An LP1 analogue, selective MOR agonist with a peculiar pharmacological profile, used to scrutiny the ligand binding domain

Article abstract of DOI:10.1016/j.bmc.2016.08.057

The hypothesis that central analgesia with reduced side effects is obtainable by occupying an ‘allosteric’ site in the MOR ligand binding domain requires the development of new ligands with peculiar pharmacological profile to be used as tools. New benzomorphan derivatives, analogues of LP1, a multitarget MOR agonist/DOR antagonist, were designed to examine in depth MOR ligand binding domain. Compound 5, bearing a diphenylic N-substituent on the benzomorphan nucleus, showed an affinity (K_i^μ?=?0.5?±?0.2?nM) comparable to that of LP1 and a better selectivity versus DOR and KOR. It elicits antinociceptive effects in ex vivo (GPI) and in vivo. This new compound engages receptor amino acidic residues not reached by LP1 and by other established MOR ligands. Molecular modeling studies, conducted on 5 and on several reference compounds, allowed us to propose possible residues in the MOR ligand binding domain essential for their interactions with ‘orthosteric’ and ‘allosteric’ binding sites.

Full text of DOI:10.1016/j.bmc.2016.08.057

Bioorganic & Medicinal Chemistry 24 (2016) 5280–5290
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
An LP1 analogue, selective MOR agonist with a peculiar
pharmacological profile, used to scrutiny the ligand binding domain
a
a
a
a
Simone Ronsisvalle a, , Giuseppina Aricò , Federica Panarello , Angelo Spadaro , Lorella Pasquinucci ,
⇑
Maria S. Pappalardo a, Carmela Parenti b, Nicole Ronsisvalle c
a Department of Drug Sciences, Medicinal Chemistry Section, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
b Department of Drug Sciences, Pharmacology and Toxicology Section, University of Catania, Italy
c Department of Biomedical and Biotechnological Sciences, University of Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The hypothesis that central analgesia with reduced side effects is obtainable by occupying an ‘allosteric’
site in the MOR ligand binding domain requires the development of new ligands with peculiar pharma-
cological profile to be used as tools. New benzomorphan derivatives, analogues of LP1, a multitarget MOR
agonist/DOR antagonist, were designed to examine in depth MOR ligand binding domain. Compound 5,
Received 19 May 2016
Revised 26 August 2016
Accepted 27 August 2016
Available online 30 August 2016
l
bearing a diphenylic N-substituent on the benzomorphan nucleus, showed an affinity (Ki = 0.5 0.2 nM)
comparable to that of LP1 and a better selectivity versus DOR and KOR. It elicits antinociceptive effects in
ex vivo (GPI) and in vivo. This new compound engages receptor amino acidic residues not reached by LP1
and by other established MOR ligands. Molecular modeling studies, conducted on 5 and on several refer-
ence compounds, allowed us to propose possible residues in the MOR ligand binding domain essential for
their interactions with ‘orthosteric’ and ‘allosteric’ binding sites.
Keywords:
MOR agonists
6,7-Benzomorphan scaffold
Modeling studies
Radioligand competition-binding assay
Tail-flick test
Ó 2016 Elsevier Ltd. All rights reserved.
GPI and MVD assays
1. Introduction
existence of two different MOR subpopulations (MOR1 and
MOR2).9 It was suggested that they could differentially mediate
Opioid analgesics are still the most commonly used drugs for
the treatment of acute and chronic pain, from moderate to
severe.1,2 They are considered to produce analgesia through three
analgesic response and unwanted respiratory depression.18,19
However, only one
l receptor has been cloned so far and the abla-
tion of a single receptor gene, i.e. oprm1, eliminates all MOR
GPCRs, named
l
-opioid receptor (MOR), d-opioid receptor (DOR)
responses.20–23,16,24
and
j
-opioid receptor (KOR)3,4, which are activated by several
From the same perspective, a number of researchers reported
that GPCRs can form dimers or oligomers and the MOR/DOR
heterodimer attracted a lot of attention. Opioids combining MOR
agonist–DOR antagonist activity may in fact be effective antinoci-
ceptive agents being able to attenuate MOR-mediated side
effects.25–29 In a previous paper, we showed that the multitarget
MOR agonist-DOR antagonist LP1, 3-[(2R,6R,11R)-8-hydroxy-
6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benazocin-3
(2H)-yl]-N-phenylpropanamide, is a central acting antinociceptive
agent with low potential to induce tolerance and potentially useful
for persistent pain conditions.28,30,31 In contrast, the activation of
DOR with MOR–DOR compounds led to the co-internalization
and co-degradation of both MOR and DOR, allowing to hypothesize
that the physiological dissociation of MOR from DOR signaling
in the pain pathway could enhance MOR-mediated analgesia
and reduce the associated side effects.32 Nevertheless, the
co-expression of MOR and DOR receptors in a single cell is still
controversial.32
endogenous opioid peptides.5,6 All these peptides are characterized
by an N-terminal Tyr residue and morphine analogues, because of
their stereo-specific interaction with the receptors, have always
been considered peptidomimetics and its site of action considered
the orthosteric binding site.
The search for safer and more effective analgesic drugs was ini-
tially oriented to the synthesis of high affinity and selective com-
pounds toward each of receptor subpopulations6–8 with the main
objective of understanding their physiological role9–14 and to sep-
arate antinociceptive activity from side effects. Several ‘new’ opi-
oids were developed and some of them are in clinical use, but
none is devoid of undesirable effects.15,16,4,17
Several clinical studies showed that both side effects and
antinociceptive action are mainly mediated through MOR14–16,4,17
and to take that in account, some researchers proposed the
⇑
Corresponding author. Tel.: +39 0957384209.
E-mail address: s.ronsisvalle@unict.it (S. Ronsisvalle).
http://dx.doi.org/10.1016/j.bmc.2016.08.057
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

S. Ronsisvalle et al. / Bioorg. Med. Chem. 24 (2016) 5280–5290
5281
Recently, Suh33 and Virk34 clearly showed the complexity of the
opioid receptor complex and that different biochemical mecha-
nisms are simultaneously possible after receptor activation.35,36
The development of TRV-130 allowed DeWire et al.14 to hypothe-
size that MOR may signal through at least two distinct pathways
mediated not only by G proteins but also based on b-arrestin
recruitment. At the MOR, b-arrestins seem to act as negative mod-
ulator of analgesia and positive modulator of some side effects,
desensitizing a GPCRs mediated signaling and stimulating inde-
pendent cell signaling outcomes.14
Based on the above considerations, the development of opioid
analgesics devoid of side effects clearly requires a better character-
ization of the MOR ligand binding domain (LBD) in order to identify
compounds able to specifically activate physiological signaling
pathways perhaps interacting with an allosteric binding site rather
then with the morphine orthosteric binding site.
ical properties of the most MOR selective compound. Molecular
dynamic studies in comparison with some reference ligands were
performed to identify binding interactions and to clarify structural
requirements for orthosteric and allosteric binding.37,38
2. Results and discussion
2.1. Chemistry
cis-(À)-(1R,5R,9R)-N-Normetazocine was separated from a com-
mercially available racemic mixture as reported by Brine et al.39
Compounds 1–4 were prepared by acylation of respective amines
with bromoacetyl chloride in anhydrous THF at 0 °C (30 min) and
at rt (30 min) in argon atmosphere. Racemic mixture (rac)-4 was
obtained using (RS)-1-phenyl-1,2,3,4-tetrahydroisoquinoline as
starting amine. The N-substituted cis-(À)-N-normetazocine deriva-
tives 5–8 were obtained by alkylation of cis-(À)-N-normetazocine
in anhydrous MeOH with the respective bromoamide derivatives
1–4. The alkylation procedure was performed in the dark under
argon atmosphere in anhydrous MeOH at 50 °C using NaHCO3
and KI. The diastereomeric mixture 8 was separated by HPLC into
its diastereomers 8a and 8b in milligram amounts by multiple
To identify molecular probes that allow to study the different
mechanisms of receptor activation, we designed some new LP1
analogues 5–8, 8a and 8b. Linear and rigid 3-diphenylalkyl-amide
N-substituted normetazocine derivatives have been synthesized.
Bulkier fragments were chosen to reduce DOR interaction. We
report their binding affinities and ex vivo and in vivo pharmacolog-
Scheme 1. Synthetic route to target compounds 5, 6, 7, 8, 8a, 8b. Reagents and conditions: (a) TEA, dry THF, 0 °C 30 min/rt 30 min, argon; (b) cis-(À)-(1R,5R,9R)-N-
normetazocine, NaHCO3, KI, dry MeOH, 24 h, argon, dark; (c) diastereomeric HPLC separation (Chiralpak AD-H column, 90:10 v/v hexane/2-propanol).

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S. Ronsisvalle et al. / Bioorg. Med. Chem. 24 (2016) 5280–5290
repetitive injections under overload conditions on a Chiralpak AD-
H analytical column (Scheme 1).
segments (pD2 6.8 0.05). As shown in Figures 1–3, the pre-treat-
ment with Naloxone (1 M), a non-selective opioid receptor antag-
onist, Naloxonazine (1 M), selective -opioid receptor
antagonist or norbinaltorphimine (norBNI) (1 M), a -selective
l
l
a
l
l
2.2. Biological activity
j
opioid receptor antagonist failed to affect 5-induced inhibition of
neurogenic contractions (control pD2 6.8 0.05; naloxone pD2
6.4 0.08; naloxonazine pD2 6.5 0.1; norBNI pD2 6.9 0.07).
The selective MOR agonist DAMGO concentration dependently
inhibited the electrically induced contraction. The potency (pD2)
of 5 was similar to that of DAMGO (5 pD2 6.8 0.05; DAMGO
pD2 6.2 0.08) (Fig. 4) and the association of the two compounds
led to a slight increase of the inhibition of the electrically-evoked
contractions (5-DAMGO pD2 7.0 0.06) (Fig. 4).
Compound 6 determined a concentration dependent inhibition
of electrically-evoked contractions of the guinea-pig isolated ileum
segments (pD2 5.7 0.08). As shown in Figure 5, in contrast to
what was obtained with 5, a single concentration (10À6 M) of
Naloxone displaced to the right the concentration–response curve
of 7 with a pD2 of 4.1 0.2 (Fig. 5).
2.2.1. In vitro radioligand binding assay
Binding affinities of new compounds were evaluated by radioli-
gand competition-binding assays in HEK293, CHO human and rat
cells. The results of these experiments on compounds 5, 6, 7, 8
l
and 8a,b are summarized in Table 1. Compounds 5 (Ki = 0.5) and
l
6 (Ki = 160) demonstrate to have a good affinity and selectivity
l
for MOR. Compound 7 showed a reduced affinity (Ki = 84), but is
still selective for MOR. Compounds 8a and 8b, synthesized to ana-
lyze possible stereospecific interaction of the fragment with recep-
tor, showed a reduced affinity with respect to 5, while 6, which
with its methyl group mimics the piperidine fragment, showed a
binding affinity comparable to 8. Compound 5 showed a binding
affinity similar to LP1 with a stronger
l/d selectivity. Compound
5 was selected for further pharmacological studies.
2.2.2. In ex vivo pharmacology
2.2.3. In vivo pharmacology
Pharmacological activities were evaluated in vitro using iso-
lated guinea pig ileum (GPI) to establish their agonist/antagonist
functional activity.
Compound 5 caused a concentration dependent inhibition of
electrically-evoked contractions of the guinea-pig isolated ileum
Compound 5 was further tested to evaluate its antinociceptive
effects in the mouse tail flick test. As shown in Figure 6 (panel
A), 5, in dosage range from 2.5 up to 10 mg/kg intraperitoneally
injected (ip), increased tail flick latency (TFL) in a dose-dependent
manner, with significant values at 30 min of observation. As shown
Table 1
Binding affinities of synthesized compounds*
Compd
MOR (Ki, nM)
KOR (Ki, nM)
DOR (Ki, nM)
Ki ratio
j/
l
Ki ratio d/l
5
6
7
8( )
8a
0.5 0.2
160 0.09
84 0.1
96 0.1
470 0.1
190 0.13
370 0.09
680 0.13
ND
440 0.1
1400 0.4
2900 0.3
ND
380
2.31
8.09
ND
880
8.75
34.52
ND
1900 0.3
7900 1.7
4.04
16.8
8b
LP1
380 0.1
0.83 0.05
900 0.1
2900 0.6
12.5
132.5
34.95
33.8
110
6
29.01
1
TRV-130**
DPDPE
U-50488
DAMGO
6
ND
ND
1.7
<10,000
ND
0.6 0.7
ND
ND
1.7 0.9
ND
1666.67
ND
ND
ND
ND
ND
ND
0.6 0.6
ND
ND
ND not determined.
*
The values are the means SEM of three independent experiments. Ki values were obtained as [3H]DAMGO displacement for the MOR, [3H]DPDPE displacement for the
DOR receptor, and [3H]U69,593 displacement for the KOR receptor.
**
[3H]Diprenorphine 14.
Figure 1. Effects of 5 in the electrically stimulated guinea pig ileum in the absence
and presence of naloxone (10À6 M). Data represent means SEM of 6–8 separate
experiments.
Figure 2. Effects of 5 in the electrically stimulated guinea pig ileum in the absence
and presence of naloxonazine (10À6 M). Data represent means SEM of 6–8
separate experiments.

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5283
show a docking score of À8.4 and À7.2, respectively. Compounds
8a and 8b present similar values (À7.7 and À7.4).
In the dynamic studies their positions in the Ligand Binding
Domain (LBD) were carefully evaluated. Most recurring interac-
tions are observed with some second extracellular loop (ECL 2)
amino acids, such as aspartic acid (Asp216), with the Cys217-
Cys140 residues, which form the disulfide bond between ECL2
and the third transmembrane domain, and with asparagine in
the 3rd transmembrane domain (TM) (Asn127) (Fig. 12).
In addition to the above mentioned amino acids, all compounds
in the series show an interesting arene–arene interaction between
the tyrosines in the 6th (Tyr299) and 7th (Tyr326) transmembrane
domains and their diphenyl portion, likewise important, because it
stabilizes the molecules in the LBD. These tyrosines seem to work
like a gate allowing or not the reaching of Asp147 (3rd TM) binding
region, the purporting morphine binding site (Fig. S1).
Trying to understand why compound 5 carried particular phar-
macological results, we compared it with morphine but also with
other established fully agonist ligands, such as DAMGO, LP1 and
TRV-130.42–44,30,45,46
Figure 3. Effects of 5 in the electrically stimulated guinea pig ileum in the absence
and presence of nor-BNI (10À6 M). Data represent means SEM of 6–8 separate
experiments.
Morphine shows a clear interaction with Asp147, confirming
the results obtained by a number of researchers.42–44,30 Specifi-
cally, it establishes a hydrogen bond between the protonated nitro-
gen moiety and the carboxylic portion of Asp147, and interactions
with Ile144 and Asn127 with its phenolic portion. The interaction
with Asp147 is stable for all the 20 ns calculated by dynamic sim-
ulations (Fig. 8). As observed in Figure S6, the distance between
His297 and the phenolic portion is always higher than 19 Å.
Moreover, some similarities between 5 and morphine are evi-
dent. Infact, looking at the RMSD of transmembrane I-III, both com-
pounds are able to activate the same conformational changes
(Fig. S2a–c). Nevertheless, morphine provokes an RMSD reduction
on TM4, in comparison with the free protein. Opposite, 5 com-
pletely prevents this variability (Fig. S2d–f). It is possible to
observe a morphine-like evolution with DAMGO (Fig. 9a–c). On
TM6, morphine and DAMGO both influence the RMSD spectra in
the same mode. Therefore, they could be able to promote similar
biological effects on MOR. Compound 5, however, provokes a
strong stabilization of TM4 caused by its interaction with ECL2
amino acids.
Even more interesting resulted the evaluation of ligands RMSD
(Fig. 9c). Infact, morphine binds Asp147, thus stabilizing itself in
the orthosteric binding site. Compound 5 shows an undulatory
movement, justified by its interaction not only with Asp216 on
Figure 4. Effects of DAMGO and 5 in the electrically stimulated guinea pig ileum.
Data represent means SEM of 6–8 separate experiments.
in panel B (Fig. 6), 5 and morphine showed a similar potency at the
dose of 5 mg/kg ip, but morphine maintained its antinociceptive
effect until 60 min of observation (*P <0.05 vs saline treated-mice,
**P <0.05 vs saline treated-mice).
Pretreatment with naloxone (3 mg/kg ip), 45 min prior to 5
(5 mg/kg ip), decreased the antinociceptive effect of the compound,
confirming the interaction with opioid receptors in vivo (Fig. 7).
2.3. Molecular modeling studies
Ability to resolve X-ray of MOR, DOR and KOR receptors was a
fundamental step toward the comprehension of protein conforma-
tional changes and to study protein activation cascades and activa-
tion or modulation of internalization processes.
MOR, DOR and KOR receptors crystals were presented in 2012
by Manglik et al. (PDB code 4DKL, resolution: 2.8 Å),44 Granier
et al. (4EK4)36 and Wu et al. (4DJH),40 respectively. Recently,
another MOR crystal was published by Huang et al. (PDB code
5C1M, resolution: 2.1 Å).41
Molecular modeling studies on compounds 5–7 were first per-
formed. In line with its binding data, compound 5 demonstrates
to have the best docking score (À10.6) compared to 6 and 7, which
Figure 5. Effects of DFE in the electrically stimulated guinea pig ileum in the
absence and presence of naloxone (10À6 M). Data represent means SEM of 6–8
separate experiments.

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S. Ronsisvalle et al. / Bioorg. Med. Chem. 24 (2016) 5280–5290
Figure 6. Antinociceptive effect of different doses of compound 5 Panel A. Comparison between analgesic effect of morphine (5 mg/kg ip) and 5 (5 mg/kg ip) Panel B. Data are
expressed as the mean SD. *p <0.05 versus saline-treated rats (n = 8); #p <0.05 versus saline-treated rats (n = 8).
also presents an arene-arene stabilization between its diphenylic
fragment and Tyr326 on TM7 (Fig. 10). Noteworthly, compound 5
interacts with its diphenylic portion with His297, proposed as rel-
evant residue in the site directed mutagenesis studies conducted
on MOR by Bot et al. in 1997.51
DAMGO shows an unstable evolution up to 10 ns. During this
period of time it interacts by its tyrosine fragment with Asp147.
After 10 ns, it appears to reach a stabilization by posing close to
Ile144 and the aforementioned cysteines with its tyrosine group,
to Trp318 with its phenolic portion and to Tyr75 with an H–arene
interaction.
Altogether the above consideration allow us to hypothesize that
compound 5 might not be sensitive to Naloxone antagonism
because of the strong interaction with ECL2 amino acids and tyro-
sine residues. This interaction might be responsible of a slow dis-
Figure 7. Effect of naloxone (3 mg/kg ip) on 5 analgesia. Data are expressed as the
sociation rate from receptor as observed by Virk et al. for
mean SD. *p <0.05 versus saline-treated rats (n = 8); **p <0.05 versus 5-treated rats
buprenorphine.34
(n = 8).
To evaluate possible evidence of different pathways for MOR
activation, the change in the distance between Asp147 and
Tyr326 was carefully examined and compared with that observed
for the free protein. As reported in Figure S5, a stable reduction of
about 2 Å is observed for compound 5 and DAMGO.
ECL2 by the protonated nitrogen moiety on benzomorphan
nucleus, (Fig. 12) but also with cysteines 140 and 217 with which
interacts with the phenolic group. More interesting, compound 5,
To better understand the role of the benzormophan pharma-
cophoric group of 5, we compared the poses in the LBD of LP1,
whose molecular dynamic studies were recently published.14
LP1, displaced by Naloxone despite the pharmacophoric similari-
ties with 5, shows remarkable diversities in the docking poses
within the MOR. First of all, the shorter amminic spacer and the
presence of only one benzene ring makes it able to directly bind
the receptor on ECL2 with its amidic group, thus greatly reducing
its LBD occupancy. Probably for this reason, LP1 is able to have also
a good interaction with DOR, whose LBD is smaller than that of
MOR. LP1 shows an unstable interaction between its phenolic
group and Asp147, for a few ns (Fig. 8). However, a non classical
interaction seems to be imposed by the benzomorphan moiety
with cysteines 140 and 217 in the LBD. LP1 shows a disposition
similar to that of 5 in the interaction of Asp216 with the ben-
zomorphan protonated amino group and of the above mentioned
cysteines with the phenolic portion (Figs. S3b and S4a).
Trying to understand the pharmacological role of this ‘allosteric’
site with respect to the ‘orthosteric’ of morphine, we thought to
evaluate, in our system, the dynamic characteristics of TRV-130,
Figure 8. RMSD of Asp147 in presence of morphine, LP1 and TRV130 and free
protein. Simulation times are expressed in ps.

S. Ronsisvalle et al. / Bioorg. Med. Chem. 24 (2016) 5280–5290
5285
Figure 9. (a) RMSD of TM4 in presence of morphine, DAMGO, compound 5 and without ligand (b) RMSD of TM6 in presence of morphine, DAMGO and compound 5 (c) RMSD
of heavy atoms of morphine, DAMGO, compound 5 and TRV130. Simulation times are expressed in ps.
After 7.5 ns, TRV-130 moves with this fragment to a stable pose
superimposable to that of the benzomorphan group of 5 (Fig. 13).
The interaction of 5 with Asn127 and Tyr128 remains peculiar.47
The ability of TRV-130, compared to morphine (and purportedly
morphine-like compounds), to occupy a non-classical site could be
the cause of a different activation of protein pathway (Fig. 14).
In Figures 13 and 15, it is possible to evaluate TRV-130 posi-
tional changes in comparison with 5, in the LBD, before (magenta)
and after (purple) 7.5 ns. Only after 7.5 ns an engagement of the
Cys140 residue is observed for both compounds (Fig. 9c). It is note-
worthy to observe that the RMSD of Thr208, which is located on
ECL2 close to the 4th TM domain, shows an important difference
between TRV-130 and compound 5 (Fig. 13). As observed in Fig-
ure 15, a strong change is observed only for 5 after 7.5 ns, thus
allowing to hypothesize a possible relevant role of Thr208 in the
MOR allosteric LBD, as a consequence of ECL2 conformational
changes after activation. Placement differences of TRV-130 and 5
with respect to the ECL2 are a possible justification of naloxone
Figure 10. RMSD of Tyr326 in presence of morphine, DAMGO, compound 5.
failure in antagonizing compound 5 but not TRV-130.
Simulation times are expressed in ps.
In conclusion, 5 seems to establish a fundamental hydrogen
bonding interaction with Asp216 in ECL2. Compound 5 also pos-
sesses
a critical arene–arene interactions with Tyr326 and
a recently synthesized MOR agonist, able to activate b-arrestins cir-
cuit with a limited activation of the G-protein mediated pathway.14
TRV-130 results clearly allocated in the region of the LBD close to
the ECL2 (Fig. 11) for 7.5 ns. This behavior is confirmed by a hydro-
gen bonding interaction between Asp216 and the protonated
nitrogen on 3-methoxy-tiophen-2-ilic moiety (Fig. 12).
Tyr128 with a benzomorphan phenol ring, and also strongly binds
cysteines 217 and 140, which are involved in the disulfur bridge.
This perturbation, relevant to provoke receptor conformational
changes, was also observed with TRV-130 (Fig. S3a). Similarly to
morphine, LP1 and DAMGO showed no changes (Figs. 3b, S4a–b).

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S. Ronsisvalle et al. / Bioorg. Med. Chem. 24 (2016) 5280–5290
Figure 11. Pose of TRV130 in the binding pocket of mu opioid receptor and its interaction with Asp216 on ECL2 (figure captured at 2 ns).
3. Conclusions
In this study, the synthesis of 6,7-benzomorphanic derivatives,
analogues of LP1 and bearing a diphenylic fragment and the pecu-
liar pharmacological properties of compound 5 were reported.
Specifically, compound 5 was a selective high affinity MOR agonist
with in vivo antinociceptive activity. In the GPI, it was able to block
electric stimulation not reversed by Naloxone. Molecular dynamic
studies were conducted on compound 5 and on various established
MOR agonists, in order to detect possible analogies and differences
in their interactions with the amino acid residues present in the
MOR LBD. Morphine, LP1, DAMGO and TRV130 were selected for
the study.
As is widely known from mutagenesis data, we confirmed the
interaction of morphine with Asp147, considered critical for high
agonist binding affinity and full inhibition of cAMP.52 Interactions
with Asn127 and Ile 144 were also observed.
In line with what has been recently observed for the d peptides,
and considered possible also for the
, by Fenalti et al.,54 in our
experiments, DAMGO, initially interacts with Asp147 with a high
Figure 12. RMSD of Asp216 in presence of LP1, TRV130 and compound 5.
Simulation times are expressed in ps.
l
Figure 13. Two poses of TRV-130, at 2 ns (magenta) and at 8 ns (purple), showing a movement of about 7.6 Å. In blue compound 5 captured at 5 ns.

S. Ronsisvalle et al. / Bioorg. Med. Chem. 24 (2016) 5280–5290
5287
Figure 14. TRV130 (purple) and morphine (green) with respect to Asp147, captured at 8 ns.
assume that DAMGO might play a double role in the biochemical
signaling of MOR.53
The present dynamic simulation also indicates that ECL2, with
its disulfide bridge, may play an important role on MOR conforma-
tional stabilization. ECL2 seems to be involved in the maintenance
of ligands in the LBD. Possibly, the lack of the ligand initial interac-
tion with the tyrosines plays a role in naloxone capability to dis-
place agonists.
In conclusion, these studies seem to suggest the possibility that
complete MOR signaling can be obtained by occupying sites in the
MOR LBD other than those occupied by morphine and its ana-
logues. Further biochemical and pharmacological studies with
allosteric ligands are necessary to investigate antinociceptive path-
ways and to have information on signal transduction and on the
processes of receptor activation/deactivation.
4. Experimental
4.1. Chemistry
Figure 15. Involvement of TRV130 and compound
Simulation times are expressed in ps.
5 on RMSD of Thr208.
4.1.1. General procedure (A) for the preparation of 3-bromo-N-
substituted- and 3-bromo-N,N-disubstituted propanamide
derivatives (1–3)
occupancy value displayed for 3 ns (data not showed) and then sta-
bilizes itself in an allosteric binding pocket interacting with Cys217
and Cys140 of the disulfide bridge. Interestingly, DAMGO, in its
final pose, superimposes compound 5, which binding site was
defined by the a stable interaction with the following residues:
Asn127(TM2), Tyr128(TM2), Asp216(ECL2), Tyr148(TM3), Tyr299
(TM6) and Tyr326(TM7).
Dynamic studies conducted on TRV-130 showed that, similarly
to compound 5, it interacts with the above mentioned cysteines
and ECL2, but less strongly than 5 coherently with the different
behavior observed toward naloxone antagonism. LP1, the proto-
typic benzomorphan derivative acting as MOR agonist/DOR antag-
onist, interacts, as recently reported,46 with the above mentioned
cysteines, but neither does interact with Asp147 nor shows the
ability to form a stable arene–arene interaction with the tyrosines
on 6th and 7th TM domain.
To a solution of bromoacetyl chloride (1.40 g, 8.17 mmol)
in10 mL of dry THF cooled at 0 °C, under argon atmosphere, was
added dropwise, under vigorous stirring, a solution of the appro-
priate amine (5.45 mmol) and triethylamine (0.276 g, 2.72 mmol)
in dry THF (10 mL). After 30 min at 0° and 30 min at room temper-
ature the reaction mixture was quenched by the addition of 50 ml
of H2O and extracted with dichloromethane (3 Â 50 ml). The com-
bined organic extracts were washed with saturated NaHCO3 and
brine, and then dried over anhydrous Na2SO4. Evaporation under
reduced pressure gave the crude 3-bromopropanamide derivatives
(1–3) which were purified by flash chromatography on silica gel,
using CH2Cl2 and cyclohexane as eluent.
4.1.1.1. 3-Bromo-N-(2,2-diphenylethyl)propanamide (1). White
solid (93%). mp: 98–100 °C. 1H NMR (CDCl3) d ppm: 7.28–7.35 (m,
4H); 7.20–7.27 (m, 6H); 5.55 (br s,1H); 4.20 (t, J = 8.0 Hz, 1H); 3.92
(dd, J = 8.0, 6.0 Hz, 2H); 3.55 (t, J = 6.50 Hz, 2H); 2.61 (t, J = 6.50 Hz,
2H). Anal. Calcd for C17H18BrNO: C, 61.46; H, 5.46; N, 4.22. Found:
C, 61.51; H, 5.51; N, 4.29.
If we assume that morphine activates the G-protein pathway
and the b-arrestin2 circuit alone with its orthosteric occupancy
in the LBD and that TRV-130 is able to activate the G-protein path-
way and both the b-arrestin1 and 2 circuits without any interac-
tion with Asp147 but occupying the allosteric LBD, we can

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S. Ronsisvalle et al. / Bioorg. Med. Chem. 24 (2016) 5280–5290
4.1.1.2.
3-Bromo-N-(diphenylmethyl)-N-methylpropanamide
154–156 °C; 1H NMR (CDCl3) d ppm: 7.26 (m. 3H); 7.23 (m, 3H);
7.19 (m, 2H); 7.08 (m, 1H); 6.98 (m, 1H); 6.66 (m, 1H); 6.1 (s,
0.4H); 5.30 (s, 0.6H); 3.85 (m, 1H); 3.53 (m, 1H); 3.46 (m, 1H);
3.17 (m, 2H); 2.99 (m, 4H); 2.81 (m, 5H); 2.76 (m, 2H); 2.19 (m,
2H); 2.15 (d, 2H); 2.01 (s, 1H); 1.31 (s, 3H); 0.81 (d, 3H). Anal. Calcd
for C32H38N2O2: C, 79.63; H, 7.93; N, 5.80. Found: C, 79.58; H, 8.02;
N, 5.73.
HPLC separations were performed on a Perkin Elmer Flexar FX-
10 UHPLC System equipped with a single-wavelength UV–visible
detector (Perkin Elmer, Italy). Separations were performed on Chi-
(2). White solid (73%). mp: 102–104 °C. 1H NMR (CDCl3) d ppm:
7.13–7.41 (m, 10H); 6.27 (s, 1H); 3.72 (s, J = 7.00 Hz, 2H); 3.03 (t,
J = 7.00 Hz, 2H); 2.82 (s, 3H). Anal. Calcd for C17H18BrNO: C,
61.46; H, 5.46; N, 4.22. Found: C, 61.59; H, 5.53; N, 4.23.
4.1.1.3. 3-Bromo-N-(diphenylmethyl)propanamide (3). White
solid (74%). mp: 96–98 °C. 1H NMR (CDCl3) d ppm: 7.19–7.37 (m,
10H); 6.25 (s, 1H); 3.64 (t, J = 6.60 Hz, 2H); 2.81 (t, J = 6.6 Hz, 2H).
Anal. Calcd for C16H16BrNO: C, 60.39; H, 5.07; N, 4.40. Found: C,
60.45; H, 5.06; N, 4.53.
ralpak AD-H analytical column (250 mm  4.6 mm, 5
l particle
size, Chiral Technology Europe, Illkirch Cedex France). The mobile
phase consisted of hexane/2-propanol (90:10 v/v) at flow rate of
0.5 ml/min. The wavelength was set at 254 nm and the column
was maintained at 23 °C.
4.1.1.4. 3-Bromo-1-(1-phenyl-1,2,3,4-tetrahydronaphthalen-2-
yl)propan-1-one (4). White solid (71%). mp: 110–112 °C. 1H
NMR (CDCl3) ppm: 7.04–7.37 (m, 10H); 6.93 (s, 1H); 3.64–3.81 (m,
3H); 3.40–3.59 (m, 1H); 2.75–3.19 (m, 4H). Anal. Calcd for C18H18
-
BrNO: C, 62.80; H, 5.27; N, 4.07. Found: C, 62.88; H, 5.29; N, 4.13.
4.1.2.4.
3-[(1R,13R)-4-Hydroxy-1,13-dimethyl-10-azatricyclo
[7.3.1.02,7]trideca-2,4,6-trien-10-yl]-1-[(1R)-1-phenyl-1,2,3,4-
4.1.1.5. General procedure (B) for the preparation of N-substi-
tuted cis-(À)-N-normetazocine derivatives (5–7). A mixture
tetrahydronaphthalen-2-yl]propan-1-one (8a).
Yellowish
solid;(63%); mp: 152–154 °C; [
a]
20: À50° (c 1.0, MeOH); 1H NMR
D
of cis-(À)-(1R,5R,9R)-N-normetazocine (150.00 mg, 0.69 mmol),
the appropriate 3-bromoamide derivatives (1–3, 1.04 mmol),
NaHCO3 (87.37 mg, 1.04 mmol) and a catalytic amount of KI was
stirred in dry methanol (10 mL) at 50 °C for 24 h under an argon
atmosphere in the dark. The reaction mixture was filtered and
the solid residue was rinsed with methanol. Concentration under
reduced pressure of the combined filtrate and washing methanol
solutions gave a solid/semisolid residue which was purified by
flash chromatography on silica gel using as eluent CH2Cl2/MeOH
(95:5, v/v). Immediately before the purification process the crude
product was treated with an appropriate amount of eluent, filtered
and loaded in the column for the chromatographic separation.
(CDCl3) d ppm: 7.26 (m, 3H); 7.23 (m, 3H); 7.19 (m, 2H); 7.08
(m, 1H); 6.98 (m, 1H); 6.66 (m, 1H); 6.1 (s, 0.4H); 5.30 (s, 0.6H);
3.85 (m, 1H); 3.53 (m, 1H); 3.46 (m, 1H); 3.17 (m, 2H); 2.99 (m,
4H); 2.81 (m, 5H); 2.76 (m, 2H); 2.19 (m, 2H); 2.15 (d, 2H); 2.01
(s, 1H); 1.31 (s, 3H); 0.81 (d, 3H). Anal. Calcd for C32H38N2O2: C,
79.63; H, 7.93; N, 5.80. Found: C, 79.60; H, 8.00; N, 5.69.
4.1.2.5.
[7.3.1.02,7]trideca-2,4,6-trien-10-yl]-1-[(1S)-1-phenyl-1,2,3,4-
tetrahydronaphthalen-2-yl]propan-1-one (8b). Yellowish
solid; (52%); mp: 150–152 °C; [
20: 52° (c 1.0, MeOH); 1H NMR
3-[(1R,13R)-4-hydroxy-1,13-dimethyl-10-azatricyclo
a]
D
(CDCl3) d ppm: 7.26 (m. 3H); 7.23 (m, 3H); 7.19 (m, 2H); 7.08
(m, 1H); 6.98 (m, 1H); 6.66 (m, 1H); 6.1 (s, 0.4H); 5.35 (s, 0.6H);
3.85 (m, 1H); 3.53 (m, 1H); 3.46 (m, 1H); 3.17 (m, 2H); 2.99 (m,
4H); 2.81 (m, 5H); 2.76 (m, 2H); 2.19 (m, 2H); 2.15 (d, 2H); 2.01
(s, 1H); 1.25 (s, 3H); 0.75 (d, 3H). Anal. Calcd for C32H38N2O2: C,
79.63; H, 7.93; N, 5.80. Found: C, 80.02; H, 7.88; N, 5.88.
4.1.2. N-(2,2-Diphenylethyl)-3-[(2R,6R,11R)-8-hydroxy-6,11-
dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-
yl]propanamide (5)
Yellowish solid (65%); mp: 156–158 °C; À49° (c 1.0, MeOH); 1H
NMR (CDCl3) d ppm: 8.83 (br s, 1H); 7.33–7.17 (m, 10H); 6.86–6.81
(m, 1H); 6.65–6.61 (m, 1H); 6.69–6.73 (m, 1H); 4.22 (t, J = 8 Hz.
1H); 3.97–3.80 (m, 2H); 2.73–2.50 (m, 5H); 2.41–2.20 (m, 3H);
1.95–1.84 (m, 1H); 1.37–1.14 (m, 3H); 1.23 (s, 3H); 0.72 (d,
J = 7 Hz. 3H). Anal. Calcd for C31H36N2O2: C, 79.45; H, 7.74; N,
5.98. Found: C, 79.71; H, 7.79; N, 6.01.
4.2. In vitro radioligand binding assay
MOR, KOR and DOR affinities were investigated by CEREP in 500
competition experiments with radioligands. In the Mor assay, the
selective agonist ligand DAMGO was used as radioligand. To label
Kor receptors was employed U 50488 as agonist radioligand. For
Dor was used DPDPE as radioligand.
In Mor assay, HEK-293 cells were used as a source of receptors.
For Kor assay rat CHO cells and for Dor human CHO cells were
used. Non-specific binding was determined in the presence of nal-
4.1.2.1.
dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-
yl]-N-methylpropanamide (6). Yellowish solid (42%); mp:
N-(Diphenylmethyl)-3-[(2R,6R,11R)-8-hydroxy-6,11-
172–174 °C; À52° (c 1.0, MeOH); 1H NMR (CDCl3) d ppm: 7.41–
7.11 (m, 10H); 6.94–6.86 (m, 1H); 6.80–6.76 (m, 1H); 6.75–6.70
(m, 1H); 6.46–6.41 (m, 1H); 2.68–2.41 (m, 5H); 2.40–2.20 (m.
3H); 2.07–1.99 (m, 1H); 1.21–1.46 (m, 3H); 1.34 (s, 3H); 0.85 (d,
J = 6.5 Hz), 3H). Anal. Calcd for C31H36N2O2: C, 79.45; H, 7.74; N,
6.83. Found: C, 80.15; H, 7.78; N, 6.03.
trexone (10 lM). Eight concentrations of each compound (0.3–
1000 nM) were used in the assays.
4.3. In ex vivo pharmacology
4.3.1. Guinea-pig ileum preparation
4.1.2.2.
N-(Diphenylmethyl)-3-[(2R,6R,11R)-8-hydroxy-6,11-
Sections of guinea-pig ileum longitudinal muscle/myenteric
plexus were prepared according to Kinney et al. (1995) with minor
modifications. Male Dunkin–Hartley guinea-pigs (200–300 g)
(Harlan Laboratories, S.Pietro al Natisone (UD) were killed by
decapitation. The intestines were exteriorized, the ileo-caecal junc-
tion located and 5 cm of the terminal ileum discarded. Approxi-
mately 30 cm of the terminal ileum was removed and the lumen
flushed with Krebs solution (in mM: 118 NaCl, 4.75 KCl, 2.45 CaCl2,
1.71 MgCl2, 25.0 NaHCO3, 0.93 KH2PO4, 11 glucose). Four 2 cm long
segments of the ileum were secured on to a perspex holder sup-
porting two parallel platinum wire electrodes and placed in a
dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-
yl]propanamide (7). Yellowish solid (48%); mp: 168–170 °C; À58°
(c 1.0, MeOH); 1H NMR (CDCl3) d ppm: 9.98 (br s, 1H); 7.38–7.20 (m.
10H); 6.89–6.84 (m, 1H); 6.68–6.54 (m, 2H); 6.25–6.20 (m, 1H); 2.89–
2.70 (m, 4H); 2.67–2.32 (m, 4H); 2.05–1.97 (m, 1H); 1.18–1.44 (m,
3H); 1.23 (s, 3H); 0.67 (d, J = 7 Hz, 3H). Anal. Calcd for C30H34N2O2:
C, 79.26; H, 7.54; N, 6.16. Found: C, 79.58; H, 7.55; N, 6.19.
4.1.2.3.
3-{4-Hydroxy-1,13-dimethyl-10-azatricyclo[7.3.1.02,7
]
trideca-2,4,6-trien-10-yl}-1-(1-phenyl-1,2,3,4-tetrahydronaph-
thalen-2-yl)propan-1-one (8 ). Yellowish solid (84%); mp:

S. Ronsisvalle et al. / Bioorg. Med. Chem. 24 (2016) 5280–5290
5289
20 ml isolated organ bath containing Krebs solution at 37.0 °C and
bubbled with 5% CO2/95% O2. The strips were placed under a 2 g
load and contractility measured using an appropriate transducer
connected to a PowerLab 4/20 recorder (ADInstruments, Castle
Hill, NSW, Australia). After 60 min equilibration, maximal contrac-
tions of the tissue were elicited by transmural stimulation using
single pulses (0.1 Hz, 0.3 ms, 200 mA) delivered by a Digitimer
multisystem D330 stimulator.
Following a 60–90 min equilibrium period, during which the
Krebs solution was changed several times, test compounds were
added cumulatively, allowing a minimum of 5 min before addi-
tional compound was added to the bath.
ware (developed by Chemical Computing Group).48 The obtained
proteins have undergone relaxing dynamic cycles for 20 ns by
NAMD software in NVT and NPT ensemble (T = 300°K;
P = 1.01325 bar). The same conditions were applied to all other
simulations. Receptors were then inserted in a phospholipidic
bilayer, made by 260 dipalmitoylphosphatidylcholine (POPC) resi-
dues, and a water bilayer (11485 water molecules) was created
(grid dimension: 113 Â 112 Â 79 Å). Both procedures were effectu-
ated by charmm-gui.org (default settings), a web-based graphical
user interface able to generate various molecular systems.49 Addi-
tional relaxing cycles were further accomplished for other 20 ns.
Checked the stability of the systems, docking studies were con-
ducted by MOE with a ‘Triangle Matcher’ placement methodology.
Ligands were minimized with FFMM94X forcefield (developed for
the MOE software) with lowest minimization gradient. Charges
were calculated by LigX, MOE subprogram. Conformational studies
were then performed, using systematic and stochastic methods.
Databases were created with more than 15.000 conformations.
Rescoring on databases was then effectuated (London dG and
GBVI/WSA dG). After evaluation of docking scores and interaction
ligand energies, starting poses were chosen. During protein-mem-
brane relaxing phases and docking evaluations, only one angstrom
of RMSD gap through modified 4DKL and 5C1M was observed. The
first was however selected to accomplish our studies. Subse-
quently, initial steps to run molecular dynamic studies were car-
ried out. NAMD software50 was chosen to conduct these studies
and input files were prepared by tLeap developed in Amber12
and AmberTools 12 with ‘ff12SB’ and ‘ff99SBildn’ forcefield for pro-
tein and ‘gaff’ forcefield for ligands.44 AmberTools 14 was utilized
for membrane forcefield, in particular ‘lipid11’ and ‘lipid14’. Ligand
charges were obtained by Gaussian09. tLeap was used to neutralize
complex charge. In an initial phase, protein and ligand are fixed to
obtain a good merge between ligand–protein system and phospho-
lipidic bilayer. After this initial step, all components were slowly
4.3.2. Data analysis
Electrically-evoked contractions have been expressed as a per-
centage of the contraction. The effectiveness of a given compound
to inhibit electrically induced contraction was measured as the
percentage change from baseline. The concentration of a given test
compound eliciting half-maximal inhibition of the electrically
induced contraction (EC50) was determined by non-linear curve fit-
ting (Prism v. 3.0, GraphPad) using the mean response of at least
three separate trials as the given response for a single concentra-
tion. The potency of the opioid receptor agonists in the absence
and presence of the antagonists was assessed as the negative log-
arithm of the concentration required to cause 50% of the maximum
response (pD2).
4.4. In vivo pharmacology
4.4.1. Animals
Male Swiss CB1 mice (Harlan Laboratories, S.Pietro al Natisone
(UD)) weighing 25–30 g were housed six to a cage. Animals were
kept at a constant room temperature (25 1 °C) under a 12:12 h
light and dark cycle with free access to food and water. Each mouse
was used for only one experiment. Experimental procedures were
approved by the Local Ethical Committee (IACUC) and conducted in
accordance with international guidelines as well as European Com-
munities Council Directive and National Regulations (CEE Council
86/609 and DL 116/92).
released (backbone and ligand in a first phase and later a-carbon).
POPC is always completely free. Every final production cycle was
conducted 10 times and the mean was utilized to create a RMSD
curves (see in the text). Measurements up to 50 ns not showed rel-
evant variations.
4.4.2. Tail-Flick Test
Acknowledgments
Nociception was evaluated by the radiant heat tail-flick test.
Briefly, it consisted of irradiation of the lower third of the tail with
an infrared source (Ugo Basile, Comerio, Italy). The day before the
experiment, mice were habituated to the procedure for measuring
nociception threshold. Experiments were performed at room tem-
perature (25 1 °C). The basal pre-drug latency was established
between 3 and 4 s and was calculated as the average of the first
three measurements, which were performed at 5 min intervals. A
cutoff latency of 10 s was established to minimize damage to the
tail. Post-treatment tail flick latencies (TFLs) were determined at
30, 60 and 90 min after intraperitoneal (ip) injection.
This work was supported by a FP6 European Grant ‘Normolife’
(LSHC-CT-2006-037733) and by ‘Finanziamento della Ricerca’
FIR2014 granted by University of Catania. The authors gratefully
acknowledge Fabbrica Italiana Sintetici (Italy) for providing ( )-
cis-N-normetazocine.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.08.057.
4.4.3. Statistical Analysis
References and notes
Data are expressed as mean values (SEM analysis of variance
(ANOVA) followed by the post hoc ‘Student–Newman–Keuls’ test
were performed to assess significance using the Instat 3.0 software
(GraphPad Software, San Diego, CA). P <0.05 was considered
significant.
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