Discovery of 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido [2,3-d]pyrimidine, an orally active, non-nucleoside adenosine kinase inhibitor

Article abstract of DOI:10.1021/jm000314x

Adenosine (ADO) is an endogenous homeostatic inhibitory neuromodulator that reduces cellular excitability at sites of tissue injury and inflammation. Inhibition of adenosine kinase (AK), the primary metabolic enzyme for ADO, selectively increases ADO concentrations at sites of tissue trauma and enhances the analgesic and antiinflammatory actions of ADO. Optimization of the high-throughput screening lead, 4-amino-7-aryl-substituted pteridine (5) (AK IC₅₀ = 440 nM), led to the identification of compound 21 (4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido [2,3-d]pyrimidine, ABT-702), a novel, potent (AK IC₅₀ = 1.7 nM) nonnucleoside AK inhibitor with oral activity in animal models of pain and inflammation.

Full text of DOI:10.1021/jm000314x

J . Med. Chem. 2001, 44, 2133-2138
2133
Discover y of 4-Am in o-5-(3-br om op h en yl)-7-(6-m or p h olin o-p yr id in -
3-yl)p yr id o[2,3-d ]p yr im id in e, a n Or a lly Active, Non -Nu cleosid e Ad en osin e
Kin a se In h ibitor
Chih-Hung Lee,* Meiqun J iang, Marlon Cowart, Greg Gfesser, Richard Perner, Ki Hwan Kim, Yu Gui Gu,
Michael Williams, Michael F. J arvis, Elizabeth A. Kowaluk, Andrew O. Stewart, and Shripad S. Bhagwat†
Neurological and Urological Diseases Research, Global Pharmaceutical Research and Development, Abbott Laboratories,
Abbott Park, Illinois 60064-6125
Received J uly 24, 2000
Adenosine (ADO) is an endogenous homeostatic inhibitory neuromodulator that reduces cellular
excitability at sites of tissue injury and inflammation. Inhibition of adenosine kinase (AK),
the primary metabolic enzyme for ADO, selectively increases ADO concentrations at sites of
tissue trauma and enhances the analgesic and antiinflammatory actions of ADO. Optimization
of the high-throughput screening lead, 4-amino-7-aryl-substituted pteridine (5) (AK IC50 ) 440
nM), led to the identification of compound 21 (4-amino-5-(3-bromophenyl)-7-(6-morpholino-
pyridin-3-yl)pyrido [2,3-d]pyrimidine, ABT-702), a novel, potent (AK IC50 ) 1.7 nM) non-
nucleoside AK inhibitor with oral activity in animal models of pain and inflammation.
In tr od u ction
413 (Figure 1). The present article describes the opti-
mization of a novel non-nucleoside AK inhibitor, com-
pound 5, identified as a high-throughput screening lead,
to yield 4-amino-5-(3-bromophenyl)-7-(6-morpholino-py-
Extracellular concentrations of the endogenous neu-
romodulator, adenosine (ADO), are increased under
conditions of metabolic stress and trauma (e.g., pain,
inflammation, tissue damage, ischemia, seizure activity,
etc.) and act to limit tissue damage and restore normal
function1 by activating members of the P1 receptor
family, A1, A2A, A2B, and A3 receptors.2 Increased extra-
cellular ADO concentrations can result in the inhibition
of excitatory amino acid (glutamate) release, suppres-
sion of free radical formation, and neutrophil adhesion,
depending on the phenotype of the tissue involved.3
Since ADO has a half-life on the order of seconds in
physiological fluids,4 its extracellular actions are re-
stricted to the tissue and cellular sites where it is
released.5 The effects of extracellular ADO are termi-
nated by its reuptake and phosphorylation by ADO
kinase (AK; ATP: adenosine 5′-phosphotranferase, EC
2.7.1.20) and via deamination by adenosine deaminase
(ADA).6 By preventing ADO phosphorylation, AK inhi-
bition increases intracellular ADO concentrations, alter-
ing the equilibrium of the bidirectional transport sys-
tems responsible for ADO reuptake with the net effect
of increasing the local concentration of ADO in the
extracellular compartment.7 AK inhibition is a more
effective mechanism for increasing extracellular ADO
levels than inhibition of the catabolic enzyme ADA, as
shown by the comparative efficacy of AK and ADA
inhibitors in reducing seizure and nociceptive activity
in vivo.8,9
ridin-3-yl)pyrido[2,3-d]pyrimidine 21, a potent (IC50
)
1.7 nM) and selective AK inhibitor with oral activity in
animal models of pain and inflammation.
Ch em istr y
Compounds 5-10 were prepared from the condensa-
tion of 4,5,6-triamonopyrimidine and phenylglyoxals in
refluxing 1,4-dioxane as previously described14 and
which is shown in Scheme 1. The synthesis of 6-substi-
tuted pteridine 11 was accomplished using similar
synthetic methods as illustrated in Scheme 1. The
diketone was prepared by oxidation of the corresponding
substituted acetophenone by selenium oxide in refluxing
1,4-dioxane.
Synthesis of the 6-substituted pyridopyrimidine ana-
logues (12-16) was prepared through iodination of 4,6-
diaminopyrimidine in DMF to yield 4,6-diamino-5-
iodopyrimidne and followed by Suzuki coupling reaction
with substituted vinylboronic acid to yield 4,6-diamino-
5-vinylpyrimidine. The final cyclization process was
furnished by aza-Cope rearrangement of 4,6-diamino-
5-vinylpyrimidine and benzaldehyde in diphenyl ether
to yield 6-substituted pyridopyrimidine analogues, which
is illustrated by Scheme 2.
The 4-amino-5-(3-bromophenyl)-7-(6-morpholino-py-
ridin-3-yl)pyrido[2,3-d]pyrimidine (21) was prepared
from the condensation of 3-bromobenzylidene-malono-
nitrile with acetopyridine and ammonium acetate to
produce the amino cyano pyridine intermediate 22. The
final cyclization of 22 to the product 21 was furnished
All published AK inhibitors are purine nucleoside
analogues and include 5′-deoxy-5′-amino-ADO 1,10 5′-
deoxy-5-iodotubercidin 2,11 clitocine 3,12 and GP-3269
15
by heating in formamide (Scheme 3).
* Correspondence: Dr. Chih-Hung Lee, D-4PM, AP9A, Room L-15,
Neurological and Urological Diseases Research, Pharmaceutical Prod-
ucts Division, Abbott Laboratories, Abbott Park, IL 60064-6125. Ph:
847-938-1849. Fax: 847-937-9195. E-mail: lance.lee@abbott.com.
† Present address: Drug Discovery, Signal Pharmaceuticals, 555
Oberlin Drive #100, San Diego, CA 92121.
Biology
AK inhibition was measured at 23 °C in a 100 µL
reaction mixture in triplicate containing 64 mM Tris
10.1021/jm000314x CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/26/2001

2134 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Lee et al.
Sch em e 3
described.16 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 radioactivity was
determined by standard scintillation spectrometry. A
96-well plate high-throughput screening assay was
derived from this methodology using [3H]-6-methyl-
mercaptopurineribofuranoside (Amersham, Arlington
Heights, IL) as an AK substrate that is not susceptible
to catabolism by ADO deaminase. Assays for ADO
phosphorylation in intact cells were conducted using
confluent IMR-32 human neuroblastoma cells (ATCC,
Gaithersburg, 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 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. Nociceptive paw
flinching in rats (Formalin test) was assessed 30 min
following an intraplantar injection of 5% formalin (50
µL) into the right hind paw.17
F igu r e 1. Structures of nucleoside AK inhibitors and 21
(ABT-702).
Sch em e 1a
a
Reagents and conditions for A: (a) dioxane/H2O/∆. For B: (a)
SeO2/dioxane/∆.
Sch em e 2a
Resu lts a n d Discu ssion
The non-nucleoside AK inhibitor 5 was identified as
the result of screening the Abbott compound library for
inhibition of AK. Compound 5 inhibited AK activity
(IC50 ) 440 nM, Table 1) but was considerably weaker
in inhibiting ADO phosphorylation in intact cells (IC50
) 3600 nM), indicating poor ability to penetrate the cell
membrane. Initial SAR studies on 5 replacing the
dimethylamino group with different functional groups
failed to improve AK inhibitory activity. For example,
4-H (6), 4-NH2 (7), 4-NO2 (8), 4-OCH3 (9), and 4-mor-
pholino (10) showed either weaker or similar in vitro
potency compared to 5 (Table 1). However, phenyl
substitution in the 6-position of 5 resulted in a greater
than 15-fold increase in in vitro activity (Table 1,
compound 11, IC50 ) 25 nM). Given that compound 11
showed improved AK inhibitory activity relative to 5,
comparison of 5 and 11 with the known nucleoside AK
inhibitor, 5′deoxy-5-iodotubercidin 2, suggested that the
6-phenyl ring of 11 might overlap with the lipophilic
a
Reagents and conditions: (a) I2/DMF; (b) Pd(PPh3)4/DMF; (c)
PhOPh/∆; (d) air.
HCl (pH 7.5), 0.2 mM MgCl2, 1 mM ATP, 0.2 µM
[U-14C]-adenosine or [2-3H]-adenosine (Amersham In-
ternational, Bucks, U.K.), and appropriate volumes of
rat brain cytosol as a source of AK as previously

Orally Active, Non-Nucleoside AK Inhibitor
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2135
Ta ble 1. In Vitro Biological Activity of ADO Kinase Inhibitorsa
a
All values are the mean ( SEM of at least three separate observations run in triplicate.
iodo substituent of 2 (Figures 2 and 3). Replacement of
the 5-position nitrogen of 5 with carbon was considered
an alternative scaffold to the pteridine pharmacophore
and had the potential to offer better access to the iodo
lipophilic pocket defined by compound 2 (Figure 3).
Further SAR investigation between the 6-substituted
pridopyrimidines 12-16 and the 5-substituted pyri-
dopyrimidines 18-20 suggested that these compounds
interacted with AK in similar relative orientations.
However, 5-substituted pyridopyrimidines showed a
greater than 80-fold improvement in in vitro potency
as compared to 5 and were at least 10-fold more active
as AK inhibitors as compared to the 6-substitutents
analogues. These data indicate that the 5-position
substituted pyridopyrimidines offered improved align-
ment relative to 2 as compared to the 6-position
substitutions (Figure 4). That the 5-substituent provided
an additional lipophilic interaction was confirmed by the
increased AK inhibitor potency of the 5-cyclohexyl
derivative 18 as compared to 13. Extensive SAR studies
using the 4-amino-7-(4-dimethylaminophenyl)-pyridopy-
rimidine core led to the identification of the 5-(3-
bromophenyl) analogue 19 as an optimal substituent to
improve in vitro potency with high affinity to inhibit

2136 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Lee et al.
ring was considered to be the possible cause of the
metabolic liability of compound 20 (t1/2 ) 0.4 h after iv
dosing). Additional SAR was focused on the amino
substituent of the pyridyl ring with the finding that the
morpholine analogue 21 had significantly enhanced in
vivo activity (formalin induced nociception ED50 ) 60
µmol/kg, po)17 and the plasma half-life was extended to
1 h after iv dosing (data not shown).
4-Amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-
3-yl)pyrido[2,3-d]pyrimidine 21 was active both in in-
hibiting AK (IC50 ) 1.7 nM) and ADO phosphorylation
in the intact cells (IC50 ) 50 nM) (Table 1). Compound
21 was also highly selective for AK inhibition as
compared to other sites of ADO action including ADA,
ADO receptors, and ADO transport sites.16 Compound
21 had dose-dependent antinociceptive and antiinflam-
matory actions in a variety of animal models of nocice-
ptive, inflammatory, and neuropathic pain.16, 17
In conclusion, compound 21 is the first of a novel class
of potent, selective, non-nucleoside, orally active AK
inhibitors that have potent antinociceptive effects in
animal models.
F igu r e 2. Superposition of compounds 2 (green) and 5
(yellow).
Exp er im en ta l Section
Melting points (uncorrected) were determined in open
capillary tubes with a Buchi 510 apparatus. 1H NMR was
measured on X-300 Hz spectrometers, using tetramethylsilane
as internal standard. Positive ion fast atom bombardment
(FAB), electron spray ionization (ESI), or dissolvable chemical
ionization (DCI) were obtained on Finnigin-4000 instruments.
High-resolution mass spectra were measured on a Finnigin-
4000 spectrometer. Flash column chromatography was carried
out on EM science silica gel 66 (230-400 mesh). Elemental
combustion analyses were within (0.4% of theoretical values
and obtained from Roberson Microlit Laboratories, Inc. The
following abbreviations are used in the Experimental Sec-
tion: THF for tetrahydrofuan, CDCl3 for deuteriochloroform,
DMSO-d6 for deuteriodimethyl sulfoxide, CH2Cl2 for methylene
chloride, DMF for N,N-dimethylformamide, and TFA for
trifluoroacetic acid. Reactions were routinely conducted under
inert gas (N2) unless otherwise indicated.
F igu r e 3. Superposition of compounds 2 and 11 (yellow).
General procedures for compounds 5-11 were followed as
described in ref 14.
Gen er a l P r oced u r es for Com p ou n d s 13-16: 5-Iod o-
4,6-d ia m in op yr im id in e. 4,6-Diaminopyrimidine hemisulfate
monohydrate (26.13 g, 147.5 mmol) and K2CO3 (30.58 g, 221.3
mmol) were suspended in water (400 mL), and iodine (41.19g,
162.3 mmol) and DMF (100 mL) were added. The solution was
heated at 45 °C for 23 h. After cooling, a 2 M solution of
Na2S2O3 (15 mL) was added, and the solution turned clear.
The white solid was then collected by filtration and washed
with water (3 × 20 mL), and the solid was dried under high
vacuum to yield 33.1 g of product (90%): 1H NMR (300 MHz,
DMSO-d6) d; MS (DCI/NH3) m/z 237 (M + H)+.
p-Meth yl-styr ylbor on ic Acid . 4-Ethynyl-4-methyl-ben-
zene (5 mmol) was dissolved in 5 mL of dry THF, and
catecholborane (5 mL, 1 M in THF) was added dropwise at 0
°C. The solution was heated to reflux for 1.5 h, and the solvent
was removed under vacuum. The solution was quenched with
1 M HCl (10 mL), and this solution was used directly for the
next step.
5-(p-Meth yl-styr yl)-4,6-d ia m in op yr im id in e. To a solu-
tion of 5-iodo-4,6-diaminopyrimidine (1 mmol) in 50 mL of
dioxane (or DME) were added substituted boronic acid (5
mmol), 5% of Pd(PPh3)4, and 1 M Na2CO3 (10 mL). The reaction
mixture was heated at 80 °C for 12 h. After workup, the crude
mixture was purified by column chromatography (using 5%
MeOH/CH2Cl2 as eluent) to give the product (yield 45-86%).
1H NMR (300 MHz, DMSO-d6) δ 7.75 (s, 1H), 7.6 (d, 2 H, J )
7.5 Hz), 7.4 (d, 2 H, J ) 7.5 Hz), 6.9 (q, 2 H, J ) 16.5, 15 Hz),
2.45 (s, 3 H); MS (DCI/NH3) m/z 215 (M + H)+.
F igu r e 4. Superposition of compounds 2 and 19 (yellow).
enzymatic AK activity and to inhibit ADO phosphory-
lation in intact cells (Table 1). Compound 19 also dose
dependently reduced nociception in the persistent phase
of the formalin test (ED50 ) 1 µmol/kg; ip).17 However,
compound 19 was not active following oral administra-
tion. In an attempt to improve oral activity, heteroatoms
were introduced into the 7-phenyl ring. The 7-pyridyl
analogue 20, like 19, potently inhibited AK and ADO
phosphorylation in intact cells (Table 1); however, oral
analgesic activity was not improved (21% reduction of
formalin-induced nociception, 100 µmol/kg, po). Dealky-
lation of the dimethylamino substituent on the 7-aryl

Orally Active, Non-Nucleoside AK Inhibitor
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2137
4-Am in o-6-(4-m e t h ylp h e n yl)-7-(4-(d im e t h yla m in o)-
p h en yl)p yr id o[2,3-d ]p yr im id in e (13). 4,6-Diamino-5-(2-
phenylethenyl)pyrimidine (150 mg) was suspended in 10 mL
of phenylether with 1.2 equiv of 4-(dimethylamino)benzalde-
hyde and 1.5 g of 4 Å molecular sieves. The solution was heated
to 170 °C for 4 h and then cooled to room temperature. The
reaction mixture was loaded on the top of a silica gel column
and eluted with hexane to remove phenylether followed by 5%
MeOH/CH2Cl2 as eluent to give 130 mg of the product. 1H
NMR (300 MHz, CDCl3) δ 8.75 (s, 1 H), 7.98 (s, 1 H), 7.5 (d, 2
H, J ) 9 Hz), 7.18 (m, 4 H), 6.55 (d, 2H, J ) 9 Hz), 5.9 (bs,
2H), 2.8 (s, 6H), 2.49 (s, 3H); MS (DCI/NH3) m/z (M + NH4)+,
156 (M + H)+. Anal. Calcd for C22H21N5: C, 74.34; H, 5.96; N,
19.7. Found: 74.52; H, 5.84; N, 19.7.
6-Ch lor o-3-a cetylp yr id in e. As per the procedure de-
scribed in Tetrahedron 1992, 48, 9233, we have prepared 100
g of this material. Triethylamine (EM Chemical Company,
183.2 g, 1.8 mol) followed by dimethyl malonate (Aldrich
Chemical Company, 119 g, 0.9 mol) were added to a 2 L three-
neck round-bottom flask with mechanical stirring containing
magnesium chloride (Aldrich Chemical Company, 51 g, 0.53
mol) in dry tolene (700 mL). The resulting gray and hetero-
geneous mixture was stirred at 25 °C for 1.5 h, and then
6-chloronicotinic acid chloride (Lancaster Ltd., 134 g, 0.75 mol)
was added in the solid form slowly in small portions over 45
min. Stirring was continued for 40 min before concentrated
HCl (261.0 g, 2.32 mol) was carefully added to quench the
reaction. The toluene layer was separated and removed in
vacuum to give a white needle-like solid. The solid was directly
treated with DMSO (650 mL) and water (29 mL). The mixture
was heated at 155 °C for approximately 3 h and cooled. It was
then quenched with water, and the solid was collected by
filtration. The solid was redissolved in CH2Cl2 and dried over
MgSO4, and the solvent was removed under vacuum to give
the product as a white solid 94 g (80% yield). 1H NMR (300
MHz, CDCl3) δ 8.94 (dd, 1 H, J ) 2.5, 0.7 Hz), 8.20 (dd, 1 H,
J ) 8.4, 2.6 Hz), 7.45 (dd, 1 H, J ) 8.1, 0.7 Hz), 2.62 (s, 3 H);
13C NMR (75 MHz, CDCl3) δ 195.3, 155.6, 150.1, 138.0, 131.1,
124.5, 26.6; MS (DCI/NH3) m/z 173 (M + NH4)+, 156 (M + H)+.
dine was suspended in formamide (25 mL). The reaction was
heated to reflux (using a heating mantel). After 1-2 h of
refluxing (usually TLC monitoring is necessary: product Rf
at 0.4-0.5 and SM at 0.7 with 10% MeOH/CH2Cl2), the
reaction mixture was cooled to room temperature, and the
reaction mixture was quenched with water to give a brown
solid. The solid was then filtered and collected. There was
product still remaining in the mother liquor. The solid was
then purified by column chromatography using 10% MeOH/
CH2Cl2 as eluent to give the desired product (1.2 g as a yellow
solid, 31% yield). Futher purification of this compound by flash
column chromatography using 5% H2O/CH3CN as eluent (Rf
0.3) gave 98% (HPLC: 35% 0.1 M NH4OAc/65% CH3CN) pure
product in 89% yield. 1H NMR (300 MHz, DMSO-d6) δ 9.08
(d, 1 H, J ) 2.4 Hz), 8.53 (s, 1 H), 8.47 (dd, 1 H, J ) 9.2, 2.4
Hz), 7.85 (m, 2 H), 7.78 (m, 1 H), 7.56 (m, 2 H), 6.98 (d, 1 H,
J ) 9.1 Hz), 3.72 (m, 4 H), 3.62 (m, 4 H); 13C NMR (100 MHz,
DMSO-d6) δ 161.9, 159.7, 159.6, 158.8, 158.1, 148.0, 147.6,
140.4, 136.5, 132.0, 131.3, 130.9, 127.9, 122.2, 122.1, 119.1,
106.3, 104.5, 65.9, 44.7. HRMS calcd for C22H2079BrN6O,
463.0882; observed, 463.0888. Anal. Calcd for C22H19BrN6O‚
0.5H2NC(O)H: C, 55.62; H, 4.25; N, 18.74. Found: C, 55.43;
H, 4.27; N, 18.76.
Ack n ow led gm en t. We thank H. Yu, K. Alexander,
K. Kohlhaas, J . Mikusa, C. Wismer, and C. Zhu for the
in vitro and in vivo biological screening data. We also
thank the high-throughput screening group of the
Advanced Technology Research Division.
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1
yield. H NMR (300 MHz, CDCl3) δ 8.75 (d, 1 H, J ) 2.6 Hz),
8.03 (dd, 1 H, J ) 9.2, 2.6 Hz), 6.61 (d, 1 H, J ) 9.2 Hz), 3.80
(m, 4 H), 3.67 (m, 4 H), 2.50 (s, 3 H); 13C NMR (75 MHz, CDCl3)
δ 195.1, 160.4, 150.5, 137.1, 123.0, 105.3, 66.4, 44.8, 25.8; MS
(DCI/NH3) m/z 207 (M + H)+.
Gen er a l P r oced u r es for Com p ou n d s 17-21: 2-Am in o-
3-cya n o-4-(3-b r om op h en yl)-6-(4-(2-m or p h lin oop yr id o)-
p yr id in e. In
a Dean-Stark apparatus, 6-morpholinyl-3-
acetylpyridine (6.95 g, 33.7 mmol), m-bromo-benzaldehyde
(6.24g, 33.7 mmol), malononitrile (2.22 g, 33.7 mmol), and
ammonium acetate (4.2 g) were combined in 40 mL of benzene
and heated to reflux for 3 h. After cooling, the solvent was
removed under vacuum to give a dark solid. The solid was
triturated with methanol, filtered, and washed with methanol
twice (20 mL) to give a light yellow solid 4.85 g (33% yield).
1H NMR (300 MHz, DMSO-d6) δ 8.94 (d, 1 H, J ) 2.0 Hz),
8.29 (dd, 1 H, J ) 9.1, 2.7 Hz), 7.86 (dd, 1 H, J ) 1.7, 1.7 Hz),
7.71 (m, 2 H), 7.51 (dd, 1 H, J ) 7.8, 7.8 Hz), 7.27 (s, 1 H),
6.94 (m, 3 H), 3.71 (m, 4 H), 3.59 (m, 4 H); 13C NMR (100 MHz,
DMSO-d6) δ 160.7, 159.4, 157.2, 152.7, 147.6, 139.3, 136.2,
132.2, 130.9, 130.7, 127.4, 122.2, 121.8, 117.0, 107.7, 106.1,
85.2, 65.9, 44.7; MS (DCI/NH3) m/ z 436/438 (M + H)+. TLC
using 5% MeOH/CH2Cl2 as eluent: Rf ∼ 0.65). The product
TLC produced a light blue, highly fluorescent spot.
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