Development of Nociceptin Receptor

Development of Nociceptin Receptor
(NOP) Agonists and Antagonists
Carlo Mustazza and Giuditta Bastanzio

Dipartimento del Farmaco, Istituto Superiore di Sanita, Viale Regina Elena 299, I-00161 Roma, Italy
Published online 22 January 2010 in Wiley Online Library (wileyonlinelibrary.com).

DOI 10.1002/med.20197
.
Abstract: The nociceptin opioid (NOP) receptor is the most recently discovered member of the family
of the opioid receptors; its endogenous agonist is the peptide nociceptin. Due to the subsequent elu-
cidation of its physiological role in both central and peripheral nervous system and in some non-neural
tissues, there is a rapidly growing interest in the pharmacological application of substances active on
this receptor. Despite the current clinical use of a morphinane-based NOP/MOP mixed ligand (bu-
prenorphine) as an analgesic and in the treatment of drug addictions, so far just a few clinical trials have
been made with selective NOP ligands. However, the perspective of their utilization is rapidly growing.
Agonists can nd applications in the treatment of neuropathic pain, anxiety, cough, drug addition,
urinary incontinence, anorexia, congestive heart failure, hypertension; and antagonists for pain, de-
pression, Parkinsons disease, obesity, and as memory enhancers. Besides peptide ligands, which are still
subjected to many pharmacological investigations, many different chemical classes of NOP ligands have
been discovered: piperidines, nortropanes, spiropiperidines, 4-amino-quinolines and quinazolines, and
others. The new advances in establishing structureactivity relationships, also with the help of modeling
studies, can permit the development of more active and selective molecules. & 2010 Wiley Periodicals, Inc.
Med Res Rev, 31, No. 4, 605648, 2011
Key words: nociceptin; NOP receptor ligands; opioids; structureactivity relationship
1. INTRODUCTION
The nociceptin receptor was discovered in 1994,1 following the identication and sequencing
from the human and mouse genome of the cDNA expressing it. It belongs to the trans-
membrane G-protein coupled receptor family and initially was called opioid-receptor like-1
receptor (ORL-1) because of its remarkable sequence homology with the classical opioid
receptors. Since 2000 the former denominations ORL-1, OP4, and LC132 have denitively
been replaced by nociceptin opioid (NOP) receptor, as established by the International
Union of Basic and Clinical Pharmacology (IUPHAR).2 Its endogenous agonist, discovered
in 1995,3,4 is the heptadecapeptide nociceptin/orphanin FQ (N/OFQ), which does not have
Correspondence to: Carlo Mustazza, Dipartimento del Farmaco, Istituto Superiore di Sanita,Viale Regina Elena 299, I-00161Roma,
Italy, E-mail: mustazza@iss.it
Medicinal Research Reviews, 31, No. 4, 605--648, 2011

& 2010 Wiley Periodicals, Inc.
606 K MUSTAZZA AND BASTANZIO
appreciable afnity to the other opioid receptors and whose sequence is Phe-Gly-Gly-Phe-
Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln.
The intracellular events triggered by the binding of N/OFQ with the NOP receptor, and
mediated by the activation of a G-protein, include inhibition of adenylyl cyclase, activation
of phospholipase C (PLC) and of K1 channels, inhibition of Ca21 channels, and activation
of mitogen-activated protein-kinases, which is only partially dependent on PLC.5 In neural
tissues, the main effect is the inhibition of the release of neurotransmitters (acetylcholine,
catecholamines, GABA, glutamate, 5-hydroxytryptamine, tachykinines).6,7 N/OFQ stimu-
lates neural nitric oxid synthase activity initiated by the activation of NDMA receptors in the
spinal cord. This action is related to the pronociceptive spinal effect at low doses, which is
reversed at higher doses.8
2. THE NOP RECEPTOR AS A PHARMACOLOGICAL TARGET
The NOP receptor is widely distributed in the central nervous system, with the highest
density in the forebrain, brainstem, and dorsal and ventral horns of the spinal cord. It is also
present in the peripheral nervous system and in some non-neural tissues (epidermis, im-
munocytes, and vascular endothelium).911 Its activation by N/OFQ produces many biolo-
gical effects,12,13 including hyperalgesia or analgesia (N/OFQ has a hyperalgesic effect in the
brain and an analgesic effect in the spinal cord and in the periphery), blockage of analgesia
produced by opioids, attenuation of behavioral response to stress, hypolocomotion, brady-
cardia, hypotension, vasodilation, diuresis, antinatriuresis, increased bladder capacity, in-
hibition of micturition reex, counteraction of the rewarding properties of drugs (e.g.
morphine, cocaine, alcohol), immunodepression, inhibition of tachykinergic bronchocon-
striction, inhibition of gastrointestinal motility, increasing food intake, impairment of
learning and memory, and erectile activity. Both NOP agonists and antagonists have a broad
therapeutic potential,14 the former as antitussives, anxiolytics, vasodilators, hypotensives, in
the treatment of neuropathic pain, drug dependence, urinary incontinence, congestive heart
failure, and anorexia, the latter as analgesics devoid of tolerance, antidepressants, nootropics,
and in the treatment of obesity and Parkinsons disease. At present, only very few clinical
trials have been done on NOP selective ligands.14,15 The antagonist JTC-801 (Japan Tobacco
Inc.) has been used in clinical trials for pain (Phases I and II, as reported in some reviews,14,15
but clinical data are not available in the literature) and a Phase I clinical trial has been
proposed for the antagonist SB-612111 (GlaxoSmithKline) in the treatment of Parkinsons
disease.14 N/OFQ has been used in clinical trials by intravesical administration in patients
with overactive bladder16 and the peptidic peripherally acting NOP partial agonist ZP-120
(Zealand Pharma) has been used in a Phase II study as a diuretic in the treatment of acute
heart failure.15 There are, nevertheless, a lot of clinical data available for opioid drugs
analogous to morphine which are also active on the NOP receptor: the mixed NOP/MOP
partial agonist buprenorphine is in clinical use for pain and opiate addiction17 and has been
used in clinical trials for cocaine addiction,18 and the antagonist TRK 820 (Acologix) is in
Phase III as an antipruritic.19 In fact there is a growing interest in mixed NOP/MOP ligands
as analgesics and in the treatment of drug abuse.20
The subject of the development of NOP ligands has been reviewed in 2000 by Calo
et al.21 in 2001 by Barlocco et al.22 and by Ronzoni et al.23 for nonpeptidic ligands, in 2003 by
Zaveri,24 in 2005 by Bignan et al.15 This review covers, along with all the necessary references
to previous works, all the recent advances in the design of NOP ligands belonging to the
already well established chemical classes of peptides, morphinans, piperidines,
4-amino-quinolines and quinazolines, cyclohexylamines, pyrroles, and compounds strictly
Medicinal Research Reviews DOI 10.1002/med
NOCICEPTIN RECEPTOR (NOP) AGONISTS AND ANTAGONISTS K 607
related to the above mentioned, together with more recent ligands based on new templates,
such as the spirotetracyclic NOP/MOP mixed ligands, 6-piperazinylbenzimidazoles, and
2-(1,2,4-oxadiazolyl)-indoles and pyrrolopiperidines, focusing mainly on relationships
between the chemical structure and activity or other pharmacological relevant features
(e.g. selectivity, metabolic stability).
3. NOCICEPTIN-RELATED PEPTIDES
Early structureactivity relationship (SAR) studies on N/OFQ2528 established that the ac-
tivity was at least partially maintained in its truncated analogues until N/OFQ(113)
(Table I). Besides, also the N/OFQ amide and the truncated amidated analogues until
N/OFQ(113)NH2 1 maintained almost all the biological activity of the natural peptide
(Table II), whereas the amino-terminal portion of N/OFQ resulted to be the most sensitive to
the structural and conformational modications. This was evidenced by substituting single
amino acids with alanine (Table III), thus showing that Phe1, Phe4, and Arg8 and, to a lesser
extent, Gly2 are the most important residues for afnity and activity. Also, Phe1 is essential
for selectivity, as its substitution with Tyr gave a peptide active on NOP but also on opioid
receptors.29,30 The same substitution for Phe4 gave a peptide with appreciable NOP binding
afnity but no activity. The substitution of Gly2 with Ala was detrimental for activity,
whereas the substitution of this residue with D-Ala gave a peptide with a substantially
maintained potency. On the contrary, the substitution of Gly3 with D-Ala suppressed ac-
tivity, whereas if it was replaced with Ala, the activity was partially maintained. This trend is
connected to different conformational effects of a-methylation according to the position. By
replacing Thr5 with Ala or D-Ala, afnity was lowered and activity suppressed; instead, Gly6
can be changed with either Ala or D-Ala with only a slight loss of potency. As it concerns the
other residues, the substitution of Ala7 with its D-enantiomer gave a partial agonist, whereas
by substituting Leu14 with Tyr or iodotyrosine, the biological activity of N/OFQ was fully
maintained. This is at the base of the now common use of [125I-Tyr14]N/OFQ as a radi-
oligand for the NOP receptor.30
Many of the following investigations evidenced the importance of the rst four residues
as the message domain, whereas the remaining part of the molecule can be indicated as the
address domain, as in the other opioid peptides. A study by Guerrini et al.31 showed that
the depletion of Gly2 in 1 gave an inactive peptide. This also happened by changing Phe4
with Leu, whereas if the same substitution was made from Phe1 the activity was maintained,
Table I. Afnities and Activities of Nociceptin and its Truncated Analogues
Compound pIC50 pEC50 Receptor source Ref.

27
N/OFQ 9.9 9.1 CHO
27
N/OFQ(113) 8.4 8.1 CHO
27
N/OFQ(112) 6.8 7.1 CHO
27
N/OFQ(111) 5.9 5.5 CHO
27
N/OFQ(110) 5.7 5.4 CHO
N/OFQ(19) 5.4 o5 CHO 27
N/OFQ(18) 5.0 o5 CHO 27
N/OFQ(17) o5 o5 CHO 26

Table II. Afnities and Activities of Nociceptin Amide and Nociceptin Truncated Amidated
Analogues

25
N/OFQ 8.2 Rat brain
N/OFQ 8.7 7.8 () 28
25
N/OFQ-NH2 7.9 Rat brain
N/OFQ-NH2 9.1 7.7 () 28
25
N/OFQ(116)NH2 7.8 Rat brain
25
25
25
N/OFQ(113)NH2 9.1 7.7 () 28
25
N/OFQ(112)NH2 7.6 6.1 () 28
25
N/OFQ(111)NH2 5.7 5.5 () 28
25
25
N/OFQ(19)NH2 o5 o5 () 28
N/OFQ(18)NH2 o4 Rat brain 25

25
25
25
N/OFQ(15)NH2 o5 o5 () 28
N/OFQ(14)NH2 o3.8 Rat brain 25
N/OFQ(14)NH2 o5 o5 () 28
()Receptor binding was determined on mouse brain, activity on mouse vas deferens.
showing that Phe4 and not Phe1 is involved in receptor activation. Another paper from the
same group32 describes the peptide [Phe1c(CH2NH)Gly2]N/OFQ(113)NH2, obtained from
1 by reducing the rst peptidic bond to CH2NH. This peptide was initially claimed as the rst
NOP antagonist to be found, but was revealed to be a partial agonist by further pharma-
cological investigations.33 From studies of analogues of 1 substituted on the rst residue28,34
(Table IV), it was shown that the substitution of Phe1 with Leu or cyclohexylalanine
maintained activity, whereas the same replacement with analogous aminoacids like p-me-
thylphenylalanine or N-methylphenylalanine decreased it consistently. This can be explained
as a fundamental role of the spatial disposition of the aromatic group, conrmed by the fact
that also replacing the rst residue with its enantiomer D-Phe or with the bulky N,N-dia-
llylphenylalanine or 1,2,3,4-tetrahydroquinoline-3-carboxylic acid led to inactive peptides.
The positive amino-terminal charge also plays a fundamental role, as showed by the scarce
activity of the N-acetylderivative of 1.
The substitution of the rst residue with N-benzylglycine gave the antagonist [Nphe1]N/
OFQ(113)NH2. This latter compound was in turn the template for a SAR study on the
substitution of the rst residue35 (Table V), showing the critical role of both sterical and
electronic properties of the aromatic group for the activity. In fact, the activity was strongly
reduced or suppressed by introducing various substituents on the phenyl of N-benzylglycine, as
well as by replacing it with various aliphatic, aromatic or heteroaromatic substituents, or else
introducing or removing a CH2 group, as in N-phenylglycine, N-phenethylglycine or N-benzyl-
b-alanine, or eventually replacing N-benzylglycine with its cyclic analogues (2,3-dihydroindole-
1-acetic acid, 2,3-dihydroisondole-2-acetic acid, 1,2,3,4-tetrahydroisoquinoline-2-acetic acid).

Table III. Afnity and Activity Data for Nociceptin or Nociceptin Amide Analogues Substituted at a
Single Amino Acid Residue

25
N/OFQ 8.2 Rat brain
26
N/OFQ 9.7 9.0 CHO
41
N/OFQ 9.1 7.9 COS-7
50
N/OFQ-NH2 7.8 Mouse VD
[Ala1]N/OFQ 7.7 o5 CHO 26
26
[Tyr1]N/OFQ 9.9 9.0 CHO
26
[D-Phe1]N/OFQ 8.6 7.0 CHO
26
[D-Ala2]N/OFQ 9.2 8.3 CHO
26
[Ala3]N/OFQ 10.0 7.4 CHO
[D-Ala3]N/OFQ 7.8 o5 CHO 26

26
[D-Phe4]N/OFQ 8.6 o6 CHO 26
[D-Thr5]N/OFQ 7.8 o6 CHO 26

26
[Ala6]N/OFQ 6.8 CHO
26
[D-Ala6]N/OFQ 7.0 CHO
26
[Ser7]N/OFQ 6.9 CHO
26
[D-Ala7]N/OFQ 9.9 7.5 CHO
[D-Arg8]N/OFQ 8.8 o6 CHO 26

25
[Ala9]N/OFQ 7.4 Rat brain
25
25
26
[Ala12]N/OFQ 8.9 6.8 CHO
25
41
[Ala14]N/OFQ 8.8 8.0 COS-7
41
[Arg14]N/OFQ 9.5 8.4 COS-7
41
[Lys14]N/OFQ 9.4 8.4 COS-7
41
[Val14]N/OFQ 9.0 7.8 COS-7
41
[Phe14]N/OFQ 9.0 8.0 COS-7
41
[Trp14]N/OFQ 9.4 8.2 COS-7
26
26
[I-Tyr14]N/OFQ 9.3 8.9 CHO
26
[I2-Tyr14]N/OFQ 9.0 8.7 CHO
41
[Arg15]N/OFQ 9.5 8.5 COS-7
41
[Lys15]N/OFQ 9.4 8.2 COS-7
41
[Leu15]N/OFQ 9.3 8.0 COS-7
41
[Val15]N/OFQ 8.7 7.5 COS-7
41
[Phe15]N/OFQ 8.0 7.9 COS-7
41
[Tyr15]N/OFQ 8.0 7.4 COS-7
41
[Trp15]N/OFQ 8.7 7.3 COS-7
25
49
[Aib7]N/OFQ-NH2 10.3 10.2 HEK-293
49
[Aib11]N/OFQ-NH2 10.1 9.9 HEK-293
49
[Aib15]N/OFQ-NH2 10.7 9.7 HEK-293
49
[Nma7]N/OFQ-NH2 8.7 8.0 HEK-293
49
[Nma11]N/OFQ-NH2 8.2 7.3 HEK-293
49
[Nma15]N/OFQ-NH2 10.2 9.1 HEK-293

Table III. Continued

53
[Glu16]N/OFQ-NH2 7.4 Mouse VD
53
[Pro6]N/OFQ-NH2 6.6 Mouse VD
53
53
Table IV. SAR of the First Aminoacid Residue in N/OFQ(113)NH2
Compound pIC50 pEC50 Ref.

34,31
N/OFQ(113)NH2 9.1 7.8
31
[D-Phe1]N/OFQ(113)NH2 Inactive
34,28
[p-MePhe]N/OFQ(113)NH2 6.7 5.6
28
[N-MePhe]N/OFQ(113)NH2 6.0
31
[(N,N-diallyl)Phe] N/OFQ(113)NH2 Inactive
[Tyr1]N/OFQ(113)NH2 8.4 7.6 () 34,28
[(20 ,60 diMe)Tyr1] N/OFQ(113)NH2 7.9 () 28

28
[Cha1] N/OFQ(113)NH2 9.0 7.9
34
[Leu1] N/OFQ(113)NH2 8.6 7.6
31
Ac-N/OFQ(113)NH2 5.8
28
[Tic1]N/OFQ(113)NH2 Inactive
Cha, 3-cyclohexyl-L-alanine; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. Affinities were determined on
mouse brain membranes and activities on mouse vas deferens; where indicated by () activity was affected by
1 mM naloxone.
Also, methylation either on the nitrogen or on the glycine CH2 of the residue (to give N-benzyl-
L-or D-Ala) suppressed activity, whereas antagonistic activity was maintained if the methyl was
introduced in the benzyl CH2 of the residue, in the case of the stereoisomer [(S)(bMe)Nphe1]N/
OFQ(113)NH2.
A study on the variation of the fourth residue in 136,28 (Table VI) showed that by
substituting it with analogues with altered aromaticity, such as cyclohexylalanine, trypto-
phan, a- or b-naphthylalanine, or different for the position of the phenyl group (e.g.
a-phenylglycine, homophenylalanine, N-benzylglycine, D-phenylalanine) or by bringing a
sterical constraint as in the case of a-methylphenylalanine or 2-aminotetraline-2-carboxylic
acid, activity was suppressed or greatly lowered, with the exception of the substitution with
N-methylphenylalanine which caused only a slight decrease of potency. The p-substitution on
Phe4 gave compounds more active than 1 if the substituent was F, NO2, or CN, whereas
other substituents had a deactivating effect, almost complete in the case of NH2 or OH.
Especially interesting was the peptide [(pF)Phe4]N/OFQ(113)NH2, which was a template
for further studies (see below).
As a further development, a SAR study on the peptidic bond between the rst two residues
in 137 (Table VII) showed that the NOP activity was partly maintained by replacing that bond
with CH2O. Instead, the substitution to CH2S led to a partial agonist, the substitution to
COCH2 greatly diminished activity, and the inversion to NHCO or the methylation to
CON(CH3) suppressed it completely. Another study on analogues of 138 showed that its
N-terminal extension with an Arg residue giving [Arg0]N/OFQ(113)NH2 diminished both

Table V. SAR of the First Aminoacid Residue in [Nphe1]N/OFQ(113)NH2
Compound pIC50 Antagonist pKB Agonist pEC50 Ref.

34,35
[Nphe1]N/OFQ(113)NH2 7.0 6.4 Inactive
34,35
[Ntyr1]N/OFQ(113)NH2 5.5 Inactive Inactive
34
[Ncha1]N/OFQ(113)NH2 5.6 Inactive Inactive
34
[Nleu1]N/OFQ(113)NH2 5.6 Inactive Inactive
34,35
[N(p-Me)phe1]N/OFQ(113)NH2 5.6 Inactive Inactive
35
[N(o-Me)phe1]N/OFQ(113)NH2 5.8 Inactive
35
[N(m-Me)phe1]N/OFQ(113)NH2 5.6 Inactive
35
[N(p-NO2)phe1]N/OFQ(113)NH2 Inactive Inactive
35
[N(p-F)phe1]N/OFQ(113)NH2 Inactive Inactive
35
[N1nal1]N/OFQ(113)NH2 Inactive Inactive
35
[N2nal1]N/OFQ(113)NH2 Inactive Inactive
35
[N2thi1]N/OFQ(113)NH2 5.8 Inactive
35
[N3thi1]N/OFQ(113)NH2 5.7 Inactive
35
[N2fur1]N/OFQ(113)NH2 Inactive Inactive
35
[N3fur1]N/OFQ(113)NH2 Inactive Inactive
35
[N(Bn)Sar1]N/OFQ(113)NH2 Inactive Inactive
35
[(S)(bMe)Nphe1]N/OFQ(113)NH2 6.5 Inactive
35
[(R)(bMe)Nphe1]N/OFQ(113)NH2 5.9 Inactive
35
[N(Bn)Ala1]N/OFQ(113)NH2 Inactive Inactive
35
[N(Bn)D-Ala1]N/OFQ(113)NH2 Inactive Inactive
35
[Nphg1]N/OFQ(113)NH2 5.4 Inactive
35
[Nhphe1]N/OFQ(113)NH2 Inactive Inactive
35
[N(Bn)bAla1]N/OFQ(113)NH2 Inactive Inactive
35
[Tia1]N/OFQ(113)NH2 5.3 Inactive
35
[Dsa1]N/OFQ(113)NH2 5.3 Inactive
35
[Dia1]N/OFQ(113)NH2 5.3 Inactive
Ncha, N-cyclohexylmethylglycine; N1nal, N-(1-naphthylmethyl)glycine; N2nal, N-(2-naphthylmethyl)glycine; N2thi,
N-(2-thienylmethyl)glycine; N3thi, N-(3-thienylmethyl)glycine; N2fur, N-(2-furylmethyl)glycine; N3fur, N-(3-furyl-
methyl)glycine; Nphg, N-phenylglycine; Nhphe, N-phenethylglycine; Tia, 1,2,3,4-tetrahydroquinoline-2-acetic acid;
Dsa, 1,2-dihydroisoindole-2-acetic acid; Dia, 2,3-dihydroindole-1-acetic acid. Affinities were determined on mouse
brain membranes and activities on mouse vas deferens.
afnity and selectivity, and that the substitution of Gly3 with polar residues, such as Arg, Asn,
or formyllisine caused a very relevant drop in potency (complete in the case of Asn). The
substitution of Ser10 with D-Ser, Pro, or D-Pro lowered afnity due to the changes in backbone
conformation of the peptides (Tables VIII and IX). The importance of the Gly2Gly3 portion for
the biological activity of 1 has also been demonstrated in the above mentioned work by Calo
et al.32 (Table VIII), which showed that the conformation of this portion is very important for
the interaction with the receptor, just as the substitution of Gly2 or Gly3 with Phe or D-Phe was
detrimental for activity. The distance between the two a-carbons of Phe1 and Phe4 also plays a
fundamental role, as showed by the suppression of activity by substituting the Gly2Gly3
sequence with a b-alanine, GABA, d-aminovaleric, or proline residue. Additionally, the inser-
tion of a third glycine residue greatly diminished activity. In another work,39 it was shown that
the substitution of Gly2 in 1 with sarcosine reduced both agonistic activity and selectivity. If
Gly3 was substituted with sarcosine, the agonistic activity was suppressed. These effects are
related to the increased exibility of the amino-terminal portion of N/OFQ due to methylation.
All these results, as well as many of the following, depicted the activity of N/OFQ
as related to its amino-terminal portion, but a test on the carboxy-terminal fragments of

Table VI. SAR of the 4th Aminoacid Residue in N/OFQ(113)NH2

36
N/OFQ(113)NH2 7.5 Mouse VD
N/OFQ(113)NH2 9.0 9.5 () 36
28
[Leu4]N/OFQ(113)NH2 Inactive Mouse VD
28
[Trp4] N/OFQ(113)NH2 6.4 Mouse VD
36
[Cha4]N/OFQ(113)NH2 34% Emax at 10 mM Mouse VD
36
[(pPh)Phe4]N/OFQ(113)NH2 Inactive Mouse VD
36
[1Nal4]N/OFQ(113)NH2 5.3 Mouse VD
36
[2Nal4]N/OFQ(113)NH2 5.6 Mouse VD
36
[C(bPh)Phe4]N/OFQ(113)NH2 Inactive Mouse VD
36
[Phg4]N/OFQ(113)NH2 Inactive Mouse VD
36
[hPhe4]N/OFQ(113)NH2 Inactive Mouse VD
36
[Nphe4]N/OFQ(113)NH2 16% Emax at 10 mM Mouse VD
36
[N-MePhe4]N/OFQ(113)NH2 7.1 Mouse VD
28
[D-Phe4]N/OFQ(113)NH2 Inactive Mouse VD
[Tyr4]N/OFQ(113)NH2 6.3 6.9 () 36
[(pF)Phe4]N/OFQ(113)NH2 9.4 9.8 () 36
[(pCl)Phe4]N/OFQ(113)NH2 9.1 9.6 () 36
[(pBr)Phe4]N/OFQ(113)NH2 8.7 8.8 () 36
[(pI)Phe4]N/OFQ(113)NH2 7.8 8.1 () 36
[(mF)Phe4]N/OFQ(113)NH2 9.0 9.6 () 36
[(oF)Phe4]N/OFQ(113)NH2 8.4 9.0 () 36
[(pNO2)Phe4]N/OFQ(113)NH2 9.5 9.9 () 36
[(pCN)Phe4]N/OFQ(113)NH2 9.2 9.5 () 36
[(pCF3)Phe4]N/OFQ(113)NH2 9.0 7.5 () 36
[(pMeO)Phe4]N/OFQ(113)NH2 7.0 7.2 () 36
[(pMe)Phe4]N/OFQ(113)NH2 7.8 8.0 () 36
[(pNH2)Phe4]N/OFQ(113)NH2 5.7 6.6 () 36
Cha, cyclohexylalanine; 1Nal, (1-naphthyl)alanine; 2Nal, (2-naphthyl)alanine; Phg, L-a-phenylglycine; hPhe, homo-
phenylalanine. () Receptor binding determined in mouse forebrain membranes and activity in CHO cell transfected
with human NOP receptor. All the compounds showed no antagonistic activity except [Tyr4]N/OFQ(1--13)NH2,
showing antagonistic activity on mouse VD (pKB 5 5.5) but not on CHO cells.
N/OFQ27 (Table X) unexpectedly revealed a signicant agonist activity for peptides

N/OFQ(617) and, to a lesser degree, N/OFQ(1217), whereas N/OFQ(217) is less active.
These results can be rationalized by considering the role of the rst aminoacids in the
sequence as relating more to the conformation of the peptidic chain than to the binding with
residues on the receptor.
The relevant importance of the two couples of basic residues (Arg8Lys9 and Arg12-
Lys13) for the afnity and activity of N/OFQ or 1, which has been successfully exploited in
further works, has been evidenced since the early studies which showed that the substitution
of any of these four residues with Ala residues was detrimental for activity.31 The in-
troduction of a third Arg-Lys couple in the sequence of N/OFQ led to the discovery of
[Arg14,Lys15]N/OFQ, which was the rst peptide agonist more potent than N/OFQ itself to
be found. Instead, its analogues [Arg6,Lys7]N/OFQ and [Arg10,Lys11]N/OFQ had weaker
activities (especially the rst one)40 (Table XI). A further work revealed a similar activity also
for [Lys14,Lys15]N/OFQ, [Arg14,Arg15]N/OFQ, and [Lys14,Arg15]N/OFQ, showing that
the superagonistic activity is related to the synergistic effect of two basic residues in positions
14 and 15. Instead, the introduction of only a Lys or Arg on position 14 or 15 produced no
Table VII. SAR of the Modication of the First Residue and of the Bond Between the Two First
Residues in [Xaa1c(X)Gly2]N/OFQ(113)NH2
Compound pKi Antagonist pKB Agonist pEC50 Ref.

37
Xaa 5 Phe; X 5 CONH Inactive 7.8
32,34,37
Xaa 5 Phe; X 5 CH2NH 8.0 6.8 Inactive
32
Xaa 5 D-Phe; X 5 CH2NH Inactive Inactive
34
Xaa 5 Tyr; X 5 CH2NH 7.0 5.7 (a)
34
Xaa 5 p-MePhe; X 5 CH2NH 6.2 5.7 Inactive
34
Xaa 5 Cha; X 5 CH2NH 6.5 Inactive Inactive
34
Xaa 5 Leu; X 5 CH2NH 7.8 5.4 (a)
37
Xaa 5 Phe; X 5 CH2N(CH3) Inactive (a)
37
Xaa 5 Phe;X 5 CH2O Inactive 7.4
37
Xaa 5 Phe;X 5 CH2S 6.2 6.8
37
Xaa 5 Phe;X 5 COCH2 Inactive 6.2
37
Xaa 5 Phe;X 5 NHCO Inactive Inactive
(a) Only a slight effect at 10 mM. Affinities were determined in mouse brain membranes and activities in mouse vas
deferens.
Table VIII. SAR of the Gly2-Gly3 Portion in N/OFQ(113)NH2
Compound pEC50 Ref.

31,32
N/OFQ(113)NH2 7.7
31
[des-Gly2]N/OFQ(113)NH2 Inactive
32
[des-Gly2,3]N/OFQ(113)NH2 Inactive
28
[D-Ala2]N/OFQ(113)NH2 6.0
28
[Sar2] N/OFQ(113)NH2 5.7
32
[Phe2]N/OFQ(113)NH2 Inactive
32
[D-Phe2]N/OFQ(113)NH2 Inactive
32
[Phe3]N/OFQ(113)NH2 Inactive
39
[Sar3]N/OFQ(113)NH2 (a)
32
[D-Phe3]N/OFQ(113)NH2 5.7
31
[Ala2,3]N/OFQ(113)NH2 Inactive
28
[Arg3]N/OFQ(113)NH2 Inactive
32
[Pro2,des-Gly3]N/OFQ(113)NH2 Inactive
31
[D-Ala2,des-Gly3]N/OFQ(113)NH2 (a)
32
[b-Ala2,des-Gly3]N/OFQ(113)NH2 Inactive
32
[Gaba2,des-Gly3]N/OFQ(113)NH2 Inactive
32
[Vra2,des-Gly3]N/OFQ(113)NH2 Inactive
32
[Phe-(Gly)3-Phe]-N/OFQ(513)NH2 5.5
38
[Lys(For)3] N/OFQ(513) NH2 3.2
Vra, 5-aminovaleric acid. (a) Only a slight effect at 10 mM. Activities were determined on mouse vas deferens.
more than a moderate increase of activity.41 In the same study afnities and activities were
also determined for N/OFQ substituted only at position 14 or 15 (Table III). The result was
that introducing a tryptophan in position 14 gave effects similar to a basic residue, whereas a
similar trend was not observed for position 15. This can be explained in terms of cation/p
interaction for Lys or Arg in position 14 with aromatic residues on receptor, which can be
replaced by a hydrophobic interaction. The considerable activity of [Trp14,Lys15]N/OFQ
and [Trp14,Arg15]N/OFQ (both 10 times more potent than N/OFQ in vitro), shown in a

Table IX. Afnities and Activities of Variously Substituted Analogues of N/OFQ(113)NH2 and of
N/OFQ(114)NH2
Compound pKi pEC50 Receptor source Ref.

38,28
N/OFQ(113)NH2 9.7 7.8 CHO
43
N/OFQ(113)NH2 6.2 Rat VD
55
N/OFQ(113)NH2 9.5 9.0 HEK-293
28
[Pro6]N/OFQ(113)NH2 5.9 CHO
28
[D-Ala7]N/OFQ(113)NH2 5.8 CHO
28
[Lys8]N/OFQ(113)NH2 5.0 CHO
28
[Arg9] N/OFQ(113)NH2 7.6 CHO
28
[Lys8,Arg9] N/OFQ(113)NH2 5.4 CHO
28
[Ala8,Ala9] N/OFQ(113)NH2 Inactive CHO
38
[D-Ser10] N/OFQ(113)NH2 8.8 5.1 CHO
38
[Pro10]N/OFQ(113)NH2 8.4 5.1 CHO
38
[D-Pro10]N/OFQ(113)NH2 7.0 7.2 CHO
38
[Arg3, D-Ser10] N/OFQ(113)NH2 6.0 5.2 CHO
38
[Arg3, Pro10]N/OFQ(113)NH2 5.8 5.3 CHO
38
[Arg3, D-Pro10]N/OFQ(113)NH2 (a) 4.6 CHO
38
[Asn3, D-Ser10] N/OFQ(113)NH2 (a) 6.0 CHO
38
[Asn3,Pro10]N/OFQ(113)NH2 (a) 4.6 CHO
38
[Asn3, D-Pro10]N/OFQ(113)NH2 (a) 4.0 CHO
38
[Arg0]N/OFQ(113)NH2 8.3 CHO
38
[Arg0,Arg3]N/OFQ(113)NH2 6.6 4.1 CHO
38
[Arg0, D-Ser10]N/OFQ(113)NH2 8.7 CHO
28
[Pro11]N/OFQ(113)NH2 5.7 CHO
28
[Ala12]N/OFQ(113)NH2 Inactive CHO
28
[Lys12]N/OFQ(113)NH2 7.1 CHO
28
[Arg13]N/OFQ(113)NH2 7.4 CHO
28
[Lys12,Arg13]N/OFQ(113)NH2 7.2 CHO
43
[Orn9]N/OFQ(113)NH2 6.6 Rat VD
43
[Orn13]N/OFQ(113)NH2 4.9 Rat VD
43
[Orn9,Orn13]N/OFQ(113)NH2 6.4 Rat VD
43
[Dab9,Dab13]N/OFQ(113)NH2 5.2 Rat VD
43
[Dap9,Dap13]N/OFQ(113)NH2 5.6 Rat VD
[Cys0,Cys7]N/OFQ(113)NH2 5.6 o5 HEK-293 55
55
[Cys0,Cys11]N/OFQ(113)NH2 5.9 6.5 HEK-293
55
55
55
55
57
[Asp6,Lys10]N/OFQ(113)NH2 9.3 7.7 CHO
57
[D-Asp7,Lys10]N/OFQ(113)NH2 8.7 7.0 CHO
(a) Only a slight effect at 10 mM.
subsequent work,42 gave further conrmation of this interpretation. In comparison, only a

slight enhancement in both afnity and activity compared to N/OFQ was obtained in the
case of [Lys14,Trp15]N/OFQ and [Arg14,Trp15]N/OFQ.
A SAR study on the two original basic couples28 (Table IX) showed that Arg8 plays a
critical role in the interaction with the receptor and cannot be replaced even with another
positively charged residue, such as Lys, without a strong decrease in activity, as shown by the
mouse vas deferens assays on [Lys8]N/OFQ(113)NH2 and [Lys8,Arg9] N/OFQ(113)NH2.
On the other hand, an exchange of Arg with Lys or vice versa in the positions 9, 12, or 13 did
Table X. Afnities and Activities of Carboxyterminal Fragments of Nociceptin
Compound pKi pEC50 Receptor source Ref.

27
N/OFQ 9.9 9.1 CHO
27
N/OFQ(2-17) 6.6 5.9 CHO
27
N/OFQ(617) 7.7 7.4 CHO
N/OFQ(1017) o6 o5 CHO 26
27
N/OFQ(1217) 7.3 6.1 CHO
N/OFQ(1317) o5 o5 () 28
N/OFQ(217)NH2 o5 () 28
N/OFQ(1317)NH2 o5 () 28
() Affinities were determined on mouse brain membranes and activities on mouse vas deferens.
not give a signicant effect. In another work,43 a peptide with a slightly enhanced activity
compared to 1 was obtained by substitution of both Lys9 and Lys13 in 1 with ornitine (Orn).
However, the substitution on these positions with the shorter chain homologues 1,4-diami-
nobutyric and 1,3-diaminopropionic acid reduced activity. The substitution of Lys with
ornitine was more effective if it concerned position 9 only, whereas [Orn13]N/
OFQ(113)NH2 resulted to be much less active than 1. The chelating analogue derived from
[Arg14,Lys15]N/OFQ-NH2 by binding on Lys15 a diethylenetriaminepentaacetic acid
(DTPA) unit, as well as the dimeric analogue with the DTPA unit linking the Lys15 of two
peptidic portions, had similar afnities to N/OFQ and were even more potent as agonists in
the in vitro assays and on mouse vas deferens.44
The above mentioned effect of the introduction of a third couple of basic residues has
been successfully exploited (Table XI). This has allowed the conversion of the potent agonist
[(pF)Phe4]N/OFQ (113)NH2, the low-potency antagonist [Nphe1]N/OFQ(113)NH2 and
the selective partial agonist [Phe1c(CH2NH)Gly2] N/OFQ (113)NH2 (see above), respec-
tively, into the superagonist UFP 102 [(pF)Phe4,Arg14,Lys15]N/OFQ-NH2, the very potent
and selective antagonist UFP 101 [NPhe1,Arg14,Lys15]N/OFQ-NH2 and the high-afnity
partial agonist UFP 103 [Phe1c(CH2NH)Gly2,(pF)Phe4,Arg14,Lys15]N/OFQ-NH2,45 all of
which were developed by the University of Ferrara. UFP 101 is a useful tool in pharma-
cological in vitro and in vivo studies,46 and its tritiated derivative is a good radioligand for
the NOP receptor.47
Other peptides have been designed with the aim of introducing conformational
restraints, as in the case of the strong agonists [Aib7]N/OFQ-NH248 and [Aib7,Aib11]N/
OFQ-NH2,49 both restricted to an a-helix conformation because of the a-aminoisobutyric
acid (Aib) residues (Table III). The introduction in position 7 or 15 of other C-a,a-dis-
ubstituted linear, branched, or cyclic aminoacids gives poorer results, with activities in most
cases lesser than N/OFQ amide itself, because of the distortion of the a-helix.50 The con-
formational effect has been further exploited by carrying out the same substitution of Ala7
with Aib in UFP 101, UFP102, and UFP103, giving, respectively, the strong antagonist UFP
111, the superagonist UFP 112, and the partial agonist UFP 113 (University of Ferrara).50
By substituting both Ala7 and Ala11 with Aib, were obtained the agonist [(pF)Phe4,
Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 and the antagonist [NPhe1,(pF)Phe4,Aib7,Aib11,
Arg14,Lys15]N/OFQ-NH2, both endowed with a very strong activity,51 which was also
displayed in the in vivo assays in mice.52
The effect of the constraint to a-helix conformation on the activity of N/OFQ analogues
on ORL-1 receptor has been conrmed by the high-agonist effect of [Leu11,Leu15]N/OFQ-
NH2, whereas [Leu7,Leu11]N/OFQ-NH2 was less active than 1, despite the presence of the
same conformational effect. This latter result can be rationalized in terms of steric crowding
Table XI. Afnities and Activities of Variously Substituted Analogues of Nociceptin and Nociceptin
Amide
Antagonist Receptor
Compound pIC50 pEC50 pKb source Ref.
40
N/OFQ 9.0 7.8 HEK-293
41
N/OFQ 9.1 7.9 COS-7
50
N/OFQ 7.4 Mouse VD
40
[Arg6,Lys7]N/OFQ 8.0 6.6 HEK-293
40
[Arg10,Lys11]N/OFQ 8.8 7.7 HEK-293
40
[Arg14,Lys15]N/OFQ 9.5 9.0 HEK-293
41
[Arg16,Lys17]N/OFQ 9.3 8.6 COS-7
41
[Lys14,Arg15]N/OFQ 9.7 8.9 COS-7
41
[Arg14,Arg15]N/OFQ 9.6 9.0 COS-7
41
[Lys14,Lys15]N/OFQ 9.8 9.0 COS-7
41
[Arg14,Lys15,Arg16,Lys17]N/OFQ 9.4 8.6 COS-7
45
N/OFQ-NH2 10.3 9.0 CHO
45
[(p-F)Phe4]N/OFQ-NH2 10.7 9.5 CHO
45
[Arg14,Lys15]N/OFQ-NH2 11.2 9.8 CHO
45
[(p-F)Phe4,Arg14,Lys15]N/OFQ-NH2 11.3 10.1 CHO
45
[Phe1C(CH2NH)Gly2]N/OFQ-NH2 9.9 8.3 CHO
45
[Phe1C(CH2NH)Gly2,(p-F)Phe4]N/OFQ-NH2 10.3 9.1 CHO
45
[Phe1C(CH2NH)Gly2,Arg14,Lys15]N/OFQ-NH2 10.5 9.0 CHO
45
[Phe1C(CH2NH)Gly2,(p-F)Phe4,Arg14,Lys15]N/OFQ-NH2 10.7 9.7 CHO
45
[Nphe1]N/OFQ-NH2 9.4 Inactive 7.5 CHO
45
[Nphe1,(p-F)Phe4]N/OFQ-NH2 9.6 8.2 8.3 CHO
45
[Nphe1,Arg14,Lys15]N/OFQ-NH2 9.9 Inactive 9.1 CHO
45
[Nphe1,(p-F)Phe4,Arg14,Lys15]N/OFQ-NH2 10.6 9.4 9.7 CHO
49
[Aib7,Aib11]N/OFQ-NH2 10.3 10.1 HEK-293
50
[Nphe1,Aib7,Arg14,Lys15]N/OFQ-NH2 9.7 Inactive 8.7 CHO
50
[Nphe1,Aib7,Arg14,Lys15]N/OFQ-NH2 Inactive 7.5 Mouse VD
50
[Nphe1,(p-F)Phe4,Aib7,Arg14,Lys15]N/OFQ-NH2 10.5 10.5 CHO
50
[Phe1C(CH2NH)Gly2,(p-F)Phe4,Aib7,Arg14,Lys15]N/OFQ-NH2 10.3 9.7 CHO
50
[Phe1C(CH2NH)Gly2,(p-F)Phe4,Aib7,Arg14,Lys15]N/OFQ-NH2 Variable 9.1 Mouse VD
effect
[(p-F)Phe4,Aib7,Aib11]N/OFQ-NH2 10.1 8.5 () 51
[(p-F)Phe4,Arg14,Lys15]N/OFQ-NH2 10.2 9.0 () 51
[(p-F)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 10.3 9.1 () 51
[Nphe1,Aib7,Aib11]N/OFQ-NH2 9.1 7.1 () 51
[Nphe1,(p-F)Phe4,Aib7,Aib11]N/OFQ-NH2 9.2 7.7 () 51
[Nphe1,(p-F)Phe4,Arg14,Lys15]N/OFQ-NH2 9.5 7.8 () 51
[Nphe1,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 9.6 8.0 () 51
[Nphe1,(p-F)Phe4Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 10.0 8.4 () 51

53
[Leu7,Leu11]N/OFQ-NH2 7.6 Mouse VD
53
[Leu11,Leu15]N/OFQ-NH2 8.1 Mouse VD
53
[Leu11,Leu15,Glu16]N/OFQ-NH2 7.6 Mouse VD
Aib, a-aminoisobutyric acid. () Affinities determined on rat forebrain membranes and activities on mouse VD.
in the interaction between the receptor and the so-called hinge region of the peptide, span-
ning from the fth to the seventh residue.53 The presence of such a exible b-turn has been
conrmed by NMR conformational studies54 and is consistent with the fact that both
[Pro6]N/OFQ-NH2 and [D-Ala7]N/OFQ-NH2 showed fairly good afnity but scarce activity.
This can be due to the conformational constraints between the message domain and the
region bearing the positively charged residues. Another situation where an enhanced pro-
pensity to the a-helix is not favorable to activity is represented by [Glu6]N/OFQ-NH2 and
[Glu6,Leu11,Leu15]N/OFQ-NH2, where this conformation is favored by the electrostatic
Table XII. Afnity and Activity Data for Cyclic Analogues of Nociceptin
Compound pKi pEC50 pKb() (antagonist) Receptor source Ref.

55
N/OFQ 9.5 8.7 HEK-293
56
N/OFQ 9.2 8.3 CHO
59
N/OFQ 9.6 BHK
c.[Cys0,Cys7]N/OFQ(113)NH2 6.1 o5 HEK-293 55
55
c.[Cys0,Cys11]N/OFQ(113)NH2 6.4 6.4 HEK-293
55
55
55
55
58
c.[Cys10,Cys14]N/OFQ(114)NH2 9.7 8.3 CHO
56
c.[Cys7,Cys10]N/OFQ(114)NH2 10.0 9.2 CHO
57
c.[Asp6,Lys10]N/OFQ(113)NH2 9.5 8.4 CHO
57
c.[D-Asp7,Lys10]N/OFQ(113)NH2 9.6 8.8 CHO
58
c.[Nphe1,Cys10,Cys14]N/OFQ(114)NH2 7.9 Inactive 5.7 CHO
59
c.[D-Cys2,Cys5]N/OFQ-NH2 9.0 BHK
() determined on mouse VD as an effect on inhibitory action of N/OFQ(1--13)NH2 on an electrically stimulated
mouse VD.
interaction between the acidic residue and Lys12, but there is a prevailing effect of the
diminished afnity due to negative charge in the address domain.53
Whereas the introduction of helix structure in 1 is generally favorable to activity, a
conformational constraint to a b-strain diminishes both afnity and activity, as in [Nma7]N/
OFQ and [Nma11]N/OFQ (Nma, N-methylalanine), which showed only a residual agonistic
activity (EC50 5 96 and 407 nM, respectively). Instead, the activity was almost maintained
with respect to N/OFQ in [Nma15]N/OFQ (EC50 5 5.3 nM), giving further proof of the
fact that the conformational effects are more important in the amino-terminal portion of
N/OFQ.49
Other compounds derived from 1, which also showed an increased activity due to con-
formational restraints, are some cyclic analogues, such as the agonists cyclo[Cys10,Cys14]N/
OFQ(114)NH2,55 cyclo[Cys7,Cys10]N/OFQ(113)NH2,56 cyclo[Asp6,Lys10]N/OFQ(113)NH2
and cyclo[D-Asp7,Lys10]N/OFQ(113)NH2,57 and the partial agonist [Nphe1]cyclo[Cys10,
Cys14]N/OFQ(114)NH2.58 The high-afnity ligand cyclo[d-Cys2,Cys5]N/OFQNH2 is reported
in another work59 (see Table XII for cyclopeptides and Table IX for some acyclic counterparts).
Another ligand derived from N/OFQ is retronociceptin methyl ester, with poor afnity
in vitro (IC50 5 480 mM) but active on mice by i.c.v. administration as an analgesic and
memory enhancer at the dose of 0.1 mmol per mouse.60 The incorporation in 1 of a carbamic
acid residue gave peptides, such as as bHph-Gly-NHCO-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-
Arg-Lys-NH2 and Phe-NHCO-Gly-bHph-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-NH2
(bHph, b-homophenylalanine), which had a good resistance to aminopeptidase M and rat
membrane homogenates, but nevertheless had a low afnity to NOP (pKi 5 5.71 and 5.75,
respectively).61
4. OTHER PEPTIDES
A screening of a combinatorial library led to ve hexapeptides with high NOP afnity

and active in vitro as partial agonists: Ac-RYYRWR-NH2 (2), Ac-RYYRWK-NH2 (3),
Ac-RYYRIK-NH2 (4), Ac-RYYLWR-NH2 (5), and Ac-RYYKWK-NH2 (6).62 They
resulted to be inactive in vivo because of their instability to metabolism,63 but were the lead
compounds for the development of many other NOP ligands; moreover, tritiated 3 is a
selective radioligand.59 The partial agonist Ac-RYYRWKKKKKKK-NH2 (ZP120, devel-
oped by Zealand Pharma)64 is interesting because of its inability to penetrate the CNS, after
iv administration, thus its action is limited to peripheral NOP receptors, producing pro-
longed diuretic and antinatriuretic effects.65,66
Many SAR studies have been performed on hexapeptides 26 (Table XIII). Cyclic
analogues of 3 like cyclo-(-RYYRWK-) had only a scarce afnity.59 A SAR study on 4
showed that the removal of acetyl or Ala substitution of Arg1, Tyr2, or Tyr3 almost sup-
pressed afnity, whereas this effect was much smaller for the same substitution of one of the
other three residues or the removal of Lys6. Moreover, the removal of the terminal amide
group had almost no effect.67 The substitution the of C-terminal amide group in 4 with a
hydroxymethyl gave the partial agonist Ac-RYYRIK-ol,68 which displayed in vivo effects in
mice similar to N/OFQ.69 Tritiated Ac-RYYRIK-ol is a radioligand for NOP endowed with
high afnity and selectivity, reversibility, and biological stability.70
Other analogues have been obtained by modiyng the N-terminal acyl of 271 and 4.72
These are the compounds which in most cases maintained a very high afnity, unless there
was a bulky acyl group as in the nipecotyl analogue of 2 or in the 2-ethylbutyryl, 2-me-
thylvaleryl, and 2,2-dimethylbutyryl analogues of 4. Compact acyl groups are more tolerated,
as in the case of the t-butylacetyl, benzoyl, and adamantylcarbonyl analogues of 4. Activities
are nevertheless remarkably affected by the acyl group, with a general trend toward antag-
onism for peptides bearing an N-terminal acyl with a longer and b-branched chain, thereby
showing the role of the terminal acyl portion in the activation or inactivation of the receptor.
In fact, whereas the formyl analogue of 4 has only a slightly higher afnity and almost the
same activity of 4, we nd among these derivatives the low-potency antagonist valeryl-
RYYRWR-NH271 and the potent antagonist isovaleryl-RYYRIK-NH2.72
In another study,73 the substitution of tryptophan in 3 with various aromatic amino acid
residues, such as homophenylalanine, 1-naphthylalanine, and (NCH3)tryptophan, gave
partial agonists with similar potency compared to 3. This SAR study on the tryptophan
residue showed that the distance and the spatial orientation of the six-membered ring in
regard to the peptide backbone play a relevant role in the binding, as showed by the low or
absent afnities of peptides bearing, instead of Trp, a residue where this distance is reduced
(as in 2-, 3-, or 4-pyridylalanine, and as already known in the case of Phe62) or enhanced (as
in 2-naphthylalanine). The same effect is observed if there is a different disposition of the
benzene ring (as in D-Trp or in 2,3,4,9-tetrahydro-b-carboline-3-carboxylic acid) or even a
sterical crowding (as in p-phenyl-phenylalanine and diphenylalanine). This series of com-
pounds had a partial agonistic prole similar to 3, showing that Trp residue is involved in
binding but not in receptor activation.
A SAR study on 274 conrmed that Arg1 is essential for both afnity and activity, thus
its substitution with e-aminocaproic, diaminopropionic, or diaminobutyric acid greatly di-
minished afnity and, in the case of the two latter substitutions, gave antagonistic peptides.
The substitution of Arg4 or Arg6 with e-aminocaproic acid was also detrimental for both
afnity and activity. Instead, the substitution by various groups of either hydroxyls or H-3 on
Tyr2 andTyr3 gave compounds with comparable afnity (or even stronger as in the case of
(3-Cl-Tyr)2-(2)), but at least one of the hydroxyl groups of Tyr2 and Tyr3 must be main-
tained for agonistic efcacy, thus Ac-R(p-F-Phe)(p-F-Phe)RWR-NH2 is a pure antagonist. If
an acidic moiety, such as COOH or SO3H, replaced these hydroxyls, the result was a drop in
both afnity and activity. The above mentioned (3-Cl-Tyr)2-(2) (Syn-120, developed by
Synvax) is a potent and selective partial NOP agonist (EC50 5 0.5 nM) that was able to
reverse morphine-induced analgesia in mice and to produce analgesic and antiallodynic
Table XIII. Afnities and Activities of Peptides 26 and Their Analogues
Max. Receptor
Compound pKi pEC50 stimulation (%) source Ref.
62
N/OFQ 9.4 8.4 100 CHO(m)
59
N/OFQ 9.6 BHK
67,72
N/OFQ 9.1 8.4 COS-7
68
N/OFQ 8.9 Rat brain
71
N/OFQ 10.4 9.0 100 CHO
62
Ac-RYYRWR-NH2 9.2 8.7 52 CHO(m)
71,74
Ac-RYYRWR-NH2 10.0 8.8 58 CHO
62
Ac-RYYRWK-NH2 9.1 8.7 57 CHO(m)
73
Ac-RYYRWK-NH2 8.3 61 Mouse VD
62
Ac-RYYRIK-NH2 8.8 8.3 30 CHO(m)
62
Ac-RYYKWR-NH2 8.8 8.1 49 CHO(m)
62
Ac-RYYKWK-NH2 9.1 8.2 52 CHO(m)
59
c(RYYRWK) 6.4 BHK
59
c(GRYYRWK) 6.4 BHK
67
Ac-AYYRIK-NH2 5.1 COS-7
67
Ac-RAYRIK-NH2 5.6 COS-7
67
Ac-RYARIK-NH2 6.0 COS-7
67
Ac-RYYAIK-NH2 7.5 COS-7
67
Ac-RYYRAK-NH2 7.8 COS-7
67
Ac-RYYRIA-NH2 7.8 COS-7
67
Ac-RYYRI-NH2 8.3 COS-7
67
Ac-RYYR-NH2 5.6 COS-7
67
Ac-RYY-NH2 Inactive COS-7
67
Ac-RYYRIK 8.8 COS-7
67
RYYRIK-NH2 7.0 COS-7
67
RYYRIK 6.7 COS-7
71
Propionyl-RYYRWR-NH2 10.0 8.2 51 CHO
71
Butyryl-RYYRWR-NH2 9.5 7.8 22 CHO
Valeryl-RYYRWR-NH2 9.9 n.m. o10 CHO 71
Hexanoyl-RYYRWR-NH2 9.6 n.m. o15 CHO 71

71
Heptanoyl-RYYRWR-NH2 9.6 8.2 28 CHO
72
Formyl-RYYRIK-NH2 9.2 7.6 61 COS-7
72
Ac-RYYRIK-NH2 9.1 7.9 58 COS-7
72
Propionyl-RYYRIK-NH2 8.8 7.6 46 COS-7
72
Butyryl-RYYRIK-NH2 8.7 7.5 21 COS-7
72
Isobutyryl-RYYRIK-NH2 8.6 7.4 14 COS-7
72
Valeryl-RYYRIK-NH2 8.2 Inactive 7 COS-7
72
Isovaleryl-RYYRIK-NH2 8.1 Inactive 0 COS-7
Ac-R-(p-Me-Phe)-(p-Me-Phe)-RWR-NH2 7.9 o5 CHO 74
74
Ac-R-(p-NO2-Phe)-YRWR-NH2 9.4 7.8 59 CHO
74
Ac-R-(p-F-Phe)-YRWR-NH2 9.3 7.4 54 CHO
Ac-R-(p-F-Phe)-(p-F-Phe)-RWR-NH2 8.8 o5 CHO 74
74
Ac-R-(O-Me-Tyr)-YRWR-NH2 9.3 7.3 52 CHO
Ac-R-(O-Me-Tyr)-(O-Me-Tyr)RWR-NH2 8.2 o5 CHO 74
74
Ac-R-(3-Cl-Tyr)-YRWR-NH2 10.5 9.3 75 CHO
74
Ac-R-(p-CN-Phe)-YRWR-NH2 9.7 7.5 50 CHO
74
Ac-RY-(p-F-Phe)-RWR-NH2 10.3 8.7 70 CHO
74
Ac-RY-(p-NO2-Phe)-RWR-NH2 9.8 8.5 48 CHO
74
Ac-R-(p-Ph-Phe)-YRWR-NH2 9.6 7.7 46 CHO
74
Ac-RY-(2,6-di-Me-Tyr)-RWR-NH2 10.4 8.3 53 CHO
n.m., not measurable; CHO(m), CHO cell transfected with mouse NOP.
effects in mice and rats.75 Similar results have been reported for the analogue with Tyr2
substituted with O-benzyl-3-chlorotyrosine (EC50 5 0.3 nM). This compound was described
in a patent by Synvax,76 together with various analogues of 2 where Tyr2 and/or Tyr3 are
replaced with various 4-substituted phenylalanines, giving peptides whose activity ranges
from full agonism to antagonism.
In another work,77 a SAR study on a peptide strictly related to the above compounds,
Ac-RYYRIR-NH2 (7), is reported. Peptide 7 was endowed with an afnity comparable to
26 and a strong antagonistic activity towards N/OFQ in mouse vas deferens bioassay. Ala
substitution of one of the amino acid residues in 7 gave a decrease in afnity, which was very
marked in the case of the rst three residues (especially Arg1), and only moderate for the
other, according to a trend already observed for 26. The substitution of Arg1 with citrulline
was detrimental for afnity, whereas only a slight reduction in afnity and a substantial
maintaining of the antagonistic activity was observed if the same substitution was made for
Arg4 or Arg6. If Ile5 in 7 was substituted with other aliphatic residues (Val, Leu, norleucine,
tert-leucine) the antagonism was also conserved, but was strongly reduced if this residue was
replaced with aromatic residues (Phe, Tyr, 2,3,4,9-tetrahydro-b-carboline-3-carboxylic acid).
Furthermore, replacing this position with achiral residues like Aib or with D-aminoacids
shifted the activity from antagonism to agonism. In particular, the analogues of 7 with a
D-Trp or a D-Arg in the 5 position resulted as being potent agonists.
Also related to 2 is the tetrapeptide OS-461 (Fig. 1) and its analogues OS-462 and OS-
500 developed by Nippon Shinyaku78 with NOP agonistic activity and analgesic effects in
mice; the two latter peptides produced hyperphagic effect in rats.79
Some chimeric peptides derived from 4 and the MOP ligand dermorphine80 with mixed
MOP/NOP activity showed a strong analgesic effect by intrathecal administration in mice.81
Also, chimeric peptides N/OFQ/dynorphin A, active on both KOP and NOP receptors, have
been reported.82,83
A screening of b-structure constrained peptides led to Peptide III-BTD (Fig. 1),84
showing antagonistic NOP activity and agonist activity on opioid receptors; it was in turn the
lead compound for the rather selective antagonists 8 and 9,85 obtained by substitution of the
central thiaindolizidinone moiety with a indolizidinone or quinolizidinone system. From 9,
the very selective NOP antagonist 1086 was obtained by changing the rst arginine residue
with citrulline.
5. MORPHINANES
Morphinane derivatives developed as NOP ligands (Fig. 2) are poorly selective. Some of the
classic opioid drugs are also active on the NOP receptor: etorphine has a moderate afnity
(Ki 5 530 nM) and a full agonist activity;27 buprenorphine is a mixed NOP/MOP partial
agonist in clinical use for pain and drug addiction (see Introduction); naloxone benzoylhy-
drazone is a MOP antagonist, KOP agonist and NOP partial antagonist with an anti-
nociceptive effect in mice, which is lost in NOP knockout mice.87
Other further developed morphinane derivatives include: (a) the hydroxamic acids 11
(Pzer), active as NOP antagonists and opioid agonists;88 (b) the KOP agonist and NOP
antagonist TRK-820 (nalfurane, developed by Toray and now produced by Acologix),89 a
compound that had been evaluated in mice by s.c. administration and showed an anti-
nociceptive effect devoid of adverse effects and dependence90 and is under clinical trial as an
antipruritic (see Introduction); (c) compound 12 patented by Euro-Celtique,91 which differs
from buprenorphine only for the substituent on nitrogen and is more selective on MOP; (d) a
series of compounds developed by Gruenenthal,92 exemplied by 13, and characterized by
Figure 1. Some peptidic NOP ligands.
ring fusion of the morphinane system with an indole moiety. The NOP activity of the latter
compounds can be shifted from agonism to antagonism depending on the substituent on the
indole nitrogen and the two hydroxyls.
6. 4-ARYLPIPERIDINES AND 3-ARYLNORTROPANES
The 4-arylpiperidine framework (Fig. 3) was proposed for the NOP ligands because of its
structural afnity with known active spiropiperidines (see below). A combinatorial chemistry
study93 led to nd some 4-aryl-4-hydroxypiperidines with good afnity on NOP but scarcely
selective over MOP, like the agonist 14 and the antagonist 15. The most important result of
this study was the development of a pharmacophore model whose main features are: (a) a
large hydrophobic group on the piperidine nitrogen, (b) a small hydrophobic group like
phenyl at the 4 position of the piperidine ring, and (c) a hydrogen bond donor or acceptor
bound at the same carbon as the previous group.
The 4-hydroxy-4-phenylpiperidine motif is present in the selective agonist 16,94 devel-
oped from the lead 17, which in turn comes from a high-throughput screening. A SAR study
on the benzhydryl substituent showed that both afnity and selectivity were enhanced by
ortho-monosubstitution on one or both phenyls with methyl, chlorine, or uorine. Fusion of
the two rings in rigid systems, such as uorenyl or dibenzosuberyl, gave instead less active
and unselective products. This can lead to the conclusion that afnity is enhanced if the
bulky lipophilic group in the above-described pharmacophore is distorted from planarity.
Figure 2. Morphinanes.
Alkylation of the hydroxyl group diminished the afnity, especially for larger substituents.
A further SAR study on the 4-aryl group95 conrmed the predicted requirements for the
smaller lipophilic group according to the above pharmacophore, since phenyl could be re-
placed with saturated substituents with a comparable size such as butyl or cyclohexyl,
whereas larger groups, such as p-ethylphenyl or benzyl, or more polar substituents, such as
2-pyridyl or 2-thiazolyl, gave compounds with remarkably reduced afnities. The study of
the effect of ortho-substitution on phenyl led to develop 18; optimization of the benzhydryl
substituent led to 19, which was tested in vivo along with some derivatives for antitussive oral
activity in guinea pigs. The best in vivo activity was obtained for the sulfamido derivative 20
(ED50 5 0.06 mg/kg at 2 hr).
A series of 3-(aryloxymethyl)-4-arylpiperidines with NOP antagonist properties, in-
cluding the selective antagonist NNC-63-0780, have been developed by Novo Nordisk.96
Another compound, which only partly corresponds to the above pharmacophore (since
the hydroxyl group is not on the piperidine ring but on the bulky hydrophobic group)
is the selective and potent antagonist SB-612111 (GlaxoSmithKline). By intravenous
administration in mice, it was able to reverse tolerance to morphine and thermal hyper-
algesia, and produced an antidepressant effect reversed by i.c.v. injection of N/OFQ.9799
This compound was the lead for the aza-substituted compound 21, characterized by a
considerably lesser afnity to the hERG K1 channel (binding on this protein is related to
Figure 3. 4-Arylpiperidines and 3-arylnortropanes.
adverse cardiovascular side effects). The hERG K1 channel afnity was further reduced by
the contraction of the saturated ring to 6 members and, in a more consistent way, by the
introduction of a hydroxyl in the trans-3 position of the piperidine ring. These results,
combined with a SAR study on the 4-aryl, led to the potent and selective antagonist 22,
which was tested in vivo in mice, giving the reversal of the reduction of locomotor
activity produced by a NOP agonist, at the dose of 10 mg/kg, and showed the advantages
of oral availability and lack of cardiovascular effects.100 A series of NOP ligands, strictly
related to these compounds with general formula 23, is described in a patent from the
same group.101
Euro-Celtique has developed a series of 4-phenyl-4-(tetrazol-5-yl)piperidines with mixed

NOP/MOP agonistic activity, with the general formula 24 or 25, which were unable to pass
through the bloodbrain barrier and thus had analgesic properties limited to periphery.102
Strictly related to these compounds are the bicyclic analogues 3-arylnortropanes, like
SCH 221510 (Schering), a potent NOP agonist, orally active on rodents as an anxiolytic,103
and the analogous agonists developed by Schering like 26,104 also orally active as an an-
xiolytic in rats and mice, 27105 and 28,106 both showing a potent oral antitussive activity in
guinea pigs (ED50 5 0.04 and 0.19 mg/kg, respectively, at 2 hr). Further studies on this che-
mical class has recently led to 29107 and 30,108 both displaying a very potent antitussive
activity in rats (ED50 5 0.02 and 0.03 mg/kg, respectively, at 2 hr).
7. 4-HETEROCYCLIC SUBSTITUTED PIPERIDINES AND 2-HETEROCYCLIC

SUBSTITUTED QUINOLIZIDINES
The most relevant part of this class of ligands (Fig. 4) is constituted by piperidines bearing a
2-oxobenzimidazol-1-yl moiety in the 4 position, which were developed because of the NOP
afnity of the neuroleptic pimozide.109 Starting from the lead 31, a low-afnity unselective
agonist, Banyu Pharmaceutical Co. developed J-113397, the rst nonpeptidic selective NOP
antagonist,110 which still nds application in pharmacological studies.111 This stimulated the
development of new synthetic strategies for this compound,112114 also in a radiolabeled
form, which nevertheless resulted as not to being suitable for PET imaging because of its
relevant nonspecic binding.115 J-113397 has in turn been a lead for the preparation of new
NOP ligands, including its achiral analogue Trap-101 (University of Ferrara),116 the ligands
with the general formula 32 (Purdue Pharma),117 the potent antagonists 33118 and 34119
(Banyu) and a series of agonists with general formula 35 (Pzer).120
Another series of 4-(2-oxobenzimidazol)-1-ylpiperidines with NOP agonist activity has
been developed by Organon Laboratories Ltd. (Newhouse, UK). Starting from the screening
hit 36, showing a good NOP afnity (Ki 5 12 nM) and the advantage of the solubility in
water as hydrochloride, but scarce potency and selectivity, the moderately selective agonist
37121 was developed. The latter showed an antinociceptive effect in mice by intravenous
administration (ED50 5 1.07 mmol/kg). A SAR study of the substitution on N-3 benzimida-
zole showed that polar substituents enhanced afnity, selectivity, and functional potency.
The selective agonist 38 displayed an antinociceptive and sedative effect in mice by in-
travenous administration.122 A further work on the substitution of the N-methylacetamide
moiety with various bioisosteric heterocycles led to 39, bearing a 1,2,4-triazole substituent.
This compound is a potent and selective NOP agonist which was also tested in vivo in mice
and showed antinociceptive and antiallodynic effects.123
The selective NOP agonist W-212393, derived from 38 by replacing the N-piperidine
substituent with an 1-acenaphthenyl group, has been developed by Mitsubishi Pharma as an
agent for the treatment of drug abuse.124 By intraperitoneal administration, it provoked
entrainment of body temperature circadian rhythm in rats.125
Incorporating the piperidine ring in the bicyclic quinolizidine framework gave the potent
but unselective agonist SR 14136 (SRI International).126 In the recent literature, there have
been several reports of piperidines bearing a variety of heterocyclic substituents in the 4
position, as NOP ligands:
(a) trans (or cis)-octahydro-2-oxobenzimidazol-1-yl in the antagonists 40 and 41127 and in
the ligands with the general formula 42 (Euro-Celtique)128;
Figure 4. Other piperidines and quinolizidines active on NOP receptors.
(b) 2-substituted benzimidazol-1-yl in the potent and selective agonists MCOPPB129,130 and
PCPB,131 both developed by Pzer and orally active as anxiolytics in mice. The former,
resulting from an extensive SAR study on the 2-substituent on benzimidazole, is the most
potent nonpeptidic NOP receptor agonist in vitro so far found (Ki 5 0.0858 nM);
(c) 2-oxoindol-1-yl in compounds as 43 and 45, where the modication of the substituent
on the piperidine nitrogen permitted to shift the activity from agonists to partial
Figure 4. Continued.
agonists and to antagonists.132 One of these compounds, the mixed MOP/NOP partial
agonist SR 16435 (SRI International) (43), showed analgesic properties in mice after
i.c.v.injection.133 The possibility, in this series of indolinone derivatives, of varying the
activity from agonism to antagonism by changing the N-1 substituent, established the
base for a further modeling study.134 The latter, in addition to conrming the main
features of the pharmacophore described above for 4-arylpiperidines, and substantially
valid for the vast majority of NOP nonpeptidic ligands including spiroderivatives,
assumes a trigger site on the receptor that is responsible for agonism, and can be
activated by substituents in a-position in respect to nitrogen on this bulky hydrophobic
moiety. This can rationalize the fact that by elongating this substituent with a
methylene unit some agonists like 44 can be converted into antagonists like 45;
(d) indol 3-yl and pyrrolo[2,3-b]pyridin-3-yl as in the full agonist 46, in the high-afnity
ligand 47135 and in a series of agonists and antagonists with general formula 48
patented by Johnson & Johnson, also comprising 1,2,5,6-tetrahydropyridines136;
(e) 1,3-dihydro-2,1,3-benzothiadiazol-2,2,dion-1-yl and 1,3-dihydro-2,1,3-benzothiadia-
zin-2,2,dion-1-yl in compounds such as 49 and 50 which are potent agonists, but
unstable to metabolism137;
(f) quinoxalin-2,3-dion-(or dithion)-1-yl, 1,5-benzodiazepin-2,3-dion-(or dithion)-1-yl and
1,3,5-benzotriazepin-2,3-dion(or dithion)-1-yl in a series of compounds with general
formulas 51, 52 developed by Purdue Pharma and Shionogi, which include both
agonists and antagonists138;
(g) benzoxazol-2-on-3-yl and 2-cyanoiminobenzimidazol-1-yl in a series of ligands like 53
and 54 described in a patent by Euro-Celtique,139 which also reports compounds
bearing the above mentioned units of 2-indolinone and 2-benzimidazolone;
(h) 3,4-dihydroquinolin-2-on-yl in the ligands with general formula 55 patented by Euro-
Celtique140;
(i) 1,2,4-oxadiazol-3-yl in a series of agonists with general formula 56 developed by Euro-
Celtique.141
8. SPIROPIPERIDINES AND SPIRONORTROPANES
Many of the NOP receptor ligands so far developed bear a spiropiperidine scaffold (Fig. 5).
Of those, the most studied are the 1,3,8-triazaspiro[4,5]decan-4-ones, like the 5-HT partial
agonist spiroxatrine that showed a signicant NOP afnity in the early screenings.109 From
the lead 57 (potent full agonist but unselective), Hoffman La Roche developed the potent and
selective full agonist Ro 65-6570, showing anxiolytic properties in rats.142,143 Other analogues
were obtained by Roche by the variation of the lipophilic substituent on the piperidine
nitrogen, as 58,142 59144 and Ro 64-6198.145 Currently, the latter is the nonpeptidic NOP
agonist most extensively used in pharmacological and preclinical studies. Its therapeutical
potential concerns the treatment of anxiety, drug abuse, neuropathic pain, cough, and an-
orexia.146 Subcutaneous administration of Ro 64-6198 in rhesus monkeys (dose
Figure 5. Spiropiperidines and spironortropanes.

0.0010.06 mg/kg) produced an antinociceptive effect that is devoid of the typical side effects
of MOP agonists (respiratory depression, itch, reinforcing effects).147 A radiolabeled form of
the N-methyl analogue of Ro 64-6198 has been tested as a radio tracer in mice, with poor
results because of the scarce brain uptake.148
Many other 1,3,8-triazaspirodecan-2-ones have been developed by varying the sub-
stituent on the piperidine nitrogen and, in many cases, by introducing a substituent in N-3.
Novo Nordisk obtained NNC 63-0532, an agonist with scarce selectivity149 and its analogues
with general formula 60.150 The same motif is present in agonists with general formula 61
(Pzer)151 and antagonists like 62 (Banyu),152 as well as in other high-afnity ligands ob-
tained by Johnson & Johnson like the agonist 63, showing anxiolytic activity in rats,153 and
the agonists with general formula 64 (Solvay).154 In a further study by Schering, lead 65 was
developed into the selective and orally available full agonist 66.155 This compound in turn,
throughout a SAR study on the variation of substituents on N-8 and on the substitution on
the amidic nitrogen led to 67, a selective full agonist, orally available and active as an
antitussive in guinea pigs.156 Other ligands bearing the same moiety are the agonists 68
(Purdue Pharma),157 and 69, recently obtained by Johnson & Johnson,158 as well as agonists
bearing an aminoalkyl moiety in position 3 such as 70, or a dimeric structure such as 71, both
showing an aquaretic effect by i.v. administration in rats.159 It should also be mentioned
that nonpeptide/peptide chimeric ligands, such as 72, displayed a NOP agonistic activity
in vitro.160
Strictly related to the above compounds (because they present a different kind of spiro
junction between the same units of piperidine and 4-imidazolinone), are the 1,4,8-triaza-
spiro[4,5]decan-2-ones developed by Banyu, including antagonists such as those with the
general formula 73,161 and potent agonists such as 74.162

There are also many other NOP ligands where piperidine is spiranized in the 4 position
with different moieties. The isosteric system 2,3,8-triazaspiro[4,5]decan-4-one is present in
ligands such as 75, patented by Euro-Celtique,163 whereas the contracted scaffold of the 1,7-
diazaspiro[3,5]nonane characterizes the high-afnity ligands 76 (Roche).164
Other compounds present a piperidine ring connected by a 4-spiro junction with var-
ious bicyclic (and sometimes tricyclic) systems, such as isobenzofuran-1-one in 77 and
isobenzofuran in 78, (both antagonists patented by Roche),165 indane and isobenzofuran in
79 and 80 (the rst one a screening hit, the second a potent and selective antagonist)166 and
in the recently developed antagonist 81, very potent (EC50 5 0.53 nM) and also selective
also over hERG K1 channel,167 indene and indane in the ligands like 82 developed by
Euro-Celtique,168 hexahydropyrrole[c]pyrrole in the potent and selective agonist 83,169 2,3-
dihydroindole and 2-indolinone in a series of ligands with general formula 84 patented by
Johnson & Johnson,170 2-indolinone in the antagonists such as 85 and 86,171 1,2-dihy-
droisoquinolin-3-one in the potent and selective agonist 87,172 2,3-dihydroquinazolin-4-one
and 5,6-dihydro[1,2,4]triazolo[1,5-c]quinazoline in a series of derivatives with moderate
afnity, whose activity ranged from partial agonism to pure antagonism, and in some
cases displayed a good selectivity as in the case of 88.173 A series of NOP antagonists,
recently developed by Pzer, is constituted of piperidines spiranized with the systems of
indane, isobenzofuran, 2,3-dihydroindole, 2-indolinone, and isochromane, with general
formula 89.174
Strictly related to the latter are the antagonists spironortropane acids such as 90175 and
the corresponding amides such as 91,176 also developed by Pzer (the patents do not specify
the endo or exo conguration for these compounds).
9. 4-AMINO-QUINOLINES AND QUINAZOLINES
Although this chemical class of NOP ligands (Fig. 6) has not been as extensively studied as
the former ones, it is nevertheless one of the most important as it includes JTC-801, the rst
nonpeptidic selective NOP ligand to be used in clinical trials (see above). The NOP an-
tagonist JTC-801 was developed by Japan Tobacco Inc. from the lead 92, which came from
a random screening.177 In the original SAR study it was evidenced that the removal of the
4-amino group, or the introduction of substituents on it, suppressed afnity. The same
effect was obtained if the amidic group was substituted or inverted; the ortho-substitution
on the benzoyl moiety was also essential. In vivo pharmacological testing showed that JTC-
801 was orally bioavailable and had analgesic effects in mice.178 A further study, which also
reported a new synthesis of JTC-801,179 showed that the afnity was strongly reduced
by the introduction of an electron-withdrawing or sterically hindering group in the 3 po-
sition of quinoline, or by aza-substitution on the same position. Instead, a 3-methyl
substituent only slightly enhanced afnity, but reduced selectivity. In this work, an NMR
Figure 6. 4-Amino-quinolines and quinazolines.
conformational study was also performed on JTC-801 and its analogues, showing that the
distance between quinoline C-2 and the C-4 of the phenoxy group is a critical parameter for
activity. Moreover, a Schild-plot analysis showed that this compound is an allosteric an-
tagonist, unlike piperidine and spiropiperidine ligands that compete with N/OFQ on the
same receptorial site. Other JTC-801 analogues with NOP antagonistic properties, such as
93, have been described in a patent by Japan Tobacco Inc.180
A series of N,2-disubstituted 4-aminoquinazolines with general formula 94 and NOP
antagonistic properties has been developed by Dreier LLP and has been tested on mice as
antipruritics,181 and others antagonists with the same general formula have been obtained
by Nippon Shinyaku.182,183 They include compound 95, developed from the moderately
active and selective lead 96, bearing a benzo[g]quinazoline system and coming from an
high-throughput screening. The subsequent SAR studies led to the substitution of benzene
annulation with a 6-methyl substituent, then to the cyclization of the aminoalkyl chain to
its rigid analogue 1,2-disubstituted cyclohexane, and to the introduction of a guanidine
group, instead of the amino, to simulate the Arg residue in the natural ligand. Also, the
substitution of the alkenyl moiety with an amide group led to a further increase of activity
because of its ability to hydrogen bond. This was evidenced by a docking model study on 95
with the NOP receptor, which also evidenced many features common to most competitive
NOP ligands: the cyclohexane and methylbenzene moieties interact with a larger lipophilic
pocket and the chlorophenyl substituent with a smaller one and there is a strong electro-
static interaction between the positively charged guanidine group and Asp130. Besides,
hydrogen bonding between the guanidine group and the oxygen of the side-chain of
Thr305, and between carbonyl and CONH2 of the side-chain of Gln280 play a role in the
interaction.
10. 6-PIPERAZINYL-BENZIMIDAZOLES
A rst lead compound bearing the 6-(1-piperazinyl)benzimidazole moiety (Fig. 7), was the
ligand with moderate afnity 97 (IC50 5 910 nM), obtained from a library screening. It was
optimized in a rst SAR study,184 which showed that the afnity could be increased by
replacing the substituent on sulfur with secondary or tertiary alkyl groups, whereas the
oxidation to sulphoxide or sulphone considerably lowered afnity. Further improvements
were obtained by the substitution of the 5-uorine with chlorine and the introduction of a
2-hydroxyethyl substituent on the piperazine nitrogen. The result of this work was compound
98, a potent and selective NOP antagonist, which, however, resulted as being unstable to
metabolism.
A further study from the same group185 showed that metabolic stability (determined by
incubation with human liver microsomes) could be enhanced by introducing a methyl in the
2-position of piperazine and replacing the 2-methylpentyl substituent with t-butyl. In order to
maintain good selectivity, it was necessary to isolate the (R) enantiomer by a racemate
resolution, as the (S) enantiomer had a strong afnity to KOP. The result of this study was
compound 99, a potent, selective, orally available, and brain penetrable NOP antagonist.
11. 2-(1,2,4-OXADIAZOLYL)-INDOLES AND PYRROLOPYRIDINES
This novel class of NOP ligands has only recently been reported in a paper by Sugimoto and
coworkers.186
The indolic lead 100 (Fig. 8), resulting from high-throughput screening from a com-
pound library, had a fair afnity (95 nM) and a measurable activity as a NOP antagonist
(632 nM). A SAR study, aimed at its optimization, showed that in the alkylaryl moiety a
shorter or longer polymethylene linker reduced afnity. Other structural modications giving
the same results were the introduction on this moiety of nitrogen- or oxygen-bearing sub-
stituents, and the substitution of the aryl with linear or branched alkyl moieties. Instead,
afnity was maintained by replacing the aryl with some cycloalkyl substituents. The
Figure 7. 6-Piperazinylbenzimidazoles.

5-aminoalkyl moiety could not be shifted to the positions 6,7, or 8 without losing afnity,
and also a 5,6-annulation resulted to be detrimental for it. Moreover, 5-aza-substitution on
the indole ring increased afnity, whereas 7-aza-substitution suppressed it. Afnity was also
Figure 8. 2-(1,2,4-Oxadiazolyl)indoles and pyrrolopyridines.
Figure 9. Miscellaneous nonpeptidic NOP ligands.

lost if the oxadiazole linker was replaced by other ve-membered heteroaromatic rings or by
an amide linker. The result of this SAR study was the pyrrolo[3,2-c]pyridine derivative 101,
endowed with potent NOP antagonistic activity (IC50 5 40 nM).186
12. OTHER NONPEPTIDIC LIGANDS
Other NOP receptor ligands characterized by various chemical scaffolds are represented in
Figure 9.
Some 4-acylaminomethylcyclohexylamines such as 102,187 4-alkoxymethylcyclohex-
ylamines such as 103,188 4-aminocyclohexyl- and cyclohexylideneacetamides such as as
104,189 as well as ureas or amides substituted on nitrogen by a 4-aminocyclohexyl group, such
as 105,190 4-alkyl (or alkenyl-or-alkinyl) substituted 1-arylcyclohexylamines as 106,191 and
4-aminocyclohexanols as 107,192 all developed by Gruenenthal, are mixed NOP/MOP li-
gands, active in vivo as analgesics in mice. Recently, Gruenenthal has developed new 1,4-
cyclohexyldiamine derivatives that are active as NOP ligands and as analgesics in mice, such
as 108193 and 109.194 A further series of mixed NOP/MOP ligands bearing the same scaffold,
with general formula 110,195 has been developed; these compounds are also actives as in-
hibitors of serotonin and noradrenaline reuptake.
Some cyclohexylamines spiranized in the 4 position with tricyclic systems, which are
active as agonists on both MOP and NOP receptors and have general formula 111, have been
tested on animal models of drug withdrawal196,197 and of acute and neuropathic pain.198
A further series of similar compounds with the same general formula, also developed by
Gruenenthal, includes selective NOP agonists and antagonists.199 A series of 4-propionyla-
minopiperidines, with mixed MOP/NOP agonistic activity and general formula 112 has been
developed by Purdue Pharma.200
Other novel chemical classes of NOP ligands are the aminoalkylazetidines like the agonist
113,201 the mixed NOP/MOP ligands 5-aminomethylpyrrole-2-carboxamides with general formula
114, active also as noradrenaline and serotonine uptake inhibitors,202 and the antagonists 3-ami-
nomethyl-pyrroles, pyrazoles, and imidazoles with general formula 115, developed by Banyu.203
13. CONCLUSION AND PERSPECTIVES
Although the discovery of the NOP receptor and its endogenous ligand is rather recent and to
date there have been just a few clinical trials on compounds active on this system, there is
rapidly increasing pharmacological research on the therapeutical potential of both NOP ago-
nists and antagonists. This interest is also extending to peptides and is not limited to pure NOP
agonists and antagonists, but also to mixed NOP/MOP ligands. Along with the increasing
exploitation of the original chemical classes of nonpeptide NOP ligands, such as piperidines and
spiropiperidines, compounds with completely different scaffolds are being developed and mo-
lecular modeling studies are providing further details of ligand/receptor interactions.
Selectivity is a major topic in order to obtain compounds that are as much as possible
devoid of side effects. Therefore, the most promising compounds have been screened on a
wide range of receptors besides the other opiate receptors. In this respect, an especially
relevant requirement is the lack of activity on the hERG potassium channel in order to
address cardiac liabilities.
As it concerns adverse effects directly related to the activity on NOP, the most relevant
literature report has been made by Shoblock146 for the agonist Ro 64-6198. These effects,
observed in rats and mice, include hypolocomotion, ataxia, memory impairment and hy-
pothermia and are absent in NOP-knockout mice. They also vary considerably by species: so
in rats, unlike in mice, there is a therapeutical window between the anxiolytic dose and the
dose producing adverse effects.
ACKNOWLEDGMENTS
We thank Dr. Maria Rosaria del Giudice for helpful discussions.
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Carlo Mustazza was born in 1961 in Enna, Italy. He was graduated in Chemistry in 1986 at the
University of Catania and has been working as a technician at the University of Udine from
1989 to 1991, where he collaborated to research in heterogeneous catalysis; since 1991 he is a
researcher at Istituto Superiore di Sanita (Rome). His research interests concern
pharmaceutical organic synthesis.
Giuditta Bastanzio was born in 1980 in Italy. She was graduated in Pharmaceutical Chemistry
in 2005 at the University of Naples. Since 2008 she is a Ph.D. fellow at University of Rome La
Sapienza, carrying on her research at Istituto Superiore di Sanita. Her research interests
concern pharmaceutical organic synthesis and application of NMR techniques to biological
systems.

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Development of Nociceptin Receptor

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Development of Nociceptin Receptor

(NOP) Agonists and Antagonists

Carlo Mustazza and Giuditta Bastanzio

Published online 22 January 2010 in Wiley Online Library (wileyonlinelibrary.com).

Key words: nociceptin; NOP receptor ligands; opioids; structureactivity relationship

Medicinal Research Reviews, 31, No. 4, 605--648, 2011

2. THE NOP RECEPTOR AS A PHARMACOLOGICAL TARGET

Table I. Afnities and Activities of Nociceptin and its Truncated Analogues

Compound pIC50 pEC50 Receptor source Ref.

N/OFQ(18) 5.0 o5 CHO 27

Medicinal Research Reviews DOI 10.1002/med

Compound pIC50 pEC50 Receptor source Ref.

N/OFQ(18)NH2 o4 Rat brain 25

N/OFQ(14)NH2 o3.8 Rat brain 25

Medicinal Research Reviews DOI 10.1002/med

Compound pIC50 pEC50 Receptor source Ref.

[Ala4]N/OFQ 7.4 o5 CHO 26

[Ala5]N/OFQ 8.3 o5 CHO 26

[D-Thr5]N/OFQ 7.8 o6 CHO 26

[D-Arg8]N/OFQ 8.8 o6 CHO 26

Medicinal Research Reviews DOI 10.1002/med

Table III. Continued

Compound pIC50 pEC50 Receptor source Ref.

Table IV. SAR of the First Aminoacid Residue in N/OFQ(113)NH2

Compound pIC50 pEC50 Ref.

[(20 ,60 diMe)Tyr1] N/OFQ(113)NH2 7.9 () 28

Medicinal Research Reviews DOI 10.1002/med

Table V. SAR of the First Aminoacid Residue in [Nphe1]N/OFQ(113)NH2

Compound pIC50 Antagonist pKB Agonist pEC50 Ref.

Medicinal Research Reviews DOI 10.1002/med

Table VI. SAR of the 4th Aminoacid Residue in N/OFQ(113)NH2

Compound pIC50 pEC50 Receptor source Ref.

[(pF)Phe4]N/OFQ(113)NH2 9.4 9.8 () 36

[(pCl)Phe4]N/OFQ(113)NH2 9.1 9.6 () 36

[(pBr)Phe4]N/OFQ(113)NH2 8.7 8.8 () 36

[(pI)Phe4]N/OFQ(113)NH2 7.8 8.1 () 36

[(mF)Phe4]N/OFQ(113)NH2 9.0 9.6 () 36

[(oF)Phe4]N/OFQ(113)NH2 8.4 9.0 () 36

[(pNO2)Phe4]N/OFQ(113)NH2 9.5 9.9 () 36

[(pCN)Phe4]N/OFQ(113)NH2 9.2 9.5 () 36

[(pCF3)Phe4]N/OFQ(113)NH2 9.0 7.5 () 36

[(pMeO)Phe4]N/OFQ(113)NH2 7.0 7.2 () 36

[(pMe)Phe4]N/OFQ(113)NH2 7.8 8.0 () 36

[(pNH2)Phe4]N/OFQ(113)NH2 5.7 6.6 () 36

N/OFQ27 (Table X) unexpectedly revealed a signicant agonist activity for peptides

Compound pKi Antagonist pKB Agonist pEC50 Ref.

Table VIII. SAR of the Gly2-Gly3 Portion in N/OFQ(113)NH2

Compound pEC50 Ref.

Medicinal Research Reviews DOI 10.1002/med

Compound pKi pEC50 Receptor source Ref.

subsequent work,42 gave further conrmation of this interpretation. In comparison, only a

Table X. Afnities and Activities of Carboxyterminal Fragments of Nociceptin

Compound pKi pEC50 Receptor source Ref.

[(p-F)Phe4,Arg14,Lys15]N/OFQ-NH2 10.2 9.0 () 51

[(p-F)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 10.3 9.1 () 51

[Nphe1,Aib7,Aib11]N/OFQ-NH2 9.1 7.1 () 51

[Nphe1,(p-F)Phe4,Aib7,Aib11]N/OFQ-NH2 9.2 7.7 () 51

[Nphe1,(p-F)Phe4,Arg14,Lys15]N/OFQ-NH2 9.5 7.8 () 51

[Nphe1,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 9.6 8.0 () 51

[Nphe1,(p-F)Phe4Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 10.0 8.4 () 51

Compound pKi pEC50 pKb() (antagonist) Receptor source Ref.

A screening of a combinatorial library led to ve hexapeptides with high NOP afnity

Hexanoyl-RYYRWR-NH2 9.6 n.m. o15 CHO 71