Endomorphin-1 analogues containing β-proline are μ-opioid receptor agonists and display enhanced enzymatic hydrolysis resistance

Article abstract of DOI:10.1021/jm011059z

In this paper we describe the synthesis and affinity toward the μ-opioid receptor of some tetrapeptides obtained from endomorphin-1, H-Tyr-Pro-Trp-Phe-NH₂ (1), by substituting each amino acid in turn with its homologue. The ability to bind μ -opioid receptors depends on the β-amino acid, and in particular 4, which contains β-L-Pro, has a K_I, in the nanomolar range. The peptides 4 and 5 are significantly more resistant to enzymatic hydrolysis than 1. The same compounds, as well as the μ-opioid receptor agonist DAMGO, produced a concentration-dependent inhibition of forskolin-stimulated cyclic AMP formation, thus behaving as μ-opioid agonists. These features suggest that this novel class of endomorphin-1 analogues may represent suitable candidates for the in vivo investigation as potential μ-opioid receptor agonists.

Full text of DOI:10.1021/jm011059z

J . Med. Chem. 2002, 45, 2571-2578
2571
En d om or p h in -1 An a logu es Con ta in in g â-P r olin e Ar e µ-Op ioid Recep tor Agon ists
a n d Disp la y En h a n ced En zym a tic Hyd r olysis Resista n ce
Giuliana Cardillo,*,† Luca Gentilucci,*,† Ahmed R. Qasem,‡ Fabio Sgarzi,† and Santi Spampinato‡,§
Dipartimento di Chimica “G. Ciamician” and “CSFM”, via Selmi 2, Universita` degli Studi di Bologna, 40126-Bologna, Italy,
and Dipartimento di Farmacologia, via Irnerio 48, Universita` di Bologna, 40126-Bologna, Italy
Received October 12, 2001
In this paper we describe the synthesis and affinity toward the µ-opioid receptor of some
tetrapeptides obtained from endomorphin-1, H-Tyr-Pro-Trp-Phe-NH2 (1), by substituting each
amino acid in turn with its homologue. The ability to bind µ-opioid receptors depends on the
â-amino acid, and in particular 4, which contains â-L-Pro, has a KI in the nanomolar range.
The peptides 4 and 5 are significantly more resistant to enzymatic hydrolysis than 1. The
same compounds, as well as the µ-opioid receptor agonist DAMGO, produced a concentration-
dependent inhibition of forskolin-stimulated cyclic AMP formation, thus behaving as µ-opioid
agonists. These features suggest that this novel class of endomorphin-1 analogues may represent
suitable candidates for the in vivo investigation as potential µ-opioid receptor agonists.
In tr od u ction
selectivity could depend on the presence of spatially
distinct hydrophobic pockets in different receptors.22,23
The comparison of several endogenous and synthetic
tetrapeptide opioid ligands showed that µ-selectivity and
affinity strongly depend on the amino acid sequence. In
particular, the presence of a Tyr in the first position, a
Pro or D-Ala in the second position, lipophilic residues
in the third and fourth positions, and amidation at the
C-terminus seem to be very important features.1,15-17,22-24
A more subtle requirement is the spatial disposition
of each residue.23 Indeed, conformational differences
may be responsible for large variation in affinity and
selectivity. Several attempts have recently been made
to determine the bioactive conformation of endomor-
phin-1. These studies, using molecular modeling and
2-dimensional NMR in different solvents and environ-
ments, have indicated that proline is a key residue, on
the basis of its role as a spacer that fixes the peptide
shape and induces the other residues to assume the
proper spatial orientation for ligand-receptor interac-
tion.17,18,22,24,25
In a recent study of peptides containing â-amino acids,
we examined modified endomorphins with a â-amino
acid in their sequence. This change could give tetrapep-
tides which are degraded or inactivated by enzymes less
rapidly than endogenous peptides. We previously re-
ported the preparation of some tetrapeptides in which
each residue at a time in the series H-Tyr-Pro-Trp-
PheNH2 was replaced by the corresponding â-isomer,
to obtain new tetrapeptides the same size as the parent
endomorphin, and tested their affinities for µ-opioid
receptors through binding assays in rat brain mem-
branes.26 These tetrapeptides showed different affinities
for µ-opioid receptors. Our results stress that proline is
important for fixing the conformation of the other
residues.
The discovery of the endogenous peptides endomor-
phin-1, H-Tyr-Pro-Trp-Phe-NH2, and endomorphin-2,
H-Tyr-Pro-Phe-Phe-NH2,1-3 in the mammalian brain
encouraged the application of natural or synthetic
peptides as analgesics instead of morphine.4-8 Indeed,
both morphine and endomorphins mainly act as agon-
ists at the same µ-opioid receptors, but the latter are
thought to inhibit pain without some of the undesirable
side effects of morphine.6-10 Quite often, however,
endomorphins, as well as opioid peptides in general,
have a limited in vivo efficacy, since they are easily
degraded by different proteases.11-14 To increase their
efficacy, the concomitant use of some peptidase inhibi-
tors can be considered.15 On the other hand, several
peptidomimetics or more stable peptide analogues, in
which the original structures of endogenous peptides
have been modified,15,16 including endomorphin-117 and
endomorphin-218 diastereoisomers, have been synthe-
sized and investigated. In several cases, the peptido-
mimetics exhibit long-term toxicity and have difficulty
in penetrating the blood-brain barrier.19,20 For this
reason, the search for new peptidomimetics or peptide
analogues is of current interest.15,16,21
In addition, the structure-activity relationships of
endomorphins which have been systematically modified
through the introduction of modified residues within the
sequence are of particular interest, since the basis of
ligand recognition and the selectivity of opioid receptors
are only partially known. Indeed, there is still no
conclusive evidence of the presence of a µ-opioid address
in ligands, even though it seems plausible that the
* To whom correspondence should be addressed. (G.C.) E-mail:
cardillo@ciam.unibo.it. Phone: +39 051 2099570. Fax: +39 051
2099456. (L.F.) E-mail: gentil@ciam.unibo.it. Phone: +39 051 2099575.
Fax: +39 051 2099456.
Another possible modification is the introduction of
homo-â-amino acids to the sequence. â-Peptides formed
by homologated â-amino acids have been studied for
years to discover stable secondary structures.27-34 In
several cases, the substitution of R-amino acids by their
† Dipartimento di Chimica “G. Ciamician” and “CSFM”, Universita`
degli Studi di Bologna.
‡ Dipartimento di Farmacologia, Universita` di Bologna.
§ E-mail: spampi@biocfarm.unibo.it. Phone: +39 051 2091851.
Fax: +39 051 248862.
10.1021/jm011059z CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/09/2002

2572 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Sch em e 1. Synthesis of Homo-Amino Acidsa
Cardillo et al.
Sch em e 3. Synthesis of Homo-Tetrapeptides 1-6a
a
Reagents and conditions: (a) EtOCOCl (1.1 equiv), triethyl-
amine (TEA) (2.1 equiv), THF, -15 °C, 15 min; (b) CH2N2, Et2O,
-5 °C, 1 h, 85-92%; (c) CF3COOAg (0.11 equiv)/TEA (2.8 equiv),
THF/H2O (9:1), -25 °C, 4 h, 70-82%.
Sch em e 2. Synthesis of D-Homo-Prolinea
a
Reagents and conditions: (a) N-methylmorpholine (1.2 equiv),
isobutylchloroformate (1.2 equiv), THF, -5 °C, 10 min (not
isolated); (b) NaBH4 (1.2 equiv), I2 (0.48 equiv), THF, 0 °C, 2 h,
75%; (c) TsCl (1.2 equiv), TEA (1.2 equiv), catalytic dimethylami-
nopyridine (DMAP), CH2Cl2, 0 °C, 3 h, 78%; (d) KCN (3 equiv),
DMSO, 90 °C, 4 h, 60%; (e) 12 N HCl/AcOH (5:1), reflux, 4 h, 80%;
(f) 2 N NaOH/acetone, BOC2O (1.5 equiv), 0 °C, 2 h, 98%.
â-isomers in biologically active peptides resulted in
increased activity and enzymatic stability.35-37
In this paper we report the synthesis of tetrapeptides
based on endomorphin-1, in which one residue at a time
was replaced by its â-homologue. Most of these homo-
amino acids are commercially available, or can be
readily prepared in optically pure form. We report the
affinities displayed by the modified endomorphins for
µ-opioid receptors as tested by displacement assays
performed on µ-opioid receptors in rat brain membranes
labeled with [3H]DAMGO. In conjunction with this, we
also present the effects of these peptides displaying the
higher affinities to µ-opioid receptors on cyclic AMP
formation in whole SH-SY5Y human neuroblastoma
cells to better understand if they act as µ-receptor
agonist or antagonist. Finally, we examined the degra-
dation rates of some tetrapeptides in the presence of
commercially available proteolytic enzymes to deter-
mine if the structural and conformational changes gave
improved resistance in comparison to native endomor-
phin-1.
a
Reagents and conditions: (a) TEA (3 equiv), hydroxybenzo-
triazole (HOBt) (1.5 equiv), 1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide (EDCI)/HCl (1.5 equiv), CHCl3/DMF (9:1), 0 °C, N2,
8 h, 60-90%; (b) saturated HCl/dioxane, 0 °C, 45 min, 100%.
rolidylacetonitrile.42 Acid hydrolysis simultaneously
gave Boc deprotection and cyano group hydrolysis to
afford (R)-homo-Pro in good yield. Final treatment with
di-tert-butyl dicarbonate gave N-Boc-(R)-homo-Pro. In
a similar way, N-Boc-(S)-homo-Pro could be obtained
starting from N-Boc-(S)-Pro.
Peptides 1-6 were prepared in solution using a
convergent approach, by coupling the amino amide and
the N-tert-butyloxycarbonyl-amino acid in the presence
of EDCI/HOBt (Scheme 3).38 The resulting Boc-protect-
ed peptides were purified by flash chromatography over
silica gel, with yields of 60-90%. Deprotection was
achieved by treatment with saturated HCl in dioxane,38
and the resulting HCl peptide salts were used without
purification for the next coupling. Finally, HCl tet-
rapeptide salts were purified by reversed-phase pre-
parative HPLC, to give 1-6 as TFA salts. Purities were
determined to be 94-97% by analytical reversed-phase
HPLC (Table 2).
The final tetrapeptide TFA salts were characterized
by MALDI-MS and/or FAB-MS analysis (Table 2).
In this manner, we prepared a series of tetrapeptides
based on the endomorphin-1 sequence, H-Tyr-Pro-Trp-
Phe-NH2, containing a â-homo-amino acid. Since Pro is
thought to be responsible for the spatial disposition of
the other residues, we prepared tetrapeptides containing
both homo-L-Pro and homo-D-Pro.43
R a d ioliga n d Bin d in g St u d ies a n d Discu ssion .
The tetrapeptides were incubated with rat brain mem-
brane homogenates containing µ-opioid receptors and
using [3H]DAMGO as a µ-specific radioligand. All
experiments were performed in triplicate. From the
Resu lts a n d Discu ssion
Ch em istr y: Syn th esis a n d Ch a r a cter iza tion of
Tetr a p ep tid es. Most of the optically pure (S)-N-tert-
butyloxycarbonyl-â-amino acids were purchased, or
readily prepared from N-tert-butyloxycarbonyl-R-amino
acid by means of the Arndt-Eistert homologation, with
overall yields of 60-75% (Scheme 1).38
(R)-N-tert-Butyloxycarbonyl-homo-Pro was obtained
starting from D-proline using a multistep homologation
procedure which was slightly modified with respect to
the literature,39 since in this case the Arndt-Eistert40
reaction gave us poor yields (Scheme 2).
The N-Boc-(R)-Pro mixed anhydride was reduced with
sodium borohydride41 to Boc-prolinol, which was tosyl-
ated and treated with KCN to give the N-Boc-pyr-

Endomorphin-1 Analogues Containing â-Proline
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2573
Ta ble 1. Affinities and Hill Slopes of DAMGO and Ligands 1-6 for [3H]DAMGO Binding Sites in Rat Brain Membranesa
compd
sequence
KI (nM)
IC50 (nM)
nH
DAMGO
1.6((0.3)
9.9((0.6)
0.5((0.2)
0.90((0.05)
0.74((0.03)
1
Tyr-Pro-Trp-Phe-NH2
0.16((0.02)
(endomorphin-1)b
b
b
2
3
4
5
6
Tyr-Pro-Trp-â-Phe-NH2
Tyr-Pro-â-Trp-Phe-NH2
79.1((0.3)
73.0((0.5)
2.1((0.3)
67((6)
80.1((0.8)
23.3((0.6)
4((2)
79((2)
990((40)
0.8((0.1)
0.7((0.1)
0.69((0.03)
0.6 ((0.2)
0.8((0.1)
b
b
Tyr-L-â-Pro-Trp-Phe-NH2
Tyr-D-â-Pro-Trp-Phe-NH2
b
â-Tyr-Pro-Trp-Phe-NH2
26((4)
a
b
Means ( SE of three experiments performed in triplicate. TFA salts.
Ta ble 2. Purities and Masses of Tetrapeptides 1-6
purity peptide mass
purity peptide mass
compd
(%)a
[M + H]b
compd
(%)a
[M + H]b
1
2
3
96
94
96
611.2c
625.0c
625.2c
4
5
6
95
97
94
625.0,c 625.4d
625.3,c 625.6d
625.1c
a
b
Average value from HPLC of methods 1 and 2. Calculated
values: 1, 610.3; 2-6, 624.3. c FAB-MS. MALDI-MS.
d
F igu r e 2. Effects of DAMGO and endomorphin-1 analogues
4 and 5 on forskolin-stimulated cyclic AMP production in SH-
SY5Y cells.
spacer that keeps the other residues in the correct
spatial disposition, our results seem to indicate that
homo-Pro still permits 4 to assume a geometry similar
to that of 1, probably due to the enhanced conforma-
tional freedom given by the extra carbon. Although it
is possible that the different tetrapeptides interact with
the receptor in distinct ways, it seems likely that they
would fit in a similar way, due to flexibility of the
tetrapeptide chain. In addition, a certain degree of
“induced fit” should also be considered for receptor
conformation, which would enable molecules with dif-
ferent shapes to bind to the same part of a receptor.45
The significantly lower affinity observed by substituing
Pro with homo-D-Pro in 5 confirms that the absolute
configuration remains a very important aspect of the
role of Pro as a spacer, despite the greater flexibility
gained by the peptide.17,26
F igu r e 1. Competition curves of 1, 4, and 5 with [3H]DAMGO
for its specific binding sites in rat brain membranes. The
membranes were incubated with various concentrations of
ligands (10-12 to 5 × 10-2) and 1 nM DAMGO for 60 min at
room temperature.
displacement binding assays, KI, IC50, and Hill factors
were obtained for DAMGO, endomorphin-1 (1), and
tetrapeptides 2-6, and are reported in Table 1. The
competition curves of the most active peptides 4 and 5
with [3H]DAMGO are reported in Figure 1 and com-
pared with that of 1.
The affinity values for DAMGO and 1 agree with the
literature (KI ) 1.9 and 0.36 nM, respectively).1,15,22,44
All the compounds displayed a concentration-dependent
displacement of [3H]DAMGO. In all cases the slope
factor (nH) was less than unity. By comparing the values
for peptides 2-6, it is clear that the peptides 2, 3, 5,
and 6 display a low affinity for µ-opioid receptors, while
4 was the most potent in displacing [3H]DAMGO, (Table
1). In particular, 4 has a KI value in the nanomolar
range, although it is slightly less potent than the
reference compound 1 (Table 1, Figure 1). These results
indicate that substituting Phe, Trp, and Tyr with homo-
Phe, homo-Trp, and homo-Tyr, respectively, in the
tetrapeptide sequence caused a significant loss of affin-
ity with respect to the reference compound 1. These
results can be explained by considering that these
residues are responsible for specific hydrophobic ligand-
receptor interactions, and thus a change in their spatial
disposition is detrimental for a good ligand-receptor fit.
On the other hand, substituting Pro with homo-L-Pro
gives the new homo-peptide 4, which retains a good
µ-receptor affinity. Since Pro is thought to act as a
Compound 4, which was the most effective in displac-
ing [3H]DAMGO from rat brain membranes, and com-
pound 5 were further investigated to evaluate any effect
on cyclic AMP production in intact SH-SY5Y cells which
constitutively express abundant µ-opioid receptors and
fewer δ-opioid receptors.44,46 The potent µ-opioid agonist
DAMGO, as well as 4 and 5, suppressed forskolin (10
µM)-stimulated cyclic AMP production in a concentra-
tion-dependent manner (Figure 2). The IC50 values of
DAMGO, 4, and 5 were 1.10 ( 0.27, 6.53 ( 0.25, and
45.0 ( 9.2 nM, respectively. The potency of DAMGO was
in the nanomolar range as well as previously reported
by Toll et al. for the same cell line.46 Harrison et al.,44
adopting the same assay system, have reported that
endomorphin-1 produced concentration-dependent in-
hibition of foskolin-stimulated cyclic AMP production
and the pIC50 was 7.72 ( 0.13. Further, naloxone (100
nM) significantly reversed the effect elicited by the
DAMGO, 4 (1 µM), and 5 (10 µM) (data not shown).
These findings indicate that 4 and 5 act in the µ-opioid
receptor as agonists, and their IC50 values for the
inhibitory effects in cyclic AMP production are well

2574 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Cardillo et al.
Ta ble 3. HPLC Data of Peptides Derived from Enzymatic Hydrolysis vs Authentic Samples in Two Different Solvent Systems
Rf (peptide
vs authentic,
method 1)
Rf (peptide
vs authentic,
method 2)
Rf (peptide
vs authentic,
method 1)
Rf (peptide
vs authentic,
method 2)
compd
compd
1
19.38/19.40
16.88/16.89
13.83/13.83
20.47/20.46
10.71/10.68
13.43/13.39
19.54/19.53
16.50/16.45
35.11/35.05
28.92/28.89
14.10/14.09
34.80/34.86
12.85/12.83
16.11/16.13
35.17/35.12
24.75/24.70
Tyr-L-â-Pro-Trp-Phe
5
Tyr-D-â-Pro-Trp
6
â-Tyr-Pro-Trp
â-Tyr-Pro
â-Tyr-Pro-Trp-Phe
20.40/20.40
20.12/20.10
17.25/17.28
19.87/19.92
17.28/17.30
12.01/12.00
20.99/21.01
36.72/36.76
36.22/36.20
27.60/27.63
36.19/36.22
29.18/29.21
15.61/15.58
37.57/37.54
Tyr-Pro-Trp
Phe-NH2
Tyr-Pro-Trp-Phe
Tyr-Pro
Trp-Phe-NH2
4
Tyr-L-â-Pro-Trp
F igu r e 3. Kinetic curve of 1 digestion and digestion products
upon incubation with R-chymotrypsin.
F igu r e 4. Kinetic curve of 1 and 4-6 digestion upon incuba-
tion with R-chymotrypsin.
correlated to their KI values for the µ-opioid receptor
obtained in binding studies.
Tetr a p ep tid e En zym a tic Digestion . A very im-
portant topic to examine in the field of biologically active
peptides is their resistance to enzymatic degradation.
Thus, we tested the resistance of the modified tetrapep-
tide 4, which showed the higher µ-opioid receptor
affinity (Table 1), to some common, commercially avail-
able enzymes.47 To verify the effect of residue substitu-
tion, we also examined the hydrolysis of 5 and 6 under
the same conditions. The peptides were incubated in a
buffered solution (pH 7.4) with R-chymotrypsin, ami-
nopeptidase-M, and carboxypeptidase-Y at 37 °C.47 At
designated intervals, we quenched an aliquot of the
incubated mixture, and the mixture was analyzed by
reversed-phase HPLC. Sampling times were chosen so
that a representative kinetic curve could be constructed.
Peak attributions were determined by comparison with
authentic fragments prepared by taking into account
the more plausible47 tetrapeptide decompositions (Table
3). To prevent incorrect attributions, Rf values were
compared for two distinct HPLC systems, as reported
in the Experimental Section. Experiments were per-
formed in parallel, taking portions of the same enzyme
solution. Data were shown in plots of area vs time. To
appreciate the resistances displayed by the different
peptides, areas were normalized to 100 at t ) 0 and the
curves were superimposed.
F igu r e 5. Kinetic curve of 1 digestion and digestion products
upon incubation with aminopeptidase-M.
other hand, 4 was extremely resistant under the same
conditions, with 93% intact after 3 h.
The digestion of 1 with aminopeptidase-M gave a
mixture of Tyr-Pro and Trp-Phe-NH2.47 After 3 h,
further hydrolysis of the latter dipeptide to Trp and Phe-
NH2 was evident (Figure 5). Comparison of the degra-
dation rates for 1, 4, 5, and 6 (Figure 6) showed that 1
and 6 have basically the same resistance, with 17% and
14% remaining after 3 h, respectively, while 4 and 5
both show excellent resistance, 89% and 96% remaining
after 3 h, respectively. In fact these two peptides can
be considered stable during the time period investigated,
since no trace of any product arising from their enzy-
matic digestion was detected by HPLC.
The enzymatic digestion of endomorphin-1 by carbox-
ypeptidase-Y begins with hydrolysis of the C-terminus
amide Phe-NH2, to give the tetrapeptide Tyr-Pro-Trp-
Phe, which is in turn hydrolyzed to the tripeptide Tyr-
Pro-Trp upon cleavage of the Trp-Phe bond (Figure 7).47
Comparison of the rates of digestion of 1 and 4 by
carboxypeptidase-Y indicates a comparable hydrolysis
resistance, within the error ranges. Indeed, after 24 h
The digestion of 1 with R-chymotrypsin proceeded
very rapidly, and gave a mixture of the tripeptide Tyr-
Pro-Trp and Phe-NH2 (Figure 3). Indeed, only 14% of
the original tetrapeptide quantity was intact after 3 h
(Figures 3 and 4). The degradation of 5 and 6 showed a
similar trend; although 23% of 5 and 17% of 6 remained
after 3 h (Figure 4), this was not considered enhanced
resistance to hydrolysis with respect to 1, since these
values are within the estimated error ranges. On the

Endomorphin-1 Analogues Containing â-Proline
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2575
enhance peptide stability. Again, Pro seems to influence
the conformation of the entire peptide, and the resis-
tance of 4 and 5 can be attributed to a significant change
in peptide-enzyme recognition.
Con clu sion s
The tetrapeptides described in this paper can be
regarded as promising modified endomorphins and
interesting models for the study of ligand-receptor
interaction. Indeed, they display different affinities for
µ-opioid receptors, depending on the homo-amino acid
introduced and its absolute stereochemistry. Good af-
finities were found for 4-6. In particular, 4 (Tyr-L-homo-
Pro-Trp-Phe-NH2) showed affinity comparable to that
of DAMGO. The cyclic AMP assay confirmed that 4 and
5 act as agonists, and probably bind in the same way
as 1. Due to the presence of an extra backbone carbon,
4 and 5 should be more flexible than endomorphin-1.
Nevertheless, it appears that they can assume an active
conformation. We should emphasize that the binding
modes of flexible DAMGO and more conformationally
constrained endomorphins are suspected to be the same,
although additional binding modes can be considered.48
Moreover, a certain degree of flexibility should also be
attributed to the receptor itself, thus enabling molecules
with different shapes to interact in the same way.45
Finally, we tested the resistance of 1 and 4-6 to the
proteolytic enzymes R-chymotrypsin, aminopeptidase-
M, and carboxypeptidase-Y. Our results indicate that
the presence of homo-Pro gives the peptides 4 and 5 a
good resistance, while homo-Tyr seems to have little
effect. In particular, due to its enzyme resistance, and
affinity for the opioid receptor, peptide 4 may validate
further study for the development of a peptide analgesic
based on the endomorphin sequence.
F igu r e 6. Kinetic curve of 1 and 4-6 digestion upon incuba-
tion with aminopeptidase-M.
F igu r e 7. Kinetic curve of 1 digestion and digestion products
upon incubation with carboxypeptidase-Y.
Exp er im en ta l Section
Gen er a l Meth od s. Unless stated otherwise, chemicals were
obtained from commercial sources and used without further
purification. CH2Cl2 was distilled from P2O5. THF was distilled
from sodium benzophenone ketyl. Flash chromatography was
performed on Merck silica gel 60 (230-400 mesh), and solvents
were simply distilled. Preparative reversed-phase HPLC was
performed on a Waters Delta Prep 4000 Millipore, with a C18
column RP-18 (40-63 µm). The FAB-MS instrument employed
was a Micromass ZMD spectrometer equipped with a single
quadrupole analyzer and a Z-spray ionspray source outfitted
with a 50 mm deactivated fused Si capillary connected to a
Harvard Apparatus pump 11 for sample injection. Data
acquisition and spectral analysis were conducted with Mass-
lynx 3.3 software running on a Digital Equipment Corp.
personal computer. Nitrogen was used as both desolvation and
nebulizer gas. The desolvation temperature was set at 200 °C
and the capillary voltage at 3.0 kV. MALDI-TOFMS analysis
was performed on a Bruker Biflex III apparatus, equipped with
a reflectron and pulse ion extraction (PIE). The instrument
was run in positive ion reflectron mode (voltage 20 kV) with
PIE set at “short”. The cutoff was set at m/z 300 using the
deflection mode, and the investigated range was 288-3590.
Laser power (N2, 337 nm) was adjusted for optimal resolution,
and each spectrum was obtained by addition of 100-400 shots.
The peptides were diluted in 1:1 matrix solution, which was
R-cyano-4-hydroxycinnamic acid in water/acetonitrile (2:1) +
0.1% TFA. Analytical HPLC was performed on an HP Series
1100, with an HP Hypersil ODS column (4.6 µm particle size,
100 Å pore diameter, 250 mm), DAD 215.8 nm. Enzymatic
hydrolysis was performed in an MGM Lauda RC20 thermo-
static bath. Carboxypeptidase-Y lyophilized powder, 20%
F igu r e 8. Kinetic curve of 1 and 4-6 digestion upon incuba-
tion with carboxypeptidase-Y.
only 23% of the initial amount of endogenous 1 was
detected, while 29% of the homopeptide 4 was found
(Figure 7). Compound 6 was slightly more resistant,
35% remaining after 24 h. More appreciably, peptide 5
showed the higher resistance, with 50% of the starting
amount intact after 24 h.
Overall, our results indicate that no hydrolysis of the
amide bond next to the substituted amino acid occurred.
Peptide 6, which contains homo-Tyr, maintains almost
the same resistance as native 1 upon digestion with each
enzyme. On the contrary, 4 and 5, which contain homo-
Pro, show enhanced resistance against aminopeptidase-
M, which hydrolyzes the Pro-Trp bond. Interestingly,
a good resistance enhancement is observed for 4 also
against R-chymotrypsin, which hydrolyzes the Trp-Phe
bond, while 5 shows resistance against carboxypepti-
dase-Y, which hydrolyzes the Phe-NH2 bond. Compari-
son of 4 and 5 with 6 also suggests that the simple
substitution of a generic residue is not sufficient to

2576 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Cardillo et al.
protein content, pH 5, 19 units/mg of solid, 100 units/mg of
protein, aminopeptidase-M suspension in 3.5 M (NH4)2SO4
solution, pH 7.4, 5.1 mg of protein/mL, 29 units/mg, and
R-chymotripsin, 60 U/mg, were purchased from Sigma. Ho-
mogenates were centrifuged in Beckman J 6B and Beckman
J 2-21 centrifuges. Radioactivity was measured by liquid
scintillation spectrometry using a Beckman apparatus.
ylpyrrolidine (0.48 g, 78%). To a solution of (R)-N-Boc-2-
tosyloxymethylpyrrolidine (1.1 g, 3.0 mmol) in DMSO (15 mL)
was added KCN (0.58 g, 9.0 mmol), and the mixture was
stirred at 90 °C for 4 h, then EtOAc was added, and the organic
layer was washed three times with small portions of water.
The collected water layers were treated with KMnO4 before
elimination. The organic layer was dried over Na2SO4, and
solvent was evaporated at reduced pressure, giving crude (R)-
N-Boc-pyrrolidylacetonitrile as an oil, purified (0.38 g, 60%)
by flash chromatography over silica gel (eluant 50:50 cyclo-
Syn th esis of Boc-â-Tyr , Boc-â-Tr p , a n d Boc-â-P h e by
Ar n d t-Eister t Hom ologa tion . To a stirred solution of Boc-
amino acid (10 mmol) in anhydrous THF (35 mL) were added
EtOCOCl (11 mmol) and TEA (21 mmol) under an inert
atmosphere at -15 °C. After 15 min, a solution of CH2N2 in
ether was added at -5 °C until the pale yellow color persisted.
After 1 h the CH2N2 excess was destroyed with glacial acetic
acid (0.5 mL), and the mixture was diluted with ether (30 mL)
and washed with saturated NaHCO3, saturated NH4Cl, and
brine. The organic layer was dried over Na2SO4, and solvent
was removed at reduced pressure. The residue was purified
by flash chromatography over silica gel (eluant 3:7 EtOAc/
cyclohexane), giving the diazoketone as an oil (85-92%). To a
stirred solution of the diazoketone (5 mmol) in 9:1 THF/H2O
(15 mL) was added a solution of CF3COOAg (0.55 mmol) in
TEA (14 mmol) at -25 °C under an inert atmosphere. The
mixture was stirred at room temperature for 4 h, and then
solvent was removed at reduced pressure. The residue was
diluted with saturated NaHCO3, and the mixture was ex-
tracted twice with ether. HCl (1 M) was added to the aqueous
layer at 0 °C until the pH was around 2-3, and the mixture
was extracted three times with EtOAc. The organic layers were
collected and dried over Na2SO4, and solvent was evaporated
at reduced pressure. Boc-homo-amino acid was obtained pure
after flash chromatography over silica gel (eluant 1:1 EtOAc/
cyclohexane) with a yield of 70-82%.
20
hexane/EtOAc): [R]D ) -96.4° (c ) 1, CHCl3).
(R)-â-P r o-HCl. A mixture of (R)-N-Boc-pyrrolidylacetoni-
trile (0.21 g, 1.0 mmol), 12 N HCl (10 mL), and glacial CH3-
COOH (2 mL) was refluxed for 6 h. Then the reaction was
allowed to reach room temperature, and it was washed twice
with Et2O. Water was evaporated at reduced pressure, giving
(R)-â-Pro as hydrochloric salt (0.13 g, 80%), used without
20
further purification: [R]D ) -35.5° (c ) 0.7, 2 N HCl); for
(S)-homo-Pro, lit.50 [R]D ) +35° (c ) 1, 2 N HCl).
20
(R)-Boc-â-P r o. (R)-â-Pro-HCl (0.17 g, 1.0 mmol) was dis-
solved in water (5 mL), and the pH was adjusted to around 10
at 0 °C with NaOH. Then the solution was diluted with acetone
(10 mL), and di-tert-butyl dicarbonate (0.33 g, 1.5 mmol) was
added at 0 °C with stirring. After 15 min the temperature was
allowed to reach room temperature. After 2 h acetone was
evaporated at reduced pressure, and 3 M HCl was added at 0
°C until pH 3. The mixture was extracted three times with
EtOAc, and the collected organic layers were dried over Na2-
SO4. Solvent was evaporated at reduced pressure, giving (R)-
Boc-â-Pro (0.34 g, 98%), which was used without further
purification: [R]D ) +42.3° (c ) 2.0, DMF) (lit.40 [R]D
)
20
20
+40.5° (c ) 1.9, DMF)).
Syn th esis a n d Ch a r a cter iza tion of Tetr a p ep tid es. As
a general procedure, the peptide coupling was performed by
stirring overnight the HCl salt of the amino amide, the N-tert-
butyloxycarbonyl-amino acid (1.2 equiv), triethylamine (3
equiv), 1-hydroxy-1H-benzotriazole (1.5 equiv), and the HCl
salt of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (1.5
equiv), in a 9:1 mixture of CHCl3 and DMF at 0 °C and under
a nitrogen atmosphere. After 8 h, the solvent was evaporated
at reduced pressure, and the residue was dissolved in EtOAc.
The solution was washed with 0.5 M HCl, saturated NaHCO3,
and brine. The organic layer was dried over Na2SO4, and
solvent was removed at reduced pressure. Peptides were
obtained pure by flash chromatography over silica gel (eluant
96:4 EtOAc/MeOH) with yields from 60% to 90%.
Syn th esis of Boc-â-P h e-NH2. A solution of Boc-â-Phe (0.28
g, 1.0 mmol) in CH2Cl2 (10 mL), TEA (0.31 mL, 2.2 mmol),
and trimethylacethyl chloride (0.12 mL, 1.0 mmol) was stirred
under an inert atmosphere at 0 °C. After 30 min, the solution
was saturated with NH3 at 0 °C, and stirring was maintained
for 30 min. The reaction was quenched with water, and the
mixture was extracted three times with EtOAc. The organic
layers were collected and dried over Na2SO4. Boc-â-Phe-NH2
was obtained after solvent evaporation at reduced pressure
(0.25 g, 90%), and used without further purification.
Syn th esis of Boc-D-â-P r o. (R)-N-Boc-p r olin ol. A mixture
of Boc-D-proline (0.75 g, 3.5 mmol), N-methylmorpholine (0.46
mL, 4.2 mmol), isobutylchloroformate (0.55 mL, 4.2 mmol), and
THF (4 mL) was stirred at -5 °C. After 10 min the mixture
was filtered over Celite, and the precipitate was washed with
THF. In a round-bottom flask a solution of NaBH4 (0.16 g, 4.2
mmol) in dry THF (10 mL) was stirred at 0 °C under an inert
atmosphere. Iodine (0.44 g, 1.7 mmol) was slowly added over
30 min, and after an additional 10 min the collected organic
layers containing the Boc-D-Pro mixed anhydride were added
with stirring. After 30 min at 0 °C the temperature was
allowed to reach room temperature, and the mixture was
stirred for 5 h. Then solvent was evaporated at reduced
pressure, the residue was diluted with EtOAc, and the mixture
was washed with water (5 mL). The water layer was extracted
twice with EtOAc, and the organic layers were collected and
dried over Na2SO4. Solvent was evaporated at reduced pres-
sure. The residue was separated by flash chromatography over
silica gel (eluant 75:25 cyclohexane/EtOAc), giving pure (R)-
N-tert-Butyloxycarbonyl group deprotection was performed
by treatment with HCl in dioxane at 0 °C. After 45 min the
solvent was evaporated at reduced pressure, and the resulting
HCl peptide salt, obtained in quantitative yield, was used
without purification for the next coupling. HCl-tetrapeptide
salts were prepurified by recrystallization from MeOH/Et2O.
Final purification was performed by semipreparative reversed-
phase HPLC on a Waters Delta Prep 4000 Millipore, with a
C18 column RP-18 (40-63 µm, 250 mm) with solvent systems
A (0.1% TFA in water) and B (0.1% TFA in acetonitrile),
gradient 100% A to 50% B in 50 min at a 5.0 mL/min flow.
Purities were determined by analytical reversed-phase HPLC
under two distinct systems (Table 2).
Sp ectr oscop ic Ch a r a cter iza tion of Tyr -L-â-P r o-Tr p -
1
P h e-NH2 (4). H NMR (CD3OD, 400 MHz, major conformer)
20
N-Boc-prolinol (0.53 g, 75%): ([R]D ) +47.0° (c ) 1, CHCl3)
δ 1.70-1.80 (m, 1H), 1.80-1.88 (m, 2H), 1.92-2.08 (m, 1H),
2.77 (dd, J ) 8.1, 14.4 Hz, 1H), 2.89 (d, J ) 6.6 Hz, 2H), 2.93
(dd, J ) 5.4, 8.7 Hz, 1H), 3.02-3.15 (m, 3H), 3.22 (d, J ) 6.6
Hz, 2H), 3.48-3.59 (m, 1H), 4.24 (dd, J ) 6.6, 7.2 Hz, 1H),
4.44 (dd, J ) 4.8, 8.4 Hz, 1H), 4.53 (t, J ) 6.9 Hz, 1H), 4.58 (t,
J ) 6.6 Hz, 1H), 6.71 (d, J ) 7.0 Hz, 1H), 6.76 (d, J ) 6.8 Hz,
2H), 6.93 (d, J ) 7.0 Hz, 1H), 7.01-7.31 (m, 7H), 7.60 (d, J )
7.1 Hz, 2H), 7.82 (d, J ) 6.5 Hz, 1H); 13C NMR (CD3OD) δ
24.7, 25.4, 28.5, 29.6, 38.4, 39.4, 41.2, 46.3, 55.4, 56.0, 56.5,
110.5, 112.1, 116.5, 119.0, 122.2, 124.5, 125.4, 127.5, 128.1,
129.1, 130.1, 131.8, 137.5, 137.8, 157.8, 162.0, 168.2, 173.5,
176.8.
20
(lit.49 [R]D ) +47.5° (c ) 1, CHCl3)).
(R)-N-Boc-p yr r olid yla ceton itr ile. A mixture of (R)-N-
Boc-prolinol (0.35 g, 1.75 mmol), TEA (0.29 mL, 2.1 mmol),
catalytic DMAP (0.071 g, 0.58 mmol), and tosyl chloride (0.40
g, 2.1 mmol) in CH2Cl2 (20 mL) was stirred at 0 °C. After 3 h,
the mixture was washed with saturated Na2CO3, and the
water layer was extracted twice with CH2Cl2. Organic layers
were collected and dried over Na2SO4, and solvent was
evaporated at reduced pressure. The resulting crude oil was
purified by flash chromatography over silica gel (eluant 80:20
cyclohexane/EtOAc), giving pure (R)-N-Boc-2-tosyloxymeth-

Endomorphin-1 Analogues Containing â-Proline
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2577
Recep tor Bin d in g Assa ys. Rat brain, without cerebellum,
was weighed and homogenized in 10 volumes of ice-cold 0.32
M sucrose/10 mM Tris-HCl (pH 7.4 at 4 °C). The homogenate
was centrifuged at 2000 rpm, for 10 min, at 4 °C, and the
supernatant was in turn centrifuged at 19000 rpm, for 20 min,
at 4 °C. The resulting pellet was suspended in 10 volumes of
50 mM Tris-HCl/100 mM NaCl (pH 7.4 at 4 °C) as incubation
buffer and incubated for 1 h at room temperature (in a water
bath at 37 °C) to remove any endogenous opioid ligands. After
a final centrifugation at 19000 rpm, for 20 min, at 4 °C, the
pellet was stored at -80 °C for up to two weeks.
The protein concentration was determined according to
Lowry et al.51 [3H]DAMGO was used as µ-selective radioligand
(1 nM); the specific activity was 64 Ci/mmol, Kd ) 4.85 nM
and Bmax ) 48 fmol/mg of protein, n ) 3. Nonspecific binding
was determined in the presence of 100 µM DAMGO. The
incubation buffer consisted of 50 mM Tris-HCl, 0.1% BSA (pH
7.4 at 4 °C), and 2 mM EDTA. To prevent any peptidase
degradation, the following protease inhibitors were added to
the binding buffer: captopril (25 µg/mL), bacitracin (0.2 mg/
mL), leupeptin (10 µg/mL), phenylmethylsulfonyl floride (0.19
mg/mL), and aprotinin (5 KIU/mL). δ- and κ-opioid receptors
were blocked with 0.01 M DADLE and 0.01 M U50,488,
respectively.
The mixture (1 mL) was incubated for 1 h at room temper-
ature, and then it was filtered under vacuum through glass
fibers (GFB, Whatman, soaked for 1 h in 0.1% polyethylene-
imine) and washed with ice cold washing buffer (50 mM Tris-
HCl, pH 7.4, at 4 °C). The ligand-receptor complex radioac-
tivity retained in the filter was measured by liquid scintillation
spectrometry using a scintillator after 12 h of incubation in
the scintillation cocktail. All assays were performed in tripli-
cate, and repeated at least three times. Stock solutions (10-2
M) were made in dimethyl sulfoxide or methanol/HCl (0.1 N)
(1:1 v/v).
mL (0.5 mg)/5 mL), carboxypeptidase-Y (0.5 mg/5 mL). Experi-
ments were performed in parallel, by adding 1.0 mL portions
of the same enzyme solution to each peptide solution. At
designated intervals, a 0.50 mL aliquot of incubated mixture
was quenched with 15 µL of 1 N HCl and diluted with 0.2 mL
of CH3CN. Sampling times were chosen so that a representa-
tive kinetic curve could be constructed. The resulting solution
was filtered over PTFE syringe filters, pore diameter 0.20 µm.
Samples were analyzed by HPLC. Blanks were obtained by
incubation of peptides in Tris (50 nM), pH 7.4/CH3CN (5:2)
with 1.5 µL of 1 N HCl for 6 h.
Hydrolysis results were collected in graphics: the amount
of starting peptide remaining vs time, given in area % of the
initial value calculated from HPLC. To appreciate the different
resistances displayed by the peptides, the areas were normal-
ized to 100 at t ) 0 and the curves were superimposed. Each
hydrolysis experiment was repeated at least twice, and the
reported data are mean values. Error ranges were estimated
on the basis of the standard deviation, and are substantially
similar for the different peptides. Peak attributions were
determined by comparison with authentic fragments prepared
by coupling the proper R- or â-amino acids by taking into
account the more plausible tetrapeptide decompositions. To
confirm the coincidence between authentic fragments and
fragments resulting from enzymatic digestion, we used ana-
lytical reversed-phase HPLC under two distinct column and
solvent systems (Table 3).
HP LC P u r ity a n d P ea k Attr ibu tion s. Method 1: HP
Hypersil ODS column (4.6 µm particle size, 100 Å pore
diameter, 250 mm) with solvent systems A (0.1% TFA in
water) and B (0.1% TFA in acetonitrile), gradient 100% A to
50% B in 20 min at a 1.0 mL/min flow, followed by 20 min at
50% B. Method 2: Phenomenex Luna C18 column (5 µm
particle size, 100 Å pore diameter, 250 mm) with solvent
systems A (0.1% TFA in water) and B (0.1% TFA in acetoni-
trile), gradient 95% A to 80% B in 50 min at 1.0 mL/min flow.
Deter m in a tion of In h ibition of Cyclic AMP Accu m u -
la tion . The activity of endomorphin-1 analogues was deter-
mined by measuring the potency for the inhibition of forskolin-
stimulated cyclic AMP accumulation in SH-SY5Y cells (obtained
from the European Collection of Cell Cultures, passage no. 12).
Cells were routinely grown as indicated by the purchaser. A
75 cm flask at 95-100% confluence was split into 24 wells and
incubated overnight. When the confluence arrived at 85-100%,
the medium was removed and the cells were washed three
times with PBS. Then 240 µL of medium without serum (F12/
DMEM (1:1) + 2 mM glutamine + 1% nonessential amino
acids (NEAA)) with 0.5 mM 3-isobutyl-1-methylxanthine
(IBMX; Sigma), the following protease inhibitors (captopril 25
(µg/mL), bacitracin (0.2 mg/mL), leupeptin (10 µg/mL), phen-
ylmethylsulfonyl fluoride (0.19 mg/mL), and aprotinin (5 IU/
mL)), and 0.01 M DADLE (δ-opioid receptors blocker) were
added, and the cells were incubated at 37 °C for 10 min.
Forskolin (10 µM) and the ligand of interest at the concentra-
tion range studied were added (30 µL of each, to a final volume
of 300 µL), and the cells were incubated at 37 °C for 15 min.
The cells were washed three times with PBS, then 500 µL of
95% ethanol was added to each well, the cells were incubated
at 37 °C for 5 min, then transferred to 1.5 mL tubes, and
centrifuged at 6000 rpm, for 5 min, at 4 °C, and then the
resulting supernatant was transferred to a new tube. The
pellets were washed with 500 µL of ethanol/water (2:1 v/v) and
centrifuged again at 6000 rpm, for 5 min, at 4 °C, and the
supernatant was added to the first one. Then the samples were
dried under vacuum by speed vac for 4-6 h until completely
dried, and then cyclic AMP levels were determined using a
commercial kit (Amersham). Each well was determined indi-
vidually, and the triplicates were averaged, IC50 values were
determined (Graph PAD Software, San Diego, CA.).
Ack n ow led gm en t. We thank MURST (60% and
Cofin ‘00) and Bologna University (Funds for Selected
Topics) for providing financial support. We thank Dr.
Paola Bocchini for MALDI-MS analysis and Dr. Marco
Lucarini for FAB-MS analysis.
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