Design and synthesis of 1-aminocycloalkane-1-carboxylic acid-substituted deltorphin analogues: unique delta and mu opioid activity in modified peptides.

Article abstract of DOI:10.1021/jm950490j

Deltorphin analogues were substituted by a series of achiral C alpha,alpha-dialkyl cyclic alpha-amino acids (1-aminocycloalkane-1-carboxylic acids, Ac chi c, where chi = a hexane, pentane, or propane cycloalkane ring) in position 2, 3, 4, or 2 and 3 in de

Full text of DOI:10.1021/jm950490j

J . Med. Chem. 1996, 39, 773-780
773
Design a n d Syn th esis of 1-Am in ocycloa lk a n e-1-ca r boxylic Acid -Su bstitu ted
Deltor p h in An a logu es: Un iqu e δ a n d µ Op ioid Activity in Mod ified P ep tid es
Angela Breveglieri,† Remo Guerrini,† Severo Salvadori,† Clementina Bianchi,‡ Sharon D. Bryant,§
Martti Attila,| and Lawrence H. Lazarus*,§
Department of Pharmaceutical Sciences and Institute of Pharmacology, University of Ferrara, I-44100 Ferrara, Italy, Peptide
Neurochemistry Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709,
and Department of Pharmacy, Division of Pharmacology and Toxicology, University of Helsinki, SF-00014 Helsinki, Finland
Received J uly 3, 1995X
Deltorphin analogues were substituted by a series of achiral CR,R-dialkyl cyclic R-amino acids
(1-aminocycloalkane-1-carboxylic acids, Acxc, where
) a hexane, pentane, or propane
x
cycloalkane ring) in position 2, 3, 4, or 2 and 3 in deltorphin C, and in position 2 in [Ac6c2,-
des-Phe3]deltorphin C hexapeptide. Receptor assays indicated that even though Ac6c2 and Ac6c3
exhibited a diminished Kiδ by ca. 20-fold (2.5-3.3 nM) relative to deltorphin C (Kiδ ) 0.15
nM), selectivity was marginally elevated (Kiµ/Kiδ ) 1250) or enhanced by about 70%, and both
peptides fitted stringent iterative calculations for a two-site binding model (η ) 0.625 and
0.766, respectively, P < 0.0001). The disubstituted [Ac6c2,3]- or [Ac6c2,des-Phe3]deltorphin
analogues yielded peptides with decreased Kiδ, such that the latter peptide was essentially
inactive. The presence of Ac5c or Ac3c in place of Phe3 further diminished Kiδ (15.4 to 19.0
nM), yet δ selectivity only fell about one-half (Kiµ/Kiδ ) 440 and 535, respectively), and only
the former peptide fitted a two-site binding model (η ) 0.799). The replacement of Asp4 by
Ac6c, Ac5c, or Ac3c produced essentially nonselective analogues through the acquisition of high
µ affinities (2.5, 0.58, and 0.27 nM, respectively) while maintaining high δ affinities (Kiδ )
0.045-0.054 nM) which were about 3-fold greater than that of deltorphin C. Using
pharmacological assays in vitro (mouse vas deferens and guinea pig ileum), position
3-substituted analogues all indicated substantial losses in bioactivity, whereas substitution
by 1-aminocycloalkanes at the fourth position retained high δ activity. In fact, the bioactivity
of [Ac3c4]deltorphin C indicated a peptide with relatively weak δ selectivity, which was
comparable to the observations with the receptor binding data. In summary, the data confirmed
that (i) δ selectivity occurs in the absence of D-chirality at position 2, (ii) the aromaticity of
Phe3 is replaceable by an achiral residue with a hydrophobic ring-saturated side chain, and
(iii) the acquisition of dual high-affinity analogues occurs through the elimination of the anionic
function at position 4 and replacement by an amino acid with a hydrophobic side chain.
In tr od u ction 1
example, through the extensive alteration to or replace-
ment of Phe3.8,9,12,13 On the other hand, analogues in
which the acidic function at position 4 was substituted
by amino acids with aliphatic side chains produced
peptides with minor changes in δ affinities, although δ
selectivity was nearly ablated due to the acquisition of
greatly enhanced µ affinities.6,7,14-16 Other studies on
sequentially C-terminal abbreviated peptides indicated
that the N-terminal sequence tetrapeptides contained
the crucial elements required for the expression of opioid
activity since receptor selectivity underwent a reversal
from δ to µ.4,7,14,17 In toto, the observations with opioid
agonists re-enforced the universality that the opioid
“message” concept was confined within the N-terminal
sequence, which came into prominence through the
development of opioidmimetic di-,18 tri-,18,19 and tet-
rapeptide antagonists.19
Deltorphins are a group of opioid heptapeptides from
amphibian skin that interact with high affinity and
selectivity for the δ opioid receptor. In contrast to the
enkephalin analogues, the deltorphins and µ specific
dermorphins contain the common N-terminal tripeptide
sequence H-Tyr1-D-Xaa2-Phe3, where D-Xaa2 is either
D-Met2 in deltorphin A (termed deltorphin,2 dermorphin
gene-associated peptide,3 or dermenkephalin4), D-Ala2
in dermorphins and deltorphins B and C ([Glu4]- and
[Asp4]deltorphins II and I, respectively2), or D-Leu2 in
a deltorphin-like tridecapeptide which is not an active
opioid ligand.5 An L-isomer at the second residue
virtually inactivates these peptides.2,3 Investigations in
which normal or unusual amino acid analogues were
substituted for residues in the N-terminal domain
(positions 1-4) of deltorphin revealed that δ affinity and
discrimination between δ and µ receptors (δ selectivity)
depended on the properties of the side chain and the
chirality of the amino acid,8-13 as seen, by way of
Ra tion a le
The essential amino acid sequence in deltorphin B or
C lies within the N-terminal tetrapeptide region H-Tyr1-
D-Ala2-Phe3-(Glu/Asp)4, in which the two essential aro-
matic side chains are separated by a key D-isomer.
However, the introduction of Tic in deltorphin at the
second position12,20 and the formation of abbreviated
Tic2-containing opioidmimetic peptides provided new
insights that the residue at the second position could
* Author to whom correspondence should be addressed at NIEHS,
P.O. Box 12233, Research Triangle Park, NC 27709. Fax: 919-541-
0626.
† Department of Pharmaceutical Sciences, University of Ferrara.
‡ Institute of Pharmacology, University of Ferrara.
§ National Institute of Environmental Health Sciences.
| University of Helsinki.
X Abstract published in Advance ACS Abstracts, J anuary 1, 1996.
0022-2623/96/1839-0773$12.00/0 © 1996 American Chemical Society

774 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Breveglieri et al.
Resu lts a n d Discu ssion
Recep tor Bin d in g An a lyses. The effect of substi-
tuting the 1-aminocycloalkane-1-carboxylic acids at
position 2 (compound 2), 3 (compounds 3, 7, and 9), 4
(compounds 4, 8, and 10), or 2 and 3 (compound 5) in
deltorphin C heptapeptides or position 2 in [Ac6c2,des-
Phe3]deltorphin C hexapeptide (compound 6) resulted
in distinct differences in receptor binding parameters
(Table 1).
The inclusion of Ac6c at position 2 (peptide 2) yields
a peptide lacking D-chirality and induced analogous
decreases in both δ and µ affinities (ca. 20-30-fold) to
that of Ac6c at position 3: That peptide (3) lacks one of
the two aromatic side chains and increased in δ selec-
tivity. However, in comparison to peptide 1, the δ
selectivity of peptide 2 increased ca. 30% and that of
peptide 3 rose 70%. The simultaneous inclusion of Ac6c
at positions 2 and 3 (compound 5), which concomitantly
lost both chirality and the aromaticity associated with
Phe3, yielded a peptide with substantially diminished
δ affinity and δ selectivity.
F igu r e 1. Structures of the 1-aminocycloalkane-1-carboxylic
acids showing the alkane rings of propane, pentane, and
hexane.
be replaced by a heterocyclic residue to produce opioid
peptides that behaved as δ antagonists.18,19 Further,
modification of the physicochemical properties of the
phenylalanyl side chain, such as alteration of the
electronic configuration of Phe through para substitu-
tion by halogens and amino and nitro groups13 or spatial
displacement of the aromatic ring and replacement by
various amino acids,8,9 supported the view that aroma-
ticity at position 3 was responsible for receptor binding.
The [Ac6c2,des-Phe3]deltorphin hexapeptide 6 enabled
direct observation on the effect of 1-aminocyclohexane
in the absence of an aromatic side chain at the third
residue, being substituted by a branched aliphatic side
chain in Val; δ receptor binding activity was essentially
eliminated, more so than in hexapeptides containing
Phe3 in which the C-terminal Gly7 residue was omit-
ted.4,6,17 These binding data would tend to suggest that
even though 1-aminocyclohexane can effectively replace
D-Ala2 (peptide 2), an aliphatic side chain containing
substituent at position 3 fails to allow proper alignment
with the ligand-binding domain of the δ receptor with
a hexapeptide. Whereas the substitutions of Phe3 by
Ac5c (peptide 7) and Ac3c (peptide 9) also decreased δ
and µ affinities, δ selectivities only diminished by about
one-half.
The receptor binding data demonstrate marginal
affects on affinity under the following conditions: (i) the
loss of chirality of the residue at the second position
occurs in the presence of Phe3 and (ii) the replacement
of Phe3 by a ring-saturated hydrophobic side chain led
to only moderate changes in δ selectivity. However, an
aromatic residue in the third position of deltorphin C
heptapeptides enhanced δ affinity (peptide 1).8,9,15
The elimination of the negatively charged Asp4 by
either Ac6c (peptide 4), Ac5c (peptide 8), or Ac3c (peptide
10) produced deltorphin analogues with remarkable and
unanticipated properties, namely, the simultaneous
attainment of both high δ and µ affinities (Table 1). The
augmentation of µ affinities relative to deltorphin C
(peptide 1) was 60-, 250-, and 540-fold for Ac6c, Ac5c,
and Ac3c, respectively, such that the latter two ana-
logues were essentially nonselective for either receptor
type. Thus, replacement of the acidic function produced
peptides with dual high affinities,21 a phenomenon we
termed “opioid infidelity”;16 this was similarly observed
with other position 4 deltorphin analogues containing
hydrophobic side chain residues with aliphatic,15,16,21
cyclic imino,21 indole,14 or aromatic properties.14 How-
ever, charge neutralization by the substitution of Asp
or Glu by Asn or Gln, respectively, which moderately
enhanced µ affinity, failed to eliminate δ selectivity even
though δ affinity remained high.6,15,30 The inference
provided by these data is that a negatively charged
1
Molecular dynamics conformations13,21,22 and H-NMR23
analyses provided evidence on the existence of â-turns
involving H-Tyr-D-Ala-Phe and revealed that the D-
chirality of the Ala side chain apparently stabilized the
â-turn.
In our attempt to further understand the structural
and chemical features of the residues in the 2-4
positions in deltorphin heptapeptides that influence
binding to opioid receptors, we systematically substi-
tuted the achiral ring-saturated CR,R-dialkyl cyclic R-ami-
no acids (1-aminocycloalkane-1-carboxylic acids) for
D-Ala2, Phe3, or the anion at position 4; the cycloalkane
contained either a three-, five-, or six-membered satu-
rated side chain ring (Figure 1). (It is noteworthy that
1-aminocyclopropane-1-carboxylic acid is a natural amino
acid found in plants24 as an intermediate in the conver-
sion of methionine to ethylene25,26 and interesting in
that it acts as a partial agonist on the NMDA receptor
and prevents tolerance to µ and δ agonists in vivo.27)
Studies with model peptides have shown that the
1-aminocyclopropane substitution can be accommodated
into γ as well as helical turns (310- or R-helices) in small
peptides,28,29 although all CR,R-dialkylated glycyl-con-
taining peptides folded into a type II â-turn (C10-helix)
based on crystallographic data.29 Therefore, since it is
well established by 1H-NMR studies23 and conforma-
tional motifs developed by molecular dynamics simula-
tions21,22 that deltorphins contain a â-turn in their
N-terminus, the possibility exists that the cyclic CR,R
-
dialkyl residues might then represent ring-saturated
analogues of Phe3 without major disruptive effects on
receptor properties. Our data demonstrate several
unique properties imparted to deltorphin analogues by
the incorporation of 1-aminocycloalkane-1-carboxylic
amino acids in the N-terminal sequence of positions
2-4.

Cycloalkanecarboxylic Acid Deltorphin Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 775
Ta ble 1. Receptor Binding Parameters of 1-Aminocycloalkane-1-carboxylic Acid-Substituted Deltorphin Analogues
no.
peptide
Kiδ (nM)
η
Kiµ
η
δ selectivity (µ/δ)
1
2
3
4
5
6
7
8
9
Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2
Tyr-Ac6c-Phe-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Ac6c-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Phe-Ac6c-Val-Val-Gly-NH2
Tyr-Ac6c-Ac6c-Asp-Val-Val-Gly-NH2
Tyr-Ac6c-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Ac5c-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Phe-Ac5c-Val-Val-Gly-NH2
Tyr-D-Ala-Ac3c-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Phe-Ac3c-Val-Val-Gly-NH2
0.15 ( 0.03 (8)
2.48 ( 0.58 (5)
3.28 ( 0.76 (7)
0.045 ( 0.02 (4)
87.8 ( 28.6 (4)
5264 ( 1073 (6)
15.4 ( 1.9 (4)
-0.951
-0.625
-0.766
-1.025
nda
147 ( 29 (11)
3110 ( 88.0 (2)
5465 ( 1259 (4)
2.45 ( 0.37 (4)
nda
980
1250
1670
54
170
84
440
10
535
5
nda
nda
-1.012
14 824 ( 2787 (3)
440 667 ( 49 512 (3)
6822 ( 1041 (4)
0.58 ( 0.08 (5)
10 167 ( 2241 (3)
0.27 ( 0.03 (3)
nda
nda
nda
-0.799
-1.128
-0.956
-0.929
nda
0.054 ( 0.01 (4)
19.0 ( 2.7 (4)
-1.004
nda
10
0.054 ( 0.012 (3)
-1.076
a
nd ) not determined. The numbers in parentheses indicate the n values.
Ta ble 2. Bioassays of Position 3-Substituted
residue causes an electrostatic repulsion between the
ligand and the µ receptor which apparently contains an
Asp residue at the binding site.31 A corollary to this
proposal is that an anion in deltorphin or the opioid-
mimetic peptides18 plays a minor role in the acquisition
of δ affinity,6,14,30 serving only to discriminate between
δ and µ receptors.
1-Aminocycloalkane-1-carboxylic Acid Deltorphin Analoguesa
IC50 (nM)
peptide
MVD
GPI
1, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2
3, Tyr-D-Ala-Ac6c-Asp-Val-Val-Gly-NH2
0.46 ( 0.24 337 ( 68
540 ( 130 >10 µM
4, Tyr-D-Ala-Phe-Ac6c-Val-Val-Gly-NH2 0.52 ( 0.06 558 ( 10
7, Tyr-D-Ala-Ac5c-Asp-Val-Val-Gly-NH2 1640 ( 260 >10 µM
8, Tyr-D-Ala-Phe-Ac5c-Val-Val-Gly-NH2 0.19 ( 0.01 140 ( 3.9
In spite of the reduction of δ affinity with deltorphin
analogue 2, the maintenance of δ selectivity succinctly
implies that the D-stereoconfiguration at position 2 is
not an absolute requirement as previously consid-
ered.2,3,7,20 Moreover, the configuration at the R carbon
of this residue may impart new properties on the
peptide; i.e., the change in chirality of Tic2 from D to L
configuration reversed the selectivity in dermorphin
analogues.32 The aromaticity of the side chain at
position 3 (peptides 3, 7, and 9) is partially replaceable
by a saturated alkane ring as long as the heptapeptide
contains D-Ala at position 2. In other words, the alkane
ring cannot concurrently replace both residues (vis-a`-
vis peptide 5). This bisubstitution in the N-terminal
region could adversely affect the secondary structure,
perhaps by the elimination of the existing â-turn.21,22
Despite the decrease in high δ affinities in peptides 2
and 3, their receptor binding capabilities were quite
comparable to that seen with many enkephalin-derived
analogues.
Bin d in g Site Mod els. The analyses of Hill coef-
ficients (η) and fits to one- or two-site binding model
paradigms based on iterative calculations according to
the stringent criteria of Attila et al.33 and Bryant et al.21
[η < 0.85 with narrow log 95% confidence intervals (ca.
0.1) and P > 0.0001] indicated that, with the exception
of peptides 2, 3, and 7, the analogues fitted one-site
binding models; the η for peptides 2, 3, and 7 ranged
from 0.625 to 0.799, in which compound 2 contains a
cyclohexane residue in lieu of D-Ala2 and the latter two
analogues contain cyclohexane and cyclopentane sub-
stitutions for Phe3, respectively. These values are in
keeping with the η observed for [Pro4]- and [D-Asp4]-
deltorphin and deltorphin B,21 in addition to several
Dmt-Tic-Ala tripeptides,18b which were found previously
to be the only analogues that fit a two-site binding model
based on these stringent criteria.21 A low-energy con-
former of [Ac6c3]deltorphin C (compound 3)34 determined
using molecular dynamics stimulations indicated that
the solution conformation resembles that of peptides
which fit two-site binding models21 and differed from
those interacting with a single binding site paradigm.
Furthermore, even though peptides 3 and 7 interact
with the δ receptor in a complex, heterogeneous manner,
they nonetheless associate with µ sites through a simple
bimolecular interaction of a one-site binding model as
9, Tyr-D-Ala-Ac3c-Asp-Val-Val-Gly-NH2
10, Tyr-D-Ala-Phe-Ac3c-Val-Val-Gly-NH2 0.43 ( 0.05 15 ( 1
458 ( 40 >10 µM
a
The IC50 values (nM) represent the mean ( SE of four
independently conducted experiments using mouse vas deferens
(MVD) and guinea pig ileum (GPI) as described in the Experi-
mental Section. The Ke values for the antagonists naloxone and
ICI 174,864 were both within the range of 1-2 nM.
observed with other deltorphin analogues.21 In this
respect, deltorphin analogues with high µ affinities
(peptides 8 and 10) interact with µ receptors in a
manner comparable to that of dermorphin.33
Bioa ctivity. The replacement of Phe3 (peptide 1) by
Ac6c (peptide 3), Ac5c (peptide 7), and Ac3c (peptide 9)
on the activity recorded with mouse vas deferens proved
to be more detrimental than binding to brain membrane
receptor sites. On the other hand, substitution by
1-aminocycloalkanes at position 4 in place of Asp
produced peptide analogues (4, Ac6c; 8, Ac5c; and 10,
Ac3c) in which the bioactivity on mouse vas deferens
reflected their high δ affinities (Table 1). However, the
dual high δ and µ receptor affinities observed with
peptides 4 and 8 were not replicated in the guinea pig
ileum assay suggesting the possible existence of a
different peptide conformation which is poorly recog-
nized by the µ receptors associated with this tissue. In
contrast, peptide 10 (Ac3c4) demonstrated relatively high
affinity in both bioassays (Table 2). Whereas δ affinity
maximally decreased about 120-fold in peptide 9 (Table
1), its bioactivity fell ca. 1000-fold relative to deltorphin
C (Table 2). This further suggests the possibility that
peripheral δ receptors differ from those in the central
nervous system or that the lack of agonist activity could
indicate possible acquisition of antagonist activity,
which is observed in analogues modified at position 3
due to alteration of their conformation determined by
molecular dynamics simulations.33
Con clu sion s
The CR,R-dialkylated cyclic amino acid-substituted
deltorphin analogues provide sufficient data to conclude
the following interactions with the ligand-binding do-
main of opioid receptors: (i) the retention of δ selectivity
occurred in the absence of D-chirality at position 2, and
the requirement for a D-isomer is no longer consider

776 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Breveglieri et al.
Sch em e 1. Synthetic Scheme for the Synthesis of
crucial for receptor binding; (ii) the partial replacement
of the aromaticity associated with Phe3 by ring-
saturated side chain suggests that hydrophobic interac-
tion per se may be an equal or more important element
than the aromatic side chain, although peptide confor-
mation may impart an additional effect on the associa-
tion between the aminocycloalkane-containing ligand
and the receptor; (iii) the elimination of the anionic
function which yielded heptapeptides with high affinity
for both δ and µ receptor sites with the concomitant loss
of δ selectivity provides further evidence that the
peptide anion causes a repulsion from the µ receptor,
confirming the presence of a negatively charged group
in the receptor binding pocket;31 (iv) compounds 2, 3,
and 7 fit a two-site binding model which is considered
to be related to the δ2 receptor subtype due to modifica-
tion in the topographical conformation of the peptide21
in confirmation that CR,R-dialkylated R-amino acids
induce and maintain helical conformation in model
peptides;28,29 and (v) the simultaneous deletion of second
position D-chirality and third position aromaticity in a
hexapeptide analogue that lost both δ and µ affinities
suggests that the even though the hydrophobic nature
of this sequence is maintained, the change in conforma-
tion is incompatible with that of the receptor pocket.
1-Aminocycloalkane-1-carboxylic Acid Analogues of
Deltorphin C (Protective groups or substituents (P, P′,
P′′) on the Tyr residue are designated in the key)
A saturated alkane ring at position 2 or 3 enabled the
peptide to acquire the specific conformation necessary
for receptor recognition through the maintenance of
appropriate â-turns in the N-terminal region.13,21,22
Since data established that 1-aminocyclopropane-1-
carboxylic acid, and CR,R-dialkyl R-amino acids in gen-
eral,29 permitted the formation of helical turns in model
peptides,28 retention of opioid binding parameters in the
absence of the essential D-chirality in the second
position2-4 suggests that if a substituted amino acid side
chain permits the N-terminal sequence to sustain the
necessary â-turn found in deltorphin,21,22 the analogue
will exhibit appropriate binding parameters. The un-
precedented presence of high dual affinity in heptapep-
tide analogues upon elimination of the negative charge
at position 4 was similarly observed by substitution with
amino acids containing an aliphatic side chain21 and
supported by in vitro pharmacological bioassays with
peptide 10. The enhancement of µ affinity confirms
earlier observations that hydrophobic factors strongly
influence binding to µ receptor sites.15,17,18 The ability
to bind with near equal avidity between two distinct
receptor molecules (δ and µ) provides valuable clues on
conformational differences that are brought into play
during binding of opioid ligands by their respective
receptors.16,21,22,34 Furthermore, the high-affinity in-
teraction with the µ ligand-binding domain by peptides
8 and 10 through a one-site binding model also implies
that their conformation must partially resemble that of
dermorphin. Whereas spatial orientation must also be
ideally suited for interaction with the ligand-binding
domain of the receptors, peptide sequence6,15 and amino
acid composition4,6-9,12,14,15 allows for differentiation.
Further, Hill coefficients based on stringent iterative
calculations in combination with molecular dynamics
simulations21,34 provide additional clues on the potential
conformation of peptides that are recognized by common
factors in the disparate δ and µ receptors of rat brain
membranes.
Exp er im en ta l Section
Ma ter ia ls. Enkephalin-derived peptides, cyclic [D-Pen2,5
]
(DPDPE) and [D-Ala2,NRMePhe4,Gly-ol5] (DAGO), were prod-
ucts of Bachem (Torrance, CA); [3H]DPDPE (60.0 Ci/mmol)
was from Amersham (Arlington Heights, IL) and [3H]DAGO
(37.1 Ci/mmol) from NEN-DuPont (Billrica, MA). The follow-
ing compounds were purchased from Sigma Chemical Co. (St.
Louis, MO): bacitracin, bestatin, bovine serum albumin (RIA
grade), HEPES [N-(2-hydroxyethyl)piperazine-N′-2-ethane-
sulfonic acid], phenylmethanesulfonyl fluoride, soybean trypsin
inhibitor, and ultrapure sucrose. Glass microfiber (GF/C)
filters were from Whatman International, Ltd. (Maidstone,
U.K.). Amino acids and protected amino acids containing the
protecting groups Fmoc [NR-[(9-fluorenylmethyl)oxy]carbonyl],
Boc (NR-tert-butoxycarbonyl), OtBu (tert-butyl ester), and OBzl
(benzyl ester) were products of Bachem (Switzerland), and the
resin Fmoc-PAL-PEG-PS was from Millipore (Waltham, MA).
All other chemicals were of the highest purity grade available.
P ep tid e Syn th esis. All deltorphin C ([Asp4]deltorphin I)
analogues were synthesized by solution methods and, in some
cases, by mixed solution-solid phase methods as depicted in
Scheme 1.
The cyclic CR,R-dialkylated residues examined included Ac3c,
Ac5c, and Ac6c. Analogues modified at position 3 (peptides 3,
7, and 9) required esterification of the CR,R-dialkylated residue
followed by soluble carbodiimide condensation with Boc-D-Ala-
OH to give a protected dipeptide. The dipeptides were
deblocked at the N- and C-termini by treatment with NaOH
and TFA and condensed with a protected Tyr residue. Final
condensation was achieved between N-protected tripeptides
and C-terminal tetrapeptide of deltorphin C by soluble car-
bodiimide.
Deltorphin C analogues modified at position 4 (peptides 4,
8, and 10) were synthesized adopting different chemical
strategies. The Ac5c analogue was NR-protected with Z and
condensed with the common C-terminal tripeptide Val-Val-
Gly-NH2 of deltorphins I and II. Catalytic removal of Z by
H2/Pd, followed by condensation with N-terminal tripeptide
Boc-Tyr-D-Ala-Phe-OH, gave the protected heptapeptide ana-
logue. Synthesis of deltorphin peptides containing Ac3c and
Ac6c at position 4 both required esterification of CR,R residues
as reported above and were sequentially condensed with Boc-
Phe-OH and Boc-Tyr-D-Ala-OH. Chemical hydrolysis of the
ester function and final condensation with the C-terminal
tripeptide gave the protected [Ac3c4]deltorphin analogue, while

Cycloalkanecarboxylic Acid Deltorphin Analogues
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3 777
Ta ble 3. Analytical Properties of 1-Aminocycloalkane-1-carboxylic Acid-Containing Deltorphin Analogues
chromatographic parametersa
b
no.
peptide
I
II
[R]20
K′
MH+ c
D
2
3
4
5
6
7
8
9
10
Tyr-Ac6c-Phe-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Ac6c-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Phe-Ac6c-Val-Val-Gly-NH2
Tyr-Ac6c-Ac6c-Asp-Val-Val-Gly-NH2
Tyr-Ac6c-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Ac5c-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Phe-Ac5c-Val-Val-Gly-NH2
Tyr-D-Ala-Ac3c-Asp-Val-Val-Gly-NH2
Tyr-D-Ala-Phe-Ac3c-Val-Val-Gly-NH2
0.33
0.34
0.39
0.27
0.28
0.31
0.60
0.21
0.67
0.64
0.53
0.76
0.51
0.41
0.48
0.68
0.46
0.70
-46.4
-25.2
+30.0
-13.7
-32.3
-20.9
+29.4
-23.5
+13.2
14.02
10.60
11.23
14.15
9.00
9.20
13.11
6.69
823
747
779
801
676
733
765
705
737
11.59
a
TLC determination of Rf was conducted with the following solvent systems: I, 1-butanol/acetic acid/H2O (3:1:1:1, v/v/v); II, ethyl
acetate/pyridine acetate/acetic acid/H2O (12:4:2:2.2, v/v/v/v). K′ is the capacity factor determined by analytical HPLC. c The mass ion
b
(MH+) was obtained by mass spectrometry.
the intermediate Boc-Tyr-D-Ala-Phe-Ac6c-OH was used in the
solid phase of the acylation of the Val-Val-Gly-resin.
Analogues substituted in position 2 (peptide 2) required
protection as an ester function of Ac6c as reported and
condensation with Boc-Tyr-OH through IBCF. The dipeptide
ester function Boc-Tyr-Ac6c-OMe was normally removed and
condensed with Phe-OMe; again the ester function of Boc-Tyr-
Ac6c-Phe-OMe was removed, and the N-protected tripeptide
Boc-Tyr-Ac6c-Phe-OH was condensed with the C-terminal
tetrapeptide Asp(OtBu)-Val-Val-Gly-NH2 of deltorphin C.
The doubly substituted analogue (peptide 5) was obtained
first by condensation via EDC/HOBt with Boc-Ac6c-OH and
H-Ac6c-OMe to obtain a protected dipeptide. Boc was removed
followed by Boc-Tyr(But)-OH condensation to give the pro-
tected tripeptide Boc-Tyr(But)-Ac6c-Ac6c-OMe, which was hy-
drolyzed at the C-terminal function followed by one pot-step
solid phase acylation of Asp(OtBu)-Val-Val-Gly-resin. Peptide
6 was obtained during the first solid phase synthesis of the
doubly substituted analogue; under the conditions outlined
below, the coupling between Fmoc-Ac6c-OH and the H-Ac6c-
resin normally does not proceed in an efficient manner.
Solid P h a se Syn th esis. Solid phase synthesis was per-
formed in a Milligen 9050 synthesizer using a Rink resin (0.47
mmol/g; 0.1 g in all syntheses) obtained from Bachem (Tor-
rance, CA). The resin was mixed with glass beads (1:15, w/w)
purchased from Sigma (St. Louis, MO). Protected amino acids
were from either Bachem or NovaBiochem. Peptides were
assembled using Fmoc-protected amino acids (4-fold excess)
and DIPCDI (4-fold excess) and HOBt (4-fold excess) as
coupling agents and coupled for 1 h at each step. The
incorporation of Val5 required double coupling. Final acylation
step with Boc-Tyr-
the amino acid derivatization reagent. Ac6c and Ac5c were not
quantitatively determined during the amino acid analysis.
Lyophilized samples of peptides (50-1000 pmol) were placed
in heat-treated borosilicate tubes (50 × 4 mm), sealed, and
hydrolyzed using 200 mL of 6 N HCl containing 1% phenol in
the Pico-Tag workstation for 1 h at 150 °C. A Pico-Tag column
(15 × 3.9 mm) was used to separated the PITC-amino acid
derivatives.
TLC was performed in precoated plates of silica gel F254
(Merck, Darmstadt, Germany) using the following solvent sys-
tems: (I) 1-butanol/acetic acid/H2O (3:1:1, v/v/v), (II) ethyl
acetate/pyridine acetate/acetic acid/H2O (12:4:2:2.2, v/v/v/v),
and (III) CH2Cl2/methanol/toluene (17:2:1, v/v/v). Ninhydrin
(1%) or chlorine iodine spray reagents were employed to detect
the peptides.
Melting points were determined on a Reicher-Kofler ap-
paratus and are uncorrected. Optical rotations used a Perkin-
Elmer 241 polarimeter with a 10 cm cell using methanol at a
peptide concentration of 1%. Chemical characteristics of each
peptide were confirmed using 1H-NMR spectroscopy with a 200
MHz Bruker instrument and are recorded in δ units. Molec-
ular weights were determined by a triple-stage quadrapole
mass spectrometer (TSQ 700, Finnigan MAT) equipped with
a pneumatic electrospray (ionspray) interface, and the data
were compiled using a DEC 5000/125 computer (Table 3).
Cou p lin g P r oced u r es. Meth od A. To a solution of the
carboxy component (3.50 mmol) in DMF (15 mL) were added
the amino acid or amino peptide component (3.26 mmol), TEA
(3.26 mmol, if the amino component was in the protonated
form), HOBt (3.52 mmol) to reduce racemate formation, and
coupling reagent, DCC, or EDC (3.52 mmol), in the above order
at 0 °C. The reaction mixture was stirred for 1 h at 0 °C and
for 12 h at room temperature. N′,N′-Dicyclohexylurea was
filtered off (if DCC was added) and the solvent evaporated in
vacuo. The residue was dissolved in ethyl acetate (30 mL) and
washed with 10% citric acid, brine, 5% NaHCO3, and brine,
successively. The organic phase was dried over Na2SO4,
filtered, and evaporated to dryness. The residue was crystal-
lized from the appropriate solvents.
D-Ala-Phe-Ac6c-OH and Boc-Tyr(But)-Ac6c-Ac6c-OH always
required 4-fold excess and 14 h during the recycling coupling
step. The two analogues were cleaved from the resin by
treatment with TFA/H2O/Et2SiH (88:5:7, v/v/v) at room tem-
perature for 1 h.
P u r ifica tion . Crude peptides were purified by preparative
reversed-phase HPLC using a Waters Delta Prep 3000 system
with a Delta Pak C18 (30 × 3 cm, 300 Å, 15 µm spherical
Meth od B. The amino component (amino acid or peptide,
0.62 mmol) and the activated carboxy component (OSu, 0.56
mmol) were dissolved in DMF (6 mL containing 0.62 equiv of
TEA if the amino component was protonated), and the mixture
was allowed to react at room temperature for 4 h. The solvent
was evaporated in vacuo, and the residue was worked up as
in method A.
Meth od C. To a solution of the carboxy component (1
mmol) in DMF (2 mL) were added IBCF (1 mmol), the amino
acid or the amino peptide component (1 mmol), and TEA (1 or
2 mmol if the amino component was in the protonated form)
in the above order at -15 °C. The reaction mixture was stirred
for 1 h at -5 °C and for 12 h at room temperature. The solvent
was evaporated in vacuo and the residue worked up as
described in method A.
particle size) column. The peptides were eluted with
a
gradient of 0-100% B in 25 min at a flow rate of 30 mL/min
using mobile phase A (10% acetonitrile in 0.1% TFA, v/v) and
mobile phase B (60% acetonitrile in 0.1% TFA, v/v).
Analytical HPLC analyses were performed on a Bruker
liquid chromatography LC 21-C instrument fitted with a Vydac
C18 (218 TP 5415, 5 µm, 4.5 × 175 mm) particle column and
equipped with a Bruker LC 313 UV variable-wavelength
detector. Recording and quantification were accomplished
with a chromatographic data processor coupled to an Epson
computer system (BX-10). Analytic determination and capac-
ity factor (K′) of the peptides were determined using HPLC
conditions in the above solvent systems programmed at flow
rates of 1 mL/min: (a) linear gradients from 50% to 100%
solvent B in 25 min and (b) linear gradients from 0% to 100%
B in 25 min. All analogues showed <1% impurities when
monitored at 220 nm.
P r otection P r oced u r es. Meth od D. To a solution of
amino acid (20 mmol) in tert-butyl alcohol (10 mL) were added
1 N NaOH (20 mL) and a solution of Boc2O (22 mmol) in tert-
butyl alcohol (10 mL) in the above order at 0 °C. The reaction
mixture was stirred overnight. The turbid solution was diluted
An a lytica l Deter m in a tion s. Amino acid analyses were
carried out using PITC (Pico-Tag) methodology (Millipore) as

778 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 3
Breveglieri et al.
with 20 mL of water and extracted with pentane (30 mL). The
aqueous phase was acidified to pH 2-3 with solid citric acid
and extracted with three 50 mL portions of ethyl acetate. The
organic phase was dried over Na2SO4. The solvent was
evaporated in vacuo, and the residue was crystallized from
the appropriate solvents.
J ) 7.00 Hz), 1.13-1.68 (10H, 5CH2, ms), 1.42 (9H, 3CH3, s),
2.81-3.22 (4H, 2CH2, ms), 3.65 (3H, CH3, s), 4.17-4.23 (2H,
2CH, m), 4.64 (1H, CH, m), 5.28 (1H, NH, d, J ) 7.69 Hz),
6.62 (1H, NH, d, J ) 7.36 Hz), 6.72 and 6.98 (2H + 2H, C6H4,
2d, J ) 8.20 Hz), 7.24 (5H, C6H5, s), 7.20-7.25 (1H, NH, br),
7.46 (1H, NH, s), 6.16 (1H, NH, s).
Meth od E. To a solution of the amino acid (5 mmol) in
dioxane (5 mL) were added 1 N NaOH (5 mL) and a solution
of Z2O (5.5 mmol) in dioxane (5 mL) in the above order at 0
°C. The reaction mixture was stirred overnight. The turbid
solution was worked up as described in method D.
Meth od F . To a suspension of the amino acid (7 mmol) in
methanol (7 mL) was added dichlorosulfoxide (1.7 mL) drop-
wise at -15 °C. The mixture was stirred at room temperature
for 3 h and than refluxed for 4 h. The solvent was evaporated
in vacuo, and the residue was triturated with diethyl ether.
The resulting solid was collected and dried.
4. An a lytica l Da ta of P r otected In ter m ed ia tes Re-
la ted to [Acxc3] P ep tid es. H-Ac3c-OMe HCl: 91% yield; mp
184-186 °C; Rf 0.48 (III).
Boc-D-Ala -Ac3c-OH: 73% yield; mp 168-170 °C; [R]20
D
+20.6°; Rf 0.79 (II); 1H-NMR (CDCl3 + DMSO) 1.39 (9H, 3CH3,
s), 1.35 (3H, CH3, d, J ) 6.5 Hz), 1.47 (4H, 2CH2, 2m), 2.91
(2H, CH2, m), 4.12 (1H, CH, m), 4.39 (1H, CH, m), 5.76 (1H,
NH, br), 6.74 and 7.00 (2H + 2H, 2d, J ) 8.28 Hz), 7.29 (1H,
NH, br), 7.84 (1H, NH, s).
Boc-D-Ala -Ac3c-Asp (OtBu )-Va l-Va l-Gly-NH2: 82% yield;
mp 138-140 °C; [R]20 +2.1°; Rf 0.26 (III).
D
An a lyses of th e P ep tid e An a logu es. 1. An a lytica l
H-Ac5c-OMe‚HCl: 99% yield; mp 190-192 °C; Rf 0.45 (III).
Da ta of P r otected In ter m ed ia tes Rela ted to [Ac6c2]
Boc-D-Ala -Ac5c-OH: 97% yield; mp 148-150 °C; [R]20
D
+24.5°; Rf 0.86 (II); 1H-NMR (CDCl3) 1.35 (3H, CH3, d, J )
7.0 Hz), 1.44 (9H, 3CH3, s), 1.77 and 2.28 (8H, 4CH2, m), 4.18
(1H, CH, m), 5.23 (1H, NH, d, J ) 7.53 Hz), 7.09 (1H, NH, s).
P ep t id es. Boc-Tyr -Ac6c-OH: 79% yield; mp 210-212 °C;
1
[R]20 -26.6°; Rf 0.90 (II); H-NMR (CDCl3) 1.38 (9H, 3CH3,
D
s), 1.46 and 1.73-2.02 (10H, 5CH2, ms), 2.91 (2H, CH2, m),
4.25 (1H, CH, m), 6.08 (1H, NH, d, J ) 7.29 Hz), 6.70 and
7.03 (2H + 2H, 2d, J ) 8.33 Hz), 7.27 (1H, NH, s).
Boc-Tyr -D-Ala -Ac5c-OH: 94.7% yield; mp 96-98 °C; [R]20
D
+35.4°; Rf 0.88 (II); 1H-NMR (CDCl3 + DMSO) 1.38 (9H, 3CH3,
s), 1.43 (3H, CH3, d, J ) 6.1 Hz), 1.73-2.22 (8H, 4CH2, m),
2.88 (2H, CH2, m), 4.20 (1H, CH, m), 4.42 (1H, CH, m), 5.35
(1H, NH, br), 6.75 and 6.99 (2H + 2H, 2d, J ) 8.38 Hz), 7.08
(1H, NH, br), 7.46 (1H, NH, s).
Boc-Tyr -Ac6c-P h e-OH: 89% yield; mp 128-130 °C; [R]20
D
+4.2°; Rf 0.88 (II); 1H-NMR (CDCl3 + DMSO) 1.38 (9H, 3CH3,
s), 1.22-2.08 (10H, 5CH2, ms), 2.94 (2H, CH2, m), 3.10 (2H,
CH2, m), 4.24 (1H, CH, m), 5.70 (1H, NH, d, J ) 6.83 Hz),
6.75 and 7.03 (2H + 2H, 2d, J ) 8.28 Hz), 6.83 (1H, NH, s),
7.07 (1H, NH, br).
Boc-Tyr -D-Ala -Ac5c-Asp (Ot Bu )-Va l-Va l-Gly-NH 2: 80%
yield; mp 144-146 °C; [R]20 +3.3°; Rf 0.20 (III).
D
Boc-Tyr -Ac6c-P h e-Asp(OtBu )-Val-Val-Gly-NH2: 71% yield;
H-Ac6c-OMe HCl: 94% yield; mp 192-194 °C; Rf 0.49 (III).
mp 125-127 °C; [R]20 -26.7°; Rf 0.33 (III).
Boc-D-Ala -Ac6c-OH: 87% yield; mp 125-126 °C; [R]20
D
D
+38.1°; Rf 0.83 (II); 1H-NMR (CDCl3) 1.36 (3H, CH3, d, J )
7.0 Hz), 1.45 (9H, 3CH3, s), 1.77-2.28 (10H, 5CH2, m), 4.21
(1H, CH, m), 5.2 (1H, NH, br), 7.0 (1H, NH, s).
2. An a lytica l Da ta of P r otected In ter m ed ia tes Re-
la ted to [Ac6c2-Ac6c3] P ep tid es. Boc-Ac6c-Ac6c-OMe: 56%
yield; mp 130-132 °C; Rf 0.76 (III); 1H-NMR (CDCl3) 1.46 (9H,
3CH3, s), 1.30-2.11 (20H, 10CH2, ms), 3.67 (3H, CH3, s), 4.67
(1H, NH, s), 7.43 (1H, NH, br s).
Boc-Tyr -D-Ala -Ac6c-OH: 93% yield; mp 110-113 °C; [R]20
D
+40.6°; Rf 0.80 (II); 1H-NMR (CDCl3 + DMSO) 1.38 (9H, 3CH3,
s), 1.43 (3H, CH3, d, J ) 6.1 Hz), 1.73-2.22 (10H, 5CH2, m),
2.88 (2H, CH2, m), 4.20 (1H, CH, m), 4.42 (1H, CH, m), 5.35
(1H, NH, br), 6.75 and 6.99 (2H + 2H, 2d, J ) 8.38 Hz), 7.08
(1H, NH, br), 7.46 (1H, NH, s).
Boc-Tyr (OtBu )-Ac6c-Ac6c-OH: 61% yield; mp 145-147 °C;
[R]20 -1.7°; Rf 0.27 (III); 1H-NMR (CDCl3) 1.33 (9H, 3CH3,
D
s), 1.39 (9H, 3CH3, s), 1.49-2.24 (20H, 10CH2, ms), 2.90 and
3.15 (2H, CH2, AB or ABX, J AB ) 14.16 Hz, J AX ) 8.45 Hz,
J BX ) 5.95 Hz), 4.28 (1H, CH, X of ABX, m), 5.09 (1H, NH,
br), 6.56 (1H, NH, s), 6.94 and 7.13 (2H + 2H, C6H4, 2d, J )
8.42 Hz), 7.59 (1H, NH, s).
Dep r otection P r oced u r es. Meth od G. Boc and OtBu
protecting groups were removed by treating the peptide with
aqueous 95% TFA for 30 min. The solvent was evaporated in
vacuo, and the residue was triturated with diethyl ether. The
resulting solid was collected and dried.
Meth od H (Hyd r ogen a tion ). Hydrogenation was per-
formed in methanol or acetic acid (10 mL/mmol of peptide) at
atmospheric pressure and room temperature in the presence
of 5% or 10% palladium on charcoal (catalyst to peptide ratio,
1:9, w/w) generally for 1 h (testing with TLC using solvent I).
The catalyst was filtered through paper and evaporated to
dryness. The residue was treated as described in method C
(supra vide).
Meth od I. The peptide methyl ester (5 mmol) was dissolved
in methanol (20 mL) and treated with 1.2 mol equiv of 1 N
NaOH for 2 h at room temperature (TLC tests used solvent
II). The solution was diluted with water and concentrated in
vacuo to remove methanol. After cooling at 0 °C, it was
acidified with 1 N HCl and the product extracted with ethyl
acetate. The organic solution was washed with brine, dried
over Na2SO4, filtered, and evaporated to dryness. The result-
ing solid peptide was crystallized from the appropriate sol-
vents.
Ra d ior ecep tor Assa ys. Receptor affinities of deltorphin
C analogues were assessed using competitive binding assays
labeled either with [3H]DPDPE (0.63 nM) for the δ sites or
with [3H]DAGO (1.28 nM) for the µ sites according to published
methods.13,15,35 Excess unlabeled peptides (2 µM) saturated
the opioid binding sites in order to obtain a base-line value.
Duplicate tubes contained preincubated rat brain synaptoso-
mal membranes3 in equilibrium assays containing 50 mM
HEPES, pH 7.5, 5 mM MgCl2, glycerol, and protease inhibi-
tors3,35 for 120 min at room temperature (22-23 °C). Incuba-
tion mixtures were trapped in the filters and rapidly washed
within 5 s with 3 × 2 mL of ice-cold buffer containing 0.01%
Boc-Tyr -D-Ala -Ac6c-Asp (Ot Bu )-Va l-Va l-Gly-NH 2: 80%
yield; mp 123-126 °C; [R]20 +1.6°; Rf 0.20 (III).
D
3. An a lytica l Da ta of P r otected In ter m ed ia tes Re-
la ted to [Acxc4] P ep tid es. Boc-P h e-Ac3c-OMe: 85% yield;
1
mp 128-130 °C; [R]20 -1.84°; Rf 0.76 (III); H-NMR (CDCl3)
D
0.85-1.00 (1H, 1/2CH2, m), 1.05-1.15 (1H, 1/2CH2, m), 1.41 (9H,
3CH3, s), 1.40-1.73 (2H, CH2, m), 3.03-3.09 (2H, CH2, AB of
ABX, J AB ) 13.5 Hz, J BX ) 7.5 Hz), 3.66 (3H, CH3, s), 4.31
(1H, CH, X of ABX, m), 5.12 (1H, NH, br), 6.39 (1H, NH, s),
7.27 (5H, C6H5, s).
Boc-Tyr -D-Ala -OH: 99% yield; mp 190-193 °C; [R]20
D
+5.7°; Rf 0.89 (I); 1H-NMR (DMSO) 1.16 (3H, CH3, d, J ) 9.7
Hz), 1.29 (9H, 3CH3, s), 2.50-1.85 (2H, CH2, m), 3.95-4.22
(2H, 2CH, m), 6.64 and 7.01 (2H + 2H, C6H4, 2d, J ) 8.18
Hz), 6.75 (1H, NH, d, J ) 6.75 Hz), 8.14 (1H, NH, d, J ) 7.40
Hz), 12.45 (1H, COOH, vbr).
Boc-Tyr -D-Ala -P h e-Ac3c-OMe: 85% yield; mp 110-112 °C;
[R]20 +24.0°; Rf 0.25 (III).
D
Boc-Tyr -D-Ala -P h e-Ac3c-Va l-Va l-Gly-NH2: 78% yield; mp
130-132 °C; [R]20 +5.6°; Rf 0.22 (III).
D
Z-Ac5c-Va l-Va l-Gly-NH2: 55% yield; mp 150-153 °C; [R]20
D
-6.3°; Rf 0.30 (III).
Boc-Tyr -D-Ala -P h e-Ac5c-Va l-Va l-Gly-NH2: 42% yield; mp
200-205 °C; [R]20 +0.39°; Rf 0.35 (III); K′ 6.68 (a).
D
Boc-P h e-Ac6c-OMe: 84% yield; mp 112-113 °C; [R]20
D
-17.0°; Rf 0.76 (III); 1H-NMR (CDCl3) 1.43 (9H, 3CH3, s), 1.45-
2.00 (10H, 5CH2, ms), 3.02 and 3.12 (2H, CH2, AB of ABX,
J AB ) 14.2 Hz, J AX ) 6.50 Hz, J BX ) 7.52 Hz), 3.69 (3H, CH3,
s), 4.33 (1H, CH, X of ABX, m), 5.07 (1H, NH, d, J ) 6.54 Hz),
7.28 (5H, C6H5, s).
Boc-Tyr -D-Ala -P h e-Ac6c-OMe: 46% yield; mp 100-102 °C;
[R]20 +15.8°; Rf 0.57 (III); 1H-NMR (CDCl3) 1.07 (3H, CH3, d,
D

Cycloalkanecarboxylic Acid Deltorphin Analogues
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BSA or lactalbumin. In the duplicate assays, labeled peptides
were displaced using 6-14 concentrations of each analogue
to cover a 1000-fold range in peptide concentration. For
determination of the Hill coefficients and statistical analyses
of the data, however, assays were conducted in triplicate using
25-35 concentrations of peptide and tested for fits to one- or
two-site binding models according to Attila et al.32 and Bryant
et al.21 using Prism (v. 1.03, GraphPad, San Diego, CA).
Competitive inhibition constants (Ki) were derived from the
IC50 values based on the equations of Cheng and Prusoff.36
Bioa ctivity. Bioassays were conducted according to Sal-
vadori et al.13 using a 2-3 cm portion of guinea pig ileum (GPI)
in a 20 mL organ bath containing 70 µM hexamethonium
bromide and 0.125 µM mepyramine maleate aerated with 95%
O2/5% CO2 at 36 °C for µ receptors as follows: GPI was
stimulated transmurally with 0.5 ms square-wave pulses at
0.1 Hz in which the stimulus was 1.5 times that necessary to
produce a maximal twitch (∼30 V) and recorded at a magni-
fication ratio of 1:15. For δ receptors, a single mouse vas
deferens (MVD) was used suspended in 4 mL of modified
Kreb’s solution aerated with 95% O2/5% CO2 at 33 °C while
the twitch induced by field stimulation (0.1 Hz for 1 ms at 40
V) was recorded with a isometric transducer. Dose-response
curves were prepared for each analogue in comparison to
known compounds for each tissue preparation (dermorphin or
morphine for GPI and deltorphin for MVD). The Ke values
for naloxone or N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI
174,864) were in the range of 1-2 nM as detailed previously.13
Ack n ow led gm en t. S. Salvadori was supported in
part by grants from CNR Progetto Finalizzato Chimica
Fine e Secondaria II and Murst. We appreciate the
library services of F. T. Lyndon and her support in
providing the retrieval of published material.
Refer en ces
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Chem. 1972, 247, 977). Other abbreviations: Acxc, CR,R-dialky-
lated R-amino acids (1-aminocycloalkane-1-carboxylic acid) (hex-
ane, ) 6; pentane, ) 5; propane ) 3); Boc, tert-butoxycar-
x
x
x
bonyl; Boc2O, Boc-anhydride (di-tert-butyl dicarbonate); DCC,
dicyclohexylcarbodiimide; DIPCDI, 1,3-diisopropylcarbodiimide;
DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; EDC,
ethyldicarbodiimide; Fmoc, (fluoren-9-ylmethoxy)carbonyl; HOBt,
1-hydroxybenzotriazole; IBCF, isobutyl chloroformate; OtBu,
tert-butyl ester; 1H-NMR, proton nuclear magnetic resonance
spectrometry; Rink resin, 4-(2′,4′-dimethoxyphenyl)-Fmoc-ami-
nomethylphenoxy-resin; TEA, triethylamine; TFA, trifluoroacetic
acid; Z, benzyloxycarbonyl.
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