Synthesis and antinociceptive activity of pyrrolidinylnaphthalenes

Article abstract of DOI:10.1016/S0968-0896(00)00117-6

In this paper the synthesis of the racemates (2R,3S/2S,3R)-1,2-dimethyl-3-[2-(6-substituted naphthyl)]-3-hydroxypyrrolidine 1b-d [(2R,3S/2S,3R)-1b-d] are reported. Compounds 1b-d were prepared by reaction of the racemic 1,2-dimethyl-3-pyrrolidone 2 with the lithiation product obtained from 2-bromo-6-substituted naphthalene 3b-d. Pharmacological properties of (2R,3S/2S,3R)-1a-d are also described. Analgesic activity was investigated by the hot plate test and binding affinities towards μ, δ and κ opioid receptors were evaluated. A preliminary evaluation of the in vivo side-effects was also accomplished using the rota-rod test. Interesting antinociceptive activity was shown by all compounds and in particular by 1d, which is the most active compound, since it is six-fold more potent than morphine and has lower side effects on the locomotory activity. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00117-6

Bioorganic & Medicinal Chemistry 8 (2000) 1925±1930
Synthesis and Antinociceptive Activity of Pyrrolidinylnaphthalenes
Simona Collina,a,* Ornella Azzolina,a Dina Vercesi,a Massimo Sbacchi,b
Mark Albert Scheideler,b Annalisa Barbieri,c Enrica Lanzac and Victor Ghislandia
a
Á
Dipartimento di Chimica Farmaceutica, Universita di Pavia, viale Taramelli, 12, I-27100, Pavia, Italy
bDepartment of Biology, SmithKline Beecham, via Zambeletti, I-20021 Baranzate di Bollate, Milan, Italy
Á
Dipartimento di Farmacologia sperimentale ed applicata, Universita di Pavia, viale Taramelli, 14, I-27100, Pavia, Italy
c
Received 27 January 2000; accepted 14 April 2000
AbstractÐIn this paper the synthesis of the racemates (2R,3S/2S,3R)-1,2-dimethyl-3-[2-(6-substituted naphthyl)]-3-hydroxypyrrolidine
1b±d [(2R,3S/2S,3R)-1b±d] are reported. Compounds 1b±d were prepared by reaction of the racemic 1,2-dimethyl-3-pyrrolidone 2 with
the lithiation product obtained from 2-bromo-6-substituted naphthalene 3b±d. Pharmacological properties of (2R,3S/2S,3R)-1a±d are
also described. Analgesic activity was investigated by the hot plate test and binding anities towards m, d and k opioid receptors were
evaluated. A preliminary evaluation of the in vivo side-e€ects was also accomplished using the rota-rod test. Interesting antinociceptive
activity was shown by all compounds and in particular by 1d, which is the most active compound, since it is six-fold more potent than
morphine and has lower side e€ects on the locomotory activity. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Now we report our study on the structure±activity rela-
tionships of 1,2-dimethyl-3-[2-(6-hydroxynaphthyl)]-3-
hydroxypyrrolidine (2R,3S/2S,3R)-1a,7 and its structural
analogues 1,2-dimethyl-3-[2-(6-substituted naphthyl)]-3-
hydroxypyrrolidines 1b±d (Fig. 1).
Our research during recent years has addressed the study of
compounds with antinociceptive activity that interact with
the opioid receptor system. The analgesia process of many
opioid compounds is accompanied by multiple undesired
side e€ects often due to the activation of multiple subtypes
of receptors. Therefore, it is not always possible to
establish clear relationships between the activation of
speci®c opioid receptors and consequent numerous phar-
macological e€ects. The development of ligands highly
selective for each receptor type is an important goal since
these ligands could be very useful for investigating the
biological e€ects produced by the involvement of di€erent
receptors and could also be potential therapeutic agents.
In this paper the synthesis of compounds 1b±d and the
evaluation of the pharmacological properties of com-
pounds 1a±d by in vivo and in vitro methods are
described. Antinociceptive activity was evaluated by the
hot plate test (HPT). Binding anities of the com-
pounds to m, d and k opioid receptors were investigated
in vitro, by opioid receptor binding assays, and in vivo
by means of HPT. Rota-rod test (RRT) was also per-
formed to investigate the e€ects of the compounds on
motor coordination.
Many peptide ligands with opioid activities have been
investigated and the role of the topochemical features has
already been emphasized.1 3 Nonpeptide ligands have
also been studied.4 In this ®eld we have examined the non-
peptide ligands dialkylaminoalkylnaphthalenes5,6 and
cycloaminoalkylnaphthalenes.7 These compounds are
structurally related to the heterosteroid 17-methyl-17-aza-
equilenine (Fig. 1) that was already investigated in opioid
analgesia studies and was found to possess the most
analgesic properties among phenolic heterosteroids.8,9
Results and Discussion
The synthesis procedure of 1,2-dimethyl-3-[2-(6-sub-
stitutednaphthyl)]-3-hydroxypyrrolidines 1b±d is reported
.
in Scheme 1. The pyrrolidinols (2R,3S/2S,3R)-1b±d HCl
were prepared by reaction of the pyrrolidone (R,S)-2
with the lithiation products obtained by treating the
appropriate 2-bromo-6-substituted-naphthalenes 3b±d
with tert-BuLi and by successive treatment with 10%
HCl. Using tert-BuLi instead of n-BuLi and less drastic
.
cooling of the reaction mixuture, 1b±d HCl were
7
*Corresponding author. Tel.: +39-382-507376; fax: +39-382-422975;
e-mail: collina@chifar.unipv.it
.
obtained in higher yields than 1a HCl.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00117-6

1926
S. Collina et al. / Bioorg. Med. Chem. 8 (2000) 1925±1930
Figure 1. 17-Methyl-17-azaequilenine and pyrrolidinylnaphthalenes.
Scheme 1. Synthesis route of 1b±d.
The con®guration of the new chiral center C3 was pre-
dicted on the basis of our previous experience with the
synthesis of the analogous 1,2-dimethyl-3-[2-(6-hydroxy-
bear any substituent on the naphthalene nucleus. Thus
the activity in vivo seems to be related to the structural
features of this moiety since substantial di€erences were
recorded in the response to painful stimulus, according
to the following sequence: OH<F<OCH3<H, as
shown in Figure 2a.
7
.
naphthyl)]-3-hydroxypyrrolidine (2R,3S/2S,3R)-1a HCl.
As already shown, the reaction occurs via a nucleophy-
lous attack of the naphthalenic anion at the prochiral
center of the pyrrolidone 2 on the less hindered side, con-
.
sequentely 100% pure (2R,3S/2S,3R)-1b±d HCl diaster-
The antinociceptive activity of the 1a±d compounds was
found to be completely reversed by the non speci®c opioid
antagonist naloxone (NLX) administered at 10mg/kg,
con®rming that they are opioid analgesics. In order to
identify which opioid receptor type was mainly involved in
the antinociceptive activity, a preliminary investigation on
selective opioid antagonists was performed by the HPT (a
model that produces low variability). As shown in Table
3 and in Figure 2b, the activities of all compounds were
in¯uenced to a various extent by naloxone 0.5 mg/kg
(NLX, a m speci®c antagonist when used at low dose),
naltrindole 1 mg/kg (NTN, a d speci®c antagonist) and
nor-binaltorphimine 5 mg/kg (nor-BNI, a k speci®c
antagonist). In particular it is evident that the anti-
nociceptive activity of 1a, 1c and 1d is highly antag-
onized by NTN and consequently we can suppose that
the activation of mainly d receptors is involved in the
analgesic properties of these compounds. However,
eomers were obtained as shown from 1H NMR analysis of
the crude products. The structures of the compounds were
con®rmed by means of elemental analysis (Table 1) and
spectroscopic measurements (IR, 1H NMR, 13C NMR).
In our recent work7 we found that the 1,2-dimethyl-3-[2-
(6-hydroxynaphthyl)]-3-hydroxypyrrolidine 1a displayed
in vivo an analgesic activity comparable to that of
morphine, exerting antinociceptive e€ects in HPT per-
formed on mice. Therefore, the analogous compounds
that we synthesized for this study were tested in the
HPT and the results are shown in Table 2.
It is interesting to note that all compounds showed
strong analgesic properties with potency relative to
morphine ranging from 0.78 to 6.12. The analgesic
activity is particularly noticeable for 1d which does not
Table 2. Analgesic activity in the hot plate test
Table 1. Analytical data
Compound
AD50
mg/kg
Conf.
limits
AD50
mmol/kg
Relative
potency
(vs morphine)
Calcd.%
H
Found%
H N
Compound
Formula
C
N
C
.
Morphine HCl 3H2O 5.38 3.89±7.43
(2R, 3S/2S, 3R)-1a HCl 5.41 1.65±3.41
(2R, 3S/2S, 3R)-1b HCl 4.12 2.56±6.62
.
(2R, 3S/2S, 3R)-1c HCl 2.11 1.11±4.03
14.3
18.4
13.9
6.8
1.00
0.78
1.03
2.09
6.12
.
.
.
1b HCl
C
C
16H19NOFCl 64.97 6.47 4.74 65.02 6.54 4.48
17H22NO2Cl 66.33 7.20 4.55 66.50 7.15 4.28
C16H20NOCl 69.18 7.26 5.04 69.24 7.45 4.91
10H6BrF 53.37 2.69 53.06 2.58
.
1c HCl
.
1d HCl
3b
.
(2R, 3S/2S, 3R)-1d HCl 0.65 0.21±1.99
C
Ð
Ð
2.3

S. Collina et al. / Bioorg. Med. Chem. 8 (2000) 1925±1930
1927
Table 3. Ecacy of NLX, NTN, nor-BNI as selective antagonists of
m, d and k opioid receptors
either by an interaction with a receptor having a stimu-
latory activity on an opioid neuron, such as Cholecys-
tokinin and neuropeptide FF analogues.10,11
Compound
% of inhibitiona
NTN
NLX
(0.5 mg/kg)
nor-BNI
Furthermore, in order to assess the possible non-speci®c
muscle-relaxant or sedative e€ects of the compounds
1a±d (which could generate false positives in the HPT)
the integrity of motor coordination of the mice was eval-
uated with the rota-rod test (RRT) to distinguish analge-
sia from drug-induced motor changes. The monitoring
was e€ected after 30 s and was repeated after 120 s.
After 30 s all compounds were found to in¯uence the
locomotory activity to a lesser extent than morphine. In
fact, the percentage of mice that remained on the bar
ranges from 75% (compound 1c) to 100% (compounds
1a±b±d), whereas only 65% of the mice treated with mor-
phine remained on the bar. When motor coordination
was tested for a longer period of time (120 s) the seda-
tive e€ects of compound 1d were found again to be
inferior to those of morphine (75% instead of 50% of
the mice remained on the bar). Experimental data are
reported in Table 4.
(2R, 3S/2S, 3R)-1a.HCl 46.0 (Æ7.1)
(2R, 3S/2S, 3R)-1b.HCl 73.8 (Æ5.8)
(2R, 3S/2S, 3R)-1c.HCl 86.1 (Æ3.3)*
(2R, 3S/2S, 3R)-1d.HCl 22.1 (Æ5.3)
100**
51.8 (Æ4.6) 54.5 (Æ5.7)
91.3 (Æ4.8)**
100**
100**
16.2 (Æ5.11)
28.9 (Æ5.4)
aIn brackets the standard error is reported. *P<0.05; **P< 0.01.
there is evidence from the results of the in vitro binding
assays that all compounds exhibit very poor anity for
opioid receptors (Ki values of the mmolar order).
This knowledge suggests that compounds 1a±d produce
antinociceptive e€ects by an indirect activation of the
opioid system. This hypothesis could justify the results of
HPT in the presence of receptor subtype selective antago-
nists (NLX, NTN and nor-BNI) if an inhibition of speci®c
enzymes, responsible for degradation of endogenous
opioid peptides (endorphins, dynorphins and enkephalins)
is invoked.
In conclusion, the pyrrolidinylnaphthalenes investigated in
this work possess very interesting pharmacological prop-
erties. It has been shown that both the analgesic activity
(HPT) as well as the side e€ects on locomotory activity
(RRT) are related to the features of the naphthalene
moiety. Nevertheless, on the basis of actual knowledge,
any correlation can not be done between the structural
features of the compounds and their intrinsic activity.
Di€erences in biological e€ects could depend also on dif-
ferent metabolic stability or pharmacokinetic properties.
A wide pharmacological screening in vitro is in progress
on the most potent compound 1d, to investigate the mode
of action of our compounds and to establish whether and
which enzymatic or receptor system is involved in the
biological activities. Antinociceptive properties of our
compounds may result either by an inhibition at enzy-
matic level of endogenous opioid peptides degradation
Anyway, owing to its pharmacological pro®le (2.3 mmol/
kg the AD50 in the HPT and low side e€ects evident
from the RRT), 1d is the most interesting compound,
thus it will be considered for further investigation in
order to elucidate the mode of action of these novel
compounds. Furthermore, since the stereoselectivity can
play an important role on the drug±receptor interaction,
our purpose in the near future will be the study of the
single stereoisomers.
Experimental
Chemistry
General methods. Melting points were determined with a
Buchi apparatus and are uncorrected. Elemental analyses
Table 4. Locomotor activity in the rota-rod test: percentage of mice
which remain on the bar after 30 or 120 s
Compound
.
30 s
120 s
65a
100
100
75
50a
25b
12.5b
37.5b
75
.
Morphine HCl 3H2O
.
(2R, 3S/2S, 3R)-1a HCl
(2R, 3S/2S, 3R)-1b HCl
(2R, 3S/2S, 3R)-1c HCl
.
.
.
(2R, 3S/2S, 3R)-1d HCl
100
Figure 2. (a) In¯uence of the substituent on the antinociceptive activ-
ity of 1a±d; (b) Inhibition of the antinociceptive activity of 1a±d from
selective opioid antagonists.
aP<0.05.
bP<0.01.

1928
S. Collina et al. / Bioorg. Med. Chem. 8 (2000) 1925±1930
were carried out with a Perkin±Elmer 240 C,H,N analyzer
and were within Æ0.4% of the theoretical values. IR
spectra were obtained on a Perkin±Elmer 682 spectro-
photometer and 1H NMR and 13C NMR spectra [TMS as
internal standard (d=0.00)] were obtained using a Bruker
AMX 400 (1H 400 MHz, 13C 100.617 MHz) apparatus.
Di€erential scanning calorimetry was carried out with a
Mettler TA 4000 apparatus equipped with DSC 25 cell.
Detection of compounds in TLC was done with UV light
or iodine vapor. ICN silica gel 60 (70±230 mesh) was used
for ¯ash chromatography. Anhydrous sodium sulfate was
always used to dry organic solutions. Evaporation was
performed in vacuum with a rotatory evaporator. All
reagents and solvents were purchased from commercial
suppliers and employed without further puri®cation.
evidenced only an endothermic process corresponding
to the melting point of the substances; no thermal phe-
nomena attributable to the evaporation of the crystal-
lization solvent were present.
ꢀ
.
(2R,3S/2S,3R)-1b HCl. 44.1% yield; mp 202±204 C
(isopropyl alcohol); TLC analysis [stationary phase:-
Merck silica gel 60 F254; mobile phase: hexane 87/isopro-
pyl alcohol 13/methyl alcohol/(C2H5)2NH 2]:Rf 0.34. IR
(nujol) main absorptions (cm 1): 3250, 2675, 1610, 1510,
1
1407, 1190, 1103, 969, 949, 897, 862, 802; H NMR (in
CD3OD) d: 1.15 (d, 3H, CH3CH, J=6.5); 2.28 (ddd, 1H,
HCHCH2N, Jgem=13.7, Jvic=3.4±8.6); 2.75 (ddd, 1H,
HCHCH2N, Jgem=13.7, Jvic=8.6±11.6); 2.95 (s, 3H,
CH3N); 3.42 (t, 1H, HCHN, Jgem=11.26, Jvic=4); 3.70 (q,
1H, CH3CH, J=6.4); 3.90 (dt, 1H, HCHN, Jgem=11.26,
Jvic=8.4±8.4); 7.28 (dt, 1H, aromatic, CH 7, J=2.5±9.0±
9.0); 7.49 (d, 1H, aromatic, CH 5, J=9.8); 7.62 (d, 1H,
aromatic, CH 3, J=8.7); 7.85 (d, 1H, aromatic, CH 4,
J=8.7); 7.90 (dd, 1H, aromatic, CH 8, J=5.6); 8.03 (s,
1H, aromatic, CH 1); 13C NMR (in CD3OD) d: 164.32
(C4, aromatic); 132.31, 132.19, 129.32, 126.10, 125.46,
118.18, 117.84, 111.96 and 111.68 (9C, aromatic, C8,
C10, C9, C1, C2, C6, C7, C3 and C5); 82.27 (C3); 73.66
(C2); 55.05 (C5); 39.93 and 39.70 (NCH3 and C4); 8.70
(C2CH3). Anal. C16H18FNO.HCl (C,H,N).
2-Bromo-6-¯uoronaphthalene 3b. The synthesis of 3b
was essentially accomplished according to Newman et
al.12 The reaction of 2-bromo-6-naphthol with ammo-
nium sul®te and 30% ammonium hydroxide under
pressure (27 h at 150 ꢀC) produced the crude brown-
yellow 2-bromo-6-naphthylamine (mp 118±120 ꢀC).
This compound was puri®ed by ¯ash chromatography
(mobile phase CH2Cl2 70/hexane 30) and the recovered
colorless solid (mp 120±122 ꢀC) was converted to the
corresponding diazonium hexa¯uorophosphate (mp
107±110 ꢀC) by treatment at 0 ꢀC with 3.5% HCl, 50%
NaNO2 and 60% hexa¯uorophosphoric acid. The ther-
mal decomposition of the diazonium salt produced the
crude 3b which was puri®ed by sublimation and crys-
tallization (90% aqueous MeOH); mp 63±65 ꢀC; TLC
analysis [stationary phase: Merck RP-18, F254; mobile
phase: 85% (v/v) aqueous MeOH]: Rf 0.31. IR (nujol)
main absorptions (cm 1): 3070, 1625, 1598, 1572, 1505,
1260, 1250, 1200, 1140, 1118, 1062, 960, 905, 890, 878,
ꢀ
.
(2R,3S/2S,3R)-1c HCl. 70.7% yield; mp 204±206 C (iso-
propyl alcohol 90/H2O 10); TLC analysis [stationary
phase: Merck silica gel 60 F254; mobile phase: hexane 87/
isopropyl alcohol 13/methyl alcohol 3/(C2H5)2NH 2]:Rf
0.28. IR (nujol) main absorptions (cm 1): 3220, 2662, 1630,
1605, 1200, 1028, 962, 905, 850, 812; 1H NMR (in CD3OD)
d: 1.15 (d, 3H, CH3CH, J=6.5); 2.28 (ddd, 1H, HCH
CH2N, Jgem=13.5, Jvic=3.5±8.5); 2.74 (ddd, 1H, HCH
CH2N, Jgem=13.5, Jvic=8.5±11.5); 2.95 (s, 3H, CH3N);
3.42 (dt, 1H, HCHN, Jgem=11.3, Jvic=3.5); 3.68 (q, 1H,
CH3CH, J=6.5); 3.85 (s, 3H, OCH3); 3.895 (dt, 1 H,
HCHN, Jgem=11.3, Jvic=8.5, 8.5); 7.10 (dd, 1H, aro-
matic, CH 5, J=3.0±9.0); 7.19 (dt, 1H, aromatic, CH 7,
J=2.5); 7.53 (dd, 1H, aromatic, CH 3, J=2.0±9.0); 7.73
(d, 1H, aromatic, CH 4, J=9.0); 7.77 (d, 1H, aromatic,
CH 8, J=8.7); 7.91 (s, 1 H, aromatic, CH 1). 13C NMR
(in CD3OD) d: 151.49 (C4, aromatic); 128.46, 127.42,
122.42, 121.83, 120.22, 117.22, 116.22, 112.06 and 98.32
(9 C, aromatic, C8, C10, C1, C9, C2, C6, C7, C3 and
C5); 73.75 (C3); 65.10 (C2); 47.46 (C5); 46.36 (OCH3);
41.27±39.99 (C2CH3); 31.06 and 31.29 (NCH3 and C4).
1
868, 815. H NMR (in CD3OD) d: 7.30 (dt, 1H, CH 7,
J=2.5±9.0±9.0; 7.47 (dd, 1H, CH 5, J=2.5±9.5); 7.53
(ddd, 1H, CH 3, J=9.0±2.0±1.0); 7.75 (d, 1H, CH 4,
J=9.0); 7.83 (dd, 1H, CH 8, J=5.5±9.0); 8.02 (d, 1H,
CH 1, J=2.0). Anal. C10H6BrF (C,H).
General procedure for the synthesis of (2R,3S/2S,3R)-
1,2-dimethyl-3-[2-(6-substituted-naphthyl)]-3-hydroxypyr-
.
rolidines hydrochlorides [(2R,3S/2S,3R)-1b±d HCl]. The
synthesis of 1b±d was performed essentially according to
the procedure that we have already reported for the
preparation of 1a, suitably modi®ed, as described.7 A
1.7 M solution of tert-BuLi (21 mL, 35.7 mmol) in pen-
tane was added under nitrogen at 40 ꢀC to a solution
of 3b, 3c and 3d respectively (17.8 mmol) in anhydrous
ether (60 mL). After stirring at 40 ꢀC for 1 h the tem-
perature was allowed to warm to 5 ꢀC and then freshly
prepared (R,S)-1,2-dimethyl-3-pyrrolidone [(R,S)-2]13
[1.7 g, 15 mmol, bp18 47±48 ꢀC, purity=99.9% (GC)] in
anhydrous ether (20 mL) was added dropwise while
maintaining the temperature at 5 ꢀC. The mixture was
stirred for a further 3 h. A 10% solution of HCl
(approximately 25 mL) was added to bring the pH to 2;
after 15 min a ®ne white solid precipitated. The organic
phase was then separated from the aqueous phase con-
taining the white solid in the form of a very ®ne suspen-
sion. The crude product was recovered by ®ltration and
recrystallised. Di€erential scanning calorimetry (DSC)
.
Anal. C17H21NO2 HCl (C,H,N).
ꢀ
.
(2R,3S/2S,3R)-1d HCl. 77.7% yield; mp 204±206 C
(isopropyl alcohol 95/H2O 5); TLC analysis [stationary
phase:Merck silica gel 60 F254; mobile phase: hexane 87/
isopropyl alcohol 13/methyl alcohol 3/(C2H5)2NH 2] : Rf
0.45. IR (nujol) main absorptions (cm 1): 3300, 2680,
1602, 1505, 1112, 1075, 960, 900, 860, 819, 750; 1H NMR
(in CD3OD) d: 1.15 (d, 3H, CH3CH, J=6.50); 2.285
(ddd, 1H, HCHCH2N, Jgem=14.0, Jvic=3.5±8.7); 2.78
(ddd, 1H, HCHCH2N, Jgem=14.0, Jvic=8.3±10.5); 2.97
(s, 3H, CH3N); 3.45 (dt, 1H, HCHN, Jgem=11.0,
Jvic=3.4); 3.75 (q, 1H, CH3CH, J=6.3); 3.91 (dt, 1H,
HCHN, Jgem=11.0, Jvic=8.0); 7.45 (m, 2H, aromatic);
7.61 (dd, 1H, aromatic, CH 4, J=8.5); 7.83±8.01 (s+m,

S. Collina et al. / Bioorg. Med. Chem. 8 (2000) 1925±1930
1929
4 H, aromatic); 13C NMR (in CD3OD) d: 167.00 (C8,
aromatic); 162.53, 162.24, 157.39, 157.10, 156.48, 155.41,
155.36, 153.62 and 151.91 (9C, aromatic, C9, C10, C4,
C6, C1, C2, C5, C3 and C7); 110.01 (C3); 101.36 (C2);
82.71 (C5); 67.63 and 67.38 (NCH3 and C4);36.34
The apparatus consisted of a platform equipped with a
rotating rod (3 cm diameter) with a non-slippery sur-
face. The rod was placed at a 15 cm height from the base
and was divided by six disks into ®ve equal sections in
order to simultaneously test up to ®ve mice. The speed
of rota-rod was ®xed at 17 rpm. The animals were
selected randomly and divided in groups of 10 mice for
any experiment.
.
(C2CH3). Anal. C16H19NO HCl (C,H,N).
Pharmacology
Pharmacological studies in vivo were performed on
male adult Swiss mice weighting 30Æ5 g. To assess the
antinociceptive e€ects the hot plate test (HPT) was uti-
lized. Compounds were dissolved in saline solution and
administered within 1 h from dissolution.
Before the beginning of the test the mice were trained
to remain on the rod for the maximum time used
(120 s) and those animals that did not remain on the
bar during this time were rejected. Performance time
was monitored before (control animals) and 15 min
after treatment with the substances, tested at AD50 of
HPT. The integrity of motor coordination was eval-
uated by counting the number of mice of each group
that fell from the rod during the periods of 30 and
120 s.
The HPT experiments of all compounds 1a±d were run
simultaneously.
Hot plate test (HPT)
The response to a thermal stimulus was evaluated using
a copper plate heated to 55 ꢀC. The reaction time was
measured in seconds.
In all sets of experiments a group of mice was treated
with morphine as a standard antinociceptive agent in
order to make a suitable comparison with the tested
compounds.
The response of the mouse included the sitting on its
hind legs and licking.14
Results are expressed as the percentage of mice that fell
from the bar during the ®xed period of time. Statistical
analysis was performed by Student t test for grouped
data. P Values less than 0.05 were considered signi®cant.
Experimental data are reported in Table 4.
Once the basal animal reaction time was determined,
groups of 10 mice were treated (via ip injection) with
increasing doses of compound. Control animals received
the same volume of saline solution. The reaction time to
the pain stimulus was measured 20 min after ip injection.
The reaction time of the control animals was 23Æ2 s.
Radioligand binding test
Opioid receptor binding experiments were carried out
using membranes prepared by hypotonic lysis from
CHO (m and d) or HEK-293 (k) cells expressing cloned
human opioid receptors.21 [3H]-U-69593, [3H]-DADLE
(d-Ala2, d-Leu5 enkephalin) and [3H]-DAMGO (d-
Ala2, MePhe4, Gly-ol5 enkephalin) were used as the
radioligands to label k, d and m receptors respectively.
AD50 values were determined using a computerized
program.15
Experimental data are reported in Table 2.
Antagonist activity test
To assess an involvement of the opioid system (m, k and
d receptors) in the antinociceptive activity (determined
by HPT), the following opioid antagonists were used:
naloxone (NLX) at high doses (10 mg/kg) as a non
selective opioid inhibitor, naloxone at low doses (0.5 mg/
kg) as a m-preferential antagonist,16,17 nor-binaltorphi-
mine (nor-BNI) 5 mg/kg as a k preferential antagonist18
and naltrindole (NTN) 1 mg/kg, as a d selective
antagonist.19
The assay was carried out in Tris bu€er 50 mM pH 7.4
with ®nal volume of 1 or 2 mL. The assay tubes were
incubated at 25 ꢀC for 60 min. The non speci®c receptor
binding was evaluated in the presence of Naloxone,
10 mM. Bound ligand was separated from free ligand by
®ltration through Whatman GF/B ®lters using a Bran-
dell Cell Harvester. The radioactivity on the ®lters was
measured by liquid scintillation counting.
The data obtained from competition experiments were
analyzed using non linear ®tting analysis according to
Benfenati and Guardabasso22 and using the RS/1 soft-
ware. Ki values were determined from IC50 using the
Cheng and Pruso€ equation and were >1000 nM for all
compounds.
Antagonists were administered ip before agonists at the
following times: NLX 30 min, nor-BNI 24 h, NTN 5 min.
Experimental data are reported in Table 3 and in Figure
2b.
Rota-rod test
In order to assess the possible non-speci®c muscle-
relaxant or sedative e€ects of the investigated com-
pounds, the integrity of motor coordination of the mice
was tested on the rota-rod apparatus by using a light
modi®cation of the method described by Vaught et al.20
Acknowledgements
This work was supported by a grant from MURST. The
authors wish to thank Dr. Laura Linati for obtaining
the NMR spectra.

1930
S. Collina et al. / Bioorg. Med. Chem. 8 (2000) 1925±1930
11. Gouarderes, C.; Jhamandas, K.; Sutak, M.; Zajac, J. M.
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