Discovery and SAR of a novel series of non-MPEP site mGlu5 PAMs based on an aryl glycine sulfonamide scaffold

Article abstract of DOI:10.1016/j.bmcl.2012.10.068

Herein we report the discovery and SAR of a novel series of non-MPEP site metabotropic glutamate receptor 5 (mGlu₅) positive allosteric modulators (PAMs) based on an aryl glycine sulfonamide scaffold. This series represents a rare non-MPEP site

Full text of DOI:10.1016/j.bmcl.2012.10.068

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7388–7392
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR of a novel series of non-MPEP site mGlu5 PAMs based on
an aryl glycine sulfonamide scaffold
Alice L. Rodriguez a,b, Ya Zhou a,b,c, Richard Williams a,b, C. David Weaver a,e, Paige N. Vinson a,b,c
,
Eric S. Dawson a,b,c, Thomas Steckler f, Hilde Lavreysen f, Claire Mackie g, José M. Bartolomé f,
Gregor J. Macdonald f, J. Scott Daniels a,b,c, Colleen M. Niswender a,b,c, Carrie K. Jones a,b,c
,
P. Jeffrey Conn a,b,c, Craig W. Lindsley a,b,c,d, Shaun R. Stauffer a,b,c,d,
⇑
a Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
d Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
e Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA
f Neuroscience, Janssen Research and Development, Beerse, Belgium
g CREATe ADME/Tox, Janssen Research and Development, Beerse, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report the discovery and SAR of a novel series of non-MPEP site metabotropic glutamate recep-
tor 5 (mGlu5) positive allosteric modulators (PAMs) based on an aryl glycine sulfonamide scaffold. This
series represents a rare non-MPEP site mGlu5 PAM chemotype.
Received 27 July 2012
Revised 12 October 2012
Accepted 15 October 2012
Available online 23 October 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Metabotropic glutamate receptor 5 (mGlu5)
Positive allosteric modulator (PAM)
Non-MPEP
Allosteric modulation of metabotropic glutamate receptor sub-
type 5 (mGlu5), with positive allosteric modulators (PAMs) is an
increasingly popular approach for selective receptor activation.1–5
Targeting NMDA hypofunction,6 as opposed to classical hyperdop-
aminergia,7 mGlu5 PAMs have provided robust preclinical valida-
tion in multiple schizophrenia and cognition models.8–15
Recently, mGlu5 PAMs have been reported representing diverse
chemotypes (Fig. 1);16 however, the majority of these, such as
1–6, bind at the MPEP (an mGlu5 negative allosteric modulator,
or NAM) binding site,1–5,8–14 and in vivo efficacy has yet to be dem-
onstrated for a non-MPEP site PAM, such as 717,18 or 8.19 Moreover,
a new phenomenon has emerged where very subtle structural
changes, that is, a ‘molecular switch’, to multiple MPEP-site PAMs
can modulate either the mode of pharmacology (PAM to NAM) or
mGlu subtype selectivity, raising concerns over the pharmacology
of metabolites in vivo.11,13,20–22 Importantly, this phenomenon of
‘molecular switches’ has not been observed with the two known
non-MPEP site PAM chemotypes, CPPHA (7)17,18 and VU0357121
(8),19 though the SAR has proven to be far steeper than the MPEP
site congeners. Thus, our lab has been focused on the identification
and optimization of additional non-MPEP site mGlu5 PAMs to
ascertain the presence or absence of ‘molecular switches’, and to
determine if non-MPEP site ligands afford the same in vivo efficacy
profile as MPEP-site PAMs.
Herein, we describe the synthesis and SAR of a novel series of
potent, non-MPEP site mGlu5 PAMs based on an aryl glycine sul-
fonamide scaffold identified in a functional HTS.12
A high-throughput functional screen, employing a triple-add
calcium mobilization assay of 160,000 compounds, identified
1400 confirmed mGlu5 PAMs, and over 60 with EC50s below
500 nM in our high-expressing rat mGlu5 HTS cell line.12 From this,
we identified VU0034403 (9), an unprecedented mGlu5 PAM
chemotype with potent (rat EC50 = 98 nM, 48% Glu Max, 7.9-fold
shift) ago-PAM activity (Fig. 2 top panel), and amenable to chemi-
cal optimization via an iterative library approach. Significantly, 9
was selective versus the other mGluRs (mGlu1–4,6–8 >10
l
M), and
afforded minimal displacement of [3H]-methoxyPEPy (Ki >10
l
M,
Fig. 2 bottom panel), suggesting that 9 binds at a site distinct from
the MPEP site.
Synthesis of first generation analogs 13 proceeded smoothly
through a five step process (Scheme 1) surveying diversity at the
⇑
Corresponding author.
E-mail address: shaun.stauffer@vanderbilt.edu (S.R. Stauffer).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.068

A. L. Rodriguez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7388–7392
7389
MPEP Site
F
Cl
Cl
a
Cl
b
c
Ph
N
OMe
H
N
N
S
NH2
NH
S
N
F
N
CN
N
O
O
O
O
O
O
F
Ph
1
2
11
12
13
, DFB
, CDPPB
N
N
N
Cl
Cl
NHCH3
N
O
d
N
H
N
OH
F
N
S
N
S
R
3
4
, ADX-47273
O
O
O
O O
14
O
O O
15
O
NR1R2
OBn
CN
N
X
N
Scheme 1. Library synthesis of analogs 15. Reagents and conditions: (a) MeSO2Cl,
pyridine, CH2Cl2, 99%, (b) (i) NaH, DMF, (ii) methyl bromoacetate, DMF, 94–98%, (c)
LiOH, MeOH, THF, 99%, (d) PS-HOBt, HATU, 2,6-lutidine, DMF, RR’NH, CH2Cl2, 26–
68%. All library compounds were purified by mass-directed prep LC where required.
Ar
5
6
, VU0364289
Non-MPEP Site
Cl
F
O
PAMs, the chemical optimization program transitioned to human
mGlu5 cell lines with expression levels more closely aligned with
native expression.12,23 Potency was maintained for freshly pre-
pared 9 in the rat cell-line while maximum efficacy increased
(rat EC50 = 78 nM, 74% Glu Max), and Table 1 shows data for se-
lected analogs 15 in both rat and human mGlu5 cell lines. Against
the human receptor, the potency of HTS lead 9 was tenfold less po-
tent relative to the rat receptor cell line with an EC50 of 780 nM.
Similar to the non-MPEP PAM CPPHA (7), SAR was extremely
flat, with this first generation library affording very few active
PAMs, and a ꢀthreefold difference between rat and human po-
tency. Of the ꢀ50 analogs tested, only four showed good PAM
activity, and these are all closely related phenyl 15a (rat
EC50 = 72 nM, 61% Glu Max), and fluoro-pyridine congeners 15b–d
O
N
O
O
N
H
HN
OH
F
O
7
8
, VU0357 121
, CPPHA
Figure 1. Representative MPEP site (1–6) and non-MPEP site (7 and 8) mGlu5 PAMs.
Alternate Aryl
Substituents/Cyclize
Alternate Amides
Cl
R4
N
iterative
parallel
H
R1
N
Cl
(rat EC50s 1.7–4.5 lM, 64–72% Glu Max). Basic and acidic analogs
N
R3
N
S
were inactive, as were 3° amide congeners including a constrained
indoline. In general cyclic aliphatic amides were weakly active and
acyclic amides were inactive. Like 9, 15a possessed strong allosteric
agonist activity on rat mGlu5 (an ago-PAM) which precluded the
determination of an accurate fold shift measurement; however,
the fold-shift is estimated to be minimal (ꢀ1.2). Within this series
no ago-PAM activity was detected in the lower expressing human
mGlu5 cell line. Based upon previous studies wherein receptor
expression level was found to impact allosteric agonist activity23
we believe this phenomenon to be expression level dependent
rather than due to a species difference. While 15a possessed the
O
O O
synthesis
O
S
O O
R2
Alternate sulfonamides
10
9
, VU0034403
120
100
80
60
40
20
0
potency, selectivity (mGlu1–4,6–8 >10
plasma protein binding, Fu = 0.13) required for in vivo studies in rats
for a non-MPEP PAM ([3H]-methoxyPEPy Ki >10
M), 15a suffered
lM) and free fraction (rat
MPEP
l
VU0034403
poor metabolic stability (64% remaining after 15 min).24 The
pyridyl congener 15b displayed an equivalent extent of plasma
protein binding (rat Fu = 0.17, human Fu = 0.09) and improved
metabolic stability in rat liver microsomes (39% remaining after
15 min); however, the rat mGlu5 potency was not optimal for
in vivo studies.
-11 -10
-9
-8
-7
-6
-5
-4
Log Compound, [M]
Figure 2. Top panel: Structure of non-MPEP mGlu5 PAM HTS hit VU0034403 (9)
and the chemical optimization plan leading to analogs 10. Bottom panel: Radio-
ligand displacement assay [3H]-methoxyPEPy using VU0034403 and MPEP.
The next library iteration surveyed branching at the a-position
with alkyl substituents. Analogs were easily prepared (Scheme 2),
beginning with alkylation of 12 using the appropriate ethylbro-
moacetate (Me, Et, and n-Pr branched) and saponification to afford
acid intermediates 16a–c. Amide formation was performed as pre-
viously described to generate racemic analogs 17. The methyl
derivative 17a was active (rat EC50 = 68 nM), but displayed dimin-
ished efficacy (43% Glu Max). Larger alkyl groups (Et 17b and n-Pr
Eastern amide moiety. Beginning with 2-Cl aniline 11, sulfonyla-
tion delivered 12 in quantitative yield. Alkylation of 12 with
methyl bromoacetate provided ester 13, which was saponified to
give acid 14. A two-step amide coupling procedure, employing a
diverse collection of amines (aromatic, benzylic, 1°, 2°, aliphatic,
with basic and acidic moieties) generated analogs 15 in isolated
yields ranging from 26% to 68%.
17c) were weak (17b rat EC50 = 3.3
>10 M).
Based on these data, we then introduced a cyclic constraint
between the -position and the 2-Cl moiety to generate a racemic
lM) or inactive (17c rat EC50
l
While it was important to employ the high expressing rat
mGlu5 cell line in the HTS to ensure we identified even weak mGlu5
a

7390
A. L. Rodriguez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7388–7392
Table 1
Structures and activities of analogs 15
Cl
H
N
N
S
R
O
O O
15
a
Compd
R
rmGlu5 pEC50/EC50
% Glu Max
74
hmGlu5 pEC50/EC50
% Glu Max
63
Cl
7.11
0.08
6.11
0.78
9
7.14
0.07
5.86
1.38
15a
15b
61
66
77
N
N
5.43
3.72
NT
NT
F
F
5.76
1.74
<5.0
>10
15c
15d
64
72
42
35
F
N
5.35
4.47
<5.0
>10
a
pEC50 are the average of three independent determinations and represent a coefficient of variation (CV) <0.1, EC50 units are in
l
M; NT = not tested.
Cl
Cl
Cl
R
R
a,b
c
H
N
H
N
H
OH
Cl
N
NH
S
N
S
N
S
N
S
N
S
O
O
O O
O
O O
O
O
O O
O
O O
F
12
16a, R = Me
16b, R = Et
17a, R = Me
17b, R = Et
Cl
Cl
16c,
17c,
R = n-Pr
R = n-Pr
20
21
rat EC50 = 2.5 µM
glu max = 74%
rat EC50 = 6.7 µM
glu max = 78%
Scheme 2. Library synthesis of analogs 17. Reagents and conditions: (a) NaH, RBr,
DMF, 17-quant.% (b) LiOH, MeOH, THF, 99%, (c) PS-HOBt, HATU, 2,6-lutidine, DMF,
(e) m-ClPhNH2, CH2Cl2, 73%. All library compounds were purified by mass-directed
prep LC where required.
H
H
N
N
N
N
N
S
O
S
O O
O
O O
a,b,c
H
N
Cl
O
N
S
N
H
Cl
N
O
O O
O
N
18
19
22
23
rat EC50 > 10 µM
glu max = 75%
rat EC50 = 1.3 µM
glu max = 68%
Scheme 3. Synthesis of racemic tetrahydroisoquinoline derivative 19. Reagents and
conditions: (a) MeSO2Cl, pyridine, CH2Cl2, 99%, (b) LiOH, MeOH, THF, 99%, (d) PS-
HOBt, HATU, 2,6-lutidine, DMF, (e) m-ClPhNH2, CH2Cl2, 74%.
Figure 3. Structures of non-MPEP mGlu5 PAM (S)-tetrahydroisoquinoline analogs
20–22.
tetrahydroisoquinoline derivative 19 (Scheme 3). In the high
expressing HTS rat mGlu5 cell line, this provided a moderately po-
tent and efficacious PAM (EC50 = 1.0 lM, 60% Glu Max). Based on
the tetrahydroisoquinoline series as illustrated by these examples
(68–78%), they displayed weak potency in the micromolar range.
In parallel, additional libraries were focused on identifying
alternative aryl R1 moieties (Fig. 2) and non-aromatic amides R2
in an attempt to improve metabolic stability while maintaining
acceptable potency and efficacy following the routes outlined in
Schemes 1 and 2. Similar to CPPHA 7, SAR was shallow, with only
ꢀ10% of compounds assayed displaying mGlu5 PAM activity. Of
these, VU0400100 (24), an analog with an additional 5-CF3 on
the western 2-ClPh ring and an eastern cyclopropyl methyl amide,
was studied extensively (Fig. 4). PAM 24 was moderately potent on
both human and rat mGlu5, did not displace [3H]-methoxyPEPy,
the commercial availability of enantiopure (R)- and (S)-18, subse-
quent libraries (consisting of 80 analogs) explored stereochemistry
as well as diversity at both the sulfonamide and the amide moie-
ties following the chemistry outlined in Scheme 3. Enantioselective
mGlu5 potentiation was observed, with the (S)-enantiomer gener-
ally >10-fold more potent than the (R)-enantiomer. Overall little
improvement in potency was observed, SAR remained relatively
flat, and metabolic stability remained an issue. Figure 3 highlights
representative racemic and (S)-enantiomer analogs 20–23.
Although efficacy would appear to be overall improved within

A. L. Rodriguez et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7388–7392
rmGlu5 EC50 = 398 nM, 61% glu max
7391
Cl
H
N
hmGlu5 EC50 = 4.5 µM, 70% glu max
[3H]-methoxyPEPy (Ki > 30 µM)
mGlu1-4,6-8 > 10 µM
in vitro CLHEP (h, r): 15, 53 mL/min/kg
plasma Fu (human, rat): 0.10, 0.13
rat brain Fu = 0.10
F3C
N
O S OO
24, VU0400100
Figure 4. Structure, pharmacological, and DMPK profile of VU0400100, 24, a non-
MPEP site mGlu5 PAM.
was highly selective versus the other mGlus and displayed signifi-
cantly reduced plasma protein binding (rat, human) and nonspe-
cific binding (Fu, 0.10) in rat brain homogenate binding.
Moreover, 24 lacked significant activity in a Ricerca radioligand
binding panel of 68 GPCRs, ion channels and transporters (<50%
inhibition at 10
panel of ion channels relevant to cardiovascular safety (hERG, Ca,
and Na, IC50 >10 M).
lM) and 24 was devoid of functional activity in a
l
Consistent with the in vitro predicted clearance (Fig. 4), rat
pharmacokinetics indicated 24 suffered extensive first-pass hepa-
tic metabolism (EH, 0.98) following an oral administration, as sig-
nificantly lower exposure was observed in systemic supply
relative to hepatic portal vein blood supply (AUCHPV:AUCplasma
,
Figure 6. Suflex-Sim overlay of CPPHA (7, grey surface, ball and stick) and 15a (gold
52). Exposures were improved with intraperitoneal or subcutane-
ous dosing routes, achieving CNS exposure relative to the systemic
circulation (AUCbrain:AUCplasma, 0.4) sufficient to suggest brain pen-
etration is possible for this class of non-MPEP based mGlu5 PAMs.
Currently, in vitro studies are underway to establish if the pan-
P450 inactivator, 1-aminobenzotriazole (ABT), will have an impact
on the clearance of 24 in rat microsomes.25 If sufficient reduction of
P450-mediated clearance of 24 is observed in vitro, it may be
possible to enhance exposure in vivo and establish proof-of-concept
in rat behavioral models with a non-MPEP site mGlu5 PAM by
administering 24 to rats that receive an oral dose of ABT.
surface, capped sticks).
these hybrid PAMs, the activity observed suggests that it may be
possible to identify additional sulfonamide containing mGlu5
PAMs.
In addition to the above exercise preparing sulfonamide hybrids
using known MPEP-site mGlu5 PAM scaffolds we have also under-
taken a computational evaluation of low energy, ligand-based con-
formational ensembles of MPEP and non-MPEP ligands. Mutual
flexible low energy shape-based alignment of sulfonamide 15a
with CPPHA (7) using the Surflex-Sim algorithm suggests a pre-
ferred fit for CPPHA (7) versus other MPEP modulators (Fig. 6).26
Studies are ongoing to develop a comprehensive model based upon
this approach as an entry point to identify additional novel non-
MPEP modulators.
Significantly, this is only the third known non-MPEP site mGlu5
PAM chemotype, and like CPPHA (7) and VU0357121 (8), SAR was
steeper than that of MPEP site PAMs. Importantly, no ‘molecular
switches’ were observed within this non-MPEP site series.16 While
DMPK properties currently preclude in vivo studies, key PAMs
within this series are valuable tools for detailed in vitro pharmaco-
logical and electrophysiology studies. VU0400100 (24) has now
been declared MLPCN probe ML332 and is freely available upon re-
quest.27 Additional studies and refinements are in progress and
will be reported in due course.
It is interesting to note that to our knowledge this is a rare
example of a chemical series of mGlu5 PAMs containing a sulfon-
amide moiety. Analogs of 9 wherein the sulfonamide was replaced
with N-alkyl, N-aryl or a N-acetyl moiety were devoid of PAM
activity. These observations prompted us to replace the amide moi-
ety in the known mGlu5 MPEP-site PAM series 4 and 5 (Fig. 1) with
sulfonamides (Fig. 5). While potency was diminished at least an or-
der of magnitude relative to the direct amide counterpart scaffolds
(4 and 5, Fig. 1),12,15 the sulfonamide congeners 25 and 26 were
weak to moderately active PAMs in the high expressing rat cell
line, suggesting additional avenues for optimization. Within the
human cell line the acetylene PAM 25 was inactive and the Addex
analog 26 a weak PAM. Competition binding studies using mem-
branes isolated from rmGlu5 demonstrated no displacement of
[3H]-methoxyPEPy with up to 30
tent analogs are needed to establish an allosteric binding site for
lM 25 or 26. Although more po-
Acknowledgments
O
O
N
N
O
S
F
This work was supported in part by grants from the NIH
(NS031373, MH062646, and MH89870) and from an industry
sponsored contract from Johnson & Johnson. Vanderbilt is a mem-
ber of the MLPCN and houses the Vanderbilt Specialized Chemistry
Center for Accelerated Probe Development (U54MH084659). The
authors thank Daryl F. Venable and Kiran K. Gogi for technical
assistance.
N
O
F
N
S
O
O
F
25
26
rmGlu5 EC50 = 457 nM, glu max = 55%
hmGlu5 Inactive
rmGlu5 EC50 = 3.4 µM, glu max = 84%
hmGlu5 > 10 µM, glu max = 43%
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the amide moiety replaced with the analogous sulfonamide linker.

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24. See Ref. 12 for a description of in vitro DMPK assay methods (plasma protein
binding, metabolic stability).
25. Balani, S. K.; Zhu, T.; Yang, T. J.; Liu, Z.; He, B.; Lee, F. W. Drug Metab. Dispos.
2002, 30, 1059.
26. Analysis and surface figure generated using SybylX v1.2 on
a Linux
workstation. For Suflex Sim background see: Jain, A. N. J. Med. Chem. 2004,
47, 947.
27. For information on the MLPCN and information on how to request probe
compounds, such ML332, see: http://mli.nih.gov/mli/mlpcn/.
15. Liu, F.; Grauer, S.; Kelley, C.; Navarra, R.; Graf, R.; Zhang, G.; Atkinson, P. J.;
Popiolek, M.; Wantuch, C.; Khawaja, X.; Smith, D.; Olsen, M.; Kouranova, E.; Lai,