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

Article abstract of DOI:10.1016/j.bmcl.2010.01.109

This Letter describes a chemical lead optimization campaign directed at VU0119498, a pan G_q mAChR M₁, M₃, M₅ positive allosteric modulator (PAM) with the goal of developing a selective M₁ PAM. An iterative library synthesis approach delivered a potent (M₁ EC₅₀ = 830 nM) and highly selective M₁ PAM (>30 μM vs M₂-M₅).

Full text of DOI:10.1016/j.bmcl.2010.01.109

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1972–1975
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
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
Thomas M. Bridges a,d, J. Phillip Kennedy b,d, Meredith J. Noetzel a, Micah L. Breininger a,d
,
Patrick R. Gentry a,c,d, P. Jeffrey Conn a,c,d, Craig W. Lindsley 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 Program in Drug Discovery, Nashville, TN 37232, USA
d Vanderbilt Specialized Chemistry Center(MLPCN), 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 a chemical lead optimization campaign directed at VU0119498, a pan Gq mAChR M1,
M3, M5 positive allosteric modulator (PAM) with the goal of developing a selective M1 PAM. An iterative
Received 21 December 2009
Revised 15 January 2010
Accepted 20 January 2010
Available online 1 February 2010
library synthesis approach delivered a potent (M1 EC50 = 830 nM) and highly selective M1 PAM (>30
vs M2–M5).
lM
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
mAChR
Muscarinic
Acetylcholine
PAM
Recently, we described the identification of VU0119498, a pan Gq
mAChR M1, M3, M5 positive allosteric modulator (PAM), from a func-
tional high throughput screen (Fig. 1).1 In subsequent Letters, we de-
scribed chemical lead optimization efforts based on VU0119498 (1)
that delivered the first highly M5-preferring PAM (VU0238429 (2))
and a highly M5-selective PAM (VU0400265 (3)).2,3
was a key compound (calcium mobilization assay M1 EC50 = 845 nM,
100% ACh Max, 100-fold left-shift of ACh CRC at 100 lM), brain pen-
etration was acceptable, but not optimal, due presumably to the car-
boxylic acid moiety.17,18 Our initial report on the discovery of
VU0119498 also described three other series of weak M1 PAMs,
andidentifiedthatdifferentM1 PAMchemotypesdisplayeddifferent
modes of activity on downstream receptor signaling.1 Thus, all allo-
Incorporation of a 5-OCF3 moiety on the isatin ring was essen-
tial for M5 PAM activity and can be viewed as a ‘molecular switch’
to modulate mAChR subtype selectivity.1–3 As we described previ-
ously, other substituents on the isatin ring led to pan mAChR PAMs
with varying degree of potency and efficacy across M1–M5.2,3
Selective M1 activation is an attractive therapeutic approach for
the treatment of cognitive impairment, Alzheimer’s disease, schizo-
phrenia and a number of other CNS disorders.4–14 Until recently, no
highly selective M1 activators existed, and those that claimed to be
highly M1 selective were either not centrally penetrant or possessed
significant ancillary pharmacology which prohibited their use as
probes to study M1 receptor function.15,16 We have disclosed three
selective M1 activators (Fig. 2): BQCA (4),17,18 a highly selective M1
PAM, TBPB (5) a second generation M1 allosteric agonist19–21 and
VU0357017 (6), a best-in-class M1 allosteric agonist.22 While BQCA
Figure 1. HTS lead VU0119498, a pan Gq mAChR M1, M3, M5 PAM, VU0238441,
VU0238429, a highly M5-preferring PAM and VU0400265, a highly selective M5
PAM. Data represent means from at least three independent determinations with
similar results using mobilization of intracellular calcium in M1–M5 CHO cells (M2
and M4 cells co-transfected with Gqi5).
* Corresponding author. Tel.: +1 615 322 8700; fax: +1 615 343 6532.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.109

T. M. Bridges et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1972–1975
1973
Figure 2. BQCA, a highly selective M1 PAM, TBPB, a second generation M1 allosteric agonist, and VU0357017, a best-in-class M1 allosteric agonist.
steric M1 activation is not equivalent, and additional tool com-
pounds representing diverse chemotypes are required to truly dis-
sect and study M1 function in the CNS. Based on our ability to
develop an M5-selective PAM from a pan Gq M1, M3, M5 PAM,2,3 we
initiated an effort to optimize VU0119498 for M1 PAM activity in
an attempt to add a unique chemotype to our tool kit of selective
M1 activators.
Our initial optimization strategy is outlined in Figure 3, and as
SAR with allosteric ligands is often shallow, we employed an iter-
ative parallel synthesis approach. From our M5 work where we
counter-screened on M1, we quickly learned that most substitu-
tions on the isatin ring led to pan mAChR activation profiles with
various degrees of potency, efficacy, and subtype-selectivity.2,3
Thus, our first libraries employed a naked isatin core and surveyed
diversity on the southern benzyl moiety.
Scheme 1. Reagents and conditions: (a) p-bromobenzylbromide, K2CO3, KI, ACN, rt,
16 h (97%); (b) RB(OH)2, Pd(Pt-Bu3)2, Cs2CO3, THF/H2O, mw, 120 °C, 20 min (15–
90%); (c) K2CO3, KI, ACN, rt, 16 h (50–90%).
Libraries were prepared according to Scheme 1, wherein com-
mercial indoline-2,3-dione 7 was alkylated with p-bromobenzyl-
bromide to deliver key intermediate 8. A 12-member Suzuki
library was then prepared to explore the effect of introduction of
biaryl and heterobiaryl motifs into VU0119498 providing analogs
9 (Fig. 4). In parallel, 7 was alkylated with functionalized phenethyl
bromides 10 to probe the effect of chain homologation in analogs
11. Compound libraries were triaged by a single point (10 lM)
screen for their ability to potentiate an EC20 concentration of ACh
on M1 CHO cells. SAR was extremely shallow, with only one analog
9a demonstrating robust M1 potentiation (Fig. 5). VU0365137 (9a),
possessing an N-methyl pyrazole in the 4-position of the southern
benzyl ring displayed an M1 EC50 of 2.3
versus M3 and M5. Moreover, 9a afforded a ꢀ5-fold leftward shift
of the M1 ACh CRC at 10
M, and a larger ꢀ14-fold shift at
30
lM, and good selectivity
Figure 4. Representative analogs 9 comprising the first generation M1 PAM library.
EC50s >10 lM.
l
l
M, with ꢀ30% intrinsic allosteric agonism. Intriguingly, the
5-OCF3 congener of 9a is an equipotent M5-preferring PAM,2,3
highlighting the aforementioned ‘molecular switch’ to engender
M5 preference. However, it was exciting to see that we could devel-
op an M1-preferring PAM from our initial pan Gq M1, M3, M5 PAM
lead.1
playing potentiation of M1, and two analogs provided M1 EC50
s
below 1 M. Fluorine substitution was well tolerated on both the
l
isatin core (4,7-difluoro or 7-fluoro) and on the benzyl ring (2-fluoro
and 2,6-difluoro). The addition of a single fluorine atom to the 2-po-
sition of the benzyl ring delivered 12a, with an M1 EC50 of 830 nM
(65% ACh Max)—comparable to BQCA (M1 EC50 = 845 nM), but
without the carboxylic acid moiety. This single change afforded a
threefold increase in potency over VU0365137 (9a). A 2,6-difluorob-
enzyl congener 12b provides equivalent M1 potency with a slightly
diminished ACh Max (60%). As fluorine content increased 12c–12e
(fluoro-substitution on both the isatin core and benzyl ring) pro-
vided comparable M1 potency, but lower ACh Max (40–55%).
VU0366369 (12a) was studied further (Fig. 6). Gratifyingly, 12a
was found to be a highly selective M1 PAM, with minimal/no activa-
Since SAR was incredibly shallow, we next incorporated subtle
changes, in the form of fluorine atoms, to the VU0365137 (9a) scaf-
fold, as we had previously shown was productive in optimizing
BQCA, 4.23 Interestingly, there was some, but highly limited SAR
overlap between these two series of M1 PAMs. Following the syn-
thetic route outlined in Scheme 1, analogs with fluorine on both
the isatin scaffold and the benzyl ring were readily prepared and
evaluated for their ability to potentiate an EC20 of ACh at M1. This ef-
fort was more productive (Table 1) with five of the analogs 12 dis-
tion of M2–M5 up to 30 lM (Fig. 5A and B). However, in M1 ACh CRC
fold-shift experiments, 12a as well as the difluoro congener 12b dis-
played only a subtle effect, increasing the potency of ACh by only 3Â
and 2Â, respectively, at 30
lM (Fig. 5C). The smaller fold-shift ap-
pears to correlate with the lower overall ACh Max for this ser-
ies.1,15,16 Lack of correlation between PAM potency and fold-shift is
commonly observed within series of mAChR allosteric modulators
and underscores the importance of determining both parameters
when establishing SAR.15 Nonetheless, VU366369 (12a) represents
Figure 3. Initial optimization strategy for VU0119498, a pan Gq M1, M3, M5 PAM.

1974
T. M. Bridges et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1972–1975
Figure 5. (A) Structure and activity of VU0365137 (9a); (B) CRCs for VU0365137 (9a) in the presence of a submaximal (ꢀEC20) concentration of ACh at M1, M3 and M5; (C)
Fold-shift experiments of the ACh CRC at M5 with both 10 M and 30 M concentrations of 9a, providing an approximately fivefold and 14-fold shift, respectively. Data
l
l
represent means of at least three independent determinations with similar results using mobilization of intracellular calcium in M1, M3, or M5 CHO cells.
Table 1
Structures and activities of analogs 12
a
Compd
VU number
0366369
Compound
M1EC50
0.83
(
lM)
% ACh maxa
12a
65
12b
12c
0366368
0366370
0.86
2.3
60
55
12d
12e
0366367
0366372
1.1
1.2
40
50
a
Average of at least three independent determinations. All compounds M1EC50 >30 lM.

T. M. Bridges et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1972–1975
1975
potent M1 PAM. VU0366369 possesses comparable potency to
BQCA and represents only the second known chemotype to provide
highly selective M1 potentiation. Efforts to develop an M3 PAM
from this chemotype have thus far proven unsuccessful; however,
the ability to dial in or out M1 and M5 PAM activity within a single
scaffold is unprecedented. Further in vitro and in vivo characteriza-
tion of VU0400265 and VU0366369 is in progress with exciting re-
sults, which will be reported in due course.
Acknowledgments
The authors thank NIMH (1RO1 MH082867), NIH, the MLPCN
(1U54 MH084659) and the Alzheimer’s Association (IIRG-07-
57131) for support of our Program in the development of subtype
selective allosteric ligands of mAChRs.
References and notes
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Figure 6. (A) and (B) CRCs for VU0366368 (12b) and VU0366369 (12a) in the
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and M5; (C) Fold-shift experiments of the ACh CRC at M5 with 30 lM of 12a and
12b, providing an approximately 3- and 2-fold-shift, respectively. Data represent
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the second knownchemotype toprovide potent and selectiveM1 po-
sitive allosteric modulation.
Having been able to optimize a pan Gq M1, M3, M5 PAM to deli-
ver a potent and selective M1 PAM (VU0366369, 12a) and a potent
and selective M5 PAM (VU0400265, 3),2,3 we hoped to identify
‘molecular switches’ within this chemotype that would engender
M3 PAM selectivity. We began by evaluating all analogs synthe-
sized to date, that did not potentiate an EC20 of ACh at M1 or M5,
for their ability to potentiate an EC20 of ACh at M3 at a 10 lM con-
centration. Surprisingly, identification of an M3 PAM within this
chemotype remains elusive.
Thus, optimization of a pan Gq mAChR M1, M3, M5 PAM, which
previously led to the discovery of the first selective M5 PAM
(VU0400265), provided VU0366369 (12a), a highly selective and