Design of novel neurokinin 1 receptor antagonists based on conformationally constrained aromatic amino acids and discovery of a potent chimeric opioid agonist-neurokinin 1 receptor antagonist

Article abstract of DOI:10.1021/jm1016285

A screening of conformationally constrained aromatic amino acids as base cores for the preparation of new NK1 receptor antagonists resulted in the discovery of three new NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-3′, 5′-(CF₃)₂-Bn], 20 [Ac-Aba-Gly-NMe-3′,5′- (CF₃)₂-Bn], and 23 [Ac-Tic-NMe-3′,5′-(CF ₃)₂-Bn], which were able to counteract the agonist effect of substance P, the endogenous ligand of NK1R. The most active NK1 antagonist of the series, 20 [Ac-Aba-Gly-NMe-3′,5′-(CF₃) ₂-Bn], was then used in the design of a novel, potent chimeric opioid agonist-NK1 receptor antagonist, 35 [Dmt-d-Arg-Aba-Gly-NMe-3′,5′- (CF₃)₂-Bn], which combines the N terminus of the established Dmt¹-DALDA agonist opioid pharmacophore (H-Dmt-d-Arg-Phe-Lys-NH₂) and 20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-d-Arg-Aba-Gly-NH₂ (36), also proved to be an extremely potent and balanced μ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.

Full text of DOI:10.1021/jm1016285

ARTICLE
pubs.acs.org/jmc
Design of Novel Neurokinin 1 Receptor Antagonists Based on
Conformationally Constrained Aromatic Amino Acids and Discovery
of a Potent Chimeric Opioid Agonist-Neurokinin 1 Receptor
Antagonist
Steven Ballet,*,† Debby Feytens,† Koen Buysse,† Nga N. Chung,‡ Carole Lemieux,‡ Suneeta Tumati,§
Attila Keresztes,§ Joost Van Duppen,|| Josephine Lai,§ Eva Varga,§ Frank Porreca,§ Peter W. Schiller,‡
Jozef Vanden Broeck,|| and Dirk Tourwꢀe†
†Department of Organic Chemistry, Vrije Universiteit Brussel, B-1050 Brussels, Belgium
‡Department of Chemical Biology and Peptide Research, Clinical Research Institute, Montreal, Quꢀebec H2W 1R7, Canada
§Department of Pharmacology, University of Arizona, Tucson, Arizona 85724, United States
Animal Physiology and Neurobiology, Zoological Institute, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
S Supporting Information
b
ABSTRACT: A screening of conformationally constrained
aromatic amino acids as base cores for the preparation of new
NK1 receptor antagonists resulted in the discovery of three new
NK1 receptor antagonists, 19 [Ac-Aba-Gly-NH-30,50-(CF3)2-
Bn], 20 [Ac-Aba-Gly-NMe-30,50-(CF3)2-Bn], and 23 [Ac-Tic-
NMe-30,50-(CF3)2-Bn], which were able to counteract the
agonist effect of substance P, the endogenous ligand of
NK1R. The most active NK1 antagonist of the series, 20 [Ac-
Aba-Gly-NMe-30,50-(CF3)2-Bn], was then used in the design of
a novel, potent chimeric opioid agonist-NK1 receptor antago-
nist, 35 [Dmt-D-Arg-Aba-Gly-NMe-30,50-(CF3)2-Bn], which
combines the N terminus of the established Dmt1-DALDA agonist opioid pharmacophore (H-Dmt-D-Arg-Phe-Lys-NH2) and
20, the NK1R ligand. The opioid component of the chimeric compound 35, that is, Dmt-D-Arg-Aba-Gly-NH2 (36), also proved to
be an extremely potent and balanced μ and δ opioid receptor agonist with subnanomolar binding and in vitro functional activity.
’ INTRODUCTION
resulted in a hypothesis for the receptor-bound conformation of
NK1R antagonists.9,10 In most cases, the two aromatic groups,
crucial for high receptor affinity, are in proximity of each other
and are proposed to adopt either a parallel face-to-face9 or a
perpendicular “T” or edge-on “L” arrangement.10
Biologically, prolonged pain states lead to neuroplastic
changes in the CNS that modify/increase the release of certain
neurotransmitters, such as the pronociceptive neurotransmitter
SP, and increase the expression of the corresponding receptors
for these released pain-enhancing ligands.11-13 The current
treatment of chronic pain cannot counteract these changes;
hence, the currently used analgesics are not effective in these
pathological conditions. For this reason, an approach was taken
to design bifunctional ligands that act as agonists at opioid
receptors and as antagonists at NK1 receptors.11,16-18 The
concept for this type of designed multiple ligand (DML)19 was
originally developed by Lipkowski and co-workers.18,20-22
Substance P (SP, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-
Met-NH2), an undecapeptide neurotransmitter or neuromodulator,
is a member of the tachykinin family of peptides. These peptides
mediate their biological functions through binding to three neuro-
kinin G protein-coupled receptors, NK1, NK2, and NK3, which
have as endogenous ligands the peptides SP, neurokinin A, and
neurokinin B, respectively.1 The NK1 receptor is expressed both in
the central nervous system (CNS) and in peripheral tissues. The
observation that the release of SP is linked to the transmission of
pain and inflammatory responses related to noxious stimuli2 makes
SP receptor antagonists (NK1R antagonists) potential therapeutic
agents for pathologies such as postoperative pain, migraine,3
arthritis,4 asthma, nausea, and emesis.1,5,6
Small peptidomimetic NK1R antagonists have been devel-
oped and often possess a bis-aromatic motif, typically consisting
of a substituted (-OMe or -bis-CF3) phenyl group next to
another aromatic ring that is coupled to a central scaffold (e.g.,
1-4, Figure 1).7-9 The relative disposition of the aromatics in
such ligands was investigated in a large set of SP antagonists and
Received: December 22, 2010
Published: March 17, 2011
r
2011 American Chemical Society
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Figure 1. Structures of reported peptidomimetic NK1R antagonists.
Figure 2. Examples of reported bifunctional opioid-NK1 ligands 6-8.
Figure 3. General structures of the 9 (Aba), 10 (Aia), 11 (Tic), and 12
(Tcc) scaffolds.
Antagonism at the NK1 receptors blocks the signals induced by
SP, the native ligand for NK1R, and would potentially inhibit the
increased secretion of the peptide hormone SP and the enhanced
expression of NK1 receptors upon sustained opioid administra-
tion or in prolonged pain states, while the opioid subunit of these
DMLs, on the other hand, would be responsible for the activation
of μ opioid (MOR) and δ opioid (DOR) receptors to produce
the analgesic effect. Moreover, the administration of morphine to
NK1 knockout mice did not show the morphine-related reward-
ing properties.23
In general, the advantages of multitarget agents include enhanced
efficacy, relative to drugs that use the “one active agent, one target”
approach by modulating multiple targets simultaneously and lower
risk of drug-drug interaction, which can potentially cause unex-
pected side effects and/or toxicity. Because poor safety and a lack in
efficacy are the main causes of failure of compounds in clinical trials,
the approach of multitarget drug discovery is a research area of
increasing interest in drug discovery programs.24 Other advantages
of a single ligand with multiple biological activities over a cocktail of
individual drugs are (i) easy administration, (ii) single biodistribu-
tion, (iii) simple pharmacokinetic profile, and (iv) higher concen-
trations at the synaptic cleft, which may lead to significant synergies
in potency and efficacy.24
Hruby and Lipkowski synthesized three lead chimeras, 6 (TY
027, [H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-30,50-Bn-
(CF3)2]), 7 (H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-30,50-
Bn(CF3)2), and 8 (H-Tyr D-Ala-Gly-Phe-NH-NH-Trp-Cbz)
(Figure 2), which contain an enkephalin-based opioid pharma-
cophore at the N terminus and a NK1 receptor-binding subunit
at the C terminus.11,16,22,24-27 Gratifyingly, the dual opioid-
NK1R activity resulted in both enhanced antinociception in
acute pain models and prevention of opioid-induced tolerance in
chronic trials for the peptidic bifunctional ligand 6 and its ester
analogue 7.16,25 Compounds 6 and 7 were shown to potently
attenuate neuropathic pain without producing opioid-induced
tolerance; hence, they seem to validate the concept or approach
of using bifunctional opioid agonist-NK1 antagonist chimeras to
tackle this type of pathology.28
Previously, we have suggested the 4-amino-1,2,4,5-tetrahydro-
2-benzazepin-3-one 9 (Aba) and amino indoloazepinone 10
(Aia) (Figure 3) to represent privileged templates. The appro-
priate derivatization of the central core scaffolds 9 and 10 allowed
to obtain (selective) ligands for several G protein-coupled
receptors. Examples include opioid GPCRs,29,30 bradykinin
B2,31 and somatostatin receptors.32,33
In this work, we present the 30,50-bistrifluoromethyl benzyl
derivatization of the conformationally constrained amino acids
Aba 9 and Aia 10 and, in addition, of the 1,2,3,4-tetrahydroiso-
quinoline-3-carboxylic acid (Tic) 11 and Tcc 12 templates for
the development of new NK1R antagonists. The 1-phenyl-
benzazepine core 9 (R1 = Ph) was chosen as an alternative for
the benzodiazepine core in 4 (Figure 1),14 whereas the indoloa-
zepine core 10 could be considered to lead to a constrained
analogue of the potent antagonist 5.15
The biological evaluation of all newly prepared compounds for
NK1R antagonism led to the identification of three potent
antagonists. This discovery allowed us to integrate the novel
NK1R pharmacophore into the design of a compact peptidomi-
metic bifunctional opioid-NK1R ligand containing an opioid
agonist component derived from the potent μ opioid agonist
[Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2; Dmt =20,60-
tyrosine).34 Here, we present the design rationale, synthesis,
and in vitro biological evaluation of both NK1R ligands and a
dual opioid-NK1R ligand.
’ RESULTS AND DISCUSSION
Synthesis. Using an earlier reported methodology the phtha-
loyl (Phth)-(1S/R)-(4S)-amino-1,2,4,5-tetrahydro-2-benzaze-
pin-3-ones stereoisomers 13 and 14 were obtained.35 N-
Methyl-30,50-bistrifluoromethyl benzylamine was prepared by
N-methylation of the corresponding, commercially available,
benzyl chloride.36 Coupling of this secondary amine, or the
primary 30,50-bistrifluoromethyl benzylamine, with O-(benzo-
triazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate
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(TBTU) as a coupling reagent in the presence of Et3N gave the
N-protected Phth-(1R)-4-amino-(4S/R)-benzazepinone inter-
mediates. Removal of the Phth protective group via hydrazino-
lysis was completed in refluxing EtOH and followed by a
standard acetylation using acetic acid anhydride and DIPEA as
a base. The final compounds 15-17 were purified by preparative
RP-HPLC followed by lyophilization (purity g95%).
These compounds were structurally analogous to investigated
benzodiazepines of type 4 (Figure 1) that possessed the desired
NK1R antagonism.14 In this benzodiazepine series, as described
by Armour et al., the role and necessity of the third aromatic
moiety were questioned, and this motivated us to make the
compounds without the 1-phenyl substitution (Scheme 2). As
mentioned in the Introduction, numerous examples of NK1R
antagonists containing only two aromatics are published
(selected examples: 1-3 and 5 in Figure 1).
We started from the dipeptide mimetic Boc-(S)-Aba-Gly-OH
18,37 which was again coupled to both N-methyl- and 30-50-
bistrifluoromethyl benzylamine. Removal of the Boc group by
classic acidolysis (TFA/CH2Cl2/anisole 49:49:2) and acetyla-
tion, followed by HPLC purification, gave aminobenzazepinones
19 and 20. The identical pathway starting from commercial
Boc-(S)-Tic-OH 21 yielded tetrahydroisoquinoline analogues
22 and 23.
Scheme 2. Synthesis of Aba and Tic Analogues 19, 20, 22,
and 23a
Scheme 1. Synthesis of 1-Phenyl Aba Analogues 15-17a
a Reaction conditions: (a) 1.1 equiv of 30,50-bistrifluoromethyl benzyl-
amine or N-methyl-30,50-bistrifluoromethyl benzylamine, 1.1 equiv of
TBTU, 3 equiv of TEA, CH2Cl2, room temperature, 2 h; (b) 2 equiv of
a Reaction conditions: (a) 1.1 equiv of 30,50-bistrifluoromethyl benzyl-
amine or N-methyl-30,50-bistrifluoromethyl benzylamine, 1.1 equiv of
TBTU, 3 equiv of TEA, CH2Cl2, room temperature, 2 h; (b) TFA/
CH2Cl2/anisole 49:49:2, room temperature, 3 h; (c) 5 equiv of Ac2O,
TEA, EtOH, room temperature, 3 h.
NH2NH2 H2O, EtOH, reflux, 1.5 h; c) 5 equiv of Ac2O, TEA, EtOH,
3
room temperature, 3 h.
Scheme 3. Synthesis of Aia and Tcc Analogues 28a/b, 30, and 32a/ba
a Reaction conditions: (a) 1.05 equiv of H-GlyOMe HCl, 20 wt % MgSO4, 2.5 equiv of NaBH3CN, CH2Cl2, pH 6, room temperature, 4 h; (b) 1.5 equiv
3
of EDC HCl, 2.5 equiv of pyridine, acetonitrile:water 1:1, room temperature, 2 days; (c) 4 equiv of 1 M aqueous LiOH, MeOH, room temperature,
3
1.5 h; (d) 1.1 equiv of N-methyl-30,50-bistrifluoromethyl benzylamine, 1.1 equiv of TBTU, 3 equiv of NEt3, room temperature, 2 h; (e) 5% H2O in
TFA:acetonitrile 4:1, room temperature, 1 h; (f) acetonitrile:H2O 1:1, NEt3, pH 6, 5 equiv of Ac2O, room temperature, 2 h; (g) 1.05 equiv of
30,50-bistrifluoromethyl benzylamine, 20 wt % MgSO4, 2.5 equiv of NaBH3CN, CH2Cl2, pH 6, room temperature, 4 h.
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The seven- and six-membered Trp-based constrained Aia and
Tcc analogues were prepared from the Boc-20-formyl-(S/R)-Trp-
OH 24a/b32 and Boc-(S/R)-Tcc-OH 25a/b,38 respectively
(Scheme 3). Reductive amination with methyl glycinate and sodium
cyanoborohydride was immediately followed by 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC)-mediated lactamiza-
tion. The latter reaction was slow and needed 48 h to give complete
conversion to 26a/b. Saponification with LiOH and standard
coupling using TBTU gave 27a/b. The final acetylated structures
28a/b were obtained using the procedure for the Aba and Tic
ligands, after acidolysis and treatment with acetic anhydride.
A constrained analogue of 5 [Ac-Trp-O-30,50-(CF3)2Bn,39
Figure 1] was synthesized starting from 24a via reductive
alkylation with 30,50-bistrifluoromethyl benzylamine and carbo-
diimide-induced cyclization, to afford indoloazepinone 29. Stan-
dard Boc deprotection and acetylation gave 30. Moreover, both
(S)- and (R)-Tcc derivatized structures 32a and 32b were
obtained after a coupling/deprotection/acetylation sequence
similar to that used for the Tic analogues 22 and 23.
N-terminal fragment Boc-Dmt-D-Arg(Pbf)-Aba-Gly-OH was
first prepared using a Fmoc solid phase peptide synthesis strategy
on 2-chlorotrityl resin with DIC as a coupling reagent and HOBt
as an additive (Scheme 4). Boc-Dmt-OH was coupled as the final
residue, so that the N-terminal Boc group would be cleaved
simultaneously with the acid labile Pbf side chain protective
group. First, the protected sequence Boc-Dmt-D-Arg(Pbf)-Aba-
Gly-OH 33 was removed from the 2-Cl-trityl resin under mild
acidic conditions (1% TFA in CH2Cl2 for 30 min). Introduction
of the C-terminal pharmacophoric unit, the N-methyl-30,50-
bistrifluoromethyl benzylamide was performed by a coupling
the secondary amine with 33 by means of BOP in the presence of
diisopropylethylamine in dichloromethane over 3 h at room
temperature. Final cleavage of the protection groups in 34 was
achieved by treatment with TFA/CH2Cl2 49/49 (2 h at room
temperature) and anisole (2%) to scavenge the released carboca-
tions during deprotection. The final compound 35 was purified
by preparative HPLC (g95% purity).
The peptidic sequence for verification of the opioid activity of the
N-terminal opioid component, H-Dmt-D-Arg-Aba-Gly-NH2 36,
was prepared by standard SPPS using NR-Fmoc chemistry on Rink
resin with DIC/HOBt as the coupling mixture (not shown). After
cleavage from the solid support, the solvent was removed in vacuo,
the resulting crude mixture was washed with chilled Et2O, and the
precipitate was purified via preparative HPLC.
Biological Evaluation and Structure-Activity Relation-
ships. The functional activity of the potential NK1 receptor
ligands was evaluated for NK1R agonism and antagonism using
an assay measuring the agonist-induced calcium-dependent
aequorine luminescence of cells expressing NK1 receptors.40
None of the derivatized 1-phenyl-Aba (Scheme 1), Aba and Tic
(Scheme 2), and Aia and Tcc analogues (Scheme 3) showed any
significant agonism up to a concentration of 10-4 M (not shown).
This is in contrast to SP, which gave the expected NK1R activation
and served as the positive agonist control compound. Next, the
targeted neurokinin 1 receptor (NK1R) antagonism of all com-
pounds was evaluated. Three compounds (19, 20, and 23) were
able to counteract the effect of SP, as proven by the rightward shift of
the dose-response curve of SP in the functional receptor assay (see
the Supporting Information for graphical data).
Finally, the chimeric opioid-NK1R DML 35 (Scheme 4) was
prepared after identification of the most potent NK1R antagonist,
proven to be compound 20 (see the section Biological Evaluation
and Structure-Activity Relationships). For the synthesis of
the double pharmacophore-containing peptidomimetic struc-
ture, we combined an adapted (constrained) version of Dmt1-
[DALDA] (Dmt-D-Arg-Phe-Lys-NH2) with 20. As such, the
Scheme 4. Synthesis of Bifunctional Chimera 35a
These results demonstrate that both Tic and Aba can serve as a
core scaffold for the development of NK1R antagonists and that
for the Aba type central scaffolds, the 1-phenyl substituent
hinders antagonism at N1KR for both (4S) and (4R) configura-
tions (15-17). In contrast, the constrained tryptophan analogues
28a/b, 30, and 32a/b, obtained by derivatization of both 24a/b
and 25a/b, did not show any antagonist properties. This
indicates that these types of conformational constraints are not
mimicking the bioactive conformation of 5 or that the change of
a Reaction conditions: (a) Fmoc-Aba-Gly-OH, diisopropylethylamine,
CH2Cl2, room temperature, overnight; (b) 20% piperidine/DMF (5 þ
15 min); (c) coupling of Fmoc-D-Arg(Pbf)-OH and DIC/HOBt in
DMF room temperature, 2 h; (d) coupling of Boc-Dmt-OH and DIC/
HOBt in DMF, room temperature, 2 h; (e) 1% TFA in CH2Cl2 for 30
min; (f) coupling of N-methyl-30,50-bistrifluoromethyl benzylamine with
BOP/DIPEA in DMF, room temperature, 3 h; (g) TFA/CH2Cl2/
anisole 49:49:2, room temperature, 2 h.
Figure 4. Three lowest energy conformations of compound 20 showing a stacked orientation of the aromatics (A), a T-perpendicular orientation (B,
3.94 kJ/mol above A), and a L-perpendicular orientation (C, 4.72 kJ/mol above A).
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Table 1. Structures, Functional Activities, and Affinities of NK1, Opioid, and Opioid-NK1 Ligands
a The pA2 values were calculated using Schild's equation.41 b Inhibitory constants (Ki) of NK1 receptor ligands, measured for the receptor prototype
[3H]-SP in the presence of hNK1-CHO membranes. Results are means ( SEMs of three independent experiments. Binding data were calculated using
the nonlinear regression/one site competition fitting options of the GraphPad Prism Software. c Values represent means of 3-6 experiments ( SEMs.
The GPI functional assay is representative of MOR activation, whereas the MVD is a δ receptor-representative assay. Binding affinities of compounds for
μ and δ opioid receptors were determined by displacing [3H]DAMGO and [3H]DSLET, respectively, from rat brain membrane binding sites, and
binding affinities for κ opioid receptors were measured by displacement of [3H]U69,593 from guinea pig brain membrane binding sites.
the ester function in 5 into an amide function is not tolerated.
These structural features are, however, also present in the most
potent NK1R antagonist in the series, analogue 20. The preferred
conformations of 20 was studied by molecular modeling using
Macromodel.54 The three lowest energy conformations, which
differ by only 4.72 kJ/mol, have orientations of the aromatic rings
corresponding to the stacked (Figure 4A), the perpendicular T
(Figure 4B), or L orientations (Figure 4C).9,10 The distances
between the centroids of the aromatic rings (4.25, 5.33, and 5.49
Å, respectively) are within the range proposed for 30,50-bistri-
fluoromethyl benzyl-containing NK1R antagonists.10 This
suggests that 20 is able to orient its aromatic rings for the most
efficient interaction with the NK1R. In the indole analogue of 20,
compound 28a, similar conformations were found. However, the
pyrrole ring in 28a shifts the annulated benzene ring to a further
distance from the 30,50-bistrifluoromethyl benzene ring than in 20.
This might account for the difference in NK1R antagonist potency.
The calculated pA2 values (Table 1) allowed to rank the
relative potency of the Aba- and Tic-derived structures 19, 20,
and 23.41,42 These values indicate that a N-methylation at the level of
the C-terminal amide bond resulted in improved antagonist activity
[pA2(19) = 6.2 vs pA2(20) = 8.4 and pA2(23) = 7.5] and that the
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lactam constraint of 20, in combination with the carboxymethyl
chain of Gly, was favored over the isoquinoline constraint in 23.
In a next step, the binding affinity (Ki) of the active com-
pounds was determined on CHO membranes expressing the
human NK1 receptor. These data show that the antagonist
potency, indicated as pA2, of these compounds is in accordance
with the receptor binding affinities and gives the relative rank
order of 19 < 23 < 20. The moderate nanomolar binding affinity
of 19 and lack of antagonism of 22 offers proof that, for
compounds of type 19 and 20 and 22 and 23, the presence of
a hydrogen bond donor at the C-terminal benzyl amide, is
unfavorable for receptor binding and antagonist activity. Both
20 and 23 possess a tertiary amide linkage that results in
increased hydrophobicity and eliminates the possibility for
hydrogen bond formation at the amide bond site.
assay indicate that DOR agonist activity is still maintained but
only with a two digit nanomolar IC50 value.
The creation of bifunctional ligands of the type of compound
35 in general is a difficult task because the connected pharma-
cophores can interfere with each other to change or reduce their
binding and activity at the corresponding target receptors.
Therefore, the merged DML 35 is a very successful example of
a dual ligand in which a large overlap of pharmacophores is
present. The central Aba structure is part of both pharmaco-
phores. In addition, our earlier research had shown that a benzyl
amide moiety was tolerated in the shorter opioid ligand Dmt-
Aba-Gly-NH-Bn for both MOR and DOR,30 although this
subunit was shifted by one position (D-Arg in position 2 is
inserted) in hybrid 35 described here.
Earlier results have demonstrated that the use of the Aba
scaffold in opioid peptides results in very potent agonists with
high receptor affinity.30,43-45 Several examples were reported in
’ CONCLUSIONS
Different constrained Phe and Trp amino acid mimics were
used as central scaffolds in the design of new NK1R analogues.
Unfortunately, all Trp derivatives were inactive, both as agonists
and antagonists, in the functional assay measuring the agonist-
induced calcium-dependent aequorine luminescence of cells
expressing NK1 receptors. Gratifyingly, the derivatization of
the Ac-Tic and Ac-Aba-Gly scaffolds as the 30,50-bistrifluoro-
methyl benzylamides resulted in moderate (structure 19) to
good (structures 20 and 23) NK1R antagonists. The common-
ality of the Aba ring in both opioid and NK1R ligands prompted
us to combine both pharmacophores and produce successfully a
compact bifunctional opioid-NK1R ligand (structure 35) that
targets both the opioid and the neurokinin system. This study
also identified opioid control compound 36 as an extremely
potent and balanced μ and δ opioid receptor agonist in vitro,
which deserves further investigation.
Chimeric ligand 35 can be considered as a small DML with
highly merged frameworks. In general, medicinal chemists try to
maximize the degree of overlap and, hence, minimize molecular
weight, to have a better chance at oral activity.24 Ongoing
research consists of the structural fine-tuning of this new lead
compound. More extensive in vitro and in vivo assays are
scheduled to verify the membrane permeability of this dual
opioid agonist-NK1 receptor antagonist through important
biological barriers, such as the blood-brain barrier (BBB).
which Aba serves as the third residue in synthetic peptidic opioid
39
ligands (e.g., Tyr1-D-Ala2-Aba3-Gly4-NH2 and
Tyr1-D-Ala2-
Aba3-Gly4-Tyr5-Pro6-Ser7-NH2).46 This conformational con-
straint yields a substantial enhancement in DOR affinity, while
maintaining MOR binding and functional activity. In addition,
the presence of a positive charge in the side chain of a D-Arg
residue in position 2 is suggested to play a role in the excellent
membrane transport properties of Dmt1-D-Arg2-Phe3-Lys4-
NH2.47,48 For these reasons, we prepared the opioid “control”
compound Dmt-D-Arg-Aba-Gly-NH2 36 via solid phase pep-
tide synthesis using Fmoc chemistry with Rink amide resin as a
solid support. As can be seen from Table 1, both the opioid
receptor affinities and the functional activities of 36 are
extremely high and balanced for the μ and δ receptor, as
indicated by subnanomolar values in all assays. On the other
hand, this compound showed weak κ receptor binding affinity.
These data also indicate that D-Arg in position 2 can be
combined with the conformational constraint imposed by
Aba3 to provide a potent and balanced μ/δ opioid component
for the DML.
The framework combination24 of potent ligands for the two
separate targets resulted in multitarget ligand 35, which is a
combination of the most potent NK1R antagonist of the series,
Ac-Aba-Gly-NMe-30,50-(CF3)2Bn 20, and the very potent opioid
ligand Dmt-D-Arg-Aba-Gly-NH2 36. Although a modest loss in
NK1R antagonism was observed for 35 (pA2 8.4 f 7.8),
subnanomolar hNK1R binding (Ki = 0.5 nM) was determined
and proves that the presence of the opioid subunit even adds
favorable features for efficient binding to this G protein-coupled
receptor. As for the opioid in vitro characteristics, one can see
that MOR binding is less disrupted, as compared to DOR affinity,
when both frameworks are combined. A 3-fold loss in μ opioid
receptor (MOR)—but still subnanomolar—binding affinity was
determined [Ki(μ) 0.15 f 0.416 nM], whereas a 17-fold loss in
DOR affinity was observed [Ki(δ) 0.6 f 10.4 nM], relative to the
opioid “control” compound 36. As expected on the basis of the
weak κ receptor binding affinity of 36, chimera 35 showed weak κ
receptor binding affinity as well. The receptor binding affinities
are in agreement with the results obtained in the in vitro
functional GPI and MVD tissue bioassays. In the guinea pig
ileum (GPI) assay, reflecting mainly MOR activation, a low
nanomolar IC50 value of 8.51 nM indicates that, even though a
potency loss is observed relative to 36, DML 35 still is a very
potent agonist at the opioid μ receptor. The results of the MVD
’ EXPERIMENTAL SECTION
General. Thin-layer chromatography (TLC) was performed on
plastic sheet precoated with silica gel 60F254 (Merck, Darmstadt,
Germany) using specified solvent systems. Mass Spectrometry (MS)
was recorded on a Micromass Q-Tof Micro spectrometer using electro-
spray (ESP) ionization (positive or negative ion mode). Data collection
was done with Masslynx software. Analytical RP-HPLC was performed
using an Agilent 1100 Series system (Waldbronn, Germany) with a
Supelco Discovery BIO Wide Pore (Bellefonte, PA) RP C-18 column
(25 cm ꢀ 4.6 mm, 5 μm) using UV detection at 215 nm. The mobile
phase (water/acetonitrile) contained 0.1% TFA. The gradient consisted
of a 20 min run from 3 to 97% acetonitrile at a flow rate of 1 mL/min.
Preparative HPLC was performed on a Gilson apparatus and controlled
with the software package Unipoint. The Reverse phase C18 column
(Discovery BIO Wide Pore 25 cm ꢀ 21.2 mm, 10 μm) was used under
the same conditions as the analytical RP-HPLC but with a flow rate of
20 mL min-1. A purity of more than 95% was determined for all
compounds by analytical RP-HPLC using the conditions described
1
above. H NMR and 13C NMR spectra were recorded at 250 and 63
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Journal of Medicinal Chemistry
ARTICLE
MHz, respectively, on a Bruker Avance 250 spectrometer or at 500 and
125 MHz on a Bruker Avance II 500. Calibration was done with TMS
(tetramethylsilane) or residual solvent signals as an internal standard.
The solvent used is mentioned in all cases, and the abbreviations used are
as follows: s (singlet), d (doublet), dd (double doublet), t (triplet), br s
(broad singlet), and m (multiplet).
[3H] SP (135 Ci/mmol, Perkin-Elmer, United States) 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) SP at 10 μM was used to define the
nonspecific binding. The samples were incubated in a shaking water bath
at 25 °C for 20 min. The [3H] SP concentration and the incubation time
were selected based on the studies of Yamamoto et al.26 The reaction
was terminated by rapid filtration through Whatman grade GF/B filter
paper (Gaithersburg, MD) presoaked in 1% polyethyleneimine, washed
four times each with 2 mL of cold saline, and the filter bound radio-
activity was determined by liquid scintillation counting (Beckman
LS5000 TD). The media and chemicals listed above were purchased
from Sigma (Sigma-Aldrich, St. Louis, MO) unless otherwise stated.
Data Analysis. Analysis of data collected from three independent
experiments performed in duplicates is done using GraphPad Prizm 4
software (GraphPad, San Diego, CA). Log IC50 values for each test
compound were determined from nonlinear regression. The inhibition
constant (Ki) was calculated from the antilogarithmic IC50 value by the
Cheng and Prusoff equation.49
Functional NK1R Assay40. Cell Line and Cell Culture Condi-
tions. The Chinese hamster ovary K1 (CHO-K1) cell line, stably
expressing human NK1 receptor (hereafter referred to as CHO-NK1
cells), was transfected with an apoaequorin expression vector (pER2)
using Fugene6 (Roche Applied Science). The cell line and expression
vector were obtained from Euroscreen (Belgium). The CHO-NK1 cells
were cultured in sterile DMEM/HAM's F12 medium (Sigma) supple-
mented with 10% fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL
streptomycin, and 400 μg/mL G 418 (Geneticin, Gibco) at 37 °C with
5% CO2 and were trypsinized every 3 days.
Aequorin Charging Protocol. Transfected cells in the midlog phase
were detached by changing the growth medium for PBS buffer supple-
mented with 5 mM EDTA (pH 8). The cells were spun down and
incubated for 4 h at a concentration of 5 ꢀ 106 cells/mL in DMEM-F12
medium without phenol red (Gibco) supplemented with 0.1% BSA
(BSA medium) and 5 μM coelenterazine h (Molecular Probes). After
coelenterazine loading, the cells were diluted 10-fold in the same
medium and incubated for an additional period of 30 min. The cells
were mildly shaken during the incubation periods.
Functional GPI and Mouse Vas Deferens (MVD) Assays.
The GPI50 and MVD51 bioassays were carried out as described in detail
elsewhere.52,53
A dose-response curve was determined with
[Leu5]enkephalin as standard for each ileum and vas preparation, and
IC50 values of the compounds being tested were normalized according
to a published procedure.54
Aequorin Luminescence Assay. A dilution series of peptide agonist
(SP was purchased from Sigma) ranging from 10-11 to 10-4 M was
distributed in a white 96-well plate. For investigating antagonism, the
synthetic compounds were added to these wells to obtain the desired
concentrations (ranging from 10-8 to 10-4 M). One negative control
sample (BSA medium only) was included in each row of the 96-well
plate. The plate was loaded in a “Multimode Reader Mithras, LB940”
(Berthold). The wells were screened one by one, and each measurement
started at the moment of injection of 50 μL of the coelenterazine-loaded
cell suspension, containing 2.5 ꢀ 104 cells. Light emission was measured
every second for 30 s after which 50 μL of 10 nM ATP solution (positive
control) was injected. Each measurement was carried out in duplicate.
Light emission was recorded for an additional period of 10 s per well, and
the data were presented in relative light units (RLU).
Opioid Receptor Binding Assays. Opioid receptor binding
studies were performed as described in detail elsewhere.52 Binding
affinities for μ and δ opioid receptors were determined by displacing,
respectively, [3H]DAMGO (Multiple Peptide Systems, San Diego, CA)
and [3H]DSLET (Multiple Peptide Systems) from rat brain membrane
binding sites, and κ opioid receptor (KOR) binding affinities were
measured by displacement of [3H]U69,593 (Amersham) from guinea
pig brain membrane binding sites. Incubations were performed for 2 h at
0 °C with [3H]DAMGO, [3H]DSLET, and [3H]U69,593 at respective
concentrations of 0.72, 0.78, and 0.80 nM. IC50 values were determined
form log-dose displacement curves, and Ki values were calculated from
the IC50 values by means of the equation of Cheng and Prusoff,49 using
values of 1.3, 2.6, and 2.9 nM for the dissociation constants of
[3H]DAMGO, [3H]DSLET, and [3H]U69,593, respectively.
Molecular Modeling. The calculations were carried out using
Macromodel 5.054 with Maestro 8.0 as a graphic interface. The MM3*
force field55 was used for energy minimization in combination with the
GB/SA solvation model of Still et al.,56 using MacroModel's default
parameters for an aqueous medium. Conformational searches were
carried out using the Pure Low Mode Search.57 Structures were
generated and minimized by means of the Polak-Ribiꢁere conjugate
gradient method as implemented in MacroModel, using a gradient
convergence criterion of 0.1 kJ/mol Å. The resulting conformations
were again minimized to an energy convergence of 0.01 kJ/mol Å.
Duplicate structures and those greater than 50 kJ/mol above the global
minimum were discarded. The remaining structures were clustered into
families using Xcluster 1.7 (the aromatic carbons were used as compar-
ison atoms). A rmsd value of 0.2 Å was used.
General Synthetic Procedures. Boc Deprotection. Boc-pro-
tected amine (1.8 mmol) was dissolved in a mixture of TFA:water
95:5 (13 mL), and acetonitrile was added (6 mL). The reaction was
stirred for 1 h, and the mixture was evaporated. Crude TFA salts were
used in the next reactions.
Formation of Amide Bonds. Carboxylic acid (54 mmol) was dis-
solved in dry CH2Cl2 (250 mL). NEt3 (22.5 mL, 162 mmol, 3 equiv) and
TBTU (19.07 g, 59 mmol, 1.1 equiv) were added, and the mixture was
stirred at room temperature for 10 min. Amine TFA salt (59 mmol, 1.1
equiv) was added, and the pH was kept at 8 by the addition of NEt3. The
reaction was stirred for 1 h. The solution was extracted with HCl (1 M, 3
Data Analysis. Luminescence data (peak integration) were calculated
using MikroWin 2000 software (Berthold), which was linked to the
Microsoft Excel program. All statistical and curve-fitting analyses were
performed using Prism 4.0 (GraphPad) software. Data are expressed in
percentage (% RLU) of the maximal luminescence that was detected with
10-4 M SP (without antagonist). The competitive nature of antagonism
was evaluated using the Schild plot method.41 All antagonists analyzed in
this study provided linear regression plots and were considered competitive.
The pA2 values were calculated using Schild's equation.42
hNK1/CHO Cell Membrane Preparation and Radioligand
Binding Assay. Recombinant hNK1/CHO cells were grown to
confluency in 37 °C, 95% air and 5% CO2, humidified atmosphere, in
a Forma Scientific (Thermo Forma, OH) incubator in Ham's F12
medium supplemented with 10% fetal bovine serum, 100 U/mL
penicillin,100 μg/mL streptomycin, and 500 μg/mL geneticin. The
confluent cell monolayers were then washed with Ca2þ, Mg2þ-deficient
phosphate-buffered saline (PD buffer) and harvested in the same buffer
containing 0.02% EDTA. After centrifugation at 2700 rpm for 12 min,
the cells were homogenized in ice-cold 10 mM Tris-HCl and 1 mM
EDTA, pH 7.4, buffer. A crude membrane fraction was collected by
centrifugation at 18000 rpm for 12 min at 4 °C, the pellet was suspended
in 50 mM Tris-Mg buffer, and the protein concentration of the
membrane preparation was determined by using Bradford assay.
Six different concentrations of the test compound were each incu-
bated, in duplicates, with 20 μg of membrane homogenate, and 0.4 nM
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Journal of Medicinal Chemistry
ARTICLE
ꢀ 80 mL), NaHCO3 (saturated, 3 ꢀ 80 mL), and brine (3 ꢀ 80 mL).
The organic layer was dried over MgSO4, and after filtration, the residue
was evaporated. No further purification was necessary.
Hz), 5.29(m, 2H), 4.44(t, 2H,J=6.5Hz),4.28(d, 1H,J=15.8Hz), 4.07(d,
1H, J = 15.8 Hz), 3.45 (dd, 1H, J1 = 17.2 Hz, J2 = 5.0 Hz), 2.96 (dd, 1H, J1 =
17.3 Hz, J2 = 14.4 Hz), 1.45 (s, 9H). 13C NMR (CDCl3): δ 173.0, 168.8,
155.3, 140.6, 135.2, 132.6, 132.1, 131.7, 130.9, 128.8, 128.6, 127.8, 126.6,
121.5, 80.4, 53.6, 52.6, 50.4, 42.6, 36.9, 28.5
Acetylation. Amine TFA salt (0.89 mmol, 1 equiv) was dissolved in
acetonitrile:water 1:1 (10 mL), and the pH was adjusted to 6 by the
addition of NEt3. Ac2O (0.42 mL, 4.5 mmol, 5 equiv) was added in three
portions, while the pH was kept at 6. The reaction was stirred for 2 h at
room temperature. The solution was evaporated, and after the addition
of EtOAc (25 mL), it was extracted with HCl (1 M, 3 ꢀ 15 mL),
NaHCO3 (saturated, 3 ꢀ 15 mL), and brine (3 ꢀ 15 mL). The organic
layer was dried over MgSO4, and after filtration, the solution was
evaporated.
Saponification. Methyl ester (4.1 mmol) was dissolved in MeOH
(96 mL), and aqueous LiOH (1 M, 4 equiv, 16 mmol, 16.2 mL) was added.
The reaction was monitored by TLC (silica, EtOAc), and after 1.5 h,
it was complete. MeOH was evaporated, and water (100 mL) and
EtOAc (50 mL) were added. After extraction, the aqueous layer was
acidified to pH 4 and extracted with EtOAc (3 ꢀ 50 mL). The organic
layers were combined and washed with brine (3 ꢀ 100 mL). The
organic phase was dried over MgSO4, and after filtration, the mixture
was evaporated.
Ac-(S)-Aba-Gly-NH-30,50-(CF3)2-Bn (19). Preparative HPLC yielded
the desired compound (white powder, 43%). HPLC (standard
gradient): tret, 15.85 min. TLC Rf (EtOAc), 0.39. MS (ESPþ) found
m/z 502.14 [M þ H]þ, C23H21F6N3O3 requires 501.15. 1H NMR 250
MHz (CDCl3): δ 8.27 (br s, 1H), 7.78 (m, 3H), 7.37-6.99 (m, 5H),
5.41 (m, 1H), 5.29 (d, 1H, J = 16.8 Hz), 4.53 (m, 3H), 4.00 (d, 1H, J =
16.8 Hz), 3.79 (d, 1H, J = 16.8 Hz), 3.45 (dd, 1H, J1 = 17.7 Hz, J2 = 5.5
Hz), 2.95 (dd, 1H, J1 = 17.3 Hz, J2 = 13.7 Hz), 2.04 (s, 3H). 13C NMR
(CDCl3): δ 171.8, 169.0, 168.2, 141.2, 135.0, 132.4, 131.2, 130.6, 130.3,
128.2, 127.6, 127.3, 125.7, 120.3, 52.6, 50.9, 48.3, 41.6, 35.7, 22.6
Boc-(S)-Aba-Gly-NMe-30,50-(CF3)2-Bn. After flash chromatography
(EtOAc/hexane 1:1), the desired compound was obtained (white solid,
81%). HPLC (standard gradient): tret, 18.9 min. TLC Rf (EtOAc/
hexane 1:1): 0.49. MS (ESPþ) found m/z 574 [M þ H]þ,
C27H29F6N3O4 requires 573.21. 1H NMR 250 MHz (CDCl3): δ 7.81
(s, 1H), 7.66 (s, 2H), 7.00-7.25 (m, 4H), 5.89 (d, 1H, J = 5.3 Hz), 5.34
(d, 1H, J = 15.9 Hz), 5.26 (m, 1H), 4.78 (s, 1H), 4.71 (s, 1H), 4.51 (d,
1H, J = 15.9 Hz), 4.00 (m, 2H), 3.51 (dd, 1H, J1 = 16 Hz and J2 = 4.4 Hz),
3.02 (m, 1H), 2.96 (s, 3H), 1.47 (s, 9H). 13C NMR (CDCl3): δ 173.1,
168.5, 155.2, 139.3, 135.7, 132.4, 132.7, 131.9, 130.9, 128.6, 128.1, 126.5,
126.2, 121.8, 79.9, 53.2, 51.0, 49.5, 49.3, 37.2, 34.7, 28.3
Peptide Synthesis. Peptide 36 was synthesized manually by NR-Fmoc
solid phase methodology on Rink amide resin (0.189 mmol scale) using
DIC and HOBt as the coupling reagents. A 3-fold excess of the building
blocks [Fmoc-Aba-Gly-OH, Fmoc-D-Arg(Pbf)-OH, and Fmoc-Dmt-OH]
and activating agents was applied in the presence of a 9-fold excess of
diisopropylethylamine, and dry DMF was used as a solvent. Fmoc
deprotections were carried out by treating the resin twice (5 and
15 min) with 20% piperidine in DMF. Final cleavage of the peptide as
well as the Pbf side chain protection group removal was accomplished
by treatment with TFA/TES/water 90:5:5 for 90 min. The peptide
was isolated and purified by RP-HPLC on a Supelco DiscoveryBIO
wide pore preparative C18 column in 45% overall yield and was >95%
pure as determined by analytical RP-HPLC. The structure of pure
compound 36 was confirmed by high-resolution electrospray ioniza-
tion (ESP) mass spectrometry.
Ac-(S)-Aba-Gly-NMe-30,50-(CF3)2-Bn (20). Preparative HPLC yielded
the desired compound 20 (white powder, 47%). HPLC (standard
gradient): tret, 15.69 min. TLC Rf (EtOAc/hexane 1:1), 0.40. MS
(ESPþ) found m/z 516 [M þ H]þ, C24H23F6N3O3 requires 515.16.
1H NMR 250 MHz (CDCl3): δ 7.82 (s, 1H), 7.69 (d, 2H), 7.31-6.96
(m, 4H), 5.49 (m, 1H), 5.36 (d, 1H, J = 17.2 Hz), 4.80 (d, 1H, J = 7.2
Hz), 4.74 (d, 1H, J = 7.2 Hz), 4.51 (d, 1H, J = 17.2 Hz,), 4.51 (d, 1H, J =
17.0 Hz), 3.99 (m, 2H), 3.58 (dd, 1H, J1 = 17.6 Hz, J2 = 5.6 Hz), 2.98 (s,
3H), 2.10 (s, 3H). 13C NMR (CDCl3): δ 172.2, 170.2, 168.2, 139.3,
135.5, 132.4, 132.5, 131.9, 131.0, 128.6, 128.3, 128.0, 126.3, 121.4, 53.5,
51.0, 48.7, 49.5, 36.3, 34.8, 23.1
Hybrid 35 was prepared through a two-step approach. First, Boc-
Dmt-D-Arg(Pbf)-Aba-Gly-OH was prepared on 2-chlorotrityl resin
(0.15 mmol). Fmoc-Aba-Gly-OH (1.2 equiv) and DIPEA (5 equiv) in
CH2Cl2 were added to the swollen solid support, and the reaction
mixture was shaken overnight. The resin was washed three times with
DMF and three times with CH2Cl2. Subsequently, Fmoc-D-Arg(Pbf)-
OH and Boc-Dmt-OH were coupled as described above. The protected
C-terminal free acid Boc-Dmt-D-Arg(Pbf)-Aba-Gly-OH 33 was ob-
tained after treatment of the resin with 1% v/v TFA in CH2Cl2 for 30
min. The obtained crude peptide was obtained after removal of the
solvent in vacuo. The crude peptide was pure enough to be used directly
in the second step, the coupling step with N-methyl-30,50-bistrifluor-
omethyl benzylamine. This reaction was carried out on 33 (0.043 mmol)
in CH2Cl2 (10 mL) after adding BOP (1.1 equiv) in the presence of
DIPEA (2.5 equiv) and using CH2Cl2 as a solvent. After the reaction
mixture was stirred for 3 h, an extraction with saturated sodium
bicarbonate (5 mL) was performed, and the organic phase was dried
over magnesium sulfate, and the solvent was removed. After final
treatment with TFA/CH2Cl2/anisole 49:49:2 for 2 h, evaporation of
the solvent under vacuum yielded crude compound 35, which was
purified and characterized by preparative RP-HPLC and HRMS as
mentioned above.
Boc-Tic-NMe-30,50-(CF3)2-Bn. After flash chromatography (EtOAc/
hexane 1:1), the desired compound was obtained with a yield of 90%
(white solid, 65 mg). HPLC (standard gradient): tret, 19.85 min. TLC Rf
(EtOAc/hexane 1:1), 0.75. MS (ESPþ) found m/z 517 [M þ H]þ,
C25H26F6N2O3 requires 516.18. 1H NMR 250 MHz (CDCl3): δ (cis/
trans) 7.80 (s, 1H), 7.68 (s, 2H), 7.07-7.25 (m, 4H), 5.31 (t, 1H, J = 5.5
Hz), 5.15 (t, 1H, J = 5.5 Hz), 4.80 (m, 2H), 4.53 (m, 2H), 3.14 (s, 3H),
3.05 (m, 2H), 1.50 and 1.37 (2s, 9H). 13C NMR (CDCl3): δ (cis/trans)
172.6, 155.2, 140.0, 134.4, 132.1, 131.8, 128.4, 127.8, 127.0, 126.7, 125.8,
124.3, 121.4, 80.7, 50.8, 50.7, 46.0, 45.0, 35.2, 34.9, 31.2, 29.8, 28.3
Ac-Tic-NMe-30,50-(CF3)2-Bn (23). Purification was performed by
preparative HPLC (51%, white powder). HPLC (standard gradient):
tret, 16.82 min. TLC Rf (EtOAc/hexane 1:1), 0.72. MS (ESPþ) found m/
z 459 [M þ H]þ, C22H20F6N2O2 requires 458.14. 1H NMR 250 MHz
(CDCl3): δ(cis/trans mixture) 7.77 (s, 1H), 7.65 (s, 2H), 7.29-7.14 (m,
4H), 5.41 (pseudo t, 1H, J = 6.0 Hz), 4.80-4.63 (m, 4H), 3.18 (s, 3H),
3.12 (m, 2H), 2.26 (s, 3H). 13C NMR (CDCl3): δ(cis/trans mixture)
172.4, 170.6, 139.8, 133.7, 132.9, 132.7, 130.5, 127.9, 127.8, 127.0, 125.6,
121.5, 51.8, 50.4, 47.4, 35.6, 30.9, 21.8
H-Dmt-D-Arg-Aba-Gly-NH2 (36). Preparative HPLC yielded the
desired compound (white powder, 45%). HPLC (standard gradient):
tret, 8.73 min. TLC Rf (EBAW), 0.19. HRMS (ESPþ) found m/z
581.3199 [M þ H]þ, C29H41N8O5 requires 581.3195.
Compound Characterization. Boc-(S)-Aba-Gly-NH-30,50-(CF3)2-
Bn. Flash chromatography (EtOAc/hexane 1:1) yielded the desired com-
pound (white solid, 83%). HPLC (standard gradient): tret, 19.3 min. TLC Rf
(EtOAc/hexane 1:1), 0.43. MS (ESPþ) found m/z 560 [M þ H]þ,
H-Dmt-D-Arg-Aba-Gly-NMe-30,50-(CF3)2Bn (35). Preparative HPLC
yielded the desired compound (white powder, 54%). HPLC (standard
gradient): tret, 14.19 min. TLC Rf (EBAW), 0.33. HRMS (ESPþ) found
m/z 821.3580 [M þ H]þ, C39H47F6N8O5 requires 821.3574.
1
C26H27F6N3O4 requires 559.19. H NMR 250 MHz (CDCl3): δ 7.78
(s, 1H), 7.65 (s, 2H), 7.30-7.02 (m, 4H), 6.72 (br s, 1H), 5.73 (d, 1H, J=7.2
2474
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Journal of Medicinal Chemistry
ARTICLE
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’ ACKNOWLEDGMENT
S.B. and D.F. are postdoctoral fellows of the Research
Foundation—Flanders (F.W.O.), which also supported the work
by Grant G.0008.08. The Institute for Innovation through
Science and Technology (IWT Vlaanderen) granted K.B. a Ph.
D. scholarship. This work was also supported by grants from the
MDEIE (to D.T. and P.W.S.), NIH (DA-25196 to P.W.S.), and
CIHR (MOP-89716 to P.W.S.). We gratefully thank Sofie Van
Soest and Hans Peter Vandersmissen for technical support
(NK1R aequorin assays) and Euroscreen (Belgium) for the kind
gift of CHO-NK1 cells and apoaequorin expression construct.
We gratefully acknowledge the Interuniversity Attraction Poles
program (Belgian Science Policy Grant P6/14) for financial
support (J.V.d.B.).
’ ABBREVIATIONS USED
Aba, 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one; Aia, 4-
amino-1,2,4,5-tetrahydro-indolo[2,3-c]azepin-3-one; CHO cells,
Chinese hamster ovary cells; CNS, central nervous system; DML,
designed multiple ligand; DOR, δ opioid receptor; EBAW, ethyl
acetate/n-butanol/acetic acid/water 1:1:1:1 (v/v); EDC, 1-ethyl-
3-(3-dimethylaminopropyl) carbodiimide; KOR, κ opioid receptor;
MOR, μ opioid receptor; NK1R, neurokinin 1 receptor; SP, sub-
stance P; TBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluro-
nium tetrafluoroborate; Tcc, 1,2,3,4-tetrahydro-β-carboline-3-carboxy-
lic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Pbf, 2,2,
4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Phth, phthaloyl
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