Identification of a novel spiropiperidine opioid receptor-like 1 antagonist class by a focused library approach featuring 3D-pharmacophore similarity

Article abstract of DOI:10.1021/jm0509851

A focused library approach identifying novel leads to develop a potent ORL1 antagonist is described. Beginning from a compound identified by random screening, an exploratory library that exhibited a diverse display of pharmacophores was designed. After evaluating ORL1 antagonistic activity, a highly focused library was designed based on 3D-pharmacophore similarity to known actives. A novel D-proline amide class was identified in this library and was found to possess potent ORL1 antagonistic activity.

Full text of DOI:10.1021/jm0509851

J. Med. Chem. 2006, 49, 847-849
847
Scheme 1. Design of Libraries Focusing on Pharmacophore
Similarity
Identification of a Novel Spiropiperidine Opioid
Receptor-like 1 Antagonist Class by a Focused
Library Approach Featuring 3D-Pharmacophore
Similarity
Yasuhiro Goto, Sachie Arai-Otsuki, Yukari Tachibana,
Daisuke Ichikawa, Satoshi Ozaki, Hiroyuki Takahashi,
Yoshikazu Iwasawa, Osamu Okamoto, Shoki Okuda,
Hisashi Ohta, and Takeshi Sagara*
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd.,
Okubo 3, Tsukuba 300-2611, Japan
ReceiVed October 4, 2005
Abstract: A focused library approach identifying novel leads to
develop a potent ORL1 antagonist is described. Beginning from a
compound identified by random screening, an exploratory library that
exhibited a diverse display of pharmacophores was designed. After
evaluating ORL1 antagonistic activity, a highly focused library was
designed based on 3D-pharmacophore similarity to known actives. A
novel D-proline amide class was identified in this library and was found
to possess potent ORL1 antagonistic activity.
Scheme 2. Preparation of Libraries
Table 1. Hit Compounds from the Diverse Library
The fourth opioid receptor, opioid receptor-like 1 (ORL1),
was discovered in 1994 by homology cloning.1 Subsequently,
its endogenous agonist, a 17-amino acid peptide termed noci-
ceptin or orphanin FQ (NC/OFQ), was identified.2 Pharmaco-
logical studies using NC/OFQ and ORL1-deficient mice showed
that the NC/OFQ-ORL1 system may have important roles in
the regulation of pain response,3 morphine tolerance,4 learning
and memory,5 food intake,6 anxiety,7 the cardiovascular system,8
locomotor activity,9 and so on.10
These results prompted many pharmaceutical companies to
identify potent and selective ORL1 agonists and antagonists.
However, few structural classes were reported as ORL1
antagonists.11 Therefore, additional structurally diverse ORL1
antagonists with distinct physicochemical properties and im-
proved antagonistic activity from known ORL1 antagonists are
required for better understanding of the physiological roles of
ORL1 receptors and for examining the therapeutic potential of
its antagonists.
As previously reported, we identified a small molecular ORL1
antagonist, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-
piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (1),12 and
the search for structurally diverse ORL1 antagonists among our
in-house chemical collection led to identification of spiropip-
eridine analogue 2, which exhibited moderate but complete
ORL1 antagonistic activity (IC50: 8.3 nM in GTPγS assay).
a Displacement of [125I]Tyr14-nocicpetin binding from the CHO cells
stably expressing cloned human ORL1 receptors.
binding activity and the optimal distance between the two
pharmacophores by investigating a range of aromatic groups
in the hydrophobic acyl part of 2. In addition, introduction of
hydrophilicity was attempted using this library approach in an
effort to reduce the high hydrophobicity of 2 (log D >5).
The library was designed as follows. First, a virtual library
was constructed by combining amine 3 possessing spiropiperi-
dine and n-propyl spacer group and all available aromatic COOH
building blocks in the company reagent database. From this
virtual library of 480 compounds, the diverse library (36
compounds)13 was selected by visual inspection to exhibit a
diverse 3D distribution of aromatic groups in the acyl region.
To simplify understanding of the required pharmacophore
On the basis of preliminary SAR analysis of analogues in
our chemical collection, an aromatic ring and cationic amine
group in the spiropiperidine portion and an additional aromatic
ring in the ortho position of terminal benzoic acid were predicted
to be important pharmacophores for antagonistic activity in
ORL1 receptors. We wanted to examine the relationship between
* To whom correspondence should be addressed. Phone: 81-29-877-
2000. Fax: 81-29-877-2029. E-mail: takeshi_sagara@merck.com.
10.1021/jm0509851 CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/19/2006

848 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Letters
Table 2. Hit Compounds (purified) from Diverse and Focused Libraries
a Displacement of [125I]Tyr14-nociceptin, b [3H]diprenorphin, c [3H]U-69593, d [3H]naltrindole binding from CHO cells stably expressing cloned human
ORL1, opioid µ-, opioid κ-, and opioid δ-receptors, respectively. e IC50 values on nociceptin-produced [35S]GTPγS binding to ORL1-expressed in CHO
cells. f EC50 values relative to the maximal [35S]GTPγS binding produced by nociceptin in ORL1 expressed CHO cells. All values are means of three
independent determinations performed in duplicate.
Table 3. In Vitro and in Vivo Profiles for ORL1 Antagonist 24
binding affinity lC50 (nM)
GTPγS
brain penetrability
antagonism
agonism
in vivo antagonism
(% reversal)g
plasma
brain levels
cmpd
ORL1a
µb
κc
δd
IC50 (nM)e
EC50 (nM)f
(nM)h
0.88
(nmol/g brain)h
24
0.27 ( 0.038
6700 ( 4300
2500 ( 200
>10000
0.15 ( 0.0067
>1000
88 ( 9.7%
4.37
a Displacement of [125I]Tyr14-nociceptin, b [3H]diprenorphin, c [3H]U-69593, d [3H]naltrindole binding from CHO cells stably expressing cloned human
ORL1, opioid µ-, opioid κ-, and opioid δ-receptors, respectively. e IC50 values for nociceptin-produced [35S]GTPγS binding to ORL1-expressed in CHO
cells. f EC50 values relative to the maximal [35S]GTPγS binding produced by nociceptin in ORL1-expressed CHO cells. a-f All values are means of three
independent determinations performed in duplicate. g Data shows antagonistic activity of analogue 24 (10 mg/kg, sc) against the reduction in locomotor
activity produced by an ORL1 agonist for 60 min in mice. Values are expressed as % reversal of the agonist response (mean ( SEM). h Plasma and blains
of rats were collected 1 h after the drug administration (10 mg/kg, sc) and drug concentrations were measured (n ) 3 mice/group).
geometry, only simple aromatic COOH building blocks without
additional pharmacophores (i.e., H-bonding donor or acceptor)
were selected. In addition, as the n-propyl spacer is a highly
flexible structure, less flexible COOH building blocks were
selected with higher priority.
After testing the diverse library, the focused library was
designed based on 3D-pharmacophore similarity to known
actives. From the remainder of the virtual library, analogues
that showed similar pharmacophore orientation to 2 and hit
compounds from the diverse library were selected using a
computational method (20 compounds).13 In this case, aromatic
COOH building blocks with heteroatoms were employed with
higher priority in order to decrease the hydrophobicity of the
molecule.
It should be noted that additional aromatic COOH building
blocks were designed to show similar pharmacophore orientation
to known actives and prepared by simple modification of
commercially available reagents in order to enrich the structural
variation of the original virtual library. Eleven aromatic COOH
reagents13 were thus prepared as custom-made building blocks
and were added to the focused library.
Analogues were prepared by simple condensation reaction
performed in parallel (Scheme 2), and all samples were tested
by LC-MS and were confirmed to show more than 85% purity.
Library members were tested for their inhibitory effects on
ligand binding to the human ORL1 receptors and on GTPγS
binding to proteins using membrane fractions of CHO cells
expressing ORL1. Binding affinities for ORL1 were determined
by displacement of [125I]Tyr14-NC/OFQ, and agonist/antagonist
activities were measured by the [35S]GTPγS binding method.14
Cross reactivity to other opioid receptors was also tested.
Affinities for human µ-, κ-, and δ-receptors were assayed
similarly to ORL1 using membrane fractions of CHO cells
expressing in each receptor.
The results of representative library members are summarized
in Table 1. From the diverse library, compounds 12 and 14
showed comparable binding activity (IC50 <100 nM) to 2. On
the basis of structure-activity relationship analysis of the diverse
library set, it was clear that particular pharmacophore geometry
in the acyl part was required for good affinity. The regio- and
stereochemistry in the acyl part (compounds 12 vs 13 and 14
vs 15) were important for ORL1 binding. Consequently, in the

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 849
References
focused library, analogues showing similar pharmacophore
geometry to compounds 2, 12, and 14 were prioritized and
tested.
Compounds with IC50 values of <100 nM from both the
diverse and focused libraries were purified and subjected to
further characterization. Binding activities for ORL1, µ-, κ-, and
δ-receptors and functional activities for ORL1 receptors of
purified compounds are summarized in Table 2.
All purified compounds showed potent ORL1 antagonistic
activity and no agonistic activity. Among these, N-benzyl-D-
proline analogue 23 showed substantially improved binding and
antagonistic activity when compared with 2. The stereochemistry
of the proline structure was very important to potency (23 vs
22), and thus 23 was thought to successfully meet the required
pharmacophore geometry by forming a stereospecific interaction
with ORL1 receptor. Analogue 23 showed a 12.5-fold higher
antagonistic activity than 1 and a more than 4000-fold higher
selectivity over µ-, κ-, and δ-receptors. In addition, by introduc-
ing an additional tert-amino structure in the acyl moiety, 23
showed decreased hydrophobicity (log D of 23 and 2 were 2.7
and 3.1, respectively).
Analogue 23 showed novel structural features when compared
with reported ORL1 antagonists, and thus we have identified a
novel class of ORL1 antagonist with substantially improved
antagonistic activity by searching chemotypes of random
screening hit 2.
In the course of our medicinal chemistry efforts to assess the
potential of N-benzyl-D-proline derivatives as leads for CNS
drugs, close analogue 24 with an isobenzofuran structure in the
spiropiperidine portion was prepared.
Analogue 24 showed comparable activity and selectivity as
23 and good brain permeability (Table 3). In addition, 24
showed statistically significant in vivo antagonistic activity
against the reduction in locomotor activity produced by an ORL1
agonist (control; n ) 7: 1844 ( 104 counts/1 h, agonist; n )
7: 187 ( 20counts/1 h, agonist + 24 (3 mg/kg); n ) 8: 1619
( 168 counts/1 h (p < 0.05 from agonist treated group), agonist
+ 24 (10 mg/kg); n ) 8: 1647 ( 160 counts/1 h, (p < 0.05
from agonist treated group)).13
In conclusion, we discovered a novel class of ORL1
antagonist using a focused library approach starting from a
moderately active hit compound found in our chemical collec-
tion. The N-benzyl-D-proline analogue showed significantly
improved antagonistic activity when compared with other
reported ORL1 antagonists and showed good brain penetrability
and in vivo antagonistic activity. This newly identified class
may serve as useful pharmacological tool in vivo to investigate
the physiological roles of the NC/OFQ-ORL1 system as well
as the therapeutic potential of ORL1 antagonists. In particular,
its significantly improved potency, appropriate selectivity, and
hydrophilicity would be suitable for development of a PET tracer
to examine the in vivo pharmacodynamics of ORL1 antagonists.
Further details of the SAR and pharmacological profiles of the
analogues will be discussed in due course.
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Supporting Information Available: Building blocks for fo-
cused and diverse libraries, synthetic procedures, pharmacophore
similarity search, and in vivo pharmacological experiment. This
material is available free of charge via the Internet at http://
pubs.acs.org.
JM0509851