Discovery of the first highly M5-preferring muscarinic acetylcholine receptor ligand, an M5 positive allosteric modulator derived from a series of 5-trifluoromethoxy N-benzyl isatins

Article abstract of DOI:10.1021/jm900286j

This report describes the discovery and initial characterization of the first positive allosteric modulator of muscarinic acetylcholine receptor subtype 5 (mAChR5 or M5). Functional HTS, identified VU0119498, which displayed micromolar potencies for poten

Full text of DOI:10.1021/jm900286j

J. Med. Chem. 2009, 52, 3445–3448
3445
Chart 1. M1 and M4 Positive Allosteric Modulators
Discovery of the First Highly M5-Preferring
Muscarinic Acetylcholine Receptor Ligand,
an M5 Positive Allosteric Modulator Derived
from a Series of 5-Trifluoromethoxy
N-Benzyl Isatins
Thomas M. Bridges, Joy E. Marlo, Colleen M. Niswender,
Carrie K. Jones, Satyawan B. Jadhav, Patrick R. Gentry,
Hyekyung C. Plumley, C. David Weaver, P. Jeffrey Conn,
and Craig W. Lindsley*
deficits in long-term potentiation (LTP) at the hippocampal
mossy fiber-CA3 synapse and show deficits in hippocampal-
dependent behavioral cognitive tests.8
Vanderbilt Program in Drug DiscoVery, Departments of
Pharmacology and Chemistry, Vanderbilt UniVersity Medical
Center, 1205 LH, NashVille, Tennessee, 37232-0697
In light of these and related findings, activation of M5 has
been suggested as a potential target for treatment of Alzheimer’s
disease, perhaps in combination with M1 activation.9 Consistent
with the putative postsynaptic localization of M5 in the ventral
tegmental area (VTA), other M5-KO data suggest this subtype
plays an important role in regulation of mesolimbic dopamine
transmission.3,9 Indeed, M5-KO mice exhibit decreased reward
responses to morphine, decreased self-administration of cocaine,
and less pronounced drug withdrawal symptoms, suggesting that
M5 antagonists or negative modulators may have therapeutic
value in treatment of illicit drug addiction.9-11 Further phar-
macological exploration of these and related hypotheses greatly
depends on the discovery of novel M5-preferring or selective
small molecule tools.
We recently reported on a diverse group of novel mAChR
positive allosteric modulators (PAMs), some of which were
highly selective for M1 or M4 (Chart 1, Figure 1).6,12,13 Other
mAChR PAMs displayed mixed subtype-selectivity profiles.12
These compounds enhanced receptor activation in response to
ACh in Ca2+ mobilization assays and did not compete with the
orthosteric antagonist [3H]-N-methylscopolamine (NMS) in
radioligand binding experiments performed with M1-CHO
membranes, which strongly suggests an allosteric mechanism
of modulation.12 Interestingly, the p-bromobenzyl-substituted
isatin screening hit VU0119498 (113) was found to exhibit
allosteric potentiator activity at the natively Gq-coupled M1, M3,
and M5 receptors with comparable potency and efficacy in Ca2+
mobilization assays but lacked potentiator effects at the natively
Gi-coupled M2 and M4 receptors in cells cotransfected with
ReceiVed March 6, 2009
Abstract: This report describes the discovery and initial characteriza-
tion of the first positive allosteric modulator of muscarinic acetylcholine
receptor subtype 5 (mAChR5 or M5). Functional HTS, identified
VU0119498, which displayed micromolar potencies for potentiation
of acetylcholine at M1, M3, and M5 receptors in cell-based Ca2+
mobilization assays. Subsequent optimization led to the discovery of
VU0238429, which possessed an EC50 of approximately 1.16 µM at
M5 with >30-fold selectivity versus M1 and M3, with no M2 or M4
potentiator activity.
The five cloned muscarinic acetylcholine receptor subtypes
(mAChR1-5 or M1-5a) are known to play highly important and
diverse roles in many basic physiological processes including
gastrointestinal, cardiovascular, motor, attention, learning, memory,
pain, sleep, and other functions.1-3 Correspondingly, muscarinic
agonists and antagonists targeting one or more subtypes have
been used preclinically and clinically for research and treatment
of a wide range of pathologies.3,4 Given the high sequence
homology of the mAChRs across subtypes and particularly
within the orthosteric acetylcholine (ACh) binding site, discov-
ery of truly subtype-selective compounds has proven historically
difficult. Because of the paucity of selective compounds, a
detailed understanding of the precise roles of each subtype in
neurobiology and in various central nervous system (CNS)
disorders has thus remained challenging.3,4
Recently, a number of novel highly subtype-selective allos-
teric ligands for M1 and M4 have emerged from functional cell-
based screening efforts.5,6 However, no ligands have been
reported to date as being highly M5-preferring or selective.
Relative to the other mAChRs, little is known about M5, which
is expressed at very low levels in the CNS and peripheral
tissues.2-4
Interestingly, data from studies using mAChR5 knockout
(M5-KO) mice suggest that M5 is the sole mediator of ACh-
induced vasodilation in the cerebral vasculature and thereby may
have therapeutic relevance for cerebrovascular diseases or acute
ischemic stroke.7,8 M5-KO mice have also been found to exhibit
Figure 1. HTS hit 113 potentiates responses in M1, M3, and M5-
expressing CHO cells. In the presence of a fixed submaximal
concentration of ACh (∼EC20) in Ca2+, assays performed with CHO
cells stably expressing each of the five mAChR subtypes (M2 and M4
cotransfected with Gqi5). M1 EC50 ) 6.04 µM, %max ) 82.4; M3 EC50
) 6.38 µM, %max ) 65.5; M5 EC50 ) 4.08 µM, %max ) 74.4;
inactive at M2 and M4 (data shown are means ( SEM, N g 3).
* To whom correspondence should be addressed. Phone: 001-615-322-
8700. Fax: 001-615-343-3088. E-mail: craig.lindsley@vanderbilt.edu.
a Abbreviations: mAChR, muscarinic acetylcholine receptor; ACh,
acetylcholine; CNS, central nervous system; KO, knockout; LTP, long-
term potentiation; VTA, ventral tegmental area; PAM, positive allosteric
modulator; NMS, N-methylscopolamine; AUC, area under curve; CRC,
concentration-response curve.
10.1021/jm900286j CCC: $40.75
2009 American Chemical Society
Published on Web 05/13/2009

3446 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Scheme 1. Library Synthesis of VU0119498 Analoguesa
Letters
Table 1. Structures and Activities at M1 and M5 of Analogues of
Compound 113
compd
R1
R2
M1 EC50 (µM)
M5 EC50 (µM)
22
25
33
38
41
42
44
46
47
49
50
56
63
64
103
110
111
112
H
7-F
7-Cl
4,7-Cl
5-OCF3
5-OCF3
H
5-F
5-F
7-F
7-F
7-Cl
5-OCF3
5-OCF3
7-Cl
5-OCF3
5-OCF3
5-OCF3
4-OMe
4-Br
>10
3.99
5.38
>30
>30
>30
5.88
6.21
8.55
>30
5.18
3.20
7.09
>30
7.14
>30
>30
>30
>30
1.93
3.96
7.15
1.70
1.16
3.19
4.09
7.13
>30
3.26
2.11
1.85
4.07
4.66
1.86
3.28
>10
a Reagents and conditions: (a) K2CO3, KI, ACN, microwave 160°C, 10
min, 20-95%.
4-OMe
3-OMe
3-OMe
4-OMe
4-CF3
2-CF3
4-CF3
2-CF3
4-CF3
4-CF3
4-CF3
2-Cl
2-F, 4-Br
4-OCF3
2-Me
chimeric Gqi5 protein, which facilitates coupling to the PLC/
Ca2+ pathway (Figure 1).12 In similar assays, a high fixed 30
µM concentration of 113 induced a 14-fold leftward shift of a
full ACh concentration response curve (CRC), signifying a
robust potentiation effect. This hit was therefore chosen as an
attractive starting point for subsequent optimization efforts
directed toward increasing M5 selectivity and potency while
simultaneously decreasing M1 and M3 activity.
To rapidly explore SAR around 113, we generated an
analogue library in matrix format wherein each of eight
commercially available isatins (1-8) was reacted with ap-
proximately 12 benzyl halides (9-20) under standard microwave
alkylation conditions (Scheme 1).14 Resulting analogues
(21-112) were then screened in a single point format at a 30
µM final concentration in Ca2+ mobilization assays using M5
and M1 cells receiving a fixed submaximal concentration
(∼EC20) of ACh (Figure 2, see full SAR table in Supporting
Information).14 This method efficiently triaged analogues dis-
playing high M1 vs M5 or M5 vs M1 preference. Interestingly,
some analogues displayed robust potentiation effects at M5 (i.e.,
elevation of ACh ∼EC20 to >50-60% of maximum ACh
response) with absent or weak potentiation at M1, thus exhibit-
ing strong preference for M5 versus M1 activity.
2-F, 4-Br
In terms of maximal potentiation efficacy, SAR from the
initial 30 µM screen of the entire library suggested that 5-OCF3
substitution of the isatin core (R1) was generally favored for
increased M5 versus M1 activity, while halogen substitutions
at the 7-position conferred a more dual M1/M5 activity (Table
1).14 Indeed, all 5-OCF3 substituted compounds chosen from
the initial screen possessed 1-5 µM potencies at M5 and >30
µM potencies at M1, with the exception of compunds 63 and
112, which nonetheless possessed greater M5 versus M1
activities (Table 1).14 Numerous benzylic substitutions (R2) were
tolerated for M1 and M5 activity, depending on the isatin core
(R1).14 Methoxybenzyl was generally favored for M5 versus
Figure 2. Screen of analogue library (compounds 21-112) at 30 µM for potentiation of submaximal acetylcholine (∼EC20) in M1 (A) and M5 (B)
cells by Ca2+ assay (data shown are means ( SEM, N g 3).14

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3447
Figure 3. Concentration response curve for 42 performed in the
presence of a fixed ACh ∼EC20 in Ca2+ assays performed with CHO
cells stably expressing each of the five mAChR subtypes (M2 and M4
cotransfected with Gqi5). M1 EC50 ) >30 µM; M3 EC50 ) >30 µM;
M5 EC50 ) 1.16 µM, %max ) 91; inactive at M2 and M4 (data shown
are means ( SEM, N g 3).
Figure 5. Compound 42 CRC alone or in the presence of fixed ACh
EC20 demonstrating an absence of intrinsic agonist activity at M5 by
Ca2+ assay (top left), effect of fixed 30 µM 42 on an ACh CRC
demonstrating a 14-fold increase in ACh potency at M5 by Ca2+ assay
(top right), lack of competition with [3H-NMS] binding to M5 by 42
versus atropine control (Ki ) 0.21 nM, bottom left), and the effect of
fixed 30 µM 42 on ACh competition with [3H-NMS] binding
demonstrating a 10-fold increase in ACh affinity for M5 (ACh + vehicle
Ki ) 1.61 µM; ACh + 42 Ki ) 159 nM; data shown are means (
SEM, N g 3).
Figure 4. Concentration response curve for 56 performed in the
presence of a fixed ACh ∼EC20 in Ca2+ assays performed with CHO
cells stably expressing each of the five mAChR subtypes (M2 and M4
cotransfected with Gqi5). M1 EC50 ) 3.20 µM, %max ) 87; M3 EC50
) 2.22 µM, %max ) 78; M5 EC50 ) 2.11 µM, %max ) 86; M2 EC50
) 2.84 µM, %max ) 55; M4 EC50 ) >10 µM (data shown are means
( SEM, N g 3).
hit 113 when evaluated in a similar assay at M1, as previously
reported.12 Moreover, 42 lacked intrinsic agonist activity at M5
when tested up to 30 µM in the absence of ACh (Figure 5, top
left).
We also evaluated HTS hit 113 and the highly M5-preferring
analogue 42 in competition-binding experiments with M5-CHO
membrane preparations using the orthosteric radioligand [3H]-
N-methylscopolamine ([3H]-NMS), a potent pan-mAChR an-
tagonist. At up to 30 µM, 42 lacked competition with [3H]-
NMS, consistent with an allosteric mode of binding to M5
(Figure 5, bottom left). By contrast, 113 exhibited approximately
50% inhibition of radioligand binding at 30 µM (Figure 5,
bottom left). Compound 42 also caused a 10-fold increase in
ACh affinity for M5 at 30 µM, indicating that its mechanism
of potentiation is in part due to enhancement of ACh binding
to M5.
We then evaluated pharmacokinetics and brain penetration
for the M5 PAM to evaluate its ability to serve as an in vivo
probe. Unfortunately, 42 is characterized by poor systemic
absorption after intraperitoneal administration with maximum
concentration in plasma (161.7 ng/mL) being achieved within
1 h.14 However, it is slowly eliminated from systemic circulation
and has elimination half-life of 4.7 h.14 Although quickly taken
M1 activity, whereas trifluoromethylbenzyl was generally
favored for dual M1/M5 potentiation activity (Table 1).14 Most
other simple congeners, including those with various methyl
substitutions at R1 or R2, displayed weak or no potentiation
activity at either receptor.14 In light of the high M5 versus M1
potentiation preference displayed by analogue 42 (1.16 µM M5
EC50 and >30 µM M1 EC50) in these assays, the full subtype-
selectivity profile of this compound was obtained in similar Ca2+
assays using M2, M3, and M4 cells. Likewise, full subtype-
selectivity determination was also performed with compound
56, which was the most potent dual M1/M5 potentiator in this
initial evaluation at both receptors (2.11 µM M5 EC50 and 3.20
µM M1 EC50).
Remarkably, 42 (VU0238429) had similarly poor potency at
M3 as at M1, with potentiation of the ACh ∼EC20 beginning
to emerge only in the double-digit micromolar range (Figure
3). Gratifyingly, this compound maintained its lack of potentia-
tion activity at M2 and M4 in cells cotransfected with Gqi5 at
up to 30 µM (Figure 3). Given these data, the approximately
1.16 µM M5 potency of compound 42 provides >30× selectivity
for M5 versus the other four subtypes, thus representing the
first highly M5-preferring muscarinic receptor ligand ever
reported. Conversely, compound 56 (VU0238441) was found
to retain the M1/M3/M5 potentiation profile of the parent HTS
hit 113 and interestingly possessed modest potentiation at M2
and M4 at high concentrations (Figure 4).
up in the brain, it exhibits poor brain penetration with AUCbrain
/
AUCplasma value of 0.25.14 Future lead optimization will focus
on improving brain penetration.
Although a growing number of allosteric modulators have
been recently documented for mAChR1 and mAChR4, to date
no selective mAChR5 ligand has been reported. Compound 42,
a congener of 113 identified from an analogue library synthesis
approach, serves as the first highly mAChR5-preferring small
molecule. Despite the relatively limited scope of this analogue
library in terms of structural modification, we found clear SAR
associated with this novel mAChR chemotype, with 5-OCF3
modification of the isatin core and 3- or 4- position OMe
substitutions of the benzyl ring conferring strong M5-preference
versus M1-M4. In contrast, virtually all modifications confer-
ring increased M1 activity simultaneously increased M5 activ-
ity.14 As exemplified in the case of 56, 7-Cl substitution of the
To further evaluate the ability of 42 to potentiate ACh-induced
activation of mAChR5, we obtained full CRCs for ACh in M5
cells in the presence of either vehicle or a fixed (30 µM)
concentration of 42 (Figure 5, top right). In these assays,
compound 42 induced a robust 14-fold left-shift of the ACh CRC,
which was virtually identical to that found with the parent HTS

3448 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Letters
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isatin core carried similar and increased M1/M5 potency and
efficacy but also caused degeneration into a pan-muscarinic
potentiator when profiled at all five subtypes. Further chemical
modification of compound 42 to improve M5 PAM potency
and characterization in electrophysiology and in vivo experi-
ments are underway and will be reported in due course.
Acknowledgment. We thank the NIH and NIMH for support
of our programs. We specifically acknowledge the support of
the Alzheimer’s Association (IIRG-07-57131) and NIMH
(1RO1MH082867-01 and 5R01MH073673). T.M.B. acknowl-
edges an ITTD (T90-DA022873) predoctoral training grant.
Supporting Information Available: Experimental procedures
and analytical data for compounds 113, 42, and 56 are provided as
kinetics study. This material is available free of charge via the
Internet at http://pubs.acs.org.
(11) Thomsen, M.; Woldbye, D. P. D.; Wortwein, G.; Fink-Jensen, A.;
Wess, J.; Caine, S. B. Reduced Cocaine Self-Administration in
Muscarinic M5 Acetylcholine Receptor-Deficient Mice. J. Neurosci.
2005, 25, 8141–8149.
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