Synthesis and evaluation of new endomorphin analogues modified at the Pro2 residue

Article abstract of DOI:10.1016/j.bmcl.2009.06.008

Six new endomorphin analogues, incorporating constrained amino acids in place of native proline have been synthesized. Residues of (S)-azetidine-2-carboxylic acid (Aze), 3,4-dehydro-(S)-proline (Δ³Pro), azetidine-3-carboxylic acid (3Aze) and de

Full text of DOI:10.1016/j.bmcl.2009.06.008

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4115–4118
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of new endomorphin analogues modified
at the Pro2 residue
Domenica Torino a, Adriano Mollica b, Francesco Pinnen b, Gino Lucente a, Federica Feliciani b,
Peg Davis d, Josephine Lai d, Shou-Wu Ma d, Frank Porreca d, Victor J. Hruby c,
*
a Dipartimento di Chimica e Tecnologie del Farmaco e Istituto di Chimica Biomolecolare, CNR Sezione di Roma, ‘Sapienza’, Università di Roma, P.le A. Moro 5, 00185 Roma, Italy
b Dipartimento di Scienze del Farmaco, Università di Chieti-Pescara ‘G. d’Annunzio’, Via dei Vestini 31, 66100 Chieti, Italy
c Department of Chemistry, University of Arizona, Tucson, AZ 85721, USA
d Department of Pharmacology, University of Arizona, Tucson, AZ 85719, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Six new endomorphin analogues, incorporating constrained amino acids in place of native proline have
been synthesized. Residues of (S)-azetidine-2-carboxylic acid (Aze), 3,4-dehydro-(S)-proline (
azetidine-3-carboxylic acid (3Aze) and dehydro-alanine ( Ala) have been used to prepare [
3Pro2]EM-
2 (1), [Aze2]EM-1 (2), [Aze2]EM-2 (3), [3Aze2]EM-1 (4), [3Aze2]EM-2 (5) and [ Ala2]EM-2 (6). Binding
assays and functional bioactivities for - and d-receptors are reported. The highest affinity, bioactivity
and selectivity are shown by peptides 2 and 3 containing the Aze residue.
Ó 2009 Elsevier Ltd. All rights reserved.
D
3Pro),
Received 21 April 2009
Revised 27 May 2009
Accepted 1 June 2009
Available online 6 June 2009
D
D
D
l
Keywords:
Azetidine carboxylic acids
3,4-Didehydro-(S)-proline
Didehydro-alanine
Endomorphins
l-Receptors
The endogenous neuropeptides endomorphin-1 (Tyr-Pro-Trp-
Several chemical modifications performed on EMs have been
described previously and many of them were focused on the Pro
at position 2.12–20 The presence of Pro, the sole proteinogenic
amino acid possessing a cyclic structure and a secondary amino
group, represents a crucial factor in determining the structural
and conformational properties of proteins and peptides. In the spe-
cific context of studies on EMs analogues, the Pro2 is considered a
stereochemical spacer, capable of favouring proper spatial orienta-
tion of the aromatic side chain groups, a key factor for ligand
recognition and interaction with the receptor.21,22
This Letter reports on the EM analogues given in Figure 1, which
are obtained by replacing the Pro in position 2 with one of the four
amino acid residues depicted in Figure 2. These amino acids, char-
acterized by different constraints and structural features, can dee-
ply alter the tetrapeptide backbone conformation and thus
modulate in new ways the bioactivity of the peptide.
Phe-NH2; EM-1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2;
EM-2), originally isolated from bovine brain1 and subsequently from
the human brain cortex,2 continue to attract considerable attention
in the field of opioid peptides. In comparison with the other endog-
enous peptide ligands of opioid receptors, endomorphins (EMs)
combine potency and efficacy with high affinity and selectivity
towards the l
-opioid receptor,1,3,4 the receptor most involved with
acute analgesic effects in the central nervous system.5 Furthermore,
the EMs proteolytic stability, although low on a general scale, is the
highest among the known endogenous opioid peptides6,7 and their
activity is accompanied by reduced cardiorespiratory side effects,
as compared with the reference alkaloid ligand morphine.8,9 All of
these properties, render EMs the object of continuous investigations
anda promisingtargetforthedevelopment ofnewopioidanalgesics.
However, as found in the case of most short linear natural peptides,
native EMs lack critical therapeutic characteristics such as bioavail-
ability, duration of action and oral activity.10 Research efforts are
then focused on the design of analogues and peptidomimetics in
order to improve EMs properties as potential drugs as well as to
better understand the key structural and conformational features
on which receptor recognition and binding are based.11
A first modification, which leads to the EM-2 analogue 1, is
based on the introduction of the 3,4-dehydro-(S)-proline (D
3Pro)
residue. This amino acid, as compared with Pro, has an almost
planar and less flexible cyclic side chain ring, which does not allow
rapid interconversion between ring-puckered forms typical of
Pro residues and enhances, at the same time, the population of
cis conformers around the CO–N tertiary amide bond.23,24 In
addition to this, the presence of the double bond may significantly
improve, through
p
–
D
p
interactions, its binding to opioid recep-
* Corresponding author. Tel.: +1 520 621 6332; fax: +1 520 621 8407.
E-mail addresses: hruby@email.arizona.edu, hruby@u.arizona.edu (V.J. Hruby).
tors.25,26 A related
3Pro2 containing EM-2 analogue, namely
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.008

4116
D. Torino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4115–4118
OH
OH
HN
O
O
H
N
H
N
Xaa
Xaa
H2N
N
H
NH2
H2N
N
H
NH2
O
O
O
O
Pro2]EM-2
: Xaa = Aze [Aze2]EM-1
1
Δ3
: Xaa = Pro
Δ3
2
[
3: Xaa = Aze
[Aze2]EM-2
4: Xaa = 3Aze [3Aze2]EM-1
5: Xaa = 3Aze [3Aze2]EM-2
2
Δ
Δ
6
: Xaa = Ala
[ Ala ]EM-2
Figure 1. Molecular structure of the EM analogues 1–6.
conformational restriction imposed on the backbone by the planar
structure of Ala moiety, as well as its electronic distribution,
significantly influences the properties of this compound. Although
a variety of molecular modifications performed on native EMs have
D
CO2H
CO2H
N
H
N
H
Aze
Δ3Pro
(3,4-dehydro-(S)-proline)
[(S)-Azetidine-2-carboxylic acid]
been reported, analogues containing
a,b-unsaturated amino acid
residues are very scarce.36 Thus, the reported data on compound
6 represent, to the best of our knowledge, the first information
available in the literature on receptor binding affinity and selectiv-
ity of this type of EM analogues.
CO2H
N
H
3Aze
H2N
CO2H
Ala
Δ
The solution phase synthesis of peptides 1–5 were performed
following standard literature methods37which are given in Scheme
1. The synthesis of the dehydro-alanine-containing analogue 6,
was accomplished by exploiting the reactivity of the methylcys-
teine sulfoxide38 as illustrated in Scheme 2. All new compounds
were characterized by 1H NMR data and elemental analysis.39
(dehydro-alanine)
(Azetidine-3-carboxylic acid)
Figure 2. Molecular structure of the amino acids used in the present paper to
replace the native Pro2 residue in endomorphins: (S)-azetidine-2-carboxylic (Aze);
3,4-dehydro-(S)-proline (
nine ( Ala).
D
3Pro); azetidine-3-carboxylic acid (3Aze); dehydro-ala-
D
Receptor binding affinities to the d- and
l-opioid receptors
were performed using cell membrane preparations from transfec-
ted cells that stably express the respective receptors. The radiola-
beled ligands used were [3H]DPDPE and [3H]DAMGO for d- and
[Dmt1,
D
3Pro2]EM-2, has been previously reported by Toth and
Szemenyei27 and utilised for the synthesis of the corresponding tri-
tium labelled [3H2]Pro2 analogue. Two of the other modified li-
gands here reported analogues contain a cyclic lower homologue
of Pro. In particular, in analogues [Aze2]EM-1 (2) and [Aze2]EM-2
(3) the (S)-azetidine-2-carboxylic acid (Aze) has been incorporated.
Literature data show that the four-membered ring system of N-
acylated (S)-azetidine-2-carboxylic acid (Aze) is either planar or
only slightly puckered28–30 and presents, as compared with pro-
line, reduced side chain steric effects, with increased backbone
flexibility.31 This latter property generates a larger number of
low energy conformers with a population of the cis rotamers
around the CO–N amide bond which has been suggested to be
higher than that observed in Pro-containing peptides.32 Further-
more, the different ring size may significantly influence the type
l-receptors, respectively (for more details about the procedure
see Ref. 40). The in vitro tissue bioassays (MVD and GPI/LMMP)
were performed as described previously.41 IC50 values represent
means of no less than four experiments. IC50 values, relative
potency estimates, and their associated standard errors were
determined by fitting the data to the Hill equation by a computer-
ised non-linear least-squares method.
As shown in Table 1 all analogues, except [3Aze2]EM-1 (4), which
can be considered a l/d mixed agonist, show high selectivity for l-
receptors and functional bioactivities in good agreement with the
binding values. The highest affinity and selectivity is shown by the
Aze-containing peptides 2 and 3. This finding, together with the
good activity of the [
the residue at position 2 as a stereochemical spacer.
D
3Pro2]EM-2 analogue 1, confirms the role of
of folding with b-turns and c-turns preferentially induced by Pro
and Aze, respectively.31
The azetidine ring system also has been incorporated into the
backbone of the analogues [3Aze2]EM-1 (4) and [3Aze2]EM-2 (5).
Azetidine-3-carboxylic acid (3Aze) is an achiral conformationally
restricted analogue of b-alanine which, although not used in opioid
chemistry, has been employed for the synthesis of other pharma-
ceutical compounds.33–35 As compared with Aze and Pro residues,
3Aze maintains the cyclic structure and the secondary amino
group in the ring but is devoid of both chirality and restricted rota-
Yaa
Phe
Tyr
Xaa
Boc
Boc
H
OH H
OMe
OMe
EDC/HOBT
TFA/CH Cl 1:1
Boc
Boc
OH
OMe
OMe
OMe
EDC/HOBT
a
tion around the N–C bond. Among 5-membered chiral b-residues
TFA/CH Cl 1:1
Boc
Boc
Boc
H
OH
H
related to 3Aze, both b-proline (pyrrolidine-3-carboxylic acid) and
b-homoproline (2-pyrrolidineacetic acid) have already been used
to replace Pro2 in EM analogues.14,15 Furthermore, EM-2 analogues
containing cyclic 6-membered higher homologues of b-proline and
3Aze [viz nipecotic acid (piperidine-3-carboxylic acid) and isonip-
ecotic acid (piperidine-4-carboxylic acid), respectively)] have been
recently reported.20
EDC/HOBT
OMe
NH2
MeOH 0ºC/NH sat.
TFA/CH Cl 1:1
NH2
Scheme 1. Synthesis of EM analogues 1–5. Xaa =
D
3Pro; Yaa = Phe (1); Xaa = Aze;
The last analogue under study here is [
contains the rigid skeleton of dehydro-alanine (
D
Ala2]EM-2 (6) which
Ala). The local
Yaa = Trp (2); Xaa = Aze; Yaa = Phe (3); Xaa = 3Aze; Yaa = Trp (4); Xaa = 3Aze;
Yaa = Phe (5).
D

D. Torino et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4115–4118
4117
OH
OH
O
S
CH3
OH
S
CH3
OMe
O
a
b, c
d, e
H
N
H
N
f, g
HCl.
H2N
H2N
OMe
Boc
N
H
OH
Boc
N
H
N
H
O
O
O
1a
O
O
S
2a
S
CH3
CH3
3a
O
OH
O
OH
OH
O
O
O
O
H
N
H
N
H
N
i
H
H
N
h
OH
N
Boc
Boc
N
NH2
N
H
N
H
N
H
TFA.
N
NH2
H2N
H
H
O
O
O
O
O
O
5a
4a
6a
Scheme 2. Reagents and conditions: (a) SOCl2, MeOH, 15 min at 0 °C then 3 h at rt; (b) Boc-Tyr-OH, EDC, HOBT, DIPEA, CH2Cl2 30 min at 0 °C then 12 h at rt; (c) 1 N NaOH,
MeOH 5 h at rt; (d) HClÁPhe-OMe, EDC, HOBT, DIPEA, CH2Cl2 30 min at 0 °C then 12 h at rt; (e) NaIO4, H2O, dioxane 1 h at 0 °C; (f) DBU, MeOH, 1.5 h at rt; (g) 1 N NaOH, MeOH
5 h at rt; (h) HClÁPhe-NH2, EDC, HOBT, DIPEA, CH2Cl2 30 min at 0 °C then 12 h at rt; (i) TFA, CH2Cl21.5 h at rt under N2.
Table 1
Binding affinity and in vitro activity for compounds 1–6
Compound
Receptor affinitya,b (nM)
Ki
Selectivity
Functional bioactivity
Kid
l
d
l
MVDb (IC50
)
GPIb (IC50
)
1 [
D
3Pro2]EM-2
n.c.c
6.5 2.3
2.3 0.23
5.6 1.2
34 5.1
210 51
34 6.3
—
70 12
14 2.1
7.9 0.83
6.4 1.2
140 32
1400 280
170 23
2 [Aze2]EM-1
3 [Aze2]EM-2
4 [3Aze2]EM-1
5 [3Aze2]EM-2
3500 360
5100 600
40 8.5
6900 1200
710 130
—
1500
920
1.2
32
21
—
140 21
150 60
170 32
13% at 1
490 150
1710 110
510 35
lM
6 [D
Ala2]EM-2
EM-1d
EM-2d
10.70 1.5
9.56 0.98
28
15
3
2
—
—
a
Displacement of [3H]DAMGO ( -selective) and [3H]DPDPE (d-selective) from rat brain membrane binding sites.
l
S.E.M.
n.c. = data not collected.
Data from Ref. 42
b
c
d
The two analogues containing a b-residue, namely [3Aze2]EM-1
achiral 3Aze are detrimental features which prevail over the
advantages which, in larger cyclic systems, derive from the pres-
ence of a b-positioned carboxylic group.
(4) and [3Aze2]EM-2 (5) exhibit highly different activity profiles
(Table 1). The much lower activity shown by 5, as compared to
the Trp3 containing analogue [3Aze2]EM-1 (4), can be interpreted
on the basis of the well recognised role of the aromatic residues
on ligand-receptor interactions. Several studies on EM-1 and EM-
2 clearly show that aromatic side chains with different size, dis-
tinct noncovalent interactions and H-bonding capability, can
highly influence conformations43 and binding.21,44,45 Results of
ligands 4 and 5 indicate that the beneficial effect on the activity,
constantly observed in EM analogues containing the indole ring
system at position 3, gives results that are particularly efficient
as compared to the Phe3 residue in models containing the achiral
3Aze residue.
Peptide (6), containing the rigid skeleton of
DAla residue, has
only modest affinity and bioactivity at the -opioid receptor, and
l
this suggests that an efficient constraint at position 2 involves an
amino acid residue with a cyclic structure.
Further studies on the synthesis and activity of EM analogues
obtained by replacing native amino acid residues with synthetic
mimetics are under way in our laboratories and will be reported
in due course.
Acknowledgements
The low activity shown by [3Aze2]EM-2 (5) appears, however, is
quite unexpected when compared to the high activity shown by
EM analogues containing other cyclic b-residues in place of the
native Pro2. In particular, among the recently examined EM-2 ana-
logues containing positional isomers of the 6-membered piperi-
dine-carboxylic acid, replacement of Pro2 with the chiral
nipecotic acid (viz piperidine-3-carboxylic acid) led, in accordance
with the results concerning other related cyclic b-residues,14,15 to a
highly active analogue.20 On the contrary, Pro2 replacement with
the achiral piperidine-4-carboxylic acid (viz isonipecotic acid)
resulted, as in the case of the here reported analogue 5, to a prac-
tically inactive ligand.20 It can be argued that the high conforma-
tional restriction and the reduced flexibility of the 4-membered
This research was supported in part by grants from the U.S.
Public Health Service, National Institute of Drug Abuse DA06284
and DA13449 (V.J.H.).
References and notes
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D
3Pro-Phe-
a
a
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a
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}
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4.04 (2H, two m, AzeC4H2), 3.94 (1H, m, Tyr a-CH), 4.41 (1H, m, Trp a-CH), 4.48
(1H, m, Phe a-CH), 4.60 (1H, m, Aze a-CH), 6.55–7.35 (16H, m, aromatics and
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32. Kern, D.; Schutkowski, M.; Drakenberg, T. J. Am. Chem. Soc. 1997, 119, 8403.
33. Macdonald, S. J. F.; Dowle, M. D.; Harrison, L. A.; Clarke, G. D. E.; Inglis, G. G. A.;
Johnson, M. R.; Shah, P.; Smith, R. A.; Amour, A.; Fleetwood, G.; Humphreys, D.
C.; Molloy, C. R.; Dixon, M.; Godward, R. E.; Wonacott, A. J.; Singh, O. M.;
Hodgson, S. T.; Hardy, G. W. J. Med. Chem. 2002, 45, 3878.
(1H, m, Tyr a-CH), 4.42 (1H, m, Trp a-CH), 4.52 (1H, m, Phe a-CH), 6.58–7.59
(16H, m, aromatic and CONH2), 8.04–8.33 (5H, m, Trp NH-CO, Phe NH and Tyr
NH3+), 9.4 (1H, s, Tyr OH), 10.79 (1H, s, Trp NH). Anal. Calcd for C35H37F3N6O7:
C, 59.15; H, 5.25; N, 11.82. Found: C, 59.29; H, 5.27; N, 11.78.
TFAÁH-Tyr-3Aze-Phe-Phe-NH2 (5): 1H NMR (DMSO-d6) d: 2.55–3.90 (11H, m,
3Aze C3H2, 3Aze C4H2, Tyr b-CH2, Phe3 b-CH2, Phe4 b-CH2 and 3Aze
a-CH), 3.99
(1H, m, Tyr
a a a-CH), 6.61–7.36
-CH), 4.47–4.53 (2H, m, Phe3 -CH and Phe4
(16H, m, aromatics and CONH2), 8–8.31(5H, m, Phe3 NH, Phe4 NH and Tyr
NH3+), 9.41 (1H, s, Tyr OH). Anal. Calcd for C33H36F3N5O7: C, 59.01; H, 5.40; N,
10.43. Found: C, 59.22; H, 5.41; N, 10.45.
TFAÁH-Tyr-
b-CH2, Phe3 b-CH2, Phe4 b-CH2), 4.24 (1H, m, Tyr
-CH, Phe4
-CH), 5.54 and 6.17 (2H, two s,
aromatics and CONH2), 7.46 (1H, s,
Ala NH), 8.09 (3H, br, Tyr NH3+), 8.18 and
D
Ala-Phe-Phe-NH2 (6): 1H NMR (DMSO-d6) d: 2.74–3.07 (6H, m, Tyr
a
-CH), 4.50–4.58 (2H, m, Phe3
DAla CH2), 6.69–7.29 (16H, m,
a
a
D
8.56 (2H, two d, Phe3 NH, Phe4 NH), 9.69 (1H, s, Tyr OH). Anal. Calcd for
C32H34F3N5O7: C, 58.44; H, 5.21; N, 10.65. Found: C, 58.37; H, 5.26; N, 10.61.
40. Wang, Z.; Gardell, L. R.; Ossipov, M. H.; Vanderah, T. W.; Brennan, B. B.;
Hochgeschwender, U.; Hruby, V. J.; Malan, T. P., Jr.; Lai, J.; Porreca, F. J. Neurosci.
2001, 21, 1779.
41. Kramer, T. H.; Davis, P.; Hruby, V. J.; Burks, T. F.; Porreca, F. J. Pharmacol. Exp.
Ther. 1993, 266, 577.
42. Biondi, B.; Giannini, E.; Negri, L.; Melchiorri, P.; Lattanzi, R.; Rosso, F.; Ciocca, L.;
Rocchi, R. Int. J. Pept. Res. Ther. 2006, 2, 145.
34. Fish, P.; Barber, C. G.; Brown, D. G.; Butt, R.; Collis, M. J.; Dickinson, R. P.; Henry,
B. T.; Horne, V. A.; Huggins, J. P.; King, E.; O’Gara, M.; McCleverty, D.; McIntosh,
F.; Phillips, C.; Webster, R. J. Med. Chem. 2007, 50, 2341.
43. Gentilucci, L.; Tolomelli, A. Curr. Top. Med. Chem. 2004, 4, 105.
44. Yu, Y.; Shao, X.; Cui, Y.; Liu, H.-M.; Wang, C.-L.; Fan, Y.-Z.; Liu, J.; Dong, S.-L.;
Cui, Y.-X.; Wang, R. Chem. Med. Chem. 2007, 2, 309.
35. Klein, S. I.; Czekaj, M.; Molino, B. F.; Chu, V. Bioorg. Med. Chem. Lett. 1997, 7,
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45. Podlogar, B. L.; Paterlini, M. G.; Ferguson, D. M.; Leo, G. C.; Demeter, D. A.;
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