χ-Space Screening of Dermorphin-Based Tetrapeptides through Use of Constrained Arylazepinone and Quinolinone Scaffolds

Article abstract of DOI:10.1021/acsmedchemlett.7b00347

Herein, the synthesis of novel conformationally constrained amino acids, 4-amino-8-bromo-2-benzazepin-3-one (8-Br-Aba), 3-amino-3,4-dihydroquinolin-2-one, and regioisomeric 4-amino-naphthoazepinones (1- and 2-Ana), is described. Introduction of these cons

Full text of DOI:10.1021/acsmedchemlett.7b00347

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Letter
Chi-Space Screening of Dermorphin-Based Tetrapeptides Through
Use of Constrained Arylazepinone and Quinolinone Scaffolds
Olivier Van der Poorten, Robin Van Den Hauwe, Emilie Eiselt, Cecilia Betti,
Karel Guillemyn, Nga N. Chung, François Hallé, Frederic BIHEL, Peter W.
Schiller, Dirk Tourwé, Philippe Sarret, Louis Gendron, and Steven Ballet
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00347 • Publication Date (Web): 04 Oct 2017
Downloaded from http://pubs.acs.org on October 7, 2017
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χ-Space Screening of Dermorphin-Based Tetrapeptides Through Use
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of Constrained Arylazepinone and Quinolinone Scaffolds
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†
†
‡
†
†
‡
Olivier Van der Poorten, Robin Van Den Hauwe, Emilie Eiselt, Cecilia Betti, Karel Guillemyn,
§
Ψ
Ψ
§
†
Nga N. Chung, François Hallé, Frédéric Bihel, Peter W. Schiller, Dirk Tourwé, Philippe Sarret,
Louis Gendron, Steven Ballet*
‡
,†
†
Research Group of Organic Chemistry, Departments of Chemistry and Bioengineering Sciences, Vrije Universiteit Brussel,
Pleinlaan 2, 1050 Brussels, Belgium
‡
Département de PharmacologieꢀPhysiologie, Université de Sherbrooke, Centre de Recherche du CHU de Sherbrooke,
Centre d’Excellence en Neurosciences de l’Université de Sherbrooke, and Institut de Pharmacologie de Sherbrooke, Sherꢀ
brooke, Québec, Canada
§
Department of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Avenue Des Pins
Ouest, Montreal, QC, H2W1R7, Canada
Ψ
UMR7200, CNRS, Université de Strasbourg, Faculté de Pharmacie, 74 Route du Rhin, 67401 Illkirch, France
KEYWORDS Opioid dermorphin ligands, conformational constraints, 4ꢀamino ꢀ2ꢀbenzazepinꢀ3ꢀone, 3ꢀaminoꢀ3ꢀ4ꢀ
dihydroquinolinꢀ2ꢀone, 4ꢀaminoꢀnaphthoazepinones
ABSTRACT: Herein, the synthesis of novel conformationally constrained amino acids, 4ꢀaminoꢀ8ꢀbromoꢀ2ꢀbenzazepinꢀ3ꢀone (8ꢀ
BrꢀAba), 3ꢀaminoꢀ3ꢀ4ꢀdihydroquinolinꢀ2ꢀone (Dhq), and regioisomeric 4ꢀaminoꢀnaphthoazepinones (1ꢀ and 2ꢀAna), is described.
Introduction of these constricted scaffolds into the Nꢀterminal tetrapeptide of dermorphin (i.e., HꢀTyrꢀDꢀAlaꢀPheꢀGlyꢀNH ) induced
significant shifts in binding affinity, selectivity, and in vitro activity at the µꢀ and δꢀopioid receptors (MOP and DOP, respectively).
2
A reported constrained µꢀ/δꢀopioid lead tetrapeptide HꢀDmtꢀDꢀArgꢀAbaꢀGlyꢀNH was modified through application of various
2
constrained building blocks to identify optimal spatial orientations in view of activity at the opioid receptors. Interestingly, when
the aromatic moieties were turned towards the Cꢀterminus of the peptide sequences, (partial) (ant)agonism at MOP and weak
(ant)agonism at DOP were noticed, whereas the incorporation of the 1ꢀAna residue led towards balanced low nanomolar MOP/DOP
binding
and
in
vitro
agonism.
Nowadays, morphine remains the gold standard analgesic
for the treatment of severe to moderate pain. Besides
morphine, opioid ligands such as the fentanyl family of
Opioid peptides have a common Nꢀterminal message part
and a variable Cꢀterminal address segment, which is
responsible for receptor subtype selectivity (e.g., Figure 1).
Receptor selectivity is dependent on the conformational space
available to the peptide and the relative orientation of the Tyr
painkillers are commonly used in
a clinical context.
Regrettably, the development of analgesic tolerance and
physical dependence, induced by chronic administration of
and Phe side chains is undisputedly an important feature of
1
9ꢀ10
common opioids, limits their longꢀterm use. Despite these
opioid peptide pharmacophores.
Since different receptors
severe drawbacks, they remain widely in use to date.
may require slightly different side chain orientations for
optimal binding events, receptor (sub)type selectivity can be
achieved via introduction of conformationally constrained
amino acids. Such conformational constraints provide control
In the search of nonaddictive analgesics with a prolonged
duration of action and reduced side effects, a plethora of
structural analogues of endoꢀ and exogenous opioid peptides
2ꢀ4
1
2
has been explored. Many peptidomimetic techniques have
over the χ ꢀ and χ ꢀspace via favoring, disfavoring or
5
been applied in this field, including peptoids, retroꢀinverso
excluding the gauche (ꢀ), the gauche (+), or the trans
6
7
8
2, 4, 11ꢀ15
analogues, amide bond isosteres, and macrocyclizations.
conformation.
In this way, useful information about the
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2,
preferred and bioactive side chain topology can be obtained.
conformationally constrained scaffolds were incorporated into
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dermorphinꢀlike tetrapeptides using conventional Fmocꢀbased
solidꢀphase peptide synthesis (SPPS). The resulting
dermorphin sequences were evaluated in vitro using both
opioid receptor binding and functional GPI and MVD assays.
In particular, the exogenous opioid peptide dermorphin 1 (Hꢀ
TyrꢀDꢀAlaꢀPheꢀGlyꢀTyrꢀProꢀSerꢀNH ) exhibits a high potency
2
and selectivity for the µꢀopioid receptor (MOP). Cꢀterminal
truncation of sequence 1 revealed that the Nꢀterminal
tetrapeptide HꢀTyrꢀDꢀAlaꢀPheꢀGlyꢀNH 2 (Figure 1) was the
2
16ꢀ17
minimal segment with full opiateꢀlike activity in vivo.
To
explore the χꢀspace in 1, the PheꢀGly dipeptidic segment was
previously substituted by the constrained AbaꢀGly
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dipeptidomimetic. Introduction of the 4ꢀaminoꢀ1,2,4,5ꢀ
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tetrahydroꢀ3Hꢀ2ꢀbenzazepinꢀ3ꢀone (Aba) scaffold into
tetrapeptide 2 at position 3 (i.e. Phe exchanged for Aba)
1
8
resulted in tetrapeptide 3 (Figure 1). While the Abaꢀ
containing ligand (3) showed no dramatic change in MOP
affinity and activity (determined by the guinea pig ileum (GPI)
assay), as compared to the native tetrapeptide sequence, DOP
binding and activity significantly increased. DOP agonism was
determined via the isolated mouse vas deferens (MVD) test.
Figure 2. 8ꢀBrꢀAba 5, Dhq 6, and 1/2ꢀAna 7a-c scaffolds as
conformationally constrained phenylalanine derivatives.
The constrained phenylalanine building block Aba (scaffold
5
with H instead of Br) can be prepared via different synthetic
pathways, such as attack of an aromatic ring onto a Nꢀ
acyliminium ion intermediate, reductive aminations of oꢀ
formylꢀPhe derivatives, followed by intramolecular cyclization
22
or even the Ugiꢀ4ꢀcomponent reaction. In order to prepare a
diversifiable analogue, the 8ꢀBrꢀAba ring was synthesized
either via a Nꢀacyliminium ion precursor 11 (Scheme 1A) or
via a reductive amination and subsequent cyclization reaction
starting from Phthꢀ2ꢀformylꢀLꢀPheꢀOH 15 (Scheme 1B). To
access 11, commercially available 4ꢀBrꢀLꢀPheꢀOH was Nꢀ
phthaloylated to give Phthꢀ4ꢀBrꢀLꢀPheꢀOH 8 (Scheme 1A, see
supporting information for experimental details). Coupling
with ethyl glycinate hydrochloride was achieved by TBTUꢀ
activation in the presence of Et N to give dipeptide Phthꢀ4ꢀBrꢀ
3
LꢀPheꢀGlyꢀOEt 9 in 75% yield after reꢀcrystallization from hot
EtOH. Subsequent ethyl ester hydrolysis, to form Phthꢀ4ꢀBrꢀ
LꢀPheꢀGlyꢀOH 10, was followed by the formation of
oxazolidinone 11 by means of paraformaldehyde using a
DeanꢀStark azeotropic distillation in refluxing toluene. The 7ꢀ
membered lactam ring in 12 (Phthꢀ8ꢀBrꢀAbaꢀGlyꢀOH) was
quantitatively formed via Nꢀacyliminium ion cyclization using
9
Figure 1. Native dermorphin 1, and truncated analogues HꢀTyrꢀ
1
6ꢀ17
18
DꢀAlaꢀPheꢀGlyꢀNH2 2,
HꢀTyrꢀDꢀAlaꢀAbaꢀGlyꢀNH
3
and
2
1
9
trifluoromethanesulfonic acid (TFMSA) in dry CH Cl .
2 2
HꢀDmtꢀDꢀArgꢀAbaꢀGlyꢀNH 4.
2
Phthaloyl deprotection of 12 by hydrazinolysis and final Fmoc
protection resulted in Fmocꢀ8ꢀBrꢀAbaꢀGlyꢀOH 13. The
enantiomeric ratio was determined with FDAA (Marfey’s
1
Additionally, the replacement of Tyr with the unnatural
amino acid 2’,6’ꢀdimethyltyrosine (Dmt) resulted in improved
binding to and activity at the opioid receptors. Inspired by
2
0
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reagent) derivatization of dephthaloylated 12 (structure not
1
21
2
[
Dmt ]DALDA (HꢀDmtꢀDꢀArgꢀPheꢀLysꢀNH ), DꢀAla was
2
2
shown). Since LCꢀMS analysis indicated only one peak
corresponding to the desired mass, and to make sure that no
racemization occurred in this synthetic route, it was decided to
replaced by DꢀArg , to give HꢀDmtꢀDꢀArgꢀAbaꢀGlyꢀNH 4
2
(
Figure 1). The latter analogue exhibited excellent MOP/DOP
potency and was shown to cross the bloodꢀbrain barrier (BBB)
couple Phthꢀ8ꢀBrꢀAbaꢀGlyꢀOH 12 with HCl.NH ꢀLꢀValꢀOtBu
2
19
after intravenous or subcutaneous administration.
ester via EDCꢀactivation in presence of HOBt. Gratifyingly, a
single enantiomer of the resulting tripeptide was observed via
The present study employs novel conformationally
constrained PheꢀGly dipeptidomimetics to screen the χꢀspace
1
LCꢀMS and H NMR analysis (dr ≥ 99:1).
3
of the aromatic Phe side chain within dermorphin’s
Alternatively, commercially available 2ꢀcyanoꢀLꢀPheꢀOH
was protected as Phthꢀ2ꢀcyanoꢀLꢀPheꢀOH 14 using the same
conditions applied for 8. It was subsequently converted to the
corresponding Phthꢀ2ꢀformylꢀLꢀPheꢀOH 15 by means of
tetrapeptide sequence. Additionally, an extension of the side
chain is realized aiming at improved binding with the opioid
receptors, but also in an attempt to modulate receptor
selectivity and activity. In continuation of our efforts in
peptidomimetic design, 4ꢀaminoꢀ8ꢀbromoꢀ2ꢀbenzazepinꢀ3ꢀone
(8ꢀBrꢀAba), 3ꢀaminoꢀ3ꢀ4ꢀdihydroquinolinꢀ2ꢀone 6 (Dhq),
and regioisomeric 4ꢀaminoꢀnaphthoazepinone 7a-c (1/2ꢀAna)
scaffolds (Figure 2) were synthesized in solution. Next, these
24
hydrogen and Raney nickel (Scheme 1B). Phthꢀ2ꢀformylꢀ
PheꢀOH 15 was converted to Phthꢀ4ꢀBrꢀ2ꢀformylꢀPheꢀOH 16
5
using bromoisocyanuric acid monosodium salt (BICAꢀNa) in
25
concentrated sulfuric acid,
since other bromination
26
conditions making use of bromine/Lewis acid systems or
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brominating ionic liquids, proved unsuccessful. During the
bromination step, the minor regioisomer Phthꢀ6ꢀBrꢀ2ꢀformylꢀ
PheꢀOH (structure not shown) was also formed (ratio 84:16
based on RPꢀHPLC peak area) and separated from the desired
product 16 by reversedꢀphase column chromatography which
was isolated in 54% yield. Reductive amination with ethyl
glycinate hydrochloride followed by intramolecular
cyclization by means of DCCꢀactivation in acetonitrile
afforded compound 17. After ethyl ester hydrolysis, the
dipeptide mimetic Phthꢀ8ꢀBrꢀAbaꢀGlyꢀOH 12 was obtained.
Considering the overall yield and undetected racemization of
pathway A, this route was preferred. The second pathway is,
however, more broadly applicable, since pathway A is
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restricted
to
αꢀamino
acid
use.
Scheme 1. Synthesis of Fmocꢀ8ꢀBrꢀAbaꢀGlyꢀOH 13. a) HCl.NH ꢀGlyꢀOEt, TBTU, Et N, dry CH Cl , rt, 16 h; b) 1 N
2
3
2
2
HCl /acetone, 90 °C, 16 h; c) (CH O) , pꢀTosOH, DeanꢀStark apparatus, dry toluene, 115 °C, 5 h; d) TFMSA, dry CH Cl , rt, Ar
aq
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atmosphere, 16 h; e) NH NH .H O, EtOH, 90 °C, 2 h; f) FmocꢀOSu, Na CO , acetone/H O (1:1 v/v), rt, 19 h; g) H 50 psi, RaNi,
2 2 2 2 3 2 2
Py/AcOH/H O (1:1:1 v/v), 50 °C, 19 h; h) BICAꢀNa, 60% H SO , rt, 6 h, (Phthꢀ6ꢀBrꢀ2ꢀformylꢀPheꢀOH separated via RP column
2
2
4
chromatography); i) HCl.NH ꢀGlyꢀOEt, NaCNBH , dry CH Cl , rt, 3 h; j) DCC, CH CN, rt, 2 h.
2
3
2
2
3
Scheme 2. Synthesis of FmocꢀDhqꢀGlyꢀOH 22. a) HCl.NH2ꢀ
GlyꢀOEt, TBTU, Et N, dry CH Cl , rt, 4 h; b) CuI, N,N'ꢀ
phthaloyl deprotectionꢀFmoc protection provided FmocꢀDhqꢀ
GlyꢀOH 22 in good yield. The enantiomeric ratio was
determined via coupling of PhthꢀDhqꢀGlyꢀOH 19 with
3
2
2
dimethylethylenediamine, K PO , dry and degassed toluene,
3
4
1
20 °C, Ar atmosphere, 67 h; c) 1 N HCl /acetone, 90 °C, 45
HCl.NH ꢀLꢀValꢀOtBu. A single enantiomer of the resulting
tripeptide was observed via LCꢀMS and H NMR analysis (dr
aq
2
1
h; d) NH NH .H O, EtOH, 90 °C, 4 h; e) FmocꢀOSu, Na CO ,
2
2
2
2
3
acetone/H O (1:1 v/v), rt, 20 h.
≥ 99:1), indicating that no racemization of Phe’s original αꢀ
carbon occurred during the Cu(I)ꢀcatalyzed amidation reaction
under the conditions described in Scheme 2.
2
Molecular modeling of the NꢀacetylꢀN’ꢀmethylamide
tetrapeptide model of dipeptide 6 (i.e., AcꢀDhqꢀGlyꢀNHMe)
indicated that the lowest energy conformer showed a trans
1
conformation along the C ꢀC bond (χ dihedral angle) and
α
β
was consistent with an (inverse) γꢀturn conformation, leading
to a pseudo 7ꢀmembered ring (i → i±2) (see supporting
information). In contrast, the above described Aba moieties
had previously been shown to prefer extended
32
conformations.
Moreover,
4ꢀaminoꢀnaphthoazepinones
7a-c
were
synthesized in complete analogy to 8ꢀBrꢀAba 5 (cfr. Scheme
A). Commercially available 1ꢀ and 2ꢀLꢀNalꢀOH were Nꢀ
phthaloylated to provide Phthꢀ1/2ꢀLꢀNalꢀOH 23-24 (see
supporting information). Dipeptide formation, followed by
ethyl ester hydrolysis gave Phthꢀ1/2ꢀLꢀNalꢀGlyꢀOH 27-28
1
Whereas we have recently published the synthesis of 3ꢀ
aminoꢀ3,4ꢀdihydroꢀ1Hꢀquinolinꢀ2ꢀones (Dhq) through
regioselective Pdꢀcatalyzed intramolecular cyclization, we
propose here another method to access the 3ꢀaminoꢀ3ꢀ4ꢀ
28
(Scheme 3). Next, oxazolidinones 29-30 were formed in the
dihydroquinolinꢀ2ꢀone
6
(Dhq) core through
a
key
presence of paraformaldehyde and a catalytic amount of pꢀ
TosOH. Recrystallization of crude oxazolidinones 29-30 from
hot toluene resulted in rather low yields (39 and 30% resp.)
and both products 29-30 appeared unstable during silica gel
flash chromatography. Switching to benzene as crystallization
medium improved significantly the final yields (72 and 65%,
resp.). The 7ꢀmembered lactam rings of 31 (1ꢀAna) and 32a,b
intramolecular Cu(I)ꢀcatalyzed Goldberg amidation reaction
2
9ꢀ31
(Scheme 2).
Therefore, commercially available 2ꢀIꢀLꢀPheꢀ
OH was Nꢀphthaloylated to afford Phthꢀ2ꢀIꢀLꢀPheꢀOH 18 (see
supporting information). Subsequently, dipeptide formation
was followed by the Cu(I)ꢀcatalyzed Goldberg reaction using
CuI/N,N’ꢀdimethylethylenediamine/K PO as
a
catalytic
3
4
system in dry and degassed toluene. This resulted in a clean
conversion to the desired δꢀlactam 20 in 63% yield. Ethyl ester
hydrolysis gave PhthꢀDhqꢀGlyꢀOH 21 and subsequent
(
2ꢀAna) were again accessed via Nꢀacyliminium ion
cyclization using TFMSA. Despite the higher reactivity of
position versus position in naphthyl groups for
1
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electrophilic aromatic substitutions, two regioisomers 32a and
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binding affinities, while DOP affinity was lost for HꢀDmtꢀDꢀ
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32b were obtained from the educt Phthꢀ2ꢀLꢀNalꢀoxazolidinone
30 (9:1 ratio based on RPꢀHPLC peak area). Lowering the
reaction temperature from room temperature to respectively 0
°C and ꢀ78 °C during the Nꢀacyliminium cyclization reaction
did not improve overall conversion into the major 2ꢀAna
component 32a. Phthaloyl deprotection and Fmoc protection
of both 31 and 32a,b afforded Fmocꢀ1ꢀAnaꢀGlyꢀOH 33 and
the two regioisomers Fmocꢀ2ꢀAnaꢀGlyꢀOH 34a,b in good
yields. Since regioisomers (32a,b and) 34a,b were inseparable
via silica gel column chromatography nor via preparative RPꢀ
HPLC, it was decided to use them as a mixture in Fmocꢀbased
SPPS of the dermorphin tetrapeptides and attempt a separation
after peptide assembly.
Argꢀ[2ꢀAnaꢀGly]ꢀNH 39 (DOP IC 153 nM) and its DꢀAla
analogues, HꢀDmtꢀDꢀAlaꢀ[2ꢀAnaꢀGly]ꢀNH 40a,b (DOP IC
167 nM for 40a; 40b was not tested).
2 50
2
50
Scheme 3. A. Synthesis of Fmocꢀ1ꢀAnaꢀGlyꢀOH 33 and B.
Synthesis of Fmocꢀ2ꢀAnaꢀGlyꢀOH 34a,b. a) HCl.NH ꢀGlyꢀ
2
OEt, TBTU, Et N, dry CH Cl , rt; b) 1 N HCl /acetone, 90
3
2
2
aq
°C; c) (CH O) , pꢀTosOH, DeanꢀStark apparatus, dry toluene,
2
n
115 °C; d) TFMSA, dry CH Cl , rt, Ar atmosphere; e)
2
2
NH NH .H O, EtOH, 90 °C; f) FmocꢀOSu, Na CO ,
2
2
2
2
3
acetone/H O (1:1 v/v), rt.
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The conformationally constrained PheꢀGly dipeptidomimetic
building blocks 13, 22, 33 and 34a,b were then used to prepare
analogues of the tetrapeptide 4 using standard Fmocꢀbased
SPPS on Rink amide AM resin via DIC/HOBt activation. The
tetrapeptide analogues 35-41 were evaluated in vitro in order
to determine their pharmacological profile. The peptide
analogues containing either 34a or 34b could be separated by
reversedꢀphase preparative HPLC.
The binding affinities of tetrapeptides 35-41 for MOP and
DOP receptors were determined by competitive displacement
1
25
125
of [ I]ꢀDAMGO and [ I]ꢀDeltorphin II, respectively. The in
vitro activity data of peptides 35-41 were obtained by
measuring the inhibition of electrically induced contractions in
the isolated GPI and MVD functional assays (Table 1). Taking
into consideration that lead tetrapeptide HꢀDmtꢀDꢀArgꢀ[Abaꢀ
Gly]ꢀNH 4 possesses K values of 0.15 and 0.60 nM for MOP
2
i
and DOP, respectively, and IC50 values of 0.32 nM (GPI) and
1
9
0
.42 nM (MVD) in the functional assays, data in Table 1
indicate that the insertion of 8ꢀBrꢀAba, Dhq and 1/2ꢀAna
constraints at position 3 in the dermorphin sequence did not
dramatically change the low nanomolar MOP affinity and all
analogues proved good ligands for this receptor (IC50 ranging
from 0.33 to 2.19 nM). In contrast, DOP affinity was
significantly decreased for all tetrapeptides apart from 37, 38
and 41, all possessing the 1ꢀAnaꢀGly constraint. These
observations were nicely reflected in the GPI and MVD
functional assays. Opioid tetrapeptide HꢀDmtꢀDꢀArgꢀ[8ꢀBrꢀ
AbaꢀGly]ꢀNH 35 (DOP IC 60.7 nM; MVD IC50 8.51 nM)
2
50
displayed decreased DOP binding affinity and activity, as
compared to the AbaꢀGly lead peptide 4 (DOP K 0.60 nM;
i
19
MVD IC50 0.42 nM), while MOP affinity was retained (MOP
IC50 0.76 nM). This observation indicates that DOP tolerates
the bromine substituent less than MOP does in view of
efficient binding. Similarly, introduction of the DhqꢀGly
While the incorporation of the 1ꢀAna scaffold in 37 and 38
led to in vitro agonism at MOP and DOP, the 2ꢀAna
constrained peptides 39 and 40a,b showed moderate
MOP/DOP agonism (39), and even potent MOP/weak DOP
antagonism (40a,b). Similarly to the recently reported
dipeptidomimetic, to give HꢀDmtꢀDꢀArgꢀ[DhqꢀGly]ꢀNH 36,
2
resulted in dramatically decreased DOP binding affinity (DOP
IC50 630 nM) and activity (MVD IC50 90.5 nM). This could
indicate that the conformation induced by the γꢀturn promoting
δꢀlactam constraint in 36 was not beneficial towards DOP
binding and receptor activation, and/or means that beneficial
interactions with the Dhq aromatic ring is lost in this type of
ligand. However, both tetrapeptides 35 and 36 showed only
partial agonist activity at MOP in the GPI functional assay,
and hence full MOP agonism was lost through these
1
Cyclodals (cyclic Dmt [DALDA] analogues, structures not
33
shown), MOP antagonists are of key interest for reversing
morphineꢀinduced, centrally mediated analgesia in the
treatment of opioid abuse and overdose. From these data, it
seems that the 1ꢀ versus 2ꢀAnaꢀGly dipeptidomimetics are
excellent tools to i) examine the importance of the relative side
chain orientations of key pharmacophore aromatic residues,
and ii) modulate receptor subtype recognition and activation.
modifications.
Interestingly,
incorporation
of
the
regioisomeric 4ꢀaminoꢀnaphthoazepinones (Figure
2
and
Scheme 3) resulted in distinct biological profiles (Table 1): Hꢀ
Since HꢀDmtꢀNMeꢀDꢀAlaꢀ[AbaꢀGly]ꢀNH is reported to be a
2
34 2
DmtꢀDꢀArgꢀ[1ꢀAnaꢀGly]ꢀNH 37 and HꢀDmtꢀDꢀAlaꢀ[1ꢀAnaꢀ
superpotent opioid, ligand 38 was Nꢀmethylated at DꢀAla , to
afford HꢀDmtꢀNMeꢀDꢀAlaꢀ[1ꢀAnaꢀGly]ꢀNH 41, by coupling
2
Gly]ꢀNH 38 showed excellent low nanomolar MOP and DOP
2
2
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ACS Medicinal Chemistry Letters
dihydroquinolinꢀ2ꢀone 6 (Dhq), and regioisomeric 4ꢀaminoꢀ
of commercially available FmocꢀNMeꢀDꢀAlaꢀOH. Nꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
methylation of the peptide backbones is a powerful strategy to
enhance overall bioavailability, improve metabolic stability
towards protease activity, and differentiate receptor subtype
selectivity via conformational modulation. HꢀDmtꢀNMeꢀDꢀ
Alaꢀ[1ꢀAnaꢀGly]ꢀNH2 41 showed excellent low nanomolar
MOP (IC50 0.35 nM) and DOP (IC50 1.47 nM) affinities. These
binding affinities were translated into improved functional
activities, as determined in the GPI (IC50 0.43 nM) and MVD
naphthoazepinones 7a-c (1/2ꢀAna) dipeptidomimetics into the
optimized dermorphin tetrapeptide lead sequence has resulted
in compact and high affinity MOP/DOP opioid receptor
ligands. Whereas insertion of the conformationally constrained
8ꢀBrꢀAbaꢀGly, DhqꢀGly, or 2ꢀAnaꢀGly dipeptides resulted in
decreased DOP recognition, application of the regioisomeric
1ꢀAnaꢀGly building block led towards excellent low
nanomolar MOP and DOP binding affinities and in vitro
functional activities. The described cyclic constrained
aromatic amino acids can be regarded as additional tools to
modulate receptor selectivity and activity, but they also
3
5
(
IC50 1.09 nM) tissue bioassays. Compound 41 is also the most
hydrophobic analogue among the ligands encompassing the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[
(
1ꢀAnaꢀGly] constraint, based on RPꢀHPLC retention times
see supporting information). Therefore, the direct comparison
provide
a
way to enhance proteolytic stability and
of structures 37, 38, 41, and a reference tetrapeptide HꢀDmtꢀ
bioavailability of lead peptides. Bulky Nal residues often
19
37
DꢀArgꢀ[AbaꢀGly]ꢀNH 4, in terms of in vivo antinociception,
provide a means of modulating receptor selectivity. The
2
will provide insight into the importance of hydrophobicity for
eventual in vivo analgesia. In a previous report, we indeed
identified hydrophobicity to be a key determinant for activity,
rather than the presence of a conformational azepinone
current building blocks present the additional feature to
control χ dihedral angles and thus presenting specific ligand
topologies. The present study clearly showcases the
advantages of such constrained residues for improving ligand
potency and opioid receptor selectivity. It is expected that
these building blocks will find application in other biologically
active peptides.
36
constraint.
In conclusion, the insertion of constricted 4ꢀaminoꢀ8ꢀbromoꢀ
ꢀbenzazepinꢀ3ꢀone (8ꢀBrꢀAba), 3ꢀaminoꢀ3ꢀ4ꢀ
2
5
Table 1. In vitro opioid receptor affinity and activity of dermorphin tetrapeptides 35-41.
a
a
b
b
No.
Compounds
GPI
MVD
MOP
DOP
Selectivity
IC50 DOP
/IC50 MOP
(IC50 nM)
(IC50 nM)
(IC50 nM) (IC50 nM)
c
35
HꢀDmtꢀDꢀArgꢀ[8ꢀBrꢀAbaꢀGly]ꢀNH
2
22.0±2.4
8.51±0.70
90.5±14.4
1.42±0.18
0.86±0.16
114±7.0
0.76±0.49 60.7±13.7
1.62±0.03 630±65.0
0.33±0.01 1.00±0.45
0.39±0.07 2.07±0.30
2.19±0.70 153±12.0
1.78±0.40 167±38.0
80
389
3
5
70
94
d
3
3
3
3
6
7
8
9
HꢀDmtꢀDꢀArgꢀ[DhqꢀGly]ꢀNH
HꢀDmtꢀDꢀArgꢀ[1ꢀAnaꢀGly]ꢀNH
HꢀDmtꢀDꢀAlaꢀ[1ꢀAnaꢀGly]ꢀNH
2
33.7±3.6
0.252±0.014
1.13±0.13
21.4±1.9
2
2
HꢀDmtꢀDꢀArgꢀ[2ꢀAnaꢀGly]ꢀNH
HꢀDmtꢀDꢀAlaꢀ[2ꢀAnaꢀGly]ꢀNH
major
major
2
4
0a
2
2.88±0.10
306±12.0
(
K ,antagonist)
e
(K ,antagonist)
e
40b
41
HꢀDmtꢀDꢀAlaꢀ[2ꢀAnaꢀGly]ꢀNH
2
minor
5.53±1.12
360±58.0
/
/
/
(K ,antagonist)
(K ,antagonist)
e
e
HꢀDmtꢀNMeꢀDꢀAlaꢀ[1ꢀAnaꢀGly]ꢀNH
0.43±0.06
1.09±0.04
0.35±0.04 1.47±0.04
4
2
a
The GPI functional assay is representative of MOP activation, whereas the MVD is a DOP receptorꢀrepresentative assay.
b
125
Binding affinities of compounds for MOP and DOP opioid receptors were determined by competitive displacement of [ I]ꢀDAMGO
1
25
(
MOPꢀselective) and [ I]ꢀDeltorphin II (DOPꢀselective) from HEK293 cells expressing MOP or DOP.
c
d
IC , partial agonist, IC , partial agonist
2
5
35
O.V.d.P., R.V.D.H., C.B., KG, D.T. and S.B. are grateful to the
Research Foundation Flanders (FWOꢀVlaanderen) and to the
ASSOCIATED CONTENT
Supporting Information
Flanders Innovation
& Entrepreneurship (VLAIO) for the
financial support. In addition, CB and SB thank the Strategic
Research Program (SRP) of the VUB for funding. The work of
P.W.S. was supported by grants from the U.S. National Institute
on Drug Abuse (NIH, DA004443) and the Canadian Institutes of
Health Research (MOPꢀ89716). L.G. and P.S. thank the Canadian
Institutes of Health Research (CIHR) for supporting their
research.
Complete experimental details along with the characterization of
the synthesized compounds. The Supporting Information is
available free of charge on the ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
*
Phone: +32 2 6293292. Eꢀmail: sballet@vub.ac.be.
ABBREVIATIONS
Author Contributions
Aba, 4ꢀaminoꢀ1,2,4,5ꢀtetrahydroꢀ3Hꢀ2ꢀbenzazepinꢀ3ꢀone; Ana, 4ꢀ
aminoꢀnaphthoazepinone; BBB, bloodꢀbrain barrier; DALDA, Hꢀ
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
TyrꢀDꢀArgꢀPheꢀLysꢀNH ; Dhq, 3ꢀaminoꢀ3ꢀ4ꢀdihydroquinolinꢀ2ꢀ
2
one;
diisopropylcarbodiimide; Dmt, 2’,6’ꢀdimethyltyrosine; DOP, δꢀ
opioid receptor; EDC.HCl, 1ꢀethylꢀ3ꢀ(3ꢀ
DCC,
1,3ꢀdicyclohexylcarbodiimide;
DIC,
1,3ꢀ
Notes
The authors declare no competing financial interest.
dimethylaminopropyl)carbodiimide hydrochloride; FDAA, 1ꢀ
fluoroꢀ2,4ꢀdinitrophenylꢀ5ꢀLꢀalanine amide; GPI, guinea pig
ileum; HEK, human embryonic kidney; HOBt, 1ꢀ
ACKNOWLEDGMENT
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ACS Medicinal Chemistry Letters
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hydroxybenzotriazole; IC , concentration needed to replace 50%
18. Ballet, S.; Frycia, A.; Piron, J.; Chung, N. N.; Schiller, P. W.;
5
0
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3
4
5
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9
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1
1
1
1
1
1
1
1
1
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2
2
2
2
2
2
2
2
3
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4
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4
4
4
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4
4
4
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5
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5
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5
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6
of a receptorꢀbound ligand; MOP, µꢀopioid receptor; MSB,
methyl 2ꢀ((succinimidooxy)carbonyl)benzoate; MVD, mouse vas
Kosson, P.; Lipkowski, A. W.; Tourwé, D., Synthesis and Biological
Evaluation of Constrained Analogues of the Opioid Peptide HꢀTyrꢀDꢀ
AlaꢀPheꢀGlyꢀNH2 Using the 4ꢀAminoꢀ2ꢀbenzazepinꢀ3ꢀone Scaffold.
J. Pept. Res. 2005, 66, 222ꢀ230.
deferens; NaCNBH , sodium cyanoborohydride; SPPS, solidꢀ
3
®
phase peptide synthesis; RaNi, Raney Nickel; TBTU, Oꢀ
1
9. Guillemyn, K.; Kleczkowska, P.; Novoa, A.; Vandormael, B.;
(benzotriazolꢀ1ꢀyl)ꢀN,N,N’,N’ꢀtetramethyluronium
Van den Eynde, I.; Kosson, P.; Asim, M. F.; Schiller, P. W.; Spetea,
M.; Lipkowski, A. W.; Tourwé, D.; Ballet, S., In Vivo
Antinociception of Potent Mu Opioid Agonist Tetrapeptide
Analogues and Comparison with a Compact Opioid Agonist ꢀ
Neurokinin 1 Receptor Antagonist Chimera. Mol. Brain 2012, 5, 1ꢀ
11.
20. Bryant, S. D.; Jinsmaa, Y.; Salvadori, S.; Okada, Y.; Lazarus, L.
H., Dmt and Opioid Peptides: a Potent Alliance. Biopolymers 2003,
71, 86ꢀ102.
21. Schiller, P. W.; Nguyen, T. M. D.; Berezowska, I.; Dupuis, S.;
Weltrowska, G.; Chung, N. N.; Lemieux, C., Synthesis and In Vitro
Opioid Activity Profiles of DALDA Analogues. Eur. J. Med. Chem.
2000, 35, 895ꢀ901.
22. Ballet, S.; Guillemyn, K.; Van der Poorten, O.; Schurgers, B.;
Verniest, G.; Tourwé, D., AzepinoneꢀConstrained Amino Acids in
Peptide and Peptidomimetic Design. Top. Heterocycl. Chem. 2015, 1ꢀ
33.
tetrafluoroborate; TFMSA, trifluoromethanesulfonic acid
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