Novel highly potent μ-opioid receptor antagonist based on endomorphin-2 structure

Article abstract of DOI:10.1016/j.bmcl.2008.01.009

The μ-opioid agonists endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) exhibit an extremely high selectivity for the μ-opioid receptor and thus represent a potential framework for modification into μ-antagonists. Here we report on the synthesis and biological evaluation of novel [d-2-Nal⁴]endomorphin-2 analogs, [Sar²,d-2-Nal⁴]endomorphin-2 and [Dmt¹,Sar²,d-2-Nal⁴]endomorphin-2 (Dmt = 2′6′-dimethyltyrosine; Sar = N-methylglycine, sarcosine; d-2-Nal = 3-(2-naphthyl)-d-alanine). [Dmt¹,Sar²,d-2-Nal⁴]endomorphin-2 possessed very high affinity for the μ-opioid receptor (IC₅₀ = 0.01 ± 0.001 nM) and turned out to be a potent and extremely selective μ-opioid receptor antagonist, as judged by the in vitro aequorin luminescence-based calcium assay (pA₂ = 9.19). However, in the in vivo hot plate test in mice this analog was less potent than our earlier μ-opioid receptor antagonist, [Dmt¹,d-2-Nal⁴]endomorphin-2 (antanal-2). The exceptional μ-opioid receptor in vitro activity and selectivity of [Dmt¹, Sar²,d-2-Nal⁴]endomorphin-2 makes this analog a valuable pharmacological tool, but further modifications are needed to improve its in vivo profile.

Full text of DOI:10.1016/j.bmcl.2008.01.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1350–1353
Novel highly potent l-opioid receptor antagonist based
on endomorphin-2 structure
Jakub Fichna,a Jean-Claude do-Rego,b Tomasz Janecki,c Renata Staniszewska,a
Jeroen Poels,d Jozef Vanden Broeck,d Jean Costentin,b
Peter W. Schillere and Anna Janeckaa,*
aLaboratory of Biomolecular Chemistry, Institute of Biomedicinal Chemistry, Medical University of Lodz, Mazowiecka 6/8,
92-215 Lodz, PL, Poland
bLaboratoire de Neuropsychopharmacologie Expe´rimentale, CNRS-FRE 2735, IFRMP 23, Universite´ de Rouen, Rouen, France
cInstitute of Organic Chemistry, Technical University of Lodz, Lodz, Poland
dLaboratory for Developmental Physiology, Genomics and Proteomics, Zoological Institute, Catholic University, Leuven, Belgium
eLaboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal, Canada
Received 21 December 2007; accepted 3 January 2008
Available online 8 January 2008
Abstract—The l-opioid agonists endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) exhibit an
extremely high selectivity for the l-opioid receptor and thus represent a potential framework for modification into l-antagonists.
Here we report on the synthesis and biological evaluation of novel [D-2-Nal4]endomorphin-2 analogs, [Sar2,D-2-Nal4]endomor-
phin-2 and [Dmt1,Sar2,D-2-Nal4]endomorphin-2 (Dmt = 2060-dimethyltyrosine; Sar = N-methylglycine, sarcosine; D-2-Nal = 3-(2-
naphthyl)-D-alanine). [Dmt1,Sar2,D-2-Nal4]endomorphin-2 possessed very high affinity for the l-opioid receptor
(IC50 = 0.01 0.001 nM) and turned out to be a potent and extremely selective l-opioid receptor antagonist, as judged by the
in vitro aequorin luminescence-based calcium assay (pA2 = 9.19). However, in the in vivo hot plate test in mice this analog was less
potent than our earlier l-opioid receptor antagonist, [Dmt1,D-2-Nal4]endomorphin-2 (antanal-2). The exceptional l-opioid receptor
in vitro activity and selectivity of [Dmt1, Sar2,D-2-Nal4]endomorphin-2 makes this analog a valuable pharmacological tool, but fur-
ther modifications are needed to improve its in vivo profile.
ꢀ 2008 Elsevier Ltd. All rights reserved.
The development of selective and potent opioid pep-
tide antagonists for the major receptor types (l, d,
and j) continues to be a key objective in pharmaco-
logical research. In particular, l-opioid receptor
antagonists are of interest both, as pharmacological
tools and as potential therapeutic agents in the treat-
ment of opioid abuse and overdose. l-Antagonists of
alkaloid structure, such as naloxone and naltrexone,
lack a high degree of selectivity,1 while b-funaltrexam-
ine, which is l-opioid receptor selective, behaves as an
irreversible antagonist.2 For that reason, the search
for potent and selective l-opioid antagonists of a pep-
tide structure is a challenge for peptide chemists. The
l-opioid agonists, endomorphin-1 (Tyr-Pro-Trp-Phe-
NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2), ex-
hibit an extremely high selectivity for the l-opioid
receptor,3 and thus represent a potential framework
for modification into l-antagonists.4,5
In our earlier studies we have shown that the introduc-
tion of 3-(2-naphthyl)-D-alanine (D-2-Nal) into position
4 of endomorphins resulted in obtaining analogs with
weak antagonist activity.6 Further modification of these
analogs, involving the replacement of Tyr1 by 2060-dim-
ethyltyrosine (Dmt), resulted in obtaining two analogs,
antanal-1 ([Dmt1,D-2-Nal4]endomorphin-1) and ant-
anal-2 ([Dmt1,D-2-Nal4]endomorphin-2), which dis-
played a high degree of l-antagonist potency, both
in vitro and in vivo.7
In this study we described the synthesis and biological
activities of two novel [D-2-Nal4]endomorphin-2 ana-
logs: [Sar2,D-2-Nal4]endomorphin-2 and [Dmt1,Sar2,D-
2-Nal4]endomorphin-2, in which Pro in position 2 was
Keywords: Endomorphin-2 analog; Binding assay; Aequorin lumines-
cence-based calcium assay; Hot plate test.
*
Corresponding author. Tel.: +48 42 679 04 50x259; fax: +48 42 678
42 77; e-mail: ajanecka@zdn.am.lodz.pl
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.009

J. Fichna et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1350–1353
1351
replaced by N-methylglycine (sarcosine, Sar). The ratio-
nale for this modification was based on our earlier
observations that Sar2 increased the l-receptor affinity
and the metabolic stability, when introduced into the se-
quence of endomorphin-2.8,9
CHO-MOR-Aeq and CHO-DOR-Aeq, expressing,
respectively, the l- or d-opioid receptors and apoaequo-
rin, were used to study the agonist-induced biolumines-
cent responses caused by the release of intracellular Ca2+
ions. The EC50 and pA2 values and maximal calcium rise
for the l- and d-mediated intracellular calcium re-
sponses induced by endomorphin-2 and its analogs are
shown in Table 3. Endomorphin-2 produced a potent
agonist effect in the CHO-MOR-Aeq cells, with the
EC50 value of 0.040 0.001 nM and a maximal Ca2+
rise of 85.89 0.30%. For comparison, deltorphin-II
was used as a potent d-agonist in the CHO-DOR-Aeq
cell line (EC50 value <0.01 nM and a maximal Ca2+
stimulation of 86.59 0.04%). Both analogs 4 and 5
did not produce any significant calcium responses in
either cell line.
Peptides were prepared by manual solid phase synthesis,
using Rink Amide resin and Fmoc protection, according
to a protocol published earlier.7 After cleavage from the
resin by TFA/TIS treatment, the crude peptides were
purified by preparative RP HPLC. Their purity and
structural identity were established by analytical RP
1
HPLC and FAB-MS (Table 1). The H NMR data for
analog 5 are given in Ref. 10. H NMR spectrum re-
1
vealed the 3:7 ratio of stereoisomers derived from a con-
formational equilibrium around the Dmt-Sar amide
bond.
In our earlier studies we have shown that [D-2-
Nal4]endomorphin-2 (2) and [Dmt1,D-2-Nal4]endomor-
phin-2 (antanal-2, 3) were l-opioid receptor antago-
nists with pA2 values of 6.42 and 8.89, respectively.7
In order to examine the l-antagonist properties of
the peptide analogs 4 and 5 in the aequorin lumines-
cence-based calcium assay, competition experiments
against the l-selective agonist endomorphin-2 were per-
formed. The pA2 values were then calculated and com-
pared with the value for a well-known universal opioid
antagonist of the alkaloid structure, naloxone. Analog
5 produced the concentration-dependent rightward
shift of the concentration–response curve of endomor-
phin-2 (Fig. 1), with the pA2 value of 9.19 and was
slightly less potent than naloxone (pA2 = 9.66). This
analog did not produce any antagonist effect at the
Affinities for the l- and d-opioid receptors were deter-
mined in rat brain membrane binding assays, as de-
scribed elsewhere.11 The results in Table 2 show that
changing Pro with Sar in 4 caused a total loss of bind-
ing affinity (IC50 = 1560 160 and 15800 3200 nM,
for the l- and d-opioid receptors, respectively). An out-
standing increase of the l-opioid affinity and selectivity
in comparison with [Dmt1,D-2-Nal4]endomorphin-2
(antanal-2, 3) and the parent peptide, endomorphin-2,
was observed for 5 (IC50 = 0.01 0.001, 1.52 0.38,
and 0.79 0.05 nM, respectively).
For the determination of the in vitro opioid activities,
compounds were tested in aequorin luminescence-based
calcium assay.7 Recombinant mammalian cell lines,
Table 1. Physicochemical data of endomorphin-2 analogs
a
HPLCtr (min)
Compound
Sequence
FAB-MS
MW
Purity (%)
Formula
[M+H]+
1
2
3
4
5
Tyr-Pro-Phe-Phe-NH2 (endomorphin-2)
Tyr-Pro-Phe-D-2-Nal-NH2
12.51
10.43
16.60
14.73
15.06
C32H37N5O5
C36H39N5O5
C38H43N5O5
C34H37N5O5
C36H41N5O5
571
621
649
595
623
572
622
650
596
624
98
97
98
98
98
Dmt-Pro-Phe-D-2-Nal-NH2 (antanal-2)
Tyr-Sar-Phe-D-2-Nal-NH2
Dmt-Sar-Phe-D-2-Nal-NH2
a HPLC elution on a Vydac C18 column (0.46 · 25 cm, 5 lm) using the solvent system of 0.1% TFA in water (A)/80% acetonitrile in water containing
0.1% TFA (B) and a linear gradient of 20–80% solvent B over 25 min at flow rate of 1 mL/min.
Table 2. Opioid receptor binding assays of endomorphin-2 analogs
Compound
Sequence
IC50 (nM)
l
d
d/l
1
2
3
4
5
Tyr-Pro-Phe-Phe-NH2 (endomorphin-2)
Tyr-Pro-Phe-D-2-Nal-NH2
0.79 0.05a
19.5 2.10b
1.52 0.38a
1560 160a
0.01 0.001a
—
>10,000c
ND
7.74 0.22d
>10,000c
>10,000c
0.56 0.05c
>10,000
—
5.09
Dmt-Pro-Phe-D-2-Nal-NH2 (antanal-2)
Tyr-Sar-Phe-D-2-Nal-NH2
Dmt-Sar-Phe-D-2-Nal-NH2
>6.41
>10,000
—
Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin-II)
All values are expressed as means SEM of three to six determinations.
a Determined against [3H]DAMGO.
b Determined against [3H]naloxone.
c Determined against [3H]naltrindole.
d Determined against [3H]DSLET. Data for 2 from Ref. 6 data for 3 from Ref. 7. ND, no data.

1352
J. Fichna et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1350–1353
Table 3. The EC50 and pA2 values and maximal calcium rise for the l- and d-mediated intracellular calcium response induced by endomorphin
analogues
Compound
Sequence
CHO-MOR-Aeq
CHO-DOR-Aeq
a
b
EC50 SEM
(nM)
pA2
Max Ca2+-rise
(%)
EC50 SEM
(nM)
pA2
Max Ca2+-rise
(%)
1
Tyr-Pro-Phe-Phe-NH2
(endomorphin-2)
0.040 0.001
—
85.89 0.30
>1000
—
5.75 0.11
2
3
Tyr-Pro-Phe-D-2-Nal-NH2
Dmt-Pro-Phe-D-2-Nal-NH2
(antanal-2)
—
—
6.42
8.89
35.75 1.66
22.87 0.02
>1000
15.5 0.44
—
—
42.62 0.60
55.71 0.58
4
5
Tyr-Sar-Phe-D-2-Nal-NH2
Dmt-Sar-Phe-D-2-Nal-NH2
Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2
(deltorphin-II)
—
—
—
7.50
9.19
—
0.03 0.01
26.77 4.40
—
>1000
>1000
<0.01
—
—
—
0.18 0.11
7.49 0.13
86.59 0.04
Naloxone
—
9.66
0.10 0.01
—
—
—
All values are expressed as means SEM of three independent experiments performed in duplicate.
a Determined against endomorphin-2.
b Determined against deltorphin-II. Data for 2 and 3 from Ref. 7.
Endomorphin-2
A
d-opioid receptor, thus being extremely l-selective. It is
interesting to notice that the replacement of Tyr1 by
Dmt in this analog did not result in a loss of l-opioid
receptor selectivity, as usually observed with this
unnatural amino acid.12,13
Endomorphin-2 + Analog 3 (10-5 M)
Endomorphin-2 + Analog 3 (10-6 M)
Endomorphin-2 + Analog 3 (10-7 M)
Endomorphin-2 + Analog 3 (10-8 M)
100
80
60
40
20
The in vivo experiments for analog 5, using the hot
plate test in mice, were performed as described previ-
ously.7 Analog 5 alone, at the dose of 10 lg/animal
(icv), did not produce any analgesic effect (Table 4).
The co-administration of 5 reversed the antinocicep-
tion induced by endomorphin-2 (Table 5), but surpris-
ingly, the observed effect was weaker than expected.
Judging from the results in Tables 2 and 5, it seems
that higher l-opioid receptor selectivity results in low-
er in vivo antagonist activity. However, the number of
compared analogs is too low to draw more general
conclusions.
-20
-16
-12
-8
-4
logC, M
B
Endomorphin-2
Endomorphin-2 + Analog 5 (10-5 M)
Endomorphin-2 + Analog 5 (10-6 M)
Endomorphin-2 + Analog 5 (10-7 M)
Endomorphin-2 + Analog 5 (10-8 M)
In summary, further modification of [Dmt1,D-2-
Nal4]endomorphin-2 (antanal-2), involving the replace-
ment of Pro2 by Sar, resulted in a great increase of the
l-opioid receptor affinity and selectivity, as well as the
l-antagonist potency, as judged by the in vitro tests.
However, in vivo antanal-2 remained the most potent
l-opioid receptor antagonist among [D-2-Nal4]endo-
morphin-2 analogs.
100
80
60
40
20
Acknowledgments
-20
-16
-12
-8
-4
logC, M
This work was supported by the grants from the Medi-
cal University of Lodz (Nos. 502-11-460, 502-11-461,
and 503-1099-1), a grant for Bilateral Scientific Cooper-
ation between Flanders and Poland (BIL05/17), a grant
‘Start’ from the Foundation for Polish Science (to J.F.),
a grant from the Centre National de la Recherche Scien-
tifique (CNRS, France), and grants from the Canadian
Institutes of Health Research (MOP-5655) and the
NIH, USA (DA-004443 and DA-008924). The authors
wish to thank Jozef Cieslak for his excellent technical
assistance.
Figure 1. Concentration-dependent effect of (A) [Dmt1, D-2-Nal4]end-
omorphin-2 (antanal-2, 3) and (B) [Dmt1, Sar2, D-2-Nal4]endomor-
phin-2 (5) on the concentration–response curve for the calcium rise
induced by endomorphin-2 in the CHO-MOR-Aeq cells. The data
represent means SEM of three independent experiments carried out
in duplicate. Results are expressed as the fractional luminescence, that
is, the ratio of the agonist generated signal (A) and the total
luminescence (agonist + lysed cells, A + L), thereby correcting for
potential well-to-well variation in the number of injected cells. Calcium
(100%) rise corresponds to the amount of calcium ions released during
cell lysis.

J. Fichna et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1350–1353
1353
Table 4. Antinociceptive effect of endomorphin-2 analogs in the mouse hot plate test after intracerebroventricular injection (10 lg/animal)
Compound
Sequence
Latencies (%MPE) to
Rearing
Paw licking
Jumping
1
3
5
Tyr-Pro-Phe-Phe-NH2 (endomorphin-2)
Dmt-Pro-Phe-D-2-Nal-NH2 (antanal-2)a
Dmt-Sar-Phe-D-2-Nal-NH2
13.70 2.30
0.09 0.03
0.04 0.42
24.80 3.00
0.18 0.07
0.09 0.53
64.70 8.00
1.54 0.32
1.42 2.55
All values are expressed as means SEM (n = 10).
aData from Ref. 7. To evaluate the hot plate test responses, the control latencies (t0) and test latencies (t1) were determined after injection of saline
and a peptide, respectively. The percentage of maximal possible effect (%MPE) was calculated as %MPE = (t1 ꢀ t0)/(t2 ꢀ t0) · 100, where the cutoff
time (t2) was 240 s.
Table 5. Determination of antagonist activity of endomorphin-2 analogs in the mouse hot plate test after intracerebroventricular injection
Sequence
Latencies (%MPE) to
Rearing
Paw licking
Jumping
Endomorphin-2a
5.25 0.90
0.34 0.04
2.94 0.06
0.26 0.15
1.89 0.92
12.0 2.0
0.22 0.09
5.26 0.10
0.13 0.62
3.63 1.83
34.7 4.7
2.19 0.26
17.9 0.2
2.26 3.25
11.5 5.7
Endomorphin-2 + naloxoneb
Endomorphin-2 + Tyr-Pro-Phe-D-2-Nal-NH2 (2)c
Endomorphin-2 + Dmt-Pro-Phe-D-2-Nal-NH2 (3)c
Endomorphin-2 + Dmt-Sar-Phe-D-2-Nal-NH2 (5)c
To evaluate the hot plate test responses, the control latencies (t0) and test latencies (t1) were determined after injection of saline and a peptide,
respectively. The percentage of maximal possible effect (%MPE) was calculated as %MPE = (t1 ꢀ t0)/(t2 ꢀ t0) · 100, where the cutoff time (t2) was
240 s.
a Endomorphin-2 was injected at the dose of 3 lg/animal.
b Endomorphin-2 and naloxone were injected at the dose of 3 and 5 lg/animal, respectively.
c Endomorphin-2 and each analog were injected at the dose of 3 and 10 lg/animal, respectively. Data for 2 from Ref. 6, data for 3 from Ref. 7.
Chemical shifts are reported as d values. The assignment
of resonances was based on COSY experiment. H NMR
References and notes
1
(DMSO-d6): 2.03 (1.8H, s, Dmt, Ar–CH3), 2.11 (4.2H, s,
Dmt, Ar–CH3), 2.25–2.35 (0.6H + 0.7H, m, Phe b-
CHAHB, Phe b-CHA), 2.60 (0.7H, dd, J = 13.8 Hz,
J = 8.5 Hz, Phe b-CHB), 2.88–2.96 (2H + 0.3H,
+0.7H + 0.3H, m, Dmt b-CH2, Sar a-CHA, Nal b-CHA,
Nal b-CHB), 3.07 (0.3H, d, J = 16.8 Hz, Sar a-CHB), 3.13
(0.7H, d, J = 16.1 Hz, Sar a-CHB), 3.15–3.22
(0.7H + 0.3H, m, Nal b-CHA, Nal b-CHB), 3.97 (0.3H,
quintet, J = 5.2 Hz, Dmt a-CH), 4.30 (0.7H, d,
J = 16.1 Hz, Sar a-CHA), 4.37 (0.7H, quintet, J = 4.9 Hz,
Dmt a-CH), 4.46–4.53 (1H + 1H, m, Phe a-CH, Nal a-
CH), 6.41 (2H, s, Dmt, Ar–H), 6.80–7.18 (5H, m, Phe, Ar–
H), 7.40–7.85 (9H, m, Nal, Ar–H, CONH2), 8.01 (0.7H, d,
J = 8.7 Hz, Phe, NH), 8.07 (0.3H, d, J = 8.3 Hz, Phe, NH),
8.21 (2.1H, br s, Dmt, NH3þ), 8.25 (0.9H, br s, Dmt,
NH3þ), 8.44 (0.3H, d, J = 8.4 Hz, Nal, NH), 8.43 (0.7H, d,
J = 8.6 Hz, Nal, NH), 9.19 (0.7H, s, Dmt, OH), 9.22
(0.3H, s, Dmt, OH).
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