Discovery of N-sulfonyl-7-azaindoline derivatives as potent, orally available and selective M4 muscarinic acetylcholine receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2014.04.083

We designed and synthesized novel N-sulfonyl-7-azaindoline derivatives as selective M₄ muscarinic acetylcholine receptor agonists. Modification of the N-carbethoxy piperidine moiety of compound 2, an M₄ muscarinic acetylcholine receptor (mAChR)-preferring agonist, led to compound 1, a selective M₄ mAChR agonist. Compound 1 showed a highly selective M₄ mAChR agonistic activity with weak hERG inhibition in vitro. A pharmacokinetic study of compound 1 in vivo revealed good bioavailability and brain penetration in rats. Compound 1 reversed methamphetamine-induced locomotor hyperactivity in rats (1-10 mg/kg, po).

Full text of DOI:10.1016/j.bmcl.2014.04.083

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of N-sulfonyl-7-azaindoline derivatives as potent, orally
available and selective M4 muscarinic acetylcholine receptor agonists
Atsushi Suwa a, Yasuko Konishi a, Yoshiharu Uruno a, Kentaro Takai a, Tomokazu Nakako b,
Mutsuko Sakai a, Takeshi Enomoto b, Yoshiaki Ochi b, Harumi Matsuda b, Atsushi Kitamura b,
Yasuaki Uematsu a, Akihiko Kiyoshi b, Takaaki Sumiyoshi a,
⇑
a Drug Research Division, Dainippon Sumitomo Pharma Co., Ltd, 3-1-98, Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan
b Drug Research Division, Dainippon Sumitomo Pharma Co., Ltd, 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized novel N-sulfonyl-7-azaindoline derivatives as selective M4 muscarinic
acetylcholine receptor agonists. Modification of the N-carbethoxy piperidine moiety of compound 2, an
M4 muscarinic acetylcholine receptor (mAChR)-preferring agonist, led to compound 1, a selective M4
mAChR agonist. Compound 1 showed a highly selective M4 mAChR agonistic activity with weak hERG
inhibition in vitro. A pharmacokinetic study of compound 1 in vivo revealed good bioavailability and
brain penetration in rats. Compound 1 reversed methamphetamine-induced locomotor hyperactivity
in rats (1–10 mg/kg, po).
Received 28 January 2014
Revised 14 April 2014
Accepted 22 April 2014
Available online xxxx
Keywords:
Muscarinic acetylcholine receptor agonist
M4 muscarinic acetylcholine receptor
Subtype-selective agonist
Schizophrenia
Ó 2014 Elsevier Ltd. All rights reserved.
N-Sulfonyl-7-azaindoline
Psychosis is the key features in schizophrenia, and is currently
treated with dopamine D2 receptor antagonists.1 However, there
is an ongoing need for alternative approaches considering non-
responders to D2 receptor antagonists.2 A pilot clinical trial with
xanomeline (Fig. 1), an M1 and M4 muscarinic acetylcholine recep-
tor (mAChR)-preferring agonist, has demonstrated the efficacy of
this agent as both an antipsychotic and a cognition-enhancing
agent in schizophrenic patients who had exhibited poor response
with previous antipsychotic treatment.3 While it has been widely
accepted that M1 mAChR is key subtype for cognitive function,4
animal studies with M1 or M4 mAChR-knockout mice have sug-
gested that M4 mAChR, but not M1 mAChR, predominantly medi-
ates antipsychotic efficacy of xanomeline.5 It is further supported
by recent studies showing that M4 mAChRs regulates dopaminer-
gic neurotransmission in the nucleus accumbens that is the key
brain region for psychosis.4 Although the development of xanome-
line has been halted by the peripheral side effects via M3 mAChR
activation,6 M1 and M4 mAChR-dual agonist would be promising
both for psychosis and cognitive deficits in schizophrenia. How-
ever, there is a large hurdle to achieve the optimal balance
between M1 and M4 mAChR agonism for developing dual-agonists.
It is essential because a recent clinical study have shown that even
M1 mAChR-selective agonism might induce some dose-related
muscarinic side effects.7 Furthermore, animal studies have shown
that over-activation of M1 mAChR might have a risk for seizure.8
Alternative approach especially for psychosis is M4 mAChR-selec-
tive agonist or positive allosteric modulators (PAM) such as
LY2033298.9 Brady and colleagues have identified, VU0152100
(Fig. 1), a brain-penetrant M4 mAChR PAM, reversed amphet-
amine-induced locomotor hyperactivity without causing sedation
in rats.10 On the other hand, identifying agonists selective for M4
mAChR has so far been challenging because of limited resources
in the literature. Here, we describe the identification of novel N-
sulfonyl-7-azaindoline derivatives as potent, orally available, and
selective M4 mAChR agonists (Fig. 1).
Among the compounds synthesized in our drug discovery pro-
gram for M1 and M4 mAChRs selective agonists,11 we picked up
the N-sulfonyl-7-azaindoline compound 2 as an M4 mAChR prefer-
ring agonist. A calcium mobilization assay12 confirmed that com-
pound 2 M4 mAChR agonistic activity (88% at 0.3
potent than its M1 mAChR agonistic activity (20% at 0.3
l
M) is more
M)
l
(Table 1). This finding indicates that the N-methansulfonyl-7-
azaindoline scaffold increases M4 mAChR preference. However,
the selectivity of compound 2 for M4 (EC50 = 93 nM, IA = 83%)
versus M1 mAChR (EC50 = 352 nM, IA = 59%) was not so high (Fig. 2).
⇑
Corresponding author at present address: Department of Life Science and
Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai Univer-
sity, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan. Tel.: +81 663681773.
E-mail address: t-sumiyo@kansai-u.ac.jp (T. Sumiyoshi).
http://dx.doi.org/10.1016/j.bmcl.2014.04.083
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Suwa, A.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.083

2
A. Suwa et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
O
Me
N
Me
N
NH2
N
OEt
Me
O
N
N
O
Me
N
S
NH2
Cl
O
S
HN
Me
N
S
HN
MeO
N
S
O
Me
N
OMe
Me
O
Xanomeline
LY2033298
1
VU0152100
Figure 1. Structures of M4 mAChR agonists and modulators.
O
Table 1
N
SAR at the N-carbamoyl piperidine moiety of the N-sulfonyl-7-azaindoline derivatives
O
OEt
R
N
N
or
OEt
Me
O
N
N
OEt
N
N
Introduction of bulky substituents
N
S
O
S
Me
N
O
O
Me
O
Compound
R
Agonistic activity
(% effect)
hERG
inhibition12
2
(IC50: lM)
a
a
a
a
b
M1
20
M2
12
NT
M3
M4
88
4
M5
3
Figure 3. Strategy to increase selectivity for M4 mAChR.
NCO2Et
NCO2Et
2
3
7
1.90
NT
Table 2
NT
NT
NT
PK and pharmacological profiles of compound 1
Me
Me
Me
Me
Compound 1
1
4
5
6
5
3
4
94
52
3
3
3.63
17
NCO2Et
PK profiles
3
2
1
NT
NT
NT
NCO2Me
NCO2i-Pr
NCO2t-Bu
P-gp efflux ratio
BA (%)
Brain/plasma ratio
Pharmacological profiles
0.9
49
0.9
NT
NT
NT
NT
NT
NT
2.75
NT
M4 EC50 (nM)
M4 IA(%)
13
81
2
Binding assay
NT: Not tested.
a
a
Maximum efficacy of each receptor subtype was defined as 100%. Concentration
M.
Concentration of the test compound was 10
a1DR inhibition (%)
0
0
a
of the test compound was 0.3
l
D2R inhibition (%)
H1R inhibition (%)
b
a
l
M.
27
a
Concentration of the test compound was 3 lM.
To further increase selectivity for the M4 mAChR, we introduced into
compound 2 another moiety that decreases M1 mAChR agonistic activ-
ity. We focused on the N-carbethoxy piperidine moiety (Fig. 3) because
Lindsley and co-workers reported that a tropane unit partializes M1
mAChR agonistic activity.13
The structure–activity relationships (SARs) of the prepared N-
sulfonyl-7-azaindoline derivatives are summarized in Table 1.
Replacement of the piperidine by a tropane depleted M4 mAChR
agonistic activity (compound 3). Assuming that a tropane unit is
too large to maintain M4 mAChR agonistic activity, we designed
compound 1 with a methyl group at the 4-position of the N-car-
bethoxypiperidine. As expected, compound 1 decreased M1 mAChR
agonistic activity, while maintaining M4 mAChR agonistic activity.
A further SAR study of the carbethoxy moiety revealed that rela-
tively small alkyl group can maintain M4 mAChR agonistic activity
O
N
OEt
N
N
S
O
N
Me
O
2
Figure 2. Functional activity of compound 2. Agonistic activity on M1–5 mAChRs. The maximum efficacy of each subtype of ACh was defined as 100%.
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A. Suwa et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
(compound 4), while bulky alkyl groups diminish M4 mAChR ago-
nistic activity (compounds 5 and 6). Considering that compounds 1
showed only micromolar inhibition of human Ether-Go-Go-
Related-Gene (hERG), we selected this compound for further eval-
uation as a potent M4 mAChR-selective agonist.
Next, we determined the pharmacokinetic (PK) of compound 1
both in vitro and in vivo (Table 2). Compound 1 was not a substrate
of p-glycoprotein (P-gp). As for PK in vivo, compound 1 showed
high bioavailability (BA, 49%, 1 mg/kg, po) in rats with good brain
penetration (brain/plasma ratio: 0.9). These findings indicate that
compound 1 is a druggable candidate.
Finally, we evaluated compound 1 pharmacological potential. In
a radioligand-binding panel assay, compound 1 had weak affinity
to human dopamine D2, histamine H1 and adrenaline a1D receptors
(Table 2). In addition, compound 1 showed a highly selective M4
mAChR agonistic activity (EC50 = 13 nM, IA = 81%) with no activa-
tion of the M1–3 and M5 mAChRs (Fig. 4A). In addition, compound
1 competitively antagonizes the M1 mAChR functional response
to ACh (Fig. 4B). These results indicate that introduction of a
methyl group at the 4-position of the N-carbethoxypiperidine led
to a completely abolished M1 mAChR agonistic activity. Next, we
evaluated the antipsychotic potency of compound 1 in rats. Oral
administration of compound
1 reversed methamphetamine-
induced hyperlocomotion in dose dependent manner (1–10 mg/kg,
Fig. 4C).12 As far as we know, this is the first agonist highly selective
for M4 mAChR which reverses psychosis-like behavior in rodents.
Compound 1 is a potent, orally available, brain-penetrant and highly
selective M4 mAChR agonist with a druggable safety profile.
Although further investigation is necessary, the results of this study
demonstrate that M4 mAChR-selective agonist compound 1 is a
promising candidate for the treatment of schizophrenia. Further-
more, it would be useful as a pharmacological tool for investigating
the physiological role of M4 mAChR in vivo.
The 3-spiro-7-azaindoline derivatives 2–6 were synthesized
as shown in Scheme 1. Compound 714 was converted to the
sulfonamide 8. Deprotection of the benzyl group of 8 by catalytic
reduction afforded compound 9. Reductive amination with the
N-Boc-4-formylpiperidine 11 and aldehyde 12 afforded compounds
3 and 6. Boc deprotection followed by introduction of substituents
at the final step using appropriate carbamoyl chloride afforded
compounds 1, 4 and 5.15
Figure 4. Functional activity of compound 1. (A) Agonistic activity on M1-5 mAChRs.
The maximum efficacy of each subtype of ACh was defined as 100%. (B) Antagonism
for ACh-stimulated activity on M1 mAChR. M1 mAChR was expressed in CHO cells.
Both compound 1 and ACh (0.01
lM) were added at the same time. The maximum
efficacy of ACh (0.3 M) was defined as 100%. (C) Effects on methamphetamine-
l
induced hyperactivity in rats. Compound 1 or vehicle was administered into rats
60 min before injection with methamphetamine (0.1 mg/kg, ip). Locomotor activity
was measured for 80 min from 10 min after the methamphetamine injection.
Values correspond to the mean SEM (n = 6). ⁄⁄P <0.01 versus vehicle/metham-
phetamine-treated group (Dunnett test).
In summary, we have identified N-sulfonyl-7-azaindoline
derivatives as selective M4 mAChR agonists. Modification of the
Me
N
Me
N
Me
N
Bn
N
Me
OHC
NBoc
d
NH
NCO2R
H
N
Bn
N
NBoc
11
e
a
b
c
N
S
O
N
S
O
N
N
N
S
O
N
S
O
N
N
H
N
N
N
N
Me
Me
O
O
4
: R = Me
S
Me
O
Me
Me
O
O
1: R = Et
O
5: R = i-Pr
7
8
9
6
10
NCO2Et
H
N
OHC
NCO2Et
N
12
f
N
S
O
N
N
S
O
N
Me
O
Me
O
9
3
Scheme 1. Reagents and conditions: (a) methanesulfonyl chloride, N,N-diisopropylethylamine, CH2Cl2, rt, 17 h, 78%; (b) 10% Pd/C, HCO2NH4, MeOH, reflux, 12 h, 79%; (c) tert-
butyl 4-formyl-4-methylpiperidine-1-carboxylate 11, Ti(O-iPr)4, NaBH(OAc)3, CH2Cl2, reflux, 19 h, quant.; (d) trifluoroacetic acid, CH2Cl2, rt, 17 h, 87%; (e) methyl
chloroformate, ethyl chloroformate, or isopropyl chloroformate, N,N-diisopropylethylamine, CH2Cl2, rt, 1.5 h, 78%; (f) 12, NaBH(OAc)3, CH2Cl2, rt, 19 h, quant.
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4
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N-carbethoxy piperidine moiety of compound 2 led to the discov-
ery of compound 1, which showed selective M4 mAChRs agonistic
activity, high oral bioavailability, and good brain penetration in
rats. In addition, compound
1 reversed methamphetamine-
induced psychosis-like behavior in rats. These findings indicate
that compound 1 is a promising antipsychotic candidate with a
new mechanism of action.
Acknowledgment
8. Bymaster, F. P.; Carter, P. A.; Yamada, M.; Gomeza, J.; Wess, J.; Hamilton, S. E.;
Nathanson, N. M.; McKinzie, D. L.; Felder, C. C. Eur. J. Neurosci. 2003, 17, 1403.
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C.; Christopoulos, A.; Lazareno, S.; Birdsall, N. J.; Bymaster, F. P.; Felder, C. C.
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We are grateful to Keiko Bando for performing the elemental
analysis and Norio Fujiwara for his useful discussion.
10. Brady, A. E.; Jones, C. K.; Bridges, T. M.; Kennedy, J. P.; Thompson, A. D.;
Heiman, J. U.; Breininger, M. L.; Gentry, P. R.; Yin, H.; Jadhav, S. B.; Shirey, J. K.;
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11. Takai, K.; Inoue, Y.; Konishi, Y.; Suwa, A.; Uruno, Y.; Matsuda, H.; Nakako, T.;
Sakai, M.; Nishikawa, H.; Hashimoto, G.; Enomoto, T.; Kitamura, A.; Uematsu,
Y.; Kiyoshi, A.; Sumiyoshi, T. Bioorg. Med. Chem. Lett. 2014. in press. http://
dx.doi.org/10.1016/j.bmcl.2014.04.085.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.04.
083.
12. The procedures for calcium mobilization assays, hERG inhibition and
methamphetamine-induced hyperlocomotion in rats are described in
Sumiyoshi, T.; Enomoto, T.; Takai, K.; Takahashi, Y.; Konishi, Y.; Uruno, Y.;
Tojo, K.; Suwa, A.; Matsuda, H.; Nakako, T.; Sakai, M.; Kitamura, A.; Uematsu,
Y.; Kiyoshi, A. ACS Med. Chem. Lett. 2013, 4, 244.
13. Melancon, B. J.; Gogliotti, R. D.; Tarr, J. C.; Saleh, S. A.; Chauder, B. A.; Lebois, E.
P.; Cho, H. P.; Utley, T. J.; Sheffler, D. J.; Bridges, T. M.; Morrison, R. D.; Daniels, J.
S.; Niswender, C. M.; Conn, P. J.; Lindsley, C. W.; Wood, M. R. Bioorg. Med. Chem.
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15. Analytical data of compound 1 is described in Supporting information.
Please cite this article in press as: Suwa, A.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.04.083