Conformationally constrained deltorphin analogs with 2-aminotetralin-2- carboxylic acid in position 3

Article abstract of DOI:10.1021/jm9602726

Two approaches to the design of very active and highly selective δ opioid peptides were used to obtain new deltorphin analogs with altered hydrophobic and stereoelectronic properties. Deltorphin I and II analogs were synthesized involving the substitution of Ile instead of Val at positions 5 and 6 in the address domain and 2-aminotetralin-2-carboxylic acid (Atc) instead of Phe in the message domain. The peptides were agonists in the subnanomolar range in the MVD assay and in the micromolar or higher range in the GPI assay, showing a very high selectivity for δ receptors. A very similar trend could be observed in radioreceptor binding assays in which selective tritiated opioid ligands were used. (R)- and (S)-Atc-deltorphins exhibited similar K(i) values in the binding assay, with almost complete loss of the stereospecificity of the binding. Conformational studies provided evidence for little disturbance of the backbone conformational equilibrium when Phe³ is replaced by (S)- or (R)-Atc. The use of the Atc constraint gives additional evidence that, during its interaction with the δ receptor, the side chain of residue 3 adopts the trans conformation at χ₁.

Full text of DOI:10.1021/jm9602726

990
J . Med. Chem. 1997, 40, 990-995
Con for m a tion a lly Con str a in ed Deltor p h in An a logs w ith
2-Am in otetr a lin -2-Ca r boxylic Acid in P osition 3
Ge´za To´th,*,† Zsuzsa Darula,† Antal Pe´ter,‡ Ferenc Fu¨lo¨p,§ Dirk Tourwe´, Hendrika J aspers,
Patricia Verheyden, Zsolt Bo¨cskey,| Zolta´n To´th,# and Anna Borsodi@
Isotope Laboratory and Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences,
P.O. Box 521, H-6701 Szeged, Hungary, Department of Inorganic and Analytical Chemistry, Attila J o´zsef University,
P.O. Box 440, H-6701 Szeged, Hungary, Institute of Pharmaceutical Chemistry, Albert Szent-Gyo¨rgyi Medical University,
P.O. Box 121, H-6701 Szeged, Hungary, Eenheid Organische Chemie, Vrije Universiteit Brussel, Pleinlaan 2,
B-1050 Brussels, Belgium, CHINOIN Ltd., P.O. Box 110, H-1045 Budapest, Hungary, and Alkaloida Chemical Factory Ltd.,
P.O. Box 1, H-4440 Tiszavasva´ri, Hungary
Received April 8, 1996X
Two approaches to the design of very active and highly selective δ opioid peptides were used
to obtain new deltorphin analogs with altered hydrophobic and stereoelectronic properties.
Deltorphin I and II analogs were synthesized involving the substitution of Ile instead of Val at
positions 5 and 6 in the address domain and 2-aminotetralin-2-carboxylic acid (Atc) instead of
Phe in the message domain. The peptides were agonists in the subnanomolar range in the
MVD assay and in the micromolar or higher range in the GPI assay, showing a very high
selectivity for δ receptors. A very similar trend could be observed in radioreceptor binding
assays in which selective tritiated opioid ligands were used. (R)- and (S)-Atc-deltorphins
exhibited similar Ki values in the binding assay, with almost complete loss of the stereospeci-
ficity of the binding. Conformational studies provided evidence for little disturbance of the
backbone conformational equilibrium when Phe3 is replaced by (S)- or (R)-Atc. The use of the
Atc constraint gives additional evidence that, during its interaction with the δ receptor, the
side chain of residue 3 adopts the trans conformation at ø1.
In tr od u ction
Modifications in the side chains of the individual
amino acids in deltorphins demonstrated that changes
in the electronic6 and hydrophobic structure signifi-
cantly influenced the receptor binding properties. Al-
terations in the hydrophobicity of the positions 5 and 6
revealed a pivotal hydrophobic core in the deltorphins.
Sasaki et al.7 synthesized Ile5,6-deltorphin II, replacing
Val at positions 5 and 6 by Ile and obtained more
lipophilic peptide, with eight times higher affinity and
five times more δ selectivity. Ile5,6-deltorphin II in
tritiated form gave us8 a useful tool to investigate the
binding properties of different opioid compounds. This
compound was very stable against protolytic enzymes8,9
and during storage. The specific/nonspecific binding
ratio was also excellent in the binding assay.
The incorporation of conformational constraints in
analogs of biologically active peptides is a well-known
approach for enhancing receptor selectivity and modu-
lating efficacy. This approach has been especially
successful in the opioid peptide area, where the natural
small opioid peptides are known to be structurally
flexible molecules whose conformations are strongly
dependent on the environment. This is the main reason
why these peptides lack significant selectivity toward
one or the other opioid receptor type or subtype (µ, δ,
and κ and their subtypes). In efforts to obtain better
conformational integrity, various conformationally re-
stricted opioid peptide analogs have been developed (for
reviews, see Hruby and Gehrig, Schiller et al.2,3) for
agonists and antagonists as well.
Recently, conformational restriction of the phenyla-
lanine residue in deltorphins has resulted in drastic
effects on receptor affinity and selectivity and on signal
transduction. Tetrahydroisoquinoline-3-carboxylic acid
(Tic),10-12 different â-methylphenylalanines,13,14 2-ami-
notetralin-2-carboxylic acid (Atc), 2-aminoindan-2-car-
boxylic acid (Aic),10,11 and 4-aminotetrahydro-2-benza-
zepin-3-one12 were used instead of Phe at position 3 to
investigate the effect of the side-chain conformation on
the binding properties of deltorphins. Especially the Atc
and Aic analogs displayed extraordinary δ receptor
affinity and selectivity. Identification of the configura-
tion of Atc in the diastereomeric peptides was not
attempted, however.
Currently, the most potent and most selective δ
agonists are the deltorphins. H-Tyr-D-Met-Phe-His-
Leu-Met-Asp-NH2 (deltorphin), 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.4,5 In general deltorphin I and its analogs are
more active in the δ opioid binding assay than deltor-
phin and deltorphin II analogs, while deltorphin II and
its analogs are more δ selective compared to deltorphin
and deltorphin I molecules because of their lower
affinity for µ receptors.
† Isotope Laboratory, Hungarian Academy of Sciences.
‡ Attila J o´zsef University.
In the present paper we describe new deltorphin
analogs involving modifications in the side chains at
positions 3, 5, and 6 causing changes in the hydrophobic
and stereoelectronic properties. The new analogs were
obtained by substitution of Atc at position 3, and Ile at
positions 5 and 6 in deltorphin I and II. We describe
§ Albert Szent-Gyo¨rgyi Medical University.
Vrije Universiteit Brussel.
| CHINOIN Ltd.
#
Alkaloida Chemical Factory Ltd.
@ Institute of Biochemistry, Hungarian Academy of Sciences.
X Abstract published in Advance ACS Abstracts, February 15, 1997.
S0022-2623(96)00272-5 CCC: $14.00 © 1997 American Chemical Society

Conformationally Constrained Deltorphin Analogs
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 991
Ta ble 1. Opioid Receptor Binding Affinities of Deltorphin Analogsa
K
iδ (nM)
K
iµ (nM)
Kiκ (nM)
peptides
[3H]NTI
[3H]DIDII
[3H]TIPPΨ
[3H]DAGO
[3H]U-69593
Kiµ/Kiδ
(1) Tyr-D-Ala-Phe-Asp-Ile-Ile-Gly-NH2
(2) Tyr-D-Ala-Phe-Glu-Ile-Ile-Gly-NH2
(3) Tyr-D-Ala-(R)-Atc-Asp-Ile-Ile-Gly-NH2
(4) Tyr-D-Ala-(S)-Atc-Asp-Ile-Ile-Gly-NH2
(5) Tyr-D-Ala-(R)-Atc-Glu-Ile-Ile-Gly-NH2
(6) Tyr-D-Ala-(S)-Atc-Glu-Ile-Ile-Gly-NH2
0.047 ( 0.011
5.750 ( 0.354
3.950 ( 0.778
1.050 ( 0.071
0.515 ( 0.106
0.745 ( 0.007
0.056 ( 0.010
0.385 ( 0.078
0.435 ( 0.021
0.030 ( 0.008
0.035 ( 0.007
0.310 ( 0.050
0.018 ( 0.002
0.535 ( 0.100
1.650 ( 0.212
0.153 ( 0.030
0.380 ( 0.028
0.028 ( 0.011
3224 ( 1037
3188 ( 1039
4526 ( 483
660 ( 47
>10000
>10000
>10000
>10000
>10000
>10000
57571
8281
10405
22000
20886
5803
731 ( 16
1799 ( 723
a
Kiµ/Kiδ ratios: Kiδ of [3H]DIDII was used to calculate the selectivity ratio. Values in the table represent means of three to five
measurements, each containing two parallels. SD is included.
the determination of the configuration of Atc in the
peptides prepared from racemic Boc-Atc.
from racemic Atc-methyl ester when R-chymotrypsin
was used.
Other supporting evidence for the identification of (R)-
Atc and (S)-Atc was provided by chiral TLC. In general,
L-amino acids have higher Rf values than those of
D-amino acids on chiral TLC plates with an eluent
mixture of acetonitrile-methanol-water, 4:1:1.18 In
this case, (S)-Atc has a higher Rf than that of (R)-Atc.
Resu lts a n d Discu ssion
Syn th esis. Peptides were synthesized by usual solid-
phase techniques with Boc strategy, using dicyclohexy-
lcarbodiimide (DCC)/hydroxybenzotriazole (HOBt) as
coupling agents. Atc was prepared by a modified
Strecker synthesis.15,16 Boc-Atc was obtained in racemic
form and incorporated as such into the peptides. The
diastereomeric peptides were isolated separately by
semipreparative HPLC. The configuration of Atc in the
peptides was determined from the amino acid mixture
obtained from the peptides after hydrolysis by chiral
TLC and by HPLC of the amino acid mixture derivatized
by GITC (details in the Experimental Section). (R)- and
(S)-Atc were prepared by enzymatic digestion using
N-trifluoroacetyl or methyl ester derivatives of Atc and
carboxypeptidase A or R-chymotrypsin, respectively. The
absolute configuration of the Atc enantiomer which was
obtained from enzymatic digestion with carboxypepti-
dase A was determined by X-ray crystallography. This
Atc enantiomer was treated with 48% HBr in water and
crystallized from water and its absolute configuration
was shown to be R by the Flack x value of 0.10(7). The
substituted ring of (R)-Atc is in a screw-boat conforma-
tion with the ammonium group in a pseudoequatorial
and the carboxyl group in a pseudoaxial position (Figure
1, Supporting Information). The packing of the mol-
ecules in the crystal structure presents a noticeable
separation of the nonpolar and polar constituents into
layers, of which the hydrophilic layer is stabilized by
six different hydrogen bonds, i.e. all of the hydrogen
bond donor possibilities are fully utilized (Figure 2,
Supporting Information). The bromide ions sit in the
center of a distorted tetrahedron, accepting hydrogen
bonds from the carboxylic and ammonium moieties and
from two separate water molecules. The water donates
two hydrogen bonds to two separate bromide ions and
accepts two from two separate ammonium ions from its
crystallographic environment. The dihedral angles of
the saturated ring are reported in Table 6 (Supporting
Information). The angles corresponding to ø1 and ø2 of
a conformationally constrained phenylalanine are 153.2°
and -46.1°, respectively, in this crystal structure.
Biologica l Da ta . The synthetic deltorphin I and II
analogs were examined with regard to their binding
properties to rat brain opioid receptors and in vitro
bioactivities via their abilities to inhibit an electrically
induced contraction of the mouse vas deferens (MVD)
and guinea pig ileum (GPI).
[3H]Ile5,6-deltorphin II,8 [3H]TIPP[Ψ],19 and [3H]nal-
trindole20,21 were used as δ radioligands, and [3H]DAGO
and [3H]U-69,593 as µ and κ radioligands, respectively.
The opioid receptor binding affinities of the deltorphin
analogs are summarized in Table 1. All six peptides
exhibited very high affinity for δ receptors (Ki values
were in the subnanomolar range). We used tritiated
agonist
([3H]DIDII) and
tritiated
antagonist
([3H]TIPP[Ψ] and [3H]naltrindole) in the binding assays.
These three relatively new tritiated ligands are very
selective for δ opioid receptors. In the competition
studies the peptides were less active against [3H]-
naltrindole, compared to data against the two peptide
ligands. We used Ki values from [3H]DIDII binding
study to calculate the selectivity (Kiµ/Kiδ). As can be
seen from Table 1, the most δ-selective compound was
the Ile5,6-deltorphin I (compound 1), but the selectivities
of compounds 4 and 5 were also more than 20 000. In
the Atc analogs of deltorphin I, the (S)-Atc analog 4 is
the more potent and selective, whereas in the Atc
analogs of deltorphin II, the (R)-Atc analog 5 is better
in potency and in selectivity. µ Receptor binding
affinities were in the interval 660-4526 nM, while all
Ki values obtained for the κ receptors were higher than
10 000 nM. Deltorphin I with D-Phe at position 3 was
20-50 times less potent than the parent peptide,10 while
the Atc analogs had more comparable Ki values in the
case of (R)- or (S)-Atc-deltorphin I and II analogs.
In the in vitro bioassay (Table 2), all six peptides
displayed properties similar to those in the binding
assay. In the MVD assay (where the δ opioid receptors
are predominant), the analogs were agonists in the
subnanomolar range, while in the GPI assay (which
constains µ and κ opioid receptors) the IC50 values were
in the micromolar or higher range. These results
indicate that the δ opioid receptor selectivity of these
peptides is higher than that of the parent peptide.4 The
ratio IC50(GPI)/IC50(MVD) for the deltorphin II analogs
was >100 000.
Atc can be regarded as a constrained analog of both
Phe and Hfe (with a reversed configurational relation-
ship).17 It is interesting to note that the resolution of
racemic Atc by enzymatic methods resulted in Atc with
different configurations, depending on the enzyme used.
With carboxypeptidase A and N-trifluroacetyl-Atc as
substrate, Atc with the R configuration could be ob-
tained, while Atc with the S configuration was formed

992 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Ta ble 2. In Vitro Bioactivities of Synthetic Deltorphin Analogs
To´th et al.
IC50 (nM)
peptides
MVD
GPI
g3000
g1500
4210 ( 470
67250 ( 27600
6618 ( 1750
2162 ( 754
19570 ( 3180
31270 ( 5360
GPI/MVD ratios
b
b
Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2
0.32
0.21
g9375
g7143
51342
185841
91917
56895
217444
115815
(1) Tyr-D-Ala-Phe-Asp-Ile-Ile-Gly-NH2
(2) Tyr-D-Ala-Phe-Glu-Ile-Ile-Gly-NH2
(3) Tyr-D-Ala-(R)-Atc-Asp-Ile-Ile-Gly-NH2
(4) Tyr-D-Ala-(S)-Atc-Asp-Ile-Ile-Gly-NH2
(5) Tyr-D-Ala-(R)-Atc-Glu-Ile-Ile-Gly-NH2
(6) Tyr-D-Ala-(S)-Atc-Glu-Ile-Ile-Gly-NH2
0.082 ( 0.010
0.500 ( 0.080
0.070 ( 0.008
0.038 ( 0.004
0.090 ( 0.030
0. 270 ( 0.010
a
b
Values in the table are mean ( SD. Reference 4.
As can be seen from Table 2, peptides containing Atc
with R or S configuration were almost equipotent in the
MVD assay (only a 2-3-fold difference). An almost
complete loss of stereospecificity was earlier observed
for the (R)- and (S)-Atc3 analogs of Tyr-D-Orn-Phe-Glu-
NH2, a slightly µ selective peptide17 and (R)- and (S)-
Atc3 analogs of deltorphin I.10,11 The bioselectivity of
Ile5,6-deltorphin I with (R)-Atc at position 3 (compound
3) was about 2 times higher than that of Ile5,6-deltorphin
I. In the cases of Ile5,6-deltorphin I and Ile5,6-deltorphin
II (compounds 1 and 2), [(S)-Atc]3 and [(R)-Atc]3 sub-
stitutions, respectively, resulted in the increased affinity
in the MVD assay. The most selective was (R)-Atc3,
Ile5,6-deltorphin II (compound 5), where the ratio GPI/
MVD was higher than 200 000.
The sequence of potency and selectivity of the analogs
in the in vitro bioassay differs from the one in the
binding assay, which was already observed for other
opioids.10,17 A possible difference between peripheral
and central opioid receptors has been proposed as an
explanation.22
These results confirmed that an increase in the
lipophilicity of deltorphins in the address part at posi-
tions 5 and 6 results in more active and more δ selective
analogs. Very recently, Sasaki and Chiba reported new
deltorphin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) ana-
logs with NR-alkylglycine at positions 5 and 6 wich also
exhibit higher affinity and δ selectivity as compared to
the parent peptide.23
gauche (+).12 Although it was shown that trans was
preferred in solution, a receptor-induced change to
gauche (+) cannot be excluded. Earlier Tic3-deltorphin
II was synthesized by us12 and Tic3-deltorphin I by
Shiller and Salvadori and their co-workers.10,11 The Tic
residue can only adopt gauche (-) and gauche (+)
conformations in these peptides. The binding and
bioactivity were significantly decreased by this substitu-
tion. This finding could be an indirect evidence that
the preferred bioactive conformation of Phe3 in deltor-
phin is trans.
Replacement of Phe3 with Atc in the deltorphins can
produce conformational constraints both in the side
chains and in the backbone. The Atc residue is es-
sentially able to adopt two side chain conformations,17
which are characterized by the torsion angles ø1 ) 180°
(t); ø2 ) +25° and ø1 ) -60° (g(-)); ø2 ) -25° for (S)-
Atc and ø1 ) 180° (t); ø2 ) -25° and ø1 ) +60° (g(+));
ø2 ) +25° for (R)-Atc. The (R)-amino acid adopts a
conformation in the crystal which is close to trans: ø1
) 153.2° and ø2 ) -46.1° (Table 6, Supporting Informa-
tion). This does not, however, exclude a conformational
change when this residue is incorporated into a peptide
or during receptor interaction. However, since the
benzazepin3-deltorphin analog has a high potency (IC50
) 5.0 nM, despite the absence of the important Glu4
side chain),12 and since also the (S)-Atc3-deltorphins are
very potent, and considering that the only common side
chain conformation which is possible in both analogs is
trans, this argues in favor of trans as the δ receptor-
bound conformation. Our results, therefore, do not
support the model proposed by Mosberg26 for the cyclic
peptide J OM-13, in which Phe3 has a gauche (-)
conformation. It is, however, consistent with the model
proposed by Nikiforovich27,28 for J OM-13 and other δ
agonists.
It was previously demonstrated that the NMR pa-
rameters (chemical shift, nuclear Overhauser effects
(NOE’s), and temperature dependence) of the D-Ala2
methyl group in deltorphins are very good probes for
detecting changes in side chain or backbone conforma-
tions in the N-terminal.12,25 We have determined these
NMR parameters for Ile5,6-deltorphin II and for the (S)-
and (R)-Atc analogs in dimethyl sulfoxide (DMSO)
solution. The chemical shifts (Table 7, Supporting
Information) were assigned by using two-dimensional
homonuclear Hartmann-Hahn and rotating frame
NOE’s spectroscopy. A complete sequential analysis
was possible on the basis of the CRH (residue i)-NH
(residue i + 1) NOE’s. The temperature dependence of
the NH chemical shifts, together with the NH-CRH
vicinal coupling constants, are reported in Table 8
(Supporting Information). The conformationally indica-
Con for m a tion a l Stu d ies. Besides the lipophilicity,
our results and those of several other studies10,11,24
proved that properly oriented side chains are needed
for high affinity and selectivity.
From molecular modeling studies, Salvadori et al.11
concluded that in deltorphin I the preferred side chain
conformation of Phe3 is trans (ø1 ) 138.3°), whereas in
the [(S)-Atc3] analog it is gauche (-) (ø1 ) -56.8°).
Moreover, the low energy backbone conformation of the
[(S)-Atc3] analog is calculated to be distinctly different
from that of deltorphin I. We also investigated the effect
of the side chain conformation of the aromatic amino
acids in deltorphins and dermorphins.12,25 NMR studies
comparing deltorphin I and the [(S,S)-âMePhe3] and
[(S,R)-âMePhe3] analogs indicated that the preferred
side chain conformation of Phe3 in solution is gauche
(-).13 With 4-aminotetrahydro-2-benzazepin-3-one as
a conformational constraint for Phe3-Gly4 in the deltor-
phin sequence, and also in the dermorphin sequence,
high δ affinity was observed for both analogs.12,25 This
finding indicates that the preferred gauche (-) confor-
mation for Phe3 in deltorphin cannot be the bioactive
one. The benzazepine constraint excludes the gauche
(-), but still allows two conformations at ø1: trans and

Conformationally Constrained Deltorphin Analogs
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 993
tive NMR parameters for the D-Ala2 methyl protons are
listed in Table 9 (Supporting Information). The chemi-
cal shifts reveal only very minor differences between the
(S)- and (R)-Atc3 analogs. Moreover, chemical shift
differencies with the values for the parent Ile5,6-deltor-
phin II are limited to the D-Ala2 methyl signal. These
similarities do not support significantly different con-
formational behavior as predicted by Salvadori.11 The
observed low-field shift of the D-Ala methyl signal and
the small temperature dependence indicate that the
shielding effect due to the aromatic side chain at
position 3 is absent in both [Atc3] analogs. Together
with the absence of NOE’s for the Atc residue (Table 9,
Supporting Information), this argues in favor of a
common trans side chain orientation for both Atc
residues although an influence of the fixation of the ø2
in Atc, compared to a freely rotating phenyl ring in the
[Phe3] analog, cannot be excluded. Because of the
absence of an R proton, this conformation cannot be
confirmed by coupling constants with the â protons.
Radioligands except DAGO (Amersham) were prepared in
our laboratory as described earlier for naltrindole,21 Ile5,6
-
deltorphin II,8 and TIPP[ψ].19
2-Am in otetr a lin -2-Ca r boxylic Acid (Atc, 1). (R,S)-Atc
was synthesized as described by Schiller et al.17 Briefly, a hot
mixture of aqueous sodium hydroxide (50 mL, 40%) and
propylene glycol (100 mL) was added to 7,8-benzo-1,3-diazaspiro-
[4.5]decane-2,4-dione (21.6 g, 0.1 mol), and the mixture was
stirred and refluxed for 20 h. The solution was diluted with
water (100 mL). The solution was acidified with aqueous HCl
(5 N) to pH 5.5. The desired amino acid slowly precipitated
as a white powder: 16.8 g (88%); mp 309-310 °C; MS m/ e
191 (M+). The purity of the crude product was 98%, and this
was used for further transformations.
N-r-Boc-(R,S)-2-am in otetr alin -2-car boxylic acid (2) was
prepared by literature procedure:29,30 mp 182 °C; 1H-NMR
(ppm) (CDCl3) δ 1.43 (s, 9H), 2.13 (m, 2H), 2.85 (s, 3H), 3.38
(d, 1H), 7.13 (m, 4H), 7.25 (s, 1H).
(R,S)-N-(Tr iflu or oa cet yl)-2-a m in ot et r a lin -2-ca r b oxy-
lic Acid (3). To a solution of 1 (2.87 g, 15 mmol) in TFA (20
mL) was added dropwise trifluoroacetic anhydride (5.21 mL),
and the mixture was vigorously stirred at 0 °C for 2 h and
then for 2 days at room temperature. After the solvent and
excess anhydride had been removed under reduced pressure,
the residue was diluted with water (30 mL), extracted with
ethyl acetate (two times), brine (two times), and water (two
times), dried over MgSO4, and concentrated. The resulting
yellow oil was crystallized from EtOAc and petroleum ether:
yield 2.80 g (65%); mp 186 °C; TLC (chloroform-ethanol-
acetic acid, 90:10:1) Rf ) 0.48; MS m/ e 287 (M+); 1H-NMR
(DMSO) δ 2.05 (m, 1H), 2.40 (m, 1H), 2.73 (m, 2H), 3.12 (d,
1H), 3.25 (d, 1H), 7.1 (m, 4H), 9.5 (s, 1H), 13 (s, 1H).
Con clu sion
The present paper describes the simultaneous ap-
plication of two approaches to the design of highly
selective opioid peptides. In the address domain of
deltorphins with the substitution of Ile instead of Val
at positions 5 and 6, and in the message domain with
the substitution of Atc instead of Phe at position 3, we
synthesized very active and highly selective δ opioid
ligands, which are the most active and most selective
deltorphin analogs reported to date. Conformational
studies provide evidence for little disturbance of the
backbone conformational equilibrium when Phe3 is
replaced by (S)- or (R)-Atc. These conformational stud-
ies in solution cannot rule out that a higher energy
conformer may be the one interacting with the recep-
tor.10 However, the use of the Atc constraint, in
conjunction with Tic and the 4-amino-2-benzazepin-3-
one constraint, allows the conclusion that, during its
interaction with the δ opioid receptor, the side chain of
residue 3 adopts the trans conformation at ø1.
En zym a tic Sep a r a tion of 1 by Ca r boxyp ep tid a se A. 3
(1.5 g) was suspended in 150 mL of water. The pH was
adjusted to 7.5-8 with NaOH, 200 µL of carboxypeptidase A
(∼2-3 mg) was added, and the mixture was stirred for 1 day.
From time to time the pH was adjusted to 7.5-8. After
completition of the reaction, charcoal was added to the solution
to remove the enzyme. After filtration, the pH of the solution
was adjusted to 3 with 1 N HCl. Unreacted 3 was extracted
with ethyl acetate. The aqueous solution was evaporated to
∼50 mL, and the pH was adjusted to 5-6. The precipitated
crystals were filtered off and dried: yield 478 mg, 93%; [R]25
D
) -6.77° (c ) 1.0, 0.3 M HCl/water). The ethyl acetate
solution after drying over MgSO4 was evaporated, and the oil
was crystallized from ethyl acetate/petroleum ether: yield 734
mg (98%); [R]25 ) +4.1° (c ) 1.0, ethanol). The resulting
D
N-TFA-(S)-Atc was hydrolyzed by refluxing with 6 N HCl (40
mL) for 6 h. The solution was evaporated. The residue was
dissolved in 20 mL of water. The pH was adjusted to 6 with
1 N NH4OH. The crystals were filtered off: yield 210 mg
(43%); [R]25D ) +6.3° (c ) 1.0, 0.3 M HCl/water). (R)-Atc‚HBr
was prepared by treating (R)-Atc with 48% HBr/water and
crystallized from water: yield 170 mg (25%); mp 268-270 °C;
Exp er im en ta l Section
Gen er a l Meth od s. Protected and unprotected amino acids
(except Atc), coupling reagents, 4-methylbenzhydrylamine
resin, carboxypeptidase A, R-chymotrypsin, and 7,8-benzo-1,3-
diazaspiro[4.5]decane-2,4-dione were purchased from Sigma-
Aldrich Kft, Budapest, Hungary; Bachem Feinchemikalien AG,
Bubendorf, Switzerland. Precoated plates (silica gel F254
(Merck, Darmstadt, Germany) were used for TLC, while for
the optical purity control of Atc isomers, chiralplate (Macherey-
Nagel, Du¨ren, Germany) was used. The following solvent
systems were used: (I) butanol-acetic acid-water (4:1:1); (II)
butanol-acetic acid-pyridine-water (15:3:10:12); (III) aceto-
nitrile-methanol-water (4:1:1). Ninhydrin, UV light, and I2
were applied to detect the peptides. Reversed-phase HPLC
was performed on a Merck-Hitachi liquid chromatograph with
a Vydac 218TP54 C18 column for analytical purposes or a
Vydac 218TP1010 C18 column for semipreparative separations.
[R]25 ) -7.16° (c ) 1.0, water); chiral TLC, eluent acetoni-
D
trile-methanol-water (6:1:1), Rf (Atc from aqueous solution)
0.57; Rf (Atc from organic solution) 0.63.
En zym a tic Sep a r a tion of 1 w ith r-Ch ym otr yp sin .
(R,S)-Atc-OCH3 (600 mg) was prepared by a known method,15
using methanol and SOCl2 dissolved in 10 mL of methanol and
40 mL of water. The pH was adjusted to 7.9 with NaOH, 6
mg of enzyme (R-chymotrypsin) was added, and the mixture
was stirred for 1 day. The unreacted ester was extracted with
ethyl acetate. The aqueous layer was treated with charcoal,
and after evaporation to 10 mL, the pH was adjusted to 3.
The crystals were filtered off: yield 108 mg (38.6%). The ethyl
acetate solution was dried on MgSO4 and evaporated. The oil
was crystallized as the HCl salt from ether-HCl. The crystals
were filtered off and dried: yield 135 mg (45%). According to
chiral TLC, the Atc formed from aqueous solution was an S/R
mixture, but the S isomer predominanted: ∼3:1.
Quantitative amino acid analyses were performed on an HP
1090 Amino Quant amino acid analyzer (Hewlett-Packard,
Waldbronn, Germany). A Hypersyl ODS C18 column (200 × 2
mm i.d., 5 µm particle size, Shandon Scientific) was used.
Molecular weights of peptides were determined by FAB mass
spectrometry on a double-focusing MS 902S spectrometer
(A.E.I. Scientific Division, Manchester, U.K.).
Solid -P h a se Syn th esis a n d P u r ifica tion of th e Dia s-
ter eom etr ic P ep tid es. Peptide synthesis was performed by
the manual solid-phase technique using 4-methylbenzhydry-
lamine resin (0.8 mmol/g of titratable amine). Boc-protected
1H-NMR spectra were measured on a Brucker AM 400
spectrometer (Brucker, Zug, Switzerland).

994 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
To´th et al.
amino acids and dicyclohexylcarbodiimide (DCC) and 1-hy-
droxybenzotriazole (HOBt) as coupling agents were used. Side
chain protections were benzyl for Asp and Glu and dichloro-
carbobenzyloxy for Tyr. 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,31 (9) Boc
deprotection with 50% TFA in DCM (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 lyophilized. Crude peptides were purified to
homogenity by semipreparative reversed-phase high-perfor-
mance chromatography (RP-HPLC) on a Vydac 218TP1010 C18
column with a linear gradient of 0.1% TFA of water (eluent
A) and acetonitrile-0.1% TFA (eluent B); starting 25% aceto-
nitrile to 30% acetonitrile for 25 min. After the column was
washed with 80% acetonitrile for 10 min, it was equilibrated
with 25% acetonitrile to be ready for the next run. The final
products were obtained as lyophilizates. Peptide purity was
assessed by analytical RP-HPLC and TLC in three different
solvent systems. Fast atom bombardment mass spectrometry
confirmed the appropriate molecular weight (Table 10, Sup-
porting Information). Amino acid analyses gave the expected
amino acid patterns.
Det er m in a t ion of t h e Con figu r a t ion of At c in t h e
P ep tid es. After RP-HPLC purification, two diastereomeric
peptides were obtained. One milligram of each peptide was
hydrolyzed separately in 6 N HCl under argon pressure in
Teflon bombs in a microwave oven.32 The solvent was removed
by flushing with argon. The dried samples were used for
separation of the amino acid mixture, and the Atc was collected
(Vydac column 218TP54 C18, 254 nm, linear gradient from 10
to 15% of acetonitrile in 0.1% TFA solution within 30 min (k′
of Atc: 2.73)). Half of the amount of Atc was spotted onto a
chiral TLC plate to determine the Rf value in acetonitrile-
methanol-water, 5:1:1. These Rf values were compared with
those of standard (R)- and (S)-Atc (Rf of (R)-Atc 0.37; Rf of (S)-
Atc 0.43). The other part of the Atc from the HPLC separation
was used for GITC derivatization.33,34 When this derivatiza-
tion was performed with (R)- and (S)-Atc standards, the k′
values of the Atc derivatives (Vydac 218TP54 column, eluent
0.1% TFA in water-methanol (55:45), k′ of S isomer 2.41; k′
of R isomer 2.97) indicated that the first eluting peptide
contains (R)-Atc. This is true for deltorphin I and deltorphin
II analogs, too.
concentration was determined by the method of Bradford36).
In competition experiments, the following conditions were used
for incubations: [3H]DAGO (35 °C, 45 min); [3H]naltrindole
(25 °C, 60 min); [3H]Ile5,6-deltorphin II (35 °C, 45 min); [3H]-
TIPP[ψ] (25 °C, 30 min). Incubations were started by the
addition of membrane suspension, continued under gentle
vortexing and shaking in a thermal water bath, and termi-
nated by rapid vacuum filtration through Whatman GF/C
filters using a Brandell cell harvester. After two washings
with 10 mL portions of ice-cold buffer, the filters were dried
for 3 h at 37 °C and the radioactivity was measured in a
toluene-based scintillation cocktail, using a Beckman LS
5000TD scintillation spectrometer. The extent of nonspecific
binding was determined in the presence of 10 µM naloxone.
Experimental data were calculated by using the LIGAND
program of Munson and Rodbard.37
GP I a n d MVD in Vitr o Bioa ssa ys. The bioassays of all
the peptides were based on electrically induced smooth muscle
contractions of mouse vas deferens (MVD) and guinea pig
ileum (GPI) myenteric plexus-longitudinal muscle strips.38
The tissues were suspended between platinum axial wire
electrodes in warm (37 °C), oxygenated (95% O2, 5% CO2)
Krebs bicarbonate solution (34 °C and Mg-free for MVD) in
an 18 mL bath and stretched to an optimal tension of 1 g for
GPI and 0.5 g for MVD. The tissues were then allowed to
equilibrate for 15 min and stimulated transmurally with
square-wave impulses (0.1 Hz, 1 ms duration for GPI and 2
ms for MVD) at supramaximal (40 V) voltage. The GPI
preparation was equilibrated for 60 min before the application
of the peptides; the contraction then was stable. The opioid
agonist activity of the drugs was determined by constructing
dose-response curves with the single dose method. The
concentration causing a 50% reducting the contractions was
also established: thus, the IC50 values were estimated. The
IC50 estimates and their associated standard errors were
determined by fitting the mean data to the Hill equation, using
a computerized least squares method.39
Ack n ow led gm en t. This work was supported by
grants from Hungarian Research Fundation (OTKA) T
16991, A42, and T 14898 and from European Com-
munity Copernicus (CIPA-CT94-0226).
Su p p or tin g In for m a tion Ava ila ble: Crystal structure
determination, crystal data and structure refinement, atomic
coordinates and equivalent isotropic displacement parameters,
bond length, bond angles, torsion angles, anisotropic displace-
ment parameters, and hydrogen coordinates for (R)-Atc; two
figures for the crystal structure of (R)-Atc‚HBr and the packing
of the (R)-Atc‚HBr molecules in the crystal structure, as well
as chemical shifts, temperature dependence of NH chemical
shifst and NH-CRH coupling constants, D-Ala2 methyl param-
eters and physicochemical data for deltorphin analogs (12
pages). Ordering information is given on any current mast-
head page.
NMR Stu d y of Ile5,6-Deltor p h in II a n d Its [Atc3] An a -
logs. Samples were prepared by dissolving 2 mg of peptide
in 0.5 mL of 0.1% TFA in water (pH ) 2). This solution was
lyophilized, and the remaining powder was dried over P2O5
in vacuo and then dissolved in 0.5 mL of DMSO-d6 (>99.96%
Aldrich). 1D spectra were measured at 303, 313, 323, and 333
K. 2D HOHAHA, ROESY, and E. COSY spectra were
measured at 313 K.
2D HOHAHA spectra were recorded with an MLEV 17
spin-lock pulse of 60 ms, preceded and followed by 2.5 ms
trim pulses. For NOE spectroscopy (ROESY spectra), a mixing
time of 200 ms was used. The 2D ROESY and HOHAHA
spectra were measured as 256 free induction decays of 2K data
points. Processing was done by multiplication with a π/3
shifted sine-bell window in both dimensions, zero filling to 2K
in ω1, Fourier transformation, phase correction, and baseline
correction in both dimensions.
Refer en ces
(1) Symbols and abbreviations are in accord with the recommenda-
tions of the IUPAC-IUB Commission on Nomenclature (J . Biol.
Chem. 1972, 247, 977-983). All optically active amino acids are
of the L variety unless otherwise noted. Other abbreviations used
are Hfe, homophenylalanine; Atc, 2-aminotetralin-2-carboxylic
acid; TFA, trifluroacetic acid; DCM, dichloromethane; DIEA,
diisopropylethylamine; DMF, dimethylformamide, DCC, dicy-
clohexylcarbodiimide; HOBt, 1-hydroxybenzotriazole; HPLC,
high-performance liquid chromatography; GITC, 2,3,4,6-tetra-
O-acetyl-â-D-glucopyranosylisothiocyanate; DAGO, Tyr-D-Ala-
Gly-NMe-Phe-Gly-ol; TIPP[Ψ], Tyr-TicΨ[CH2NH]Phe-Phe-OH;
U69593, (5R,7R,8R)-(-)-N-methyl-[7-(1-pyrrolidinyl)-1-oxaspiro-
[4.5]dec-8-yl]benzeneacetamide; NTI, naltrindole; DIDII, Ile5,6
deltorphin II.
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method of Simon et al.35 The binding experiments were
performed in 50 mM TRIS-HCl buffer, pH 7.4, at a final
volume of 1.0 mL containing 300-500 µg of protein (protein

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