Synthesis and SAR of selective muscarinic acetylcholine receptor subtype 1 (M1 mAChR) antagonists

Article abstract of DOI:10.1016/j.bmcl.2007.12.051

This Letter describes the synthesis and SAR, developed through an iterative analogue library approach, of a novel series of selective M1 mAChR antagonists for the potential treatment of Parkinson's disease, dystonia and other movement disorders. Compounds

Full text of DOI:10.1016/j.bmcl.2007.12.051

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 885–890
Synthesis and SAR of selective muscarinic acetylcholine
receptor subtype 1 (M1 mAChR) antagonists
L. Michelle Lewis,c, Douglas Sheffler,a,c, Richard Williams,a,c Thomas M. Bridges,a
J. Phillip Kennedy,b J. T. Brogan,b Matthew J. Mulder,a,c Lyndsey Williams,a,c
Natalia T. Nalywajko,c Colleen M. Niswender,a,c Charles D. Weaver,a,c
P. Jeffrey Conna,c and Craig W. Lindsleya,b,c,
*
aDepartment of Pharmacology, Vanderbilt University Medical Center, 802 Robinson Research Building,
Nashville, TN 37232-6600, USA
bDepartment of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
cVanderbilt Program in Drug Discovery, Vanderbilt Institute of Chemical Biology, Nashville, TN 37232, USA
Received 29 November 2007; revised 18 December 2007; accepted 19 December 2007
Available online 4 January 2008
Abstract—This Letter describes the synthesis and SAR, developed through an iterative analogue library approach, of a novel series
of selective M1 mAChR antagonists for the potential treatment of Parkinson’s disease, dystonia and other movement disorders.
Compounds in this series possess M1 antagonist IC50s in the 441 nM–19 lM range with 8- to >340-fold functional selectivity versus
rM2–rM5.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The muscarinic acetylcholine receptors (mACHRs) are
members of the G protein-coupled receptor (GPCR)
family A that mediate the metabotropic actions of the
neurotransmitter acetylcholine.1,2 To date, five distinct
subtypes of mAChRs (M1–M5) have been cloned and
sequenced. M1, M3, and M5 activate phospholipase C
and calcium through Gq whereas M2 and M4 block
the action of adenylyl cyclase through Gi/o.1,2 The cho-
linergic system, mediated by mAChRs, plays a critical
role in a wide variety of CNS and peripheral functions
including memory and attention mechanisms, motor
control, nociception, regulation of sleep wake cycles,
cardiovascular function, renal and gastrointestinal func-
tion, and many others.1–4 As a result, agents that can
selectively modulate the activity of mAChRs have the
potential for therapeutic use in multiple pathological
states. However, due to high sequence conservation
within the orthosteric binding site of the five mAChR
subtypes, it has been historically difficult to develop
mAChR subtype selective ligands.1–5
To date, the majority of reported muscarinic antagonists
are unselective, such as a scopolamine, 1.6 Recently, pir-
enzapine, 2, has emerged as a relatively selective M1
receptor antagonist (20- to 50-fold versus M2–M5)
and there are numerous reports of moderately selective
M3 antagonists (20- to 50-fold versus M2) such as 3.7
Interestingly, the most selective M1 antagonist, MT7,
4, the 65 amino acid peptide (>1000-fold versus M2–
M5) was derived from venom extracts of the green
mamba snake (Fig. 1).8 Based on brain expression and
cellular localization, data from mAChR knock-out
mice, and clinical trials with muscarinic agents, the M1
mAChR subtype is an attractive molecular target for
the treatment of Alzheimer’s disease (AD), Parkinson’s
disease (PD), and dystonia due to its role in cognition
and motor control.9 Indeed, pan-muscarinic agonists,
such as the M1/M4 preferring xanomeline, showed effi-
cacy in Phase III clinical trials in AD patients; however,
activation of peripheral M2 and M3 receptors led to
intolerable adverse side effects.10 Moreover, anti-cholin-
ergic agents have also demonstrated efficacy in both PD
and dystonia patients, and this benefit is believed to be
derived from antagonism of the M1 mAChR subtype;
Keywords: Muscarinic acetylcholine receptors; M1; Antagonist; Dys-
tonia; G protein-coupled receptors.
*
Corresponding author. Tel.: +1 615 322 8700; fax: +1 615 343
6532; e-mail: craig.lindsley@vanderbilt.edu
These authors contributed equally to this work.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.051

886
L. Michelle Lewis et al. / Bioorg. Med. Chem. Lett. 18 (2008) 885–890
were then counter-screened against an mGluR4/CHO
O
H
H3C
N
N
cell line which eliminated 9 hits. The remaining com-
pounds were tested in triplicate in 10-point concentra-
tion–response curves against both rat M1/CHO and
rat M4/CHO cells to identify compounds with ꢀ10-fold
selectivity for M1 versus M4, our initial cutoff for a lead.
While the vast majority of compounds displayed no
selectivity for M1 versus M4, we identified two related
structures based on a N-(4-(4-ethylpiperazin-1-yl)phenyl
O
N
N
OH
O
O
N
O
N
CH3
amide scaffold, 5 (rM1 IC50 = 0.49 lM, rM4 IC50
7.9 lM) and 6 (rM1 IC50 = 0.58 lM, rM4 IC50
=
=
1, scopolamine
2, pirenzepine
5.1 lM), which displayed ꢀ16- to ꢀ9-fold selectivity,
respectively, for rM1 versus rM4 and displayed compa-
rable inhibition of human M1 (Fig. 2).
OH
O
H
CH3
N
HN
Analogues of 5 and 6 were synthesized in a library for-
mat according to Scheme 1. Both requisite anilines 7
and 8, 3-chloro-(4-(4-ethylpiperazin-1yl)aniline and (4-
(4-ethylpiperazin-1yl)aniline, were commercially avail-
able and acylated under standard conditions employing
polymer-supported reagents and scavengers to afford
24-member libraries of analogues 9 and 10, respec-
tively.14 In the initial lead optimization phase, we pre-
pare a 24-member library employing a diversity set of
acid chlorides containing aromatic, alphatic, polar, ba-
sic, and acidic moieties in order to rapidly probe the
breadth and scope of the SAR; subsequent libraries will
be more focused. As the chemistry was straightforward,
we elected to re-synthesize the parent compounds 5 and
6 within the library. All analogues were purified by
mass-guided HPLC to analytical purity.15 Surprisingly,
all analogues 10, as well as the re-synthesized parent 6,
were found to be inactive on rM1. Moreover, upon re-
synthesis in the library, 5 lost considerable efficacy as
an M1 antagonist (rM1 IC50 = 13 lM), but still dis-
H
3
LTCVKSNSIWFPTSEDCPDGQNLCFKRWQYISPRMY
DFTRGCAATCPKAEYRDVINCCGTDKCNK
4, MT7
Figure 1. Structures of representative mAChR antagonists.
however, the relative contributions from M4 are
unclear.1–10 In order to probe the role of M1 antagonism
as a potential therapeutic approach for Parkinson’s dis-
ease, dystonia, and other movement disorders, potent
small molecule mAChR antagonists are required with
a high degree of M1 versus M4 selectivity for study in
preclinical models.
played ꢀ10-fold selectivity versus rM4 (IC50
>
150 lM).16 Not surprisingly, analysis of the original
screening samples 5 and 6 indicated that there were sev-
eral impurities in the wells, and we elected not to pursue
a complex deconvolution exercise. Despite these find-
ings, the strategy of employing library synthesis and
exploding SAR around a primary HTS hit proved
advantageous for 5, as analogues 9 proved to possess
intriguing mAChR selectivity profiles.
The Vanderbilt Screening Center for GPCRs, Ion Chan-
nels and Transporters, and the companion Chemistry
Center, were established as members of the Molecular
Libraries Screening Center Network (MLSCN) initiated
and supported by the NIH Molecular Libraries Road-
map.11,12 The MLSCN is a nationwide consortium of
facilities that provide high-throughput small molecule
screening and medicinal chemistry expertise for the
development of chemical probes for use as tools to ex-
plore biological targets/pathways for which small mole-
cule tools are unavailable.12 One such target which lacks
the appropriate small molecule tools are the muscarinic
acetylcholine receptors (mAChRs).1–10
Based on this unmet need in the scientific community,
our MLSCN Center initiated an effort to identify potent
small molecule mAChR antagonists with high specificity
for M1 for use as a chemical probe and lead for further
optimization toward a novel therapeutic. Toward this
goal, we optimized a real-time cell-based calcium-mobi-
lization assay employing a rat M1/CHO cell line (Z0
averaged 0.7), screened a 63,656 member MLSCN com-
pound library, and identified 2179 primary M1 antago-
nist hits.13 Of these primary hits, 1665 were available
from Biofocus-DPI for re-test, and duplicate testing
afforded 723 confirmed hits (43%). These compounds
Figure 2. HTS leads 5 and 6, rM1 antagonists with selectivity versus
rM4 of ꢀ10-fold in the primary assays.

L. Michelle Lewis et al. / Bioorg. Med. Chem. Lett. 18 (2008) 885–890
Table 1. Structures and mAChR activities of analogues 9
887
Cl
HN
O
N
N
R
9
a
a
a
a
a
Compound
R
M1 IC50 (lM)
M2 IC50 (lM)
M3 IC50 (lM)
M4 IC50 (lM)
M5 IC50 (lM)
5
13.2
>150
4.6
>150
>150
>150
>150
>150
>150
24
>150
>150
>150
66
>150
9a
9b
9c
9d
9e
9f
>150
>150
>150
>150
29
>150
>150
>150
>150
13
5.0
F
5.6
>150
20
1.15
1.1
Ph
Ph
52
70
18
7.6
O
O
9g
9h
9i
3.3
>150
>150
3.5
>150
>150
3.1
>150
>150
>150
>150
>150
>150
1.1
Ph
18.8
0.44
>150
Ph
9j
>150
>150
>150
a IC50s are an average of three independent experiments using rat mAChR (CHO) cell lines.
Table 1 highlights SAR and mAChR selectivity for ana-
logues 9 of HTS hit 5. In general, SAR was rather flat
for this series. Truncation of the pentyl side chain of 5
to simpler aliphatic chains, such as n-propyl 9a, led to
a total loss of rM1 antagonist activity. Cyclization to
form a cyclohexyl ring, as in 9b, afforded a selective
rM1 antagonist (rM1 IC50 = 4.6 lM, >32-fold selective
versus rM2–rM5), and a 3-fold increase in potency rela-
tive to HTS lead 5. The phenyl analogue 9c maintained
M1 activity relative to 9b, but mAChR selectivity at
rM4 began to erode. However, conversion to a benzyl
moiety 9d once again maintained rM1 activity (rM1
IC50 = 5.6 lM) and also displayed >26-fold selectivity
for rM2–rM5 (IC50s > 150 lM). Further chain homolo-
gation to the phenethyl congener 9f afforded a low
(47-fold versus rM2, 63-fold versus rM3, 16-fold versus
rM4 and 6.9-fold versus rM5). Introduction of a cyclic
constraint in the form of a cyclopropyl moiety in the
phenethyl chain as in 9e provided a compound with an
in vitro profile roughly equivalent to 9f. Incorporation
of an oxygen atom in the phenylether as in 9g provided
an M1 antagonist of modest potency (rM1
IC50 = 3.3 lM), but with >45-fold selectivity versus
rM2–rM5 (Fig. 3). Replacement of the phenyl moiety
with a cyclopentyl group afforded compound 9i, with
an rM1 IC50 of 441 nM and with >340-fold selectivity
versus M4, but modest selectivity versus rM2, rM3,
and rM5 (7.9-fold, 7-fold, and 2.4-fold, respectively).
Compound 9i possessed the potentcy requirements for
an MLSCN M1 antagonist probe molecule (affinity/
activity >500 nM) as well as the required selectivity
(>10-fold selectivity) versus rM4 (>340-fold selectiv-
micromolar
potency
rM1
antagonist
(rM1
EC50 = 1.1 lM) with high mAChR subtype selectivity

888
L. Michelle Lewis et al. / Bioorg. Med. Chem. Lett. 18 (2008) 885–890
ity).11,12 When evaluated against other receptors and en-
zymes, 9i displayed no significant ancillary pharmacol-
ogy (see Fig. 4).
N
N
N
N
a or b
Our attention now focused on examining mAChR sub-
type selectivity in binding assays to determine if the
functional selectivity was mirrored in competition radi-
oligand binding experiments and to determine whether
9i was binding at the orthosteric versus an allosteric
binding site. For these experiments, we evaluated the
ability of 9i to displace [3H]-N-methylscopolamine
([3H]-NMS), an orthosteric radioligand, versus all five
mAChR subtypes with atropine as a positive control
(Fig. 5).17 In the event, 9i was shown to possess an
rM1 Ki of 12.7 nM with selectivity versus rM2–rM5
(6- to 35-fold) and atropine controls demonstrated
pan-mAChR antagonism as anticipated (Table 2). Grat-
ifyingly, the functional rM1 versus rM4 selectivity was
X
X
NH2
HN
O
R
9, X=Cl
10, X=H
7, X=Cl
8, X =H
Scheme 1. Library synthesis of analogues 9 and 10. Reagents and
conditions: (a) i—PS-DCC, HOBt, RCOOH, ii—MP-CO32ꢁ, 62–98%
or (b) i—RCOCl, PS-DIEA, ii—PS-trisamine, 79–98%. All library
compounds were purified by mass-guided HPLC to >98% purity.15
Figure 3. Concentration–response curves for 9g on rat M1–M5.
Compound 9g displays >45-fold selectivity versus M2–M5. Curves
represent the average of three separate experiments.
Figure 5. [3H]-NMS competition binding experiments for 9i on rat
M1–M5. Compound 9i displays 27-fold selectivity versus M2, 6-fold
selectivity versus M3, 35-fold selectivity versus M4, and 7-fold
selectivity versus M5. Curves represent the average of three separate
experiments.
Table 2. Ki determinations and binding fold selectivity for 9i
Cl
HN
O
N
N
9i
mAChR 9i Kia (nM)
Fold selectivity Atropine Ki (nM)a
(vs M1)
M1
M2
M3
M4
M5
12.7 1.7
338.0 13.5
74.8 4.3
0.88 0.04
27
6
2.69 0.20
0.96 0.03
0.56 0.01
1.80 0.11
445.1 23.8
85.7 15.9
35
7
Figure 4. Concentration–response curves for 9i on rat M1–M5.
Compound 9i displays 7.9-fold selectivity versus M2, 7-fold selectivity
versus M3, >340-fold selectivity versus M4, and 2.4-fold selectivity
versus M5. Curves represent the average of three separate experiments.
a Kis are an average of three independent experiments using rat
mAChR (CHO) cell lines.

L. Michelle Lewis et al. / Bioorg. Med. Chem. Lett. 18 (2008) 885–890
889
they do not rule out a binding mode wherein 9i partially
overlaps with the orthosteric binding site which could
account for the observed competitive binding with
[3H]-NMS and high rM1 versus rM4 subtype selectiv-
ity.17 Nor do these data rule out the possibility that 9i
is in fact binding to a non-overlapping allosteric site
which causes a conformational exclusion of the orthos-
teric ligand binding site. Mutagenesis and off-rate exper-
iments are planned to address these possibilities.
In summary, an MLSCN M1 antagonist chemical probe
development project afforded 9i, a selective rM1 versus
rM4 orthosteric antagonist which meets the criteria for
a small molecule MLSCN chemical probe. Our hit-to-
lead strategy of iterative library synthesis to explode
SAR and to re-synthesize HTS hits within the first gen-
eration libraries proved highly beneficial, as the intial
HTS ‘hits’ lost considerable activity upon re-synthesis
and evaluation. Had we employed a more traditional
approach wherein HTS ‘hits’ were first re-synthesized
and evaluated prior to generating analogues, these series
would not have been pursued further, and 9i would not
have been identified. Clearly, serendipity played a major
role in the success of this lead optimization strategy, but
this is a high risk approach that must be judiciously em-
ployed based on the chemistries involved, the assay
capacity, and the overall cost. Further refinements and
in vitro/in vivo pharmacology will be reported for this
class of M1 antagonists in due course.
Acknowledgments
The authors thank the NIH, NIMH and the MLSCN for
funding of the Vanderbilt Screening Center for GPCRs,
Ion Channels & Transporters (3U54MH074427), the
Chemistry Supplement (3U54MH074427-02S1) and the
XO1 (1XO1MH077606-01) M1 antagonist screening
application which enabled this work.
Figure 6. PI hydrolysis studies and Schild analysis for 9i on rat M1.
These data strongly support an orthosteric mode of binding for 9i.
Data represent the average of three separate experiments.
References and notes
1. (a) Bonner, T. I.; Buckley, N. J.; Young, A. C.; Brann, M.
R. Science 1987, 237, 527; (b) Bonner, T. I.; Young, A. C.;
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Neurochem. Res. 2003, 28, 437.
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ashi, S.; Lazareno, S.; Sugimoto, M. Mol. Pharmacol.
1999, 55, 778.
mirrored in the radioligand competition binding experi-
ment, but the fold selectivity had diminished ꢀ10-fold.
We often observe shifts in potency and selectivity be-
tween binding and functional assays, and we view the
functional activity/selectivity as a more important mea-
sure of mAChR selectivity as a binding event does not
dictate
inhibition.
a
functional response, that is, mAChR
5. Eglen, R. M.; Choppin, A.; Dillon, M. P.; Hedge, S. Curr.
Opin. Chem. Biol. 1999, 3, 426.
Phosphoinostitide (PI) hydrolysis studies and Schild
analysis were performed on 9i to confirm its activity in
an alternate signaling pathway modulated by M1 and
to further elucidate its binding mode. As shown in Fig-
ure 6, 9i causes a dose-dependent rightward shift of the
ACh concentration–response curve in a PI hydrolysis
experiment which translates in a Schild analysis to a
Kd of 10 nM and a slope of 0.98 0.10. These data
strongly support the [3H]-NMS binding data and indi-
cate that 9i is an orthosteric M1 antagonist; however,
6. (a) Burke, R. E. Mov. Disord. 1986, 1, 135; (b) Buckley, N.
J.; Bonner, T. I.; Buckley, C. M.; Brann, M. R. Mol.
Pharmacol. 1989, 35, 469; (c) Walebroeck, M.; Tastenoy,
M.; Camus, J.; Christophe, J. Mol. Pharmacol. 1990, 36,
267; (d) Dei, S.; Bellucci, C.; Buccioni, M.; Ferraroni, M.;
Guandalini, L.; Manetti, L.; Martini, E.; Marucci, G.;
Matucci, R.; Nesi, M.; Romanelli, M. N.; Scapecchi, S.;
Teodori, E. J. Med. Chem. 2007, 50, 1409.
7. Hammer, R.; Berrie, C. P.; Birdsall, N. J. M.; Burgen, A.
S. V.; Hulme, E. C. Nature 1980, 283, 90.

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8. Bradley, K. N. Pharmacol. Ther. 2000, 85, 87.
9. Birdsall, N. J. M.; Nathanson, N. M.; Schwarz, R. D.
Trends Pharmacol. Sci. 2001, 22, 215.
10. Bodick, N. C.; Offen, W. W.; Levey, A. I.; Cutler, N. R.;
Gauthier, S. G.; Satlin, A.; Shannon, H. E.; Tollefson, G.
D.; Rasmussen, K.; Bymaster, F. P.; Hurley, D. J.; Potter,
W. Z.; Paul, S. M. Arch. Neurol. 1997, 54, 465; Bodick, N.
C.; Offen, W. W.; Shannon, H. E.; Satterwhite, J.; Lucas,
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1997, 11, S16.
temperature for 12 h at which time it was determined
complete by LC/MS. The reaction mixture was concen-
trated in situ, redissolved in 1 mL DMSO, and purified on
the Agilent 1200 preparative LCMS. Concentration of
purified fractions afforded the TFA salt of N-(3-chloro-4-
(4-ethylpiperazin-1-yl)phenyl)-3-cyclopentylpropanamide
9i as a white solid (80 mg, 42%). 1H NMR (400 MHz,
DMSO-d6)d10.03 (br s, 1H), 7.84 (d, J = 2.0 Hz, 1H), 7.44
(dd, J = 2.0, 8.4 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 3.57 (m,
2H), 3.35 (m, 2H), 3.21 (m, 2H), 3.13 (m, 2H), 2.99 (m, 2H),
2.29 (t, J = 7.6 Hz, 2H), 1.73 (m, 3H), 1.58 (m, 4H), 1.47
(m, 2H), 1.25 (t, J = 7.2 Hz, 2H), 1.08 (m, 3H). 13C NMR
(100 MHz, DMSO-d6)d 171.4, 142.3, 136.2, 127.5, 121.1,
120.6, 118.5, 50.8, 50.7, 48.1, 39.3, 35.7, 32.0, 31.3, 24.7,
8.9; LC–MS, single peak, 2.79 min, m/e, 364.2 (M+1).
17. Phosphoinositide (PI) hydrolysis. Hamster Ovary (CHO)
cells containing rat M1 (rM1) were plated at 120,000 cells
per well in standard growth media (F12 (HAM), supple-
mented with 10% fetal bovine serum, and 20 mM HEPES)
in 24-well plates 24 h prior to assay. Cell media were
replaced late in the day with standard growth media
containing 1 lCi/mL [3H]inositol (Perkin-Elmer LAS) and
cells were incubated overnight at 37 ꢁC in 5% CO2.
[3H]inositol-containing media were removed and the rM1
cells were treated with either vehicle or fixed concentra-
tions of antagonist (2·, 500 lL) in a modified Krebs’-
bicarbonate buffer (108 mM NaCl, 4.7 mM KCl, 2.5 mM
CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaH-
CO3, and 10 mM Glucose, pH 7.4) equilibrated to 37 ꢁC
and 5% CO2, and supplemented with 30 mM LiCl.
Following vehicle or antagonist addition, agonist was
added (2·, 500 lL) in modified Krebs’-bicarbonate buffer
and cells were incubated for 1 h at 37 ꢁC and 5% CO2. The
accumulation of phosphoinositides was terminated by
aspiration, followed by the addition of 1 mL of 10 mM
formic acid. Cells were incubated in the 10 mM formic
acid for at least 30 min at room temperature to insure
extraction of phosphoinositides. Columns packed with a
1 mL bed of AGꢂ 1-X8 Resin 100–200 mesh anion-
exchange resin (formate form) (Bio-Rad; Hercules, CA)
were washed with 10 mL of water twice. Following the
water washes, the entire 1 mL sample volumes were added
to columns, avoiding the transfer of any cells. Columns
were washed with 2 mL of water, then with 10 mL of
water, followed by 10 mL of 5 mM myo-inositol made up
in water. Total phosphoinositides (PIs) were eluted with
10 mL of 0.1 M formic acid/0.2 M ammonium formate
into vials containing 3a70B liquid scintillation cocktail
(Research Products International; Elk Grove Village, IL)
and radioactivity was measured by liquid scintillation
counting. Following use, columns were regenerated with
10 mL of 0.1 M formic acid/1 M ammonium formate and
washed with 30 mL of water. Data were fit with GraphPad
Prism version 4.0 to a 4 parameter logistic equation to
determine IC50 values and Schild Dose-Ratios.
11. For information on the Molecular Library Screening
http://nihroadmap.nih.gov/
Center Network,
molecularlibraries.
see:
12. For information on the Vanderbilt Molecular Library
Screening Center Network Center please refer to: http://
www.vanderbilt.edu/mlscn/Templates/index.htm.
13. Functional assay. Chinese Hamster Ovary (CHO) cells
containing rat M1 (rM1) receptor were plated at
10,000 cells/well and rM2/Gqi5, rM3, rM4/Gqi5, and
rM5 were all plated in 384-well plates at 25,000 cells/well
in assay media 1 (F12 (Ham), 10% FBS, 2 mM Gluta-
MAX (Invitrogen), and 20 mM HEPES), except rM4/
Gqi5 which used assay media 2 (DMEM, 20 mM HEPES,
10% FBS, 2 mM L-glutamine, 1· non-essential amino
acids (NEAA), and 1 mM Na pyruvate). The plates were
incubated overnight at 37 ꢁC in 5% CO2. Media were
removed and assay buffer (Hanks’ Balanced Salt Solution,
20 mM HEPES, and 2.5 mM probenecid, pH 7.4) con-
taining 4.0 lM Fluo4-AM dye (Invitrogen) was added.
Cells were incubated for 45 min (37 ꢁC, 5% CO2) for dye
loading. Cell plates were loaded into the Hamamatsu
FDSS equipped with 480 nm excitation and 540 nm
emission filters. Test compound in assay buffer + 0.1%
DMSO was added at 5 s and simultaneously the plate was
kinetically imaged. Subsequently, 8 nM acetylcholine
(EC80) in assay buffer was added at 197 s and imaging
continued for a total of 4 min acquisition time. DMSO
(0.1%), compound vehicle, and 80 nM acetylcholine
(ECMax) were added to each plate as controls. Compounds
were tested in concentrations ranging from 150 lM to
2.5 nM in triplicate on three different days. Kinetic data
were transformed and fit with GraphPad Prism version 4.0
to a 4 parameter logistic equation to determine IC50
values.
14. (a) Lindsley, C. W.; Weaver, D.; Conn, P. J.; Marnett, L.
ACS Chem. Biol. 2007, 2, 17; (b) Lindsley, C. W.; Weaver,
D.; Bridges, T. M.; Kennedy, J. P. Wiley Encyclopedia of
Chemical Biology, in press.
15. Leister, W. H.; Strauss, K. A.; Wisnoski, D. D.; Zhao, Z.;
Lindsley, C. W. J. Comb. Chem. 2003, 5, 322.
16. Typical experimental for the synthesis of 9i: To a solution
of 3-chloro-4-(4-ethylpiperazin-1-yl)aniline
7 (100 mg,
0.417 mmol) in 9:1 DMF:DIEA (2 mL) was added 3-
cyclopentyl propanoyl chloride (63.9 lL, 0.417 mmol) all
at once. The reaction mixture was stirred at room