Discovery, Synthesis, and Preclinical Characterization of N-(3-Chloro-4-fluorophenyl)-1H-pyrazolo[4,3-b]pyridin-3-amine (VU0418506), a Novel Positive Allosteric Modulator of the Metabotropic Glutamate Receptor 4 (mGlu4)

Article abstract of DOI:10.1021/acschemneuro.6b00035

The efficacy of positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 4 (mGlu₄) in preclinical rodent models of Parkinson's disease has been established by a number of groups. Here, we report an advanced preclinically characterized mGlu₄ PAM, N-(3-chloro-4-fluorophenyl)-1H-pyrazolo[4,3-b]pyridin-3-amine (VU0418506). We detail the discovery of VU0418506 starting from a common picolinamide core scaffold and evaluation of a number of amide bioisosteres leading to the novel pyrazolo[4,3-b]pyridine head group. VU0418506 has been characterized as a potent and selective mGlu₄ PAM with suitable in vivo pharmacokinetic properties in three preclinical safety species.

Full text of DOI:10.1021/acschemneuro.6b00035

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Article
Discovery, Synthesis and Pre-Clinical Characterization of N-(3-chloro-4-
fluorophenyl)-1H-pyrazolo[4,3-b]pyridin-3-amine (VU0418506), a novel positive
allosteric modulator of the metabotropic glutamate receptor 4 (mGlu4)
Darren W. Engers, Anna L. Blobaum, Rocco D. Gogliotti, Yiu-Yin Cheung, James M.
Salovich, Pedro M. Garcia-Barrantes, J. Scott Daniels, Ryan Morrison, Carrie K. Jones,
Matthew G. Soars, Xiaoliang Zhuo, Jeremy Hurley, John E. Macor, Joanne J. Bronson,
P. Jeffrey Conn, Craig W Lindsley, Colleen M Niswender, and Corey R. Hopkins
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00035 • Publication Date (Web): 13 Apr 2016
Downloaded from http://pubs.acs.org on April 20, 2016
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Discovery, Synthesis and Pre-Clinical Characterization of N-(3-
chloro-4-fluorophenyl)-1H-pyrazolo[4,3-b]pyridin-3-amine
(VU0418506), a novel positive allosteric modulator of the
metabotropic glutamate receptor 4 (mGlu4)
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Darren W. Engers†,€, Anna L. Blobaum†,€, Rocco D. Gogliotti†,€, YiuꢀYin Cheng†,€, James M.
Salovich†,€, Pedro M. GarciaꢀBarrantes€, J. Scott Daniels†,€, Ryan Morrison†,€, Carrie K. Jones†,€,
Matthew G. Soars§, Xiaoliang Zhuo§, Jeremy Hurley§, John E. Macor§, Joanne J. Bronson§, P.
Jeffrey Conn†,€,#, Craig W. Lindsley†,‡,€, Colleen M. Niswender†,€,#, Corey R. Hopkins†,‡,€,*
‡
€
†Department of Pharmacology, Department of Chemistry, Vanderbilt Center for Neuroscience
#
Drug Discovery, Vanderbilt Kennedy Center, Vanderbilt University, Nashville, TN 37232.
§Bristol-Myers Squibb Co., Research and Development, 5 Research Parkway, Wallingford, CT
06492.
Abstract: The efficacy of positive allosteric modulators (PAMs) of the metabotropic glutamate
receptor 4 (mGlu4) in preꢀclinical rodent models of Parkinson’s disease has been established by a
number of groups. Here, we report an advanced preꢀclinically characterized mGlu4 PAM, Nꢀ(3ꢀ
chloroꢀ4ꢀfluorophenyl)ꢀ1Hꢀpyrazolo[4,3ꢀb]pyridinꢀ3ꢀamine (VU0418506).
We detail the
discovery of VU0418506 starting from a common picolinamide core scaffold and evaluation of a
number of amide bioisosteres leading to the novel pyrazolo[4,3ꢀb]pyridine head group.
VU0418506 has been characterized as a potent and selective mGlu4 PAM with suitable in vivo
pharmacokinetic properties in three preclinical safety species.
Keywords: metabotropic glutamate receptor 4, mGlu4, CYP induction, Parkinson’s disease,
pyrazolo[4,3-b]pyridine
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INTRODUCTION
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Parkinson’s disease (PD) is a chronic movement disorder resulting from the loss of dopaminergic
neurons in the basal ganglia (BG).1 The four core motor symptoms of PD are tremor, rigidity,
bradykinesia, and postural instability, all of which can impact simple task completion as the
disease progresses. At present there is no cure for PD, and there are only medications that
provide symptomatic relief. Generally, afflicted individuals are given dopamine receptor
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agonists or levodopa (
L
ꢀDOPA) to combat motor symptoms, the latter in combination with
ꢀDOPA to reach the brain before being converted to dopamine.2
carbidopa, which allows
L
Unfortunately, not all symptoms are responsive to drug treatment (with rigidity and bradykinesia
responding the best), leaving a significant portion of the disease population poorly treated;
additionally, drug responsiveness wanes over disease progression.3 Other therapeutics can aid in
disease management (e.g., anticholinergics); however, a small, but significant, portion of PD
patients remain unresponsive to current treatment options. In certain individuals, deep brain
stimulation (DBS), a surgical procedure wherein electrodes are implanted within nuclei of the
BG, has been shown to have dramatic results.4 However, careful patient selection is an
important step for DBS success and only a small number of patients qualify for this surgery.
Thus, the treatment options for the symptomatic and diseaseꢀmodifying intervention for PD
remain limited, particularly as the disease progresses.
Motor function is controlled by the BG through two distinct pathways from the striatum –
the direct and indirect pathways.5,6 These pathways have opposite effects on motor activity –
activation of the direct pathway promotes movement and the indirect pathway inhibits it. In PD
patients, the balance between these two pathways is lost, leading to reduced inhibitory input to
the internal globus pallidus and substantia nigra pars reticulata via the direct pathway and an
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overall increase in the excitatory flow to these output nuclei via the polysynaptic indirect
pathway.5,6 Over the past several years, the metabotropic glutamate receptor 4 (mGlu4) has
gained attention as a potential target for pharmacological intervention to normalize the BG
circuitry in PD patients.7,8 This receptor is expressed presynaptically on terminals projecting
from the striatum to the globus pallidus external segment (GPe), the first synapse of the BG
indirect pathway (the “striatoꢀpallidal” synapse).9 Activation of mGlu4 at these terminals has
been shown to reduce inappropriate GABA release onto the GPe, normalizing motor output.5,6,9
Additionally, expression of mGlu4 in the striatum and other BG structures has been postulated to
be involved in positive responses on motor output induced by activation or potentiation of
mGlu4.
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Over the past several years, our laboratories and others have disclosed a series of
picolinamideꢀbased mGlu4 positive allosteric modulators (PAMs) (Figure 1).10 This scaffold has
proven to deliver potent and selective mGlu4 PAMs and several of these compounds have been
used in preclinical antiꢀParkinsonian animal models.11,12,13,14 Compounds 315,16 and 716,17 were
shown to reverse haloperidolꢀinduced catalepsy after systemic administration (administered
either subcutaneously (SC) or via an oral (PO) route). Within the picolinamide series of mGlu4
PAMs, we have extensively characterized compound 7 and have shown that this ligand is
effective when administered alone in reversing haloperidolꢀinduced catalepsy, forelimb
asymmetryꢀinduced by unilateral 6ꢀhydroxydopamine (6ꢀOHDA) lesions in the median forebrain
bundle, and deficits in attention induced by bilateral 6ꢀOHDA nigrostriatal lesions in rats.17 In
addition to activity when administered alone, 7 was shown to enhance the activity of preladenant
(an A2A antagonist previously in clinical trials for PD therapy) in the reversal of haloperidolꢀ
induced catalepsy.17 Lastly, when administered with an inactive dose of
LꢀDOPA, 7 potentiated
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the effects of LꢀDOPA in reversing forelimb asymmetry, suggesting that mGlu4 PAMs may also
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provide L
ꢀDOPA sparing activity.17 Unfortunately, the picolinamide scaffold suffered from poor
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stability in vivo due to, some extent, the instability of the amide bond. Thus, we initiated a
medicinal chemistry effort to find a suitable replacement for the picolinamide head group with
the goal of identifying a potent and selective mGlu4 PAM with improved pharmacokinetic
properties.
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O
O
O
N
S
O
O
O
N
H
O
N
N
N
Cl
Cl
N
Cl
N
H
H
1, 4PAM1
2, 4PAM2
O
O
Cl
O
S
O
N
H
N
N
N
Cl
H
H
3, VU0361737 (ML128)
hmGlu4 EC50 = 240 nM
4, VU0364439
hmGlu4 EC50 = 19.8 nM
O
H
N
N
O
O
O
N
O
Cl
N
N
Cl
N
Cl
H
H
5, VU0366037
hmGlu4 EC50 = 517 nM
6, VU0400195 (ML182)
hmGlu4 EC50 = 291 nM
O
N
N
Cl
H
7, VU0364770 (ML292)
hmGlu4 EC50 = 1100 nM
Figure 1. Previously disclosed mGlu4 PAMs from the picolinamide series.
RESULTS AND DISCUSSION
We have previously explored the SAR around the rightꢀhand portion of the molecule and
have identified several groups that are tolerated (amides, sulfonamides, imides).12ꢀ14 Thus, our
initial SAR optimization in this report started with the evaluation of known amide bioisosteres18
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while keeping the 2ꢀpyridine moiety intact, as our previous work has shown that this group is
optimal for activity (Table 1). We have previously evaluated the reduction of the carbonyl
group to the benzyl amine, the reversal of the amide, and the introduction of a cyclopropyl group
as a carbonyl replacement.14 However, each of these substitutions led to significant loss of
activity. Replacing the picolinamide moiety with the 2ꢀpyridyl sulfonamide, 8, led to a complete
loss of activity against mGlu4. To test the hypothesis that the planarity around this group needed
to be maintained, we next evaluated two cyclic amide bioisosteres.18 First, the 1,2,4ꢀoxadiazole
group was introduced, 9, but this replacement led to an inactive compound. Next, we introduced
the triazine moiety, 10, to keep the hydrogen bond donor group available as in the secondary
amide; however, this change led to an inactive compound as well. To introduce steric bulk to the
amide bond, we alkylated the secondary amide, 11; that, too, led to an inactive compound.
Lastly, an amino group was introduced to generate an internal hydrogen bond with the carbonyl
group, and this was a productive change (12, 578 nM). Taking into account our previous
attempts,14,16 as well as this current endeavor, it appears that very little change is tolerated around
the picolinamide portion of this scaffold.
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Table 1. Amide replacements.
mGlu4 pEC50 mGlu4 EC50 %GluMax
Cmpd
(± SEM)a
6.62c
(nM)a
240c
(± SEM)a,b
3
7
8
5.96c
1,100c
Inactived
46d
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<4.5
<4.5
<4.5
>30,000
15.3 ± 2.7
25.1 ± 2.7
16.1 ± 1.9
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>30,000
>30,000
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6.24 ± 0.03
578
88.1 ± 2.0
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aCalcium mobilization mGlu4 assay; values are average of n = 3. bAmplitude of response
in the presence of 30 ꢀM test compound, normalized to a standard compound, PHCCC
(100% GluMax). cData taken from Ref. 16. dn = 1.
Having established that the aminopicolinamide head group imparted significant mGlu4
PAM activity, we next examined other cyclized head groups to systematically explore the SAR
around this portion of the molecule. We first looked to cyclize the amide NH portion with the
pyridine moiety as this region was thought to participate in an internal hydrogen bond.
However, cyclizing this portion of the molecule, 13, led to an inactive compound, confirming the
previous results observed with alkylated compounds. Efforts were then directed at cyclization of
compounds while keeping the NH bond intact. The azabenzimidazole compound, 14, was
inactive; however, the 2ꢀpyridyl benzimidazole, 15, was active, although 5ꢀfold less potent than
7. A productive change was introduction of the benzoxazole head group, 16, which was
equipotent with 7, but 4ꢀfold less active than 3. The two quinazolinꢀ4ꢀone analogs were not well
tolerated (17 and 18), even though both compounds possessed the carbonyl and NH components
of the amide bond. Other 5ꢀ and 6ꢀmembered cyclized moieties that either kept the nitrogen of
the pyridine constant (20 and 22) or the heteroatom of the carbonyl (19 and 21), or both, 23,
were also not tolerated. However, when we contracted the 6ꢀmembered ring of 23 and
introduced an additional heteroatom in the form of an isoxazolo[4,5ꢀb]pyridine group19, we were
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pleased to see a significant improvement in mGlu4 PAM activity (24, EC50 = 339 nM). This
compound has the elements of the pyridine and carbonyl; however, it does not contain the
additional hydrogen bond found in the aminopyridine. Synthesis of a compound that retains an
NH in the form of the indazole group led to a potent PAM (25, EC50 = 67 nM). Interestingly,
this compound does not contain the nitrogen of the putative pyridine group as present in the
previous active compounds. Introduction of the pyrazolo[4,3ꢀb]pyridine group20 did not
significantly increase activity (26, EC50 = 68 nM). In addition, inclusion of a 4ꢀfluoro atom in
order to block oxidative metabolism in this position was also well tolerated. Overall, however,
these compounds represent a novel chemical scaffold for mGlu4 PAMs.
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Table 2. Aminopicolinamide replacements.
mGlu4 pEC50 mGlu4
EC50 %GluMax
(± SEM)a,b
Cmpd
(± SEM)a
(nM)a
<4.5
>30,000
14.4 ± 2.2
34.8 ± 7.3
13
<4.5
>30,000
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5.25 ± 0.07
6.04 ± 0.04
5645
915
113.6 ± 7.6
35.7 ± 1.8
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<5
>10,000
>30,000
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<4.5
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<4.5
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>30,000
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<4.5
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339
23.5 ± 2.6
106.5 ± 1.9
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aCalcium mobilization mGlu4 assay; values are average of n = 3. bAmplitude of
response in the presence of 30 ꢀM test compound, normalized to a standard compound,
PHCCC (100% GluMax).
Having identified replacement head groups for the picolinamide that were potent mGlu4
PAMs, we next determined if these compounds displayed improved pharmacokinetic properties
compared to the picolinamides 3 and 7 in a battery of in vitro assays (Table 3). Compounds 3
and 7, unsubstituted picolinamides, were unstable in liver microsomes with high intrinsic
clearance in rat that approached the rat hepatic blood flow rate.21,22 However, both compounds
displayed moderate free fraction in both rat and human plasma.22,23 Compounds were also
evaluated for their inhibition of the cytochrome P450 (CYP450) enzymes using a cocktail
approach in human liver microsomes as a firstꢀtier screen for potential drugꢀdrug interaction
liability. Each of these compounds was a potent inhibitor of CYP1A2 (3, 130 nM; 7, 550 nM),
but showed no significant activity against CYP2C9, CYP2D6 or CYP3A4 (>30 ꢀM). The
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aminopicolinamide compound, 12, showed a very similar profile to both 3 and 7, with high
predicted clearance in liver microsomes, moderate free fraction in rat and human plasma, and
potent inhibition of CYP1A2, but with inactivity against the other CYPs. Neither the
isoxazolo[4,5ꢀb]pyridine, 24, nor the indazole, 25, provided any improvement in the in vitro PK
properties; in fact, while each of these were more highly protein bound in rat and human, other
portions of their overall profile were comparable to the previous compounds profiled.
Significant improvement, however, was seen with the pyrazolo[4,3ꢀb]pyridine compound, 26.
Compound 26 had significantly lower intrinsic clearance in both rat (CLHEP = 12.1 mL/min/kg)
and human (CLHEP = 10.1 mL/min/kg) liver microsomes. In addition, 26 displayed moderate
free fraction in both rat (Fu = 0.028) and human (Fu = 0.017) plasma. 26 was a potent inhibitor
of CYP1A2 but was much less active against CYP2C9 (IC50 = 8.9 ꢀM) and CYP2D6 and
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CYP3A4 (>30 ꢀM).
To establish an in vitro/in vivo correlation (IV/IVC), we next evaluated both 24 and 26 in
in vivo PK experiments to assess clearance (Table 3). Both compounds demonstrated an
excellent correlation in rats between in vitro and in vivo parameters, with 24 showing
suprahepatic clearance (CLp = 117 mL/min/min) and a short halfꢀlife (t1/2 = 31 min), while 26
showed moderate clearance (CLp = 29 mL/min/kg) and a moderate halfꢀlife (t1/2 = 91 min) in rats
(Tables 3 and 4).24 To better understand the metabolic instability difference between 24 and 26,
we performed a metabolic softꢀspot analysis (Figure 2). Analysis of the two compounds
revealed that the major metabolite of 24 was N–O bond cleavage of the isoxazolo[4,5ꢀb]pyridine
moiety, whereas the major metabolite of 26 was oxoꢀdefluorination, albeit in low abundance.
Based on these in vitro and in vivo PK parameters, Compound 26, VU0418506, was chosen for
advancement.
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Table 3. In vitro and in vivo PK properties of selected compounds (predicted hepatic clearance, PPB,
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CYP inhibition).
Plasma Protein
Rat CLHEP Human CLHEP
(mL/min/kg)a
Binding (Fu)b
CYP450 IC50 (ꢀM)c
Rat IV (1 mg/kg)
CL
T1/2
Rat
Human
1A2
2C9
2D6
3A4
Cmpd
3
(mL/min/kg) (min)
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58.7
64.1
44
48.6
12.1
17.2
18.2
20.3
ND
16.7
10.1
0.024
0.019
0.024
0.004
0.009
0.028
0.023
0.021
0.026
0.002
0.009
0.017
0.13
0.55
0.2
<0.1
<0.1
<0.1
>30
>30
>30
>30
3.6
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
13.2
>30
7
12
24
25
26
117
71.7
29
30.6
144
91
8.9
aPredicted hepatic clearance based on intrinsic clearance in rat and human liver microsomes. bFu = fraction unbound. cIC50
determinations of major CYP enzymes in human liver microsomes using specific probe substrateꢀmetabolite pairs for each CYP.
ND = not determined.
OH
NH
Ring-opened
parent
N
Cl
H
N
O
N
Parent +1
oxygen
OH
O
N
N
H
Cl
N
N
Cl
H
N
OH
Cl
HN
N
Oxidative
defluorination
N
H
N
F
HN
N
N
F
HN
N
N
H
Cl
Hydroxylation
HO
N
Cl
H
N
Figure 2. Met ID in Rat Liver Microsomes
To further profile 26, we prepared >10 grams as outlined in Scheme 1. 3ꢀFluoroꢀ2ꢀ
formylpyridine, 27, was heated in the presence of hydrazine which led to the pyrazolo[4,3ꢀ
b]pyridine which was then brominated (2 M NaOH, Br2) yielding 3ꢀbromoꢀ1Hꢀpyrazolo[4,3ꢀ
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b]pyridine, 28, in 29% yield over the two steps. Next, the nitrogen was protected as the Boc
amide (Boc2O, DMAP, Et3N) in good yield (88%). Finally, the target compound 26 was
completed via a BuchwaldꢀHartwig amination of 29 and 30 (Pd2(dba)3, XꢀPhos, Cs2CO3)
followed by Boc deprotection (TFA, CH2Cl2) in good overall yield (70% for two steps).25,26
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Scheme 1. Synthesis of 26 (VU0418506).
We next evaluated 26 in a functional assay using the rat mGlu4 protein and demonstrated
that its activity was similar to that observed for the human receptor (pEC50 = 7.34 ± 0.04, 46
nM). To assess the selectivity of 26 for mGlu4 versus the other mGlu receptors, we performed
selectivity assays in foldꢀshift format for the eight mGlu receptors.14 26 was found to be
selective versus the Group I and Group II mGlu receptors (Figure 3 A-C, E) and against mGlu7
and mGlu8 (Figure 3, G and H), two of the Group III receptors. The only other mGlu receptor
that appears to functionally interact (with equipotency) with 26 is mGlu6 (Figure 3F), a receptor
primarily expressed in the retina. In addition to mGlu selectivity, 26 was tested against a wider
panel of GPCRs, enzymes, ion channels, nuclear hormone receptors and transporters (Suppl.
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Table 1) and was found to be inactive against all of the targets, except monoamine oxidaseꢀB
(MAOꢀB, 330 nM). A scan against a panel of 96 kinase targets (DiscoveRx, KINOMEscan,
www.discoverx.com) revealed no significant activity (<30%). To further deꢀrisk 26 for potential
advancement into preꢀclinical toxicological studies, we progressed it into a cardiac channel panel
IC50 screen (ChanTest Cardiac Channel PanelTM, www.chantest.com), and tested for activity
versus nine cardiac ion channels (including hERG); 26 was inactive up to 100 ꢀM against all
nine channels (Suppl. Table 2). Lastly, 26 was negative in a miniꢀAMES test (EuroFins,
formerly Ricerca; www.eurofins.com) against TA98 and TA100 with and without S9.
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Figure 3. Compound 26 potentiates the glutamate response in mGlu4 expressing cell lines (D) and is
inactive against other mGlu receptors, except mGlu6 (F).
Further in vivo PK profiling of 26 revealed that the compound had excellent oral
bioavailability in both rats (F = 95%) and dogs (F = 36%) (Table 4). In addition, evaluation of
26 in a nonꢀhuman primate (using a rhesus IV cassette) revealed a low clearance molecule (CLp
= 13 mL/min/kg) with a moderate halfꢀlife (t1/2 = 3 hrs) (Table 4). Lastly, a plasma:brain PO
snapshot study in rats showed substantial uptake of 26 in both the plasma and brain with a Kp
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(brain:plasma ratio (0 – 6 h)) of 2.10 (Table 4). A more detailed discussion of the CSF levels
and in vivo efficacy are detailed in the companion article, Niswender et al.
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Table 4. In vivo PK for lead compounds:
26
26, IV/PO PK in Beagle Dog
26, IV/PO PK in SD Rat
26, IV PK in Rhesus Monkey
PK Parameters (IV)
CLp (mL/min/kg)
Vss (L/kg)
Rat, 1 mg/kg
29
4.0
Dog, 1 mg/kg
Rhesus, Cassette
36
2.4
221
66
13
2.5
173
184
t
(min)
91
½
MRT (min)
132
AUC (hr·ng/mL)
608
515
Rat
3 mg/kg
PK Parameters (PO)
Dog, 3 mg/kg
10 mg/kg
C
T
(ng/mL)
(hr)
429
1
875
375
max
2
0.75
max
AUC PO (hr·ng/mL)
%F
1895
100
7418
95
556
36
Plasma:Brain (PO, 0 – 6 h, 10 mg/kg)
AUCplasma0ꢀ6
AUCbrain0ꢀ6
B:P
8771
18415
2.1
Before moving Compound 26 into extensive in vivo testing and chronic dosing
paradigms, we investigated the potential of the potent CYP1A2 inhibitory activity observed with
Compound 26 to affect dosing strategies. Activity against CYP1A2 is not surprising for these
compounds as it is well known that small polyꢀaromatic groups inhibit this enzyme.27 However,
as CYP1A2 is also the primary enzyme responsible for the metabolism of 26, we evaluated the
potential of 26 to autoꢀinduce CYP1A2 expression or activity in cryopreserved hepatocytes.28 26
was shown to be a potent inducer of CYP1A2 activity (EC50 = 9.8 ꢀM and Emax = 104 foldꢀ
induction) and this manifested in a multiꢀday pharmacokinetic study in rodents where, upon day
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4 of dosing, significantly reduced plasma levels of 26 were detected in both male and female
Sprague Dawley (SD) rats (15 mg/kg PO, 1x daily) (Supp. Figure 1). Total reductions in
plasma AUC in male SD rats were from 1148 ꢀM*hr (Day 1) to 200 ꢀM*hr (Day 4); similar
results were observed in female SD rats at the same dose. Therefore, due to this CYP1A2 autoꢀ
induction profile in rodents, 26 is not suitable for chronic dosing, but still remains a valuable tool
compound for acute studies as highlighted in our companion manuscript, Niswender et al.
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CONCLUSION
In summary, we report the discovery and characterization of a novel chemical scaffold as
an advanced preꢀclinical positive allosteric modulator of mGlu4, VU0418506 (26), the overall
profile of which is shown in Table 5. We have shown that VU0418506 is potent against both the
human (EC50 = 68 nM) and rat (EC50 = 46 nM) receptors and is selective against the other mGlu
receptors, except retinallyꢀrestricted mGlu6. While VU0418506 autoꢀinduced CYP1A2 activity
which limits its use in chronic dosing assays, it is anticipated that it will remain a valuable
research tool compound for acute dosing. Extensive in vivo PK studies show that VU0418506
displays good bioavailability in two preꢀclinical species (rat and dog) and is not a substrate for Pꢀ
glycoprotein (Pgp, Table 5). Further selectivity profiling of VU0418506 revealed that it is
selective against a number of kinases and other target families (GPCRs, ion channels, etc.).
Initial examination of potential toxicological endpoints showed that VU0418506 is clean against
a panel of ion channels associated with cardiac toxicity and it negative in a miniꢀAMES assay
with and without S9. Further in vivo examination of VU0418506 in a number of preꢀclinical
animal models of Parkinson’s disease and other CNS disease models is reported in our
companion manuscript.
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Table 5. Summary of 26 (VU0418506).
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26, VU0418506
68 nM
46 nM
15.5
262.7
3.96
Human mGlu4 EC50 (nM)
Rat mGlu4 EC50 (nM)
Rat Fold Shift (@ 30 ꢀM)
MW
cLogP
TPSA
53.6
Intrinsic Clearance (mL/min/kg)
Human CLHEP
Rat CLHEP
Dog CLHEP
Mouse CLHEP
Rhesus CLHEP
10.1
12.1
18.9
50.1
11.5
Plasma Protein Binding (%Fu)
Human
Rat
Dog
Mouse
Rhesus
CYP450 Inhibition (ꢀM)
1A2
2C9
2D6
3A4
2C19
1.7
2.8
0.9
1.9
1.4
<0.1
8.9
>30
>30
6.1
In vivo PK Parameters (Rat IV, 1 mg/kg)
CL (mL/min/kg)
Vss (L/kg)
AUC (ng·h/mL)
MRT (min)
29
4.0
608
132
91
T1/2 (min)
In vivo PK Parameters (Rat PO, 10 mg/kg)
Cmax (ng/mL)
Tmax (hr)
875
2
AUC PO (ng·h/mL) 7418
%F
95%
Rat CNS PO PBL (0 – 6 hr)
Plasma (ng·h/mL)
Brain (ng·h/mL)
B:P
8771
18415
2.1
24
14
Papp, AꢀB (10ꢀ6)
Papp, BꢀA (10ꢀ6)
Efflux Ratio
0.6
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METHODS
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General. All NMR spectra were recorded on a 400 MHz AMX Bruker NMR
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spectrometer. H and 13C chemical shifts are reported in δ values in ppm downfield with the
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deuterated solvent as the internal standard. Data are reported as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, b = broad, m = multiplet),
integration, coupling constant (Hz). Low resolution mass spectra were obtained on an Agilent
6120 or 6150 with ESI source. Method A: MS parameters were as follows: fragmentor: 70,
capillary voltage: 3000 V, nebulizer pressure: 30 psig, drying gas flow: 13 L/min, drying gas
temperature: 350 ºC. Samples were introduced via an Agilent 1290 UHPLC comprised of a
G4220A binary pump, G4226A ALS, G1316C TCC, and G4212A DAD with ULD flow cell.
UV absorption was generally observed at 215 nm and 254 nm with a 4 nm bandwidth. Column:
Waters Acquity BEH C18, 1.0 x 50 mm, 1.7 um. Gradient conditions: 5% to 95% CH3CN in
H2O (0.1% TFA) over 1.4 min, hold at 95% CH3CN for 0.1 min, 0.5 mL/min, 55 ºC. Method B:
MS parameters were as follows: fragmentor: 100, capillary voltage: 3000 V, nebulizer pressure:
40 psig, drying gas flow: 11 L/min, drying gas temperature: 350 ºC. Samples were introduced via
an Agilent 1200 HPLC comprised of a degasser, G1312A binary pump, G1367B HPꢀALS,
G1316A TCC, G1315D DAD, and a Varian 380 ELSD (if applicable). UV absorption was
generally observed at 215 nm and 254 nm with a 4 nm bandwidth. Column: Thermo Accucore
C18, 2.1 x 30 mm, 2.6 um. Gradient conditions: 7% to 95% CH3CN in H2O (0.1% TFA) over 1.6
min, hold at 95% CH3CN for 0.35 min, 1.5 mL/min, 45 ºC. High resolution mass spectra were
obtained on an Agilent 6540 UHD QꢀTOF with ESI source. MS parameters were as follows:
fragmentor: 150, capillary voltage: 3500 V, nebulizer pressure: 60 psig, drying gas flow: 13
L/min, drying gas temperature: 275 ºC. Samples were introduced via an Agilent 1200 UHPLC
comprised of a G4220A binary pump, G4226A ALS, G1316C TCC, and G4212A DAD with
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ULD flow cell. UV absorption was observed at 215 nm and 254 nm with a 4 nm bandwidth.
Column: Agilent Zorbax Extend C18, 1.8 µm, 2.1 x 50 mm. Gradient conditions: 5% to 95%
CH3CN in H2O (0.1% formic acid) over 1 min, hold at 95% CH3CN for 0.1 min, 0.5 mL/min, 40
ºC. For compounds that were purified on a Gilson preparative reversedꢀphase HPLC, the system
comprised of a 333 aqueous pump with solventꢀselection valve, 334 organic pump, GXꢀ271 or
GXꢀ281 liquid hander, two column switching valves, and a 155 UV detector. UV wavelength for
fraction collection was userꢀdefined, with absorbance at 254 nm always monitored. Method:
Phenomenex Axiaꢀpacked Luna C18, 30 x 50 mm, 5 µm column. Mobile phase: CH3CN in H2O
(0.1% TFA). Gradient conditions: 0.75 min equilibration, followed by user defined gradient
(starting organic percentage, ending organic percentage, duration), hold at 95% CH3CN in H2O
(0.1% TFA) for 1 min, 50 mL/min, 23 ºC. Solvents for extraction, washing and chromatography
were HPLC grade. All reagents were purchased from Aldrich Chemical Co. and were used
without purification.
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3-bromo-1H-pyrazolo[4,3-b]pyridine (28). A mixture of 3ꢀfluoroꢀ2ꢀformylpyridine,
27, (1.0 eq) and anhydrous hydrazine (8.0 eq) was heated to 110 °C. After 16h, the reaction was
cooled to rt and slowly poured onto ice water. After extracting with EtOAc (3 x), the collected
organic layers were washed with Brine (100 mL), dried (MgSO4), filtered and concentrated to
provide a dark brown oil. LCMS (method A): single peak (214 and 254 nm), RT = 0.102 min,
MS (ESI+) m/z = 120.2 [M + H]+. The crude residue was dissolved in 2 M NaOH (0.5 M) and a
solution of Br2 (1.0 eq) in 2 M NaOH (1.0 M) was added dropwise. After 3 h at rt, NaHSO3
(aqueous) (1 mL) was added followed by 4 N HCl (~1.0M). A solid precipitated formed and
was collected by filtration and air dried affording 28 as an offꢀwhite solid (29% yield over 2
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steps). LCMS (method A): single peak (214 and 254 nm), RT = 0.270 min, MS (ESI+) m/z =
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200.0 [M + H]+.
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tert-Butyl 3-bromo-1H-pyrazolo[4,3-b]pyridine-1-carboxylate (29). To a solution of
28 (1.0 eq), DMAP (10 mg) and Et3N (1.15 eq) in dry DMF (0.25 M) was added Boc2O (1.1 eq)
at rt. After 16 h at rt, the reaction was added to EtOAc: H2O (1:1). The separated organic layer
was washed with water (2x), brine, dried (MgSO4), filtered and concentrated to provide desired
product 29 (88% yield). Material was taken through without further purification. LCMS
(method A): single peak (214 and 254 nm), RT = 0.793 min, MS (ESI+) m/z = 298.2 [M + H]+.
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N-(3-chloro-4-fluorophenyl)-1H-pyrazolo[4,3-b]pyridin-3-amine (25): A mixture of
29 (1.0 eq), Cs2CO3 (2.0 eq), Pd2(dba)3 (5 mol%) and 2ꢀDicyclohexylphosphinoꢀ2′,4′,6′ꢀ
triisopropylbiphenyl (XPhos) (10 mol%) in a roundꢀbottomed flask were subjected to evacuation
and purging with nitrogen (3x). A solution of 3ꢀchloroꢀ4ꢀfluoroaniline, 30, (1.1 eq) in 1,4ꢀ
dioxane (0.1 M) was added. The reaction mixture was heated to 100 °C. After 12h, the reaction
was added to EtOAc:H2O (1:1) and the organic layer was separated. The aqueous layer was
reextracted with EtOAc (2x). The collected organic layers were washed with brine, dried
(MgSO4), filtered and concentrated to afford a crude solid. The material was taken through to
the next step. The crude residue was dissolved in DCM (0.1 M) at 0 °C and TFA (1.0 M) was
added. The ice bath was removed. After an additional 30 min, LCMS confirmed the loss of
starting material. The solvent was removed under reduced pressure and the residue was purified
by reverse phase liquid column chromatography (35ꢀ65% acetonitrile: water with 0.1%
trifluoroacetic acid) to yield 26 as an orange solid (70% over two steps). LCMS (method B):
single peak (214 and 254 nm), RT = 0.753 min, MS (ESI+) m/z = 263.1 [M + H]+; 1H NMR (400
MHz, d6ꢀDMSO): δ 9.55 (br s, 1H), 8.49 (s, 1H), 8.11ꢀ8.10 (m, 1H), 7.99 (d, J = 8.0 Hz, 1H),
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7.66 (m, 1H), 7.46 (m, 1H), 7.30 (dd, J = 9.2, 8.8 Hz, 1H). HRMS, calc’d for C12H9N4FCl [M +
H]+, 263.0500; found 263.0499.
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at DOI:
Chemical synthesis of all analogs, in vitro Pharmacology procedures, in vitro PK
methods, in vivo PK methods, Selectivity and initial toxicology evaluation of 26, 4ꢀday
in vivo PK study of 26, human hepatocyte induction methods and in vitro
biotransformation protocols.
AUTHOR INFORMATION
Corresponding Author
*Phone: 615ꢀ936ꢀ6892. Fax: 615ꢀ778ꢀ1414. Eꢀmail: corey.r.hopkins@vanderbilt.edu
Author Contributions
C.R.H. and C.W.L. oversaw and designed the chemistry. D.W.E., R.D.G., Y.Y.C., J.M.S., and
P.M.B. performed the synthetic chemistry work. A.L.B., J.S.D. and R.M. designed the
pharmacokinetic experiments. P.J.C. and C.M.N. designed and analyzed the in vitro
pharmacology experiments. C.R.H. and C.M.N. wrote the manuscript with input from all
authors.
ACKNOWLEDGMENTS
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The authors would like to thank the Michael J. Fox Foundation for Parkinson’s Research and
BristolꢀMyers Squibb for their support of the program to develop mGlu4 PAMs.
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REFERENCES
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progress in the development of mGluR4 positive allosteric modulators for the treatment
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For Table of Contents Use Only
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Discovery, Synthesis and Pre-Clinical Characterization of N-(3-chloro-4-fluorophenyl)-1H-
pyrazolo[4,3-b]pyridin-3-amine (VU0418506), a novel positive allosteric modulator of the
metabotropic glutamate receptor 4 (mGlu4)
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Darren W. Engers, Anna L. Blobaum, Rocco D. Gogliotti, YiuꢀYin Cheng, James M. Salovich, Pedro M.
GarciaꢀBarrantes, J. Scott Daniels, Ryan Morrison, Carrie K. Jones, Matthew G. Soars, Xiaoliang Zhuo,
Jeremy Hurley, John E. Macor, Joanne J. Bronson, P. Jeffrey Conn, Craig W. Lindsley, Colleen M.
Niswender, Corey R. Hopkins*
H
F
N
N
Cl
N
H
N
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VU0418506
hmGlu4 EC50 = 68 nM
rmGlu4 EC50 = 46 nM
B:P = 2.1
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