Synthesis and structure-activity relationships of an orally available and long-acting analgesic peptide, Nα-amidino-Tyr-D-Arg-Phe-MeβAla-OH (ADAMB)

Article abstract of DOI:10.1021/jm010357t

A novel dermorphin tetrapeptide N^α-amidino-Tyr-D-Arg-Phe-MeβAla-OH (ADAMB) was designed based on the structures of several dermorphin tetrapeptide analogues, including N^α-amidino-Tyr-D-Arg-Phe-Gly-OH (ADA-DER), H-Tyr-D-Arg-Phe-βAla-O

Full text of DOI:10.1021/jm010357t

J . Med. Chem. 2002, 45, 5081-5089
5081
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of a n Or a lly Ava ila ble a n d
Lon g-Actin g An a lgesic P ep tid e, Nr-Am id in o-Tyr -D-Ar g-P h e-MeâAla -OH (ADAMB)
Tadashi Ogawa,† Tetsuhisa Miyamae,† Kimie Murayama,‡ Kaori Okuyama,§ Toru Okayama,†,*
Masaki Hagiwara,† Shinobu Sakurada,§ and Tadanori Morikawa†
Research Institute, Daiichi Fine Chemical Co. Ltd., 530 Chokeiji, Takaoka, Toyama 933-8511, J apan, Department of
Biochemistry, J untendo University School of Medicine, Tokyo 113-8421, J apan, and Department of Physiology and
Anatomy, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, J apan
Received J uly 30, 2001
A novel dermorphin tetrapeptide NR-amidino-Tyr-D-Arg-Phe-MeâAla-OH (ADAMB) was de-
signed based on the structures of several dermorphin tetrapeptide analogues, including
NR-amidino-Tyr-D-Arg-Phe-Gly-OH (ADA-DER), H-Tyr-D-Arg-Phe-âAla-OH (TAPA), and
H-Tyr-D-Arg-Phe-Sar-OH (DAS-DER). These parent compounds were known to show a weak
oral analgesic activity in animals and/or to possess a different mechanism of analgesia from
other µ-opioid peptides. Six analogues of ADAMB were also synthesized to investigate the effect
on potency of N-terminal amidination and N-methyl-â-alanine (MeâAla) substitution at position
4. Compounds were assessed using the tail pressure test in mice after subcutaneous and oral
administration. Among the peptides tested, ADAMB showed the strongest oral antinociceptive
activity, with an ED50 of 5.8 vs 22.2 mg/kg for morphine, as well as a 38-fold stronger activity
after subcutaneous administration. ADAMB also showed long-lasting antinociceptive activity,
with 50% of the maximum effect persisting in the tail pressure test at 10 h after oral
administration (10 mg/kg). In contrast, orally administered morphine (80 mg/kg) showed a
rapid decrease of activity in the same test and its antinociceptive effect disappeared within 4
h. When the antinociceptive effect of ADAMB was compared with that of analogues possessing
âAla4 (1) or Sar4 (2), as well as analogues with N-substitution (3-6), it was found that both
the NR-amidino substitution and the MeâAla4 were synergistically involved in creating ADAMB’s
exceptionally high antinociceptive activity.
In tr od u ction
selective µ-opioid receptor agonist. Salvadori et al.11
have extensively studied dermorphin analogues and
have reported that NR-amidination strengthens their
antinociceptive activity. We recently found NR-amidino-
Tyr-D-Arg-Phe-Gly-OH (ADA-DER) as one of the most
potent dermorphin tetrapeptides.12 An analogue with
N-methylation at position 4, H-Tyr-D-Arg-Phe-Sar-OH
(DAS-DER),13 shows selectivity for the µ1-opioid recep-
tor subtype and has oral antinociceptive activity in
rats without inducing respiratory depression.14 Sato
et al.15 recently reported that H-Tyr-D-Arg-Phe-âAla-
OH (TAPA),16 in which the amino acid residue at
position 4 was substituted with â-alanine, had a more
potent and longer-lasting antinociceptive effect than
morphine after local or systemic administration and was
a selective µ1-opioid receptor agonist. In addition, the
typical withdrawal symptoms that occurred upon ces-
sation of administration or after treatment with nalox-
one, a classical µ-opioid antagonist, were less severe for
TAPA than for morphine.17 These observations encour-
aged us to explore the possibility of developing novel
and orally active analgesic peptides without the adverse
effects of morphine. On the basis of the structures of
ADA-DER, DAS-DER, and TAPA, a compound featuring
both NR-amidination and N-methyl-â-alanine (MeâAla)
at position 4 (ADAMB) was designed (Figure 1). This
paper reports on the synthesis and the in vivo antinoci-
ceptive activity of ADAMB. To assess its potential as
an oral analgesic, antinociceptive activity was evaluated
in mice by the tail pressure test and was compared with
Since the isolation and identification of enkephalins1
and other endogenous opioid peptides,2 numerous pep-
tidic analogues have been synthesized in attempts to
develop an analgesic without the serious side effects of
narcotics such as morphine.3 However, these efforts
have met with little success because of problems with
side effects, low bioavailability, and high cost of produc-
tion. Dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-
NH2) was recently isolated from the skin of amphibians4
and was reported to possess a potent and long-lasting
opioid-like activity.5 The N-terminal tetrapeptide of
dermorphin is known to be the minimum sequence
required for opioid activity,6 although this fragment
shows a lower potency than that of the parent hep-
tapeptide.7 Synthetic peptides derived from the dermor-
phin tetrapeptide, which have the sequence Tyr-D-AA2-
Phe-AA4 (where AA2 and AA4 represent a certain amino
acid), have been reported to show a potent agonist
activity for the µ-opioid receptor.8 Substitution of D-Ala
at position 2 with D-Arg has been widely applied since
the studies on D-Arg-kyotorphin were reported,9 in order
to increase the potency and the duration of action of the
peptide. For example, Schiller10 reported that DALDA
(D-AA2 ) D-Arg, AA4 ) Lys-NH2) is a potent and highly
* To whom correspondence should be addressed. Tel: +81(766)21-
3456. Fax: +81(766)26-4439. E-mail: okayama@daiichi-fcj.co.jp.
† Daiichi Fine Chemical Co. Ltd.
‡ J untendo University School of Medicine.
§ Tohoku Pharmaceutical University.
10.1021/jm010357t CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/08/2002

5082 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Ogawa et al.
F igu r e 1. Design of ADAMB based on the structure of several dermorphin tetrapeptide analogues.
Sch em e 1a
a
Reagents: (a) WSCI-HOBt. (b) 4 N HCl in EtOAc. (c) 1-(Bis-benzyloxycarbonylguanyl)pyrazole, Et3N in DMF. (d) H2/Pd-C in AcOH.
that of morphine. In addition, six analogues of ADAMB
were synthesized to clarify the influence of N-amidina-
tion and MeâAla4 substitution on antinociceptive activ-
ity and the data obtained regarding the structure-
activity relationships (SARs) are also discussed here.
chloride (WSCI‚HCl) with HOBt as the coupling reagent
(Scheme 1). The side chain functional groups and the
C-terminal carboxylic acid were fully protected by Cbz
or Bzl protection groups, which were simultaneously
removed by catalytic hydrogenolysis in the presence
of Pd on charcoal. Thus, starting with H-MeâAla-OBzl,
NR-Boc-protected amino acids were coupled sequen-
tially to give the fully protected tetrapeptide (7). An
NR-amidino group was introduced in a bis-Cbz-protected
form using (benzyloxycarbonylimino-pyrazol-1-yl-meth-
yl)carbamic acid benzyl ester.20 Then, all of the protect-
ing groups were cleanly removed by catalytic hydroge-
nation to give the desired product (ADAMB) with a high
purity. This method is used to facilitate the large-scale
synthesis of compounds containing the highly polar and
hydrophilic functional groups. All final products were
purified by reverse phase C18 flush column chromatog-
raphy with elution using aqueous CH3CN containing
0.1% acetic acid to afford the purified products (>95%
purity by high-performance liquid chromatography
(HPLC) analysis in two diverse systems). The structure
Ch em istr y
ADAMB was successfully synthesized by both solid
phase and solution phase methods. The NR-Fmoc strat-
egy was used for solid phase synthesis, employing a
p-alkoxybenzyl alcohol resin (Wang resin).18 NR-Fmoc-
protected amino acids were added sequentially, using
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-
fluorophosphate (HBTU) with 1-hydroxybenzotriazole
(HOBt) as the coupling reagents in 1-methyl-2-piperi-
done (NMP). N-Terminal amidination was performed on
the resin after assembly of the peptide chain by removal
of the Fmoc group followed by treatment with a mixture
of 1H-pyrazole-1-carboxamidine hydrochloride19 and
diisopropylethylamine in dimethylformamide (DMF).
Analogues bearing âAla (1) or Sar (2) at position 4 were
also synthesized using the same method. To investigate
the effect of N-terminal substitution on antinociceptive
activity, four other analogues of ADAMB were also
synthesized by the solid phase method. These analogues
had structures that could be represented by the general
formula of R-Xaa-D-Arg-Phe-MeâAla-OH, where R and
Xaa are the N-terminal substituent and the amino acid
residue, respectively. They were designated as com-
pounds 3 (R ) H, Xaa ) Tyr), 4 [R ) H, Xaa )
N-methyltyrosine (MeTyr)], 5 [R ) none, Xaa ) N,N-
dimethyltyrosine (Me2Tyr)], and 6 (R ) acetyl, Xaa )
Tyr, âAla4 instead of MeâAla4). To obtain a substantial
amount of the compound for animal tests, ADAMB was
subsequently synthesized by a solution method using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
1
of the final product was confirmed by H NMR and by
high-resolution mass spectral (HRMS) analysis.
Biologica l Resu lts
The synthesized compounds were tested to determine
their relative opioid receptor binding affinity by dis-
placement of selective radioligands from mice spinal
cord or guinea pig brain membrane preparations.
[3H][D-Ala2,MePhe4,Gly-ol5]enkephalin ([3H]DAMGO),
[3H]deltorphin-II, and [3H]U-69593 were used for de-
termining the µ-, δ-, and κ-affinity, respectively. The
results of these binding assays are summarized in Table
1. ADAMB showed a high binding affinity for the
µ-opioid receptor, which was comparable to that of
DAMGO in the [3H]DAMGO binding assay. When com-

Orally Available and Long-Acting Analgesic Peptide
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5083
Ta ble 1. Receptor Binding Assay of ADAMB and Its Analogues
[3H]DAMGO (µ)
relative potencyb
[3H]deltolphin-II (δ)
[3H]U-69593 (κ)
a
compd
R1
R2
AA4
IC50 (nM)
IC50 (nM)
IC50 (nM)
ADAMB
H2NC(dNH)-
H2NC(dNH)-
H2NC(dNH)-
H-
H-
H-
H-
H-
H-
CH3-
H-
MeâAla
âAla
12.9 ( 3.4
0.90
0.56
0.35
1.14
0.26
0.11
<0.01
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
1
2
3
4
5
6
20.8 ( 3.2
32.9 ( 12
10.2 ( 4.2
45.0 ( 11
101 ( 20
>1000
Sar
MeâAla
MeâAla
MeâAla
âAla
CH3-
CH3-
CH3CO-
DAMGO
11.6 ( 1.7
1.00
>1000
>1000
(5.07 ( 0.68)c
deltorphin-II
U-69593
nd
nd
13.7 ( 2.3c
nd
nd
3.17 ( 0.35c
a
b
Concentration that gives half-maximal effect. Data are given as the mean ( SEM (n ) 3). Relative potencies are on a molar basis
(DAMGO ) 1). c Ki value.
Ta ble 2. In Vivo Antinociceptive Activity of ADAMB and Its Analogues (Tail Pressure Test)
MPE %maxa
po (10 mg/kg)
ED50 (mg/kg)b
po
compd
sc (1 mg/kg)
sc
sc/po (%)
ADAMB
1
2
3
4
5
6
100 ( 0.0
93 ( 6.9
90 ( 5.0
91 ( 5.1
87 ( 9.7
29 ( 11
<10
82 ( 6.4
18 ( 4.8
26 ( 11
31 ( 8.8
34 ( 9.7
13 ( 5.5
0.089 (0.06-0.15)
0.32 (0.22-0.46)
0.39 (0.25-0.61)
0.39 (0.22-0.69)
0.21 (0.13-0.34)
nd
5.8 (3.6-9.4)
1.5
1.7
2.2
19 (9.3-37)
18 (11-29)
nd
15 (9.0-25)
nd
nd
1.5
<10
nd
nd
morphine
nd
3.3 (2.2-4.9)
22 (14-36)
15
a
b
Data were taken at 1.5 h after drug administration and are given as the mean ( SEM for groups of 10 mice. The ED50 value was
estimated at the time of peak activity and are given as the mean value with its 95% confidence limit for 10 mice.
pared to a compound without N-terminal amidination
(3), ADAMB had almost the same binding affinity,
whereas N-terminal methylation (4) or dimethylation
(5) caused a significant decrease of affinity for the
µ-opioid receptor. On the other hand, modifications at
position 4 caused a decrease of µ-affinity. Thus, each of
the analogues in which MeâAla4 was substituted by
âAla4 (1) or Sar4 (2) showed a decline of µ-affinity to
one-half or one-third of that for ADAMB, respectively.
An analogue with N-terminal acetylation (6) did not
show any strong affinity (IC50 > 1000 nM) for the
µ-opioid receptor. None of the compounds showed any
significant affinity (IC50 > 1000 nM) for either the δ- or
the κ-opioid receptor, which indicated that these ana-
logues show specific affinity for the µ-opioid receptor
relative to the δ- and the κ-opioid receptors.
The compounds were tested to assess their in vivo
antinociceptive potency (Table 2) using the tail pressure
test21 after subcutaneous (sc) and oral (po) administra-
tion to mice. The maximum possible effect (MPE %) was
initially measured at fixed doses of 1 subcutaneously
and 10 mg/kg orally, after which analogues with a high
potency were assessed for antinociceptive activity on the
basis of ED50 values. ADAMB showed a very strong
antinociceptive activity in mice after sc administration,
and its ED50 value was 37 times higher than that of
morphine. ADAMB was significantly antagonized by sc
pretreatment with naloxone (data not shown) in the
same mouse test. As expected from the design of
ADAMB, its po antinociceptive activity was also strong
and the ED50 value was 3.8 times higher than that of
morphine. To assess bioavailability, the ED50 dose ratio
(sc/po) of ADAMB was calculated and compared with
that of morphine. The ratio for ADAMB was 1.5%, while
that for morphine was 15%. Substitution of MeâAla at
position 4 with either âAla (1) or Sar (2) (corresponding
to TAPA and DAS-DER with NR-amidination) caused a
significant decrease in potency to one-third of that for
ADAMB after both sc and po administration. However,
both compound 1 and compound 2 still had a stronger
sc antinociceptive activity than morphine and compa-
rable po activity. The compound without N-terminal
substitution (3) also showed a significant decrease of
antinociceptive activity when compared with ADAMB.
N-Terminal monomethylation (4) did not affect the
antinociceptive activity when compared to the ana-
logue with an N-terminal primary amine (3), whereas
substitution of both hydrogens at the amino terminal
with methyl groups (5) caused a substantial decrease
of activity. Comparison of the ED50 ratio (sc/po) for
ADAMB with those of compounds 1, 2, and 4 showed
that the bioavailability was unchanged by modification
at the N-terminal and position 4. To further investigate
the role of the amino terminal, an analogue bearing an
N-acetyl group (6) was synthesized. This modification
canceled the basicity and nucleophilicity of the amino
group and resulted in no measurable antinociceptive

5084 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Ogawa et al.
Ta ble 3. In Vivo Antinociceptive Activity of ADAMB and
Selected Analogues (Hot Plate Test)
attempt to develop new peptide analgesics as well as to
determine their pharmacological properties.22 Among
them, TAPA and DAS-DER, in which the C-terminal
amino acid residue at position 4 is substituted with
â-Ala or Sar, respectively, are known to have an
antinociceptive activity even after oral administration.
Therefore, we expected that a tetrapeptide bearing
MeâAla at position 4 could show increased oral anti-
nociceptive activity by combining homologation and
N-methylation. We have recently synthesized more than
50 compounds with the general structure of NR-amidino-
Tyr-D-Arg-Phe-X (where X represents various amino
acids, amides, amino esters, and amino alcohols) and
have found that they show strong antinociceptive activ-
ity comparable to that of morphine after oral adminis-
tration.12 Therefore, a combination of these modifica-
tions seemed likely to amplify oral activity and to be a
promising approach for the development of an orally
active analgesic peptide. To confirm our hypothesis, we
performed an in vivo test using mice for initial evalu-
ation of the compounds synthesized. As expected,
ADAMB showed very potent oral antinociceptive activ-
ity and was approximately 4 times as strong as mor-
phine. In our preliminary experiment using a similar
mouse test that we employed in this study, the anti-
nociceptive effect of ADAMB was five times stronger
than that of TAPA after sc administration and seven
times stronger after po administration (based on ED50
values). However, the bioavailability of ADAMB may
not be sufficient for an oral analgesic since its dose ratio
(sc/po) was 10-fold smaller than that of morphine (based
on ED50 values). Comparison of ADAMB with analogue
3 (no NR-substitution) showed that NR-amidination
markedly improved the antinociceptive effect after both
sc and po administration, while the dose ratio (sc/po) of
the ED50 values did not change as compared with that
of analogue 4. This indicated that N-amidination is
important for antinociceptive activity but not for bio-
availability. Likewise, the combination of N-methylation
and homologation of the amino acid residue at position
4 did not achieve any increase in bioavailability, al-
though significant improvement of antinociceptive ac-
tivity was observed (ADAMB vs compounds 1 and 2).
Therefore, further structural modification of ADAMB,
including alteration of other parts of the molecule, will
be necessary before this type of compound can be used
as an oral analgesic.
compd
ED50 (sc, mg/kg)a
compd
ED50 (sc, mg/kg)a
ADAMB
1
2
0.07 (0.02-0.24)
0.21 (0.12-0.36)
0.08 (0.3-0.21)
3
4
0.15 (0.08-0.21)
0.11 (0.05-0.24)
1.7 (0.7-3.8)
morphine
a
The ED50 value was estimated at the time of peak activity
and was given as the mean value with its 95% confidence limit
for 10 mice.
effect, even though the N-acetamino group was virtually
the same size and shape as the N-terminal guanidino
group.
ADAMB and several analogues (1-4), which showed
strong antinociceptive activity in the mouse tail pres-
sure test, were assessed for their antinociceptive potency
in a mouse hot plate test after sc administration (Table
3). Like the tail pressure test, ADAMB showed the
strongest antinociceptive activity among the compounds
tested. Morphine was 24 times less potent in terms of
the ED50 value than ADAMB. The Sar4 analogue 2
showed a comparable ED50 value to that of ADAMB, but
2 was significantly less active than ADAMB in the tail
pressure test. With the exception of compound 2, which
is an R-amino acid analogue showing a difference at
position 4, the antinociceptive results obtained in the
hot plate test showed a good correlation with the data
from the tail pressure test.
Figure 2 shows the time course of the antinociceptive
effect of sc administered morphine and ADAMB in the
mouse tail pressure test. ADAMB (Figure 2B) produced
dose-related stronger antinociceptive activity than mor-
phine (Figure 2A). The total duration of action for
ADAMB was over 10 h at a maximally active dose of
0.2 mg/kg, whereas the activity of morphine disappeared
within 4 h at a dose of 10 mg/kg.
As shown in Figure 3B, oral ADAMB also had dose-
related and significant antinociceptive activity, with
50% of MPE being maintained at 10 h after administra-
tion at the maximally active dose of 10 mg/kg. The peak
effect appeared 4-6 h after administration at this dose.
Morphine showed its maximum antinociceptive activity
at a dose of 80 mg/kg with a peak at 60 min after
administration. Oral ADAMB had a far longer duration
of action than morphine, which was effective for less
than 4 h after administration (Figure 3A).
To clarify the role of the µ-opioid receptor subtypes,
µ1 and µ2, in the antinociceptive effect of ADAMB on
noxious stimuli, pretreatment (sc) with a µ1 selective
opioid receptor antagonist, naloxonazine, or a nonselec-
tive antagonist, naloxone, was done in the mouse tail
pressure test (Figure 4). Then, the results were com-
pared with those for morphine (Figure 5). Either nalox-
onazine (35 mg/kg) or naloxone (0.2 mg/kg) pretreat-
ment at 24 h before administration of ADAMB (0.5
mg/kg) markedly attenuated its antinocieptive action,
and the difference in the extent of attenuation was not
significant. In contrast, pretreatment with naloxonazine
showed only partial attenuation of the antinociceptive
effect of morphine (10 mg/kg), while naloxone completely
suppressed it.
The presence of an N-terminal amino group in opioid
peptides is crucial for a strong agonist effect on the
opioid receptors.23 N-Terminal amidination of dermor-
phin tetrapeptides is known to decrease the in vitro
affinity for opioid receptors, while it strengthens the in
vivo antinociceptive acitvity.24 However, little is known
about the SARs of N-terminal amidination as well as
the effect of this modification on other opioid peptides.
In the present study, the antinociceptive activity of the
N,N-dimethylated analogue (5) showed a considerable
decrease, whereas the monomethylated analogue (4)
retained similar activity to that of the unsubstituted
analogue (3). On the other hand, N-acetylation led to a
decrease of activity. These observations suggest the
importance of an N-terminal amino group that retains
basicity and, unlike most of the nonpeptide µ-agonists
Discu ssion
Since the isolation of dermorphin, many analogues
have been synthesized and extensively investigated in

Orally Available and Long-Acting Analgesic Peptide
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5085
F igu r e 2. Time course of the antinociceptive effect of subcutaneous morphine (A) and ADAMB (B) in the mouse tail pressure
test. The doses used are shown in the figure. Data are given as the mean ( SEM for groups of 10 mice.
F igu r e 3. Time course of the antinociceptive effect of oral morphine (A) and ADAMB (B) in the mouse tail pressure test. The
doses used are shown in the figure. Data are given as the mean ( SEM for groups of 10 mice.
F igu r e 4. Effect of ADAMB (0.5 mg/kg, sc) after pretreatment
with naloxone or naloxonazine in the mouse tail pressure test.
Naloxone (0.2 mg/kg, sc) was given 5 min before ADAMB.
Naloxonazine (35 mg/kg, sc) was given 24 h before ADAMB.
Data are the mean ( SE for 10 mice. *** < 0.001 vs ADAMB
alone (Tukey’s test).
F igu r e 5. Effect of morphine (10 mg/kg, sc) after pretreat-
ment with naloxone or naloxonazine in the mouse tail pressure
test. Naloxone (0.2 mg/kg, sc) was given 5 min before mor-
phine. Naloxonazine (35 mg/kg, sc) was given 24 h before
morphine. Data are the mean ( SE for 10 mice. *** < 0.001
vs morphine alone; # < 0.05 for pretreatment with naloxone
vs pretreatment with naloxonazine (Tukey’s test).
that commonly possess a tertiary amine, at least one
unsubstituted amine hydrogen in Tyr1 as well.
is partly due to enhancement of the compound’s stabil-
ity. It could be also explained by the slow and continu-
ous influx of ADAMB into the central nervous system
across the blood-brain barrier (BBB) in addition to tight
binding of the peptide with the µ-opioid receptors. These
properties would be related to a low clearance rate from
the blood as well as the peptide’s extremely high
stability to enzymatic hydrolysis. Nevertheless, this
feature may be advantageous in combination with the
higher efficacy of the compound relative to morphine,
even though slow release morphine formulations such
as 12 h oral preparations3 have been widely accepted
for clinical use. The high efficacy and long duration of
ADAMB has an exceptionally long-lasting antinoci-
ceptive activity after both sc and po administration as
does TAPA.15 Comparison of the time-course of the
antinociceptive effect of ADAMB with that of morphine
showed that the onset was slower for ADAMB after
either sc or po administration. The peak time of
analgesia after administration was accordingly shifted
to later than that for morphine, being 1.5-2 (sc) and
3-4 h (po) for ADAMB vs 0.5 (sc) and 1 h (po) for
morphine, respectively. The reason for the stronger and
longer antinociceptive activity of ADAMB along with its
slow onset is still unclear, although we assume that it

5086 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Ogawa et al.
products was achieved by the flush chromatography using a
C-18 reverse phase silica gel Chromatorex DM1020T, Fuji
Silysia Chemical Ltd., Aichi, J apan, eluted by a stepwise
gradient of acetonitrile (starting from 1% and stepwise gradi-
ent by 2%) in 0.1 M acetic acid. Analytical HPLC was on a
reverse phase Nucleosil 5C18 column (4.6 mm × 150 mm) in a
Shimadzu LC-10A HPLC system. The products were analyzed
with the two different eluting systems; A: a linear gradient
of 10-70% acetonitrile in 0.1% aqueous TFA over 15 min; B:
an isocratic elution with 12% acetonitrile in 0.1% aqueous TFA
over 30 min. The flow rate was 1 mL/min, and the chromato-
gram was recorded by UV detection at 230 and 280 nm. 1H
NMR spectra were recorded with a J EOL AL-300 (300 MHz)
spectrometer, using tetramethylsilane (TMS) as an internal
standard. HRMS were obtained with J EOL mass spectrometer
model J MS-700.
Solid P h a se Syn th esis of Nr-Am id in o-Tyr -D-Ar g-P h e-
MeâAla -OH (ADAMB). The peptide was synthesized using
an NR-Fmoc strategy and Wang resin. The peptides were
carried using a Perkin-Elmer model 430A automated synthe-
sizer on a 0.25 mmol scale. Fmoc-D-Arg(Pmc)OH and Fmoc-
Tyr(t-Bu) were used for the side chain protection. NR-Fmoc-
protected amino acids (4 equiv) were added sequentially, using
HBTU with HOBt as coupling reagents in NMP. After the
peptide assembly was completed, the Fmoc group was removed
from the Fmoc-Tyr(t-Bu)-D-Arg(Pmc)Phe-MeâAla-Aliko resin,
and the peptide resin was mixed with 1H-pyrazole-1-cabox-
amidine hydrochloride (0.99 g, 6.75 mmol) and diisopropyl-
ethylamine (1.29 mL, 7.40 mmol) in DMF (6 mL) and agitated
at room temperature overnight. The peptides were cleaved
using standard techniques and a cleavage mixture of 90% TFA,
5% anisole, 2.5% methyl sulfide, and 2.5% 1,2-ethandithiol.
The aqueous solution of the crude products was neutralized
with NaHCO3 and then purified by the method shown above
to give the desired compound (60 mg, 39%) as acetic acid salts
after lyophilization. [R]25D +28.8° (c 0.58, 1 M AcOH). 1H NMR
(CD3OD, 300 MHz): δ 7.23 (m, 5H), 7.05 (dd, J ) 8.4, 2.4 Hz,
2H), 6.72 (d, J ) 8.4 Hz, 2H), 5.17 (t, J ) 7.2 Hz, 1H), 4.41
(m, 1H), 4.26 (m, 1H), 3.59 (m, 1H), 3.45 (m, 1H), 3.10-2.85
(m, 6H), 2.88 (s, 2H), 2.85 (s, 1H), 2.35 (m, 2H), 1.50 (br m,
2H), 1.23 (m, 2H). FAB-HRMS: calcd for C29H42N9O6 [M +
1]+, 612.3258; found, 612.3233.
Solu tion P h ase Syn th esis of ADAMB. Boc-P h e-MeâAla-
OBzl. To a stirred suspension of H-MeâAla-OBzl‚p-TsOH (8.04
g, 22.0 mmol) in EtOAc (20 mL), Boc-Phe-OH (5.31 g, 20.0
mmol), HOBt (2.97 g, 22.0 mmol), and Et3N (3.07 mL, 22.0
mmol) were added at -10 °C. Then, WSCI‚HCl (4.60 g, 24.0
mmol) was added to the solution. The mixture was stirred for
30 min at the same temperature and overnight at room
temperature. The reaction mixture was washed with 1 N HCl,
saturated NaHCO3, and brine. The organic phase was dried
over MgSO4, filtered, and evaporated in vacuo to give colorless
oil (8.45 g, 95.9%). [R]25D +7.2° (c 1.05, DMF). 1H NMR (CDCl3,
300 MHz): δ 7.50-7.12 (m, 10H), 5.38 (t, J ) 8.7 Hz, 1H),
5.10 (s, 2H), 4.77 (dt, J ) 15, 7.5 Hz, 1H), 3.63 (dt, J ) 14, 6.6
Hz, 0.67H), 3.46 (dt, J ) 6.9, 6.6 Hz, 1H), 3.42 (m, 0.33H),
2.94 (dd, J ) 8.1, 3.0 Hz, 2H), 2.81 (s, 1H), 2.67 (s, 2H), 2.53
(dt, J ) 13, 6.6 Hz, 1H), 2.42 (m, 0.67H), 2.04 (m, 0.33H), 1.40
(s, 9H). FAB-HRMS: calcd for C25H33N2O5 [M + 1]+, 441.2389;
found, 441.2391.
Boc-D-Ar g(Cbz2)P h e-MeâAla-OBzl. Boc-Phe-MeâAla-OBzl
(6.54 g, 14.8 mmol) was dissolved into 4 N HCl/EtOAc (20 mL)
and stirred for 30 min at room temperature. Et2O and hexane
were added, and the precipitated material was separated by
decantation. The residual gum was dissolved in DMF (30 mL)
and neutralized with Et3N at 0 °C. Boc-D-Arg(Cbz2)OH (7.60
g, 14.0 mmol) and HOBt (1.89 g, 14.0 mmol) were added to
the solution, and Et3N (2.10 mL, 15.0 mmol) was added. Then,
WSCI‚HCl (3.22 g, 16.8 mmol) was added to the solution at
-10 °C. This mixture was stirred for 30 min at -10 °C and
overnight at 4 °C. The reaction mixture was diluted with
EtOAc (150 mL) and washed with 1 N HCl, 10% Na2CO3, and
brine. The organic phase was dried over MgSO4, filtered, and
evaporated in vacuo. The residue was solidified from hexane
action of ADAMB should markedly reduce the effective
dose for once or twice daily administration without any
manipulation of the drug formulation to extend its
duration of action.
Although µ-opioid receptor agonists are very impor-
tant for the control of pain in the clinical setting, these
drugs have several undesirable side effects, including
respiratory depression,25 constipation,26 and depen-
dence.27 Some of these effects are thought to be related
to one of the µ-receptor subtypes, either the µ1- or the
µ2-receptor. For example, opioid analgesia has been
suggested to be mediated by the µ1-receptor, whereas
the µ2-receptor seems to be involved in respiratory
depression and effects on gastrointestinal transit.28
TAPA and DAS-DER are known to have a high affinity
for the µ1-opioid receptor subtype since their antinoci-
ceptive activity is strongly antagonized by pretreatment
with naloxonazine, an irreversible µ1-opioid receptor
antagonist.29 Therefore, it can be expected that these
compounds may show a substantial reduction in the
adverse effects of µ-opioid peptides that are elicited
through the µ2-opioid receptor. This seems to be the case
for ADAMB. The antinociceptive activity of sc ADAMB
in mice was markedly attenuated by pretreatment with
naloxonazine, a selective µ1-antagonist, and the same
level of attenuation was achieved as that seen with
nonselective naloxone. In contrast, morphine was par-
tially antagonized by naloxonazine but was completely
antagonized by naloxone. These results imply that
ADAMB has a strong antinociceptive action mainly
through interaction with the µ1-opioid receptor subtype.
In fact, we have obtained preliminary experimental
results in animals to support our assumption that
ADAMB inherits promising characteristics as an anal-
gesic from its parent compounds. Less constipation in
comparison to that caused by morphine and negligible
psychological dependence were observed in mice, as well
as low cross-tolerance for morphine (the details will be
reported elsewhere).
In conclusion, ADAMB was synthesized based on the
structures of several dermorphin tetrapeptide analogues
and showed more potent antinociceptive activity than
morphine in the mouse tail pressure test after sc and
po administration. This strong and long-lasting activity
after oral administration may qualify ADAMB as a
candidate for the development as a new analgesic.
Further investigations are now underway to confirm
whether ADAMB also avoids side effects owing to its
µ1-subtype selective agonism.
Exp er im en ta l Section
Commercial NR-Fmoc-protected amino acids, NR-Boc-pro-
tected amino acids, trifluoroacetic acid (TFA), WSCI‚HCl, and
HOBt were obtained from Peptide Institute, Inc., Osaka,
J apan. HBTU was purchased from Calbiochem-Novabiochem
J apan Ltd., Tokyo, J apan. Wang resin (100-200 mesh, 0.37
mmol/g) was obtained from Watanabe Chemical Industries
Ltd., Hiroshima, J apan, and 1H-pyrazole-1-carboxamidine
hydrochloride was purchased from Aldrich Chemicals, WI.
Fmoc-MeâAla-OH was prepared according to the reported
method30 with Fmoc-OSu and MeâAla. Boc-D-Arg(Cbz2)OH was
prepared according to the procedure reported by J etten.31 Thin-
layer chromatography was performed on silica plates (0.25
mm; Merck, 60 F254). Flash column chromatography for the
intermediates of solution phase synthesis was performed using
Merck silica gel 60 (230-400 mesh). Purification of the final

Orally Available and Long-Acting Analgesic Peptide
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5087
D
to give the tripeptide as white amorphous (11.7 g, 96.7%). [R]25
Nr-Am id in o-Tyr -D-Ar g-P h e-âAla -OH (1). The same solid
phase procedure for the synthesis of ADAMB was employed,
but Fmoc-âAla-Wang resin was used instead of Fmoc-MeâAla-
Wang resin as a starting material to give 1 as a white powder
+3.9° (c 1.00, DMF). 1H NMR (CDCl3, 300 MHz): δ 9.45 (br s,
1H), 9.26 (br s, 1H), 7.43-7.04 (m, 20H), 6.90 (br t, J ) 7.0
Hz, 1H), 5.46-4.97 (m, 6H), 5.23 (s, 2H), 5.10 (s, 2H), 4.15
(m, 1H), 3.97 (m, 1H), 3.92 (m, 1H), 3.62(dt, J ) 14, 6.9 Hz,
0.67H), 3.40 (m, 0.33H), 3.50 (m, 1H), 2.86 (br d, J ) 7.5 Hz,
2H), 2.78 (s, 1H), 2.65 (s, 2H), 2.49 (dt, J ) 6.9, 3.0 Hz, 1H),
2.39 (m, 0.67H), 2.18 (m, 0.33H), 1.92 (m, 1H), 1.70 (m, 1H),
1.56 (m, 2H), 1.41 (s, 9H). FAB-MS 865 [M + 1]+. Anal.
(C47H56N6O10) C, H, N.
(60 mg, 43%). [R]25 +20.5° (c 1.11, 1M AcOH). 1H NMR
D
(CD3OD, 300 MHz): δ 7.24 (m, 5H), 7.06 (d, J ) 8.4 Hz, 2H),
6.70 (d, J ) 8.4 Hz, 2H), 4.58 (dd, J ) 11, 4.2 Hz, 1H), 4.36 (t,
J ) 7.2 Hz, 1H), 3.97 (t, J ) 7.2 Hz, 1H), 3.57-3.32 (m, 3H),
3.07-2.83 (m, 4H), 2.76 (dd, J ) 14, 11 Hz, 1H), 2.38 (t, J )
6.3 Hz, 2H), 1.41 (m, 2H), 1.14 (m, 1H), 0.93 (m, 1H). FAB-
HRMS: calcd for C28H40N9O6 [M + 1]+, 598.3102; found,
598.3125.
Nr-Am id in o-Tyr -D-Ar g-P h e-Sa r -OH (2). The same solid
phase procedure for the synthesis of ADAMB was employed,
but Fmoc-Sar-Wang resin was used instead of Fmoc-MeâAla-
Wang resin as a starting material to give 2 as a white
powder (63 mg, 42%). [R]25D +26.5° (c 0.35, 1M AcOH). 1H NMR
(CD3OD, 300 MHz): δ 7.17 (m, 5H), 7.01 (d, J ) 8.1 Hz, 2H),
6.67 (d, J ) 8.1 Hz, 2H), 5.08 (t, J ) 6.3 Hz, 1H), 4.35 (br s,
1H), 4.19 (m, 1H), 3.98 (t, J ) 17 Hz, 1H), 3.83 (d, J ) 18 Hz,
1H), 3.67 (m, 1H), 3.14-2.73 (m, 9H), 1.51 (m, 1H), 1.40 (m,
1H), 1.16 (m, 2H). FAB-HRMS: calcd for C28H40N9O6 [M +
1]+, 598.3102; found, 598.3108.
Boc-Tyr (Bzl)-D-Ar g(Cbz2)P h e-MeâAla -OBzl (7). Boc-D-
Arg(Cbz2)Phe-MeâAla-OBzl (4.76 g, 5.50 mmol) was dissolved
into 4 N HCl/EtOAc (20 mL) and stirred for 25 min at room
temperature. Et2O was added to the solution, and the precipi-
tated solid was separated by filtration to give the Boc-removed
tripeptide quantitatively. This amine component, Boc-Tyr(Bzl)-
OH (1.86 g, 5.00 mmol), and HOBt (743 mg, 5.50 mmol) were
dissolved in DMF (10 mL), and WSCI‚HCl (1.15 g, 6.00 mmol)
was added to the solution at -8 °C. The pH of the solution
was adjusted to 5 with Et3N (1.35 mL). This mixture was
stirred for 30 min at -8 °C and overnight at 4 °C. The reaction
mixture was diluted with EtOAc (100 mL) and washed with
10% citric acid, saturated NaHCO3, and brine. The organic
phase was dried over MgSO4, filtered, and evaporated in vacuo.
The residue was solidified from EtOAc and hexane to give
H-Tyr -D-Ar g-P h e-MeâAla -OH (3). The same solid phase
procedure for the synthesis of ADAMB was employed, but the
amidination process was skipped to give 3 as a white pow-
D
white amorphous (4.76 g, 85.8%). [R]25 -3.2° (c 1.04, DMF).
D
1
der (76 mg, 53%). [R]25 +47.4° (c 0.84, 1 M AcOH). H NMR
(CD3OD, 300 MHz): δ 7.25 (m, 5H), 7.11 (d, J ) 8.4 Hz, 2H),
6.74 (d, J ) 8.4 Hz, 2H), 5.12 (t, J ) 7.2 Hz, 1H), 4.22 (m,
1H), 4.09 (m, 1H), 3.53 (br t, J ) 7.2 Hz, 2H), 3.17-2.89 (m,
6H), 2.89 (s, 2H), 2.85 (s, 1H), 2.42 (m, 2H), 1.42 (m, 2H), 1.18
(m, 2H). FAB-HRMS: calcd for C28H40N7O6 [M + 1]+, 570.3040;
found, 570.3021.
1H NMR (CDCl3, 300 MHz): δ 9.49 (br s, 1H), 9.29 (br s, 1H),
7.60-7.00 (m, 26H), 7.00 (d, J ) 8.7 Hz, 2H), 6.81 (d, J ) 8.7
Hz, 2H), 6.79 (br s, 1H), 5.29-4.88 (m, 6H), 5.08 (s, 2H), 4.95
(s, 2H), 4.49 (m, 1H), 4.29 (m, 1H), 3.80 (m, 1H), 3.67 (m, 1H),
3.42 (m, 1H), 3.03-2.79 (m, 5H), 2.76 (s, 1H), 2.65 (s, 2H), 2.46
(br t, J ) 7.5 Hz, 1.33H), 2.40 (m, 0.67H), 2.16 (m, 2H), 1.54
(m, 2H), 1.36 (s, 9H). FAB-MS 1119 [M + 1]+. Anal.
(C63H71N7O12) C, H, N.
H-MeTyr -D-Ar g-P h e-MeâAla-OH (4). The same solid phase
procedure for the synthesis of ADAMB was employed, but
Fmoc-MeTyr(t-Bu)OH was used instead of Fmoc-Tyr(t-Bu)OH,
and the amidination process was skipped to give 4 as a white
powder (80 mg, 55%). [R]23D +44.4° (c 1.01, 1M AcOH). 1H NMR
(CD3OD, 300 MHz): δ 7.21 (m, 5H), 7.00 (d, J ) 8.4 Hz, 2H),
6.70 (dd, J ) 8.4, 2.7 Hz, 2H), 5.18 (t, J ) 7.2 Hz, 0.5H), 5.00
(t, J ) 7.5 Hz, 0.5H), 4.24 (dt, J ) 6.0, 6.0 Hz, 1H), 3.63 (br m,
1H), 3.42 (br m, 2H), 3.10-2.63 (m, 6H), 2.87 (d, J ) 6.3 Hz,
3H), 2.38 (br m, 2H), 2.29 (d, J ) 6.6 Hz, 3H), 1.43 (br m, 2H),
1.09 (m, 2H). FAB-HRMS: calcd for C29H42N7O6 [M + 1]+,
584.3197; found, 584.3203.
Nr-(N,N′-Bis-b en zyloxyca r b on yl)a m id in o-Tyr (Bzl)-D-
Ar g(Cbz2)P h e-MeâAla -OBzl (8). Tetrapeptide 7 (2.43 g, 2.20
mmol) was dissolved in 4 N HCl/dioxane (20 mL) at -8 °C
and stirred for 30 min at room temperature. Et2O (80 mL) was
added to the solution, and the precipitated oil was separated
by decantation. The residue was solidified from Et2O (40 mL).
The solid was collected by filtration and dried under a reduced
pressure to give Boc-removed tetrapeptide (2.23 g, 97.4%) as
a white powder. This amine hydrochloride (1.04 g, 1.00 mmol)
and (benzyloxycarbonylimino-pyrazol-1-yl-methyl)carbamic acid
benzyl ester (416 mg, 1.10 mmol) were dissolved in DMF (2
mL). The pH of the solution was adjusted to 8 with Et3N (210
µL) at 0 °C. The mixture was stirred for 6 h at room
temperature, and Et3N (50 µL) was added in the middle of
the reaction to keep the pH 8. The reaction mixture was poured
into 10% citric acid (15 mL), and the precipitated oil was
extracted with EtOAc (40 mL). The EtOAc solution was
washed with brine, dried over MgSO4, filtered, and evaporated
in vacuo. The residual oil was purified by flush chromatogra-
phy eluted with CHCl3:MeOH (100:1) followed by precipitation
from Et2O to give white amorphous powder (0.93 g, 71.0%).
[R]25D +8.9° (c 1.01, DMF). 1H NMR (CDCl3, 300 MHz): δ 11.50
(s, 1H), 9.48 (br s, 1H), 9.26 (br s, 1H), 8.81 (t, J ) 7.5 Hz,
1H), 7.47-7.09 (m, 35H), 7.06 (d, J ) 8.4 Hz, 2H), 6.87 (d, J
) 8.1 Hz, 1H), 6.81 (d, J ) 8.4 Hz, 2H), 5.23-4.86 (m, 14H),
4.76 (dt, J ) 7.2, 7.2 Hz, 1H), 4.42 (dt, J ) 7.0, 7.0 Hz, 1H),
3.59 (m, 1H), 3.70 (m, 1H), 3.59 (m, 1H), 3.27 (m, 1H), 2.99
(br t, J ) 6.0, 2H), 2.80 (br d, J ) 7.2 Hz, 2H), 2.70 (s, 1H),
2.57 (s, 2H), 2.42 (t, J ) 7.2 Hz, 1.33H), 2.35 (m, 0.67H), 1.99
(br s, 2H), 1.49 (m, 2H). FAB-MS 1329 [M + 1]+. Anal.
(C75H77N9O14) C, H, N.
Me2Tyr -D-Ar g-P h e-MeâAla -OH (5). The same solid phase
procedure for the synthesis of ADAMB was employed, but
Me2Tyr(t-Bu)OH was used instead of Fmoc-Tyr(t-Bu)OH, and
the amidination process was skipped to give 5 as a white
powder (68 mg, 45%). [R]23 +44.4° (c 1.08, 1 M AcOH). 1H
D
NMR (CD3OD, 300 MHz): δ 7.21 (m, 5H), 7.00 (d, J ) 8.4 Hz,
2H), 6.70 (dd, J ) 8.4, 1.5 Hz, 2H), 5.15 (t, J ) 6.9 Hz, 0.5H),
4.99 (t, J ) 7.5 Hz, 0.5H), 4.14 (m, 1H), 3.54 (m, 1H), 3.43 (m,
2H), 3.10-2.80 (m, 6H), 2.89 (s, 2H), 2.85 (s, 1H), 2.53 (s, 3H),
2.51 (s, 3H), 2.35 (br m, 2H), 1.38 (m, 1H), 1.24 (m, 1H), 0.95
(m, 2H). FAB-HRMS: calcd for C30H44N7O6 [M + 1]+, 598.3353;
found, 598.3321.
Nr-Ac-Tyr -D-Ar g-P h e-âAla -OH (6). The same solid phase
procedure for the synthesis of 1 was employed, but N-terminal
was acetylated with acetic anhydride instead of amidination
to give 6 as a white powder (84 mg, 56%). [R]25D +30.6° (c 0.87,
1
1M AcOH). H NMR (DMSO-d6, 300 MHz): δ 9.73 (t, 5.1 Hz,
1H), 8.29 (m, 1H), 8.09 (d, J ) 8.4 Hz, 1H), 7.89 (t, J ) 5.1
Hz, 1H),7.01 (m, 6H), 7.00 (d, J ) 8.1 Hz, 2H), 6.64 (d, J ) 8.1
Hz, 2H), 4.38 (dt, J ) 7.2, 7.2 Hz, 1H), 4.29 (m, 1H), 4.03 (dd,
J ) 14, 7.5 Hz, 1H), 3.30-3.05 (m, 3H), 2.96 (br d, J ) 3.9 Hz,
2H), 2.98-2.60 (m, 3H), 2.14 (m, 2H), 1.77 (s, 3H), 1.57 (m,
1H), 1.42 (m, 1H), 1.22 (m, 2H). FAB-HRMS: calcd for
Nr-Am id in o-Tyr -D-Ar g-P h e-MeâAla -OH (ADAMB). To
the solution of protected tetrapeptide 8 (657 mg, 0.50 mmol)
in AcOH (10 mL) was added 10% Pd-C (50% wet). This
solution was vigorously stirred under H2 atmosphere for 4 h
at room temperature. The catalyst was removed by filtration,
and the filtrate was concentrated in vacuo. The residue was
precipitated from Et2O to give a white powder (0.30 g, 91.2%)
as acetate salt.
C
29H40N7O7 [M + 1]+, 598.2989; found, 598.2967.
Bin d in g Assa ys. Synaptosomal fractions were prepared
from mice spinal cord and guinea pig cerebella according to
the method of Chang et al.32 Briefly, spinal cord or cerebella
was homogenized in a 0.32 M sucrose solution at 0 °C and
centrifuged at 6000g for 15 min. The supernatant was centri-

5088 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Ogawa et al.
(6) Broccardo, M.; Erspamer, V.; Erspamer, G. F.; Improta, G.;
Linari, G.; Melchiorri, P.; Montecucchi, P. C. Pharmacological
Data on Dermorphins, a New Class of Potent Opioid Peptides
from Amphibian Skin. Br. J . Pharmacol. 1981, 73, 625-631.
(7) Sato, T.; Sakurada, S.; Sakurada, T.; Furuta, S.; Chaki, K.;
Kisara, K.; Sasaki, Y.; Suzuki, K. Opioid Activities of D-Arg2-
Substituted Tetrapeptides. J . Pharmacol. Exp. Ther. 1987, 242,
654-659.
fuged at 40 000g for 30 min, and the pellets were homogenized
in 5 mM Tris-HCl buffer (pH 7.4) at 0 °C. The suspension was
centrifuged at 6000g for 15 min. The supernatant was then
centrifuged twice at 40 000g for 30 min each. After the second
centrifugation, the membranes were resuspended in 50 mM
Tris-HCl buffer (pH 7.4).
[3H]DAMGO, [3H]deltorphin-II, and [3H]U-69593 were used
for the determination of relative affinities for µ-, δ-, and
κ-receptors, respectively. Binding assays were carried out by
incubating an aliquot of the membrane fraction (250 µg for
[3H]DAMGO, 350 µg for [3H]deltorphin-II, and 180 µg for
[3H]U-69593) containing protease inhibitors and the labeled
ligand (3 nM for [3H]DAMGO or [3H]deltorphin-II, and 2 nM
for [3H]U-69593) in 50 mM Tris-HCl buffer (pH 7.4). After 60
(8) de Castiglione, R.; Rossi, A. C. Structure-Activity Relationships
of Dermorphin Synthetic Analogues. Peptides 1985, 6 (Suppl.
3), 117-125.
(9) Takagi, H.; Shiomi, H.; Ueda, H.; Amano, H. Morphine-Like
Analgesia by a New Dipeptide, L-Tyrosyl-L-Arginine (Kyotor-
phin) and its Analogue. Eur. J . Pharmacol. 1979, 55, 109-111.
(10) Schiller, P. W.; Nguyen, T. M.-D.; Chung, N. N.; Lemieux, C.
Dermorphin Analogues Carrying an Increased Positive Net
Charge in their “Message” Domain Display Extremely High
µ-Opioid Receptor Selectivity. J . Med. Chem. 1989, 32, 698-
703.
(11) Salvadori, S.; Marastoni, M.; Tomatis, R. Opioid Peptides.
Structure-Activity Relationships in Dermorphin Tetrapeptides.
Il Farmaco Ed. Sci. 1982, 37, 514-518.
(12) Sakurada, S.; Murayama, K.; Nakano, M.; Hongo, K.; Takeshi-
ma, S.; Take, N. Peptide Derivatives. PCT Int. Appl. WO
9524421, 1995; Chem. Abstr. 1996, 124, 146854.
(13) Sasaki, Y.; Matsui, M.; Taguchi, M.; Suzuki, K.; Sakurada, S.;
Sato, T.; Sakurada, T.; Kisara, K. D-Arg2-Dermorphin Tetrapep-
tide Analogues. A Potent and Long-Lasting Analgesic Activity
after Subcutaneous Administration. Biochem. Biophys. Res.
Commun. 1984, 120, 214-218.
(14) Paakkari, P.; Paakkari, I.; Bonhof, S.; Feuerstein, G.; Sire´n, A.-
L. Dermorphin analogue Tyr-D-Arg2-Phe-Sarcosine-Induced Opi-
oid Analgesia and Respiratory Stimulation. The Role of Mu1-
receptors? J . Pharmacol. Exp. Ther. 1993, 266, 544-550.
(15) Sato, T.; Takahashi, N.; Tan-No, K.; Kisara, K.; Sakurada, T.;
min at 25 °C, the reaction mixture was filtered over
a
Whatman GF/B filter soaked with 0.1% polyethyleneimine and
the filter was washed twice with cold Tris-HCl buffer. Filters
were counted in a liquid scintillation counter after overnight
extraction with liquid scintillation fluid (3 mL). Specific
binding was determined from the differences between total
binding and that in the presence of excess (10 µM) unlabeled
ligand. Kd and Bmax values were obtained using Scatchard
analysis.
An tin ocicep tive Assa y. Male mice of ddY strain weighing
10-32 g were used in the experiment. They were purchased
from J apan SLC Inc. (Shizuoka, J apan) and housed in cages
of 5-6 animals, matched for weight, and placed in a colony
room. Animals were given standard food (MM-3, J apan SLC
Inc.) and tap water ad libitum in an air-conditioned room at
23 ( 2 °C and 50 ( 10% relative humidity with a standard 12
h light-dark cycle (lights on 6:00-18:00). The tail pressure
test was performed according to the reported method by
Sakurada et al.33 with a slight modification. Thus, mechanical
pressure was applied to the base of the tail at a rate of 32
g/sec using an automated tail pressure unit (Ugo Basile, Italy).
Biting or struggling behavior in mice was used as an indication
of response threshold, and only mice responding behaviorally
to a tail pressure of 100-300 g were selected for this experi-
ment. The trials were terminated at the level of 500 g to
prevent tail tissue damage. The mean ( SEM of the pressure
level was plotted. To obtain the response curve, the dose was
plotted against percentage of MPE (% MPE ) (P2 - P1/500 -
P1) × 100, where P1 is the response pressure before drug
administration (g) and P2 is the response pressure after drug
administration (g). In the hot plate test,34 the animals were
placed on a 55 °C hot plate (Ugo Basil, model-DS37) sur-
rounded by a glass tube (φ14 × 13 H cm), and the latency of
either a jump or a hindpaw lick was measured. Prior to drug
administration, the nociceptive response of each mouse was
measured and mice showing the response in 10-20 s after
placement were chosen for the test. A cutoff time of 40 s was
used to minimize tissue damage. The peptides administered
were dissolved in saline solution (Fuso Chemical Industries,
Osaka, J apan). Saline solution was used as the control. ED50
vales were obtained by the method of Litchfield and Wil-
coxon.35
Sakurada, S. Comparison of Opioid Activity Between
a N-
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