Discovery and SAR of a novel series of potent, CNS penetrant M4PAMs based on a non-enolizable ketone core: Challenges in disposition

Article abstract of DOI:10.1016/j.bmcl.2016.07.042

This Letter describes the chemical optimization of a novel series of M₄PAMs based on a non-enolizable ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selective and CNS penetrant; however, the com

Full text of DOI:10.1016/j.bmcl.2016.07.042

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR of a novel series of potent, CNS penetrant M PAMs
4
based on a non-enolizable ketone core: Challenges in disposition
a,c,y
a,b,y
a,y
d
a
a
Michael R. Wood
, Meredith J. Noetzel
, James C. Tarr , Alice L. Rodriguez , Atin Lamsal ,
Sichen Chang , Jarrett J. Foster , Emery Smith , Peter Chase , Peter S. Hodder , Darren W. Engers a,b,
a
a
d
e
a,b,g
, Nicholas J. Brandon , Michael W. Wood , Mark E. Duggan , P. Jeffrey Conn a,b,g,
f
f
f
Colleen M. Niswender
a,b,
⇑
a,b,c,
⇑
Thomas M. Bridges
, Craig W. Lindsley
a
Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
The Scripps Research Institutes Molecular Screening Center, Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA
Amgen Inc., Thousand Oaks, CA 91320, USA
b
c
d
e
f
Neuroscience Innovative Medicines, Astra Zeneca, 141 Portland Street, Cambridge, MA 02139, USA
Vanderbilt Kennedy Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
g
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 chemical optimization of a novel series of M4 PAMs based on a non-enolizable
ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selec-
tive and CNS penetrant; however, the compound was highly cleared in vitro and in vivo. SAR provided
Received 18 July 2016
Accepted 19 July 2016
Available online xxxx
analogs for which M
4
PAM potency and CNS exposure were maintained; yet, clearance remained high.
Metabolite identification studies demonstrated that this series was subject to rapid, and near quantita-
Keywords:
tive, reductive metabolism to the corresponding secondary alcohol metabolite that was devoid of M
PAM activity.
4
M
4
Muscarinic acetylcholine receptor
Positive allosteric modulator (PAM)
Schizophrenia
Ó 2016 Elsevier Ltd. All rights reserved.
Structure–Activity Relationship (SAR)
Selective activation of the M
has emerged as a very promising, and mechanistically distinct,
therapeutic approach for the treatment of numerous neuropsychi-
4
muscarinic acetylcholine receptor
4
MLSMR collection, and four structurally novel M PAMs were iden-
tified. Of the hits, VU0009153 (7, CID: 682264) represented an
interesting lead (Fig. 2), as it was similarly potent on both human
4
and rat M (EC50s of 120 nM (76% ACh Max) and 76 nM (71% ACh
2
2
1–8
9
atric and rare genetic CNS disorders.
Until recently, the M
4
PAMs reported to date have been primarily within a single
Max), respectively (using DMSO stock from the source plates). In
addition to no apparent species differences, 7 was also inactive
amide-bearing chemotype (Fig. 1),1
0–19
wherein non-obvious, sub-
tle structural modifications engender steep SAR, species differ-
on both human M
re-synthesis of 7,
EC50 = 138 nM, pEC50 = 6.86 ± 0.07, ACh Max = 76.8 ± 4.1 and rM
1
and M
5
(built-in HTS counterscreen). Upon
ences in PAM potency as well as affinity/cooperativity and
M
4
PAM potency remained intact (hM
4
subtype selectivity, solubility, and/or P-gp efflux.1
0–21
As such, all
4
of the known chemotypes of M
4
PAMs (1–6) pose one or more
EC50 = 145 nM, pEC50 = 6.84 ± 0.10, ACh Max = 80.5 ± 6.3). More-
over, it was a des-NH variant of the prototypical M PAM chemo-
major challenges en route to a clinical development candidate. In
this Letter, we detail the identification, SAR and DMPK profile of
a new, non-enolizable ketone chemotype, related to 1, 2 and 4,
identified in a high-throughput screening (HTS) campaign.
Under the auspice of the MLPCN, we performed a functional M
HTS in 1536-well assay format against the ꢀ360,000 compound
4
types 1, 2, and 4, replacing the amide linker with a non-
enolizable ketone. PAM 7 displayed very high in vitro intrinsic
clearance (CLint) in both rat and human hepatic microsomes with
predicted hepatic clearance (CLhep) near hepatic blood flow rates
(Qhep), but was this due to the trimethyl moiety, or an inherent fea-
ture of the ketone-linker? Figure 2 also highlights the three regions
of 7 targeted for SAR exploration with focus on alternate aryl/het-
eroaryl moieties, evaluating our preferred aza systems of 2 and 4,
while also exploring ketone bioisosteres.
2
2
4
⇑
Corresponding authors.
E-mail addresses: thomas.m.bridges@vanderbilt.edu (T.M. Bridges), craig.lind-
sley@vanderbilt.edu (C.W. Lindsley).
y
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2016.07.042
0
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wood, M. R.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.042

2
M. R. Wood et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Me
N
Me
N
NH2
OMe
NH2
O
Cl
O
N
S
O
S
HN
Me
S
HN
MeO
HN
N
Me
1
, LY2033298
2, VU0152100 (ML108) OMe
3, VU0490524 (ML293)
N
N
Me
N
NH2
O
O
Me
O
N
S
HN
N
N
N
F
N
SO CF3
S
2
N
N
4
, VU0467154
5, VU6002703
6, VU6003130
Figure 1. Structures of reported M
4
PAMs 1–6.
Alternative
NH2
Alternate/Preferred
AzaSystems
Ar/Het/Alkyl
CN
SH
Ar (Het)
Ar (Het)
hM4 EC50 = 120 nM (76% AChmax
rM4 EC50 = 76 nM (71% AChmax
inactive on hM /hM
Plasma fu (h, r) = 0.007, 0.008
Microsomal CLint (h, r) = 429, 2070
Pred. CLhep (h, r) = 20, 68 mL/min/kg
)
N
N
N
S
O
)
NH2
8
12
13
14
1
5
NH2
S
N
Cl
CN
SH
O
Cl
O
a
+
Br
Alternatives
Ar (Het)
S
N
O
7
, VU0009153
9
11
Figure 2. Structure and key pharmacology and in vitro DMPK data for the M
4
PAM
NH
2
HTS hit VU0009153 (7), and three areas to address in the lead optimization
campaign. Potency, efficacy, and selectivity was determined via calcium mobiliza-
tion assays with hM4/Gqi5-CHO or rM4/Gqi5-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. Plasma protein binding
and hepatic microsomal intrinsic clearance values represent data from a single
CN
SH
Ar (Het)
N
N
S
N
N
O
(
10
(n = 1) experiment performed in triplicate.
Scheme 1. Reagents and conditions: (a) KOH, EtOH, rt, 6 h, 55–90%.
The chemistry to produce analogs of 7 was straightforward
(
Scheme 1), and employed advanced mercaptonitrile intermedi-
7; however, unpredictable species differences emerged (cf, 14a
versus 14d–e) with a trend towards diminished potency at rat
3,15–19
ates 8–10 previously described
in a one-step protocol. Here,
intermediates 8–10 are treated with a diverse array of commercial
bromomethylketones 11 under basic conditions to afford, via a
one-pot alkylation/cyclization sequence analogs 12–14, respec-
tively in yields ranging from 55% to 90%.
M
4
. Encouragingly, analogs within these three subseries retained
selectivity for M versus M1–3,5 (data not shown).
From our ongoing M PAM program, we had additional mercap-
4
4
tonitrile intermediates beyond 8–10, so we elected to convert
them into ketone analogs 15–17 and assess their profiles (Fig. 3).
Gratifyingly, significant diversity was tolerated, providing, in the
Table 1 highlights select SAR for the first generation of analogs
1
7
2 surveying diverse aryl/heteroaryl moieties. The direct analog of
, 12a, lost ꢀ7- to 10-fold potency at both human and rat M
4
,
case of 15, our most potent M
(hM EC50 = 37 nM, pEC50 = 7.12 ± 0.09, ACh Max = 63.8 ± 5.9 and
rM EC50 = 182 nM, pEC50 = 6.74 ± 0.05, ACh Max = 52.7 ± 5.4).
Initial in vitro and in vivo DMPK evaluation of analogs 12–17
showed a range of low to moderate fraction unbound in rat and
human plasma (f s from 0.005 to 0.180) coupled with high CNS
distribution (K s of 0.83 to 6.95 and Kp,uus of 0.48 to 2.12, but
4
PAM in the ketone-linked series
whereas the direct ketone congener of 2 (12c) was essentially
equipotent at both human and rat M , but with diminished efficacy
ꢀ50% ACh Max). The 2,4-difluorophenyl analog 12e and the 4-pyr-
idyl congener 12f displayed good activity at human M , but exhib-
ited a significant (4- to 7-fold) rightward shift in potency at rat M
4
4
4
(
4
4
.
u
In general, ketone-linked subseries 12 possessed weaker efficacy
than the corresponding amide-linked derivatives. Relative to 7,
these findings suggest substitution in the 5-position may be
p
2
low absolute concentrations in plasma and brain). In addition,
analogs 12–17 showed generally clean CYP450 inhibition profiles
with the exception of often potent CYP1A2 inhibition (data not
shown). However, 12–17 all shared one common property, pre-
dicted in vitro clearance in hepatic microsomes near hepatic blood
flow rates (hCLhep P 20 mL/min/kg and rCLhep P 68 mL/min/kg)
despite their structural diversity. The only commonality amongst
them was the non-enolizable ketone linker. In vitro metabolite
identification (Met ID) studies performed with 12f (as a represen-
tative of the series) in rat and human cryopreserved hepatocytes
demonstrated a rapid, and near quantitative, reductive metabolism
required for M
While in a very limited number of cases (Table 2), substitution
at the 5-position with a chlorine atom afforded active M PAMs
4
PAM potency.
4
with good efficacy as in 13a–b, solubility with other analogs
proved poor (driven by cLogPs > 5) and hindered evaluation. Thus,
our attention focused on the 5,6-dimethyl pyridazine core of 4 and
ketone analogs 14.
4 4
As shown in Table 3, analogs 14 restored M PAM potency (hM
EC50s 102 nM to 891 nM) and efficacy (ACh Max 55–76%), akin to
Please cite this article in press as: Wood, M. R.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.042

M. R. Wood et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 1
4
Structures and activities for M PAM analogs 12
NH
2
Ar (Het)
S
N
O
12
Compd
Ar (Het)
hM
EC50 (nM)a
% ACh Max ± SEM]
4
hM
(±SEM)
4
pEC50
rM
4
4
rM pEC50
(±SEM)
EC50 (nM)a
[
[% ACh Max ± SEM]
12a
12b
12c
12d
12e
12f
Ph
891
47 ± 6%]
355
70 ± 6%]
479
48 ± 6%]
240
45 ± 9%]
562
39 ± 3%]
589
71 ± 5%]
6.05 ± 0.12
6.45 ± 0.04
6.32 ± 0.11
6.62 ± 0.11
6.25 ± 0.28
6.23 ± 0.12
1500
[46 ± 4%]
380
[73 ± 4%]
490
[54 ± 4%]
195
[41 ± 2%]
3980
[35 ± 7%]
2239
5.82 ± 0.03
6.42 ± 0.02
6.31 ± 0.05
6.71 ± 0.03
5.40 ± 0.31
5.65 ± 0.22
[
4-OHPh
4-OMePh
3-Cl,4-FPh
2,4-diFPh
4-pyridyl
[
[
[
[
[
[64 ± 6%]
a
Calcium mobilization assays with hM4/Gqi5-CHO or rM4/Gqi5-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.
Table 2
route affording the generic secondary alcohol 18 as the single
major metabolite (Fig. 4). We synthesized these known and puta-
tive metabolites by simple reduction of the ketones, and found that
Structures and activities for M
4
PAM analogs 13
NH
2
Cl
unfortunately, analogs 18 were devoid of M
4
PAM activity (human
Ar (Het)
and rat EC50s > 10 M), i.e., a primary pharmacology inactivation
l
S
N
metabolism pathway. Subsequent rat IV PK studies using a subset
O
13
of representative compounds demonstrated that these PAMs were
p
also highly cleared in vivo (CL s > 70 mL/min/kg) indicating a good
in vitro:in vivo correlation (IVIVC). Interestingly, even the sec-
ondary alcohols 18 had high predicted hepatic clearance
Compd Ar
hM
EC50 (nM)a
% ACh
4
hM
(±SEM)
4
pEC50 rM
4
4
rM pEC50
(±SEM)
(Het)
EC50 (nM)a
[% ACh
[
(
CLhep > 15 and > 55 mL/min/kg for human and rat, respectively).
Based on these data, we decided to pursue two divergent
Max ± SEM]
Max ± SEM]
1
3a
3b
Ph
372
6.43 ± 0.13 692
[43 ± 4%]
6.98 ± 0.16 417
[95 ± 7%]
6.16 ± 0.12
6.38 ± 0.06
courses of action: (1) survey enolizable ketone congeners, and (2)
survey alternatives for the ketone functional group. Figure 5 high-
lights two representative examples, 19 and 20, of the first
approach employing a phenethylketone derivative. Despite the sig-
[45 ± 5%]
1
3-
105
[74 ± 5%]
pyridyl
a
Calcium mobilization assays with hM4/Gqi5-CHO or rM4/Gqi5-CHO cells per-
nificant structural change, 19 (hM
0.12, ACh Max = 59.1 ± 4.8 and rM
4
EC50 = 479 nM, pEC50 = 6.32 ±
EC50 = 540 nM, pEC50 = 6.27 ±
formed in the presence of an EC20 fixed concentration of acetylcholine; values
represent means from three (n = 3) independent experiments performed in
triplicate.
4
0
.02, ACh Max= 67.9 ± 5.9) and 20 (hM
4
EC50 = 380 nM,
Table 3
4
Structures and activities for M PAM analogs 14
NH
2
Ar (Het)
N
S
N
O
14
Compd
Ar (Het)
hM
EC50 (nM)a
% ACh Max ± SEM]
4
hM
(±SEM)
4
pEC50
rM
4
4
rM pEC50
(±SEM)
EC50 (nM)a
[
[% ACh Max ± SEM]
1
1
1
1
1
4a
4b
4c
4d
4e
Ph
102
76 ± 3%]
646
63 ± 6%]
331
55 ± 5%]
105
74 ± 5%]
891
76 ± 4%]
6.99 ± 0.04
6.19 ± 0.21
6.48 ± 0.28
6.98 ± 0.16
6.05 ± 0.05
166
[67 ± 8%]
1450
[70 ± 7%]
380
[66 ± 4%]
417
[95 ± 7%]
3550
[68 ± 3%]
6.78 ± 0.04
5.84 ± 0.20
6.42 ± 0.05
6.38 ± 0.06
5.45 ± 0.07
[
4-OMePh
2,4-diFPh
3-pyridyl
4-pyridyl
[
[
[
[
a
Calcium mobilization assays with hM4/Gqi5-CHO or rM4/Gqi5-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.
Please cite this article in press as: Wood, M. R.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.07.042

4
M. R. Wood et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
NH
2
N
NH
2
N
NH2
N
N
O
N
S
O
N
S
S
HO
N
O
O
N
O
O
15
16
17
hM4 EC50 = 37 nM
rM4 EC50 = 182 nM
hM4 EC50 = 76 nM
rM4 EC50 = 417 nM
hM4 EC50 = 295 nM
rM4 EC50 = 1,230 nM
Figure 3. More highly functionalized M
4
PAMs 15–17 in the ketone series. Potencies were determined via calcium mobilization assays with hM4/Gqi5-CHO or rM4/Gqi5-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.
R2
N
R2
N
In summary, an HTS campaign identified a new M
type, based on a non-enolizable ketone moiety, as a substitute for
the classical amide linker in 1–4. Despite attractive M PAM
4
PAM chemo-
NH
2
rat or human
hepatocytes
NH
2
R1
R1
Ar (Het)
Ar (Het)
OH
4
X
or
X
S
S
O
potency and CNS penetration, the compounds uniformly were
unstable in hepatic microsomes as well as in vivo, and underwent
a rapid and near quantitative reductive metabolism to the corre-
sponding secondary alcohol – an inactive metabolite, confirmed
via synthesis and biological evaluation. All attempts to replace
the ketone were unsuccessful, rendering this new core not viable
to deliver a clinical candidate. Further study and optimization with
other HTS series are ongoing, and will be reported in due course.
NaBH
4
, MeOH, rt
1
2-17
18
M4 EC50s >10 µM
hCLhep>15 mL/min/kg
M
4
EC50s = 37 nM to >10 µM
hCLhep>20 mL/min/kg
rCLhep >60 mL/min/kg
rCLhep >55 mL/min/kg
Figure 4. Metabolite identification studies in rat and human cryopreserved
hepatocytes with 12f suggest 12–17 undergo rapid and near quantitative reductive
metabolism to the corresponding inactive secondary alcohols, 18. Predicted hepatic
clearance values (from microsomal CLint assays) represent data from a single (n = 1)
experiment performed in triplicate.
Acknowledgments
We thank the NIH for funding via the NIH Roadmap Initiative
1
X01 MH077607 (C.M.N.), the Molecular Libraries Probe Center
Network (U54MH084512 to P.S.H. and U54MH084659 to C.W.L.)
We also thank William K. Warren, Jr. and the William K. Warren
Foundation who funded the William K. Warren, Jr. Chair in Medi-
cine (to C.W.L.).
F
OMe
N
NH
2
NH
2
N
S
S
N
N
O
O
References and notes
1
9
20
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Acad. Sci. U.S.A. 2008, 105, 10978.
hM4 EC50 = 479 nM
rM4 EC50 = 540 nM
hM4 EC50 = 380 nM
rM4 EC50 = 254nM
2.
Leach, K.; Loiancono, R. E.; Felder, C. C.; McKinize, D. L.; Mogg, A.; Shaw, D. B.;
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4
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4
EC50 = 254 nM,
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highly cleared in vitro (predicted CLhep = 18 and 62 mL/min/kg
for human and rat, respectively). Thus, this approach was termi-
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A number of substitutions for the ketone moiety were prepared
9.
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mono-fluoro, gem-difluoro, ether and amino (1° and 2°) deriva-
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4
PAMs. In short, we synthesized and assessed
1
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4
l
1
1
1
1
All attempts to make the 2- and 3-spirooxetane ketone bioisosteres
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that the ketone series was not tractable for the development of a
clinical candidate, despite similar non-enolizable ketone moieties
1
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found in commercial drugs wherein ketone reduction is not a
major route of metabolism.2
4,25
In the present system, the presence
PAM activity, likely serves
1
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