Chemical lead optimization of a pan Gq mAChR M1, M3, M5 positive allosteric modulator (PAM) lead. Part I: Development of the first highly selective M5 PAM

Article abstract of DOI:10.1016/j.bmcl.2009.11.089

This Letter describes a chemical lead optimization campaign directed at VU0238429, the first M₅-preferring positive allosteric modulator (PAM), discovered through analog work around VU0119498, a pan G_q mAChR M₁, M₃, M₅ PAM. An iterative library synthesis approach delivered the first selective M₅ PAM (no activity at M₁-M₄ @ 30 μM), and an important tool compound to study the role of M₅ in the CNS.

Full text of DOI:10.1016/j.bmcl.2009.11.089

Bioorganic & Medicinal Chemistry Letters 20 (2010) 558–562
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 I: Development of the first highly
selective M5 PAM
Thomas M. Bridges a,d, J. Phillip Kennedy b,d, Hyekyung P. Cho a,c,d, Micah L. Breininger a,d
,
Patrick R. Gentry a,c,d, Corey R. Hopkins 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 VU0238429, the first M5-prefer-
ring positive allosteric modulator (PAM), discovered through analog work around VU0119498, a pan Gq
mAChR M1, M3, M5 PAM. An iterative library synthesis approach delivered the first selective M5 PAM
Received 30 October 2009
Revised 17 November 2009
Accepted 18 November 2009
Available online 22 November 2009
(no activity at M1–M4 @ 30
lM), and an important tool compound to study the role of M5 in the CNS.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
M5
Muscarinic receptor
Allosteric
Positive allosteric modualtor
PAM
The muscarinic acetylcholine receptors (mAChRs) are members
of the family A G-protein-coupled receptors (GPCRs) and include
five subtypes denoted M1–M5. M1, M3 and M5 couple to Gq and
activate phospholipase C, whereas M2 and M4 couple to Gi/o and
associated effector systems. All five of the mAChRs are known to
play critical roles in multiple basic physiological processes.1–3 As
such cholinergic agents that activate or inhibit one or more sub-
types have found success both preclinically and clinically for a
number of peripheral and CNS pathologies.3,4 Within the mAChRs,
a major challenge has been a lack of subtype selective ligands to
study the specific contribution of discrete mAChRs in various
disease states. To address this limitation, we have focused on tar-
geting allosteric sites on mAChRs as a means to develop subtype
selective small molecules, both allosteric agonists and positive
allosteric modulators (PAMs).5–11
Relative to M1–M4, little is known about the precise role(s) of
M5 in the CNS. However, localization data and studies using
M5-knockout (KO) mice suggest that M5 activation is highly impor-
tant to regulation of central dopaminergic pathways and to ACh-
induced cerebrovasodilation. In light of these findings, drugs
targeting M5 may have therapeutic potential for numerous CNS
disorders, including cerebrovascular dementia, stroke, Parkinson’s
disease, Alzheimer’s disease, and Schizophrenia.2,4,12–14 Histori-
cally, lack of selective pharmacological tools available to confirm
the putative role(s) of M5 has seriously hindered progress in this
area.
Starting from a pan M1, M3, M5 PAM, VU0119498, we felt it may
be possible to maintain M5 PAM activity and dial out M1 and M3
PAM activity through a chemical lead optimization campaign. In
a recent Letter,15 we reported on the discovery and characteriza-
From a functional cell-based high-throughput screen (HTS) to
identify M1 positive allosteric modulators (PAMs) we identified
VU0119498, an M1, M3, M5 PAM (Fig. 1). This was a unique hit from
the screen, as it was not selective for M1, but the first example of a
pan-Gq mAChR PAM, devoid of activity at the Gi/o-coupled M2 and
tion of VU0238429, the first M5-preferring PAM. At 30 lM,
VU0238429 displayed a 14-fold leftward shift of the ACh concen-
tration-response-curve (CRC), increased ACh affinity for M5 by
15
ꢀ11-fold and did not displace [3H]-NMS from binding to M5.
While this was a major advance in the field, we hoped to develop
a truly M5 selective PAM to dissect the role of M5 in the CNS. In this
Letter, we describe an iterative parallel synthesis approach16 for
the further optimization of VU0238429, and the discovery of M5
10
M4.
* Corresponding author. Tel.: +1 615 322 8700; fax: +1 615 343 6532.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
PAMs with unprecedented mAChR selectivity (ꢁ30
lM vs M1–M4).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.089

T. M. Bridges et al. / Bioorg. Med. Chem. Lett. 20 (2010) 558–562
559
Figure 2. Optimization strategy for VU0238429 (1), a highly M5-preferring PAM.
Our optimization strategy is outlined in Figure 2, and as SAR
with allosteric ligands is often shallow,5,6,17 we employed an itera-
tive parallel synthesis approach.16 From our earlier work, the
5-OCF3 group was essential for M5-preferring activity, so this moi-
ety was maintained.15 Libraries were prepared according to
Scheme 1, wherein commercial 5-(trifluoromethoxy)indoline-2,3-
dione 4 was alkylated with p-bromobenzylbromide to deliver key
intermediate 5. A 11-member Suzuki library was then prepared
to explore the effect of introduction of biaryl and heterobiaryl
motifs into VU0238429 providing analogs 6. In parallel, 4 was
alkylated with functionalized phenethyl bromides 7 to probe the
effect of chain homologation in analogs 8. Compound libraries
were then triaged by a single point (10 lM) screen for their ability
to potentiate an EC20 of ACh in M5-CHO cells (Fig. 3).15 Based on
this screen, select compounds were assayed in 8-point CRCs based
on their potentiation efficacy.
In general, chain homologation in analogs 8 proved unsuccess-
ful as potency was compromised despite retention of PAM efficacy
Figure 1. HTS hit VU0119498, a pan Gq mAChR M1, M3, M5 PAM, VU0238441, a pan
mAChR PAM and VU0238429, a highly M5-preferring PAM. Data represent means of
at least three independent determinations with similar results using mobilization of
intracellular calcium as a functional readout for receptor activation M1–M5 CHO
cells (M2 and M4 cells co-transfected with Gqi5).
Scheme 1. Reagents and conditions: (a) p-bromobenzylbromide, K2CO3, KI, ACN, rt, 16 h (99%); (b) RB(OH)2, Pd(Pt-Bu3)2, Cs2CO3, THF:H2O, mw, 120 oC, 20 min (10–90%); (c)
K2CO3, KI, ACN, rt, 16 h (50–90%).
Figure 3. ACh EC20 triage screen of libraries of analogs 6 and 8 at 10 lM in M5 CHO cells by intracellular calcium mobilization assay. Data represent means from at least three
independent determinations with similar results.

560
T. M. Bridges et al. / Bioorg. Med. Chem. Lett. 20 (2010) 558–562
Table 1
for all of these analogs (M5 EC50s 2.7– 4.8 lM) with similar ACh
Structures and activities of analogs 6
Max values (70–85%). Shallow SAR was again noted with com-
pounds either being active in this potency range or inactive as
M5 PAMs.
Analogs 6a (VU0365114) and 6b (VU0365117) were selected for
additional follow-up. Figure 4 depicts Gq mAChR (M1, M3 and M5)
CRCs for 6a and 6b. Note 6a possesses improved M5 selectivity ver-
sus VU0238429 (3), with only modest activation of M3 at 30 lM.
Compd
6a
VU number
0365114
R
hM5 EC50
2.7
(lM)
%ACh Maxa
a
Both analogs 6a and 6b elicit significant leftward shifts (>50-fold)
of the ACh CRC, as compared to the 14-fold shift of VU0238429 (3).
As seen with the M1 PAM BQCA18–20 and other ago-potentiators for
class C GPCRs,21–23 Figure 4C indicates moderate intrinsic allosteric
85
85
agonism at 30 lM.
6b
0365117
2.8
Encouraged by the potency and mAChR selectivity of VU03
65114 (6a) and VU0238429 (3), we synthesized (Scheme 2) a hy-
brid analog possessing a biphenyl ethermoiety, VU0400265 (10).
6c
0365118
4.8
85
VU0400265 possessed an M5 EC50 of 1.9
Max. Importantly, VU0400265 was completely selective versus
M1–M4, affording no elevation of an ACh EC20 at M1–M4 at 30
(Fig. 5). Notably, VU0400265 (10) represents the most selective
M5 PAM described to date; however, unlike 6a and 6b, analog 10
lM with a 75% ACh
6d
6e
6f
0365121
0365123
0365116
3.6
3.3
3.9
80
85
70
l
M
only afforded a ꢀ5-fold shift of the ACh CRC at 30
lM.
Efforts next centered on maintaining mAChR selectivity and M5
potency, while attempting to improve fold-shift. Subtle structural
changes have been shown in this series to have dramatic effects
on potency, selectivity and fold-shift. Therefore, we next explored
the effect of moving the 5-OCF3 moiety to the 6-position of the
a
Average of at least three independent determinations. All compounds M1 EC50
M.
>30
l
at higher concentrations. For example, 8a, the direct phenethyl
analog of VU0238429 possessed an M5 EC50 of 4.9 M and 80%
l
ACh maximum response (data not shown). Biaryl and heterobiaryl
analogs 6 proved far more productive, affording a number of M5
PAMs with high selectivity versus M1 (>30
micromolar M5 EC50s (Table 1). All other analogs 6 possessed M5
EC50s > 10 M. In general, both 5- (6b and 6e) and six-membered
lM EC50s) and low
l
heterocycles (6c and 6d) were tolerated as were simple phenyl
(6a) and substituted phenyl (6f). Potency was virtually identical
Scheme 2. Reagents and conditions: (a) K2CO3, KI, ACN, mw, 160 oC, 10 min (68%).
Figure 4. (A) CRCs for VU0365114 (6a) at M1, M3 and M5 CHO cells; (B) CRCs for VU0365117 (6b) at M1, M3 and M5 CHO cells; (C) M5 Fold-shift experiments of the ACh CRC
with 30
M of either 6a or 6b (both >50Â) in M5 CHO cells. Data represent means from at least three independent determinations with similar results.
l

T. M. Bridges et al. / Bioorg. Med. Chem. Lett. 20 (2010) 558–562
561
Figure 5. CRCs for VU0400265 (10) at M1, M2, M3, M4 and M5 CHO cells (EC50 = 1.9
reported to date. Data represent means from at least three independent determinations with similar results (M2 and M4 cells co-transfected with Gqi5).
lM) with a submaximal (ꢀEC20) of ACh. VU0400265 (10) is the most selective M5 PAM
Figure 6. CRCs for VU0400267 (11) and VU0400266 (12) at M1, M2, M3, M4 and M5 CHO Cells with a submaximal (EC20) of ACh. Data represent means from at least three
independent determinations with similar results (M2 and M4 cells co-transfected with Gqi5).
isatin core. Following the route in Scheme 1, we quickly prepared
the 6-OCF3 analogs 11 and 12 of our initial M5-preferring PAM
VU0119498 (3) and the biphenyl congener VU0365114 (6a),
respectively. Interestingly, M5 potency was relatively maintained
6a is highly selective for M5 and displays a >50-fold shift of the
ACh CRC. These selective tool compounds will finally allow
researchers to dissect the role of M5 in the CNS, the one mAChR
that has remained a mystery due to the lack of tool compounds.
Since selective M5 PAMs could be obtained from a pan Gq PAM
lead, we are currently optimizing VU0119498 to provide highly
selective M1 PAMs as well as M3 PAMs. Efforts in this arena are
in progress with exciting results, which will be reported in due
course.
(M5 EC50s of 5.7 lM and 2.7 lM, for 11 (VU0400267) and 12
(VU0400266), respectively); however, both afforded ꢀ95% of ACh
Max, suggesting the fold-shift might be improved with this modi-
fication. Quite unexpectedly, mAChR selectivity was lost (Fig. 6),
once again highlighting the difficulty in developing SAR for alloste-
ric ligands.5,6,17 A 6-OCF3 congener of VU0400265 (10) provided
similar erosion in mAChR selectivity. Due to the loss in M5 selectiv-
ity, ACh fold-shift experiments were not performed.
All of these analogs displayed moderate to poor PK in rats with
limited brain exposure (AUCBrain/AUCPlasma ꢀ0.25), presumably due
to the bis-carbonyl of the isatin moiety. However, these are impor-
tant tools to study M5 function in cells, in electrophysiology and by
icv injection. We did not examine the brain exposure when a
DMSO-containing vehicle was employed, and this may improve
brain levels.21,22
Acknowledgement
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.
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