Pyrido[2,3-d]pyrimidin-7-one inhibitors of cyclin-dependent kinases

Article abstract of DOI:10.1021/jm000271k

The identification of 8-ethyl-2-phenylamino-8H-pyrido [2,3-d]pyrimidin-7-one (1) as an inhibitor of Cdk4 led to the initiation of a program to evaluate related pyrido [2,3-d]pyrimidin-7-ones for inhibition of cyclin-dependent kinases (Cdks). Analysis of m

Full text of DOI:10.1021/jm000271k

4606
J . Med. Chem. 2000, 43, 4606-4616
P yr id o[2,3-d ]p yr im id in -7-on e In h ibitor s of Cyclin -Dep en d en t Kin a ses
Mark Barvian,§ Diane H. Boschelli,§ J ennifer Cossrow,§ Ellen Dobrusin,§ Ali Fattaey,† Alex Fritsch,† David Fry,‡
Patricia Harvey,‡ Paul Keller,‡ Michelle Garrett,† Frances La,§ Wilbur Leopold,‡ Dennis McNamara,§
Maire Quin,† Susanne Trumpp-Kallmeyer,§ Peter Toogood,*,§ Zhipei Wu,§ and Erli Zhang§
Departments of Chemistry and Cancer Research, Parke-Davis Pharmaceutical Research,
Division of Warner Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan 48105, and Onyx Pharmaceuticals,
3031 Research Drive, Richmond, California 94806
Received J une 22, 2000
The identification of 8-ethyl-2-phenylamino-8H-pyrido[2,3-d]pyrimidin-7-one (1) as an inhibitor
of Cdk4 led to the initiation of a program to evaluate related pyrido[2,3-d]pyrimidin-7-ones for
inhibition of cyclin-dependent kinases (Cdks). Analysis of more than 60 analogues has identified
some clear SAR trends that may be exploited in the design of more potent Cdk inhibitors. The
most potent Cdk4 inhibitors reported in this study inhibit Cdk4 with IC50 ) 0.004 µM ([ATP]
= 25 µM). X-ray crystallographic analysis of representative compounds bound to the related
kinase, Cdk2, reveals that they occupy the ATP binding site. Modest selectivity between Cdks
is exhibited by some compounds, and Cdk4-selective inhibitors block pRb+ cells in the G1-
phase of the cell division cycle.
In tr od u ction
venting cell proliferation.3 Indeed, the endogenous Cdk
inhibitor proteins of the INK4A (e.g. p16) and CIP/KIP
families (e.g. p27) inhibit G1 cyclin/Cdk complexes and
cause cells to accumulate in late G1-phase.
Uncontrolled cell proliferation is a hallmark of tum-
origenesis and cancer as well as other disorders such
as restenosis. In normal tissues, eukaryotic cell prolif-
eration and cell division are highly regulated. Cells
traverse a series of coordinated events known col-
lectively as the cell cycle. This cell cycle is generally
regarded as comprising four primary phases known as
G1, S, G2, and M. DNA replication occurs during
S-phase, and mitosis (cell division) occurs in the sub-
sequent M-phase. Transition between these four phases
is controlled by the action of cyclin-dependent kinases
(Cdks). These serine-threonine kinases are activated by
regulatory units called cyclins. Cyclin levels fluctuate
during the cell cycle in response to a variety of extra-
cellular and intracellular signals, and Cdk activities are
turned on and off in response to raised and lowered
cyclin levels.1
At least 9 Cdks and 16 cyclins have been described
in the literature.2 The essential enzymes for cell cycle
control appear to be cyclin D/Cdk4, cyclin D/Cdk6, cyclin
E/Cdk2, cyclin A/Cdk2, and cyclin B/Cdk1 (also known
as Cdc2/cyclin B). Cyclin D/Cdk4, cyclin D/Cdk6, and
cyclin E/Cdk2 control passage through the G1-phase and
the G1- to S-phase transition by phosphorylation of the
retinoblastoma phosphoprotein, pRb. Cyclin A/Cdk2
regulates passage through the S-phase, and cyclin
B/Cdk1 controls the G2 checkpoint and regulates entry
into mitosis. The activation of G1 cyclins such as cyclin
D occurs in response to growth factors. Once cells exit
the G1-phase and enter S-phase they are committed to
cell division. Thus, inhibition of cells in the G1-phase
would appear to be an important mechanism for pre-
Further support for the importance of G1 cyclin/Cdk
enzymes may be found by genetic analysis of human
tumor cell lines. Greater than 90% of all human tumors
analyzed exhibit aberrant up-regulation of the Cdk4/
Rb pathway including: overexpression of cyclin D1,
mutation of Cdk4, mutation of Rb, or deletion of p16.4
These observations argue strongly that restoration of
cyclin D/Cdk activity to “normal” by use of a specific Cdk
inhibitor might counteract the tumorigenic effect of
misregulation in this pathway and provide a treatment
for disorders characterized by cell proliferation.
Encouraged by the prior discovery of EGF receptor
kinase-selective inhibitors,5 we and others set out to
identify specific Cdk inhibitors that could be used to test
the hypothesis that inhibition of cyclin D/Cdk4 will
inhibit cell proliferation. Recently, attention has shifted
more toward inhibitors of cyclin E/cdk2 as a result of
observations that p16 may cause a G1 cell cycle block
because it displaces p27 from the cyclin D/Cdk4 com-
plex, making it available to inhibit cyclin E/Cdk2.6 The
protein p27 exhibits activity both as a Cdk inhibitor and
as a catalyst to cyclin D/Cdk4 assembly. Despite reports
of several potent Cdk inhibitors in the literature,7-10
the majority of these compounds display potent inhibi-
tion of Cdk1 and/or Cdk2. Consequently, the hypothesis
that a selective inhibitor of cyclin D/Cdk4 will inhibit
cell proliferation and ultimately tumor growth still has
not been tested. Here we describe our efforts toward this
goal with the development of selective Cdk4 inhibitors
based on the pyrido[2,3-d]pyrimidine template.
* Corresponding author, current address: Pfizer Global Research
and Development, Ann Arbor Laboratories, 2800 Plymouth Rd., Ann
Arbor, MI 48105. Tel: (734) 622-1335. Fax: (734) 622-1407. E-mail:
peter.toogood2@pfizer.com.
In itia l Lea d Id en tifica tion
§ Department of Chemistry, Parke-Davis Pharmaceutical Research.
‡ Department of Cancer Research, Parke-Davis Pharmaceutical
Research.
Recently, our laboratories have reported a class of
highly potent tyrosine kinase inhibitors based on a
6-aryl-substituted pyrido[2,3-d]pyrimidin-7-one tem-
† Onyx Pharmaceuticals.
10.1021/jm000271k CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/11/2000

Inhibitors of Cdks
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24 4607
Sch em e 1a
a
i
(a) RNH2, Et3N, THF; (b) LiAlH4, THF; (c) MnO2, CHCl3; (d) Ph3PCdCO2Et, THF, reflux; (e) Pr2EtN, DBU, heat; (f) 8, CHCl3; (g)
R′′-NH2, heat.
Sch em e 2a
a
(a) 8, CH2Cl2; (b) aniline, heat; (c) NaH, R-I, DMF; (d) R′-NH2, (DMSO), heat.
plate.11,12 These compounds are broadly active against
several tyrosine kinases including EGFr, FGFr, PDGFr,
and c-Src but are inactive against Cdks. A model for
the binding mode of these inhibitors bound to tyrosine
kinases was proposed using homology-derived three-
dimensional models and inhibitor SAR data.13 In this
model, the pyrido[2,3-d]pyrimidin-7-one forms two hy-
drogen bonds to Met-341 of cSrc kinase and the 6-aryl
substituent is located in a deep pocket adjacent to the
ATP binding site. In EGFr, FGFr, PDGFr, and cSrc
kinases this pocket is relatively large. In contrast, in
Cdks, this pocket is occluded by bulky amino acid side
chains including the phenyl ring from Phe-80 (Cdk2
numbering). The proposed binding site model suggested
that pyrido[2,3-d]pyrimidines lacking a 6-aryl substitu-
ent would be able to bind in the ATP binding site of
Cdks. This hypothesis was confirmed with the discovery
that compound 1 (Table 1) inhibits cyclin D/Cdk4 with
an IC50 ) 0.62 µM. In addition, compound 1 exhibits
some selectivity for Cdks vs other kinases including
FGFr (IC50 ) 3.7 µM) and cSrc (IC50 ) 21 µM). With
this encouraging result in hand, we embarked upon a
full investigation of the SAR of the C6-hydrogen pyrido-
[2,3-d]pyrimidin-7-one template in order to identify
more selective and more potent Cdk4 inhibitors.
condensation of commercially available 4-chloro-2-me-
thylthio-5-pyrimidinecarboxylic acid ethyl ester (2) with
a primary alkylamine in THF containing triethylamine
provided intermediates with structure 3.11 It was found
that reduction of the ester using lithium aluminum
hydride to alcohol 4, followed by reoxidation with MnO2,
provided aldehyde 5 more reliably than the correspond-
ing one-step reduction. Reaction of the aldehyde with
(carbethoxymethylene)triphenylphosphorane in THF
under reflux gave the undesired (E)-acrylates 6, which
could be conveniently isomerized with ring closure to
provide the pyrido[2,3-d]pyrimidine core molecules 7 by
heating in the presence of DBU. Oxidation of the methyl
sulfide in compounds 7 with (()-trans-2-(phenylsulfo-
nyl)-3-phenyloxaziridine (8)14 or m-chloroperbenzoic acid
provided the corresponding sulfoxide 9a or sulfone 9b.
Introduction of amines at the C2-position then was
achieved by heating the sulfoxide or sulfone with at
least 2 equiv of amine, in the presence or absence of
solvent, at temperatures ranging from 100 to 175 °C,
to provide the general structure 10.
When the first step of this synthesis was performed
with ammonium hydroxide instead of a primary alky-
lamine then the example of structure 7 obtained was
compound 11. This intermediate could be oxidized with
oxaziridine 8 and reacted with aniline to produce
analogue 12, which in turn was alkylated at N8 by
treatment with sodium hydride and alkyl halides to
produce compounds with the structure 13. Alternatively,
compound 11 was alkylated at N8 to give structure 7,
Ch em istr y
The pyrido[2,3-d]pyrimidin-7-one analogues used in
this study were prepared by following the general routes
illustrated in Schemes 1 and 2. As shown in Scheme 1,

4608 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24
Ta ble 1. Variation of the C2-Side Chain
Barvian et al.
then oxidized and treated with amines to produce
compounds 10.
Specific examples of compounds prepared by all three
of these routes are detailed in the Experimental Section.
Similarly, the pyrimidine N3 nitrogen could form a
hydrogen bond to the NH of Leu-83. Based on this model
it was anticipated that the N-methyl derivative 19
would be a significantly weaker inhibitor of Cdk4.
Indeed, 19 failed to inhibit Cdk4 at all concentrations
up to 40 µM.
Resu lts a n d Discu ssion
Electron-poor anilines were generally found to be
worse inhibitors of Cdk4 than the initial lead. For
example, the p-fluoroaniline (20) and m-fluoroaniline
(21) derivatives were both approximately 2-fold worse
than compound 1. The compound with a 4-aminopyri-
dine side chain at C2 (22) was similarly worse than
compound 1, while a compound possessing a pentafluo-
roethoxy substituent on the aniline ring (23) lost over
an order of magnitude of activity.
Mixed results were obtained with compounds con-
taining electron-rich anilines at C2. The Cdk4 inhibitory
activity of oxygen-substituted anilines was sensitive to
the position and nature of the oxygen substituent. For
example, the o-methoxyaniline 24 was inactive vs Cdk4.
Indeed, substitution in this position was found generally
to be detrimental to Cdk inhibitory activity. The p-
methoxyaniline 25 was equivalent to compound 1, while
Str u ctu r e-Activity P r ofile. Compound 1 possesses
an aniline at the C2-position. To investigate whether
an aromatic amine was important for potent inhibition
of Cdk4, we prepared a small number of alkylamines
(14-18) and measured their potency in purified enzyme
assays. Strikingly, the cyclohexylamine derivative 17
displayed a 5-fold drop in potency with IC50 ) 3.3 µM.
Other alkylamine derivatives fared no better with IC50
values ranging from 4.3 µM for the isopropylamine 15
to > 40 µM for the benzylamine 18 (see Table 1). These
results discouraged us from further probing alkylamine
derivatives and allowed us to focus on C2-anilines.
Additional information regarding the C2-amine func-
tion was gleaned from the N-methylaniline derivative
19. The proposed binding model for compound 1 sug-
gested that the aniline NH might form a hydrogen bond
to the carbonyl of Leu-83 in Cdk2 (Val-96 in Cdk4).

Inhibitors of Cdks
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24 4609
Ta ble 3. Optimizing for Potency vs Cyclin D/Cdk4
Ta ble 2. Variation of the N8-Side Chain
compd
R
Cdk4/D IC50 (µM)
38
1
methyl
5.480
0.620
0.145
0.553
0.190
1.495
0.297
0.159
0.210
0.047
0.182
0.038
1.667
13.500
0.940
4.150
2.860
7.800
5.000
31.000
Cdk4/D
IC50 (µM)
ethyl
compd R(a, b, or c)
R′
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
isopropyl
n-propyl
sec-butyl
n-butyl
isobutyl
isopentyl
cyclopentyl
cyclohexyl
cycloheptyl
exo-1-bicyclo[2.2.1]hept-2-yl
phenyl
cyclohexylmethyl
CH2Ph
30
57
58
59
60
61
62
63
64
65
37
66
67
68
69
70
36
71
72
R(a)
R(a)
R(a)
R(a)
R(a)
R(a)
R(b)
R(b)
R(b)
R(b)
R(b)
R(b)
R(b)
R(b)
R(b)
R(b)
R(c)
R(c)
R(c)
ethyl
0.160
0.045
0.0007
0.011
0.004
0.450
2.100
1.650
0.175
0.140
0.085
0.032
0.013
0.009
0.006
0.004
0.900
0.034
0.008
isopropyl
cyclopentyl
cyclohexyl
cycloheptyl
exo-1-bicyclo[2.2.1]hept-2-yl
2-benzyloxyethyl
CH2(CHOH)CH2OH
phenyl
cyclopropyl
ethyl
isopropyl
isopentyl
methoxymethyl
2-methoxyethyl
2-ethoxyethyl
C-oxiranylmethyl
CH2CO2Me
cyclopentyl
exo-1-bicyclo[2.2.1]hept-2-yl
cyclohexyl
ethyl
cyclopentyl
exo-1-bicyclo[2.2.1]hept-2-yl
the p-hydroxyaniline 26 was marginally better, possibly
due to partial ionization of the phenol. This trend was
reinforced by comparison of the 3,4-dimethoxyaniline 27
(IC50 ) 4.08 µM) and the 4-hydroxy-3-methoxyaniline
28 (IC50 ) 1.83 µM) which was twice as potent. Exten-
sion of the alkyl substituent appeared to be detrimental
by comparison of 29 and 25; however, the amine-
containing alkyl chain in 30 provided a significant leap
in potency with IC50 ) 0.16 µM.
Amine-substituted anilines in general proved to yield
more potent Cdk4 inhibitors than oxygen-substituted
anilines or halogenated anilines. For example, 31 was
twice as potent as 25 and 4 times as potent as 20.
Examination of compounds 32-36 indicated that this
effect was not solely electronic; constraint of the nitrogen
substituents into a six-membered heterocycle (34-36),
but not a five-membered aromatic ring (32), significantly
improved activity against Cdk4 compared to the diethy-
lamine derivative 33. A further improvement in potency
was exhibited by the N-methylpiperazine-substituted
aniline 37, which possesed an IC50 ) 0.085 µM, 7-fold
better than compound 1 and 3.5-fold more potent than
the morpholine analogue 34. Similar to 30, the tertiary
nitrogen in 37 appeared to participate in a specific
beneficial interaction with the Cdk protein, possibly by
electrostatically countering the presumed negative charge
on Asp-86 (Cdk2 numbering).
Having identified a C2-side chain that greatly im-
proved the potency of these Cdk inhibitors, attention
was turned to the N8-substituent. Clear trends rapidly
became evident regarding the SAR at this position of
the molecule (Tables 2 and 3). First, oxygenated sub-
stituents were typically poor choices for this position
(see compounds 52-56). [Note: This was true even for
groups designed to crudely mimic the ribose ring of ATP
that would occupy the same region of the Cdk active
site (e.g. 63, Table 3).] Second, it was obvious that a
fairly large and predominantly hydrophobic space was
available for occupation by the N8-substituent (e.g.
norbornyl in 48). In contrast to the C2-position, alkyl
Ta ble 4. Selectivity of Cdk Inhibitors
IC50 (µM)
compd
Cdk1/B
Cdk2/A
Cdk2/E
Cdk4/D
FGFr
1
23
40
42
66
70
72
1.015
0.091
0.299
2.000
0.675
0.079
>40
0.129
0.238
0.050
0.037
0.058
0.015
0.209
0.41
0.620
7.833
0.553
1.495
0.032
0.004
0.008
3.295
44.200
6.600
5.290
0.070
0.051
8.62
1.200
0.112
0.058
0.270
0.020
0.165
groups generally were preferred to aryl groups at N8,
and among this set, cycloalkyl groups fared better than
simple linear or branched chain alkyl groups (compare
39-44 to 45-48). A definite trend in potency may be
observed in Table 3. With either 4-(diethylaminoethoxy)-
phenylamino or 4-(N-methylpiperazinyl)phenylamino at
C2, potency improved in going from simple alkyl to
cyclopentyl to cyclohexyl in a reasonably predictable
fashion. Compounds 60 and 70 were among the most
potent Cdk4 inhibitors prepared to date with IC50
)
0.004 µM. These compounds represent a 150-fold im-
provement in potency when compared to the initial
chemical lead (1).
En zym e Selectivity. All new Cdk inhibitors were
routinely screened against cyclin B/Cdk1, cyclin A/Cdk2,
and cyclin E/Cdk2 as well as representative tyrosine
kinases such as FGFr, c-Src, and PDGFr. In general, it
was found that the potency of our inhibitors against all
of these kinases tended to increase with increased
potency against Cdk4. For example 70 is an 8-fold more
effective Cdk4 inhibitor than 66; however, it also is more
potent against each of the other kinases examined
(Table 4). Despite this trend, 70 is 3-fold more potent
against cyclin D/Cdk4 than against cyclin A/Cdk2 and
at least an order of magnitude less potent against FGFr.
In contrast, 66 is only 2-fold more potent against Cdk4
than either cyclin A/Cdk2 or FGFr. One of the most
interesting inhibitors of Cdk4 among the compounds

4610 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24
Barvian et al.
that we have described is compound 72. This analogue
displays greater than 15-fold selectivity for cyclin
D/Cdk4 vs cyclin E/Cdk2 and is inactive vs cyclin
B/Cdk1. It is more than 1000-fold selective for cyclin
D/Cdk4 vs the tyrosine kinase FGFr (IC50 ) 8.62 µM).
In addition, 72 fails to inhibit another serine-threonine
kinase, protein kinase C, at concentrations up to 50 µM.
Further details regarding the biochemical behavior of
this compound are described in an accompanying manu-
script.15
Distinguishing between Cdk4 and Cdk2 was typically
the most difficult challenge for this class of Cdk inhibi-
tors. Indeed, several analogues are of interest precisely
because they are selective for Cdk2 vs other kinases.
Of particular note, compounds 40 and 42 display quite
respectable selectivity for Cdk2 vs Cdk1, Cdk4, or FGFr,
and compound 42 in particular is 40-fold selective for
cyclin A/Cdk2 vs cyclin D/Cdk4. A quite different
selectivity was discovered for compound 23 which is
unusual in inhibiting cyclin B/Cdk1 better than other
Cdks or FGFr. In summary, compounds have been
identified in the pyrido[2,3-d]pyrimidin-7-one class that
are selective for cyclin B/Cdk1, cyclin A/Cdk2, or cyclin
D/Cdk4 as a function of the choice of N8- and C2-
substituents. Compounds with even marginal selectivity
for cyclin D/Cdk4 in vitro were found to cause a specific
G1 block in an Rb-positive cell line (MDA MB453)
consistent with Cdk4 inhibition in cells. Overall, how-
ever, higher levels of selectivity are likely to be neces-
sary in order to draw conclusions regarding the effect
of inhibiting a single Cdk in vivo.
F igu r e 1. Electron density around compound 66 at 2.0 Å
resolution in a complex of compound 66 bound to Cdk2. The
2fo - fc electron density map was produced using Quanta and
displayed at 1.0σ.
Bin d in g of Cd k In h ibitor s to Cd k 2. The initially
proposed binding mode for pyrido[2,3-d]pyrimidin-7-
ones binding to the Cdk catalytic subunit is supported
by kinetic studies that indicate that 66 is competitive
with ATP. In addition, crystal structure data confirm
that 66 binds in the ATP site as predicted and that the
C2-side chain projects out of the binding pocket toward
solution (Figure 1). X-ray crystallographic analysis
further revealed that 66 binds to Cdk2 with nitrogen
N3 accepting a hydrogen bond from the amide nitrogen
of Leu-83 in the protein and the C2-NH donating a
hydrogen bond to the oxygen of Leu-83. Thus, although
the pyrido[2,3-d]pyrimidin-7-one ring is positioned in a
fashion similar to the purine ring of ATP bound to
cdk2,16,17 its precise hydrogen-bonding pattern is dif-
ferent. In crystal structures of Cdk2-ATP, Cdk2-stauro-
sporine, and other inhibitor-bound Cdk2 complexes,18-20
the ATP or inhibitor donates a hydrogen bond to the
main chain carbonyl oxygen of residue Glu-81, and no
such H-bond is observed with compound 66.
Exp er im en ta l Section
Gen er a l Meth od s. NaH refers to 60 wt % NaH in mineral
oil. All solvents and reagents were used as obtained. Anhy-
drous solvents were obtained commercially and used without
further drying. Melting points were determined with a Thomas-
Hoover capillary melting point apparatus or a MEL-TEMP
melting point apparatus and are uncorrected. 1H NMR spectra
were recorded using a Varian Unity 400-MHz spectrometer.
Chemical shifts are in parts per million (δ) referenced to Me4-
Si (0.00 ppm) or CHCl3 (7.20 ppm). The amount of solvent or
water present in the molecular formula was determined by
1H NMR and microanalysis. Chemical ionization mass spectra
(CI) were recorded on a VG Trio 2 mass spectrometer instru-
ment using a reagent gas of 1% NH3 in CH4. Atmospheric
pressure chemical ionization mass spectra (APCIMS) were
recorded using a VG Trio 2000 mass spectrometer in a matrix
of MeOH/MeCN/DMSO. Combustion analyses (CHN) were
determined by Robertson Microlit Laboratories, Inc., Madison,
NJ .
4-Eth yla m in o-2-m eth a n esu lfa n ylp yr im id in e-5-ca r box-
ylic Acid Eth yl Ester (3; R ) Et). To a room-temperature
solution of 4-chloro-2-methanesulfanylpyrimidine-5-carboxylic
acid ethyl ester (2; 10.00 g, 43.10 mmol) in 150 mL of
tetrahydrofuran was added triethylamine (18.5 mL, 133 mmol)
followed by 9 mL of a 70% aqueous solution of ethylamine.
The solution was stirred for 30 min then concentrated in vacuo
and partitioned between chloroform and saturated aqueous
sodium bicarbonate. The organic layer was dried over mag-
nesium sulfate, filtered, and concentrated to provide 9.32 g
(90%) of 4-ethylamino-2-methanesulfanylpyrimidine-5-carbox-
ylic acid ethyl ester as an oil: 1H NMR (DMSO-d6) δ 1.70 (t,
J ) 7 Hz, 3H), 1.30 (t, J ) 7 Hz, 3H), 2.48 (s, 3H), 3.51 (q, J
) 7 Hz, 2H), 4.27 (q, J ) 7 Hz, 2H), 8.31 (t, J ) 5 Hz, 1H),
8.53 (s, 1H); MS (CI) m/z 242 (M + 1). Anal. (C10H15N3O2S) C,
H, N.
In conclusion, a class of pyrido[2,3-d]pyrimidin-7-one
inhibitors of Cdks has been identified and some mem-
bers of this class display modest selectivity for Cdk4 vs
other kinases. In addition, representative compounds
inhibit the proliferation of human tumor cells in tissue
culture; for example, compound 72 inhibits the HCT116
human colon carcinoma cell line with IC50 ) 0.213 µM.
Such data suggests that 72 can enter cells and indicates
the potential of this class of compounds for treating
cancer in vivo. SAR analysis together with structural
data are currently being employed to facilitate the
design of even more selective and potent Cdk inhibitors.
(4-Eth ylam in o-2-m eth an esu lfan ylpyr im idin -5-yl)m eth -
a n ol (4; R ) Et). A solution of 4-ethylamino-2-methanesulfa-

Inhibitors of Cdks
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24 4611
nylpyrimidine-5-carboxylic acid ethyl ester (8.93 g, 37.1 mmol)
in 100 mL of tetrahydrofuran was added dropwise to a room
temperature suspension of lithium aluminum hydride (2.30
g, 60.5 mmol) in 100 mL of tetrahydrofuran. After 10 min, the
reaction was carefully quenched with 4.5 mL of water, 4.5 mL
of 15% NaOH, and an additional 16 mL of water, and the
mixture was stirred for 1.5 h. The white precipitate was
removed by filtration, washing with ethyl acetate. The filtrate
was concentrated in vacuo and 1:1 hexane:ethyl acetate was
added. The solids were collected to give 6.77 g (92%) of (4-
ethylamino-2-methanesulfanylpyrimidin-5-yl)methanol: mp
152-156 °C; 1H NMR (DMSO-d6) δ 1.13 (t, J ) 7 Hz, 3H),
2.41 (s, 3H), 3.39 (q, J ) 6 Hz, 2H), 4.29 (d, J ) 5 Hz, 2H),
5.09 (t, J ) 5 Hz, 1H), 6.79 (t, J ) 5 Hz, 1H), 7.82 (s, 1H); MS
(CI) m/z 200 (M + 1). Anal. (C8H13N3OS) C, H, N.
4-Eth yla m in o-2-m eth a n esu lfa n ylp yr im id in e-5-ca r box-
a ld eh yd e (5; R ) Et). To (4-ethylamino-2-methanesulfa-
nylpyrimidin-5-yl)methanol (6.44 g, 32.4 mmol) in 600 mL of
chloroform was added manganese oxide (21.0 g, 241 mmol).
The suspension was stirred at room temperature for 2 h and
an additional 5.5 g of manganese oxide was added. Stirring
was continued for 4.5 h. The mixture was filtered through
Celite, washing with chloroform. The filtrate was concentrated
in vacuo to give 6.25 g (97%) of 4-ethylamino-2-methanesulfa-
nylpyrimidine-5-carboxaldehyde: mp 58-61 °C; 1H NMR
(DMSO-d6) δ 1.17 (t, J ) 7 Hz, 3H), 2.51 (s, 3H), 3.54 (q, J )
6 Hz), 8.52 (s, 1H), 8.69 (b, 1H), 9.74 (s, 1H); MS (CI) m/z 198
(M + 1). Anal. (C8H11N3OS) C, H, N.
4-Am in o-2-m et h a n esu lfa n ylp yr im id in e-5-ca r b oxylic
Acid Eth yl Ester (3; R ) H). To a room-temperature solution
of 4-chloro-2-methanesulfanylpyrimidine-5-carboxylic acid eth-
yl ester (15.0 g, 65 mmol) in 200 mL of tetrahydrofuran was
added 25 mL of triethylamine followed by 35 mL of aqueous
ammonium hydroxide. After stirring at room temperature for
1.5 h, an additional 30 mL of aqueous ammonium hydroxide
was added, and stirring was continued for 1 h. The reaction
mixture was concentrated in vacuo and partitioned between
ethyl acetate and saturated aqueous sodium bicarbonate. The
organic layer was washed with brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. Ethyl acetate and
hexane were added, and the resultant solid was collected by
filtration to provide 10.84 g (79%) of 4-amino-2-methanesulfa-
nylpyrimidine-5-carboxylic acid ethyl ester which was used
directly without further purification.
(4-Am in o-2-m e t h a n e su lfa n ylp yr im id in -5-yl)m e t h a -
n ol (4; R ) H). A solution of 4-amino-2-methanesulfanylpy-
rimidine-5-carboxylic acid ethyl ester (13.36 g, 63 mmol) in
250 mL of tetrahydrofuran was added dropwise to a room-
temperature suspension of lithium aluminum hydride (3.82
g, 100 mmol) in 250 mL of tetrahydrofuran. After 30 min, the
reaction was cooled to 0 °C, and isopropyl alcohol was added
until bubbling diminished. The reaction was quenched with
15 mL of water, 15 mL of 15% NaOH, and 50 mL of water,
and the mixture was stirred for 1 h. The white precipitate was
removed by filtration, washing with ethyl acetate. The filtrate
was concentrated in vacuo and 3:1 hexane:ethyl acetate was
added. The solids were collected, washed with 3:1 hexane:ethyl
acetate, followed by hexane. The solid was dissolved in ethyl
acetate, and the solution was dried over magnesium sulfate.
Filtration followed by concentration in vacuo gave 8.14 g (76%)
of (4-amino-2-methanesulfanylpyrimidin-5-yl)methanol: 1H
NMR (DMSO-d6) δ 2.33 (s, 3H), 4.23 (s. 2H), 5.05 (br s, 1H),
6.63 (br s, 2H), 7.83 (s, 1H); MS (CI) m/z 172 (M + 1). Anal.
(C6H9N3OS) C, H, N.
4-Am in o-2-m eth a n esu lfa n ylp yr im id in e-5-ca r boxa ld e-
h yd e (5; R ) H). To (4-amino-2-methanesulfanylpyrimidin-
5-yl)methanol (8.14 g, 48 mmol) in 1 L of chloroform was added
manganese oxide (33.13 g, 381 mmol). The suspension was
stirred at room temperature overnight then filtered through
Celite washing with 300 mL of chloroform. The filtrate was
concentrated in vacuo to give 8.14 g (quantitative yield) of
4-amino-2-methanesulfanylpyrimidine-5-carboxaldehyde: mp
185-187 °C (lit.21 mp 183-184 °C); 1H NMR (DMSO-d6) δ 2.43
(s, 3H), 7.97 (s, 1H), 8.24 (s, 1H), 8.51 (s, 1H), 9.71 (s, 1H); MS
(CI) m/z 170 (M + 1). Anal. (C6H7N3OS) C, H, N.
Eth yl 3-(4-Am in o-2-m eth a n esu lfa n ylp yr im id in -5-yl)-
a cr yla te (6; R ) H). To a room-temperature solution of
4-amino-2-methanesulfanylpyrimidine-5-carboxaldeyde (4.08
g, 24.14 mmol) in 100 mL of tetrahydrofuran was added
(carbethoxymethylene)triphenylphosphorane (10.80 g, 31 mmol).
The reaction mixture was heated at reflux for 3 h then stirred
at room-temperature overnight. The reaction mixture was
concentrated in vacuo, and the residue was purified by flash
chromatography, eluting with 1:1 ethyl acetate:hexane, to
provide 4.30 g (75%) of ethyl 3-(4-amino-2-methanesulfanylpy-
rimidin-5-yl)acrylate: mp softens at 108 °C; 1H NMR (DMSO-
d6) δ 1.25 (t, J ) 7 Hz, 3H), 2.43 (s, 3H), 4.17 (q, J ) 7 Hz,
2H), 6.51 (d, J ) 6 Hz, 1H), 7.38 (s, 2H), 7.70 (d, J ) 6 Hz,
1H), 8.44 (s, 1H); MS (CI) m/z 240 (M + 1). Anal. (C10H13N3O2S)
C, H, N.
E t h yl 3-(4-E t h yla m in o-2-m et h a n esu lfa n ylp yr im id in -
5-yl)a cr yla te (6; R ) Et). To a room-temperature solution
of 4-ethylamino-2-methanesulfanylpyrimidine-5-carboxalde-
hyde (6.34 g, 32.14 mmol) in 100 mL of tetrahydrofuran was
added (carbethoxymethylene)triphenylphosphorane (14.32 g,
41.14 mmol). The reaction mixture was heated at reflux for
70 min. The reaction mixture was concentrated in vacuo, and
the residue was purified by flash chromatography, eluting with
ethyl acetate, to provide 7.04 g (82%) of ethyl 3-(4-ethylamino-
1
2-methanesulfanylpyrimidin-5-yl)acrylate: mp 79-80 °C; H
NMR (DMSO-d6) δ 1.15 (t, J ) 7 Hz, 3H), 1.25 (t, J ) 7 Hz,
3H), 2.45 (s, 3H), 3.42,(q, J ) 6 Hz, 2H), 4.18 (q, J ) 7 Hz,
2H), 6.50 (d, J ) 6 Hz, 1H), 7.70 (d, J ) 6 Hz, 1H), 7.78 (b,
1H), 8.37 (s, 1H); MS (CI) m/z 268 (M + 1). Anal. (C12H17N3O2S)
C, H, N.
8-Eth yl-2-m eth a n esu lfa n yl-8H-p yr id o[2,3-d ]p yr im idin -
7-on e (7; R ) Et). To a room-temperature solution of ethyl
3-(4-ethylamino-2-methanesulfanylpyrimidin-5-yl)acrylate (6.62
g, 24.78 mmol) in 30 mL of N,N-diisopropylethylamine was
added 1,8-diazabicyclo[5.4.0]undec-7-ene (4.25 mL). The reac-
tion mixture was heated at reflux overnight then cooled to
room temperature. The resultant solid was collected by filtra-
tion and washed with 1:1 hexane:ethyl acetate to give 1.83 g
(33%) of 8-ethyl-2-methanesulfanyl-8H-pyrido[2,3-d]pyrimidin-
7-one. The filtrate was concentrated in vacuo and upon the
addition of hexane, a solid formed that was collected, washed
with hexane, and purified by flash chromatography eluting
with ethyl acetate to provide an additional 2.22 g (40%) of title
product: 1H NMR (DMSO-d6) δ 1.15 (t, J ) 7 Hz, 3H), 2.53 (s,
3H), 4.25 (q, J ) 6 Hz, 2H), 6.56 (d, J ) 9 Hz, 1H), 7.86 (d, J
) 9 Hz, 1H), 8.82 (s, 1H); MS (CI) m/z 222 (M + 1). Anal.
(C10H11N3OS) C, H, N.
8-Eth yl-2-m eth a n esu lfin yl-8H-p yr id o[2,3-d ]p yr im id in -
7-on e (9a ; R ) Et). To a room-temperature solution of 8-ethyl-
2-methanesulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one (2.22 g,
10.04 mmol) in 100 mL of chloroform was added (()-trans-2-
(phenylsulfonyl)-3-phenyloxaziridine (3.17 g, 12.15 mmol). The
solution was stirred at room temperature overnight then
concentrated in vacuo. The residue was treated with ethyl
acetate to give a solid that was collected by filtration and
washed with ethyl acetate to provide 2.21 g (93%) of 8-ethyl-
2-methanesulfinyl-8H-pyrido[2,3-d]pyrimidin-7-one: mp 202-
2-Meth an esu lfan yl-8H-pyr ido[2,3-d]pyr im idin -7-on e (7;
R ) H). To a room-temperature solution of ethyl 3-(4-amino-
2-methanesulfanylpyrimidin-5-yl)acrylate (368 mg, 1.53
mmol) in 3 mL of N,N-diisopropylethylamine was added 1,8-
diazabicyclo[5.4.0]undec-7-ene (380 µL). The reaction mixture
was heated at reflux for 3 h then cooled to room temperature
and concentrated. The residue was purified by flash chroma-
tography eluting with ethyl acetate. The fractions containing
the product were partially concentrated in vacuo, and the
solids were removed by filtration to provide 134 mg (45%) of
2-methanesulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one: mp 269-
271 °C; 1H NMR (DMSO-d6) δ 2.56 (s, 3H), 6.50 (d, J ) 9 Hz,
1
203 °C; H NMR (DMSO-d6) δ 1.17 (t, J ) 7 Hz, 3H), 2.88 (s,
3H), 4.30 (q, J ) 7 Hz, 2H), 6.80 (d, J ) 9 Hz, 1H), 8.03 (d, J
) 9 Hz, 1H), 9.17 (s, 1H); MS (CI) m/z 238 (M + 1). Anal.
(C10H11N3O2S) C, H, N.

4612 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24
Barvian et al.
1H), 7.90 (d, J ) 9 Hz, 1H), 8.85 (s, 1H), 12.40 (s, 1H); MS
(CI) m/z 194 (M + 1). Anal. (C8H7N3OS) C, H, N.
NMR (DMSO-d6) δ 6.12 (d, J ) 9 Hz, 1H), 7.00 (t, J ) 7 Hz,
1H), 7.79 (d, J ) 9 Hz, 1H), 7.89 (d, J ) 7 Hz, 1H), 8.73 (s,
1H), 9.95 (s, 1H), 12.09 (s, 1H); MS (CI) m/z 239 (M + 1). Anal.
(C13H10N4O‚0.15H2O) C, H, N.
8-Eth yl-2-m eth a n esu lfa n yl-8H-p yr id o[2,3-d ]p yr im idin -
7-on e (7; R ) Et) by Alk yla tion . To a suspension of NaH
(80 mg of a 60% suspension of NaH in mineral oil) in 10 mL
of dimethylformamide was added 2-methanesulfanyl-8H-py-
rido[2,3-d]pyrimidin-7-one (262 mg, 1.35 mmol). The reaction
mixture was heated to 50 °C resulting in a brown solution.
The solution was cooled slightly and iodoethane (150 µL, 1.88
mmol) was added. The reaction was heated to 50 °C for 10
min, then cooled to room temperature and partitioned between
cold water and ethyl acetate. The organic layer was dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
residue was purified by flash chromatography, eluting with
1:1 ethyl acetate:hexane to 100% ethyl acetate, to provide the
title compound 192 mg (64%).
Gen er a l P r oced u r e for th e P r ep a r a tion of 2-Am in o-
8-eth yl-8H-p yr id o[2,3-d ]p yr im id in -7-on es fr om 8-Eth yl-
2-m eth a n esu lfin yl-8H-p yr id o[2,3-d ]p yr im id in -7-on e (9a ;
R ) Et). To 8-ethyl-2-methanesulfinyl-8H-pyrido[2,3-d]pyri-
midin-7-one was added 1-10 equiv of an amine. In those cases
where the amine was aniline, a substituted aniline, or a
tertiary amine, the reaction mixture was heated at 100-175
°C for 10-60 min. In the case of primary or secondary
alkylamines, the reaction was performed at room temperature
for 10-60 min. A typical workup involved dilution of the cooled
reaction mixture with ethyl acetate followed by an aqueous
wash with sodium bicarbonate solution. The organic layer was
dried over magnesium sulfate, filtered then evaporated to
dryness. The products were purified by crystallization from
ethyl acetate and hexanes, or by silica gel chromatography.
Yields ranged from 40-75%.
8-E t h yl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p yr im id in -7-
on e (1): mp 203-204 °C; 1H NMR (DMSO-d6) δ 1.13 (t, J ) 7
Hz, 3H), 4.25 (q, J ) 7 Hz, 2H), 6.33 (d, J ) 9 Hz, 1H), 6.97 (t,
J ) 8 H, 1H), 7.28 (t, J ) 8 Hz, 2H), 7.75 (m, 3H), 8.72 (s,
1H), 10.05 (s, 1H); MS (CI) m/z 267 (M + 1). Anal. (C15H14N4O‚
0.05EtOAc) C, H, N.
8-E t h yl-2-e t h yla m in o-8H -p yr id o[2,3-d ]p yr im id in -7-
on e (14): mp 160-161 °C; 1H NMR (DMSO-d6) δ 1.19 (m, 6H),
3.39 (q, J ) 6 Hz, 2H), 4.26 (q, J ) 7 Hz, 2H), 6.23 (d, J ) 9
Hz, 1H), 7.70 (d, J ) 9 Hz, 1H), 7.87 (s, 1H), 8.56 (s, 1H); MS
(CI) m/z 219 (M + 1). Anal. (C11H14N4O) C, H, N.
8-Isop r op yl-2-m eth a n esu lfa n yl-8H-p yr id o[2,3-d ]p yr i-
m id in -7-on e (7; R ) iP r ). To a suspension of NaH (48 mg of
a 60% suspension of NaH in mineral oil) in 6 mL of dimeth-
ylformamide was added 2-methanesulfanyl-8H-pyrido[2,3-d]-
pyrimidin-7-one (158 mg, 0.82 mmol). The reaction mixture
was heated to 50 °C resulting in a yellow solution. The solution
was cooled slightly and 2-iodopropane (120 µL, 1.20 mmol) was
added. The reaction was heated at 50 °C for 30 min then cooled
to room temperature and partitioned between water and ethyl
acetate. The organic layer was dried over magnesium sulfate,
filtered, and concentrated in vacuo. The residue was purified
by flash chromatography, eluting with a gradient of 1:3 ethyl
acetate:hexane to 100% ethyl acetate, to provide 140 mg (69%)
of 8-isopropyl-2-methanesulfanyl-8H-pyrido[2,3-d]pyrimidin-
1
7-one: mp 101-102 °C; H NMR (DMSO-d6) δ 1.54 (d, J ) 7
Hz, 6H), 2.60 (s, 3H), 5.69 (b, 1H), 6.57 (d, J ) 9 Hz, 1H), 7.88
(d, J ) 9 Hz, 1H), 8.86 (s, 1H); MS (CI) m/z 236 (M + 1). Anal.
(C11H13N3OS) C, H, N.
8-Eth yl-2-isop r op yla m in o-8H-p yr id o[2,3-d ]p yr im id in -
1
7-on e (15): mp 119-120 °C; H NMR (DMSO-d6) δ 1.20 (t, J
) 9 Hz, 3H), 2.50 (d, 6H), 4.04-4.09 (m, 1H), 4.25 (q, J ) 9
Hz, 2H), 6.23 (d, J ) 9 Hz, 1H), 7.69 (d, J ) 9 Hz, 1H), 7.68-
7.88 (m, 1H), 8.57 (s, 1H); MS (CI) m/z 233 (M + 1). Anal.
(C12H16N4O) C, H, N.
8-Isop r op yl-2-m et h a n esu lfin yl-8H -p yr id o[2,3-d ]p yr i-
i
m id in -7-on e (9a ; R ) P r ). To a room-temperature solution
of 8-isopropyl-2-methanesulfanyl-8H-pyrido[2,3-d]pyrimidin-
7-one (1.19 g, 5.08 mmol) in 50 mL of chloroform was added
(()-trans-2-(phenylsulfonyl)-3-phenyloxaziridine (1.76 g, 6.75
mmol). The solution was stirred at room temperature over-
night then concentrated in vacuo. The residue was treated with
ethyl acetate and hexane to give a solid which was collected
by filtration and purified by flash chromatography, eluting
with a gradient of ethyl acetate to 10% methanol in ethyl
acetate, to provide 1.00 g (78%) of 8-isopropyl-2-methanesulfi-
nyl-8H-pyrido[2,3-d]pyrimidin-7-one: mp 132-133 °C; 1H
NMR (DMSO-d6) δ 1.57 (d, J ) 7 Hz, 6H), 2.94 (s, 3H), 5.72
(m, 1H), 6.81 (d, J ) 9 Hz, 1H), 8.04 (d, J ) 9 Hz, 1H), 9.19 (s,
1H); MS (CI) m/z 252 (M + 1). Anal. (C11H13N3O2S) C, H, N.
2-ter t-Bu tyla m in o-8-eth yl-8H-p yr id o[2,3-d ]p yr im id in -
1
7-on e (16): mp 103-104 °C; H NMR (DMSO-d6) δ 1.19 (t, J
) 7 Hz, 3H), 1.44 (s, 9H), 4.26 (q, J ) 7 Hz, 2H), 6.24 (d, J )
9 Hz, 1H), 7.52 (br s, 1H), 7.70 (d, J ) 9 Hz, 1H), 8.58 (1H, s).
Anal. (C13H18N4O1‚0.25H2O) C, H, N.
2-Cycloh exylam in o-8-eth yl-8H-pyr ido[2,3-d]pyr im idin -
1
7-on e (17): mp 135-136 °C; H NMR (DMSO-d6) δ 1.13 (t, J
) 7 Hz, 3H), 1.24-1.93 (m, 10H), 3.67-3.77 (m, 1H), 4.18 (q,
J ) 7 Hz, 2H), 6.16 (d, J ) 9 Hz, 1H), 7.63 (d, J ) 9 Hz, 1H),
7.58-7.74 (m, 1H), 8.50 (s, 1H); MS (CI) m/z 273 (M + 1). Anal.
(C15H20N4O) C, H, N.
2-Ben zyla m in o-8-et h yl-8H -p yr id o[2,3-d ]p yr im id in -7-
on e (18): mp 96-97 °C; 1H NMR (DMSO-d6) δ 1.00 (t, J ) 7
Hz, 3H), 4.18 (q, J ) 7 Hz, 2H), 4.55 (d, J ) 8 Hz, 2H), 6.23
(d, J ) 9 Hz, 1H), 7.19-7.39 (m, 5H), 7.70 (d, J ) 9 Hz, 1H),
8.45 (b, 1H), 8.59 (s, 1H); MS (CI) m/z 281 (M + 1). Anal.
(C16H16N4O) C, H, N.
8-Eth yl-2-(m eth ylp h en yla m in o)-8H-p yr id o[2,3-d ]p yr i-
m id in -7-on e (19): mp 139-141 °C; 1H NMR (DMSO-d6) δ
1.11 (t, J ) 7 Hz, 3H), 3.56 (s, 3H), 4.13 (q, J ) 7 Hz, 2H),
6.32 (d, J ) 9 Hz, 1H), 7.27-7.29 (m, 1H), 7.40-7.44 (m, 4H),
7.77 (d, J ) 9 Hz, 1H), 8.66 (s, 1H); MS (CI) m/z 281 (M + 1).
Anal. (C16H16N4O‚0.75EtOAc) C, H, N.
2-Meth an esu lfin yl-8H-pyr ido[2,3-d]pyr im idin -7-on e (9a;
R ) H). To a room-temperature solution of 2-methanesulfanyl-
8H-pyrido[2,3-d]pyrimidin-7-one (120 mg, 0.62 mmol) in 20 mL
of chloroform was added (()-trans-2-(phenylsulfonyl)-3-phe-
nyloxaziridine (8; 200 mg, 0.77 mmol). The solution was stirred
at room temperature overnight. The solid was collected by
filtration and found to be 2-methylthio-8H-pyrido[2,3-d]pyri-
midin-7-one. The filtrate was stirred at room temperature for
2 days then concentrated. Addition of ethyl acetate resulted
in the formation of a solid that was collected by filtration to
provide 64 mg (76% based on recovered starting material) of
2-methanesulfinyl-8H-pyrido[2,3-d]pyrimidin-7-one: mp 237-
242 °C; 1H NMR (DMSO-d6) δ 2.90 (s, 3H), 6.73 (d, J ) 9 Hz,
1H), 8.05 (d, J ) 9 Hz, 1H), 9.16 (s, 1H), 12.96 (s, 1H); MS
(CI) m/z 210 (M + 1). Anal. (C8H7N3O2S‚0.2H2O) C, H, N.
8-E t h yl-2-(4-flu or op h en yla m in o)-8H-p yr id o[2,3-d ]p y-
1
r im id in -7-on e (20): mp 215-217 °C; H NMR (DMSO-d6) δ
0.24 (t, J ) 7 Hz, 3H), 4.29 (q, J ) 7 Hz, 2H), 6.39 (d, J ) 9
Hz, 1H), 7.20 (t, J ) 9 Hz, 2H), 7.80 (d, J ) 9 Hz, 1H), 7.81 (d,
J ) 9 Hz, 2H), 8.78 (s, 1H), 10.13 (s, 1H). Anal. (C15H13N4O1F1)
C, H, N.
2-P h en yla m in o-8H-p yr id o[2,3-d ]p yr im id in -7-on e (12).
A suspension of 204 mg of the mixture of 2-methanesulfinyl-
8H-pyrido[2,3-d]pyrimidin-7-one and 2-methanesulfonyl-8H-
pyrido[2,3-d]pyrimidin-7-one in 1 mL of aniline was heated
at reflux for 10 min resulting in a dark brown solution. Upon
cooling to room temperature, a solid formed. Ethyl acetate was
added, and the solid was collected by filtration, washed with
ethyl acetate, then suspended in methanol and filtered, and
washed with additional methanol to provide 175 mg of 2-phe-
8-E t h yl-2-(3-flu or op h en yla m in o)-8H-p yr id o[2,3-d ]p y-
1
r im id in -7-on e (21): mp 210-212 °C; H NMR (DMSO-d6) δ
1.27 (t, J ) 7 Hz, 3H), 4.33 (q, J ) 7 Hz, 2H), 6.43 (d, J ) 9
Hz, 1H), 6.84 (dtr, J ) 3, 9 Hz, 1H), 7.36 (q, J ) 7 Hz, 1H),
7.54 (d, J ) 7 Hz, 1H), 7.84 (d, J ) 9 Hz, 1H), 7.89 (s, 1H),
8.83 (s, 1H), 10.33 (s, 1H). Anal. (C15H13N4O1F1‚0.1H2O‚
0.1EtOAc) C, H, N.
1
nylamino-8H-pyrido[2,3-d]pyrimidin-7-one: mp >350 °C; H

Inhibitors of Cdks
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24 4613
8-Eth yl-2-(p yr id in -4-yla m in o)-8H-p yr id o[2,3-d ]p yr im i-
d in -7-on e (22): mp 259-260 °C; 1H NMR (DMSO-d6) δ 1.28
(t, J ) 7 Hz, 3H), 4.35 (q, J ) 7 Hz, 2H), 6.49 (d, J ) 9 Hz,
1H), 7.81 (q, J ) 2 Hz, 2H), 7.88 (d, J ) 9 Hz, 1H), 8.43 (q, J
) 2 Hz, 2H), 8.88 (s, 1H), 10.50 (s, 1H); MS (CI) m/z 268 (M +
1). Anal. (C14H13N5O‚0.25H2O) C, H, N.
8-Eth yl-2-[3-(1,1,2,2-tetr a flu or oeth oxy)p h en yla m in o]-
8H-p yr id o[2,3-d ]p yr im id in -7-on e (23): mp 175-176 °C; 1H
NMR (DMSO-d6) δ 1.19 (t, J ) 7 Hz, 3H), 4.32 (q, J ) 7 Hz,
2H), 6.44 (d, J ) 9 Hz, 1H), 6.70-6.97 (m, 2H), 7.43 (t, J ) 8
Hz, 1H), 7.62 (d, J ) 8 Hz, 1H), 7.85 (d, J ) 9 Hz, 1H), 8.08 (s,
1H), 8.83 (s, 1H), 10.39 (s, 1H); MS (CI) m/z 383 (M + 1). Anal.
(C17H14N4F4O2) C, H, N.
8-Eth yl-2-(2-m eth oxyp h en yla m in o)-8H-p yr id o[2,3-d ]-
p yr im id in -7-on e (24): mp 126-128 °C; 1H NMR (DMSO-d6)
δ 1.19 (t, J ) 7 Hz, 3H), 3.85 (s, 3H), 4.24 (q, J ) 7 Hz, 2H),
6.38 (d, J ) 9 Hz, 1H), 6.97-7.14 (m, 3H), 7.81 (d, J ) 9 Hz,
1H), 8.03 (d, J ) 8 Hz, 1H), 8.66 (s, 1H), 8.75 (s, 1H); MS (CI)
m/z 297 (M + 1). Anal. (C16H16N4O2‚0.2H2O) C, H, N.
8-Eth yl-2-(4-m eth oxyp h en yla m in o)-8H-p yr id o[2,3-d ]-
p yr im id in -7-on e (25): mp 196-197 °C; 1H NMR (DMSO-d6)
δ 1.23 (t, J ) 7 Hz, 3H), 3.74 (S, 3h), 4.29 (q, J ) 7 Hz, 2H),
6.35 (d, J ) 9 Hz, 1H), 6.93 (d, J ) 9 Hz, 2H), 7.70 (d, J ) 9
Hz, 2H), 7.79 (d, J ) 9 Hz, 1H), 6.73 (s, 1H), 9.95 (s, 1H); MS
(CI) m/z 297 (M + 1). Anal. (C16H16N4O2‚0.5H2O) C, H, N.
8-E t h yl-2-(4-h yd r oxyp h en yla m in o)-8H -p yr id o[2,3-d ]-
p yr im id in -7-on e (26). A mixture of compound 25 (133 mg,
0.45 mmol) and 1 mL of 48% aqueous HBr in 10 mL of
propionic acid was heated at reflux for 3 h. After cooling to
room temperature, the reaction mixture was partitioned
between ethyl acetate and saturated sodium bicarbonate. The
aqueous layer was further extracted with ethyl acetate and
the organic layers were combined and washed with brine, dried
over magnesium sulfate, filtered, and concentrated in vacuo.
The resultant solid was purified by dissolving in ethyl acetate
and passing the solution through silica gel to provide 26 (58
mg, 46%): mp 222-224 °C; 1H NMR (DMSO-d6) δ 1.21 (t, J )
7 Hz, 3H), 4.28 (q, J ) 7 Hz, 2H), 6.33 (d, J ) 9 Hz, 1H), 6.73
(d, J ) 9 Hz, 2H), 7.54 (d, J ) 8 Hz, 2H), 7.77 (d, J ) 9 Hz,
1H), 8.70 (s, 1H), 9.19 (s, 1H), 9.83 (s, 1H); MS (CI) m/z 283
(M + 1). Anal. (C15H14N4O2‚0.25H2O) C, H, N.
3H), 2.52 (q, J ) 7 Hz, 4H), 2.76 (t, J ) 6 Hz, 2H), 3.99 (t, J
) 6 Hz, 2H), 4.28 (q, J ) 7 Hz, 2H), 6.35 (d, J ) 9 Hz, 1H),
6.92 (d, J ) 9 Hz, 2H), 7.68 (d, J ) 9 Hz, 2H), 7.78 (d, J ) 9
Hz, 1H), 8.73 (s, 1H), 8.95 (s, 1H); MS (CI) m/z 382 (M + 1).
Anal. (C21H27N5O2‚0.5H2O) C, H, N.
2-(4-Dim eth yla m in op h en yla m in o)-8-eth yl-8H-p yr id o-
[2,3-d ]p yr im id in -7-on e (31): mp 189-191 °C; 1H NMR
(DMSO-d6) δ 1.23 (t, J ) - 7 Hz, 3H), 2.87 (s, 6H), 4.28 (q, J )
7 Hz, 2H), 6.31 (d, J ) 9 Hz, 1H), 6.74 (d, J ) 9 Hz, 2H), 7.60
(br d, J ) 7 Hz, 2H), 7.76 (d, J ) 9 Hz, 1H), 8.69 (s, 1H), 9.81
(s 1H). Anal. (C17H19N5O1) C, H, N.
8-Eth yl-2-(4-p yr r ol-1-ylp h en yla m in o)-8H-p yr id o[2,3-d ]-
p yr im id in -7-on e (32): mp 220-222 °C; 1H NMR (DMSO-d6)
δ 1.27 (t, J ) 7 Hz, 3H), 4.33 (q, J ) 7 Hz, 2H), 6.25 (t, J ) 2
Hz, 2H), 6.40 (d, J ) 9 Hz, 1H), 7.33 (t, J ) 2 Hz, 2H), 7.56 (d,
J ) 9 Hz, 1H), 7.83 (d, J ) 9 Hz, 1H), 7.89 (d, J ) 9 Hz, 1H),
8.80 (s, 1H), 10.20 (s, 1H). Anal. (C19H17N5O1‚0.1H2O‚0.3EtOAc)
C, H, N.
2-(4-Dieth ylam in oph en ylam in o)-8-eth yl-8H-pyr ido[2,3-
1
d ]p yr im id in -7-on e (33): mp 108-109 °C; H NMR (DMSO-
d6) δ 1.08 (t, J ) 7 Hz, 6H), 1.23 (t, J ) 7 Hz, 3H), 3.30 (q, J
) 7 Hz, 4H), 4.28 (q, J ) 7 Hz, 2H), 6.30 (d, J ) 9 Hz, 1H),
6.67 (d, J ) 9 Hz, 2H), 7.55 (br s, 2H), 7.75 (d, J ) 9 Hz, 1H),
8.68 (s, 1H), 9.77 (s, 1H). Anal. (C19H23N5O1) C, H, N.
8-Eth yl-2-(4-(m or p h olin -4-yl)p h en yla m in o)-8H-p yr id o-
[2,3-d ]p yr im id in -7-on e (34): mp 227-228 °C; 1H NMR
(DMSO-d6) δ 1.18 (t, J ) 7 Hz, 3H), 3.07 (t, J ) 5 Hz, 4H),
3.74 (t, J ) 5 Hz, 4H), 4.29 (q, J ) 7 Hz, 2H), 6.34 (d, J ) 9
Hz, 1H), 6.94 (d, J ) 9 Hz, 2H), 7.66 (d, J ) 9 Hz, 2H), 7.78
(d, J ) 9 Hz, 1H), 8.72 (s, 1H), 9.91 (s, 1H). Anal. (C19H21N4O2‚
0.25H2O) C, H, N.
8-E t h yl-2-(4-p ip er id in -1-ylp h en yla m in o)-8H -p yr id o-
1
[2,3-d ]p yr im id in -7-on e (35): mp 188 °C; H NMR (DMSO-
d6) δ 1.19 (t, J ) 7 Hz, 3H), 1.47 (d, J ) 4 Hz, 2H), 1.58 (s,
4H), 3.02 (d, J ) 5 Hz, 4H), 4.24 (4, J ) 7 Hz, 2H), 6.28 (d, J
) 9 Hz, 1H), 6.87 (d, J ) 9 Hz, 2H), 7.58 (d, J ) 8 Hz), 7.73 (d,
J ) 9 Hz, 1H), 8.67 (s, 1H), 9.84 (s, 1H); MS (CI) m/z ) 350.1
(M + 1). Anal. (C20H23N5O1) C, H, N.
8-E t h yl-2-{4-[4-(3-h yd r oxyp r op yl)p ip er a zin -1-yl]p h e-
n ylam in o}-8H-pyr ido[2,3-d]pyr im idin -7-on e (36): 1H NMR
(DMSO-d6) δ 1.13-1.28 (m, 7H), 1.38-1.44 (m, 2H), 1.70 (d, J
) 12 Hz, 2H), 2.53 (quin., J ) 11 Hz, 2H), 3.35 (q, J ) 6 Hz,
2H), 3.57 (d, J ) 12 Hz, 2H), 4.25 (q, J ) 7 Hz, 2H), 4.35 (t, J
) 5 Hz, 1H), 6.29 (d, J ) 9 Hz, 1H), 6.88 (d, J ) 9 Hz, 2H),
7.58 (d, J ) 8 Hz, 2H), 7.73 (d, J ) 9 Hz, 1H), 8.67 (s, 1H),
9.85 (br s, 1H).
8-E t h yl-2-[4-(4-m et h ylp ip er a zin -1-yl)p h en yla m in o]-
8H-p yr id o[2,3-d ]p yr im id in -7-on e (37): mp 185-186 °C; 1H
NMR (DMSO-d6) δ 1.24 (t, J ) 7 Hz, 3H), 2.22 (s, 3H), 2.45 (t,
J ) 5 Hz, 4H), 3.09 (t, J ) 5 Hz, 4H), 4.29 (q, J ) 7 Hz, 2H),
6.33 (d, J ) 9 Hz, 1H), 6.93 (d, J ) 9 Hz, 2H), 7.64 (d, J ) 9
Hz, 2H), 7.77 (d, J ) 9 Hz, 1H), 8.71 (s, 1H), 9.90 (s, 1H); MS
(CI) m/z 365 (M + 1). Anal. (C20H24N6O‚1.0H2O) C, H, N.
The following compounds were prepared from 2 via a similar
route to the one described for compound 1.
8-Meth yl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p yr im id in -7-
on e (38): mp 244-247 °C; 1H NMR (DMSO-d6) δ 3.59 (s, 3H),
6.40 (d, J ) 9.4 Hz, 1H), 7.03 (t, J ) 7.2 Hz, 1H), 7.35 (t, J )
7.8 Hz, 2H), 7.82 (d, J ) 9.2 Hz, 3H), 7.35 (t, J ) 7.9 Hz, 2H),
7.82 (d, J ) 9.2 Hz, 3H), 8.78 (s, 1H), 10.11 (s, 1H); MS (CI)
m/z ) 253 (M + 1). Anal. (C14H12N4O1‚0.20H2O) C, H, N.
8-Isopr opyl-2-ph en ylam in o-8H-pyr ido[2,3-d]pyr im idin -
7-on e (39): mp 170-171 °C; 1H NMR (DMSO-d6) δ 1.53 (d, J
) 7 Hz, 6H), 5.72 (br s, 1H), 6.31 (d, J ) 9 Hz, 1H), 7.04 (t, J
) 7 Hz, 1H), 7.34 (t, J ) 7 Hz, 2H), 7.72-7.77 (m, 3H), 8.75
(s, 1H), 10.00 (s, 1H); MS (CI) m/z 281 (M + 1). Anal.
(C16H16N4O) C, H, N.
2-(3,4-Dim eth oxyp h en yla m in o)-8-eth yl-8H-p yr id o[2,3-
1
d ]p yr im id in -7-on e (27): H NMR (DMSO) δ 1.17 (t, J ) 7
Hz, 3H), 3.68 (s, sH), 3.72 (s, 3H) 4.27 (q, J ) 7 Hz, 2H), 6.31
(d, J ) 9 Hz, 1H), 6.88 (d, J ) 9 Hz, 1H), 7.23 (dd, J ) 2, 9 Hz,
1H), 7.48 (br s, 1H), 7.74 (d, J ) 9 Hz, 1H), 8.69 (s, 1H), 9.89
(s, 1H). Anal. (C17H18N4O3‚0.54H2O) C, H, N.
8-Eth yl-2-(4-h yd r oxy-3-m eth oxyp h en yla m in o)-8H-p y-
r id o[2,3-d ]p yr im id in -7-on e (28): mp 196-200 °C; 1H NMR
(DMSO) δ 1.16 (t, J ) 8 Hz, 3H), 3.73 (s, 3H), 4.25 (q, J ) 7
Hz, 2H), 6.29 (d, J ) 9 Hz, 1H), 6.68 (d, J ) 9 Hz, 1H), 7.05
(d, J ) 8 Hz, 1H), 7.46 (br s, 1H), 7.73 (d, J ) 9 Hz, 1H), 8.67
(d, J ) 4 Hz, 1H), 9.80 (br s, 1H); MS (CI) m/z 313.1 (M + 1).
Anal. (C16H16N4O3‚0.23H2O) C, H, N.
8-E t h yl-2-[4-(2-m et h oxyet h oxy)p h en yla m in o]-8H -p y-
r id o[2,3-d ]p yr im id in -7-on e (29). Phenol 26 (0.099 g, 0.326
mmol), 2-bromoethyl methyl ether (0.055 g, 0.398 mmol) and
potassium carbonate (0.36 g, 2.61 mmol) in DMF (5 mL) were
heated to reflux for 5 min. The reaction mixture was allowed
to cool then H2O (40 mL) was added and a precipitate formed.
This precipitate was collected by filtration then dissolved in
ethyl acetate and dried over magnesium sulfate. Following
removal of the drying agent and evaporation of the solvent,
column chromatography provided the product as a yellow oil.
Crystallization from hexanes-ethyl acetate gave a white solid
(0.093 g, 83%: mp 169-171 °C; 1H NMR (DMSO-d6) δ 1.23 (t,
J ) 7 Hz, 3H), 3.31 (s, 3H), 3.65 (t, J ) 5 Hz, 2H), 4.07 (t, J
) 5 Hz, 2H), 4.29 (q, J ) -7 Hz, 2H), 6.35 (d, J ) 9 Hz, 1H),
6.94 (d, J ) 9 Hz, 2H), 7.68 (d, J ) 9 Hz, 2H), 7.79 (d, J ) 9
Hz, 1H), 8.73 (2, 1 H), 9.95 (s, 1H). Anal. (C18H20N4O3‚0.25H2O)
C, H, N.
8-Isopr opyl-2-[4-(4-m eth ylpiper azin -1-yl)ph en ylam in o]-
8H-p yr id o[2,3-d ]p yr im id in -7-on e (40): mp 221-222 °C; 1H
NMR (DMSO-d6) δ 1.51 (d, J ) 7 Hz, 6H), 2.45 (t, J ) 5 Hz,
4H), 3.09 (t, J ) 5 Hz, 4H), 5.69 (b, 1H), 6.26 (d, J ) 9 Hz,
1H), 6.92 (d, J ) 9 Hz, 2H), 7.54 (d, J ) 9 Hz, 2H), 7.71 (d, J
) 9 Hz, 1H), 8.68 (s, 1H), 9.77 (s, 1H); MS (CI) m/z 379 (M +
1). Anal. (C21H26N6O‚0.25H2O) C, H, N.
2-[4-(2-Dieth yla m in oeth oxy)p h en yla m in o]-8-eth yl-8H-
p yr id o[2,3-d ]p yr im id in -7-on e (30): mp 128-129 °C; 1H
NMR (DMSO-d6) δ 0.975 (t, J ) 7 Hz, 6H), 1.23 (t, J ) 7 Hz,

4614 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24
Barvian et al.
8-sec-Bu tyl-2-ph en yla m in o-8H-p yr id o[2,3-d ]p yr im idin -
7-on e (41): mp 155-156 °C; 1H NMR (DMSO-d6) δ 0.78 (br s,
3H), 1.50 (d, J ) 6.5 Hz, 3H), 2.15 (br s, 1H), 1.93 (dq, J )
6.7, 3.7 Hz, 1H), 5.54 (br s, 1H), 6.32 (d, J ) 8.4 Hz, 1H), 7.04
(t, J ) 7.2 Hz, 1H), 7.34 (t, J ) 7.5 Hz, 2H), 7.73 (d, J ) 8.0
Hz, 1H), 7.67 (d, J ) 9.4 Hz, 2H), 8.76 (s, 1H), 10.02 (br s,
1H); MS (CI) m/z ) 295 (M + 1). Anal. (C17H18N4O) C, H, N.
8-Bu t yl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p yr im id in -7-
on e (42): mp 183-184 °C; 1H NMR (DMSO-d6) δ 0.94 (t, J )
7 Hz, 3H), 1.38-1.44 m, 2H), 1.63-1.67 (m, 2H), 4.25 (t, J )
8 Hz, 2H), 6.39 (d, J ) 9 Hz, 1H), 7.04 (t, J ) 7 Hz, 1H), 7.33
(t, J ) 7 Hz, 2H), 7.80-7.83 (m, 3H), 8.78 (s, 1H), 10.12 (s,
1H); MS (CI) m/z 295 (M + 1). Anal. (C17H18N4O‚0.25H2O) C,
H, N.
8-Isobu tyl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p yr im id in -
7-on e (43): mp 170-171 °C; 1H NMR (DMSO-d6) δ 0.89 (d, J
) 7 Hz, 6H), 2.26-2.29 (m, 1H), 4.14 (d, J ) 7 Hz, 2H), 6.39
(d, J ) 9 Hz, 1H), 7.04 (t, J ) 7 Hz, 1H), 7.34 (t, J ) 7 Hz,
2H), 7.81-7.83 (m, 3H), 8.78 (s, 1H), 10.11 (s, 1H); MS (CI)
m/z 295 (M + 1). Anal. (C17H18N4O‚0.10EtOAc) C, H, N.
8-(1-Eth ylp r op yl)-2-p h en yla m in o-8H-p yr id o[2,3-d ]p y-
r im id in -7-on e (44): mp 116-118 °C; major conformer: 1H
NMR (DMSO-d6) δ 0.76 (t, J ) 7 Hz, 6H), 1.83-1.90 (m, 4H),
5.40-5.55 (m, 1H), 6.30 (d, J ) 9 Hz, 1H), 7.04 (t, J ) 7 Hz,
1H), 7.32-7.36 (m, 2H), 7.75-7.81 (m, 3H), 8.77 (s, 1H), 10.07
(s, 1H); MS (CI) m/z 309 (M + 1). Anal. (C18H20N4O‚0.2H2O)
C, H, N.
8-Cyclop en tyl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p yr im i-
d in -7-on e (45): mp 188-192 °C; 1H NMR (DMSO-d6) δ 1.59-
1.64 (m, 2H), 1.73-1.78 (m, 2H), 1.81-1.92 (m, 2H), 2.22-
2.30 (m, 2H), 5.81-6.03 (m, 1H), 6.33 (d, J ) 9 Hz, 1H), 7.04
(t, J ) 7 Hz, 1H), 7.34 (t, J ) 7 Hz, 2H), 7.71 (d, J ) 8 Hz,
2H), 7.77 (d, J ) 9 Hz, 1H), 8.76 (s, 1H), 10.00 (s, 1H); MS
(CI) m/z 307.3 (M + 1). Anal. (C18H18N4O‚0.28H2O) C, H, N.
8-Cycloh exyl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p yr im i-
d in -7-on e (46): mp 202-204 °C; 1H NMR (DMSO-d6) δ 1.21-
1.39 (m, 3H), 1.57-1.99 (m, 5H), 2.49-2.60 (m, 2H), 5.35 (br
s, 1H), 6.31 (d, J ) 9 Hz, 1H), 7.06 (t, J ) 7 Hz, 1H), 7.35 (t,
J ) 7 Hz, 2H), 7.59-7.77 (m, 3H), 8.75 (s, 1H), 10.04 (s, 1H);
MS (CI) m/z 321 (M + 1). Anal. (C19H20N4O‚0.20H2O) C, H, N.
8-Cycloh ep tyl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p yr im i-
d in -7-on e (47): mp 156-158 °C; 1H NMR (DMSO-d6) δ 1.52-
1.77 (m, 10H), 2.41-2.43 (m, 2H), 5.59 (br s, 1H), 6.31 (d, J )
9 Hz, 1H), 7.06 (t, J ) 7 Hz, 1H), 7.34 (t, J ) 7 Hz, 2H), 7.74-
7.77 (m, 3H), 8.75 (s, 1H), 10.11 (s, 1H); MS (CI) m/z 335 (M
+ 1). Anal. (C20H22N4O) C, H, N.
brine. The organic layer was dried over magnesium sulfate,
filtered, and concentrated in vacuo.
8-Cycloh exylm eth yl-2-p h en yla m in o-8H-p yr id o[2,3-d ]-
p yr im id in -7-on e (50): mp 230-231 °C; 1.00-1.20 (m, 5H),
1.57-1.60 (m, 3H), 1.67 (br s, 2H), 1.97 (br s, 1H), 1.45 (d, J
) 7 Hz, 2H), 6.39 (d, J ) 9 Hz, 1H), 7.04 (t, J ) 7 Hz, 1H),
7.33 (t, J ) 8 Hz, 2H), 7.80-7.84 (m, 3H), 8.78 (s, 1H), 10.11
(s, 1H); MS (CI) m/z 335.2 (M + 1). Anal. (C20H22N4O) C, H,
N.
8-Ben zyl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p yr im id in -7-
on e (51): mp 215-216 °C; 1H NMR (DMSO-d6) δ 5.49 (s, 2H),
6.48 (d, J ) 9.4 Hz, 1H), 6.99 (t, J ) 7.4 Hz, 1H), 7.20-7.31
(m, 7H), 7.60 (d, J ) 8.0 Hz, 2H), 7.90 (d, J ) 9.4 Hz, 1H),
8.82 (s, 1H), 10.09 (s, 1H); MS (CI) m/z ) 329 (M + 1). Anal.
(C20H16N4O1‚0.25H2O) C, H, N.
8-Meth oxym eth yl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p y-
1
r im id in -7-on e (52): mp 173-174 °C; H NMR (DMSO-d6) δ
3.34 (s, 3H), 5.64 (s, 2H), 6.40 (d, J ) 9 Hz, 1H), 7.03 (t, J )
7 Hz, 1H), 7.34 (t, J ) 7 Hz, 2H), 7.81 (d, J ) 7 Hz, 2H), 7.86
(d, J ) 9 Hz, 1H), 8.80 (s, 1H), 8.83 (s, 1H), 10.14 (s, 1H); MS
(CI) m/z 283 (M + 1). Anal. (C15H14N4O2) C, H, N.
8-(2-Met h oxyet h yl)-2-p h en yla m in o-8H -p yr id o[2,3-d ]-
p yr im id in -7-on e (53): mp 179-180 °C; 1H NMR (DMSO-d6)
δ 3.26 (s, 3H), 3.62 (t, J ) 6.4 Hz, 2H), 4.47 (t, J ) 6.4 Hz,
2H), 6.39 (d, J ) 9.4 Hz, 2H), 7.03 (t, J ) 7.3 Hz, 1H), 7.33 (t,
J ) 7.8 Hz, 2H), 7.80 (d, J ) 6.8 Hz, 2H), 7.82 (d, J ) 9.2 Hz,
1H), 8.78 (s, 1H), 10.11 (s, 1H); MS (CI) m/z ) 297 (M + 1).
Anal. (C16H16N4O2‚0.12H2O) C, H, N.
8-(2-Eth oxyeth yl)-2-p h en yla m in o-8H-p yr id o[2,3-d ]p y-
1
r im id in -7-on e (54): mp 151-153 °C; H NMR (DMSO-d6) δ
0.99 (t, J ) 7.0 Hz, 3H), 3.40 (q, J ) 6.9 Hz, 2H), 3.58 (t, J )
6.7 Hz, 2H), 4.40 (t, J ) 6.7 Hz, 2H), 6.34 (d, J ) 9.3 Hz, 1H),
6.98 (t, J ) 7.3 Hz, 1H), 7.27 (t, J ) 7.8 Hz, 2H), 7.74-7.78
(m, 3 H), 8.73 (s, 1H), 10.06 (s, 1H); MS (CI) m/z ) 311.1 (M
+ 1). Anal. (C17H18N4O2) C, H, N.
8-Oxir a n ylm eth yl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p y-
1
r im id in -7-on e (55): mp 163-165 °C; H NMR (DMSO-d6) δ
2.61-2.62 (m, 1H), 2.73-2.75 (m, 1H), 3.30-3.32 (m, 1H),
4.34-4.39 (m, 1H), 4.52-4.57 (m,1H), 6.42 (d, J ) 9 Hz, 1H),
7.04 (t, J ) 7 Hz, 1H), 7.35 (t, J ) 7 Hz, 2H), 7.79 (d, J ) 7
Hz, 2H), 7.86 (d, J ) 9 Hz, 1H), 8.80 (s, 1H), 10.13 (s, 1H); MS
(CI) m/z 295 (M + 1). Anal. (C16H14N4O2‚0.05EtOAc) C, H, N.
(7-Oxo-2-p h en yla m in o-7H -p yr id o[2,3-d ]p yr im id in -8-
yl)a cetic a cid m eth yl ester (56): mp 232-233 °C; 1H NMR
(DMSO-d6) δ 3.68 (s, 3H), 5.04 (s, 2H), 6.45 (d, J ) 9 Hz, 1H),
7.04 (t, J ) 7 Hz, 1H), 7.32 (t, J ) 7 Hz, 2H), 7.69 (d, J ) 7
Hz, 2H), 7.91 (d, J ) 9 Hz, 1H), 8.83 (s, 1H), 10.18 (s, 1H); MS
(CI) m/z 311 (M + 1). Anal. (C16H14N4O3) C, H, N.
8-Bicyclo[2.2.1]h ept-2-yl-2-ph en ylam in o-8H-pyr ido[2,3-
1
d ]p yr im id in -7-on e (48): mp 225-226 °C; H NMR (DMSO-
The following compounds were prepared from 2 via a similar
route to the one described for compound 1.
d6) δ 1.16 (d, J ) 9 Hz, 1H), 1.25-1.29 (m, 1H), 1.40 (t, J ) 9
Hz, 1H), 1.51-1.62 (m, 2H), 1.69 (t, J ) 10 Hz, 1H), 2.17-
2.20 (m, 1H), 2.37 (br s, 1H), 2.41 (br s, 1H), 2.62 (d, J ) 9 Hz,
1H), 5.32 (t, J ) 8 Hz, 1H), 6.28 (d, J ) 9 Hz, 1H), 7.05 (t, J
) 7 Hz, 1H), 7.33 (t, J ) 9 Hz, 2H), 7.73 (d, J ) 9 Hz, 1H),
7.77 (d, J ) 9 Hz, 2H), 8.74 (s, 1H), 10.06 (s, 1H). Anal.
(C20H20N4O1‚0.2H2O) C, H, N.
2-[4-(2-Dieth ylam in oeth oxy)ph en ylam in o]-8-isopr opyl-
8H -p yr id o[2,3-d ]p yr im id in -7-on e (57): mp 84-85 °C; 1H
NMR (DMSO-d6) δ 0.975 (t, J ) 7 Hz, 6H), 1.50 (d, J ) 6 Hz,
6H), 2.55 (q, J ) 7 Hz, 4H), 2.76 (t, J ) 6 Hz, 2H), 3.99 (t, J
) 6 Hz, 2H), 5.45 (b, 1H), 6.27 (d, J ) 9 Hz, 1H), 6.92 (d, J )
9 Hz, 2H), 7.58 (d, J ) 9 Hz, 2H), 7.72 (d, J ) 9 Hz, 1H), 8.70
(s, 1H), 9.82 (s, 1H); MS (CI) m/z 396 (M + 1). Anal.
(C22H29N5O2) C, H, N.
8-P h en yl-2-p h en yla m in o-8H-p yr id o[2,3-d ]p yr im id in -7-
on e (49): mp 300-302 °C; 1H NMR (DMSO-d6) δ 6.49 (d, J )
- 9 Hz, 1H), 6.84 (t, J ) 7 Hz, 1H), 6.96 (t, J ) 8 Hz, 2H), 7.29
(d, J ) 8 Hz, 2H), 7.35 (dd, J ) 1, 4 Hz, 2H), 7.55-7.63 (m,
3H), 7.94 (d, J ) 9 Hz, 1H), 8.83 (s, 1H), 10.01 (s, 1H). Anal.
(C19H14N4O1‚0.25H2O) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 8-Su bsti-
t u t e d -2-p h e n y la m in o -8H -p y r id o [2,3-d ]p y r im id in -7-
on es fr om 2-P h en yla m in o-8H-p yr id o[2,3-d ]p yr im id in -7-
on e (7). To a suspension of NaH (1.0-1.5 equiv of a 60%
suspension of NaH in mineral oil) in 5 mL of dimethylforma-
mide was added 7 (1 equiv). The reaction mixture was heated
to 50-60 °C resulting in a yellow solution. The solution was
cooled slightly and the desired alkyl halide (1.1-2.0 equiv) was
added. The reaction mixture was heated at 50 °C, for a time
ranging from 5 min to 1 h, then cooled to room temperature
and partitioned between water and ethyl acetate. In some
cases, the organic layer was washed with additional water or
8-Cyclop en tyl-2-[4-(2-d ieth yla m in oeth oxy)p h en yla m i-
n o]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (58): mp 105-107
1
°C; H NMR (DMSO-d6) δ 0.975 (t, J ) 7 Hz, 6H), 1.56-1.87
(m, 6H), 2.21-2.28 (m, 2H), 2.50 (q, J ) 7 Hz, 4H), 2.76 (t, J
) 6 Hz, 2H), 3.99 (t, J ) 6 Hz, 2H), 5.82 (br s, 1H), 6.29 (d, J
) 9 Hz, 1H), 6.92 (d, J ) 9 Hz, 2H), 7.55 (d, J ) 9 Hz, 2H),
7.74 (d, J ) 9 Hz, 1H), 8.70 (s, 1H), 9.81 (s, 1H); MS (CI) m/z
422 (M + 1). Anal. (C24H31N5O2‚0.25H2O) C, H, N.
8-Cycloh exyl-2-[4-(2-dieth ylam in oeth oxy)ph en ylam in o]-
8H-p yr id o[2,3-d ]p yr im id in -7-on e (59): mp 135-137 °C; 1H
NMR (DMSO-d6) δ 0.975 (t, J ) 7 Hz, 6H), 1.29-1.82 (m, 8H),
2.50 (q, J ) 7 Hz, 4H), 2.76 (t, J ) 6 Hz, 2H), 4.01 (t, J ) 6
Hz, 2H), 5.35 (br s, 1H), 6.27 (d, J ) 9 Hz, 1H), 6.92 (d, J ) 9
Hz, 2H), 7.59 (br s, 2H), 7.72 (d, J ) 9 Hz, 1H), 8.69 (s, 1H),
9.90 (s, 1H); MS (CI) m/z 436 (M + 1). Anal. (C25H33N5O2) C,
H, N.

Inhibitors of Cdks
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 24 4615
8-Cycloh ep tyl-2-[4-(2-d ieth yla m in oeth oxy)p h en yla m i-
n o]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (60): mp 119-121 °C;
1H NMR (DMSO-d6) δ 0.99 (t, J ) 7 Hz, 6H), 1.30-1.85 (m,
10H), 2.18 (s, 2H), 2.56 (q, J ) 7 Hz, 4H), 2.80 (t, J ) 6 Hz,
2H), 4.01 (t, J ) 6 Hz, 2H), 5.38 (br s, 1H), 6.26 (d, J ) 9 Hz,
1H), 6.91 (d, J ) 9 Hz, 2H), 7.40 (br s, 2H), 7.71 (d, J ) 9 Hz,
1H), 8.68 (s, 1H), 9.80 (s, 1H); MS (CI) m/z 450 (M + 1). Anal.
(C26H35N5O2‚0.15H2O) C, H, N.
8-Bicyclo[2.2.1]h ep t-2-yl-2-[4-(2-d ieth yla m in oeth oxy)-
p h en yla m in o]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (61): mp
133-134 °C; 1H NMR (DMSO-d6) δ 0.97 (t, J ) 7 Hz, 6H),
1.13 (d, J ) 9 Hz, 1H), 1.25 (br s, 1H), 1.35 (br s, 1H), 1.49-
1.69 (m, 3H), 2.17 (br s, 1H), 2.37 (d, J ) 11 Hz, 2H), 2.60 (d,
J ) 9 Hz, 1H), 2.75 (t, J ) 6 Hz, 2H), 4.00 (t, J ) 6 Hz, 2H),
5.27 (t, J ) 7 Hz, 1H), 6.24 (d, J ) 9 Hz, 1H), 6.90 (d, J ) 9
Hz, 2H), 7.60 (d, J ) 9 Hz, 2H), 7.69 (d, J ) 9 Hz, 1H), 8.68 (s,
1H), 9.87 (s, 1H); MS (CI) m/z 484.4 (M + 1). Anal. (C26H33N5O2‚
0.4H2O) C, H, N.
(m, 1H), 2.22 (s, 3H), 2.37-2.41 (m, 2H), 2.45 (t, J ) 5 Hz,
4H), 2.61-2.63 (m, 1H), 3.09 (t, J ) 9 Hz, 4H), 5.32 (b, 1H),
6.28 (d, J ) 9 Hz, 1H), 6.92 (d, J ) 9 Hz, 2H), 7.54 (d, J ) 9
Hz, 2H), 7.71 (d, J ) 9 Hz, 1H), 8.67 (s, 1H), 9.82 (s, 1H); MS
(CI) m/z 431 (M + 1). Anal. (C25H30N6O‚0.15H2O) C, H, N.
8-Cycloh exyl-2-[4-(4-m eth ylp ip er a zin -1-yl)p h en yla m i-
n o]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (70): mp 205-207 °C;
1H NMR (DMSO-d6) δ 1.30-1.82 (m, 8H), 2.22 (s, 3H), 2.52-
2.56 (m, 2H), 2.45 (t, J ) 5 Hz, 4H), 2.52-2.56 (m, 2H), 3.09
(t, J ) 5 Hz, 4H), 5.35 (br s, 1H), 6.26 (d, J ) 9 Hz, 1H), 6.92
(d, J ) 9 Hz, 2H), 7.54 (d, J ) 9 Hz, 2H), 7.71 (d, J ) 9 Hz,
1H), 8.68 (s, 1H), 9.77 (s, 1H); MS (CI) m/z 419 (M + 1). Anal.
(C24H30N6O‚0.1EtOAc) C, H, N.
8-Cyclop en tyl-2-{4-[4-(3-h yd r oxyp r op yl)p ip er id in -1-yl]-
p h en yla m in o}-8H-p yr id o[2,3-d ]p yr im id in -7-on e (71): mp
1
223 °C; H NMR (DMSO-d6) δ 1.18-1.42 (m, 4H), 1.44-1.50
(m, 2H), 1.57 (br s, 2H), 1.74 (d, J ) 11 Hz, 4H), 1.86 (br s,
2H), 2.23 (br s, 2H), 2.58 (t, J ) 11 Hz, 2H), 3.40 (t, J ) 6 Hz,
2H), 3.62 (d, J ) 12 Hz, 2H), 4.39 (t, J ) 5 Hz, 1H), 5.80 (br
s, 1H), 6.27 (d, J ) 9 Hz, 1H), 6.91 (d, J ) 9 Hz, 2H), 7.47 (d,
J ) 9 Hz, 2H), 7.73 (d, J ) 9 Hz, 1H), 8.68 (s, 1H), 9.73 (br s,
1H); MS (CI) m/z 448.3 (M + 1). Anal. (C26H33N5O2‚0.55H2O)
C, H, N.
8-(2-Ben zyloxyeth yl)-2-[4-(4-m eth ylp ip er a zin -1-yl)p h e-
n ylam in o]-8H-pyr ido[2,3-d]pyr im idin -7-on e (62): mp 183-
1
185 °C; H NMR (DMSO-d6) δ 2.21 (s, 3H), 2.42 (t, J ) 5 Hz,
4H), 3.01 (br s, 4H), 3.72 (t, J ) 7 Hz, 2H), 4.49-4.53 (m, 4H),
6.34 (d, J ) 9 Hz, 1H), 6.86 (d, J ) 9 Hz, 2H), 7.22-7.30 (m,
5H), 7.62 (d, J ) 9 Hz, 2H), 7.79 (d, J ) 9 Hz, 1H), 8.72 (s,
1H), 9.90 (s, 1H); MS (CI) m/z 470.9 (M + 1). Anal. (C27H38N6O2)
C, H, N.
8-Bicyclo[2.2.1]h ep t-2-yl-2-{4-[4-(3-h yd r oxyp r op yl)p i-
p er id in -1-yl]p h en yla m in o}-8H-p yr id o[2,3-d ]p yr im id in -7-
on e (72): mp 223 °C; 1H NMR (DMSO-d6) δ 1.12-1.75 (m,
16H), 2.17 (br s, 1H), 2.36 (d, J ) 9 Hz, 2H), 2.58 (t, J - 12
Hz, 2H), 3.39 (q, J ) 6 Hz, 2H), 3.60 (d, J ) 12 Hz, 2H), 4.38
(t, J ) 5 Hz, 1H), 5.28 (t, J ) 8 Hz, 1H), 6.22 (d, J ) 9 Hz,
1H), 6.89 (d, J ) 9 Hz, 2H), 7.54 (d, J ) 9 Hz, 2H), 7.68 (d, J
) 9 Hz, 1H), 8.66 (s, 1H), 9.80 (br s, 1H); MS (CI) m/z 474.3
(M + 1). Anal. (C28H36N5O2‚0.09H2O) C, H, N.
8-(2,3-Dih yd r oxyp r op yl)-2-[4-(4-m eth ylp ip er a zin -1-yl)-
p h en yla m in o]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (63): mp
188-195 °C; 1H NMR (DMSO-d6) δ 2.08 (s, 3H), 2.25-3.35
(m, 4H), 3.02-3.35 (m, 4H), 3.14-3.38 (m, 2H), 3.80-4.60 (m,
4H), 4.62 (d, J ) 6 Hz, 1H), 6.27 (d, J ) 9 Hz, 1H), 6.85 (d, J
) 9 Hz, 2H), 7.65-7.68 (m, 2H), 7.71 (d, J ) 9 Hz, 1H), 8.64
(s, 1H); MS (CI) m/z 411.2 (M + 1).
Cd k Assa ys (Cd k 4/cyclin D1, Cd k 2/cyclin E, Cd k 2
cyclin A, a n d Cd c2/cyclin B). All Cdks were human recom-
binant proteins expressed in insect cells through baculovirus
infection. Enzyme assays for IC50 determinations and kinetic
evaluation were performed in 96-well filter plates (Millipore
MADVN6550). The total volume was 0.1 mL containing a final
concentration of 20 mM Tris (tris[hydroxmethyl]aminomethane),
pH 7.4, 50 mM NaCl, 1 mM dithiothreitol, 10 mM MgCl2, 25
µM ATP (for Cdk4) or 12 µM ATP (for Cdk2/E, Cdk2/A and
Cdc2/B) containing 0.25 µCi of [32P]ATP, 20 ng of enzyme, 1
µg of GST-retinoblastoma and appropriate dilutions of inhibi-
tor. All components except the ATP were added to the wells
and the plate was placed on a plate mixer for 2 min. The
reaction was started by adding [32P]ATP and the plate was
incubated at 25 °C for 15 min. The reaction was terminated
by addition of 0.1 mL of 20% trichloroacetic acid (TCA). The
plate was kept at 4 °C for at least 1 h to allow the substrate
to precipitate. The wells were then washed 5 times with 0.2
mL of 10% TCA and 32P incorporation was determined with a
beta plate counter (Wallac Inc., Gaithersburg, MD).
2-[4-(4-Meth ylp ip er a zin -1-yl)p h en yla m in o]-8-p h en yl-
8H-p yr id o[2,3-d ]p yr im id in -7-on e (64): mp 259-262 °C; 1H
NMR (DMSO-d6) δ 2.21 (s, 3H), 2.43 (t, J ) 5 Hz, 4H), 2.97
(br s, 4H), 6.43 (d, J ) 9 Hz, 1H), 6.54 (br s, 2H), 7.13 (br s,
2H), 7.32 (dd, J ) 1, 8 Hz, 2H), 7.56-7.62 (m, 3H), 7.89 (d, J
) 9 Hz, 1H), 8.76 (s, 1H), 9.84 (s, 1H); MS (CI) m/z 413.1 (M
+ 1). Anal. (C24H24N5O‚0.3H2O) C, H, N.
8-Cyclopr opyl-2-[4-(4-m eth ylpiper azin -1-yl)ph en ylam i-
n o]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (65): mp 210-211
1
°C; H NMR (DMSO-d6) δ 0.79-0.83 (m, 2H), 1.22-1.27 (m,
2H), 2.22 (s, 3H), 2.45 (t, J ) 5 Hz, 4H), 2.83-2.86 (m, 1H),
3.08 (t, J ) 5 Hz, 4H), 6.26 (d, J ) 9 Hz, 1H), 6.94 (d, J ) 9
Hz, 2H), 7.71 (d, J ) 9 Hz, 1H), 7.79 (br s, 2H), 8.66 (s, 1H),
9.95 (br s, 1H); MS (CI) m/z 376.9 (M + 1). Anal. (C21H24N5O)
C, H, N.
8-Isopr opyl-2-[4-(4-m eth ylpiper azin -1-yl)ph en ylam in o]-
8H-p yr id o[2,3-d ]p yr im id in -7-on e (66): mp 221-222 °C; 1H
NMR (DMSO-d6) δ 1.51 (d, J ) 7 Hz, 6H), 2.22 (s, 3H), 2.45
(t, J ) 5 Hz, 4H), 3.09 (t, J ) 5 Hz, 4H), 5.69 (br s, 1H), 6.26
(d, J ) 9 Hz, 1H), 6.92 (d, J ) 9 Hz, 2H), 7.54 (d, J ) 9 Hz,
2H), 7.71 (d, J ) 9 Hz, 1H), 8.68 (s, 1H), 9.77 (s, 1H); MS (CI)
m/z 379 (M + 1). Anal. (C21H26N6O‚0.25H2O) C, H, N.
8-(1-Eth ylp r op yl)-2-p r op yl-2-[4-(4-m eth ylp ip er a zin -1-
yl)p h en yla m in o]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (67):
mp 155-157 °C; major conformer: 1H NMR (DMSO-d6) δ 0.69
(t, J ) 8 Hz, 6H), 1.75-1.82 (m, 4H), 2.16 (s, 3H), 2.39 (t, J )
5 Hz, 4H), 3.03 (t, J ) 5 Hz, 4H), 5.40 (br s, 1H), 6.19 (d, J )
9 Hz, 1H), 6.86 (d, J ) 9 Hz, 2H), 7.53 (d, J ) 9 Hz, 2H), 7.70
(d, J ) 9 Hz, 1H), 8.64 (s, 1H), 9.80 (br s, 1H); MS (CI) m/z
406 (M + 1). Anal. (C23H30N6O‚0.1H2O) C, H, N.
8-Cyclopen tyl-2-[4-(4-m eth ylpip er a zin -1-yl)p h en yla m i-
n o]-8H-p yr id o[2,3-d ]p yr im id in -7-on e (68): yield 43%; mp
175-177 °C; 1H NMR (DMSO-d6) δ 1.56-1.87 (m, 6H), 2.22-
2.26 (m, 5H), 2.45 (t, J ) 5 Hz, 4H), 3.09 (t, J ) 5 Hz, 4H),
5.82 (b, 1H), 6.27 (d, J ) 9 Hz, 1H), 6.92 (d, J ) 9 Hz, 2H),
7.50 (d, J ) 9 Hz, 2H), 7.72 (d, J ) 9 Hz, 1H), 8.69 (s, 1H),
9.75 (s, 1H); MS (CI) m/z 405 (M + 1). Anal. (C23H28N6O) C,
H, N.
Tyr osin e Kin a se Assa ys. PDGF, FGF and SRC were
obtained and assayed as previously described.22
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