Development of novel LP1-based analogues with enhanced delta opioid receptor profile

Article abstract of DOI:10.1016/j.bmc.2017.07.021

Pain relief achieved by co-administration of drugs acting at different targets is more effective than that obtained with conventional MOR selective agonists usually associated to relevant side effects. It has been demonstrated that simultaneously targeting different opioid receptors is a more effective therapeutic strategy. Giving the promising role for DOR in pain management, novel LP1-based analogues with different N-substituents were designed and synthesized with the aim to improve DOR profile. For this purpose, we maintained the phenyl ring in the N-substituent of 6,7-benzomorphan scaffold linked to an ethyl spacer bearing a hydroxyl/methyl or methoxyl group at carbon 2 or including it in a 1,4-benzodioxane ring. LP1 analogues were tested by competition binding assays. Compounds 6 (K_i^MOR = 2.47 nM, K_i^DOR = 9.6 nM), 7 (K_i^MOR = 0.5 nM and K_i^DOR = 0.8 nM) and 9 (K_i^MOR = 1.08 nM, K_i^DOR = 6.6 nM) retained MOR affinity but displayed an improved DOR binding capacity as compared to LP1 (K_i^MOR = 0.83 nM, K_i^DOR = 29.1 nM). Moreover, GPI and MVD functional assays indicated that compounds 6 (IC₅₀ = 49.2 and IC₅₀ = 10.8 nM), 7 (IC₅₀ = 9.9 and IC₅₀ = 11.8 nM) and 9 (IC₅₀ = 21.5 and IC₅₀ = 4.4 nM) showed a MOR/DOR agonist profile, unlike LP1 that was a MOR agonist/DOR antagonist (IC₅₀ = 1.9 and IC₅₀ = 1240 nM). Measurements of their antinociceptive effect was evaluated by mice radiant tail flick test displaying for compounds 6, 7 and 9 ED₅₀ values of 1.3, 1.0 and 0.9 mg/kg, i.p., respectively. Moreover, the antinociceptive effect of compound 9 was longer lasting with respect to LP1. In conclusion the N-substituent nature of compounds 6, 7 and 9 shifts the DOR profile of LP1 from antagonism to agonism.

Full text of DOI:10.1016/j.bmc.2017.07.021

Accepted Manuscript
Development of novel LP1-based analogues with enhanced delta opioid receptor
profile
Lorella Pasquinucci, Rita Turnaturi, Orazio Prezzavento, Emanuela Arena,
Giuseppina Aricò, Zafiroula Georgoussi, Rosalba Parenti, Giuseppina
Cantarella, Carmela Parenti
PII:
S0968-0896(17)31128-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.021
BMC 13858
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
25 May 2017
5 July 2017
10 July 2017
Please cite this article as: 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 profile, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.07.021
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Development of novel LP1-based analogues with enhanced delta opioid
receptor profile
a
a*
a
Lorella Pasquinucci , Rita Turnaturi , Orazio Prezzavento , Emanuela
a
a
b
c
Arena , Giuseppina Aricò , Zafiroula Georgoussi , Rosalba Parenti ,
d
e
Giuseppina Cantarella , Carmela Parenti .
a
Department of Drug Sciences, Medicinal Chemistry Section, University of
Catania, Viale A. Doria 6, 95125 Catania, Italy.
b
Laboratory of Cellular Signalling and Molecular Pharmacology, Institute of
Biosciences and Applications, National Center for Scientific Research
“
Demokritos”, Ag. Paraskevi 15310, Athens, Greece.
c
Department of Biomedical and Biotechnological Sciences, Physiology
Section, University of Catania, Catania, Italy
d
Department of Biomedical and Biotechnological Sciences, Pharmacology
Section, University of Catania, Catania, Italy
e
Department of Drug Sciences, Pharmacology and Toxicology Section,
University of Catania, Viale A. Doria 6, 95125 Catania, Italy.
*
Corresponding author. Address: Department of Drug Sciences, University of
Catania, Viale A. Doria 6, 95125 Catania, Italy. Phone: +39 0957384017; e-
mail address: rita.turnaturi@tiscali.it (R.Turnaturi)
KEYWORDS: Mu opioid receptor; Delta opioid receptor; 6,7-Benzomorhan
scaffold; Radioligand competition-binding; GPI and MVD; Pain.
1

Highlights
1
2
3
. Synthesis of LP1 analogues with different N-substituents.
. Compounds 6, 7 and 9 retained MOR but improved DOR affinity.
. Identification of MOR/DOR agonists assessed by GPI and MVD functional
assays.
. Antinociception effects were assessed for compounds 6, 7 and 9 by tail flick
test.
4
5
.The N-substituent nature maintains the LP1 MOR profile but improved the
DOR profile.
2

Abstract
Pain relief achieved by co-administration of drugs acting at different targets is
more effective than that obtained with conventional MOR selective agonists
usually associated to relevant side effects. It has been demonstrated that
simultaneously targeting different opioid receptors is a more effective
therapeutic strategy. Giving the promising role for DOR in pain management,
novel LP1-based analogues with different N-substituents were designed and
synthesized with the aim to improve DOR profile. For this purpose, we
maintained the phenyl ring in the N-substituent of 6,7-benzomorphan scaffold
linked to an ethyl spacer bearing a hydroxyl/methyl or methoxyl group at
carbon 2 or including it in a 1,4-benzodioxane ring. LP1 analogues were
MOR
tested by competition binding assays. As demonstrated Compounds 6 (K
i
=
=
DOR
i
MOR
DOR
i
MOR
2
1
.47 nM, K
= 9.6 nM), 7 (K
i
= 0.5 nM and K
= 0.8 nM) and 9 (K
i
DOR
i
.08 nM, K
= 6.6 nM) retained MOR affinity but displayed an improved
DOR binding capacity as compared to LP1. Moreover, GPI and MVD
functional assays indicated that compounds 6 (IC50= 49.2 and IC50= 10.8 nM),
7
(IC50= 9.9 and IC50= 11.8 nM) and 9 (IC50= 21.5 and IC50= 4.4 nM) showed
a MOR/DOR agonist profile, unlike LP1 that was a MOR agonist/DOR
antagonist (IC50= 1.9 and IC50= 1240 nM). Measurements of their
antinociceptive effect was evaluated by mice radiant tail flick test displaying
for compounds 6, 7 and 9 ED50 values of 1.3, 1.0 and 0.9 mg/kg, i.p.,
respectively. Moreover, the antinociceptive effect of compound 9 was longer
lasting with respect to LP1. In conclusion the N-substituent nature of
compounds 6, 7 and 9 shifts the DOR profile of LP1 from antagonism to
agonism.
3

1
. Introduction
Currently available analgesic drugs are not always efficacious, and pain
control remains a therapeutic need and a major challenge in medical
1
research. Morphine and other opioids, widely used analgesic drugs for the
treatment of moderate to severe pain, elicit their effects primarily through
a
MOR , whose activation determines not only analgesia but also a sequel of
side effects such as sedation, nausea, respiratory depression and
constipation. Moreover, long-term MOR agonist used for management of
chronic pain can lead to exacerbation of analgesic tolerance and physical
2
dependence, all of which limit their clinical application.
DOR activation is becoming an attractive target for treatment of chronic pain
3-8
conditions. DOR and MOR are co-localized and interact both physically and
9
functionally. Simultaneously MOR/DOR targeting by agonist/agonist or
agonist/antagonist ligands results in synergism in terms of increased
10
antinociception and reduces the undesirable side effects. Dual MOR/DOR
ligands, such as the fentanyl-based RV-Jim-C3 and the
benzylideneoxymorphone derivative UMB 246, have been recently developed
1
1-13
as possible approach for acute and chronic pain management.
We have previously synthesized and characterized the benzomorphan-based
1
1
compound LP1 (Figure 1) featured by MOR agonist/DOR antagonist
14-18
profile.
Based upon the interesting pharmacological properties of LP1, our
a
Abbreviations: MOR, mu opioid receptor; DOR, delta opioid receptor; KOR,
kappa opioid receptor; GPI, guinea pig ileum; MVD, mouse vas deferens; K
inhibitory constants; i.p., intraperitoneal; s.c., subcutaneous; TLC, thin-layer
chromatography; GCMS, gas chromatography-mass spectroscopy; Bu SnO,
dibutyltin oxide; pTsCl, p-toluenesulfonyl chloride; TEA, trimethylamine; IC50
concentration for 50% inhibition; K , equilibrium dissociation constant; TFLs,
i
,
2
,
e
tail flick latencies; ED50, effective dose-50; NLX, naloxonazine; nBNI, nor-
Binaltorphimine; NTI, naltrindole.
4

research was thus focused on the identification of its analogues exhibiting an
improved profile towards DOR. A plethora of experimental evidence
demonstrates the unpredictability of agonist and antagonist properties
conferred by N-substituents on 6,7-benzomorphan ligands, we thus explored
their influence in the (−)-cis-N-normetazocine scaffold. We have previously
shown that the replacement of phenyl ring in N-propanamide spacer of LP1
11
with saturated groups resulted in unsuccessful DOR binding. On the other
hand, phenyl replacement with bulkier aromatic groups, such as naphthyl,
19
quinoline and isoquinoline rings, was also not tolerated for interaction with
the DOR binding pocket. Moreover, the bulkier size of the N-substituents
moves the functional profile from agonism to antagonism versus MOR. Thus,
in N-substituent the aromatic ring size and its orientation, in respect to the (−)-
cis-N-normetazocine scaffold, affects the binding and functional properties.
In the present contribution, we designed and synthesized LP1 analogues (6-
1
0, Figure 1) that maintained the phenyl ring in the N-substituent of (−)-cis-N-
normetazocine linked to an ethyl spacer bearing a hydroxyl/methyl or
methoxyl group at carbon 2 or including it in a 1,4-benzodioxane ring. Shorter
and more flexible N-substituent side chain with H-bonding groups at carbon 2
were introduced in order to investigate their influence on the phenyl ring
orientation with respect to (−)-cis-N-normetazocine scaffold. Moreover, two
compounds (6-7) with a determined stereochemistry at carbon 2 were
synthesized to explore the importance of spatial determinants in the proximity
of useful phenyl ring. All synthesized analogues were tested by competition
binding assays in order to assess their affinity and selectivity on MOR, DOR
and KOR binding. Compounds with relevant K
i
values were evaluated by GPI
5

and MVD assays. The antinociceptive effect of most interesting analogues
was assessed by mice tail flick test.
Figure 1. LP1 and its N-analogues 6-10 structures.
2
. Results and Discussion
2
.1. Chemistry
The synthesis of the novel 6,7-benzomorphan compounds is illustrated in
Scheme 1. By means of resolution of commercially available racemic mixture,
the (−)-cis-N-normetazocine was obtained separated from a, as previously
20
reported. Compounds 1-5 were prepared by primary alcohol sulfonylation
2
1
with pTsCl, and TEA in CH
2
Cl
2
with catalytic Bu
2
SnO. Target compounds 6-
1
0 were obtained by alkylation of (−)-cis-N-normetazocine with the respective
16
tosylated alcohols 1-5 (Scheme 1). All synthesized compounds possess a
chiral center at the N-substituent of (−)-cis-N-normetazocine scaffold and
compounds 6 and 7 are single isomers obtained through use of chiral (R) or
(
S) primary alcohols. All other products (8-10) are a racemic mixture. All
1
13
synthesized compounds were characterized by IR, H NMR, C NMR, mass
spectroscopy and elemental analysis.
6

Scheme 1. Synthetic pathway.
2
.2. Evaluation of opioid receptor binding.
All synthesized compounds were tested in vitro by radioligand competition
binding assays to measure affinity and selectivity for MOR, DOR and KOR.
Calculated K are listed in Table 1.
i
Table 1. Opioid receptor binding affinity of N-substituted (−)-cis-N-
normetazocine analogues.
a,b
K
i
(nM) ± SEM
DOR
K
i
ratio
Comp
6
MOR
KOR
DOR/MOR KOR/MOR
2.47 ± 0.3
9.6 ± 0.5
7.30 ± 0.2
3.88
1.6
2.66
6.1
-
2.95
4.44
0.17
14.09
-
7
8
9
0.5 ± 0.2
60 ±1.3
0.8 ± 0.2
160 ± 2.8
6.6 ± 0.6
2190 ± 53
29.1 ± 1
2.22 ± 0.2
10.51 ± 0.9
15.22 ± 0.8
2276 ± 86
110 ± 6
1.08 ± 0.1
1036 ± 34
0.83 ± 0.05
10
LP1
33.8
132.5
a
Values are means ± SEM of three separate experiments, each carried out
in duplicate.
b
3
3
K values were obtained as [ H]DAMGO displacement for MOR, [ H]DPDPE
i
3
displacement for DOR, and [ H]U69,593 displacement for KOR.
7

All compounds showed MOR, DOR and KOR nanomolar affinity with the
exception of ligand 10 that exhibited micromolar affinity versus all three opioid
receptor subtypes tested. In particular, ligands 6-9 retained MOR affinity of
the lead compound LP1, while DOR and KOR affinity is increased. In fact, the
DOR K
i
values of compounds 6, 7 and 9 were three-, thirty-six- and four-times
values of LP1.Moreover, the KOR affinity of compounds 6-9
lower than the K
i
was fifteen-, forty-nine-, ten- and seven-times higher respectively as
compared to LP1. Additionally, the rank order of MOR binding was
7
>9>6>8>>10; the rank order for DOR binding affinity was 7>9>6>>8>>10;
whereas that of KOR was 7>6>8>9>>10.
Tested compounds 6-10 differ from LP1 for the nature and chiral feature of
the N-substituent. As previously reported, the N-phenylpropanamide
substituent of LP1 confers high MOR, significant DOR and negligible KOR
affinity. In compounds 6-10 the lack of the amide group in the side chain of
the N-substituent is well tolerated by MOR and KOR and significantly
improved DOR interaction. Moreover, the increased flexibility of N-substituent,
in comparison to that of LP1, could allow an optimal interaction with the opioid
receptor binding pocket.
The analogue with the (S)-2-hydroxy-2-phenylethyl as N-substituent (7)
showed the best MOR, DOR and KOR affinity profile, while its (R)-isomer (6)
resulted to have K values for MOR, DOR and KOR five-, twelve- and three-
i
times higher, respectively. The hydroxyl group in alpha respect to the phenyl
ring seems to play a role for the binding interaction at MOR, DOR and KOR
probably by a hydrogen bond with the binding pocket of opioid receptors.
Furthermore, by these data emerged the pivotal role of the sterocenter in N-
8

substituent of these isomers. Indeed, it is well known that the stereochemistry
affects the parameters such as potency, intrinsic efficacy and binding affinity
in ligand-opioid receptor interaction. For instance, the MOR/DOR agonist DPI-
3
290, featured by three chiral centers, generates eight isomers each of them
22
with a peculiar profile of affinity. Analogously, the benzomorphan-based
ligand MR2266 and its enantiomer MR2267 showed an opposite profile of
23
efficacy versus KOR.
Compound 9, bearing as N-substituent the (2R/S)-2-methoxy-2-phenylethyl
group, in comparison to compound 7, retained MOR binding profile but
showed a DOR and KOR affinity eight and seven times lower, respectively.
However, in comparison to compound 7 the 2-methoxy-2-phenylethyl
analogue (9) is a diastereoisomeric mixture. Thus, in this case only the
importance of the methoxyl group as hydrogen bond acceptor for the
interaction with opioid receptors emerged.
Compounds 8 and mainly 10 exhibited a lower binding profile for the MOR
and DOR. Specifically, compound 8 with an (2R/S)-2-hydroxy-2-phenylpropyl
group as N-substituent displayed K
hundred, two-hundred and five-times higher, respectively compared with
compound 7. The K values for all opioid receptors were in the micromolar
range for compound 10 that possesses a (2R/S)-2,3-dihydro-1,4-benzodioxin-
-ylmethyl group as N-substituent. These data highlighted that the steric
i
values for MOR, DOR and KOR one-
i
2
hindrance of the carbon in alpha respect to the phenyl group is detrimental
mainly for MOR and DOR binding interaction. Moreover, in compound 10 the
1
,4-benzodioxane ring led to a loss of flexibility of the N-substituent.
2
.3. Evaluation of functional opioid receptor in GPI and MVD.
9

Opioid agonist potency (IC50 values) was determined in GPI (for MOR and
19
KOR) or in MVD assays (for DOR), using previously described protocols. K
e
values for antagonists were determined from the ratio of IC50 values obtained
with tested compounds in the presence and in absence of a fixed known
MOR, DOR and KOR antagonist concentration.
Apart from compound 8, the other ligands listed in Table 2 showed high opioid
agonist potency in GPI and MVD assays. These effects were reversed by
selective opioid antagonists NLX, norBNI and NTI (see K values in Table 2).
e
The agonist potency determined in isolated tissues was in accordance with
the affinity values measured in the binding assays. The minor discrepancies
detected are possibly due to the different structural requirements between
central and peripheral receptors or to the different ability of these compounds
to reach the receptors in the isolated tissue preparations.
Increasing concentrations of compounds 6-9 produced significant and
concentration-related inhibition in electrically stimulated GPI contractions as
summarized in Table 2. The rank order of activity was 7>9>6>>8.
Analogously, except compound 8, increasing concentrations of compounds 6-
9
produced significant and concentration-related decrease in electrically
stimulated MVD contractions (Table 2). The rank order of activity was
>6>7>>8.
9
Compounds 6, 7 and 9 retained a significative MOR potency that resulted
twenty-six-, five- and eleven-times lower, respectively, in comparison to LP1.
18
Notably, regarding the lead compound LP1 , the N-substituent nature of
compounds 6, 7 and 9 shifts the DOR profile from antagonism to agonism. In
particular, the highest IC50 value in MVD assay was detected for compound 9.
1
0

Moreover, high functional selectivity was demonstrated by compound 9 in GPI
versus MOR.
Table 2. IC50 values for opioid effects on the contractility of electrically
stimulated GPI and MVD.
a
b
IC50 (nM) ± SEM
K
e
(nM) ± SEM
NLX
nBNI
NTI
MVD
Comp
6
GPI
MVD
GPI
49.2 ± 2
10.8± 1.3
35 ± 1.6
21 ± 1.3
−
20 ± 1
9 ± 0.5
−
4 ± 0.1
7
8
9
9.9 ± 0.9
194.4 ± 33
21.5 ± 1.3
1.9 ± 0.6
11.8 ± 1
793.5± 43
4.4± 0.7
5.4 ± 0.3
−
3.9 ± 0.3
−
19 ± 0.7
2.3 ± 0.3
>3000
LP1
1240± 44
10.8 ± 0.9
a
b
50% inhibitory concentrations;
e
K were determined from the ratio of IC50
values obtained with tested compounds in the presence and in absence of a
fixed known MOR, DOR and KOR antagonist concentration. The results
summarized represent the mean ± SEM from four independent tissues.
2
.4. In vivo pharmacological evaluation in mouse tail flick assay.
Based on in vitro results, the antinociceptive effect induced by i.p.
administration of compounds 6, 7 and 9, using mice tail-flick test, was
evaluated. All tested compounds, produced a dose-dependent analgesic
effect in the range dose of 0.5-5 mg/kg compared to the group of rats treated
with saline (*p< 0.05 vs saline-treated rats).
Evaluation of antinociceptive effect of compounds 6 and 7 revealed a maximal
effect at 30 min post-administration (Figure 2 and 3, panel A). A much lower
ED50 was measured for compound 6 (1.3 mg/kg i.p.; 0.9-2.1 95% C.l.) and
compound 7 (1.0 mg/kg i.p.; 0.7-1.5 95% C.l.) (Figure 2 and 3, panel B) as
compared to the ED50 value of morphine (2.7 mg/kg s.c.) and LP1 (2.03
1
4
mg/kg s.c.) , respectively.
1
1

Figure 2. (A) Time-course (min) of compound 6-induced analgesia measured
by tail flick test. Results are expressed in seconds (s). Data are means ± SEM
from 6 to 8 mice. *P<0.05 vs saline-treated mice. (B) Analgesic dose-
response curve ± SEM for compound 6 was plotted at 30 min post-treatment.
Figure 3. (A) Time-course (min) of compound 7-induced analgesia measured
by tail flick test. Results are expressed in seconds (s). Data are means ± SEM
from 6 to 8 mice. *P<0.05 vs saline-treated mice. (B) Analgesic dose-
response curve ± SEM for compound 7 was plotted at 30 min post-treatment.
1
2

In vivo evaluation of compound 9 established that the maximal analgesic
threshold was reached between 45 and 60 min after injection and lasted until
1
80 min after treatment (*p< 0.05 vs saline-treated rats) (Figure 4, panel A).
Compared to morphine and LP1, compound 9 determined a significant
analgesic effect but a much lower ED50 of 0.9 mg/kg i.p. (0.6-1.2 95% C.l.)
(
Figure 4, panel B). Moreover, although the morphine analgesic effect started
to decline from 60 min post-administration, the antinociceptive effect of
compound 9 maintained higher threshold values for longer periods of time.
Longer lasting analgesic effects were also measured at 180 and 240 min after
administration (data not shown).
Figure 4. (A) Time-course (min) of compound 9-induced analgesia measured
by tail flick test. Results are expressed in seconds (s). Data are means ± SEM
from 6 to 8 mice. *P<0.05 vs saline-treated mice. (B) Analgesic dose-
response curve ± SEM for compound 9 was plotted at 45 min post-treatment.
Pretreatment with naloxone (3 mg/kg s.c.) 30 min prior to 6, 7 and 9
administrations prevented the analgesic effect induced by these compounds
1
3

at the tested doses (data not shown). These results, consistent with
radioligand binding and GPI and MVD data suggest that compounds 6, 7 and
9
analgesic effect is mediated by the opioid receptors and are much more
improved with respect to morphine and LP1.
. Conclusion
3
In this study we showed that N-substituent nature in 6,7-benzomorphan
scaffold could modulate the affinity and activity towards MOR and DOR. In our
series of ligands, 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, allow an optimal interaction with the opioid binding
pocket. Importantly, it was also highlighted for compounds 6 and 7 the
influence of stereochemistry in the ligand-opioid receptor interaction.
Compounds 6, 7 and 9 exhibit a strong antinociceptive effect consistent with
the in vitro data herein outlined. All compounds retained MOR efficacy of LP1
with improved DOR affinity and shifted DOR functional profile from
antagonism to agonism. An improved activity at DOR gains importance when
3,5
pain develops. Nociceptive stimuli induce DOR trafficking and it has been
demonstrated a possible regulatory role for DOR in the functional interaction
between MOR and DOR. DOR seems to modulate MOR downstream
signaling increasing antinociception and reducing the side effects. Thus, our
new developed compounds 6, 7 and 9 characterized as MOR and DOR
agonists could be good candidates for treatment of chronic pain due to their
potential ability to decrease the MOR induced side effects such as tolerance
9
development.
4
. Experimental section
1
4

4
.1. Chemistry
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. Melting points were determined in open capillary tubes with a Büchi
5
30 apparatus 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 CHCl
3
or MeOH solution with a Perkin-
Elmer 241 polarimeter. Infrared spectra were recorded on a 1600 FT-IR
1
13
Perkin-Elmer instrument. H and C NMR spectra were routinely recorded on
a Varian Inova-200 spectrometer in CDCl solution; chemical shifts δ are
3
expressed in ppm with reference to tetramethylsilane as an internal standard.
GCMS analysis was recorded using a Shimadzu QP500 EI 171 (70 eV).
Elemental analyses (C, H, N) were performed on a Carlo Erba 1106 analyzer
and the analysis results were within ±0.4% of the theoretical values. All
reported compounds had a purity of at least 95%.
4
.2. General procedure for tosylate intermediates (1-5).
To a solution of the appropriate alcohol (10 mmol) in dry CH
added Bu SnO (0.2 mmol) followed by the addition of pTsCl e (10 mmol) and
TEA (10 mmol). The reaction mixture was kept under vigorous stirring at rt for
-6 h. The reaction mixture was quenched by adding water. The solution was
2
Cl (20 mL) was
2
2
1
extracted with dichloromethane and then combined organic phase were
washed with a saturated aqueous solution of brine and dried over anhydrous
1
5

Na
2
SO . The solvent removing in vacuo give a residue that was crystallized or
4
chromatographed to afford the desired the compounds (1-5).
4
.2.1. (R)-2-hydroxy-2-phenylethyl 4-methylbenzenesulfonate (1).
Waxy solid (70%); mp 67-68 °C; ꢀꢁ ꢃꢂ ꢄ = - 50.1° (c 1.0, CHCl
). IR (CHCl )
3
3
-
1 1
3
220, 3020, 1595, 1451, 1355 cm . H NMR (CHCl
.26 (m, 7H), 4.98 (dd, J = 8.4, 3.4 Hz,1H), 4.15 (dd, J = 10.4, 3.4
Hz,1H), 4.04 (dd, J = 8.4, 10.4 Hz,1H), 2.45 (s, 3H).
3
) δ 7.77 (d, 2H), 7.35-
7
4
.2.2. (S)-2-hydroxy-2-phenylethyl 4-methylbenzenesulfonate (2).
Waxy solid (74%); mp 65-67 °C; ꢀꢁ ꢃꢂ ꢄ = + 51.2° (c 1.0, CHCl
). IR (CHCl )
3
3
-
1
1
3
7
4
4
220, 3019, 1597, 1453, 1355 cm . H NMR (CHCl ) δ 7.77 (d, 2H), 7.36-
3
.26 (m, 7H), 4.99 (dd, J = 8.4, 3.4 Hz,1H), 4.15 (dd, J = 10.4, 3.4 Hz,1H),
.04 (dd, J = 8.4, 10.4 Hz,1H), 2.45 (s, 3H).
.2.3. (2R/S)-2-hydroxy-2-phenylpropyl 4-methylbenzenesulfonate (3).
Waxy solid (66%); mp 52-54 °C. IR (CHCl
3
) 3215, 3020, 1598, 1455, 1360
) δ 7.71 (d, 2H), 7.35-7.26 (m, 7H), 4.09-4.04 (m,
H), 2.45 (s, 3H), 1.56 (s, 3H).
.2.4. (2R/S)-2-methoxy-2-phenylethyl 4-methylbenzenesulfonate (4).
-
1
1
cm . H NMR (CHCl
3
2
4
-
1 1
Colorless oil (70%). IR (CHCl ) 3020, 1598, 1451, 1358, 1176 cm . H NMR
3
(
CHCl
3
) δ 7.74 (d, 2H), 7.32-7.26 (m, 7H), 4.44-4.38 (m, 1H), 4.15-4.03
m, 2H), 3.23 (s, 3H), 2.44 (s, 3H).
.2.5. (2R/S)-2,3-dihydro-1,4-benzodioxin-2-ylmethyl
(
4
4-
methylbenzenesulfonate (5).
Waxy solid (74%); mp 79-81 °C. IR (CHCl
3
) 3034, 1595, 1497, 1370, 1269,
-
1
1
1
4
176 cm . H NMR (CHCl
3
) δ 7.8 (d, 2H), 7.35 (d, 2H), 6.84-6.79 (m, 4H),
.38 (m, 1H), 4.26 (m, 3H), 4.05 (m, 1H), 2.46 (s, 3H).
1
6

4
.3. General procedure for synthesis of N-Substituted (−)-cis-N-
normetazocine analogue (6-10).
A mixture of (−)-cis-N-normetazocine (1 equiv), the appropriate tosylate
intermediates (1-5, 1.5 equiv), NaHCO (1.5 equiv) and a catalytic amount of
3
KI was stirred in DMF at 65 °C for 24h. After cooling, the reaction mixture was
filtered and concentrated under vacuum to remove DMF. The resulting
residue was purified by flash chromatography on silica gel column using as
eluent CH
hydrochloride salts by dissolving the free bases in minimum amount of
methanol and adding 1N HCl in Et O to the solution.
.3.1. (2R,6R,11R)-3-((R)-2-hydroxy-2-phenylethyl)-6,11-dimethyl-1,2,3,4,5,6-
2
Cl
2 3
/CH OH (97:3, v/v). The free bases were converted to the
2
4
hexahydro-2,6-methanobenzo[d]azocin-8-ol (6).
White solid (65%); mp: 167-169 °C; ꢀꢁ ꢃꢂ ꢄ - 54.8° (c 1.0, MeOH). H NMR
1
(
CDCl
.96 (m, 1H, J = 8.2 Hz), 6.68 (m, 1H, J = 2.0 Hz), 6.58 (dd, 1H, J = 8.0, 2.0
Hz), 5.09 (d, 1H), 3.41-3.32 (m, 2H), 3.21-3.05 (m, 4H), 2.72-2.67 (m, 1H),
3
, free base) δ 7.42-7.40 (m, 2H), 7.32-7.29 (m, 2H), 7.25-7.22 (m, 1H),
6
2
3
1
4
.13-2.12 (m, 1H), 2.06-2.00 (m, 1H), 1.50-1.48 (m, 1H), 1.34 (s, 3H), 0.90 (d,
13
H). C NMR (CHCl , free base) δ 154.09, 143.06, 142.34, 128.65, 128.44,
3
27.98, 127.4, 126.01, 112.96, 112.18, 68.69, 61.68, 55.57, 46.95, 41.62,
+
1.24, 36.31, 25.42, 25.18, 14.07. MS m/z [M] 337,5. Anal. Calcd for
22
C H
27NO ·HCl: C, 70,7%; H, 7,5%; N, 3,7%. Found: C, 70,4%; H, 7,7%; N,
2
3
,6%.
.3.2. (2R,6R,11R)-3-((S)-2-hydroxy-2-phenylethyl)-6,11-dimethyl-1,2,3,4,5,6-
4
hexahydro-2,6-methanobenzo[d]azocin-8-ol (7).
ꢃꢄ
1
White solid (68%); mp: 168-169 °C; ꢀꢁ - 33.4° (c 1.0, MeOH). H NMR
ꢂ
1
7

(
CDCl
.96 (m, 1H, J = 8.2 Hz), 6.68 (m, 1H, J = 2.0 Hz), 6.58 (dd, 1H, J = 8.2, 2.0
Hz), 6.97-6.95 (m, 1H), 6.68-6.67 (m, 1H), 6.59-6.58 (m, 1H), 5.10 (d, 1H),
3
, free base) δ 7.42-7.39 (m, 2H), 7.32-7.30 (m, 2H), 7.25-7.22 (m, 1H),
6
3
2
.41-3.32 (m, 2H), 3.21-3.05 (m, 4H), 2.71-2.67 (m, 1H), 2.14-2.12 (m, 1H),
13
.06-2.00 (m, 1H), 1.50-1.48 (m, 1H), 1.33 (s, 3H), 0.89 (d, 3H). C NMR
, free base) δ 154.10, 143.07, 142.33, 128.65, 128.44, 127.99, 127.4,
(
CHCl
3
1
2
7
4
26.03, 112.96, 112.17, 68.69, 61.69, 55.56, 46.95, 41.62, 41.25, 36.31,
+
5.43, 25.18, 14.09. MS m/z [M] 337,6. Anal. Calcd for C22
H
27NO ·HCl: C,
2
0,7%; H, 7,5%; N, 3,7%. Found: C, 70,5%; H, 7,6%; N, 3,5%.
.3.3. (2R,6R,11R)-3-(2-hydroxy-2-phenylpropyl)-6,11-dimethyl-1,2,3,4,5,6-
hexahydro-2,6-methanobenzo[d]azocin-8-ol (8).
White solid (66%); mp: 134-136 °C; ꢀꢁ ꢃꢂ ꢄ - 61.7° (c 1.0, MeOH). H NMR
1
(
CDCl
3
, free base) δ 7.65-7.53 (m, 2H), 7.42-7.24 (m, 3H), 6. 90 (m, 1H, J =
.2 Hz), 6.62 (m, 1H, J = 2.2 Hz), 6.38 (m, 1H, J = 8.2, 2.2 Hz), 3.38-3.32 (m,
H), 3.12-2.7 (m, 2H), 2.49-2.39 (m, 2H), 1.96-1.85 (m, 1H), 1.65 (d, 3H), 1.28
8
4
13
(
s, 3H), 1.11-1.04 (m, 1H), 0.79 (d, 3H). C NMR (CDCl
3
, free base) δ
1
1
1
56.71, 146.42, 140.50, 128.88, 128.56, 127.54, 125.32, 123.21, 114.21,
12.17, 65.34, 60.29, 48.57, 42.92, 38.31, 35.02, 29.83, 27.11, 24,51, 23,37,
+
3.43. MS m/z [M] 351.5. Anal. Calcd for C23
H
29NO ·HCl: C, 71,2%; H, 7,8%;
2
N, 3,6%. Found: C, 71,0%; H, 7,9%; N, 3,5%.
.3.4. (2R,6R,11R)-3-(2-methoxy-2-phenylethyl)-6,11-dimethyl-1,2,3,4,5,6-
4
hexahydro-2,6-methanobenzo[d]azocin-8-ol (9).
White solid (62%); mp: 177-180 °C; ꢀꢁ ꢃꢂ ꢄ - 59.9° (c 1.0, MeOH). H NMR
1
(
CHCl
3
, free base) δ 7.24-7.19 (m, 5H), 6.82 (d, 1H, J = 8.2 Hz), 6.65 (d, 1H, J
=
2.2 Hz), 6.55 (dd, 1H, J = 8.0, 2.4 Hz), 4.36 (d, 1H), 3.13 (s, 3H), 3.12-3.07
1
8

(
m, 1H), 2.93-2.54 (m, 5H), 2.35-2.11 (m, 1H), 1.93-1.89 (m, 2H), 1.24 (s, 3H),
1
3
1
1
8
.18-1.10 (m, 1H), 0.74 (d, 3H). C NMR (CHDl , free base) δ 154.47,
3
42.97, 140.85, 128.42, 128.13, 127.71, 126.61, 125.85, 113.18, 112.36,
1.62, 62.10, 58.08, 56,72, 46.78, 41.41, 40.67, 36.05, 25.30, 24.05, 14.11.
+
MS m/z [M] 351,4. Anal. Calcd for C23
H
29NO ·HCl: C, 71,2%; H, 7,8%; N,
2
3
,6%. Found: C, 71,1%; H, 7,9%; N, 3,5%.
.3.5. (2R,6R,11R)-3-((2,3-dihydrobenzo[b][1,4]dioxin-2-yl)methyl)-6,11-
4
dimethyl-1,2,3,4,5,6-hexahydro-2,6-methanobenzo[d]azocin-8-ol (10).
ꢃꢄ
1
White solid (48%); mp: 176 °C dec.; ꢀꢁ - 42.8°(c 1.0, MeOH). H NMR
ꢂ
(
CDCl
3
, free base) δ 6.87 (d, 1H, J = 8.2 Hz), 6.83-6.78 (m, 4H), 6.65 (m, 1H,
J = 2.2 Hz), 6.54 (dd, 1H, J = 8.0, 2.2 Hz), 4.27-4.18 (m, 2H), 3.99-3.90 (dd,
1
2
H), 2.96-2.82 (m, 4H), 2.57-2.39 (m, 2H), 2.24-2.18 (m, 1H), 1.83-1.73 (m,
13
H), 1.26 (s, 3H), 1.15-1.14 (m, 1H), 0.76 (d, 3H). C NMR (CDCl , free
3
base) δ 156.60, 145.95, 145.77, 131.05, 130.84, 124.09, 123.85, 119.99,
1
3
19.67, 115.54, 114.86, 74.61, 69.60, 60.36, 57.66, 49.58, 44.52, 43.99,
+
8.78, 28.07, 26.86, 16.80. MS m/z [M] 365.5. Anal. Calcd for
23
C H
27NO ·HCl: C, 68,7%; H, 7.0%; N, 3,5%. Found: C, 68,5%; H, 7.3%; N,
3
3
4
4
,3%.
.4. Biological Evaluation.
.4.1 Drugs.
3
3
3
[
H]-DAMGO, [ H]-DPDPE and [ H]-(+)-U69,593 were purchased by Perkin-
Elmer. NTI, U50,488, NLX, Morphine sulfate, nor-BNI and naloxone
chlorhydrate were purchased from Tocris. Tested compounds were dissolved
in pyrogen-free isotonic saline (Baxter Healthcare, Deerfield, IL) and DMSO
(
5%) and were administered to mice i.p.
1
9

4
.4.2.
Animals. Male Swiss CB1 mice (Harlan Laboratories, S.Pietro al Natisone
UD)) weighing 25-30 g were housed six to a cage. Animals were kept at a
(
constant room temperature (25 ± 1 °C) under a 12:12 h light and dark cycle
with free access to food and water. Each mouse was used for only one
experiment. Experimental procedures were approved by the Local Ethical
Committee (IACUC) and conducted in accordance with international
guidelines as well as European Communities Council Directive and National
Regulations (CEE Council 86/609 and DL 116/92).
4
.4.3. In vitro competitive radioligand binding assay.
MOR, DOR and KOR binding experiments were performed on rat or guinea
pig brain membranes according to experimental protocol described by Spetea
24
et al. Protein concentration was determined by Lowry’s method using bovine
25,26
serum albumin as standard.
Binding experiments at MOR and DOR were
carried out by incubating 0.4 mg/ml and 0.5 mg/ml of rat brain membrane
3
proteins, respectively for 45 min at 35 °C either with 1 nM [ H]-DAMGO (48.4
3
Ci/mM) or 2 nM [ H]-DPDPE (52.7 Ci/mM) in 50 mM Tris-HCl (pH 7.4).
Regarding KOR binding assays, guinea pig brain membranes (0.5 mg/ml)
3
were incubated for 30 min at 30 °C with 1 nM [ H]-(+)-U69,593 (42.69 Ci/mM).
−5
−11
Test compounds were added in concentration ranging from 10 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 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 cocktail and counted on
2
0

a Beckman LS 6500 liquid scintillation counter. K
using the EBDA/ LIGAND program purchased from Elsevier/Biosoft.
.4.4. In vitro functional assay.
GPI preparation. Sections of GPI longitudinal muscle/myenteric plexus were
i
values were calculated
4
27
19
prepared according to Kinney et al. with minor modifications. Male
Dunkin–Hartley guinea pigs (200–300 g) (Harlan Laboratories, S.Pietro al
Natisone (UD)) were killed by decapitation. The intestines were exteriorized,
the ileo-caecal junction located and 5 cm of the terminal ileum discarded.
Approximately 30 cm of the terminal ileum was removed and the lumen
flushed with Krebs solution (in mM: 118 NaCl, 4.75 KCl, 2.45 CaCl
2
, 1.71
MgCl , 25.0 NaHCO , 0.93 KH PO , 11 glucose). Four 2 cm long segments of
2
3
2
4
the ileum were secured on to a Perspex holder supporting two parallel
platinum wire electrodes and placed in a 20 ml isolated organ bath containing
Krebs solution at 37.0 °C and bubbled with 5% CO
2
/95% O . The strips were
2
placed under a 2 g load and contractility measured using an appropriate
transducer connected to a PowerLab 4/20 recorder (ADInstruments, Castle
Hill, NSW, Australia). After 60 min equilibration, maximal contractions of the
tissue were elicited by transmural stimulation using single pulses (0.1 Hz, 0.3
ms, 200 mA) delivered by a Digitimer multisystem D330 stimulator. Following
a 60-90 min equilibrium period, during which the Krebs solution was changed
several times, test compounds were added cumulatively, allowing a minimum
of 5 min before additional compound was added to the bath.
4
.4.5. MVD preparation.
Preparation of MVD was carried out as previously described by Hughes et
28
19
al. with minor modifications. CD1 mice (30–35 g) (Harlan Laboratories,
2
1

S.Pietro al Natisone (UD)) were killed by stunning and both vasa deferentia
were dissected out. They were gently stripped of adhering fat, connective
tissue and blood vessels and mounted on to a Perspex holder supporting two
parallel platinum wire electrodes and placed in a 20 ml isolated organ bath
containing modified Krebs solution (in mM: 118 NaCl, 4.75 KCl, 2.45 CaCl
5.0 NaHCO , 0.93 KH PO , 11 glucose) at 37.0 °C and bubbled with 5%
CO /95% O . Contractions were recorded at a constant tension of 0.5 g by an
appropriate transducer connected to PowerLab 4/20 recorder
ADInstruments, Castle Hill, NSW, Australia). After 30 min equilibration,
2
,
2
3
2
4
2
2
a
(
stimulation pulses were delivered at 20 Hz, 0.5 ms, 1 s duration at 40 s
intervals by a Digitimer multisystem D330 stimulator. Following a 60 min
equilibrium period, during which the Krebs solution was changed several
times, test compounds were added cumulatively, allowing a minimum of 5 min
before additional compound was added to the bath.
4
.4.6. Data analysis.
The effectiveness of a given compound to inhibit electrically induced
contraction was measured as the percentage change from baseline. IC50 were
determined by linear regression of logarithmic concentration-effect curves
(
Prism v. 3.0, GraphPad) using the mean response of at least three separate
trials as the given response for a single concentration.
The pre-incubation with selective opioid antagonists (NLX, norBNI and NTI, 1
μM) to block opioid effects on the tissue, was 20 min with the exception of
norBNI, where it was 30 min. K values of antagonists were calculated
e
29
according to the single dose method. Data are presented as the mean ±
SEM. Statistical analysis was performed with one-way ANOVA followed by
2
2

Newman–Keuls post hoc test. The results were considered significantly
different when p value was less than 0.05.
4
.5. In vivo pharmacology. Nociceptive Test.
24
Nociception was evaluated by the radiant heat tail-flick test. Briefly, it
consisted of irradiation of the lower third of the tail with an infrared source
(
Ugo Basile, Comerio, Italy). The day before the experiment, rats 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 4 s and was calculated as the average of the
first three measurements, which were performed at 5 min intervals. A cutoff
latency of 20 s was established to minimize damage to the tail. Post-treatment
TFLs were determined at 30, 60, 90, 120, 180, and 240 min after i.p. injection.
4
.3.8. Statistical Analysis. All values are 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
30, 31
calculation of 95% confidence limits (95% CL) by the method of Bliss.
Acknowledgements
This work was supported by University of Catania (FIR2014) to Carmela
Parenti. The authors gratefully acknowledge Fabbrica Italiana Sintetici (Italy)
for providing (±)-cis-N-normetazocine.
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[
6
2
8

Figure legends
Figure 1. LP1 and its N-analogues 6-10 structures.
Figure 2. (A) Time-course (min) of compound 6-induced analgesia measured
by tail flick test. Results are expressed in seconds (s). Data are means ± SEM
from 6 to 8 mice. *P<0.05 vs saline-treated mice. (B) Analgesic dose-
response curve ± SEM for compound 6 was plotted at 30 min post-treatment.
Figure 3. (A) Time-course (min) of compound 7-induced analgesia measured
by tail flick test. Results are expressed in seconds (s). Data are means ± SEM
from 6 to 8 mice. *P<0.05 vs saline-treated mice. (B) Analgesic dose-
response curve ± SEM for compound 7 was plotted at 30 min post-treatment.
Figure 4. (A) Time-course (min) of compound 9-induced analgesia measured
by tail flick test. Results are expressed in seconds (s). Data are means ± SEM
from 6 to 8 mice. *P<0.05 vs saline-treated mice. (B) Analgesic dose-
response curve ± SEM for compound 9 was plotted at 45 min post-treatment.
Scheme 1. Synthetic pathway.
Graphical abstract
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