Drug design and synthesis of ε opioid receptor agonist: 17-(cyclopropylmethyl)-4,5α-epoxy-3,6β-dihydroxy-6, 14-endoethenomorphinan-7α-(N-methyl-N-phenethyl)carboxamide (TAN-821) inducing antinociception mediated by putative ε opioid receptor

Article abstract of DOI:10.1016/j.bmc.2004.05.024

Here we report the new drug design and synthesis of a series of 6,14-endoethenomorphinan-7-carboxamide derivatives as a putative ε opioid receptor agonist. One of these compounds, 17-(cyclopropylmethyl)-4,5α- epoxy-3,6β-dihydroxy-6,14-endoethenomorphinan-

Full text of DOI:10.1016/j.bmc.2004.05.024

Bioorganic & Medicinal Chemistry 12 (2004) 4133–4145
Drug design and synthesis of e opioid receptor agonist:
17-(cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-6,14-
endoethenomorphinan-7a-(N-methyl-N-phenethyl)carboxamide
(TAN-821) inducing antinociception mediated by
putative e opioid receptor
Hideaki Fujii,a,* Minoru Narita,b,c Hirokazu Mizoguchi,b,d Miho Murachi,a
Toshiaki Tanaka,a Koji Kawai,a Leon F. Tsengb and Hiroshi Nagasea,*,ꢀ
aPharmaceutical Research Laboratories, Toray Industries, Inc., 1111, Tebiro, Kamamura, Kanagawa 248-8555, Japan
bDepartment of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA
cDepartment of Toxicology, School of Pharmacy, Hoshi University, Tokyo 142-8501, Japan
dDepartment of Physiology and Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
Received 7 April 2004; revised 18 May 2004; accepted 18 May 2004
Available online 15 June 2004
Abstract—Here we report the new drug design and synthesis of a series of 6,14-endoethenomorphinan-7-carboxamide derivatives as
a putative e opioid receptor agonist. One of these compounds, 17-(cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-6,14-endo-
ethenomorphinan-7a-(N-methyl-N-phenethyl)carboxamide (TAN-821), showed agonistic activity for a putative e opioid receptor
(IC50 ¼ 71.71 nM) in the rat vas deferens (RVD) preparations. TAN-821 stimulated the binding of the nonhydrolyzable guanosine
50-triphosphate analog, guanosine 50-(c-thio)-triphosphate (GTPcS), to the mouse pons/medulla membrane via the activation of
putative e opioid receptor. Moreover, TAN-821 given intracerbroventricularly (i.c.v.) produced a marked antinociception in the tail-
flick test (ED50 ¼ 1.73 lg) and the hot-plate test (ED50 ¼ 2.05 lg) in a dose-dependent manner. The antinociception induced by TAN-
821 administered i.c.v. was blocked by the i.c.v.-pretreatment with a putative e opioid receptor partial agonist b-endorphin [1–27],
but not a l opioid receptor antagonist b-FNA, a d opioid receptor antagonist NTI, or a j opioid receptor antagonist nor-BNI. The
present results suggest that TAN-821 may be a useful tool for the investigation on the pharmacological properties of the putative e
opioid receptor.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
endogenous ligand for the l opioid receptor.6;7 How-
ever, pharmacological effects of b-endorphin seem to be
mediated by not only l but also other opioid receptors.
The detailed studies on b-endorphin provided evidence
that the antinociceptive effect of b-endorphin is medi-
ated by the b-endorphin-sensitive non-l, non-d, and
non-j opioid receptors, so-called putative e opioid
receptor.8–10 Unlike l, d, and j opioid receptors, which
have been more precisely characterized and cloned, the
putative e opioid receptor has not yet been cloned, be-
cause selective agonists and antagonists for the putative
e opioid receptor are not available so far described.
Although several compounds, like etorphine11 or bre-
mazocine12 as well as b-endorphin produce the antino-
ciception mediated through the putative e opioid
receptor, those actions can be also regulated by the l or
b-Endorphin is a nonselective endogenous opioid pep-
tide consisted of 31 amino acid residues, and has a
moderate affinity for the l and d opioid receptors.1;2
Until the discovery of endomorphins,3 which were re-
cently identified as endogenous l opioid peptides,4;5 b-
endorphin had been proposed to be a prototypical
Keywords: Putative e opioid receptor; TAN-821; Analgesics; Message–
address concept.
* Corresponding authors. Tel.: +81-467-32-9643; fax: +81-467-32-47-
91; e-mail: hideaki_fujii@nts.toray.co.jp
ꢀ
Present address: School of Pharmaceutical Sciences, Kitasato Uni-
versity, 5-9-1 Shirokane, Minatoku, Tokyo 108-8641, Japan.
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.05.024

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H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
j opioid receptors. We report here for the first time that
the newly synthesized compounds, 6,14-endo-
ethenomorphinan-7a-carboxamide derivatives are likely
to be classified as a selective agonist for the putative e
opioid receptor.
HO
Me
OH
Me
N
N
Me
Me
D
C
OMe
B
O
A
OH
OH
Bremazocine
2. Design rationale
Etorphine
Several selective opioid ligands such as nor-BNI,13
Figure 2. Stereo model of etorphine and bremazocine. Ring letters in
6,14-endoethenomorphinan are shown in etorphine structure.
NTI,14;15 TRK-820,16 and ())-TAN-6717–19 were discov-
ered by use of
a
message–address concept.15;20;21
According to the concept, opioid ligands can be viewed
to contain two elements: an essential message site that is
recognized by the receptor subsite responsible for the
signal transduction process and an address site that is
recognized by a subsite that is unique to a single
receptor type such as l, d, j, or e and functions to en-
hance binding to the subsite. Since the Tyr1 amine
moiety in opioid peptide structures or the tertiary amine
moiety in nonpeptidic opioid structures is known to be
important for activity, an identical element in the amine
moiety of opioid peptidic or nonpeptidic ligands can be
viewed as the message.21 The message–address concept
is useful for rational drug design of selective opioid
receptor ligands.
sage and address sites may play a crucial role to elicit
agonistic activity for the putative e opioid receptor.
According to this hypothesis, location of the address site
is important in order to design the putative e opioid
receptor agonist.
With respect to nonpeptidic ligands, it has been reported
that etorphine11 and bremazocine12 showed antinoci-
ceptive effect partially mediated by the putative e opioid
receptor. A stereo model of these compounds is shown
in Figure 2. The substituent of etorphine, the 1-hy-
droxyalkyl moiety, is held above the C-ring of the
morphinan skeleton by a bicyclo[2.2.2]octene structure
and seems to extend in a direction toward the a-helical
C-terminal region of b-endorphin, that is, the address
site. On the contrary, bremazocine has no substituent
located in the spatial area. Therefore, etorphine’s
skeleton would be more suitable than bremazocine’s
skeleton for the design of a selective e opioid receptor
agonist. On the other hand, many 7a-(1-hydroxyalkyl)-
6,14-endoethenomorphinan derivatives such as etor-
phine have already been synthesized and their antino-
ciceptive effects have been evaluated.24–26 Consequently,
6,14-endoethenomorphina-7a-carboxamide derivatives
3 were selected because the derivatives 3 have a 7a-
carboxamide substituent as a possible address site and
there are few examples synthesizing the 7-carboxamide
derivatives.
On the basis of the stereostructure of b-endorphin,22
a
binding model of b-endorphin to the putative e opioid
receptor has been proposed (Fig. 1).23 According to the
model, the C-terminal part, which has an a-helical
structure, and the N-terminal part are in close contact
with each other. As a result, the C-terminal part is held
above the Gly2-Gly3-Phe4-Met5 sequence by some, most
probably, hydrophobic or hydrogen bonding interac-
tions, and may interact with the putative e opioid
receptor. It is considered that the Tyr1 residue (bold
lines in Fig. 1) and the C-terminal part may correspond
to the message and the address sites, respectively, in
terms of the message–address concept.15;20;21 This model
suggests that the spatial relationship between the mes-
ε opioid receptor
3. Chemistry
C-terminal part (= address site)
Lys28
Tyr27
The target compounds 3a–j were prepared (Schemes 1–
3) from 17-(cyclopropylmethyl)northebaine (1)27–29 by
Diels–Alder reaction with acrylamide derivatives, ethyl
acrylate,30 or acrylonitrile. The carboxylic acid 5 was
obtained by hydrolysis of the Diels–Alder adduct 4. The
carboxylic acid 5 was converted to the intermediates
2b,c, or 2e–i via the acid chloride 6, which was obtained
by treatment of the carboxylic acid 5 with oxalyl chlo-
ride, followed by amidation with the corresponding
amines. The intermediate 2j was prepared (Scheme 3)
from the 7a-carbonitrile 7 by Ritter reaction with
t-BuOH. The O-methyl groups of the intermediates 2a–j
were removed by treatment with BBr3 to afford the
target compounds 3a–j. The ethanomorphinan deriva-
tives 10, 7b-carboxamide 9, and 7a-acylamino derivative
15 were also synthesized in order to study the role of
Glu31
Gly30
Lys29
O
H2N
Gly2 Gly3
Met5
Phe4
message site
OH
Figure 1. Binding model of b-endorphin to putative e opioid receptor.
The model was taken from Ref. 23 and modified. Bold lines and dotted
lines express the message site and the hydrophobic or hydrogen
bonding interactions between the amino acid residues, respectively.
The interactions between the address site and the putative e opioid
receptor are indicated by wavy lines.

H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
4135
17
N
9
14
16
10
N
N
11
8
1
15
O
O
a
b
7
6
2
12
4
13
NR1R2
NR1R2
3
5
O
O
O
MeO
OMe
MeO
OMe
HO
OH
1
2a-e
3a-e
a: R1=Ph, R2=H
b: R1=Bn, R2=H
d: R1=Ph(CH2)4, R2=H
e: R1=allyl, R2=H
c: R1=Ph(CH2)2, R2=H
Scheme 1. Reagents and conditions: (a) N-R1-N-R2-acrylamide, xylene, reflux; (b) BBr3/CH2Cl2, CH2Cl2, rt. Numbering of atoms in 6,14-endo-
ethenomorphinan is shown in formula 2.
N
N
N
HCl
O
O
a
b
OEt
OMe
OH
OMe
O
O
O
MeO
OMe
N
MeO
MeO
1
4
5
N
N
O
O
O
d
c
e
Cl
NR1R2
NR1R2
O
O
O
MeO
OMe
MeO
OMe
HO
OH
6
2b, c, e-i
3b, c, e-i
b: R1=Bn, R2=H
c: R1=Ph(CH2)2, R2=H
e: R1=allyl, R2=H
f: R1=Ph(CH2)3, R2=H
g: R1=Ph(CH2)2, R2=Me
h: R1=allyl, R2=Me
i: R1=propargly, R2=H
Scheme 2. Reagents and conditions: (a) ethyl acrylate, reflux; (b) 6 M HCl, reflux; (c) (COCl)2, CHCl3, reflux; (d) HNR1R2, Et3N, CH2Cl2, 0 °C; (e)
BBr3/CH2Cl2, CH2Cl2, rt.
N
N
N
a
+
CN
OMe
CN
OMe
O
O
O
MeO
OMe
MeO
N
MeO
1
7
8
N
O
O
b
c
7
HN
OMe
HN
OH
O
O
MeO
HO
2j
3j
Scheme 3. Reagents and conditions: (a) acrylonitrile, reflux; (b) t-BuOH, 85% H2SO4, AcOH, rt; (c) BBr3/CH2Cl2, CH2Cl2, rt.
the etheno-bridge or the 7a-carbamoyl group for the
putative e opioid receptor activity. The epimer 9 (Fig. 3)
of 7a-carboxyamide 3c was obtained by Diels–Alder
reaction with N-phenethylacrylamide as a by-product
followed by demethylation.
The target compounds 10c and 10g were prepared
(Scheme 4) from compounds 3c and 3g by the catalytic
hydrogenation, respectively. The target compound 15
was prepared by Curtius rearrangement as a key reac-
tion (Scheme 5).31;32 Amine 13 was obtained from

4136
H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
tions (Table 1). In the present study, compounds 3b,c,g,
N
10c, and 10g showed agonistic activity in the RVD
preparation. Compound 3g (TAN-821) was the most
potent agonist among them and its potency
(IC50 ¼ 71.71 nM) is comparable to that of b-endorphin
(IC50 ¼ 73.91 nM). All these compounds possess a phe-
nyl group in their 7-substituents.
O
HN
O
HO
OH
9
Compounds 3e and 3h–j, which have no phenyl group in
their 7-substituents, exhibited no agonistic activity in
this preparation. Compounds 3a,d, and 3f also did not
show agonistic activity, though they possess a phenyl
group in their 7-substituents. The length between the
carbamoyl group and the phenyl group in these com-
pounds is longer or shorter than that of the compounds
showing the agonistic activity. Both conversions of the
7a-carboxamide 3c into the 7b-carboxamide 9 and the
7a-acylamino derivative 15 led to no agonistic activity.
Figure 3. Structure of compound 9.
N
N
O
NR1R2
O
a
NR1R2
O
3c, 3g
O
HO
OH
HO
OH
10c, 10g
c: R1=Ph(CH2)2, R2=H, g: R1=Ph(CH2)2, R2=Me
Table 1. Effects of compounds 3a–j, 9, 10c,g, and 15 on the electrically-
stimulated RVD contractions
Scheme 4. Reagents and conditions: (a) H2, Pd/C, CH3SO3H, MeOH,
rt.
Compound
IC50 (nM) or %Max response
b-Endorphin
73.91 nM (38.35–109.46)a
No inhibition at 0.1 mM
hydrazide 11 by Curtius rearrangement. Compound 15
was prepared by amidation of amine 13 with 3-phenyl-
propionyl chloride followed by demethylation.
Morphineb
Etorphineb
3a
50 nM
––c
3b
3c
45.8% (28.7–62.8)a at 10 lM
34.4% (22.6–46.2)a at 8 lM
3d
3e
––c
4. Inhibition of smooth muscle contraction
––c
3f
3g (TAN-821)
––c
It is postulated that the putative e opioid receptor, but
not l, d, and j opioid receptors, is present in the rat vas
deferens (RVD). This contention is supported by the
evidence that b-endorphin exhibits agonistic activity
(inhibition of the electrically-stimulated RVD contrac-
tions) in the RVD preparation, while the l opioid
receptor agonist morphine, the endogenous d opioid
peptide enkephalin, or the endogenous j opioid peptide
dynorphin does not show agonistic activity in the same
preparation.33;34 The agonistic activities of compounds
3a–j, 9, 10c,g, and 15 for putative e opioid receptor were
evaluated on the electrically-stimulated RVD contrac-
71.71 nM (51.21–100.41)a
3h
3i
––c
––c
3j
––c
9
––c
10c
10g
15
44.2% (18.4–70.1)a at 1 lM
60.9% (45.1–76.7)a at 2 lM
––c
a Numbers in parentheses are 95% confidence intervals.
b Ref. 35.
c The inhibition of electorically-stimulated contraction was not ob-
served.
N
N
N
O
NH
OMe
O
O
OBn
b, c
a
OEt
OMe
NHNH2
O
O
O
MeO
MeO
OMe
MeO
4
11
12
N
N
N
O
O
R
R
f
d
e
NH2
OMe
R=(CH2)2Ph
NH
OMe
NH
OH
O
13
O
14
O
15
MeO
MeO
HO
Scheme 5. Reagents and conditions: (a) NH2NH2ÆH2O, 2-ethoxyethanol, reflux; (b) NaNO2 aq, 6 M HCl; (c) BnOH, reflux, (d) AcOH, concd HCl,
80 °C; (e) RCOCl, Et3N, CH2Cl2, 0 °C; (f) BBr3/CH2Cl2, CH2Cl2, rt.

H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
4137
Table 2. Effects of compounds 3b,c,g, 10c, and 10g on the electrically-stimulated MVD contractions
Compound
IC50 (nM)
IC50 ratioa (l)b
IC50 ratioa (d)c
IC50 ratioa (j)d
b-Endorphine
Morphinee
Etorphinee
3b
89
890
––f
––f
––f
2.0
1.8
1.3
1.8
1.0
––f
––f
––f
2.6
2.8
2.6
1.7
1.4
––f
––f
––f
2.0
0.94
1.6
2.4
5.8
0.25
0.36 (0.26–0.52)g
0.038 (0.034–0.043)g
0.0029 (0.0017–0.0048)g
0.0015 (0.0011–0.0019)g
0.059 (0.0038–0.0090)g
3c
TAN-821 (3g)
10c
10g
a IC50 ratio ¼ (IC50 value of the agonist in the presence of the antagonist)/( IC50 value of the agonist in the absence of the antagonist).
b Naloxone (30 nM).
c NTI (10 nM).
d nor-BNI (10 nM).
e Ref. 35.
f Not evaluated.
g Numbers in parentheses are 95% confidence intervals.
However, the difference between the etheno-bridge
(compounds 3c and 3g) and the ethano-bridge (com-
pounds 10c and 10g) had little or no effect on their
agonistic activities. These results suggest that the spatial
position of the phenyl group in the 7-substituent may
play an important role to exhibit the agonistic activity
for putative e opioid receptor.
The agonistic activity of compounds 3b,c,g, 10c, and
10g, which are agonists in the RVD preparation, for
other opioid receptors was evaluated on the electrically-
stimulated mouse vas deferens (MVD) contractions. It is
generally accepted that the MVD contains the l, d, and
j opioid receptors and their agonists inhibit the elec-
trically stimulated MVD contraction.33 In the present
study, these compounds exhibited the potent agonistic
activity in MVD preparation (Table 2). However, their
agonistic activities were not well attenuated by the l
opioid receptor antagonist naloxone (30 nM), the d
opioid receptor antagonist NTI (10 nM), or the j opioid
receptor antagonist nor-BNI (10 nM). At present, it is
not clear by what receptor the potent agonistic activity
of these compounds in the MVD preparation is medi-
ated.
Figure 4. [35S]GTPcS binding by TAN-821 and the effects of opioid
receptor antagonists on TAN-821-stimulated [35S]GTPcS binding in
the mouse pons/medulla. The statistical significance of differences be-
tween the groups was assessed with one-way ANOVA followed by
Newman–Kenls’s test. ꢀp < 0:05 and ꢀꢀp < 0:01 versus 10 lM TAN-
821 alone.
duced by selective l, d, and j opioid receptor agonists,
respectively, attenuated the TAN-821-stimulated
[35S]GTPcS binding. In the presence of all antagonists,
b-FNA, NTI, and nor-BNI, the stimulation of
[35S]GTPcS binding induced by TAN-821 still remained
(Fig. 4). Unlike TAN-821, the b-endorphin-stimulated
[35S]GTPcS binding was partially attenuated by b-FNA,
however, no further attenuation of TAN-821-stimulated
[35S]GTPcS binding was observed in the presence of all
b-FNA, NTI, and nor-BNI. Taken together, the present
results suggest that TAN-821-stimulated [35S]GTPcS
binding, rather than b-endorphin-stimulated [35S]GTP-
cS binding, may be more selectively mediated by the
putative e opioid receptor.
5. [35S]GTPcS binding
The intrinsic activity of TAN-821, the most potent
agonist in the RVD preparation, was evaluated by the
[35S]GTPcS binding in the mouse pons/medulla mem-
branes.9;36 The stimulation of the [35S]GTPcS binding
reflects the activation of G-protein coupled with a
receptor such as opioid receptors. b-Endorphin has been
reported to stimulate the [35S]GTPcS binding in the
mouse pons/medulla membranes via the activation of
both l and putative e opioid receptors. In the present
study, TAN-821 stimulated the [35S]GTPcS binding to
the mouse pons/medulla membranes in a concentration-
dependent manner. Neither the l opioid receptor
antagonist b-FNA (10 lM), NTI (0.03 lM), nor nor-
BNI (0.1 lM) at the concentration, which completely
attenuated the stimulation of [35S]GTPcS binding in-
6. Antinociception
The antinociceptive effect of TAN-821 was evaluated
using the tail-flick37 and the hot-plate38 tests. Groups
of mice were injected i.c.v. with 1, 2.5, or 5.0 lg of

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H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
TAN-821 and the tail-flick and the hot-plate responses
were measured at various times after the injection.
TAN-821 given i.c.v. produced a dose-dependent inhi-
bition of the tail-flick response. The inhibition of the
tail-flick response reached a peak at 2 h after the injec-
tion, and lasted more than 24 h (Fig. 5A). Similarly,
TAN-821 administered i.c.v. inhibited the hot-plate re-
sponse in a dose-dependent manner. The inhibition of
the hot-plate response reached at 2 h after the injection,
and lasted more than 24 h (Fig. 5B). The ED50 value of
TAN-821 for the antinociception using the tail-flick and
the hot-plate tests was 1.73 and 2.05 lg, respectively.
The antinociception induced by the i.c.v.-administered
TNA-821 was significantly blocked by the i.c.v.-pre-
treatment with the putative e opioid receptor partial
agonist b-endorphin [1–27],39;40 while the i.c.v.-pre-
treatment with b-FNA (1 lg), NTI (5 lg), nor nor-BNI
(5 lg) failed to attenuate the antinociception induced by
TAN-821 given i.c.v. (Fig. 6). These results clearly
suggest that the antinociception induced by TNA-821 at
the supraspinal site may be mediated by the putative e
Figure 6. Effects of opioid receptor antagonists on the antinociception
induced by TAN-821 in the mouse tail-flick test. The statistical sig-
nificance of differences between the groups was assessed with one-way
ANOVA followed by Newman–Kenls’s test. ꢀꢀp < 0:01 versus 10 min
pretreatment with saline.
opioid receptor. Although it has been already reported
that b-endorphin, etorphine,11 and bremazocine12 show
the antinociception mediated by the putative e opioid
receptor, these actions are also partially mediated by the
l or j opioid receptors. TAN-821 is the first compound
to produce the antinociception selectively mediated by
the putative e opioid receptor. TAN-821 may be a useful
tool for the investigation on the pharmacological
properties of the putative e opioid receptor.
7. Discussion
We designed and synthesized a selective agonist for the
putative e opioid receptor on the basis of the message–
address concept. The results of evaluation of the syn-
thesized compounds using RVD preparation suggested
that the 7-substituent may play a critical role to produce
the agonistic activity for the putative e opioid receptor
and indicated the structure–activity relationship as fol-
lows: (1) a phenyl group in the 7a-carbamoyl substituent
would be essential. (2) The suitable length between the
carbamoyl and phenyl groups would be an ethylene
length. One of the synthesized compounds, TAN-821,
produced the potent antinociceptive effect selectively
mediated by the putative e opioid receptor. Taken to-
gether, it is reasonable that the 7a-(N-methyl-N-phen-
ethyl)carbamoyl moiety could be viewed as the
accessory site. The accessory site, the 7a-substituent,
would interact with the receptor as shown in a binding
model of TAN-821 to the putative e opioid receptor
(Fig. 7). The morphinan skeleton can generally be
viewed as the message site (bold lines in Fig. 7). The
address site is held above the message site by a rigid
bicyclo[2.2.2]octene structure. On the other hand, the C-
terminal part of b-endorphin, the address site, is also
held above the message site, the N-terminal part by
some, most probably, hydrophobic or hydrogen bond-
ing interactions (Fig. 1). Comparison between binding
Figure 5. Antinociceptive effect of TAN-821 in the mouse tail-flick test
(A) and hot-plate test (B). The statistical significance of differences
between the groups was assessed with two-way ANOVA followed by
Bonferroni’s test. ꢀp < 0:05, ꢀꢀp < 0:01, and ꢀ ꢀ ꢀp < 0:001 versus saline-
treated mice. Numbers in parentheses are 95% confidence intervals.

H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
4139
determined with a Heraeus CHN-ORAPID for carbon,
hydrogen, and nitrogen, Yokogawa IC-7000 for sulfur.
Elemental analyses were within 0.4% of the theoretical
values. The progress of the reaction was determined on
Merck Silica Gel Art.5715 or Fuji silysia NH Silica Gel.
All the column chromatographies were carried out using
Merck Silica Gel Art.9385, Merck Silica Gel Art.7734,
Fuji Silysia Silica Gel DM-2010, or Fuji Silycia Silica Gel
DM-2035. All the experiments were carried out under an
argon atmosphere except for the catalytic hydrogenation.
ε opioid receptor
O
N
Me
OH
address site
N
O
message site
OH
9.1. Ethyl 17-(cyclopropylmethyl)-4,5a-epoxy-3,6b-di-
methoxy-6,14-endoethenomorphinan-7a-carboxylate (4)
Figure 7. Binding model of TAN-821 to putative e opioid receptor.
Bold lines express the message site. The interactions between the ad-
dress site and the putative e opioid receptor are indicated by wavy
lines.
A stirred solution of 17-(cyclopropylmethyl)northebaine
(1) (6.66 g, 19.0 mmol) in ethyl acrylate (20 mL) was
refluxed for 15 h. After cooling to rt, the reaction mixture
was concentrated in vacuo. The residue was crystallized
from AcOEt to give 5.88 g (69%) of 4: mp 139–142 °C. IR
(KBr) cmꢁ1: 3078, 2998, 2938, 2836, 2816, 1721, 1630,
1603, 1499, 1454, 1441, 1377, 1350, 1284, 1261, 1209,
1166, 1100, 1060, 1017, 901, 774, 700. 1H NMR (CDCl3,
300 MHz) d: 0.10–0.20 (2H, m), 0.46–0.59 (2H, m), 0.78–
0.90 (1H, m), 1.25 (3H, t, J ¼ 7:2 Hz), 1.47 (1H, dd,
J ¼ 6:3, 12.6 Hz), 1.84–1.90 (1H, m), 1.97 (1H, dt,
J ¼ 5:7, 12.6 Hz), 2.32–2.51 (4H, m), 2.70 (1H, dd,
J ¼ 4:8, 11.7 Hz), 2.85 (1H, dd, J ¼ 6:9, 9.0 Hz), 3.07–
3.17 (2H, m), 3.55 (1H, d, J ¼ 6:6 Hz), 3.62 (3H, s), 3.82
(3H, s), 4.12 (1H, dq, J ¼ 7:2, 10.8 Hz), 4.16 (1H, dq,
J ¼ 7:2, 10.8 Hz), 4.61 (1H, s), 5.56 (1H, d, J ¼ 8:7 Hz),
5.85 (1H, d, J ¼ 8:4 Hz), 6.52 (1H, d, J ¼ 8:1 Hz), 6.62
(1H, d, J ¼ 8:1 Hz). HREI-MS [M]þ m=z calcd for
C27H33NO5: 451.2359. Found: 451.2358.
models of b-endorphin and TAN-821 suggests that the
relative location between the message and the address
sites in TAN-821 seems to be very similar to that in b-
endorphin. As mentioned in the design rationale section,
the spatial relationship between the message and the
address sites may be important to exhibit agonistic
activity for the putative e opioid receptor.
8. Conclusion
We have designed and synthesized a series of 6,14-endo-
ethenomorphinan-7a-carboxamide
derivatives
for
the putative e opioid receptor agonist. 17-(Cycloprop-
ylmethyl)-4,5a-epoxy-3,6b-dihydroxy-6,14-endoetheno-
morphinan-7a-(N-methyl-N-phenethyl)carboxamide,
TAN- 821 (3g), one of the synthesized compounds,
showed agonistic activity for the putative e opioid
receptor on the electrically-stimulated RVD contractions
and the [35S]GTPcS binding to the mouse pons/medulla
membrane. Furthermore, TAN-821 given i.c.v. produced
the b-endorphin-like antinociceptive effect. These find-
ings support the idea that TAN-821 may be a useful tool
for investigation on the pharmacological properties of the
putative e opioid receptor.
9.2. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dimethoxy-
6,14-endoethenomorphinan-7a-carboxylic acidꢂhydro-
chloride (5ꢂHCl)
A stirred solution of 4(2.02 g, 4.47 mmol) in 6 M HCl
(30 mL) was refluxed for 7 h. After cooling to rt, the
reaction mixture was cooled in an ice-bath. Cold water
(20 mL) was added to the solution to give a precipitate.
The precipitate was recrystallized from MeOH to give
1.56 g (76%) of 5ÆHCl: mp 190–195 °C (dec.). IR (KBr)
cmꢁ1: 3596, 3320, 2932, 2666, 2634, 1736, 1702, 1632,
1508, 1475, 1458, 1437, 1292, 1270, 1214, 1195, 1172,
1127, 1104, 1054, 948, 816. 1H NMR (DMSO-d6,
300 MHz) d: 0.36–0.48 (1H, m), 0.54–0.78 (3H, m), 1.07–
1.22 (1H, m), 1.44 (1H, dd, J ¼ 6:0, 12.6 Hz), 1.97 (1H,
d, J ¼ 12:5 Hz), 2.32 (1H, dt, J ¼ 3:7, 13.9 Hz), 2.79–
3.26 (5H, m), 3.32–3.54 (3H, m), 3.46 (3H, s), 3.72 (3H,
s), 4.43 (1H, d, J ¼ 6:2 Hz), 4.94 (1H, s), 5.59 (1H, d,
J ¼ 8:8 Hz), 5.62 (1H, d, J ¼ 8:8 Hz), 6.60 (1H, d,
J ¼ 8:4 Hz), 6.73 (1H, d, J ¼ 8:1 Hz), 9.76 (1H, br s),
12.27 (1H, br s). HRESI-MS [MþH]þ m=z calcd for
C25H29NO5: 424.2124. Found: 424.2122.
9. Experimental
Melting points were determined on a Yanaco MP-500D
melting point apparatus and were uncorrected. Nuclear
magnetic resonance (NMR) spectra were recorded on a
Varian GEMINI 300 (300 MHz), JEOL GX-400
(400 MHz), Varian UNITY plus 500 (500 MHz), or
Varian UNITY INOVA 600 (600 MHz) spectrometers
and the chemical shifts are reported as d values (ppm)
related to tetramethylsilane (TMS). Infrared (IR) spectra
were obtained using a JASCO FT/IR-5000 as KBr pellets
or neat. Mass spectra (MS) were obtained on a JEOL
JMS-D300, JEOL JMS-DX303, VG ZAB-HF, or
micromass LCT (HP1100) (A-MS-4) instruments by
applying an electric ionization (EI) method, a fast atom
bombardment (FAB) ionization method, or an electro-
spray ionization (ESI) method. Elemental analyses were
9.3. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethenomorphinan-7a-(N-phenethyl)carboxamideꢂ
methane sulfonate (3cꢂMeSO3H)
Method A: To a solution of 17-(cyclopropylmethyl)-
northebaine (1) (354 mg, 0.99 mmol) in dichlorobenzene

4140
H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
(10 mL) was added N-phenethylacrylamide (365 mg,
2.1 mmol) and the mixture was then refluxed for 20 h.
After cooling to rt, the reaction solution was poured
into distilled water, then extracted with AcOEt. The
combined organic layer was washed with brine and
dried over MgSO4. After removing the solvent in vacuo,
the residue was chromatographed on silica gel to give a
mixture of 2c and N-phenethylacrylamide. To the stirred
solution of the mixture in dry CH2Cl2 (20 mL) was
added dropwise a 1.0 M solution of BBr3 in dry CH2Cl2
(6.0 mL, 6.0 mmol) at 0 °C. After stirring at rt for 4 h,
the reaction mixture was cooled to 0 °C. To the reaction
mixture was added 6% aqueous ammonia (20 mL), then
stirred vigorously at rt. The resulting mixture was sep-
arated and extracted with CHCl3. The combined organic
layers were dried over MgSO4 and evaporated in vacuo.
The residue was chromatographed on silica gel to give
130 mg (26% two-step yield) of 3c and 16 mg (3% two-
step yield) of 9. To the stirred suspension of 3c or 9 in
MeOH was added dropwise MeSO3H until the solution
became acidic or a maleic acid (3.7 mg, 31.9 lmol)
solution in MeOH at 0 °C, respectively. Ethyl acetate
was then added to the solution. The precipitated
amorphous salt was filtered to give 3cÆMeSO3H or
9ÆHOOCCH@CHCOOH. Compound 3cÆMeSO3H: IR
(KBr) cmꢁ1: 3422, 1657, 1649, 1638, 1562, 1543, 1460,
1323, 1199, 1048, 785, 555. 1H NMR (DMSO-d6,
600 MHz) d: 0.40–0.45 (1H, m), 0.47–0.42 (1H, m), 0.61–
0.67 (1H, m), 0.70–0.76 (1H, m), 1.07–1.15 (1H, m), 1.52
(1H, dd, J ¼ 6:4, 13.5 Hz), 2.01–2.13 (2H, m), 2.33
(3.3H, s), 2.50–2.56 (1H, m), 2.66–2.78 (3H, m), 2.91–
3.06 (3H, m), 3.23 (1H, dt, J ¼ 6:7, 12.8 Hz), 3.27–3.40
(3H, m), 3.48 (1H, quintet, J ¼ 6:7 Hz), 4.27 (1H, s),
4.36 (1H, d, J ¼ 7:0 Hz), 5.46 (1H, d, J ¼ 8:9 Hz), 5.62
(1H, d, J ¼ 8:9 Hz), 5.72 (1H, br s), 6.45 (1H, d,
J ¼ 7:9 Hz), 6.53 (1H, d, J ¼ 7:9 Hz), 7.18–7.24 (3H, m),
7.26–7.52 (2H, m), 7.74 (1H, br t, J ¼ 5:0 Hz), 8.36 (1H,
br s), 9.10 (1H, br s). MS (FAB) m=z 499 [MþH]þ. Anal.
Calcd for C31H34N2O4Æ0.2H2OÆ1.3MeSO3H: C, 61.86;
H, 6.36; N, 4.47; S, 6.65. Found: C, 61.88; H, 6.56; N,
4.44; S, 6.51. Compound 9ÆHOOCCH@CHCOOH: IR
(free base, KBr) cmꢁ1: 3418, 2946, 1642, 1547, 1502,
prepared 6 in dry CHCl3 (10 mL) at 0 °C. After stirring
at rt for 1 h, the reaction mixture was poured into a
saturated NaHCO3 solution, and then extracted with
AcOEt. The combined organic layer was washed with
brine and dried over MgSO4. After removing the solvent
in vacuo, the residue was chromatographed on silica gel
to give 509 mg (quantitative yield) of 2c. To the stirred
solution of 2c (509 mg, 0.97 mmol) in dry CH2Cl2
(20 mL) was added dropwise a 1.0 M solution of BBr3 in
dry CH2Cl2 (6.0 mL, 6.0 mmol) at 0 °C. After stirring at
rt for 1 h, the reaction mixture was cooled to 0 °C. To
the reaction mixture was added 6% aqueous ammonia
(20 mL), then stirred vigorously at rt. The resulting
mixture was separated and extracted with CHCl3. The
combined organic layers were dried over MgSO4 and
evaporated in vacuo. The residue was chromatographed
on silica gel to give 331 mg (69%) of 3c. To the stirred
suspension of 3c in MeOH was added dropwise
MeSO3H at 0 °C until the solution became acidic. Ethyl
acetate was then added to the solution. The precipitated
amorphous salt was filtered to give 6cÆMeSO3H.
The following compounds were synthesized by method
A:
9.4. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethenomorphinan-7a-(N-phenyl)carboxamideꢂ
methane sulfonate (3aꢂMeSO3H)
Two-step yield (29%). IR (KBr) cmꢁ1: 3400, 1678, 1671,
1657, 1601, 1545, 1499, 1446, 1321, 1209, 1195, 1048. 1H
NMR (DMSO-d6, 400 MHz) d: 0.39–0.52 (2H, m), 0.60–
0.78 (2H, m), 1.07–1.17 (1H, m), 1.64 (1H, dd, J ¼ 8:5,
17.3 Hz), 2.11 (2H, br d, J ¼ 5:9 Hz), 2.33 (3.6H, s),
2.76–2.90 (2H, m), 2.92–3.11 (3H, m), 3.28–3.58 (3H,
m), 4.36 (1H, s), 4.43 (1H, d, J ¼ 6:8 Hz), 5.54 (1H, d,
J ¼ 8:8 Hz), 5.65 (1H, d, J ¼ 8:8 Hz), 5.92 (1H, br s),
6.50 (1H, d, J ¼ 8:3 Hz), 6.57 (1H, d, J ¼ 8:3 Hz), 7.05
(1H, t, J ¼ 7:3 Hz), 7.30 (2H, t, J ¼ 7:8 Hz), 7.59 (2H, d,
J ¼ 8:3 Hz), 8.41 (1H, br s), 9.13 (1H, br s), 9.84 (1H, s).
MS (FAB) m=z 471 [MþH]þ. Anal. Calcd for
C29H30N2O4Æ0.4H2OÆ1.2MeSO3H: C, 61.16; H, 6.05; N,
4.72; S, 6.49. Found: C, 61.05; H, 6.13; N, 4.84; S, 6.45.
1
1471, 1461, 1321, 1204, 1177, 1048, 784. H NMR (free
base, CDCl3, 300 MHz) d: 0.05–0.20 (2H, m), 0.41–0.59
(2H, m), 0.74–0.92 (1H, m), 1.48 (1H, t, J ¼ 12:4 Hz),
1.62 (1H, d, J ¼ 10:7 Hz), 2.27–2.57 (6H, m), 2.63–2.76
(1H, m), 2.86 (2H, t, J ¼ 7:0 Hz), 3.09 (2H, d,
J ¼ 18:1 Hz), 3.42–3.68 (3H, m), 4.68 (1H, br s), 4.93
(1H, s), 5.37 (1H, d, J ¼ 8:5 Hz), 5.70 (1H, dd, J ¼ 1:2,
8.5 Hz), 6.32 (1H, br s), 6.46 (1H, d, J ¼ 8:2 Hz), 6.62
(1H, d, J ¼ 8:2 Hz), 7.12–7.35 (5H, m). MS (EI) m=z 498
[M]þ Anal. calcd for C31H34N2O4Æ0.2H2OÆC4H4O4: C,
67.99; H, 6.26; N, 4.53. Found: C, 67.63; H, 6.51; N,
4.92.
9.5. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethenomorphinan-7a-[N-(4-phenylbutyl)]carb-
oxamideꢂmethane sulfonate (3dꢂMeSO3H)
Two-step yield (39%). IR (KBr) cmꢁ1: 3450, 2938, 2368,
1649, 1638, 1562, 1460, 1323, 1209, 1195, 1046, 783, 555.
1H NMR (DMSO-d6, 300 MHz) d: 0.37–0.53 (2H, m),
0.57–0.78 (2H, m), 1.04–1.17 (1H, m), 1.33–1.46 (2H,
m), 1.47–1.64 (3H, m), 1.97–2.12 (2H, m), 2.35 (4.5H, s),
2.50–2.61 (3H, m), 2.75 (1H, dd, J ¼ 8:5, 12.6 Hz), 2.87–
3.19 (5H, m), 3.26–3.36 (1H, m), 3.36 (1H, d,
J ¼ 19:2 Hz), 3.40–3.54 (1H, m), 4.26 (1H, s), 4.36 (1H,
d, J ¼ 6:6 Hz), 5.46 (1H, d, J ¼ 8:8 Hz), 5.60 (1H, d,
J ¼ 8:8 Hz), 6.45 (1H, d, J ¼ 8:2 Hz), 6.53 (1H, d,
J ¼ 8:0 Hz), 7.12–7.23 (3H, m), 7.23–7.33 (2H, m),
7.66 (1H, br t, J ¼ 5:4 Hz), 8.36 (1H, br s). MS (FAB)
m=z 527 [MþH]þ. Anal. Calcd for C33H38N2
Method B: To a stirred suspension of 5ÆHCl (445 mg,
0.97 mmol) in dry CHCl3 (20 mL) was added dropwise
oxalyl dichloride (0.3 mL, 3.4 mmol) at 0 °C. The reac-
tion mixture was refluxed gently for 2 h. After cooling to
rt, the mixture was concentrated in vacuo to give crude
acid chloride 6. To the stirred solution of phenethyl-
amine (0.50 g, 4.1 mmol) and Et3N (0.28 mL, 2.0 mmol)
in dry CHCl3 (10 mL) was added dropwise a solution of

H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
4141
O4Æ0.6H2OÆ1.5MeSO3H: C, 60.79; H, 6.68; N, 4.11; S,
7.06. Found: C, 60.61; H, 6.61; N, 4.41; S, 7.06.
3.26–3.52 (3H, m), 4.28 (1H, s), 4.37 (1H, d, J ¼ 6:8 Hz),
5.46 (1H, d, J ¼ 8:8 Hz), 5.62 (1H, d, J ¼ 8:8 Hz), 5.70
(1H, br s), 6.45 (1H, d, J ¼ 8:3 Hz), 6.54 (1H, d,
J ¼ 8:3 Hz), 7.13–7.24 (3H, m), 7.324–7.32 (2H, m), 7.70
(1H, br t, J ¼ 5:4 Hz), 8.37 (1H, br s), 9.09 (1H, br s).
MS (FAB) m=z 513 [MþH]þ. Anal. Calcd for
C32H36N2O4Æ0.5H2OÆMeSO3H: C, 64.16; H, 6.69; N,
4.53; S, 5.19. Found: C, 64.04; H, 6.84; N, 4.59; S, 5.28.
The following compounds were synthesized by both
methods A and B:
9.6. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethenomorphinan-7a-(N-benzyl)carboxamideꢂ
methane sulfonate (3bꢂMeSO3H)
9.9. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethenomorphinan-7a-(N-methyl-N-phenethyl)-
carboxamideꢂmethane sulfonate (3gꢂMeSO3H, TAN-
821ꢂMeSO3H)
Two-step yield (58%, method A), 92% (the first step in
method B), 49% (the second step in method B). IR
(KBr) cmꢁ1: 3400, 3058, 1640, 1551, 1502, 1473, 1458,
1
1437, 1361, 1323, 1195, 1120, 1046, 702, 555. H NMR
(DMSO-d6, 300 MHz) d: 0.37–0.54 (2H, m), 0.57–0.77
(2H, m), 1.04–1.18 (1H, m), 1.56 (1H, dd, J ¼ 6:2,
12.6 Hz), 1.98–2.18 (2H, m), 2.33 (3H, s), 2.60–2.69 (1H,
m), 2.76–2.87 (1H, m), 2.88–3.10 (3H, m), 3.21–3.49
(3H, m), 4.19–4.40 (4H, m), 5.48 (1H, d, J ¼ 8:7 Hz),
5.65 (1H, d, J ¼ 8:7 Hz), 6.45 (1H, d, J ¼ 8:1 Hz), 6.54
(1H, d, J ¼ 8:1 Hz), 7.17–7.37 (5H, m), 8.19 (1H, br t,
J ¼ 5:8 Hz), 8.40 (1H, br s), 9.13 (1H, br s). MS (FAB)
m=z 485 [MþH]þ. Anal. Calcd for C30H32N2O4Æ
0.4H2OÆMeSO3H: C, 63.33; H, 6.31; N, 4.76; S, 5.45.
Found: C, 63.18; H, 6.39; N, 4.68; S, 5.75.
Quantitatively (the first step), 74% (the second step). IR
(KBr) cmꢁ1: 3422, 2940, 1629, 1501, 1464, 1414, 1320,
1209, 1194, 1119, 1057, 934, 783. H NMR (DMSO-d6,
1
400 MHz) d: 0.4–0.54 (2H, m), 0.6–0.68 (1H, m), 0.70–
0.77 (1H, m), 1.06–1.15 (1H, m), 1.30 (0.6H, dd, J ¼ 6:4,
12.2 Hz), 1.46 (0.4H, dd, J ¼ 6:4, 12.2 Hz), 1.98 (0.6H,
br d, J ¼ 11:7 Hz), 2.04 (0.4H, br d, J ¼ 11:7 Hz), 2.18–
2.37 (1H, m), 2.32 (1.8H, s), 2.33 (1.2H, s), 2.67–2.78
(1H, m), 2.75 (1.2H, s), 2.82–3.08 (5H, m), 3.05 (1.8H,
s), 3.12 (0.6H, t, J ¼ 7:8 Hz), 3.26–3.40 (2.4H, m), 3.44–
3.64 (3.6H, m), 3.81–3.89 (0.4H, m), 4.33 (0.6H, d,
J ¼ 6:8 Hz), 4.36 (0.4H, d, J ¼ 6:8 Hz), 4.43 (0.4H, s),
4.46 (0.6H, s), 5.40 (0.6H, d, J ¼ 8:8 Hz), 5.43 (0.4H, d,
J ¼ 8:8 Hz), 5.64 (0.4H, d, J ¼ 8:8 Hz), 5.70 (0.6H, d,
J ¼ 8:8 Hz), 6.45 (1H, d, J ¼ 8:3 Hz), 6.54 (0.6H, d,
J ¼ 8:3 Hz), 6.55 (0.4H, d, J ¼ 8:3 Hz), 7.17–7.38
(5H, m), 8.36 (1H, br s), 9.07 (1H, br s). MS (FAB)
m=z 513 [MþH]þ. Anal. Calcd for C32H36N2O4Æ
0.7H2OÆMeSO3H: C, 63.79; H, 6.72; N, 4.51; S, 5.16.
Found: C, 63.66; H, 6.79; N, 4.58; S, 5.29.
9.7. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethenomorphinan-7a-(N-allyl)carboxamideꢂ
methane sulfonate (3eꢂMeSO3H)
Two-step yield (44%, method A), quantitatively (the first
step in method B), 87% (the second step in method B).
IR (KBr) cmꢁ1: 3398, 1657, 1640, 1543, 1502, 1468,
1421, 1323, 1210, 1195, 1060, 1052, 785. 1H NMR
(DMSO-d6, 300 MHz) d: 0.36–0.564 (2H, m), 0.56–0.77
(2H, m), 1.03–1.18 (1H, m), 1.53 (1H, dd, J ¼ 6:1,
12.3 Hz), 1.98–2.1 8 (2H, m), 2.34 (3.3H, s), 2.55–2.64
(1H, m), 2.71–2.85 (1H, m), 2.87–3.11 (3H, m), 3.20–
3.49 (3H, m), 3.53–3.80 (2H, m), 4.28 (1H, s), 4.37 (1H,
d, J ¼ 6:6 Hz), 5.04 (1H, ddd, J ¼ 2:1, 3.9, 10.3 Hz),
5.18 (1H, ddd, J ¼ 2:1, 4.0, 17.2 Hz), 5.47 (1H, d,
J ¼ 8:7 Hz), 5.62 (1H, d, J ¼ 8:7 Hz), 5.71–5.86 (1H, m),
6.45 (1H, d, J ¼ 8:1 Hz), 6.54 (1H, d, J ¼ 8:1 Hz), 7.83
(1H, br t, J ¼ 5:8 Hz), 8.41 (1H, br s), 9.13 (1H, br s).
MS (FAB) m=z 453 [MþH]þ. Anal. Calcd for
C26H30N2O4Æ0.3H2OÆ1.1MeSO3H: C, 59.65; H, 6.47; N,
5.13; S, 6.46. Found: C, 59.70; H, 6.47; N, 5.13; S, 6.32.
9.10. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethenomorphinan-7a-(N-allyl-N-methyl)carbox-
amideꢂmethane sulfonate (3hꢂMeSO3H)
Quantitatively (the first step), 59% (the second step). IR
(KBr) cmꢁ1: 3424, 3022, 2942, 1627, 1503, 1471, 1418,
1
1321, 1209, 1195, 1059, 785, 561. H NMR (free base,
CDCl3, 300 MHz) d: 0.08–0.24 (2H, m), 0.43–0.62 (2H,
m), 0.74–1.00 (1H, m), 1.11–1.31 (1H, m), 1.84–2.08
(2H, m), 2.29–2.49 (4H, m), 2.64–2.70 (2H, m), 2.88
(1.2H, s), 2.96–3.36 (2H, m), 3.03 (1.8H, s), 3.51 (1H, d,
J ¼ 6:3 Hz), 3.90–4.05 (2H, m), 4.41 (0.4H, s), 4.45
(0.6H, s), 4.65 (1H, br s), 5.09–5.33 (2H, m), 5.37 (1H, d,
J ¼ 8:5 Hz), 5.44 (1H, br s), 5.64–5.88 (1H, m), 5.89–
6.02 (1H, m), 6.46 (1H, d, J ¼ 8:0 Hz), 6.61 (0.4H, d,
J ¼ 8:2 Hz), 6.62 (0.6H, d, J ¼ 8:0 Hz). MS (free base,
EI) m=z 448 [M]þ. Anal. Calcd for C27H32N2O4Æ
0.8H2OÆMeSO3H: C, 60.15; H, 6.78; N, 5.01; S, 5.74.
Found: C, 60.09; H, 6.65; N, 5.06; S, 5.90.
The following compounds were synthesized by method
B:
9.8. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethenomorphinan-7a-[N-(3-phenylpropyl)]car-
boxamideꢂmethane sulfonate (3fꢂMeSO3H)
Quantitatively (the first step), 58% (the second step). IR
(KBr) cmꢁ1: 3356, 2938, 1645, 1547, 1502, 1461, 1321,
1210, 1195, 1046, 784, 556. 1H NMR (DMSO-d6,
400 MHz) d: 0.38–0.53 (2H, m), 0.59–0.77 (2H, m), 1.05–
1.18 (1H, m), 1.55 (1H, dd, J ¼ 6:6, 12.5 Hz), 1.62–1.75
(2H, m), 2.00–2.17 (2H, m), 2.33 (3H, s), 2.52–2.63 (3H,
m), 2.78 (1H, dd, J ¼ 9:3, 12.2 Hz), 2.90–3.12 (5H, m),
9.11. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethenomorphinan-7a-(N-propargyl)carboxamideꢂ
methane sulfonate (3iꢂMeSO3H)
Quantitatively (the first step), 56% (the second step). IR
(KBr) cmꢁ1: 3442, 3394, 2944, 1657, 1541, 1504, 1471,
1
1321, 1209, 1193, 1049, 931, 783. H NMR (DMSO-d6,

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H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
300 MHz) d: 0.36–0.54 (2H, m), 0.58–0.78 (2H, m), 1.03–
1.19 (1H, m), 1.52 (1H, dd, J ¼ 6:3, 12.4 Hz), 1.99–2.16
(2H, m), 2.33 (3H, s), 2.53–2.63 (1H, m), 2.72–2.84 (1H,
m), 2.87–3.13 (3H, m), 3.10 (1H, t, J ¼ 2:5 Hz), 3.25–
3.55 (3H, m), 3.78 (1H, ddd, J ¼ 2:5, 5.0, 15.6 Hz), 3.92
(1H, ddd, J ¼ 2:5, 5.6, 15.6 Hz), 4.26 (1H, s), 4.37 (1H,
d, J ¼ 6:9 Hz), 5.48 (1H, d, J ¼ 8:5 Hz), 5.62 (1H, d,
J ¼ 8:5 Hz), 6.46 (1H, d, J ¼ 8:2 Hz), 6.54 (1H, d,
J ¼ 8:2 Hz), 8.10 (1H, t, J ¼ 5:3 Hz), 8.38 (1H, br s),
9.11 (1H, br s). MS (free base, EI) m=z 432 [M]þ. Anal.
Calcd for C26H28N2O4Æ0.7H2OÆMeSO3H: C, 59.92; H,
6.22; N, 5.18; S, 5.92. Found: C, 59.76; H, 6.34; N, 5.11;
S, 6.00.
was chromatographed on silica gel to give 460 mg (67%)
of 2j. To the stirred solution of 2j (208 mg, 0.44 mmol) in
dry CH2Cl2 (5 mL) was added dropwise a 1.0 M solution
of BBr3 in dry CH2Cl2 (4.4 mL, 4.4 mmol) at 0 °C. After
stirring at rt for 2 h, the reaction mixture was cooled to
0 °C. To the solution was added 6% aqueous ammonia
(5 mL), then stirred vigorously at rt. The resulting mix-
ture was separated and extracted with CHCl3. The
combined organic layers were dried over MgSO4 and
evaporated in vacuo. The residue was chromatographed
on silica gel to give 121 mg (62%) of 3j. To the stirred
suspension of 3j in MeOH was added dropwise a solu-
tion of maleic acid (31.2 mg, 0.27 mmol) in MeOH at
0 °C. Ethyl acetate was then added to the solution. The
precipitated amorphous salt was filtered to give
3cÆmaleate. IR (free base, KBr) cmꢁ1: 3388, 2924, 1644,
1538, 1455, 1364, 1322, 1223, 1025. 1H NMR (free base,
CDCl3, 400 MHz) d: 0.08–0.20 (2H, m), 0.45–0.59 (2H,
m), 0.77–0.91 (1H, m), 1.27–1.44 (2H, m), 1.33 (9H, s),
1.81–1.90 (1H, m), 1.92–2.05 (1H, m), 2.27–2.51(5H, m),
2.67–2.79 (1H, m), 3.09 (1H, d, J ¼ 18:3 Hz), 3.10–3.20
(1H, m), 3.54 (1H, d, J ¼ 5:9 Hz), 4.37 (1H, s), 4.86
(1H, br s), 5.43 (1H, d, J ¼ 8:3 Hz), 5.50–5.64 (1H, m),
5.81 (1H, d, J ¼ 8:3 Hz), 6.46 (1H, d, J ¼ 8:1 Hz),
6.61 (1H, d, J ¼ 8:1 Hz). MS (free base, EI) m=z 450
[M]þ. Anal. Calcd for C27H34N2O4Æ2.1H2OÆC4H4O4: C,
61.60; H, 7.04; N, 4.63. Found: C, 61.59; H, 6.88; N,
4.63.
9.12. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dimethoxy-
6,14-endoethenomorphinan-7a-carbonitrile (7) and 17-
(cyclopropylmethyl)-4,5a-epoxy-3,6b-dimethoxy-6,14-
endoethenomorphinan-7b-carbonitrile (8)
A stirred solution of 17-(cyclopropylmethyl)northebaine
(1) (1.01 g, 2.9 mmol) in acrylonitrile (15 mL) was ref-
luxed for 18 h. After cooling to rt, the reaction mixture
was concentrated in vacuo. The residue was chromato-
graphed on silica gel to give 581 mg (50%) of 7 and
518 mg (44%) of 8 as an amorphous substance. Com-
pound 7: IR (KBr) cmꢁ1: 2908, 2243, 1632, 1503, 1445,
1283, 1208, 1165, 1102, 1060. 1H NMR (CDCl3,
300 MHz) d: 0.06–0.19 (2H, m), 0.45–0.60 (2H, m), 0.75–
0.90 (1H, m), 1.55 (1H, dd, J ¼ 5:8, 13.2 Hz), 1.80–1.98
(2H, m), 2.30–2.45 (3H, m), 2.45 (1H, dd, J ¼ 6:7,
18.8 Hz), 2.67–2.77 (1H, m), 2.89 (1H, dd, J ¼ 5:6,
9.7 Hz), 3.11 (1H, d, J ¼ 18:4 Hz), 3.32 (1H, dd, J ¼ 9:7,
13.3 Hz), 3.56 (1H, d, J ¼ 6:3 Hz), 3.68 (3H, s), 3.82
(3H, s), 4.52 (1H, d, J ¼ 1:4 Hz), 5.66 (1H, d,
J ¼ 9:1 Hz), 5.98 (1H, d, J ¼ 8:8 Hz), 6.54 (1H, d,
J ¼ 8:2 Hz), 6.63 (1H, d, J ¼ 8:2 Hz). HREI-MS [M]þ
m=z calcd for C25H28N2O3: 404.2100. Found: 404.2095.
Compound 8: IR (KBr) cmꢁ1: 2936, 2835, 2235, 1629,
1506, 1457, 1263, 1167, 1132, 1103, 1056. 1H NMR
(CDCl3, 300 MHz) d: 0.09–0.18 (2H, m), 0.46–0.59 (2H,
m), 0.77–0.92 (1H, m), 1.71 (1H, dd, J ¼ 11:8, 13.5 Hz),
1.89 (1H, d, J ¼ 9:9 Hz), 2.27–2.48 (5H, m), 2.75–2.85
(1H, m), 2.80 (1H, dd, J ¼ 3:6, 11.8 Hz), 3.13 (1H, d,
J ¼ 19:0 Hz), 3.18 (1H, dd, J ¼ 3:8, 13.2 Hz), 3.51 (1H,
d, J ¼ 6:6 Hz), 3.64 (3H, s), 3.83 (3H, s), 5.03 (1H, d,
J ¼ 1:6 Hz), 5.57 (1H, d, J ¼ 9:1 Hz), 5.94 (1H, dd,
J ¼ 1:5, 8.9 Hz), 6.52 (1H, d, J ¼ 8:2 Hz), 6.63 (1H, d,
J ¼ 8:0 Hz). HREI-MS [M]þ m=z calcd for C25H28N2O3:
404.2100. Found: 404.2117.
9.14. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethanomorphinan-7a-(N-phenethyl)carboxamideꢂ
methane sulfonate (10cꢂMeSO3H)
A solution of 3c (131 mg, 0.26 mmol) in MeOH (5 mL)
with MeSO3H (20 lL, 0.31 mmol) was stirred for 48 h
with 10% Pd/C (50% wet, 20 mg) under H2 atmosphere
(1 atm) at rt. The catalyst was removed by filtration, and
the filtrate was stirred for 20 h with 10% Pd/C (50% wet,
67 mg) under H2 atmosphere (1 atm) at rt. The catalyst
was removed by filtration, and the filtrate was concen-
trated in vacuo to half the volume. The resulting solu-
tion was poured into a saturated NaHCO3 solution, and
extracted with AcOEt. The extract was washed with
brine, dried over MgSO4, and evaporated in vacuo. The
residue was chromatographed on silica gel to give 40 mg
(30%) of 10c. To a stirred solution of 10c in MeOH was
added dropwise MeSO3H at 0 °C until the solution be-
came acidic. Ethyl acetate was then added to the solu-
tion. The precipitated amorphous salt was filtered to
give 10cÆMeSO3H: IR (KBr) cmꢁ1: 3404, 2942, 1653,
1
1547, 1504, 1463, 1323, 1209, 1048, 783, 703. H NMR
9.13. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihydroxy-
6,14-endoethenomorphinan-7a-(N-t-butyl)carboxamideꢂ
maleate (3jꢂHOOCCH@CHCOOH)
(DMSO-d6, 500 MHz) d: 0.35–0.48 (2H, m), 0.55–0.73
(3H, m), 1.03–1.13 (2H, m), 1.33 (1H, dt, J ¼ 6:4,
12.5 Hz), 1.73 (1H, t, J ¼ 12:5 Hz), 1.86–1.93 (2H, m),
2.14 (1H, dt, J ¼ 4:4, 13.8 Hz), 2.32 (3H, s), 2.40–2.63
(2H, m), 2.68–2.81 (3H, m), 2.82–2.97 (2H, m), 3.20–
3.32 (3H, m), 3.34–3.45 (2H, m), 3.89 (1H, d,
J ¼ 6:8 Hz), 4.17 (1H, s), 5.02 (1H, br s), 6.54 (1H, d,
J ¼ 8:3 Hz), 6.68 (1H, d, J ¼ 7:8 Hz), 7.17–7.33 (5H, m),
7.80 (1H, br t, J ¼ 5:1 Hz), 8.11(1H, br s), 9.28 (1H, br
s). HRESI-MS [MþH]þ m=z calcd for C31H36N2O4:
501.2753. Found: 501.2758.
A solution of 7 (581 mg, 1.4 mmol) in t-BuOH (3.2 mL),
AcOH (4.3 mL), and an 85 vol % H2SO4 (2.0 mL) solu-
tion was stirred for 8 h at rt. After removing the solvent
in vacuo, the reaction mixture was poured into a satu-
rated NaHCO3 solution and extracted with CH2Cl2. The
organic layer was washed with brine and dried over
MgSO4. After removing the solvent in vacuo, the residue

H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
4143
9.15. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dihy-
droxy-6,14-endoethanomorphinan-7a-(N-methyl-N-phen-
ethyl)carboxamideꢂmethane sulfonate (10gꢂMeSO3H)
2838, 1715, 1506, 1454, 1259, 1230, 1104, 1058, 1006,
911, 733. 1H NMR (CDCl3, 300 MHz) d: 0.08–0.18 (2H,
m), 0.46–0.56 (2H, m), 0.76–0.90 (1H, m), 1.06 (1H, dd,
J ¼ 4:4, 13.7 Hz), 1.83 (1H, dd, J ¼ 2:2, 12.9 Hz), 2.05
(1H, dt, J ¼ 5:6, 12.6 Hz), 2.27–2.46 (4H, m), 2.73 (1H,
dd, J ¼ 5:1, 12.2 Hz), 3.08 (1H, d, J ¼ 18:4 Hz), 3.42–
3.56 (2H, m), 3.51 (3H, s), 3.81 (3H, s), 4.05 (1H, br s),
4.71 (1H, s), 4.81 (1H, br d, J ¼ 7:7 Hz), 5.10 (2H, s),
5.56 (1H, d, J ¼ 8:8 Hz), 5.64 (1H, d, J ¼ 8:8 Hz), 6.51
(1H, d, J ¼ 8:0 Hz), 6.61 (1H, d, J ¼ 8:0 Hz), 7.27–7.42
(5H, m). HREI-MS [M]þ m=z calcd for C32H36N2O5:
528.2624. Found: 528.2602.
Yield (53%). IR (KBr) cmꢁ1: 3454, 3428, 2940, 1625,
1498, 1462, 1421, 1204, 1048. 1H NMR (DMSO-d6,
300 MHz) d: 0.33–0.51 (2H, m), 0.51–0.75 (3H, m), 0.98–
1.15 (2H, m), 1.29–1.48 (1H, m), 1.72–1.94 (3H, m),
2.25–2.40 (1H, m), 2.32 (3H, s), 2.52–3.00 (4.5H, m),
2.79 (1.5H, s), 3.06 (1.5H, s), 3.09–3.30 (3H, m), 3.30–
3.76 (4H, m), 3.84–4.04 (1.5H, m), 4.38 (1H, s), 6.54
(0.5H, d, J ¼ 8:2 Hz), 6.55 (0.5H, d, J ¼ 8:2 Hz), 6.68
(0.5H, d, J ¼ 8:0 Hz), 6.69 (0.5H, d, J ¼ 8:0 Hz), 7.17–
7.35 (5H, m), 8.14 (1H, br s), 9.32 (1H, br s). MS (FAB)
m=z 515 [MþH]þ. Anal. Calcd for C32H38N2O4Æ
0.7H2OÆ1.1MeSO3H: C, 62.81; H, 6.97; N, 4.43; S, 5.57.
Found: C, 62.90; H, 6.70; N, 4.43; S, 5.65.
9.18. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dimeth-
oxy-6,14-endoethenomorphinan-7a-amine (13)
To a stirred solution of 12 (300 mg, 0.53 mmol) in AcOH
(15 mL) and concentrated HCl (7.5 mL) was heated at
80 °C for 1 h. After cooling to rt, the reaction mixture
was concentrated in vacuo. The residue was poured into
15% aqueous ammonia and extracted with AcOEt. The
combined organic layer was dried over MgSO4 and
evaporated in vacuo. The reside was chromatographed
on silica gel to give 171 mg (82%) of 13 as a viscous oil:
IR (neat) cmꢁ1: 3418, 2948, 1633, 1505, 1455, 1289,
9.16. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dimeth-
oxy-6,14-endoethenomorphinan-7a-carbohydrazide (11)
To a stirred solution of 4 (870 mg, 1.9 mmol) in 2-eth-
oxyethanol (10 mL) was added NH2NH2ÆH2O (10 mL)
and the mixture was refluxed for 30 h. After cooling to
rt, the reaction solution was poured into distilled water
and extracted with AcOEt. The combined organic layer
was dried over MgSO4 and evaporated in vacuo. The
residue was chromatographed on silica gel to give
763 mg (91%) of 11 as an amorphous substance: IR
(KBr) cmꢁ1: 3456, 3322, 2932, 1655, 1633, 1503, 1443,
1
1266, 1167, 1105, 947. H NMR (CDCl3, 300 MHz) d:
0.88–0.19 (2H, m), 0.44–0.58 (2H, m), 0.76 (1H, dd,
J ¼ 4:7, 13.5 Hz), 0.77–0.92 (1H, m), 1.79 (1H, dd,
J ¼ 2:5, 13.2 Hz), 2.00 (1H, dt, J ¼ 5:7, 12.6 Hz), 2.29–
2.46 (4H, m), 2.70 (1H, dd, J ¼ 5:1, 11.9 Hz), 3.03 (1H,
ddd, J ¼ 1:4, 4.7, 8.8 Hz), 3.08 (1H, d, J ¼ 19:0 Hz),
3.31 (1H, dd, J ¼ 8:8, 13.5 Hz), 3.49 (1H, d, J ¼ 6:6 Hz),
3.64 (3H, s), 3.83 (3H, s), 4.53 (1H, d, J ¼ 1:1 Hz), 5.57
(1H, d, J ¼ 8:8 Hz), 5.74 (1H, d, J ¼ 8:8 Hz), 6.51 (1H,
d, J ¼ 8:2 Hz), 6.62 (1H, d, J ¼ 8:0 Hz). HREI-MS [M]þ
m=z calcd for C24H30N2O3: 394.2256. Found: 394.2252.
1
1285, 1260, 1208, 1166, 1103, 1055. H NMR (CDCl3,
300 MHz) d: 0.06–0.18 (2H, m), 0.43–0.57 (2H, m), 0.75–
0.90 (1H, m), 1.58 (1H, dd, J ¼ 6:3, 13.2 Hz), 1.84 (1H,
dd, J ¼ 2:5, 13.2 Hz), 1.99 (1H, dt, J ¼ 5:5, 12.5 Hz),
2.28–2.48 (4H, m), 2.59 (1H, dd, J ¼ 6:3, 9.3 Hz), 2.71
(1H, dd, J ¼ 4:8, 11.7 Hz), 3.11 (1H, d, J ¼ 19:0 Hz),
3.13 (1H, dd, J ¼ 9:9, 12.9 Hz), 3.57 (1H, d, J ¼ 6:6 Hz),
3.64 (3H, s), 3.82 (3H, s), 3.87 (2H, d, J ¼ 4:1 Hz), 4.54
(1H, d, J ¼ 1:4 Hz), 5.60 (1H, d, J ¼ 8:8 Hz), 5.90 (1H,
d, J ¼ 8:8 Hz), 6.52 (1H, d, J ¼ 8:0 Hz), 6.62 (1H, d,
J ¼ 8:2 Hz), 7.28 (1H, br s). HREI-MS [M]þ m=z calcd
for C25H31N3O4: 437.2315. Found: 437.2335.
9.19. 17-(Cyclopropylmethyl)-4,5a-epoxy-7a-[(3-phenyl-
propionyl)amino]-6,14-endoethenomorphinan-3,6b-diolꢂ
methane sulfonate (15ꢂMeSO3H)
To a solution of 13 (393 mg, 1.0 mmol) in CH2Cl2
(10 mL) with Et3N (0.85 mL, 6.1 mmol) was added
dropwise a solution of 3-phenylpropionyl chloride
(0.74 mL, 5.0 mmol) in CH2Cl2 (5 mL) at 0 °C and then
stirred for 1 h at the same temperature. The reaction
mixture was poured into a saturated NaHCO3 solution
and extracted with AcOEt. The combined organic layer
was dried over MgSO4 and evaporated in vacuo. The
residue was chromatographed on silica gel to give
440 mg (84%) of 14. To the stirred solution of 14
(440 mg, 0.83 mmol) in dry CH2Cl2 (15 mL) was added
dropwise a 1.0 M solution of BBr3 in dry CH2Cl2 (5 mL,
5.0 mmol) at 0 °C. After stirring at rt for 1 h, the reaction
mixture was cooled to 0 °C. To the reaction mixture was
added 6% aqueous ammonia (100 mL), then stirred
vigorously at rt. The resulting mixture was separated
and extracted with CHCl3. The combined organic layers
were dried over MgSO4 and evaporated in vacuo. The
residue was chromatographed on silica gel to give 61 mg
(15%) of 15. To the stirred suspension of 15 in MeOH
9.17. 17-(Cyclopropylmethyl)-4,5a-epoxy-3,6b-dimeth-
oxy-6,14-endoethenomorphinan-7a-(N-benzyloxycar-
bonyl)amine (12)
To a mixture of 11 (752 mg, 1.7 mmol) in 6 M HCl
(10 mL) and Et2O (15 mL) was added dropwise an
aqueous solution of NaNO2 (202 mg, 2.9 mmol) at 0 °C
and stirred vigorously for 30 min at the same tempera-
ture. After being neutralized with NaHCO3, the reaction
mixture was extracted with Et2O and dried over MgSO4.
Benzyl alcohol (5 mL) was added to the solution, and
then Et2O was removed in vacuo. The resulting solution
was refluxed for 1 h, then cooled to rt. The reaction
mixture was poured into distilled water and extracted
with AcOEt. The combined organic layer was dried over
MgSO4 and evaporated in vacuo. The residue was
chromatographed on silica gel to give 696 mg (72%) of
12 as a viscous oil: IR (neat) cmꢁ1: 3434, 3316, 2940,

4144
H. Fujii et al. / Bioorg. Med. Chem. 12 (2004) 4133–4145
was added dropwise MeSO3H at 0 °C until the solution
became acidic. Ethyl acetate was then added to the
solution. The precipitated amorphous salt was filtered to
give 16Æ MeSO3H: IR (KBr) cmꢁ1: 3426, 1639, 1546,
was homogenized with 15 volume (w/v) of ice-cold
0.32 M sucrose using a Potter–Elvehjem tissue grinder
with a Teflon pestle. The homogenate was centrifuged at
4 °C for 10 min at 1000g. The pellet was discarded and
supernatant was centrifuged at 4 °C for 20 min at
20,000g. The pellet was resuspended in 15 volumes of an
ice-cold 50 mM Tris–HCl buffer (pH 7.4) and centri-
fuged at 4 °C for 20 min at 20,000g. The resultant pellet
was resuspended in an ice-cold assay buffer [Tris–HCl
(pH 7.4), 50 mM, MgCl2, 5 mM, EGTA, 1 mM, NaCl,
100 mM] and then stored at ꢁ70 °C until used. The
homogenized membrane fractions (3–8 lg of protein/
assay) were incubated at 25 °C for 2 h in assay buffer
with various concentrations of the agonist, 30 lM gua-
nosine-50-diphosphate and 50 pM [35S]GTPcS in a total
volume of 1 mL. The reaction was terminated by filter-
ing through Whatman GF/B glass filters, which had
been previously soaked in a soaking buffer [Tris–HCl
(pH 7.4), 50 mM, MgCl2, 5 mM] at 4 °C for 2 h, using a
Brandel cell harvester. The filters were then washed
three times with 5 mL of an ice-cold Tris–HCl buffer
(pH 7.4) and transferred to scintillation counting vials.
Then 0.5 mL of the tissue solubilizer Soluene-350 and
4 mL of the scintillation cocktail Hionic Fluor were
added to the vials. After a 12 h equilibration period, the
radioactivity in the samples was determined with a
liquid scintillation analyzer. Nonspecific binding was
measured in the presence of 10 lM unlabeled GTPcS.
1
1502, 1461, 1322, 1210, 1195, 1048, 931, 785, 703. H
NMR (DMSO-d6, 400 MHz) d: 0.37–0.53 (2H, m), 0.59–
0.77 (2H, m), 0.92 (1H, dd, J ¼ 4:4, 13.2 Hz), 1.05–1.17
(1H, m), 1.96–2.05 (1H, m), 2.18 (1H, dt, J ¼ 4:4,
13.9 Hz), 2.32 (3.3H, s), 2.42 (1H, dt, J ¼ 3:1, 7.8 Hz),
2.79 (2H, t, J ¼ 7:8 Hz), 2.87–3.05 (3H, m), 3.19 (1H,
dd, J ¼ 8:8, 13.2 Hz), 3.28–3.40 (2H, m), 3.42–3.52 (1H,
m), 3.91–4.00 (1H, m), 4.26 (1H, d, J ¼ 6:8 Hz), 4.41
(1H, s), 5.58 (1H, d, J ¼ 8:3 Hz), 5.65 (1H, d,
J ¼ 8:3 Hz), 6.45 (1H, d, J ¼ 7:8 Hz), 6.55 (1H, d,
J ¼ 8:3 Hz), 7.13–7.31 (5H, m), 7.52 (1H, d, J ¼ 7:3 Hz),
8.38 (1H, br s), 9.11 (1H, br s). MS (FAB) m=z 499
[MþH]þ. Anal. Calcd for C31H34N2O4Æ0.5H2OÆ
1.1MeSO3H: C, 62.86; H, 6.47; N, 4.57; S, 5.75. Found:
C, 62.64; H, 6.51; N, 4.56; S, 5.99.
All pharmacological experiments were approved by and
conformed to the guidelines for Care and Use of Lab-
oratory Animals in Toray Pharmaceutical Research
Laboratories or the guidelines of the Medical College of
Wisconsin Animal Care Committee.
9.20. Inhibition of smooth muscle contraction
Male SD rats were sacrificed by bleeding under anes-
thesia or after clubbing the head, and the vas deferens
were excised. Male ICR mice were sacrificed by cervical
vertebra dislocation, and the vasa deferentia were ex-
cised. For the assay using RVD or MVD, a Magnus
tube was filled with Ringer’s solution (NaCl 154 mM,
KCl 5.66 mM, CaCl2 2.54 mM, glucose 2.77 mM,
NaHCO3 5.95 mM, tyrosine 0.0018 mM) or Krebs
Henseleit solution without Mg (NaCl, 120.7 mM, KCl,
5.9 mM, NaH2PO4Æ2H2O 1.2 mM, glucose 11.5 mM,
NaHCO3 15.5 mM, CaCl2 2.5 mM), respectively, kept at
36–37 °C, and gassed with 5% CO2 and 95% O2. Elec-
trical stimulation was given through ring-shaped plati-
num electrodes located above and below the RVD or the
MVD preparations at 70–100 mA, 0.2 Hz for 5 mS or
150–250 mA, 0.1 Hz for 1 mS, respectively. The con-
traction of the preparations was recorded on a poly-
graph using an isometric transducer. Compounds were
added into the Magnus tube cumulatively. The agonistic
activity was shown in terms of IC50 value or the degree
of inhibition of contraction with the concentration of
compounds. For the assay using MVD, effects of com-
pounds in the presence of naloxone (30 nM), NTI
(10 nM), or nor-BNI (10 nM), were also described. IC50
ratio was calculated from the equation: IC50
ratio ¼ (IC50 value of the agonist in the presence of the
antagonist)/(IC50 value of the agonist in the absence of
the antagonist).
9.22. Assessment of antinociception
Antinociception was determined by the tail-flick and
hot-plate tests. For measurement of the latency of the
tail-flick response, male ICR mice were gently held by
hand with their tail positioned in an apparatus for
radiant heat stimulation on the dorsal surface of the tail.
The intensity of heat stimulus was adjusted so that the
animal flicked its tail after 3–5 s. For the hot-plate test,
male ICR mice were individually placed on the hot-plate
(55 °C) and the reaction time starting from the place-
ment of the mouse on a 30 ꢃ 30 ꢃ 30 cm hot-plate to the
time of licking the paw was measured. Control latencies
for the paw-licking response were 7–10 s. The inhibition
of the tail-flick and paw-licking responses was expressed
as percent maximum possible effect, %MPE, which was
calculated as: [ðT 1 ꢁ T 0Þ=ðT 2 ꢁ T0Þ] ꢃ 100, where T 0 and
T 1 were the tail-flick or hot-plate latencies before and
after the treatments and T 2 was the cutoff time. Cutoff
times were set at 10 and 30 s for the tail-flick and hot-
plate tests, respectively.
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