Discovery of novel arylpyrazole series as potent and selective opioid receptor-like 1 (ORL1) antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.04.116

The synthesis and biological evaluation of new potent opioid receptor-like 1 antagonists are presented. A structure-activity relationship (SAR) study of arylpyrazole lead compound 1 obtained from library screening identified compound 31, (1S,3R)-N-{[1-(3-

Full text of DOI:10.1016/j.bmcl.2009.04.116

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3627–3631
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel arylpyrazole series as potent and selective opioid
receptor-like 1 (ORL1) antagonists
Kensuke Kobayashi, Minaho Uchiyama, Hirokatsu Ito, Hirobumi Takahashi, Takashi Yoshizumi,
Hiroki Sakoh, Yasushi Nagatomi, Masanori Asai, Hiroshi Miyazoe, Tomohiro Tsujita, Mioko Hirayama,
*
Satoshi Ozaki, Takeshi Tani, Yasuyuki Ishii, 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 antagonists are presented. A
structure–activity relationship (SAR) study of arylpyrazole lead compound 1 obtained from library
screening identified compound 31, (1S,3R)-N-{[1-(3-chloropyridin-2-yl)-5-(5-fluoro-6-methylpyridin-3-
yl)-4-methyl-1H-pyrazol-3-yl]methyl}-3-fluorocyclopentanamine, which exhibits high intrinsic potency
and selectivity against other opioid receptors and hERG potassium channel.
Received 30 March 2009
Revised 22 April 2009
Accepted 24 April 2009
Available online 3 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
ORL1 antagonist
Nociceptin/orphanin FQ
Arylpyrazole
hERG channel
Opioid receptor-like 1 (ORL1), was discovered in 1994 based on
its high degree of amino acid sequence homology to the classical
opioid receptors.1 Despite this homology, it was shown that this
receptor did not bind to classical opioids with appreciable affinity.
Subsequently, its endogenous agonist, a 17-amino acid peptide
known as nociceptin or orphanin FQ (NC/OFQ), was identified.2
A
number of reports have since demonstrated the possible involve-
ment of the NC/OFQ-ORL1 system in pain reguration,3 morphine
tolerance,4 learning and memory,5–7 food intake,8 anxiety,9 the car-
diovascular system,10,11 locomotor activity,12 and so on.13
Several small-molecule antagonists with high affinity and selec-
tivity for ORL1 have recently been reported.14–19 We also reported
novel classes of orally active and brain penetrable ORL1 antago-
nists that exhibit a wide safety window with regard to adverse car-
diovascular effects.20,21 However, additional structurally diverse
ORL1 antagonists are required for better understanding of the
physiological roles of ORL1 receptor and the therapeutic potential
of its antagonists. We report here a series of structurally novel aryl-
pyrazole ORL1 antagonists that demonstrate high affinity for the
human ORL1 receptor, while possessing desirable in vitro features.
Screening of the sample collection identified arylpyrazole deriv-
ative 1 (Fig. 1), which has a structure distinct from various pub-
lished ORL1 antagonists. This derivative showed sub-micromolar
ORL1 binding affinity and antagonistic activity, as well as potent
Figure 1. Biological profiles of lead compound 1.
human ether-a-go-go related gene (hERG) K+ channel binding
affinity. Thus, we attempted to enhance its intrinsic potency while
removing its hERG inhibitory activity.
Synthesis of arylpyrazole ORL1 antagonists followed the general
procedure shown in Scheme 1. Aryl ethyl ketone 2 was coupled
with 2-(1H-benzotriazol-1-yl)-2-oxoethyl acetate 3 to afford the
desired b-diketone 4. Treatment of 4 with the corresponding aryl-
hydrazine in acetic acid followed by saponification gave 3-hydrox-
ymethylpyrazole 5. The key intermediate 3-formylpyrazole 6 was
prepared via oxidation of alcohol function with MnO2. Reductive
* 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.2009.04.116

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K. Kobayashi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3627–3631
crease in binding activity; however, further prolongation of the
alkyl group (8) showed no effect on ORL1 activity. Significant
enhancement was observed when a cyclohexane was installed
at the amine region (9). Further introduction of the substituent
at the amine nitrogen in 9 resulted in a dramatic loss of potency
(10 and 11). Piperidine analogue 12 also showed weak ORL1
activity, suggesting that the tertiary amine was unfavourable in
terms of binding affinity. Thus, this series has an extremely differ-
ent feature from an ORL1 antagonist, because, to our knowledge,
a tertiary amine is essential for ORL1 affinity among most of the
reported small-molecule ORL1 ligands.26 Among the synthesized
compounds, cyclopentylamine analogue 13 exhibited the best
intrinsic potency.
By modifying the amine part, ORL1 activity was enhanced;
however, hERG inhibitory activity of 13 remained high (Table 2).
We then directed our SAR efforts toward the modification of two
aryl moieties using cyclopentylamine derivative 13 as a template.
As reduction of the lipophilicity of the molecule was essential in
removing affinity for hERG,27 SAR studies were carried out using
analogues that incorporated hydrophilic pyridine ring(s). Although
the 2- and 4-pyridine derivatives at the 5-position of the pyrazole
ring (14 and 16, respectively) showed significantly impaired ORL1
binding affinity, 3-pyridine analogue 15 was equipotent to com-
pound 13. In addition, binding affinity of 15 for hERG was 100-fold
less than that of 13, which can be attributed to the increased
hydrophilicity of 15 (log D7.4 = 2.9) in comparison to 13
(log D7.4 = >4). Introduction of an additional pyridine ring at the
1-position of the pyrazole ring (17) resulted in negligible hERG
inhibitory activity while retaining the desired intrinsic potency.
Next, the effects of substituents on the two pyridine rings were
investigated (Table 3). Cl-substitution at the 6-position of the left
pyridine ring (18) resulted in increased ORL1 binding affinity, but
hERG binding affinity was also enhanced. In contrast, 6-methylpyr-
idine analogue 19 displayed potent ORL1 binding affinity and
antagonistic activity, while retaining good selectivity over hERG.
Introduction of a bulkier ethyl (20) or trifluoromethyl (21) group
at the 6-position, or a methyl group (22) at the 5-position impaired
binding affinity. We thus examined the effect of substituents on
the bottom pyridine ring using 6-methylpyridine 19. Replacement
of 30-chlorine with a methyl (23), trifluoromethyl (24) or methane-
sulfonyl (25) group resulted in a decrease in intrinsic potency. For
compound 26, which has two chlorine atoms at the 30- and 50-posi-
tions, binding affinity was good, while affinity for hERG binding
was enhanced. The SAR results (Table 3) also demonstrated a good
correlation between lipophilicity and hERG affinity.
Scheme 1. Reagents and conditions: (a) LHMDS, THF, ꢀ78 °C to rt; (b) Ar2NHNH2,
AcOH, 80 °C; (c) NaOH aq, MeOH, rt; (d) MnO2, CHCl3, rt; (e) R1R2NH, Zn(BH3CN)2,
MeOH, rt.
amination of 6 with the related amine provided arylpyrazole deriv-
atives 1, 7–31.
Analogues were tested for their inhibitory effects on ligands for
human ORL1 receptor and on GTPcS binding to proteins using
membrane fractions of CHO cells expressing ORL1. Binding affini-
ties for ORL1 were determined by displacement of [125I]Tyr14-NC/
OFQ, and agonist/antagonist activities were measured by the
[
35S]GTP S binding method.22 Binding affinity to hERG K+ channel
c
was measured by the displacement of [35S]-radiolabeled MK499 in
membranes derived from HEK 293 cells stably transfected with the
hERG gene and expressing the IKr channel protein.23,24
Our first objective was to examine the effects of substituents
at the pyrazolylmethylamine region. A rapid analogue synthesis
was carried out in order to prepare a series of arylpyrazoles with
a variety of secondary or tertiary amines. The ORL1 binding affin-
ity of selected compounds from the approximately 40 pyrazoles
prepared (data not shown) are listed in Table 1.25 Replacement
of an isopropyl (1) with an isobutyl (7) resulted in a twofold in-
Extensive SAR studies resulted in 19, which demonstrated po-
tent ORL1 activity and good selectivity over hERG binding; how-
ever, further evaluation revealed that this compound was subject
to human P-glycoprotein (P-gp) efflux (transport ratio, 4.1) (Table
4).28 Therefore, we focused our efforts on overcoming this issue.
Based on a report by Cox et al.,29 we speculated that lowering
the pKa of two amine moieties, cyclopentylamine (N1) and/or
methylpyridine (N2), would remove P-gp susceptibility. To confirm
this hypothesis, analogues of 19 with fluorine installed directly on
the cyclopentane or methylpyridine rings were prepared and eval-
uated. Consequently, 2-fluoro- (27) and 3,3-difluoro- (29) cyclo-
pentylamine analogues (calculated pKa of 6.9 and 6.5,
respectively) were not subject to P-gp efflux, although the intrinsic
potency decreased two- to threefold. In contrast, incorporation of a
fluorine at the 3-position on the cyclopentane ring (pKa 7.4) re-
sulted in enhanced ORL1 binding affinity; however, the fluorine
atom was ineffective against P-gp efflux (28). With regard to the
basicity of the left pyridine, 5-fluorination resulted in 30 with re-
duced pKa (from 4.5 to 2.1) and susceptibility to P-gp efflux with-
out any loss of potency toward ORL1. Combination of 30 with the
3-fluorocyclopentylamine moiety led to identification of com-
Table 1
Effects of N-substitution on binding affinity at human ORL1 receptor
Compounds
R1
R2
ORL1 bindinga IC50 (nM)
1
7
8
9
10
11
12
13
iPr
H
H
H
H
350
150
150
75
>1000
>1000
560
28
CH2iPr
CH2CH2iPr
Cyclohexyl
Cyclohexyl
Cyclohexyl
Me
iPr
–(CH2)5–
Cyclopentyl
H
a
n = 1 (Ref. 25).

K. Kobayashi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3627–3631
3629
Table 2
Binding affinities of 13–17 toward ORL1 and hERG
R1
R2
ORL1 bindinga IC50 (nM)
hERG binding IC50 (nM)
Log D7.4
b
Compounds
13
14
15
16
28
23
>4
"
"
"
530
37
2900
2.9
1.3
>1000
61
17
23,000
a
n = 1 (Ref. 25).
Measured by shake-flask method.
b
pound 31, which was not a human P-gp substrate while possessing
affinity for human ORL1 receptor and good selectivity over binding
affinity for hERG and other opioid receptors. In addition, 31 is not
subject to human P-gp efflux, suggesting that its brain penetrabil-
ity may be suitable for humans. Further evaluation of this com-
pound is currently underway.
sub-nanomolar ORL1 activity.
Further evaluation revealed that compound 3130 was
a
highly selective ORL1 antagonist against
(Table 5).
l- and j-opioid receptors
In conclusion, SAR investigation of a novel class of arylpyrazoles
as ORL1 antagonists identified analogue 31, which exhibits high
Table 3
SAR of substituents on the left and bottom pyridine rings
R1
R2
ORL1 bindinga IC50 (nM)
Antagonisma IC50 (nM)
hERG binding IC50 (nM)
Log D7.4
b
Compounds
17
18
19
20
21
22
23
24
25
26
—
6-Cl
6-Me
6-Et
6-CF3
5-Me
6-Me
6-Me
6-Me
6-Me
30-Cl
61
11
4.0
67
270
13
33
36
72
8.2
23,000
2700
16,000
1.3
2.1
1.7
2.0
2.2
1.6
1.2
1.9
30-Cl
12
4.7
30-Cl
30-Cl
30-Cl
30-Cl
9000
1200
30-Me
30-CF3
30-SO2Me
30-Cl, 50-Cl
2.5
a
n = 1 (Ref. 25).
Measured by shake-flask method.
b

3630
K. Kobayashi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3627–3631
Table 4
Effects of fluorine atom on in vitro profiles
R1
R2
H
H
H
H
F
ORL1 bindinga IC50 (nM)
Antagonisma IC50 (nM)
hERG binding IC50 (nM)
log D7.4
human P-gp transport ratioc
pKa
b
d
Compounds
N1
N2
19
4.0
11
4.7
7.1
1.0
4.5
1.2
0.31
16,000
30,000
29,000
21,000
23,000
41,000
1.7
4.1
1.9
4.4
1.4
2.3
1.7
8.2
6.9
7.4
6.5
8.2
7.4
4.5
4.5
4.5
4.5
2.1
2.1
27e
28e
29e
30
2.0
2.5
9.3
1.1
0.52
1.3
2.1
2.1
31
F
1.8
eMore potent enantiomers.
a
n = 1 (Ref. 25).
Measured by shake-flask method.
b
c
Transport ratio: B ꢀ A/A ꢀ B (Ref. 28).
d
Calculated pKa values.
Table 5
Off-target activities (
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j
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Compounds
Binding IC50 (nM)
ORL1
0.52
la
ja
31
5600
530
a
Displacement of a [3H]diprenorphin (
l
) and [3H]U69593 (
j
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