2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 1. Structure-activity relationships and optimization of heterocyclic substituents

Article abstract of DOI:10.1021/jm701185v

Previously we have described a novel series of potent and selective A _2A receptor antagonists (e.g., 1) with excellent aqueous solubility.¹ While these compounds are efficacious A_2A antagonists in vivo, the presence of an

Full text of DOI:10.1021/jm701185v

J. Med. Chem. 2008, 51, 1719–1729
1719
2-Amino-N-pyrimidin-4-ylacetamides as A2A Receptor Antagonists: 1. Structure-Activity
Relationships and Optimization of Heterocyclic Substituents
Deborah H. Slee,*,† Yongsheng Chen,† Xiaohu Zhang,† Manisha Moorjani,† Marion C. Lanier,† Emily Lin,† Jaimie K. Rueter,†
John P. Williams,† Sandra M. Lechner, Stacy Markison, Siobhan Malany,‡ Mark Santos,‡ Raymond S. Gross,§ Kayvon Jalali,#
Yang Sai,# Zhiyang Zuo,# Chun Yang,# Julio C. Castro-Palomino,| María I. Crespo,| Maria Prat,| Silvia Gual,| José-Luis Díaz,|
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 September 20, 2007
Previously we have described a novel series of potent and selective A2A receptor antagonists (e.g., 1) with
excellent aqueous solubility.1 While these compounds are efficacious A2A antagonists in vivo, the presence
of an unsubstituted furyl moiety was a cause of some concern. In order to avoid the potential metabolic
liabilities that could arise from an unsubstituted furyl moiety, an optimization effort was undertaken with
the aim of replacing the unsubstituted furan with a more metabolically stable group while maintaining potency
and selectivity. Herein, we describe the synthesis and SAR of a range of novel heterocyclic systems and the
successful identification of a replacement for the unsubstituted furan moiety with a methylfuran or thiazole
moiety while maintaining potency and selectivity.
Introduction
react and form protein adducts that can result in liver toxicity,
among other adverse reactions.8,9 To address this concern, other
heterocycles and substituted furyl moieties were explored as
alternatives to a simple furan, with the aim of finding a moiety
with an in vitro profile similar to that of compound 1 but with
less likelihood of producing reactive metabolites.9
The adenosine receptors are G-protein-coupled receptors of
which four subtypes have so far been identified: A1, A2A, A2B,
and A3. Through these receptors, adenosine regulates a wide
range of physiological functions such as motor function, sleep,
anxiety, pain, and psychomotor activity.2 While all adenosine
receptor subtypes are present in the brain, A2A receptor
expression is the highest in a few brain regions, including the
striatum and nucleus accumbens.3 A2A receptors have been
shown to modulate the release of GABA in the striatum, which
appears to regulate the activity of medium spiny neurons. By
reduction of GABA output, A2A antagonism helps restore normal
function in the basal ganglia following dopamine depletion.
Thus, A2A receptor antagonists may be a useful treatment for
neurodegenerative movement disorders such as Parkinson’s and
Huntington’s diseases,4 dyskinesias such as those caused by
prolonged use of neuroleptic and dopaminergic drugs,5 and
dystonias such as restless leg syndrome.6
Interestingly, many potent A2A receptor antagonists including
compounds 1, 26, and 271 (Table 1), and many of the non-
xanthine A2A receptor antagonists described in the literature,
contain an unsubstituted furan moiety including the clinical
candidate 2 (SCH420814, Figure 1).7 While compound 1 is a
potent and selective (hA2A) receptor antagonist (human A2A Ki
) 2.7 nM, 100-fold selective for human A2A over human A1)
with in vivo efficacy,1 there was some concern that the presence
of an unsubstituted furyl moiety could be a metabolic liability.
It is well documented that unsubstituted furans can be metabo-
lized to form reactive intermediates that have the potential to
Chemistry
The methods used to synthesize pyrimidine analogues 1 and
26-40 are described below. In most cases the appropriate
commercially available nitrile 3 was converted to the carboxya-
midine 4 via treatment with sodium methoxide in methanol at
room temperature, followed by reaction with ammonium
chloride. These carboxyamidines were then converted to the
6-chloropyrimidin-4-amines 8a-d via one of two routes. The
first route was via the 6-hydroxypyrimidin-4-amine intermedi-
ates 5b-d, through reaction of the carboxyamidines 4b-d with
ethyl cyanoacetate in the presence of sodium methoxide. The
resulting intermediates 5b-d were then treated with phosphorus
oxychloride to give the key intermediates 8b-d. Alternatively,
the carboxyamidine 4a was reacted with diethyl malonate in
methanol, in the presence of sodium ethoxide, to yield the
corresponding pyrimidine-4,6-diol 6a. Reaction with phosphorus
oxychloride in the presence of N,N-diisopropylethylamine
yielded the 4,6-dichloropyrimidine intermediate 7a, which was
then treated with ammonium hydroxide in methanol under reflux
to yield intermediate 8a as shown in Scheme 1. These
intermediates were then further derivatized to install R2 as
described in Schemes 2 and 3.
The chlorine moiety of 8a-d can be displaced with pyrazole
(Scheme 2) to give analogues 9a-d. This reaction was slower,
and yields were reduced when substituted pyrazoles were used.
To overcome this, an alternative approach was used for the
synthesis of the methyl-substituted pyrazole analogues, which
consisted of first displacing the chloro moiety of 8a-d with
hydrazine to give intermediates 10a-d, followed by cyclization
with pentane-2,4-dione to give the dimethylpyrazole analogues
11a-d. Intermediate 10d was also reacted with 4-dimethylami-
* To whom correspondence should be addressed. Phone: 858-617-7849.
Fax: 858-617-7619. Email: dslee@neurocrine.com.
†
Department of Medicinal Chemistry, Neurocrine Biosciences.
Department of Neuroscience, Neurocrine Biosciences.
Department of Pharmacology and Lead Discovery, Neurocrine Bio-
‡
sciences.
§
Department of Chemical Development, Neurocrine Biosciences.
Department of Preclinical Development, Neurocrine Biosciences.
Almirall Research Center.
#
|
10.1021/jm701185v CCC: $40.75
2008 American Chemical Society
Published on Web 02/29/2008

1720 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Slee et al.
Figure 1. Representative analogue 1 of lead series and a clinical compound 2.
Scheme 1a
a Reagents and conditions: (a) NaOMe (1.1 equiv), NH4Cl, MeOH, room temp up to 68 h, 87–96%; (b) ethyl cyanoacetate, NaOMe, EtOH, 70 °C, 6–12
h, 60–80%; (c) NaOEt, diethylmalonate, room temp to reflux, 12–60 h, 78%; (d) POCl3, 90 °C, 3–12 h, 33–87%; (e) NH4OH, MeOH, 80 °C, 20 h, 76%.
Scheme 2a
4. While this second route was less versatile, it was more
convenient for the analogues when R2 is a carbon-linked thiazole
or pyridine.
Additional analogues were synthesized from the commercially
available 4-amino-2,6-dichloropyrimidine 18 (Scheme 5). In-
termediate 18 was reacted with pyrazole in dimethylformamide
in the presence of cesium carbonate to give the 2,6-disubstituted
analogue 19. The N-[6-chloro-2-(3,5-dimethylpyrazol-1-yl)py-
rimidin-4-yl]acetamide 20 was made from compound 18 by
reaction with hydrazine followed by cyclization with pentan-
2,4-dione and subsequent acetylation using acetic anhydride.
Suzuki coupling of 20 with phenylvinylboronic acid yielded the
olefin intermediate 21, which upon oxidative cleavage with
ozone/dimethyl sulfide afforded aldehyde 22,10 which was used
without purification and cyclized with TosMIC or methyl
TosMIC in MeOH in the presence of potassium carbonate, to
give intermediate 23 or 24 respectively, with in situ removal of
the N-acetyl group.11 Negishi coupling12 of 20 with thiazole in
THF in the presence of BuLi, ZnCl2, and tetrakis(triphenylphos-
phine)palladium(0) afforded 25 with concurrent cleavage of the
N-acetyl group.
a Reagents and conditions: (a) pyrazole; CsCO3, DMF, 85 °C, 21 h, 55%.
nobut-3-en-2-one at room temperature to give the 5-methylpyra-
zole analogue 12.
For the synthesis of carbon-linked heterocycles at R2, the
carboxyamidines 4a, and 4d were reacted with 3-oxo-3-thiazol-
2-ylpropionic acid ethyl ester 13a, and the commercially
available thiophene carboxyamidine 4e was reacted with 3-oxo-
3-pyridin-2-ylpropionic acid ethyl ester 13b to give intermedi-
ates 14a-c. Ester 13b was commercially available; however,
compound 13a was synthesized through reaction of the methyl
ketone 17 with diethyl carbonate, using sodium hydride as the
base. The resulting pyrimidin-4-ol intermediates of formula
14a-c could then be converted to the amine in two steps via
treatment with phosphorus oxychloride to give 15a-c, followed
by reaction with ammonium hydroxide, to yield the correspond-
ing 6-heteroarylpyrimidin-4-amines 16a-c as outlined in Scheme
The aminopyrimidine intermediates 9a-d, 11a-d, 12,
16a-c, 19, and 23-25 described above were then converted
to the final compounds via the route outlined in Scheme 6. Each
aminopyrimidine was reacted with chloroacetyl chloride to give
the corresponding intermediate, which was then used crude and
reacted with methylpiperazine to give compounds 1 and 26-40
(Table 1).
Results and Discussion
After the initial discovery of compound 1, further investiga-
tion into the in vitro metabolism of this compound identified
what were believed to be small amounts of the downstream

Acetamides as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1721
Scheme 3a
a Reagents and conditions: (a) N2H4, EtOH, 90 °C, 2 h, 80%; (b) pentane-2,4-dione, 0–90 °C, 2 h, 72%; (c) 4-dimethylaminobut-3-en-2-one, HCl, dioxane,
room temp, 30%.
Scheme 4a
a Reagents and conditions: (a) KOt-Bu, (1.5 equiv), t-BuOH, 135 °C, 12 h, 50%; (b) POCl3, 90 °C, 24 h, 66%; (c) NH4OH, EtOH, 120 °C, 3 h, 53%; (d)
diethyl carbonate (5 equiv), NaH, 90 °C, 2 h, 56% after distillation.
Scheme 5a
a Reagents and conditions: (a) pyrazole, Cs2CO3, DMF, 120 °C, 15 h, 60%; (b) N2H4, NMP, 60 °C, 2 h; (c) pentane-2,4-dione, 0–60 °C, 2 h, 66% (two
steps); (d) Ac2O, AcOH, 90 °C, 18 h, 83%; (e) trans-2-phenylvinylboronic acid, tetrakis(triphenylphosphine)palladium(0), Na2CO3, H2O/dioxane, 90 °C,
16 h, 85%; (f) O3, Me2S, CH2Cl2/MeOH; (g) TosMIC, K2CO3, MeOH, 80 °C, 16 h, 50% (over two steps); (h) Me-TosMIC, K2CO3, MeOH, 80 °C, 16 h,
50% (over two steps); (i) thiazole, BuLi, ZnCl2, THF, -78 °C, then tetrakis(triphenylphosphine)palladium(0), 80 °C, 2 h, 70%.
products that would result from the metabolism of the furan
moiety (Figure 2). While this was not observed in human
hepatocytes and does not always lead to toxicity, the literature
is rich in reports of drugs containing furan moieties that cause
toxicities that are attributed to reactive intermediates derived
from metabolism of the furan moiety.13
To address this concern, alternative heterocycles and substi-
tuted furyl moieties were explored with the goal of finding an

1722 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Slee et al.
Scheme 6a
reactive metabolites. As can be seen from Table 1, this proved
to be somewhat challenging. Simply adding a methyl group to
the furan of compound 1 to give compound 28 had a large
negative effect on potency, reducing binding to the hA2A
receptor from 2.7 to 135 nM (45-fold). Interestingly, the addition
of methyl groups to the pyrazole at R2 of compound 28, as in
examples 29 and 30, counteracts this effect and brings the
potency back from 135 nM to 14 and 12 nM, respectively. In
addition, the selectivity for the hA2A receptor over the human
A1 (hA1) receptor also improves, from 6-fold to 28-fold for
compound 29 and to 71-fold for compound 30. Selectivity over
the hA1 receptor was desired because the A1 receptor is present
in cardiac muscle and, as a result, may represent a cardiotoxicity
risk.14 Replacing the pyrazole at R2 of compound 29 with
thiazole (compound 31) increased potency slightly, but selectiv-
ity remained poor. The combination of thiophene at R1 with
2-pyridyl at R2 gave a quite potent compound (32) (Ki ) 24
nM), but again, selectivity over the hA1 receptor was modest.
The symmetrical bis-pyrazole analogue 33 was less active than
compound 1 (hA2A Ki ) 131 vs 2.7 nM), indicating that the
nitrogen of a pyrazole was less tolerated than the oxygen of
the furan moiety. In some cases the heterocycles appear to be
interchangeable as with compounds 34 and 40, which have
similar in vitro profiles (Ki values of 27 and 9 nM, respectively).
For these compounds it is possible that the bulky dimethylpyra-
zole moiety dictates the binding mode, and forces the pyrimidine
core to rotate to accommodate these substituents. In other words,
the heterocycles and side chain are still able to bind to the same
regions of the A2A receptor.
a Reagents and conditions: (a) chloroacetyl chloride, pyridine, DCM,
room temp, 2–12 h; (b) methylpiperazine, DIPEA, room temp, 6–12 h,
14–82%.
Table 1
The oxazole analogues 35 and 36 were poor hA2A receptor
antagonists, indicating that the presence of a nitrogen atom at
the 3-position was not tolerated. A pyrazole at R2 in combination
with a 2-thiazole (compound 37) or 2-pyridyl (compound 38)
at R1 gave moderately potent analogues with activities of 17
and 53 nM, respectively. Consistent with previous observations,
both activity and selectivity were significantly improved when
these heterocycles (2-pyridyl and 2-thiazole) were combined
with dimethylpyrazole at R2. The analogue with 2-pyridyl at
R1 in combination with dimethylpyrazole (compound 39) had
a Ki of 26 nM against the hA2A receptor and was 173-fold
selective for hA2A over the hA1 receptor. The analogue with
2-thiazole at R1 in combination with dimethylpyrazole at R2
(compound 40) had a Ki of 9 nM against the hA2A receptor and
was 222-fold selective over the hA1 receptor. In general the
compounds described were found to be functional antagonists
in a cAMP assay15 (compound 26, IC50 of 103 nM); however,
the binding assay was used routinely for lead optimization
because of the variability and low throughput of the functional
assay.
Overall, compounds 30 and 40 appeared to represent the most
promising leads. While compound 30 still contained a furyl
moiety, the methyl group would be expected to significantly
attenuate the minor metabolism of the furan that was observed
with compound 1. Additional metabolite identification to support
this will be discussed in the following paper. Compound 40
showed some weak inhibition of CYP3A4 (IC50 of 13 µM);
however, compound 30 appeared to have no significant CYP
inhibition potential in the same dose range. Therefore compound
30 was chosen for further lead optimization for in vivo efficacy,
as will be discussed in the accompanying paper.
a Displacement of specific [3H]-DPCPX binding at human A1 receptors
expressed in HEK293 cells. Displacement of specific [3H]-ZM241385
binding at human A2A receptors expressed in HEK293. Data are expressed
as mean values of at least three runs ( SEM.
Conclusion
analogue with a similar in vitro and in vivo profile to the lead
compound 1, but with reduced risk of producing chemically
We have developed a novel series of potent and selective
human adenosine A2A receptor antagonists, which have excellent

Acetamides as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1723
5% D (95% C) to 95% D (5% C) in 6.43 min with a 1.02 min hold
at 95% D followed by a return and hold at 5% D for 1.52 min.
Analytical HPLC-MS method 6 was as follows: platform, Agilent
equipped with an autosampler, an UV detector (220 and 254 nm),
a MS detector (APCI); HPLC column, Waters XTerraMS C18, 3.0
mm × 250 mm; HPLC gradient, 1.0 mL/min from 10% acetonitrile
in water to 90% acetonitrile in water in 46 min, maintaining 90%
for 7.0 min. Both acetonitrile and water have 0.025% TFA.
Analytical HPLC-MS method 7 was as follows: platform, Agilent
equipped with an autosampler, an UV detector (220 and 254 nm),
a MS detector (APCI); HPLC column, Waters XTerraMS C18, 3.0
mm × 250 mm; HPLC gradient, 1.0 mL/min from 10% acetonitrile
in water to 90% acetonitrile in water in 46 min, maintaining 90%
for 7.0 min. Acetonitrile has 0.1% NH4OH, and water has 0.05%
NH4OH. Preparative HPLC-MS was as follows: 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 mm × 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 (HCl) (4a). To a solution of sodium
methoxide (55.5 mmol) in methanol (50 mL) was added 2-furoni-
trile 3a (5.0 g, 53.2 mmol). The mixture was stirred at room
temperature for 3 h. To the resulting solution was slowly added
ammonium chloride (3.14 g, 58.7 mmol), and the mixture was
stirred at room temperature until the reaction was complete (up to
68 h). The resulting suspension was filtered and the solvent removed
under reduced pressure. The solid obtained was washed with ethyl
ether (3 × 25 mL) to give compound 5 as an off-white solid (7.5
g, 96%). 1H 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).
Figure 2. Metabolites of compound 1 after incubation with human
liver microsomes. Tentative assignment of peaks observed by LC-MS.
physical properties for drug development. These compounds
were optimized to remove an unsubstituted furan moiety with
the objective of reducing the risk of forming chemically reactive
metabolites. From this SAR study a number of potential lead
compounds were identified. Compounds 30 and 40 were the
most potent and had promising selectivity. While these com-
pounds were not quite as potent as compound 1, the potency
and selectivity were in a suitable range for these compounds to
be considered for further characterization and lead optimization.
Experimental Section
Pyridine-2-carboxamidine (HCl) (4b). The title compound 4b
was obtained as a pale-yellow solid starting from 2-pyridinenitrile
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 chro-
matography 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 spectrometer. The elemental analysis was done
by Robertson Microlit Laboratory, Madison, NJ. Analytical
HPLC-MS method 1 was as follows; platform, Agilent 1100 series
equipped with an autosampler, a UV detector (220 and 254 nm), a
MS detector (APCI); HPLC column, YMC ODS AQ, S-5, 5 µm,
2.0 mm × 50 mm cartridge; HPLC gradient, 1.0 mL/min from 10%
acetonitrile in water to 90% acetonitrile in water in 2.5 min,
maintaining 90% for 1 min. Both acetonitrile and water have
0.025% TFA. Analytical HPLC-MS method 2 was as follows:
platform, Agilent 1100 series equipped with an autosampler, an
UV detector (220 and 254 nm), a MS detector (APCI); HPLC
column, Phenomenex Synergi-Max RP, 2.0 mm × 50 mm column;
HPLC gradient, 1.0 mL/min from 5% acetonitrile in water to 95%
acetonitrile in water in 13.5 min, maintaining 95% for 2 min. Both
acetonitrile and water have 0.025% TFA. Analytical HPLC-MS
method 3 was as follows: platform, Agilent 1100 series equipped
with an autosampler, an UV detector (220 and 254 nm), a MS
detector (electrospray); HPLC column, XTerra MS, C18, 5 µm, 3.0
mm × 250 mm column; HPLC gradient, 1.0 mL/min from 10%
acetonitrile in water to 90% acetonitrile in water in 46 min, jump
to 99% acetonitrile and maintain 99% acetonitrile for 8.0 min. Both
acetonitrile and water have 0.025% TFA. Analytical HPLC-MS
method 4: platform, Dionex equipped with an autosampler, an UV
detector (220 and 254 nm), a MS detector (APCI); HPLC column,
Phenomenex C18 4.6 mm × 150 mm; HPLC gradient, 2.5 mL/
min from 5% acetonitrile in water to 90% acetonitrile in water in
9.86 min, 12.30 min run. Both acetonitrile and water have 0.04%
NH4OH. Analytical HPLC-MS method 5 was as follows: platform
Agilent 1100 series equipped with an autosampler, an UV detector
(220 and 254 nm), a MS detector (APCI); HPLC column,
Phenomenex Fusion RP, 2.0 mm × 50 mm column; HPLC gradient,
1.0 mL/min. Solvent C is 6 mM ammonium formate in water, and
solvent D is 25% acetonitrile in methanol. The gradient runs from
1
using the procedure described for 4a above. H NMR (300 MHz,
DMSO-d6): δ 8.81 (m, 1H), 8.40 (d, J ) 7.8, 1H), 8.02 (dt, J )
8.0, 1.8, 1H), 7.78 (m, 1H).
Thiazole-2-carboxamidine (HCl) (4c). See ref 16.
5-Methyl-2-furancarboxamidine (HCl) (4d). The title com-
pound 4d (3.71 g, 87%) was obtained as a pale-yellow solid starting
from 5-methyl-2-furonitrile (2.85 g) using the procedure described
1
for 4a above. H NMR (300 MHz, DMSO-d6): δ 8.49 (bs, 4 H),
7.64 (d, J ) 3.6, 1H), 6.36 (d, J ) 3.6, 1H), 2.27 (s, 3H).
Thiophene-2-carboxamidine (HCl) (4e). 4e is commercially
available.
6-Hydroxy-2-(pyridin-2-yl)pyrimidin-4-amine (5b). To ethanol
(100 mL) was added pyridine-2-carboxamidine (HCl) 4b (20.8 g,
130 mmol, 1.1 equiv) and NaOMe/MeOH solution (100 mL, 30%
W, 5 equiv). The mixture was stirred at room temperature for 30
min. Ethyl cyanoacetate (13 mL, 120 mmol, 1 equiv) was added,
and the reaction mixture was heated at 90 °C for 16 h. The reaction
mixture was concentrated to dryness and resuspended in water (70
mL), and 4 M HCl was added to adjust the pH to 5. The mixture
was stirred at 0 °C for 30 min, filtered, and washed with cold water
(2 × 20 mL) and the solid obtained dried under vacuum at 90 °C
for 12 h. Intermediate 5b was obtained as a golden solid (18.1 g,
73%). 1H NMR (300 MHz, DMSO-d6): δ 8.70 (m, 1H), 8.20 (dd,
J ) 8.1, 0.9, 1H), 8.02 (dt, J ) 7.6, 1.8, 1H), 7.47 (ddd, J ) 7.4,
4.9, 1.3, 1H), 6.64 (s, 1H). LCMS-1: tR ) 1.55 min. MS: m/z 189.0
[M + H]+, expected 189 [M + H]+.
6-Hydroxy-2-(1,3-thiazol-2-yl)pyrimidin-4-amine (5c). Com-
pound 5c was prepared using the same procedure as for 5b above
except that the amidine 4c was used instead of 4b. 1H NMR (300
MHz, DMSO-d6): δ 8.07 (d, J ) 3.0, 1H), 8.05 (d, J ) 3.0, 1H),
5.46 (s, 1H).
6-Hydroxy-2-(5-methyl-2-furyl)pyrimidin-4-amine (5d). Com-
pound 5d was prepared using the same procedure as for 5b above
except that the amidine 4d was used instead of 4b. 1H NMR (300
MHz, DMSO-d6): δ 7.35 (d, J ) 3.6, 1H), 6.29 (d, J ) 3.3, 1H),
4.95 (s, 1H), 2.32 (s, 3H).
2-(2-Furyl)pyrimidine-4,6-diol (6a). To a solution of sodium
ethoxide (19.1 mmol) in ethanol (90 mL) was slowly added

1724 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Slee et al.
compound 4a (5.6 g, 38.2 mmol). The mixture was stirred at room
temperature for 30 min, and then diethyl malonate (4.87 g, 30.4
mmol) was added. 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 M HCl.
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 6a 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).
7.92–7.86 (m, 1H), 7.79–7.78 (m, 1H), 7.46–7.42 (m, 1H), 7.04
(s, 1H), 6.49 (dd, J ) 2.7, 1.5, 1H), 6.36 (bs, 1H, NH2). LCMS-1:
tR ) 1.83 min. MS: m/z 239 [M + H]+, expected 239 [M + H]+.
N-(6-Pyrazol-1-yl-2-thiazol-2-ylpyrimidin-4-yl)amine (9c). In-
termediate 9c was prepared from 8c according to the procedure
described for 9a. 1H NMR (300 MHz, CDCl3 + 10% CD3OD): δ
8.56 (d, J ) 2.4, 1H), 7.86 (d, J ) 3.0, 1H), 7.69 (m, 1H), 7.50 (d,
J ) 3.0, 1H), 6.54 (m, 1H), 6.98 (s, 1H), 6.41 (m, 1H). LCMS-5:
tR ) 4.02 min. LCMS-2: tR ) 4.32 min. MS: m/z 245 [M + H]+,
expected 245 [M + H]+.
2-(5-Methyl-2-furyl)-6-(1H-pyrazol-1-yl)pyrimidin-4-amine (9d).
Intermediate 9d was prepared from 8d according to the procedure
described for 9a. 1H NMR (300 MHz, CDCl3): δ 8.63 (d, J ) 2.7,
1H), 7.74 (d, J ) 1.5, 1H), 7.23 (d, J ) 3.6, 1H), 6.85 (s, 1H),
6.46 (dd, J ) 2.7, 1.8, 1H), 6.18 (m, 1H), 5.36 (bs, 2H), 2.46 (s,
3H).
6-(3,5-Dimethylpyrazol-1-yl)-2-(furan-2-yl)pyrimidin-4-
ylamine (11a). To a solution of intermediate 8a (1.0 g, 5.1 mmol)
in absolute EtOH (4.5 mL) was added anhydrous hydrazine (0.32
mL, 10.2 mmol). The mixture was heated at 90 °C for 22 h to give
10a. The mixture was then cooled to 0 °C. 2,4-Pentanedione (1.05
mL, 10.2 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 to dryness. The crude 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 11a as a white solid (0.94 g, 72%). LCMS-
1: tR ) 2.44 min. MS: m/z 256 [M + H]+, expected 256 [M +
H]+.
4,6-Dichloro-2-(2-furyl)pyrimidine (7a). To a suspension of
compound 6a (3.0 g, 16.8 mmol) was added N,N-diisopropylethy-
lamine (3.85 g, 29.8 mmol) and phosphorus oxychloride (17 mL).
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), a saturated solution of sodium bicarbonate (2 × 25 mL), and
brine and dried (Na2SO4). The solvent was removed under reduced
1
pressure to give compound 7a as a gray solid (3.15 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 (8a). A suspension of
compound 7a (2.0 g, 9.3 mmol) in methanol (14 mL) and 30%
ammonium hydroxide (27 mL) was heated at 80 °C 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 8a was obtained
as an off-white solid (1.48 g, 76%). 1H NMR (300 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).
6-Chloro-2-(pyridin-2-yl)pyrimidin-4-amine (8b). To 5b (14.0
g, 74.5 mmol) in a sealed tube (350 mL) was added POCl3 (75
mL). The mixture was heated at 90 °C for 16 h. The mixture was
cooled to room temperature and poured onto ice (500 mL volume).
Solid sodium carbonate was added to adjust the pH to 7. The
mixture was extracted twice with 300 mL of ethyl acetate (300
mL). The organic layers were combined, washed with sodium
bicarbonate and brine, dried (Na2SO4), and concentrated to a yellow
6-(3,5-Dimethylpyrazol-1-yl)-2-pyridin-2-ylpyrimidin-4-
ylamine (11b). Intermediate 11b was prepared from 8b according
1
to the procedure described for 11a. H NMR (300 MHz, DMSO-
d6): δ 8.80 (d, J ) 4.5, 1H), 8.41 (d, J ) 7.8, 1H), 8.18 (ddd, J )
7.8, 7.5, 1.2, 1H), 7.71–7.75 (m, 1H), 7.04 (s, 1H), 6.22 (s, 1H),
2.77 (s, 3H), 2.22 (s, 3H). LCMS-4: tR ) 6.59 min. MS: m/z 267
[M + H]+, expected 267 [M + H]+.
1
solid (5.1 g, 33%). H NMR (300 MHz, CDCl3 + 10% CD3OD):
δ 8.70 (m, 1H), 8.41 (m, 1H), 7.90 (dt, J ) 7.7, 1.6, 1H), 7.45
(ddd, J ) 7.5, 4.8, 1.2, 1H), 6.44 (s, 1H). LCMS-1: tR )1.71 min.
MS: m/z 206.9 [M + H]+, expected 207 [M + H]+.
6-(3,5-Dimethylpyrazol-1-yl)-2-thiazol-2-ylpyrimidin-4-
ylamine (11c). Intermediate 11c was prepared from 8c according
1
to the procedure described for 11a. H NMR (300 MHz, DMSO-
6-Chloro-2-(thiazol-2-yl)pyrimidin-4-amine (8c). Intermediate
8c was prepared from 5c according to the procedure described for
8b above. 1H NMR (300 MHz, CDCl3 + 10% CD3OD): δ 7.92 (d,
J ) 3.3, 1H), 7.52 (d, J ) 3.0, 1H), 6.44 (s, 1H).
d6): δ 8.10 (d, J ) 3.3, 1H), 7.90 (d, J ) 3.6, 1H), 6.86 (s, 1H),
6.15 (s, 1H), 2.74 (s, 3H), 2.20 (s, 3H). LCMS-1: tR ) 2.43 min.
MS: m/z 273 [M + H]+, expected 273 [M + H]+.
6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyrimidin-
4-ylamine (11d). Intermediate 11d was prepared using the proce-
dure described for 11a except that intermediate 8d was used instead
6-Chloro-2-(5-methylfuran-2-yl)pyrimidin-4-ylamine (8d). In-
termediate 8d was prepared from 5d according to the procedure
1
1
described for 8b above. H NMR (300 MHz, DMSO-d6): δ 7.07
of intermediate 8a. H NMR (300 MHz, CDCl3): δ 7.12 (d, J )
(d, J ) 3.3, 1H), 6.40 (s, 1H), 6.21 (dd, J ) 3.0, 1.2, 1H), 2.86
(bs, 2H), 2.36 (s, 3H). LCMS-1: tR ) 2.18 min. MS: m/z 210 [M
+ H]+, expected 210 [M + H]+.
3.0, 1H), 6.80 (s, 1H), 6.15 (m, 1H), 6.00 (s, 1H), 5.10 (bs, 2H),
2.76 (s, 3H), 2.44 (s, 3H), 2.28 (s, 3H). LCMS-2: tR ) 5.75 min.
MS: m/z 270 [M + H]+, expected 270 [M + H]+.
2-(2-Furyl)-6-(1H-pyrazol-1-yl)pyrimidin-4-amine (9a). To a
solution of compound 8a (1.0 g, 5.1 mmol) in anhydrous DMF
(20 mL) was added pyrazole (0.7 g, 10.2 mmol) and cesium
carbonate (3.34 g, 10.2 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 chromatography with silica gel, eluting
with methylene chloride/methanol (3%) to give compound 9a as
2-(5-Methylfuran-2-yl)-6-(5-methylpyrazol-1-yl)pyrimidin-4-
ylamine (12). To 10 mL of ethanol was added intermediate 8d
(3.0 g, 14.3 mmol), DIEA (4.6 g, 35.8 mmol, 2.5 equiv), and
anhydrous hydrazine (0.92 g, 28.6 mmol, 2.0 equiv). The reaction
mixture was stirred at 90 °C for 48 h. The mixture was cooled to
room temperature and concentrated under vacuum. The residue was
dissolved in anhydrous dioxane (30 mL). A solution of 4-dimethy-
laminobut-3-en-2-one (8.22 g, 72.6 mmol, 5 equiv) in 10 mL of
dioxane was added slowly at room temperature. Then 1 M aqueous
HCl (2.4 mL) was added and the mixture stirred at room
temperature overnight. Solvents were evaporated, the residue
dissolved in ethyl acetate, quenched with saturated sodium bicar-
bonate (20 mL) and extracted with ethyl acetate (2 × 30 mL). The
aqueous solution was washed with an additional aliquot of ethyl
acetate (20 mL). The organic layers were combined and dried
(Na2SO4), then concentrated under reduced pressure. The residue
was purified by column chromatography, eluting with ethyl acetate/
hexanes, 30–70%, to give 12 as a pale-yellow solid (1.2 g, 30%).
1H NMR (300 MHz, CDCl3 + 10% CD3OD): δ 7.61 (d, J ) 3.3,
1
an off-white solid (0.64 g, 55%). H 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 ) 4.24 min (98%). LCMS-3: tR ) 6.40
min. MS: m/z 228 [M + H]+, expected 228 [M + H]+.
6-Pyrazol-1-yl-2-pyridin-2-ylpyrimidin-4-ylamine (9b). Inter-
mediate 9b was prepared from 8b according to the procedure
1
described for 9a. H NMR (300 MHz, CDCl3): δ 8.86–8.83 (m,
1H), 8.71 (dd, J ) 2.4, 0.6, 1H), 8.57 (dt, J ) 8.1, 1.2, 1H),

Acetamides as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1725
1H), 7.58 (d, J ) 1.2, 1H), 6.93 (s, 1H), 6.26 (d, J ) 3.3, 1H),
6.20 (bs, 1H), 2.73 (s, 3H), 2.44 (s, 3H). LCMS-1: tR ) 2.09 min.
MS: m/z 256 [M + H]+, expected 256 [M + H]+.
2-(5-Methyl-2-furyl)-6-(1,3-thiazol-2-yl)pyrimidin-4-amine (16b).
Intermediate 16b was prepared from 15b according to the procedure
1
described for 16a. H NMR (250 MHz, DMSO-d6): δ 8.03 (dd, J
3-Oxo-3-thiazol-2-ylpropionic Acid Ethyl Ester (13a). To a
solution of 60% sodium hydride (95.4 mmol) in diethyl carbonate
(90 mL) was slowly added 2-acetylthiazole 17 (5.0 g, 39.4 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 title compound 13a as a pale-orange oil
) 3.0, 1.0, 1H), 7.93 (dd, J ) 3.0, 1.0, 1H), 7.28 (bs, 2H), 7.08 (d,
J ) 3.4, 1H), 6.99 (m, 1H), 6.29 (dd, J ) 3.0, 1.0, 1H), 2.38 (s,
3H).
6-Pyridin-2-yl-2-thiophen-2-ylpyrimidin-4-ylamine (16c). In-
termediate 16c was prepared from 15c according to the procedure
described for 16a. 1H NMR (300 MHz, CDCl3 + 10% CD3OD): δ
8.54 (d, J ) 4.2, 1H), 8.44 (d, J ) 7.8, 1H), 7.94 (s, 1H), 7.84 (t,
J ) 7.6, 1H), 7.40 (d, J ) 5.4, 1H), 7.36 (m, 1H), 7.18 (m, 1H),
7.06 (m, 1H). LCMS-1: tR ) 1.94 min. MS: m/z 255 [M + H]+,
expected 255 [M + H]+.
1
(4.4 g, 56%). H NMR (250 MHz, CDCl3): δ 7.99 (d, J ) 5.3,
2,6-Bis(1H-pyrazol-1-yl)pyrimidin-4-amine (19). To a solution
of 2,6-dichloro-pyrimidin-4-amine 18 (81 mg, 0.5 mmol) in
anhydrous DMF (1 mL) was added pyrazole (68 mg, 1.0 mmol)
and cesium carbonate (0.32 g, 1.0 mmol). The mixture was heated
at 120 °C for 15 h. The solution was poured into water (10 mL)
and extracted with ethyl acetate (2 × 5 mL). The organic layer
was washed with water (2 × 5 mL) and brine (5 mL) and dried
(Na2SO4), and the solvent was removed under reduced pressure.
The residue was purified by preparative TLC plate, eluting with
3% methanol in methylene chloride, to give the title compound 19
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 (14a). To a solu-
tion of potassium tert-butoxide (0.57 g, 6.03 mmol) in butanol (2
mL) was added compound 4a (0.85 g, 4.26 mmol) and 3-oxo-3-
thiazol-2-ylpropionic acid ethyl ester 13a (0.75 g, 4.69 mmol). The
mixture was heated at 135 °C for 3 h. The crude reaction was poured
into water (20 mL) and acidified with 10% HCl (25 mL). The
resulting solid was filtered, washed with water (2 × 25 mL), and
dried. The title compound 14a was obtained as an off-white solid
(0.64 g, 50%). 1H 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).
2-(5-Methyl-2-furyl)-6-(1,3-thiazol-2-yl)pyrimidin-4-ol (14b).
Intermediate 14b was prepared according to the procedure described
for 14a except that 5-methyl-2-furancarboxamidine 4d was used
1
as an off-white solid (66 mg, 60%). H NMR (300 MHz, CDCl3
+ 10% CD3OD): δ 8.61 (m, 1H), 8.50 (m, 1H), 7.71 (m, 1H),
6.68 (m, 1H), 6.72 (s, 1H), 6.42 (m, 2H). LCMS-5: tR ) 3.82 min.
LCMS-2: tR ) 4.10 min. MS m/z 228 [M + H]+, expected 228 [M
+ H]+.
1
N-[6-Chloro-2-(3,5-dimethylpyrazol-1-yl)pyrimidin-4-yl]ac-
etamide (20). 4-Amino-2,6-dichloropyrimidine 18 (40 g, 0.24 mol,
1 equiv) was dissolved in N-methylpyrrolidinone (200 mL). The
slurry was heated to 60 °C, and anhydrous hydrazine (19 mL, 0.61
mol, 2.5 equiv) was added slowly over 1.5 h. The mixture was
cooled to room temperature, and 2,4-pentanedione (63 mL, 0.61
mol, 2.5 equiv) was added slowly, with cooling to keep the reaction
temperature below 50 °C. After 1 h, the mixture was heated to 50
°C and then ethanol (200 mL) was added, followed by water (400
mL). Once the water addition was complete, the reaction mixture
was cooled to room temperature, and the solid was collected by
filtration. The solid was washed with ethanol/water (3 × 200 mL)
and dried under vacuum at 60 °C overnight. The tan solid obtained
was a mixture of the desired regioisomer (43.2 g, 85% purity by
HPLC area at 254 nm) and the 4-dimethylpyrazole regioisomer.
The product was recrystallized from hot THF/i-PrOAc to give
6-chloro-2-(3,5-dimethyl-pyrazol-1-yl)pyrimidin-4-ylamine as a
white solid (66%). LCMS-3: tR ) 1.97 min. MS: m/z 223.9 [M +
H]+, expected 224 [M + H]+.
instead of 4a. H NMR (250 MHz, CDCl3): δ 8.03 (d, J ) 2.8,
1H), 7.98 (d, J ) 2.8, 1H), 7.44 (d, J ) 2.8, 1H), 6.77 (s, 1H),
6.38 (d, J ) 2.8, 1H), 2.45 (s, 3H).
2-(Thien-2-yl)-6-(pyridin-2-yl)pyrimidin-4-ol (14c). Intermedi-
ate 14c was prepared according to the procedure described for 14a
except that thiophen-2-carboxamidine 4e was used instead of 4a
and that 3-oxo-3-pyridin-2-ylpropionic acid ethyl ester 13b was
used instead of 3-oxo-3-thiazol-2-ylpropionic acid ethyl ester 13a.
1H NMR (300 MHz, DMSO-d6): δ 8.74 (d, J ) 4.8, 1H), 8.38 (d,
J ) 7.8, 1H), 8.22 (d, J ) 3.9, 1H), 8.11 (m, 1H), 7.91 (d, J ) 4.8,
1H), 7.62 (m, 1H), 7.25 (dd, J ) 4.8, 3.9, 1H), 7.17 (s, 1H).
4-Chloro-2-(2-furyl)-6-(1,3-thiazol-2-yl)pyrimidine (15a). A
suspension of compound 14a (0.63 g) in phosphorus oxychloride
(20 mL) was refluxed for 24 h. The solvent was removed under
reduced pressure, and ice and water were slowly added. The
resulting solid was filtered, washed with 2 M sodium hydroxide,
and dried. Purification by column chromatography with silica gel
and methylene chloride as eluent gave compound 15a as an off-
white solid (0.44 g, 66%). LCMS-1: tR ) 2.73 min. MS: m/z 263.9
[M + H]+, expected 264 [M + H]+.
The 6-chloro-2-(3,5-dimethylpyrazol-1-yl)pyrimidin-4-ylamine
(40 g, 0.18 mol, 1 equiv) was dissolved in acetic acid (200 mL,
0.9 mol, 0.5 equiv) and stirred. Acetic anhydride (80 mL, 0.8 mol,
4.7 equiv) was added and the mixture heated to 90 °C overnight.
Once the reaction was complete, the mixture was cooled to room
temperature and water (16 mL) was added over 30 min. The product
was collected via filtration, washed with water (4 × 75 mL), and
dried under vacuum at 50 °C overnight. Compound 20 was obtained
as an off-white crystalline solid (AcOH solvate) (48.2 g, 0.15 mol,
4-Chloro-2-(5-methyl-2-furyl)-6-(1,3-thiazol-2-yl)pyrimi-
dine (15b). Intermediate 15b was prepared from 14b according to
1
the procedure described for 15a. H NMR (250 MHz, CDCl3): δ
8.03 (d, J ) 4.8, 1H), 7.81 (s, 1H), 7.53 (d, J ) 3.2, 1H), 7.31 (d,
J ) 3.2, 1H), 6.15 (d, J ) 4.8, 1H), 2.41 (s, 3H).
4-Chloro-2-(thien-2-yl)-6-(pyridin-2-yl)-pyrimidine (15c). In-
termediate 15c was prepared from 14c according to the procedure
1
described for 15a. H NMR (300 MHz, CDCl3): δ 8.74 (td, J )
1
83%). H NMR (300 MHz, CDCl3): δ 9.07 (s, 1H), 8.03 (s, 1H),
4.8, 0.9, 1H), 8.59 (d, J ) 8.1, 1H), 8.23 (s, 1H), 8.13 (dd, J )
3.9, 1.2, 1H), 7.93 (dt, J ) 7.6, 1.7, 1H), 7.54 (dd, J ) 5.2, 1.4,
1H), 7.47 (ddd, J ) 7.6, 4.7, 1.0, 1H), 7.17 (dd, J ) 5.0, 3.8, 1H).
2-(2-Furyl)-6-(1,3-thiazol-2-yl)pyrimidin-4-amine (16a). A
suspension of compound 15a (0.25 g) in ethanol (22 mL) and 30%
ammonium hydroxide (22 mL) was heated at 120 °C in a pressure
reactor for 3 h. 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
16a as an off-white solid (0.12 g, 53%). 1H 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).
6.03 (s, 1H), 2.65 (s, 3H), 2.30 (s, 3H), 2.22 (s, 3H), 2.12 (s, 3H).
LCMS-1: tR ) 2.11 min. MS: m/z 265.9 [M + H]+, expected 265.9
[M + H]+.
N-[6-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-((E)-styryl)pyrimidin-
4-yl]acetamide (21). A mixture of intermediate 20 (3.26 g, 10
mmol), phenylvinylboronic acid (2.3 g, 15 mmol), and sodium
carbonate (4.3 g, 40 mmol) in dioxane/water (9/1, 100 mL) was
degassed with bubbling N2 for 15 min. Tetrakis(triphenylphos-
phine)palladium(0) (0.58 g, 0.5 mmol) was added and the mixture
heated at 90 °C for 16 h. The solution was poured into water (20
mL) and extracted with ethyl acetate (2 × 50 mL). The organic
layer was washed with water (2 × 15 mL) and brine (15 mL) and
dried (Na2SO4), and the solvent was removed under reduced
pressure. The residue was purified by flash column chromatography

1726 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Slee et al.
with 20-35% acetone in methylene chloride to give the title
compound 21 as a yellow solid (2.83 g, 85%). LCMS-1: tR ) 2.41
min. MS: m/z 334 [M + H]+, expected 334 [M + H]+.
2.3, 1H), 6.21 (s, 1H), 6.09 (s, 1H), 4.01 (s, 2H), 3.82–3.74 (m,
4H), 3.60–3.48 (m, 4H), 3.02 (s, 3H). LCMS-7: tR ) 13.25 min
(99%). MS: m/z 368 [M + H]+, Expected 368 [M + H]+. Anal.
(C18H21N7O2 ·2HCl·0.9H2O) C, H, N.
N-[2-Formyl-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyrimidin-4-
yl]acetamide (22). Intermediate 21 (0.2 g, 0.8 mmol) was dissolved
in MeOH/DCM (4/1, 10 mL) and cooled to -78 °C, and O3 was
bubbled in for 5 min. After the solution was flushed with N2 for
10 min, dimethyl sulfide (0.2 mL) was added, and the mixture was
allowed to warm to room temperature. The solution was evaporated
with N2 purging to give crude aldehyde 22, which was used directly
without purification in the following reactions.
2-(1,3-Oxazol-5-yl)-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyrimi-
din-4-amine (23). A mixture of intermediate 22 (104 mg, 0.4
mmol), TOSMIC (160 mg, 0.8 mmol), and potassium carbonate
(170 mg, 1.2 mmol) in MeOH (10 mL) was heated at 80 °C for
16 h. MeOH was evaporated, and the residue was partitioned
between ethyl acetate and water. The organic layer was washed
with brine and dried (Na2SO4), and the solvent was removed under
reduced pressure. The residue was purified by preparative TLC with
5% methanol in methylene chloride to give 23 as a yellow solid
(51 mg, 50% over two steps). 1H NMR (300 MHz, CDCl3 + 10%
CD3OD): δ 7.99 (s, 1H), 7.65 (s, 1H), 6.58 (s, 1H), 6.00 (s, 1H),
2.62 (s, 3H), 2.25 (s, 3H). LCMS-5: tR ) 4.04 min. LCMS-2: tR )
3.87 min. MS: m/z 257 [M + H]+, expected 257 [M + H]+.
2-(4-Methyl-1,3-oxazol-5-yl)-6-(3,5-dimethyl-1H-pyrazol-1yl)py-
rimidin-4-amine (24). Intermediate 22 (208 mg, 0.8 mmol) was
reacted with Me-TOSMIC (336 mg, 1.6 mmol) according to the
procedure described for intermediate 23 to give intermediate 24 as
a yellow solid (108 mg, 50% over two steps). LCMS-1: tR ) 1.88
min. MS: m/z 271 [M + H]+, expected 271 [M + H]+.
The following compounds were prepared using the same general
method as described for compound 1:
N-(2-Furan-2-yl-6-thiazol-2-ylpyrimidin-4-yl)-2-(4-methylpip-
erazin-1-yl)acetamide (26). Intermediate 16a was reacted with
chloroacetyl chloride followed by methylpiperazine according to
the procedure described for compound 1 to give compound 26 as
a slightly yellow solid (59%). 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-
4: tR ) 22.58 min (100%). MS: m/z 385 [M + H]+. Anal.
(C18H20N6O2S·2HCl) C, H, N.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-furan-2-ylpyrimidin-4-yl]-
2-(4-methylpiperazin-1-yl)acetamide (27). Intermediate 11a was
reacted with chloroacetyl chloride followed by methylpiperazine
according to the procedure described for compound 1 to give
1
compound 27 as an off-white solid (76%). H NMR (free base)
(300 MHz, CD3OD): δ 8.45 (s, 1H), 7.76 (dd, J ) 1.5, 0.6, 1H),
7.31 (dd, J ) 3.6, 0.6, 1H), 6.65 (dd, J ) 3.6, 1.5, 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-4: tR ) 17.00 min (100%). LCMS-6: tR
)
26.5. MS: m/z 396 [M + H]+, expected 396 [M + H]+
.
N-[2-(5-Methylfuran-2-yl)-6-pyrazol-1-ylpyrimidin-4-yl]-2-(4-
methylpiperazin-1-yl)acetamide (28). Intermediate 9d was reacted
with chloroacetyl chloride followed by methylpiperazine according
to the procedure described for compound 1 to give compound 28
1
as a yellow solid (64%). H NMR (300 MHz, CDCl3): δ 9.63 (s,
2-(1,3-Thiazol-2-yl)-6-(3,5-dimethyl-1H-pyrazol-1-yl)pyrimi-
din-4-amine (25). To a solution of thiazole (70 µL, 1.0 mmol) in
anhydrous THF (2 mL) at -78 °C was added n-BuLi (1.6 M in
hexane, 1.0 mL, 1.6 mmol) followed by stirring for 15 min. ZnCl2
(0.5 M in THF, 6.6 mL, 3.3 mmol) was added, and the mixture
was stirred for 1 h and then allowed to warm to -20 °C.
Intermediate 20 (65 mg, 0.2 mmol) and tetrakis(triphenylphos-
phine)palladium(0) (46 mg, 0.04 mmol) were added, and the
mixture was heated at 80 °C for 2 h. The solution was then poured
into 1 M HCl (10 mL) and extracted with ethyl acetate (2 × 10
mL). The organic layer was washed with water (2 × 5 mL) and
brine (5 mL) and dried (Na2SO4), and the solvent was removed
under reduced pressure. The residue was purified by preparative
TLC plate, eluting with 5% methanol in methylene chloride to give
1H), 8.65 (s, 1H), 8.55 (s, 1H), 7.80 (s, 1H), 7.30 (s, 1H), 6.49 (s,
1H), 6.21 (s, 1H), 3.23 (s, 2H), 2.48 (s, 4H), 2.33 (s, 4H), 2.17 (s,
3H). LCMS-2: tR ) 4.52 min. LCMS-5: tR ) 5.43 min. MS: m/z
382 [M + H]+.
N-[2-(5-Methylfuran-2-yl)-6-(5-methylpyrazol-1-yl)pyrimidin-
4-yl]2-(4-methylpiperazin-1-yl)acetamide (29). Intermediate 12
was reacted with chloroacetyl chloride followed by methylpipera-
zine according to the procedure described for compound 1 to give
compound 29 as a yellow solid (55%). 1H NMR (300 MHz, DMSO-
d6): δ 8.45 (s, 1H), 7.72 (s, 1H), 7.21 (d, J ) 3.6, 1H), 6.38 (s,
2H), 6.35 (d, J ) 3.6, 1H), 2.79 (bs, 4H), 2.76 (s, 3H), 2.45–2.50
(m, 4H), 2.38 (s, 3H). LCMS-2: tR ) 4.79 min. LCMS-5: tR
)
5.74 min. MS: m/z 396 [M + H]+, expected 396 [M + H]+.
1
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-(4-methylpiperazin-1-yl)acetamide (30). Intermedi-
ate 11d was reacted with chloroacetyl chloride followed by
methylpiperazine according to the procedure described for com-
25 as yellow solid (0.19 g, 70%). H NMR (300 MHz, CDCl3 +
10% CD3OD): δ 7.90 (d, J ) 3.3, 1H), 7.53 (d, J ) 3.0, 1H), 7.06
(s, 1H), 6.09 (s, 1H), 2.72 (s, 3H), 2.32 (s, 3H). LCMS-5: tR
)
4.90 min. LCMS-2: tR ) 4.16 min. MS: m/z 273 [M + H]+,
1
expected 273 [M + H]+.
pound 1 to give compound 30 as a white solid (82%). H NMR
(300 MHz, CD3OD): δ 8.42 (s, 1H), 7.20 (d, J ) 3.3, 1H), 6.26
(dd, J ) 3.0, 0.9, 1H), 6.13 (bs, 1H), 3.28 (s, 2H), 2.76 (s, 3H),
2.58–2.76 (m, 8H), 2.43 (s, 3H), 2.35 (s, 3H), 2.27 (s, 3H). LCMS-
2: tR ) 5.27 min. LCMS-6: tR ) 19.17 min. MS: m/z 410 [M +
H]+, expected 410 [M + H]+.
General Method for Final Compounds 1 and 26–40. To
dichloromethane (5 mL) were added compound 9a (0.3 g 1.3
mmol), chloroacetyl chloride (0.22 g, 0.20 mmol, 1.5 equiv), and
pyridine (0.16 g). The reaction mixture was stirred at room
temperature for 2 h. The reaction was quenched with saturated
sodium bicarbonate (5 mL) and extracted with dichloromethane (3
× 15 mL). The organic layers were combined and dried (Na2SO4),
then concentrated to give 2-chloro-N-(2-furan-2-yl-6-pyrazol-1-
ylpyrimidin-4-yl)acetamide as a yellow solid (0.4 g, 100% crude
yield), which was used crude in the next reaction.
N-[2-(5-Methylfuran-2-yl)-6-thiazol-2-ylpyrimidin-4-yl]-2-(4-
methylpiperazin-1-yl)acetamide (31). Intermediate 16b was re-
acted with chloroacetyl chloride followed by methylpiperazine
according to the procedure described for compound 1. The product
was purified by HPLC to give compound 31 as the TFA salt (15%).
1H NMR (300 MHz, CDCl3): δ 9.62 (s, 1H), 8.74 (s, 1H), 8.02 (d,
J ) 3.0, 1H), 7.54 (d, J ) 3.0, 1H), 7.34 (d, J ) 3.6, 1H), 6.22
(dd, J ) 3.6, 0.9, 1H), 3.24 (s, 2H), 2.62–2.80 (m, 8H), 2.48 (s,
3H), 2.43 (s, 3H). LCMS-5: tR ) 5.36 min (100%). LCMS-2: tR )
4.57 min. MS: m/z 399 [M + H]+, expected 399 [M + H]+.
N-[2-(Thiophen-2-yl)-6-pyridin-2-ylpyrimidin-4-yl]-2-(4-me-
thylpiperazin-1-yl)acetamide (32). Intermediate 16c was reacted
with chloroacetyl chloride followed by methylpiperazine according
to the procedure described for compound 1. The product was
To dichloromethane (10 mL) were added 2-chloro-N-(2-furan-
2-yl-6-pyrazol-1-ylpyrimidin-4-yl)acetamide (1.6 g, 5.3 mmol),
DIPEA (1.85 mL, 10.6 mmol, 2.0 equiv), and methylpiperazine
(2.0 equiv). The reaction mixture was stirred at room temperature
overnight. The reaction was quenched with water (5 mL) and
extracted with dichloromethane (3 × 15 mL). The organic layers
were combined and dried (Na2SO4), then concentrated. The residue
was purified by column chromatography on silica gel (DCM with
methanol gradient from 0 to 5%) to give N-(2-Furan-2-yl-6-pyrazol-
1-ylpyrimidin-4-yl)-2-(4-methylpiperazin-1-yl)acetamide (1) as a
1
purified by HPLC to give compound 32 as a TFA salt (48%). H
1
pale yellow solid (70%). H NMR (500 MHz, D2O): δ 7.56 (d, J
NMR (300 MHz, CDCl3 + 10% CD3OD): δ 8.86 (s, 1H), 8.69 (td,
J ) 4.8, 0.9, 1H), 8.49 (d, J ) 8.1, 1H), 8.02 (dd, J ) 3.4, 1.4,
) 1.2, 1H), 7.39 (s, 1H), 7.24 (s, 1H), 7.02 (s, 1H), 6.45 (d, J )

Acetamides as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1727
1H), 7.87 (dt, J ) 7.6, 1.8, 1H), 7.47 (dd, J ) 5.1, 0.9, 1H), 7.41
(ddd, J ) 7.4, 4.7, 1.4, 1H), 7.13 (dd, J ) 5.1, 3.9, 1H), 3.39 (s,
2H), 3.18 (m, 4H), 3.02 (m, 4H), 2.84 (s, 3H). LCMS-2: tR ) 4.5
min. LCMS-5: tR ) 5.7 min. MS: m/z 395 [M + H]+, expected
395 [M + H]+.
(s, 1H), 8.39 (dd, J ) 0.9, 8.1, 1H), 8.05 (ddd, J ) 7.8, 7.5, 1.8,
1H), 7.60 (m, 1H), 6.26 (s, 1H), 4.47 (s, 2H), 2.82 (s, 3H), 2.25 (s,
3H). LCMS-2: tR ) 4.31 min. LCMS-5: tR ) 5.53 min. MS: m/z
343 [M + H]+, expected 343 [M + H]+.
2-Chloro-N-[6-(3,5-dimethylpyrazol-1-yl)-2-pyridin-2-ylpyrimi-
din-4-yl]acetamide was then reacted with methylpiperazine accord-
ing to the procedure described for compound 1. The product was
purified by HPLC to give compound 39 as the TFA salt (50%).
LCMS-2: tR ) 3.81 min. LCMS-5: tR ) 5.53 min. MS: m/z 407
[M + H]+, expected 407 [M + H]+.
N-(2,6-Dipyrazol-1-ylpyrimidin-4-yl)-2-(4-methylpiperazin-1-
yl)acetamide (33). Intermediate 19 was reacted with chloroacetyl
chloride followed by methylpiperazine according to the procedure
described for compound 1. The product was purified by HPLC to
1
give compound 33 as the TFA salt (35%). H NMR (300 MHz,
CDCl3): δ 8.56 (t, J ) 3.0, 2H), 8.51 (s, 1H), 7.75 (m, 2H), 6.48
(m, 2H), 3.36 (s, 2H), 3.32 (m, 4H), 3.00 (m, 4H), 2.79 (s, 3H).
LCMS-2: tR ) 3.65 min. LCMS-5: tR ) 4.71 min. MS: m/z 368
[M + H]+, expected 368 [M + H]+.
N-[2-(3,5-Dimethylpyrazol-1-yl)-6-thiazol-2-ylpyrimidin-4-yl]-
2-(4-methylpiperazin-1-yl)acetamide (34). Intermediate 25 was
reacted with chloroacetyl chloride followed by methylpiperazine
according to the procedure described for compound 1. The product
was purified by HPLC to give compound 34 as the TFA salt (38%).
1H NMR (300 MHz, CDCl3): δ 9.64 (bs, 1H), 8.82 (s, 1H), 8.04
(d, J ) 3.3, 1H), 7.56 (d, J ) 3.0, 1H), 6.10 (s, 1H), 3.29 (s, 2H),
2.99–2.89 (m, 8H), 2.80 (s, 3H), 2.62 (s, 3H), 2.36 (s, 3H). LCMS-
2: tR ) 4.24 min. LCMS-5: tR ) 5.07 min. MS: m/z 413 [M +
H]+, expected 413 [M + H]+.
N-[2-(3,5-Dimethylpyrazol-1-yl)-6-oxazol-5-ylpyrimidin-4-yl]-
2-(4-methylpiperazin-1-yl)acetamide (35). Intermediate 23 was
reacted with chloroacetyl chloride followed by methylpiperazine
according to the procedure described for compound 1. The product
was purified by HPLC to give compound 35 as the TFA salt (14%).
1H NMR (300 MHz, CDCl3 + 10% CD3OD): δ 8.41 (s, 1H), 8.11
(s, 1H), 8.08 (s, 1H), 6.31 (s, 1H), 3.36 (s, 2H), 2.87 (m, 8H), 2.70
(s, 3H), 2.56 (s, 3H), 2.25 (s, 3H). LCMS-2: tR ) 3.74 min. LCMS-
5: tR ) 4.85 min. MS: m/z 397 [M + H]+, expected 397 [M +
H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-thiazol-2-ylpyrimidin-4-yl]-
2-(4-methylpiperazin-1-yl)acetamide (40). Intermediate 11c was
reacted with chloroacetyl chloride followed by methylpiperazine
according to the procedure described for compound 1. The product
was purified by HPLC to give compound 40 the TFA salt (31%).
1H NMR (300 MHz, CD3OD): δ 8.63 (s, 1H), 8.05 (d, J ) 3.0,
1H), 7.85 (d, J ) 3.6, 1H), 6.14 (s, 1H), 3.31 (s, 2H), 2.80 (s, 3H),
2.60–2.80 (m, 8H), 2.35 (s, 3H), 2.26 (s, 3H). LCMS-6: tR ) 15.96
min. LCMS-7: tR ) 22.66 min. MS: m/z 413 [M + H]+, expected
413 [M + H]+.
Biology Experimental Section. Pharmacology. Adenosine
A1 and A2A Receptor Binding 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 amplicon was cloned into the pcDNA5/FRT/V5-His-TOPO
expression vector (Invitrogen) and sequence confirmed using an
ABI 3100 automated sequencer (Applied Biosystems). Each expres-
sion 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, catolog no. 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.
N-[2-(3,5-Dimethylpyrazol-1-yl)-6-(4-methyloxazol-5-yl)pyri-
midin-4-yl]-2-(4-methylpiperazin-1-yl)acetamide (36). Intermedi-
ate 24 was reacted with chloroacetyl chloride followed by meth-
ylpiperazine according to the procedure described for compound
1. The product was purified by HPLC to give compound 36 as the
1
TFA salt (28%). H NMR (300 MHz, CDCl3): δ 9.50 (bs, 1H),
8.39 (s, 1H), 7.96 (s, 1H), 6.09 (s, 1H), 3.34 (s, 2H), 3.17 (m, 4H),
2.80 (s, 3H), 2.71 (s, 3H), 2.66 (s, 3H), 2.35 (s, 3H), 1.74 (m, 4H).
LCMS-2: tR ) 4.02 min. LCMS-5: tR ) 5.13 min. MS: m/z 411
[M + H]+, expected 411 [M + H]+.
Binding Assay. An aliquot of membranes (1–2 µg of protein)
was preincubated for 30 min at room temperature in the presence
of 10 µg/mL adenosine deaminase (type IV calf spleen, Sigma).
Membranes 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 presence of
varying concentrations of competing ligand. Nonspecific binding
was determined in the presence of excess (1 µM) of 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
(PerkinElmer) that had been pretreated with 0.5% polyethylene-
imine. The filter plates were then washed with 3 × 200 µL of 50
mM Tris-HCl, 50 mM NaCl, pH 7.4. Bound radioligand was
determined by scintillation 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).17 (1) For the
A1 membrane assay, the results are as follows: 3H-DPCPX
measured Kd ) 1.0 ( 0.5 nM; Bmax ) 8 ( 4 pmol/mg by Scatchard
analysis; control DPCPX (Tocris, 0439) Ki ) 1.6 ( 0.7 nM. (2)
For the human A2A membrane assay, the results are as follows:
3H-ZM241385 measured Kd ) 0.22 ( 0.20 nM; Bmax ) 33 ( 8
pmol/mg by Scatchard analysis; control NBI 80634 Ki ) 0.25 (
0.04 nM. (3) For the rat A2A membrane assay, the results are as
2-(4-Methylpiperazin-1-yl)-N-(6-pyrazol-1-yl-2-thiazol-2-ylpy-
rimidin-4-yl)acetamide (37). Intermediate 9c was reacted with
chloroacetyl chloride followed by methylpiperazine according to
the procedure described for compound 1. The product was purified
1
by HPLC to give compound 37 as the TFA salt (40%). H NMR
(300 MHz, CDCl3 + 10% CD3OD): δ 8.65 (d, J ) 3.0, 1H), 8.64
(s, 1H), 7.99 (d, J ) 3.6, 1H), 7.78 (d, J ) 1.5, 1H), 7.59 (d, J )
3.0, 1H), 6.49 (dd, J ) 2.7, 1.8, 1H), 3.42 (s, 2H), 3.36 (m, 4H),
3.07 (m, 4H), 2.82 (s, 3H). LCMS-2: tR ) 3.78 min. LCMS-5: tR
) 4.75 min. MS: m/z 385 [M + H]+, expected 385 [M + H]+.
2-(4-Methylpiperazin-1-yl)-N-(6-pyrazol-1-yl-2-pyridin-2-ylpy-
rimidin-4-yl)acetamide (38). Intermediate 9b was reacted with
chloroacetyl chloride followed by methylpiperazine according to
the procedure described for compound 1. The product was purified
1
by HPLC to give compound 38 as the TFA salt (32%). H NMR
(DMSO): δ 8.88 (d, J ) 2.7, 1H), 8.76 (d, J ) 4.2, 1H), 8.60 (s,
1H), 8.55 (d, J ) 7.8, 1H), 8.03 (dt, J ) 8.1, 1.8, 1H), 7.93 (d, J
) 0.9, 1H), 7.59 (dd, J ) 4.8, 0.9, 1H), 6.66 (s, 1H), 3.50 (s, 2H),
3.35–3.45 (m, 4H), 3.0–3.2 (m, 4H), 2.47 (s, 3H). LCMS-2: tR )
2.81 min. LCMS-5: tR ) 6.34 min. MS: m/z 379 [M + H]+,
expected 379 [M + H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-pyridin-2-ylpyrimidin-4-
yl]-2-(4-methylpiperazin-1-yl)acetamide (39). Intermediate 11b
was reacted with chloroacetyl chloride according to the procedure
described for compound 1 to give 2-chloro-N-[6-(3,5-dimethyl-
pyrazol-1-yl)-2-pyridin-2-ylpyrimidin-4-yl]acetamide. 1H NMR
(TFA salt) (300 MHz, DMSO): δ 8.77 (dd, J ) 0.9, 4.2, 1H), 8.52

1728 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Slee et al.
3
follows: H-ZM241385 measured Kd ) 0.33 ( 0.20 nM; Bmax
)
pellets are gently resuspended with 2 mL Krebs Henseleit buffer
(Sigma) by inversion. The cells suspension is adjusted with Krebs
Henseleit buffer to get a cell concentration of approximately 2.5
× 106 cells/mL. If rat hepatocytes are used or the incubation time
is over 2 h, it is suggested that Waymouth’s medium be used instead
of Krebs Henseleit buffer.
2.4 pmol/mg by Scatchard analysis, control NBI 80634 Ki ) 1.2
( 0.3 nM.
Preclinical Assays. Metabolic Stability Assay in Rat and
Human Liver Microsomes. Pooled male human and rat 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 containing 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. Precipitated proteins were removed by centrifuga-
tion for 15 min at 3000 rpm, and the supernatant fluid (∼0.1 mL)
was analyzed by LC/MS for the percentage of parent compound
remaining. The in vitro initial rates of metabolism were scaled using
constants, such as microsomal protein/g of liver, g of liver/kg of
body weight, and liver blood flow, to predict systemic clearance
and maximum predicted percent bioavailability. These calculations
from nonlinear regression assume that liver metabolism alone is
the determinant of bioavailability.
Metabolite Identification Assay in Human Liver Micro-
somes. Human liver microsomes (HLM) were purchased from a
commercial source (XenoTech LLC, Kansas City, KS) as a mixed
gender pool from 10 donors. Liver microsomal incubations for
metabolite identification were conducted using a 50 µM concentra-
tion of the NCE at a microsomal protein concentration of 0.5 mg/
mL in a 50 mM potassium phosphate buffer in the presence of an
NADPH-generating system composed of 1.0 mM NADP+, 3.0 mM
MgCl2, 5.0 mM G6P, and 3.0 units/mL G6PDH. A 50 mM stock
solution of the NCE in DMSO was used to achieve a final
concentration 50 µM, with the final concentration of DMSO being
less than 0.1% v/v. All concentrations are relative to a final
incubation volume of 1 mL.
Acknowledgment. We thank Shawn Ayube, Paddi Ekhlassi,
and John Harman for analytical support.
Supporting Information Available: NMR and LCMS data on
key final compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
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Incubations were conducted for 0, 30, 60, and 120 min at 37 °C
in a shaking water bath and were terminated by adding 1 mL of
ice-cold ACN. After the incubation suspensions are thoroughly
vortexed for 1 min and centrifuged at 3000 rpm for 20 min. The
resultant supernatant fractions were kept at -80 °C before LC/MS
analysis.
LC/MS analysis was carried out on an Agilent 1100 LC system
coupled to a Finnigan LCQ ion trap mass spectrometer. The Agilent
1100 LC systems consisted of a binary pump, a diode array detector,
a column heater, and a vacuum degasser/mobile phase tray. The
LC columns were typically a YMC ODS-AQ column, 150 mm ×
2 mm i.d., 5 µm particle size, and were operated at 45 °C. A typical
mobile phase used consisted of mobile phase A (0.1% formic acid
in deionized water) and mobile phase B (0.1% formic acid in
acetonitrile). Gradients were generally about 25 min long and were
optimized for separation of each NCE and its metabolites. Flow
rates were typically 0.40 mL/min. The mass spectrometer was
operated in (+)-ESI mode, and data dependent MS-MS spectra
were obtained for structural characterization.
In Human Liver Hepatocytes. Human hepatocytes were
purchased from a commercial source (XenoTech LLC, Kansas City,
KS). Cryopreservation vials containing hepatocytes are removed
from the liquid nitrogen storage unit and quickly placed in a water
bath at 37 °C for 2.0 min. Hepatocytes are prepared with Hepatocyte
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resuspended with 5 mL of tube B by inversion. The viability of
hepatocytes is recorded. The volume of cell suspension is adjusted
with tube B to obtain a cell concentration of approximately 1.0 ×
106 to 4.0 × 106 cells/mL. The cell suspension is centrifuged again
at 400–600 rpm for 3 ( 0.5 min at room temperature. The cell
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