Unique high-affinity synthetic μ-opioid receptor agonists with central- and systemic-mediated analgesia

Article abstract of DOI:10.1021/jm020459z

Unique opioid mimetic substances containing identical N-terminal aromatic residues separated by an unbranched alkyl chain containing two to eight methylene groups were developed. Regardless of the length of interposing alkyl chain, the bis-Tyr and bis-Phe

Full text of DOI:10.1021/jm020459z

J . Med. Chem. 2003, 46, 3201-3209
3201
Articles
Un iqu e High -Affin ity Syn th etic µ-Op ioid Recep tor Agon ists w ith Cen tr a l- a n d
System ic-Med ia ted An a lgesia
Yoshio Okada,*,† Yuko Tsuda,† Yoshio Fujita,† Toshio Yokoi,† Yusuke Sasaki,‡ Akihiro Ambo,‡ Ryoji Konishi,§
Mitsuhiro Nagata,§ Severo Salvadori,| Yunden J insmaa, Sharon D. Bryant, and Lawrence H. Lazarus*,
Faculty of Pharmaceutical Sciences, Department of Medicinal Chemistry and High Technology Research Center,
Kobe Gakuin University, Nishi-ku, Kobe 651-2180, J apan, Department of Biochemistry, Tohoku Pharmaceutical
University, 4-1, Komatsushima 4-chome, Aoba-ku, Sendai 981-8558, J apan, Teikoku Seiyaku Co., Ltd., Sanbonmatsu,
Oochi-cho, Ohkawa-gun, 769-2695, J apan, Department of Pharmaceutical Sciences, University of Ferrara,
I-44100 Ferrara, Italy, and Peptide Neurochemistry, LCBRA, National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina 27709
Received October 17, 2002
Unique opioid mimetic substances containing identical N-terminal aromatic residues separated
by an unbranched alkyl chain containing two to eight methylene groups were developed.
Regardless of the length of interposing alkyl chain, the bis-Tyr and bis-Phe compounds were
inactive; however, replacement by a single Dmt (2′,6′-dimethyl-L-tyrosine) residue enhanced
activity by orders of magnitude. Moreover, the bis-Dmt compounds were another 10-fold more
potent with an optimum intra-aromatic ring distance of about four to six methylene units.
1,4-Bis(Dmt-NH)butane (7) had high µ-opioid receptor affinity (Ki ) 0.041 nM) and functional
µ-opioid agonist bioactivity (IC50 ) 5.3 nM) with in vivo central (intracerebroventricular) and
systemic (subcutaneous) analgesia in mice (1.5- to 2.5-fold greater than and 10-12% relative
to morphine, respectively); these activities were reversed by naloxone to the same degree. It
appears that the bis-Dmt compounds indiscriminately act as both message and address domains.
In tr od u ction 1
opiates,18-20 stipulated that structurally distinct regions
of the molecule exert different functions. In other words,
the “message domain” in opioid peptides was considered
to include the N-terminal Tyr with its free amine and
hydroxyl groups, and a spacer consisting of one or two
amino acids (D-Ala, D-Met, Pro, or Gly-Gly). On the other
hand, the “address domain” would contain the subse-
quent C-terminal residues beginning with the second
aromatic residue (usually Phe or Trp3 in the case of
endomorphin-121 and hemorphin,22 respectively) that
trigger the biological response. Our data, however,
demonstrate that symmetric opioid mimetic substances,
which contain two identical dimethylated tyrosylamides
separated by a simple unbranched alkyl chain (Table
1), are able to serve as both the message and address
domains when binding within the µ-opioid receptor to
yield high-affinity compounds to produce morphine-like
analgesia that was naloxone-reversible. In this report,
we present opioid receptor affinity values (Ki), classical
functional bioassays in vitro, biological activity in vivo
with mice, and 1H NMR analysis results that permit
us to present a new interpretation or variation on the
message-address domain hypothesis concerning opioid
ligand-receptor interaction using these unique bis-Dmt
substances.
Opioid receptors play a role in relaying information
for a variety of physiological events,2 the effects of which
are transmitted by a variety of peptides for δ-, µ-, and
κ-opioid receptors.3 Despite the structural diversity of
opioid peptides, an N-terminal Tyr is a common struc-
tural element except in the nociceptin/orphanin ligand
for ORL receptors, which has an N-terminal Phe
residue.4 Although replacement of Tyr by 2′,6′-dimethyl-
L-tyrosine (Dmt) initially yielded a weakly active µ-re-
ceptor peptide,5 incorporation of Dmt subsequently and
dramatically altered the activity of numerous unrelated
opioids by elevating affinities, affecting receptor selec-
tivity, and changing the spectrum of their bioactivity.6-11
The utilization of the Dmt-Tic pharmacophore7 in lieu
of the TIP(P) series of pseudopeptides12 produced opioid
ligand families consisting of highly potent δ-receptor
antagonists, δ-receptor agonists, and bifunctional ana-
logues exhibiting δ-receptor antagonism or δ-receptor
agonism with µ-receptor agonism depending on the
C-terminal constituents.13-15
The concept of message and address domains,16 as
applied to opioid peptide agonists17 and non-peptide
* To whom correspondence should be addressed. For Y.O.:
phone, +78-974-1551; fax, +78-974-5689; e-mail, okada@
pharm.kobegakuin.ac.jp. For L.H.L.: phone, (919) 541-3238; e-mail,
lazarus@niehs.nih.gov.
Ra tion a le
† Kobe Gakuin University.
‡ Tohoku Pharmaceutical University.
The design of a simple opioid mimetic containing
minimal functional groups for receptor recognition,
namely Dmt,5-7 enabled the investigation of the role of
§ Teikoku Seiyaku Co., Ltd.
| University of Ferrara.
LCBRA, National Institute of Environmental Health Sciences.
10.1021/jm020459z CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/17/2003

3202 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Okada et al.
Ta ble 1. Opioid Receptor Binding and Functional Biological Activity of Opioid Mimetics Containing an Alkyl Spacera
receptor binding, Ki (nM)
functional bioactivity
compd
peptide
δ
µ
δ/µ
13
81
21
GPI IC50 (nM)
MVD IC50 (µM)
pA2(δ)
1
2
3
4
5
6
7
8
9
Tyr-NH-(CH2)2-NH-Tyr
Dmt-NH-(CH2)2-NH-Dmt
Tyr-NH-(CH2)4-NH-Tyr
Phe-NH-(CH2)4-NH-Phe
Dmt-NH-(CH2)4-NH-Phe
Dmt-NH-(CH2)4-NH-Tyr
Dmt-NH-(CH2)4-NH-Dmt
Tyr-NH-(CH2)6-NH-Tyr
Dmt-NH-(CH2)6-NH-Dmt
Tyr-NH-(CH2)8-NH-Tyr
Dmt-NH-(CH2)8-NH-Dmt
8290 ( 433 (3)
115.7 ( 10.6 (6)
6500 ( 278 (3)
46190 ( 458 (3)
62.0 ( 5.7 (3)
133 ( 18 (3)
53.4 ( 14.8 (5)
21880 ( 4020 (3)
46.1 ( 8.8 (5)
6150 ( 525 (3)
14.8 ( 3.0 (7)
648 ( 95 (5)
1.43 ( 0.01 (3)
309 ( 104 (5)
1530 ( 122 (3)
0.52 ( 0.089 (4)
0.38 ( 0.02 (3)
0.041 ( 0.003 (4)
410 ( 85 (5)
nd
nd
>10
nd
2844 ( 517
5.5
nd
nd
30
nd
119
349
1302
53
870
34
181 ( 36
255 ( 11
5.33 ( 0.65
nd
3.08 ( 0.53
nd
>10
>10
>10
nd
>10
nd
5.5
5.3
5.8
0.053 ( 0.01 (6)
399 ( 41 (7)
0.19 ( 0.024 (4)
6.1
6.4
10
11
78
53.7 ( 7
>10
a
Opioid receptor binding data of the bis-amide compounds are listed as the mean ( SE and determined using rat brain P2 synaptosomal
preparations with [3H]DPDPE for δ-opioid receptors and [3H]DAGO for µ-opioid receptors as detailed elsewhere.7 The number of independent
repetitions is noted in parentheses (n). Receptor selectivity is the ratio (δ/µ) of the affinity constants (Ki), determined according to Cheng
and Prusoff.39 µ-Opioid agonism used GPI (guinea pig ileum), while MVD (mouse vas deferens) defines δ bioactivity; n ) 5-6 for bioassays.
The pA2 is the negative log of the molar concentration required to double the agonist concentration to achieve the original response and
defines antagonism. nd ) not determined.
alkane linkers on selectivity for the µ-opioid receptor
subtype. These symmetrical compounds suggest inter-
action at both of the hypothesized message and address
regions in the receptor and supply prototypes for the
design of opioid mimetics with novel and distinct
linkers.15,23
receptor affinity another 10-fold (Ki ) 0.041-0.053 nM)
with high receptor selectivity (δ/µ ) 1302 and 870,
respectively). Thus, the optimal distance between the
Dmt residues for maximum µ-opioid receptor binding
appeared to be butyl (7) = hexyl (9) > octyl (11) . ethyl
(2), i.e., an unbranched aliphatic chain < ethyl < octyl
in length. As suggested elsewhere,7-10 the key residue
in this transformation toward increased opioid receptor
interaction was due solely to the presence of Dmt.
Ch em istr y
All the opioid mimetic compounds in this study were
synthesized by standard solution methodology for pep-
tide synthesis using Fmoc- and Boc-amino protection
groups and using PyBop as the coupling reagent.23,24
Symmetrical ligands (1-4, 7-11) were prepared by
coupling Boc-amino acids with a diamine followed by
TFA (1.0 mL, 13 mmol) containing anisole (0.10 mL,
0.90 mmol) for 1 h at room temperature. The compounds
were then precipitated with diethyl ether, filtered, and
purified by HPLC (vide infra). Asymmetric compounds
(5, 6) were prepared using mono-Boc-protected di-
amine25 and Fmoc- and Boc-amino acids. The Fmoc
group was removed by 20% piperidine, and the crude
compounds were precipitated with ether and purified
using RP-HPLC on a COSMOSIL C18 column (4.6 mm
× 250 mm) or a YMC R & D R-ODS-5 Å column (4.6
mm × 250 mm) using a Waters model 600E for analyti-
cal and preparative HPLC. The compounds were eluted
from the columns using linear gradients starting from
10% acetonitrile in 0.05% TFA with a 1% increase in
acetonitrile concentration per minute at a flow rate of
1 mL/min; detection was at 220 nm. Retention times
were recorded as either tR(C) or tR(Y), respectively.
Purity was greater than 98%. 1H, 13C, and 2D NMR
spectra were measured using a 30 mg sample of each
compound dissolved in 0.5 mL of DMSO-d6 and mea-
sured on a Bruker ARX-500 spectrometer at 25 °C.
F u n ction Bioa ctivity in Vitr o. The high µ-receptor
agonism of 7 and 9 (IC50 ) 5.3 and 3.1 nM, respectively)
and undetectable δ agonism coupled with their low
δ-receptor antagonism (pA2 ) 6.4-5.3) and remarkable
opioid receptor affinities (Table 1) support the following
conclusions: (i) The compounds predominately act at
µ-opioid receptor sites. (ii) The bioactivities of 2, 7, 9,
and 11 suggest that the optimum distance for µ-receptor
agonism between the Dmt amides was greater than two
but less than eight methylene groups. The effect of a
spacer between aromatic centers was observed with
other opioids containing N-terminal Dmt14,15 or Tyr.26
(iii) The increase in both µ- and δ-opioid receptor
affinities with Dmt suggests a degree of structural
similarity in their binding sites. The asymmetric ana-
logues (5, 6), particularly H-Dmt-NH-(CH2)4-NH-Phe-H
(5), could be considered an analogue of known opioid
peptides containing a single D-amino acid,21,27,28 a dipep-
tide spacer,29,30 or a heteroaromatic residue (e.g., a Tic
residue12) between aromatic amino acids.26 (iv) The
weak opioid receptor binding affinities of the bis-Tyr and
bis-Phe cognates (1, 3, 4, 8, 10) demonstrate an inability
of these residues to promote ligand interaction. Whereas
the methyl groups at the 2′ and 6′ positions of tyrosine
might affect the ø2 angle of Dmt, comparative modeling
data suggest that they might reduce rotational freedom
of the aromatic ring.31-34 We hypothesize that these
methyl groups stabilize the interaction with the phar-
macophoric groups (hydroxyl, N-terminal amine, and
the aromatic ring through hydrophobic forces, ring
stacking, or π-π interactions) within the receptor that
could enhance its bioactivity. While this mechanism is
not yet fully understood, our data as well as those in
the literature9,11,26,30,31,33,34 point in this direction.
Resu lts a n d Discu ssion
Op ioid Recep tor Affin ity. The opioid mimetics
containing bis-Tyr (1, 3, 8, 10) and bis-Phe (4) interacted
poorly with δ- and µ-opioid receptors (Table 1). Substi-
tution by a single Dmt residue (5, 6) enhanced µ-opioid
receptor affinities several-hundred-fold (Ki ) 0.38-0.52
nM), supporting observations with a large variety of
opioid substances containing N-terminal Dmt.6-10 The
bis-Dmt-containing ligands 7 and 9 increased µ-opioid
In Vivo Biologica l Activity. In mice, 7 rapidly
produced centrally mediated (icv) analgesia induced by

µ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3203
F igu r e 1. Analgesia of H-Dmt-NH-(CH2)4-NH-Dmt-H (7) in mice. Dose-dependent appearance of analgesia by the tail-flick test
is expressed as a function of time (a, d) and area under the curve (AUC) (b, c, e). Intracerebroventricular (icv) injections (a-c):
saline ([) or 7 at 0.03 nmol (0), 0.1 nmol (2), 0.3 nmol (O), or 1 nmol (9) in 4 µL of saline per mouse. (c) Naloxone (2 mg/kg) was
injected sc 30 min before 7. (d, e) Subcutaneous (sc) injections: saline ([) or 7 at 10 mg/kg (0), 30 mg/kg (2), 100 mg/kg (O) in
saline, or 3 mg/kg morphine (4). Data are the mean ( SE with n ) 5-7 animals per time point. Statistical significance used
Student’s t-test. Asterisks denote significant differences between mice treated with saline and 7 (/, p < 0.05; //, p < 0.01; ///,
p < 0.001). The symbol # indicates a significant difference (p < 0.05) between the effect of 7 and treatment with naloxone or that
between morphine and the naloxone-treated animals.
a spinal nociceptive mechanism (tail-flick test) that was
1.5- to 2.2-fold greater than morphine and was naloxone-
reversible; naloxone inhibited analgesia by 7 and mor-
phine to the same degree (68% and 71%, respectively)
(Figure 1). The supraspinal nociceptive pathway (hot
plate test) revealed equivalent analgesia to morphine,
and both substances were completely blocked by nalox-
one (Figure 2). Subcutaneous (sc) injection of 7 produced
an analgesia in a dose-dependent mechanism that was
10-12% as potent as morphine (Figure 1), verifying that
7 indeed crossed the blood-brain barrier. While the
receptor binding affinity and functional bioactivity are
essentially similar for 7 and 9 (Table 1), the icv and sc
administration of 7 and 9 in rats indicated, however,
that 7 was about twice as potent as 9 in both experi-
mental paradigms and statistically significant above the
saline controls (p < 0.05) (data not shown).
molecular symmetry of 7 precluded its use for NMR
analysis) revealed an absence of cross-peaks between
N- and C-terminal protons indicative of an extended,
open structure (Figure 3). The strongest cross-peaks
detected occurred between the protons of the 2′ and 6′
methyl groups and those at 3′ and 5′ positions of the
tyramine ring of Dmt. The nuclear Overhauser effect
(NOE) cross-peak intensities became progressively
weaker thereafter (Figure 3).
Molecu la r Mod elin g. The lowest energy structure
of 7 was derived using systematic conformational search-
ing and energy minimization based on the J coupling
values and NOE cross-peaks of 6 (Figure 4). That
structure was among 8323 stable conformations that fit
the criteria defined by the NMR data reflecting the
flexible nature of these compounds. The length of the
alkyl chain maintains the distance and positioning of
the two aromatic centers, the putative message and
address regions; the flexible nature of the chain facili-
1H NMR An a lysis. Solution structures of 5 and 6
1
determined by 2D H and 13C NMR spectroscopy (the

3204 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Okada et al.
F igu r e 2. Supraspinal analgesia of H-Dmt-NH-(CH2)4-NH-Dmt-H (7) using the hot plate test in mice. See the caption to Figure
1 for details. Mice were injected intracerebroventricularly (icv), and the response was measured identically without (a, b) and
with naloxone (c) and after subcutaneous (sc) injections (d, e). Statistical analyses are given in the caption to Figure 1.
tates the ability of the compound to conform to the
topography of the receptor cleft. In general, the extended
conformations of 7 were similar to extended structures
reported for δ- and µ-opioid tetra-, penta-,32 and hep-
tapeptide agonists32,33 and differed substantially from
the X-ray derived structures of three Dmt-Tic ana-
logues.34
as well as the N-terminal amine of the ligand and
functional groups of the receptor. Perhaps a single Dmt
residue interacts at one site of the receptor through
combined hydrophobic/aromatic ring associations as well
as hydrogen bonding, while Phe (5), Tyr (6), or the
second Dmt (7, 9) associates with another similar region
of the opioid receptor.
The small dimensions of the bis-Dmt compounds
appear to fit the µ-opioid receptor binding site (pocket)
with ease and precludes binding between two adjacent
receptors in the cell membrane, as suggested with the
larger dimeric enkephalin36 and dimeric dermorphin
analogues.37 Furthermore, the ability of bis-(Dmt-NH)-
alkyl opioid mimetic compounds to transit the blood-
brain barrier is an important characteristic of this class
of compounds. In many ways, the simple design of our
final products resembles the minimalist themes pre-
sented in music by Philip Glass, the early artwork by
Frank Stella, or a single note played to perfection on
the J apanese bamboo flute. Combination of Dmt and
an alkyl chain might also enhance proteolytic stability
Con clu sion s
Dmt enhanced the receptor affinities of opioid ligands
for both δ-and µ-opioid receptors.8,13-15,35 The agonist
activity measured in vitro and analgesic effects in vivo
of the bis-Dmt compounds (7, 9) appear to be mediated
through µ-opioid receptors. Furthermore, the symmetry
of these compounds verified that these molecules con-
tain identical “message” and “address” domains. Our
results suggest the following: the effectiveness of Dmt
may involve not only hydrophobic forces that interact
within a lipid milieu but also its ability to align and
stabilize the tyrosyl ring, permitting more effective H
bonding to occur between the required hydroxyl group

µ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3205
1
F igu r e 3. (a) NOE cross-peaks of H-Dmt-NH-(CH2)4-NH-Phe-H (5) measured in DMSO using H NMR spectrometry. The cross-
peaks are indicated by the arrows, and the cross-peak intensities are summarized as being strong (S) (<2.25 Å), medium (M)
1
(2.25-3.0 Å), or weak (W) (3.0-4.0 Å). (b) 1H NMR analysis of H-Dmt-NH-(CH2)4-NH-Tyr-H (6) in DMSO. H, 13C, and 2D NMR
spectra were measured using a 30 mg sample of each compound dissolved in 0.5 mL of DMSO-d6 and measured on a Bruker
ARX-500 spectrometer at 25 °C.
Billerica, MA, and [3H]DAGO (58.0 Ci/mmol) was obtained
from Amersham, Arlington Heights, IL, for use in δ- and
µ-opioid receptor binding assays, respectively.
1,4-Bis(Nr-Boc-Dm t-a m in o)bu ta n e. BOP reagent (700
mg, 1.6 mmol) was added to a solution of NR- Boc-Dmt-OH
(500 mg, 1.6 mmol) and 1,4-diaminobutane (60 mg, 0.68 mmol)
in DMF (15 mL) containing Et3N (0.66 mL, 4.7 mmol) at room
temperature. The reaction mixture was stirred for 18 h at room
temperature. After removal of the solvent, the residue was
extracted with AcOEt, which was washed with 10% citric acid,
5% Na2CO3, and water, dried over Na2SO4, and evaporated
down. Petroleum ether was added to the residue to obtain a
precipitate, which was collected by filtration. The crude
product in CHCl3 (5 mL) was applied to a silica gel column
(BW-127ZH, 3 cm × 16 cm), which was equilibrated, and
eluted with CHCl3 (2100 mL). After removal of the solvent of
the effluent (1500-2100 mL), petroleum ether was added to
the residue to yield crystals, which were collected by filtra-
F igu r e 4. Low-energy conformation of H-Dmt-NH-(CH2)4-NH-
Dmt-H (7). Molecular modeling was performed on a Silicon
Graphics Octane2 computer system using software from
Accelrys described previously.34 Extensive conformational
searching was performed utilizing conformational constraints
based on 1H NMR data for H-Dmt-NH-(CH2)4-NH-Tyr-H (6).
Minimizations were performed in the AMBER force field,
stopping with a root-mean-square gradient of 0.001 kcal/(mol‚
Å2). A total of 8232 conformations were analyzed with relative
energies ranging from 59 to 443 kcal/mol. The lowest energy
conformer is displayed with carbon atoms in green, oxygen
atoms in red, and nitrogen atoms in blue. Hydrogen atoms are
not displayed.
tion: yield 300 mg (58.4%); mp 214-217 °C; Rf1 ) 0.46, Rf2
)
0.32. Anal. Calcd for C36H54N4O8‚0.5H2O: C, 63.6; H, 8.15; N,
8.24. Found: C, 63.8; H, 8.16; N, 8.04.
1,4-Bis(Dm t-a m in o)bu ta n e‚2HCl (7). A solution of 1,4-
bis-(NR- Boc -Dmt-amino)butane (200 mg, 0.30 mmol) in TFA
(1.0 mL, 13 mmol) containing anisole (0.10 mL, 0.90 mmol)
was stirred for 1 h at room temperature. Ether was added to
a solution to form a precipitate, which was collected by
filtration and dried in vacuo. The solution of the product in 1
M HCl (0.6 mL) was lyophilized to give a fluffy powder: yield
110 mg (68%); Rf4 ) 0.19, Rf5 ) 0.32, tR(C) ) 14.97 min. TOF-
MS m/z: calcd for C26H38N4O4 (M + H)+ 471.6; found 471.9.
1H NMR (DMSO-d6) δ: 7.78 (2H, t, J ) 5.5 Hz, -CO-NH-),
6.43 (4H, s, aromatic 3,5-H), 3.69 (2H, dd, J ) 11.1, 4.6 Hz,
R-H), [3.02 (2H, dd, J ) 13.7, 11.1 Hz) and 2.89 (2H, dd, J )
13.7, 4.6 Hz), â-H2], [2.94 (2H, dd, J ) 12.7, 6.2 Hz) and 2.89
(2H, m), NH-CH2-], 2.18 (12H, s, 3,6-CH3), 0.95-0.85 (4H,
m, NH-CH2-CH2-). 13C NMR (DMSO-d6) δ: 167.9 (q, >Cd
O), 155.3 (q, aromatic C-4), 138.3 (q, aromatic C-2,6), 122.1
(q, aromatic C-1), 114.9 (t, aromatic C-3,5), 51.8 (t, C-R), 38.1
and increase the therapeutic potential of these deriva-
tives for human or veterinary applications.
Exp er im en ta l Section
Ma ter ia ls. The amino acid Dmt was synthesized according
to the procedure of Dygos et al. in this laboratory.38 [3H]-
DPDPE (32.0 Ci/mmol) was purchased from NEN-DuPont,

3206 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Okada et al.
(s, NH-CH2-) 30.4 (s, C-â), 25.1 (s, NH-CH2-CH2-), 19.7 (p,
1-Nr-Boc-Dm t -a m in o-4-Nr-F m oc-Tyr (Br Z)-a m in ob u -
ta n e. NR-Boc-Dmt-OH (550 mg, 1.80 mmol), 1-amono-4-NR-
Fmoc-Tyr(BrZ)-aminobutane‚TFA [prepared from Boc-amino-
4-NR-Fmoc-Tyr(BrZ)-aminobutane (1.2 g, 1.5 mmol), anisole
(0.25 mL, 2.3 mmol), and TFA (2.2 mL, 30 mmol) as usual],
and PyBOP (1.12 g, 2.2 mmol) were dissolved in DMF (20 mL)
containing DIEA (0.88 mL, 5.1 mmol). The reaction mixture
was stirred for 15 h at room temperature. After removal of
the solvent, the residue was extracted with AcOEt, which was
washed with 10% citric acid, 5% Na2CO3, and water, dried over
Na2SO4, and evaporated down. Ether was added to the residue
to yield crystals, which were collected by filtration and
recrystallized from EtOH: yield 1.1 g (63%); mp 186-190 °C;
Rf1 ) 0.77. Anal. Calcd for C52H57N4O10Br: C, 63.7; H, 5.87;
N, 5.73. Found: C, 63.6; N, 5.84; N, 5.50.
1-(Dm t-a m in o)-4-Tyr -a m in obu ta n e‚2HCl (6). 1-NR-Boc-
Dmt-amino-4-NR-Fmoc-Tyr(BrZ)-aminobutane (600 mg, 0.61
mmol) was prepared according to the procedure for 5 (vide
supra) with a 100 mg (32%) yield of a white fluffy amorphous
powder: Rf4 ) 0.20, Rf5 ) 0.36, tR(C) ) 15.78 min. TOF-MS
m/z: calcd for C24H34N4O4 (M + H)+ 443.5; found 443.8. 1H
NMR (free compound, DMSO-d6) δ: 8.541 (1H, t, J ) 5.4 Hz,
NH of Tyr amide), 7.972 (1H, t, J ) 5.4 Hz, NH of Dmt amide),
7.046 (2H, d-like, J ) 8.5 Hz, 2,6-H of Tyr), 6.717 (2H, d-like,
J ) 8 5 Hz, 3,5-H of Tyr), 6.434 (2H, s, 3,5-H of Dmt), 3.923
(1H, t, J ) 7.0 Hz, R-H of Tyr), 3.701 (1H, dd, J ) 10.6 Hz,
R-H of Dmt), 3.02-2.79 (8H, m, 1,4-CH2 + â-CH2 of Tyr and
Dmt), 2.184 (6H, s, diMe of Dmt), 1.18-1.03 (4H, m, 2,3-CH2).
13C NMR (DMSO-d6) δ: 167.91 (q, >CdO of Dmt), 167.63 (q,
>CdO of Tyr), 156.40 (q, >Cd, 4 of Tyr), 155.52 (q, >Cd, 4 of
Dmt), 138.16 (q, >Cd, 2,6 of Dmt), 130.31 (t, H-Cd, 2,6 of
Tyr), 124.89 (q, >Cd, 1 of Tyr), 122.08 (q, 1 of Dmt), 115.15
(t, H-Cd, 3,5 of Tyr), 114.81 (t, H-Cd, 3,5 of Dmt), 53.69 (t,
H-C<, R of Tyr), 51.67 (t, H-C<, R of Dmt), 38.11 (s, -CH2-,
1 or 4 CH2), 38.05 (s, -CH2-, 4 or 1 CH2), 36.12 (s, â of Tyr),
30.45 (s, â of Dmt), 25.56 (s, -CH2-, 2 or 3 CH2), 25.52 (s,
-CH2-, 3 or 2 CH2), 19.88 (p, -CH3, diMe of Dmt).
1,4-Bis(Nr-Boc-P h e-a m in o)bu ta n e. NR-Boc-Phe-OH (1.1
g, 4.0 mmol), 1,4-diaminobutane (180 mg, 2.0 mmol), and
PyBOP (2.5 g, 4.8 mmol) were dissolved in DMF (25 mL)
containing Et3N (0.67 mL, 4.8 mmol). The reaction mixture
was stirred for 15 h at room temperature. After removal of
the solvent, AcOEt and water were added to the residue to
obtain crystals, which were collected by filtration and recrys-
tallized from EtOH: yield 0.75 g (32%); mp 186-189 °C; Rf1
) 0.65, Rf2 ) 0.45. Anal. Calcd for C32H46N4O6: C, 66.0; H,
7.96; N, 9.62. Found: C, 65.9; H, 7.75; N, 9.61.
1,4-Bis(P h e-a m in o)bu t a n e‚2HCl (4). A solution of 1,4-
bis(NR-Boc-Phe-amino)butane (580 mg, 1.0 mmol) and anisole
(0.15 mL, 1.4 mmol) in TFA (1.5 mL, 20 mmol) was stirred for
1 h at room temperature. Ether was added to the solution to
form a precipitate, which was collected by filtration. The crude
product in 3% AcOH (3 mL) was applied to a Sephadex G-15
column (2.5 cm × 43 cm), which was equilibrated, and eluted
with 3% AcOH. Fractions (8 g each) were collected, and the
solvent of the effluent (nos. 8-11) was removed. The residue
was lyophilized from 1 M HCl to yield an amorphous powder:
yield 340 mg (75%), Rf4 ) 0.27, Rf5 ) 0.47, tR(C) ) 21.04 min.
TOF-MS m/z: calcd for C22H30N4O2 (M + H)+ 383.4; found
383.3.
2,6-CH3).
1-Boc-a m in o-4-(Nr-F m oc-P h e-a m in o)b u t a n e. 1-(Boc-
amino)-4-aminobutane3 (2.0 g, 10 mmol), NR-Fmoc-Phe-OH (4.6
g, 12 mmol), PyBOP (6.24 g, 10 mmol), and HOBt (1.6 g, 12
mmol) were dissolved in DMF (50 mL) containing DIEA (4.2
mL, 24 mmol). The reaction mixture was stirred for 15 h at
room temperature. After removal of the solvent, AcOEt was
added to the residue to obtain a precipitate, which was
collected by filtration and recrystallized from EtOH: yield 3.0
g (45%); mp 165-167 °C; Rf1 ) 0.66. Anal. Calcd for C33H39N3O5‚
0.25H2O: C, 70.5; H, 7.08; N, 7.47. Found: C, 70.5; H, 7.06;
N, 7.64.
1-Nr-Boc-Dm t-am in o-4-(Nr-Fm oc-P h e-am in o)bu tan e. NR-
Boc-Dmt-OH (460 mg, 1.50 mmol), 1-amino-4-(NR-Fmoc-Phe-
amino)butane‚TFA [prepared from 1-Boc-amino-4-(NR-Fmoc-
Phe-amino)butane (830 mg, 1.50 mmol), anisole (0.25 mL, 2.3
mmol), and TFA (2.2 mL, 30 mmol) as usual], PyBOP (930
mg, 1.8 mmol), and HOBt (270 mg, 1.8 mmol) were dissolved
in DMF (10 mL) containing DIEA (0.50 mL, 3.80 mmol). The
reaction mixture was stirred for 15 h at room temperature.
After removal of the solvent, the residue was extracted with
AcOEt, which was washed with 10% citric acid, 5% Na2CO3,
and water, dried over Na2SO4, and evaporated down. Petro-
leum ether was added to the residue to yield a precipitate.
The crude product in CHCl3 (5 mL) was applied to a silica gel
column (YMC 70-230 mesh, 3 cm × 16 cm), which was
equilibrated, and eluted with CHCl3. After removal of the
solvent of the effluent (210 mL), petroleum ether was added
to the residue to give crystals, which were collected by
filtration: yield 600 mg (63%); mp 201-203.5 °C; Rf1 ) 0.52.
Anal. Calcd for C44H52N4O7‚0.3H2O: C, 70.0; H, 7.03; N, 7.42.
Found: C, 70.0; H, 7.24; N, 7.48.
1-Dm t-a m in o-4-P h e-a m in obu ta n e‚2HCl (5). 1-NR-Boc-
Dmt-amino-4-(NR-Fmoc-Phe-amino)butane (500 mg, 0.78 mmol)
was treated with 20% piperidine in DMF (11.5 mL) for 2 h at
room temperature. After removal of the solvent, ether was
added to the residue to obtain a precipitate, which was
collected by filtration (Rf1 ) 0.16, Rf3 ) 0.70). This product
(300 mg, 0.57 mmol) was dissolved in TFA (1.0 mL, 13 mmol)
containing anisole (0.10 mL, 0.90 mmol), and the solution was
stirred for 1 h at room temperature. Ether was added to the
solution to give a precipitate, which was collected by filtration.
The crude product was purified with HPLC and lyophilized
from 1 M HCl to obtain a fluffy amorphous powder: yield 220
mg (56%); Rf4 ) 0.30, Rf5 ) 0.46, tR(C) ) 17.07 min. TOF-MS
m/z: calcd for C24H34N4O3 (M + H)+ 427.6; found 427.5. 1H
NMR (free compound, DMSO-d6) δ: 8.648 (1H, t, J ) 5.5 Hz,
NH of Phe amide), 7.997 (1H, t, J ) 5.5 Hz, NH of Dmt amide),
7.32-7.23 (5H, m, aromatic H of Phe), 6.441 (2H, s, aromatic
H of Dmt), 4.043 (1H, t, J ) 7.0 Hz, R-H of Phe), 3.711 (1H,
dd, J ) 9.3, 6.4 Hz, R-H of Dmt), 3.071 (2H, d, J ) 7.0 Hz,
â-CH2 of Phe), 3.01-2.70 (6H, m, 1,4-CH2 + â-CH2 of Dmt),
2.187 (6H, s, diMe of Dmt), 1.16-1.01 (4H, m, 2,3-CH2). 13C
NMR (DMSO-d6) δ: 167.91 (q, >CdO of Dmt), 167.52 (q, >Cd
O of Tyr), 155.54 (q, >Cd, 4 of Dmt), 138.14 (q, >Cd, 2,6 of
Dmt), 135.10 (t, >Cd, 1 of Phe), 129.42 (t, H-Cd, 2,6 of Phe),
128.25 (t, H-Cd, 3,5 of Phe), 126.84 (t, H-Cd, 4 of Phe),
122.08 (q, >Cd, 1 of Dmt), 114.80 (t, H-Cd, 3, 5 of Dmt), 53.39
(t, H-C<, R of Phe), 51.65 (t, H-C<, R of Dmt), 38.09 (s,
-CH2-, 1 or 4 CH2), 38.05 (s, -CH2-, 4 or 1 CH2), 36.83 (s, â
of Phe), 30.44 (s, â of Dmt), 25.52 (s, -CH2-, 2 or 3 CH2), 25.48
(s, -CH2-, 3 or 2 CH2), 19.90 (p, -CH3, diMe of Dmt).
1,4-Bis(Nr-Boc-Tyr -a m in o)bu ta n e. NR-Boc-Tyr-OH (5.6 g,
20 mmol), 1,4-diaminobutane (880 mg, 10 mmol), BOP (10.6
g, 24 mmol), and HOBt (3.0 g, 20 mmol) were dissolved in DMF
(50 mL) containing Et3N (5.6 mL, 40 mmol). The reaction
mixture was stirred for 15 h at room temperature. After
removal of the solvent, the residue was extracted with AcOEt,
which was washed with 5% Na2CO3 and water, dried over Na2-
SO4, and evaporated down. Ether was added to the residue to
form a precipitate, which was collected by filtration: yield 4.2
g (68%); mp 123-126 °C; Rf1 ) 0.40. Anal. Calcd for C32H46N4O8‚
0.7H2O: C, 61.3; H, 7.56; N, 8.93. Found: C, 61.4. H, 7.81; N,
8.78.
1-Boc-a m in o-4-Nr-F m oc-Tyr (Br Z)-a m in obu ta n e. 1-Boc-
amino-4-aminobutane (0.94 g, 5 mmol), NR-Fmoc-Tyr(BrZ)-OH
(3.1 g, 12 mmol), and PyBOP (6.24 g, 12 mmol) were dissolved
in DMF (30 mL) containing DIEA (1.7 mL, 10 mmol). The
reaction mixture was stirred for 15 h at room temperature.
After removal of the solvent, AcOEt and 5% Na2CO3 were
added to the residue to give crystals, which were collected by
filtration and recrystallized from EtOH: yield 3.1 g (80%); mp
145-148 °C; Rf1 ) 0.80, Rf2 ) 0.80. Anal. Calcd for C41H44N3O8-
Br: C, 62.6; H, 5.63; N, 5.34. Found: C, 62.6; H, 5.65; N, 5.25.
1,4-Bis(Tyr -a m in o)bu ta n e‚2HCl (3). A solution of 1,4-bis-
(NR-Boc-Tyr-amino)butane (550 mg, 0.90 mmol) and anisole

µ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15 3207
(0.28 mL, 2.6 mmol) in TFA (2.75 mL, 36 mmol) was stirred
for 1 h at room temperature. Ether was added to the solution
to form a precipitate, which was collected by filtration and
lyophilized from 1 M HCl: yield 340 mg (70%), Rf4 ) 0.27,
tR(C) ) 7.1 min. TOF-MS m/z: calcd for C22H30N4O4 (M + H)+
414.4; found 414.1.
Anal. Calcd for C34H50N4O8‚0.5H2O: C, 62.7; H, 7.88; N, 8.59.
Found: C, 62.7; H, 7.80; N, 8.59.
1,2-Bis(Dm t-a m in o)eth a n e‚2TF A (2). A solution of 1,2-
bis(NR-Boc-Dmt-amino)ethane (200 mg, 0.30 mmol) and anisole
(0.1 mL, 0.90 mmol) in TFA (1.0 mL, 13 mmol) was stirred for
1 h at room temperature. Ether was added to the solution to
form a precipitate, which was collected by filtration, dried in
1,6-Bis(Nr-Boc-Dm t-a m in o)h exa n e. NR-Boc-Dmt-OH (500
mg, 1.6 mmol), 1,6-diaminohexane (92 mg, 0.80 mmol), and
PyBOP (1.0 g, 1.9 mmol) were dissolved in DMF (15 mL)
containing Et3N (0.27 mL, 1.9 mmol). The reaction mixture
was stirred for 18 h at room temperature. After removal of
the solvent, the residue was extracted with AcOEt, which was
washed with 10% citric acid, 5% Na2CO3, and water, dried over
Na2SO4, and evaporated down. Petroleum ether was added to
the residue to give a precipitate, which was collected by
filtration. The crude product in AcOEt/n-hexane (1:1, 5 mL)
was applied to a silica gel column (BW-127ZH, 3 cm × 16 cm),
which was equilibrated, and eluted with AcOEt/n-hexane (1:
1, 300 mL). After removal of the solvent of the effluent (1-
200 mL), petroleum ether was added to the residue to form a
precipitate, which was collected by filtration: yield 300 mg
(53%); mp 183-186 °C; Rf1 ) 0.52, Rf2 ) 0.08. Anal. Calcd for
C38H58N4O8‚0.25H2O: C, 64.9; H, 8.38; N, 7.96. Found: C, 65.0;
H, 8.22; N, 7.78.
vacuo, and lyophilized from water: yield 120 mg (60%), Rf4
)
0.14, Rf5 ) 0.58, tR(C) ) 13.8 min. TOF-MS m/z: calcd for
C
24H34N4O4 (M + H)+ 443.5; found 443.6.
1,2-Bis(Nr-Boc-Tyr -a m in o)eth a n e. NR-Boc-Tyr-OH (4.1 g,
15 mmol), 1,2-diaminoethane (0.36 g, 6.0 mmol), and BOP (7.6
g, 17 mmol) were dissolved in DMF (40 mL) containing Et3N
(2.4 mL, 17 mmol). The reaction mixture was stirred for 15 h
at room temperature. After removal of the solvent, the residue
was extracted with AcOEt, which was washed with 5% Na2-
CO3 and water, dried over Na2SO4, and concentrated. Petro-
leum ether was added to the residue to form a precipitate. The
crude product in CHCl3 (5 mL) was applied to a silica gel
column (BW-127ZH, 3 cm × 34 cm), which was equilibrated,
and eluted with CHCl3. After removal of the solvent of the
effluent (300-2300 mL), petroleum ether was added to the
residue to obtain a precipitate: yield 1.0 g (30%); mp 203-
206 °C; Rf1 ) 0.49. Anal. Calcd for C30H42N4O8‚0.7H2O: C, 60.1;
H, 7.29; N, 9.34. Found: C, 60.2; H, 6.96; N, 9.01.
1,6-Bis(Dm t-a m in o)h exa n e‚2HCl (9). A solution of 1,6-
bis(NR-Boc-Dmt-amino)hexane (150 mg, 0.21 mmol) and ani-
sole (0.10 mL, 0.90 mmol) in TFA (1.0 mL, 13 mmol) was
stirred for 1 h at room temperature. Ether was added to the
solution to form a precipitate, which was collected by filtration,
dried in vacuo, and lyophilized from 1 M HCl: yield 90 mg
(75%), Rf4 ) 0.21, Rf5 ) 0.42, tR(C) ) 18.15 min. TOF-MS m/z:
calcd for C28H42N4O4 (M + H)+ 499.6; found 499.2. 1H NMR
(DMSO-d6) δ: 7.96 (2H, t, J ) 5.5 Hz, CO-NH-), 6.40 (4H, s,
aromatic 3,5-H), 3.70 (2H, dd, J ) 10.7, 5.0 Hz, R-H), [3.00
(2H, m) and 2.81 (2H, m) NH-CH2-], [2.98 (2H, dd, J ) 13.9,
10.8 Hz) and 2.93 (2H, dd, J ) 13.9, 5.0 Hz), â-H2], 2.18 (12H,
s, 2,6-CH3), 1.13 (4H, m, NH-CH2-CH2-), 0.90 (4H, m, NH-
CH2-CH2-CH2-). 13C NMR (DMSO-d6) δ: 167.8 (q, >CdO),
155.5 (q, aromatic C-4), 138.1 (q, aromatic C-2,6), 122.0 (q,
aromatic C-1), 114.8 (t, aromatic C-3,5), 51.6 (t, C-R), 38.5 (s,
NH-CH2-), 30.5 (s, C-â), 28.3 (s, NH-CH2-CH2-), 25.7 (s,
NH-CH2-CH2-CH2-), 19.9 (p, 2,6-CH3).
1,6-Bis(Nr-Boc-Tyr -a m in o)h exa n e. NR-Boc-Tyr-OH (5.6
g, 20 mmol), 1,6-diaminohexane (1.9 g, 10 mmol), BOP (11 g,
24 mmol), and HOBt (3.0 g, 20 mmol) were dissolved in DMF
(50 mL) containing Et3N (5.6 mL, 40 mmol). The reaction
mixture was stirred for 15 h at room temperature. After
removal of the solvent, the residue was extracted with AcOEt,
which was washed with 5% Na2CO3 and water, dried over Na2-
SO4, and evaporated down. Ether was added to the residue to
form a precipitate, which was collected by filtration: yield 5.0
g (79%); mp 146-151 °C; Rf1 ) 0.56. Anal. Calcd for C34H50N4O8‚
2.5H2O: C, 59.4; H, 8.06; N, 8.15. Found: C, 59.2; H, 7.84; N,
8.11.
1,2-Bis(Tyr -a m in o)eth a n e‚2TF A (1). A solution of 1,2-bis-
(NR-Boc-Tyr-amino)ethane (500 mg, 0.90 mmol) and anisole
(0.14 mL, 1.3 mmol) in TFA (1.4 mL, 18 mmol) was stirred for
1 h at room temperature. Ether was added to the solution to
form a precipitate. The precipitate was purified with HPLC
and lyophilized from water: yield 350 mg (63%), Rf4 ) 0.30,
tR(C) ) 5.5 min. TOF-MS m/z: calcd for C20H26N4O4 (M + H)+
387.4; found 387.7.
1,8-Bis(Nr-Boc-Dm t-a m in o)octa n e. NR-Boc-Dmt-OH (500
mg, 1.6 mmol), 1,8-diaminooctane (120 mg, 1.80 mmol), BOP
(840 mg, 1.9 mmol), and HOBt (260 mg, 1.9 mmol) were
dissolved in DMF (15 mLl) containing Et3N (0.54 mLl, 3.8
mmol). The reaction mixture was stirred for 18 h at room
temperature. After removal of the solvent, the residue was
extracted with AcOEt, which was washed with 10% citric acid,
5% Na2CO3, and water, dried over Na2SO4, and concentrated.
Petroleum ether was added to the residue to give crystals,
which were collected by filtration and recrystallized from
AcOEt: yield 310 mg (53%); mp 142-144 °C; Rf1 ) 0.56. Anal.
Calcd for C40H62N4O8‚0.5H2O: C, 65.3; 8.62; N, 7.61. Found:
C, 65.2; H, 8.40; N, 7.68.
1,8-Bis(Dm t-a m in o)octa n e‚2TF A (11). A solution of 1,8-
bis(NR-Boc-Dmt-amino)octane (260 mg, 1.6 mmol) and anisole
(0.1 mL, 0.90 mmol) in TFA (1.0 mL, 13 mmol) was stirred for
1 h at room temperature. Ether was added to the solution to
form a precipitate, which was collected by filtration, dried in
vacuo, and lyophilized from water: yield 120 mg (60%), Rf4
)
0.56, Rf5 ) 0.68, tR(C) ) 20.7 min. TOF-MS m/z: calcd for
C
30H46N4O4 (M + H)+ 527.7; found 527.7.
1,8-Bis(Nr-Boc-Tyr -a m in o)octa n e. NR-Boc-Tyr-OH (5.6 g,
1,6-Bis(Tyr -a m in o)h exa n e‚2TF A (8). A solution of 1,6-
bis(NR-Boc-Tyr-amino)hexane (500 mg, 1.6 mmol) and anisole
(0.12 mL, 1.1 mmol) in TFA (1.2 mL, 16 mmol) was stirred for
1 h at room temperature. Ether was added to the solution to
form a precipitate, which was collected by filtration, dried in
20 mmol), 1,8-diaminooctane (1.4 g, 10 mmol), and BOP (11
g, 24 mmol) were dissolved in DMF (50 mL) containing Et3N
(3.4 mL, 24 mmol). The reaction mixture was stirred for 15 h
at room temperature. After removal of the solvent, the residue
was extracted with AcOEt, which was washed with 5% Na2-
CO3 and water, dried over Na2SO4, and concentrated. Petro-
leum ether was added to the residue to form a precipitate,
which was collected by filtration: yield 6.1 g (91%); mp 159-
160 °C; Rf4 ) 0.60. Anal. Calcd for C36H54N4O8‚0.5H2O: C, 63.6;
H, 8.09; N, 8.23. Found: C, 63.9; H, 8.23; N, 7.97.
vacuo, and lyophilized from water: yield 250 mg (48%), Rf4
)
0.31, tR(Y) ) 20.1 min. TOF-MS m/z: calcd for C24H34N4O4 (M
+ H)+ 442.5; found 443.1.
1,2-Bis(Nr-Boc-Dm t-a m in o)eth a n e. NR-Boc-Dmt-OH (500
mg, 1.6 mmol), 1,2-diaminoethane (50 µL, 0.80 mmol), BOP
(840 mg, 1.9 mmol), and HOBt (260 mg, 1.9 mmol) were
dissolved in DMF (15 mL) containing Et3N (0.54 mL, 3.8
mmol). The reaction mixture was stirred for 18 h at room
temperature. After removal of the solvent, the residue was
extracted with AcOEt, which was washed with 10% citric acid,
5% Na2CO3, and water, dried over Na2SO4, and evaporated
down. Petroleum ether was added to the residue to yield
crystals, which were collected by filtration and recrystallized
from EtOH: yield 300 mg (58%); mp 201-205 °C; Rf1 ) 0.48.
1,8-Bis(Tyr -a m in o)octa n e‚2TF A (10). A solution of 1,8-
bis(NR-Boc-Tyr-amino)octane (1.0 g, 1.5 mmol) and anisole
(0.45 mL, 4.2 mmol) in TFA (4.5 mL, 60 mmol) was stirred for
1 h at room temperature. Ether was added to the solution to
form
a precipitate, which was purified with HPLC and
lyophilized from water: yield 320 mg (30%), Rf4 ) 0.30, tR(C)
) 18.3 min. TOF-MS m/z: calcd for C26H38N4O4 (M + H)+
471.6; found 471.2.

3208 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 15
Okada et al.
Biologica l Activity in Isola ted Tissu e P r ep a r a tion s.
Preparations of the myenteric plexus longitudinal muscle were
obtained from the small intestine of guinea pigs (GPI) for
µ-opioid agonism; mouse vas deferens (MVD contains δ-opioid
receptors. Both tissues were used for field stimulation with
bipolar rectangular pulses of supramaximal voltage. Agonists
were tested for their inhibition of the electrically evoked twitch,
and the results are expressed as the IC50 values obtained from
concentration-response curves. The IC50 values represent the
mean ( SE of five to six separate assays. [D-Ala2]deltorphin I
and dermorphin were used as internal standards with MVD
and GPI, respectively.
(2 µM) established nonspecific binding. Labeled membranes
were rapidly filtered on Whatman GF/C glass fiber filters,
washed three times with 2 mL amounts, and dried at 75 °C
for 60 min. The radioactivity was determined using CytoScint
(ICN). All analogues were analyzed in duplicate using five to
eight dosages and three to five independent repetitions;
different synaptosomal preparations were frequently used to
ensure statistical significance (n values are listed in Table 1
in parentheses and results are mean ( SE) The affinity
constants (Ki) were calculated according to Cheng and Pru-
soff.39
Molecu lar Modelin g. Low-energy conformations of H-Dmt-
NH-(CH2)4-NH-Dmt-H (7) were generated using a Silicon
Graphics Octane2 computer system and Insight II software
(version 98) from Accelrys. Twenty-four structures were gener-
ated on the basis of 1H NMR data and NOE cross-peaks
(Figure 3b) for H-Dmt-NH-(CH2)4-NH-Tyr-H (6) from which
the values of 29.9° and 190.5° for φ1 and φ2, 47.9°, 120.1°, and
Deter m in a tion of An a lgesia . Spinal analgesia of H-Dmt-
NH-(CH2)4-NH-Dmt-H (7) was determined using the tail-flick
test in mice (Figure 2). Radiant heat was applied on the dorsal
surface of the tail using a tail-flick apparatus (Columbus
Instruments, Columbus, OH). Baseline tail withdrawal latency
was adjusted between 2 and 3 s, and a cutoff time was set at
8 s to avoid external heat-related damage. The analgesic
response was measured after 10 or 15 min following intrac-
erebroventricular (icv) or subcutaneous (sc) injections, respec-
tively, and groups of five to seven animals were tested at each
time point. The dose-dependent appearance of analgesia
following icv administration of 7 is expressed as the time
course of its appearance (a) and area under the curve (AUC)
(b). The AUC was obtained by plotting the response time (s)
on the ordinate and time (min) on the abscissa after admin-
istration of 7. Mice were injected icv with either saline ([) or
0.03 nmol (0), 0.1 nmol (2), 0.3 nmol (O), or 1 nmol (9) of 7 in
4 µL of saline per mouse. (c) The reversal by the µ-opioid
antagonist naloxone (2 mg/kg sc) on analgesia induced by 7
was observed by injection 30 min before 7. (d) The sc injection
of 7 is expressed as the time course of action and AUC (e).
Mice were injected either with saline ([) or with 10 mg/kg
(0), 30 mg/kg (2), 100 mg/kg (O) of 7 in saline, or 3 mg/kg
morphine (0). Male Swiss-Webster mice weighing 20-25 g
(Taconic, NC) were used throughtout. All data are reported
as the mean ( SE. Statistical significance was determined by
Student’s t-test. Asterisks denote significant differences be-
tween mice treated with saline and 7 (/, p < 0.05; //, p < 0.01;
///, p < 0.001).
Methods for the determination of analgesia reported in
Figure 2 for the hot plate test, which is a measure of
supraspinal analgesia of H-Dmt-NH-(CH2)4-NH-Dmt-H (7) in
mice. The animals were placed on an electrically heated plate
(IITC model 39D hot plate analgesia meter, IITC Inc., Wood-
land Hills, CA) at 55 ( 0.1 °C after 10 or 15 min following icv
administration or sc injection, respectively. The response time
was measured by observing movements consisting of jumping,
licking, or shaking their hind paws with a baseline latency of
15 s and maximal cutoff time of 30 s. Groups of five to seven
mice were tested at each time point. Analgesia of 7 after icv
administration is expressed as a time course (a) and AUC (b).
Mice were injected either with saline ([) or with 0.03 nmol
(0), 0.1 nmol (2), 0.3 nmol (O), or 1 nmol (9) of 7 in 4 µL of
saline per mouse. (c) Naloxone (2 mg/kg sc) was injected 30
min before icv administration of 7. (d) The sc analgesic effect
of 7 is expressed as a time course and AUC (e). Mice were
injected either with saline ([) or with 10 mg/kg (0), 30 mg/kg
(2), 60 mg/kg (O) of 7, or 3 mg/kg morphine (0). The mice
strain and statistical analyses used are given in the caption
to Figure 2.
(2)
151.2° for ø1(1), and 21.9° and 139.4° for ø1 were derived.
Extensive conformational searching was performed utilizing
constraints on φ1, φ2, ø1(1), and ø1(2) and rotating the remaining
amino acid and carbon-chain bonds in 60° and 180° increments
for the peptide bonds within the range of 0-360°. The energy
threshold was set at 10.0 kcal/mol with a derivative of 0.01
kcal/(mol‚Å), the energy tolerance was set at 1.0 kcal/mol, and
the rms was set at 1.0 Å. Minimizations were performed in
the AMBER force field, stopping with a root-mean-square
gradient of 0.001 kcal/(mol‚Å2). On the basis of these param-
eters and computations, 343 conformations were generated for
each of the 24 starting structures and a total 8232 conforma-
tions were analyzed with relative energies ranging from 59 to
443 kcal/mol.
Ack n ow led gm en t. This work was supported in part
by a Grant-in-Aid for Scientific Research from J apan
Society for the Promotion of Science (Grant C-11694326).
The authors appreciate the critical comments by the
anonymous reviewers.
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