Lead optimization of 4-acetylamino-2-(3,5-dimethylpyrazol-1-yl)-6- pyridylpyrimidines as A2A adenosine receptor antagonists for the treatment of Parkinson's disease

Article abstract of DOI:10.1021/jm800851u

4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-pyrimidines bearing substituted pyridyl groups as C-6 substituents were prepared as selective adenosine hA _2A receptor antagonists for the treatment of Parkinson's disease. The 5-methoxy-3-pyridyl derivative 6g (hA_2A K_i 2.3 nM, hA₁ K_i 190 nM) was orally active at 3 mg/kg in a rat HIC model but exposure was poor in nonrodent species, presumably due to poor aqueous solubility. Follow-on compound 16a (hA_2A K_i 0.83 nM, hA₁ K_i 130 nM), bearing a 6-(morpholin-4-yl)-2-pyridyl substituent at C-6, had improved solubility and was orally efficacious (3 mg/kg, HIC) but showed time-dependent cytochrome P450 3A4 inhibition, possibly related to morpholine ring metabolism. Compound 16j (hA_2A K_i 0.44 nM, hA₁ K_i 80 nM), bearing a 6-(4-methoxypiperidin-1-yl)- 2-pyridyl substituent at C-6, was sparingly soluble but had good oral exposure in rodent and nonrodent species, had no cytochrome P450 or human ether-a-go-go related gene channel issues, and was orally efficacious at 1 mg/kg in HIC and at 3 mg/kg for potentiation of L-dopa-induced contralateral rotations in 6-hydroxydopamine-lesioned rats.

Full text of DOI:10.1021/jm800851u

J. Med. Chem. 2008, 51, 7099–7110
7099
Lead Optimization of 4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-6-pyridylpyrimidines as A2A
Adenosine Receptor Antagonists for the Treatment of Parkinson’s Disease
Xiaohu Zhang,†,2 John E. Tellew,*,† Zhiyong Luo,† Manisha Moorjani,† Emily Lin,† Marion C. Lanier,† Yongsheng Chen,†
John P. Williams,† John Saunders,† Sandra M. Lechner,‡ Stacy Markison,‡ Tanya Joswig,‡ Robert Petroski,‡ Jaime Piercey,‡
William Kargo,‡ Siobhan Malany,§ Mark Santos,§ Raymond S. Gross,†,3 Jenny Wen,| Kayvon Jalali,| Zhihong O’Brien,|
Carol E. Stotz, Mar´ıa I. Crespo,# Jose´-Luis D´ıaz,# and Deborah H. Slee*,†
Departments of Medicinal Chemistry, Pharmacology, Neuroscience, Chemical DeVelopment, Preclinical DeVelopment, and Pharmaceutical
DeVelopment, Neurocrine Biosciences, 12780 El Camino Real, San Diego, California 92130, Almirall Research Center, Almirall, Ctra. Laurea`
Miro´, E-08980 St. Feliu de Llobregat, 408-410 Barcelona, Spain
ReceiVed July 10, 2008
4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-pyrimidines bearing substituted pyridyl groups as C-6 substituents
were prepared as selective adenosine hA2A receptor antagonists for the treatment of Parkinson’s disease.
The 5-methoxy-3-pyridyl derivative 6g (hA2A Ki 2.3 nM, hA1 Ki 190 nM) was orally active at 3 mg/kg in
a rat HIC model but exposure was poor in nonrodent species, presumably due to poor aqueous solubility.
Follow-on compound 16a (hA2A Ki 0.83 nM, hA1 Ki 130 nM), bearing a 6-(morpholin-4-yl)-2-pyridyl
substituent at C-6, had improved solubility and was orally efficacious (3 mg/kg, HIC) but showed time-
dependent cytochrome P450 3A4 inhibition, possibly related to morpholine ring metabolism. Compound
16j (hA2A Ki 0.44 nM, hA1 Ki 80 nM), bearing a 6-(4-methoxypiperidin-1-yl)-2-pyridyl substituent at C-6,
was sparingly soluble but had good oral exposure in rodent and nonrodent species, had no cytochrome
P450 or human ether-a-go-go related gene channel issues, and was orally efficacious at 1 mg/kg in HIC and
at 3 mg/kg for potentiation of L-dopa-induced contralateral rotations in 6-hydroxydopamine-lesioned rats.
Introduction
Extracellular adenosine regulates a wide range of functions
in higher organisms, in which the effects are mediated by a
family of four class A (rhodopsin-like) GPCRs,a,1 adenosine
receptors known as A1, A2A, A2B, and A3.2 Caffeine, a
nonselective adenosine receptor antagonist, has been used for
millennia for its effects on wakefulness and alertness, and in
recent years, epidemiological studies have shown that consump-
tion of caffeine is associated with a decreased risk of developing
Parkinson’s disease, although a true neuroprotective effect has
not been demonstrated.3 The neurological effects of caffeine
are due in large part to its activity at human adenosine 2A (hA2A)
receptors, which are abundant in the nucleus accumbens,
olfactory tubercle, and striatum, where they are colocalized with
dopamine D2 receptors.4 It has been shown that antagonists of
Figure 1. Lead acylaminopyrimidine hA2A antagonists. R ) phenyl,
substituted phenyl, pyridyl or substituted pyridyl, R1 ) small alkyl or
alkoxy.
A2A receptors in the striatum can potentiate the response of
dopamine agonists acting at D2 receptors in the same location.
This has led to the hypotheses that A2A antagonists, either alone
or in combination with dopamine agonists, can have a role in
the treatment of neurodegenerative movement disorders such
as Parkinson’s disease and Huntington’s disease, and there is
substantial preclinical evidence in support of this hypothesis.5
Accordingly, the pharmaceutical industry has made a substantial
investment in recent years to develop selective, orally available
A2A antagonists.6 At least three A2A antagonists are currently
undergoing clinical trials for Parkinson’s symptomology.7
Previously we reported work on a series of 4-acylaminopy-
rimidine A2A antagonists based on 1 (Figure 1).8 This compound
has high affinity for the hA2A and rat A2A (rA2A) receptors but
has poor selectivity over the human A1 (hA1) receptor (hA2A
Ki 0.6 nM, hA1 Ki 9.2 nM), blockade of which could lead to
diuresis and various cardiovascular effects.9 The simple furan
substituent in this series of compounds also represents a
toxicology risk.10 Recently, we reported on a series of active
* To whom correspondence should be addressed. For D.H.S.: phone,
858-617-7849; fax, 858-617-7925; E-mail: dslee@neurocrine.com. For
J.E.T.: phone, 858-812-1628; fax, 858-332-4513; E-mail: jtellew@gnf.org;
current address, Genomics Institute of the Novartis Research Foundation,
10675 John Jay Hopkins Drive, San Diego, California 92121.
†
Department of Medicinal Chemistry, Neurocrine Biosciences.
Present address: BioDuro Co., No.29 Life Science Park Road,
2
Changping District, Beijing, P.R.China 102206. Email: xiaohu.zhang@
bioduro.com.
3
Department of Chemical Development, Neurocrine Biosciences.
Department of Neuroscience, Neurocrine Biosciences.
Department of Pharmacology, Neurocrine Biosciences.
‡
§
|
Department of Preclinical Development, Neurocrine Biosciences.
Department of Pharmaceutical Development, Neurocrine Biosciences.
#
Almirall Research Center.
a hA2A, human adenosine 2A; hA1, human adenosine 1; HIC, haloperidol
induced catalepsy; GPCR, G-protein coupled receptor; MED, minimal
effective dose; hERG, human ether-a-go-go related gene; CYP, cytochrome
P450.
10.1021/jm800851u CCC: $40.75
2008 American Chemical Society
Published on Web 10/24/2008

7100 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Zhang et al.
Table 1. Human A2A and A1 Binding Affinity and Other Data for Compounds 6a-6h, 7, and 9a-9d
Ki ( SEM (nM)a
Clint (mL/min kg)
b
c
compd
R
R1
hA2A
hA1
hA1/hA2A
HLM
RLM
CYP-3A4 IC50(µM)
6a
6b
6c
6d
6e
6f
6g
6h
7
Ph
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
87 ( 9
290 ( 110
2100 ( 20
820 ( 150
65 ( 8
3.3
18
21
ND
ND
ND
ND
36
9.3
35
41
ND
ND
ND
ND
360
110
120
140
43
43
3-pyridyl
4-pyridyl
3-MeO-Ph
2-F-3-MeO-Ph
3,5-di-MeO-Ph
5-MeO-3-pyridyl
5-OH-3-pyridyl
5-(2-methoxy-ethoxy)-3-
pyridyl
120 ( 4
>20
8.5
0.74
>20
0.38
18
40 ( 0
0.92 ( 0.13
0.60 ( 0.04
0.19 ( 0.05
2.3 ( 0.2
2.9 ( 0.5
3.1 ( 0.6
71
110 ( 9
187
116
82
67
36
22 ( 2
190 ( 20
195 ( 100
110 ( 13
18
14
3.0
9a
5-MeO-3-pyridyl
(morpholin-4-yl)-
methyl
4.0 ( 0.1
940 ( 170
235
3.0
150
>20
9b
9c
9d
5-MeO-3-pyridyl
5-MeO-3-pyridyl
5-MeO-3-pyridyl
Et
OMe
OEt
1.8 ( 0.4
7.4 ( 0.6
7.8 ( 1.8
70 ( 22
780 ( 60
620 ( 100
39
106
79
29
16
38
110
93
59
17
16
20
a Data are expressed as geometric means of at least two runs ( the standard error of measurement (SEM). b Displacement of specific [3H]-27 ([3H]-
ZM241385, Figure 3)21 binding at hA2A receptors expressed in HEK293 cells. c Displacement of specific [3H]-28 ([3H]-DPCPX, Figure 3)22 binding at hA1
receptors expressed in HEK293 cells; HLM ) human liver microsomes; RLM ) rat liver microsomes; ND ) not determined.
Scheme 1a
and selective compounds such as 2 (hA2A Ki 12 nM, hA1 Ki
850 nM), in which the furyl group has been substituted or
replaced entirely. During the course of that work, it became
clear that one of the preferred pyrimidine C-6 substituents, 3,5-
dimethylpyrazol-1-yl, could also serve well as a C-2 furan
replacement, as in 3 (hA2A Ki 27 nM, hA1 Ki 1700 nM).11
Compounds with amine-bearing substituents on the acyl group
such as 2 tend to be blockers of the hERG channel,12-14
blockade of which has been associated with cardiovascular
safety risks.15 Neutral lipophilic acyl groups yield active and
selective compounds16 with reduced hERG liability, but these
compounds tend to be poorly soluble and are not generally orally
active in rat haloperidol-induced catalepsy (HIC), an in vivo
pharmacological model used to assess reversal of the effects of
the dopamine D2 antagonist haloperidol.17 Elaborate acyl groups,
whether neutral or basic, tend to improve selectivity over hA1,
but they also tend to decrease activity at the rA2A receptor,
complicating interpretation of in vivo results. We reasoned that
combining the C-2 dimethylpyrazole substituent of 3 with a
simple 4-acylamino group, as in 1, might allow us to discover
compounds that had a combination of good human and rat A2A
activity, good selectivity over hA1, and low hERG inhibitory
activity. We recently reported preliminary results from this
approach, in which we explored substituted phenyl substituents
at the C-6 position.18 We now report the extension of this work
to pyridyl compounds of general structure 4 (Figure 1), in which
R1 is a small alkyl or alkoxy group. This work resulted in the
discovery of a highly active and selective series of compounds,
the best of which (6g and 16j) displayed good in vivo oral
activity in rat models for Parkinson’s disease and were advanced
to preclinical development. The details of this investigation are
disclosed herein.
Chemistry. The compounds shown in Table 1 were prepared
from 511 according to the methods described in Scheme 1.
Compounds 6a-6h bear simple 6-aryl substituents and were
prepared in one step by Suzuki coupling with suitable com-
mercially available boronic acids or esters (Scheme 1). Com-
pound 7 was prepared by Mitsunobu alkylation of 6h.
Compound 9a was prepared by acylation of 8 with chloroacetyl
chloride, followed by displacement with morpholine. Compound
a Reagents and conditions: (a) RB(OR′)2, Pd(Ph3P)4, K2CO3, dioxane/
water, 90 °C; (b) 2-methoxyethanol, DEAD, Ph3P; (c) K2CO3, MeOH, r.t.;
(d) ClCH2COCl, pyridine; morpholine; (e) EtCOCl, pyridine, DCM; (f)
triphosgene, DCM; methanol or ethanol.
9b was prepared by acylation of 8 with propionyl chloride.
Because direct reaction of 8 with chloroformates was unsuc-

Pyridylpyrimidines as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7101
Scheme 2a
a Reagents and conditions: (a) RB(OR′)2, Pd(Ph3P)4, K2CO3, dioxane/water, 90 °C; (b) amine, DMA, 190 °C, microwave; (c) amine, DMSO, 100 °C; (d)
amine hydrochloride, NMP, TEA, 160 °C, 80 min, microwave; (e) amine hydrochloride, dioxane, Pd2dba3, K3PO4, ligand 13, 100 °C; (f) morpholine,
DMSO, CuI, L-proline, K2CO3, 100 °C.
cessful, we prepared 9c and 9d by reaction of 8 with triphosgene,
followed by treatment with methanol or ethanol, respectively.
The aminopyridine-substituted compounds of Table 4 were
generally prepared from halopyridyl intermediates 10, 11, and
12 by either thermal or metal-catalyzed amine displacement
reactions (Scheme 2). In the thermal reaction, under a variety
of conditions, the desired acetylamino products were typically
accompanied by the corresponding deacetylated primary amine
products. The halopyridyl intermediates were obtained by Suzuki
reaction of 5 with commercially available halopyridyl boronate
esters. Compound 16b was obtained by direct Suzuki reaction
of 5 with 6-aminopyridin-2-ylboronic acid pinacol ester
(Scheme 1).
In the case of compound 17, the displacement routes of
Scheme 2 failed, so an alternate route involving de novo
synthesis of the pyrimidine nucleus was used (Scheme 3).
Potassium tert-butoxide promoted reaction of acetonitrile with
cyanopyridine 20b provided enaminonitrile 21b. Subsequent
condensation19 with thiourea, methylation, and oxidation with
oxone provided sulfone 24b. Displacement of the methane-
sulfonyl group with 3,5-dimethylpyrazole was problematic;
however, displacement with hydrazine, followed by condensa-
tion with 2,4-pentanedione, provided the pyrazole 26b in
acceptable yield. Acetylation furnished final compound 17.
Variations of the route in Scheme 3 were used to perform scale-
up syntheses of 16a (Table 4) and other compounds in this
general series. Depending on the substrate, the oxidation of
sulfide 23 to sulfone 24 could be accompanied by oxidation of
the amine side chain to generate an N-oxide. The parent amine
was regenerated via reduction with hydrazine (or another
reaction component) during the subsequent sulfone displacement
step.
the pyrimidine C-6 position with the goal of improving potency
and selectivity against the hA1 receptor. This survey revealed
that a variety of substitution patterns gave compounds with hA2A
activity in the 10-50 nM range but typically with low to fair
selectivity over hA1 (5-30×). The key finding from this survey
was that a hydrogen bond accepting group in the phenyl
3-position, such as fluoro or methoxy, improved both hA2A
binding activity and selectivity over hA1. Thus compounds such
as 6d, 6e, and in particular 6f were among the most active and
selective compounds in the series (Table 1). As expected for
these very weakly basic compounds, little or no hERG inhibition
was seen in this series.20
Further profiling of 6e and 6f (Tables 1 and 2) indicated that
both compounds had moderate to good stability in human
(HLM) and rat (RLM) liver microsomal assays and had good
human functional antagonist activity in a cAMP assay,23
validating the design premise for the series. Compound 6f was
poorly soluble and had poor permeability in an in vitro Caco-2
cell permeability assay (p ) 24 nm/s), while 6e had reasonable
solubility in a high-throughput solubility assay in pH 7.4
phosphate-buffered saline (PBS). Compound 6e also was found
to have good Caco-2 cell permeability (p ) 260 nm/s). In the
rat HIC model, however, orally dosed (PO) 6e was inactive,
presumably due to lack of exposure (Table 2). Compound 6f,
in spite of its in vitro profile, had good oral exposure and was
quite efficacious in the HIC model, having a minimal effective
dose (MED) of 1 mg/kg (Table 2). Unfortunately, these and
other 3-methoxyphenyl compounds interacted with CYP3A4,
either through direct inhibition or in the case of 6f, through
time-dependent increase of CYP3A4 inhibition, indicating potent
inhibition by a metabolite.
Our discovery efforts thus turned to finding a compound with
the efficacy and selectivity profile of 6f but with a more
favorable metabolism profile. Because the simple 3-pyridyl
compound 6b was similar in activity to phenyl compound 6a
and appeared to have somewhat less CYP3A4 inhibitory activity,
we made methoxypyridine 6g. This compound was a potent
antagonist of hA2A and was reasonably selective versus hA1
Results and Discussion
The prototypical phenyl and pyridyl compounds 6a, 6b, and
6c were found to be moderately potent binders at the hA2A
receptor but had poor selectivity versus hA1 (Table 1). We
performed an extensive survey of substituted phenyl groups at

7102 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Zhang et al.
Scheme 3a
solubility and exposure difficulties.26 Compound 6g (pKa 3.5)
could be formulated as a hydrochloride salt, but this resulted in
no overall improvement in pharmacokinetics.
We sought a followup to 6g that would have improved
nonrodent exposure, which we hoped to achieve by increasing
aqueous solubility and/or decreasing the melting point.27 We
reasoned that introducing a weakly basic or polar substituent
onto the pyridine ring could increase solubility by disrupting
crystal packing and/or increasing polarity. To this end, we
prepared a series of morpholine-substituted pyridine isomers
14, 15, 16a, and 17 (Table 4). Compound 17 had only moderate
binding affinity, while 14 had poor functional activity (cAMP
IC50 213 nM). Compound 15 was reasonably active and
selective; however, the 3-morphinyl-2-pyridyl isomer 16a
stood out, having very potent hA2A binding affinity, high
selectivity over hA1, and very good functional activity (Table
2). Solubility of 16a (0.14 mg/mL in PBS) was also improved
over that of 6g.
Fixing on the 3-substituted-2-pyridyl isomeric relationship,
we then surveyed other amines in place of the morpholine of
16a, with illustrative examples shown in Table 4. A simple
amino group (16b) gave weaker binding, while some acyclic
amines such as dimethylamine (16c) gave compounds with high
binding affinity and selectivity although with relatively poor
human functional activity (cAMP IC50 156 nM, Table 2). Cyclic
aliphatic amines such as pyrrolidine (16d) or piperidine (16e)
gave potent compounds but with reduced selectivity. Homo-
morpholine 16f was less active and had poor RLM stability.
N-Methylpiperazine derivative 16g was the most potent com-
pound in the series, both in terms of binding affinity and also
in terms of functional activity (Table 2). In addition, it was
highly selective over hA1 (175×) and its weakly basic amino
group (measured pKa 7.6) provided options for formulation,
although actual solubility (0.24 mg/mL, PBS) was similar to
that of 16a. Compound 16g was studied in the rat HIC model,
where, disappointingly, it showed no inhibition of catalepsy.
Exposure was somewhat low (Table 2) in spite of good stability
in the RLM assay and reasonable in vitro permeability (MDCK-
MDR assay, p ) 154 nm/s). However, comparison with 6g
indicates that the poor efficacy for 16g is clearly not due to
modest exposure alone and is consistent with a trend we have
observed in which basic compounds, in spite of good plasma
and gross brain exposure, have generally weak efficacy in the
rat HIC model.17 Compound 16g was also found to be a potent
blocker of the hERG channel (IC50 540 nM, electrophysiology
assay).
a Reagents and conditions: (a) morpholine, DMA, Et3N, 100 °C; (b) NH3,
MeOH, sealed tube, 75 °C; (c) (CF3CO)2O, DCM, Et3N, 0 °C to r.t.; (d)
t-BuOK, MeCN, toluene, r.t.; (e) thiourea, NaOEt, EtOH, sealed tube, 100
°C; (f) NaOH, H2O, MeI, r.t.; (g) oxone, MeOH, H2O, NaHCO3; (h)
H2NNH2, EtOH, reflux; (i) 2,4-pentanedione, EtOH, r.t.; (j) AcCl, pyridine,
DCM, r.t.
Returning to the relatively nonbasic morpholine 16a, we
found that it had a good overall in vitro profile (Tables 2 and
4) and showed good efficacy in the HIC model with a MED of
3 mg/kg. This compound was inactive at the hERG channel
and was not a potent inhibitor of CYP3A4 under normal assay
conditions. However, 16a was found to have time-dependent
inhibition of CYP3A4 and was further found to form glutathione
adducts when incubated with HLM in the presence of glu-
tathione, implying metabolic generation of a reactive species.
Metabolite identification studies (HLM) indicated that a sub-
stantial portion of the metabolism took place on the morpholine
ring.28
yet was only a weak inhibitor of CYP3A4 and a weak blocker
of the hERG channel (IC50 24 µM, electrophysiology assay). It
had good functional activity and was orally efficacious at 1 mg/
kg in HIC. We prepared a series of analogues of 6g in which
we varied the methoxy group (6h, 7) or the acyl group (9a-9d);
however, none of these compounds was superior overall to 6g,
having either lower selectivity (7, 9b) or poor in vivo efficacy
in the HIC model (6h, 9a, 9c, 9d Table 2). Compound 6g had
poor aqueous solubility (<0.01 mg/mL at pH g 2) but had
reasonable oral exposure in the rat (AUC0-24 1430 ng h/mL at
10 mg/kg, free base, 25% PEG-400 in D5W).25 Exposure at
elevated doses (30-300 mg/kg) in rat was sufficient for 14 day
toxicological studies in that species (Table 3). However,
exposure in nonrodent species (dog, cynomolgus monkey,
marmoset) was extremely low using a variety of formulations,
precluding further development. The extremely high melting
point (283 °C) of 6g free base no doubt contributed to the
We then set out to replace the morpholine ring with a group
that would not be susceptible to the metabolic issues seen with
16a. Since we had observed that an oxygen atom (presumably
a hydrogen bond acceptor) distal to the amine nitrogen improved
selectivity, we incorporated a number of oxygen-bearing cyclic
and acyclic amines in place of the morpholine. In particular,

Pyridylpyrimidines as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7103
Table 2. Secondary Data for Select Compounds
a
a
b
Rat HIC
MED (mg/kg)
HIC %
Cplasma
Cbrain
rA2A
hcAMPc
IC50 (nM)
solubilityd
(mg/mL)
compd
reversala
(ng/mL)
(ng/g)
Ki (nM)
measured logD7.4
measured pKa
6ee
6f
ND
1330
ND
264
ND
860
ND
ND
150
53
ND
1150
ND
154
ND
1380
ND
ND
170
91
12
3
32
17
57
64
49
0.17
<0.02
ND
ND
ND
ND
ND
ND
0.14
0.24
ND
ND
0.03
0.20
2.0
3.2
ND
2.4
2.4
2.8
ND
ND
ND
ND
ND
ND
3.6
ND
ND
ND
ND
3.5
5.3, 3.6
3.6
ND
ND
2.9
1f
1
49
64
6he
6g
62
32
30
21
68
82
10
7
20
14
1.5
10
9ae
9b
9c
1
10
69
46
31
167
196
13
7
156
70
9de
16a
16ge
16c
16h
16j
16k
3f
55
7.6
30
10
1f
290
220
150
16
180
870
560
9
ND
ND
3.4
70
88
18
22
30
ND
a At time ) 2 h after 10 mg/kg oral dose. b Displacement of specific [3H]-27 binding at rA2A receptors expressed in CHO cells. c hA2A receptor antagonism
of 3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoic acid (CGS-21680)24 stimulated
cAMP. d Solubility in PBS at pH 7.4. e Inactive at a dose of 30 mg/kg PO. f Lowest dose tested; ND ) not determined.
Table 3. Plasma Exposure of 6g in Rats Following Oral Dosing
(Vehicle: 25% PEG-400 in D5W)
3.7 as vehicle (AUC0-24 25,700 ng h/mL at 300 mg/kg PO).
Solubility in this formulation was 1 mg/mL. In dogs, acceptable
exposure was also observed (AUC0-24 3,400 ng h/mL at 30
mg/kg PO) using 10% PEG400 in 0.25% methylcellulose as
vehicle. On the basis of the above data, 16j was selected for
further preclinical evaluation.
Hydroxypiperidine metabolite 16k had very low exposure
when dosed orally at 10 mg/kg (Table 2) and thus had low
efficacy in the rat HIC model at this dose. It is interesting to
note, however, that 16k strongly inhibited catalepsy at a dose
of 30 mg/kg PO (90% inhibition, Table 2). Exposure at this
dose was moderate (plasma and brain levels 180 ng/mL and 91
ng/g, respectively, Table 2), suggesting that circulating 16k
could contribute to the efficacy of 16j in vivo.
dose (mg/kg)
Cmax (ng/mL)
AUC0-24 (ng h/mL)
10
30
100
300
410
2600
3400
4200
1430
18700
28900
36800
we chose methoxyalkyl-substituted amines, reasoning that the
metabolically “soft” methoxy group might divert metabolism
from the aminopyridine center. Selected compounds are listed
in Table 4. Acyclic derivative 16h was a potent and reasonably
selective hA2A antagonist and was efficacious in the HIC model
at 10 mg/kg. The methoxyazetidine 16i was similarly selective
and had even more potent binding affinity. However, the
4-methoxypiperidine compound 16j had the most interesting
in vitro profile and was selected for detailed characterization.
Compound 16j was a highly potent A2A antagonist at both
the human and rat receptors with greater than 100× selectivity
over hA1. Compound 16j did not show time-dependent inhibi-
tion of CYP3A4 and did not generate glutathione adducts upon
incubation with HLM in the presence of glutathione. Metabolic
stability in HLM and RLM assays was decreased relative to
16a but remained reasonable. As anticipated, the major me-
tabolite observed from incubation with HLM was the O-
demethylated compound 16k, which turned out to be a potent
and selective hA2A antagonist as well (Table 4). Solubility for
16j was still poor (0.03 mg/mL, PBS); however, its melting
point was 140 °C, substantially lower than for 6g, indicating
weaker crystal packing and the potential for more rapid
absorption.
In spite of its relatively poor solubility, 16j demonstrated
efficacy in two Parkinson’s related behavioral models. 16j was
efficacious down to a dose of 1 mg/kg PO in HIC and
demonstrated 88% inhibition of catalepsy at 10 mg/kg, a dose
at which plasma exposure (150 ng/mL) was moderate but brain
exposure (560 ng/g) was relatively high (Table 2). We also
examined the ability of 16j to potentiate L-dopa induced
rotational behavioral in unilaterally 6-hydroxydopamine-lesioned
rats (Figure 2). 16j dose-dependently potentiated L-dopa induced
rotations with an MED of 3 mg/kg when dosed orally.
In escalating dose pharmacokinetic studies in the rat, exposure
of 16j reached a plateau at moderate doses using a variety of
formulations, limiting the scope of safety studies. However,
adequate exposure of 16j, for safety screening, was achieved
in the mouse with 5% TPGS29 in 0.5% methylcellulose at pH
Conclusion
In summary, we have prepared a series of potent and selective
4-acetylamino-2-(3,5-dimethylpyrazol-1-yl)pyrimidine hA2A an-
tagonists with substituted pyridines as the C-6 substituent.
Selected compounds had good in vivo activity in the rat HIC
model of Parkinson’s symptomology. The advanced compound
6g was precluded from further development due to solubility-
related low exposure in nonrodent species. Successor compound
16a, with a more elaborate morpholine substituent on the
pyridine ring, was more soluble and showed promising activity
but also showed time-dependent CYP3A4 inhibition and gener-
ated glutathione adducts when incubated with HLM in the
presence of glutathione. Methoxypiperidine 16j, with a readily
metabolized methoxy group, was free of the reactive metabolite
issues seen with 16a and had a superior in vitro and in vivo
profile. Although 16j still suffered from poor thermodynamic
aqueous solubility, its melting point was significantly lower than
that of 6g, allowing for more rapid absorption with proper
formulation. Exposure in mouse and dog was sufficient for 16j
to be chosen for further preclinical development, which included
14-day safety screening. The results of these studies will be
reported in due course.
Experimental Section
Biology. Human and rat A2A and human A1 binding assays,
metabolism studies, hERG assays, membrane permeability assays,
pharmacokinetic assays, and HIC efficacy studies were performed
as described previously.14
L-dopa Induced Rotations in 6-Hydroxydopamine Lesioned
Rats. Male Wistar rats (Charles River; 250 g) were pretreated 30
min before surgery with desipramine and pargyline to ensure a

7104 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Table 4. Data for Compounds 17, 14, 15, and 16a-16k
Zhang et al.
Ki ( SEM (nM)a
Clint (mL/min kg)
b
c
compd
NR2R3
morpholin-4-yl
morpholin-4-yl
morpholin-4-yl
morpholin-4-yl
amino
N,N-dimethyl-amino
pyrrolidin-1-yl
piperidin-1-yl
homomorpholin-4-yl
4-methylpiperazin-1-yl
(2-methoxy-ethyl)-methyl-amino
3-methoxy-azetidin-1-yl
4-methoxy-piperidin-1-yl
4-hydroxy-piperidin-1-yl
hA2A
hA1
hA1/hA2A
HLM
RLM
CYP-3A4 IC50(µM)
17
14
15
87 ( 7
4600 ( 350
220 ( 90
580 ( 170
130 ( 13
440 ( 15
81 ( 12
53
74
48
155
57
133
28
ND
12
ND
3.0
ND
54
ND
ND
94
3.0
52
ND
150
ND
25
ND
1600
ND
ND
1000
25
ND
>20
ND
>20
>20
25
>20
ND
>20
>20
2.5
3.0 ( 0.8
12 ( 1
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
0.83 ( 0.1
7.9 ( 0.5
0.61 ( 0.07
0.26 ( 0.01
0.56 ( 0.05
2.3 ( 0.4
0.71 ( 0.11
0.72 ( 0.13
0.47 ( 0.06
0.44 ( 0.1
1.9 ( 0.2
7.2 ( 4
31 ( 5
54
280 ( 20
120 ( 10
55 ( 17
121
175
76
72
193
247
490
160
540
230
34 ( 15
25
41
43
11
>20
>20
85 ( 24
470 ( 110
a Data are expressed as geometric means of at least two runs ( the standard error of measurement (SEM); b Displacement of specific [3H]-27 binding at
human A2A receptors expressed in HEK293 cells; c Displacement of specific [3H]-28 binding at human A1 receptors expressed in HEK293 cells; HLM )
Human liver Microsomes; RLM ) Rat liver Microsomes; ND ) not determined.
chambers for 30 min, injected with carbidopa (12 mg/kg ip), 30
min later they were given the treatment, then 30 min later treated
with L-dopa (10 mg/kg ip). Rotational behavioral was then measured
over the next 5 h.
CYP2D6 and CYP3A4 Inhibition Screen. Recombinant
CYP2D6 or CYP3A4 were incubated with a selective marker
substrate (AMMC (3-[2-(N,N-diethyl-N-methylammonium)-ethyl]-
7-methoxy-4-methylcoumarin) for CPY2D6 and BFC (7-benzyloxy-
4-trifluoromethylcoumarin) for CYP3A4) in a NADPH generating
system in 96-well assay plates with a total incubation volume of
200 µL/well. Incubations were carried out using NCE concentrations
of 0.16, 0.8, 4.0, and 200 µM. The NADPH generating system for
the CYP2D6 assay consisted of 75 mM potassium phosphate buffer
(pH 7.4), 0.5 mM magnesium chloride, 0.01 mM nicotinamide
adenine dinucleotide phosphate (NADP), 0.5 mM glucose-6-
phosphate (G-6-P), 1 unit/mL glucose-6-phosphate dehydrogenase
(G-6-PD). The NADPH generating system for the CYP3A4 assay
consisted of 75 mM potassium phosphate buffer (pH 7.4), 3.0 mM
magnesium chloride, 1.0 mM NADP, 5.0 mM gluc G-6-P, and 1
unit/mL G-6-PD.
Figure 2. Potentiation of L-dopa induced rotations in unilaterally
6-hydroxydopamine-lesioned rats by orally dosed 16j.
Reactions were initiated with the addition of the enzyme solution
and the samples are incubated at 37 °C for 30 min. Reactions were
stopped by adding 75 µL of ice-cold 80% acetonitrile/20% 0.5 M
Tris Base. The amount of AHMC formed from CYP2D6 catalyzed
O-demethylation of AMMC and the amount of 7-HFC formed from
CYP3A4 catalyzed dealkylation of BFC were detected using a 96-
well microplate fluorescence reader.
Metabolic Stability Screen. Pooled human liver microsomes
(0.5 mg/mL; n > 10; mixed gender) were incubated at 37 °C with
the NCE in the presence of an NADPH-generating system contain-
ing 50 mM, potassium phosphate buffer (pH 7.4), 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 in six 1.2 mL titer tube plates using
a 1 µM concentration of each NCE (containing up to 0.01% DMSO)
with a total volume of 250 µL per incubation. Each plate
representing a single time point contains 96 Titertube Micro Tubes
allowing for duplicates of 48 compounds 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 an internal standard for LC-MS).
Figure 3. Structures of antagonists used in binding assays.
maximal effect of the 6-hydroxydopamine (6-OHDA). Then 8 µg
of 6-OHDA was injected over 4 min into the medial forebrain
bundle (+2.4 mm lateral to the midline, -2.2 mm anterior to
bregma (AP), -8.1 mm ventral to the dura surface). After a 3 week
recovery, the lesion was verified by measuring rotational behavior
in response to apomorphine hydrochloride (0.5 mg/kg sc). Rats that
failed to make more than 300 rotations in a 1 h test period were
excluded from the study. Rats that passed the apomorphine screen
underwent L-dopa sensitization; they were treated with L-dopa (10
mg/kg ip) three times separated by 3-5 days. If rats did not respond
to L-dopa by increasing rotations on two of the three sensitization
days, they were excluded from the experiment. Each rat underwent
each treatment in a randomized design. Test days were separated
by 2-3 days. On each test day, rats were habituated to the rotational

Pyridylpyrimidines as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7105
Precipitated proteins were removed by centrifugation for 35 min
at 3500 rpm at 5 °C, and the supernatant fluid (∼0.1 mL) was
analyzed by LC-MS-MS to determine the amount of parent
compound remaining. The in vitro initial rates of metabolism are
scaled using constants such as 45 mg microsomal protein/gm liver,
20 g liver/kg body weight, and 20 mL/min/kg liver blood flow to
predict systemic clearance and % bioavailability. These calculations
from nonlinear regression assume that liver metabolism and not
absorption is the determinant of Bioavailability.
General Suzuki Coupling Method: N-[6-(6-Chloropyridin-
2-yl)-2-(3,5-dimethyl-pyrazol-1-yl)-pyrimidin-4-yl]-acetamide (12).
A mixture of 5 (1.8 g, 6.8 mmol), 6-chloropyridin-2-boronic acid
pinacol ester (2.27 g, 9.5 mmol), potassium carbonate (1.88 g, 13.6
mmol), dioxane (35 mL), and water (3.5 mL) was sparged with
nitrogen gas for 5 min. Tetrakis(triphenylphosphine)palladium(0)
(785 mg, 0.68 mmol) was added, the mixture was sparged with
nitrogen gas for an additional 10 min, then the vessel was sealed
and heated with stirring at 90 °C for 16 h. Water (50 mL) was
added, and the resulting solid was collected, rinsed with water and
ethyl acetate, and then dried under vacuum to provide the title
compound (1.53 g, 4.5 mmol, 66%) as a pink solid. 1H NMR (300
MHz, CDCl3): δ 8.94 (s, 1H), 8.87 (s, 1H), 8.26 (d, J ) 8, 1H),
7.81 (t, J ) 8, 1H), 7.44 (d, J ) 8, 1H), 6.14 (s, 1H), 2.83 (s, 3H),
2.43 (s, 3H), 2.28 (s, 3H). LCMS-2: tR ) 6.39 min. MS: m/z 343,
345 [M + H]+, expected 343, 345 [M + H]+.
Time-Dependent Inhibition of CYP450 Enzymes (1A2, 2C9,
2C19, 2D6, 3A4). Pooled human liver microsomes (N ) 10) were
preincubated with the NCE at 37 °C for 0 and 30 min in mixtures
containing potassium phosphate buffer (50 mM, pH 7.4), MgCl2
(3 mM), and EDTA (1 mM) with an NADPH-generating system
(1 mM NADP, 5 mM G-6-P, and 1 unit/mL G-6-PD) at the final
concentrations indicated. After the preincubation period, the marker
substrates (1A2: phenacetin at 50 µM; 2C9: diclofenac at 4 µM;
2C19: S-mephenytoin at 35 µM; 2D6: dextromethorphan at 5 µM;
3A4: midazolam at 3 µM) were added, and the incubation was
continued for 10 min (30 min for 1A2). Several concentrations (e.g.,
5-7 concentrations) of the NCE were studied, which normally
cover a range that spans at lease 2 orders of magnitude (e.g.,
0.5-100 µM). Incubations were stopped by the addition of a stop
reagent (e.g., acetonitrile with internal standard). Precipitated
proteins were removed by centrifugation (3500 rpm for 35 min at
5 °C). Aliquots of supernatant fractions were analyzed by LC-MS-
MS to determine the amounts of the metabolites of the marker
substrates (acetaminophen, 4′-hydroxydiclofenac, 4′-hydroxyme-
phenytoin, dextrorphan, 1′-hydroxymidazolam, respectively).
In Vitro Absorption Studies in Caco-2 Cells. Caco-2 cells
(approximately 1.35 × 106 cells/mL) were thawed and transferred
to a Corning Costar T-75 flask (Cambridge, MA) with 25 mL of
DMEM supplemented with 10 mM HEPES buffer, 1 mM sodium
pyruvate, 2 mM L-glutamine, 0.1 mM MEM nonessential amino
acids solution, and 10% FBS for subculture. After subculture to a
minimum of 69 passages, Caco-2 cells were seeded (∼67000 cells)
onto the apical side of microporous polycarbonate membranes in
24-well Costar Transwell plates and cultured for at least 21 days
to allow for formation of a confluent monolayer (diffusional barrier)
and optimal expression of P-glycoprotein at 37 °C, 95% humidity
and 5% CO2.
N-[6-(2-Chloropyridin-4-yl)-2-(3,5-dimethyl-pyrazol-1-yl)-py-
rimidin-4-yl]-acetamide (10). Prepared according to the procedure
for 12 from 6-chloropyridin-2-boronic acid pinacol ester (40% crude
1
yield). H NMR (300 MHz, CDCl3): δ 8.58 (d, J ) 5, 1H), 8.55
(s, 1H), 7.99 (s, 1H), 7.87 (dd, J ) 1,5, 1H), 6.19 (s, 1H), 6.03 (s,
1H), 2.84 (s, 3H), 2.47 (s, 3H), 2.30 (s, 3H). LCMS-2: tR ) 5.99
min. MS: m/z 343, 345 [M + H]+, expected 343, 345 [M + H]+.
N-[6-(5-Bromopyridin-3-yl)-2-(3,5-dimethyl-pyrazol-1-yl)-py-
rimidin-4-yl]-acetamide (11). Prepared according to the procedure
for 12, from 3-bromopyridine-5-boronic acid. Purification by
chromatography (9:1 DCM/methanol eluant) yielded the title
compound as a white solid (11% yield). 1H NMR (300 MHz,
CDCl3): δ 9.26 (d, J ) 2, 1H), 8.80 (d, J ) 2, 1H), 8.65 (s, 1H),
8.50 (t, J ) 2, 1H), 8.47 (s, 1H), 6.09 (s, 1H), 2.78 (s, 3H), 2.35
(s, 3H), 2.26 (s, 3H). LCMS-2: tR ) 6.04 min. MS: m/z 387, 389
[M + H]+, expected 387, 389 [M + H]+.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-phenyl-pyrimidin-4-yl]-ac-
etamide (6a). Prepared according to the procedure for 12 from
phenylboronic acid. Purification by HPLC yielded the title com-
pound (10 mg, 30% yield). A sample was further purified by
preparative TLC (1:1 hexanes/acetone eluant). 1H NMR (300 MHz,
DMSO-d6): δ 11.26 (br s, 1H), 8.45 (s, 1H), 8.06-8.09 (m, 2H),
7.56-7.59 (m, 3H), 6.16 (s, 1H), 2.68 (s, 3H), 2.19 (s, 3H), 2.18
(s, 3H). LCMS-2: tR ) 6.68 min. (97%); MS: m/z 308 [M + H]+,
expected 308 [M + H]+.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-pyridin-3-yl-pyrimidin-4-
yl]-acetamide (6b). Prepared according to the procedure for 12
from 3-pyridylboronic acid. Purification by HPLC yielded the title
compound (5 mg, 6% yield) as the TFA salt. LCMS-2: tR ) 3.69
min. (95%); MS: m/z 309 [M + H]+, expected 309 [M + H]+.
LCMS-4: tR ) 6.15 min (100%). MS: m/z 309 [M + H]+, expected
309 [M + H]+.
Incubation of Caco-2 cell monolayer with 20 µM of a NCE
(containing up to 0.1% DMSO) in a solution containing 99% HBSS,
1% HEPES, 0.1 µM propranolol, and 25 µM PEG 400 (1.0 mL for
basolateral chamber and 0.3 mL for apical chamber) was conducted
at 37 °C, 95% humidity with gentle rotary shaking for 60 min.
After the incubation the contents of the apical and basolateral
chambers were collected in a 96-deep well plate and analyzed by
LC-MS-MS to determine the amount of parent compound in each
chamber. Digoxin was used as a positive control for P-glycoprotein-
mediated transport, and propranolol and PEG 400 were used as
high permeability internal standard and low permeability internal
standard for quantitation, respectively.
Chemistry. Reagents, starting materials, and solvents were
purchased from commercial suppliers and used as received.
Concentration refers to evaporation under vacuum using a Bu¨chi
rotatory evaporator. Reaction products were purified, when neces-
sary, by flash chromatography on silica gel (40-63 µm) with the
solvent system indicated. Spectroscopic data were recorded on a
Varian Mercury 300 MHz spectrometer or a Bruker Avance 500
MHz spectrometer. Elemental analyses were done by Robertson
Microlit Laboratory, Madison, NJ, or Numega Resonance Labo-
ratories, San Diego, CA. High-resolution mass spectrometry was
performed by the Scripps Research Institute Mass Spectrometry
Facility, La Jolla, CA. Melting points were recorded on a Bu¨chi
535 apparatus. Preparative HPLC-MS: Platform, Dionex equipped
with a Gilson 215 autosampler/fraction collector, UV detector, and
a Dionex MSQ mass detector; HPLC column, Phenomenex Synergy
Max-RP, 21.2 mm × 50 mm. HPLC gradient: 35 mL/min,
acetonitrile/water, 8-30 min run times. Both acetonitrile and water
have 0.05% TFA.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-pyridin-4-yl-pyrimidin-4-
yl]-acetamide (6c). Prepared according to the procedure for 12 from
4-pyridylboronic acid. Purification by HPLC yielded the title
1
compound (17 mg, 21%) as the TFA salt. H NMR (300 MHz,
DMSO-d6): δ 8.85-8.87 (m, 2H), 8.55 (s, 1H), 8.10-8.12 (m, 2H),
6.20 (s, 1H), 2.70 (s, 3H), 2.21 (s, 6H). LCMS-4: tR ) 6.13 min
(99%). MS: m/z 309 [M + H]+, expected 309 [M + H]+.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(3-methoxyphenyl)-pyri-
midin-4-yl]-acetamide (6d). Prepared according to the procedure
for 12 from 3-methoxyphenylboronic acid. Purification by HPLC
1
yielded the title compound (34 mg, 39%). H NMR (300 MHz,
CDCl3): δ 8.51 (br s, 1H), 8.46 (s, 1H), 7.69-7.72 (m, 2H), 7.42
(t, J ) 8, 1H), 7.05-7.08 (m, 1H), 6.07 (s, 1H), 3.89 (s, 3H), 2.81
(s, 3H), 2.35 (s, 3H), 2.24 (s,3H). LCMS-2: tR ) 6.78 min (99%).
MS: m/z 338 [M + H]+, expected 338 [M + H]+.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(2-fluoro-3-methoxy-phen-
yl)-pyrimidin-4-yl]-acetamide (6e). Prepared according to the
procedure for 12 from 2-fluoro-3-methoxyphenylboronic acid. The
crude product was taken up in 9:1 DCM/methanol, filtered, and
then the filtrate was concentrated to yield a solid. This solid was
triturated with ether to yield the title compound as a pale-yellow
solid (230 mg, 43%). 1H NMR (300 MHz, DMSO-d6): δ 11.28 (s,
1H), 8.45 (s, 1H), 7.50 (m, 1H), 7.35 (m, 2H), 6.17 (s, 1H), 3.90

7106 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
(s, 3H), 2.62 (s, 3H), 2.20 (s, 3H), 2.19 (s, 3H). LCMS-3: tR
Zhang et al.
)
6-(5-Methoxypyridin-3-yl)-2-(3,5-dimethyl-pyrazol-1-yl)-py-
rimidin-4-ylanine (8). A mixture of 6g (1.05 g, 3.1 mmol),
potassium carbonate (325 mesh, 1.29 g, 9.3 mmol), and methanol
(45 mL) was stirred at rt for 16 h and then was filtered and
concentrated. The residue was chromatographed (9:1 DCM/
methanol eluant) to provide the title compound (845 mg, 92%) as
21.16 min (100%). MS: m/z 356 [M + H]+, expected 356 [M +
H]+. Anal. (C18H18FN5O2) C, H, N.
N-[6-(3,5-Dimethoxy-phenyl)-2-(3,5-dimethyl-pyrazol-1-yl)-
pyrimidin-4-yl]-acetamide (6f). Prepared according to the proce-
dure for 12 from 3,5-dimethoxyphenylboronic acid pinacol ester.
Purification as for 6e yielded the title compound as a white solid
1
a solid. H NMR (300 MHz, CDCl3): δ 8.79 (d, J ) 1, 1H), 8.40
1
(296 mg, 54%). H NMR (300 MHz, DMSO-d6): δ 8.42 (s, 1H),
(d, J ) 3, 1H), 7.87 (dd, J ) 1,3, 1H), 6.72 (s, 1H), 6.05 (s, 1H),
5.40 (br s, 2H), 3.92 (s, 3H), 2.76 (s, 3H), 2.35 (s, 3H). LCMS-4:
tR ) 3.08. MS: m/z 297 [M + H]+, expected 297 [M + H]+.
7.18 (m, 2H), 6.75 (m, 1H), 6.17 (s, 1H), 3.86 (s, 6H), 3.35 (s,
3H), 2.68 (s, 3H), 2.19 (s, 3H). LCMS-3: tR ) 22.43 min (100%).
MS: m/z 368 [M + H]+, expected 368 [M + H]+. Anal.
(C19H21N5O3) C, H, N.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(5-methoxy-pyridin-3-yl)-
pyrimidin-4-yl]-2-morpholin-4-yl-acetamide (9a). A solution of
8 (1.0 g, 3.4 mmol) and pyridine (0.32 mL, 4.1 mmol) in
dichloromethane (70 mL) was treated at rt with chloroacetyl chloride
(0.30 mL, 3.7 mmol). The mixture was stirred at rt for 2 h then
partitioned against 10% aqueous sodium hydrogensulfate solution.
The aqueous layer was extracted with dichloromethane, and then
the combined dichloromethane layers were washed with brine, dried
over sodium sulfate, filtered, and concentrated. The crude product
was triturated with ethyl acetate to provide a solid, which was
dissolved in dichloromethane (30 mL). Morpholine (0.57 g, 6.6
mmol) was added, and the resulting solution was stirred at rt for
16 h. The mixture was concentrated and the residue purified by
silica gel chromatography (95/3/0.1 DCM/methanol/aqueous am-
monia eluant) to provide the title compound as a beige solid (1.1
g, 76% yield). The free base was dissolved in dichloromethane and
treated with an excess of hydrogen chloride in ether. Concentration
provided the HCl salt as a beige solid (1.09 g) 1H NMR (300 MHz,
DMSO-d6): δ 8.85 (s, 1H), 8.55 (s, 1H), 8.38 (s, 1H), 7.98 (s, 1H),
6.20 (s, 1H), 4.37 (s, 2H), 3.94 (s, 3H), 3.80-4.00 (m, 4H),
3.20-3.60 (m, 4H), 2.70 (s, 3H), 2.20 (s, 3H). LCMS-4: tR ) 7.00
min (98.4%). MS: m/z 424 [M + H]+, expected 424 [M + H]+;
Anal. (C21H25N7O3 ·HCl·H2O) C, H, N.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(5-methoxy-pyridin-3-yl)-
pyrimidin-4-yl]-propionamide (9b). A mixture of 8 (300 mg, 1.0
mmol), pyridine (0.15 mL, 1.8 mmol), and DCM (3 mL) was treated
with propionyl chloride (188 mg, 2.0 mmol) at rt and stirred at rt
for 68 h. Aqueous sodium bicarbonate was added, and the mixture
was extracted with four portions of DCM. The combined organic
extracts were washed with aqueous sodium hydrogensulfate and
brine, dried over magnesium sulfate, and then a few drops of
methanol were added and the mixture was filtered and concentrated.
The residue was purified by chromatography (97:3 DCM/MeOH
eluant) to provide the title compound as a solid (200 mg, 52%).
The free base was dissolved in DCM (30 mL) and then was treated
with 2 M hydrogen chloride in ether (0.285 mL, 0.57 mmol). The
mixture was concentrated and dried under vacuum to provide the
title compound (200 mg) as the HCl salt. 1H NMR (300 MHz,
CD3OD): δ 9.35 (d, J ) 2, 1H), 8.96 (m, 1H), 8.78 (d, J ) 3, 1H),
8.73 (s, 1H), 6.32 (s, 1H), 4.19 (s, 3H), 2.81 (s, 3H), 2.60 (q, J )
8, 2H), 2.41 (s, 3H), 1.24 (t, J ) 8, 3H). LCMS-3: tR ) 15.08 min
(98.6%). MS: m/z 353 [M + H]+, expected 353 [M + H]+.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(5-methoxy-pyridin-3-yl)-
pyrimidin-4-yl]-acetamide (6g). Prepared according to the pro-
cedure for 12 from 4.8 g (18 mmol) 5 and 5-methoxypyridyl-3-
boronic acid pinacol ester. The crude reaction mixture was
concentrated and the residue suspended in a mixture of ethyl acetate
and water. The mixture was filtered, and then the resulting solid
was slurried in ether. Filtration yielded the title compound (2.93 g,
48%). A portion of this material (1.5 g, 4.4 mmol) was suspended
in hexafluoroisopropanol (20 mL) and treated with 2 M hydrogen
chloride in ether (2.7 mL, 5.4 mmol) at rt. The sample was
concentrated, then twice taken up in 100 mL water and lyophilized
to dryness to provide the title compound as the hydrochloride salt
1
dihydrate. H NMR (300 MHz, CD3OD): δ 9.27 (d, J ) 1, 1H),
8.85 (m, 1H), 8.70 (d, J ) 3, 1H), 8.65 (s, 1H), 6.23 (s, 1H), 4.15
(s, 3H), 2.76 (s, 3H), 2.35 (s, 3H), 2.29 (s, 3H). LCMS-3: tR
)
13.50 min (100%). MS: m/z 339 [M + H]+, expected 339 [M +
H]+. Anal. (C17H18N6O2 ·HCl·2H2O) C, H, N.
N-[6-(5-Hydroxypyridin-3-yl)-2-(3,5-dimethyl-pyrazol-1-yl)-
pyrimidin-4-yl]-acetamide (6h). A mixture of 3-bromo-5-hydroxy-
pyridine (1.0 g, 5.7 mmol), bis(pinacolato)diboron (1.75 g, 6.9
mmol), potassium acetate (1.7 g, 17 mmol), [1,1′-bis(diphenylphos-
phino)ferrocene]dichloropalladium(II) (PdCl2 ·dppf, 380 mg, 0.47
mmol), and anhydrous dioxane (15 mL) was sparged with nitrogen
and then heated in a sealed tube for 5 h. Additional bis(pinacola-
to)diboron (1.75 g, 6.9 mmol) and PdCl2 ·dppf (380 mg, 0.47 mmol)
were added and the reaction heated for an additional 24 h. Ethyl
acetate was added, and the reaction mixture was poured onto celite
and washed with ethyl acetate. The filtrate was discarded. The solid
cake was then washed with 1:1 DCM/methanol, and then methanol.
The filtrate was concentrated to provide a crude mixture of
5-hydroxypyridyl-3-boronic acid and its pinacol ester (400 mg).
A mixture of the crude boronic acid/ester from above (400 mg),
5 (765 mg, 2.9 mmol), tris(dibenzylideneacetone)dipalladium(0)
(132 mg, 0.14 mmol), cesium carbonate (1.9 g, 5.8 mmol), tri-tert-
butylphosphonium tetrafluoroborate salt (84 mg, 0.30 mmol), THF
(10 mL), and water (2 mL) was sparged with nitrogen for 10 min
and then heated at reflux under nitrogen for 5 h. The cooled reaction
mixture was filtered and the solid rinsed with THF and water. The
collected solid (200 mg) was taken up in 1:1 DCM/methanol, then
filtered and the filtrate concentrated. The residue was taken up in
THF (10 mL) and water (2 mL) and stirred with Smopex-110 resin
(Alfa-Aesar no. 44724) for 16 h. The mixture was filtered to remove
the resin, rinsed with THF/water, methanol, and DCM. The filtrate
was concentrated, then azeotroped with ethanol, and then dried
under vacuum to provide the product as a yellow solid (158 mg,
27% yield). A sample was purified by preparative HPLC to provide
[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(5-methoxy-pyridin-3-yl)-py-
rimidin-4-yl]-carbamic acid methyl ester (9c). A solution of
triphosgene (300 mg, 1.0 mmol) in dichloromethane (3 mL) was
added to an ice-cold suspension of 8 (300 mg, 1.0 mmol) and
pyridine (0.60 mL, 7.4 mmol) in DCM (3 mL). The mixture was
stirred at 0 °C for 90 min, then methanol (0.30 mL, 7.4 mmol)
was added and the mixture was stirred at rt for 68 h. The reaction
mixture was concentrated and the residue partially purified by
preparative HPLC. The product was converted to the free base by
extraction with DCM from aqueous sodium bicarbonate, then the
residue was purified by preparative TLC (95:5 DCM/MeOH eluant)
1
the TFA salt. H NMR (300 MHz, DMSO-d6): δ 11.41 (s, 1H),
8.87 (br s, 1H), 8.53 (m, 1H), 8.48 (s, 1H), 8.34 (br s, 1H), 6.20 (s,
1H), 2.69 (s, 3H), 2.21 (s, 6H). LCMS-3: tR ) 10.96 min (100%).
MS: m/z 325 [M + H]+, expected 325 [M + H]+.
N-[6-(6-Amino-pyridin-2-yl)-2-(3,5-dimethyl-pyrazol-1-yl)-py-
rimidin-4-yl]-acetamide (16b). Prepared according to the procedure
for 12 from 6-aminopyridin-2-ylboronic acid pinacol ester. HPLC
1
1
to provide the title compound as a white solid (52 mg, 15%). H
purification provided the TFA salt (68 mg, 28%). H NMR (300
NMR (300 MHz, DMSO-d6): δ 11.21 (s, 1H), 8.84 (d, J ) 1, 1H),
8.50 (d, J ) 2, 1H), 8.25 (s, 1H), 7.95 (br s, 1H), 6.17 (s, 1H),
3.95 (s, 3H), 3.76 (s, 3H), 2.67 (s, 3H), 2.21 (s, 3H). LCMS-2: tR
) 4.49 min (100%). MS: m/z 355 [M + H]+, expected 355 [M +
H]+.
MHz, CDCl3): δ 8.38 (dd, J ) 1,5, 1H), 8.19 (s, 1H), 8.09 (s, 1H),
7.70-7.61 (m, 2H), 7.48 (d, J ) 8, 1H), 6.97-7.01 (m, 1H), 6.02
(s, 1H), 2.63 (s, 3H), 2.33 (s, 3H), 2.21 (s, 3H). LCMS-2: tR
)
3.65 min (100%). MS: m/z 324 [M + H]+, expected 324 [M +
H]+.

Pyridylpyrimidines as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7107
[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(5-methoxy-pyridin-3-yl)-py-
rimidin-4-yl]-carbamic acid ethyl ester (9d). Prepared according
to the procedure for 9c from 8 (80 mg, 0.27 mmol) and ethanol.
Following HPLC purification, the product was dissolved in DCM
and treated with 2 M HCl in ether (0.30 mL, 0.6 mmol). The
mixture was concentrated to provide the title compound as the HCl
salt (57 mg, 52% yield). 1H NMR (300 MHz, DMSO-d6): δ 11.14
(s, 1H), 8.86 (br s, 1H), 8.54 (d, J ) 2, 1H), 8.25 (s, 1H), 8.02 (br
s, 1H), 6.16 (s, 1H), 4.21 (q, J ) 7, 2H), 3.95 (s, 3H), 2.65 (s, 3H),
2.19 (s, 3H), 1.26 (t, J ) 7, 3H). LCMS-4: tR ) 7.62 min (100%).
MS: m/z 369 [M + H]+, expected 369 [M + H]+.
7.76-7.62 (m, 2H), 7.06 (d, J ) 9, 1H), 6.16 (s, 1H), 4.04-4.08
(m, 2H), 3.44-3.46 (m, 1H), 3.34 (s, 3H), 3.24-3.34 (m, 2H),
2.69 (s, 3H), 2.20 (s, 3H), 2.18 (s, 3H), 1.92-1.96 (m, 2H),
1.47-1.49 (m, 2H). LCMS-3: tR ) 22.07 min (100%). MS: m/z
422 [M + H]+, expected 422 [M + H]+. Anal. (C22H27N7O2 ·
1/4HOAc) C, H, N.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(5-morpholin-4-yl-pyridin-
3-yl)-pyrimidin-4-yl]-acetamide (14). A mixture of 11 (150 mg,
0.39 mmol), morpholine (150 mg, 1.7 mmol), potassium carbonate
(150 mg, 1.1 mmol), L-proline (50 mg, 0.43 mmol), copper(I) iodide
(50 mg, 0.26 mmol), and DMSO (3 mL) was heated in a sealed
tube with stirring at 100 °C for 16 h. The cooled reaction mixture
was diluted with methanol, filtered, and purified by prep HPLC.
Extraction of product fractions (DCM/aqueous sodium bicarbonate),
followed by two preparative TLC purifications (9:1 DCM/methanol
eluant, then ethyl acetate eluant) provided the free base (yield 3.4
mg, 2%). LCMS-2: tR ) 3.88 min (95%). MS: m/z 394 [M + H]+,
expected 394 [M + H]+.
N-{2-(3,5-Dimethyl-pyrazol-1-yl)-6-[5-(2-methoxy-ethoxy)-py-
ridin-3-yl]-pyrimidin-4-yl}-acetamide (7). Diethyl azodicarboxy-
late (DEAD, 61 mg, 0.35 mmol) was added to a solution of 6h (75
mg, 0.23 mmol), 2-methoxyethanol (26 mg, 0.74 mmol), and
triphenylphosphine (91 mg, 0.34 mmol) in THF (1 mL) at rt. The
mixture was stirred at rt for 16 h. Additional DEAD (22 mg),
triphenylphosphine (30 mg), and 2-methoxyethanol (10 mg) were
added, and the mixture was stirred at rt for an additional 4 h. The
mixture was concentrated and purified by preparative HPLC to yield
N-[6-(6-Dimethylamino-pyridin-2-yl)-2-(3,5-dimethyl-pyrazol-
1-yl)-pyrimidin-4-yl]-acetamide (16c). A mixture of 12 (400 mg),
dimethylamine hydrochloride (300 mg), and DMF (6 mL) was
heated in a microwave reactor at 200 °C for 20 min. The cooled
reaction mixture was diluted with methanol, filtered, and partially
purified by preparative HPLC. Extraction of product fractions
(DCM/aqueous sodium bicarbonate), followed by preparative TLC
purification (9:1 CHCl3/MeOH eluant) provided the free base as a
1
the product as the TFA salt (22 mg, 19%). H NMR (300 MHz,
DMSO-d6): δ 11.41 (s, 1H), 8.82 (d, J ) 1, 1H), 8.50 (d, J ) 1,
1H), 8.46 (s, 1H), 7.95 (s, 1H), 6.19 (s, 1H), 4.29-4.32 (m, 2H),
3.70-3.73 (m, 2H), 3.33 (s, 3H), 2.69 (s, 3H), 2.21 (s, 3H), 2.20
(s, 3H). LCMS-2: tR ) 4.35 min (95.9%). MS: m/z 383 [M + H]+,
expected 383 [M + H]+.
1
yellow solid (70 mg, 17% yield). H NMR (300 MHz, CDCl3): δ
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(2-morpholin-4-yl-pyridin-
4-yl)-pyrimidin-4-yl]-acetamide (15). A mixture of 10 (50 mg,
0.15 mmol), morpholine (26 mg, 0.30 mmol), and DMA (1 mL)
was heated in a microwave reactor at 190 °C for 20 min. Direct
purification of the reaction mixture by preparative HPLC furnished
the title compound as the TFA salt. This material was converted to
the free base by extraction with DCM from aqueous sodium
bicarbonate to yield the free base as a yellow solid (10 mg, 17%
8.99 (s, 1H), 8.32 (s, 1H), 7.57-7.65 (m, 2H), 6.66 (dd, J ) 9,1,
1H), 6.06 (s, 1H), 3.19 (s, 6H), 2.81 (s, 3H), 2.36 (s, 3H), 2.23 (s,
3H). LCMS-2: tR ) 4.57 min (100%). MS: m/z 352 [M + H]+,
expected 352 [M + H]+.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(6-pyrrolidin-1-yl-pyridin-
2-yl)-pyrimidin-4-yl]-acetamide (16d). A mixture of 12 (100 mg,
0.38 mmol), pyrrolidine (0.8 mL), and DMSO (1 mL) was heated
in a sealed tube at 90 °C for 64 h. Preparative HPLC purification
yielded the deacetylated product (90 mg, 69%) as the TFA salt.
The salt was converted to the free base by extraction with DCM
from aqueous sodium bicarbonate (yield 65 mg). This material was
reacetylated by treatment with pyridine (32 mg) and acetyl chloride
(17 mg) in DCM (5 mL) at rt for 1 h. The reaction mix was
concentrated and subjected to preparative HPLC purification.
Extraction (DCM/aqueous sodium bicarbonate) provided the free
base (17 mg, 12% yield) as a yellow solid. LCMS-2: tR ) 4.43
min (100%). MS: m/z 378 [M + H]+, expected 378 [M + H]+.
ESHRMS m/z 378.2040, [M + H]+ requires 378.2037.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(3,4,5,6-tetrahydro-2H-
[1,2′]bipyridinyl-6′-yl)-pyrimidin-4-yl]-acetamide (16e). Prepared
according to the procedure for 15 from 12 (50 mg, 0.15 mmol)
and piperidine. Direct purification of the reaction mixture by
preparative HPLC furnished the title compound (3 mg, 5% yield)
as the TFA salt. LCMS-2: tR ) 6.22 min (98%). MS: m/z 392 [M
+ H]+, expected 392 [M + H]+. ESHRMS m/z 392.2188, [M +
H]+ requires 392.2193.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(6-[1,4]oxazepan-4-yl-py-
ridin-2-yl)-pyrimidin-4-yl]-acetamide (16f). Prepared according
to the procedure for 15 from 12 (50 mg, 0.15 mmol), homomor-
pholine hydrochloride (42 mg, 0.3 mmol), and triethylamine (0.042
mL, 0.3 mmol). Direct purification of the reaction mixture by
preparative HPLC furnished the title compound (17 mg, 28% yield)
as the TFA salt. 1H NMR (300 MHz, CDCl3): δ 8.92 (s, 1H), 8.44
(s, 1H), 7.59-7.63 (m, 2H), 6.68 (d, J ) 8, 1H), 6.06 (s, 1H),
3.84-3.92 (m, 4H), 3.71 (t, J ) 6, 2H), 2.80 (s, 3H), 2.35 (s, 3H),
2.23 (s, 3H), 2.10 (t, J ) 5, 2H), 0.8-1.2 (m, 2H). LCMS-1: tR )
2.33 min (100%). MS: m/z 408 [M + H]+, expected 408 [M +
H]+.
N-{2-(3,5-Dimethyl-pyrazol-1-yl)-6-[6-(4-methyl-piperazin-1-
yl)-pyridin-2-yl]-pyrimidin-4-yl}-acetamide (16g). Prepared ac-
cording to the procedure for 15 from 12 (45 mg, 0.13 mmol) and
N-methylpiperazine. Direct purification of the reaction mixture by
preparative HPLC furnished the title compound as the TFA salt.
This material was converted to the free base by extraction with
1
yield). H NMR (300 MHz, CDCl3): δ 8.62 (s, 1H), 8.49 (s, 1H),
8.38 (d, J ) 6, 1H), 7.39-7.31 (m, 2H), 6.11 (s, 1H), 3.87 (t, J )
5, 4H), 3.63 (t, J ) 5, 4H), 2.79 (s, 3H), 2.38 (s, 3H), 2.26 (s, 3H).
LCMS-2: tR ) 3.91 min (100%). MS: m/z 394 [M + H]+, expected
394 [M + H]+.
N-(2-(3,5-Dimethyl-pyrazol-1-yl)-6-{6-[(2-methoxyethyl)-meth-
ylamino]-pyridin-2-yl}-pyrimidin-4-yl)-acetamide (16h). Pre-
pared according to the procedure for 15 starting from 12 (500 mg,
1.5 mmol) and N-(2-methoxyethyl)methylamine (1.0 g, 11 mmol).
Aqueous sodium bicarbonate was added to the crude reaction
mixture, and the mixture was extracted with DCM. The combined
organic extracts were dried over sodium sulfate, filtered, and
concentrated. The residue was purified by preparative TLC (9:1
DCM/MeOH with 1% NH4OH as eluant) to provide the title
1
compound (70 mg 12%). H NMR (300 MHz, CDCl3): δ 8.93 (s,
1H), 8.31 (s, 1H), 7.65-7.57 (m, 2H), 6.68 (d, J ) 8, 1H), 6.06 (s,
1H), 3.87 (t, J ) 6, 3H), 3.72 (t, J ) 5, 3H), 3.39 (s, 3H), 3.19 (s,
3H), 2.81 (s, 3H), 2.35 (s, 3H), 2.22 (s, 3H). LCMS-2: tR ) 4.96
min (100%). MS: m/z 396 [M + H]+, expected 396 [M + H]+.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(4-methoxy-3,4,5,6-tet-
rahydro-2H-[1,2′]bipyridinyl-6′-yl)-pyrimidin-4-yl]-acetamide
(16j). A mixture of 12 (300 mg, 0.85 mmol), powdered potassium
phosphate (650 mg, 3.1 mmol), tris(dibenzylideneacetone)di-
palladium(0) (39 mg, 0.038 mmol), 4-methoxypiperidine hydro-
chloride (264 mg, 1.74 mmol), 2-dicyclohexylphosphino-2′,6′-
dimethoxybiphenyl (72 mg, 0.18 mmol), and dioxane (10 mL) was
sparged with nitrogen gas for 5 min, and then the reaction vessel
was sealed and the mixture heated with stirring at 100 °C for 16 h.
Water and ethyl acetate were added to the cooled reaction mixture,
and then the mixture was filtered and the aqueous phase extracted
twice with ethyl acetate. The combined organic extracts were dried
over sodium sulfate, filtered, and concentrated to provide a yellow
oil. Preparative HPLC, followed by neutralization of product
fractions with solid sodium bicarbonate and extraction with ethyl
acetate, provided a brown solid. Trituration with ethyl acetate
provide the title compound as a yellow-brown solid (80 mg, 22%).
1H NMR (300 MHz, DMSO-d6): δ 11.20 (s, 1H), 8.87 (s, 1H),

7108 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Zhang et al.
DCM from aqueous sodium bicarbonate to yield the free base as a
yellow solid (12 mg, 23%). H NMR (300 MHz, CDCl3): δ 8.92
4H). LCMS-1: tR ) 2.38 min. MS: m/z 190 [M + H]+, expected
190 [M + H]+.
1
(s, 1H), 8.33 (s, 1H), 7.72 (d, J ) 8, 1H), 7.64 (d, J ) 8, 1H), 6.80
(d, J ) 8, 1H), 6.07 (s, 1H), 3.71 (t, J ) 5, 4H), 2.80 (s, 3H), 2.55
(t, J ) 5, 4H), 2.36 (s, 3H), 2.35 (s, 3H), 2.23 (s, 3H). LCMS-3:
tR ) 15.11 min (100%). MS: m/z 407 [M + H]+, expected 407 [M
+ H]+.
N-{2-(3,5-Dimethyl-pyrazol-1-yl)-6-[6-(3-methoxy-azetidin-1-
yl)-pyridin-2-yl]-pyrimidin-4-yl}-acetamide (16i). Prepared ac-
cording to the procedure for 15 from 12 (50 mg, 0.15 mmol),
3-methoxyazetidine hydrochloride (37 mg, 0.30 mmol), and DIPEA
(0.053 mL, 0.30 mmol). Direct purification of the reaction mixture
by preparative HPLC furnished the title compound (3 mg, 5% yield)
as the TFA salt. LCMS-2: tR ) 4.67 min (90%). MS: m/z 394 [M
+ H]+, expected 394 [M + H]+. ESHRMS m/z 394.1995, [M +
H]+ requires 394.1986.
3-Amino-3-(6-morpholin-4-yl-pyridin-2-yl)-acrylonitrile (21a).
A mixture of 20a (7.43 g, 39 mmol, 1.0 equiv), anhydrous toluene
(100 mL), and potassium t-butoxide (13.2 g, 118 mmol, 3.0 equiv)
was stirred without external heating or cooling while anhydrous
acetonitrile (4.1 mL, 79 mmol, 2.0 equiv) was added dropwise over
10 min. The thick yellow mixture was stirred for an additional 2 h.
Aqueous sodium bicarbonate solution was added (exothermic), and
then the mixture was extracted with ethyl acetate. The combined
organic layers were dried over sodium sulfate, filtered, and
concentrated to provide the title compound as a brown oil (9.6 g),
which was used without purification. LCMS-1: tR ) 2.38 min. MS:
m/z 231 [M + H]+, expected 231 [M + H]+.
4-Amino-6-(6-morpholin-4-yl-pyridin-2-yl)-1H-pyrimidine-2-
thione (22a). A suspension of 21a (9.5 g, est 35 mmol) and thiourea
(5.6 g, 74 mmol) in absolute ethanol (100 mL) was treated with
sodium ethoxide solution (21 wt %, Aldrich, 27.5 mL, 74 mmol).
The brown mixture was heated in a sealed tube at 100 °C for 49.5 h.
The cooled reaction mixture was poured onto 10% aqueous sodium
dihydrogen phosphate solution (200 mL) chilled in an ice bath.
The mixture was filtered. The filter cake was washed with water
and then air-dried briefly to provide the title compound (34.6 g) as
a tan gummy solid. This material was used without further
purification. A sample was dried further for analysis. 1H NMR (300
MHz, DMSO-d6): δ 10.9 (br s, 1H), 7.7-7.8 (m, 3H), 7.37 (d, J )
8, 1H), 7.05 (d, J ) 8, 1H), 6.62 (s, 1H), 3.72 (m, 4H), 3.52 (m,
4H). LCMS-1: tR ) 1.97 min. MS: m/z 290 [M + H]+, expected
290 [M + H]+.
2-Methylsulfanyl-6-(6-morpholin-4-yl-pyridin-2-yl)-pyrimidin-
4-ylamine (23a). A suspension of 22a (34.5 g, est 30 mmol) in
5% aqueous sodium hydroxide (100 mL, 125 mmol) was stirred at
rt while iodomethane (1.86 mL, 30 mmol) was added in one portion.
A light-tan precipitate formed. After 45 min, the mixture was treated
with 10% aqueous sodium dihydrogen phosphate solution (final
pH 9), and then the solid was collected, washed with water, and
air-dried to provide the title compound (9.95 g) as a tan solid, still
wet with water. A sample was dried further for analysis. 1H NMR
(300 MHz, DMSO-d6): δ 7.59-7.72 (m, 2H), 7.08 (s, 1H), 6.99
(brs, 2H), 6.92 (d, J ) 8, 1H), 3.75 (m, 4H), 3.52 (m, 4H), 2.48 (s,
3H). LCMS-1: tR ) 2.06 min. MS: m/z 304 [M + H]+, expected
304 [M + H]+.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(4-hydroxy-3,4,5,6-tetrahy-
dro-2H-[1,2′]bipyridinyl-6′-yl)-pyrimidin-4-yl]-acetamide (16k).
Prepared according to the procedure for 15 from 12 (500 mg, 1.5
mmol) and 4-hydroxypiperidine (300 mg, 2.9 mmol). Direct
purification of the reaction mixture by preparative HPLC furnished
the title compound as the TFA salt. This material was converted to
the free base by extraction with DCM from aqueous sodium
bicarbonate to yield the free base as a yellow solid (45 mg, 8%
1
yield). H NMR (300 MHz, DMSO-d6): δ 11.19 (s, 1H), 8.85 (s,
1H), 7.60-7.73 (m, 2H), 7.04 (d, J ) 9, 1H), 6.16 (s, 1H), 4.72
(d, J ) 5, 1H), 4.09-4.14 (m, 2H), 3.70-3.75 (m, 1H), 3.15-3.22
(m, 2H), 2.67 (s, 3H), 2.19 (s, 3H), 2.17 (s, 3H), 1.79-1.85 (m,
2H), 1.37-1.44 (m, 2H). LCMS-3: tR ) 16.99 min (100%). MS:
m/z 408 [M + H]+, expected 408 [M + H]+. Anal. (C21H25N7O2)
C, H, N.
Ethyl 6-(Morpholin-4-yl)pyridine-2-carboxylate (18a). Mor-
pholine (4.17 mL, 47.9 mmol, 1.05 equiv) was added to a solution
of ethyl 6-bromopyridine-2-carboxylate (10.5 g, 45.6 mmol, 1.0
equiv) and triethylamine (9.5 mL, 68.4 mmol, 1.5 equiv) in N,N-
dimethylacetamide (20 mL). The mixture was heated at 100 °C in
a sealed tube for 14.5 h. Aqueous sodium bicarbonate was added,
and the mixture was extracted with ethyl acetate. The organic layer
was dried over sodium sulfate, filtered, and concentrated to a brown
oil. Chromatography (3:1 hexanes/EtOAc eluant) gave the product
as a yellow solid (6.5 g, 60% yield). 1H NMR (300 MHz, CDCl3):
δ 7.62 (t, J ) 7, 1H), 7.47 (d, J ) 7, 1H), 6.85 (d, J ) 7, 1H), 4.39
(q, J ) 7, 2H) 3.85 (t, J ) 5, 4H), 3.59 (t, J ) 5, 4H), 1.42 (t, J
) 7, 3H). LCMS-1: tR ) 2.35 min. MS: m/z 237 [M + H]+,
2-Methanesulfonyl-6-[6-(4-oxy-morpholin-4-yl)-pyridin-2-yl]-
pyrimidin-4-ylamine (24a). A suspension of 23a (9.9 g, est 25
mmol) in methanol (200 mL) and water (50 mL) was cooled in an
ice bath and treated dropwise with a solution of oxone (20.7 g) in
water (100 mL) over 10 min. The temperature was kept below 20
°C during the addition. Saturated sodium bicarbonate solution (75
mL) was then added dropwise over 5 min (effervescence), and the
ice bath was removed. After 1 h, an additional portion of solid
oxone (5.5 g) was added. After an additional 2 h, dichloromethane
(200 mL) was added and the mixture was filtered. The filter cake
was washed with water and air-dried to provide the first product
fraction as a tan solid (7.0 g). The two phases of the filtrate were
separated. The aqueous phase was adjusted to pH > 8 and was
extracted with dichloromethane and ethyl acetate. The combined
organic extracts were dried over sodium sulfate, filtered, and
concentrated to provide the second product fraction (1.3 g) as a
yellow solid. A slurry of product fractions one (6.8 g) and two
(1.3 g) in ethanol was concentrated to dryness to provide the title
expected 237 [M + H]+
.
6-(Morpholin-4-yl)pyridine-2-carboxamide (19a). 18a (6.3 g,
26.7 mmol) was dissolved in 7N ammonia in methanol (150 mL).
The solution was heated with stirring in a sealed tube at 75 °C for
16 h. The mixture was concentrated, and the solid residue was
coevaporated with methanol, then ethyl acetate. The solid product
was dried under vacuum to provide the title compound (5.46 g) as
an off-white solid, which was used without further purification. 1H
NMR (300 MHz, CDCl3 + 1 drop CD3OD): δ 7.60 (t, J ) 8, 1H),
7.59 (d, J ) 8, 1H), 6.89 (d, J ) 8, 1H), 3.85 (t, J ) 5, 4H), 3.54
(t, J ) 5, 4H). LCMS-1: tR ) 1.97 min. MS: m/z 208 [M + H]+,
expected 208 [M + H]+
.
6-(Morpholin-4-yl)pyridine-2-carbonitrile (20a). A suspension
of 19a (5.4 g, 26 mmol) and triethylamine (7.3 mL, 52 mmol) in
dichloromethane (100 mL) was cooled in an ice bath and treated
dropwise with trifluoroacetic anhydride (4.0 mL, 29 mol). The
mixture was allowed to stir at 0 °C for 15 min, then was allowed
to warm to rt. After 30 min, additional trifluoroacetic anhydride
(0.5 mL, 3.6 mmol) was added, and the mixture was stirred for
another 30 min. Aqueous sodium bicarbonate solution was added,
and the mixture was extracted with dichloromethane. The organic
layer was dried over sodium sulfate, filtered, and concentrated to a
solid, which was triturated with 3:1 hexanes/ethyl acetate to provide
the title compound (3.69 g) as a white solid. A second crop (0.66
1
compound (7.3 g) as a tan solid. H NMR (300 MHz, DMSO-d6)
major species: δ 8.58 (d, J ) 8, 1H), 8.41 (d, J ) 8, 1H), 8.27 (t,
J ) 8, 1H), 7.98 (br s, 2H), 7.60 (s, 1H), 4.41 (t, J ) 11, 2H), 4.19
(t, J ) 11, 2H), 3.82 (d, J ) 11, 2H), 3.38 (s, 3H), 2.95 (d, J ) 11,
2H). LCMS-1: tR ) 1.60 min. MS: m/z 352 [M + H]+, expected
352 [M + H]+.
2-(3,5-Dimethyl-pyrazol-1-yl)-6-(6-morpholin-4-yl-pyridin-2-
yl)-pyrimidin-4-ylamine (26a). A suspension of 24a (7.3 g, 19.4
mmol) in ethanol (200 mL) was treated with anhydrous hydrazine
(6 mL, 189 mmol). The mixture was refluxed for 16 h. The mixture
(containing crude arylhydrazine 25a) was allowed to cool to rt,
1
g, slightly yellow solid) was obtained from the mother liquor. H
NMR (300 MHz, CDCl3): δ 7.56 (dd, J ) 7,9, 1H), 7.02 (d, J )
7, 1H), 6.83 (d, J ) 9, 1H), 3.82 (t, J ) 5, 4H), 3.56 (t, J ) 5,

Pyridylpyrimidines as A2A Receptor Antagonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7109
and then pentane-2,4-dione (20.5 mL, 200 mmol) was added
dropwise over 30 min without external cooling. The mixture was
heated at 70 °C for 1 h and then was allowed to cool and the solvent
was removed. The resulting tan solid (30.5 g) was slurried with
water (150 mL) at 40 °C for 15 min and then was allowed to cool.
The cooled mixture was filtered, and the filter cake was rinsed with
water. The resulting filter cake was slurried with ethyl acetate and
then concentrated to dryness to provide the product 26a (5.9 g) as
a tan solid, which contained the desired product in addition to 3,5-
dimethylpyrazole (LCMS analysis). A sample was further purified
for analysis as follows: The crude product was dissolved in 1 M
hydrochloric acid and then the solution was washed three times
with EtOAc. 1N Sodium hydroxide solution was added until a white
precipitate formed and then the mixture was extracted with DCM.
The DCM extract was dried over sodium sulfate, filtered, and then
concentrated to provide 26a as a yellow solid. 1H NMR (300 MHz,
DMSO-d6) major species: δ 7.72 (t, J ) 8, 1H), 7.60 (d, J ) 8,
1H), 7.28 (br s, 2H), 7.25 (s, 1H), 6.97 (d, J ) 8, 2H), 6.05 (s,
1H), 3.74 (m, 4H), 3.54 (m, 4H), 2.60 (s, 3H), 2.16 (s, 3H). LCMS-
1: tR ) 2.27 min. MS: m/z 352 [M + H]+, expected 352 [M +
H]+.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(6-morpholin-4-yl-pyridin-
2-yl)-pyrimidin-4-yl]-acetamide (16a). Acetyl chloride (0.035 mL,
0.45 mmol) was added at rt to a stirred mixture of the amine (115
mg, 0.33 mmol) and pyridine (0.053 mL, 0.66 mmol) in DCM (3
mL). The mixture was stirred at rt for 2.5 h, and then brine was
added and the mixture was extracted with dichloromethane. The
combined extracts were dried over sodium sulfate, filtered, and
concentrated. The residue was purified by chromatography (1:1
hexanes/THF eluant) to provide the title compound (110 mg, 85%
yield) as a yellow solid. 1H NMR (300 MHz, DMSO-d6): δ 11.21
(s, 1H), 8.85 (s, 1H), 7.79-7.68 (m, 2H), 7.05 (d, J ) 8.3, 1H),
6.15 (s, 1H), 3.75 (t, J ) 45.7, 4H), 3.55 (t, J ) 4.9, 4H), 3.31 (s,
3H), 2.22 (s, 3H), 2.16 (s, 3H). LCMS-2: tR ) 6.07 min (100%).
MS: m/z 394 [M + H]+, expected 394 [M + H]+.
Methyl 2-(morpholin-4-yl)pyridine-4-carboxylate (18b). Pre-
pared from methyl 4-chloropyridin-2-carboxylate (4.5 g, 26 mmol)
and morpholine according to the procedure of 18a, except that the
reaction product was not purified by chromatography. Brown solid
(yield 3.3 g, 54%), LCMS-1: tR ) 0.43 min. MS: m/z 223 [M +
H]+, expected 223 [M + H]+.
2-(Morpholin-4-yl)pyridine-4-carboxamide (19b). Prepared
from 18b (3.3 g, 15 mmol) according to the procedure of 19a. The
crude solid product was washed with methanol and then ether to
provide the title compound as an off-white solid (yield 2.0 g, 65%).
LCMS-1: tR ) 0.33 min. MS: m/z 208 [M + H]+, expected 208
[M + H]+.
2-(Morpholin-4-yl)pyridine-4-carbonitrile (20b). Prepared from
19b (2.0 g, 9.6 mmol) according to the procedure of 20a, except
that the crude solid was triturated with EtOAc/ether. White solid
(1.45 g, 79%). LCMS-1: tR ) 0.98 min. MS: m/z 190 [M + H]+,
expected 190 [M + H]+.
3-Amino-3-(4-morpholin-4-yl-pyridin-2-yl)-acrylonitrile (21b).
Prepared from 20b (1.45 g, 7.6 mmol) according to the procedure
of 21a. The crude product was triturated with ether to provide the
to provide a yellow foam (370 mg, 100%). The major species
present was tertiary amine title compound; the N-oxide was minor.
LCMS-1: tR ) 1.71 min. MS: m/z 336 [M + H]+, expected 336
[M + H]+.
2-(3,5-Dimethyl-pyrazol-1-yl)-6-(4-morpholin-4-yl-pyridin-2-
yl)-pyrimidin-4-ylamine (26b). Prepared from 24b (370 mg, 1.1
mmol) according to the procedure for 26a, with modifications.
Following treatment with hydrazine, the reaction mixture was
filtered and the solid washed with methanol. The combined filtrate
was concentrated to provide the intermediate hydrazinopyrimidine
25b as a yellow foam. This was then treated with pentane-2,4-
dione in ethanol according to the procedure of 26a. Final purification
by chromatography (9:1 DCM/methanol eluant) provided the title
compound 26b (110 mg, 29% from 21b) as a yellow solid. LCMS-
1: tR
) 2.05 min. MS: m/z 352 [M + H]+, expected 352 [M +
H]+
.
N-[2-(3,5-Dimethyl-pyrazol-1-yl)-6-(4-morpholin-4-yl-pyridin-
2-yl)-pyrimidin-4-yl]-acetamide (17). Prepared according to the
procedure for 16a from 26b (110 mg, 0.31 mmol). Purification of
the crude material by preparative HPLC, followed by conversion
to the free base (DCM/aqueous sodium bicarbonate extraction)
yielded the title compound (15 mg, 12% yield) as a yellow solid.
1H NMR (300 MHz, CDCl3): δ 8.94 (s, 1H), 8.44 (d, J ) 6.0,
1H), 8.31 (s, 1H), 7.85 (d, J ) 2.4, 1H), 6.77 (dd, J ) 6.0, 2.4,
1H), 6.08 (s, 1H), 3.87 (t, J ) 4.8, 4H), 3.37 (t, J ) 4.8, 4H), 2.79
(s, 3H), 2.36 (s, 3H), 2.23 (s, 3H). LCMS-2: tR ) 3.99 min (100%).
MS: m/z 394 [M + H]+, expected 394 [M + H]+.
Acknowledgment. We thank Shawn Ayube, Paddi Ekhlassi,
Jamie Rueter, and John Harman for analytical support.
Supporting Information Available: Copies of NMR and LCMS
data are available for key final compounds, along with detailed
descriptions of HPLC conditions for compound analysis. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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