N-Acyl-N′-arylpiperazines as negative allosteric modulators of mGlu1: Identification of VU0469650, a potent and selective tool compound with CNS exposure in rats

Article abstract of DOI:10.1016/j.bmcl.2013.05.020

Development of SAR in an N-acyl-N′-arylpiperazine series of negative allosteric modulators of mGlu₁ using a functional cell-based assay is described in this Letter. Characterization of selected compounds in protein binding assays was used to aid in selecting VU0469650 for further profiling in ancillary pharmacology assays and pharmacokinetic studies. VU0469650 demonstrated an excellent selectivity profile and good exposure in both plasma and brain samples following intraperitoneal dosing in rats.

Full text of DOI:10.1016/j.bmcl.2013.05.020

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3713–3718
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Acyl-N0-arylpiperazines as negative allosteric modulators of mGlu1:
Identification of VU0469650, a potent and selective tool compound
with CNS exposure in rats
Kimberly M. Lovell a,b, Andrew S. Felts a,b, Alice L. Rodriguez a,b, Daryl F. Venable a,b
,
Hyekyung P. Cho a,b, Ryan D. Morrison a,b, Frank W. Byers a,b, J. Scott Daniels a,b
,
Colleen M. Niswender a,b, P. Jeffrey Conn a,b, Craig W. Lindsley a,b,c, Kyle A. Emmitte a,b,c,
⇑
a Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA
b Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Development of SAR in an N-acyl-N0-arylpiperazine series of negative allosteric modulators of mGlu1
using a functional cell-based assay is described in this Letter. Characterization of selected compounds
in protein binding assays was used to aid in selecting VU0469650 for further profiling in ancillary phar-
macology assays and pharmacokinetic studies. VU0469650 demonstrated an excellent selectivity profile
and good exposure in both plasma and brain samples following intraperitoneal dosing in rats.
Ó 2013 Elsevier Ltd. All rights reserved.
Article history:
Received 2 April 2013
Revised 29 April 2013
Accepted 7 May 2013
Available online 17 May 2013
Keywords:
Glutamate
GPCR
mGlu1
Allosteric modulator
CNS
Piperazine
L
-Glutamic acid (glutamate) is the major excitatory neurotrans-
An area within the mGlu allosteric modulator field that has gar-
nered substantial interest has been the design and application of
selective small molecule negative allosteric modulators (NAMs)
of mGlu1.3 Several tool compounds have been discovered during
recent years further establishing the link between mGlu1 antago-
nism and the potential treatment of CNS related disorders such
as addiction,4 anxiety,5 epilepsy,6 pain,5a,7 and psychotic disor-
ders.5a,8 Compounds 1–6 (Fig. 1) are representative of the mGlu1
NAM chemotypes that have been discovered to date and examples
of molecules with demonstrated efficacy in vivo. For example,
JNJ16259685 (1) was efficacious in a rat model of anxiety9 as well
as rat10 and squirrel monkey11 models of addiction. A-841720 (2)
was established as an analgesic in rat models of neuropathic pain12
and post-operative pain.13 Inhibition of nociceptive pain was ob-
served with 3 in rats14 and with YM298198 (4) in mice.15 Antipsy-
chotic activity as measured by acoustic prepulse inhibition models
was noted with 516 and 617 in rats and with 18 and 48 in mice. Also
of interest, recent research has identified a potential role for mGlu1
inhibition in the treatment melanoma18 and certain types of breast
cancer.19
mitter in the mammalian central nervous system (CNS). Glutamate
produces its effects through binding to both ionotropic and metab-
otropic glutamate receptors. The metabotropic glutamate recep-
tors (mGlus) belong to family
C of the G protein-coupled
receptors (GPCRs). Further classification of the mGlus discovered
to date has been according to their structure, preferred signal
transduction mechanisms, and pharmacology (Group I: mGlu1
and mGlu5; Group II: mGlu2 and mGlu3; Group III: mGlu4, mGlu6,
mGlu7, and mGlu8).1 Many of these receptors have attracted signif-
icant attention as promising targets for the treatment of a variety
of CNS related disorders. The design of drug-like compounds that
selectively target a specific mGlu has been complicated by the fact
that the orthosteric binding site of the mGlus is highly conserved.
An alternative strategy that has proven successful for overcoming
such selectivity hurdles has been the optimization of compounds
that modulate the function of the receptor through interaction
with an allosteric binding site.2
We have recently become interested in the discovery and devel-
opment of structurally novel mGlu1 NAMs with properties suitable
⇑
Corresponding author. Tel.: +1 615 936 8401; fax: +1 615 322 8577.
E-mail address: kyle.a.emmitte@Vanderbilt.edu (K.A. Emmitte).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.020

3714
K. M. Lovell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3713–3718
Figure 1. mGlu1 NAM compounds with in vivo efficacy.
for evaluation in rodent models of a variety of CNS disorders. A po-
tential starting point for a hit to lead optimization program focused
on mGlu1 NAMs was identified through cross screening during the
course of our recent work in the mGlu5 NAM field (Fig. 2).20 The
adamantyl carboxamides 7 and 8 were mGlu5 NAMs that were also
found to behave as mGlu1 NAMs in a functional cell based assay.21
Our mGlu1 assay measures the ability of the compound to block
the mobilization of calcium by an EC80 concentration of glutamate
in cells expressing human mGlu1.
One attractive feature of this scaffold was the fact that robust
chemistry would allow to us to rapidly vary three distinct portions
of the chemotype and evaluate considerable synthetic diversity
(Scheme 1). For instance, commercially available and enantiopure
piperazines 9 (R1 = CH3) were useful for evaluating substitution
of the piperazine core. For the purpose of evaluating SAR about
the aryl portion of the chemotype (R2), substituted piperazines 9
were reacted with adamantoyl chloride to afford amide intermedi-
ates 10. Removal of the carbamate protecting group under acidic
conditions gave amine intermediates 11. Target compounds were
accessible from amines 11 through nucleophilic aromatic substitu-
tion reactions with aryl fluorides or Buchwald–Hartwig22 amina-
tion reactions with suitable aryl halides.23 For evaluation of SAR
around the amide portion of the scaffold, a simple reorganization
of the synthetic transformations was required. Reaction of amines
9 with either 2-fluoropyridine or 2-bromopyridine under appropri-
ate conditions provided intermediate 12. Following cleavage of the
protecting group, amines 13 were readily converted to the target
amide compounds using well established methods.24
Scheme 1. Reagents and conditions: (a) 1-adamantoyl chloride, DIEA, CH2Cl2; (b)
HCl, MeOH, dioxanes; (c) R2F, NMP, wave, 250 °C, 10 min; (d) R2X (X = Cl, Br, or I),
l
Pd2(dba)3 or Pd(OAc)2, Xantphos, NaOtBu or Cs2CO3, dioxanes,
l wave, 120 °C,
10 min or 100 °C, 18 h; (e) R3COCl, DIEA, CH2Cl2; (f) R3CO2H, HATU, DIEA, CH2Cl2.
for one enantiomer versus the other in the case of both the
2-methyl and 3-methyl analogs. With the 2-methyl analogs a 15-
fold preference was observed for the R-enantiomer (15 vs 14),
while with the 3-methyl analogs a sixfold preference was observed
for the R-enantiomer (17 vs 16). Furthermore, both 15 and 17 were
approximately fourfold more potent than the unsubstituted com-
parator 7. Moreover, an equally substantial discovery was noted
upon testing of 15 and 17 in our functional mGlu5 assay.25 Both
compounds were only weak antagonists in this assay (IC50
>10 lM). In short order, we had moved from a hit compound with
little to no group I mGlu selectivity to two compounds with more
than 60-fold selectivity for mGlu1 versus mGlu5.
We next chose to evaluate SAR around the amide portion of the
chemotype in the context of both the (R)-2-methyl piperazine (ser-
ies I) and the (R)-3-methyl piperazine (series II) cores (Table 2). A
number of analogs (18–35) were prepared in an attempt to identify
replacements for the 1-adamantyl group. This was not generally
successful; however, there were some notable exceptions. In the
context of the (R)-2-methyl piperazine but not the (R)-3-methyl
piperazine, the cyclooctyl (26) and the trans-(tert-butyl)cyclohexyl
(30) groups demonstrated weak to moderate NAM activity against
mGlu1. Additionally, the bicyclo[3.3.1]nonan-3-yl (32) group was
essentially equipotent to 15, though this was again not effective
in the case of the (R)-3-methyl piperazine (33 vs 17). It has been
reported that the cubyl group can function as an effective and less
lipophilic isosteric replacement for the adamantyl group.26 Unfor-
tunately, such a modification was not effective in our chemotype
(34 and 35).
Evaluation of methyl substituted piperazines was one of our
first areas of investigation (Table 1). New compounds were pre-
pared with the 2-pyridyl group found in hit 7 (14–17). Evaluation
of these enantiopure analogs yielded some interesting results. A
preference with regard to the mGlu1 NAM activity was observed
Our attention next moved from the amide to the aryl portion of
the template (Table 3). Using an approach similar to that previ-
ously described, we again observed only weak antagonists (37,
41, 43, 45) and a compound (39) with no activity at the top concen-
tration tested (30 lM) in the context of the (R)-3-methyl pipera-
zine. Fortunately, the same was not observed with the (R)-2-
methyl piperazine analogs. Though 4-pyridyl analog 38 demon-
strated only weak mGlu1 NAM activity, 3-pyridyl analog 36 exhib-
ited good potency. Good to moderate potency was also noted with
Figure 2. mGlu1 NAM starting points for hit to lead optimization.

K. M. Lovell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3713–3718
3715
Table 1
Chiral piperazine analogs
a
Compd
R1
R2
mGlu1 pIC50 ( SEM)
mGlu1 IC50 (nM)
% Glu maxa,b ( SEM)
7
H
H
H
H
6.20 0.21
5.62 0.18
6.79 0.15
6.01 0.13
6.78 0.14
630
2420
161
988
166
1.1 0.8
ꢀ1.4 2.5
2.5 0.5
1.8 1.0
2.0 0.4
14
15
16
17
(S)-CH3
(R)-CH3
H
(S)-CH3
(R)-CH3
H
a
Calcium mobilization mGlu1 assay; values are average of n P 3.
b
Amplitude of response in the presence of 30
lM test compound as a percentage of maximal response (100
lM glutamate); average of n P 3.
pyrimidines 40 and 44 and pyrazine 42. Finally, the 2-cyanophenyl
analog 46 had potency within twofold of that observed with 15 and
represented an improvement over the unsubstituted comparator
piperazine 8 (Fig. 2).
Having seen little progress in identifying improved analogs with
the (R)-3-methyl piperazine core, we chose to focus our remaining
efforts on attempting to improve upon the results observed with
(R)-2-methyl piperazine analogs 15 and 46 (Table 4). Knowing that
very subtle structural modifications can dramatically affect activity
with allosteric modulators, we prepared fluorinated analogs of 15
(48–50). Though all three compounds exhibited good potency,
6-fluoropyridine 48 was the most potent and only slightly less po-
tent than 15. We also prepared heteroaryl analogs (51–54) of 46 as
a means to reduce lipophilicity. Most of these derivatives demon-
strated good (51 and 53) to moderate (52) potency; however, ana-
log 54 represented a nearly threefold improvement relative to 46
and the most potent compound from within the chemotype iden-
tified to date.
Calculation of the ligand-lipophilicity efficiency (LLE) values for
our most potent analogs identified analog 54 as the compound
with the best balance of potency and lipophilicity, two factors
linked to drug-likeness (Table 5).27 We also decided to examine
Table 2
Amide SAR
% Glu maxa,b ( SEM)
a
Compd
Series
R
mGlu1 pIC50 ( SEM)
mGlu1 IC50 (nM)
15
17
I
II
6.79 0.15
6.78 0.14
161
166
2.5 0.5
2.0 0.4
18
19
I
II
<4.5
<4.5
>30,000
>30,000
—
—
20
21
I
II
<4.5
<4.5
>30,000
>30,000
—
—
22
23
I
II
<5.0c
<4.5
>10,000
>30,000
51.8 3.3
—
24
25
I
II
<5.0c
<5.0c
>10,000
>10,000
29.9 2.1
62.6 3.3
26
27
I
II
5.18 0.06
<5.0c
6630
>10,000
0.0 8.2
52.9 3.3
28
29
I
II
<5.0c
<4.5
>10,000
>30,000
32.1 3.1
—
30
31
I
II
5.44 0.08
<5.0c
3610
>10,000
0.3 0.4
44.1 3.7
32
33
I
II
6.78 0.11
<5.0c
167
>10,000
1.3 0.2
12.2 10.6
34
35
I
II
<5.0c
<4.5
>10,000
>30,000
62.7 9.6
—
a
b
c
Calcium mobilization mGlu1 assay; values are average of n P 3.
Amplitude of response in the presence of 30
CRC does not plateau.
lM test compound as a percentage of maximal response (100
lM glutamate); average of n P 3.

3716
K. M. Lovell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3713–3718
Table 3
First generation aryl SAR
% Glu maxa,b ( SEM)
a
Compd
Series
R
mGlu1 pIC50 ( SEM)
mGlu1 IC50 (nM)
15
17
I
II
6.79 0.15
6.78 0.14
161
166
2.5 0.5
2.0 0.4
36
37
I
II
6.22 0.06
<5.0c
596
>10,000
1.8 0.2
32.3 5.2
38
39
I
II
<5.0c
<4.5
>10,000
>30,000
49.2 4.5
—
40
41
I
II
6.15 0.07
<5.0
714
>10,000
0.9 0.4
17.0 5.7
42
43
I
II
5.93 0.08
<5.0
1190
>10,000
ꢀ0.3 0.8
34.0 5.1
44
45
I
II
5.88 0.07
<5.0
1330
>10,000
ꢀ1.0 2.2
34.7 7.2
46
47
I
II
6.58 0.06
5.61 0.09
264
2460
2.1 0.3
5.3 1.7
a
Calcium mobilization mGlu1 assay; values are average of n P 3.
b
c
Amplitude of response in the presence of 30
CRC does not plateau.
lM test compound as a percentage of maximal response (100
lM glutamate); average of n P 3.
these same compounds for their propensity to non-specifically
bind to rat plasma proteins.28 Binding to proteins can limit the
amount of drug available to interact with the target, and thus im-
pact efficacy in an in vivo setting. Since we are primarily interested
in CNS applications of an mGlu1 NAM, we also chose to further pro-
file interesting compounds by measuring their binding to rat brain
homogenates. Not surprisingly, the measured fraction unbound
tracked well with the calculated logP values. In the instances
where brain homogenate binding was measured, there was re-
duced fraction unbound in brain homogenates relative to plasma.
Our experience is that such a relationship between the unbound
fraction in brain homogenates versus plasma is often but not
always the case. Gratifyingly, the most potent analog 54 also dem-
onstrated an increased fraction unbound compared to most of the
other compounds tested.
and a half-life of approximately 1 hour. We reasoned that such
a clearance profile was supportive of further studies and pro-
ceeded to evaluate the compound in a plasma-brain level exper-
iment using intraperitoneal (IP) dosing. Given the moderate to
high clearance observed in the IV experiment, the IP dosing
route was chosen in an attempt to maximize exposure. The
30 min post-dose time point was selected as it would be a rele-
vant time point for many behavioral experiments of interest. We
were pleased to see that the CNS penetration for VU0469650
was excellent and that the absolute levels in the brain were
quite good. Taking into account the aforementioned brain
homogenate binding data, we estimate the unbound fraction in
the brain to be 32 nM. If that is indeed the case, a threefold in-
crease in dose (30 mg/kg) should provide unbound drug levels
near the functional IC50 for mGlu1.
Compound 54 (VU0469650) was also examined in cell based
functional assays for its selectivity against the other members of
the mGlu family and was determined to be inactive against
In conclusion, we have identified a new mGlu1 NAM tool com-
pound from within a series of N-acyl-N0-arylpiperazines using a hit
to lead optimization plan initiated from a cross screening hit.
VU0469650 can be prepared using a simple three step procedure
and readily available starting materials. The compound is also po-
tent and selective with excellent CNS penetration in rats. Future
plans include evaluation of the compound in rodent behavioral as-
says associated with CNS disorders linked to mGlu1 signaling. Re-
sults and observations from such studies will be the subject of
future communications.
mGlu2–8 at 10 l
M.29 Additionally, we tested the compound in a
functional cell based assay against rat mGlu1 and found little
difference across species.30 With over 100-fold selectivity against
the other mGlus, we also wanted to assess the selectivity of the
compound against
a wider variety of targets. As such, we
submitted the compound to a commercially available radioligand
binding assay panel of 68 clinically relevant GPCRs, ion channels,
kinases, and transporters,31 and no significant responses were
found at a concentration of 10
l
M.32
Acknowledgments
Given the results summarized herein, we chose to evaluate
VU0469650 in a rat pharmacokinetic study (Table 6).33 In order
to understand the rate of metabolism in vivo, a study was con-
ducted using intravenous (IV) dosing. VU0469650 exhibited
moderate to high clearance, a moderate volume of distribution,
We thank Seaside Therapeutics (VUMC36176) for their support
of our programs in the development of mGlu1 NAMs. We also
thank Tammy S. Santomango for technical contributions with the
protein binding assays.

K. M. Lovell et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3713–3718
3717
Table 4
Second generation aryl SAR
a
Compd
R
mGlu1 pIC50 ( SEM)
mGlu1 IC50 (nM)
% Glu maxa,b ( SEM)
15
6.79 0.15
161
255
414
712
2.5 0.5
2.0 0.4
1.3 0.9
2.4 0.7
48
49
50
6.59 0.11
6.38 0.07
6.15 0.05
46
51
6.58 0.06
6.46 0.14
264
344
2.1 0.3
3.3 1.0
52
53
5.86 0.04
1400
ꢀ0.8 3.3
6.40 0.06
7.01 0.03
398
99
0.3 2.2
2.8 0.3
54
a
Calcium mobilization mGlu1 assay; values are average of n P 3.
b
Amplitude of response in the presence of 30
lM test compound as a percentage of maximal response (100 lM glutamate); average of n P 3.
Table 6
Table 5
Rat PK results for 54 (VU0469650)
Protein binding results
Compd
cLogPa
mGlu1 IC50 (nM)
LLEb
Rat PPBc (Fu)
Rat BHBc (Fu)
15
17
46
48
49
51
53
54
4.49
4.49
4.76
4.74
4.65
4.22
3.51
3.60
161
166
264
255
414
344
398
99
2.30
2.29
1.82
1.85
1.73
2.24
2.89
3.41
0.016
0.017
0.003
0.006
0.012
0.016
0.081
0.071
0.011
0.008
—
—
—
—
—
0.016
IV PKa,c
IP PKb,c
Dose
Half-life
MRT
CL
0.2 mg/kg
55 min
43 min
52 mL/min/kg
2.2 L/kg
Dose
10 mg/kg
30 min
509 ng/mL
727 ng/mL
1.4
Time point
Plasma concd
Brain concd
B/P ratio
a
b
c
Calculated using ADRIANA. Code (www.molecular-networks.com).
LLE (ligand-lipophilicity efficiency) = pIC50—cLogP.
Fu = fraction unbound.
VSS
a
b
c
Formulation = 10% EtOH, 40% PEG400, 50% DMSO.
Formulation = 10% Tween80 in water.
Male Sprague–Dawley rats (n = 2).
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Moreland, R. B.; Brioni, J. D.; Stewart, A. O. Bioorg. Med. Chem. Lett. 2006, 16,
4936.
13. Zhu, C. Z.; Baker, S.; El-Kouhen, O.; Lehto, S.; Hollingsworth, P. R.; Gauvin, D.
M.; Hernandez, G.; Zheng, G. Z.; Chang, R.; Moreland, R. B.; Stewart, A. O.;
Brioni, J. D.; Honore, P. Eur. J. Pharmacol. 2008, 580, 314.
14. Mantell, S. J.; Gibson, K. R.; Osborne, S. A.; Maw, G. N.; Rees, H.; Dodd, P. G.;
Greener, B.; Harbottle, G. W.; Million, W. A.; Poinsard, C.; England, S.; Carnell,
P.; Betts, A. M.; Monhemius, R.; Prime, R. L. Bioorg. Med. Chem. Lett. 2009, 19,
2190.
365.2341; found 365.2344. ½a D25
ꢀ45.7° (c = 6.0, MeOH).
ꢂ
24. Synthesis of 17 is representative of route II. (R)-4-N-Boc-2-methyl piperazine
(490 mg, 2.44 mmol), 2-bromopyridine (257
(212 mg, 0.366 mmol), Pd2(dba)3 (112 mg, 0.112 mmol), and NaOtBu
(705 mg, 7.34 mmol) were added to 5.0 mL of dioxanes in microwave
lL, 2.69 mmol), Xantphos
a
reaction vial. The reaction was heated for 10 min in the microwave at 120 °C
and monitored by LCMS. Upon completion, the mixture was cooled and filtered
through a pad of celite and concentrated in vacuo. The crude material was
purified by flash chromatography and immediately dissolved in 1.0 mL of
MeOH with stirring. 4 N HCl in dioxanes (4.0 mL, 16 mmol) was added. Once
the protecting group had cleaved as judged by LCMS, an aqueous solution of
saturated sodium bicarbonate was added until the pH was basic. The mixture
was extracted with dichloromethane, and the organic layers were
concentrated in vacuo to afford 260 mg (60%) of (R)-2-methyl-1-(pyridin-2-
15. Kohara, A.; Toya, T.; Tamura, S.; Watabiki, T.; Nagakura, Y.; Shitaka, Y.;
Hayashibe, S.; Kawabata, S.; Okada, M. J. Pharmacol. Exp. Ther. 2005, 315, 163.
16. Satoh, A.; Nagatomi, Y.; Hirata, Y.; Ito, S.; Suzuki, G.; Kimura, T.; Maehara, S.;
Hikichi, H.; Satow, A.; Hata, M.; Ohta, H.; Kawamoto, H. Bioorg. Med. Chem. Lett.
2009, 19, 5464.
17. Ito, S.; Hirata, Y.; Nagatomi, Y.; Satoh, A.; Suzuki, G.; Kimura, T.; Satow, A.;
Maehara, S.; Hikichi, H.; Hata, M.; Ohta, H.; Kawamoto, H. Bioorg. Med. Chem.
Lett. 2009, 19, 5310.
18. (a) Martino, J. J.; Wall, B. A.; Mastrantoni, E.; Wilimczyk, B. J.; La Cava, S. N.;
Degenhardt, K.; White, E.; Chen, S. Oncogene 2012. http://dx.doi.org/10.1038/
onc.2012.471. advance online publication; (b) Ohtani, Y.; Harada, T.; Funasaka,
Y.; Nakao, K.; Takahara, C.; Abdel-Daim, M.; Sakai, N.; Saito, N.; Nishigori, C.;
Aiba, A. Oncogene 2008, 27, 7162; (c) Namkoong, J.; Shin, S.-S.; Lee, H. J.; Marín,
Y. E.; Wall, B. A.; Goydos, J. S.; Chen, S. Cancer Res. 2007, 67, 2298.
19. Speyer, C. L.; Smith, J. S.; Banda, M.; DeVries, J. A.; Mekani, T.; Gorski, D. H.
Breast Cancer Res. Treat. 2012, 132, 565.
yl)piperazine as
(260 mg, 1.47 mmol), 1-adamantoyl chloride (437 mg, 2.20 mmol), and DIEA
(508 L, 2.93 mmol) were dissolved in 1.0 mL of dichloromethane and
a colorless liquid. (R)-2-Methyl-1-(pyridin-2-yl)piperazine
l
monitored by LCMS. Upon completion, 5.0 mL of MeOH was added, and the
mixture was concentrated in vacuo. The crude product was purified via reverse
phase chromatography (0.1% TFA in H2O/MeCN). The fractions containing the
product were combined and neutralized by the addition of aqueous saturated
sodium bicarbonate. The mixture was extracted with dichloromethane, and
the organic layers were combined and concentrated in vacuo to afford 200 mg
(40% yield) of the product as a white solid. 1H NMR 1H NMR (400 MHz, DMSO-
d6) d 8.12–8.07 (m, 1H), 7.52 (t, J = 8.0 Hz, 1H), 6.79 (Rotamer 1, d, J = 8.6, 0.65
H) 6.73 (Rotamer 2, d, J = 8.6 Hz, 0.35 H), 6.64–6.59 (m, 1H), 4.64 (Rotamer 1,
m, 0.65 H), 4.49 (Rotamer 2, m, 0.35 H), 4.4–4.22 (m, 1H), 4.16–3.95 (m, 2H),
3.27 (m, 2.35 H), 2.80–2.69 (m, 0.65H), 2.02–1.85 (m, 9H), 1.75–1.62 (m, 6H),
1.12 (Rotamer 1, d, J = 6.6 Hz, 2H), 0.95 (Rotamer 2, d, J = 6.6 Hz, 1H); HRMS
(TOF, ES+) C21H30N3O [M+H]+ calculated 340.2389; found 340.2389. a 2D5
½ ꢂ
20. Rodriguez, A. L.; Williams, R.; Zhou, Y.; Lindsley, S. R.; Le, U.; Grier, M. D.;
Weaver, C. D.; Conn, P. J.; Lindsley, C. W. Bioorg. Med. Chem. Lett. 2009, 19, 3209.
21. HEK 293A TREx cells stably expressing human mGlu1 were plated in black-
ꢀ20.2° (c = 6.0, MeOH).
25. HEK293A cells expressing rat mGlu5 were cultured and plated. The cells were
loaded with a Ca2+ sensitive fluorescent dye and the plates were washed and
placed in the Functional Drug Screening System (Hamamatsu). Test compound
was applied to cells 3 s after baseline readings were taken. Cells were
incubated with the test compounds for 140 s and then stimulated with an
EC20 concentration of glutamate; 60 s later an EC80 concentration of agonist
was added and readings taken for an additional 40 s. Allosteric modulation by
the compounds was measured by comparing the amplitude of the responses at
the time of glutamate addition plus and minus test compound. For a more
detailed description of the assay, see Sharma, S.; Rodriguez, A. L.; Conn, P. J.;
Lindsley, C. W. Bioorg. Med. Chem. Lett. 2008, 18, 4098.
walled, clear-bottomed, poly-D-lysine coated 384-well plates in 20 lL of assay
medium (DMEM containing 10% dialyzed FBS, 20 mM HEPES, and 1 mM
sodium pyruvate) at a density of 20 K cells/well. The cells were induced with
50 ng/mL tetracycline and grown overnight at 37 °C in the presence of 5% CO2.
The next day, medium was exchanged to assay buffer (Hank’s balanced salt
solution, 20 mM HEPES, and 2.5 mM probenecid) using an ELX405 microplate
washer, leaving 20
(2.3 M final) indicator dye (prepared as a DMSO stock and mixed in a 1:1 ratio
with pluronic acid F-127) in assay buffer, and incubation for 45 m at 37 °C. The
dye was then exchanged to assay buffer using an ELX405, leaving 20 L/well.
lL/well, followed by addition of 20 lL/well 2ꢁ Fluo-4 AM
l
l
26. Gunosewoyo, H.; Guo, J. L.; Bennett, M. R.; Coster, M. J.; Kassiou, M. Bioorg. Med.
Chem. Lett. 2008, 18, 3720.
27. Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881.
28. Binding to rat plasma proteins and brain homogenates were measured using
equilibrium dialysis according to methods similar to those described in
Kalvass, J. C.; Maurer, T. S. Biopharm. Drug Dispos. 2002, 23, 327.
29. mGlu selectivity assays are described in Noetzel, M. J.; Rook, J. M.; Vinson, P. N.;
Cho, H.; Days, E.; Zhou, Y.; Rodriguez, A. L.; Lavreysen, H.; Stauffer, S. R.;
Niswender, C. M.; Xiang, Z.; Daniels, J. S.; Lindsley, C. W.; Weaver, C. D.; Conn,
P. J. Mol. Pharmacol. 2012, 81, 120.
Ca2+ flux was measured using the Functional Drug Screening System
(FDSS7000, Hamamatsu, Japan). Compounds were serially diluted 1:3 into 10
point concentration response curves and transferred to daughter plates using
the Echo acoustic plate reformatter. Compounds were diluted into assay buffer
to a 2ꢁ stock using a Thermo Fisher Combi which was applied to cells at t = 3 s.
Cells were incubated with the test compounds for 2.3 min and then stimulated
with an EC20 concentration of glutamate; 1.9 min later an EC80 concentration of
glutamate was added and readings taken for an additional 1.7 min. Data were
collected at 1 Hz. Concentration response curves were generated using a four
point logistical equation with XLfit curve fitting software for Excel.
30. Analogous to the method described in Ref.21 with the exception that HEK 293A
TREx cells stably expressing rat mGlu1 were used.
22. (a) Surry, D. S.; Buchwald, S. L. Angew. Chem., Int. Ed. 2008, 47, 6338; (b)
Schlummer, B.; Scholz, U. Adv. Synth. Catal. 2004, 346, 1599.
31. LeadProfilingScreenÒ, Eurofins Panlabs, Inc. http://www.eurofinspanlabs.com.
32. Significant responses are defined as those that inhibited more than 50% of
radioligand binding. In the case of VU0469650, no inhibition greater than 41%
23. Synthesis of 54 (VU0469650) is representative of route I. (R)-4-N-Boc-2-methyl
piperazine (665 mg, 3.32 mmol), 1-adamantoyl chloride (990 mg, 4.98 mmol),
and DIEA (1.15 mL, 6.64 mmol) were dissolved in 2.0 mL of dichloromethane,
and the reaction was monitored by LCMS. Upon completion, 5.0 mL of MeOH
was added, and the mixture was concentrated in vacuo. The residue was
purified by flash chromatography. The purified product was immediately
dissolved in 1.0 mL of MeOH and excess 4 N HCl in dioxanes (4.0 mL, 16 mmol).
Once the protecting group had cleaved as judged by LCMS, an aqueous solution
of saturated sodium bicarbonate was added until the pH was basic. The
mixture was extracted with dichloromethane, and the organic layers were
concentrated in vacuo to afford 814 mg (94% yield) of adamantan-1-yl((R)-2-
(adenosine A1 and sigma r1) was observed.
33. Male Sprague–Dawley rats weighing between 250 and 300 g were purchased
from Harlan Laboratories (Indianapolis, IN). VU0469650 was used as its mono-
HCl salt for these studies. For the IV study, cannulated animals with catheters
surgically implanted in the carotid artery and jugular vein were used. Blood
collections via the carotid artery were performed at 0.033, 0.117, 0.25, 0.5, 1, 2,
4, 7, and 24 h after administration via the jugular vein catheter. For the IP
study, rats were euthanized and decapitated 30 min after compound
administration, and both blood and brain samples were collected. Following
protein precipitation, the supernatants of all plasma and brain homogenate
samples were analyzed by means of LC–MS/MS. All animal studies were
approved by the Vanderbilt University Medical Center Institutional Animal
Care and Use Committee.
methylpiperazin-1-yl)methanone as
a white solid. Adamantan-1-yl((R)-2-
methylpiperazin-1-yl)methanone (355 mg, 1.35 mmol) and 2-cyano-3-
fluoropyridine (1.42 mL, 5.41 mmol) were dissolved in 4.0 mL of NMP in a
microwave reaction vial and heated in the microwave for 10 min at 250 °C. The