Hybrid peptides endomophin-2/DAMGO: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2013.07.044

Endomorphin-2 [Tyr-Pro-Phe-Phe-NH₂] and DAMGO [Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol] are natural (EM2) and synthetic (DAMGO) opioid peptides both selective for μ opioid receptor with high analgesic activity. In this work we report synthesis, in vitro and in vivo biological evaluation of five new hybrid EM2/DAMGO analogues, with the aim to obtain compounds with high affinity at μ-opioid receptor, high activity in animal nociception tests (hot plate and tail flick) and improved enzymatic stability. Double N-methylation on both Phe residues and C-terminal ethanolamide led to analogue 6e, which possesses a good in vitro μ affinity (K_i^μ = 34 nM), combined with a remarkable in vivo antinociceptive activity.

Full text of DOI:10.1016/j.ejmech.2013.07.044

European Journal of Medicinal Chemistry 68 (2013) 167e177
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Hybrid peptides endomorphin-2/DAMGO: Design, synthesis
and biological evaluation
,
a
a
a
Adriano Mollica a , Roberto Costante , Azzurra Stefanucci , Francesco Pinnen ,
*
Grazia Luisi a, Stefano Pieretti b, Anna Borsodi c, Engin Bojnik c, Sándor Benyhe c
a Dipartimento di Farmacia, Università degli Studi “G. d’Annunzio” di Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy
b Istituto superiore di Sanità, Dipartimento del Farmaco, Viale Regina Elena 299, 00161 Rome, Italy
c Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Temesvari krt 62, 6726 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Endomorphin-2 [Tyr-Pro-Phe-Phe-NH2] and DAMGO [Tyr-D-Ala-Gly-(NeMe)Phe-Gly-ol] are natural
Received 15 March 2013
Received in revised form
3 July 2013
Accepted 5 July 2013
Available online 11 August 2013
(EM2) and synthetic (DAMGO) opioid peptides both selective for
activity. In this work we report synthesis, in vitro and in vivo biological evaluation of five new hybrid
EM2/DAMGO analogues, with the aim to obtain compounds with high affinity at -opioid receptor, high
m opioid receptor with high analgesic
m
activity in animal nociception tests (hot plate and tail flick) and improved enzymatic stability. Double
N-methylation on both Phe residues and C-terminal ethanolamide led to analogue 6e, which possesses a
good in vitro
m
affinity (Kmi ¼ 34 nM), combined with a remarkable in vivo antinociceptive activity.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Analgesics
DAMGO
Endomorphins
Hot plate
Nociception
Opioid
Tail flick
1. Introduction
compared to a poor affinity for d and k receptors [2,9e12]. Endo-
morphins exhibit potent in vivo antinociceptive activity, which
duration depends on the species, pain tests applied, and adminis-
tration route [13e16]. In contrast to other opioids, including
morphine, EMs could also be effective in treatment of neuropathic
pain [17,18].
According to the message/address concept [19], endomorphins’
structure can be divided into two parts: the N-terminal sequence
Tyr-Pro-Trp/Phe, which represents the message sequence and pro-
vides the correct conformation in the receptor binding and the
Phe4-NH2 moiety, which is the address sequence which contributes
to peptide stability and selectivity [20e22]; the Pro2 residue acts as
stereochemical spacer, inducing the correct orientation of the other
residues necessary for receptoreligand interaction [23e26]. The
Tyr1-Pro2 amide bond exists in an equilibrium mixture of cis/trans
conformations [27]; the isomerization around this bond seems to
Morphine and other opioid drugs are the most used analgesics
for the relief of moderate and severe pain [1]. However, prolonged
use of these compounds leads to a well-known range of side-
effects, including tolerance, physical dependence and respiratory
depression [2,3].
Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, EM1) and Endomor-
phin-2 (Tyr-Pro-Phe-Phe-NH2, EM2) are endogenous
m-selective
opioid peptides originally isolated from bovine brain [4] and sub-
sequently from human brain cortex [5] by Zadina and Hackler.
Endomorphins (EMs) present a unique N-terminal sequence Tyr-
Pro-Trp/Phe structurally related to opioid peptide morphiceptin
[6,7], different from the typical N-terminal sequence Tyr-Gly-Gly-
Phe of other opioid peptides, such as enkephalins and dynorphins
[8]. This structural features deeply influence the activity, hence EMs
label
m
opioid receptor (MOR) with a high affinity and selectivity
be a crucial factor to control pep interaction between the three
aromatic rings, determining the preferred EM conformation
[23,28,29]. Keller et al. demonstrated that the bioactive confor-
mation is cis [30], after estimation of cis/trans EMs ratios in various
conditions [20e23,25].
* Corresponding author. Tel.: þ39 08713554476; fax: þ39 08713554470.
E-mail address: a.mollica@unich.it (A. Mollica).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.07.044

168
A. Mollica et al. / European Journal of Medicinal Chemistry 68 (2013) 167e177
Endomorphin-related ligands show favorable profiles in terms
of analgesia and tolerance/dependence, and seem to produce less
respiratory depression than other agonists [31], nevertheless the
susceptibility to enzymatic degradation [32,33] and a limited up-
take in the central nervous system of opioid peptides strongly re-
duces a possible therapeutic application [34].
To obtain more stable and efficacious
m selective agonists,
several chemical modifications of EMs were performed, and a large
number of products were synthesized [16,35e46].
DAMGO [Tyr-D-Ala-Gly-(N-Me)Phe-Gly-ol] is a highly
m selective
Scheme 1. Synthesis of intermediate 1a. Reagents and conditions: (a) SOCl2, MeOH.
opioid peptide, synthetized for the first time in 1980 [47]. Its
structural features are different from those of endomorphins being
more similar to enkephalins, in fact Tyr1 is conserved, D-Ala2 was
introduced in place of Gly, while N-methyl Phe in position 3 and the
C-terminus ethanolamine represent the distinctive features. In
mouse writhing nociception test, DAMGO was more than 100-fold
potent than morphine [47]. In addition, DAMGO displays an
increased enzymatic stability compared to other opioid peptides.
These characteristics make DAMGO excellent as radiolabeled ligand
2. Chemistry
All products were synthesized in solution phase [53]. For com-
pounds 6a and 6e, HCl$(NeMe)Phe-OMe was obtained starting
from commercially available Boc-(NeMe)Phe-OH by treatment
with SOCl2 in MeOH for 3 h at r.t. (Scheme 1) [54]. Coupling re-
actions were performed using EDC/HOBt/DIPEA in DMF, with the
exception of compound 6e, for which Bop-Cl/NMM in DMF were
used in the first two couplings. Deprotection of Na-tert-butylox-
ycarbonyl group was performed using 1:1 TFA/CH2Cl2 mixture for
1 h, under N2 atmosphere (Scheme 2).
for
m
receptors for in vitro binding assays, used as a standard [48,49].
In this work we report synthesis, in vitro and in vivo biological
evaluation of five new hybrid EM2/DAMGO analogues (Fig. 1). The
aim of this project is to combine the distinctive features of EMs and
DAMGO in order to obtain compounds with a remarkable affinity at
m-opoid receptor, high activity in animal nociception tests (hot
3. Results and discussion
plate and tail flick), together with an improved resistance toward
enzymatic degradation due to the presence of N-methylated resi-
dues as previously reported [50e52]. In this paper the typical EM
Pro2 residues have been maintained, as well as the DAMGO C-ter-
minal ethanolamine. N-methylation was performed on the Phe3
and Phe4 residues, with the exception for compound 6d, in which
Sarcosine (NeMe Gly) was used in place of Phe3.
The in vitro biological evaluation of novel EM2-DAMGO ana-
logues 6aee was performed as previously described [55e57] and
results are shown in Table 1. The binding affinity of the hybrid
peptides 6aee toward rat d- and rat m-opioid receptors was deter-
mined by competitive binding against [3H]Ile5,6deltorphin-II [58]
and [3H]DAMGO [49] respectively. For functional characterization
Fig. 1. Structures of Met/Leu-enkephalins, Endomorphin-2, DAMGO and analogues 6aee.

A. Mollica et al. / European Journal of Medicinal Chemistry 68 (2013) 167e177
169
Scheme 2. Synthesis of products 6aee. Reagents and conditions: (a) Boc-Phe-OH for compounds 2aeb; Boc-N(Me)Phe-OH for compounds 2c and 2e; Boc-Sar-OH for compound 2d;
EDC, HOBt$H2O, DIPEA, DMF for compounds 2aed; Bop-Cl, NMM, CH2Cl2 for compound 2e. (b) TFA 50% in CH2Cl2. (c) Boc-Pro-OH, EDC, HOBt$H2O, DIPEA, DMF for compounds 3ae
d; Boc-Pro-OH, Bop-Cl, NMM, CH2Cl2 for compound 3e. (d) TFA 50% in CH2Cl2. (e) Boc-Tyr-OH, EDC, HOBt$H2O, DIPEA, DMF for compounds 4aed; Boc-Tyr-OH, Bop-Cl, NMM, CH2Cl2
for compound 4e. (f) 2-aminoethanol, 60 ꢀC. (g) TFA 50% in CH2Cl2.
of the compounds, the dose-effect response for DOR and MOR
activation was evaluated by MVD and GPI assays respectively. As
shown in Table 1, compound 6d which presents sarcosine at position
MDV/GPI bioassays. This data is not surprising, being in full accor-
dance with a previous study showing that modifications of the ar-
omatic moiety in position 3 may deeply influence the activity of EM2
derivatives [39]. Compounds 6a and 6c, with a single N-MePhe
3, shows no affinity for both
m and d receptors, and no activity in
Table 1
Binding affinity and in vitro functional bioactivity of compounds 6aee.
Compound
rMOR,a [3H]DAMGO
rDOR,a [3H]Ile5,6-Deltorphin-II
Selectivity
Functional bioactivity
Kmi (nM)c
IC50
Kdi (nM)c
Kdi /Kmi
MVD (IC50
)
GPI (IC50
)
b
b
d,e
d,e
IC50
EM2
8.33 ꢁ 0.06
8.62 ꢁ 0.04
NAf
6.54 ꢁ 0.04
6.94 ꢁ 0.03
6.71 ꢁ 0.03
NAf
3.56 ꢁ 0.3
1.7 ꢁ 0.03
>10,000
224 ꢁ 18
90 ꢁ 4
NAf
>10,000
>10,000
1.6 ꢁ 0.02
3864 ꢁ 180
799 ꢁ 35
>10,000
e
510 ꢁ 35
e
15 ꢁ 2
DAMGO
NAf
e
e
e
[3H]Ile5,6 Deltorphin-II
8.51 ꢁ 0.06
5.28 ꢁ 0.04
5.97 ꢁ 0.06
NAf
e
e
6a
6b
6c
6d
6e
17.2
8.9
e
>10,000
1909 ꢁ 353
>10,000
>10,000
3641 ꢁ 470
717 ꢁ 58
165 ꢁ 14
503 ꢁ 26
>10,000
74 ꢁ 9
155 ꢁ 10
>10,000
34 ꢁ 2
NAf
>10,000
6401 ꢁ 122
e
7.35 ꢁ 0.04
5.23 ꢁ 0.02
188.3
a
Displacement of [3H]Ile5,6-Deltorphin-2 (
d
-selective) and [3H]DAMGO (
m-selective) from rat brain membrane binding site.
b
The logIC50 ꢁ standard error are expressed as logarithmic values determined from the nonlinear regression analysis of data collected from at least two independent
experiments performed in duplicate.
c
The Ki values are calculated using the Cheng and Prusoff equation to correct for the concentration of the radioligand used in the assay.
d
Concentration at 50% inhibition of muscle contraction at electrically stimulated isolated tissues (n ¼ 4).
e
ꢁS.E.M.
f
NA ¼ not binding affinity.

170
A. Mollica et al. / European Journal of Medicinal Chemistry 68 (2013) 167e177
residue, have a low affinity (>100) but a comparable activity with
EM2 at -receptor; no substantial affinity and activity at receptor
m
d
was detected. Compound 6b, with unsubstituted NH-amide bond,
shows a good affinity, with Kmi of 90 nM and the highest Kid of the
series, correlated with a slight activity in MVD assay. Analogue 6e,
with two NeMe residues, displays the highest affinity (34 nM) and
selectivity (188-fold) toward MOR, with a great activity in GPI
bioassay. As previously reported, N-methylation of EM2 is signifi-
cantly associated with the bioactive conformation of the modified
peptides and their interaction with the m-opioid receptor [50]. None
of analogues 6aee reached the affinity of parent compounds and
unexpectedly, the combination of structural features of two selective
m
agonists, such as EM2 and DAMGO, provided compounds with
residual
d
affinity (e.g. compound 6b, Kdi ¼ 799 nM). The
m binding
affinity and selectivity increase with grade of N-methylation, con-
firming that introduction of N-methylated amino acids, in addition
to well-known protection against proteolysis [44], produces an
increment in MOR binding in contrast to DOR, due to different
conformational requirement of these receptors.
Fig. 3. Antinociceptive effect (peak time) of EM2 and EM2 analogues 6aee at the dose
of 7 nmol after i.c.v administration in the tail flick test. The antinociceptive activity is
expressed as percentage of the maximum possible effect (% MPE.) ꢁ s.e.m. ** is for
P < 0.01 and *** is for P < 0.001 vs V (vehicle-treated animals). N ¼ 8e11.
It is worth to note that the presence of a single methyl on the
nitrogen in position 3 and 4 respectively (6a and 6c), does not
produce some changes in activity and receptor selectivity, while
their simultaneous presence determines an increase of potency and
selectivity toward
m receptor.
antinociceptive efficacy in the tail flick test and the lowest potency,
as demonstrated by the AD50 value, while in the hot plate test 6c
proves to be the less efficacious compound. This effect was previ-
ously observed by Goldber and Tseng [9,14] in their studies on C-
terminal amide to alcohol conversion, in which high-affinity and
low intrinsic efficiency agonists were reported. Compound 6d was
confirmed to be devoid of activity.
By comparing in vivo and in vitro activities with in vitro receptor
affinity, it should be observed that, despite a generally low affinity
toward ORs if compared with parent peptides, novel hybrid EM2/
DAMGO peptides, with the exception of compound 6d, have a good
antinociceptive profile. In particular, tail flick graphic (Fig. 3) shows
that peptides 6aec produce moderate analgesia, over 50% of the
maximum possible effect, and 6e almost reaches the level of EM2,
although it has a lower MOR affinity. This result can be explained
considering the better enzymatic stability of product 6e that
partially compensates the lower binding affinity. In order to better
evaluate the effects of the N-methylation and C-terminal ethano-
lamide conversion, the plasma stability of product 6e was tested
and compared to the parent peptide EM2 (Fig. 4).
Compounds 6aee were evaluated through in vivo tail flick and
hot plate tests, as previously reported [59]. Mice response to heat
was recorded after intracerebroventricular administration of EM2
and compounds 6aee (7 nmol). Maximum effects for each peptide
(efficacy) are shown in Figs. 2 and 3. Table 2 shows also the dose
that produces 50% of the maximum antinociceptive effect
(potency).
Analogue 6e, resulted the most potent compound in eliciting
antinociception in both the tests according to m-binding and GPI
assays data. In the hot plate test 6e reaches an MPE of 74%, lower
than EM2 (92%), but clearly higher than other analogues. These data
are further confirmed by tail flick nociception test, in which peptide
6e shows a more comparable efficacy respect to EM2. AD50 values
indicate that, in addition to the compound 6e, analogues 6b and 6c
have a discrete potency, in particular in the tail flick assay, with 3.44
and 3.03 nmol respectively. Compound 6a has the lowest
4. Conclusion
Systematic investigation of the effect of N-methylation joined to
C-terminal modification was performed in this study.
We synthetized and tested five new peptides with common
features of
m selective agonists EM2 and DAMGO. Compounds 6aec
Table 2
In vivo antinociceptive activity of EM2 and compounds 6aee.
a
a
Cmpd.
Hot plate, AD50
,
Tail flick, AD50,
(95% confidence limits),b nmol
(95% confidence limits),b nmol
EM2
6a
6b
6c
6d
6e
2.29 (1.20e4.37)
9.44(5.44e16.38)
6.66 (3.08e14.36)
9.60 (3.14e29.35)
NAc
1.49 (0.86e2.57)
5.56 (2.36e13.12)
3.44 (1.59e7.41)
3.03 (0.80e11.41)
NAc
3.77 (1.75e8.07)
2.65 (1.41e4.96)
a
Fig. 2. Antinociceptive effect (peak time) of EM2 and EM2 analogues 6aee at the dose
of 7 nmol after i.c.v administration in the hot plate test. The antinociceptive activity is
expressed as percentage of the maximum possible effect (% MPE.) ꢁ s.e.m. *** is for
P < 0.001 vs V (vehicle-treated animals). N ¼ 8e11.
AD50 ¼ dose that produces 50% of maximum antinociceptive effect.
b
The AD50 values and their 95% confidence limits were determined by using the
graded doseeresponse procedure described by Tallarida and Murray [60].
c
Not activity.

A. Mollica et al. / European Journal of Medicinal Chemistry 68 (2013) 167e177
171
(1.1 eq.), HOBt (1.1 eq.) and DIPEA (1.65 eq.) were added at 0 ꢀC.
After 10 min the amino acid or the intermediate peptide as TFA salt
(1 eq.) was added together with DIPEA (1.65 eq.). The reaction was
stirred for additional 10 min at 0 ꢀC, allowed to warm at r.t. and
stirred overnight. The solvent was evaporated under reduced
pressure, the residue was suspended in EtOAc and washed with
three portions of citric acid 5%, NaHCO3 s.s., and brine. The organic
layers were combined, dried under Na2SO4, filtered and evaporated
under reduced pressure to give a crude solid.
5.3. General N-terminal ethanolamide introduction procedure (B)
The N-Boc protected tetrapeptide was dissolved in a large excess
of ethanolamine and the reaction mixture was stirred at 60 ꢀC for
12 h. The ethanolamine was evaporated under reduced pressure,
and the residue was extracted with two portions of EtOAc. The
organic layers were combined, dried under Na2SO4, filtered and
evaporated under vacuum to give a crude solid.
Fig. 4. Stability curves of EM2 (red line) and compound 6e (purple line) in human
plasma. The samples were tested in three independent experiments (n ¼ 3) and
represent the mean ꢁ SEM. The significance among groups was evaluated with the
analysis of variance (two-way ANOVA test) followed by Bonferroni’s posthoc com-
parisons between compound 6e and EM2 using the statistical software GraphPad
Prism v.4. Statistical significance was P < 0.05 (**P < 0.01; ***P < 0.001); times from t0
to t5 present no statistical significance (P > 0.05). (For interpretation of the references
to color in this figure legend, the reader is referred to the web version of this article.)
5.4. HCl$(NeMe)Phe-OMe (1a)
A solution of Boc-(NeMe)-Phe-OH (1 eq.) was dissolved in
MeOH, then SOCl2 (2.2 eq.) was added dropwise at 0 ꢀC. The re-
action mixture was stirred at r.t. for 3 h, then it was concentrated
and treated several times with Et2O. Finally it was evaporated un-
der vacuum until to obtain the final product (quantitative).
display a moderate in vivo antinociceptive activity; compound 6d
was inactive at both in vitro and in vivo tests; conversely peptide 6e
showed the greatest analgesic potency, with m-receptor affinity and
Rf ¼ 0.63 (CH2Cl2/MeOH 9:1). 1HNMR (DMSO-d6)
d: 2.95e3.02 (2H,
m, b-CH2), 3.30 (3H, s, NeCH3), 3.75 (3H, s, COOCH3), 4.82 (1H, m, a-
selectivity comparable to EM2. Structureeactivity relationships
concluded that N-methylation is significantly associate with the
bioactive conformation of EM2 analogues and their interaction
with opioid receptors. In addition, the C-terminal amide conversion
to alcohol leads to high-affinity but low intrinsic efficiency agonists.
Metabolic stability studies, carried out on product 6e, confirmed
that multi-N-methylation and C-terminal modification with etha-
nolamide moiety confer to the derivative 6e a better plasma sta-
bility than parent peptide, with half life around 40 min, and could
be a useful tools to develop stable candidates as opioid drugs. In
conclusion, this work allowed to obtain compounds with prolonged
persistence on the opioid receptors. Nevertheless the two different
key features of EM2 and DAMGO were found to be not synergic for
binding, hence no enhancement of potency or efficacy have been
recorded. N-methylated analogues of endomorphin-2-ol described
in this paper can be viewed as interesting models for the study of
ligandereceptor interactions.
CH), 7.05e7.41 (5H, m, Ar), 9.36 (3H, s, NHþ3 ). ESI-HRMS calcd for
C12H17ClO2 m/z: 229.0995 [M þ H]þ; found 229.0992.
5.5. N-Boc-Phe-(NeMe)Phe-OMe (2a)
Coupling reaction was performed between Boc-Phe-OH and
HCl$(NeMe)-Phe-OMe (1a) according to general procedure A. The
product was purified by silica gel column chromatography (CH2Cl2/
EtOAc 9:1 to CH2Cl2/EtOAc 7:3) to obtain the pure product (58%).
Rf ¼ 0.63 (CH2Cl2/EtOAc 9:1). 1H NMR (CDCl3)
d: 1.37 (9H, s, Boc),
2.80e3.38 (5H, m, Phe2 -CH2 and Phe2 NeCH3), 3.44e3.56 (2H, m,
b
Phe1 -CH2), 3.69 (3H, s, COOCH3), 4.70 (1H, m, Phe1
b a-CH), 5.05
(1H, d, Phe1 NH), 5.22 (1H, m, Phe2
a-CH), 7.10e7.35 (10H, m, Ar).
ESI-HRMS calcd for C25H32N2O5 m/z: 441.2389 [M þ H]þ; found
441.2393.
5.6. N-Boc-Pro-Phe-(NeMe)Phe-OMe (3a)
5. Experimental
N-Boc deprotection and coupling reaction between Boc-Pro-OH
and TFA$Phe-(NeMe)-Phe-OMe were performed according to
general procedure A. The product was purified by silica gel column
chromatography (CH2Cl2/EtOAc 9:1 to CH2Cl2/EtOAc 6:4) to obtain
the pure product (32%). Rf ¼ 0.26 (CH2Cl2/EtOAc 8:2). 1H NMR
5.1. General
All the starting materials and reagents were purchased from
SigmaeAldrich (Sigma Aldrich Corp., Saint Louis, MO, USA). 1H
NMR spectra were determined in CDCl3 or DMSO-d6 solution with a
Varian 300 MHz (Varian Medical Systems, Inc., Palo Alto, CA, USA),
and chemical shifts were referred to TMS. The mass spectra were
performed on a TOF-ESI spectrometer. Thin layer chromatography
was performed on silica gel Merck (Merck & Co., Inc., Whitehouse
Station, NJ, USA) 60 F254 plates.
(DMSO-d6)
Pro
-CH2), 2.78e2.98 (4H, m, Phe2
(3H, s, Phe3 NeCH3), 3.24 (2H, m, Pro
d
: 1.35 (9H, s, Boc), 1.67 (2H, m, Pro
g
-CH2), 1.95 (2H, m,
-CH2 and Phe3
-CH2), 3.10
-CH2), 3.60 (3H, s, COOCH3),
-CH and Phe3
-CH),
b
b
b
d
4.05 (1H, m, Pro
a
-CH), 4.90 (2H, m, Phe2
a
a
7.00e7.35 (10H, m, Ar), 7.77 (1H, d, Phe2 NH). ESI-HRMS calcd for
C30H39N3O6 m/z: 538.2917 [M þ H]þ; found 538.2919.
5.7. N-Boc-Tyr-Pro-Phe-(NeMe)Phe-OMe (4a)
5.2. General coupling procedure (A)
N-Boc deprotection and coupling reaction between Boc-Tyr-OH
and TFA$Pro-Phe-(NeMe)-Phe-OMe were performed according to
general procedure A. The product was purified by silica gel column
chromatography (CH2Cl2/EtOAc 6:4 to CH2Cl2/EtOAc 1:1) to obtain
the pure product (72%). Rf ¼ 0.25 (CH2Cl2/EtOAc 1:1). 1H NMR
The Na terminal Boc-protected peptides were all deprotected by
a mixture of TFA in CH2Cl2 1:1 at r.t. The intermediate TFA salts were
used for subsequent reactions without further purification. The Na
Boc-protected amino acid (1.1 eq) was dissolved in DMF, then EDC

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A. Mollica et al. / European Journal of Medicinal Chemistry 68 (2013) 167e177
(CDCl3)
d
: 1.31 (9H, s, Boc), 1.80 (2H, m, Pro
-CH2), 2.77e3.02 (5H, m, Phe3
-CH2), 3.50 (2H, m, Pro
-CH2), 3.58 (3H, s, COOCH3), 4.38 (1H, m, Pro -CH), 4.47 (1H, m,
-CH), 5.20 (2H, m, Phe4
-CH and Tyr
b
-CH2), 2.00 (2H, m, Pro
procedure A. The product was purified by silica gel column chro-
matography (CH2Cl2/EtOAc 8:2 to CH2Cl2/EtOAc 3:7) to obtain the
pure product (10%). Rf ¼ 0.39 (CH2Cl2/EtOAc 1:1). 1H NMR (DMSO-
g
-CH2), 2.71e2.80 (2H, m, Tyr
b
b-CH2
and Phe4 NeCH3), 2.90e3.30 (2H, m, Phe4
b
d
Tyr
a
d6)
d
: 1.32 (9H, s, Boc), 1.90 (2H, m, Pro
g
-CH2), 2.70e2.80 (2H, m,
-CH2,
-CH2), 3.60 (3H, s, COOCH3), 4.12 (1H, m, Tyr
-CH e Phe4 -CH), 4.73 (1H, m, Phe3
a
-CH), 4.92 (1H, m, Phe3
a
a
Tyr
b
b
-CH2), 2.89 (2H, m, Pro
b-CH2), 2.90e3.10 (6H, m, Pro d
NH), 6.88 (1H, d, Phe3 NH), 6.90e7.30 (14H, m, Ar), 8.15 (1H, s, Tyr
OH). ESI-HRMS calcd for C39H48N4O8 m/z: 701.3550 [M þ H]þ;
found 701.3548.
Phe3
-CH2 e Phe4
b
a-
CH), 4.52 (2H, m, Pro
a
a
a-CH),
5.16 (1H, d, Tyr NH), 6.50 (1H, d, Phe3 NH), 6.65e7.30 (15H, m, Phe4
NH e Ar), 9.18 (1H, s, Tyr OH). ESI-HRMS calcd for C38H46N4O7 m/z:
671.3445 [M þ H]þ; found 671.3441.
5.8. N-Boc-Tyr-Pro-Phe-(NeMe)Phe-NH(CH2)2OH (5a)
Reaction was performed according to procedure B. The product
was purified by silica gel column chromatography (EtOAc/MeOH
98:2 to EtOAc/MeOH 94:6) to obtain the pure product (41%).
5.13. N-Boc-Tyr-Pro-Phe-Phe-NH(CH2)2OH (5b)
Reaction was performed according to procedure B. The product
was purified by silica gel column chromatography (CH2Cl2/EtOAc
3:7 to EtOAc/MeOH 92:8) to obtain the pure product (38%).
Rf ¼ 0.46 (EtOAc/MeOH 9:1). 1H NMR (DMSO-d6)
d: 1.35 (9H, s, Boc),
1.80 (2H, m, Pro g
-CH2), 2.65 (3H, s, Phe4 NeCH3), 2.68e2.72 (2H, m,
-CH2), 2.76e3.36 (12H, m, Phe3
-CH2), 4.24 (1H, m, Tyr
-CH and Phe3
b
-CH2, Phe4
b
-CH2, Pro
-CH), 4.36
-CH),
b-CH2,
Rf ¼ 0.39 (EtOAc/MeOH 9:1). 1H NMR (DMSO-d6)
d
: 1.39 (9H, s, Boc),
Tyr
ethanolamine 2 ꢂ CH2 and Pro
(1H, s, ethanolamine OH), 4.64 (2H, m, Pro
b
d
a
1.60 (2H, m, Pro
g
-CH2), 2.63 (2H, m, Tyr
b
-CH2), 2.90 (2H, m, Pro
-CH2 and ethanolamine
-CH2), 4.20 (1H, m, Pro -CH), 4.31 (2H,
a-CH), 4.45 (1H, s, ethanolamine OH), 4.68
b-
CH2), 2.95e3.10 (8H, m, Phe3
2 ꢂ CH2), 3.40 (2H, m, Pro
m, Tyr
-CH and Phe2
(1H, m, Phe4
b
-CH2, Phe4
b
a
a
4.80 (1H, m, Phe4
a-CH), 5.40 (1H, m, Tyr NH), 6.50e7.30 (15H, m,
d
a
Ar and Phe4 NH), 8.00 (1H, t, ethanolamine NH), 9.2 (1H, s, Tyr OH).
ESI-HRMS calcd for C40H51N5O8 m/z: 730.3816 [M þ H]þ; found
730.3820.
a
a
-CH), 5.03 (1H, d, Tyr NH), 6.60e7.30 (16H, m, Phe3
NH, Phe4 NH and Ar), 7.60 (1H, t, etanolamine NH), 9.22 (1H, s, Tyr
OH). ESI-HRMS calcd for C39H49N5O7 m/z: 700.3710 [M þ H]þ;
found 700.3711.
5.9. TFA$Tyr-Pro-Phe-(NeMe)Phe-NH(CH2)2OH (6a)
Deprotection of N-Boc-Tyr-Pro-Phe-(NeMe)Phe-NH(CH2)2OH
(5a) was performed by a mixture of TFA in CH2Cl2 1:1 to give the
5.14. TFA$Tyr-Pro-Phe-Phe-NH(CH2)2OH (6b)
final product (quantitative). 1H NMR (DMSO-d6)
d
: 1.23 (2H, m, Pro
-CH2), 2.62e3.47
-CH2 Pro -CH2 and
-CH), 4.53e4.60 (2H, m,
-CH), 4.66 (1H, s, ethanolamine OH), 4.88 (1H,
Deprotection of N-Boc-Tyr-Pro-Phe-Phe-NH(CH2)2OH (5b) was
performed by a mixture of TFA in CH2Cl2 1:1 to give the final
g
-CH2), 2.28 (2H, m, Tyr
b
-CH2), 2.44 (2H, m, Pro
b
(13H, m, Phe3
b
-CH2, Phe3 NeCH3, Phe4
b
d
product (quantitative). 1H NMR (DMSO-d6)
d
: 1.28 (2H, m, Pro
-CH2), 2.44 (2H, m, Pro -CH2), 2.73-3.24
-CH2, Phe4
-CH2 Pro -CH2 and ethanolamine
2 ꢂ CH2), 4.13 (1H, m, Pro -CH), 4.50e4.53 (2H, m, Tyr -CH and
Phe2 -CH), 4.65 (1H, s, ethanolamine OH), 4.67 (1H, m, Phe4
-CH),
g-
ethanolamine 2 ꢂ CH2), 4.31 (1H, m, Pro
Tyr
-CH and Phe2
m, Phe4
a
CH2), 2.28 (2H, m, Tyr
b
b
a
a
(10H, m, Phe3
b
b
d
a
-CH), 6.68e7.23 (16H, m, Ar), 7.58 (1H, t, ethanolamine
a
a
NH), 7.99 (2H, m, Phe3 NH), 9.35 (3H, s, Tyr NHþ3 ), 9.56 (1H, s, Tyr
OH). ESI-HRMS calcd for C37H44F3N5O8 m/z: 744.3220 [M þ H]þ;
found 744.3224.
a
a
6.68e7.25 (16H, m, Ar), 7.69 (1H, t, ethanolamine NH), 7.90e8.13
(2H, m, Phe3 NH and Phe4 NH), 9.36 (3H, s, Tyr NHþ3 ), 9.43 (1H, s, Tyr
OH). ESI-HRMS calcd for C36H42F3N5O8 m/z: 730.3064 [M þ H]þ;
found 730.3065.
5.10. N-Boc-Phe-Phe-OMe (2b)
Coupling reaction was performed between Boc-Phe-OH and
HCl$Phe-OMe (1b) according to general procedure A, obtaining the
pure product (quantitative). Rf ¼ 0.59 (CH2Cl2/EtOAc 9:1). 1H NMR
5.15. N-Boc-(NeMe)Phe-Phe-OMe (2c)
(CDCl3) d b b-
: 1.39 (9H, s, Boc), 3.04 (4H, m, Phe1 -CH2 and Phe2
Coupling reaction was performed between Boc-(NeMe)-Phe-
OH and HCl$Phe-OMe (1b) according to general procedure A,
obtaining the pure product (90%). Rf ¼ 0.59 (CH2Cl2/EtOAc 9:1). 1H
CH2), 3.66 (3H, s, COOCH3), 4.30 (1H, m, Phe2
a-CH), 4.70 (1H, m,
Phe1 -CH), 4.90 (1H, m, Phe2 NH), 6.25 (1H, d, Phe2 NH), 6.90e7.30
a
NMR (CDCl3)
3.30 (4H, m, Phe1
4.74e4.85 (2H, m, Phe1
d
: 1.28 (9H, s, Boc), 2.42 (3H, s, Phe1 NeCH3), 2.70e
(10H, m, Ar). ESI-HRMS calcd for C24H30N2O5 m/z: 427.2233
b
-CH2 and Phe2
b
-CH2), 3.63 (3H, s, COOCH3),
[M þ H]þ; found 427.2230.
a
-CH and Phe2
a
-CH), 6.34 (2H, d, Phe2 NH),
7.00e7.26 (10H, m, Ar). ESI-HRMS calcd for C25H32N2O5 m/z:
5.11. N-Boc-Pro-Phe-Phe-OMe (3b)
441.2389 [M þ H]þ; found 441.2386.
N-Boc deprotection and coupling reaction between Boc-Pro-OH
and TFA$Phe-Phe-OMe were performed according to general proce-
dure A, obtaining the pure product (quantitative). Rf ¼ 0.57 (CH2Cl2/
5.16. N-Boc-Pro-(NeMe)Phe-Phe-OMe (3c)
EtOAc 1:1). 1H NMR (CDCl3)
d: 1.37 (9H, s, Boc), 1.83 (2H, m, Pro
g-
N-Boc deprotection and coupling reaction between Boc-Pro-OH
and TFA$(NeMe)Phe-Phe-OMe were performed according to gen-
eral procedure A. The product was purified by silica gel column
chromatography (CH2Cl2/EtOAc 9:1 to CH2Cl2/EtOAc 1:1) to obtain
the pure product (32%). Rf ¼ 0.77 (CH2Cl2/EtOAc 8:2). 1H NMR
CH2), 2.10 (2H, m, Pro
b
-CH2), 2.80e3.10 (4H, m, Phe2
b
-CH2 and Phe3
b
a
-CH2), 3.16 (2H, m, Pro -CH2), 3.61 (3H, s, COOCH3), 4.10 (1H, m, Pro
d
-CH), 4.12 (1H, m, Phe2 -CH), 4.58 (1H, m, Phe3
a a-CH), 6.11 (1H, d,
Phe3 NH), 6.58 (1H, d, Phe2 NH), 6.90e7.30 (10H, m, Ar). ESI-HRMS
calcd for C29H37N3O6 m/z: 524.2761 [M þ H]þ; found 524.2765.
(CDCl3)
NeCH3), 2.80e3.26 (6H, m, Phe2
3.45 (2H, m, Pro -CH2), 3.67 (3H, s, COOCH3), 4.20 (1H, m, Pro
CH), 4.80 (2H, m, Phe2 -CH and Phe3
a-CH), 7.05e7.35 (10H, m, Ar),
d
: 1.44 (9H, s, Boc), 1.85 (2H, m, Pro
g
-CH2), 2.38 (3H, s, Phe2
-CH2 and Pro -CH2),
b
-CH2, Phe3
b
b
5.12. N-Boc-Tyr-Pro-Phe-Phe-OMe (4b)
d
a-
a
N-Boc deprotection and coupling reaction between Boc-Tyr-OH
and TFA$Pro-Phe-Phe-OMe were performed according to general
8.70 (1H, d, Phe3 NH). ESI-HRMS calcd for C30H39N3O6 m/z:
538.2917 [M þ H]þ; found 538.2918.

A. Mollica et al. / European Journal of Medicinal Chemistry 68 (2013) 167e177
173
5.17. N-Boc-Tyr-Pro-(NeMe)Phe-Phe-OMe (4c)
a-CH2), 7.00e7.35 (5H, m, Ar), 8.50 (1H, d, Phe NH). ESI-HRMS calcd
for C23H33N3O6 m/z: 448.2448 [M þ H]þ; found 448.2447.
N-Boc deprotection and coupling reaction between Boc-Tyr-OH
and TFA$Pro-(NeMe)Phe-Phe-OMe were performed according to
general procedure A. The product was purified by silica gel column
chromatography (CH2Cl2/EtOAc 9:1 to CH2Cl2/EtOAc 6:4) to obtain
the pure product (77%). Rf ¼ 0.67 (CH2Cl2/EtOAc 1:1). 1H NMR
5.22. N-Boc-Tyr-Pro-Sar-Phe-OMe (4d)
N-Boc deprotection and coupling reaction between Boc-Tyr-OH
and TFA$Pro-Sar-Phe-OMe were performed according to general
procedure A. The product was purified by silica gel column chro-
matography (EtOAc/MeOH 98:2 to EtOAc/MeOH 94:6) to obtain the
(CDCl3)
NeCH3), 2.68e3.18 (8H, m, Tyr
-CH2), 3.45 (2H, m, Pro -CH2), 3.71 (3H, s, COOCH3), 4.22 (1H, m,
Pro -CH), 4.55 (1H, m, Tyr -CH), 4.81 (1H,
-CH), 4.72 (1H, m, Phe4
m, Phe3
d
: 1.39 (9H, s, Boc), 1.85 (2H, m, Pro
g
-CH2), 2.51 (3H, s, Phe3
-CH2, Phe3 -CH2 e Phe4
b
-CH2, Pro
b
b
pure product (56%). Rf ¼ 0.52 (EtOAc/MeOH 9:1). 1H NMR (CDCl3)
d
:
b
d
a
a
a
1.35 (9H, s, Boc), 1.89 (2H, m, Pro g-CH2), 2.10 (2H, m, Pro b-CH2),
a
-CH), 5.20 (1H, d, Tyr NH), 6.70e7.30 (14H, m, Ar), 8.15
2.70e3.25 (7H, m, Tyr
b
-CH2, Phe
-CH2), 3.55 (3H, s COOCH3), 3.65 (2H, m, Sar CH2), 4.46
-CH), 4.68 (1H, m, Pro -CH), 5.03 (1H, m, Phe -CH),
b-CH2, Sar NeCH3), 3.42 and 2.67
(1H, s, Tyr OH), 8.75 (1H, d, Phe4 NH). ESI-HRMS calcd for
(2H, m, Pro
(1H, m, Tyr
d
a
C39H48N4O8 m/z: 701.3550 [M þ H]þ; found 701.3552.
a
a
6.65e7.30 (9H, m, Ar), 7.45 (1H, d, Tyr NH), 7.90 (1H, s, Tyr OH), 8.42
(1H, d, Phe NH). ESI-HRMS calcd for C32H42N4O8 m/z: 611.3081
[M þ H]þ; found 611.3078.
5.18. N-Boc-Tyr-Pro-(NeMe)Phe-Phe-NH(CH2)2OH (5c)
Reaction was performed according to procedure B. The product
was purified by silica gel column chromatography (EtOAc/MeOH
98:2 to EtOAc/MeOH 94:6) to obtain the pure product (20%).
5.23. N-Boc-Tyr-Pro-Sar-Phe-NH(CH2)2OH (5d)
Rf ¼ 0.65 (EtOAc/MeOH 9:1). 1H NMR (CDCl3)
d: 1.35 (9H, s, Boc),
Reaction was performed according to procedure B. The product
was purified by silica gel column chromatography (EtOAc/MeOH
95:5 to EtOAc/MeOH 9:1) to obtain the pure product (48%).
1.70 (2H, m, Pro g
-CH2), 2.52 (3H, s, Phe3 NeCH3), 2.55e3.24 (12H,
m Tyr
2 ꢂ CH2), 3.50 (2H, m, Pro
m, Phe3
-CH), 4.40 (1H, m, Tyr
OH), 4.80 (1H, m, Phe4
b
-CH2, Pro
b
-CH2, Phe3
-CH2), 4.16 (1H, m, Pro
-CH), 4.56 (1H, s, ethanolamine
b
-CH2, Phe4
b
-CH2 and ethanolamine
-CH), 4.36 (1H,
Rf ¼ 0.52 (EtOAc/MeOH 9:1). 1H NMR (CDCl3)
d
: 1.35 (9H, s, Boc),
d
a
a
a
1.90 (2H, m, Pro g-CH2), 2.14 (2H, m, Pro b-CH2), 2.93e3.50 (11H, m,
a
-CH), 5.61 (1H, s, Tyr NH), 6.65e7.26 (15H,
Tyr
(2H, m, Sar CH2), 3.76 (2H, m, Pro
4.63 (1H, s, ethanolamine OH), 4.70 (2H, m, Pro
b
-CH2, Phe
b
-CH2, Sar NeCH3 and ethanolamine 2 ꢂ CH2), 3.75
-CH2), 4.60 (1H, m, Tyr -CH),
-CH and Phe
m, ethanolamine NH and Ar), 8.05 (1H, d, Phe4 NH), 8.24 (1H, s, Tyr
OH). ESI-HRMS calcd for C40H51N5O8 m/z: 730.3816 [M þ H]þ;
found 730.3819.
d
a
a
a-
CH), 5.28 (1H, d, Tyr NH), 6.65e7.35 (10H, m, ethanolamine NH and
Ar), 7.76 (1H, d, Phe NH), 8.38 (1H, s, Tyr OH). ESI-HRMS calcd for
C33H45N5O8 m/z: 640.3346 [M þ H]þ; found 640.3342.
5.19. TFA$Tyr-Pro-Phe-(NeMe)Phe-NH(CH2)2OH (6c)
Deprotection of N-Boc-Tyr-Pro-Phe-(NeMe)Phe-NH(CH2)2OH
(5c) was performed by a mixture of TFA in CH2Cl2 1:1 to give the
5.24. TFA$Tyr-Pro-Sar-Phe-NH(CH2)2OH (6d)
final product (quantitative). 1H NMR (DMSO-d6)
d
: 1.24 (2H, m, Pro
-CH2), 2.62e3.22
-CH2 Pro -CH2 and
-CH), 4.51e4.58 (2H, m,
a-CH), 4.66 (1H, s, ethanolamine OH), 4.88 (1H,
Deprotection of N-Boc-Tyr-Pro-Sar-Phe-NH(CH2)2OH (5d) was
performed by a mixture of TFA in CH2Cl2 1:1 to give the final
g
-CH2), 2.26 (2H, m, Tyr
(13H, m, Phe3 -CH2, Phe4 NeCH3, Phe4
ethanolamine 2 ꢂ CH2), 4.29 (1H, m, Pro
Tyr
-CH and Phe2
m, Phe4
b-CH2), 2.46 (2H, m, Pro b
product (quantitative). 1H NMR (DMSO-d6)
d
: 1.77 (2H, m, Pro
g
b
-
-
b
b
d
a
CH2), 2.25 (2H, m, Pro
b-CH2), 2.92e3.46 (11H, m, Tyr -CH2, Phe
b
a
CH2, Sar NeCH3 and ethanolamine 2 ꢂ CH2), 3.72 (2H, m, Sar CH2),
3.78 (2H, m, Pro -CH2), 4.57 (1H, m, Tyr -CH), 4.64 (1H, s, etha-
nolamine OH), 4.74 (2H, m, Pro -CH and Phe -CH), 5.28 (1H, d, Tyr
a
-CH), 6.68e7.28 (16H, m, Ar), 7.66 (1H, t, ethanolamine
d
a
NH), 7.92e8.15 (2H, m, Phe3 NH and Phe4 NH), 9.38 (3H, s, Tyr NHþ3 ),
9.53 (1H, s, Tyr OH). ESI-HRMS calcd for C37H44F3N5O8 m/z:
744.3220 [M þ H]þ; found 744.3222.
a
a
NH), 6.65e7.26 (10H, m, ethanolamine NH and Ar), 7.96 (1H, d, Phe
NH), 8.13 (1H, s, Tyr OH), 9.37 (3H, s, Tyr NHþ3 ), 9.56 (1H, s, Tyr OH).
ESI-HRMS calcd for C30H38N3F5O8 m/z: 654.2751 [M þ H]þ; found
654.2748.
5.20. N-Boc-Sar-Phe-OMe (2d)
Coupling reaction was performed between Boc-Sar-OH and
HCl$Phe-OMe (1b) according to general procedure A, obtaining the
pure product (99%). Rf ¼ 0.67 (CH2Cl2/EtOAc 1:1). 1H NMR (CDCl3)
5.25. N-Boc-(NeMe)Phe-(NeMe)Phe-OMe (2e)
Boc-(NeMe)Phe-OH (1.1 eq) was dissolved in CH2Cl2, than
Bop$Cl (1.1 eq.), and DIPEA (1.65 eq.) were added at ꢃ15 ꢀC. After
30 min. HCl$(NeMe)-Phe-OMe (1a, 1 eq.) was added together with
DIPEA (1.65 eq.). The reaction was stirred at r.t. overnight. The
solvent was evaporated under reduced pressure; the residue was
suspended in EtOAc and washed with three portions of citric acid
5%, NaHCO3 s.s., and brine. The organic layers were combined, dried
under Na2SO4, filtered and evaporated under reduced pressure to
give a crude solid. The product was purified by silica gel column
chromatography (CH2Cl2/EtOAc 95:5 to EtOAc/MeOH 9:1) to obtain
the pure product (42%). Rf ¼ 0.39 (CH2Cl2/EtOAc 95:5). 1H NMR
d
: 1.27 (9H, s, Boc), 2.82 (3H, m, Sar NeCH3), 3.04e3.20 (2H, m, Phe
b
-CH2), 3.72 (3H, s, COOCH3), 3.83 (2H, m, Sar CH2), 4.90 (1H, m, Phe
a
-CH2), 6.36e6.57 (1H, d, Phe NH), 7.00e7.35 (5H, m, Ar). ESI-HRMS
calcd for C18H26N2O5 m/z: 351.31920 [M þ H]þ; found 351.1923.
5.21. N-Boc-Pro-Sar-Phe-OMe (3d)
N-Boc deprotection and coupling reaction between Boc-Pro-OH
and TFA$Sar-Phe-OMe were performed according to general pro-
cedure A. The product was purified by silica gel column chroma-
tography (CH2Cl2/EtOAc 2:8 to CH2Cl2/EtOAc 1:9) to obtain the pure
(CDCl3)
Phe1 NeCH3), 2.70e3.40 (4H, m, Phe1
(3H, s, COOCH3), 4.37 and 5.20 (1H, m, Phe2
a
d
: 1.38 (9H, s, Boc), 2.36 (3H, s, Phe2 NeCH3), 2.42 (3H, s,
-CH2 and Phe2
-CH2), 3.66
-CH), 4.80 and 5.20
product (76%). Rf ¼ 0.35 (EtOAc). 1H NMR (CDCl3)
d
: 1.35 (9H, s Boc),
b
b
1.80 (2H, m, Pro
g
-CH2), 2.10 (2H, m, Pro
b
-CH2), 2.90e3.30 (5H, m,
-CH2), 3.50 (2H, m, Sar
-CH), 4.90 (1H, m, Phe
Phe
b-CH2eSar NeCH3), 3.40 (2H, m, Pro
d
(1H, m, Phe1
a-CH), 6.80e7.35 (10H, m, Ar). ESI-HRMS calcd for
CH2), 3.60 (3H, s, COOCH3), 4.50 (1H, m, Pro
a
C26H34N2O5 m/z: 455.2546 [M þ H]þ; found 455.2547.

174
A. Mollica et al. / European Journal of Medicinal Chemistry 68 (2013) 167e177
5.26. N-Boc-Pro-(NeMe)Phe-(NeMe)Phe-OMe (3e)
9.43 (1H, s, Tyr OH). ESI-HRMS calcd for C38H46F3N5O8 m/z:
758.3377 [M þ H]þ; found 758.3380.
N-Boc-(NeMe)Phe-(NeMe)Phe-OMe (2e) was deprotected by a
mixture of TFA in CH2Cl2 1:1 at r.t. The intermediate TFA salt was
used for subsequent reaction without further purification. Boc-Pro-
OH (1.1 eq.) was dissolved in CH2Cl2, then Bop$Cl (1.1 eq.), and
DIPEA (1.65 eq.) were added at ꢃ15 ꢀC. After 30 min. TFA$(NeMe)
Phe-(NeMe)Phe-OMe (1 eq.) was added together with DIPEA (1.65
eq.). The reaction was stirred at r.t. overnight. The solvent was
evaporated under reduced pressure, the residue was suspended in
EtOAc and washed with three portions of citric acid 5%, NaHCO3 s.s.,
and brine. The organic layers were combined, dried under Na2SO4,
filtered, and evaporated under reduced pressure to give a crude
solid. The product was purified by silica gel column chromatog-
raphy (CH2Cl2/EtOAc 9:1 to EtOAc/MeOH 1:1) to obtain the pure
6. In vitro biological assays
6.1. Chemicals and radioligands
[3H]DAMGO ([D-Ala2, NMePhe4, Gly5-ol]enkephalin; 41 Ci/
mmol) and [3H]Ile5,6deltorphin-2; 48 Ci/mmol) were radiolabeled
in the Isotope Laboratory of BRC (Radiolab), Szeged as described
previously [58]. Tris(hydroxymethyl)amino-methane (Tris), and
the peptidase inhibitors bestatin and phosphoramidon were pur-
chased from SigmaeAldrich (St. Louis, MO, USA). Captopril was
obtained from The Squibb Institute for Medical Research (Prince-
ton, NJ).
product (34%). Rf ¼ 0.67 (CH2Cl2/EtOAc 1:1). 1H NMR (CDCl3)
d: 1.37
(9H, s, Boc), 1.60 (2H, m, Pro
g
-CH2), 2.01 (2H, m, Pro
b
-CH2), 2.52
6.2. Animals
(3H, s, Phe3 NeCH3), 2.55 (3H, s, Phe2 NeCH3), 2.90e3.20 (4H, m,
Phe2 -CH2 and Phe3
-CH2), 3.50 (2H, m, Pro -CH2), 3.67 (3H, s,
COOCH3), 4.51 (1H, m, Pro -CH), 5.60 (1H,
-CH), 4.70 (1H, m, Phe3
m, Phe2
b
b
d
Inbred Wistar rats (250e300 g body weight) were housed in the
local animal house of the Biological Research Center (BRC, Szeged,
Hungary). Animals were kept in groups of four, allowed free access
to standard food and tap water and maintained on a 12:12-h light/
dark cycle until the time of sacrifice. Animals were handled ac-
cording to the European Communities Council Directives (86/609/
ECC) and the Hungarian Act for the Protection of Animals in
Research (XXVIII.tv. 32.x). Accordingly, the number of animals and
their suffering were minimized.
a
a
a
-CH), 6.80e7.37 (10H, m, Ar). ESI-HRMS calcd for
C31H41N3O6 m/z: 552.3074 [M þ H]þ; found 552.3077.
5.27. N-Boc-Tyr-Pro-(NeMe)Phe-(NeMe)Phe-OMe (4e)
N-Boc deprotection and coupling reaction between Boc-Tyr-OH
and TFA$Pro-(NeMe)Phe-(NeMe)Phe-OMe were performed ac-
cording to general procedure A. The product was purified by silica
gel column chromatography (CH2Cl2/EtOAc 1:1) to obtain the pure
6.3. Rat brain membrane preparations
product (45%). Rf ¼ 0.53 (CH2Cl2/EtOAc 7:3). 1H NMR (CDCl3)
d: 1.36
Crude membrane fractions from brains of Wistar rats and guinea
pigs were prepared as described earlier [61]. Animals were
decapitated and the brains without cerebellum were quickly
removed and washed several times to remove any unwanted blood
or tissue particles with chilled 50 mM TriseHCl (pH 7.4) buffer. The
brains were blotted dry, weighed and suspended in 5 volumes/
weight of the original brain tissue with ice-cold 50 mM TriseHCl
(pH 7.4) buffer. The brains were than homogenized at 1000ꢂ rpm
with an electrically driven Braun Teflon-glass rota-homogenizer at
(9H, s, Boc), 1.95 (2H, m, Pro g-CH2), 2.001 (2H, m, Pro b-CH2), 2.53
(3H, s, Phe4 NeCH3), 2.75 (3H, s, Phe3 NeCH3), 2.80e3.40 (4H, m,
Phe3 -CH2 and Phe4
-CH2), 2.81 and 3.97 (2H, m, Tyr -CH2), 3.50
(2H, m, Pro -CH2), 3.69 (3H, s, COOCH3), 4.60 (1H, m, Tyr -CH),
4.74 (1H, m, Pro -CH), 5.15 (1H, d, Tyr NH),
-CH), 5.14 (1H, m, Phe3
5.53 (1H, m, Phe4
b
b
b
d
a
a
a
a
-CH), 6.58e7.40 (14H, m, Ar), 8.06 (1H, s, Tyr
OH). ESI-HRMS calcd for C40H50N4O8 m/z: 715.3707 [M þ H]þ;
found 715.3705.
4
ꢀC, using 10e15 strokes of the homogenizer. The final volume of
5.28. N-Boc-Tyr-Pro-(NeMe)Phe-(NeMe)Phe-NH(CH2)2OH (5e)
the homogenate was made up to 30 volume/weight of the brain and
filtered through four layers of gauze to remove any larger aggre-
gates. After centrifugation with a Sorvall RC5C centrifuge at
40,000ꢂ g (18,000ꢂ rpm) for 20 min at 4 ꢀC, the resulting pellet
was resuspended in fresh buffer (30 volumes/weight) by using a
vortex. The suspension was incubated for 30 min at 37 ꢀC to remove
any endogenous opioids. Centrifugation was repeated under the
same conditions as described above, and final pellet was re-
suspended in 5 volumes of 50 mM TriseHCl (pH 7.4) buffer con-
taining 0.32 M sucrose to give a final concentration of 3e4 mg/ml
protein. The presence of sucrose is necessary for stabilization of
proteins for storage. The membranes were kept in 5 ml aliquots
at ꢃ70 ꢀC until use. The binding activity of the protein remained
stable for at least two months. Membranes were thawed and re-
suspended in 50 mM TriseHCl (pH 7.4) buffer and centrifuged at
40,000ꢂ g for 20 min at 4 ꢀC to remove the sucrose. The resulting
pellets were taken up in appropriate fresh buffer and immediately
used in binding assays.
Reaction was performed according to procedure B, obtaining the
pure product (96%). Rf ¼ 0.64 (EtOAc/MeOH 9:1). 1H NMR (CDCl3)
d:
1.53 (9H, s, Boc), 1.85 (2H, m, Pro g-CH2), 2.07 (2H, m, Pro b-CH2),
2.37 (2H, m, ethanolamine
(3H, s, Phe3, NeCH3), 2.70e3.30 (10H, m, Tyr
-CH2, Phe4
-CH2, ethanolamine -CH2), 4.39 (1H, s, ethanolamine
OH), 4.60 (1H, m, Tyr -CH), 4.85 (1H, m, Pro -CH), 5.10 (1H, m,
Phe3 -CH), 5.20 (1H, d, Tyr NH), 5.40 (2H, m, Phe4
-CH and
a
-CH2), 2.50 (3H, s, Phe4 NeCH3), 2.60
-CH2, Pro
-CH2, Phe3
b
d
b
b
b
a
a
a
a
ethanolamine NH), 6.50e7.45 (14H, m, Ar), 8.17 (1H, s, Tyr OH). ESI-
HRMS calcd for C41H53N5O8 m/z: 744.3972 [M þ H]þ; found
744.3969.
5.29. TFA$Tyr-Pro-(NeMe)Phe-(NeMe)Phe-NH(CH2)2OH (6e)
Deprotection
NH(CH2)2OH (6e) was performed by a mixture of TFA in CH2Cl2 1:1
to give the final product (quantitative). 1H NMR (DMSO-d6)
: 1.28
(2H, m, Pro -CH2), 2.28 (2H, m, Tyr -CH2), 2.44 (2H, m, Pro -CH2),
2.53 (3H, s, Phe4 NeCH3), 2.65 (3H, s, Phe3, NeCH3), 2.73e3.24 (10H,
m, Phe3 -CH2, Phe4
-CH2 Pro
-CH2 and ethanolamine 2 ꢂ CH2),
4.13 (1H, m, Pro -CH), 4.50e4.53 (2H, m, Tyr -CH),
-CH and Phe2
4.65 (1H, s, ethanolamine OH), 4.67 (1H, m, Phe4
-CH), 6.68e7.25
(16H, m, Ar), 7.69 (1H, t, ethanolamine NH), 9.36 (3H, s, Tyr NHþ3 ),
of
N-Boc-Tyr-Pro-(NeMe)Phe-(NeMe)Phe-
d
g
b
b
6.4. Receptor binding assays
b
b
d
All binding assays were performed at 25 ꢀC for 30 min in 50 mM
TriseHCl buffer (pH 7.4) in a final volume of 1 ml, containing 1 mg
BSA and 0.2e0.4 mg/ml membrane protein. Rat brain membranes
were incubated with the type-specific radioprobes, such as the
a
a
a
a

A. Mollica et al. / European Journal of Medicinal Chemistry 68 (2013) 167e177
175
selective
m
receptor agonist [3H]DAMGO (0.9e1.2 nM) and
d
recep-
609/EEC on laboratory animal protection in Italy. Animal welfare
was routinely checked by veterinarians from the Service for
Biotechnology and Animal Welfare.
tor selective agonist [3H]Ile5,6deltorphin-2 (0.8e1.3 nM) in the
presence of unlabeled test ligands (their concentrations ranged
from 10e5 to 10e11 M). Conditions for incubations are given in the
figure legends. Non-specific binding was determined in the pres-
7.2. Nociception assay
ence of 10
mM naloxone. Three peptidase inhibitors, 1
mM captopril,
1
m
M bestatin and 1
m
M phosphoramidon were included in the
Antinociceptive responses were determined with the hot plate
test and the tail flick test as previously reported [60]. In the case of
hot plate test, the thermal nociception was assessed with a
commercially available apparatus consisting in a metal plate
25 ꢂ 25 cm (Ugo Basile, Italy) heated to a constant temperature of
55.0 ꢁ 0.1 ꢀC, on which a plastic cylinder (20 cm diameter, 18 cm
high) was placed. The time of latency (s) was recorded from the
moment the animal was inserted inside the cylinder up to when it
licked its paws or jumped off the hot plate; the latency exceeded
the cut-off time of 60 s. The baseline was calculated as mean of
three readings recorded before testing at intervals of 15 min. The
time course of latency was then determined at 5, 10, 15, 20 and
30 min after treatment. The tail flick latency was obtained using a
commercial unit (Ugo Basile, Italy), consisting of an infrared
radiant light source (100 W, 15 V bulb) focused onto a photocell
utilizing an aluminum parabolic mirror. During the trials the mice
were gently hand-restrained with a glove. Radiant heat was
focused 3e4 cm from the tip of the tail, and the latency (s) of the
tail withdrawal recorded. The measurement was interrupted if the
latency exceeded the cut off time (15 s at 15 V). Also in this case,
the baseline was calculated as mean of three readings recorded
before testing at intervals of 15 min and the time course of latency
determined at 5, 10, 15, 20 and 30 min after treatment. In both the
hot plate and tail flick tests, data were expressed as time course of
assay buffer to prevent metabolic inactivation of the compounds
tested [62]. Reaction was terminated and bound and free radio-
ligands were separated by rapid filtration under vacuum through
Whatman GF/C (radiolabeled peptides) filters by using Brandel
M24R Cell Harvester. Filters were washed three times with 5 ml ice-
cold 50 mM TriseHCl (pH 7.4) buffer. After filtration and separation
procedure had been completed, fiber-disks were dried under an
infrared lamp, and then removed from the filter-sheet by tweezers.
Each disk was inserted into UltimaGoldÔ environment friendly,
non-volatile, toluene-free scintillation cocktail, and placed into
individual sample vials (transparent glass, Packard). Bound radio-
activity was determined in Packard Tricarb 2300TR liquid scintil-
lation analyzer. Receptor binding experiments were performed in
duplicate and repeated at least three times. Experimental data were
analyzed and graphically processed by GraphPad Prism research
software package with standard office computers.
6.5. GPI and MVD in vitro bioassays
Electrically induced smooth muscle contractions of mouse vas
deferens and strips of guinea pig ileum longitudinal muscle
myenteric plexus were used as bioassays and performed as
described previously [63,64]. Tissues came from male ICR mice
weighing 25e30 g and from male Hartley guinea pigs weighing
150e400 g. The tissues were first 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 the
MVD), and allowed to equilibrate for 15 min. Tissues were then
stretched to optimal length previously determined to be 1 g ten-
sion, allowed to equilibrate for 15 min. The tissues were stimulated
transmurally between platinum plate electrodes at 0.1 Hz, 0.4 ms
pulses (2.0 ms pulses for MVD) at supramaximal voltage.
Endomorphin-2 and analogues 1e5 at five to seven different con-
the percentage of maximum effect (% MPE)
¼
(post drug
latency ꢃ baseline latency)/(cut-off time ꢃ baseline latency) ꢂ 100.
Various doses of endomorphin-2 and analogues 6aed and 6e were
injected i.c.v according to the following procedure: mice were
lightly anesthetized with isoflurane, and an incision was made in
the scalp. Injections were performed using a 10
ml Hamilton
microsyringe at a point 2-mm caudal and 2-mm lateral from the
bregma. Compounds were injected at a depth of 3 mm in a volume
of 5 mL. Complete doseeresponses curves were then established for
the analysis of 50% antinociceptive dose (AD50) values and slope
function. The AD50 values and their 95% confidence limits were
determined by using the graded doseeresponse procedure
described by Tallarida and Murray [60].
centrations were added to the baths in 20e60 mL volumes to pro-
duce cumulative doseeresponse curves. Percent inhibition was
calculated by using an average contraction height for 1 min pre-
ceding the addition of the peptide divided by contraction height
3 min after the exposure to the peptide. IC50 values are the mean of
not less than four separate assays. IC50 estimates and their associ-
ated standard errors were determined by fitting the mean data to
the Hill equation using a computerized least-squares method.
7.3. Metabolic stability in human plasma
The evaluation of the metabolic stability of product 6e
compared to that of EM2 was carried out following a previously
reported method [65].
7. In vivo nociception tests
495
m
L of thawed human plasma (previously frozen at ꢃ70 ꢀC)
was spiked with 5 mL EM2 and analogue 6e in DMSO to achieve a
7.1. Animals
final concentration of 100
m
g/mL, and then incubated at 37 ꢀC
(ꢁ1 ꢀC).
Male CD-1 mice weighing 25e30 g and male guinea-pigs
weighing 300e400 g (Harlan, Milan, Italy) were used for the ex-
periments. Animals were housed for at least 1 week before exper-
imental sessions in colony cages (7 mice in each cage; 4 guinea-pigs
in each cage) under standard light (light on from 7.00 a.m. to 7.00
p.m.), temperature (21 ꢁ 1 ꢀC), relative humidity (60 ꢁ 10%) with
food and water available ad libitum. The experiments were con-
formed to the guidelines for pain research with laboratory animals.
The research protocol was approved by the Service for Biotech-
nology and Animal Welfare of the Istituto Superiore di Sanità and
authorized by the Italian Ministry of Health, according to Legisla-
tive Decree 116/92, which implemented the European Directive 86/
Prepared samples were removed at several designated time
points and incubation was stopped by adding an equal volume of
the blocking solution 5% aqueous ZnSO4 solution, MeOH, and ACN
(5:3:2) which precipitated proteins. The mixture was vortexed and
centrifuged at 12,000 ꢂ g for 5 min, then 50
mL of clear supernatant
was directly injected into the HPLC system (Waters model 600
solvent pump and 2996 photodiode array detector, with XBridge
BEH 130 C18, 4.6 ꢂ 250 mm, 5
mm). Empower v.2 Software (Waters
Spa, Milford, MA, USA) was used for data acquisition and elabora-
tion. The samples were tested in three independents experiments
(n ¼ 3) and reported values represent the mean ꢁ the Standard
Error (SEM). The significance among groups was evaluated with the

176
A. Mollica et al. / European Journal of Medicinal Chemistry 68 (2013) 167e177
analysis of variance (two-way ANOVA test) followed by Bonferro-
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