(+)-cis-N-ethyleneamino-N-normetazocine derivatives. Novel and selective sigma ligands with antagonist properties.

Article abstract of DOI:10.1021/jm970333f

A series of (+)-cis-N-normetazocine derivatives has been described, and their affinities for sigma1, sigma2, and phencyclidine (PCP) sites and opioid, muscarinic (M2), dopamine (D2), and serotonin (5-HT2) receptors were evaluated. The effect of the N-subs

Full text of DOI:10.1021/jm970333f

1
574
J . Med. Chem. 1998, 41, 1574-1580
Articles
(
+)-cis-N-Eth ylen ea m in o-N-n or m eta zocin e Der iva tives. Novel a n d Selective σ
Liga n d s w ith An ta gon ist P r op er ties
,
†
†
†
†
†
Giuseppe Ronsisvalle,* Agostino Marrazzo, Orazio Prezzavento, Lorella Pasquinucci, Franco Vittorio,
†
†
‡
‡
Valeria Pittal a` , Maria S. Pappalardo, Silvia Cacciaguerra, and Santi Spampinato
Department of Pharmaceutical Sciences, University of Catania, Viale Andrea Doria, 6, 95125 Catania, Italy, and Department
of Pharmacology, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy
Received May 19, 1997
A series of (+)-cis-N-normetazocine derivatives has been described, and their affinities for σ
, and phencyclidine (PCP) sites and opioid, muscarinic (M ), dopamine (D ), and serotonin
5-HT ) receptors were evaluated. The effect of the N-substitution with a substituted ethylamino
spacer was investigated. Compounds 8c-11c displayed high affinities for σ sites and for opioid
receptors. Substitution of the second basic nitrogen either with alkyl or cycloalkyl substituents
give compounds (1a -6a ) with high affinity and selectivity for σ binding sites. Compounds
a -5a were further characterized in vivo, and their agonist/antagonist activity was evaluated.
1
,
σ
(
2
2
2
2
1
1
1
In mouse, compound 1a and 2a as well as haloperidol suppressed in a dose-related manner
the stereotyped behavior induced by (+)-SKF 10,047. Compounds 3a -5a and (+)-pentazocine
do not affect the stereotyped behavior induced by ip injection of (+)-SKF 10,047. Therefore,
from this series of compounds we identified potent and selective σ
useful to unveil the functional role of σ sites.
1
ligands which might prove
1
In tr od u ction
is still difficult to define which σ ligands act as agonists
or antagonists at these sites. Several researchers have
proposed that σ ligands which are associated with
psychotomimetic effects are agonists (for instance, (+)-
pentazocine and (+)-SKF 10,047), while compounds
such as haloperidol, rimcazole, and NE-100 (N,N-
dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethyl-
amine monohydrochloride), which block the binding and
σ binding sites display a unique drug selectivity
pattern and anatomical distribution in the central
nervous system and in peripheral tissues. Proteins
1
with the characteristics of σ recognition sites have been
2
-5
purified,
and recently a 24 kD protein was cloned
using degenerate oligonucleotides and cDNA library
6
screening. Thus far, at least two subtypes of σ binding
several pharmacological actions of these agonists are
7
,8
sites have been pharmacologically characterized.
cording to this classification, σ1 binding sites display
high affinity for haloperidol, 1,3-di(2-tolyl)guanidine
Ac-
25-28
considered antagonists.
In agreement with this
29-31
hypothesis, Pasternak and co-workers
have exten-
sively investigated possible functional interactions be-
tween σ1 and opioid receptors. They have suggested
that in addition to other activities, σ1 sites comprise a
tonically active antiopioid system. In fact, they have
found that (+)-pentazocine, acting as σ1 agonist, an-
tagonizes opioid-induced analgesia; on the contrary,
haloperidol, acting as σ antagonist, greatly enhances
this latter effect. Nevertheless, there is still controversy
regarding this classification; for instance, Matsumoto
(
DTG), and (+)-cis-benzomorphans, such as (+)-pent-
azocine and (+)-N-allyl-N-normetazocine (NANM or (+)-
SKF10,047), whereas σ2 binding sites are characterized
by high affinity for haloperidol and DTG, but low affinity
for (+)-cis-benzomorphans.
σ binding sites seem to be implicated in a wide variety
of functions, ranging from the motor control to the
synthesis and/or release of several neurotransmitters
or cell proteins.9
-13
Recent observations suggest that
24
et al. have defined BD-1047 (N-[2-(3,4-dichlorophenyl)-
σ binding sites may be also implicated in the cell growth
ethyl]-N-methyl-2-(dimethylamino)ethylamine) and BD-
and proliferation.1
4,15
Although their physiological sig-
1
063 (1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpipera-
nificance is not yet completely known, the potential
involvement of σ sites in affective disorders and in
schizophrenia has been suggested.
pharmacological studies have been carried out with
ligands showing reduced selectivity; thus, the results
are difficult to interpret. In addition, although selective
zine) as σ antagonists capable of attenuating the
dystonia produced by DTG and haloperidol. Therefore,
the lack of specific antagonists has been a relevant
impediment to the progress in research on σ sites.
As previously mentioned, (+)-cis-benzomorphan de-
rivatives are generally considered σ agonists; although,
only for (+)-SKF 10,047 and (+)-pentazocine is there
pharmacological evidence supporting their agonistic
1
6,17
Many of these
1
8-24
ligands have been synthesized in recent years,
it
*
To whom correspondence should be addressed.
University of Catania.
University of Bologna.
1
,32
†
properties.
Moreover, studies of σ sites using (+)-
‡
SKF 10,047 resulted in confusion with phencyclidine
S0022-2623(97)00333-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/17/1998

(+)-cis-N-Ethyleneamino-N-normetazocines Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10 1575
Ta ble 1. Physical Properties of N-Alkylated cis-(+)-N-Normetazocine Derivatives
formulac
[R]23D, deg
d
compd
R
R1
method of synthesis
yield (%)
mp (°C)
1
2
3
4
5
6
7
8
9
1
1
a
a
a
a
a
a
c
c
-(CH2)5-
A
A
A
A
A
A
B
B
B
B
B
62
70
35
67
63
70
34
85
61
77
35
184-186a
205-206a
187-189a
C21H32N2O‚C2H2O4‚2.5H2O
C20H30N2O2‚C2H2O4‚0.5H2O
C22H34N2O‚C2H2O4‚1.5H2O
C20H30N2O‚C2H2O4‚1.5H2O
C20H32N2O‚C2H2O4‚1.5H2O
C18H28N2O‚C2H2O4‚2 H2O
C22H34N2O‚2C2H2O4‚0.5H2O
C22H28N2O‚H2O
+40
+40
+60
+44
+48
+52
+78
+74
+90
+88
+84
-(CH2)2-O-(CH2)2-
-(CH2)6-
a
-(CH2)4-
181-182
C2H5
CH3
C6H11
C6H5
C6H5
C2H5
CH3
H
182-184a
180-182a
a
174-175
H
64-65b
b
c
0c
1c
CH3
C2H5
H
67-68
C23H30N2O‚0.5H2O
C6H5
220a
C24H32N2O‚C2H2O4‚0.5H2O
C23H30N2O‚2C2H2O4
CH2C6H5
178-179a
a
Oxalate salt. b Free amine. c Elemental analyses were within (0.4% of the theoretical values. d All optical rotations were determined
in ethanol (c ) 1.00).
Sch em e 1. General Method for the Preparation of
N-Alkyl-cis-(+)-N-normetazocine Derivatives (1a -6a )a
Sch em e 2. General Method for the Preparation of
N-Alkyl-cis-(+)-N-normetazocine Derivatives (7c-11c)a
a
(i) CH3OH, NaHCO3.
(PCP) site of the N-methyl-D-aspartate (NMDA), since
this (+)-benzomorphan binds both σ and PCP binding
3
3
sites.
Several studies on cis-(+)- and cis-(-)-N-
normetazocine derivatives have been carried out in
order to clarify the structure-affinity relationship and
have confirmed that the nucleus of cis-(+)-N-normetazo-
cine with suitable N-substituents is a good pharma-
a
(ii) THF, 4-(dimethylamino)pyridine; (iii) CH3OH, NaHCO3;
(
iv) THF, diborane.
cophore to probe σ1 binding sites.3
4-39
Carroll et al.
34
cyclic ring system, such as piperidine, morpholine,
azepine, or pyrrolidine, respectively, range between 7.4
and 20.1 nM. Compounds 5a and 6a , supporting a
diethyl or a dimethyl N-substituent, have an affinity of
5.2 and 19.8 nM, respectively, while 7c with the
cyclohexyl N-substituent shows reduced affinity (Ki )
1.6 nM). The affinities for σ2 sites are lower than those
for σ1 sites, ranging between 114 and 378 nM, and 4a
shows the greatest selectivity for σ1 with respect to σ2
subtype sites (σ1/σ2 ratio ) 35). All of these compounds
have a very low affinity for opioid receptors (Ki ranging
between 1466 and 12 290 nM). Moreover, 1a and 2a
do not display any significant affinity for κ opioid
receptors (Ki > 25 000 nM) (data not shown). Com-
pounds 1a , 2a , 4a , and 7c are also highly selective for
the σ sites relative to PCP, whereas 3a displays the
highest affinity for PCP site (Ki ) 50.2 nM) and N-ethyl
and N-methyl derivatives (5a and 6a , respectively) show
low affinity for this binding site (Table 2).
have examined a series of enantiomeric (+)-cis-benzo-
morphans at σ and PCP sites and opioid receptors. The
results have indicated that, in general, lipophilic groups
on the nitrogen may improve selectivity and potency at
σ sites, and the greatest improvement was observed
with a N-benzyl substituent. However, it has not been
deeply investigated if any constituent of this class may
act as a σ antagonist. Thus, to obtain new insights on
structural requirements, we synthesized a novel series
of substituted N-ethyleneamines of cis-(+)-N-normeta-
zocine to discriminate among σ sites and other receptor
systems, which may represent additive binding sites for
several σ ligands, and to evaluate their agonist and
antagonist properties.
1
4
Ch em istr y
cis-(+)-(1S,5S,9S)-N-Normetazocine was separated by
4
0
racemic mixture as reported by Brine et al.
Com-
pounds 1a -6a were prepared by alkylation of cis-(+)-
N-normetazocine with commercially available chloro-
ethylamines (Scheme 1). Compounds 7a and 11a were
prepared by acylation of respective amines with bro-
moacetyl chloride in anhydrous THF, at -5 °C (Scheme
Compounds 8c and 9c have the highest affinity for
σ1 sites (Ki ) 5.9 and 5.6 nM, respectively). However,
these compounds do not show notable selectivity for σ1
binding sites despite the presence of a cis-(+)-N-
normetazocine nucleus. In fact, they also have signifi-
cant affinity for opioid receptors (Ki ) 79.8 and 28.8 nM,
respectively). Moreover, the substitution of methyl with
ethyl group or phenyl with benzyl on the second basic
nitrogen provides compounds 10c and 11c with a lower
affinity for σ1 sites and opioid receptors.
2
). The respective bromo amides 7a -11a gave the
amides 7b-11b by alkylation of cis-(+)-N-normetazo-
cine in MeOH at 50 °C using NaHCO3. Reduction of
the resulting amides with diborane in anhydrous THF
provided the final compounds 7c-13c. Physical and
analytical data for compounds 1a -6a and 7c-11c are
shown in Table 1.
With regard to other receptors systems, compounds
a -6a and 7c, displaying moderate to high affinity for
σ1 binding sites, have a very low or a negligible affinity
Resu lts
1
As shown in Table 2, σ1 binding affinities of com-
pounds 1a -4a with the second nitrogen atom part of a
for muscarinic (M2), dopamine (D2), and serotonin (5-

1
576 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10
Ronsisvalle et al.
Ta ble 2. Binding Affinities of N-Substituted cis-(+)-N-Normetazocine Derivatives to σ, Opioid, and PCP Binding Sites
Ki ( SEM (nM)a
3
b
3
b
[3H]Nalox. (opioid)
[3H](+)-SKF 10,047b,d (PCP)
compd
R
R1
[ H]-(+)-Pentaz. (σ1)
[ H]DTG (σ2)
1
2
3
4
5
6
7
8
9
1
1
a
a
a
a
a
a
c
c
-(CH2)5-
9.6 ( 0.8
13.4 ( 1.3
20.1 ( 1.8
7.4 ( 0.7
114 ( 10
300 ( 12
151 ( 10
260 ( 11
378 ( 13
347 ( 15
328 ( 12
111 ( 9
124 ( 10
151 ( 11
187 ( 14
12.7 ( 2.4
1440 ( 293
c
12000 ( 340
8168 ( 170
9128 ( 197
3800 ( 120
12290 ( 210
2888 ( 98
1466 ( 45
79.8 ( 8.4
28.8 ( 2.3
211 ( 20
>10000
-(CH2)2O(CH2)2-
-(CH2)6-
>10000
50.2 ( 0.9
>10000
-(CH2)4-
C2H5
CH3
C2H5
CH3
H
15.2 ( 2.1
19.8 ( 2.8
41.6 ( 3.3
5.9 ( 0.2
7645 ( 228
3380 ( 123
>10000
C6H11
C6H5
e
H
nd
nd
nd
nd
e
c
0c
1c
C6H5
C6H5
CH3
C2H5
H
5.6 ( 0.2
e
42.2 ( 3.8
29.6 ( 3.2
1.6 ( 0.3
e
CH2C6H5
366 ( 22
e
haloperidol
>10000
nd
(
+)-pentaz.
6.1 ( 0.9
169 ( 9.82
2370 ( 95
1900 ( 39
4320 ( 140
448 ( 33
(+)-SKF 10,047
a
Values are the mean of three separate experiments each carried out in duplicate. b All curves were best fit to a one-site model. c σ2
Binding assays were carried out in the presence of 200 nM of (+)-SKF 10,047 to mask σ1 binding sites; thus, it was not feasible to evaluate
d
e
the Ki of this compound. PCP binding assays were carried out in the presence of 5 µM of haloperidol to mask σ binding sites. nd ) not
determined.
Ta ble 3. Binding Affinities of N-Substituted
cis-(+)-N-Normetazocine Derivatives to Muscarinic (M2),
Dopamine (D2), and Serotonin (5-HT2) Receptors
kg, as evaluated adopting the scoring scale described
by Tanaka et al. (data not shown).
41
Ki ( SEM (nM)a
Discu ssion
[3
H]-N-methyl- [3H]spiroperidol [3H]ketanserine
(D2) (5-HT2)
Previous reports showed that the cis-(+)-N-normet-
azocine nucleus represents an established pharmaco-
phore for σ ligands with fair to good selectivity with
respect to opioid and PCP binding sites. Moreover, it
has been also observed in other series, such as N-[2-
compd
scopolamine (M2)
1
2
3
4
5
6
7
a
a
a
a
a
a
c
7800 ( 350 >10000 >10000
419 ( 34
>10000
>10000
>10000
>10000
>10000
>10000
>10000
1840 ( 130 >10000
1950 ( 110 >10000
(
3,3-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolinyl)eth-
1990 ( 80
>10000
>10000
238 ( 13
>10000
>10000
>10000
>10000
1.95 ( 0.2
>10000
4
2
ylamine derivatives, that optimal σ binding affinity
and selectivity was mainly due to the presence of an
diaminoethylene spacer. Here, we reported the synthe-
sis, binding affinities [for σ1, σ2, PCP, opioid, muscarinic
haloperidol
34 ( 8
>10000
(
+)-SKF
1
0047
(M2), dopamine (D2), and serotonin (5-HT2) receptors],
a
Values are the mean of three separate experiments each
and in vivo experiments to fully evaluate the agonist/
antagonist properties of a new series of N-ethylamino-
substituted cis-(+)-N-normetazocine derivatives.
All (+)-cis-N-normetazocine derivatives synthesized
maintain a notable affinity for σ1 binding sites in
comparison with the reference compounds [(+)-pent-
azocine and (+)-SKF 10,047]. Compounds 1a -6a and
7c suggest that an optimal σ1 binding affinity can be
achieved with the presence of the second basic nitrogen
tied into a cycloalkyl ring. However, an increase in the
cycloalkyl ring size (1a -4a ) reduces σ1 affinity.
In the series 8c-11c with an aromatic N-substituent,
8c and 9c show high σ1 affinity. The introduction of
lipophilic bulky substituents provides compounds 10c
and 11c with a lower affinity.
carried out in duplicate. All curves were best fit to a one-site model.
HT2) receptors (Table 3). Only 2a has appreciable
affinity for the muscarinic M2 receptor (Ki ) 419 nM).
According to the above-reported binding assays, com-
pounds 1a -5a , displaying little affinity for opioid recep-
tors, were selected to be assayed in an in vivo model
developed to evaluate the potential antipsychotic activ-
ity of σ1 antagonists.41 Compounds 1a and 2a , when
administered ip in the mouse, suppressed in a dose-
related manner the stereotyped behavior observed fol-
lowing ip injection of the σ1 agonist (+)-SKF 10,047. The
ED50 values were 0.82 mg/kg for 1a and 0.88 mg/kg for
2
a , respectively, while for haloperidol, chosen as refer-
ence compound, the ED50 was 0.12 mg/kg. On the
contrary, compounds 3a -5a and (+)-pentazocine, when
administered ip in the mouse, up to the dose of 5 mg/
kg, do not affect the stereotyped behavior induced by
ip injection of (+)-SKF 10,047.
Nevertheless, all cis-(+)-N-normetazocine derivatives
synthesized show a σ2/σ1 binding ratio (ranging from 35
to 3.6) lower than that of (+)-pentazocine (σ1/σ2 ) 236).
Thus, the presence of an ethyleneamino spacer on (+)-
cis-N-normetazocine nucleus seems to increase σ2 bind-
ing affinity in comparison with (+)-pentazocine and
other reported compounds. However, the substituents
on the ethyleneamino spanner chain were capable to
modulate the selectivity with the respect to opioid and
PCP binding sites. In fact, alkyl and cycloalkyl deriva-
Compounds 1a and 2a do not impair rotarod perfo-
mance in mice when administered ip up to the dose of
3
4
3
0 mg/kg. Moreover, these compounds, contrary to the
+)-SKF 10,047, do not cause any significant behavioral
effects and ataxia in the mouse up to the dose of 30 mg/
(

(+)-cis-N-Ethyleneamino-N-normetazocines Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10 1577
tives do not have affinity for opioid receptors, in contrast
with the analogues with aromatic substituents. These
compounds show significant opioid affinities, and this
effect is clearly represented by the compound 8a which
shows an higher affinity for opioid receptors (79.8 nM)
when compared with other already reported analogues
h. The mixture reaction was cooled and diluted with 85 mL
of ethyl acetate and 15 mL of water. The organic layer was
separated, washed with saturated aqueous brine solution,
dried over anhydrous sodium sulfate, and filtered. The filtrate
was evaporated in vacuo to yield the free bases, which were
purified by silica gel flash column chromatography using
3
CHCl :EtOH (95:5) as eluent. The compounds 1a -6a were
3
4
devoid of the second basic nitrogen.
dissolved in THF and treated with a solution of H
in THF to give the oxalate salts as white solids. The analyti-
cally pure samples were obtained by recrystallization.
2
C
2
O
4
2
‚2H O
According with a previous study,3 compounds 1a , 2a ,
4
4
a -6a , and 7c show that more lipophilic N-substituents
(
+)-6(S ),11(S )-Dim e t h yl-3-[2-(p ip e r id in -1-yl)e t h yl]-
seem to reduce the affinity for PCP sites. Compound
1
,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-ol ox-
3
a represents an exception to this trend.
23
a la te (1a ): 62% yield; mp 184-186 °C; [R]
EtOH); MS (EI) m/z 328 (M ). Anal. (C21
2.5H O) C, H, N.
D
) +40° (c 1.0,
2 2 4
O‚C H O ‚
With respect to agonist/antagonist assays, among the
+
H
32
N
2
tested compounds (1a -5a ), 1a and 2a supporting a
piperidine and a morpholine ring, respectively, antago-
nize the (+)-SKF 10,047-induced stereotyped behavior,
while compounds 4a and 5a do not affect this behavior
at all.
2
(+)-6(S),11(S)-Dim et h yl-3-[2-(m or p h olin -4-yl)et h yl]-
1,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-ol ox-
a la te (2a ):44 70% yield; mp 205-206 °C; [R]
23
) +40° (c 1.0,
D
+
EtOH); MS (EI) m/z 330 (M ). Anal. (C20
H
30
N
2
O
2
2 2 4
‚C H O ‚
0
.5H
+)-6(S ),11(S )-D im e t h y l-3-[2-(Aze p a n -1-y l)e t h y l]-
,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-ol ox-
2
O) C, H, N.
It is noteworthy that compounds 1a and 2a suppress
the (+)-SKF 10,047-induced stereotyped behavior as
well as the purported σ antagonist haloperidol. The in
vivo experiments seem to suggest that these compounds
display antagonist properties with respect to (+)-SKF
(
1
23
a la te (3a ): 35% yield; mp 187-189 °C; [R]
D
) +60 (c 1.0,
+
EtOH); MS (EI) m/z 342 (M ). Anal. (C22
H
34
N
2
O‚C H O ‚
2 2 4
2
1.5H O) C, H, N.
1
0,047. It has been reported that (+)-SKF 10,047 and
(+)-6(S),11(S)-Dim et h yl-3-[2-(p yr r olid in -1-yl)et h yl]-
,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-ol ox-
1
other benzomorphan derivatives may cause stereotyped
behavior and ataxia in rodents acting as “agonists” at
σ-like sites.42 However, compounds 1a and 2a do not
produce any ataxia or stereotyped behavior in the
mouse, thus further supporting the hypothesis that it
is unlikely that they may act as an “agonists” at σ sites
to cause psychotomimetic effects.43
2
3
a la te (4a ): 67% yield; mp 181-182 °C; [R]
D
) +44° (c 1.0,
+
EtOH); MS (EI) m/z 314 (M ). Anal. (C20
H
30
N
2
O‚C H O ‚
2 2 4
1
.5H
+)-6(S ),11(S )-Dim e t h yl-3-[2-(Die t h yla m in o)e t h yl]-
,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-ol ox-
2
O) C, H, N.
(
1
2
3
a la te (5a ): 63% yield; mp 182-184 °C; [R]
D
) +48° (c 1.0,
+
EtOH); MS (EI) m/z 316 (M ). Anal. (C20
H
32
N
2
O‚C H O ‚
2 2 4
1
.5H
+)-6(S),11(S)-Dim et h yl-3-[2-(Dim et h yla m in o)et h yl]-
,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-ol ox-
2
O) C, H, N.
The high selectivity of 1a and 2a for σ binding sites,
with their lack of significant affinity for other receptor
systems, including PCP, opioid, and dopamine D2, which
may represent other additive binding sites for several
σ ligands,33 provide strong support that these antagonist
effect could be mediated by σ1 sites.
(
1
2
3
a la te (6a ): 70% yield; mp 180-182 °C; [R]
D
) +52° (c 1.0,
+
EtOH); MS (EI) m/z 288 (M ). Anal. (C18
H
28
N
2
O‚C H O ‚
2 2 4
2
H
2
O) C, H, N.
Meth od B. Gen er a l P r oced u r e for th e Syn th esis of
In conclusion, we presented evidence that the N-
substitution of cis-(+)-N-normetazocine nucleus with an
ethylamino spacer provide compounds with a reduced
σ2/σ1 selectivity, but with the retention of a good affinity
for σ1 sites and selectivity with respect to other binding
sites. Moreover, we presented evidence that suitable
functionalization may afford compounds with antagonist
σ1 properties.
2-Br om o-N,N-d isu bstitu ted -a ceta m id e Der iva tives 7a -
1
1a . To a solution of bromoacetyl chloride (4.7 g, 30 mmol) in
1
0 mL of dry THF cooled at 0 °C was added dropwise, under
vigorous stirring, a solution of amine (20 mmol) and 4-(dim-
ethylamino)pyridine (1.1 g, 9.4 mmol) in dry THF (20 mL).
After 1 h the reaction mixture was quenched with H
extracted with CHCl . The organic extract was washed with
a saturated NaHCO solution, dried with anhydrous Na SO
and concentrated under reduced pressure. The crude products
2
O and
3
3
2
4
,
7
a -11a were used with no further purification.
Exp er im en ta l Section
Gen er a l P r oced u r e for N-Su bstitu tion of cis-(+)-N-
Nor m eta zocin es 7b-11b. These compounds were prepared
by the same procedure described for 1a -6a , starting from 7a -
Reagents were purchased from Aldrich Chemical Co. unless
otherwise specified. Melting points were determined on a
Buchi 530 capillary apparatus and are uncorrected. Analytical
thin-layer chromatography (TLC) was performed on precoated
silica gel 60 F254 aluminum sheets (Merck); visualization was
accomplished under UV or in an iodine chamber. Merck silica
gel 60, 230-400 mesh, was used for flash column chromatog-
raphy. Optical rotations were determined in MeOH solution
with a Perkin-Elmer 241 polarimeter. Infrared spectra were
recorded on a 1600 FT-IR Perkin-Elmer instrument and are
consistent with the assigned structures. Elemental analyses
were measured on an elemental analyzer (Model 1106, Carlo
Erba). Molecular weights of the obtained products were
determined by MS on a Kratos 2S RFA spectrometer using a
Tektronix 4205 computer system.
1
1a . The crude products 7b-11b were purified by silica gel
flash chromatography using cyclohexane:ethyl acetate (70:30)
as eluent.
(
+)-N-Cycloh exyl-2-(8-h yd r oxy-6(S),11(S)-d im et h yl-
2
,4,5,6-t et r a h yd r o-1H -2(S),6-m et h a n ob en za zocin -3-yl)-
a ceta m id e (7b): 91% yield; mp 233-237 °C; MS (EI) m/z 356-
+
(
M ).
(
+)-2-(8-Hydr oxy-6(S),11(S)-dim eth yl-2,4,5,6-tetr ah ydr o-
1
H -2(S ),6-m e t h a n ob e n za zocin -3-yl)-N -p h e n yla ce t a m -
id e (8b): 69% yield; mp 256-258 °C; MS (EI) m/z 350 (M ).
+
(
+)-2-(8-Hydr oxy-6(S),11(S)-dim eth yl-2,4,5,6-tetr ah ydr o-
H-2(S),6-m eth a n oben za zocin -3-yl)-N-m eth yl-N-p h en yl-
a ceta m id e (9b): 88% yield; mp 224-226 °C; MS (EI) m/z 364
1
+
(
M ).
Meth od A. Gen er a l P r oced u r e for N-Alk yla tion of cis-
(
+)-N-Nor m eta zocin e Der iva tives 1a -6a . cis-(+)-N-Nor-
metazocine (200 mg, 0.92 mmol), chloroethylamine monohy-
(115.9 mg, 1.38 mmol),
(+)-N-E t h yl-2-(8-h yd r oxy-6(S),11(S)-d im et h yl-2,4,5,6-
tetr a h yd r o-1H-2(S),6-m eth a n oben za zocin -3-yl)-N-p h en -
yla ceta m id e (10b): 71% yield; mp 96-98 °C; MS (EI) m/z 378
drochloride 1-6 (1.38 mmol), NaHCO
3
+
and a catalytic amount of potassium iodide were added in 5
mL of dry MeOH, and the mixture was stirred at 50 °C for 4
(M ).
(+)-N-Ben zyl-2-(8-h yd r oxy-6(S),11(S)-d im eth yl-2,4,5,6-

1
578 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10
Ronsisvalle et al.
t et r a h yd r o-1H -2(S),6-m et h a n ob en za zocin -3-yl)a cet a m -
id e (11b): 91% yield; mp 103-106 °C; MS (EI) m/z 364 (M ).
out on membranes obtained from guinea pig cerebella and
+
3
using [ H]U69,593 (62 Ci/mmol; K
d
) 1.98 ( 0.4; n ) 3) in the
2
4
5
Gen er a l P r oced u r e for th e Red u ction of cis-(+)-N-
presence of 300 nM D-Ala ,N-MePhe ,Gly-ol -enkephalin and
2
5
Su bstitu ted -N-n or m eta zocin e Der iva tives 7c-11c. To a
300 nM D-Ala -D-Leu -enkephalin to block µ and δ receptors,
respectively, and to direct the binding of the radioligand to κ
receptors.49
4
5
1
M solution of diborane in THF, cooled at 0 °C and under
nitrogen atmosphere, a solution of the appropriate cis-(+)-N-
substituted-N-normetazocine 7b-11b in anhydrous THF (5
mL) was slowly added. The reaction, under nitrogen atmo-
sphere, was heated to reflux for 8 h and cooled at room
temperature, and 2 mL of a 6 M hydrochloric acid solution
was added slowly. THF was removed by distillation at
Bin d in g to P CP Sites. PCP binding assay was carried
out on rat brain membranes following the procedure described
50
by Largent et al. In a final assay volume of 0.25 mL, 10 nM
3
[
H](+)-SKF 10,047 (49.2 Ci/mmol) was incubated in the
presence of various concentrations of each compound with
tissue homogenate (450 µg of protein/assay tube) for 30 min
at room temperature. Binding assays were carried out in the
presence of 5 µM haloperidol to specifically block σ sites.
atmospheric pressure. NaHCO
was added to aqueous phase, and the latter was extracted with
CHCl (30 mL). The organic mixture was dried with anhy-
drous Na SO and evaporated in vacuo to give the free bases
c-11c, which were purified by silica gel flash chromatogra-
phy using CHCl :cyclohexane:EtOH (50:45:5) as eluent. The
purified compounds 7c-11c dissolved in THF were treated
with a solution of H O in THF to give the oxalate
3
saturated solution (15 mL)
3
2
4
Nonspecific binding was estimated with 100 µM (+)-SKF
7
50
1
0,047. According to Largent et al., this assay procedure
3
allows to estimate binding affinity of compounds interacting
with PCP sites.
2
C
2
O
4
‚2H
2
Bin d in g to Dop a m in er gic (D
gic (D ) receptor binding assay was performed using 0.5 nM
H]spiroperidol (18.5 Ci/mmol; K ) 0.5 ( 0.08 nM; n ) 3)
and rat striatal membranes according to Briley and Langer;
2
) Recep tor s. Dopaminer-
salts as white solids. The analytically pure samples were
obtained by recrystallization.
2
3
[
d
(
+)-3-(2-Cycloh exyla m in oet h yl)-6(S),11(S)-d im et h yl-
5
1
1
,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-ol ox-
a la te (7c): 34% yield; mp 174-175 °C; [R]
EtOH); MS (EI) m/z 342(M ). Anal. (C22
2
3
nonspecific binding assay was measured in the presence of 10
µM haloperidol.
D
) +78° (c 1.0,
2 2 4
O‚2C H O ‚
+
H
34
N
2
Bin d in g to 5-HT
receptor binding assay was performed using 1 nM [ H]-
ketanserin (77.1 Ci/mmol; K ) 1.49 ( 0.4; n ) 3) and guinea
2 2
Recep tor s. Serotoninergic (5-HT )
0
.5H
+)-6(S ),11(S )-D im e t h y l-3-(2-p h e n y la m in o e t h y l)-
,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-ol ox-
2
O) C, H, N.
3
(
d
1
2
3
pig frontal cortex membranes according to the procedure
described by Tam et al.;52 nonspecific binding was measured
in the presence of 1 µM methisergide.
a la te (8c): 85% yield; mp (free base) 64-65 °C; [R]
c 1.0, EtOH); MS (EI) m/z 336 (M ). Anal. (C22
D
) +74°
+
(
H
28
N
2
O‚H O)
2
C, H, N.
+)-6(S),11(S)-Dim et h yl-3-[2-(m et h ylp h en yla m in o)et -
h yl]-1,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-
(
2 2
Bin d in g to Mu sca r in ic (M ) Recep tor s. Muscarinic (M )
receptor binding assay was carried out in rat heart membranes
2
3
53
ol oxa la te (9c): 61% yield; mp (free base) 67-68 °C; [R]
D
)
O‚
prepared by the method of Waelbroeck et al. Binding assay
+
3
+
90° (c 1.0, EtOH); MS (EI) m/z 350 (M ). Anal. (C23
H
30
N
2
was performed in the presence of 0.5 nM of [ H]-N-methylsco-
0
.5H O) C, H, N.
2
polamine (79.5 Ci/mmol; K
d
) 0.24 (0.06 nM; n ) 3).
(
+)-6(S),11(S)-Dim eth yl-3-[2-(eth ylph en ylam in o)eth yl]-
Nonspecific binding was determined in the presence of 10 µM
atropine for 120 min; the reaction was carried at room
temperature and terminated by filtration on Whatman GF/B
filter.
1
,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-ol ox-
a la te (10c): 77% yield; mp 220 °C; [R]
MS (EI) m/z 364 (M ). Anal. (C24
H, N.
(
,2,3,4,5,6-h exa h yd r o-2(S),6-m eth a n oben za zocin -8-ol ox-
a la te (11c): 35% yield; mp 178-179 °C; [R]
EtOH); MS (EI) m/z 350 (M ). Anal. (C23
2
3
D
) +88° (c 1.0, EtOH);
O‚C ‚0.5H O) C,
+
H
32
N
2
2
H
2
O
4
2
The concentration of the test compounds causing 50%
inhibition of radioligand (IC ) was determined from concen-
+)-6(S ),11(S )-D im e t h y l-3-(2-b e n zy la m in o e t h y l)-
5
0
1
tration-response curves in which at least 10 concentrations
of test compounds were examined. Inhibition constants (Ki
values) for the binding of test compounds were calculated using
the EBDA/LIGAND program,54 purchased from Elsevier/
Biosoft.
2
3
D
) +84° (c 1.0,
+
H
30
N
2
O‚2C
2
H
2
O
4
)
C, H, N.
Ra d ioliga n d Bin d in g Assa ys. Bin d in g to σ
binding assay was carried out on guinea pig brain membranes
prepared by the method of Matsumoto et al., and protein
content was evaluated.
described by DeHaven et al. Briefly, each tube contains 500
µg of membrane protein, 3 nM [ H]pentazocine (31.6 Ci/mmol;
the value of the apparent dissociation constant (K
.8 nM, n ) 3). Nonspecific binding was determined by adding
0 µM haloperidol. The reaction was performed for 150 min
at 37 °C and terminated by filtration over Whatman GF/B
glass filters that were presoaked in a 0.5% polyethylenimine
solution.
1
Sites. σ
1
Beh a vior a l Stu d ies. Male Swiss mice (25-30 g, Charles
River, Italy) were placed individually into clear acrylic cages
and left for 1 h to acclimatize to the new environment. Thirty
minutes after the ip administration of haloperidol, compounds
2
4
4
6
Binding assay was performed as
4
7
3
1
a -5a , or (+)-pentazocine (dissolved in the vehicle) or vehicle
alone (0.5% carboxymethyl cellulose in water, w/v), (+)-SKF
0,047 (30 mg/kg, ip) was administrated. Behavioral scores
d
) was 4.3 (
0
1
1
for 5-min periods started 10 min after (+)-SKF 10,047 admin-
istration and lasted for 40 min. The scoring scale adopted is
41
reported by Tanaka et al.
Ten mice were treated with
vehicle, and groups of 10 or 6 mice were treated with
haloperidol (0.01-1 mg/kg) and compounds 1a -5a (0.01-5
mg/kg) or (+)-pentazocine. The total score of the vehicle-
treated group was defined as 100% and ED50 values for treated
mice were determined using log-probit conversion of data.
Bin d in g to σ
2
Sites. σ
2
binding assay was carried out on
guinea pig brain membranes prepared as described by Mach
4
8
3
et al. These were incubated with 3 nM [ H]DTG (35 Ci/mmol;
) 9.9 ( 0.8 nM; n ) 3) in the presence of 200 nM (+)-SKF
0,047 to mask σ sites. Incubation was carried out in 50 mM
K
1
d
1
Rota r od Test. Groups of six Swiss male mice (25-30 g)
were treated with the vehicle (0.5% carboxymethyl cellulose
in water, w/v) or test compound administered intraperitoneally
Tris‚HCl (pH 8.0) for 120 min at room temperature, and assays
were terminated by the addition of ice-cold 10 mM Tris‚HCl
(
pH 8.0) followed by filtration through Whatman GF/B glass
3
0 min before being placed on an accelerated rotarod (Ugo
fibers. Nonspecific binding was evaluated in the presence of
µM DTG.
Bin d in g to Op ioid Recep tor s. Total opioid receptor
binding was assessed on rat brain membranes prepared as
previously reported and incubated in the presence of [ H]-
naloxone (55.5 Ci/mmol; K
cific binding was evaluated in the presence of 10 µM naloxone.
In the case of κ opioid receptor assays, binding was carried
Basile, Milan, Italy) and provided three opportunities to
maintain balance on the bar. Rats that failed to maintain
balance in the least one of three separate trials were consid-
ered impaired.
5
4
9
3
d
) 6.6 ( 0.7 nM; n ) 3). Nonspe-
Ack n ow led gm en t. We thank Italian MURST and
CNR for financial support. The authors thank Dr.

(+)-cis-N-Ethyleneamino-N-normetazocines Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 10 1579
Salvatore Di Marco for the elemental analyses. (()-cis-
N-normetazocine was obtained from Fabrica Italiana
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