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-