Effect of 2′,6′-dimethyl-l-tyrosine (Dmt) on pharmacological activity of cyclic endomorphin-2 and morphiceptin analogs

Article abstract of DOI:10.1016/j.bmc.2011.10.040

This study reports the synthesis and biological evaluation of a series of new side-chain-to-side-chain cyclized endomorphin-2 (EM-2) and morphiceptin analogs of a general structure Tyr-c(Xaa-Phe-Phe-Yaa)NH₂ or Tyr-c(Xaa-Phe-d-Pro-Yaa)NH₂

Full text of DOI:10.1016/j.bmc.2011.10.040

Bioorganic & Medicinal Chemistry 19 (2011) 6977–6981
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effect of 20,60-dimethyl-L-tyrosine (Dmt) on pharmacological activity of cyclic
endomorphin-2 and morphiceptin analogs
Jakub Fichna a, Renata Perlikowska a, Anna Wyre˛bska a, Katarzyna Gach a, Justyna Piekielna a,
Jean Claude do-Rego b,c, Geza Toth d, Alicja Kluczyk e, Tomasz Janecki f, Anna Janecka a,
⇑
a Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland
b Institut Fédératif de Recherches Multidisciplinaires sur les Peptides (IFRMP 23), Laboratoire de Neuropsychopharmacologie Expérimentale EA 4359, Faculty of Medicine
and Pharmacy, University of Rouen, Rouen, France
c Centre National de la Recherche Scientifique (CNRS), France
d Institute of Biochemistry, Biological Research Centre of Hungarian Academy of Sciences, Szeged, Hungary
e Faculty of Chemistry, University of Wrocław, Wroclaw, Poland
f Institute of Organic Chemistry, Technical University of Lodz, Lodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
This study reports the synthesis and biological evaluation of a series of new side-chain-to-side-chain
cyclized endomorphin-2 (EM-2) and morphiceptin analogs of a general structure Tyr-c(Xaa-Phe-Phe-
Received 13 July 2011
Revised 9 October 2011
Accepted 14 October 2011
Available online 20 October 2011
Yaa)NH2 or Tyr-c(Xaa-Phe-
D
-Pro-Yaa)NH2, respectively, where Xaa and Yaa were
L
/
D
Asp or
L/D Lys. Fur-
ther modification of these analogs was achieved by introduction of 20,60-dimethyl-
L
-tyrosine (Dmt)
instead of Tyr in position 1. Peptides were synthesized by solid phase method and cleaved from the resin
by a microwave-assisted procedure.
Keywords:
Binding studies
Dmt1-substituted analogs displayed high affinity at the
l-opioid receptors, remained intact after incu-
bation with the rat brain homogenate and showed remarkable, long-lasting
l-opioid receptor-mediated
l- and d- opioid receptors
antinociceptive activity after central, but not peripheral administration.
Hot plate test
Our results demonstrate that cyclization is a promising strategy in the development of new opioid anal-
gesics, but further modifications are necessary to enhance the blood–brain barrier permeability.
Ó 2011 Published by Elsevier Ltd.
Antinociception
Solid phase peptide synthesis
1. Introduction
synthesized over the years through a variety of strategies. The
endogenous
l-opioid receptor selective endomorphins are more
Cyclization is a well recognized powerful tool in peptide chem-
istry for generating analogs with improved bioactivity. Cyclic pep-
tides, compared to linear peptides, have been considered to have
greater potential as therapeutic agents due to their increased
chemical and enzymatic stability, receptor selectivity, and im-
proved pharmacodynamic properties.1 Cyclization can be achieved
through various bridging bonds between peptide ends and/or side
chains.2 Cyclic peptides adopt conformations that are better de-
fined than those of their linear counterparts, which often exist in
a conformational equilibrium. Indeed, cyclization reduces the
molecular conformational freedom, which is responsible for the
contemporary activation of different receptors, increases metabolic
stability and may increase lipophilicity, which often improves the
blood–brain barrier (BBB) permeability of peptides.
difficult to cyclize due to their short, only tetrapeptide sequence
and the lack of the reactive side-chain groups. Therefore, to obtain
cyclic analogs of endomorphins different approaches have been
used, such as extension of a peptide chain by an additional amino
acid or/and functionalization of amino acid side-chains.
Cardillo et al. synthesized a library of cyclic analogs of endomor-
11
phin-1 (EM-1) containing a Gly5 bridge between Tyr1 and Phe4
.
From this library, the cyclopeptide c[Tyr-
duced significant antinociception in a visceral pain model.12
Purington et al. synthesized a series of cyclic pentapeptides of a
general structure Tyr-c[ -Cys-Xaa-Yaa-Cys]-NH2, cyclized via a
disulfide bridge, which displayed -opioid receptor agonist and
D-Pro-D-Trp-Phe-Gly] pro-
D
l
d-opioid receptor partial agonist/antagonist properties.13
In our earlier papers we have described the synthesis and antin-
ociceptive activity of a series of endomorphin-2 (EM-2) and mor-
phiceptin analogs obtained by cyclization through an amide bond
between the side-chain amino and carboxy groups of diamino
and dicarboxy amino acids introduced into the peptide chain into
positions 2 and 5.14,15
Cyclization has been successfully used in the field of opioid
peptides. A great number of cyclic analogs of d-selective enkepha-
lins and deltorphins3–8 and
j
-selective dynorphins9,10 have been
In this report we describe further modifications of these cyclic
peptides, in particular introduction of highly lipophilic unnatural
⇑
Corresponding author. Tel.: +48 42 679 04 50x261; fax: +48 42 678 42 77.
E-mail address: anna.janecka@umed.lodz.pl (A. Janecka).
0968-0896/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2011.10.040

6978
J. Fichna et al. / Bioorg. Med. Chem. 19 (2011) 6977–6981
amino acid 20,60-dimethyl-
L
-tyrosine (Dmt) into position 1.
2.3. Peptide characterization
2.3.1. Tyr-c( -Lys-Phe-Phe-Asp)-NH2 (2)
Replacement of Tyr in opioid peptides by Dmt paved the way for
achieving increased bioactivities. Introduction of methyl groups
into the aromatic ring of Tyr influences the conformation of the
analogs because of the increased bulkiness of the side-chain. The
methyl groups on the aromatic ring of Dmt undoubtedly play a
dominant role in the interaction within the opioid binding domain
by either direct interaction with hydrophobic side-chains of recep-
tor residues in order to align the critical OH group, or by stabiliza-
tion of a favored cis-conformer prior to and during binding.16
However, Dmt enhances affinities relative to Tyr cognates for both,
D
RT: 20.50 min. ESI-MS: calcd for C37H45N7O7 699, found [M+H]+
700. 1H NMR (DMSO-d6) d 0.75–0.81 (m, 1H), 1.01–1.09 (m, 1H),
1.21–1.34 (m, 4H), 2.34–2.44 (m, 2H), 2.78–2.82 (m, 1H), 2.92–
3.15 (m, 7H), 3.85–3.88 (m, 1H), 3.90–3.95 (m, 1H), 4.15–4.19
(m, 1H), 4.32–4.36 (m, 1H), 4.46–4.50 (m, 1H), 6.62 (d, J = 8.5,
2H), 7.01–7.29 (m, 12H), 7.64 (t, J = 5.5, 1H), 7.75 (d, J = 6.5, 1H),
7.79 (d, J = 8.5, 1H), 7.92 (d, J = 6, 1H), 7.98 (d, J = 8.5, 1H), 8.49
(br s, 2H), 9.19 (s, 1H).
l
- and d-receptors, which results in decreased selectivity of Dmt1
analogs.17–19 Here we wanted to study the impact of the incorpo-
ration of Dmt1 into the sequence of cyclic opioid peptides on their
binding affinity and antinociceptive activity.
2.3.2. Dmt-c(D-Lys-Phe-Phe-Asp)-NH2 (3)
RT: 14.59 min. ESI-MS: calcd for C39H49N7O7 728, found [M+H]+
729. 1H NMR (DMSO-d6) d 0.70–0.76 (m, 1H), 0.92–1.00 (m, 1H),
1.20–1.31 (m, 4H), 2.19 (s, 6H), 2.36–2.47 (m, 2H), 2.75–2.79 (m,
1H), 2.87–3.07 (m, 7H), 3.82–3.85 (m, 1H), 3.94–3.99 (m, 1H),
4.14–4.18 (m, 1H), 4.38–4.42 (m, 1H), 4.47–4.51 (m, 1H), 6.43 (s,
2H), 7.05–7.27 (m, 10H), 7.67 (t, J = 5.5, 1H), 7.78 (d, J = 6.5, 1H),
7.84 (d, J = 8.5, 1H), 7.88 (d, J = 6.0, 1H), 8.01 (d, J = 8.5, 1H), 8.33
(br s, 2H), 9.11 (s, 1H).
2. Material and methods
2.1. General
All reagents, unless otherwise stated, were purchased from Sig-
ma–Aldrich (Poznan, Poland). t-Butyloxycarbonyl (Boc)-protected
amino acids and p-methylbenzhydrylamine (MBHA) resin were
purchased from Bachem AG (Bubendorf, Switzerland).
Analytical and semi-preparative RP-HPLC used were Waters
Breeze (Milford, MA, USA) with Vydac C18 column (5
2.3.3. Tyr-c(D-Lys-Phe-D-Pro-Asp)-NH2 (5)
RT: 18.35 min. ESI-MS: calcd for C33H43N7O7 649, found [M+H]+
650. 1H NMR (DMSO-d6) d 0.71–1.86 (m, 10H), 2.23–2.30 (m, 1H),
2.66–2.71 (m, 1H), 2.81–3.05 (m, 3H), 3.13–3.18 (m, 2H), 3.23–3.28
(m, 1H), 3.54–3.58 (m, 1H), 3.75–3.79 (m, 1H), 4.12–4.16 (m, 1H),
4.30–4.34 (m, 1H), 4.44–4.59 (m, 3H), 6.74 (d, J = 8.5, 2H), 7.02–
7.29 (m, 8H), 7.54 (t, J = 5.0, 1H), 7.64 (d, J = 6.0, 1H), 7.73 (d,
J = 5.6, 1H), 8.42 (br s, 2H), 9.37 (s, 1H).
l
m,
4.6 ꢀ 250 mm) and Vydac C18 column (10
lm, 22 ꢀ 250 mm),
respectively. Mass spectra of peptides were recorded on Bruker
Apex Ultra 7T FT-ICR mass spectrometer with electrospray ioniza-
tion (ESI-MS; Billerica, MA, USA). 1H NMR (d) spectra were recorded
in DMSO-d6 solution on Bruker Avance II+ 700 MHz spectrometer
with TMS as an internal standard. J values are given in Hz.
2.3.4. Dmt-c(D-Lys-Phe-D-Pro-Asp)-NH2 (6)
RT: 12.69 min. ESI-MS: calcd for C35H47N7O7 678, found [M+H]+
679. 1H NMR (DMSO-d6) d 0.65–1.85 (m, 10H), 2.18 (s, 6H), 2.27–
2.31 (m, 1H), 2.66–2.70 (m, 1H), 2.79–3.05 (m, 3H), 3.13–3.19
(m, 2H), 3.23–3.28 (m, 1H), 3.55–3.59 (m, 1H), 3.76–3.81 (m,
1H), 4.12–4.16 (m, 1H), 4.31–4.36 (m, 1H), 4.44–4.58 (m, 3H),
6.44 (s, 2H), 7.08 (d, J = 7.5, 1H), 7.13–7.32 (m, 5H), 7.61 (t,
J = 5.0, 1H), 7.68 (d, J = 6.3, 1H), 7.74 (d, J = 5.6, 1H), 8.42 (br s,
2H), 9.18 (s, 1H).
2.2. Peptide synthesis
Peptides were synthesized by manual solid-phase procedure as
described before, using techniques for Boc-protected amino acids
on MBHA resin (100–200 mesh, 0.8 mM/g, Novabiochem).14
e
N -amino group of Lys and
D
-Lys was protected by 9-fluorenylm-
ethyloxycarbonyl (Fmoc), b-carboxy group of Asp and
D
-Asp by
fluorenylmethyl ester (OFm), and hydroxy group of Tyr by 2-bro-
mo-benzyloxycarbonyl (2-Br-Z). 50% trifluoroacetic acid (TFA) in
dichloromethane (DCM) was used for deprotection of Boc-groups
and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetra-
fluoroborate (TBTU) was employed to facilitate coupling. Fully
assembled Boc-protected peptides were treated with 20% piperi-
dine in dimethylformamide (DMF) to remove base-labile groups
(Fmoc and OFm), followed by cyclization (TBTU).
Simultaneous deprotection and cleavage of peptides from the re-
sin was accomplished using TFA in a microwave-assisted procedure,
described elsewhere.20 Shortly, a sample of the resin (50 mg) in a
2 ml polypropylene syringe reactor was treated with TFA/TIS/H2O
(95:2.5:2.5; 1 ml) for 15 min at room temperature. The reactor
was then transferred into the microwave synthesizer and irradiated
with gas cooling at 30 W with magnetic stirring and temperature
limit of 50 °C for 30 s at a time (total irradiation time 32.5 min). Be-
tween irradiations, the resin was cooled in an ice-water bath for
2 min. Every 10 cycles the solution was removed, the resin washed
with TFA, and a new portion of cleavage mixture added to the syr-
inge. Thecombinedfiltrates were evaporatedin a stream of nitrogen.
Crude peptides were purified by RP-HPLC using the solvent sys-
tem of 0.1% TFA in water (A)/80% acetonitrile in water containing
0.1% TFA (B) and a linear gradient of 0–100% B over 15 min. The
purity of peptides was verified by analytical RP-HPLC, employing
the solvent system of 0.1% TFA in water (A) and 80% acetonitrile
in water containing 0.1% TFA (B). A linear gradient of 0–100% of
solvent B over 25 min was used.
2.3.5. Tyr-c(D-Asp-Phe-D-Pro-Lys)-NH2 (7)
RT: 16.25 min. ESI-MS: calcd for C33H43N7O7 649, found [M+H]+
650. 1H NMR (DMSO-d6) d 1.08–1.73 (m, 10H), 2.13–2.17 (m, 1H),
2.65–2.70 (m, 1H), 2.83–3.02 (m, 4H), 3.15–3.20 (m, 1H), 3.20–3.24
(m, 1H), 3.58–3.61 (m, 1H), 3.77–3.80 (m, 1H), 4.00–4.08 (m, 2H),
4.31–4.35 (m, 1H), 4.41–4.46 (m, 1H), 4.58–4.62 (m, 1H), 6.64 (d,
J = 8.5, 2H), 6.75 (d, J = 7.7, 1H), 6.98 (d, J = 8.5, 2H), 7.18–7.27
(m, 5H), 7.56 (d, J = 5.0, 1H), 7.73 (t, J = 5.6, 1H), 8.09 (d, J = 9.0,
1H), 8.56 (br s, 2H), 9.10 (s, 1H).
2.3.6. Dmt-c(D-Asp-Phe-D-Pro-Lys)-NH2 (8)
RT: 12.44 min. ESI-MS: calcd for C35H47N7O7 678, found [M+H]+
679. 1H NMR (DMSO-d6) d 1.03–1.67 (m, 10H), 2.09–2.13 (m, 1H),
2.18 (s, 6H), 2.67–2.71 (m, 1H), 2.85–3.04 (m, 4H), 3.14–3.18 (m,
1H), 3.26 (dd, J = 6.3, J = 10.5, 1H) 3.55–3.58 (m, 1H), 3.78–3.81
(m, 1H), 4.01–4.10 (m, 2H), 4.31–4.35 (m, 1H), 4.44–4.49 (m,
1H), 4.53–4.58 (m, 1H), 6.42 (s, 2H), 6.68 (d, J = 7.7, 1H), 7.17–
7.27 (m, 5H), 7.58 (d, J = 5.0, 1H), 7.70 (t, J = 5.6, 1H), 8.11 (d,
J = 9.0, 1H), 8.44 (br s, 2H), 9.11 (s, 1H).
2.4. Opioid receptor binding assays
Receptor-binding assays were performed as described previ-
ously.15 Briefly, binding affinities for
l- and d-opioid receptors were
determined during incubation with crude membrane preparation

J. Fichna et al. / Bioorg. Med. Chem. 19 (2011) 6977–6981
6979
isolated from Wistar rat brains, in the presence of [3H]DAMGO or
[3H][Ile5,6]deltorphin-2, used as the
- and d-selective radioligands,
respectively. Non-specific binding was determined in the presence
of 1 M naloxone. Incubations were terminated by rapid filtration
through GF/B Whatman glass fiber strips, soaked in 0.5% polyethyl-
amine, using Sampling Manifold (Millipore, Billerica, MA, USA). The
filters were washed twice with ice-cold TrisHCl buffer. The bound
radioactivity was measured in Packard Tri-Carb 2100 TR liquid scin-
tillation counter (Ramsey, MN, USA) after overnight extraction of
the filters in 4 ml of Perkin Elmer Ultima Gold scintillation fluid
(Wellesley, MA, USA). Three independent experiments for each as-
say were carried out in duplicate.
hot plate and the latencies to paw licking, rearing and jumping
were measured. A cut-off time of 240 s was used to avoid tissue in-
jury. The percentage of the maximal possible effect (%MPE) was
calculated as: %MPE = (t1ꢁt0)/(t2ꢁt0) ꢀ 100, where t0–control la-
tency, t1–test latency, and t2–cut-off time. The median antinocicep-
tive dose (ED50) was calculated as described earlier.15
To determine the in vivo blood–brain barrier permeability, mor-
phiceptin and analogs (0.01 and 1 mg/kg) were injected intrave-
nously (iv) 30 min before the experiment and the hot plate test
was performed as described above.
l
l
2.8. Statistical analysis
2.5. Metabolic stability
Statistical and curve-fitting analyses were performed using
Prism 5.0 (GraphPad Software Inc.). The data are expressed as
means SEM. Differences between groups were assessed by one-
way analysis of variance (ANOVA), followed by a post-hoc multiple
comparison Student–Newman–Keuls test. Antagonist effects in the
combination experiments were analyzed using two-way analysis of
variance (ANOVA) and a post-hoc multiple comparison Student-
Newman-Keuls test was used for multiple comparisons between
groups. A probability level of 0.05 or lower was considered as statis-
tically significant.
Enzymatic degradation studies of endomorphin-2, morphicep-
tin and their cyclic analogs were performed using rat brain homog-
enate, following a protocol reported previously.15 Briefly, rat brains
were isolated, pooled and homogenized in a Polytron with 20 vol-
umes of Tris–HCl (50 mM, pH 7.4), and the homogenate was then
stored at ꢁ80 °C. The aliquots (100
incubated with 100 l of a peptide (0.5 mM) for 0, 7.5, 15, 22.5,
30 and 60 min at 37 °C in a final volume of 200 l. The reaction
was stopped at the required time by placing the tube on ice and
acidifying the content with 20 l of aqueous HCl (1 M). The ali-
ll, 10 mg protein/ml) were
l
l
l
3. Results and discussion
quots were centrifuged at 20,000g for 10 min at 4 °C. The obtained
supernatants were filtered over Millex-GV syringe filters (Milli-
pore, Billerica, MA, USA) and analyzed by RP-HPLC on a Vydac
EM-2 and morphiceptin were used as parent compounds for the
design of new cyclic pentapeptides of a general structure: Xaa-
C18 column (5
l
m, 4.6 ꢀ 250 mm), using the solvent system of
c(Yaa-Phe-Phe-Zaa)-NH2 (Xaa = Tyr or Dmt, Yaa =
and Zaa = Asp or Lys) or Xaa-c(Yaa-Phe- -Pro-Zaa)-NH2 (Xaa = Tyr
or Dmt, Yaa = -Lys or -Asp and Zaa = Asp or Lys). Peptides were
D-Lys or D-Asp
0.1% TFA in water (A) and 80% acetonitrile in water containing
0.1% TFA (B) and a linear gradient of 0–100% B over 25 min. Three
independent experiments for each assay were carried out in dupli-
cate. The rate constants of degradation (k) were obtained by a least
square linear regression analysis of logarithmic peak areas [ln(A/
AD)], where A is the amount of the remaining peptide and AD is
the initial amount of peptide, versus time courses. Degradation
half-lives (t0.5) were calculated from the rate constants as ln2/k.
D
D
D
synthesized using standard Boc/Fmoc orthogonal protection on p-
methylbenzhydrylamine (MBHA) resin. Cyclization was carried
out between the side-chain amino and carboxy groups of diamino
and dicarboxy amino acids introduced at positions 2 and 5 of the
sequence, when a protected peptide was still bound to the resin.
Cyclic peptides were cleaved from the resin by mixture of TFA/
TIS/H2O (95:2.5:2.5:1), using a microwave-assisted procedure.
The microwave-assisted cleavage of peptides was used earlier
exclusively for analytical purposes.20 Here we demonstrated that
this procedure can be successfully applied also for the cleavage
of cyclic peptides from the MBHA resin in quantities sufficient
for biological experiments. Since cleavage yields in this novel pro-
cedure were about 25% lower than those in traditional methods,
additional optimization of cleavage conditions is necessary.
2.6. Animals
Male Swiss albino mice (CD1 IFFA-CREDO/Charles River,
France), weighing 20–26 g, were used for the study. The animals
were housed at a constant temperature (22 °C) and maintained un-
der a 12-h light/dark cycle in sawdust coated plastic cages with ac-
cess to standard laboratory chow and tap water ad libitum.
Opioid receptor binding affinities of new analogs at
l- and d-
2.7. Assessment of antinociception
receptors were determined in rat brain membrane preparations
in vitro against [3H]DAMGO and [3H][Ile5,6]deltorphin-2, respec-
tively and are reported in Table 1. The affinities of the cyclic ana-
The procedures used in this study were in accordance with the
European Communities Council Directive of 24 November 1986
(86/609/EEC), and were approved by the Local Ethical Committee
for Animal Research with the following numbers: N/10-04-04-12
and N/12-04-04-14.
Peptides or vehicle, in a final volume of 10 ll, were adminis-
trated intracerebroventricularly (i.c.v.) in the left brain ventricle
of manually immobilized mice, using a Hamilton microsyringe
logs at the
l-opioid receptors were in general higher than those
of parent compounds, EM-2 and morphiceptin. The introduction
of Dmt in position 1 resulted in further significant enhancement
of the l-opioid receptor affinity, which was more pronounced for
morphiceptin analogs. Additionally, cyclic analogs acquired some
affinity at the d-opioid receptors.
The resistance of cyclic analogs against proteolytic degradation
was evaluated in vitro, using the rat brain homogenate. The calcu-
lated half-lives are summarized in Table 2. The cyclic analogs were
more resistant to enzymatic degradation than their parent com-
pounds, which are particularly vulnerable to proteolysis. Analogs
3, 6 and 8, with Dmt in position 1, remained practically intact after
the incubation with the homogenate.
The antinociceptive effect of peptides was assessed in the hot
plate test in mice (supraspinally-mediated analgesia). The i.c.v.
injection of all tested analogs produced a dose-dependent analgesic
(50 ll, Hamilton Company, Remo, NV, USA) connected to a needle
(diameter 0.5 mm). In dose–response experiments, the i.c.v. injec-
tions were performed 5 min before the beginning of the test. All
compounds were dissolved in DMSO and further diluted in saline
to desired concentration. Vehicle alone did not influence the ob-
served parameters.
The analgesic activity was assessed in the hot plate test in mice,
as described earlier.15 A transparent plastic cylinder (14 cm diam-
eter, 20 cm height) was used to confine the mouse on the heated
(55 0.5 °C) surface of the plate. The animals were placed on the

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J. Fichna et al. / Bioorg. Med. Chem. 19 (2011) 6977–6981
Table 1
Receptor binding affinities of endomorphin-2 (EM-2), morphiceptin and their cyclic analogs at
l
- and d-receptors in rat brain homogenate
No.
Sequence
IC50 SEM (nM)
la
db
d/l
1
2
3
4
5
6
7
8
Tyr-Pro-Phe-Phe-NH2 (EM-2)c
0.99 0.08
0.56 0.03
0.26 0.02
56.6 4.5
89.5 7.2
5.60 0.90
15.3 0.9
4.40 0.60
—
>1000
>1000
498
>1000
>17.7
>11.2
23.5
>65.3
13.2
—
c
Tyr-c(
Dmt-c(
Tyr-Pro-Phe-Pro-NH2 (morphiceptin)
Tyr-c( -Lys-Phe- -Pro-Asp)-NH2
Dmt-c( -Lys-Phe- -Pro-Asp)-NH2
Tyr-c( -Asp-Phe- -Pro-Lys)-NH2
Dmt-c( -Asp-Phe- -Pro-Lys)-NH2
Tyr- -Ala-Phe-Glu-Val-Val-Gly-NH2 (DLT)
D-Lys-Phe-Phe-Asp)-NH2
279 21
400 10
>1000
D-Lys-Phe-Phe-Asp)-NH2
D
D
>1000
D
D
132
9
D
D
>1000
58.3 5.0
0.080 0.004
D
D
D
All values are mean SEM of three independent experiments performed in duplicate.
a
Displacement of [3H]DAMGO.
b
Displacement of [3H][Ile5,6]deltorphin-2.
c
Data from Ref. 15.
action, significantly stronger than that of EM-2 and morphiceptin
(Fig. 1). Exceptionally strong antinociceptive effect was observed
for analogs 6 and 8, with the ED50 values lower than 0.01 lg/animal
(i.c.v.) (Table 3).
The antinociception produced by the cyclic analogs was further
investigated in the time-course experiments. The maximal analge-
sic action of analogs 6 and 8 (both 10 ng/animals, i.c.v.) was ob-
served up to 30 min after administration, compared to 5–10 min
for morphiceptin (10
lg/animal, i.c.v.) and EM-2 (3 lg/animal,
i.c.v.), and the effect lasted up to 90 min (Fig. 2).
To characterize the involvement of opioid receptors in the anal-
gesic action of analogs 6 and 8, co-administration studies with opi-
oid receptor antagonists were performed. The antinociceptive
effect of both analogs (3 ng/animal, i.c.v.) was blocked by b-funaltr-
examine (b-FNA, 1
opioid receptors (Fig. 3). d-Opioid receptor antagonist naltrindole
(NTL, 1 g/animal, i.c.v.) and -opioid receptor antagonist nor-
binaltorphimine (nor-BNI, 5 g/animal, i.c.v.) did not influence
lg/animal), showing the involvement of the l-
l
j
l
the analgesic action of analogs 6 and 8 (Fig. 3).
The hot plate test in mice was then used to examine the perme-
ability of analogs 6 and 8 through BBB after peripheral administra-
tion. However, intravenous (iv) injection of these peptides (0.01
and 1 mg/kg) did not produce any significant central effects, indi-
cating that they did not cross the BBB.
In conclusion, this is the first report on the synthesis of a new
series of cyclic analogs based on EM-2 and morphiceptin structure,
incorporating Dmt in position 1. We also demonstrated that the cyc-
lic peptides can be cleaved from the resin using the microwave-
assisted procedure.
Table 2
Degradation rates and half-lives of endomorphin-2 (EM-2), morphiceptin and their
cyclic analogs
No. Sequence
Brain homogenate
100 ꢀ k (per min) t1/2 (min)
1
2
3
4
5
6
7
8
Tyr-Pro-Trp-Phe-NH2 (EM-2)
Tyr-c( -Lys-Phe-Phe-Asp)-NH2
Dmt-c( -Lys-Phe-Phe-Asp)-NH2
4.83 0.37
0.19 0.01
0.06 0.01
14.3 1.1
363 22
>1000
16.7 2.2
117 12
>1000
D
Figure 1. Dose–response curves for the inhibition of paw licking (a), rearing (b),
and jumping (c) induced by i.c.v. injection of endomorphin-2 (EM-2), morphiceptin
and their cyclic analogs determined in the hot plate test in mice. Data are
mean SEM of 10 animals per group.
D
Tyr-Pro-Phe-Pro-NH2 (morphiceptin) 4.13 0.27
Tyr-c(
Dmt-c(
Tyr-c(
Dmt-c(
D
-Lys-Phe-
-Lys-Phe-
-Asp-Phe-
-Asp-Phe-
D
-Pro-Asp)-NH2
-Pro-Asp)-NH2
-Pro-Lys)-NH2
-Pro-Lys)-NH2
0.60 0.05
0.020 0.001
1.00 0.03
0.11 0.01
D
D
D
D
63.3 2.5
627 38
D
D
All values are mean SEM of three independent experiments performed in
duplicate.
Biological evaluation of the new analogs revealed that the cycli-
zation improves the l-opioid receptor binding affinity and signifi-
RP-HPLC elution on a Vydac C18 column (5
lm, 4.6 ꢀ 250 mm) using the solvent
cantly enhances stability against enzymatic degradation, which is
system of 0.1% TFA in water (A)/80% acetonitrile in water containing 0.1% TFA (B)
and a linear gradient of 0–100% solvent B over 25 min at flow rate of 1 ml/min.
in line withearlier observations of our group, as well as others.12,14,15

J. Fichna et al. / Bioorg. Med. Chem. 19 (2011) 6977–6981
6981
Table 3
The introduction of Dmt into position 1 increased peptide affinity at
the -opioid receptors, drastically increased stability in vitro and
antinociceptive activity, therefore produced similar effects as shown
in linear peptides.21–23 Selective opioid antagonists confirmed that
the long-lasting analgesia after central administration was medi-
ED50 values for endomorphin-2 (EM-2), morphiceptin and their cyclic analogs in the
hot plate test in mice
l
No.
Sequence
ED50 (jumping) (lg/animal)
1
2
3
4
5
6
7
8
Tyr-Pro-Phe-Phe-NH2 (EM-2)
5.48 0.61
0.05 0.01
0.020 0.003
5.11 0.42
0.020 0.002
<0.01
Tyr-c(
Dmt-c(
Tyr-Pro-Phe-Pro-NH2 (morphiceptin)
Tyr-c( -Lys-Phe- -Pro-Asp)-NH2
Dmt-c( -Lys-Phe- -Pro-Asp)-NH2
Tyr-c( -Asp-Phe- -Pro-Lys)-NH2
Dmt-c( -Asp-Phe- -Pro-Lys)-NH2
D-Lys-Phe-Phe-Asp)-NH2
ated exclusively by the l-opioid receptors.
D-Lys-Phe-Phe-Asp)-NH2
The extra-ordinary antinociceptive activity of cyclic analogs
incorporating Dmt1 confirms their potential as useful tools for
D
D
the pharmacological studies of the l-opioid receptors.
D
D
D
D
0.07 0.01
<0.01
D
D
Acknowledgments
Data are mean SEM of 10 animals per group.
This work was supported by a grant POLONIUM, a grant from
Polish Ministry of Science No. 730/N-POLONIUM/2010/0, a grant
from the Medical University of Lodz No. 503/1-156-02/503-01,
and a grant from the Centre National de la Recherche Scientifique
(CNRS, France). The authors wish to thank Jozef Cieslak for his
excellent technical assistance.
References and notes
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Figure 3. Antagonist effect of b-funaltrexamine (b-FNA, 1
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i.c.v.), on the inhibition of jumping induced by morphiceptin (10 g/animal, i.c.v.)
and analogs 6 and 8 (both 3 ng/animal) determined in the hot plate test in mice.
Data are mean SEM of 10 animals per group. ###p <0.001 for agonist + b-FNA
versus agonist alone using two-way ANOVA, followed by the Student–Newman–
Keuls test.
l
g/animal, i.c.v.), nal-
l
l
g/animal,
l
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