Development of a highly potent, novel M5positive allosteric modulator (PAM) demonstrating CNS exposure: 1-((1H-indazol-5-yl)sulfoneyl)-N-ethyl-N-(2-(trifluoromethyl)benzyl)piperidine-4-carboxamide (ML380)

Article abstract of DOI:10.1021/jm500995y

A functional high throughput screen identified a novel chemotype for the positive allosteric modulation (PAM) of the muscarinic acetylcholine receptor (mAChR) subtype 5 (M₅). Application of rapid analog, iterative parallel synthesis efficiently optimized M₅potency to arrive at the most potent M₅PAMs prepared to date and provided tool compound 8n (ML380) demonstrating modest CNS penetration (human M₅EC₅₀= 190 nM, rat M₅EC₅₀= 610 nM, brain to plasma ratio (K_p) of 0.36).

Full text of DOI:10.1021/jm500995y

Brief Article
pubs.acs.org/jmc
Open Access on 08/22/2015
Development of a Highly Potent, Novel M5 Positive Allosteric
Modulator (PAM) Demonstrating CNS Exposure: 1‑((1H‑Indazol-5-
yl)sulfoneyl)‑N‑ethyl‑N‑(2-(trifluoromethyl)benzyl)piperidine-4-
carboxamide (ML380)
Patrick R. Gentry,†,‡,§ Masaya Kokubo,†,‡,§ Thomas M. Bridges,†,‡,§ Meredith J. Noetzel,†,‡,§
Hyekyung P. Cho,†,‡,§ Atin Lamsal,†,‡ Emery Smith,⊥ Peter Chase,⊥ Peter S. Hodder,⊥
Colleen M. Niswender,†,‡,§ J. Scott Daniels,†,‡,§ P. Jeffrey Conn,†,‡,§ Craig W. Lindsley,†,‡,∥,§
and Michael R. Wood*,†,‡,∥,§
†Department of Pharmacology, ‡Vanderbilt Center for Neuroscience Drug Discovery, and §Vanderbilt Specialized Chemistry Center
for Accelerated Probe Development (MLPCN), Vanderbilt University Medical Center, Nashville, Tennessee 37232, United States
∥Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States
⊥Lead Identification Division, Molecular Screening Center, and Translational Research Institute, The Scripps Research Institute, 130
Scripps Way, Jupiter, Florida 33458, United States
S
* Supporting Information
ABSTRACT: A functional high throughput screen identified a novel chemotype for the positive allosteric modulation (PAM) of
the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5). Application of rapid analog, iterative parallel synthesis
efficiently optimized M5 potency to arrive at the most potent M5 PAMs prepared to date and provided tool compound 8n
(ML380) demonstrating modest CNS penetration (human M5 EC50 = 190 nM, rat M5 EC50 = 610 nM, brain to plasma ratio
(Kp) of 0.36).
(NAM) at many of the CNS-important mAChRs: M1 PAMs,6
INTRODUCTION
■
M4 PAMs,7 M5 PAMs,8 and a novel M5 NAM.9
As a vital neurotransmitter, acetylcholine (ACh) activates ion
Currently, M5 is the least characterized of the mAChRs
channels and G protein coupled receptors (GPCRs) through its
because of its low expression level14 and until recently an
interactions with nicotinic and muscarinic (mAChR) acetylcho-
line receptors, respectively.1,2 Among the five mAChRs,
subtypes 1, 4, and 5 (M1, M4, and M5) are most strongly
absence of selective activators and inhibitors. Nevertheless,
phenotypic observations of M5 knockout (KO) mice,3 M5
receptor localization studies, and experiments utilizing non-
selective, orthosteric muscarinic ligands highlight this receptor’s
therapeutic potential.2 M5 KO mice display decreased prepulse
inhibition (a model of psychosis)10 and cognitive deficits
associated with CNS neuronal and cerebrovascular abnormal-
ities.11 The loss of M5 mAChRs in KO mice prevents their
CNS vasculature from dilating in response to ACh,12 which
could have implications for cerebral hypoperfusion as related to
Alzheimer’s disease,13 schizophrenia, ischemic stroke, and
migraine. Collectively, these data support the role of a M5
PAM in the treatment of numerous CNS diseases. Here we
report the development of the first CNS penetrant M5 PAM,
associated with normal central nervous system (CNS)
functioning.2 The M2 and M3 subtypes are more broadly
expressed in the periphery on smooth muscle and glandular
tissues3 such that aberrant overactivation of these receptors
leads to the adverse effects associated with nonselective
muscarinic agonists. Designing orthosteric small-molecule
muscarinic ligands with sufficient selectivity over the other
four mAChRs has long been a problem due to the highly
conserved environment of the ACh binding site (the
orthosteric site). A prudent response to instances such as this
has been to abandon orthosteric-acting molecules in favor of
ligands that interact at allosteric sites (sites that are topo-
graphically and structurally distinct from the endogenous
agonist binding site).4,5 We have employed this approach to
identify a range of high quality muscarinic ligands with positive
allosteric modulation (PAM) or negative allosteric modulation
Received: July 2, 2014
Published: August 22, 2014
© 2014 American Chemical Society
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Journal of Medicinal Chemistry
Brief Article
a
which is structurally distinct from our previously reported
isatin-containing M5 PAMs.8
Scheme 1. Synthesis of M5 PAM HTS Hit 1 and Its Analogs
RESULTS AND DISCUSSION
■
High-Throughput Screen. Our initial foray into M5 PAMs
began with the identification of a nonselective M1, M3, M5 PAM
as a confirmed hit from an M1-focused high throughput
screening (HTS) campaign.6 Although very high levels of M5
selectivity were engendered through a strategically placed
substituent on the isatin core, we were unable to detect these
M5 PAMs in rodent CNS. As such, we performed a high
throughput screen directly interrogating M5 functional activity
in conjunction with the Scripps Research Institute Molecular
Screening Center (SRIMSC). For this campaign, we used a
triple addition protocol (compound addition followed by low
and high concentrations of orthosteric agonist (ACh)) to
screen the MLPCN14 collection of ∼360 000 compounds. This
screening strategy allows for the identification of activators
(agonists and PAMs) while also surveying for inhibitors
(NAMs and antagonists). Single concentration-point screening
experiments in Chinese hamster ovary (CHO) cells stably
expressing the human M1, M4, and M5 receptors identified 3920
M5 hits (1.07% hit rate). Hits were triaged based on activity in
untransfected cells, structural tractability, and the elimination of
frequent hitters.15 The most attractive M5 activators were
purchased from commercial sources and reconfirmed using 10-
point concentration−response curves (CRC). These “triple-
add” CRC experiments resulted in the identification of nine
confirmed M5 PAMs, nine M5 antagonists,9,16 and zero M5
agonists.
a
Reagents and conditions: (a) benzylamine, HATU, DCM, DIPEA,
95%; (b) NaOtBu, Et-I, 15-crown-5, THF, 88%; (c) HCl, dioxane,
99%; (d) 1,4-benzodioxan-6-sulfonyl chloride, DCM, DIPEA, 70%.
was quite flexible, when applied to many of the modifications
proposed in Figure 1, the SAR was disappointingly rigid. A
litany of modifications were not tolerated and resulted in a
complete loss of M5 activity: (1) removal of N-benzyl or N-
ethyl moiety to provide a secondary amide, (2) cyclizing the
ethyl group back to the piperidine, (3) relocating the carbonyl
to produce the acetamide or benzamide analogs, (4) replacing
the amide with a sulfonamide, (5) replacing the piperidine with
an azetidine or [1.3.0] bicycle, (6) replacing the sulfonamide
with an amide, urea, or carbamate, and (7) any alkylsulfona-
mide in place of an arylsulfonamide. The failure of these global
modifications suggested that an improvement in potency would
require a better understanding of the M5 PAM SAR brought
about by more modest modifications.
Initial improvements in PAM potency were provided by
modifications to the arylsulfonamide (Table 1). The unadorned
phenylsulfonamide 6a showed slightly improved potency over
the HTS hit; however, potency could be further improved
through the introduction of methoxy groups at the para and
meta positions. Interestingly, the 3,4-dimethoxy analog 6e
displayed slightly reduced efficacy (AChmax) relative to the HTS
hit and may speak to the preference for sulfonamides with more
planar aryl groups at this location. Substitution at the ortho
position was clearly disfavored as indicated by 6d and the
sulfonamide regioisomer of the HTS hit 6f. A sampling of
alternative monocyclic heteroaryl groups (6g−m) did not
provide improved activity. Building on the importance of
substituents at the 3 and 4 positions, we explored a range of
bicyclic heteroarylsulfonamides with annulated rings spanning
these two positions. A number of heterocycles showed
significant improvements over the HTS hit. In particular, the
benzofuranyl (6o) and indazolylsulfonamides (6p and 6q)
provided hM5 PAMs with EC50 of 1.6−2.4 μM, representing an
approximately 3-fold improvement over the naked phenyl-
sulfonamide.
Chemistry. Structurally, the most promising of the M5 PAM
hits, 1 (Figure 1), represented a novel chemotype for an M5
Figure 1. Structure and initial plans to modify HTS hit 1.
PAM and offered a wide range of straightforward modifications
due to its highly modular appearance. The broad range of
planned modifications to 1, shown in Figure 1, could readily be
accomplished by employing the synthesis route shown in
Scheme 1. Starting from the N-Boc protected carboxylic acid
core 2, peptide coupling with an amine introduced the first
alkyl group on amide 3. Deprotonation of this amide was
followed by the addition of an alkylating agent (e.g., ethyl
iodide) and a crown ether, necessary to facilitate the
introduction of the second alkyl group, providing 4. Removal
of the Boc protecting group under standard anhydrous HCl
conditions provided the salt 5. The amine of 5 was then
sulfonylated to provide the HTS hit 1. Alternatively, this
secondary amine could be functionalized through reactions
with a wide range of electrophiles (e.g., acid chlorides,
isocyanates, alkyl halides, etc.). Although the synthesis route
Employing sulfonamides structurally related to those
appearing in Table 1, we explored changes to the western
region of 1 and were gratified that introduction of a methyl
group at the benzylic position improved potency while
demonstrating enantiospecific activity (Table 2). While the
(S)-enantiomer 7a was inactive at hM5, the (R)-methyl
enantiomer 7b demonstrated a 3-fold improvement in potency
relative to its des-methyl analog 6b. This improvement in
potency was maintained across the benzofuranyl (7c) and 2,3-
dihydrobenzofuranyl (7d) analogs. Although slight, improve-
ments in hM5 PAM potency were realized with the
incorporation of 2,3-dihydroindenyl and the 6- and 5-
indazolylsulfonamides (7e, 7f, and 7g, respectively).
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Table 1. Structures and Activities of Analogs 6
a
a
compd
Ar
hM5 pEC50
hM5 EC50 (μM)
ACh max (%)
6a
6b
6c
6d
6e
6f
phenyl
5.21 0.06
6.2
69
84
77
−
57
53
−
3
3
4
4-methoxyphenyl
5.48 0.06
3.3
3-methoxyphenyl
5.51 0.09
3.1
2-methoxyphenyl
−
<5
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.4
3,4-dimethoxyphenyl
2,3-dihydrobenzo[b][1,4]dioxin-5-yl
imidazol-4-yl
3
2
<5
6g
6h
6i
5-methylthiophen-2-yl
3,5-dimethylisoxazol-4-yl
6-(trifluoromethyl)pyridin-3-yl
pyridin-4-yl
<5
64
3
−
−
−
−
−
<5
−
−
−
−
6j
6k
61
6m
6n
6o
6p
6q
6r
pyridin-3-yl
pyridin-2-yl
−
quinolin-7-yl
66
80
86
86
81
−
3
5
4
4
5
benzofuran-5-yl
5.63 0.11
5.81 0.08
5.77 0.09
5.53 0.11
−
1H-indazol-5-yl
1.6
1H-indazol-6-yl
1.7
benzo[c][1,2,5]thiadiazol-5-yl
benzo[c][1,2,5]thiadiazol-4-yl
benzo[d][1,3]dioxol-5-yl
3.0
6s
6t
>10
7.4
5.13 0.16
93 12
a
hM5 pEC50 and ACh max data reported as averages SEM from our calcium mobilization assay; n = 3−4 determinations; −, not determined.
Table 2. Structures and Activities of Analogs 7
a
a
compd
∗
Ar
hM5 pEC50
hM5 EC50 (μM)
ACh max (%)
7a
7b
7c
7d
7e
7f
S
4-methoxyphenyl
−
inactive
0.97
−
R
R
R
R
R
R
4-methoxyphenyl
6.01 0.07
6.03 0.06
5.95 0.05
6.14 0.05
6.12 0.05
6.13 0.04
90
80
88
97
87
86
3
2
2
2
2
1
benzofuran-5-yl
0.93
2,3-dihydrobenzofuran-5-yl
2,3-dihydro-1H-inden-5-yl
1H-indazol-6-yl
1.11
0.73
0.76
7g
1H-indazol-5-yl
0.74
a
hM5 pEC50 and ACh max data reported as averages SEM from our functional calcium mobilization assay; n = 3−4 determinations; −, not
determined.
Simultaneously, we were exploring modifications to the
western aryl ring in the context of the indazole sulfonamides
and identified a number of productive alterations depicted in
Table 3. Systematically moving a fluorine around the phenyl
ring revealed that substitution at the 2 and 3 positions was
favored, with a trend in preference for the 3-fluoro 8b. As such,
small groups were introduced at the meta position. Most
notably, the 3-methyl analog 8f yielded a submicromolar
potency (hM5 EC50 = 0.87 μM). This potency was mirrored by
the analogous 2-methyl analog 8g and prompted a further
exploration of substituents at the ortho position. The 2-chloro
(8i) and 2-trifluoromethyl (8j) groups provided further
improvements in potency, but interestingly the 3- and 4-
trifluoromethyl analogs (8k and 8l, respectively) possessed
greatly reduced activity and illustrated the frequently steep
nature of allosteric SAR. The two most potent ortho-
substituted analogs (Cl and CF3) with the 6-indazolyl-
sulfonamide were also examined in the context of the 5-
indazolylsulfonamide (8m and 8n) and found to be among the
most potent hM5 PAMs prepared to date. Specifically, 8n,
displaying a hM5 PAM EC50 = 0.19 μM, was 8-fold more potent
than its des-CF3 congener (6p, Table 1). Disappointingly,
addition of a methyl group in the (R) configuration to 8n,
analogous to the compounds in Table 2, resulted in a 10-fold
decrease in potency (hM5 EC50 = 2.4 μM, structure not
shown).
In vitro metabolite identification experiments performed on
7g implicated the N-ethyl moiety as the primary site of
metabolism. While we were simultaneously exploring mod-
ification to this N-ethyl group with variations at other locations,
a concise but still representative description of these efforts can
be summarized in the context of the highly optimized 8n and
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Journal of Medicinal Chemistry
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activity at hM5 (respectively, structures not shown). The ethyl
group in 8n could be extended without incurring a loss in
potency as demonstrated by the n-propyl analog 9a. However,
the other three-carbon isomers (9b−d) all suffered a >10-fold
drop in activity. Attempts to mitigate metabolism through the
introduction of polarity on the terminus of the ethyl group in
8n similarly engendered an unacceptable decrease in activity
upon introduction of a hydroxyl (9f) or even a single fluorine
atom (9g). Given the complete absence of activity demon-
strated by the cyclopropyl analog 9d, it was surprising to find
that the cyclobutyl version (9e) displayed mid-micromolar
potency. Supporting the hypothesis that alkyl branching is not
well tolerated directly adjacent to the amide nitrogen (i.e., 9c−
e) but that larger alkyl groups could be present more distally,
the sec-butyl analog 9h was equipotent to its n-propyl conger
(9a). Furthermore, even larger alkyl groups at this location
(9i−k) displayed consistently high levels of activity. Unfortu-
nately, none of these modifications could shift the primary
route of metabolism away from this region of these molecules
while maintaining high levels of M5 PAM activity, nor could
they attenuate an inherently high rate of in vitro metabolism
(i.e., rat hepatic microsomal CLint > 500 mL min−1 kg−1).
However, 9d was able to reduce intrinsic microsomal clearance
by an order of magnitude, but this came at the expense of losing
all hM5 activity.
Table 3. Structures and Activities of Analogs 8
a
indazolyl
hM5 EC50 ACh max
a
compd
R
attachment
hM5 pEC50
<5
(μM)
(%)
8a
8b
8c
8d
8e
8f
4-F
6
6
6
6
6
6
6
6
6
6
6
6
5
5
>10
1.5
55
3
2
5
2
5
2
3-F
5.82 0.04
5.67 0.12
5.79 0.06
5.58 0.10
6.06 0.07
6.06 0.07
6.12 0.06
6.19 0.05
6.32 0.04
5.26 0.09
−
89
84
77
85
78
103
92
93
92
59
−
2-F
2.1
3-Cl
1.6
3-MeO
3-Me
2-Me
2-MeO
2-Cl
2.6
0.87
0.87
0.75
0.64
0.48
5.6
8g
8h
8i
3
3
2
2
3
8j
2-CF3
3-CF3
4-CF3
2-Cl
8k
81
8m
8n
inactive
0.35
0.19
6.46 0.08
78
2
2
2-CF3
6.71 0.06
96
a
hM5 pEC50 and ACh max data reported as averages SEM from our
calcium mobilization assay; n = 3−5 determinations; −, not
determined.
Pharmacology and Selectivity. A subset of M5 PAMs
were further assessed for their ability to enhance the potency of
ACh at the hM5 receptor using a fluorescence based calcium
mobilization assay. Experimentally, a fixed concentration of
PAM (10 μM) or vehicle was added prior to the addition of a
concentration response curve (CRC) of ACh, and the left shift
in potency of ACh was determined as the ratio (fold shift) of
the potency in the absence and presence of PAM. As shown in
Table 5, the HTS hit 1 produced a fold shift of 2.3, while the
more potency-optimized analogs showed at least twice that
value. Four of the most potent compounds from Tables 2 and 3
(7b, 7g, 8j, and 8n) gave fold shift values in the 7- to 12-fold
range, similar to earlier M5 PAMs.8
Although 7g and 8n displayed similar fold shift values, 8n
was superior to 7g with respect to hM5 PAM potency and by
virtue of its superior muscarinic subtype selectivity profile.17
The muscarinic subtype selectivity profile for 8n across the five
human and rat receptor subtypes can be seen in Figure 2. 8n
shows no activity at hM2 or hM4 (the natively Gi/o coupled
mAChRs; our assays employed cells co-transfected with
chimeric Gqi5 to facilitate M2/M4 coupling to Ca2+
mobilization) and displays greater than 10-fold selectivity
over hM1 and hM3 (the Gq coupled mAChRs). The lower
potencies, combined with lower efficacies, at hM1 (hM1 PAM
its analogs appearing in Table 4. Although not bearing the
optimized 5-indazolylsulfonamide shown in Table 4, early
Table 4. Structures and Activities of Analogs 9
a
hM5 EC50
(μM)
ACh max
(%)
a
compd
R
hM5 pEC50
9a
9b
9c
9d
9e
9f
n-propyl
6.81 0.06
5.74 0.04
<5
0.16
1.8
94
2
2
3
allyl
79
isopropyl
>10
inactive
1.8
54
cyclopropyl
cyclobutyl
−
−
84
5.74 0.09
4
3
4
2-hydroxyethyl
2-fluoroethyl
sec-butyl
<5
>10
1.3
60
9g
9h
9i
5.90 0.07
6.82 0.06
6.91 0.06
6.92 0.06
6.89 0.05
95
0.15
0.12
0.12
0.13
100
104
102
103
2
2
2
2
neopentyl
9j
cyclopropylmethyl
cyclobutylmethyl
9k
a
hM5 pEC50 and ACh max data reported as averages SEM from our
calcium mobilization assay; n = 3−5 determinations; −, not
determined.
EC50 = 5.4 μM, AChmax = 52%) and hM3 (hM3 PAM EC50
=
2.1 μM, AChmax = 67%) when compared to those for 8n at hM5
(hM5 PAM EC50 = 0.19 μM, AChmax = 96%) may actually
afford a greater than 10-fold selectivity window. Interestingly,
when assessed at the rat muscarinic receptors, the level of
results indicated that groups smaller than ethyl (i.e., hydrogen
or methyl) resulted in a complete loss of, or greatly diminished,
Table 5. ACh Fold-Shift Values for Select M5 PAMs
compd
1
6p
6q
7b
7g
12
8j
8n
a
ACh fold shift
2.3 0.1
5.4 0.7
4.8 0.4
7
1
3
7
2
9
4
a
ACh fold-shift data, for compounds at 10 μM, reported as averages SEM from our calcium mobilization assay and represent leftward shifts in
ACh potency; n = 3−4.
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Journal of Medicinal Chemistry
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similarity points to the possibility of a common allosteric
binding site and the high probability that further SAR will
reveal completely selective M5 PAMs from this series. To more
broadly explore this new scaffold’s potential for nonmuscarinic,
off-target activity, 8n was submitted to Eurofin’s Pan Labs lead
profiling screen. This battery of radioligand binding assays
consists of 68 common GPCRs, ion channels, and transporters
where the test compound (8n) was present at 10 μM.
Remarkably, 8n showed a significant response (>49%
radiologand displacement; see Supporting Information for
complete results) in just two assays: cannabinoid CB1 receptor
(50% displacement) and σ1 receptor (53% displacement).
However, these binding results do not guarantee functional
activity and the mid-range values did not prompt functional
determinations.
Figure 2. Muscarinic subtype selectivity profile of 8n: (A) human
selectivity (hM5 EC50 = 0.19 μM, hM4 EC50 > 30 μM, hM3 EC50 = 2.1
μM, hM2 EC50 > 30 μM, hM1 EC50 = 5.4 μM); (B) rat selectivity (rM5
EC50 = 0.61 μM, rM4 EC50 > 30 μM, hM3 EC50 = 3.1 μM, hM2 EC50
>
30 μM, hM1 EC50 = 2.0 μM). Data represent the mean SEM from at
least three independent determinations employing highly expressing
cell lines with similarly high expression levels of muscarinic receptors.
Metabolism and Disposition. Encouraged by these initial
results, 8n was characterized in a variety of DMPK assays
(Table 6). From an in vitro standpoint, 8n displayed minimal
subtype selectivity diminished and rM1 was now closest in
potency to rM5.
The pharmacology of 8n was further profiled in radioligand
binding experiments (Figure 3). Increasing concentrations of
Table 6. DMPK Profile of 8n
in vitro
in vivo
microsome CLint
rat: 2600
(male, Sprague−Dawley, n = 3)
(mL/min/kg)
human: 540
rat: 68
CLplasma (mL/min/kg)
elimination t1/2 (min)
Vdss (L/kg)
66
a
predicted CLhep
(mL/min/kg)
22
human: 20
rat: 0.014
human: 0.015
rat: 0.011
1.6
0.36
0.28
fu plasma
brain/plasma Kp
(at 15 min) Kp,uu
fu brain
a
Determined using CLint values in the well-stirred model of organ
clearance not corrected for fraction unbound in plasma.
Figure 3. (A) [3H]NMS competition binding. 8n has no inhibitory
effect on [3H]NMS binding (97.1% max), while the control (atropine)
inhibits [3H]NMS binding in a concentration dependent manner (Ki =
1.47 nM, 1.8% max). (B) Acetylcholine affinity shift profile of 8n.
Increasing fixed concentrations of 8n result in progressive left shifts of
the ACh inhibition curve, with a maximal shift of approximately 15.
Experiments were performed using membranes prepared from hM5
metabolic stability with a very high hepatic microsomal intrinsic
clearance in rat and human (CLint; rat, 2600 mL min−1 kg−1;
human, 540 mL min−1 kg−1) and a correspondingly high
predicted hepatic clearance in both species (CLhep; rat, 68 mL
min−1 kg−1; human, 20 mL min−1 kg−1), on par with hepatic
blood flow. A low fraction unbound in plasma was observed for
rat and human (fu,plasma; rat, 0.014; human, 0.015) and in rat
brain homogenate (fu,brain; rat, 0.011). In rodents (male,
Sprague−Dawley rats, 1 mg/kg iv, n = 3), similar instability was
observed after intravenous dosing; 8n displayed high clearance
(66 mL min−1 kg−1), a moderate volume of distribution (1.6 L
kg−1), and a short half-life (t1/2, 22 min). A modest total brain-
plasma partition coefficient (Kp = 0.36) was also determined
from these experiments (15 min after administration); however,
the unbound brain-plasma partition coefficient (Kp,uu = 0.28)
tempers the attractiveness of 8n as a highly CNS penetrant
compound. Still, the high permeability determined in Caco-2
cells (Papp = 2.5 × 10−5 cm/s) represents an attractive starting
point to further optimize CNS exposure.
CHO cells. Data represent the mean
independent determinations.
SEM from at least three
8n or atropine (control) were incubated with a fixed
concentration of [3H]N-methylscopolamine (NMS, 0.3 nM,
an orthosteric antagonist) and membranes expressing the hM5
receptor. While atropine displaced [3H]NMS binding in a
concentration dependent manner, 8n had no effect on
[3H]NMS binding (Figure 3A), suggesting that 8n interacts
with the hM5 receptor via an allosteric mechanism. To further
characterize the interaction of 8n with the hM5 receptor,
increasing fixed concentrations of 8n were incubated with a
CRC of ACh in the presence of a fixed concentration of
[3H]NMS (0.4 nM) to determine the effect of 8n on the
affinity of ACh. 8n shifted the ACh competition curve leftward
by ∼15-fold (Figure 3B), further demonstrating its function as
a PAM, acting through modulation of the potency and affinity
of ACh. However, it has yet to be defined what in vitro
properties are required for a hM5 PAM to generate a specific in
vivo outcome. Only now are we beginning to amass the
necessary tool compounds to explore this question.
CONCLUSION
■
In summary, we have developed 8n (also referred to as ML380
or VU0481443), which is among the most potent M5 PAMs
reported to date (hM5 EC50 = 190 nM, rM5 EC50 = 610 nM)
and is M5 preferring with some functional activity remaining at
M1 and M3. This compound will be a useful tool to further
investigate the in vitro properties of the M5 receptor as more
advanced PAMs are identified. This novel chemotype
distinguishes itself from our previously published isatin-
containing M5 PAMs in its ability to be detected within the
CNS even though its partition coefficients (Kp and Kp,uu) are
Interestingly, this novel class of hM5 PAMs showed a clear
preference for the Gq coupled mAChRs over the Gi/o coupled
receptors, which is reminiscent of the nonselective pan-Gq
PAM HTS hit8 that served as the progenitor for three previous
M5 PAM probe molecules8 and an M1 selective PAM.6 This
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Journal of Medicinal Chemistry
Brief Article
less than optimal. However, the highly modular nature of this
ligand will allow for continued structural optimization to
further improve potency, selectivity, metabolic stability, and
CNS penetration. Continuing efforts around this scaffold are in
progress and will be reported in due course.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and spectroscopic data for selected
compounds, detailed pharmacology, and DMPK methods. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
S
EXPERIMENTAL SECTION
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 615-322-0670. Fax: 615-343-3088. E-mail: michael.r.
wood@vanderbilt.edu.
Chemistry. The general chemistry, experimental information, and
syntheses of key compounds are supplied in the Supporting
Information. Purity for all final compounds was >95%, and each
showed a parent mass ion consistent with the desired structure
(LCMS).17
■
Notes
1-((1H-Indazol-5-yl)sulfonyl)-N-ethyl-N-(2-(trifluoromethyl)-
benzyl)piperidine-4-carboxamide (8n). To a solution of 1-Boc-4-
piperidinecarboxylic acid (2.00 g, 8.72 mmol, 1 equiv) and DIPEA
(4.48 mL, 26.2 mmol, 3 equiv) in DCM (30 mL, 0.3 M) was added 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (2.51 g, 13.1
mmol, 1.5 equiv), hydroxybenzotriazole (1.77 g, 13.1 mmol, 1.5
equiv), and ethylamine HCl (1.42 g, 17.5 mmol, 2.0 equiv). The
mixture was stirred for 2 h at room temperature before being
quenched with aqueous NaHCO3. The organic layer was separated,
and the aqueous layer was extracted with DCM. The combined
organic layers were washed with brine, dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified via
silica gel column chromatography to give 1.89 g of 1-Boc-4-
(ethylcarbamoyl)piperidine (83% yield). To a solution of 1-Boc-4-
(ethylcarbamoyl)piperidine (50.0 mg, 0.195 mmol, 1 equiv) and 15-
crown-5 (77.4 μL, 0.390 mmol, 2 equiv) in THF (2 mL, 0.1 M) was
added NaOtBu (28.1 mg, 0.293 mmol, 1.5 equiv). The mixture was
stirred for 30 min at room temperature before adding 2-
(trifluoromethyl)benzyl bromide (59.4 μL, 0.39 mmol, 2 equiv).
After 16 h, the mixture was concentrated under reduced pressure and
the residue partitioned between H2O and DCM. The organic layer was
separated, and the aqueous layer was extracted with DCM. The
combined organic layers were concentrated under reduced pressure
and the residue was purified via Gilson preparative LC (MeCN/water/
0.1% TFA gradient as the mobile phase through a c-18 column) to
obtain 1-Boc-4-(ethyl(2-(trifluoromethyl)benzyl)carbamoyl)-
piperidine. To a solution of 1-Boc-4-(ethyl(2-(trifluoromethyl)-
benzyl)carbamoyl)piperidine in DCM was added MP-TsOH (5
equiv). The mixture was heated to 100 °C under microwave
irradiation for 10 min. The mixture was filtered, and the resin was
rinsed with MeOH before washing with NH3/MeOH to elute product.
Solvent was removed under reduced pressure to give 43 mg of pure 4-
(ethyl(2-(trifluoromethyl)benzyl)carbamoyl)piperidine (70% yield,
two steps). To a solution of 4-(ethyl(2-(trifluoromethyl)benzyl)-
carbamoyl)piperidine (20 mg, 0.063 mmol, 1 equiv) and DIPEA (33
μL, 0.20 mmol, 3 equiv) in DCM was added 1H-indazole-5-sulfonyl
chloride (21 mg, 0.095 mmol, 1.5 equiv). The mixture was allowed to
stir for 2 h at room temperature and was then quenched with MeOH
and concentrated under reduced pressure. The residue was purified via
Gilson preparative LC (MeCN/water/0.1% TFA gradient as the
mobile phase through a c-18 column) to obtain 4.2 mg of 8n (15%
yield). HRMS (TOF, ES+) C23H26N4O3F3S [M + H]+ calcd mass
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was generously supported by Grant NIH/MLPCN
U54 MH084659 (C.W.L.) and Grant U54 MH084512
(Scripps).
ABBREVIATIONS USED
■
hM5, human muscarinic acetylcholine receptor subtype 5;
MLPCN, Molecular Libraries Probe Production Centers
Network; NMS, N-methylscopolamine
REFERENCES
■
(1) Smythies, J. Section I. The cholinergic system. Int. Rev. Neurobiol.
2005, 64, 1−122.
(2) Dencker, D.; Thomsen, M.; Wortwein, G.; Weikop, P.; Cui, Y.;
Jeon, J.; Wess, J.; Fink-Jensen, A. Muscarinic acetylcholine receptor
subtypes as potential drug targets for the treatment of schizophrenia,
drug abuse, and Parkinson’s disease. ACS Chem. Neurosci. 2011, 3, 80−
89.
(3) Wess, J.; Eglen, R. M.; Gautam, D. Muscarinic acetylcholine
receptors: mutant mice provide new insights for drug development.
Nat. Rev. Drug Discovery 2007, 6, 721−733.
(4) Melancon, B. J.; Hopkins, C. R.; Wood, M. R.; Emmitte, K. A.;
Niswender, C. M.; Christopoulos, A.; Conn, P. J.; Lindsley, C. W.
Allosteric modulation of 7 transmembrane spanning receptors: theory,
practice and opportunities for CNS drug discovery. J. Med. Chem.
2012, 55, 1445−1464.
(5) Conn, P. J.; Jones, C.; Lindsley, C. W. Subtype selective allosteric
modulators of muscarinic receptors for the treatment of CNS
disorders. Trends Pharmacol. Sci. 2009, 30, 148−156.
(6) Bridges, T. M.; Kennedy, J. P.; Noetzel, M. J.; Breininger, M. L.;
Gentry, P. R.; Conn, P. J.; Lindsley, C. W. Chemical lead optimization
of a pan Gq mAChR M1, M3, M5 positive allosteric modulator (PAM)
lead. Part II: Development of a potent and highly selective M1 PAM.
Bioorg. Med. Chem. Lett. 2010, 20, 1972−1975.
(7) Byun, N. E.; Grannan, M.; Bubser, M.; Barry, R. L.; Thompson,
A.; Rosanelli, J.; Gowrishankar, R.; Kelm, N. D.; Damon, S.; Bridges,
T. M.; Melancon, B. J.; Tarr, J. C.; Brogan, J. T.; Avison, M. J.; Deutch,
A. Y.; Wess, J.; Wood, M. R.; Lindsley, C. W.; Gore, J. C.; Conn, P. J.;
Jones, C. K. Antipsychotic drug-like effects of the selective M4
muscarinic acetylcholine receptor positive allosteric modulator
VU0152100. Neuropsychopharmacology 2014, 39, 1578−1593.
(8) Gentry, P. R.; Bridges, T. M.; Lamsal, A.; Vinson, P. N.; Smith,
E.; Chase, P.; Hodder, P. S.; Engers, J. L.; Niswender, C. M.; Daniels, J.
S.; Conn, P. J.; Wood, M. R.; Lindsley, C. W. Discovery of ML326: the
first sub-micromolar, selective M5 PAM. Bioorg. Med. Chem. Lett. 2013,
23, 2996−3000.
1
495.1678, found 495.1679. H NMR (1:1.25 rotamer ratio, ∗ denotes
minor rotamer, 400.1 MHz, CDCl3) δ (ppm): 8.31 (s, 1H); 8.25 (m,
1H); 7.78, 7.72* (d, J = 8.8 Hz, 1H); 7.68−7.57 (m, 2H); 7.52*, 7.46
(t, J = 7.6 Hz, 1H); 7.38*, 7.32 (t, J = 7.6 Hz, 1H); 7.21−7.13 (m,
1H); 4.76, 4.64* (s, 2H); 3.95−3.86, 3.85−3.76* (m, 2H); 3.41*, 3.22
(q, J = 7.2 Hz, 2H); 2.60−2.46 (m, 2H); 2.37−2.26 (m, 1H); 2.11−
1.92 (m, 2H); 1.91−1.81, 1.74−1.65* (m, 2H); 1.16−1.05 (m, 3H).
13C NMR (1:1.35 rotamer ratio, ∗ denotes minor rotamer, 100.6 MHz,
CDCl3) δ (ppm): 174.70; 141.40, 141.34*; 136.29, 135.77*; 135.95;
132.67*, 132.31; 129.31*, 129.19; 127.94, 127.86*; 127.90 (q, J = 30.3
Hz); 126.65 (q, J = 5.3); 126.47; 126.26 (q, JCF = 245 Hz); 126.04 (q,
J = 5.6 Hz); 122.81, 122.73*; 122.66; 110.92, 110.89*; 46.94*, 44.23;
45.64, 45.46*; 42.12, 41.87*; 37.96*, 37.68; 28.48, 28.27*; 14.44,
12.67*.
(9) Gentry, P. R.; Kokubo, M.; Bridges, T. M.; Kett, N. R.; Harp, J.
M.; Cho, H. P.; Smith, E.; Chase, P.; Hodder, P. S.; Niswender, C. M.;
Daniels, J. S.; Conn, P. J.; Wood, M. R.; Lindsley, C. W. Discovery of
the first M5-selective and CNS penetrant negative allosteric modulator
(NAM) of a muscarinic acetylcholine receptor: (S) 9b-(4-chlor-
7809
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ophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo[2,1-a]-
isoindol-5(9bH)one (ML375). J. Med. Chem. 2013, 56, 9351−9355.
(10) Thomsen, M.; Wortwein, G.; Fink-Jensen, A.; Woldbye, D. P.
D.; Wess, J.; Caine, S. B. Decreased prepulse inhibition and increased
sensitivity to muscarinic, but not dopaminergic drugs in M5 muscarinic
acetylcholine receptor knockout mice. Psychopharmacology 2007, 192,
97−110.
(11) Araya, R.; Noguchi, T.; Yuhki, M.; Kitamura, N.; Higuchi, M.;
Saido, T. C.; Seki, K.; Itohara, S.; Kawano, M.; Tanemura, K.;
Takashima, A.; Yamada, K.; Kondoh, Y.; Kanno, I.; Wess, J.; Yamada,
M. Loss of M5 muscarinic acetylcholine receptors leads to
cerebrovascular and neuronal abnormalities and cognitive deficits in
mice. Neurobiol. Dis. 2006, 24, 334−344.
(12) Yamada, M.; Lamping, K. G.; Duttaroy, A.; Zhang, W.; Cui, Y.;
Bymaster, F. P.; McKinzie, D. L.; Felder, C. C.; Deng, C.-X.; Faraci, F.
M.; Wess, J. Cholinergic dilation of cerebral blood vessels is abolished
in M5 muscarinic acetylcholine receptor knockout mice. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 14096−14101.
(13) Kim, H. A.; Miller, A. A.; Drummond, G. R.; Thrift, A. G.;
Arumugam, T. V.; Phan, T. G.; Srikanth, V. K.; Sobey, C. G. Vascular
cognitive impairment and Alzheimer’s disease: role of cerebral
hypoperfusion and oxidative stress. Naunyn-Schmiedeberg’s Arch.
Pharmacol. 2012, 385, 953−959.
(14) For the MLPCN see http://mli.nih.gov/mli/mlpcn/. ML380 is
an MLPCN probe and freely available upon request.
(15) Baell, J. B.; Holloway, G. A. New substructure filters for removal
of pan assay interference compounds (PAINS) from screening libraries
and for their exclusion in bioassays. J. Med. Chem. 2010, 53, 2719−
2740.
(16) Gentry, P. R.; Kokubo, M.; Bridges, T. M.; Cho, H. P.; Smith,
E.; Chase, P.; Hodder, P. S.; Utley, T. J.; Rajapakse, A.; Byers, F.;
Niswender, C. M.; Morrison, R. D.; Daniels, J. S.; Wood, M. R.; Conn,
P. J.; Lindsley, C. W. Discovery, synthesis and characterization of a
highly mAChR selective M5 orthosteric antagonist, VU0488130
(ML381): a novel molecular probe. ChemMedChem 2014, 9, 1677−
1682.
(17) See Supporting Information for full details.
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