Design and synthesis of novel hydrazide-linked bifunctional peptides as δ/μ opioid receptor agonists and CCK-1/CCK-2 receptor antagonists

Article abstract of DOI:10.1021/jm050851n

A series of hydrazide-linked bifunctional peptides designed to act as agonists for δ/μ opioid receptors and antagonists for CCK-1/CCK-2 receptors was prepared and tested for binding to both opioid and CCK receptors and in functional assays. SAR studies in the CCK region examined the structural requirements for the side chain groups at positions 1′, 2′, and 4′ and for the N-terminal protecting group, which are related to interactions not only with CCK, but also with opioid receptors. Most peptide ligands that showed high binding affinities (0.1 - 10 nM) for both δ and μ opioid receptors generally showed lower binding affinities (micromolar range) at CCK-1 and CCK-2 receptors, but were potent CCK receptor antagonists in the GPI/LMMP assay (up to Ke = 6.5 nM). The results indicate that it is reasonable to design chimeric bifunctional peptide ligands for different G-protein coupled receptors in a single molecule.

Full text of DOI:10.1021/jm050851n

J. Med. Chem. 2006, 49, 1773-1780
1773
Design and Synthesis of Novel Hydrazide-Linked Bifunctional Peptides as δ/µ Opioid Receptor
Agonists and CCK-1/CCK-2 Receptor Antagonists
Yeon Sun Lee,† Richard S. Agnes,† Hamid Badghisi,‡ Peg Davis,‡ Shou-wu Ma,‡ Josephine Lai,‡ Frank Porreca,‡ and
Victor J. Hruby*,†
Departments of Chemistry and Pharmacology, UniVersity of Arizona, Tucson, Arizona 85721
ReceiVed August 26, 2005
A series of hydrazide-linked bifunctional peptides designed to act as agonists for δ/µ opioid receptors and
antagonists for CCK-1/CCK-2 receptors was prepared and tested for binding to both opioid and CCK receptors
and in functional assays. SAR studies in the CCK region examined the structural requirements for the side
chain groups at positions 1′, 2′, and 4′ and for the N-terminal protecting group, which are related to interactions
not only with CCK, but also with opioid receptors. Most peptide ligands that showed high binding affinities
(0.1-10 nM) for both δ and µ opioid receptors generally showed lower binding affinities (micromolar
range) at CCK-1 and CCK-2 receptors, but were potent CCK receptor antagonists in the GPI/LMMP assay
(up to Ke ) 6.5 nM). The results indicate that it is reasonable to design chimeric bifunctional peptide
ligands for different G-protein coupled receptors in a single molecule.
Introduction
Nerve injury associated pain, neuropathic pain, is very
difficult to treat, and the underlying mechanisms of this
condition are not fully understood. Preclinical studies have
shown that a significant consequence of injuries to peripheral
nerves is an increase in the expression of the endogenous peptide
cholecystokinin (CCKa) in rodents. CCK is known to act via
two types of receptors, termed CCK-1 and CCK-2. CCK has
been shown to reduce the antinociceptive effects of opioids at
a number of CNS sites, particularly in the spinal cord and in
the brainstem, as well as eliciting a direct pronociceptive
action.1-5 Among the three (δ, µ, κ) opioid receptor subtypes,
the activation of µ and δ opioid receptors produces antinoci-
ception; most clinically used opioid drugs, including morphine,
are µ opioid agonists. Acute blockade of spinal and supraspinal
CCK receptors enhances the antinociceptive potency of mor-
phine and may also prevent or reverse the development of
tolerance to opioids.6-9
From this background, we have hypothesized that the
development of novel ligands that possess properties of antago-
nists at CCK receptors and of agonists at the opioid receptors,
within the same molecule, may present a significant therapeutic
advantage in the treatment of pain states and enhanced analgesic
effect in chronic pain states such as neuropathies and analgesia
without the development of tolerance.10,11 This novel idea came
Figure 1. Design of hydrazide-linked bifunctional peptides.
Figure 2. Structure of hydrazide-linked bifunctional peptides synthe-
sized in this study.
out of our earlier discovery that suggested that CCK-2 and δ
opioid receptors have overlapping topographical structural
pharmacophores for their agonist ligands6,7 and led us to develop
SNF9007, which interacts with both the CCK-2 and δ opioid
receptors.14
We have designed novel bifunctional peptide ligands where
both opioid and CCK pharmacophores are fused (Figure 1). We
combined opioid and CCK pharmacophores through a hydrazide
linker so that the designed bifunctional peptide ligands maintain
topologically related structures to both pharmacophores. For the
opioid agonist pharmacophore, the half-structure of biphalin
(Tyr-D-Ala-Gly-Phe-NH-), which has remarkable efficacy at
both δ and µ opioid receptors,15 was chosen. And for the CCK
antagonist pharmacophore, CCK30-33 (Trp-Met-Asp-Phe-NH2)
was selected and modified in different positions to optimize
CCK activities while opioid activities are retained (Figure 2).
Our aim was to develop novel CCK antagonist and opioid
agonist activities in one structure using two known pharma-
cophores as the starting point. A desired requirement was
balanced CCK-1 and CCK-2 antagonist activities, together with
* To whom correspondence should be addressed. Tel: (520)-621-6332.
Fax: (520)-621-8407. E-mail: hruby@u.arizona.edu.
† Department of Chemistry.
‡ Department of Pharmacology.
a Abbreviations: Ac, acetyl; AcOH, acetic acid; Adoc, adamantyloxy-
carbonyl; Boc, tert-butyloxycarbonyl; BOP, (benzotriazole-1-yloxy)tris-
(dimethylamino)phosphonium hexafluorophosphate; BuOH, butanol; Bzl,
benzyl; CCK, cholecystokinin; CHO, Chinese hamster ovary; CNS, central
nervous system; Cpac, o-chlorophenylaminocarbonyl; DCC, N,N-dicyclo-
hexylcarbodiimide; DCU, N,N-dicyclohexylurea; DMF, N,N-dimethylform-
amide; DMSO, dimethyl sulfoxide; hDOR, human δ opioid receptor;
DPDPE, c[D-Pen2,D-Pen5]enkephalin; DAMGO, [D-Ala2,NMePhe4,Gly5-
ol]enkephalin; EtOAc, ethyl acetate; GPCR, G-protein coupled receptor;
GPI, guinea pig isolated ileum; HEK, human embryonic kidney; HOBt,
1-hydroxybenzotriazole; LMMP, longitudinal muscle with myenteric plexus;
rMOR, rat µ opioid receptor; MVD, mouse vas deferens; NMM, N-
methylmorpholine; PG, protecting group; Pya, pyrenylalanine; SAR,
structure-activity relationships; TFA, trifluoroacetic acid; Tac, toluenyl-
aminocarbonyl.
10.1021/jm050851n CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/09/2006

1774 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Lee et al.
Structure-Activity Relationships. As shown in Figure 1,
rational design of the peptides that demonstrate activity as an
opioid agonist and a CCK antagonist was accomplished by
combining opioid and CCK pharmacophores through a hy-
drazide linker. The hydrazide linker should not interfere with
binding interactions with both receptors. And key side chain
groups of both pharmacophores could serve for molecular
recognition at both opioid and CCK receptors. For the opioid
receptors, the two key pharmacophore elements are the pheny-
lalanine and tyrosine residues and, for the CCK receptors, the
indole group of tryptophan, the phenyl group for phenylalanine,
and possibly a carboxyl group from aspartic acid. Considering
these points, the C-terminus of a opioid agonist structure (Tyr-
D-Ala-Gly-Phe-), which is the half-structure of biphalin,15 and
the CCK agonist structure (Trp-Met-Asp-Phe-), which is
CCK30-33, were linked through a hydrazide. To ensure an
antagonist function for CCK, the Trp residue was replaced by
D-Trp.16 Met was substituted by Nle in the way not to change
their character but to avoid unnecessary side reactions. The 2′
and 4′ positions of the CCK portion were N-methylated in some
cases to give increased backbone constraint, and the N-terminal
protecting groups were modified to optimize the biological
activity and physicochemical properties. The N-terminus of the
Tyr residue on the opioid part was not blocked because it was
known that the free amine group plays an important role in
opioid activity.
Figure 3. Synthesis of hydrazide-linked bifunctional peptides.
agonist activities at both δ and µ opioid receptors. The designed
and synthesized peptide ligands were evaluated for (1) their
opioid binding affinities at the human δ opioid receptor (hDOR)
and the rat µ opioid receptor (rMOR) in competition assays
against standard radioligands using cells that express with these
receptors; (2) their opioid efficacies using the [35S]GTP-γ-S
binding assay; (3) their δ/µ opioid agonist activities in stimulated
isolated mouse vas deferens (MVD) and guinea pig isolated
ileum (GPI) bioassays, respectively; (4) their CCK-1 and CCK-2
receptor binding affinities in competition assays against standard
radioligands using transfected cells that express the hCCK-1
or hCCK-2 receptors; and (5) their antagonism and agonism of
CCK receptors in the unstimulated GPI/longitudinal muscle with
myenteric plexus (LMMP). The success of this approach is
reflected in the biological activity of the series of hydrazide-
linked bifunctional peptide ligands. This new series was
optimized to give a number of desirable bifunctional activities
for opioid and CCK receptors.
Opioid binding affinities of these ligands for the human δ
opioid receptor (hDOR) or the rat µ opioid receptor (rMOR)
were determined by radioligand competition analysis using
[3H]c[D-Pen2,D-Pen5]enkephalin (DPDPE) to label the δ opioid
receptor and [3H][D-Ala2,NMePhe4,Gly5-ol]enkephalin (DAM-
GO) to label the µ opioid receptor in cell membrane preparations
from transfected cells that stably express the respective receptor
type. CCK binding affinities were evaluated by competition
analysis using [125I]CCK-8(SO3) to label membranes from
HEK293 cells that express either the human CCK-1 or human
CCK-2 receptors. In Table 2, most of the synthesized peptide
ligands showed high binding affinities at both δ and µ opioid
receptors in the nanomolar range with slight δ or µ selectivity
and lower but well-balanced binding affinities at CCK-1 and
CCK-2 receptors in the micromolar range. No selectivity (0.5-
2, except for 6) for CCK-1 or CCK-2 binding affinities was
seen, which can be significant when considering the possible
role of balanced CCK-1 and CCK-2 antagonists in the treatment
of pain states.17 There have been observations18,19 that show
that although both CCK-1 and CCK-2 receptors may exist in
the brain, the CCK-2 receptor predominates in the dorsal horn
of the spinal cord of rodents, whereas the CCK-1 receptor
Results and Discussion
Synthesis. Bifunctional peptide ligands were prepared step-
wise by the solution-phase method using Boc/Bzl-chemistry.
The synthetic strategy for preparation of ligands involved four
key steps: (1) stepwise chain elongation starting from Phe-OEt,
(2) hydrazine substitution, (3) stepwise chain elongation on the
tetrapeptide hydrazide, and (4) hydrogenation (Figure 3). During
the chain elongation, peptide intermediates were isolated by
precipitation from appropriate organic solvents or 5% NaHCO3
solution with high purity and finally deprotected by hydrogena-
tion to give target peptide ligands with more than 90% crude
purity. These were purified by preparative RP-HPLC to give
pure (>98%) hydrazide-linked peptides (see Table 1 for
analytical data).
Table 1. Analytical Data of Hydrazide-Linked Bifunctional Peptide Ligands
MS (M - TFA + H)+
HRMS (M - TFA + H)+
HPLC
TLC (Rf)c
tR
(min)b
A
tR
(min)b
B
%
purity
%
purity
no.
observed
calcd
observed
calcd
I
II
III
1
2
3
4
5
6
7
8
9
1132.3
1210.3
1210.3
1032.3
1132.2
1074.3
1146.1
1088.3
1256.1
1132.3
1210.4
1210.4
1032.2
1132.3
1074.2
1146.3
1088.2
1256.8
1154.5296a
1232.5711a
1232.5888a
1032.4899
1132.5487
1074.5016
1146.5639
1110.5007a
1278.5169a
1154.5282a
1232.5751a
1232.5751a
1032.4943
1132.5468
1074.5049
1146.5619
1110.5020a
1278.5115a
23.1
26.3
26.5
19.4
26.5
21.9
23.7
19.4
20.9
98
98
99
99
99
99
98
99
100
10.3
13.5
13.6
10.8
13.6
9.8
10.9
7.1
8.4
98
98
98
99
99
99
98
99
100
0.13
0.16
0.14
0.00
0.13
0.05
0.14
0.07
0.04
0.86
0.90
0.86
0.20
0.87
0.55
0.83
0.74
0.71
0.93
0.94
0.93
0.79
0.92
0.80
0.93
0.82
0.87
a (M - TFA + Na)+. b (A) 10-90% of acetonitrile containing 0.1% TFA within 40 min and up to 100% within additional 5 min, 1 mL/min, (B) 35-75%
of acetonitrile containing 0.1% TFA within 20 min and up to 100% within additional 5 min, 1 mL/min. c (I) CHCl3:MeOH:AcOH ) 90:10:3, (II) EtOAc:
n-BuOH:water:AcOH ) 5:3:1:1, (III) n-BuOH:water:AcOH ) 4:1:1.

Hydrazide-Linked Bifunctional Peptides
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1775
Table 2. Binding Affinities of Bifunctional Peptides at δ/µ Opioid Receptors and CCK-1/CCK-2 Receptors
HDOR,a [3H]DPDPEb
RMOR,a [3H]DAMGOc
hCCK-1,d [125I]CCK-8(SO3)e
hCCK-2,d [125I]CCK-8(SO3)f
log
IC50
log
IC50
log
IC50
log
IC50
g,h
g,h
g,h
g,h
no.
Ki, nMi
45
0.23
3.4
1.4
59
23
0.82
Ki, nMi
Ki, nMi
Ki, nMi
1
2
3
4
5
6
7
8
9
-7.03 ( 0.11
-9.38 ( 0.27
-8.11 ( 0.21
-8.49 ( 0.11
-6.86 ( 0.08
-7.27 ( 0.09
-8.70 ( 0.31
-7.97 ( 0.42
-8.95 ( 0.07
-7.19 ( 0.30
-6.71 ( 0.34
-7.43 ( 0.07
-8.92 ( 0.06
-7.26 ( 0.11
-7.21 ( 0.11
-7.32 ( 0.19
-7.96 ( 0.13
-8.67 ( 0.08
31
88
16
-4.45 ( 0.11
-6.07 ( 0.09
-6.24 ( 0.12
-4.99 ( 0.27
-5.39 ( 0.21
nc
>10000
840
560
10000
4100
nc
1900
nc
-5.00 ( 0.19
-6.25 ( 0.21
-6.03 ( 0.14
-5.03 ( 0.23
-5.4 ( 0.36
-5.88 ( 0.09
-5.81 ( 0.10
nc
>10000
560
930
9300
3800
1300
1500
nc
0.51
24
26
20
5.3
0.92
-5.72 ( 0.13
5.2
0.48
nc
-5.99 ( 0.14
1000
-5.76 ( 0.13
1700
a Competition analyses were carried out using membrane preparations from transfected HN9.10 cells that constitutively expressed the respective receptor
types. b Kd ) 0.50 ( 0.1 nM. c Kd ) 0.85 ( 0.2 nM. d Competition analyses were carried out using whole cell lysate preparations from transfected HEK293
cells that constitutively expressed the respective receptor types. e Kd ) 1.9 ( 0.2 nM. f Kd ) 1.3 ( 0.4 nM. g Logarithmic values determined from the
nonlinear regression analysis of data collected from at least two independent experiments. h Competition against radiolabeled ligand. i Antilogarithmic value
of the respective IC50. nc: incomplete competition of the radioligand at the highest test concentration of the peptide. An IC50 value cannot be determined.
Table 3. Opioid Agonist Functional Activities in [35S]GTP-γ-S Binding
Assays
Table 4. Functional Assay Result for Bifunctional Peptide Ligands at
Opioid and CCK Receptors
hDORa
rMORa
opioid, IC50 (nM)a
CCK GPI/LMMP
log
EC50
EC50
Emax
log
EC50
EC50
Emax
antagonist,
(nM)c
(%)d
(nM)c
(%)d
b
b
no.
MVD (δ)
GPI (µ)
agonist (A50)b
Ke (nM)c
no.
1
2
3
4
5
6
7
8
9
-7.13 ( 0.27 74
-7.56 ( 0.42 27
-8.25 ( 0.24 5.6 61 ( 4
-8.03 ( 0.16 9.4 65 ( 3
-7.57 ( 0.17 27
-7.26 ( 0.15 54
-7.60 ( 0.30 26
-9.28 ( 0.68 0.53 70 ( 10 -8.29 ( 0.40 5.2
-8.88 ( 0.05 1.3 81 ( 1 -7.84 ( 0.31 15
100 ( 8 -6.69 ( 0.11 210
170 ( 4
103 ( 9
60 ( 5
82 ( 3
83 ( 4
90 ( 4
1
2
3
4
5
6
7
8
9
24 ( 5
38 ( 6
84 ( 11
26 ( 5
460 ( 130
640 ( 200
0% at 1 µM
0% at 1 µM
6.5 ( 0.8
44 ( 12
150 ( 15
59 ( 13
14 ( 3
25 ( 3
-7.98 ( 0.34 10
-6.62 ( 0.26 240
-8.01 ( 0.11 9.6
-8.02 ( 0.16 9.5
-7.12 ( 0.15 76
1200 ( 270 0% at 1 µM
81 ( 21
230 ( 31 55 ( 6
0% at 1 µM
0% at 1 µM
0% at 1 µM
54 ( 3
72 ( 4
36 ( 18
42 ( 15
5.1 ( 0.3 62 ( 13
28 ( 5
0% at 1 µM 5% at 1 µM
33 ( 8
120 ( 12 -9.19 ( 0.37 0.64 130 ( 13
none at 1 µM
38 ( 7
150 ( 34
-
84 ( 10
33 ( 3
-
0% at 1 µM
0% at 1 µM
-
26 ( 3
2.7 ( 1.5 8.8 ( 0.3
SNF9007 29 ( 10 220 ( 81
200 ( 30
biphalin -8.95 ( 0.17 1.1 83 ( 4
DPDPE -8.80 ( 0.25 1.6 69 ( 4
DAMGO
-
-
-
-
biphalin
-
0% at 300 nM 66 ( 14
-
-
-
-7.44 ( 0.19 37
150 ( 9
a Concentration at 50% inhibition of muscle concentration at electrically
stimulated isolated tissues. b Contraction of isolated tissue relative to initial
muscle contraction with KCl. c Inhibitory activity against the CCK-8 induced
muscle contraction. Ke: concentration of antagonist needed to inhibit CCK-8
to half its activity.
a Expressed from CHO cell. b Logarithmic values determined from the
nonlinear regression analysis of data collected from at least two dependent
experiments. c Antilogarithmic value of the respective EC50. d Net total
bound/basal binding × 100 ( SEM.
predominates in that of the primate. This observation is
important in that it suggests that blockade of both CCK-1 and
CCK-2 receptors may be critical for efficacy in humans.
antagonist activity at CCK receptors, though the CCK binding
affinities were quite low. Interestingly, this ligand showed the
highest agonist activity for the δ opioid receptor both in the
[35S]GTP-γ-S binding assay and in the functional assay (MVD)
with EC50 ) 0.53 nM and IC50 ) 5.1 nM, respectively. It also
had somewhat better antagonist activity with a Ke ) 38 nM at
the GPI/LMMP than expected on the basis of the binding data.
It is likely that the enhanced δ opioid receptor activity and
selectivity over the µ receptor is because of the NMe modifica-
tion on the Phe4′ residue, which is closer to the opioid portion
of the ligand. The structure-activity relationships for the δ and
µ opioid receptors in the CCK pharmacophore will be discussed
later in detail.
We used [35S]GTP-γ-S binding to examine opioid agonist
efficacy, for functional characterization of the ligands at the δ
and µ opioid receptors.20 Classical functional assays also were
performed to evaluate their opioid agonist activities in the GPI
(µ) and MVD (δ). The assay result was generally comparable
with the [35S]GTP-γ-S binding assay. Most ligands exhibited
better agonist activity for δ opioid receptor (EC50 ) 0.53-74
nM, IC50 ) 5.1-230 nM) than µ opioid receptor (EC50 ) 0.64-
210 nM, IC50 ) 28 to none) in functional assays and GTP
binding assays (Tables 3 and 4).
In vitro functional bioactivity was determined by measuring
the inhibition against the CCK-8-induced muscle contraction
using unstimulated GPI/LMMP for CCK receptor antagonist
activity (Table 4). The Ke values, calculated from the IC50 values
of the reference agonist CCK-8, of ligands 1-9 (Table 4) were
between 1 and 100 nM, except for ligand 7, which is only a
partial CCK agonist with no antagonist activity. All other ligands
tested had no CCK agonist activity.
The best result in all three assays occurred when Phe4′ was
replaced with NMePhe and when the D-Trp1′ was replaced with
Trp1′ and the amino function of the residue was protected with
an acetyl group to give 8. As can be seen in Tables 2-4, ligand
8, which satisfies many of the criteria we set out in our design,
showed agonist activity at δ and µ opioid receptors and
Ligand 1, in which the Nle and D-Trp residues were replaced
at positions 2′ and 1′ in the CCK region instead of Met and
Trp of CCK30-33 with a Boc protecting group, showed moder-
ately high opioid binding affinities at both δ and µ receptors
but low CCK-1 and CCK-2 binding affinities (Ki > 10 µM). It
was interesting that very good CCK antagonist activity (6.5 nM;
see Table 4) was shown in the unstimulated GPI/LMMP
assay,21,22 which is a commonly used index for CCK-1, even
though low binding affinities at CCK receptors were obtained.
There were some differences in binding affinities for CCK
receptors and for CCK antagonist activity at the unstimulated
GPI/LMMP. While the ligands showed CCK antagonist activity
at the unstimulated GPI/LMMP, several ligands did not bind
to the CCK receptors well in the cloned receptors. These

1776 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Lee et al.
differences are considered to be from possible allosteric effects
and need further investigation. As a starting point, ligand 1 was
modified in different positions to increase CCK activity. For
structure-activity relationships of the CCK pharmacophore of
these hydrazide-linked bifunctional peptides, the following
structural and positional modifications were made: (1) variation
of the N-terminal protecting group, (2) inversion of chirality of
the N-terminal amino acid, (3) N-methylation of the Nle in
position 2′ and the Phe in position 1′, and (4) introduction of
known amino acid derivatives in position 3′.23 Aspartic acid in
the 2′ position of CCK pharmacophore was not changed,
because it has been known that its modification leads to a
marked decreases in CCK potency.24-25
In our earlier studies10,11 on structure-activity relationships,
we showed that N-methylation of the Nle residue in the 2′
position of the CCK pharmacophore was necessary for balanced
and high CCK-1/CCK-2 binding affinities depending on 1′
position modification. NMR studies27 showed that cis/trans
isomerization of the NMeNle residue may be related to the
selectivity. The N-methylation of Nle2′ in 7 led to increases of
binding affinities at both CCK receptors (5- and 7-fold at CCK-1
and CCK-2 receptors, respectively) and at opioid receptors (55-,
and 2-fold at δ and µ opioid receptors, respectively), relative
to 1. Also, this modification gave the highest efficacy (EC50
)
0.64 nM) at the µ opioid receptor. However, despite its relatively
high CCK and opioid binding affinities and very high efficacy
at µ opioid receptor, ligand 7 lost CCK antagonist activity and
µ opioid agonist activity while unexpectedly showing partial
agonist activity at the CCK receptor. This result suggests that
CCK and opioid pharmacophores have a common backbone
requirement for both receptors and that substitution of D-Trp4′-
NMeNle2′ is not tolerated at both receptors. Trp was introduced
instead of D-Trp, leading to the ligand 5, which showed
improved binding affinities at CCK receptors while retaining
moderate opioid affinities. Also a large increase in its efficacy
for the µ opioid receptor in GTP-γ-S binding and µ opioid
agonist activity in the GPI assay were shown for this analogue
with reversed µ selectivity over δ. While the chirality of position
1′ does not affect CCK binding affinities and activities by itself,
the constraint from N-methylation of 2′ position restricts this
moiety within a topographical space to give either a favorable
or unfavorable conformation to CCK and opioid receptors.
Considering the biological data, the backbone conformation
achieved by the Trp1′-Nle2′ combination seems to favor both
the CCK and opioid receptors.
It was shown that the N-terminal protecting group of the CCK
pharmacophore plays an important role in CCK binding
affinities.26 The N-terminal of the CCK pharmacophore was
substituted with different protecting groups such as Boc, Ac,
and adamantyloxycarbonyl (Adoc) groups. Interestingly, these
modifications affected not only binding affinities at CCK
receptors (more than 18-fold) but also (up to 200-fold) binding
affinities for opioid receptors (see 1-4 in Table 2). Substitution
of hydrophobic and bulky group such as 1-Adoc or 2-Adoc
resulted in increased binding affinities at both CCK-1 and
CCK-2 receptors. In the case of the 1-Adoc group modification,
much more increased δ opioid activity (Ki ) 0.23 nM) and
selectivity (µ/δ ) 430) over µ opioid receptor was seen in
binding assays. Although there was no clear explanation for
the lowest µ opioid binding affinity of ligand 2 with the 1-Adoc
substitution, compared with ligand 3 containing the 2-Adoc
substitution, ligand 2 still showed the best functional activity
in the GTP-γ-S binding assay (Table 3). Deletion of the
N-terminal protecting group decreased both CCK-1 and CCK-2
binding affinities somewhat but increased both δ and µ opioid
binding affinities, especially at the µ receptor. The binding result
coincided with GTP-γ-S binding and even functional assays in
the MVD and GPI (Table 4), which demonstrated that the
N-terminal protecting group in CCK pharmacophore plays an
important role in determining opioid activities. Ligand 4, without
an N-terminal protecting group in the CCK region, showed high
δ/µ opioid activities in both binding and GTP-γ-S assays and
a distinct increase in µ opioid activity in the GPI functional
assay (IC50 ) 81 nM). Even though the ligand showed reduced
CCK binding affinities, it still retained moderate CCK antagonist
activity (Ke ) 60 nM). Derivatization of peptides at the N-or
C-terminus has frequently been carried out in attempts to
improve the activity, bioavailability, and physicochemical
properties of potential drug candidates, often with much success.
In our case, these modifications seemed to have the greatest
effect on both CCK and opioid activities of the hydrazide-linked
bifunctional peptides as measured by in vitro binding and
functional assays. A free amine moiety in the CCK region
seemed to play a role in making a favorable conformation for
the opioid, especially for µ receptor activity, whereas the
protecting group did not seem to be essential for CCK activities.
N-Terminal modification was done where chirality was reversed.
There did not appear to be a great difference in CCK and opioid
activities between ligands 5 and 6, which have a Boc group
and an Ac group at the N-terminus, respectively. This result
coincides with the previous observation that the acetyl protected
tetrapeptide (Ac-Trp-Lys(Tac)-Asp-NMePhe-NH2) is equi-
potent to the Boc protected analogue for CCK receptors.23,26
Both Boc- or Ac-protected tetrapeptides showed the same good
potency and selectivity for the CCK-1 receptor, even with a
big difference in physicochemical properties.
The o-chlorophenylaminocarbonyl (Cpac) substituted lysine
derivative, Lys(Cpac),23 was introduced into the 2′ position
instead of Nle in compound 8 to give 9, which has more than
6-fold increased CCK-1/CCK-2 binding affinities with high
binding (0.48 nM) and good efficacy in the GTP-γ-S binding
(EC50 ) 1.3, Emax ) 81%) for the δ opioid receptor. It is likely
that this is because the NMePhe substitution in the 4′ position
of CCK pharmacophore plays a role in making a favorable
structure for δ opioid receptor. Comparing 6, which has a 4′
Phe, with ligand 8, which has a NMePhe in the 4′ position,
again showed that 4′ position modification affected not only
CCK activities but also opioid activities. The CCK antagonist
activity of 8 changed slightly in the functional assays, but the
efficacy at opioid receptors was increased greatly with distinct
δ selectivity (µ/δ ) 10). In both GTP-γ-S and functional assays,
δ opioid selectivity was greatly increased, which demonstrated
the role of this position in determining δ opioid receptor activity.
The backbone change by N-methyl modification of the 4′
position is more favorable for the δ opioid receptor conforma-
tion. It is interesting that the 1′ position of the CCK pharma-
cophore affects the µ opioid activity more than δ activity and
vice versa for the 4′ position. In an earlier study,28 we replaced
this 4′ position by the very bulky pyrenylalanine (Pya), which
resulted in a loss of both CCK antagonist and µ and δ opioid
agonist activities in functional assays, even though a slight
decrease in both binding affinities was seen. From this observa-
tion, it appears that small differences in structure might cause
great changes in the biological activity of the ligand.
In summary, most ligands retained their opioid activities, but
often not their CCK activities, even though some of them
showed good antagonist function for the CCK receptor.
Comparing the two individual pharmacophores, one for the

Hydrazide-Linked Bifunctional Peptides
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1777
opioid receptors (Tyr-D-Ala-Gly-Phe-NH2)29 and the other for
the CCK receptors (CCK-4),30 respectively, the hydrazide-linked
bifunctional peptides were found to have the same high activities
for the opioid receptors, but relatively lower activities for CCK
receptors. The conformation of the opioid portion of the
hydrazide-linked bifunctional peptides was not changed on the
basis of the biphalin half-structure. The hydrazide linker
appeared to play a role in maintaining the conformation for
binding to the opioid receptors. On the contrary, the CCK
portion of the bifunctional peptide possessed different confor-
mations. In this case, CCK receptor binding was reduced
because the rigidity of the hydrazide linker resulting in a
decrease in CCK activity.
1.1 equiv) and NMM (2 equiv) were added, and the mixture was
stirred for 30 min at 0 °C and for 1 day at room temperature. After
checking the disappearance of the starting amine, DCU formed was
filtered off. The filtrate was concentrated and diluted with 100 mL
of EtOAc. The organic layer was washed with 2 N HCl (20 mL),
saturated NaHCO3 (40 mL), and finally brine (40 mL), followed
by drying with anhydrous Na2SO4. The solution was evaporated
under reduced pressure and dried in vacuo to give Boc-Gly-Phe-
OEt as an oil. The oil was dissolved in cold TFA (40 mL) and
stirred for 20 min at 0 °C. The mixture was evaporated off and
coevaporated with toluene (50 mL, two times). The solid was
filtered off, washed with toluene, and dried in vacuo to afford 8.2
g (96%) of TFA‚H-Gly-Phe-OEt as a very hygroscopic powder:
MS m/z 251.0 (M - TFA + H)+.
TFA‚H-D-Ala-Gly-Phe-OEt. TFA‚H-Gly-Phe-OEt (7.30 g, 20
mmol) and Boc-D-Ala (4.16 g, 22 mmol) were dissolved in DMF
(100 mL) and cooled in ice-bath for 10 min. BOP (9.7 g, 22 mol),
2.97 g (22 mmol) of HOBt, and 4.4 mL (40 mmol) of NMM were
added to the reaction mixture, which stirred for 30 min at 0 °C and
for 7 h at room temperature. After checking for disappearance of
the starting amine, the mixture was concentrated under reduced
pressure, followed by dilution with EtOAc (150 mL) and 2 N HCl
(70 mL). The organic layer was washed with saturated NaHCO3
(2 × 15 mL) and brine (20 mL) and dried over anhydrous Na2-
SO4. After filtering, the solution was concentrated and dried in
vacuo to give Boc-D-Ala-Gly-Phe-OEt as a sticky solid. The
powder (about 19.7 mmol) was dissolved in cold TFA (20 mL)
and stirred for 20 min at 0 °C. The mixture was evaporated and
coevaporated with toluene (2 × 50 mL). The solid was washed
with EtOAc (2 × 50 mL) and dried to give 5.38 g (63%, two-step
yield) of TFA‚H-D-Ala-Gly-Phe-OEt as a white powder: MS m/z
321.9 (M - TFA + H)+.
Z-Tyr(Bzl)-D-Ala-Gly-Phe-OEt. Z-Tyr(Bzl)-D-Ala-Gly-Phe-
OEt (5.30 g, 81% yield) as a white power was prepared by the
same coupling method as described above and isolated from
EtOAc: analytical HPLC tR 29.9 min, purity >95%; MS m/z 708.9
(M + H)+, 731.3 (M + Na)+.
Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH2. Z-Tyr(Bzl)-D-Ala-Gly-
Phe-OEt (730 mg) in 10 mL of DMF was treated with 55%
hydrazine (1 mL) for 1 day and triturated with water to give 660
mg of pure Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH2 as a white powder
in 95% yield: analytical HPLC tR 24.0 min, purity >95%; MS
m/z 695.1 (M + H)+.
Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle-Xxx-
PG. Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH2 was coupled stepwise
with 1.1 equiv of Boc-Phe, Boc-Asp(Bzl), and Boc-Nle using the
standard BOP/HOBt procedure to afford pure Z-Tyr(Bzl)-D-Ala-
Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle‚TFA [MS (M - TFA + H)+
1160.2; analytical HPLC tR 28.7 min, purity > 95%] in 90% yield.
The heptapeptide was coupled with Boc-D-Trp, 1-Adoc-D-Trp,
2-Adoc-D-Trp, Boc-Trp, and Ac-Trp, respectively, and triturated
with 5% NaHCO3 to give crude Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-
NH-Phe-Asp(OBzl)-Nle-D-Trp-Boc, Z-Tyr(Bzl)-D-Ala-Gly-Phe-
NH-NH-Phe-Asp(OBzl)-Nle-D-Trp-1-Adoc, Z-Tyr(Bzl)-D-Ala-
Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle-D-Trp-2-Adoc, Z-Tyr(Bzl)-
D-Ala-Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle-Trp-Boc, and Z-Tyr-
(Bzl)-D-Ala-Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle-Trp-Ac in quan-
titative yields in greater than 80% purity in all cases.
TFA‚H-Tyr-D-Ala-Gly-Phe-NH-NH-Phe-Asp-Nle-D-Trp-
Boc (1). Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle-
D-Trp-Boc was hydrogenated using Pd-C as a catalyst for 2 days
at room temperature, purified by preparative RP-HPLC (20-70%
of acetonitrile within 25 min), and lyophilized to give pure 1 as a
white powder in 67% yield.
TFA‚H-Tyr-D-Ala-Gly-Phe-NH-NH-Phe-Asp-Nle-D-Trp-1-
Adoc (2). Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle-
D-Trp-1-Adoc was hydrogenated using Pd-C as a catalyst for 2
days at room temperature, purified by preparative RP-HPLC (20-
50% of acetonitrile within 30 min), and lyophilized to give pure 2
as a white powder in 57% yield.
Conclusions
A series of hydrazide-linked bifunctional peptides have been
synthesized and tested in an effort to optimize both opioid and
CCK activities for the treatment of pain. These hydrazide-linked
bifunctional peptides produced very potent δ and µ opioid
agonist activities and moderately potent and very balanced CCK-
1/CCK-2 antagonist activities. From structure-activity relation-
ships, the hydrazide linker, which is inserted to combine opioid
and CCK pharmacophores, is considered to play a role in
making a desirable conformation for opioid receptors more so
than for CCK receptors. Further, CCK pharmacophore modi-
fications affect not only CCK potency but also opioid potency,
which suggests that the two pharmacophores interact with each
other to make one conformation or two different conformations
for opioid and CCK receptors. From the biological profile of
these peptide ligands, it was demonstrated that it is reasonable
to build agonist function for opioid receptors and antagonist
function for CCK receptors in a single structure. It is worth
noting that two different pharmacophores for different G-protein
coupled receptors (GPCRs) can be designed into one structure
by rational design.
Experimental Section
All amino acid derivatives except 1-Adoc-D-Trp, 2-Adoc-D-Trp,
Phe-OEt, and Boc-Lys(Cpac) were purchased from Novabiochem,
Bachem, and Chem Impex International. 1-Adoc-D-Trp, 2-Adoc-
D-Trp, and Phe-OEt were prepared by well-established procedures.
Boc-Lys(Cpac) was synthesized by the reaction of Boc-Lys with
2-chlorophenyl isocyanate in the presence of base.23 myo-[2-3H-
(N)]-inositol, [tyrosyl-3,5-3H(N)], D-Ala2-MePhe4-glyol5-enkephalin
(DAMGO), [tyrosyl-2,6-3H(N)]-(2-D-penicillamine,5-D-penicil-
lamine)enkephalin (DPDPE), Bolton-Hunter radioiodinated chole-
cystokinin octapeptide [CCK-8(s)], and [35S]guanosine 5′-(γ-thio)
triphosphate were purchased from Perkin-Elmer. Cholecystokinin
[CCK-8(s)] was purchased from American Peptide Company Inc.
Bovine serum albumin (BSA), protease inhibitors, Tris, and other
buffer reagents were obtained from SIGMA. Culture medium
(MEM, DMEM, IMDM), penicillin/streptomycin, and fetal calf
serum (FCS) were purchased from Gibco. Coupling reactions were
monitored by TLC. TLC was performed on aluminum sheets coated
with a 0.2 mm layer of silica gel 60 F254 Merck using the following
solvent systems: (1) CHCl3:MeOH:AcOH ) 90:10:3, (2) EtOAc:
n-BuOH:water:AcOH ) 5:3:1:1, and (3) n-BuOH:water:AcOH )
4:1:1; ninhydrin spray was used for detection. Analytical HPLC
was performed on a Hewlett-Packard 1090 (C-18, Vydac, 4.6 ×
250 mm, 5 µm) and preparative RP-HPLC on a Hewlett-Packard
1100 (C-18, Vydac, 10 × 250 mm, 10 µm). 1H NMR spectra were
recorded on a Bruker DRX-500 spectrometer. Mass spectra were
taken in the positive ion mode under ESI methods.
TFA‚H-Gly-Phe-OEt. HCl‚H-Phe-OEt (5.4 g, 23.4 mmol) was
dissolved in 60 mL of DMF. Boc-Gly (4.56 g, 26 mmol, 1.1 equiv)
and HOBt (3.52 g, 26 mmol, 1.1 equiv) were added to the mixture,
which was cooled in an ice-bath for 10 min. DCC (5.36 g, 26 mmol,

1778 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Lee et al.
TFA‚H-Tyr-D-Ala-Gly-Phe-NH-NH-Phe-Asp-Nle-D-Trp-2-
Adoc (3). Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle-
D-Trp-2-Adoc was hydrogenated using Pd-C as a catalyst for 2
days at room temperature, purified by preparative RP-HPLC (20-
50% of acetonitrile within 25 min), and lyophilized to give pure 3
as a white powder in 62% yield.
CO2, humidified atmosphere in a Forma Scientific incubator in
DMEM with 10% BSA and 100 U mL penicillin/100 µg mL
streptomycin.
Radioligand Labeled Binding Assays. Opioid Receptors.
Crude membranes were prepared as previously described31 from
the transfected cells that express the MOR or the DOR. The protein
concentration of the membrane preparations was determined by the
Lowry method, and the membranes were stored at -80 °C until
use. Membranes were resuspended in assay buffer [50 mM Tris,
pH 7.4, containing 50 µg/mL bacitracin, 30 µM bestatin, 10 µM
captopril, 100 µM phenylmethylsulfonylfluoride (PMSF), 1 mg/
mL BSA]. For saturation analysis, six concentrations of [3H]-
DAMGO (0.02-6 nM, 47.2 Ci/mmol) or six concentrations of
[3H]DPDPE (0.1-10 nM, 44 Ci/mmol) were each mixed with 200
µg of membranes from MOR or DOR expressing cells, respectively,
in a final volume of 1 mL. For competition analysis, 10 concentra-
tions of a test compound were each incubated with 50 µg of
membranes from MOR- or DOR-expressing cells and the Kd
concentration of [3H]DAMGO (1.0 nM, 50 Ci/mmol) or of [3H]-
DPDPE (1.0 nM, 44 Ci/mmol), respectively. Naloxone at 10 µM
was used to define the nonspecific binding of the radioligands in
all assays. All samples were carried out in duplicate. The samples
were incubated in a shaking water bath at 25 °C for 3 h, followed
by rapid filtration through Whatman grade GF/B filter paper
(Gaithersburg, MD) presoaked in 1% polyethyleneimine and
washing four times each with 2 mL of cold saline, and the
radioactivity was determined by liquid scintillation counting (Beck-
man LS5000 TD).
TFA‚H-Tyr-D-Ala-Gly-Phe-NH-NH-Phe-Asp-Nle-D-Trp-H (4).
To the solution of Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH-Phe-Asp-
(OBzl)-Nle-D-Trp-Boc in DCM were added 1 mL of TFA and 0.1
mL of anisole. After stirring for 30 min at 0 °C, the mixture was
evaporated to an oil and triturated with ether to give Z-Tyr(Bzl)-
D-Ala-Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle-D-Trp-H as a white
powder. The Z-, Bzl-protected octapeptide was hydrogenated using
Pd-C as a catalyst for 2 days at room temperature, purified by
preparative RP-HPLC (10-60% of acetonitrile within 25 min), and
lyophilized to give pure 4 as a white powder in 45% yield.
TFA‚H-Tyr-D-Ala-Gly-Phe-NH-NH-Phe-Asp-Nle-Trp-Boc (5).
Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle-Trp-Boc
was hydrogenated using Pd-C as a catalyst for 2 days at room
temperature, purified by preparative RP-HPLC (20-60% of
acetonitrile within 25 min), and lyophilized to give pure 5 as a
white powder in 32% yield.
TFA‚H-Tyr-D-Ala-Gly-Phe-NH-NH-Phe-Asp-Nle-Trp-Ac (6).
Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH-Phe-Asp(OBzl)-Nle-Trp-Ac was
hydrogenated using Pd-C as a catalyst for 2 days at room
temperature, purified by preparative RP-HPLC (20-60% of
acetonitrile within 25 min), and lyophilized to give pure 6 as a
white powder in 35% yield.
Z-Tyr-D-Ala-Gly-Phe-NH-NH-Phe-Asp-NMeNle-D-Trp-
Boc (7). Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH2 was coupled stepwise
with 1.1 equiv of Boc-Phe, Boc-Asp(Bzl), and Boc-NMeNle using
the standard BOP/HOBt procedure to afford pure Z-Tyr(Bzl)-D-
Ala-Gly-Phe-NH-NH-Phe-Asp(OBzl)-NMeNle‚TFA [MS m/z 1174.3
(M + H)+; analytical HPLC tR 28.4 min] in 90% yield. The
heptapeptide was coupled with Boc-D-Trp and triturated with 5%
NaHCO3 to give crude Z-Tyr(Bzl)-D-Ala-Gly-Phe-NH-NH-Phe-
Asp(OBzl)-NMeNle-D-Trp-Boc [MS m/z 1488.3 (M + Na)+;
analytical HPLC tR 36.6 min] in quantitative yield. The octapeptide
was hydrogenated using Pd-C as a catalyst for 2 days at room
temperature, purified by preparative RP-HPLC (20-60% of ac-
etonitrile within 25 min), and lyophilized to give pure 7 as a white
powder in 23% yield.
TFA‚H-Tyr-D-Ala-Gly-Phe-NH-NH-NMePhe-Asp-Yyy-Trp-
Ac (8, 9). H-Tyr-D-Ala-Gly-Phe-NH-NH2 was coupled stepwise
with 1.1 equiv of Boc-NMePhe, Boc-Asp(Bzl), Boc-Nle (or Boc-
Lys(Cpac)), and finally Ac-Trp to give Z-Tyr(Bzl)-D-Ala-Gly-Phe-
NH-NH-NMePhe-Asp(OBzl)-Yyy-Trp-Ac as a white powder in
55% yield. The coupling of Boc-Asp(Bzl) to Z-Tyr(Bzl)-D-Ala-
Gly-Phe-NH-NH-NMePhe‚TFA [MS m/z 856.2 (M + H)+; analyti-
cal HPLC tR 25.3 min] was not completed for 1 day. There was
still starting peptide, which was removed by solid-phase extraction
using Sep-Pak Catridges (Waters). After completion of chain
elongation, the protected peptides were hydrogenated using Pd-C
as a catalyst for 2 days at room temperature, purified by preparative
RP-HPLC (20-50% of acetonitrile within 25 min for 8, 10-50%
of acetonitrile within 30 min for 9), and lyophilized to give pure 8
and 9 as a white powder in 56% and 20%, respectively.
Cell Lines. The cDNA for the human DOR was a gift from Dr.
Brigitte Kieffer (IGBMC, Illkirch, France). The cDNA for the rat
MOR was a gift from Dr. Huda Akil (University of Michigan).
The cDNA for the human CCK-1 and CCK-2 was a gift from Dr.
Alan Kopin (Tufts University). Stable expression of the human
CCK-1 (CMV Tag2) and CCK-2 (pCDNA HisXpress) receptors
in HEK 293 cells was produced by transfecting the cells with the
respective cDNA by calcium phosphate precipitation followed by
clonal selection in neomycin. Stable expression of the rat MOR
(pCDNA3) and the human DOR (pCDNA3) in the neuroblastoma
cell line HN9.10 was achieved with the same method. Expression
of the respective receptors was initially verified and the level of
expression periodically monitored by radioligand saturation analysis
(see below). All cells were maintained at a 37 °C, 95% air/5%
CCK Receptors. All assays were performed using whole cells
preparations expressing either the human CCK-1 or CCK-2
receptors. The assay buffer used was low-glucose DMEM, pH 7.4,
in the presence of 0.5 mg/mL BSA and protease inhibitors, as above.
For saturation analysis, 12 concentrations (12.5 pM-4 nM) of
unlabeled CCK-8(s)/[125I]CCK-8 (labeled-to-unlabeled ratios 1:25
to 1:65) were incubated with 200 000 cells/assay tube in a final
volume of 1 mL. For [125I]CCK-8 (2200 Ci/mmol)/ligand competi-
tion analysis, 10 different concentrations (from 10-13 to 10-4 M)
of each test peptide were mixed with [125I]CCK-8 (1.0-1.5 pM)
and 200 000 cells/assay tube in a final volume of 1 mL in assay
buffer. Nonspecific binding was defined as that of [125I]CCK-8
bound in the presence of 1 µM unlabeled CCK-8. All samples were
done in duplicate. Incubations were carried out at 30 °C for 60
min in a shaking water bath, and the reaction was terminated by
rapid filtration through Whatman GF/ B filter paper (presoaked in
polyethyleneimine) and washed four times with 2 mL of ice-cold
saline. The radioactivity was determined by γ-counting (Packard
Cobra II γ-Counter). The log IC50 ( SEM of a peptide for each
receptor type was determined by nonlinear regression analysis of
data pooled from at least two independent experiments using
GraphPad Prism4 (Graph Pad, San Diego, CA). The Ki values were
calculated as the antilogarithmic value of the IC50. For competition
analysis using [3H]DAMGO or [3H]DPDPE, the Ki values were
calculated from the IC50 by the Cheng and Prusoff equation.
[35S]GTP-γ-S Binding Assay. The method was carried out
according to that previously described.32 Membrane preparation (10
µg) to a final volume of 1 mL incubation mix (50 mM Hepes, pH
7.4, 1 mM EDTA, 5 mM MgCl2, 30 µM GDP, 1 mM dithiothreitol,
100 mM NaCl, 0.1 mM PMSF, 0.1% BSA, 0.1 nM [35S]GTP-γ-S)
was added along with various concentrations, in duplicate or
triplicate, of the test drug and incubated for 90 min at 30 °C in a
shaking water bath. Reactions were terminated by rapid filtration
through Whatman GF/B filters (presoaked in water), followed by
four washes with 4 mL of ice-cold wash buffer (50 mM Tris, 5
mM MgCl2, 100 mM NaCl, pH 7.4). The radioactivity was
determined by liquid scintillation counting as above. The basal level
of [35S]GTPγS binding was defined as the amount bound in the
absence of any test drug. Nonspecific binding was determined in
the presence of 10 µM unlabeled GTP-γ-S. Total binding was
defined as the amount of radioactivity bound in the presence of
test drug. The effect of the drug at each concentration on [35S]-
GTP-γ-S binding was calculated as a percentage by the following

Hydrazide-Linked Bifunctional Peptides
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1779
equation: [total bound - basal]/[basal - nonspecific] × 100. Data
were expressed as log EC50 ( standard error from at least two
independent experiments analyzed by nonlinear regression analysis
using GraphPad Prism4. The Emax values were expressed as mean
( standard error.
exposure to the dose of agonist. IC50 values represent the mean to
not less than three tissues. A50, IC50, and Emax estimates were
determined by computerized nonlinear least-squares analysis.
Acknowledgment. The work was supported by a grant from
the USDHS, National Institute on Drug Abuse, DA-12394. We
thank Ms. Adrienne Begaye for technical assistance and Ms.
Margie Colie for assistance with the manuscript.
Supporting Information Available: 1H NMR data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
GPI and MVD in Vitro Bioassays. The in vitro tissue bioassays
were performed as described previously.33-35 IC50 values represent
the mean of no less than four tissues. IC50 estimates, relative potency
estimates, and their associated standard errors were determined by
fitting the data to the Hill equation by a computerized nonlinear
least-squares method. In the MVD assay, male ICR mice under
ether anesthesia were sacrificed by cervical dislocation, and the
vasa deferentia was removed. The tissue were tied to a gold chain
with suture silk and mounted between platinum wire electrodes in
20-mL organ baths at a tension of 0.5 g and bathed in oxygenated
(95% O2, 5% CO2) magnesium-free Kreb’s buffer at 37 °C. They
were stimulated electrically (0.1 Hz, single pulses, 2.0 ms duration)
at supramaximal voltage. Following an equilibrium period, com-
pounds were added to the bath cumulatively in volumes of 14-16
mL until maximum inhibition is reached. Response to an IC50 dose
of DPDPE (10 nM) was measured to determine tissue integrity
before compound testing begins.
In the GPI bioassay, male Hartley guinea pigs under anesthesia
were sacrificed by decapitation and a nonterminal portion of the
ileum was removed. The LMMP were carefully separated from the
circular muscle and were cut into strips. The tissues were tied to a
gold chain with suture silk, mounted between platinum wire
electrodes in 20-mL baths at a tension of 1 g containing 37 °C
oxygenated (95% O2, 5% CO2) Kreb’s buffer (118 mM NaCl, 4.7
mM KCl, 2.5 mM CaCl2, 1.19 mM KH2PO4, 1.18 mM MgSO4, 25
mM NaHCO3, and 11.48 mM glucose), and allowed to equilibrate
for 15 min. The tissues were stimulated electrically (0.1 Hz, 0.4
ms duration) at supramaximal voltage. Following an equilibration,
the compound was added to the baths in 15-60 µL aliquots until
maximum inhibition was observed. Percent inhibition was calculated
by using the average contraction height for 1 min preceding the
addition of the compound divided by the contraction height 3 min
after exposure to the dose of the compound. Response to an IC50
dose of PL-017 (10 nM) was measured to determine tissue integrity
before compound testing begins.
Functional Assays for CCK. Male Hartley guinea pigs under
ether anesthesia were killed by decapitation, and a nonterminal
portion of the ileum was removed. The longitudinal muscle with
myenteric piexus (LMMP) was carefully separated from the circular
muscle and cut into strips as described previously.36 These tissues
were tied to gold chains with suture silk and mounted between
platinum wire electrodes in 20-mL organ baths at a tension of 1 g
and bathed in oxygenated (95% O2, 5% CO2) Kreb’s bicarbonate
buffer at 37 °C. Tissues were stimulated electrically (0.1 Hz, 0.4
ms duration) at supramaximal voltage to stabilize baseline force
and tissue health. Response to an IC50 dose of PL-017 (100 nM)
was measured to determine tissue integrity before analogue testing
began. Following an equilibration period, tissues were challenged
with KCl (67 mM) to determine initial maximal muscle contractility.
An initial noncumulative CCK-8 dose-response curve was
constructed using concentrations from 1 to 100 mM. The test
compound was added to the bath in concentrations from 1 to 1000
mM. If no agonist activity was observed, 3 min later a dose of
CCK-8 was added to determine the test compound’s antagonist
activity until a complete CCK-8 dose-response curve had again
been reconstructed using a dose of antagonist that seemed to cause
a 3-fold shift rightward. Tissues were again challenged with KCl
to determine tissue changes during the assay. After thorough
washing, electrical stimulation was again applied, tissue resiliency
tested with 100 nM PL-017, and an opioid dose-response curve
was constructed with the test compound.
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