Identification of MK-1925: A selective, orally active and brain-penetrable opioid receptor-like 1 (ORL1) antagonist

Article abstract of DOI:10.1016/j.bmcl.2009.06.051

Structure-activity relationship studies directed toward improving the metabolic stability of compound 1 resulted in the identification of 3-[5-(3,5-difluorophenyl)-3-({[(1S,3R)-3-fluorocyclopentyl]amino}methyl)-4-methyl-1H-pyrazol-1-yl]propanenitrile 39 (

Full text of DOI:10.1016/j.bmcl.2009.06.051

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4729–4732
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of MK-1925: A selective, orally active and brain-penetrable
opioid receptor-like 1 (ORL1) antagonist
Kensuke Kobayashi, Tomohiro Tsujita, Hirokatsu Ito, Satoshi Ozaki, Takeshi Tani, Yasuyuki Ishii,
*
Shoki Okuda, 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:
Structure–activity relationship studies directed toward improving the metabolic stability of compound 1
resulted in the identification of 3-[5-(3,5-difluorophenyl)-3-({[(1S,3R)-3-fluorocyclopentyl]amino}-
methyl)-4-methyl-1H-pyrazol-1-yl]propanenitrile 39 (MK-1925) as a selective, orally available and
brain-penetrable opioid receptor-like 1 (ORL1) antagonist. The compound also showed in vivo efficacy
after oral dosing. Therefore, compound 39 was selected to undergo further studies as a clinical candidate.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 16 May 2009
Revised 12 June 2009
Accepted 15 June 2009
Available online 17 June 2009
Keywords:
ORL1 antagonist
Nociceptin/orphanin FQ
Arylpyrazole
In 1994, opioid receptor-like 1 (ORL1) was identified as a fourth
opioid receptor using cloning techniques.1–4 Subsequently, its
endogenous agonist, a 17-amino acid peptide termed nociceptin
or orphanin FQ (NC/OFQ), was identified.5,6 Nociceptin and ORL1
receptors are widely distributed in the central nervous system,
and the physiological roles of the NC/OFQ–ORL1 system have been
the focus of intense research. In addition to investigations using
NC/OFQ, studies involving ORL1-deficient mice showed that this
system may play important roles in pain regulation,7 learning
and memory,8–10 food intake,11 anxiety12 and the cardiovascular
system,13,14 as well as other areas,15 thus prompting pharmaceuti-
cal companies to identify small molecules as potent and selective
ORL1 agonists and antagonists. However, additional structurally
diverse ORL1 antagonists are required for better understanding of
the physiological roles of the ORL1 receptor and the therapeutic
potential of its antagonists.16–19
aryl moiety with smaller alkyl groups, aiming to simplify the
molecular structure, as illustrated in Figure 1.
Analogues 25–33, 36 and 39 were prepared as described previ-
ously.20 The synthesis of representative compound 39 is depicted
in Scheme 1. 3,5-Difluorophenyl ethyl ketone 2 was coupled with
2-(1H-benzotriazol-1-yl)-2-oxoethyl acetate 3 to afford b-diketone
4. Cyclization of 4 with 2-cyanoethylhydrazine followed by sapon-
ification provided 3-hydroxymethylpyrazole 5. Finally, reductive
amination of (1S,3R)-3-fluorocyclopentanamine 7 and the alde-
hyde 6, obtained through oxidation of the alcohol 5, gave the
desired compound 39.
4-Chloro and 4-methoxymetyl analogues, 34 and 35 were
prepared according to Scheme 2. 4-Unsubstituted pyrazole 9
prepared by coupling of starting material 8 with (CO2Et)2 followed
by cyclization was halogenated with N-bromosuccinimide (NBS) or
N-chlorosuccinimide (NCS) to afford 4-chloro (10) and 4-bromop-
yrazole (11), respectively, in almost quantitative yield. Cross-cou-
In previous communications,20 we described the identification
of potent and selective ORL1 antagonist 1 (Fig. 1). However, further
evaluation showed that 1 had a poor pharmacokinetic (PK) profile
in rats (F = 3.2%, t1/2 = 0.5 h, CLp = 68 ml/min/kg) with unaccept-
able metabolic stability in human hepatocytes (39% remaining),21
thus suggesting that its oral availability in humans was insuffi-
cient. Herein, we report structure–activity relationship (SAR) stud-
ies that we conducted to address this issue in the arylpyrazole
series of ORL1 antagonists. As the first step, we replaced the lower
* Corresponding author.
E-mail address: osamu_okamoto@merck.com (O. Okamoto).
Figure 1. Compound 1 and modification design.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.051

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K. Kobayashi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4729–4732
Scheme 1. Reagents and conditions: (a) LHMDS, THF, À78 °C to rt, 61%; (b) 2-
cyanoethylhydrazine, AcOH, 90 °C; (c) aq. NaOH, MeOH, rt, 77% (2 steps); (d) Dess–
Martin periodinane, CHCl3, rt, 75%; (e) 7, Zn(BH3CN)2, MeOH, rt, 85%.
Scheme 3. Reagents and conditions: (a) hydrazine hydrate or ethyl hydrazinoac-
etate, 4 N HCl–dioxane, EtOH, reflux, 74–80%; (b) TBSCl, imidazole, DMF, 80 °C; (c)
bromoacetone, Et3N, toluene–DMF, reflux, 6% (2 steps); (d) LiAlH4, THF, rt, 83% (2
steps); (e) TBAF, THF, rt; (f) Dess–Martin periodinane (for 18) or MnO2 (for 20),
CHCl3, rt, 27–38% (2 steps); (g) 7, Zn(BH3CN)2, MeOH, rt, 80–90%.
by alkylation with bromoacetone or reduction with LiAlH4, respec-
tively. The yield of the alkylation with bromoacetone was low due
to the major formation of the N-alkyl regioisomer. After deprotec-
tion of the TBS group, the final products 37 and 38 were prepared
via the same procedure for the compound 39.
Firstly, we explored the effect of alkyl substituents upon the
intrinsic potency and the metabolic stability (Table 1). Replace-
ment of the chloropyridine moiety with an isopropyl group (25)
resulted in no significant decrease in potency toward ORL1. Smal-
ler ethyl (26) or methyl (27) analogues were tolerable in terms of
Table 1
SAR of alkyl substituents at 1 position on pyrazole ring
Scheme 2. Reagents and conditions: (a) LHMDS, (CO2Et)2, THF, À78 °C to rt; (b)
MeNHNH2, EtOH, rt, 58% (2 steps); (c) NCS or NBS, MeCN, 80 °C, 98%–quant.; (d)
(methoxymethyl)tributyltin, PdCl2(PPh3)2, DMF, 90 °C, 18%; (e) LiAlH4, THF, 0 °C; (f)
MnO2, CHCl3, rt, 60–70% (2 steps); (g) 7, Zn(BH3CN)2, MeOH, rt, 65–67%.
Compds
R
ORL1 bindinga
IC50 (nM)
Antagonisma
IC50 (nM)
HH stabilityb
remaining
%
pling22 of 11 with (methoxymethyl)tributyltin gave 12. Ethyl ester
of 10 and 12 was converted in two steps, LiAlH4 reduction and
MnO2 oxidation, into the corresponding pyrazole-4-carbaldehydes
13 and 14. Finally, 13 and 14 underwent reductive amination with
(1S,3R)-3-fluorocyclopentanamine 7 to produce the desired chiral
analogues.
1
0.52
0.31
39
25
26
27
28
i-Pr
Et
Me
H
0.92
2.0
4.3
0.61
0.61
2.2
25
21
Synthesis of analogues 37 and 38 are outlined in Scheme 3.
Alcohols 15 and 16 obtained in a similar manner as shown in
9.9
7.8
a
See Ref. 23 for detailed description. n = 1 (Ref. 24).
See Ref. 21 for detailed description.
Scheme
1 were protected using tert-butyldimethylsilyl (TBS)
b
groups to afford 17 and 19, which were converted to 18 and 20

K. Kobayashi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4729–4732
4731
Table 2
Table 3
SAR of 4- and 5-substituents on pyrazole ring
SAR of hydrophilic substituents at 1 position on pyrazole ring
Compds
R1
R2
ORL1
Antagonisma
IC50 (nM)
HH
bindinga
IC50 (nM)
stabilityb
%
Compds
R
ORL1 bindinga Antagonisma HH stabilityb Log
c
remaining
IC50 (nM)
IC50 (nM)
% remaining
D7.4
31
36
37
38
39
Me
3.1
2.5
56
1.9
<1
1.4
1.3
1.5
CH2CH2SO2Me 33
27
29
Me
Me
4.3
6.4
2.2
6.5
25
CH2C(O)Me
CH2CH2OH
CH2CH2CN
12
15
8.2
4.6
77
a
See Ref. 23 for detailed description. n = 1 (Ref. 24).
See Ref. 21 for detailed description.
Measured by shake-flask method.
b
c
30
31
32
Me
Me
Me
15
3.1
17
13
improved by only this approach, we shifted our efforts toward
the modification of other sites using 27 as a template.
Metabolite identification study of 27 revealed that the major
metabolic pathway in human hepatocytes was hydroxylation on
the left pyridine moiety or the methyl group at the 4 position of
the pyrazoles moiety. Therefore, modification of these parts was
performed (Table 2). We first replaced the metabolically labile
methylpyridine with a fluorobenzene ring. As a result, 3,5-difluoro-
benzene analogue 31 exhibited potent ORL1 activity in comparison
with 3,4-difluoro (29) or 3,4,5-trifluoro (30) analogues. This modi-
fication led to the improvement of metabolic stability in human
hepatocytes as expected. Further optimization of 3,5-disubstituted
benzene moiety resulted in the impaired intrinsic potency (32 and
33). We thus examined the effects of 4-substituents on the pyra-
zole ring. Although 4-chloro (34) and 4-methoxymethyl (35)
analogues showed equipotent ORL1 affinity as compound 31, the
metabolic stability decreased slightly.
2.5
56
33
34
Me
210
9.3
1.4
At this stage, we again examined the effects of the lower alkyl
region. SAR studies were carried out using analogues that incorpo-
rated hydrophilic alkyl groups (Table 3). Introduction of the substi-
tuent with a sulfone (36), a ketone (37), and an alcohol (38)
reduced the ORL1 binding affinity. In contrast, when a cyanoethyl
group was introduced, compound 39 displayed better metabolic
stability than 31 while retaining good ORL1 binding affinity and
antagonistic activity, which can be attributed to the increased
hydrophilicity of 39 (log D7.4 = 1.5) in comparison to 31
(log D7.4 = 1.9). Consequently, we selected compound 39 for further
evaluation.
Cl
2.5
1.0
40
39
35
CH2OMe
a
See Ref. 23 for detailed description. n = 1 (Ref. 24).
See Ref. 21 for detailed description.
b
As shown in Table 4, compound 3925 showed excellent selectiv-
ity over other opioid receptors and hERG potassium channel.26,27 In
a standard panel for off-target activity, compound 39 did not
display significant affinity for the other 163 receptors and ion
ORL1 affinity. Surprisingly, analogue 28 without any substituents
at the 1 position also exhibited nanomolar binding affinity. How-
ever, analogues 27 and 28 still had poor metabolic stability in
human hepatocytes. Because the metabolic stability was not
Table 4
Biological profiles of 39
Binding IC50 (nM)
da
>10,000 9800 >10,000 9500
Human P-gp transport ratioc
1.2
Pharmacokinetic parametersd
ORL1 la
ja
hERGb
Species iv CLp (ml/min/Kg) Vdss (L/Kg) PO AUC0-inf
(
lM h) Cmax (lM) t1/2 (h) F (%)
8.2
Rat
Dog
83
37
5.2
3.5
0.3
1.3
0.40
2.6
0.7
2.8
21
62
a
Displacement of [3H]diprenorphin (
l
j l-, j- and d-opioid receptors,
), [3H]U69593 ( ) and [3H]naltrindole binding in CHO cells stably expressing cloned human
respectively.
b
Displacement of [35S]-radiolabeled MK499 in membranes derived from HEK 293 cells stably transfected with hERG gene and expressing the IKr channel protein (Ref.
26,27).
c
Transport ratio: B–A/A–B (Ref. 28).
Oral dose 3 mg/kg, and IV dose 1 mg/kg. The values represent the mean (n = 3).
d

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K. Kobayashi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4729–4732
Table 5
was moved forward as a clinical development candidate, labeled
as MK-1925.
In vivo antagonistic activity of 39
po dose
(mg/kg) (% reversal)
In vivo antagonisma
Brain penetrabilityb
Acknowledgements
Plasma (
l
M) Brain (nmol/g brain) b/p ratio
3
10
30
17
81
112
0.016
0.315
1.627
0.035
0.516
3.017
3.42
1.62
1.84
We would like to acknowledge the contributions of the follow-
ing individuals to this work: T. Azuma-Kanoh, H. Nambu, N. Sakai,
T. Inoue, D. Ichikawa, S. Okuda, N. Ami, M. Fukushima and M.
Nishino.
a
Data shows antagonistic activity of 39 against the reduction in locomotor
activity produced by ORL1 agonist for 60 min in mice. Values are expressed as %
reversal of the agonist response.
b
At 60 min after oral administration in mice.
References and notes
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nist (the receptor that reached over 50% at 10 lM was the choline
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out, and 39 displayed fair PK profiles in rats and dogs. Our modifi-
cation to increase metabolic stability resulted in improved oral bio-
availability in rats (F = 21%) in comparison with the lead compound
1 (F = 3.2%).
Furthermore, in vivo antagonist activity against the reduction in
locomotor activity induced by ORL1 agonist29 was determined. As
shown in Table 5 and Figure 2, when dosed in mice orally, 39
exhibited in vivo antagonistic activity in a dose-dependent man-
ner, and complete reversal was observed at 30 mg/kg po, thus
reflecting its good brain penetrability.
In conclusion, SAR studies performed in the arylpyrazole series
related to 1 with the aim of reducing metabolism led to the iden-
tification of compound 39, which exhibited good ORL1 activity and
improved metabolic stability. This was achieved by replacing the
lower pyridine moiety with a cyanoethyl group and substituting
the left 3-fluoro-2-methylpyridine with 3,5-difluorobenzene. Com-
pound 39 showed good selectivity over other opioid receptors and
the hERG channel. In addition, 39 was orally active in the in vivo
locomotor test as well as good brain penetrability in mice. Based
on the profiles reported in this communication, this compound
20. Kobayashi, K.; Uchiyama, M.; Ito, H.; Takahashi, H.; Yoshizumi, T.; Sakoh, H.;
Nagatomi, Y.; Asai, M.; Miyazoe, H.; Tsujita, T.; Hirayama, M.; Ozaki, S.; Tani, T.;
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hepatocytes.
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23. Binding affinities to ORL1 were determined by displacement of [125I]Tyr14-NC/
OFQ, and antagonist activities were measured by the
method.
[ cS binding
35S]GTP
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Kato, T.; Ohta, H.; Iwasawa, Y. J. Med. Chem. 1999, 42, 5061) was used as an
internal control across all assay plates for data validation.
25. Analytical data of 39 L
-tartaric acid salt: 1H NMR (400 MHz, CD3OD) d [ppm]
1.99 (m, 3H), 2.07 (s, 3H), 2.20 (m, 2H), 2.34 (m, 1H), 2.51 (m, 1H), 3.04 (t, 2H,
J = 6.3 Hz), 3.87 (m, 1H), 4.32 (s, 2H), 4.35 (t, 2H, J = 6.3 Hz), 5.21 (m, 1H), 7.11
(m, 2H), 7.18 (m, 1H). MS (ESI+): m/z 363.2 [M+H]+.
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S. D.; Salata, J. J.; Jurkiewicz, N. K.; Sanguinetti, M. C.; Stein, R. B.; Gehret, J. R.;
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29.
A
non-peptidyl synthetic agonist (WO03/10168; 0.3 mg/kg) was
subcutaneously administered.