SAR inspired by aldehyde oxidase (AO) metabolism: Discovery of novel, CNS penetrant tricyclic M4 PAMs

Article abstract of DOI:10.1016/j.bmcl.2019.06.032

This letter describes progress towards an M₄ PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M₄ PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.

Full text of DOI:10.1016/j.bmcl.2019.06.032

Accepted Manuscript
SAR inspired by aldehyde oxidase (AO) metabolism: Discovery of novel, CNS
penetrant tricyclic M4 PAMs
Trevor C. Chopko, Changho Han, Alison R. Gregro, Darren W. Engers, Andrew
S. Felts, Mike S. Poslusney, Katrina A. Bollinger, Ryan D. Morrison, Michael
Bubser, Atin Lamsal, Vincent B. Luscombe, Hyekyung P. Cho, Nathalie C.
Schnetz-Boutaud, Alice L. Rodriguez, Sichen Chang, J. Scott Daniels, Donald
F. Stec, Colleen M. Niswender, Carrie K. Jones, Michael R. Wood, Michael W.
Wood, Mark E. Duggan, Nicholas J. Brandon, P. Jeffrey Conn, Thomas M.
Bridges, Craig W. Lindsley, Bruce J. Melancon
PII:
S0960-894X(19)30413-5
https://doi.org/10.1016/j.bmcl.2019.06.032
BMCL 26510
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 May 2019
14 June 2019
19 June 2019
Please cite this article as: Chopko, T.C., Han, C., Gregro, A.R., Engers, D.W., Felts, A.S., Poslusney, M.S., Bollinger,
K.A., Morrison, R.D., Bubser, M., Lamsal, A., Luscombe, V.B., Cho, H.P., Schnetz-Boutaud, N.C., Rodriguez,
A.L., Chang, S., Scott Daniels, J., Stec, D.F., Niswender, C.M., Jones, C.K., Wood, M.R., Wood, M.W., Duggan,
M.E., Brandon, N.J., Jeffrey Conn, P., Bridges, T.M., Lindsley, C.W., Melancon, B.J., SAR inspired by aldehyde
oxidase (AO) metabolism: Discovery of novel, CNS penetrant tricyclic M4 PAMs, Bioorganic & Medicinal
Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.06.032
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SAR driven by aldehyde oxidase (AO) metabolism:
Discovery of novel, CNS penetrant tricyclic M4 PAMs
Leave this area blank for abstract info.
Trevor Chopko, Changho Han, Alison R. Gregro, Darren W. Engers, Andrew S. Felts, Mike S. Poslusney, Katrina A.
Bollinger, Ryan D. Morrison, Michael Bubser, Atin Lamsal, Vincent B. Luscombe, Hyekyung P. Cho, Nathalie C. Schnetz-
Boutaud, Alice L. Rodriguez, Sichen Chang, J. Scott Daniels, Donald F. Stec, Colleen M. Niswender, Carrie K. Jones,
Michael R. Wood, Michael W. Wood, Mark E. Duggan, Nicholas J. Brandon, P. Jeffrey Conn, Thomas M. Bridges, Craig
W. Lindsley and Bruce J. Melancon
NH2
NH2
NH2
O
O
O
F
N
HN
S
HN
S
HN
S
HN
N
F
N
N
O
N
aldehyde
oxidase (AO)
cyclization to
heterocycles
hM4 EC50 = 100 nM
rM4 EC50 = 14 nM
75% rev. of rat AHL
(10 mg/kg PO)
SO2CF3
SO2CF3
hM4 EC50 = 1.6 µM
rM4 EC50 = 290 nM
hM4 EC50 > 10 µM
rM4 EC50 = 260 nM
rat brain:plasma Kp,uu < 0.03
rat brain:plasma Kp,uu ~1.0

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
SAR inspired by aldehyde oxidase (AO) metabolism: Discovery of novel, CNS
penetrant tricyclic M4 PAMs
Trevor C. Chopko,a† Changho Han,a† Alison R. Gregro,a Darren W. Engers,a,b Andrew S. Felts,a,b Mike S.
Poslusney,a Katrina A. Bollinger,a Ryan D. Morrison,a Michael Bubser,a,b Atin Lamsal,a Vincent B.
Luscombe,a Hyekyung P. Cho,a Nathalie C. Schnetz-Boutaud,a,b Alice L. Rodriguez,a,b Sichen Chang,a,b J.
Scott Daniels,a,b,g Donald F. Stec,b,c,g Colleen M. Niswender,a,b,f,g Carrie K. Jones,a,b Michael R. Wood,a,b
Michael W. Wood,e Mark E. Duggan,e Nicholas J. Brandon,e P. Jeffrey Conn,a,b,f,g Thomas M. Bridges,a,b
Craig W. Lindsleya,b,c,d,g* and Bruce J. Melancona,b*
aVanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, TN 37232, USA
bDepartment of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
cDepartment of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
dDepartment of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA
eAstraZeneca Neuroscience, IMED Biotech Unit, R&D Boston, MA 02451, USA.
fVanderbilt Kennedy Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
gVanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
† These authors contributed equally to this work.
*To whom correspondence should be addressed: craig.lindsley@vanderbilt.edu or bruce.melancon@vanderbilt.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
This letter describes progress towards an M4 PAM preclinical candidate driven by an unexpected
aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an
equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design
efforts yielded two novel tricyclic cores that enhanced M4 PAM potency, regained CNS
penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in
reversing amphetamine-induced hyperlocomotion in rats.
Keywords:
M4
2019 Elsevier Ltd. All rights reserved.
Muscarinic acetylcholine receptor
Positive allosteric modulator (PAM)
Structure-Activity Relationship (SAR)
Aldehyde oxidase (AO)
Positive allosteric modulators (PAMs) that act on the
muscarinic acetylcholine receptor subtype 4 (M4) continue to
be the focus of programs developing new antipsychotic
therapeutics, and other indications including Huntington’s
disease (HD) and Parkinson’s disease (PD).1-2, 5-7 In many of
these studies, structure-activity relationships (SAR) of the
thieno[2,3-b]pyridine 2-carboxamides and the thieno[2,3-
c]pyridazine 6-carboxamides were derived from varied
functionalization of these cores and driven by potency results
from cell-based calcium efflux assays.3-4 Recently, we have
reported on our efforts to identify novel M4 PAM chemotypes
through scaffold hopping and a reexamination of hydrogen-
bonding motifs contained within these privileged scaffolds.8-
23Concurrent efforts also identified a series of ligands where an
aldehyde oxidase (AO) metabolite of the thieno[2,3-c]pyridine
2-carboxamides drove the SAR towards new tricyclic
analogues, culminating in the identification of a thieno[3,2-
e][1,2,4]triazolo-[1,5-a]pyridine-7-carboxamide system. In
this Letter, we report on the syntheses of these tricyclic ligands
and their varied SAR and DMPK results that generated this
novel class25-27 of selective M4 PAMs with excellent in vitro
potency, good ADME properties, and one of the most
pronounced
reversals
of
amphetamine-induced
hyperlocomotion (AHL) in an in vivo model reported for this
class of compounds.

NH2
NH2
CNS penetration (brain:plasma Kp = 1.8, Kp,uu = 0.76).
Encouraged by the potential of the thieno[2,3-c]pyridine core,
we performed a metabolic soft-spot experiment in rat hepatic
microsomes (+/- NADPH) to inform the rational design of
analogs possessing increased metabolic stability. As shown in
Figure 2, interestingly, we observed only minor
Cl
O
O
S
S
HN
HN
MeO
N
N
NH2
1,
2,
VU0469830
LY2033298
O
SO2CF3
N
S
HN
NH2
O
NH2
O
5,
VU0467206
N
N
S
S
HN
HN
N
N
SO2CF3
NH2
- Extensive NADPH-independent
F
O
H2N
3,
4,
VU0476406
VU0467154
oxidation of the western pyridine
SO2CF3
CONHR
HN
N
S
NH
- Minor NADPH-dependent
hydroxylation of benzylic carbon
S
O
5a
5b
N
O
- Co-incubations with AO inhibitors
block formation of the major oxidative
metabolite
S
Figure 1. Historical M4 PAMs showing a conserved ß-amino
carboxamide (in red) with poor physicochemical properties.
Compound 5 (VU0467206, in blue) has potential to improve solubility
via salt formation.
NH2
O
F
O
F
5
CONHR
F
HN
S
While the dominant M4 PAM chemotype is exemplified by
either a thieno[2,3-b]pyridine 2-carboxamide or a thieno[2,3-
c]pyridazine 6-carboxamide, these cores typically suffer from
poor physicochemical properties and variable species
muscarinic potency. To overcome solubility limited
absorption, we explored other, related congeners with the
potential for salt formation via a more basic nitrogen atom.
Despite steep SAR that precluded the majority of targeted
cores, a thieno[2,3-c]pyridine 2-carboxamide chemotype,
exemplified by 5 (VU0467206), was identified as a novel M4
PAM harboring a more basic nitrogen, with the potential to
modulate physicochemical and DMPK properties.
Figure 2. Soft-spot analysis of 5. Confirmation of aldehyde oxidase-
dependent (AO-dependent) metabolism was confirmed through AO
inhibition in vitro suggesting either 5a or 5b as the core of the major
AO metabolite.
NADPH-dependent metabolism (hydroxylation of the benzylic
carbon), but noted extensive NADPH-independent oxidation of
the western pyridine ring, later confirmed to be via aldehyde
oxidase (AO). Based on previous experience with AO
oxidation of heteroarenes,24 we suspected that the major
metabolite was either the thieno[2,3-c]pyridine-7(6H)-one 5a
or the thieno[2,3-c]pyridine-5(6H)-one 5b.
The synthesis of 5 is illustrated in Scheme 1. Condensation
of methyl 2-mercaptoacetate with isonicotinitrile 6 proceeds to
provide 7 in 83% yield. Suzuki cross coupling reaction
provides the 4-methylthieno[2,3-c]pyridine scaffold 8 in 56%
yield. Saponification followed by HATU mediated coupling
with the privileged trifluoromethylsulfone amine 9, delivers
compound 5 in 35% yield after reverse phase HPLC.
After exhaustive 2D NMR experiments (1H-1H COSY,
HSQC, HMBC), we identified the putative AO-mediated
metabolite of M4 PAM 5, as the thieno[2,3-c]pyridine-7(6H)-
one 5a core, and specifically, compound 15 (VU6016365). We
initiated the synthesis of compound 15 in order to
independently confirm its structure. In Scheme 2, pyridone 10
was bis-brominated using N-bromosuccinimide to afford 11
and it was subsequently condensed with methyl thioglycolate
to provide 12. Protection of the penultimate aminothiophene
with methoxymethyl chloride (MOMCl) proceeded in an
efficient 56% yield over three steps, providing compound 13.
Saponification of the ester followed by standard amide
coupling with our preferred amine provided compound 14 in
good yield. Synthesis of the AO metabolite 15 was completed
via palladium-mediated installation of the methyl group
followed by removal of the hemi-aminal ether under aqueous
acidic conditions.
Scheme 1. Synthesis of thieno[2,3-c]pyridine M4 PAM 5.a
Br
Br
NH2
NH2
N
O
O
O
O
a
b
N
N
N
S
S
Br
6
7
8
NH2
O
c, d
N
S
HN
Scheme 2. Synthesis of AO metabolite 15 (VU6016365).a
F F
H2N
F
S
F
O
O
5,
VU0467206
F
F
9
O
Br
Br
O
Br
O
NH2
HN
S
N
N
S
O
O
O
O
O
a
b
c
O
HN
HN
HN
HN
S
Br
O
O
aReagents and conditions: (a) methyl 2-mercaptoacetate, K2CO3, IPA,
65 °C, 83%; (b) CH3-BF3K, Pd(dppf)Cl2·DCM, Cs2CO3, THF/H2O
(10:1), MW 145 °C, 56%; (c) aq. KOH, MeOH/H2O (3:2), 50 °C,
>99%; (d) R-NH2, HATU, DIEA, DMF, rt, 35%.
10
11
12
13
O
NH2
Br
O
HN
O
O
d
e,f
HN
S
HN
HN
S
HN
O
Compound 5 proved to be a potent rat M4 PAM (EC50 = 290
nM), but with a moderate to high predicted hepatic clearance
(rat CLhep = 51 mL/min/kg; rat microsomal CLint = 200
mL/min/kg), which was confirmed in vivo (rat CLp = 56
mL/min/kg), and thus a good in vitro/in vivo correlation
(IVIVC). In our standard rat IV PBL cassette, 5 showed
excellent brain penetration (brain:plasma Kp = 2.5, Kp,uu = 1.0;
0.2 mg/kg, 10% EtOH, 40% PEG 400, 50% DMSO 2 mg/mL).
Next, we dosed 5 in a discrete rat 10 mg/kg IP PBL study (10%
Tween 80 in water, 4 mg/ mL) and observed similarly high
F
F
14
15
, VU6016365
AO Metabolite
F
F
F
S
F
O
S
O
O
O
aReagents and conditions: (a) NBS, ACN, 65 °C; (b)
methylthioglycolate, K2CO3, IPA, 65 °C; (c) MOMCl, DIEA, DCM,
50 °C, 56% over 3 steps; (d) i. LiOH, THF/H2O (3:2) 50 °C; ii. (4-
((trifluoromethyl)sulfonyl)phenyl)methanamine, HATU, DIEA,
DMF, rt, 31% over 2 steps; (e) CH3BF3K, Pd(dppf)Cl2, Cs2CO3, 1,4-

NH2
dioxane/H2O (10:1) 100 °C; (f) 4M HCl in 1,4-dioxane/H2O, 85 °C,
3% over 2 steps.
N
N
R1
S
R1
Cl
O
O
R1
S
a
b
c,d
S
N
N
Cl
N
Cl
16
17
18
R1
NH2
NH2
NH2
O
R1
S
R1
O
O
e
f
S
HN R3
N
S
HN R3
S
HN R3
N
N
S
N
N
O
R2
19
20
21
aReagents and conditions: (a) NaSMe, MeOH, 91%; (b)
methylthioglycolate, K2CO3, IPA, 50 °C, 65%; (c) KOH, H2O, 100
°C, 98%; (d) R3NH2, HATU, DIEA, DMF, rt, 22-54%; (e) 30% H2O2,
AcOH, 41%; (f) i. hydrazine hydrate, DMSO, MW 140 °C; ii.
Triethylorthoformate or triethylorthoacetate, NMP, MW 180 °C, 21-
53% over 2 steps.
Figure 3. HPLC-UV (reverse phase) chromatograms of parent
standard alone (5, retention = 14.7-14.8 min), metabolite standard
alone (15, retention = 13.2-13.3 min), and parent (5) incubated (25
µM, 1 hr, 37 ºC) in rat hepatic S9 (5 mg/mL, absence of NADPH).
The 1,3,4-triazolo tricyclic series of compounds (Table 1)
was was separated into two classes where R1 = H or R1 = Cl.
For compounds 21a-e, we expected that R1= Cl would be very
potent M4 PAMs as chloro-substituted methylpyridine
scaffolds1-23 were typically more potent than their proteo-
counterparts: 21a (EC50 = 16 nM), 21b (EC50 = 12 nM), 21c
(EC50 = 20 nM), 21d (EC50 = 22 nM), and 21e (EC50 = 8 nM).
However, despite their exquisite potency for the human M4
receptor, these compounds exhibited low fraction unbound in
With an authentic sample of 15 in hand, we compared the
HPLC-UV chromatograms of the parent compound 5 to that of
the AO metabolite 15 (Figure 3), and to compound 5 subjected
to rat hepatic S9 incubation (without NADPH). There was
excellent retention time correlation of the standards to the
observed peaks from the hepatic S9 incubation. Confirmation
of the metabolite identity was also provided by simultaneous
mass spectrometric (MSn) analysis, which revealed matching
fragmentation patterns and ion spectra for the respective
standards and S9 peaks. Further validation was then obtained
by analysis of the sample from the S9 incubation after addition
of the authentic standard of 15 (10 µM final concentration),
which produced an increase in the corresponding peak area
with the same MSn spectra observed prior to addition. In light
of these findings and the NMR data collected, we concluded
that we had correctly identified the structure of 15. Evaluation
of the in vitro pharmacology of 15 showed it was similarly
potent to 5 (rat M4 EC50 = 260 nM) but had an increased
fraction unbound in both rat and human plasma relative to 5
(15: human ƒu plasma = 0.024, rat ƒu plasma = 0.051), but was not
centrally penetrant (brain:plasma Kp < 0.13, Kp,uu < 0.03),
likely as a result of an additional hydrogen bond donor
conferring activity as a substrate for efflux transporter(s).
While not a productive scaffold for CNS indications, we
envisioned constraining the amide in 15 into five-membered
heterocycles might maintain favorable properties while
restoring CNS.
brain homogenate (21b, rat ƒu brain = 0.002; 21c, rat ƒu brain
=
0.011) and many were substrates for P-gp efflux (MDCK-
MDR1 cell line, efflux ratios from 5 to 73 for 21a-e). Similarly,
21f-m showed undesirable profiles with a 2-10 fold decrease in
potency and high efflux ratios (MDCK-MDR1 cell line, efflux
ratios > 15 for 21f-m), which precluded their advancement.
Table 1. Structures and activities for M4 PAM 1,3,4-triazole tricyclic
analogs 21.a
NH2
R1
O
S
HN R3
N
N
N
R2
21
Cmpd
R1
R2
R3
hM4
EC50 (M)a
[% ACh Max]
0.26
N
H
F
15
-
-
H
F
F
S
[84]
0.016
[90]
0.012
[82]
0.020
[73]
0.022
[88]
0.008
[94]
0.11
[79]
O
O
21a
21b
21c
21d
21e
21f
21g
21h
21i
Cl
Cl
Cl
Cl
Cl
H
N
H
H
All of the potential tricyclic scaffolds required de novo
syntheses. The first tricyclic series that we investigated
mimicked the halogenation pattern of 1 (LY2033298, Figure
1). Starting with dichloropyridine (R1 = H) or trichloropyridine
(R1 = Cl) 16, SNAr with sodium methanethiolate afforded
compound 17 in good yield. Condensation with methyl
thioglycolate, followed by saponification and amide coupling
under standard conditions yielded a series of compounds with
the generic structure of 19. Oxidation of the sulfide to the
sulfoxide 20 proceeded in moderate yield using hydrogen
peroxide under acidic conditions. Treatment of 20 with
HN
H
N
H
CH3
CH3
H
N
H
HN
HN
N
H
H
H
0.017
[87]
0.22
[84]
0.12
H
H
HN
N
H
hydrazine and triethylorthoacetate (R2
=
Me) or
N
H
H
triethylorthoformate (R2 = H) in one pot under microwave
conditions produced a compound library of the structure 21,
Scheme 3.
[71]
0.089
[78]
0.99
[60]
21j
21k
21l
H
H
N
H
O
F
N
H
H
F
F
H
S
O
O
Scheme 3. Synthesis of 1,3,4-triazole tricycles of 21.a
F
H
H
0.012
[84]
HN
N
F
F
F
21m
H
H
0.026
[87]
HN
N

aFor SAR determination, calcium mobilization human M4/Gqi5 assays were
performed n = 1 independent times in triplicate with an EC20 fixed concentration
of acetylcholine.
NH2
O
S
HN R2
N
N
Undaunted, other tricyclic scaffolds were investigated,
including the imidazo- and the 1,3,5-triazolo tricyclic variants.
The synthesis of either scaffold began with dichloropyridine 22
(Scheme 4, panel A). Nucleophilic aromatic substitution with
ammonia in methanol provided aminopyridine 23 with high
regioselctivity in excellent yield. Condensation with
methylthioglycolate at high temperature afforded diamino-4-
methylthieno[2,3-b]pyridine 24. In Scheme 4, panel B, the
imidazo-tricyclic scaffold was constructed via condensation
with chloroacetaldehyde or 1-chloropropan-2-one to provide
either the unsubstituted hydrogen version or the methyl
substituted congener 25, (R1 = Me or H). Saponification
followed by amide coupling using HATU conditions provided
a small library of compounds 26. Similarly from compound 24,
1,3,5-triazolo tricycles were synthesized via condensation with
26
R1
Cmpd
R1
R2
hM4
EC50 (M)a
[% ACh Max]
0.13
26a
26b
26c
26d
26e
26f
H
H
N
H
[77]
0.27
[80]
0.24
[66]
2.1
[63]
0.31
[65]
1.2
[76]
0.30
[71]
2.0
[47]
HN
H
HN
N
H
N
H
H
N
H
O
CH3
CH3
CH3
HN
HN
26g
26h
N,N-dimethylformamide
triethylorthoacetate in the presence of hydroxylamine.
Cyclization/dehydration was accomplished using
dimethyl
acetal
or
N
H
N
aFor SAR determination, calcium mobilization human M4/Gqi5 assays were
performed n = 1 independent times in triplicate with an EC20 fixed
concentration of acetylcholine.
trifluoroacetic anhydride to afford 27. Saponification with
sodium hydroxide followed by amide coupling under standard
HATU conditions provided a library of 1,3,5-triazolo tricycles
28.
no compounds which were improved over previous library
iterations with respect to potency (26b-h, EC50 s range from
240 nM to 2.1 µM).
Scheme 4. Synthesis of tricyclic analogs 26 and 28.a
N
NH2
N
O
O
a
b
A
B
Table 3 highlights the final tricyclic series investigated, the
1,3,5-triazolo tricycles. A limited set of unsubstituted triazoles
(R1 = H) was synthesized, 28a-28c, with amides that possess
good potency and were representative of well tolerated analogs
across multiple scaffolds. These same amides were utilized in
the substituted case (R1 = Me) for compounds 28d-r. Many
compounds containing privileged M4 PAM amides (28d, EC50
= 46 nM; 28e, EC50 = 60 nM; 28g, EC50 = 84 nM) had desirable
potencies, but once again possessed poor physicochemical
properties (xLogPs > 3.5). N-aryl-azetidine amides were
previously reported to be potent analogs with the potential to
be centrally penetrant. Here, compounds 28j-r were
synthesized and showed comparable potencies to previously
described M4 PAMs. 28j is a moderately potent M4 PAM (EC50
= 140 nM) with low in vivo clearance in rat (rat CLp = 13
mL/min/kg); however, it suffered from low brain distribution
(rat brain:plasma Kp,uu = 0.02) and off-target activity at human
muscarinic M2 receptor (hM2 EC50 = 650 nM). 28l has similar
properties to others in its series (hM4 EC50 = 140 nM, rat CLp
= 13 mL/min/kg, rat brain:plasma Kp,uu = 0.22, hM2 EC50 > 10
µM), but ultimately suffered from relatively low brain
penetration and poor physicochemical properties. Compound
28n while a potent M4 PAM, was also active at human M2 (hM2
EC50 = 600 nM), typifying the unpredictable SAR with respect
to hM2 for this series. Finally, 28p is a potent M4 PAM (hM4
H2N
N
Cl
S
Cl
N
Cl
H2N
N
22
23
24
NH2
NH2
O
O
O
c
d, e
S
S
HN R2
N
N
N
N
25
26
R1
R1
24
NH2
NH2
O
O
O
h, i
f, g
S
HN R2
N
N
S
N
N
N
N
R1
27
28
R1
aReagents and conditions: Panel A. Synthesis of diamino-4-
methylthieno[2,3-b]pyridine 24. Reagents and conditions: (a) 7 N NH3
in MeOH, MW 150 °C, 90%; (b) methylthioglycolate, K2CO3, IPA,
MW 120 °C, 51%. Panel B. Synthesis of imidazo-tricycles 26 and
1,3,5-triazolo tricycles 28: (c) chloroacetaldehyde (R1 = H) or 1-
chloropropan-2-one (R1 = Me), NaHCO3, EtOH, 80 °C, 98%; (d) 6M
NaOH, THF/MeOH (3:2), 53%; (e) R2NH2, HATU, DIEA, DMF, 18-
43%; (f) DMF-DMA (R1 = H) or triethylorthoacetate (R1 = Me),
HONH2, IPA, 99%; (g) TFAA, THF, 0 °C, 90%; (h) NaOH, EtOH,
63%; (i) R2NH2, HATU, DIEA, DMF, 11-28%.
EC50 = 110 nM) and had low predicted clearance (rat CLhep
=
As shown in Table 2, imidazo-tricyclic analogs 26 proved
to be less than optimal with a significant loss of potency
compared to previous triazolo tricycles. While small alkyl
amines (26a, EC50 = 130 nM) were still preferred, providing a
potent compound, there were
14 mL/min/kg based on microsomal CLint), low in vivo
clearance (rat CLp = 15 mL/min/kg), and moderate brain
distribution and low unbound brain concentration (rat
brain:plasma Kp = 0.47, Kp,uu = 0.04).28 28p was evaluated in a
bidirectional MDCK-MDR1 assay for its potential to be a
substrate for human P-gp efflux and showed a low efflux ratio
(ER = 1.2). While 28p did show moderate potency at human
M2 (hM2 EC50 = 1.2 µM), we chose to move it forward to
Table 2. Structures and activities for M4 PAM 1,3,4-triazolo tricyclic
analogs 26.a

reversal studies of amphetamine-induced hyperlocomotion
(AHL) in SD rats.
Table 3. Structures and activities for M4 PAM 1,3,5-triazolo tricyclic
analogs 28.a
NH2
O
S
HN R2
N
N
N
R1
28
Cmpd
R1
R2
hM4
EC50 (M)a
[% ACh Max]
0.037
[85]
Figure 4. Reversal of amphetamine-induced hyperlocomotion in SD
rats (male, n > 4 per dose group) by 28p (VU6001852). M4 PAM or
vehicle (10% tween-80 90% water [v/v]) was administered orally
30 min after habituation in the chamber, and then 0.75 mg/kg
amphetamine was administered subcutaneously 30 min later
(t = 60 min). Total ambulations were measured over the subsequent
1 h interval (t = 60–120 min) and used to calculate % reversal of AHL
for each dose group. Data represent means ± SEM and were analyzed
by a one-way ANOVA; post hoc comparisons were made by
Dunnett’s test compared to amphetamine-vehicle conditions with
statistical significance determined as p < 0.05.
28a
28b
28c
28d
28e
28f
H
H
HN
0.086
[76]
0.095
[80]
0.046
[80]
0.060
[75]
HN
N
N
H
H
O
CH3
CH3
CH3
HN
HN
N
H
N
0.12
[63]
In summary, we identified several novel tricyclic scaffolds
with excellent potency at M4, derived from a putative AO
metabolite.24 While some of the SAR data are reminiscent of
our previous work highlighting the importance of certain
structural motifs in the ß-amino carboxamide class of M4
PAMs, there are disconnects based on the nature of the tricyclic
core. Efforts towards development candidates in this, and
other, novel scaffolds are underway, and will be reported in due
course.
28g
28h
28i
CH3
CH3
CH3
CH3
N
H
0.084
[84]
0.16
[90]
1.9
[56]
0.14
[89]
O
F
N
H
O
N
H
F
F
F
S
O
O
Cl
N
28j
HN
HN
N
N
N
Cl
CF3
N
28k
CH3
0.25
[90]
Cl
N
28l
CH3
CH3
0.14
[91]
0.16
[94]
Acknowledgments
HN
HN
Cl
N
Cl
N
28m
We thank the NIH and NIMH for funding (U19MH097056).
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.).
N
N
N
N
28n
28o
28p
28q
28r
CH3
CH3
CH3
CH3
CH3
0.12
[86]
0.27
[87]
0.11
[91]
0.41
[97]
0.62
[86]
HN
HN
N
N
N
CF3
F
N
F
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HN
HN
N
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