Discovery of N-substituted 7-azaindoline derivatives as potent, orally available M1 and M4 muscarinic acetylcholine receptors selective agonists

Article abstract of DOI:10.1016/j.bmcl.2014.04.085

We designed and synthesized novel N-substituted 7-azaindoline derivatives as selective M₁ and M₄ muscarinic acetylcholine receptors (mAChRs) agonists. Hybridization of compound 2 with the HTS hit compound 5 followed by optimization of the N-substituents of 7-azaindoline led to identification of compound 1, which showed highly selective M₁ and M₄ mAChRs agonistic activity, weak human ether-a-go-go related gene inhibition, and good bioavailability in multiple animal species.

Full text of DOI:10.1016/j.bmcl.2014.04.085

Accepted Manuscript
Discovery of N-substituted 7-azaindoline derivatives as potent, orally available
M1 and M4 muscarinic acetylcholine receptors selective agonists
Kentaro Takai, Yasunao Inoue, Yasuko Konishi, Atsushi Suwa, Yoshiharu
Uruno, Harumi Matsuda, Tomokazu Nakako, Mutsuko Sakai, Hiroyuki
Nishikawa, Gakuji Hashimoto, Takeshi Enomoto, Atsushi Kitamura, Yasuaki
Uematsu, Akihiko Kiyoshi, Takaaki Sumiyoshi
PII:
S0960-894X(14)00442-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.085
BMCL 21578
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 January 2014
14 April 2014
22 April 2014
Please cite this article as: 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., Discovery
of N-substituted 7-azaindoline derivatives as potent, orally available M1 and M4 muscarinic acetylcholine receptors
selective agonists, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.
2014.04.085
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Discovery of N-substituted 7-azaindoline derivatives as potent, orally available M1 and M4 muscarinic acetylcholine receptors
selective agonists
Kentaro Takai,a Yasunao Inoue,b,‡ Yasuko Konishi,a Atsushi Suwa,a Yoshiharu Uruno,a Harumi Matsuda,b Tomokazu Nakako,b
Mutsuko Sakai,a Hiroyuki Nishikawa,b Gakuji Hashimoto,b Takeshi Enomoto,b Atsushi Kitamura,b Yasuaki Uematsu,a Akihiko
Kiyoshib and Takaaki Sumiyoshia,†,
*
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
‡ Present address: R & D Department, DSP Gokyo Food & Chemical Co. Ltd., 33-94 Enoki-cho, Suita, Osaka 564-0053, Japan
†Present address: Department of Life Science and Biotechnology, Fuculty of Chemistry, Materials and Bioenginieering, Kansai
University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan
Abstract
We designed and synthesized novel N-substituted 7-azaindoline derivatives as selective M1 and M4 muscarinic acetylcholine
receptors (mAChRs) agonists. Hybridization of compound 2 with the HTS hit compound 5 followed by optimization of the
N-substituents of 7-azaindoline led to identification of compound 1, which showed highly selective M1 and M4 mAChR agonistic
activity, weak human ether-a-go-go related gene inhibition, and good bioavailability in multiple animal species.
Keywords:
M1 muscarinic acetylcholine receptor, M4 muscarinic acetylcholine receptor, muscarinic acetylcholine receptor subtype-selective
agonist, schizophrenia, antipsychotic agents, 7-azaindoline derivative
Schizophrenia is a heterogeneous disease with a diverse symptomatology including positive symptoms, negative symptoms, and
cognitive deficits.1 However, current antipsychotics, dopamine and serotonin receptors antagonists, have limited efficacy especially
for negative symptoms and cognitive deficits.2, 3 In addition to dopaminergic and serotonergic systems, multiple line of evidence
have indicated that abnormality of neurotransmission via muscarinic acetylcholine receptors (mAChRs) is involved in the
pathophysiology of schizophrenia.4 Among the identified mAChR subtypes (M1–M5), M1 and M4 mAChRs are the most heavily
expressed in the central nervous system (CNS)5 and are considered attractive therapeutic targets for the treatment of schizophrenia
and Alzheimer’s disease.6 In fact, clinical trials with xanomeline (Figure 1), an M1 and M4 mAChR-preferring agonist, have
demonstrated the efficacy of this agent as both an antipsychotic and a cognition-enhancing agent in schizophrenia and Alzheimer
disease.7 It has been generally believed that M1 mAChR agonists would be beneficial for cognitive dysfunction,8 whereas M4
mAChR agonists would be useful for the treatment of psychosis.9 Therefore, M1 and M4 mAChR-dual agonism would represent
both antipsychotic and cognitive efficacy of xanomeline. In addition, the selectivity for M1 and M4 mAChRs versus M2 and M3
mAChRs would be essential, since M2 and M3 mAChRs could be mainly mediate peripheral cholinergic side effects. However,
identification of subtype-selective mAChRs agonists has been challenging, because of the high sequence homology and
conservation of the orthosteric ACh binding site among mAChR subtypes.10 A major breakthrough in this field is the recent
discovery of AC-42 as an allosteric M1 agonist,11 which led to the identification of several other selective allosteric M1 mAChR
ligands (Figure 1) like TBPB12, VU035701713 (M1 mAChR agonists), and VU045694014 (a M1 mAChR PAM). In addition, several
M4 mAChR positive allosteric modulators (PAMs) like LY203329815 and VU015210016 have been identified (Figure 1). On the
other hand, there are only a few compounds that show dual-agonism for M1 and M4 mAChRs.17, 18 Indeed, discovery of selective M1
and M4 mAChRs-dual activators with druggable safety and pharmacokinetic profiles is a big challenge.

Figure 1: Structures of various M1 and M4 mAChRs agonists and modulators
In our drug discovery program, we have discovered and reported 8-azadihydroquinolone derivatives as potent M1 and M4
mAChR agonists.18 Especially, compound 2 (Figure 1) showed selective M1 and M4 mAChRs agonistic activity,19 weak human
ether-a-go-go related gene (hERG) inhibition,19 and oral in vivo antipsychotic-like efficacy.19 However, pharmacokinetic (PK)
studies of this compound revealed low bioavailability (BA) (2.6% at 2.5 mg/kg, po) and quick clearance (CL: 49.3 mL/min/kg) in
rats. Based on our analysis of the metabolites of compound 3 in urinary excretion, we assumed that oral administration of
compound 3 afforded the dealkylated metabolite 4 through first-pass effect (Figure 2). Relatively low CL of compound 4 (17.5
mL/min/kg) indicates that removal of N-alkyl group improve bioavailability of compound 2. However, these alkyl groups are
essential to increase subtype selectivity for M1 and M4 mAChRs over M2 mAChR. Based on these results, we stopped to develop
8-azadihydroquinolone derivatives because it is difficult to discover another substituent stable in vivo. To discover another lead
compound with both high subtype-selectivity and stability in vivo, we initiated lead generation study for finding new scaffold. Here,
we describe the design, synthesis, and evaluations of the N-substituted 7-azaindoline derivative 1 (Figure 1) as a potent,
metabolically stable and selective M1 and M4 mAChRs agonist (Figure 1).
Figure 2. Removal of N-alkyl group of compound 3 through first-pass effect.
Structure-activity relationships (SARs) of 8-azadihydroquinolone derivatives indicated that the alkyl substituent at the 1-nitrogen
atom of 8-azadihydroquinolone is necessary for subtype selectivity over M2 mAChR. Based on this finding, we selected the HTS
hit compound 5 with a common spiropiperidine scaffold as compound 2, because we hypothesized that this common structure
would maintain pharmacological activity. In addition, we assumed that replacement of the N-alkylated amide moiety of compound
2 by a pyrrolidine urea would avoid quick clearance. These assumptions led us to design and synthesize compound 6 (Figure 3) as
an initial lead compound.

Figure 3. Our concept for lead generation
Compound 6 showed moderate M1 and M4 mAChR agonistic activity with potent hERG inhibition (Table 1). First, we modified
the alkylamino group of the urea moiety to reduce compound 6 hERG inhibition without lowering its M1 and M4 mAChR agonistic
activity. A series of related urea compounds (Table 1) were therefore synthesized and their SARs were evaluated. Replacement of
the monoalkylamino groups of compound 6 and 8 with a dialkylamino group (compound 7 and 1) improved hERG inhibition.
Carbamoyl compound 9 showed potent hERG inhibition. Modification of the alkylamine to an azetidine (compound 10) or a
pyrrolidine (compound 11) led to decreased M4 mAChR agonistic activity. Replacement of the urea group by a methanesulfonyl
group (compound 12) lowered M1 mAChR agonistic activity (M1: 20% at 0.3 µM, M4: 88% at 0.3 µM). On the other hand, the use
of a methoxycarbnyl group (compound 13) or an ethyl group (compound 14) resulted in decreased M4 mAChR agonistic activity
compared with compound 1. We speculate that hERG inhibition of compound 1 was improved because dialkylamino urea disturb
the formation an intramolecular hydrogen bond between the NH proton of the urea and the nitrogen atom of the pyridine (Figure 4).
As reported in the literature,20 compounds 8 and 9 showed relatively high logP values (2.5 and 2.0, respectively) and strong hERG
inhibition (IC50 = 0.30 and 0.19 µM, respectively). We selected compound 1 as a potent candidate M1 and M4 mAChR agonist.
Table 1: SARs of N-substituents in 7-azaindoline derivatives
mAChR agonistic activitya
hERG inhibition
Compound
R
M1
at 1µM
M2
at 1µM
M3
at 1µM
M4
at 1µM
M5
at 10µM
(IC50: µM)
2
6
7
8
9
1
–
91%
55%
52%
74%b
99%b
76%b
20%
13%
23%
7%b
2%
5%
134%
77%
3%
11%
9%
2.89
0.23
2.54
0.30
0.19
> 10
-CONHEt
-CONEt2
-CONHMe
-CONH2
-CONMe2
4%
132%
98%b
8%b
11%b
2%b
17%
8%
17%b
10%b
103%b
118%b
11%
10
11
NT
NT
NT
29%b
10%b
NT
NT
NT
NT
NT
NT
NT
12
13
14
-Ms
-CO2Me
-Et
20%b
NT
12%b
NT
7%b
NT
6%
88%b
7%
3%
NT
1.90
NT
34%
14%
58%
17%
NT
a Maximum activity for each receptor subtype was defined as 100% in the calcium mobilization assay.
b Concentration of the test compound was 0.3µM.
NT: Not tested.

Figure 4: Relationship between intramolecular hydrogen bond formation and lipophilicity
In PK studies, compound 1 showed improved BA and CL in rats (Table 2) with high bioavailability in dogs (49.3%, 1 mg/kg, po)
and monkeys (39.8%, 1 mg/kg, po). In addition, compound 1 exhibited good blood brain barrier penetration (Kp = 0.7) in rats.
These results indicate that compound 1 could overcome the PK issues of compound 2.
Table 2: PK profile of compound 1 in vivo.
CL
(mL/min/kg)
Cmax
(ng/mL)
T1/2
(h)
Vdss
(L/kg)
Species
BA(%)
Kp
Rata
Dogb
51.1
49.3
39.8
28.4
–
206.6
–
0.9
–
1.8
–
0.7
–
Monkeyb
16.5
30.7
4.76
2.3
–
a Test compound was dosed intravenously at 0.25 mg/kg and orally at 2.5 mg/kg.
b Test compound was dosed intravenously at 1 mg/kg and orally at 1 mg/kg.
Finally, we evaluated the pharmacological profile of compound 1 in detail. Compound 1 showed high subtype-selectivity for M1
and M4 mAChRs over M2, M3, and M5 mAChRs in the calcium mobilization assay (Figure 5 and Table 3). In addition, a
radioligand-binding panel assay using a broad range of G protein-coupled receptors, ion channels, enzymes and transporters,
revealed that compound 1 (3 µM) shows no more than 40% binding inhibition against 61 targets in the panel, except for sigma
receptors (57% at 3 µM, Table S1). Furthermore, compound 1 showed antipsychotic-like effect at a dose lower than that of
xanomeline in a rat methamphetamine-induced locomotor hyperactivity test (Table 3). Although the efficacy of compound 1 on
animal models for negative and cognitive symptoms remains to be investigated, these results indicate that compound 1 has a good
potential as an orally available antipsychotic.
Figure 5. Agonistic activity of compound 1 on M1–M5 mAChRs in the calcium mobilization assay. The maximum efficacy of each
subtype of mAChRs was defined as 100%.

Table 3. Pharmacological data of xanomeline and compound 1.
mAChR agonistic activitiesa, EC50/intrinsic activity
Antipsychotic-like activity b
ED50
M1
M2
M3
M4
M5
xanomelinec
1
16 nM / 100％
379 nM / 36%
44 nM / 86%
126 nM / 85％
46% at 30 µM
8.1 mg/kg (sc)
5% at 10 µM
2% at 10 µM
11% at 10 µM
1.44 mg/kg (po)
133 nM / 76％
47 nM / 108％
a
b
mAChR agonistic activity was measured in the calcium mobilization assay. Antipsychotic-like effect was evaluated in the
methamphetamine-induced locomotor hyperacitivity test in rats. c Data for xanomeline was reported in our previous report17.
The 3-spiro-7-azaindoline derivatives, i.e. compound 1 and compounds 6‒14, were synthesized as shown in Scheme 1. The
commercially available (2-chloro-3-pyridinyl)methanol (15) was converted to the cyanide 17 via the chloride 16. Introduction of a
spiropiperidine part to 17 using an N-benzyl-bis(2-chloroethyl)amine followed by cyclization with LiAlH(Ot-Bu)3 afforded the key
intermediate 19. In the urea formation step of 20, reproducibility of the yield was poor. The optimization of this step allowed us to
accomplish a multi-gram scale synthesis of 20 in quantitative yield.21 Deprotection of the benzyl group of 20 by catalytic reduction
followed by reductive amination with ethyl 4-formylpiperidine-1-carboxylate afforded compound 1.22 Meanwhile, a series of
compounds having other substituents at the 1-nitrogen atom were synthesized by introducing the substituent at the final step using
an appropriate carbamoyl chloride (6‒11), methanesulfonyl chloride (12), methyl chloroformate (13), or ethyl iodide (14).
Scheme 1. Reagents and conditions: (a) SOCl2, CH2Cl2-toluene, rt, 16 h, 97%; (b) KCN, EtOH-H2O, reflux, 1.5 h, 98%; (c)
N-benzyl-bis(2-chloroethyl)amine hydrochloride, KOH, DMSO, 50 °C, 2.5 h, 67%; (d) LiAlH(Ot-Bu)3, THF, reflux, 20 h, 84%; (e)
N,N-dimethylcarbamoyl chloride, N,N-diisopropylethylamine, toluene, reflux, 6 h, 99%; (f) Pd/C, HCO2NH4, MeOH, reflux, 22:
quant; (g) ethyl 4-formylpiperidine-1-carboxylate, NaBH(OAc)3, AcOH, CH2Cl2, rt, 2 h, 23: 55% (2 steps), 1: 86%; (h) 6‒11:
appropriate carbamoyl chloride, Et3N, toluene, rt, 18–42%; 12: methanesulfonyl chloride, Et3N, 56%; 13: methyl chloroformate,
Et3N, CH2Cl2, rt, 48%; 14: EtI, NaH, DMF, rt, 16%.
In summary, we have identified 7-azaindoline derivatives as selective M1 and M4 mAChRs agonists. Optimization of the
substituent at the anilinic nitrogen atom of 7-azaindoline led to compound 1, which showed selective M1 and M4 mAChRs agonistic
activity, good blood brain barrier penetration, and high bioavailability in rats, dogs and monkeys. In addition, oral administration of
compound 1 reversed methamphetamine-induced hyperlocomotion in rats. These results indicate that compound 1 has a good
potential as an orally available antipsychotic with a novel mechanism of action.
Acknowledgement
We are grateful to Keiko Bando for performing the elemental analysis and Shinya Usui and Norio Fujiwara for their useful

discussions.
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22. Analytical data of compound 1 is described in supporting information.

Graphical Abstract