Novel ORL1-selective antagonists with oral bioavailability and brain penetrability

Article abstract of DOI:10.1016/j.bmcl.2008.04.037

Following the discovery of 5-chloro-6-[piperazin-1-yl]-1H-benzimidazole as a novel pharmacophore for potent and selective ORL1 antagonist activity, optimization of this new lead by introduction of a methyl substitution on the piperazine ring resulted in a highly potent and selective, orally available, and brain penetrable ORL1 antagonist, 2-(tert-butylthio)-5-chloro-6-[(2R)-4-(2-hydroxyethyl)-2-methylpiperazin-1-yl]-1H-benzimidazole. Stereochemistry of the methyl substituent on the piperazine ring to control the functional activity of other opioid receptors is also described.

Full text of DOI:10.1016/j.bmcl.2008.04.037

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 3282–3285
Novel ORL1-selective antagonists with oral bioavailability
and brain penetrability
Osamu Okamoto,* Kensuke Kobayashi, Hiroshi Kawamoto, Satoru Ito,
Takashi Yoshizumi, Izumi Yamamoto, Masaya Hashimoto, Atsushi Shimizu,
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 14 April 2008
Available online 28 April 2008
Abstract—Following the discovery of 5-chloro-6-[piperazin-1-yl]-1H-benzimidazole as a novel pharmacophore for potent and selec-
tive ORL1 antagonist activity, optimization of this new lead by introduction of a methyl substitution on the piperazine ring resulted
in a highly potent and selective, orally available, and brain penetrable ORL1 antagonist, 2-(tert-butylthio)-5-chloro-6-[(2R)-4-(2-
hydroxyethyl)-2-methylpiperazin-1-yl]-1 H-benzimidazole. Stereochemistry of the methyl substituent on the piperazine ring to con-
trol the functional activity of other opioid receptors is also described.
ꢀ 2008 Elsevier Ltd. All rights reserved.
In a previous communication,1 we reported the prepara-
tion and biological evaluation of a series of 6-(piperazin-
ORL1 antagonists, which exhibit enhanced affinity and
improved metabolic stability. In addition, we report an
interesting finding regarding the opioid j functional
activity of this class. The SAR study culminated in the
identification of orally available, brain penetrable, and
selective ORL1 antagonist 8.
1-yl)-1H-benzimidazoles
as
nociceptin
receptor
antagonists. The fourth opioid receptor, opioid recep-
tor-like 1 (ORL1), was discovered in 1994 by homology
cloning.2–5 Subsequently, its endogenous 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 have important roles
in the regulation of pain response,8 morphine tolerance,9
learning and memory,10–12 food intake,13 anxiety,14 the
cardiovascular system,15,16 locomotor activity,17 etc.18
Benzimidazole analogs were prepared as described pre-
viously.1 The preparation of representative analog 8 is
depicted in Scheme 1.
In the previous SAR study,1 we found that structural
bulkiness around the sulfur atom significantly enhanced
in vitro potency (Table 1).19–21 However, these modifica-
tions resulted in decreased metabolic stability. Meta-
bolic stability decreases with increasing lipophilicity of
the S-alkyl substituent. To determine potent antagonists
with good metabolic stability, we expanded the SAR
study on the hitherto-unmodified region, the piperazine
ring. A series of compounds with various substituted
piperazines were prepared while retaining a t-Bu or 3-
pentyl group at the thioether moiety due to the relatively
good metabolic stability and in vitro potency. Table 219–21
summarizes the results from SAR studies. Introduction
of methyl or dimethyl groups on the 3-position of the
piperazine resulted in a significant decrease in metabolic
stability (compounds 4 and 5). A bridged piperazine
structure, 6, showed better metabolic stability without
a significant loss of potency.
These results prompted many pharmaceutical compa-
nies to identify potent and selective ORL1 agonists
and antagonists. However, to elucidate the biological
role of the ORL1 receptor and investigate the therapeu-
tic potential of ORL1 antagonists, there remains a need
to develop orally available and brain penetrable ORL1
antagonists.
In this communication, we report an extensive SAR
investigation on a benzimidazole series to elaborate
Keywords: Nociceptin/orphanin FQ; Nociceptin receptor; Opioid rec-
eptor-like 1(ORL1) antagonist.
*
Corresponding author. E-mail: osamu_okamoto@merck.com
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.037

O. Okamoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3282–3285
3283
Cl
NO2
NH2
and d receptors, respectively. On the other hand,
compound 8 with an (R)-methyl configuration showed
100-fold higher selectivity for j receptors. It has been
reported that j agonists have adverse effects such as
psychotomimetic effects and aversion.22,23 Therefore,
we selected compound 8 for further evaluation.
Cl
a
b, c
d
F
F
NH2
F
NH2
Cl
NO2
NH2
Cl
NO2
NH2
g, h
e, f
N
N
N
BocN
TBDMSO
Me
Me
In a standard panel for off-target activity, compound 8
did not display significant affinity for the other 163
receptors and ion channels, indicating that compound
8 is a selective ORL1 antagonist (the receptors and
channels that reached over 50% at 10 lM were the
monoamine transporter (52%), potassium channel-
HERG (86%), serotonin 5-HT2B (52%), and sodium
channel site 2 (68%)). Pharmacokinetic (PK) studies of
compound 8 were carried out in mice, rats and monkeys
and showed moderate PK profiles (Table 4). Despite
high clearance and short half-life in rodents, relatively
low clearance and good plasma half-life were observed
in monkeys.
Me Me
Me
H
N
Cl
Cl
N
N
S
S
i
N
H
N
H
N
N
N
HO
Me
TBDMSO
Me
8
j
BocN
NH
Me
HN NH
Me
A
Scheme 1. Synthesis of representative derivative 8. Reagents and
conditions: (a) NCS, CHCl3, 0 ꢁC, 12 h (29%); (b) KNO3, TFAA, rt,
12 h; (c) K2CO3, MeOH, rt, 0.5 h (90%, 2 steps); (d) A, i-Pr2NEt,
DMSO, 140 ꢁC, 18 h (52%); (e) HCl, dioxane, rt, 16 h; (f) TBDMS-
OCH2CH2CHO, NaBH(OAc)3, MeOH, rt, 1 h (85%, 2 steps); (g) Fe,
aq NH4Cl, MeOH, THF, reflux, 3 h; (h) CS2, aq NaOH, MeOH,
50 ꢁC, 2 h (quant., 2 steps); (i) t-BuOH, TFA, rt, 16 h (78%); (j) Boc2O,
THF, rt, 12 h (48%).
With regard to brain penetrability in ICR mice, the
brain–plasma ratio was 0.47, which represents modest
penetrability; at 60 min after po administration (30 mg/
kg) of 8, brain concentration was 1.04 nmol/kg brain
and plasma level was 2.38 lM. Further study revealed
that compound 8 was subject to mouse P-gp mediated
efflux (mdr1a directional transport ratio (B/A)/(A/B) is
6.2; passive permeability is 25.3 · 10À6 cm/s). It is likely
that the limited brain penetrability in mice is due to sus-
ceptibility to P-gp efflux. On the other hand, compound
8 was not a substrate for human P-gp efflux ((B/A)/(A/B)
is 2.2). Hence, compound 8 should be a good brain pe-
netrant in humans.
We then shifted our attention to the introduction of a
substituent on the 2-position of the piperazine ring. In
this modification, we utilized the t-Bu group at the thio-
ether part due to its good metabolic stability. A finding
to be noted is that introduction of a methyl group at the
2-position of the piperazine ring leads to an approxi-
mately 10-fold improvement in potency and the reten-
tion of good metabolic stability. In addition, there is
no difference in potency or metabolic stability between
the enantiomers (Compounds 7 and 8). Hence, we con-
ducted further evaluation of compounds 7 and 8 and
made observations with regard to selectivity against
other opioids (Table 3).19
In conclusion, we have optimized a novel benzimidazole
structure in a series of potent ORL1 antagonists. The re-
sult of the SAR study in this class led to the identifica-
tion of antagonist 8, which exhibited high affinity for
the human ORL1 receptor and a selectivity over 100-
fold higher than other opioid receptors. Compound 8
presents fair pharmacokinetic properties in mice, rats,
and monkeys and moderate brain penetrability in mice.
Compound 7 having an (S)-methyl configuration on the
piperazine ring showed moderate affinity for other opi-
oid receptors, and exerted agonistic activity at the j
Table 1. Binding affinity, functional activity, and metabolic stability of benzimidazole analogs
Cl
N
S
N
H
R
N
N
HO
Bindinga IC50 (nM)
Compound
R
Antagonisma IC50 (nM)
3.3
HM stabilityb % remaining
51
clogP
Me
Me
1
9.7
3.94
Me
Me
2
3
17
21
71
36
3.19
4.16
Me
Me
Me
1.2
0.66
Me
a See Ref. 19 for detailed description. n = 1 (Ref. 21).
b See Ref. 20 for detailed description.

3284
O. Okamoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3282–3285
Table 2. Binding affinity, functional activity, and metabolic stability of benzimidazole analogs modified at the piperazine ring
R2
Cl
N
S
R1
N
H
Compound
R1
R2
Me
Bindinga IC50 (nM)
Antagonisma IC50 (nM)
HM stabilityb % remaining
Me
Me
*
N
Me
Me
Me
4
8.0
22
17
N
HO
Me
N
Me
Me
N
N
5
6
7
8
73
59
7
63
70
87
HO
HO
**
18
27
N
N
N
Me
Me
Me
N
N
1.1
2.6
2.2
0.65
HO
HO
Me
Me
Me
Me
Me
*Racemate.
**Chiral isomer.
a See Ref. 19 for detailed description. n = 1 (Ref. 21).
b See Ref. 20 for detailed description.
Table 3. Effects of Me substitution on selectivity over other opioid receptors
Me Me
Me
S
Cl
N
N
H
R
Functional profiles in [35S]GTPcS binding assaya
a
Binding IC50 (nM)
Compound
R
ORL1
l
j
d
ORL1
l
j
d
N
2
7
8
17
4400 120
>1000 IC50 21 nM
IC50 2.2 nM
NT
NT
NT
N
HO
N
N
1.1
2.6
67
5.3 238
IC50 25 nM EC50 18 nM Emax 80% EC50 14 nM Emax 94%
N
N
HO
HO
Me
Me
1900 120
>1000 IC50 0.65 nM IC50 97 nM IC50 180 nM
IC50 1200 nM
a See Ref. 19 for detailed description. n = 1.
Acknowledgments
Table 4. Pharmocokinetics of compound 8 in mouse, rat, and monkey
F (%)
T1/2 (h)
Cl (ml/min/kg)
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
grateful to Dr. J. Sakaki and Dr. N. Ohtake for critically
reading the manuscript.
ICR mouse
SD rat
Rhesus monkey
33
29
37
0.81
0.92
3.1
121
81
20
Oral dose 3 mg/kg and IV dose 1 mg/kg.
The overall profiles of 824 are complementary and
appropriate for pharmacological evaluation of ORL1
antagonism. Further pharmacological studies of this
antagonist are currently underway.
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125I]Tyr14-NC/OFQ, and agonist/antagonist
activities were measured by the [35S]GTPcS binding
method. Cross reactivity to other opioid receptors was
also tested. Affinities and agonist/antagonist activities for
human l-, j-, and d-receptors were assayed similarly to
ORL1 using membrane fractions of CHO cells expressed
in each receptor (Affinities were measured by displacement
of a [3H]diprenorphin for l, [3H]U-69593 for j and
[3H]naltrindole for d binding, respectively. Antagonist
activities were measured by DAMGO for l, U-69593 for j
and DADLE for d, respectively).
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24. Analytical data for compound 8. 1H NMR (400 MHz,
CDCl3) d [ppm] 0.91 (3H, d, J = 6.4 Hz), 1.50 (9H, s),
2.25–2.96 (8H, m), 3.14–3.22 (1H, m), 3.35–3.45 (1H, m),
3.69 (2H, t, J = 5.2 Hz), 7.20–7.90 (2H, m), 9.50 (1H, br s).
MS (ESI+): m/z 383.2 (M+H)+.