Discovery, synthesis, and structure-activity relationship development of a series of N-(4-acetamido)phenylpicolinamides as positive allosteric modulators of metabotropic glutamate receptor 4 (mGlu4) with CNS exposure in rats

Article abstract of DOI:10.1021/jm101271s

Herein we report the discovery, synthesis, and evaluation of a series of N-(4-acetamido)-phenylpicolinamides as positive allosteric modulators of mGlu₄. Compounds from the series show submicromolar potency at both human and rat mGlu₄. In addition, pharmacokinetic studies utilizing subcutaneous dosing demonstrated good brain exposure in rats.

Full text of DOI:10.1021/jm101271s

1106 J. Med. Chem. 2011, 54, 1106–1110
DOI: 10.1021/jm101271s
Discovery, Synthesis, and Structure-Activity Relationship Development of a Series of
N-(4-Acetamido)phenylpicolinamides as Positive Allosteric Modulators of Metabotropic
Glutamate Receptor 4 (mGlu4) with CNS Exposure in Rats
Darren W. Engers,†,‡,1 Julie R. Field,† Uyen Le,† Ya Zhou,†,‡ Julie D. Bolinger,† Rocio Zamorano,†,‡ Anna L. Blobaum,†,‡
Carrie K. Jones,†,‡,§ Satyawan Jadhav,†,‡ C. David Weaver,†,^ P. Jeffrey Conn,†,‡,^ Craig W. Lindsley,†, ,‡,^,1
Colleen M. Niswender,*,†,‡ and Corey R. Hopkins*,†,‡,1
†Department of Pharmacology, ‡Vanderbilt Program in Drug Discovery, Vanderbilt University Medical Center, Nashville, Tennessee 37232,
United States, §Tennessee Valley Healthcare System, U.S. Department of Veterans Affairs, Nashville, Tennessee 37212, United States,
Department of Chemistry, ^Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232, United States, and
1Vanderbilt Specialized Chemistry Center for Accelerated Probe Development (MLPCN), Nashville, Tennessee 37232, United States
Received October 12, 2010
Herein we report the discovery, synthesis, and evaluation of a series of N-(4-acetamido)-phenylpico-
linamides as positive allosteric modulators of mGlu4. Compounds from the series show submicromolar
potency at both human and rat mGlu4. In addition, pharmacokinetic studies utilizing subcutaneous
dosing demonstrated good brain exposure in rats.
Introduction
One subtype that has received much attention of late due to
the discovery of selective allosteric ligands and its implication
in a number of disease states, such as Parkinson’s disease (PD),
epilepsy, and anxiety, is mGlu4.10,11 The interest in targeting
this receptor using allosteric modulation was initially sparked
via studies with the compound N-phenyl-7-(hydro-
xyimino)cyclopropa[b]chromen-1a-carboxamide, PHCCC,12,13
1, a partially selective mGlu4 positive allosteric modula-
tor (PAM). With the exception of partial antagonist activity
at mGlu1, 1 was shown to be selective versus the other mGlus.
Compound 1 has been an invaluable tool compound for the
study of mGlu4 and has been shown to be active in acute
rodent models of Parkinson’s disease (PD) when administered
either via intracerebroventricular (icv) injection or systemi-
cally in a 50% DMSO vehicle. Although 1 has been a break-
through compound for the study of mGlu4, it has substantial
drawbacks due to its poor pharmacokinetic properties and
relatively flat structure-activity relationship. Over the past
three years, our laboratories and others have added to the list
of selective and novel tool compounds for the study of mGlu4
via positive allosteric modulation (Figure 1). Thus far, only
two disclosed mGlu4 PAMs (3 (VU0361737)14 and 415) have
been shown to be CNS penetrant when dosed systemically in a
nontoxic vehicle. In this communication, we herein disclose a
second series of N-(4-acetamido)-phenylpicolinamides that
act as mGlu4 positive allosteric modulators and show that
representative compounds are also CNS penetrant.
G-Protein coupled receptors (GPCRsa) comprise a large
protein family of transmembrane receptors and have histori-
cally been the most studied class of drug targets. GPCRs are
activated by orthosteric ligand binding, which induce macro-
molecular protein changes and stimulation of signal transduc-
tion pathways and cellular responses inside the cell. The
GPCR superfamily is grouped into three classes (class A
(rhodopsin-like), class B (secretin family), and class C
(metabotropic glutamate)).1 The mGlus belong to the class C
GPCR subfamily and are further subdivided into three
classes.2 Group I consists of mGlu1 and mGlu5, group II
consists of mGlu2 and mGlu3, and group III consists of
mGlu4, mGlu6, mGlu7, and mGlu8. Because of the lack of
selectivity and physicochemical properties of orthosteric li-
gands of the mGlus, significant effort has been initiated to
identify compounds that act via allosteric sites (i.e., sites
different from the orthosteric site) on the receptors.3,4 This
approach has been successful for the group I and II glutamate
8
receptors5 (mGlu1,6,7 mGlu5,8,9 and mGlu2 ) with multiple
tool compounds associated with each receptor; however, in
contrast, the group III family has been studied in much less
detail due to the paucity of selective ligands, including allo-
steric modulators.
*To whom correspondence should be addressed. For C.M.N.:
phone, 615-343-4303; fax, 615-343-3088; E-mail, colleen.niswender@
vanderbilt.edu. For C.R.H.: phone, 615-936-6892; fax, 615-936-4381;
E-mail, corey.r.hopkins@vanderbilt.edu. Mailing address for C.M.N.
and C.R.H.: Department of Pharmacology, Vanderbilt University
Medical Center, 2213 Garland Avenue, Nashville, Tennessee 37232.
a Abbreviations: SAR, structure-activity relationship; mGlu, met-
abotropic glutamate receptor; PAM, positive allosteric modulator;
HTS, high-throughput screening; Clint, intrinsic clearance; PPB, plasma
protein binding; AUC, area under the curve; GPCR, G-protein coupled
receptor; PHCCC, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-
1a-carboxamide; CNS, central nervous system; PD, Parkinson’s disease;
GIRK, G protein-coupled inwardly rectifying potassium channel.
Starting from a functional high-throughput screening
(HTS) initiated at Vanderbilt University, a number of N-(4-
acetamido)-phenylcarboxamide compounds were discovered,
as exemplified by compound 5 (Figure 2, Pubchem Com-
pound ID (CID): 1314024; ChemBridge ID: 7637085). This
compound represented a large class of hits that were identified
by our HTS and signaled a starting point for a lead identifica-
tion program in our laboratories.
r
pubs.acs.org/jmc
Published on Web 01/19/2011
2011 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1107
(9e, 2-thiazole, 627 nM). Next, six-membered heterocycles
(9g-i) were evaluated with the 2-pyridyl compound, 9g,
exhibiting the best potency (99.5 nM). Modulation of
the basicity of the pyridine nitrogen led to a 10-fold loss in
potency (pyrazine, 9h, 1780 nM; 4-pyrimidine, 9i, 1330 nM).
Replacement of the heterocyclic amides with carbocyclic
amides also led to much reduced potency (cyclohexyl, 9k,
1120 nM).
Having discovered a more potent replacement for the initial
5-bromo-2-furyl amide, we next turned our attention to the
central core as this contains a 3-methoxy moiety which, when
contained on the 2,4-dianiline species, could lead to bis-
quinone formation. To this end, a number of substituted
phenyl analogues were evaluated (Table 2). The 3-methoxy
was replaced by both halogens (3-Cl, 10a, 517 nM; 3-F, 10d,
556 nM), trifluoromethyl (10b, 2090 nM), and ultimately
removed (10c, 1605 nM). All of these substitutions led to a
significant decrease in activity; however, we were encouraged
to see that halogen substitution was tolerated and generated
compounds with submicrolar potency.
Figure 1. Structures of 1 and other recently reported mGlu4 PAMs.
Next, our attention turned to the amide portion that
originally was the 2-chlorophenyl amide (Table 3). Noting
that the fluoro- and chloro-phenyl compounds were equipo-
tent, we utilized both of these cores when assessing the right-
hand amide SAR and a series of aryl, cycloalkyl, alkyl, and
heteroaromatic amides were evaluated. A number of interest-
ing observations regarding the SAR were gleaned from this
study. The 2-chlorophenyl (10a, 517 nM) was superior to
other 2-subsituted compounds (2-fluorophenyl, 12f, 2380 nM;
2-methylphenyl, 12a, 958 nM; 2-methoxyphenyl, 12i, in-
active). Moving the halogen around the ring or adding a
second halogen generally led to compounds with much re-
duced potency (12g, inactive and 12h, 2150 nM); however, the
2-chloro-4-fluoro compound (12d, 114 nM) was more active
than the 2-chloro analogue (10a, 517 nM). However, when the
2-chloro-4-fluoro was replaced with 2,4-difluoro (12g, in-
active), there was a significant loss of potency (similar to the
loss seen in 10a vs 12f). Carbocycles were of equal potency
(cyclohexyl, 12b, 426 nM vs phenyl, 12c, 361 nM); however,
the smaller isopropyl group lost significant activity (12e, 4270
nM). All of the heterocyclic substituents were much less active
with the exception of 2-thiazole (12k, 403 nM).
Figure 2. Structure of initial HTS hit.
Results
The compounds were prepared via a common intermediate
such that the phenyl core and the 2-chlorophenyl amide could
be held constant while the furyl amide portion was evaluated
(Scheme 1). To this end, the nitro aniline, 6, was acylated with
2-chlorophenyl acid chloride to give 8. Hydrogenation of the
nitro group (Ra-Ni, H2) followed by amide formation led to
compounds 9a-k and 10a-d. An alternative approach was
employed for compounds12a-n. First, the aniline of 6 was di-
Boc protected (Boc2O, DMAP), followed by nitro reduction
(H2 (1 atm), Pd/C) and picolinamide formation (picolinyl acid
chloride, diisopropyl ethyl amine) leading to 11. Finally, Boc
deprotection (4 M HCl, dioxane) followed by amide forma-
tion yielded 12a-n.
Discussion
Last, as this structural class represents a 2,4-dianiline, we
were interested to determine if we could replace the internal
core structure with a nonaniline compound (see Table 1,
Supporting Information). We utilized an indole core struc-
ture, and when the NH of either amide was replaced with an
alkyl substituent, a significant loss of potency was seen.
Alkylation of either amide also led to inactive compounds
(data not shown).
With our best compounds evaluated for human and rat
potency, we next turned our attention to evaluation of the
compounds’ ability to shift the glutamate response curve to
the left, i.e. glutamate potentiation (fold shift, FS), as well as
to assess the microsomal stability (intrinsic clearance) and
the ability of the compounds to bind plasma proteins (see
Table 3, Supporting Information). For the most part, the
GluMax (% 1) was mirrored in the fold shift data, although a
few exceptions are worth noting. First, the 2-thiazole (9e) and
2-pyridyl (9g) both had high GluMax values (133.9% and
147.0%, respectively); however, this relatively small differ-
ence produced a significant difference in fold shift (32.2 vs
72.0, respectively). Both of these values are the largest fold
shifts reported to date for an mGlu4 PAM. As observed for
The initial SAR was centered around the furyl amide
portion (9a-k) in order to replace the 5-bromo-2-furyl amide
portion, as this is a high molecular weight compound (MW=
449.7). Several heterocyclic replacements, as well as aryl and
carbocycles, were evaluated (Table 1). The two pharmacol-
ogical assays we utilized to assess mGlu4 PAM potency:
(1) Chinese hamster ovary cells expressing human mGlu4 and
the chimeric G protein Gqi5 to induce calcium mobilization
and (2) HEK cellsexpressing rat mGlu4 in conjunctionwithG
protein regulated inwardly rectifying potassium (K) channels
(GIRK) to induce thallium flux18 show some day-to-day
variability in the maximal PAM response; for this reason, all
data have been normalized to the % response of the control
PAM, 1, to compare relative efficacy.19,20 Initial removal of
the bromine resulted in a more potent compound (9a, furyl,
977 nM), while the addition of a large phenyl group led to
complete loss of activity (9b). Moving from the furyl com-
pound to the saturated tetrahydrofuran (9c) also led to an
inactive compound. Substituting the furan for sulfur contain-
ing heterocycles, such as thiophene and thiazole, resulted in
compounds that were equipotent (9d and 9f) or more potent

1108 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Engers et al.
Scheme 1. Synthesis of N-(4-Acetamido)-phenylcarboxamides, 9, 10, and 12a
a All library compounds were purified by mass-directed prep LC where required.16,17 Reagents and conditions: (a) 2-chlorobenzoyl chloride, 7, DIEA,
dichloroethane, μW, 100 ꢀC; (b) Ra/Ni, H2 (1 atm) (85-94% yield, 2 steps); (c) RCO2H, HOBt, DIEA, EDCI, 1,4-dioxane; (d) ROC1, DMF:DIEA
(4:1); (e) Boc2O, DMAP (cat.) (95-99%); (f) H2 (1 atm) (80-99%); (g) picolinoyl acid chloride HC1, dichloroethane, DIEA (90-98%); (h) 4 M HCl/
dioxane (90-95%).
Table 1. Human and Rat mGlu4 Potency and % GluMax Response (as Normalized to Standard 1) for Selected Furyl Amide Analogues
hEC50
(nM)a
GluMax
(%compd 1)b
rEC50
(nM)a
GluMax
(%compd 1)b
compd
R
5
5-bromo-2-furan
2-furan
1440 ( 100
977 ( 99
87.0 ( 1.2
62.7 ( 2.4
16.3 ( 1.5
21.3 ( 2.9
63.4 ( 1.5
77.6 ( 1.7
86.8 ( 2.3
79.4 ( 1.7
74.3 ( 0.4
77.9 ( 3.5
1000 ( 90
1010 ( 200
inactivec
125.4 ( 6.7
122.3 ( 2.9
16.3 ( 2.7
27.1 ( 3.0
96.7 ( 8.5
133.9 ( 6.6
132.4 ( 6.6
147.0 ( 4.3
111.7 ( 6.6
133.1 ( 6.2
9a
9b
9c
9d
9e
9f
9g
9h
9i
5-phenyl-2-furan
2-tetrahydrofuran
2-thiophene
2-thiazole
inactivec
inactivec
>10000
860 ( 90
377 ( 101
753 ( 164
106 ( 28
1120 ( 180
894 ( 203
cannot fitd
1520 ( 630
2820 ( 44
1170 ( 150
627 ( 80
845 ( 39
4-thiazole
2-pyridine
pyrazine
99.5 ( 9
1780 ( 140
1330 ( 90
cannot fitd
1120 ( 270
4740 ( 51
4-pyrimidine
phenyl
cyclohexyl
9j
9k
1
26.9 ( 5.3
43.2 ( 13.2
a EC50 and GluMax, are the average of at least three independent determinations performed in triplicate (mean ( SEM shown in table). b 1 is run as a
control compound each day, and the maximal response generated in mGlu4 CHO cells in the presence of mGlu4 PAMs varies slightly in each experiment.
Therefore, data were further normalized to the relative 1 response obtained in each day’s run. c Inactive compounds are defined as %GluMax did not
surpass 2ꢀ the EC20 value at the highest concentration (30 μM) for that day’s run. d Curve could not be fit due to insolubility at higher concentrations.
other allosteric modulators, the fold shift data, representative
of compound efficacy, did not always track with the potency
values (10a, 570 nM, 18.3 FS vs 12d, 169 nM, 6.7 FS).
the compounds showed weak activity against CYP2C9 and
CYP2D6 (>10 μM). Last, selectivity versus the other mGlu’s
was performed on 10a and this compound was selective versus
the group I and group II mGlu’s but showed weak PAM
activity (as assessed by the ability of 10a to left-shift the
glutamate response curve at 30 μM) against mGlu7 (5.3 FS)
and mGlu8 (3.8 FS) (see Table 2, Supporting Information).
While the compound is not completely selective for mGlu4, this
compound seems to be group III mGlu preferring, which may
allow for future rationally designed site directed mutagenesis
studies to help confirm the binding location on the receptor.
Although there is a large range of fold shift values within
compounds in this series, all other properties appear to be
equivalent (intrinsic clearance, PPB, CYP inhibition profile);
thus, three representative compounds were chosen to further
advanced into in vivo PK studies (see Table 4, Supporting
Information). The hydrochloride salts of these compounds
(9g, 10a, 12a) were synthesized and dosed subcutaneously
(10 mg/kg) as an aqueous microsuspension containing 10%
Tween-80, and the amount of compound present in brain and
Compounds were further evaluated by assessing their rate
of intrinsic clearance in vitro as well as their ability to bind to
plasma proteins (PPB) (see Table 3, Supporting Information).
Two compounds possessed poor metabolic stability (9e and
15). These compounds possess a labile methoxy group which
could be the contributing factor to their instability. Interest-
ingly, 9g also possesses a methoxy group but exhibits accep-
table intrinsic clearance values (42.6 mL/min/kg). All of the
other compounds tested exhibited acceptable intrinsic clear-
ance (<50 mL/min/kg). All compounds evaluated were
highly protein bound in rats (>99.5% bound, <0.5%
free unbound, fu). In addition to establishing in vitro
pharmacology, metabolic stability, and protein binding pro-
files, representative compounds (9g, 10a, and 12a) were tested
for their inhibition of the major CYP450 enzymes (CYP1A2,
2C9, 3A4, and 2D6). These compounds showed no significant
activityagainstCYP1A2and CYP3A4(>30 μM), but each of

Brief Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1109
plasma was determined at 0.5, 1, 3, and 6 h after administration
(see Table 4, Supporting Information). Uptake of 10a
(VU0366037) was substantial in both plasma and brain with
a brain:plasma ratio of 1.04. Plasma exposure of VU0366038,
12a, and VU0415374, 9g, were comparable to 10a; however,
these compounds exhibited lower brain exposure with a brain:
plasma ratio of 0.2 and 0.33, respectively.
10a possessing fold shifts >15. In addition, compounds 10a
and 12a showed acceptable in vitro PK parameters (intrinsic
clearance) and were further evaluated for CNS exposure. 10a
showed good exposure in both plasma and brain, with a ratio
of 1.04.
Experimental Section
General. The syntheses of selected compounds are described
below. The general chemistry, experimental information, and
syntheses of all other compounds are supplied in the Supporting
Information. Purity of all final compounds was determined by
HPLC analysis and is >95%.
Conclusion
In summary, we have discovered and evaluated a novel
series of N-(4-acetamido)-phenylpicolinamides as mGlu4
positive allosteric modulators suitable as in vivo tool com-
pounds. Compounds 9g, 10a, and 12a exhibited submicro-
molar EC50s against both human and rat mGlu4, and all three
robustly left-shifted the glutamate response curve with 9g and
N-(4-(2-Chlorobenzamido)-3-methoxyphenyl)picolinamide
(9g). To a solution of 8g (0.91 g, 2.98 mmol) in ethanol
(EtOH):ethyl acetate (EtOAc) (200 mL, 1:1) was added Ra-Ni
(∼0.1 equiv). An H2 atmosphere (1 atm) was applied to the
reaction (rxn) mixture. After 16 h, the rxn was filtered through
Celite and concentrated. The crude reside was deemed >85%
pure by LCMS and carried through without further purifica-
tion. To a solution of the aforementioned aniline in dimethyl-
formamide (DMF) (25 mL) and diisopropyl ethylamine (DIEA)
(5 mL, 28.7 mmol) was added 2-pyridinecarbonyl chloride (0.53
g, 2.98 mmol). After 12 h at rt, the rxn was added to EtOAc:H2O
(150 mL, 1:1). The organic layer was washed with H2O (2 ꢀ
50 mL) and brine (50 mL) and dried (MgSO4). After filtration
and concentration, the residue was purified by mass-guided
preparative HPLC to afford amide 9g (0.66 g, 58% yield over
2 steps). Analytical LCMS (method 2): single peak (220 nm),
RT = 1.167 min, m/z 382.0 [M þ H]þ. 1H NMR (400 MHz,
DMSO-d6): δ 10.64 (s, 1H), 9.58 (s, 1H), 8.74 (d, J=4.4 Hz,
1H), 8.16 (d, J=8.0 Hz, 1H), 8.07 (ddd, J=8.0, 8.0, 1.6 Hz, 1
H), 7.83 (d, J = 8.8 Hz, 1H), 7.75 (d, J = 2.0 Hz, 1H), 7.68
(dd, J = 6.4, 4.8 Hz, 1H), 7.58-7.46 (m, 5H), 3.82 (s, 3H).
HRMS: calcd for C20H17N3O3Cl (M þ Hþ), 382.0958; found,
382.0958.
Table 2. Human and Rat mGlu4 Potency and % GluMax Response (as
Normalized to Standard 1) for Selected Central Phenyl Modification
Analogues
hEC50
(nM)a
GluMax
(%compd 1)b
rEC50
(nM)a
GluMax
(%compd 1)b
compd
R1
9g
10a 3-Cl
3-OMe 99.5 ( 9
517 ( 57
79.4 ( 1.7
77.9 ( 1.6
106 ( 28
570 ( 131 128.2 ( 5.6
147.0 ( 4.3
10b 3-CF3 2090 ( 190 69.5 ( 1.5 2360 ( 230 101.9 ( 7.4
10c
10d 3-F
H
1605 ( 200 33.6 ( 0.5 2260 ( 260
556 ( 76 59.7 ( 1.1 739 ( 92
49.6 ( 5.4
91.4 ( 2.9
a EC50 and GluMax are the average of at least three independent
determinations performed in triplicate (mean ( SEM shown in table).
b Compound 1 is run as a control compound each day, and the maximal
response generated in mGlu4 CHO cells in the presence of mGlu4 PAMs
varies slightly in each experiment. Therefore, data were further normal-
ized to the relative 1 response obtained in each day’s run.
N-(3-Chloro-4-(2-chlorobenzamido)phenyl)picolinamide (10a).
Following the general procedure above for 9g, compound 10a
(0.62 g, 81%) was obtained as a tan solid: Analytical LCMS
(method 2): single peak (214 nm); RT = 1.496 min. 1H NMR (400
MHz, DMSO-d6):δ10.91 (s, 1H), 10.18 (s, 1H), 8.77 (d, J=4.8Hz,
Table 3. Human and Rat mGlu4 Potency and % GluMax Response (as Normalized to Standard 1) for Selected Acetamido Amide Analogues
hEC50
(nM)a
GluMax
(%compd 1)b
rEC50
(nM)a
GluMax
(%compd 1)b
compd
R2
12a
12b
12c
12d
12e
12f
12g
12h
12i
2-methylphenyl
cyclohexyl
phenyl
958 ( 48
426 ( 58
361 ( 40
114 ( 26
4270 ( 540
2380 ( 170
inactivec
72.5 ( 1.1
59.2 ( 1.0
37.9 ( 1.3
61.1 ( 4.1
60.0 ( 1.1
34.5 ( 1.0
24.6 ( 1.5
37.4 ( 3.2
16.4 ( 1.3
86.7 ( 2.0
61.5 ( 2.0
57.8 ( 1.8
49.9 ( 4.1
24.9 ( 0.8
915 ( 161
592 ( 70
518 ( 43
123.1 ( 2.9
106.0 ( 0.7
74.2 ( 8.7
118.8 ( 7.3
109.0 ( 3.4
71.6 ( 10.0
43.5 ( 12.0
61.0 ( 9.0
19.0 ( 2.1
124.8 ( 8.7
105.5 ( 2.4
93.8 ( 3.8
87.1 ( 3.9
45.6 ( 10.8
2-chloro-4-fluorophenyl
isopropyl
169 ( 28
4820 ( 1160
4390 ( 1140
2100 ( 780
2340 ( 817
inactivec
2-fluorophenyl
2,4-difluorophenyl
3,5-dichlorophenyl
2-methoxyphenyl
5-thiazole
2150 ( 448
inactivec
12j
2180 ( 310
403 ( 28
1040 ( 100
4720 ( 900
inactivec
1730 ( 170
533 ( 145
1130 ( 140
2970 ( 290
3350 ( 290
12k
12l
2-thiazole
2-thiophene
12m
12n
N-methyl-2-indole
5-phenyl-2-furyl
a EC50 and GluMax are the average of at least three independent determinations performed in triplicate (mean ( SEM shown in table). b Compound 1
is run as a control compound each day, and the maximal response generated in mGlu4 CHO cells in the presence of mGlu4 PAMs varies slightly in each
experiment. Therefore, data were further normalized to the relative 1 response obtained in each day’s run. c Inactive compounds are defined as %
GluMax did not surpass 2ꢀ the EC20 value at the highest concentration (30 μM) for that day’s run.

1110 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Engers et al.
1H), 8.23 (d, J = 2.0 Hz, 1H), 8.19 (d, J = 7.6 Hz, 1H), 8.10 (ddd,
J = 8.0, 1.6, 1.6 Hz, 1H), 7.93 (dd, J = 8.8, 2.0 Hz, 1H), 7.73-
7.70 (m, 1H), 7.63-7.46 (m, 5H). HRMS: calcd for C19H14N2-
O2Cl2 [M þ H]þ, 386.0463; found, 386.0467.
(2) Schoepp, D. D.; Jane, D. E.; Monn, J. A. Pharmacological agents
acting at subtypes of metabotropic glutamate receptors. Neuro-
pharmacology 1999, 38, 1431–1476.
(3) Conn, P. J.; Christopoulos, A.; Lindsley, C. W. Allosteric mod-
ulators of GPCRs: a novel approach for the treatment of CNS
disorders. Nature Rev. Drug. Discovery 2009, 8, 41–54.
N-(3-Chloro-4-(2-methylbenzamido)phenyl)picolinamide (12a).
Toa solutionof6 (1.67g, 9.69mmol) anddimethylaminopyridine
(DMAP) (0.12 g, 0.97 mmol) in tetrahydrofuran (THF) (50 mL)
at rt was added (Boc)2O (6.33 g, 29.0 mmol). After 16 h, the rxn
was added to EtOAc:water (200 mL, 1:1) and the organic layer
was separated and washed with brine (50 mL), dried (MgSO4),
filtered, and concentrated. To a solution of the crude residue in
EtOAc (200 mL) was added Rainey-Ni (∼0.1 equiv), and an H2
atmosphere via balloon (1 atm) was applied. After 16 h, LCMS
confirmed loss of starting material, and the mixture was filtered
through a Celite filter and concentrated and was carried on with-
out purification. To a solution of the crude residue in dichloro-
methane (DCM) (20 mL) was added DIEA (3.4 mL, 24.2 mmol),
followed by picolinoyl acid chloride hydrochloride (1.72 g, 9.69
mmol). After 16 h, the rxn was added to DCM:NaHCO3 (aq)
(200 mL, 1:1) and the organic layer was separated. The organic
layerwas washed withbrine (50mL), dried(MgSO4), filtered, and
concentrated to afford amide 11, which was carried on without
purification. To a solution of amide 11 in DCM (100 mL) at 0 οC
was added 4 M HCl in 1,4-dioxane (20 mL). After 15 min, the ice
bath was removed. The violet-red solution was stirred for 12 h at
rt, and after TLC confirmed loss of starting material, the solvent
was removed under vacuo. To a mixture of the crude residue in
DMF (20 mL) at 0 οC was added DIEA (4.78 mL, 34.0 mmol)
followed by o-toluoyl chloride (1.26 mL, 9.69 mmol). After 15
min, the ice bath was removed. After an additional 12 h, the rxn
was added to EtOAc:H2O (200 mL, 1:1). The organic layer was
washed with H2O (2 ꢀ 50 mL) and brine (50 mL) and dried
(MgSO4). After filtration and concentration, the residue was pu-
rified by mass-guided preparative HPLC to afford amide 12a
(1.45 g, 41% overall yield). Analytical LCMS (method 2): single
peak (214 nm); RT=1.482 min. 1H NMR (400 MHz, DMSO-d6):
δ 10.90 (s, 1H), 9.94 (s, 1H), 8.77 (d, J=4.0 Hz, 1H), 8.22 (d, J =
2.0 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.10 (ddd, J = 8.0, 2.0, 2.0
Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.71 (ddd, J = 7.6, 4.8, 1.6 Hz,
1H), 7.58 (d, J = 8.8 Hz, 5H), 7.54 (d, J = 7.6 Hz, 1H), 7.40
(dd, J = 7.2, 7.2 Hz, 1H), 7.33-7.30 (m, 2H), 2.46 (s, 3H).
HRMS: calcd for C20H17N3O2Cl [M þ H]þ, 366.1009; found,
366.1009.
(4) May, L. T.; Leach, K.; Sexton, P. M.; Christopoulos, A. Allosteric
modulation of G protein-coupled receptors. Annu. Rev. Pharmacol.
Toxicol. 2007, 47, 1–51.
(5) Johnson, M. P.; Nisenbaum, E. S.; Large, T. H.; Emkey, R.; Baez,
M.; Kingston, A. E. Allosteric modulators of metabotropic glutamate
receptors: lessons learnt from mGlu1, mGlu2 and mGlu5 potentiators
and antagonists. Biochem. Soc. Trans. 2004, 32, 881–887.
(6) Gasparini, F.; Kuhn, R.; Pin, J.-P. Allosteric modulation of group 1
metabotropic glutamate receptors: novel subtype-selective ligands and
therapeutic perspectives. Exp. Opin. Pharmacol. 2002, 2, 43–49.
(7) Knoflach, F.; Mutel, V.; Jolidon, S.; Kew, J. N. C.; Malherbe, P.;
Vieira, E.; Wichman, J.; Kemp, J. A. Positive allosteric modulators
of metabotropic glutamate 1 receptor: characterization, mecha-
nism of action, and binding site. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 13402–13407.
(8) Schlumberger, C.; Pietraszek, M.; Gravius, A.; Klein, K.-U.; Greco,
ꢀ
S.;More, L.; Danysz, W. ComparisonofthemGlu5 receptorpositive
allosteric modulator ADX47273 and the mGlu2/3 receptor agonist
LY354740 in tests for antipyschotic-like activity. Eur. J. Pharmacol.
2009, 623, 73–83.
(9) Felts, A. S.; Lindsley, S. R.; Lamb, J. P.; Rodriguez, A. L.; Menon,
U. N.; Jadhav, S.; Jones, C. K.; Conn, P. J.; Lindsley, C. W.; Emmitte,
K. A. 3-Cyano-5-fluoro-N-arylbenzamides as negative allosteric mod-
ulators of mGlu5: identification of easily prepared tool compounds with
CNS exposure in rats. Bioorg. Med. Chem. Lett. 2010, 20, 4390–4394.
(10) Hopkins, C. R.; Lindsley, C. W.; Niswender, C. M. mGluR4
Positive Allosteric Modulation as Potential Treatment of Parkinson’s
Disease. Future Med. Chem. 2009, 1, 501–513.
(11) Lindsley, C. W.; Niswender, C. M.; Engers, D. W.; Hopkins, C. R.
Recent Progress in the Development of mGluR4 Positive Allosteric
Modulators for the Treatment of Parkinson’s Disease. Curr. Top.
Med. Chem. 2009, 949–963.
(12) Maj, M.; Bruno, V.; Dragic, Z.; Yamamoto, R.; Battaglia, G.;
Inderbitzin, W.; Stoehr, N.; Stein, T.; Gasparini, F.; Vranesic, I.;
Kuhn, R.; Nicoletti, F.; Flor, P. J. (-)-PHCCC, a positive allosteric
modulator of mGluR4: characterization, mechanism of action, and
neuroprotection. Neuropharmacology 2003, 45, 895–906.
(13) Marino, M. J.; Williams, D. L., Jr.; O’Brien, J. A.; Valenti, O.;
McDonald, T. P.; Clements, M. K.; Wang, R.; DiLella, A. G.
Kinney, G. G.; Conn, P. J. Allosteric modulation of group III
metabotropic glutamate receptor 4: a potential approach to Parkinson’s
disease treatment. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,13668–13673.
(14) Engers, D. W.; Niswender, C. M.; Weaver, C. D.; Jadhav, S.; Menon,
U. N.; Zamorano, R.; Conn, P. J.; Lindsley, C. W.; Hopkins, C. R.
Synthesis and evaluation of a series of heterobiarylamides that are
centrally penetrant metabotropic glutamate receptor 4 (mGluR4)
positive allosteric modulators (PAMs). J. Med. Chem. 2009, 52,
4115–4118.
Acknowledgment. We thank Emily L. Days, Tasha
Nalywajko, Cheryl A. Austin, and Michael Baxter Williams
for their critical contributions to the HTS portion of the proj-
ect. In addition, we thank Katrina Brewer, Ryan Morrison,
and Matt Mulder for technical assistance with the PK assays
and Chris Denicola, Nathan Kett, and Sichen Chang for the
purification of compounds utilizing the mass-directed HPLC
system. This work was supported by the National Institute of
Mental Health, National Institute of Neurological Disorders
and Stroke, the Michael J. Fox Foundation, the Vanderbilt
Department of Pharmacology, and the Vanderbilt Insti-
tute of Chemical Biology. Vanderbilt is a member of the
MLPCN and houses the Vanderbilt Specialized Chemis-
try Center for Accelerated Probe Development (NIH/
MLPCN; 5U54MH084659-02).
(15) East, S. P.; Bamford, S.; Dietz, M. G. A.; Eickmeier, C.; Flegg, A.;
Ferger, B.; Gemkow, M. J.; Heilker, R.; Hengerer, B.; Kotey, A.; Loke,
€
P.; Schanzle, G.; Schubert, H.-D.; Scott, J.; Whittaker, M.; Williams,
M.; Zawadzki, P.; Gerlach, K. An orally bioavailable positive allosteric
modulator of the mGlu4 receptor with efficacy in an animal model of
motor dysfunction. Bioorg. Med. Chem. Lett. 2010, 20, 4901–4905.
(16) Leister, W.; Strauss, K.; Wisnoski, D.; Zhao, Z.; Lindsley, C.
Development of a custom high-throughput preparative liquid
chromatography/mass spectrometer platform for the prepara-
tive purification and analytical analysis of compound libraries.
J. Comb. Chem. 2003, 5, 322–329.
(17) All new compounds were characterized by LCMS and/or 1H NMR
and found to be in agreement with their structures (>95% purity).
(18) Niswender, C. M.; Johnson, K. A.; Luo, Q.; Ayala, J. E.; Kim, C.;
Conn, P. J.; Weaver, C. D. A novel assay of Gi/o-linked G protein-
coupled receptor coupling to potassium channels provides new
insights into the pharmacology of the group III metabotropic
glutamate receptors. Mol. Pharmacol. 2008, 73, 1213–1224.
(19) Williams, R.; Zhou, Y.; Niswender, C. M.; Luo, Q.; Conn, P. J.;
Lindsley, C. W.; Hopkins, C. R. Re-exploration of the PHCCC
scaffold: discovery of improved positive allosteric modulators of
mGluR4. ACS Chem. Neurosci. 2010, 1, 411–419.
Supporting Information Available: Experimental procedures,
spectroscopic data, and NMR data for selected compounds, and
biological procedures. This material is available free of charge
via the Internet at http://pubs.acs.org.
(20) Engers, D. W.; Gentry, P. R.; Williams, R.; Bolinger, J. D.;
Weaver, C. D.; Menon, U. N.; Conn, P. J.; Lindsley, C. W.;
Niswender, C. M.; Hopkins, C. R. Synthesis and SAR of novel,
4-(phenylsulfamoyl)phenylacetamide mGlu4 positive allosteric
modulators (PAMs) identified by functional high-throughput
screening (HTS). Bioorg. Med. Chem. Lett. 2010, 20, 5175–5178.
References
(1) Conn, P. J.; Pin, J.-P. Pharmacology and functions of metabotropic
glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 205–
237.