A novel pyrazolo[1,5- a ]pyrimidine is a potent inhibitor of cyclin-dependent protein kinases 1, 2, and 9, which demonstrates antitumor effects in human tumor xenografts following oral administration

Article abstract of DOI:10.1021/jm100732t

Cyclin-dependent protein kinases (CDKs) are central to the appropriate regulation of cell proliferation, apoptosis, and gene expression. Abnormalities in CDK activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Here, we report the identification of a pyrazolo[1,5-a]pyrimidine derived compound, 4k (BS-194), as a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7, and CDK9 (IC₅₀ = 3, 30, 30, 250, and 90 nmol/L, respectively). Cell-based studies showed inhibition of the phosphorylation of CDK substrates, Rb and the RNA polymerase II C-terminal domain, down-regulation of cyclins A, E, and D1, and cell cycle block in the S and G₂/M phases. Consistent with these findings, 4k demonstrated potent antiproliferative activity in 60 cancer cell lines tested (mean GI₅₀ = 280 nmol/L). Pharmacokinetic studies showed that 4k is orally bioavailable, with an elimination half-life of 178 min following oral dosing in mice. When administered at a concentration of 25 mg/kg orally, 4k inhibited human tumor xenografts and suppressed CDK substrate phosphorylation. These findings identify 4k as a novel, potent CDK selective inhibitor with potential for oral delivery in cancer patients.

Full text of DOI:10.1021/jm100732t

8508 J. Med. Chem. 2010, 53, 8508–8522
DOI: 10.1021/jm100732t
A Novel Pyrazolo[1,5-a]pyrimidine Is a Potent Inhibitor of Cyclin-Dependent Protein
Kinases 1, 2, and 9, Which Demonstrates Antitumor Effects in Human Tumor
Xenografts Following Oral Administration
Dean A. Heathcote,† Hetal Patel,† Sebastian H. B. Kroll,‡ Pascale Hazel,§ Manikandan Periyasamy,†
Mary Alikian,† Seshu K. Kanneganti,† Ashutosh S. Jogalekar, Bodo Scheiper,‡ Marion Barbazanges,‡ Andreas Blum,‡
Jan Brackow,‡ Alekasandra Siwicka,‡ Robert D. M. Pace,‡ Matthew J. Fuchter,‡ James P. Snyder, Dennis C. Liotta,
Paul. S. Freemont,§ Eric O. Aboagye,† R. Charles Coombes,† Anthony G. M. Barrett,‡ and Simak Ali*,†
†Dept of Oncology, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, England,
‡Department of Chemistry, Imperial College London, London SW7 2AZ, England, §Division of Molecular Biosciences,
Imperial College London, London SW7 2AZ, England, and Department of Chemistry, Emory University, Atlanta GA30322, United States
Received June 17, 2010
Cyclin-dependent protein kinases (CDKs) are central to the appropriate regulation of cell proliferation,
apoptosis, and gene expression. Abnormalities in CDK activity and regulation are common features of
cancer, making CDK family members attractive targets for the development of anticancer drugs. Here,
we report the identification of a pyrazolo[1,5-a]pyrimidine derived compound, 4k (BS-194), as a
selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7, and CDK9 (IC50
=
3, 30, 30, 250, and 90 nmol/L, respectively). Cell-based studies showed inhibition of the phosphorylation
of CDK substrates, Rb and the RNA polymerase II C-terminal domain, down-regulation of cyclins A,
E, and D1, and cell cycle block in the S and G2/M phases. Consistent with these findings, 4k
demonstrated potent antiproliferative activity in 60 cancer cell lines tested (mean GI50 = 280 nmol/L).
Pharmacokinetic studies showed that 4k is orally bioavailable, with an elimination half-life of 178 min
following oral dosing in mice. When administered at a concentration of 25 mg/kg orally, 4k inhibited
human tumor xenografts and suppressed CDK substrate phosphorylation. These findings identify 4k as
a novel, potent CDK selective inhibitor with potential for oral delivery in cancer patients.
Introduction
mutational or epigenetic alterations, for example, p16 loss has
been observed in skin, lung, breast, and colorectal cancer (for
review see ref 5). Synthetic inhibitors of CDK activity there-
fore present as a logical approach in the development of new
cancer therapies. However, the development of inhibitors
selective for a particular CDK has been questioned by the
finding that inhibition of some CDKs may lead to compensa-
tion by other CDKs. For example, cells from mice that have
been ablated for CDK2 are able to cycle, and CDK2-/- mice
are viable,6,7 while evidence for compensation of CDK2
by CDK1 has been forthcoming.8,9 Similarly, CDK4-/- and
CDK6-/- mice are viable, althoughthe double null mice show
late embryonic lethality.10-12 Hence, a highly selective inhib-
itor of CDK2, 4, or 6 may be subject to clinical resistance due
to the potential for compensation. However, acquisition of
CDK dependence seems to feature in some tumor types. For
example, despite the fact that CDK4 is not required for
normal mammary gland development, it does appear to be
important for induction of mammary tumors in mouse
models.13-15 The CDK4/CDK6 partner cyclin D1 is similarly
required for HER2- and Ras-induced mammary tumors, but
not for Myc and Wnt1 induced mammary tumors,16 indicat-
ing that, atleast in sometumor types, requirementfor selective
CDKs is acquired during the process of tumorigenesis in a
process that is dependent on the pathway leading to tumor
formation.3,17
Cyclin-dependent kinases (CDKa) are serine/threonine ki-
nases that regulate progression through the cell cycle. CDK4
and CDK6 mediateprogression through the G1 growth phase,
CDK2 is required for entry into and transition through the
DNA replication or synthesis (S) phase, and CDK1 controls
progression through G2 and mitosis. CDK7, together with its
partners cyclinH andMAT1, form the CDKactivating kinase
(CAK), which activates the cell cycle CDKs by phosphorylat-
ing them at a threonine residue in the activation segment
(T-loop).1-3
Deregulation of cell cycle progression is a universal char-
acteristic of cancer, and the majority of human cancers have
abnormalities insomecomponentofCDK activity, frequently
through elevated and/or inappropriate CDK activation. Ex-
amples include cyclin overexpression, for example, cyclin D1
overexpression is commonly observed in breast cancer,4 and
loss of expression of CDK inhibitory proteins (CKI) through
*To whom correspondence should be addressed. Phone: þ44 20 8383
3789. Fax: þ44 20 8383 5830. E-mail: simak.ali@imperial.ac.uk.
a Abbreviations: CDK, cyclin dependent kinase; Rb, retinoblastoma;
PolII, RNA polymerase II; CKI, CDK inhibitory protein; HUVEC,
human normal vascular endothelial cells; PBMC, peripheral blood
mononuclear cells; SRB, sulforhodamine B; FACS, fluorescence-activated
cell sorter, ADME-Tox, absorption, distribution, metabolism, excretion,
and toxicity; PO, per oral, IV, intravenous; IP, intraperitoneal.
r
pubs.acs.org/jmc
Published on Web 11/16/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8509
Figure 1. Structures of some CDK inhibitors currently in clinical evaluation. See text for references.
Scheme 1. Synthesis of Pyrazolopyrimidinesa
a (a) BnNH2, EtOH, reflux, 16 h, 95%; (b) Boc2O, THF, cat. DMAP, 25 °C, 20 h, 96%; (c) amine, Pd2(dba)3, rac-BINAP, NaOtBu, 100 °C, 16 h,
40-99%; (d) MeOH, HCl, 25 °C, 3 h, 67-99%.
We reasoned that inhibitors of CDK7 could be potentially
important in cancer therapy, as they would inhibit activation
of many of the cyclin-dependent kinases involved in cell cycle
progression. Moreover, as part of the TFIIH general tran-
scription factor complex, CDK7/CycH/MAT1 mediates the
phosphorylation of the C-terminal domain of the largest
subunit of RNA polymerase II, an event required for promo-
ter clearance and transcription initiation.18-20 In carrying out
a screen for CDK7 inhibitors, we identified a compound,
4k, which inhibits CDK7 but is also a potent inhibitor of
CDK1, CDK2, CDK5, and CDK9. In further evaluating
the activity of 4k, we show that 4k potently inhibits growth
of the great majority of cancer cell lines examined and
inhibits phosphorylation of CDK substrates at submicro-
molar concentrations. A number of inhibitors that target
cell cycle CDKs have been identified and some have been
evaluated clinically, including roscovitine (Seliciclib), SCH-
727965, and AT7519 (Figure 1).21-23 4k is a pyrazolopyr-
imidine as is SCH-727965. Unlike many other CDK inhi-
bitors, however, we show that 4k is orally bioavailable and
inhibits tumor growth in vivo. As such, 4k is an important new
CDK inhibitor with clinical potential.
the C-7 chloride using benzylamine followed by palladium-
catalyzed displacement of the C-5 chloride under Buch-
wald-Hartwig reaction conditions, as described previously
(Scheme 1).24 Protection of the benzylic amine as a carbamate
was essential for successful C-5 amination. Following depro-
tection, the analogues were assayed for CDK activity by
measurement of free ATP remaining in the kinase reaction,
as determined using a luciferase assay.
The high potency offered by the pyrazolopyrimidine core
was highlighted by the effective inhibition of several CDKs
by 4a, which bears the roscovitine side chain (Supporting
Information Table 1). Deletion of the free hydroxyl group in
4b had a large detrimental effect on the potency. Interest-
ingly, chain elongation and replacement of the hydroxyl by a
terminal amine 4c selectively increases potency toward
CDK7. Compound 4c, which we have previously reported
(BS-181), inhibits CDK7 activity up to 83% while demon-
strating only weak inhibition of the other CDKs. Compound
4c inhibited CDK7 activity, with an IC50 of 21 nm/L, with
greater than 40-fold selectivity over other CDKs and a wider
range of 70 kinases and additionally showed antitumor
activity in vivo.24
Further long chain C-5 analogues were synthesized, and
nitrile 4d showed a similar inhibition of CDK7 activity (IC50
20 nm/L) while also showing increased CDK2 inhibition.
Similarly, amidine 4e showed only a slight decrease in CDK7
inhibition (IC50 40 nm/L), however, this also proved to be a
highly potent CDK2 inhibitor.
Having identified 4c as a potent and selective CDK7 inhib-
itor, it was decided to further investigate the SAR around the
C-5 side chain against CDK potency and selectivity. Several
analogues containing a terminal basic residue in combination
with a free hydroxyl group were investigated. These were
readily synthesized from alkene 6 (see Scheme 2) via cross
metathesis, or oxidation/Wittig reactions followed by reduction
of the resultant alkene prior to Buchwald-Hartwig coupling.
However, the parent alkene 4f showed decreased inhibition
compared to the analogue with the roscovitine side chain 4a,
Results
Identification of 4k. Of the CDK inhibitors currently
undergoing clinical trials (Figure 1), a structural class that
has yielded several CDK-selective ATP antagonists is the
2,6,9-trisubstituted purines, exemplified by roscovitine,
which shows good biological and pharmacological proper-
ties. However, our computational analysis has shown that
the pyrazolo[1,5-a]pyrimidine core has a less favorable aque-
ous solvation energy than the corresponding purine, sug-
gesting that this compound class would be more readily
transferred into the hydrophobic kinase active site.24,25 In
this study, a range of pyrazolopyrimidines were synthesized
and tested for CDK activity.
The compounds were readily accessible from dichloropyra-
zolo[1,5-a]pyrimidine 1 by sequential selective substitution of

8510 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Scheme 2. First Generation Synthesis of 4k and 4la
Heathcote et al.
a (a) t-BuMe2SiCl, Et3N, cat. DMAP, CH2Cl2, 25 °C, 16 h, 99%; (b) 2, Pd2(dba)3, rac-BINAP, NaOtBu, 100 °C, 16 h, 43%; (c) cat. OsO4, NMO,
MeCN, H2O, 25 °C, 45 min, 3k 42%, 3l 33%; (d) MeOH, HCl, 25 °C, 3 h, 4k 79%, 4l 67%.
Table 1. Inhibition of Cyclin-Dependent Protein Kinase Activity by 4ka
roscovitine
IC50 (μM) (SD)
4k
IC50 (μM) (SD)
kinase
CDK1
CDK2
CDK4
CDK5
CDK6
CDK7
CDK9
2.1 (0.5)
0.1 (0.05)
13.5 (0.2)
0.16 (0.2)
23.5 (1.3)
0.54 (0.09)
0.95 (0.17)
0.033 (0.01)
0.003 (0.001)
20 (1.3)
0.03 (0.006)
35.5 (1.3)
0.25 (0.04)
0.09 (0.01)
a The mean IC50 values ( μM) for roscovitine and 4k, obtained from
three experiments, are shown, together with the standard deviation (SD)
from the mean. The line graphs from which IC50 values were obtained
are shown in Supporting Information Figure 1.
and none of the analogues with the functionalized side chains
4g, 4h, or 4i showed any significant inhibition of the CDKs.
The importance of free hydroxyl groups however, was
reinforced by the high CDK7 inhibition and good selectivity
demonstrated by the 2-(2-hydroxyethoxy)-ethylamino ana-
logue 4j. Accordingly, further hydroxylated compounds
were synthesized by dihydroxylation of the vinyl residue
present in 3f, which after diastereoisomer separation and
deprotection yielded the two amino triols 4k and 4l (BS-195).
Both diastereoisomers proved to be highly active against a
range of CDKs.
4k was chosen for further study because it inhibited CDK2
with an IC50 = 3 nmol/L (Table 1; Supporting Information
Figure 1), an IC50 more than 30-fold lower than that showed
by roscovitine (IC50 = 100 nmol/L).26 4k also inhibited
CDK1 (IC50 = 33 nmol/L), CDK5 (IC50 = 30 nmol/L),
and CDK9 (IC50 = 90 nmol/L) and was a less potent
inhibitor of CDK7 (IC50 = 250 nmol/L), whereas CDK4
and CDK6 were not inhibited by 4k. Compound 4l gave a
similar profile of CDK inhibition, however, 4k had the
advantage of being a crystalline solid. Compound 4k was
therefore selected for development due to both potency and
ease of purification. In addition, the relative configuration of
the molecule and by analogy its absolute stereochemistry was
readily assigned by an X-ray crystal structure determination
(Figure 2).
Figure 2. Crystal structure of 4k.
enantiomerically and diastereomerically pure 4k was inves-
tigated. We decided to introduce a fully functionalized C-5
side chain through the existing Buchwald-Hartwig coupling
reaction, therefore a synthesis of the requisite fully protected
amino-triol was required.
(-)-Diethyl tartrate (8) was converted to the correspond-
ing cyclic sulfate using reaction conditions developed by
Sharpless, by conversion to the cyclic sulfite with thionyl
chloride, followed by ruthenium catalyzed oxidation to the
cyclic sulfate.27 Subsequent ring SN2 opening using sodium
azide gave the requisite azido-alcohol 9. However, the oxida-
tion reaction proved to be capricious, and as a consequence
of difficulties encountered with handling of the cyclic sulfate,
a more reliable method was investigated. Direct SN2 ring-
opening of the cyclic sulfite with sodium azide with careful
control of temperature was highly effective, giving azide 9 in
70% yield.28 The diester was readily reduced to triol 10 by
lithium borohydride generated in situ in EtOH.28,29 It was
necessary to protect the free hydroxyl groups to prevent
competitive O-arylation in the Buchwald-Hartwig cou-
pling, and it was hoped to achieve this in a single operation.
However, attempts to protect all the hydroxyl groups as an
orthoester by condensation with trimethyl orthoacetate or
trimethyl orthobenzoate with acid catalysis failed. Attempts
to prepare the triple methoxymethyl ether were complicated
by formation of cyclic acetals and were only low yielding. A
two-step procedure was therefore employed with the vicinal
diol first being protected as an acetonide and the primary
alcohol subsequently protected by methoxymethylation.
The initial synthesis of 4k using a nonselective late stage
alkene dihydroxylation reaction usefully gave access to both
diastereoisomers, however, the diastereoisomer separation
was laborious and unsuitable once 4k had been selected for
development (Scheme 2). Accordingly, an alterative route to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8511
Scheme 3. Second Generation Synthesis of 4ka
a (a) SOCl2, NEt3, CH2Cl2, 0 °C, 1 h; (b) NaN3, DMF, 12 h, 70% (2 steps); (c) NaBH4,LiCl, EtOH, 0 °C, 12 h; (d) (MeO)2CMe2, TsOH, Me2CO, 50 °C,
3 h; (e) MOMCl,iPr2NEt, CH2Cl2, 0 °C, 12 h, 70% (3 steps); (f) H2, Pd/C, MeOH, 1 h, 97%; (g) 2,Pd2(dba)3, rac-BINAP, NaOtBu, 100 °C, 16 h, 95%; (h)
MeOH, HCl, 25 °C, 3 h, 90%.
Table 2. Inhibition of Protein Kinases by 4ka
Catalytic hydrogenation over palladium on charcoal gave
the required fully protected amine 12, which underwent high
yielding Buchwald-Hartwig coupling to produce the pro-
tected 4k precursor 13. Global deprotection under acidic
conditions gave 4k as a single stereoisomer (Scheme 3). The
1H and 13C NMR spectra of 4k are shown in Supporting
Information Figures 2 and 3.
4k (10 μM)
% activity
remaining SD
4k (10 μM)
% activity
enzyme remaining SD
enzyme
MKK1
ERK1
65
42
15 AMPK
15 MARK3
88
82
108
88
19
89
15
75
88
102
87
80
103
94
101
62
86
92
73
55
65
85
95
85
77
86
32
116
92
120
89
94
87
67
92
91
70
72
1
4
ERK2
JNK1
JNK2
26
93
4
3
BRSK2
MELK
14
1
4k Is a Potent Inhibitor of CDK1, CDK2, CDK7, and CDK9.
Determination of the CDK selectivity of 4k, performed by
assaying inhibition of the activities of 76 proteins kinases
representing members of different kinase classes (Table 2),
showed significant inhibition of CAMKKβ, CK1, DYRK1A,
ERK1, ERK2, and IRR at 10000 nmol/L but a less than 50%
inhibition of these kinases when 4k was used at a concentration
of 1000 nmol/L (data not shown), suggestive of IC50 values
>1000 nmol/L. In confirmation of this, the IC50 values for
CAMKKβ, CK1, DYRK1A, ERK1, ERK2, and IRR were
determined as 2450, 1040, 2100, 3730, 3110, and 1800 nmol/L,
respectively. The only exception was ERK8, whose activity was
inhibited by 74% with 10 μmol/L 4k and the IC50 was deter-
mined as 330 nmol/L (data not shown), 100-fold higher than the
IC50 for CDK2, about 10-fold higher IC50 than that obtained
for CDK1 or CDK5.
94
98
12 CK1
12 CK2
1
p38a MAPK
P38b MAPK
p38g MAPK
p38s MAPK
ERK8
7
94
3
DYRK1A
14 DYRK2
3
112
86
1
0
4
DYRK3
NEK2a
1
26
75
3
RSK1
RSK2
15 NEK6
IKKb
11 PIM1
15
3
90
84
1
PDK1
PKBa
6
103
105
92
6
9
1
0
2
PIM2
PIM3
SRPK1
MST2
EFK2
1
PKBb
SGK1
5
14
8
S6K1
PKA
77
93
1
ROCK 2
PRK2
PKCa
102
106
96
14 HIPK2
6
3
4
PAK4
PAK5
7
3
˚
A 1.8 A crystal structure of 4k bound to the inactive form
PKC ζ
PKD1
64
97
14 PAK6
15 Src
6
of CDK2 was obtained by soaking the ligand into preformed
CDK2 crystals. After molecular replacement with the pub-
lished CDK2 structure, clear and unambiguous density for
the ligand was found within the CDK2 ATP binding site
(Figure 3A,D). Compound 4k binds in a similar fashion to
roscovitine, with the core heterocyclic ring occupying ap-
proximately the same position as the ATP purine ring.30 The
phenyl ring substituent occupies a hydrophobic pocket out-
side the ATP binding site, while N1 and N6 each form a
hydrogen bond to the L83 main chain, as seen in the structure
of roscovitine bound to CDK2 (Figure 3B,C). In addition,
the three side chain hydroxyl groups of 4k form several
water-mediated interactions with CDK2 backbone and side
chain atoms, involving residues E12, T14. D86, Q131, and
D145 (Figure 3B). This is in contrast to roscovitine, where
the number of stabilizing water-mediating interactions be-
tween the single ligand hydroxyl group and CDK2 is limited
(Figure 3C). The structure suggests that the increased po-
tency of 4k in CDK2 inhibition assays compared to roscov-
itine could be due to tighter binding of the former to the
inactive state of CDK2. The increased affinity of 4k com-
pared to roscovitine stems from its ability to form a network
13
2
MSK1
MNK1
96
98
1
3
Lck
CSK
1
MNK2
MAPKAP-K2
PRAK
136
111
97
15 FGF-R1
14 IRR
2
11
10
14
11
7
8
1
5
EPH A2
MST4
SYK
CAMKKb
CAMK1
SmMLCK
PHK
23
93
101
62
13 YES1
8
8
5
IKKe
TBK1
IGF1-R
7
CHK1
CHK2
98
61
8
15
8
GSK3b
CDK2-cyclin A
PLK1
63
15 VEG-FR
3
68
0
4
BTK
IR-HIS
8
2
13
PLK1 (okadaic acid)
97
14 EPH-B3
a Inhibition of 76 protein kinases by 4k at a concentration of 10 μmol/L.
The assay was carried out in duplicate. Percentage of kinase activity
remaining is shown, together with the standard deviations (SD).
of water mediated interactions within the CDK2 ATP bind-
ing pocket. Due to a major movement of the glycine loop
(residues 12-20) upon cyclin binding to CDK2, it is expected
that the 4k hydroxyl substituents will form different interactions

8512 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Heathcote et al.
Figure 3. Crystal structure of 4k bound to CDK2. (A) Shown is the 4k difference electron density map after refinement, contored at 2.9σ. (B)
4k binding site in CDK2 is shown with hydrogen bonding interactions depicted as dashed lines. Water-mediated interactions are also shown.
Carbon atoms are colored in cyan, nitrogen in blue, and oxygen in red. Water molecules are represented as red crosses. (C) Roscovitine binding
site in CDK2 (PDB code 2A4L), as (B). (D) Summary of CDK2-4k data collection and refinement statistics.
in the active complex, and we are currently progressing studies
to obtain a structure for 4k bound to the active CDK2-cyclin A
complex. Notwithstanding, the crystallography data confirm 4k
structure and binding to CDK2.
4k Promotes Cell Cycle Arrest and Inhibits Cancer Cell
Growth. A panel of cell lines representing a range of tumor
types, including breast, lung, prostate, and colorectal cancer,
were treated with increasing concentrations of 4k for 72 h.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8513
Table 3. In Vitro Growth Inhibitory Activity of 4ka
B, and cyclin D1 were dramatically reduced in the case of 4k
at a concentration of 1 μmol/L. Reduction in the levels of
these cyclins was also observed with higher concentrations
(30 and 40 μmol/L) of roscovitine. CDK2 levels were not
influenced by 4k treatment, although its levels were reduced
at high concentrations of roscovitine. Phosphorylation of
Thr-170 of CDK2 was, however, reduced following 24 h
treatment with 4k. Together, these results indicate a rapid
inhibition of CDK activity following 4k addition, with
slower acting effects on cyclin levels.
In Vivo Pharmacokinetic Studies and Tumor Growth In-
hibition. To determine whether 4k inhibits tumor growth in
vivo, nude mice bearing MCF-7 tumors were injected in-
traperitoneally twice daily with 5 or 10 mg/kg, to give total
daily doses of 10 mg/kg or 20 mg/kg, over a period of 14 days.
Tumor growth was inhibited in a dose-dependent manner,
with 30% and 40% reduction in tumor growth, compared
with the control group, for 10 and 20 mg/kg/day doses of 4k,
respectively (Figure 5A). At these doses, there was no
apparent toxicity, as judged by lack of significant adverse
effects on animal weights (Figure 5B).
roscovitine
4k
cell type
breast
cell line
MCF-7
GI50 (μM) (SD) GI50 (μM) (SD)
7.8 (0.2)
22.6 (0.1)
16.3 (0.1)
24.9 (0.2)
15.5 (0.1)
15 (0.2)
0.3 (0.1)
0.3 (0.1)
0.1 (0.1)
0.12 (0.1)
0.1 (0.1)
0.2 (0.1)
0.25 (0.1)
0.5 (0.1)
1.0 (0.1)
0.3 (0.2)
6.3 (0.1)
MDA-MB-231
MCF-10A
COLO-205
HCT-116
A549
colorectal
lung
osteosarcoma
prostate
liver
SaOS2
PC3
13.9 (0.1)
25.2 (0.1)
25.9 (0.2)
35.7 (0.2)
3.7 (0.1)
HepG2
Sk-Ov-3
ovarian
human normal
umbilical vein
endothelial
cells (HUVEC)
a The mean IC50 values ( μM) were obtained using the sulforhodamine
B (SRB) assay. Shown are the mean values derived from at least three
replicates, together with the standard deviations (SD) from the means.
Determination of proliferation using the sulforhodamine B
(SRB) assay showed that growth was inhibited for all cell
lines tested, with 50% growth inhibition (GI50) values of
100-500 nmol/L (Table 3). By contrast, GI50 values for
roscovitine ranged between 7.8-35.7 μmol/L for the same
cell lines. Interestingly, human normal vascular endothelial
cells (HUVEC) (GI50 = 6.3 μmol/L) were somewhat less
sensitive to 4k than the cancer lines.
On the basis of our findings, 4k was submitted to the
Developmental Therapeutics Program at the NCI (http://
dtp.nci.nih.gov) for testing using the NCI60 cancer cell line
screen. In this screen, treatment with 4k showed potent
inhibition of all 60 cancer lines (Figure 4A, Supporting
Compound 4k was found to have favorable absorption,
distribution, metabolism, elimination, and toxicity (ADME-
Tox) properties. The drug had an aqueous solubility (PBS,
pH 7.4) of 54.0 μmol/L (range, 47.16-60.89 μmol/L) and parti-
tioncoefficient (Log D, n-octanol: PBS pH 7.4) of 3.0. In vitro
cellular absorption (A-B permeability, TC7, pH 6.5/7.4) of
4k was 9.7 ꢀ 10-6 cm s-1 (range, 9.32-10.06 ꢀ 10-6 cm s-1
;
3
3
recovery = 91(%) at a test concentration of 10 μmol/L. In the
same assay, the absorption of propranolol used as control
was 54.2 ꢀ 10-6 cm s-1 (range, 53.64-54.81 ꢀ 10-6 cm s-1
;
3
3
recovery = 85(%). Whereas plasma protein binding did not
differ significantly between mouse and human, microsomal
metabolism was found to be more favorable for human
compared to mouse liver microsomes (Table 4). Injection
of 4k at 10 mg/kg into mice via the intraperitoneal (IP),
intravenous (IV), and oral (PO) routes of administration of
resulted in terminal elimination half-lives of 147, 210, and
178 min, respectively, with the measured plasma concentra-
tion at 15 min of 10474 (SEM = 5634), 25351 (SEM = 780),
and 9347 (SEM = 551) ng/mL, respectively (Table 5; Figure 6).
One of two metabolites detected after intravenous adminis-
tration of 4k was identified as an oxidative (hydroxylated)
metabolite (data not shown).
Information Figure 4), with GI50 < 1 μmol/L (mean GI50
=
2.81 ꢀ 10-7 mol/L). For the great majority of the lines, the
50% lethal concentration (LC50) was >100 μmol/L (mean
LC50 > 10 ꢀ 10-4 mol/L) (Supporting Information Table 2).
For comparison, in the same cell panel, flavopiridol is a
potent inhibitor of cancer cell growth (mean GI50 = 7.47 ꢀ
10-8 mol/L) but lethal concentration for flavopiridol is
reached at submicromolar concentrations (LC50 = 9.04 ꢀ
10-7 mol/L) (http://dtp.nci.nih.gov).
Flow cytometric analysis of HCT116 cells treated with 1 and
10 μmol/L 4k showed a significant reduction in cells in G1, with
a concomitant increase in cells in S and G2/M phases of the cell
cycle (Figure 4B; Supporting Information Figure 5), as would
be expected from the potent inhibition of CDK1 and CDK2,
but not of CDK4 and CDK6, by 4k. Similar effects were
observed in MCF-7 cells (data not shown). There was also an
increase in the sub-G1 population, suggestive of a small, but
significant amount of apoptosis at 1 and 10 μmol/L 4k, which
was confirmed by annexin V staining (data not shown).
4k Inhibits Phosphorylation of CDK Substrates and Pro-
motes Loss of Cyclin Expression. Immunoblotting of whole
cell lysates prepared from HCT116 colon cancer cells treated
with 4k for 4 and 24 h showed inhibition of the phosphor-
ylation of the CDK2 substrate RB at Ser-780, Ser-795, Ser-
801, Ser-807/Ser-811, and Thr-821 (Figure 4C,D), the CDK1
substrate protein phosphatase type 1R (PP1a) at Thr-320,
and the CDK9 substrate Ser-2 in the C-terminal domain
(CTD) of RNA polymerase II (PolII). Altered levels of
CDKs or cyclins did not accompany inhibition of RB
and PolII phosphorylation at 4 h following 4k addition
(Figure 4E). By contrast, at 24 h, levels of cyclin A, cyclin
Pharmacokinetic studies indicated good oral bioavailabil-
ity (88%; n = 3 animals for 8 time points analyzed for each of
PO and IV administration) for 4k. To determine whether
treatment with 4k elicits responses in vivo, 4k was adminis-
tered by oral gavage to nu/nu-BALB/c athymic nude mice.
Immunostaining and flow cytometric analysis of peripheral
blood mononuclear cells (PBMC) isolated 6 h following
administration showed that 4k at concentrations of 25 mg/
mL induced a 60-70% reduction in RB phosphorylation at
Thr-821 (Figure 7). There were also 25-50% fall in PolII
Ser-2 and Ser-5 phosphorylation with 25 mg/mL 4k, with
reductions in phosphorylation of PolII being maximal at
100 mg/mL 4k, although at concentrations of 50 mg/mL or
higher, PolII levels also fell. No difference in RB Thr-821 or
PolII phosphorylation was evident 24 h following adminis-
tration with 50 mg/mL 4k, although RB Thr-821 phosphor-
ylation was reduced by 50% ( p<0.01), 24 h after adminis-
tration of 75 mg/mL 4k (data not shown).
The above results indicated that oral administration of 4k
results in rapid down-regulation of RB and PolII phosphorylation

8514 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Heathcote et al.
Figure 4. Compound 4k treatment of HCT116 cells leads to cancer cell growth inhibition, cell cycle arrest, and inhibition of CDK substrate
phosphorylation. (A) Growth inhibition profiles for the NCI 60 cancer cell panel. For details of the growth profiles of the individual cell lines
see Supporting Information. (B) Effect of 4k on cell cycle kinetics. 4k at the concentrations shown was added to HCT116 cells for 24 h prior to
fixation, staining with propidium iodide (PI), and flow cytometric analysis. The percentage of cells in the sub-G1 (apoptosis), G1, S-phase, and
G2/M, as determined from three independent experiments, are shown. Error bars represent the standard errors of the mean (SEM). P-Values
were determined using Student’s t test, in which comparison between the DMSO control, and 4k treatment at each concentration was carried
out. Asterisks (*) denote p < 0.05. (C-E) Molecular pharmacology of 4k on HCT116 cells. Cell lysates prepared from HCT116 cells treated
with 4k or roscovitine for 4 or 24 h were immunoblotted for various proteins or phosphorylated forms, as labeled.
but recovery within 24 h. To determine if oral administration of
4k inhibits the growth of HCT116 tumor xenografts, animals
were treated once daily with 25 mg/kg 4k by oral gavage.
Tumor growth was inhibited in a dose-dependent manner, with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8515
Figure 5. Compound 4k inhibits growth in human breast tumor xenografts. (A) Randomized MCF-7 tumor bearing mice were injected
intraperitoneally twice daily with vehicle (control, n = 11) or with 5 or 10 mg/kg of 4k, giving a total daily dose of 10 mg/kg/day (n = 8) or 20
mg/kg/day (n = 9), respectively, over a period of 14 days. The change in tumor volume was determined for each animal, as tumor volume
relative to the tumor volume of each animal at day 1. The line graphs show the mean tumor volumes for the animals in each treatment group. (B)
Shown are the weights of the animals used over the time course of the study. Error bars represent the standard errors of the mean.
Table 4. In Vitro Plasma Protein Binding and Microsomal Metabolism
of 4ka
reduced PolII phosphorylation at Ser-2 and Ser-5 in vitro and in
vivo. Phosphorylation of Rb regulates its interaction with E2F
transcription factors, interaction with hypophosphorylated
Rb repressing the expression of E2F-regulated genes. Many
other interactions have been described, in particular, with
proteins containing LXCXE motifs, including histone deacety-
lases HDAC1 and HDAC2 and chromatin remodelling pro-
teins, with Rb phosphorylation also being critical for these
interactions.32,33 Rb phosphorylation was inhibited at all sites
studied, with the observed inhibition being considerably greater
for 4k than that observed for equivalent concentrations of
roscovitine, in keeping with the much better growth inhibition
seen for 4k compared with roscovitine. Phosphorylation of all
Rb sites was abolished with 10 μmol/L of 4k, with substantial
inhibition being obtained at concentrations as low as 0.1 μmol/L
for Thr821, although complete inhibition of Thr821 phosphor-
ylation was not achieved even at high concentrations of 4k,
which may be a reflection of that fact that in addition to being
phosphorylated by CDK2, Thr821 can be phosphorylated by
CDK6.34,35 Phosphorylation of PP1a at Thr-320 was inhibited
in a dose-dependent manner, likely reflecting the inhibition of
CDK1 activity by 4k. These analyses were reflected in the
accumulation of cells in the S and G2/M phases of the cell
cycle, with a concomitant reduction in cells in the G1 phase.
As expected from the potent inhibition of CDK1, CDK2,
and CDK9 by 4k and the inhibition of Rb and PolII phos-
phorylation, the growth of diverse cancer cell lines was
inhibited, with GI50 values ranging between 0.1 and 1.0 μmol/L.
This was confirmed by growth studies performed with the
NCI60 cancer cell line panel, GI50 values ranging between
0.1 and 0.5 μmol/L, with the exception of the multidrug
resistant NCI/Adr-Res ovarian cancer cell line, which was
inhibited somewhat less well (GI50 = 2.0 μmol/L). The
growth of human normal vascular endothelial cells was
also inhibited less well than the growth of the cancer lines
(GI50 = 6.3 μmol/L), suggestive of some selectivity for
cancer cells over normal cells.
plasma protein binding
test concentration, μM
% protein bound
(human)
% protein bound
(mouse)
4k, 10
quinidine, 10
94.3 (94.0 -94.5)
71.1 (65.3-76.9)
88.3 (86.0-90.6)
71.1 (70.0-72.2)
in vitro metabolism (metabolic stability) (liver microsomes)
test concentration,
μM
parent remaining
(human) (%)
parent remaining
(human) (%)
4k, 1.0
propranolol, 1.0
83 (80.6-84.8)
74 (73.5-75.2)
29 (28.8-29.1)
37 (34.7-39.6)
a Data are mean values (range). Incubation: 0 and 60 min at 37 °C.
Substrate/co-factor: test compound (1 μM), NADP (1 mM), G6P (5 mM),
G6PDHase (1 U/mL) with 0.6% methanol, 0.6% acetonitrile (n = 2).
50% reduction in tumor growth in the 25 mg/kg/day group
(Figure 8A), with no significant loss in animal weights in the
vehicle or 4k treatment groups. (Figure 8B).
Immunohistochemical staining of resected tumors showed no
difference in Rb level in the vehicle or 4k treatment groups
(Figure 8C,D). By contrast. levels of Rb phosphorylation at
Ser807/811 and Thr821 were decreased in the 4k treated tumors
when compared with the vehicle treated group ( p < 0.005).
Discussion
Recent studies show that inhibition of individual CDKs
required for cell cycle progression may be ineffective cancer
treatments either because they are not essential or due to
potential compensation by other CDKs.3 For example, in the
case of CDK2, a popular target for drug discovery due to its role
in progression through the S phase, inhibition of this kinase was
insufficient to prevent cancer cell proliferation,31 whereas com-
bined depletion of CDK2 and CDK1 in cancer cells resulted in
greater cell cycle arrest than seen with depletion of CDK2 or
CDK1 alone.9 In this context, the potent CDK1 and CDK2
inhibitory activity of 4k provides an attractive therapeutic
approach. Further, inhibition of CDK9 activity and consequent
reduction in PolII activity has been shown to augment the
growth inhibitory effects of CDK1 and CDK2 inhibition in
cancer cell lines;9 here, 4k inhibited CDK9, as well as CDK7 and
The physicochemical and predictive ADME-Tox proper-
ties of 4k, including aqueous solubility, cell permeability, and
plasma protein binding were consistent with utility for in vivo
studies. Pharmacokinetic studies demonstrated good oral
bioavailability, which was confirmed by single oral dose
administration, followed by collection of PBMC and FACS

8516 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Table 5. Pharmacokinetic Parameters for 4ka
Heathcote et al.
route of admin-
istration
bioavail-ability
(%)
Cmax
(min)
AUClast
(min ng/mL)
AUCINF
(min mg/mL)
terminal
points
dose (mg/kg)
T
max (min)
T1/2 (min)
3
1583520
1922912
3
158188
1926751
PI
PO
10
10
73
88
30
15
15604
9347
147
178
3
3
route of
admin-istration
dose
(mg/kg)
Cl (mL/min/
kg)
Vz
(mL/kg)
AUClast
(min ng/mL)
AUCINF
(min mg/mL)
terminal
points
T1/2 (min)
Vss (mL/kg)
754
3
2174209
3
2182968
IV
10
210
5
1391
3
a The studies were carried out in mice and the pharmacokinetic parameters were derived from noncompartmental model using WinNonlin 5.2.
T1/2=elimination half-life. Cl=total body clearance. Vz=volume of distribution. Vss=an estimate of the volume of distribution at steady state.
AUClast = area under the concentration-time curve from the time of dosing to the time of last observation that is greater than the limit of quantitation.
AUCINF = area under the concentration-time curve from the time of dosing, extrapolated to infinity. Tmax = time of maximum observed
concentration. Cmax=concentration corresponding to Tmax. The bioavailability was calculated based on AUCINF. Terminal points = the number of
observations used to calculate the terminal slope.
identification and preclinical evaluation of a potent orally
active CDK inhibitor, which demonstrates selectivity for
CDK1, CDK2, CDK5, and CDK9, with moderate inhibition
in vitro of CDK7 and which is a poor inhibitor of CDK4 and
CDK6. Compound 4k selectively exhibited nanomolar enzy-
matic potency, potently inhibited all cell lines tested, and
demonstrated inhibition of phosphorylation of CDK sub-
strates. A pyrazolopyrimidine compound (Dinaciclib; SCH
727965) which is similar to 4k in selectively inhibiting CDK1,
CDK2, CDK5, and CDK9 with IC50 values of 1-4 nmol/L,
which shows tumor regression in mouse models25 and is well
tolerated in patients following IV administration in a phase I
setting,23 has recently been described. Our previous descrip-
tion of a CDK7 specific pyrazolopyrimidine-based com-
ound,24 together with the identification of 4k, show that
pyrazolopyrimidine-based compounds have considerable
potential for development as potent CDK inhibitors. Finally,
the oral bioavailability of 4k highlights it as an important new
Figure 6. Pharmacokinetic determination of 4k levels. The mean
plasma concentration of 4k over time was determined for four mice
at each time point following 4k administration. Error bars represent
the standard errors of the mean.
CDK inhibitor, with potential for clinical development.
analysis of RB and PolII phosphorylation after 6 h. Inhibition
of Rb Thr-821 phosphorylation was maximal with a dose of
Materials and Methods
25 mg/kg. Although PolII Ser-2 phosphorylation was de-
creased in a dose-dependent manner from 10 to 100 mg/kg,
PolII levels were also seen to fall at doses of 50 and 100 mg/kg.
Importantly, even at doses of 75 and 100 mg/kg, no significant
reduction in PolII or Rb phosphorylation was noted 24 h
following 4k administration, which may be a reflection of the
fact that the plasma concentration of 4k are likely to be below
the concentrations that demonstrate inhibition of Rb and
PolII phosphorylation in cell lines beyond 6 h post adminis-
tration. Nevertheless, single daily ip and oral administration
of 4k showed tumor growth inhibition in xenograft models at
doses of 10 mg/kg ip in MCF-7 xenografts and 25 mg/kg po in
HCT116xenografts. Further, using Rbphosphorylation asan
immunohistochemical marker of CDK activity, we have been
able to observe that oral 4k administration results in reduced
Rb phosphorylation in the tumors. This is indicative of a
relationship between pharmacodynamic factors and thera-
peutic outcome for 4k.
General Synthetic Methods. All manipulations of air or moi-
sture sensitive materials were carried out in oven- or flame-dried
glassware under an inert atmosphere of nitrogen or argon. Syringes,
which were used to transfer reagents and solvents, were purged with
nitrogen prior to use. Reaction solvents were distilled from CaH2
(CH2Cl2, PhMe, Et3N), Na/Ph2CO (THF, Et2O), or obtained as
dry or anhydrous from Aldrich Chemical Co. (DMF, MeCN) or
BDH (EtOH). Other solvents and all reagents were obtained from
commercial suppliers and were used as obtained if purity was
>98%. All flash column chromatography was carried out on silica
gel 60 particle size 0.040-0.063 mm, unless otherwise stated. Thin
layer chromatography (TLC) was performed on precoated alumi-
num backed or glass backed plates and visualized under ultraviolet
light (254 nm) or following spraying with potassium permanganate,
vanillin, or phosphomolybdic acid (PMA) stains as deemed appro-
priate. LC-MS analysis was performed using a Waters LCT
Premier XE equipped with a Waters Atlantis C18-reverse phase
column of length 30 mm, inner diameter 2.1 mm and particle size
3 μm using a mobile phase of water (0.1% formic acid):acetonitrile,
monitoring at 254 nm. Compound purity was determined by
combustion analysis (elemental analysis) or LC-MS analysis and
was confirmed to be >95% for all compounds.
Conclusions
Several selectiveand broad-specificityCDK inhibitors have
now entered clinical trials. Phase I studies show that they can
generally be safely administered. Of particular note is
PD0332991, a selective CDK4/CDK6 inhibitor, as well as
agents with broader selectivity, a few of which have been
progressed to phase II studies either as single agents or in
combination with chemotherapeutic agents such as cisplatin,
gemcitabine, and capecitabine.23 Here we have described the
Dichloride 1 was prepared according to methods described by
ref 51.
Benzyl-(5-chloro-3-iso-propylpyrazolo[1,5-a]pyrimidin-7-yl)-
amine. 5,7-Dichloro-3-iso-propyl-pyrazolo[1,5-a]pyrimidine (1)
(15.5 g, 67.0 mmol) and benzylamine (14.7 mL, 134 mmol) in
EtOH (500 mL) were heated at reflux for 16 h. The reaction
mixture was cooled to ambient temperature, concentrated in
vacuo, and the residue recrystallized from EtOAc. The mother

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8517
Figure 7. Oral administration of 4k inhibits cyclin-dependent kinase substrate phosphorylation in mouse peripheral blood mononuclear cells.
Mouse PBMCs were collected 6 h following dosing of female nude mice by oral gavage at the dose levels shown. PBMCs were incubated with
the appropriate primary antibodies, followed by fluorescently labeled secondary antibodies and flow cytometric analysis (n = 4, except for the
100 mg/kg dose where n = 3) to determine the status of (A) Rb, (B) Rb phosphorylated at threonine-821 (Rb Thr821), (C) RNA polymerase II
Ser-2 phosphorylation (PolII Ser-2), and (D) total PolII. Error bars represent the standard errors of the mean. P-Values were determined using
Student’s t test; # represents p < 0.05.
liquor was chromatographed on silica (EtOAc:hexanes 1:8) to
yield the title pyrazolo[1,5-a]pyrimidine as a white solid. The
combined yield of the crystallized and chromatographed material
was 19.3 g, 96%; mp 78-79 °C (EtOAc); TLC Rf 0.16 (EtOAc:
hexanes 1:16). IR (neat) 3380, 3239, 1616, 1585 cm-1. 1H NMR
(CDCl3, 400 MHz) δ 7.84 (s, 1H), 7.40-7.32 (m, 5H), 6.88 (m,
1H), 5.91 (s, 1H), 4.55 (m, 2H), 3.28 (sept, J = 6.9 Hz, 1H), 1.33
(d, J = 6.9 Hz, 6H). 13C NMR (CDCl3, 100 MHz) δ 150.2, 146.9,
144.2, 141.6, 135.8, 129.1, 128.3, 127.3, 117.0, 84.7, 46.2, 23.5,
23.4. MS m/z (ESI) 301 (M þ H)þ. HRMS (ESI) calcd for
C16H18ClN4þ, 301.1215; found, 301.1227. Anal. Calcd for
C16H17ClN4: C, 63.89; H, 5.70; N, 18.63. Found: C, 63.95; H,
5.78; N, 18.59.
in PhMe (0.30 M) and stirred for 15 min. The amine (1.1 equiv) was
added and the mixture heated to 95 °C for 16 h and cooled and
dilutedwithEtOAc.Theorganicphasewaswashedwithwater(3ꢀ),
brine, dried (MgSO4), and concentrated in vacuo. Chromatography
on silica (EtOAc:hexanes) gave the pure coupled products.
General Procedure for the Final Acid Deprotection. AcCl was
dissolved in MeOH (5.0 M) with stirring at 0 °C. After 15 min,
the solution was warmed to room temperature and stirred for a
further 15 min. The ambient was added and the mixture stirred
at ambient temperature for 3 h and concentrated in vacuo. The
residue was dissolved in CH2Cl2, washed with saturated aque-
ous sodium bicarbonate, dried (Na2SO4), and concentrated in
vacuo. Crystallization (MeCN or MeOH) or chromatography
(CH2Cl2: MeOH) gave the pure analogues.
tert-Butyl-benzyl-(5-chloro-3-iso-propylpyrazolo[1,5-a]pyrimidin-
7-yl)-carbamate Monohydrate (2). Benzyl-(5-chloro-3-iso-propyl-
pyrazolo[1,5-a]pyrimidin-7-yl)-amine (17.0 g, 56.7 mmol), Boc2O
(16.1 g, 73.7 mmol), and DMAP (100 mg) in THF (400 mL) were
stirred for 20 h at ambient temperature. EtOAc (300 mL) was
added, and the organic layer was washed with water (2 ꢀ 400 mL)
and saturated aqueous NaHCO3 (400 mL) and dried (Na2SO4).
Concentration in vacuo, chromatography (EtOAc:hexanes 1:8),
and recrystallization from EtOAc and hexanes (1: 8) gave carba-
mate 2 (22.6 g, 100%) as a pale-yellow solid; mp 100-103 °C
Compound 5 was prepared according to Campbell et al.36
(R)-1-(tert-Butyldimethylsilyloxy)-3-buten-2-amine (6). Et3N
(5.00 mL, 35.6 mmol), DMAP (20.0 mg), and t-BuMe2SiCl (2.70
g, 17.8 mmol) were added to amino-alcohol 5 (2.00 g, 16.2
mmol) in CH2Cl2 (80 mL). The mixture was stirred overnight at
ambient temperature. Water (80 mL) was added, and the
mixture was vigorously stirred for 10 min. The organic phase
was separated, washed with water (60 mL) and brine (60 mL),
and dried (Na2SO4). Concentration in vacuo gave amine 6 (2.83
(EtOAc); TLC Rf 0.40 (EtOAc:hexanes 1:8). IR (neat) 1612 cm-1
.
g, 43%) as a yellow oil; TLC Rf 0.55 (EtOAc:hexanes 1:1); [R]25
D
1H NMR (CDCl3, 400 MHz) δ 8.02 (s, 1H), 7.34-7.21 (m, 5H),
6.48 (s, 1H), 5.03 (s, 2H), 3.31 (sept, J = 6.9 Hz, 1H), 1.52 (s, 2H),
1.39 (s, 9H), 1.37 (d, J = 6.9 Hz, 6H). 13C NMR (CDCl3, 100
MHz) δ 152.8, 148.0, 145.1, 144.3, 142.6, 136.8, 128.7, 127.8, 127.8,
118.3, 106.3, 83.0, 51.4, 28.0, 23.6, 23.4. MS m/z(ESI) 401(Mþ H)þ.
HRMS (ESI) calcd for C21H26ClN4O2þ, 401.1739; found,
401.1735. Anal. Calcd for C21H27ClN4O3: C, 60.21; H, 6.50; N,
13.37. Found: C, 60.28; H, 6.58; N, 13.43.
1
(c 1.09, CH2Cl2) þ 22.8. IR (neat) 1471, 1254, 1095 cm-1. H
NMR (CDCl3, 300 MHz) δ 5.80 (m, 1H), 5.14 (m, 2H), 3.60 (m,
1H), 3.42 (m, 2H), 1.61 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H). 13
C
NMR (CDCl3, 75 MHz) δ 139.2, 115.1, 67.8, 55.8, 25.9, 18.3,
-5.4. MS m/z (CI) 202 (M þ H)þ. HRMS (CI) calcd for
C10H24NOSiþ, 202.1622; found, 202.1622.
(R)-tert-Butyl Benzyl(5-((1-((tert-butyldimethylsilyl)oxy)but-
3-en-2-yl)amino)-3-iso-propylpyrazolo[1,5-a]pyrimidin-7-yl)car-
bamate (3f ). Synthesized according to the general procedure for
Buchwald-Hartwig coupling, 2 (100 mg, 0.25 mmol), Pd2dba3
(12.0 mg, 0.013 mmol), BINAP (24.0 mg, 0.039 mmol), sodium
tert-butoxide (36.0 mg, 0.38 mmol), amine 6 (58.0 mg, 0.27
mmol), and PhMe (0.80 mL) yielded carbamate 3f (72.0 mg,
General Procedure for Buchwald-Hartwig Coupling Reaction.
tert-Butyl-benzyl-(5-chloro-3-iso-propylpyrazolo[1,5-a]pyrimidin-
7-yl)-carbamate (1.0 equiv), tris(dibenzylideneacetone)dipalla-
dium (0.05 equiv), 2,20-bis(diphenylphosphino)-1,10-binaphthalene
(0.15 equiv), and sodium tert-butoxide (1.1 equiv) were suspended

8518 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Heathcote et al.
Figure 8. Oral administration of 4k inhibits human tumor xenografts. (A) Female nude mice bearing HCT116 tumor xenografts were dosed by
oral gavage with vehicle (n = 8) or 4k at a concentration of 25 mg/kg (n = 6). (B) The weights of the animals used over the time course of the
study are shown. Error bars represent the standard errors of the mean. P-Values were determined using Student’s t test; # represents p < 0.05, *
represents p < 0.01, and ** represents p < 0.001. (C) Shown are representative images for tumor xenografts immunostained using antibodies
for Rb, Rb pSer807/811, and Rb pThr821. (D) One thousand nuclei were scored for determining percentage of stained cells in sections
from each tumor. The results of the scoring are shown as box plots. Student’s t test analysis showed that pSer807/811 and pThr821 staining
was significantly different (p < 0.005) between the vehicle and 4k treatment groups, whereas there was no significant difference in levels of
total Rb.
0.13 mmol, 51%). Purification EtOAc:hexanes (1:10): [R]D (c
0.59, CH2Cl2) þ 16.3. IR (neat) 3370, 1722, 1642, 1581, 1516,
45 min at ambient temperature, and the aqueous phase was
extracted with EtOAc (3 ꢀ 200 mL). The combined organic
phases were dried (Na2SO4), concentrated in vacuo, and chro-
matographed (EtOAc:hexanes 1:20) to yield carbamate 3k (1.00 g,
1.67 mmol, 42%) and carbamate 3l (800 mg, 1.34 mmol, 33%).
3k: IR (neat) 3363, 1721, 1644, 1518 cm-1. 1H NMR (CDCl3, 400
MHz) δ 7.80 (s, 1H), 7.36-7.27 (m, 5H), 5.72 (s, 1H), 5.24-5.22
(m, 1H), 4.98-4.94 (m, 3H), 4.18-4.11 (m, 1H), 3.95-3.90 (m,
1H), 3.79-3.76 (m, 1H), 3.64-3.57 (m, 3H), 3.10 (hept, J = 6.9
Hz, 1H), 2.84-2.81 (m, 1H), 1.43 (s, 9H), 1.33 (d, J = 6.9 Hz,
6H), 0.93 (s, 9H), 0.13-0.12 (m, 6H). 13C NMR (CDCl3, 100
MHz) δ 154.8, 153.4, 141.7, 137.6, 128.5, 127.9, 127.6, 113.8, 97.4,
83.8, 82.5, 70.4, 62.5, 61.8, 53.5, 51.7, 28.0, 25.9, 23.6, 23.5,
23.2, -5.4. MS m/z (CI) 600 (M þ H)þ. HRMS (CI) calcd for
C31H50N5O5Siþ, 600.3576; found, 600.3578.
1
1368, 1158, 1107, 837, 777, 699 cm-1. H NMR (CDCl3, 300
MHz) δ 7.75 (s, 1H), 7.27 (m, 5H), 5.85 (m, 1H), 5.71 (s, 1H),
5.19 (m, 2H), 4.97 (br s, 1H), 4.95 (br s, 2H), 4.54 (m, 1H), 3.75
(m, 2H), 3.08 (m, 1H), 1.40-1.31 (m, 15H), 0.88 (s, 9H), 0.05 (s,
6H). 13C NMR (CDCl3, 75 MHz) δ 154.0, 153.5, 146.1, 142.8,
141.5, 137.7, 136.4, 128.4, 127.9, 127.4, 116.2, 113.3, 97.2, 82.0,
65.0, 54.6, 51.3, 28.0, 25.9, 23.8, 23.1, 18.3, -5.4. MS m/z (CI)
566 (M þ H)þ. HRMS (CI) calcd for C31H48N5O3Siþ, 566.3526;
found, 566.3538.
tert-Butyl Benzyl(5-(((2S,3S)-1-((tert-butyldimethylsilyl)oxy)-
3,4-dihydroxybutan-2-yl)amino)-3-iso-propylpyrazolo[1,5-a]pyri-
midin-7-yl)carbamate (3k). tert-Butyl Benzyl(5-(((2S,3R)-1-((tert-
butyldimethylsilyl)oxy)-3,4-dihydroxybutan-2-yl)amino)-3-iso-propyl-
pyrazolo[1,5-a]pyrimidin-7-yl)carbamate (3l). OsO4 (5.40 mL, 15
mol %, 2.5 wt % in tBuOH) was added to alkene 3f (2.27 g, 4.00
mmol) and NMO H2O (0.97 g, 8.3 mmol) in MeCN and H2O (4:
1, 80 mL). After 16 h, reaction was quenched by the addition of
saturated aqueous Na2SO3 (20 mL). The mixture was stirred for
3l: IR (neat) 3361, 1719, 1644, 1518 cm-1. 1H NMR (CDCl3,
400 MHz) δ 7.77 (s, 1H), 7.32-7.23 (m, 5H), 5.78 (s, 1H),
5.27-5.25 (m, 1H), 4.98-4.89 (m, 2H), 4.41 - 4.31 (m, 2H),
4.14-4.09 (m, 1H), 4.00-3.90 (m, 2H), 3.79-3.75 (m, 1H),
3.61-3.57 (m, 1H), 3.40-3.36 (m, 1H), 3.11-3.04 (m, 1H), 1.40

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8519
(s, 9H), 1.32-1.28 (m, 6H), 0.89 (s, 9H), 0.08-0.06 (m, 6H). 13
C
addition of MeOCH2Cl (3.26 mL, 43.2 mmol) and the mixture was
stirred overnight. The solution was poured into water and ex-
tracted into CH2Cl2 (3ꢀ). The combined organic layers were
washed with brine, dried (Na2SO4), and concentrated in vacuo.
Chromatography (hexanes:EtOAc 10:1) gave azide 11 (3.49 g, 15.1
mmol, 70%); [R]25D (c 1.2, CHCl3) þ 18.6. IR (neat) 3229, 2100,
NMR (CDCl3, 100 MHz) δ 141.7, 137.6, 128.5, 127.9, 127.6,
113.8, 97.4, 82.5, 70.4, 62.4, 61.8, 60.4, 53.5, 51.6, 28.0, 25.9, 23.6,
-5.4. MS m/z (CI) 600 (M þ H)þ. HRMS (CI) calcd for
C31H50N5O5Siþ, 600.3576; found, 600.3574.
(2S,3S)-3-(7-(Benzylamino)-3-iso-propylpyrazolo[1,5-a]pyrim-
idin-5-ylamino)-1,2,4-butanetriol (4k). Synthesized according to
the general procedure for acidic deprotection. 3k (605 mg,
1.01 mmol), HCl/MeOH (50.0 mL). Yield triol 4k (310 mg,
0.81 mmol, 79%). Purification crystallization from MeCN: mp
1
1373, 1259 cm-1. H NMR (CDCl3, 400 MHz) δ 4.68 (s, 2H),
4.14-4.06 (m, 2H), 3.97-3.94 (m, 1H), 3.84-3.78 (m, 1H),
3.71-3.64 (m, 2H), 3.42 (s, 3H), 1.47 (s, 3H), 1.37 (s, 3H). 13C
NMR (CDCl3, 100 MHz) δ 109.8, 96.7, 74.8, 67.3, 66.6, 63.0, 55.5,
26.5, 25.2. MS m/z (ESI) 249 (M þ NH4)þ. HRMS (ESI) calcd for
C9H18N3O4þ, 232.1297; found, 232.1303.
(S)-1-((S)-2,2-Dimethyl-1,3-dioxolan-4-yl)-2-(methoxymethoxy)-
ethanamine (12). Azide 11 (3.49 g, 15.1 mmol) in MeOH (100 mL)
and added to Pd/C (10 wt %, 350 mg). The atmosphere was
replaced by hydrogen and hydrogen bubbled through the suspen-
sion for 1 h. The mixture was filtered through Celite and concen-
trated in vacuo to yield amine 12 (3.02g,14.7mmol,97%).IR(neat)
3330, 1600, 1461 cm-1. 1H NMR (CDCl3, 400 MHz) δ 4.65 (s, 2H),
4.07-4.01 (m, 2H), 3.93-3.87 (m, 1H), 3.66 (dd, J = 9.7, 3.5 Hz,
1H), 3.51 (dd, J= 9.7, 6.2 Hz, 1H), 3.38 (s, 3H), 3.10-3.06 (m, 1H),
1.45 (br. s, 2H), 1.43 (s, 3H), 1.36 (s, 3H). 13C NMR (CDCl3, 100
MHz) δ 108.9, 96.7, 77.1, 69.8, 66.3, 55.3, 53.2, 26.6, 25.3. HRMS
(ESI) calcd for C9H20NO4, 206.1392; found, 206.1395.
(2S,3S)-3-(7-(Benzylamino)-3-iso-propylpyrazolo[1,5-a]pyrimidin-
5-ylamino)-1,2,4-butanetriol (4k). Synthesized according to the
general procedure for Buchwald-Hartwig coupling, 2 (500 mg,
1.25 mmol), Pd2dba3 (57.0 mg, 0.062 mmol), BINAP (117.0 mg, 0.19
mmol), sodium tert-butoxide (132 mg, 1.38 mmol), 12 (283 mg, 1.38
mmol), and PhMe (5.0 mL). Purification EtOAc:hexanes (1:4).
Analytically pure material could not be obtained at this stage.
General procedure for acidic deprotection followed. HCl/MeOH
(50.0 mL) yield 4k (412 mg, 1.07 mmol, 86%). Purification:
crystallization from MeCN.
184-186 °C; [R]25 (c 0.20, CH3OH) -25.0. IR (neat) 3229,
D
1639, 1581 cm-1. 1H NMR (CD3OD, 400 MHz) δ 7.69 (s, 1H),
7.42-7.27 (m, 5H), 5.32 (s, 1H), 4.56 (s, 2H), 4.10-4.04 (m,
1H), 3.87 (dd, J = 11.4, 4.1 Hz, 1H), 3.84 (dd, J = 11.1, 5.5
Hz), 3.63-3.56 (m, 3H), 3.05 (hept, J = 6.9 Hz, 1H), 1.31 (d,
J = 6.9 Hz, 3H), 1.29 (d, J = 6.9 Hz, 3H). 13C NMR (CD3OD,
100 MHz) δ 157.6, 146.9, 144.9, 140.6, 137.7, 128.4, 127.1, 126.6,
112.1, 72.8, 72.0, 63.0, 61.7, 54.3, 45.1, 23.3, 22.6, 22.2. MS
m/z (ESI) 386 (M þ H)þ. HRMS (ESI) calcd for C20H28N5O3
þ
,
386.2187; found, 386.2181.
(2R,3S)-3-(7-(Benzylamino)-3-iso-propylpyrazolo[1,5-a]pyrim-
idin-5-ylamino)-1,2,4-butanetriol (4l). Synthesized according to the
general procedure for acidic deprotection. 3l (765 mg, 1.28 mmol),
HCl/MeOH (50.0 mL). Yield 4l (338 mg, 0.86 mmol, 67%). Puri-
fication chromatography (EtOAc): [R]25D (c 0.20, CH3OH) -60.0.
IR (neat) 3307, 1637, 1579 cm-1. 1H NMR (CD3OD, 400 MHz) δ
7.68 (s, 1H), 7.40-7.25 (m, 5H), 5.34 (s, 1H), 4.54 (s, 2H),
4.23-4.20 (m, 1H), 3.93-3.89 (ddd, J = 8.3, 5.9, 2.2 Hz, 1H),
3.80 (dd, J = 11.0, 6.2 Hz, 1H), 3.77 (dd, J = 11.0, 7.0 Hz, 1H),
3.52 (dd, J = 11.5, 6.1 Hz, 1H), 3.39 (dd, J = 11.0, 8.7 Hz, 1H),
3.03 (hept, J = 6.9 Hz, 1H), 1.31 (d, J = 6.9 Hz, 3H), 1.29 (d, J =
6.9 Hz, 3H). 13C NMR (CD3OD, 100 MHz) δ158.0, 146.9, 144.5,
140.1, 137.7, 128.3, 127.1, 126.7, 112.0, 72.8, 70.8, 62.2, 62.0, 53.4,
45.1, 23.2, 22.6, 22.2. MS m/z (ESI) 386 (M þ H)þ. HRMS (ESI)
calcd for C20H28N5O3þ, 386.2187; found, 386.2189.
Second Generation Synthesis of 4k. (2R,3S)-Diethyl 2-azido-
3-hydroxysuccinate (9). (-)-Diethyl tartrate (10.0 g, 48.5 mmol)
in CH2Cl2 (250 mL) was cooled to -5 °C. Et3N (13.5 mL, 97.0
mmol) was added, followed by the dropwise addition of SOCl2
(5.28 mL, 72.7 mmol). After 30 min, the mixture was poured into
water and the aqueous layer extracted into CH2Cl2 (3ꢀ). The
combined organic layerswerewashedwithbrine, dried(Na2SO4),
and concentrated in vacuo to yield the cyclic sulfite which was
directly dissolved in DMF (400 mL), and sodium azide (6.30 g,
97.0 mmol) was added. The mixture was stirred for 12 h and then
poured into water and extracted into Et2O (3ꢀ). The combined
organic layers were washed with brine, dried (MgSO4), and
concentrated in vacuo. Chromatography (hexanes:EtOAc 4:1)
gave azide 9 (7.79 g, 33.7 mmol, 70%): [R]D (c 1.0, EtOH) -39.4.
1H NMR (CDCl3, 400 MHz) δ 4.66 (dd, J = 5.5, 2.7 Hz, 1H),
4.32 (m, 4H), 3.31 (d, J = 5.5 Hz, 1H), 1.35 (t, J = 7.1 Hz, 3H),
1.34 (t, J 7.1 Hz, 3H). 13C NMR (CDCl3, 100 MHz) δ 170.6,
166.9, 71.5, 64.2, 62.6, 62.0, 14.0.
Expression, Purification, and Crystallography of CDK2 Com-
plexed with 4k. A cDNA encoding human CDK237 was cloned
into a pProEx-HTb vector (Invitrogen) containing a 6ꢀHis-
SUMO-1 N-terminal tag and expressed in E. coli BL21 (DE3).
To cleave the SUMO tag, SuPr-1/Senp2 (Imagenes, Berlin,
Germany) was cloned into pET28b (Novagen) with both
N- and C-terminal 6ꢀHis tags and expressed in E. coli BL21(DE3).
After a first nickel affinity purification, 6ꢀHis-SUMO-CDK2
was incubated with partially purified SUMO protease at 4 °C
overnight. Cleaved CDK2 was separated from the 6ꢀHis-
SUMO tag and the SUMO protease using a second nickel
affinity column and further purified on a Superdex 75 gel
filtration column (GE Healthcare). This purification method
takes advantage of the fact that the SUMO protease cleaves the
SUMO tag without leaving any extra N-terminal residues,
which would hinder crystallization. Purified CDK2 was stored
at -80 °C in the crystallization buffer (10 mM Hepes pH 7.4, 0.5
mM DTT). Crystallization and ligand soaking conditions were
as previously described.38 A 1.8 A diffraction data set was
˚
collected at beamline I02 (Diamond Light Source, Didcot,
England) and the structure solved by molecular replacement
with Molrep4,39 using the 1HCK PDB structure as the search
model. 4k was fitted automatically using ARP/wARP ligand40
and the structure refined using Refmac5.41 Coordinates and
structure factors have been deposited to the Protein Data Bank,
PDB code 3NS9.
In Vitro Kinase Assays. Purified recombinant CDK1/cycB1,
CDK2/cycE, CDK4/cycD1, CDK5/p35NCK, CDK6/CycD1,
CDK7/CycH/MAT1, and CDK9/CycT were purchased from
Proqinase GmbH (Freiburg, Germany). Kinase assays were
performed according to manufacturer’s protocols, using sub-
strate peptides purchased from Proqinase GmbH. Measurement
of the amount of ATP remaining at the end of the reaction,
carried out with a luciferase assay (PKLight assay; Cambrex,
England), was used to determine inhibition of kinase activity by
compounds.
(S)-4-((S)-1-Azido-2-(methoxymethoxy)ethyl)-2,2-dimethyl-
1,3-dioxolane (11). Diester 9(5.00 g, 21.6 mmol) in EtOH (80.0 mL)
was cooled to -10 °C and lithium chloride (909 mg, 21.6 mmol)
was added, followed by portionwise addition of sodium borohy-
dride (4.80 g, 130 mmol). The suspension was warmed overnight,
recooled to -10 °C, and acidified to pH4 by the dropwise
addition of 1 M HCl in H2O. The resultant clear solution was
concentrated in vacuo to yield crude triol 10, which was directly
suspended in Me2CO (80.0 mL) and 2,2-dimethoxypropane
(25.0 mL). p-TsOH.H2O (400 mg, 2.1 mmol) was added and the
mixture heated to 50 °C for 2.5 h, cooled, and saturated ammo-
nium chloride solution (60.0 mL) was added. The suspension was
stirred for 1 h, poured into water, and extracted into Et2O (3ꢀ).
The combined organic layers were washed with brine, dried
(MgSO4), and concentrated in vacuo. The residue was dissolved
in CH2Cl2 (25.0 mL), cooled to 0 °C, and N,N-di-iso-propylethy-
lamine (7.51 mL, 43.2 mmol) was added followed by the slow

8520 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Heathcote et al.
Cell Growth Assays. Cell lines were obtained from the CRUK
cell bank facility and were routinely cultured in DMEM supple-
mented with 10% fetal calf serum (FCS) (First Link, England).
Cell growth was assessed using the Sulforhodamine B (SRB)
assay, as described.42 Human normal umbilical vein endothelial
cells (HUVEC) and the appropriate culture media were pur-
chased from Lonza Wokingham Ltd., England, and the cells
were cultured as per supplier’s protocols.
Flow Cytometry. HCT116 colon cancer cells were seeded (4 ꢀ
105) in 6-well plates in DMEM containing 10% FCS and allowed
to adhere for 24 h. Compounds prepared in DMSO were added,
and after 24 h incubation, cells were trypsinized, centrifuged at
1100 rpm for 5 min, and resuspended in 5 mL of ice-cold PBS,
centrifuged as above, gently resuspended in 2 mL ice-cold 70%
ethanol, and incubated at 4 °C for 1 h. Cells were washed twice
with 5 mL of ice-cold PBS and resuspended in 100 μL of PBS
containing 100 μg/mL RNase (Sigma-Aldrich, England) and 1
mL of 50 μg/mL propidium iodide (Sigma-Aldrich, England) in
PBS. Following incubation overnight in the dark at 4 °C and
filtering through 70 μm muslin gauze into FACS tubes (Becton-
Dickinson, England) to remove cell clumps, stained cells were
processed using the RXP cytomics software on a Beckman
Coulter Elite ESP (Beckman Coulter, High Wycombe, United
Kingdom). The data were analyzed using Flow Jo v7.2.5 (Tree
Star Inc., San Calos, CA), and statistical analysis was performed
using the unpaired Student’s t test to determine p-values.
were processed using the RXP cytomics software on a Beckman
Coulter Elite ESP, and the data were analyzed using Flow Jo
v7.2.5.
Absorption, Distribution, Metabolism, Elimination and Toxi-
city (ADME-Tox) and Pharmacokinetic Studies. Studies were
performed under contract by Cerep Inc. (USA), using described
methods.45-48
Tumor Xenografts. HCT116 cells (5 ꢀ 106) were injected
subcutaneously in not more than 0.1 mL volume into the flank
of the animals (female nu/nu-BALB/c athymic nude mice).
Tumor measurements were performed twice per week, and
1
volumes were calculated using the formula /2[length (mm)] ꢀ
[width (mm)]2. The animals were randomized, and when tumors
had reached a volume of 100-200 mm3, animals were entered
into the different treatment groups and treatment with 4k or
vehicle control was initiated. Animals were treated with 4k
prepared as abovedaily by oral gavage for a total of 14 days.
At the end of the 14-day treatment period, the mice were
sacrificed, and the tumors were fixed by formalin fixation and
paraffin embedding. Throughout the 14-day treatment period,
animal weights were determined each day and tumor volumes on
alternate days. The MCF-7 xenograft study was performed
exactly as described previously.24
Immunohistochemistry. Tumors were dissected from mice and
fixed in 10% formalin and embedded in paraffin blocks. Im-
munohistochemical staining was performed as described
previously,49 except that antigen retrieval was performed by
microwaving for 10 min using 10 mmol/L citrate buffer (pH 6.0).
Antibodies used were Rb pThr821 (ab4787) from Abcam
(England) or Rb (9309) and Rb pSer807/811 (9308) from New
England Biolabs, England. Images were acquired using the
Automated Cellular Imaging System (ACIS) (Carl Zeiss Ltd.,
Welwyn Garden City, England). Cell positivity was determined
by counting >1000 cells per section, and the results were
tabulated using GraphPad Prism software, with statistical sig-
nificance being determined using the Student’s t test (GraphPad
Prism).
Immunoblotting. Cells (8 ꢀ105), plated in 10 cm plates, were
treated with compounds after 24 h. Four hours later, cell lysates
were prepared by the addition of 500 μL of RIPA buffer (Sigma-
Aldrich, England), containing phosphatase inhibitor cocktail
and complete protease inhibitor cocktail, both from Roche
Diagnostics, England. Protein concentrations were determined
using the BCA protein assay (Thermo-Scientific, England).
Immunoblotting was performed as described previously.43 Anti-
body details are available upon request.
In Vivo Assessment of Molecular Pharmcology. All procedures
were approved by the Imperial College London Animal Ethics
Committee. The experiments were done by licensed investiga-
tors in accordance with the United Kingdom Home Office’s
regulations under the Animal (Scientific Procedures) Act 1986
and were within guidelines set out by the United Kingdom
Coordinating Committee for Cancer Research’s Ad hoc Com-
mittee on the Welfare of Animals in Experimental Neoplasia.44
Seven-week-old female nu/nu-BALB/c athymic nude mice were
purchased from Harlan Olac Ltd. (England). 4k, prepared in
28% (w/v) PEG300/5% (v/v) dimethylacetamide/10% (w/v)
HPB cyclodextrin in H2O, was administered by oral gavage.
Drug was administered by exact body weight, with the injection
volume being not more than 0.2 mL. After 6 h, the mice were
anaesthetized and blood collected by cardiac puncture into tubes
containing 200 μL of PBS, 100 U of Heparin, and 10 mM EDTA.
Blood was mixed with 1 mL of red blood cell lysis solution
(Flowgen Bioscience, Nottingham, England) for 15 min at room
temperature, followed by centrifugation for 5 min at 1,000 rpm to
pellet the peripheral blood mononuclear cells (PBMC). PMBC
were washed twice in PBS, and the cells were stained using the Fix
and Perm kit (Caltag Laboratories, Burlingame, CA, USA)
following the manufacturer’s instructions. Briefly, after washing
the PBMCs in PBS, 2 ꢀ 104-105 cells were fixed using 50 μL of
reagent A (fixation medium) for 15 min at room temperature. At
the end of the incubation, the cells were washed in PBS, and then
50 μL of reagent B (permeabilization medium) was added
together with antibodies for RNA polymerase II (PolII), PolII
P-Ser2, RB, or RB P-Thr821 (purchased from Abcam, England)
at a dilution of 1:100 for 1 h at room temperature. Cells were
washed three times with PBS and were incubated for 1 h with goat
antirabbit Alexafluor 488 (Invitrogen, Paisley, England) diluted
at 1:200 in PBS in the dark at room temperature. Finally cells
were washed in PBS three times and resuspended in 200 μL of
PBS and stored at 4 °C for flow cytometric analysis. Labeled cells
Acknowledgment. This work was funded by grants from
the Engineering and Physical Sciences Research Council and
Cancer Research UK. Our thanks go to the MRC Protein
Phosphorylation unit for the 76-kinase screening and to
Cerep, Inc. for the ADME-Tox and PK studies. The Devel-
opmental Therapeutics Program at NCI carried out the
NCI60 screening of 4k. We are grateful for support from
the NIHR Biomedical Research Centre funding scheme. We
also thank the CR-UK and the Dept of Health funded
Imperial College Experimental Cancer Medicine Centre
(ECMC) grant.
Supporting Information Available: In vitro kinase and cell
growth inhibition data, cell cycle profiles, and spectral data for
4k, as well as additional synthetic methods. This material is
available free of charge via the Internet at http://pubs.acs.org.
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