Identification of novel benzimidazole series of potent and selective ORL1 antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.04.054

Structure-activity studies on benzimidazole lead 1 obtained from library screening led to the discovery of potent and selective ORL1 antagonist 28, 5-chloro-2-[(1-ethyl-1-methylpropyl)thio]-6-[4-(2-hydroxyethyl)piperazin-1-yl]-1H-benzimidazole, which is s

Full text of DOI:10.1016/j.bmcl.2008.04.054

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3278–3281
Identification of novel benzimidazole series of potent and selective
ORL1 antagonists
Osamu Okamoto,* Kensuke Kobayashi, Hiroshi Kawamoto, Satoru Ito, Atsushi Satoh,
Tetsuya Kato, Izumi Yamamoto, Sayaka Mizutani, Masaya Hashimoto, Atsushi Shimizu,
Hiroki Sakoh, Yasushi Nagatomi, Yoshikazu Iwasawa, Hiroyuki Takahashi,
Yasuyuki Ishii, Satoshi Ozaki and Hisashi Ohta
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co. Ltd., Okubo-3, Tsukuba, Ibaraki 300-2611, Japan
Received 12 December 2007; revised 19 March 2008; accepted 19 April 2008
Available online 25 April 2008
Abstract—Structure–activity studies on benzimidazole lead 1 obtained from library screening led to the discovery of potent and
selective ORL1 antagonist 28, 5-chloro-2-[(1-ethyl-1-methylpropyl)thio]-6-[4-(2-hydroxyethyl)piperazin-1-yl]-1H-benzimidazole,
which is structurally distinct from conventional non-peptide antagonists known to date.
Ó 2008 Elsevier Ltd. All rights reserved.
Opioid receptor like 1 (ORL1) is a G-protein-coupled
receptor with a high degree of amino acid sequence
homology to the classical opioid receptors. Since the
identification of nociceptin (orphanin FQ or NC/OFQ)
as the endogenous ligand of ORL1,1,2 a number of
reports have demonstrated the possible involvement of
the NC/OFQ-ORL1 system in pain regulation,3 cogni-
tion,4–6 anxiety7 and cardiovascular function.8,9 Until
now, few classes of non-peptidic ORL1 antagonists with
fairly good selectivity towards opioid receptors have
been developed,10–12 namely benzimidazolyl piperi-
dines,13,14 spiropiperidines15 and 4-aminoquinolines.16
The biological role of the ORL1 receptor and therapeu-
tic roles for ORL1 antagonists will be established
through the development of drug-like molecules with
good bioavailability and good brain penetration. Here,
we report the discovery and SAR study of a novel series
of selective ORL1 antagonists. A sample collection
screening directed at identifying new and structurally di-
verse leads provided 6-(piperazin-1-yl) benzimidazole 1
as a sub-micromolar receptor antagonist. Modification
of the 6-piperazinyl benzimidazole lead and their effects
on in vitro measures of ORL1 antagonism are also
addressed.
6-(Piperazin-1-yl)-benzimidazole derivatives (F) were
synthesized according to the general procedure illus-
trated in Scheme 1. N-substituted piperazines were con-
densed with 5-fluoro-2-nitroaniline (A) in the presence
of N,N-diisopropylethylamine at 140 °C to afford the de-
sired N-phenylpiperazines (B). Reduction of the nitro
function with Fe followed by condensation with carbon
disulfide gave the key intermediate benzimidazole 2-thi-
one (D). Alkylation of D with appropriate alkyl and
arylalkyl halides in the presence of potassium carbonate
provided the corresponding thioether analogs (F). Com-
pounds derived from N-Boc piperazine were deprotected
under acidic conditions. Analogs incorporating a ter-
NH
R2
N
NH2
NH2
R2
F
NO2
NH2
R2
N
NO2
NH2
N
R3
b
a
N
S
R3
N
N
R3
A
B
C
H
R2
N
R2
N
R1
S
c
d or e
N
H
N
H
N
N
N
R3
D
F
R3
Scheme 1. Synthesis of 6-(piperazin-1-yl)-benzimidazole derivatives.
Reagents and conditions: (a) iPr2NEt, DMSO; (b) Fe, NH4Cl, THF–
MeOH–H2O; (c) CS2, 1 N NaOH, EtOH; (d) R1-X, K2CO3, DMF; (e)
R1-OH, TFA.
Keywords: Nociceptin/orphanin FQ; Nociceptin receptor (NOP);
Opioid receptor-like 1 (ORL1) antagonist.
*
Corresponding author. E-mail: osamu_okamoto@merck.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.054

O. Okamoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3278–3281
3279
Table 2. Binding affinity and functional activity of 4 and its analogs at
human ORL1 receptor
tiary carbon adjacent to the sulfur atom were synthe-
sized by coupling the benzimidazole 2-thione with the
corresponding tertiary alcohol in TFA at room
temperature.
F
N
R
N
H
N
Analogs were tested for their inhibitory effects on ligand
binding to the human ORL1 receptor, and on GTPcS
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]GTPcS binding method.17 Cross reactivity to other
opioid receptors was also tested. Affinities for human l-,
j- and d-receptors were assayed similarly to ORL1 using
membrane fractions of CHO cells expressing each
receptor.
HN
Compound
R
Binding*
IC50 (nM)
Antagonism*
IC50 (nM)
F
4
63
S
S
5
6
2900
140
Me
S
Structural modification was performed at three individ-
ual sites of the benzimidazole core of compound 1. Our
initial interest was to examine the effects of substituents
at the piperazine nitrogen atom (Table 1).18 It is known
that all the reported small-molecule ORL1 ligands
incorporating a piperidine framework have a bulky sub-
stituent at the nitrogen, such as cyclooctylmethyl, naph-
thylmethyl and acenaphthenyl groups, which confers
high affinity for the ORL1 receptor.10–12 In contrast to
these reports, introduction of a bulky substituent at
the piperazine nitrogen in our benzimidazole core re-
sulted in a drastic loss of the binding affinity for
ORL1 (2 and 3 vs 1). N-unsubstituted piperazine ana-
logue 4 showed the best binding affinity. This suggested
that the new series represents a different type of ORL1
antagonist.
Me
S
7
78
1700
Me
8
9
180
31
S
S
Me
Cl
S
10
23
380
Cl
S
11
12
13
55
2700
Cl
Cl
We then directed our SAR efforts towards the modifi-
cation of the thioether portion using the N-unsubsti-
tuted piperazine analogue 4 as a template (Table
2).18 Replacement of p-fluorobenzylthioether with
methylthioether resulted in a significant decrease in
potency for ORL1 (Compound 5); further exploration
of the SAR around the p-fluorobenzylthioether 4 was
conducted using commercially available benzyl halides
for exploration of preliminary SAR (6–12). We found
that 2,3-dichlorobenzyl derivative 12 appeared to be
the most potent antagonist among the derivatives syn-
thesized. It is likely that some structural bulkiness in
the thioether part is essential for the antagonistic
activity. Conversion of the thioether to sulfone or
Cl
S
6.0
140
Cl
Cl
Cl
2900
S
O
Cl
O
S
14
15
240
17
O
930
690
S
S
Me
16
4.7
Table 1. Effects of N-substitution of the piperazine ring on binding
affinity at human ORL1 receptor
Me
* n = 1 (Ref. 18).
F
F
sulfoxide was found to markedly decrease the potency
for ORL1 (13 and 14).
N
S
N
H
N
N
R
We then incorporated bulky branched alkyl groups
into this region in order to examine its steric effect.
The cyclohexylmethyl analogue 15 and (1-ethylpro-
pyl)thioether analogue 16 showed good binding affin-
ity for ORL1 but a loss in antagonist activity. Next,
we focused on the effects of 5-substitution (X) of the
benzimidazole ring (Table 3).18 Replacement of the
Compound
R
Binding* IC50 (nM)
1
2
3
4
Me
Benzyl
910
8200
Cyclooctylmethyl
H
>10000
63
* n = 1 (Ref. 18).

3280
O. Okamoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3278–3281
Table 3. Effects of 5-substitution of the benzimidazole ring on binding affinity and functional activity at human ORL1 receptor
Me
Me
X
N
N
S
N
H
N
R
Compound
R
X
Binding* IC50 (nM)
Antagonism* IC50 (nM)
690
16
17
18
19
20
21
22
H
F
4.7
320
3100
15
H
H
H
Ac
CN
Cl
Cl
Cl
H
78
H
Et
2.1
10
9.1
3.9
3.3
Hydroxyethyl
9.7
* n = 1 (Ref. 18).
Table 4. Binding affinity and functional activity of 22 and its analogs at human ORL1 receptor
Cl
N
S R
N
H
N
N
HO
Compound
R
Binding* IC50 (nM)
Antagonism* IC50 (nM)
Binding IC50 (nM)**
l
j
d
Me
Me
22
23
24
9.7
120
17
3.3
3100
60
>1000
Me
Me
3100
4400
60
NT
NT
Me
Me
21
120
Me
Me
Me
25
5.6
2.5
3500
160
NT
Me
26
27
20
20
520
31
88
NT
NT
Me
Me
1.8
1.8
1400
Me
28
1.2
0.66
2800
170
>1000
Me
* n = 1 (Ref. 18).
** Displacement of [3H]diprenorphin (l), [3H]U-69593 (j and [3H]naltrindole for (d) binding in CHO cells stably expressing cloned human opioid l-,
opioid j- and opioid d-receptors, respectively. n = 1.
fluorine atom with a hydrogen (17) or an acetyl (18)
resulted in a loss of potency for ORL1. Binding affin-
ity for ORL1 was recovered with a cyano (19) or a
chloride (20–22), resulting in excellent functional activ-
ity for ORL1 (ORL1 IC50 = 3–9 nM). The increase in
potency can be explained by the torsion angle between
the benzimidazole core ring and the piperazine ring
produced by the bulky Cl atom or by the electronic
factor of the Cl atom.
At this stage, we again examined the effects of the sub-
stituents at the piperazine nitrogen atom. Although the
binding affinity of N-alkyl analogs (21 and 22) decreased
slightly when compared to N–H analogue 20, as ob-
served in the initial modification (Table 1), their func-
tional activity was unexpectedly enhanced. The reason
for this difference was not clear. Further evaluation of
this series showed better metabolic stability for the N-
hydroxyethyl analogue when compared to the N-ethyl

O. Okamoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3278–3281
3281
2. Reinscheid, R. K.; Nothacker, H. P.; Bourson, A.; Ardati,
A.; Henningsen, R. A.; Bunzow, J. R.; Grandy, D. K.;
Langen, H.; Monsma, F. J.; Civelli, O. Science 1995, 270,
792.
3. Mogil, J. S.; Grisel, J. E.; Reinscheid, K. K.; Civelli, O.;
Belknap, J. K.; Grandy, D. K. Neuroscience 1996, 75, 333.
4. Manabe, T.; Noda, Y.; Mamiya, T.; Katagiri, H.; Hou-
tani, T.; Nishi, M.; Noda, T.; Takahashi, T.; Sugimoto, T.;
Nabeshima, T.; Takeshima, H. Nature 1998, 394, 577.
5. Sandin, J.; Georgieva, J.; Schott, P. A.; Ogren, S. O.;
Terenius, L. Eur. J. Neurosci. 1997, 9, 194.
6. Yu, T. P.; Fein, J.; Phan, T.; Evans, C. J.; Xie, C. W.
Hippocampus 1997, 7, 88.
7. Jenck, F.; Moreau, J. L.; Martin, J. R.; Kilpatrick, G.
J.; Reinscheid, R. K.; Monsma, F. J.; Nothacker, H.
P.; Civelli, O. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
14854.
8. Champion, H. C.; Kadowitz, P. J. Life Sci. 1997, 60, L241.
9. Gumusel, B.; Hao, Q. Z.; Hyman, A.; Chang, J. K.;
Kapusta, D. R.; Lippton, H. Life Sci. 1997, 60, L141.
10. Ronzoni, S.; Peretto, L.; Giardina, G. A. M. Expert Opin.
Ther. Patents 2001, 11, 525.
11. Zaveri, N. Life Sci. 2003, 73, 663.
12. Bignan, G. C.; Connolly, P. J.; Middleton, S. A. Expert
Opin. Ther. Patents 2005, 15, 357.
analogue (e.g. 51% for 22 vs 31% for 21, as measured by
the percentage of parent compound remaining after
30 min of 37 °C incubation with human liver micro-
somes). Therefore, we selected the 2-hydroxyethyl group
as the preferred substituent at the piperazine nitrogen
and revisited the SAR at the thioether position (Table
4).18
It was found that structural hindrance around the sulfur
atom significantly enhanced the potency for ORL1. This
steric factor was confirmed with the introduction of a
methyl group (23 vs 24, 25 and 26 vs 27). Lead optimi-
zation of the benzimidazole series resulted in the identi-
fication of analogue 28;19 the compound had single digit
nanomolar binding affinity for ORL1, excellent antago-
nist activity in the functional assay and very good selec-
tivity over other opioid receptors.
In conclusion, we optimized the novel benzimidazole
structure of potent ORL1 antagonists. The results of
SAR study in this class led to the identification of
ORL1-selective antagonist 28, which exhibited high
affinity for the human ORL1 receptor. Further SAR
studies of this series are currently underway.
13. Kawamoto, H.; Ozaki, S.; Itoh, Y.; Miyaji, M.; Arai, S.;
Nakashima, H.; Kato, T.; Ohta, H.; Iwasawa, Y. J. Med.
Chem. 1999, 42, 5061.
14. Kyle, D. WO Patent 2001039775, 2001; Chem. Abst. 2001
.135, 33477.
Acknowledgments
15. Goto, Y.; Arai-Otsuki, S.; Tachibana, Y.; Ichikawa, D.;
Ozaki, S.; Takahashi, H.; Iwasawa, Y.; Okamoto, O.;
Okuda, S.; Ohta, H.; Sagara, T. J. Med. Chem. 2006, 49,
847.
16. Shinkai, H.; Ito, T.; Iida, T.; Kitao, Y.; Yamada, H.;
Uchida, I. J. Med. Chem. 2000, 43, 4667.
17. Ozaki, S.; Kawamoto, H.; Itoh, Y.; Miyaji, M.; Azuma,
T.; Ichikawa, D.; Nambu, H.; Iguchi, T.; Iwasawa, Y.;
Ohta, H. Eur. J. Pharmacol. 2000, 402, 45.
We acknowledge the contributions of the following sci-
entists to this work: T. Azuma-Kanoh, H. Nambu, N.
Sakai, T. Inoue, D. Ichikawa, S. Okuda, N. Ami, M.
Fukushima and M. Nishino. The authors are also grate-
ful to Dr. J. Sakaki and Dr. N. Ohtake for critically
reading the manuscript.
18. J-113,397 (Ref. 13) was used as an internal control across
all assay plates for data validation.
References and notes
19. Analytical data for HCl salt of compound 28. 1H
NMR(400 MHz, CD3OD) d ppm] 1.26 (6H, t, J = 7.2 Hz),
1.41 (3H, s), 1.65–1.85 (4H, m), 3.20–3.50 (6H, m), 3.50–
3.68 (2H, m), 3.70–3.84 (2H, m), 3.90–4.00 (2H, m), 7.60
(1H, s), 7.92 (1H, s). MS (ESI+): m/z 397.2 (M+H)+.
1. Meunier, J. C.; Mollereau, C.; Toll, L.; Suaudeau, C.;
Moisand, C.; Alvinerie, P.; Butour, J. L.; Guillemot, J. C.;
Ferrara, P.; Monsarrat, B.; Mazarguil, H.; Vassart, G.;
Parmentier, M.; Costentin, J. Nature 1995, 377, 532.