The cis-4-amino-l-proline residue as a scaffold for the synthesis of cyclic and linear endomorphin-2 analogues

Article abstract of DOI:10.1021/jm201402v

Endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH₂) is an endogenous tetrapeptide that combines potency and efficacy with high affinity and selectivity toward the μ opioid receptor, the most responsible for analgesic effects in the central nervous system

Full text of DOI:10.1021/jm201402v

Article
pubs.acs.org/jmc
The cis-4-Amino-L-proline Residue as a Scaffold for the Synthesis of
Cyclic and Linear Endomorphin-2 Analogues
Adriano Mollica,*,† Francesco Pinnen,† Azzurra Stefanucci,† Federica Feliciani,† Cristina Campestre,†
Luisa Mannina,‡,§ Anatoly P. Sobolev,§ Gino Lucente,⊥ Peg Davis,# Josephine Lai,# Shou-Wu Ma,#
Frank Porreca,# and Victor J. Hruby▽
†Dipartimento di Scienze del Farmaco, Universita
̀
di Chieti-Pescara “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy
‡Dipartimento di Chimica e Tecnologie del Farmaco, “La Sapienza” Universita
̀
di Roma, P.le A. Moro 5, 00185 Rome, Italy
§Istituto di Metodologie Chimiche, Laboratorio di Risonanza Magnetica “Annalaura Segre”, CNR, Via Salaria Km 29.300, 00015
Monterotondo, Rome, Italy
⊥Dipartimento di Chimica e Tecnologie del Farmaco e Istituto di Chimica Biomolecolare, CNR Sezione di Roma, “La Sapienza”
̀
Universita di Roma, P.le A. Moro 5, 00185 Roma, Italy
#Department of Pharmacology and ▽Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721,
United States
S
* Supporting Information
ABSTRACT: Endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-
NH2) is an endogenous tetrapeptide that combines potency
and efficacy with high affinity and selectivity toward the μ
opioid receptor, the most responsible for analgesic effects in
the central nervous system. The presence of the Pro2
represents a crucial factor for the ligand structural and
conformational properties. Proline is in fact an efficient
stereochemical spacer, capable of inducing favorable spatial
orientation of aromatic rings, a key factor for ligand
recognition and interaction with receptors. Here the Pro2
has been replaced by 4(S)-NH2-2(S)-proline (cAmp), a
proline/GABA cis-chimera residue. This bivalent amino acid
maintains the capacity to influenc the tetrapeptide con-
formation and offers the opportunity to generate new linear models and unusually constrained cyclic analogues characterized by
an N-terminal Tyr bearing a free α-amino group. The results indicate that the new analogues do not show affinity for both δ and
κ opioid receptors and bind only poorly to the μ receptors (for cyclopeptide 9: Kiμ = 660 nM; GPI (IC50) = 1.4% at 1 μM; for
linear tetrapeptide acid 13: Kiμ = 2000 nM; GPI (IC50) = 0% at 1 μM; for linear tetrapeptide amide 15: Kiμ = 310 nM; GPI
(IC50) = 894 nM).
As found in the case of enkephalins and many other opioid
peptides, EMs contain a free and protonable Tyr NH2 which is
a key element for the μ-receptor recognition. However, a
distinctive structural characteristic of endomorphins is the
presence of the Pro residue in position 2 of the backbone.2 The
cis/trans isomerization around the Tyr1-Pro2 peptide bond
associated with the π−π interactions involving the three
aromatic residues represents a relevant factor in controlling
the preferred conformations of the tetrapeptide ligand. As
previously noted,3,4 the effect of this structural feature, together
with the presence of the free Tyr NH2, is enhanced by the
location of the Tyr1-Pro2 moiety within the biologically relevant
N-terminal tripeptide message sequence (Tyr-Pro-Phe) of this
class of opioid tetrapeptides.
INTRODUCTION
■
The two tetrapeptide amides endomorphin 1 (H-Tyr-Pro-Trp-
Phe-NH2; EM-1) and endomorphin 2 (H-Tyr-Pro-Phe-Phe-
NH2; EM-2) are endogenous opioid tetrapeptides isolated from
bovine and human brain.1 The high interest for these two
ligands is mainly based on their high selectivity and affinity for
μ-opioid receptors. Furthermore, EMs exhibit a strong
antinociceptive effect on acute pain similar to that of morphine
and are devoid of some undesirable side effects typical of
morphine. The neuropathic pains are also strongly relieved with
efficacy higher than that shown by the majority of the other
opioid peptides. Thus, both the simple chemical structure and
the beneficial effects shown by EMs in various pathological
conditions continue to stimulate design and synthesis of
analogues to improve the biological activity, the conformational
heterogeneity, and the other limitations typical of peptides used
as drugs.
Received: October 18, 2011
Published: March 7, 2012
© 2012 American Chemical Society
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Figure 1. Previously reported endomorphin-1 and endomorphin-2 cyclic analogues obtained by adopting the “head-to-tail strategy” (compounds A
and B)31,28 and the “side chain-to-side chain” strategy (compound C).32 Only the latter analogue maintains a free, biologically relevant, N-terminal
Tyr primary amino group.
In order to study structure−activity relationships and
develop potent and selective analgesics, a great number of
opioid peptide analogues have been synthesized and evaluated
in the past decade. Native opioid peptides show poor
bioavailability mainly due to the inability to penetrate the
blood−brain barrier5,6 and to the rapid degradation by
peptidases such as aminopeptidases and dipeptidyl peptidase
IV.7−9 To overcome these limitations, different strategies have
been developed and exhaustively reviewed.10−20 Results from
these studies suggest that the stabilization of folded structures
may enhance endomorphin activity and underscore at the same
time the relevant role that the spatial orientation of the three
aromatic side chains may play.3,21−27 In light of these
observations and taking into account the intrinsic limitations
of peptides as therapeutic agents, the synthesis of cyclic
analogues appears particularly appealing. Although the adopted
synthetic strategies may greatly differ, the most common
methods are based on the introduction of bivalent amino acids
so as to close lactam or disulfide bridges21,26−29 or, more
recently, on the adoption of ring closing metathesis.30
As compared with enkephalins, only few cyclic analogues of
endomorphins have been reported so far.28,31,32 A first
approach adopts the head-to tail type of cyclization and leads
to c[-Tyr-Pro-Phe-Phe-]28 or c[-Tyr-Pro-Trp-Phe-Gly-]31
models (Figure 1) characterized by a lactam bridge between
a C-terminal carboxyl group and the N-terminal Tyr NH2. In a
second strategy a side chain-to-side chain approach is adopted
and models of the type Tyr-c[-Lys-Phe-Phe-Asp-] or Tyr-c[-
Asp-Phe-Phe-Lys-] are obtained through incorporation of two
bivalent amino acids in positions 2 and 5 of a pentapeptide
backbone.32,33 In both cases some of the obtained cyclic EM
analogues show sensibly improved activity and selectivity and
this, despite the lack of elements, is considered relevant for the
activity, such as the protonable N-terminal Tyr NH2 or the Pro-
residue in position 2. These results strongly suggest to apply
the cyclization approach to EM analogues characterized by the
presence of a free N-terminal Tyr NH2 together with the rigid
skeleton of the Pro residue at position 2.
On the basis of the above considerations, we report here
synthesis and activity of new linear and cyclic models based on
the sequence of EM-2 and containing modification at the Pro2
(See Schemes 1 and 2). The relevant structural feature of the
new models resides on the utilization of cis-4-amino-L-proline
(cAmp) to replace the native Pro. This synthetic residue34,35
combines the conformational rigidity of the pyrrolidine ring
structure of the native Pro with the primary amino group of a γ-
amino-n-butyric acid skeleton, thus realizing a chimera amino
acid,36 namely proline/GABA cis-chimera. The presence of the
cAmp at position 2 of the tetrapeptide backbone does not alter
significantly the EM-2 amino acid sequence and offers at the
same time, as compared with the native Pro residue, an
additional amino group available for a cyclization reaction. This
leads to a “side-chain to tail” type of cyclization, namely an
amide bond between the cAmp-γNH2 and the Phe4 C-terminal
carboxyl (see Figure 2). The obtained cyclic system gives rise to
an 11-membered cyclotripeptide further constrained by the
presence of the pyrrolidine inside the ring. This is a very
unusual structure which resembles already reported small ring
cyclic tripeptides obtained by using β-amino acids, γ-amino
acids, or mixed systems of α- and β-residues15,37−43 as building
blocks.
The biological consequences of the insertion of a cAmp
residue in the linear EM analogues 13 (Tyr-cAmp-Phe-Phe-
OH) and 15 (Tyr-cAmp-Phe-Phe-NH2) (Scheme 2) possess-
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a
Scheme 1. Chemical Synthesis of Intermediates 1−8 and Final Product 9
a
Reagents and conditions: (a) 1, HCl·Phe-OMe, EDC, HOBt·H2O, NMM, DMF, 0 °C, 20 min, then room temperature, 24 h, 90−95%. (b) 2,
TFA/DCM 1:1, room temperature, 3 h, quantitative. (c) 3, Boc-cAmp (Cbz)-OH, EDC, HOBt·H2O, NMM, DMF, 0 °C, 20 min, then room
temperature, 24 h, 77−88%. (d) 4, 1 N NaOH, MeOH, room temperature, 3 h, quantitative. (e) 5, 10% Pd/C, MeOH, H2, room temperature, 2−6
h, 73−81%. (f) 6, PyBop, DIPEA, DMF, room temperature, 12 h, 58−60%. (g) 7, TFA/DCM 1:1, room temperature, 3 h, quantitative. (h) 8, Boc-
Tyr-OH, EDC, HOBt·H2O, NMM, DMF, 0 °C, 20 min, then room temperature, 24 h, 80−83%. (i) 9, TFA/DCM 1:1, room temperature, 3 h, 76−
93%.
ing a C-terminal free carboxyl or amide group, respectively,
have also been examined. The receptorial affinity of all cyclic
and linear analogues was determined by means of displacement
binding and in vitro bioactivity assays (Table 1).
concentration needed to shift the dose−response curve of PL-
017 2-fold to the right).
DISCUSSION AND CONCLUSIONS
■
In this study, we focused attention on the design of a new class
of linear and cyclic EM-2; namely, the 11-membered cyclic
peptide 9 and the two corresponding linear COOH terminal
and CONH2 terminal tripeptides 13 and 15 are reported. As
mentioned previously, attempts to synthesize cyclotripeptides
made up of α-amino acids (i.e., nine-membered cyclic systems)
are not successful because dimerization leading to cyclo-
hexapeptides or formation of more complex systems (aza-
cyclols) deriving from endoannular CO−NH interaction is,
with the only exception being the cyclization of N-substituted
α-amino acids such as proline, the unique result. In the present
case, due to the presence of cis-4-amino-L-proline, a new,
unusual, and stable 11-membered cyclotripeptide system has
been isolated and tested for its bioactivity as an EM analogue.
Data reported in Table 1 indicate that cyclization associated
with high restriction of the backbone flexibility produces
RESULTS
■
Biological tests (Table 1) performed on the linear tetrapeptide
containing a C-terminal free carboxylic group 13 showed that
the product was inactive at μ, δ, and κ opioid receptors. The
corresponding linear amide derivative 15 showed some activity
for μ opioid receptors. Also, the cyclic compound 9 showed a
certain affinity for μ receptors and no affinity for δ and κ
receptors. Furthermore, the second cationic center in position 4
of the cAmp moiety in products 13 and 15, in general, seems to
be less favorable for binding to μ, δ, and κ opioid receptors,
probably due to repulsive interactions with another cationic
center present in the binding pocket. The products were also
tested for μ and δ opioid antagonism; in particular, product 15
was the only one of this series of compounds with any
antagonist activity: 15 at 10 mM showed Ke = 1800 220 (the
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a
Scheme 2. Chemical Synthesis of Intermediates 10−12, 14 and Final Products 13 and 15
a
Reagents and conditions: (a) 10, TFA/DCM 1:1, room temperature, 3 h, quantitative. (b) 11, Cbz-Tyr-OH, EDC, HOBt·H2O, NMM, DMF, 0 °C,
20 min, then 24 h at room temperature, 80−89%. (c) 12, 1 N NaOH, MeOH, room temperature, 3 h, quantitative. (d) 13, HBr/AcOH 33%, room
temperature, 3 h, 70−75%. (e) 14, IBCF, NMM, THF, NH4OH, 30 min, −10 °C, then 3 h at room temperature, 85−87%. (f) 15, 33% HBr/AcOH,
3 h at room temperature, 65−73%.
Figure 2. Structure of 4-amino-n-butyric acid (GABA), cis-4-amino-(S)-proline (cAMP), and the here-reported cAmp-containing endomorphin-2
analogues.
analogue 9, which possesses a strongly altered biological profile.
9 displays potencies 70 times lower than that of the parent, thus
indicating that the new hydrogen bond pattern and the 3D
shape of the molecule is drastically modified and ultimately
affects the receptor binding mode of action.
With regard to the linear analogues 13 and 15, the
replacement of the proline residue in position 2 with a more
hydrophilic residue, namely cAmp, produces a drastic affinity
loss at both μ and δ receptors. To the best of our knowledge,
this is the first information about the influence exerted by an
additional cationic center positioned at the second position of
endomorphin-2. This result seems to indicate that the repulsion
between two closely located electric charges (namely on Tyr1
and cAmp2 residues, respectively) significantly and negatively
affects the properties bound to the backbone conformation and
the receptor recognition.
Further studies are currently in progress to investigate the
relationship between the conformation of different stereo-
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Table 1. Binding Affinity and in Vitro Activity for Compounds 9, 13, and 15
a
c
rMor, [3H]DAMGO
functional bioactivity
g
,
f
a
d
b
e
g
g
compound
log IC50
Kiμ
hDor, [3H]DPDPE, Kiδ
hKor, [3H]U69593, Kiκ
GPI (IC50)
15
MVD (IC50)
h
EM-2
9.6 0.98
660 79
2
510 35
0% at 1 μM
3.6% at 1 μM
20% at 1 μM
i
9
−5.86
−5.38
−6.18
nb
nb
nb
nb
1.4% at 1 μM
0% at 1 μM
894.50 126
13
15
2000 224
310 81
nb
nb
a
Competition analyses was carried out using membrane preparations from transfected HN9.10 cells that constitutively expressed the DOR and
b
c
d
MOR, respectively. Competition analyses for κ receptors were carried out using rat recombinant CHO cells. Kd = 0.85 0.2 nM. Kd = 0.50 0.1
nM. Kd = 2.0 0.05 nM. The Ki values are calculated using the Cheng and Prusoff equation to correct for the concentration of the radioligand used
in the assay. SEM. Data from ref 3. nb = no binding detected.
e
f
g
h
i
The linear products 13 and 15 were obtained from product 3 (see
Scheme 2). Product 3 was deprotected at the terminal N-Boc by
treatment with TFA in DCM 1:1 to give the TFA salt 10 that was
coupled to Cbz-Tyr-OH to give the tetrapeptide 11. This was
deprotected at the C-terminal by hydrolysis of the methyl ester group
to obtain the intermediate 12 as a common precursor of both 13 and
15. Compound 13 was obtained as hydrobromide salt by treatment
with HBr in glacial acetic while carboxy activation via mixed anhydride
followed by treatment with NH4OH gave the fully protected
tetrapeptide amide 14. This was deprotected with HBr in glacial
acetic acid to give the final amide 15 as hydrobromide salt.
isomeric analogues of the cyclotripeptide 9 and receptor
affinity, in particular concerning the relative spatial orientation
of the three aromatic side chains.
EXPERIMENTAL SECTION
■
General. All solvents, reagents, and starting materials were
obtained from commercial sources unless otherwise indicated. All
reactions were performed under N2 unless otherwise noted.
Intermediate products 1, 3, 6, and 8 were purified by silica gel
chromatography. Products 9, 13, and 15 used for the biological assay
were purified by RP-HPLC using a semipreparative Vydac (C18-
bonded, 300 Å) column and a gradient elution at a flow rate of 10 mL/
min. The gradient used was 10−90% acetonitrile in 0.1% aqueous TFA
over 40 min. Approximately 10 mg of crude peptide was injected each
time, and the fractions containing the purified peptide were collected
and lyophilized to dryness. The purity of the final products,
determined by NMR analysis and by analytical RP-HPLC (C18-
bonded 4.6 × 150 mm) at a flow rate of 1 mL/min on a Waters Binary
pump 1525 using an isocratic elution of 20% CH3CN/H2O 0.1% TFA,
monitored with a Waters 2996 Photodiode Array Detector, was found
to be >95%.
TFA·NH2-Phe-Phe-OMe (2). To a solution of N-Boc-Phe-OH (1
mmol) in DMF were added EDC (1.1 equiv), HOBt·H2O (1.1 equiv),
and one portion of NMM (2.2 equiv) at 0 °C under stirring. After 10
min, HCl·Phe-OMe (1 equiv) and a second portion of NMM (1.1
equiv) were added, the mixture was warmed to room temperature
overnight, DMF was evaporated under reduced pressure, the oily
residue was dissolved in EtOAc, and the organic layer was washed with
5% citric acid, NaHCO3 saturated solution (s.s.), and brine. The
organic layers were combined, dried under Na2SO4, filtered, and
evaporated under reduced pressure to give the crude product, which
1
was purified with silica gel column chromatography (90−95%). 1: H
1H NMR spectra were performed in CDCl3 or DMSO-d6 solution
on a Varian Inova operating at the 1H frequency of 300 MHz and on a
NMR (CDCl3) δ: 1.39 (s, 9H, C(CH3)3), 2.98−3.08 (m, 4H, βCH2
Phe), 3.68 (s, 3H, OCH3), 4.22−4.39 (m, 1H, αCH Phe1), 4.70−4.83
(m, 1H, αCH Phe2), 4.85 (d, 1H, Boc-NH), 6.24 (d, 1H, NH Phe2),
6.91−7.01 (m, 10H, Ar). ESI-HRMS for C24H31N2O5 [MH+], calcd
427.2254; found 427.2251. LRMS (ESI) m/z = 427.2.
1
Bruker AVANCE AQS600 operating at the H frequency of 600.13
MHz. Chemical shifts were referred to TMS as internal standard in the
case of CDCl3 solution and to the residual proton signal of DMSO at
2.5 ppm in the case of DMSO-d6 solution. Peptide structures were
determined by means of 2D NMR experiments namely 1H−1H
Compound 1 was dissolved in a 1:1 TFA/DCM mixture for 3 h at
r.t. under N2 atmosphere. Then the mixture was evaporated under
reduced pressure until complete elimination of TFA to give the crude
intermediate product 2 that was used in the next step without further
purification (88%): ESI-HRMS for C19H23N2O3 [MH+], calcd
327.1735; found 327.1733. LRMS (ESI) m/z = 327.2.
1
TOCSY and H−1H NOESY. Peptide structures were also confirmed
by high resolution-mass spectra (HR-MS)
2 ppm. For the final
products 9, 13, and 15 elemental analyses (within 0.4% of the
theoretical values) were performed.
Synthesis. Nα-Boc-cAmp(Cbz)-OH was synthesized based on a
well-defined protocol as previously reported.35,36 All couplings of the
linear intermediates and linear products were performed using the
standard coupling method of carbodiimide (EDC/HOBt/NMM) in
DMF as described below. The synthesis of the cyclic peptide 9 begins
with the coupling between Boc-Phe-OH and HCl·H-Phe-OMe (see
Scheme 1) to obtain a dipeptide 1. The dipeptide obtained was
deprotected at the N-terminal moiety by TFA 1:1 DCM. The resulting
TFA salts 2 were then coupled with Nα -Boc-cAmp(Cbz)-OH to yield
the tripeptide 3. This peptide was used in two different synthetic
pathways: to give the cyclic product 9 (see Scheme 1) and to give the
linear products 13 and 15 (see Scheme 2). The cyclic product 9 (see
Scheme 1) was obtained by deprotecting the tripeptide 3 at the
COOMe terminal by hydrolysis with 1 N NaOH in MeOH followed
by deprotection of the cAmp 4-amino group by hydrogenolysis with
10% Pd/C in MeOH to give 5. The deprotected tripeptide 5 was
cyclized using PyBop coupling reagent in a highly diluted DMF
solution (10−3 M). The reaction provided the cyclic products 6 in
good yields. Then 6 was N-terminal deprotected in TFA/DCM
mixture and the resulting TFA salt 7 coupled to Boc-Tyr-OH to give
8. Product 8 was N-terminal deprotected in TFA/DCM mixture to
give the resulting TFA salt 9.
N-Boc-cAmp(Cbz)-Phe-Phe-OMe (3). N-Boc-cAmp(Cbz)-OH
(1.2 equiv) was dissolved in DMF, and EDC (1.35 equiv) and
HOBt·H2O (1.35 equiv) were added to the mixture. Then TFA·Phe-
Phe-OMe (2) (1.30 equiv) and NMM (0.56 mL) were added. The
mixture was kept 12 h at room temperature, DMF was evaporated
under reduced pressure, the oily residue was dissolved in EtOAc, and
the organic layer was washed with 5% citric acid, NaHCO3 s.s., and
brine. The organic layers were combined, dried under Na2SO4, filtered,
and evaporated under reduced pressure to give the crude product that
1
was purified with silica gel column chromatography (84%): H NMR
(CDCl3) δ: 1.39 (s, 9H, C(CH3)3), 1.97−2.19 (m, 2H, Pro C3H2),
2.80−3.22 (m, 4H, βCH2Phe), 3.39−3.44 (m, 2H, Pro C5H2), 3.60 (s,
3H, OCH3), 4.15 (m, 1H, αCHPro), 4.24 (m, 1H, Pro C4H), 4.53 (m,
1H, αCHPhe2), 4.72 (m, 1H, αCHPhe3), 5.02 (m, 2H, CH2 Cbz),
6.22−7.72 (m, 18H, Ar and NH Cbz and NH Phe2,3). ESI-HRMS for
C37H45N4O8 [MH+], calcd 673.3225; found 673.3222. LRMS (ESI)
m/z = 673.3.
N-Boc-cAmp(Cbz)-Phe-Phe-OH (4). NaOH (1 N, 3 equiv) was
added slowly to a mixture of methyl ester 3 (0.54 equiv) in MeOH at 0
°C, and then the mixture was stirred 3 h at room temperature. Then
the organic solvent was evaporated, and distilled water was added to
the residue and extracted with one portion of diethyl ether. Then the
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aqueous layer was acidified by 1 N HCl to pH = 3, the precipitated
product was extracted with three portions of EtOAc. The organic
layers were combined, dried under Na2SO4, filtered, and evaporated
under reduced pressure to give the free C-terminal acid that was used
into the next step without further purification. 4: ESI-HRMS for
C36H43N4O8 [MH+], calcd 659.3127; found 659.3130. LRMS (ESI)
m/z = 659.3.
Cbz-Tyr-cAmp(Cbz)-Phe-Phe-OMe (11). To a solution of Cbz-
Tyr-OH (0.32 equiv) in DMF were added EDC (0.32 equiv),
HOBt·H2O (0.32 equiv), and NMM (0.075 mL) at 0 °C. Then
TFA·H-cAmp(Cbz)-Phe-Phe in DMF and NMM were added, and the
reaction mixture was kept 12 h at room temperature. Then DMF was
evaporated under reduced pressure, the oily residue was dissolved in
EtOAc, and the organic layer was washed with 5% citric acid, NaHCO3
s.s., and brine. The organic layers were combined and dried under
Na2SO4, filtered, and evaporated under reduced pressure to give the
crude product, purified with silica gel column chromatography (83%):
1H NMR ((CD3)2SO) δ: 1.68−2.33 (m, 2H, Pro C3H2), 2.61−2.77
(m, 2H, βCH2 Tyr1), 2.85−2.97 (m, 2H, βCH2 Phe3), 2.93−2.99 (m,
2H, βCH2 Phe4), 3.30−3.99 (m, 2H, Pro C5H2), 3.54 (s, 3H, OCH3),
4.11 (m, 1H, Pro C4H), 4.29 (m, 1H, αCH Tyr1), 4.35 (m, 1H, αCH
Pro), 4.45 (m, 1H, αCH Phe4), 4.47 (m, 1H, αCH Phe3), 5.03 (m, 2H,
CH2 Cbz), 6.64 (d, 2H, C3,5H Tyr1), 7.04 (d, 2H, C2,6H Tyr1), 7.47
(d, 1H, NH Pro), 7.55 (d, 1H, NH Tyr1), 8.10 (d, 1H, NH Phe3), 8.36
(d, 1H, NH Phe4). ESI-HRMS for C49H52N5O10 [MH+], calcd
870.3715; found 870.3718. LRMS (ESI) m/z = 870.4.
Cbz-Tyr-cAmp(Cbz)-Phe-Phe-OH (12). NaOH (1 N, 3 equiv)
was added slowly to a mixture of methyl ester 11 (0.54 equiv) in
MeOH at 0 °C and then stirred 3 h at room temperature. Then the
organic solvent was evaporated, distilled water was added to the
residue, and the mixture was extracted with one portion of diethyl
ether. Then the aqueous layer was acidified with 1 N HCl to pH = 3,
and the precipitated product was extracted with three portions of
EtOAc. The organic layers were combined, dried under Na2SO4,
filtered, and evaporated under reduced pressure to give the free C-
terminal acid, used in the next step without further purification. 12: 1H
NMR ((CD3)2SO) δ: 1.68−2.32 (m, 2H, Pro C3H2), 2.61−2.75 (m,
2H, βCH2 Tyr1), 2.84−2.99 (m, 2H, βCH2 Phe3), 2.91−3.03 (m, 2H,
βCH2 Phe4), 3.28−3.98 (m, 2H, Pro C5H2), 4.10 (m, 1H, Pro C4H),
4.28 (m, 1H, αCH Tyr1), 4.36 (m, 1H, αCH Pro), 4.41 (m, 1H, αCH
Phe4), 4.47 (m, 1H, αCH Phe3), 5.03 (m, 2H, CH2 Cbz), 6.64 (d, 2H,
C3,5H Tyr1), 7.04 (d, 2H, C2,6H Tyr1), 7.46 (d, 1H, NH Pro), 7.55 (d,
1H, NH Tyr1), 8.08 (d, 1H, NH Phe3), 8.18 (d, 1H, NH Phe4). ESI-
HRMS for C48H50N5O10 [MH+], calcd 856.3624; found 856.3627.
LRMS (ESI) m/z = 856.4.
N-Boc-cAmp-Phe-Phe-OH (5). Product 4 was dissolved in
MeOH, Pd/C 10% was added, and the mixture was kept under H2
atmosphere for 2−6 h at room temperature stirring vigorously. The
catalyzer was removed by paper filtration and the solvent evaporated
under reduced pressure. The obtained crude product 5 was used in the
next step without further purification. ESI-HRMS for C28H37N4O6
[MH+], calcd 525.2789; found 525.2791. LRMS (ESI) m/z = 535.3.
c[-4-NH-Pro-Phe-Phe-] (6). A mixture of the tetrapeptide Nα-Boc
protected 5 (10−3 mol) in DMF and DIPEA (0.5 mL) was added
dropwise to a solution of PyBop (1.5 equiv) in DMF and DIPEA (3
equiv) under stirring. The mixture was kept 12 h at room temperature,
DMF was evaporated under reduced pressure, and the oily residue was
dissolved in EtOAc. The organic layer was washed with 5% citric acid,
NaHCO3 s.s., and brine. The organic layers were combined and dried
under Na2SO4, filtered, and evaporated under reduced pressure to give
the crude product 6, which was purified by silica gel column
chromatography (60%): 1H NMR (CDCl3) δ: 1.29 (s, 9H, C(CH3)3),
2.04−2.27 (m, 2H, Pro C3H2), 2.95−3.25 (m, 4H, βCH2Phe), 3.44
(m, 1H, Pro C5Ha), 3.60 (m, 1H, Pro C5Hb), 3.78 (m, 1H, αCHPhe3),
4.18 (m, 1H, Pro C4H), 4.45 (m, 1H, αCHPhe2), 4.56 (m, 1H,
αCHPro), 6.00−7.30 (m, 13H, Ar and NH Phe2 and NH Phe3 and
γNHPro). ESI-HRMS for C28H35N4O5 [MH+], calcd 507.2637; found
507.2638. LRMS (ESI) m/z = 507.3.
TFA·c[-4-NH-Pro-Phe-Phe] (7). 6 (0.27 equiv) was dissolved in a
TFA/DCM 1:1 mixture for 3 h at r.t. under N2 atmosphere. Then the
mixture was evaporated under reduced pressure until complete
elimination of TFA to give the crude product 7 which was used in
the next step without further purification (89%): ESI-HRMS for
C23H27N4O3 [MH+], calcd 407.2158; found 407.2160. LRMS (ESI)
m/z = 407.2.
Boc-Tyr-c[-4-NH-Pro-Phe-Phe] (8). To a solution of Boc-Tyr-
OH (0.32 equiv) in DMF were added EDC (0.32 equiv), HOBt·H2O
(0.32 equiv), and NMM (0.075 mL) at 0 °C. Then TFA·c[4-NH-Pro-
Phe-Phe] in DMF and NMM were added to the reaction mixture to
2HBr·Tyr-cAmp-Phe-Phe-OH (13). 12 was dissolved in 33%
HBr/acetic acid mixture for 3 h at room temperature, and then the
mixture was evaporated under reduced pressure. The crude HBr salt
obtained was purified by HPLC C18 (H2O/CH3CN) (72%): 1H NMR
((CD3)2SO) δ: 1.85−2.49 (m, 2H, Pro C3H2), 2.85−3.00 (m, 2H,
βCH2 Phe3), 2.93−3.06 (m, 2H, βCH2 Phe4), 2.96−3.02 (m, 2H,
βCH2 Tyr1), 3.34−3.95 (m, 2H, Pro C5H2), 3.85 (m, 1H, Pro C4H),
4.38 (m, 1H, αCH Tyr1), 4.44 (m, 1H, αCH Phe4), 4.56 (m, 1H, αCH
Phe3), 4.62 (m, 1H, αCH Pro), 6.70 (d, 2H, C3,5H Tyr1), 7.06 (d, 2H,
C2,6H Tyr1), 7.96 (d, 3H, NH Pro), 8.17 (d, 3H, NH Tyr1), 8.45 (d,
1H, NH Phe4), 8.68 (d, 1H, NH Phe3). ESI-HRMS for C32H38N5O6
[MH+], calcd 588.2875; found 588.2873. LRMS (ESI) m/z = 588.3.
Cbz-Tyr-cAmp(Cbz)-Phe-Phe-NH2 (14). To the mixture of
tetrapeptide COOH terminal 12 (1 mmol) in THF at −10 °C were
added NMM (2.2 mmol) and isobutyl chloroformate (IBCF) (1.1
mmol) under stirring. After 15 min, NH4OH s.s. was added in excess,
and the mixture was kept for 15 min at −10 °C and then at room
temperature for 3 h. Then the solvent was evaporated under reduced
pressure, and the oily residue was dissolved in EtOAc. The organic
layer was washed with 5% citric acid, NaHCO3 s.s., and brine. The
organic layers were combined and dried under Na2SO4, filtered, and
evaporated under reduced pressure to give the crude product 14,
1
give 8 (83%): H NMR ((CD3)2SO) δ: 1.29 (s, 9H, C(CH3)3), 2.09
(m, 2H, Pro C3H2), 2.60−2.94 (m, 2H, βCH2 Tyr1), 2.86−3.05 (m,
2H, βCH2 Phe3), 3.08−3.20 (m, 2H, βCH2 Phe4), 3.48−4.00 (m, 2H,
Pro C5H2), 3.77 (m, 1H, αCH Phe4), 4.16 (m, 1H, αCH Tyr1), 4.34
(m, 1H, αCH Phe3), 4.40 (m, 1H, Pro C4H), 4.64 (m, 1H, αCH Pro),
6.59 (d, 1H, NH Pro), 6.65 (d, 2H, C3,5H Tyr1), 6.99 (d, 1H, NH
Tyr1), 7.10 (d, 2H, C2,6H Tyr1), 7.72 (d, 1H, NH Phe4), 8.27 (d, 1H,
NH Phe3). ESI-HRMS for C37H44N5O7 [MH+], calcd 670.3228; found
670.3231. LRMS (ESI) m/z = 670.3.
TFA·Tyr-c[-4-NH-Pro-Phe-Phe] (9). 8 (0.22 equiv) was dissolved
in a TFA/DCM 1:1 mixture for 3 h at r.t. under N2 atmosphere. Then
the mixture was evaporated under reduced pressure until complete
elimination of TFA to give the crude intermediate product 9, used in
the next step without further purification (87%): 1H NMR
((CD3)2SO) δ: 2.04−2.19 (m, 2H, Pro C3H2), 2.79−3.14 (m, 2H,
βCH2 Tyr1), 2.89−3.07 (m, 2H, βCH2 Phe3), 3.06−3.18 (m, 2H,
βCH2 Phe4), 3.24−3.97 (m, 2H, Pro C5H2), 3.71 (m, 1H, αCH Phe4),
4.12 (m, 1H, αCH Tyr1), 4.38 (m, 1H, Pro C4H), 4.40 (m, 1H, αCH
Phe3), 4.69 (m, 1H, αCH Pro), 6.44 (d, 1H, NH Pro), 6.72 (d, 2H,
C3,5H Tyr1), 7.18 (d, 2H, C2,6H Tyr1), 7.87 (d, 1H, NH Phe4), 8.04 (d,
1H, NH Tyr1), 8.27 (d, 1H, NH Phe3). ESI-HRMS for C32H36N5O5
[MH+], calcd 570.2712; found 570.2710. LRMS (ESI) m/z = 570.3.
TFA·H-cAmp(Cbz)-Phe-Phe-OMe (10). 3 (0.27 equiv) was
dissolved in a TFA/DCM 1:1 mixture for 3 h at r.t. under N2
atmosphere. Then the mixture was evaporated under reduced pressure
until complete elimination of TFA to give the crude intermediate
product 10, used in the next step without further purification (92%):
ESI-HRMS for C32H37N4O6 [MH+], calcd 573.2783; found 573.2780.
LRMS (ESI) m/z = 573.3.
1
which was purified with silica gel column chromatography. H NMR
((CD3)2SO) δ: 1.68−2.32 (m, 2H, Pro C3H2), 2.61−2.76 (m, 2H,
βCH2 Tyr1), 2.83−3.02 (m, 2H, βCH2 Phe3), 2.85−2.94 (m, 2H,
βCH2 Phe4), 3.30−3.99 (m, 2H, Pro C5H2), 4.11 (m, 1H, Pro C4H),
4.29 (m, 1H, αCH Tyr1), 4.34 (m, 1H, αCH Pro), 4.39 (m, 1H, αCH
Phe3), 4.39 (m, 1H, αCH Phe4), 5.03 (m, 2H, CH2 Cbz), 6.64 (d, 2H,
C3,5H Tyr1), 7.04 (d, 2H, C2,6H Tyr1), 7.50 (d, 1H, NH Pro), 7.58 (d,
1H, NH Tyr1), 7.99 (d, 1H, NH Phe3), 8.13 (d, 1H, NH Phe4). ESI-
HRMS for C48H51N6O9 [MH+], calcd 855.3744; found 855.3743.
LRMS (ESI) m/z = 855.4.
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Article
2HBr·Tyr-cAmp-Phe-Phe-NH2 (15). 14 was dissolved in 33%
HBr/acetic acid mixture for 3 h at room temperature, and then the
mixture was evaporated under reduced pressure. The crude HBr salt
obtained was purified by HPLC C18 (H2O/CH3CN) (75%): 1H NMR
((CD3)2SO) δ: 1.88−2.51 (m, 2H, Pro C3H2), 2.80−2.97 (m, 2H,
βCH2 Tyr1), 2.84−3.01 (m, 2H, βCH2 Phe4), 2.88−2.96 (m, 2H,
βCH2 Phe3), 3.47−3.98 (m, 2H, Pro C5H2), 3.87 (m, 1H, Pro C4H),
4.27 (m, 1H, αCH Tyr1), 4.45 (m, 1H, αCH Phe4), 4.51 (m, 1H, αCH
Phe3), 4.61 (m, 1H, αCH Pro), 6.70 (d, 2H, C3,5H Tyr1), 7.06 (d, 2H,
C2,6H Tyr1), 8.06 (d, 3H, NH Tyr1), 8.06 (d, 3H, NH Pro), 8.18 (d,
1H, NH Phe4), 8.62 (d, 1H, NH Phe3). ESI-HRMS for C32H39N6O5
[MH+], calcd 587.3047; found 587.3045. LRMS (ESI) m/z = 587.3.
Functional GPI and Mouse Vas Deferens (MVD) Assays. In
vitro biological assays were performed on 9 as TFA salts, and 13, 15 as
hydrobromide salts. GPI and MVD in vitro bioassays were performed
as described previously.43−45 Electrically induced smooth muscle
contractions of mouse vas deferens and guinea pig ileum longitudinal
muscle-myenteric plexus were used as bioassays. Tissue came from
male ICR mice weighing 25−30 g and from male Hartley guinea pigs
weighing 150−400 g. The tissues were tied to gold chains with suture
silk, suspended in 20 mL baths containing 37 °C oxygenated (95% O2,
5% CO2) Krebs bicarbonate solution (magnesium-free for MVD), and
allowed to equilibrate for 15 min. The tissues were then stretched to
optimal length previously determined to be 1 g tension (0.5 g for
MVD), allowed to equilibrate for 15 min, and stimulated transmurally
between platinum plate electrodes at 0.1 Hz for 0.4 ms pulses (2.0 ms
pulses for MVD) and supramaximal voltage. Drugs were added to the
baths in 20−60 μL volumes. The agonists remained in contact with the
tissue for 3 min, and the baths were then rinsed several times with
fresh Krebs solution until tissues regained predrug contraction height.
IC50 values represent the mean of not less than four tissues. IC50
estimates and relative potency estimates were determined by fitting the
mean data to the Hill equation by using a computerized nonlinear
least-squares method. All biological data are summarized in Table 1.
Radioligand Labeled Binding Assays. μ and δ Opioid
Receptors. Crude membranes were prepared as previously
described44,45 from transfected cells that express the MOR or
the DOR. The protein concentration of the membrane
preparations was determined by the Lowry method, and the
membranes were stored at −80 °C until use. Membranes were
resuspended in assay buffer [50 mM Tris, pH 7.4, containing
50 μg/mL bacitracin, 30 μM bestatin, 10 μM captopril, 100 μM
PMSF, 1 mg/mL BSA]. For saturation analysis, six concen-
trations of [3H]DAMGO (0.02−6 nM, 47.2 Ci/mmol) or six
concentrations of [3H]DPDPE (0.1−10 nM, 44 Ci/mmol)
were each mixed with 200 μg of membranes from MOR- or
DOR-expressing cells, respectively, in a final volume of 1 mL.
For competition analysis, 10 concentrations of a test compound
were each incubated with 50 μg of membranes from MOR- or
DOR-expressing cells and a Kd concentration of [3H]DAMGO
(1.0 nM, 50 Ci/mmol) or of [3H]DPDPE (1.0 nM, 44 Ci/
mmol), respectively. Naloxone at 10 μM was used to define the
nonspecific binding of the radioligands in all assays. All samples
were carried out in duplicate. The samples were incubated in a
shaking water bath at 25 °C for 3 h, followed by a rapid
filtration through Whatman grade GF/B filter paper (Gaithers-
burg, MD) presoaked in 1% polyethyleneimine and washing
four times each with 2 mL of cold saline, and the radioactivity
was determined by liquid scintillation counting (Beckman
LS5000 TD).
saturation binding assays, cell membrane suspensions were incubated
for 60 min at 25 °C with various concentrations [3H]U69593.
Nonspecific binding was determined in the presence of 10 μM of
naloxone. For the competitive binding assays, the cell membrane
suspensions were incubated for 60 min at RT with 3 nM [3H]U69593
in the presence of various concentrations of ligands. After incubation
for 60 min, the membrane suspensions were rapidly filtered, and the
radioactivity of each filter was then measured by liquid scintillation
counting. The binding assay results are the mean
SEM of four
independent experiments, each performed in duplicate.
ASSOCIATED CONTENT
* Supporting Information
■
S
Elemental analysis data for compounds 1−15 are reported.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39-085-3554476. Fax: +39-085-3554477. E-mail: a.
mollica@unich.it.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
Boc, tert-butyloxycarbonyl; BSA, bovine serum albumin; Cbz,
carbobenzoxy; GPI, guinea pig ileum; [3H]DAMGO, [3H]-[D-
Ala(2),N-Me-Phe-(4),Gly-ol(5)]enkephalin; [3H]-U69593,
[3H]-(+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-
oxaspiro[4.5]dec-8-yl]benzeneacetamide; DCM, dichlorome-
thane; DIPEA, diisopropylethylamine; [3H]-DPDPE, [3H]-
c[2-D-penicillamine,5-D-penicillamine]enkephalin; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide; DOR, δ
opioid receptor; EDC, 1-ethyl-(3-dimethylaminopropyl)-
carbodiimide; HOBt, 1-hydroxybenzotriazole; IBCF, isobutyl
chloroformate; MOR, μ opioid receptor; MVD, mouse vas
deferens; NMM, N-methylmorpholine; PMSF, phenylmethyl-
sulfonyl fluoride; RP-HPLC, reversed phase high performance
liquid chromatography; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; TMS, tetramethylsilane; PyBop, benzotriazol-
1-yloxytripyrrolidinophosphonium hexafluorophosphate
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