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G. Duvey et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4523–4527
Table 6
[3H]MPEP binding and ADMET profiling of key compounds
Cpd ID
1
6c
2b
4h
4i
3a
21
23
FLIPR rat mGlu5 IC50 (nM)a
1292
1180
118/97
0.17/0.18
>10 all
isoforms
77
401
384/386
0.11/0.02
<0.75 on 1A2
316
299
42/85
0.16/0.10
>10 all
isoforms
90
60
166
53
139
108/46
0.08/0.03
>10 all
isoforms
2620
n.d.
<5/<5
n.d.
108
n.d.
649/79
n.d.
>10 all
isoforms
Rat cortex [3H]MPEP binding (nM)
151
CLint Rat/Human (
Solubility (mg/mL)c pH 1/pH 7.4
CYP inhibition ( M)
3A4/2C9/2D6/1A2
l
L/min/mg.prot)
—b/83
n.d.
—b/31
0.10/0.04
>10 all
isoforms
l
>10 all
isoforms
n.d.
a
b
c
All compounds displayed full (>99%) inhibition of mGlu5 at 30
Compound unstable in rat plasma matrix.
Kinetic solubility measurement using DMSO solution.
lM.
many of the other changes attempted resulted in complete loss of
activity; such moieties and steep SAR and are often observed in
mGlu5 receptor NAM chemical series, with fluoro-, chloro- and cy-
ano-substituted pyridines being regularly found amongst the most
potent molecules in other mGlu5 receptor NAM series.3–5 This fur-
ther reinforces the likely overlap of pharmacophoric space be-
tween this series and those of the acetylene-containing series.
Transfer of these active motifs to the ketone linker resulted in
the most potent compound in this series, 3a.
Finally, investigation of the 4,5,6,7-tetrahydropyrazolo[1,5-
a]pyridine core was conducted (Scheme 2, Table 5). Substitution
of the 3-position of the 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine
for ketone linked compounds showed that whilst substitution of
this position was tolerated, a general trend towards decreased
activity was observed for such compounds (23–25). It is also
shown that aromatization or ring opening of the 6 membered sat-
urated ring of the 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine is det-
rimental to potency (20, 21).
Plasma exposure of 2b (Phase II)
Dose (mg/kg)
1
3
10
46
30
501
Mean plasma exposure (ng/mL)
2.4
5.3
Figure 2. Effect of 2b (1, 3, 10 and 30 mg/kg in 80% PEG400, p.o., 3 mL/kg, 30 min
pre-treatment time) in the formalin test in Sprague–Dawley rats (n = 10/group).
Each point represents the observed mean (+SEM). ⁄⁄p <0.01 compared with 80%
PEG400 in Phase II.
Rat cortex binding studies were performed on key compounds
using known mGlu5 allosteric ligand [3H]MPEP.6 Similar levels of
potency, were seen between functional activity in the FLIPR Ca2+
release assay (Table 6), suggesting that this series of compound
occupies or perturbs the classical allosteric MPEP site onmGlu5.
Profiling of key compounds from this SAR investigation in pre-
liminary in vitro ADME screens showed that the amide, keto and
direct branched linker compounds suffered significant metabolic
instability in rat microsomes (Table 6). Furthermore, certain
amide-linked compounds were shown to be unstable in the assay
medium, suggesting rapid hydrolysis of the amide bond. Stability
of the compounds profiled in human microsomes suggests discrep-
ancy between the two species, with most compounds, amide-
linked included, being significantly more stable in human. The
much improved stability of 21 demonstrates that the saturated
ring of the core heterocycle is likely a major site of metabolism.
Only compound 2b of the ether linked compounds displayed a suf-
ficiently acceptable in vitro intrinsic microsomal clearance in rat to
progress to PK studies. All compounds displayed good solubilities
and with the exception of direct-linked compound 6c there were
no recurring major flags in CYP inhibition on 4 major isoforms.
3 mg/kg iv administration of compound 2b to rat showed
this compound to have a high clearance and moderate volume of
distribution resulting in a short half-life (Table 7). 30 mg/kg sc with
continuous CSF sampling suggested a one compartmental behav-
iour between plasma and CSF, with a moderate-high CSF/plasma
ratio similar to the in vitro measured plasma free fraction (rat
PPB fu = 32%). Therefore, it was decided to profile compound 2b
in the biphasic rat formalin model of nociception.8 mGlu5 receptor
is implicated in the processing of nociceptive behavior,9 and
mGlu5 receptor NAM MTEP has been shown to be active in reduc-
ing nociceptive behavior induced by formalin.10 Indeed, orally
dosed compound 2b (1, 3, 10, 30 mg/kg) showed dose dependent
reduction of nociceptive behaviour in Phase II (1 h post injection)
but not Phase I of the formalin test (Fig. 2), with significant effect
being observed at 30 mg/kg dose, the maximum effect being of
equal magnitude to that seen with positive control MTEP. Terminal
plasma concentrations of compound 2b showed dose-proportional
exposure of the compound, with only the 30 mg/kg dose demon-
strating an exposure of sufficient magnitude for compound 2b to
be exposed at levels close to the IC50 value in the brain.11 This con-
firms that compound 2b is orally bioavailable and strongly sug-
gests the compound is capable of blocking mGlu5 receptor
in vivo. It is postulated that the anti-nociceptive trend observed
at each dose may be due to exposure of the compound during
the early stages of the experiment, and that mGlu5 blockade
has a delayed-response anti-nociceptive effect in the formalin test
in rat.
Table 7
Pharmacokinetics of 2b in rats
Rat PK 3 mg/kg iva
Rat PK 30 mg/kg scb
CL (mL/min/kg)
Vss (L/kg)
72
1.1
0.2
2896
416
14.4
T
(h)
½
CMax (ng/mL) Plasma
CMax (ng/mL) CSF
CMax CSF/Plasma (%)
In conclusion, we have identified a novel mGlu5 receptor NAM
chemotype with postulated overlap with the MPEP/MTEP pharma-
cophoric space. Optimization of each area of the hit molecule re-
sulted in identification of several sub-100 nM compounds.
Molecules from this series are highly soluble and brain penetrant,
a
Results are mean of 3 animals.
Results are mean of 4 animals.
b