Novel N-Substituted Benzomorphan-Based Compounds: From MOR-Agonist/DOR-Antagonist to Biased/Unbiased MOR Agonists

Article abstract of DOI:10.1021/acsmedchemlett.9b00549

Modifications at the basic nitrogen of the benzomorphan scaffold allowed the development of compounds able to segregate physiological responses downstream of the receptor signaling, opening new possibilities in opioid drug development. Alkylation of the phenyl ring in the N-substituent of the MOR-agonist/DOR-antagonist LP1 resulted in retention of MOR affinity. Moreover, derivatives 7a, 7c, and 7d were biased MOR agonists toward ERK1,2 activity stimulation, whereas derivative 7e was a low potency MOR agonist on adenylate cyclase inhibition. They were further screened in the mouse tail flick test and PGE2-induced hyperalgesia and drug-induced gastrointestinal transit.

Full text of DOI:10.1021/acsmedchemlett.9b00549

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Letter
Novel N-substituted benzomorphan-based compounds: from
MOR-agonist/DOR-antagonist to biased/unbiased MOR agonists.
Lorella Giuseppina Pasquinucci, Carmela Parenti, M Carmen Ruiz-Cantero, Zafiroula
Georgoussi, Paschalina Pallaki, Enrique J Cobos, Emanuele Amata, Agostino Marrazzo,
Orazio Prezzavento, Emanuela Arena, Maria Dichiara, Loredana Salerno, and Rita Turnaturi
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00549 • Publication Date (Web): 28 Jan 2020
Downloaded from pubs.acs.org on January 30, 2020
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Novel N-substituted benzomorphan-based compounds: from MOR-
agonist/DOR-antagonist to biased/unbiased MOR agonists.
Lorella Pasquinuccia, Carmela Parentib*, M. Carmen Ruiz-Canteroc, Zafiroula Georgoussid, Paschalina
Pallakid, Enrique J. Cobosc, Emanuele Amataa, Agostino Marrazzoa, Orazio Prezzaventoa, Emanuela
Arenaa, Maria Dichiaraa, Loredana Salernoa, Rita Turnaturia*
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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, Viale A. Doria 6, 95125
Catania, Italy.
c Department of Pharmacology, Faculty of Medicine and Institute of Neuroscience, Biomedical Research Center, University
of Granada, Parque Tecnológico de Ciencias de la Salud, 18100 Armilla, Granada, Spain; Teófilo Hernando Institute for
Drug Discovery, 28029, Madrid, Spain.
d Laboratory of Cellular Signaling and Molecular Pharmacology, Institute of Biosciences and Applications, National Center
for Scientific Research "Demokritos", Ag. Paraskevi 15310, Athens, Greece.
* Corresponding authors.
E-mail address: cparenti@unict.it (Carmela Parenti)
E-mail address: rita.turnaturi@unict.it (Rita Turnaturi)
KEYWORDS Multitarget; dual-target; pain; SAR; benzomorphan; opioid.
ABSTRACT: Modifications at the basic nitrogen of the benzomorphan scaffold allowed the development of compounds able to
segregate physiological responses downstream of the receptor signaling, opening new possibilities in opioid drug development.
Alkylation of the phenyl ring in the N-substituent of the MOR-agonist/DOR-antagonist LP1, resulted in retention of MOR affinity.
Moreover, derivatives 7a, 7c and 7d were biased MOR agonists towards ERK1,2 activity stimulation, whereas derivative 7e was a
low potency MOR agonist on adenylate cyclase inhibition. They were further screened in the mouse tail flick test and PGE2-
induced hyperalgesia and drug-induced gastrointestinal transit.
During the last decade efforts were made to develop
effective multitarget opioid ligands as an alternative strategy
to overcome the typical side effects associated to opioid
selective agonists.1-3 For instance, valid analgesic effect with
lower propensity to produce tolerance and physical
dependence was reported for both dual MOR/DOR agonist4-6
and MOR agonist/DOR antagonist ligands given in persistent
pain models.7-9
Recently, the concept of biased agonists,10 able to
differentially activate GPCR downstream pathways, became a
new approach in the design of novel drug candidates. It was
reported that opioid compounds promoting G-protein signaling
produce analgesia, while β-arrestin recruitment is responsible
for opioid side effects such as constipation.11-13
Benzomorphan nucleus represents a versatile template14,15
for the development of a specific functional profile by
modifying N-substituent or 8-OH group. In this context, the
introduction of a tertiary N-Methyl-N-phenylethylamino group
as N-substituent conferred a MOR agonist profile in vitro and
in vivo (1, Figure 1).16 The replacement of the N-ethylamino
spacer with the N-acetamido one was detrimental for MOR,
DOR and KOR recognition,17 while an N-propanamido spacer
improved the opioid binding profile. In particular, LP1 (2,
Figure 1), with an N-phenylpropanamido substituent, resulted
in vitro and in vivo a potent MOR agonist/DOR antagonist18
able to counteract nociceptive pain and behavioral signs of
persistent pain with low tolerance-inducing capability.19,20 The
phenyl replacement with the bulkier N-naphthyl ring (3,
Figure 1), switched the MOR efficacy profile from agonism to
antagonism.21 Analogously, the increased steric hindrance of
the aromatic moiety with indoline, tetrahydroquinoline or
diphenylamine group affected the shift from MOR agonism to
antagonism.22 More recently, a dual MOR/DOR agonist,
endowed of a significant long-lasting antinociceptive effect,23-
25
was developed through the introduction of the short and
flexible 2R/S-methoxy ethyl spacer as N-substituent (LP2 4,
Figure 1). Moreover, the 2S diastereoisomer of LP2 was found
a potent G-protein biased MOR/DOR agonist with a 3-times
lower ED50 value.26
Since minor structural modifications often result in
significant changes in the pharmacological profile of opioid
ligands, we expanded our SAR studies by the synthesis of LP1
derivatives 7a-e, variously alkylated at the phenyl ring of the
N-propanamido substituent, and 11a-e, featured also by a
tertiary N-propanamido substituent. Finally, derivatives 14a-c,
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reflux, 3 h; b) NaBH4 (0.5 M solution in EtOH) reflux, 6 h; c) 3-
bearing a secondary or tertiary N-ethylamino spacer, were
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3
4
5
6
7
8
bromopropionyl chloride (1.5 eq), 4-(dimethylamino)pyridine
(DMAP) (0.47 eq), dry THF, rt, 3h; d) (–)-cis-(1R,5R,9R)-N-
normetazocine (1 eq), NaHCO3 (1.5 eq), KI, DMF, 65 °C, 20 h.
synthesized (Figure 1).
H3C
O
O
N
N
H
N
H
N
N
N
N-benzyl anilines 9a-e, obtained by reductive amination
with NaBH4, were acylated with 3-bromopropionyl chloride to
obtain the respective amides 10a-e. Derivatives 11a-e were
prepared according to the synthetic route shown in Scheme 2.
CH3
CH3
CH3
CH3
CH3
CH3
HO
HO
HO
1
2
(LP1)
3
R3
The N-(2-chloroethyl)anilines 13a-c were obtained by
alkylation with 1-bromo-2-chloroethane.29 Then, the next step
to get target derivatives 14a-c was carried out as reported in
Scheme 3. All newly synthesized compounds were
characterized by IR, 1H NMR, 13C NMR, and elemental
analysis.
R2
R4
O
9
R
R3
R2
N
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N
R1
*
O
N
N
R
N
CH3
R1
CH3
CH3
CH3
CH3
CH3
CH3
HO
HO
HO
4
(LP2)
7a-e, 11a-e
14a-c
R2
R
N
R3
R3
R2
Figure 1. Benzomorphan-based compounds structures.
Cl
a
According to the previously reported method,17,28 we
prepared derivatives 7a-e, 11a-e and 14a-c as reported in
Schemes 1-3. After cis-(±)-N-normetazocine resolution,27 the
target compounds 7a-e were obtained by alkylation of cis-(−)-
(1R,5R,9R)-N-normetazocine with the respective amides 6a-e
(Scheme 1).
R1
R1
HN
R
12a-c
13a-c
b
R
R3
R2
R
Bn
H
R1
H
R2
H
R3
H
N
R2
R4
H
N
N
R3
R4
Br
12a, 13a, 14a
12b, 13b, 14b
12c, 13c, 14c
R3
R2
R1
a
CH3
CH3
O
CH3
CH3
CH3
H
H
R1
R1
NH2
H
CH3
HO
5a-e
6a-e
14a-c
b
Scheme 3. Synthesis of N-substituted normetazocine derivatives
14a-c. Reagents and conditions: a) 1-bromo-2-chloroethane (0.3
eq), CH3CN, 110 °C in sealed tube, 10 min; b) (–)-cis-
(1R,5R,9R)-N-normetazocine (1 eq), NaHCO3 (1.5 eq), KI, DMF,
50 °C, 12 h.
R3
R2
R1
R2
R3
R4
R4
O
5a, 6a, 7a
5b, 6b, 7b
5c, 6c, 7c
5d, 6d, 7d
5e, 6e, 7e
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
CH3
CH3
H
N
H
CH3
CH3
H
R1
N
CH3
CH3
7a-e
To investigate the SAR of synthesized novel derivatives,
their binding and efficacy profile at MOR, DOR and KOR was
explored. Binding at MOR, DOR and KOR was evaluated by
competitive displacement of [3H]DAMGO, [3H]DPDPE and
[3H]U69,593, respectively.30 Ki values of derivatives 7a-e,
11a-e and 14a-c, calculated using nonlinear regression
analysis (GraphPad Prism), are listed in Table 1.
C2H5
H
HO
H
CH3
Scheme 1. Synthesis of N-substituted normetazocine derivatives
7a-e. Reagents and conditions: a) 3-bromopropionyl chloride (1.5
eq), 4-(dimethylamino)pyridine (DMAP) (0.47 eq), dry THF, rt,
3h; b) (–)-cis-(1R,5R,9R)-N-normetazocine (1 eq), NaHCO3 (1.5
eq), KI, DMF, 65 °C, 20 h.
The synthesized derivatives showed a broad range of
binding affinity for MOR (Ki= 7.4-1,540 nM) and low or no
affinity for DOR and KOR. Derivatives 7a and 7e, having
methyl groups in position 2’,6’ and 2’,5’ respectively,
possessed the highest MOR affinity, followed by derivatives
7b, 7d and 7c having slight less affinity for this receptor. A
third methyl group in position 4’ (7b), as well as an ethyl
group in position 6’ (7d), reduced MOR affinity by 6- and 2-
times compared to 7a and 7e. The dimethyl alkylation in
position 2’ and 4’ (7c) resulted in a worse MOR binding
profile. Thus, methylation in orto and meta is well tolerated
while the para-methylation was unfavorable. In MOR-ligand
interaction the negative influence of para substitution, with
both electron-withdrawing or electron-donor groups, was
outlined.22 A worse DOR and KOR binding profile was
recorded for derivatives 7a-e. Indeed, in comparison to LP1
their DOR and KOR affinity were from 7- to 69-times and
from 7- to 61-times lower, respectively. The introduction of a
benzyl pendant at the amidic nitrogen (11a) and the
R2
R2
R1
N
R2
O
a, b
c
R3
R1
R3
R1
Br
NH2
HN
R3
8a-e
10a-e
9a-e
d
R2
R3
O
R1
R2
H
R3
H
N
8a, 9a, 10a, 11a
8b, 9b, 10b, 11b
8c, 9c, 10c, 11c
8d, 9d, 10d, 11d
8e, 9e, 10e, 11e
H
R1
N
CH3
CH3
CH3
CH3
H
CH3
CH3
H
CH3
CH3
CH3
CH3
H
HO
11a-e
C2H5
Scheme 2. Synthesis of N-substituted normetazocine derivatives
11a-e. Reagents and conditions: a) benzaldehyde (1 eq), MeOH,
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simultaneous phenyl ring methylation (11b-e) resulted derivatives 7a-e. The steric hindrance at the amine nitrogen in
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2
3
4
detrimental for opioid binding affinity, mainly at DOR and
KOR (Table 1). Such modifications hindered the ligands to
adopt a compatible ligand-receptor conformation. Derivatives
14a-c, featured by an N-ethylamino spacer, showed MOR
affinity higher than derivatives 11a-e and lower than
derivative 14a resulted in a dramatically loss of opioid
receptor affinity, mainly at MOR, respect to compound 1
(KiMOR = 6.1 nM), featured by a tertiary N-Methyl-N-
phenylethylamino group.
5
6
7
8
9
Table 1. Opioid receptor binding affinity of LP1 (2) derivatives 7a-e, 11a-e and 14a-c.
R3
R2
R4
O
R
R3
R2
N
N
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R1
N
N
R
R1
CH3
CH3
CH3
CH3
HO
HO
14
7
11
and
Ki (nM) ± SEM [a,b]
DOR
Cmp
7a
R
H
H
H
H
R1
CH3
CH3
R2
H
CH3
CH3
H
H
H
H
CH3
CH3
H
H
CH3
H
R3
H
H
H
H
CH3
H
H
H
H
R4
CH3
CH3
H
C2H5
H
MOR
7.4 ± 0.75
43.7 ± 2
KOR
252 ± 10
313 ± 15
340 ± 13
320 ± 11
480 ± 22
> 5,000
> 3,000
444 ± 24
> 2,000
151 ± 6
> 3,000
> 1,000
> 2,000
31 ± 1.300
110 ± 6.00
277 ± 12
> 2,000
7b
7c
7d
7e
11a
11b
11c
11d
11e
14a
14b
14c
1[c]
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
H
20.8 ± 1
14.9 ± 0.92
7.9 ± 0.65
606 ± 34
400 ± 24
504 ± 27
165 ± 7
1,540 ± 59
244 ± 12
129 ± 10
182 ± 13
6.1 ± 0.50
0.83 ± 0.05
0.90 ± 0.04
-
474 ± 20
198 ± 8
478 ± 22
> 5,000
> 5,000
> 5,000
> 5,000
> 5,000
> 1,000
> 5,000
> 5,000
147 ± 5.700
29 ± 1.00
-
H
Bn
Bn
Bn
Bn
Bn
Bn
H
H
CH3
CH3
H
C2H5
H
H
H
H
CH3
CH3
CH3
H
H
H
2 (LP1)[d]
DAMGO
Naltrindole
U50,488
[a]
-
0.83 ± 0.04
-
0.27±0.03
-
-
[b]
Values are means ± SEM of three separate experiments, each carried out in duplicate.
[Ki values were obtained as
[3H]DAMGO displacement for MOR, [3H]DPDPE displacement for DOR, and [3H]U69,593 displacement for KOR. [c] Ref. [16].
[d] Ref. [17].
To examine the functional significance of derivatives 7a-e,
displaying the best MOR binding profile, we tested their
ability to affect agonist-mediated AC inhibition. Opioid
receptors signal through Gi/Go proteins to inhibit AC,31-34
which is known to be one of their major pathway to induce
analgesia.12 For that reason, HEK293 cells stably expressing
the MOR were treated with increasing concentrations of
derivatives 7a-e, and the levels of forskolin-stimulated AC
activity were tested. Derivatives 7a-7d were unable to inhibit
AC even at high concentrations up to 10-5 M (data not shown).
Treatment of HEK293 cells with compound 7e resulted with
50±3 % inhibition of cAMP accumulation at a concentration
of 10 μΜ (Figure 2). However, this effect was much lower
than that detected with LP1 (Figure 2). These results suggest
that derivative 7e could be considered as an effective MOR
agonist with low potency on AC inhibition. To further identify
whether derivatives 7a, 7c, 7d and 7e behave as MOR agonists
we measured alterations of ERK1,2 phosphorylation mediated
by these derivatives upon MOR activation. It was previously
demonstrated that opioid receptors stimulate ERK1,2 via
pertussis toxin-sensitive Gi/o-protein signaling mechanism32-34
and regulate additional effectors by interacting with other
scaffolding proteins.35 In addition, it is well known that
signaling of opioid receptors through ß-arrestin pathway leads
to ERK1,2 activation.36 Serum-starved HEK293 cells
expressing stably the MOR were challenged with derivatives
7a, 7c, 7d and 7e with different time intervals ranging from 5-
15 min. As shown in Figure 3, western blotting with a specific
phospho-ERK1,2 antibody revealed an increase in ERK1,2
phosphorylation reaching a peak within 5 min administration
for tested derivatives, which decreased after 15 min compound
exposure. The same pattern of increased ERK1/2
phosphorylation was also shown for LP1. However, the levels
of ERK1,2 phosphorylation mediated by derivative 7e retain
even after 15 min of receptor stimulation. These results
suggest that 7a, 7c, 7d and 7e act as potent MOR agonists with
derivatives 7a, 7c, 7d exerting biased agonist properties
towards ERK1,2 activity stimulation.
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To determine if in vitro biased and unbiased G-protein profile
administration of PGE2 do not influence gastrointestinal
transit (Figure 6).
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7
8
could reflect what is happening in animal pain models,
derivatives 7a and 7e were further screened in the mouse-tail
flick test. 7a and 7e, in a dose range from 2.5 up to 7.5 mg/kg
i.p., did not significantly modify TFLs, during the entire time
of observation (90 min, Figure 4 panel A and B, respectively)
compared to the group of mice treated with saline (p > 0.05 vs
saline-treated mice).
Morphine already induced significant gastrointestinal transit
inhibition at a dose devoid of antihyperalgesic effect (1
mg/kg), and this effect dose-dependently increased reaching
values of distance travelled by the charcoal meal as low as
10.5 cm at the highest tested dose of the opioid (3 mg/kg)
(compare Figures
5
and 6). Instead, LP1 inhibited
gastrointestinal transit only at the highest dose tested (4
mg/kg), which induced a maximal antihyperalgesic effect
(compare Figures 5 and 6). These results indicate that the
MOR agonist/DOR antagonist LP1 has a more favorable
safety profile than morphine.
Considering that PGE2-induced hyperalgesia is well
known to be triggered by cAMP accumulation and the
consequent protein kinase A activation,37 we selected this
assay as a suitable index of the behavioral effects of the
derivatives 7a, 7c, 7d and 7e through cAMP inhibition. The
administration of PGE2 induced a marked decrease in the
withdrawal latency of the injected paw to heat stimulation,
in comparison to saline-injected controls, denoting the
development of thermal hyperalgesia. There were no
statistically significant differences between the values
obtained in the paw contralateral to PGE2 or saline (data
not shown). Both morphine (1-3 mg/kg, s.c.) and LP1 (1-4
mg/kg, s.c.) induced a dose-dependent increase in paw
withdrawal latency in PGE2-treated mice, reaching values
similar to control animals (i.e. a full antihyperalgesic
effect) (Figure 5) at the highest doses tested. In
contradiction, the administration of 7a, 7c, 7d or 7e (8-16
mg/kg, s.c.) did not induce any significant antihyperalgesic
effect (Figure 5). These results are in agreement with the
inhibition of cAMP accumulation by morphine and LP1
and the absence of any effect detected by 7a, 7c, 7d or 7e
in the same experiments.
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Figure 2. Effect of derivative 7e and LP1 on MOR-mediated
cAMP accumulation. The inhibition of cAMP accumulation was
measured as described in Material and methods in HEK293 cells
stably expressing the MOR in the presence of various
concentrations of LP1 and 7e, in response to treatment with 50
μΜ forskolin. The IC50 values of LP1 and 7e are 4.8 x10-9 ± 0.5
M and 2.4 x10-4 ± 0.83 M respectively. Data represent as cAMP
accumulation (% of maximum) and are the average of ± SEM of
triplicate determinations from three independent experiments.
As constipation is a known opioid-induced side effect
dependent on the activation of ß-arrestin pathway,12,13 we also
tested the effects of derivatives 7a, 7c, 7d and 7e on
gastrointestinal transit. Immediately after the evaluation of the
behavioral responses to heat stimulus, mice received
intragastrically an activated charcoal solution. The charcoal
meal travelled about 30 cm of the small intestine in either
mouse treated i.pl. with saline or PGE2, indicating that the
Figure 3. Effect of derivatives 7a, 7c, 7d and 7e on ERK1,2 phosphorylation mediated upon MOR activation. Stably transformed HEK293
cells expressing the MOR were challenged with 1 μΜ of derivatives 7a, 7c, 7d and 7e for 5, 10 and 15 min and cell lysates were resolved
in SDS-PAGE (10%). The ERK1,2 phosphorylation mediated by DAMGO and LP1 (1 μΜ) after 5 min exposure was used as positive
controls. Phosphorylation of ERK1,2 was abolished upon pretreatment of the cells with naloxone (10 μΜ, 30 min), prior to 5 min DAMGO
administration (negative control). The phosphorylated-ERK1,2 was visualized by immunoblotting with a phosphor-ERK1,2 (upper panel).
Equal loading was verified by stripping and reprobing the PVDF membrane with a specific α-tubulin antibody (lower panel). Results are
representative of three independent experiments.
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Figure 4. Time-course (min) of derivatives 7a and 7e-induced antinociceptive effect measured by tail flick test (panel A and B,
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respectively). Results are expressed in seconds (s). Data are means ± SEM from 6 to 8 mice. *P < 0.05 vs saline-treated mice.
Figure 5. Effects of morphine, LP1 and derivatives 7a, 7c, 7d and
7e on PGE2-induced heat hyperalgesia. The results represent the
latency to hindpaw withdrawal in response to radiant heat in mice
treated intraplantarly (i.pl.) with PGE2 or saline (S). Mice were
tested (in the paw injected with PGE2 or its solvent) 10 min after
the intraplantar injection. Morphine, LP1, 7a, 7c, 7d, 7e or their
solvent (S) were administered subcutaneously (s.c.) 20 min before
the i.pl. injection. Statistically significant differences between the
values obtained in mice i.pl. injected with saline and PGE2:
*p<0.05, **pꢀ<ꢀ0.01, and between the values obtained in mice
treated with PGE2 alone or associated with morphine or LP1:
##pꢀ<ꢀ0.01 (one-way ANOVA followed by Bonferroni test).
Figure 6. Effects of morphine, LP1 and derivatives 7a, 7c, 7d and
7e on gastrointestinal transit. Immediately after the evaluation of
PGE2-induced hyperalgesia [i.e. 30 minutes after the
subcutaneous (s.c.) administration of morphine, LP1, 7a, 7c, 7d or
7e or saline (S)], mice were given a 0.5% charcoal suspension
intragastrically. Transit of the charcoal was measured 30 min after
its ingestion. Each bar and vertical line represents the mean ±
SEM of values obtained in 6-7 mice. Statistically significant
differences between the values obtained in saline-treated group
and mice treated with morphine or LP1: *p<0.05, **pꢀ<ꢀ0.01
(one-way ANOVA followed by Bonferroni test).
In summary, we have repurposed the N-modified
benzomorphan scaffold to develop novel LP1 derivatives to
further understanding the requirements for MOR interaction.
A secondary amido N-substituent, as well as an orto- and/or
orto/meta-methyl introduction to the phenyl ring, resulted in
retention of MOR agonism. Derivatives 7a, 7e, 7c and 7d
resulted MOR agonists with a peculiar functional profile,
being 7e a biased MOR agonist, able to stimulate G-protein
pathway, and 7a, 7c and 7d unbiased MOR agonists, able to
stimulate ERK1,2 activity.
However, the administration of 7a, 7c, 7d or 7e (8-16
mg/kg, s.c.) did not alter gastrointestinal transit distances, as
the charcoal meal travelled approximately 30 cm of the small
intestine in all cases (Figure 6). Although these derivatives
were able to act in vitro as opioid agonists mediating ERK1,2
phosphorylation, someone could assume that they may
activate the ß-arrestin pathway and thus are unable to decrease
gastrointestinal transit. Animals administered with these
derivatives did not show either a Straub tail response (data not
shown), which is a known centrally-induced opioid effect.38
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ACKNOWLEDGMENT
Page 6 of 17
ERKs activation can be facilitated by distinct pathways
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mediated by G-proteins or β-arrestins dependent pathways.
Fast activation of ERKs (2 min) is usually mediated by G-
proteins resulted in the nuclear translocation of
phosphorylated ERKs, whereas a slower activation of ERKs
(10 min), the time sets that was used in our studies, is
mediated by β-arrestins and resulted in the cytosolic retention
of the phosphorylated ERKs. Different MOR agonists activate
ERKs via β-arrestins dependent or independent pathways, thus
resulting in differential subcellular localization of activated
ERKs and altering their effect on gene transcription driven by
the agonist [36]. In addition to opioid receptor-mediated
activation of ERKs via β-arrestins, β2AR stimulation resulted
in ERKs activation via a β-arrestin dependent pathway.39
This work was supported by University of Catania (PdR 2016-
2018) to Lorella Pasquinucci. The authors acknowledge Fabbrica
Italiana Sintetici (Italy) for cis-(±)-N-normetazocine and Dr
Raffaele Morrone (CNR, ICB Catania) for MS spectra. We
acknowledge support by the OPENSCREEN-GR (MIS 5002691)
funded by the Operational Programme NSRF 2014-2020 and co-
financed by Greece and the European Union (European Regional
Development Fund). We also acknowledge Spanish Ministry of
Economy and Competitiveness (MINECO, grant SAF2016-
80540-R). MC Ruiz-Cantero was supported by an FPU grant from
the Spanish Ministry of Education, Culture, and Sports.
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ABBREVIATIONS
Compounds provided with functional selectivity could open
new possibilities in opioid drug development. Indeed, biased
MOR agonists toward G-protein are analgesics with low side
effects incidence while biased MOR agonist toward β-arrestin
could be useful to treat hypermotility disorders.
MOR, mu opioid receptor; DOR, delta opioid receptor; GPCR, G-
protein coupled receptor; KOR, kappa opioid receptor; Ki,
inhibition constant; AC, adenylyl cyclase; HEK293, human
embryonic kidney 293; ERK1,2, extracellular regulated kinase 1
and 2; TFLs, tail flick latencies; PGE2, Prostaglandin E2; β-
arrestins, β2AR, β-adrenergic receptor; s.c., subcutaneous; i.p.,
intraperitoneal; i.pl. intraplantar.
Besides the notable antinociceptive and antihyperalgesic
effect, the dual-target profile of LP1 conferred a safer profile
resulting in a less gastrointestinal transit inhibition than
morphine. In accordance with in vitro data, synthesized
derivatives did not elicit any significant antinociceptive and
antihyperalgesic effect. Differences of pharmacokinetic could
explain the low correlation between the in vivo inability to
decrease gastrointestinal transit and the in vitro evidence. In
conclusion, we found hits able to segregate physiological
responses downstream of the receptor signaling that could be
optimized.
REFERENCES
(1) Zhu, L.; Cui, Z.; Zhu, Q.; Zha, X.; Xu, Y. Novel Opioid Receptor
Agonists with Reduced Morphine-like Side Effects. Mini Rev. Med.
Chem. 2018, 18, 1603−1610.
(2) Turnaturi, R.; Aricò, G.; Ronsisvalle, G.; Parenti, C.; Pasquinucci, L.
Multitarget opioid ligands in pain relief: New players in an old game. Eur.
J. Med. Chem. 2016, 108, 211−228.
(3) Turnaturi, R.; Aricò, G.; Ronsisvalle, G.; Pasquinucci, L.; Parenti, C.
Multitarget Opioid/Non-opioid Ligands: A Potential Approach in Pain
Management. Curr. Med. Chem. 2016, 23, 4506−4528.
(4) Nastase, A. F.; Griggs, N. W.; Anand, J. P.; Fernandez, T. J.; Harland,
A. A.; Trask, T. J.; Jutkiewicz, E. M.; Traynor, J. R.; Mosberg, H. I.
Synthesis and Pharmacological Evaluation of Novel C-8 Substituted
Tetrahydroquinolines as Balanced-Affinity Mu/Delta Opioid Ligands for
the Treatment of Pain. ACS Chem. Neurosci. 2018, 9, 1840−1848.
(5) Podolsky, A. T.; Sandweiss, A.; Hu, J.; Bilsky, E. J.; Cain, J. P.;
Kumirov, V. K.; Lee, Y. S.; Hruby, V. J.; Vardanyan, R. S.; Vanderah, T.
W. Novel fentanyl-based dual μ/δ-opioid agonists for the treatment of
acute and chronic pain. Life Sci. 2013, 93, 1010−1016.
(6) Lei, W.; Vekariya, R. H.; Ananthan, S.; Streicher J. M. A Novel Mu-
Delta Opioid Agonist Demonstrates Enhanced Efficacy With Reduced
Tolerance and Dependence in Mouse Neuropathic Pain Models. J. Pain
2019, Jun 12. pii: S1526-5900(19)30745-X.
(7) Harland, A. A.; Yeomans, L.; Griggs, N. W.; Anand, J. P.; Pogozheva,
I. D.; Jutkiewicz, E. M.; Traynor, J. R.; Mosberg, H. I. Further
Optimization and Evaluation of Bioavailable, Mixed-Efficacy μ-Opioid
Receptor (MOR) Agonists/δ-Opioid Receptor (DOR) Antagonists:
Balancing MOR and DOR Affinities. J. Med. Chem. 2015, 58, 8952−69.
(8) Anand, J. P.; Kochan, K. E.; Nastase, A. F.; Montgomery, D.; Griggs,
N. W.; Traynor, J. R.; Mosberg, H. I.; Jutkiewicz, E. M. In vivo effects of
μ-opioid receptor agonist/δ-opioid receptor antagonist peptidomimetics
following acute and repeated administration. Br. J. Pharmacol. 2018, 175,
2013−2027.
(9) Dietis, N.; Niwa, H.; Tose, R.; McDonald, J.; Ruggieri, V.: Filaferro,
M.; Vitale, G.; Micheli, L.; Ghelardini, C.; Salvadori, S.; Calo, G.;
Guerrini, R.; Rowbotham, D. J.; Lambert, D. G. In vitro and in vivo
characterization of the bifunctional μ and δ opioid receptor ligand UFP-
505. Br. J. Pharmacol. 2018, 175, 2881−2896.
ASSOCIATED CONTENT
Supporting Information
Experimental procedures for the synthesis and characterization of
the compounds, radioligand binding, adenylyl cyclase inhibition,
ERK1,2 activations, tail-flick, PGE2-induced hyperalgesia, drug-
induced gastrointestinal transit inhibition assays. This material is
available free of charge via the Internet.
AUTHOR INFORMATION
Corresponding Authors
* Dr Rita Turnaturi
rita.turnaturi@unict.it
ORCID 0000-0002-5895-7820
Prof. Carmela Parenti
cparenti@unict.it
ORCID 0000-0003-1412-2597
Author contributions
L.P., R.T. and C.P. designed all paper experiments, analyzed and
discussed results and wrote the paper. L.P., R.T. and E.A.
designed and synthesized new compounds. C.P. performed in
vivo experiment. O.P. and E.Ar. performed and analyzed
radioligand binding experiments. A.M., L.S. and M.D.
participated to the statistical analysis and characterized
compounds. Z.G. and P.P. performed and analyzed in vitro
functional experiments. E.J.C. and M.C.R.-C. performed and
analyzed in vivo experiments. All authors have participated in the
writing refinement and given approval to the final version of the
manuscript.
(10) Stein, C. New concepts in opioid analgesia. Expert. Opin. Investig.
Drugs. 2018, 7, 1−11.
(11) Turnaturi, R.; Chiechio, S.; Salerno, L.; Rescifina, A.; Pittalà, V.;
Cantarella, G.; Tomarchio, E.; Parenti, C.; Pasquinucci, L. Progress in the
development of more effective and safer analgesics for pain management.
Eur. J. Med. Chem. 2019, 183, 111701.
(12) Manglik, A.; Lin, H.; Aryal, D. K.; McCorvy, J. D.; Dengler, D.;
Corder, G.; Levit, A.; Kling, R. C.; Bernat, V.; Hübner, H.; Huang, X. P.;
Sassano, M. F.; Giguère, P. M.; Löber, S.; Duan, D.; Scherrer, G.;
Kobilka, B. K.; Gmeiner, P.; Roth, B. L.; Shoichet, B. K. Structure-based
Note
The authors declare no conflict of interest.
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ACS Medicinal Chemistry Letters
discovery of opioid analgesics with reduced side effects. Nature 2016,
537, 185−190.
Allodynia and Astrocytic Connexin 43 in an Animal Model of
Neuropathic Pain. Mol. Neurobiol. 2019, 56, 7338−7354.
1
2
3
4
5
6
7
8
(13) Bond, R. A.; Lucero Garcia-Rojas, E. Y.; Hegde, A.; Walker, J. K. L.
Therapeutic Potential of Targeting ß-Arrestin. Front. Pharmacol. 2019,
10, 124.
(14) Turnaturi, R.; Montenegro, L.; Marrazzo, A.; Parenti, R.;
Pasquinucci, L.; Parenti, C. Benzomorphan skeleton, a versatile scaffold
for different targets: A comprehensive review. Eur. J. Med. Chem. 2018,
155, 492−502.
(15) Turnaturi, R.; Marrazzo, A.; Parenti, C.; Pasquinucci, L.
Benzomorphan scaffold for opioid analgesics and pharmacological tools
development: A comprehensive review. Eur. J. Med. Chem. 2018, 148,
410−422.
(16) Turnaturi, R.; Parenti, C.; Prezzavento, O.; Marrazzo, A.; Pallaki, P.;
Georgoussi, Z.; Amata, E.; Pasquinucci, L. Synthesis and Structure-
Activity Relationships of LP1 Derivatives: N-Methyl-N-phenylethylamino
Analogues as Novel MOR Agonists. Molecules 2018, 23, pii: E677.
(17) Pasquinucci, L.; Prezzavento, O.; Marrazzo, A.; Amata, E.;
Ronsisvalle, S.; Georgoussi, Z.; Fourla, D. D.; Scoto, G. M.; Parenti, C.;
Aricò, G.; Ronsisvalle, G. Evaluation of N-substitution in 6,7-
benzomorphan compounds. Bioorg. Med. Chem. 2010, 18, 4975−1482.
(18) Parenti, C.; Turnaturi, R.; Aricò, G.; Marrazzo, A.; Prezzavento, O.;
Ronsisvalle, S.; Scoto, G. M.; Ronsisvalle, G.; Pasquinucci, L.
Antinociceptive profile of LP1, a non-peptide multitarget opioid ligand.
Life Sci. 2012, 90, 957−961.
(19) Pasquinucci, L.; Parenti, C.; Turnaturi, R.; Aricò, G.; Marrazzo, A.;
Prezzavento, O.; Ronsisvalle, S.; Georgoussi, Z.; Fourla, D. D.; Scoto, G.
M.; Ronsisvalle, G. The benzomorphan-based LP1 ligand is a suitable
MOR/DOR agonist for chronic pain treatment. Life Sci. 2012, 90, 66−70.
(20) Parenti, C.; Turnaturi, R.; Aricò, G.; Gramowski-Voss, A.;
Schroeder, O. H.; Marrazzo, A.; Prezzavento, O.; Ronsisvalle, S.; Scoto,
G. M.; Ronsisvalle, G.; Pasquinucci, L. The multitarget opioid ligand
LP1's effects in persistent pain and in primary cell neuronal cultures.
Neuropharmacology 2013, 71, 70−82.
(21) Pasquinucci, L.; Turnaturi, R.; Aricò, G.; Parenti, C.; Pallaki, P.;
Georgoussi, Z.; Ronsisvalle, S. Evaluation of N-substituent structural
variations in opioid receptor profile of LP1. Bioorg. Med. Chem. 2016, 24,
2832−42.
(22) Pasquinucci, L.; Parenti, C.; Amata, E.; Georgoussi, Z.; Pallaki, P.;
Camarda, V.; Calò, G.; Arena, E.; Montenegro, L.; Turnaturi, R. Synthesis
and Structure-Activity Relationships of (−)-cis-N-Normetazocine-Based
LP1 Derivatives. Pharmaceuticals 2018, 11, pii: E40.
(23) Pasquinucci, L.; Turnaturi, R.; Prezzavento, O.; Arena, E.; Aricò, G.;
Georgoussi, Z.; Parenti, R.; Cantarella, G.; Parenti, C. Development of
novel LP1-based analogues with enhanced delta opioid receptor. Bioorg.
Med. Chem. 2017, 25, 4745−4752.
(24) Pasquinucci, L.; Turnaturi, R.; Montenegro, L.; Caraci, F.; Chiechio,
S.; Parenti, C. Simultaneous targeting of MOR/DOR: A useful strategy for
inflammatory pain modulation. Eur. J. Pharmacol. 2019, 847, 97−102.
(25) Vicario, N.; Pasquinucci, L.; Spitale, F. M.; Chiechio, S.; Turnaturi,
R.; Caraci, F.; Tibullo, D.; Avola, R.; Gulino, R.; Parenti, R.; Parenti, C.
Simultaneous Activation of Mu and Delta Opioid Receptors Reduces
(26) Pasquinucci, L.; Turnaturi, R.; Calò, G.; Pappalardo, F.; Ferrari, F.;
Russo, G.; Arena, E.; Montenegro, L.; Chiechio, S.; Prezzavento, O.;
Parenti,
C.
(2S)-N-2-methoxy-2-phenylethyl-6,7-benzomorphan
compound (2S-LP2): Discovery of a biased mu/delta opioid receptor
agonist. Eur. J. Med. Chem. 2019, 168, 189−198.
(27) Brine, G. A.; Berrang, B.; Hayes, J. P.; Carroll, F. I. An improved
resolution of (±)-cis-N-normetazocine. J. Heterocyclic Chem. 1990, 27,
2139−2143.
(28) Accolla, M. L.; Turnaturi, R.; Sarpietro, M. G.; Ronsisvalle, S.;
Castelli, F.; Pasquinucci, L. Differential scanning calorimetry approach to
investigate the transfer of the multitarget opioid analgesic LP1 to
biomembrane model. Eur. J. Med. Chem. 2014, 77, 84−90.
(29) Romera, J. L.; Cid, J. M.; Trabanco, A. A. Potassium iodide catalysed
monoalkylation of anilines under microwave irradiation. Tetrahedron Lett.
2004, 45, 8797–8800.
(30) Prezzavento, O.; Arena, E.; Sánchez-Fernández, C.; Turnaturi, R.;
Parenti, C.; Marrazzo, A.; Catalano, R.; Amata, E.; Pasquinucci, L.;
Cobos, E. J. (+)-and (-)-Phenazocine enantiomers: Evaluation of their dual
opioid agonist/σ(1) antagonist properties and antinociceptive effects. Eur.
J. Med. Chem. 2017, 125, 603–610.
(31) Childers, S. R. Opioid receptor-coupled second messenger systems.
Life Sci. 1991, 48, 1991-2003.
(32) Fourla, D. D.; Papakonstantinou, M. P.; Vrana, S.; Georgoussi, Z.
Selective interactions of spinophilin with the C-terminal domains of the δ-
and μ-opioid receptors and G-proteins differentially modulate opioid
receptor signaling. Cell Signal. 2012, 24, 2315–2328.
(33) Morou, E.; Georgoussi, Z. Expression of the third intracellular loop
of the delta-opioid receptor inhibits signaling by opioid receptors and
other G protein coupled receptors. J. Pharm. Exp. Ther. 2005, 315, 1368–
1379.
(34) Papakonstantinou, M. P.; Karoussiotis, C.; Georgoussi, Z. RGS2 and
RGS4 proteins: New modulators of the κ-opioid receptor signaling. Cell
Signal. 2015, 27, 104–114.
9
10
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13
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15
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17
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21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(35) Georgoussi, Z.; Georganta, E.-M.; Milligan, G. The other side of
opioid receptor signaling: Regulation by protein-protein interaction. Curr.
Drug Targets 2012, 13, 80-102.
(36) Zheng, H.; Loh, H. H.; Law, P.Y. Beta-arrestin-dependent mu-opioid
receptor-activated extracellular signal-regulated kinases (ERKs)
Translocate to Nucleus in Contrast to
G protein-dependent ERK
activation. Mol. Pharmacol. 2008, 73, 178–190.
(37) Grösch, S.; Niederberger, E.; Geisslinger, G. Investigational drugs
targeting the prostaglandin E2 signaling pathway for the treatment of
inflammatory pain. Expert. Opin. Investig. Drugs. 2017, 26, 51–61.
(38) Nath, C.; Gupta, M. B.; Patnaik, G. K.; Dhawan, K. N. Morphine-
induced straub tail response: mediated by central mu2-opioid receptor.
Eur. J. Pharmacol. 1994, 263, 203–205.
(39) Shenoy, S. K.; Drake, M. T.; Nelson, C. D.; Houtz, D. A.; Xiao, K.;
Madabushi, S.; Reiter, E.; Premont, R. T.; Lichtarge, O.; Lefkowitz. R. J.
beta-arrestin-dependent, G protein-independent ERK1/2 activation by the
beta2 adrenergic receptor. J. Biol. Chem. 2006, 281, 1261–1273.
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