5,6,7-Trisubstituted 4-Aminopyrido[2,3-d]pyrimidines as Novel Inhibitors of Adenosine Kinase

Article abstract of DOI:10.1021/jm030327l

The synthesis and structure-activity relationship of a series of 5,6,7-trisubstituted 4-aminopyrido[2,3-d]pyrimidines as novel nonnucleoside adenosine kinase inhibitors is described. A variety of alkyl, aryl, and heteroaryl substituents were found to be tolerated at the C5, C6, and C7 positions of the pyridopyrimidine core. These studies have led to the identification of analogues that are potent inhibitors of adenosine kinase with in vivo analgesic activity.

Full text of DOI:10.1021/jm030327l

J . Med. Chem. 2003, 46, 5249-5257
5249
5,6,7-Tr isu bstitu ted 4-Am in op yr id o[2,3-d ]p yr im id in es a s Novel In h ibitor s of
Ad en osin e Kin a se
Richard J . Perner,* Yu-Gui Gu, Chih-Hung Lee, Erol K. Bayburt, J effery McKie,† Karen M. Alexander,
Kathy L. Kohlhaas, Carol T. Wismer, J oe Mikusa, Michael F. J arvis, Elizabeth A. Kowaluk, and
Shripad S. Bhagwat†
Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, Illinois 60064-6115
Received J uly 3, 2003
The synthesis and structure-activity relationship of a series of 5,6,7-trisubstituted 4-ami-
nopyrido[2,3-d]pyrimidines as novel nonnucleoside adenosine kinase inhibitors is described.
A variety of alkyl, aryl, and heteroaryl substituents were found to be tolerated at the C5, C6,
and C7 positions of the pyridopyrimidine core. These studies have led to the identification of
analogues that are potent inhibitors of adenosine kinase with in vivo analgesic activity.
In tr od u ction
Adenosine kinase (AK) is a ubiquitous intracellular
enzyme which catalyzes the phosphorylation of adenos-
ine (ADO) to adenosine monophosphate and therefore
is a key enzyme in the control of cellular concentrations
of ADO. Adenosine has been characterized as a homeo-
static modulator of cellular activity1 and, as such, has
several important physiological effects in the central
nervous system, functioning as an endogenous anticon-
vulsant2,3 and a neuroprotective agent.4 Adenosine has
also been implicated in modulating transmission in pain
pathways in the spinal cord.5 During periods of exces-
sive cellular activity or tissue trauma extracellular ADO
release is enhanced which activates specific P1 puri-
nergic receptors (A1, A2A, A2B, and A3) to elicit a variety
of responses which tend to decrease cellular activity and
restore cellular function toward normal.6,7 Because ADO
has a half-life measured in seconds8 in extracellular
fluids, its endogenous actions are highly localized.
Therefore, selective inhibition of AK represents an
attractive approach to enhance the release of endog-
enous ADO to the extracellular space, thus benefiting
from its neuroprotective, anticonvulsant, and antinoci-
ceptive effects.
High-throughput screening of the Abbott compound
library aimed at identifying novel, nonnucleoside AK
inhibitors produced two structurally related pteridine-
like molecules, 1 and 2 (Figure 1), with low micromolar
potency as AK inhibitors (AK(enzyme): IC50 ) 0.4 µM).
Both compounds were inactive at other receptor, enzyme
and kinase screens, suggesting selectivity for AK.9 The
ability of 1 and 2 to inhibit adenosine phosphorylation
by AK was confirmed and their selectivity further
characterized within the AK program. Although com-
pounds 1 and 2 were identified as AK inhibitors
with modest potency, they were considerably weaker at
inhibiting ADO phosphorylation in intact cells
Figu r e 1. Adenosine kinase inhibitor high-throughput screen-
ing hits (1 and 2).
(AK(intact cell): IC50 ) 22 µM and 3.6 µM, respectively),
indicating poor ability to penetrate the cell membrane.
Screening of analogues of 1 and 2 as well as medicinal
chemistry efforts directed at optimizing the potency and
improving the membrane permeability of these AK
inhibitors was initiated. While no other analogue was
found to be more potent than the original hits, initial
SAR studies of compound 2 yielded several important
observations. Replacement of the dimethylamino group
of 2 with a variety of other functionality (e.g., NH2, NO2,
OCH3) failed to improve AK inhibitory activity. How-
ever, installation of a phenyl substituent in the 6-posi-
tion of the pteridine nucleus resulted in a greater than
15-fold increase in in vitro activity (IC50 ) 25 nM vs
440 nM).10 Additionally, it was found through synthetic
efforts that the 5-nitrogen was not required, which not
only resulted in increased compound output due to an
improved synthetic scheme, but also yielded an alterna-
tive scaffold that allowed for substitution at that posi-
tion as well. It was found that placement of a phenyl
substituent at the 5-position gave a compound with very
good AK inhibitory activity (IC50 ) 7 nM, compound 22,
Table 1). These results opened the door to new chem-
istry strategies for optimizing the potency, as well as
characterizing the physical chemical properties of these
types of molecules, one of which was to substitute the
5-, 6-, and 7-positions around the pyridopyrimidine
* Corresponding author: Richard J . Perner, Department R4PM
AP9A/L-19, Abbott Laboratories, 100 Abbott Park Rd., Abbott Park,
IL 60064-6115, Ph: (847)-937-1023, FAX: (847)-937-9195, e-mail:
richard.j.perner@abbott.com.
† Present address: Celgene, Signal Research Division, 5555 Oberlin
Dr., San Diego, CA 92130.
10.1021/jm030327l CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/21/2003

5250 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Ta ble 1. In Vitro Biological Activity of Adenosine Kinase Inhibitorsa
Perner et al.

4-Aminopyrido[2,3-d]pyrimidine Inhibitors of Adenosine Kinase J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5251
Ta ble 1 (Continued)
a
All values are the mean ( SEM of at least three separate observations run in triplicate.
Sch em e 1a
a
Reagents and conditions: (a) NH4OAc, n-butanol, reflux; (b) formamidine acetate, diglyme, 155 °C.
Sch em e 2a
nucleus in a single molecule. It was this investigation
which evolved into the trisubstituted pyridopyrimidine
series 3.
Ch em istr y
The synthesis of 5,6,7-trisubstituted 4-aminopyrido-
[2,3-d]pyrimidines was accomplished using two dis-
tinctly different synthetic routes. In the first route
a
Reagents and conditions: (a) n-BuLi; (b) -78 °C, THF.
shown in Scheme 1, ketone 4 and dicyanoethylene 5
were combined with ammonium acetate in n-butanol at
reflux to give the intermediate aminocyanopyridine 6
in 20-50% yield, presumably via Michael addition of
the in situ formed enamine of 4 to 5 followed by
cyclization on one of the nitrile groups and air oxidation
leading to the aromatic pyridine ring. Intermediate 6
was then subsequently reacted with formamidine ac-
etate in diglyme at 155 °C to form the aminopyrimidine
ring of the desired trisubstituted pyridopyrimidine 3 in
15-50% yield.
eral different synthetic routes were utilized for the
preparation of 4 as shown in Schemes 2 and 3. The first
method (Scheme 2), which was useful for analogues
where the 7-position substituent was phenyl or thienyl,
involved addition of an aryllithium reagent 7b, prepared
via direct lithiation or metal-halogen exchange, to an
appropriately substituted Weinreb amide11 8 to give
after workup the desired ketone 4. For cases where the
Ar7 substituent was 2-amino-substituted pyridyl, two
procedures were employed, both starting with 6-chlo-
ronicotinyl chloride as shown in Scheme 3. The first
procedure, when R6 was frequently aryl, utilized a
condensation reaction between an appropriately sub-
The requisite ketones 4 and dicyanoethylenes 5 were
prepared by standard methods in separate steps. Sev-

5252 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Perner et al.
Sch em e 3a
a
Reagents and conditions: (a) LiN(TMS)2, THF; (b) DMSO/H2O, 155 °C; (c) NH(Me)OMe‚HCl, Et3N, CH2Cl2, 0 °C; (d) -78 °C to room
temp., THF; (e) NRR′, ethanol/reflux or H2O/100 °C sealed tube.
Sch em e 4
perature in 1,2,4-trichlorobenzene or diphenyl ether as
described previously.10
Resu lts a n d Discu ssion
Table 1 summarizes the effect of placing three sub-
stituents simultaneously around the pyridopyrimidine
core, and in general these data indicate good tolerability.
A variety of aryl or heteroaryl groups at the 5-position
could be incorporated into the molecule, leading to an
increase in AK potency as compared to the unsubsti-
tuted derivatives 21 and 23. Increasing the lipophilicity
of these groups by adding substituents such as the
isopropyl moiety in compound 25 tended to yield more
potent analogues, indicating the importance of this
interaction. Additionally, although it was found that
incorporation of a phenyl substituent at the 6-position
of the pteridine ring system of 2 boosted the in vitro
activity more than 15-fold (IC50 ) 25 nM vs 440 nM),10
a comparison of compounds 21, 22, and 23 suggested
that in the pyridopyrimidine series the 5-substituent
played a more important role than the 6-substituent
with regard to enzymatic AK inhibitory activity. Per-
haps this was due to better alignment of the 5-substitu-
ent in the lipophilic pocket of the binding site in the
AK enzyme. Thus, the tolerability of a group at the
6-position provided the potential for utilizing this sub-
stituent not only for improving AK activity but also to
alter the physical chemical characteristics of the mol-
ecule to ultimately improve such properties as cell
penetration, solubility, and pharmacokinetics.
Substituents at the 6-position could be aryl or alkyl.
Substitution on the 6-phenyl ring which yielded potent
analogues ranged from lipophilic groups such as iso-
propyl (compound 25) and fluoro (compound 35) to
hydrophilic groups such as 3,4-dimethoxy and dimethyl-
amino (e.g., 31 and 36). Alkyl groups at the 6-position
gave potent analogues provided they were not too
sterically demanding. For example, smaller groups such
as n-pentyl (compound 40) and cyclopropyl (compound
37) had good AK inhibitory activity (IC50 ) 55 nM and
60 nM, respectively), albeit approximately 12-fold lower
than the case where R6 is aryl (e.g., 33). However,
branched alkyl groups, such as isopropyl, or larger
cycloalkyl groups such as cyclohexyl, led to a 60 to 100-
fold decrease in AK potency as compared to aryl (Table
1, compounds 38 and 39). In addition, the 6-alkyl
analogues tended to have weaker ability to inhibit ADO
Sch em e 5a
a
Reagents and conditions: (a) (PPh3)2PdCl2, CuI, Et3N; (b)
(aq)K2CO3/MeOH; (c) Ar5I, (PPh3)2PdCl2, CuI, Et3N; (d) cat-
echolborane, THF, reflux; (e) (PPh3)4Pd, (aq)Na2CO3, (aq)NaOH,
reflux; (f) Ar7CHO, 1,2,4-trichlorobenzene, 4, air.
stituted ethyl acetate 9 and acyl chloride 10a to yield
â-ketoester 11 which was decarboxylated at elevated
temperature to give the desired ketone 13. Alterna-
tively, when R6 was frequently alkyl, an appropriate
Grignard reagent 12 was reacted with 10 after Weinreb
amide formation to give intermediate 13. Ketone 13 was
then converted to the desired ketone 4 by reaction with
the requisite amine in ethanol at reflux or in water at
100 °C in a sealed tube. The dicyanoethylenes 5 were
prepared by reacting an aryl aldehyde with malononi-
trile using glycine as the catalyst (Scheme 4).12
Scheme 5 illustrates an alternate route for preparing
pyridopyrimidines 3 wherein the substituents at C5 and
C6 were the same moiety. A bis-substituted acetylene
derivative 18, prepared via tandem palladium coupling
reactions of trimethylsilylacetylene and the appropri-
ately substituted iodobenzene 17, was treated with
catecholborane followed by Suzuki coupling of the
resulting boronic acid with 4,6-diamino-5-iodopyrimi-
dine (19) to give the trisubstituted alkene 20. The
desired products 3 were then obtained via cyclization
by aza-Cope rearrangement of the intermediate formed
by reaction of 20 with an aryl aldehyde at high tem-

4-Aminopyrido[2,3-d]pyrimidine Inhibitors of Adenosine Kinase J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5253
Ta ble 2. In Vivo Analgesic Activity of Selected
In conclusion, we have developed a series of 5,6,7-
trisubstituted 4-aminopyrido[2,3-d]pyrimidines as novel
5,6,7-Trisubstituted Pyridopyrimidinesa
AK IC50
(nM)
intact cell IC50
(nM)
hotplate analgesia,
% MPE
nonnucleoside AK inhibitors. These compounds showed
very potent inhibitory activity at the AK enzyme, some
with sub-nanomolar potency. A wide variety of substi-
tuted aryl groups at the 5-position, and substituted aryl
and alkyl groups at the 6-position were evaluated and
yielded potent analogues. At the 7-position, the 2-thio-
phene moiety generally gave the most potent analogues,
often in the sub-nanomolar range. In an effort to
improve the solubility of these compounds a variety of
amino-substituted pyridyl groups were installed at the
7-position which resulted in potent AK inhibitors, but
had only a modest effect on the aqueous solubility. In
addition, some analogues showed statistically significant
analgesic activity in the mouse hotplate and rat forma-
lin animal models of pain.
compd
24
32
33
16
3.2
5.0
250
53
130
77*
50*
56*
a
% MPE ) maximal protective effect ([postdrug latency] -
[vehicle latency])/([maximum latency] - [vehicle latency]) × 100%,
where maximum (cutoff) latency was 180 s. Values represent mean
within ( 8%. *Indicates p < 0.05 compared to vehicle-treated
mice.15
phosphorylation in intact cells, indicating poor cell
penetration. Other groups that could be placed at the
6-position included benzyl (compound 41, IC50 ) 63 nM)
as well as the ethyl acetate derivative 29 which gave a
single-digit nanomolar analogue.
A variety of aromatic and heteroaromatic groups at
the 7-position were equally tolerated, with the 2-thio-
phene moiety generally giving the most potent ana-
logues. Compound 26, for example, was 29-fold more
potent than the corresponding dimethylaminophenyl
derivative 24 and about 6-fold more potent than the
dimethylaminopyridyl analogue 32. In fact, in many
cases such compounds as 26, 27, and 28 had sub-
nanomolar affinity to inhibit enzymatic AK activity,
while essentially all others were in the single-digit
nanomolar range. Despite the high potency of the
7-thienyl compounds, the amino-substituted pyridines,
for example compounds 30, 33, and 34, were the most
significant 7-position substituents, as these groups not
only yielded potent analogues but also provided the
potential for improving the aqueous solubility of these
compounds, a problem that plagued the earlier ana-
logues. From the developing structure-activity rela-
tionship studies of this and other series, it was found
that a wide variety of substituents could be placed on
the 7-aryl ring para to the pyridopyrimidine core
without any loss of AK activity.14 As a result groups
such as dimethylamino, morpholino, and N-methyl-N-
methoxyethyl were incorporated into the 7-pyridyl sub-
stituent which yielded very potent AK inhibitors, while
at the same time more readily allowing for hydrochlo-
ride salt formation and somewhat improved aqueous
solubility.
Exp er im en ta l Section
1H NMR spectra were obtained at 300 MHz using tetra-
methylsilane as internal standard. The mass spectra (electron
spray ionization (ESI) and dissolvable chemical ionization
(DCI)) and high-resolution mass spectra were recorded on
Finnigin-4000 instruments. Elemental combustion analyses
were within (0.4% of theoretical values and were performed
by Robertson Microlit Laboratories. Flash column chromatog-
raphy was carried out on EM Science silica gel 60 (230-400
mesh). Reactions were routinely conducted under inert atmo-
sphere (N2) using commercial high purity solvents as received.
General procedures for the preparation of compounds 21-
23 were followed as described in ref 10.
Gen er a l P r oced u r es for th e P r ep a r a tion of Com -
p ou n d s 24-27: 1,2-Bis(4-isop r op ylp h en yl)a cetylen e (18;
Ar 5 ) 4-isop r op ylp h en yl). To a solution of 4-iodoisopropy-
lbenzene (12.3 g, 50 mmol) in triethylamine (150 mL) were
added trimethylsilylacetylene (5.89 g, 60 mmol), dichlorobis-
(triphenylphosphine)palladium(II) (0.70 g, 1 mmol), and cop-
per(I) iodide (1.5 g, 7.9 mmol). The reaction was stirred at room
temperature for 18 h, diluted with hexanes, and filtered. The
filtrate was evaporated under reduced pressure to give crude
1-(4-isopropylphenyl)-2-trimethylsilylacetylene. This crude prod-
uct was dissolved in methanol (100 mL), aqueous 1 M potas-
sium carbonate solution (25 mL) was added, and the reaction
was stirred at room temperature for 2 h. The reaction mixture
was then diluted with water and extracted with pentane. The
organic layers were combined, dried with magnesium sulfate,
and evaporated under reduced pressure without heating to
give crude 4-isopropylphenyl acetylene. This crude acetylene
was then dissolved in triethylamine (100 mL). 4-Iodoisopro-
pylbenzene (12.3 g, 50 mmol), dichlorobis(triphenylphosphine)-
palladium(II) (0.70 g, 1 mmol), and copper(I) iodide (1.5 g, 7.9
mmol) were added. The reaction was stirred at room temper-
ature for 2 days, heated to reflux for 1 h, cooled, diluted with
hexanes, and filtered. The filtrate was evaporated under
reduced pressure. The residue was filtered through a pad of
silica gel with hexanes, and the solvent was evaporated to give
Selected compounds were screened for analgesic
activity in animal pain models such as the mouse
hotplate test and rat formalin test, and some showed
significant effects. For example, compounds 24, 32, and
33 all showed statistically significant acute analgesic
effects in the mouse hotplate test at 30 µmol/kg ip (Table
2). In addition, 32 and 33 showed a statistically signifi-
cant (p < 0.05) 38% and 39% reduction in formalin-
induced nociception at the 10 µmol/kg ip dose, respec-
tively.
1
11.40 g (87%) of the title compound. H NMR (CDCl3): δ 7.45
(m, 4H), 7.20 (m, 4H), 2.91 (m, 2H), 1.25 (d, 12H, J ) 7.0 Hz).
MS (DCI/NH3): m/z 263 (M + H)+.
4,6-Dia m in o-5-(1,2-bis(4-isop r op ylp h en yl)eth en yl)p y-
r im id in e (20; Ar 5 ) 4-isop r op ylp h en yl). 1,2-Bis(4-isopro-
pylphenyl)acetylene (11.40 g, 43 mmol) was dissolved in 50
mL of THF, catecholborane (1 M, 50 mL) in THF was added,
and the mixture was heated at reflux for 30 h. The mixture
was cooled, then 4,6-diamino-5-iodopyrimidine,10 30 mL of
saturated aqueous sodium bicarbonate, 20 mL of 3 N aqueous
sodium hydroxide, and 1.00 g (0.87 mmol) tetrakis(triph-
enylphosphine)palladium(0) were added. The mixture was
heated to reflux for 18 h, cooled, diluted with water, then
extracted with ethyl acetate. The organic layers were com-
bined, dried with magnesium sulfate, and the solvent evapo-
Only very modest improvements in aqueous solubility
were able to be achieved through various substitutions
at the 6-position or by incorporation of the aminopyridyl
substituents at Ar7. Therefore, despite the excellent in
vitro and encouraging in vivo data associated with this
series of compounds, their poor solubility and the
presumed poor pharmacokinetic properties precluded
further development of the trisubstituted pyridopyrim-
idines.

5254 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Perner et al.
rated. The residue was chromatographed on silica gel with
2.5% to 5% (19:1 ethanol:ammonium hydroxide) in ethyl
acetate to give the desired product (4.53 g, 28% yield). 1H NMR
(DMSO-d6): δ 7.81 (s, 1H), 7.26 (d, 2H, J ) 8.1 Hz), 7.12 (m,
4H), 7.06 (d, 2H, J ) 8.1 Hz), 6.57 (s, 1H), 5.71 (bs, 4H), 2.84
(m, 2H), 1.18 (m, 12H). MS (DCI/NH3): m/z 273 (M + H)+.
4-Am in o-5,6-bis(4-isop r op ylp h en yl)-7-(4-d im eth yla m i-
n op h en yl)p yr id o[2,3-d ]p yr im id in e (25). A sample of 4,6-
diamino-5-(1,2-bis(4-isopropylphenyl)ethenyl)pyrimidine (745
mg, 2 mmol) was dissolved in 20 mL of 1,2,4-trichlorobenzene
containing 4-dimethylaminobenzaldehyde (0.89 g, 6 mmol),
and approximately 1 g of 4 Å molecular sieves was added to
the reaction mixture. The mixture was heated to reflux for 20
h, cooled, and filtered through a pad of Celite. The filtrate was
applied directly to a silica gel chromatography column, which
was eluted with 2.5% (19:1 ethanol:ammonium hydroxide) in
ethyl acetate to give the desired product (186 mg, 18.5% yield).
1H NMR (DMSO-d6): δ 8.51 (s, 1H), 7.70 (bs, 1H), 7.26 (d, 2H,
J ) 9 Hz), 7.17 (d, 2H, J ) 8 Hz), 7.12 (d, 2H, J ) 8 Hz), 6.90
(d, 2H, J ) 8 Hz), 6.83 (d, 2H, J ) 8 Hz), 6.49 (d, 2H, J ) 9
Hz), 4.70 (bs, 1H), 2.87 (s, 6H), 2.84 (m, 1H), 2.72 (m, 1H),
1.12 (d, 6H, J ) 6.8 Hz), 1.06 (d, 6H, J ) 6.8 Hz). MS (DCI/
NH3): m/z 502 (M + H)+. Anal. Calcd (C33H35N5‚0.25EtOAc):
C, H, N.
22.0 mmol) in 75 mL of anhydrous THF at -78 °C. The
reaction was allowed to proceed 90 min., then diluted with 100
mL of Et2O and poured into 1 N aq HCl. The aqueous phase
was extracted with Et2O, and the combined organic fraction
was washed with brine, dried (Na2SO4), and concentrated in
vacuo. Flash chromatography (25% EtOAc/hexanes) yielded
1
2.91 g (50%) of the title compound. H NMR (CDCl3): δ 7.77
(dd, 1H, J ) 3.6, 1.2 Hz), 7.64 (dd, 1H, J ) 5.1, 1.2 Hz), 7.13
(dd, 1H, J ) 5.1, 3.6 Hz), 6.84 (m, 3H), 4.14 (s, 2H), 3.88 (s,
3H), 3.86 (s, 3H). MS (DCI/NH3): m/z 263 (M + H)+, 280 (M
+ NH4)+.
3-Cyan o-4-(4-br om oth ien -2-yl)-5-(3,4-dim eth oxyph en yl)-
6-(th ien -2-yl)-2-p yr id in ea m in e (6; Ar 5 ) 4-br om oth ien -
2-yl, R6 ) 3,4-d im eth oxyp h en yl, Ar 7 ) th ien -2-yl). 2-(3,4-
Dimethoxyphenyl)-1-(thien-2-yl)ethanone (1.56 g, 5.95 mmol),
4-bromo-2-(2,2-dicyanoethenyl)thiophene (1.71 g, 7.13 mmol),
and NH4OAc (1.15 g, 14.9 mmol) were combined in n-BuOH
(10 mL) and heated to reflux. After 24 h the reaction mixture
was cooled, diluted with EtOAc, and washed with water, brine,
dried over Na2SO4, and concentrated in vacuo. Flash chroma-
tography (40% EtOAc/hexanes) gave the desired product (0.76
g) which was shown to contain minor unidentifiable side
products which could not be separated. 1H NMR (CDCl3): δ
7.40-6.60 (m, 8H), 5.36 (bs, 2H), 3.91 (s, 3H), 3.72 (s, 3H).
MS (DCI/NH3): m/z 498/500 (M + H)+.
4-Am in o-5-(4-br om oth ien -2-yl)-6-(3,4-dim eth oxyph en yl)-
7-(th ien -2-yl)pyr ido[2,3-d]pyr im idin e Hydr och lor ide (28).
3-Cyano-4-(4-bromothien-2-yl)-5-(3,4-dimethoxyphenyl)-6-(thien-
2-yl)-2-pyridineamine (750 mg, 1.50 mmol) and formamidine
acetate (312 mg, 3.00 mmol) were taken up in 10 mL of
diglyme and heated to 155 °C. Additional formamidine acetate
(1 equiv) was added at 90 min intervals over a total of 6 h,
then heating was continued overnight. The cooled reaction
mixture was then partitioned between EtOAc and H2O. The
organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. Flash chromatography (3.5% MeOH/
CH2Cl2) gave a brown residue which was dissolved in a small
amount of CH2Cl2 followed by addition of Et2O to precipitate
the desired product (209 mg, 26%). This material was con-
verted to the hydrochloride salt using ethanolic HCl followed
by precipitation with Et2O and filtration of the product. 1H
NMR (DMSO-d6): δ 9.91 (bs, 1H), 8.89, (s, 1H), 7.88 (m, 1H),
7.83 (d, 1H, J ) 5.1 Hz), 7.27 (m, 1H), 7.02 (m, 1H), 6.96 (d,
1H, J ) 9.0 Hz), 6.85 (d, 1H, J ) 2.1 Hz), 6.70 (m, 2H), 6.47
(bs, 1H), 3.78 (s, 3H), 3.64 (s, 3H). MS (DCI/NH3): m/z 525/
527 (M + H)+. Anal. Calcd (C23H17BrN4O2S2‚1.1HCl): C, H,
N.
4-Am in o-5,6-d ip h en yl-7-(4-d im et h yla m in op h en yl)p y-
r id o[2,3-d ]p yr im id in e (24). Compound 24 was synthesized
using iodobenzene and the process described for the synthesis
of 25. 1H NMR (DMSO-d6): δ 8.53 (s, 1H), 7.75 (bs, 1H), 7.37-
7.25 (m, 5H), 7.24 (d, 2H, J ) 9 Hz), 7.15 (m, 3H), 6.98 (m,
2H), 6.50 (d, 2H, J ) 9 Hz), 4.50 (bs, 1H), 2.88 (s, 6H). MS
(DCI/NH3): m/z 418 (M + H)+. Anal. Calcd (C27H23N5‚
0.5H2O): C, H, N.
4-Am in o-5,6-d ip h en yl-7-(th ien -2-yl)p yr id o[2,3-d ]p yr i-
m id in e (26). Compound 26 was synthesized using iodoben-
zene, 2-thiophenecarboxaldehyde, and the process described
for the synthesis of 25. 1H NMR (DMSO-d6): δ 8.55 (s, 1H),
7.85 (bs, 1H), 7.65 (m, 1H), 7.40-7.20 (m, 8H), 7.16 (m, 2H),
6.84 (m, 1H), 6.14 (m, 1H), 4.50 (bs, 1H). MS (DCI/NH3): m/z
381 (M + H)+. Anal. Calcd (C23H16N4S‚0.3H2O): C, H, N.
4-Am in o-5,6-b is(3-flu or o-4-m et h ylp h en yl)-7-(t h ien -2-
yl)p yr id o[2,3-d ]p yr im id in e H yd r och lor id e (27). Com-
pound 27 was synthesized using 3-fluoro-4-methyliodobenzene,
2-thiophenecarboxaldehyde, and the process described for the
1
synthesis of 25. H NMR (DMSO-d6): δ 9.77 (bs, 1H), 8.90 (s,
1H), 7.84 (m, 1H), 7.43-6.85 (m, 7H), 6.52 (m, 1H), 5.83 (bs,
1H), 2.21 (s, 6H). MS (DCI/NH3): m/z 445 (M + H)+. Anal.
Calcd (C25H18F2N4S‚1.9HCl): C, H, N.
4-Am in o-5-(3-br om op h en yl)-6-eth oxyca r bon ylm eth yl-
7-(th ien -2-yl)pyr ido[2,3-d]pyr im idin e Hydr och lor ide (29).
Compound 29 was synthesized using ethylsuccinyl chloride,
3-bromo-(2,2-dicyanoethenyl)benzene,12 and the process de-
scribed for the synthesis of 28. 1H NMR (DMSO-d6): δ 9.88
(bs, 1H), 8.87 (s, 1H), 8.0 (m, 1H), 7.9 (m, 1H), 7.75-7.6 (m,
3H), 7.43 (m, 1H), 7.32 (m, 1H), 6.13 (bs, 1H), 4.01 (q, 2H, J )
7 Hz), 3.75 (m, 2H), 1.09 (t, 3H, J ) 7 Hz). MS (DCI/NH3):
m/z 469/471 (M + H)+. Anal. Calcd (C21H17BrN4O2S‚HCl): C,
H, N.
4-Am in o-5-(4-br om oth ien -2-yl)-6-(3,4-dim eth oxyph en yl)-
7-(4-(N-m eth yl-N-(2-m eth oxyeth yl)a m in o)p h en yl)p yr id o-
[2,3-d ]p yr im id in e Hyd r och lor id e (30). Compound 30 was
synthesized using 1-bromo-4-(N-methyl-N-(2-methoxyethyl)-
amino)benzene and the process described for the synthesis of
28. 1H NMR (DMSO-d6): δ 9.71 (bs, 1H), 8.85 (s, 1H), 7.86
(m, 1H), 7.40 (m, 1H), 7.34 (d, 2H, J ) 9 Hz), 6.79 (m, 2H),
6.59 (d, 2H, J ) 9 Hz), 6.48 (dd, 1H, J ) 8.1, 1.7 Hz), 6.31 (bs,
1H), 3.69 (s, 3H), 3.60 (s, 3H), 3.55-3.40 (m, 4H), 3.22 (s, 3H),
2.92 (s, 3H). MS (DCI/NH3): m/z 606/608 (M + H)+. Anal.
Calcd (C29H28BrN5O3S‚1.4HCl): C, H, N.
Gen er a l P r oced u r es for th e P r ep a r a tion of Com -
pou n ds 28-31: 4-Br om o-2-(2,2-dicyan oeth en yl)th ioph en e
(5; Ar 5 ) 4-br om oth ien -2-yl). Compound 5 was synthesized
following the method of Bastus.12 4-Bromo-2-thiophenecarbox-
aldehyde (6.92 g, 36.2 mmol) and malononitrile (2.39 g, 36.2
mmol) were dissolved in 100 mL of 1:1 EtOH:H2O. A small
spatula of glycine was added, and the reaction was stirred at
ambient temperature for 30 min. The precipitated product was
collected by suction filtration, washed with water, and dried
under vacuum overnight. The result was 8.38 g (97%) of the
desired product. 1H NMR (CDCl3): δ 7.77 (m, 1H), 7.74 (m,
2H). MS (DCI/NH3): m/z 238/240 (M + H)+.
2-(3,4-Dim eth oxyp h en yl)-1-(th ien -2-yl)eth a n on e (4; R6
) 3,4-d im eth oxyp h en yl, Ar 7 ) th ien -2-yl). (3,4-Dimethoxy-
phenyl)acetic acid (13.0 g, 66.4 mmol) was suspended in 200
mL of anhydrous CH2Cl2 followed by addition of EDCI (15.3
g, 79.7 mmol), HOBt (20.6 g, 152 mmol), triethylamine (8.06
g, 79.7 mmol), and N,O-dimethylhydroxylamine hydrochloride
(6.48 g, 66.4 mmol). The reaction was stirred 3 days at ambient
temperature after which the solvent was evaporated at reduced
pressure. The residue was partitioned between EtOAc and
water. The organic layer was washed with aq HCl, sat.
NaHCO3, brine, dried (Na2SO4), and concentrated in vacuo to
give 10.5 g (66%) of N-methyl-N-methoxy-(3,4-dimethoxyphen-
yl)acetamide as a pale brown oil.
4-Am in o-5-(3-br om op h en yl)-6-(3,4-d im eth oxyp h en yl)-
7-(4-(d im eth yla m in o)p h en yl)p yr id o[2,3-d ]p yr im id in e Di-
h yd r och lor id e (31). Compound 31 was synthesized using
3-bromo-(2,2-dicyanoethenyl)benzene,12 1-bromo-4-(dimethyl-
amino)benzene, and the process described for the synthesis of
28. 1H NMR (DMSO-d6): δ 9.90 (bs, 1H), 8.92 (s, 1H), 7.61
2-Lithiothiophene (1.0 M in THF, 33.0 mL, 33.0 mmol) was
added dropwise to a solution of the oil obtained above (5.26 g,

4-Aminopyrido[2,3-d]pyrimidine Inhibitors of Adenosine Kinase J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5255
(m, 1H), 7.48 (d, 2H, J ) 9 Hz), 7.45-7.3 (m, 2H), 6.80-6.67
(m, 2H), 6.60 (d, 2H, J ) 9 Hz), 6.45-6.35 (m, 2H), 5.96 (bs,
1H), 3.65 (s, 3H), 3.55 (bd, 3H), 2.93 (s, 6H). MS (DCI/NH3):
m/z 556/558 (M + H)+. Anal. Calcd (C29H26BrN5O2‚2HCl): C,
H, N.
J ) 9.5 Hz), 6.12 (bs, 1H), 3.64 (m, 4H), 3.50 (m, 4H). MS (DCI/
NH3): m/z 539/541 (M + H)+. Anal. Calcd (C28H23BrN6O‚
2HCl): C, H, N.
4-Am in o-5,6-d ip h en yl-7-(6-(d im eth yla m in o)p yr id in -3-
yl)p yr id o[2,3-d ]p yr im id in e H yd r och lor id e (32). Com-
pound 32 was synthesized using (2,2-dicyanoethenyl)ben-
zene,12 ethyl phenylacetate, dimethylamine, and the process
described for the synthesis of 34. 1H NMR (DMSO-d6): δ 10.1
(bs, 1H), 8.98 (s, 1H), 8.13 (d, 1H, J ) 2.0 Hz), 7.55 (dd, 1H, J
) 9.2, 2.0 Hz), 7.43 (m, 3H), 7.29 (m, 2H), 7.18 (d, 3H), 7.08
(m, 2H), 6.83 (m, 1H), 5.80 (bs, 1H), 3.14 (s, 6H). MS (DCI/
NH3): m/z 419 (M + H)+. Anal. Calcd (C26H22N6‚1.8HCl): C,
H, N.
Gen er a l P r oced u r e for P r ep a r a tion of Com p ou n d s
32-37: 1-(6-Ch lor op yr id in -3-yl)-2-p h en yleth a n on e (13;
R6 ) p h en yl). A solution of ethyl phenylacetate (10.3 g, 62.8
mmol, neat) was added dropwise to a solution of lithium bis-
(trimethylsilyl)amide (75 mL, 75 mmol) in 150 mL of THF at
-78 °C. The reaction was stirred for 60 min followed by
addition of 6-chloronicotinyl chloride (13.3 g, 75 mmol, solid)
in one portion. The reaction was stirred an additional 60 min,
then quenched with saturated ammonium chloride solution.
The mixture was diluted with Et2O, poured into water, and
the aqueous phase was extracted with Et2O. The combined
organic layers were washed with brine, dried (Na2SO4), and
concentrated in vacuo to 22 g crude product as a light yellow
solid. This material was dissolved in DMSO (200 mL) and H2O
(10 mL), and the solution was heated to 155 °C for 3 h. The
reaction was then cooled and poured into water, and the
product was extracted with Et2O. The combined Et2O layers
were washed with water, brine, dried (MgSO4), and concen-
trated under vacuum. The product was purified by trituration
with 30% EtOAc/hexanes which gave 7.79 g (54%) of the title
4-Am in o-5-(3-ch lor op h en yl)-6-p h en yl-7-(6-(d im et h yl-
a m in o)-p yr id in -3-yl)-p yr id o[2,3-d ]p yr im id in e Dih yd r o-
ch lor id e (33). Compound 33 was synthesized using 3-chloro-
(2,2-dicyanoethenyl)benzene,12 ethyl phenylacetate, dimethyl-
1
amine, and the process described for the synthesis of 34. H
NMR (DMSO-d6): δ 10.06 (bs, 1H), 8.98 (s, 1H), 8.10 (d, 1H,
J ) 2.4 Hz), 7.55 (dd, 1H, J ) 9.6, 2.4 Hz), 7.43 (m, 2H), 7.36
(m, 1H), 7.30-7.15 (m, 4H), 7.09 (m, 1H), 7.03 (m, 1H), 6.76
(bd, 1H), 6.15 (bs, 1H), 3.10 (s, 6H). MS (DCI/NH3): m/z 453
(M + H)+. Anal. Calcd (C26H21ClN6‚2HCl): C, H, N.
4-Am in o-5-(3-b r om op h en yl)-6-(4-flu or op h en yl)-7-(6-
m or p h olin op yr id in -3-yl)p yr id o[2,3-d ]p yr im id in e Dih y-
d r och lor id e (35). Compound 35 was synthesized using
3-bromo-(2,2-dicyanoethenyl)benzene,11 ethyl 4-fluoropheny-
1
compound. H NMR (CDCl3): δ 8.99 (d, 1H, J ) 2.1 Hz), 8.21
(dd, 1H, J ) 8.4, 2.1 Hz), 7.43 (d, 1H, J ) 8.4 Hz), 7.39-7.22
(m, 5H), 4.27 (s, 2H). MS (DCI/NH3): m/z 232 (M + H)+.
1
lacetate, and the process described for the synthesis of 34. H
1-(6-Mor p h olin op yr id in -3-yl)-2-p h en yleth a n on e (4; R 6
) p h en yl, Ar 7 ) 6-m or p h olin op yr id in -3-yl). The ketone 13
from above (2.18 g, 9.41 mmol) and morpholine (2.46 mL, 28.3
mmol) were dissolved in 20 mL of absolute ethanol, and the
mixture was heated to reflux for 18 h. The volatiles were then
removed under vacuum, and the residue was partitioned
between Et2O and saturated NaHCO3. The Et2O layer was
washed with brine, dried (Na2SO4), and concentrated under
vacuum to give the title compound (2.62 g, 99%). 1H NMR
(CDCl3): δ 8.85 (d, 1H, J ) 2.7 Hz), 8.08 (dd, 1H, J ) 9.6, 2.7
Hz), 7.36-7.19 (m, 5H), 6.60 (d, 1H, J ) 9.6 Hz), 4.17 (s, 2H),
3.80 (m, 4H), 3.66 (m, 4H). MS (DCI/NH3): m/z 283 (M + H)+.
NMR (DMSO-d6): δ 10.05 (bs, 1H), 8.97, (s, 1H), 8.17 (d, 1H,
J ) 2.7 Hz), 7.61 (m, 1H), 7.50 (m, 2H), 7.37 (t, 1H, J ) 7.8
Hz), 7.31 (m, 1H), 7.15-7.0 (m, 4H), 6.79 (d, 1H, J ) 9.5 Hz),
6.16 (bs, 1H), 3.65 (m, 4H), 3.52 (m, 4H). MS (DCI/NH3): m/z
557/559 (M + H)+. Anal. Calcd (C28H22BrFN6O‚2.1HCl): C, H,
N.
4-Am in o-5-(3-ch lor oph en yl)-6-(4-dim eth ylam in oph en yl)-
7-(6-m or ph olin opyr idin -3-yl)pyr ido[2,3-d]pyr im idin e (36).
Compound 36 was synthesized using 3-chloro-(2,2-dicyano-
ethenyl)benzene,12 ethyl 4-(dimethylamino)phenylacetate, and
the process described for the synthesis of 34. 1H NMR (DMSO-
d6): δ 8.72 (bs, 1H), 8.28 (d, 1H, J ) 2.6 Hz), 7.80 (dd, 1H, J
) 8.8, 2.6 Hz), 7.30 (m, 2H), 7.20 (bs, 1H), 7.06 (m, 1H), 6.70
(d, 2H, J ) 8.5 Hz), 6.45 (m, 3H), 5.15 (bs, 2H), 3.78 (m, 4H),
3.51 (m, 4H), 2.88 (s, 6H). MS (DCI/NH3): m/z 538 (M + H)+.
Anal. Calcd (C30H28ClN7O‚0.1H2O): C, H, N.
4-Am in o-5-(4-br om oth ien -2-yl)-6-cyclop r op yl-7-(6-(d i-
m eth yla m in o)p yr id in -3-yl)p yr id o[2,3-d ]p yr im id in e Di-
h yd r och lor id e (37). Compound 37 was synthesized using
4-bromo-2-(2,2-dicyanoethenyl)thiophene,12 ethyl 2-cyclopropyl
acetate, dimethylamine, and the process described for the
synthesis of 34. 1H NMR (CDCl3): δ 8.71 (d, 1H, J ) 2 Hz),
8.67 (s, 1H), 8.23 (dd, 1H, J ) 9, 2 Hz), 7.58 (d, 1H, J ) 1.5
Hz), 7.14 (d, 1H, J ) 1.5 Hz), 6.63 (d, 1H, J ) 9 Hz), 5.45 (b,
2H), 3.20 (s, 6H), 1.98 (m, 1H), 0.73 (m, 2H), 0.13 (m, 2H). MS
(DCI/NH3): m/z 467/469 (M + H)+. Anal. Calcd (C21H19BrN6S‚
2.3HCl): C, H, N.
3-Cya n o-4-(3-b r om op h en yl)-5-p h en yl-6-(6-m or p h oli-
n op yr id in -3-yl)-2-p yr id in ea m in e (6; Ar 5 ) 3-br om op h en -
yl, R6 ) p h en yl, Ar 7 ) 6-m or p h olin op yr id in -3-yl). A
solution of 1-(6-morpholinopyridin-3-yl)-2-phenylethanone (1.30
g, 4.60 mmol), 3-bromo-(2,2-dicyanoethenyl)benzene (1.61 g,
6.91 mmol), and NH4OAc (0.89 g, 11.5 mmol) in 15 mL of
n-butanol was heated to reflux. After 24 h the reaction mixture
was cooled to ambient temperature followed by addition of
Et2O. The resulting precipitate was collected by filtration,
washed with Et2O, and dried under vacuum. The result was
0.57 g of the desired product which was found to also contain
1
some of the starting ketone. H NMR (CDCl3): δ 8.20 (d, 1H,
J ) 2.1 Hz), 7.40-7.20 (m, 3H), 7.10 (m, 4H), 7.02 (m, 1H),
6.83 (m, 2H), 6.36 (d, 1H, J ) 9 Hz), 5.30 (bs, 2H), 3.77 (m,
4H), 3.47 (m, 4H). MS (DCI/NH3): m/z 512/514 (M + H)+.
4-Am in o-5-(3-b r om op h en yl)-6-p h en yl-7-(6-m or p h oli-
n op yr id in -3-yl)p yr id o[2,3-d ]p yr im id in e Dih yd r och lor id e
(34). 3-Cyano-4-(3-bromophenyl)-5-phenyl-6-(6-morpholinopy-
ridin-3-yl)-2-pyridineamine (566 mg, 1.10 mmol) and forma-
midine acetate (573 mg, 5.50 mmol) were taken up in 15 mL
of diglyme and heated to 150 °C. Additional formamidine
acetate (1 equiv) was added at 90 min intervals over a total of
6 h, then heating was continued overnight. The cooled reaction
mixture was then partitioned between EtOAc and H2O. The
organic layer was washed with brine, dried over Na2SO4, and
concentrated in vacuo. Flash chromatography (5% MeOH/CH2-
Cl2) gave a brown residue which was dissolved in a small
amount of CH2Cl2 followed by addition of Et2O to precipitate
the product. This material was converted to the hydrochloride
salt using ethanolic HCl followed by precipitation with Et2O
Gen er a l P r oced u r e for P r ep a r a t ion of Com p ou n d s
38-41: 1-(6-Ch lor op yr id in -3-yl)-3-p h en ylp r op a n on e (13;
R6 ) ben zyl). A sample of 6-chloronicotinyl chloride (15.4 g,
87.4 mmol) was added to a mixture of N,O-dimethylhydroxyl-
amine hydrochloride (9.38 g, 96.2 mmol) and triethylamine
(36.6 mL, 262 mmol) in 200 mL of CH
2Cl2 cooled to 0 °C. The
reaction was stirred for 2 h, then poured into water. The
separated organic layer was washed with brine, dried (Na2SO4),
and concentrated under vacuum to give 14.6 g of the inter-
mediate Weinreb amide as a light brown oil. A sample of the
intermediate amide (4.09 g, 20.4 mmol) in 100 mL of THF was
cooled to -78 °C followed by dropwise addition of phenethyl-
magnesium chloride (30.6 mL, 30.6 mmol, 1 M in THF). The
reaction was allowed to warm to ambient temperature and stir
3 h after which it was quenched by 1 N aq HCl. The mixture
was partitioned between Et2O and saturated NaHCO3. The
organic layer was washed with brine, dried (Na2SO4), and
concentrated in vacuo. The crude product was purified by flash
chromatography eluting with 30% EtOAc/hexanes which gave
1
and filtration of the product (102 mg, 15%). H NMR (DMSO-
d6): δ 10.05 (bs, 1H), 8.97 (s, 1H), 8.15 (d, 1H, J ) 2.4 Hz),
7.58 (dt, 1H, J ) 7.5, 1.7 Hz), 7.53 (d, 1H, J ) 2.4 Hz), 7.49
(m, 1H), 7.34 (m, 2H), 7.20 (s, 3H), 7.05 (m, 2H), 6.76 (d, 1H,

5256 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24
Perner et al.
1
3.77 g (75%) of the desired product. H NMR (CDCl3): δ 8.92
4-Am in o-5-(3-br om oph en yl)-6-pen tyl-7-(6-(dim eth ylam i-
n o)-p yr id in -3-yl)-p yr id o[2,3-d ]p yr im id in e Dih yd r och lo-
r id e (40). Compound 40 was synthesized using 3-bromo-(2,2-
dicyanoethenyl)benzene,12 n-hexylmagnesium chloride, and the
process described for the synthesis of 41. 1H NMR (DMSO-
d6): δ 8.23 (d, 1H, J ) 2.4 Hz), 7.69 (m, 2H), 7.63 (bs, 1H),
7.49 (t, 1H, J ) 8 Hz), 7.40 (d, 1H, J ) 8 Hz), 6.78 (bs, 2H),
6.70 (d, 1H, J ) 9 Hz), 3.07 (s, 6H), 2.31 (m, 2H), 1.02 (m,
2H), 0.85 (m, 4H), 0.59 (t, 3H, J ) 7 Hz). MS (DCI/NH3): m/z
491/493 (M + H)+. Anal. Calcd (C25H27BrN6‚2HCl): C, H, N.
AK In h ibition Assa y. AK inhibition was measured at 23
°C in a 100 µL reaction mixture in triplicate containing 64
mM Tris HCl (pH 7.5), 0.2 mM MgCl2, 1 mM ATP, 0.2 µM
[U-14C]-adenosine or [2-3H]-adenosine (Amersham Interna-
tional, Bucks, United Kingdom) and appropriate volumes of
rat brain cytosol as a source of AK as previously described.9
After incubation for 15 min, the reaction was terminated by
aliquoting 40 µL of the reaction mixture onto DE-81 anion
exchange filter disks. The filter disks were air-dried, washed
in 2 mM ammonium formate, and dried again. Bound radio-
activity was determined by standard scintillation spectrom-
etry. A 96-well plate high-throughput screening assay was
derived from this methodology using [3H]-6-methylmercap-
topurine (Amersham) as an AK substrate that is not suscep-
tible to catabolism by ADO deaminase.
(d, 1H, J ) 2.7 Hz), 8.18 (dd, 1H, J ) 8.4, 2.7 Hz), 7.44 (d, 1H,
J ) 8.4 Hz), 7.37-7.18 (m, 5H), 3.30 (m, 2H), 3.08 (m, 2H).
MS (DCI/NH3): m/z 246 (M + H)+.
1-(6-Mor p h olin op yr id in -3-yl)-3-p h en ylp r op a n on e (4;
R6 ) ben zyl, Ar 7 ) 6-m or p h olin op yr id in -3-yl). A solution
of ketone 13 from above (3.77 g, 15.3 mmol) and morpholine
(5.35 mL, 61.4 mmol) in 30 mL of absolute ethanol was heated
to reflux for 18 h. The volatiles were then removed under
vacuum, and the residue was partitioned between Et2O and
saturated NaHCO3. The Et2O layer was washed with brine,
dried (Na2SO4), and concentrated under vacuum to give 4.50
1
g (100%) of the title compound. H NMR (CDCl3): δ 8.78 (m,
1H), 8.05 (dd, 1H, J ) 9.3, 2.1 Hz), 7.34-7.16 (m, 5H), 6.60
(d, 1H, J ) 9.3 Hz), 3.81 (m, 4H), 3.67 (m, 4H), 3.19 (m, 2H),
3.05 (m, 2H). MS (DCI/NH3): m/z 297 (M + H)+.
3-Cya n o-4-(3-b r om op h en yl)-5-b en zyl-6-(6-m or p h oli-
n op yr id in -3-yl)-2-p yr id in ea m in e (6; Ar 5 ) 3-br om op h en -
yl, R6 ) ben zyl, Ar 7 ) 6-m or p h olin op yr id in -3-yl). A
solution of 1-(6-morpholinopyridin-3-yl)-3-phenylpropanone
(1.68 g, 5.67 mmol), 3-bromo-(2,2-dicyanoethenyl)benzene12
(1.98 g, 8.50 mmol), and NH4OAc (1.09 g, 14.2 mmol) in 15
mL of n-butanol was heated to reflux. After 16 h the reaction
mixture was cooled and concentrated in vacuo. Flash chroma-
tography (50% EtOAc/hexanes) gave 0.84 g (28%) of the desired
product. 1H NMR (CDCl3): δ 8.36 (d, 1H, J ) 2.1 Hz), 7.61
(dd, 1H, J ) 9.3, 2.1 Hz), 7.48 (m, 1H), 7.20-7.07 (m, 5H),
6.97(m, 1H), 6.67-6.56 (m, 3H), 5.25 (bs, 2H), 3.83 (m, 6H),
3.55 (t, 4H, J ) 5.1 Hz). MS (DCI/NH3): m/z 526/528 (M +
H)+.
4-Am in o-5-(3-b r om op h en yl)-6-b en zyl-7-(6-m or p h oli-
n op yr id in -3-yl)p yr id o[2,3-d ]p yr im id in e Dih yd r och lor id e
(41). A solution of 3-cyano-4-(3-bromophenyl)-5-benzyl-6-(6-
morpholinopyridin-3-yl)-2-pyridineamine (830 mg, 1.58 mmol)
and formamidine acetate (822 mg, 7.90 mmol) in 15 mL of
diglyme was heated to 150 °C. Additional formamidine acetate
(1 equiv) was added at 90 min intervals over a total of 6 h and
heating was continued overnight. The cooled reaction mixture
was then partitioned between EtOAc and H2O. The organic
layer was washed with brine, dried over Na2SO4, and concen-
trated in vacuo. Flash chromatography (5% MeOH/CH2Cl2)
gave a brown residue which was dissolved in a small amount
of CH2Cl2 followed by addition of Et2O to precipitate the
desired product. This material was converted to the hydro-
chloride salt using ethanolic HCl followed by precipitation with
Et2O and filtration of the product. (620 mg, 63%). 1H NMR
(DMSO-d6): δ 9.94 (bs, 1H), 8.92, (s, 1H), 8.40 (d, 1H, J ) 2.4
Hz), 7.83 (dd, 1H, J ) 8.8, 2.4 Hz), 7.74 (m, 1H), 7.46 (m, 2H),
7.34 (m, 1H), 7.18-7.05 (m, 3H), 6.93 (d, 1H, J ) 9.2 Hz), 6.67
(m, 2H), 6.10 (bs, 1H), 4.00 (m, 2H), 3.68 (m, 4H), 3.55 (m,
4H). MS (DCI/NH3): m/z 553/555 (M + H)+. Anal. Calcd
(C29H25BrN6O‚2HCl): C, H, N.
In ta ct Cell ADO P h osp h or yla tion Assa y. Assays for
ADO phosphorylation in intact cells were conducted using
confluent IMR-32 human neuroblastoma cells (ATCC, Gaith-
ersburg, MD). Appropriate concentrations of test compounds
(10-11 to 10-4 M) were added to each cell culture well and
incubated in 400 µL of warm Gey’s Balanced Salt Solution for
10 min. The reaction run in triplicate was initiated by the
addition of 50 µL of 2 µM [U-14C]-adenosine. After a 20 min
incubation, the assay buffer was rapidly aspirated and the cells
were quickly frozen by the addition of excess liquid nitrogen.
A 50 µL aliquot of the thawed supernatant was placed onto
DE-81 filter disks and processed as described above.
In Vivo Eva lu a tion of AK In h ibitor s. Acute thermal
nociception was assessed in mice (n ) 6-8 per group) using
the 55 °C hotplate test as previously described.9 Nociceptive
paw flinching in rats (Formalin test, n ) 6 per group) was
assessed 30 min following an intraplantar injection of 5%
formalin (50 µL) into the right hindpaw.13 Compounds were
administered intraperitoneally 30 min before nociceptive test-
ing.
Ack n ow led gm en t. We thank the Abbott Structural
Chemistry group for excellent NMR and mass spec
support. We also thank the high-throughput screening
group of the Advanced Technology Research Division.
4-Am in o-5-(4-b r om ot h ien -2-yl)-6-isop r op yl-7-(6-m or -
p h olin op yr id in -3-yl)p yr id o[2,3-d ]p yr im id in e Dih yd r o-
ch lor id e (38). Compound 38 was synthesized using 3-bromo-
(2,2-dicyanoethenyl)benzene,12 isobutylmagnesium chloride,
and the process described for the synthesis of 41. 1H NMR
(DMSO-d6): δ 10.0 (bs, 1H), 8.90 (s, 1H), 8.32 (d, 1H, J ) 2
Hz), 7.92 (m, 1H), 7.80 (m, 2H), 7.66 (t, 1H, J ) 8 Hz), 7.58 (d,
1H, J ) 8 Hz), 7.04 (d, 1H, J ) 9 Hz), 5.84 (bs, 1H), 3.74 (m,
4H), 3.59 (m, 4H), 3.35-3.05 (m, 1H), 0.9 (m, 6H). MS (DCI/
NH3): m/z 505/507 (M + H)+. Anal. Calcd (C25H25BrN6O‚
2.2HCl): C, H, N.
Refer en ces
(1) Newby, A. C. Adenosine and the concept of retaliatory metabo-
lites. Trends Biochem. Sci. 1984, 9, 42-4.
(2) Dragunow, M. Endogenous anticonvulsant substances. Neurosci.,
Biobehav. Rev. 1986, 10, 229-44.
(3) During, M. J .; Spencer, D. D. Adenosine: a potential mediator
of seizure arrest and postictal refractoriness. Anal. Neurol. 1992,
32, 618-24.
(4) Zhou, J . G.; Meno, J . R.; Hsu, S. S.; Winn, H. R. Effects of
theophylline and cyclohexyladenosine on brain injury following
normo- and hyperglycemic ischemia: a histopathologic study in
the rat. J . Cereb. Blood Flow Metab. 1994, 14, 166-73.
(5) Sawynok, J . Purines in pain management. Curr. Opin. CPNS
Invest. Drugs. 1999, 1, 27-38.
4-Am in o-5-(3-br om op h en yl)-6-cycloh exyl-7-(6-(d im eth -
yla m in o)-p yr id in -3-yl)p yr id o[2,3-d ]p yr im id in e Dih yd r o-
ch lor id e (39). Compound 39 was synthesized using 3-bromo-
(2,2-dicyanoethenyl)benzene,12 cyclohexylmethylmagnesium
chloride, and the process described for the synthesis of 41. 1H
NMR (DMSO-d6): δ 8.48 (s, 1H), 8.19 (d, 1H, J ) 2.6 Hz), 7.86
(dt, 1H, J ) 7.4, 1.8 Hz), 7.81 (bs, 1H), 7.66-7.52 (m, 3H),
6.76 (d, 1H, J ) 8.8 Hz), 4.4 (bs, 1H), 3.11 (s, 6H), 2.65 (m,
1H), 1.65-1.35 (m, 5H), 1.15-0.95 (m, 2H), 0.85-0.55 (m, 3H).
(6) Bruns, R. F. Role of adenosine in energy supply/demand balance.
Nucleosides Nucleotides 1991, 10, 931-43.
(7) Miller, L. P.; Hsu, C. Therapeutic potential for adenosine
receptor activation in ischemic brain injury. J . Neurotrauma
1992, 9, Suppl 2 S563-77.
(8) Moser, G. H.; Schrader, J .; Duessen, A. Turnover of adenosine
in plasma of human and dog blood. Am. J . Physiol. 1989, 25,
799-806.
(9) J arvis, M. F.; Yu, H.; Kohlhaas, K.; Alexander, K.; Lee, C.-H.;
J iang, M.; Bhagwat, S. S.; Williams, M.; Kowaluk, E. A. ABT
702 (4-amino-5- (3-bromophenyl)-7-(6-morpholinopyridin-3-yl)
MS (DCI/NH3): m/z 503/505 (M + H)+. Anal. Calcd (C26H27
BrN6‚2HCl): C, H, N.
-

4-Aminopyrido[2,3-d]pyrimidine Inhibitors of Adenosine Kinase J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 24 5257
pyrido [2,3,-d] pyrimidine), a novel orally effective adenosine
kinase inhibitor with analgesic and antiinflammatory properties.
I. In vitro characterization and acute antinociceptive effects in
the mouse. J . Pharmacol. Exp. Ther. 2000, 295, 1156-64.
(10) Lee, C.-H.; J iang M.; Cowart, M.; Gfesser, G.; Perner, R.; Kim,
K. H.; Gu, Y. G.; Williams, M.; J arvis, M. F.; Kowaluk, E. A.;
Stewart, A. O.; Bhagwat, S. S. Discovery of 4-amino-5-(3-
bromophenyl)-7-(6-morpholinopyridin-3-yl)pyrido[2,3-d]pyrimi-
dine, an orally active, nonnucleoside adenosine kinase inhibitor.
J . Med. Chem. 2001, 44, 2133-2138.
(11) Nahm, S.; Weinreb, S. M. N-Methoxy-N-methylamides as effec-
tive acylating agents. Tetrahedron Lett. 1981, 22, 3815-18.
(12) Bastus, J . B. The reaction of diazomethane with double bonds.
III. A stepwise and improved Buchner-Curtius-Schlottenbeck
reaction. Tetrahedron Lett. 1963, 4, 955-8.
(13) Kowaluk, E. A.; Mikusa, J .; Wismer, C.; Zhu, C.; Alexander, K.;
Kohlhaas, K.; Lee, C.-H.; J iang, M.; Bhagwat, S. S.; Williams,
M.; J arvis, M. F. ABT-702 (4-amino-5- (3-bromophenyl)-7-(6-
morpholino-pyridin-3-yl) pyrido [2,3,-d]pyrimidine): a novel
orally effective adenosine kinase inhibitor with analgesic and
antiinflammatory properties. II. In vivo characterization in the
rat. J . Pharmacol. Exp. Ther. 2000, 295, 1165-74.
(14) Zheng, G. Z.; Lee, C.-H.; Pratt, J . K.; Perner, R. J .; J iang, M.;
Gomtsyan, A.; Matulenko, M. A.; Mao, Y.; Koenig, J . R.; Kim,
K. H.; Muchmore, S.; Yu, H.; Kohlhaas, K.; Alexander, K. M.;
McGaraughty, S.; Chu, K.; Wismer, C. T.; Mikusa, J .; J arvis,
M. F.; Marsh, K.; Kowaluk, E. A.; Bhagwat, S. S.; Stewart, A.
O. Pyridopyrimidine analogs as novel adenosine kinase inhibi-
tors. Bioorg. Med. Chem. Lett. 2001, 11, 2071-74.
(15) Kowaluk, E. A.; Kohlhaas, K. L.; Bannon, A.; Gunther, K.; Lynch,
J . J ., III; J arvis, M. F. Characterization of the effects of
adenosine kinase inhibitors on acute thermal nociception in
mice. Pharmacol. Biochem. Behav. 1999, 63, 83-91.
J M030327L