Chiral effect of a Phe residue in position 3 of the Dmt1-l (or d)-Tic2 analogues on opioid functional activities

Article abstract of DOI:10.1021/ml400115n

In this letter, we describe a structure-activity relationships study, specifically related to the chirality of third amino acid residue in our H-Dmt-l(or d)-Tic analogues, of which C-terminus is attached to a piperidinyl moiety. Observed selectivities and functional activities of these analogues demonstrated that the chiralities of the second and third position residues are crucial for determining whether these ligands act as antagonists or agonists at the δ opioid receptor, but not at the μ opioid receptor.

Full text of DOI:10.1021/ml400115n

Letter
pubs.acs.org/acsmedchemlett
Chiral Effect of a Phe Residue in Position 3 of the Dmt1‑L(or D)‑Tic2
Analogues on Opioid Functional Activities
Yeon Sun Lee,† HongChang Qu,† Peg Davis,‡ Shou-Wu Ma,‡ Ruben Vardanyan,† Josephine Lai,‡
Frank Porreca,‡ and Victor J. Hruby*,†
‡
†Department of Chemistry and Biochemistry and Department of Pharmacology, University of Arizona, Tucson, Arizona 85721,
United States
S
* Supporting Information
ABSTRACT: In this letter, we describe a structure−activity
relationships study, specifically related to the chirality of third
amino acid residue in our H-Dmt-L(or D)-Tic analogues, of
which C-terminus is attached to a piperidinyl moiety. Observed
selectivities and functional activities of these analogues
demonstrated that the chiralities of the second and third
position residues are crucial for determining whether these
ligands act as antagonists or agonists at the δ opioid receptor,
but not at the μ opioid receptor.
KEYWORDS: Dmt-Tic, opioid functional activities, structure−activity relationship, δ opioid receptor, chirality
a
Scheme 1. Synthesis of Dmt-L(or D)-Tic Analogues
Dmt-Tic has been known as the most potent δ opioid receptor
antagonist with high selectivity and a minimum peptide unit.1
In many studies, modifications of Dmt-Tic at the C-terminus
changed the biological profile dramatically, for example, from
selective δ opioid receptor antagonist to mixed μ/δ opioid
receptor agonists.2−5 These big changes in the biological profile
might be the result of topographical changes caused mainly by
the conformational preference of the Tic residue resulting from
the modifications because it has been known that close
proximity and parallel orientation of the two aromatic rings of
Dmt-Tic are key features of δ opioid antagonist activity.2,6,7
In our recent studies, we observed that the attachment of a D-
Phe(p-Cl) to the dipeptide structure in analogue 5 changed
opioid receptor function from a δ antagonist to a δ agonist.8
This result suggests a profound influence of D-amino acid
substitution in position 3 on the potency of Dmt-Tic analogues.
It is certainly possible that the bulky aromatic group of the D-
Phe(p-Cl) residue can cause a conformational change of the Tic
residue, which is responsible for the δ opioid receptor
antagonist activity. It is also possible that the chirality of the
third amino acid residue plays a role in a conformational change
of the Tic residue because analogues in which two Phe residues
are attached to the C-terminus of Dmt-Tic (H-Dmt-Tic-Phe-
Phe-OH and H-Dmt-Tic-Phe-Phe-NH2) still possess antagonist
activities at the δ opioid receptor (Ke = 0.20 and 0.21 nM,
respectively).9,10
a
Reagents and conditions: (a) stepwise coupling (1.1 equiv BOP/1.1
equiv HOBt/2.5 equiv NMM, DMF, room temperature, 2−3 h),
followed by deprotection (TFA, 0 °C, 20 min).
Thanks to the piperidinyl moieties and the Dmt residue, this
series of analogues exhibits high lipophilicity (aLOGPs of
4.02−4.91 in Table 1), which is a key factor for penetrating the
blood−brain barrier (BBB). All analogues were evaluated for
their affinity and selectivity for μ and δ opioid receptors using
transfected HN9.10 cells. The Dmt-Tic (3−5) and Dmt-D-Tic
(6−10) analogues generally exhibited high binding affinity (Ki
= 0.11−15 nM) at both μ and δ opioid receptors, but distinct
subtype selectivity. Only Dmt-D-Tic-D-Phe(p-Cl or p-F)
analogues 9 and 10 resulted in lower affinities (Ki = 130 and
On the basis of these observations of the chiral effect of the
Phe residue, we have further investigated the chiral effect of the
Phe3 residue with a series of analogues in which the
configurations of Tic and Phe(p-Cl) (or Phe, or Phe(p-F)]
residues are D or L and the C-terminus is linked to the
piperidinyl moieties 1 or 2 (Scheme 1 and Table 1).
Received: March 21, 2013
Accepted: May 23, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml400115n | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
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Table 1. Structure of Dmt-L(or D)-Tic Analogues
Table 2. Binding Affinities of Dmt-L(or D)Tic Analogues at μ
and δ Opioid Receptors
H-Dmt1-Xxx2-Yyy3-Z4
a
b
a
c
hDOR [3H]DPDPE
rMOR [3H]DAMGO
a
analogue
aLOGPs
Xxx2
Yyy3
Z4
Ki
Ki
3
4
4.30
4.91
4.30
4.91
4.91
4.41
4.91
4.85
Tic
phe
compound 1
compound 1
compound 1
compound 1
compound 1
compound 1
compound 1
compound 2
d
e
d
e
no.
log IC50
(nM)
log IC50
(nM)
Kiμ/Kiδ
Tic
Phe(p-Cl)
D-Phe(p-Cl)
phe
b
3
4
−9.37 0.11
−9.17 0.10
0.20
0.43
0.38
0.76
5.5
−7.70 0.12
−7.49 0.13
9.5
15
48
5
Tic
35
6
D-Tic
D-Tic
D-Tic
D-Tic
D-Tic
f
5
22
58
7
Phe(p-Cl)
Phe(p-F)
D-Phe(p-Cl)
D-Phe(p-Cl)
6
−8.80 0.11
−7.93 0.07
−7.69 0.15
−6.56 0.08
−9.02 0.06
−7.54 0.10
−8.39 0.08
−7.60 0.08
−7.87 0.04
0.45
14
0.59
2.5
8
7
9
8
9.3
1.9
12
0.20
0.09
0.026
230
0.067
10
10
9
130
250
1.2
a
b
http://www.vcclab.org/lab/alogps/. See ref 8.
10
−6.28 0.10
6.4
280
3.8
1.2
g
Dmt-Tic-NH2
250 nM, respectively) at the δ receptor. All analogues were
tested for their efficacy in GTP-γ-S assay and for antagonist (or
agonist) activity in functional assays, respectively, and both
assay results were correlated with each other. As predicted, the
functional assays resulted in dissimilar prototypes of functional
activities for the δ opioid receptor depending upon the
chiralities of positions 2 and 3. All the series of analogues
showed a broad range of agonist activities but no antagonist
activities at the μ opioid receptor. In this series, analogue 4
exhibited the best δ opioid antagonism (Ke = 0.79 0.6 nM)/
moderate μ opioid agonism (IC50 = 350 90 nM), which can
be an interesting clinical analgesic on the basis of its low
propensity to cause tolerance and dependence during chronic
use.9−11
The H-Dmt-L(or D)-Tic analogues were prepared by
stepwise solution-phase peptide syntheses starting from
commercially available N-phenyl-N-piperidin-4-yl-propiona-
mide (for analogues 3−9) or 4-((1H-1,2,4-triazol-1-yl)-
methyl)-4-cyclohexylpiperidine (for analogue 10) using Nα-
Boc-strategy in high yields (overall yields in up-to 8 steps >
45%) (Scheme 1).12,13 Each coupling reaction used 1.1 equiv
BOP/1.1 equiv HOBt/2.5 equiv NMM for 2−3 h at room
temperature. During the chain elongations, pure peptide
intermediates were obtained by simple precipitation from
appropriate organic solvents, usually diethyl ether. The
syntheses proceeded very well resulting in high purity of
peptides. The crude peptides showed more than 90% purity
and could be further purified by prep. RP-HPLC using a
gradient system composed of acetonitrile and a 0.1% aqueous
TFA solution. The structures were confirmed by HR-MS
spectroscopy and 1H NMR spectroscopy (see Supporting
Information).
g
Dmt-D-Tic-NH2
57
Dmt-Tic-Phe-Phe
-NH2
0.12
h
a
Competition analyses were carried out using membrane preparations
from transfected HN9.10 cells that constitutively expressed the
respective receptor types. Kd = 0.50 0.10 nM. Kd = 0.85 0.20
nM. Logarithmic values determined from the nonlinear regression
analysis of data collected from at least three independent experiments.
b
c
d
e
f
g
Antilogarithmic value of the respective IC50. See ref 7. See ref 1.
h
See refs 9 and 10.
which have an L-Tic2 residue, bind to the δ opioid receptor
better than the μ opioid receptor by 35−58 fold. The change of
configuration from L- to D-Tic2 abolished high δ opioid affinity
while simultaneously increasing μ affinity to yield ligands with
either reduced δ selectivity or the acquisition of μ selectivity,
similar to changes in affinity and selectivity observed with
isomeric modifications in Dmt-Tic analogues. It is well-known
that the chirality of Tic residue plays an important role in the
activity of Dmt-Tic moiety contained tripeptides as well as in
the TIP(P′) peptides.1,15 Our result supports the fact that,
while Dmt-Tic represents the δ opioid message domain, Dmt-
D-Tic better defines a μ opioid message domain.1,7,15
In ligands 3−5, halogenations of the aromatic ring in the
Phe3 residue and substituting the L-Phe(p-Cl)3 residue with D-
residue did not affect binding affinities and selectivities at either
receptor. Therefore, it is considered that the Dmt-Tic moiety
plays a dominate role in the affinity, and the third amino acid
residue does not affect it critically. This result is consistent with
earlier reports that cis−trans equiliblium of the peptide
backbone between positions 1 and 2 in Tyr-Tic-(L- or D-)Phe
depends mostly on the configurations of Tyr and Tic residues
forming the peptide bond, but not the Phe residue following
the sequence.16
In contrast, in ligands 7 and 8, which contain the Dmt-D-Tic
moiety, p-chloro or p-fluoro substitutions on the aromatic ring
of the Phe3 residue led to reduced bining affinities at both μ and
δ receptors. In previous studies, we showed that halogenation
of the aromatic ring of a Phe4 residue in enkephalin-like ligands
enhanced activities at both opioid receptors.13 Our current SAR
result was different than the known effect of halogenation on
enkephalin analogues. It is clear that this conflicting result is
due to the absence of the Gly3 residue, which can separate the
D-Tic2 residue from the aromatic ring of a Phe residue.
Whereas most ligands showed good binding affinities at the δ
opioid receptor in the subnanomolar to the nanomolar range,
ligand 9, in which the configuration of the third amino acid
residue was the D-form, lost its binding affinitiy (Ki = 130 nM)
24-fold. However, its binding affinity at the μ receptor was
In vitro bioassays were carried out as previously described.14
Opioid binding affinities at the human δ opioid receptor
(hDOR) and the rat μ opioid receptor (rMOR) were
determined by competition analyses against [3H]DPDPE (δ)
and [3H]DAMGO (μ) using membrane preparations from
transfected HN9.10 cells that constitutively express the
respective receptors. Opioid agonist efficacy was examined by
monitoring [35S]GTP-γ-S binding. Functional assays to
evaluate their opioid agonist activities were performed using
the stimulated isolated mouse vas deferens (MVD, δ) and
guinea pig ileum (GPI, μ) bioassays. In general, the functional
assay results correlated with those from the [35S]GTP-γ-S
binding assay.
In the competition binding assay, all the Dmt-L(or D)Tic
analogues except 9 and 10 showed high binding affinities for
both μ and δ receptors, but with distinct selectivities depending
upon the configuration of Tic3 residue (Table 2). Ligands 3−5,
B
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ACS Medicinal Chemistry Letters
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Table 3. Functional activities of Dmt-L(or D)Tic analogues
[35S]GTP-γ-S binding
MVD(δ)
antagonist
GPI(μ)
a
a
hDOR
rMOR
agonist
agonist
antagonist
b
c
d
b
c
d
e
f
e
f
no.
log EC50
EC50 (nM)
Emax (%)
log EC50
EC50 (nM)
Emax (%)
IC50 (nM)
Ke (nM)
5.0
IC50 (nM)
Ke (nM)
3
4
nr
nr
20%
3
740 220
350 90
0%
nr
nr
1.6%
0.79 0.6
g
5
0.48
8.1
nr
18
6.2
0.90
nr
16
40
74 14
42+6
14%
nr
nr
6
−8.09 0.07
−7.84 0.14
128
−9.04 0.28
−8.35 0.17
82
9
7
150 20
27%
8
15
nr
47
4.5
nr
82
86 29
0%
720 210
4%
9
nr
nr
nr
10
nr
nr
810 140
nr
6%
h
Dmt-Tic-NH2
42
>10 μM
i
Dmt-Tic-Phe-Phe-NH2
0.21 0.04
18
2
a
b
Expressed in CHO cells. Logarithmic values determined from the nonlinear regression analysis of data collected from at least three independent
c
d
e
experiments. Antilogarithmic value of the respective EC50 values. Net total bound/basal binding ×100 SEM. Concentration at 50% inhibition of
muscle contraction at electrically stimulated isolated tissues or % inhibition of contraction height at 1 μM. Antagonist. See ref 7. See ref 1. See refs
f
g
h
i
9 and 10. nr: no response.
maintained in a similar range (Ki = 12 nM) to those of the
other ligands. Apparently replacement of the piperidinyl ring in
position 4 of ligand 9 with the triazoly containing ring in ligand
10 did not affect the binding affinities but slightly changed the
selectivity. On the basis of these SAR results, it is clear that, in
this series of analogues, the configurations of Tic2 and Phe3 play
an important role in determining their binding affinities and
receptor selectivities.
biological profile from ligands 6 and 8 and the ligand was not
an agonist for the δ and μ receptors, but a weak antagonist (Ke
= 150 nM) for the δ receptor. It was shown that p-chloro
substitution of the aromatic ring of Phe3 residue in Dmt-Tic
analogues did not change its biological activity, and thus,
ligands 3 and 4 showed very similar biological profiles.
Therefore, it is clear that Dmt-D-Tic analogues showed distinct
SAR compared to Dmt-Tic analogues.
As predicted, Dmt-Tic analogues 3 and 4 did not show
agonist stimulation at both δ and μ receptors in the [35S]GTP-
γ-S binding assay but exhibited strong δ antagonist (Ke = 5.0
3 nM)/weak μ agonist (IC50 = 740 220 nM) activities in
MVD/GPI assays, respectively (Table 3). As mentioned earlier,
surprisingly, the ligand 5, in which the chirality of third amino
acid residue was changed from L- to D-, exhibited δ agonist
activity instead of antagonist activity in the [35S]GTP-γ-S (EC50
= 0.48 nM, Emax = 18%) and the MVD (IC50 = 74 14 nM)
assays. According to a previous report, the distance between the
Dmt-Tic and the third aromatic nucleus is an important
criterion in converting Dmt-Tic from a highly potent δ
antagonist into a potent δ agonist or into ligands with mixed δ
and μ opoioid properties.3 This fact can help us to elucidate
how ligand 5 has a moderate partial δ agonist activity instead,
which is inconsistent with other Dmt-Tic analogues containing
a D-amino acid residue, for example, D-Ala, D-Asp, and D-Gln, in
position 3.14 Even with moderate binding affinity (Ki = 22 nM)
and efficacy (EC50 = 6.2 nM) at the μ opioid receptor, ligand 5
did not show agonist activity in the GPI assay. This may be the
result of the conflicting tendencies of the Dmt-Tic message
domain to impart δ antagonism. It is possible that, for the same
reason, the ligand showed partial agonist activity with low Emax
(18%) but with high affinity (Ki = 0.38 nM).
As published before, the Dmt1-D-Tic2-Gly3-Phe(p-Cl)4
analogue did not show δ opioid antagonist activity, but a
potent agonist activity in the MVD assay (IC50 = 0.21 nM, μ/δ
= 23) unlike ligand 7, which includes a Phe(p-Cl)3 residue and
exhibits weak antagonist activity (IC50 = 170 nM). Again, this
functional assay result confirms that insertion of a Gly residue
into position 3 of Dmt-D-Tic analogues can distinguish a
topographical structure from the other ligands.
On the basis of the binding affinities of ligands 9 and 10, the
consecutive change of configuration (L,L to D,D) was not
tolerated for the δ receptor, and thus, the ligands 9 and 10 have
reduced binding affinities at the δ receptor. While the ligands
showed moderate binding affinities (Ki = 12 and 6.4 nM) and
selectivity for the μ receptor, no functional activity was
observed in the [35S]GTP-γ-S assay and the GPI assay, and
instead, low δ opioid agonist function (IC50 = 810 140 nM)
was observed in the MVD assay. Overall, the opioid agonist
activities (IC50) in the GPI assay were relatively less than the
one in the MVD assay, and the discrepancy seems to be the
result caused by conflict between the Dmt-Tic (or Dmt-D-Tic)
message and the analogues.18
It was shown that the Tic2 residue in Dmt-contained
peptides plays an important role in their biological activities for
the δ opioid receptor, and thus a significant loss in the receptor
binding affinity and bioactivity occurred when D-Tic is
utilized.17 In most cases, Dmt-D-Tic-Phe analogues behave
like Tyr-D-Xaa-Phe peptides and have μ agonism. As predicted,
Dmt-D-Tic analogues 6 and 8 showed higher binding affinity
(Ki = 0.45 and 1.9 nM, respectively) and agonist efficacy (EC50
= 0.90 and 4.5 nM, respectively), but relatively lower agonist
activity (IC50 = 82 and 720 nM) at the μ receptor. However,
ligand 7, as observed in the binding assay, exhibited a different
In conclusion, we observed that the chiralities of Tic and Phe
residues in positions 2 and 3 have a large influence on the
binding affinity and efficacy profile of Dmt-L-(or D-)Tic
analogues. Whereas Dmt-Tic-Phe analogues are δ receptor
selective antagonists and Dmt-Tic-D-Phe analogues are δ
receptor selective agonists, Dmt-D-Tic-Phe and Dmt-D-Tic-
Gly analogues are nonselective δ/μ agonists, except for the
analogue that includes a Phe(p-Cl)3 residue and shows δ
antagonist activity. In contrast, no functional activity except low
δ agonist activity was observed in Dmt-D-Tic-D-Phe analogues.
C
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ACS Medicinal Chemistry Letters
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ASSOCIATED CONTENT
* Supporting Information
Analytical data. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*(V.J.H.) E-mail: hruby@email.arizona.edu. Phone: (520)-621-
6332. Fax: (520)-621-8407.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
Funding was provided by USDHS, National Institute of Health
(DA-12394 and DA-06284).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
Boc, tert-butyloxycarbonyl; BOP, (benzotriazole-1-yloxy)-tris-
(dimethylamino)-phosphonium hexafluorophosphate; CHO,
Chinese hamster ovary; DMF, N,N-dimethylformamide;
hDOR, human δ opioid receptor; DPDPE, c[D-Pen2,D-Pen5]-
enkephalin; DAMGO, [D-Ala2,NMePhe4,Gly5-ol]enkephalin;
Dmt, 2,6-dimethyltyrosine; GPI, guinea pig isolated ileum;
HOBt, 1-hydroxybenzotriazole; rMOR, rat μ opioid receptor;
MVD, mouse vas deferens; NMM, N-methylmorpholine; RP-
HPLC, reverse phase high performance liquid chromatography;
SAR, structure−activity relationships; TFA, trifluoroacetic acid
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