 2.6 Hz, 1H, ArH), 7.51 (d, J = 8.4 Hz, 2H, ArH), 7.43 (t, J = 8.8 Hz, 1H, ArH), 7.28 (d,
J = 8.0 Hz, 2H, ArH), 7.15–7.12 (m, 1H, ArH), 2.78 (t, J = 5.6 Hz, 4H, NCH ), 2.17 (s, 3H, CH ), 1.59 (quint,
2
3
J = 5.2 Hz 4H, CH ), 1.41–1.30 (m, 2H, CH ).
2
2
1
3
C NMR (100 MHz, DMSO-d ): δ 159.4, 156.4 (d, J = 246.8 Hz), 144.2, 140.1, 136.1 (d, J = 3.4 Hz), 133.8,
6
131.8 (2C), 128.9 (2C), 127.7, 127.6, 125.9 (d, J = 7.9 Hz), 119.9 (d, J = 18.9 Hz), 117.7, 117.1 (d, J = 22.4
Hz), 55.4, 25.4, 23.1, 9.0.
1
3
EI-MS m/z 446 (M+), 347 (M-99). HR-EI-MS m/z 446.1031 (calcd for C H O N Cl F : 446.1071).
2
2
21
1
4
2 1
4.1.3.6.
5-(4-Chlorophenyl)-1-(7-chloroquinolin-4-yl)-4-methyl-N-(1-piperidinyl)-1H-pyrazole-3-
carboxamide (6)
1
Yellowish solid with a yield of 78 % yield, mp 231–232 °C. H NMR (400 MHz, CDCl ): δ 8.79 (d, J = 4.8
3
Hz, 1H, ArH), 8.12 (d, J = 2 Hz, 1H, ArH), 7.64 (d, J = 9.2 Hz, 1H, ArH), 7.46 (dd, J = 9,2 and 2.0 Hz, 1H,
ArH), 7.16 (d, J = 8.8 Hz, 2H, ArH), 6.99 (d, J = 4.8 Hz, 1H, ArH), 6.91 (d, J = 8.4 Hz, 2H, ArH), 2.96 (bs,
1H, NCH ), 2.36 (s, 3H, CH )1.73 (quint, J = 5.6 Hz, 4H, CH ), 1.40 (m, 1H, CH ).
2
3
2
2
1
3
C NMR (100 MHz, CDCl ): δ 159.9, 151.1, 150.0, 143.7, 143.69, 143.4, 136.9, 135.5, 130.7 (2C), 129.33
3
(2C), 129.28, 128.8, 126.9, 125.0, 122.9, 119.6, 119.4, 57.0, 25.4, 23.2, 9.5.
FAB-MS m/z 480 [(M+1)+], 482 [(M+3)+], 380 [(M+1)-100]. HR-FAB-MS m/z 480.1336 (calcd for
1
3
C H O N Cl : 480.1352).
2
5
24
1
5
2

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
4.1.3.7.
5-(4-Chlorophenyl)-4-methyl-N-(1-piperidinyl)-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-
carboxamide (7)
1
Yellowish solid with a yield of 60 % yield, mp 174–175 °C. H NMR (400 MHz, DMSO-d ): δ 11.14, 7.82
6
(d, J = 8.4 Hz, 2H, ArH), 7.54 (d, J = 8.4 Hz, 2H, ArH), 7. 53 (d, J = 8.8 Hz, 2H, ArH), 7.30(d, J = 8.4 Hz,
2H, ArH), 3.34 (bs, 4H, NCH ), 2.23 (s, 3H, CH ), 1.82 (quint, J = 5.2 Hz, 4H, CH ), 1.53–1.44 (m, 2H,
2
3
2
CH2).
1
3
C NMR (100 MHz, DMSO-d ): δ 159.9, 142.4, 141.9, 141.2, 134.1, 131.8 (2C), 129.1 (2C), 128.4 (q, J =
6
32.2 Hz), 127.2, 126.5 (q, J = 270.8 Hz), 126.4 (q, J = 3.6 Hz, 2C), 125.8 (2C), 119.1, 56.1, 23.4, 21.5, 8.9.
FAB-MS m/z 463 [(M+1)+], 465 [(M+3)+], 363 [(M+1)-100]. HR-FAB-MS m/z 463.1491 (calcd for
3
5
C H O N ClF : 463.1507).
2
3
23
1
4
3
4.1.3.8. 1-(2,4-Difluorophenyl)-5-(4-fluorophenyl)-4-methyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide
(8)
1
White solid with a yield of 74 % yield, mp 193–195 °C. H NMR (400 MHz, DMSO-d ): δ 9.06 (s, 1H, NH),
6
7.76–7.7 (m, 1H, ArH), 7.41–7.36 (m, 1H, ArH), 7.28–7.21 (m, 5H, ArH), 2.78 (t, J = 5.4 Hz, 4H, NCH2),
2.20 (s, 3H, CH ), 1.58 (quint, J = 5.4 Hz, 4H, CH ), 1.39–1.28 (m, 2H, CH ).
3
2
2
1
3
C NMR (100 MHz, DMSO-d ): δ 162.11 (dd, J = 248.1 and 11.5 Hz), 162.10 (d, J = 245.3 Hz), 159.4,
6
156.4 (dd, J = 251.4 and 13.3 Hz), 144.6, 142.6, 131.6 (d, J = 8.5 Hz, 2C), 131.3 (d, J = 10.1 Hz), 124.8 (d, J
= 3 Hz), 123.7 (dd, J = 12.3 and 3.7 Hz), 116.4, 115.7 (d, J = 21.7 Hz, 2C), 112.3 (dd, J = 22.7 and 3.6 Hz),
104.9 (dd, J = 27.1 and 23.9 Hz), 55.3, 25.4, 23.0, 9.0.
FAB-MS m/z 415 [(M+1)+], 315 [(M+1)-100]. HR-FAB-MS m/z 415.1728 (calcd for C H O N F :
2
2
22
1
4 3
415.1740).
4.1.3.9.
1-(3-Chloro-4-fluorophenyl)-5-(4-fluorophenyl)-4-methyl-N-(1-piperidinyl)-1H-pyrazole-3-
carboxamide (9)
1
White solid with a yield of 56 % yield, mp 177–178 °C. H NMR (400 MHz, DMSO-d ): δ 9.09 (s, 1H, NH),
6
7.69 (dd, J = 6.6 and 2.4 Hz, 1H, ArH), 7.42 (t, J = 8.8 Hz, 1H, ArH), 7.33–7.26 (m, 4H, ArH), 7.17–7.13 (m,

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
1H, ArH), 2.80 (t, J = 5.2 Hz, 4H, NHCH ), 2.16 (s, 3H, CH ), 1.59 (quint, J = 5.2 Hz, 4H, CH ), 1.40–1.30
2
3
2
(m, 2H, CH2).
1
3
C NMR (100 MHz, DMSO-d ): δ 162.2 (d, J = 245.7 Hz), 159.5, 156.4 (d, J = 249.6 Hz), 144.1, 141.0,
6
136.1 (d, J = 3.4 Hz), 132.3 (d, J = 8.5 Hz, 2C), 127.5, 125.8 (d, J = 7.9 Hz), 125.2 (d, J = 3.2 Hz), 119.8 (d, J
= 15.9 Hz), 117.6, 117.1 (d, J = 22.4 Hz), 115.9 (d, J = 21.6 Hz, 2C), 55.4, 25.4, 23.1, 9.0.
FAB-MS m/z 431 [(M+1)+], 433 [(M+3)+], 331 [(M+1)-100]. HR-FAB-MS m/z 431.1425 (calcd for
3
5
C H O N Cl F : 431.1445).
2
2
22
1
4
1 3
4.1.3.10.
carboxamide (10)
1-(7-Chloroquinolin-4-yl)-5-(4-fluorophenyl)-4-methyl-N-(1-piperidinyl)-1H-pyrazole-3-
1
Light yellow solid with a yield of 72 % yield, mp 242–243 °C. H NMR (400 MHz, CDCl ): δ 8.83 (d, J = 4.8
3
Hz, 1H, ArH), 8.15 (d, J = 2.1 Hz, 1H, ArH), 7.69 (d, J = 9 Hz, 1H, ArH), 7.50 (dd, J = 9.0 and 2.1 Hz, 1H,
ArH), 7.04–6.98 (m, 3H, ArH), 6.96–6.88 (m, 2H, ArH), 2.85 (bs, 4H, NCH ), 2.41 (s, 3H, CH ), 1.73 (quint,
2
3
J = 5.4 Hz, 4H, CH ), 1.40 (m, 2H, CH ).
2
2
1
3
C NMR (100 MHz, CDCl ): δ 162.8 (d, J = 246.3 Hz), 159.8, 151.1, 150.1, 145.1, 143.6, 143.4, 136.5,
3
131.2 (d, J = 8.3 Hz, 2C), 128.9, 128.8, 124.9, 124.52, 124.5 (d, J = 3.4 Hz), 122.9, 119.3, 119.1, 116.1 (d, J =
21.7 Hz), 57.1, 25.4, 23.3, 9.3.
FAB-MS m/z 464 [(M+1)+], 465 [(M+3)+], 364 [(M+1)-100]. HR-EI-MS m/z 463.1554 (calcd for
1
3
C H O N Cl F : 463.1570).
2
5
23
1
5
1
1
4.1.3.11.
5-(4-Bromophenyl)-4-methyl-N-(1-piperidinyl)-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-
carboxamide (11)
1
Reddish solid with a yield of 70 % yield, mp 168–169°C. H NMR (400 MHz, DMSO-d ): δ 9.12 (s, 1H, NH),
6
7.79 (d, J = 8.8 Hz, 2H, ArH), 7.65 (d, J = 8.4 Hz, 2H, ArH), 7.50 (d, J = 8.4 Hz, 2H, ArH), 7.21 (d, J = 8.4
Hz, 2H, ArH), 2.807 (t, J = 5.2 Hz, 4H, NCH ), 2.17 (s, 3H, CH ), 1.59 (quint, J = 5.2 Hz, 4H, CH ), 1.40–
2
3
2
1.32(m, 2H, CH2).
1
3
C NMR (100 MHz, DMSO-d ): δ159.4, 144.8, 142.1, 140.5, 131.9 (4C), 128.1, 127.9 (q, J = 32.1 Hz),
6
126.3 (q, J = 3.7 Hz, 2C), 125.403 (2C), 123.8 (q, J = 270.9 Hz), 122.6, 118.2, 55.3, 25.4, 23.1, 8.9.

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
FAB-EI m/z 506 (M+1), 508 [(M+2)+], 407 (M-99). HR-FAB-MS m/z 506.0926 (calcd for
7
9
C H O N BrF : 506.0924).
2
3
23
1
4
3
4.1.3.12.
1-(2,4-Dichlorophenyl)-4-methyl-N-(1-piperidinyl)-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-
carboxamide (12)
1
Light pink solid with a yield of 54 % yield, mp 208–210 °C. H NMR (400 MHz, DMSO-d ): δ 9.11 (s, 1H,
6
NH), 7.80–7.75 (m, 3H, ArH), 7.59 (dd, J = 6.6 and 2.4 Hz, 1H, ArH), 7.44 (d, J = 8.4 Hz, 2H, ArH), 2.78 (t,
J = 5.6 Hz, 4H, NHCH ), 2.25 (s, 3H, CH ), 1.58 (quint, J = 5.2 Hz, 4H, CH ), 1.38–1.29 (m, 2H, CH ).
2
3
2
2
1
3
C NMR (100 MHz, DMSO-d ): δ 159.3, 144.6, 142.1, 135.6, 135.2, 132.6, 132.02, 132.00, 130.3 (2C),
6
129.6, 129.0 (q, J = 31.9 Hz), 128.4, 125.5 (q, J = 3.8 Hz, 2C), 123.9 (q, J = 270.9), 117.1, 55.3, 25.4, 23.0,
9.1.
FAB-MS m/z 497 [(M+1)+], 499 [(M+3)+], 501 [(M+5)+], 397 [(M+1)-100]. HR-FAB-MS m/z 497.1098
3
5
(calcd for C H O N Cl F : 497.1117).
2
3
22
1
4
2 3
4.1.3.13.
1-(2,4-Difluorophenyl)-4-methyl-N-(1-piperidinyl)-5-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-
carboxamide (13)
1
Brown solid with a yield of 81 % yield, mp 157 °C. H NMR (400 MHz, DMSO-d ): δ 9.12 (s, 1H, NH),
6
7.80–7.74 (m, 1H, ArH), 7.77 (d, J = 8.4 Hz, 2H, ArH), 7.45 (d, J = 8.4 Hz, 2H, ArH), 7.42–7.37 (m, 1H,
ArH), 7.28–7.23 (m, 1H, ArH), 2.78 (t, J = 5.2 Hz, 4H, NCH ), 2.24 (s, 3H, CH ), 1.58 (quint, J = 5.2 Hz, 4H,
2
3
CH ), 1.34 (m, 1H, CH ).
2
2
1
3
C NMR (100 MHz, DMSO-d ): δ 162.2 (dd, J = 248.5 and 11.4 Hz), 159.2, 156.2, (dd, J = 251.5 and 13.5),
6
144.8, 142.0, 132.6, 131.2 (d, J = 10.3 Hz), 130.1 (2C), 129.0 (q, J = 31.5 Hz), 125.6 (q, J = 14.8 Hz), 123.9
(q, J = 271.0 Hz), 123.6 (dd, J = 12.1 and 4.1 Hz), 117.1, 112.5 (d, J = 22.7 and 3.7 Hz), 105.0 (dd, J = 27.3
and 23.7), 55.3, 25.4, 23.0, 9.0.
FAB-MS m/z 465 [(M+1)+], 365 [(M+1)-100]. HR-FAB-MS m/z 465.1685 (calcd for C H O N F :
2
3
22
1
4 5
465.1708).
5
5
88
89
4.2. Animals

ACCEPTED MANUSCRIPT
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
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).
4.3. Induction of diabetes
Diabetes was induced in overnight fasted rats by intraperitoneal (i.p.) injection of 110 mg/kg of
nicotinamide 15 minutes 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 200-350 mg/dL) were detected after
72 h using a glucometer and these were used for antidiabetic screening.
4.4 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 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 hours 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 T1, T3, T5 and T7 hours 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 [29].
4.5. Homology modeling
Since the crystallographic structure of human CB1 was not available, a three dimensional structure
was built using homology modeling. The amino acid sequence of human CB1 (GenBank: EAW48576) [30]
was retrieved from the NCBI-Protein database [31]. For the comparative analysis and homologous sequences
search, PSI-BLAST [32] and UniProt [33] servers were used against the Protein Data Bank (PDB) database
[34]. The crystallographic structure of β-1 adrenergic receptor of Meleagris gallopavo (PDB ID: 4AMJ_B)

ACCEPTED MANUSCRIPT
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
[35] was selected as the template protein. Three-dimensional models were constructed with three different
programs: MODELLER 9v11 [36], SWISS-MODEL [37], and I-TASSER [38]. Based on previous studies
[39, 40], the N-terminal (residues 1–109) and C-terminal (residues 413–472) domains of human CB1
sequence were omitted during the modeling construction. From SWISS-MODEL workspace, the estimation
of the local quality and the protein stereochemistry quality of the models were evaluated by QMEAN6 [41]
and PROCHECK [42] respectively. The best evaluated structure was selected for further refinement using the
WHAT IF Web Interface (http://swift.cmbi.ru.nl/servers/html/index.html). Finally, a disulfide bridge between
C257 and C264 was generated for the final CB1 model [40, 43].
4.6. Construction of the receptor-membrane complex
Energy minimization (EM) of the final CB1 model was performed using the GROMOS96 43a1 force
field [44] implemented in the GROMACS 4.5.3 package [45]. The receptor-membrane (CB1-POPC) complex
of the CB1 model with a 1-1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC) lipid bilayer was
carried out following the same workflow implemented by Gonzalez, et al. [40]. First, the model was
immersed in a pre-equilibrated POPC bilayer (128 molecules) employing the Berger lipid parameters [46]
retrieved
from
the
D.
P.
Tieleman’s
web
site
(http://moose.bio.ucalgary.ca/index.php?page=Structures_and_Topologies) [47]. Subsequently, the POPC
bilayer was properly packed around the CB1 model (CB1-POPC) using the experimental area per lipid (0.675
2
nm ) to correct density and system dimensions, resulting in a box of 68 Å X 68 Å X 130 Å. The Z-axis was
extended in order to fully immerse the intra and extracellular loop regions [48]. Electrostatic forces were
calculated with the PME implementation of the Ewald summation method [49], and the Lennard-Jones and
-
Coulomb interactions were set at 1.0 nm. A total of 16 Cl atoms were added randomly in order to neutralize
the charge of the system. The system was equilibrated by 3500 steps of energy minimization with position
restraints on the protein, enabling the relaxation of the POPC and the solvent. Finally, a flexible energy
minimization was performed on the CB1-POPC complex employing the steepest-descent algorithm, until the
-
1
-2
maximum force decayed to 100 kJ mol nm . It is noteworthy that the CB1-POPC complex was built to
equilibrate the system and to show a more realistic environment during the optimization; nevertheless, the
POPC bilayer was removed during the docking and molecular dynamics studies.

ACCEPTED MANUSCRIPT
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
4.7. Docking studies
Docking studies were performed in order to identify different theoretical binding modes of the
synthesized compounds into the minimized CB1-POPC complex. The compounds were constructed and
submitted to a geometry optimization employing the MMFF94x force field in Spartan 10 [50] The torsional
root and branches of the ligands were chosen using MGLTools 1.5.4 [51], allowing flexibility for all rotatable
bonds of the ligand except for the amide bond, which was fixed. In addition, MGLTools 1.5.4 was used to
assign Gasteiger-Marsilli atomic charges to all ligands.
Subsequently, the compounds were docked to the binding site performing the Lamarckian genetic algorithm
of AutoDock 4.2 package [52]. A grid box of 60 Å x 60 Å x 60 Å with 0.375 Å spacing and centered at the
lysine 192 (Lys192) was used to calculate the atom types: H, HD, C, A, N, NA, OA, SA, S, Br, Cl and F. A
total of 25 runs were undertaken with a maximal number of 5,000,000 energy evaluations and initial
populations of 150 conformers. The best binding mode of each molecule was selected based on the lowest
calculated free binding energy and the cluster size.
4.8. Molecular dynamics studies
In order to assess the stability of the compounds into the binding pocket, ligand-receptor complexes
with the selected binding modes were submitted to a short molecular dynamics simulation. First, energy
-
1
-2
minimization of the system was carried out applying positional restraints (100 kJ mol nm ) to the α-carbons
of the transmembrane helices (TMH), using the same force field and parameters described above. Following
energy minimization, the system was equilibrated by 500 ps of MD simulation while gradually reducing all
restraints to zero. Finally, the complexes were subjected to a 2 ns MD simulation without any restraints and
using the SHAKE algorithm. [53]. Rescoring of the binding free energy was calculated based on the linear
interaction energy (LIE) method equation [54, 55]. The simulations were carried out at a constant pressure of
1 bar and 300 K temperature.
Acknowledgments

ACCEPTED MANUSCRIPT
6
6
6
6
73
74
75
76
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 Anuar Flores Gahona from Facultad de Química, UNAM, for the review of this paper
and the helpful suggestions
6
6
77
78
References
6
6
6
6
79
80
81
82
[1] R.H. Eckel, S.C. Grundy, P.Z. Zimmet, The metabolic sindrome, Lancet 365 (2005) 1415–1428.
[2] WHO report factsheet 311.
[3] K.J. Alberti, P. Zimmet, J. Shaw, The metabolic syndrome--a new worldwide definition, Lancet 366
(2005) 1059–1062
6
6
83
84
[4] A.O. Ogbera, Diabetol. Prevalence and gender distribution of the metabolic syndrome, Metab. Syndr. 2
(2010) 1–5
6
6
6
85
86
87
[5] R. B. Ervin National health statistics reports; no 13, MD: National Center for Health Statistics. 2009.
[6] R.S: Padwal, S.R: Majumdar, Drug treatments for obesity: orlistat, sibutramine, and rimonabant.Lancet
369 (2007) 71–77
6
88
[7] L.M. Neff, L.J. Aronne, Pharmacotherapy for obesity, Curr. Atheroscler. Rep. 9 (2007) 454–462.
6
6
6
89
90
91
[8] F.X. Pi-Sunyer, L.J. Aronne, H.M. Heshmati. J. Devin, J. Rosentock, Effect of rimonabant, a cannabinoid-
1 receptor blocker, on weight and cardiometabolic risk factors in overweight or obese patients: RIO-North
America: a randomized controlled trial, JAMA 295 (2006) 761–775.
6
6
6
92
93
94
[9] I. Crespo, R. Gómez de Heras, F. Rodríguez de Fonseca, M. Navarro. Pretreatment with subeffective
doses of Rimonabant attenuates orexigenic actions of orexin A-hypocretin 1, Neuropharmacology 54 (2008)
219–225.
6
6
6
95
96
97
[10] J. Tam, V.K. Verumi, J. Liu, S. Bátkau, B. Mukhopadhyay, G. Godlewsky, D. Osei-Hyiaman, S.
Ohnuma, S.V. Ambudkar, J. Pickel, A. Makriyannis, G. Kunos, Peripheral CB1 cannabinoid receptor
blockade improves cardiometabolic risk in mouse models of obesity, J. Clin. Invest. 120(2010) 2953–2966.

ACCEPTED MANUSCRIPT
6
6
98
99
[11] L. Bellocchio, C. Cervino, R. Pasquali, U. Pagotto, Cannabinoid type 1 receptor: another arrow in the
adipocytes' bow, J. Neuroendocrinol. 20 (2008) 130–138.
7
7
7
7
00
01
02
03
[12] S. Engeli, Peripheral metabolic effects of endocannabinoids and cannabinoid receptor blockade, Obesity
Facts 1 (2008) 8–15.
[13] E. Hernández-Vázquez, O Méndez-Lucio, F. Hernández-Luis, Activity landscape analysis, CoMFA and
CoMSIA studies of pyrazole CB1 antagonists, Med. Chem. Res. In press. DOI 10.1007/s00044-012-0418-y
7
7
7
04
05
06
[14] J.Z. Chen, X. W. Han, Q. Liu, A. Makriyannis, J. Wang, X. Q. Xie, 3D-QSAR studies of arylpyrazole
antagonists of cannabinoid receptor subtypes CB1 and CB2. A combined NMR and CoMFA approach, J.
Med.Chem. 49 (2006) 625–636.
7
7
7
7
7
7
07
08
09
10
11
12
[15] P.H. Reggio, Toward the design of cannabinoid CB receptor inverse agonists and neutral antagonists,
1
Drug Develop. Res. 70 (2009) 585–600.
[16] D.P. Hurst, D.L. Lynch, J. Barnett-Norris, S.M. Hyatt, H.H. Seltzman, M. Zhong, Z. Song, J. Nie, D.
Lewis, P.H. Reggio, N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-
carboxamide (SR141716A) Interaction with LYS 3.28(192) Is Crucial for Its Inverse Agonism at the
Cannabinoid CB1 Receptor, Mol. Pharmacol. 62 (2002) 1274–1287.
7
7
7
13
14
15
[17] S. D. McAllister, G. Rizvi, S. Anavi-Gofer, D. P. Hurst, J. Barnett-Norris, D. L. Lych, P. H. Reggio, M.
E. Abood, An aromatic microdomain at the cannabinoid CB(1) receptor constitutes an agonist/inverse agonist
binding region, J. Med. Chem. 46 (2003) 5139–5152.
7
7
7
7
7
7
7
16
17
18
19
20
21
22
[18] R. Lan, Q. Liu, P. Fan, S. Lin, S. R. Fernando, D. McCallion, R. Pertwee, A. Makriyannis, Structure-
activity relationships of pyrazole derivatives as cannabinoid receptor antagonists, J. Med. Chem. 42 (1999)
769–776.
[19] V.K. Kotagiri, S. Suthrapu, J.M. Reddy, C.P. Rao, V. Bollugoddu, A. Bhattacharya, R. Bandichhor, An
Improved Synthesis of Rimonabant: Anti-Obesity Drug, Org. Process Res. Dev. 11 (2007) 910–912.
[20] P.Masiello, C. Broca, R. Gross, M. Roye, M. Manteghetti, D. Hillaire-Buys, M. Novelli, G. Ribes.
Diabetes 47 (2008) 224–229

ACCEPTED MANUSCRIPT
7
7
7
23
24
25
[21] A.V. Stepanchikova, A.A. Lagunin, D.A: Filimonov, V.V.Poroikov, Prediction of biological activity
spectra for substances: evaluation on the diverse sets of drug-like structures, Curr. Med. Chem. 10 (2003)
225–233.
7
7
26
27
[22] A. Marwaha, R.K. Goelb, M.P. Mahajan, PASS-predicted design, synthesis and biological evaluation of
cyclic nitrones as nootropics, Bioorg. Med. Chem. Lett. 17 (2007) 5251–5255.
7
7
28
29
[23] C.L. Worth, G. Kleinau, G. Krause, Comparative sequence and structural analyses of G-protein-coupled
receptor crystal structures and implications for molecular models, PLoS One, 16 (2009) e70
7
7
7
7
7
7
7
30
31
32
33
34
35
36
[24] C.L. Worth, A.Kreuchwig, G. Kleinau, GPCR-SSFE: A comprehensive database of G-protein-coupled
receptor template predictions and homology models, BMC Bioinformatics, 12 (2011), 185.
[25] J. Nilsson, B. Persson, G. von Heijne, Comparative analysis of amino acid distributions in integral
membrane proteins from 107 genomes, Proteins, 60 (2005), 606–616
[26] X. Dong, Y. Zhao, X. Huang, K. Lin, J. Chen, E. Wei, T. Liu, Structure-based drug design using GPCR
homology modeling: Toward the discovery of novel selective CysLT2 antagonists, Eur. J. Med. Chem. 62
(2013), 754-763
7
7
7
7
37
38
39
40
[27] S. Ortega-Gutierrez, M.L. Lopez-Rodriguez, CB1 and CB2 Cannabinoid Receptor Binding Studies
Based on Modeling and Mutagenesis Approaches, Mini Rev. Med. Chem. 5 (2005), 651-658
[28] K. Stierand, M. Rarey, Drawing the PDB: Protein−Ligand Complexes in Two Dimensions, ACS Med.
Chem. Lett. 1 (2010), 540-545. (http://www.poseview.de/poseview)
7
7
7
7
7
7
41
42
43
44
45
46
[29] J.J. Ramírez-Espinosa, M.Y. Rios, S. López-Martínez, F. López-Vallejo, J.L. Medina-Franco, P. Paoli,
G. Camici, G. Navarrete-Vázquez, R. Ortiz-Andrade, S. Estrada-Soto, Eur. J. Med. Chem. 46 (2011) 2243-
2251.
[30] J. C. Venter, The sequence of the human genome, Science 291 (2001), 1304-1351
[31] K. D. Pruitt, T. Tatusova, D.R. Maglott, NCBI reference sequences (RefSeq): a curated non-redundant
sequence database of genomes, transcripts and proteins, Nucl. Acids Res. 35 (2007) D61-D65
7
7
7
47
48
49
[32] S. F. Altschul, T. L. Madden, A. A. Schäffer, J. Zhang, Z. Zhang, W. Miller, D. J: Lipman, Gapped
BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25 (1997)
3389-3402.

ACCEPTED MANUSCRIPT
7
7
50
51
[33] R. Apweiler, A. Bairoch, C.H. Wu, Protein sequence databases, Cur. Op. Chem. Biol. 8 (2004) 76-80
7
7
52
53
[34] F.C. Bernstein, T.F. Koetzle, G.J.B. Williams, E.F. Meyer Jr., The protein data bank: A computer-based
archival file for macromolecular structures, Arch. Biochem. Biophys. 185 (1978) 584-591.
7
7
54
55
[35] T.C. Warne, P.C. Edwards, A.G. Leslie, C.G. Tate, Crystal structures of a stabilized β1-adrenoceptor
bound to the biased agonists bucindolol and carvedilol, Structure 20 (2012) 841-849.
7
7
7
56
57
58
[36] N. Eswar, B. Webb, M.A. Marti-Renom, M.S. Madhusudhan, D. Eramian, M.Y Shen, U. Pieper, A. Sali,
Comparative protein structure modeling using MODELLER in Current Protocols in Protein Science, 50
(2007) 2.9.1–2.9.31.
7
7
7
7
7
7
59
60
61
62
63
64
[37] T. Schwede, J. Kopp, N. Guex, M.C. Peitsch, SWISS-MODEL: an automated protein homology-
modeling server, Nucl. Acids Res. 31 (2003) 3381-3385
[38] A. Roy, A. Jucukural, Y. Zhang, I-TASSER: a unified platform for automated protein structure and
function prediction, Nat. Protoc. 5 (2010), 725-738.
[39] R. Ai, C.A. Chang, Ligand-specific homology modeling of human cannabinoid (CB1) receptor, J. Mol.
Graph. Model. 38 (2012) 155-164.
7
7
7
65
66
67
[40] A. Gonzalez, L.S. Duran, R. Araya-Secchi, J.A. Garate, C.D. Pessoa-Mahana, C.F. Lagos, T. Perez-Acle,
Computational modeling study of functional microdomains in cannabinoid receptor type 1, Bioorg. Med.
Chem. 16 (2008) 4378-4389.
7
7
7
7
7
68
69
70
71
72
[41] P. Benkert, S.C.E. Tosatto, D. Schomburg, QMEAN: A comprehensive scoring function for model
quality assessment, Proteins, 71 (2008), 261-277.
[42] R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PROCHECK: a program to check the
stereochemical quality of protein structures, J. Appl. Crystallogr. 26 (1993) 283-291.
[43] J.F. Fay, T.D. Dunham, D.L. Farrens, Cysteine Residues in the Human Cannabinoid Receptor:ꢀ Only
7
7
73
74
C257 and C264 Are Required for a Functional Receptor, and Steric Bulk at C386 Impairs Antagonist
SR141716A Binding, Biochemistry, 44 (2005), 8757-8769.

ACCEPTED MANUSCRIPT
7
7
7
7
7
7
7
7
7
7
75
76
77
78
79
80
81
82
83
84
[44] W.F. van Gunsteren, S.R. Billeter, A.A. Eising, P.H. Hünenberger, P. Krüger, A.E. Mark, W.R.P. Scott,
and I. G. Tironi, Biomolecular Simulation: The GROMOS96 Manual and User Guide, Vdf Hochschulverlag
AG an der ETH Zürich, Zürich, Switzerland, 1996, pp. 1-1042.
[45] D. Van Der Spoel, E. Lindahl, B. Hess, G. Groenhof, A.E. Mark, H.J.C. Berendsen, GROMACS: Fast,
flexible, and free, J. Comput. Chem. 26 (2005), 1701-1718.
[46] O. Berger, O. Edholm, F. Jähnig, Molecular dynamics simulations of a fluid bilayer of
dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature, Biophys. J. 72
(1997) 2002-2013.
[47] D.P. Tieleman, S.J. Marrink, H.J.C. Berendsen, A computer perspective of membranes: Molecular
dynamics studies of lipid bilayer systems, Biochim. Biophys. Acta 1331 (1997) 235–270.
7
7
85
86
[48] A. Ghosh, U. Sonavane, S.K. Andhirka, G.K. Aradhyam, R. Joshi, Structural insights into human GPCR
protein OA1: a computational perspective, J. Mol. Model. 18 (2012) 2117-2133.
7
87
[49] T. Darden, Y. Darrin, L. Pedersen, Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large
7
88
systems, J. Chem. Phys. 98 (1993) 10088-10093.
7
7
7
89
90
91
[50] Y. Zhao, N.E. Schultz, D.G. Truhlar, Design of density functionals by combining the method of
constraint satisfaction with parametrization for thermochemistry, thermochemical kinetics, and noncovalent
interactions, J. Chem. Theory Comput, 2 (2006), 364-382
7
7
92
93
[51] M. F. Sanner. Python: A Programming language for software integration and development. J. Mol.
Graphics Mod., 17 (1999)57–61.
7
7
7
94
95
96
[52] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, R.S. Goodsell, A.J. Olson, AutoDock4
and AutoDockTools4: Automated docking with selective receptor flexibility, J. Comput. Chem. 30 (2009)
2785–2791.