Discovery, synthesis and characterization of a highly muscarinic acetylcholine receptor (mAChR)-selective M5-orthosteric antagonist, VU0488130 (ML381): A novel molecular probe

Article abstract of DOI:10.1002/cmdc.201402051

Of the five G-protein-coupled muscarinic acetylcholine receptors (mAChRs; M₁-M₅), M₅ is the least explored and understood due to a lack of mAChR subtype-selective ligands. We recently performed a high-throughput functional screen and identified a number of weak antagonist hits that are selective for the M₅ receptor. Here, we report an iterative parallel synthesis and detailed molecular pharmacologic profiling effort that led to the discovery of the first highly selective, central nervous system (CNS)-penetrant M₅-orthosteric antagonist, with sub-micromolar potency (hM₅ IC₅₀=450 nM, hM₅ K _i=340 nM, M₁-M₄ IC₅₀ >30 μM), enantiospecific inhibition, and an acceptable drug metabolism and pharmacokinetics (DMPK) profile for in vitro and electrophysiology studies. This compound will be a powerful tool and molecular probe for the further investigation into the role of M₅ in addiction and other diseases.

Full text of DOI:10.1002/cmdc.201402051

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DOI: 10.1002/cmdc.201402051
Discovery, Synthesis and Characterization of a Highly
Muscarinic Acetylcholine Receptor (mAChR)-Selective M5-
Orthosteric Antagonist, VU0488130 (ML381): A Novel
Molecular Probe
Patrick R. Gentry,[a] Masaya Kokubo,[a] Thomas M. Bridges,[a] Hyekyung P. Cho,[a]
Emery Smith,[b] Peter Chase,[b] Peter S. Hodder,[b] Thomas J. Utley,[a] Anuruddha Rajapakse,[a]
Frank Byers,[a] Colleen M. Niswender,[a] Ryan D. Morrison,[a] J. Scott Daniels,[a]
Michael R. Wood,[a] P. Jeffrey Conn,[a] and Craig W. Lindsley*[a]
Of the five G-protein-coupled muscarinic acetylcholine recep-
tors (mAChRs; M1–M5), M5 is the least explored and understood
due to a lack of mAChR subtype-selective ligands. We recently
performed a high-throughput functional screen and identified
a number of weak antagonist hits that are selective for the M5
receptor. Here, we report an iterative parallel synthesis and de-
tailed molecular pharmacologic profiling effort that led to the
discovery of the first highly selective, central nervous system
(CNS)-penetrant M5-orthosteric antagonist, with sub-micromo-
lar potency (hM5 IC50 =450 nm, hM5 Ki =340 nm, M1–M4 IC50
>30 mm), enantiospecific inhibition, and an acceptable drug
metabolism and pharmacokinetics (DMPK) profile for in vitro
and electrophysiology studies. This compound will be a power-
ful tool and molecular probe for the further investigation into
the role of M5 in addiction and other diseases.
esis that M5 modulates dopaminergic neurotransmission and
À/À
that M5 functions in addiction/reward mechanisms, with M5
mice displaying decrease cocaine place preference and self-ad-
ministration, without affecting food intake.[4–10] Further bio-
chemical studies with M5-targeted antisense oligonucleotides,
or scopolamine, infused into the VTA have recapitulated the
genetic findings,[11,12] strongly suggesting that selective M5 in-
hibition might provide a novel therapeutic approach for the
treatment of addiction.[1–12]
Only recently have M5-preferring and M5-selective ligands
begun to emerge. By targeting allosteric sites[13,14] on mAChRs,
we recently described the first M5-selective ligands, a series of
isatin-based M5 positive allosteric modulators (PAMs) (1–3,
Figure 1).[15–17] Shortly thereafter, Dwoskin and co-workers de-
scribed an M5-preferring orthosteric antagonist (4, Ki =2.2 mm)
with 11-fold selectivity for M5 over M1, but no functional (IC50)
or drug metabolism and pharmacokinetics (DMPK) data was re-
ported.[18] Last year, we reported the first results from a func-
tional M5 high-throughput screen (HTS), where we identified
and optimized the first M5-selective negative allosteric modula-
tor (NAM), 5, with sub-micromolar potency (hM5 IC50 =300 nm,
hM1–4 IC50 >30 mm).[19] Based on the Dwoskin work[18] and our
earlier studies that had yielded a highly (>75-fold versus M2–
M5) subtype-selective M1-orthosteric antagonist 6,[20] we revisit-
ed our HTS hits in an attempt to identify and optimize a highly
M5-selective orthosteric antagonist to complete our tool kit of
M5 probes to dissect the role of M5 in the CNS.
Numerous studies have suggested that subtype-selective mus-
carinic acetylcholine receptor (mAChR) antagonists could pro-
vide novel approaches to treat a number of central nervous
system (CNS) disorders;[1–7] however, the majority of existing
antagonists are not selective for specific mAChR subtypes, forc-
ing the field to rely on biochemical and or genetic approaches
to discern therapeutic potential.[1–7] Of the central mAChRs, M5
expression is the lowest (<2%), and M5 is predominantly local-
ized in dopaminergic neurons originating in the substantia
nigra pars compacta (SNc) and the ventral tegmental area
À/À
(VTA).[4–10] Studies with M5 mice have confirmed the hypoth-
Under the auspice of the Molecular Libraries Probe Centers
Network (MLPCN),[21] a triple-add, functional HTS was per-
formed evaluating 360000 compounds for M5 modulatory ac-
tivity. In parallel, we screened the same collection in triple-add,
functional mode against M1, M4 and the parental Chinese ham-
ster ovary (CHO) cell line, resulting in nine confirmed and se-
lective inhibitors of M5, from which the M5 NAM 5 resulted.[19]
Evaluation of the eight remaining hits led us to quickly focus
on a weak, yet selective, isoxazole-based amide 7 (Figure 2),
which was confirmed in the HTS center as having an IC50 value
of 9.3 mm against M5 and selectivity versus M1 and M4 (IC50
>30 mm). Resynthesis of 7 (VU0480131) and screening of this
freshly prepared powder significantly increased our enthusiasm
for the hit, as this sample of 7 displayed improved potency for
both human and rat M5 (hM5 IC50 =1.12 mm (pIC50 =5.94Æ
[a] P. R. Gentry, Dr. M. Kokubo, Dr. T. M. Bridges, Dr. H. P. Cho, T. J. Utley,
A. Rajapakse, F. Byers, Prof. C. M. Niswender, R. D. Morrison,
Prof. J. S. Daniels, Prof. M. R. Wood, Prof. P. J. Conn, Prof. C. W. Lindsley
Department of Pharmacology
Vanderbilt Center for Neuroscience Drug Discovery
Vanderbilt Specialized Chemistry Center (MLPCN)
Vanderbilt University Medical Center
Nashville, TN 37232-6600 (USA)
E-mail: craig.lindsley@vanderbilt.edu
[b] E. Smith, P. Chase, Dr. P. S. Hodder
Lead Identification Division, Translational Research Institute
Scripps Research Institute Molecular Screening Center
130 Scripps Way, Jupiter, FL 33548 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402051.
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0.03), rM5 IC50 =3.47 mm (pIC50 =
5.46Æ0.07)) and high selectivity
versus human M1–M4 (IC50
>30 mm). We then assessed the
mechanism of action of 6 and
performed competition binding
studies with [3H]-N-methylsco-
polamine (NMS) and an atropine
control, and observed that 7 dis-
placed [3H]-NMS in a concentra-
tion-dependent manner with a Ki
value of 1.3 mm (pKi =5.88Æ0.05,
comparable to its IC50 value of
1.1 mm), indicating competitive,
and thus, orthosteric, antago-
nism reminiscent of the highly
selective M1-orthosteric antago-
nist 6.[19,20]
Figure 3 highlights the chemi-
cal optimization strategy for 7,
wherein we evaluated multiple
dimensions of the ligand simul-
taneously through iterative par-
Figure 1. Structures of recently reported M5 positive allosteric modulators (1–3), an M5-preferring orthosteric an-
tagonist (4), the first highly selective M5 negative allosteric modulator (5), and a highly M1-selective orthosteric an-
tagonist (6).
Figure 2. Structure and pharmacological profile of HTS hit 7 (VU0480131). a) Structure and pharmacology of 7, as determined from the HTS stock solution
(hM5 IC50 =9.3 mm) and as a resynthesized, fresh powder (hM5 IC50 =1.12 mm). b) Concentration–response curves of 7 for human (h) and rat (r) M5, as well as
hM1–4 (IC50 >30 mm; n=3). c) [3H]-N-methylscopolamine (NMS) competition binding (n=3) in membranes prepared from human M5-expressing cells, showing
competitive displacement (Ki =1.3 mm).
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(NosCl) provides sulfonamides 13 in good yield
(Scheme 3). The N-methyl moiety was installed via
a Mitsunobu reaction with methanol to afford tertiary
sulfonamides 14. Deprotection was accomplished by
treatment with 3-chlorothiophenol in a modest 20%
yield to provide chiral secondary amines 15. Finally,
a HATU-mediated coupling with acid 8 afforded the
single enantiomers of 7, (S)-16 and (R)-16, in overall
yields of ~15%.[22]
When screened in the human M5 cell-based assay,
enantiospecific inhibition was observed, with (R)-16
possessing no activity (hM5 IC50 >10 mm), yet (S)-16
displayed sub-micromolar potency (hM5 IC50 =
Figure 3. Chemical optimization plan for HTS hit 7 (VU0480131) via multidimensional,
iterative parallel synthesis.
allel synthesis, as the structure–activity relationship (SAR) for
the analogous 6 was very steep. The first round of library syn-
thesis surveyed alternate amides; thus, commercial acid 8 was
acylated under HATU (1-[bis(dimethylamino)methylene]-1H-
1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate)
conditions to afford analogues 9 in yields ranging from 56–
90% (Scheme 1). SAR was extremely steep, with all analogues
Scheme 1. Preparation of amide analogues 9. Reagents and conditions:
a) R1R2NH, HATU, CH2Cl2, RT, 12 h, 56–90%.
tested, even regioisomeric pyridines, possessing hM5 IC50
values >10 mm. This effort only afforded one weakly active
compound, 9a (hM5 IC50 =5.6 mm, rM5 IC50 >10 mm), an N-ethyl
congener of 7 (Figure 4).
Figure 4. Structures and activities of amide analogues 9. All were uniformly
inactive, except for N-ethyl analogue 9a.
With the amide region of 7 intolerant to modification, we
next prepared a library of diverse aryl ethers to assess SAR and
potentially identify a replacement for the ketone (Scheme 2).
Alcohol 10 smoothly reacted with a variety of phenols under
Mitsunobu conditions to provide a library of ether analogues
11, in yields ranging from 35–70%.[22] Nothing in this collection
showed activity (hM5 IC50 >10 mm), despite surveying nitro,
halogen, alkoxy and alkyl moieties around the pendant phenyl
ring (for full listing of analogues, see Experimental Section in
the Supporting Information). Replacement of the ether oxygen
with an NH, or acylated congener, also led to a complete loss
of M5 inhibitory activity, as did amide analogues in place of the
aryl ether moiety. Replacement of the central isoxazole with
a phenyl ring also led to inactive compounds. Again, akin to
the steep SAR noted for the highly selective M1-orthosteric an-
tagonist 6, very little modification to 7 was tolerated. Thus, we
elected to next synthesize the single enantiomers of 7 and
assess whether enantioselective inhibition was observed.
Starting with commercial (S)- or (R)-1-(pyridin-2-yl)ethana-
mine 12, treatment with 4-nitrobenzenesulfonyl chloride
Scheme 2. Preparation of aryl ether analogues 11. Reagents and conditions:
a) ArOH, PPh3, DIAD, CH2Cl2, RT, 2 h, 35–70%.
450 nm, pIC50 =6.34Æ0.05) and exceptional mAChR selectivity
(hM1–4 IC50 >30 mm) (Figure 5). These findings led us to then
profile (S)-16 against rat mAChRs, and once again, (S)-16 was
selective (rM1–4 IC50 >30 mm), but a fourfold diminution in po-
tency was noted for rat M5 (rM5 IC50 =1.65 mm, pIC50 =5.78Æ
0.04). Based on the potency and selectivity of (S)-16, it was ap-
proved as an MLPCN probe and given the identifier ML381
(VU0488130).[21] As expected, competition binding with [3H]-
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Next, we began to assess the physiochemical and
DMPK profile of ML381. ML381 is a low-molecular
weight probe (MW=379) with favorable lipophilicity
(cLogP=2.74) and an acceptable topological polar
surface area (TPSA) of 86. Moreover, ML381 was
found to be both soluble in phosphate-buffered
saline (PBS) (96 mm at pH 7.4 and 238C) and stable
(99% parent remaining after 48 h in PBS at 238C). In
rat and human hepatic microsomes, ML381 exhibited
high intrinsic clearance (rat CLint: 770 mLminÀ1 kgÀ1
;
human CLint: 93 mLminÀ1 kgÀ1) with predicted hepatic
clearance values (rat CLhep: 64 mLminÀ1 kgÀ1; human
CLhep: 17 mLminÀ1 kgÀ1) near the respective rates of
hepatic blood flow in each species (Table 1). In
plasma protein binding experiments, ML381 exhibit-
ed exceptionally high fraction unbound (fuplasma) in
rat (58.4%) and moderately high fuplasma in human
(11%), which prompted investigation into the com-
pound’s stability over time (4 h; 378C) in plasma of
Scheme 3. Preparation of (S)-16 and (R)-16, single enantiomers of racemic 7. Reagents
and conditions: a) NosCl, DIEPA, CH2Cl2, RT, 2 h, 78–90%; b) MeOH, PPh3, DIAD, CH2Cl2, RT,
1 h, 74–90%; c) 3-ClPhSH, K2CO3, MeCN, RT, 4 h, 20%; d) 8, HATU, DIEPA, CH2Cl2, RT, 2 h,
89–96%. Enantiopurity: >99% ee as analyzed by chiral supercritical fluid chromato-
graphy.
NMS indicated that ML381 is an orthosteric antagonist, with
a Ki value of 341 nm (pKi =6.47Æ0.03), comparable to its IC50
value of 450 nm (Figure 5b).
both species, ultimately revealing a pronounced instability in
rat plasma (approximately 1% of parent remaining) and a mod-
erate instability in human plasma (~76% of parent remaining),
Figure 5. Structure and pharmacological profile of (S)-16, ML381 (VU0480131). a) Structure and pharmacology of ML381. b) [3H]-N-methylscopolamine (NMS)
competition binding (n=3) in membranes prepared from human M5-expressing cells, showing competitive displacement (Ki =340 nm). Concentration–
response curves of ML381 for c) human M5 (IC50 =450 nm) and M1–M4 (IC50 >30 mm), and for d) rat M5 (IC50 =1.65 mm) and M1–M4 (IC50 >30 mm).
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cies (Figure 6). These analyses revealed hydrolytic cleavage of
the amide as the principle pathway of biotransformation in mi-
crosomes of both species as well as in rat plasma. Additionally,
a number of NADPH-dependent mono-oxidation pathways
were also identified in rat and human microsomes, including
O-dearylation (M2), phenyl hydroxylation (M3), keto-reduction
(M4), and N-dealkylations at the amide (M5, M6; Figure 6).
In order to gauge distribution to the central nervous system
(CNS), concentrations of ML381 in whole brain and plasma at
a single time point (0.25 h) were measured following a single
intravenous (IV) administration (0.2 mgkgÀ1) to male Sprague–
Dawley rats (n=2). This study revealed a brain/plasma parti-
tion coefficient (Kp) of 0.58, suggesting moderate distribution
to the CNS (Table 1); determination of an unbound brain/
plasma partition coefficient (Kp,uu) was precluded by an ab-
sence of accurate fuplasma data due to the aforementioned
plasma instability. In a bidirectional MDCK-MDR1 trans-well
assay, ML381 (5 mm) exhibited an efflux ratio (ER) of 1.6, sug-
gesting an absence of P-glycoprotein (P-gp)-mediated active
efflux liabilities at the blood–brain barrier (BBB; Table 1). Fur-
thermore, favorably low inhibition of four major human cyto-
chrome P450 enzymes by ML381 was observed in human hep-
atic microsomes, with inhibition of 2C9 (6.3 mm) representing
the predominant liability (Table 1). Moreover, ML381 was
screened in a Eurofins radioligand binding panel of 68 GPCRs,
ion channels, and transporters at a concentration of 10 mm,[23]
and no significant activity was noted (no inhibition
>50% at 10 mm). Thus, in addition to unprecedented
selectivity versus M1–M4 receptors, ML381 possesses
clean ancillary pharmacology against a diverse array
of discrete molecular targets. Together, these findings
suggest that ML381 possesses an overall acceptable
DMPK profile for pharmacodynamic studies in rat,[24]
with the exception of poor metabolic stability and
a potential for amide hydrolysis in plasma; as such,
ML381 is best suited to use as an in vitro/electrophy-
siological probe.
Table 1. Drug metabolism and pharmacokinetics (DMPK) profile of
ML381 in Sprague–Dawley rat and human.[a]
Property
Rat
Human
Sprague–Dawley rat
Hepatic microsomal CLint
Predicted CLhep
fuplasma
770 mLminÀ1 kgÀ1
93 mLminÀ1 kgÀ1
64 mLminÀ1 kgÀ1
17 mLminÀ1 kgÀ1
0.58[b]
0.14
0.58[c]
–
–
–
–
–
0.112
–
–
fubrain
Cbrain/Cplasma (Kp)
MDCK-MDR1 ER
P450 1A2 IC50
P450 2C9 IC50
P450 2D6 IC50
P450 3A4 IC50
1.6
>30 mm
6.3 mm
>30 mm
27 mm
[a] Values represent the mean of at least two replicates with similar re-
sults. [b] fuplasma values were not accurate due to instability in rat plasma.
[c] Kp value was determined at 0.25 h following a 0.2 mgkgÀ1 IV dose (n=
2).
which ultimately prevented accurate determination of fuplasma
.
A high fraction unbound in brain (fubrain: 0.14) was observed
for ML381 in rat whole-brain homogenate.
In order to determine the relevant biotransformation path-
way(s) contributing to the poor metabolic stability observed in
vitro, metabolite identification (Met ID) experiments were per-
formed using hepatic microsomes and plasma from both spe-
In summary, we have developed (S)-16 (also
known as ML381 or VU0480131), the most potent
and selective M5-
orthosteric antagonist (hM5 IC50 =450 nm, M5 Ki =
340 nm, hM1–hM4 IC50 >30 mm) reported to date.
Moreover, ML381 possesses a favorable DMPK profile
and CNS penetration; however, instability in rat
plasma indicates the utility of ML381 will be primarily
as a molecular probe for in vitro and electrophysiolo-
gy studies. SAR was extremely steep for this series,
with subtle modifications leading to complete loss of
M5 inhibitory activity, reminiscent of the M1 ligand 6;
however, enantiospecific inhibition was observed,
with all of the activity residing in the (S)-enantiomer.
Current efforts are focused on further chemical opti-
mization to address the plasma instability identified
in Met ID studies and to improve PK and CNS pene-
tration. Studies are underway and will be reported in
due course. ML381 is an MLPCN probe and is freely
available upon request.
Figure 6. Metabolite identification for 7, ML381 (VU0480131), in rat and human, indicat-
ing six major routes of metabolism (M1–M6).
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Experimental procedures for the medicinal chemistry, pharmacolo-
gy and drug metabolism studies, as well as compound characteri-
zation data, are provided in the Supporting information, available
at http://dx.doi.org/10.1002/cmdc.201402051.
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Acknowledgements
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This work was generously supported by the US National Institutes
of Health/Molecular Libraries Probe Centers Network (MLPCN)
grant U54 MH084659 (to C.W.L.) and grant U54 MH084512 (to
the Scripps Research Institute Molecular Screening Center). Dr.
Lindsley acknowledges the Warren Family and Foundation for
funding the William K. Warren, Jr. Chair in Medicine.
Keywords: acetylcholine · antagonists · molecular probes ·
muscarinic acetylcholine receptors · orthosteric ligands
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Received: February 28, 2014
Published online on April 1, 2014
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