Novel Cyclic Biphalin Analogues by Ruthenium-Catalyzed Ring Closing Metathesis: In Vivo and in Vitro Biological Profile

Article abstract of DOI:10.1021/acsmedchemlett.8b00495

In this work we report the application of the ring-closing metathesis (RCM) to the preparation of two cyclic olefin-bridged analogues of biphalin (Tyr-d-Ala-Gly-Phe-NH-NH a Phe a Gly a d-Ala a Tyr), using the second generation Grubbs' catalyst. The resulting cis- and trans-cyclic isomers were identified, fully characterized, and tested in vitro at μ (IR), ? (DOR), and ?° (KOR) opioid receptors and in vivo for antinociceptive activity. Both were shown to be full agonists at MOR and potential partial antagonists at DOR, with low potency KOR agonism. They also share a strong antinociceptive effect after intracerebroventricular (i.c.v.) and intravenous (i.v.) administration, higher than that of the cyclic biphalin analogues containing a disulfide bridge between the side chains of two d-Cys or d-Pen residues, previously described by our group.

Full text of DOI:10.1021/acsmedchemlett.8b00495

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Letter
Novel cyclic biphalin analogues by Ruthenium-catalysed
ring closing metathesis: in vivo and in vitro biological profile
Azzurra Stefanucci, Wei Lei, Stefano Pieretti, Marilisa Pia Dimmito, grazia
luisi, Ettore Novellino, Micha# Eligiusz Nowakowski, Wiktor Ko#mi#ski,
Sako Mirzaie, Gokhan Zengin, John M. Streicher, and Adriano Mollica
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/
acsmedchemlett.8b00495 • Publication Date (Web): 08 Mar 2019
Downloaded from http://pubs.acs.org on March 11, 2019
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Novel cyclic biphalin analogues by Ruthenium-catalysed ring closing
metathesis: in vivo and in vitro biological profile
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Azzurra Stefanucci,1 Wei Lei,2 Stefano Pieretti,3 Marilisa Pia Dimmito,1 Grazia Luisi,1 Ettore
Novellino,4 Michał Nowakowski,5 Wiktor Koźmiński,5 Sako Mirzaie,6 Gokhan Zengin,7 John M.
Streicher,2 Adriano Mollica1,*
1 Dipartimento di Farmacia, Università di Chieti-Pescara “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy.
2 Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ USA.
3 Istituto Superiore di Sanità, Centro Nazionale Ricerca e Valutazione Preclinica e Clinica dei Farmaci, Viale Regina
Elena 299, 00161, Rome, Italy.
4 Dipartimento di Farmacia, Università di Napoli “Federico II”, Via D. Montesano 49, 80131 Naples, Italy.
5 Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, 02-089
Warsaw, Poland.
6 Department of Biochemistry, Islamic Azad University, Sanandaj, Iran.
7 Department of Biology, Science Faculty, Selcuk University, Konya, Turkey.
KEYWORDS: biphalin, ring closing metathesis, opioids, olefin, antinociception
ABSTRACT: In this work we report the application of the Ring-Closing Metathesis (RCM) to the preparation of two cyclic
olefin bridged analogues of biphalin (Tyr-D-Ala-Gly-Phe-NH-NH<-Phe<-Gly<-D-Ala<-Tyr), using the second generation
Grubbs’ catalyst. The resulting cis and trans cyclic isomers were identified, fully characterized and tested in vitro at  R),
 (DOR) and  (KOR) opioid receptors and in vivo for antinociceptive activity. Both were shown to be full agonists at MOR
and potential partial antagonists at DOR, with low potency KOR agonism. They also share a strong antinociceptive effect after
intracerebroventricular (i.c.v.) and intravenous (i.v.) administration, higher than that of the cyclic biphalin analogues containing
a disulfide bridge between the side chains of two D-Cys or D-Pen residues, previously described by our group.
Peptide cyclization represents a common strategy for the
development of compounds with enhanced conformational
stability, compared to their linear analogues. Cyclic peptides
may act as mimetics of protein secondary structures, and are
often able to freeze the molecule in the bioactive
conformation, leading to the optimization of ligand’s
activity in terms of binding affinity, potency, selectivity and
protease stability.1,2 Disulfide-bridge incorporation into a
linear peptide allows the design of synthetic models
characterized by the limitation of conformational flexibility
to form a well-defined secondary structure, but the disulfide
bond itself is labile and sensitive to the activity of
reductases, and can be cleaved by various nucleophiles.
Moreover, the stereoelectronic hindrance of sulfur atoms’
lone pairs guide the geometry of the bond fixing the dihedral
angle C-S-S-C at a value of ±90°.3
counterparts.7 The cyclization reaction utilized ruthenium-
catalyzed ring-closing olefin metathesis (RCM), affording
cis and trans olefin analogues of enkephalins, that resulted
in metabolic stability and rigid conformation.8
Despite the amount of literature reporting the use of RCM in
peptide chemistry,9 and a great variety of enkephalin
derivatives,10 this cyclization strategy has not yet been
proven on bivalent analogues such as biphalin. Biphalin is
an octapeptide characterized by two enkephalin-derived
pharmacophores joined together by a hydrazine bridge;
biphalin is able to bind  and -opioid receptors with an
EC50 of 1-5 nM, with a strong antinociceptive effect
associated with few side-effects and low dependence upon
chronic use.11 This profile makes biphalin a strong lead
compound for further modification. The combination of the
entropic factors related to the two pharmacophore branches
in biphalin with the favorable properties of a cyclic
compound, could result in the design of more stable and
potent opioid peptide analogues.
In searching for more stable and versatile products, the
replacement of the labile S-S junction in cyclic peptides
with an uncleavable C-C bond appears to be a promising
strategy. Dicarba-analogues of oxytocin incorporating a 1,6-
’-diamino-suberic acid residue, in place of the cysteine
residue,4,5 and cyclic olefin analogues prepared starting from
linear D-allylglycine containing peptides,6 are notable
applications of this strategy. In the field of opioid peptide
analogues, the incorporation of a C-C bond into a cyclic
structure resulted in highly active and potent  analogues
of H-Tyr-c[D-Cys-Gly-Phe-Cys]-OH, with increased
chemical and biological stability compared to the disulfide
Indeed, our research group described a series of cyclic
biphalin analogues containing a disulfide bridge between the
side chains of Cys and D-Pen residues in place of the D-Ala
amino acid,12 different xylene isomers,13 and a fluorescein-
maleimide moiety (Figure 1).14 The first two classes of
compounds are characterized by mixed -opioid agonism,
while the analogue containing the o-xylene moiety shows an
improved human plasma stability compared to biphalin.
While an improvement, these compounds may still be
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ACS Medicinal Chemistry Letters
limited in their pharmacokinetic (PK) properties by the
labile sulfur bonds. We then hypothesized that cyclic
biphalin analogues with a covalent C-C bond would further
improve the PK properties of our earlier generations of
cyclic biphalin analogues. Thus in this work we describe the
synthesis of cyclic analogues of biphalin in which the D-Ala
amino acids in position 2,2’ have been replaced with D-
allylglycine (D-allyl-Gly) residues to promote cyclization
mediated by the RCM reaction.
Page 2 of 7
Their remarkable stability to air, water, acid and functional
group tolerance render these group of catalysts suitable for
applications in peptide chemistry.16,17 We first explored
RCM using Zhan-1B catalyst and Grubbs I generation under
several reaction conditions,18 leading to major recovery of
the starting material and undetermined byproducts, probably
due to the low solubility of the linear compound in the
reaction solvent. The desired cyclic product 4 was finally
obtained in good yield as a mixture of cis/trans isomers,
using the more stable and reactive Grubbs II generation
catalyst in DCE under reflux for 78 h. The ring closure was
achieved with a catalytic amount of catalyst (10%) to a
solution of peptide 3 in DCE at reflux, monitoring by TLC
(AcOEt/MeOH = 97:3) until reaction completion.
1
2
3
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H
N
H
H
N
H
N
N
HN
NH
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HN
NH
O
O
O
O
S
O
O
O
O
O
O
O
O
HN
NH
NH
HN
NH
NH
S
HN
HN
R'
R'
S
S
R
R
O
O
a
O
O
H2
N
NH2
OH
H2
N
The mixture of the Boc protected cyclic products is
inseparable by C18-HPLC, thus the two isomeric peptides
cis-5a (ABAM-A) and trans-5b (ABAM-B) were obtained
by HPLC separation after Boc removal, in about 1.4:1 ratio,
respectively. The cis and trans isomers of the TFA salts
were clearly distinguishable in preparative C-18 column
showing different retention times, thus they were separated
and characterized. The two novel compounds were obtained
in good overall yields (52% for cis-5a and 36% for trans-
5b) and ≥ 95% purity following analytical RP-HPLC.
Because of the symmetry of the two isomers, the analysis of
the J coupling constant of the olefin group was not possible.
Thus in this case the assignment was based on the chemical
shift of the carbon in alpha position to the double bond (C
Table 1S see SI). Their chemical shifts differ markedly by
around 5 ppm, due to the gauche -substituent effect and
permit identification of ABAM-B as the trans isomer and
ABAM-A as the cis isomer.19,20
NH2
OH
HO
R, R' = H or CH3
HO
b
ref. 12
ref. 13
DISULFIDE BOND
I GENERATION CLIPS
O
O
O
O
H
H
H
H
N
N
N
N
NH2
H2
N
N
H
N
N
N
H
H
H
O
O
O
O
BIPHALIN
OH
HO
H
H
N
N
HN
NH
O
O
O
O
O
O
HN
NH
NH
HN
S
S
O
O
H2N
NH2
OH
HO
O
O
N
III GENERATION CLIPS
HOOC
O
ref. 14
OH
O
c
Figure 1. Cyclic biphalin analogues prepared via different
strategies: (a) disulfide bond containing analogues,12 (b)
xylene moiety containing analogue via I generation CLIPS
technology,13 (c) fluorescein-maleimide containing analogue
via III generation CLIPS technology.14
Competition radioligand binding tests were then performed
against the non-selective opioid antagonist [3H]-
Diprenorphine to measure compound affinity for opioid
receptors: Ki values for ABAM-A and ABAM-B are 75.8
and 53.6 nM respectively at MOR and 7.3 and 5.2 nM for
DOR, while both have low affinity for KOR (495 and 686
nM) (Figure 3S, Table 1). The compounds are modestly
selective for DOR>MOR>>>KOR, and there is no apparent
difference between the two ABAM compounds in their
binding profile.
H
N
H
N
H
H
N
N
HN
NH
HN
NH
O
O
O
O
O
O
O
O
O
O
O
O
HN
NH
NH
HN
HN
NH
NH
H
H
HN
O
O
O
O
4
. H2
TFA
N
NH2
BocHN
.TFA
NHBoc
OH
OH
HO
HO
trans-5b
ABAM-B
cis-5a
ABAM-A
We then tested the ABAM compounds for their functional
activity at the opioid receptors using 35S-GTPγS coupling
(Figure 4S, Table 2). Both compounds showed full efficacy
and high potency at the MOR, with a very similar profile to
the prototypical high efficacy full agonist DAMGO. The
compounds further showed a higher potency than binding
affinity (Table 1), suggesting that the compounds possess
high intrinsic efficacy at the MOR. Interestingly, the
compounds showed no detectable agonist activity at the
DOR, despite the high binding affinity detected in Table 1.
The prototypical DOR agonist SNC80 exerted the expected
activity, validating the assay. Lastly, the compounds showed
full efficacy but low potency agonist activity at the KOR, in
line with the low binding affinity of the compounds for the
KOR.
Figure 2. Structures of 5a (ABAM-A), 5b (ABAM-B) and
Boc-protected precursor 4.
Two cyclic geometric isomers of biphalin cis-5a and trans-
5b (Figure 2) were separated and purified by RP-HPLC,
then characterized by NMR analysis and identified by
HRMS. The in vitro binding affinity and G protein
stimulation on  R),  (DOR) and  (KOR) opioid
receptors combined with in vivo antinociceptive efficacy
were then evaluated.
RESULTS AND DISCUSSION
The linear intermediate peptides were prepared in solution
using N-Boc strategy, EDC.HCl/anhydrous HOBt/NMM in
DMF for coupling reactions and a solution of TFA/DCM =
1:1 for Boc deprotection (Scheme 1S, see SI).15 All the
intermediate products were purified by trituration in diethyl
ether and then characterized by LRMS and NMR
spectroscopy (see SI). For the preparation of the cyclic Boc-
protected analogue 4 via RCM, the reaction conditions were
extensively optimized; Ruthenium carbene complexes are
easier to prepare and handle than molybdenum complexes.
Both ABAM compounds showed high DOR affinity (Table
1) but no DOR agonist activity (Table 2), suggesting that the
compounds could be antagonists for the DOR. We thus
tested the antagonist activity of the ABAM compounds vs.
100 nM SNC80 at the DOR using 35S-GTPγS coupling
(Figure 3). Intriguingly, both compounds showed potent
(44.8 nM for ABAM-A and 0.36 nM for ABAM-B) but
partial (25.6% and 27.3%) antagonist activity at the DOR.
The positive control antagonist naloxone displayed full
(100%) antagonist activity, validating the assay. These
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ACS Medicinal Chemistry Letters
results are unexpected; as weak partial antagonism would
classically be correlated with relatively high efficacy partial
agonism. However, the ABAM compounds showed no
detectable partial agonist activity (Table 2). We further
confirmed the lack of ABAM antagonist activity at the DOR
using a forskolin-induced cAMP assay; neither ABAM
compound produced antagonism, while the selective DOR
antagonist naltrindole did (Figure 5S, SI).
experiments. A) The summary curves combined from each
independent experiment are shown. B) The antagonist
potency (IC50) and efficacy (IMAX) was calculated separately
from each experiment and reported as the mean ± SEM. The
IMAX of each compound was normalized to the inhibition
caused by the positive control antagonist naloxone (100%).
It is unclear why ABAM compounds show this profile at
DOR, which could include multiple explanations. The
ABAM compounds also show their strongest potency
difference in this assay, with ABAM-B showing a ~100-fold
potency improvement over ABAM-A. This difference was
not observed in the other assays, and the compounds should
be further investigated for their unique molecular
pharmacology at DOR. Also of note, these results show a
strong difference from the profile of biphalin, which is a
high potency full agonist at both MOR and DOR. These
results suggest that the ligands exhibit high potency as MOR
agonist/DOR partial antagonist compounds, with a binding
affinity for DOR higher than that of MOR with very low
binding affinity and potency for KOR. This profile is in
contrast with the cyclic amide analogues of enkephalins
described by Purington,21 Prydzial,22 and Schiller,23 for
which a high binding affinity has been demonstrated for all
three opioid receptors. In particular, peptide Tyr-c[D-Cys-
Phe-2-Nal-Cys]NH2 exerted agonism at MOR and KOR,
while displaying antagonism at DOR in the GTPγS binding
assay;21 our novel compounds have lost the high potency
KOR agonism exhibiting a biological profile that closely
resemble that of the KSK-103 compound.24 Notably, the two
geometrical isomers do not show any significant difference
in their opioid profile with the exception of their DOR
antagonist potency (Figure 3). Encouraged by these data, the
antinociceptive effect of ABAM-A and ABAM-B was
measured by the hot plate test in mice after i.v. and i.c.v.
administration (Figure 4). Biphalin was used as a reference
compound, with vehicle administration as a negative
control. ABAM-B produced an MPE% ≥ 50% that lasted
over 60 min after i.c.v. and 45 min after i.v. administration,
implying a longer lasting analgesic effect than that of
ABAM-A, which fell under the 50% mark at both of these
timepoints. Both compounds exerted a strong analgesic
effect higher than that of biphalin after i.c.v. and i.v.
administration; this result suggests that both ABAM ligands
are resistant to enzymatic degradation and are also able to
cross the BBB to reach the CNS. Analogous behavior has
been observed previously with other biphalin analogues
published by our research group.12,13 These results support
our hypothesis that replacement of the labile disulfide link in
the cyclic analogs further improves on the in vivo
performance of the parent ligand biphalin. The cyclic MOR
agonists/DOR antagonists DIPPΨNH2 described by Schiller
et al.23 and KSK-103 reported by Purington et al.24 showed
poor BBB penetration and have poor bioavailability. In this
case glycosylation of peptides has been demonstrated to
improve metabolic stability and CNS activity following
peripheral administration.24-26 However, our compounds
show high efficacy after i.v. administration, suggesting high
BBB penetration. Our compounds may thus improve on
these earlier generations of compounds.
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2
3
4
5
6
7
8
9
Ligands
Compounds Ki (nM)
DOR
MOR
KOR
-
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Naloxone
U50,488
ABAM-A
ABAM-B
51.2 ± 2.5
-
75.8 ± 11.9
53.6 ± 2.7
70.0 ± 2.9
-
7.3 ± 0.7
5.2 ± 2.7
23.9 ± 4.2
495 ± 43
686 ± 53
Table 1. Opioid receptor affinity of ABAM-A and ABAM-
B. ABAM compounds or naloxone (MOR, DOR) or
U50,488 (KOR) positive control were competed against 3H-
diprenorphine in membranes from CHO cells expressing one
of the human opioid receptors. Data reported as the mean ±
SEM of N = 3 independent experiments. The affinity values
(Ki) are shown for all compounds, calculated separately
from each experiment and reported as the mean ± SEM. The
naloxone and U50,488 positive controls showed expected
values at the receptors, validating the assays.
MOR
DOR
EC50
(nM)
KOR
EMAX
Ligands
DAMGO
SNC80
EC50
EMAX
(%)
100
EMAX
(%)
-
EC50
(nM)
-
(nM)
19.7
± 3.4
-
(%)
-
-
-
-
210 ±
69
-
100
-
-
-
U50,488
-
7.8 ±
0.4
100
ABAM-
A
19.6
± 4.7
91.0
± 3.0
NC
NC
NC
1843
± 169
69.0 ±
3.6
ABAM-
B
17.7
± 2.6
85.7
± 2.6
NC
969 ±
64
73.3 ±
0.3
Table 2. Opioid receptor functional activity of ABAM-A
and ABAM-B. The functional agonist activity of the
compounds was measured using 35S-GTPγS coupling in the
membranes of CHO cells expressing one of the human
opioid receptors. Data reported as the mean ± SEM of N = 3
independent experiments. The data for each set of assays
was normalized to the maximum stimulation (100%) caused
by positive control agonist (DAMGO for MOR; SNC80 for
DOR; U50,488 for KOR) or vehicle (0%). The potency
(EC50) and efficacy (EMAX) of each compound from each
receptor calculated separately from each experiment and
reported as the mean ± SEM. Positive control agonists
behaved as expected, validating the assays. The EMAX was
normalized to the maximum stimulation (100%) caused by
positive control agonist.
In conclusion, the substitution of the disulfide bridge in the
first cyclic analogue of biphalin27 with an olefin bridge leads
to geometrical isomer analogues with less affinity at MOR
and DOR in respect to the previously reported
analogues.12,13,27 Surprisingly the novel compounds show an
increased potency and efficacy in G protein stimulation at
MOR, with no observed activation of the DOR. Indeed, our
compounds show a slightly improved DOR binding
selectivity and a strong analgesic activity higher than that of
Figure 3. DOR antagonist activity of ABAM-A and
ABAM-B. ABAM compounds or naloxone positive control
was competed against 100 nM of SNC80 at the DOR using
35S-GTPγS coupling. Data was normalized to the stimulation
caused by 100 nM SNC80 (100%) or vehicle (0%) and
reported as the mean ± SEM of N = 3 independent
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the cyclic analogue containing D-Pen,12 in the hot-plate test
Page 4 of 7
2000 Da at 5 sec/scan; capillary voltage 3.5 kV, cone
voltage 10 V, desolvation temperature 180, desolvation gas
flow (N2) 200 L/h, source temperature 110 °C. The purity of
each sample was established by analytical RP-HPLC with a
C18-bonded column 4.6 mm × 150 mm at 236 and 268 nm,
flow rate of 1 mL/min, gradient of H2O/ACN-0.1% TFA
from 5% to 95% ACN in 30 min, and was found to be
≥95%.
1
2
3
4
5
6
7
8
after i.v. and i.c.v. administration. These results indicate that
the ABAM compounds are MOR agonists /DOR partial
antagonists, different from the mixed R/DOR agonist
activity exerted by biphalin and its cyclic analogues reported
in the literature so far.12-14,27 In this work we have described
a cyclic biphalin analogue endowed with MOR agonist
/DOR partial antagonist activity, which retains a good
binding affinity for MOR and DOR, avoiding KOR binding;
this latest aspect could be crucial in the design of safer drugs
without undesirable side effects, such as tolerance and
dependence, that hamper the clinical use of opioid
analgesics.
Chemistry. Intermediate compound 1 has been previously
described and characterized in literature.27 The linear
intermediate peptides 2 and 3 were synthesized in solution
using N-Boc strategy, coupling reactions have been
performed using EDC.HCl/anhydrous HOBt /NMM in
DMF, and Boc removal with a mixture of TFA/DCM = 1:1
at r.t. for 1 h (Scheme 1S, see SI).15 For the RCM reaction, a
catalytic amount of the second generation Grubbs catalyst
(10%) was added to a solution of peptide 3 (100 mg, 0.086
mmoL) in DCE (80 mL) at 80 °C for 78 h. The ring closure
was controlled by monitoring with TLC (AcOEt/MeOH =
97:3) until reaction completion. Then the solvent was
evaporated by rotavapor and the crude residue was triturated
with diethyl ether (3 times). The solid residue was dried in
high vacuum to give product 4 as inseparable mixture of
cis/trans isomers in quantitative yield (see SI). N-Boc
protection was removed with a solution of TFA/DCM = 1/1,
the two isomeric peptides cis-5a (ABAM-A) and trans-5b
(ABAM-B) were isolated by RP-HPLC and obtained in
about 1.4:1 ratio, in 52% and 36% overall yields
respectively. Assignment of NMR signals was performed
using standard set of NMR spectra (ROESY, COSY,
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TOCSY, and H-13C HSQC) (Table 1S, see SI); relevant
1
parameters are given in Table 2S (see SI). The spectra were
measured on DDR2 Agilent 600 MHz spectrometer
equipped with a Triax probe. The chemical shifts were
calibrated on DMSO signal.
Figure 4. In vivo hot-plate tests on ABAM-A and ABAM-
B after i.c.v. and i.v. administrations. In these experiments,
peptides were administered at the dose of 0.15 nmol/mouse
for i.c.v. administration and at the dose of 1.5 mol/kg for
i.v. administration. * is for p<0.05, ** is for p<0.01, **** is
for p<0.0001 vs B. N=10. Data reported as the mean ± SEM
of the maximum possible effect (MPE) in the hot plate
assay.
c[TFA.NH2-Tyr-D-allyl-Gly-Gly-Phe-NH]2 (cis-5a). HPLC
1
rt = 16.30 (min); H-NMR  DMSO-d6): 2.22-2.32 (4H, m,


D-allyl-Gly CH2), 2.67-2.91 (4H, m, Tyr CH2), 2.83-3.07


(4H, m, Phe CH2), 3.31-3.94 (4H, dd, Gly CH), 3.84 (2H,


q, Tyr CH), 4.32 (2H, q, D-allyl-Gly CH), 4.55 (2H, q,


Phe CH), 5.18 (2H, q, D-allyl-Gly CH), 6.68 (4H, dd, Tyr
aromatics), 7.02 (4H, dd, Tyr aromatics), 7.20-7.26 (10H, m,
Phe aromatics), 7.99 (2H, d, Phe NH), 8.51 (2H, t, Gly NH),
8.57 (2H, d, D-allyl-Gly NH), 9.29 (2H, s, OH Tyr); 13C-
NMR (DMSO-d6): 29.9, 36.9, 42.3, 51.6, 52.2, 114.9,
126.1, 126.6, 127.8, 128.9, 130.1. ESI-HRMS: Calcd exact
mass without TFA for C48H56N10O10 m/z = 933.4259
[M+H]+; found m/z 933.4221.
EXPERIMENTAL PROCEDURES
General Information.
All reagents, solvents, and starting materials were purchased
by Sigma-Aldrich (MI, Italy) and VWR International
(Pennsylvania, US). All the intermediate products have been
purified by trituration in diethyl ether and then characterized
c[TFA.NH2-Tyr-D-allyl-Gly-Gly-Phe-NH]2
(trans-5b).
1
by LRMS and H-NMR spectroscopy. The final TFA salts
1
5a and 5b were purified by RP-HPLC on a VP 250/16
Nucleosil 100-7 C18, 5.0 μm, 250 mm × 10 mm column at a
flow rate of 7 mL/min with a Waters Binary pump 600,
linear gradient of H2O/ACN-0.1% TFA from 5% to 95%
ACN in 30 min. The identity of the Nα-Boc-protected
HPLC rt = 17.04 (min); H-NMR  DMSO-d6): 2.14-2.19
(4H, m, D-allyl-Gly CH2), 2.68-2.89 (4H, m, Tyr CH2),
2.80-3.09 (4H, m, Phe CH2), 3.25-3.99 (4H, dd, Gly CH),






3.89 (2H, q, Tyr CH), 4.33 (2H, q, D-allyl-Gly CH), 4.53


(2H, q, Phe CH), 5.22 (2H, q, D-allyl-Gly CH), 6.68 (4H,
dd, Tyr aromatics), 7.02 (4H, dd, Tyr aromatics), 7.20-7.27
(10H, m, Phe aromatics), 8.15 (2H, d, Phe NH), 8.46 (2H, t,
Gly NH), 8.52 (2H, d, D-allyl-Gly NH), 9.30 (2H, s, OH
Tyr); 13C-NMR (DMSO-d6): 35.2, 36.8, 42.0, 51.6, 52.3,
114.9, 126.1, 127.8, 127.9, 128.8, 130.1. ESI-HRMS: Calcd
exact mass without TFA for C48H56N10O10 m/z = 933.4259
[M+H]+; found m/z 933.4241.
1
products was confirmed by H-NMR analysis on a Varian
Oxford 300 MHz and ESI-LRMS using a LCQ Finnigan-
Math mass spectrometer. Peptide structures of the final
compounds were determined by NMR spectrometry (Tables
1S,2S see SI) and confirmed by ESI-HRMS mass
spectrometry (Table 3S, Figures 1S,2S see SI). Samples
were diluted to about 200 fmol/uL in 50:50 ACN-water with
0.1% formic acid. They were infused to an ESI ionization
source on a Waters Q-Tof Premier quadrupole-time of flight
mass spectrometer operating in positive ion mode. Glu-
fibrinopeptide B was used as a lock mass standard. MS data
were acquired for 10 minutes with ms scans from 400 to
In vitro studies
Cell lines and cell culture
Chinese hamster ovary (CHO) cells expressing the human
MOR, DOR, or KOR were used for all experiments. The
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3
details for these cell lines, including their KD values for H-
sessions, the mice were housed in colony cages (7 mice per
cage) under standard light (from 7:00 AM to 7:00 PM),
temperature (21±1°C) and relative humidity (60±10%) for at
least 1 week. Food and water were available ad libitum. The
research protocol was approved by the Service for
Biotechnology and Animal Welfare of the Istituto Superiore
di Sanità and authorized by the Italian Ministry of Health,
according to Legislative Decree 26/14, which implemented
the European Directive 2010/63/UE on laboratory animal
protection in Italy. Animal welfare was routinely checked by
veterinarians from the Service for Biotechnology and
Animal Welfare. Animal studies are reported in compliance
with the ARRIVE guidelines.29
diprenorphine binding, can be found in Stefanucci et al.14
The cells were maintained in 1:1 DMEM/F12 culture media
(Gibco), with 1X penicillin/streptomycin, and 10% heat-
inactivated fetal bovine serum (Gibco) in a 5% CO2
atmosphere 37°C incubator. For experiments, cells were
harvested using 5 mM EDTA in PBS, collected, centrifuged,
and stored at -80°C. Membrane preparations for binding or
GTPγS coupling were created using the same protocol as
reported.14
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Competition radioligand binding
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Competition radioligand binding was performed exactly as
reported in Stefanucci et al.14 Membrane preparations of
receptor-containing CHO cells were combined with
concentration curves of ABAM compounds or positive
Drugs and treatment procedure. DMSO was purchased
from Merck (Italy). On each test day, peptides solutions
were freshly prepared using saline containing 0.9% NaCl
and DMSO in the ratio DMSO: saline 1:5 v/v. These
solutions were injected at a volume of 10 μL/mouse for
intracerebroventricular (i.c.v.) administrations, or at a
volume of 10 ml/kg for intravenous (i.v.) administrations.
3
control, and a fixed concentration (4.33 – 5.25 nM) of H-
diprenorphine (PerkinElmer). Vehicle concentrations were
equalized between each reaction. Reactions were incubated
for 1 h at r.t. The resulting plates were read using a
PerkinElmer MicroBeta2 6-detector 96-well format
scintillation counter. The data was normalized to 3H-
diprenorphine alone (100%) or non-specific binding
measured by the inclusion of 10 μM naloxone (0%). IC50
values for each curve and the calculated Ki using the
Surgery for i.c.v. injections. For i.c.v. injections, mice were
lightly anesthetized with isoflurane, and an incision was
made in the scalp. Injections were performed using a 10 L
Hamilton microsyringe at a point 2-mm caudal and 2-mm
lateral from the bregma.
3
established KD of H-diprenorphine in each cell line was
calculated using GraphPad Prism 7.0, and reported as the
mean ± SEM.
35S-GTPγS coupling assay
Hot plate test. Thermal nociception was assessed as
previously reported, using
a commercially available
apparatus consisting in a metal plate 25x25 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 15, 30, 45, 60, 90 and 120 min after treatment.
Data from the hot plate test, were reported as time course of
the percentage of maximum effect (%MPE) = (post drug
latency – baseline latency) / (cut-off time – baseline latency)
x 100.30
The GTPγS assay was again performed exactly as reported
in Stefanucci et al.14 Membrane preparations of receptor-
containing CHO cells were combined with concentration
curves of ABAM compound or positive control along with
0.1 nM 35S-GTPγS (PerkinElmer), and incubated at room
temperature for 1 h. Vehicle concentrations were normalized
between each reaction. The resulting plates were read as
above, and the data normalized to the stimulation caused by
positive control compound (100%) or vehicle (0%). The
potency (EC50) and efficacy (EMax) values were calculated
for each curve using GraphPad Prism 7.0 and reported as the
mean ± SEM. The efficacy was defined for each compound
in relation to the maximum efficacy of the positive control
compound, defined as 100%. The antagonist experiments
with DOR cells were performed as above, except the
membranes were incubated with concentration curves of
antagonist for 5 minutes before the addition of 100 nM
SNC80. The data was normalized to 100 nM SNC80 (100%)
or vehicle (0%), and analyzed as above, using naloxone as a
positive control antagonist.
Data analysis and statistics. Experimental data were
expressed as mean ± s.e.m. Significant differences among
the groups were evaluated with an analysis of variance
followed by Tukey’s post-hoc comparisons using the
GraphPad Prism 6.03 software. Statistical significance was
set at P<0.05.
Supporting Information
Forskolin induced [3H]cAMP assay
The Supporting Information is available free of charge on the
ACS Publications website. Details of compounds synthesis,
characterization, in vitro assays (PDF).
The cAMP assay was performed as reported in Stevenson et
al.28 The same DOR-expressing CHO cells were used as
above, plated live in 96 well plates (20,000 cells/well) and
recovered for 24 hrs. Cells serum starved for 4 hours,
followed by 500 μM IBMX for 20 min (IBMX in all
treatment solutions). ABAM compounds or naltrindole
positive control incubated on cells for 15 minutes, followed
by agonist (16.7 nM SNC80) combined with forskolin (100
μM) for 10 minutes. The cells were then processed as
reported above, and cAMP levels determined by
competition with [3H]-cAMP for binding to recombinant
PKA. The resulting signal was normalized to percent of
forskolin stimulation (100%) or vehicle alone (0%), and
analyzed using Prism 7.0.
AUTHOR INFORMATION
Corresponding Author
** Corresponding author email: a.mollica@unich.it
Author Contributions
AS: synthesis, characterization and writing of the text. WL and
JS: in vitro binding and function experiments. SP: in vivo
assays; MPD: purification of the novel compounds. GL and
EN: writing and revision of the text; MN and WK: NMR
analysis. SM and GZ: design of the molecules. JS: co-wrote the
manuscript. AM: design, co-wrote and coordinated all the
research units.31
In vivo studies
Animals. Male CD-1 mice (Harlan, Italy) weighing 25-30 g
were used in all the experiments. Before the experimental
Funding Sources
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ACS Medicinal Chemistry Letters
Page 6 of 7
14. Stefanucci, A.; Lei, W.; Hruby, V. J.; Macedonio, G.; Luisi, G.;
This paper was supported in part by Institutional funds from the
University of Arizona.
Carradori, S.; Streicher, J. M.; Mollica, A. Fluorescent-labeled
bioconjugates of the opioid peptides biphalin and DPDPE
incorporating fluorescein-maleimide linkers. Future Med. Chem.
2017, 9, 859-869.
15. Mollica, A.; Pinnen, F.; Feliciani, F.; Stefanucci, A. New potent
biphalin analogues containing p-fluoro-L-phenylalanine at the 4,4’
positions and non-hydrazine linker. Amino Acids 2010, 40, 503-
1511.
1
2
3
4
5
6
ABBREVIATIONS
DOR, δ-opioid receptor; MOR, μ-opioid receptor; KOR, k-
opioid receptor; RCM, Ring Closing Metathesis; PK,
pharmacokinetic properties; DCE, dichloroethane, EDC, 1-
ethyl-(3-(dimethylamino)propyl)-carbodiimide;
DAMGO,
7
8
9
[DAla(2), N-Me-Phe-(4), Gly-ol(5)] enkephalin; SNC80, (+)-4-
[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-
16. Arisawa, M. Development of environmentally benign
organometallic catalysis for drug discovery and its application.
Chem Pharm Bull (Tokyo) 2007, 55, 1099-1118.
17. Binder, J. B.; Raines, R. T. Olefin metathesis for chemical
biology. Curr Opin Chem Biol. 2008, 12, 767-773.
18. Montgomery, T. P.; Ahmed, T. S.; Grubbs, R. H.
Stereoretentive olefin metathesis: An avenue to kinetic selectivity.
Angew Chem Int. Ed Engl. 2017, 56, 11024-11036.
19. Dounis, P.; Feast, W. J.; Kenwright, A. M. Ring-opening
metathesis polymerization of monocyclic alkenes using
molybdenum and tungsten alkylidene (Schrock-type) initiators and
13C nuclear magnetic resonances studies of the resulting
polyalkenamers. Polymer 1995, 36, 2787-2796.
20. Beierbeck, H.; Saunders, J. K. A reinterpretation of beta,
gamma, and delta substituent effects on 13C chemical shifts. Can.
J. Chem. 1976, 54, 2985-2995.
21. Purington, L. C.; Pogozheva, I. D.; Traynor, J. R.; Mosberg, H.
I. Pentapeptides displaying μ opioid receptor agonist and δ opioid
receptor partial agonist/antagonist properties. J. Med. Chem. 2009,
52, 7724-7731.
22. Przydzial, M. J.; Pogozheva, I. D.; Ho, J. C.; Bosse, K. E.;
Sawyer, E.; Traynor, J. R.; Mosberg, H. I. Design of high affinity
cyclic pentapeptide ligands for kappa-opioid receptors. J. Pept. Res.
2005, 66, 255-262.
23. Schiller, P. W.; Fundytus, M. E.; Merovitz, L.; Weltrowska, G.;
Nguyen, T. M.; Lemieux, C.; Chung, N. N.; Coderre, T. J. The
opioid mu agonist/delta antagonist DIPP-NH(2)[Psi] produces a
potent analgesic effect, no physical dependence, and less tolerance
than morphine in rats. J. Med. Chem. 1999, 42, 3520-3526.
24. Purington, L. C.; Sobczyk-Kojiro, K. S.; Pogozheva, I. D.;
Traynor, J. R.; Mosberg, H. I. Development and in vitro
characterization of a novel bifunctional mu-agonist/delta antagonist
opioid tetrapeptide. ACS Chem Biol. 2011, 6, 1375-1381.
25. Polt, R.; Dhanasekaran, M.; Keyari, C. M. Glycosylated
neuropeptides: a new vista for neuropsychopharmacology?. Med.
Res. Rev. 2005, 25, 557-585.
26. Mosberg, H. I.; Yeomans, L.; Anand, J. P.; Porter, V.; Sobczyk-
Kojiro, K.; Traynor, J. R.; Jutkiewicz, E. M. Development of a
bioavailable μ opioid receptor (MOPr) agonist, δ opioid receptor
(DOPr) antagonist peptide that evokes antinociception without
development of acute tolerance. J. Med. Chem. 2014, 57, 3148-
3153.
27. Mollica, A.; Davis, P.; Ma, S.-W.; Porreca, F.; Lai, J.; Hruby,
V. J. Synthesis and biological activity of the first cyclic biphalin
analogues. Bioorg. Med. Chem. Lett. 2006, 16, 367-372.
28. Stevenson, G.W.; Luginbuhl, A.; Dunbar, C.; LaVigne, J.;
Dutra, J.; Atherton, P.; Bell, B. Cone, K.; Giuvelis, D.; Polt, R.;
Streicher, J.M.; Bilsky, E.J. Pharmacol. Biochem. Behav. 2015,
132, 49-55. The mixed-action delta/mu opioid agonist MMP-2200
does not produce conditioned place preference but does maintain
drug self-administration in rats, and induces in vitro markers of
tolerance and dependence.
29. McGrath, J. C.; Lilley, E. Implementing guidelines on reporting
research using animals (ARRIVE etc.): new requirements for
publication in BJP. Br J Pharmacol 2015, 172, 3189-3193.
30. Piekielna-Ciesielska, J.; Mollica, A.; Pieretti, S.; Fichna, J.;
Szymaszkiewicz, A.; Zielińska, M.; Kordek, R.; Janecka, A.
Antinociceptive potency of a fluorinated cyclopeptide Dmt-c[D-
Lys-Phe-p-CF3-Phe-Asp]NH2. J Enz. Inhib Med Chem. 2018, 33,
560-566.
methoxy
hydroxybenzotriazole; i.c.v., intracerebroventricular; i.v.,
intravenous; NMM, N-methylmorpholine; DMF,
benzyl]-N,N-diethylbenzamide;
HOBt,
1-
10
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60
dimethylformamide;; RP-HPLC, reversed phase high
performance liquid chromatography; ACN, acetonitrile; NMR,
nuclear magnetic resonance; ROESY, rotating-frame nuclear
Overhauser effect correlation spectroscopy; COSY, correlation
spectroscopy; TOCSY, total correlation spectroscopy; HSQC,
heteronuclear single quantum correlation; LRMS, low
resolution mass spectrometry; HRMS, high resolution mass
spectrometry;
TFA,
trifluoroacetic
acid;
DCM,
dichloromethane; BBB, blood brain barrier; CNS, central
nervous system; GTP, guanosine triphosphate; MPE, maximum
possible effect; DMSO, dimethylsulfoxide; CHO, chinese
hamster ovary; DMEM/F12,
medium/nutrient mixture
Dulbecco’s modified eagle
F-12; EDTA,
ethylenediaminetetraacetic acid; PBS, phosphate buffered
saline.
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