The discovery of VU0486846: steep SAR from a series of M1 PAMs based on a novel benzomorpholine core

Article abstract of DOI:10.1016/j.bmcl.2018.05.009

This letter describes the chemical optimization of a new series of M₁ positive allosteric modulators (PAMs) based on a novel benzomorpholine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0486846 (7), devoid of adverse effect liability. This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0486846 and details all of the challenges faced in allosteric modulator programs (both steep and flat SAR, as well as subtle structural changes affecting CNS penetration and overall physiochemical and DMPK properties).

Full text of DOI:10.1016/j.bmcl.2018.05.009

Accepted Manuscript
The discovery of VU0486846: Steep SAR from a series of M PAMs based on
1
a novel benzomorpholine core
Jeanette L. Bertron, Hyekyung P. Cho, Pedro M. Garcia-Barrantes, Joseph D.
Panarese, James M. Salovich, Kellie D. Nance, Darren W. Engers, Jerri M.
Rook, Anna L. Blobaum, Colleen M. Niswender, Shaun R. Stauffer, P. Jeffrey
Conn, Craig W. Lindsley
PII:
S0960-894X(18)30399-8
https://doi.org/10.1016/j.bmcl.2018.05.009
BMCL 25826
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
21 March 2018
26 April 2018
5 May 2018
Please cite this article as: Bertron, J.L., Cho, H.P., Garcia-Barrantes, P.M., Panarese, J.D., Salovich, J.M., Nance,
K.D., Engers, D.W., Rook, J.M., Blobaum, A.L., Niswender, C.M., Stauffer, S.R., Conn, P.J., Lindsley, C.W., The
discovery of VU0486846: Steep SAR from a series of M PAMs based on a novel benzomorpholine core, Bioorganic
1
&
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.009
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of M PAMs based on a benzomorpholine core
1
Jeanette L. Bertron, Hyekyung P. Cho, Pedro M. Garcia-Barrantes, Joseph D. Panarese, James M. Salovich, Kellie D.
Nance, Darren W. Engers, Jerri M. Rook, Anna L. Blobaum, Colleen M. Niswender, Shaun R. Stauffer, P. Jeffrey Conn,
and Craig W. Lindsley

Bioorganic & Medicinal Chemistry Letters
The discovery of VU0486846: Steep SAR from a series of M PAMs based on a
1
novel benzomorpholine core
a,c,†
a,b,†
a,c
a,b
Jeanette L. Bertron, Hyekyung P. Cho,
Pedro M. Garcia-Barrantes, Joseph D. Panarese, James M.
a,b
a,b
a,b
a,b
a,b
Salovich, Kellie D. Nance, Darren W. Engers, Jerri M. Rook, Anna L. Blobaum, Colleen M.
a,b,e
a,b,c
a,b,e
a,b,c,d*
Niswender,
Shaun R. Stauffer,
P. Jeffrey Conn, , and Craig W. Lindsley
a
Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
b
c
Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
d
Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA
e
Vanderbilt Kennedy Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
†
These authors contributed equally
To whom correspondence should be addressed: craig.lindsley@vanderbilt.edu
*
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
1
This letter describes the chemical optimization of a new series of M positive allosteric
modulators (PAMs) based on a novel benzomorpholine core, developed via iterative parallel
synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0486846 (7),
devoid of adverse effect liability. This is the first report of the optimization campaign (SAR and
DMPK profiling) that led to the discovery of VU0486846 and details all of the challenges faced
in allosteric modulator programs (both steep and flat SAR, as well as subtle structural changes
affecting CNS penetration and overall physiochemical and DMPK properties).
Keywords:
M
1
Muscarinic acetylcholine receptor
Positive allosteric modulator (PAM)
Schizophrenia
2018 Elsevier Ltd. All rights reserved.
Structure-Activity Relationship (SAR)
1
5
M (muscarinic acetylcholine receptor subtype 1) positive
highly efficacious in multiple rodent cognition models. Here,
we describe for the first time the optimization campaign (SAR
and DMPK profiles
1
allosteric modulators (PAMs) represent an exciting
therapeutic strategy to treat multiple domains of cognitive
dysfunction in CNS disorders such as schizophrenia and
1
-5
Alzheimer’s disease. However, the great potenital of this
target has been hindered, originally by nonselective
6
orthosteric ligands (1), and then by potent ago-PAMs 2-5 (in
cell lines (M PAM EC s <100 nM) and M agomist potency
1
50
1
<
1 M) and native tissues) that proved to be cognitive
disrupting and proconvulsive due to over stimulation of the
7
-12
M receptor (Figure 1). Recently, we disclosed the first M
1
1
13
PAM free from adverse events, VU6004256 (6), and with
robust efficacy in NMDA receptor 1 (NR1) knock-down
14
mice. With the goal of providing the community with an
improved, and much needed, M PAM in vivo tool compound,
1
here we report on VU0486846 (7). PAM 7 (M PAM EC50
1
~
250 nM for human and rat), based on a novel
benzomorpholine core, is a significant structural departure
within the M PAM field that has proven to be a valuable
1
Figure 1. Structures representative of a classical ‘M agonist’ (1),
rodent in vivo M PAM tool compound, devoid of agonist
1
1
that engenders cholinergic side effects, M ago-PAMs 2-5, that are
1
activity in native systems, free from adverse effects and
M agonists in native systems and engender adverse effects, and M
1
1

PAMs 6 and 7, which are devoid of adverse effects.
provide derivatives 12 as mixtures of diastereomers. Finally,
1
5
As briefly detailed in the initial disclosure of 7, we
scaffold-hopped from the prototypical 6,6-fused ring system
of 2 to a novel benzomorpholine core (as in 7) wherein we
either a copper-mediated coupling with heterocycles, or a
Suzuki coupling protocol, produced the putative M PAMs 13
1
in 35-86% yield. To facilitate more rapid SAR, we initially
screened analogs 13 as racemates, and used the racemic
3
simultaneously increased sp character while bringing the
Lewis basic oxygen of the quinolone into the 6,6-ring system.
These modifications of the core also created a new sterogenic
center, which, in the case of 7, the (R)-enantiomer displayed
enantiopreference. Here, we will detail the synthesis, SAR,
and DMPK profiles of a multi-dimensional optimization
campaign within the benzomorpholine series that ultimately
led to the discovery of 7, an M PAM with a balance of
1
overall properties as a new in vivo tool compound, free from
adverse effect liability.
version of 7, 14 (VU0484043, M EC = 0.92 M, pEC =
1
50
50
6.10±0.13, ACh Max 77±7; M -M EC s >30 M) as a
2
5
50
1
5
reference compound.
Initial SAR around 14 with analogs 15-20 showed overall
‘flat’ SAR in terms of M PAM potency, but significant
1
5
1
impact on physiochemical and DMPK properties. Of the
structural changes in Figure 2, introduction of a quaternary
carbon at the chiral center, as in 16, led to a diminution in
potency (M EC50 = 7.3 M, 61% ACh Max); however, all
1
other modifications were within 2- to 3-fold of 14. A major
finding was that the des-oxy tetrahydroisoquinoline analog 15
was essentially equipotent to 14, suggesting that the key
a
1
Scheme 1. Synthesis of M PAM analogs 13.
7
-14
intramolecular hydrogen bond (IMHB) of PAMs 4-7 may
not be a key tenet in this new series (however, this will be
1
6
disclosed in a subsequent publication). Other changes,
represented by 17-20, while active as M PAMs, negatively
impacted plasma protein binding (f <0.001) and/or decreased
CNS penetration (brain/plasma K s <0.05) relative to 14 (f
.11, rat K = 0.17, mouse K = 0.7).
Based on these data, we next explored the ‘fluorine walk’,
a strategy that has been highly successful in allosteric
modulator optimization, particularly for other M PAM
This exercise led to intriguing SAR (Figure 3)
1
u
p
u
=
0
p
p
1
1
7,18
scaffolds.
relative to 14. While incorporation of a fluorine atom at the 5-
positon (21) led to an approximate 3-fold decrease in activity
(
7
M
1
PAM EC50 = 3.2 M, pEC50 = 5.51±0.12, ACh Max
6±2), installation at the 6-position (22) led to a ~3-fold
increase in M PAM potency (M PAM EC = 0.32 M,
a
Reagents and conditions: (a) substituted 2-aminophenols, CH
3
CN, K
2
CO
CN, 80 C, 66-74%;
, HATU, DIEA, DMF, rt,
6-80%; (e) Het-NH, (1S,2S)-N ,N -dimethylcyclohexane-1,2-diamine, CuI,
PO , dioxane, rt, 35-50%, or Ar(Het)-B(OH) , 5 mol% Pd(PPh
THF:H
3
,
o
o
6
(
6
K
0 C, 52-80%; (b) 4-bromoAr(Het)bromide, K
2 3 3
CO , CH
1
1
50
c) KOH, THF/H O (2:1), rt, 95-98%; (d) HN R
2
2 3
pEC50 = 6.51±0.08, ACh Max 88±1). Further movement to
the 7-position (23) led to a diminution in potency (M PAM
EC50 = 2.2 M, pEC50 = 5.66±0.03, ACh Max 80±2). An
almost 7-fold increase in M PAM potency (M PAM EC50
0.14 M, pEC = 6.85±0.06, ACh Max 89±1) was realized by
incorporation of a single fluorine atom at the 8-position (24).
Moreover, the rat CNS penetration was improved, relative to
14 (K = 0.17), with K s ranging from 0.3 to 0.4. Despite the
1
2
1
3
4
2
3 4
) ,
o
2
O, 45 C, 54-86%.
1
1
=
The synthesis of diverse analogs 13 was straightforward
50
and starting materials were readily available from commercial
1
5
sources. Ethyl 2,3-dibromopropanoate 8 was condensed with
various substituted 2-aminophenols to provide the racemic
heterocyclic cores 9 in 52-80% yields. Alkylation with
substituted 4-bromo benzyl bromides or the analogous
heterocyclic congeners proceeded smoothly delivering 10 in
p
p
improvement in M PAM potency and modest gains in CNS
penetration, incorporation of a single
1
Figure 2. Initial SAR around 7, surveying multiple dimensions with analogs
of 13, M PAMs 15-20.
1
yields ranging from 66-74%. A quantitative hydrolysis of the
ester gave 11, which then underwent a HATU-mediated
amide coupling with diverse primary and secondary amines to
Figure 3. Impact of the ‘fluorine walk’ around the benzomorpholine core of

1
4, to deliver M PAM analogs 21-24.
presence of an EC20 fixed concentration of acetylcholine; values represent
1
fluorine atom negatively impacted the DMPK profiles of 21-
4. Unlike 14 (with good fraction unbound, f > 0.11 in rat
and human plasma, and moderate predicted hepatic clearance,
CLhep = 11.1 and 43.2 mL/min/kg for human and rat,
means from three (n=3) independent experiments performed in triplicate.
Total brain:plasma partition coefficients determined at 0.25 h post-
administration of an IV cassette dose (0.20–0.25 mg/kg) to male, SD rats (n =
b
2
u
1
), ND = not determined.
respectively), analogs 21-23 all showed unacceptable fraction
Removal of the chiral hydroxyl moiety (25a) led to a
unbound (f
CLheps of ~20 and >65 mL/min/kg, for human and rat,
respectively). Thus, the ‘fluorine walk’ improved potency,
but at the cost of disposition; therefore, a ‘balanced’ M PAM
u
<0.01) and high predicted hepatic clearance
(
complete loss of M
the rat K for 25a was >1, suggesting that this key hydroxyl
pharmacophore engendered rat P-gp susceptibility and was
responsible for the generally low brain levels (K <0.4)
observed with this moiety across multiple M PAM
1
PAM activity (EC50 >10 M). However,
p
1
in vivo tool compound would not be derived from this sub-
series, and alternate regions of 14 needed to be evaluated.
We returned to the unsubstituted benzomorpholine core of
p
1
chemotypes (note, the ER for human is generally less than
1.2, suggesting this is rat specific). Interestingly, all these
analogs possess favorable CNS MPO scores (>4.5), despite
1
4, and surveyed alternative amide moieties for the (1S,2S)-2-
aminocyclohexan-1-ol amide with analogs 25 (Table 1).
19
rodent CNS exposure issues. The ring in 14 could be
contracted to a cyclopentyl congener (25b) with comparable
activity, but the well-known pyran homolog (25c) afforded a
1
Table 1. Structures and human activities for M PAM analogs 25.
substantial increase in M
1 1
PAM potency (M PAM EC50 =
0
.19 M, pEC50 = 6.73±0.13, ACh Max 91±4). Deletion of
the hydroxyl group on the pyran ring as in 25d similarly led to
a ~34-fold loss in activity. Replacement of the hydroxyl with
either a primary amine (25e) or a ketone (25f) retained
reasonable M
the (1S,2S)-2-aminocyclohexan-1-ol amide of 14 could begin
to buy back M PAM activity as steric bulk increased, but
proved inferior to their cyclic counterparts. We then evaluated
functionalized pyridinyl amides (25j-l), and were pleased to
1
PAM activity. Acyclic congeners (25g-i) of
1
b
Cpd
R
hM
EC50 (M)
% ACh Max
SEM]
1
hM
pEC50
(±SEM)
1
Rat K
p
a
[
±
1
see that these too retained M PAM activity despite the
significant structural departure. Of these, the 3-hydroxy
pyridinyl amide (25j) was equipotent to 14. For all of these
new analogs 25, their DMPK profiles were inferior compared
to the parent 14, displaying an undesired combination of high
predicted hepatic clearance (CLhep ~20 and >65 mL/min/kg in
human and rat, respectively), unacceptable plasma protein
u
1
4
0
.92
6.10+0.13
0.17
1.1
[
77+7]
2
2
2
2
2
5a
5b
5c
5d
5e
>
10
>
5.0
[35]
binding (human and rat f <0.01)
1
.2
5.93+0.07
0.09
0.08
1.0
[
77+5]
0
.19
6
.73+0.03
[91+4]
6
.6
5.20+0.07
[
69+6]
2
.3
5.65+0.07
ND
[
62+3]
2
2
2
2
2
2
2
5f
2
.7
5.58+0.06
5.23+0.19
5.67+0.02
5.72+0.07
6.16+0.04
5.64+0.04
5.79+0.10
ND
ND
ND
ND
0.11
ND
ND
[
78+5]
5g
5h
5i
6
.4
[
60+3]
2
.2
[
78+3]
1
.9
[
80+4]
5j
Figure 4. Divergent metabolic pathways leading to high predicted human
0
.71
and rat hepatic clearance for M PAMs 14 and 25j.
1
[
75+7]
5k
2
.3
p
and/or poor CNS penetration (K s <0.1). Interestingly,
[
81+2]
metabolite identification studies with representative analogs,
14 and 25j, showed that the route of metabolism differed
between the saturated amides (driven by P450-mediated
debenzylation to afford 26) and pyridinyl amides (amide
hydroylsis by esterases liberating 27). Of all the analogs
5l
1
[
.7
80+1]
Calcium mobilization assays with hM
a
1
-CHO cells performed in the

evaluated thus far in this benzomorpholine series, 7 was an
outlier with a balance of M PAM potency, pharmacological,
and DMPK properties that made it an ideal in vivo tool
compound.
As the pyridine biaryl derivative 18 (Figure 2) was
equipotent to 14, we elected to evaluate analogs of this motif
in an effort to identify an in vivo tool. Simple substituents,
[85+3]
28e
CF
3
ND
ND
0.11
1
3
.7
5.44+0.06
5.65+0.13
6.17+0.10
6.15+0.14
6.55+0.12
6.18+0.06
6.32+0.08
5.90+0.02
6.75+0.17
[84+5]
2
2
8f
2
.5
[
90+3]
8g
such as Cl, Br and N(CH
modifications were indeed tolerated as M
methyl group (28d) proved equipotent (M
3
)
2
(28a-c) demonstrated that such
0.70
[89+3]
1
PAMs, but a
1
PAM EC50 = 0.85
18
0
.78
0.11

M, pEC50 = 6.09±0.07, ACh Max 85±3) to 14, but with
reduced molecular weight and hydrogen bond acceptors.
[
92+3]
2
2
8h
Interestingly, bromine (28b) was more potent than chlorine
0
.29
0.08
ND
(
27a), yet CF
methyl (28d), suggesting size alone was not a driving factor
for M PAM potency. A simple vinyl group (28g) was also
equipotent (M PAM EC50 = 0.70 M, pEC50 = 6.17±0.10,
3
(28e) and cyclopropyl (28f) were weaker than
[
87+2]
8i
1
0.67
91+5]
[
1
28j
ND
ND
0.10
ACh Max 89±3) to 14. However, similar to analogs 25, the
DMPK profiles of these compounds precluded further
advancement as in vivo tools. Thus, we decided to evaluate
additional heterobiaryl derivatives of 18. Moving from the N-
linked pyrazole (18) to an N-linked imidazole (28h), and
0
.50
[
94+4]
2
2
8k
1
.3
[
94+4]
introduction of basic amine, led to an increase in M
1
PAM
8l
potency (M PAM EC50 = 0.29 M, pEC50 = 6.55±0.12, ACh
1
0.22
84+5]
Calcium mobilization assays with hM
[
Max 87±2), as did replacement with a pyrrole (28j). Non-N-
linked, N-methyl pyrazoles (28k and 28l) also proved to be
active, but regioisomeric preference was noted. Here, 28l was
a
1
-CHO cells performed in the
presence of an EC20 fixed concentration of acetylcholine; values represent
means from three (n=3) independent experiments performed in triplicate.
Total brain:plasma partition coefficients determined at 0.25 h post-
administration of an IV cassette dose (0.20–0.25 mg/kg) to male, SD rats (n =
), ND = not determined.
the most potent analog in the series (M
M, pEC50 = 6.75±0.07, ACh Max 84±5). Once again,
however, none of these analogs possessed a balanced profile
that positioned them to serve as M PAM in vivo tools, and 7
VU0486846) remained the most attractive compound from
1
PAM EC50 = 0.22
b

1
1
(
the overall series, offering robust efficacy without adverse
effect liability or cholinergic side effects in multiple
preclinical species. As recently detailed, 7 was devoid of M
agonism in cells and native systems, was devoid of
In summary, we have detailed for the first time the
progression and SAR from the prototypical quinolone-based M
PAM core to a novel benzomorpholine core. A multi-
dimensional optimization campaign extensively surveyed diverse
regions of the racemic counterpart 14 of 7 (VU0486846). While
1
5
1
1
cholinergic toxicity in mice, rats and non-human primates,
afforded robust efficacy in a rat novel object recognition assay
MED = 3 mg/kg p.o.) and for the first time, demosntarted
that M1 PAMs can reverse the cognitive deficits induced by
many new pharmacophores were identified that enhanced M
1
(
PAM potency, and challenged some notions regarding the critical
need for an IMHB, physiochemical properties and DMPK
profiles preclude their advancement as in vivo tool compounds.
Importantly, the scaffold hopping exercise from the quinolone
core to the benzomorpholine core gave rise to the highly valuable
15
atypical antipsychotics, such as risperidone.
Table 2. Structures and human activities for M PAM analogs 28.
1
1
rodent M PAM in vivo tool compound 7 (VU0486846), with a
balanced profile; this compound demonstrated utility in target
validation studies without adverse effect liability. Further
1
optimization efforts in route to M PAM clinical candidates, and
further evaluation of the tetrahydroisoquinoline core, will be
reported in due course.
Acknowledgments
b
Cpd
R
hM
EC50 (M)
% ACh Max
SEM]
1
hM
(±SEM)
1
pEC50
Rat K
p
We thank the NIH and NIMH for funding (MH082867 and
MH106839). We also thank William K. Warren, Jr. and the
William K. Warren Foundation who funded the William K.
Warren, Jr. Chair in Medicine (to C.W.L.).
a
[
±
2
2
2
2
8a
8b
8c
8d
Cl
7
.6
5.15+0.12
5.97+0.17
ND
ND
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1
1
6
7

Highlights



Detailed SAR leading to an M1 PAM
devoid of cholinergic liability
Enhanced sp3 character and
enantiospecific M PAM activity
1
Unexpected SAR form the fluorine walk
strategy