Synthesis and structure-activity relationships of LP1 derivatives: N-methyl-N-phenylethylamino analogues as novel MOR agonists

Article abstract of DOI:10.3390/molecules23030677

The opioid pharmacological profile of cis-(-)-N-normetazocine derivatives is deeply affected by the nature of their N-substituents. Here, our efforts were focused on the synthesis and pharmacological evaluation of novel derivatives of the lead LP1, a multitarget opioid analgesic compound featuring an N-phenylpropanamido substituent. LP1 derivatives 5a-d and 6a-d were characterized by flexible groups at the N-substituent that allow them to reposition themselves relative to cis-(-)-N-normetazocine nucleus, thus producing different pharmacological profiles at the mu, delta and kappa opioid receptors (MOR, DOR and KOR) in in vitro and in vivo assays. Among the series, compound 5c, with the best in vitro and in vivo profile, resulted a MOR agonist which displays a K_i^MOR of 6.1 nM in a competitive binding assay, and an IC₅₀ value of 11.5 nM and an I_max of 72% in measurement of cAMP accumulation in HEK293 cells stably expressing MOR, with a slight lower efficacy than LP1. Moreover, in a mouse model of acute thermal nociception, compound 5c, intraperitoneally administered, exhibits naloxone-reversed antinociceptive properties with an ED₅₀ of 4.33 mg/kg. These results expand our understanding of the importance of N-substituent structural variations in the opioid receptor profile of cis-(-)-N-normetazocine derivatives and identify a new MOR agonist useful for the development of novel opioid analgesics for pain treatment.

Full text of DOI:10.3390/molecules23030677

molecules
Article
Synthesis and Structure-Activity Relationships of LP1
Derivatives: N-Methyl-N-phenylethylamino
Analogues as Novel MOR Agonists
Rita Turnaturi 1 ID , Carmela Parenti 2, Orazio Prezzavento 1, Agostino Marrazzo 1
,
ID
Paschalina Pallaki 3, Zafiroula Georgoussi 3, Emanuele Amata 1 and Lorella Pasquinucci 1,
*
ID
1
Department of Drug Sciences, Medicinal Chemistry Section, University of Catania, Viale A. Doria 6,
95125 Catania, Italy; rita.turnaturi@unict.it (R.T.); prezzave@unict.it (O.P.); marrazzo@unict.it (A.M.);
eamata@unict.it (E.A.)
Department of Drug Sciences, Pharmacology and Toxicology Section, University of Catania, Viale A. Doria 6,
95125 Catania, Italy; cparenti@unict.it
Laboratory of Cellular Signaling and Molecular Pharmacology, Institute of Biosciences and Applications,
National Centre for Scientific Research “Demokritos” Agia Paraskevi-Attikis, 15310 Athens, Greece;
ppallaki@bio.demokritos.gr (P.P.); iro@bio.demokritos.gr (Z.G.)
2
3
*
Correspondence: lpasquin@unict.it; Tel.: +39-095-738-4273
Received: 21 February 2018; Accepted: 15 March 2018; Published: 16 March 2018
Abstract: The opioid pharmacological profile of cis-( )-N-normetazocine derivatives is deeply
−
affected by the nature of their N-substituents. Here, our efforts were focused on the synthesis
and pharmacological evaluation of novel derivatives of the lead LP1, a multitarget opioid analgesic
compound featuring an N-phenylpropanamido substituent. LP1 derivatives 5a
characterized by flexible groups at the N-substituent that allow them to reposition themselves relative
to cis-( )-N-normetazocine nucleus, thus producing different pharmacological profiles at the mu,
–d and 6a–d were
−
delta and kappa opioid receptors (MOR, DOR and KOR) in in vitro and in vivo assays. Among the
series, compound 5c, with the best in vitro and in vivo profile, resulted a MOR agonist which displays
a KiMOR of 6.1 nM in a competitive binding assay, and an IC50 value of 11.5 nM and an Imax of 72%
in measurement of cAMP accumulation in HEK293 cells stably expressing MOR, with a slight
lower efficacy than LP1. Moreover, in a mouse model of acute thermal nociception, compound 5c,
intraperitoneally administered, exhibits naloxone-reversed antinociceptive properties with an ED50
of 4.33 mg/kg. These results expand our understanding of the importance of N-substituent structural
variations in the opioid receptor profile of cis-( )-N-normetazocine derivatives and identify a new
−
MOR agonist useful for the development of novel opioid analgesics for pain treatment.
Keywords: 6,7-benzomorphan derivatives; MOR agonist; pain; radioligand competitive binding;
cAMP accumulation assay; tail-flick test
1. Introduction
The cis-(−)-N-normetazocine nucleus, derived by structure-activity relationships (SARs) during
morphine skeleton simplification [1,2], has been the subject of medicinal chemistry exploration for
the development of new drugs and pharmacological tools to explore opioid pharmacology in vitro
and in vivo ]. This nucleus is a rigid scaffold able to support the phenolic ring and the basic
nitrogen in a correct conformation mimicking the tyramine of opioid peptides [ ]. A plethora
of experimental evidence demonstrated the unpredictability of agonist and antagonist properties
conferred by N-substituents on 6,7-benzomorphan ligands [ ]. Thus, our approach consisted in
[
3
4
5–9
the introduction at the basic nitrogen of the cis-(
−
)-N-normetazocine nucleus of different functional
Molecules 2018, 23, 677; doi:10.3390/molecules23030677
www.mdpi.com/journal/molecules

Molecules 2018, 23, 677
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groups with the aim to explore their influence on the mu, delta and kappa opioid receptor (MOR, DOR
and KOR) affinity, selectivity and activity in the resulting compounds.
Previously, we synthesized a series of compounds by introducing at the N-substituent of
cis-( )-N-normetazocine an aromatic ring and/or alkyl residues linked by an N-propanamido or
−
N-acetamido spacers (Figure 1) [10]. Among them, the multitarget opioid ligand LP1, featured by an
N-phenylpropanamido substituent, possessed affinity and intrinsic activity for MOR (Ki = 0.83 nM,
IC50 = 4.8 nM) and DOR (Ki = 29.1 nM, IC50 = 12 nM). LP1, in vivo tested, resulted a potent opioid
analgesic compound (ED50 = 2.03 mg/kg) with low tolerance-inducing capability [11,12] also suitable
for the management of persistent pain conditions such as neuropathic and inflammatory pain [13].
Figure 1. Structures of LP1 and its analogues.
Here, our efforts were focused on the evaluation of the effect of N-substituent structural
variations on the opioid receptor profile of LP1 through the design and synthesis of two new series of
derivatives. The new LP1 derivatives designed possessed different secondary and tertiary ethylamino
or propylamino side chains bearing phenyl or cyclohexyl rings (Figure 2). Thus, they may help our
understanding of opioid ligand-receptor interactions. All synthesized LP1 derivatives were tested
by opioid receptor competitive binding assays to determine their affinity at MOR, DOR and KOR.
Moreover, the compounds were further evaluated in vitro by measuring their adenylyl cyclase (AC)
activity in order to establish their functional profile. Finally, the compound with the best functional
profile was also tested in vivo through a mouse tail flick test.
In the new synthesized benzomorphan ligands flexible groups at the N-substituent position
allow them to reposition themselves relative to the cis-( )-N-normetazocine nucleus thus producing a
−
different pharmacological profile at MOR, DOR and KOR. In particular, compound 5c, with the
best in vitro and in vivo profile, resulted a MOR agonist suitable for the development of novel
analgesic drugs.
Figure 2. Structures of LP1 N-modified derivatives 5a–d and 6a–d.

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2. Results and Discussion
2.1. Chemistry
The synthesis of the novel compounds 5a
–
d
and 6a
–d
was carried out as illustrated in Scheme 1.
Amides 1a
chloride and 3-bromopropionyl chloride, respectively, and compounds 3a
by alkylation of cis-( )-(1R,5R,9R)-N-normetazocine with the respective amides, as previously
reported [10 14]. Then, reduction of compounds 3a and 4a with diborane in anhydrous THF
provided the final compounds 5a and 6a . The new synthesized derivatives were characterized by
IR, 1H-NMR, 13C-NMR and elemental analysis.
–
d
and 2a
–
d
were prepared by acylation of the respective amines with 2-chloroacetyl
–d
and 4a were obtained
–d
−
,
–
d
–d
–
d
–d
Scheme 1. Synthetic pathway.
2.2. Pharmacology
2.2.1. In Vitro Radioligand Binding Assay
The MOR, DOR and KOR binding affinity of compounds 5a–d and 6a–d were determined by
radioligand competitive binding experiments as previously reported [15]. Calculated inhibition
constant (Ki) values are listed in Table 1.
As shown in Table 1, LP1 derivatives displayed a broad range of binding affinity for MOR (Ki
= 6.1–160 nM) and KOR (Ki = 19.8–56 nM) and lesser or no affinity towards DOR. The first series of
ligands 5a
although in comparison to the lead LP1 their Ki values were 7- and 10-times higher. With the exception
of compound 5a, an improved KOR affinity was reported for compounds 5b . Indeed, their Ki values
–d, with an N-ethylamino spacer, retained a significant MOR affinity, in particular 5c and 5b,
–
d

Molecules 2018, 23, 677
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were 5-, 3- and 4-times lower than the LP1 Ki value. A worse DOR affinity profile was recorded for
compounds 5a–d.
For the second series of ligands 6a
was recorded. The binding profile of compounds 6a
values were 10-, 58-, 82- and 103 times higher in comparison to LP1. In addition, in comparison with
the series containing the N-ethylamino substituent, the Ki values of compounds 6a for MOR were
higher. Analogously to the series 5a , an improved KOR binding affinity was recorded also for the
series with the N-propylamino substituent 6a . Their Ki values for KOR were lower in comparison to
LP1 and higher in comparison to the series 5a 5d. The introduction of the N-propylamino substituent
was significantly detrimental for DOR binding affinity.
–
d, with an N-propylamino side chain, a similar trend of affinity
–
d
versus MOR got worse, and in fact their Ki
–d
–d
–d
–
Table 1. Opioid receptor binding affinity of compounds 5a–d and 6a–d.
Ki (nM) ± SEM a,b
0
Compound
n
R
R
MOR
DOR
KOR
5a
5b
5c
5d
6a
6b
6c
6d
LP1
0
0
0
0
1
1
1
1
H
H
CH3
C2H5
H
H
CH3
C2H5
Ph
C6H11
Ph
Ph
Ph
C6H11
Ph
Ph
83 ± 30
8.3 ± 0.8
6.1 ± 0.5
160 ± 7.0
7.0 ± 0.6
48 ± 1.4
68 ±1.6
86 ± 3.0
0.83 ± 0.05
1.16 ± 0.1
-
270 ± 10
70 ± 2.0
147 ± 5.7
411 ±14
117 ± 5.2
>5000
100 ± 5.0
19.8 ±0.9
31 ± 1.3
28.7 ± 1.0
71 ± 2.0
126 ± 4.5
94 ± 3.5
56 ±1.7
110 ± 6.0
-
>5000
1060 ± 152
29.1 ± 1.0
-
DAMGO
U50,488
Naltrindole
-
0.34 ± 0.1
-
1.13 ± 0.1
a
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.
In general, in N-amino derivatives 5a–d and 6a–d the introduction of a second positive
charge at the nitrogen substituent retained MOR affinity, improved KOR affinity and
dramatically reduced DOR affinity. Among the series, compounds 5b and 5c, with the
N-cyclohexylethylamino and N-methyl-N-phenylethylamino substituent, respectively, showed a
significant MOR (Ki = 8.3 and 6.1 nM, respectively) and KOR (Ki = 19.8 and 31 nM, respectively)
affinity and the compound 6a, with the N-phenylpropanamino substituent, showed similar MOR/KOR
binding values of compounds 5b and 5c (Table 1).
In comparison to the respective previously synthesized and in vitro evaluated N-amido series
(Figure 1), an opposite trend emerged [10]. Indeed, the shorter N-acetamido spacer produced a
significant loss of MOR affinity relative to their N-propanamido homologs. All N-acetamido and
N-propanamido compounds displayed a low KOR affinity with the partial exception of the compound
bearing the N-ethyl-N-phenylpropanamido substituent (KiKOR = 70 nM). Moreover, the N-secondary
amido substituent was preferred for MOR interaction while the N-tertiary amido substituent was
unfavorable. These evidences suggested that, in comparison to the N-acetamido and N-propanamido
series, the increased flexibility of N-substituent of compounds 5a–d and 6a–d and the presence of a

Molecules 2018, 23, 677
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second positive charge could allow a different interaction with the opioid receptor binding pocket.
In fact, all new synthesized compounds possess orientable groups at the N-substituent that allow to
reposition themselves relative to the cis-( )-N-normetazocine nucleus giving differences in binding
−
versus MOR, DOR and KOR.
2.2.2. Adenylyl Cyclase-Mediated Effects
As typical Gi/o coupled receptor, MOR, DOR and KOR signals are mediated by AC inhibition.
Given the significant MOR and moderate KOR affinity, to examine the functional role of target
compounds, in opioid receptor signaling, the ability of 5a–d and 6a–d to affect forskolin-stimulated
AC activity in HEK293 cells stably expressing the MOR or KOR was tested and the results are reported
in Table 2.
Table 2. Effect of compounds 5a–d and 6a–d on cAMP accumulation by the KOR and MOR.
Imax (%) ± SD c
IC50 (nM) ± SD a,b
Compound
MOR
KOR
MOR
KOR
5a
5b
5c
5d
6a
6b
6c
6d
55.3 ± 7.0
74.0 ± 3.5
11.5 ± 2.5
66 ± 1.3
1000 ± 65
180 ± 50
ND d
ND
1400 ± 69
>5000
ND
60 ± 4
28 ± 1
72 ± 5
55 ± 3
50 ± 3
-
50 ± 3
40 ± 2
73 ± 3.8
73 ± 0.3
-
58 ± 4
-
-
7.4 ± 1.1
53 ± 4
ND
44 ± 3
21.61 ± 3.5
9.51 ± 2.0
4.8 ± 0.5
3.18 ± 0.3
-
-
-
-
ND
-
-
LP1
DAMGO
U50,488
0.82 ± 0.03
68 ± 5
a
Agonist properties of compounds in the inhibition of forskolin-stimulated cAMP accumulation by MOR and KOR.
The inhibition of cAMP accumulation was measured as described in Section 4.3. IC50 value is the concentration
b
of the compound needed to produce half maximal inhibition, with all values presented as the average
±
SD of
c
triplicate determinations from three independent experiments. ICmax value is the maximal percent inhibition
d
obtained with the compound. ND, not determined.
In HEK293 cells stably expressing the MOR, compounds 5a–d and 6a–d, with the exception of
compound 6b, dose-dependently inhibited cAMP accumulation although with marked differences
in potency and efficacy. Compound 5c resulted a MOR agonist that, in comparison to LP1, showed
a 2-times higher IC50 value but comparable efficacy. Compounds 5a, b, d and 6a, c, d resulted weak
MOR partial agonists with IC50 values ranging from 7.4 nM to 74.0 nM and Imax values ranging from
28% to 60% inhibition over control. In parallel experiments in HEK293 cells stably expressing KOR,
target compounds exhibited a different trend. In particular, 5a
with IC50 values ranging from 180 nM to 5000 nM, and efficacy with Imax values ranging from 44%
to 58% inhibition over control. Compounds 5c and 6c were not able to dose-dependently inhibit
,b and 6a,b showed a very low potency,
,d
,d
cAMP accumulation and only at very high concentrations determined 30% cAMP inhibition.
2.2.3. Tail Flick Test
The MOR agonist 5c was evaluated for acute agonistic effects in mice. In detail, it was tested
for its ability to produce antinociception in the mouse tail flick test at the dose-range of 1–7 mg/kg
intraperitoneal (ip) injected. The compound produced a dose-dependent analgesic effect compared
to the group of mice treated with saline (* p < 0.05 vs. saline-treated mice). In vivo evaluation of 5c
established that the maximal antinociceptive activity was reached at 45 min (Figure 3, panel A) after ip
injection and significantly lasted, at the highest dose, until 75 min after injection. Pretreatment with
naloxone (3 mg/kg sc), 30 min prior to 5c, prevented its antinociceptive effect (data not shown).
Figure 3, panel B shows the analgesic dose-response curve with the ED50 value (4.33 mg/kg ip) and its
confidence limits (CL) (3.35–5.59). These results are in agreement with our in vitro functional assays.

Molecules 2018, 23, 677
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(A)
(B)
Figure 3.
(A) Time-course (min) of compound 5c induced analgesia measured by tail flick latency
(TFL). Results are expressed in seconds (s). Data are means
±
±
SEM from six to eight mice. * p < 0.05
SEM for compound 5c was plotted at
vs. saline-treated mice. (
45 min post-treatment.
B) Analgesic dose-response curve
3. Discussion
In this study, our attention was focused on the evaluation of N-substituents in
)-N-normetazocine-based compounds to extend the knowledge about this structural region
cis-(
−
in the opioid receptor interaction. LP1 derivatives produced a pharmacological profile at MOR, DOR
and KOR different one from another as consequence of the N-substituent nature whose flexibility
allow them to reposition themselves differently relative to the cis-( )-N-normetazocine nucleus.
−
Moreover, the presence of a second positive charge at the N-substituent, as well as the shortening of
the N-substituent spacer, seem to address the ligand-opioid receptor interaction mainly at MOR and
KOR. Almost all synthesized compounds, in HEK293 cells stably expressing MOR, resulted MOR
partial agonists and compound 5c exhibited an IC50 value of 11.5 nM and an Imax of 72% with a slight
lower efficacy than LP1. In tail flick test compound 5c confirmed its in vitro profile with an analgesic
activity naloxone-reversed and an ED50 of 4.33 mg/kg. Moreover, in compound 5c the second positive
charge at the N-substituent could affect its pharmacokinetic properties and could, in part, justify its
reduced antinociceptive effect compared to LP1 after systemic administration.
Our results corroborated a plethora of experimental data [
5,
7,
8] revealing the unpredictability
of pharmacological properties conferred by N-substituents on (
−
)-cis-N-normetazocine scaffold.
For instance, the introduction at the basic nitrogen of an aromatic ring and/or alkyl residues linked
by an N-acetamido spacer was detrimental for opioid receptors affinity (KiMOR 722–2930 nM, KiDOR
> 5000 nM, KiKOR 335–5000 nM) [10]. Contrarily, aromatic ring and/or alkyl residues linked by
the N-propanamido spacer, as well as the presence of a secondary amido group, improved opioid
interaction mainly at MOR [10]. Bulkier aromatic groups, such as naphthyl, quinoline and isoquinoline
rings, at the N-propanamido spacer, were not tolerated for interaction with the opioid receptors binding
pocket mainly at DOR [16]. Moreover, the bulkier size of the N-substituent moved the functional
profile from agonism to antagonism versus MOR [16]. A shorter and more flexible ethyl spacer with
H-bonding groups at carbon 2 in N-substituent of (
interaction with opioid binding pocket mainly at MOR and DOR [15].
−
)-cis-N-normetazocine nucleus allowed an optimal

Molecules 2018, 23, 677
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4. Materials and Methods
4.1. General Experimental Procedures
All commercial chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or
Merck (Darmstadt, Germany) and were used without further purification. ( )-cis-N-normetazocine
±
was obtained from Fabbrica Italiana Sintetici (Milano, Italy). Melting points were determined in
open capillary tubes with a Büchi 530 apparatus (Büchi, Flawil, Switzerland) and are uncorrected.
Analytical TLC was performed on silica gel 60 F254 aluminum sheets (Merck) with fluorescent
indicator. Components were visualized by UV light (λ = 254 nm) and iodine vapors. Flash column
chromatography was carried out on Merck silica gel 60 (230–400 mesh). Optical rotations were
determined in CHCl3 or MeOH solution with a Perkin-Elmer 241 polarimeter (Llantrisant, UK).
1
Infrared spectra were recorded on a 1600 FT-IR Perkin-Elmer instrument. H- and 13C-NMR spectra
were routinely recorded on an Inova-200 spectrometer (Varian, Palo Alto, CA, USA) in CDCl3 solution;
chemical shifts
Elemental analyses (C, H, N) were performed on a Carlo Erba 1106 analyzer (Milan, Italy) and the
analysis results were within 0.4% of the theoretical values. All reported compounds had a purity of
δ are expressed in ppm with reference to tetramethylsilane as an internal standard.
±
at least 95%. Synthesis and analytical data of compounds 1a–d, 2a–d, 3a–d and 4a–d were described in
reference [10].
4.2. Chemistry
General procedure for the reduction of cis-(
5a and 6a
atmosphere, a solution of the appropriate cis-(
and 4a (1 mmol) in THF (4 mL) was slowly added. The reaction mixture was refluxed for 12 h and
−
)-N-substituted-N-normetazocine derivatives to afford compounds
. To a 1 M solution of diborane (2 mL, 2 mmol) in THF, cooled at 0 C and under nitrogen
◦
–
d
–d
−
)-N-substituted-N-normetazocine derivatives 3a–d
–d
cooled at room temperature, and 2 mL of a 6 M hydrochloric acid solution was added slowly. THF was
removed by distillation at atmospheric pressure. NaHCO3 saturated solution was added to aqueous
phase, and the latter was extracted with CHCl3. The organic mixture was dried over anhydrous
Na2SO4 and evaporated in vacuo to give the free base. The compounds 5a
–d and 6a–d were dissolved
in THF and treated with a solution of H2C2O4 2H2O in THF to give the oxalate salts as white solids.
·
The analytical pure samples were obtained by recrystallization.
(2R,6R,11R)-3-(2-Anilinoethyl)-6◦,11-dimethyl-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-8-ol
(
5a).
◦
White solid (87%); m.p. 145–146 C; [α]D25
=
−
60 (c 1.0, MeOH); 1H-NMR (CDCl3, free base)
δ
7.13–7.06
(2H, m), 6.96–6.92 (1H, m), 6.67–6.54 (5H, m), 3.62–3.56 (1H, m), 3.42–3.01 (7H, m), 2.56–2.49 (1H, m),
2.14–1.88 (2H, m), 1.46–1.39 (1H, m), 1.29 (3H, s), 0.79 (3H, d); 13C-NMR (CDCl3, free base)
156.16,
147.90, 140.24, 128.97, 128.37, 123.19, 116.38, 113.74, 112.27, 111.88, 57.95, 51.54, 45.67, 40.15, 39.32, 37.61,
δ
34.70, 28.94, 24.26, 12.93; anal. C 64.72, H 7.21, N 6.35%, calcd. for C22H28N2O
C 64.85, H 7.26, N 6.30%.
·
C2H2O4 H2O (444.521),
·
(2R,6R,11R)-3-[2-(Cyclohexylamino)ethyl)-6,11-dimethyl-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-8-ol
5b). White solid (80%); m.p. 153–155 ◦C; [α]D25 59◦ (c 1.0, MeOH); 1H-NMR (CDCl3, free base)
(
δ
=
−
6.89–6.85 (1H, m), 6.69–6.56 (2H, m), 2.86–2.39 (8H, m), 2.10–1.71 (8H, m), 1.31 (3H, s), 1.28–1.18 (7H,
m), 0.80 (3H, d); 13C-NMR (CDCl3, free base)
δ 155.02, 142.95, 127.98, 127.42, 113.27, 112.49, 58.79,
56.98, 53.40, 45.17, 43.20, 42.13, 41.74, 36.36, 32.74, 25.81, 25.45, 24.95, 24.16, 14.08; anal. C 63.94, H 8.52,
N 6.27%, calcd for C22H34N2O·C2H2O4·H2O (450.568), C 63.98, H 8.50, N 6.22%.
(2R,6R,11R)-6,11-Dimethyl-{2-[methyl(phenyl)amino]ethyl}-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-
8-ol (5c). White solid (84%); m.p. 162–163 ◦C; [α]D25
=
−
58◦ (c 1.0, MeOH); 1H-NMR (CDCl3, free base)
δ
7.63–7.40 (2H, m), 7.15–6.85 (1H, m), 6.74–6.51 (5H, m), 3.33 (3H, s), 3.26–2.89 (9H, m), 2.57–2.49
(2H, m), 1.33–1.16 (1H, m), 1.29 (3H, s), 0.76 (3H, d); 13C-NMR (CDCl3, free base)
156.29, 148.69,
140.53, 129.52, 128.89, 123.55, 116.93, 114.08, 112.55, 112.14, 59.09, 51.36, 48.95, 46.97, 45.71, 38.24, 35.10,
δ

Molecules 2018, 23, 677
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34.95, 24.47, 23.14, 13.20; anal. C 65.44, H 7.37, N 6.12%, calcd for C23H30N2O
C 65.48, H 7.47, N 6.11%.
·
C2H2O4
·
H2O (458.547),
(2R,6R,11R)-6,11-Dimethyl-{2-[ethyl(phenyl)amino]ethyl}-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-8-ol
5d). White solid (85%); m.p. 158–160 ◦C; [α]D25 59◦ (c 1.0, MeOH); 1H-NMR (CDCl3, free base)
7.15–7.07 (2H, m), 6.86–6.82 (1H, m), 6.63–6.47 (5H, m), 3.50–3.38 (2H, m), 3.31 (2H, q, J = 6.8 Hz),
2.84–2.47 (7H, m), 2.17–1.64 (2H, m), 1.42–1.07 (1H, m), 1.25 (3H, s), 1.06 (3H, t, J = 7.0 Hz), 0.74 (3H,
d); 13C-NMR (CDCl3, free base)
155.51, 147.46, 142.20, 129.11, 127.81, 123.19, 114.93, 112.93, 111.82,
(
δ
=
−
δ
111.28, 60.74, 58.11, 51.81, 45.31, 44.39, 40.21, 39.79, 35.73, 25.23, 23.57, 13.78, 12.17; anal. C 66.17, H 7.66,
N 5.95%, calcd. for C24H32N2O·C2H2O4·H2O (472.574), C 66.08, H 7.68, N 5.93%.
(2R,6R,11R)-3-(3-Anilinopropyl)-6,11-dimethyl-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-8-ol (6a).
◦
◦
45 (c 1.0, MeOH); 1H-NMR (CDCl3, free base)
δ 7.09–7.02
White solid (91%); m.p. 149–150 C; [α]D25
=
−
(2H, m), 6.94–6.90 (1H, m), 6.65–6.47 (5H, m), 3.41–3.36 (1H, m), 3.04–2.78 (7H, m), 2.35–2.24 (1H, m),
1.98–1.84 (2H, m), 1.84–1.65 (2H, m), 1.39–1.28 (1H, m), 1.28 (3H, s), 0.77 (3H, d); 13C-NMR (CDCl3,
free base)
δ 156.08, 148.75, 140.87, 128.95, 128.75, 128.26, 124.08, 115.69, 113.58, 112.03, 57.55, 51.35,
45.32, 40.57, 39.25, 38.50, 35.16, 24.60, 24.50, 22.85, 13.28; anal. C 65.28, H 7.54, N 6.18%, calcd. for
C23H30N2O·C2H2O4·H2O (458.547), C 65.48, H 7.47, N 6.11%.
(2R,6R,11R)-3-[3-(Cyclohexylamino)propyl)-6,11-dimethyl-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-8-ol
(
6b). White solid (94%); m.p. 136–138 ◦C; [α]D25
=
−
47◦ (c 1.0, MeOH); 1H-NMR (CDCl3, free base)
δ
6.96–6.92 (1H, m), 6.66–6.59 (2H, m), 3.44–3.41 (1H, m), 2.97–2.88 (6H, m), 2.49–2.35 (1H, m), 2.07–1.98
(7H, m), 1.74–1.56 (3H, m), 1.43–1.28 (6H, m), 1.28 (3H,s), 1.11–1.08 (1H, m), 0.79 (3H, d); 13C-NMR
(CDCl3, free base)
δ 156.26, 140.39, 128.35, 123.30, 113.80, 112.00, 60.08, 57.70, 56.04, 55.75, 54.92, 50.13,
45.26, 41.20, 34.87, 28.51, 24.76, 24.36, 23.93, 21.02, 13.09; anal. C 64.55, H 8.64, N 6.09%, calcd. for
C23H36N2O·C2H2O4·H2O (464.595), C 64.63, H 8.68, N 6.03%.
(2R,6R,11R)-6,11-Dimethyl-{3-[methyl(phenyl)amino]propyl}-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-
8-ol (6c). White solid (91%); m.p. 140–142 ◦C; [α]D25
=
−
52◦ (c 1.0, MeOH); 1H-NMR (CDCl3, free base)
δ
7.19–7.12 (3H, m), 6.73–6.60 (5H, m), 3.63–3.57 (1H, m), 3.44–2.89 (7H, m), 2.85 (3H, s), 2.49–2.40
(1H, m), 2.09–1.88 (4H, m), 1.45–1.38 (1H, m), 1.28 (3H, s), 0.78 (3H, d); 13C-NMR (CDCl3, free
base) 156.29, 148.88, 140.13, 129.04, 128.42, 122.99, 115.96, 113.86, 112.22, 111.99, 57.69, 49.12, 40.75,
δ
39.91, 39.13, 39.00, 38.25, 37.73, 34.77, 24.24, 21.14, 12.92; anal. C 66.10, H 7.64, N 5.99%, calcd. for
C24H32N2O·C2H2O4·H2O (472.574), C 66.08, H 7.68, N 5.93%.
(2R,6R,11R)-6,11-Dimethyl-{3-[ethyl(phenyl)amino]propyl}-1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-
8-ol (6d). White solid (93%); m.p. 138–139 ◦C; [α]D25
=
−
55◦ (c 1.0, MeOH); 1H-NMR (CDCl3, free base)
δ
7.18–6.92 (3H, m), 6.69–6.52 (5H, m), 3.52–3.37 (2H, m), 3.31 (2H, q, J = 6.8 Hz), 2.97–2.91 (4H, m),
2.64–2.45 (3H, m), 2.07–1.74 (4H, m), 1.43–1.36 (1H, m), 1.28 (3H, s), 1.06 (3H, t, J = 6.8 Hz), 0.79 (3H,
d); 13C-NMR (CDCl3, free base)
156.16, 147.44, 140.44, 129.14, 128.32, 123.48, 115.34, 113.70, 111.95,
δ
111.84, 57.73, 50.53, 46.80, 45.33, 44.07, 39.75, 37.89, 34.93, 25.61, 24.37, 22.56, 13.03, 11.97; anal. C 66.65,
H 7.81, N 5.72%, calcd. for C25H34N2O·C2H2O4·H2O (486.601), C 66.64, H 7.87, N 5.76%.
4.3. Receptor Binding Assays
Drugs and reagents. Radioligands [3H]-DAMGO, [3H]-DPDPE and [3H]-(+)-U69,593 were
purchased by Perkin-Elmer Life Sciences (Boston, MA, USA). Naloxone hydrochloride was purchased
from Tocris Bioscience (Bristol, UK).
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. [17], as reported elsewhere [18]. Protein concentration was determined by Lowry’s method
using bovine serum albumin as standard [19]. 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 either with 1 nM [3H]-DAMGO (48.4 Ci/mM) or 2 nM [3H]-DPDPE (52.7 Ci/mM) in 50 mM

Molecules 2018, 23, 677
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Tris-HCl (pH 7.4). Regard◦ing KOR binding assays, guinea pig brain membranes (0.5 mg/mL) were
incubated for 30 min at 30 C with 1 nM [3H]-(+)-U69,593 (42.69 Ci/mM). Test compounds were added
in concentration ranging from 10−5 to 10−11 M. Nonspecific binding was assessed in the presence of
10
GF/B glass fiber filters, which were presoaked for 1 h 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
µM of unlabeled naloxone. The reaction was terminated by filtering the solution through Whatman
×
cocktail and counted on a Beckman LS 6500 liquid scintillation counter.
Data analysis. Ki values were calculated using the EBDA/LIGAND program (version 4) purchased
from Elsevier/Biosoft (Cambridge, UK).
4.4. cAMP Accumulation Assay
Cell culture. HEK293 cells stably expressing either the EE-tagged MOR or the flag-tagged KOR [20
22] were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf
serum, 2 mM glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin under 5% CO2 at 37 ◦C.
Measurements of cAMP accumulation. Measurements of AC activity were performed as described
by Papakonstantinou et al. [22] or the GloSensor cAMP Assay (Promega) following the Manufacturer’s
instructions. The generation of cAMP was assessed in response to treatment of the cells with various
–
concentrations of the appropriate ligand (10 pM–50
means SD of triplicate determinations from three independent experiments. Analysis of the data
µM) using 50 µ
M forskolin at 37 ◦C. Values are
±
was performed using Origin 7.5 software (OriginLab Corporation, Northampton, MA, USA).
4.5. In Vivo Pharmacology
Animals. Male Swiss CB1 mice (Envigo Laboratories, S. Pietro al Natisone (UD), Italy) 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 Communities Council
Directive and National Regulations (CEE Council 86/609 and DL 116/92).
Nociceptive Test. Nociception was evaluated by the radiant heat tail-flick test [11,23]. 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 performed at 5 min intervals. A cut-off latency of 15 s was established to minimize damage
to the tail. All tested compounds were dissolved in pyrogen-free isotonic saline (Baxter Healthcare,
Deerfield, IL, USA) and DMSO (5%) and were administered to mice ip. Post-treatment tail flick
latencies (TFLs) were determined at 30, 45, 60, 75 and 90 min after ip injection.
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 (ED50)
values were calculated using least-squares linear regression analysis followed by calculation of 95%
confidence limits (95% CL) by the method of Bliss [12,24].
5. Conclusions
This study expands the understanding on the importance of N-substituent structural variations in
opioid receptor profile of cis-( )-N-normetazocine derivatives and identify compound 5c as a new
−
MOR agonist useful for the development of novel opioid analgesics for pain treatment.
Acknowledgments: This work was supported by University of Catania (PdR 2016-2018) to Lorella Pasquinucci.
The authors gratefully acknowledge Fabbrica Italiana Sintetici (Milan, Italy) for providing (
We thank Thomas Fazio for his assistance with English language editing.
±
)-cis-N-normetazocine.

Molecules 2018, 23, 677
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Author Contributions: R.T., C.P. and L.P. designed and performed chemistry and in vivo experiments; O.P.
designed and performed the radioligand binding assays; Z.G. and P.P. designed and performed the cAMP assay;
L.P., C.P., R.T., O.P., E.A. and A.M. participated in the analysis and interpretation of data. R.T., C.P. and L.P. wrote
the manuscript. All the authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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