Deltorphin II analogues with 6-hydroxy-2-aminotetralin-2-carboxylic acid in position 1

Article abstract of DOI:10.1021/jm9911534

Two approaches to the design of very active and highly selective δ opioid peptides were used to obtain new deltorphin analogues with altered hydrophobic and stereoelectronic properties. Deltorphin II analogues were synthesized with the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 6-hydroxy-2-aminotetralin-2-carboxylic acid (Hat) instead of Tyr¹ in the message domain. In the radioreceptor-binding studies, in which type-specific tritiated opioid ligands were used, (R)- and (S)-Hat- deltorphins exhibited similar K(i) values, revealing high δ selectivity. The peptides displayed agonist properties in the in vitro bioassay, with IC₅₀ values in the subnanomolar range in the mouse vas deferens assay and in the micromolar or higher range in the guinea pig ileum assay, again demonstrating a high selectivity toward δ receptors. The agonist property of the new ligands was confirmed by means of [³⁵S]GTPγS-binding experiments in membranes of the rat frontal cortex.

Full text of DOI:10.1021/jm9911534

J . Med. Chem. 2000, 43, 1359-1366
1359
Deltor p h in II An a logu es w ith 6-Hyd r oxy-2-a m in otetr a lin -2-ca r boxylic Acid in
P osition 1
Zsuzsanna Darula,† Katalin E. Ko¨ve´r,# Krisztina Monory,† Anna Borsodi,† EÄ va Mako´,§ Andra´s Ro´nai,§
Dirk Tourwe´,| Antal Pe´ter,‡ and Ge´za To´th*,†
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary,
Department of Organic Chemistry, Lajos Kossuth University, P.O. Box 20, H-4010 Debrecen, Hungary, Department of
Pharmacology, Semmelweis University of Medicine, P.O. Box 370, H-1445 Budapest, Hungary, Eenheid Organische Chemie,
Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium, and Department of Inorganic and Analytical Chemistry,
Attila J o´zsef University, P.O. Box 440, H-6701 Szeged, Hungary
Received August 25, 1999
Two approaches to the design of very active and highly selective δ opioid peptides were used
to obtain new deltorphin analogues with altered hydrophobic and stereoelectronic properties.
Deltorphin II analogues were synthesized with the substitution of Ile instead of Val at positions
5 and 6 in the address domain and 6-hydroxy-2-aminotetralin-2-carboxylic acid (Hat) instead
of Tyr1 in the message domain. In the radioreceptor-binding studies, in which type-specific
tritiated opioid ligands were used, (R)- and (S)-Hat-deltorphins exhibited similar Ki values,
revealing high δ selectivity. The peptides displayed agonist properties in the in vitro bioassay,
with IC50 values in the subnanomolar range in the mouse vas deferens assay and in the
micromolar or higher range in the guinea pig ileum assay, again demonstrating a high selectivity
toward δ receptors. The agonist property of the new ligands was confirmed by means of [35S]-
GTPγS-binding experiments in membranes of the rat frontal cortex.
In tr od u ction 1
dan-2-carboxylic acid (Aic),7,8 and 4-aminotetrahydro-
2-benzazepin-3-one9 were used to investigate the effects
of the Phe3 side-chain conformation on the binding
properties of deltorphins. Especially the Atc and Aic
analogues displayed extraordinary δ receptor affinities
and selectivities.
The incorporation of conformational constraints into
biologically active peptides is a well-known approach for
enhancing their receptor selectivity and modulating
their efficacy. This approach has been especially suc-
cessful in the opioid peptide area, where the conforma-
tion of the small, structurally flexible natural ligands
is strongly dependent on the environment. This is the
main reason these peptides lack significant selectivity
toward one or other opioid receptor types (µ, δ, κ) or
subtypes. To obtain better conformational integrity,
various conformationally restricted opioid peptide ana-
logues, agonists, and antagonists have been developed.2-4
The most potent and most selective δ agonists belong
in the deltorphin family. H-Tyr-D-Met-Phe-His-Leu-Met-
Asp-NH2 (dermenkephalin), H-Tyr-D-Ala-Phe-Asp-Val-
Val-Gly-NH2 (deltorphin I), and H-Tyr-D-Ala-Phe-Glu-
Val-Val-Gly-NH2 (deltorphin II) were isolated from frog
skin.5,6 As discussed below, structure-activity relation-
ship studies revealed the roles of the individual amino
acid residues in the receptor-binding abilities of the
ligands.
Systematic replacement of residue 4 in deltorphins I
and II revealed the great importance of this residue.
Although Asn-, Gln-,13,14 Ser-, and Cys15-substituted
deltorphin I or II analogues retained the high δ affinity
of the parent compounds, the selectivities of these
ligands were significantly lower. The location of the
charged group relative to the hydrophobic residues in
the address domain of the peptide is also critical for δ
affinity and selectivity.13 The role of this charged residue
is to prevent µ site recognition and/or binding16 or to
stabilize the appropriate conformation of the ligands for
δ receptors.17,18
Replacement of the Val residues in positions 5 and 6
by Ile in deltorphin II19 resulted in a more lipophilic
compound, with 8 times higher affinity and 5 times
better δ selectivity than that of the parent compound.
Conformational restriction of the Phe3 residue in
deltorphins has resulted in drastic effects on the affinity,
selectivity, and ability of the ligands to generate intra-
cellular responses. 1,2,3,4-Tetrahydroisoquinoline-3-car-
boxylic acid (Tic),7-9 different â-methylphenylalanines,10,11
2-aminotetralin-2-carboxylic acid (Atc),7,8,12 2-aminoin-
The Tyr moiety is one of the key pharmacophores of
opioid peptides. Replacement of L- with D-Tyr1 in der-
menkephalin resulted in a 1200-fold lower δ affinity.20
Replacement of Tyr1 in deltorphin I with Phe1 produced
a 32-fold decrease in δ receptor affinity, but only a 7-fold
drop in antinociceptive potency,21 which indicates that
the phenolic hydroxy group of Tyr contributes to the
effective interaction with the opioid receptor, but is not
an absolute requirement for opioid activity. The effect
of conformational restriction of the first residue has not
been investigated as widely as that of the Phe3 residue.
Qian et al.22 synthesized (2S,3S)-2′,6′-dimethyl-â-me-
thyltyrosine-substituted deltorphin I and found that the
* To whom correspondence should be addressed. Tel: +36 62
432080/189. Fax: +36 62 432576. E-mail: geza@nucleus.szbk.u-
szeged.hu.
† Hungarian Academy of Sciences.
#
Lajos Kossuth University.
§ Semmelweis University of Medicine.
| Vrije Universiteit Brussel.
‡ Attila J o´zsef University.
10.1021/jm9911534 CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/10/2000

1360 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Darula et al.
Ta ble 1. 1H Chemical Shifts (δ in ppm), Coupling Constants (J in Hz), and Temperature Coefficients of NH Protons (-ppb/K) for (S)-
and (R)-Hat1,Ile5,6-deltorphin II (T ) 300 K, DMSO-d6)
NH
Hr
Hâ
Hγ a
Hδ a
HAr
residue
(S)
(R)
(S)
(R)
(S)
(R)
(S)
2.78
(R)
2.74
(S) (R)
(S)
(R)
6.92
6.61
6.54
((S)/(R))-Hat1
3.29 â
2.75 â′
J ââ′ ) 17.0 J ââ′ ) 17.1
0.95 0.95
3.29 â
2.77 â′
2.25 2.31 6.91
1.88 1.94 6.59
6.54
2.71
D-Ala2
Phe3
Glu4
Ile5
8.09
8.07
4.35
4.34
J Râ ) 7.0
J NHR ) 8.2 J NHR ) 7.5 J Râ ) 7.2
(5.3)
8.17
NHR ) 8.7
(5.1)
8.29
NHR ) 7.9
(5.7)
7.90
NHR ) 8.7
(4.3)
7.98
(4.2)
8.18
4.60
4.61
J Râ ) 3.8
3.02 â (S) 3.05 â (S)
2.69 â′ (R) 2.72 â′ (R)
J ââ′ ) 13.7 J ââ′ ) 13.8
1.87 â
1.77 â′
7.1-7.3 7.1-7.3
J
J
NHR ) 8.7 J Râ ) 3.6
(4.4)
8.26
NHR ) 7.8 J Râ ) 6.2
(5.7)
7.88
NHR ) 8.9 J Râ ) 8.1
(4.2)
7.93
NHR ) 8.3 J Râ ) 7.8
(6.0)
8.05
J Râ′ ) 10.4 J Râ′ ) 9.9
4.32
4.34
J Râ ) 7.0
1.90 â
1.79 â′
2.25
2.26
J
J
J Râ′ ) 7.8
4.23
4.24
J Râ ) 7.5
1.72
1.74
1.44 γ
1.06 γ′
0.81 CH3 0.82 CH3
1.44 γ
1.09 γ′
0.81 CH3 0.82 CH3
1.45 γ
1.09 γ′
0.81 0.82
0.81 0.82
J
J
Ile6
4.12
4.13
J Râ ) 7.6
1.73
1.74
1.45 γ
1.10 γ′
J
NHR ) 8.2
(6.2)
8.09
J
Gly7
3.67 R
3.68 R
3.57 R′
J NHR ) 6.1 J NHR ) 6.2 3.57 R′
J NHR′ ) 5.5 J NHR′ ) 5.5 J RR′ ) 16.7 J RR′ ) 16.8
(6.0) (5.6)
a
For Hat, Cγ corresponds to C(4) and Cδ to C(3); see also Figure 1. Temperature coefficients of NH protons (-ppb/K) are given in
parentheses.
new analogue was 2 and 5 times less potent in the
Syn th esis
binding assay and in the MVD bioassay, respectively,
while retaining the high selectivity of the parent ligand.
Results of NMR studies suggested that the most popu-
lated side-chain conformation of the first residue is the
gauche(-). Guerrini et al.23 synthesized 2′,6′-dimethyl-
L-tyrosine-substituted deltorphin II, and the new ligand
was found to exhibit high dual affinity and bioactivity
toward δ and µ opioid receptors. Enhanced interaction
with the µ receptor was interpreted in terms of the
increased hydrophobicity of the first residue and/or the
possible changes in the peptide topography. It seems
that the degree of methylation of the Tyr1 affects the
conformation of the peptide and its ability to interact
differentially with δ and µ opioid receptors.
Hat, a conformationally restricted bicyclic Tyr ana-
logue, in which rotation about ø1 and ø2 is restricted,
was incorporated into enkephalin24,25 and DPDPE ana-
logues.26,27 These ligands displayed high affinity toward
δ receptors tested either as a mixture24,25 or after
separation of the diastereomers on HPLC.26,27 This
indicates that both (R) and (S) stereochemistries allow
superimposition of the key elements of the δ pharma-
cophore within the Tyr residue, i.e., the R-amino group
and the phenolic aromatic and hydroxy elements. These
findings made this amino acid a promising candidate
to incorporate into deltorphin II to obtain more informa-
tion about its bioactive conformation by comparing the
accessible conformations and biological activities of the
new ligands.
Hat was synthesized according to well-known meth-
ods described in the literature.28 Racemic Hat was built
into Ile5,6-deltorphin II by standard solid-phase peptide
chemistry. However, RP-HPLC separation of the result-
ing diastereomeric peptides was not successful, although
acetonitrile, methanol, and tetrahydrofuran were tried
as organic modifiers in TFA/water at different flow rates
in order to separate the peptides. Thus, the amino acid
had to be synthesized in enantiomerically pure form
first. Mosberg et al.27 applied R-chymotrypsin to resolve
the methyl ester of Hat. However, this approach re-
sulted only in enantiomerically enriched amino acid.
Similar results were obtained in the resolution of Atc
with R-chymotrypsin by To´th et al.12 In contrast, resolu-
tion of N-trifluoroacetyl-Atc with carboxypeptidase A
resulted in high enantiomeric purity. For this reason,
carboxypeptidase A was applied to digest N-trifluoro-
acetyl-6-methoxy-Atc. According to chiral TLC and
GITC derivatization,29 the products of the resolution
have high optical purity (ee g 95%). The absolute
configurations of the enantiomers were not determined
but can be assigned unambiguously by comparison of
the [R]D values with literature data where crystal-
lographic assignment was carried out.30 It is interesting
to note that, while R-chymotrypsin follows the “normal”
stereochemistry in the resolution of Atc12 and Hat27
,
carboxypeptidase A digests the (R) enantiomer of these
amino acids.
NMR Exp er im en ts
In the present paper, we describe the synthesis and
conformational evaluation of two new conformationally
constrained Ile5,6-deltorphin II analogues in which the
Tyr1 is replaced by (R)- or (S)-Hat1. The new peptides
were tested in different in vitro assays to investigate
their receptor affinity, specificity, and agonist potency.
Finally, on the basis of the NMR data, a proposal is put
forward as concerns the preferred solution conformation.
One-dimensional (1D) proton and band-selective TOC-
SY spectra and also two-dimensional (2D) TOCSY31,32
and ROESY33,34 spectra of the (S)- and (R)-Hat1 ana-
logues of Ile5,6-deltorphin II were recorded in DMSO-d6
at 500 MHz. A complete sequential assignment35 of the
proton resonances could be achieved in a straightfor-
ward manner by the combined use of TOCSY and
ROESY correlation maps. All 1H chemical shifts and

Deltorphin II Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1361
Ta ble 2. NOEs Observed in ROESY Spectra of (S) and (R)
Stereoisomers of Hat1,Ile5,6-deltorphin II (T ) 300 K, DMSO-d6)
from
to
NOE intensitiesb
residue
protona
residue
protona
(S) (R)
Hat1
Hat1
Hat1
Hat1
Hat1
Ala2
Ala2
Ala2
Phe3
Phe3
Phe3
Phe3
Phe3
Phe3
Phe3
Phe3
Phe3
Phe3
Glu4
Glu4
Ile5
âH
Ala2
Ala2
Hat1
Hat1
Hat1
Phe3
Phe3
Phe3
Glu4
Glu4
Glu4
Glu4
Phe3
Phe3
Phe3
Phe3
Ala2
Ala2
Ile5
NH
NH
δH
sd
sd
wd
md
wd
s
w
w
s
sd
sd
δH
âH
wd
wd
wd
s
âH′
δH′
RH
NH2
NH2
NH
NH
NH
NH
NH
NH
NH
NH
RH
RH
NH
âH
NH
NH
NH
NH
NH
NH
NH
F igu r e 1. NOE patterns used to assign the gauche(-)
conformation in the (S)-Hat1-deltorphin analogue.
âH
w
w
s
w-m
w
NH
RH
1
The large similarities of the H NMR parameters and
âH
w-m
w
âH′
NH
âH′
âH′
âH
the ROE patterns observed for the two analogues
indicate their very similar conformational characteris-
tics. The side-chain conformations of the Hat1 and Phe3
residues, which have a crucial impact on the receptor-
binding affinity and/or selectivity, were deduced from
the observed NOE effects and also, in the case of Phe3,
from the measured homonuclear J Râ vicinal coupling
constants. For Phe3, the gauche(-) conformation was
found to be the most populated in both analogues (66%
and 62% for (S) and (R) analogues, respectively).
However, the gauche(+) conformer also made a signifi-
cant contribution to the conformational equilibrium
(33% and 36% for (S) and (R) analogues, respectively).
Analysis of the NOE patterns involving Hat1 and D-Ala2
resonances (see correlations denoted by footnote d in
Table 2) allowed an unambiguous assignment of the
preferred rotamer for Hat1, namely gauche(-) for the
(S) and gauche(+) for the (R) analogue. These NOE
patterns are indicated on the gauche(-) conformer in
Figure 1. These patterns cannot be fitted to the trans
conformer without creating major discrepancies.
w
vw
sc
sc
wc
sc
wc
sc
Ar-H
Ar-H
Ar-H
RH
wc
wc
vwc
s
wc
wc
vwc
s
NH
RH
Ile5
w
s
w
s
w
s
Ile6
Ile5
NH
RH
Ile6
Ile6
Gly7
Gly7
s
w
Ile6
NH
w
a
High-field protons of nonequivalent CH2’s are denoted as H′.
b
NOE cross-peak intensities are qualitatively classified as strong
(s), medium (m), or weak (w), corresponding to distance limits of
1.8-2.5, 1.8-3.0, and 1.8-3.5 Å, respectively. c For the Phe3
residue, these NOE correlations, together with the 3J HRHâ coupling
constants, indicate the dominance of the gauche(-) conformation:
gauche(-) 66%, gauche(+) 33%, trans 1% for the (S) analogue;
gauche(-) 62%, gauche(+) 36%, trans 2% for the (R) analogue.d For
the Hat1 residue, these NOE correlations corroborate the presence
of the gauche(-) and gauche(+) conformations for the (S) and (R)
analogues, respectively.
The gauche(-) conformation of the (S)-Hat-deltorphin
analogue is in agreement with the absence of an NOE
effect between the Hat1 aromatic protons and the D-Ala2
methyl protons and with the chemical shift of 0.95 ppm
of these methyl protons. In deltorphin II, the corre-
sponding signal is observed at the unusually high field
position of 0.62 ppm, which is ascribed to shielding by
the Tyr1 aromatic ring in the trans orientation. How-
ever, it is close to the value of 0.91 ppm observed for
the Htc1-deltorphin II, which also has a preferred
gauche(-) conformation of the Htc residue.9
coupling constants, together with the temperature coef-
ficients of NH protons, are collected in Table 1. The H
1
NMR spectra remained the same after 10-fold dilution
which excludes the occurrence of peptide self-aggrega-
tion. For the (R) analogue, where severe signal overlap
between the NH and NH2 resonances hampered the
conventional temperature shift measurement (simply
acquiring 1D spectra at several temperatures), a series
of 1D TOCSY spectra with band-selective excitation36
of the aliphatic proton resonances were recorded. The
TOCSY transfer from aliphatic to NH protons resulted
in clean, NH2-free proton spectra and consequently
allowed an accurate evaluation of the temperature
dependence of the NH resonances. The temperature
coefficients were found to vary between -4.2 and -6.2
ppb/K, which indicates that none of the amide protons
are involved in stable hydrogen bonding. ROE peak
intensities measured for 120 ms of mixing are reported
in Table 2.
The observed NOE effects, and in particular the
intense CRHi-NHi+1 and weak NHi-NHi+1 NOEs de-
tected simultaneously for residues 2-7, indicate a time-
averaged backbone conformation in solution. The con-
secutive NHi-NHi+1 NOEs, and also the medium-range
NOE observed between Phe3-Hâ and Ile5-NH, strongly
suggest that some kind of folded conformers may exist
in conformational equilibrium with the extended ones.
The assumption of fast exchange between partially
folded and extended conformations is supported by the
measured J NHR couplings, ranging from 7.9 to 8.3 Hz.
Biologica l Da ta
The new analogues were tested with regard to their
binding properties to rat brain opioid receptors, their
ability to activate the GTP-binding proteins coupled to
the opioid receptors, and their in vitro bioactivities to
inhibit the electrically induced contractions of MVD and
GPI preparations.
The results of the radioligand-binding assays are
shown in Table 3. Both new ligands exhibited high
affinities toward δ opioid receptors, with Ki values in
the subnanomolar range. The (S) analogue was more
potent against high-affinity and specificity δ ligands,
[3H]pClPhe4-DPDPE and [3H]Ile5,6-deltorphin II, but
also displayed a several times higher µ affinity too.
Thus, despite the higher Ki values observed, the (R)
analogue proved to be the more selective (µ/δ selectivi-
ties of 2176 and >10000 for the (S) and the (R) analogue,
respectively). Moreover, the (R) analogue demonstrated
a higher selectivity as compared to the parent peptide

1362 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Darula et al.
Ta ble 3. Ki (nM) Valuesa of Deltorphin II Analogues in the Radioligand-Binding Assay
[3H]pClPhe4-DPDPE
[3H]Ile5,6-deltorphin II
[3H]DAMGO
[3H]norBNI
Ki (nM)
Hill slope
Ki (nM)
0.39
Hill slope
Ki (nM)
3188
Hill slope
Ki (nM)
Ile5,6-deltorphin IIb
5a
5b
0.16 ( 0.02
0.94 ( 0.10
-0.34 ( 0.03
-0.42 ( 0.08
0.34 ( 0.02
1.00 ( 0.18
-0.69 ( 0.09
-0.50 ( 0.08
740 ( 72.8
>10000
-1.01 ( 0.04
-1.094 ( 0.22
>10000
>10000
a
b
Values listed are arithmetic means ( SEM of 3-5 measurements carried out in duplicate. Cited from ref 12.
Ta ble 4. In Vitro Bioactivities of Deltorphin II Analogues
Ta ble 5. Stimulation of [35S]GTPγS Binding by Different
Concentrations of Deltorphin II Analogues
isolated organ IC50 (nM)a MVD/GPI potency ratio
% stimulation of [35S]GTPγS bindinga
ligand
MVD
GPI
IC50(GPI)/IC50(MVD)
concn (µM)
Ile5,6-deltorphin II
5a
5b
deltorphin II 0.39 ( 0.05 >1000b
>2564
2490
>1522
5a
5b
2.06 ( 0.47 5129 ( 816
0.1
1
10
152.2 ( 13.0
179.6 ( 12.8
173.9 ( 6.5
155.4 ( 16.3
176.1 ( 19.6
173.0 ( 26.0
126.1 ( 7.8
136.9 ( 8.7
160.9 ( 6.1
6.57 ( 0.87 >10000c
a
50% inhibitory concentrations; arithmetic means ( SEM of
b
a
4-6 measurements are listed. 12.0 ( 0.8% (mean ( SEM, n )
4) inhibition at 10-6 M. c 11.0 ( 0.9% (mean ( SEM, n ) 4)
inhibition at 10-5 M.
Stimulation is given as a percentage of the specific binding.
Data were calculated from three independent experiments per-
formed in triplicate and are presented here as means ( SEM.
Nonspecific binding was 63%. Nonstimulated [35S]GTPγS binding
was 86.65 ( 13.42 fmol/mg of protein.
Ile5,6-deltorphin II, too. None of the ligands revealed a
detectable affinity toward κ receptors, as measured with
[3H]norBNI.
[3H]pClPhe4-DPDPE and [3H]Ile5,6-deltorphin II are
presumed to label δ1 and δ2 opioid receptors, respec-
tively. The new deltorphin analogues seem to label both
sites equipotently. Furthermore, the values of the Hill
slope (Table 3) are far from unity in experiments
involving the use of δ radioligands, which suggests the
labeling of a heterogeneous receptor population, too.
Similar results were obtained in the in vitro bioassays
(Table 4), where the (S) analogue was about 4 times
more potent than the (R) analogue in the MVD test. The
maximum effects in MVD were identical for the two
analogues. However, the slope function calculated from
the logarithmic regressions of dose-response curves was
significantly higher for the (S) analogue (36.7 (31.8-
42.2) versus 20.7 (19.5-27.0)). In accordance with the
results obtained in the radioligand-binding assays, the
(S) analogue acted more potently in the GPI prepara-
tions, where predominantly µ receptors are present.
Thus, both in bioassays and in radioligand-binding
experiments, the (S) analogue is the more potent while
the (R) analogue is the more selective.
F igu r e 2. Superposition of the gauche(-) and gauche(+)
conformations of (S)-Hat and (R)-Hat, respectively.
the slope function may support this notion. The (S)
analogue showed very similar effect to the parent
compound Ile5,6-deltorphin II. In summary, the two new
analogues and the parent peptide all have about the
same intrinsic efficacy.
Con clu sion
The effects of agonists on the activation of opioid
receptors and their potency and efficacy in the activation
can be determined in functional assays. Similarly to
other G protein-coupled receptors, the δ opioid receptor
achieves its cellular activity by first activating G
proteins. Thus, receptor-activated [35S]GTPγS binding
serves as a valuable marker of the ability of an agonist
to activate a receptor quantitatively. The G protein-
activating properties of the new ligands were tested in
[35S]GTPγS-binding experiments in membranes of the
rat frontal cortex, a brain area known to be rich in δ
opioid-binding sites. This assay reflects the GDP-GTP
exchange reaction evoked by receptor agonists on the R
subunit of the G protein, by measurement of the binding
of the nonhydrolyzable GTP analogue.37 [35S]GTPγS-
binding experiments (Table 5) confirmed that the new
peptides have agonist properties since both of them
stimulated [35S]GTPγS binding (i.e. activated G pro-
teins) significantly (about 1.7 times the control value).
However, different activation pattern by the two ana-
logues may suggest a partial agonist property for the
(R) analogue. In the MVD bioassay, the differences in
The two ligands display quite similar affinities and
selectivities, indicating the similarity of the topography
of the ligands during their binding to the opioid receptor.
This also suggests that the bioactive side-chain confor-
mation of the Hat1 residue should be available for both
the (S) and (R) analogues. Indeed, a superposition of
the nitrogen, CR, and carbonyl groups of (S)- and (R)-
Hat in the gauche(-) and gauche(+) conformations,
respectively, indicates that topologically the two enan-
tiomers differ by a translation of the aromatic rings
relative to each other (Figure 2). However, the phenolic
hydroxy groups are pointing in the same direction. This
suggests that the δ receptor is able to interact equally
well with both orientations.
However, the high δ affinity of the Hba1-deltorphin
II analogue,38 which cannot adopt a gauche(-) confor-
mation, strongly suggests that the Hat1-deltorphin
analogues switch from their preferred conformation in
solution to a trans conformation during receptor inter-
action.

Deltorphin II Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1363
1
crystals in 85% yield. Mp: 231-232 °C. H NMR (DMSO-d6/
Exp er im en ta l Section
TFA): 7.05 (d, J ) 8.4 Hz, 1 arom H); 6.76, 6.74 (dd, J 1 ) 8.4
Hz, J 2 ) 2.2 Hz; 1 arom H); 6.72 (m, 1 arom H); 3.72 (s, 3
OCH3 H); 3.25 (d, J ) 16.7 Hz, 1 aliph H); 2.90 (d, J ) 16.7
Hz, 1 aliph H); 2.84 (t, J ) 6.4 Hz, 2 aliph H); 2.19 (m, 1 aliph
H); 2.04 (m, 1 aliph H). MS (ESI): 222 (MH+). TLC (3) Rf: 0.60.
HPLC tR: 13.2 min.
Gen er a l Meth od s. Protected and unprotected amino acids
(except Hat), coupling reagents, 4-methylbenzhydrylamine
resin, carboxypeptidase A, and 2,3,4,6-tetra-O-acetyl-â-D-glu-
copyranosyl isothiocyanate (GITC) were purchased from Sigma-
Aldrich Kft, Budapest, Hungary, or from Bachem Feinchemi-
kalien AG, Bubendorf, Switzerland. Precoated plates (silica
gel F254 (Merck, Darmstadt, Germany)) were used for TLC with
the following solvent systems: (1) acetonitrile-methanol-
water (4:1:1), (2) chloroform-methanol-acetic acid (90:10:1),
(3) n-butanol-acetic acid-water (2:1:1), (4) n-butanol-acetic
acid-water (4:1:1). UV light, I2, and ninhydrin were applied
to detect the compounds. Chiral purity control of Hat isomers
was performed on Chiralplate (Macherey-Nagel, Du¨ren, Ger-
many) and by GITC derivatization. Reversed-phase HPLC was
performed on a Merck-Hitachi liquid chromatograph with a
Vydac 218TP54 C18 column for analytical or a Vydac 218TP1010
C18 column for semipreparative separations. Analytical RP-
HPLC gradients (0-80% of organic component over 30 min
at a flow rate of 1 mL/min) were run with the solvent system
0.1% (v/v) TFA in water/0.1% (v/v) TFA in acetonitrile; peaks
were monitored at 215 nm.
Syn th esis of N-Tr iflu or oa cetyl-6-m eth oxy-2-a m in ote-
tr a lin -2-ca r boxylic Acid (N-TF A-6-OCH3-Atc, 4). A mix-
ture of 1.75 g (7.91 mmol) of 3 and 7.5 mL (54.0 mmol, 6.8
equiv) of trifluoroacetic anhydride in 28 mL of TFA was stirred
in an icebath for 2 h, followed by stirring at room temperature
for 21 h. The brown solution was evaporated, and water was
added to the resulting brown oil. The oil solidified, and the
white precipitate was crystallized from EtOH-water (1:1) to
give 1.67 g of white crystals in 68% yield. Mp: 145-147 °C.
1H NMR (CD3OD): 6.98 (d, J ) 8.4 Hz, 1 arom H); 6.72, 6.70
(dd, J 1 ) 8.4 Hz, J 2 ) 2.6 Hz, 1 arom H); 6.67 (d, J ) 2.5 Hz,
1 arom H); 3.75 (s, 3 OCH3 H); 3.25 (d, J ) 16.5 Hz, 1 aliph
H); 3.10 (d, J ) 16.4 Hz, 1 aliph H); 2.81 (m, 2 aliph H); 2.54
(m, 1 aliph H); 2.18 (m, 1 aliph H). MS (ESI): 318 (MH+). TLC
(3) Rf: 0.71. HPLC tR: 21.4 min.
Resolu tion of N-TF A-6-OCH3-Atc. 7.32 g (23.1 mmol) of
4 was dissolved in 500 mL of water, the pH was adjusted to
7-8 with 2 M NaOH, 500 µL of carboxypeptidase A (Sigma
C-0386; 22 mg of protein/mL, 70 U/mg of protein) was added
and the reaction mixture was stirred for 4 days at 37 °C. The
mixture was acidified to pH 2 with concentrated HCl, the
solution was boiled, charcoal was added, the mixture was
boiled again, the charcoal was filtered off, the solution was
extracted with 4 × 100 mL of EtOAc, and the combined organic
fraction was extracted with 3 × 80 mL of water. Both solutions
were evaporated to dryness, 50 mL of 48% HBr was added to
each residue, the solutions were refluxed for 24 h under
stirring and then evaporated, the residues were dissolved in
30 mL of hot water, the pH was adjusted to 6-7 with 25%
aqueous NH3 solution, and the precipitate was filtered off and
crystallized from MeOH-water (1:1).
Molecular weights of peptides were determined by ESI mass
spectrometry on a double focusing MS 902S spectrometer
(A.E.I. Scientific Division, Manchester, U.K.). Routine 1H NMR
spectra were measured on a Bruker AC 250-P 250 MHz
spectrometer. Radioligands, except for DAMGO (Amersham)
and [pClPhe4]-DPDPE (NEN), were prepared in our laboratory
as described earlier for Ile5,6-deltorphin II39 and norBNI.40
Syn th esis of 6-Hyd r oxy-2-a m in otetr a lin -2-ca r boxylic
Acid (Ha t, 1).28 A mixture of 6-methoxy-2-spiro[hydantoin-
tetralin] (8.79 g, 35.7 mmol), prepared from 6-methoxy-2-
tetralone41 and aqueous 48% HBr (120 mL), was refluxed for
48 h. The aqueous HBr was then evaporated off, the residue
was dissolved in 200 mL of hot water and the pH was adjusted
to 5-6 with 25% aqueous NH3 solution. The resulting brown
solid was crystallized from ethanol to give 4.96 g of white
1
powder in 67% yield. Mp: 280-282 °C. H NMR (DMSO-d6):
(R)-Ha t (Ha t fr om th e w a ter la yer ): Yield: 1.26 g (26%).
7.02, 6.70 ppm (2d, J ) 8.2 Hz, 2 arom H); 6.67 ppm (s, 1 arom
H); 3.33 ppm, 2.96 ppm (2d, J ) 16.8 Hz); 2.96-2.72 ppm (m,
2 aliph H); 2.28-2.40 ppm (m, 1 aliph H); 1.99-2.15 ppm (m,
1 aliph H). MS (ESI): 208 (MH+). TLC (1) Rf: 0.44. HPLC tR:
10.6 min.
1
Mp: >280 °C. H NMR (DMSO-d6): 6.84 ppm (d, J ) 8.2 Hz,
1 arom H); 6.54 ppm (2d, J 1 ) 8.2 Hz, J 2 ) 2.3 Hz, 1 arom H);
6.49 ppm (d, J ) 2.0 Hz, 1 arom H); 3.18 ppm, 2.61 ppm (2d,
J ) 16.9 Hz, aliph CH2); 2.64-2.73 ppm (m, 2 aliph H); 2.02-
2.08 ppm (m, 1 aliph H); 1.77-1.80 ppm (m, 1 aliph H). MS
(ESI): 208 (MH+). TLC (1) Rf: 0.37; (4) Rf: 0.50. Chiral TLC
(1) Rf: 0.48 (identical with the lower spot of racemic Hat).
[R]D20: -4.1° × mL/g × dm (c ) 6.67 g/100 mL of 1 M HCl
(0.32 M)). HPLC tR: 10.7 min.
Syn th esis of ter t-Bu tyloxyca r bon yl-Ha t (Boc-Ha t, 2).
2 was prepared from 11.73 g (57 mmol) of 1 by a standard
literature procedure42 in dioxane-water (2:1) at pH 10 with
1.1 equiv of di-tert-butyl dicarbonate (Boc2O), but after stirring
for 3 h, an additional portion of Boc2O (0.3 equiv) was added
and stirring was continued for 4 days at room temperature.
After extraction, the resulting brownish oil was crystallized
from EtOAc/petroleum ether. In accordance with the high
excess of Boc2O, about 2% of bis-Boc-Hat was also formed,
which was revealed by RP-HPLC to crystallize together with
Boc-Hat. This mixture of the protected amino acid was used
for peptide synthesis. Yield: 10.08 g (58%). Mp: 141 °C. 1H
NMR (CD3OD): 6.87, 6.54 ppm (2d, J ) 8.0 Hz, 2 arom H);
6.53 ppm (s, 1 arom H); 3.30-3.35 ppm (m, aliph CH2); 3.14,
2.92 ppm (2d, J ) 16.1 Hz, aliph CH2) 2.68-2.81 ppm (m, 2
aliph H); 1.42 ppm (s, 9 tBu-H). MS (ESI): 307 (MH+). TLC
(1) Rf: 0.74; (2) Rf: 0.32. HPLC tR: 18.8 and 27.5 min for bis-
Boc-Hat.
Syn th esis of 6-Meth oxy-2-a m in otetr a lin -2-ca r boxylic
Acid (6-OCH3-Atc, 3). A mixture of 5 g (20.3 mmol) of
6-methoxy-2-spiro[hydantointetralin] and 35 g (111 mmol, 5.5
equiv) of Ba(OH)2‚8H2O in 150 mL of hot water was refluxed
in a pyrolysis tube for 47 h. The reaction mixture was then
transferred to a flask, 2-300 mL of water was added, and the
insoluble white residue was filtered off. The pH of the solution
was adjusted to 2 with 1 M H2SO4 and stirred in a waterbath
for 1 h, the precipitated BaSO4 was filtered off and washed
two times with hot water, and the pH of the combined filtrate
was set to 6 with 25% aqueous NH3 solution. The mixture was
evaporated to half-volume and the resulting white precipitate
was crystallized from EtOH-water (1:1) to give 3.81 g of white
(S)-Ha t (Ha t fr om th e or ga n ic la yer ): Yield: 1.59 g
1
(33%). Mp: >280 °C. H NMR (DMSO-d6) in close agreement
with that of (R)-Hat: 6.84 ppm (d, J ) 8.2 Hz, 1 arom H);
6.55 ppm (2d, J 1 ) 8.1 Hz, J 2 ) 2.2 Hz, 1 arom H); 6.49 ppm
(s, 1 arom H); 3.18 ppm, 2.62 ppm (2d, J ) 16.9 Hz, aliph CH2);
2.66-2.72 ppm (m, 2 aliph H); 2.02-2.08 ppm (m, 1 aliph H);
1.78-1.81 ppm (m, 1 aliph H). MS (ESI): 208 (MH+). TLC (1)
Rf: 0.37; (4) Rf: 0.50. Chiral TLC (1) Rf: 0.56 (identical with
the upper spot of racemic Hat). [R]D20: +3.7° × mL/g × dm (c
) 6.67 g/100 mL of 1 M HCl (0.32 M)). HPLC tR: 10.7 min.
Solid -P h a se Syn th esis a n d P u r ifica tion of (S)-Ha t-D-
Ala -P h e-Glu -Ile-Ile-Gly-NH2 (5a ) a n d (R)-Ha t-D-Ala -P h e-
Glu -Ile-Ile-Gly-NH2 (5b). Peptide synthesis was performed
manually by the solid-phase technique, using 4-methylben-
zhydrylamine resin (0.8 mmol/g of titratable amine). Boc-
protected amino acids were used and dicyclohexylcarbodiimide
(DCC) and 1-hydroxybenzotriazole (HOBt) as coupling agents.
Side-chain protection was benzyl for Glu. The deprotection
solution was 50% TFA and 2% anisole in dichloromethane
(DCM). The protocol for peptide synthesis in each cycle was
as follows: (1) addition of Boc-amino acid in DCM (2 equiv),
(2) addition of DCC (2 equiv), (3) addition of HOBt (2 equiv),
(4) shaking for 1-3 h, (5) washing with DCM (3 × 2 min), (6)
washing with EtOH (3 × 2 min), (7) washing with DCM (3 ×
2 min), (8) monitoring completion of the reaction with the
ninhydrin test,43 (9) Boc deprotection with 50% TFA in DCM

1364 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7
Ta ble 6. Physicochemical Data on Deltorphin II Analogues
Darula et al.
Ra d ioliga n d -Bin d in g Assa ys. Membranes were prepared
from Wistar rat brain (minus cerebellum) according to the
method of Simon et al.50 The binding experiments were
performed in 50 mM Tris-HCl buffer, pH 7.4, in a final volume
of 1.0 mL containing 300-500 µg of protein, different concen-
trations of (S)- and (R)-Hat1,Ile5,6-deltorphin II, and type-
specific radioligands. Protein concentration was determined
by the method of Bradford.51 The following conditions were
used for incubations: [3H]DAMGO (0.5 nM, 35 °C, 45 min);
[3H]Ile5,6-deltorphin II (0.5 nM, 35 °C, 45 min); [3H]pClPhe4-
DPDPE (0.8 nM, 25 °C, 90 min); and [3H]norBNI (0.1 nM, 25
°C, 60 min). Incubations were started by the addition of
membrane suspension, continued under gentle shaking in a
thermal water bath, and terminated by rapid vacuum filtration
through Whatman GF/B or C filters using a Brandell cell
harvester. After three washings with 6-mL portions of ice-cold
buffer, the filters were dried and the radioactivity was
measured in a toluene-based scintillation cocktail, using a
Wallac 1409 scintillation spectrometer (Turku, Finland; η )
0.55). The extent of nonspecific binding was determined in the
presence of 10 µM naloxone. Data were evaluated with the
use of the software GraphPad Prism 2.01.
HPLCa
TLC
Rf (1)
ESI MS
MW
peptide
k′
Rf (4)
Ile5,6-deltorphin IIb
5a
5b
0.51
0.58
0.59
0.61
0.60
0.59
811
837
837
4.73
4.75
a
b
Solvent front breakthrough at 3.0 min. Cited from ref 12.
(2 min + 20 min), (10) washing with DCM (3 × 2 min), (11)
neutralization with DIEA in DCM (2 × 2 min), and (12)
washing with DCM (3 × 2 min). Simultaneous deprotection
and cleavage from the resin were accomplished by treatment
with 90% HF and 10% anisole (9 mL of HF and 1 mL of
anisole/g peptide resin) at 0 °C for 1 h. After evaporation of
the HF, the peptide resin was washed with diethyl ether and
the peptide was extracted with glacial acetic acid and lyoph-
ilized. Crude peptides were purified by semipreparative RP-
HPLC. Peptide purity was assessed by analytical RP-HPLC
and TLC. ESI mass spectrometry confirmed the appropriate
molecular weight (Table 6).
NMR Exp er im en ts.35 These were carried out with
a
[
35S]GTP γS Bin d in g. Agonist-stimulated [35S]GTPγS bind-
Bruker Avance DRX 500 (500.13 MHz for 1H) spectrometer
equipped with a 5-mm triple-resonance probe (1H/13C/15N) and
an actively shielded z-gradient coil. The samples contained 2.3
and 3.1 mg of (S) and (R) analogue, respectively, dissolved in
0.5 mL of DMSO-d6. Proton chemical shifts are referred to the
residual signal of the solvent, δDMSO-d6 ) 2.49 ppm. All 1H
NMR data, including the chemical shifts, coupling constants,
and NH temperature coefficients reported in Table 1, were
extracted from resolution-enhanced 1D spectra or, in the case
of signal overlap, from 1D band-selective TOCSY spectra or
the highly digitized 1D traces of gradient-enhanced TOCSY
correlation maps.31,32 Gradient-enhanced 2D TOCSY experi-
ments were run by using the MLEV 17 sequence32 for isotropic
mixing with a duration of 62 ms. High-power spin-lock pulses
of 2-3 ms with simultaneously switched gradients31 were
applied to generate pure absorption signals for coupling
constant measurement. The 2D data matrix consisted of 4K
× 320 complex data points. Zero-filling in F1 and a squared
cosine function in both F1 and F2 were applied prior to Fourier
transformation. Eight transients were accumulated for each
of the t1 increments, with a relaxation delay of 1.8 s. A spin-
lock field of 8300 Hz was used for the TOCSY transfer. A
spectral width of 4740 Hz was set in both dimensions for the
2D homonuclear experiments. The 1D band-selective TOCSY
experiments were performed by using the double-pulsed field
gradient spin-echo sequence.44 Band-selective excitation of
aliphatic proton resonances was executed by means of a pulse-
width-modulated DANTE-Z pulse,36 using an I-BURP-1/
DANTE train with 32 elementary pulses (P ) 20.6-µs 90°
pulse, τ ) 50-µs interpulse delay, resulting in a (2 ppm
excitation bandwidth).
ing was measured in crude membranes of the rat frontal
cortex, a brain area known to be enriched in δ opioid recep-
tors.52 After dissection of the frontal cortex, the procedure of
membrane preparation was the same as in the case of the
radioligand-binding assays.50 Experiments were carried out in
50 mM Tris-HCl buffer containing 1 mM EGTA, 3 mM MgCl2,
and 100 mM NaCl in a final volume of 1 mL. Assay tubes,
containing 10 µg of protein, 30 µM GDP, 0.1/1/10 µM opioid
ligands, and 0.05 nM [35S]GTPγS, were incubated for 1 h at
30 °C. For positive control, Ile5,6-deltorphin II was utilized.
Nonstimulated activity was measured in the absence of tested
compounds, while nonspecific binding was measured in the
presence of 10 µM nonlabeled GTPγS. The incubation was
terminated by filtering the samples through Whatman GF/F
glass fiber filters in a Millipore filtration instrument. Filters
were washed three times with 5 mL of ice-cold 50 mM Tris-
HCl buffer (pH 7.4) and then dried, and the radioactivity was
measured in a Wallac 1409 scintillation counter (Turku,
Finland), using a toluene-based scintillation cocktail.
GP I a n d MVD Bioa ssa ys. The bioassays of the peptides
were based on the measurement of the inhibition of electrically
induced smooth muscle contractions of MVD and GPI myen-
teric plexus-longitudinal muscle strips.53 Longitudinal muscle
strips taken from the ileum of male guinea pigs (GPI) weighing
400-600 g and the vas deferens of CFLP mice (MVD) weighing
35-40 g were prepared and used as described previously.54
The experimental conditions for GPI were quite similar to the
international routine. The technical parameters for the experi-
ments with the MVD preparation were either a modified
version54 or the original description.55 In brief, a single vas
was used instead of a pair. The preparations were mounted
in Mg2+-free Krebs solution at 31 °C under an initial tension
of 0.05-0.08 g. The field electrical stimulation parameters
were as follows: rectangular pulses of 1-ms duration, 9 V/cm
intensity. Twin pulses (100-ms pulse distance) were repeated
at 10 s.
The populations of side-chain rotamers of the Phe residues
were quantitatively assessed from the homonuclear coupling
45
constants, J Râ
,
using the Pachler equations46,47 with Cung’s
parametrization proposed for aromatic amino acids.48 The
stereospecific assignment of the prochiral â-protons of Phe3
indicated in Table 1 could be deduced from the concerted use
of homonuclear vicinal couplings (J Râ) and intraresidue ROE
effects observed between NH and Hâ and between HR and Hâs,
respectively.
Ack n ow led gm en t. This work was supported by
Grant OTKA 030086 (G.T.), Grants OMFB 969748/1205
and OTKA T 022104 (A.B.), and the Flemish-Hungar-
ian Bilateral Intergovermental S&T Cooperation B-3/
96 (G.T., A.P., D.T.). K.E.K. thanks the National Re-
search Foundation for support (OTKA D 23749, T
029089). The purchase of the spectrometer used in the
study was supported by OMFB Mec-930098, Phase-
Accord H-9112-0198, and OTKA A 084. Zs. Darula was
supported by the Soros Fundation. The authors grate-
fully acknowledge the technical assistance of Ms. Zs.
Canjavec.
ROESY spectra33,34 were recorded for a mixing time of 120
ms with a CW spin-lock field of 3300 Hz. A relaxation delay
of 1.8 s was allowed between successive transients. Sixty-four
transients were recorded with 2K complex data points each
for a total number of 512 experiments. For processing, the
matrices were zero-filled and apodized by a squared cosine
function in both dimensions. A polynomial baseline correction
was also applied. Volume intensities of ROE peaks were
measured and classified as strong (s), medium (m), or weak
(w), corresponding to inter-proton distance ranges of 1.8-2.5,
1.8-3.0, and 1.8-3.5 Å, respectively.49 All NOE correlations
of conformational relevance are reported in Table 2.

Deltorphin II Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 7 1365
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The Aspartic Acid in Deltorphin I and Dermenkephalin Pro-
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Su p p or tin g In for m a tion Ava ila ble: Analytical data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Refer en ces
(1) Abbreviations and definitions are those recommended by the
IUPAC-IUB Commission on Nomenclature (J . Biol. Chem.
1972, 247, 977-983). All optically active amino acids are of the
L configuration unless otherwise noted. Other abbreviations: Tic,
tetrahydroisoquinoline-3-carboxylic acid; Htc, 7-hydroxytetrahy-
droisoquinoline-3-carboxylic acid; Atc, 2-aminotetralin-2-car-
boxylic acid; Aic, 2-aminoindan-2-carboxylic acid; Hat, 6-hydroxy-
2-aminotetralin-2-carboxylic acid; Hba, 8-hydroxy-4(S)-amino-
1,3,4,5-tetrahydro-3-oxo-2H-2-benzazepine-2-acetic acid; dermen-
kephalin, H-Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2; deltorphin I,
H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2; deltorphin II, H-Tyr-D-
Ala-Phe-Glu-Val-Val-Gly-NH2; DPDPE, Tyr-D-Pen-Gly-Phe-D-
Pen-OH; norBNI, norbinaltorphimine; DAMGO, Tyr-D-Ala-Gly-
NMe-Phe-Gly-ol; GTP, guanosine 5′-triphosphate; GDP, guanosine
5′-diphosphate; GTPγS, guanosine 5′-O-(γ-thio)triphosphate;
MVD, mouse vas deferens; GPI, guinea pig ileum; TFA, trifluo-
roacetic acid; Boc2O, di-tert-butyl dicarbonate; DCM, dichlo-
romethane; DIEA, diisopropylethylamine; HOBt, 1-hydroxyben-
zotriazole; DCC, dicyclohexylcarbodiimide; DMSO, dimethyl
sulfoxide; GITC, 2,3,4,6-tetra-O-acetyl-â-D-glucopyranosyl isothio-
cyanate; HPLC, high-performance liquid chromatography; TLC,
thin-layer chromatography.
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