Novel non-peptide nociceptin/orphanin FQ receptor agonist, 1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl] -1H-benzimidazole: Design, synthesis, and structure-activity relationship of oral receptor occupancy in the brain for orally potent

Article abstract of DOI:10.1021/jm7012979

An endogenous heptadecapeptide, nociceptin/orphanin FQ (N/OFQ), and a G-protein-coupled receptor, N/OFQ peptide (NOP) receptor [or opioid-receptor-like-1 (ORL1) receptor], have been described in terms of its structure, distribution, and pharmacology. Thus

Full text of DOI:10.1021/jm7012979

610
J. Med. Chem. 2009, 52, 610–625
Novel Non-Peptide Nociceptin/Orphanin FQ Receptor Agonist,
1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole: Design,
Synthesis, and Structure-Activity Relationship of Oral Receptor Occupancy in the Brain for
Orally Potent Antianxiety Drug1,2
Shigeo Hayashi,* Akiko Hirao, Aki Imai, Hiroshi Nakamura, Yoshinori Murata, Katsuyo Ohashi, and Eriko Nakata
Pfizer Global Research & DeVelopment Nagoya Laboratories, Pfizer Japan Inc., 5-2 Taketoyo, Aichi 470-2393, Japan
ReceiVed October 29, 2007
An endogenous heptadecapeptide, nociceptin/orphanin FQ (N/OFQ), and a G-protein-coupled receptor, N/OFQ
peptide (NOP) receptor [or opioid-receptor-like-1 (ORL1) receptor], have been described in terms of its
structure, distribution, and pharmacology. Thus, the N/OFQ and NOP receptor are located in the central
nervous systems in humans, primates, and rodents, and are involved in the integration of the emotional
components in the brain; e.g., N/OFQ displays anxiolytic activity in the brain. For identifying orally potent
anxiolytic, drug-design studies were performed with a series of 1,2-disubstituted benzimidazole derivatives,
which resulted in the identification of various chemotypes of highly potent NOP selective full agonists in
vitro with high or moderate NOP receptor occupancy in the mice brain per os such as 1-[1-(1-
methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole 1 (MCOPPB), the most potent
novel non-peptide NOP full agonist in vitro and an orally potent anxiolytic in the mice.
Chart 1. Structure of Novel Orally Potent NOP Receptor Agonist,
1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-
benzimidazole 1 (MCOPPB)
Introduction
Anxiety disorders3-5 such as generalized anxiety disorder
(GADa),6 social anxiety disorder (SAD),7 post-traumatic stress
disorder (PTSD),8 panic disorder (PD),9 specific phobias,10 and
obsessive-compulsive disorder (OCD),11 caused by continuous
or acute stress or fear, comprise the majority of all psychiatric
disorders for a long period. For the acute treatment of anxiety,
benzodiazepines remain the drug of choice because of their
effectiveness and rapid onset of action, in spite of having several
liabilities that limit their therapeutic utility,12,13 namely, amne-
sia,14 muscle relaxation,15 daytime or driving-time sleepiness,16,17
alcohol interaction,15 and abuse potential by drug dependence.18
Meanwhile, an opioid receptor that was regarded as one of
orphan G-protein-coupled receptors (GPCRs) and has high
homology to classical opioid receptors (such as µ-, κ-, and
δ-receptors) that belong to the superfamily of GPCRs, opioid-
receptor-like-1 receptor (ORL1 receptor, or as the fourth opioid
peptide receptor, OP4 receptor) has been described in terms of
structure, distribution, and pharmacology, recently.19-23 As well,
the isolation of a heptadecapeptide, named nociceptin24 (or
orphanin FQ),25 has subsequently been reported with its
structure, location, and physiological roles with the receptor.
Nowadays the ORL1 receptor is referred to as the N/OFQ
peptide receptor, NOP receptor. The NOP receptor and N/OFQ
are located at high levels in forebrain that includes cortical and
limbic regions and in brainstem, e.g., cerebral cortex, hippoc-
ampus, hypothalamus, thalamus, amygdala, septum, nucleus
accumbens, and locus coeruleus, which involve the integration
of the emotional components of fear and stress in human,
primate, and rodent.26-30 Furthermore, N/OFQ31-38 and other
NOP receptor agonist39-41 have demonstrated anxiolytic activi-
ties in some laboratory animals after intracerebroventricular (icv)
or intraperitoneal (ip) administration. The aim of this study is
to find orally potent, metabolically stable, and side effect free
or minimized NOP receptor agonist with new chemotype as a
new-class anxiolytic, together with differentiation from diaz-
epam that is a representative of benzodiazepine-type anxiolytic.
* Towhomcorrespondenceshouldbeaddressed.E-mail:Shigeo_Hayashi@
nifty.com.
a Abbreviations: GPCR, G-protein-coupled receptor; N/OFQ, nociceptin/
orphanin FQ; [35S]GTPγS, [35S]guanosine 5′-γ-thiotriphosphate; HEK,
human embryonic kidney; CHO, Chinese hamster ovary; GAD, generalized
anxiety disorder; SAD, social anxiety disorder; PTSD, post-traumatic stress
disorder; PD, panic disorder; OCD, obsessive-compulsive disorder;
MCOPPB, 1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-
1H-benzimidazole; LAH, lithium aluminium hydride; WSCI, water soluble
carbodiimide, i.e., 1-ethyl-3-(3-dimethylaminopropylcarbodiimide) hydro-
chloride; ee, enantiomeric excess (or enantiomer excess); SAR, structure–
activity relationship. The following are in accordance with Nomenclature
Committee of the International Union of Pharmacology (NC-IUPHAR):
N/OFQ peptide receptor, NOP receptor (or opioid-receptor-like-1 (ORL1)
receptor); MOP receptor, µ-opioid receptor; KOP receptor, κ-opioid receptor;
DOP receptor, δ-opioid receptor.
Chemistry
The retrosynthesis of the NOP receptor agonist 1, 1-[1-(1-
methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-ben-
zimidazole (MCOPPB) (Chart 1), was outlined in Scheme 1.
Thus, the optically pure (R)-enantiomer 1 was prepared from
N-protected chiral (R)-nicopetic acid 2 and N-[1-(1-methylcy-
clooctyl)-4-piperidinyl]-1,2-benzenediamine 3.
First, the (R)-enantiomer of N-protected nicopetic acid 2 was
synthesized as follows (Scheme 2). Thus, the amino portion of
ethyl (R)-nipecotate 5, prepared from commercially available
10.1021/jm7012979 CCC: $40.75
2009 American Chemical Society
Published on Web 01/06/2009

Nociceptin/Orphanin FQ Receptor Agonist
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 611
Scheme 1. Retrosynthesis of 1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole 1 (MCOPPB) from 2
and 3
Scheme 2. Synthesis of (R)-N-Boc-Nipecotic Acid 2a
a Reagents and conditions: (a) neutralization; (b) Boc2O, CH2Cl2, 0 °C to room temp; (c) LiOH/H2O-MeOH/THF, 0∼6 °C.
Scheme 3. Synthesis of N-[1-(1-Methylcyclooctyl)-4-piperidinyl]-1,2-benzenediamine 3a
a Reagents and conditions: (a) 2,5-hexanedione, AcOH, toluene, reflux; (b) Pd(OH)2, H2 (4.5 atm), MeOH; (c) cyclooctanone, KCN, TsOH, H2O, 45 °C;
(d) MeMgBr/THF, CH2Cl2, -78 °C to room temp; (e) NH2OH·HCl, EtOH/H2O, reflux; (f) 1-fluoro-2-nitrobenzene, K2CO3, CH3CN, reflux; (g) Zn, NH4Cl,
MeOH/H2O.
ethyl (R)-nipecoate L-tartrate 4 (Aldrich) by basic extraction,
was protected with tert-butoxycarbonyl (Boc) group, followed
by alkalic hydrolysis of its ester portion to give (R)-N-Boc-
nipecotic acid 2.42,43
peridin-4-amine 13. Then the nitro group of 13 was reduced to
the amino group with zinc powder48 to afford the corresponding
1,2-phenylenediamine derivative, e.g., N-[1-(1-methylcyclooc-
tyl)-4-piperidinyl]-1,2-benzenediamine 3.
N-[1-(1-Methylcyclooctyl)-4-piperidinyl]-1,2-benzenedi-
amine 3 was prepared as follows (Scheme 3). First, the primary
amino group of commercially available 4-amino-1-benzylpip-
eridine 7 was protected as a pyrrole skeleton transformed by
acidic condensation with 2,5-hexanedione to give 4-(1-ben-
zylpiperidinyl)-2,5-dimethylpyrrole 844 and then its benzyl group
at the nitrogen portion of the piperidine ring was removed by
hydrogenolysis with Pd(OH)2 to afford 4-piperidinyl-2,5-dim-
ethylpyrrole 9. Second, the cyclooctylmethyl group was intro-
duced onto nitrogen atom of the piperidine ring of the pyrrole
derivative 9 by Strecker reaction with cyclooctanone and
potassium cyanide followed by methylation with Grignard
reagent45,46 to give 4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(1-me-
thylcyclooctyl)piperidine 11. Third, the pyrrole protective group
of 11 was removed with hydroxylammonium to generate 1-(1-
methylcyclooctyl)piperidin-4-amine 12, which was introduced
onto the 1-position of 1-fluoro-2-nitrobenzene by substitution
reaction47 to obtain 1-(1-methylcyclooctyl)-N-(2-nitrophenyl)pi-
By use of 2 and 3, benzimidazole skeleton formation and
the final conversion to compound 1 were consecutively per-
formed as follows (Scheme 4). Thus, (R)-N-Boc-nipecotic acid
2 was amidated with N-[1-(1-methylcyclooctyl)-4-piperidinyl]-
1,2-benzenediamine 3 by water soluble carbodiimide, i.e.,
1-ethyl-3-(3-dimethylaminopropylcarbodiimide) hydrochloride
(WSCI) and 1-hydroxybenzotriazole (HOBT) method42,43 to
give tert-butyl (3R)-3-{[(2-{[1-(1-methylcyclooctyl)-4-piperi-
dinyl]amino}phenyl)amino]carbonyl}-1-piperidinecarboxylate
14. In succession, the resulting amide portion and the internal
secondary amino portion of 14 were condensed with mild acidic
dehydration in the presence of AcOH in toluene after reaction
condition studies49 to give tert-butyl (3R)-3-{1-[1-(1-methyl-
cyclooctyl)-4-piperidinyl]-1H-benzimidazol-2-yl}-1-piperidin-
ecarboxylate 15. Finally, the N-Boc portion of the 2-(3-
pipeidinyl)benzimidazole derivative 15 was deprotected by
acidic condition, followed by neutralization to afford the
requisite (3R)-NH-piperidine derivative, 1-[1-(1-methylcyclooc-

612 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Hayashi et al.
Scheme 4. Synthesis of 1-[1-(1-Methylcyclooctyl)-4-
piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole 1 and Its
N-Me Derivative 16a
As well, 2-arylbenzimidazole analogues bearing active-
hydrogen of the functional groups at 3′-position of the aryl
portion, e.g., 3′-(HOCH2CH2O)-phenyl analogue 40 and 3′-
(H2NCH2)-phenyl analogue 43, were prepared as follows
(Scheme 7). In the former case (40), the corresponding ethyl
(3-formylphenoxy)acetate 38 for the construction of (HOCH2-
CH2O)-phenyl portion was prepared from 3-hydroxybenzalde-
hyde 37 with ethyl 1-bromoacetate, which was converted into
benzimidazole derivative 39 by oxidative condensation in the
52
presence of Pb(OAc)4 with the above 1-substituted 1,2-
phenylenediamine 3, followed by reduction of its ester group
with LAH to afford 3′-(HOCH2CH2O)-phenyl analogue 40. In
the latter case (43), the phenylenediamine derivative 3 was
amidated with 3-cyanobenzoyl chloride 41, followed by benz-
imidazole skeleton formation with POCl3 to give 42, whose
nitrile portion on its phenyl core was reduced with LAH to
afford 3′-(H2NCH2)-phenyl analogue 43.53
All of these 1,2-disubstituted benzimidazole derivatives 1,
16, 21-26, 36, 40, and 43 were converted into tri- or
dihydrochloride using HCl in MeOH. The purities of the
hydrochlorides were confirmed by elementary analysis to be
within (0.3% of calculated values, respectively. These salts
were used for SAR studies with pharmacological evaluations.
Results and Discussion
For structure-activity relationship (SAR) study of NOP
receptor agonist in vitro, various types of 1,2-disubstituted
benzimidazole derivatives were designed and synthesized for
our in vitro criteria as NOP receptor agonists, i.e., the Ki value
for the binding affinity to human NOP receptor (hNOP receptor)
was less than 2 nM; the EC50 value for the functional activity
of [35S]GTPγS binding in response to the agonist–hNOP
receptor binding was less than 10 nM; and the selectivities of
the Ki and EC50 values for NOP receptor against other opioid
receptors such as human µ-receptor (hMOP receptor), human
κ-receptor (hKOP receptor), and human δ-receptor (hDOP
receptor) were over 10-fold.
a Reagents and conditions: (a) WSCI, HOBT, Et3N, CH2Cl2, -30 °C to
room temp; (b) AcOH, toluene, 100 °C; (c) 10% HCl in MeOH; (d) LAH,
THF, room temp to reflux.
tyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole 1,
as the sole product as a solid. The analysis of chemical purity
and enantiomeric excess (ee) [i.e., (R*-enantiomer – S*-
enantiomer)/(R*-enantiomer + S*-enantiomer)] of compound
1 showed it to be 98% and 97% ee, respectively, which indicated
that the total synthesis had no chemical and racemization
problem.
The biological assays in vitro were performed as follows.
First, the binding affinities of the synthetic NOP agonists to
hNOP receptors were measured by the displacement of tritium-
labeled N/OFQ for the recombinant hNOP receptors expressed
in human embryonic kidney (HEK)-293 cells. Second, the
biological responses were determined by the induction of the
binding of radiolabeled ligand [35S]GTPγS to the R-unit of the
G-protein due to the binding of the compounds to hNOP
receptors in the HEK-293 cells.54,55 As for the in vitro selectivity
evaluation of the NOP agonists, binding affinities and biological
functional activities for other opioid receptors were determined
as well. Thus, the binding affinities of the NOP agonists to other
human opioid receptors, i.e., µ-, κ-, and δ-receptors that
expressed in Chinese hamster ovary (CHO)-K1 cells, HEK-293
cells, and CHO-K1 cells, respectively, were measured by the
displacement of corresponding radiolabeled ligands such as
[3H]DAMGO for human µ-receptors, [3H]enadoline for human
κ-receptors, [3H]DPDPE for human δ-receptors, respectively.
And the functional activities were determined by the induction
of [35S]GTPγS binding stimulated by the binding of the
compounds to the each opioid receptors (µ-, κ-, or δ-receptors)
expressed in the above respective cell lines. In these function
studies, the EC50 (potency) value was the concentration produc-
ing a half-maximal response of its own and the Emax (efficacy)
value was the maximal response calculated as the percentage
of the maximal response produced by each control such as
N/OFQ, DAMGO, enadoline, and DPDPE.
As well, compound 16, the (3R)-N-Me piperidinyl analogue
of the compound 1, was prepared from the above (3R)-N-Boc
piperidine derivative 15 by reduction of its N-Boc moiety with
lithium aluminium hydride (LAH)50 (Scheme 4).
On the other hand, compound 21, the (3S)-enantiomer of the
NH-piperidine derivative 1, and compound 22, the (3S)-
enantiomer of the N-Me piperidine derivative 16, were synthe-
sized in the same preparation manners for their respective (3R)-
enantiomers. Thus, utilizing (S)-N-Boc-nipecotic acid 18 prepared
from commercially available (S)-nipecotic acid hydrochloride
17 (Digital Specialty Chemicals), and N-[1-(1-methylcyclooc-
tyl)-4-piperidinyl]-1,2-benzenediamine 3, the corresponding
2-[(3S)-N-Boc-piperidinyl]benzimidazole derivative 20 was
prepared by amidation, then deprotection of its N-Boc portion
to obtain 21 and LAH reduction of the N-Boc portion to obtain
22 were conducted, respectively (Scheme 5).
Furthermore, various 2-aryl- or 2-bicycloarylbenzimidazole
analogues 23-26 and 36 were prepared as shown below
(Scheme 6). Thus, N-[1-(1-methylcyclooctyl)-4-piperidinyl]-1,2-
benzenediamine 3 was amidated with a variety of arylcarboxylic
acids 27-30 or arylcarbonyl chloride 35, followed by internal
condensation with POCl3 to afford the corresponding 1-[1-(1-
methylcyclooctyl)-4-piperidinyl]-2-aryl-1H-benzimidazole de-
rivatives, e.g., 3′-MeSO2-phenyl analogue 23, 3′-Cl-4′-F-phenyl
analogue 24, 6′-indolyl analogue 25, 2′-benzofuranyl analogue
26, and 3′-CF3-phenyl analogue 36, respectively.42,49,51

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Scheme 5. Synthesis of 1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3S)-3-piperidinyl]-1H-benzimidazole 21 and Its N-Me Derivative
22a
a Reagents and conditions: (a) Boc2O, Et3N, MeOH, 0 °C to room temp; (b) WSCI, HOBT, Et3N, CH2Cl2, -30 °C to room temp; (c) AcOH, toluene, 100
°C; (d) 10% HCl in MeOH; (e) LAH, THF, room temp to reflux.
In general as summarized in Table 1, various 1,2-disubstituted
benzimidazole analogues, e.g., most of the 1-[1-(1-alkylcy-
clooctyl)-4-piperidinyl]-2-substituted-1H-benzimidazole deriva-
tives in the Table were satisfied for the above in vitro criteria.
First, the Ki values of the compounds 1, 16, 21–26, 36, 40, and
43 for the binding affinities to hNOP receptor were less than 2
nM, i.e., 0.0858-1.70 nM. Second, the EC50 values of the
compounds 1, 16, 21–24, 26, 36, 40, and 43 for the activity of
[35S]GTPγS binding in response to the respective agonist–hNOP
receptor binding were less than 10 nM, i.e., 0.39-5.7 nM, with
greater maximal effects than that of N/OFQ. Furthermore, the
selectivities of the binding (Ki) and function (EC50) of the NOP
agonists, including (3R)-enantiomers 1 and 16, for hNOP
receptor against other opioid receptors were greater than 10-
fold except for (3S)-enantiomers 21 and 22 that were partially
less selective (Tables 1 and 2). In the function studies, their
maximal effects were low or partial to other opioid receptors
except for the δ-receptor response with very low binding
affinities (Table 1). Taken together, these were selective full
NOP receptor agonists with comparative potency to N/OFQ,
although their selectivities were not compatible to N/OFQ.56
From the viewpoint of SAR, these 1,2-disubstituted benz-
imidazole derivatives which possess a six-member ring such
as the cycloalkylamino ring (1, 16, 21, and 22), phenyl ring
(23, 24, 36, 40, and 43), and bicyclic ring such as benzo-fuzed
heteroaryl ring (25 and 26) were well tolerable as 2-substituents
of the benzimidazole. In addition, the structural characteristics
at the ꢀ-position on the six-member ring were significant and
allowed for a variety of modifications of bulkiness and different
chemotypes with diverse polarity, lipophilicity, basicity, and size
for potent and selective NOP receptor agonists with the
1-(methylcyclooctyl-piperidinyl)benzimidazole skeleton, i.e.,
secondary amine (1 and 21) or tertiary amine portions (16 and
22) as part of the piperidine ring, and methanesulfonyl (23),
halogen (24), trifluoromethyl (36), hydroxyethoxy (40), or
aminomethyl (43) group as a substituent on phenyl core. In the
5′-indole case (25), NH-portion of the indole core is possible
as a substituent at the ꢀ-position of phenyl core, like the above
2-aryl substituted benzimidazole analogues. Also, another
bicycloaryl type, i.e., 2′-benzofuran skeleton (26), is allowable
as a 2-substituent of benzoimidazole.
The rank of the orders of the binding affinities (Ki values)
for the 2-substituents on benzimidazole portion is 3′-NH-
piperidinyl (1 and 21) > 3′-N-Me-piperidinyl (16 and 22), 3′-
(MeSO2)-phenyl (23), 3′-Cl-4′-F-phenyl (24), 3′-(H2NCH2)-
phenyl (43) > 3′-(HOCH2CH2O)-phenyl (40) > 2′-benzofuranyl
(26) > 3′-CF3-phenyl (36) > 6′-indolyl (25). In comparison,
the order of the potencies of the biological response (EC50
values) is approximately the same as the above Ki values. To
be more precise, there are highly significant positive correlations
of the EC50 values of NOP agonists for [35S]GTPγS binding to
the respective Ki values of NOP agonists for inhibition of [3H]N/
OFQ binding as shown in Figure 1 (r2 ) 0.984, p < 0.0001),
indicating that the binding of NOP agonist to NOP receptor is
the primary mechanism of the G-protein activation response and
the potency of the functional activity depends on the potency
of the binding affinity for each analogue.
For clarifying SAR, the 2-piperidinylbenzimidazole analogues
are particularly noteworthy. Comparison among the respective
optical isomers of the piperidinyl analogues, (R)-NH-piperidine
derivative 1 was more selective to NOP receptor over other
opioid receptors (µ, κ, and δ) than the corresponding (S)-NH-

614 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Hayashi et al.
Scheme 6. Synthesis of 1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-aryl-1H-benzimidazole Derivatives 23-26 and 36a
a Reagents and conditions: (a) WSCI, THF, -20 °C to room temp; (b) POCl3, reflux or 100 °C; (c) pyridine; (d) POCl3, reflux.
Scheme 7. Synthesis of Compounds 40 and 43a
a Reagents and conditions: (a) ethyl 1-bromoacetate, K2CO3, CH3CN, 70 °C; (b) (i) 38, EtOH, reflux; (ii) Pb(OAc)4, benzene, reflux; (c) LAH, THF,
0 °C to room temp; (d) pyridine; (e) POCl3, reflux; (f) LAH, THF, 0 °C to room temp.
enantiomer 21, and the (R)-N-Me form 16 was more NOP
receptor selective than the (S)-N-Me form 22 over κ- and
δ-receptors, as mentioned above generally. And the most
superior NOP receptor agonist of this study, the (R)-NH-
piperidine analogue 1, was determined to have a Ki value of
0.0858 nM for hNOP receptor, whereas that of (R)-N-Me
analogue 16 was 0.20 nM. Compared with the highly potent
hNOP receptor binding, the binding affinities to other human
opioid receptors of 1 were less potent; i.e., the Ki values were
1.052 nM (µ), 23.1 nM (κ), and greater than 667 nM (δ),

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 615
Table 1. Structure-Activity Relationships of in Vitro Binding Affinities and Functional Activities to Human Recombinant NOP Receptor and Other
Opioid Receptors for 1,2-Disubstituted Benzimidazole Analoguesa
a Ki values for these compounds were measured by the displacement of [3H]N/OFQ binding to hNOP receptors expressed in HEK-293 cells, [3H]DAMGO
binding to human µ-receptors expressed in CHO-K1 cells, [3H]enadoline binding to human κ-receptors expressed in HEK-293 cells, and [3H]DPDPE binding
to human δ-receptors expressed in CHO-K1 cells. Ki ) IC50/(1+[radioligand]/KD). Radioligands: [3H]N/OFQ as hNOP receptor agonist, concentration 0.4
nM, KD ) 0.135 nM; [3H]DAMGO as µ-receptor agonist, concentration 1.0 nM, KD ) 0.821 nM; [3H]enadoline as κ-receptor agonist, concentration 0.5 nM,
KD ) 0.683 nM; [3H]DPDPE as δ-receptor agonist, concentration 2.0 nM, KD ) 2.00 nM. b EC50 values for these compounds were determined by the
induction of the binding of [35S]GTPγS to R-unit of G-protein due to binding of the compounds to hNOP receptors expressed in HEK-293 cells, human
µ-receptors expressed in CHO-K1 cells, human κ-receptors expressed in HEK-293 cells, and human δ-receptors expressed in CHO-K1 cells. Emax (efficacy)
value was the maximal response calculated as the percentage of the maximal response produced by each control (N/OFQ, DAMGO, enadoline, and DPDPE).
c NT: not tested. d Data from ref 56.

616 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
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Table 2. Ki Values and Selectivity Profiles for Binding Affinities of (3R)-NH-Piperidine Analogue 1 and (3R)-N-Me-Piperidine Analogue 16 to Human
NOP, µ-, κ-, and δ-Receptorsa
binding affinity Ki (nM)
selectivity ratio
compd
hNOP
µ
κ
δ
µ/hNOP
κ/hNOP
δ/hNOP
1
16
0.0858
0.20
1.052
3.7
23.1
25
>667
>500
12.26
18.5
269
125
>7770
>2500
a The selectivities of NOP receptor agonists against µ-, κ-, or δ-receptor were calculated as the ratios of the Ki values for human µ-, κ-, or δ-receptor to
the Ki values for hNOP receptor.
the blood-brain barrier and the druglikeness concept for oral
activity. For example, to confirm essential factors for the
purpose, appropriate physicochemical properties such as mo-
lecular size and bulkiness (molecular weight), shape, acceptable
lipophilicity, basicity, and numbers of heteroatoms, hydrogen
bond donors, hydrogen bond acceptors, and rotatable bonds were
evaluated. And as a primary assessment for this study, NOP
receptor occupancies of the NOP receptor agonists in the mice
brain per os (po) were evaluated by their displacement of
radioligand [3H]N/OFQ from NOP receptors in the brain.
The flow of the procedure for the ex vivo NOP receptor
binding is as follows. First, male ddY mice were deprived of
Figure 1. Correlation between Ki and EC50 for NOP receptor agonists:
Ki values for binding affinity to hNOP receptor and EC50 values for
[
water for 24 h and then given the NOP receptor agonist orally
at 10 mg/kg. After 1 h, the brains were quickly removed in
their entirety. The brain tissues except cerebellum and medulla
were homogenized, and the resulting membranes were then used
to test for the specific bindings of [3H]N/OFQ to determine the
occupation ratios of the NOP receptor agonists to NOP receptor
in the brain.
35S]GTPγS binding in response to hNOP receptor binding.
respectively. Consequently, for the Ki values, the binding
selectivities of 1 as NOP receptor agonist against other opioid
receptors were 12.26-fold over µ-receptor, 269-fold over
κ-receptor, and greater than 7770-fold over δ-receptor (Table
2). Furthermore, the functional activity of 1 for hNOP receptor
was greater than other human opioid receptors, i.e., EC50 ) 0.39
nM, Emax ) 140% (NOP); EC50 ) 34 nM, Emax ) 30% (µ);
Through the design and synthetic study with various chemo-
types, NOP receptor agonists that displayed high or moderate
level NOP occupancies in the mice brain (10 mg/kg, po, 1 h)
were identified. The results of the structure-NOP receptor
occupancy relationships of the NOP receptor agonists for orally
potent brain activity are illustrated in Table 3 with respective
in vitro hNOP binding affinities and characteristic physico-
chemical properties.58-60
EC50 ) 80 nM, Emax ) 43% (κ); EC50 ) 4596 nM, Emax
)
79% (δ), and the functional selectivities of the hNOP agonism
against that of other human opioid receptors for EC50 values
were 87-fold over µ-receptor, 205-fold over κ-receptor, and
12041-fold over δ-receptor.
In general, various types of NOP receptor agonists were
categorized by the level of oral NOP receptor occupancy in the
mice brain (10 mg/kg, 1 h), i.e., to be greater than 50% as the
high level, to be less than 50% and greater than 31% as the
moderate level, and to be 31% or less as the low level.
Therefore, from the viewpoint of structural feature, these
analogues that have 1-[(cyclooctylmetyl)piperidinyl]benzimidazole
moiety as a common portion were classified with the respective
characteristic portion at 2-position of benzimidazole moiety for
each molecule, e.g., 3′-NH-piperidinyl (1) and 3′-N-Me-pip-
eridinyl (16) analogues in the high level category, 3′-MeSO2-
phenyl (23), 2′-benzofuranyl (26), and 3′-(HOCH2CH2O)-phenyl
(40) analogues in the moderate level category, and 3′-Cl-4′-F-
phenyl (24), 6′-indolyl (25), 3′-CF3-phenyl (36), and 3′-
(H2NCH2)-phenyl (43) analogues in the low level category,
while all these structural variations of the substituents at 3′-
position of piperidinyl ring as well as at 3′-position of aryl ring
were well allowable for the potent hNOP receptor binding
affinity in vitro as described above.
Additionally, the binding affinity of NOP agonist 1 as well
as the other NOP agonists described above is greater than that
of 2-(3,5-dimethylpiperazin-1-yl)-1-[1-(1-methylcyclooctyl)pi-
peridin-4-yl]-1H-benzimidazole (PCPB), a probe for NOP
agonist study, which was very recently reported (Hirao et al.,
2008),57 whose Ki value for NOP binding affinity was 2.1 nM.
In addition, the Ki values of PCPB for other opioids were 21
nM (µ), 15 nM (κ), and 29% inhibition at 1 µM (δ), and the
functional activities of PCPB for opioid receptors were EC50
6.0 nM and Emax ) 188% (NOP), EC50 ) 193 nM and Emax
)
)
11% (µ), EC50 ) 18 nM and Emax ) 26% (κ). Hence, PCPB
was also a less selective NOP agonist than the selective NOP
agonist 1. As well, a 2-unsubstituted benzimidazole analogue,
1-[1-(1-methylcyclooctyl)piperidin-4-yl]-1H-benzimidazole,showed
less selective NOP receptor binding over µ-receptor binding;
i.e., the Ki values were 2.5 nM (NOP) and 11 nM (µ) (further
data not shown).
Overall, the present series of 1,2-disubstituted benzimidazole
analogues showed highly potent and selective hNOP binding
affinities and functional activities as synthetic non-peptide NOP
agonists. Significantly, NOP agonist 1 is the most potent full
agonist among known 1,2-disubstituted benzimidazole derivative
agonists for now.
The relationships of the actual oral NOP receptor occupancy
in the mice brain with in vitro hNOP binding affinities as well
as physicochemical properties were significant as shown below.
First of all, it is noteworthy that the NOP receptor agonists
that show high (1 and 16) or moderate (23, 26, and 40) NOP
occupancy levels (10 mg/kg, po) have potent NOP binding
affinities as a requirement, of which Ki values are greater than
0.53 nM in vitro. In contrast, there were no high or moderate
NOP occupancy analogues of which Ki values are less than 0.6
Subsequently, SAR of the NOP agonist to identify appropriate
structure was efficiently investigated for orally active anxiolytic.
Originally, the NOP receptor agonists in this study were
rationally designed from the viewpoint of brain-active anxiolytic
after oral administration, e.g., the well brain penetration through

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 617
Table 3. SAR of NOP Occupancies for 1,2-Disubstituted Benzimidazole Analogues: ex Vivo NOP Occupancies in the Mice Brain (po) versus in Vitro
hNOP Binding Affinities and Physicochemical Propertiesa
a Data are expressed as inhibition of ex vivo specific binding of [3H]N/OFQ in the mice brain for drug-treated mice versus vehicle-treated mice (mean
( SEM of three mice). The ex vivo binding was measured 1 h after oral administration of the NOP receptor agonists in male ddY mice (10 mg/kg, po, 0.1%
methylcellulose). b Predicted by ACD/Laboratories 9.0.

618 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Hayashi et al.
nM in vitro. Therefore, it is essential for high or moderate NOP
occupancy in the mice brain (10 mg/kg) of orally administered
NOP receptor agonist to have highly potent intrinsic binding
affinity to hNOP receptor in vitro; i.e., the Ki value is greater
than 0.6 nM, at least.
On the other hand, the physicochemical properties for the
NOP occupancy have been meaningful for drug design and
succeeding optimization.
For example, the range of molecular weight is less than 480
and the range of TPSA is less than 64 Å, as a whole. Nowadays,
these values are acceptable in many cases as the features for
marketable oral drugs58-60 and were useful for successful results
as expected.
By comparision of NOP occupancy with lipophilicity, it is
suggested that the appropriately lower lipohilic analogues were
more suitable than higher lipophilic analogues for higher NOP
occupancy per os. Specifically, for the high NOP occupancy
analogues (53-61%) such as NH and N-Me piperidine deriva-
tives (1 and 16), the lipophilicity values at pH 7.4 as ACDlogD
calculated by ACD software (ACDlogD7.4) were appropriately
low, i.e., 2.57 and 3.57, respectively. And the moderate NOP
occupancy aryl analogues (34-38%) such as 3′-MeSO2-phenyl
(23), 2′-benzofuranyl (26), and 3′-(HOCH2CH2O)-phenyl (40)
analogues had moderate ACDlogD7.4 values, i.e., 5.03, 6.40,
and 5.49, respectively, while the low NOP occupancy analogues
(31% to no NOP occupancy) such as 3′-Cl-4′-F-phenyl (24),
3′-CF3-phenyl (36), and 3′-(H2NCH2)-phenyl (43) analogues had
much higher ACDlogD7.4 values, i.e., 7.38-7.49. In the case
of the 6′-indolyl analogue (25), no NOP occupancy in the mice
brain per os was observed owing to its reduced NOP binding
affinity in vitro, although the lipophilicity was a moderate
ACDlogD7.4 value, 6.18.
In addition, the range of the number of hydrogen bond donors
of these NOP receptor agonists is 0-1 except for a primary
amine analogue (43), whose number is 2, that showed low level
NOP receptor occupancy.
In other physicochemical properties for the high or moderate
NOP occupancy analogues, diverse characteristics such as
numbers of hydrogen bond acceptors (2-4) and rotatable bonds
(3-6), and basicity of functional group for 2-substituent of
benzimidazole (neutral group, e.g., 23, 26, and 40; basic group,
e.g., 1 and 16) were acceptable. As well, characteristic of ring
skeleton for the 2-substituent of benzimidazole was variable;
namely, both nonaromatic ring and aromatic ring were tolerable.
These studies will be published elsewhere as a subsequent
article.1a,2 As well, on the basis of these facts, further pharma-
cological characterization of 1 as a new-class anxiolytic was
performed for differentiation in terms of various potential side
effects from diazepam that is a representative of classical
benzodiazepine-type anxiolytic, which was already illustrated
(Hayashi et al., 2006; Hirao et al., 2008).1a,61
Experimental Section
Chemistry. General Procedures. In general, reagents, solvents,
reagents, and other chemicals were used as purchased without
further purification unless noted otherwise. All reactions with air-
or moisture-sensitive reactants and solvents were carried out under
nitrogen atmosphere. Flash column chromatography (medium
pressure liquid chromatography) purifications were carried out using
Merck silica gel 60 (230-400 mesh ASTM). Preparative thin-layer
chromatography (PTLC) purifications were carried out on Merck
silica gel 60 F254 precoated glass plates at a thickness of 0.5 or 1.0
mm. The structures of all isolated compounds were ensured by
NMR, IR, MS, or elementary analysis. Nuclear magnetic resonance
(1H and 13C NMR) data were determined at 270 MHz on a JNM-
LA 270 (JEOL) spectrometer, at 300 MHz on a JNM-LA300
(JEOL) spectrometer, or at 150 or 600 MHz on an AVANCE
(Bruker) spectrometer. Chemical shifts are expressed in δ (ppm).
1H NMR chemical shifts were determined relative to tetramethyl-
silane (TMS) as internal standard. 13C NMR chemical shifts were
determined relative to internal TMS at δ 0.0 or to the 13C signal of
solvent: CDCl3 δ 77.04 or CD3OD δ 49.8. NMR data are reported
as follows: chemical shift, number of atoms, multiplicities (s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br,
broadened), and coupling constants. Distortionless enhancement by
polarization transfer (DEPT), heteronuclear multiple quantum
coherence (HMQC), and heteronuclear multiple bond connectivity
(HMBC) were measured for the determination of 1H-13C correla-
tion, and the multiplicities by the proton interaction were expressed
in 13C NMR data. Infrared spectra were measured by an IR-470
(Shimadzu) infrared spectrometer. Low-resolution mass spectral
data (EI) were obtained on an Automass 120 (JEOL) mass
spectrometer. Low-resolution mass spectral data (ESI) were obtained
on a Quattro II (Micromass) mass spectrometer Agilent 1100 HPLC
system. Optical rotations were measured on a P-1020 (JASCO)
polarimeter. The compounds 1, 16, 21-26, 36, 40, and 43 were
used for pharmacological evaluations after hydrochloride formation
using HCl in MeOH, respectively.
Ethyl (R)-(1-tert-Butoxycarbonyl)-3-piperidinecarboxylate
(6). Ethyl (R)-nipecotate L-tartrate {[R]D +10 (c 5, H2O), 5.04 g,
16.4 mmol} was dissolved in saturated aqueous NaHCO3 (250 mL),
then extracted with CH2Cl2 (300 mL × 10). The combined extracts
were concentrated in vacuo. The residue was diluted with CH2Cl2
(200 mL), washed with saturated aqueous NaHCO3 (80 mL), dried
over anhydrous Na2SO4, filtered, then concentrated in vacuo.
Subsequently, the resulting salt-free ethyl (R)-nipecotate 5 (2.62
g) was dissolved in CH2Cl2 (24 mL) under N2, di-tert-butyl
dicarbonate (3.58 g, 16.4 mmol) was added at 0 °C, and the mixture
was stirred at the temperature for 15 min. Then the reaction mixture
was warmed to room temperature, stirred for 18 h, and concentrated
in vacuo to give 4.84 g of the title compound 6 as a colorless oil
that including a small amount of CH2Cl2 on 1H NMR analysis.
This compound 6 was used for the next reaction without further
Conclusion
In the present strategy, brain-penetrative and oral-active potent
NOP receptor agonists with requisite structures were efficiently
developed and identified utilizing drug design and synthetic study
with NOP occupancy evaluation in the mice brain after oral
administration. Indeed, a novel non-peptide 1,2-disubstituted ben-
zimidazole analogue 1, namely, 1-[1-(1-methylcyclooctyl)-4-pip-
eridinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole (MCOPPB), dem-
onstrated the most potent and selective NOP full agonism with
high NOP occupancy in the mice brain per os.
1
purification. H NMR (300 MHz, CDCl3) δ 4.14 (2H, q, J ) 7.1
Hz), 4.34-2.76 (4H, m), 2.48-2.37 (1H, m), 2.09-2.00 (1H, m),
1.74-1.38 (3H, m), 1.46 (9H, s), 1.26 (3H, t, J ) 7.1 Hz).
(R)-(1-tert-Butoxycarbonyl)-3-piperidinecarboxylic Acid (2).
To a stirred solution of the above ethyl (R)-(1-tert-butoxycarbonyl)-
3-piperidinecarboxylate 6 (4.84 g) in MeOH (24 mL)-THF (10
mL) was added dropwise 4 N LiOH (8.4 mL, 33.6 mmol)-H2O
(15.0 mL) solution at 0 °C under N2. After being stirred below 6
°C for 17 h, the reaction mixture was concentrated on a rotary
evaporator. To the residue was added H2O (10 mL), and the mixture
was cooled to 0 °C and acidified by adding 2 N HCl to adjust the
As a succeeding step of this series of study, SAR and
optimization for orally potent anxiolytic efficacy, long lasing
metabolic stability, and little hHERG channel binding affinity
with the above NOP agonists were investigated. As a result,
the potent NOP agonist 1 was identified as a potential anxiolytic
that demonstrated the most potent oral efficacy for the Vogel
anticonflict test among the NOP agonists in our study, e.g., a
significant Vogel anticonflict activity at 10 mg/kg (po, mice)
as minimum effective dose with advantageous characteristics.

Nociceptin/Orphanin FQ Receptor Agonist
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 619
pH to 1. The mixture was extracted with CH2Cl2 (50 mL × 8).
The combined extracts were dried over anhydrous MgSO4, filtered,
and concentrated in vacuo to give 3.57 g of the title compound 2
1.5 h. After the resulting solution was cooled to 0 °C, saturated
aqueous NH4Cl (5 mL) was added, and then the resulting mixture
was stirred at the same temperature for 30 min. The organic layer
was separated, and the aqueous layer was extracted with CH2Cl2
(10 mL × 2). The combined extracts were dried over anhydrous
MgSO4 and filtered. The filtrate was concentrated in vacuo, and
the residue was purified by flash column chromatography (silica
gel, hexane/EtOAc ) 15/1) to give 149 mg of the title compound
1
in 95% yield (two steps) as a white solid. H NMR (270 MHz,
CDCl3) δ 4.23-4.03 (1H, m), 3.94-3.84 (1H, m), 3.14-2.97 (1H,
m), 2.93-2.81 (1H, m), 2.57-2.43 (1H, m), 2.12-2.02 (1H, m),
1.78-1.40 (3H, m), 1.46 (9H, s).
1-Benzyl-4-(2,5-dimethyl-1H-pyrrol-1-yl)piperidine (8). In a
1 L flask with an azeotropic apparatus under N2, a solution of
4-amino-1-benzylpiperidine (57.1 g, 300 mmol), 2,5-hexanedione
(34.6 g, 303 mmol), and glacial acetic acid (4.5 mL) in dry toluene
(400 mL) was heated under reflux conditions.44 After no more
aqueous layer was observed in a water trap (∼3 h), the reaction
solution was cooled to room temperature. To the solution, hexane
(300 mL), saturated aqueous NaHCO3 (300 mL), and H2O (100
mL) were added. The resulting slightly basic aqueous layer was
separated, and the red-brown organic layer was washed with H2O
(100 mL) and brine (50 mL), dried over anhydrous MgSO4, then
filtered. The filtrate was concentrated in vacuo to afford 82 g of
the title compound 8 (crude), which became gradually solid
1
11 in 49% yield as a white solid. H NMR (270 MHz, CDCl3) δ
5.74 (2H, s), 3.92-3.78 (1H, s), 3.08 (2H, br d, J ) 6.8 Hz), 2.31
(6H, s), 2.23-2.05 (4H, m), 1.87-1.28 (16H, m), 0.84 (3H, s).
MS (EI direct) m/z: M+ 302.
1-(1-Methylcyclooctyl)piperidin-4-amine (12). Typical Pro-
cedure. In a 200 mL flask with a reflux condenser under N2, a
solution of 4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-(1-methylcyclooc-
tyl)piperidine 11 (1.22 g, 4.03 mmol) and NH2OH·HCl (2.2 g, 32
mmol) in EtOH (12 mL)-H2O (6 mL) was heated under reflux
conditions for 19 h, then cooled to room temperature. The resulting
orange solution was poured into ice-cold 2 N NaOH (∼200 mL),
and then CH2Cl2 (50 mL) was added. The organic layer was
separated, and the basic aqueous layer was extracted with CH2Cl2
(50 mL × 2). The combined extracts were dried over anhydrous
Na2SO4 and filtered. The filtrate was concentrated in vacuo to give
0.86 g of the title compound 12 in 95% yield (crude) as an oil
including a small amount of starting material 11 based on 1H NMR.
Compound 12: 1H NMR (270 MHz, CDCl3) δ 2.86 (2H, br d, J )
11.3 Hz), 2.65-2.50 (1H, m), 2.08 (2H, td, J ) 11.3 Hz, J ) 1.8
Hz), 1.86-1.17 (20H, m), 0.80 (3H, s).
1-(1-Methylcyclooctyl)-N-(2-nitrophenyl)piperidin-4-amine (13).
To a solution of 1-(1-methylcyclooctyl)piperidin-4-amine 12 (3.46
g, 15.4 mmol) and 1-fluoro-2-nitrobenzene (2.39 g, 16.9 mmol) in
dry CH3CN (77 mL) was added anhydrous K2CO3 (2.77 g, 20.0
mmol) at room temperature under N2. The resulting yellow
suspension was stirred under reflux conditions using an oil bath
(∼100 °C) overnight. After the mixture was cooled to room
temperature, the solvent was evaporated. The residue was partitioned
between CH2Cl2 (50 mL) and saturated aqueous NaHCO3 (50 mL).
The organic layer was separated, and the aqueous layer was
extracted with CH2Cl2 (15 mL × 3). The combined extracts were
washed with brine, dried over anhydrous K2CO3, filtered, and
concentrated in vacuo to give 5.75 g of orange oil, which was
purified by flash column chromatography (silica gel, CH2Cl2/MeOH
) 20/1) to give 4.95 g of the title compound 13 in 93% yield as an
orange solid. 1H NMR (270 MHz, CDCl3) δ 8.17 (1H, dd, J ) 8.6
Hz, J ) 1.5 Hz), 8.11 (1H, br s), 7.40 (1H, ddd, J ) 8.6 Hz, J )
7.1 Hz, J ) 1.7 Hz), 6.86 (1H, br d, J ) 8.6 Hz), 6.59 (1H, ddd,
J ) 8.6 Hz, J ) 7.2 Hz, J ) 1.2 Hz), 3.60-3.40 (1H, m), 2.97-2.85
(2H, m), 2.33-2.24 (2H, m), 2.10-2.00 (2H, m), 1.90-1.20 (16H,
m), 0.84 (3H, s).
1
including a small amount of toluene based on H NMR analysis.
This compound 8 was used for the next step without further
1
purification. H NMR (270 MHz, CDCl3) δ 7.36-7.14 (5H, m),
5.74 (2H, s), 3.96-3.82 (1H, m), 3.55 (2H, s), 3.02 (2H, br d, J )
11.5 Hz), 2.30 (6H, s), 2.32-2.20 (2H, m), 2.08 (2H, td, J ) 11.9
Hz, J ) 1.9 Hz), 1.79 (2H, br d, J ) 12.0 Hz).
4-(2,5-Dimethyl-1H-pyrrol-1-yl)piperidine (9). In a 500 mL
hydrogenation apparatus, to a solution of 1-benzyl-4-(2,5-dimethyl-
1H-pyrrol-1-yl) piperidine 8 (30 g, crude) in MeOH (150 mL),
Pd(OH)2 (6 g, 20 wt%) was added. The resulting dark-brown
mixture was stirred at room temperature under H2 (4.5 atm) for
16 h. The resulting mixture was filtered through a Celite pad with
MeOH washings (20 mL × 5). The filtrate was concentrated to
give 18.5 g of the title compound 9 in 95% yield (two steps, crude)
as an oil. This compound 9 was used for the next step without
1
further purification. H NMR (270 MHz, CDCl3) δ 5.74 (2H, s),
4.06-3.90 (1H, m), 3.23 (2H, d, J ) 11.9 Hz), 2.71 (2H, t, J )
12.2 Hz), 2.31 (6H, s), 2.22-2.04 (2H, m), 1.83 (2H, br d, J ) 10
Hz), 1.61 (1H, br s).
1-[4-(2,5-Dimethyl-1H-pyrrol-1-yl)-1-piperidinyl]cyclooctan-
ecarbonitrile (10). In a 300 mL flask with a reflux condenser under
N2, to a mixture of the above crude 4-(2,5-dimethyl-1H-pyrrol-1-
yl)piperidine 9 (15.6 g, 87.5 mmol) in H2O (30 mL), a solution of
TsOH·H2O (20 g, 105 mmol) in H2O (20 mL) was added at room
temperature. The reaction mixture was stirred and then turned red.
This reaction was exothermic, and the internal temperature reached
33 °C. The resulting solution was maintained at that temperature
with stirring for 10 min, then cyclooctanone (13.2 g, 105 mmol)
was added. The resulting mixture was heated at 45 °C, a solution
of KCN (11.4 g, 175 mmol) in H2O (30 mL) was added, and the
resulting mixture was stirred at the same temperature for 1 day.
The resulting solid was collected by filtration and washed with H2O
(50 mL × 2). A mixture of the solid (∼23 g) and hexane (75 mL)
was stirred in an ice-cold bath for 15 min to remove excess
cyclooctanone from the solid. The resulting mixture was filtered,
and the obtained cake was washed with hexane (15 mL × 2) and
then dried in a vacuum oven (45 °C, 13 h) to give 19.8 g of the
N-[1-(1-Methylcyclooctyl)-4-piperidinyl]-1,2-benzenedi-
amine (3). To a solution of the above 1-(1-methylcyclooctyl)-N-
(2-nitrophenyl)piperidin-4-amine 13 (4.95 g, 14.3 mmol) in THF
(128 mL) was added MeOH (41 mL), H2O (12.8 mL), zinc powder
(9.37 g, 143 mmol), and NH4Cl (3.83 g, 71.6 mmol) at room
temperature under N2. The reaction mixture was stirred at that
temperature, and the yellow color disappeared during 15 min. Then
the reaction mixture was filtered through a Celite pad with THF
washing, and the filtrate was concentrated in vacuo to give a mixture
of a brown solid and aqueous solution. To the mixture was added
saturated aqueous NaHCO3 (50 mL), and then the mixture was
extracted with CH2Cl2. The combined extracts were dried over
anhydrous K2CO3, filtered, and concentrated to give 4.54 g of the
1
title compound 10 in 72% yield. H NMR (270 MHz, CDCl3) δ
5.75 (2H, s), 4.04-3.88 (1H, m), 3.19 (2H, br d, J ) 7.1 Hz), 2.31
(6H, s), 2,32-1.99 (10H, m), 1.75-1.43 (10H, m). MS (EI direct)
m/z: M+ 313. Anal. Calcd for C20H31N3: C, 76.63; H, 9.97; N, 13.40.
Found: C, 76.39; H, 9.89; N, 13.53.
1
title compound 3 in 100% yield as a dark-brown solid. H NMR
4-(2,5-Dimethyl-1H-pyrrol-1-yl)-1-(1-methylcyclooctyl)piperi-
dine (11). Typical Procedure. In a 30 mL flask with a septum
under N2, a solution of 1-[4-(2,5-dimethyl-1H-pyrrol-1-yl)-1-
piperidinyl]cyclooctanecarbonitrile 10 (313 mg, 0.998 mmol) in dry
CH2Cl2 (3.1 mL) was cooled in a dry ice solmix bath. To this
solution, 0.90 M MeMgBr in anhydrous THF (1.7 mL, 1.5 mmol)
was added dropwise via syringe at -78 °C. The resulting mixture
was stirred at that temperature for 30 min, then allowed to warm
to room temperature gradually, and stirred at room temperature for
(300 MHz, CDCl3) δ 6.82-6.62 (4H, m), 3.40-3.10 (3H, m),
2.95-2.85 (2H, m), 2.30-2.15 (2H, m), 2.10-2.00 (2H, m),
1.90-1.20 (17H, m), 0.83 (3H, s).
tert-Butyl
(3R)-3-{[(2-{[1-(1-Methylcyclooctyl)-4-piperi-
dinyl]amino}phenyl)amino]carbonyl}-1-piperidinecarboxylate
(14). To a stirred solution of (R)-(1-tert-butoxycarbonyl)-3-pip-
eridinecarboxylic acid 2 (756.6 mg, 3.30 mmol), N-[1-(1-methyl-
cyclooctyl)-4-piperidinyl]-1,2-benzenediamine 3 (800 mg, 2.54

620 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Hayashi et al.
mmol), and 1-hydroxybenzotriazole (HOBT) (567.5 mg, 4.20 mmol)
in dry CH2Cl2 (17 mL) was added Et3N (1.1 mL, 7.9 mmol)
followed by water soluble carbodiimide, i.e., 1-ethyl-3-(3-dimethyl-
aminopropylcarbodiimide) hydrochloride (WSCI) (805.1 mg, 4.20
mmol) at -30 °C under N2, and the reaction mixture was allowed
to warm to room temperature. After being stirred at room
temperature for 18 h, the reaction mixture was partitioned between
CH2Cl2 (80 mL) and H2O (80 mL). The organic layer was separated,
washed with H2O (50 mL), dried over anhydrous MgSO4, filtered,
and concentrated in vacuo to give 1.443 g of the title compound
14 as a brown solid (crude) including small amount of CH2Cl2 based
on 1H NMR, which was used for the next reaction without further
purification. 1H NMR (300 MHz, CDCl3) δ 7.60 (1H, br s),
7.36-7.34 (1H, m), 7.12-7.05 (1H, m), 6.79-6.70 (2H, m),
4.07-1.20 (33H, m), 1.47 (9H, s), 0.83 (3H, s).
31.6 (2C, t), 30.4 (1C, t), 28.4 (2C, t), 25.9 (1C, t), 25.0 (1C, t),
21.6 (2C, t), 20.6 (1C, q).
HCl Salt Formation. General Procedure. Compound 1 (730.7
mg) was dissolved in CH2Cl2 (30 mL) and excess equivalent of
10% HCl/MeOH solution (120 mL). The mixture was stirred at
room temperature for 1 h and then concentrated in vacuo. The
resulting white solid was collected and dried under vacuum at 50
°C to give 769.3 mg of the corresponding trihydrochloride as a
white solid: [R]2D3 -4.0 (c 0.39, MeOH). IR (KBr): 3412, 2930,
2712, 1626, 1479, 1468, 1308, 1107, 1042, 1009, 856, 770 cm-1
.
MS (ESI positive) m/z: [M + H]+ 409. Anal. (C26H40N4 ·3HCl·
0.8H2O) C, H, N.
1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3R)-1-methyl-3-
piperidinyl]-1H-benzimidazole (16). To a solution of tert-butyl
(3R)-3-{1-[1-(1-methylcyclooctyl)-4-piperidinyl]-1H-benzimidazol-
2-yl}-1-piperidinecarboxylate 15 (849.6 mg, 1.67 mmol) in anhy-
drous THF (25.0 mL) was added lithium aluminium hydride (LAH)
(228.2 mg, 6.01 mmol) at room temperature under N2. The reaction
mixture was stirred at that temperature under N2 for 1 h, then
warmed up to reflux conditions and stirred for 2 h. The reaction
mixture was cooled to room temperature, more LAH (90.0 mg,
2.37 mmol) was added, and the mixture was refluxed with stirring
for 3 h. After cooling to 0 °C, AcOEt (10 mL) was added dropwise
to the reaction mixture, stirred at 0 °C for 10 min, then stirred at
room temperature for 20 min. The mixture was cooled to 0 °C,
then H2O (30.0 mL) was added dropwise. The mixture was stirred
at 0 °C for 30 min, and stirred at room temperature for 30 min.
The mixture was poured into saturated aqueous NaHCO3 (5 mL),
then extracted with CH2Cl2 (60 mL × 3). The combined extracts
were dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The residue was purified by flash column chromatography
(silica gel, CH2Cl2/MeOH/25% aqueous ammonia ) 100/10/2) to
give the title product as a solid, which was repurified by flash
column chromatography (silica gel, CH2Cl2/MeOH/25% aqueous
ammonia ) 1900/50/2.5) to give 517.5 mg of the title compound
16 in 73% yield as a slight brownish white solid. [R]2D3 -15 (c
0.13, MeOH). 1H NMR (600 MHz, CDCl3) δ 7.75-7.73 (1H, m),
7.65-7.62 (1H, m), 7.23-7.18 (2H, m), 4.26-4.20 (1H, m),
3.21-3.14 (3H, m), 3.06-3.04 (2H, m), 2.94 (1H, d, J ) 10.1
Hz), 2.56-2.44 (3H, m), 2.39 (3H, s), 2.26-2.21 (2H, m),
2.09-1.36 (23H, m). 13C NMR (150 MHz, CDCl3) δ 155.8 (1C,
s), 143.2 (1C, s), 133.5 (1C, s), 121.7 (1C, d), 121.4 (1C, d), 119.6
(1C, d), 112.2 (1C, d), 60.2 (1C, t), 58.1 (1C, s), 55.7 (1C, t), 55.0
(1C, d), 46.5 (1C, q), 45.3 (2C, t), 35.7 (1C, d), 34.9 (2C, t), 31.6
(2C, t), 29.7 (1C, t), 28.4 (2C, t), 25.6 (1C, t), 25.0 (1C, t), 21.6
(2C, t), 20.5 (1C, q). According to the general procedure to form
the HCl salt of 1 described above, compound 16 (490.0 mg) was
converted into 554.3 mg of the corresponding trihydrochloride. IR
(KBr): 3450, 2928, 2702, 1628, 1474, 1308, 1252, 1171, 1105, 968,
937, 854, 772 cm-1. MS (ESI positive) m/z: [M + H]+ 423. Anal.
(C27H42N4 ·3HCl·H2O) C, H, N.
(S)-(1-tert-Butoxycarbonyl)-3-piperidinecarboxylic Acid (18).
To a stirred solution of (S)-nipecotic acid hydrochloride {[R]D +3.4
(c 1, H2O), 596.2 mg, 3.60 mmol} in MeOH (17 mL) was added
Et3N (17 mL) followed by di-tert-butyl dicarbonate (942.8 mg, 4.32
mmol) at 0 °C under N2. The reaction mixture was warmed to room
temperature, stirred for 2 days, and concentrated in vacuo. The
residue was diluted with CH2Cl2 (50 mL)-H2O (10 mL), and the
mixture was cooled to 0 °C, acidified by adding 2 N HCl to adjust
to pH 3. The organic layer was separated, and the aqueous layer
was extracted with CH2Cl2 (60 mL). The organic layers were
combined, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo to give 862.1 mg of the title compound 18 as a white
solid in quantitative yield (crude). 1H NMR of this compound was
the same as that of (R)-enantiomer 2.
tert-Butyl (3S)-3-{[(2-{[1-(1-Methylcyclooctyl)-4-piperidiny-
l]amino}-phenyl)amino]carbonyl}-1-piperidinecarboxylate (19).
According to the procedure for the corresponding (3R)-enantiomer,
i.e., tert-butyl (3R)-3-{[(2-{[1-(1-methylcyclooctyl)-4-piperi-
dinyl]amino}phenyl)amino]carbonyl}-1-piperidinecarboxylate 14,
332.0 mg of the title compound 19 was prepared as a crude product,
tert-Butyl (3R)-3-{1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-
1H-benzimidazol-2-yl}-1-piperidinecarboxylate (15). A solution
of
tert-butyl
(3R)-3-{[(2-{[1-(1-methylcyclooctyl)-4-piperi-
dinyl]amino}phenyl)amino]carbonyl}-1-piperidinecarboxylate 14
(1.443 g, crude) and glacial acetic acid (0.70 mL, 12.2 mmol) in
dry toluene (70 mL) was stirred at 100 °C under N2 for 16 h, cooled
to room temperature, and then concentrated in vacuo. The residue
was dissolved in CH2Cl2 (50 mL), the mixture was cooled to 0 °C,
and saturated aqueous NaHCO3 (50 mL) was added. The organic
layer was separated, and the aqueous layer was extracted with
CH2Cl2 (50 mL). The organic layers were combined, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The residue
was purified by flash column chromatography (silica gel, CH2Cl2/
MeOH ) 32/1) to give 998.7 mg of the title compound 15 in 77%
yield (two steps) as a pale-brown solid. 1H NMR (270 MHz, CDCl3)
δ 7.76-7.71 (1H, m), 7.65-7.60 (1H, m), 7.25-7.19 (2H, m),
4.40-4.10 (3H, m), 3.25-2.70 (5H, m), 2.60-2.45 (2H, m),
2.35-2.20 (2H, m), 2.15-1.30 (20H, m), 1.47 (9H, s), 0.89 (3H,
s).
1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-
1H-benzimidazole (1), MCOPPB. A mixture of tert-butyl (3R)-
3-{1-[1-(1-methylcyclooctyl)-4-piperidinyl]-1H-benzimidazol-2-yl}-
1-piperidinecarboxylate 15 (998.7 mg, 1.96 mmol) and 10% HCl/
MeOH solution (100 mL) was stirred at room temperature under
N2 for 16 h, then concentrated in vacuo. To the residue was added
CH2Cl2 (60 mL). The mixture was cooled to 0 °C and basified by
adding saturated aqueous NaHCO3. The organic layer was separated,
and the aqueous layer was extracted with CH2Cl2 (70 mL × 3).
The organic layers were combined, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The residue was purified by
flash column chromatography (silica gel, CH2Cl2/MeOH/25%
aqueous ammonia ) 120/10/1) to give 735.1 mg of the title
compound 1 in 92% yield as a brown oil. HPLC analysis: (1)
Chemical purity of 98% with no impurity over 0.5% (apparatus,
Alliance 2690 with PDA detector, Waters; analytical column,
Kromasil 5C4, 250 mm × 4.6 mm, 5 µm (particle size); eluent,
CH3CN/0.5% HClO4 ) 32/68 to 90/10 gradient; flow rate, 1.0 mL/
min; column temperature, 40 °C; UV detection, 210 nm; retention
time, 12 min. (2) Chiral analysis, 97% enantiomeric excess (ee),
determined by HPLC employing a chiral stationary phase (ap-
paratus, Alliance 2690 with PDA detector, Waters; analytical
column, CHIRALCEL OG, 250 mm × 4.6 mm, Daicel Chemical
Industries; eluent, hexane/EtOH/2-propanol/diethylamine ) 95/2.5/
2.5/0.1; flow rate, 1 mL/min; room temperature (27-28 °C);
dissolving solvent, hexane/EtOH ) 1:1; UV detection, 250 nm;
retention time; peak of (R)-form, 12 min; peak of (S)-form, 10 min;
(R)/(S) ) 98.5:1.5. 1H NMR (600 MHz, CDCl3) δ 7.75-7.71 (1H,
m), 7.64-7.62 (1H, m), 7.23-7.18 (2H, m), 4.24-4.18 (1H, m),
3.26-3.05 (6H, m), 2.80 (1H, dt, J ) 11.7 Hz, J ) 2.64 Hz),
2.58-2.47 (2H, m), 2.29-2.21 (2H, m), 2.11-2.05 (1H, m),
2.05-1.92 (4H, m), 1.89-1.79 (3H, m), 1.77-1.51 (11H, m),
1.44-1.35 (2H, m), 0.89 (3H, s). 13C NMR (150 MHz, CDCl3) δ
155.9 (1C, s), 143.2 (1C, s), 133.5 (1C, s), 121.7 (1C, d), 121.5
(1C, d), 119.6 (1C, d), 112.2 (1C, d), 58.2 (1C, s), 55.0 (1C, d),
51.4 (1C, t), 46.5 (1C, t), 45.4 (2C, t), 36.0 (1C, d), 35.0 (2C, t),

Nociceptin/Orphanin FQ Receptor Agonist
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 621
including a small amount of CH2Cl2, from (S)-(1-tert-butoxycar-
bonyl)-3-piperidinecarboxylic acid 18 (95.4 mg, 0.416 mmol) and
N-[1-(1-methylcyclooctyl)-4-piperidinyl]-1,2-benzenediamine 3 (101.0
mg, 0.320 mmol). 1H NMR of this compound 19 was the same as
that of (R)-enantiomer 14.
1-[1-(1-Methylcyclooctyl)piperidin-4-yl]-2-[3-(methylsulfo-
nyl)phenyl]-1H-benzimidazole (23). To a stirred solution of
3-(methylsulfonyl)benzoic acid (84.1 mg, 0.420 mmol) and N-[1-
(1-methylcyclooctyl)-4-piperidinyl]-1,2-benzenediamine 3 (126.2
mg, 0.400 mmol) in anhydrous THF (3.5 mL) was added WSCI
(99.7 mg, 0.520 mmol) at -20 °C under N2. The reaction mixture
was stirred at that temperature for 10 min, then stirred at 0 °C for
30 min, and then allowed to warm to room temperature. After being
stirred at room temperature for 3 days, the reaction mixture was
cooled to 0 °C, H2O (3 mL) and saturated aqueous NaHCO3 (3
mL) were added, then the mixture was extracted with CH2Cl2 (15
mL × 2). The combined extracts were dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. Subsequently, the
residue was mixed with POCl3 (15 mL) under N2, and the reaction
mixture was stirred at 100 °C for 7 h, then cooled to room
temperature, and then concentrated in vacuo. The residue was
diluted with CHCl3 (20 mL), cooled to 0 °C, H2O (15 mL) was
added, and the mixture was basified by adding 25% aqueous
ammonia. The organic layer was separated, and the aqueous layer
was extracted with CHCl3 (15 mL × 3). The organic layers were
combined, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by PTLC (silica gel, CH2Cl2/
MeOH ) 20/1) to give 83.8 mg of the title compound 23 in 44%
yield. 1H NMR (270 MHz, CDCl3) δ 8.25-8.24 (1H, m),
8.13-8.09 (1H, m), 7.96-7.92 (1H, m), 7.85-7.73 (3H, m),
7.39-7.28 (2H, m), 4.29-4.12 (1H, m), 3.18-2.80 (5H, m), 2.59
(2H, dq, J ) 11.9 Hz, J ) 3.46 Hz), 2.09 (2H, t, J ) 10.9 Hz),
1.94-1.33 (16H, m), 0.82 (3H, s). Compound 23 (83.8 mg) was
tert-Butyl (3S)-3-{1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-1H-
benzimidazol-2-yl}-1-piperidinecarboxylate (20). According to
the procedure for the corresponding (3R)-enantiomer, i.e., tert-butyl
(3R)-3-{1-[1-(1-methylcyclooctyl)-4-piperidinyl]-1H-benzimidazol-
2-yl}-1-piperidinecarboxylate 15, 132.0 mg of the title compound
20 was prepared in 78% yield (two steps) as a slight brownish white
solid from the above tert-butyl (3S)-3-{[(2-{[1-(1-methylcyclooc-
tyl)-4-piperidinyl]amino}phenyl)amino]carbonyl}-1-piperidinecar-
1
boxylate 19 (332.0 mg). H NMR of this compound 20 was the
same as that of (R)-enantiomer 15.
1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3S)-3-piperidinyl]-
1H-benzimidazole (21). According to the procedure for the
corresponding (3R)-enantiomer, i.e., 1-[1-(1-methylcyclooctyl)-4-
piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole 1, 52.0 mg of
the title compound 21 was prepared in 92% yield from tert-butyl
(3S)-3-{1-[1-(1-methylcyclooctyl)-4-piperidinyl]-1H-benzimidazol-
2-yl}-1-piperidinecarboxylate 20 (70.7 mg, 0.139 mmol). 1H NMR
of this compound was the same as that of (R)-enantiomer 1.
According to the general procedure to form HCl salt, compound
21 (52.0 mg) was converted into 63.3 mg of the corresponding
trihydrochloride. HPLC analysis: 96% ee, determined by employing
a chiral stationary phase (apparatus, Alliance 2690 with PDA
detector, Waters; analytical column, CHIRALCEL OG, 250 mm
× 4.6 mm, Daicel Chemical Industries; eluent, hexane/EtOH/2-
propanol/diethylamine ) 97/1.5/1.5/0.1; flow rate, 1 mL/min; room
temperature; dissolving solvent, hexane/EtOH ) 1:1; UV detection,
252 nm; retention time, peak of (R)-form, 22 min; peak of (S)-
form, 15 min; (R)/(S) ) 2:98. [R]2D4 ) +5.3 (c 0.53, MeOH). 13C
NMR (150 MHz, CD3OD) δ 153.9 (1C, s), 135.1 (1C, s), 132.9
(1C, s), 128.2 (1C, d), 128.0 (1C, d), 117.2 (1C, d), 116.7 (1C, d),
73.7 (1C, s), 55.0 (1C, d), 48.3 (1C, t), 48.3 (1C, t), 48.2 (1C, t),
45.9 (1C, t), 33.9 (1C, d), 33.5 (1C, t), 33.4 (1C, t), 30.0 (1C, t),
29.9 (1C, t), 29.1 (2C, t), 28.6 (1C, t), 27.2 (1C, t), 24.8 (2C, t),
23.7 (1C, t), 23.1 (1C, q). IR (KBr) was the same spectrum as that
of trihydrochloride of 1. MS (ESI positive) m/z: [M + H]+ 409.
Anal. (C26H40N4 ·3HCl·2.5H2O) C, H, N.
1-[1-(1-Methylcyclooctyl)-4-piperidinyl]-2-[(3S)-1-methyl-3-
piperidinyl]-1H-benzimidazole (22). To a solution of tert-butyl
(3S)-3-{1-[1-(1-methylcyclooctyl)-4-piperidinyl]-1H-benzimidazol-
2-yl}-1-piperidinecarboxylate 20 (61.3 mg, 0.120 mmol) in anhy-
drous THF (3.0 mL) was added LAH (22.9 mg, 0.603 mmol) at
room temperature under N2. The reaction mixture was stirred for
30 min, then warmed up to reflux conditions, stirred for 5 h, and
then cooled to 0 °C. AcOEt (3.0 mL) was added dropwise to the
reaction mixture at 0 °C, stirred at 0 °C for 10 min, then stirred at
room temperature for 20 min. After the mixture was cooled to 0
°C, H2O (4.0 mL) was added dropwise. After being stirred at 0 °C
for 1 h, the mixture was poured into saturated aqueous NaHCO3
(5 mL), then the mixture was extracted with CH2Cl2 (25 mL × 3).
The combined extracts were dried over anhydrous MgSO4, filtered,
and concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, CH2Cl2/MeOH/25% aqueous ammonia
) 1000/100/8) to give 42.0 mg of the title compound 22 in 83%
yield as a slight brownish white solid. 1H NMR of this compound
22 was the same as that of (R)-enantiomer 16. According to the
general procedure to form HCl salt described above, compound 22
(42.0 mg) was converted into 47.4 mg of the corresponding
trihydrochloride: [R]2D4 +4.6 (c 0.43, MeOH). 13C NMR (150 MHz,
CD3OD) δ 153.2 (1C, s), 135.3 (1C, s), 132.9 (1C, s), 128.2 (1C,
d), 128.0 (1C, d), 117.2 (1C, d), 116.8 (1C, d), 73.6 (1C, s), 57.8
(1C, t), 56.0 (1C, t), 55.0 (1C, d), 48.4 (1C, t), 48.3 (1C, t), 45.3
(1C, q), 34.9 (1C, d), 33.5 (1C, t), 33.4 (1C, t), 30.0 (1C, t), 29.9
(1C, t), 29.1 (2C, t), 27.4 (1C, t), 27.2 (1C, t), 24.8 (3C, t), 23.2
(1C, q). MS (ESI positive) m/z: [M + H]+ 423. Anal.
(C27H42N4 ·3HCl·3.3H2O) C, H, N.
converted into 86.9 mg of the corresponding dihydrochloride: 13
C
NMR (150 MHz, CD3OD) δ 152.2 (1C, s), 144.5 (1C, s), 137.1
(1C, d), 136.8 (1C, s), 133.5 (1C, s), 133.0 (1C, d), 133.0 (1C, d),
131.2 (1C, d), 128.2 (1C, s), 128.1 (1C, d), 127.9 (1C, d), 118.1
(1C, d), 117.0 (1C, d), 73.7 (1C, s), 56.2 (1C, d), 48.2 (2C, t), 45.1
(1C, q), 33.4 (2C, t), 29.9 (2C, t), 29.1 (2C, t), 27.1 (1C, t), 24.7
(2C, t), 22.9 (1C, q). IR (KBr): 3406, 2928, 2692, 1624, 1464,
1300, 1148, 966, 860, 777 cm-1. MS (ESI positive) m/z: [M +
H]+ 480. Anal. (C28H37N3O2S·2HCl·1.25H2O) C, H, N.
2-(3-Chloro-4-fluorophenyl)-1-[1-(1-methylcyclooctyl)piperi-
din-4-yl]-1H-benzimidazole (24). To a stirred solution of 3-chloro-
4-fluorobenzoic acid (110.0 mg, 0.630 mmol) and N-[1-(1-
methylcyclooctyl)-4-piperidinyl]-1,2-benzenediamine 3 (189.3 mg,
0.600 mmol) in anhydrous THF (4.0 mL) was added WSCI (149.5
mg, 0.780 mmol) at -20 °C under N2. The reaction mixture was
stirred at the temperature under N2 for 10 min, then stirred at 0 °C
for 30 min, and then allowed to room temperature. After being
stirred at room temperature for 2 days, the reaction mixture was
poured into saturated aqueous NaHCO3 (15 mL), then the mixture
was extracted with CH2Cl2 (15 mL × 4). The combined extracts
were dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. Subsequently, to the residue was added POCl3 (20 mL) at
room temperature under N2, and the reaction mixture was stirred
under reflux conditions for 5 h, then allowed to room temperature,
and concentrated in vacuo. The residue was cooled to 0 °C and
diluted with CHCl3 (30 mL). H2O (20 mL) was added at 0 °C,
then the mixture was basified by adding 25% aqueous ammonia at
0 °C. The organic layer was separated, and the aqueous layer was
extracted with CHCl3 (30 mL × 4). The organic layers were
combined, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by PTLC (silica gel, CH2Cl2/
MeOH ) 10/1) to give 155.6 mg of the title compound 24 in 57%
1
yield as a white solid. H NMR (270 MHz, CDCl3) δ 7.82-7.72
(3H, m), 7.52-7.46 (1H, m), 7.34-7.25 (3H, m), 4.30-4.20 (1H,
m), 3.12 (2H, br d, J ) 11.2 Hz), 2.65-2.51 (2H, m), 2.15-2.06
(2H, m), 1.92-1.30 (16H, m), 0.83 (3H, s). MS (ESI positive) m/z:
[M + H]+ 454. Compound 24 was converted into the corresponding
trihydrochloride: 13C NMR (150 MHz, CD3OD) δ 162.7 (1C, s,
JC-F ) 254.9 Hz, CF), 151.8 (1C, s), 136.7 (1C, s), 134.8 (1C, d),
133.4 (1C, s), 133.0 (1C, d, JC-F ) 8.5 Hz), 128.0 (1C, d), 127.8
(1C, d), 124.4 (1C, s, JC-F ) 18.4 Hz), 124.3 (1C, s), 120.1 (1C,
d, JC-F ) 22.0 Hz), 118.0 (1C, d), 116.9 (1C, d), 73.7 (1C, s),
56.0 (1C, d), 48.1 (2C, t), 33.3 (2C, t), 29.9 (2C, t), 29.0 (2C, t),

622 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Hayashi et al.
27.1 (1C, t), 24.7 (2C, t), 22.9 (1C, q). IR (KBr): 3422, 2930, 1611,
1-[1-(1-Methylcyclooctyl)piperidin-4-yl]-2-[3-(trifluorometh-
yl)phenyl]-1H-benzimidazole (36). To a solution of N-[1-(1-
methylcyclooctyl)-4-piperidinyl]-1,2-benzenediamine 3 (250 mg,
0.792 mmol) in dry pyridine (3.5 mL) was added dropwise
3-(trifluoromethyl)benzoyl chloride (198 mg, 0.951 mmol) at room
temperature under N2. The resulting mixture was stirred at room
temperature overnight. The volatile materials were removed in
vacuo to give a black powder, which was dissolved in CH2Cl2 (8
mL), washed with saturated aqueous NaHCO3, dried over anhydrous
K2CO3, filtered, then concentrated in vacuo. Subsequently, to the
residue was added POCl3 (8 mL) under N2, and the reaction mixture
was stirred under reflux conditions for 4.5 h. After the mixture was
cooled to room temperature, the volatile materials were removed
in vacuo to give a dark-brown oil. The residue was dissolved in
CH2Cl2, washed with saturated aqueous NaHCO3 and brine, dried
over anhydrous K2CO3, filtered, and concentrated in vacuo. The
residue was purified by PTLC (silica gel, CH2Cl2/acetone ) 12/1)
to give 79.3 mg of the title compound 36 in 21% yield as a white
1487, 1468, 1269, 1086, 1061, 764, 644 cm-1
. Anal.
(C27H33N3FCl·2HCl·0.82H2O) C, H, N.
2-(1H-Indol-6-yl)-1-[1-(1-methylcyclooctyl)piperidin-4-yl]-1H-
benzimidazole (25). To a stirred solution of 1H-indole-6-carboxylic
acid (84.6 mg, 0.525 mmol) and N-[1-(1-methylcyclooctyl)-4-
piperidinyl]-1,2-benzenediamine 3 (157.8 mg, 0.500 mmol) in
anhydrous THF (3.5 mL) was added WSCI (124.6 mg, 0.650 mmol)
at -20 °C under N2. The reaction mixture was stirred at that
temperature under N2 for 10 min, warmed to 0 °C, stirred for 30
min, and then allowed to warm to room temperature. After being
stirred at room temperature for 3 days, the reaction mixture was
cooled to 0 °C, H2O (3 mL) and saturated aqueous NaHCO3 (3
mL) were added, then the mixture was extracted with CH2Cl2 (15
mL × 2). The combined extracts were dried over anhydrous
MgSO4, filtered, and concentrated in vacuo. Subsequently, the
residue was mixed with POCl3 (15 mL) under N2, and the reaction
mixture was stirred at 100 °C for 7 h and then cooled to room
temperature and concentrated in vacuo. The residue was diluted
with CHCl3 (20 mL), cooled to 0 °C, H2O (15 mL) was added,
and the mixture was basified by adding 25% aqueous ammonia.
The organic layer was separated, and the aqueous layer was
extracted with CHCl3 (15 mL × 3). The organic layers were
combined, dried over anhydrous MgSO4, filtered, and concentrated
in vacuo. The residue was purified by PTLC (silica gel, CH2Cl2/
MeOH ) 20/1) to give 98.0 mg of the title compound 25 in 44%
1
powder. H NMR (270 MHz, CDCl3) δ 7.95 (1H, s), 7.84-7.64
(5H, m), 7.33-7.30 (2H, m), 4.32-4.17 (1H, m), 3.13 (2H, br d,
J ) 12 Hz), 2.66-1.30 (20H, m), 0.82 (3H, s). MS (EI direct)
m/z: M+ 469. Compound 36 was converted into the corresponding
dihydrochloride: 13C NMR (150 MHz, CD3OD) δ 153.1 (1C, s),
139.4 (1C, s), 135.5 (1C, d), 134.0 (1C, s), 133.7 (1C, s, JC-F
33.3 Hz), 132.5 (1C, d), 130.4 (1C, d), 129.7 (1C, s), 128.9 (1C, d,
JC-F ) 3.6 Hz), 127.1 (1C, d), 127.1 (1C, d), 125.9 (1C, s, JC-F
)
)
271.6 Hz, CF3), 119.2 (1C, d), 116.3 (1C, d), 73.6 (1C, s), 55.6
(1C, d), 48.3 (2C, t), 33.4 (2C, t), 29.9 (2C, t), 29.1 (2C, t), 27.1
(1C, t), 24.7 (2C, t), 22.8 (1C, q). Anal. (C28H34N3F3 ·2HCl·0.7H2O)
C, H, N.
1
yield. H NMR (300 MHz, CDCl3) δ 10.59 (1H, br s), 8.01 (1H,
s), 7.87-7.71 (3H, m), 7.32-7.23 (4H, m), 6.56-6.55 (1H, m),
4.59-4.46 (1H, m), 3.08 (2H, d, J ) 11.4 Hz), 2.67-2.52 (2H,
m), 2.10-1.30 (18H, m), 0.79 (3H, s). Compound 25 was converted
into the corresponding dihydrochloride. IR (KBr): 3387, 2928, 1624,
1558, 1464, 1398, 1306, 1171, 1117, 943, 835, 762 cm-1. MS (ESI
positive) m/z: [M + H]+ 441. Anal. (C29H36N4 ·2HCl·1.8H2O) C,
H, N.
Ethyl (3-Formylphenoxy)acetate (38). To a solution of 3-hy-
droxybenzaldehyde (500 mg, 4.09 mmol) in dry CH3CN (5 mL)
was added anhydrous K2CO3 (679 mg, 4.91 mmol) followed by
ethyl 1-bromoacetate (684 mg, 4.09 mmol) at room temperature
under N2. The reaction mixture was stirred at 70 °C for 4 h, cooled
to room temperature, and then H2O was added. The mixture was
extracted with AcOEt (10 mL × 3). The combined extracts were
washed with saturated aqueous NaHCO3, dried over anhydrous
K2CO3, filtered, and concentrated to give 1.21 g of pale-yellow
oil. The residue was purified by flash column chromatography
(hexane/AcOEt ) 5:1) to give 890 mg of the title compound 38 in
quantitative yield as a colorless oil including small amount of
2-(1-Benzofuran-2-yl)-1-[1-(1-methylcyclooctyl)piperidin-4-
yl]-1H-benzimidazole (26). To a stirred solution of 1-benzofuran-
2-carboxylic acid (95.7 mg, 0.590 mmol) and N-[1-(1-methylcy-
clooctyl)-4-piperidinyl]-1,2-benzenediamine 3 (176.7 mg, 0.560
mmol) in anhydrous THF (4.0 mL) was added WSCI (139.6 mg,
0.728 mmol) at -20 °C under N2, and the reaction mixture was
stirred at the temperature for 20 min, then stirred at 0 °C for 1 h,
and then allowed to warm to room temperature. After being stirred
at room temperature for 5 days, the reaction mixture was suspended
with CHCl3 (2 mL) and saturated aqueous NaHCO3 (1 mL). The
mixture was filtered through Extrelut NT3 with CHCl3 (10 mL),
and the filtrate was concentrated in vacuo. Subsequently, the residue
was mixed with POCl3 (4.0 mL) under N2, and the reaction mixture
was stirred at 100 °C for 8 h in a sealed tube, and then cooled to
room temperature, and concentrated in vacuo. The residue was
diluted with CHCl3 (4 mL), cooled to 0 °C, basified by adding
25% aqueous ammonia. The mixture was filtered through Extrelut
NT3 with CHCl3 (10 mL), then the filtrate was concentrated in
vacuo. The residue was purified by flash column chromatography
(silica gel, hexane/CH2Cl2/acetone ) 7/1/1) followed by PTLC
purification (silica gel, hexane/acetone ) 5/1) to give 53.9 mg of
the title compound 26 in 22% yield. 1H NMR (300 MHz, CDCl3)
δ 7.88-7.82 (1H, m), 7.78-7.73 (1H, m), 7.72-7.69 (1H, m),
7.64-7.60 (1H, m), 7.43-7.29 (5H, m), 4.93-4.82 (1H, m), 3.19
(2H, d, J ) 11.9 Hz), 2.60 (2 H, dq, J ) 12.3 Hz, J ) 3.66 Hz),
2.28-2.20 (2H, m), 2.04-1.33 (16H, m), 0.88 (3H, s). Compound
26 was converted into the corresponding dihydrochloride: 13C NMR
(150 MHz, CD3OD) δ 158.2 (1C, s), 143.2 (1C, s), 140.1 (1C, s),
135.0 (1C, s), 133.3 (1C, s), 130.6 (1C, d), 129.4 (1C, s), 128.9
(1C, d), 128.4 (1C, d), 126.7 (1C, d), 125.2 (1C, d), 117.7 (1C, d),
117.6 (1C, d), 117.1 (1C, d), 114.0 (1C, d), 73.7 (1C, s), 56.9 (1C,
d), 48.4 (2C, t), 33.4 (2C, t), 29.9 (2C, t), 28.8 (2C, t), 27.1 (1C,
t), 24.8 (2C, t), 23.1 (1C, q). IR (KBr): 3406, 2924, 2654, 1612,
1466, 1443, 1344, 1306, 1259, 1229, 1192, 1144, 1109, 1076, 1009,
947, 889, 839, 752, 648, 615 cm-1. MS (ESI positive) m/z: [M +
H]+ 442. Anal. (C29H35N3O·2HCl·0.5H2O) C, H, N.
1
1
AcOEt based on H NMR. H NMR (270 MHz, CDCl3) δ 9.97
(1H, s), 7.53-7.44 (2H, m), 7.37 (1H, s), 7.27-7.22 (1H, m), 4.69
(2H, s), 4.29 (2H, q, J ) 7.1 Hz), 1.33-1.25 (3H, m).
Ethyl (3-{1-[1-(1-Methylcyclooctyl)piperidin-4-yl]-1H-benz-
imidazol-2-yl}phenoxy)acetate (39). To a solution of ethyl (3-
formylphenoxy)acetate 38 (198 mg, 0.951 mmol) in EtOH (6 mL)
was added N-[1-(1-methylcyclooctyl)-4-piperidinyl]-1,2-benzene-
diamine 3 (300 mg, 0.951 mmol) at room temperature under N2.
The resulting solution was stirred under reflux conditions for 1 h.
The volatile materials were removed in vacuo to give black oil,
which was diluted with CH2Cl2 (10 mL). The organic layer was
washed with saturated aqueous NaHCO3, dried over anhydrous
K2CO3, filtered, and concentrated in vacuo. Subsequently, the
residue was dissolved in dry benzene (6 mL), Pb(OAc)4 (464 mg,
1.05 mmol) was added under N2, then the mixture was stirred under
reflux conditions for 1 h.52 After the mixture was cooled, CH2Cl2
(10 mL) was added to the mixture, which was then washed with
saturated aqueous NaHCO3, dried over anhydrous K2CO3, filtered,
and concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, CH2Cl2/MeOH ) 20/1) followed by
PTLC purification (silica gel, CH2Cl2/MeOH ) 20/1) to give 304.6
1
mg of the title compound 39 in 64% yield as a white powder. H
NMR (270 MHz, CDCl3) δ 7.82-7.73 (2H, m), 7.43-7.07 (6H,
m), 4.69 (2H, s), 4.31-4.23 (3H, m), 3.10 (2H, br d, J ) 10 Hz),
2.60-1.27 (23H, m), 0.82 (3H, s).
2-(3-{1-[1-(1-Methylcyclooctyl)piperidin-4-yl]-1H-benzimida-
zol-2-yl}phenoxy)ethanol (40). To a solution of ethyl (3-{1-[1-
(1-methylcyclooctyl)piperidin-4-yl]-1H-benzimidazol-2-yl}phe-
noxy)acetate 39 (304 mg, 0.604 mmol) in anhydrous THF (6 mL)

Nociceptin/Orphanin FQ Receptor Agonist
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3 623
was added LAH (23.0 mg, 0.606 mmol) at 0 °C under N2. The
reaction mixture was stirred for 15 min at the temperature, then
stirred at room temperature overnight. To the reaction mixture was
added dropwise H2O (23 µL), 15% aqueous NaOH (23 µL), and
H2O (23 µL × 3). The resulting mixture was diluted with THF (20
mL), filtered through a Celite pad, and the filtrate was concentrated
in vacuo. The residue was purified by flash column chromatography
(silica gel, CH2Cl2/MeOH ) 20/1) to give 228.4 mg of the title
Research & Development Nagoya Laboratories. The animal work
was also approved by the Pfizer Institutional Animal Care and Use
Committee (IACUC).
In Vitro Characterization of NOP Receptor Agonists.54 In
vitro studies on NOP receptor binding affinities and functional
efficacies ([35S]GTPγS assays) as well as on other opioid (µ-, κ-,
and δ-) receptors binding affinities and functional efficacies were
conducted.
1
compound 40 in 82% yield as a white solid. H NMR (270 MHz,
Materials. The human NOP receptor transfected human embry-
onic kidney (HEK)-293 cell membranes, the human µ-receptor
transfected Chinese hamster ovary (CHO)-K1 cell membranes, the
human κ-receptor transfected HEK-293 cell membranes, and the
human δ-receptor transfected CHO-K1 cell membranes were
purchased from Receptor Biology Inc. [3H]N/OFQ (150 Ci/mmol),
[3H]enadoline (45.0 Ci/mmol), [35S]GTPγS (1060-1150 Ci/mmol),
and wheatgerm agglutinin (WGA)-scintillation proximity assay
(SPA) beads were obtained from Amersham Pharmacia Biotech
K.K. [3H]DAMGO (54.0 Ci/mmol) and [3H]DPDPE (45.0 Ci/
mmol) were provided from NEN Life Science Products Inc. N/OFQ
was from Peptide Institute Inc. DAMGO and DPDPE were from
Sigma Chemical.
Evaluation of Receptor Binding Affinities to NOP Receptor
and Other Opioid (µ-, K-, and δ-) Receptors. All competitive
displacement analyses (IC50 and Ki) for the NOP receptor as well
as the µ-, κ-, and δ-receptors were performed in duplicate in a 96-
well plate using a scintillation proximity assay (SPA). After the
reaction, the assay plate was centrifuged at 1000 rpm for 1 min,
then the radioactivity was measured by a 1450 MicroBeta (Wallac)
liquid scintillation counter. IC50 values were calculated by nonlinear
regression with the software GraphPad Prism, version 4.0 (Graph-
Pad Software, Inc., San Diego, CA). Ki value was calculated by
the following equation: Ki ) IC50/(1 + [L]/KD), where [L] is the
radiolabeled ligand concentration and KD is the dissociation
constant.55
CDCl3) δ 7.81-7.73 (2H, m), 7.43-7.07 (6H, m), 4.30 (1H, m),
4.18-4.14 (2H, m), 4.01-3.95 (2H, m), 3.15-3.03 (2H, m),
2.75-2.42 (2H, m), 2.18-1.30 (19H, m), 0.82 (3H, s). MS (EI
direct) m/z: M+ 461. Compound 40 was converted into the
corresponding trihydrochloride: 13C NMR (150 MHz, CD3OD) δ
162.0 (1C, s), 153.4 (1C, s), 135.2 (1C, s), 133.1 (1C, s), 133.1
(1C, d), 128.4 (1C, d), 128.0 (1C, d), 126.9 (1C, s), 124.0 (1C, d),
121.1 (1C, d), 117.9 (1C, d), 117.2 (1C, d), 117.2 (1C, d), 73.7
(1C, s), 72.0 (1C, t), 62.4 (1C, t), 56.2 (1C, d), 48.2 (2C, t), 33.3
(2C, t), 29.9 (2C, t), 29.0 (2C, t), 27.1 (1C, t), 24.7 (2C, t), 22.9
(1C, q). IR (KBr): 3387, 2930, 2719, 1466, 1242, 1173, 1045, 955,
766, 694 cm-1. Anal. (C29H39N3O2 ·2HCl·1.2H2O) C, H, N.
3-{1-[1-(1-Methylcyclooctyl)piperidin-4-yl]-1H-benzimidazol-
2-yl}benzonitrile (42). To a mixture of N-[1-(1-methylcyclooctyl)-
4-piperidinyl]-1,2-benzenediamine 3 (300 mg, 0.951 mmol) and
3-cyanobenzoyl chloride (189 mg, 1.14 mmol) was added dry
pyridine (4 mL) at room temperature under N2. The resulting
mixture was stirred at room temperature overnight. The volatile
materials were removed in vacuo. The residue was dissolved in
CH2Cl2 (25 mL), washed with saturated aqueous NaHCO3 (10 mL),
dried over anhydrous K2CO3, filtered, then concentrated in vacuo.
Subsequently, the resulting solid was dissolved in POCl3 (10 mL)
under N2, and the reaction mixture was stirred under reflux
conditions for 4 h. After the mixture was cooled to room
temperature, the volatile materials were removed in vacuo. The
residue was dissolved in CH2Cl2 (20 mL), washed with saturated
aqueous NaHCO3 and brine, dried over anhydrous K2CO3, filtered,
then concentrated in vacuo. The residue was purified by flash
column chromatography (silica gel, CH2Cl2/MeOH ) 60/1 to 30/
1) to give 186.2 mg of the title compound 42 in 46% yield as a
white powder. 1H NMR (270 MHz, CDCl3) δ 7.95 (1H, s),
7.90-7.63 (5H, m), 7.35-7.30 (2H, m), 4.27-4.12 (1H, m), 3.14
(2H, br d, J ) 12 Hz), 2.70-2.52 (2H, m), 2.14-1.31 (18H, m),
0.83 (3H, s).
NOP Receptor Binding Assay. Methods. The NOP membranes
(8.3 µg) were incubated for 45 min at 22 °C with 0.4 nM [3H]N/
OFQ, 1.0 mg of WGA-SPA beads, and six different concentrations
of compounds (10-11-10-5 M, 10-fold) in a final volume of 0.2
mL of 50 mM HEPES buffer, pH 7.4, containing 10 mM MgCl2
and 1 mM EDTA. Nonspecific binding was determined by the
addition of 1 µM unlabeled N/OFQ. Under these conditions,
approximately 900 cpm of total binding were obtained, of which
30 cpm were nonspecific.
µ-Receptor Binding Assay. Methods. The µ-membranes (18
µg) were incubated for 45 min at 25 °C with 1.0 nM [3H]DAMGO,
1.0 mg of WGA-SPA beads, and six different concentrations of
compounds (10-fold) in a final volume of 0.2 mL of 50 mM Tris-
HCl buffer, pH 7.4, containing 5 mM MgCl2. Nonspecific binding
was determined by the addition of 1 µM of unlabeled DAMGO.
Under these conditions, approximately 240 cpm of total binding
were obtained, of which 23 cpm were nonspecific.
K-Receptor Binding Assay. Methods. The κ-membranes (13
µg) were incubated for 45 min at 22 °C with 0.5 nM [3H]enadoline,
1.0 mg of WGA-SPA beads, and six different concentrations of
compounds (10-fold) in a final volume of 0.2 mL of 50 mM Tris-
HCl buffer, pH7.4, containing 10 mM MgCl2 and 1 mM EDTA.
Nonspecific binding was determined by the addition of 1 µM
unlabeled enadoline. Under these conditions, approximately 270
cpm of total binding were obtained, of which 15 cpm were
nonspecific.
1-(3-{1-[1-(1-Methylcyclooctyl)piperidin-4-yl]-1H-benzimida-
zol-2-yl}phenyl)methanamine (43). To 3-{1-[1-(1-methylcyclooc-
tyl)piperidin-4-yl]-1H-benzimidazol-2-yl}benzonitrile 42 (136 mg,
0.319 mmol) in anhydrous THF (1.6 mL) was added LAH (12.0
mg, 0.316 mmol) at 0 °C under N2. The reaction mixture was stirred
for 5 min at that temperature, then stirred at room temperature
overnight. To the reaction mixture was added dropwise saturated
aqueous NH4Cl, then the mixture was extracted with CH2Cl2 (10
mL × 3). The combined extracts were dried over anhydrous K2CO3,
filtered, and concentrated in vacuo. The residue was purified by
PTLC (silica gel, CH2Cl2/MeOH/acetone ) 1/1/1) to give 73.6 mg
as a pale-yellow oil. The residue was recrystallized from
CH2Cl2-hexane to afford 35.2 mg of the title compound 43 in 26%
yield as a white powder. 1H NMR (270 MHz, CDCl3) δ 7.85-7.73
(2H, m), 7.67 (1H, s), 7.52-7.25 (5H, m), 4.42-4.28 (1H, m),
3.97 (2H, s), 3.10 (2H, br d, J ) 10.8 Hz), 2.67-2.49 (2H, m),
2.16-1.23 (20H, m), 0.81 (3H, s). Compound 43 was converted
into the corresponding trihydrochloride: 13C NMR (150 MHz,
CD3OD) δ 153.8 (1C, s), 139.0 (1C, s), 136.8 (1C, s), 134.3 (1C,
d), 134.0 (1C, s), 132.6 (1C, d), 132.5 (1C, d), 132.2 (1C, d), 129.1
(1C, s), 127.1 (1C, d), 127.0 (1C, d), 118.8 (1C, d), 116.4 (1C, d),
73.6 (1C, s), 55.5 (1C, d), 48.3 (2C, t), 44.7 (1C, q), 33.4 (2C, t),
29.9 (2C, t), 29.2 (2C, t), 27.2 (1C, t), 24.7 (2C, t), 23.1 (1C, q).
IR (KBr): 3427, 2930, 2926, 2748, 2363, 773, 700 cm-1. Anal.
(C28H38N4 ·3HCl·3.6H2O) C, H, N.
δ-Receptor Binding Assay. Methods. The δ-membranes (18
µg) were incubated for 45 min at 22 °C with 2.0 nM [3H]DPDPE,
1.0 mg of WGA-SPA beads, and six different concentrations of
compounds (10-fold) in a final volume of 0.2 mL of 50 mM Tris-
HCl buffer, pH 7.4, containing 5 mM MgCl2. Nonspecific binding
was determined by the addition of 1 µM unlabeled DPDPE. Under
these conditions, approximately 800 cpm of total binding were
obtained, of which 70 cpm were nonspecific.
Biology. General. All animal experiments were conducted
according to the guideline of animal care and use, and all procedures
were approved by the Animal Ethics Committee in Pfizer Global
Evaluation of Functional Activity: [35S]GTPγS Binding
Assays. Methods. Agonist stimulated binding of [35S]GTPγS was
investigated according to the method of SPA G-protein-coupled

624 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 3
Hayashi et al.
receptor assay provided by Amersham Pharmacia Biotech with
slight modification. Each human NOP, µ-, κ-, and δ-receptor
expressing cell membrane was suspended in assay buffer (20 mM
HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 5 µM GDP,
1 mM DTT, pH 7.4). The membranes (the same amount as for
their respective receptor binding assays) were incubated for 30 min
at 22 °C with 0.4 nM [35S]GTPγS, 1.5 mg WGA-SPA beads, and
various concentrations of compounds in duplicate in a 0.2 mL total
volume. After the reaction, the assay plate was centrifuged at 1000
rpm for 2 min, and then the radioactivity was measured by a 1450
MicroBeta (Wallac) liquid scintillation counter. Nonspecific binding
was assessed with 10 µM of unlabeled GTPγS. Agonist stimulated
binding was determined as the difference between total binding in
the presence of compound and basal binding determined in the
absence of compound. EC50 (potency) value was the concentration
producing a half-maximal response of its own. Emax (efficacy) value
was the maximal response calculated as the percentage of the
maximal response produced by each control (N/OFQ, DAMGO,
enadoline, and DPDPE). All the binding data were analyzed by
nonlinear regression with the software GraphPad Prism, version
4.0 (GraphPad Software, Inc., San Diego, CA).
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is available free of charge via the Internet at http://pubs.acs.org.

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