Adamantyl analogues of paracetamol as potent analgesic drugs via inhibition of TRPA1

Article abstract of DOI:10.1371/journal.pone.0113841

Paracetamol also known as acetaminophen, is a widely used analgesic and antipyretic agent. We report the synthesis and biological evaluation of adamantyl analogues of paracetamol with important analgesic properties. The mechanism of nociception of compoun

Full text of DOI:10.1371/journal.pone.0113841

RESEARCH ARTICLE
Adamantyl Analogues of Paracetamol as
Potent Analgesic Drugs via Inhibition of
TRPA1
Nieves Fresno1, Ruth Pe´rez-Ferna´ndez1, Carlos Goicoechea2, Ibon Alkorta1*,
Asia Ferna´ndez-Carvajal3, Roberto de la Torre-Mart´ınez3, Susana Quirce3,
Antonio Ferrer-Montiel3, M. Isabel Mart´ın2, Pilar Goya1, Jose´ Elguero1
1. Instituto de Qu´ımica Me´dica, IQM-CSIC, Madrid, Spain, 2. Departamento de Farmacolog´ıa y Nutricio´n,
Unidad Asociada de I+D+i al CSIC, Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos,
Alcorco´n, Madrid, Spain, 3. Institute of Molecular and Cellular Biology, Universidad Miguel Herna´ndez,
Alicante, Spain
*ibon@iqm.csic.es
Abstract
OPEN ACCESS
Citation: Fresno N, Pe´rez-Ferna´ndez R,
Goicoechea C, Alkorta I, Ferna´ndez-Carvajal A,
et al. (2014) Adamantyl Analogues of Paracetamol
as Potent Analgesic Drugs via Inhibition of
TRPA1. PLoS ONE 9(12): e113841. doi:10.1371/
journal.pone.0113841
Paracetamol also known as acetaminophen, is a widely used analgesic and
antipyretic agent. We report the synthesis and biological evaluation of adamantyl
analogues of paracetamol with important analgesic properties. The mechanism of
nociception of compound 6a/b, an analog of paracetamol, is not exerted through
direct interaction with cannabinoid receptors, nor by inhibiting COX. It behaves as
an interesting selective TRPA1 channel antagonist, which may be responsible for
its analgesic properties, whereas it has no effect on the TRPM8 nor TRPV1
channels. The possibility of replacing a phenyl ring by an adamantyl ring opens new
avenues in other fields of medicinal chemistry.
Editor: Shang-Zhong Xu, University of Hull, United
Kingdom
Received: May 29, 2014
Accepted: October 31, 2014
Published: December 1, 2014
Copyright: ß 2014 Fresno et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and repro-
duction in any medium, provided the original author
and source are credited.
Introduction
Data Availability: The authors confirm that all data
underlying the findings are fully available without
restriction. All relevant data are within the paper
and its Supporting Information files.
Although paracetamol (4-hydroxyacetanilide, acetyl-p-aminophenol, acetamino-
phen, APAP) [1–3] was found to be an effective analgesic more than a century
ago, its mechanism of action is complex and the subject of continuous research
mainly due to its extensive metabolism in animals and humans [4].
Funding: The present work has been supported
by grants RTA (RED Trastornos Adictivos RD06/
001/0014), SAF2012-40075-C02, BFU2009-08346,
and BFU2012-39092-C02-01, CONSOLIDER
INGENIO2010 Program (CSD2008-00005), and
PROMETEO/2010/046. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Several reports have described pathways of APAP to exert its analgesic activity.
Unlike non-steroidal anti-inflammatory drugs (NSAIDs), whose analgesic and
anti-inflammatory effects are related to their inhibition of the cyclooxygenase
enzymes (COX-1 and COX-2), paracetamol is a weak anti-inflammatory agent
with an absence of COX-related adverse effects [5–6]. In the brain and spinal cord
paracetamol is metabolized by fatty amide hydrolase (FAAH) to N-arachido-
Competing Interests: The authors have declared
that no competing interests exist.
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
noylphenolamine (AM404). AM404 is a known activator of the capsaicin receptor
(TRPV1) [7] and the cannabinoid CB1 receptor system [8–9] both of which
confer analgesia in the central nervous system. The main drawback found in the
use of paracetamol is the mechanism-based inactivation (MBI) of cytochrome
P450 enzymes (CYPs) [10]. APAP metabolites (e.g. N-acetyl-p-benzoquinonei-
mine (NAPQ1) have centered the attention because of their toxic actions causing
hepatotoxicity [11–12]. Many articles have been devoted to this topic [13–17] and
different attempts have been made to circumvent this problem. For instance, 3-
hydroxyacetanilide is non hepatotoxic because the oxygen atoms of quinones can
only be at 1,2-,ortho, or, 4-positions, para [18]. Other authors have designed new
analgesics derived from the paracetamol metabolite (AM404) having an
anandamide chain instead of the acetamido group [19]. The replacement of the
methyl group by saccharin or an open form of it (SCP-1 and SCP-123) improves
considerably its water solubility [20]. A proline prodrug was developed with this
purpose [21] and a paracetamol analogue known as propacetamol is currently in
the market. Note that paracetamol presents polymorphism [22–23]. Whereas
researchers have been trying to avoid the formation of the metabolite responsible
for APAP toxicity designing non-hepatotoxic paracetamol analogues [24], a new
antinociceptive mechanism driven by activation of TRPA1 in the spinal cord by
APAP metabolites such as NAPQ1 has been reported [25].
In August 2013, the FDA warned of rare but serious skin reaction associated
with paracetamol. Despite the fact that it has been on the market for decades,
paracetamol can be considered a ‘‘standalone’’ drug of which no effective
analogues are known [26]. Some compounds which have been reported include
the N-methyl derivative, and N-(1H-indazolo-5-yl), N-(4-hydroxy)acetamides
and recently pyridinol-fused ring derivatives, but in all of them, the aromatic ring
is present [27–29].
The approach proposed in the present paper is totally different being based on
the hypothesis that the replacement of the phenyl ring of paracetamol by an
adamantane ring with the same substituents in 1,4-positions, will yield analogues
of paracetamol. Adamantyl derivatives are found in medicinal chemistry in very
different domains [30–32]. Adamantane is one of the few singular hydrocarbon
moieties that have been successfully employed in pharmaceutical industry. The
most popular ‘‘hit’’ is amantadine used for the treatment of influenza and
Parkinson’s disease [33]. Related to analgesia and/or inflammation some
analogues of D8-THC such as compound 1 have been reported (Fig. 1) [31–32].
Quinolone 2 binds selectively to the cannabinoid CB2 receptor being endowed
with analgesic activity in vivo [34]. Some derivatives from 3 reported as P2X7
receptor antagonists acted as agents against rheumatoid arthritis [35–36].
Amongst the carbohydrazides, the most interesting is 4 developed by Abbott [37].
As can be seen, none of the structures represented in Fig. 1 is related to our
compounds.
In this work we report the synthesis and biological evaluation of compounds 5,
6a and 6b (Fig. 2) in which the phenyl ring has been substituted by an adamantyl.
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
Figure 1. Some adamantyl derivatives with antiinflamatory and analgesic properties.
doi:10.1371/journal.pone.0113841.g001
Results and Discussion
1.1 Chemistry
The preparation of compound 5 is summarized in Fig. 3. Commercially available
3-amino-1-adamantanol (7) [38–39] was acetylated in water, the solvent of choice
for green chemistry.
Compounds 6a and 6b were synthesized following the route summarized in
Fig. 4 [40]. These compounds were obtained after a reductive amination and in
three steps from the commercially available 5-hydroxy-2-adamantanone (8). The
synthesis was carried out using the pure enantiomer of the S-a-methylbenzyla-
mine and the 5-hydroxy-2-adamantanone refluxing in absolute ethanol. The
intermediate compound 9 was hydrogenated. This hydrogenation with the
catalyst Pd/C 10% could have been performed in two steps, isolating the
benzylamino derivatives, but it was preferred an overnight hydrogenation to yield
compounds 10a and 10b. The last synthetic step consisted of the acetylation in
water of the mixture using the same conditions as for compound 5 yielding an E/Z
mixture of compounds 6a and 6b in a 7:3 ratio, with the E isomer being the major
´
product. Compounds 6a and 6b were found in a paper by Gonzales-Nunez et al.
[41] but with a different enriched mixture (Z:E 75/25) and not separated for fully
˜
Figure 2. Adamantyl analogues of paracetamol.
doi:10.1371/journal.pone.0113841.g002
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
Figure 3. Synthesis of compound 5. Reagents and conditions: i) acetic anhydride, water, 0 C2rt.
˚
doi:10.1371/journal.pone.0113841.g003
characterization. In our case, compounds 6a and 6b were purified by automated
semipreparative LC-MS for their characterization.
The 13C and 1H NMR spectroscopy in DMSO-d6 of the mixture of 6a and 6b is
interesting. The signals were assigned taking into account that 6a was the major
isomer (about 70%) and by means of 2D experiments. To check the assignments
as well as the isomerism, we calculated the absolute shielding [42-43] (s, ppm) at
the GIAO/B3LYP/6-311+G(d,p) level and transform the absolute shielding into
chemical shifts (d, ppm, see Table S1).
1.2 Pharmacology
The analgesic activity of compounds 5 and the 6a/b mixture was compared to that
of paracetamol and morphine in a model of visceral pain (Fig. 5). The
intraperitoneal (i.p.) administration of acetic acid (2%) induced a typical writhing
reaction, characterized by a wave of contraction of the abdominal musculature
followed by extension of the hind limbs. After acetic acid administration, mice
were placed in individual transparent containers and, after 5 min, the number of
¨
writhes was counted during a 10 min period. In naive animals, the number of
stretches induced by acetic acid was 25.4¡0.8. Morphine (5 mg/kg, i.p.), an
opioid accepted as the gold standard of analgesic drugs, notably decreased the
number of writhes by 75%. Paracetamol (100 mg/kg), administered i.p.
Figure 4. Synthesis of compounds 6a and 6b. Reagents and conditions: i) (i) S-a-methylbenzylamine,
EtOH, reflux, 2 days; (ii) H , 10% Pd-C, THF, rt; 12 h; (iii) acetic anhydride, water, 0 C2rt.
˚
2
doi:10.1371/journal.pone.0113841.g004
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
Figure 5. Analgesic activity of compounds 5 and 6a/b in comparison with paracetamol and morphine.
Bars show the mean number of stretches induced by acetic acid (i.p., 2%) in mice treated with paracetamol
(100, 200 mg/kg), morphine (5 mg/kg), compound 5 (100, 200 or 500 mg/kg), or compound 6a/b (250, 375,
500 and 750 mg/kg). Data are given as mean ¡ SEM, with n515, and (* p,0.05, ** p,0.01 One way ANOVA
to compare with control group.
doi:10.1371/journal.pone.0113841.g005
30 minutes before acetic acid, was less effective than morphine reducing only by
40% the nociceptic behavior. Furthermore, a 2-fold increment in the paracetamol
dose did not result in an increase of its analgesic effect in this pain model (Fig. 5).
Akin to the reference analgesic agents, compounds 5 and 6a/b were
administered i.p. 30 minutes before the injection of acetic acid (Fig. 5). The
solubility of these derivatives was significantly higher than paracetamol allowing
their testing at higher doses, which is a desirable property. Both compounds (5
and 6a/b) were able to induce a significant antinociceptive effect that was more
potent for compound 6a/b. As illustrated in Fig. 5, compound 5 displays a similar
40% antinociceptive activity to that recorded with paracetamol. This analgesic
effect of compound 5 displayed some dose-dependency and saturated at 200 mg/
kg. In contrast, compound 6a/b exhibited stronger analgesic activity than
compound 5, and a clear dose-dependent analgesic activity that saturated at
750 mg/kg. At saturation, compound 6a/b displays an analgesic efficacy
significantly higher than paracetamol and similar to morphine. Taken together,
these results suggest that compound 6a/b is a more potent analgesic than
paracetamol.
We next investigated the mechanism of action underlying the analgesic activity
of analogue 6a/b. First, we evaluated a possible action on cannabinoid receptors.
For this purpose, compound 6a/b was administered 30 minutes before the
selective CB1 AM251 (3 mg/kg i.p.) and CB2 AM630 (1 mg/kg, i.p) antagonists,
and the writhing test was carried out 30 minutes after the administration of the
antagonists. Neither the CB1 nor the CB2 antagonist was able to block the
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
antinociceptive activity of compound 6a/b (number of stretches 13¡1 (AM251)
and 15.5¡1.7 (AM630). Thus, the cannabinoid system does not appear to play a
significant role in the antinociception induced by the paracetamol derivatives 5
and 6a/b.
An alternative mechanism that we evaluated was the potential inhibition of
cyclooxygenase enzymes COX-1 or COX-2. Our findings indicate that, while
paracetamol inhibited COX-2 with an IC50 of 7.08¡1.62 mM, compound 6a/b did
not affect the enzymatic activity at 10 mM. This result implies that inhibition of
COX enzymes does not underlie the in vivo analgesic activity of compound 6a/b.
Because of the pivotal role of thermoTRP channels in pain transduction [44–
46], we hypothesized that the analgesic activity of compound 6a/b may be due to a
direct inhibition of some these channels. Among the thermoTRPs, TRPM8
(‘‘melastatin’’), TRPV1 (‘‘vanilloid’’) and TRPA1 (‘‘ankyrin’’) are the most
validated in pain signaling. TRPV1 is considered a molecular integrator of
noxious heat stimuli in nociceptors [47], TRPM8 is a pivotal sensor for cold
stimuli, and TRPA1 is a unique sensor of noxious environmental stimuli [45–48].
Therefore, we selected these channels to evaluate if any of them was the target of
compound 6a/b (Fig. 6).
The channels were stably expressed in eukaryotic cells, and a Ca2+ fluorographic
assay used to monitor their activity upon instillation of their respective agonists in
the absence and presence of compound 6a/b at 50 mM. As illustrated in Fig. 7a,
only the activity of the TRPA1 channels was selectively blocked up to 85% at this
concentration of 6a/b. No significative effect was recorded for the other
thermoTRPs. A dose-response curve reveals that compound 6a/b displayed an
IC50 of 2.6 mM, indicating that compound 6a/b is a moderate antagonist of
TRPA1 channels (Fig. 7b). The inhibitory activity of compound 6a/b was further
demonstrated electrophysiologically as evidenced by the blockade of the AITC-
evoked ionic currents (Figures S1 and S2 in the presence and in the absence of
extracellular Ca2+). Collectively, these findings indicate that TRPA1 is a molecular
target of compound 6a/b, and could participate in the antinociceptive effect
showed in the writing test.
Conclusions
We have described the synthesis and pharmacological evaluation of new
paracetamol analogs derived from an adamantane scaffold. Compounds 5 and 6a/
b represent attractive leads to be developed since they show an improved
antinociceptive effect compared to paracetamol. In addition, adamantane
derivatives have proved to be very biocompatible, so possible toxic effects due to
chronic treatment should not be expected.
The main result of the present communication is that phenyl ring, ubiquitous
in medicinal chemistry, in some cases could be replaced by an adamantyl ring
without loss but improvement of the biological properties. To the ortho, meta and
para positions of a phenyl ring correspond to 1,2, 1,3 and 1,4-substituents on an
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
Figure 6. Functional expression of the TRP channels in the cell lines. Fluorescence time course for cells
treated with TRP antagonists (squares) or agonist (circle). TRP-mediated Ca2+ influx was observed upon
addition of agonist in the seven cycle, while the addition of the antagonist (squares) antagonized this
response. The effect of the compound 6a/b at 50 mM is shown (triangles). TRPA1 was activated with 100 mM
AITC, TRPV1 was activated by 10 mM capsaicin and TRPM8 was activated with 100 mM menthol. Ruthenium
red (RR) was at 10 mM, and AMTB was used at 100 mM.
doi:10.1371/journal.pone.0113841.g006
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
Figure 7. Fluorometric Ca2+ assay of thermoTRP channels to evaluate the inhibitory activity of
compound 6a/b. a) Activity of compound 6a/b at 50 mM on TRPA1, TRPV1 and TRPM8. b) Dose-response
curve for TRPA1 inhibition by compound 6a/b. Solid lines depict the best fit to a binding isotherm, with IC50
52.6¡1.1 mM, and nH (hill coefficient) 50.21. TRPA1 was activated with 100 mM AITC, TRPV1 was activated
by 10 mM capsaicin and TRPM8 was activated with 100 mM menthol. Data are given as mean ¡ SEM, with n
(number of experiments) >5.
doi:10.1371/journal.pone.0113841.g007
adamantyl ring. This is in agreement with our findings since the 1,4-derivative,
with a similar substitution to that of paracetamol, has shown greater potency than
the 1,3-derivative.
Even though more pharmacological research is needed (using TRPA1 agonists
for example), compound 6a/b, an analogue of paracetamol, able to block TRPA1
channel, is an interesting, new, antinociceptive drug.
Experimental
Chemistry
All chemicals were purchased from commercial suppliers and used without
further purification. TLC: precoated silica-gel 60 254 plates (Merck), detection by
UV light (254 nm). Flash-column Chromatography (FC): Kieselgel 60 (230–400
mesh; Merck). Melting points (mp) were determined in open capillaries with a
1
Gallenkamp capillary melting-points apparatus. H and 13C NMR spectra were
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
recorded on Bruker Advance 300 spectrometer operating at 300.13 MHz and
75.47 MHz respectively, in CDCl3 or DMSO-d6 as solvents. Chemical shifts are
reported in ppm on the d scale. In the case of multiplets, the signals are reported
as intervals. Signals were abbreviated as s, singlet; d, doublet; t, triplet; and m,
multiplet. Coupling constants are expressed in hertz. Hydrogenation reactions
were carried out in a Shaker type hydrogenation apparatus (Parr). Elemental
analysis was determined with a LECO Elemental Analyzer CHNS-932. LC-MS
analyses were performed using an Alliance 2695 (Waters) with a diode array UV/
Vis detector Waters 2996 and interfaced to a Micromass ZQ mass spectrometer.
Analyses were performed using reversed phase HPLC silica based columns:
column Bridge C18 3.5 mm (2.16100 mm). Using an injection volume of 3 mL,
a flow rate of 0.25 mL/min and gradient elution (2 to 30% over 15 min) of
acetonitrile in water. Acetonitrile contains 0.08% v/v formic acid and water
contains 0.1% v/v formic acid. Analyses were monitored at 254 nm wavelength.
Synthesis of N-3-hydroxyadamantan-1-yl acetamide (5)
To a solution of 3-amino-1-adamantol (0.61 g, 3.7 mmol) in water (7 mL) was
added acetic anhydride (0.45 mL) at 0 C. The reaction mixture was kept at 0 C in
˚
˚
an ice bath for 1 hour. Then it was taken to room temperature and stirred for
2 hours. When the reaction was completed, the mixture was extracted with
CH2Cl2. The organic layers were dried over MgSO4 and the solvent evaporated
under reduced pressure yielding a white solid (0.343 g, 45%). M.p. 224.4 C.
˚
Elemental analysis calculated for C12H19NO2; C, 68.87; H, 9.15; N, 6.69; found: C,
1
68.59; H, 9.27; N, 6.86. RMN H (300 MHz, CDCl3) d 5.25–5.17 (s, 1H), 2.29–
2.22 (m, 2H), 2.03–1.95 (m, 2H), 1.95–1.88 (m, 7H), 1.72–1.67 (m, 4H), 1.58–
1.53 (m, 2H). 13C (75 MHz, CDCl3) d 169.8 (C5O), 69.6 (C-OH), 54.7 (C-NH),
49.5 (CH2), 44.5 (2CH2), 40.7 (2CH2), 35.3 (CH2), 31.0 (2CH), 25.0 (CH3).
Synthesis of 4-((E)-(1-phenylethylidene)amino)adamantan-1-ol (9)
5-Hydroxy-2-adamantanone (8) (1.8 g, 10 mmol) and S-a-methylbenzylamine
(1.3 g, 10 mmol) were dissolved in absolute ethanol (50 mL) and refluxed for
64 h. The reaction mixture was concentrated obtaining yellow oil as the resulting
imine 9 (2.8 g). It was used without further purification in the next step. LC-MS
retention time 5.52 min [M+H]+ 5270.
Synthesis of E and Z-4-aminoadamantan-1-ol (10a) and (10b)
Benzylimine 9 (0.33 g, 1.2 mmol) was dissolved in THF (15 mL) and kept at 0 C
˚
in an ice bath for 5 min. Then, Pd-C 10% (0.24 g) was added and the mixture was
hydrogenated at atmospheric pressure for 12h. The catalyst was removed by
filtration and the solvent evaporated yielding isomers 10a and 10b as a white
powder (0.13 g, 67%). M.p. 250 C. LC-MS retention time isomer E 0.64 min
˚
[M+H]+ 5168; Z isomer 0.95 min [M+H]+ 5168. Elemental analysis calculated
for C10H17NO: C, 71.81; H, 10.25; N, 8.37; found: C, 71.60; H, 10.32; N, 8,28. E-
1
isomer (10a) RMN H (300 MHz, CDCl3) d 3.02–2.98 (m, 1H, CH-NH2), 2.11–
2.05 (m, 1H, CH(7)), 1.95–1.87 (m, 4H, CH2 (6,10), CH (5,3)), 1.80–1.69 (m, 6H,
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
CH2 (8,2,9)), 1.41–1.34 (m, 2H, CH2 (6,10)). Z-isomer (10b) RMN 1H (300 MHz,
CDCl3) d 2.87–2.85 (m, 1H, CH-NH2), 2.11–2.05 (m, 1H, CH(7)), 1.99–1.96 (m,
4H, CH (5,3), CH2 (2,9)), 1.80–1.69 (m, 2H, CH2 (8)), 1.68–1.65 (m, 2H, CH2
(6,10)), 1.63–1.58 (m, 2H, CH2 (6,10)), 1.53–1.46 (m, 2H, CH2 (2,9)). E-isomer
(10a) 13C (75 MHz, CDCl3) d 69.6 (C-OH, C1), 54.6 (C-NH, C4), 45.9 (CH2,
C8), 45.1 (2CH2, C2, C9), 37.1 (2CH, C5, C3), 30.3 (2CH2, C6, C10), 29.5 (CH,
C7). Z-isomer (10b) 13C (75 MHz, CDCl3) d 69.6 (C-OH, C1), 54.0 (C-NH, C4),
45.7 (CH2, C8), 38.9 (2CH2, C2, C9), 37.9 (2CH, C5, C3), 36.3 (2CH2, C6, C10),
29.8 (CH, C7).
Synthesis of N-5-hydroxyadamantan-2-yl acetamide (6a) and (6b)
To a solution of isomers 10a and 10b (0.50 g, 3 mmol) in water (8 mL) was
added acetic anhydride (0.4 mL, 3.5 mmol) at 0 C. The reaction mixture was
˚
stirred for 1h at 0 C and then for 2h at room temperature. The mixture was
˚
extracted with CH2Cl2 (10 mL). The organic layers were dried over MgSO4 and
the solvent evaporated under reduced pressure. The crude was successively washed
with ether yielding a mixture of isomers 6a and 6b as a white powder (0.482 g,
77%). M.p. 184 C. LC-MS: LC-MS retention time isomer E 7.34 min [M+H]+
˚
5210; isomer Z 7.83 min [M+H]+ 5210. Elemental analysis calculated for
C12H19NO2: C, 68.87; H, 9.15; N, 6.69; found: C, 68.70; H, 9.35; N, 6.39.
1
E-isomer (6a) RMN H (300 MHz, DMSO-d6) d 3.75-3.70 (m, 1H, CH-NH),
2.52–2.48 (m, 1H, CH(7)), 1.99–1.91 (m, 2H, CH (5,3)), 1.87–1.81 (m, 5H, CH2
(6,10), CH3), 1.67–1.60 (m, 2H, CH2 (2,9)), 1.60–1.53 (m, 4H, CH2 (8,2,9)),
1.28–1.21 (m, 2H, CH2 (6,10)). Z-isomer (6b) RMN 1H (300 MHz, DMSO-d6) d
3.65–3.60 (m, 1H, CH-NH), 2.52–2.48 (m, 1H, CH(7)), 2.00–1.92 (m, 2H, CH
(5,3), 1.87–1.81 (m, 5H, CH2 (2,9), CH3), 1.60–1.53 (m, 6H, CH2 (8,6,10)), 1.36–
1.29 (m, 2H, CH2 (2,9)). E-isomer (6a) 13C (75 MHz, DMSO-d6). d 169.8
(C5O), 69.6 (C-OH, C1), 52.9 (C-NH, C4), 46.3 (CH2, C8), 45.2 (2CH2, C2, C9),
34.0 (2CH, C5, C3), 30.5 (2CH2, C6, C10), 29.9 (CH, C7) 23.4 (CH3, C12). Z-
isomer (6b) 13C (75 MHz, DMSO-d6). d 169.8 (C5O), 69.6 (C-OH, C1), 52.4 (C-
NH, C4), 46.0 (CH2, C8), 39.8 (2CH2, C2, C9), 36.3 (2CH, C5, C3), 34.9 (2CH2,
C6, C10), 29.8 (CH, C7) 23.3 (CH3, C12).
Pharmacological evaluation
The animals, CD-1 male mice (25–27 g), were redistributed in groups, two of
which were used as the control group. The remaining twelve groups were treated
with paracetamol 100 and 200 mg/kg (two different groups), compound 5 (100,
200, 500 mg/kg) and compound 6a/b (250, 375, 500, 750 mg/kg). An additional
group received morphine 5 mg/kg. All drugs were intraperitoneally (i.p.)
administered, 30 minutes before the algogen agent. Control mice were treated
with 10 ml/kg of saline solution. Each group included 10–12 animals; those
animals showing behavioral alterations were previously discarded. Each animal
was used only once. An observer who was unaware of the different treatments
carried out the collection of data.
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
The experimental test used was the acetic acid writhing test. The effect of the
i.p. administration of 10 ml/kg of acetic acid (2%) was measured by quantifying
the number of writhing responses in the three groups of animals over a
10 minutes period. Responses were measured starting 5 minutes after the algogen
agent was administered. The animals used were supplied by the animal house of
Universidad Rey Juan Carlos. Room temperature was kept at 22¡1 C. A 12 hours
˚
light - dark cycle was started at 8:00 a.m., at constant humidity. All animals were
given free access to food and water.
Experimental protocols used in this investigation were approved by the Ethical
Committee of Rey Juan Carlos University and were conducted in accordance with
the guidelines of the International Association for the Study of Pain [49].
Inhibition of Isolated Cyclooxygenases COX-1 and COX-2
A COX inhibitor screening assay (kit 700100) was used to determine the activity
of isolated ovine COX-1 and human recombinant COX-2 as described by the
manufacturer (Cayman Chemical Company, USA).
Calcium fluorography
For fluorescence assays, cells expressing TRP channels (TRPV1-SH-SY5Y,
TRPM8-HEK and TRPA1-IMR90) were seeded in 96-well plates (Corning
Incorporated, Corning, NY) at a cell density of 40,000 cells 2 days before
treatment. The day of treatment the medium was replaced with 100 mL of the dye
loading solution Fluo-4 NW supplemented with probenecid 2.5 mM. Then the
compounds dissolved in DMSO were added at the desired concentrations and the
plate(s) were incubated at 37 C in a humidified atmosphere of 5% CO for
˚
2
60 minutes.
The fluorescence was measured using instrument settings appropriate for
excitation at 485 nm and emission at 535 nm. (POLARstar Omega BMG LAB
tech). A baseline recording of 7 cycles was recorded prior to stimulation with the
agonist (10 mM capsaicin for TRPV1, 100 mM menthol for TRPM8, and 100 mM
AITC for TRPA1). The corresponding antagonist (10 mM Ruthenium Red for
TRPV1 and TRPA1, 100 mM AMTB for TRPM8) was added for the blockade. The
changes in fluorescence intensity were recorded during 15 cycles more. DMSO, at
the higher concentration used in the experiment, was added to the control wells.
The degree of blockage (%) of TRP channel activity was calculated by:
(F0{FI)
%Blockage~
(Fc0{FcI)
Where:
F0 is the fluorescence after the addition of agonist in the presence of the
compound,
FI is the fluorescence before the addition of agonist in the presence of the
compound,
PLOS ONE | DOI:10.1371/journal.pone.0113841 December 1, 2014
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
Fc0 is the fluorescence after the addition of agonist in the absence of the
compound,
FcI is the fluorescence before the addition of agonist in the absence of the
compound.
The Z factor was calculated using the following equation:
3 Ã (SDmaxzSDmin
)
Z~1{
Meanmax{Meanmin
Where:
Meanmax is the mean of the maximum fluorescence in the presence of agonist,
Meanmin is the mean of the maximum fluorescence in the presence of agonist
and antagonist.
Computational details
The geometries of compounds 5, 6a and 6b were optimized at the B3LYP/6-
31G(d) level and the corresponding frequencies (all 0) proved that they are
minima. These geometries were reoptimized at the B3LYP/6-311++G(d,p) level.
Absolute shieldings (s, ppm) and SSCC (Hz) were calculated on these geometries
using the GIAO approximation for the s values. The corresponding chemical
shifts (d, ppm) were obtained using the empirical transformation equations we
have established for a large collection of compounds. The used equations
transform calculated values in the gas phase to experimental values in solution
(usually CDCl3 or DMSO-d6). See supplementary data.
Supporting Information
Figure S1. Compound 6a/b strongly inhibits TRPA1- mediated currents in the
presence of extracellular Ca2+. A) Representative whole-cell voltage clamp
recording from hTRPA1-expressing IMR-90. Currents were measured every
second during a holding potential of -60 mV. Pre-application of 6a/b (20 s) was
followed by co-application with 100 mM AITC for 60s in presence of 2.0 mM
extracellular Ca2+. Current traces of different colours denote the different
concentrations of compound 6a/b tested. B) Dose response of compound 6a/b
blockade activity. Solid line depicts the fitting to a Michaelis isotherm. The
estimated IC50 value was 10.6¡0.7 mM. Data are given as mean ¡ sem, with n>4
cells) per data point.
doi:10.1371/journal.pone.0113841.s001 (TIF)
Figure S2. Compound 6a/b weakly inhibits TRPA1 currents in the absence of
extracellular Ca2+. A) Representative whole-cell voltage clamp recording from
hTRPA1-expressing IMR-90 cells in the absence of extracellular Ca2+. Ionic
currents, at positive and negative potentials were measured every 2s during a
350 ms voltage ramp from 260 mV to +60 mV and evoked with 100 mM AITC
PLOS ONE | DOI:10.1371/journal.pone.0113841 December 1, 2014
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Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs
followed by the addition of 100 mM compound 6a/b for 20s. AITC was present
with drug application and after application to evaluated current reversibility B)
Current-voltage relationships of TRPA1 in the absence and presence of 100 mM
compound 6a/b in Ca2+ free medium. The inferred IC50 value was >80 mM, as
saturation of blockade was not reached in the absence of Ca2+. Data were obtained
from n>4 cells.
doi:10.1371/journal.pone.0113841.s002 (TIF)
Table S1. Absolute chemical shielding for 6a and 6b calculated at GIAO/
B3LYP/6-311+G(d,p) level. The relative values have been obtained using the
equation described in ref. 1–2.
doi:10.1371/journal.pone.0113841.s003 (DOC)
Acknowledgments
´ ´
We thank Dr. Concepcion Perez for her technical assistance in the COX inhibition
studies.
This paper is dedicated to Professor Sergio Erill who inspired it.
Author Contributions
Conceived and designed the experiments: PG AFC MIM JE AFM. Performed the
experiments: NF CG RTM SQ. Analyzed the data: RPF. Contributed reagents/
materials/analysis tools: CG AFC RTM SQ. Wrote the paper: RPF JE.
Computational calculations: IA.
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