Synthesis and biological activity of new series of N-modified analogues of the N/OFQ(1-13)NH2 with aminophosphonate moiety

Article abstract of DOI:10.1007/s00726-011-1177-7

New series of N-modified analogues of the N/OFQ(1-13)NH₂ with aminophosphonate moiety have been synthesized and investigated for biological activity. These peptides were prepared by solid-phase peptide synthesis-Fmoc-strategy. The N/OFQ(1-13)NH₂ analogues were tested for agonistic activity in vitro on electrically stimulated rat vas deferens smooth-muscle preparations isolated from Wistar albino rats. Our study has shown that the selectivity of the peptides containing 1-[(methoxyphosphono) methylamino]cycloalkanecarboxylic acids to the N-side of Phe is not changed-they remain selective agonists of NOP receptors. The derivative with the largest ring (NOC-6) demonstrated efficacy similar to that of N/OFQ(1-13)NH₂, but in a 10-fold higher concentration. The agonistic activity of newly synthesized N-modified analogues of N/OFQ(1-13)NH₂ with aminophosphonate moiety was investigated for the first time.

Full text of DOI:10.1007/s00726-011-1177-7

Amino Acids (2012) 43:1217–1223
DOI 10.1007/s00726-011-1177-7
ORIGINAL ARTICLE
Synthesis and biological activity of new series of N-modified
analogues of the N/OFQ(1–13)NH2 with aminophosphonate
moiety
•
Petar T. Todorov Polina I. Mateeva
•
•
•
Rositza N. Zamfirova Nikola D. Pavlov
Emilia D. Naydenova
Received: 25 July 2011 / Accepted: 22 November 2011 / Published online: 6 December 2011
Ó Springer-Verlag 2011
Abstract New series of N-modified analogues of the
N/OFQ(1–13)NH2 with aminophosphonate moiety have
been synthesized and investigated for biological activity.
These peptides were prepared by solid-phase peptide syn-
thesis—Fmoc-strategy. The N/OFQ(1–13)NH2 analogues
were tested for agonistic activity in vitro on electrically
stimulated rat vas deferens smooth-muscle preparations
isolated from Wistar albino rats. Our study has shown that
the selectivity of the peptides containing 1-[(methoxy-
phosphono)methylamino]cycloalkanecarboxylic acids to
the N-side of Phe is not changed—they remain selective
agonists of NOP receptors. The derivative with the largest
ring (NOC-6) demonstrated efficacy similar to that of
N/OFQ(1–13)NH2, but in a 10-fold higher concentration.
The agonistic activity of newly synthesized N-modified
analogues of N/OFQ(1–13)NH2 with aminophosphonate
moiety was investigated for the first time.
(1995), which binds selectively to its own receptors (NOP
receptors), previously referred to as ‘orphan’ receptor.
Nociceptin is a neuropeptide of 17 amino acids (Phe-Gly-
Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-
Asn-Gln), derived from a larger precursor, prepronociceptin
(ppN/OFQ), which is similar in humans, rats and mice
(Mollereau et al. 1996; Guerrini et al. 2000a, b, c). Despite
its structural similarity to opioids, namely dinorphin A,
N/OFQ does not interact with classical opioid receptors
(Henderson and McKnight 1997; Darland et al. 1998;
Guerrini et al. 2001a, b). This shows that the N/OFQ/NOP-
system is a separate circuit with its own biological functions.
Nociceptin is known mainly for its involvement in the
transmission of pain signals and such physiological as
nociception, locomotion, feeding behaviour, and respira-
tory, gastrointestinal and urogenital functions (Civelli,
2008; Pan et al. 2000; Dzambazova et al. 2008; Xie et al.
¨
2008; Koster et al. 1999; Rizzi et al. 2001). Structure–
Keywords Nociceptin analogues Á Nociceptin/orphanin
FQ Á NOP receptor Á SPPS Á Aminophosphonates Á
Rat vas deferens
activity relationship studies identified strategies to render
N/OFQ ligands less susceptible to enzymatic degrada-
tion. These include truncation and C-terminal amidation,
creating N/OFQ(1–13)NH2 and modifications of the
N-terminal region of N/OFQ as with [Phe1w(CH2–NH)Gly2]
N/OFQ(1–13)NH2 (Guerrini et al. 1998, 2001a, b; Okada
et al. 2000; Calo et al. 2000a, b; Kitayama et al. 2007;
Arduinetal. 2007). Guerrinietal. (1998, 2000a, b, c, 2001a, b)
have synthesized a series of analogues of the NOP-receptor
antagonist [Nphe1]-NC(1–13)NH2 and have tested them for
agonistic and antagonistic activities on the mouse vas
deferens. The N-terminal tridecapeptide sequence of
nociceptin molecule, N/OFQ(1–13)NH2, was discovered to
exert all the biological activities of N/OFQ, with arginine
and lysine being crucial for the receptor binding (Nayde-
nova et al. 2006). Identification of novel NOP-receptor
ligands of either peptide or non-peptide nature has been the
Introduction
Nociceptin/orphanin (N/OFQ) is an endogenous peptide,
identified by Meunier et al. (1995) and Reinscheid et al.
P. T. Todorov Á N. D. Pavlov Á E. D. Naydenova (&)
Department of Organic Chemistry, University of Chemical
Technology and Metallurgy, Sofia 1756, Bulgaria
e-mail: e_naydenova@abv.bg
P. I. Mateeva Á R. N. Zamfirova
Institute of Neurobiology, Bulgarian Academy of Sciences,
1113 Sofia, Bulgaria
123

1218
P. T. Todorov et al.
subject of intense research activity (Bignan et al. 2005;
Zaveri 2003). The replacement of Ala with the Ca,a-dime-
thylated amino acid Aib at positions 7, 11 and 15 in N/OFQ
sequence in the case of [Aib7]N/OFQ-NH2 produced a
NOP-receptor ligand that was seven-folds more potent than
the parent peptide, as determined in the [35S]GTPcS func-
tional assay (Zhang et al. 2002). Many analogues were also
examined in search of variations of their functions, com-
pared to the ‘original’ molecule (Rizzi et al. 2002).
Fmoc-Ser(t-Bu)-OH, Thr—as Fmoc-Thr(t-Bu)-OH and
Lys—as Fmoc-Lys(Boc)-OH. All coupling reactions were
performed, using for amino acid/TBTU/HOBt/DIEA/resin
a molar ratio of 3/2.9/3/6/1. A 20%-piperidine solution in
N,N-dimethylformamide (DMF) was used to remove the
Fmoc group at every step. The coupling and deprotection
reactions were checked by the Kaiser test. We have pre-
pared the corresponding aminophosphonates, according to
recently published papers (Naydenova et al. 2008, 2010a, b).
All the 1-[(dimethoxyphosphono)methylamino]cycloalkan-
ecarboxylic acids (3 equiv) were coupled to the growing
peptide chain by using TBTU (3 equiv) in the presence of
an equimolar concentration of 1-hydroxy benzotriazole
(HOBt) dissolved in excess of N,N-diisopropylethylamine
(DIEA) and DMF. The coupling reaction time was 15 h.
The cleavage of the synthesized peptide from the resin was
done, using a mixture of 95% trifluoroacetic acid (TFA),
2.5% triisopropylsilan (TIS) and 2.5% water. During the
cleavage, one of the methoxy groups from cyclic amino-
phosphonic residue was removed. After filtration of the
exhausted resin, the solvent was concentrated in vacuum
and the residue triturated with cool ether. The purity of the
peptides was checked by HPLC analysis, column: Sym-
metryShieldTM RP-18, 3.5 l (50 9 4.6 mm), flow: 1 ml/
min, H2O (0.1% TFA)/CH3CN (0.1% TFA), gradient
0 ? 100% (15 min) and 100% (4 min). The crude pep-
tides were purified using semi-preparative HPLC, column
XBridgeTM Prep C18 10 lm (10 9 250 mm), flow: 5 ml/
min, H2O (0.1% TFA)/CH3CN (0.1% TFA), gradient
20 ? 70% (20 min). The ESI mass spectra were recorded
with a platform II quadrupole mass spectrometer fitted with
an electrospray source. Optical rotations were measured
with a Perkin Elmer 341 polarimeter. Amino acid analysis
was performed on a HITACHI L-8500 amino acid analyser
on samples hydrolysed in 6 N HCl at 110°C for 24 h.
Amino acid analysis of the peptide acid hydrolysates gave
the expected amino acid ratios. The analytical data of the
synthetic peptides prepared were as follows: compound
NOC-1 tR 4.10 min,[98% pure, 1,452.6 calculated (MH?),
1,452.8 observed (MH?); compound NOC-2 tR 4.60 min,
[99% pure, 1,599.8 calculated (MH?), 1,599.5 observed
(MH?); compound NOC-3 tR 4.40 min, [99% pure,
1,480.8 calculated (MH?), 1,480.3 observed (MH?); com-
pound NOC-4 tR 5.10 min,[98% pure, 1,627.8 calculated
(MH?), 1,627.5 observed (MH?); compound NOC-5 tR
4.80 min, [98% pure, 1,494.8 calculated (MH?), 1,494.9
observed (MH?); compound NOC-6 tR 5.20 min, [98%
pure, 1,641.9 calculated (MH?), 1,641.7 observed (MH?).
Phosphonopeptides are phosphorus analogues of natu-
rally occurring peptides containing a tetrahedral phospho-
rus atom. Their importance is obvious from the fact that
they are being widely used as enzyme inhibitors and, as
haptens in catalytic antibody research, because they can be
considered as stable mimetics of tetrahedral transition
states in ester and amide hydrolysis and formation
(Hirschmann et al. 1994; Kafarski and Lejczak 2000a, b;
Palacios et al. 2003; Cunningham et al. 2008). To date,
several efficient synthetic routes have been developed for
synthesis of phosphonopeptides and phosphinopeptides,
containingC-terminala-aminoalkylphosphinic acids (Kukhar
et al. 1994; Soloshonok et al. 1992, 2010; Xu and Yu 1999;
Meng and Xu 2010). As part of our research, the synthesis,
the characterisation and the biological activity of new
series of small peptides with aminophosphonate moiety as
NOP-receptor ligands, have previously been described
(Naydenova et al. 2010a, b).
Herein, we report the synthesis and biological activity of
novel NOP-receptor ligands. The new N/OFQ(1–13)NH2
derivatives with modification at 1st position by 1-[(meth-
oxyphosphono)methylamino]cycloalkanecarboxylic acids
were prepared using SPPS Fmoc-chemistry. In this study,
we tested the new ligands in vitro on electrically stimulated
smooth-muscle preparations from rat vasa deferentia. The
biological activity of the new compounds and the rela-
tionship ‘structure–activity’ were discussed.
Materials and methods
Synthesis
The protected amino acids were purchased from IrisBio-
tech (Germany). All other reagents and solvents were
analytical or HPLC grade and were bought from Merck
(Germany).
The solid-phase peptide synthesis by Fmoc (9-fluor-
enylmethoxy-carbonyl) chemistry was used to obtain new
NOP-receptor ligands. Rink-amide resin was used as a solid-
phase carrier, and 2-(1-OH-benzotriazole-1-yl)1,1,3,3-
tetramethyl-carbamide tetrafluoroborat (TBTU) was used
as a coupling reagent. The 3-functional amino acids were
embedded as follows: Arg—as Fmoc-Arg(Pbf)-OH, Ser—as
Biological tests
The biological activity of the newly synthesized analogues
of N/OFQ (1–13)NH2 was tested in vitro on electrically
123

Synthesis and biological activity of N/OFQ(1–13)NH2 analogues
1219
stimulated preparations of rat vas deferens. The smooth
muscles were isolated from Wistar albino rats, weighing
180–200 g. The prostatic parts of the vasa deferentia,
12–15 mm long, were carefully cleaned from the sur-
rounding connective tissues and blood vessels, fixed in
4-ml organ bath and connected to electronic transducers.
Before the experiment, the preparations were preloaded
with 1 g and left for a 60-min adaptation at 32.5°C in
Krebs solution, aerated with 95% O2 and 5% CO2.
Smooth-muscle contractions were evoked by low-fre-
quency electrical stimulation (LFES) with parameters
0.05 Hz frequency, 1 ms pulse duration, sub-maximal
voltage. The smooth-muscle tone and the electrically
induced contractions were isometrically registered. The
tested compounds were cumulatively applied to the organ
bath in concentrations of 1 9 10-11 M–1 9 10-5 M. In
some experiments, 1 9 10-6 M naloxone (to block l-, d-
and j- opioid receptors) or 3 9 10-5 M naloxone ben-
zoylhydrazone (to block opioid and nociceptin receptors)
was administered 10 or 15 min, respectively, before the
examined compounds.
we prepared and evaluated for agonistic activity in vitro
new N-modified analogues of the N/OFQ(1–13)NH2 with
aminophosphonate moiety containing five-, seven- and
eight-membered cycloalkane rings (Table 1). The peptides
(NOC 1–NOC 6) were obtained with good yield by solid
phase peptide synthesis—Fmoc (9-fluorenylmethoxy-car-
bonyl) chemistry, according to the previously described
procedure (Naydenova et al. 2010a, b). The optical rota-
tions and amino acid analysis data of the newly synthesized
compounds are shown in Table 1. The 1-[(dimethoxy-
phosphono) methylamino]cycloalkanecarboxylic acids were
previously prepared using Kabachnik-Fields reaction
(Naydenova et al. 2008, 2010a, b).
In order to estimate the ‘structure–activity’ relation, we
prepared the compounds NOC-1, NOC-3, and NOC-5 by
replacement of the N-terminal Phe in N/OFQ(1–13)NH2 with
1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic
acids. Aiming to additionally prove the importance of Phe,
we introduced the 1-[(methoxyphosphono)methylamino]
cycloalkanecarboxylic acids to the N-side of N/OFQ(1-13)
NH2 (NOC-2, NOC-4, NOC-6). The new analogues are
with the following sequences:
In some experiments, cumulative concentration–response
curves of N/OFQ(1–13)NH2 were built in the absence or in
the presence of NOC-3 (in three different concentrations—
1 9 10-7, 1 9 10-6 and 1 9 10-5 M), applied 30 min
before N/OFQ(1–13)NH2. In order to protect the pep-
tides from degradation, a cocktail of peptidase inhibi-
tors (amastatin, bestatin, phosphoramidon and captopril
(Nicholson et al. 1998)) in a final concentration of 3 9
10-5 M, was added to the incubation medium.
X-GGFTGARKSARK-NH2
(NOC-1, NOC-3, NOC-5)
X-FGGFTGARKSARK-NH2
(NOC-2, NOC-4, NOC-6)
where X is:
O
C
O
P
HO
CH2 NH
H3CO
Animals
n = 0, 2, 3
(CH2)n
In the experiments, male Wistar rats (180–200 g), housed
at 22–25°C, were used. The animals were allowed an
adaptation period with free access to food and water, and a
natural day/night light cycle. All the animals were killed
under light ether anaesthesia.
The crude peptides were purified on a reversed-phase
high-performance liquid chromatography (HPLC) and the
molecular weights were determined, using electrospray
ionisation mass-spectrometry. The analytical data are
shown in the ‘‘Materials and methods’’.
The experiments were performed according to the
requirements of the European Convention for the Protec-
tion of Experimental Animals (Protection of animals used
for experimental purposes, Council Directive 86/609/EEC
of November 1986) and the rules of the Ethics Committee
of the Institute of Neurobiology, BAS.
Biological activity
Intensive structure–activity studies (Dooley et al. 1997;
Reischeid et al. 1996; Butour et al. 1997; Guerrini et al.
2000a, b, c) have shown that the sequence Phe1-Gly–Gly-
Phe4 is critical for the message domain of N/OFQ, involved
in receptor binding and activation. The newly synthesized
peptides, tested in our laboratory, could be divided in two
groups. In the first one, aminophosphonate substituents
with varying ring (C5, C7, C8) are linked to Phe1 and the
indicated sequence remains intact. The peptides, including
the natural sequence (NOC-2, NOC-4, NOC-6), exert very
similar agonist activity (pEC50 = 6.17–6.51) and strong
Results and discussion
Chemistry
We have recently reported structure–activity relationship
of hexapeptides analogues with aminophosphonates moiety
as NOP-receptor ligands (Naydenova et al. 2010a, b). Now,
123

1220
P. T. Todorov et al.
Table 1 Physicochemical data for synthetic peptides
No.
Peptides
[a]aD (°)
-17.5
Amino acid analysisb
Gly
Phe
Thr
Ala
Arg
Ser
Lys
NOC-1
3.07
1.04
1.03
2.04
2.00
1.04
2.04
O
HO
CH2 NH
CO GGFTGARKSARK-NH2
CO FGGFTGARKSARK-NH2
P
H3CO
NOC-2
NOC-3
-22.6
-16.9
3.06
3.00
2.00
1.09
1.07
1.00
2.08
2.02
2.04
2.08
1.00
1.06
2.02
2.00
O
P
HO
CH2 NH
H3CO
O
HO
CO
P
CH2 NH
GGFTGARKSARK-NH2
H3CO
NOC-4
NOC-5
O
P
-21.7
-18.1
3.01
3.05
2.10
1.10
1.06
1.00
2.01
2.00
2.10
2.00
1.06
1.04
2.01
2.00
HO
CO
CO
CH2 NH
FGGFTGARKSARK-NH2
GGFTGARKSARK-NH2
H3CO
O
P
HO
CH2
NH
NH
H3CO
NOC-6
O
P
-23.2
3.08
2.07
1.07
2.05
2.10
1.10
2.02
HO
CO
CH2
FGGFTGARKSARK-NH2
H3CO
a
Optical rotation in 1% AcOH (c = 1) at 20°C
b
After hydrolysis with 6 N HCl at 110°C for 24 h. The corresponding 1-[(dimethoxyphosphono)methylamino]cycloalkanecarboxylic acids do not give ninhydrin
detection and cannot be proved by this method
120
100
80
60
40
20
0
inhibition of smooth-muscle contractions (Emax = -70 to
-90%, comparable to that of N/OFQ(1–13)NH2). In this
group (and for the whole study), NOC-6 is the most active
compound (Fig. 1). Unexpectedly, the larger cycle (C8) in
NOC-6 increased the activity and efficacy of the com-
pound, compared to peptides NOC-2 and NOC-4. Nalox-
one had no effect, whilst the pre-incubation of the
preparations with naloxone benzoylhydrazone completely
prevented the inhibition of smooth-muscle contractions,
thus showing that these three modified peptides selectively
bound to NOP receptors (Figs. 1, 2, 3).
NOC_2
NOC_2 + Nal
NOC_2 + Nal-B
When Phe at position 1 is substituted by the same am-
inophosphonate residues (NOC-1, NOC-3, NOC-5), the
obtained derivatives showed much lower activity and
affinity in the experiments on electrically induced smooth-
muscle contractions (Table 2). The effects are prevented to
a great extent by pre-incubation of the smooth-muscle
preparations with naloxone (Figs. 4, 5, 6); this indicates
that these new derivatives have gained affinity for classical
opioid receptors. Based on structure–activity relationship
study of N/OFQ, Reinscheid et al. (1996) have suggested
-11
-10
-9
-8
-7
-6
-5
log conc. (M)
Fig. 1 Effects of N/OFQ(1-13)NH2 and NOC-6 on LFES-induced
contractions
that more bulky side chain in this position prevents the
formation of a favourable secondary structure. In the
derivatives of the second group, a very big side chain is
123

Synthesis and biological activity of N/OFQ(1–13)NH2 analogues
1221
linked to Gly2, which could be the possible reason for the
low activity. The replacement of Phe at position 1 by
1-[(methoxyphosphono)methylamino]cycloalkanecarboxy-
lic acids goes along with a loss of receptor affinity and
biological activity of the obtained derivatives (Table 2,
Figs. 4, 5, 6).
The observed reduced efficacy (Emax) of the compounds
in this group gave us reason to test the antagonistic
120
100
80
120
100
80
60
60
40
40
NOC_1
NOC_4
NOC_1 + Nal
NOC_4 + Nal
20
20
NOC_1 + Nal-B
NOC_4 + Nal-B
0
0
-8
-7
-6
-5
-4
-11
-10
-9
-8
-7
-6
-5
log conc. (M)
log conc. (M)
Fig. 4 Effects of NOC-1 on LFES-induced contractions
Fig. 2 Effects of NOC-2 on LFES-induced contractions
120
100
80
120
100
80
60
60
NOC_6
40
20
0
40
NOC_3
NOC_6 + Nal
NOC_6 + Nal-B
N/OFQ(1-13)NH2
NOC_3 + Nal
20
0
NOC_3 + Nal-B
-11
-10
-9
-8
-7
-6
-5
-11
-10
-9
-8
-7
-6
-5
log conc. (M)
log conc. (M)
Fig. 3 Effects of NOC-4 on LFES-induced contractions
Fig. 5 Effects of NOC-3 on LFES-induced contractions
Table 2 Biological activity of the newly synthesized peptides on LFES-evoked contractions of rat vas deference
Peptide tested
Peptide alone
pEC50
Peptide ? naloxone
Emax (%)
pEC50
Emax (%)
N/OFQ(1-13)NH2
NOC-1
7.42 0.11
5.37 0.31
6.17 0.12
6.28 0.33
6.16 0.13
5.92 0.17
6.51 0.15
-85
2
7.18 0.13
4.85 0.72
5.92 0.08
5.83 0.36
6.06 0.06
5.74 0.72
6.55 0.10
-86
-31
-55
-17
-68
-19
-87
3
7
9
5
5
8
5
-54 10
NOC-2
-68
-26
-69
4
6
8
NOC-3
NOC-4
NOC-5
-48 10
-90
NOC-6
3
The maximal inhibitory effect of N/OFQ(1–13)NH2 is reached at a concentration of 10-6 M. Compared to N/OFQ(1–13)NH2, the newly
synthesized analogues have shown much lower activity. With them, the initial effect, as well as the maximal one, developed at higher
concentrations. Therefore, in Table 2, the equi-effective maximal concentrations have been used
123

1222
P. T. Todorov et al.
120
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properties of NOC-3 on the response to N/OFQ(1-13)NH2
(data not shown). In the presence of NOC-3 in any of the
concentration tested (1 9 10-7, 1 9 10-6, 1 9 10-5 M),
there was no concentration-dependent rightward shift of
the nociceptin response curve, indicating that the peptide
has no inhibitory properties but rather is a very week
agonist.
`
Guerrini R, Calo G, Bigoni R, Rizzi A, Varani K, Toth G, Gessi S,
Hashiba E, Hashimoto Y, Lambert DG, Borea PA, Tomatis R,
Salvadori S, Regoli D (2000a) Further studies on nociceptin-
related peptides: discovery of a new chemical template with
antagonist activity on the nociceptin receptor. J Med Chem
43:2805–2813
We describe the synthesis and biological investigation
of novel N-modified analogues of the N/OFQ(1–13)NH2
with aminophosphonate moiety. The newly synthesized
compounds were obtained by solid-phase peptide synthe-
sis—Fmoc-strategy. All of the peptides were tested for
agonistic activity in vitro. Our results have shown that the
selectivity of the peptides containing 1-[(methoxyphos-
phono)methylamino]cycloalkanecarboxylic acids at the
N-side of Phe is not changed—they remain selective agonists
of NOP receptors. The derivative with the largest ring (NOC-
6) demonstrated efficacy similar to that of N/OFQ(1–13)NH2,
but in a 10-fold higher concentration. Attachment of ami-
nophosphonates to Gly2 goes along with a loss of biolog-
ical activity of the obtained derivatives and an expressed
affinity to the classic opioid receptors.
Guerrini R, Rizzi A, Varani K, Toth G, Gessi S, Hashiba E,
Hashimoto Y, Lambert DG (2000b) Further studies on nocicep-
tin-related peptides: discovery of a new chemical template with
antagonist activity on the nociceptin receptor. J Med Chem
43:2805–2813
Guerrini R, Calo G, Rizzi A, Bigoni R, Rizzi D, Regoli D, Salvatory S
(2000c) Structure-activity relationships of nociceptine and
related peptides: comparition with dynorphin A. Peptides
7:923–933
Guerrini R, Calo’ G, Bigoni R, Rizzi A, Regoli D, Salvadori S
(2001a) Structure-activity relationship of [Nphe1]-NC-(1–13)-
NH2, a pure and selective nociceptin/orphanin FQ receptor
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Acknowledgments The study was supported by Grant DTK 02/61
of the National Research Fund, Sofia, Bulgaria.
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