Discovery of VU6027459: A First-in-Class Selective and CNS Penetrant mGlu7Positive Allosteric Modulator Tool Compound

Article abstract of DOI:10.1021/acsmedchemlett.0c00432

Herein, we report the discovery of the first selective and CNS penetrant mGlu7 PAM (VU6027459) derived from a "molecular switch"within a selective mGlu7 NAM chemotype. VU6027459 displayed CNS penetration in both mice (Kp = 2.74) and rats (Kp= 4.78), it was orally bioavailable in rats (%F = 69.5), and undesired activity at DAT was ablated.

Full text of DOI:10.1021/acsmedchemlett.0c00432

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Letter
Discovery of VU6027459: a first-in-class selective
and CNS penetrant mGlu7 PAM tool compound
Carson W Reed, Jacob J Kalbfeisch, Madison J Wong, Jordan P Washecheck, Ashton Hunter,
Alice L Rodriguez, Anna L. Blobaum, P. Jeffrey Conn, Colleen M Niswender, and Craig W Lindsley
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00432 • Publication Date (Web): 20 Aug 2020
Downloaded from pubs.acs.org on August 24, 2020
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Discovery of VU6027459: a first-in-class selective and CNS
penetrant mGlu7 PAM tool compound
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†,
†
†
║
Carson W. Reed,†, Jacob J. Kalbfleisch, Madison J. Wong, Jordan P. Washecheck, Ashton Hunter, Alice L.
π
π
,║
ɸ
,║
ɸ
,║,
ɸ
,║,
ɸ
†,
ɸ
ψ
ψ
Rodriguez, Anna L. Blobaum, P. Jeffrey Conn,
Colleen M. Niswender,
* and Craig W. Lindsley
,║
*
†Department of Chemistry, Vanderbilt University, Nashville, TN 37232, United States
ɸDepartment of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, United States
║Warren Center for Neuroscience Drug Discovery, Vanderbilt University School of Medicine, Nashville, TN 37232, United States
ψVanderbilt Kennedy Center, Vanderbilt University, Nashville, TN 37232, USA
πThese authors contributed equally
ABSTRACT: Herein, we report the discovery of the first selective and CNS penetrant mGlu7 PAM (VU6027459) derived from a
‘molecular switch’ within a selective mGlu7 NAM chemotype. VU6027459 displayed CNS penetration in both mice (Kp = 2.74) and
rats (Kp = 4.78), was orally bioavailable in rats (%F = 69.5) and undesired activity at DAT was ablated.
KEYWORDS: metabotropic glutamate receptor, mGlu7, positive allosteric modulator (PAM), VU6027459, Rett syndrome
Holding the distinction of being the most widely expressed
metabotropic glutamate receptor in the CNS, mGlu7 is a Group
III mGlu receptor (along with mGlu4,6,8) that has an extremely
low affinity for the endogenous agonist glutamate, and thus, is
believed to act as an ‘emergency brake’ when glutamate levels
become elevated.1,2 There is currently a lack of highly selective
and CNS penetrant mGlu7 positive allosteric modulators
(PAMs). While an mGlu7 allosteric agonist does exist,3 its rapid
metabolism makes interpretation of in vivo effects difficult to
establish.4 Thus, the therapeutic potential of mGlu7 activation
has been ascertained from studies in mGlu7 knock-out mice and
with mGlu7 negative allosteric modulators. These studies
suggest relevance in treating depression, schizophrenia,
cognitive disorders, autism and ADHD.5-15 Human genetics and
GRM7 polymorphisms have further strengthened these disease
associations, while also highlighting the key role of mGlu7 in
neurodevelopmental disorders, notably, Rett syndrome.16-30
Using non-selective Group III PAMs, such as 1-4 (Figure 1),
in combination with mGlu7 NAMs (e.g., 5 and 6) a
pharmacological role for mGlu7 PAMs in Rett syndrome in
correcting apneas, as well as social and cognitive dysfunction
in mouse models of Rett syndrome, has been discovered.31-35
ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinolone
carboxylate scaffold exemplified by 7 (VU6009339 mGlu7 IC50
= 6.1 M, >30 M vs. mGlu1-5,8), all moieties surveyed were
inactive, save for the cyano group, which engendered a
‘molecular switch’ to a highly selective (>30 M vs. mGlu1-5,8
)
and CNS penetrant (rat Kp = 4.7, Kp,uu = 0.96) mGlu7 PAM 8
(VU6014181, mGlu7 EC50 = 879 nM (pEC50 = 6.06±0.18,
51.3±6.6% L-AP4 max (rat GIRK assay)).38 Notably, this was
the first ‘molecular switch’ ever encountered with mGlu7
allosteric ligands (Figure 2). Based on these exciting data, we
also
O
N
Cl
HN
O
N
S
MeO
O
O
N
NH
N
S
N
N
N
N
Cl
H
NHAc
1
2
, ADX88178
, VU0155094
3
, VU0422288
mGlu4 EC50
mGlu7 EC50 >10 M
mGlu8 EC50 = 1.2 M
=
52 nM
mGlu4 EC50
mGlu7 EC50
=
=
3.2 M
1.5 M
mGlu4 EC50 = 108 nM
mGlu7 EC50 = 146 nM
mGlu8 EC50 = 125 nM
mGlu8 EC50 = 900 nM
OCF3
O
F3C
N
O
N
F
N
N
OCH3
O
F
N
O
H
N
N
OMe
N
4
5
, ADX71743
mGlu7 EC50 = 650 nM
other mGlus > 10 M
, VU6005649
While highly selective and CNS penetrant mGlu7 NAMs,
e.g. 6 and related congeners, have been realized, mGlu7-
selective PAMs have remained elusive despite multiple high-
throughput screening campaigns.34,36-37 During an mGlu7 NAM
optimization campaign surveying ester replacements within an
6, VU6012962
mGlu7 EC50 = 347 nM
other mGlus > 10 M
mGlu4 EC50 >10 M
mGlu7 EC50 = 649 nM
mGlu8 EC50 = 2.6 M
Figure 1. Structures of reported mGlu Group III PAMs 1-3, mGlu7/8 PAM
4 and highly selective mGlu7 NAMs 5 and 6.
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reacted with the conjugate base of acetonitrile (formed from the
evaluated PAM 8 in our rat mGlu7 calcium assay, where similar
potency and efficacy were noted (mGlu7 EC50 = 1.1 M (pEC50
= 5.98±0.11), 85.1±18.5% L-AP4 max), further confirming
mGlu7 PAM activity. PAM 8 also displayed moderate rat in
vivo PK (Clp = 47.5 mL/min/kg, t1/2 = 0.75 hr, Vss = 4.1 L/kg),
suitable as a lead for further optimization (rat fu = 0.032; mouse
fu = 0.051); however, while it was uniformly clean (no
inhibition >50%@10 M) in a Eurofin ancillary radioligand
binding panel of 68 different GPCRs, ion channels and
transporters, it displayed activity at one ‘no go’ target – the
dopamine transporter (DAT 85%@10 M, Ki = 430 nM), as
global increase in dopamine would mask target validation
efforts for mGlu7. Thus, our optimization work-flow would add
DAT as a key anti-target to be evaluated and eliminated from a
potential in vivo probe.
1
2
3
4
5
6
7
8
deprotonation of acetonitrile with n-BuLi) followed by
subsequent treatment with acetic acid to form quinolones 12 in
moderate to good yields (58-77%). Quinolones 12 were then
heated to 100 °C in neat POCl3 to form chloroquinolines 13 in
78-93% yield. Then, an SNAr reaction between 13 and 2-
fluorophenylpiperazine yielded analogs 14 in good yields (60-
82%). Alternatively, a number of 5,6-fused systems 15 were
commercially available, and they underwent the SNAr smoothly
to afford analogs 16.
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SAR was steep, with all analogs 16 devoid of mGlu7 PAM
activity (see Supporting Information), and only fluorinated con-
Scheme 1. Synthesis of alternate heterocyclic core analogs 14 and 16 of 8.a
O
O
O
c
OH
NH2
a
b
OMe
NH2
OMe
N
Rn
Rn
Rn
N
9
10
11
NC
F
O
Cl
N
N
N
CN
CN
d
e
Rn
Rn
N
H
N
Rn
12
13
14
Figure 2. A “Molecular Switch’ changes the mode of pharmacology from
mGlu7 NAM (7) to mGlu7 PAM (8). The mGlu7 PAM activity of 8 is
comparable in both mGlu7 GIRK (EC50= 880 nM, 51.3% L-AP4 max) and
calcium (EC50= 1.1 M, 85.1% L-AP4 max) assays.
NC
F
Cl
CN
e
N
N
N
Het
N
Het
15
16
o
aReagents and conditions: (a) TMSCHN2, PhH:MeOH, 0 C, 1h, 76-90%;
(b) DMF●DMA (Dimethylformamide Dimethylacetal), MeOH, 150 oC
(µW), 1 h, 99%; (c) (i) n-BuLi, MeCN, THF, -78 oC,30 min; (ii) AcOH, -
78 oC,30 min, 1 hr, 58-77% over two steps; (d) POCl3, 100 oC, 78-93%; (e)
2-Fphenylpiperazine, DMF, 150 oC (µW), 15 min, 60-82%.
In order to optimize 8 for mGlu7 PAM activity, DMPK
profile and to ablate DAT activity, we elected to perform a
multi-dimensional optimization campaign, surveying a wide
range of chemical diversity (Figure 3). For the first round of
SAR, we elected to hold the 2-fluorophenyl piperazine moiety
constant and survey alternatives for the cyanoquinolone core
employing the route depicted in Scheme 1. Here, anthranilic
geners 14a-d of 8 (i.e., the ‘fluorine walk’) displayed mGlu7
PAM activity (Figure 4). Of note, 14c displayed comparable
mGlu7 PAM activity (EC50 = 1.4 M, 99% L-AP4 max) to 8, but
also eliminated DAT activity, indicating that the 6-fluoro
moiety was essential to eliminate DAT activity. However, 14c
was moderate to highly cleared in vitro (rat CLHEP = 56
ml/min/kg, mouse CLHEP = 87 mL/min/kg), and displayed high
protein binding (rat/mouse plasma fu = 0.010/0.012). In
contrast, 14d, a 6,7-difluro congener, displayed comparable
DAT inhibition (Ki = 2.2 M).
e-deficient heterocycles
other E-withdrawing groups
Chiral Me groups
[3.3.0]
[3.1.0]
piperazine
bioisosteres
F
N
N
other substituents
additional Ns
NC
N
other E-withdrawing groups
small heterocycles
larger ethers
OMe
8
alternate substituents
Figure 3. Optimization plan for mGlu7 PAM 8, surveying broad SAR.
acids 9 are reacted with TMS diazomethane to form methyl
esters 10 in 76-90% yield. Treatment of esters 10 with
DMF●DMA under microwave irradiation resulted in the
formation of amidines 11, which were carried on crude and
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F
F
1
2
3
4
5
6
7
8
9
10
F
F
F
F
N
N
N
N
N
N
N
N
N
N
R
CN
NC
F
NC
NC
NC
F
N
OR1
N
F
N
N
F
N
OMe
OMe
OMe
OMe
20
21
, R = H
, R = F
14a
14b
14c
14d
EC50 > 10 M
35% L-AP4 Max
DAT IC50 = 4 M
EC50 = 1.8 M
53% L-AP4 Max
DAT IC50 = 3 M
EC50 = 1.4 M
98% L-AP4 Max
DAT IC50 > 10 M
EC50 = 1.1 M
62% L-AP4 Max
DAT IC50 = 2.2 M
Cmpd
No.
18
R
R1
mGlu7 EC50
(M)
>10
mGlu7
%L-AP4 max
H
F
H
F
H
F
H
F
H
H
90
90
73
98
68
98
67
88
19
>10
1.5
1.1
>10
3.8
1.25
0.69
Figure 4. SAR of fluorinated analogs 14 of PAM 8.
20a
21a
20b
21b
20c
21c
CD3
CD3
CF2H
CF2H
i-Pr
11
12
13
14
15
16
17
18
19
20
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In parallel, we evaluated a wide-range of piperazine
bioisoteres, once again maintaining the 2-fluorophenyl moiety,
as well as the parent 8-methoxy quinolone core of 8, or the 6-
fluoro core of 14c. Here, we were surprised to find that only
the parent piperazine had mGlu7 PAM activity- all other analogs
17 were inactive (Figure 5). Similarly, alternatives to the 2-
fluorophenyl moiety in 8/14c proved inactive, further
narrowing the scope of the SAR of this PAM series.
i-Pr
20d
H
1.8
>10
1.3
67
23
74
85
70
71
21d
F
20e
H
H
H
O
O
O
F
N
(+)-20e
(-)-20e
21e
1.1
link
N
NC
H, F
1.1
N
OMe
0.51
F
O
17
20f
(+)-20f
(-)-20f
21f
H
1.3
1.9
88
77
89
76
O
O
O
O
H
H
F
Figure 5. Inactive piperazine bioisosteric analogs 17 of 8/14c.
These data turned our attention to the 8-OMe moiety as a
potential site of diversification as well as a potential metabolic
soft spot (e.g., oxidative dealkylation). Starting from either 8
or 14c, demethylation with BBr3 provided the corresponding
phenols 18/19 in good yields (Scheme 2). A microwave-
assisted alkylation reaction afforded the desired ether analogs
20/21 in 47-68% yield.
1.3
0.65
a Calcium mobilization assays with rat mGlu7/Gqi5-HEK cells performed in the
presence of an EC20 fixed concentration of L-AP4; values represent means from
one independent experiment performed in triplicate.
Scheme 2. Synthesis of 8-ether analogs 20 and 21.a
Structures and mGlu7 PAM activities of selected analogs
20/21 are highlighted in Table 1. The phenolic congeners 18
and 19 were inactive (EC50s > 10 M), whereas the –OCD3
analogs 20a/21a were equipotent to the parent proteo
compounds. PAM activity varies based on the presence or
absence of the 6-F moiety, as highlighted in 20b versus 21b and
20d versus 21d. Sterically bulky and/or lipophilic ethers were
generally inactive, save for the iospropoxy derivatives 20c
(EC50 = 1.3 M, 67% L-AP4 max) and 21c (EC50 = 0.69 M,
88% L-AP4 max). Incorporation of Lewis basic sites, as in the
case of tetrahydrofuranyl ethers 20e-f/21e-f showed potency
comparable to the parent PAMs. However, the racemic 20e and
21e were as potent and efficacious as the separated (chiral SFC)
single enantiomers (e.g., (rac)-20e compared to (+)-20e, (-)-20e
and (rac)-20f compared to (+)-20f, (-)-20f). Overall, very little
F
F
F
N
N
N
N
N
N
a
b
R
CN
R
CN
R
CN
N
N
N
OMe
OH
OR1
18
19
20
21
, R = H
, R = F
, R = H
, R = F
8
, R = H
14c
, R = F
aReagents and conditions: (a) BBr3, DCM, -78 C to rt, 16h, 54-76%; (b)
o
R1Br, K2CO3, MeCN, 150 oC (µW), 1 h, 47-68%.
Table 1. Structures and mGlu7 PAM activities of selected analogs 20/21.a
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texture to the SAR was observed, and the vast majority of
Page 4 of 18
1
2
3
4
5
6
7
8
analogs evaluated were inactive.
F
F
F
F
N
N
Scheme 3. Synthesis of 8-heterocyclic analogs 23.a
N
N
N
N
N
N
F
CN
F
CN
F
CN
F
CN
N
N
N
N
N
a
N
N
N
N
N
O
O
O
CN
CN
O
O
D3C
N
Br
20e
VU6027271
21a
21c
VU6027446
23g
Het
VU6027459
VU6027452
9
22
23
aReagents and conditions: (a) Pd2dba3, rac-BINAP, NaOtBu, toluene, 110
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58
59
60
Figure 6. mGlu7 PAM tool compound candidates 20e, 21a, 21c and 23g.
oC,16h, 32-61%.
At this point, we elected to perform deeper molecular
pharmacology (n = 3 on rat calcium and GIRK HEK lines) and
DMPK profiling (Table 3) of a selection of mGlu7 PAMs
identified thus far (to assess if any had the overall profile to be
useful as in vitro and/or in vivo tool compounds to probe
selective mGlu7 activation (Figure 6)). All four PAMs showed
good CNS penetration in rats, with Kps >4 and Kp,uus >0.79, no
DAT activity, and moderate predicted hepatic clearance in rats
(comparably high in mouse). All four were highly protein
bound in both rat and mouse, as well as in rat/mouse brain
homogenate binding. Of these, 21a (VU6027459) emerged as
the most attractive mGlu7 PAM for further profiling with good
potency (EC50s of 1.6 M and 0.99 M in Ca and GIRK,
respectively) and the best efficacy (116% and 72% for Ca and
GIRK activity, respectively) in both rat cell lines. Furthermore,
21a displayed ~1% free fraction (fu = 0.01) in both rat and
mouse, modest rat predicted hepatic clearance (CLHEP = 46.9
mL/min/kg) and favorable CNS exposure in rat (Kp = 4.78, Kp,uu
= 0.96) and mouse (Kp = 2.74, Kp,uu = 0.23). In a discrete IV/PO
PK study, 21a displayed a 7.5 hour half-life and good oral
bioavailability (69.5% F).39
Based on the activity of furans 20e/21e, we decided to
explore other saturated and aromatic heterocyclic groups in the
8-position via Buchwald-Hartwig chemistry (Scheme 3)
employing 22 to deliver analogs 23. As before, SAR was steep,
with few active mGlu7 PAMs (Table 2). Here, only a single
morpholine derivative, 23g, proved potent (EC50 = 1.3 M, 74%
L-AP4 max), and the importance of the Lewis basic oxygen was
pronounced, as the analogous piperidine 23e was inactive.
Table 2. Structures and mGlu7 PAM activities of selected analogs 23.a
F
N
N
CN
N
Het
23
Cmpd
No.
Het
mGlu7 EC50
mGlu7
%L-AP4 max
(M)
N
23a
23b
23c
>10
6.7
22
81
23
N
Table 3. In vitro Pharmacology, DMPK and plasma:brain level (PBL)
data for select mGlu7 PAMs 20e, 21a, 21c and 23g.
N
N
Property
MW
cLogP
TPSA
20e
21a
383
4.38
51.8
21c
23g
417
3.83
55.1
N
>10
N
418
3.98
61.1
408
5.22
52.8
N
N
23d
23e
23f
>10
>10
>10
22
24
22
In vitro
Pharmacologya
Rat Calcium Assay
EC50 (M)
pEC50±SEM
[%L-AP4 max±SEM]
Rat GIRK Assay
EC50 (M)
pEC50±SEM
[%L-AP4 max±SEM]
DAT (Ki/IC50) M
In vitro PK
parameters
Rat CLHEP
(mL/min/kg)
mouse CLHEP
(mL/min/kg),
N
N
1.6
5.81±0.1
84.8±17.1 116.6±12.3
1.6
5.80±0.1
0.83
6.08±0.11
82.0±3.6
1.3
5.89±0.13
75.7±14.1
1.0
0.99
0.75
0.71
6.15±0.14
29.5±7.1
>10
F
F
5.99±0.1
62.2±9.5
>10
6.00±0.1
62.8±3.4
>10
6.12±0.07
54.5±3.8
>10
N
O
23g
23h
1.3
7.1
74
84
N
54.0
75.8
46.9
84.4
50.7
76.2
58.6
76.2
O
a Calcium mobilization assays with rat mGlu7/Gqi5-HEK cells performed in the
presence of an EC20 fixed concentration of L-AP4; values represent means from
one independent experiment performed in triplicate.
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Rat fu (plasma)
0.03
[0.006]
0.02
[0.01]
0.01
[0.002]
0.01
[0.001]
0.005
[0.001]
0.004
[0.001]
0.01
[0.002]
0.01
[0.002]
CWL and CMN drafted/corrected the manuscript and directed the
[Rat fu (brain)]
Mouse fu (plasma)
[Mouse fu (brain)]
Rat PBL
(IV, 0.2 mg/kg)
Kp
1
2
3
4
science. CWR, JJK, MJW, JPW performed the chemical synthesis.
CWL and CMN oversaw the target selection and interpreted the
biological data. AH, YM, ALR and PJC performed the in vitro
molecular pharmacology studies. ALB performed the in vitro and in
vivo DMPK studies. All authors have given approval to the final
version of the manuscript.
4.36
0.79
4.78
0.96
4.99
1.00
6.84
1.24
5
Kp,uu
6
7
8
9
Mouse PBL
(IP, 10 mg/kg)
Kp
Acknowledgement
ND
ND
2.74
0.23
ND
ND
ND
ND
The authors would also like to thank the NIH (NIMH,
R01MH113543 to CMN and CWL), R01MH104158 (to CMN),
William K. Warren, Jr. and the William K. Warren Foundation who
funded the William K. Warren, Jr. Chair in Medicine (to C.W.L.)
and endowed the Warren Center for Neuroscience Drug Discovery.
Kp,uu
aND = not determined.
10
11
12
13
14
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17
18
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20
21
22
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Figure 7A highlights the mGlu7 PAM activity of 21a in our
rat Ca and GIRK lines, as well as human mGlu7, where 21a
displays comparable potency and efficacy and no agonist
ASSOCIATED CONTENT
activity; thus a pure mGlu7 PAM.
Importantly, 21a was
Supporting Information. General methods for the synthesis and
characterization of all compounds, and methods for the in vitro and
in vivo DMPK protocols and supplemental figures. This material
is available free of charge via the Internet at http://pubs.acs.org.
inactive (EC50 > 30 M; >10 μM at mGlu8) at the other mGlu
receptors (Figure 7B), and thus representing the first-in-class,
selective mGlu7 PAM. Beyond mGlu selectivity, 21a was
evaluated in a Eurofin Lead Profiling Screen of 68 GPCRs, ion
channels and transporters and found to possess no ancillary
pharmacology (no inhibition >50%@10 M) except for at
sigma 1 (77%@10 M, binding IC50 = 3.5 M).
ABBREVIATIONS
PAM, positive allosteric modulator; PBL, plasma:brain level; DMPK, drug
metabolism and pharmacokinetics; DAT, dopamine transporter
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Figure 7. Molecular pharmacology profile of mGlu7 PAM 21a
(VU6027459). A) mGlu7 PAM concentration-response curves on human
mGlu7, rat mGlu7 (calcium) and rat mGlu7 (GIRK). B) PAM concentration
response curves for 21a on mGlu1,2,3,4,5,8 showing no activity up to 30 M;
the exception was week PAM activity at mGlu8 above 10 μM.
In summary, by virtue of the first described ‘molecular
switch’ within a series of mGlu7 NAMs, the first-in-class mGlu7
PAMs were identified. Steep SAR in the optimization
campaign eventually led to the discovery of 21a
(VU06027459), a highly selective and CNS penetrant tool
compound, suitable for exploring the role of selective mGlu7
activation in vitro and in vivo. Results from ongoing in vivo
work in Rett models and other rodent disease models will be
reported in due course.
AUTHOR INFORMATION
Corresponding Authors
*(CWL). Phone: 1 615-322-8700. Fax: 1 615-936-4381. Email:
craig.lindsley@vanderbilt.edu. *(CMN). Phone: 1-615-343-4303. E-
mail: colllen.niswender@vanderbilt.edu
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Vansteenwegen, D.; Hermans, D.; van der Putten, H.; D’Hooge, R. Deficits
Author Contributions
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97x64mm (300 x 300 DPI)
ACS Paragon Plus Environment