[Dmt1]DALDA analogues with enhanced μ opioid agonist potency and with a mixed μ/κ opioid activity profile

Article abstract of DOI:10.1016/j.bmc.2014.02.011

Analogues of [Dmt¹]DALDA (H-Dmt-d-Arg-Phe-Lys-NH₂; Dmt = 2′,6′-dimethyltyrosine), a potent μ opioid agonist peptide with mitochondria-targeted antioxidant activity, were prepared by replacing Phe³ with various 2′,6′-dialkylated Phe analogues, including 2′,6′-dimethylphenylalanine (Dmp), 2′,4′, 6′-trimethylphenylalanine (Tmp), 2′-isopropyl-6′- methylphenylalanine (Imp) and 2′-ethyl-6′-methylphenylalanine (Emp), or with the bulky amino acids 3′-(1-naphthyl)alanine (1-Nal), 3′-(2-naphthyl)alanine (2-Nal) or Trp. Several compounds showed significantly increased μ agonist potency, retained μ receptor selectivity and are of interest as drug candidates for neuropathic pain treatment. Surprisingly, the Dmp³-, Imp³-, Emp³- and 1-Nal³-containing analogues showed much increased κ receptor binding affinity and had mixed μ/κ properties. In these cases, molecular dynamics studies indicated conformational preorganization of the unbound peptide ligands due to rotational restriction around the C ^βC^γ bond of the Xxx³ residue, in correlation with the observed κ receptor binding enhancement. Compounds with a mixed μ/κ opioid activity profile are known to have therapeutic potential for treatment of cocaine abuse.

Full text of DOI:10.1016/j.bmc.2014.02.011

Bioorganic & Medicinal Chemistry 22 (2014) 2333–2338
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
[Dmt1]DALDA analogues with enhanced
l
opioid agonist potency
and with a mixed
l/j
opioid activity profile
Longxiang Bai a, Ziyuan Li a, Jiajia Chen a, Nga N. Chung c, Brian C. Wilkes c, Tingyou Li a,b,
,
⇑
Peter W. Schiller c,d,
⇑
a School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
b Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China
c Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, QC H2W 1R7, Canada
d Department of Pharmacology, Université de Montréal, Montreal, QC H3C 3J7, Canada
a r t i c l e i n f o
a b s t r a c t
Analogues of [Dmt1]DALDA (H-Dmt- -Arg-Phe-Lys-NH2; Dmt = 20,60-dimethyltyrosine), a potent
D l opioid
Article history:
agonist peptide with mitochondria-targeted antioxidant activity, were prepared by replacing Phe3 with
various 20,60-dialkylated Phe analogues, including 20,60-dimethylphenylalanine (Dmp), 20,40,60-trimethyl-
phenylalanine (Tmp), 20-isopropyl-60-methylphenylalanine (Imp) and 20-ethyl-60-methylphenylalanine
(Emp), or with the bulky amino acids 30-(1-naphthyl)alanine (1-Nal), 30-(2-naphthyl)alanine (2-Nal) or
Received 5 December 2013
Revised 22 January 2014
Accepted 3 February 2014
Available online 19 February 2014
Trp. Several compounds showed significantly increased
and are of interest as drug candidates for neuropathic pain treatment. Surprisingly, the Dmp3-, Imp3-,
Emp3- and 1-Nal3-containing analogues showed much increased
receptor binding affinity and had
mixed properties. In these cases, molecular dynamics studies indicated conformational preorganiza-
tion of the unbound peptide ligands due to rotational restriction around the CbAC bond of the Xxx3
residue, in correlation with the observed receptor binding enhancement. Compounds with a mixed
opioid activity profile are known to have therapeutic potential for treatment of cocaine abuse.
Ó 2014 Elsevier Ltd. All rights reserved.
l agonist potency, retained l receptor selectivity
Keywords:
l
opioid agonists
j
[Dmt1]DALDA
l/j
Alkylated phenylalanine analogues
Opioid activity profiles
c
j
l/j
1. Introduction
compound was 3000-fold more potent than morphine with intra-
thecal (i.th.) administration.2 The extraordinary potency of [Dmt1]-
DALDA as a spinal analgesic is due to its triple action as a l opioid
The dermorphin-derived tetrapeptide [Dmt1]DALDA (H-Dmt-
D
-
Arg-Phe-Lys-NH2), containing 20,60-dimethyltyrosine (Dmt), is a
agonist, as a norephinephrine uptake inhibitor and as a releaser of
endogenous opioid peptides.2,3 Surprisingly, [Dmt1]DALDA also
produced a potent antinociceptive effect in the mouse tail-flick test
when given subcutaneously (sc) (40–220 times more potent than
morphine), indicating that it is capable of crossing the blood–brain
barrier.4,5 The long-lasting antinociceptive effect of this compound
in the acute pain models observed with both i.th. and sc adminis-
tration is due to its high stability against enzymatic degradation
and slow clearance.2,6 The favorable drug-like properties of [Dmt1]-
DALDA as a systemically active analgesic have been reviewed.7
highly potent
l
opioid agonist.1 It has subnanomolar
l
receptor
receptor binding selec-
) = 1:14,700:156]. In the rat tail-flick assay this
l
binding affinity (Ki = 0.143 nM) and high
l
tivity [Ki ratio (
l
/d/j
Abbreviations: Boc,
Phe(NMe)-Gly-ol; Dmp, 20,60-dimethylphenylalanine; Dmt, 20,60-dimethyltyrosine;
[Dmt1]DALDA, H-Dmt-
-Arg-Phe-Lys-NH2; DSLET, H-Tyr- -Ser-Gly-Phe-Leu-Thr-
tert-butyloxycarbonyl;
DAMGO,
H-Tyr-D-Ala-Gly-
D
D
OH; EDT, 1,2-ethanediol; Emp, 20-ethyl-60-methylphenylalanine; Fmoc, 9-fluore-
nylmethoxycarbonyl; GPI, guinea pig ileum; HOBt, 1-hydroxybenzotriazole; Imp,
20-isopropyl-60-methylphenylalanine; IMM, inner mitochondrial membrane; MVD,
mouse vas deferens; 1-Nal, 30-(1-naphthyl)alanine; 2-Nal, 30-(2-naphthyl)alanine;
Pbf, 2,3-dihydro-2,2,4,6,7-pentamethyl-5-benzofuranyl)sulfonyl; PyBOP, (ben-
zotriazol-1-yl-oxy)tris(pyrrolidino)phosphonium hexafluorophosphate; RP-HPLC,
reversed-phase high performance liquid chromatography; TLC, thin layer chro-
Confocal laser scanning microscopy carried out with a fluores-
8
cent [Dmt1]DALDA analogue, H-Dmt-
D-Arg-Phe-atn-Dap-NH2
and mitochondrial fractionation studies using a tritiated [Dmt1]-
DALDA analogue showed that the compound was taken up by cells
and was distributed to the inner mitochondrial membrane (IMM).9
The ability of [Dmt1]DALDA to penetrate the cellular membrane
and target the IMM is due to its structural motif of alternating aro-
matic and basic residues. Because the Dmt residue has antioxidant
matography; Tmp, 20,40,60-trimethylphenylalanine; U69,593, (5
a,7a,8b)-(ꢀ)-N-
methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide.
⇑
Corresponding authors. Tel./fax: +86 25 8686 8475 (T.L.); tel.: +1 514 987 5576;
fax: +1 514 987 5513 (P.W.S.).
E-mail addresses: l_tingyou@njmu.edu.cn (T. Li), schillp@ircm.qc.ca (P.W. Schiller).
http://dx.doi.org/10.1016/j.bmc.2014.02.011
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2334
L. Bai et al. / Bioorg. Med. Chem. 22 (2014) 2333–2338
properties,9 [Dmt1]DALDA acts as a IMM-targeted antioxidant.
Since mitochondrial reactive oxygen species (ROS) in spinal cord
2. Results and discussion
2.1. Chemistry
dorsal horn neurons play
a key role in neuropathic pain
mechanisms,10 [Dmt1]DALDA was examined for its antinociceptive
effectiveness in the rat spinal nerve ligation model and was found
to be more effective than morphine in this experimental model of
neuropathic pain.11
Dmp, Tmp, Imp and Emp were synthesized as reported.19
Peptides were prepared by the solid-phase peptide synthesis meth-
od on a Rink amide resin, using 9-fluorenyl-methoxycarbonyl
(Fmoc)-protected amino acids and (benzotriazol-1-yl-oxy)tris
Introduction of conformational constraints into opioid peptides
has been shown to have interesting effects on potency and opioid
receptor binding selectivity. Global conformational restriction
(pyrr-olidino)phosphonium
hexafluorophosphate
(PyBOP)/1-
hydroxybenzotriazol (HOBt) as coupling agents. The side chains of
Lys and Arg were protected with tert-butyloxycarbonyl (Boc) and
(2,3-dihydro-2,2,4,6,7-pentamethyl-5-benzofuranyl)sulfonyl (Pbf),
respectively. Peptides were cleaved from the resin by treatment
with TFA/EDT/anisole = 3.8:0.1:0.1(v/v), purified by semi-
preparative HPLC, and lyophilized.
through various types of cyclization resulted in
selective opioid agonists.12–14 Conformational restriction of the
side chains of Phe and Tyr ( constraints, reviewed in Ref. 15) in
l or d receptor-
v
opioid peptides has also been shown to have significant effects
on opioid receptor binding affinities and selectivities. b-Methyla-
tion of the Phe residue in cyclic enkephalin analogues produced
compounds that retained high d receptor binding affinity16 or
showed improved d receptor selectivity.17 Substitution of Dmt for
Tyr1 in opioid peptides generally enhances opioid receptor binding
affinity, as was the case with the d-selective analogue
Analytical data of the peptides are presented in Table 1.
2.2. 2In vitro opioid activity determinations
In the opioid receptor binding assays (Table 2), H-Dmt-D-Arg-
[Dmt1]DPDPE (H-Dmt-c[ -Pen]-OH)18 and the
D-Pen-Gly-Gly-Phe-D
Dmp-Lys-NH2 (1) showed 15-fold higher
l receptor binding affin-
agonist [Dmt1]DALDA.1 Introduction of the 20,60-dimethyl groups
l
ity than the [Dmt1]DALDA parent (8) with a Ki value in the low
l
c
in Dmt restricts rotation around the CbAC bond.
l
picomolar range (Ki = 9.35 pM). Similar to 8, analogue 1 displayed
In an effort to obtain [Dmt1]DALDA analogues with enhanced
l
l
high
l
receptor binding selectivity (selectivity ratio K =Kdi =Kij
=
i
opioid agonist potency or possibly altered opioid receptor selectiv-
ity profiles, we replaced the Phe3 residue with various alkylated
phenylalanine residues, in which rotational mobility around the
1:11200:350). Despite its increased
l receptor binding affinity,
the agonist potency of 1 in the GPI assay was similar to that of 8
(Table 3). A Dmp3-analogue of the dermorphin-derived tetrapep-
c
CbAC bond is expected to be restricted. These include 20,60-dim-
tide H-Tyr-
D
-Arg-Phe-b-Ala-NH2 had also been reported to have
ethylphenylalanine (Dmp) (1), 20,40,60-trimethylphenylalanine
(Tmp) (2), 20-isopropyl-60-methylphenylalanine (Imp) (3) and 20-
ethyl-60-methylphenylalanine (Emp) (4) (Fig. 1).19 Furthermore,
analogues containing the bulky aromatic amino acids 3-(1-naph-
thyl)alanine (1-Nal) (5), 3-(2-naphthyl)alanine (2-Nal) (6) and
Trp (7) in place of Phe3 were also synthesized. The in vitro opioid
increased
l
opioid receptor binding affinity.20 The enhanced
agonist potency of 1 as compared to parent 8 in the MVD assay
is due to its 15-fold higher receptor binding affinity, resulting
in increased activation of receptors that are also present in the
vas preparation in addition to the predominant d receptors. In
comparison with the [Dmt1]DALDA parent (8), H-Dmt-
-Arg-
Tmp-Lys-NH2 (2) had comparable receptor binding affinity, 6-
fold higher d receptor affinity and 2-fold lower affinity, and thus
also retained high receptor preference. In the GPI assay it was a
l
l
D
activity profiles of the compounds were determined in
-opioid receptor binding assays and in the functional guinea pig
ileum (GPI) and mouse vas deferens (MVD) assays. The GPI
contains both and opioid receptors, whereas in the MVD d
l-, d- and
l
j
j
l
l
j
12-fold less potent agonist than 8. Compounds 1 and 2 both
opioid receptors are predominant with
l and j receptors also pres-
showed lower potency in the GPI assay than was expected on the
ent at lower concentration.
basis of their
l receptor binding affinities. This could be explained
with their reduced access to receptors in this tissue as a conse-
quence of their increased lipophilic character resulting from the
Dmp and Tmp substitutions.. In comparison with the [Dmt1]-
DALDA parent (8), the Imp3-analogue (3) had similar
l
receptor
receptor affinity in the
subnanomolar range (Kji = 0.236 nM) and higher d receptor binding
binding affinity, but 100-fold increased
j
affinity (Kdi = 77.1 nM). Thus, it showed no
selectivity, but still quite high preference for
l
versus
and
j
receptor
l
j
receptors
over d receptors. The high agonist potency of 3 in the MVD assay
(IC50 = 1.22 nM) was unexpected, but may be due to its activation
of
l and j receptors in the vas in addition to the agonist effect at
Table 1
Analytical parameters of [Dmt1] DALDA analogues
No.
Compound
TLCa
HPLC
tR (min)
Mass
(M+H)+
b
1
2
3
4
5
6
7
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
D-Arg-Dmp-Lys-NH2
D-Arg-Tmp-Lys-NH2
D-Arg-Imp-Lys-NH2
D-Arg-Emp-Lys-NH2
D-Arg-1-Nal-Lys-NH2
D-Arg-2-Nal-Lys-NH2
D-Arg-Trp-Lys-NH2
0.63
0.62
0.62
0.61
0.59
0.58
0.56
7.52
8.31
9.11
8.40
8.44
8.32
6.42
668.1
682.1
696.2
682.1
690.1
690.1
679.1
a
Solvent: n-BuOH/Py/HOAc/H2O = 15:10:3:12.
See Section 4.1 for conditions.
b
Figure 1. Structural formulas of Dmp, Tmp, Emp, Imp, 1-Nal and 2-Nal.

L. Bai et al. / Bioorg. Med. Chem. 22 (2014) 2333–2338
2335
Table 2
Opioid receptor binding affinities of [Dmt1]DALDA analoguesa
l
j
Kdi (nM)
Compound
Potency ratio
/d/
Ki (nM)
Ki (nM)
l
j
1
2
3
4
5
6
7
8
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
D
D
D
D
D
D
D
D
-Arg-Dmp-Lys-NH2
-Arg-Tmp-Lys-NH2
-Arg-Imp-Lys-NH2
-Arg-Emp-Lys-NH2
-Arg-1-Nal-Lys-NH2
-Arg-2-Nal-Lys-NH2
-Arg-Trp-Lys-NH2
0.00935 0.0015
0.158 0.013
0.155 0.007
0.0958 0.0101
0.109 0.012
0.235 0.057
0.0991 0.0019
0.143 0.015
105 13
329
77.1 3.8
50.0 6.7
173 19
258 29
4.70 0.11
48.0 0.6
0.236 0.002
0.961 0.089
1.60 0.09
18.9 5.6
1:11,200:350
1:2080:304
1:497:2
9
1:522:10
1:1590:15
1:1100:80
1:1880:88
1:14,700:156
186
2
16.5 4.6
22.3 4.2
b
-Arg-Phe-Lys-NH2
2100 310
a
Values represent means of 3–4 determinations SEM.
Data taken from Ref. 1.
b
Table 3
states (
v
2 = +90°, ꢀ90°) (Fig. 2a), with a rotational barrier of
GPI and MVD assays of [Dmt1]DALDA analogues
3.3 kcal/mol (Table 4). With Dmp, Emp and Imp, the
v2 (ꢀ90°) did
not change in the course of the simulation, indicating that rotation
Compound
GPI
IC50 (nM)
MVD
IC50 (nM)
around the CbAC bond is impeded. In these cases the barrier of rota-
c
a
a
tion was 9–10 kcal/mol for this bond. Rotation around the CbAC
c
1
2
3
4
5
6
7
8
H-Dmt-
D
D
D
D
D
D
D
D
-Arg-Dmp-Lys-NH2
-Arg-Tmp-Lys-NH2
-Arg-Imp-Lys-NH2
-Arg-Emp-Lys-NH2
-Arg-1-Nal-Lys-NH2
-Arg-2-Nal-Lys-NH2
-Arg-Trp-Lys-NH2
2.02 0.02
17.4 1.7
1.72 0.20
19.1 1.3
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
H-Dmt-
bond of 1-Nal was also somewhat restricted, as only two transitions
between
simulation with a barrier of rotation of 5.2 kcal/mol. In contrast, four
0.828 0.114
0.474 0.042
1.90 0.05
71.5 3.9
1.22 0.21
0.220 0.010
0.755 0.065
64.8 16.1
1.67 0.26
23.1 2.0
v2 = ꢀ90° and
v2 = +90° were observed during the 1 ns
transitionsbetween these two conformational states were seen with
2-Nal, indicating greater rotational flexibility around the CbAC
c
1.07 0.11
1.41 0.29
b
-Arg-Phe-Lys-NH2
bond (barrier of rotation of 3.6 kcal/mol) as compared to 1-Nal.
Interestingly, rotational flexibility of the isopropyl substituent on
the phenyl ring of Imp was also restricted, with only one conforma-
tional state being occupied in the course of the stimulation, whereas
theethyl substituentin Emp enjoyedunhinderedrotationalfreedom
a
Values represent means of 3–4 determinations SEM.
Data taken from Ref. 1.
b
(Fig. 2b,
s rotation).
the predominant d receptors. The opioid receptor binding profile of
the Emp3-analogue, H-Dmt-
-Arg-Emp-Lys-NH2 (4), was similar to
It is well documented that di-ortho groups on phenyl rings in
ligands of various proteins can promote a favorable conformational
preorganization due to increased rotational barriers, resulting in
increased binding affinity (for a review, see Ref. 21). Interestingly,
the four peptides containing Dmp3 (1), Imp3 (3), Emp3 (4) and
D
that of 3, but it did show modest preference for
l receptors over j
l
receptors (Kji =K = 10). It was the most potent agonist of the series
i
both in the GPI assay (IC50 = 0.474 nM) and in the MVD assay
(IC50 = 0.220 nM), in agreement with its very high binding affinities
1-Nal3 (5) show markedly increased
j receptor binding affinity
at both
l and j receptors and its highest d receptor binding affinity
as compared to the [Dmt1]DALDA parent (8). As described above,
among all compounds.
side chain rotational mobility around the CbAC bond of the 3-po-
c
Substitution of 1-Nal for Phe3 in [Dmt1]DALDA produced a
sition residue in these four peptides is restricted and the resulting
conformational preorganization in the unbound state may be
compound (5) which retained high
l
receptor binding affinity
receptor affinity
l
Ki = 0.109 nM) and also showed high
j
(Kji = 1.60 nM). Similar to peptides 3 and 4, compound 5 was a
potent agonist in both functional assays. The 2-Nal3-analogue (6)
showed lower binding affinity than the 1-Nal3-analogue (5) at all
favorable for their interaction with the
j opioid receptor. The
nearly 100-fold
j receptor binding affinity increase seen with the
Imp3-analogue (3) may be due to the combined effect of rotational
restriction around the CbAC bond and of the isopropyl substitu-
c
three opioid receptors, particularly at the
j
receptor (Kji = 18.9
ent, with both the phenyl ring and its isopropyl substituent
engaging in favorable hydrophobic receptor interactions.
nM). In agreement with its profile of lower opioid receptor binding
affinities, compound 6 displayed significantly reduced agonist
potencies both in the GPI assay and in the MVD assay. In compar-
ison with the [Dmt1]DALDA (8) parent, the Trp3-analogue (7) had
3. Conclusions
similar
l and j receptor binding affinities, but 11-fold higher d
receptor affinity. In agreement with the receptor binding data,
compounds 7 and 8 were about equipotent in the GPI assay, but
7 was 13-fold more potent than 8 in the MVD assay.
The performed amino acid substitutions at the 3-position resi-
due of [Dmt1]DALDA resulted in several analogues with enhanced
l
opioid agonist potency with one of them, H-Dmt-
D-Arg-Dmp-
Lys-NH2 (1), showing low picomolar receptor binding affinity.
l
2.3. Molecular modeling studies
These peptides retain the alternating aromatic-basic structural
motif required for mitochondria-targeted antioxidant activity and
may have improved therapeutic potential for the treatment of neu-
ropathic pain states. In general, the new analogues had somewhat
increased, but still modest d receptor binding affinity (Kdi = 50–
300 nM). Previously, it had been demonstrated that endomor-
phin-2 analogues containing Dmp, Tmp, Emp or Imp in place of
Phe3, or Dmp, Tmp, 1-Nal or 2-Nal in place of Phe4 also showed
To assess the side chain flexibility of the various amino acids
substituted for Phe3 in [Dmt1]DALDA, a molecular dynamics study
(1 ns duration) was performed at 300 K with some of these amino
acids in their N-acetylated and carboxamidated form
[AcANHACH(R)ACONH2]. In all amino acids, rotation around v1
was not impeded and frequent transitions between the g+, gꢀ and t
states were observed (data not shown). As expected, conformational
enhanced
most of the peptides containing 20,60-disubstituted phenylalanine
analogues (1, 3, 4) or 1-Nal (5) displayed much enhanced
l
and d receptor binding affinities.22,23 Unexpectedly,
c
flexibility around the CbAC bond was observed in the case of Phe
with multiple transitions occurring between the two rotational
j

2336
L. Bai et al. / Bioorg. Med. Chem. 22 (2014) 2333–2338
Figure 2. Molecular dynamics simulation of NAc-Xxx3-CONH2. (a) v2 of Xxx3 = Phe, Dmp, Emp, Imp, 1-Nal and 2-Nal. (b)
s of ethyl and isopropyl substituents of Emp and
Imp.
Table 4
liquid chromatograph using the following solvent system: solvent
A, 0.05% TFA in water; solvent B, 0.05% TFA in CH3CN. Semi-prepara-
tive RP-HPLC was performed on a SunFire™ Prep C18 column
(20 ꢁ 150 mm) with a linear gradient of 90% A to 50% A over
15 min at a flow rate of 10 ml/min. Analytical RP-HPLC was per-
formed on a SunFire™ Prep C18 column (4.6 ꢁ 150 mm) with a lin-
ear gradient of 90% A to 10% A over 30 min at a flow rate of 1.2 ml/
min and the retention time, tR (min), was determined. 1H and 13C
NMR spectra were recorded on an AVANCE AV-500 or AV-300 spec-
trometer at 25 °C. Chemical shifts are indicated as d values (ppm)
relative to tetramethylsilane (TMS). Molecular masses of peptides
were determined by electron spray mass spectrometry on a Agilent
1100 mass spectrometer.
Barriers of v2 rotation of Phe, Dmp, Emp, Imp, 1-Nal and 2-Nal
Amino acid
Energy (
(kcal/mol)
v
2 = +90°)
Energy (
(kcal/mol)
v
2 = +180°)
D energy
(kcal/mol)
Phe
2.30
4.01
4.38
5.67
3.88
2.15
5.60
13.41
14.30
16.41
9.03
3.30
9.40
9.92
10.74
5.15
3.61
Dmp
Emp
Imp
1-Nal
2-Nal
5.76
receptor binding affinities and had a mixed
l/j opioid profile. A
molecular dynamics study revealed rotational restriction around
c
the CbAC bond of the 3-position residue in these peptides. The
4.2. Peptide synthesis
resulting conformational preorganization of the unbound ligands
selectively strengthens
j receptor binding. Compounds with a
Peptide synthesis was performed by the manual solid phase
mixed
l/j opioid activity profile have therapeutic potential for
technique using
a
Rink Amide resin (1% cross-linked,
the treatment of cocaine abuse.24
100–200 mesh, 0.24 m equiv/g) obtained from GL Biochem (Shang-
hai) Ltd. Peptides were assembled using Fmoc-protected amino
acids and PyBOP and HOBt as coupling agents. The side chain pro-
tection was Boc for Lys and Pbf for Arg. The phenolic hydroxyl
group of Dmt was unprotected. The following steps were per-
formed in each cycle: (1) addition of Fmoc-amino acid (2.5 equiv)
in DMF; (2) addition of HOBt (2.5 equiv); (3) addition of PyBOP
(2.5 equiv); (4) addition of DIEA (2.5 equiv) and mixing for 2.5 h
at 26 °C; (5) washing with DMF (5 ꢁ 5 ml); (6) monitoring
4. Experimental section
4.1. General methods
Precoated plates (silica gel F254, Qiangdao, China) were used for
ascending TLC in the following solvent systems: n-BuOH/Py/HOAc/
H2O (15:10:3:12). RP-HPLC was performed on a Waters Delta 600

L. Bai et al. / Bioorg. Med. Chem. 22 (2014) 2333–2338
2337
completion of the reaction with the ninhydrin test; (7) Fmoc
deprotection with 20% (v/v) piperidine in DMF (5 min); (8) Fmoc
deprotection with 20% (v/v) piperidine in DMF (30 min). After
complete peptide assembly, Fmoc protection was removed by
treatment with piperidine in DMF. The resin was washed with
DMF (3 ꢁ 5 ml), CH2Cl2 (3 ꢁ 5 ml), MeOH (3 ꢁ 5 ml), CH2Cl2
(3 ꢁ 5 ml), MeOH (3 ꢁ 5 ml) and was dried in a desiccator. Peptides
were cleaved from the resin by treatment with TFA/EDT/anisole
(3.8:0.1:0.1) for 2 h at 26 °C (8 ml of the mixture/g of resin), and
the cleavage procedure was repeated twice. The filtrates were
combined and precipitated with ice-cold ether/hexane (1:1). The
crude peptides were purified by semi-preparative HPLC. Each pep-
tide was at least 98% pure as assessed by analytical reversed-phase
HPLC. Molecular weights were confirmed by MS.
(s, 1H). 13C NMR (125 MHz, DMSO-d6) d (ppm): 15.45, 19.85,
20.04, 22.22, 23.88, 25.22, 26.66, 28.77, 30.70, 31.40, 31.49,
51.35, 52.27, 52.56, 114.85, 115.99, 118.38, 121.92, 125.91,
126.26, 127.73, 133.27, 136.92, 138.30, 142.83, 155.58, 156.71,
157.83, 158.07, 158.32, 170.00, 170.61, 172.99.
4.2.5. H-Dmt-D-Arg-1-Nal-Lys-NH2 (5)
170.4 mg. 1H NMR (300 MHz, DMSO-d6) d: 0.76 (s, 2H),
0.94–1.07 (m, 2H), 1.34 (s, 2H), 1.53–1.69 (m, 4H), 2.15 (s, 6H),
2.78–2.84 (m, 5H), 2.95–3.03 (t, J = 11.49 Hz, 1H), 3.08–3.18 (t,
J = 10.20 Hz, 1H), 3.63–3.66 (d, J = 10.53 Hz, 1H), 3.88 (s, 1H),
4.15–4.24 (m, 2H), 4.63 (s, 1H), 6.38 (s, 2H), 7.15 (s, 4H), 7.33–
7.41 (m, 4H), 7.48–7.58 (m, 2H), 7.75–7.77 (d, J = 7.02 Hz, 2H),
7.87–7.90 (d, J = 7.62 Hz, 2H), 7.97–8.00 (d, J = 8.10 Hz, 1H), 8.19–
8.24 (t, J = 6.18 Hz, 4H), 8.55–8.58 (d, J = 8.4 Hz, 1H), 9.05 (s, 1H).
13C NMR (75 MHz, DMSO-d6) d: 19.83, 22.30, 24.02, 26.67, 28.54,
30.67, 31.39, 34.73, 51.36, 52.24, 52.49, 53.41, 114.84, 121.89,
123.62, 125.11, 125.48, 125.99, 127.05, 127.55, 128.53, 131.39,
133.39, 133.44, 138.29, 155.57, 156.68, 158.44, 168.45, 170.29,
170.66, 173.35.
4.2.1. H-Dmt-D-Arg-Dmp-Lys-NH2 (1)
73.1 mg. 1H NMR (300 MHz, DMSO-d6) d: 0.82 (s, 2H), 1.00–1.12
(m, 2H), 1.29 (s, 2H), 1.51–1.55 (t, J = 6.66 Hz, 3H), 1.63–1.65 (m,
1H), 2.16 (s, 6H), 2.28 (s, 6H), 2.77–2.85 (m, 6H), 2.96–3.11 (m,
2H), 3.89 (s, 1H), 4.12–4.24 (m, 2H), 4.57–4.65 (q, J = 7.65 Hz,
1H), 6.40 (s, 2H), 6.86–6.94 (m, 3H), 7.07 (s, 2H), 7.17 (s, 2H),
7.44 (s, 1H), 7.92–7.95 (d, J = 8.04 Hz, 1H), 8.19–8.22 (d,
J = 7.68 Hz, 1H), 8.33–8.36 (d, J = 7.98 Hz, 3H), 9.07 (s, 1H). 13C
NMR (75 MHz, DMSO-d6) d: 19.87, 19.96, 22.26, 23.82, 26.65,
28.75, 30.61, 31.36, 32.28, 51.29, 51.90, 52.22, 52.35, 114.86,
121.86, 125.99, 127.79, 134.05, 136.88, 138.31, 155.60, 156.75,
158.06, 158.47, 168.24, 170.00, 170.73, 173.04.
4.2.6. H-Dmt-D-Arg-2-Nal-Lys-NH2 (6)
58 mg. 1H NMR (300 MHz, DMSO-d6) d: 0.65 (s, 2H), 0.93–1.06
(m, 2H), 1.30–1.33 (d, J = 7.53 Hz, 2H), 1.52–1.67 (m, 4H), 2.14 (s,
6H), 2.74–2.18 (t, J = 7.71 Hz, 3H), 2.86–3.02 (m, 2H), 3.32 (s, 3H),
3.87 (s, 1H), 4.17–4.21 (t, J = 6.06 Hz, 2H), 4.66 (s, 1H), 6.36 (s,
2H), 7.10 (s, 4H), 7.27 (s, 1H), 7.41–7.49 (m, 4H), 7.71–7.84 (m,
6H), 8.13–8.15 (t, J = 3.57 Hz, 4H), 8.37–8.40 (d, J = 8.73 Hz, 1H),
9.00 (s, 1H). 13C NMR (75 MHz, DMSO-d6) d (ppm): 20.33, 22.79,
24.18, 27.18, 29.40, 31.21, 31.21, 31.92, 51.86, 52.83, 54.31,
115.32, 122.44, 125.80, 126.35, 127.66, 127.81, 128.09, 128.18,
132.20, 132.27, 135.84, 138.73, 156.03, 157.13, 158.44, 158.83,
170.57, 171.20, 173.80.
4.2.2. H-Dmt-D-Arg-Tmp-Lys-NH2 (2)
22.4 mg. 1H NMR (300 MHz, DMSO-d6)d: 0.77 (s, 2H), 1.02–1.08 (m,
2H), 1.26–1.34 (m, 5H), 1.50 (s, 1H), 2.16–2.23 (m, 13H), 2.59–2.65
(t, J = 7.80 Hz, 2H), 2.73–2.89 (m, 2H), 2.94–3.05 (m, 4H), 3.89–
3.92 (m, 2H), 4.23–4.26 (m, 1H), 4.35 (s, 1H), 6.42 (s, 2H), 6.74 (s,
2H), 7.04 (s, 2H), 7.17 (s, 2H), 7.27 (s, 1H), 7.44 (s, 1H), 7.78–7.81
(d, J = 7.89 Hz, 4H), 8.24–8.34 (m, 4H), 9.10 (s, 1H). 13C NMR
(75 MHz, DMSO-d6) d: 19.70, 19.87, 20.40, 21.73, 24.11, 26.43,
28.98, 30.38, 30.77, 31.64, 51.55, 52.31, 52.37, 52.82, 114.91,
122.04, 128.53, 130.56, 134.70, 136.73, 138.30, 155.58, 156.75,
170.48, 170.71, 173.63.
4.2.7. H-Dmt-D-Arg-Trp-Lys-NH2 (7)
99.9 mg. 1H NMR (500 MHz, DMSO-d6) d: 0.73–0.82 (m, 2H),
1.05–1.15 (m, 2H), 1.26–1.31 (m, 2H), 1.49–1.54 (m, 3H),
1.62–1.68 (m, 1H), 2.16 (s, 6H), 2.76–2.79 (m, 4H), 2.81–2.85 (dd,
J1 = 14.05 Hz, J2 = 4.85 Hz, 1H), 2.88–2.93 (dd, J1 = 14.65 Hz,
J2 = 9.45 Hz, 1H), 2.97–3.02 (t, J = 10.90 Hz, 1H), 3.15–3.18 (dd,
J1 = 14.50 Hz, J2 = 4.45 Hz, 1H), 3.90 (s, 1H), 4.15–4.24 (m, 2H),
4.51–4.55 (m, 1H), 6.39 (s, 2H), 6.94–6.97 (t, J = 7.50 Hz, 2H),
7.02–7.07 (dd, J1 = 14.15 Hz, J2 = 7.15 Hz, 3H), 7.11–7.12 (d,
J = 2.15 Hz, 2H), 7.30–7.32 (d, J = 8.10 Hz, 1H), 7.34–7.36 (m, 1H),
7.60–7.61 (d, J = 7.85 Hz, 1H), 7.78 (s, 3H), 7.91–7.93 (d,
J = 8.00 Hz, 1H), 8.19–8.21 (d, J = 7.75 Hz, 1H), 8.27–8.31 (m, 4H),
9.04 (s, 1H), 10.70 (s, 1H). 13C NMR (125 MHz, DMSO-d6) d:
19.85, 22.26, 23.87, 26.70, 27.83, 28.80, 30.61, 31.40, 51.29,
52.21, 52.32, 53.45, 109.80, 111.17, 114.86, 118.09, 118.39,
120.78, 121.84, 123.81, 127.81, 135.99, 138.30, 155.59, 156.69,
158.07, 158.32, 168.24, 170.11, 171.07, 173.37.
4.2.3. H-Dmt-D-Arg-Imp-Lys-NH2 (3)
151.8 mg. 1H NMR (500 MHz, DMSO-d6) d: 0.79–0.82 (m, 2H),
1.02–1.05 (m, 1H), 1.12–1.14 (dd, J1 = 6.55 Hz, J2 = 4.60 Hz, 7H),
1.27–1.33 (m, 2H), 1.51–1.57 (m, 3H), 1.65–1.70 (m, 1H), 2.16 (s,
6H), 2.29 (s, 3H), 2.76–2.85 (m, 7H), 2.98–3.03 (m, 1H), 3.13–
3.17 (dd, J1 = 14.10 Hz, J2 = 6.50 Hz, 1H), 3.88 (s, 1H), 4.18–4.23
(m, 2H), 4.51–4.56 (q, J = 7.00 Hz, 1H), 6.40 (s, 2H), 6.87–6.89 (d,
J = 6.45 Hz, 1H), 6.99–7.04 (m, 3H), 7.08 (s, 1H), 7.18 (s, 2H),
7.40–7.43 (t, J = 5.30 Hz, 1H), 7.80–7.82 (m, 4H), 8.22–8.24 (d,
J = 7.80 Hz, 1H), 8.31–8.35 (m, 4H), 9.05 (s, 1H). 13C NMR
(125 MHz, DMSO-d6) d: 19.84, 20.50, 22.19, 23.74, 23.86, 24.37,
26.67, 27.90, 28.69, 30.63, 30.99, 31.53, 51.29, 52.15, 52.39,
53.21, 114.86, 121.84, 122.77, 126.39, 127.44, 132.57, 136.77,
138.31, 155.59, 156.72, 157.85, 158.09, 158.34, 158.59, 168.31,
170.05, 170.49, 173.07.
4.3. Molecular dynamics studies
All calculations were performed using SYBYL version 7.0 (Tripos
Associates, St. Louis, MO). The Tripos force field was used for
energy calculations with a dielectric constant of 78. Phenylalanine
was taken from a fragment library and modified as needed to gen-
erate the desired amino acid derivatives. In all cases the N-terminal
amino group was acetylated and the C-terminal carboxylic acid
group was amidated. Molecular dynamics simulations were carried
4.2.4. H-Dmt-D-Arg-Emp-Lys-NH2 (4)
96.5 mg. 1H NMR (500 MHz, DMSO-d6) d: 0.84 (s, 2H), 1.02–1.10
(m, 4H), 1.15–1.18 (m, 1H), 1.25–1.30 (m, 2H), 1.50–1.54 (m, 3H),
1.64–1.68 (m, 1H), 2.16 (s, 6H), 2.29 (s, 3H), 2.61–2.69 (m, 2H),
2.74–2.77 (t, J = 7.10 Hz, 2H), 2.79–2.87 (m, 4H), 2.97–3.02 (m,
1H), 3.07–3.11 (q, J = 6.65 Hz, 1H), 3.87–3.88 (d, J = 5.60 Hz, 1H),
4.15–4.25 (m, 2H), 4.54–4.59 (q, J = 7.65 Hz, 1H), 6.39 (s, 2H),
6.89–6.91 (m, 2H), 6.93–6.98 (m, 1H), 7.06–7.07 (d, J = 6.20 Hz,
3H), 7.42 (s, 1H), 7.77 (s, 2H), 7.85–7.87 (d, J = 8.15 Hz, 2H),
8.19–8.20 (d, J = 7.65 Hz, 2H), 8.33–8.34 (d, J = 8.80 Hz, 3H), 9.04
c
out at 300 K for 1 ns. The barrier of rotation around the CbAC bond
of the amino acids Phe, Dmp, Emp, Imp, 1-Nal and 2-Nal was deter-
mined using the torsion driver subroutine of SYBYL, with v1 set at
ꢀ60°. The v2 bond was rotated by 5° increments and each struc-
ture was minimized. Calculations using the standard v1 values of

2338
L. Bai et al. / Bioorg. Med. Chem. 22 (2014) 2333–2338
+60° and 180° were also performed with select amino acids and
References and notes
produced very similar results (data not shown).
1. Schiller, P. W.; Nguyen, T. M.-D.; Berezowska, I.; Dupuis, S.; Weltrowska, G.;
Chung, N. N.; Lemieux, C. Eur. J. Med. Chem. 2000, 35, 895.
2. Shimoyama, M.; Shimoyama, N.; Zhao, G. M.; Schiller, P. W.; Szeto, H. H. J.
Pharmacol. Exp. Ther. 2001, 297, 364.
4.4. In vitro bioassays and receptor binding assays
3. Szeto, H. H.; Soong, Y.; Wu, D.; Quian, X.; Zhao, G. M. J. Pharmacol. Exp. Ther.
2003, 305, 696.
4. Neilan, C. L.; Nguyen, T. M.-D.; Schiller, P. W.; Pasternak, G. W. Eur. J. Pharmacol.
2001, 419, 15.
5. Zhao, G. M.; Soong, Y.; Shimoyama, M.; Berezowska, I.; Schiller, P. W.; Szeto, H.
H. J. Pharmacol. Exp. Ther. 2002, 302, 188.
6. Szeto, H. H.; Lovelace, J. L.; Fridland, G.; Soong, Y.; Fasolo, J.; Wu, D.; Desiderio,
D. M.; Schiller, P. W. J. Pharmacol. Exp. Ther. 2001, 298, 57.
7. Schiller, P. W. Life Sci. 2010, 86, 598.
8. Schiller, P. W.; Berezowska, I.; Weltrowska, G.; Chen, H.; Lemieux, C.; Chung, N.
N. J. Pept. Res. 2005, 65, 556.
9. Zhao, K.; Zhao, G.-M.; Wu, D.; Soong, Y.; Birk, A. V.; Schiller, P. W.; Szeto, H. H. J.
Biol. Chem. 2004, 279, 34682.
10. Kim, H. K.; Park, S. K.; Zhou, J. L.; Tagliatela, G.; Chung, K.; Coggeshall, R. E.;
Chung, J. M. Pain 2004, 111, 116.
11. Shimoyama, M.; Schiller, P. W.; Shimoyama, N.; Toyama, S.; Szeto, H. H. Chem.
Biol. Drug Des. 2012, 80, 771.
12. Schiller, P. W.; DiMaio, J. Nature 1982, 297, 74.
13. Mosberg, H. I.; Hurst, R.; Hruby, V. J.; Gee, K.; Yamamura, H. I.; Galligan, J. J.;
Burks, T. F. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 5871.
14. Mosberg, H. I.; Omnaas, J. R.; Smith, C. B.; Medzihradsky, F. Life Sci. 1988, 43,
1013.
15. Hruby, V. J.; Li, G.; Haskell-Luevano, C.; Shenderovich, M. Biopolymers 1997, 43,
219.
16. Hruby, V. J.; Toth, G.; Gehrig, C. A.; Kao, L.-F.; Knapp, R.; Lui, G. K.; Yamamura,
H. I.; Kramer, T. H.; Davis, P.; Burks, T. F. J. Med. Chem. 1991, 34, 1823.
17. Mosberg, H. I.; Omnaas, J. R.; Lomize, A.; Hey, D. L.; Nordan, I.; Mousigian, C.;
Davis, P.; Porreca, F. J. Med. Chem. 1994, 37, 4384.
18. Hansen, D. W.; Stapelfeld, A.; Savage, M. A.; Reichman, M.; Hammond, D. L.;
Haaseth, R. C.; Mosberg, H. I. J. Med. Chem. 1992, 35, 684.
19. Li, T.; Tsuda, Y.; Minoura, K.; In, Y.; Ishida, T.; Lazarus, L. H.; Okada, Y. Chem.
Pharm. Bull. 2006, 54, 873.
20. Ambo, A.; Niizuma, H.; Sasaki, A.; Kohara, H.; Sasaki, Y. Bioorg. Med. Chem. Lett.
2003, 13, 1269.
21. Leung, C. S.; Leung, S. S. F.; Tirado-Rives, J.; Jorgensen, W. L. J. Med. Chem. 2012,
55, 4489.
The GPI25 and MVD26 bioassays were carried out as described
elsewhere.27,28 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.29 Opioid receptor binding studies were per-
formed as described in detail elsewhere.27 Binding affinities for
l
and d 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
j opioid receptor 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
concentration of 0.72, 0.78 and 0.80 nM. IC50 values were deter-
mined from log–dose displacement curves, and Ki values were calcu-
lated from the IC50 values by means of the equation of Cheng and
Prusoff,30 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.
Agonist potencies determined with compounds in the GPI assay
are often significantly lower than their binding affinities measured
in the
l
opioid receptor binding assay.1,31 This could be due to dif-
receptors or to some-
ferences between central and peripheral
l
what impeded receptor access of compounds in the tissue
preparation (Kosterlitz, personal communication). Furthermore,
compounds with high
l agonist potency tend to show higher ago-
nist potency in the MVD assay than is expected on the basis of their
d receptor binding affinities determined in the binding assay. This
22. Li, T.; Shiotani, K.; Miyazaki, A.; Tsuda, Y.; Ambo, A.; Sasaki, Y.; Jinsma, Y.;
Marczak, E.; Bryant, S. D.; Lazarus, L. H.; Okada, Y. J. Med. Chem. 2007, 50, 2753.
23. Zhang, L.; Chang, L.; Yu, L. L.; Liu, J. C.; Chen, J. J.; Li, X. W.; Lazarus, L. H.; Li, T.
Chin. Chem. Lett. 2011, 22, 907.
is due to activation of
l receptors that are also present in the vas
preparation, albeit at lower concentration than the predominant
d receptors.32
24. Mello, N. K.; Negus, S. S. Ann. N.Y. Acad. Sci. 2000, 909, 104.
25. Paton, W. D. M. Br. J. Pharmacol. Chemother. 1957, 12, 119.
26. Henderson, G.; Hughes, J.; Kosterlitz, H. W. Br. J. Pharmacol. 1972, 46, 764.
27. Schiller, P. W.; Lipton, A.; Horrobin, D. F.; Bodanszky, M. Biochem. Biophys. Res.
Commun. 1978, 85, 1332.
28. DiMaio, J.; Nguyen, T. M.-D.; Lemieux, C.; Schiller, P. W. J. Med. Chem. 1982, 25,
1432.
29. Waterfield, A. A.; Leslie, F. M.; Lord, J. A. H.; Ling, N.; Kosterlitz, H. W. Eur. J.
Pharmacol. 1979, 58, 11.
30. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.
31. Hruby, V. J.; Gehrig, C. A. Med. Res. Rev. 1989, 9, 343.
32. Corbett, A. D.; Paterson, S. J.; Kosterlitz, H. W. In Handbook of Experimental
Pharmacology; Herz, A., Ed.; Springer: Berlin, 1993; pp 645–679. Vol. 104/1.
Acknowledgments
The work was supported in part by grants from the U.S. National
Institutes of Health (DA004443) and the Canadian Institutes of
Health Research (MOP-89716) to P.W.S., and supported in part by
grants to T.L. from Nanjing Medical University (KY109RC01110801)
and from the Collaborative Innovation Center for Cardiovascular
Disease Translational Medicine.