Development of a more highly selective M1 antagonist from the continued optimization of the MLPCN Probe ML012

Article abstract of DOI:10.1016/j.bmcl.2011.11.110

This Letter describes the continued optimization of an MLPCN probe molecule (ML012) through an iterative parallel synthesis approach. After exploring extensive modifications throughout the parent structure, we arrived at a more highly M₁-selective antagonist, compound 13l (VU0415248). Muscarinic subtype selectivity across all five human and rat receptors for 13l, along with rat selectivity for the lead compound (ML012), is presented.

Full text of DOI:10.1016/j.bmcl.2011.11.110

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1044–1048
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of a more highly selective M1 antagonist
from the continued optimization of the MLPCN Probe ML012
Bruce J. Melancon a,b,c,d, Alexander P. Lamers b,d, Thomas M. Bridges a,c,d, Gary A. Sulikowski b,d
,
Thomas J. Utley a,c,d, Douglas J. Sheffler a,c, Meredith J. Noetzel a,c,d, Ryan D. Morrison a,c,d
,
J. Scott Daniels a,c,d, Colleen M. Niswender a,c,d, Carrie K. Jones a,c,d,e, P. Jeffrey Conn a,c,d
,
Craig W. Lindsley a,b,c,d, Michael R. Wood a,b,c,d,
⇑
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
c Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
d Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
e U.S. Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes the continued optimization of an MLPCN probe molecule (ML012) through an iterative
parallel synthesis approach. After exploring extensive modifications throughout the parent structure, we
arrived at a more highly M1-selective antagonist, compound 13l (VU0415248). Muscarinic subtype selec-
tivity across all five human and rat receptors for 13l, along with rat selectivity for the lead compound
(ML012), is presented.
Received 8 November 2011
Revised 23 November 2011
Accepted 28 November 2011
Available online 6 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Muscarinic acetylcholine receptor 1
M1
Antagonist
ML012
VU0415248
Acetylcholine (ACh) is a critical neurotransmitter with diverse
functions both within the central nervous system (CNS) and in
peripheral signaling pathways.1–4 ACh carries out its functions by
interacting with two very distinct groups of receptors; a set of
ligand-gated ion channels—the nicotinic acetylcholine receptors
(nAChRs)- and a set of family A, G protein-coupled receptors
(GPCRs)—the muscarinic acetylcholine receptors (mAChRs). The
muscarinic cohort of acetylcholine receptors are further divided into
five subtypes (M1–5).5 These five subtypes can be further classified
into two subsets based on their G protein-coupling partners, with
the M1, 3, 5 receptors preferentially coupling to Gq/11 which stimu-
lates PLC and calcium mobilization, and with the M2,4 receptors pref-
erentially coupling to Gi/o to inhibit adenylate cyclase (AC), thereby
decreasing cAMP production.4 These mAChRs are widely expressed
throughout the body with varying degrees of expression levels for
specific subtypes based on the particular site or organ.6 As a result,
mAChRs play significant roles in a wide range of physiological func-
tions such as memory and attention, motor control, nociception,
regulation of sleep-wake cycles, cardiovascular function, secretory
functions, and mediators of inflammation, renal and GI function,
among many others.6,7
While it is believed that the M1,4,5 receptors are the most impor-
tant for CNS targets, what remains unknown are the specific func-
tions that are regulated by each individual receptor subtype.7 This
is a direct result of the highly conserved orthosteric binding site
for the endogenous ligand (ACh), which is shared across all five
mAChRs; this high conservation has severely confounded the devel-
opment of muscarinic ligands with a high degree of selectivity for a
single subtype.8 A lack of subtype selectivity, however, has not pre-
cluded the use of mAChR-acting pharmaceuticals for a wide range of
indications but many of the undesirable side effects for these non-
selective medications can be attributed to activity at the other
mAChRs (often M2 and M3), limiting their clinical utility. Addition-
ally, the limited selectivity displayed by earlier mAChR ligands has
complicated the interpretation of both animal model studies as well
as human clinical trials with respect to the specific mAChR subtype
being responsible for the given desired (or undesired) result. For
example, xanomeline, a reported M1- and M4-selective agonist,
showed clinical efficacy in Phase II trials for Alzheimer’s disease
and schizophrenia,9,10 but also has nearly equivalent agonist activity
at M3.8 Even in the absence of off-target mAChR activity, the debate
⇑
Corresponding author.
E-mail address: michael.r.wood@vanderbilt.edu (M.R. Wood).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.110

B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1044–1048
1045
There are a number of ways to potentially engender mAChR subtype
selectivity among synthetic ligands. First, the more classical
approach, by simultaneously binding to the orthosteric site while
at the same time extending beyond this highly conserved region of
the receptor and into adjacent areas which may be less structurally
conserved among the other mAChRs (vide infra).11 Second, by bind-
ing to a completely distinct region of the mAChR (an allosteric site),
which is distinct from the orthosteric binding site and which might
also show less amino acid similarity between the five mAChRs. This
allosteric approach has been highly successful for a number of the
individual mAChRs: M1,12–14 M4,15 and M5.16
O
S
N
N
N
N
N
O
S
O
NH
ML012
(VU0255035)
hM1 IC50 = 690 nM
Figure 1. The MLPCN probe molecule ML012.
still remains as to whether a single mAChR subtype (M1 or M4) is
responsible for the very encouraging positive outcomes, in these
trials.
Single and double mAChR knock out (KO) mice for each of the
muscarinic receptor subtypes have yielded a great deal of insight
into the specific roles played by each receptor, but these results must
always be viewed with the caveat that compensatory changes may
have occurred during the development of the mutant KO mice.8
More highly selective mAChR ligands would allow for a better
understanding of the individual roles for each of the five mAChRs.
In a previous publication from our laboratories, we described the
highly selective M1 antagonist ML012 (VU0255035, Fig. 1).11
Although acting at the orthosteric site on all five mAChRs, ML012
achieved M1 selectivity in the range of 45- to 159-fold over M2–5
.
Additionally, ML012reducedpilocarpine-induced seizures in rodent
models at doses that did not induce deficits in contextual freezing,
demonstrating that selective M1 antagonists may have greater ther-
apeutic potential than the non-selective muscarinic antagonists
presently available. Given the potential for M1 antagonists in such
CO2Me
CO2Me
a
c
b, c
O
H2N
ArSO2HN
O
N
N Ar'
1
2
d, a
ArSO2HN
3
CO2H
N
N Ar'
BocHN
BocHN
4
5
Scheme 1. Reagents: (a) ArSO2Cl, NEt3, DCM; (b) NaOH/H2O/THF; (c) amine, EDCI, HOBt, NEt3; (d) 4 M HCl in dioxane.
Table 1
Structures and activities of M1 antagonist analogs 6a–o
O
S
N
N Ar'
N
N
O
S
O
NH
6a-o
Compd
Ar0
hM1 IC50
4.5
(l
M)a
Cl
Br
Cl
N
ML012
0.69
2
6e
6j
0.32
N
N
N
Me
F
N
6a
6b
6f
>10
>10
6k
6l
>10
N
F
N
N
Cl
Me
N
>10
6g
>10
>10
N
N
N
Cl
6c
>10
>10
6h
6i
>10
>10
6m
S
N
N
N
Me
N
S
N
O
6d
6n
6o
>10
>10
N
a
Average of at least three independent determinations. Standard deviations ranging from 9–19% of the value reported.

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B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1044–1048
Table 2
Structures and activities of M1 antagonist analogs 13a–r
O
N
N
Ar'
O
S
NH
13a-r
Ar
O
Ar' =
Br
Ar' =
N
N
a
a
Compd
Ar0
hM1 IC50
(lM)
Compd
Ar0
hM1 IC50
2.6
(lM)
N
N
13a
1.1
>10
13b
13d
13f
O
O
O
O
O
O
S
N
N
Me
Cl
Me
Me
N
N
N
N
13c
13e
>10
>10
N
N
N
N
>10
N
N
N
N
13g
4.8
13h
>10
S
F
N
13i
>10
2.1
13j
13l
>10
0.4
S
13k
N
N
N
13m
>10
13n
>10
Me2N
N
13o
13q
>10
>10
13p
13r
>10
>10
Me
O
N
N
Me
S
N
N
N
AcHN
a
Average of at least three independent determinations. Standard deviations ranging from 5–23% of the value reported.
indications as Parkinson’s disease, movement disorders, and Fragile
X syndrome,17,18 we have continued the optimization of ML012 and
now detail those efforts in this Letter.
on the 4-pyridyl ring only resulted in decreased antagonist activity
(6a–c), as measured by their ability to inhibit the calcium mobili-
zation induced by ACh. Very few analogs around a 3-pyridyl moi-
ety (structures and data not shown) were prepared, but these
compounds only served to confirm exploratory SAR demonstrating
that a 3-pyridyl was the least tolerated.11 Alternatively, substitu-
tions around a 2-pyridyl scaffold were more fruitful. As a starting
point, the unsubstituted 2-pyridyl analog 6d possessed inhibitory
activity roughly 14-fold less than ML012, but a modest improve-
ment resulted from chlorination para to the nitrogen, as in com-
pound 6e. Halogenation at the ortho-position caused a decrease
in activity (6f and 6g), as did methylation at either the para- or
the ortho-position (6h and 6i). The largest improvement in activity
resulted from the substitution of a bromine at the meta-position
shown in compound 6j. This M1 antagonist displayed activity
With ML012 as our starting point, we utilized an iterative par-
allel synthesis approach to explore structure–activity-relationships
(SAR) at both ends of the molecule by employing the synthesis
routes depicted in Scheme 1, while simultaneously making more
speculative modifications within single compounds. To explore
SAR around the Eastern portion, we started with b-alanine methyl
ester 1. Sulfonation to provide 2, was then followed by saponifica-
tion and amide coupling to provide amides (3) appearing in Table 1
containing numerous variations in aryl groups (Ar’). Alternatively,
starting from the N-Boc protected b-alanine 4, peptide coupling
with a limited number of optimized Eastern portions provided
compound 5, which was then treated with HCl subsequent to sul-
fonation with a variety of sulfonyl chlorides to provide the sulfon-
amides (3) appearing in Table 2, which now display a wide range of
Ar groups on the Western side.
(IC50 = 0.32 lM) 2-fold greater than ML012 and provided an alter-
native Eastern aryl group for additional rounds of SAR. Replacing
the terminal pyridine with a variety of heterocycles met with sim-
ilarly unforgiving steep SAR (6k–o).
While not conducted in an exhaustive fashion, various replace-
ments for the piperazine heterocycle were explored. However,
Table 1 shows the SAR developed around a variety of aryl
groups attached to the piperazine. With ML012 as our benchmark
(hM1 IC50 = 0.69 l
M),19 it can be seen that additional substitutions

B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1044–1048
OH
1047
concentration tested (30
l
M), while the (S)-enantiomer (11) dem-
N
N
N
N
N
N
onstrated about 30% inhibition of the acetylcholine response at the
same concentration. The free N–H of the sulfonamide was important
for M1 antagonist activity since N-methylation of ML012 to provide
compound 12 resulted in a greater than 14-fold loss in potency (hM1
Figure 2. Piperazine replacements that did not maintain potency (hM1
IC50 >10
l
M).
IC50 >10 lM).
Now focusing on the Western sulfonamide region, and employ-
ing our two best Eastern aryl groups, SAR was similarly restrictive
(Table 2—Ar0 = 4-pyridyl (analogs on the right) and 5-bromo-2-pyr-
idyl (analogs on the left)). The slight change associated with replac-
ing the benzothiadiazole with a benzoxadiazole in compounds 13a
and 13b resulted in a 3-fold and 4-fold decrease in activity relative
to their sulfur analogs 6j and ML012, respectively. Substitution on
either of these bicyclic heterocycles at the 5- or 7-positions resulted
in weakly active or inactive compounds (13c–g). Moving the sulfon-
amide from the 4-position to the 5-position on the benzothiadiazole
was also not tolerated, as can be seen with compound 13h. Although
it was not surprising that removing all heteroatoms from the sulfon-
amide aryl ring would result in a complete loss of activity (13i), it
was somewhat surprising that the benzothiazole sulfonamide 13j
was also inactive. This was particularly unexpected considering
the discovery that a single nitrogen atom reintroduced into the
naphthalene sulfonamide, to arrive at the quinoline sulfonamides
13k and 13l, could provide compounds with activity on par with,
or better than, their benzothiadiazole and benzoxadiazole analogs,
and highlights the importance of this nitrogen’s positioning. This
nitrogen effect was very specific for mimicking the interactions pre-
sumably occurring with the N-1 nitrogen of the benzothiadiazole.
Hence, the introduction of a nitrogen atom on the other side of the
naphthalene, to mimic the N-3 nitrogen of the benzothiadiazole,
resulted in a very weakly active compound (quinoline 13m). Further
attempts to present heteroatoms in this approximate location gen-
erally met with no success (13n–r).
H
O
O
O
H
N
N
N
O
S
O
O
S
O
O
S
O
Me
NH
NH
NH
F
7
8
9
O
O
Me
Me
N
N
N
O
S
O
O
S
O
O
S
O
N
NH
NH
Me
12
11
10
Figure 3. A collection of central linker modifications.
none of the more commonly employed surrogates shown in Fig. 2
provided antagonists with appreciable activity.
The b-alanine central linker was also explored with respect to the
various modifications shown in Fig. 3. The reduced amide analog of
ML012, compound 7 (Fig. 3), while still an antagonist, showed a
greater than 14-fold loss in potency (hM1 IC50 >10
17% ACh activity remaining at the highest concentration tested
(30 M)). Methylation at the -position of compound 13b (See
Table 2) provided a pair of inactive enantiomers (8). However, fluo-
rination at this location did not remove all activity, but instead
provided a racemic mixture with somewhat reduced inhibitory
lM, but only
l
a
efficacy (9, 25% activity remaining at 30
l
M) relative to the non-
Ultimately, 6j and 13l represented the only compounds with
improved antagonist activity relative to ML012. The quinoline
sulfonamide 13l was deemed more attractive due to more pro-
nounced improvements in its calculated and physical properties
when compared to either ML012 or 6j. Although not a major differ-
ence, 13l possesses the lowest molecular weight among the three
compounds (MW ML012 = 432, 6j = 511 and 13l = 425). Both 6j
and 13l displayed more moderate cLogP values (1.88 and 1.04,
respectively) relative to ML012 (cLogP = 0.37). The quinoline
sulfonamide compound 13l no longer contained the electron defi-
cient benzothiadiazole found in both ML012 and 6j, which subse-
quently reduced its polar surface area to 96 Å2 and favorably
compared to the other two compounds which both had a polar sur-
face area of 108 Å2. With these properties in mind, compound 13l
fluorinated analog, 13b (hM1 IC50 = 4.5 M). It should not be over-
l
looked that compound 9 was a mixture of two enantiomers while
compound 13b was a single achiral molecule. Given the roughly
2-fold decrease in potency for 9, there remains the possibility that
one enantiomer of 9 had about the same potency as its non-fluori-
nated progenitor 13b, while the other enantiomer of 9 could be
inactive. Regardless, substitution by either of these groups was not
obviously beneficial with respect to potency. The individual
b-substituted enantiomers, 10 and 11, were prepared from commer-
cially available starting materials, and while both enantiomers were
less potent than their unsubstituted analog 13b, there was some
evidence for enantiospecific activity between the two compounds.
The (R)-enantiomer (10) was completely inactive at the highest
Figure 4. Compound 13l (VU0415248) and ML012 (VU0255035) selectively antagonize the M1 mAChR relative to the M2-5 mAChRs. CRCs were performed in the presence of
an EC80 concentration of ACh for each receptor in a calcium mobilization assay. Data were normalized to the maximum response to 30 M ACh and are presented as a
percentage of the EC80 ACh response. A, selectivity for 13l across the rat mAChRs (rM1 EC50 = 0.18 M). B, selectivity for ML012 across the rat mAChRs (rM1 EC50 = 0.24 M).
l
l
l

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B. J. Melancon et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1044–1048
was profiled against both the rat and human M1–5 receptors in a
functional assay for calcium mobilization. Figure 4A shows these
results for the rat (r) M1–5 receptors. Despite being presumably
an orthosteric ligand, based on its close structural relationship to
ML012, 13l shows very high selectivity for the M1 receptor over
the M2–5 subtypes with a similar level of selectivity observed
Program in the development of subtype selective mAChR antago-
nists.
References and notes
1. Bonner, T. I.; Buckley, N. J.; Young, A. C.; Brann, M. R. Science 1987, 237, 527.
2. Bonner, T. I.; Young, A. C.; Brann, M. R.; Buckley, N. J. Neuron 1988, 1, 403.
3. Wess, J. Annu. Rev. Pharmacol. Toxicol. 2004, 44, 423.
across the human subtypes (hM2–5 IC50s >30 lM, data not shown).
For comparison, ML012 was profiled against the complete set of rat
M1-5 receptors and these results appear in Figure 4B (data not
available at the time of our initial publication on ML012). Gratify-
ingly 13l represented an improvement over ML012 with respect to
muscarinic subtype selectivity at the rat receptor, and to a lesser
4. Langmead, C. J.; Watson, J.; Reavill, C. Pharmacol. Ther. 2008, 117, 232.
5. Wess, J. Crit. Rev. Neurobiol. 1996, 10, 69.
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Lindsley, C. W. Drug News Perspect. 2010, 23, 229.
8. Heinrich, J. N.; Butera, J. A.; Carrick, T.; Kramer, A.; Kowal, D.; Lock, T.; Marquis,
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Calligaro, D. O.; Shipley, L. A.; Buelke-Sam, J. L.; Bodick, N. C.; Farde, L.;
Sheardown, M. J.; Olesen, P. H.; Hansen, K. T.; Suzdak, P. D.; Swedberg, M. D. B.;
Sauerberg, P.; Mitch, C. H. Drug Dev. Res. 1997, 40, 158.
10. Shekhar, A.; Potter, W. Z.; Lightfoot, J.; Lienemann, J.; Dube, S.; Mallinckrodt, C.;
Bymaster, F. P.; McKinzie, D. L.; Felder, C. C. Am. J. Psychiatry 2008, 165, 1033.
11. Sheffler, D. J.; Williams, R.; Bridges, T. M.; Lewis, L. M.; Xiang, Z.; Kane, A. S.;
Byun, N. E.; Jadhav, S.; Mock, M. M.; Zheng, F.; Lewis, L. M.; Jones, C. K.;
Niswender, C. M.; Weaver, C. D.; Lindsley, C. W.; Conn, P. J. Mol. Pharmacol.
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12. Bridges, T. M.; Kennedy, J. P.; Noetzel, M. J.; Breininger, M. L.; Gentry, P. R.;
Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2010, 20, 1972.
13. Reid, P. R.; Bridges, T. M.; Sheffler, D. J.; Cho, H. P.; Lewis, L. M.; Days, E.;
Daniels, J. S.; Jones, C. K.; Niswender, C. M.; Weaver, C. D.; Conn, P. J.; Lindsley,
C. W.; Wood, M. R. Bioorg. Med. Chem. Lett. 2011, 21, 2697.
extent
IC50 = 0.18
a
small improvement with respect to potency (rM1
M for 13l and rM1 IC50 = 0.24 M for ML012). Addi-
l
l
tionally, the quinoline nitrogen at its specific location indicated
the presence of an important interaction between compound 13l
and the M1 receptor which does not appear to be present in the
M2–5 receptor subtypes. We next evaluated 13l for its potential
as an improved in vivo tool, but were disappointed in the com-
pound’s poor pharmacokinetic properties. Consistent with
in vitro DMPK predictions of high clearance, 13l displayed an IV
clearance value of 77 mL/min/kg, roughly hepatic blood flow, and
a shorter half life (t1/2 = 40 min) relative to ML012 in male Spra-
gue–Dawley rats.11In tandem, 13l was found to display very low
estimated bioavailability and undetectable CNS exposure in the
rat (% F <10 and undetectable CNS concentrations when dosed or-
ally at 10 mg/kg).
14. Kuduk, S. D.; Chang, R. K.; Di Marco, C. N.; Ray, W. J.; Ma, L.; Wittmann, M.;
Seager, M. A.; Koeplinger, K. A.; Thompson, C. D.; Hartman, G. D.; Bilodeau, M.
T. ACS Med. Chem. Lett. 2010, 1, 263.
15. Brady, A. E.; Jones, C. K.; Bridges, T. M.; Kennedy, J. P.; Thompson, A. D.;
Heiman, J. U.; Breininger, M. L.; Gentry, P. R.; Yin, H.; Jadhav, S. B.; Shirey, J. K.;
Conn, P. J.; Lindsley, C. W. J. Pharmacol. Exp. Ther. 2008, 327, 941.
16. Bridges, T. M.; Kennedy, J. P.; Hopkins, C. R.; Conn, P. J.; Lindsley, C. W. Bioorg.
Med. Chem. Lett. 2010, 20, 5617.
17. Veeraragavan, S.; Bui, N.; Perkins, J. R.; Yuva-Paylor, L. A.; Carpenter, R. L.;
Paylor, R. Psychopharmacology 2011, Article ASAP. doi: 10.1007/s00213-011-
2276-6.
In summary, we have expanded the SAR surrounding ML012,
resulting in the development of 13l (VU0415248) as a more potent
in vitro tool with improved selectivity for the M1 receptor.
On-going work which builds on the SAR described herein may pro-
vide compounds with both improved selectivity and PK parame-
ters. This work will be reported in due course. ML012 is an
MLPCN probe and freely available upon request.20
18. Healy, A.; Rush, R.; Ocain, T. ACS Chem. Neuro., 2011, Article ASAP. doi: 10.1021/
cn200019z.
19. Although the IC50 for ML012 has been previously reported as 132nM (Ref. 11),
the inherent variability associated with functional assays supports that the
most recent IC50 = 690nM for ML012 is equally valid and can serve as a
baseline comparator between the two papers.
20. For information on the MLPCN and information on how to request probe
compounds, such as ML012, see: http://mli.nih.gov/mli/mlpcn/.
Acknowledgments
The authors thank Seaside Therapeutics, NIMH (RO1MH082867),
NIH (U54MH084659) and NINDS (P50NS071669) for support of our