A structure-activity relationship study and combinatorial synthetic approach of C-terminal modified bifunctional peptides that are δ/μ opioid receptor agonists and neurokinin 1 receptor antagonists

Article abstract of DOI:10.1021/jm070332f

A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance, and to provide better compliance than coadministration of two drugs. Compounds were synthesized using a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free carboxyl peptide, Boc-Tyr(tBu)-D-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or amidated in solution phase. The structure-activity relationships (SAR) showed that the C-terminus acted as not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl (3) demonstrated high binding affinities at both δ and μ receptors (K_i = 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens (MVD) and guinea pig isolated ileum (GPI) assays (IC ₅₀ = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation (K_e = 26 nM) and good affinity for the hNK1 receptor (K_i = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities.

Full text of DOI:10.1021/jm070332f

J. Med. Chem. 2008, 51, 1369–1376
1369
A Structure–Activity Relationship Study and Combinatorial Synthetic Approach of C-Terminal
Modified Bifunctional Peptides That Are δ/µ Opioid Receptor Agonists and Neurokinin 1
Receptor Antagonists
Takashi Yamamoto,† Padma Nair,† Josef Vagner,† Tally Largent-Milnes,‡ Peg Davis,‡ Shou-wu Ma,‡ Edita Navratilova,‡
Sharif Moye,‡ Suneeta Tumati,‡ Josephine Lai,‡ Henry I. Yamamura,‡ Todd W. Vanderah,‡ Frank Porreca,‡ and
Victor J. Hruby*,†
Departments of Chemistry and Pharmacology, UniVersity of Arizona, Tucson, Arizona 85721
ReceiVed March 22, 2007
A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed
with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected
to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the
NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance,
and to provide better compliance than coadministration of two drugs. Compounds were synthesized using
a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free
carboxyl peptide, Boc-Tyr(tBu)-D-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared
intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or
amidated in solution phase. The structure–activity relationships (SAR) showed that the C-terminus acted as
not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the
opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr-
D-Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl (3) demonstrated high binding affinities at both δ and µ receptors (Ki
) 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens
(MVD) and guinea pig isolated ileum (GPI) assays (IC50 ) 50 and 13 nM, respectively). Compound 3 also
showed a good antagonist activity in the GPI assay with substance P stimulation (Ke ) 26 nM) and good
affinity for the hNK1 receptor (Ki ) 14 nM). Consequently, compound 3 is expected to be a promising and
novel type of analgesic with bifunctional activities.
Introduction
Opioid analgesics are the mainstay for treatment of moderate
to severe pain. Research on opioids and their receptors has been
active over the past decades.1 However, the clinical use of opioid
drugs for the treatment of chronic pain states still encounters
many obstacles, including side effects and analgesic tolerance,
which is explained as a reduction of pain relief during prolonged
administration of opioids.2 Repeated morphine exposure resulted
Figure 1. Design of bifunctional peptides possessing a δ/µ opioid
in enhanced levels of substance P, a transmitter of pain signals,
both in vitro and in vivo.3,4 This induces increased pain that
could require a higher dose of pain-relief and thus be manifested
as “antinociceptive tolerance”.5 Supporting this concept, NK1
knockout mice do not show opioid-induced antinociceptive
tolerance, and coadministration of opioid agonist and substance
P antagonist showed enhanced antinociceptive potency in acute
pain states and in prevention of opioid-induced tolerance.6–11
Moreover, studies have shown that the distributions of opioids
and substance P containing neurons overlap, and NK1 and
opioid receptors exist nearby in a synaptic cleft.12–14 We
hypothesized that bifunctional peptides with opioid agonist and
substance P antagonist pharmacophores in one molecule will
interact with each receptor separately. Such interaction can lead
to a potent antinociceptive activity without development of
tolerance.15,16 In fact, several bifunctional compounds have
showed concerted efficacy due to successful combination of the
pharmacophore and neurokinin-1 pharmacophore.
functions.17–19 Our design is based on the overlapping phar-
macophore concept that has been developed in our group mainly
for bifunctional peptides with opioid agonist and cholecystokinin
(CCK) antagonist activities.20–22 The enkephalin-based tetrapep-
tide Tyr-D-Ala-Gly-Phe was used as an opioid agonist
pharmacophore.23,24 As for a pharmacophore for a substance P
antagonist, the 3′,5′-bis(trifluoromethyl)-benzyl ester of the
tetrapeptide Phe-Pro-Leu-Trp was chosen and was placed at the
C-terminal (Figure 1).15,16,25,26 Because both pharmacophores
share a Phe, an initial sequence for the bifunctional peptide was
designed as a 3′,5′-bis(trifluoromethyl)-benzyl ester of H-Tyr-
D-Ala-Gly-Phe-Pro-Leu-Trp with the Phe overlapped (Figure
1). However, because the enzymatic hydrolysis of 3′,5′-
bis(trifluoromethyl)-benzyl ester has been reported, and the two
highly lipophilic trifluoromethyl groups might lead to low
solubility in aqueous solutions, our design approach has focused
on modifications of the 3′,5′-bis(trifluoromethyl)-benzyl ester
at the C-terminal to replace its ester by an amide or by complete
removal of the trifluoromethyl groups from the C-terminal.27,28
* 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.
‡
10.1021/jm070332f CCC: $40.75
2008 American Chemical Society
Published on Web 02/12/2008

1370 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Yamamoto et al.
Figure 2. Synthesis of bifunctional peptides on 2-chlorotrityl resin.
(a) Fmoc-Trp(Boc)-OH, diisopropylethylamine (DIEA), dichloromethane
(DCM), 2 h. (b) Stepwise chain elongation using 2-(6-Chloro-1H-
benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate
(HCTU)/20% piperidine chemistry. (c) 1% Trifluoroacetic acid (TFA)
in DCM, 30 min. (d) benzyl bromide or 3,5-bis(trifluoromethyl)benzyl
bromide, Cs2CO3, dimethylformamide (DMF), 2 h. (e) benzyl amine
or 3,5-bis(trifluoromethyl)benzyl amine or (3,5-Bis-trifluoromethyl-
benzyl)-methyl-amine, 6-Chloro-1-Hydroxybenzotriazole (Cl-HOBt),
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), DMF, 1 h. (f)
82.5% v/v TFA, 5% water, 5% thioanisol, 2.5% 1,2-ethanedithiol, 5%
phenol, 1 h.
To date, many classes of C-terminal modified peptides have
attracted our interest, and a number of approaches to modifying
the C-terminal have been reported.29–31 These approaches can
be classified into many categories, including nucleophilic
cleavage of protected peptides bound from appropriate resins,
attachment with a C-terminal functional group, side chain
anchoring followed by normal solid phase N-to-C peptide
synthesis, backbone amide attachment onto a solid support,
inverse C-to-N solid phase peptide synthesis, and conventional
solution phase synthesis (see ref 30 for a review). However, it
is difficult to synthesize C-terminal esters or tertiary amides by
the first two methods, and our designed peptides did not have
a suitable side chain moiety to anchor on a resin. Repeated
inverse C-to-N coupling leads to severe racemization, and
conventional Boc solution phase peptide synthesis is very labor
intensive for large amounts of longer peptides. Therefore, we
combined solid phase and solution phase chemistry to develop
a two-step combinatorial approach. In this method, a side chain-
protected peptide with a free C-terminal carboxylate was
synthesized using a NR-Fmoc solid phase peptide synthesis
(SPPS) on 2-chlorotrityl resin, followed by esterification or
amidation in solution phase without any detectable racemization
(Figure 2). Here, we report on the design, synthesis, and
biological activities of novel bifunctional peptides with opioid
agonist and substance P antagonist activities.
Results and Discussion
Peptide Synthesis. A series of peptides was obtained through
a two-step synthetic approach. The first step was the synthesis
of Boc-Tyr(tBu)-D-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH as a
shared intermediate using NR-Fmoc chemistry on a 2-chlorotrityl
resin, which is a common support for batch SPPS of protected
peptides with a free C-terminal.28 First, Fmoc-Trp(Boc)-OH was
introduced on a resin in the presence of diisopropylethylamine
(DIEA) in dimethylformamide (DMF). Resin-bound Fmoc-
Trp(Boc) was treated with 20% piperidine to remove a NR-
Fmoc protecting group. Couplings of the following amino acids
were carried out with standard in situ activating reagents used
in routine Fmoc SPPS with 2-(6-Chloro-1H-benzotriazole-1-
yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU),
in the presence of DIEA, to generate 6-Chloro-1-Hydroxyben-

C-Terminal Modified Bifunctional Peptides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1371
Table 2. Binding Affinities of Bifunctional Peptides at δ/µ Opioid Receptors and NK1 Receptors
hDORa, [3H]DPDPEb rMORa, [3H]DAMGOc hNK1d, [3H]substance Pe rNK1d, [3H]substance Pf
g
g
g
g
No.
log IC50
Ki (nM)
log IC50
Ki (nM) Ki(µ)/Ki(δ)
log IC50
-10.0 ( 0.14
-6.7 ( 0.07 100
Ki (nM)
log IC50
Ki (nM) Ki(rNK1)/Ki(hNK1)
1.6 35
270 2.7
1
2
3
4
5
6
-7.0 ( 0.10
-7.2 ( 0.14
-7.7 ( 0.09
-6.8 ( 0.24
-6.8 ( 0.08
-6.5 ( 0.09
50
31
10
77
72
31
2.6
-6.4 ( 0.20 180
3.6
0.046 -8.3 ( 0.06
-7.2 ( 0.17
-8.9 ( 0.09
-7.0 ( 0.33
-7.7 ( 0.21
-7.8 ( 0.13
29
0.65
46
9.5
6.8
1.4
0.94
0.065
0.60
0.13
0.22
0.54
-6.1 ( 0.04
30 ( 9.9%h
33 ( 7.6%h
-7.0 ( 0.06
-7.7 ( 0.03
-7.5 ( 0.03
-8.5 ( 0.04
-8.9 ( 0.06
-8.5 ( 0.06
14
1.6
0.61
1.4
33
6.1
54
4.4
Biphalini
L-732,138
-8.8 ( 0.02
0.73
-6.4 ( 0.12
130
180
a Competition analyses were carried out using membrane preparations from transfected HN9.10 cells that constitutively expressed the δ and µ opioid
receptors, respectively. b Kd ) 0.45 ( 0.1 nM. c Kd ) 0.50 ( 0.1 nM. d Competition analyses were carried out using membrane preparations from transfected
CHO cells that constitutively expressed the human or rat NK1 receptors. e Kd ) 0.40 ( 0.17 nM. f Kd ) 0.16 ( 0.03 nM. g The IC50 ( standard errors are
expressed as logarithmic values determined from the nonlinear regression analysis of data collected from two independent experiments (four independent
experimental values per drug concentration). The Ki values are calculated using the Cheng and Prusoff equation to correct for the concentration of the
radioligand used in the assay. h Inhibition % at 1µM. i Reference 49.
Table 3. Opioid Agonist Functional Activities in [35S]GTP-γ-S Binding Assays
hDORa
rMORa
EC50 (nM)c
Emax (%)d
log EC50
EC50 (nM)c
Emax (%)d
b
b
No.
log EC50
1
2
3
4
5
6
-7.5 ( 0.28
-7.1 ( 0.14
-7.8 ( 0.28
-6.8 ( 0.16
-7.1 ( 0.13
-6.9 ( 0.21
-9.0 ( 0.17
-8.8 ( 0.25
35
85
17
150
80
120
1.1
1.6
16 ( 0.7
131 ( 6
56 ( 6
70 ( 4
161 ( 7
137 ( 7
83
-6.8 ( 0.24
-7.4 ( 0.21
-8.2 ( 0.44
-7.5 ( 0.36
-7.2 ( 0.26
-7.1 ( 0.17
140
36
0.71
29
57
72
26 ( 2
66 ( 4
100 ( 16
120 ( 16
61 ( 4
72 ( 3
Biphalin
DPDPE
DAMGO
69
-7.4 ( 0.19
37
150
a Expressed from HN9.10 cell. b The log EC50 ( standard error are logarithmic values determined from the nonlinear regression analysis of data collected
from two independent experiments performed in duplicates. c Antilogarithmic value of the respective EC50. d The Emax value is the saturable, maximum level
of [35S]GTPγS binding in the cell membranes upon incubation with the peptide, expressed as [Net [35S]GTPγS bound/basal [35S]GTPγS bound] × 100 (
standard error.
Table 4. Functional Assay Result for Bifunctional Peptide Ligands at
Opioid and Substance P Receptors
zotriazole (Cl-HOBt) esters. The obtained resin-bound Boc-
Tyr(tBu)-D-Ala-Gly-Phe-Pro-Leu-Trp(Boc) was cleaved off the
resin with 1% TFA in dichloromethane (DCM) in 30 min. The
protected peptide was obtained after evaporation, followed by
precipitation with chilled petroleum ether. The resulting white
solid was washed twice with chilled petroleum ether, then dried
under vacuum to give the protected peptide with good purity
(98.8%) and moderate yield (57.0%: based on the substitution
of the resin).
opioid agonista,b
substance P antagonistb
MVD (δ),
GPI (µ),
GPI, Ke
No.
IC50 (nM)c
IC50 (nM)c
(nM)d
1
2
3
4
5
6
400 ( 23
40 ( 4.4
50 ( 10
41 ( 8.6
45 ( 6.3
150 ( 26
2.7 ( 1.5
520 ( 40
74 ( 25
13 ( 3.3
9.0 ( 0.5
350 ( 91
52 ( 9.4
8.8 ( 0.3
3.6 ( 1.1
150 ( 17
26 ( 3.9
59 ( 18
8.5 ( 2.1
6.9 ( 1.1
The second step was esterification or amidation of the
protected intermediate, followed by side-chain deprotection in
the solution phase. The esterification was performed employing
cesium carbonate to form the cesium salt of the protected peptide
to react with benzyl bromide or 3′,5′-bis(trifluoromethyl)-benzyl
bromide.25,26 The crude esters, H-Tyr-D-Ala-Gly-Phe-Pro-Leu-
Trp-O-3,5-Bzl(CF3)2 (1) and H-Tyr-D-Ala-Gly-Phe-Pro-Leu-
Trp-O-Bzl (2), were obtained quantitatively by one hour
treatment with the cleavage cocktail (82.5% v/v TFA, 5% water,
5% thioanisole, 2.5% 1,2-ethanedithiol, and 5% phenol) to
quench the highly stabilized carbocations released from per-
manent protecting groups. Some acid-mediated hydrolysis of
C-terminal benzyl ester occurred during the final cleavage, but
the purities of the final crude peptides were still moderate to
good (84% for 1 and 67% for 2). For the amidation, standard
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/Cl-
HOBt coupling chemistry with two equivalents of reactant amine
was used. The crude amides 3-6 were obtained with 98%
quantitative yield and good purity (75∼93%). The crude
peptides 1-6 were purified by RP-HPLC (>99% purity) and
were characterized by analytical HPLC, 1H NMR, HRMS, and
TLC (Table 1). 1H NMR studies showed cis/trans isomerization
at the Pro5 residue. The ratios of two amide rotamers and their
assignments are available in the Supporting Information. Pep-
Biphalin
L-732,138
250 ( 87
a All the tested compounds were full agonists compared to the standards
DPDPE and PL-017 for MVD and GPI assays, respectively. b Calculated
from four isolated tissues (n ) 4). c Concentration at 50% inhibition of
muscle concentration at electrically stimulated isolated tissues. d Inhibitory
activity against the substance P-induced muscle contraction in the presence
of 1 µM naloxone, Ke: concentration of antagonist needed to inhibit
substance P to half of its activity. The Ke values were obtained from full
concentration-effect curve for substance P in the absence and presence of
the test compounds in the presence of 1 µM naloxone to block opioid effects
on the tissue.
tides 1-6 were also prepared using NR-Boc solution phase
chemistry with a previously reported protocol.15 The two-step
1
approach described in this publication showed the same H
NMR spectra with the ones through NR-Boc solution chemistry,
implying that the reported approach led to the undetectable
amounts of racemization.
Structure–Activity Relationships. All receptor binding
affinities and functional activities of peptides 1–6 were evaluated
as previously described (Tables 2–4).15 For evaluating lipophi-
licity of peptides, the logarithm of distribution coefficient
between 1-octanol and saline (log D7.4) was measured (Table
1).32

1372 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Yamamoto et al.
In evaluating the test peptides’ antagonist activity at the NK1
receptor, radioligand analysis indicated that these peptides
showed moderate selectivity between the NK1 receptors from
rat and human. A number of NK1 antagonists have been
previously reported to exhibit selectivity among NK1 receptors
from different species. For example, CP-96,345 and RP-67,580,
both of which are well-defined NK1 antagonists, showed
significant selectivity between the human and rat NK1 receptor,
and their affinity at the guinea pig NK1 receptor was similar to
that for the human NK1 receptor. The selectivity of these
compounds among species homologues has been proposed to
be due to structural differences identified in the primary
sequences of the receptors. The rat NK1 receptor is known to
have 95% homology with the human NK1. Twenty-one out of
407 amino acid residues differ between the two receptor
homologues, including 6 amino acid residues in the transmem-
brane domains, whereas the guinea pig NK1 shares 97%
homology with the human NK1 homologue with 100% identical
transmembrane domains. These structural differences, especially
in the critical transmembrane segments, have been proposed to
account for differences in the inhibition constants of NK-1
antagonists for the rat NK1 when compared with that for human
NK1 or guinea pig NK14,33–36 Consistent with the earlier
observations, L-732,138 (Ac-Trp-3,5-O-Bzl(CF3)2), which was
a part of substance P antagonist pharmacophore of 1 and shared
the same binding pocket with CP-96,345 and RP-67,580,
showed high affinity at the human NK1 receptor when compared
with that for the rat NK1 receptor (Table 2).37 As shown in
Table 2, peptide 1, with a 3′,5′-bis(trifluoromethyl)-benzyl ester
at the C-terminal, had good binding affinity for both human
and rat NK1 receptors and exhibited functional antagonism
against substance P stimulation in guinea pig ileum (Ke ) 3.6
nM, GPI). This peptide had a longer peptide sequence than
L-732,138. Compared with L732,138, peptide 1 showed much
less selectivity between the human and rat NK1 receptor
homologues (35-fold selectivity for the human NK1 receptor
vs 200-fold selectivity in the case of L732,138). Peptide 1 also
had moderate binding affinities at the DOR and MOR (Ki ) 50
and 180 nM, respectively). In the MVD, GPI, and [35S]GTPγS
binding assays, the opioid agonist activities of 1 were consistent
with the binding assays. As expected, 1 showed a lipophilic
character with its log D7.4 value being above the detectable limit
(greater than 4.0). Interestingly, the removal of the two
trifluoromethyl groups of 1 (2) resulted in increasing opioid
affinities especially at the MOR (Ki ) 31 nM, DOR; 29 nM,
MOR) (Table 2). In the functional tissue assays, both the
activities in the MVD and GPI assays also were increased from
those of 1 (IC50 ) 40 nM, MVD; 74 nM, GPI). However, the
removal of trifluoromethyl groups at the C-terminal ester
resulted in reduced antagonist activities for the NK1 receptors,
indicating a much more critical role for the trifluoromethyl
groups for the substance P antagonist pharmacophore. For the
derivative without trifluoromethyl groups (2), the affinities at
the NK1 receptors were much decreased from those of 1, and
these results were consistent with the Ke value in the GPI assay
(Ki ) 270 nM, rNK1; Ki ) 100 nM, hNK1; Ke ) 150 nM,
GPI).
guinea pig and the rat should be enhanced from that of 2 (more
than 40-fold). As expected from the result of the GPI assay, 3
showed a good affinity at hNK1 (Ki ) 14 nM). Surprisingly,
the opioid activities of 3 showed large increases from those of
both 1 and 2, especially at the MOR. Its affinity at the DOR
was 10 nM, and subnanomolar-level affinity was found at the
MOR (Ki ) 0.65 nM), which were consistent with the results
in the [35S]GTPγS binding (EC50 ) 17 nM, DOR; 0.71 nM,
MOR) and tissue assays (IC50 ) 50 nM, MVD; 13 nM, GPI)
(Tables 3 and 4). These results indicated that 3 is expected to
work as a potent bifunctional peptide with µ-preferable opioid
agonist and substance P antagonist activities in humans, but not
in rats. However, a simple N-methylation in the C-terminal
amide of 3 (4) decreased affinities at both the opioid receptors
(Ki ) 77 nM, DOR; 46 nM, MOR) from those of 3, and this
biological trend was maintained in the [35S]GTPγS binding
assay. Contrary to the results from both the radioligands and
the [35S]GTPγS binding assays, 4 showed good potencies in
the MVD and GPI assays (IC50 ) 41 nM, MVD; 9.0 nM, GPI)
(Table 4). The functional activity of 4 as a substance P
antagonist was more than two times lower than 3 (Ke ) 59
nM, GPI), although its Ki value at hNK1 was improved (1.6
nM). These biological results of the compounds without
trifluoromethyl groups (2-4) proved that the C-terminus may
act as an address region for opioid activities, although it is
structurally apart from the opioid pharmacophore. In fact, simple
ester-to-amide modifications at the C-terminus changed the
opioid selectivity into µ-preference. The log D7.4 values of the
peptides without trifluoromethyl groups (2-4) showed moder-
ately lipophilic values (3.8, 2; 3.3, 3; 3.5, 4), which were
decreased from that of 1 (Table 1).
Because the introduction of amide at the C-terminus was
successful in the benzyl amide derivative 3, we next prepared
the 3′,5′-bis(trifluoromethyl)-benzyl amide derivative (5). It also
showed a µ-selective binding affinities with nanomolar-level
affinity at the MOR and a moderate affinity at the DOR (Ki )
72 nM, DOR; 9.5 nM, MOR) (Table 2). However, in the tissue
assays, the IC50 value in the MVD assay (45 nM) was better
than that in the GPI assay (350 nM). Therefore, 5 could strongly
bind at the MOR, but its functional activity to regulate a µ opioid
signal in the tissue was apparently decreased. As for its
substance P antagonist activity, ligand 5 had decreased affinities
at both the hNK1 and rNK1 receptors from those of the
C-terminal ester 1 (Ki ) 0.61 and 33 nM, respectively) (Table
2). In the GPI assay, the substitution of a benzyl ester to amide
with trifluoromethyl groups showed less influence on sub-
stance P antagonist activity compared to the results of 2 and 3
(Ke ) 3.6 nM for 1 and 8.5 nM for 5) (Table 4). Finally, the
N-methylation in the C-terminal amide (6) was also tested.
Relatively small differences (less than 2.5-fold) were found in
the binding affinities at rMOR, hDOR, and hNK1 receptors from
those of 5. For opioids, 6 showed moderate binding at the DOR
and good affinity for the MOR (Ki ) 31 nM, DOR; 6.8 nM,
MOR), and this trend was maintained in the IC50 values in MVD
and GPI assays (150 and 52 nM, respectively), although its
functional activities were relatively low in the [35S]GTPγS
binding (EC50 ) 120 nM, DOR; 72 nM, MOR). The binding
affinity of 6 at the hNK1 receptor was two times less than that
of 5 (Ki ) 1.4 nM), but its Ke value in the GPI assay with
substance P stimulation was 6.9 nM, which was almost
equipotent to 5 (Tables 2 and 4). Consequently, 6 was also found
as a potent bifunctional peptide derivative with opioid agonist
In contrast to the large decreased activities of the C-terminal
benzyl ester derivative without trifluoromethyl groups (2) for
the substance P antagonist activity in the GPI assay, the Ke value
of its benzyl amide derivative (3) was 26 nM, which was 6 and
10 times more active than those of 2 and L-732,138, respectively
(Table 4). Because 3 had only low affinity for the rNK1 receptor
(30% inhibition at 1.0 µM), the species difference between the

C-Terminal Modified Bifunctional Peptides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1373
piperidine in DMF (1 × 2 min and 1 × 20 min). The deprotected
resin was washed with DMF (3 × 15 mL) DCM (3 × 15 mL) and
then with DMF (3 × 15 mL). Fmoc-Leu-OH (3 equiv) and HCTU
(2.9 equiv) were dissolved in 30 mL of DMF, then DIEA (6 equiv)
was added. The coupling mixture was transferred into the syringe
with the resin, and then the mixture was shaken for 2 h. All the
other amino acids, Pro, Phe, Gly, D-Ala, and Tyr were consecutively
coupled using the procedure described above, using the TNBS test
(all amino acids except for Phe) or chloranil test (only for Phe) to
check the extent of coupling. In case of a positive test result, the
coupling was repeated until a negative test result was obtained.
The resulting batch of the resin-bound protected Boc-Tyr(tBu)-D-
Ala-Gly-Phe-Pro-Leu-Trp(Boc) was carefully washed with DMF
(3 × 15 mL), DCM (3 × 15 mL), DMF (3 × 15 mL), and DCM
(3 × 15 mL) and then dried under vacuum. The dry resin was placed
in 10 mL fritted polypropylene syringes and was swollen with DCM
for 1 h. The peptide was cleaved off the solid support with 1% v/v
TFA in DCM (30 mL) for 30 min, and most of the organic solvent
was removed under vacuum. The obtained crude peptide was
precipitated out by the addition of chilled petroleum ether (45 mL)
to give a white precipitate. The suspension was centrifuged for 20
min at 7000 rpm, and then the liquid was decanted off. The crude
peptide was washed with petroleum ether (2 × 50 mL), and after
the final centrifugation, the intermediate peptide was dried under
vacuum (2 h) to obtain the title compound (1.99 g, 57.0%). The
purity of the final products (99.8%) was checked by analytical RP-
HPLC using a Hewlett-Packard 1090m system (230 nm) on a
reverse phase column (Vydac 218TP104, C-18, 4.6 × 75 mm, 5
µm). The peptide was eluted with a linear gradient of aqueous
CH3CN/0.1% CF3CO2H (30–100% in 40 min) at a flow rate of 1.0
mL/min. The crude peptide was used for subsequent reactions
without further purification. MS (ESI) m/z 1109 (MH)+.
H-Tyr-D-Ala-Gly Phe-Pro-Leu-Trp-O-3,5-Bzl(CF3)2 ·TFA (1).
Boc-Tyr(tBu)-D-Ala-Gly-Phe-Pro-Leu-Trp(Boc)-OH (150 mg, 0.135
mmol) and 3,5-bis(trifluoromethyl)benzyl bromide (104 mg, 0.338
mmol) were dissolved in DMF (2 mL). Cesium carbonate (220 mg,
0.676mmol) was added to the solution at 0 °C. After being stirred
for 2 h at room temperature (rt), saturated aqueous sodium
bicarbonate (50 mL) was added to the solution, and the product
was extracted with ethyl acetate (30 mL) three times. The combined
organic phases were washed with 5% aqueous citrate and saturated
aqueous sodium chloride (50 mL each) and were dried over sodium
sulfate. The solvent was evaporated, and the protected peptide was
precipitated in cold petroleum ether (PE, 45 mL). The PE suspension
was centrifuged for 20 min at 7000 rpm, and then the liquid was
decanted off. The crude peptide was washed with petroleum ether
(2 × 50 mL), and after the final centrifugation, the intermediate
peptide was dried under vacuum. The obtained protected peptide
was treated with 82.5% v/v TFA, 5% water, 5% thioanisole, 2.5%
1,2-ethanedithiol, and 5% phenol (1.5 mL, 1 h). The crude peptide
was precipitated out by the addition of chilled diethyl ether (45
mL) to give white precipitates. The suspension was centrifuged
for 20 min at 7000 rpm, and then the liquid was decanted. The
crude peptides were washed with diethyl ether (2 × 45 mL), and
after the final centrifugation, the peptides were dried under vacuum
(2 h). The resulting white residues (116 mg, quantitative) were
dissolved in a 3:1 mixture of acetonitrile and distilled water (5 mL),
and the insoluble impurities were removed by passing the solutions
through syringe filters (Gelman Laboratory, Ann Arbor, Michigan,
Acrodisc 13 mm syringe filter with 0.45 µM PTFE membrane).
Final purification was accomplished by preparative RP-HPLC,
followed by lyophilization.
and substance P antagonist activities that are expected to work
in both human and rat.
Conclusions
A series of linear peptides was designed based on the concept
of overlapping pharmacophores with direct connection of the
enkephalin-derived pharmacophore for opioid agonist (Tyr-D-
Ala-Gly-Phe) and the pharmacophore for substance P antagonist
(Phe-Pro-Leu-Trp-O-3,5-Bzl(CF3)2). The modifications in this
study focused on the C-terminus of the peptides, and our two-
step combinatorial approach was applied to the synthesis with
good yields and with no detectable racemization. These
modifications at the C-terminus, which are a part of the
pharmacophore for substance P antagonist, resulted in different
affinities as well as bioactivities not only for the NK1 receptors,
but also for the opioid receptors. Among the obtained peptides,
the benzyl amide derivative 3 (H-Tyr-D-Ala-Gly-Phe-Pro-Leu-
Trp-NH-Bzl) exhibited potent and µ-preferable opioid agonist
activities together with good substance P antagonist activity in
the GPI assay. Although 3 showed low affinity for the rNK1
receptor, which implies that 3 has limited substance P antagonist
activity in rat, 3 is expected to be a potent and novel type of
analgesic in humans due to its bifunctional activities. The
N-methyl benzyl amide derivative with trifluoromethyl groups
in the C-terminus (6) showed efficient potency in both opioid
agonist and substance P antagonist activities together with the
nanomolar affinities at both rNK1 and hNK1 receptors, indicat-
ing the importance of trifluoromethyl groups especially for the
rNK1 affinity. It should be noted that the C-terminus of peptide
derivatives acted as not only a critical pharmacophore for the
substance P antagonist, but also as an address region for
the opioid agonist pharmacophore, and its modification could
lead to potent µ opioid selective bifunctional compounds from
the δ-selective enkephalin-based pharmacophore.
Experimental Section
Materials. All amino acid derivatives and HCTU were purchased
from EMD Biosciences (Madison, Wisconsin), Bachem (Torrance,
California), SynPep (Dublin, California) and Chem Impex Inter-
national (Wood Dale, Illinois). 2-Chlorotrityl resin was acquired
from Iris Biotech GmbH (Marktredwitz, Germany). ACS grade
organic solvents were purchased from VWR Scientific (West
Chester, Pennsylvania), and other reagents were obtained from
Sigma-Aldrich (St. Louis, Missouri) and were used as obtained.
The polypropylene reaction vessels (syringes with frits) were
purchased from Torviq (Niles, Michigan). 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-penicillamine) enkepha-
lin (DPDPE); [3H]-substance P; and [35S]-guanosine-5′-(γ-thio)
triphosphate were purchased from Perkin-Elmer (Wellesley, Mas-
sachusetts). Bovine serum albumin (BSA), protease inhibitors, Tris,
and other buffer reagents were obtained from Sigma (St. Louis,
Missouri). Culture medium (MEM, DMEM, and IMDM), penicillin/
streptomycin, and fetal calf serum (FCS) were purchased from
Invitrogen (Carlsbad, California).
Boc-Tyr(tBu)-D-Ala-Gly-Phe-Pro-Leu-Trp(Boc)-OH (7). The
peptide was synthesized manually by the NR-Fmoc solid-phase
methodology using HCTU as the coupling reagents. 2-Chlorotrityl
resin (2.0 g, 1.56 mmol/g) was placed into a 50 mL polypropylene
syringe with the frit on the bottom and swollen in DMF (20 mL)
for 1 h. The resin was washed with DMF (3 × 15 mL) and then
with DCM (3 × 15 mL). Fmoc-Trp(Boc)-OH (1.2 equiv) was
dissolved in 30 mL of DCM, and then DIEA (5 equiv) was added.
The reaction mixture was transferred into the syringe with the resin
and then shaken for 2 h. The resin was washed three times with
DMF (15 mL), three times with DCM (15 mL), and then with DMF
(3 × 15 mL). The Fmoc protecting group was removed by 20%
H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-O-Bzl·TFA (2). The title
peptide was prepared using the same method as described for
H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-O-3,5-Bzl(CF3)2 ·TFA (1). The
yield of obtained crude peptide was 98.3%.
H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl·TFA (3). Boc-
Tyr(tBu)-D-Ala-Gly-Phe-Pro-Leu-Trp(Boc)-OH (50 mg, 0.045 mmol)
and Cl-HOBt (8.7 mg, 0.054 mmol) were dissolved in DMF (1
mL). Benzyl amine (5.8 mg, 0.090 mmol) and EDC (10.4 mg, 0.054
mmol) were added to the solution at room temperature and were

1374 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Yamamoto et al.
stirred until the starting material disappeared in TLC; then, saturated
aqueous sodium bicarbonate (50 mL) was added to the mixture.
The reaction mixture was extracted three times with ethyl acetate
(30 mL). The combined organic phases were washed with 5%
aqueous citrate and saturated aqueous sodium chloride (50 mL each)
and then dried over sodium sulfate. The solvent was evaporated,
and the crude peptide was precipitated in cold petroleum ether (45
mL). The product was twice dispersed in cold petroleum ether,
centrifuged, and decanted, and then dried under reduced pressure.
The obtained protected peptide was treated with 82.5% v/v TFA,
5% water, 5% thioanisole, 2.5% 1,2-ethanedithiol, and 5% phenol
(1.5 mL, 1 h). The crude peptide was precipitated out by the addition
of chilled diethyl ether (45 mL) to give a white precipitate. The
resulting peptide suspensions were centrifuged for 20 min at 7000
rpm, and the liquid was decanted. The crude peptides were washed
with diethyl ether (2 × 45 mL), and after a final centrifugation,
the peptides were dried under vacuum (2 h). The resulting white
residues (53 mg, quantitative) were dissolved in a 3:1 mixture of
acetonitrile and distilled water (1 mL), and the insoluble impurities
were removed by passing the solutions through syringe filters
(Gelman Laboratory, Acrodisc 13 mm syringe filter with 0.45 µM
PTFE membrane). Final purification was accomplished by prepara-
tive RP-HPLC. The pure title compound was obtained after
lyophilization.
centrifuged at 6500 rpm in a VanGuard V6500 (GlaxoSmithKline,
Research Triangle Park, North Carolina) for 15 min. The layers
were separated, and each layer was once again centrifuged. The
peptide concentrations of the obtained layers were analyzed by
HPLC (30–70% of acetonitrile containing 0.1% TFA within 20 min
and up to 95% within additional 5 min, 1 mL/min, 230 nm, Vydac
218TP104 C-18 column). The logarithm of 1-octanol/saline dis-
tribution (log D7.4) was calculated as the ratio of peptide concentra-
tion in the 1-octanol and saline phases.
Cell Lines.15 For opioid receptors, the cDNA for the human δ
opioid receptor (DOR) was a gift from Dr. Brigitte Kieffer (IGBMC,
Illkirch, France). The cDNA for the rat µ opioid receptor (MOR)
was a gift from Dr. Huda Akil (University of Michigan, Michigan).
Stable expression of the rat MOR (pCDNA3) and the human DOR
(pCDNA3) in the neuroblastoma cell line, HN9.10 were achieved
with the respective cDNAs by calcium phosphate precipitation
followed by clonal selection in neomycin. Expression of the
respective receptors was initially verified, and the level of expression
was periodically monitored by radioligand saturation analysis (see
below). All cells were maintained at 37 °C, with a 95% air/5%
CO2 humidified atmosphere in a Forma Scientific incubator in
Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS and
100 U mL penicillin/100 µg mL streptomycin.
For the NK1 receptor, the hNK1/CHO and rNK1/CHO cell lines
were obtained from Dr. James Krause (University of Washington
Medical School, St. Louis, Missouri). Expression of the receptor
was verified as previously described by Krause et al.45 All cells
were maintained at a 37 °C, 95% air and 5% CO2, humidified
atmosphere, in a Forma Scientific incubator in Ham’s F12 with
2.5 mM HEPES, 10% fetal bovine serum, and 100 U mL penicillin/
100 µg mL streptomycin/ 500 µg mL Geneticin.
H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-NMe-Bzl·TFA (4). The
title peptide was prepared using same method as described for
H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-NH-3,5-Bzl ·TFA (2). The crude
peptide was obtained quantitatively.
H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-NH-3,5-Bzl(CF3)2 ·TFA
(5). The title peptide was prepared using same method as described
for H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-NH-3,5-Bzl·TFA (2). The
yield of obtained crude peptides was 98.1%.
Radioligand Labeled Binding Assays. For the opioid receptors,
crude membranes were prepared as previously described46 from
the transfected cells that express either the rat MOR (rMOR) or
the human DOR (hDOR). 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), and 1 mg/mL BSA). The
dissociation constant (Kd) of tritiated [D-Ala2, NMePhe4, Gly5-ol]-
enkephalin ([3H]DAMGO) at the rMOR and that of tritiated c[D-
Pen2, D-Pen5]-enkephalin ([3H]DPDPE) at the hDOR were as
previously described.15 For competition analysis, 10 concentrations
of a test compound were each incubated, in duplicates, 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 assays were carried out in duplicates and were
repeated. 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% polyethyl-
eneimine, washed 4 times each with 2 mL of cold saline, and then
radioactivity determined by liquid scintillation counting (Beckman
LS5000 TD).
For the NK1 receptor, competition binding assays for the human
and rat NK1 receptors were carried out on crude membranes
prepared from transfected CHO cells expressing the corresponding
NK1 receptor. Ten concentrations of a test compound were each
incubated, in duplicates, with 50–100 µg of membrane homogenate
and 0.3-0.4 nM [3H] substance P (135 Ci/mmol, Perkin-Elmer)
in 1 mL final volume of assay buffer (50 mM Tris, pH 7.4,
containing 5 mM MgCl2, 50 µg/mL bacitracin, 30 µM bestatin, 10
µM captopril, and 100 µM phenylmethylsulfonylfluoride (PMSF))
at 25 °C for 20 min. Substance P at 10 µM was used to define the
nonspecific binding. Membrane concentrations used in the assay
were within the tissue linearity range. The [3H] substance P
concentration was selected based on the saturation binding experi-
ments that showed a high affinity binding with Kd ) 0.40 ( 0.17
nM or Kd ) 0.16 ( 0.03 nM for the hNK1 and rNK1 receptors,
H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-NMe-3,5-Bzl(CF3)2 ·TFA
(6). The title peptide was prepared using same method as described
for H-Tyr-D-Ala-Gly-Phe-Pro-Leu-Trp-NH-3,5-Bzl·TFA (2). The
crude peptide was obtained quantitatively.
Characterization of Peptides. Preparative RP-HPLC was
performed on Waters Delta Prep 4000 with a Waters XTerra C-18
column (19 × 250 mm, 10 µm, a linear gradient of 33–53% or
40–60% acedtonitrile/0.1% TFA at a flow rate of 15.0 mL/min).
The purified peptides were characterized by HRMS, TLC, analytical
1
HPLC, and H-1D-NMR. Sequential assignment of proton reso-
nances was achieved by 2D-TOCSY NMR experiments.38–42 High-
resolution MS were taken in the positive ion mode using ESI
methods at the University of Arizona Mass Spectrometry Facility.
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. TLC
chromatograms were visualized by UV light and by ninhydrin spray
followed by heating (hot plate). Analytical HPLC was performed
on a Hewlett-Packard 1100 or Hewlett-Packard 1090m with a
Waters NOVA-Pak C-18 column (3.9 × 150 mm, 5 µm, 60Å) or
a Vydac 218TP104 C-18 column (4.6 × 250 mm, 10 µm, 300 Å).
1H-1D-NMR spectra were recorded on Bruker DRX-500 or DRX-
600 spectrometer. 2D-TOCSY NMR spectra were performed on a
Bruker DRX-600 spectrometer equipped with 5 mm Nalorac triple-
resonance single-axis gradient probe. The NMR experiments were
conducted in DMSO-d6 solution at 298 K. Spectra were referenced
to residual solvent protons as 2.49 ppm. The processing of NMR
data was performed with the XwinNmr software (Bruker BioSpin,
Fremont, California) and the Felix2000 package (Accelrys Inc., San
Diego, California). In TOCSY experiments, the TPPI mode43 with
MLEV-17 Mixing Sequence44 was used with a mixing time of 62.2
ms, at a spin-lock field of 8.33 kHz. TOCSY spectra were acquired
with 2k complex pairs in t2 and 750 FIDs using a 90°-shifted sine-
squared window function in both dimensions.
Octanol/Saline Distribution (log D7.4). HEPES buffer (0.05 M
HEPES buffer in 0.1 M NaCl, pH 7.4, 500 µL) was added to 2 mg
of peptide and was mixed with 500 µL of 1-octanol. The sample
was shaken at rt for 12 h to allow equilibrating. The sample was

C-Terminal Modified Bifunctional Peptides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1375
respectively. The incubation times correspond to the binding
equilibrium as determined from the kinetics experiments. The
reaction was terminated by rapid filtration and was washed as
described above. The filter-bound radioactivity was measured by
liquid scintillation counting (Beckman LS 6000SC). Log IC50 values
for each test compound were determined from nonlinear regression
analysis of data collected from two independent experiments
performed in duplicates (40 independent experimental values) using
GraphPad Prizm 4 software (GraphPad, San Diego, California). The
inhibition constant (Ki) was calculated from the antilogarithmic IC50
value by the Cheng and Prusoff equation.
Mouse Isolated Vas Deferens (MVD) Assay. The in vitro tissue
bioassay was performed as described previously.15 Male ICR mice
under ether anesthesia were sacrificed by cervical dislocation, and
the vasa deferentia were removed. Tissues were tied to gold chains
with suture silk, mounted between platinum wire electrodes in 20
mL of organ baths at a tension of 0.5 g, bathed in oxygenated (95:5
O2:CO2) magnesium free Kreb’s buffer at 37 °C, and stimulated
electrically (0.1 Hz, single pulses, 2.0 msec duration) at supra-
maximal voltage as previously described.48 Following an equilibra-
tion period, compounds were added to the bath cumulatively in
volumes of 14–60 µL until maximum inhibition was reached.
Response to an IC50 dose of DPDPE (10 nM) was measured to
determine tissue integrity before a compound testing.
Analysis of the GPI and MVD Assays. For opioid data analysis,
percentage inhibition was calculated using the average tissue
contraction height for 1 min preceding the addition of the agonist
divided by the contraction height 3 min after exposure to the dose
of agonist. IC50 values represent the mean of not less than four
tissues. IC50 and Emax estimates were determined by computerized
linear regression analysis over a typical drug concentration range
of 100-fold (2 logarthmic dose range) (the pharmacological statistics
package FlashCalc: Dr. Michael Ossipov, University of Arizona,
Tucson, Arizona).
[35S]GTPγS Binding Assay. The method was carried out as
previously described.15,46 Membrane from transfected rMOR or
hDOR cells (10 µg) in a final volume of 1 mL reaction 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, and
0.1 nM [35S]GTPγS) was incubated with various concentrations
of the test drug ranging from 1 × 10-12 to 1 × 10-5 M, in
duplicates, 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, and
pH 7.4). The radioactivity was determined by liquid scintillation
counting as above. 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 GTPγS.
Total binding was defined as the amount of radioactivity bound in
the presence of test drug. The effect of the drug at each concentra-
tion on [35S]GTPγS binding was calculated as a percentage by the
following equation: [Total bound - Basal]/[Basal - Nonspecific]
× 100. Data were analyzed by nonlinear least-squares regression
analysis using GraphPad Prism4 and using data collected from two
independent experiments in duplicates. The EC50 for a test drug is
converted from the logarithmic EC50 value derived from the best
fit curve, and the maximum stimulatory effect is expressed as Emax
( standard error.
For substance P data analysis, the height of the maximum peak
produced during the control substance P dose–response curve was
used as a 100% response, and other values were calculated as a
percentage. Ke values represent the mean of not less than four
tissues. Ke estimates were determined by computerized linear
regression analysis as above (FlashCalc).
Acknowledgment. This work was supported by grants from
the USDHS, National Institute on Drug Abuse, DA-13449 and
DA-06284. We thank Dr. Neil E. Jacobsen for assistance with
the NMR measurements and the University of Arizona Mass
Spectrometry Facility with the mass spectra measurements. We
express appreciation for Ms. Margie Colie and Ms. Brigid
Blazek for assistance with the manuscript.
For both radioligand and [35S]GTPγS binding, we adopted two
independent experiments as our standard procedure for the initial
evaluation of all compounds using cell lines because of the large
number of compounds we routinely screen on multiple receptor
types to determine both affinity and biological activity. These initial
analyses were designed to identify trends of structure–activity
relationship as rapidly as possible and were not intended for
determining geometric mean values of Ki or EC50. The data pooled
from two independent experiments indicated that the data are
reproducible.
Supporting Information Available: 1H NMR data and in vivo
result presented in reference 16. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) Yaksh, T. L. Spinal opiate analgesia: characteristics and principles of
action. Pain 1981, 11, 293–346.
Guinea Pig Isolated Ileum Assay. The in vitro tissue bioassays
were performed as previously described.15 Male Hartley guinea pigs
under ether anesthesia were sacrificed by decapitation, a nonterminal
portion of the ileum was removed, and the longitudinal muscle with
myenteric plexus (LMMP) was carefully separated from the circular
muscle as previously described.47 These tissues were tied to gold
chains with suture silk, mounted between platinum wire electrodes
in 20 mL organ baths at a tension of 1 g, bathed in oxygenated
(95:5 O2:CO2) Kreb’s bicarbonate buffer at 37 °C, stimulated
electrically (0.1 Hz, 0.4 msec duration) at supramaximal voltage.
Following an equilibration period, compounds were added cumu-
latively to the bath in volumes of 14–60 µL until maximum
inhibition was reached. A baseline for PL-017 was constructed to
determine tissue integrity and to allow calculation of antagonist
activity before opioid analogue testing began. If no agonist activity
was observed at 1 µM, a repeat PL-017 dose–response curve was
constructed to test for antagonist qualities.
All substance P antagonist compounds testing was performed in
the presence of 1 µM naloxone to block opioid effects on the tissue.
Two minutes after naloxone was added to the bath, the test
compound was added. Four minutes after naloxone was added, the
test dose of substance P was added to the bath, the peak height
was noted, and the tissues were washed. Agonist activity of the
analog was also observed during this period. Testing stopped at 1
mM concentrations of the test compound.
(2) Ossipov, M. H.; Lai, J.; King, T.; Vanderah, T. W.; Porreca, F.
Underlying mechanisms of pronociceptive consequences of prolonged
morphine exposure. Biopolymers 2005, 80, 319–324.
(3) Kalso, E. Improving opioid effectiveness: from ideas to evidence. Eur.
J. Pain 2005, 9, 131–135.
(4) Datar, P.; Srivastava, S.; Coutinho, E.; Govil, G. Substance P: structure,
function, and therapeutics. Curr. Top Med. Chem. 2004, 4, 75–103.
(5) King, T.; Ossipov, M. H.; Vanderah, T. W.; Porreca, F.; Lai, J. Is
paradoxical pain induced by sustained opioid exposure an underlying
mechanism of opioid antinociceptive tolerance? Neurosignals 2005,
14, 194–205.
(6) King, T.; Gardell, L. R.; Wang, R.; Vardanyan, A.; Ossipov, M. H.;
Malan, T. P., Jr.; Vanderah, T. W.; Hunt, S. P.; Hruby, V. J.; Lai, J.;
Porreca, F. Role of NK-1 neurotransmission in opioid-induced
hyperalgesia. Pain 2005, 116 (3), 276–288.
(7) King, T.; Ossipov, M. H.; Vanderah, T. W.; Porreca, F.; Lai, J. Is
paradoxical pain induced by sustained opioid exposure an underlying
mechanism of opioid antinociceptive tolerance. Neurosignals 2005,
14 (4), 194–205.
(8) Ma, W.; Zheng, W. H.; Kar, S; Quirion, R. Morphine treatment induced
calcitonin gene-related peptide and substance P increases in cultured
dorsal root ganglion neurons. Neuroscience 2000, 99 (3), 529–539.
(9) Powell, K. J.; Quirion, R.; Jhamandas, K. Inhibition of neurokinin-
1-substance P receptor and prostanoid activity prevents and reverses
the development of morphine tolerance in vivo and the morphine-
induced increase in CGRP expression in cultured dorsal root ganglion
neurons. Eur. J. Neurosci. 2003, 18 (6), 1572–1583.

1376 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Yamamoto et al.
(10) Misterek, K.; Maszczynska, I.; Dorociak, A.; Gumulka, S. W.; Carr,
D. B.; Szyfelbein, S. K.; Lipkowski, A. W. Spinal co-administration
of peptide substance P antagonist increases antinociceptive effect of
the opioid peptide biphalin. Life Sci. 1994, 54 (14), 939–944.
(11) Gu, G.; Kondo, I.; Hua, X. Y.; Yaksh, T. L. Resting and evoked spinal
substance P release during chronic intrathecal morphine infusion:
parallels with tolerance and dependence. J. Pharmacol. Exp. Ther.
2005, 314 (3), 1362–1369.
(12) Hökfelt, T.; Kellerth, J.; Nilsson, G.; Pernow, B. Substance P:
localization in the central nervous system and in some primary sensory
neurons. Science, 1975, 190, 889–890.
(13) Marchand, J. E.; Kream, R. M. The glycine-extended SP precursor,
SP-G, is normally expressed in SP-containing neurons, in Substance
P and Related Peptides: Cellular and Molecular Physiology. Leeman
S. E., Krause J. E., Lembeck, F. eds.; New York Academy of Sciences:
New York, 1990.
(28) Lewis, R. T.; Macleod, A. M.; Merchant, K. J.; Kelleher, F.; Sanderson,
I.; Herbert, R. H.; Cascieri, M. A.; Sadowski, S.; Ball, R. G.;
Hoogsteen, K. Tryptophan-derived NK1 antagonists: conformationally
constrained heterocyclic bioisosteres of the ester linkage. J. Med.
Chem. 1995, 38, 923–933.
(29) Sasubilli, R.; Gutheil, W. General Inverse Solid Phase Synthesis
Method for C-Terminally Modified Peptide Mimetics. J. Comb. Chem.
2004, 6, 911–915.
(30) Alsina, J.; Albercio, F. Solid-phase synthesis of C-terminal modified
peptides. Biopolymres 2003, 71, 454–477.
(31) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis A
Practical Approach; Oxford University Press: New York, 2000.
(32) Egleton, R. D.; Mitchell, S. A.; Huber, J. D.; Janders, J.; Stropova,
D.; Polt, R.; Yamamura, H. I.; Hruby, V. J.; Davis, T. P. Improved
bioavailability to the brain of glycosylated Met-enkephalin analogs.
Brain Res. 2000, 881, 37–46.
(14) Aicher, S. A.; Sharma, S.; Cheng, P. Y.; Chen, L. Y. L.; Pickel, V. M.
Dual Ultrastructural Localization of µ-Opiate Receptors and Substance
P in the Dorsal Horn. Synapse 2000, 36, 12–20.
(15) Yamamoto, T.; Nair, P.; Davis, P.; Ma, S. W.; Navratilova, E.; Moye,
M.; Tumati, S.; Vanderah, T. W.; Lai, J.; Porreca, F.; Yamamura,
H. I.; Hruby, V. J. Design, Synthesis and Biological Evaluation of
Novel Bifunctional C-terminal Modified Peptides for δ/µ Opioid
Receptor Agonists and Neurokinin-1 Receptor Antagonists. J. Med.
Chem. 2007, 50 (12), 2779–2786.
(16) Yamamoto, T.; Nair, P.; Davis, P.; Ma, S. W.; Moye, S.; Largent, T.;
Vanderah, T. W.; Lai, J.; Porreca, F.; Yamamura, H. I.; Hruby, V. J.
Design, Structure-Activity Relationships and Biological Evaluation of
Novel Bifunctional C-terminal Modified Peptides for δ/µ Opioid
Receptor Agonists and Neurokinin-1 Receptor Antagonists. 232nd
ACS National Meeting, San Francisco, CA, United States, Sep. 10–
14, 2006; p MEDI-7. The in vivo result presented at the conference is
also available in the Supporting Information.
(17) Foran, S. E.; Carr, D. B.; Lipkowski, A. W.; Maszczynska, I.;
Marchand, J. E.; Misicka, A.; Beinborn, M.; Kopin, A. S.; Kream,
R. M. Inhibition of morphine tolerance development by a substance
P-opioid peptide chimera. J. Pharmacol. Exp. Ther. 2000, 295 (3),
1142–1148.
(18) Bonney, I. M.; Foran, S. E.; Marchand, J. E.; Lipkowski, A. W.; Carr,
D. B. Spinal antinociceptive effects of AA501, a novel chimeric
peptide with opioid receptor agonist and tachykinin receptor antagonist
moieties. Eur. J. Pharmacol. 2004, 488 (1–3), 91–99.
(19) Morphy, R.; Rankovic, Z. Designed multiple ligands. An emerging
drug discovery paradigm. J. Med. Chem. 2005, 48 (21), 6523–6543.
(20) Lee, Y. S.; Agnes, R. S.; Badghisi, H.; Davis, P.; Ma, S. W.; Lai, J.;
Porreca, F.; Hruby, V. J. Design and synthesis of novel hydrazide-
linked bifunctional peptides as delta/mu opioid receptor agonists and
CCK-1/CCK-2 receptor antagonists. J. Med. Chem. 2006, 49 (5),
1773–1780.
(33) Saria, A. The tachykinin NK1 receptor in the brain: pharmacology
and putative functions. Eur. J. Pharmacol. 1999, 375 (1–3), 5160.
and references therein.
(34) Tooneya, P. A.; Andersona, W. B.; Lynch-Frameb, A.; Chahla, L. A.
The effects of haloperidol treatment on the distribution of NK1 receptor
immunoreactive neurons in guinea-pig brain. Neurosci. Lett. 2005,
383 (1–2), 155–159.
(35) Field, M. J.; McCleary, S.; Boden, P.; Suman-Chauhan, N.; Hughes,
J.; Singh, L. Involvement of the central tachykinin NK1 receptor during
maintenance of mechanical hypersensitivity induced by diabetes in
the rat. J. Pharmacol. Exp. Ther. 1998, 285 (3), 1226–1232.
(36) Barr, A. J.; Watson, S. P. Non-peptide antagonists, CP-96,345 and
RP 67580, distinguish species variants in tachykinin NK1 receptors.
Br. J. Pharmacol. 1993, 108 (1), 223–227.
(37) Cascieri, M. A.; Macleod, A. M.; Underwood, D.; Shiao, L. L.; Ber,
E.; Sadowski, S.; Yu, H.; Merchant, K. J.; Swain, C. J.; Strader, C. D.;
Fong, T. M. Characterization of the interaction of N-acyl-L-tryptophan
benzyl ester neurokinin antagonists with the human neurokinin-1
receptor. J. Biol. Chem. 1994, 269 (9), 6587–6591.
(38) Braunschweiler, L.; Ernst, R. R. Coherence transfer by isotropic
mixing: Application to proton correlation spectroscopy. J. Magn.
Reson. 1983, 53, 521–528.
(39) Davis, D. G.; Bax, A. Assignment of complex 1H-NMR spectra via
two-dimensional homonuclear Hartmann-Hahn spectroscopy. J. Am.
Chem. Soc. 1985, 107, 2820–2821.
(40) Subramanian, S.; Bax, A. Generation of pure phase NMR subspectra
for measurement of homonuclear coupling constants. J. Magn. Reson.
1987, 71, 325–330.
(41) Rance, M. Improved techniques for homonuclear rotating-frame and
isotropic mixing experiments. J. Magn. Reson. 1987, 74, 557–564.
(42) Bax, A.; Davis, D. G. MLEV-17-based two-dimensional homonuclear
magnetization transfer spectroscopy. J. Magn. Reson. 1985, 65, 355–
360.
(21) Agnes, R. S.; Lee, Y. S.; Davis, P.; Ma, S. W.; Badghisi, H.; Porreca,
F.; Lai, J.; Hruby, V. J. Structure-activity relationships of bifunctional
peptides based on overlapping pharmacophores at opioid and chole-
cystokinin receptors. J. Med. Chem. 2006, 49 (10), 2868–2875.
(22) Hruby, V. J.; Agnes, R. S.; Davis, P.; Ma, S. W.; Lee, Y. S.; Vanderah,
T.W.; Lai, J.; Porreca, F. Design of novel peptide ligands which have
opioid agonist activity and CCK antagonist activity for the treatment
of pain. Life Sci. 2003, 73 (6), 699–704.
(23) Horan, P. J.; Mattia, A.; Bilsky, E. J.; Weber, S.; Davis, T. P.;
Yamamura, H. J.; Malatynska, E.; Appleyard, S. M.; Slaninova, J.;
Misicka, A.; Lipowski, A. W.; Hruby, V. J.; Porreca, F. Antinocice-
ptive Profile of Biphalin, a Dimeric Enkephalin Analog. J. Pharmacol.
Exp. Ther. 1993, 265, 1446–1454.
(43) Marion, D.; Wüthrich, K. Application of phase sensitive two-
dimensional correlated spectroscopy (COSY) for measurements of
1H-1H spin-spin coupling constants in proteins. Biochem. Biophys.
Res. Commun. 1983, 113, 967–974.
(44) Braunschweiler, L.; Ernst, R. R. Coherence transfer by isotropic
mixing: Application to proton correlation spectroscopy. J. Magn.
Reson. 1983, 53, 521–528.
(45) Boyd, N. D.; Kage, R.; Dumas, J. J.; Krause, J. E.; Leeman, S. E.
The peptide binding site of the substance P (NK-1) receptor localized
by a photoreactive analogue of substance P: presence of a disulfide
bond. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 433–437.
(46) Lorenzen, A.; Fuss, M.; Vogt, H.; Schwabe, U. Measurement of
guanine nucleotide-binding protein activation by A1 adenosine receptor
agonists in bovine brain membranes: stimulation of guanosine-5′-O-
(3-[35S]thio)triphosphate binding. Mol. Pharmacol. 1993, 44 (1), 115–
123.
(24) Gentilucci, L. New Torends in the Development of Opioid Peptide
Analogues as Advanced Remedies for pain Relief. Curr. Top. Med.
Chem. 2004, 4, 19–38.
(25) Millet, R.; Goossens, L.; Goossens, J. F.; Chavatte, P.; Bertrand-
Caumont, K.; Houssin, R.; Henichart, J. P. Conformation of the
tripeptide Cbz-Pro-Leu-Trp-OBzl(CF3)2 deduced from two-dimensional
1H-NMR and conformational energy calculations is related to its
affinity for NK1-receptor. J. Pept. Sci 2001, 7 (6), 323–330.
(26) Millet, R.; Goossens, L.; Bertrand-Caumont, K.; Chavatte, P.; Houssin,
R.; Henichart, J. P. Synthesis and biological evaluation of tripeptide
derivatives of Cbz-Gly-Leu-Trp-OBzl(CF3)2 as NK1/NK2 ligands. Lett.
Pep. Sci. 1999, 6, 255–262.
(47) Porreca, F.; Burks, T. F. Affinity of normorphine for its pharmacologic
receptor in the naive and morphine-tolerant guinea-pig isolated ileum.
J. Pharmacol. Exp. Ther 1983, 225 (3), 688–693.
(48) Porreca, F.; LoPresti, D.; Ward, S. J. Opioid agonist affinity in the
guinea-pig ileum and mouse vas deferens. Eur. J. Pharmacol. 1990,
179 (1–2), 129–139.
(49) Lipkowski, A. W.; Misicka, A.; Davis, P.; Stropova, D.; Janders, J.;
Lachwa, M.; Porreca, F.; Yamamura, H. I.; Hruby, V. J. Biological
activity of fragments and analogues of the potent dimeric opioid
peptide, biphalin. Bioorg. Med. Chem. Lett. 1999, 9 (18), 2763–2766.
(27) Macleod, A. M.; Merchant, K. J.; Cascieri, M. A.; Sadowski, S.; Ber,
E.; Swain, C. J.; Baker, R. N-Acyl-L-tryptophan benzyl esters: potent
substance P receptor antagonists. J. Med. Chem. 1993, 14, 2044–2045.
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