Discovery of a potent and selective inhibitor of cyclin-dependent kinase 4/6

Article abstract of DOI:10.1021/jm049354h

A pharmacological approach to inhibition of cyclin-dependent kinases 4 and 6 (Cdk4/6) using highly selective small molecule inhibitors has the potential to provide novel cancer therapies for clinical use. Achieving high levels of selectivity for Cdk4/6, versus other ATP-dependent kinases, presents a significant challenge. The pyrido[2,3-d]pyrimidin-7-one template provides an effective platform for the inhibition of a broad cross-section of kinases, including Cdks. It is now demonstrated that the modification of pyrido[2,3-d]pyrimidin-7-ones to include a 2-aminopyridine side chain at the C2-position provides inhibitors with exquisite selectivity for Cdk4/6 in vitro. This selectivity profile is recapitulated in cells where the most selective inhibitors create a G₁ block at concentrations up to 100-fold the IC₅₀ for cell proliferation. On the basis of its selectivity profile and pharmacokinetic profile, compound 43 (PD 0332991) was identified as a drug candidate for the treatment of cancer.

Full text of DOI:10.1021/jm049354h

2388
J. Med. Chem. 2005, 48, 2388-2406
Discovery of a Potent and Selective Inhibitor of Cyclin-Dependent Kinase 4/6
Peter L. Toogood,*,† Patricia J. Harvey,‡ Joseph T. Repine,† Derek J. Sheehan,† Scott N. VanderWel,†
Hairong Zhou,† Paul R. Keller,‡ Dennis J. McNamara,† Debra Sherry,† Tong Zhu,§ Joanne Brodfuehrer,§
Chung Choi,† Mark R. Barvian,† and David W. Fry‡
Medicinal Chemistry, Cancer Pharmacology, and Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and
Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105
Received August 6, 2004
A pharmacological approach to inhibition of cyclin-dependent kinases 4 and 6 (Cdk4/6) using
highly selective small molecule inhibitors has the potential to provide novel cancer therapies
for clinical use. Achieving high levels of selectivity for Cdk4/6, versus other ATP-dependent
kinases, presents a significant challenge. The pyrido[2,3-d]pyrimidin-7-one template provides
an effective platform for the inhibition of a broad cross-section of kinases, including Cdks. It
is now demonstrated that the modification of pyrido[2,3-d]pyrimidin-7-ones to include a
2-aminopyridine side chain at the C2-position provides inhibitors with exquisite selectivity for
Cdk4/6 in vitro. This selectivity profile is recapitulated in cells where the most selective
inhibitors create a G1 block at concentrations up to 100-fold the IC50 for cell proliferation. On
the basis of its selectivity profile and pharmacokinetic profile, compound 43 (PD 0332991) was
identified as a drug candidate for the treatment of cancer.
Introduction
Inhibition of cyclin-dependent kinases (Cdks) associ-
ated with cell cycle regulation is anticipated to provide
an effective approach to the control of tumor growth in
the clinic.1 To this end, we2 and others3-8 have been
studying inhibitors of Cdks 1, 2, and 4 in vitro, with
the goal of identifying Cdk-selective small molecule
inhibitors with physical properties amenable to achiev-
ing high exposure in vivo. Such agents may then be used
to test the hypothesis, first in animal models and then
in patients, that pharmacological inhibition of one or
more Cdks will inhibit tumor growth. Although much
biochemical evidence supports targeting Cdk4/D as the
most important Cdk for regulating cell proliferation,1f,9
many chemical series and programs have actually
tended to favor inhibition of Cdk1 and 2 over inhibition
of Cdk4/D.3-7 Consequently, the more advanced Cdk
Figure 1. Cdk inhibitors CYC-202 (1), BMS 387032 (3) and
inhibitors in terms of preclinical and clinical evaluation,
flavopiridol (3).
such as CYC-202 (1)10 and BMS-387032 (2),11 may be
better characterized as inhibitors of Cdk2 rather than
inhibitors of Cdk4/D. Flavopiridol (3),12 a Cdk inhibitor
In addition, Cdk2 inhibition by siRNA fails to halt the
that has been studied clinically for several years and is
currently being evaluated in combination therapy, prob-
ably exerts its activity through inhibition of Cdk9 and
inhibition of cyclin D1 expression.13 While these com-
pounds are all effective at inhibiting the growth of
human tumor xenografts in mice, none has yet shown
efficacy for the treatment of cancer in human patients.14
Moreover, as more has been learned about the roles of
various Cdks in regulating cell-cycle transitions, evi-
dence has been presented that Cdk2 activity is not
essential for cells to undergo mitosis,15 and may be
substituted by another kinase, possibly Cdk4/D or Cdk6.
proliferation of osteosarcomas and Rb-negative cervical
cancers, suggesting that Cdk2 may not be a good target
for inhibition by small molecules intended to treat
cancer.16 These recent results have shifted attention
back toward Cdk4/D as the primary cell cycle Cdk target
for cancer drug discovery.
Selective inhibitors of Cdk4/D have been described in
the literature8 and it is noteworthy that higher selectiv-
ity can be achieved for inhibition of Cdk4/D versus other
Cdks than for inhibition of Cdk2 versus Cdk1. This
latter pair appears to present a higher level of structural
similarity in three dimensions than Cdk4/D and Cdk2,
making the design of truly selective Cdk2 inhibitors
especially challenging. Some Cdk2 inhibitors also inhibit
extraneous kinases such as Cdk5 and Gsk3, further
complicating their biochemical profile. Among the Cdk4/D
inhibitors that have been described, CINK4 (4),8g is
* Corresponding author. Tel: 734 622 1335. Fax: 734 622 5165.
E-mail: Peter.toogood2@pfizer.com.
† Medicinal Chemistry.
‡ Cancer Pharmacology.
§ Pharmacokinetics, Dynamics and Metabolism.
10.1021/jm049354h CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/02/2005

Inhibitor of Cyclin-Dependent Kinase 4/6
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2389
Scheme 2
Chemistry
The compounds described in this report were prepared
following the general approach that has been disclosed
previously.2 Introduction of the aminopyridyl side chains
by displacement of a sulfoxide at the C2 position of the
pyrido[2,3-d]pyrimidin-7-one core was achieved in mod-
erate to low yields by heating the starting sulfoxides in
toluene to 110 °C overnight (Scheme 1). In most cases
the product precipitated from the reaction mixture upon
cooling, considerably facilitating the isolation of pure
material. The reaction mixtures also contained several
side products, including significant quantities of the
corresponding 2-hydroxy pyrido[2,3-d]pyrimidin-7-ones
(9) and a 2:1 adduct tentatively identified as structure
10 (Scheme 1). Attempts to exclude water from the
reaction mixtures failed to eliminate the formation of
hydroxy-pyrimidinones suggesting that a non-hydrolytic
pathway to these compounds exists, possibly involving
rearrangement of the starting sulfoxide. The 2:1 adduct
could be minimized but never entirely eliminated by
increasing the ratio of aminopyridine to sulfoxide in the
reaction mixture. Changes in reaction temperature and
solvent failed to substantially improve the yield of
desired product. tert-Butyl carbamate protected side
chains such as piperazines and aminopyrrolidines were
deprotected by acidolysis in ethereal hydrochloric acid
and isolated as their hydrochloride salts. Compounds
containing a C6-acetyl group (12) were accessed from
the corresponding C6 bromides (11) via Stille coupling
with (ethoxyvinyl)tributylstannane, followed by hy-
drolysis, either before or after installation of the C2-
side chain (Scheme 2). In some cases, it was possible to
install the C2 side chain late in the synthesis via a
palladium-mediated arylation reaction of pyrimidine
Figure 2. Selective Cdk4 inhibitors.
somewhat selective for Cdk4/D and inhibits the growth
of HCT116 tumors in mice. However, the relatively
weak potency of this compound (IC50 ) 1.5 µM) raises
questions regarding its mechanism of action in vivo.
Urea derivatives from Banyu (e.g. 5),8j,k also are some-
what selective for Cdk4/D in vitro, but only limited cell-
based activity data and no in vivo data have been
disclosed for these compounds. Scientists from Lilly
have reported two series of related carbazoles (e.g.
6)8c-f,n that display selectivity for Cdk4/D, and selected
examples are active versus HCT116 tumors in vivo.
However, many of these compounds retain significant
potency against Cdk2/cyclin E.
Our goal is to identify potent (nanomolar) inhibitors
of Cdk4/D that possess sufficient selectivity against
other kinases to test the hypothesis that pharmacologi-
cal inhibition of Cdk4/D will provide a mild and effective
treatment for the inhibition of tumor growth. Here, we
describe structural modifications to a series of pyrido-
[2,3-d]pyrimidin-7-ones that have led to the discovery
of some of the most potent and selective Cdk4/D inhibi-
tors yet reported.17 Specifically, modification of the C2-
side chain to a 2-aminopyridine confers exquisite selec-
tivity for Cdk4/D versus all other kinases tested.
Moreover, distinct biochemical differences have been
identified between selective Cdk4/D inhibitors and
mixed inhibitors that inhibit both Cdk4/D and Cdk2.
These observations provide confidence that the com-
pounds described here also act selectively upon Cdk4/D
in the more challenging context of the intracellular
environment.
Scheme 1

2390 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Toogood et al.
Scheme 3
amines 13 to provide products 14 as shown in Scheme
3. This latter approach was explored as an alternative
to sulfoxide displacement but was not as general and
provided no improvement in overall yields. All target
Figure 3. Initial leads in the discovery of compound 43.
1
compounds were characterized by H NMR and mass
spectroscopy. Purity was assessed by combustion analy-
sis or by reversed phase HPLC and high-resolution mass
spectroscopy.
Results and Discussion
The first pyrido[2,3-d]pyrimidin-7-one prepared con-
taining a 2-aminopyridine substituent at C2 was com-
pound 15a. A comparison of 15a with its aniline
counterpart 15b identified a remarkable difference in
selectivity profile between the two analogues, with the
pyridyl analogue (15a) substantially favoring inhibition
of Cdk4/D over inhibition of Cdk2/A (Figure 3). Con-
temporaneously, the nonspecific Cdk inhibitor, com-
pound 16 (Figure 3), was identified as a potent inhibitor
of Cdk4/D (IC50 ) 0.002 µM), Cdk2A (IC50 ) 0.043 µM)
and the fibroblast growth factor receptor (FGFr: IC50
) 0.080 µM). Bromide 16 was shown to inhibit the
growth of human tumor xenografts in mice (not shown),
demonstrating an ability to reach its molecular target-
(s) in vivo. Consequently, this compound was selected
as a starting point for addressing whether replacing
aniline side chains with aminopyridine-containing side
chains would provide a general method for achieving
selective inhibition of Cdk4/D, with compounds possess-
ing the potential to inhibit this target in vivo.
To our great satisfaction, the next compound pre-
pared, compound 17 (Figure 4), was found to exhibit
excellent selectivity for Cdk4/D versus other Cdks and
the receptor tyrosine kinases FGFr and PDGFr (platelet-
derived growth factor receptor). Moreover, compound 17
imposed a G1 block on asynchronously growing Rb-
positive MDA-MB453 cells that was maintained at
concentrations of inhibitor up to 10 µM. This remarkable
observation led to a more thorough exploration of
pyrido[2,3-d]pyrimidin-7-ones containing 2-aminopyri-
dine side chains at the C2 position and the resulting
SAR trends are detailed below.
A brief examination of the N8 substituent was per-
formed to determine whether changing the nature of the
C2 side chain would alter the previously observed
preference for a cycloalkyl group at N8. A limited survey
of N8 substituents (Table 1) indicated that a cyclopentyl
group at N8 provided the best combination of potency
and selectivity for Cdk4/D. Consequently, all remaining
analogues were prepared holding this group constant.
Attention was turned next to the C6 substituent.
Although the bromine atom in compound 17 conferred
good potency for Cdk4/D inhibition, the size and lipo-
philicity of this atom suggested that a more optimal
substituent might exist for this position. Specifically,
an improvement in overall physical properties was
anticipated with substituents that could lower the total
Figure 4. A potent and selective Cdk4 inhibitor.
Table 1. SAR at the N8-Position of
Pyrido-[2,3-d]-pyrimidin-7-ones
compd
R
Cdk4/D IC50 (µM)
Cdk2A IC50 (µM)
18
19
20
21
isopropyl
>5
>5
>5
cyclopropyl
cyclopentyl
cyclohexyl
0.92
0.015
0.013
2.5
0.835
Table 2. SAR at the C6-Position of
Pyrido-[2,3-d]-pyrimidin-7-onesa
Cdk4/D
Cdk2A
MDA-MB435
compd
X
IC50 (µM) IC50 (µM)
IC50 (µM)
17
22
23
24
25
26
27
28
29
30
31
32
33
34
Br
0.016
0.051
0.019
0.027
0.022
0.013
0.013
0.018
0.031
0.124
0.037
1.760
0.123
0.595
6.05
>5
>5
0.09
1.10
0.19
0.29
1.08
0.56
0.33
1.00
2.15
NA
F
NH2
Me
4.05
Et
>5
>5
3.8
>5
>5
>5
>5
>5
>5
>5
CH2OH
CH2OMe
CH2OEt
CH2O(CH2)2OMe
(CH2)2OEt
O(CH2)2OEt
NA
i
OCH2 Pr
NA
Ac
2.77
NA
CO2Et
a NA means data not available.
molecular weight and introduce some polarity at the C6
position, while retaining potency for Cdk4/D. Therefore,
the substituents examined were generally small and
polar in nature as shown in Table 2. Historical data for

Inhibitor of Cyclin-Dependent Kinase 4/6
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2391
peared to be tolerated at the C6 position, including small
polar groups such as NH2 (23), nonpolar substituents
such as ethyl (25), and extended chains such as meth-
oxyethoxymethyl (29). Four compounds displayed po-
tency for Cdk4/D comparable to compound 17, namely
compounds 23, 26, 27, and 28. Strikingly, the branched
sec-butoxy substituent in compound 32 was not well
tolerated and resulted in a drop in potency against
Cdk4/D of 2 orders of magnitude, indicating a possible
size limitation in this region of the binding site. These
observations are consistent with expectations based on
a structural model in which the C6 substituent projects
into the back of the ATP binding pocket.2a
Figure 5. Selective Cdk4 inhibitors containing aniline side
chains.
Selected examples from Table 2 were evaluated in
cell-based assays for inhibition of [14C]-thymidine in-
corporation into MDA-MB435 human breast carcinoma
cells. Cdk4/D inhibitors cause cells to accumulate in G1
and since DNA synthesis occurs in S-phase, inhibition
of DNA synthesis, and consequently thymidine incor-
poration, provides a useful surrogate for inhibition of
cell proliferation. Of the compounds examined, only
compound 23 was considered sufficiently potent in cell-
based assays for further studies. Unfortunately, this
compound displayed very low solubility (less than 3 µg/
mL) and was not pursued further. Nonetheless, encour-
aged that the effect of the pyridyl side chain on
selectivity was general, SAR studies were continued in
search of compounds more suitable for in vivo studies.
As described in the accompanying manuscript,18
another structural modification in the pyrido[2,3-d]-
pyrimidin-7-one series that led to improved selectivity
for Cdk4/D was the introduction of a methyl substituent
at the C5 position. To determine whether the C5-methyl
and the C2-pyridyl groups could work together to
provide even greater selectivity for Cdk4/D, compounds
were prepared containing both of these structural
features. Since compounds 35, 36, and 37 (Figure 5) had
already been identified as potent and quite selective
inhibitors of Cdk4/D,18 these structures were chosen as
Table 3. Varying the C5, C6, and C2 Side Chain Substituents
Cdk4/D
IC50 (µM)
Cdk2A
IC50 (µM)
compd
R′′
R′
X
38
39
40
17
41
42
33
43
34
44
Me
H
piperazine
H
H
0.580
0.95
>5
>5
>5
6.05
>5
>5
>5
>5
>5
>5
Br
Br
Br
Br
Ac
Ac
Ac
Me
H
H
>5
piperazine
piperazine
H
piperazine
piperazine
piperazine
piperazine
0.016
0.16
0.440
0.123
0.011
0.595
0.049
Me
Me
H
Me
H
CO2Et
CO2Et
Me
pyrido[2,3-d]pyrimidin-7-one inhibitors of Cdks indi-
cated that selectivity for Cdk4/D was most difficult to
achieve versus Cdk2/A. Thus, inhibition of Cdk4/D and
Cdk2/A was measured routinely for these compounds
to provide a first-pass indication of inhibitor selectivity.
In all cases in Table 2, significant selectivity for Cdk4/D
was retained. In general, a range of substituents ap-
Table 4. Optimizing the C2 Side Chaina
Cdk4/D
IC50 (µM)
Cdk2A
IC50 (µM)
MDA-MB435
IC50 (µM)
compd
X
R′
41
45
46
47
48
49
50
51
43
52
53
54
55
56
57
58
59
60
61
Br
Br
Br
Br
Br
Br
Br
Br
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
piperazine
0.16
1.1
>5
>5
>5
>5
>5
>5
>5
>5
>5
2.05
>5
>5
>5
>5
>5
>5
>5
>5
>5
NA
(CH3OCH2CH2)2N-
3,5-dimethylpiperazine
N-methylpiperazine
homopiperazine
NA
0.063
0.136
>5
0.570
NA
NA
NA
piperidine
>5
0.074
1.95
4-hydroxypiperidine
morpholine
1.400
NA
piperazine
0.011
0.051
0.021
0.037
0.005
0.014
0.012
0.005
0.019
0.004
0.030
0.160
NA
(CH3OCH2CH2)2N
3,3-dimethylpiperazine
3,5-dimethylpiperazine
N-methylpiperazine
3-aminopyrrolidine
homopiperazine
0.420
0.570
0.325
0.340
0.070
1.930
0.200
0.220
2.040
piperidine
4-hydroxypiperidine
morpholine
3,5-dimethylmorpholine
a NA means data not available.

2392 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Toogood et al.
starting points for the study of C5-methyl/C2-pyridyl
additivity. In each case, changing the aniline to a
2-aminopyridine in the C2 side chain rendered the
compounds less potent for Cdk4/D, (compare 35 to 41,
36 to 43, and 37 to 44: Figure 5 and Table 3). Where it
could be determined, an increase in selectivity for
Cdk4/D versus Cdk2/A was observed (compare 36 to 43).
The effect of the C5 methyl group depended very much
on the exact nature of the C6 substituent. Addition of a
C5 methyl group to compounds that contained a 2-ami-
nopyridine in the C2 side chain led to an increase in
potency when there was an acetyl or ethyl ester sub-
stituent at C6 (compare 33 to 43 and 34 to 44), but a
drop in potency when there was a bromine atom at C6
(compare 17 to 41). This disparity suggests that the C6
acetyl or ester carbonyl group is forming an additional
interaction with the protein when it is oriented by the
adjacent methyl group out of the plane defined by the
pyrido[2,3-d]pyrimidin-7-one rings.
The data in Table 3 identified compound 43 as a
remarkably potent and selective Cdk4/D inhibitor,
exhibiting a near optimal biochemical profile. However,
to this point there had been no investigation of the
heterocyclic group attached to the pyridine ring to
determine whether the piperazine substituent was
optimal with regard to potency and physical properties.
Thus, a series of piperazine replacements was investi-
gated using both the C5-methyl/C6-acetyl pyrido[2,3-
d]pyrimidin-7-one, and the C5-methyl/C6-bromo pyrido-
[2,3-d]pyrimidin-7-one cores. These compounds are listed
in Table 4. It is noteworthy that the C6 bromides are
less potent than the C6-acetyl derivatives in every case,
although the 3,5-dimethylpiperazine derivative 46 dis-
plays an IC50 ) 0.063 µM for Cdk4/D, which is only
2-fold less potent than acetyl analogue 54. Several of
the C6-acetyl derivatives displayed potencies for Cdk4/
D, and selectivities against Cdk2/A comparable to
compound 43. In cell based assays, four compounds
displayed IC50 values of ∼0.200 µM or less. Of these
four, compounds 43 and 57 are both more potent and
more soluble (data not shown) than compounds 59 and
60, identifying them as excellent biochemical tools for
studying the effects of selective Cdk4/D inhibition in
cells.
Figure 6. Cell cycle flow cytometry histograms for a selection
of Cdk inhibitors exhibiting various levels of selectivity for
Cdk4. Data were recorded using MDA-MB453 cells as de-
scribed in Experimental Methods. Gray bars: cells in G1 phase;
unfilled bars: cells in S phase; filled bars: cells in G2 phase.
Table 5.
Cdk4/D
Cdk2/A
MDA-MB435 MDA-MB468
compd IC50 (µM) IC50 (µM)
IC50 (µM)
IC50 (µM)
Selective Cdk4 inhibitors are expected to block cell
cycling by imposing a G1 block in an Rb-dependent
manner. Five compounds, including compound 43, were
compared by flow cytometry for their effect on cell
cycling in MDA-MB453 human breast carcinoma cells
(Figure 6, Table 5). The five compounds were chosen to
span a range of selectivity for Cdk4/D vs Cdk2A as well
as to represent a variety of different structural features.
Thus, compound 16 is a nonselective Cdk inhibitor
containing an aniline side chain; compounds 36 and 37
contain aniline side chains and are moderately to highly
selective for Cdk4/D in enzyme assays. Compounds 17
and 43 contain pyridyl side chains and are both highly
selective for Cdk4/D. Compound 16 imposed a G1 block
on cells at 0.1 µM, at which concentration it may
functionally inhibit Cdk4/D more than Cdk2. At higher
concentrations, however, substantial populations of cells
in G2 were observed. Compound 36 is approximately
100-fold selective for Cdk4/D versus Cdk2A, but this
compound only produced a clean G1 block in cells at
16
36
37
17
43
0.002
0.002
0.006
0.016
0.011
0.043
0.230
0.020
0.030
0.170
0.090
0.160
0.600
1.1
>5
>3
>5
>5
>3
>3
concentrations less than 3 µM. Compound 37 appears
to be highly selective for Cdk4/D versus other Cdks
based on comparative enzyme inhibition data obtained
in vitro. It also lacks antiproliferative activity against
the Rb-negative cell line, MDA MB468, however, in
MDA-MB453 cells, concentrations of this compound
greater than or equal to 3 µM, similarly produced a
significant population of cells in G2. In contrast, com-
pounds 17 and 43, produced sustained G1 blocks at 3
µM and even up to 10 µM (not shown). Thus, flow
cytometry provides a method of distinguishing the most
highly selective inhibitors of Cdk4/D. Even apparently
selective Cdk4/D inhibitors such as 36 and 37 arrest
some cells in G2 at concentrations >1 µM. This profile

Inhibitor of Cyclin-Dependent Kinase 4/6
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2393
cycle block in Rb-positive cells that is maintained at
high concentrations of the inhibitor. This latter property
is unique to the most highly selective Cdk4/D inhibitors
and among the pyrido[2,3-d]pyrimidin-7-ones is only
observed with compounds possessing a 2-aminopyridyl
substituent in the C2 position. Among these highly
selective inhibitors, compound 43 displays a superior
overall profile including the combined attributes of
potency, selectivity, and pharmaceutical properties. A
more extensive biochemical and biological characteriza-
tion of compound 43 will be published separately.17
Compound 43 is currently under exploratory develop-
ment for the treatment of cancer.
Experimental Section
8-Cyclopentyl-2-(pyridin-2-ylamino)-8H-pyrido[2,3-d]-
pyrimidin-7-one (15a). 8-Cyclopentyl-2-methanesulfinyl-8H-
pyrido[2,3-d]pyrimidin-7-one (200 mg, 0.7 mmol) and 2-ami-
nopyridine (130 mg, 1.4 mmol) were combined in a 10 mL
round-bottomed flask. The flask was purged with nitrogen (10
min) and then heated in a 160 °C oil bath (30 min). After
cooling, the orange residue was triturated with water to afford
an orange solid, which was further purified by reversed-phase
HPLC to provide 15a (15 mg, 7%) as a yellow solid. mp: >250
°C. 1H NMR (400 MHz, DMSO-d6) 8.83 (s, 1H), 8.33 (d, J ) 4
Hz, 1H), 7.91 (m, 2H), 7.83 (d, J ) 9 Hz, 1H), 7.13 (m, 2H),
6.41 (d, J ) 9 Hz, 1H), 5.81 (m, 1H), 2.20 (m, 2H), 1.90 (m,
2H), 1.75 (m, 2H), 1.56 (m, 2H).
Figure 7. Pharmacokinetic profile of compound 43 in rat
following intravenous or oral dosing.
is not well understood, but is likely related to the
inhibition of kinases other than Cdk4/D in cells by the
less selective inhibitors. A sustained G1 block at con-
centrations g3 µM appears to be a hallmark of more
highly selective Cdk4/D inhibitors. This observation
provides encouragement that compounds such as 17 and
43 display functional selectivity for Cdk4/D in cells and
suggests a method for screening for Cdk4/D selectivity
that is supplementary to performing extensive in vitro
enzyme assays. To be considered selective for Cdk4/D,
inhibitor molecules must exhibit selectivity in enzyme
assays, selectively inhibit proliferation of Rb-positive
and not Rb-negative cell lines, and produce a robust G1
block at concentrations of inhibitor close to 100-fold the
IC50 for inhibition of cell proliferation.
On the basis of the data in Table 4, compounds 43,
57, 59, and 60 appeared comparable in terms of their
potency and selectivity for Cdk4/D, and activity in cells;
however, upon further evaluation, compound 43 stood
out as a superior candidate. In particular, compound
43 is equipotent versus Cdk4 and Cdk6,17 displays good
pharmacokinetic properties in rats (Figure 7, Table 6;
CL ) 37.5 ( 1.7 mL/min/kg, 56% oral bioavailability),
and excellent pharmaceutical properties (not shown).
In summary, the introduction of a 2-aminopyridyl
substituent at the C2-position of pyrido[2,3-d]pyrimidin-
7-ones substantially biases the selectivity of these
compounds toward selective inhibition of Cdk4/D versus
other serine/threonine and tyrosine kinases. This effect
appears to be general and to apply across a wide range
of kinases. The reason the pyridyl group at C2 confers
such excellent selectivity for Cdk4/D on these com-
pounds is not understood at the present time and will
likely have to await the solution of a crystal structure
for a Cdk4/D/inhibitor complex. Compound 43 specifi-
cally displays a high level of selectivity for Cdk4/D
versus over 36 other kinases.17 This selectivity is further
reflected in cell-based assays in the form of a G1 cell
6-Bromo-8-cyclopentyl-2-(5-piperazin-1-yl-pyridin-2-
ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one Hydrochlo-
ride (17). Under a dry argon atmosphere were combined
6-bromo-8-cyclopentyl-2-methanesulfinyl-8H-pyrido[2,3-d]py-
rimidin-7-one (0.78 g, 2.19 mmol) and 4-(6-amino-pyridin-3-
yl)-piperazine-1-carboxylic acid tert-butyl ester (63, 0.67 g, 2.4
mmol) without solvent. The flask was evacuated and heated
to 120 °C for 1 h. The mixture was purified by chromatography
on silica gel, eluting with chloroform, to give a yellow foam,
0.288 g. Recrystalization from acetonitrile gave 4-[6-(6-bromo-
8-cyclopentyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylami-
no)-pyridin-3-yl]-piperazine-1-carboxylic acid tert-butyl ester
(62 (0.266 g, 21%). MS (APCI) m/z 570.0 (M + 1). Compound
62 (0.26 g, 0.46 mmol) was dissolved in 1:1 chloroform/
methanol (15 mL), to which was added diethyl ether (25 mL).
The solution was purged with anhydrous hydrogen chloride
gas and stoppered for 18 h. The resulting white solid was
collected by filtration, washed with diethyl ether and dried in
1
vacuo at 60 °C to give 17 as a pale yellow solid (0.254 g). H
NMR (400 MHz, DMSO-d6) δ 9.32 (s, 2 H), 8.86 (s, 1 H), 8.47
(s, 1 H), 8.02 (s, 1 H), 7.82 (m, 1 H), 7.79 (d, J ) 9.28 Hz, 1 H),
5.88 (m, 1 H), 3.40 (s, 4 H), 3.22 (s, 4 H), 2.16 (m, 2 H), 1.93
(m, 2 H), 1.77 (m, 2 H), 1.58 (m, 2 H); MS (APCI) m/z ) 470.0
(M + 1). Anal. (C21H24BrN2O‚1.25H2O‚2.2HCl) C, H, N, Cl,
H2O.
4-[6-(8-Isopropyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrim-
idin-2-ylamino)-pyridin-3-yl]-piperazine-1-carboxylic Acid
tert-Butyl Ester (64). A mixture of 2-chloro-8-isopropyl-8H-
pyrido[2,3-d]pyrimidin-7-one (338 mg, 1.5 mmol) and com-
pound 63 (460 mg, 2.0 mmol) in toluene (6 mL) was heated at
110 °C for ∼20 h and then cooled to room temperature. The
solid was collected by filtration, washed with toluene, and
dried. The sample was dissolved in CH2Cl2 and purified by
two preparative TLC plates eluted in 10% MeOH/CH2Cl2. The
band with Rf ) 0.23 was extracted to give 64 as a yellow solid
1
(180 mg, 26%). H NMR (400 MHz, DMSO-d6) δ 9.29(s, 1H),
8.80 (br, 1H), 8.90∼8.17 (m, 2H), 7.70 (d, J ) 2.5 Hz,1H), 7.2
Table 6.
dose
(mg/kg)
Cmax
(ng/mL)
AUC(0 - ∞)
(ng‚h/mL)
AUC(0 - t)
(ng.h/mL)
CL
Vd
(L/kg)
F
(%)
route
Tmax (h)
t1/2(h)
(mL/min/kg)
iv
po
5
5
-
-
2.4 ( 0.3
2.1 ( 0.1
2230 ( 103
1200 ( 393
2160 ( 71
1140 ( 384
37.5 ( 1.7
-
7.0 ( 0.4
-
-
178 ( 47
3.5 ( 1.9
56.1

2394 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Toogood et al.
(d, J ) 9.8 Hz, 1 H), 6.88 (d, J ) 9.6 Hz,1H), 5.6∼5.5 (m, 1H),
4.06 (m, 1 H), 3.9-3.4 (m, 4H), 3.14 (d, J ) 5.2 Hz, 2H), 2.98
(m, 4H), 1.52 (s, 3H), 1.1.50 (s, 3H), 1.38 (s, 9H); MS (APCI)
m/z 466.2 (M + 1).
∼3 min. The solution was stirred at room temperature for ∼6
h then filtered to collect the solid. This solid was washed with
CH2Cl2 and dried in vacuo to provide compound 20 (98 mg,
1
66%). mp 213-215 °C; H NMR (400 MHz, DMSO-d6) δ 9.11
(br s, 2H), 8.86 (s, 1H), 8.01 (s, 1H), 7.86 (d, J ) 9.5 Hz, 1H),
7.85 (br s, 1H), 7.77 (d, J ) 9 Hz, 1H), 6.44 (d, J ) 9 Hz, 1H),
5.83-5.77 (m, 1H), 3.87 (br s, 4H), 3,23 (br s, 4H), 2.21 (br s,
2H), 1.92 (br s, 2H), 1.75 (br s, 2H), 1.58 (br s, 2H); MS (APCI)
m/z 392.1 (M + 1). Anal. (C21H25N7O‚2.0HCl‚2.5H2O): C, H,
N.
4-[6-(8-Cyclohexyl-7-oxo-7,8-dihhydro-pyrido[2,3-d]py-
rimidin-2-ylamino)-pyridin-3-yl]-piperazine-1-carboxyl-
ic Acid Tert-Butyl ester (68). A mixture of 8-cyclohexyl-2-
methanesufinyl-8H-pyrido[2,3-d]pyrimidine-7-one (430 mg,
1.47 mmol) and 63 (556 mg, 2.43 mmol) in toluene (5 mL) was
heated at 100 °C for 18 h. It was cooled to room temperature
and the solid formed was collected and washed with toluene
then dried to give 68 (105 mg, 14%). 1H NMR (400 MHz,
DMSO-d6) δ 10.02 (s, 1H), 8.70 (s, 1H), 8.04 (d, J ) 3.0 Hz,
1H), 7.72 (d, J ) 9.2 Hz, 1H), 7.44 (dd, J ) 9.2, 3.1 Hz, 1 H),
6.28 (m, 1H), 3.60 (m, 4H), 3.08 (m, 4H), 1.6-1.8 (m, 10H),
1.39 (s, 9H);. MS (APCI) m/z 506.1 (M + 1).
8-Isopropyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-
pyrido[2,3-d]pyrimidin-7-one Hydrochloride Salt (18).
HCl gas was bubbled through a solution of 64 (180 mg, 0.39
mmol) in CH2Cl2 (5 mL) at room temperature. The light yellow
solid formed was collected by filtration 5 h later. The solid was
hygroscopic so it was dissolved in MeOH and a few drops of
water were added to the solution. The solvent was then
removed under reduced pressure to generate a glass solid. The
solid was washed with acetone and dried further to yield 18
1
(101 mg, 66%). mp 237-240 °C; H NMR (400 MHz, DMSO-
d6) δ 9.38 (br s, 1 H), 9.28 (s, 1 H), 8.88 (br s, 1 H), 8.14 (d, J
) 9.5 Hz, 1 H), 8.07 (d, J ) 9.0 Hz, 1 H), 7.73 (s, 1 H), 7.23 (d,
J ) 9.5 Hz, 1 H), 6.85 (d, J ) 9.5 Hz, 1 H), 5.57-5.01 (m, 1
H), 3.23 (br s, 4 H), 3.17 (br s, 4 H), 1.49 (s, 3 H), 1.47(s, 3 H);
MS (APCI) m/z 366.2 (M + 1). Exact Mass: Calculated
(C19H23N7O1) 366.2039, found 366.2039 (M + 1); HPLC purity
93%.
8-Cyclopropyl-2-methylsulfinyl-8H-pyrido[2,3-d]pyrim-
idin-7-one (65). A solution of 8-cyclopropyl-2-methylsulfanyl-
8H-pyrido[2,3-d]pyrimidin-7-one (0.5 g, 2.1 mmol) and 2-ben-
zenesulfonyl-3-phenyl-oxaziridine (0.84 g, 3.2 mmol) in CH2Cl2
(5 mL) was stirred at room temperature for 20 h. The white
solid formed was collected by filtration and washed with
hexane, then dried to give 65 (0.388 g, 74%). 1H NMR (400
MHz, DMSO-d6) δ 9.15 (s, 1H), 8.0 (d, J ) 9.5 Hz, 1H), 6.74
(d, J ) 9.5 Hz, 1 H), 2.92 (s, 1H), 1.18-1.14 (m, 2H), 0.83-
0.79 (m, 2H).
8-Cyclohexyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-
8H-pyrido[2,3-d]pyrimidin-7-one Hydrochloride (21). HCl
gas was bubbled through a solution of 68 (105 mg, 0.21 mmol)
in CH2Cl2 (3 mL) at room temperature until a solid was
formed. The mixture was stirred at room temperature for 6 h,
and the solid formed was collected by filtration. The solid was
hygroscopic. It was recrystalized from MeOH with addition of
a few drops of water to yield 21 (40 mg, 35%). mp 228-230
1
°C; H NMR δ (400 MHz, DMSO-d6) 9.34 (br s, 2 H), 8.85 (s,
1 H), 8.03 (s, 2 H), 7.86-7.84 (m, 3 H), 6.42 (d, J ) 8 Hz, 1 H),
5.25 (br s, 1 H), 3.40 (s, 4 H), 3.39 (s, 4 H), 1.79-1.19 (m, 10
H); MS (APCI) m/z 506.1 (M + 1). Anal. (C22H27N7O‚2.0HCl‚
3.5H2O): C, H, N, Cl.
4-[6-(8-Cyclopropyl-7-oxo-7,8-dihhydro-pyrido[2,3-d]-
pyrimidin-2-ylamino)-pyridin-3-yl]-piperazine-1-carbox-
ylic Acid tert-Butyl Ester (66). A mixture of 8-cyclopropyl-
2-methylsulfinyl-8H-pyrido[2,3-d]pyrimidin-7-one (388 mg, 1.56
mmol) and compound 63 (462 mg, 2.0 mmol) in toluene (5 mL)
was heated at 100 °C for 18 h. It was cooled to room
temperature, and the solid was collected by filtration and
washed with toluene and dried to give 66 (96 mg, 13%). 1H
NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.67 (s, 1H), 8.39
(d, J ) 9.3 Hz, 1H), 8.0 (d, J ) 2.95 Hz, 1 H), 7.71 (d, J ) 9.3
Hz, 1H), 6.28 (d, J ) 9.3 Hz, 1H), 3.42 (br, 4H), 3.05 (br, 4H0,
2.80 (m, 1H), 1.37 (s, 9H), 1.20 (d, J ) 6.1 Hz, 2H), 0.76 (br,
2H).
8-Cyclopentyl-6-fluoro-2-methanesulfinyl-8H-pyrido-
[2,3-d]pyrimidin-7-one (69). 8-Cyclopentyl-6-fluoro-2-meth-
ylsulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one (10.5 g, 37.9 mmol)
and 2-benzenesulfonyl-3-phenyl-oxaziridine (11.8 g, 45.4 mmol)
were combined in dichloromethane (120 mL) and stirred at
room temperature for 18 h. The mixture was evaporated to
an oil, crystallized from ethyl acetate/diethyl ether, filtered
and dried in vacuo to provide 69 as a white solid (8.88 g, 80%).
1H NMR (400 MHz, CDCl3) δ 8.94 (s, 1H), 7.25 (d, 1H), 6.06-
5.99 (m, 1H), 2.98 (s, 3H), 2.28-2.21 (m, 2H), 2.18-2.12 (m,
2H), 2.02-1.94 (m, 2H), 1.74-1.67 (m, 2H).
4-[6-(8-Cyclopentyl-6-fluoro-7-oxo-7,8-dihydro-pyrido-
[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-piperazine-1-
carboxylic Acid tert-Butyl Ester (70). Compounds 69 (2.0
g, 6.77 mmol) and 63 (6.0 g, 21 mmol) were added to toluene
(8 mL) and heated to 98 °C for 18 h. The mixture was filtered
and washed with toluene and the solid suspended in diethyl
ether. The mixture was filtered and the solid was dissolved in
chloroform, washed with 1 N citric acid, brine and dried over
anhydrous magnesium sulfate. The crude product was tritu-
rated with diethyl ether and dried in vacuo to provide 70 as a
solid (0.88 g, 25%). MS (APCI) m/z 510.2 (M + 1). 1H NMR
(400 MHz, CDCl3) δ 8.58 (s, 1 H) 8.20 (d, J ) 9.04 Hz, 1 H)
8.01 (d, J ) 2.69 Hz, 1 H) 7.36 (dd, J ) 9.28 Hz, 1 H) 7.23 (s,
1 H) 5.91 (m, 1 H) 3.60 (m, 4 H) 3.11 (m, 4 H) 2.34 (m, 2 H)
2.09 (m, 2 H) 1.90 (m, 2 H) 1.69 (m, 2 H) 1.47 (s, 9 H).
8-Cyclopropyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-
8H-pyrido[2,3-d]pyrimidin-7-one Hydrochloride Salt (19).
HCl gas was bubbled through a solution of 66 (96 mg, 0.21
mmol) in CH2Cl2 (5 mL) for a few min until a solid started to
form. The mixture was stirred at room temperature for 18 h
and then the solid was collected by filtration and washed with
CH2Cl2 then dried in vacuo to give 19 (83 mg, 85%). mp >300
1
°C. H NMR (400 MHz, DMSO-d6) δ 9.07 (br s, 2H), 8.80 (s,
1H), 8.08 (br s, 1H), 8.07 (d, J ) 2 Hz, 1H), 7.86 (br s, 1H),
7.88 (d, J ) 9 Hz, 1H), 6.40 (d, J ) 9 Hz, 1H), 3.37 (br s, 4H),
3.21 (br s, 4H), 2.92-2.90 (m, 1H), 1.23 (d, J ) 6 Hz, 2H),
0.81-0.77 (m, 2H). Anal. (C19H21N7O‚2.1HCl‚1.5H2O): C, H, N.
4-[6-(8-Cyclopentyl-7-oxo-7,8-dihydro-pyrido[2,3-d]py-
rimidin-2-ylamino)-pyridin-3-yl]-piperazine-1-carboxyl-
ic Acid tert-Butyl Ester (67). A mixture of 8-cyclopentyl-2-
methanesulfinyl-8H-pyrido[2,3-d]pyrimidine-7-one (416 mg,
1.5 mmol) and compound 63 (460 mg, 2.0 mmol) in toluene (6
mL) was heated at 110 °C for ∼20 h then cooled to room
temperature. The solid formed was collected by filtration,
8-Cyclopentyl-6-fluoro-2-(5-piperazin-1-yl-pyridin-2-
ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one Hydrochlo-
ride (22). Compound 70 (0.195 g, 0.38 mmol) was dissolved
in 1:1 chloroform/methanol (8 mL), purged with anhydrous
hydrogen chloride gas and stirred for 2.5 h at room temper-
ature. To the mixture was added diethyl ether (15 mL) giving
a precipitate that was filtered, washed with ether and dried
in vacuo to provide 22 as a yellow solid (0.177 g, 88%). MS
1
washed with toluene and dried to give 67 (143 mg, 19%). H
NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.72 (s, 1H), 8.03
(d, J ) 3.0 Hz, 1H), 7.85 (m, 1H), 7.74 (d, J ) 9.2 Hz, 1 H),
7.25 (m, 1H), 6.31 (d, J ) 9.3 Hz, 1H), 5.80 (m, 1 H), 3.4 (m,
4H), 3.28 (m, 4H), 2.47 (m, 2H), 1.9 (m, 2H), 1.87 (br, 2H),
1.6-1.8 (br, 2H), 1.6-1.5 (m, 2H), 1.39 (s, 9H); MS (APCI) m/z
492.2 (M + 1).
1
(APCI) m/z 410.3 (M + 1). H NMR (400 MHz, DMSO-d6) δ
8-Cyclopentyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-
8H-pyrido[2,3-d]pyrimidin-7-one Hydrochloride (20). A
solution of 67 (143 mg, 0.29 mMol) in CH2Cl2/MeOH (6 mL/
1.5 mL) was treated with HCl gas at room temperature for
9.54 (s, 2 H) 8.91 (s, 1 H) 8.01 (s, 1 H) 7.92 (d, J ) 9.04 Hz, 1
H) 7.77 (d, J ) 9.77 Hz, 1 H) 5.85 (m, 1 H) 3.43 (m, 4 H) 3.21
(s, 4 H) 2.20 (m, 2 H) 1.96 (m, 2 H) 1.80 (m, 2 H) 1.58 (m, 2 H).
Anal. (C21H24N7O‚1.0H2O‚2.0HCl): C, H, N.

Inhibitor of Cyclin-Dependent Kinase 4/6
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2395
(8-Cyclopentyl-2-methylsulfanyl-7-oxo-7,8-dihydro-py-
rido[2,3-d]pyrimidin-6-yl)-carbamic Acid tert-Butyl Es-
ter (71). To anhydrous tert-butyl alcohol (30 mL) were added
8-cyclopentyl-2-methylsulfanyl-7-oxo-7,8-dihydro-pyrido[2,3-d]-
pyrimidine-6-carboxylic acid, 72 (2.48 g, 8.02 mmol), trieth-
ylamine (0.974 g, 9.63 mmol) and over 5 min, diphenylphos-
phoryl azide (2.65 g, 9.63 mmol) with stirring. This mixture
was heated at 75 °C for 18 h. The mixture was filtered, and
the solid was washed with ethyl acetate. The washings were
concentrated to an oil enriched in the desired product. The oil
was triturated with hexane/diethyl ether and the washings
were filtered through silica gel and Celite. The filtrate was
concentrated in vacuo yielding 71 as a crystalline solid (1.37
8-Cyclopentyl-2-methanesulfinyl-6-methyl-8H-pyrido-
[2,3-d]pyrimidin-7-one (77). Compound 75 (2.56 g, 9.30
mmol) was dissolved in dichloromethane (17 mL) and metha-
nol (17 mL) to which 2-benzenesulfonyl-3-phenyl-oxaziridine
was added and the reaction mixture was stirred for 16 h. The
solvent was removed and diethyl ether added. The precipitated
solid was collected by filtration to give 77 as a white solid (2.30
g, 85%). 1H NMR (400 MHz, CDCl3) δ 8.85 (s, 1H), 7.54 (s,
1H), 5.99, (m, 1H), 2.95 (s, 3H), 2.27 (d, J ) 1.2 Hz, 3H), 2.24
(m, 2H), 2.13 (m, 2H), 1.94 (m, 2H), 1.70 (m, 2H).
8-Cyclopentyl-6-methyl-2-(5-piperazin-1-yl-pyridin-2-
ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one Hydrochlo-
ride (24). Compound 77 (1.0 g, 3.43 mmol) was added to 63
(1.91 g, 6.86 mmol) in toluene (5 mL). The mixture was heated
to 100 °C over 18 h then treated with diethyl ether to produce
a precipitate. This precipitate was collected by filtration then
dried in vacuo to provide 4-[6-(8-cyclopentyl-6-methyl-7-oxo-
7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-
piperazine-1-carboxylic acid tert-butyl ester (78) as a yellow
solid (0.411 g). MS (APCI) m/z 506.2 (M + 1). Compound 78
(0.411 g, 0.813 mmol) was dissolved in a 1:1 mixture of
methanol:chloroform, purged with anhydrous hydrogen chlo-
ride gas and stirred for 2 h at room temperature. A solid was
precipitated by addition of diethyl ether. The suspension was
filtered and the residue dried in vacuo yielding 24 as a yellow
solid (0.393 g). (APCI) m/z 406.2 (M + 1). 1H NMR (400 MHz,
DMSO-d6) δ 9.34 (s, 2 H) 8.75 (s, 1 H) 8.02 (d, J ) 2.93 Hz, 1
H) 7.82 (d, 1 H) 7.75 (s, 1 H) 7.71 (d, J ) 1.22 Hz, 1 H) 5.84
(m, 1 H) 3.39 (s, 4 H) 3.21 (s, 4 H) 2.20 (m, 2 H) 2.04 (m, 3 H)
1.93 (m, 2 H) 1.74 (m, 2 H) 1.58 (m, 2 H). Anal. (C22H27N7O‚
2.85H2O‚2.2HCl): C, H, N, Cl.
1
g, 45%). MS (APCI) m/z 377.2 (M + 1). H NMR (400 MHz,
CDCl3) δ 8.58 (s, 1 H) 8.12 (s, 1 H) 7.77 (s, 1 H) 5.99 (m, 1 H)
2.57 (s, 3 H) 2.26 (m, 2 H) 2.04 (m, 2 H) 1.87 (m, 2 H) 1.68 (m,
2 H) 1.47 (s, 9 H).
6-Amino-8-cyclopentyl-2-(5-piperazin-1-yl-pyridin-2-
ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one Hydrochlo-
ride (23). Compound 71 (1.3 g, 3.45 mmol) was added to 1:1
dichloromethane:methanol (12 mL) followed by 2-benzene-
sulfonyl-3-phenyl-oxaziridine (1.08 g, 4.14 mmol). The mixture
was stirred at 25 °C for 3.5 h, evaporated to an oil and eluted
through silica gel with chloroform. Fractions containing the
product were evaporated to yield (8-cyclopentyl-2-methane-
sulfinyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidin-6-yl)-carbam-
ic acid tert-butyl ester (73) as a solid (1.2 g, 89%). MS (APCI)
m/z 393.1 (M + 1). Compound 73 (1.2 g, 3.06 mmol) and 63
(2.36 g, 8.48 mmol) were combined in toluene (4 mL) and
heated to 105 °C for 12 h. The resulting paste was diluted with
toluene, filtered, washed with toluene and partitioned between
diethyl ether and 1 N citric acid. The mixture was filtered,
and the solid was washed with water and diethyl ether. The
solid then was dissolved in chloroform, dried over anhydrous
magnesium sulfate, and filtered, and the filtrate diluted with
diethyl ether, giving a solid precipitate. This solid was collected
by filtration and dried in vacuo to give 4-[6-(6-tert-butoxycar-
bonylamino-8-cyclopentyl-7-oxo-7,8-dihydro-pyrido[2,3-d]py-
rimidin-2-ylamino)-pyridin-3-yl]-piperazine-1-carboxylic acid
tert-butyl ester (74) as a solid (0.311 g, 17%). MS (APCI) 607.2
(M + 1). Compound 74 (0.31 g, 0.511 mmol) was added to 1:1
chloroform:methanol (20 mL), and the mixture was purged
with anhydrous hydrogen chloride gas then stirred at room
temperature for 18 h. The resulting solid was collected by
filtration, washed with diethyl ether, and dried in vacuo to 23
as a yellow solid (0.202 g, 100%). MS (APCI) m/z 407.4 (M +
1). 1H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 2 H) 8.72 (s, 1 H)
8.05 (d, 1 H) 7.91 (s, 1 H) 7.58 (d, 1 H) 6.75 (s, 1 H) 5.94 (m,
1 H) 3.37 (m, 4 H) 3.23 (m, 4 H) 2.20 (m, 2 H) 2.00 (m, 2 H)
1.85 (s, 2 H) 1.61 (m, 2 H). Anal. (C21H26N8O‚1.25H2O‚2HCl):
C, H, N.
8-Cyclopentyl-6-methyl-2-methylsulfanyl-8H-pyrido-
[2,3-d]pyrimidin-7-one (75). 2-(Diethoxy-phosphoryl)-propi-
onic acid ethyl ester (15.24 g, 64 mmol) was dissolved in
tetrahydrofuran (100 mL) to which n-butyllithium (47.7 mL,
119 mmol, 2.5 M in hexanes) was slowly added at -70 °C.
4-Cyclopentylamino-2-methylsulfanyl-pyrimidine-5-carbalde-
hyde (76) (15 g, 63 mmol) was dissolved in tetrahydrofuran
(70 mL) then added to the reaction mixture allowing the
reaction to warm to -40 °C. After 3 h the reaction was warmed
to room temperature, poured into cold 1 N citric acid and
extracted with diethyl ether. The organic layer was washed
with brine, dried over MgSO4, filtered and concentrated to give
a yellow oil which was purified by silica gel chromatography.
The resulting oil was dissolved to 1,8-diazabicyclo[5.4.0]undec-
7-ene (75 mL) and heated to 150 °C for 4 h. The reaction
mixture was cooled to room temperature, diluted with ethyl
acetate (350 mL), washed with 5% HCl and brine, dried over
MgSO4, then filtered and concentrated in vacuo. The remain-
ing residue was diluted with diethyl ether and the precipitated
solid was filtered off to give 75 as a white solid (6.33 g, 31%).
1H NMR (400 MHz, CDCl3) δ 8.52 (s, 1H), 7.39 (d, J ) 1.2 Hz,
1H), 5.96, (m, 1H), 2.59 (s, 3H), 2.30 (m, 2H), 2.19 (d, J ) 1.2
Hz, 3H), 2.07 (m, 2H), 1.86 (m, 2H), 1.67 (m, 2H).
8-Cyclopentyl-6-ethyl-2-methanesulfonyl-8H-pyrido-
[2,3-d]pyrimidin-7-one (79). To a cooled (0 °C, ice bath)
solution of 8-cyclopentyl-6-ethyl-2-methylsulfanyl-8H-pyrido-
[2,3-d]pyrimidin-7-one, 80 (5.0 g, 17.28 mmol), in dichlo-
romethane (25 mL) under nitrogen was added m-chloroper-
benzoic acid (MCPBA) (7.4 g, 30.0 mmol). The cold bath was
removed, and the reaction mixture was stirred at room
temperature for 3 h. The reaction mixture was poured into
aq. NaHCO3 (saturated solution, 100 mL) and extracted three
times with dichloromethane (300 mL total). The organic layers
were combined and dried over magnesium sulfate. Removal
of the drying agents and evaporation of the solvent gave a dark
orange oil which was chromatographed on silica gel eluting
with an ethyl acetate/dichloromethane gradient to give 79 as
a white powder. Recrystalization from dichloromethane/hex-
anes gave pure 79 as white needles (3.56 g, 11.1 mmol). mp
1
174-176 °C. H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 7.50
(s, 1H), 5.98-5.89 (m, 1H), 3.36 (s, 3H), 2.68 (q, J ) 7.3 Hz,
2H), 2.30-2.22 (m, 2H), 2.16-2.11 (m, 1H), 1.97-1.89 (m, 1H),
1.72-1.68 (m, 1H), 1.26 (t, J ) 7.3 Hz, 3H); MS (APCI) 322
(M + 1, 100).
8-Cyclopentyl-6-ethyl-2-(5-piperazin-1-yl-pyridin-2-
ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one Hydrochlo-
ride (25). Compounds 79 (0.80 g, 2.62 mmol) and 63 (1.82 g,
6.55 mmol) in toluene (10 mL) were heated to 105 °C over 10
h. The resulting suspension was filtered and the solid washed
with toluene and dried in vacuo yielding 4-[6-(8-cyclopentyl-
6-ethyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino)-
pyridin-3-yl]-piperazine-1-carboxylic acid tert-butyl ester (81)
as a solid (0.204 g). MS (APCI) m/z 520.1 (M + 1). Compound
81 (0.204 g, 0.39 mmol) was dissolved in 1:1 chloroform:
methanol (16 mL) and purged with anhydrous hydrogen
chloride gas. After stirring for 3.5 h, addition of diethyl ether
(8 mL) gave a solid precipitate. The solid was filtered, washed
with diethyl ether and dried in vacuo yielding 25 (0.180 g) as
1
a yellow solid. MS (APCI) m/z 420.2 (M + 1). H NMR (400
MHz, DMSO-d6) δ 9.61 (s, 1 H) 8.90 (s, 1 H) 8.09 (d, J ) 11.72
Hz, 1 H) 7.99 (d, J ) 2.93 Hz, 1 H) 7.79 (s, 1 H) 7.73 (d, J )
9.53 Hz, 1 H) 5.86 (m, 1 H) 3.43 (m, 4 H) 3.21 (m, 4 H) 2.19
(m, 2 H) 1.99 (m, 2 H) 1.77 (m, 2 H) 1.58 (d, J ) 4.88 Hz, 2 H)
1.14 (t, J ) 7.45 Hz, 3H). Anal. (C34H29N7O‚1.2H2O‚2.1HCl):
C, H, N, Cl.

2396 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Toogood et al.
6-Bromomethyl-8-cyclopentyl-2-methylsulfanyl-8H-
pyrido[2,3-d]pyrimidin-7-one (82). Compound 77 (3.5 g,
12.7 mmol) and N-bromosuccinimide (2.6 g, 14.6 mmol) in
carbon tetrachloride (100 mL) were irradiated with ultraviolet
light allowing the temperature to reach 45 °C over 3 h. The
mixture was filtered, washed with dilute sodium sulfite
solution, then brine, and dried over anhydrous magnesium
sulfate. The crude product was chromatographed on silica gel
eluting with 1:1 ethyl acetate:hexane to provide 82 as a
crystalline solid (1.46 g, 32%), mp 103-105 °C. 1H NMR (400
MHz, CDCl3) δ 8.59 (s, 1 H), 7.72 (s, 1 H), 5.94 (m, 1 H), 4.45
(s, 2 H), 2.60 (s, 3 H), 2.32 (m, 2 H), 2.06 (m, 2 H), 1.87 (m, 2
H), 1.67 (m, 2 H).
Acetic Acid 8-Cyclopentyl-2-methylsulfanyl-7-oxo-7,8-
dihydro-pyrido[2,3-d]pyrimidin-6-ylmethyl Ester (83).
Compound 82 (1.33 g, 3.75 mmol) and silver acetate (1.03 g,
6.2 mmol) were added to glacial acetic acid (10 mL) and heated
to 110 °C for 5 h. The solvents then were evaporated at reduced
pressure, and the resulting residue was suspended in ethyl
acetate and filtered. The solid obtained was recrystallized from
ethyl acetate to provide 83 as a solid (0.89 g, 71%). MS (APCI)
m/z 334.2 (M + 1). 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1 H),
7.57 (s, 1 H), 5.93 (m, 1 H), 5.09 (s, 2 H), 2.61 (s, 3 H), 2.32 (m,
2 H), 2.15 (s, 3 H), 2.06 (m, 2 H), 1.88 (m, 2 H), 1.68 (m, 2 H).
and diluted with hexanes to give a precipitate which was
collected by filtration to give 86 as an off-white solid (3.11 g,
24%). MS (APCI) m/z 306.0 (M + 1). 1H NMR (400 MHz,
CDCl3) δ 8.46 (s, 1H), 7.49 (t, J ) 1.7 Hz, 1H), 5.81 (m, 1H),
4.28 (d, J ) 1.7 Hz, 1H), 3.37 (s, 3H), 2.47 (s, 3H), 2.18 (m,
2H), 1.93 (m, 2H), 1.73 (m, 2H), 1.55 (m, 2H).
8-Cyclopentyl-2-methanesulfinyl-6-methoxymethyl-
8H-pyrido[2,3-d]pyrimidin-7-one (87). Compound 86 (4.44
g, 14.54 mmol) and 2-benzenesulfonyl-3-phenyl-oxaziridine
(4.94 g, 18.90 mmol) were dissolved in dichloromethane (100
mL) and stirred at ambient temperature for 12 h. The solvent
volume was reduced to approximately 50 mL and was then
purified by silica gel chromatography to give 87 as an off-white
solid (2.51 g, 54%). MS (APCI) m/z 322.0 (M + 1). 1H NMR
(400 MHz, CDCl3) δ 8.93 (s, 1H), 7.78 (t, J ) 1.7 Hz, 1H), 5.99
(m, 1H), 4.46 (d, J ) 1.7 Hz, 1H), 3.53 (s, 3H), 2.96 (s, 3H),
2.23 (m, 2H), 2.12 (m, 2H), 1.93 (m, 2H), 1.69 (m, 2H).
4-[6-(8-Cyclopentyl-6-methoxymethyl-7-oxo-7,8-dihydro-
pyrido[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-pipera-
zine-1-carboxylic Acid tert-Butyl Ester (88). Compounds
87 (2.5 g, 7.78 mmol) and 63 (2.99 g, 10.73 mmol) were heated
to reflux in toluene (25 mL) for 16 h. The reaction mixture
was cooled to room temperature and purified by silica gel
chromatography to give 88 as a yellow solid (1.24 g, 31%). MS
1
(APCI) m/z 536.4 (M + 1). H NMR (400 MHz, CDCl3) δ 8.59
Acetic Acid 8-Cyclopentyl-2-methanesulfinyl-7-oxo-
7,8-dihydro-pyrido[2,3-d]pyrimidin-6-ylmethyl Ester (84).
Compound 83 (0.85 g, 2.55 mmol) and 2-benzenesulfonyl-3-
phenyl-oxaziridine (0.8 g, 3.06 mmol) were mixed in dichlo-
romethane (20 mL) and stirred at room temperature for 5 h.
To this mixture was added diethyl ether giving a solid
precipitate, which was filtered and dried in vacuo to provide
(s, 1H), 8.26 (d, J ) 9.3 Hz, 1H), 7.97 (d, J ) 2.7 Hz, 1H), 7.6
(t, J ) 1.5 Hz, 1H), 7.38 (dd, J ) 2.7, 9.0 Hz, 1H), 5.89 (m,
1H), 4.55 (d, J ) 1.2 Hz, 1H), 3.66 (q, J ) 7.1 Hz, 2H), 3.60
(m, 4H), 3.11 (m, 4H), 2.34 (m, 2H), 2.07 (m, 2H), 1.88 (m,
2H), 1.69 (m, 2H), 1.48 (s, 9H), 1.30 (t, J ) 6.8 Hz, 3H).
8-Cyclopentyl-6-methoxymethyl-2-(5-piperazin-1-yl-
pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one (27).
Compound 88 (0.110 g, 0.205 mmol) was dissolved in dichlo-
romethane (2 mL). 2 N HCl in diethyl ether (2 mL) was added,
and the reaction mixture was stirred at room temperature for
18 h. The solvent was evaporated to give 27 as a yellow
hydrochloride salt (0.096 g, 92%). mp >300 °C. MS (APCI) m/z
1
84 as a solid (0.81 g, 91%). MS (APCI) m/z 350.2 (M + 1). H
NMR (400 MHz, CDCl3) δ 8.94 (s, 1 H), 7.69 (s, 1 H), 5.96 (m,
1 H), 5.14 (s, 2 H), 2.96 (s, 3 H), 2.22 (m, 2 H), 2.18 (s, 3 H),
2.11 (m, 2 H), 1.93 (m, 2 H), 1.69 (m, 2 H).
4-[6-(6-Acetoxymethyl-8-cyclopentyl-7-oxo-7,8-dihydro-
pyrido[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-pipera-
zine-1-carboxylic Acid tert-Butyl Ester (85). Compounds
84 (0.80 g, 2.29 mmol) and 63 (1.17 g, 4.20 mmol) were added
to toluene (8 mL) and heated to 96 °C for 6 h. The reaction
mixture was allowed to cool then filtered and the residue
washed with toluene. The resulting solid was dried in vacuo
then recrystallized from chloroform/diethyl ether to provide
85 as a solid (0.213 g, 17%). MS (APCI) m/z 564.3 (M + 1). 1H
NMR (400 MHz, CDCl3) δ 8.60 (s, 1 H), 8.29 (d, J ) 9.04 Hz,
1 H), 7.96 (s, 1 H), 7.56 (s, 1 H), 7.41 (d, J ) 9.28 Hz, 1 H),
5.89 (m, 1 H), 5.08 (s, 2 H), 3.60 (m, 4 H), 3.11 (m, 4 H), 2.34
(m, 2 H), 2.14 (s, 3 H), 2.09 (m, 2 H), 1.88 (m, 2 H), 1.69 (m, 2
H), 1.47 (s, 9 H).
1
436.4 (M + 1). H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 2H),
8.92 (s, 1H), 7.95 (s, 1H), 7.92 (m, 1H), 7.85 (s, 1H), 7.70 (d, J
) 9.0 Hz, 1H), 5.81 (m, 1H), 4.26 (s, 2H), 3.36 (m, 7H), 3.20 (s,
4H), 2.17 (m, 2H), 1.91 (m, 2H), 1.73 (m, 2H), 1.55 (m, 2H).
Exact Mass: Calculated (C23H29N7O21) 436.2461, found 436.2467
(M + 1); HPLC purity 95%.
8-Cyclopentyl-6-ethoxymethyl-2-methylsulfanyl-8H-
pyrido[2,3-d]pyrimidin-7-one (89). 3-Ethoxy-propionic acid
ethyl ester (12.31 g, 84.2 mmol) was dissolved in tetrahydro-
furan (40 mL) to which LiHMDS (89 mL, 88.9 mmol, 1.0 M in
THF) was slowly added. Aldehyde 76 (10.0 g, 42.2 mmol) was
then added neat, and the reaction mixture was stirred at
ambient temperature for 17 h then brought to reflux for 7 h.
The reaction mixture was diluted with ethyl acetate and water,
the layers were separated, organic layer was dried over MgSO4
and the solvent was evaporated to give a crude oil. The crude
product was dissolved in ethyl acetate and diluted with
hexanes to give a precipitate, which was collected by filtration
to give 89 as an off-white solid (4.70 g, 35%). MS (APCI) m/z
320.1 (M + 1). 1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 7.52
(t, J ) 1.5 Hz, 1H), 5.82 (m, 1H), 4.32 (d, J ) 1.7 Hz, 1H),
3.53 (q, J ) 7.1 Hz, 2H), 2.47 (s, 3H), 2.17 (m, 2H), 1.93 (m,
2H), 1.73 (m, 2H), 1.54 (m, 2H), 1.15 (t, J ) 7.1 Hz, 3H).
8-Cyclopentyl-6-hydroxymethyl-2-(5-piperazin-1-yl-
pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one Hy-
drochloride (26). Compound 85 (0.21 g,0.36 mmol) was
dissolved in 1:1 chloroform:methanol (8 mL), and the solution
was purged with anhydrous hydrogen chloride gas then
allowed to stir for 3 h at room temperature. This mixture was
added to diethyl ether (50 mL) to give a solid, which was
collected by filtration, washed with diethyl ether, then dried
in vacuo to provide 26 as a solid (0.17 g, 93%). MS (APCI) m/z
1
422.2 (M + 1). H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 2 H),
8.96 (s, 1 H), 7.99 (d, J ) 2.93 Hz, 1 H), 7.93 (d, J ) 8.30 Hz,
1 H), 7.88 (s, 1 H), 7.75 (d, J ) 9.52 Hz, 1 H), 5.85 (m, 1 H),
4.36 (d, J ) 1.47 Hz, 2 H), 3.39 (m, 4 H), 3.23 (m, 4 H), 2.20
(m, 2 H), 1.95 (m, 2 H), 1.76 (m, 2 H), 1.58 (m, 2 H). Anal.
(C22H27N7O2‚1.0H2O‚2.0HCl): C, H, N, Cl.
8-Cyclopentyl-6-ethoxymethyl-2-methanesulfinyl-8H-
pyrido[2,3-d]pyrimidin-7-one (90). Compound 89 (4.60 g,
14.40 mmol) and 2-benzenesulfonyl-3-phenyl-oxaziridine (4.89
g, 18.72 mmol) were dissolved in dichloromethane (30 mL) and
stirred at ambient temperature for 12 h. The crude product
was then purified by silica gel chromatography to give 90 as
a white waxy solid (2.67 g, 55%). MS (APCI) m/z 336.1 (M +
8-Cyclopentyl-6-methoxymethyl-2-methylsulfanyl-8H-
pyrido[2,3-d]pyrimidin-7-one (86). 3-Methoxy-propionic acid
methyl ester (9.95 g, 84.2 mmol) was dissolved in tetrahydro-
furan (40 mL) to which LiHMDS (89 mL, 88.9 mmol, 1.0 M in
THF) was slowly added. Aldehyde 76 (10.0 g, 42.2 mmol) was
then added neat and the reaction mixture brought to reflux
for 7 days. The reaction mixture was diluted with ethyl acetate
and water, the layers were separated, the organic layer was
dried over MgSO4 and the solvent was evaporated to give a
crude oil. The crude product was dissolved in ethyl acetate
1
1). H NMR (400 MHz, CDCl3) δ 8.94 (s, 1H), 7.81 (t, J ) 1.7
Hz, 1H), 5.98 (m, 1H), 4.50 (d, J ) 1.7 Hz, 1H), 3.68 (q, J )
7.1 Hz, 2H), 2.96 (s, 3H), 2.22 (m, 2H), 2.12 (m, 2H), 1.94 (m,
2H), 1.69 (m, 2H), 1.31 (t, J ) 7.1 Hz, 3H).
4-[6-(8-Cyclopentyl-6-ethoxymethyl-7-oxo-7,8-dihydro-
pyrido[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-pipera-
zine-1-carboxylic Acid tert-Butyl Ester (91). Compounds

Inhibitor of Cyclin-Dependent Kinase 4/6
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2397
90 (1.0 g, 2.86 mmol) and 63 (1.10 g, 3.95 mmol) were heated
to reflux in toluene (10 mL) for 16 h. The reaction mixture
was cooled to room temperature and purified by silica gel
chromatography to give 91 as a yellow solid (0.140 g, 15%).
8-Cyclopentyl-6-(2-ethoxy-ethyl)-2-methylsulfanyl-8H-
pyrido[2,3-d]pyrimidin-7-one (95). To a cooled (-78 °C)
solution of 4-ethoxy-butyric acid ethyl ester (9.85 g, 61.47
mmol) in THF (25 mL) was added lithium bis(trimethylsilyl)-
amide (77.0 mL, 76.85 mmol, 1 M solution in THF). The
reaction mixture was stirred for 10 min to form the anion.
Aldehyde 76 (7.29 g, 30.7 mmol) was then added and the
reaction allowed to warm to room temperature and stirred
overnight. The reaction mixture was quenched with 10%
aqueous HCl (100 mL). The aqueous layer was extracted with
ethyl acetate (150 mL total), and the organic layers were
combined and concentrated to give a yellow oil. Chromato-
graphic purification on silica gel (chloroform/ethyl acetate
gradient) gave 95 (3.22 g, 9.65 mmol, 31%). MS (APCI) m/z
334 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H),
7.47-7.46 (m, 1H), 5.99-5.90 (m, 1H), 3.69 (t, J ) 6.25 Hz,
2H), 3.49 (q, J ) 7.03 Hz, 2H), 2.84 (t, J ) 6.25 Hz, 2H), 2.59
(s, 3H), 2.34-2.29 (m, 2H), 2.08-2.02 (m, 2H), 1.88-1.83 (m,
2H), 1.69-1.65 (m, 3H), 1.17 (t, J ) 7.04 Hz, 3H).
1
MS (APCI) m/z 550.4 (M + 1). H NMR (400 MHz, CDCl3) δ
8.59 (s, 1H), 8.26 (d, J ) 9.3 Hz, 1H), 7.97 (d, J ) 2.7 Hz, 1H),
7.6 (t, J ) 1.5 Hz, 1H), 7.38 (dd, J ) 2.7, 9.0 Hz, 1H), 5.89 (m,
1H), 4.55 (d, J ) 1.2 Hz, 1H), 3.66 (q, J ) 7.1 Hz, 2H), 3.60
(m, 4H), 3.11 (m, 4H), 2.34 (m, 2H), 2.07 (m, 2H), 1.88 (m,
2H), 1.69 (m, 2H), 1.48 (s, 9H), 1.30 (t, J ) 6.8 Hz, 3H).
8-Cyclopentyl-6-ethoxymethyl-2-(5-piperazin-1-yl-py-
ridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one (28).
Compound 91 (0.140 g, 0.242 mmol) was dissolved in dichlo-
romethane (2 mL). 2 N HCl in diethyl ether (2 mL) was added,
and the reaction mixture was stirred at room temperature for
18 h. The solvent was evaporated to give 28 as a yellow solid
(0.116 g, 86%). Foams >190 °C. MS (APCI) m/z 450.1 (M + 1).
1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 2H), 8.34 (s, 1H), 8.01
(d, J ) 2.7 Hz, 1H), 7.86 (s, 1H), 7.83 (s, 1H), 7.76 (d, J ) 9.5
Hz, 1H), 5.84 (m, 1H), 4.32 (d, J ) 1.2 Hz, 1H), 3.57 (q, J )
6.8 Hz, 2H), 3.38 (m, 4H), 3.23 (m, 4H), 2.26 (m, 2H), 1.89 (m,
2H), 1.75 (m, 2H), 1.58 (m, 2H), 1.19 (t, J ) 6.8 Hz, 3H). Exact
Mass: Calculated (C23H29N7O2) 450.2617, found 450.2633 (M
+ 1); HPLC purity 96%.
8-Cyclopentyl-6-(2-methoxy-ethoxymethyl)-2-methyl-
sulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one (92). Compound
82 (1.33 g, 3.75 mmol) was dissolved in 2-methoxyethanol (10
mL) to which potassium carbonate (0.778 g, 5.63 mmol) was
added, and the mixture was stirred at room temperature for
2 h. The reaction mixture was then filtered and the salts
washed with ethyl acetate. The combined organics were
evaporated to give 92 as a waxy solid (1.00 g, 76%). MS (APCI)
m/z 350.2 (M + 1). 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H),
7.71 (t, J ) 1.6 Hz, 1H), 5.95 (m, 1H), 4.52 (d, J ) 1.6 Hz,
1H), 3.76 (m, 2H), 3.63 (m, 2H), 3.41 (s, 3H), 2.60 (s, 3H), 2.32
(m, 2H), 2.06 (m, 2H), 1.87 (m, 2H), 1.68 (m, 2H).
8-Cyclopentyl-2-methanesulfinyl-6-(2-methoxy-ethoxy-
methyl)-8H-pyrido[2,3-d]pyrimidin-7-one (93). Compound
92 (1.46 g, 4.18 mmol) and 2-benzenesulfonyl-3-phenyl-oxa-
ziridine (1.31 g, 5.01 mmol) were dissolved in dichlorometh-
ane (10 mL) and stirred at ambient temperature for 12 h. The
reaction mixture was then purified by silica gel chromatog-
raphy to give 93 as a white waxy solid (0.60 g, 39%). MS (APCI)
m/z 366.0 (M + 1). 1H NMR (400 MHz, CDCl3) δ 8.94 (s, 1H),
7.88 (t, J ) 1.7 Hz, 1H), 5.95 (m, 1H), 4.52 (d, J ) 1.7 Hz,
1H), 3.80 (m, 2H), 3.65 (m, 2H), 3.43 (s, 3H), 2.98 (s, 3H), 2.25
(m, 2H), 2.13 (m, 2H), 1.94 (m, 2H), 1.70 (m, 2H).
4-{6-[8-Cyclopentyl-6-(2-ethoxy-ethyl)-7-oxo-7,8-dihy-
dro-pyrido[2,3-d]pyrimidin-2-ylamino]-pyridin-3-yl}-pi-
perazine-1-carboxylic Acid tert-Butyl Ester (96). Com-
pounds 95 (1.0 g, 2.86 mmol) and 63 (1.10 g, 3.95 mmol) were
heated to reflux in toluene (10 mL) for 16 h. The mixture was
cooled to room temperature and purified by silica gel chroma-
tography to give 96 as an orange solid (0.328 g, 20%). MS
1
(APCI) m/z 564.2 (M + 1). H NMR (400 MHz, CDCl3) δ 8.54
(s, 1H), 8.26 (d, J ) 9.0 Hz, 1H), 7.98 (d, J ) 2.9 Hz, 1H), 7.45
(s, 1H), 7.38 (dd, J ) 2.9, 9.3 Hz, 1H), 5.90 (m, 1H), 3.70 (t, J
) 6.3, 1H), 3.61 (m, 4H), 3.51 (q, J ) 7.1, 1H), 3.11 (m, 4H),
2.84 (t, J ) 5.9, 1H), 2.33 (m, 2H), 2.08 (m, 2H), 1.87 (m, 2H),
1.69 (m, 2H), 1.48 (s, 9H), 1.19 (t, J ) 7.1, 1H).
8-Cyclopentyl-6-(2-ethoxy-ethyl)-2-(5-piperazin-1-yl-
pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one Hy-
drochloride (30). Compound 96 (0.325 g, 0.577 mmol) was
dissolved in dichloromethane (4 mL). 2 N HCl in diethyl ether
(4 mL) was added and the mixture was stirred at room
temperature for 18 h. The solvent was evaporated to give 30
as a yellow solid (0.292 g, 98%). MS (APCI) m/z 464.1 (M +
1). 1H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 2H), 8.86 (s, 1H),
7.97 (d, J ) 2.68 Hz, 2H), 7.78 (s, 1H), 7.71 (d, J ) 10.01 Hz,
1H), 5.80-5.87 (m, 1H), 3.54 (t, J ) 6.59 Hz, 2H), 3.36-3.46
(m, 6H), 3.20 (s, 4H), 2.67 (t, J ) 6.47 Hz, 2H), 2.10-2.23 (m,
2H), 1.90-1.98 (m, 2H), 1.71-1.79 (m, 2H), 1.52-1.59 (m, 2H),
1.05 (t, J ) 6.95 Hz, 3H). Anal. (C25H33N7O2‚2.6HCl‚0.35H2O):
C, H, N.
8-Cyclopentyl-6-(2-ethoxy-ethoxy)-2-methylsulfanyl-
8H-pyrido[2,3-d]pyrimidin-7-one (97). To a suspension of
sodium hydride (45 mg, 1.1 mmol, 60% oil dispersion) in THF
(10 mL), under nitrogen, was added 2-ethoxyethanol (113 mg,
1.25 mmol). The reaction mixture was stirred at room tem-
perature for 30 min. To this mixture, 8-cyclopentyl-6-fluoro-
2-methylsulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one (98, 280 mg,
1.0 mmol) was added. The reaction mixture was then heated
to reflux and stirred overnight. The cooled solution was
quenched with water (25 mL) and extracted with ethyl acetate
(50 mL). The organic layer was subsequently washed twice
with aq. NH4Cl (20 mL each) and brine (20 mL). The organic
layer was dried over magnesium sulfate. Removal of the drying
agents and evaporation of the solvent gave a yellow oil, which
was chromatographed on silica gel eluted with an ethyl
acetate/hexane gradient to 97 as a colorless oil (289 mg, 0.83
mmol, 83%). MS (APCI) m/z 350 (M + 1). 1H NMR (400 MHz,
CDCl3) δ 8.52 (s, 1H), 6.77 (s, 1H), 6.04-5.95 (m, 1H), 4.16 (t,
J ) 4.0 Hz, 2H), 3.86 (t, J ) 4.0 Hz, 2H), 3.58 (q, J ) 8.0 Hz,
2H), 2.59 (s, 3H), 2.34-2.25 (m, 2H), 2.13-2.03 (m, 2H), 1.91-
1.82 (m, 2H), 1.71-1.60 (m, 2H), 1.20 (t, J ) 8.0 Hz, 3H).
4-{6-[8-Cyclopentyl-6-(2-methoxy-ethoxymethyl)-7-oxo-
7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino]-pyridin-
3-yl}-piperazine-1-carboxylic Acid tert-Butyl Ester (94).
Compounds 93 (1.0 g, 2.86 mmol) and 63 (1.10 g, 3.95 mmol)
were heated to reflux in toluene (10 mL) for 16 h. The reaction
mixture was cooled to room temperature and purified by silica
gel chromatography to give 94 as a yellow solid (0.140 g, 15%).
1
MS (APCI) m/z 580.5 (M + 1). H NMR (400 MHz, CDCl3) δ
8.60 (s, 1H), 8.34 (m, 1H), 7.95 (s, 1H), 7.69 (t, J ) 1.4 Hz,
1H), 7.42 (m, 1H), 5.91 (m, 1H), 4.53 (d, J ) 1.2 Hz, 1H), 3.78
(m, 1H), 3.63 (m, 6H), 3.43 (s, 3H), 3.11 (m, 4H), 2.35 (m, 2H),
2.08 (m, 2H), 1.88 (m, 2H), 1.69 (m, 2H), 1.48 (s, 9H).
8-Cyclopentyl-6-(2-methoxy-ethoxymethyl)-2-(5-piper-
azin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-
7-one hydrochloride (29). Compound 94 (0.140 g, 0.242
mmol) was dissolved in dichloromethane (2 mL). 2 N HCl in
diethyl ether (2 mL) was added, and the reaction mixture was
stirred at room temperature for 18 h. The solvent was
evaporated to give 29 as a yellow solid (0.116 g, 86%). Foams
1
>190 °C. H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 2H), 8.93
(s, 1H), 8.01 (d, J ) 2.69 Hz, 1H), 7.83 (s, 2H), 7.78 (d, J )
9.04 Hz, 1H), 5.80-5.87 (m, 1H), 4.36 (d, J ) 1.22 Hz, 2H),
3.65 (dd, J ) 5.62, 3.66 Hz, 2H), 3.46-3.52 (m, 2H), 3.35-
3.42 (m, 4H), 3.24 (d, J ) 10.01 Hz, 7H), 2.16-2.24 (m, 2H),
1.90-1.98 (m, 2H), 1.72 - 1.79 (m, 2H), 1.53-1.59 (m, 2H).
MS (APCI) m/z 480.2 (M + 1). Anal. (C25H33N7O2‚2.16HCl):
C, H, N.
8-Cyclopentyl-6-(2-ethoxy-ethoxy)-2-methanesulfinyl-
8H-pyrido[2,3-d]pyrimidin-7-one (99). To a solution of 97
(289 mg, 0.83 mmol) in chloroform (5 mL) was added 2-ben-
zenesulfonyl-3-phenyl-oxaziridine (281 mg, 1.07 mmol). The
reaction mixture was stirred at room-temperature overnight,
under nitrogen. The solvents were removed, and the crude
product was chromatographed on silica gel, eluting with a 5%

2398 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Toogood et al.
methanol-ethyl acetate/hexane gradient to give 99 as a
colorless oil (210 mg, 67%). 1H NMR (400 MHz, CDCl3) δ 8.84
(s, 1H), 6.89 (s, 1H), 6.06-5.98 (m, 1H), 4.23 (t, J ) 4.0 Hz,
2H), 3.89 (t, J ) 4.0 Hz, 2H), 3.60 (q, J ) 6.9 Hz, 2H), 2.95 (s,
3H), 2.28-2.19 (m, 2H), 2.15-2.10 (m, 2H), 1.97-1.88 (m, 2H),
1.71-1.64 (m, 2H), 1.21 (t, J ) 6.9 Hz, 3H).
butyl ester (104) (440 mg, 0.77 mmol) and tetrakis(triphenyl-
phosphine)palladium(0) (88 mg, 0.077 mmol) in toluene (5 mL).
The reaction mixture was heated at 110 °C for 1 h then cooled
to room temperature. The solid so formed was collected by
filtration and washed with toluene, then dried to give 103. MS
1
(APCI) m/z 534.2 (M + 1). H NMR (400 MHz, DMSO-d6) δ
9.07 (s, 1H), 8.46 (s, 1H), 7.97 (s, 1H), 7.90 (br s, 1H), 7.72 (d,
J ) 9 Hz, 1H), 5.83 (m, 1H), 3.47 (br s, 4H), 3.16 (br s, 4H),
2.55 (s, 3H), 2.20 (br s, 2H), 1.94 (br s, 2H), 1.78 (br s, 2H),
1.58 (br s, 2H), 1.39 (s, 9H).
4-{6-[8-Cyclopentyl-6-(2-ethoxy-ethoxy)-7-oxo-7,8-dihy-
dro-pyrido[2,3-d]pyrimidin-2-ylamino]-pyridin-3-yl}-pi-
perazine-1-carboxylic Acid tert-Butyl Ester (100). Com-
pounds 99 (1.2 mL of a 0.46 M solution in toluene, 0.552 mmol)
and 63 (0.307 g, 1.1 mmol) were combined in toluene under
nitrogen and heated to 110 °C. After 4 h the toluene was
replaced by xylenes (1 mL) and heating was continued under
reflux overnight. After cooling to room temperature, the crude
reaction mixture was dissolved in CH2Cl2 and washed with
saturated aqueous ammonium chloride solution then with
brine. The organic layer was dried (MgSO4), filtered and
evaporated to dryness. Chromotagraphy on silica gel eluting
with 5% CH3OH in CH2Cl2 followed by a second chromatog-
raphy step eluting with ethyl acetate provided 100 (70 mg,
22%) as a yellow solid. MS (APCI) m/z 580.2 (M + 1). 1H NMR
(400 MHz, DMSO- d6) δ 8.50 (s, 1H), 8.26 (d, J ) 9 Hz, 1H),
7.94 (d, J ) 3 Hz, 1H), 7.39 (dd, J ) 3, 9 Hz, 1H), 6.78 (s, 1H),
5.89-5.98 (m, 1H), 4.15 (t, J ) 5 Hz, 2H), 3.86 (t, J ) 5 Hz,
2H), 3.56-3.62 (m, 6H), 3.09 (br t, J ) 5 Hz, 4H), 2.29-2.33
(m, 2H), 2.07-2.10 (m, 2H), 1.84-1.92 (m, 2H), 1.63-1.69 (m,
2H), 1.47 (s, 9H), 1.22 (t, J ) 7 Hz, 3H).
8-Cyclopentyl-6-(2-ethoxy-ethoxy)-2-(5-piperazin-1-yl-
pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one Hy-
drochloride (31). Compound 100 (70 mg, 0.12 mmol) was
dissolved in CH2Cl2 (2.5 mL), and 2 M HCl in ether (2.5 mL)
was added. This mixture was stirred for 2 h at room temper-
ature, and a yellow precipitate formed. The solvents were
removed under reduced pressure, and the resulting solid was
suspended in ether and collected by filtration then dried
overnight in vacuo at 50 °C to give 31 (30 mg, 52%). MS (APCI)
m/z 480.4 (M + 1). Anal. (C25H33N7O3‚2HCl‚3.44H2O): C, H,
N. 1H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 2H), 8.80 (s, 1H),
7.96 (s, 2H), 7.71 (d, J ) 9 Hz, 1H), 7.31 (s, 1H), 5.93-5.85
(m, 1H), 4.08 (t, J ) 4 Hz, 2H), 3.72 (t, J ) 4 Hz, 2H), 3.50 (q,
J ) 7 Hz, 2H), 3.39 (s, 4H), 3.38 (s, 4H), 2.21-2.16 (m, 2H),
1.97 (s, 2H), 1.80-1.78 (m, 2H), 1.61-1.58 (m, 2H), 1.11 (t, J
) 7 Hz, 3H).
8-Cyclopentyl-6-isobutoxy-2-(5-piperazin-1-yl-pyridin-
2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one hydrochlo-
ride (32). 60% Sodium hydride in oil (0.182 g, 4.4 mmol) was
washed with hexane and added to 2-methyl-1-propanol (10
mL). This mixture effervesced and formed a solution. To this
solution was added 4-[6-(8-cyclopentyl-6-fluoro-7-oxo-7,8-di-
hydro-pyrido[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-pipera-
zine-1-carboxylic acid tert-butyl ester, 102 (0.225 g, 0.44 mmol),
and the mixture was heated at 95 °C for 72 h. The solvents
were evaporated, and the residue was dissolved in diethyl
ether then filtered. The filtrate was evaporated to provide 4-[6-
(8-cyclopentyl-6-isobutoxy-7-oxo-7,8-dihydro-pyrido[2,3-d]py-
rimidin-2-ylamino)-pyridin-3-yl]-piperazine-1-carboxylic acid
tert-butyl ester (101) as a crystalline solid (0.092 g, 37%). MS
(APCI) m/z 564.3 (M + 1). Compound 101 (0.067 g, 0.119
mmol) was dissolved in chloroform (5 mL), cooled to 0 °C. This
solution was purged with anhydrous hydrogen chloride gas and
stoppered for 3 h. Diethyl ether was added to the mixture
giving a precipitate that was filtered and dried in vacuo to 32
as a solid (0.056 g, 36%). MS (APCI) m/z 464.3 (M + 1). 1H
NMR (400 MHz, DMSO-d6) δ 9.25 (s, 2 H), 8.80 (s, 1 H), 7.95
(s, 1 H), 7.71 (s, 1 H), 7.29 (s, 1 H), 5.89 (m, 1 H), 3.73 (s, 4 H),
3.38 (m, 4 H), 3.23 (d, 2 H), 2.19 (s, 2 H), 1.97 (m, 2 H), 1.78
(m, 2 H), 1.58 (m, 2 H), 0.98 (d, J ) 5.86 Hz, 6 H). Anal.
(C25H33N7O2‚1.0H2O‚2.0HCl): C, H, N, Cl.
6-Acetyl-8-cyclopentyl-2-(5-piperazin-1-yl-pyridin-2-
ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one hydrochlo-
ride (33). Anhydrous HCl gas was bubbled through a solution
of 103 (398 mg, 0.74 mmol), in MeOH/CH2Cl2 (10 mL/10 mL)
at room temperature for ∼5 min. The reaction mixture was
stirred overnight, and then solvent was removed under
reduced pressure. The remaining solid was triturated with hot
ethyl acetate and dried to provide 33 (329 mg, 76%). mp >300
°C. MS (APCI) m/z 434.2 (M + 1). 1H NMR (400 MHz, DMSO-
d6) δ 8.99 (s, 1H), 8.39 (s, 1H), 8.08 (s, 1H), 7.83 (d, J ) 9 Hz,
1H), 7.65 (br s, 1H), 5.84-5.79 (m, 1H), 3.37 (br s, 4H), 2.54
(s, 3H), 2.24 (br s, 2H), 1.89 (br s, 2H), 1.76 (br s, 2H), 1.58 (br
s, 2H). Anal. (C23H27N7O2‚4.25HCl): C, H, N.
2-[5-(4-tert-Butoxycarbonyl-piperazin-1-yl)-pyridin-2-
ylamino]-8-cyclopentyl-7-oxo-7,8-dihydro-pyrido[2,3-d]-
pyrimidine-6-carboxylic Acid Ethyl Ester (105). 8-Cyclo-
pentyl-2-methylsulfanyl-7-oxo-7,8-dihydro-pyrido[2,3-d]py-
rimidine-6-carboxylic acid ethyl ester, 106 (1.2 g, 3.6 mmol),
was dissolved in CH2Cl2 (20 mL) and treated with 2-benzene-
sulfonyl-3-phenyl-oxaziridine (1.13 g, 4.32 mmol) at room
temperature and stirred for 1 day. Following concentration
under reduced pressure, the crude reaction mixture was
chromatographed on silica gel eluting with ethyl acetate to
give 8-cyclopentyl-2-methanesulfinyl-7-oxo-7,8-dihydro-pyrido-
[2,3-d]pyrimidine-6-carboxylic acid ethyl ester (107) as a white
solid (0.85 g, 68%). MS (APCI) m/z 350.0 (M + 1). Compounds
107 (0.936 g, 2.68 mmol) and 63 (3.0 g, 10.8 mmol) were added
to toluene (5 mL) and heated to 100 °C for 1 h. Diethyl ether
(10 mL) was added causing a solid to precipitate. This solid
was collected by filtration, washed with diethyl ether, and
dried in vacuo yielding 105 as a yellow solid (0.42 g, 28%).
1
MS (APCI) m/z 564.3 (M + 1). H NMR (400 MHz, CDCl3) δ
8.67 (s, 1 H), 8.22 (s, 1 H), 8.16 (d, J ) 9.04 Hz, 1 H), 8.05 (d,
J ) 2.44 Hz, 1 H), 7.30 (dd, J ) 9.28, 2.93 Hz, 1 H), 5.85 (m,
1 H), 4.34 (q, J ) 7.08 Hz, 2 H), 3.57 (m, 4 H), 3.09 (m, 4 H),
2.33 (m, 2 H), 2.05 (m, 2 H), 1.82 (m, 2 H), 1.62 (m, 2 H), 1.44
(s, 9 H), 1.34 (t, J ) 7.08 Hz, 3 H).
8-Cyclopentyl-7-oxo-2-(5-piperazin-1-yl-pyridin-2-ylami-
no)-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carboxylic Acid
Ethyl Ester Hydrochloride (34). Compound 105 (0.40 g,
0.709 mmol) was dissolved in a mixture of chloroform (15 mL)
and ethanol (15 mL) and the solution was purged with
anhydrous hydrogen chloride gas. After 2 h, the addition of
ethyl acetate precipitated a solid that was filtered, washed
with diethyl ether and dried in vacuo to yield 34 (0.4 g) as a
yellow solid. MS (APCI) m/z 464.4 (M + 1). 1H NMR (400 MHz,
DMSO-d6) δ 9.46 (s, 2 H), 9.00 (s, 1 H), 8.47 (s, 1 H), 8.03 (d,
J ) 2.4 Hz, 1 H), 7.89 (s, 1 H), 7.79 (s, 1 H), 5.79 (m, 1 H),
4.23 (q, J ) 7.0 Hz, 4 H), 3.41 (m, 4 H), 2.17 (m, 2 H), 1.91 (m,
2 H), 1.74 (m, 2 H), 1.55 (d, J ) 4.6 Hz, 2 H), 1.25 (t, J ) 7.0
Hz, 3 H). Anal. (C24H29N7O3‚0.75H2O‚2.0HCl): C, H, N.
4-[6-(8-Cyclopentyl-5-methyl-7-oxo-7,8-dihydro-pyrido-
[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-piperazine-1-
carboxylic Acid tert-Butyl Ester (108). 8-Cyclopentyl-2-
methanesulfinyl-5-methyl-8H-pyrido[2,3-d]pyrimidin-7-one (109)
(0.40 g, 1.37 mmol) and 63 (0.497 g, 1.78 mmol) were heated
to reflux in toluene (4 mL) for 16 h. The reaction mixture was
cooled to room temperature, and the precipitate that formed
was collected by filtration and washed on the funnel with
toluene (3 × 10 mL) to give 108 as a dark brown-gray solid
4-[6-(6-Acetyl-8-cyclopentyl-7-oxo-7,8-dihydro-pyrido-
[2,3-d]pyrimidin-2-ylamino)-pyridin-3yl]-piperazine-1-
carboxylic Acid tert-Butyl Ester (103). Tributyl(1-ethoxy-
vinyl)tin (0.39 mL, 1.15 mmol) was added to a mixture of 4-[6-
(6-bromo-8-cyclopentyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimi-
din-2-ylamino)-pyridin-3-yl]-piperazine-1-carboxylic acid tert-
1
(0.100 g, 16%). MS (APCI) m/z 506.2, (M + 1). H NMR (400
MHz, DMSO-d6) δ 9.92 (s, 1H), 8.78 (s, 1H), 8.02 (d, J ) 2.9
Hz, 1H), 7.87 (d, J ) 9.3 Hz, 1H), 7.50 (dd, J ) 2.9, 9.0 Hz,
1H), 6.18 (s, 1H), 5.77 (m, 1H), 3.44 (m, 4H), 3.07 (m, 4H),

Inhibitor of Cyclin-Dependent Kinase 4/6
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2399
2.39 (s, 3H), 2.20 (m, 2H), 1.85 (m, 2H), 1.71 (m, 2H), 1.55 (m,
2H), 1.39 (s, 9H).
at room temperature for a total of 3 days, then the solvents
were removed by evaporation at reduced pressure. The re-
maining solid was suspended in ether and filtered to give 41
as a yellow solid (0.04 g, 100%), which was dried in vacuo at
8-Cyclopentyl-5-methyl-2-(5-piperazin-4-yl-pyridin-2-
ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one hydrochlo-
ride (38). Compound 108 (0.093 g, 0.184 mmol) was dissolved
in dichloromethane (3 mL) to which 2 N HCl in diethyl ether
(2 mL) was added, and the resulting mixture was stirred for
2 days. Additional 2 N HCl was added and stirring was
continued for 16 h. The solvent was removed to 38 as a yellow
solid (0.080 g, 91%). Decomposes >212 °C. MS (APCI) m/z
1
50 °C. mp > 235 °C. MS (APCI) m/z 486.1 (M + 1). H NMR
(400 MHz, DMSO-d6) δ 9.17 (s, 2H), 8.98 (s, 1H), 8.04 (s, 1H),
7.84-7.79 (m, 2H), 5.92-5.87 (m, 1H), 3.39 (br s, 4H), 3.29
(br s, 4H), 2.58 (s, 3H), 2.15 (br s, 2H), 1.91 (br s, 2H), 1.76 (br
s, 2H), 1.57 (br s, 2H). Anal. (C23H26N7OBr‚2.64H2O‚2.0HCl):
Calc′d: C, 43.68; H, 5.55; N, 16.21, Cl (ionic), 11.72. Found:
C, 44.08; H, 5.32; N, 15.23, Cl (ionic), 11.65.
1
406.1, (M + 1). H NMR (400 MHz, DMSO-d6) δ 9.27 (s, 2H),
8.85 (s, 1H), 8.02 (d, J ) 2.9 Hz, 1H), 7.91 (d, J ) 9.3 Hz, 1H),
7.78 (d, J ) 9.3 Hz, 1H), 6.33 (s, 1H), 5.86-5.74 (m, 1H), 3.44-
3.35 (m, 4H), 3.26-3.19 (m, 4H), 2.39 (s, 3H), 2.23-2.15 (m,
2H), 1.94-1.86 (m, 2H), 1.74 (m, 2H), 1.59-1.51 (m, 2H). Exact
Mass: Calculated (C22H27N7O1) 406.2355, found 450.2671 (M
+ 1); HPLC purity 97%.
6-Acetyl-8-cyclopentyl-5-methyl-2-(pyridin-2-ylamino)-
8H-pyrido[2,3-d]pyrimidin-7-one (42). 6-Acetyl-2-amino-8-
cyclopentyl-5-methyl-8H-pyrido[2,3-d]pyrimidin-7-one (115, 195
mg, 0.681 mmol) and sodium tert-butoxide (92 mg, 0.953 mmol)
were suspended in N2-purged toluene (5 mL). To this suspen-
sion were added 2-bromo pyridine (78 µL), tris(dibenzyli-
deneacetone)-dipalladium(0) (25 mg, 0.027 mmol) and BINAP
(34 mg, 0.054 mmol). The reaction vial was purged with argon,
and the reaction was heated at 70 °C overnight. The reaction
mixture was diluted with ether and methanol, filtered through
a pad of Celite and concentrated under reduced pressure. The
crude product was chromatographed on silica gel eluting with
a gradient of 40% to 100% ethyl acetate in hexanes. Compound
42 as a solid (40 mg, 16%). mp >210 °C. MS (APCI) m/z 364.1
(M + 1). 1H NMR (400 MHz, CDCl3) δ 8.84 (s, 1H), 8.35-8.32
(m, 2H), 8.21 (bs, 1H), 7.75-7.71 (m, 1H), 7.03-7.01 (m, 1H),
5.89-5.85 (m, 1H), 2.54 (s, 3H), 2.37 (s, 3H), 2.03-2.08 (m,
2H), 1.92-1.87 (m, 2H), 1.73-1.67 (m, 2H).
6-Bromo-8-cyclopentyl-2-methanesulfinyl-8H-pyrido-
[2,3-d]pyrimidin-7-one (110). Prepared from 6-bromo-8-cy-
clopentyl-2-methylsulfanyl-8H-pyrido[2,3-d]pyrimidin-7-one
(111) following the procedure described for 69. MS (APCI) m/z
358 (M + 1). 1H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H),
8.63 (s, 1H), 5.91-5.86 (m, 1H), 2.89 (s, 3H), 2.15 (br s, 2H),
2.04 (br s, 2H), 1.87-1.79 (m, 2H), 1.61-1.58 (m, 2H).
6-Bromo-8-cyclopentyl-2-(pyridin-2-ylamino)-8H-pyrido-
[2,3-d]pyrimidin-7-one (39). Compound 110 and 2-aminopy-
ridine were reacted according to the procedure described for
compound 108 to 39 in 37% yield. mp: 273∼275 °C. MS (APCI)
1
m/z 385.9 (M + 1). H NMR (400 MHz, DMSO-d6) δ 10.33 (s,
1H), 8.80 (s, 1H), 8.38 (s, 1H), 8.31 (dd, J ) 5 Hz, 1 Hz, 1H),
8.04 (d, J ) 8.5 Hz, 1H), 7.81-7.77 (m, 1H), 7.07-7.04 (m,
1H), 5.90-5.71 (m, 1H), 2.27 (br s, 2H), 1.91 (br s, 2H), 1.79
(br s, 2H), 1.58 (br s, 2H). Anal. (C17H16BrN5O‚0.1H2O): C, H,
N.
6-Bromo-8-cyclopentyl-2-methanesulfinyl-5-methyl-
8H-pyrido[2,3-d]pyrimidin-7-one (112). Compound 112 was
prepared from 6-bromo-8-cyclopentyl-5-methyl-2-methylsul-
fanyl-8H-pyrido[2,3-d]pyrimidin-7-one (113) following the pro-
cedure described for compound 69. MS (APCI) m/z 372.9 (M
+ 1). 1H NMR (400 MHz, CDCl3) δ 9.01 (s, 1H), 6.06-5.97 (m,
1H), 2.93 (s, 3H), 2.67 (s, 3H), 2.21-2.11 (m, 2H), 2.10-2.04
(m, 2H), 1.94-1.87 (m, 2H), 1.67-1.62 (m, 2H).
6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-py-
ridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one Hy-
drochloride (43). A suspension of 114 (5.93 g, 0.010 mol),
tetrakis(triphenylphosphine)palladium(0) (1.40 g, 0.00121 mol)
and tributyl(1-ethoxyvinyl)tin (5.32 mL, 0.0157 mol) in toluene
(30 mL) was heated under reflux for 3.5 h. The mixture was
cooled and filtered to give a solid. Purification of the solid by
silica gel chromatography using a gradient of 5-66% ethyl
acetate/hexane over 15 min gave 4-{6-[8-cyclopentyl-6-(1-
ethoxy-vinyl)-5-methyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimi-
din-2-ylamino]-pyridin-3-yl}-piperazine-1-carboxylic acid tert-
butyl ester, 116, as a yellow foam (4.50 g, 78%). MS (APCI)
m/z 576.3 (M + 1). This foam was dissolved in dichloromethane
(100 mL) and cooled using an ice-water bath. Hydrogen
chloride gas was bubbled into the solution, and the resulting
suspension was stoppered and stirred at room-temperature
overnight, then diluted with diethyl ether (200 mL). The
resulting solid was collected by filtration, washed with diethyl
ether and dried to give 43 as a yellow solid (4.01 g, 92%). mp
200 °C. MS (APCI) m/z 448.3 (M + 1). 1H NMR (400 MHz,
DMSO-d6) δ 9.12 (br s, 1H), 8.97 (s, 1H), 8.05 (d, J ) 1 Hz,
1H), 7.83 (d, J ) 9 Hz, 1H), 7.76-7.74 (m, 1H), 5.83-5.79 (m,
1H), 3,37 (br s, 4H), 3.22 (br s, 4H), 2.40 (s, 3H), 2.30 (s, 3H),
2.21 (br s, 2H), 1.88 (br s, 2H), 1.77 (br s, 2H), 1.56 (br s, 2H).
Anal. (C24H29N7O2‚2.4H2O‚1.85HCl): C, H, N, Cl. HPLC purity
) 99% (C18 reverse phase, 10-95% gradient of 0.1%TFA/CH3-
CN in 0.1%TFA/H2O). Exact Mass: Calculated (C25H31N7O3)
478.2556, found 478.2570 (M + 1).
2-[5-(4-tert-Butoxycarbonyl-piperazin-1-yl)-pyridin-2-
ylamino]-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydro-pyrido-
[2,3-d]pyrimidine-6-carboxylic Acid Ethyl Ester (117). A
mixture of 113 (442 mg, 1.25 mmol), Pd(OAc)2 (312 mg, 1.4
mmol), bis(diphenylphosphinic)propane (400 mg, 0.97 mmol)
and N,N-diisopropylethylamine (1.1 g, 8.87 mmol) in EtOH
(20 mL) was stirred under ∼600 psi of CO and heated to 100
°C for 16 h. The solution thus obtained was filtered, and the
filtrate was concentrated under reduced pressure to yield an
orange oil, which was purified by chromatography (20% ethyl
acetate/hexane) to give 8-cyclopentyl-5-methyl-2-methylsulfa-
nyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carboxylic acid
ethyl ester, 118, as an oil (138 mg, 36%). MS (APCI) m/z 348.2
(M + 1). Compound 118 (138 mg, 0.40 mmol) was dissolved in
CH2Cl2 (6 mL), and 2-benzenesulfonyl-3-phenyl-oxaziridine
(155 mg, 0.6 mmol) was added. The reaction mixture was
stirred at room temperature for 18 h, then the solvent was
6-Bromo-8-cyclopentyl-5-methyl-2-(pyridin-2-ylamino)-
8H-pyrido[2,3-d]pyrimidin-7-one (40). A mixture of 112
(370 mg, 1 mmol) and 2-aminopyridine (140 mg, 1.5 mmol) in
toluene (5 mL) was heated at 110 °C for 18 h then cooled to
room temperature. The solid formed was collected by filtration
and washed with toluene, then acetone, and dried in vacuo to
give 40 as a beige solid (22 mg, 30%). mp 267-268 °C. MS
1
(APCI) m/z 402.0 (M + 1). H NMR (400 MHz, DMSO-d6) δ
10.29 (s, 1 H), 8.98 (s, 1 H), 8.29 (br s, 1 H), 8.04 (t, J ) 7 Hz,
1 H), 7.02 (t, J ) 7 Hz, 1 H), 5.92-5.87 (m, 1 H), 2.55 (s, 3 H),
2.16 (br s, 2 H), 1.90 (br s, 2 H), 1.75 (br s, 2 H), 1.56 (br s, 2
H). Anal. (C18H18BrN5O‚0.33H2O): C, H, N.
4-[6-(6-Bromo-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydro-
pyrido[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-pipera-
zine-1-carboxylic Acid tert-Butyl Ester (114). A suspen-
sion of 112 (10.00 g, 0.027 mol) and 63 (10.37 g, 0.0373 mol)
in toluene (100 mL) was heated under nitrogen in an oil bath
for 7 h. Thin-layer chromatography (SiO2, 10% MeOH/DCM)
indicated that both starting materials remained. The suspen-
sion was heated under reflux for a further 18 h. The resulting
suspension was cooled to room temperature and filtered to give
114 (5.93 g, 38%). mp >250 °C. MS (APCI) m/z 584.2 (M + 1).
1H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H), 8.36 (br s, 1H), 7.48
(t, J ) 8 Hz, 1H), 5.99-5.92 (m, 1H), 3.61 (t, J ) 5 Hz, 4H),
3.12 (t, J ) 5 Hz, 4H), 2.61 (s, 3H), 2.29 (br s, 2H), 2.12 (br s,
2H), 1.88 (br s, 2H), 1.68 (br s, 2H), 1.48 (s, 9H).
6-Bromo-8-cyclopentyl-5-methyl-2-(5-piperizin-1-yl-py-
ridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one Hy-
drochloride (41). Compound 114 (0.04 g, 0.07 mmol) was
suspended in CH2Cl2 (10 mL) and MeOH was added in order
to produce a solution (up to ∼6 mL). 2 M HCl in ether (10
mL) was added with stirring. The reaction mixture was stirred

2400 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Toogood et al.
removed under reduced pressure and the remaining residue
was purified by prepative TLC (50% ethyl acetate/hexane). The
more polar, product-containing fraction was extracted into
CH2Cl2 and the solvent evaporated to provide 8-cyclopentyl-
2-methanesulfinyl-5-methyl-7-oxo-7,8-dihydro-pyrido[2,3-d]py-
rimidine-6-carboxylic acid ethyl ester, 119, as a white solid
(110 mg, 76%). MS (APCI) m/z 364.2 (M + 1). A solution of
119 (110 mg, 0.30 mmol) and 63 (310 mg, 1.1 mmol) in toluene
was heated at 100 °C for 10 h and then cooled to room
temperature. Diethyl ether was added to the reaction mixture
and the product precipitated. This precipitate was collected
by filtration and dried to provide 117 (50 mg, 29%). MS (APCI)
11.2 Hz, 2H), 3.35 (m, 2H), 2.76 (dd, J ) 12.0, 12.0 Hz 2H),
2.58 (s, 3H), 2.14 (m, 2H), 1.92 (m, 2H), 1.77 (m, 2H), 1.58 (m,
2H), 1.28 (d, J ) 6.4 Hz, 6H). Exact Mass: Calculated (C24H30-
BrN7O) 512.1773, found 512.1792 (M + 1); HPLC purity 95%.
6-Bromo-8-cyclopentyl-5-methyl-2-[5-(4-methyl-piper-
azin-1-yl)-pyridin-2-ylamino]-8H-pyrido[2,3-d]pyrimidin-
7-one (47). Compound 112 (1.0 g, 2.7 mmol) and 5-(4-methyl-
piperazin-1-yl)-pyridin-2-ylamine (1.48 g, 7.7 mmol) were
combined in toluene (3.0 mL) under nitrogen. The reaction
mixture was heated to reflux and stirred for 4 h. The reaction
mixture was cooled to room temperature and filtered. The
solids were washed with additional toluene (25 mL total) and
dried in vacuo to produce 47 as a yellow powder (338 mg, 29%).
mp 278-280 °C (dec); MS (APCI) m/z 500, 498 (M + 1); 1H
NMR (400 MHz, CDCl3) δ 10.71-10.64 (m, 2H), 9.01 (s, 1H),
8.10-8.09 (m, 1H), 7.89 (d, J ) 0.10 Hz, 1H), 7.52-7.30 (m,
1H), 5.97-5.89 (m, 1H), 3.87-3.84 (m, 2H), 3.53-3.50 (m, 2H),
3.22-3.09 (m, 4H), 2.83-2.82 (m, 3H), 2.60 (s, 3H), 2.21-2.15
(m, 2H), 1.94 (br, 2H), 1.81-1.78 (m, 2H), 1.62-1.60 (M, 2H);
Anal. (C23H28BrN7O1‚3.00H2O‚1.65HCl‚0.60C2H5OH): C, H, N.
1
m/z 578.4 (M + 1). H NMR (400 MHz, DMSO-d6) δ 10.10 (s,
1H), 8.89 (s, 1H), 8.04 (d, J ) 1 Hz, 1H), 7.84 (d, J ) 2 Hz,
1H), 7.48-7.44 (m, 1H), 5.78 (m, 1H), 4.26 (q, J ) 7 Hz, 2H),
3.45 (br s, 4H), 3.10 (br s, 4H), 2.32 (s, 3H), 2.29 (br s, 2H),
1.85 (br s, 2H), 1.73 (br s, 2H), 1.55 (br s, 2H), 1.25 (t, J ) 7
Hz, 3H).
8-Cyclopentyl-5-methyl-7-oxo-2-(5-piperazin-1-yl-pyri-
din-2-ylamino)-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-
carboxylic Acid Ethyl Ester Hydrochloride (44). Anhy-
drous HCl gas was bubbled through a solution of 117 (50 mg,
0.086 mmol) in CH2Cl2/EtOH at room temperature, and the
reaction was stirred for 24 h. Diethyl ether was added to the
reaction mixture, and a solid precipitated which was isolated
and dried to 44 as a yellow solid (12 mg, 29%). mp 216-218
°C. MS (APCI) m/z 478.1 (M + 1). 1H NMR (400 MHz, DMSO-
d6) δ 8.91 (s, 1H), 8.83 (br s, 1H), 8.07 (s, 1H), 7.85 (d, J ) 9
Hz, 1H), 7.59 (br s, 1H), 5.81-5.76 (m, 1H), 4.26 (q, J ) 8 Hz,
2H), ∼3.39 (br s, 4H - buried under HDO peak), 3.23 (br s,
4H), 2.34 (s, 3H), 1.85 (br s, 2H), 2.85 (br s, 2H), 2.75 (br s,
2H), 1.25 (t, J ) 8 Hz, 3H); HPLC purity 95%.
4-[6-(6-Bromo-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydro-
pyrido[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-azepane-
1-carboxylic Acid tert-Butyl Ester (121). A solution of 4-(6-
amino-pyridin-3-yl)-azepane-1-carboxylic acid tert-butyl ester
(614 mg, 2.10 mmol) in toluene (10 mL) was refluxed in a
Dean-Stark apparatus for 3 h. The heat was removed, and
when the reflux subsided, compound 112 (700 mg, 1.89 mmol)
was added. This mixture was refluxed for 12 h under N2.
Succinic anhydride (500 mg) was added and the reflux
continued for 3 h. The reaction mixture was cooled and
dissolved in ethyl acetate (100 mL), and the organic layer was
washed with water (100 mL total). The organic layer was dried
over magnesium sulfate, and the solvents were evaporated.
The crude product was subjected to chromatography on silica
gel and eluted with a chloroform/2-propanol gradient to give
121 as a yellow powder (414 mg, 39%). MS (APCI) m/z 600 (M
2-{5-[Bis-(2-methoxy-ethyl)-amino]-pyridin-2-ylamino}-
6-bromo-8-cyclopentyl-5-methyl-8H-pyrido[2,3-d]pyrimi-
din-7-one (45). Compound 112 (0.4 g, 1.08 mmol) and N5,N5-
Bis-(2-methoxy-ethyl)-pyridine-2,5-diamine (0.5 g, 2.2 mmol)
were combined in toluene (3.5 mL) and heated to 110 °C. After
5 h the reaction mixture was allowed to cool, and the crude
product was directly chromatographed on silica gel eluting
with a gradient of 25% to 100% ethyl acetate in hexanes to
provide 45 (0.49 g, 85%) as an orange gum. mp 94-95 °C. MS
1
+ 1). H NMR (400 MHz, MeOH-d6) δ 9.08 (m, 1 H), 8.00 (m,
1 H), 7.74 (m, 1 H), 7.49 (m, 1 H), 6.08 (m, 1 H), 3.68 (m, 6 H),
3.40 (m, 2 H), 2.69 (s, 3 H), 2.24 (m, 2 H), 2.09 (m, 2 H), 1.90
(m, 4 H), 1.69 (m, 2 H), 1.33 (m, 9 H).
6-Bromo-8-cyclopentyl-2-(5-[1,4]diazepan-1-yl-pyridin-
2-ylamino)-5-methyl-8H-pyrido[2,3-d]pyrimidin-7-one Hy-
drochloride (48). Hydrogen chloride gas was bubbled through
a solution of 121 (80 mg, 0.13 mmol) in chloroform (5 mL) for
30 min. The solvents were evaporated, and the residue was
triturated with ethanol (5 mL). Compound 48 was obtained
as a yellow powder (44 mg, 68%). MS (APCI) m/z 501, 499 (M
+ 1); 1H NMR (400 MHz, DMSO-d6) δ 8.84 (s, 2H), 8.13 (s,
1H), 7.66-7.64 (m, 1H), 7.42-7.39 (m, 1H), 5.86-5.82 (m, 1H),
4.36 (s, 1H), 3.81 (s, 2H), 3.60 (s, 2H), 3.16 (s, 2H), 2.09 (s,
4H), 1.99 (s, 2H), 1.79 (br, 2H), 1.60 (s, 2H), 1.05 (s, 2H); Anal.
(C23H28Br1N7O1‚0.15HCl‚2.55C2H5OH‚0.45CHCl3): C, H, N.
6-Bromo-8-cyclopentyl-5-methyl-2-(3,4,5,6-tetrahydro-
2H-[1,3′]bipyridinyl-6′-ylamino)-8H-pyrido[2,3-d]pyrimi-
din-7-one (49). Compound 112 (1.0 g, 2.70 mmol) and 3,4,5,6-
tetrahydro-2H-[1,3′]bipyridinyl-6′-ylamine (0.668 g, 3.73 mmol)
were heated to reflux in toluene (10 mL) for 16 h. The reaction
mixture was cooled to room temperature, and the precipitate
that formed was collected by filtration and washed on the
funnel with toluene (3 × 10 mL) to give 49 as a brown solid
(0.358 g, 27%). mp 293-295 °C. MS (APCI) m/z 485.0, 483.0
(M + 1). 1H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H), 8.27 (s,
1H), 8.17 (d, J ) 9.0 Hz, 1H), 8.01 (s, 1H), 7.38 (d, J ) 6.8 Hz,
1H), 5.98 (m, 1H), 3.1 (m, 4H), 2.60 (s, 3H), 2.30 (m, 2H), 2.11
(m, 2H), 1.88 (m, 2H), 1.57-1.75 (m, 8H). Anal. (C23H27-
BrN6O1): C, H, N.
1
(APCI) m/z 530.1 (M + 1). H NMR (400 MHz, CDCl3) δ 8.66
(s, 1H), 8.18 (s, 1H), 7.64 (dd, J ) 9, 2 Hz, 1H), 7.05 (br s, 1H),
6.62 (d, J ) 9 Hz, 1H), 5.88 (br s, 1H), 3.73 (t, J ) 6 Hz, 4H),
3.58 (t, J ) 6 Hz, 4H), 3.34 (s, 6H), 2.55 (s, 3H), 2.25-2.22 (m,
2H), 1.93 (br s, 2H), 1.79 (br s, 2H), 1.58 (br s, 2H). Anal.
(C24H32N6O3Br1‚0.13H2O): C, H, N.
4-[6-(6-Bromo-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydro-
pyrido[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-2,6-di-
methyl-piperazine-1-carboxylic Acid tert-Butyl Ester
(120). Compounds 112 (1.0 g, 2.70 mmol) and 63 (1.14 g, 3.73
mmol) were heated to reflux in toluene (10 mL) for 16 h. The
reaction mixture was cooled to room temperature, and the
precipitate that formed was collected by filtration and washed
on the funnel with toluene (3 × 10 mL) to give 120 as a dark
brown-gray solid (0.620 g, 38%). MS (APCI) m/z 614.4, 612.4
1
(M + 1). H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H), 8.23 (d, J
) 8.8 Hz, 1H), 7.99 (d, J ) 2.7 Hz, 1H), 7.36 (dd, J ) 2.7, 8.8
Hz, 1H), 5.99 (m, 1H), 4.28 (m, 2H), 3.30 (m, 2H), 2.93 (dd, J
) 4.4, 11.7 Hz 2H), 2.61 (s, 3H), 2.30 (m, 2H), 2.11 (m, 2H),
1.89 (m, 2H), 1.68 (m, 2H), 1.49 (s, 9H), 1.38 (d, J ) 6.8 Hz,
6H).
6-Bromo-8-cyclopentyl-2-[5-(3,5-dimethyl-piperazin-1-
yl)-pyridin-2-ylamino]-5-methyl-8H-pyrido[2,3-d]pyrimi-
din-7-one Hydrochloride (46). Compound 120 (0.051 g,
0.083 mmol) was dissolved in dichloromethane (3 mL) to which
2 N HCl (2 mL) was added, and the mixture was stirred at
room temperature for 2 h. This mixture was concentrated and
allowed to sit for 10 days, and it was then dissolved in 2 N
HCl (2 mL) and stirred at room temperature for 5 h. The
solvent was removed to give 46 as a yellow solid (0.039 g, 71%).
decomposes >225 °C. MS (APCI) m/z 514.0, 512.0 (M + 1). 1H
NMR (400 MHz, DMSO-d6) δ 9.51 (m, 1H), 9.02 (m, 1H), 8.98
(s, 1H), 8.07 (s, 1H), 7.83 (s, 2H), 5.90 (m, 1H), 3.85 (d, J )
6-Bromo-8-cyclopentyl-2-(4-hydroxy-3,4,5,6-tetrahydro-
2H-[1,3′]bipyridinyl-6′-ylamino)-5-methyl-8H-pyrido[2,3-
d]pyrimidin-7-one (50). Compound 112 (2.50 g, 6.76 mmol)
and 6′-amino-3,4,5,6-tetrahydro-2H-[1,3′]bipyridinyl-4-ol (1.96
g, 10.13 mmol) were combined in toluene (10 mL) under
nitrogen. The reaction mixture was heated to reflux and stirred
for 4 h. The reaction mixture was cooled to RT and filtered.
The solids were washed with additional toluene (75 mL total)

Inhibitor of Cyclin-Dependent Kinase 4/6
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2401
and dried in vacuo to produce 50 as a yellow powder (566 mg,
tetrakis(triphenylphosphine)palladium (0.093 g, 0.081 mmol)
and tributyl-(1-ethoxy-vinyl)-stannane (0.379 g, 1.05 mmol)
were dissolved in toluene (3 mL) and slowly brought to reflux
for 1 h. The solvent was evaporated and the solid was
redissolved in dichloromethane (8 mL) and purified by silica
gel chromatography to give 123 as a yellow solid (0.405 g, 99%).
1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 8.15 (d, J ) 9.0 Hz,
1H), 8.00 (s, 1H), 7.85 (d, J ) 2.9 Hz, 1H), 7.18 (m, 1H), 5.90
(m, 1H), 4.52 (d, J ) 2.4 Hz, 1H), 4.18 (d, J ) 2.4 Hz, 1H),
3.93 (q, J ) 7.1 Hz, 2H), 3.80 (m, 2H), 3.38 (m, 2H), 3.26 (s,
2H), 2.41 (s, 3H), 2.35 (m, 2H), 2.06 (m, 2H), 1.85 (m, 2H),
1.64 (m, 2H), 1.49 (s, 9H), 1.45 (s, 6H), 1.36 (t, J ) 7.1 Hz,
3H).
1
17%). MS (APCI) m/z 501, 499 (M + 1). H NMR (400 MHz,
DMSO-d6) δ 10.06 (s, 1H), 8.96 (s, 1H), 8.04 (s, 1H), 7.83 (d, J
) 9.3 Hz, 1H), 7.46 (d, J ) 7.3 Hz, 1H), 5.93-5.89 (m, 1H),
4.71 (s,1H), 3.65-3.60 (m, 1H), 3.53-3.51 (m, 2H), 2.88-2.83
(m, 2H), 2.57 (s, 3H), 2.18 (br, 2H), 1.90-1.81 (m, 5H), 1.59-
1.48 (m, 3H). Anal. (C23H27Br1N6O2, 0.45 H2O): C, H, N.
6-Bromo-8-cyclopentyl-5-methyl-2-(5-morpholin-4-yl-
pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one (51).
Compound 112 (1.0 g, 2.70 mmol) and 5-morpholin-4-yl-
pyridin-2-ylamine (0.668 g, 3.73 mmol) were heated to reflux
in toluene (10 mL) for 16 h. The reaction mixture was cooled
to room temperature, and the precipitate that formed was
collected by filtration and washed on the funnel with toluene
(3 × 10 mL). The solid obtained was refluxed in ethyl acetate
(15 mL), cooled and filtered to give 51 as a dark brown-gray
solid (0.350 g, 27%). mp >300 °C. 1H NMR (400 MHz, CDCl3)
δ 8.78 (s, 1H), 8.17 (d, J ) 9.0 Hz, 1H), 8.02 (d, J ) 2.7 Hz,
1H), 7.97 (s, 1H), 7.32 (dd, J ) 2.9, 9.0 Hz, 1H), 5.99 (m, 1H),
3.89 (m, 4H), 3.16 (m, 4H), 2.61 (s, 3H), 2.30 (m, 2H), 2.10 (m,
2H), 1.88 (m, 2H), 1.68 (m, 2H). m/z 487.0, 485.0 (M + 1). Anal.
(C22H25BrN6O2‚1.37H2O): C, H, N.
6-Acetyl-8-cyclopentyl-2-[5-(3,3-dimethyl-piperazin-1-
yl)-pyridin-2-ylamino]-5-methyl-8H-pyrido[2,3-d]pyrimi-
din-7-one Hydrochloride (53). Compound 123 (0.400 g,
0.663 mmol) was dissolved in ethyl acetate (10 mL) and 6 N
HCl (10 mL) and stirred at room temperature for 2 h. The
solvent was evaporated to give a yellow solid, which was dried
in a vacuum oven for 5 h at 50 °C. The solid was triturated
with EtOH (20 mL) and filtered to yield 53 as a yellow solid
1
(0.120 g, 38%). MS (APCI) m/z 476.1 (M + 1). H NMR (400
MHz, DMSO-d6) δ 9.15 (s, 2H), 8.93 (s, 1H), 8.04 (d, J ) 3.2
Hz, 1H), 7.82 (d, J ) 9.1 Hz, 1H), 7.64 (m, 1H), 5.78 (m, 1H),
3.31 (m, 2H), 3.24 (m, 2H), 3.18 (s, 2H), 2.38 (s, 3H), 2.28 (s,
3H), 2.18 (m, 2H), 1.85 (m, 2H), 1.73 (m, 2H), 1.54 (m, 2H),
1.35 (s, 6H). Anal. Calc′d for C26H33N7O2‚2.89HCl‚3.0H2O: C,
49.18; H, 6.65; N, 15.44. Found; C, 49.55; H, 6.80; N, 14.76.
6-Acetyl-2-{5-[bis-(2-methoxy-ethyl)-amino]-pyridin-2-
ylamino}-8-cyclopentyl-5-methyl-8H-pyrido[2,3-d]pyri-
midin-7-one (52). Compound 45 (0.4 g, 0.75 mmol), tributyl-
(1-ethoxy-vinyl)-stannane (0.42 g, 1.175 mmol) and palladium
tetrakistriphenylphosphine (0.1 g, 0.09 mmol) were combined
in N2-purged toluene (4 mL) and heated to 110 °C. After 2 h
the reaction mixture was allowed to cool, and solid 40% KF
on alumina (0.2 g) was added. This mixture was diluted with
toluene (15 mL) and mixed by swirling for 2 min. After filtering
and removal of the solvents, the crude product was chromato-
graphed on silica gel eluting with 50-65% ethyl acetate in
hexanes to give an orange gum (0.298 g). This gum was
dissolved in CH2Cl2 and washed with 10% KF in H2O, then
brine and dried (MgSO4). Following removal of the drying
agent and evaporation of the solvent, the remaining material
was dissolved in ethyl acetate (10 mL) and treated with 1 M
HCl (aqueous). The resulting mixture was stirred vigorously
for 1 h at room temperature. Sufficient CH2Cl2 was added to
dissolve the precipitate that had formed, and the organic
solution was washed with saturated aqueous sodium bicarbon-
ate solution. The aqueous layer was back extracted twice with
CH2Cl2, and the combined organic layers were dried (MgSO4).
Removal of the drying agent and evaporation of the solvent
gave a foamy solid, which was dissolved in ethyl acetate (20
mL) and filtered then diluted with an equal volume of hexanes
and stored at 4 °C. The yellow crystals that formed were
collected by filtration and dried in vacuo to give 62 (120 mg,
32%). mp 138-138 °C. MS (APCI) m/z 495.3 (M + 1). 1H NMR
(400 MHz, CDCl3) δ 9.75 (s, 1H), 8.85 (s, 1H), 8.17 (s, 1H),
7.60 (br s, 1H), 6.65 (d, J ) 9 Hz, 1H), 5.9 (m, 1H), 3.62 (t, J
) 6 Hz, 4H), 3.46 (t, J ) 6 Hz, 4H), 3.22 (s, 6H), 2.38 (s, 3H),
2.25 (s, 3H), 2.17 (br s, 2H), 1.67 (br s, 2H), 1.45 (br s, 2H).
Anal. (C26H34N6O4): C, H, N.
4-[6-(6-Bromo-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydro-
pyrido[2,3-d]pyrimidin-2-ylamino)-pyridin-3-yl]-2,2-di-
methyl-piperazine-1-carboxylic Acid tert-Butyl Ester
(122). Compound 112 (1.0 g, 2.70 mmol) and 4-(6-amino-
pyridin-3-yl)-2,2-dimethyl-piperazine-1-carboxylic acid tert-
butyl ester (1.14 g, 3.73 mmol) were heated to reflux in toluene
(10 mL) for 16 h. The reaction mixture was cooled to room
temperature, and the precipitate that formed was collected by
filtration and washed on the funnel with toluene (3 × 10 mL)
to give 122 as a dark brown-gray solid (0.525 g, 32%). 1H NMR
(400 MHz, DMSO-d6) δ 9.96 (s, 1H), 8.91 (s, 1H), 7.89 (d, J )
2.7 Hz, 1H), 7.74 (d, J ) 8.8 Hz, 1H), 7.26 (dd, J ) 3.2, 9.3 Hz,
1H), 6.18 (s, 1H), 5.86 (m, 1H), 3.67 (m, 2H), 3.37 (m, 4H),
2.54 (s, 3H), 2.15 (m, 2H), 1.84 (m, 2H), 1.71 (m, 2H), 1.53 (m,
2H), 1.39 (s, 9H), 1.33 (s, 6H).
4-{6-[8-Cyclopentyl-6-(1-ethoxy-vinyl)-5-methyl-7-oxo-
7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino]-pyridin-
3-yl}-2,6-dimethyl-piperazine-1-carboxylic Acid tert-
Butyl Ester (124). Compound 120 (0.450 g, 0.735 mmol),
tetrakis(triphenylphosphine)palladium (0.102 g, 0.088 mmol)
and tributyl-(1-ethoxy-vinyl)-stannane (0.414 g, 1.15 mmol)
were dissolved in toluene (4 mL) and slowly brought to reflux
for 2 h. The solvent was evaporated and the solid redissolved
in dichloromethane (8 mL). This solution was purified by silica
gel chromatography to give 124 as a yellow solid (0.275 g, 62%).
1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 8.20 (d, J ) 9.0 Hz,
1H), 8.06 (s, 1H), 8.00 (d, J ) 2.7 Hz, 1H), 7.32 (dd, J ) 2.7,
9.0 Hz, 1H), 5.89 (m, 1H), 4.51 (d, J ) 2.4 Hz, 1H), 4.26 (m,
2H), 4.17 (d, J ) 2.4 Hz, 1H), 3.93 (q, J ) 6.8 Hz, 2H), 3.28 (d,
J ) 11.7, 2H), 2.90 (dd, J ) 4.2, 11.7 Hz, 1H), 2.41 (s, 3H),
2.35 (m, 2H), 2.06 (m, 2H), 1.85 (m, 2H), 1.65 (m, 2H), 1.48 (s,
9H), 1.45 (s, 6H), 1.36 (m, 9H).
6-Acetyl-8-cyclopentyl-2-[5-(3,5-dimethyl-piperazin-1-
yl)-pyridin-2-ylamino]-5-methyl-8H-pyrido[2,3-d]pyrimi-
din-7-one Hydrochloride (54). Compound 124 (0.250 g,
0.414 mmol) was dissolved in dichloromethane (3 mL) to which
2 N HCl in diethyl ether (3 mL) was added, and the mixture
was stirred at room temperature for 16 h. The solvent was
evaporated, and the solid was dried in a vacuum oven for 24
h at 50 °C to give 54 as a yellow solid (0.120 g, 38%). MS
1
(APCI) m/z 476.1 (M + 1). H NMR (400 MHz, DMSO-d6) δ
9.51 (m, 2H), 9.0 (m, 1H), 8.97 (s, 1H), 8.08 (d, J ) 2.7 Hz,
1H), 7.84 (d, J ) 9.3 Hz, 1H), 7.78 (m, 1H), 5.80 (m, 1H), 3.35
(d, J ) 11.5 Hz, 2H), 3.35 (m, 2H), 2.75 (dd, J ) 12.2, 2H),
2.40 (s, 3H), 2.30 (s, 3H), 2.19 (m, 2H), 1.88 (m, 2H), 1.76 (m,
2H), 1.57 (m, 2H), 1.29 (d, J ) 6.6 Hz, 6H). Anal. Calc′d for
C26H33N7O2‚2.70HCl‚0.10H2O: C, 54.23, H, 6.28, N, 17.03.
Found: C, 54.60; H, 6.68; N, 16.57.
8-Cyclopentyl-6-(1-ethoxy-vinyl)-5-methyl-2-[5-(4-meth-
yl-piperazin-1-yl)-pyridin-2-ylamino]-8H-pyrido[2,3-d]-
pyrimidin-7-one (125). A 6-dram vial was charged with 47
(266 mg, 0.53 mmol) and tetrakis(triphenylphosphine) pal-
ladium(0) (61 mg, 0.053 mmol) and the atmosphere replaced
with argon. Toluene (5 mL) was added followed by tributyl-
(1-ethoxy-vinyl)-stannane (289 mg, 0.80 mmol). The vial was
heated to 110 °C and stirred for 12 h. The reaction mixture
was diluted with chloroform (25 mL) and adsorbed onto silica
gel. Chromatographic purification on silica gel (chloroform/2-
propanol + 1%TEA gradient) gave 125 (237 mg, 91%), which
was used without further purification. MS (APCI) m/z 490 (M
+ 1).
4-{6-[8-Cyclopentyl-6-(1-ethoxy-vinyl)-5-methyl-7-oxo-
7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino]-pyridin-
3-yl}-2,2-dimethyl-piperazine-1-carboxylic Acid tert-
Butyl Ester (123). Compound 122 (0.412 g, 0.673 mmol),

2402 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Toogood et al.
6-Acetyl-8-cyclopentyl-5-methyl-2-[5-(4-methyl-piper-
azin-1-yl)-pyridin-2-ylamino]-8H-pyrido[2,3-d]pyrimidin-
7-one (55). To a solution of 125 (237 mg, 0.48 mmol) in
chloroform (5 mL) was added hydrogen chloride (2 M ethereal
solution, 2.0 mL, 4.0 mmol). The reaction mixture was stirred
at room temperature for 12 h. The solvents were evaporated,
and the residue was dissolved in ethanol. The ethanol was
evaporated to give 55 (239 mg, 0.52 mmol). MS (APCI) m/z
8-Cyclopentyl-6-(1-ethoxy-vinyl)-5-methyl-2-(3,4,5,6-
tetrahydro-2H-[1,3′]bipyridinyl-6′-ylamino)-8H-pyrido-
[2,3-d]pyrimidin-7-one (128). Compound 51 (0.310 g, 0.641
mmol), tetrakis(triphenylphosphine)palladium (0.089 g, 0.077
mmol) and tributyl-(1-ethoxy-vinyl)-stannane (0.361 g, 1.0
mmol) were dissolved in toluene (3 mL) and slowly brought to
reflux for 2 h. The reaction mixture was allowed to cool, then
purified by silica gel chromatography to give 128 as a yellow
1
1
462 (M + 1). H NMR (400 MHz, DMSO-d6) δ 10.83 (m, 2H),
solid (0.180 mg, 59%). H NMR (400 MHz, CDCl3) δ 8.73 (s,
9.00 (s, 1H), 8.1 (m, 1H), 7.88-7.82 (m, 2H), 5.89-5.80 (m,
1H), 3.88-3.85 (m, 2H), 3.54-3.51 (m, 2H), 3.23-3.11 (m, 4H),
2.83-2.82 (m, 3H), 2.43 (s, 3H), 2.34 (s, 3H), 2.23-2.11 (m,
2H), 1.93 (br, 2H), 1.81-1.77 (m, 2H), 1.60-1.59 (m, 2H); Anal.
(C25H31N7O2‚2.70HCl‚1.05C2H5OH): C, H, N.
1H), 8.16 (d, J ) 9.0 Hz, 1H), 8.05 (s, 1H), 8.01 (d, J ) 2.9 Hz,
1H), 7.36 (dd, J ) 2.9, 9.3 Hz, 1H), 5.90 (m, 1H), 4.52 (d, J )
2.4 Hz, 1H), 4.18 (d, J ) 2.2 Hz, 1H), 3.93 (q, J ) 7.1 Hz, 2H),
3.14 (m, 4H), 2.41 (s, 3H), 2.36 (m, 2H), 2.06 (m, 2H), 1.84 (m,
2H), 1.56-1.77 (m, 8H), 1.21 (t, J ) 7.1 Hz, 3H).
(1-{6-[8-Cyclopentyl-6-(1-ethoxy-vinyl)-5-methyl-7-oxo-
7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino]-pyridin-
3-yl}-pyrrolidin-3-yl)-carbamic Acid tert-Butyl Ester (126).
A 6-dram vial was charged with {1-[6-(6-bromo-8-cyclopentyl-
5-methyl-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino)-
pyridin-3-yl]-pyrrolidin-3-yl}-carbamic acid tert-butyl ester
(prepared by the method described for compound 122) (379
mg, 0.65 mmol) and tetrakis(triphenylphosphine) palladium-
(0) (75 mg, 0.065 mmol) and the atmosphere replaced with
argon. Toluene (5 mL) was added followed by tributyl-(1-
ethoxy-vinyl)-stannane (352 mg, 0.97 mmol). The vial was
heated to 110 °C and stirred for 12 h. The reaction mixture
was diluted with chloroform (25 mL) and adsorbed onto silica
gel. Chromatographic purification on silica gel (chloroform/2-
propanol + 1% TEA gradient) gave 126 as a yellow solid (394
mg, 100%) that was used without further purification. MS
(APCI) m/z 576 (M + 1).
6-Acetyl-8-cyclopentyl-5-methyl-2-(3,4,5,6-tetrahydro-
2H-[1,3′]bipyridinyl-6′-ylamino)-8H-pyrido[2,3-d]pyrimi-
din-7-one (58). Compound 128 (0.180 g, 0.379 mmol) was
dissolved in ethyl acetate (10 mL), and 6 N HCl (10 mL) was
added then the mixture was stirred at room temperature for
2 h. The mixture was diluted with dichloromethane and
aqueous NaHCO3. The layers were separated, and the organic
layer was dried over MgSO4, filtered, and the solvent evapo-
rated to give 58 as a yellow solid (0.120 g, 71%). mp 251-253
°C. MS (APCI) m/z 447.2 (M + 1). 1H NMR (400 MHz, CDCl3)
δ 8.78 (s, 1H), 8.20 (d, J ) 8.5 Hz, 1H), 7.95 (s, 1H), 7.39 (m,
1H), 5.85 (m, 1H), 3.15 (m, 4H), 2.53 (s, 3H), 2.36 (s, 3H), 2.33
(m, 2H), 2.05 (m, 2H), 1.87 (m, 2H), 1.77-1.56 (m, 8H). MS
(APCI) m/z 447.2. Anal. (C25H30N6O2‚0.35H2O): C, H, N.
8-Cyclopentyl-6-(1-ethoxy-vinyl)-2-(4-hydroxy-3,4,5,6-
tetrahydro-2H-[1,3′]bipyridinyl-6′-ylamino)-5-methyl-8H-
pyrido[2,3-d]pyrimidin-7-one (129). A 6-dram vial was
charged with 50 (316 mg, 0.63 mmol) and tetrakis(triphenyl-
phosphine) palladium(0) (72 mg, 0.063 mmol) and the atmo-
sphere replaced with argon. Toluene (5 mL) was added
followed by tributyl-(1-ethoxy-vinyl)-stannane (343 mg, 0.95
mmol). The vial was heated to 110 °C and stirred for 12 h.
The reaction mixture was diluted with chloroform (25 mL) and
adsorbed onto silica gel. Chromatographic purification on silica
gel (chloroform/2-propanol + 1% TEA gradient) gave 129 (255
mg, 83%). 1H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1 H), 8.85
(s, 1 H), 8.03 (m, 1 H), 7.82 (t, J ) 8.42 Hz, 1 H), 7.44 (dd, J
) 9.03, 3.17 Hz, 1 H), 5.81 (m, 1 H), 4.70 (d, J ) 4.15 Hz, 1
H), 4.44 (d, J ) 1.95 Hz, 1 H), 4.04 (d, J ) 1.95 Hz, 1 H), 3.81
(q, J ) 6.91 Hz, 2 H), 3.62 (m, 1 H), 3.50 (m, 2 H), 2.84 (m, 2
H), 2.36 (s, 3 H), 2.22 (m, 2 H), 1.78 (m, 6 H), 1.50 (m, 4 H),
1.24 (t, J ) 6.95 Hz, 3 H).
6-Acetyl-2-[5-(3-amino-pyrrolidin-1-yl)-pyridin-2-ylami-
no]-8-cyclopentyl-5-methyl-8H-pyrido[2,3-d]pyrimidin-
7-one (56). To a solution 126 (394 mg, 0.68 mmol) in
chloroform (5 mL) was added hydrogen chloride (2 M ethereal
solution, 2.0 mL, 4.0 mmol). The reaction mixture was stirred
at room temperature for 12 h. The solvents were evaporated,
and the residue was dissolved in ethanol. The ethanol was
evaporated to give 56 (239 mg, 76%). MS (APCI) m/z 487 (M
1
+ 1). H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.34 (br,
2H), 7.78-7.73 (m, 2H), 7.51 (br, 1H), 5.89-5.80 (m, 1H), 3.98
(br, 2H), 3.62-3.51 (m, 4H), 2.40-3.23 (m, 2H), 2.44 (s, 3H),
3.34 (s, 3H), 2.25-2.20 (m, 2H), 2.16-2.13 (m, 1H), 1.93 (br,
2H), 1.80-1.78 (m, 2H), 1.61-1.58 (m, 2H); Anal. (C24H29N7O2‚
2.10HCl‚2.85H2O‚0.45C2H5OH): C, H, N, Cl.
4-{6-[8-Cyclopentyl-6-(1-ethoxy-vinyl)-5-methyl-7-oxo-
7,8-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino]-pyridin-
3-yl}-[1,4]diazepane-1-carboxylic Acid tert-Butyl Ester
(127). A 6-dram vial was charged with 121 (123 mg, 0.25
mmol) and tetrakis(triphenylphosphine) palladium(0) (29 mg,
0.025 mmol) and the atmosphere replaced with argon. Toluene
(5 mL) was added followed by tributyl-(1-ethoxy-vinyl)-stan-
nane (137 mg, 0.37 mmol). The vial was heated to 110 °C and
stirred for 12 h. The reaction mixture was diluted with
chloroform (25 mL) and adsorbed onto silica gel. Chromato-
graphic purification on silica gel (chloroform/ethyl acetate
gradient) gave 127 as a yellow solid (116 mg, 80%), which was
used without further purification. MS (APCI) m/z 590 (M +
1).
6-Acetyl-8-cyclopentyl-2-(4-hydroxy-3,4,5,6-tetrahydro-
2H-[1,3′]bipyridinyl-6′-ylamino)-5-methyl-8H-pyrido[2,3-
d]pyrimidin-7-one (59). To a solution of 129 (255 mg, 0.52
mmol) in chloroform (2 mL) was added hydrogen chloride (2
M ethereal solution, 5.0 mL, 10.0 mmol). The reaction mixture
was stirred at room temperature for 12 h. The solvents were
evaporated, and the residue was dissolved in ethanol. The
ethanol was evaporated to give 59 (213 mg, 88%). MS (APCI)
1
m/z 463 (M + 1). HNMR (400 MHz, DMSO-d6) δ 10.90 (br,
1H), 9.07 (s, 1H), 8.19 (s, 1H), 7.91 (br, 2H), 5.91-5.89 (m,
1H), 3.77 (br, 1H), 3.62 (br, 2H), 3.07 (br, 2H), 2.58 (s, 3H),
2.40 (s, 3H), 2.30 (br, 2H), 1.98-1.86 (m, 5H), 1.65 (br, 4H);
Anal. (C25H30N6O3, 1.76 C3H8O1, 0.36 CHCl3): C, H, N.
6-Acetyl-8-cyclopentyl-2-(5-[1,4]diazepan-1-yl-pyridin-
2-ylamino)-5-methyl-8H-pyrido[2,3-d]pyrimidin-7-one hy-
drochloride (57). To a solution 127 (116 mg, 0.20 mmol) in
chloroform (5 mL) was added hydrogen chloride (2 M ethereal
solution, 5.0 mL, 10.0 mmol). The reaction mixture was stirred
at room temperature for 12 h. The solvents were evaporated,
and the residue was dissolved in ethanol. The ethanol was
evaporated to give 57 (47 mg, 50%). MS (APCI) m/z 462 (M +
1). 1H NMR (400 MHz, DMSO-d6) δ 9.19 (br, 2H), 8.99 (s, 1H),
7.91 (s, 1H), 7.78-7.75 (m, 2H), 5.88-5.80 (m, 1H), 3.80-3.77
(m, 3H), 3.25 (br, 3H), 3.16 (br, 2H), 2.44 (s, 3H), 2.34 (s, 3H),
2.49-2.18 (m, 2H), 2.12-2.10 (m, 2H), 1.93 (br, 2H), 1.81-
1.78 (m, 2H), 1.61-1.58 (m, 2H); Anal. (C25H31N7O2‚2.80HCl‚
0.45C3H8O2): C, H, N.
8-Cyclopentyl-6-(1-ethoxy-vinyl)-5-methyl-2-(5-mor-
pholin-4-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimi-
din-7-one (130). Compound 51 (0.290 g, 0.597 mmol), tetrakis-
(triphenylphosphine)palladium (0.083 g, 0.072 mmol) and
tributyl-(1-ethoxy-vinyl)-stannane (0.336 g, 0.932 mmol) were
dissolved in toluene (4 mL) and slowly brought to reflux for 3
h. The reaction mixture was purified by silica gel chromatog-
1
raphy to give 130 as a yellow solid (0.110 g, 39%). H NMR
(400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.83 (s, 1H), 8.02 (d, J )
2.9 Hz, 1H), 7.86 (d, J ) 9.0 Hz, 1H), 7.44 (dd, J ) 3.2, 9.3 Hz,
1H), 5.79 (m, 1H), 4.42 (d, J ) 2.0 Hz, 1H), 4.01 (d, J ) 2.0
Hz, 1H), 3.79 (q, J ) 6.8 Hz, 2H), 3.72 (m, 4H), 3.09 (m, 4H),
2.34 (s, 3H), 2.17 (m, 2H), 1.85 (m, 2H), 1.71 (m, 2H), 1.55 (m,
2H), 1.21 (m, 3H).

Inhibitor of Cyclin-Dependent Kinase 4/6
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2403
6-Acetyl-8-cyclopentyl-5-methyl-2-(5-morpholin-4-yl-
pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one (60).
Compound 130 (0.490 g, 1.03 mmol) was dissolved in dichlo-
romethane (5 mL). 2 N HCl in diethyl ether (3 mL) was added,
and the resulting mixture was stirred at room temperature
for 4 h. Then, additional 2 N HCl in diethyl ether (2 mL) was
added, and the mixture was stirred for an additional 12 h. The
reaction mixture was diluted with dichloromethane and aque-
ous NaHCO3. The layers were separated, the organic layer was
dried over MgSO4 and filtered and the solvent evaporated to
give a yellow solid. The solid was recrystallized from a mixture
of hexanes, ethyl acetate and dichloromethane to give 60 as a
yellow solid (0.280 g, 61%). mp 268-270 °C. MS (APCI) m/z
containing a final concentration of 20 mM Tris-HCl (pH 7.4),
50 mM NaCl, 1 mM dithiothreitol (DTT), 10 mM MgCl2, 25
µM ATP (for Cdk4-cyclin D1 and D3 and Cdk6-cyclin D2) or
12 µM ATP (for Cdk2-cyclin E, Cdk2-cyclin A and Cdk1-cyclin
B) containing 0.25 µCi of [γ-32P]ATP, 20 ng of enzyme, 1 µg of
GST-Rb-Cterm and appropriate dilutions of inhibitor. All
components except the [γ-32P]ATP were added to the wells,
and the plate was placed on a plate mixer for 2 min. The
reaction was then started by adding the [γ-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), and the plate was kept at 4 °C for at least 1 h to allow
the substrate to precipitate. The wells were then washed five
times with 0.2 mL of 10% TCA, and radioactive incorporation
was determined with a beta plate counter (Wallac Inc.,
Gaithersburg, MD). Enzyme assays for other protein kinases
were performed as described previously.21-23 The IC50 values
were determined from five-point titrations performed in
duplicate. The mean of two determinations is provided in the
tables. When the duplicates differed by greater 2-fold, a third
determination was made.
Cell Culture. All cell lines were obtained from ATCC and
maintained at 37 °C, 5% CO2 in Dulbecco’s modified Eagle’s
medium containing 10% fetal bovine serum (FBS) (Life
Technologies, Inc). To measure thymidine incorporation into
DNA, cells were seeded at 2 × 104 per well in a 96-well
Cytostar T plate (Amersham Corp.) and incubated overnight
to allow cells to attach. Predetermined concentrations of test
compound were added to the wells and incubated for 24 h at
37 °C. Then, [14C]thymidine (0.1 µCi) was added to each well
and incorporation of the radiolabel allowed to proceed for 72
h. Incorporated radioactivity was determined with a beta plate
counter (Wallac Inc., Gaithersburg, MD). IC50 values were
determined from five-point titration curves by line fitting using
SigmaPlot.
1
449.2 (M + 1). H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1H),
8.17 (d, J ) 9.0 Hz, 1H), 8.02 (d, J ) 2.7 Hz, 1H), 7.31 (dd, J
) 2.9, 9.0 Hz, 1H), 5.86 (m, 1H), 3.88 (m, 4H), 3.15 (m, 4H),
2.54 (s, 3H), 2.36 (s, 3H), 2.32 (m, 2H), 2.05 (m, 2H), 1.87 (m,
2H), 1.68 (m, 2H). Exact Mass: Calculated (C24H28N6O3)
449.2301, found 449.2313 (M + 1); HPLC purity 95%.
6-Bromo-8-cyclopentyl-2-[5-(2,6-dimethyl-morpholin-
4-yl)-pyridin-2-ylamino]-5-methyl-8H-pyrido[2,3-d]py-
rimidin-7-one (131). Compound 112 (1.0 g, 2.70 mmol) and
5-(2,6-dimethyl-morpholin-4-yl)-pyridin-2-ylamine (0.668 g,
3.73 mmol) were heated to reflux in toluene (10 mL) for 16 h.
The reaction mixture was cooled to room temperature, and the
precipitate that formed was collected by filtration and washed
on the funnel with toluene (3 × 10 mL) to give 131 as a brown
solid (0.358 g, 27%). mp 287-289 °C. MS (APCI) m/z 515.1,
513.1 (M + 1). 1H NMR (400 MHz, CDCl3) δ 8.80 (s, 1H), 8.21
(d, J ) 9.3 Hz, 1H), 7.98 (d, J ) 2.9 Hz, 1H), 7.34 (dd, J ) 3.2,
9.3 Hz, 1H), 5.98 (m, 1H), 3.84 (m, 2H), 3.40 (dd, J ) 2.9, 10.2
Hz, 2H), 2.61 (s, 3H), 2.46 (dd, J ) 10.2, 10.2 Hz, 2H), 2.30
(m, 2H), 2.11 (m, 2H), 1.88 (m, 2H), 1.68 (m, 2H), 1.28 (d, J )
6.3 Hz, 6H). Anal. (C24H29BrN6O2): Calc. C, 56.14, H, 5.69, N,
16.37. Found: C, 55.90; H, 5.62; N, 16.10.
Flow Cytometry. Cells were harvested and washed in
phosphate buffered saline (PBS) containing 5 mM EDTA. They
were then washed in PBS containing 1% FBS (1% FBS/PBS),
fixed in 85% ethanol and stored at 4 °C for at least 16 h and
up to 5 days. Cells were then washed again in 1% FBS/PBS
and incubated at 37 °C for 30 min in 1% FBS/PBS containing
40 mg/mL propidium iodide (PI) (Molecular Probes) and 250
mg/mL RNase A (Roche Diagnostics Ltd.). Data were collected
using a Coulter EPICS Elite ESP equipped with a Spectra-
physics argon-ion laser and analyzed using ModFit (Verity
Software House, Inc.). Results represent a minimum of 15 000
cells assayed for each sample.
Pharmacokinetics Analysis. Male Sprague-Dawley rats
(n ) 3 or 4) were administered a single 5 mg/kg dose as a 5
min iv infusion of the HCl salt or a single 5 mg/kg po dose of
the isethionate salt by gavage. The iv dose was administered
as a solution in 10% DMA/50% Propylene glycol/40% D5W
vehicle, and the po dose was administered as a suspension in
5%:95% PEG200/0.5% methylcellulose vehicle. Serial blood
samples were collected from each rat over a 24 h period after
dosing. Plasma samples were analyzed for compound 43
concentrations by LC/MS/MS (Column: MetaSil Basic 3.0 cm
× 2.0 mm; Mobil phase: 90:10/10:90 (10 mM ammonium
formate w/0.1% formic acid: acetonitrile); ionization: electro-
spray positive ion mode; data acquisition: MassChrom V 1.1/
Analyst V 1.2). Pharmacokinetic parameters for compound 43
were determined from the plasma concentration-time data
using noncompartmental methods.
8-Cyclopentyl-2-[5-(2,6-dimethyl-morpholin-4-yl)-pyri-
din-2-ylamino]-6-(1-ethoxy-vinyl)-5-methyl-8H-pyrido-
[2,3-d]pyrimidin-7-one (132). Compound 131 (0.062 g, 0.121
mmol), tetrakis(triphenylphosphine)palladium (0.017 g, 0.015
mmol) and tributyl-(1-ethoxy-vinyl)-stannane (0.068 mg, 0.188
mmol) were dissolved in toluene (2 mL) and slowly brought to
reflux for 12 h. Additional tetrakis(triphenylphosphine)-pal-
ladium (0.010 g) was added and the reaction brought to reflux
for 16 h. The reaction mixture was cooled and purified by silica
gel chromatography to give 132 as a yellow solid (0.055 g, 90%).
1H NMR (400 MHz, CDCl3) δ 8.72 (s, 1H), 8.17 (d, J ) 9.0 Hz,
1H), 7.99 (d, J ) 2.9 Hz, 1H), 7.83 (s, 1H), 7.29 (dd, J ) 2.9,
9.0 Hz, 1H), 5.89 (m, 1H), 4.51 (d, J ) 2.5 Hz, 1H), 4.17 (d, J
) 2.4 Hz, 1H), 3.93 (q, J ) 7.1 Hz, 2H), 3.83 (m, 2H), 3.37 (d,
J ) 10.3 Hz, 2H), 2.44 (dd, J ) 10.5, 10.5, 2H), 2.41 (s, 3H),
2.34 (m, 2H), 2.06 (m, 2H), 1.84 (m, 2H), 1.65 (m, 2H), 1.36 (t,
J ) 7.1 Hz, 3H), 1.26 (d, J ) 6.4 Hz, 6H).
6-Acetyl-8-cyclopentyl-2-[5-(2,6-dimethyl-morpholin-4-
yl)-pyridin-2-ylamino]-5-methyl-8H-pyrido[2,3-d]pyrimi-
din-7-one (61). Compound 132 (0.055 g, 0.109 mmol) was
dissolved in ethyl acetate (3 mL) and 1 N aqueous HCl (2 mL)
and stirred at room temperature for 48 h. The reaction mixture
was diluted with dichloromethane and aqueous NaHCO3. The
layers were separated, the organic layer was dried over MgSO4
and filtered and the solvent was evaporated to give 61 (0.020
1
g, 38%). MS (APCI) m/z 477.2 (M + 1). H NMR (400 MHz,
CDCl3) δ 8.79 (s, 1H), 8.1 (d, J ) 9.0 Hz, 1H), 8.00 (d, J ) 2.7
Hz, 1H), 7.90 (s, 1H), 7.30 (dd, J ) 3.1, 9.3 Hz, 1H), 5.87 (m,
1H), 3.83 (m, 2H), 3.37 (d, J ) 10.0 Hz, 2H), 2.54 (s, 3H), 2.46
(dd, J ) 11.7, 11.7, 2H), 2.37 (m, 2H), 2.05 (m, 2H), 1.87 (m,
2H), 1.68 (m, 2H), 1.27 (d, J ) 6.3 Hz, 6H). Anal. (C24H29-
BrN6O2‚1.45H2O‚0.15EtOAc): C, H, N.
Acknowledgment. The authors would like to ac-
knowledge Dr. Vladimir Beylin and Mr. Michael Waldo
for providing scaled-up quantities of many key inter-
mediates, and Dr. Garrett Hoge, Mr. Norman Colbry
and Mr. Mark Lovdahl for performing all of the high-
pressure reactions. HRMS data were provided by Dana
DeJohn. For support and helpful discussions, the au-
thors are grateful to Dr. Alexander Bridges and Dr. Dick
Leopold.
Cyclin Dependent Kinase Assays and Other Protein
Kinases. Cdk assays for IC50 determinations were performed
in 96-well filter plates (Millipore, MADVN6550). All Cdk-cyclin
complexes were expressed in insect cells through baculovirus
infection and purified as described previously.19 The substrate
for the assays was amino acids 792-928 of pRb fused to GST
(GST-RbCterm).20 The total volume for each well was 0.1 mL

2404 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Toogood et al.
M.; Schow, S. R.; Schultz, P. G.; Wang, X.; Wick, M. M.;
Shiffman, D. CVT-313, A Specific and Potent Inhibitor of CDK2
That Prevents Neointimal Proliferation. The J. Biol. Chem. 1997,
272, 29207-29211. (j) Havl´ıcˇek, L.; Hanusˇ, J.; Vesely´, J.; Leclerc,
S.; Meijer, L.; Shaw, G.; Strnad, M. Cytokinin-Derived Cyclin-
Dependent Kinase Inhibitors: Synthesis and cdc2 Inhibitory
Activity of Olomoucine and Related Compounds. J. Med. Chem.
1997, 40, 408-412. (k) Meijer, L.; Borgne, A.; Mulner, O.; Chong,
J. P. J.; Blow, J. J.; Inagaki, N.; Inagaki, M.; Delcros, J.-G.;
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Supporting Information Available: Purity data for
target compounds. Additional experimental details for the
synthesis of aminopyridine side chains. This information is
available free of charge via the Internet at http://pubs.acs.org.
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