Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4): Part I. Discovery of pyrazolo[3,4-d]pyrimidines as novel mGluR4 positive allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2008.08.087

This letter describes the synthesis and SAR, developed through an iterative analogue library approach, of an mGluR4 positive allosteric modulator lead based on a pyrazolo[3,4-d]pyrimidine scaffold. Despite tremendous therapeutic potential, Compound 7, VU0080421, and related congeners represent only a handful of mGluR4 positive allosteric modulators ever described.

Full text of DOI:10.1016/j.bmcl.2008.08.087

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5626–5630
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Positive allosteric modulators of the metabotropic glutamate receptor
subtype 4 (mGluR4): Part I. Discovery of pyrazolo[3,4-d]pyrimidines
as novel mGluR4 positive allosteric modulators
Colleen M. Niswender a,c, , Evan P. Lebois a,c, , Qingwei Luo a,c, Kwangho Kim d, Hubert Muchalski b,
Huiyong Yin c, P. Jeffrey Conn a,c,d, Craig W. Lindsley a,b,c,d,
*
a Department of Pharmacology, Vanderbilt University Medical Center, 802 Robinson Research Building, Nashville, TN 37232-6600, USA
b Department of Chemistry, Vanderbilt University Medical Center, 802 Robinson Research Building,Nashville, TN 37232-6600, USA
c Vanderbilt Program in Drug Discovery, Vanderbilt University, Nashville, TN 37232, USA
d Vanderbilt Institute of Chemical Biology, 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
Article history:
This letter describes the synthesis and SAR, developed through an iterative analogue library approach, of
an mGluR4 positive allosteric modulator lead based on a pyrazolo[3,4-d]pyrimidine scaffold. Despite tre-
mendous therapeutic potential, Compound 7, VU0080421, and related congeners represent only a hand-
ful of mGluR4 positive allosteric modulators ever described.
Received 6 August 2008
Revised 22 August 2008
Accepted 26 August 2008
Available online 29 August 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
mGluR4
PAM
Positive allosteric modulator
Parkinson’s disease
GPCR
Glutamateis the major excitatory neurotransmitter in the central
nervous system, exerting its effects through both ionotropic and
metabotropic glutamate receptors. The metabotropic glutamate
receptors (mGluRs)are members of the GPCR family C, characterized
by a large extracellular amino-terminal agonist binding domain. To
date, eight mGluRs have been cloned, sequenced and assigned to
three groups (Group I: mGluR1 and mGluR5; Group II: mGluR2
and mGluR3; Group III: mGluRs 4, 6, 7, 8) based on their sequence
homology, pharmacology and coupling to effector mechanisms.1
The Group III mGluRs are the least explored and characterized of
the mGluRs, but despite this fact, mGluR4 has garnered a great deal
of attention as a therapeutic target for multiple indications.2
mGluR1 negative allosteric modulator (NAM) (À)-CPCCOEt 4.5 (À)-
PHCCC possesses an EC50 of 4.1 lM, with a 5.5-fold leftward shift of
the glutamate response curve and selectivity versus mGluRs 2, 3, 5,
6, 7, 8.5,6 However, (À)-PHCCC is a partial antagonist (30%) of mGluR1.
SAR for (À)-PHCCC is very ‘flat’, with virtually any chemical
modifications resulting in a complete loss of mGluR4 PAM activity
(Fig. 2), a finding common with several series of mGluR PAMs.7–10
Despite this, (À)-PHCCC has been a very important proof of concept
(POC) compound demonstrating a therapeutic role for selective
mGluR4 activation in Parkinson’s disease,6,11 anxiety,12 depres-
sion,13 neuroprotection14 and oncology.15
In all of these pioneering POC experiments, PHCCC was either
administered through intracerebroventricular injection (icv) or by
employing toxic 50% DMSO vehicles which disrupt the blood–brain
barrier, as PHCCC possesses poor physiochemical properties and
limited brain penetration.6,11–15 In order to advance this field,
new mGluR4 PAMs are required with improved efficacy, physio-
chemical properties and novel molecular architectures. In this let-
ter, we describe the discovery and SAR of a novel mGluR4 PAM,
based on a pyrazolo[3,4-b]pyrimidine scaffold, derived from an
HTS campaign.
The reason for the slower pace of development within Group III
mGluRs concerns the availability of ligands.2,3 Most pharmacologi-
cal studies employ prototypical Group III agonists such as
L-(+)-2-
amino-4-phosphonobutryic acid, -AP4, or functionalized
L
1
carboxyphenylglycines 2, which have limited CNS penetration
(Fig. 1).4 A major breakthrough in the field occurred when Maj and
co-workers reported on the discovery of (À)-PHCCC 3, the first
mGluR4 positive allosteric modulator (PAM), derived from the
Our mGluR4 PAM HTS identified three pyrazolo[3,4-d]pyrimi-
dines that afforded a concentration-dependent potentiation of an
EC20 of glutamate in mGluR4/Gqi5 CHO cells (Fig. 3).7 When HTS
* Corresponding author. Tel.: +1 615 322 8700; fax: +1 615 343 6532.
E-mail address: craig.lindsley@vanderbilt.edu (C.W. Lindsley).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.087

C. M. Niswender et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5626–5630
5627
Figure 4. Compound 7, potentiates mGluR4 activation by glutamate. In the absence
of glutamate, 7 does not activate mGluR4. In the presence of an EC20 concentration
of glutamate, 7 caused a concentration-dependent potentiation of mGluR4 with an
EC50 for potentiation of 4.6
represents the average of at least three independent determinations.
lM, equivalent to PHCCC, EC50 ꢀ 4.1 l
M.5,6 Data
220
Control
180
140
100
60
PHCCC, 3
20
Figure 1. Chemical structures of orthosteric mGluR4 agonists L-AP4 (1), function-
alized phenylglycines (2) and the mGluR4 PAM (À)-PHCCC (3) which was derived
-20
-10 -9 -8 -7 -6 -5 -4 -3 -2
from the mGluR1 NAM (À)-CPCCOEt (4).
Log glutamate, [M]
220
180
140
100
60
Control
7
20
-20
-10 -9 -8 -7 -6 -5 -4 -3 -2
Log glutamate, [M]
Figure 5. Both PHCCC (3) and 7 shift the glutamate agonist response curves to the
left 5.5- and 27.2(+8.5)-fold, respectively, at 30 M (EC50 shifts from 7.5 + 1.6 M to
l
l
317 + 100 nM). Interestingly, PHCCC increases maximal response whereas 7 affords
no increase in the glutamate max, akin to other mGluR5 PAMs reported from our
laboratory.7
Figure 2. Chemical modifications and the resulting ‘flat’ SAR for (À)-PHCCC.
stocks were evaluated with full concentration–response curves, 7,
1-(2,4-diphenyl)-4-(3-methylpiperdin-1-yl)-1H-pyrazolo[3,4-d]-
pyrimidine, was a stand-out compound with an EC50 for potenti-
ation of 4.6
lM while having no effect on mGluR4 in the absence
of glutamate (Fig. 4).16
HN
N
N
HN
N
PHCCC, 3
N
N
200
N
N
N
5
6
7
N
N
N
N
N
150
5
6
7
100
50
0
1
3
10 30
1
3
10 30
1
3
10 30
1
3
10 30
μ
Compound, [ M]
Figure 3. Concentration-dependent potentiation of glutamate in mGluR4/Gqi5 CHO cells by pyrazolo[3,4-d]pyrimidine compounds 5, 6 and 7 in the high throughput
screening campaign (HTS).

5628
C. M. Niswender et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5626–5630
Table 1
Glutamate fold shifts by VU0080421 analogues 12
NR1R2
N
N
N
N
Ar
12
Compound
NR1R2
Ar
Fold shifta
5.5
(À)-PHCCC, 3
7 VU0080241
27.2 11.8
9.9
N
CF3
12a
12b
N
O
N
3.5
12c
2.7
N
Cl
HN
O
N
12d
2.4
N
Figure 6. Monomers employed in the diversity library of pyrazolo[3,4-d]pyrimi-
dines 12 (7 arylhydrazines  18 amines) based on HTS lead 7, VU0080421.
Monomers in boxes indicate that they produced analogues 12 that potentiated an
EC20 of glutamate in mGluR4/Gqi5 CHO cells.
a
Fold shifts represent the average of at least three independent experiments
performed in quadruplicate at 30 M.
Additionally, 7 was superior to PHCCC in terms of fold shift of
the glutamate concentration–response curve. As shown in Figure
5, PHCCC induces a 5.5-fold leftward shift of the glutamate re-
sponse curve with an elevation in the glutamate max, whereas 7
elicits a 27.2( 8.5)-fold shift with no increase in the glutamate
max. Compound 7 was selective versus mGluR5, but was a full
sence of radioligands for mGluR4, this issue will have to be ad-
dressed at a later time.
With a bona fide novel mGluR4 PAM lead in hand, we employed
an iterative analogue library synthesis approach to rapidly evalu-
ate SAR for 7. Despite HTS stocks of 7 confirming structure and pur-
ity, re-synthesis of 7 (VU0080241) resulted in a compound with
antagonist of mGluR1 (IC50 = 2.6 lM). This finding was surprising
slightly lower potency and a less robust fold shift (EC50 ꢀ 5
lM,
since PHCCC was derived from a series of mGluR1 antagonists
(the (À)-CPCCOEt 4 series), it was not surprising that 3 was a
30% partial antagonist of mGluR1, but 7 is structurally unrelated.
It is possible that PHCCC and 7 share a common allosteric binding
site on mGluR4 that is also conserved in mGluR1, but in the ab-
fold shift 11.8 3.3, Table 1); however, VU0080241 still represents
an improvement over PHCCC. In light of the ‘flat’ SAR observed by
ourselves and others in the field with PHCCC,5–7 we initially syn-
thesized multi-dimensional diversity libraries to determine the
scope and breadth of the SAR. However, classical synthetic ap-
proaches to pyrazolo[3,4-d]pyrimidines yields were typically mod-
erate (<50%) with prolonged reaction times at high temperatures
(multiple steps requiring >48 h at reflux).17 As recently reported,
we have developed a rapid, high yielding and general micro-
wave-assisted approach to access diverse pyrazolo[3,4-d]pyrimi-
dines, and this expedited protocol was employed to produce
analogues of VU0080421 (Scheme 1).18 The synthesis began by
reacting 2-(ethoxymethylene)malononitrile 8 with seven arylhydr-
azines under microwave irradiation to deliver 5-amino-1-aryl-1H-
pyrazole-4-carbonitriles 9. The nitrile analogues 9 were then
hydrolyzed with H2SO4 to produce the corresponding 5-amino-1-
aryl-1H-pyrazole-4-carboxamides 10. A microwave-assisted con-
densation employing carboxamides 10 in neat formamide
smoothly afforded the corresponding analogous 1-aryl-1H-pyraz-
olo[3,4-d]pyrimidines 11. MAOS conditions (POCl3, DMF, 120 °C,
45 min, mw) were used for the conversion to the chloro congeners,
which were then subjected to a microwave-assisted SNAr reaction
to provide the final analogues 12 based on HTS lead 7,
VU0080421.19 In short order, over 126 analogues 12 (7 arylhydra-
zines  18 amines) were prepared and evaluated as mGluR4 PAMs
(Fig. 6).
O
NC
H2N
NC
NC
H2N
H2N
a
b
N
N
N
OEt
N
Ar
Ar
8
9
10
NR1R2
OH
N
c
d
N
N
N
N
Ar
N
N
N
Ar
11
12
Scheme 1. Reagents and conditions: (a) ArNHNH2, EtOH, mw, 105 °C, 10 min, 54–
77%; (b) i—H2SO4, 0 °C; ii—H2NCHO, mw, 200 °C, 20 min, 48–91%; (c) i—POCl3, DMF,
mw, 120 °C, 45 min, ii—HNR1R2, DMF, PSDIEA, mw, 90 °C, 20 min, 84–99%.18,19

C. M. Niswender et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5626–5630
5629
12. Stachowicz, K.; Klak, K.; Klodzinska, A.; Chojnacka-Wojcik, E.; Pilc, A. Eur. J.
Pharmacol. 2004, 498, 153.
13. Klak, K.; Palucha, A.; Branski, P.; Pilc, A. Amino Acids 2007, 32, 169.
14. Canudas, A. M.; Di Giorgi-Gerevini, V.; Iacovelli, L.; Nano, G.; D’Onofrio, M.;
Arcella, A.; Giangaspero, F.; Busceti, C.; Ricci-Vitiani, L.; Battaglia, G.; Nicoletti,
F.; Melchiorri, D. J. Neurosci. 2004, 24, 10343.
15. Iacovelli, L.; Arcella, A.; Battaglia, G.; Pazzaglia, S.; Aronica, E.; Spinsanti, P.;
Caruso, A.; De Smaele, E.; Saran, A.; Gulino, A.; D’Onofrio, M.; Giangaspero, F.;
Nicoletti, F. J. Neurosci. 2006, 26, 8388.
16. Assay details: Cell culture-human mGluR4/Gqi5/CHO line. Human mGluR4
(hmGluR4)/CHO cells were stably transfected with the chimeric G protein
Gqi5 and single clones were selected via hygromycin resistance and
screened for mGluR4-mediated calcium mobilization using the method
described below. hmGluR4/CHO cells were cultured in 90% Dulbecco’s
modified Eagle media (DMEM), 10% fetal bovine serum (FBS), 100 U/ml
penicillin/streptomycin, 20 mM Hepes (pH 7.3), 1 mM sodium pyruvate,
As in the case of PHCCC, analogues 12 of VU0080241 were
uniformly inactive on mGluR4 with only the parent HTS lead 7,
the re-synthesized VU0080421 and four other analogues (12a–
d) showing any activity as mGluR4 PAMs. SAR for this series
was ‘flat’ with only a 3.9% active rate. With one exception (12c,
containing a 2-chlorophenyl group), the 2,4-dimethylphenyl moi-
ety (6, 7, 12a, 12b, 12d) was required for activity, and little diver-
sity was tolerated with respect to the nature of the NR1R2 moiety.
As shown in Table 1, analogues 12 lost efficacy, EC50s > 10 lM,
but provided robust leftward shifts of the glutamate response
curve 2.4- to 9.9-fold. One explanation for the ‘flat’ SAR is that
the allosteric binding sites which VU0080421 and (À)-PHCCC oc-
cupy are very shallow, similar to the second, non-MPEP, allosteric
binding site on mGluR5 that CPPHA occupies.8,9 In addition,
in vitro DMPK studies identified stability issues with
VU0080241. Importantly, VU0080241 was found to be unstable
in fortified liver microsome preparations, with only 9% of the par-
ent compound remaining after 90 min.
Despite the disappointing SAR and microsomal instability,
VU0080421 (7) represents a significant advance in the mGluR4 PAM
field.VU0080421(7)possessesalarge11.8-to27.2-foldshift, thelarg-
est we have observed for an mGluR PAM, and it does not contain the
oxime or amide NH moieties that are speculated to contribute to the
observed lack of brain penetration for PHCCC in vehicles other than
DMSO. Moreover, VU0080421 represents a novel chemotype for a
PAM of mGluRs and is one of only a handful of reported PAMs of
mGluR4. Further refinements to VU0080241 and related series of
mGluR4 PAMs are in progress and will be reported in due course.
2 mM glutamine, 400 lg/ml G418 (Mediatech, Inc., Herndon, VA) and 5 nM
methotrexate (Calbiochem, EMD Chemicals, Gibbstown, NJ). Culturing
conditions for other mGluR cell lines are described below. All cell culture
reagents were purchased from Gibco/Invitrogen (Carlsbad, CA).Calcium
fluorescence assay. Primary high throughput screening details were
described in Ref. 7. Briefly, human mGluR4/Gqi5/CHO cells (30,000 cells/
20 lL/well) were plated in black-walled, clear-bottomed 384 well plates
(Greiner Bio-One, Monroe, NC) in DMEM containing 10% dialyzed FBS,
20 mM Hepes, and 100 U/ml penicillin/streptomycin (Assay Media). The cells
were grown overnight at 37 °C in the presence of 5% CO2. The next day,
media was removed and the cells were incubated with 20 lL of 1 lM Fluo-
4AM (Invitrogen, Carlsbad, CA) prepared as a stock in DMSO and mixed in a
1:1 ratio with pluronic acid F-127 and diluted in Assay Buffer (Hank’
Balanced Salt Solution, 20 mM Hepes and 2.5 mM Probenecid (Sigma–
Aldrich, St. Louis, MO) for 45 min at 37 °C. Dye was removed and 20
Assay Buffer was added. Test compounds were diluted into Assay Buffer to
generate a 20
M stock. Ca2+ flux was measured using the Functional Drug
lL of
l
Screening System 6000 (FDSS6000, Hamamatsu, Japan). Appropriate baseline
readings were taken (10 images at 1 Hz, excitation, 470 20 nm emission,
540 30 nm) and test compounds were added at
10 M.)Confirmation/selectivity studies for other mGluRs. Rat mGluRs 1 and 5.
Compounds were assessed using 10 point concentration–response curves
starting at a 30 M final concentration and diluted by 1:3. For fold shift
experiments, glutamate concentration–response curves were performed in
the presence of 30 final concentration of compound. Calcium
a final concentration
l
l
Acknowledgments
a
lM
The authors thank C. David Weaver, Emily L. Days, Tasha Naly-
wajko, Cheryl A. Austin and Michael Baxter Williams for their crit-
ical contributions to the HTS portion of the project as well as the
National Institute of Mental Health, the Michael J. Fox Foundation,
the Vanderbilt Department of Pharmacology and the Vanderbilt
Institute of Chemical Biology for support of this research.
fluorescence assays were employed for counterscreening rat mGluR1/Baby
Hamster Kidney (mGluR1/BHK) and rat mGluR5/HEK cells using a similar
triple-addition protocol employing appropriate EC20 and EC80 glutamate
concentration for each receptor, the exceptions being that cells were plated
at 20,000 cells/well and 15,000 cells/well in Assay Media, respectively.
Maximum calcium fluorescence, compared to control, was calculated for
the EC20 and EC80 peaks, respectively, after exporting raw plate data to
Microsoft Excel.
17. (a) Katsuhiko, N.; Kawano, H.; Sasaoka, S.; Ukawa, C.; Hirama, T.; Takada, A.;
Cottam, H. B.; Robins, R. K. J. Heterocycl. Chem. 1994, 31, 239; (b) Tiberghien, N.;
Lumley, J.; Reynolds, K.; Angell, R. M.; Matthews, N.; Cockerill, G. S.; Barnes,
M.C. WO 047288, 2005.
18. Daniels, R. N.; Kim, K.; Lewis, J. A.; Lebois, E. P.; Muchalski, H.; Lindsley, C. W.
Tetrahedron Lett. 2008, 49, 305.
19. Experimental for the synthesis of VU0080241, 7: 2,4-Dimethylhydrazine
hydrochloride was partitioned between 2 M sodium hydroxide solution
and dichloromethane. The organic layer was separated, dried and reduced
under vacuum to give the free hydrazine (1.90 g, 14 mmol). The free
References and notes
1. (a) Schoepp, D. D.; Jane, D. E.; Monn, J. A. Neuropharmacology 1999, 38, 1431;
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Marino, M. J.; Conn, P. J. Curr. Opin. Pharm. 2006, 8, 98.
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Jones, J. K.; Conn, P. J. Curr. Top. Med. Chem. 2005, 5, 847.
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Pharmacol. 1992, 227, 361; (b) Gasparini, F.; Bruno, V.; Battaglia, G.; Lukic, S.;
Leonhardt, T.; Inderbitzen, W.; Laurie, D.; Sommer, B.; Varney, M. A.; Hess, S.
D.; Johnson, E. C.; Kuhn, R.; Urwyler, S.; Sauer, D.; Poret, C.; Schmutz, M.;
Nicoletti, F.; Flor, P. J. J. Pharmacol. Exp. Ther. 1999, 289, 1678.
5. 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.
Neuropharmacology 2003, 45, 895.
hydrazine and ethoxymethylenemalononitrile
8
(1.70 g, 14 mmol) in
ethanol were irradiated at 105 °C for 10 min by microwave. The crude
product was recrystallized from ethanol to yield a yellow solid product 9
(2.30 g, 77%). 1H NMR (CDCl3, 400 MHz) d (ppm) 7.59 (s, 1H), 7.17 (s, 1H),
7.13 (s, 1H) 7.12 (s, 1H), 4.47 (s, 2H), 2.38 (s, 3H), 2.07 (s, 3H); 13C NMR
(CDCl3, 100 MHz) d (ppm) 150.51, 140.93, 140.59, 136.20, 132.27, 132.20,
127.85, 127.28, 114.34, 74.35, 21.13, 17.13; LCMS, single peak, 2.57 min,
m/e, 213.95 (M+1). Compound 9 (4.6 g, 21.7 mmol) was then treated with
concentrated H2SO4 (30 ml) at 0 °C. The reaction mixture was stirred at
room temperature for 1H then quenched with ice. The solution was
neutralized with aqueous NH4OH and filtered to provide yellow solid
product (4.77 g, 95%). A suspension of 5-amino-1-(2,4-dimethylphenyl)-1H-
pyrazole-4-carboxamide 10 (2.30 g, 10 mmol) in formamide was irradiated
at 200 °C for 20 min by microwave. The cooled solution was diluted with
water. The product was filtered, washed with water and dried over in
6. Marino, M. J.; Willaim, D. L., Jr.; O’Brien, J. A.; Valenti, O.; McDonald, T. P.;
Clements, M. K.; Wang, R.; DiLella, A. G.; Hess, J. F.; Kinney, G. G.; Conn, P. J.
PNAS 2003, 100, 13668.
7. Niswender, C. M.; Johnson, K. A.; Weaver, C. D.; Jones, C. K.; Xiang, Z.; Luo, Q.;
Rodriguez, A. L.; Marlo, J. E.; de Paulis, T., Thompson, A. D.; Days, E. L.;
Nalywajko, T.; Austin, C. A; Williams, M. B.; Ayala J. E.; Williams, R.; Lindsley, C.
W.; Conn, P. J. Mol Pharmacol, in press; doi: 10.1124/mol.108.049551.
8. Zhao, Z.; Wisnoski, D. D.; O’Brien, J. A.; Lemiare, W.; Williams, D. L., Jr.;
Jacobson, M. A.; Wittman, M.; Ha, S.; Schaffhauser, H.; Sur, C.; Pettibone, D. J.;
Duggan, M. E.; Conn, P. J.; Hartman, G. D.; Lindsley, C. W. Bioorg. Med. Chem.
Lett. 2007, 17, 1386.
vacuo to afford
a gray solid 1-(3,5-dimethylphenyl)-1H-pyrazolo[3,4-
d]pyrimidin-4-ol 11 (2.18 g, 91%). 1H NMR (DMSO, 400 MHz) d (ppm) 8.27 (s,
1H), 8.03 (s, 1H), 7.23 (d, J = 7.6 Hz, 2H), 7.23 (d, J = 7.6 Hz, 1H), 2.35 (s, 3H), 1.99
(s, 3H); 13C NMR (CDCl3, 100 MHz) d (ppm) 157.75, 153.10, 148.85, 139.28,
135.85, 135.02, 134.39, 131.73, 127.91, 127.40, 106.42, 21.07, 17.74; LCMS,
9. O’Brien, J. A.; Lemaire, W.; Wittmann, M.; Jacobson, M. A.; Ha, S. N.; Wisnoski,
D. D.; Lindsley, C. W.; Schaffhauser, H. J.; Sur, C.; Duggan, M. E.; Pettibone, D. J.;
Conn, J.; Williams, D. L. J. Pharmacol. Exp. Ther. 2004, 309, 568.
single peak, 2.36 min, m/e, 242.96 (M+1).
A suspension of 1-(2,4-
dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ol 11 (2.36 g, 9.82 mmol)
and POCl3 (3 ml) in dichloroethane (7 ml) was irradiated at 120 °C for 45 min
by microwave. The solvent was removed in vacuo to yield gray solid product. To a
solution of 4-chloro-1-(3,5-dimethylphenyl)-1H-pyrazolo[3,4-d]pyrimidine
(2.32 g, 9 mmol) and 3-methylpiperidine (3.76 g, 27 mmol) in DMF (24 ml)
was added triethylamine (3.16 ml, 27 mmol) at room temperature. The reaction
10. Lindsley, C. W.; Wisnoski, D. D.; Leister, W. H.; O’Brien, J. A.; Lemiare, W.;
Williams, D. L., Jr.; Burno, M.; Sur, C.; Kinney, G. G.; Pettibone, D. J.; Tiller, P. R.;
Smith, S.; Duggan, M. E.; Hartman, G. D.; Conn, P. J.; Huff, J. R. J. Med. Chem.
2004, 47, 5825.
11. Battaglia, G.; Busceti, C. L.; Molinaro, G.; Biagioni, F.; Traficante, A.; Nicoletti, F.;
Bruno, V. J. Neurosci. 2006, 26, 7222.

5630
C. M. Niswender et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5626–5630
mixture was irradiated in microwave at 90 °C for 15 min. The cooled solution
1H), 2.86 (t, J = 11.6 Hz, 1H), 2.38 (s, 3H), 2.17 (s, 3H), 1.98–1.60 (m, 4H), 1.35–
1.24 (m, 1H), 1.03 (d, J = 6.4 Hz, 3H); 13C NMR (CDCl3, 100 MHz) d (ppm) 155.83,
155.20,153.82, 140.04, 135.09, 133.61, 133.00, 132.08, 127.50, 127.22, 113.81,
33.06, 31.26, 25.18, 21.21, 21.18, 19.12, 17.88; LCMS, single peak, 2.87 min, m/e,
322.12 (M+1).
was treated with water to provide yellow solid VU 0080241 (7), 1-(2,4-
dimethylphenyl)-4-(3-methylpiperidin-1-yl)-1H-pyrazolo[3,4-d]pyrimidine (2.67 g,
84%). 1H NMR (CDCl3, 400 MHz) d (ppm) 8.35 (s, 1H), 8.12 (s, 1H), 7.24 (d,
J = 8.0 Hz, 1H), 7.17 (s, 1H), 7.13 (d, J = 8.0 Hz, 1H), 4.66 (s, 2H), 3.21 (t, J = 11.6 Hz,