Identification of novel, water-soluble, 2-amino-N-pyrimidin-4-yl acetamides as A2A receptor antagonists with in vivo efficacy

Article abstract of DOI:10.1021/jm070623o

Potent adenosine hA_2A receptor antagonists are often accompanied by poor aqueous solubility, which presents issues for drug development. Herein we describe the early exploration of the structure-activity relationships of a lead pyrimidin-4-yl a

Full text of DOI:10.1021/jm070623o

400
J. Med. Chem. 2008, 51, 400–406
Articles
Identification of Novel, Water-Soluble, 2-Amino-N-pyrimidin-4-yl Acetamides as A2A Receptor
Antagonists with In Vivo Efficacy
Deborah H. Slee,*,† Xiaohu Zhang,† Manisha Moorjani,† Emily Lin,† Marion C. Lanier,† Yongsheng Chen,† Jaimie K. Rueter,†
Sandra M. Lechner,‡ Stacy Markison,‡ Siobhan Malany,§ Tanya Joswig,‡ Mark Santos,§ Raymond S. Gross, John P. Williams,†
Julio C. Castro-Palomino,0 María I. Crespo,0 Maria Prat,0 Silvia Gual,0 José-Luis Díaz,0 Jenny Wen,# Zhihong O’Brien,# and
John Saunders†
Departments of Medicinal Chemistry, Pharmacology and Lead DiscoVery, Neuroscience, Chemical DeVelopment and Preclinical DeVelopment,
Neurocrine Biosciences, 12790 El Camino Real, San Diego, California 92130, and Almirall Research Center, Almirall, Ctra. Laureà Miró,
408-410, E-08980 St. Feliu de Llobregat, Barcelona, Spain
ReceiVed May 31, 2007
Potent adenosine hA2A receptor antagonists are often accompanied by poor aqueous solubility, which
presents issues for drug development. Herein we describe the early exploration of the structure–activity
relationships of a lead pyrimidin-4-yl acetamide series to provide potent and selective 2-amino-N-pyrimidin-
4-yl acetamides as hA2A receptor antagonists with excellent aqueous solubility. In addition, this series of
compounds has demonstrated good bioavailability and in vivo efficacy in a rodent model of Parkinson’s
disease, despite having reduced potency for the rat A2A receptor versus the human A2A receptor.
Introduction
The effects of adenosine are mediated through at least four
specific cell membrane receptors classified as A1, A2A, A2B, and
A3. These receptors belong to the G protein-coupled receptor
family.1 The A1 and A3 receptors decrease cellular cAMP levels
through their coupling to Gi proteins, which inhibit adenylate
cyclase. In contrast, A2A and A2B receptors couple to Gs/olf
proteins that activate adenylate cyclase and increase intracellular
levels of cAMP. While A1 receptors are widely distributed in
most areas of the brain and peripheral organs,1 A2A receptors
Figure 1. hA2A antagonists in clinical development (1 and 2) and lead
are highly concentrated in the dorsal striatum, nucleus accum-
bens, and olfactory tubercle.2 This unique cellular location of
the A2A receptor provides the anatomical basis for the
adenosine-dopamine interaction that underlies the motor
symptomatic benefits of A2A receptor antagonists in Parkinson’s
disease.
Adenosine A2A receptors modulate the release of GABA in
the striatum, which appears to regulate the activity of medium
spiny neurons. By reducing GABA output, A2A antagonism
helps restore normal function in the basal ganglia following
dopamine depletion. Thus, hA2A receptor antagonists may be a
useful treatment for neurodegenerative movement disorders such
as Parkinson’s and Huntington’s disease,3 dystonias such as
restless leg syndrome,4 and dyskinesias such as those caused
by prolonged use of neuroleptic and dopaminergic drugs.5
compound 3.
community for a number of years,6 however, several potent and
selective structures are now emerging. Kyowa Hakko Kogyo,
Schering-Plough, and Vernalis/Biogen have taken hA2A receptor
antagonist drugs into the clinic for the treatment of Parkinson’s
disease. Kyowa has recently filed an NDA for compound 1,7
(KW6002, Figure 1), Schering-Plough has taken compound 2
(SCH420814, Figure 1)8 into phase II clinical trials, and
Vernalis/Biogen have entered phase II trials with V2006
(structure not yet disclosed).9
Poor solubility is a common issue with many adenosine
antagonists, for example, 2 is reported to have solubility of less
than 10 ng/mL at pH 7.4.8 The work herein describes a novel
series of potent, selective, and highly soluble hA2A antagonists
for the treatment of Parkinson’s disease, as represented by
compound 3 in Figure 1.
The development of potent and selective antagonists of the
hA2A receptor has challenged the medicinal chemistry research
Chemistry
* To whom correspondence should be addressed. Tel.: 858 617-7849.
Fax: 858 617-7619. E-mail: dslee@neurocrine.com.
The pyrimidine analogues, 3, 17, 18, and 20–23 were
synthesized according to a number of methods, as described
below in Schemes 1–4. The method used to make key
intermediates involved conversion of the commercially available
nitrile 4 to the corresponding carboxyamidine 5 via treatment
with sodium methoxide in methanol at room temperature,
†
Medicinal Chemistry, Neurocrine Biosciences.
Neuroscience, Neurocrine Biosciences.
Pharmacology, Neurocrine Biosciences.
‡
§
Chemical Development, Neurocrine Biosciences.
Almirall Research Center.
Preclinical Development, Neurocrine Biosciences.
0
#
10.1021/jm070623o CCC: $40.75
2008 American Chemical Society
Published on Web 01/12/2008

Water-Soluble 2-Amino-N-pyrimidin-4-yl Acetamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 401
Scheme 1a
a Reagents and conditions: (a) NaOMe, (1.1 equiv), NH4Cl, MeOH, rt, 6–12 h, 96%; (b) ethyl cyanoacetate, NaOMe, EtOH, 67%; (c) NaOEt, diethylmalonate,
rt-reflux, 12–60 h, 78%; (d) POCl3, DIPEA, 90 °C, 3–12 h, 87%; (e) NH4OH, MeOH, 80 °C, 12 h, 76%.
Scheme 2a
a Reagents and conditions: (a) diethyl carbonate (5 equiv), NaH, 80 °C, 12 h, 56%; (b) KOtBu, (1.5 equiv), tBuOH, 90 °C, 12 h, 50%; (c) POCl3, DIPEA,
90 °C, 3–12 h, 66%; (d) NH4OH, MeOH, 80 °C, 12 h, 53%.
Scheme 3a
Figure 2. Lead structures 17 and 18, with three groups R1, R2, and
R3 for optimization.
worked well, but on larger scale, the phosphorus oxychloride
a Reagents and conditions: (a) N2H4, EtOH, 90 °C; (b) pentane-2,4-dione,
0-90 °C, 2 h, 72%; (c) pyrazole, Cs2CO3, DMF, 85 °C, 21 h, 55%.
reaction step was cleaner with intermediate 7 than with 6,
therefore, this slightly longer route was preferred. Further
reaction of 9 to install R2 (as defined in Figure 2) is illustrated
in Scheme 3.
Scheme 4a
There are a number of commercially available ethyl 3-oxo-
propanoates, however, 11 was synthesized via the reaction of
the desired methyl ketone, in this case, 10, with diethyl
carbonate, using sodium hydride as the base to give 3-oxo-3-
thiazol-2-yl-propionic acid ethyl ester 11 in good yield (Scheme
2). Compound 11 was then reacted with the carboxyamidine 5
to install R2 directly. After treatment with phosphorus oxychlo-
ride to give 13, reaction with ammonium hydroxide yielded the
corresponding 6-heteroaryl-pyrimidin-4-amine intermediate 14
(Scheme 2). While this second route was less versatile, it was
more convenient for the thiazole analogue. Intermediate 14 was
further manipulated, as described in Scheme 4.
a Reagents and conditions: (a) acetylchloride, pyridine, DCM, rt, 12 h,
80–95%; (b) KOH (aq), dioxane, 90 °C, 30 min-2 h, 90–95%; (c)
chloroacetyl chloride, pyridine, DCM, rt, 2–12 h, 60–85%; (d) primary or
secondary amine, Et3N, rt, 6–12 h.
Derivatization of the key 6-chloropyrimidin-4-amine inter-
mediate 9 was carried out to give a number of analogues, as
shown in Scheme 3. The dimethyl-substituted pyrazole analogue
was synthesized by first displacing the chloride with hydrazine
to give 15, followed by cyclization with pentane-2,4-dione to
give intermediate 16. Alternatively, the chloride can be displaced
with pyrazole directly to give intermediate 17.
followed by reaction with ammonium chloride. The resulting
carboxyamidine was then reacted with diethyl malonate in
methanol in the presence of sodium ethoxide to yield the
corresponding pyrimidine-4,6-diol 7. Intermediate 7 was then
reacted with phosphorus oxychloride, in the presence of N,N-
diisopropyl-ethylamine to yield the 4,6-dichloropyrimidine 8,
which was then treated with refluxing ammonium hydroxide in
methanol to yield the 6-chloropyrimidin-4-amine intermediate
9. Alternatively, 9 can be formed in two steps (from 5) via
reaction with ethyl cyanoacetate to give intermediate 6, followed
by treatment with phosphorus oxychloride. This latter route
The aminopyrimidine intermediates 14, 16, and 17 described
above were then converted to the final compounds via the routes
outlined in Scheme 4. Compound 17 was acylated using
acetylchloride and pyridine to give compound 18. Compounds

402 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Slee et al.
Table 1.
22, and 23 were selected to be further studied for in vivo
efficacy. The haloperidol-induced catalepsy (HIC) model in rat
was used as the primary assay to screen compounds for efficacy.
Compound 1 is reported to work in this model down to 0.3
mg/kg.12 As shown in Figure 3, all three analogues 3, 22, and
23 were efficacious at the screening dose of 10 mg/kg p.o. It is
worth noting that the hA2A receptor antagonist activity for these
compounds was on the order of 10-fold less for the rat A2A
(rA2A) receptor than for the hA2A receptor (Table 3). In contrast,
compound 1 was found to be slightly less potent against hA2A
than rA2A (Ki 6 and 2 nM, respectively).
Ki (nM) ( SEM
a
b
cmpd
hA2A
hA1
hA1/hA2A
17
18
2.9 ( 0.1
0.6 ( 0.4
56 ( 1
9.2 ( 0.6
20
15
a Displacement of specific [3H]-ZM241385 binding at human A2A
receptors expressed in HEK293. b Displacement of specific [3H]-DPCPX
binding at human A1 receptors expressed in HEK293 cells. Data are
expressed as mean values of at least three runs ( SEM.
3 and 20-23 (Table 2) were made via reaction of the
corresponding intermediates 19a, b, or c with the appropriate
amine.
Conclusions
We have developed a novel series of potent and selective
adenosine hA2A antagonists that contain basic amines that confer
excellent physical properties for drug development. Although
in most cases the compounds were less potent against the rA2A
receptor, we were able to demonstrate in vivo efficacy for this
series in a rat model of haloperidol-induced catalepsy. Further
optimization and evaluation of this series in other models of
Parkinson’s disease will be reported in due course.
Results and Discussion
Compound 17 was identified as an antagonist of the hA2A
receptor with a Ki of 2.9 nM (Figure 2).
The main deficiencies of this molecule appeared to be the
lack of selectivity for hA2A versus hA1 (20-fold), the relatively
poor metabolic stability in human liver microsomes (scaled
intrinsic clearance 73 mL/min/kg), and the limited aqueous
solubility (0.08 mg/mL in PBS). Interestingly, acylation of
compound 17 to give compound 18 led to a significant increase
in potency (Ki 0.6 nM, Table 1). Although the selectivity for
hA2A over hA1 was not significantly enhanced, this finding
opened up an area for further exploration, and a series of
modifications were made via acylation of the aromatic amine.
Incorporation of polar substituents in the R3 region, in an attempt
to immediately address the issues of solubility, gave encouraging
results.
Experimental Section
Solubility Assay Protocol. To determine solubility of com-
pounds, approximately 1 mg of sample was weighed into a Falcon
centrifuge tube (15 mL), and the weight was recorded to 0.001
mg. Assay medium (200 µL, phosphate buffered saline (PBS), pH
7.4, or water, etc.) was added, and the sample was sonicated for
10 min and then shaken overnight. The sample was then centrifuged,
and the supernatant was analyzed by HPLC to determine the
concentration of sample in solution. The concentration in solution
was then calculated based on a standard curve generated from
known dilutions of authentic sample.
The addition of a single amino moiety to give compound 20
(Table 2) improved physical properties and potency against hA2A
was maintained. Further analogues were made and it was found
that the incorporation of a morpholine ring (compound 21) both
maintained potency and enhanced the selectivity for hA2A over
hA1. Replacement of the morpholine with a piperazine ring to
give compound 3 further increased selectivity and gave a highly
soluble compound when converted to the HCl salt (>25 mg/
mL in water). In addition, compound 3 had a measured Log
D of 2.2 and pKa of 7.910 (distal piperazine nitrogen), with
a molecular weight of 367, which are good physical properties
for achieving oral bioavailability and cell permeability.
Metabolic stability, measured in human liver microsomes,
was also much improved (scaled intrinsic clearance 6 mL/
min/kg) over that of 17, presumably due to the increased
hydrophilic nature of this compound. Indeed, compound 3
had good bioavailability in rat (%F 79, AUC 2870 ng · h/
mL, from a 10 mg/kg oral dose, unpublished data). As can
be seen from the brain exposure at 2 h (Table 3), compound
3 and the closely related and physically similar analogue
compound 22 also have good exposure in the brain. In
addition, these compounds showed no significant inhibition
of the major cytochrome P450 enzymes (>10 µM, CYP2D6
and CYP3A4). Compound 3 was also assayed against the
human A2B and human A3 receptors at CEREP11 and was
found to bind with Ki values of 35 and 660 nM, respectively.
The effect of subtle variations to the R2 region was also
explored (see Figure 2 and Scheme 4). Both the thiazole
(compound 22) and the dimethyl pyrazole (compound 23) ring
system variants were well tolerated at the R2 position, with
compound 23 having 864-fold selectivity for hA2A over hA1.
This is presumably due to the ability of hA2A to tolerate the
increased steric bulk of the methyl groups, whereas for hA1,
this increase in bulk cannot be accommodated. Compounds 3,
Biology Experimental
Pharmacology. Adenosine A1 and A2A Receptor Bind-
ing Assays. Receptor Cloning. The coding sequence of the
human A1 and A2A receptor was amplified from a human brain
cDNA library by the polymerase chain reaction. Each ampli-
con was cloned into the pcDNA5/FRT/V5-His-TOPO expression
vector (Invitrogen), and the sequence was confirmed using an
ABI 3100 automated sequencer (Applied Biosystems). Each
expression construct was transfected into Flp-In HEK cells
(Invitrogen) using Lipofectamine 2000 (Invitrogen). Cells stably
expressing either the human A1 or A2A receptor were selected
using 1 mg/mL hygromycin in complete DMEM.
Membrane Preparation. Crude membranes were prepared
from Flp-In HEK cells transfected with either the human A1 or
A2A receptor by resuspending cells in lysis buffer (50 mM Tris-
HCl pH 7.4, 5 mM EDTA, 10 mM MgCl2) and disrupting under
N2 at a pressure of 900 psi (Parr Cell disruption bomb, cat.
#4639) for 30 min on ice, followed by differential centrifugation.
The resulting crude membrane pellet was resuspended in assay
buffer (50 mM Tris-HCl pH 7.4, 1 mM EDTA, 10 mM MgCl2).
Membrane protein concentration was determined by Bradford
assay. Membranes of cloned rat A2A receptor produced in CHO
cells were obtained from Perkin-Elmer (SignalScreen, Lot no.
6110536-09). Membrane aliquots were stored at -80 °C.
Binding Assay. An aliquot of membranes (1–2 µg of protein)
was preincubated for 30 min at RT in the presence of 10 µg/
mL adenosine deaminase (type IV calf spleen, Sigma). Mem-
branes were then incubated for 90 min with either 1.0 nM
[3H]DPCPX (120.00 Ci/mmol Perkin-Elmer NET 974) for the
A1 membranes or 2.0 nM [3H]-ZM 241385 (27.40 Ci/mmol
Tocris R1036) for the A2A human and rat membranes in the

Water-Soluble 2-Amino-N-pyrimidin-4-yl Acetamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 403
Table 2.
a See Table 1. Data are expressed as mean values of at least three runs ( SEM. b See Table 1. Data are expressed as mean values of at least three runs
( SEM.
Table 3.
counting using a TopCount-NXT (Packard). Binding data were
analyzed by nonlinear, least-squares curve fitting algorithms
using GraphPad Prism (GraphPad Software, Inc., San Diego,
CA) or ActivityBase (IDBS, Guildford, Surrey, U.K.). Ki values
were calculated from IC50 values using the Cheng-Prusoff
equation (Cheng and Prusoff, 1973).13
a
b
hA2A Ki
rA2A Ki
concn in rat
brainc (ng/g)
cmpd
(nM) ( SEM
(nM) ( SEM
3
22
23
2.7 ( 0.2
0.9 ( 0.1
2.0 ( 0.2
26 ( 4
11 ( 2
14 ( 2
2700
3200
600
a See Table 1. b Displacement of specific [3H]-ZM241385 binding at rat
2A receptors expressed in CHO cells. Data are expressed as mean values
3
For A1 membrane assay, H-DPCPX measured Kd ) 1.0 (
A
0.5 nM and Bmax ) 8 ( 4 pmol/mg by Scatchard Analysis.
Control DPCPX (Tocris, 0439) Ki ) 1.6 ( 0.7 nM. For human
of at least three runs ( SEM. c At 2 h after dosing with a 10 mg/kg oral
(p.o.) dose.
3
A2A membrane assay, H-ZM241385 measured Kd ) 0.22 (
0.20 nM and Bmax ) 33 ( 8 pmol/mg by Scatchard Analysis.
Control NBI 80634 Ki ) 0.25 ( 0.04 nM. For rat A2A
3
membrane assay, H-ZM241385 measured Kd ) 0.33 ( 0.20
nM and Bmax ) 2.4 pmol/mg by Scatchard Analysis. Control
NBI 80634 Ki ) 1.2 ( 0.3 nM.
In Vivo Assays. Selected compounds were tested for their
effects on haloperidol-induced catalepsy, measured with the bar
test. Separate groups of rats were used to test each compound
(n ) 24/experiment). Male Wistar rats (Charles River) were
habituated to the vivarium for 1 week prior to testing. Rats were
weighed (∼220 g at time of testing) and administered test
compound orally (p.o.) 2 h prior to the bar test. The vehicle for
all test compounds was 5% cremophor in sterile water given at
a volume of 5 mL/kg. One hour prior to the bar test, rats were
given an intraperitoneal (i.p.) injection of haloperidol (1.5 mg/
kg in a 10 mL/kg of sterile water). The bar test consisted of 3
and 30 s trials spaced 3 min apart. During a test, the rat’s
forepaws were placed on an 8.0 cm high metal bar. The descent
latency of one paw from the bar was measured and recorded
(in seconds) as the score for a given trial. The descent latency
from each of the three trials was averaged to obtain the mean
descent latency for each rat. Plasma and brain were collected
following the haloperidol-induced catalepsy test (samples col-
lected 2 h post dose), and samples were analyzed to determine
exposure (the method is described below in Preclinical Assays).
Figure 3. Effect of compounds 3, 22, and 23 in the haloperidol-induced
catalepsy bar test. Mean descent latency ((standard error) in seconds.
The vehicle bar represents the mean and standard error from 3
experiments. *Vehicle vs treated rats, p < 0.05.
presence of varying concentrations of competing ligand. Non-
specific binding was determined in the presence of excess (1
µM) DPCPX or CGS15943 for the A1 and A2A membranes,
respectively. Bound and free ligands were separated by rapid
vacuum filtration using a Packard 96-well cell harvester onto
UniFilter GF/C filter plates (Perkin Elmer) that had been
pretreated with 0.5% polyethyleneimine. The filter plates were
than washed 3 × 200 µL with 50 mM Tris-HCl and 50 mM
NaCl, pH 7.4. Bound radioligand was determined by scintillation
Preclinical Assays. Metabolic Stability Assay in Rat
and Human Liver Microsomes. Pooled male human and rat

404 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Slee et al.
liver microsomes (0.5 mg/mL for human and 0.1 mg/mL for
rat; n > 10; mixed gender) were incubated at 37 °C with the
NCE in the presence of an NADPH-generating system contain-
ing 50 mM, pH 7.4, potassium phosphate buffer, 3 mM
magnesium chloride, 1 mM EDTA, 1 mM NADP, 5 mM G-6-
P, and 1 unit/mL G-6-PD. Incubations were conducted with 1
µM of each NCE (0.01% DMSO), with a total volume of 250
µL in duplicate at each time point (0, 5, 10, 20, 40, and 60
min). Reactions were stopped by the addition of 0.3 mL of
acetonitrile containing a proprietary internal standard. Precipi-
tated proteins were removed by centrifugation for 15 min at
3000 rpm, and the supernatant fluid (∼0.1 mL) was analyzed
by LC/MS for the % of parent compound remaining. The in
vitro initial rates of metabolism were scaled using constants,
such as microsomal protein/gm liver, gm liver/kg body weight,
and liver blood flow, to predict systemic clearance and %
bioavailability. These calculations from nonlinear regression
assume that liver metabolism, not absorption, is the determinant
of bioavailability.
Büchi 535 apparatus. Descriptions of analytical HPLC-MS
methods 1–6 are given in Supporting Information; preparative
HPLC-MS; platform, Dionex, equipped with a Gilson 215
autosampler/fraction collector, UV detector, and a Dionex MSQ
mass detector; HPLC column, Phenomenex Synergy Max-RP,
21.2 × 50 mm. HPLC gradient, 35 mL/min, 5% acetonitrile in
water to 95% acetonitrile in water in 17.7 min. Both acetonitrile
and water have 0.05% TFA.
2-Furancarboxamidine (5). To a solution of sodium meth-
oxide (3.0 g, 56 mmol) in methanol (50 mL) in a round-
bottomed flask equipped with a stirrer bar was added 2-furoni-
trile (5.0 g, 53 mmol). The mixture was stirred at room
temperature for 3 h. To the resulting solution was slowly added
ammonium chloride (3.0 g, 60 mmol), and the mixture was
stirred at room temperature for 68 h. The resulting suspension
was filtered, and the solvent was removed under reduced
pressure. The solid obtained was washed with diethyl ether (3
× 25 mL) to give compound 5 as an off-white solid (HCl salt;
1
7.5 g, 96%). H NMR (300 MHz, DMSO-d6): δ 9.22 (s, 3H),
8.19 (s, 1H), 7.89 (d, J ) 3.8, 1H), 6.88–6.86 (m, 1H).
Screening PK Protocol for Sample Extraction and Anal-
ysis To Determine Brain Exposure in Rat at the 2 h Time
Point. Sample extraction and analysis to determine brain
exposure in rat at the 2 h time point was carried out as follows.
Brain tissue sample analysis was conducted with a liquid
chromatography in tandem with a mass spectrometric detector
(LC/MS/MS) method. Half of the brain (left or right) was
weighed and homogenized in 2.0 mL of ACN/H2O/FA (formic
acid; v/v/v 60/40/0.1) containing 50 µL internal standard (10
µg/mL NBI 67377). The homogenates were centrifuged at 3000
rpm for 20 min, and the supernatant was collected for LC/MS/
MS analysis. Brain standards were prepared by spiking 1.0 mL
of a series of premade calibration standard aqueous solutions
(prepared in 60/40 ACN/H2O), 1.0 mL of ACN/H2O/FA (v/v/v
60/40/0.2), and 50 µL internal standard (10 µg/ml) into half-
untreated rat brains. Standards for brain were processed and
analyzed at the same time and in exactly the same way as the
analytical samples.
2-(2-Furyl)pyrimidine-4,6-diol (7). To a solution of sodium
ethoxide (10.8 g, 200 mmol) in ethanol (90 mL) was slowly
added compound 5 (5.6 g, 38 mmol). The mixture was stirred
at room temperature for 30 min, and then diethyl malonate (5.0
g, 30 mmol) was added. The flask was fitted with a condenser
and the suspension was refluxed for 32 h. The solvent was
removed under reduced pressure, and the residue was suspended
in water (100 mL) and acidified to pH ) 6 with 5 N
hydrochloric acid. The resulting solid was filtered and washed
with water (50 mL), ethanol/ethyl ether (4:1, 25 mL), and ethyl
ether (2 × 25 mL). Compound 7 was obtained as a pale yellow
solid (4.2 g, 78%). 1H NMR (300 MHz, DMSO-d6): δ 7.80 (s,
1H), 7.40 (d, J ) 3.4, 1H), 6.60–6.70 (m, 1H), 5.00 (s, 1H).
4,6-Dichloro-2-(2-furyl)pyrimidine (8). To a suspension of
compound 7 (3.0 g, 17 mmol) was added N,N-diisopropyleth-
ylamine (3.9 g, 30 mmol) and phosphorus oxychloride (17 mL).
The flask was fitted with a condenser and the mixture was
refluxed for 3 h. The solvent was removed under reduced
pressure and methylene chloride (50 mL) and ice were slowly
added. The organic layer was washed with water (2 × 25 mL),
saturated solution of sodium bicarbonate (2 × 25 mL), and brine
and dried (Na2SO4). The solvent was removed under reduced
Bioanalytical Analysis. The LC/MS/MS system was equipped
with an Agilent 1100 HPLC system coupled to a CTC PAL
autoinjector (Leap Technologies, Carrboro, NC) and a Sciex
API 4000 triple quadruple mass spectrometer (Toronto, Canada).
Samples were eluted at 0.45 mL/min using a mobile phase
consisting of solution A (0.1% formic acid in water) and solution
B (0.1% formic acid in acetonitrile) with a fast gradient (0–0.1
min, 5% B; 0.1–1.0 min, 5 to 95% B; hold to 2.5 min; 2.5–2.7
min, 95–5% B; hold to 3.5 min) and a column temperature of
20 °C. The HPLC column used for this assay was Agilent
Zorbax SB-C18 column, 2.1 × 50 mm, with a particle size of
5 µm. Electrospray ionization source was used for detection at
a positive MRM (multiple reaction monitoring) mode with the
ion pair of 368.18f113.05. Quantification was performed by
fitting peak area ratios (peak of parent analyte vs peak of the
internal standard) to a weighted (1/x) linear calibration curve.
1
pressure to give compound 8 as a gray solid (3.2 g, 87%). H
NMR (300 MHz, CDCl3): δ 7.68 (s, 1H), 7.46 (d, J ) 3.4,
1H), 7.22 (s, 1H), 6.63–6.61 (m, 1H).
6-Chloro-2-(2-furyl)pyrimidin-4-amine (9). A suspension
of compound 8 (2.0 g, 9.3 mmol) in methanol (14 mL) and
30% ammonium hydroxide (27 mL) was heated in a pressure
reactor for 20 h. The solvent was partially removed under
reduced pressure. The resulting solid was filtered, washed with
water (25 mL) and ethyl ether (25 mL), and dried. Compound
1
9 was obtained as an off-white solid (1.5 g, 76%). H NMR
(400 MHz, CDCl3): δ 7.58 (s, 1H), 7.28 (d, J ) 3.7, 1H), 6.54
(m, 1H), 6.31 (s, 1H), 5.21 (bs, 2H).
Experimental for Intermediate Synthesis and Ana-
lytical Methods. Reagents, starting materials, and solvents were
purchased from commercial suppliers and used as received.
Concentration refers to evaporation under vacuum using a Büchi
rotatory evaporator. Reaction products were purified, when
necessary, by flash chromatography on silica gel (40–63 µm)
with the solvent system indicated. Spectroscopic data were
recorded on a Varian Mercury 300 MHz spectrometer, Bruker
DPX-250 spectrometer, or Bruker Avance 500 MHz spectrom-
eter. The elemental analysis was done by Robertson Microlit
Laboratory, Madison, NJ. Melting points were recorded on a
2-Chloro-N-(2-furan-2-yl-6-pyrazol-1-yl-pyrimidin-4-yl)-
acetamide (19c). To 5 mL of dichloromethane were added
compound 17 (0.3 g 1.3 mmol), chloroacetyl chloride (0.2 g,
0.2 mmol), and pyridine (0.2 g, 0.2 mmol). The reaction mixture
was stirred at room temperature for 2 h. The reaction was
quenched with 5 mL of saturated sodium bicarbonate and
extracted with dichloromethane (3 × 15 mL). The organic layers
were combined and dried over sodium sulfate, then concentrated
to give compound 19c as a yellow solid (0.4 g, 100% crude
yield), which was used crude in subsequent reactions.

Water-Soluble 2-Amino-N-pyrimidin-4-yl Acetamides
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3 405
3-Oxo-3-thiazol-2-yl-propionic Acid Ethyl Ester (11). To
a solution of 60% sodium hydride (3.8 g, 95 mmol) in diethyl
carbonate (90 mL) was slowly added 2-acetylthiazole 10 (5.0
g, 40 mmol). The resulting solution was stirred at room
temperature for 1 h and at 90 °C for 2 h. The reaction mixture
was poured into ice/water and acetic acid (5 mL) was added.
The mixture was extracted with ethyl acetate (2 × 75 mL). The
organic layer was washed with water (2 × 50 mL) and brine
(50 mL) and dried (Na2SO4), and the solvent was removed under
reduced pressure. Distillation under reduced pressure gave the
mixture was dissolved in CH2Cl2 (50 mL) and water (25 mL).
The layers were separated, and the aqueous layer was extracted
with CH2Cl2 (4 × 25 mL). The combined organic layers were
washed with brine (25 mL), dried over magnesium sulfate,
filtered, and concentrated. The crude product was purified by
flash chromatography using 1:1 EtOAc/hexanes to give the title
compound 16 as a white solid (0.9 g, 72%). LCMS-1: tR
)
2.44 (100%). MS: m/z 256 [M + H]+, expected 256 [M + H]+.
2-Chloro-N-[6-(3,5-dimethyl-pyrazol-1-yl)-2-furan-2-yl-py-
rimidin-4-yl]-acetamide (19b). Compound 19b was prepared
according to the procedures for 19c above, except that inter-
mediate 16 was used instead of intermediate 17 to give the title
compound 19b as a pale yellow solid (64% yield), which was
used crude in subsequent reactions. LCMS-1: tR ) 2.89 (100%).
MS: m/z 332 [M + H]+, expected 332 [M + H]+.
2-(2-Furyl)-6-(1H-pyrazol-1-yl)pyrimidin-4-amine (17). To
a solution of compound 9 (1.0 g, 5.1 mmol) in anhydrous DMF
(20 mL) was added pyrazole (0.7 g, 10 mmol) and cesium
carbonate (3.3 g, 10 mmol). The mixture was heated at 85 °C
for 21 h. The solution was poured into water (50 mL) and
extracted with ethyl acetate (2 × 25 mL). The organic layer
was washed with water (2 × 25 mL) and brine (25 mL) and
dried (Na2SO4), and the solvent was removed under reduced
pressure. The resulting solid was purified by column chroma-
tography with silica gel, eluting with methylene chloride/
methanol (3%), to give compound 17 as an off-white solid (0.6
g, 55%). 1H NMR (250 MHz, CDCl3): δ 8.63 (d, J ) 3.0, 1H),
7.75 (d, J ) 1.2, 1H), 7.61 (s, 1H), 7.31 (d, J ) 3.6, 1H), 6.90
(s, 1H), 6.57–6.55 (m, 1H), 6.48–6.46 (m, 1H), 5.12 (bs, 2H).
LCMS-2: tR ) 4.2 (98%). LCMS-3: tR ) 6.4 (98%). MS: m/z
228 [M + H]+, expected 228 [M + H]+.
1
title compound 11 as a pale orange oil (4.4 g, 56%). H NMR
(250 MHz, CDCl3): δ 7.99 (d, J ) 5.3, 1H), 7.71 (d, J ) 5.3,
1H), 4.15 (m, 4H), 1.23 (t, 3H).
2-(2-Furyl)-6-(1,3-thiazol-2-yl)pyrimidin-4-ol (12). To a
solution of potassium tert-butoxide (0.6 g, 6.0 mmol) in tert-
butanol (2 mL) were added compound 5 (0.9 g, 4.3 mmol) and
compound 11 (0.8 g, 4.7 mmol). The mixture was heated at
135 °C for 3 h. The crude reaction was poured into water (20
mL) and acidified with 10% hydrochloric acid (25 mL). The
resulting solid was filtered, washed with water (2 × 25 mL),
and dried. The title compound 12 was obtained as an off-white
1
solid (0.6 g, 50%). H NMR (250 MHz, CDCl3): δ 8.05–8.15
(m, 3H), 7.64 (d, J ) 2.8, 1H), 6.90 (s, 1H), 6.78 (d, J ) 2.8,
1H).
4-Chloro-2-(2-furyl)-6-(1,3-thiazol-2-yl)pyrimidine (13). A
suspension of compound 12 (0.6 g) in phosphorus oxychloride
(20 mL) was refluxed for 24 h. The solvent was removed under
pressure and ice and water were slowly added. The resulting
solid was filtered, washed with 2 N sodium hydroxide, and dried.
Purification by column chromatography with silica gel and
methylene chloride as eluent gave compound 13 as an off-white
solid (0.4 g, 66%).
N-(2-Furan-2-yl-6-pyrazol-1-yl-pyrimidin-4-yl)-aceta-
mide (18). To a solution of compound 17 (0.3 g, 1.3 mmol) in
methylene chloride (7 mL) was added pyridine (0.2 g, 2.6 mmol)
and acetyl chloride (0.2 g, 2.6 mmol). The mixture was stirred
at room temperature for 5 h, and more pyridine (52 mg, 0.7
mmol) and acetyl chloride (52 mg, 0.7 mmol) was added. The
reaction was allowed to stand for 1.5 h at room temperature.
The solution was diluted with methylene chloride (20 mL),
washed with 10% sodium hydroxide (2 × 10 mL) and brine
(10 mL) and dried (Na2SO4). The solvent was removed under
reduced pressure. Purification by column chromatography with
silica gel, eluting with ethyl acetate/n-hexane (1:3), gave
2-(2-Furyl)-6-(1,3-thiazol-2-yl)pyrimidin-4-amine (14). A
suspension of compound 13 (0.3 g) in ethanol (22 mL) and 30%
ammonium hydroxide (22 mL) was heated at 120 °C in a
pressure reactor for 2 h 30 min. The solvent was removed under
reduced pressure and the residue was dissolved in ethyl acetate
(50 mL). The resulting solution was washed with water (2 ×
25 mL) and brine (25 mL) and dried (Na2SO4), and the solvent
was removed under reduced pressure. Purification by trituration
with ethyl ether gave compound 14 as an off-white solid (0.1
1
g, 53%). H NMR (250 MHz, DMSO-d6): δ 8.05 (d, J ) 3.0,
1H), 7.95 (d, J ) 3.0, 1H), 7.85 (d, J ) 1.5, 1H), 7.28 (bs,
2H), 7.18 (d, J ) 3.4, 1H), 7.05 (s, 1H), 6.64 (dd, J ) 3.0, 1.0,
1H).
1
compound 18 as an off-white solid (0.33 g, 92%). H NMR
(300 MHz, CDCl3): δ 8.64 (d, J ) 2.1, 1H), 8.53 (s, 1H), 8.21
(bs, 1H), 7.81 (bs, 1H), 7.62 (bs, 1H), 7.35 (d, J ) 2.1, 1H),
6.61–6.58 (m, 1H), 6.51–6.49 (m, 1H), 2.25 (s, 3H). LCMS-2:
tR ) 5.6 (99%). LCMS-4: tR ) 4.1 (97%). MS: m/z 270 [M +
H]+, expected 270 [M + H]+.
General Method for Compounds 3, 20, and 21. To 10 mL
of dichloromethane were added compound 19c (1.6 g, 5.3
mmol), DIPEA (1.8 mL, 10 mmol), and either dimethylamine,
morpholine, or 4-methyl piperazine (2.0 equiv). The reaction
mixture was stirred at room temperature overnight. The reaction
was quenched with 5 mL of water and extracted with dichlo-
romethane (3 × 15 mL). The organic layers were combined
and dried over sodium sulfate and then concentrated. The residue
was purified by column chromatography on silica gel (DCM
with methanol gradient from 0 to 5%) to give the following:
2-Chloro-N-(2-furan-2-yl-6-thiazol-2-yl-pyrimidin-4-yl)-ac-
etamide (19a). Compound 19a was prepared according to the
procedures for 19c above, except that intermediate 14 was used
instead of intermediate 17 to give the title compound 19c, a
yellow solid (quantitative yield) that was used crude in
1
subsequent reactions. H NMR (300 MHz, DMSO-d6): δ 8.53
(s, 1H), 8.11 (d, J ) 3.0, 1H), 8.04 (d, J ) 3.0, 1H), 7.25 (d,
J ) 3.6, 1H), 6.36 (d, J ) 3.3, 1H), 4.44 (s, 2H), 2.39 (s, 3H).
LCMS-1: tR ) 2.34 (100%). MS: m/z 335 [M + H]+, expected
335 [M + H]+.
6-(3,5-Dimethyl-pyrazol-1-yl)-2-furan-2-yl-pyrimidin-4-
ylamine (16). To a solution of intermediate 9 (1.0 g, 5.0 mmol)
in absolute EtOH (4.5 mL) was added anhydrous hydrazine (0.3
mL, 10 mmol). The mixture was heated at 90 °C for 22 h in a
sealed flask. The reaction was then cooled to room temperature
and placed in an ice bath at 0 °C, while 2,4-pentanedione (1.0
mL, 10 mmol) was added slowly. The reaction mixture was
heated at 90 °C for 2 h. Upon consumption of the hydrazine
intermediate, the reaction was evaporated completely. The crude
N-(2-Furan-2-yl-6-pyrazol-1-yl-pyrimidin-4-yl)-2-(4-meth-
yl-piperazin-1-yl)-acetamide (3). Compound 3 as a pale yellow
1
solid (70% yield). H NMR (500 MHz, D2O): δ 7.56 (d, J )
1.2, 1H), 7.39 (s, 1H), 7.24 (s, 1H), 7.02 (s, 1H), 6.45 (d, J )
2.3, 1H), 6.21 (s, 1H), 6.09 (s, 1H), 4.01 (s, 2H), 3.82–3.74 (m,

406 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 3
Slee et al.
4H), 3.60–3.48 (m, 4H), 3.02 (s, 3H). MS: m/z 368 [M + H]+,
expected 368 [M + H]+. Anal. (C18H21N7O2 ·2HCl·0.9H2O)
C, H, N.
2-Dimethylamino-N-(2-furan-2-yl-6-pyrazol-1-yl-pyrimi-
din-4-yl)-acetamide (20). Compound 20 as a pale yellow solid
(50% yield). 1H NMR (300 MHz, CDCl3): δ 9.87 (s, 1H), 8.65
(d, J ) 3, 1H), 8.62 (s, 1H), 7.81 (d, J ) 1, 1H), 7.65 (d, J )
1, 1H), 7.37 (d, J ) 3.6, 1H), 6.59 (dd, J ) 3.6, 1.8, 1H), 6.50
(dd, J ) 3.0, 1.5, 1H), 3.16 (s, 2H), 2.39 (s, 6H). LCMS-2: tR
) 3.8 (98%). LCMS-4: tR ) 5.2 (100%). MS: m/z 313 [M +
H]+, expected 313 [M + H]+.
N-(2-Furan-2-yl-6-pyrazol-1-yl-pyrimidin-4-yl)-2-morpho-
lin-4-yl-acetamide (21). Compound 21 as a white foam (0.5 g,
27% yield). 1H NMR (300 MHz, CD3OD): δ 8.65 (s, 1H), 8.32
(s, 1H), 7.98 (s, 1H), 7.69 (s, 1H), 7.31 (s, 1H), 6.59 (s, 1H),
6.54 (s, 1H), 3.80–4.15 (m, 4H), 3.30–3.75 (m, 4H), 3.30 (s,
2H). LCMS-5: tR ) 13.0 (99%). LCMS-6: tR ) 22.7, MS: m/z
355 [M + H]+, expected 355 [M + H]+. Anal. (C17H18N6O3
·HCl·1/4H2O) C, H, N.
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N-(2-Furan-2-yl-6-thiazol-2-yl-pyrimidin-4-yl)-2-(4-meth-
yl-piperazin-1-yl)-acetamide (22). Compound 19a was reacted
with methyl piperazine, according to the procedure described
for compounds 3, 20, and 21 above, to give compound 22 as a
slightly yellow solid (59% yield). 1H NMR (300 MHz, CDCl3):
δ 9.70 (s, 1H), 8.79 (s, 1H), 8.02 (d, J ) 3, 1H), 7.67 (d, J )
1.8, 1H), 7.55 (d, J ) 3, 1H), 7.43 (d, J ) 3.9, 1H), 6.61 (dd,
J ) 3.6, 1.8, 1H), 3.22 (s, 2H), 2.66 (m, 4H), 2.56 (m, 4H),
2.34 (s, 3H). LCMS-5: tR ) 22.6 (100%). MS: m/z 385 [M +
H]+, expected 385 [M + H]+. Anal. (C18H20N6O2S·2HCl) C,
H, N.
N-[6-(3,5-Dimethyl-pyrazol-1-yl)-2-furan-2-yl-pyrimidin-
4-yl]-2-(4-methyl-piperazin-1-yl)-acetamide (23). Intermediate
19b was reacted with methyl piperazine according to the
procedure described for compounds 3, 20 and 21, to give
compound 23 as an off-white flaky solid (76% yield). 1H NMR
(free base; 300 MHz, CD3OD): δ 7.76 (dd, J ) 1.5, 0.6, 1H),
7.31 (dd, J ) 3.6, 0.9, 1H), 6.65 (dd, J ) 3.6, 1.8, 1H), 6.13 (s,
1H), 3.29 (s, 2H), 2.77 (s, 3H), 2.54–2.75 (m, 8H), 2.35 (s,
3H), 2.27 (s, 3H). LCMS-5: tR ) 17.0 (100%).
Acknowledgment. We thank Shawn Ayube, Paddi Ekhlassi,
and John Harman for analytical support and Kayvon Jalali for
preclinical input.
(9) Lightowler, S. Presented at the International Research Conference,
Targeting Adenosine A2A Receptors in Parkinson’s Disease and other
CNS Disorders, Boston, MA, May 2006.
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Supporting Information Available: Copies of NMR and LCMS
data are available on final compounds, along with detailed descrip-
tions of HPLC conditions for compound analysis. This material is
available free of charge via the Internet at http://www.pubs.acs.org.
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