Nociceptin/orphanin FQ(1-13)NH2 analogues modified in the Phe1-Gly2 peptide bond

Article abstract of DOI:10.1016/S0960-894X(02)01004-1

The synthesis and pharmacological activity of novel nociceptin/orphanin FQ (N/OFQ) analogues modified in the Phe¹-Gly² peptide bond are reported. The aim of the present work was to elucidate the importance of this peptide bond for th

Full text of DOI:10.1016/S0960-894X(02)01004-1

Bioorganic & Medicinal ChemistryLetters 13 (2003) 365–368
Nociceptin/Orphanin FQ(1–13)NH2 Analogues Modified in the
Phe1-Gly2 Peptide Bond
Remo Guerrini,a,* Daniela Rizzi,b Marina Zucchini,a Roberto Tomatis,a
Domenico Regoli,b Girolamo Calo’b and Severo Salvadoria
aDepartment of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, via Fossato di Mortara, 17/19,
44100 Ferrara, Italy
bDepartment of Experimental and Clinical Medicine, Section of Pharmacology, University of Ferrara,
via Fossato di Mortara, 17/19, 44100 Ferrara, Italy
Received 22 July2002; revised 23 October 2002; accepted 12 November 2002
Abstract—The synthesis and pharmacological activity of novel nociceptin/orphanin FQ (N/OFQ) analogues modified in the Phe1-
Gly2 peptide bond are reported. The aim of the present work was to elucidate the importance of this peptide bond for the N/OFQ
receptor (NOP) interaction. Our studyindicates that the first peptide bond in N/OFQ is important but not crucial for interaction
with the N/OFQ receptor; for instance, substitution with a methyleneoxy bond generates an agonist derivative just 3-fold less
potent than the reference compound.
# 2002 Elsevier Science Ltd. All rights reserved.
A few years after the identification of nociceptin/
orphanin FQ [N/OFQ: H-Phe-Gly-Gly-Phe-Thr-Gly-
Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln-OH and
its receptor (N/OFQ peptide receptor (NOP)]1 a vast
amount of information regarding this novel peptide/
receptor system are available.2 It has been demonstrated
that the N/OFQ-NOP receptor system regulates several
biological functions both at peripheral and central
levels.3 Therefore, the NOP receptor is now considered
a novel target for drug development.
selective agonists such as [Tyr1]N/OFQ(1-13)NH2,9
selective partial agonists like [Phe1É(CH2–NH)Gly2]N/
OFQ(1-13)NH2 ([F/G]NC(1–13)NH2),10,11 and pure
antagonists, as [Nphe1]N/OFQ(1–13)NH212,13 and more
recentlyUFP-101. Worthyof mention is the fact that
NOP antagonists were exclusivelyidentified bymodifi-
cations of the message domain of N/OFQ.
14
In the present study, we have further investigated the
importance of the first peptide bond of N/OFQ(1–
13)NH2 for the interaction with the NOP receptor.
To fullyunderstand the biological role(s) of this system,
selective ligands for NOP receptor are needed. To this
aim, in the last few years, we performed a series of
The synthesis of the reference compounds N/OFQ(1–
13)NH2 and [F/G] N/OFQ(1–13)NH2 have been
4,5
6,10
structure–activitystudies on N/OFQ.
We divided the
alreadyreported,
while the other peptides were
N/OFQ into a message [N/OFQ(1–4)] which includes
the two pharmacophores Phe1 and Phe4 important for
receptor activation and an address sequence [N/OFQ(5–
17)] which contains the two couple of basic residues
Arg8,12-Lys9,13 crucial for receptor occupation.6 We
identified a series of original peptide ligands for the
NOP receptor including: selective agonists such as N/
obtained bymeans of a mixed solid-phase/solution
peptide synthesis approach. In particular, compound 4
was synthetised condensing Boc-Phe-CHO on the
[Sar2]N/OFQ(2–13)-PAL-PEG-PS resin, obtained by
Fmoc standard solid-phase peptide synthesis, following
10
the procedure previouslydescribed byus.
For the
synthesis of peptides 5–7 we condensed the pseudodi-
peptides Boc-PheÉ(CH2–O)-Gly-OH,15 Boc-PheÉ(CH2–
S)-Gly-OH,16 and Boc-PheÉ(CO–CH2)-Gly-OH,17 with
N/OFQ(3–13)-PAL-PEG-PS resin in the presence of
1-ethyl-3-[30-(dimethylamino)propyl]-carbodiimide hydro-
chloride (EDCl) and 1-hydroxybenzotriazole (HOBt) as
OFQ(1–13)NH2 or [(pF)Phe4]N/OFQ(1-13)NH2,8 non
7
*Corresponding author. Tel.: +39-0532-291-281; fax: +39-0532-291-
296; e-mail: r.guerrini@unife.it
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)01004-1

366
R. Guerrini et al. / Bioorg. Med. Chem. Lett. 13 (2003) 365–368
activating agents. In an attempt to adopt a similar
scheme for the synthesis of compound 8, we approach
the synthesis of the pseudodipeptide Boc-PheÉ(NH–
CO)-Gly-OH following the method for the solid phase
synthesis of retro-inverso peptides proposed by Pessi et al.18
that considers the conversion, with bis(trifluoroacetoxy)-
iodobenzene (TIB), of the amide function of the pseudo-
dipeptide malonil-d-Phe-amide into an amine function
following protection with Boc. However, this reaction
did not produce the desired compound but a series of
other derivatives which were not further investigated.
Therefore, an alternative waywas considered for the
synthesis of compound 8: it was obtained condens-
ing the pseudotetrapeptide Boc-PheÉ(NH–CO)-Gly-
Gly-Phe-OH with the N/OFQ(5–13)-PAL-PEG-PS
resin in the presence of EDCl and HOBt as activating
agent. The pseudotrateptide Boc-PheÉ(NH–CO)-Gly-
Gly-Phe-OH was obtained following Scheme 1 con-
densing the malonyl-d-Phe-amide18 with H-Gly-Phe-
OBzl.19 The rearrangement of the amide function to
amine function with TIB, the protection with Boc and
the removal of the benzyl ester afforded the desired
pseudotetrapeptide.20
Compounds 4–8 were purified byRP-HPLC ( ꢀ98%
purity) and analysed on a MALDI-TOF mass spectro-
meter, yielding mass values in line with the expected
chemical composition.
All the peptides were tested for their abilityto inhibit
the electricallyevoked contraction of the mouse vas
deferens (mVD), a pharmacological preparation sensi-
tive to N/OFQ.7 Cumulative concentration response
curves were performed with N/OFQ(1–13)NH2 and
compounds 4–8. When the peptides were found to be
inactive as agonist, theywere assaeyd as antagonists
against the reference agonist N/OFQ(1–13)NH2. The
pharmacological results of the assays are presented in
Table 1. The agonist and antagonist potencies were
measured as pEC50 and pA2, respectively. pA2 values
were determined byapplying the Gaddum Schild equa-
tion [pA2=ꢁlog((CR-1)/[Antagonist])], assuming
a
slope equal to unity. The adopted pharmacological ter-
21
minologyis in line with IUPHAR recommendations.
N/OFQ(1–13)NH2 produced a concentration dependent
inhibition of the electricallyinduced twitch with pEC
and Emax values of 7.85 and 90%, respectively( Table 1).
50
Reduction of the Phe1-Gly2 peptide bond produced [F/
G]N/OFQ(1–13)NH2 which, in the mVD, is inactive as
agonist and behaves as a competitive antagonist with a
pA2 value of 6.75.11 The antagonist properties of this
peptide were confirmed in some in vitro preparations
while in others the peptide behaves as a partial agonist
or even as a full antagonist [for a detailed description
and discussion of the pharmacological profile of [F/
G]N/OFQ(1–13)NH2, see ref 22].
The introduction of the methyl substituent onto the
É(CH2–NH) nitrogen, as in compound 4, produced an
inactive derivative. This result is in line with that
obtained bymethylation of the first Phe 1-Gly2 peptide
bond in N/OFQ(1–13)NH2; in fact, [Sar2]N/OFQ(1–
13)NH2 was demonstrated to behave as a NOP receptor
agonist 100-fold less potent than N/OFQ(1–13)NH2.5
The nitrogen methylation of the first peptide bond
([Sar2]N/OFQ(1–13)NH2) or of the reduced peptide
bond É(CH2–NH) (4) probablylimit the possible con-
formations of the N-terminal part of N/OFQ(1–
13)NH2, in particular of the side chain of Phe1, thus
causing a reduced biological activity. Moreover, nitro-
gen methylation improves the basic character of the
reduced peptide bond and this mayalso prevent NOP
receptor occupation.
The replacement of CO–NH bond with CH2–O (5)
produced an agonist 3-fold less potent than N/OFQ(1–
13)NH2 (Table 1), while substitution with CH2–S (6)
produced a reduction of both potencyand efficacy. In
fact compound 6 behaves as a partial agonist with an
intrinsic activity( a) of 0.6. These peptide bond sub-
stitutions maintain the hydrogen bond acceptor but lose
the donor properties. Quantitativelythe CH –S moiety,
2
due to reduced electronegativityand increased size of
the sulfur atom, is inferior as hydrogen bond acceptor
compared to CH2–O modification. However, the
Scheme 1. (i) EDCl, HOBt, Et3N, DMF, 76%; (ii) TIB, CH3CN/H2O
6:4, 62%; (iii) (Boc)2O, DMF; 10% Pd/C, H2, MeOH, 78%.

R. Guerrini et al. / Bioorg. Med. Chem. Lett. 13 (2003) 365–368
367
Table 1. Effects of [Phe1É(X)Gly2]N/OFQ(1–13)NH2 peptides in the electricallystimulated mVD assay
Compd
X
Agonist
Antagonista
pEC50 (CL95%
)
Emax (%)
90ꢂ2
pA2 (CL95%)
N/OFQ(1–13)NH2
[F/G]N/OFQ(1–13)NH2
CO–NH
CH2–NH
CH2–N(CH3)
CH2–O
CH2–S
CO–CH2
NH–CO
7.85 (0.15)
NDb
6.75 (0.21)
Inactive
ND
6.25 (0.14)
ND
Inactive
Inactivec
4
5
6
7
8
Crc incompleted
7.38 (0.43)
6.79 (0.22)
6.21 (0.18)
73ꢂ7
54ꢂ5*
86ꢂ3
Inactive
aThe antagonistic properties of these compounds were tested using N/OFQ(1–13)NH2 as agonist.
bND, not determined because these compounds are full agonists.
cInactive: inactive up to 10 mM.
dCrc incomplete: onlya slight effect ( <50% inhibition) was detected at the highest concentration tested (10 mM). These data are mean of at least five
biossayexperiments.
*p<0.05 versus N/OFQ(1–13)NH2 effect, according to ANOVA followed bythe Dunnet test.
hydrogen bond donor/acceptor properties of the Phe1-
Gly2 bond do not seem to be particularlyrelevant for
the biological activity. On the other hand, the geome-
tries of the CH2–O and CH2–S moieties are comparable
to that of the trans amide bond, but the distances
between the Cai–Cai+1 appear to be slightlyincreased
in the case of the CH2–S bond23 (i.e., CO–NH: 3.8 A;
CH2–O: 3.7 A; CH2–S: 4.2 A). Being Phe1 and Phe4 the
most important residues responsible for NOP receptor
acceptor/donor properties of the peudopeptide bond.
The reduction or elimination of the activityobtained
substituting Phe1-Gly2 peptide bond might be due to
impaired abilityof the N-terminal part of the peptide to
adopt energeticallyfavorable bioactive conformations.
Worthyof mention is the finding that compound 5,
although slightlyless potent than N/OFQ(1–13)NH 2, is a
full agonist at NOP receptor; this analogue is probably
more resistant than the natural sequence towards amino-
peptidase-N action. In vivo evaluation of compound 5 is
required for defining the utilityof this latter peptide
modification.
5
activation , the increased distance between Cai-Cai+1
(and therefore between Phe1 and Phe4) could be relevant
for the decrease of efficacyof compound 6. This is sug-
1
gested bythe fact that the shift of the side chain of Phe
from the Ca to the nitrogen (which produced a bigger
increase of distance between Phe1 and Phe4) fullyelimi-
nated intrinsic activitygenerating the first pure antagonist
for the NOP receptor: [Nphe1]N/OFQ(1–13)NH2.12,13
Acknowledgements
This work was supported byfunds from the Italian
Ministryof the Universityand from the Universityof
Ferrara.
The introduction of a CO–CH2 bond (7) lead to a
weaklypotent agonist derivative. Moreover, the effects
of compound 7 were slightlysensitive to the classical
opioid receptor antagonist naloxone which at 1 mM
produced a 3-fold rightward shift of the concentration
response curves to [Phe1É(CO–CH2)Gly2]N/OFQ(1–
13)NH2, demonstrating that this modification produced
a decrease in the selectivityof action of the ligand. This
peptide bond modification eliminates the NH group
(and its hydrogen bond donor/acceptor properties) but
maintains the carbonyl function as a possible hydrogen
bond acceptor moiety. This may indicate that the nitro-
gen of the first peptide/preudopeptide bond is important
for biological activity. However, the relative high
potencyof compounds 5 and 6 (in which the N atom
was replaced with O and S, respectively) indicated that a
heteroatom is required rather than specificallynitrogen.
Finally, despite the close geometrical mimicry24 between
the standard peptide bond and the retro-inverso peptide
bond (8), this latter modification generated an inactive
analogue, indicating that the position of the nitrogen
atom is also crucial for receptor occupation.
References and Notes
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IUPHAR Compendium of Receptor Characterization and Clas-
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2. Calo’, G.; Guerrini, R.; Rizzi, A.; Salvadori, S.; Regoli, D.
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44, 3956.
In conclusion, the present results suggest that the Phe1-
Gly2 peptide bond is not crucial per se for NOP receptor
interaction. Substitutions with different peptide bond
isoster generate NOP ligands with different potencies
and efficacies, independentlyfrom the hydrogen bond

368
R. Guerrini et al. / Bioorg. Med. Chem. Lett. 13 (2003) 365–368
9. Calo’, G.; Rizzi, A.; Bodin, M.; Neugebauer, W.; Salva-
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200 MHz, d): 3.12–3.31 (m 6H), 4.23 (s 2H), 4.78–4.92 (m 2H),
5.37 (s 2H), 5.81 (bs 1H), 6.73 (bs 1H), 7.17–7.38 (m 12H),
7.53 (bs 1H).
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Regoli, D.; Salvadori, S. J. Med. Chem. 1998, 41, 3360.
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H-Phe- (NH–CO)-Gly-Gly-Phe-OBzl (2): The precursor
amide (1) (5.4 g, 10 mmol) was dissolved in acetonitrile/water
6:4 (50 mL) and flushed with N2. To this stirred solution, TIB
(4.7 g, 11 mmol) dissolved in 10 mL acetonitrile was added by
siringe. The reaction was allowed to proceed for 4 h under N2
then concentrated under reduced pressure at low temperature.
The residue was suspended in NaHCO3 (5%) (100 mL) and
the product extracted with EtOAc (4ꢄ50 mL). The organic
phase was washed with brine, dried and evaporated to dry-
ness. The oil crude product was loaded onto a silica gel col-
umn and eluted with dichloromethane/methanol 9:1. The
compound obtained from chromatographywas cristallized
from diethyl ether/light petroleum 1:1, yield 3.2 g (62%),
melting point 122–127 ꢃC, [a]2D0+5.7 (c 1 methanol), MH+
517, NMR 1H (DMSO-d6, 200 MHz, d): 3.07–3.18 (m 6H),
3.31 (m 2H), 3.82 (s 2H), 4.81 (m 1H), 5.07 (m 1H), 5.28 (s
2H), 6.42 (bs 1H), 7.09–7.32 (m 17H).
Boc-Phe-Y(NH-CO)-Gly-Gly-Phe-OH (3): To a solution of
(2) (2.6 g, 5 mmol) in DMF (20 mL), (Boc)2O (1 g, 5 mmol)
was added and the reaction stirred for 6 h. After this time the
solvent was evaporated in vacuo and the oil residue dissolved
in methanol (100 mL) and hydrogenated for 1 h in the pre-
sence of 10% Pd/C. The catalyst was removed by filtration
through Celite pad and the solvent evaporated under reduced
pressure. The residue was cristallized from diethyl ether, yield
19. Varga, L.; Horvat, S.; Lemieux, C.; Schiller, P. W. Int. J.
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ꢃ
20. Synthetic procedure: H2NCO-DPhe-Malonil-Gly-Phe-
OBzl (1): To a solution of H2NCO-Dphe-malonic acid (5 g, 20
mmol) and the trifluoacetate salt ꢃof H-Gly-Phe-OBzl (8.52 g,
20 mmol) in DMF (30 mL) at 0 C, HOBt (3.4 g, 22 mmol),
EDCl (4.22 g, 22 mmol) and triethylamine (2.8 mL, 20 mmol)
were added. The reaction was stirred for 1 h at 0 ꢃC and
overnight at room temperature. After evaporation of DMF,
the residue was solubilized in EtOAc and washed with citric
acid (10%) (3ꢄ30 mL), NaHCO3 (5%) (3ꢄ30 mL) and brine.
The organic phase was dried and concentrated under reduced
pressure. The residue was cristallized from diethyl ether/light
petroleum 1:1, yield 8.3 g (76%), melting point 161–166 ꢃC,
[a]D20 ꢁ3.6 (c 1 methanol), MH+ 545, NMR 1H (DMSO-d6,
2 g (78%), melting point 175–180 C, [a]2D0+15.5 (c 1 metha-
nol), MH+ 527, NMR 1H (DMSO-d6, 200 MHz, d): 1.38 (s
9H), 2.92–3.02 (m 4H), 3.21 (s 2H), 3.93 (s 2H), 5.76 (bs 1H),
7.12–7.27 (m 12H), 7.86 (bs 1H).
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D. C.; O’Toole, J. C.; Ratcliffe, G. S.; Rees, D. C.; Roberts, E.
J. Med. Chem. 1992, 35, 1472.
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