Antinociceptive and antidepressant-like action of endomorphin-2 analogs with proline surrogates in position 2

Article abstract of DOI:10.1016/j.bmc.2014.06.056

In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity, two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH₂) analogs, containing proline surrogates in position 2 were synthesized using commercially

Full text of DOI:10.1016/j.bmc.2014.06.056

Bioorganic & Medicinal Chemistry 22 (2014) 4803–4809
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antinociceptive and antidepressant-like action of endomorphin-2
analogs with proline surrogates in position 2
Renata Perlikowska a, Justyna Piekielna a, Marzena Mazur b, Robert Koralewski b, Jacek Olczak b,
Jean-Claude do Rego c, Jakub Fichna a, Jakub Modranka d, Tomasz Janecki d, Anna Janecka a,
⇑
a Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland
b TriMen Chemicals Ltd, 92-318 Lodz, Poland
c Service Commun d’Analyse Comportementale (SCAC), IRIB, Faculté de Médecine et Pharmacie, Université de Rouen, 76183 Rouen Cedex, France
d Institute of Organic Chemistry, Lodz University of Technology, 90-924 Lodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity,
two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2) analogs, containing proline surrogates in posi-
tion 2 were synthesized using commercially available racemic trans-4-phenylpyrrolidine-3-carboxylic
acid (4-Ph-b-Pro). The obtained mixture of two diastereoisomeric peptides (2a and 2b) was separated
by HPLC and both enantiopure analogs were used in the in vitro and in vivo studies. To assign the abso-
lute configuration to the 4-Ph-b-Pro residues in both peptides, the stereoselective synthesis of (3R,4S)-4-
phenylpyrrolidine-3-carboxylic acid was performed and this enantiomer was introduced into position 2
of EM-2 sequence. Based on the HPLC retention times we were able to assign the absolute configuration
of 4-Ph-b-Pro residues in both peptide analogs. Analog 2a incorporating (3R,4S)-4-Ph-b-Pro residue pro-
duced strong analgesia in mice after intracerebroventricular (icv) administration which was antagonized
Received 25 April 2014
Revised 23 June 2014
Accepted 30 June 2014
Available online 9 July 2014
Keywords:
Unnatural amino acids
Opioid receptors
Binding studies
Hot-plate test
by the
l-opioid receptor (MOR) antagonist, b-funaltrexamine (b-FNA). This analog also influenced an
Forced-swimming test
Tail-suspension test
Locomotor activity
emotion-related behavior of mice, decreasing immobility time in the forced swimming and tail suspen-
sion tests, without affecting locomotor activity. The antidepressant-like effect was reversed by the
d-selective antagonist, naltrindole (NLT) and
iments with selective opioid receptor antagonists revealed that analgesic action of analog 2a was medi-
ated through the MOR, while the d- and -receptors (DOR and KOR, respectively) were engaged in the
j-selective nor-binaltorphimine (nor-BNI). Thus, the exper-
j
antidepressant-like activity. Analog 2b with (3S,4R)-4-Ph-b-Pro in position 2 showed no antinociceptive
or antidepressant-like activity in animal studies.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Wang.5 Simple replacement of Pro by
in a drastic loss of affinity, indicating that the
D
-Pro in both EMs resulted
-configuration of
L
Two highly selective endogenous
l
-opioid receptor (MOR)
Pro must be vital for MOR activation.6,7 Introduction of b-(R)-Pro,
but not b-(S)-Pro residue into position 2 of EM-1 produced two
orders of magnitude increased affinity for the MOR.8,9 Incorporation
of (R)-piperidine-3-carboxylic acid [(R)-Nip], a six-membered sur-
rogate of Pro, into the structure of EM-2 produced a significant
increase in the MOR affinity.10,11 Giordano et al.12 designed a series
of EM-2 analogs with R and S isomers of b-homoproline, 2-pyrrolid-
inemethanesulphonic acid (HPrs) or 3-pyrrolidinesulphonic acid
(bPrs) instead of Pro. Among them, the highest MOR affinity (only
2-fold lower comparing to EM-2) was produced by a b-sulfonamido
analog, [(S)-bPrs2]EM-2. In the study of Borics et al.,13 replacement
of Pro2 in the sequence of EM-2 by 2-aminocyclopentene- or cyclo-
hexene-1-carboxylic acid residues produced analogs with similar to
the parent compound MOR affinity.
ligands, endomorphin-1 (EM-1, Tyr-Pro-Trp-Phe-NH2) and endo-
morphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH2)1 differ structurally from
the so called ‘typical’ opioids (enkephalins, dynorphins, endor-
phins) in their only tetrapeptide length, C-terminal amidation
and the Pro residue in the second position. NMR spectroscopy
and molecular modeling studies indicate that Pro induces the other
residues to assume the proper spatial orientation for the ligand–
receptor interaction and plays in EMs a role of a stereochemical
spacer.2–4
In order to define structural requirements for position 2, various
chemical modifications were evaluated, as reviewed by Liu and
⇑
Corresponding author. Tel.: +48 42 272 57 06; fax: +48 42 272 56 94.
E-mail address: anna.janecka@umed.lodz.pl (A. Janecka).
http://dx.doi.org/10.1016/j.bmc.2014.06.056
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

4804
R. Perlikowska et al. / Bioorg. Med. Chem. 22 (2014) 4803–4809
EMs and their receptors are present in brain regions known to
Chemicals Ltd, Lodz, Poland), as a Pro surrogate for the synthesis
of EM-2 analogs. The obtained mixture of two diastereoisomeric
peptides (2a and 2b) was separated by HPLC and both enantiopure
analogs were used in the in vitro and in vivo studies. To assign the
absolute configuration to the 4-Ph-b-Pro residues in both peptides
we performed the stereoselective synthesis of (3R,4S)-4-phenyl-
pyrrolidine-3-carboxylic acid and introduced this enantiomer into
position 2 of EM-2 sequence. Based on the HPLC retention times we
were able to assign the absolute configuration of 4-Ph-b-Pro resi-
dues in both analogs.
Synthesis of N-Fmoc (3R,4S)-4-phenylpyrrolidine-3-carboxylic
acid 5 was accomplished as shown in Scheme 1. Starting dimethyl
[(1S)-2-nitro-1-phenylethyl]malonate (1) was obtained according
to the literature procedure32 by conjugate addition of dimethyl
malonate to (E)-2-nitrostyrene with the use of de-methyl-quinine
as a chiral catalyst. The subsequent reduction of the nitro group
with concomitant intramolecular ring closure provided exclusively,
thermodynamically more stable, methyl (3R,4S)-2-oxo-4-phenyl-
pyrrolidine-3-carboxylate (2) with trans arrangement of the phenyl
and methoxycarbonyl groups. This arrangement was confirmed by
a characteristic JH3H4 coupling constant (9.6 Hz) in an 1H NMR spec-
trum of 2. This value is in full agreement with JH3H4 coupling
constants observed in racemic N-substituted methyl trans-2-oxo-
4-phenylpyrrolidine-3-carboxylates (JH3H4 = 9–10 Hz).33 Treatment
be involved in mood disorders14–17 in the proximity of monoamine
neurotransmitters (serotonin, dopamine, and noradrenaline),
which play a key role in the pathophysiology of depression. In fact,
EMs have been shown to modulate serotoninergic,18–20 dopami-
nergic,18,21–23 and noradrenergic18,20,24 transmissions.
In most of the behavioral models of depression animals are
exposed to mildly aversive situations from which there is no pos-
sibility to escape and which induce recognizable behavioral
changes. In the forced-swimming (FST) and tail-suspension (TST)
tests, which are quite sensitive and relatively specific, a prolonged
exposure to aversive situations induces immobility, interpreted as
an expression of despair which could be related to the depression
syndrome.25,26
There are many pharmacological observations suggesting the
implication of the opioid system in the pathogenesis of depression
and in the mechanism of antidepressant action.27,28 Traditional
antidepressants, which do not bind to the opioid receptors, could
cause an indirect modulation of opioid neurotransmission.27 There
is evidence, that the effects of tricyclic antidepressants (TCAs),
which for a long time were the first choice for pharmacological
treatment of clinical depression, is antagonized by the blockade
of the opioid receptors, indicating the possible participation of opi-
oid neurotransmission in the antidepressant activity of these
drugs.29 Moreover, a common pathway in the analgesic effect of
both TCAs and opioids has been described. Valvedere et al.30 sug-
gested that TCAs produce antinociception partly via the participa-
tion of the endogenous opioid system and partly by activation of
noradrenergic, serotonergic or dopaminergic pathways.
Current treatments of depression either fail to produce com-
plete recovery or induce unwanted side-effect.31 Therefore, it is
desirable to seek new antidepressant-like drug candidates.
In this study we investigated the antinociceptive and antide-
pressant-like action of new EM-2 analogs containing a modified
b-Pro residue, that is, 4-phenylpyrrolidine-3-carboxilic acid
(4-Ph-b-Pro). This synthetic amino acid combines the conforma-
tional rigidity of the pyrrolidine ring of the native Pro with the
presence of a nonpolar phenyl ring, thus realizing the increased
lipophilicity requirement for better bioavailability of a peptide.
The influence of the introduced chemical modification on enzy-
matic stability, receptor binding, and in vivo activities are reported.
of pyrrolidine-3-carboxylate
2 with lithium diisopropylamide
(LDA) resulted in reduction of both, amide and ester functionality,
to give (3R,4S)-3-hydroxymethyl-4-phenylpyrrolidine (3). Finally,
standard Fmoc protection of the amine functionality and subse-
quent oxidation of alcohol 4 yielded desired Fmoc-protected
(3R,4S)-4-phenylpyrrolidine-3-carboxylic acid 5, suitable for the
solid phase peptide synthesis on the mild acid-labile support (e.g.,
Rink resin).
2.2. Peptide synthesis
Incorporation of the optically pure (3R,4S)-4-phenylpyrrolidine-
3-carboxylic acid into the peptide sequence delivered peptide 2a,
whereas the use of the commercial racemic Fmoc-trans-4-phenyl-
pyrrolidine-3-carboxylic acid provided two diastereomeric pep-
tides that were resolved using preparative HPLC. Of these two
peptides, one was identical (retention time, MS spectrum) to
peptide 2a and therefore the other one, peptide 2b, contained
4-phenylpyrrolidine-3-carboxylic acid residue of (3S,4R) configura-
tion. 1H NMR spectra of both peptides revealed that they exist as a
E/Z mixtures of conformational isomers due to restricted rotation
around the Tyr-(4-Ph-b-Pro) amide bond. The ratio of isomers
was 60/40 and 80/20 for 2a and 2b, respectively.
2. Results and discussion
2.1. Chemistry
4-Phenylpyrrolidine-3-carboxylic acid has two stereogenic
centers and four possible stereoisomers. We used commercially
available racemic (3R,4S)- and (3S,4R)-4-phenylpyrrolidine-3-car-
boxylic acid (4-Ph-b-Pro) (Fig. 1), supplied by Dr. Olczak (TriMen
2.3. In vitro studies
The ability of the new EM-2 analogs to bind to opioid receptors
was measured by displacement of [3H]DAMGO and [3H][Ile5,6]del-
torphin-2 from MOR and d-opioid receptor (DOR) in rat brain
membranes, and [3H]nor-BNI from
j-opioid receptor (KOR) in gui-
nea pig brain membranes, respectively. The binding results,
expressed as equilibrium inhibition constants (IC50) are provided
in Table 1. Stereochemistry of the Pro surrogate played a critical
role in the affinities of analogs. Peptide 2a was equipotent with
EM-2 at the MOR but showed also high affinity at the DOR and
KOR. Analog 2b was about three times less active at the MOR as
compared with EM-2 and was inactive at the DOR and KOR.
The resistance of linear analogs to enzymatic degradation was
tested using rat brain homogenate as a source of proteolytic
enzymes. The analogs were incubated with the homogenate for
60 min and then the mixtures were analyzed by RP-HPLC. The
obtained data are summarized in Table 2. The endogenous peptide,
Figure 1. Structure of (3R,4S)- and (3S,4R)-4-phenylpyrrolidine-3-carboxylic acid
(4-Ph-b-Pro).

R. Perlikowska et al. / Bioorg. Med. Chem. 22 (2014) 4803–4809
4805
Scheme 1. Reagents and conditions: (a) CoCl2 Â H2O, NaBH4, MeOH, then EDTA, À10 °C, 26% yield. (b) LiAlH4, THF, reflux, 79% yield. (c) Fmoc-OSu, Et3N, DCM, rt, overnight
71% yield. (d) PDC, DMF, rt, overnight, 64% yield.
Table 1
Opioid receptor binding affinities of EM-2 and its analogs
No.
Sequence
IC50 SEM [nM]
KORc
MORa
DORb
DOR/MOR
KOR/MOR
1
2a
2b
Tyr-Pro-Phe-Phe-NH2 (EM-2)d
Tyr-(3R,4S)-4-Ph-b-Pro-Phe-Phe-NH2
Tyr-(3S,4R)-4-Ph-b-Pro-Phe-Phe-NH2
0.79 0.05
0.85 0.01
2.91 0.65
>1000
9.06 0.65
>1000
>1000
27.11 1.85
>1000
>1000
10.66
>1000
>1000
31.89
>1000
All values are expressed as mean SEM of three independent experiments performed in duplicate.
a
Displacement of [3H]DAMGO.
b
Displacement of [3H][Ile5,6]deltorphin-2.
c
Displacement of [3H]nor-BNI.
Data from Ref. 44.
d
EM-2, was degraded rapidly, with half-life t1/2 of about 6 min while
both analogs were 5–6 times more stable.
manner, the duration of immobility. The strongest effect
(p < 0.001) was observed at a dose of 3 and 10 g/animal. Analog
2b was tested only at 3 g/animal (in higher doses caused death)
l
l
2.4. In vivo studies
and did not produce any antidepressant effect, on the contrary,
increased the immobility time. Yet another model of depression,
Antinociceptive activity of EM-2 and its two analogs was evalu-
ated in the hot-plate test, in mice, after icv administration of the
peptides at the dose range of 0.1–10 lg/animal. Analog 2a pro-
duced a dose-dependent analgesic action, stronger than that of
EM-2 (Fig. 2a), while analog 2b failed to cause any antinociceptive
effect which may suggest antagonist activity of this peptide at the
MOR. To confirm that the analgesic action of 2a was mediated
through interaction with the opioid system, opioid receptor antag-
onists were used. The selective MOR, DOR and KOR antagonists,
b-funaltrexamine (b-FNA), naltrindole (NLT) and nor-binaltorphi-
the TST was performed for both analogs at the dose of 3 lg/animal
(Fig. 3b). In the TST analog 2a also produced antidepressant-like
activity, reducing immobility time as compared with control.
Analog 2b did not influence animal behavior.
To establish which opioid receptor was engaged in mediating
the observed antidepressant-like activity, analog 2a was
co-administered with selective antagonists of all three opioid recep-
tors. The antagonists were used in the doses chosen on the basis of
our previous studies36 and alone did not produce any effects in the
FST. As shown in Figure 3c NLT and nor-BNI, but not b-FNA, com-
pletely inhibited anti-depressant-like action of analog 2a, indicating
that the observed effect was mediated by DOR and KOR. The
obtained results were in agreement with the binding studies, since
2a had affinity also for these receptors.
In order to exclude that antidepressant-like activity of analog 2a
resulted from the stimulation of the horizontal motor activity, the
LMA test was performed. The influence of the icv administration of
analog 2a on the locomotor activity of mice was measured over
three consecutive 20 min periods (Fig. 4). Analog 2a produced a
small decrease in locomotor activity in the first two periods
(p < 0.05). This is in agreement with other studies showing that
classical antidepressants either do not affect or slightly decrease
locomotor activity.36,37
mine (nor-BNI), respectively, (all in the dose of 1 lg/animal, icv,
that was shown previously to produce no effect in the hot-plate
test34,35), were used. b-FNA reversed the analgesic effect of analog
2a, while the co-administration of 2a with NLT or nor-BNI had no
effect, indicating that the analgesic action of 2a was mediated,
exclusively by MOR (Fig. 2b).
In order to check a possible antidepressant-like activity of ana-
logs 2a and 2b, these peptides were examined in two animal
behavioral models of depression: FST and TST, as well as in loco-
motor activity assay (LMA).
In the FST, analog 2a was tested in a dose range of 0.3–10
lg/animal following the icv injection. The obtained results are
shown in Fig. 3a. Analog 2a decreased, in a dose-dependent
Table 2
Degradation rates and half-lives of EM-2 and its analogsa
No.
Sequence
Brain homogenate
100 Â k (per min)
t1/2 (min)
1
2a
2b
Tyr-Pro-Phe-Phe-NH2 (EM-2)
Tyr-(3R,4S)-4-Ph-b-Pro-Phe-Phe-NH2
Tyr-(3S,4R)-4-Ph-b-Pro-Phe-Phe-NH2
11.00 0.58
2.12 0.11***
1.80 0.09***
6.00 0.32
32.55 1.84***
38.30 1.42***
All values are expressed as mean SEM of three independent experiments performed in duplicate.
a
RP-HPLC was performed on a Vydac C18 column (5
lm, 4.6 Â 250 mm) using the solvent system of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile/water (80:20, v/v) (B)
and a linear gradient of 0–100% solvent B over 25 min at a flow rate of 1 mL/min.
***
p < 0.001 as compared to EM-2 by using one-way ANOVA followed by the Student-Newman–Keuls test.

4806
R. Perlikowska et al. / Bioorg. Med. Chem. 22 (2014) 4803–4809
Figure 2. (a) Dose–response curves for the hot-plate inhibition of jumping induced by icv injection of EM-2 and analogs 2a and 2b. (b) Antagonist effect of b-FNA
(1 g/animal), NTL (1 g/animal) and nor-BNI (5 g/animal) induced by analog 2a (3 g/animal) determined in the hot-plate test in mice after icv injection. Data are
mean SEM of 10 mice per group. ⁄⁄⁄p < 0.001 for analog + b-FNA versus analog alone using one-way ANOVA followed by the Student-Newman–Keuls’ test.
l
l
l
l
Figure 3. (a) Dose–response effect of analog 2a (0.3–10
lg/animal) and 2b (3
lg/animal) on the FST. (b) Effect of analog 2a and 2b (3 lg/animal) on the TST. (c) Comparison of
antagonist action of b-FNA (1
lg/animal), NTL (1
l
g/animal) and nor-BNI (5 lg/animal) on the analog 2a (3 lg/animal)-induced effect on the FST. Mice were injected icv and
submitted to the FST and TST 10 min after the injection. Data represent mean SEM of 10 mice per group. ⁄p < 0.05; ⁄⁄⁄p < 0.001, as compared to respective control, cp < 0.001
as compared to analog alone by using one-way ANOVA followed by the Student-Newman–Keuls’ test.
High opioid activity of EM analogs with modified Pro2 residue
may be, at least in part, the result of their increased stability
against peptidases. Opioid peptides are known to undergo a rapid
enzymatic degradation. The main enzyme responsible for EMs
degradation is dipeptidyl peptidase IV (DPP IV) which liberates
the N-terminal Tyr-Pro dipeptide with high afficiency.38,39 DPP IV
is unable to degrade peptide bonds in which Pro surrogates partic-
ipate. Thus, introduction of a modified Pro into the sequence of
EMs can play a special role in the peptide stability, both, in vitro
and in vivo.

R. Perlikowska et al. / Bioorg. Med. Chem. 22 (2014) 4803–4809
4807
(5
l
m, 4.6 Â 250 mm and 10
l
m, 22 Â 250 mm), respectively.
Mass spectra of peptides were recorded on Bruker Apex Ultra 7T
FT-ICR mass spectrometer with electrospray ionization (ESI-MS;
Billerica, MA, USA). 1H NMR and 13C NMR spectra were recorded
on Bruker Avance II+ 700 spectrometer in CDCl3 or DMSO-d6 solu-
tions. Chemical shifts were reported as d values (ppm) related to
tetramethylsilane (TMS). J values are given in Hz.
4.2. Chemistry
4.2.1. The preparation of methyl ((3R,4S)-2-oxo-4-phenylpyrr-
olidine-3-carboxylate (2)
The solution of dimethyl [(1S)-2-nitro-1-phenylethyl]malonate
(1) (10.0 g, 35.5 mmol) in MeOH (130 mL) was cooled to À10 °C
and stirred vigorously. The solution of CoCl2 Â H2O (16.9 g,
71.1 mmol) was added, followed by a portion-wise addition of
NaBH4 (6.7 g, 177.8 mmol). After stirring for 3 h at À10 °C the reac-
tion mixture was allowed to warm up to the ambient temperature
and a solution of ethylenediaminetetraacetic acid disodium salt
dihydrate (52.8, 141.2 mmol) in H2O (300 mL) was added. The
reaction mixture was extracted with Et2O (3 Â 100 mL), the com-
bined organic extracts were dried (MgSO4) and evaporated. Purifi-
cation of the crude product by column chromatography (gradient
Figure 4. Effect of analog 2a (3 lg/animal) on horizontal locomotor activity. Mice
were injected icv with vehicle (control) or analog and placed in the actimeters
10 min after the injection. Horizontal displacements were measured for three
consecutive periods of 20 min. Data represent mean SEM of 10 mice per group.
⁄p < 0.05 as compared to control by using one-way ANOVA followed by the Student-
Newman–Keuls’ test.
3. Conclusion
In our earlier study we have shown that EM-1 and EM-2 pro-
duced potent antidepressant-like effect after icv administration
in mice in FST and TST depression tests and that this effect was
mediated by the MOR.36 In the present study we have tested two
diastereoisomeric peptides, 2a and 2b, with (3R,4S)- and (3S,4R)-
4-Ph-b-Pro residues in position 2, respectively. In vitro analog 2a
displayed high affinity for all three opioid receptors. In the hot-
plate test only analog 2a showed significant antinociception, even
stronger than that produced by EM-2 and the effect was antago-
nized by the MOR-selective antagonist, b-FNA. In both, FST and
TST, 2a administered icv significantly decreased the duration of
immobility, which could be related to the antidepressant-like
responses. These effects were antagonized by DOR and KOR but
not MOR-selective antagonists, indicating that analgesic and anti-
depressant-like effects can be dissociated.
elution CH2Cl2/MeOH: 500/1–300/1–100/1–9/1) yielded pure car-
22
boxylate 2 (2.02 g, 26%) as a white solid (mp 94–96 °C), [a]
D
104.8 (c 1.00 CHCl3), IR 3369, 2920, 1730, 1694, 1432, 1281,
1158, 998 cmÀ1 1H NMR (700 MHz, CDCl3) 7.34–7.37 (m, 2H),
.
7.28–7.30 (m, 1H), 7.25–7.27 (m, 2H), 6.09 (br s, 1H), 4.14 (dt,
J = 9.5 Hz, J = 8.7 Hz, 1H), 3.83 (dd, J = 9.4 Hz, J = 8.7 Hz, 1H), 3.79
(s, 3H), 3.59 (d, J = 9.5 Hz, 1H), 3.44 (dd, J = 9.4 Hz, J = 8.7 Hz, 1H).
13C NMR (176 MHz, CDCl3) 172.5, 169.6, 139.7, 129.0, 127.6,
127.0, 55.0, 52.8, 47.7, 44.3.
4.2.2. The preparation of (3R,4S)-3-hydroxymethyl-4-phenyl-
pyrrolidine (3)
A solution of methyl 2-oxopyrrolidine-3-carboxylate 2 (3.90 g,
19 mmol) in THF (70 mL) was added to a stirred suspension of
LiAlH4 (1.47 g, 40 mmol) in THF (35 mL) at 60 °C and the reaction
mixture was refluxed for 12 h. After cooling to 0 °C, H2O (5 mL)
was added, the mixture was stirred for additional 10 min and fil-
tered through a Celite bed. The filtrate was evaporated and the
crude product was purified by column chromatography (gradient
elution with DCM/MeOH: 500/1–300/1–100/1–1/1) to give pure
In vitro analog 2b showed an order of magnitude lower affinity
for the MOR and did not bind to the other two opioid receptors.
Analog 2b failed to induce antinociception in mice at the dose up
to 10 lg/animal and in the FST and TST, did not significantly influ-
ence the immobility time of animals.
22 53.7 (c 0.50 CHCl3),
.
Our observations are in accordance with the literature data
indicating that the MOR is mostly involved in analgesia, while
DOR, localized in the brain areas responsible for motor/motiva-
tional control,27,40 and especially KOR present in the brain,41 medi-
ate antidepressant-like effects.27,40
The data obtained here confirm the results of earlier studies
mentioned in the Introduction indicating that R-configuration of
a b-Pro residue is necessary for the manifestation of agonist activ-
ity of EM analogs.
In many cases pain is accompanied by depression, but clinical
treatment still focuses on the analgesic rather than mood altering
effects.42 Since we know that the opioid system is involved in both,
the analgesic and mood-elevating effects, the analog 2a with dual
action, antinociceptive and antidepressant-like, could be consid-
ered an interesting structure in the search for new lead compounds
in drug discovery.
alcohol 3 (2.5 g, 79%) as a pale yellow oil, [a]
IR 2873, 1493, 1419, 1048, 906, 754 cmÀ1D
1H NMR (700 MHz,
CDCl3) 7.29–7.32 (m, 2H), 7.24–7.26 (m, 2H), 7.20–7.22 (m, 1H),
3.71–3.74 (m, 1H), 3.59–3.61 (m, 1H), 3.44–3.47 (m, 1H), 3.33–
3.36 (m, 1H), 3.06–3.09 (m, 1H), 2.96–3.03 (m, 2H), 2.75 (br s,
2H), 2.40–2.42 (m, 1H). 13C NMR (176 MHz, CDCl3) 142.6, 128.7,
127.4, 126.6, 64.1, 55.5, 50.5, 49.8, 48.2.
4.2.3. The preparation of (3R,4S)-1-[(9-fluorenyl)methoxycar-
bonyl]-3-hydroxymethyl-4-phenylpyrrolidine (4)
A solution of pyrrolidine 3 (2.00 g, 11.3 mmol), 9-fluorenylm-
ethyl N-succinimidyl carbonate (3.80 g, 11.3 mmol, 1 equiv) and
Et3N (3.12 mL, 33.9 mmol) in DCM (100 mL) was stirred overnight
at rt. The reaction mixture was washed with 1 M HClaq (2 Â 30 mL),
brine (30 mL), dried (MgSO4) and evaporated. Crude product was
purified by column chromatography (gradient elution with DCM/
MeOH: 500/1–300/1–100/1–1/1) to provide pure N-Fmoc-pyrroli-
22
4. Experimental
4.1. General
dine 4 (3.2 g, 71%) as pale yellow oil, [
a]
4.6 (c 0.50 CHCl3), IR
D
2883, 1676, 1422, 1353, 1197, 1125 cmÀ1
.
1H NMR (700 MHz,
CDCl3) 7.74–7.79 (m, 2H), 7.59–7.64 (m, 2H), 7.32–7.42 (m, 5H),
7.24–7.30 (m, 4H), 4.40–4.43 (m, 2H), 4.22–4.28 (m, 1H), 3.91–
3.96 (m, 1H), 3.84–3.89 (m, 1H), 3.68–3.72 (m, 1H), 3.54–3.59
(m, 1H), 3.48–3.52 (m, 1H), 3.39–3.43 (m, 1H), 3.14–3.25 (m,
The analytical and semi-preparative RP-HPLC was performed on
the Waters Breeze (Milford, MA, USA) with a Vydac C18 columns

4808
R. Perlikowska et al. / Bioorg. Med. Chem. 22 (2014) 4803–4809
1H), 2.29–2.58 (m, 1H), 1.63 (br s, 1H). 13C NMR (176 MHz, CDCl3)
154.8, 144.1, 141.3, 140.0, 129.0, 128.9, 127.6, 127.5, 127.0, 125.1,
120.0, 67.3, 62.6, 54.8, 52.9, 49.3, 47.5, 45.7.
J = 8.0 Hz, 2H, 80%), 6.87 (d, J = 8.0 Hz, 20%), 6.93–7.32 (m,
15H + 15H, 80% + 20%), 8.03 (d, J = 7.5 Hz, 1H, 80%), 8.08 (br s, 2H,
80% + 20%), 8.12–8.16 (m, 1H + 2H, 80% + 20%), 9.37 (s, 1H, 20%),
9.47 (s, 1H, 80%).
4.2.4. The preparation of (3R,4S)-1-[(9-fluorenyl)methoxycar-
bonyl]-4-phenylpyrrolidine-3-carboxylic acid (5)
4.3. Animals
Pyridinium dichromate (7.50 g, 20 mmol, 5 equiv) was added in
one portion to a stirred solution of N-Fmoc-pyrrolidine 4 (1.6 g,
4 mmol) in DMF (40 mL) and the reaction mixture was stirred at
rt overnight. Next, Et2O/AcOEt (3/1, 100 mL) was added and the
mixture was washed with 1 M HClaq (2 Â 30 mL), brine (30 mL),
dried (MgSO4) and evaporated. Crude product was purified by
column chromatography (gradient elution with DCM/MeOH: 200/
Male Wistar rats (S3, Animal House, Faculty of Pharmacy, Lodz,
Poland), weighing 200–250 g were used as a source of brain mem-
branes and Male Swiss albino mice (CD1, JANVIER LABS, Le Genest-
Saint-Isle, France), weighing 20–26 g, were used in the in vivo
experiments. Animals were housed at a constant temperature
(22 1 °C) and maintained under a 12-h light/dark cycle in saw-
dust coated plastic cages with access to standard laboratory chow
and tap water ad libitum.
1–100/1) to provide pure N-Fmoc-pyrrolidine-3-carboxylic acid 5
22
(1.05 g, 64%) as a white solid (mp 190–192 °C), [
a]
29.9 (c
D
1.00 DMSO), IR 2888, 1694, 1418, 1323, 1224, 1127, 1022,
977 cmÀ1 1H NMR (700 MHz, CDCl3) 7.84–7.89 (m, 2H), 7.61–
.
4.4. In vitro experiments
7.66 (m, 2H), 7.37–7.43 (m, 2H), 7.28–7.35 (m, 6H), 7.24–7.26
(m, 1H), 4.34–4.35 (m, 2H), 4.24–4.29 (m, 1H), 3.69–3.78 (m,
2H), 3.43–3.57 (m, 2H), 3.18–3.29 (m, 2H). 13C NMR (176 MHz,
CDCl3) 173.2, 153.7, 143.9, 140.8, 139.6, 128.7, 127.8, 127.7,
127.4, 127.1, 125.1, 120.1, 66.5, 52.6, 49.4, 48.5, 47.2, 46.8.
4.4.1. Opioid receptor binding assays
Binding affinities of peptides for the MOR, DOR and KOR were
determined by displacing [3H]DAMGO, [3H][Ile5,6]deltorphin-2, or
[3H]nor-BNI, respectively, from the rat or guinea pig brain mem-
brane binding sites, according to the method described by Misicka
et al.43
4.2.5. Solid-phase peptide synthesis
Peptides were synthesized by standard solid phase procedure,
using techniques for Fmoc-chemistry on the Rink amide 4-meth-
ylbenzhydrylamine (MBHA) resin (100–200 mesh, 0.8 mM/g) and
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoro-
borate (TBTU) as a coupling agent as described elsewhere.11 Crude
peptides were purified by RP-HPLC. using a linear gradient of
0–100% B over 40 min at the flow rate of 15 mL/min, with UV
detection at 214 nm (injection volume 0.5 mL). Solvents: (A) 0.1%
TFA in water and (B) 0.1% TFA in acetonitrile/water (80: 20, v/v).
The purity of the final peptides was verified by analytical RP-HPLC
in the same solvent system over 25 min with the flow rate 1 mL/
min. The synthesized compounds were characterized by ESI-MS
and 1H NMR. The 1H NMR spectra of both peptides revealed the
presence of the mixtures of E and Z conformational isomers in
60/40 (for 2a) and 80/20 (for 2b) ratio, due to restricted rotation
around the Tyr-(4-Ph-b-Pro) amide bond.
4.4.2. Metabolic stability
Enzymatic degradation studies of the new analogs were per-
formed using rat brain homogenate, following the method
reported in detail previously.44 Briefly, the analogs were incubated
with brain homogenate over 0, 7.5, 15, 22.5, 30 and 60 min at
37 °C. The rate constants of degradation (k) were obtained by a
least square linear regression analysis of logarithmic peak areas
[ln(A/A0)], where A—amount of peptide remaining, A0—initial
amount of peptide, versus time. Degradation half-lives (t1/2) were
calculated from the rate constants as ln 2/k.
4.5. In vivo experiments
4.5.1. Assessment of antinociception
The analgesic activity of peptides was assessed in the hot-
plate test in mice, as described earlier.35 Tested peptides were
dissolved in dimethyl sulfoxide (DMSO) and further diluted with
saline to the desired concentrations. Control experiments
showed that DMSO had no effect on the results. Intacerebroven-
4.2.5.1.
Tyr-(3R,4S)-4-Ph-b-Pro-Phe-Phe-NH2
(2a).
RT:
16.350 min. ESI-MS calcd for C38H40N4O6 647.70, found [M+H]+
648.30.
1H NMR (700 MHz, DMSO-d6) 2.66–3.08 (m, 10H + 10H,
60% + 40%), 3.17–3.22 (m, 1H + 1H, 60% + 40%), 3.35–3.44 (m,
1H + 1H, 60% + 40%), 3.59–3.65 (m, 1H + 1H, 60% + 40%), 4.36–
4.43 (m, 1H + 1H, 60% + 40%), 4.51–4.57 (m, 1H + 1H, 60% + 40%),
6.69 (d, J = 8.5 Hz, 2H, 60%), 6.74 (d, J = 8.5 Hz, 2H, 40%), 6.98 (d,
J = 8.5 Hz, 2H, 60%), 7.06 (d, J = 8.5 Hz, 2H, 40%), 7.07–7.36 (m,
15H + 15H, 60% + 40%), 8.01–8.04 (m, 1H + 1H, 60% + 40%), 8.10
(br s, 2H, 60% + 40%), 8.12 (d, J = 9.0 Hz, 1H, 40%), 8.18 (d,
J = 8.5 Hz, 1H, 60%), 9.32 (s, 1H, 40%), 9.35 (s, 1H, 60%).
tricular injections (10
performed in the left brain ventricle of manually immobilized
mice with Hamilton microsyringe (50 L) connected to
needle (diameter 0.5 mm). The latencies to jumping were
measured.
The percentage of the maximal possible effect (%MPE) was cal-
culated as: %MPE = (t1 À t0)/(t2 À t0) Â 100, where t0–control
latency, t1–test latency and t2–cut-off time (240 s).
ll/mouse) of peptides or a vehicle, were
a
l
a
4.5.2. The forced-swimming test (FST)
4.2.5.2.
Tyr-(3S,4R)-4-Ph-b-Pro-Phe-Phe-NH2
(2b).
RT:
The FST was performed as described previously.36,45 The appa-
ratus consisted of two Plexiglas cylinders (20 cm height, 14 cm
internal diameter), placed side-by-side in a Makrolon cage, sepa-
rated by an opaque screen and filled with water (22 1 °C) to a
height of 12 cm. Twenty minutes before the test, the animals were
placed in small individual cages (L: 25 cm, W: 9 cm, H: 8 cm) at an
ambient temperature of 22 1 °C. Ten minutes after the icv injec-
tion of a vehicle or an examined analog, groups of four mice were
tested simultaneously for a 6-min period. The total duration of
immobility was measured using automated FST for mice (FST
X’PERT, BIOSEB, Vitrolles, France).
17.191 min. ESI-MS calcd for C38H40N4O6 647.70, found [M+H]+
648.30.
1H NMR (700 MHz, DMSO-d6) 2.27–2.32 (m, 1H + 1H,
80% + 20%), 2.62 (dd, J = 14.0 Hz, J = 8.5 Hz, 1H, 80%), 2.68 (dd,
J = 14.0 Hz, J = 8.5 Hz, 1H, 20%), 2.78–3.02 (m, 6H + 6H,
80% + 20%), 3.22–3.27 (m, 1H + 1H, 80% + 20%), 3.36–3.41 (m,
1H + 1H, 80% + 20%), 3.66–3.72 (m, 1H + 1H, 80% + 20%), 3.85–
3,92 (m, 1H + 1H, 80% + 20%), 4.19–4.25 (m, 1H + 1H, 80% + 20%),
4.40–4.44 (m, 2H + 1H, 80% + 20%), 4.51–4.55 (m, 1H, 20%), 6.72
(d, J = 8.0 Hz, 2H, 20%), 6.73 (d, J = 8.0 Hz, 2H, 80%), 6.86 (d,

R. Perlikowska et al. / Bioorg. Med. Chem. 22 (2014) 4803–4809
4809
7. Okada, Y.; Fukumizu, A.; Takahashi, M.; Shimizu, Y.; Tsuda, Y.; Yokoi, T.; Bryant,
S. D.; Lazarus, L. H. Biochem. Biophys. Res. Commun. 2000, 276, 7.
8. Cardillo, G.; Gentilucci, L.; Melchiorre, P.; Spampinato, S. Bioorg. Med. Chem.
Lett. 2000, 10, 2755.
9. Cardillo, G.; Gentilucci, L.; Qasem, A. R.; Sgarzi, F.; Spampinato, S. J. Med. Chem.
2002, 45, 2571.
10. Staniszewska, R.; Fichna, J.; Gach, K.; Toth, G.; Poels, J.; Vanden Broeck, J.;
Janecka, A. Chem. Biol. Drug Des. 2008, 72, 91.
11. Perlikowska, R.; Gach, K.; Fichna, J.; Toth, G.; Walkowiak, B.; do-Rego, J. C.;
Janecka, A. Bioorg. Med. Chem. 2009, 17, 3789.
4.5.3. The tail-suspension test (TST)
The TST was performed as described previously.36 Twenty min-
utes before the test animals were placed in small individual cages
(as above). Ten minutes after the icv injection of a vehicle or an
examined analog, mice were suspended by the tail, using an adhe-
sive Scotch tape. Total duration of immobility was measured dur-
ing the 6-min duration of the test.
12. Giordano, C.; Sansone, A.; Masi, A.; Lucente, G.; Punzi, P.; Mollica, A.; Pinnen, F.;
Feliciani, F.; Cacciatore, I.; Davis, P.; Lai, J.; Ma, S.-W.; Porreca, F.; Hruby, V. Eur.
J. Med. Chem. 2010, 45, 4594.
13. Borics, A.; Mallareddy, J. R.; Timári, I.; Kövér, K. E.; Keresztes, A.; Tóth, G. J. Med.
Chem. 2012, 55, 8418.
14. Drevets, W. C.; Raichle, M. E. Psychopharmacol. Bull. 1992, 28, 261.
15. Drevets, W. C.; Videen, T. O.; Price, J. L.; Preskorn, S. H.; Carmichael, S. T.;
Raichle, M. E. J. Neurosci. 1992, 12, 3628.
16. Drevets, W. C. Annu. Rev. Med. 1998, 49, 341.
17. Sheline, Y. I. Biol. Psychiatry 2000, 48, 791.
18. Chen, J. C.; Liang, K. W.; Huang, E. Y. Synapse 2001, 39, 239.
19. Tao, R.; Auerbach, S. B. J. Pharmacol. Exp. Ther. 2002, 303, 549.
20. Hung, K. C.; Wu, H. E.; Mizoguchi, H.; Leitermann, R.; Tseng, L. F. J. Pharmacol.
Sci. 2003, 93, 299.
4.5.4. Locomotor activity
Locomotor activity was assessed automatically in a computer-
ized actimeter (Versamax, AccuScan Instruments, Inc., Ohio, USA)
which monitored horizontal displacements.36,45 Twenty minutes
before the test the animals were placed in individual cages (as
above). Ten minutes after the icv injection mice were placed indi-
vidually in 20 Â 20 Â 30 cm compartments, in a dimly illuminated
and quiet room, then motor activity was recorded. The responses
were expressed as the number of crossed beams by mouse during
three consecutive 20 min periods.
21. Ukai, M.; Lin, H. P. Eur. J. Pharmacol. 2002, 446, 97.
22. Bujdosó, E.; Jászberényi, M.; Gardi, A. J.; Foldesi, B. I.; Telegdy, G. Neuroscience
2003, 120, 261.
4.6. Statistical analysis
23. Huang, E. Y.; Chen, C. M.; Tao, P. L. Peptides 2004, 25, 577.
24. Al-Khrasani, M.; Elor, G.; Yusuf Abbas, M.; Rónai, A. Z. Regul. Pept. 2003, 111, 97.
25. Porsolt, R. D.; Bertin, A.; Jalfre, M. Arch. Int. Pharmacodyn. Ther. 1977, 229, 327.
26. Steru, L.; Chermat, R.; Thierry, B.; Mico, J. A.; Lenegre, A.; Steru, M.; Simon, P.;
Porsolt, R. D. Prog. Neuropsychopharmacol. Biol. Psychiatry 1987, 11, 659.
27. Berrocoso, E.; Sánchez-Blázquez, P.; Garzón, J.; Mico, J. A. Curr. Pharm. Des.
2009, 15, 1612.
28. Filho, C. B.; Del Fabbro, L.; de Gomes, M. G.; Goes, A. T.; Souza, L. C.; Boeira, S. P.;
Jesse, C. R. Eur. J. Pharmacol. 2013, 698, 286.
29. Onali, P.; Dedoni, S.; Olianas, M. C. J. Pharmacol. Exp. Ther. 2010, 332, 255.
30. Valverde, O.; Micó, J. A.; Maldonado, R.; Mellado, M.; Gibert-Rahola, J. Prog.
Neuropsychopharmacol. Biol. Psychiatry 1994, 18, 1073.
31. Baghai, T. C.; Volz, H. P.; Moller, H. J. World J. Biochem. Psychiatry 2006, 7, 198.
32. Li, H.; Wang, Y.; Tang, L.; Deng, L. J. Am. Chem. Soc. 2004, 126, 9906.
33. Berestovitskaya, V. M.; Artemova, O. V.; Vasileva, O. S.; Litvinov, I. A.;
Gubaidullin, A. T.; Krivolapov, D. B.; Ostroglyadov, E. S.; Berkova, G. A. Russ. J.
Gen. Chem. 2009, 79, 645.
34. Fichna, J.; do-Rego, J. C.; Chung, N. N.; Costentin, J.; Schiller, P. W.; Janecka, A.
Peptides 2008, 29, 633.
35. Fichna, J.; Perlikowska, R.; Wyrebska, A.; Gach, K.; Piekielna, J.; do-Rego, J. C.;
Toth, G.; Kluczyk, A.; Janecki, T.; Janecka, A. Bioorg. Med. Chem. 2011, 19, 6977.
36. Fichna, J.; Janecka, A.; Piestrzeniewicz, M.; Costentin, J.; do-Rego, J. C.
Neuropsychopharmacology 2007, 32, 813.
Statistical and curve-fitting analysis were performed using
Graph Pad PRISM 5.0 (GraphPad Software In., San Diego, U.S.A.).
Results were 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. A probability level of 0.05 or lower was considered
statistically significant.
Acknowledgments
This work was supported by a Grant from the Medical Univer-
sity of Lodz No 503/1-156-02/503-01, a Grant from the Medical
University of Lodz for young researchers No 502-03/1-156-01/
502-14-121 (to R.P.), Grants from the European Regional Develop-
ment Fund (FEDER # 35233) (to S.C.A.C.) and from the EU INTER-
REG IVA
2 Seas Program (7-003-FR_TC2N) (to S.C.A.C.). R
Perlikowska was a recipient of a fellowship from the Foundation
for Polish Science (‘Start’) during the performance of this work.
The authors wish to thank Jozef Cieslak for his excellent techni-
cal assistance.
37. Lucki, I. In Encyclopedia of psychopharmacology; Stolerman, I. P., Ed.; Springer:
Berlin, 2010; pp 202–204. Vol. 2.
38. Tomboly, C.; Peter, A.; Toth, G. Peptides 2002, 23, 1573.
39. Sakurada, C.; Sakurada, S.; Hayashi, T.; Katsuyama, S.; Tan-No, K.; Sakurada, T.
Biochem Pharmacol. 2003, 66, 653.
40. Tejedor-Real, P.; Micó, J. A.; Smadja, C.; Maldonado, R.; Roques, B. P.; Gilbert-
Rahola, J. Eur. J. Pharmacol. 1998, 354, 1.
References and notes
41. Aldrich, J. V.; McLaughlin, J. P. AAPS J. 2009, 11, 312.
1. Zadina, J. E.; Hackler, L.; Ge, L. J.; Kastin, A. J. Nature 1997, 386, 499.
2. Leitgeb, B.; Tóth, G. Eur. J. Med. Chem. 2005, 40, 674.
3. Torino, D.; Mollica, A.; Pinnen, F.; Lucente, G.; Feliciani, F.; Davis, P.; Lai, J.; Ma,
S. W.; Porreca, F.; Hruby, V. J. Bioorg. Med. Chem. Let. 2009, 19, 4115.
4. Gao, Y. F.; Zhai, M. X.; Liu, W. X.; Liu, X.; Yuan, Y.; Qi, Y. M.; Wang, R. Protein
Pept. Lett. 2008, 15, 275.
5. Liu, W. X.; Wang, R. Med. Res. Rev. 2012, 32, 536.
6. Paterlini, M. G.; Avitabile, F.; Ostrowski, B. G.; Ferguson, D. M.; Portoghese, P. S.
Biophys. J. 2000, 78, 590.
42. Ryder, S. A.; Stannard, C. F. Contin. Educ. Anaesth. Crit. Care Pain 2005, 5, 18.
43. Misicka, A.; Lipkowski, A. W.; Horvath, R.; Davis, P.; Kramer, T. H.; Yamamura,
H. I.; Hruby, V. J. Life Sci. 1992, 51, 1025.
44. Perlikowska, R.; do-Rego, J. C.; Cravezic, A.; Fichna, J.; Wyrebska, A.; Toth, G.;
Janecka, A. Peptides 2010, 31, 339.
45. Cravezic, A.; Fichna, J.; Gach, K.; Wyrebska, A.; Perlikowska, R.; Costentin, J.;
Bonnet, J. J.; Janecka, A.; do-Rego, J. C. Neuropharmacology 2011, 61, 1229.