Design and synthesis of N?(benzylpiperidinyl)?4?fluorobenzamide: A haloperidol analog that reduces neuropathic nociception via σ1 receptor antagonism

Article abstract of DOI:10.1016/j.lfs.2020.117348

Aims: Haloperidol is a neuroleptic drug with high affinity towards the σ₁ receptor (σ₁R), acting as antagonist that decreases neuropathic pain, but has CNS side effects. This work describes the design and synthesis of a novel analog

Full text of DOI:10.1016/j.lfs.2020.117348

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Design
and
synthesis
of
N‑(benzylpiperidinyl)‑4‑fluorobenzamide: A haloperidol analog
that reduces neuropathic nociception via σ1 receptor antagonism
Myrna Déciga-Campos, Luis Alberto Melo-Hernández, Héctor
Torres-Gómez, Bernhard Wünsch, Dirk Schepmann, María Eva
González-Trujano, Josué Espinosa-Juárez, Francisco Javier
López-Muñoz, Gabriel Navarrete-Vázquez
PII:
S0024-3205(20)30095-3
https://doi.org/10.1016/j.lfs.2020.117348
LFS 117348
DOI:
Reference:
To appear in:
Life Sciences
Received date:
Revised date:
Accepted date:
13 November 2019
15 January 2020
21 January 2020
Please cite this article as: M. Déciga-Campos, L.A. Melo-Hernández, H. Torres-Gómez,
et al., Design and synthesis of N‑(benzylpiperidinyl)‑4‑fluorobenzamide: A haloperidol
analog that reduces neuropathic nociception via σ1 receptor antagonism, Life
Sciences(2020), https://doi.org/10.1016/j.lfs.2020.117348
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Design and synthesis of N-(benzylpiperidinyl)-4-
fluorobenzamide: a haloperidol analog that reduces
neuropathic nociception via 1 receptor antagonism
Myrna Déciga-Camposa,*, Luis Alberto Melo-Hernándezb, Héctor Torres-
Gómezc, Bernhard Wünschc, Dirk Schepmannc, María Eva González-Trujanod,
Josué Espinosa-Juáreze, Francisco Javier López-Muñoze, Gabriel Navarrete-
Vázquezb,*
aSección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina,
Instituto Politécnico Nacional. Plan de San Luis y Díaz Mirón s/n Col. Casco de Santo
Tomás 11340, Ciudad de México, México.
bFacultad de Farmacia, Universidad Autónoma del Estado de Morelos. Av. Universidad
1001, Chamilpa, Cuernavaca, Morelos, 62209, México.
cInstitut für Pharmazeutische und Medizinische Chemie der Westfälischen Wilhelms-
Universität Münster, D-48149 Münster, Germany.
dLaboratorio de Neurofarmacología de Productos Naturales. Dirección de
Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría “Ramón de la
Fuente Muñíz”, 14370, Ciudad de México, México
eDepartamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados
(Cinvestav), Sede Sur. Ciudad de México, México.
*Corresponding authors:
Dra. Myrna Déciga Campos,
Sección de Estudios de Posgrado e Investigación, Escuela Superior de
Medicina, Instituto Politécnico Nacional. Plan de San Luis y Díaz Mirón s/n Col.
Casco de Santo Tomás 11340, Ciudad de México, México
email address: mdeciga@ipn.mx; myrnadeciga@hotmail.com
Dr. Gabriel Navarrete-Vázquez,
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos. Av.
Universidad 1001, Chamilpa, Cuernavaca, Morelos, 62209, México
email address: gabriel_navarrete@uaem.mx
* Both authors contributed equally to this work.
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ABSTRACT
Aims: Haloperidol is a neuroleptic drug with high affinity towards the 1 receptor
(1R), acting as antagonist that decreases neuropathic pain, but has CNS side
effects. This work describes the design and synthesis of a novel analog N-(1-
benzylpiperidin-4-yl)-4-fluorobenzamide
(LMH-2),
which
produced
antihyperalgesic and antiallodynic effects in rats with neuropathy induced by
chronic constriction injury of the sciatic nerve (CCI), being more active than
gabapentin (The most widely used drug for the treatment of neuropathic pain).
Main methods: LMH-2 was designed as haloperidol analog. Its structure was
characterized by spectroscopic (1H and 13C NMR) and spectrometric mass
(electronic impact) techniques. Additionally, in silico predictions of
pharmacokinetic, pharmacodynamic and toxicological properties were obtained,
with promising results. A competitive binding assay using radioligands was
employed to evaluate the in vitro affinity for 1R, whereas in vivo
antihyperalgesic and antiallodynic activities were investigated using Wistar rats
with CCI.
Key findings: LMH-2 showed high affinity for 1R in an in vitro binding assay,
with a Ki = 6.0 nM and a high σ1R/σ2R selectivity ratio. Molecular docking
studies were carried out to determine the binding energy and to analyze LMH-2-
protein interactions. Through an in silico pharmacological consensus analysis,
LMH-2 was considered safe for in vivo evaluation. Thus, LMH-2 had dose-
dependent antiallodynic and antihyperalgesic activities; its efficacy was
comparable to that of gabapentin, but its potency was 2-times higher than this
drug.
Significance: LMH-2 administration produced antihyperalgesic and
antiallodynic effects by the antagonism of 1R, suggesting its potential use as
an analgesic drug for neuropathic pain.
Keywords: N-(1-Benzylpiperidin-4-yl)-4-fluorobenzamide, haloperidol, 1
receptor, neuropathic pain, allodynia, hyperalgesia.
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1.
INTRODUCTION
The 1 receptor (1R) plays a pivotal role in the transmission of pain. These
receptors are in the plasma and subcellular membranes, particularly in the
endoplasmic reticulum. It is known that 1R plays a modulatory role in
intracellular Ca2+ signaling and in the activity of some ion channels and several
neurotransmitter systems, mainly in the glutamatergic pathway [1,2]. Preclinical
evidence supports the modulatory role of σ1R in nociception, mainly based on
the inhibition of σ1R leads to decreased amplification of pain signaling within the
CNS (dorsal spinal cord, thalamus, periaqueductal gray, basolateral amygdala
and rostroventral medulla) [3]. The σ1R KO mice is insensitive or shows
attenuated expression of pain behaviors in formalin or capsaicin-induced and
neuropathic pain models [4-6]. The antagonism of σ1R leads to decreased
amplification of pain signaling within the spinal cord (central sensitization) and
the periphery. There is considerable evidence showing that BD1047, a potent
1R antagonist, blocks nociceptive effects [7-9]. Moreover, this drug might
interact with several unknown targets and, thus, interfere with opioid
antinociception [10]. However, there is evidence showing that haloperidol, a
dopaminergic antagonist used mainly as a neuroleptic drug, decreases
neuropathic pain in humans when administered alone [11] or in combination
with tramadol [12]. Currently, it has been proposed that the therapeutic efficacy
of haloperidol on neuropathic pain involves the blockade of 1R, but not via
dopamine receptor antagonism [5, 6]. However, haloperidol presented several
neurological side effects that restrict its use as a therapeutic agent. Haloperidol
has central nervous system effects, particularly extrapyramidal symptoms
(catalepsy and motor imbalance) and tardive dyskinesia, sedation, and dulling
of cognition [13]. Other adverse effects of the typical antipsychotics include the
neuroleptic malignant syndrome, orthostatic hypotension, changes in liver
function, anticholinergic and antiadrenergic side effects, sexual dysfunction, and
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weight gain. Also, high dose of haloperidol may result in irreversible liver
damage [14,15]. These findings provided evidence to consider σ1R antagonists
as an innovative and alternative approach for treating pain, especially
neuropathic pain but also other sensitizing pain conditions. Therefore, we
decided to design a novel analog of haloperidol for its possible use in treating
neuropathic pain through the antagonism of the 1R, but without the unwanted
side effects. Here, we report the design, in vitro 1R affinity and in vivo
antiallodynic and antihyperalgesic activities of LMH-2 after its systemic
administration against CCI of the sciatic nerve model in rats.
2. MATERIALS AND METHODS
2.1. Drug design of LMH-2
The previously reported pharmacophore models suggest three major features
that are imperative for high binding affinity at 1R: I) an ionizable tertiary amino
group, II) a mainly hydrophobic region, composed of an aryl ring situated at 6–
10 Å from the amino group, and III) a small secondary hydrophobic region at
2.4–3.9 Å from the amine [16,17].
The new chemical entity LMH-2 was designed as an analog of haloperidol
(Figure 1) and fulfills the requirements of reported pharmacophore models for
high-affinity σ1R ligands: I) An ionizable tertiary amine that interacts with the
1R through electrostatic interactions, II) a main hydrophobic region situated at
7.035 Å from the amine, and III) a secondary hydrophobic moiety at 3.607 Å
from the amine (Figure 2). Both aryl groups can form additional - interactions
with the 1R.
A
B
Figure 1. Chemical structures of haloperidol (A) and LMH-2 (B).
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Figure 2. Overlay of haloperidol (green) and LMH-2 (black) and the pharmacophore pattern
found in the 1R ligands applied to LMH-2
LMH-2 (substituted with a fluorine atom at the 4-position of the benzene ring)
follows the Lipinski rule of five, compatible with good pharmacokinetic behavior
(Table 1). These physicochemical parameters or properties are similar to those
shown by haloperidol and S1RA, an advanced 1R antagonist, which has
shown promising results in phase II clinical trials for neuropathic pain [18].
Table 1. Physicochemical properties of LMH-2, haloperidol, and S1RA
Compound
MW
clogP
Hydrogen
Hydrogen Number of
Polar
surface
area
bond
bond
rotatable
acceptors
donors
bonds
<500
312
375
337
<5
<10
3
<5
1
<10
3
<60 A2
LMH-2
Haloperidol
S1RA
3.11
4.31
3.33
49.4
3
1
5
36.9
5
0
5
36.9
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2.2. Biological activity spectra prediction
The in silico biological activity spectra calculated for LMH-2 were obtained using
ACD/ToxSuite software, the OSIRIS program (http://www.organic-
chemistry.org/prog/peo/) and the admetSAR database
(http://lmmd.ecust.edu.cn:8000/). The estimation of the general biological
potential of the compound can be performed based on the structural formula, its
predicted toxicity, and drug-like pharmacokinetic properties.
2.3. Chemistry
All the starting materials and reagents were obtained commercially from Sigma-
Aldrich (St. Louis, MO, USA). Melting points were determined on an SRS EZ
Melt MPA120 automated apparatus from Stanford Research Systems and are
uncorrected. TLC monitored reactions on 2 x 5 cm precoated silica gel 60 F254
plates (Merck KGaA, Darmstadt, Germany) were visualized under 254-365 nm
UV light.
The chemical structures of the synthesized compound were confirmed based on
13
their spectral data (1H NMR, C NMR spectra, and EIMS). NMR studies were
performed on an INOVA-400 MHz instrument. Chemical shifts (H, C) and
coupling constant values (J) are given in ppm and Hz, respectively. A standard
reference of TMS (H = 0, C = 0) in CDCl3 and DMSO-d6 as solvents was used.
Mass spectra were recorded on a JEOL JMS-700 instrument (JEOL USA Inc.,
Peabody, MA, USA).
2.4. Binding assay for the σ1 receptor
The test compound solutions were prepared by dissolving approximately 10
μmol (usually 2–4 mg) of LMH-2 in the required amount of DMSO so that a 10
mM stock solution was obtained. To obtain the required test solutions for the
assay, the DMSO stock solution was diluted with the respective assay buffer. All
binding experiments were carried out in duplicates in 96-well multiplates. The
protocol of the 1R binding assay was performed with the radioligand [3H]-(+)-
pentazocine (22.0 Ci/mmol; Perkin Elmer). The thawed membrane preparation
of guinea pig brain cortex (approximately 100 g of protein) was incubated with
various concentrations of LMH-2, 2 nM [3H]-(+)-pentazocine, and TRIS buffer
(50 mM, pH 7.4) at 37 °C. The nonspecific binding was determined with 10 M
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unlabeled (+)-pentazocine. The Kd-value of (+)-pentazocine is 2.9 nM [19, 20].
The dissociation constant (Kd) describes the affinity of a ligand towards a
protein. The ligand-protein affinity is influenced by the noncovalent
intermolecular interactions between the two molecules, such as hydrogen
bonds, electrostatic interactions, hydrophobic forces, and van der Waals forces.
The smaller the dissociation constant, the more strongly the ligand is bound to
the protein.
2.5. Pharmacological evaluation in vivo
2.5.1. Animals
Young male Wistar rats of approximately 90-110 g were provided by the
bioterium of the Pharmacology Department of the CINVESTAV Coapa. The
animals were kept under standard conditions (with 12 h day/night cycles, 25 °C
and a humidity of 45-65%). The experiments were carried out in accordance
with the official Mexican standard NOM-062-ZOO-1999 and under the technical
specifications for the production, care and use of laboratory animals and
following a pain model authorized by the IASP and the National Institutes of
Health guide for the care and use of Laboratory Animals (NIH Publications No.
8023, revised 1978). Rats were evaluated after surgery at 170-180 g in adult
age, the route of administration was subcutaneous (on the dorsum of the rat in
the interscapular region), and each experimental group consisted of at least six
animals.
2.5.2. Sciatic nerve surgery
The CCI involves placing four ligatures on the sciatic nerve to interrupt
neurotransmission, which generates allodynia and hyperalgesia. The animals
were anesthetized with ketamine/xylazine (50/12 mg/kg, i.p.) followed by
dissection of the biceps femoris. The most proximal part was located at the
trifurcation of the sciatic nerve, and approximately 7 mm was ligatured with silk
thread (3-0) with a distance of 1 mm between each. Subsequently, the muscle
was sutured with chromic catgut absorbable thread (4-0) and the skin with 3-0
silk thread; 15 days after surgery, the presence of allodynia and hyperalgesia
behavioral responses was evaluated.
2.5.3. Evaluation of allodynia and hyperalgesia
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The evaluation of hyperalgesia was carried out using von Frey filaments,
applying a tactile stimulus on the plantar surface of the hind limbs with a
filament to exert a force of 15 g. A filament exerting the force of 15 g was used
to measure the pain threshold, since filaments of less strength are imperceptible
to the sensation of the rats. Each stimulus was applied 10 times with intervals of
3 seconds each. The behavior that reflects hyperalgesia is quantified as the
withdrawal latency of the paw stimulated with the filament. For assessing
allodynia, the method consisted of applying a cold stimulus; 0.1 mL of acetone
was placed on the dorsal surface of the hind limbs. The response of allodynia
was quantified as the duration of jerking and licking of the limb during a lapse of
1 minute. These behaviors were analyzed at 0, 30, 60, 90, 120 and 180 minutes
after the administration of the compound to be evaluated. To establish the
therapeutic window, a screening of the compound to be evaluated was
performed using logarithmic doses and evaluating the antiallodynic and
antihyperalgesic effects. Subsequently, a dose-response curve was generated
for both activities (0.1, 1.0, 3.2, 10.0, 31.6, 100.0 mg/kg, s.c.).
2.6. Statistical analysis
To determine the effect of the compound on the presence of allodynia and
hyperalgesia behavioral responses, the effects of each treatment were analyzed
using six animals in each group and the results are expressed as the average ±
standard error of the mean (S.E.M.). The value of the area under the curve
(AUC) was calculated from the respective time course. The AUC for each dose
of analyzed compound was calculated using the trapezoidal method. Student´s
t-test was used to compare two groups. For the analysis of the nociceptive
sensitivity to mechanical stimulation at different time points and under different
treatments, one-way ANOVA was used, followed by Dunnett's post hoc test with
p≤0.05 comparison vs. vehicle. In the AUC analysis, data were compared using
one-way ANOVA followed by Tukey's post hoc test (p≤0.05). In all statistical
analysis, a p<0.05 was considered statistically significant.
2.7. Molecular docking approach
Molecular docking predicts the binding energy (G), as well as the orientation
and three-dimensional conformation (3D) of the ligand-site binding action,
generally in a protein determined by X-ray diffraction or by modeling by
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homology when the structure (3D) is unknown [21]. All in silico calculations
were done with Molecular Operating Environment (MOE, Chemical Computing
Group Inc. http://www.chemcomp.com) version 2018.01. MOE is software that
strongly supports the design of bioactive molecules through molecular
simulation, protein structure analysis, small molecule data processing, and the
study of protein and small molecule docking [22].
The crystal structure of the sigma-1 receptor (1R) complexed with N-(1-
benzylpiperidin-4-yl)-4-iodobenzamide (5HK2) at 3.2 Å resolution was obtained
from the Protein Data Bank (http://www.rcsb.org/pdb) [23]. All water molecules
were deleted, and the hydrogen atoms and charges were adjusted with the
MMFF94 force field from the MOE suite. The 3D structures were built and
minimized in MOE, using the same force field as that mentioned above. The
docking was performed considering all residues within a 4.5 Å sphere centered
on cocrystallized ligand atoms. As a placement function, Alpha Triangle was
selected, and the scores were calculated with the Affinity DG function, which
measures the enthalpy contribution to the free energy of binding (MOE), in
accordance with a validation procedure to reproduce the docking, the same
pose of cocrystallized ligand in the crystal structure (RMSD = 0.569 Å) and a
score of -6.692 Kcal/mol. For each ligand, 100 conformations were generated,
and the top-ranked conformation based on docking score energy was selected
for further studies of molecular docking. After molecular docking, we analyzed
the best calculated binding poses, and the graphical representations were
created as surface maps and ligand interactions in MOE (Figure 3).
Figure 3. 2D and 3D interaction map of the docking validation of 1R cocrystallized with N-(1-
benzylpiperidin-4-yl)-4-iodobenzamide (5HK2). RMSD = 0.569 Å, Docking Score of -6.692
Kcal/mol.
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The cocrystallized ligand N-(1-benzylpiperidin-4-yl)-4-iodobenzamide shows
several interactions with 1R, mainly by electrostatic forces between the ionized
amino group and Glu172, Phe107 and Met 93. Additionally, the amino group
shows polar interactions with Trp89 (Figure 4).
Figure 4. Overlay of cocrystallized ligand and its validated docking posture. Red: cocrystallized,
green: validation
3.
RESULTS
3.1. In silico toxicology prediction
To calculate the potential toxicity activity of LMH-2, we employed OSIRIS
property
explorer
(https://www.organic-chemistry.org/prog/peo/),
a
computational server that predicts the risk of toxicity of the synthesized
compounds on the following criteria: mutagenic, tumorigenic, irritant and
reproductive effective. It presents numerical values and color-codes for the
different criteria, which are indicated as red, a warning of toxicity; orange for
intermediate toxicity; and green for nontoxic. The results for the final compound
LMH-2, haloperidol, and S1RA are shown in Table 2.
Table 2. Toxicological properties and drug-like qualifications of LMH-2, haloperidol, and S1RA
calculated with OSIRIS property explorer.
Compound
Mutagenic Tumorigenic
activity activity
Irritant
Effect in the
Drug
Drug
score
0.81
0.6
activity reproduction likeness
LMH-2
7.85
Haloperidol
12.32
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S1RA
1.66
0.37
Table 3 shows the theoretical results of absorption and metabolism
calculated for LMH-2, S1RA, and haloperidol based on a QSAR model by
the admetSAR server http://lmmd.ecust.edu.cn/admetsar2 [24].
Table 3. Predictive results of LMH-2, haloperidol, and S1RA computed with admetSAR
LMH-2
Haloperidol
S1RA
Intestinal absorption
Blood-brain barrier
PGp substrate
+
0.9736
+
+
1.000
+
+
1.000
+
0.979
+
0.9465
+
0.9973
-
0.6047
-
0.6673
-
0.5651
-
CYP2C9 substrate
CYP2D6 substrate
CYP3A4 substrate
AMES toxicity
0.8617
+
0.8355
+
0.7895
-
0.5497
+
0.8919
+
0.5617
+
0.5079
-
0.5796
-
0.7762
+
0.7433
-
0.9133
-
0.5171
-
Carcinogenic
0.9218
Weak
Inhibitor
0.8769
Strong
Inhibitor
0.8664
Strong
Inhibitor
hERG inhibition
The complemented toxicological profile prediction of compounds LMH-2, S1RA
and haloperidol was carried out with the ACD/ToxSuite program [25], which
reported several results, such as the percentage of the probability of an hERG
potassium channel blockage (correlated with cardiotoxicity), the inhibition of
CYP450 family isoforms (correlated with drug-drug interactions), as well as
acute toxicity (mean lethal dose = LD50) by the oral administration route for the
rat or mouse model, according to the category in the Organization for Economic
Cooperation and Development (OECD) classification. Predictions of the
toxicological profile are reported in Table 4.
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Table 4. In silico toxicity profiles predicted for LMH-2, haloperidol, and S1RA using
ACD/ToxSuite software
Compound
% of inhibition or blockage (Ki or IC50 < 10
M)
Half maximal
lethal dose
Category
OECD
(LD50, mg/kg, p.o.)
hERG
CYP450 isoforms
3A4
5
2D6
54
2C9 2C19 1A2
Mouse
540
Rat
930
180
960
LMH-2
Haloperidol
S1RA
14%
90%
66%
5
7
9
5
6
5
3
0
IV
III
IV
5
63
160
26
16
55
570
3.2. Chemistry
Figure 5 shows the synthetic route to obtain LMH-2.
CH2Cl2
LMH-2
1
3
Figure 5. Chemical synthesis of LMH-2
3.2.1. Synthesis of LMH-2
Into a 25 mL round bottom flask equipped with magnetic stirring, a gas trap, and
a liquid addition funnel, 4-amino-1-benzylpiperidine (15.8 mmol) and
triethylamine (17.4 mmol) were slowly added dropwise and stirred with 3 mL of
dry dichloromethane (CH2Cl2) for 15 minutes. Afterward, the flask was placed in
an ice bath (5° C) with the addition of 4-fluorobenzoyl chloride (17.4 mmol)
diluted in 5 mL of CH2Cl2. The solution was stirred 6 h at room temperature. The
reaction was monitored by TLC analysis until completed. The solvent was
evaporated to dryness, and the solids were washed with water, filtered and
recrystallized from ethanol, obtaining white crystals weighing 0.51 g with a
melting point of 160.5-161.9 °C and a yield of 95%.
1H NMR (600 MHz, CDCl3) δ: 1.55 and 2.00 (m, 4H, 2H-9, 2H-11), 2.16 and
2.84 (m, 4H, 2H-8, 2H-12), 3.51 (s, 2H, NCH2Ph), 3.95-4.02 (m, 1H, H-10),
7.088 (t, 2H, H-3, H-5, Jm=2, Jo=8.6 Hz), 7.23-7.35 (m, 2H, H-2’, H-6’), 7.74-
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13
7.76 (m, 2H, H-2, H-6, Jo=8.2 Hz, Jm= 5.2 Hz with the F heteroatom). C NMR
(150 MHz, CDCl3) δ: 32.2 (C-9, C-11), 47.2 (C-10), 52.3 (C-8, C-12), 63.0 (C-
13), 114.9 (C-4’), 115.5 (d, C-2´, C-6´, 2JC-F=22 Hz),127.5 (C-3, C-5), 128.6 (C-
2, C-6), 129.2 (d, C-3´, C-5´, 3JC-F=7 Hz), 130.9 (C-1’), 138 (C-1), 164.13 (d, C-
1
F, JC-F=248.5 Hz), 165.7 (C=O). EIMS m/z 312.00 (20%, M+), 295.06 (10%),
82.46 (100%).
3.3. In vitro 1R affinity
The 1 and 2 receptor affinities of LMH-2 were determined in receptor binding
studies employing the radioligands [3H]-(+)-pentazocine and [3H]-di-o-
tolylguanidine, respectively. These results are given in Table 5, including
corresponding reference compounds: haloperidol, di-o-tolylguanidine, (+)-
pentazocine, and S1RA.
Table 5. 1 and 2 receptor affinities of LMH-2 and reference compounds
Ki (nM)
Selectivity
1 /2
31.6
12
Compound
1
6.0
6.3
89
2
190
78
LMH-2
haloperidol
di-o-tolylguanidine
(+)-pentazocine
S1RA
58
0.7
5.7
17
-
-
>1000
>58
3.4. Molecular docking on the 1 receptor
To determine the interaction form of the LMH-2 with 1R, it was docked into the
receptor binding site obtained from the Protein Data Bank (PDB ID: 5HK2), as
reported by Schmidt et al. [23]. The docked complex with the lowest binding
energy was selected (−10.99 kcal/mol). Figure 6 shows a two- and three-
dimensional graph of the binding of LMH-2 to the receptor. The interaction
between the protonated amine from the heterocycle and Glu172 was observed,
as well as the  interaction between Tyr103 and fluorobenzoyl moieties
(Figure 6). Both interactions agree with those reported in the literature [23].
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Figure 6. 2D and 3D interaction map of the docking of 1R with LMH-2.
3.5. In vivo antiallodynic and antihyperalgesic assay for LMH-2
In Figure 7, we observed that the sham rats (receiving surgical incision without
the ligature of the nerve) did not increase the amount of time of licking before
the application of a cold stimulus (acetone) to the ipsilateral leg. However, rats
with CCI and vehicle administration increased the amount of time licking to
18.74 ± 0.74 s. This time increase was considered as a 100% effect [26].
However, in panel B, it is shown that rats with CCI increased the % withdrawal
response after a mechanical stimulus was applied via a 15 g von Frey filament
(10 stimuli); the number of positive responses in a series of 10 was considered
as 100% hyperalgesia.
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A
25
20
15
10
5
***
0
S h am
15 d ays after C C I
B
100
80
60
40
20
0
***
***
***
***
***
***
***
C C I
S ham
0
30
60
90
120
150
180
T im e (m in )
Figure 7. The allodynic effect was determined by ipsilateral application of a cold stimulus with
acetone. Allodynia is measured as accumulative time of licking in seconds for 1 min (panel A).
The hyperalgesic effect was established with a mechanical stimulus with a 15 g von Frey
filament. The response corresponded to the average of 10 stimuli, and the maximum response
was considered as 100% (panel B). In both panels, the rats with sciatic nerve ligation (CCI)
were compared to the rats undergoing surgery without ligation of the sciatic nerve (Sham). The
data are expressed as an average of six rats ± S.E.M. The significant difference was
determined by ANOVA Two-Way, multiple comparisons (***p≤0.001, F1-60= 1056) comparing the
CCI vs. Sham rats.
Figure 8 shows the dose-response curves of the antiallodynic and
antihyperalgesic effects administered at logarithmic doses of LMH-2 (0.1, 1.0,
3.2, 10.0, 31.6, and 100.0 mg/kg, s.c.) and gabapentin (GBP) (5.6, 10.0, 17.8,
31.6, 56.2 and 100.0 mg/kg, s.c.).
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D E 50 = 9.0 ± 1.4 m g /kg, s.c. *
D E 50 = 35.3 ± 2.3 m g/kg, s.c
LM H 2
G B P
A
250
200
150
100
50
0
0.01
0.1
1
10
100
L o g D o s e (m g /k g , s .c .)
LM H2 D E 50 = 14.8 ± 2 .1 m g/kg, s.c.*
B
G B P
D E 50 = 29.9 ± 1.1 m g/kg, s.c
250
200
150
100
50
0
0.01
0.1
1
10
100
L o g D o s e (m g /k g , s .c .)
Figure 8. Dose-response curve of the antiallodynic effect (A) and antihyperalgesic (B) effects of
the administration of LMH-2 (0.1-100.0 mg/kg, s.c.) and gabapentin (GBP, 5.6-100.0 mg/kg,
s.c.). The area under the curve (AUC) for each of the drug was calculated of the time curse
(antiallodynic or antihyperalgesic effects vs 0-180 min), AUC was determined by trapezoidal
rule. Each experimental point is shown as the % response of the average of 6 experimental
animals ± S.E.M. The ED50 was determined using the linear and logarithmic method, the
significant difference between both values was established by Student´s t-test (*p≤0.0.5).
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4. DISCUSION
4.1 In silico predictions
In tables 2 and 3, it is observed that LMH-2 has a better qualification with
respect to the standard drugs, in addition to not having mutagenic or
tumorigenic properties as does S1RA, with mutagenic properties probably
conferred by the naphthyl fragment of the molecule. Also LMH-2 undergoes
intestinal absorption in humans and could cross the blood-brain barrier. This
fact is of great importance for this project since it is desired that the compounds
reach the CNS. However, according to the program data, this compound could
be a substrate of the P glycoprotein (unfavorable data for the compounds). This
protein is widely distributed in the cells of the intestinal epithelium, and it is
responsible for expelling previously absorbed xenobiotics towards the intestinal
lumen. It creates an efflux for the expulsion of its substrate that is dependent on
ATP. Regarding metabolism, the program suggests that LMH-2 could be
metabolized by the CYP2D6 and CYP3A4 isoforms. It is worth mentioning that it
is preferable to have a compound as a substrate of several isoforms or only one
isoform. Its metabolism or excretion could be reduced or inhibited by another
xenobiotic, increasing possible toxic effects.
In table 4, we can observe that he in silico calculation of inhibition for the five
main isoforms of CYP450 (3A4, 2D6, 2C9, 2C19 and 1A2) for LMH-2 were
comparable to Haloperidol at relevant clinical concentrations (<10 M), showing
low probabilities of drug-drug interactions and undesirable adverse effects.
Also, LMH-2 had a low probability of being cardiotoxic, with less than 15% of
the hERG potassium channel blockage in comparison to the compound S1RA
and haloperidol, which had a high probability (66-90%) of being cardiotoxic.
These results were presented at clinical and relevant concentrations of 10 M.
According to the data established by the program, LMH-2 falls into category IV
established by OECD, whereas haloperidol, which showed probable toxicity in
acute administration, was in category III (LD50).
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4.2. Chemistry
Compound LMH-2 was obtained with a high yield (95%) and it chemical
structure was confirmed by spectroscopic (1H and 13C Nuclear magnetic
resonance) and spectrometric data by electronic impact of the sample.
4.3. In vitro 1R affinity
LMH-2 showed promising 1R affinity of 6.0 nM, whereas the 2R affinity was
190 nM, exhibiting outstanding 31.6-fold selectivity. In competitive radioligand
receptor binding studies, a Ki–value of 6.0 nM (pKi = 8.22) was determined for
the 1R affinity of LMH-2, whereas (+)-pentazocine and haloperidol showed the
same 1R affinity, with Ki–values of 5.7 and 6.3 nM, respectively. However,
S1RA, which is in phase II clinical trials, revealed nearly 3-fold lower 1R affinity
[Ki = 17 nM (pKi of 7.73)] than LMH-2 and haloperidol.
4.4. Molecular docking on the 1 receptor
The interaction of LMH-2 with the 1 receptor is showed in Figure 6. The
contacts between the protonated amine from the piperidine heterocycle and
residue Glu172 was observed, as well as the  interaction between residue
Tyr103 and fluorobenzoyl moiety. For haloperidol, the binding energy (G)
calculated by molecular docking was -11.1 Kcal/mol with a predicted Ki of 6.82
nM (experimental Ki = 6.3 nM). In the case of S1RA, a 3-D interaction between
the ionizable nitrogen of morpholine and the amino acid residue Glu172 was
also observed. The binding energy was calculated as -10.76 Kcal/mol and a Ki
of 12.14 nM (experimental Ki =17 nM).
4.5. In vivo antiallodynic and antihyperalgesic assay for LMH-2
These results agree with those previously reported, where acetone and the 15 g
von Frey filament have been used to generate allodynia and hyperalgesia [21].
It has been documented that the behavioral response observed in this
experimental model is generated by a cascade of events that initiate damage to
the peripheral nerves, particularly tactile allodynia, which is associated with the
ascending fiber system, while the persistence of allodynia and hyperalgesia
depends on the descending pathway from the rostral ventral cord. Apparently,
the ectopic discharges generated by the nerve ligation that correspond to a
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reorganization of ion channels such as Na+ and Ca2+ are responsible for the
hypersensitivity generated in the animal [27]. It has also been documented that
in this model, an inflammatory phenomenon is generated in which cytokines
such as IL-1 and TNF- [28], as well as NKI and AMPA receptors, participate
in nerve degeneration and sensitization at the central level [29, 30]. There is
little information related to the participation of 1R antagonists in the modulation
of neuropathic pain. Some 1R antagonist ligands related to the antiallodynic
and antihyperalgesic effects have been reported; for example, compound 3-
(6',7'-dihydro-1'H-spiro [piperidine-4,4'-pyrano [4,3-c]pyrazole]) was evaluated in
the capsaicin test, and (+)-MR200 was evaluated in the formalin test [31,32]. A
group of perhydroquinoxalines was designed to act on the 1R; however, they
could not cross the blood-brain barrier, and their antiallodynic and
antihyperalgesic effects were attributed to peripheral -opioid agonism [33].
Recently, in our working group, we designed the compound 2-(3,4-
dichlorophenoxy)-N-(2-morpholin-4-yl-ethyl)acetamide, which showed high
affinity for the 1R (Ki = 42 nM). This compound showed activity in a model of
inflammatory pain at the peripheral and spinal levels, while another of the
compounds N-(2-morpholin-4-yl-ethyl)-2-(1-naphthyloxy)acetamide showed
antihyperalgesic and antiallodynic effects in the CCI model. Because the effects
were reversed by the agonists 1 (+)-pentazocine and PRE-084, the
participation of the 1R was assumed [20, 34]. The fact that LMH-2 possesses
antihyperalgesic and antiallodynic effects contributes to the knowledge that
antagonists of 1R may be of therapeutic utility for the treatment of neuropathic
pain. In the present work, it could not be established if the effect of LMH-2 was
reversed by a 1R agonist; thus, the true interactions with this receptor remains
to be established. However, it has been documented that the antinociceptive
activity of haloperidol is due to the activation of 1R rather than dopaminergic
activity [6]. Since LMH-2 is a haloperidol analog, it could also interact only at
1R [33], but more evidence of its transduction mechanism is required. The
most widely used drugs in the clinic for the treatment of neuropathic pain are
pregabalin and gabapentin (GBP), and these do not eliminate pain. Clinically,
they have high antiallodynic and antihyperalgesic efficacies compared with
efficacies of other alternatives, such as anticonvulsants, anesthetics, and
benzodiazepines [35, 36]. For this reason, in the present work, the antiallodynic
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and antihyperalgesic effects of LMH-2 were compared with those obtained with
gabapentin. In Figure 8, the effects of LMH-2 expressed as AUC were
compared with those of gabapentin using the same route of administration
(s.c.). It can be established that the effect of LMH-2 and gabapentin is dose-
dependent because there is a significant difference between doses evaluated
(one-way ANOVA followed by Tukey´s post hoc (p≤ 0.05)), both in the
antihyperalgesic and antiallodynic effects.
On the other hand in figure 8, GBP (100 mg/kg, s.c.) showed greater efficacy
antihyperalgesic (95.9 ± 3.8%) and antiallodynic (90.23 ± 4.04%); whereas,
LMH-2 at the same dose generated less antihyperalgesic (66.02 ± 6.9%) and
antiallodynic (78.37 ± 4.6%) response. However, when comparing the potency
of the effects it can be seen that the new designed compound LMH-2 is more
potent than gabapentin because its ED50 is lower, both in the antihyperalgesic
effect (LMH-2, DE50 = 14.8 ± 2.1 mg/kg and GBP, DE50 = 29.9 ± 1.1 mg/kg) as
well in the antiallodynic effect (LMH-2, ED50 = 9.0 ± 1.4, GBP, ED50 = 35.3 ± 2.3
mg/kg) this means that a lower dose is required for LMH-2 generate the same
antihyperalgesic and antiallodynic response as gabapentin in the ICC model.
These results together suggest that LMH-2 is a new chemical entity with
potential therapeutic utility in the treatment of neuropathic pain, almost 2 times
more potent than gabapentin. It is important to mention that it is necessary to
continue exploring the pharmacological activity of this compound, not only in the
CCI model but also in other models of nociception. In addition, it is important to
characterize its transduction mechanism, as well as determine the adverse
effects and the short- and long-term toxicity.
5. CONCLUSIONS
The novel haloperidol analog LMH-2 was obtained in one step with excellent
yield (95%). LMH-2 showed adequate in silico pharmacokinetic behavior and
reasonable metabolic and toxicological properties, and low toxicity was
calculated with good drug-like qualifications. In the in vitro competitive binding
assay against 1R, LMH-2 had a Ki-value of 6 nM, being equipotent with
haloperidol but three times more potent than S1RA (Ki = 17 nM). Moreover,
LMH-2 displayed favorable σ1R/σ2R selectivity. In vivo, LMH-2 produced dose-
dependent antihyperalgesic and antiallodynic effects and showed similar
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antinociceptive efficacy, but with more potency than gabapentin, in a model of
CCI in the rat; thus, it is suggested that this compound could be acting as a 1R
antagonist. However, it is necessary to further analyze its pharmacological
potential as a possible therapeutic alternative for the treatment of neuropathic
pain.
Acknowledgments: This work was partially financed by the “Consejo Nacional
de Ciencia y Tecnología” (CONACyT) project No. 253814 (Ciencia Básica,
2015) granted to G. Navarrete-Vazquez and SIP-20190293 granted to M.
Déciga-Campos. We also appreciate the support of “Facultad de Farmacia,
UAEM” for providing some research supplies for this study.
Declaration of competing Interest: The authors declare no conflict of interest.
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