Synthesis and SAR of N-(4-(4-alklylpiperazin-1-yl)phenyl)benzamides as muscarinic acetylcholine receptor subtype 1 (M1) anatgonists

Article abstract of DOI:10.1016/j.bmcl.2010.02.041

This Letter describes the synthesis and SAR, developed through an iterative analog library approach, of a novel series of selective M₁ mAChR antagonists, based on an N-(4-(4-alkylpiperazin-1-yl)phenyl)benzamide scaffold for the potential treatm

Full text of DOI:10.1016/j.bmcl.2010.02.041

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2174–2177
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of N-(4-(4-alklylpiperazin-1-yl)phenyl)benzamides
as muscarinic acetylcholine receptor subtype 1 (M1) anatgonists
Nicole R. Miller a, , R. Nathan Daniels c,e, , David Lee e, P. Jeffrey Conn a,b,c,d, Craig W. Lindsley a,b,c,d,e,
*
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Vanderbilt Program in Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Vanderbilt MLPCN Specilaized Chemistry Center, Vanderbilt University Medical Center, Nashville, TN 37232, USA
d Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
e Department of Chemistry, Vanderbilt University, 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 synthesis and SAR, developed through an iterative analog library approach, of a
novel series of selective M1 mAChR antagonists, based on an N-(4-(4-alkylpiperazin-1-yl)phenyl)benzam-
ide scaffold for the potential treatment of Parkinson’s disease, dystonia and other movement disorders.
Received 13 January 2010
Revised 8 February 2010
Accepted 8 February 2010
Available online 13 February 2010
Compounds in this series possess M1 antagonist IC50s in the 350 nM to >10
degrees of functional selectivity versus M2–M5.
lM range with varying
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Muscarinic
Antagonist
Dystonia
Parkinson’s disease
There are five subtypes of muscarinic acetylcholine receptors
(mAChR1-5 or M1–M5), members of the G protein-coupled receptor
(GPCR) family A, that mediate the metabotropic actions of the neu-
rotransmitter acetylcholine.1,2 M1, M3, and M5 activate phospholi-
pase C and calcium mobilization through Gq whereas M2 and M4
block the action of adenylyl cyclase through Gi/o.
1,2 The cholinergic
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 function
tomentiononlyafew.1–4 As aresult, agentsthatcanselectivelymod-
ulate the activity of mAChRs have the potential for therapeutic use in
multiple peripheral and central pathological states. Due to high se-
quence conservation within the orthosteric binding site of the five
mAChR subtypes, it has been historically difficult to develop mAChR
subtype-selective ligands.1–5 Based on brain expression and cellular
localization, data from mAChR knock-out mice and clinical trials
with muscarinic agents, the M1 subtype is an attractive molecular
target for the treatment of CNS disorders. M1 has been implicated
in the pathologies of Alzheimer’s disease (AD), Parkinson’s disease
(PD), and dystonia due to its role in cognition and motor control.6
* Corresponding author. Tel.: +1 615 322 8700; fax: +1 615 343 6532.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
These authors contributed equally.
Figure 1. Structures of representative mAChR antagonists.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.041

N. R. Miller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2174–2177
2175
The majority of reported muscarinic antagonists are unselec-
tive, such as a scopolamine, 1.7 Recently, pirenzapine, 2 has
emerged as a relatively selective M1 receptor antagonist (20–50-
fold vs M2–M5) and there are numerous reports of moderately
selective M3 antagonists (20–50-fold vs M2) such as 3.8 Interest-
ingly, the most selective M1 antagonist, MT7, 4, the 65 amino acid
peptide, (>1000-fold vs M2–M5) was derived from venom extracts
of the green mamba snake (Fig. 1).9 From an M1 functional screen
within the MLSCN, we identified M1 antagonists such as 5 (M1 IC50
of 441 nM and with >340-fold selectivity versus M4, but modest
selectivity versus M2, M3, and M5 (7.9-fold, 7-fold, and 2.4-fold,
respectively)) and 6 (M1 IC50 of 5.0 lM and with >30-fold selective
Scheme 1. Library synthesis of first generation analogs 8. All library compounds
vs M2–M5).10–12 Based on the M1 selectivity of 6, attractive physio-
chemical properties (MW <350, clog P 3.6) and the fact that it was
the only benzamide-containing analog in the series, we initiated a
library synthesis effort13 to develop SAR around 6.
were purified by mass-guided HPLC to >98% purity.14
guided HPLC to analytical purity.14 To effectively screen small
libraries of potential mAChR ligands, we have adopted a strategy
to triage compounds in single-point screens (at 10 lM) at M1, M3
As shown in Scheme 1, the first round of library synthesis
focused on benzamide analogs of 6. Commercially available 3-
chloro-(4-(4-ethylpierazin-1yl)aniline
7
was acylated under
and M5—the Gq-coupled mAChRs—to identify active and selective
standard conditions employing polymer-supported reagents and
scavengers13 to afford a 24-member library of analogs 8, along
with re-synthesized 6. All analogs were then purified by mass-
compounds prior to running full concentration–response curves
(CRCs).15 Figure 2 shows the 10
l
M single-point screens for the
first 25-member library of benzamide analogs 8.
Figure 2. Single-point EC80 plus 10 lM compound triage screen at M1, M3, and M5 to select compounds for full CRCs.

2176
N. R. Miller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2174–2177
Table 1
Structures and mAChR activities of analogs 8
Compd
Ar
M1 IC50
3.2
(l
M)a
M2 IC50
>10
(l
M)a
M3 IC50
>10
(l
M)a
M4 IC50
>10
(l
M)a
M5 IC50
>10
(l
M)a
6
8a
8b
0.96
0.82
ND
ND
0.82
5.6
ND
ND
2.3
1.3
8c
2.9
2.1
6.9
ND
>10
>10
3.7
ND
>10
3.5
8d
8e
0.35
ND
3.7
ND
0.83
8f
3.2
ND
>10
2.7
>10
4.3
4.2
ND
3.7
1.5
>10
4.1
4.1
8g
8h
2.9
0.49
8i
8j
2.6
4.7
>10
>10
>10
>10
3.7
>10
>10
>10
ND = not determined.
a
IC50s are an average of three independent experiments using mAChR (CHO) cell lines.
As Shown in Table 1, re-synthesized 6 displayed comparable
potency and mAChR selectivity to the original sample (M1
IC50 = 3.2 lM, IC50 >>10 lM for M2–M5). Functionalized benzam-
Having surveyed the amide moiety while maintaining the N-
ethyl piperazine, we next generated two-dimensional libraries
wherein the nature of the alkyl group was varied (9–12) while also
surveying diverse benzamides to generate analogs 9a–f, 10a–f,
11a–f, and 12a–f (Scheme 2).
Application of the same strategy to triage compounds in single-
point screens (at 10 lM) at M1, M3, and M5 to identify active and
selective compounds prior to running full (CRCs) was employed,
but >75% of these new analogs possessed no M1 antagonist activity.
The SAR for this series was incredibly shallow, with only an N-pro-
pyl congener with the 3,5-dicholrobenzamide moiety 11i display-
ide analogs 8 possessed a wide range of M1 potency and mAChR
selectivity, and we initially evaluated analogs 8 against M1, M3,
and M5. Substitution in the 2-position, 8a (2-Cl) and 8b (2-
OMe) possessed submicromolar M1 IC50s (960 nM and 820 nM,
respectively), but also showed low micromolar activity at M3
and M5. A pentafluorophenyl congener 8e (Fig. 3A) proved to
be a submicromolar antagonist of both M1 and M5 (IC50s of
350 nM and 830 nM, respectively). Substitution at the 4-position,
as with the 4-OMe derivative 8f, was comparable to the original
ing reasonable activity (M1 IC50 = 3.7
l
M, IC50 > 10
lM for M3 and
6. Interestingly,
a
2,5-bisCF3 analog 8h had an M1 IC50 of
M5), and all other analogs possessing M1 IC50s in the 6–9 l
M range.
490 nM, with ꢀninefold functional selectivity versus M3 and
M5 (Fig. 3B). Intrigued by this potent and selective M1 antago-
nist, we screened against M2 and M4 as well, but found that
8h possessed only 3–4-fold selectivity versus the Gi/o-coupled
mAChRs (Table 1). 8i, a 3,5-bisCF3 analog possessed a unique
In summary, a two-dimensional parallel synthesis library cam-
paign was performed around 6, an M1 antagonist identified in a
functional HTS screen. SAR for this series was shallow, but we were
able to improve the M1 antagonist activity of 6 into the 350–
500 nM range with analogs 8, while maintaining good mAChR
selectivity. Interestingly, 8i is the first reported dual M1/M4-prefer-
ring antagonist, which compliments the prototypical M1/M4-pre-
ferring agonist xanomeline. Other chemical series from our M1
functional screen are currently under chemical optimization, and
further refinements will be reported in due course.
profile as
a
dual M1/M4 antagonist (IC50
M, respectively), with little effect on an ACh EC80 at
M on M2, M3 or M5. Finally, a 3,4-difluoro 8j derivative
s of 2.6 lM and
3.7
10
l
l
was also comparable to the original 6. While this library afforded
interesting results, further optimization was required.

N. R. Miller et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2174–2177
2177
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. The
authors also wish to thank the second generation of the MLSCN,
coined the MLPCN, and the Vanderbilt Specialized Chemistry
Center (U54MH084659-01).
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.; Buckley, N. J.; Brann, M. R. Neuron 1988, 1, 403.
2. Felder, C. C.; Bymaster, F. P.; Ward, J.; DeLapp, N. J. Med. Chem. 2000, 43, 4333.
3. Bymaster, F. P.; McKinzie, D. L.; Felder, C. C.; Wess, J. Neurochem. Res. 2003, 28, 437.
4. Birdsall, N. J. M.; Farries, T.; Gharagozloo, P.; Kobayashi, S.; Lazareno, S.;
Sugimoto, M. Mol. Pharmacol. 1999, 55, 778.
5. Eglen, R. M.; Choppin, A.; Dillon, M. P.; Hedge, S. Curr. Opin. Chem. Biol. 1999, 3, 426.
6. Birdsall, N. J. M.; Nathanson, N. M.; Schwarz, R. D. TRENDS Pharm. Sci. 2001, 22,
215.
7. (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.
8. Hammer, R.; Berrie, C. P.; Birdsall, N. J. M.; Burgen, A. S. V.; Hulme, E. C. Nature
1980, 283, 90.
9. Bradley, K. N. Pharmacol. Ther. 2000, 85, 87.
10. For information on the Molecular Library Production Center Network see:
http://mli.nih.gov/mli/mlpcn/
Figure 3. CRCs for M1, M3, and M5 for (A) compound 8e (M1 IC50 = 350 nM) and (B)
compound 8h (M1 IC50 = 490 nM), showing ꢀninefold functional selectivity versus
M3 and M5.
11. Lewis, L. M.; Sheffler, D.; Williams, R.; Bridges, T. A.; Kennedy, J. P.; Brogan, J. T.;
Mulder, M. J.; Williams, L.; Nalywajko, N. T.; Niswender, C.; Weaver, C. D.;
Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2008, 18, 885.
12. Sheffler, D. J.; Williams, R.; Bridges, T. M.; Lewis, L. M.; Xiang, Z.; Zheng, F.;
Kane, A. S.; Byum, N. E.; Jadhav, S.; Mock, M. M.; Zheng, F.; Lewis, L. M.; Jones, C.
K.; Niswender, C. M.; Weaver, C. D.; Conn, P. J.; Lindsley, C. W.; Conn, P. J. Mol.
Pharmacol. 2009, 76, 356.
13. Kennedy, J. P.; Williams, L.; Bridges, T. M.; Daniels, R. N.; Weaver, D.; Lindsley,
C. W. J. Comb. Chem. 2008, 10, 345.
14. Leister, W. H.; Strauss, K. A.; Wisnoski, D. D.; Zhao, Z.; Lindsley, C. W. J. Comb.
Chem. 2003, 5, 322.
15. Details of the calcium mobilization assays: Chinese Hamster Ovary (CHO-K1)
cells stably expressing human (h) M1, hM3, and hM5 were used for calcium
mobilization assays. hM2 and hM4 were adapted to this assay and signaling
pathway after stably transfecting Gqi5 chimeric G protein. To measure agonist-
induced calcium mobilization and determine effect of novel compounds, stable
muscarinic cell lines plated overnight in Costar 96-well cell culture plates
(Corning) were incubated with 50 lL of 2 lM Fluo-4 AM diluted in assay buffer
[HBSS (Invitrogen) supplemented with 20 mM HEPES and 2.5 mM probenecid,
pH 7.4] for 45 min at 37 °C. Dye was then removed and replaced with assay
buffer. Cells were pre-incubated with 10
curve of novel compound, followed by
l
a
M or a concentration–response
sub-maximal concentration of
acetylcholine or carbachol. The signal amplitude was first normalized to
baseline and then expressed as a percentage of the maximal response to
acetylcholine.
Scheme 2. Library synthesis of second generation analogs 9a–f, 10a–f, 11a–f and
12a–f. All library compounds were purified by mass-guided HPLC to >98% purity.14