Synthesis, antinociceptive activity and pharmacokinetic profiles of nicorandil and its isomers

Article abstract of DOI:10.1016/j.bmc.2014.03.011

Nicorandil (N-(2-hydroxyethyl)nicotinamide nitrate) is an antianginal drug, which activates guanylyl cyclase and opens the ATP-dependent K⁺ channels, actions that have been suggested to mediate its vasodilator activity. We synthesized nicorandil and its two isomers, which vary in the positions of the side chain containing the nitric oxide (NO) donor, and also their corresponding denitrated metabolites. The activities of these compounds were evaluated in an experimental model of pain in mice. Pharmacokinetic parameters of nicorandil and its isomers, as well as the plasma concentrations of the corresponding denitrated metabolites and also nicotinamide and nitrite were determined. Nicorandil exhibited the highest antinociceptive activity, while the ortho-isomer was the least active. Nicorandil and para-nicorandil, which induced higher plasma concentrations of nitrite, exhibited higher antinociceptive activity, which suggests that the release of NO may mediate this activity.

Full text of DOI:10.1016/j.bmc.2014.03.011

Bioorganic & Medicinal Chemistry 22 (2014) 2783–2790
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antinociceptive activity and pharmacokinetic profiles
of nicorandil and its isomers
a
a
b
b
a
Isabela C. César , Adriana M. Godin , Débora P. Araujo , Francinely C. Oliveira , Raquel R. Menezes ,
c
a
a
c
Julliana R. A. Santos , Mariana O. Almeida , Marcela M. G. B. Dutra , Daniel A. Santos ,
Renes R. Machado , Gerson A. Pianetti , Márcio M. Coelho , Ângelo de Fátima
Department of Pharmaceutical Products, School of Pharmacy, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
Department of Chemistry, Institute of Exact Sciences, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
Department of Microbiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
a
a
a
b,
⇑
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Nicorandil (N-(2-hydroxyethyl)nicotinamide nitrate) is an antianginal drug, which activates guanylyl
cyclase and opens the ATP-dependent K channels, actions that have been suggested to mediate its vaso-
Received 6 January 2014
Revised 24 February 2014
Accepted 8 March 2014
Available online 19 March 2014
+
dilator activity. We synthesized nicorandil and its two isomers, which vary in the positions of the side
chain containing the nitric oxide (NO) donor, and also their corresponding denitrated metabolites. The
activities of these compounds were evaluated in an experimental model of pain in mice. Pharmacokinetic
parameters of nicorandil and its isomers, as well as the plasma concentrations of the corresponding deni-
trated metabolites and also nicotinamide and nitrite were determined. Nicorandil exhibited the highest
antinociceptive activity, while the ortho-isomer was the least active. Nicorandil and para-nicorandil,
which induced higher plasma concentrations of nitrite, exhibited higher antinociceptive activity, which
suggests that the release of NO may mediate this activity.
Keywords:
Nicorandil
Nicotinamide
Nitric oxide
NO-releasing drugs
Nitrite
Ó 2014 Elsevier Ltd. All rights reserved.
Antinociceptive activity
1
. Introduction
1A is cleaved.3 Nicotinamide, an amide derivative of vitamin B3,
similarly to nicorandil, induces vasodilation, but its potency is
8
Nicorandil [N-(2-hydroxyethyl)nicotinamide nitrate (1; Fig. 1)
much lower than that of the parent compound.
was synthetized in 19761 and has been marketed since 1984 in
Japan and other countries for the prevention and treatment of
chronic angina pectoris.2 It has been demonstrated that nicoran-
As NO and ATP-dependent K channels are important molecules
mediating many other biological actions, including inflamma-
and nociception, it would not be surprising to identify
new activities of nicorandil beyond those in the cardiovascular sys-
tem. Indeed, the inhibitory effects induced by nicorandil on the
overactive bladder in animal models, proteinuria and glomerular
injury in a model of diabetic nephropathy and intraocular pres-
sure in the anterior chamber of the eye of humans have been
demonstrated. Although the investigation of the effects induced
by nicorandil in experimental models of pain is still too prelimin-
+
–4
9,10
11
tion
+
dil releases nitric oxide (NO) and opens ATP-dependent K chan-
nels, actions that have been suggested to mediate its vasodilator
5
12
activity. It is still unclear whether nicorandil induces its NO-inde-
5
13
pendent effects directly or indirectly. Regarding the opening of
ATP-dependent K+ channels induced by nicorandil, Simpson and
14
4
Wellington (2004) have demonstrated that N-(2-hydroxyethyl)-
nicotinamide (1A; Fig. 1), the major metabolite, may mediate this
effect. In addition, 1A enhances the vascular effects of endogenous
15–17
ary, it is warranted. Many compounds that release NO
and
6,7
+
18–20
21
vasodilators, such as adenosine and adrenomedullin.
Another
activate ATP-dependent K channels,
and also nicotinamide,
metabolite, nicotinamide, may be formed when the side chain of
a nicorandil metabolite, exhibit activities in different experimental
models of pain. In addition, we have recently demonstrated that
systemic administration of nicorandil inhibits the nociceptive re-
sponse induced by formaldehyde in mice.
In the present study, we synthesized the three isomers of nico-
randil, which vary in the positions of the side chain that contains
2
2
Abbreviations: NO, nitric oxide; p.o., per os; Tmax, maximum time; AUC0–inf, area
under curve (time 0 > infinite); CMC, carboxymethyl cellulose; NMR, nuclear
magnetic resonance; Cmax, maximum concentration; AUC0–t, area under curve
(
time 0 > last time).
2
the –ONO group (Fig. 1), and investigated their activities in an
⇑
Corresponding author. Tel.: +55 31 3409 6373; fax: +55 31 3409 5700.
E-mail address: adefatima@qui.ufmg.br (Â. de Fátima).
experimental model of nociceptive and inflammatory pain in mice.
http://dx.doi.org/10.1016/j.bmc.2014.03.011
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
0

2
784
I. C. César et al. / Bioorg. Med. Chem. 22 (2014) 2783–2790
N
2.3.1. N-(2-Hydroxyethyl)nicotinamide (1A)
Yield 64%): mp 88.0 °C. IR (ATR): 3321, 3173, 2876, 1659, 1550,
N
N
N
H
N
H
N
H
N
(
ONO
2
ONO2
ONO2
1
1
3
1
422, 1296, 1057, 1028, 702. H NMR (200 MHz, DMSO-d
.39 (m, 2H), 3.57 (t, 2H, J = 5.8 Hz), 4.83 (br s, 1H), 7.42–7.50 (m,
H), 8.19 (d, 1H, J = 8.0 Hz), 8.66–8.68 (m, 2H), 9.02 (s, 1H). 13
): 42.3, 59.8, 123.4, 130.1, 135.0, 148.5,
51.7, 165.1. Anal. Calcd for C . Calcd (%): C, 57.82; H,
6
): 3.34–
O
O
O
O
O
(
1)
(2)
(3)
C
N
NMR (50 MHz, DMSO-d
6
1
6
N
H
N
H
N
H
N
8 10 2 2
H N O
OH
OH
OH
.07; N, 16.86. Found (%): C, 56.94; H, 6.03; N, 16.88.
O
(
1A)
(2A)
(3A)
2
.3.2. N-(2-Hydroxyethyl)picolinamide (2A)
Figure 1. Chemical structures of nicorandil (1), ortho-nicorandil (2) and para-nicorandil
(Yield 92%): mp 36.0 °C. IR (ATR): 3364, 3297, 2932, 2876, 1645,
1569, 1436, 1365, 1298, 1060, 1044, 821, 752. H NMR (200 MHz,
1
(
(
3) and their respective main metabolites N-(2-hydroxyethyl)nicotinamide (NHN)
1A), N-(2-hydroxyethyl)picolinamide (NHP) (2A) and N-(2-hydroxyethyl)isonico-
3
CDCl ): 3.60–3.68 (m, 2H), 3.84 (t, 2H, J = 5.0 Hz), 4.85 (br s, 1H),
tinamide (NHI) (3A).
7
.32–7.41 (m, 1H), 7.78 (td, 1H, J = 7.6, 1.6 Hz), 8.10 (d, 1H,
13
J = 7.8 Hz), 8.47 (d, 1H, J = 4.3 Hz), 8.64 (br s,1H).
C NMR
(
1
1
50 MHz, CDCl
3
): 41.6, 60.8, 121.6, 125.7, 136.9, 147.6, 149.0,
To further explore the structure-activity relationships, we deter-
mined some pharmacokinetic parameters of the three isomers, as
well as the plasma concentrations of the corresponding main deni-
trated metabolites (Fig. 1) and also the plasma concentrations of
nicotinamide and nitrite after the administration of each isomer
in mice.
64.6. Anal. Calcd for C . Calcd (%): C, 57.82; H, 6.07; N,
6.86. Found (%): C, 57.78; H, 6.12; N, 16.85.
8 10 2 2
H N O
2
.3.3. N-(2-Hydroxyethyl)isonicotinamide (3A)
(
Yield 88%): mp 135.5 °C. IR (ATR): 3317, 3173, 3058, 2931,
1
1
660, 1548, 1410, 1314, 1237, 1065, 1041, 1003, 816, 751.
): 3.32–3.40 (m, 2H), 3.47–3.59 (m, 2H),
4.81 (t, 1H, J = 5.4 Hz), 7.76 (d, 2H, J = 4.8 Hz), 8.69-8.75 (m, 3H).
H
NMR (200 MHz, DMSO-d
6
2
2
. Material and methods
1
3
C NMR (50 MHz, DMSO-d
6
): 42.3, 59.5, 121.3, 141.5, 150.1,
.1. Chemical and reagents
164.8. Anal. Calcd for C H N O . Calcd (%): C, 57.82; H, 6.07; N,
8
10
2 2
1
6.86. Found (%): C, 58.72; H, 6.04; N, 16.97.
All chemicals were obtained from commercial suppliers and
The nitrated compounds were obtained by mixing fuming nitric
used without further purification. Melting points were determined
acid (10 mmol) and the hydroxylated precursors (1 mmol) at
À5.0 °C and stirring for 2 h. The reaction mixture was poured into
a mixture of water and ice. The pH was adjusted to 6.0 by adding
1
in a Gehaka PF 1500 apparatus and are uncorrected. H nuclear
magnetic resonance (NMR) spectra and 13C NMR spectra were re-
corded on a Bruker Avance DPX/200. Chemical shift values (d) were
given in parts per million (ppm). Infrared (IR) spectra were re-
corded on a Spectro One Perkin Elmer. The elemental analyses
were performed with a Perkin-Elmer apparatus.
CaCO . The obtained solid was vacuum filtered and recrystallized
3
2
3,24
in ethanol, which furnished a white solid in all cases.
2.3.4. N-(2-Nytroxyethyl)nicotinamide (nicorandil; 1)
Yield 40%): mp 91.0 °C. IR (ATR): 3241, 3073, 1717, 1627, 1590,
(
1
2
.2. Syntheses of nicorandil and its isomers
1554, 1372, 1361, 1319, 1286, 1012, 1000, 860, 824, 705. H NMR
200 MHz, DMSO-d ): 3.65 (q, 2H, J = 5.1 Hz), 4.67 (t, 2H,
J = 5.1 Hz), 7.46–7.55 (m, 1H), 8.18 (d, 1H, J = 7.8 Hz), 8.71 (d, 1H,
(
6
Nicorandil (1) and its positional isomers (2 and 3) were synthe-
1
3
sized, as shown in Scheme 1. In summary, the corresponding esters
were treated with ethanolamine under reflux, which furnished the
hydroxylated precursors 1A to 3A. Compounds 1A–3A were trea-
ted with fuming nitric acid at À5 °C, which lead to the nitrates 1
J = 4.4 Hz), 8.94–9.02 (m, 2H). C NMR (50 MHz, DMSO-d ): 36.8,
6
72.1, 123.5, 129.5, 135.0, 148.3, 152.0, 165.2. Anal. Calcd for
C H N O . Calcd (%): C, 45.50; H, 4.30; N, 19.90. Found (%): C,
8
9
3 4
45.62; H, 4.31; N, 20.03.
1
to 3. All synthesized compounds were characterized by H and
1
3
C NMR, infrared, melting point and elemental analysis; the data
2.3.5. N-(2-Nytroxyethyl)picolinamide (ortho-nicorandil; 2)
are in accordance with previous reports.2
3,24
(Yield 63%): mp 62.5 °C. IR (ATR): 3391, 2956, 1664, 1630, 1520,
1
1
275, 1012, 858, 752. H NMR (200 MHz, DMSO-d
6
): 3.64–3.74 (m,
2
.3. Chemistry and biological assays
2H), 4.69 (t, 2H, J = 5.1 Hz), 7.55–7.61 (m, 1H), 7.94–8.07 (m, 2H),
1
3
8
.62 (d, 1H, J = 4.4 Hz), 9.08–9.10 (m, 1H). C NMR (50 MHz,
For the synthesis of the hydroxylated precursors, ethanolamine
DMSO-d ): 36.4, 72.2, 121.9, 126.6, 137.7, 148.3, 149.5, 164.3. Anal.
6
(
1.5 mmol) was added slowly to the esters (1 mmol) at 55 °C and
Calcd for C H N O . Calcd (%): C, 45.50; H, 4.30; N, 19.90. Found
8
9
3 4
stirred for 3 h. The reaction mixture was stirred at room tempera-
ture for 15 h. The residue was purified by silica gel column chro-
matography (eluent/ethyl acetate/hexane 8:2) or recrystallized
from ethyl acetate. The progress of the reaction was monitored
by TLC.
(%): C, 45.45; H, 4.21; N, 19.86.
2.3.6. N-(2-Nytroxyethyl)isonicotinamide (para-nicorandil; 3)
(Yield 55%): mp 112 °C. IR (ATR): 3265, 1647, 1618, 1545, 1322,
1
1280, 1018, 877, 754. H NMR (200 MHz, DMSO-d ): 3.65 (q, 2H,
6
OH
N
O
O
H N
N
O
HNO3, -5 oC, 2h
N
2
O
Reflux, 3h
HN
HN
OH
ONO2
ortho -substituted (2A) (92%)
meta-substituted (1A) (64%)
para-substituted (3A) (88%)
ortho -Nicorandil (2) (63%)
Nicorandil (1) (40%)
para -Nicorandil (3) (55%)
Scheme 1. Synthesis of nicorandil (1) and its isomers 2 and 3.

I. C. César et al. / Bioorg. Med. Chem. 22 (2014) 2783–2790
2785
J = 5.1 Hz), 4.67 (t, 2H, J = 5.1 Hz), 7.75 (d, 2H, J = 5.0 Hz), 8.73 (d,
the plasma samples were transferred to plastic tubes and stored
at À70 °C until analysis. The plasma concentrations of the com-
pounds were determined by a LC–MS/MS method adapted from
César et al. HPLC–MS/MS analysis was performed on a Waters
system (Milford, MA, U.S.A.), which was composed of a 1525 m bin-
ary pump, a 2777 sample manager, a TCM/CHM column oven and a
Quattro LC triple quadrupole mass spectrometer that was equipped
with an electrospray ion source. Calibration standards were pre-
pared in plasma by adding known amounts of the test compounds
2
3
H, J = 5.0 Hz), 9.03 (br s, 1H). 13C NMR (50 MHz, DMSO-d
9 3 4
6.9, 72.0, 121.2, 140.9, 150.2, 165.1. Anal. Calcd for C H N O .
6
):
8
2
6
Calcd (%): C, 45.50; H, 4.30; N, 19.90. Found (%): C, 45.67; H,
4
.26; N, 20.05.
2
.3.7. Drugs
Suspensions of nicorandil, its isomers and their corresponding
metabolites were prepared in a 0.5% w/v carboxymethylcellulose
CMC; Sigma, USA) suspension in saline. The same vehicle was
(
to control plasma. Plasma samples (100
lL) were mixed with a
used to prepare a suspension with phenobarbital (Aventis Pharma,
Brasil). The suspensions were administered per os (p.o.) in a vol-
ume of 4 mL/kg. The solution of 0.92% formaldehyde (Sigma,
USA) was prepared in saline. The suspensions and solutions were
prepared freshly before the experiments.
25 L aliquot of internal standard solution (4 mg/mL procainamide
l
in methanol). After agitation, the samples were extracted with 2 mL
of ethyl acetate and methyl-tert-butyl ether (8:2) and centrifuged
for 5 min at 3500 rpm. A 0.8 mL aliquot of the organic layer was
evaporated to dryness and reconstituted in the mobile phase
(
320 lL). A 20 lL sample was injected into the LC–MS/MS system.
2
.3.8. Animals
Chromatographic separation was performed using a ShinPack C18
column (100 Â 4.6 mm i.d.; 5 mm particle size) from Shimadzu
(Kyoto, Japan), maintained at 30 °C. The mobile phase consisted of
methanol and 2 mM aqueous ammonium acetate with 0.03% (v/v)
formic acid (33:67 v/v), at a flow rate of 1 mL/min. For quantitation,
the ion signal was recorded by selective reaction monitoring using
the following transitions: m/z 212 ? m/z 135 for nicorandil and its
isomers, m/z 166 ? m/z 106 for nicorandil metabolite and ortho-
metabolite, m/z 166 ? m/z 121 for para-metabolite, m/z 124 ? m/
z 80 for nicotinamide and m/z 236 ? m/z 163 for procainamide.
Female Swiss mice, which weighed 25–30 g, were used and had
free access to food and water. The animals were kept in a room that
had a 12 h light/12 h dark cycle and a temperature of 28 °C, which
corresponds to the thermoneutral zone for rodents, for at least
3
days before the experiments to allow acclimatization. This study
was approved by the Ethics Committee in Animal Experimentation
of the Federal University of Minas Gerais (Protocol number 131/
1
1), and all experiments were conducted according to the ethical
guidelines for the investigation of experimental pain in conscious
animals.
2
5
2
.3.12. Measurement of plasma concentrations of nitrite
2
.3.9. Evaluation of the nociceptive response induced by
Vehicle (CMC 0.5%), nicorandil or its isomers (100 mg/kg) were
formaldehyde
administered p.o. in animals fasted for 12 h. Each drug was admin-
istered to a group of 18 animals. At different times (15 min, 1 and
3 h) after the administration of nicorandil or its isomers, a group of
six animals was anesthetized with a mixture of ketamine (100 mg/
kg) and xylazine (10 mg/kg). A blood sample was collected from
the abdominal artery of each animal, placed in a heparinized tube
and immediately centrifuged at 14,000 rpm for 10 min. We used
this method of blood collection to avoid hemolysis that could affect
the nitrite colorimetric assay. Next, the plasma samples were
transferred to plastic tubes and stored at À70 °C until analysis.
Plasma samples were analyzed using a procedure based on the Gri-
One hour after p.o. administration of the vehicle (CMC 0.5%,
4
ml/kg), nicorandil, its isomers (50, 100 and 150 mg/kg) or the
main metabolites (equimolar doses to 50, 100 and 150 mg/kg of
nicorandil), formaldehyde solution (0.92%, 20 L) was injected
l
subcutaneously (s.c.) into the dorsal surface of the right hindpaw
of the animals. In one protocol, carried out after obtaining the
pharmacokinetic data, ortho-nicorandil was administered 5 min
before formaldehyde injection. Each mouse was placed under a
transparent glass funnel (18 cm diameter, 15 cm-high) and the
amount of time that the animal licked the injected paw was
determined between 0 and 5 min (first phase) and 15 and 30 min
2
7
ess reaction. In summary, 60
nilamide and 0.1% napthylethylenediamine in 2.5% phosphoric
acid) were added to 60 L of the plasma samples. After 10 min at
lL of the Griess reagent (1% sulfa-
(
second phase) after the injection of formaldehyde.
l
2
.3.10. Evaluation of the motor activity
The motor activity of the animals was evaluated in a rota-rod
room temperature, the absorbance of the samples was measured
at 540 nm. Blank plasma samples were also submitted to the same
procedure.
apparatus. The animals were trained on the apparatus at different
occasions for 3 days before the experiment. On the day of the
experiment, the animals were placed on a rota-rod (14 rpm) and
the time they spent on it was measured. The cut-off time was
2.3.13. Data collection and statistical analysis
The results, which are presented as the mean ± standard error
mean (S.E.M.), were analyzed via one-way analysis of variance,
which was followed by Newman–Keuls post-hoc test when the
main effect was significant. A P <0.05 was considered significant.
2
min. After the determination of the baseline values, the animals
were treated with the vehicle (CMC 0.5%, 4 ml/kg), nicorandil, its
isomers (150 mg/kg) or phenobarbital (40 mg/kg; positive control)
and 1 h latter they were again tested in the apparatus.
3
. Results
2
.3.11. Measurement of the plasma concentrations of
nicorandil, its isomers and the main metabolites and
nicotinamide
3.1. Synthesis and characterization of nicorandil and its isomers
For the pharmacokinetic study, we used animals that were sub-
jected to food restriction for 12 h. Nicorandil or its isomers were
administered p.o. at a dose of 100 mg/kg. Each drug was adminis-
tered to a group of 66 animals. At different times (5, 15, 30 and
The synthesis of nicorandil (1) and its isomers, ortho-nicorandil
(2) and para-nicorandil (3), was accomplished using the reaction
sequence shown in Scheme 1. The synthesis of nicorandil (1),
ortho-nicorandil (2) and para-nicorandil (3) was completed by
the nitration, which employed fuming nitric acid as a nitrating
agent, of their respective precursors, 1A, 2A and 3A. The IR spectra
of 1A, 2A and 3A displayed absorbances indicative of an –OH group
4
5 min, 1, 2, 3, 4, 6, 8 and 10 h) after administration of the samples,
a group of six animals was euthanized by decapitation. A blood
sample from each animal and also from an additional group of six
non-treated mice (control group) was collected in a heparinized
tube and immediately centrifuged at 14,000 rpm for 10 min. Next,
À1
(3321, 3364 and 3317 cm , respectively), while these bands were
absent in the IR spectra of nicorandil (1), ortho-nicorandil (2) and

2
786
I. C. César et al. / Bioorg. Med. Chem. 22 (2014) 2783–2790
para-nicorandil (3). The success of the nitration reactions was also
validated by the presence of H NMR signals corresponding to the
second phase was inhibited by the doses of 100 and 150 mg/kg
doses (Fig. 2A).
1
methylene protons, –CH
2
–X (X = OH or ONO
compounds. The H NMR spectra displayed the signals at d 3.57
t, 2H) for (1A), d 3.84 (t, 2H) (2A) and d 3.47–3.59 (m, 2H) (3A)
2
), present in these
Next, we investigated whether the previous (1 h) p.o. adminis-
tration of the two positional nicorandil isomers, ortho- (2) and
para-nicorandil (3), at the doses of 50, 100 or 150 mg/kg, could also
induce an antinociceptive effect. ortho-Nicorandil (2) inhibited
both phases of the nociceptive response only at the highest dose
(150 mg/kg) (Fig. 2B). Because the time needed to reach the peak
concentration (Tmax) for ortho-nicorandil (2) is shorter than those
for nicorandil (1) and para-nicorandil (3), as will be described in
the next session, we carried out another protocol to evaluate
whether lower doses of ortho-nicorandil (2) could induce an antin-
ociceptive effect when administered immediately before (5 min)
the injection of formaldehyde. However, in this protocol, ortho-nic-
orandil (2) did not induce an antinociceptive effect (Fig. 3). The
other positional isomer, para-nicorandil (3), at the doses of 100
and 150 mg/kg, inhibited only the second phase of the nociceptive
response (Fig. 2C). To directly compare the effects induced by nic-
orandil (1) and its two positional isomers, we carried out another
protocol in which the three compounds were administered at a
fixed dose (100 mg/kg). Providing support to the previous results,
the greatest inhibition of the nociceptive response was induced
by nicorandil, followed by para-nicorandil (3), while ortho-nicoran-
dil (2) was devoid of activity (Fig. 4).
1
(
were shifted to d 4.67 (t, 2H) for all nitrated compounds (nicorandil
and its isomers). These data confirm the expected structures were
successfully synthesized as such results are in agreement with
those reported elsewhere.2
3,24
3
.2. Effects induced by nicorandil, its positional isomers and
main metabolites on the nociceptive response induced by
formaldehyde
Previous (1 h) p.o. administration of nicorandil, at the doses of
3
, 10 or 30 mg/kg, did not inhibit the nociceptive response induced
by formaldehyde (data not shown). However, we observed that
higher doses of nicorandil (50, 100 or 150 mg/kg) did inhibit this
response in a dose-dependent manner. The first phase of the
nociceptive response was inhibited by the three doses, while the
As the main metabolites of nicorandil (1), ortho-nicorandil (2)
and para-nicorandil (3) are NHN (1A), NHP (2A) and NHI (3A),
respectively, we subsequently investigated whether the last three
compounds could mediate the effect induced by nicorandil and
its positional isomers. However, previous (1 h) p.o. administration
of NHN (1A), NHP (2A) or NHI (3A), at doses equimolar to those of
the respective parent compounds (50, 100 and 150 mg/kg), did not
inhibit the nociceptive response induced by formaldehyde (Fig. 5).
Figure 3. Nociceptive response induced by formaldehyde in mice previously
(
5 min) treated with ortho-nicorandil (2) (50, 100 or 150 mg/kg, p.o.) (n = 9).
Figure 2. Nociceptive response induced by formaldehyde in mice previously (1 h)
treated with different doses (50, 100 or 150 mg/kg, p.o.) of (A): nicorandil (1), (B):
Figure 4. Nociceptive response induced by formaldehyde in mice previously (1 h)
treated with nicorandil (1) or its isomers ortho-nicorandil (2) and para-nicorandil
⁄
⁄⁄
⁄⁄⁄
indicate statistically
ortho-nicorandil (2) or (C): para-nicorandil (3).
significant difference compared to the vehicle-treated group (P <0.05, P <0.01 and P
0.001, respectively). n = 9.
,
and
⁄
⁄⁄
⁄⁄⁄
indicate statistically significant difference from
(3) (100 mg/kg, p.o.).
,
and
<
vehicle-treated group (P <0.05, P <0.01 and P <0.001, respectively) (n = 9).

I. C. César et al. / Bioorg. Med. Chem. 22 (2014) 2783–2790
2787
Table 2
Time spent by mice on the rotating rod after previous (1 h) p.o. treatment with
nicorandil (1), ortho-nicorandil (2), para-nicorandil (3) (150 mg/kg) or phenobarbital
(40 mg/kg)
Treatment
Time spent on the rotating rod (s)
Baseline
1 h
Vehicle
120 ± 0
120 ± 0
120 ± 0
120 ± 0
119 ± 1
119 ± 1
120 ± 0
120 ± 0
120 ± 0
26 ± 2a
Nicorandil (1)
ortho-Nicorandil (2)
para-Nicorandil (3)
Phenobarbital
a
Indicates statistically significant difference compared to the vehicle-treated
group (P <0.001, respectively) (n = 9).
are shown in Table 1. The plots of the mean plasma concentration
over time for all compounds are presented in Figure 6A. Nicorandil
and its isomers were rapidly absorbed in the gastrointestinal tract,
which was demonstrated by the Tmax being less than 1 h. Plasma
levels were low but still measurable 10 h after administration of
all compounds. Nicorandil (1) and para-nicorandil (3) exhibited
similar pharmacokinetic profiles, in spite of the greater bioavail-
ability of para-nicorandil (3) compared to that of nicorandil, as
indicated by the area under the curve (AUC) values. ortho-Nicoran-
dil (2), when compared with the other two isomers, showed a dis-
tinct profile, characterized by a faster absorption, as well as a rapid
biotransformation. This isomer was also rapidly eliminated from
plasma and exhibited the lowest plasma concentration and bio-
availability of the three compounds.
Figure 6B shows the plasma concentrations of the metabolites,
NHN (1A), NHP (2A) and NHI (3A), after p.o. administration of nic-
orandil (1), ortho-nicorandil (2) and para-nicorandil (3) (100 mg/
kg), respectively. The plasma concentration–time curves of the ma-
jor metabolites of nicorandil (1) and para-nicorandil (3) (1A and
3
A, respectively) reflected the metabolism of each parent com-
pound. Similarly to the parent compounds, the metabolites of 1A
and 3A exhibited similar profiles, with 3A reaching higher concen-
trations when compared to 1A. As expected, the formation of 2A,
the main metabolite of ortho-nicorandil (2), occurred at a lower
extent, reflecting the lower absorption and rapid metabolism of
the parent compound. Additionally, the plasma concentrations of
nicotinamide did not change markedly after p.o. administration
nicorandil or its two positional isomers (100 mg/kg; Fig. 6C).
As NO is known to be a metabolic product of nicorandil (1), we
indirectly investigated its formation after administration of nico-
Figure 5. Nociceptive response induced by formaldehyde in mice previously (1 h)
treated with (A) NHN (1A), (B) NHP (2A) or (C) NHI (3A) (50, 100 or 150 mg/kg, p.o.)
(n = 9).
To eliminate motor incoordination or muscle relaxation as pos-
sible confounding effects to the antinociceptive activity of nicoran-
dil and its two positional isomers, we evaluated the effect induced
by previous (1 h) administration of the three compounds (150 mg/
kg, p.o.) on the motor coordination of the animals. None of the
compounds altered the time mice spent on the rota-rod apparatus,
while phenobarbital (40 mg/kg) markedly reduced this parameter
randil (1), ortho-nicorandil (2) and para-nicorandil (3) by measur-
À
ing the plasma concentrations of nitrite (NO
2
), which is a primary,
stable and non-volatile product. The plasma concentrations of
nitrite were determined 15 min, 1 and 3 h after the p.o. administra-
tion of vehicle (CMC 0.5%, 4 ml/kg), 1, 2 or 3 (100 mg/kg). Nicoran-
dil (1) and para-nicorandil (3) induced similar increases in the
plasma concentrations of nitrite. The highest concentrations were
observed 15 min after the administration of the compounds and
subsequently declined. On the other hand, the plasma concentra-
tions of nitrite after the administration of ortho-nicorandil (2) did
(Table 2).
3
.3. Pharmacokinetic profiles of nicorandil and its two
positional isomers
Pharmacokinetic parameters of nicorandil (1), its two positional
isomers and their corresponding metabolites were calculated and
Table 1
Pharmacokinetic parameters of nicorandil (1), its isomers ortho-nicorandil (2) and para-nicorandil (3) and corresponding metabolites (1A, 2A and 3A) (100 mg/kg) after p.o.
administration of (1), (2) and (3) in mice (n = 6)
Pharmacokinetic parameters
Nicorandil and isomers
Metabolites
1
3
2
1A
9.46 ± 1.42
2.83 ± 0.75
41.12 ± 3.22
41.97 ± 3.04
1.4 ± 0.3
3A
2A
C
T
max
max (h)
AUC0-t
AUC0-inf
1/2 (h)
(
l
g/mL)
42.63 ± 9.58
0.67 ± 0.26
49.44 ± 7.42
0.88 ± 0.63
160.40 ± 23.49
160.44 ± 23.50
0.76 ± 0.10
30.77 ± 7.24
0.11 ± 0.07
10.11 ± 1.39
10.14 ± 1.40
1.1 ± 0.2
11.80 ± 0.66
2.00 ± 0.52
41.56 ± 2.36
41.69 ± 2.28
1.1 ± 0.4
2.64 ± 0.58
0.31 ± 0.23
2.69 ± 0.67
2.70 ± 0.66
1.2 ± 0.3
(
lg h/mL)
121.18 ± 10.63
122.64 ± 10.84
1.5 ± 0.4
(
lg h/mL)
t

2
788
I. C. César et al. / Bioorg. Med. Chem. 22 (2014) 2783–2790
Figure 7. Plasma concentrations of nitrite after p.o. administration of nicorandil (1),
⁄
⁄⁄
ortho-nicorandil (2) or para-nicorandil (3) (100 mg/kg). and indicate significant
difference from vehicle-treated group (P <0.05 and P <0.01, respectively) (n = 7).
nicorandil, which we recently demonstrated,22 and expand this
activity to its two positional isomers.
The nociceptive response induced by formaldehyde involves
inflammatory, neurogenic and central nervous system compo-
nents, clearly characterized by a biphasic profile. The first phase
begins immediately after the injection of formaldehyde and in-
volves direct activation by the nociceptive stimulus of TRPA
TRPV channels in the nociceptors.
1
1
1
and
The second phase begins
5 to 20 min after the injection of formaldehyde and lasts for 20
3
1,32
to 60 min. This phase is related to an increased excitability of dor-
sal horn neurons and the production of multiple inflammatory
mediators, which lead to the sensitization of nociceptors.3
3,34
Nicorandil (1) and its isomers, ortho-nicorandil (2) and para-
nicorandil (3), inhibited mainly the second phase of the
nociceptive response, although to different extents as nicorandil
(
1) exhibited the highest activity, while ortho-nicorandil (2) was
the least active. Regarding the first phase, the compounds induced
a less marked effect, with nicorandil (1) and ortho-nicorandil (2)
partially inhibiting the response and para-nicorandil (3) devoid
of activity. As the pharmacokinetic data demonstrated that ortho-
nicorandil (2) was immediately absorbed after p.o. administration
and rapidly removed from the blood, we investigated whether the
reduced activity of this compound during the second phase of the
nociceptive response could be related to its peculiar pharmacoki-
netic profile. However, the administration of this compound
immediately before formaldehyde did not significantly inhibit the
nociceptive response. Thus, the reduced or absent antinociceptive
effect of ortho-nicorandil (2) cannot be attributed to its possible
fast clearance. Rather, the results indicate an intrinsic lack of activ-
ity of compound 2 in the pain model used in the present study.
As nicorandil (1), ortho-nicorandil (2) and para-nicorandil (3)
inhibit mainly the second phase of the nociceptive response in-
duced by formaldehyde, their profiles resemble those of anti-
Figure 6. Plasma concentrations of nicorandil (1) and its isomers ortho-nicorandil
2) and para-nicorandil (3) (A), the corresponding metabolites NHN (1A), NHP (2A)
and NHI (3A) (B) or nicotinamide (C) after p.o. administration of 1, 2 or 3 (100 mg/
kg) (n = 6).
(
3
5
36
not statistically differ from those exhibited by the group treated
with vehicle (Fig. 7).
inflammatory drugs, such as indomethacin and diclofenac. In
addition, it is unlikely that the effects induced by nicorandil (1),
ortho-nicorandil (2) and para-nicorandil (3) on the nociceptive
behavior resulted from a muscle relaxing effect or loss of motor
coordination, because these compounds did not reduce the time
that the animals spent on the rotating rod.
4
. Discussion
Positional isomerism may markedly affect the pharmacological
properties of a given compound.28–30 In the present study, we
investigated the effects induced by the systemic administration
of nicorandil and its two positional isomers on the nociceptive re-
sponse induced by formaldehyde in mice, an experimental model
of nociceptive and inflammatory pain. We demonstrated that the
three isomers exhibit an antinociceptive activity, inhibiting mainly
the second phase of this nociceptive response. These results
provide additional support for the antinociceptive activity of
As previously described, the metabolism of nicorandil initially
4
involves a denitration process, which leads to the formation of 1A
and the release of NO. In the present study, we also observed the
quick formation of 1A after administration of nicorandil (1). The
highest concentration of this metabolite was observed 3 h after
administration of nicorandil (1) and subsequently declined.
Similarly, there was also an increase in the plasma concentrations
of nitrite, which indirectly indicates the release of NO. After p.o.

I. C. César et al. / Bioorg. Med. Chem. 22 (2014) 2783–2790
2789
administration of the para isomer 3, the formation of the corre-
sponding denitrated metabolite (3A) occurred in a similar manner
to the formation of 1A after the administration of nicorandil (1).
The highest concentration of 3A, which had a magnitude similar
to that of 1A, was observed 1 h after the administration of
ortho-nicorandil (2), and subsequently declined. We also observed
that parallel to the increase of the plasma concentrations of 3A,
there was an increase of the plasma concentrations of nitrite. These
toxicological profile. We demonstrated that nicorandil, which is
used in many countries as a vasodilator, exhibits a marked
antinociceptive activity, increasing the number of NO-releasing
compounds that are potential analgesics. In addition, structural
changes in drugs with well-established pharmacologic activities
may alter their pharmacokinetic or pharmacodynamic properties
and improve their therapeutic efficacy. We demonstrated that
the ortho- (2) and para-substituted (3) isomers of nicorandil (1)
exhibited distinct pharmacodynamic and pharmacokinetic proper-
ties when compared to nicorandil. An important structural feature
for the antinociceptive activity seems to be the side chain in the
meta position of the pyridine ring, since nicorandil was the most
effective isomer.
2
results indicate that moving the side chain with the –ONO group
to the para position results in a compound 3 that retains its ability
to release NO. However, ortho-nicorandil (2) exhibits a different
profile when compared to nicorandil (1) and para-nicorandil (3).
The highest concentration of the corresponding denitrated metab-
olite 2A occurred earlier (0.5 h) when compared to those of 1A and
3
A and had a much lower magnitude, declining thereafter. As
Author contribution
expected, the reduced formation of 2A was associated with a
decreased formation of nitrite. The pharmacokinetic profile of 2A
is very different compared to 1A and 3A and indicates that its
stability is reduced, which contributes to the lower plasma
concentration and rapid clearance. Additionally, we observed that
the plasma concentrations of nicotinamide barely changed after
the administration of nicorandil or its two positional isomers.
Although nicotinamide exhibits an antinociceptive activity, the
doses that induce an effect in the model of nociceptive response
induced by formaldehyde are much higher than those of nicorandil
I.C.C., A.M.G., R.R.M., M.O.A., M.M.G.B.D., R.R.M., G.A.P., J.R.A.S.,
D.A.S. and M.M.C. were responsible for the pharmacological assay.
D.P.A., F.C.O. and A.D.F. were responsible for the design, synthesis
and characterization of the nicorandil, its analogs and metabolites.
Acknowledgment
2
1
We thank the Brazilian agencies Fundação de Amparo à
Pesquisa do Estado de Minas Gerais, Conselho Nacional de
Desenvolvimento Científico e Tecnológico and Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior for financial support.
A.D.F. is recipient of research fellowship from CNPq.
(1). Altogether, these results clearly indicate that the main
metabolites of nicorandil (1) and its positional isomers are the
denitrated compounds and that their effects are not mediated by
nicotinamide.
The pharmacokinetic profiles of nicorandil and its two posi-
tional isomers provided the rationale to investigate the activity
of their respective denitrated metabolites, as they were rapidly
formed after the administration of the parent compounds. There-
fore, we also investigated the effects induced by equimolar doses
of 1A, 2A and 3A, which are the main metabolites of nicorandil
Supplementary data
Supplementary data (the NMR spectra for all the synthesized
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2014.03.011.
(
1), ortho-nicorandil (2) and para-nicorandil (3), respectively. How-
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