2-Cyclohexylcarbonylbenzimidazoles as potent, orally available and brain-penetrable opioid receptor-like 1 (ORL1) antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.04.023

The synthesis and biological evaluation of new potent opioid receptor-like 1 (ORL1) antagonists are presented. Conversion of the thioether linkage of the prototype [It is reported prior to this communication as a consecutive series.: Kobayashi, K.; Kato, T.; Yamamoto, I.; Shimizu, A.; Mizutani, S.; Asai, M.; Kawamoto, H.; Ito, S.; Yoshizumi, T.; Hirayama, M.; Ozaki, S.; Ohta, H.; Okamoto, O. Bioorg. Med. Chem. Lett., in press] to the carbonyl linker effectively reduces susceptibility to P-glycoprotein (P-gp) efflux. This finding led to the identification of 2-cyclohexylcarbonylbenzimizole analogue 7c, which exhibited potent ORL1 activity, excellent selectivity over other receptors and ion channels, and poor susceptibility to P-gp. Compound 7c also showed satisfactory pharmacokinetic profiles and brain penetrability in laboratory animals. Furthermore, 7c showed good in vivo antagonism. Hence, 7c was selected as a clinical candidate for a brain-penetrable ORL1 antagonist.

Full text of DOI:10.1016/j.bmcl.2009.04.023

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3096–3099
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Cyclohexylcarbonylbenzimidazoles as potent, orally available
and brain-penetrable opioid receptor-like 1 (ORL1) antagonists
Kensuke Kobayashi, Minaho Uchiyama, Hirobumi Takahashi, Hiroshi Kawamoto, Satoru Ito,
Takashi Yoshizumi, Hiroshi Nakashima, Tetsuya Kato, Atsushi Shimizu, Izumi Yamamoto, Masanori Asai,
Hiroshi Miyazoe, Akio Ohno, Mioko Hirayama, Satoshi Ozaki, Takeshi Tani, Yasuyuki Ishii, Takeshi Tanaka,
*
Takanobu Mochidome, Kiyoshi Tadano, Takahiro Fukuroda, Hisashi Ohta, Osamu Okamoto
Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Okubo-3, Tsukuba, Ibaraki 300-2611, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of new potent opioid receptor-like 1 (ORL1) antagonists are pre-
sented. Conversion of the thioether linkage of the prototype [It is reported prior to this communication as
a consecutive series.: Kobayashi, K.; Kato, T.; Yamamoto, I.; Shimizu, A.; Mizutani, S.; Asai, M.; Kawamoto,
H.; Ito, S.; Yoshizumi, T.; Hirayama, M.; Ozaki, S.; Ohta, H.; Okamoto, O. Bioorg. Med. Chem. Lett., in press]
to the carbonyl linker effectively reduces susceptibility to P-glycoprotein (P-gp) efflux. This finding led to
the identification of 2-cyclohexylcarbonylbenzimizole analogue 7c, which exhibited potent ORL1 activity,
excellent selectivity over other receptors and ion channels, and poor susceptibility to P-gp. Compound 7c
also showed satisfactory pharmacokinetic profiles and brain penetrability in laboratory animals. Further-
more, 7c showed good in vivo antagonism. Hence, 7c was selected as a clinical candidate for a brain-pen-
etrable ORL1 antagonist.
Received 4 February 2009
Revised 27 March 2009
Accepted 1 April 2009
Available online 11 April 2009
Keywords:
ORL1 antagonist
Nociceptin/orphanin FQ
Benzimidazole
P-glycoprotein (P-gp)
Ó 2009 Elsevier Ltd. All rights reserved.
A fourth opioid receptor, opioid receptor-like 1 (ORL1), was dis-
covered in 1994 by homology cloning.2–5 Subsequently, its endog-
enous agonist, a 17-amino acid peptide termed nociceptin or
orphanin FQ (NC/OFQ), was identified.6,7 Pharmacological studies
using NC/OFQ and ORL1-deficient mice showed that the NC/OFQ-
ORL1 system may play important roles in the regulation of pain re-
sponse,8 morphine tolerance,9 learning and memory,10–12 food in-
take,13 anxiety,14 the cardiovascular system,15,16 locomotor
activity,17 etc.18 These results prompted many pharmaceutical
companies to identify potent and selective antagonists. However,
in order to clarify the biological role of the ORL1 receptor and
investigate the therapeutic potential of the ORL1 antagonists, there
remains a need to develop orally available and brain penetrable
ORL1 antagonists.
We previously reported the identification of potent ORL1 antag-
onists with high selectivity over binding affinity for human ether-
a-go-go related gene (hERG).1 Further evaluation revealed that
most compounds with a hydrophilic group on a thioether part re-
lated to 1 and 2 exhibited significantly low brain penetrability (Ta-
ble 1).19–21 We demonstrated that 1 and 2 were substrates for
human and mice P-gp efflux. This suggests that the poor brain pen-
etrability is due to P-gp mediated efflux. We report here structure–
activity relationship (SAR) studies to address this issue in the benz-
imidazole series of ORL1 antagonists.
The syntheses of compounds 7aÀe followed the general proce-
dure shown in Scheme 1. Nitroanilines 3 were reduced with Fe fol-
lowed by condensation with formic acid to give benzimidazole 5,
which was protected using
a 2-(trimethylsilyl)ethoxymethyl
(SEM) group to afford intermediate 6. Coupling 6 with the corre-
sponding ester using lithium tetramethylpiperidide (LTMP) fol-
lowed
by
deprotection
of
the
SEM
group
with
tetrabutylammonium fluoride (TBAF) provided compounds 7aÀe.
The starting material 3a was prepared from 3-fluoro-4-methylani-
line 8 via nitration with potassium nitrate in trifluoroacetic anhy-
dride followed by hydrolysis of the resultant trifluoroacetamide.
Cyclohexanone analogue 9 was prepared by a similar coupling
reaction as mentioned above, followed by removal of the SEM
group and ketal in aqueous TFA (Scheme 2).
As shown in previous communications,1,22,23 we have already
performed structural modification at the piperazine ring, the thio-
ether substituent and 5-substitution on the benzimidazole ring.
Therefore, the SAR study was expanded to a hitherto unmodified
region, the linkage moiety at the 2-position on the benzimidazole
core using compound 1 as a template. Replacing the sulfur atom,
however, with methylene, a nitrogen atom and an oxygen atom re-
sulted in complete loss of potency (data not shown). In contrast,
ketone analogue 7a was tolerable in terms of binding affinity for
* Corresponding author. Tel.: +81 29 877 2000; fax: +81 29 877 2029.
E-mail address: osamu_okamoto@merck.com (O. Okamoto).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.023

K. Kobayashi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3096–3099
3097
Table 1
Binding affinity for ORL1, brain penetrability, and P-gp susceptibility of 1 and 2
Compds
R
ORL1 bindinga IC50 (nM)
Brain penetrabilityb
Brain (nmol/g)
P-gpc (transport ratio)
Plasma (nM)
b/p ratio
Human
Mice
1
2
NHCO2Me
N(Me)Ac
2.4
0.81
22.2
4.56
1.11
0.38
0.05
0.09
16
33
21
54
a
See Ref. 19 for detailed description. n = 1 (Ref. 20).
At 1 h after po administration in mice (30 mg/kg).
Transport ratio: BÀA/AÀB (Ref. 21).
b
c
Table 2
Effect of the linkage at 2-position on benzimidazole ring
Compds
R1
ORL1 bindinga
IC50 (nM)
Antagonisma
IC50 (nM)
P-gpb (transport ratio)
Human
Mice
1
7a
S
2.4
5.7
0.72
7.5
16
1.9
21
3.6
C@O
a
See Ref. 19 for detailed description. n = 1 (Ref. 20).
Transport ratio: BÀA/AÀB (Ref. 21).
b
effects of substituents at the 5-position of the benzimidazole core
were investigated. Replacement of the chlorine atom with a methyl
group (7b) resulted in a slight increase in potency for ORL1; how-
ever, the binding affinity for hERG was also enhanced, despite the
decrease in lipophilicity.26 We next carried out SAR studies to as-
sess the effects of substituents on the cyclohexane ring. Cyclohex-
Scheme 1. Reagents and conditions: (a) K2CO3, DMSO, 120 °C; (b) Fe, NH4Cl,
THFÀMeOHÀH2O, reflux; (c) HCO2H, 100 °C; (d) SEMCl, NaH, THF, 55À74% (4steps);
(e) R–CO2Et, LTMP, THF, À78 °C, 46À55%; (f) TBAF, THF, reflux, 57À93%; (g) KNO3,
TFAA, rt; (h) K2CO3, MeOHÀH2O, rt, 66% (2 steps).
anone analogue
9 showed a twofold reduced ORL1 affinity,
although affinity for hERG was slightly reduced. Incorporation of
the tert-alcohol group into the 4-position on the cyclohexane ring
led to interesting results with regard to binding affinity for ORL1
and hERG. The trans isomer 7c exhibited potent ORL1 affinity
and markedly improved selectivity over hERG, without impair-
ment of metabolic stability and susceptibility to P-gp. In compari-
son, compound 7d with a cis configuration showed higher affinity
for the hERG channel, despite the fact that the binding affinity for
ORL1 was twenty times lower than that for 7c. Replacement of the
methyl group at the 1-position with a bulky ethyl group (7e) re-
sulted in slight enhancement of the binding affinity for ORL1; how-
ever, its metabolic stability was decreased due to the increase in
the lipophilicity. Consequently, we selected compound 7c for fur-
ther evaluation.
Scheme 2. Reagents and conditions: (a) LTMP, THF, À78 °C, 56%; (b) TFAÀH2O
(10:1), rt, 99%.
ORL1 as compared with compound 1 (Table 2).19–21 Surprisingly,
this modification resulted in a marked improvement in susceptibil-
ity to human and mouse P-gp-mediated efflux. Although the rea-
son for this difference is not clear, we speculate that it correlates
with pKa value of the benzimidazole.24 As a new template, 2-car-
bonylbenzimidazoles resulted from modifying the linkage moiety,
and we then revisited the SAR at the benzimidazole and cyclohex-
ane rings.
As shown in Table 4, compound 7c27 was a highly selective
ORL1 antagonist against other opioid receptors. In a standard panel
for off-target activity, 7c showed good selectivity over the other
160 receptors and ion channels (adrenergic
(72%), 1D (74%), cannabinoid CB2 (73%), histamine H3 (50%) and
choline transporter (62%) reached over 50% at 10 M). The poten-
tial cardiovascular effects of 7c were evaluated in anesthetized
dogs. At 10 mg/kg iv dosing (Cmax = 10.7 M), no adverse treat-
ment-related cardiovascular effects were observed.
a1 non-selective
a
l
The results of SAR studies on ORL1 activity, selectivity over
hERG binding, human metabolic stability, and the susceptibility
to human P-gp efflux are summarized in Table 3.19–21,25 First, the
l

3098
K. Kobayashi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3096–3099
Table 3
In vitro profiles of 2-cyclohexylcarbonylbenzimidazoles
R1
R2
ORL1 bindinga
IC50 (nM)
Antagonisma
IC50 (nM)
hERG bindingb
IC50 (nM)
log D7.4
HM stabilityd
% remaining
P-gpe (transport ratio)
c
Compds
Human
Mice
7a
7b
9
Cl
5.7
2.0
9.9
7.5
5.3
38
4900
3000
8500
3.7
3.1
3.0
74
71
1.9
3.6
Me
Cl
7c
Cl
Cl
Cl
1.4
27
1.3
20,000
3100
3.1
82
1.7
5.5
7d
7e
0.80
2.4
11,000
3.6
57
2.0
3.0
a
See Ref. 19 for detailed description. n = 1 (Ref. 20).
b
c
Displacement of a [35S] -radiolabeled MK499 in membranes derived from HEK 293 cells stably transfected with hERG gene and expressing the IKr channel protein.
Measured by shake-flask method.
See Ref. 25 for detailed description.
Transport ratio: BÀA/AÀB (Ref. 21).
d
e
Pharmacokinetic (PK) studies were carried out in rats, dogs and
With regard to brain penetrability in mice, the brain–plasma ratio
was 2.02, which was dramatically improved as compared with the
thioether leads. As 7c was not subject to human P-gp efflux, it
should be a good brain penetrant in humans.
monkeys, and 7c exhibited moderate PK profiles. Relatively low
clearance and good plasma half-life were observed in dogs and
monkeys, despite the high clearance and short half-life in rats.
Furthermore, in vivo antagonistic activity of 7c was tested in
the rat carrageenin-induced hyperalgesia model (Fig. 1). This com-
pound, administered at a dose of 30 mg/kg sc, significantly inhib-
Table 4
Off-target activities, PK profiles, and brain penetrability of 7c
Compound
7c
Binding IC50
ORL1 (nM)
la (nM)
ja (nM)
da (nM)
1.4
>10,000
1400
>10,000
Pharmacokineticsb
Rat
F (%)
15
T1/2 (h)
Cl (ml/min/kg)
F (%)
0.62
77
38
Dog
T1/2 (h)
Cl (ml/min/kg)
F (%)
3.5
34
8
Monkey
T1/2 (h)
Cl (ml/min/kg)
4.0
22
Brain penetrabilityc
Plasma (
Brain (nmol/g)
b/p ratio
l
M)
1.08
2.21
2.02
a
Displacement of a [3H]diprenorphin (
l
), [3H]U69593 (
j
), and [3H]naltrindole
binding to CHO cells stably expressing cloned human
l-, j-, and d-opioid receptors,
Figure 1. Effect of 7c on carrageenin-induced hyperalgesia in rats. Carrageenin
(0.15 ml, 3%) was administered ipl. 3 h before test. Test was performed 1 h after 7c
injection. Rat’s responses to noxious mechanical stimuli were measured. P < 0.05
respectively.
b
*
Oral dose 3 mg/kg and IV dose 1 mg/kg.
At 30 min after sc administration in mice (3 mg/kg).
c
versus vehicle.

K. Kobayashi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3096–3099
3099
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administration.
In conclusion, we performed SAR studies, both for ORL1 activity
and for susceptibility to P-gp efflux, for a benzimidazole series of
ORL1 antagonists related to 1. Modification of the linkage moiety
at the 2-position of the benzimidazole core led to identification
of 2-cyclohexylcarbonylbenzimizole 7c, which exhibited potent
ORL1 activity and low susceptibility to P-gp efflux. 7c shows fair
pharmacokinetic properties in rats and dogs, and good brain pene-
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Acknowledgements
OFQ, and antagonist activities were measured by the
method.
[ cS binding
35S]GTP
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(2H, q, J = 7.3 Hz), 2.98–3.26 (4H, m), 7.10–7.95 (2H, m), 10.02–10.27 (1H, br).
MS (ESI): m/z 419.3 [M+H]+.