Discovery of novel N-substituted oxindoles as selective M1 and M4 muscarinic acetylcholine receptors partial agonists

Article abstract of DOI:10.1021/ml300372f

Activation of the M₁ and M₄ muscarinic acetylcholine receptors is thought to play an important role in improving the symptoms of schizophrenia. However, discovery of selective agonists for these receptors has been a challenge, consid

Full text of DOI:10.1021/ml300372f

Letter
pubs.acs.org/acsmedchemlett
Discovery of Novel N‑Substituted Oxindoles as Selective M1 and M4
Muscarinic Acetylcholine Receptors Partial Agonists
Takaaki Sumiyoshi,*,† Takeshi Enomoto, Kentaro Takai, Yoko Takahashi, Yasuko Konishi,
Yoshiharu Uruno, Kengo Tojo, Atsushi Suwa, Harumi Matsuda, Tomokazu Nakako, Mutsuko Sakai,
Atsushi Kitamura, Yasuaki Uematsu, and Akihiko Kiyoshi
Drug Discovery Division, Dainippon Sumitomo Pharma Co. Ltd., 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan
S
* Supporting Information
ABSTRACT: Activation of the M1 and M4 muscarinic
acetylcholine receptors is thought to play an important role
in improving the symptoms of schizophrenia. However,
discovery of selective agonists for these receptors has been a
challenge, considering the high sequence homology and
conservation of the orthosteric acetylcholine binding site
among muscarinic acetylcholine receptor subtypes. We report
in this study the discovery of novel N-substituted oxindoles as
potent muscarinic acetylcholine receptor partial agonists
selective for M1 and M4 over M2, M3, and M5. Among these oxindoles, compound 1 showed high selectivity for the M1 and
M4 receptors with remarkable penetration into the central nervous system. Compound 1 reversed methamphetamine- and
apomorphine-induced psychosis-like behaviors with low potency to extrapyramidical and peripheral side effects.
KEYWORDS: muscarinic acetylcholine receptor, selective agonist, partial agonist, schizophrenia, oxindole
chizophrenia is a complex psychiatric disorder character-
ized by three major symptoms: (i) positive (e.g.,
receptors modulate the dynamics of cholinergic and dopami-
nergic neurotransmission and that loss of M4 function results in
a state of dopamine hyperfunction.14 In addition, the M1
agonistic activity of N-desmethyl clozapine, a major metabolite
of clozapine, suggests that M1 may also play an important role
in the antipsychotic effects of clozapine.15 Put together, these
findings suggest that selective activators of M1 and M4 may
provide a novel treatment strategy for schizophrenia patients.
Despite the promising results shown by xanomeline in
clinical trials of Alzheimer’s disease and schizophrenia, there are
concerns over its peripheral side effects, especially gastro-
intestinal (GI) distress and syncope, which may ultimately limit
the therapeutic potential of this agent. Administration of
xanomeline resulted in vomiting in more than 40% of patients
taking part in a clinical trial, terminating further development of
this compound. High blood pressure-derived syncope was
another undesirable effect observed with xanomeline.16 These
adverse effects are thought to be primarily due to activation of
the peripheral M3 mAChR (see Table S1 in the Supporting
Information) and other off-targets. Identification of a highly
selective M1 and M4 agonist is challenging, because of the high
sequence homology and conservation of the orthosteric ACh
binding site among the mAChR subtypes.17 The discovery of
AC-42 led us to identify selective allosteric ligands to M1
mAChR.18 Christopoulos and co-workers reported that M4
S
hallucinations, delusions, and excitement), (ii) negative (e.g.,
flattened affect, apathy, and social withdrawal), and (iii)
cognitive (e.g., deficit in working memory, executive function,
attentional processing, and memory).1,2 Dopamine D2
antagonists are currently used to treat schizophrenia,
particularly its positive symptoms. However, we have an
ongoing need for alternative approaches to the current clinical
treatment of schizophrenia because of a dose limitation of D2
antagonists by extrapyramidal side effects (EPS)3 and non-
responders to D2 antagonists.4 Therefore, new antipsychotics
with a new mechanism of action have been desired in the
treatment of schizophrenia.
Many of the important central actions of acetylcholine
(ACh) are mediated by muscarinic ACh receptors (mAChRs).5
To date, five mAChR subtypes (M1−M5) have been identified
and are considered to play important roles in the peripheral and
central nervous systems (CNS).6−8 M1 and M4 receptors are
the most heavily expressed in CNS and represent attractive
therapeutic targets for cognition, Alzheimer’s disease, and
schizophrenia.9 In fact, clinical trials with xanomeline (Figure
S1 in Supporting Information), an M1 and M4 preferring
orthosteric agonist, demonstrated the efficacy of this agent as
both an antipsychotic and a cognition-enhancing agent.10,11
Data from mAChR-knockout mice have also suggested that
selective M1 agonists would be beneficial for psychosis and
cognition,12 whereas M4 agonists would provide an anti-
psychotic effect for the treatment of schizophrenia.13 This
proposal is further supported by recent studies showing that M4
Received: November 1, 2012
Accepted: January 25, 2013
Published: January 27, 2013
© 2013 American Chemical Society
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Letter
mAChR also has an allosteric binding site by using M4 mAChR
positive allosteric modulator (PAM) LY2033298.19 To date, M1
mAChR allosteric agonists, M1 PAMs, and M4 mAChR PAMs
have been reported (Figure S1 in the Supporting Informa-
tion).18 Especially, the reports of centrally active modulators
TBPB,20 VU035701721 (M1 agonist), VU045694022 (M1
PAM), and VU015210023,24 (M4 PAM) indicate that selective
activations of central M1 and M4 mAChRs are an attractive
strategy for the treatment of CNS diseases. However, PAMs are
not suitable for dual activation of M1 and M4 mAChRs due to
their high selectivity. As such, the development of agents that
are selective for M1 and M4 mAChRs with druggable safety
profiles has until now been a challenge.
Table 1. Modification of the Central Amine Linker of
Compound 3
Here, we describe a novel M1 and M4 selective partial agonist
(1) that reverses methamphetamine-induced hyperlocomotion
and improves apomorphine-induced climbing behavior and
sensorimotor gating deficits. At doses providing antipsychotic
effects, compound 1 does not produce significant GI distress,
cardiovascular side effects, and EPS. These important findings
suggest that it is possible to overcome major hurdles to the
clinical development of mAChR agonists.
a
The maximum efficacy of each subtype of ACh was defined as 100%.
The concentration of the test compound was 3 μM.
In our search for selective M1 and M4 agonists, the hit
compound 2 was identified as a potential candidate. However,
this compound showed weak M4 agonistic activity. To
overcome this problem, we replaced a dimethyl olefin unit in
compound 2 by the N-carbethoxy piperidine unit known as a
pharmacophore of M4 agonists (Figure 1). The obtained lead
antagonists (haloperidol and risperidone)-induced catalepsy
decreased in M4 knockout mice.25 This report indicates the
possibility that a combination of an M4 mAChR agonist and a
D2 antagonist may lower the threshold of catalepsy. In fact,
additional administration of xanomeline (30 mg/kg, sc)
worsened haloperidol-induced catalepsy in rats (see Figure S2
in the Supporting Information). On the basis of the importance
of D2 antagonism in the treatment of schizophrenia, our efforts
aimed at the discovery of M4 mAChR agonists that do not
worsen EPS when used in combination with D2 antagonists. To
minimize EPS risk for our M4 agonists, while maintaining
antipsychotic activity, we considered lowering compounds
intrinsic activity (IA) without significantly lowering their EC50
values. The use of M4 partial agonists can be supported by the
findings that sabcomeline, a partial M4 agonist (M4 EC50, 5.7
nM; IA, 28%), reverses methamphetamine-induced hyper-
locomotion in rats (see Table S1 in the Supporting
Information). The idea that the bulky hetero ring of
sabcomeline represents an essential structural feature of this
agent led us to modify the critical N-carbethoxy piperidine unit
in our compounds to a substituted N-carbethoxy piperidine
unit (Table 2). The report of M1 agonists with carbethoxy
tropane unit also supported our working hypothesis that
introduction of substituents to N-carbethoxy piperidine unit
was allowed to M1 and M4 mAChR agonistic activities.26
Introduction of a methyl group at the 4-position of the
piperidine did not lower IA (8). On the other hand,
introduction of hydrophilic substituents at the same position
significantly lowered both EC50 and IA (9 and 10). However,
compound 1 with an exo tropane structure showed 42% IA with
low EC50 (29 nM). This final modification achieved the desired
M4 partial agonistic activity with good selectivity for M1 and M4
mAChR over M2, M3, and M5 (see Figure S3 in the Supporting
Information). Increasing the concentration of compound 1
from 0.001 to 10 μM partially activated the M1 (EC50, 12 nM;
IA, 60%) and M4 mAChRs. On the other hand, compound 1
showed very weak or negligible activation of M2, M3, and M5
even at the highest dose tested (30 μM). Compound 1
selectivity was next evaluated in the radioligand-binding panel
assay, which consists of a broad range of G protein-coupled
receptors, ion channels, enzymes, and transporters. Compound
Figure 1. Strategy to enhance hit compound 2 M4 agonistic activity.
aM4 mAChR agonistic activity was evaluated by a calcium mobilization
assay.
compound 3 showed moderate M4 agonistic activity (33% at 3
μM) with good selectivity for M1 and M4 over M2, M3, and M5
(Table 1). To potentiate the M4 agonistic activity of compound
3, we modified the central amine linker. Replacement of the
piperidine linker of compound 3 by an azetidine linker (4)
resulted in moderate M4 agonistic activity (46% at 3 μM). On
the other hand, the use of an azepane linker (5) significantly
decreased M4 agonistic activity. Finally, the pyrrolidine linker
was found to be the most potent linker, giving the S-substituted
compound 7, which was more potent than R-substituted
compound 6. Compound 7 was therefore selected for further
optimization.
Xanomeline did not induce EPS in clinical trials, indicating
that single use of M4 mAChR agonists does not produce EPS.
However, Fink-Jensen and co-workers reported that D2
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ACS Medicinal Chemistry Letters
Letter
afforded compound 1. The aldehyde 17 was prepared by
epoxidation of the N-carbethoxy nortropinone 15 followed by
formation of a formyl group using Lewis acid.
Table 2. Structure and Activity Relationship at the N-
Carbethoxy Piperidine Unit
Next, the antipsychotic effects of compound 1 were evaluated
in rodents. Subcutaneous administration of compound 1 (1, 3,
and 10 mg/kg) significantly reversed methamphetamine-
induced hyperlocomotion in rats (Figure 2A). Furthermore,
coadministration of subeffective doses of risperidone (0.6 mg/
a
The maximum efficacy of each subtype of ACh was defined as 100%.
The concentration of the test compound was 3 μM. NT, not tested.
1 (3 μM) produced less than 30% inhibitions against all targets
in the panel, except for sigma receptor (62% inhibition at 3 μM;
see Table S2 in the Supporting Information). Furthermore,
compound 1 (3 μM) did not show any agonist and antagonist
activities in the calcium mobilization assays using the cells
transiently expressing dopamine and serotonin receptors (see
Table S3 in the Supporting Information). Compound 1
improved human ether-a-go-go related gene (hERG) channel
inhibition (IC50, 2.4 μM) over compound 7 (IC50, 0.8 μM).
Interestingly, compound 1 (1 mg/kg, sc) showed good CNS
penetration (brain/blood ratio = 2.0) in rat pharmacokinetic
study. These encouraging results made compound 1 an
excellent candidate for in vivo animal studies.
Compound 1 was synthesized as shown in Scheme 1. The N-
pyrrolidinyl oxindole 14 was prepared by amide formation of 2-
bromophenylacetic acid 11 and (3S)-1-Boc-3-aminopyrrolidine
followed by palladium coupling.27 Deprotection of the Boc
group followed by reductive amination with aldehyde 17
a
Scheme 1
Figure 2. Effects of coumpound 1 and risperidone on methamphet-
amine-induced hyperactivity (A and B), catalepsy (C), apomorphine-
induced climbing (D), and PPI deficits (E) in rats and mice. (A and B)
Compound 1 or vehicle was injected into rats 30 min before injection
with methamphetamine (0.1 mg/kg, ip). Risperidone or vehicle was
administered 60 min before the injection with methamphetamine.
Locomotor activity was measured for 80 min from 10 min after the
methamphetamine injection. Values correspond to the mean SEM
(n = 6). Veh, vehicle; **P < 0.01 vs vehicle/methamphetamine-treated
group (Dunnett test). (C) Risperidone or vehicle and compound 1 or
vehicle were administered 60 min before the catalepsy tests in rats (n =
6). NS, not significant (Dunnett test). (D) Compound 1 or vehicle
was first given to mice, and 30 min later, apomorphine (1 mg/kg, sc)
was injected. The climbing behavior was observed for 20 min from 10
min after the injection with apomorphine (n = 5). **P < 0.01 vs
vehicle/apomorphine-treated group (Dunnett test). (E) Compound 1
or vehicle was first injected to rats, and 15 min later, apomorphine (0.5
mg/kg, sc) or vehicle was injected. The PPI test was preformed 15 min
after the injection with apomorphine or vehicle (n = 8). ##P < 0.01 vs
vehicle/vehicle-treated group (unpaired t test). **P < 0.01 vs vehicle/
apomorphine-treated group (Dunnett test).
a
Reagents and conditions: (a) (3S)-1-Boc-3-aminopyrrolidine, WSC,
HOBt, DIPEA, CH2Cl2, room temperature. (b) Pd(OAc)2, X-Phos,
PhB(OH)2, K2CO3, t-BuOH, reflux. (c) TFA, CH2Cl2, room
temperature. (d) Compound 17, NaBH(OAc)3, CH2Cl2, room
temperature. (e) Me3SOI, KOtBu, THF, reflux. (f) BF3·OEt2, THF,
room temperature.
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Letter
kg, po) and compound 1 (0.6 mg/kg, sc) significantly improved
hyperlocomotion in rats (Figure 2B). On the other hand,
compound 1 given alone at 10 and 30 mg/kg (sc) did not
induce catalepsy (Figure 2C). Moreover, compound 1 did not
significantly worsen risperidone (10 mg/kg, po)-induced
catalepsy in rats (10 mg/kg, po, Figure 2C). However, further
investigation is necessary to decide the EPS potency of the
combination of M4 mAChR partial agonists and D2 antagonists.
In addition, administration of compound 1 reversed apomor-
phine-induced climbing behavior in mice (Figure 2D) and
impairment of prepulse inhibition (PPI) in rats (Figure 2E),
which are claimed to have both face and construct validity vis a
vis schizophrenia symptomology.28
AUTHOR INFORMATION
Corresponding Author
*Tel: +81-6-6337-5903. Fax: +81-6-6337-6501. E-mail: takaaki-
sumiyoshi@ds-pharma.co.jp.
■
Present Address
†33-94 Enoki-cho, Suita, Osaka 564-0053, Japan.
Author Contributions
T.S. conceived and directed medicinal chemistry. T.E. directed
pharmacology. K.T., Y.T., Y.K., Y.U., K.T., and A.S. synthesized
compounds, and T.S. wrote the manuscript. T.E., H.M., T.N.,
and M.S. performed mAChR pharmacology. A.K. contributed
to PK studies. Y.U. and A.K. performed safety studies.
Notes
Next, we evaluated the cholinergic side effects of compound
1. Administration of xanomeline (10 and 30 mg/kg, sc)
induced significant salivation in mice (Figure S5 in the
Supporting Information). On the other hand, no significant
increase in salivation was seen following treatment with
compound 1 (10 and 30 mg/kg, sc). In addition, tissue assays
showed that compound 1 (3 μM) induced only weak
contraction of guinea pig ileum as compared to sabcomeline
and xanomeline (Table S4 in the Supporting Information).
These findings confirm high selectivity of compound 1 for M1
and M4 over the M3 mAChR ex vivo and in vivo. In addition,
administration of compound 1 (100 mg/kg, sc) did not induce
seizure, a well-known side effect of mAChR agonists, such as
pilocarpine, in mice (Table S5 in the Supporting Informa-
tion).29
Finally, we evaluated the cardiac effects of xanomeline and
compound 1 in telemetered cynomolgus monkey. Admin-
istration of xanomeline at 1 and 3 mg/kg increased mean blood
pressure (MBP) and prolonged QRS duration and QTc interval
(see Figure S6 and Tables S6 and S8 in the Supporting
Information). On the other hand, single subcutaneous
administration of compound 1 at 1.5 and 6 mg/kg did not
elevate MBP and showed no effect on QRS duration or QTc
interval. Both xanomeline and compound 1 increased heart
rate. Xanomeline also induced vomiting and drowsiness,
although these side effects were not observed with compound
1. Salivation and miosis were observed with both xanomeline
and compound 1. Toxicokinetic studies in monkey supported
the plasma concentrations of compound 1 as high enough to
reverse methamphetamine-induced hyperlocomotion in rats
(Table S6 and S7 in the Supporting Information). These
findings indicate that the potential cardiac toxicity and GI
distress of compound 1 are lower than those of xanomeline.
In summary, we have found compound 1 as a structurally
novel dual M1 and M4 mAChRs selective partial agonist.
Compound 1 reversed the psychosis-like behavior and showed
low potency to EPS. In addition, compound 1 can, additively
with risperidone, reverse methamphetamine-induced hyper-
locomotion in rats. Moreover, GI distress and cardiac toxicity of
compound 1 were less than those of xanomeline. These
findings support that compound 1 is suitable for further drug
development.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Naohiro Nishimura and Chie Matsuba for HRMS
and Dr. Norio Fujiwara and Yoshiaki Ochi for useful
discussions.
ABBREVIATIONS
■
EPS, extrapyramidal side effects; ACh, acetylcholine; mAChR,
muscarinic acetylcholine receptor; CNS, central nervous
system; GI, gastrointestinal; PAM, positive allosteric modu-
lator; Boc, t-butoxy carbonyl; IA, intrinsic activity; hERG,
human ether-a-go-go related gene; WSC, water-soluble
carbodiimide; HOBt, 1-hydroxybenzotriazole; DIPEA, N,N-
diisopropylethylamine; t-BuOH, tertially butanol; TFA, tri-
fluoroacetic acid; THF, tetrahydrofuran; PPI, prepulse
inhibition; MBP, mean blood pressure
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* Supporting Information
Experimental procedures for pharmacological experiments and
synthetic procedures and characterization data for the
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