2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 2. Reduction of hERG activity, observed species selectivity, and structure-activity relationships

Article abstract of DOI:10.1021/jm701187w

Previously we have described a series of novel A_2A receptor antagonists with excellent water solubility. As described in the accompanying paper, the antagonists were first optimized to remove an unsubstituted furyl moiety, with the aim of avoiding the potential metabolic liabilities that can arise from the presence of an unsubstituted furan. This effort identified a series of potent and selective methylfuryl derivatives. Herein, we describe the further optimization of this series to increase potency, maintain selectivity for the human A_2A vs the human A₁ receptor, and minimize activity against the hERG channel. In addition, the observed structure-activity relationships against both the human and the rat A_2A receptor are reported.

Full text of DOI:10.1021/jm701187w

1730
J. Med. Chem. 2008, 51, 1730–1739
2-Amino-N-pyrimidin-4-ylacetamides as A2A Receptor Antagonists: 2. Reduction of hERG
Activity, Observed Species Selectivity, and Structure-Activity Relationships
Deborah H. Slee,*,‡ Manisha Moorjani,‡ Xiaohu Zhang,‡ 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,| María I. Crespo,| Maria Prat,| Silvia Gual,| José-Luis Díaz,| Kayvon Jalali,§
Yang Sai,§ Zhiyang Zuo,§ Chun Yang,§ Jenny Wen,§ Zhihong O’Brien,§ Robert Petroski, 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 series of novel A2A receptor antagonists with excellent water solubility. As
described in the accompanying paper, the antagonists were first optimized to remove an unsubstituted furyl
moiety, with the aim of avoiding the potential metabolic liabilities that can arise from the presence of an
unsubstituted furan. This effort identified a series of potent and selective methylfuryl derivatives. Herein,
we describe the further optimization of this series to increase potency, maintain selectivity for the human
A2A vs the human A1 receptor, and minimize activity against the hERG channel. In addition, the observed
structure-activity relationships against both the human and the rat A2A receptor are reported.
Introduction
In the accompanying paper we described the SAR that led
to the identification of compound 1. Further profiling of this
lead identified compound 1 as a weak inhibitor of the hERG
channel as measured by electrophysiology. In addition, we found
that this series of A2A receptor antagonists exhibited distinct
differences in potency between the human and rat adenosine
A2A receptors, which helped to explain the limited efficacy
observed in the rodent. The existence of different structure–ac-
tivity relationships (SARs) for the human and rat A2A receptors
is rarely discussed in the literature. In this case we believe that
the reduced activity against the rat receptor inhibited our ability
to demonstrate efficacy at reasonable doses for many analogues
in this series. Herein, we describe the lead optimization of this
series and the SAR against both the human and rat A2A
receptors.
Parkinson’s disease (PD) is a debilitating disorder for which
there have been few novel treatments developed in recent years.
To date, the market is dominated by treatments that enhance
dopaminergic activity either via administration of dopamine
agonists or by dopamine replacement therapy with levodopa
(L-dopa). These treatments are not without side effects, which
include nausea, vomiting, dry mouth, confusion, hallucinations,
and somnolence (including sleep attacks). Furthermore, as the
disease progresses, the efficacy of these drugs can diminish and
the development of motor fluctuations and peak dose dyskinesias
are common.1
A2A receptor antagonists offer a new approach to the treatment
of PD that might avoid some of these side effects. Clinical trials
evaluating the motor effects observed in PD patients exhibiting
motor fluctuations and peak dose dyskinesias have validated
the use of A2A antagonists as an adjunct therapy to neurotrans-
mitter replacement in this patient population.2 There is also the
potential for A2A receptor antagonists to be effective on
movement dysfunction in early stage patients, either alone or
with reduced doses of dopamine agonists and/or L-dopa.3 In
addition, the A2A receptor has been shown to play a role in
mediation of arousal and A2A antagonists may be able to counter
the somnolent effect of these dopaminergic therapies.4 Perhaps
most importantly, there is a growing body of preclinical evidence
that A2A receptor antagonists demonstrate neuroprotective
properties that may offer protection against the development
of dyskinesias and may ultimately result in a slowing of the
progression of the disease.5
Chemistry
In the accompanying paper6 the synthesis and characterization
of compound 1, which is derived from the key intermediate 2,
was described. A number of analogues of compound 1 were
synthesized according to the procedures outlined in Schemes 1
and 2.
Three main approaches were used to synthesize the desired
analogues as follows. The synthesis of compound 4 was
achieved through attaching the side chain via acylation of
intermediate 2, with the acyl chloride generated from treatment
of 4-carboxymethylpiperidine-1-carboxylic acid tert-butyl ester
with oxalyl chloride to give intermediate 3. Intermediate 3 was
then deprotected with TFA followed by reductive amination to
install a methyl group to give compound 4. Analogues 1 and
6–18 were derived from key intermediate 5. Key intermediate
5 was synthesized via treatment of compound 2 with chloro-
acetyl chloride. Intermediate 5 was then treated with the desired
amine to displace the chlorine to give the final products 1 and
6–18. Alternatively, intermediate 5 could be treated with the
appropriate Boc-protected amine to give intermediates 19a-e.
In a manner similar to the synthesis of compound 4 from
intermediate 3, intermediates 19a-e were then deprotected via
* To whom correspondence should be addressed. Phone: 858-617-7849.
Fax: 858-617-7619. E-mail: 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.
Almirall Research Center.
Department of Preclinical Development, Neurocrine Biosciences.
|
§
10.1021/jm701187w CCC: $40.75
2008 American Chemical Society
Published on Web 02/29/2008

Acetamides as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1731
Scheme 1a
a Reagents and conditions: (a) 1-Boc-piperidine-4-ylacetic acid, oxalyl chloride, catalyst DMF, THF, evaporate, then DCM, pyridine, room temp, 4 h,
98%; (b) TFA, DCM, room temp, 1 h; (c) BH3 ·pyridine, HCOH, catalyst acetic acid, ethanol, 18 h, 22–79%; (d) chloroacetyl chloride, pyridine, DCM,
room temp, 2 h, 100%; (e) amine, DCM, DIPEA, room temp, 6-12 h, 17–92%; (f) Boc-protected diamine, DMF, TBAI, 70–90%.
Scheme 2a
a (a) Acryloyl chloride, pyridine, DCM, room temp, 6–12 h, 80%; (b) primary or secondary amine, DMF, room temp, 18 h, 75–78%.
treatment with TFA, followed by reductive amination to give
the methylated analogues 20–24 (Table 1).
Homologated analogues 26 and 27 were synthesized by
reaction of intermediate 2 with acryloyl chloride to give
intermediate 25, followed by Michael addition with the ap-
propriate amine.
channel. Various techniques for reducing hERG activity were
explored, including changing the distance between the amino
moieties, changing the shape of the side chain and distance
between the amine and the pyrimidine moiety, and varying the
pKa of the amine moieties.7 The SAR around the amino
containing side chains (R3, Figure 1) for this series is sum-
marized in Table 1. It was found that extending the methyl on
the piperazine of lead compound 1 to ethyl to give compound
6 maintained potency and significantly enhanced selectivity for
hA2A vs human A1 (hA1) to 171-fold. Enlarging the ring from
a piperazine to a homopiperazine as in analogue 7 again
maintained potency (hA2A Ki ) 10 nM) and enhanced selectivity
against hA1 to 210-fold but also increased hERG activity (IC50
) 285 nM). Further exploration by varying the relative positions,
spacing, and shape of the amino side chain gave interesting
results. Significant enhancement of potency was observed when
the distance between the two nitrogen atoms was increased, as
exemplified by compound 21 (hA2A Ki ) 1.5 nM). Interestingly,
this increase in potency was stereospecific, with the S-isomer
21 being more potent but less selective (hA2A Ki ) 1.5 nM,
hA1/hA2A 47-fold) than the R-isomer 20 (hA2A Ki ) 10 nM,
hA1/hA2A 89-fold). It was possible to remove the chirality to
give compound 22 and maintain potency and improve selectivity
(hA2A Ki ) 2.9 nM, hA1/hA2A 131-fold). When compound 22
Results and Discussion
Compound 1 is a potent human A2A (hA2A) receptor
antagonist (Ki ) 12 nM). When profiled in secondary assays,
compound 1 did not show any inhibition of the major CYP
enzymes (CYP2D6 or 3A4) but was found to have activity
against the hERG (human ether-a-go-go related gene) channel
(IC50 ) 950 nM, hERG patch-clamp). In addition, the activity
against the rat A2A (rA2A) receptor was found to be on the order
of 10-fold less than for the human A2A receptor. Because a rat
efficacy model was used to evaluate compounds, it was
necessary to track the activity of analogues against both human
and rat A2A receptors.
The available SAR around the R3 side chain (Figure 1) led
us to believe that the basic amine containing side chain was
responsible for the hERG activity. This SAR had not been fully
explored, and optimization of this side chain was undertaken
with the aim of increasing selectivity for A2A vs the hERG

1732 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Slee et al.
Table 1d

Acetamides as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1733
Table 1. Continued
a Displacement of specific [3H]-ZM 241385 binding at human A2A receptors expressed in HEK293 cells. b Displacement of specific [3H]-DPCPX binding
at human A1 receptors expressed in HEK293 cells. c Displacement of specific [3H]-ZM 241385 binding at rat A2A receptors expressed in CHO cells. d Data
are expressed as mean values of at least two runs ( SEM. ND: not determined.
Figure 2. Metabolites of compound 22, after incubation with human
liver microsomes (tentative assignment of peaks observed by LCMS).
Figure 1
When incubated with human liver microsomes plus GSH (10 mM), no
detectable amount of GSH adducts were observed.
was profiled in the hERG assay, the IC50 was found to be 1670
nM, a potency similar to that of the lead compound 1, but the
selectivity for the hA2A receptor over the hERG channel was
improved from 79-fold to 576-fold because of the increased
potency of compound 22 against the A2A receptor. In vitro
metabolite identification via LCMS after incubation of 22 in
human liver microsomes (HLM) showed that the major me-
tabolite was mono-oxidation somewhere on the aromatic system
(Figure 2), possibly on one of the methyl groups. As expected,
no downstream metabolites resulting from metabolism of the
furyl moiety were observed, indicating that the methyl group
had blocked the metabolism observed previously when the furan
was unsubstituted.6 In addition, no glutathione (GSH) adducts
were observed when the compound was incubated with HLM
in the presence of GSH, further supporting the absence of
electrophilic reactive metabolites.
by the amide analogue 8. This distal amine could also be
replaced by oxygen as in the morpholine analogue 9 and the
homomorpholine compound 10. From cassette PK studies in
rodent, however, the brain levels were poor for these analogues,
indicating a reduced capacity to cross the blood-brain barrier.
Moving the oxygen out of the ring as in the methoxypiperidine
analogue 11 resulted in a loss in potency against the human
A2A receptor and a dramatic loss in potency against the rat A2A
receptor. A series of pyrrolidine analogues were also synthe-
sized, and it was found that the S-dimethylaminopyrrolidine
analogue 13 was potent and selective, as were a number of
aminopyrrolidine analogues as exemplified by compounds
13–18. Unfortunately, for the pyrrolidine analogues tested, the
potency at the hERG channel increased to less than 1 µM, as
illustrated by compounds 13 and 17. Increasing the distance
between the two nitrogen atoms in the pyrrolidine series, as in
The basicity of the more basic nitrogen (distal to the
pyrimidine moiety) in the side chain of compound 1 could be
attenuated without a dramatic effect on binding, as illustrated

1734 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Slee et al.
helped to explain that relatively high doses were required to
show efficacy for these compounds.
Conclusion
We have developed a series of potent and selective adenosine
A2A receptor antagonists that incorporate amine containing side
chains that confer good selectivity and potency in addition to
instilling good physical properties for drug development. The
presence of these amines, however, made the series active
against the hERG channel. SAR studies around the amine
containing side chain identified compounds with potent A2A
receptor activity and reduced activity against the hERG channel.
Removal of one of the amine moieties to give compound 4
maintained potency and selectivity for the adenosine A2A
receptor, and the activity against hERG was reduced to 4170
nM (now 772-fold selective for the hA2A receptor vs the hERG
channel). In vitro metabolism studies on a representative
compound (22) indicated that there were no reactive metabolites
formed from the methylfuran. Demonstration of efficacy in the
rodent was confounded by lack of potency against the rat
receptor. It was possible to demonstrate in vivo efficacy for
some members of this series in a rat model of haloperidol
induced catalepsy, at relatively high doses of 30 mg/kg. This
indication of efficacy increases confidence that these compounds
should be efficacious in higher species where the potency is
significantly higher.
Figure 3. Effect of compounds 13 and 27 (2 h after dose) on mean
descent latency (( standard error) in seconds on haloperidol-induced
catalepsy in the bar test. The vehicle bar represents the mean and
standard error from both experiments combined: (/) vehicle versus
treated rats, p < 0.05.
Table 2a
Ki ( SEM (nM)
b
c
compd
hA2A
rA2A
exposured in rat brain (ng/g)
13
27
3.7 ( 0.6
2.4 ( 0.1
160 ( 40
41 ( 3
1900
250
a Data are expressed as mean values of at least two runs ( SEM.
b Displacement of specific [3H]-ZM 241385 binding at human A2A receptors
expressed in HEK293 cells. c Displacement of specific [3H]-ZM 241385
binding at rat A2A receptors expressed in CHO cells. d At 2 h after dosing
with a 30 mg/kg oral (po) dose.
Experimental Section
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. Melting points
were recorded on a Büchi 535 apparatus. 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, a 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, a UV
detector (220 and 254 nm), a MS detector (APCI), and Berger FCM
1200 CO2 pump module; HPLC column, pyridine PYR 60A, 6 µm,
4.6 mm × 150 mm column; HPLC gradient, 4.0 mL/min, 120 bar,
from 10% methanol in supercritical CO2 to 60% methanol in
supercritical CO2 in 1.67 min, maintaining 60% for 1 min. Back-
pressure was regulated at 140 bar. Analytical HPLC-MS method
4 was as follows: platform, Dionex equipped with an autosampler,
a 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, a 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
compounds 23 and 24, reduced hERG activity while maintaining
the desired potency and selectivity for the hA2A receptor.
Further exploration of the piperazine series to investigate the
importance of the piperazine nitrogen atom of compound 1
nearest the pyrimidine found that replacement with a carbon to
give the carbon-linked methylpiperidine analogue 4 gave a
potent and selective molecule, which implies that the distal
amine is most important for potency and selectivity. Interestingly
the hERG activity for this compound was also attenuated to
4170 nM. In addition, as typically seen for this series, compound
4 was on the order of 20-fold less active at the rat than the
human A2A receptor.
A limited set of compounds were made where the linker
between the acyl and amino moieties was extended. Initially
these compounds looked very promising and two of the most
potent and selective analogue compounds 26 and 27 are shown
at the bottom of Table 1. These compounds were stable enough
for in vitro and in vivo evaluation but showed signs of chemical
instability on storage, arising from a retro-Michael reaction that
gives the corresponding acrylate precursor (see Scheme 2).
Because of this instability, this subseries was not pursued further.
In general, compounds in this series did not exhibit robust
efficacy in rodent at doses of less than 30 mg/kg. It is believed
that this is largely explained by a lack in potency at the rat A2A
receptor. However, in some cases it was possible to demonstrate
efficacy at doses of 30 mg/kg. Two such examples are
compounds 13 (hA2A Ki ) 3.7 nM) and 27 (hA2A Ki ) 2.4
nM), which were evaluated in the haloperidol-induced catalepsy
(HIC) assay8 and demonstrated significant efficacy at 30 mg/
kg po 2 h after dosing, as shown in Figure 3. These compounds
were 20 to 40-fold less active against the rat receptor (compound
13, rA2A Ki ) 160 nM and compound 27, rA2A Ki ) 41 nM,
Table 2), which in combination with exposure in the brain

Acetamides as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1735
solvent D is 25% acetonitrile in methanol. The gradient runs from
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, a 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, a 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.
organic layers were combined and dried (Na2SO4), then concentrated
to give 2-chloro-N-[6-(3,5-dimethylpyrazol-1-yl)-2-(5-methylfuran-
2-yl)pyrimidin-4-yl]acetamide 5 as a yellow solid (0.4 g, 100%
crude yield), which used without purification in the next subsequent
reaction. LCMS-1: tR ) 2.82 (96%). MS: m/z 346 [M + H]+,
expected 346 [M + H]+. To dichloromethane (10 mL) were added
intermediate 5 (1.6 g, 5.3 mmol), DIPEA (1.85 mL, 10.6 mmol,
2.0 equiv) and the appropriate amine (2.0 equiv). The reaction
mixture was stirred at room temperature overnight. The reaction
was quenched water (5 mL) and extracted with dichloromethane
(3 × 15 mL). The organic layers were combined and dried
(Na2SO4), then concentrated. The residue was purified either by
column chromatography on silica gel (DCM with methanol gradient
from 0 to 5%) to give the free base (or the HCl salt if converted
via treatment with HCl in ether) or by HPLC (TFA salt).
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-(4-methylpiperazin-1-yl)acetamide (1). Intermediate
5 was reacted with 4-methylpiperazine according to the above
general procedure. The product was purified by HPLC to give 1 as
a white solid (82%). 1H NMR (free base) (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),
6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyrimidin-
4-ylamine (2). Intermediate 2 was prepared according to the
methods described in the accompanying paper.
2.35 (s, 3H), 2.27 (s, 3H). LCMS-2: tR ) 5.27. LCMS-6: tR
)
19.17. MS: m/z 410 [M + H]+, expected 410 [M + H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-(4-ethylpiperazin-1-yl)acetamide (6). Intermediate
5 was reacted with 4-ethylpiperazine according to the above general
procedure. The product was purified by HPLC to give 6 as a light-
brown oil (70%). LCMS-2: tR ) 5.34. LCMS-4: tR ) 9.73 (98%).
MS: m/z 424 [M + H]+, expected 424 [M + H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-(4-methyl[1,4]diazepan-1-yl)acetamide (7). Inter-
mediate 5 was reacted with N-methylhomopiperazine according to
the above general procedure. The product was purified by column
on silica gel with DCM/methanol, 90/10, to give 7 as a white solid
when converted to the HCl salt (45%). LCMS-2: tR ) 4.96. LCMS-
5: tR ) 5.70. MS: m/z 424 [M + H]+, expected 424 [M + H]+.
2-(4-Acetylpiperazin-1-yl)-N-[6-(3,5-dimethylpyrazol-1-yl)-2-
(5-methylfuran-2-yl)pyrimidin-4-yl]acetamide (8). Intermediate
5 was reacted with 4-acetylpiperazine according to the above general
procedure. The product was purified by HPLC to give 8 as a light-
brown oil (70%). LCMS-2: tR ) 5.17. LCMS-5: tR ) 5.86. MS:
m/z 438 [M + H]+, expected 438 [M + H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-morpholin-4-ylacetamide (9). Intermediate 5 was
reacted with 4-acetylpiperazine according to the above general
procedure. The product was purified by HPLC to give 9 as the
TFA salt (light-brown oil, 85%). 1H NMR (300 MHz, DMSO-d6):
δ 8.33 (bs, 1H), 7.24 (d, J ) 3.3, 1H), 6.38 (dd, J ) 0.9, 3.3, 1H),
6.24 (s, 1H), 4.3 (brm, 2H), 3.87 (brm, 4H), 2.74 (s, 3H), 2.57
(brm, 4H), 2.40 (s, 3H), 2.23 (s, 3H). LCMS-2: tR ) 5.15. LCMS-
5: tR ) 6.06. MS: m/z 397 [M + H]+, expected 397 [M + H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-[1,4]oxazepan-4-ylacetamide (10). Intermediate 5
was reacted with homomorpholine according to the above general
procedure. The product was purified by HPLC to give 10 as the
TFA salt (light-brown oil, 85%). LCMS-3: tR ) 2.05. LCMS-5: tR
) 6.21. MS: m/z 411 [M + H]+, expected 411 [M + H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-(4-methoxypiperidin-1-yl)acetamide (11). Inter-
mediate 5 was reacted with 4-methoxypiperidine according to the
above general procedure. The product was purified by HPLC to
Synthesis of N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methyl-
furan-2-yl)pyrimidin-4-yl]-2-(1-methylpiperidin-4-yl)aceta-
mide (4). To THF (10 mL) were added 1-Boc-piperidine-4-ylacetic
acid (540 mg, 2.2 mmol, 1 equiv), followed by oxalyl chloride (300
mg, 0.24 mmol, 1.1 equiv) and a few drops of DMF. The reaction
mixture was stirred under nitrogen at room temperature for 1 h
and concentrated under vacuum to dryness. The resulting 4-chlo-
rocarbonylmethylpiperidine-1-carboxylic acid tert-butyl ester was
dissolved in dichloromethane (10 mL), and intermediate 2 (300
mg, 0.5 equiv) was added, followed by pyridine (150 mg). The
mixture was stirred at room temperature for 4 h. The reaction was
then quenched by addition of saturated sodium bicarbonate (50 mL),
extracted with dichloromethane (2 × 50 mL), the organic layers
were combined, dried (Na2SO4), concentrated, and purified by silica
gel column using 40% ethyl acetate in hexane to give 4-{[6-(3,5-
dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyrimidin-4-
ylcarbamoyl]methyl}piperidine-1-carboxylic acid tert-butyl ester 3
as clear thick oil (540 mg, 98%). LCMS-1: tR ) 3.28 (90%, crude
mixture). MS: m/z 495 [M + H]+, expected 495 [M + H]+.
Compound 3 was dissolved in dichloromethane (10 mL) followed
by addition of TFA (10 mL). The mixture was stirred at room
temperature for 1 h, and the solvent was removed by nitrogen flow
to give deprotected 3 as a clear oil, which was used without further
purification (LCMS-1: tR ) 2.32 (95%, crude). MS: m/z 395 [M +
H]+). Deprotected 3 was dissolved in EtOH (10 mL), followed by
addition of formaldehyde (2 mL, 30% in water), acetic acid (10
drops), and borane-pyridine (0.2 mL). The mixture was stirred at
room temperature overnight. The reaction was quenched by addition
of 1 M HCl (50 mL) with stirring for 10 min, neutralized by addition
of saturated sodium bicarbonate, and extracted with dichloromethane
(2 × 25 mL). The organic layers were combined, dried (Na2SO4),
concentrated, and purified by silica gel column using 5% methanol
in dichloromethane. Compound 4 was obtained as a clear oil (350
1
mg, 79%). H NMR (300 MHz, CDCl3): δ 8.49 (s, 1H), 8.03 (s,
1H), 7.16 (d, J ) 3, 1H), 6.17 (d, J ) 3, 1H), 6.01 (s, 1H), 2.83–2.87
(m, 2H), 2.76 (s, 3H), 2.45 (s, 3H), 2.83–2.87 (m, 2H), 2.31 (d, J
) 6, 1H), 2.28 (s, 3H), 2.27 (s, 3H), 1.91–1.95 (m, 2H), 1.77–1.81
(m, 2H), 1.33–1.38 (m, 2H). LCMS-6: tR ) 19.79. LCMS-7: tR )
32.4. MS: m/z 409 [M + H]+, expected 409 [M + H]+. A portion
of the product was converted to HCl salt. Anal. (C22H28-
N6O2 ·2HCl·C2H6O) C, H, N.
give 11 as the TFA salt (light-brown oil, 70%). LCMS-2: tR
)
5.40. LCMS-4: tR ) 10.23. MS: m/z 425 [M + H]+, expected 425
[M + H]+.
General Method for Final Compounds 1 and 6–18. To 5 mL
of dichloromethane were added compound 2 (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
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-pyrrolidin-1-ylacetamide (12). Intermediate 5 was
reacted with pyrrolidine according to the above general procedure.
The product was purified by HPLC to give 12 as the TFA salt (light-
brown oil, 80%). 1H NMR (300 MHz, CDCl3): δ 7.52 (d, J ) 3.6,
1H), 6.90 (s, 1H), 6.26 (d, J ) 3.6, 1H), 6.06 (s, 1H), 2.77 (s, 3H),

1736 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Slee et al.
2.47 (s, 3H), 2.29 (s, 3H), 0.08 (s, 1H). LCMS-2: tR ) 5.20. LCMS-
5: tR ) 6.36. MS: m/z 381 [M + H]+, expected 381 [M + H]+.
2-((S)-3-Dimethylaminopyrrolidin-1-yl)-N-[6-(3,5-dimethylpyra-
zol-1-yl)-2-(5-methylfuran-2-yl)pyrimidin-4-yl]acetamide (13).
Intermediate 5 was reacted with (S)-3-dimethylaminopyrrolidine
according to the above general procedure. The product was purified
by column on silica gel with DCM/methanol, 90/10, to give 13 as
water (20 mL), stirred, and filtered. The Intermediate 19 was
obtained as a white solid (1.1g, 96%). Alternatively, TEA or DIEA
can be used as base. The white solid 19 was dissolved in
dichloromethane (15 mL) followed by TFA (15 mL). The reaction
mixture was stirred at room temperature for 2 h. The solvent was
removed under nitrogen flow. The oil obtained was dissolved in
EtOH (20 mL) and formaldehyde (20 mL, 30% in water), followed
by borane-pyridine (2 mL) and 10 drops of acetic acid. The
reaction mixture was stirred at room temperature overnight, then
quenched with 1 M HCl (50 mL), stirred for 10 min, then
neutralized by addition of sodium bicarbonate. The mixture was
extracted with dichloromethane (2 × 100 mL). The organic layers
were combined, dried (Na2SO4), concentrated, and purified by silica
gel column, using dichloromethane (500 mL), followed by 10%
methanol in dichloromethane (500 mL), and finally 10% 2 M
ammonia in methanol in dichloromethane (1000 mL). Compound
22 was obtained as a clear oil (0.88 g, 93%). The oil was converted
to the HCl salt via treatment with HCl in ether, followed by
evaporation of the solvent and excess HCl to give compound 22
(HCl salt) as a light-yellow solid (0.9 g, 89%). 1H NMR (300 MHz,
CDCl3): δ 9.49 (s, 1H), 8.47 (s, 1H), 7.18 (d, J ) 3.3, 1H), 6.18
(d, J ) 3, 1H), 6.01 (s, 1H), 3.17 (s, 2H), 2.97–2.93 (m, 4H), 2.85
(s, 6H), 2.77 (s, 3H), 2.47 (s, 3H), 2.28 (s, 3H), 1.91–1.87 (m,
2H), 1.70–1.55 (m, 5H). LCMS-6: tR ) 15.87. LCMS-7: tR ) 35.09.
MS: m/z 452 [M + H]+, expected 452 [M + H]+.
1
a white solid HCl salt (75%). H NMR (300 MHz, DMSO-d6): δ
8.30 (s, 1H), 7.21 (d, J ) 3.6, 1H), 6.37 (d, J ) 3.6, 1H), 6.22 (s,
1H), 2.00–4.05 (m, 9H), 2.83 (s, 6H), 2.73 (s, 3H), 2.39 (s, 3H),
2.23 (s, 3H). LCMS-6: tR ) 16.50. LCMS-7: tR ) 30.06. MS: m/z
424 [M + H]+, expected 424 [M + H]+.
2-((R)-3-Dimethylaminopyrrolidin-1-yl)-N-[6-(3,5-dimeth-
ylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyrimidin-4-yl]aceta-
mide (14). Intermediate 5 was reacted with (R)-3-dimethylami-
nopyrrolidine according to the above general procedure. The product
1
was purified by HPLC to give 14 as the TFA salt. H NMR free
base (300 MHz, CDCl3): δ 9.57 (s, 1H), 8.47 (s, 1H), 7.16 (d, J )
3.3 Hz, 1H), 6.16 (d, J ) 3.3, 1H), 6.00 (s, 1H), 3.32 (AB syst, J
) 10.5 Hz, 2H), 3.00–2.65 (m, 5H), 2.75 (s, 3H), 2.45 (s, 3H),
2.33 (s, 6H), 2.27 (s, 3H), 2.17–2.05 (m, 1H), 1.97–1.85 (m, 1H).
LCMS-3: tR ) 2.24 (100%). LCMS-5: tR ) 6.30. MS: m/z 424 [M
+ H]+, expected 424 [M + H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-((S)-3-isopropoxypyrrolidin-1-yl)acetamide (15).
Intermediate 5 was reacted with (S)-3-hydroxypyrrolidine according
to the above general procedure followed by a reaction with
2-bromopropane (1.2 equiv) and sodium hydride (1.1 equiv) in DMF
at room temperature overnight. The product was purified by silica
gel chromatography with DCM and 1% methanol as eluent. The
HCl salt of 15 was obtained (17%). LCMS-5: tR ) 6.35. LCMS-2:
tR ) 5.85. MS: m/z 439 [M + H]+, expected 439 [M + H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-((R)-3-ethylaminopyrrolidin-1-yl)acetamide (16).
Intermediate 5 was reacted with (R)-3-ethylaminopyrrolidine ac-
cording to the above general procedure. The product was purified
by column on silica gel with DCM/methanol, 90/10, and 1%
NH4OH to give 16 as a glassy solid (69 mg, 92%). 1H NMR (300
MHz, CDCl3): δ 9.65 (s, 1H), 8.45 (s, 1H), 7.15 (d, J ) 3.6, 1H),
6.13 (d, J ) 3.6, 1H), 5.99 (s, 1H), 3.65–3.71 (m, 1H), 3.38 (d,
2H), 2.98–3.10 (m, 4H), 2.99 (q, J ) 6.9, 2H), 2.75 (s, 3H), 2.42
(s, 3H), 2.31–2.40 (m, 1H), 2.27 (s, 3H), 2.17–2.25 (m, 1H), 1.44
(t, J ) 6.9, 3H). LCMS-3: tR ) 2.17. LCMS-4: tR ) 10.06. MS:
m/z 424 [M + H]+, expected 424 [M + H]+.
2-((R)-3-Dimethylaminomethylpiperidin-1-yl)-N-[6-(3,5-di-
methylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyrimidin-4-yl]ac-
etamide (20). Compound 20 was obtained by the same method as
compound 22, using (S)-1-piperidin-3-ylmethylcarbamic acid tert-
butyl ester instead of piperidin-4-ylmethylcarbamic acid tert-butyl
ester. The title compound 20 (HCl salt) was obtained as a pale-
1
yellow solid (0.22 g, 38%). H NMR (300 MHz, CDCl3) δ 9.56
(s, 1H), 8.47 (s, 1H), 7.17 (d, J ) 3.3, 1H), 6.17 (d, J ) 3, 1H),
6.01 (s, 1H), 3.22–2.84 (m, 6H), 2.76 (s, 3H), 2.72 (s, 6H), 2.44
(s, 3H), 2.28 (s, 3H), 2.14–2.12 (m, 2H), 1.82–1.18 (m, 5H). LCMS-
6: tR ) 15.76. LCMS-7: tR ) 36.21 (100%). MS: m/z 452 [M +
H]+, expected 452 [M + H]+.
2-((S)-3-Dimethylaminomethylpiperidin-1-yl)-N-[6-(3,5-di-
methylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyrimidin-4-yl]ac-
etamide (21). Compound 21 was obtained by the same method as
compound 22, using (R)-1-piperidin-3-ylmethylcarbamic acid tert-
butyl ester instead of piperidin-4-ylmethylcarbamic acid tert-butyl
ester. The title compound 21 was obtained as the HCl salt (0.25 g,
1
42%). H NMR (300 MHz, CDCl3): δ 9.57 (s, 1H), 8.46 (s, 1H),
7.16 (d, J ) 3.3, 1H), 6.17 (d, J ) 3, 1H), 6.00 (s, 1H), 3.30–2.81
(m, 6H), 2.76 (s, 3H), 2.72 (s, 6H), 2.43 (s, 3H), 2.27 (s, 3H),
2.14–2.12 (m, 2H), 1.82–1.18 (m, 5H). LCMS-6: tR ) 15.58.
LCMS-7: tR ) 36.22. MS: m/z 452 [M + H]+, expected 452 [M +
H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-((S)-3-ethylaminopyrrolidin-1-yl)acetamide (17).
Intermediate 5 was reacted with (S)-3-ethylaminopyrrolidine ac-
cording to the above general procedure. The product was purified
by column on silica gel with DCM/methanol, 90/10, to give 17 as
1
a white solid HCl salt (35%). H NMR (300 MHz, DMSO-d6): δ
2-((R)-3-Dimethylaminomethylpyrrolidin-1-yl)-N-[6-(3,5-di-
methylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyrimidin-4-yl]aceta-
mide (23). Compound 23 was obtained by the same method as
compound 22, using (R)-1-pyrrolidin-3-ylmethylcarbamic acid tert-
butyl ester instead of piperidin-4-ylmethylcarbamic acid tert-butyl
ester. The title compound 23 was obtained as the HCl salt (150
mg, 22%, from 500 mg of starting material). 1H NMR (300 MHz,
CDCl3): δ 9.62 (s, 1H), 8.49 (s, 1H), 7.18 (d, J ) 3.3, 1H), 6.17
(d, J ) 3, 1H), 6.01 (s, 1H), 3.30 (d, J ) 5.4, 2H), 2.91–2.69 (m,
4H), 2.69 (s, 3H), 2.45 (s, 3H), 2.50–2.28 (m, 3H), 2.33 (s, 6H),
2.28 (s, 3H), 2.20–1.55 (m, 2H). LCMS-6: tR ) 15.10. MS: m/z
438 [M + H]+, expected 438 [M + H]+. Anal. (C23H31N7O2 ·2HCl)
C,H, N.
2-((S)-3-Dimethylaminomethylpyrrolidin-1-yl)-N-[6-(3,5-di-
methylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyrimidin-4-yl]aceta-
mide (24). Compound 24 was obtained by the same method as
compound 22, using (S)-1-pyrrolidin-3-ylmethylcarbamic acid tert-
butyl ester instead of piperidin-4-ylmethylcarbamic acid tert-butyl
ester. The title compound was obtained as the HCl salt (350 mg,
51%, from 500 mg of starting material). 1H NMR (300 MHz,
CDCl3): δ 9.62 (s, 1H), 8.49 (s, 1H), 7.18 (d, J ) 3.3, 1H), 6.17
(d, J ) 3, 1H), 6.01 (s, 1H), 3.30 (d, J ) 5.4, 2H), 2.91–2.69 (m,
9.01 (s, 1H), 8.32 (s, 1H), 7.22 (d, J ) 3.3, 1H), 6.38 (d, J ) 3.2,
1H), 6.23 (s, 1H), 4.17 (m, 2H), 3.94 (bs, 4H), 3.41 (s, 1H),
3.00–3.02 (m, 2H), 2.73 (s, 3H), 2.39 (s, 3H), 2.23 (s, 3H),
2.09–2.18 (m, 2H), 1.20 (t, J ) 6.9, 3H). LCMS-2: tR ) 4.86.
LCMS-7: tR ) 28.06. MS: m/z 424 [M + H]+, expected 424 [M +
H]+.
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]-2-((S)-3-diethylaminopyrrolidin-1-yl)acetamide (18).
Intermediate 5 was reacted with (S)-3-diethylaminopyrrolidine
according to the above general procedure. The product was purified
by HPLC to give 18 as the TFA salt (light-brown oil, 60%). LCMS-
2: tR ) 4.92. LCMS-5: tR ) 5.99. MS: m/z 452 [M + H]+, expected
452 [M + H]+.
General Method for Synthesis of Compounds 20-24: 2-(4-
Dimethylaminomethylpiperidin-1-yl)-N-[6-(3,5-dimethylpyrazol-
1-yl)-2-(5-methylfuran-2-yl)pyrimidin-4-yl]acetamide (22). To
DMF (10 mL) were added intermediate 5 (760 mg, 2.2 mmol, 1
equiv), piperidin-4-ylmethylcarbamic acid tert-butyl ester (450 mg,
2.2 mmol, 1equiv), potassium carbonate (260 mg, 1 equiv), and a
catalytic amount of TBAI. The reaction mixture was stirred at room
temperature overnight. The reaction was quenched by addition of

Acetamides as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1737
4H), 2.69 (s, 3H), 2.45 (s, 3H), 2.50–2.28 (m, 3H), 2.33 (s, 6H),
2.28 (s, 3H), 2.20–1.55 (m, 2H). LCMS-6: tR ) 15.12 (99%).
MS: m/z 438 [M + H]+, expected 438 [M + H]+. Anal.
(C23H31N7O2 ·2HCl) C, H, N.
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 than washed 3 × 200 µL with 50 mM
Tris-HCl and 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).9 (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
N-[6-(3,5-Dimethylpyrazol-1-yl)-2-(5-methylfuran-2-yl)pyri-
midin-4-yl]acrylamide (25) for Compounds 26 and 27. To
intermediate 2 (1.0 g, 3.7 mmol, 1 equiv) stirring in 10 mL of DCM
were added pyridine (0.389 mL, 4.8 mmol, 1.3 equiv) and acryloyl
chloride (0.390 mL, 4.8 mmol, 1.3 equiv). The reaction mixture
was stirred at room temperature overnight. The reaction was then
quenched with saturated sodium bicarbonate (50 mL) and extracted
with DCM (2 × 50 mL). The combined organic layers were dried
(MgSO4) and filtered to yield crude product 25 as a light-brown
solid (80%), which was used without further purification. LCMS-
1: tR ) 2.00. LCMS-2: tR ) 5.28. MS: m/z 324 [M + H]+, expected
324 [M + H]+.
3-Dimethylamino-N-[6-(3,5-dimethylpyrazol-1-yl)-2-(5-meth-
ylfuran-2-yl)pyrimidin-4-yl]propionamide (26). To intermediate
25 (0.80 g, 2.5 mmol, 1 equiv) stirring in DMF (2 mL) was added
2 M dimethylamine in THF (3.7 mL, 7.5 mmol, 3 equiv). The
mixture was stirred at 40 °C for 2 h. The mixture from the
completed reaction was diluted with brine (50 mL) and extracted
with DCM (2 × 50 mL). The combined organic layers were dried
(MgSO4) and filtered to yield a light-brown oil. The crude product
was purified by silica gel column using 10% methanol in dichlo-
romethane. Compound 26 was obtained as a white solid (HCl salt)
(75%). 1H NMR (300 MHz, DMSO-d6): δ 10.09 (bs, 1H), 8.33 (s,
1H), 7.20 (d, J ) 3.3, 1H), 6.37 (dd, J ) 3, 0.9, 1H), 6.22 (s, 1H),
3.37 (dd, J ) 7.2, 12.3, 2H), 3.01 (dd, J ) 6.9, 14.1, 2H), 2.79 (s,
3H), 2.77 (s, 3H), 2.72 (s, 3H), 2.39 (s, 3H), 2.22 (s, 3H). LCMS-
6: tR ) 19.29. LCMS-7: tR ) 28.51. MS: m/z 369 [M + H]+,
expected 369 [M + H]+. Anal. (C19H24N602 ·2HCl) C, H, N.
3-((S)-3-Dimethylaminopyrrolidin-1-yl)-N-[6-(3,5-dimethylpy-
razol-1-yl)-2-(5-methylfuran-2-yl)pyrimidin-4-yl]propiona-
mide (27). Compound 27 was prepared in a fashion similar to that
for compound 25 using (3S)-(-)-3-(dimethylamino)pyrrolidine in
place of dimethylamine. Compound 27 was obtained as a white
solid (HCl salt) (78%). 1H NMR (300 MHz, DMSO-d6): δ 8.34 (s,
1H), 7.19 (d, J ) 2.7, 1H), 6.35 (d, J ) 2.7, 1H), 6.20 (s, 1H),
4.15 (brm, 4H), 3.50 (brm, 5H), 2.99 (m, 2H), 2.84 (s, 6H), 2.72
(s, 1H), 2.38 (s, 1H), 2.21 (s, 1H). LCMS-6: tR ) 15.11. LCMS-7:
tR ) 29.43. MS: m/z 438 [M + H]+, expected 438 [M + H]+.
Anal. (C23H31N702 ·3HCl) C, H, N.
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, catalog 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.
follows: [3H]-ZM241385 measured Kd ) 0.33 ( 0.20 nM; Bmax
)
2.4 pmol/mg by Scatchard analysis; control NBI 80634 Ki ) 1.2
( 0.3 nM.
hERG Assay Protocol. Overview. Compounds that inhibit
hERG potassium channels have been shown to prolong the QT
interval of the ECG in man and increase the risk of cardiac
arrhythmias (Ficker et al., 1998; Kiehn et al., 1996; Mohammad et
al., 1997). A preclinical assay was used to determine whether drug
candidates inhibit hERG by examining their ability to block the
potassium tail currents in HEK293 cells stably transfected with
hERG cDNA (Zhou et al., 1997; Zhou et al., 1998). Concentra-
tion–response curves for test compounds were generated and the
maximum inhibition and IC50 value determined.10
Source and Maintenance of hERG Transfected Cells. hERG.
T.HEK (HEK293 cells stably transfected with hERG cDNA) were
obtained from the University of Wisconsin. The cells were
continuously maintained and passaged in minimum essential
medium (MEM) supplemented with 10% fetal bovine serum, 1%
nonessential amino acids, and 0.4 mg/ml Geneticin (Gibco-BRL).
For electrophysiology studies, the cells were plated onto sterile glass
coverslips in 24-well tissue culture trays (containing 0.5 mL
medium) at a density (2000–5000 cells per well) that enabled
isolated cells to be selected for patch clamping. The dishes were
stored in a humidified, gassed (7.5% CO2) incubator at 37 °C.
Electrophysiological Assay Protocol. Coverslips, upon which
cells had been plated, were transferred to the recording chamber
on an inverted microscope and continuously perfused (1–2 mL/
min) with control solution at room temperature. Test substances
were applied by bath perfusion, switching from the control solution
to one containing the appropriate concentration of test substance.
The recording chamber volume was approximately 0.5 mL, and
fluid exchange took approximately 1 min.
The hERG current was recorded using standard patch-clamp
methods. A schematic diagram of the voltage waveform used in
these experiments is indicated below. The standard voltage profile
was as follows: (1) Vstep from -80 to -50 mV for 0.5 s to deter-
mine holding current, (2) Vstep from -50 to +10 mV for 4 s to
activate hERG current, (3) Vstep from +10 to -50 mV for 4 s to
remove voltage-dependent inactivation and elicit the hERG tail
current, and (4) return to the holding potential of -80 mV. The
voltage waveform was repeated every 12 s (one trial), and the
current responses to each waveform were digitized and stored onto
a computer hard disk.
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 PerkinElmer NET 974) for the A1
The baseline hERG tail current was recorded for at least 5 min
(25 trials at 12 s intervals) in control bath solution to assess the
suitability and stability of the cell. Once a stable baseline hERG
tail current had been established, the test substance at the lowest
concentration (0.1 nM) was applied for 5 min. Then the test

1738 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Slee et al.
substance was washed out by reperfusing with the control bath
solution for at least 5 min or until the effect was completely
reversed. When the new baseline hERG tail current was established
(typically 5 min), the next highest concentration of test substance
was applied for 5 min and washed out for at least 5 min. This was
repeated until the highest concentration of test substance had been
tested. Concentrations of 0.1 nM to 10 µM were used to determine
the concentration–response relationship of test substances. This was
performed on 2–6 cells/concentration.
isolation kit (XenoTech LLC) according to the protocol provided
by the manufacture (see manufacturer’s appendix). Contents of the
cryopreservation vials are gently poured into tube A containing
Percoll solution and rinsed with 1.67 mL of tube B. The cell
suspension is mixed by gentle inversion and centrifuged at 500–700
rpm for 5 ( 0.5 min at room temperature. The cell pellets are gently
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
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.
In Vivo HIC Assay. 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 (po) 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 (ip) injection of haloperidol
(1.5 mg/kg in a 10 mL/kg sterile water). The bar test consisted of
three 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 samples were collected following the haloperidol-induced
catalepsy test (samples collected 2 h after dose), and samples were
analyzed to determine exposure (method described below in the
screening PK protocol section).
The peak tail current amplitude from each trial was measured
off-line from the digitized data files using PClamp 9 software and
entered into an Excel spreadsheet. The concentration–response data
(normalized to predrug baseline) was plotted and fitted to a
sigmoidal function (logistic four parameter) using SigmaPlot
software. The IC50 value was derived from the fitted curve.
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 LCMS 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 Microsomes.
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 identi-
fication were conducted using a 50 µM concentration of the NCE at
a microsomal protein concentrations 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 concentra-
tions are relative to a final incubation volume of 1 mL.
Screening PK Protocol for Sample Extraction and Analysis
To Determine Brain Exposure in Rat at the 2 h Time Point.
Brain Sample Extraction. Brain tissue sample analysis was
conducted by liquid chromatography in tandem with a mass
spectrometric detection (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: 60/40/0.1) containing 50 µL of a
proprietary internal standard (10 µg/mL NBI-67377). The homo-
genates 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: 60/40/0.2), and 50 µL of internal standard
(10 µg/mL) into half of a rat brain. Standards for brain were
processed and analyzed at the same time and in exactly the same
way as the analytical samples.
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, and
the resultant supernatant fractions were kept at -80 °C before
LCMS analysis.
LCMS 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. and 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.
Bioanalysis. The LC-MS/MS system equipped with Agilent 1100
HPLC system was coupled to a CTC PAL autoinjector (Leap
Technologies, Carrboro, NC) and a Sciex API 4000 triple quadruple
mass spectrometer. 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, 5-95% 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
mm × 50 mm, with a particle size of 5 µm. Electrospray ionization
source was used for detection at a positive MRM (multiple reaction
monitoring) mode. 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.
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
Acknowledgment. We thank Shawn Ayube, Paddi Ekhlassi,
and John Harman for analytical support.

Acetamides as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1739
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Supporting Information Available: Copies of NMR and LCMS
data are available on key final compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
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