(2S)-N-2-methoxy-2-phenylethyl-6,7-benzomorphan compound (2S-LP2): Discovery of a biased mu/delta opioid receptor agonist

Article abstract of DOI:10.1016/j.ejmech.2019.02.043

The pivotal role of the stereocenter at the N-substituent of the 6,7-benzomorphan scaffold was investigated combining synthetic and pharmacological approaches. 2R- and 2S-diastereoisomers of the multitarget MOR/DOR antinociceptive ligand LP2 (1) were synt

Full text of DOI:10.1016/j.ejmech.2019.02.043

Accepted Manuscript
(2S)-N-2-methoxy-2-phenylethyl-6,7-benzomorphan compound (2S-LP2): Discovery
of a biased mu/delta opioid receptor agonist
Lorella Pasquinucci, Rita Turnaturi, Girolamo Calò, Francesco Pappalardo, Federica
Ferrari, Giulia Russo, Emanuela Arena, Lucia Montenegro, Santina Chiechio, Orazio
Prezzavento, Carmela Parenti
PII:
S0223-5234(19)30157-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.043
EJMECH 11135
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 November 2018
Revised Date: 11 February 2019
Accepted Date: 11 February 2019
Please cite this article as: L. Pasquinucci, R. Turnaturi, G. Calò, F. Pappalardo, F. Ferrari, G. Russo, E.
Arena, L. Montenegro, S. Chiechio, O. Prezzavento, C. Parenti, (2S)-N-2-methoxy-2-phenylethyl-6,7-
benzomorphan compound (2S-LP2): Discovery of a biased mu/delta opioid receptor agonist, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.043.
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ACCEPTED MANUSCRIPT
(2S)-N-2-methoxy-2-phenylethyl-6,7-benzomorphan compound (2S-LP2): discovery of a
biased mu/delta opioid receptor agonist
a
a
b
c
b
Lorella Pasquinucci , Rita Turnaturi *, Girolamo Calò , Francesco Pappalardo , Federica Ferrari ,
d
a
e
c,f
a,1
Giulia Russo , Emanuela Arena , Lucia Montenegro , Santina Chiechio , Orazio Prezzavento
c,1
and Carmela Parenti
a
Department of Drug Sciences, Medicinal Chemistry Section, University of Catania, Viale A.
Doria 6, 95125 Catania, Italy
b
Department of Medical Sciences, Pharmacology section, University of Ferrara, via Fossato di
Mortara 19, 44121 Ferrara, Italy
c
Department of Drug Sciences, Pharmacology and Toxicology Section, University of Catania,
Viale A. Doria 6, 95125 Catania, Italy
d
Department of Biomedical and Biotechnological Sciences, University of Catania, Via Santa
Sofia 97, 95125 Catania, Italy
e
Department of Drug Sciences, Pharmaceutical Technology Section, University of Catania,
Viale A. Doria 6, 95125 Catania, Italy
f
Oasi Research Institute-IRCCS, Troina, Italy
*
Corresponding author.
E-mail address: rita.turnaturi@unict.it (Rita Turnaturi)
1
O.P. and C.P. contributed equally to the work.
Graphical Abstract
1

ACCEPTED MANUSCRIPT
Highligthts
1
.
The stereocenter role at the N-substituent of the 6,7-benzomorphan scaffold was
investigated.
2
3
.
.
2R- and 2S-diastereoisomers of the multitarget opioid ligand LP2 were synthesized.
2S-LP2 showed a better pharmacological profile than 2R-LP2 in in vitro and in vivo
assays.
4
5
.
.
2S-LP2 resulted a biased multitarget MOR/DOR agonist.
2S-LP2 elicited an antinociceptive potency 1.5- and 3-times higher than LP2 and R-
antipode.
Keywords Pain; Multitarget; Asymmetric synthesis; Radioligand competition binding; BRET;
G-protein; β-arrestin; Tail Flick test.
2

ACCEPTED MANUSCRIPT
Abstract
The pivotal role of the stereocenter at the N-substituent of the 6,7-benzomorphan scaffold was
investigated combining synthetic and pharmacological approaches. 2R- and 2S-diastereoisomers
of the multitarget MOR/DOR antinociceptive ligand LP2 (1) were synthesized and their
pharmacological profile was evaluated in in vitro and vivo assays. From our results, 2S-LP2 (5)
showed an improved pharmacological profile in comparison to LP2 (1) and 2R-LP2 (4). 2S-LP2
(5) elicited an antinociceptive effect with a 1.5- and 3-times higher potency than LP2 (1) and R-
antipode (4), respectively. In vivo effect of 2S-LP2 (5) was consistent with the improved
MOR/DOR efficacy profile assessed by radioligand binding assay, to evaluate the opioid
receptor affinity, and BRET assay, to evaluate the capability to promote receptor/G-protein and
receptor/β-arrestin 2 interactions. 2S-LP2 (5) was able to activate, with different efficacy, G-
protein pathway over β-arrestin 2, behaving as biased agonist at MOR and mainly at DOR.
Considering the therapeutic potential of both multitarget MOR/DOR agonism and functional
selectivity over G-protein, the 2S-LP2 (5) biased multitarget MOR/DOR agonist could provide a
safer treatment opportunity.
1
1
. Introduction
Currently used analgesic drugs are chiral compounds often marketed as racemic mixture [1] or
single eutomers rather than distomers [2]. For instance, both isomers contribute to the analgesic
effect of Tramadol, marketed as mixture 1:1 of 1R,2R-(+)- and 1S,2S-(−)-isomers, with a
different but complementary mechanism of action [3], while in Nucynta® the pharmacological
1
SAR, structure-activity relationship; MOR, mu opioid receproe; DOR, delta pioid receptor;
GPI, guinea pig ileum; MVD, mouse vas deferens; BRET, bioluminescence resonance energy
transfer; KOR, kappa opioid receptor; i.p., intraperitoneal; TFL, tail flick latency.
3

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effect is due to the 1R,2R-Tapentadol isomer [4]. Thus, drug chirality is assumed to have an
important role in both design and development of new analgesic drugs. Despite the fact that
isomers share the identical molecular formulas, atom-to-atom linkages, and bonding distances,
they are different chemical compounds. Indeed, the isomers could differ in pharmacological,
pharmacokinetic and toxicological properties [5] because of the environment of living systems
where drug targets are also chiral. Thus, the stereoselective isomer-target interaction could result
in different affinity, selectivity and activity, generating substantial differences between isomers
[
6].
Recently, we synthesized a series of 6,7-benzomorphan-based compounds bearing short and
flexible substituents at the basic nitrogen [7]. This SAR study confirmed the importance of N-
substituent nature in opioid receptors affinity and/or activity modulation. In particular, the
increased flexibility of the N-substituent and the presence of a hydroxyl or methoxyl group at
carbon 2, as hydrogen bond donor and acceptor, allowed an optimal interaction with the opioid
receptor binding pocket. Among them, the compound with the (R/S)-2-methoxy-2-phenylethyl
group as N-substituent, named LP2 (1, Fig. 1), was characterized by nanomolar affinity for MOR
(
K
i
= 1.08 nM) DOR (K
i i
= 6.61 nM) and KOR (K = 15.22 nM) (Table 1) and in vitro assays
MOR
DOR
(GPI and MVD) LP2 showed MOR/DOR agonist profile (IC50
= 21.5 nM and IC50
= 4.4
nM). In the tail flick test, after i.p. administration, LP2 produced a long-lasting antinociception,
naloxone-reversed, with an ED50 of 0.9 mg/kg. Collected data highlighted that LP2 is a
multitarget MOR/DOR antinociceptive ligand. Compounds possessing multitarget opioid activity
are effective antinociceptive agents with limited adverse effects. In fact, a growing body of
evidence [8,9] suggested that in such opioid ligands the additive or synergic antinociceptive
4

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effects coupled with an improved tolerability could be the result of MOR/DOR intermodulatory
interactions [10,11].
In the present study, to investigate the role of the stereocenter at the N-substituent of the 6,7-
benzomorphan scaffold [12,13] in drug-opioid receptor interaction, we evaluated the
pharmacological fingerprint of both 2R- (4) and 2S- (5) diastereoisomers (Fig. 1) of the
multitarget MOR/DOR agonist LP2 (1) using a combination of synthetic and pharmacological
approaches. To this aim, LP2 diastereoisomers were synthesized. Both diastereoisomers were
tested in vitro by competition binding and BRET assays, and in vivo by tail flick test. The
pharmacological profile of 2R-LP2 (4) and 2S-LP2 (5) were compared each other and with LP2
(1).
Fig. 1. LP2, 2R-LP2, 2S-LP, 2R-OH and 2S-OH structures.
2
. Results and discussion
2
.1. Chemistry
To obtain compounds 2R-LP2 (4) and 2S-LP2 (5), an asymmetric approach was performed as
previously reported (Scheme 1) [7]. By means of resolution of commercially available racemic
mixture, the (−)-cis-N-normetazocine was obtained as previously reported [14,15]. Compounds 2
and 3 were prepared by primary alcohol sulfonylation with pTsCl and TEA in CH Cl with
2
2
5

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catalytic Bu
2
SnO. Target compounds 2R-LP2 (4) and 2S-LP2 (5) were obtained by alkylation of
(–)-cis-N-normetazocine with the respective tosylated alcohols 2 and 3. All compounds were
1
13
characterized by H and C NMR, IR mass spectroscopy and elemental analysis.
Scheme 1. Synthetic pathway.
2
.2. Radioligand binding assay
MOR, DOR and KOR binding affinity was determined by radioligand competition binding
experiments on rat or guinea pig brain membranes as previously reported [16]. Inhibition
constant (K ) values, calculated using nonlinear regression analysis (GraphPad Prism, version
i
5
.0), are listed in Table 1.
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Table 1. Opioid receptor binding affinity of LP2 (1), 2R-LP2 (4) and 2S-LP2 (5) on rat or guinea
pig brain membranes.
a,b
Compd
K (nM) ± SEM
i
K ratio
i
MOR
1.08 ± 0.10
DOR
6.61 ± 0.60
KOR
15.22 ± 0.80
DOR/MOR
KOR/MOR
14.10
c
LP2 (1)
6.11
2
2
R-LP2 (4)
S-LP2 (5)
12.30 ± 0.42 151.10 ± 0.60 236.00 ± 0.83
12.28
19.20
0.50 ± 0.03
2.59 ± 0.05
26.50 ± 0.44
5.18
53.00
DAMGO
1.16 ± 0.10
-
-
-
-
-
-
-
Naltrindole
-
-
1.13 ± 0.10
-
-
U50,488
a
0.34 ± 0.10
-
Values are means ± SEM of three separate experiments, each carried out in duplicate.
b
3
3
3
K values were obtained as [ H]-DAMGO displacement for MOR, [ H]-DPDPE displacement for DOR, and [ H]-
i
(
+)-U69,593 displacement for KOR, using nonlinear regression analysis (GraphPad Prism, version 5.0).
Ref. 7
c
3
All compounds displayed the capability to inhibit [ H]-DAMGO binding at MOR in a
concentration-dependent manner. In comparison to LP2 (1), MOR K values were 11-times
i
higher for 2R-LP2 (4) and 2-times lower for the 2S-antipode (5). 2R-LP2 (4) DOR affinity was
2
5
3-times lower than that of LP2 (1). Differently, 2S-LP2 (5) showed a DOR affinity about 3- and
8-times higher than that of LP2 (1) and 2R-antipode (4), respectively. We also found that KOR
affinity of 2R-LP2 and 2S-LP2 (4 and 5) was lower than that of LP2 (1). Specifically, 2S-LP2 (5)
KOR affinity resulted 2-times lower, while 2R-LP2 (4) KOR affinity was 15-times reduced in
comparison to the LP2 (1) KOR affinity.
The 2-methoxy-2-phenylethyl N-substituent of LP2 (1) provides a high affinity for MOR and
DOR and a moderate affinity for KOR [7]. The results of this study showed that 2R
configuration (4) seemed to be tolerated by MOR but not by DOR and KOR, whereas an
improved binding profile at both MOR and DOR was recorded for compound (5) with the 2S-
configuration at the 2-methoxy-2-phenylethyl N-substituent.
7

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Previously [7], we reported an analogous trend highlighting the influence of N-substituent
stereochemistry. Indeed, the 6,7-benzomorphan compound with the 2S-hydroxy-2-phenylethyl as
N-substituent (2S-OH, 7, Figure 1) showed the best MOR (K = 0.50 ± 0.20 nM), DOR (K = 0.80
i
i
±
0.20 nM) and KOR (K = 2.22 ± 0.20 nM) affinity profile, while its 2R-antipode (2R-OH, 6,
i
Figure 1) provided MOR, DOR and KOR K values 5-, 12- and 3-times higher, respectively.
i
2
.3. Bioluminescence resonance energy transfer assay
Based on affinity data and functional selectivity demonstrated by LP2 in GPI versus MOR and
MVD versus DOR, the functional activity for MOR and DOR of LP2 (1), 2R-LP2 (4) and 2S-
LP2 (5) was investigated. The compounds were evaluated for their ability to promote receptor/G-
protein and receptor/β-arrestin 2 interaction in the BRET assay previously described by Molinari
et al. [15]. The results are summarized in Table 2.
8

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Table 2. Potencies and efficacies of DADLE, LP2 (1), 2R-LP2 (4) and 2S-LP2 (5) on MOR/G-protein and MOR/β-arrestin 2 interaction, and
DOR/G-protein and DOR/β-arrestin 2 interaction in the BRET assay.
b
c
b
c
Bias factor
CL95%
Compd
pEC50 (CL95%
)
CR
α ± SEM
pEC50 (CL95%
)
CR
α ± SEM
(
)
MOR/G-protein
MOR/β-arrestin 2
a
DADLE
6.89 (6.56-7.22)
7.90 (7.51-8.30)
1
1.00
5.86 (5.71-6.01)
1
1.00
0
LP2 (1)
0.10
0.92 ± 0.02
6.56 (6.22-6.90)
0.20
0.71 ± 0.09
0.57 (0.03-1.16)
d
2
R-LP2 (4)
S-LP2 (5)
crc incomplete
8.33 (7.90-8.76) 0.04
DOR/G-protein
inactive
0.11
DOR/β-arrestin 2
-
2
0.93 ± 0.02
6.82 (6.43-7.21)
0.72 ± 0.08
0.82 (0.22-1.41)
DADLE
7.23 (6.88-7.57)
7.02 (6.83-7.21)
1
2
1.00
7.69 (7.50-7.87)
1
110
1.00
0
LP2 (1)
0.96 ± 0.03
5.65 (5.48-5.82)
0.90 ± 0.01
2.03 (1.57-2.49)
-
d
2R-LP2 (4)
crc incomplete
inactive
91
2S-LP2 (5)
7.49 (7.24-7.75) 0.55
1.07 ± 0.03
5.73 (5.40-6.07)
1.03 ± 0.03
2.31 (1.84-2.77)
a
DADLE was used as reference agonist for calculating intrinsic activity at the MOR and DOR.
CR is the ratio between EC50 of tested compounds and EC50 of standard agonist (DADLE).
α is the ratio between the intrinsic activity of tested compounds and intrinsic activity of standard agonist (DADLE) as maximal-stimulated BRET ratio (α = 1).
b
c
d
Crc incomplete means that maximal effects could not be determined due to the low potency of the compound.
Data are the mean ± SEM of at least 5 separate experiments made in duplicate.
9

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Membrane extracts taken from SH-SY5Y cells stably co-expressing the MOR/RLuc
and Gβ1/RGFP fusoproteins were used in concentration-response experiments to
evaluate receptor/G-protein interaction. DADLE promoted MOR/G-protein
interaction in a concentration-dependent manner with potency of 6.89 and maximal
effect of 0.81 ± 0.10 stimulated BRET ratio (Fig. 2, panel A).
Fig. 2. Concentration-response curve to DADLE (panel A), LP2 (1, panel B), 2R-LP2
(4, panel C) and 2S-LP2 (5, panel D) in promoting MOR/G-protein and MOR/β-
arrestin 2 interaction. Data are the mean ± SEM of 5 separate experiments made in
duplicate.
1
0

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The intrinsic activities of tested compounds were computed as fraction of DADLE
maximal-stimulated BRET ratio (α = 1). LP2 (1) and 2S-LP2 (5) (Fig. 2, panel B and
D) mimicked the maximal effect of DADLE (pEC = 6.93) being 10- and 30-times
50
more potent (pEC = 8.06 and 8.33, respectively). Compound 2R-LP2 (4) was able to
5
0
elicit a weakly stimulatory response only at the highest concentration tested (Fig. 2,
panel C).
Whole SH-SY5Y cells stably expressing the MOR/RLuc and the β-arrestin 2/RGFP
fusoproteins were used to evaluate MOR/β-arrestin 2 interaction. DADLE stimulated
the interaction of the MOR with β-arrestin 2 in a concentration-dependent manner
with pEC50 of 5.86 and maximal effects corresponding to 0.57 ± 0.20 stimulated
BRET ratio (Fig. 2, panel A). As for G-protein studies, the intrinsic activities of the
compounds were computed as fraction of the standard agonist DADLE. Compounds
LP2 (1) and 2S-LP2 (5) mimicked the stimulatory response of DADLE with slightly
lower efficacy but 5- and 9-times higher potency, respectively (Fig. 2, panel B and C).
2
R-LP2 (4) was inactive (Fig. 2, panel C). The results showed in Fig. 2 were used for
calculating the bias factor of the ligands: LP2 (1) and 2S-LP2 (5) displayed a modest
<10 times) bias toward G-protein that reached the statistical level of significance
(
only for the latter compound (Table 2).
In SH-SY5Y cells stably co-expressing the DOR/RLuc and the Gβ1/RGFP
fusoproteins, DADLE promoted receptor/G-protein interaction in a concentration-
dependent manner displaying a pEC value of 7.23 and maximal effect of 0.42 ± 0.04
5
0
stimulated BRET ratio (Fig. 3, panel A). The intrinsic activities of tested compounds
were computed as fraction of DADLE maximal-stimulated BRET ratio (α = 1).
1
1

ACCEPTED MANUSCRIPT
Fig. 3. Concentration response curve to DADLE (panel A), LP2 (1, panel B), 2R-LP2
(4, panel C) and 2S-LP2 (5, panel D) in promoting DOR/G-protein and DOR/β-
arrestin 2 interaction. Data are the mean ± SEM of 7 separate experiments made in
duplicate.
In parallel experiments, LP2 (1) and 2S-LP2 (5) (Fig. 3, panel B and D) behaved as
full agonists with similar potency and similar efficacy as the standard (Table 2).
Compound 2R-LP2 (4) was able to elicit a weakly stimulatory response only at the
highest concentration tested (Figure 3, panel C).
Whole SH-SY5Y cells stably co-expressing the DOR/RLuc and the β-arrestin
2
/RGFP fusoproteins were used to evaluate DOR/β-arrestin 2 interaction. DADLE
1
2

ACCEPTED MANUSCRIPT
stimulated the interaction of the DOR with β-arrestin 2 in a concentration-dependent
manner with pEC50 of 7.69 and maximal effects corresponding to 0.47 ± 0.05
stimulated BRET ratio (Fig. 3, panel A). The intrinsic activities of the compounds
under study were computed as fraction of DADLE. LP2 (1) and 2S-LP2 (5) mimicked
the maximal effects of DADLE being, however, less potent (Fig. 3, panel B and D),
while 2R-LP2 (4) was inactive (Fig. 3, panel C). Fig. 3 showed the comparison of the
effects of each compound on the DOR/G-protein and DOR/β-arrestin 2 interaction.
These results were used for calculating the bias factor of LP2 (1) and 2S-LP2 (5); both
compounds showed a statistically significant and large (200-times) bias toward G-
protein (Table 2).
Figure 4 shows the bias factors for LP2 (1) and 2S-LP2 (5) at MOR and DOR
between G-protein and β-arrestin 2 recruitment.
Fig. 4. Bias factor (mean ± CL95%) of LP2 (1) and 2S-LP2 (5) toward G-protein and
β-arrestin 2. DADLE was used as reference agonist for both receptors.
1
3

ACCEPTED MANUSCRIPT
We used BRET assay to examine the capability to promote MOR and DOR/G-protein
interaction of the previously synthesized 6,7-benzomorphans with 2R-OH (6) and 2S-
OH (7, Fig. S1) [7] to compare the role of the chirality. Similarly, 2S-OH (7) showed
MOR
DOR
the best MOR/DOR agonist profile (pEC50
= 8.18, α = 0.87 and pEC50
= 7.84,
MOR
α = 1.13) in comparison with the 2R-OH (6) (pEC50
= 7.65, α = 0.82 and
DOR
pEC50
= 7.23, α = 1.03) (Table S1).
After binding at MOR and DOR, LP2 (1) and 2S-LP2 (5) are able to activate, with
different efficacy, G-protein pathway over β-arrestin 2, behaving as biased agonist at
MOR and mainly at DOR. It has been demonstrated that the G-protein pathway
induces analgesia, while the β-arrestin pathway is responsible for the opioid-related
adverse reactions [18,19]. Recently, G-protein biased opioid agonists were identified
and the relationship between in vitro bias profile and in vivo analgesia and side
effects were highlighted [20-22]. Thus, the 2S-LP2 (5) biased multitarget MOR/DOR
agonist could provide a safer treatment opportunity.
2
.4. Tail Flick Test
The antinociceptive effect of 2R-LP2 (4) and 2S-LP2 (5), i.p. administered, was
examined using mouse tail-flick test [23]. Both compounds produced a dose-
dependent analgesic effect compared to the group of mice treated with saline (*p<
0
.05 vs saline-treated mice).
In comparison to LP2 (1), featured by a maximal antinociceptive threshold reached
between 45 and 60 min after injection and a prolonged analgesic effect with an ED50
of 0.9 mg/kg [7], both isomers showed different onset and duration of the
antinociceptive effect and different ED . In vivo evaluation of 2R-LP2 (4)
5
0
established that the maximal antinociceptive activity was reached 60 min after i.p.
injection and lasted over 120 min after treatment (Fig. 5, panel A). Moreover, 2R-
1
4

ACCEPTED MANUSCRIPT
isomer (4) showed the antinociceptive effect at higher doses with an ED50 of 1.8
mg/kg i.p. (1.2-2.8, CL95%) (Fig. 5, panel B).
Fig. 5. (A) Time-course (min) of 2R-LP2 (4) induced antinociception measured by
TFL. (B) Analgesic dose-response curve ± SEM for compound 2R-LP2 (4) was
plotted at 60 min post-treatment. (C) Time-course (min) of 2S-LP2 (5) induced
antinociception measured by TFL. (D) Analgesic dose-response curve ± SEM for
compound 2S-LP2 (5) was plotted at 30 min post-treatment. Results are expressed in
seconds (s). Data are means ± SEM from 6 to 8 mice. *p<0.05 vs saline-treated mice.
A robust antinociceptive effect was achieved at very low doses of 2S-LP2 (5), with an
ED of 0.6 mg/kg i.p. (0.4-0.8, CL95%) (Fig. 5, panel D), that revealed its highest
5
0
effect already at 30 min post-administration (Fig. 5, panel C). At the tested doses, 2R-
LP2 (4) and 2S-LP2 (5) didn’t affect significantly behavior responses (i.e. locomotor
activity and sedation; unpublished data).
1
5

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Therefore, 2R-LP2 (4) and 2S-LP2 (5) exhibited a different profile in modulating
acute thermal nociception. The more potent activity of 2S-LP2 (5) translated the
MOR/DOR affinity (binding assay) and the full MOR/DOR agonist activity (BRET
assay) into the behavioral antinociceptive assay. Therefore, in vivo results confirmed
that S-configuration of N-substituent improved the pharmacological profile in
comparison with 2R-isomer (4). We demonstrated the same trend for 6,7-
benzomorphan 2S-OH (7) that showed an ED50 of 1.0 mg/kg i.p. (0.7-1.5, CL95%
)
better than the 2R-isomer (2R-OH, 6) with an ED of 1.3 mg/kg i.p. (0.9-2.1, CL95%)
5
0
[7].
2
R-LP2 (4) in vivo profile does not reflect in vitro data. Indeed, 2R-LP2 (4) exhibited
only 3-times difference in antinociceptive efficacy than 2S-LP2 (5) but about 25-times
less affinity for MOR, and about 58-times less affinity for DOR, resulting a very
weak agonist at MOR and inactive at DOR. This behavior could be due to 2R-LP2 (4)
pharmacokinetic properties or stability [1,2,24].
3
. Conclusion
The pivotal role of the stereocenter at the N-substituent of the 6,7-benzomorphan
scaffold was pointed out combining synthetic and pharmacological approaches. It is
well known that the stereochemistry could affect pharmacological parameters such as
potency, intrinsic efficacy and binding affinity in ligand-opioid receptor interaction.
The development of a single isomer in place of a previous racemic mixture, a process
known as “chiral switching”, could provide potential advantages such as an improved
therapeutic index, decreased side-effects, a faster onset of action and a reduced
liability for drug-drug interactions [25].
2
R-(4) and 2S-LP2 (5) were featured by a qualitatively similar but quantitatively
different pharmacological fingerprint in agreement with the importance of
1
6

ACCEPTED MANUSCRIPT
stereoselective drug-opioid receptor interaction. Specifically, 2S-LP2 (5) showed an
antinociceptive effect at a very low dose with potency 1.5- and 3-times increased in
comparison to LP2 (1) and R-antipode (4), respectively. The in vivo effect of 2S-LP2
(5) was consistent with its MOR and DOR affinity profile combined to the improved
multitarget MOR/DOR full agonist profile. Moreover, 2S-LP2 (5) resulted a biased
MOR/DOR agonist. It has been demonstrated that biased opioid ligands selectively
activate G-protein signalling leading to more effective antinociceptive drugs with
fewer side effects. Such biased ligands could have a major impact on modern drug
discovery and represent a new strategy for the development of more tolerated drug
candidates [26,27]. Indeed, their functional selectivity for G-protein signalling and
reduced β-arrestin 2 recruitment could be consistent with their effectiveness in
chronic pain conditions management [28,29]. Thus, the advantageous
pharmacological profile of 2S-LP2 (5) could be the result of its capability to
simultaneously target MOR and DOR, that it has been widely demonstrated to be a
valid strategy to obtain an improved antinociceptive effect coupled with an improved
tolerability, combined with its capability to selectively promote the G-protein
signalling.
4
4
4
. Experimental section
.1. Chemistry
.1.1. General methods
General methods are reported in Supporting Information. All reported compounds had
a purity of at least 95%.
4
.1.2. General procedure for the synthesis of tosylate intermediates (2-3)
1
7

ACCEPTED MANUSCRIPT
The synthesis of tosylate intermediates 2-3 was performed as previously reported [7]
using (R)-(−)-2-methoxy-2-phenylethanol or (S)-(+)-2-methoxy-2-phenylethanol (see
Supporting Information).
4
.1.2.1. (R)-2-methoxy-2-phenylethyl 4-methylbenzenesulfonate (2). Waxy solid
ꢁ
ꢂ
(
94%). Mp: 52-55 °C. [α] ꢃꢃ= − 42.9° (c 1.05, CHCl ). IR (CHCl ) 3019, 2943, 1598,
ꢀ
3
3
-1 1
1
456, 1362, 1215, 1176 cm . H NMR (200 MHz, CDCl ): δ 7.76-7.72 (m, 2H, CH
3
Ts), 7.32-7.26 (m, 7H, CH Ts and aryl), 4.42-4.39 (m, 1H, CH-O), 4.09-4.03 (m, 2H,
1
3
CH ), 3.23-3.20 (t, 3H, -OCH ), 2.44 (s, 3H, CH ). C NMR (50 MHz, CDCl ) δ
2
3
3
3
1
2
44.79, 136.63, 132.91, 129.69, 128.62, 128.55, 127.83, 126.85, 81.15, 72.79, 56.98,
1.46 (see Supporting Information Figure S2-S4).
.
4
.1.2.2. (S)-2-methoxy-2-phenylethyl 4-methylbenzenesulfonate (3). Waxy solid (74%).
ꢁ
ꢂ
Mp 54-55 °C. [α] ꢃꢃ= + 42.2° (c 1.02, CHCl ). IR (CHCl ) 3019, 2934, 1598, 1455,
ꢀ
3
3
-
1 1
1
7
3
1
361, 1215, 1176 cm . H NMR (200 MHz, CHCl ) δ 7.76-7.72 (m, 2H, CH Ts),
3
.43-7.22 (m, 7H, CH Ts and aryl), 4.44-4.39 (m, 1H, CH-O) 4.09-4.03 (m, 2H, CH2),
1
3
.23-3.21 (t, 3H, -OCH ), 2.44 (s, 3H, CH ). C NMR (50 MHz, CHDl ) δ 144.67,
3
3
3
36.73, 132.92, 129.72, 128.65, 128.57, 127.86, 126.88, 81.12, 72.85, 57.01, 21.59.
see Supporting Information Figure S5-S7).
(
4
.1.3. General procedure for the synthesis of N-Substituted (−)-cis-N-normetazocine
diastereomers (4-5)
Compounds 4-5 were obtained as previously reported [7] (see Supporting
Information).
1
8

ACCEPTED MANUSCRIPT
4
.1.3.1.
(2R,6R,11R)-3-((R)-2-methoxy-2-phenylethyl)-6,11-dimethyl-1,2,3,4,5,6-
hexahydro-2,6-methanobenzo[d]azocin-8-ol (4). White solid (62 %). Mp 176-180 °C.
ꢁ
ꢂ
1
[
α] ꢃꢃ= − 53.7° (c 1.05, EtOH). H NMR (200 MHz, CDCl , free base) δ 7.32-7.19
ꢀ
3
(
m, 5H, CH aryl), 6.83 (d, 1H, J = 8.2 Hz, CH benzomorphan), 6.65 (d, 1H, J = 2.2
Hz, CH benzomorphan), 6.55 (dd, 1H, J = 8.2, 2.2 Hz, CH benzomorphan), 4.35 (d,
H, CH-O), 3.13 (s, 3H, -OCH ), 2.89-2.55 (m, 6H, CH, CH benzomorphan and
1
3
2
CH ), 2.23-2.11 (m, 1H, CH benzomorphan), 1.90-1.82 (m, 2H, CH benzomorphan),
2
2
1
.24 (s, 3H, CH benzomorphan), 1.18-1.10 (m, 1H, CH benzomorphan), 0.73 (d, 3H,
3
1
3
CH benzomorphan). C NMR (50 MHz, CDCl , free base) δ 154.29, 143.05, 141.07,
3
3
1
4
28.33, 128.11, 127.66, 127.50, 126.66, 113.06, 112.32, 81.68, 62.12, 58.7, 56.60,
6.74, 41.12, 40.33, 36.06, 25.29, 23.89, 14.11(see Supporting Information Figure S8-
+
S9). MS m/z [M] 351.4. Anal (C H NO ·HCl) C, H, N (see Supporting Information
2
3
29
2
Table S2).
4
.1.3.2.
(2R,6R,11R)-3-((S)-2-methoxy-2-phenylethyl)-6,11-dimethyl-1,2,3,4,5,6-
hexahydro-2,6-methanobenzo[d]azocin-8-ol (5). White solid (60 %). Mp 177-180 °C;
ꢁ
ꢂ
1
[
α] ꢃꢃ= + 68.3° (c 1.05, EtOH). H NMR (200 MHz, CDCl , free base) δ 7.25-7.19
ꢀ
3
(
m, 5H, CH aryl), 6.83 (d, 2H, J = 8.2 Hz, CH benzomorphan), 6.65 (d, 1H, J = 2.0
Hz, CH benzomorphan), 6.55 (dd, 1H, J = 8.2, 2.2 Hz, CH benzomorphan), 4.42 (d,
H, CH-O), 3.15 (s, 3H, -OCH ), 2.97-2.55 (m, 6H, CH, CH benzomorphan and
1
3
2
CH ), 2.11-1.85 (m, 3H, CH and CH benzomorphan), 1.25 (s, 3H, CH
3
2
2
benzomorphan), 1.18-1.15 (m, 1H, CH benzomorphan), 0.76 (m, 3H, CH3
1
3
benzomorphan). C NMR (50 MHz, CDCl , free base) δ 154.51, 142.79, 140.55,
3
1
4
28.43, 128.15, 127.86, 127.76, 126.57, 113.16, 112.25, 81.61, 61.88, 58.79, 56.71,
6.02, 41.05, 40.31, 35.91, 25.18, 24.05, 14.01(see Supporting Information Figure
1
9

ACCEPTED MANUSCRIPT
+
S10-S11). MS m/z [M] 351.4. Anal (C H NO ·HCl) C, H, N (see Supporting
2
3
29
2
Information Table S2).
4
4
.2. Receptor binding assays
.2.1. Drugs and reagents
3
3
3
Radioligands [ H]-DAMGO, [ H]-DPDPE and [ H]-(+)-U69,593 were purchased by
Perkin-Elmer Life Sciences (Boston, MA, USA). Naloxone hydrochloride was
purchased from Tocris Bioscience (Bristol, United Kingdom).
4
.2.2. Competitive Radioligand Binding Assay
MOR, DOR and KOR binding experiments were performed on rat or guinea pig brain
membranes according to the experimental protocol described by Spetea et al. [30]
without modifications, as reported elsewhere [31]. Protein concentration was
determined by Lowry’s method using bovine serum albumin as standard [32].
Binding experiments at MOR and DOR were carried out by incubating 0.4 mg/mL
and 0.5 mg/mL of rat brain membrane proteins, respectively for 45 min at 35 °C
3
3
either with 1 nM [ H]-DAMGO (48.4 Ci/mM) or 2 nM [ H]-DPDPE (52.7 Ci/mM) in
5
0 mM Tris-HCl (pH 7.4). Regarding KOR binding assays, guinea pig brain
3
membranes (0.5 mg/mL) were incubated for 30 min at 30 °C with 1 nM [ H]-(+)-
U69,593 (42.69 Ci/mM). Test compounds were added in concentration ranging from
−
5
−11
1
0
to 10
M. Nonspecific binding was assessed in the presence of 10 µM of
unlabeled naloxone. The reaction was terminated by filtering the solution through
Whatman GF/B glass fiber filters, which were presoaked for 1h in a 0.5%
polyethylenimine solution. Filters were washed with ice cold buffer (2×4 mL), dried,
soaked in 4 mL of "Ultima Gold MV" scintillation cocktail and counted on a
Beckman LS 6500 liquid scintillation counter.
2
0

ACCEPTED MANUSCRIPT
4
.2.3. Data analysis
K values were calculated using nonlinear regression analysis to fit a logistic equation
i
to the competition data using GraphPad Prism version 5.0 (GraphPad Software In.,
San Diego, USA).
4
4
.3. Functional assay
.3.1. Drugs and reagents
All cell culture media and supplements were from Invitrogen (Thermo Fisher
Scientific Inc. MA, USA). All other reagents were from Sigma Chemical Co. (Poole,
U.K.) and were of the highest purity available. DADLE was from Sigma Chemical Co.
(
Poole, U.K.), DADLE was solubilized in bidistilled water, while compounds 1, 4 and
were solubilized in DMSO (5% v/v) at a final concentration of 10 mM. Stock
solutions of ligands were stored at -20 °C.
5
4
.3.2. BRET assay
SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium
(
DMEM)/HAMS F12 (1:1) supplemented with 10% fetal bovine serum, penicillin G
100 units/mL), streptomycin (100 µg/mL), L-glutamine (2 mM), fungizone (1
(
µg/mL), geneticin (G418; 400 µg/mL) and hygromycin B (100 µg/mL) in a
humidified atmosphere of 5% CO at 37 °C. Cell lines permanently co-expressing the
2
different pairs of fusion proteins (MOR-RLuc/Gβ1-RGFP, MOR-RLuc/β-arrestin 2-
RGFP, DOR-RLuc/Gβ1-RGFP, and DOR-RLuc/β-arrestin 2-RGFP) were prepared
using the pantropic retroviral expression system by Clontech as described previously
[33]. For G-protein experiments enriched plasma membrane aliquots from transfected
cells were prepared by differential centrifugation; cells were detached with
PBS/EDTA solution (1 mM, pH 7.4 NaOH) then, after 5 min 500 g centrifugation,
2
1

ACCEPTED MANUSCRIPT
Dounce-homogenized (30 strokes) in cold homogenization buffer (Tris 5 mM, EGTA
1
mM, DTT 1 mM, pH 7.4 HCl) in the presence of sucrose (0.32 M). Three following
centrifugations were performed at 10 min 1000 g (4 °C) and the supernatants kept.
Two 20 min 24,000 g (4 °C) subsequent centrifugations (the second in the absence of
sucrose) were performed for separating enriched membranes that after discarding the
supernatant were kept in ultrapure water at -80 °C [34]. The protein concentration in
membrane preparations was determined using the QPRO-BCA kit (Cyanagen Srl,
Bologna, IT) and the spectrophotometer Ensight (Perkin Elmer, Waltham, US).
Luminescence in membranes and cells was recorded in 96-well white opaque
microplates (PerkinElmer, Waltham, MA, USA) using the luminometer Victor 2030
(PerkinElmer, Waltham, MA, USA). For the determination of receptor/G-protein
interaction, membranes (3 µg of protein) prepared from cells co-expressing MOR-
RLuc/Gβ1-RGFP or DOR-RLuc/Gβ1-RGFP were added to wells in DPBS. For the
determination of receptor/β-arrestin 2 interaction, cells co-expressing MOR-RLuc/β-
arrestin 2-RGFP or DOR-RLuc/β-arrestin 2-RGFP were plated 24h before the
experiment (100,000 cells/well). The cells were prepared for the experiment
substituting the medium with PBS with MgCl (0.5 mM) and CaCl (0.9 mM).
2
2
Coelenterazine at a final concentration of 5 µM was injected 15 minutes prior reading
the cell plate. Different concentrations of ligands in 20 µL of PBS - BSA 0.01 % were
added and incubated 5 min before reading luminescence. All the experiments were
performed at room temperature.
4
.3.3. Data analysis and terminology
The pharmacological terminology adopted in this paper were consistent with
IUPHAR recommendations [35]. All data were expressed as the mean ± standard
error of the mean (SEM of n experiments). For potency values 95% confidence limits
2
2

ACCEPTED MANUSCRIPT
(CL95%) were indicated. BRET data were calculated as BRET ratio between CPS
measured for the RGFP and RLuc light emitted using 510(10) and 460(25) filters
PerkinElmer, Waltham, MA, USA), respectively. Maximal agonist effects were
(
expressed as fraction of the DADLE maximal effects that was determined in every
assay plate. Agonist potencies were given as pEC i.e. the negative logarithm to base
5
0
1
0 of the molar concentration of an agonist that produces 50% of the maximal effect
of that agonist. Concentration-response curves to agonists were fitted to the classical
four-parameter logistic nonlinear regression model:
E
- baseline
max
LogEC50 −Log[compound)Hillslope
Effect = baseline+
(
1
+10
Curve fitting was performed using PRISM 6.0 (GraphPad Software In., San Diego,
USA). pEC50 was the concentration of agonist producing a 50% maximal response
and n was the Hill coefficient of the concentration-response curve to the agonist. CR
was the ratio between EC50 of ligand and EC50 of standard agonist (DADLE).
Maximal effects have been statistically analyzed with one-way ANOVA followed by
the Dunnett’s post hoc test for multiple comparisons; P values less than 0.05 were
considered statistically significant.
Bias factors were calculated by choosing DADLE as standard unbiased ligand. The
concentration response curves of each compound were fitted to the Black-Leff
operational model described by Nagi & Pineyro [36]:
ꢋ
ꢋ
[
ꢊ ]ꢌ ꢍ
ꢎ
ꢄꢅꢆꢇꢈꢉꢆꢅ =
ꢋ
ꢋ
ꢋ
[
ꢊ] ꢌ + ꢏ[ꢊ] + ꢐ ꢒ
ꢑ
where [A] is the agonist concentration, the maximal response of the system is given
by E , n is a fitting parameter for the slope, the affinity of the agonist is represented
m
by the equilibrium dissociation constant of the agonist-receptor complex (K ), and the
A
efficacy of the agonist is defined by τ. τ and K are descriptive parameters of intrinsic
A
2
3

ACCEPTED MANUSCRIPT
efficacy and binding affinity and may be directly obtained by fitting experimental
data to the operational equation and can be expressed as “transduction coefficients”
log(τ/K ). The relative efficiency of agonists producing activation of any pathways
A
can thus be quantified with a “normalized” transduction coefficient, namely
∆
log(τ/K ). Finally, the bias factors were calculated as difference between ∆log(τ/K )
A
A
values for a given agonist between the pathways (G protein and β-arrestin 2):
ΔΔlogꢏτ/KAꢒꢃꢏbiasꢃfactorꢒ = Δlogꢏτ/KAꢒG proteinꢃ– Δlogꢏτ/K ꢒ
A β-arrestin 2
Bias factors are expressed as the mean ± SEM of at least 5 independent experiments.
4
4
.4. In vivo pharmacology
.4.1. Animals
Male Swiss CB1 mice (Envigo Laboratories, S.Pietro al Natisone (UD)) weighing 25-
0 g were housed six to a cage. Animals were kept at a constant room temperature (25
1 °C) under a 12:12h light and dark cycle with free access to food and water. Each
3
±
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 Communities Council Directive and
National Regulations (CEE Council 86/609 and DL 116/92).
4
.4.2. Nociceptive Test
Nociception was evaluated by the radiant heat tail-flick test [37,38]. 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
temperature (25 ± 1 °C). The basal pre-drug latency was established between 3 and 5
s and was calculated as the average of the first three measurements, which were
2
4

ACCEPTED MANUSCRIPT
performed at 5 min intervals. A cutoff latency of 20 s was established to minimize
damage to the tail. All tested compounds were dissolved in pyrogen-free isotonic
saline (Baxter Healthcare, Deerfield, IL) and DMSO (5%) and were administered to
mice i.p. Post-treatment tail flick latencies (TFLs) were determined at 30, 60, 90, 120,
1
50 and 180 min after i.p. injection.
4
.4.2. Statistical Analysis
All values were presented as means ± SD. Intergroup comparisons were assessed
using an initial two-way analysis of variance (ANOVA) followed by Duncan’s
multiple range post-hoc test. Differences were considered significant when *p < 0.05.
Effective dose-50 (ED ) values were calculated using least-squares linear regression
5
0
analysis followed by calculation of 95% confidence limits (CL95%) by the method of
Bliss [39,40].
Conflicts of interest
The authors declare no other conflicts of interest.
Acknowledgements
This work was supported by University of Catania (PdR 2016-2018 - UPB
5
7722172104) to Lorella Pasquinucci. The authors gratefully acknowledge Fabbrica
Italiana Sintetici (Italy) for providing (±)-cis-N-normetazocine.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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