Discovery of [4-amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimidin-5- yl](2,3-difluoro-6-methoxyphenyl)methanone (R547), a potent and selective cyclin-dependent kinase inhibitor with significant in vivo antitumor activity

Article abstract of DOI:10.1021/jm0606138

The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K_i > 10 μM). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K_i = 0.001, 0.003, and 0.001 μM for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC₅₀ = 0.08 μM). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.

Full text of DOI:10.1021/jm0606138

J. Med. Chem. 2006, 49, 6549-6560
6549
Discovery of [4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimidin-5-yl](2,3-difluoro-6-
methoxyphenyl)methanone (R547), A Potent and Selective Cyclin-Dependent Kinase Inhibitor
with Significant in Vivo Antitumor Activity
Xin-Jie Chu,*,† Wanda DePinto,‡ David Bartkovitz,† Sung-Sau So,† Binh T. Vu,† Kathryn Packman,‡ Christine Lukacs,†
Qingjie Ding,† Nan Jiang,† Ka Wang,§ Petra Goelzer,§ Xuefeng Yin,‡ Melissa A. Smith,‡ Brian X. Higgins,‡ Yingsi Chen,|
Qing Xiang,| John Moliterni,† Gerald Kaplan,† Bradford Graves,† Allen Lovey,*,† and Nader Fotouhi†
Departments of DiscoVery Chemistry, DiscoVery Oncology, Non-Clinical Drug Safety and DiscoVery Technologies, Hoffmann-La Roche Inc.,
340 Kingsland Street, Nutley, New Jersey 07110
ReceiVed May 23, 2006
The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since
deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition
of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer.
The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive
inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a
substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has
been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has
led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4
but are inactive against a large panel of serine/threonine and tyrosine kinases (Ki > 10 µM). As one of these
representative analogues, compound 39 (R547) has the best CDK inhibitory activities (Ki ) 0.001, 0.003,
and 0.001 µM for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting
the growth of various human tumor cell lines including an HCT116 cell line (IC50 ) 0.08 µM). An X-ray
crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is
consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human
colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its
superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial
for the treatment of cancer.
Introduction
inhibitor of any CDK or combination of CDKs has reached
Phase III clinical trials as a single agent.
Cyclin-dependent kinases (CDKs) are a family of serine-
threonine protein kinases that govern the initiation, progression,
and completion of the cell cycle. Activity of the CDKs allows
the orderly transition between phases of the cell cycle. CDK
activity is controlled by association with regulatory subunits
(cyclins) and CDK inhibitor proteins, by their phosphorylation
state and by ubiquitin-mediated proteolysis.1-4 Since the loss
of cell cycle control leading to deregulated cell proliferation is
one of the hallmarks of cancer, it is anticipated that the in-
hibition of CDKs will provide an effective approach to control
tumor growth and therefore have an impact on cancer therapy.
Inhibition of CDKs has been studied by many organizations
and has been achieved using a variety of structural templates
with varying degrees of selectivity and activities. Many of these
discoveries, including extensive patent literature, have been
reviewed.5-12
Recent siRNA experiments in which inhibiting CDK2 failed
to halt the proliferation of osteosarcomas and Rb-negative
cervical cancers suggested that CDK2 may not be a good target
for cancer therapy.18 CDK2 has also been found to be dispens-
able for regulating the inhibitory effects of p27Kip1 and p21Cip1
during the G1 phase of the cell cycle.19 Additionally, CDK2
knockout mouse experiments have implied the ability of other
kinases to circumvent CDK2 and allow other CDKs and other
cyclin partners to substitute for certain stages of the cell cycle
that are essential for cells to progress toward mitosis.20,21
Complex interactions within the cell cycle and data indicating
that mice lacking all three D cyclins or both CDK4 or CDK6
survive until relatively late in the various stages of embryo-
genesis or even up to birth further cloud the overall picture.22,23
These and other studies suggest a need to rethink existing models
of cell cycle control in development and tumorigenesis.24 The
ability of CDKs and their cyclin partners to adjust, substitute,
and compensate for one another leads to a clearer notion that
the inhibition of more than one or two CDKs might be necessary
to sustain the suppression of tumor growth in a clinical setting.
The success of imatinib (Gleevec), initially thought to target
only Abl tyrosine kinase but proved to be a somewhat less than
targeted therapy, has suggested that inhibition of a number of
targets by a single molecule could provide a useful therapy.25
CDK inhibitors such as flavopiridol (L868275) (1, Figure 1)
and 7-hydroxystaurosporine (UCN-01) have been evaluated in
clinical trials for some time.13 Recently, more selective CDK
inhibitors such as roscovitine (2) (CYC-202)14 and BMS-387032
(3),15 targeting CDK2, and PD0332991 (4),16,17 targeting CDK4,
have entered clinical trials. However, to date no small molecule
* To whom correspondence should be addressed. Phone, 973-235-5183;
Fax, 973-235-7239; E-mail: xin-jie.chu@roche.com.
† Department of Discovery Chemistry.
Our goal was to identify small molecule inhibitors of CDKs
with low nanomolar activities that were selective for the CDK
family of kinases. We recently identified a series of diamino-
‡ Department of Discovery Oncology.
§ Department of Non-Clinical Drug Safety.
| Department of Discovery Technologies.
10.1021/jm0606138 CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/30/2006

6550 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Chu et al.
Figure 1. Selected CDK inhibitors flavopiridol (1), roscovitine (2), BMS-387032 (3), and PD0332991 (4).
the geometry of the substituent vectors from the two scaffolds
such that the distance between the two substituents attached to
the diaminopyrimidine is significantly larger than that of the
corresponding diaminothiazole (Figure 4). This subtle variation
in hydrophilicity and geometric parameters suggested that we
may find a different SAR between the two series. With this
consideration in mind, a series of diaminopyrimidine analogues
with small substituents on the A ring and 4-methylpiperazinyl
on the B ring were prepared (Table 1). Pyrimidine 17 with R1
as 3-methoxy was inactive, while compound 18 having R1
as 3-fluoro showed modest CDK4 inhibitory activity (Ki )
0.450 µM). The 3-trifluoromethyl analogue 19 was inactive
while 20, having the trifluoromethyl at the C2 position, ex-
hibited modest inhibition against CDK4 (Ki ) 0.821 µM). The
2-methyl and 2-methoxy analogues 21 and 22, respectively,
demonstrated increasing CDK4 inhibition, suggesting that
electron-donating substituents were preferred at the C2 position.
The 2-methoxy derivative 22 with CDK4 inhibition of 0.159
µM (Ki) was 5-fold more potent than 20 and inhibited CDK1
and CDK2 as well.
Figure 2. Generic structures of diaminothiazole 5 and diaminopyri-
midine 6 as CDK inhibitors.
thiazole-based CDK4 inhibitors 5 (Figure 2) that were selec-
tive against CDK1 and CDK2.26,27 One of these diamino-
thiazole analogues showed modest in vivo efficacy.28 In this
report, we describe the discovery of a novel series of 2,4-
diamino-5-ketopyrimidines 6 that provide highly potent inhibi-
tors of the CDK family and block the cell cycle at both G1 and
G2 check points. Optimization of this series resulted in the
selection of a lead compound for advancement into clinical
trials.29
Chemistry
After the establishment of 2,4-diaminopyrimidines as novel
CDK inhibitors, the focus was then placed on optimizing the B
ring to improve the CDK inhibitory activity while the 2-methoxy
group was maintained on the A ring (Table 2). Systematic SAR
exploration revealed that the non-aromatic 4-aminopiperidine
group instead of an aromatic B ring (as in compounds 17-22)
was critical for improving potency of the diaminopyrimidine
series. While the tertiary amine 24 was inactive, the secondary
amine 23 showed modest enzyme inhibitory activity against
CDK4 and weak inhibition against CDK1 and CDK2. Urea 25
with a non-basic NH improved both CDK1 and CDK2 inhibitory
activities by 4-fold (Ki ) 1.5 and 1.7 µM, respectively) while
maintaining modest potency against CDK4 (Ki ) 0.409 µΜ).
Without the potential hydrogen-bond donor as in urea 25,
carbamate 26 exhibited a further 3-6-fold improvement in
potency against CDK1, CDK2, and CDK4, resulting in a multi-
targeted, submicromolar CDK inhibitor (Ki ) 0.246, 0.597,
and 0.110 µM, respectively). In an effort to improve both the
potency and the physicochemical properties (data not shown)
of the diaminopyrimidine series, we focused our attention on
modifying the carbamate moiety of compound 26. Amides 27
and 28 were found to have equal to slightly more potent kinase
inhibition against CDK1, CDK2, and CDK4 compared to
carbamate 26. However, with the introduction of a strongly
polarized sulfonyl group, sulfonamides (29 and 30), demon-
strated a significant increase in kinase inhibitory activity (CDK1,
CDK2, and CDK4) and modest improvement in cellular potency
against HCT116 human colon cell line. For example, methyl
sulfonamide 29 was nearly 1 order of magnitude more potent
than carbamate 26 in the kinase assays (Ki ) 0.028, 0.024, and
0.014 µM for CDK1, CDK2, and CDK4, respectively) and
Most 2,4-diamino-5-ketopyrimidine compounds described in
this report were prepared following a four-step synthetic route
outlined in Scheme 1. Starting from commercially available
4-amino-2-ethylsulfanylpyrimidine-5-carboxylic acid (7), stan-
dard amide bond coupling with N,O-dimethylhydroxylamine
provided N-methoxy-N-methylamide 8 as the key intermediate.
Treatment of amide 8 with appropriately functionalized aryl-
lithium or Grignard reagents 9 afforded ketones 10. Oxidation
of 10 using 2 equiv of m-chloroperoxybenzoic acid (mCPBA)
gave ethyl sulfones 11; alternatively, sulfoxides 12 were
obtained as the major product when 1 equiv of mCPBA was
used. Displacement of the sulfone or sulfoxide in 11 or 12 with
various aromatic or aliphatic amines under microwave conditions
furnished 2,4-diamino-5-ketopyrimidine derivatives 6. For the
piperidinyl substituted 2,4-diamino-5-ketopyrimidine series 16,
some analogues were directly prepared with this four-step
sequence when the appropriate amines were available, whereas
the other derivatives were synthesized from intermediate 15 as
shown in Scheme 2. Reaction of 4-amino-N-tert-(butoxycarbo-
nyl)piperidine (13) with sulfone 11 or sulfoxide 12 provided
14 which, upon treatment with trifluoroacetic acid, gave
piperidine 15. Selective N-alkylation, acylation, or sulfonylation
of the secondary amine in the piperidine ring of 15 led to the
desired 2,4-diamino-5-ketopyrimidines 16.
Results and Discussion
Molecular modeling studies revealed that the 2,4-diamino-
5-ketopyrimidine core has similar electrostatic characteristics
in its donor-acceptor-donor (DAD) region as the diaminothia-
zole core. However, the replacement of the S atom by the
CdN bond makes the new scaffold considerably more hydro-
philic (Figure 3). Moreover, the expansion of ring size alters
resulted in favorable cell growth inhibition (HCT116, IC50
)
1.6 µM). It was also noted that extending the alkyl group in

A Cyclin-Dependent Kinase Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6551
Scheme 1. General Synthesis of 2,4-Diamino-5-ketopyrimidinesa
a Reagents and conditions: (a) MeONHMe‚HCl, HOBt/HBTU, DiPEA, DMF, rt, 98%; (b) 3-4 equiv of ArLi or ArMgX, THF, -78 °C, 65∼90%; (c)
mCPBA, CHCl3, 0 °C, 90-98%; (d) i-PrOH, 100-110 °C, microwave, 20 min, >90%.
Scheme 2. Synthesis of 4-Piperidinyl Substituted 2,4-Diamino-5-ketopyrimidinesa
a Reagents and conditions: (a) i-PrOH, 100∼110 °C, microwave, 20 min, >90%; (b) TFA, CH2Cl2, 0 °C, >95%; (c) R3X, R3COCl or R3SO2Cl, DiPEA,
CH2Cl2, rt, 90-98%.
Table 1. Identification of 2,4-Diamino-5-ketopyrimidines as Inhibitors
of CDKsa
Ki (µM)b
IC50 (µM)c
HCT116
compd
R1
CDK4
CDK1
CDK2
17
18
19
20
21
22
3-MeO
3-F
3-CF3
2-CF3
2-Me
2-MeO
>1
>10
>10
>10
>10
>10
1.259
>10
>10
>10
>10
>10
5.141
NDd
ND
ND
ND
16.5
11.0
0.450
>1
Figure 3. Electrostatic potential maps of diaminopyrimidine (on the
left) and diaminothiazole (on the right). An isodensity surface is first
obtained for the two molecules, and the surface is then assigned different
colors according to their electrostatic potential. The red area denotes a
region with the most negative potential and blue areas the most positive.
The maps are calculated using Spartan′02.
0.821
0.584
0.159
a See ref 32 for a description of assay conditions. b Ki values are reported
as the mean of several determinations. The variability around the mean
value was less than 50%. c Assay were run as singlets. d ND ) not
determined.
out through the preparation of a small number of analogues with
variation at the C2 position (31-33). 2-Fluoro- and 2-methyl-
substituted compounds (31 and 32, respectively) substantially
reduced CDK inhibition compared with 2-methoxy analogue
26. We believe that preorganization of the binding confor-
mation of the ligand to CDK is promoted by the 2-methoxy
group, which enforces non-coplanarity of the A ring with re-
spect to the pyrimidine core. Surprisingly, the 2-ethoxy deriva-
tive 33 resulted in greater than 10-fold loss in CDK1, CDK2,
and CDK4 inhibitory activity (vs amide 27), suggesting that
this binding pocket is not big enough to accommodate larger
substitutions on the A ring. This was consistent with our earlier
observation outlined in Table 1 and confirmed that the 2-meth-
oxy was the optimal group for the C2 position of the aromatic
A ring.
Figure 4. Overlay of diaminopyrimidine (green) vs diaminothiazole
(orange). Distances in angstrom (Å) between the centroids of the two
phenyl substituents for each core structure are shown.
the amides (e.g. 27, 28) or sulfonamides (e.g. 29, 30) had a
very limited impact on kinase inhibition, consistent with the
observation from the crystal structure that the R2 group points
toward solvent (vide infra). With the substituted 4-piperidine
identified as the most preferred B ring, a re-examination of the
simple monosubstitution on the aromatic A ring was carried

6552 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Chu et al.
Table 2. Optimization of R1 on Ring A and R on Piperidine Ring B in 2,4-Diamino-5-ketopyrimidinesa
a See ref 32 for a description of assay conditions. b,c Ki and IC50 values are reported as the mean of several determinations. The variability around the
mean value was less than 50%. d ND ) not determined.
With the identification of 1-methanesulfonyl-4-piperidine as
the optimal B ring and 2-methoxy as the most preferred group
for the C2 position of the aromatic A ring, further exploration
of other substitutions on the A ring was carried out (Table 3).
Introduction of a fluorine to the C5 position of compound 29
yielded a more potent CDK inhibitor (35) with Ki values of
less than 10 nM with respect to CDK2 and CDK4, while the
4-fluoro analogue 34 reduced the inhibition against CDK1,
CDK2, and CDK4. Compound 35 also proved to be more potent
in our cell-based assay (HCT116 cell line) with an IC50 of 0.66
µM. A notable difference for the effect of substitution at the
C5 position was that the chloro analogue 36 was 10-fold less
active in the cell-based assay than the corresponding fluoride
35, suggesting the size of the substituent at this position may
affect potency. Similar to the 5-fluoro analogue 35, compound
37 with the fluorine at the C6 position also significantly
CDKs (Ki ) 0.001, 0.003, and 0.001 µM for CDK1, CDK2,
and CDK4, respectively) and excellent cellular potency (IC50
) 0.08 µM, HCT116 cell line). Another difluoro-substituted
analogue 40, with fluorines at both the C4 and C5 positions,
had a potency equal to that of monofluoro derivative 35 but
6-fold less than that of compound 39. A 4,5,6-trifluoro substitu-
tion was still tolerated, as seen in derivative 41, with a slight
loss of the cellular potency compared to 39. However, when
the third fluorine substitution was at the C3 position (42), the
CDK inhibitory activities dropped considerably. From these data
we concluded that the 5,6-difluoro-2-methoxy substitution on
the A ring is the most preferred pattern, resulting in excellent
CDK inhibitory activities and in vitro cellular potency for the
diaminopyrimidine series.
Kinase Selectivity Profile. The most promising 2,4-diamino-
5-ketopyrimidine analogues were examined in both our in-house
kinase selectivity panel and the Upstate kinase selectivity
screen.32 As shown in Table 4, compound 39 was found to be
a potent and ATP-competitive inhibitor of the CDK family of
protein kinases. It strongly inhibits CDK1/cyclin B, CDK2/
cyclin E, and CDK4/cyclin D and shows little or no inhibitory
activity against a panel of 18 other serine/threonine kinases.
Compound 39 was also effective in inhibiting the phosphory-
lation of retinoblastoma protein (pRb) in human tumor cells in
a time dependent manner. Treatment of HCT116 cells with
compound 39 inhibited phosphorylation at both the serine 795
and 780 sites on pRb, consistent with the two phosphorylation
sites specifically phosphorylated by CDK4. Cell cycle block in
increased CDK inhibitory activity and cellular potency (IC50
)
0.30 µM, HCT116 cell line). Previous reports have indicated
that 2,6-difluorophenyl substitution provides a beneficial effect
on the potency of the CDK inhibiton.30,31 Interestingly, substitu-
tion of the 2-methoxy group in 37 with a fluorine atom resulting
in the 2,6-difluoro analogue 38 proved to be inconsequential
with respect to CDK inhibition and yet resulted in more than
10-fold loss in the cellular potency. This distinct difference
confirmed the early observation that the 2-methoxy group is
unique for this diaminopyrimidine series. Compound 39 pos-
sessing both 5- and 6-fluoro substitution culminated in an
inhibitor with low, single-digit nanomolar potency against the

A Cyclin-Dependent Kinase Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6553
Table 3. Further Substitution Patterns on Aromatic Ring A of
2,4-Diamino-5-ketopyrimidinesa
Ki (µM)b
IC50 (µM)c
compd R2 R3 R4 R5 R6 CDK4 CDK1 CDK2 HCT116
34
35
36
37
38
39
40
41
42
OMe
OMe
OMe
OMe
F
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
H
F
F
H
F
Cl
H
H
F
F
F
F
H
H
H
F
F
F
H
F
H
0.021 0.193 0.084
0.007 0.014 0.007
0.036 0.133 0.451
0.003 0.017 0.013
0.017 0.038 0.015
0.001 0.001 0.003
0.006 0.014 0.006
0.001 0.005 0.002
0.267 1.667 0.495
3.6
0.65
7.0
0.30
3.4
0.08
0.52
0.22
NDd
H
H
H
H
H
F
Figure 5. The 1.85 Å crystal structure of compound 39 bound in the
ATP binding pocket of CDK2 (no cyclin). Nitrogen atoms are in blue,
oxygen atoms in red, sulfur in orange, and fluorine in green, and key
hydrogen bonds are depicted. Bridging waters play a crucial role in
the enhanced binding of this compound to the enzyme. The figure was
made with program PyMOL.33
F
F
a See ref 32 for a description of assay conditions. b,c Ki and IC50 values
are reported as the mean of several determinations. The variability around
the mean value was less than 50%. d ND ) not determined.
Table 4. Kinase Selectivity Profile for Compound 39a
kinases
Ki (µM)b
kinases
Ki (µM)b
CDK4/cyclin D
CDK2/cyclin E
CDK1/cyclin B
PKA
0.001 ( 0.0001
GSK3â
KDR
8
>5
>5
>5
>5
>50
>5
>5
>5
>5
0.003 ( 0.0008
0.002 ( 0.0008
FGFR
EGFR
PDGF
MAPK2
IGFR
SRC
FAK
AURORA
>5
>5
>5
>5
>5
>5
>5
>50
PKB
PKCR
PKCâ
FYN
EPHB3
SGK
p38
a See ref 32 for a description of assay conditions and inhibitory activity
data against additional kinases. b Ki ) IC50/(1 + S/Km) where S is the
substrate ATP concentration and Km is the Michaelis-Menten constant for
ATP; mean of at least two separate determinations.
Figure 6. Mean plasma concentration versus time profiles of selected
compounds 29, 35, and 39 following oral administration (100 mg/kg)
in female athymic nontumor bearing mice. The error bars represent
the percent coefficient of variation. The dashed lines A (for 35) and B
(for 39) represent the in vitro IC90 values of the cell inhibition in the
HCT116 cell line (IC90 ) 762 ng/mL and 75 ng/mL for compounds
35 and 39, respectively): compound 29 (open square), compound 35
(open circle), compound 39 (filled triangle).
G1 + G2 inducing apoptosis in a dose-dependent manner in the
same cell line has also been observed.32
Structural Study of Inhibitors bound to CDK2. The crystal
structure of compound 39 bound to CDK2, shown in Figure 5,
revealed a binding motif whereby the hinge region (residues
Phe80-Glu81-Phe82-Leu83) makes key hydrogen-bond interac-
tions with the two donors in the donor-acceptor-donor (DAD)
portion of the diaminopyrimidine core. The central nitrogen
acceptor is 3.7 Å away from the backbone NH of Leu83. The
B ring piperidine makes hydrophobic contacts similar to those
seen in many kinase inhibitors,34 with the sulfonamide making
both direct and water-mediated hydrogen bonds to Lys89,
Asp86, and the backbone carbonyl of His84. The 2,6-disubsti-
tution pattern on the A ring forces it out of the plane of the
pyrimidine core and allows the difluoro to pack into the
hydrophobic pocket near Phe80 and the alkyl chain of Lys33.
stantially. But the concentrations of the 5-fluoro analogue 35
remained above or close to its IC90 level (IC90 ) 1.8 µM or
762 ng/mL in HCT116 cell line). The plasma exposure of the
5,6-difluoro analogue 39 greatly exceeded (about 10-fold at 8
h post dose) its IC90 values (IC90 ) 0.17 µM or 75 ng/mL in
HCT116 cell line). Sufficient drug exposure can be expected
from a dose regiment of 50 and 75 mg/kg three times per week
in the experimental animals. The clearance (CL) and the volume
of distribution (Vss) of these three compounds also improved
as the number of substitutions on the aromatic A ring increased
(Table 5).
As summarized in Table 6, the systemic plasma clearance of
compound 39 in female athymic non-tumor bearing mice was
38.6 mL/min/kg. A 100 mg/kg oral dose resulted in a half-life
(t1/2) of 2.8 h, and the measured oral bioavailability (Fpo) of
compound 39 in this study was approximately 21%. The
observed large volume of distribution (1070 mL/kg) suggests
deep tissue penetration. The pharmacokinetic profile of com-
Pharmacokinetics Study. To assess the preliminary phar-
macokinetic (PK) properties of this class of 2,4-diamino-5-
ketopyrimidines, several representative lead analogues including
compounds 29, 35, and 39 were administered orally and intra-
venously (iv) to female nude mice. The mean plasma concentra-
tion versus time profiles after an oral dose of 100 mg/kg are
shown in Figure 6. All three compounds were rapidly absorbed
and their Cmax was reached within 1 h. Over the 4 h period, the
observed drug concentration of compound 29 decreased sub-

6554 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Chu et al.
Table 5. Mouse PK Data Following Single IV Administration (10
Conclusions
mg/kg) of Selected Compoundsa
In summary, we have reported the discovery of a novel class
of 2,4-diamino-5-ketopyrimidines (6) as potent, selective, and
ATP-competitive CDK inhibitors. The SAR of the diaminopy-
rimidine core proved to be unique in that it favors a 2-meth-
oxyphenyl substitution on the A ring rather than a 2,6-difluoro
substitution. Further lead optimization resulted in the identifica-
tion of compound 39 as a potent inhibitor of CDK1, CDK2,
and CDK4 (Ki ) 0.001-0.003 µM) which was selective against
a large panel of related and unrelated serine/threonine and
tyrosine kinases (Ki > 10 µM). It exhibited excellent in vitro
cellular potency, inhibiting the growth of HCT116 human
colorectal tumor cell line. Furthermore, compound 39 demon-
strated significant in vivo efficacy against the HCT116 human
colorectal tumor xenograft model. On the basis of its superior
overall profile, compound 39 (R547)29 was chosen as the clinical
candidate and is currently in Phase I clinical trial for the
treatment of cancer. An expanded SAR study of this novel series
of 2,4-diamino-5-ketopyrimidine CDK inhibitors and the related
biological evaluation of further lead compounds will be reported
soon.
CL
Vss
(mL/kg)
compd
(mL/min/kg)
29
35
39
47.1
48.3
38.6
401
885
1070
a Based on mean data from three mice/time point.
Table 6. Pharmacokinetic Properties of Compound 39 in Micea
iv
po
parameter
unit
mg/kg
ng/mL
h
ng h/mL
h
mL/min/kg
mL/kg
%
administration
administration
dose
Cmax
Tmax
AUC(0-t)
T1/2
10
100
2700
0.5
8169
2.8
-
7310
0.083
4152
0.39
38.6
1070
-
CL
Vss
Fpo
-
21
a Based on mean data from three mice/time point.
Experimental Section
Chemistry. All nonaqueous reactions were carried out under an
argon or nitrogen atmosphere at room temperature, unless otherwise
noted. All reagents and anhydrous solvents were used as obtained
commercially without further purification or distillation, unless
otherwise stated. Analytical thin-layer chromatography (TLC) was
performed on EMD Chemicals silica gel 60 F254 precoated plates
(0.25 mm). Compounds were visualized by UV light and/or stained
with either p-anisaldehyde, iodine, or phosphomolybdic acid
solutions followed by heating. Analytical high-pressure liquid
chromatography (HPLC) and LC-MS analyses were conducted
using the following two instruments and conditions. Method 1:
Hewlett-Packard HP-1090 pump and HP-1090 PDA detector set
at 215 nm with the MS detection performed with a Micromass
Platform II mass spectrometer with electrospray ionization (ESI);
Chromegabond WR C18 3 µm, 120 Å, 3.2 mm × 30 mm column;
solvent A, H2O-0.02% TFA; solvent B, MeCN-0.02% TFA; flow
rate ) 2 mL/min; start 2% B, final 98% B in 4 min, linear gradient.
Method 2: Waters 2795 pump and Waters 2996 photodiode array
detector set at 214 nm with the MS detection performed with a
Waters ZQ mass spectrometer (ESI); Epic Polar Hydrophilic 3 µm,
120 Å, 3.2 mm × 30 mm column; solvent A, H2O-0.03% HCO2H;
solvent B, MeCN-0.03% HCO2H; flow rate ) 2 mL/min; start
10% B, final 100% B in 3 min linear gradient, remaining for 1
min. Compounds were purified using either of the following
methods. Flash column chromatography was performed on EM
Science silica gel 60 (particle size of 32-63 µm, 60 Å). Preparative
reverse-phase high-pressure liquid chromatography (RP HPLC) was
performed using a Waters Delta prep 4000 pump/controller, a 486
detector set at 215 nm, and a LKB Ultrorac fraction collector. The
sample was dissolved in a mixture of MeCN/20 mM aqueous
NH4Ac, applied on a C-18 20 × 100 mm column and eluted at 30
mL/min with a 20 min linear gradient of 10%-90% B, where
solvent A ) H2O with 20 mM NH4Ac and solvent B ) MeCN.
The pooled fractions were concentrated under reduced pressure and
lyophilized to afford the desired compounds. 1H NMR spectra were
recorded using a Varian Mercury 300 MHz or Varian Inova 400
MHz spectrometer and calibrated using an internal reference. The
chemical shifts are in parts per million (δ) referenced to Me4Si
(0.00 ppm) or CHCl3 (7.26 ppm). High-resolution mass spectra
(HMRS) were recorded on a Bruker Apex II FTICR mass
spectrometers with a 4.7 T magnet (ES) or Micromass AutoSpec
(EI) mass spectrometers.
Figure 7. Effect of compound 39 on HCT116 human colorectal tumor
xenograft growth. Statistical analysis was determined by the rank sum
test and one-way Anova and a post-hoc Bonferroni t-test. * )
Statistically significant as compared to the vehicle-treated group, where
p < 0.001.
pound 39 along with the excellent in vitro cellular potency
warranted the advancement of these 2,4-diamino-5-ketopyri-
midine analogues into in vivo efficacy studies.
In Vivo Antitumor Activity. Compound 39 was evaluated
for its in vivo antitumor activity in female nude mice bearing
established HCT116 human colorectal xenografts with a
mean starting volume of about 100 mm3 (Figure 7). In this
study, compound 39 was administered orally three times per
week on Monday, Wednesday, and Friday (M, W, F) as a
suspension in 1% Klucel LF in water with 0.1% Tween 80 for
18 days (a total of eight doses). At the end of the two and a
half week study, compound 39 demonstrated significant in
vivo antitumor activity in the absence of gross or histologi-
cal indications of toxicity. A dose of 25 mg/kg three times
per week inhibited HCT116 colorectal tumor growth by 52%,
while doses of 50 and 75 mg/kg three times per week in-
hibited tumor growth by 95% and 94%, respectively, as
compared to vehicle-treated mice. A more extensive biochemical
and biological characterization of compound 39, including a
comprehensive in vivo efficacy evaluation, will be published
separately.32
Crystallization, Data Collection, and Refinement. Crystals of
CDK2 were grown at 4 °C by the vapor diffusion method. CDK2-
(1-298) at 12 mg/mL was mixed with and equilibrated against

A Cyclin-Dependent Kinase Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6555
10% PEG 3350, 0.2 M ammonium phosphate, 0.1 M Bicine, pH
9.0. â-Mercaptoethanol (2%) was added to the reservoir after mixing
of the drop. Crystals appeared overnight, after which 39 at 100
mM in DMSO was added to the drop. After soaking for 24 h,
crystals were transferred to cryoprotectant containing 20% PEG
3350, 15% ethylene glycol, 0.2 M ammonium phosphate, 0.1 M
Bicine, pH 9.0, and then cooled to liquid nitrogen temperature. Data
were collected at beamline ×8C at the National Synchrotron Light
Source at Brookhaven National Laboratories. The crystals belonged
space group P212121 with unit cell dimensions a ) 53.5 Å, b )
71.65 Å, c ) 71.7 Å. Data were processed to 1.85 Å with the HKL
package35 to an R-sym of 0.039. The structure was determined by
the difference Fourier method and was refined with CNX (CNX
v.2000.1, Molecular Simulations Inc.), with 0.05% of reflections
in the test set, to a final R-factor/R-free of 0.208/0.237. Coordinates
have been deposited to the PDB under accession code 2FVD.
Pharmacokinetic Study Methods. Female immunodeficient
nude mice (13-14 weeks old) obtained from Charles River
Laboratories (Wilmington, DE) were allowed to acclimate for a
minimum of 72 h prior to study start. Mice received a single oral
(100 mg/kg) or intravenous (10 mg/kg) dose of the test compound,
and blood samples from three mice/time point were collected over
a 24-h period in EDTA vials. The oral dose was administered as a
suspension in 1% Klucel and 0.1% Tween 80, and the iv dose was
administered as a solution in 10% PEG 400 and 90% hydroxy-
propyl-â-cylcodextrin (28%). Plasma samples were analyzed by
LC/MS for the test compound, and noncompartmental PK analysis
was performed on the mean plasma concentration-time profiles
using WinNonlin (Pharsight) or Watson.
for 10c: Anal. RP-HPLC tR ) 2.06 min (method 1, 94% purity), tR
) 1.81 min (method 2, 95% purity); 1H NMR (300 MHz, CDCl3)
δ 1.40 (t, 3H, J ) 7.3 Hz, CH3), 3.14 (q, 2H, J ) 7.3 Hz, SCH2),
5.75 (br, 1 H, NH), 7.23-7.52 (m, 4 H, aromatic), 8.41 (s, 1 H,
aromatic), 8.58 (br, 1 H, NH); HRMS for C13H13FN3OS (M + H+)
calcd 278.0761, observed 278.0758.
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)-o-tolylmethanone (10b)
1
was prepared with the same procedure as described for 10c: H
NMR (300 MHz, CDCl3) δ 1.38 (t, 3H, J ) 7.2 Hz), 2.29 (s, 3H),
3.12 (q, 2H, J ) 7.2 Hz), 5.76 (brd, 1H, NH), 7.17-7.29 (m, 3H,
aromatic), 7.34-7.41 (m, 1H, aromatic), 8.14 (s, 1H, aromatic),
8.79 (brd, 1H, NH); HRMS for C14H15N3OS (M+) calcd 273.0936,
observed 273.0933.
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)(2-fluorophenyl)meth-
anone (10d) was prepared with the same procedure as described
for 10c: Anal. RP-HPLC tR ) 2.00 min (method 1, >99% purity),
1
tR ) 1.78 min (method 2, >99% purity); H NMR (300 MHz,
CDCl3) δ 1.39 (t, 3H, J ) 7.4 Hz, CH3), 3.14 (q, 2H, J ) 7.4 Hz,
3
SCH2), 5.80 (br, 1 H, NH), 7.18 (td, 1 H, Jortho ) JHF ) 8.4 Hz,
Jmeta ) 1.1 Hz, aromatic), 7.29 (td, 1H, Jortho ) 7.6 Hz, Jmeta ) 1.1
Hz, aromatic), 7.43 (ddd, 1H, Jortho ) 7.6 Hz, 4JHF ) 6.8 Hz, Jmeta
6
) 1.8 Hz, aromatic), 7.52 (m, 1H, aromatic), 8.27 (d, JHF ) 2.9
Hz, 1 H, aromatic), 8.70 (br, 1 H, NH); HRMS for C13H13FN3OS
(M + H+) calcd 278.0758, observed 278.0761.
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)(2-ethoxyphenyl)-
methanone (10e) was prepared with the same procedure as
described for 10c: white solid; Anal. RP-HPLC tR ) 2.12 min
(method 1, 98% purity), tR ) 1.86 min (method 2, >99% purity);
1H NMR (300 MHz, CDCl3) δ 1.25 (t, 3H, J ) 7.1 Hz, CH3), 1.38
(t, 3H, J ) 7.4 Hz, CH3), 3.12 (q, 2H, J ) 7.4 Hz, SCH2), 4.03 (q,
2H, J ) 7.1 Hz, 2 H, OCH2), 5.70 (br, 1 H, NH), 6.95 (d, 1H, J )
8.4 Hz, aromatic), 7.03 (dt, 1H, Jortho ) 7.5 Hz, Jmeta ) 0.8 Hz,
aromatic), 7.28 (dd, 1H, Jortho ) 7.5 Hz, Jmeta ) 1.8 Hz, aromatic),
7.34 (ddd, 1H, Jortho ) 8.4 Hz, Jortho ) 7.5 Hz, Jmeta ) 1.8 Hz,
aromatic), 8.21 (s, 1 H, aromatic), 8.72 (br, 1 H, NH); HRMS for
C15H18N3O2S (M + H+) calcd 304.1114, observed 304.1117.
(4-Amino-2-ethanesulfanylpyrimidin-5-yl)(4-fluoro-2-meth-
oxyphenyl)methanone (10f) was prepared with the same procedure
4-Amino-2-ethylsulfanylpyrimidine-5-carboxylic Acid Meth-
oxymethylamide (8). To a solution of 4-amino-2-ethylsulfanylpy-
rimidine-5-carboxylic acid (7) (1.00 g, 5.02 mmol) and diisopro-
pylethylamine (2.79 g, 21.58 mmol, 4 equiv) in DMF (20 mL) were
added 1-hydroxybenzotriazole (1.09 g, 8.07 mmol, 1.5 equiv) and
O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophos-
phate (3.06 g, 8.07 mmol, 1.5 equiv) at 0 °C. After stirring for 15
min, N,O-dimethylhydroxylamine hydrochloride (790 mg, 8.10
mmol) was added and the reaction was allowed to stir at 0-20 °C
for 2 h. The resulting reaction mixture was treated with H2O and
extracted with EtOAc. The combined organic extracts were washed
with brine, dried over Na2SO4, and concentrated. The crude product
was purified on silica gel with hexane/EtOAc (1:1) to give 8 as a
white solid (1.19 g, 98% yield): Anal. RP-HPLC tR ) 1.15 min
(method 1, >95% purity), tR ) 0.89 min (method 2, >95% purity);
1H NMR (300 MHz, CDCl3) δ 1.38 (t, 3H, J ) 7.5 Hz), 3.11 (q,
2H, J ) 7.5 Hz), 3.34 (s, 3H), 3.62 (s, 3H), 8.62 (s, 1H); HRMS
for C9H14N4O2S (M+) calcd 242.0837, observed 242.0836.
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)(2-methoxyphenyl)-
methanone (10c). To a solution of 2-iodoanisole (20.0 g, 85.47
mmol) in anhydrous THF (130 mL) was added over 30 min a 1.6
M solution of n-butyllithium in hexane (51.0 mL, 81.6 mmol), and
the mixture was stirred for a further 40 min at -78 °C to give a
clear solution. A portion of this freshly prepared 2-methoxyphen-
yllithium reagent (70.0 mL, ∼30 mmol, ∼3.5 equiv) was added
slowly to a solution of amide 8 (2.07 g, 8.54 mmol) in anhydrous
THF (30 mL) and the mixture was stirred at -78 °C for 1-2 h
until the complete consumption of amide 8. The resulting mixture
was quenched with aqueous NH4Cl solution and extracted with
EtOAc (3×). The combined organic extracts were washed with
brine (2×), dried over Na2SO4, and evaporated. The residue was
purified on silica gel with hexane/EtOAc (80/20 f 60/40) to give
10c as a light yellow solid (1.85 g, 75% yield): Anal. RP-HPLC
tR ) 1.93 min (method 1, > 95% purity), tR ) 1.72 min (method
1
as described for 10c: white solid; H NMR (300 MHz, CDCl3) δ
1.38 (t, 3H, J ) 7.4 Hz, CH3), 3.13 (q, 2H, J ) 7.4 Hz, SCH2),
3.77 (s, 3H, OCH3), 5.78 (br, 1H, NH), 6.58-6.88 (m, 2H,
aromatic), 7.18-7.39 (m, 1 H, aromatic), 8.17 (s, 1H, aromatic),
8.73 (br, 1 H, NH); LR-MS (M + H+) 308.
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)(5-fluoro-2-meth-
oxyphenyl)methanone (10g) was prepared with the same procedure
as described for 10c: white solid; Anal. RP-HPLC tR ) 2.06 min
(method 1, >99% purity), tR ) 1.82 min (method 2, > 99% purity);
1H NMR (300 MHz, DMSO-d6) δ 1.27 (t, 3H, J ) 7.3 Hz, CH3),
3.06 (q, 2H, J ) 7.3 Hz, SCH2), 3.71 (s, 3 H, OCH3), 7.16 (dd,
4
3
1H, Jortho ) 9.2, JHF ) 4.4 Hz, aromatic), 7.20 (dd, 1H, JHF
)
)
3
8.4, Jmeta ) 3.2 Hz, aromatic), 7.34 (ddd, 1H, Jortho ) 9.2, JHF
8.7, Jmeta ) 3.2 Hz, aromatic), 7.97 (s, 1H, aromatic), 8.33 (br, 1
H, NH), 8.44 (br, 1 H,NH); HRMS for C14H15FN3O2S (M + H+)
calcd 308.0864, observed 308.0863.
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)(5-chloro-2-meth-
oxyphenyl)methanone (10h) was prepared with the same procedure
as described for 10c: light yellow solid; 1H NMR (300 MHz,
DMSO-d6) δ 1.27 (t, 3H, J ) 7.2 Hz, CH3), 3.05 (q, 2H, J ) 7.2
Hz, SCH2), 3.72 (s, 3H, OCH3), 7.17 (d, 1H, Jortho ) 8.7, aromatic),
7.36 (d, 1H, Jmeta ) 2.7 Hz, aromatic), 7.53 (dd, 1H, Jortho ) 8.7
Hz, Jmeta ) 2.4 Hz, aromatic), 7.97 (s, 1H, aromatic), 8.33 (br, 1
H, NH), 8.42 (br, 1 H,NH); HRMS for C14H15ClN3O2S (M + H+)
calcd 324.0568, observed 324.0568.
(4-Amino-2-methylsulfanylpyrimidin-5-yl)(2-fluoro-6-meth-
oxyphenyl)methanone (10i). The aryllithium reagent was generated
via deprotonation of 3-fluoroanisole in a similar manner as described
for the preparation of 10c, which was reacted with 8 to give 10i as
a yellow oil: 1H NMR (300 MHz, CDCl3) δ 2.52 (s, 3H, SCH3),
3.78 (s, 3H, OCH3), 5.82 (br, 1H, NH), 6.73-6.83 (m, 2H,
aromatic), 7.33-7.44 (m, 1H, aromatic), 8.17 (s, 1H, aromatic),
8.75 (br, 1H, NH); MS (M + H+) 307.
1
2, >95% purity); H NMR (300 MHz, CDCl3) δ 1.38 (t, 3H, J )
7.4 Hz, CH3), 3.13 (q, 2H, J ) 7.4 Hz, SCH2), 3.78 (s, 3H, OCH3),
5.74 (br, 1H, NH), 6.98 (d, 1H, J ) 8.6 Hz, aromatic), 7.04 (dt,
1H, J ) 7.8 and 0.6 Hz, aromatic), 7.26 (m, 1H, aromatic), 7.46
(m, 1H, aromatic), 8.18 (s, 1H, aromatic), 8.77 (br, 1H, NH); HRMS
for C14H16N3O2S (M + H)+ calcd 290.0958, observed 290.0961.
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)(3-fluorophenyl)meth-
anone (10a) was prepared with the same procedure as described

6556 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Chu et al.
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)(2,6-difluorophenyl)-
methanone (10j) was prepared following a similar procedure as
described for 10i: 1H NMR (300 MHz, CDCl3) δ 1.39 (t, 3H, J )
7.3 Hz, CH3), 3.13 (q, 2H, J ) 7.3 Hz, CH2), 5.83 (br, 1 H, NH),
7.01 (t, 2H, J ) 7.9 Hz, 2 H, aromatic), 7.46 (tt, 1H, J ) 8.4 and
6.4 Hz, aromatic), 8.21 (s, 1 H, aromatic), 8.68 (br, 1 H, NH);
LR-MS (M + H+) 296.
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)(2,3-difluoro-6-meth-
oxyphenyl)methanone (10k) and (4-Amino-2-ethylsulfanylpy-
rimidin-5-yl)(4,5-difluoro-2-methoxyphenyl)methanone (10l).
Following a similar procedure as described for 10c, the reaction of
the aryllithium reagent, generated from 2-bromo-4,5-difluoroanisole
and n-butyllithium, with amide 8 was allowed to slowly warm to
-35 °C for 2 h. The crude material was purified on prep HPLC to
give isomers 10k and 10l in an approximate 8:1 ratio.
(4-Amino-2-ethanesulfonylpyrimidin-5-yl)-o-tolylmetha-
none (11b): 1H NMR (300 MHz, CDCl3) δ 1.33 (t, 3H, J ) 7.5
Hz), 2.25 (s, 3H), 3.44 (q, 2H, J ) 7.5 Hz), 6.34 (brd, 1H, NH),
7.16-7.28 (m, 3H, aromatic), 7.35-7.41 (m, 1H, aromatic), 8.40
(s, 1H, aromatic), 8.93 (brd, 1H, NH); HRMS for C14H16N3O3S
(M + H) + calcd 306.0907, observed 306.0910.
(4-Amino-2-ethanesulfonylpyrimidin-5-yl)(2-ethoxyphenyl)-
methanone (11e). The same procedure was used as described for
11a: white solid; Anal. RP-HPLC tR ) 1.82 min (method 1, 95%
purity), tR ) 1.60 min (method 2, 98% purity); 1H NMR (300 MHz,
methanol-d4) δ 1.16 (t, 3H, J ) 7.0 Hz, CH3), 1.35 (t, 3H, J ) 7.4
Hz, CH3), 3.52 (q, 2H, J ) 7.4 Hz, SO2CH2), 4.06 (q, 2H, J ) 7.0
Hz, OCH2), 7.10 (dt, 1H, Jortho ) 7.5 Hz, Jmeta ) 0.8 Hz, aromatic),
7.13 (d, 1 H, J ) 8.6 Hz, aromatic), 7.40 (dd, 1H, Jortho ) 7.5 Hz,
Jmeta ) 1.8 Hz, aromatic), 7.55 (ddd, 1H, Jortho ) 8.6 Hz, Jortho
)
7.5 Hz, Jmeta ) 1.8 Hz, aromatic), 8.38 (s, 1 H, aromatic); HRMS
for C15H18N3O4S (M + H+) calcd 336.1013, observed 336.1016.
(4-Amino-2-ethanesulfonylpyrimidin-5-yl)(5-fluoro-2-methoxy-
phenyl)methanone (11g): white solid; Anal. RP-HPLC tR ) 1.71
min (method 1, 87% purity), tR ) 1.49 min (method 2, 92% purity);
1H NMR (300 MHz, CDCl3) δ 1.44 (t, 3H, J ) 7.5 Hz, CH3), 3.55
(q, 2H, J ) 7.5 Hz, SO2CH2), 3.77 (s, 3H, OCH3), 6.32 (br, 1 H,
NH), 6.97 (dd, 1H, 4JHF ) 3.5 Hz, Jortho ) 8.6 Hz, aromatic), 7.09
For compound 10k: Anal. RP-HPLC tR ) 2.12 min (method 1,
>99% purity), tR ) 1.90 min (method 2, >99% purity); 1H NMR
(300 MHz, CDCl3) δ 1.39 (t, 3H, J ) 7.4 Hz, CH3), 3.14 (q, 2H,
J ) 7.4 Hz, SCH2), 3.75 (s, 3H, OCH3), 5.91 (br, 1 H, NH), 6.68
(ddd, 1H, Jortho ) 9.2 Hz, 4JHF ) 3.2 Hz, 5JHF ) 2.0 Hz, aromatic),
7.23 (q, 1H, 3JHF ) 4JHF ) Jortho ) 9.2 Hz, aromatic), 8.16 (s, 1 H,
aromatic), 8.75 (br, 1 H, NH); HRMS for C14H14F2N3O2S (M +
H+) calcd 326.0770, observed 326.0771.
For compound 10l: 1H NMR (400 MHz, DMSO-d6) δ 1.29 (t,
3H, J ) 7.3 Hz, CH3), 3.08 (q, 2H, J ) 7.3 Hz, SCH2), 3.74 (s,
3H, OCH3), 7.34 (dd, 1H, 3JHF ) 12.7 Hz, 4JHF ) 6.3 Hz, aromatic),
7.50 (t, 1H, 3JHF ) 4JHF ) 9.7 Hz, aromatic), 8.06 (s, 1 H, aromatic),
8.34 (br, 1 H, NH), 8.43 (br, 1 H, NH); HRMS for C14H14F2N3O2S
(M + H+) calcd 326.0770, observed 326.0772
3
(dd, 1H, Jmeta ) 2.8 Hz, JHF ) 7.9 Hz, aromatic), 7.22 (m, 1H,
aromatic), 8.50 (s, 1 H, aromatic), 8.89 (br, 1 H, NH); HRMS for
C14H15FN3O4S (M + H+) calcd 340.0762, observed 340.0762.
(4-Amino-2-ethanesulfonylpyrimidin-5-yl)(5-chloro-2-meth-
oxyphenyl)methanone (11h): Anal. RP-HPLC tR ) 1.89 min
(method 1, 95% purity), tR ) 1.67 min (method 2, 92% purity); 1H
NMR (300 MHz, CDCl3) δ 1.43 (t, 3H, J ) 7.4 Hz, CH3), 3.51 (q,
2H, J ) 7.4 Hz, SO2CH2), 3.77 (s, 3 H, OCH3), 6.32 (br, 1 H,
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)(2,3,4-trifluoro-6-
methoxyphenyl)methanone (10m) was prepared following a
similar procedure as described for 10i; the aryllithium reagent was
generated from deprotonation of 3,4,5-trifluoroanisole and reacted
with 8 to give 10m: 1H NMR (300 MHz, CDCl3) δ 1.39 (t, 3H, J
) 7.3 Hz, CH3), 3.13 (q, 2H, J ) 7.3 Hz, SCH2), 3.75 (s, 3H,
NH), 6.96 (d, 1H, Jortho ) 8.8 Hz, aromatic), 7.32 (d, 1H, Jmeta
)
2.6 Hz, aromatic), 7.48 (dd, 1H, Jortho ) 8.8, Jmeta ) 2.6 Hz,
aromatic), 8.48 (s, 1 H, aromatic), 8.88 (br, 1 H, NH); HRMS for
C14H15ClN3O4S (M + H+) calcd 356.0467, observed 356.0466.
(4-Amino-2-ethanesulfonylpyrimidin-5-yl)(2,6-difluorophenyl-
)methanone (11j): 1H NMR (300 MHz, CDCl3) δ 1.43 (t, 3H, J
) 7.5 Hz, CH3), 3.51 (q, 2H, J ) 7.5 Hz, SO2CH2), 6.45 (br, 1 H,
OCH3), 5.85 (br, 1 H, NH), 6.61 (ddd, 1H, 3JHF ) 11.5 Hz, 4JHF
)
5.3 Hz, 5JHF ) 2.2 Hz, aromatic), 8.15 (s, 1H, aromatic), 8.67 (br,
1 H, NH); LR-MS (M + H+) 344.
3
NH), 7.07 (t, 2H, Jortho ) JHF ) 8.0 Hz, aromatic), 7.54 (m, 1H,
(4-Amino-2-ethylsulfanylpyrimidin-5-yl)(3,4,5-trifluoro-2-
methoxyphenyl)methanone (10n) was prepared following a similar
procedure as described for 10i; the aryllithium reagent was
generated from deprotonation of 2,3,4-trifluoroanisole and reacted
with 8 to give 10n: 1H NMR (300 MHz, CDCl3) δ 1.39 (t, 3H, J
) 7.4 Hz, CH3), 3.14 (q, 2H, J ) 7.4 Hz, SCH2), 3.91 (s, 3H,
aromatic), 8.55 (s, 1 H, aromatic), 8.93 (br, 1 H, NH); HRMS for
C13H12F2N3O3S (M + H)+ calcd 328.0562, observed 328.0566.
(4-Amino-2-ethanesulfonylpyrimidin-5-yl)(2,3-difluoro-6-meth-
oxyphenyl)methanone (11k): light yellow solid; Anal. RP-HPLC
tR ) 1.82 min (method 1, 95% purity), tR ) 1.57 min (method 2,
1
98% purity); H NMR (300 MHz, CDCl3) δ 1.43 (t, 3H, J ) 7.4
3
4
OCH3), 5.86 (br, 1H, NH), 6.78 (ddd, 1H, JHF ) 8.0 Hz, JHF
)
Hz, CH3), 3.51 (q, 2H, J ) 7.4 Hz, SO2CH2), 3.77 (s, 3H, OCH3),
5
5.4 Hz, JHF ) 2.4 Hz, aromatic), 8.26 (d, 1H, J ) 3.7 Hz,
4
6.37 (br, 1H, NH), 6.72 (ddd, 1H, Jortho ) 9.4 Hz, JHF ) 3.1 Hz,
aromatic), 8.61 (br, 1 H, NH); LR-MS (M + H+) 344.
3
4
5JHF ) 1.9 Hz, aromatic), 7.30 (q, 1H, JHF ) JHF ) Jortho )9.4
Hz, aromatic), 8.48 (s, 1H, aromatic), 8.92 (br, 1 H, NH); HRMS
for C14H14F2N3O4S (M + H+) calcd 358.0668, observed 358.0671.
(4-Amino-2-ethanesulfonylpyrimidin-5-yl)(4,5-difluoro-2-meth-
oxyphenyl)methanone (11l): off-white solid; Anal. RP-HPLC tR
) 1.82 min (method 1, 95% purity), tR ) 1.58 min (method 2,
93% purity); 1H NMR (300 MHz, methanol-d4) δ 1.65(t, 3H, J )
7.4 Hz, CH3), 3.53 (q, 2H, J ) 7.4 Hz, SO2CH2), 3.77 (s, 3H,
(4-Amino-2-ethanesulfonylpyrimidin-5-yl)(2-methoxyphenyl)-
methanone (11c). To a cooled solution of 10c (2.66 g, 9.19 mmol)
in chloroform (120 mL) was added in three portions 3-chloroper-
oxybenzoic acid (∼77% purity, 5.20 g, ∼23 mmol, 2.5 equiv) and
the mixture stirred at 0 °C for 1 h. The resulting mixture was diluted
with CH2Cl2 (80 mL), washed with 10% aqueous sodium thiosulfate
(2 × 30 mL) and brine (2 × 20 mL), dried over Na2SO4, and
evaporated. The residue was purified on silica gel with hexane/
EtOAc (60/40) to give 11c as a white solid (2.30, 78% yield): 1H
NMR (300 MHz, CDCl3) δ 1.42 (t, 3H, J ) 7.5 Hz, CH3), 3.50 (q,
2H, J ) 7.4 Hz, SO2CH2), 3.78 (s, 3H, OCH3), 6.30 (br, 1 H, NH),
7.01 (d, 1H, J ) 8.3 Hz, aromatic), 7.10 (dt, 1H, Jortho ) 7.6 Hz,
Jmeta ) 0.8 Hz, aromatic), 7.34 (dd, 1H, Jortho ) 7.6 Hz, Jmeta ) 1.8
Hz, aromatic), 7.53 (m, 1H, aromatic), 8.50 (s, 1 H, aromatic), 8.93
(br, 1 H, NH); HRMS for C14H15N3O4SNa (M + Na)+ calcd
344.0675, observed 344.0679.
3
4
OCH3), 7.18 (dd, 1H, JHF ) 12.1 Hz, JHF ) 6.2 Hz, aromatic),
7.39 (dd, 1H, 4JHF ) 8.8 Hz, 3JHF ) 12.1 Hz, aromatic), 8.44 (s, 1
H, aromatic); HRMS for C14H14F2N3O4S (M + H+) calcd 358.0668,
observed 358.0672.
[4-Amino-2-[4-(4-methylpiperazin-1-yl)phenylamino]pyrimi-
din-5-yl](2-methoxyphenyl)methanone (22). A suspension of 11c
(20.0 mg, 0.062 mmol), 4-(4-methylpiperazino)aniline (15.5 mg,
0.081 mmol, 1.3 equiv), and p-TsOH hydrate (15 mg, 0.078 mmol,
i
1.2 equiv) in PrOH (2.5 mL) was placed in a sealed vessel and
(4-Amino-2-ethanesulfonylpyrimidin-5-yl)(3-fluorophenyl)-
methanone (11a): Anal. RP-HPLC tR ) 1.70 min (method 1, 90%
purity), tR ) 1.46 min (method 2, 92% purity); 1H NMR (300 MHz,
CDCl3) δ 1.45 (t, 3H, J ) 7.2 Hz, CH3), 3.53 (q, 2H, J ) 7.2 Hz,
SO2CH2), 6.34 (br, 1H, NH), 7.30-7.58 (m, 4 H, aromatic), 8.67
(br, 1H, NH), 8.72 (s, 1H, aromatic); HRMS for C13H12FN3O3S
(M + ) calcd 309.0583, observed 309.0582.
heated at 100-110 °C under microwave irradiation for 1 h. The
resulting mixture was diluted with CH2Cl2, washed with saturated
NaHCO3 solution (2×) and brine (2×), dried, and concentrated.
The crude product was purified on silica gel with CH2Cl2/MeOH
(95:5) to give 22 as a light yellow solid (23.6 mg, 91% yield):
Anal. RP-HPLC tR ) 1.35 min (method 1, 98% purity), tR ) 0.87
1
min (method 2, 99% purity); H NMR (300 MHz, CDCl3) δ 2.37

A Cyclin-Dependent Kinase Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6557
(s, 3H, NCH3), 2.60 (brd, 4H, 2×CH2), 3.17 (m, 4H, 2×CH2), 3.78
(s, 3H, OCH3), 5.59 (brd, 1H, NH), 6.87-7.11 (m, 5H, 4 aromatic
and 1 NH), 7.25 (m, 1H, aromatic), 7.37-7.49 (m, 3H, aromatic),
8.14 (s, 1H, aromatic), 8.84 (brd, 1H, NH); HRMS for C23H27N6O2
(M + H)+ calcd 419.2195, observed 419.2190.
[4-Amino-2-[4-(4-methylpiperazin-1-yl)phenylamino]pyrimi-
din-5-yl](3-fluorophenyl)methanone (18): 1H NMR (300 MHz,
CDCl3) δ 2.30 (s, 3H, CH3), 2.54 (brd, 4H, 2×CH2), 3.13 (t, 4H,
J ) 4.8 Hz, 2×CH2), 5.57 (brd, 1H, NH), 6.86 (d, 2H, J ) 8.8
Hz), 7.04 (brd, 1H, NH), 7.10-7.44 (m, 6H), 8.31(s, 1H), 8.63
(brs, 1H, NH); HRMS for C22H24FN6O (M + H)+ calcd 407.1990,
observed 407.1994.
[4-Amino-2-[4-(4-methylpiperazin-1-yl)phenylamino]pyrimi-
din-5-yl]-o-tolylmethanone (21): Anal. RP-HPLC tR ) 1.39 min
(method 1, >99% purity), tR ) 0.97 min (method 2, >99% purity);
1H NMR (300 MHz, CDCl3) δ 2.22 (s, 3H), 2.30 (s, 3H), 2.54 (m,
4H), 3.10-3.17 (m, 4H), 5.57 (brd, 1H, NH), 6.81-6.91 (m, 2H,
aromatic), 7.07 (brd, 1H, NH), 7.12-7.21 (m, 3H, aromatic), 7.25-
7.30 (m, 1H, aromatic), 7.36-7.43 (m, 2H, aromatic), 8.02 (s, 1H,
aromatic), 8.82 (brd, 1H, NH); HRMS for C23H27N6O (M + H+)
calcd 403.2241, observed 403.2247.
2NCH2), 3.71 (s, 3H, OCH3), 3.84-4.15 (br, 1H, NCH), 7.14 (m,
1H, aromatic), 7.30 (m, 1H, aromatic), 7.51 and 8.00 (2 br, 1H,
NH), 7.79 and 8.18 (br d, 1H, NH), 7.86 and 7.88 (2 s, 1H,
aromatic), 8.30 (br, 1H, NH), 8.46 (br, 1H, NH), 8.61 (br, 1H, NH);
HRMS for C17H21FN5O2 (M + H+) calcd 346.1674, observed
346.1673.
[4-Amino-2-(1-methylpiperidin-4-ylamino)pyrimidin-5-yl](2-
methoxyphenyl)methanone (24). To a stirred mixture of compound
23 as the TFA salt (prepared above, 65.0 mg, ∼0.090 mmol
calculated from compound 14c) and K2CO3 (75 mg, 0.54 mmol, 6
equiv) in anhydrous DMF (2 mL) was added iodomethane (23 mg,
0.16 mmol, 1.8 equiv). The reaction mixture was stirred at room
temperature for 2.5 h before it was diluted with EtOAc (40 mL),
washed with water and brine, dried, and concentrated. The residue
was purified on HPLC to give amine 24 as a white solid (14.1 mg,
46% yield): Anal. RP-HPLC tR ) 1.10 min (method 1, >99%
purity), tR ) 0.52 min (method 2, >99% purity); 1H NMR (CDCl3,
300 MHz) δ 1.50 (m, 2H, CH2), 1.95 (m, 2H, CH2), 2.07 (m, 2H,
NCH2), 2.22 (s, 3H, NCH3), 2.71 (m, 2H, NCH2), 3.72 (s, 3H,
OCH3), 3.79 (m, 1H, NCH), 4.97-5.65 (m, 2H, NH), 6.97 (d, 1H,
Jortho ) 8.4 Hz), 7.01 (t, 1H, Jortho ) 7.4 Hz), 7.24 (m, 1H), 7.42
(m, 1H), 7.85-8.18 (br, 1H), 8.51-8.95 (br, 1H, NH); HRMS for
C18H24N5O2 (M + H)+ calcd 342.1925, observed 342.1927.
4-[4-Amino-5-(2-methoxybenzoyl)pyrimidin-2-ylamino]pip-
eridine-1-carboxylic Acid Ethylamide (25). To a stirred solution
of compound 23 (65.0 mg, TFA salt prepared above, ∼0.090 mmol)
and triethylamine (0.10 mL, 72.6 mg, 0.71 mmol, 8 equiv) in
CH2Cl2 (3 mL) was added ethyl isocyanate (8.0 mg, 0.11 mmol,
1.2 equiv) at 0 °C. The reaction mixture was stirred for 1 h before
it was concentrated in vacuo. The residue was purified on silica
gel with CH2Cl2/MeOH (95/5) to give 25 as a white solid (31.7
mg, 89% yield): Anal. RP-HPLC tR ) 1.30 min (method 1, >99%
4-[4-Amino-5-(2-methoxybenzoyl)pyrimidin-2-ylamino]pip-
eridine-1-carboxylic Acid tert-Butyl Ester (14c). A suspension
of 11c (1.88 g, 5.87 mmol) and 4-amino-N-tert-(butoxycarbonyl)-
i
piperidine (13) (1.41 g, 7.04 mmol, 1.2 equiv) in PrOH (50 mL)
was placed in a sealed vessel and heated at 110 °C under microwave
conditions for 1.5 h. The resulting mixture was concentrated and
the crude product was purified on silica gel with CH2Cl2/MeOH
(95/5) to give 14c as a light yellow solid (2.23 g, 89% yield): Anal.
RP-HPLC tR ) 1.76 min (method 1, >99% purity), tR ) 1.49 min
(method 2, >99% purity); 1H NMR (300 MHz, CDCl3) δ 1.38 (m,
2H, CH2), 1.47 (s, 9H, t-Bu), 2.01 (m, 2H, CH2), 2.93 (m, 2H,
NCH2), 3.80 (s, 3H, OCH3), 4.03 (brm, 3H, NCH2 & NCH), 5.00-
5.70 (br, 2H, NH), 6.97 (d, 1H, Jortho ) 8.6 Hz), 7.02 (dt, 1H, Jortho
) 7.5 Hz, Jmeta ∼ 0.8 Hz), 7.24 (m, 1H), 7.42 (m, 1H), 7.94-8.22
(br, 1H,), 8.61-9.01 (br, 1H, NH); HRMS for C22H30N5O4 (M +
H)+ calcd 428.2293, observed 428.2296.
1
purity), tR ) 1.05 min (method 2, >99% purity); H NMR (300
MHz, CDCl3) δ 1.07 (br, 3H, CH3), 1.34 (m, 2H, CH2), 1.95 (m,
2H, CH2), 2.89 (m, 2H, NCH2), 3.20 (m, 2H, NCH2), 3.70 (s, 3H,
OCH3), 3.81 (m, 2H, NCH2), 3.96 (m, 1H, NCH), 4.31 (br, 1H,
NH), 4.87-5.68 (m, 2H, NH), 6.94-7.08 (m, 2H), 7.24 (m, 1H),
7.42 (m, 1H), 7.83-8.17 (br, 1H), 8.47-8.95 (br, 1H, NH); HRMS
for C20H27N6O3 (M + H)+ calcd 399.2139, observed 399.2143.
4-[4-Amino-5-(2-methoxybenzoyl)pyrimidin-2-ylamino]pip-
eridine-1-carboxylic Acid Ethyl Ester (26). A suspension of 11c
(43.1 mg, 0.134 mmol) and ethyl 4-amino-1-piperidinecarboxylate
4-[4-Amino-5-(5-fluoro-2-methoxybenzoyl)pyrimidin-2-ylami-
no]piperidine-1-carboxylic Acid tert-Butyl Ester (14g): Anal.
RP-HPLC tR ) 1.82 min (method 1, >99% purity), tR ) 1.65 min
(method 2, >99% purity); 1H NMR (300 MHz, CDCl3) δ ppm 1.41
(m, 2H, 2×CH of 2CH2), 1.47 (s, 9H, t-Bu), 2.01(m, 2H, 2×CH
of 2CH2), 2.92 (m, 2H, 2×NCH of 2NCH2), 3.77 (s, 3H, OCH3),
4.02 (br, 3H, NCH and 2×NCH of 2NCH2), 5.04-5.68 (br,
i
(30.0 mg, 0.174 mmol, 1.3 equiv) in PrOH (4 mL) was heated at
100-110 °C under microwave irradiation for 1 h. The resulting
mixture was concentrated and the residue was purified on silica
gel with CH2Cl2/MeOH (95/5) to give 26 as a light yellow solid
(47.1 mg, 88% yield): Anal. RP-HPLC tR ) 1.52 min (method 1,
>99% purity), tR ) 1.29 min (method 2, >99% purity); 1H NMR
(300 MHz, CDCl3) δ 1.27 (t, 3H, J ) 7.1 Hz, CH3), 1.31-1.49
(m, 2H, 2×CH of 2CH2), 2.02 (m, 2H, 2×CH of 2CH2), 2.88-
3.07 (m, 2H, 2×NCH of 2NCH2), 3.79 (s, 3H, OCH3), 3.95-4.19
(m, 3H, NCH and 2×NCH of 2NCH2), 4.13 (q, 2H, J ) 7.1 Hz,
OCH2), 5.02-5.67 (br, 2H, NH), 6.97 (d, 1H, Jortho ) 8.3 Hz,
4
2H, NH’s), 6.90 (dd, 1H, JHF ) 4.0 Hz, Jortho ) 9.0 Hz, aro-
3
matic), 6.98 (dd, 1H, Jmeta ) 3.1 Hz, JHF ) 7.9 Hz, aromatic),
3
7.11 (ddd, 1H, Jmeta ) 3.1 Hz, JHF ) 7.9 Hz, Jortho ) 9.0 Hz,
aromatic), 7.97-8.22 (br, 1H, aromatic), 8.56-8.93 (br, 1H, NH);
HRMS for C22H29FN5O4 (M + H+) calcd 446.2198, observed
446.2196.
[4-Amino-2-(piperidin-4-ylamino)pyrimidin-5-yl](2-meth-
oxyphenyl)methanone (23). TFA (15 mL) was added to a stirred
solution of compound 14c (1.96 g, 4.58 mmol) in CH2Cl2 (30 mL)
at 0 °C. After the reaction mixture was stirred for 1 h, it was
concentrated in vacuo to give 23 as a TFA salt (3.32 g, light yellow
solid). A portion of this crude product was purified on HPLC to
give 23 as a free base: Anal. RP-HPLC tR ) 1.11 min (method 1,
aromatic), 7.02 (t, 1H, J ) 7.4 Hz, aromatic), 7.23 (d, 1H, Jortho
)
7.4 Hz, aromatic), 7.41 (t, 1H, J ) 8.3 Hz, aromatic), 7.97-8.21
(br, 1H, aromatic), 8.60-9.00 (br, 1H, NH); HRMS for C20H26N5O4
(M + H)+ calcd 400.1980, observed 400.1984.
1
98% purity), tR ) 0.49 min (method 2, >99% purity); H NMR
1-[4-[4-Amino-5-(2-methoxybenzoyl)pyrimidin-2-ylamino]-
piperidin-1-yl]ethanone (27). Following a similar procedure as
described for compound 25, the reaction of 23 with acetyl chloride
gave 27 as a white solid: Anal. RP-HPLC tR ) 1.27 min (method
1, >99% purity), tR ) 0.99 min (method 2, >99% purity); 1H NMR
(300 MHz, CDCl3) δ 1.26-1.37 (m, 2H, CH2), 1.92-2.09 (m, 2H,
CH2), 2.03 (s, 3H, CH3), 2.74, 3.13, 3.72, 4.43 (4x m, 4H,
2×NCH2), 3.72 (s, 3H, OCH3), 4.01 (brm, 1H, NCH), 4.93-5.61
(CDCl3, 300 MHz) δ 1.23-1.41 (m, 2H, CH2), 1.88-2.05 (br, 2H,
CH2), 2.58-2.75 (br, 2H, CH2), 2.94-3.11 (br, 2H, CH2), 3.72 (s,
3H, OCH3), 3.80-3.99 (m, 1H, CH), 4.99-5.57 (br, 2H, NH), 6.89
(d, 1H, J ) 8.2 Hz), 6.94 (t, 1H, J ) 7.5 Hz), 7.16 (dd, 1H, Jortho
) 7.5 Hz, Jmeta ) 1.8 Hz), 7.34 (dt, 1H, Jortho) 8.2 Hz, Jmeta ) 1.8
Hz), 7.91-8.12 (br, 1H, aromatic), 8.53-8.88 (br, 1H, NH); HRMS
for C17H22N5O2 (M + H)+ calcd 328.1768, observed 328.1771.
[4-Amino-2-(piperidin-4-ylamino)pyrimidin-5-yl](5-fluoro-2-
methoxyphenyl)methanone (15g): Anal. RP-HPLC tR ) 1.10 min
(method 1, >99% purity), tR ) 0.59 min (method 2, >99% purity);
1H NMR (300 MHz, CDCl3) δ 1.42-1.80 (m, 2H, 2×CH of 2CH2),
2.00 (m, 1H, CH of 2CH2), 2.47 (m, 1H, CH of 2CH2), 2.82-3.09
(m, 2H, 2×NCH of 2NCH2), 3.16-3.40 (m, 2H, 2×NCH of
(br, 2H, NH), 6.97 (d, 1H, Jortho ) 8.3 Hz), 7.02 (dt, 1H, Jortho
)
7.4 Hz, Jmeta ) ∼0.8 Hz), 7.24 (m, 1H), 7.34 (m, 1H), 7.96 (br,
1H), 8.79 (br, 1H, NH); HRMS for C19H24N5O3 (M + H)+ calcd
370.1874, observed 370.1875.
1-[4-[4-Amino-5-(2-methoxybenzoyl)pyrimidin-2-ylamino]-
piperidin-1-yl]butan-1-one (28): Anal. RP-HPLC tR ) 1.45 min

6558 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22
Chu et al.
(method 1, 98% purity), tR ) 1.18 min (method 2, >99% purity);
1H NMR (300 MHz, CDCl3) δ 0.98 (t, 3H, J ) 7.4 Hz, CH3), 1.39
(m, 2H, CH2), 1.67 (m, 2H, CH2), 2.09 (m, 2H, CH2), 2.32 (m,
2H, CH2), 2.82, 3.18, 3.83, 4.54 (4m, 4H, 2×NCH2), 3.80 (s, 3H,
OCH3), 4.08 (br, 1H, NCH), 4.98-5.67 (br, 2H, NH), 6.97 (d, 1H,
J ) 8.1 Hz), 7.02 (dt, 1H, Jortho ) 7.5 Hz, Jmeta ) 0.9 Hz), 7.24
(m, 1H), 7.42 (m, 1H), 7.98-8.21 (br, 1H), 8.57-9.03 (br, 1H,
NH); HRMS for C21H28N5O3 (M + H)+ calcd 398.2187, observed
398.2189.
[4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimi-
din-5-yl](2-methoxyphenyl)methanone (29). Following a similar
procedure as described for compound 25, the reaction of 23 with
methanesulfonyl chloride gave 29 as a white solid: Anal. RP-HPLC
tR ) 1.35 min (method 1, >99% purity), tR ) 1.09 min (method 2,
>99% purity); 1H NMR (300 MHz, CDCl3) δ 1.55 (m, 2H, CH2),
2.06 (m, 2H, CH2), 2.73 (s, 3H, SCH3), 2.82 (m, 2H, NCH2), 3.68
(m, 2H, NCH2), 3.71 (s, 3H, OCH3), 3.93 (m, 1H, NCH), 4.89-
5.64 (m, 2H, NH), 6.94-7.07 (m, 2H), 7.24 (dd, 1H, Jortho ) 7.5
Hz, Jmeta ) 1.7 Hz), 7.42 (m, 1H), 7.98 (br, 1H), 8.45-8.99 (br,
1H, NH); HRMS for C18H24N5O4S (M + H)+ calcd 406.1544,
observed 406.1546.
lows) gave 36 as a white solid: Anal. RP-HPLC tR ) 1.63 min
(method 1, >99% purity), tR ) 1.38 min (method 2, >99% purity);
1H NMR (300 MHz, CDCl3) δ 1.69 (m, 2H, CH2), 2.14 (m, 2H,
CH2), 2.83 (s, 3H, SCH3), 3.06 (m, 1H, NCH), 3.58-3.86 (m, 3H,
3×NCH), 3.78 (s, 3H, OCH3), 4.06 (m, 1H, NCH), 5.02-6.79 (br,
2H, NH), 6.93 (m, 1H), 7.23 (m, 1H), 7.40 (m, 1H), 7.90-8.20
(br, 1H), 8.59-9.09 (br, 1H, NH); HRMS for C18H23ClN5O4S (M
+ H+) calcd 440.1154, observed 440.1160.
Preparation of 1-Methanesulfonylpiperidin-4-ylamine. Meth-
anesulfonyl chloride (1.0 g, 8.8 mmol) was added to a stirred
solution of commercially available piperidin-4-ylcarbamic acid tert-
butyl ester (1.0 g, 5.0 mmol) and diisopropylethylamine (4 mL) in
THF (40 mL) at 5 °C. The reaction mixture was allowed to warm
to room temperature over 1 h before it was poured into water and
extracted with CH2Cl2 (2 × 50 mL). The combined organic extracts
were washed with 5% aqueous NaHCO3, dried over Na2SO4, and
concentrated in vacuo to give a crude solid which was purified by
trituration with ether/hexane to give (1-methanesulfonyl-piperidin-
4-yl)carbamic acid tert-butyl ester as a white solid [MS (M + H+)
278]. This solid was suspended in CH2Cl2 (15 mL), treated with
TFA (5.3 mL), and stirred for 2 h at room temperature. The resulting
mixture was concentrated and the residue was triturated with ether.
The solid was filtered off, washed with ether, and dried in a vacuum
to give a quantitative yield of 1-methanesulfonyl-piperidin-4-
ylamine trifluoroacetate. A portion of the material was dissolved
in EtOAc, washed with saturated aqueous NaHCO3 solution and
brine, dried, and concentrated to give the free base 1-methane-
sulfonylpiperidin-4-ylamine as a white waxy solid: 1H NMR (300
MHz, DMSO-d6) δ 1.24 (m, 2H, CH2), 1.73 (m, 2H, CH2), 1.88
(br, 1H, NH), 2.58-2.76 (m, 3H, NCH and NCH2), 2.81 (s, 3H,
SCH3), 3.25-3.3.35 (br, 1H, NH), 3.40 (m, 2H, NCH2).
General Procedure for Preparation of Sulfoxides 12. To a
stirred solution of sulfide 10 (1.8 mmol) in CHCl3 (16 mL) at -15
°C was added portionwise mCPBA (70% purity, 2.1 mmol, 1.2
equiv). The mixture was stirred at the same temperature for 1 h
before it was diluted with CH2Cl2 (30 mL); washed successively
with 10% aqueous sodium thiosulfate (2 × 5 mL), 10% aqueous
Na2CO3 (2 × 5 mL), brine (2 × 5 mL); dried; and concentrated to
give a quantitative yield of 9:1 ratio mixture (by NMR) of sulfoxide
12 and sulfone 11. This mixture was used for next reaction without
further purification while pure sulfoxide 12 in some examples was
obtained by chromatographic purification of a small portion of the
mixture.
[4-Amino-2-(1-ethanesulfonylpiperidin-4-ylamino)pyrimidin-
5-yl](2-methoxyphenyl)methanone (30): white solid; Anal. RP-
HPLC tR ) 1.40 min (method 1, >99% purity), tR ) 1.20 min
(method 2, >99% purity); 1H NMR (300 MHz, CDCl3) δ 1.38 (brt,
3H, CH3), 1.60 (m, 2H, CH2), 2.12 (m, 2H, CH2), 2.97 (q, 2H, J )
7.2 Hz, SCH2), 2.93-3.06 (m, 2H, NCH2), 3.78 (m, 2H, NCH2),
3.80 (s, 3H, OCH3), 4.02 (m, 1H, NCH), 4.95-5.71 (m, 2H, NH),
6.97 (d, 1H, Jortho ) 8.4 Hz), 7.02 (t, 1H, Jortho)7.4 Hz), 7.24 (dd,
1H, Jortho ) 7.4 Hz, Jmeta ) 1.8 Hz), 7.42 (dt, 1H, Jortho ) 8.4 Hz,
Jmeta ) 1.8 Hz), 8.06 (brs, 1H), 8.61-9.05 (br, 1H, NH); HRMS
for C19H26N5O4S (M + H)+ calcd 420.1700, observed 420.1704.
4-[4-Amino-5-(2-methylbenzoyl)pyrimidin-2-ylamino]piperi-
dine-1-carboxylic Acid Ethyl Ester (32): white solid; Anal. RP-
HPLC tR ) 1.63 min (method 1, >99% purity), tR ) 1.38 min
1
(method 2, >99% purity); H NMR (300 MHz, CDCl3) δ 1.27 (t,
3H, J ) 7.4 Hz, CH3), 1.40 (m, 2H, 2×CH of 2CH2), 2.03 (m, 2H,
2×CH of 2CH2), 2.29 (s, 3H, CH3), 2.98 (m, 2H, 2×NCH of
2NCH2), 3.98-4.20 (m, 3H, NCH and 2×NCH of 2NCH2), 4.14
(q, 2H, J ) 7.4 Hz, OCH2), 5.04-5.73 (4br, 2H, NH and NH of
NH2), 7.17-7.38 (m, 4H, aromatic), 7.94-8.15 (2br, 1H, aromatic),
8.67-9.03 (2br, 1H, NH of NH2); HRMS for C20H26N5O3 (M +
H)+ calcd 384.2030, observed 384.2035.
1-[4-[4-Amino-5-(2-ethoxybenzoyl)pyrimidin-2-ylamino]pip-
eridin-1-yl]ethanone (33). Following a similar procedure as
described for 14c, the reaction of 11e with 1-(4-aminopiperidin-
1-yl)ethanone under microwave irradiation gave 33 as a white
solid: Anal. RP-HPLC tR ) 1.15 min (method 1, >99% purity),
4-[4-Amino-5-(2-fluorobenzoyl)pyrimidin-2-ylamino]piperi-
dine-1-carboxylic Acid Ethyl Ester (31). Following a similar
procedure as described for compound 26, a mixture of sulfoxide
12d (major) and sulfone 11d (minor) was reacted with ethyl
4-amino-1-piperidinecarboxylate to give 31 as off-white soild: Anal.
RP-HPLC tR ) 1.58 min (method 1, >99% purity), tR ) 1.40 min
1
tR ) 1.10 min (method 2, >99% purity); H NMR (300 MHz,
1
CDCl3) δ 1.28 (t, 3H, J ) 7.0 Hz, CH3), 1.46 (m, 2H, CH2), 2.06
(m, 2H, CH2), 2.11 (s, 3H, CH3), 2.91, 3.23, 3.80, 4.46 (4m, 4H,
2×NCH2), 4.05 (q, 2H, J ) 7.0 Hz, OCH2), 4.11 (m, 1H, NCH),
4.90-6.46 (m, 2H, NH), 6.95 (d, 1H, J ) 8.4 Hz), 7.01 (t, 1H, J
) 7.6 Hz), 7.16 (m, 1H), 7.33 (m, 1H), 7.90-8.29 (br, 1H), 8.57-
9.17 (br, 1H, NH); HRMS for C20H26N5O3 (M + H+) calcd
384.2030, observed 384.2035.
(method 2, >99% purity); H NMR (300 MHz, CDCl3) δ1.19 (t,
3H, J ) 7.2 Hz, CH3), 1.34 (m, 2H, CH2), 1.95 (m, 2H, CH2),
2.91 (m, 2H, NCH2), 3.88-4.11 (m, 3H, NCH & NCH2), 4.06 (q,
2H, J ) 7.2 Hz, OCH2), 4.94-5.76 (br, 2H, NH), 7.07 (t, 1H, J )
8.9 Hz), 7.17 (m, 1H), 7.32 (m, 1H), 7.39 (m, 1H), 7.92-8.25 (br,
1H), 8.43-8.97 (br, 1H, NH); HRMS for C19H23N5O3F (M + H+)
calcd 388.1780, observed 388.1783.
[4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimi-
din-5-yl](5-fluoro-2-methoxyphenyl)methanone (35): Anal. RP-
HPLC tR ) 1.46 min (method 1, >99% purity), tR ) 1.24 min
(method 2, >99% purity); 1H NMR (300 MHz, CDCl3) δ 1.63 (m,
2H, CH2), 2.15 (m, 2H, CH2), 2.81 (s, 3H, SCH3), 2.92 (m, 2H,
NCH2), 3.74 (m, 2H, NCH2), 3.77 (s, 3H, OCH3), 4.01 (m, 1H,
NCH), 5.01-5.71 (m, 2H, NH), 6.91 (dd, 1H, Jortho ) 9.1 Hz, 4JHF
[4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimi-
din-5-yl](4-fluoro-2-methoxyphenyl)methanone (34). Similar to
the preparation of compound 31, the reaction of a mixture of
sulfoxide 12f (major) and sulfone 11f (minor) with 1-methane-
sulfonylpiperidin-4-ylamine TFA salt gave 34: white solid; Anal.
RP-HPLC tR ) 1.39 min (method 1, >99% purity), tR ) 1.20
1
min (method 2, >99% purity); H NMR (300 MHz, DMSO-d6)
3
) 4.0 Hz), 6.97 (dd, 1H, JHF ) 7.9 Hz, Jmeta ) 3.2 Hz), 7.11
δ 1.53 (m, 2H, CH2), 1.88 (m, 2H, CH2), 2.66-2.90 (m, 5H,
NCH2 & SCH3), 3.51 (m, 2H, NCH2), 3.73 (s, 3H, OCH3), 3.89
3
(ddd, 1H, Jortho ) 9.1 Hz, JHF ) 7.9 Hz, Jmeta ) 3.2 Hz), 7.93-
(m, 1H, NCH), 6.84 (dt, 1H, Jortho ) 8.6 Hz,3JHF ) 8.6 Hz, Jmeta
)
8.21 (br, 1H), 8.55-9.01 (br, 1H, NH); HRMS for C18H23FN5O4S
(M + H)+ calcd 424.1450, observed 424.1455.
3
2.3 Hz), 7.03 (dd, 1H, JHF ) 11.5 Hz, Jmeta ) 1.8 Hz), 7.24 (m,
1H), 7.40 and 7.68 (2brs, 1H, NH), 7.54 and 7.74 (2brd, 1H, NH),
7.79 and 7.86 (2s, 1H, aromatic), 8.29 and 8.49 (2brs, 1H, NH);
HRMS for C18H23FN5O4S (M + H)+ calcd 424.1450, observed
424.1453.
[4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimi-
din-5-yl](5-chloro-2-methoxyphenyl)methanone (36). Following
a similar procedure as described for 14c, reaction of 11h and
1-methanesulfonylpiperidin-4-ylamine TFA salt (prepared as fol-

A Cyclin-Dependent Kinase Inhibitor
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 22 6559
[4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimi-
din-5-yl](2-fluoro-6-methoxyphenyl)methanone (37): Anal. RP-
HPLC tR ) 1.46 min (method 1, >99% purity), tR ) 1.30 min
(method 2, >99% purity); 1H NMR (300 MHz, DMSO-d6) δ 1.54
(m, 2H, CH2), 1.89 (m, 2H, CH2), 2.78 (m, 2H, NCH2), 2.83 and
2.87 (2s, 3H, SCH3), 3.52 (m, 2H, NCH2), 3.74 (s, 3H, OCH3),
3.90 (m, 1H, NCH), 6.90 (t, 1H, J ) 8.5 Hz, aromatic), 6.97 (d,
1H, J ) 8.0 Hz, aromatic), 7.46 (q, 1H, J ) 8.0 Hz, aromatic),
7.52 and 7.75-7.90 (2br, 1H, NH), 7.64 and 7.75-7.90 (2br, 1H,
NH), 7.79 and 7.86 (2s, 1H, aromatic), 8.25 and 8.44 (2br, 1H,
NH); HRMS for C18H23FN5O4S (M + H)+ calcd 424.1450,
observed 424.1451.
Aruna Railkar for in vivo formulation, Mr. Ted Lambros for
preparative HPLC purification, Mrs. Margret Jonca for analytical
HPLC data, and Mr. Richard Szypula for HRMS analysis of
all key intermediates and target compounds.
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[4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimi-
din-5-yl](2,6-difluorophenyl)methanone (38): Anal. RP-HPLC
tR ) 1.58 min (method 1, >99% purity), tR ) 1.40 min (method 2,
1
>99% purity); H NMR (300 MHz, DMSO-d6) δ 1.57 (m, 2H,
CH2), 1.89 (m, 2H, CH2), 2.79 (m, 2H, NCH2), 2.84 and 2.87 (2s,
3H, SO2CH3), 3.52 (m, 2H, NCH2), 3.92 (m, 1H, NCH), 7.23 (t,
3
2H, Jortho ) 8.4 Hz, JHF ) 8.4 Hz, aromatic), 7.58 (m, 1H,
aromatic), 7.65 and 7.94 (2br, 1H, NH), 7.78 and 8.00 (2br, 1H,
NH), 7.72 and 7.99 (2br, 1H, aromatic), 8.82 and 8.43 (2br, 1H,
NH); HRMS for C17H20F2N5O3S (M + H)+ calcd 412.1250,
observed 412.1248.
[4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimi-
din-5-yl](2,3-difluoro-6-methoxyphenyl)methanone (39): white
solid; mp 218-219 °C; Anal. RP-HPLC tR ) 1.58 min (method 1,
>99% purity), tR ) 1.44 min (method 2, >99% purity); 1H
NMR (300 MHz, CDCl3) δ 1.68 (m, 2H, CH2), 2.14 (m, 2H, CH2),
2.82 (s, 3H, SCH3), 3.02 (m, 1H, NCH), 3.64-3.82 (m, 3H,
3×NCH), 3.76 (s, 3H, OCH3), 4.05 (m, 1H, NCH), 5.06-6.37 (br,
2H, NH), 6.68 (br, 1H, aromatic), 7.21 (m, 1H, aromatic), 7.99
and 8.10 (2s, 1H, aromatic), 8.60 and 8.88 (2br, 1H, NH); HRMS
for C18H22F2N5O4S (M + H)+ calcd 442.1355, observed 442.1358.
[4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimi-
din-5-yl](4,5-difluoro-2-methoxyphenyl)methanone (40): white
solid; Anal. RP-HPLC tR ) 1.53 min (method 1, >99% purity), tR
) 1.30 min (method 2, >99% purity); 1H NMR (300 MHz, DMSO-
d6) δ 1.54 (m, 2H, CH2), 1.88 (m, 2H, CH2), 2.79 (m, 2H, NCH2),
2.84 and 2.86 (2s, 3H, SCH3), 3.51 (m, 2H, NCH2), 3.71 (s, 3H,
OCH3), 3.90 (m, 1H, NCH), 7.29 (dd, 1H, 3JHF ) 13.0 Hz, 4JHF
)
3
4
6.7 Hz, aromatic), 7.39 (t, 1H, JHF ) 9.5 Hz, JHF ) 9.5 Hz,
aromatic), 7.43 and 7.72 (2br, 1H, NH), 7.57 and 7.77 (2br, 1H,
NH), 7.82 and7.89 (2s, 1H, aromatic), 8.24 and 8.44 (2br, 1H, NH);
HRMS for C18H22F2N5O4S (M + H)+ calcd 442.1355, observed
442.1358.
[4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimi-
din-5-yl](2,3,4-trifluoro-6-methoxyphenyl)methanone (41): white
solid; Anal. RP-HPLC tR ) 1.70 min (method 1, >99% purity), tR
) 1.51 min (method 2, >99% purity); 1H NMR (300 MHz,
DMSO-d6) δ 1.55 (m, 2H, CH2), 1.89 (m, 2H, CH2), 2.78 (m, 2H,
NCH2), 2.84 and 2.89 (2s, 3H, SCH3), 3.52 (m, 2H, NCH2), 3.73
(s, 3H, OCH3), 3.91 (br, 1H, NCH), 7.20 (m, 1H, aromatic), 7.56
and 7.84 (2br, 1H, NH), 7.71 and 7.90 (2br, 1H, NH), 7.95 and
8.02 (2s, 1H, aromatic), 8.20 and 8.39 (2br, 1H, NH); HRMS for
C18H21F3N5O4S (M + H)+ calcd 460.1261, observed 460.1267.
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1
tR ) 1.57 min (method 2, >99% purity); H NMR (300 MHz,
DMSO-d6) δ 1.55 (m, 2H, CH2), 1.90 (m, 2H, CH2), 2.79 (m, 2H,
NCH2), 2.84 and 2.87 (2s, 3H, SCH3), 3.53 (m, 2H, NCH2), 3.86
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and 7.87 (2br, 1H, NH), 7.72 and 7.93 (2br, 1H, NH), 8.05 and
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