N-methylated cyclic Enkephalin analogues retain high opioid receptor binding affinity

Article abstract of DOI:10.1111/j.1747-0285.2009.00919.x

In an effort to improve the bioavailability of the non-selective, cyclic enkephalin analogues H-Dmt-c[d-Cys-Gly-Phe-d(or L)-Cys]NH₂ (Dmt = 2',6'-dimethyltyrosine), analogues N-methylated at the Phe⁴ and/or Cys⁵ residue were synthesized. In comparison with the non-methylated parent peptides, all mono- and N-di-methylated analogues in general retained high binding affinities at all three opioid receptors and high opioid agonist potencies in functional opioid activity assays. The results indicate that the progressive conformational restriction in these compounds upon mono- and di-N-methylation did not significantly affect the in vitro opioid activity profile. A low-energy conformer identified for the conformationally most restricted analogue of the series, H-Dmt-c[D-Cys-Gly-Phe(NMe)-L-Cys(NMe)]NH ₂ (6), showed good spatial overlap of the essential pharmacophoric moieties with those in the proposed μ receptor-bound conformation of the μ-selective opioid peptide JOM-6 [H-Tyr-c(S-Et-S)[D-Cys-Phe-D-Pen]NH ₂] (Pen = penicillamine) [Mosberg M.I. and Fowler C.B. (2002) J Peptide Res; 60:329-335], in agreement with the moderate μ selectivity determined for this compound. An analogue of 6 containing (2S)-2-methyl-3-(2,6- dimethyl-4-hydroxyphenyl)propanoic acid [(2S)-Mdp] in place of Dmt¹ was an opioid antagonist with quite high opioid receptor binding affinities and can be expected to show improved bioavailability because of its further increased lipophilicity and reduced hydrogen-bonding capacity.

Full text of DOI:10.1111/j.1747-0285.2009.00919.x

ª 2009 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2009.00919.x
Chem Biol Drug Des 2010; 75: 182–188
Research Article
N-Methylated Cyclic Enkephalin Analogues Retain
High Opioid Receptor Binding Affinity
Grazyna Weltrowska1, Irena Berezowska1,
Carole Lemieux1, Nga N. Chung1, Brian C.
Wilkes1 and Peter W. Schiller1,2,*
Abbreviations: (2S)-Mdp, (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxy-
phenyl)propanoic acid; BBB, blood–brain barrier; DAMGO, H-Tyr-D-
Ala-Gly-Phe(NMe)-Gly-ol; DIC, 1,3-diisopropylcarbodiimide; DIEA,
diisopropylethylamine; Dmt, 2¢,6¢-dimethyltyrosine; DPDPE, H-Tyr-c[D-Pen-
1Laboratory of Chemical Biology and Peptide Research, Clinical
Gly-Phe-D-Pen]OH; DSLET, H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH; GPI, guinea
Research Institute of Montreal, 110 Pine Avenue West, Montreal,
pig ileum; HBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
Quebec, Canada H2W 1R7
hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; HPLC, high perfor-
2Department of Pharmacology, Universitꢀ de Montrꢀal, Montreal,
Quebec, Canada H3C 3J7
*Corresponding author: Peter W. Schiller, schillp@ircm.qc.ca
mance liquid chromatography; JOM-13, H-Tyr-c[D-Cys-Phe-D-Pen]OH;
JOM-6, H-Tyr-c(S-Et-S)[D-Cys-Phe-D-Pen]NH2; MVD, mouse vas deferens;
Pen, penicillamine; TFA, trifluoroacetic acid; U50,488, trans-3,4-dichloro-
N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide; U69,593, (5a,
7a,8b)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneac-
etamide.
In an effort to improve the bioavailability of
the non-selective, cyclic enkephalin analogues
H-Dmt-c[D-Cys-Gly-Phe-D(or L)-Cys]NH2 (Dmt = 2¢,6¢-
dimethyltyrosine), analogues N-methylated at the
Phe4 and ⁄ or Cys5 residue were synthesized. In
comparison with the non-methylated parent pep-
tides, all mono- and N-di-methylated analogues in
general retained high binding affinities at all three
opioid receptors and high opioid agonist potencies
in functional opioid activity assays. The results
indicate that the progressive conformational
restriction in these compounds upon mono- and
di-N-methylation did not significantly affect the in
Received 29 October 2009, revised 14 November 2009 and accepted for
publication 14 November 2009
Cysteine-containing cyclic opioid peptide analogues were first
reported three decades ago. The two prototype cyclic enkephalin
analogues of this type with a C-terminal carboxamide group, H-Tyr-
c[D-Cys-Gly-Phe-D-(or L)-Cys]NH2, were independently synthesized by
two groups (1,2). Both diasteroisomers showed high l and d opioid
receptor binding affinities, high l and d opioid agonist potencies
in vitro and no l versus d receptor selectivity. Cyclic tetrapeptide
analogues derived from these compounds by deletion of the Gly resi-
due, H-Tyr-c[D-Cys-Phe-D(or L)-Cys]NH2, retained l and d opioid ago-
nist activity, albeit with lower potency when compared to the parent
cyclic pentapeptides, and the L-Cys4-analogue was l-selective (3,4).
Dicarba analogues of these cyclic penta- and tetrapeptide amides,
containing a –CH=CH– (cis and trans) or a –CH2–CH2– bond in place
of the disulfide linkage, were prepared (4,5). Both the olefinic and
the saturated dicarba pentapeptide analogues retained high l and d
receptor binding affinities and high l and d opioid agonist activity
in vitro. In comparison with their respective disulfide-containing
parent tetrapeptides, the dicarba tetrapeptides displayed comparable
or reduced l and d agonist potencies. Another interesting structural
modification of the tetrapeptide H-Tyr-c[D-Cys-Phe-D-Cys]NH2 resulted
in the compound JOM-6 (H-Tyr-c(S-Et-S)[D-Cys-Phe-D-Pen]NH2), in
which D-penicillamine (D-Pen) is substituted for D-Cys4,and the disul-
fide moiety is replaced by an ethylene dithioether (6). JOM-6 turned
out to be a potent and selective l opioid receptor ligand.
vitro opioid activity profile.
A low-energy con-
former identified for the conformationally most
restricted analogue of the series, H-Dmt-c[D-Cys-
Gly-Phe(NMe)-L-Cys(NMe)]NH2 (6), showed good
spatial overlap of the essential pharmacophoric
moieties with those in the proposed l receptor-
bound conformation of the l-selective opioid pep-
tide JOM-6 [H-Tyr-c(S-Et-S)[D-Cys-Phe-D-Pen]NH2]
(Pen = penicillamine) [Mosberg M.I. and Fowler
C.B. (2002) J Peptide Res; 60:329–335], in agree-
ment with the moderate l selectivity determined
for this compound. An analogue of 6 containing
(2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)pro-
panoic acid [(2S)-Mdp] in place of Dmt1 was an
opioid antagonist with quite high opioid receptor
binding affinities and can be expected to show
improved bioavailability because of its further
increased lipophilicity and reduced hydrogen-
bonding capacity.
Key words: N-methylation of peptides, opioid activity profiles, opi-
oid peptide analogues, opioid peptide SAR, peptide synthesis, theoreti-
cal conformational analysis of peptides
N-methylation of amino acid residues in biologically active peptides
enhances their stability against enzymatic degradation and introduces
Editor’s invited manuscript to celebrate the 4th Anniversary of Chemical Biology & Drug Design.
182

N-methylated Cyclic Enkephalin Analogues
conformational constraints in the peptide backbone, with the U
angle at the N-methylated residue limited to positive values (energy
minima at U = +60ꢀ and +150ꢀ). Importantly, N-methylated peptides
have a decreased capacity to form hydrogen bonds with water mol-
ecules and, consequently, are better able to cross biological barri-
ers. This is exemplified with the naturally occurring peptide
cyclosporine that contains multiple N-methylated amino acid resi-
dues and is orally active. In the present article, we describe ana-
logues of H-Tyr-c[D-Cys-Gly-Phe-D(or L)-Cys]NH2, in which the N-
terminal tyrosine was replaced by 2¢,6¢-dimethyltyrosine (Dmt) and
which are N-methylated at the Phe4 and ⁄ or Cys5 residue (Figure 1).
N-methylation at the 4- and 5-position residues was carried out,
because linear enkephalin analogues N-methylated at the 2- and 3-
position residues are known to have in general weak opioid activity
(7). Dmt was substituted for Tyr1 in these compounds because it
has been shown that dimethylation at the 2¢,6¢-positions of Tyr1 in
opioid peptides generally results in a significant increase in opioid
agonist potency (8). These compounds have increased conforma-
tional integrity and can be expected to show improved blood–brain
barrier (BBB) penetration. Replacement of the a-amino group of
Dmt1 in opioid peptides with a methyl group, as achieved by substi-
tution of (2S)-2-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic
acid [(2S)-Mdp], is a generally applicable structural modification for
conversion of opioid peptide agonists to antagonists (9). In an effort
to obtain an opioid antagonist with improved bioavailability, we
also prepared an N-dimethylated analogue of H-Tyr-c[D-Cys-Gly-Phe-
Cys]NH2 containing (2S)-Mdp in place of Tyr1 (Figure 1).
Cys and HF ⁄ anisole treatment for peptide cleavage. In the prepara-
tion of compounds 3–6 and 9, the linear precursor peptides were
synthesized by using a Rink amide AM resin with Na-Fmoc protec-
tion, S-tert-butyl protection of Cys or Cys(NMe), and peptide cleav-
age with 98% trifluoroacetic acid (TFA) ⁄ H2O. With all peptides,
disulfide bond-formation was carried out in solution with K3Fe(CN)6
as oxidation agent. Opioid activities of the compounds in vitro were
determined using the guinea pig ileum (GPI) and mouse vas defer-
ens (MVD) bioassays, and l-, d- and j opioid receptor binding
assays.
Methods and Materials
General methods
Precoated plates (silica gel 60 F254, 250 lm; Merck, Darmstadt,
Germany) were used for ascending TLC in the following systems (all
v ⁄ v); (I) hexane ⁄ AcOEt (3:1); (II) CHCl3 ⁄ MeOH (9:1); (III) n-BuOH ⁄ -
AcOH ⁄ H2O (4:1:1); (IV) n-BuOH ⁄ pyridine ⁄ AcOH ⁄ H2O (15:10:3:12).
Preparative reversed-phase high performance liquid chromatography
(HPLC) was performed on
a
Vydac 218-TP1022 column
(22 · 250 mm) with a linear gradient of 20–40% MeOH in 0.1%
TFA (peptides 1–8) or 30–70% MeOH in 0.1% TFA (peptide 9) over
30 min at a flow rate of 12 mL ⁄ min. Analytical reversed-phase
HPLC was performed on a Vydac 218-TP54 column (5 · 250 mm) at
a flow rate of 1.0 mL ⁄ min using the same linear gradients of
MeOH in 0.1% TFA as in the preparative HPLC. The same column
was also used for the determination of the capacity factors (K¢ val-
ues) under the same conditions. Molecular masses of the com-
pounds were determined by electrospray mass spectrometry on a
Hybrid Q-Tof mass spectrometer interfaced to a MASSLYNX 4.0 data
system (Micromass Ltd. Pointe-Claire, QC, Canada).
The linear precursor peptides of the target compounds were pre-
pared by solid-phase synthesis. In the case of compounds 1, 2, 7,
and 8, peptides were assembled on a p-methylbenzhydrylamine
resin with Na-Boc or Fmoc protection, 4-methylbenzyl protection of
Synthesis of N a-methylcysteine derivatives
Fmoc-(NMe)-Cys(StBu)-OH was synthesized using the oxazolidinone
method according to a literature procedure (10), and Fmoc-(NMe)-D-
Cys(StBu)-OH was prepared in an analogous manner, as described
in the following. Fmoc-D-Cys(StBu)-OH was cyclized with formalde-
hyde and camphorsulfonic acid in benzene to afford (R)-Fmoc-4-
((tert-butyldisulfanyl)methyl)-5-oxooxazolidine-3-carboxylate that was
purified by flash chromatography on silica gel (hexane ⁄ AcOEt) and
20
was obtained as an oil in 87% yield. TLC Rƒ 0.35 (I); ½aꢀD -70.8 (c
1, CHCl3); 1H NMR (500 MHz, CDCl3) d 7.86 (d, 2H, J = 7.0 Hz),
7.58, (d, 2H, J = 7.0 Hz), 7.42 (t, 2H, J = 7.0 Hz), 7.35 (m, 2H), 5.5–
5.2 (br, m, 2H), 4.75–4.35 (br, m, 2H), 4.3 (br, s, 1H), 4.01 (br, s, 1H),
3.55 (br, s, 0.5H), 3.25 (br, s, 0.5H), 3.0 (br, s, 0.5H), 2.7 (br, s, 0.5H),
1.29 (s, 9H); 13C NMR (125 MHz, CDCl3) d 170.8, 152.2, 143.4,
141.4, 127.9, 127.2, 124.6, 120.0, 78.4, 73.9, 67.6, 55.3, 48.2, 47.2,
29.5; [high resolution mass spectrometry (HRMS) (ESI)] m ⁄ e calcd
for C23H26NO4S2 [M+H]+ 444.1303, obsd 444.1301.
Acid cleavage of the oxazolidinone with triethylsilane ⁄ TFA at room
temperature for 16 h and purification by flash chromatography on
silica gel (CHCl3 ⁄ MeOH) afforded Fmoc-(NMe)-D-Cys(StBu)-OH as a
white solid in 92% yield and in a 2.3:1.0 conformer ratio. TLC Rƒ
20
Figure 1: Structural formulas of N-methylated cyclic enkephalin
analogues.
0.40 (II); ½aꢀD +95 (c 1, CHCl3); 1H NMR (500 MHz, CDCl3) d Major:
10.0 (br, s, 1H), 7.79 (m, 2H), 7.63 (m, 2H), 7.43 (m, 2H), 7.35 (m,
Chem Biol Drug Des 2010; 75: 182–188
183

Weltrowska et al.
2H), 4.78 (d, 1H, J = 8.0 Hz), 4.58 (m, 2H), 4.32 (br, t, 1H), 3.38 (d,
1H, J = 12.0 Hz), 3.20 (d, 1H, J = 12.0 Hz), 3.07 (s, 3H), 1.37 (s,
9H); Minor: 10.0 (br, s, 1H), 7.75 (m, 2H), 7.60 (m, 2H), 7.40 (m, 2H),
7.31 (m, 2H), 4.72 (m, 1H), 4.53 (m, 1H), 4.26 (br, t, 1H), 3.10 (m,
0.5H), 2.97 (s, 3H), 2.73 (m, 0.5H), 1.33 (s, 9H); 13C NMR (125 MHz,
CDCl3) d Major: 175.6, 157.0, 144.0, 141.6, 127.5, 125.4, 68.4, 60.3,
47.4, 45.0, 39.2, 34.4, 30.2; Minor: 175.6, 157.0, 144.1, 141.6,
128.0, 125.4, 68.0, 59.1, 48.5, 45.0, 39.5, 33.5, 30.2; HRMS (ESI)
m ⁄ e calcd for C23H28NO4S2 [M+H]+ 446.1460, obsd 446.1460.
Table 1: Analytical parameters of N-methylated peptides
Compound
Rf (III)
Rƒ (IV)
K ¢a
ES ⁄ (M+H)+ (m ⁄ E)
1
2
3
4
5
6
7
8
9
0.43
0.39
0.50
0.56
0.55
0.53
0.47
0.44
0.83
0.76
0.75
0.81
0.80
0.81
0.80
0.75
0.80
0.89
4.03
3.05
3.40
2.62
4.33
3.66
3.20
3.60
6.08b
631
631
631
631
645
645
617
617
644
Peptide synthesis
High performance liquid chromatography (HPLC) conditions:
The linear precursor peptides of compounds 1, 2, 7, and 8 were
prepared by the manual solid-phase technique using Fmoc protec-
tion for the a-amino group of Dmt, Gly and Phe(NMe), and Boc pro-
tection for the a-amino group of L- and D-Cys(4-MeBzl). Peptides
were assembled on a p-methylbenzhydrylamine resin (Bachem
Americas, Torrance, CA, USA) using 1,3-diisopropylcarbodiimide ⁄ 1-
hydroxybenzotriazole as coupling agents according to a published
protocol (9). Protected amino acids were purchased from Bachem or
from RSP Amino Acids, Shirley, MA, USA. Peptides were cleaved
from the resin and completely deprotected by treatment with HF for
60 min at 0 ꢀC (10 mL of HF plus 1 mL of anisole ⁄ g resin). After
evaporation of the HF, the resin was extracted three times with
Et2O and, subsequently, three times with glacial AcOH. The pep-
tides were obtained in solid form through lyophylization of the ace-
tic acid extract. The linear precursor peptides of cyclic peptides 3,
4, 5, 6, and 9 were assembled on a Rink amide AM resin
(0.62 mmol ⁄ g) using Na-Fmoc protection according to the standard
Fmoc protocol. 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU) in the presence of diisopropylethyl-
amine was used as coupling agent, and double couplings between
Cys(NMe) and Phe [or Phe(NMe)] and between Phe(NMe) and Gly
were performed. Fmoc deprotection was carried out with 30%
piperidine in N, N-dimethyformamide (DMF), and the StBu protect-
ing group was removed by treatment with a mixture of 20% b-mer-
captoethanol in DMF added to N-methylmorpholine (final
concentration of b-mercaptoethanol = 0.1 M). Peptides were cleaved
from the resin by treatment with 98% TFA ⁄ H2O in the usual man-
ner. After evaporation, treatment with ethylether provided the pep-
tides in solid form. For disulfide bond-formation, a solution
containing K3Fe(CN)6 in 0.05 M ammonium acetate was prepared
with a fourfold excess of K3Fe(CN)6 over the peptide to be oxidized.
Peptides dissolved in MeOH were added to this solution at a rate
of 8 mg ⁄ h ⁄ L of oxidation solution. All cyclic peptides were purified
by preparative reversed-phase HPLC and were found to be at least
98% pure, as assessed by HPLC and TLC. Molecular weights were
confirmed by mass spectrometry. Analytical parameters are listed in
Table 1.
a20–40% MeOH ⁄ 0.1% trifluoroacetic acid (TFA)-H2O, linear gradient over
30 min at a flow rate of 1 mL ⁄ min.
b30–70% MeOH ⁄ 0.1% TFA-H2O, linear gradient over 30 min at a flow rate
of 1 mL ⁄ min.
binding affinities were measured by displacement of [3H]U69,593
(Amersham, Bioscience, Saint-Lourent, QC, Canada) 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 deter-
mined from log-dose displacement curves, and Ki values were cal-
culated from the obtained IC50 values by means of the equation of
Cheng and Prusoff (12), 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. The GPI (13) and MVD (14) bioassays were carried out
as reported in detail elsewhere (11,15). 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 (16). Ke values
for antagonists were determined from the ratio of IC50 values
obtained with an agonist in the presence and absence of a fixed
antagonist concentration (17). l and j antagonist Ke values of com-
pounds were determined against the l agonist TAPP (H-Tyr-D-Ala-
Phe-Phe-NH2) (18) and the j agonist U50,488, respectively, and d
antagonist Ke values were measured in the MVD assay against the
d agonist H-Tyr-c[D-Pen-Gly-Phe-D-Pen]OH (DPDPE).
Theoretical conformational analysis
All calculations were performed using the molecular modeling soft-
ware SYBYL, version 7.0 (Tripos Associates, St. Louis, MO, USA). The
standard SYBYL force field was used for energy calculations, and a
dielectric constant of 78 was chosen to simulate an aqueous envi-
ronment. A stepwise approach was used to determine low-energy
conformations of the cyclic peptides (19). For each peptide, the
'bare' ring structure consisting of only the atoms directly attached
to the ring, along with associated hydrogen atoms, was first con-
structed. After minimization, a systematic conformational grid
search was carried out to identify low-energy ring structures. Each
rotatable bound was rotated in 30ꢀ increments over all space. An
allowed conformation was obtained if in a structure without unfa-
vorable vdw contacts the ring could close within 0.4 ꢀ of a normal
bond. Each allowed ring structure was minimized, and structures
within 3.0 kcal ⁄ mol of the lowest-energy ring structure were
retained for further study. To each low-energy ring structure, the
Opioid receptor binding assays and in vitro
bioassays
Opioid receptor binding studies were performed as described in
detail elsewhere (11). Binding affinities for l and d receptors were
determined by displacing, respectively, [3H]DAMGO (Multiple Peptide
Systems, San Diego, CA, USA) and [3H]DSLET (Multiple Peptide Sys-
tems) from rat brain membrane binding sites, and j opioid receptor
184
Chem Biol Drug Des 2010; 75: 182–188

N-methylated Cyclic Enkephalin Analogues
exocyclic Dmt residue and the phenylalanine side chain were
attached, and a second systematic grid search was performed on
the exocyclic rotatable bonds. Energies were calculated, and the
resulting conformations were ranked in order of increasing energy.
l Receptor-bound conformations were identified by spatial overlap
with the proposed bioactive conformation of the cyclic l opioid
peptide agonist JOM-6 (H-Tyr-c(S-Et-S)[D-Cys-Phe-D-Pen]NH2) (20).
The N-terminal amino group and the two aromatic rings of the pep-
tide studied were superimposed on the corresponding pharmaco-
phoric moieties in JOM-6.
subnanomolar l receptor binding affinity and subnanomolar or low
nanomolar d and j receptor binding affinities, with compounds 1
and 2 showing moderate preference for l and j receptors over d
receptors. The two N-dimethylated analogues (compounds 5 and 6)
also displayed subnanomolar l receptor binding affinities, very high
j receptor binding affinities, and somewhat lower d receptor bind-
ing affinities. Consequently, these two compounds showed modest
l versus d selectivity.
In comparison with the two parent peptides (7 and 8), all N-mono-
and N-dimethylated cyclic peptides also turned out to be full
agonists in the GPI assay (l receptor-representative) and in the
MVD assay (d receptor-representative) with subnanomolar or very
low nanomolar potencies in both assays (Table 3). In general, there
is good agreement between the receptor affinities measured in the
binding assays and the agonist potencies determined in the func-
tional GPI and MVD assays, but some minor quantitative discrepan-
cies are noticed. Such quantitative discrepancies have often been
observed and could be because of possible differences in the struc-
tural requirements between central and peripheral receptors or of
differences among the compounds studied with regard to their abil-
ity to access the receptors in the isolated tissue preparations.
Results
The two parent agonist peptides H-Dmt-c[D-Cys-Gly-Phe-D-Cys]NH2
(7) and H-Dmt-c[D-Cys-Gly-Phe-L-Cys]NH2 (8) showed subnanomolar
l-, d- and j receptor binding affinities and essentially no selectivity
for any of the three opioid receptor types (Table 2). Monomethyla-
tion at the Phe4 residue (compounds 1 and 2) or at the D- or L-Cys5
residue (compounds 3 and 4) resulted in compounds that retained
Table 2: Opioid receptor binding data of N-methylated cyclic
enkephalin analogues
Compound 9, the (2S)-Mdp1 analogue of cyclic peptide 6, showed
quite high l receptor binding affinity (Kli = 14.4 € 1.0 nM) and
about twofold lower d and j receptor binding affinities (Table 2).
Ki (nM)a
Ki ratio
Compound
lb
db
2.29 € 0.09
2.36 € 0.48
0.525 € 0.059
0.776 € 0.050
6.07 € 0.39
jc
l ⁄ d ⁄ j
Table 4: Number of low-energy conformers of the 'bare' ring
structures of compounds 1–8
1
2
3
4
5
6
7
8
9
0.496 € 0.037
0.354 € 0.038
0.504 € 0.039
0.586 € 0.011
0.876 € 0.059
0.641 € 0.010
0.412 € 0.035
0.282 € 0.041
14.4 € 1.0
0.447 € 0.070
0.855 € 0.087
1.01 € 0.06
0.894 € 0.126
1.42 € 0.16
0.875 € 0.015
0.602 € 0.152
0.677 € 0.055
29.5 € 1.4
1 ⁄ 5 ⁄ 1
1 ⁄ 7 ⁄ 2
1 ⁄ 1 ⁄ 2
1 ⁄ 1 ⁄ 2
1 ⁄ 7 ⁄ 2
1 ⁄ 3 ⁄ 1
1 ⁄ 1 ⁄ 1
1 ⁄ 1 ⁄ 2
1 ⁄ 2 ⁄ 2
Ring structure
Number of low-energy ringsa
H-c[D-Cys-Gly-Ala(NMe)-D-Cys]NH2
H-c[D-Cys-Gly-Ala(NMe)-L-Cys]NH2
H-c[D-Cys-Gly-Ala-D-Cys(NMe)]NH2
H-c[D-Cys-Gly-Ala-L-Cys(NMe)]NH2
H-c[D-Cys-Gly-Ala(NMe)-D-Cys(NMe)]NH2
H-c[D-Cys-Gly-Ala(NMe)-L-Cys(NMe)]NH2
H-c[D-Cys-Gly-Ala-D-Cys]NH2
28
28
28
16
9
4
69
109
1.79 € 0.03
0.202 € 0.005
0.306 € 0.011
35.9 € 3.5
aValues represent means of 3-6 determinations d SEM.
bDisplacement of [3H]DAMGO (l-selective) and [3H]DSLET (d-selective) from
rat brain membrane binding sites.
H-c[D-Cys-Gly-Ala-L-Cys]NH2
cDisplacement of [3H]U69,593 (j-selective) from guinea pig brain membrane
binding sites.
aNumbers of low-energy conformers within 3 kcal ⁄ mol of the lowest-energy
conformation.
Table 3: Guinea pig ileum (GPI)
and mouse vas deferens (MVD)
GPI
MVD
assays of N-methylated cyclic
Compound
IC50 (nM)
Kle (nM)b
Kje (nM)c
IC50 (nM)
Kde (nM)d
enkephalin analoguesa
1
2
3
4
5
6
7
8
9
1.07 € 0.19
0.457 € 0.029
1.81 € 0.36
1.36 € 0.28
1.34 € 0.24
0.394 € 0.065
0.586 € 0.211
0.812 € 0.046
1.11 € 0.13
0.884 € 0.110
0.352 € 0.020
0.122 € 0.016
3.72 € 1.35
1.95 € 0.20
0.0530 € 0.0153
0.115 € 0.005
71.0 € 7.3
151 € 16
277 € 40
aValues represent means of 3–6 determinations € SEM.
bDetermined against TAPP (H-Tyr-D-Ala-Phe-Phe-NH2).
d
cDetermined against U50,488. Determined against DPDPE.
Chem Biol Drug Des 2010; 75: 182–188
185

Weltrowska et al.
Figure 2: Spatial overlap of the lowest-energy conformation of
H-c[D-Cys-Gly-Ala(NMe)-L-Cys(NMe)]NH2 (depicted in solid lines) with
the five lowest-energy conformers of H-c[D-Cys-Gly-Ala-L-Cys]NH2
(depicted in light lines) (two views).
Figure 3: Spatial overlap of low-energy conformer of H-Dmt-
c[D-Cys-Gly-Phe(NMe)-L-Cys(NMe)]NH2 (6, red, with N-methyl
groups in magenta) with the proposed model of the l-selective
As expected, peptide 9 showed l opioid antagonist activity in the
GPI assay with a Ke value of 71.0 € 7.3 nM (Table 3). It also dis-
played j and d opioid antagonist properties with respective Ke val-
ues of 151 € 16 nM and 277 € 40 nM.
peptide JOM-6 (H-Tyr-c(S-Et-S)[D-Cys-Phe-D-Pen]NH2) in the
receptor-bound conformation (green) (20) (two views).
l
corresponding distance in the proposed d receptor-bound conforma-
tion of the d receptor-selective d agonist JOM-13 (H-Tyr-c[D-Cys-
Phe-D-Pen]OH (6,20)) (data not shown). These results may explain
the modest l vs. d receptor selectivity of compound 6.
The numbers of low-energy conformers within 3 kcal ⁄ mol of the
lowest-energy conformation obtained for the 'bare' ring structures
of cyclic peptides 1–8 in the theoretical conformational analysis
(systematic grid search and energy minimization) are listed in
Table 4. The results indicate that the L-Cys(NMe)-containing rings
are structurally more rigid than the corresponding D-Cys(NMe)-
containing ones, as a consequence of a steric clash between the
N-methyl group of L-Cys(NMe)5 and the C-terminal carboxamide
group. The lowest-energy conformers of the ring structures in the
eight compounds all contain all-trans peptide bonds. It is evident
that N-mono- and dimethylation of the 14-membered ring structures
produced a progressive decrease in conformational flexibility. The
structurally most rigid ring structure is the one contained in cyclic
peptide 6, for which only four low-energy conformers were
obtained. As depicted in Figure 2, the lowest-energy conformer of
the latter ring structure showed considerable similarity with the five
lowest-energy conformers of the ring structure contained in com-
pound 8 (H-c[D-Cys-Gly-Ala-Cys]NH2), indicating that N-methylation
at the Ala and L-Cys residues did not significantly alter the overall
low-energy ring conformation. Furthermore, the two N-methyl
groups are oriented perpendicular to the peptide ring structure.
After addition of the exocyclic Dmt1 residue and the Phe4 side
chain to the bare ring structures and subsequent energy minimiza-
tion, the resulting low-energy conformers of the moderately l
receptor-selective cyclic peptide 6 were superimposed on the
proposed model of the l receptor-bound conformation of the
l-selective cyclic opioid peptide JOM-6 (H-Tyr-c(S-Et-S)[D-Cys-Phe-D-
Pen]NH2 (20) (Figure 3). Excellent spatial overlap was observed
between the important pharmacophoric moieties (N-terminal amino
group, Dmt ⁄ Tyr side chain, Phe side chain) in JOM-6 and in the 3rd
lowest-energy conformer of 6, which is only 1.32 kcal ⁄ mol higher
in energy than the lowest-energy conformer. The root mean square
deviation (RMSD) value for this overlap is 0.70 ꢀ. Several conform-
ers of 6 with somewhat higher energy showed a shorter intramo-
lecular distance between the two aromatic rings, similar to the
Discussion and Conclusions
In comparison with parent peptides 7 and 8, all mono- and di-N-
methylated cyclic Dmt1-peptides retained similarly high l and j
receptor binding affinities and in the case of the mono-N-methylat-
ed Cys(NMe)5-analogues (compounds 3 and 4) similarly high d
receptor binding affinity. Compounds that are N-methylated at the
Phe4 residue (1,2) or at both the Phe4 and the D(or L)-Cys5 residue
(5,6) showed somewhat lower d receptor binding affinities and
moderate l versus d receptor selectivity. In agreement with the
receptor binding data, the N-methylated Dmt1-analogues also
showed high opioid agonist potencies in the GPI and MVD bioas-
says, comparable to the activities seen with the non-methylated
parent peptides. These results indicate that the presence of the N-
methyl groups per se at the 4- and 5-position residues and the pro-
gressive conformational restriction resulting from N-methylation at
one or the other, or at both these residues do not have a major
effect on the in vitro opioid activity profile. The conformationally
most constrained peptide of this series is the moderately l recep-
tor-selective compound 6, a low-energy conformer of which showed
good spatial overlap with the proposed l receptor-bound conforma-
tion of the l-selective cyclic opioid peptide JOM-6 (20). In contrast
to the N-methylated cyclic enkephalin analogues described here,
dimethylation of the b-carbons of the D-Cys2 and D-Cys5 residues in
the cyclic enkephalin analogue H-Tyr-c[D-Cys-Gly-Phe-D-Cys]OH had a
significant effect on opioid receptor binding affinity and selectivity
(21). The resulting compound, H-Tyr-c[D-Pen-Gly-Phe-D-Pen]OH
(DPDPE; Pen = penicillamine), showed somewhat lower d receptor
binding affinity but greatly increased d receptor selectivity. In this
186
Chem Biol Drug Des 2010; 75: 182–188

N-methylated Cyclic Enkephalin Analogues
case, the altered opioid activity profile is not because of a signifi-
cant change in the topography of the molecule but rather because
of steric interference caused by the b-methyl groups of the D-Pen2
residue (22). Replacement of the disulfide moiety in the cyclic opi-
oid peptides H-Tyr-c [D-Cys-Gly-Phe-D(or L)-Cys]NH2 with a –CH=CH–
(cis or trans) or a –CH2–CH2– linkage resulted in compounds that
also retained high opioid activity but showed considerable differ-
ences in the low-energy conformations of their 14-membered ring
structures among them and in comparison with the disulfide-con-
taining parent peptide (5). Taken together, the results obtained with
these various cyclic pentapeptide enkephalin analogues indicate
that significant variation in the conformation and structural flexibil-
ity of the 14-membered ring structure is tolerated and that the ring
component mainly served as a template for the proper spatial posi-
tioning of the exocyclic Tyr1 or Dmt1 residue and the Phe4 side
chain.
analogues containing a phenylalanine residue in the 3-position.
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4. Berezowska I., Chung N.N., Lemieux C., Wilkes B.C., Schiller
P.W. (2006) Cyclic dermorphin tetrapeptide analogues obtained
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Cyclic disulfide and dithioether-containing opioid tetrapeptides:
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N-methylation of three amino acid residues in the cyclic hexapeptide
aIIbb3 integrin receptor antagonist c[-Gly-Arg-Gly-Asp-D-Phe-Leu-]
resulted in a compound which showed somewhat reduced receptor
binding affinity but improved receptor selectivity (23). In this case, the
selectivity enhancement was because of the reduced flexibility of the
peptide. N-methylation at three amino acid residues of the somato-
statin-derived hexapeptide c[-Pro-Phe-D-Trp-Lys-Thr-Phe-] somewhat
reduced binding affinity for the hsst2 and hsst5 somatostatin recep-
tors but, importantly, the resulting compound was found to be orally
active (24). A linear dermorphin-derived tetrapeptide analogue con-
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retained quite high opioid agonist activity in vitro with a l receptor
binding affinity 30- to 80-fold lower than those of the N-methylated
cyclic peptides described here and produced a centrally mediated
analgesic effect after i.v. administration (25). The cyclic enkephalin
analogues N-methylated at the 4- and 5-position residues described
here (compounds 5 and 6) can be expected to have enhanced ability
to cross the BBB when compared to their non-methylated parents.
The (2S)-Mdp1-containing antagonist 9 may show even further
improved bioavailability because it contains a methyl group in place
of the N-terminal amino group and, thus, has further enhanced lipo-
philicity and reduced hydrogen-bonding capacity.
8. Hansen D.W. Jr, Stapelfeld A., Savage M.A., Reichman M.,
Hammond D.L., Haaseth R.C., Mosberg H.I. (1992) Systemic anal-
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C., Chung N.N., Schiller P.W. (2001) [2¢,6¢-Dimethyltyrosine]dynor-
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potent and selective
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This work was supported by grants from the National Institute on
Drug Abuse, NIH (DA004443) and the Canadian Institutes of Health
Research (MOP-89716).
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