Evaluation of the analgesic effect of 4-anilidopiperidine scaffold containing ureas and carbamates

Article abstract of DOI:10.3109/14756366.2016.1160902

Fentanyl is a powerful opiate analgesic typically used for the treatment of severe and chronic pain, but its prescription is strongly limited by the well-documented side-effects. Different approaches have been applied to develop strong analgesic drugs with reduced pharmacologic side-effects. One of the most promising is the design of multitarget drugs. In this paper we report the synthesis, characterization and biological evaluation of twelve new 4-anilidopiperidine (fentanyl analogues). In vivo hot-Plate test, shows a moderate antinociceptive activity for compounds OMDM585 and OMDM586, despite the weak binding affinity on both μ and δ-opioid receptors. A strong inverse agonist activity in the GTP-binding assay was revealed suggesting the involvement of alternative systems in the brain. Fatty acid amide hydrolase inhibition was evaluated, together with binding assays of cannabinoid receptors. We can conclude that compounds OMDM585 and 586 are capable to elicit antinociception due to their multitarget activity on different systems involved in pain modulation.

Full text of DOI:10.3109/14756366.2016.1160902

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Evaluation of the analgesic effect of 4-
anilidopiperidine scaffold containing ureas and
carbamates
Ludovica Monti, Azzurra Stefanucci, Stefano Pieretti, Francesca Marzoli,
Lorenzo Fidanza, Adriano Mollica, Sako Mirzaie, Simone Carradori, Luciano
De Petrocellis, Aniello Schiano Moriello, Sándor Benyhe, Ferenc Zádor, Edina
Szűcs, Ferenc Ötvös, Anna I. Erdei, Reza Samavati, Szabolcs Dvorácskó,
Csaba Tömböly & Ettore Novellino
To cite this article: Ludovica Monti, Azzurra Stefanucci, Stefano Pieretti, Francesca Marzoli,
Lorenzo Fidanza, Adriano Mollica, Sako Mirzaie, Simone Carradori, Luciano De Petrocellis,
Aniello Schiano Moriello, Sándor Benyhe, Ferenc Zádor, Edina Szűcs, Ferenc Ötvös, Anna I.
Erdei, Reza Samavati, Szabolcs Dvorácskó, Csaba Tömböly & Ettore Novellino (2016): Evaluation
of the analgesic effect of 4-anilidopiperidine scaffold containing ureas and carbamates, Journal
of Enzyme Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2016.1160902
To link to this article: http://dx.doi.org/10.3109/14756366.2016.1160902
Published online: 11 Apr 2016.
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2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.3109/14756366.2016.1160902
!
RESEARCH ARTICLE
Evaluation of the analgesic effect of 4-anilidopiperidine scaffold
containing ureas and carbamates
Ludovica Monti1, Azzurra Stefanucci2, Stefano Pieretti3, Francesca Marzoli3, Lorenzo Fidanza3, Adriano Mollica4,
5
4
6
6
7
Sako Mirzaie , Simone Carradori , Luciano De Petrocellis , Aniello Schiano Moriello , Sandor Benyhe ,
´
7
00
7
7
7
7
7
7
¨
´
´
´
Ferenc Zador , Edina Szucs , Ferenc Otvo¨s , Anna I. Erdei , Reza Samavati , Szabolcs Dvoracsko , Csaba To¨mbo¨ly ,
and Ettore Novellino8
1
2
Dipartimento di Chimica e Tecnologia del Farmaco, Sapienza Universita di Roma, Rome, Italy, Dipartimento di Chimica, Sapienza Universita di
`
`
3
Roma, Rome, Italy, Istituto Superiore di Sanita, Dipartimento del Farmaco, Rome, Italy, Dipartimento di Farmacia, Universita di Chieti-Pescara
4
`
`
‘‘G. d’Annunzio’’, Chieti, Italy, 5Department of Biochemistry, Islamic Azad University, Sanandaj, Iran, 6Endocannabinoid Research Group, Institute of
7
Biomolecular Chemistry, National Research Council, Naples, Italy, Institute of Biochemistry, Biological Research Centre, Hungarian Academy of
8
Sciences, Szeged, Hungary, and Dipartimento di Farmacia, Universita di Napoli ‘‘Federico II’’, Naples, Italy
`
Abstract
Keywords
Fentanyl is a powerful opiate analgesic typically used for the treatment of severe and chronic
pain, but its prescription is strongly limited by the well-documented side-effects. Different
approaches have been applied to develop strong analgesic drugs with reduced pharmacologic
side-effects. One of the most promising is the design of multitarget drugs. In this paper we
report the synthesis, characterization and biological evaluation of twelve new 4-anilidopiper-
idine (fentanyl analogues). In vivo hot-Plate test, shows a moderate antinociceptive activity for
compounds OMDM585 and OMDM586, despite the weak binding affinity on both m and
d-opioid receptors. A strong inverse agonist activity in the GTP-binding assay was revealed
suggesting the involvement of alternative systems in the brain. Fatty acid amide hydrolase
inhibition was evaluated, together with binding assays of cannabinoid receptors. We can
conclude that compounds OMDM585 and 586 are capable to elicit antinociception due to
their multitarget activity on different systems involved in pain modulation.
Fentanyl, FAAH enzyme, MAGL enzyme,
opioids, pain
History
Received 3 February 2016
Revised 25 February 2016
Accepted 26 February 2016
Published online 11 April 2016
Introduction
employed in the design of bivalent drugs by Hruby et al. 5 in light
of the well-documented interaction between opioid and canna-
binoid receptors6.
Opioids have been used for years in the control of pain from
moderate to severe, and with controversial results especially in the
management of chronic and neuropathic pain.
Stimulated by the previously described SAR on fentanyl and
its bivalent analogues, which reported no general rules for the
effect of the substitution on the propionyl amide moiety,5 we
synthesised 12 novel 4-anilidopiperidine derivatives replacing
the tertiary propyl-amide group in fentanyl, with different aryl-
ureas and aryl-carbamates found in several active FAAH and
MAGL inhibitors with the intent to develop novel multitarget
analgesics.7,8 FAAH and MAGL are hydrolase enzymes,
responsible of the endocannabinoid cleveage, and their inhibitors
have shown to possess nociceptive effects in different pain
Addiction and tolerance, including nausea and vomiting,
constipation and respiratory depression are well known adverse
side effects1. Fentanyl is the prototype of the 4-anilidopiperidines
opioid analgesics and is characterized by high potency and
efficacy. Expansion of the structure-activity relationships (SAR)
of fentanyl derivatives has led to the discovery of new
morphinomimetics with diverse pharmacological profiles2.
Numerous research groups have studied the structure’s modifica-
tions of fentanyl, with the aim to reduce the pharmacologic side
effects, acting on other opioid receptors such as the d-receptor3. It
has been demonstrated that m and d-opioid receptors form
heterodimers and their simultaneous stimulation can give a
better antinociceptive activity4. Fentanyl has been previously
models, as
a
consequence of the increased level of
.
endocannabinoids9,10
A first series of compounds bearing the 4-anilidopiperidine
scaffold on fentanyl was synthesised, and several substituted O-
arylcarbamates (compounds OMDM584–OMDM589) have been
used in place of the propylamide function. A second series of N-
arylureas was designed replacing the N-propyl moiety of fentanyl
with several substituted N-aryl building blocks (compounds
OMDM590–OMDM595). The synthesis of all compounds is
depicted in Scheme 1, structural features are reported in Table 1.
Address for correspondence: Adriano Mollica, Dipartimento di Scienze
del Farmaco, Faculty of Pharmacy, University ‘‘G. d’Annunzio’’ of
Chieti-Pescara, Via dei Vestini, 31, 66100 Chieti, Italy. Tel: +39-0871-
3554476. E-mail: a.mollica@unich.it

2
L. Monti et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
substituted-phenol
Scheme 1. Reagents and conditions: (a)
1-phenethyl-N-phenylpiperidin-4-amine
(0.5 mmol), Et3N (0.6 mmol), DCM, r.t.
overnight.
Et N
3
O
in toluene
Cl
O
R
from 0°C to r.t.
a
N
X
phosgene
Y
N
O
substituted-aniline
Et N
in toluene
O
3
OMDM584-595
Cl
N
H
R
from 0°C to r.t.
Table 1. Structures and yields of products OMDM584–595.
N
X
Y
N
O
Products
X
O
Y
Yields
80%
Products
X
Y
Yields
31%
OMDM584
OMDM590
NH
OMDM585
OMDM586
OMDM587
O
O
O
63%
51%
80%
OMDM591
NH
61%
48%
79%
Cl
Cl
Cl
Cl
OMDM592
OMDM593
NH
NH
OMDM588
OMDM589
O
26%
OMDM594
OMDM595
NH
74%
F
F
F
F
F
F
F
O
13%
NH
79%
F
F
F
F
F

DOI: 10.3109/14756366.2016.1160902
Biological assays on fentanyl analogues
3
Results and discussion
range, showing poor affinity for these two opioid receptors (Fig
S2A and B). In case of [3H]WIN55 212–2 binding experiments,
the affinity of OMDM585 and 586 was weaker for CBR, since it
Chemistry
Carbamates OMDM584–589 were synthesised starting from the decreased [3H]WIN55 212–2 specific binding by about 20–30%
corresponding chloroformate intermediate, which was prepared with an IC50 value in the micromolar range (Figure S2D, Table
using phosgene in toluene with the appropriate substituted phenol S1). For comparison the unlabelled forms of the tritiated ligands
and triethylamine (Scheme 1).
reported IC50 values in the nanomolar range, indicating that the
The so obtained chloroformate was reacted with a solution of radioligands performed adequately (Figure S2, Table S1).
1-phenethyl-N-phenylpiperidin-4-amine in DCM in presence of
triethylamine at room temperature, to yield the desired crude [35S]GTPgS binding experiments
product. In the same way, ureas OMDM590–595 were obtained
In G-protein activity stimulation measurements with [35S]GTPgS
from the corresponding urethane intermediates, which were
binding tests, fentanyl activated G-protein stimulation with a
prepared using phosgene in toluene with the appropriate
maximum efficacy of 159.8% (nearly 60% above basal activity)
substituted anilines and triethylamine (Scheme 1).
and with a potency of 110.4 nM (Table S2, Figure S3A and C),
Then urethane intermediate was reacted with a solution of
which corresponds with previous studies11.
1-phenethyl-N-phenylpiperidin-4-amine in DCM in presence of
Interestingly, all fentanyl analogues showed inverse agonist
triethylamine at room temperature, to yield the desired crude
properties, since they reduced G-protein basal activity with very
product.
similar parameters: the maximum efficacy was between ꢀ80–
All crude compounds were purified on silica gel chromatog-
100%, and their potency was between ꢀ3–5 mM (Figure S3A and
raphy to give final products in good yields (Table 1).
C, Table S2). This effect however was not opioid receptor
mediated, since it could not be reversed by the antagonist
naloxone even in high, 10 mM concentrations, resulting in very
In vitro assay
Biological in vitro tests were performed on all products, for m and similar maximum efficacy and potency values to those in the
d-opioid receptor affinity, on FAAH and MAGL (Table 2).
absence of naloxone (Figure S3B and D, Table S2). For
comparison, the activity of fentanyl was markedly inhibited in
the presence of naloxone and the G-protein activity reduced back
to basal activity level (Figure S3B and D, Table S2). The inverse
agonist properties of the fentanyl analogues are most probably due
Receptor binding assays
Competition binding experiments
In competition binding experiments, fentanyl reduced the [3H] to the introduction of the benzene ring in position X, since this is
DAMGO specific binding on m-opioid receptor (MOR) with a shared by all analogues, but not by fentanyl (Scheme 1). In fact,
very low 5.99 nM IC50 value, indicating a high affinity for MOR the benzene ring together with the piperidine ring is also shared
(Figure S1), as expected from previous reports, confirming the by rimonabant, which is an inverse agonist on CB1 receptor12.
adequate performance of the radioligand11.
The fentanyl analogues showed a wide range of affinity for mediated by a yet unidentified pertussis toxin sensitive GPCR.13
Additionally, the fentanyl analogues described in this study
However, the inverse agonist effect of rimonabant can be also
MOR from mid nanomolar (IC504ꢀ500 nM) to micromolar
(IC505ꢀ20 mM) (Figure S1) concentration. Compounds also share some elements of the pharmacophore of other CB1
OMDM586, 591–593 showed the highest affinity for MOR receptor inverse agonists, such as the two aromatic moieties, a
with mid nanomolar IC50 values. Thus among the structural hydrogen acceptor unit and a cyclic lipophilic part.14
modifications of fentanyl, the combination of the amide group in
position X with 4-, 3-chlorophenyl and 4-tert-butylphenyl groups
In vivo assays
in position Y (OMDM591–593) gives good affinity (Table 3).
Hot-Plate test was performed on all compounds to study the
influence of different pharmacological properties on their anal-
activities in pharmacological assays, we further characterized
Since compounds OMDM585 and 586 showed analgesic
gesic profile. Results are shown in Figure 1.
Hot-plate data confirm that compounds OMDM585, 586 have
a good analgesic activity only at high doses. However, this cannot
be explained by the opioid activity, as the binding to m and
d-opioid receptors is very weak, furthermore the FAAH and
MAGL inhibition activity was negligible.
their binding for d-opioid receptor (DOR), k-opioid receptor
(KOR) and cannabinoid receptors (CBR), again in competition
binding assays (Figure S2). Both compounds reduced total
specific binding of the KOR selective [3H]U-69593 and DOR
selective [3H]IleDelt II with an IC50 value in the micromolar
The inverse agonist activity on the G-Protein stimulation
leaves us with the hypothesis that products OMDM585 and 586
are able to elicit their antinociceptive activity either by interaction
to block one or more excitatory circuits in the brain rather than by
their affinity to m opioid and cannabinoid receptors at micromolar
range.
Table 2. In vitro assays on FAAH and MAGL inhibition.
FAAH Max
inhibition
at 10 mM
MAGL Max
inhibition
at 10 mM
IC50 on
FAAH mM
IC50 on
MAGL mM
Compounds
OMDM 584 410
OMDM 585 410
OMDM 586 410
OMDM 587 410
OMDM 588 410
OMDM 589 410
OMDM 590 410
OMDM 591 410
OMDM 592 410
OMDM 593 410
OMDM 594 410
OMDM 595 410
0%
0%
410
410
410
410
410
410
410
410
410
410
410
410
0%
0%
0%
0.74%
0%
0.41%
0%
3.50%
7.32%
1.28%
0%
Molecular docking
1.90%
10.2%
7.40%
7.40%
7.25%
8.26%
9.76%
5.82%
7.49%
9.16%
According to the experimental results, the inactive state of
m-opioid receptor was used for docking (PDB code: 4dkl). The
lowest docking energy poses of the analogues only partly matched
with the docked pose of fentanyl15, as shown in Table S3.
Furthermore, because the analogues reported weak binding
affinity and the agonist character of the parent molecule fentanyl
changed, it is feasible to assume that their binding mode differs
from that of fentanyl. To check this assumption, a distance based
2.97%

4
L. Monti et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Table 3. The affinity values (logIC50 SEM.) of OMDM584–595 compounds and fentanyl in [3H]DAMGO competition binding assays in rat brain
membrane homogenates. The logIC50 values were calculated according to the competition binding curves (see Supplementary Figure 1S). for better
comparison the two different basic structures are presented side by side.
logIC50 S.E.M. (M) (IC50
ꢂ8.22 0.06 (5.99 nM)
X
)
Compounds
Fentanyl
Compounds
Fentanyl
Y
O
N
OMDM 584
OMDM 585
ꢂ5.84 0.04 (1442 nM)
ꢂ6.28 0.06### (516.8 nM)
OMDM 590
OMDM 591
ꢂ5.66 0.1 (2180 nM)
ꢂ6.17 0.06## (665.1 nM)
Cl
Cl
OMDM 586
OMDM 587
ꢂ6.18 0.06 (654.2 nM)
ꢂ6.18 0.04 (658.9 nM)
OMDM 592
OMDM 593
ꢂ5.73 0.33 (1830 nM)
ꢂ4.63 0.24* (23050 nM)
OMDM 588
ꢂ5.57 0.10 (2657 nM)
ꢂ5.39 0.13* (4031 nM)
OMDM 594
CF
3
OMDM 589
ꢂ5.38 0.1* (4093 nM)
ꢂ6.91 0.05* (1204 nM)
OMDM 595
CF
3
‘‘X’’ and ‘‘Y’’ indicates the chemical modifications performed in the basic structure of fentanyl (Scheme 1). Compounds with comparable lower
affinity within the fentanyl analogues are highlighted in bold.
#Indicates the significant difference between the analogues with oxygen atom and an amide group in position X (Figure 1B and C) within the
corresponding substitution in position Y (Figure 1). ###p50.001; ##p50.01 (unpaired tꢂtest with two-tailed p value).
*Indicates the significant difference compared to the compounds in bold within the analogues with oxygen heteroatom or amid group in the position X.
interaction model was matched against the docking energies. The corresponding atom pairs are shown in Table S4. The docked
interaction model was constructed as follows: the distances pose, possessing the minimum binding distance by the above
between selected atoms of the ligands and the receptor were formula, was selected for each ligand and the corresponding
measured, and the square root of the sum of the squares of the ligand efficiency and distance values were used to fit a linear
Pairwise Distances was used as a measure of binding distance, as regression model (Figure 2).
shown in Equation 1.
The Spearman rank correlation coefficients were calculated
too. All atom pair combinations were checked to qualify the
ð1Þ different interaction (binding) modes. The interaction models
based on the previously published docking pose (Table S4,
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
X
2
½d ðli, riÞꢁ
i
Where, dðli, riÞ is the distance between the ith selected atom of
column A for ligand atoms), in which the anilide ring points
toward the extracellular direction,15 resulted in very weak
the ligand (li) and the receptor (ri), respectively. The

DOI: 10.3109/14756366.2016.1160902
Biological assays on fentanyl analogues
5
regression models and mainly with negative slope. The negative in the case of the non-fentanyl-like orientation, for which the 1–2–
slope is contradictory to the expectations that the deeper binding 3 interactions resulted in almost the same regression (Figure 2,
energy is a consequence of the shorter binding distance. This gave r2 ¼0.741, Spearman’s rank correlation: 0.758).
rise the possibility that the orientation of the bound analogues
differ from that of fentanyl which can also be seen in Table S3.
The interaction modes of fentanyl analogues are shown in
supplementary Figure (Figure S4). The most important interaction
To check this assumption, interaction models with the opposite between the synthesized analogues and MOR is a hydrophobic
orientation were tested (Table S4, column B for ligand atoms). interaction. Compound OMDM584 interacts just with Tyr148 due
This type of interaction models resulted in higher correlation to ꢀ-ꢀ stacking. OMDM585 can be stable in the binding site of
between the ligand efficiencies and the calculated distances. The MOR (Tyr128, Trp293 and Tyr326) via three ꢀ–ꢀ stacking
best model is shown in Figure S4.
interactions. However, the presence of some polar or charged
The key amino acids interactions are (i) Asp147-proton on residues in proximity of Fentanyl analogues may affect the MOR
piperidine N, (ii) Tyr326 side chain-anilide aromatic ring, (iii) activation. The presence of Lys303 and Lys233, as the positive
Ile144 side chain-phenethyl aromatic ring (Figure 3).
charged residues, in the vicinity of tert-butylphenyl moiety of
These results are in agreement with the finding that there are OMDM587 can dramatically destabilize the protein:ligand com-
more sites to accommodate the analogues with the additional plex. The same cases can be found for the other fentanyl
large substituent within the binding pocket in the orientation analogues: Asp147 and Lys233 are located in the vicinity of
opposite to the literature pose of fentanyl. Comparing the different phenyl and cholorophenyl moieties of OMDM585. Also, in the
interaction models (atom pair combinations), the best linear fit case of OMDM586, Lys233, Glu229 and Asn230 can have
was obtained with two atom pairs, 1 and 2 (Table S4). Involving negative effects on MOR:ligand complex stability. In the active
any other interactions significantly decreased the linear fit, except form of MOR, Tyr148 can participate in the hydrogen bonding
network involving Lys233 and a water molecule. This study
reported that the ionic interaction between ligand and Asp147 has
been found in either active or inactive form of MOR. Based on
our docking results, no similar interaction was found for fentanyl
analogues.
Figure 1. In vivo activity of compounds OMDM584–595 by hot-Plate
test. Data from hot plate experiments. **** is for p50.001 versus
Vehicle; ** is for p50.01 versus Vehicle; * is for p50.05 versus Vehicle. Figure 3. Definition of interactions; on the left: orientation similar to that
N ¼ 10.
reported by Micovic et al.15 on the right: opposite orientation.
Figure 2. Linear regression for the best interaction model of compounds OMDM584–595.

6
L. Monti et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Conclusions
spectrometer (Perkin-Elmer, Waltham, MA). Band position and
absorption ranges are given in csup>/sup>. 1H and 13C NMR
spectra were obtained on a Bruker Avance 400 MHz (Pittsburgh,
PA), using CDCl3 as solvent.
Potential abuse, unwanted side effects and lack of analgesic
efficacy are major problems in prescribing opioid analgesics16.
Bi- or poly-functional compounds can fight chronic and
neuropathic pain at various levels, demonstrating some ability to
cross the BBB, with reduced or none sign of tolerance and
Chemistry
addiction, thus supporting the idea that a single drug with FAAH/ Synthetic procedure for the carbamates OMDM584–589
MAGL inhibitor activity plus an opioid agonist activity, could be
To a stirred 15% phosgene solution in toluene (3.3 mL, 5 mmol) a
a very attractive design for the development of a ‘‘third
solution of the appropriately-substituted phenol (1.0 mmol) and
generation’’ painkillers6.
Et3N (0.168 mL, 1.2 mmol) in dry toluene (5 mL) was added drop-
In this study, we investigated the potential of multi-target
wise at 0 ꢃC. The reaction mixture was stirred for 3 h at room
compounds combining opioid agonists and FAAH/MAGL inhibi-
temperature and evaporated under vacuum. The crude chloro-
tors. The pharmacological profile of such compounds should join
formate was dissolved in dry DCM (4 mL) and a solution of 1-
high efficacy in chronic pain states reducing the development of
phenethyl-N-phenylpiperidin-4-amine (0.140 g, 0.50 mmol) and
tolerance. Significant advantages over a multidrug approach could
Et3N (0.084 mL, 0.6 mmol) in dry DCM (2 mL) was added drop-
be: (i) oral administration, (ii) better pharmacokinetic properties,
wise at room temperature with stirring. The reaction mixture was
(iii) no drug–drug interactions and (iv) improved compartment
stirred overnight at room temperature, diluted with water, and
targeting17.
extracted with AcOEt. The organic phase was washed with
The data obtained so far, indicate that our products have weak
NaHCO3 s.s. and twice with water, dried (Na2SO4), and
affinity for m-opioid receptors, and are not strong inhibitors of
evaporated under vacuum. The residue was purified by column
FAAH/MAGL. However, two products revealed a good anti-
chromatography.
nociceptive activity but very weak potency when compared to
fentanyl, therefore we concluded that the activity wasn’t mediated
Phenyl(1-phenethylpiperidin-4-yl)(phenyl)carbamate (584)
by the opioid system or endocannabinoids. In our studies, a strong
inverse agonist activity in the stimulation of the G0/i protein was Yield 80% (hexane/AcOEt, 7:3 as chromatographic eluent); mp
revealed, which can be related to several systems present in the 138–139 ꢃC (from hexane/CH2Cl2); IR 2952, 2763, 1716, 1595,
1
brain, directly involved in pain modulation.
1494, 1320, 1199, 1032, 991 csup>/sup>; H NMR (400 MHz) d
A direct binding to CBR1 receptor has been explored, but the 1.59 (2H, m), 1.94 (2H, m), 2.15 (2H, m), 2.55 (2H, m), 2.74 (2H,
so obtained data are in accord with the observed activity.
m), 3.04 (2H, d, J ¼ 11.6 Hz), 4.33 (1H, m), 7.12–7.34 (15H, m);
Further, in silico molecular docking studies have been 13C NMR (100 MHz) d 30.79, 33.84, 53.04, 56.45, 60.41, 121.61,
performed and definitely ruled out the involvement of the known 125.18, 126.06, 127.92, 128.39, 128.63, 128.94, 129.10, 129.99,
systems in the antinociceptive activity. The comparison of the best 137.73, 140.17, 151.36, 154.07. HRMS (ESI-MS, 140 eV): m/z
regression model for the conventional, fentanyl-like orientation [M + H]+ calculated for C26H29N2O2+: 401.2151, found: 401.2168.
and the opposite one indicates that the latter is more feasible. It
suggests that the analogues may recognise MOR in a different 4-Chlorophenyl(1-phenethylpiperidin-4-yl)
manner than fentanyl, due to the size of the new substituents.
Based on these results, the most important interactions
between the ligands and the receptor are (i) Asp147-proton on
piperidine N, (ii) Tyr326 side chain-anilide aromatic ring, (iii)
Ile144 side chain- phenethyl aromatic ring. This is in agreement
with the experimental results showing that the analogues lost the
agonistic character.
We may conclude that the pharmacologic profile observed in
the murine tests may be explained by the modulation of several
receptors in the brain, related to the stimulation of the cerebral
activity and usually responsible for enhancing the noxious stimuli,
like adrenergic, glutamaergic, substance P and others. Although it
has been previously reported that the fentanyl analgesic activity
could be readily maintained into small multitarget ligands, the
incorporation of potent endocannabinoid activity is more intri-
guing and challenging18.
(phenyl)carbamate (585)
Yield 63% (hexane/AcOEt, 7:3 as chromatographic eluent); mp
134–135 ꢃC (from hexane/CH2Cl2); IR 2951, 2764, 1714, 1595,
1488, 1319, 1210, 1089, 996 csup>/sup>; H NMR (300 MHz) d
1.58 (2H, m), 1.94 (2H, m), 2.14 (2H, m), 2.54 (2H, m), 2.73 (2H,
m), 3.03 (2H, d, J ¼ 11.7 Hz), 4.31 (1H, m), 6.96–7.41 (14H, m);
13C NMR (75 MHz) d 30.82, 33.87, 53.02, 55.65, 60.39, 122.96,
126.04, 128.02, 128.38, 128.61, 128.98, 129.08, 129.89, 130.44,
1
137.56, 140.18, 149.88, 153.62. HRMS (ESI-MS, 140 eV): m/z
+
: 435.1761, found:
[M + H]+ calculated for C26H28ClN2O2
435.1765.
3-Chlorophenyl(1-phenethylpiperidin-4-yl)(phenyl)
carbamate (586)
Yield 51% (hexane/AcOEt, 75:25 as chromatographic eluent); mp
Further studies are currently underway in our laboratories to 96–97 ꢃC (from hexane/CH2Cl2); IR 2949, 2760, 1717, 1589,
design multitarget molecules with potent inhibitory activity at 1493, 1299, 1203, 1020, 993 csup>/sup>; H NMR (300 MHz) d
1
FAAH enzyme and as strong opioid agonists.
1.58 (2H, m), 1.95 (2H, m), 2.14 (2H, m), 2.55 (2H, m), 2.73 (2H,
m), 3.04 (2H, d, J ¼ 11.7 Hz), 4.30 (1H, m), 6.94–7.41 (14H, m);
13C NMR (75 MHz) d 30.78, 33.86, 53.01, 55.70, 60.38, 109.97,
119.98, 122.23, 125.40, 126.04, 128.05, 128.37, 128.61, 128.99,
129.77, 129.86, 134.30, 140.17, 151.85, 153.42. HRMS (ESI-MS,
140 eV): m/z [M + H]+ calculated for C26H28ClN2O2+: 435.1761,
found: 435.1765.
Experimental section
Material and methods
All chemical reagents were commercially available unless other-
wise indicated. TLC was performed by using Merck F254 silica
plates (Kenilworth, NJ) and components were analyzed by using
UV light and an iodine chamber. Melting points were determined
on a Buchi apparatus and are uncorrected. Column chromato-
graphies were carried out using Merck silica gel 60 (230–400
mesh). IR spectra were recorded on a Spectrum-One FT-ATR
4-(tert-Butyl)phenyl(1-phenethylpiperidin-4-yl)(phenyl)
carbamate (587)
Yield 80% (hexane/AcOEt, 75:25 as chromatographic eluent); mp
152–154 ꢃC (from hexane/CH2Cl2); IR 2944, 2795, 1710, 1597,

DOI: 10.3109/14756366.2016.1160902
Biological assays on fentanyl analogues
7
1
1494, 1292, 1223, 1022, 998 csup>/sup>; H NMR (300 MHz) d 3-(4-Chlorophenyl)-1–(1-phenethylpiperidin-4-yl)-1-phenylurea
1.27 (9H, s), 1.60 (2H, m), 1.95 (2H, m), 2.14 (2H, m), 2.54 (2H, (591)
m), 2.73 (2H, m), 3.03 (2H, d, J ¼ 11.1 Hz), 4.32 (1H, m), 6.95
Yield 61% (hexane/AcOEt, 6:4 as chromatographic eluent); mp
163–165 ꢃC (from hexane/CH2Cl2); IR 3406, 2944, 2762, 1666,
1504, 1491, 1395, 1304, 1232, 1088 csup>/sup>; 1H NMR
(300 MHz) d 1.46 (2H, m), 1.89 (2H, m), 2.17 (2H, m), 2.54 (2H,
m), 2.73 (2H, m), 3.02 (2H, d, J ¼ 11.4 Hz), 4.58 (1H, m), 5.86
(1H, s), 7.14–7.51 (14H, m); 13C NMR (75 MHz) d 31.10, 33.87,
52.92, 53.15, 60.46, 120.42, 126.02, 127.67, 128.37, 128.61,
128.66, 129.15, 130.10, 131.22, 137.02, 137.53, 140.20, 153.91.
HRMS (ESI-MS, 140 eV): m/z [M + H]+ calculated for
C26H28ClN3O+: 433.1843, found: 433.1849.
(2H, d, J ¼ 7.5 Hz), 7.14–7.39 (12H, m); 13C NMR (75 MHz) d
30.85, 31.39, 33.87, 34.34, 53.07, 55.48, 60.41, 120.83, 125.97,
126.02, 127.79, 128.36, 128.61, 128.86, 129.98, 137.87, 140.22,
147.89, 149.02, 154.19. HRMS (ESI-MS, 140 eV): m/z
+
[M + H]+ calculated for C30H37N2O2
457.2781.
:
457.2777, found:
4-(Trifluoromethyl)phenyl(1-phenethylpiperidin-4-yl)
(phenyl)carbamate (588)
Yield 26% (hexane/AcOEt, 7:3 as chromatographic eluent); mp
132–134 ꢃC (from hexane/CH2Cl2); IR 2946, 2790, 1709, 1598,
1497, 1305, 1214, 1126, 1065, 995 csup>/sup>; 1H NMR
(300 MHz) d 1.60 (2H, m), 1.95 (2H, m), 2.15 (2H, m), 2.55
(2H, m), 2.74 (2H, m), 3.04 (2H, d, J ¼ 11.4 Hz), 4.32 (1H, m),
7.14–7.42 (12H, m), 7.56 (2H, d, J ¼ 8.1 Hz); 13C NMR (75 MHz)
d 30.77, 33.86, 53.00, 55.76, 60.38, 121.92, 126.06, 126.44 (q.
J ¼ 36.75 Hz), 128.14, 128.39, 128.62, 129.05, 129.84, 137.43,
3-(3-Chlorophenyl)-1–(1-phenethylpiperidin-4-yl)-1-
phenylurea (592)
Yield 48% (CHCl3/AcOEt, 8:2 as chromatographic eluent); mp
164–166 ꢃC (from hexane/CH2Cl2); IR 3282, 2923, 2769, 1638,
1587, 1513, 1426, 1306, 1242, 1084 csup>/sup>; 1H NMR
(300 MHz) d 1.48 (2H, m), 1.90 (2H, m), 2.18 (2H, m), 2.55 (2H,
m), 2.73 (2H, m), 3.03 (2H, d, J ¼ 11.4 Hz), 4.56 (1H, m), 5.88
(1H, s), 6.92–7.49 (14H, m); 13C NMR (75 MHz) d 31.06, 33.84,
52.97, 53.14, 60.44, 117.09, 119.15, 122.76, 126.04, 128.38,
128.62, 129.23, 129.67, 130.15, 131.17, 136.92, 140.11, 140.19,
153.76. HRMS (ESI-MS, 140 eV): m/z [M + H]+ calculated for
C26H28ClN3O+: 433.1843, found: 433.1849.
140.16, 153.20, 153.91. HRMS (ESI-MS, 140 eV): m/z
+
[M + H]+ calculated for C27H28F3N2O2
469.2023.
:
469.2025, found:
3-(Trifluoromethyl)phenyl(1-phenethylpiperidin-4-yl)(phenyl)car-
bamate (589)
Yield 13% (hexane/AcOEt, 7:3 as chromatographic eluent); mp
88–90 ꢃC (from hexane/CH2Cl2); IR 2951, 2786, 1718, 1597, 1496,
1278, 1206, 1161, 1030, 995 csup>/sup>; H NMR (300 MHz) d
3-(4-(tert-Butyl)phenyl)-1–(1-phenethylpiperidin-4-yl)-1-pheny-
lurea (593)
1
Yield 79% (CH2Cl2/AcOEt, 7:3 as chromatographic eluent); mp
168–170 ꢃC (from hexane/CH2Cl2); IR 3415, 2958, 1670, 1588,
1511, 1405, 1322, 1283, 1236, 1083 csup>/sup>; 1H NMR
(300 MHz) d 1.26 (9H, s), 1.45 (2H, m), 1.88 (2H, m), 2.18 (2H,
m), 2.55 (2H, m), 2.73 (2H, m), 3.02 (2H, d, J ¼ 11.1 Hz), 4.59
(1H, m), 5.79 (1H, s), 7.13–7.49 (14H, m); 13C NMR (75 MHz)
d 31.15, 31.37, 33.88, 34.17, 52.74, 53.20, 60.50, 119.09,
125.54, 126.01, 128.36, 128.62, 128.93, 129.97, 131.32, 136.24,
137.38, 140.25, 145.74, 154.30. HRMS (ESI-MS, 140 eV): m/z
[M + H]+ calculated for C30H37ClN3O+: 455.2858, found:
455.2867.
1.60 (2H, m), 1.95 (2H, m), 2.15 (2H, m), 2.55 (2H, m), 2.74 (2H,
m), 3.04 (2H, d, J ¼ 11.7 Hz), 4.32 (1H, m), 7.15–7.41 (14H, m);
13C NMR (75 MHz) d 30.77, 33.876, 53.01, 55.78, 60.39, 118.90,
121.95, 125.22, 126.06, 128.14, 128.39, 128.62, 129.06, 129.61,
129.86, 131.37, 131.80, 137.41, 140.17, 151.42, 153.38. HRMS
+
(ESI-MS, 140 eV): m/z [M + H]+ calculated for C26H28ClN2O2
435.1761, found: 435.1765.
:
General procedure for the synthesis of ureas OMDM590–595
To a stirred 15% phosgene solution in toluene (3.3 mL, 5 mmol) a
solution of the appropriate aniline (1.0 mmol) and Et3N (0.35 mL,
2.5 mmol) in dry toluene (5 mL) was added drop-wise at 0 ꢃC. The
reaction mixture was stirred for 3 h at room temperature and
evaporated under vacuum. The residue of the crude urethane was
dissolved in dry toluene (4 mL) and a solution of 1-phenethyl-N-
phenylpiperidin-4-amine (0.140 g, 0.50 mmol) in dry toluene
(2 mL) was added drop-wise at room temperature with stirring.
The reaction mixture was stirred overnight at room temperature,
diluted with water, and extracted with AcOEt. The organic phase
was washed with NaHCO3 s.s. and twice with water, dried
(Na2SO4), and evaporated under vacuum. The residue was purified
by column chromatography.
1-(1-Phenethylpiperidin-4-yl)-1-phenyl-3–(4-(trifluoromethyl)-
phenyl)urea (594)
Yield 74% (CH2Cl2/AcOEt, 7:3 as chromatographic eluent); mp
182–184 ꢃC (from hexane/CH2Cl2); IR 3400, 2954, 2803, 1671,
1
1594, 1509, 1408, 1317, 1235, 1116, 1076 csup>/sup>; H NMR
(300 MHz) d 1.50 (2H, m), 1.90 (2H, m), 2.18 (2H, m), 2.55 (2H,
m), 2.74 (2H, m), 3.03 (2H, d, J ¼ 11.4 Hz), 4.56 (1H, m), 6.03
(1H, s), 7.15–7.51 (14H, m); 13C NMR (75 MHz) d31.07, 33.88,
53.05, 53.13, 60.46, 118.44, 125.92, 125.98, 126.05, 128.39,
128.62, 129.32, 130.21, 131.15, 136.84, 140.20, 142.07, 153.61.
HRMS (ESI-MS, 140 eV): m/z [M + H]+ calculated for
C27H28F3N3O+: 467.2106, found: 467.2116.
1-(1-Phenethylpiperidin-4-yl)-1,3-diphenylurea (590)
Yield 31% (hexane/AcOEt, 4:6 as chromatographic eluent); mp
152–155 ꢃC (from hexane/CH2Cl2); IR 3426, 2922, 2758,
1674, 1519, 1490, 1437, 1313, 1282, 1232, 1079 csup>/sup>;
1H NMR (300 MHz) d 1.48 (2H, m), 1.90 (2H, m), 2.18 (2H,
m), 2.55 (2H, m), 2.73 (2H, m), 3.00 (2H, d, J ¼ 11.4 Hz),
4.60 (1H, m), 5.86 (1H, s), 6.94–7.49 (15H, m); 13C NMR
(75 MHz) d 31.12, 33.85, 52.77, 53.16, 60.46, 119.19, 122.77,
125.99, 128.35, 128.60, 128.71, 128.99, 130.01, 131.27,
137.23, 138.88, 140.21, 154.10. HRMS (ESI-MS, 140 eV):
m/z [M + H]+ calculated for C26H29N3O+: 399.2232, found:
399.2251.
1-(1-Phenethylpiperidin-4-yl)-1-phenyl-3–(3-(trifluoromethyl)-
phenyl)urea (595)
Yield 79% (CH2Cl2/AcOEt, 7:3 as chromatographic eluent); mp
111–112 ꢃC (from hexane/CH2Cl2); IR 3419, 2949, 2805, 1668,
1526, 1491, 1336, 1277, 1116, 1097 csup>/sup>; 1H NMR
(300 MHz) d 1.46 (2H, m), 1.90 (2H, m), 2.18 (2H, m), 2.55 (2H,
m), 2.74 (2H, m), 3.03 (2H, d, J ¼ 11.4 Hz), 4.59 (1H, m), 5.99
(1H, s), 7.14–7.59 (14H, m); 13C NMR (75 MHz) d31.09, 33.89,
53.06, 53.16, 60.47, 115.82, 119.27, 122.20, 126.046, 128.39,
128.63, 129.16, 129.31, 130.21, 131.18, 131.36, 136.86, 139.47,

8
L. Monti et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
140.21, 153.81.
HRMS
(ESI-MS,
140 eV):
m/z Rat and guinea pig brain membrane homogenate
[M + H]+ calculated for C27H28F3N3O+: 467.2106, found: preparation for binding assays
467.2116.
Rats and guinea pigs were decapitated and their brains were
quickly removed. The brains were prepared for membrane
preparation according to Benyhe et al.26 and partly used for
binding experiments and partly were further prepared for the
Molecular docking
Fentanyl and its analogues were docked to the X-ray structure of
the mouse m-opioid receptor in inactive state obtained from the
protein structure repository (PDB code: 4dkl, http://
www.rcsb.org). Missing residues of the receptor were restored
by the MODELLER programme package19.
Docking the flexible ligands to rigid receptor model was
performed by AutoDock Vina20, and AutoDockTools21, using the
˚
default system setup. The size of the docking grid box was 55 A,
centered on residue Asp147. The ligand structures were drawn at
pH 7.4 and energy minimized by Avogadro22, using the
MMFF94s molecular force field.
For visualization VMD23, AutoDockTools, PMV and
MSMS24, program packages were used.
35
27
´
[ S]GTPgS binding experiments according to Zador et al. .
Briefly the brains were homogenized, centrifuged in ice-cold
50 mM Tris-HCl (pH 7.4) buffer and incubated at 37 ꢃC for 30 min
in a shaking water-bath28–30. After incubation the centrifugation
was repeated as described before and the final pellet was
suspended in 50 mM Tris-HCl pH 7.4 buffer containing 0.32 M
sucrose, and stored at ꢂ80 ꢃC. For the [35S]GTPgS binding
experiments the final pellet of rat brain membrane homogenate
was suspended in ice-cold TEM (Tris-HCl, EGTA, MgCl2) and
stored at -80 ꢃC for further use.
Receptor binding assays
Data analysis was performed by R25, and linux bash scripts.
All calculations were performed in linux environment (Ubuntu
Oneiric). To compare the binding ability of the fentanyl
analogues, the ‘‘ligand efficiency’’ values were used by dividing
the docking energy by the number of non-hydrogen atoms of the
molecule.
Radioligand competition binding experiments
Aliquots of frozen rat brain membrane homogenates were
centrifuged (18 000 rpm, 20 min, 4 ꢃC) to remove sucrose and
the pellets were suspended in 50 mM Tris-HCl buffer (pH 7.4).
Membranes containing 0.3–0.5 mg/ml of protein were incubated
in the presence of unlabelled fentanyl and fentanyl analogues
(OMDM584–595) in crescent concentrations (10 ꢂ 10–10ꢂ 5 M)
for 35 ꢃC for 45 min with ꢀ1 nM [3H]DAMGO in 35 ꢃC for
45 min. OMDM585 and 586 (10 ꢂ 10–10ꢂ 5 M) were also
incubated with ꢀ1 nM of [3H]IleDelt II, [3H]U-69593, or
[3H]WIN55 212–2 in 30 ꢃC for 30 min, in 24 ꢃC for 45 min and
in 30 ꢃC for 60 min, respectively in rat brain homogenates, while
in case of[3H]U-69593 in guinea pig brain membrane homogen-
ates (the guinea pig brain has significantly more kappa receptors
than the rat brain). Additionally, unlabeled IleDelt II, U-69593
and WIN55 212–2 were also incubated together with their labeled
counterparts in increasing concentrations (10 ꢂ 10–10 ꢂ 5 M) for
control.
For experiments performed with [3H]WIN55 212–2 the incu-
bation mixture also contained 50 mM Tris/HCl, 2.5 mM EGTA,
5 mM MgCl2 and 0.5 mg/ml fatty acid-free BSA (pH 7.4). The
level of non-specific binding was determined in the presence of
10 mM unlabelled naloxone or WIN55 212–2, while total binding
was determined in the absence of cold compounds. The reaction
was terminated by rapid filtration under vacuum (Brandel M24R
Cell Harvester; Brandel Harvesters, Gaithersburg, MD), and
washed thrice with 5 ml ice-cold 50 mM Tris-HCl or 50 mM Tris/
HCl, 2.5 mM EGTA, 5 mM MgCl2, 0.5% BSA (pH 7.4) in case of
[3H]WIN55 212–2.
In vitro biological assays
Chemicals
Tris-HCl, EGTA, NaCl, MgCl2 ꢄ 6H2O, GDP, the GTP analogue
GTPgS, fentanyl and U-69593 was purchased from Sigma-Aldrich
(Budapest, Hungary). Fatty acid free bovine serum albumin
(BSA) was purchased from Serva (Heidelberg, Germany). The
highly selective MOR agonist enkephalin analogue Tyr-D-Ala-
Gly-(NMe)Phe-Gly-ol (DAMGO), the highly selective DOR
agonist Ile5,6-deltorphin II (IleDelt II) was synthesized in the
Laboratory of Chemical Biology group of the Biological Research
Centre (BRC, Szeged, Hungary). The non-selective opioid
receptor antagonist naloxone was kindly provided by the company
Endo Laboratories DuPont de Nemours (Wilmington, DE, USA).
WIN55 212–2 was purchased from Tocris Bioscience (Bristol,
UK). Ligands were dissolved in water and were stored in 1 mM
stock solution at -20 ꢃC. The radiolabelled GTP analogue,
[35S]GTPgS (specific activity: 1000 Ci/mmol) was purchased
from
Hartmann
Analytic
(Braunschweig,
Germany).
[3H]DAMGO (specific activity: 38.8 Ci/mmol), [3H]IleDelt II
(specific activity: 19,6 Ci/mmol),18 and [3H]WIN55 212–2 (spe-
cific activity: 13,1 Ci/mmol) were radiolabeled by the Laboratory
of Chemical Biology group in BRC (Szeged, Hungary). [3H]U-
69 593 (specific activity: 43,6 Ci/mmol) was purchased from
Perkin Elmer (Perkin Elmer, Waltham, MA).
The filtration was accomplished through Whatman GF/C
([3H]DAMGO, [3H]IleDelt II or GF/B ([3H]U-69593 and
[3H]WIN55 212–2) glass fibres (GE Healthcare, Little Chalfont,
UK). In case of [3H]WIN55 212–2 the filter was pre-soaked in
0.1% polyethyleneimine 30 min before the filtration. The radio-
activity of the filters was detected in UltimaGoldÔ MV aqueous
scintillation cocktail (Perkin Elmer, Waltham, MA) with Packard
Tricarb 2300TR liquid scintillation counter (Perkin Elmer,
Waltham, MA). The competition binding assays were performed
in duplicate and repeated at least three times.
Animals
For membrane homogenate preparations male and female Wistar
rats (250–300 g body weight) and male guinea pigs (ꢀ700 g body
weight, LAL/HA/BR strain) were used. Rats were housed in the
local animal house of BRC (Szeged, Hungary), while guinea pigs
´
were housed in LAB-ALL Bt. (Budapest, Hungary). All the
animals were kept in a temperature controlled room (21–24 ꢃC)
under a 12:12 light and dark cycle and were provided with water
Functional [35S]GTPgS binding experiments
and food ad libitum. All housing and experiments were conducted In [35S]GTPgS binding experiments, we measure the GDP!GTP
according to the European Communities Council Directives (86/ exchange of the Gai/o protein in the presence of a given ligand to
606/ECC) and the Hungarian Act for the Protection of Animals in measure ligand potency and the maximal efficacy of receptors G-
Research (XXVIII.tv. 32.x). The total number of animals as well protein. The nucleotide exchange is monitored by a radioactive,
as their suffering was minimized.
non-hydrolysable GTP analogue, [35S]GTPgS.

DOI: 10.3109/14756366.2016.1160902
Biological assays on fentanyl analogues
9
The functional [35S]GTPgS binding experiments were per- plastic plates using CHCl3/MeOH/NH4OH (85/15/1 by volume) as
formed as previously described31, with modifications. Briefly the the eluting system. Bands corresponding to [3H]arachidonic acid
rat brain membrane homogenates containing ꢀ10 mg/ml protein were cut, and their radioactivity was counted with a b-counter.
were incubated at 30 ꢃC for 60 min in Tris-EGTA buffer (pH 7.4)
In vivo nociception test
composed of 50 mM Tris-HCl, 1 mM EGTA, 3 mM MgCl2,
100 mM NaCl, containing 20 MBq/0.05 cm3 [35S]GTPgS
(0.05 nM) and increasing concentrations (10 ꢂ 10–10ꢂ 5 M) of
fentanyl and fentanyl analogues (OMDM 584–595) in the
presence or absence of 10 mM naloxone and excess GDP
(30 mM) in a final volume of 1 ml. Total binding was measured
in the absence of test compounds, non-specific binding was
determined in the presence of 10 mM unlabelled GTPgS. The
bound and unbound [35S]GTPgS was separated as described in the
competition binding assays section through Whatmann GF/B
glass fibres. The radioactivity of the filters was also detected as
described above. [35S]GTPgS binding experiments were per-
formed in triplicates and repeated at least three times.
Animals
Male CD-1 mice (Harlan, Italy) weighing 25–30 g were used for all
experiments. Mice were housed for at least 1 week before the
experimental sessions in colony cages (7 mice in each cage) under
standard light (from 7.00 a.m. to 7.00 p.m.), temperature
(21 1 ꢃC), relative humidity (60 10%) with food and water
available ad libitum. The research protocol was approved by the
Service for Biotechnology and Animal Welfare of the Istituto
`
Superiore di Sanita and authorised by the Italian Ministry of
Health, according to Legislative Decree 26/14, which implemented
the European Directive 2010/63/UE on laboratory animal protec-
tion in Italy. Animal welfare was routinely checked by veterinarians
from the Service for Biotechnology and Animal Welfare.
Data analysis
The specific binding of all radiolabeled compounds was
calculated by the subtraction of non-specific binding from total
binding and was given in percentage. Data were normalized to
total specific binding, which was settled 100%, which in case of
[35S]GTPgS also represents the basal activity of the G-protein.
Experimental data were presented as means SEM in the
function of the applied ligand concentration range in logarithm
form. Points were fitted with the professional curve fitting
program, GraphPad Prism 5.0 (GraphPad Prism Software Inc.,
San Diego, CA), using non-linear regression. In the radioligand
competition binding assays the ‘One-site competition’, while in
[35S]GTPgS binding assays the ‘Sigmoid dose-response’ equation
was applied to determine IC50 (unlabelled ligand affinity) and the
maximum G-protein efficacy (Emax) and ligand potency (EC50),
respectively. For IC50 and EC50 values standard error is only given
in their logarithm form by the curve fitting program due to the
data representation. Statistical analyses were performed with
GraphPad Prism 5.0. In case of two data sets, unpaired t-test with
two-tailed p values statistical analysis was used, while for three or
more data sets one-way ANOVA with Tukey’s multiple compari-
son tests were performed to determine the significance level. Since
unlabelled ligand concentrations were presented in the logarithm
form, the curve fitting programme could only calculate SEM. for
the logarithm form of IC50 (logIC50) and EC50 (logEC50) values.
At the same time their antilogarithm form has also been indicated.
Significance was accepted at the p50.05 level.
Surgery for intracerebroventricular (i.c.v.) injections
For i.c.v. injections, mice were lightly anaesthetized with
isoflurane, and an incision was made in the scalp. Injections
were performed using a 10 ml Hamilton microsyringe at a point 2-
mm caudal and 2-mm lateral from the bregma at a depth of 3 mm
in a volume of 5 mL as previously described32.
Hot-plate test
Thermal nociception (hot-plate test) was assessed with a commer-
cially available apparatus consisting of a metal plate 25ꢄ 25 cm
(Ugo Basile, Italy) heated to
a constant temperature of
55.0 0.1 ꢃC, on which a plastic cylinder (20 cm diameter, 18 cm
high) was placed. The time of latency (s) was recorded from the
moment the animal was placed in the cylinder on the hot plate until
it licked its paws or jumped; the cut-off time was 60 s. The baseline
was calculated as mean of three readings recorded before testing at
intervals of 15 min, and was in the same order of magnitude in all
experimental groups (mean 9.8 1.2 s, N ¼ 8–10). The time course
of latency was then determined at 15, 30, 45 and 60 min after
compound treatment. Experimental data were expressed as time
course of the percentage of maximum effect (%MPE) ¼ (post-drug
latency – baseline latency)/(cut-off time-baseline latency)ꢄ 100.
On each test day compound solutions were freshly prepared using
DMSO:0.9% saline 1:5 v/v. These solutions were injected at a dose
of 10 mg/mouse in a volume of 5 mL/mouse. The significance among
the groups was evaluated with the analysis of variance followed by
Tukey’s post-hoc comparisons using GraphPad Prism 6.03 software.
Statistical significance was assumed at least for p50.05.
Biochemical assays
Fatty acid amide hydrolase (FAAH) assays
The activity of products on the enzymatic hydrolysis ofanandamide
(AEA) was obtained using membranes prepared from rat brain,
incubated with the test compounds and [14C] AEA (2.4 mM) in
50 mM Tris-HCl, pH 9, for 30 min at 37 ꢃC. [14C]Ethanolamine
produced from [14C]AEA hydrolysis was measured by scintillation
counting of the aqueous phase after extraction of the incubation
mixture with two volumes of CHCl3/CH3OH ¼ 2/1 (by volume).
Datawere expressed as the concentration exerting 50% inhibition of
AEA hydrolysis (IC50), calculated by GraphPad.
Acknowledgements
We are grateful to Prof. Giorgio Ortar and Prof. Enrico Morera
(Department of Chemistry and Technology, ‘‘Sapienza’’ University of
Rome) for their suggestions and mentoring activity.
Declaration of interest
This research was partly supported by an OTKA-108518 grant (S.
Benyhe) provided by National Research, Development and
Innovation Office (NKFIH), Budapest, Hungary.
Assay of MAGL activity
The 10 000 g cytosolic fraction from COS-7 cells was incubated in
tris HCl 50 mM, at pH 7.0 at 37 ꢃC for 20 min, with synthetic
2-[3H] arachidonoyl-glycerol (1.0 mCi/mmol, 25 mM). After incu-
bation, lipids were extracted with two volumes of chloroform/
methanol ¼ 2/1 (by volume), and the extracts were lyophilized
under vacuum. Extracts were fractionated by TLC on silica on
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Supplementary materials available online