Enhanced selectivity for inhibition of analog-sensitive protein kinases through scaffold optimization

Article abstract of DOI:10.1016/j.tet.2007.02.089

The ability to inhibit any protein kinase of interest with a small molecule is enabled by a combination of genetics and chemistry. Genetics is used to modify the active site of a single kinase to render it distinct from all naturally occurring kinases. Next, organic synthesis is used to develop a small molecule, which does not bind to wild-type kinases but is a potent inhibitor of the engineered kinase. This approach, termed chemical genetics, has been used to generate highly potent mutant kinase-specific inhibitors based on a pyrazolopyrimidine scaffold. Here, we asked if the selectivity of the resulting pyrazolopyrimidines could be improved, as they inhibit several wild-type kinases with low-micromolar IC₅₀ values. Our approach to improve the selectivity of allele-specific inhibitors was to explore a second kinase inhibitor scaffold. A series of 6,9-disubstituted purines was designed, synthesized, and evaluated for inhibitory activity against several kinases in vitro and in vivo. Several purines proved to be potent inhibitors against the analog-sensitive kinases and exhibited greater selectivity than the existing pyrazolopyrimidines.

Full text of DOI:10.1016/j.tet.2007.02.089

Tetrahedron 63 (2007) 5832–5838
Enhanced selectivity for inhibition of analog-sensitive protein
kinases through scaffold optimization
Chao Zhanga and Kevan M. Shokata,b,
*
aHoward Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco,
CA 94143, USA
bDepartment of Chemistry, University of California, Berkeley, CA 94720, USA
Received 17 January 2007; revised 20 February 2007; accepted 21 February 2007
Available online 24 February 2007
Abstract—The ability to inhibit any protein kinase of interest with a small molecule is enabled by a combination of genetics and chemistry.
Genetics is used to modify the active site of a single kinase to render it distinct from all naturally occurring kinases. Next, organic synthesis is
used to develop a small molecule, which does not bind to wild-type kinases but is a potent inhibitor of the engineered kinase. This approach,
termed chemical genetics, has been used to generate highly potent mutant kinase-specific inhibitors based on a pyrazolopyrimidine scaffold.
Here, we asked if the selectivity of the resulting pyrazolopyrimidines could be improved, as they inhibit several wild-type kinases with low-
micromolar IC50 values. Our approach to improve the selectivity of allele-specific inhibitors was to explore a second kinase inhibitor scaffold.
A series of 6,9-disubstituted purines was designed, synthesized, and evaluated for inhibitory activity against several kinases in vitro and in
vivo. Several purines proved to be potent inhibitors against the analog-sensitive kinases and exhibited greater selectivity than the existing
pyrazolopyrimidines.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
The utility of an inhibitor, which specifically targets a non-
naturally occurring protein kinase is only realized when
the WT kinase can be replaced with the engineered form in
cells or animals. Advances in genetics allow for precise in-
troduction of a single mutation in many single-cell eukar-
yotes such as the budding yeast with ease. Consequently,
the chemical genetic approach has been extensively applied
to the study of various yeast kinases enabling selective phar-
macological blockage of these kinases for the first time.7
Analogous genetic manipulation in higher eukaryotes is
technically much more challenging and thus represents a
significant barrier to the use of chemical genetics to study
mammalian protein kinases. Despite the technical barrier,
genetically engineered mouse models have been created,
which carry an as allele in place of the WT form for a number
of protein kinases allowing for the first in vivo assessment of
the effects of a mono-specific protein kinase inhibitor.8,9
While requiring the extra effort of genetic manipulation
compared to traditional pharmacology, chemical genetics
allows for a critical control experiment, which addresses
the target selectivity of a pharmacological agent that is fre-
quently a major question facing protein kinase inhibitors.
An isogeneic WT control animal or cell can be treated with
the as specific kinase inhibitor in parallel to the cell or ani-
mal which carries the mutant as kinase, thus providing an
assessment of any ‘off-target’ effects of the allele-specific
inhibitor.
Potent and selective inhibitors of protein kinases are valu-
able tools for probing the cellular functions of kinases.1,2
However, due to the large number of protein kinases in
a cell and their highly homologous active sites, it has proven
difficult to find specific inhibitors for individual kinases.2,3
Our laboratory has developed a chemical approach, which
employs genetics to circumvent the specificity problem as-
sociated with conventional small-molecule inhibitors of pro-
tein kinases.4,5 The approach exploits a conserved, large
hydrophobic residue in the kinase active site (termed the
gatekeeper),6 which makes direct contact with the N6 amino
group of ATP. When this residue is mutated from the natu-
rally occurring bulky residue (methionine, leucine, phenyl-
alanine, threonine, etc.) to glycine or alanine, a novel
pocket not found in any wild-type (WT) kinase is created
within the kinase of interest. Such engineered kinases,
termed analog-sensitive (as) alleles, can thus be potently tar-
geted by inhibitor analogs that contain substituents which
occupy this enlarged ATP binding pocket and are occluded
from binding to WT kinases because they lack the additional
pocket.
Keywords: Chemical genetics; Protein kinase inhibitor; Purine scaffold.
* Corresponding author. Tel.: +1 415 514 0472; fax: +1 415 514 0822;
e-mail: shokat@cmp.ucsf.edu
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.02.089

C. Zhang, K. M. Shokat / Tetrahedron 63 (2007) 5832–5838
5833
We initially designed and synthesized a small panel of
analogs based on the pyrazolopyrimidine scaffold [exempli-
fied by PP1 (Fig. 1A),10 a nonselective kinase inhibitor], and
showed that some PP1 analogs potently inhibit the
engineered kinases.4,5 However, PP1 analogs still affect
certain WT kinases such as v-Src and Fyn. For example,
1NA-PP1, one of the most effective allele-specific inhibitors
discovered so far, inhibits WT v-Src with an IC50 of 1 mM
(Table 1), suggesting that it could inhibit kinases like
v-Src in the cell when used at mid-micromolar or higher
concentrations. This has restricted the use of PP1-derived
inhibitors to relatively low concentrations. To overcome
this limitation, we sought to develop allele-specific kinase
inhibitors with improved selectivity over the existing PP1
analogs.
In principle, the challenge of improving allele specificity can
be met by either increasing the inhibitor potency toward as
kinases or decreasing the potency toward WT kinases. The
crystal structure of Hck–PP1 complex shows that the pyrazo-
lopyrimidine ring of PP1 occupies the base-binding pocket
within the kinase active site in a nearly identical manner to
the adenine ring of ATP while the p-tolyl group at the 3-
position projects from the pyrazolopyrimidine core into
a deep hydrophobic pocket in the kinase active site
(Fig. 1A).11 The gatekeeper residue in Hck, T338, forms
one side of this hydrophobic pocket and directly stacks on
one face of the tolyl ring. The crystal structure reveals that
the N4 amino and the 3-tolyl groups of PP1 are in direct con-
tact of T338. As one of its two N–H bonds points directly at
T338, the N4 position was first chosen for derivatization
to generate allele-specific inhibitors. However, when N4-
substituted pyrazolopyrimidines were synthesized, it was
found that due to a steric clash with the 3-tolyl group the
N4 substituent is forced into the cis conformation at the
C4–N bond, which disrupted a key H-bond between PP1
and the kinase hinge region (Fig. 1A).12 Subsequently, 3-
position expanded analogs of PP1 were created to introduce
a steric clash with the gatekeeper residue.4 This effort suc-
ceeded in providing two pyrazolopyrimidine-based allele-
specific kinase inhibitors, 1NA-PP1 and 1NM-PP1, which
have been featured in the studies of various kinases. How-
ever, the extra moiety in these PP1 derivatives such as
1NA-PP1 is not oriented directly toward the gatekeeper
and thus could only form a partial steric clash with T338
(Fig. 1A). It is conceivable that WT Hck could accommodate
1NA-PP1 through mild conformational reorganization,
which may explain why 1NA-PP1 retains the ability to
inhibit unmodified Src family kinases at micromolar concen-
trations (Table 1). The pyrazolopyrimidine scaffold dictates
the relative orientation of the 3-substituent to the gatekeeper
residue and makes it difficult to design PP1 derivatives
with a more direct clash with the gatekeeper residue than
1NA-PP1.
We reasoned that inhibitors based on a different scaffold
could possess greater selectivity with respect to as kinases
if the scaffold could provide a platform for positioning sub-
stituents directly toward the gatekeeper residue while still
maintaining unperturbed interactions with the hinge region.
After examining the various protein kinase inhibitors
reported in the literature, we selected the purine scaffold (ex-
emplified by purvalanol) because it presents a distinct bind-
ing mode from PP1 and has potential to be derivatized to
induce a direct steric clash with the gatekeeper residue.13
Structural studies reveal that despite their isosteric structures
purine and pyrazolopyrimidine are situated within the kinase
active site with completely different orientations (Fig. 1). As
a result, the frequently observed hydrogen bonds between
Figure 1. (A) Crystal structure of Hck–PP1 complex at the top and the struc-
tures of PP1, 1NM-PP1, and 1NA-PP1 at the bottom. The gatekeeper residue
(Thr338) in Hck is highlighted gray with meshed surface and the H-bonds
between PP1 and the kinase hinge region are shown in dashed line. The
red arrow indicates the point and direction of derivatization on PP1 to gen-
erate allele-specific kinase inhibitors. (B) Crystal structure of CDK2–PVA
(a purvalanol analog) complex at the top and the structures of PVA and its
N9-modified derivatives at the bottom. The gatekeeper residue (Phe80) in
CDK2 is highlighted gray with meshed surface and the hydrogen bonds be-
tween PVB and the kinase hinge region are shown in dashed line. The red
arrow indicates the point and direction of derivatization on PVA to generate
allele-specific inhibitors for protein kinases.

5834
C. Zhang, K. M. Shokat / Tetrahedron 63 (2007) 5832–5838
Table 1. IC50 values of 1NA-PP1, 1NM-PP1, and the purines 1–9 against CDK2 and v-Src kinases
Kinase
1NA-PP1 1NM-PP1
1
2
3
4
5
6
7
8
9
CDK2-as1 15 nM
CDK2-as2 ND
CDK2-WT 18 mM
5 nM
ND
29 mM
44 nM
1.5 mM
7 mM
300 nM
1 mM
>100 mM >100 mM >100 mM 90 mM
400 nM
1.3 mM
7 mM
38 nM
>100 mM 900 nM
60 nM
700 nM
22 nM
62 nM
20 nM
940 nM
2 mM
4 mM
>100 mM >100 mM >100 mM >100 mM
v-Src-as1
v-Src-as2
v-Src-WT
1.5 nM
1 nM
1 mM
4 nM
15 nM
28 mM
42 nM
250 nM
140 nM
360 nM
>100 mM >100 mM >100 mM >100 mM >100 mM >100 mM >100 mM >100 mM >100 mM
500 nM
900 nM
800 nM
14 mM
11 nM
130 nM
11 nM
140 nM
11 nM
50 nM
17 nM
150 nM
180 nM
500 nM
‘ND’: not determined.
the inhibitor and the kinase hinge region were formed
through a different set of atoms in a purvalanol analog
(PVA) from PP1. The m-chlorophenyl group at N6 of PVA
projects outside the ATP binding pocket and contacts
a loop region that is not exploited by PP1. The methyl group
at N9, on the other hand, points directly at F80, the gate-
keeper residue in CDK2. Based on this observation, we pre-
dicted that an enlarged N9-substituent on the purine scaffold
would produce a direct steric clash with a bulky gatekeeper
residue such as F80 in CDK2. Herein, we describe the syn-
thesis of a series of 6,9-disubstituted purine derivatives and
the evaluation of their inhibitory activity against a number of
kinases in vitro and in vivo.
Scheme 2. (a) RNH2 (R¼cyclohexyl or aryl) 1 equiv, triethylamine 2 equiv,
1-butanol, reflux, 8 h; (b) triethylorthoformate 10 equiv, rt, 4 h; (c) aniline
2 equiv, i-PrOH, reflux, 1 h.
2. Results and discussion
N6-phenyladenine, which was subjected to reaction with dif-
ferent alkylating reagents in the second step to yield the re-
spective N9-alkyl purines. The N9-cyclohexyl (5) and the
N9-aryl purines (6–9) could not be produced using the above
synthetic route because cyclohexyl bromide was found not
reactive enough to alkylate N6-phenyladenine and because
no direct N9 arylation transformation is available. Analogs
5–9 were thus synthesized using a different route in three
steps (Scheme 2).16 4,6-Dichloro-5-aminopyrimidine was
reacted with cyclohexylamine or different arylamines, fol-
lowed by treatment with triethylorthoformate to afford 6-
chloro purines. Finally, reaction of the intermediates with
aniline provided the desired 6,9-disubstituted purines (5–9).
We designed and synthesized a series of 6,9-disubstituted
purines as specific inhibitors of analog-sensitized kinases.
The substituents at the 6-position of the purines were fixed
as a phenylamino group (considered a CDK specificity
element, the m-chloro substituent on the phenyl ring was
removed from PVA to maximize generality), while the N9-
substituents were varied in the series to explore the gate-
keeper pocket in as kinases (Schemes 1 and 2). In order
to diminish their inhibitory activity against WT kinases, we
designed the N9-substituents of these purines to be signifi-
cantly larger than the corresponding groups (varying from
Me to i-Pr) in the known purine-based kinase inhibitors
(such as olomoucine and purvalanol) in order to produce
a major steric clash with the gatekeeper residue in WT
kinases (Fig. 1B).14
We first examined the inhibitory activity of 1–9 against a
serine/threonine kinase, cyclin-dependent kinase 2 (CDK2),
and a tyrosine kinase v-Src by determining their IC50 values
in vitro. Upon mutation of the gatekeeper residues (F80 in
CDK2 and I338 in v-Src) to glycine (as1 allele) or alanine
(as2 allele), CDK2 and v-Src were shown to be dramatically
sensitized to the PP1 analogs such as 1NA-PP1 and 1NM-
PP1 (Table 1).5 The IC50 values of 1–9 against WT CDK2,
CDK2-as1 (CDK2F80G), and CDK2-as2 (CDK2F80A) were
determined. With the exception of 1, all the purines in the se-
ries inhibit WT CDK2 poorly with IC50s over 50 mM. In con-
trast, these compounds inhibit the glycine gatekeeper mutant
CDK2-as1 potently with IC50s in the nanomolar range, and
the inhibitory selectivity toward CDK2-as1 over the WT is
over 2000-fold for 5–8. The alanine mutant, CDK2-as2, is
inhibited by these purines at an intermediate level between
the wild type and as1. When the inhibition of purines 1–9
against v-Src was measured, the IC50 values of all the pu-
rines in the series against WT v-Src were found to be over
100 mM (Table 1). In contrast, the IC50 values of 1–9 against
the glycine mutant, v-Src-as1 (v-SrcI338G), lie in the nano-
molar range with several compounds in the series (5–7)
Several N9-alkyl purines (1–4) were synthesized in two
steps using modified literature procedures (Scheme 1).15 Re-
action of 6-chloropurine with aniline in the first step afforded
Scheme 1. (a) Aniline 2 equiv, i-PrOH, reflux, 1 h; (b) alkyl halide RX
1 equiv, K2CO3 1 equiv, DMF, rt, 1 h.

C. Zhang, K. M. Shokat / Tetrahedron 63 (2007) 5832–5838
5835
approaching 10 nM. The purines 5–7 exhibit extraordinary
inhibitory selectivity (>9000-fold) toward v-Src-as1 over
the WT. Furthermore, these purines inhibit the alanine mu-
tant, v-Src-as2 (v-SrcI338A), significantly less potently than
v-Src-as1. This trend parallels that of CDK2, indicating
that the purine derivatives are generally more effective in-
hibitors against the glycine gatekeeper mutant of a kinase
than the alanine form.
Fig. 2), the most effective PP1-derived inhibitor for cdc28-
as1. Importantly, these purine derivatives produced no halo
on the WT strain under the same conditions, discounting
the possibility that they cause halos by inhibiting WT kinases
or other yeast proteins (Fig. 2).
3. Conclusions
A large number of chemical genetic studies of kinases have
focused on the budding yeast Saccharomyces cerevisiae be-
cause of the relative ease of genetic manipulation and the
fact that it is a single-cell eukaryote. As some of these pu-
rines are potent inhibitors of the as1 allele of CDK2 and
v-Src in vitro, we went on to examine their inhibition of
Cdc28p, the budding yeast homolog of CDK2, in vivo. Being
the major cyclin-dependent kinase in S. cerevisiae, Cdc28p
shares high sequence homology (65% sequence identity)
with the mammalian CDK2 and is essential for yeast viabil-
ity.17,18 We previously generated a mutant strain of cdc28-
as1 yeast by replacing WT CDC28 with its as1 allele
(cdc28-as1).5 Since the kinase activity of Cdc28p is essential
for yeast viability, inhibitor candidates can be screened for
inhibition of cdc28-as1 using a halo assay.19 A solution of
each compound (1–9) was spotted onto a circular cellulose
disk (0.6 cm diameter) laid on top of a yeast growth plate
that is covered by an even lawn of yeast cells. Inhibition of
cdc28-as1 by the compound should prevent yeast growth
near the cellulose disk, which results in the formation of
a cell-free halo surrounding the disk.
In an attempt to improve upon the selectivity of the PP1-
derived inhibitors for as kinases we have examined a new ki-
nase inhibitor scaffold for the development of more selective
allele-specific kinase inhibitors in the current study. Aided
by the crystal structure of CDK2–purvalanol complex, we
designed and synthesized a series of 6,9-disubstituted pu-
rines as allele-specific kinase inhibitors. Several purines
(1, 5, 6, and 7) exhibit potent inhibitory activity against
both CDK2-as1 and v-Src-as1 in vitro with low nanomolar
IC50 values, which approach the potency of the most effec-
tive PP1 analogs such as 1NA-PP1 and 1NM-PP1. Most im-
portantly, these purines poorly inhibit WT CDK2 and v-Src
(IC50 >100 mM) and display high selectivity toward the
as1 allele over the WT kinase. These data support the conclu-
sion that purine N9-substituents in these purines can indeed
occupy the gatekeeper pocket as designed and contribute to
the potency and selectivity of these purines toward as
kinases.
We also investigated the in vivo inhibitory activity of these
purine derivatives against the as1 allele of an essential yeast
kinase CDC28 using a growth inhibition assay. Purines 1, 5–
8 caused halos to the cdc28-as1 yeast indicating that these
molecules can cross the yeast plasma membrane and inhibit
the target kinase. Good membrane permeability is a property
critical for the practical utility of small-molecule inhibitors
in cell biology studies.20
When screened against the cdc28-as1 yeast, several purines
caused large halos indicating that they efficiently cross the
yeast plasma membrane and inhibit Cdc28-as1 in the cell
(Fig. 2). As the high sequence conservation between
Cdc28p and CDK2 predicts, the most potent inhibitors in
the purine series (1, 5–8) against cdc28-as1 in vivo turned
out to be the same as the most potent compounds found
for CDK2 in vitro (Table 1). These five compounds cause
halos of comparable size to those by 1NM-PP1 (N in
While the purine derivatives exhibit improved inhibitory
selectivity toward glycine-substituted kinases (as1), their
potency does not exceed those of 1NA-PP1 and 1NM-PP1
Figure 2. Compounds 1–9 were tested in the halo assay against the cdc28-as1 yeast and the CDC28 wild-type strain. 1NM-PP1 is included on each plate for
comparison and is labeled as N on the plates. Several purines caused large halos to cdc28-as1, but not CDC28 yeast. Approximately 2ꢀ105 cells were spread
evenly on each plate before four circular cellulose disks (0.6 cm diameter) were gently laid on top of the plate. Ten microliters of each inhibitor solution (in
DMSO at 2 mM) was subsequently added to one cellulose disk. The images of the plates were taken after 30-h incubation at 30 ꢁC.

5836
C. Zhang, K. M. Shokat / Tetrahedron 63 (2007) 5832–5838
in the examples examined so far. The key advantage of the
purine series, reported in this study, over the pyrazolopyr-
imidine series is the significant improvement in orthogonal-
ity with respect to WT kinases. Whereas the commonly used
pyrazolopyrimidine inhibitor 1NA-PP1 has a selectivity fac-
tor [IC50(WT)/IC50(as)] of 700 for v-Src, purines 5–7 exhibit
a selectivity factor over 10,000-fold. In addition, considering
the small number and simplicity of the purines synthesized
in the current study, there remain significant opportunities
for improvement. Derivatization at C2 in the purine ring
and further diversification of the substituents at N6 and N9
will likely lead to improvements in potency.13 The availabil-
ity of a second series of allele-specific kinase inhibitors will
provide additional chemical tools for interrogating the
diverse kinases in the kinome.
7.02 (t, J¼8 Hz, 1H, CH-40), 7.31 (t, J¼8 Hz, 2H, CH-30),
7.94 (d, J¼8 Hz, 2H, CH-20), 8.38 (s, 2H, CH-2,8), 9.80
(s, 1H, NH); 13C NMR (DMSO, 100 MHz) d 23.6 (CH2-
300), 31.9 (CH2-200), 55.5 (CH-100), 120.2 (CH-40), 120.7
(CH-10), 122.4 (CH-30), 128.3 (C-5), 139.8 (C-6), 140.3
(C-10), 149.6 (CH-8), 151.5 (CH-2), 152.0 (C-4); HRMS
(EI) molecular ion calculated for C16H17N5 279.14840,
found 279.14910.
4.2.3. (9-Benzyl-9H-purin-6-yl)-phenylamine (2). Color-
less crystals; IR (thin film) 3052, 1624, 1579, 1497, 1477,
1
1232, 726, 695 cmꢂ1; H NMR (DMSO, 400 MHz) d 5.44
(s, 2H, CH2), 7.02 (t, J¼8 Hz, 1H, CH-40), 7.31 (t, J¼8 Hz,
2H, CH-arom), 7.33 (m, 5H, CH-arom), 7.94 (d, J¼8 Hz,
2H, CH-arom), 8.40 (s, 1H, CH-2), 8.44 (s, 1H, CH-8), 9.87
(s, 1H, NH); 13C NMR (DMSO, 100 MHz) d 46.3 (CH2),
119.7 (C-arom), 120.8 (C-arom), 122.5 (C-arom), 127.6 (C-
arom), 127.8 (C-arom), 128.3 (C-arom), 128.7 (C-arom),
136.9 (C-arom), 139.7 (C-arom), 141.7 (C-arom), 149.6
(CH-8), 152.0 (CH-2), 152.1 (C-4); HRMS (EI) molecular
ion calculated for C18H15N5 301.13275, found 301.13289.
4. Experimental
4.1. General
Materials obtained commercially were reagent grade and
1
were used without further purification. H NMR and 13C
4.2.4. (9-Naphthalen-1-ylmethyl-9H-purin-6-yl)-phenyl-
amine (3). White powder; IR (thin film) 3050, 1621, 1581,
NMR spectra were recorded on a Varian 400 spectrometer
at 400 and 100 MHz, respectively. High-resolution electron
impact mass spectra were recorded on a MicoMass VG70E
spectrometer at the University of California-San Francisco
center for Mass Spectrometry. Reactions were monitored
by thin layer chromatography (TLC), using Merck silica
gel 60 F254 glass plates (0.25 mm thick). Flash chromato-
graphy was conducted with Merck silica gel 60 (230–400
mesh).
1498, 1476, 1232, 753, 692 cmꢂ1 1H NMR (DMSO,
;
400 MHz) d 5.93 (s, 2H, CH2), 7.03 (t, J¼7 Hz, 1H,
CH-40), 7.24 (t, J¼7 Hz, 1H, CH-arom), 7.32 (t, J¼8 Hz,
2H, CH-arom), 7.47 (t, J¼8 Hz, 1H, CH-arom), 7.58 (m, 2H,
CH-arom), 7.94 (m, 4H, CH-arom), 8.28 (d, J¼8 Hz, 1H,
CH-arom), 8.35 (s, 1H, CH-2), 8.43 (s, 1H, CH-8), 9.90 (s,
1H, NH); 13C NMR (DMSO, 100 MHz) d 44.1 (CH2),
119.6 (C-arom), 120.8 (C-arom), 122.6 (C-arom), 123.0
(C-arom), 125.6 (C-arom), 125.8 (C-arom), 126.2 (C-
arom), 126.8 (C-arom), 128.3 (C-arom), 128.5 (C-arom),
128.7 (C-arom), 130.3 (C-arom), 132.3 (C-arom), 133.3
(C-arom), 139.6 (C-arom), 141.8 (C-arom), 149.8 (CH-8),
152.1 (C-arom), 152.1 (C-arom); HRMS (EI) molecular
ion calculated for C22H17N5 351.14840, found 351.14841.
4.2. General procedures for the synthesis of 1–4
exemplified by 1
4.2.1. N6-Phenyladenine. A solution of 6-chloropurine
(1.0 g, 6.5 mmol) and aniline (1.21 g, 13.0 mmol) in 50 mL
isopropanol was refluxed for 2 h. The reaction mixture was
added to 50 mL 1 M NaHCO3 aqueous solution and ex-
tracted with ethyl acetate (3ꢀ60 mL). The extracts were
combined and evaporated in vacuo to yield 1.17 g (85%)
of yellowish product. The crude product was recrystalized
from ethyl acetate to give 1.04 g (76%) of N6-phenyladenine
as colorless crystals; 1H NMR (DMSO, 400 MHz) d 7.01 (t,
1H, CH-40), 7.31 (t, 2H, CH-30), 7.95 (d, 2H, CH-20), 8.27 (s,
1H, CH-2), 8.37 (s, 1H, CH-8), 9.72 (s, 1H, NH), 13.12
(s, 1H, NH-arom); 13C NMR (DMSO, 100 MHz) d 120.5
(CH-40), 120.5 (CH-20), 122.3 (CH-30), 128.4 (C-5), 139.8
(C-arom), 139.8 (C-arom), 151.8 (C-arom), 151.8 (C-
arom), 151.8 (C-arom); HRMS (EI) molecular ion calcu-
lated for C11H9N5 211.08580, found 211.08545.
4.2.5. (9-Naphthalen-2-ylmethyl-9H-purin-6-yl)-phenyl-
amine (4). White powder; IR (thin film) 3052, 1621, 1581,
1497, 1477, 1232, 751, 692 cmꢂ1 1H NMR (DMSO,
;
400 MHz) d 5.61 (s, 2H, CH2), 7.02 (t, J¼8 Hz, 1H, CH-
40), 7.31 (t, J¼8 Hz, 2H, CH-arom), 7.50 (m, 3H, CH-arom),
7.87 (m, 4H, CH-arom), 7.94 (d, J¼8 Hz, 2H, CH-arom),
8.40 (s, 1H, CH-2), 8.49 (s, 1H, CH-8), 9.88 (s, 1H, NH);
13C NMR (DMSO, 100 MHz) d 46.5 (CH2), 119.7 (C-
arom), 120.7 (C-arom), 122.5 (C-arom), 125.6 (C-arom),
126.2 (C-arom), 126.2 (C-arom), 126.5 (C-arom), 127.6
(C-arom), 127.7 (C-arom), 128.3 (C-arom), 128.4 (C-
arom), 132.4 (C-arom), 132.8 (C-arom), 134.4 (C-arom),
139.7 (C-arom), 141.8 (C-arom), 149.7 (CH-8), 152.0 (C-
arom), 152.1 (C-arom); HRMS (EI) molecular ion calcu-
lated for C22H17N5 351.14840, found 351.14916.
4.2.2. (9-Cyclopentyl-9H-purin-6-yl)-phenylamine (1). A
mixture of N6-phenyladenine (0.70 g, 3.3 mmol), cyclopen-
tyl iodide (0.65 g, 3.5 mmol), and K2CO3 (0.48 g, 3.5 mmol)
in 20 mL of DMF was stirred overnight. The solvent was
evaporated and the yellow residue was triturated with water
and recrystalized from ethanol/water to yield 0.73 g (81%)
of product. Colorless crystals; IR (thin film) 3327, 2964,
1622, 1582, 1498, 1476, 751, 646 cmꢂ1; 1H NMR (DMSO,
400 MHz) d 1.70 (m, 2H, CH-300), 1.88 (m, 2H, CH-300), 2.03
(m, 2H, CH-200), 2.16 (m, 2H, CH-200), 4.90 (m, 1H, CH-100),
4.3. General procedures for the synthesis of 5–9
exemplified by 5
4.3.1. 6-Chloro-9-cyclohexyl-9H-purine. A solution of
4,6-dichloropurine (1.64 g, 10.0 mmol), cyclohexylamine
(0.99 g, 10.0 mmol), and triethylamine (2.02 g, 20.0 mmol)
in 50 mL 1-butanol was refluxed. After 8 h, the solution was
concentrated in vacuo to give a brown residue. This

C. Zhang, K. M. Shokat / Tetrahedron 63 (2007) 5832–5838
5837
1
intermediate product was added to triethylorthoformate and
the solution was stirred at rt for 4 h. The solvent was evapo-
rated and the yellow residue was recrystalized from ethyl
acetate to yield 1.20 g (51%) of the product. Colorless crys-
1376, 1180, 748, 691 cmꢂ1; H NMR (DMSO, 400 MHz)
d 7.06 (t, J¼8 Hz, 1H, CH-40), 7.35 (t, J¼8 Hz, 2H, CH-30),
7.62 (m, 2H, CH-arom), 7.99 (d, J¼8 Hz, 2H, CH-20), 8.04
(d, J¼8 Hz, 2H, CH-arom), 8.10 (dd, J1¼9 Hz, J2¼2 Hz,
1H, CH-arom), 8.17 (d, J¼9 Hz, 1H, CH-arom), 8.51 (s, 2H,
CH-2), 8.90 (s, 1H, CH-8), 10.03 (s, 1H, NH); 13C NMR
(DMSO, 100 MHz) d 120.4 (C-arom), 120.9 (C-arom), 121.0
(C-arom), 121.7 (C-arom), 122.8 (C-arom), 126.7 (C-arom),
127.2 (C-arom), 127.8 (C-arom), 128.0 (C-arom), 128.5 (C-
arom), 129.4 (C-arom), 131.8 (C-arom), 132.5 (C-arom),
133.0 (C-arom), 139.6 (C-arom), 140.8 (C-arom), 149.5
(CH-8), 152.4 (CH-2), 152.7 (C-4); HRMS (EI) molecular
ion calculated for C21H15N5 337.13275, found 337.13166.
1
tals; H NMR (DMSO, 400 MHz) d 1.27 (m, 1H, CH-40),
1.45 (m, 2H, CH-30), 1.69 (br d, J¼12 Hz, 1H, CH-40),
1.86 (br d, J¼14 Hz, 2H, CH-30), 1.98 (m, 4H, CH2-20),
4.50 (m, 1H, CH-10), 8.76 (s, 1H, CH-8), 8.79 (s, 1H, CH-
2); HRMS (EI) molecular ion calculated for C11H13N4Cl
236.08287, found 236.08357.
4.3.2. (9-Cyclohexyl-9H-purin-6-yl)-phenylamine (5). A
solution of 6-chloro-9-cyclohexyl-9H-purine (0.60 g,
2.5 mmol) and aniline (0.46 g, 5.0 mmol) in 30 mL isopropa-
nol was refluxed for 2 h. The reaction mixture was added
to 50 mL 1 M NaHCO3 aqueous solution and extracted with
ethyl acetate (3ꢀ60 mL). The extracts were combined and
evaporated in vacuo to give a brown residue, which was fur-
ther purified by column chromatography (5% Et2O/CHCl3)
to yield 0.61 g (83%) of product. White powder; IR (thin film)
2934, 1622, 1582, 1497, 1475, 1233, 750,646 cmꢂ1;1HNMR
(DMSO, 400 MHz) d 1.25 (m, 1H, CH-400), 1.44 (m, 2H,
CH-300), 1.71 (br d, J¼13 Hz, 1H, CH-400), 1.86 (br d,
J¼14 Hz, 2H, CH-400), 2.00 (m, 4H, CH2-200), 4.41 (m, 1H,
CH-100), 7.02 (t, J¼8 Hz, 1H, CH-40), 7.31 (t, J¼8 Hz, 2H,
CH-30), 7.94 (d, J¼8 Hz, 2H, CH-20), 8.37 (s, 1H, CH-2),
8.39 (s, 1H, CH-8), 9.80 (s, 1H, NH); 13C NMR (DMSO,
100 MHz) d 24.8 (C-400), 25.1 (C-300), 32.3 (C-200), 53.9
(C-100), 120.7 (C-arom), 120.0 (C-arom), 122.5 (C-arom),
128.4 (C-arom), 139.8 (C-arom), 140.0 (C-arom), 149.2
(CH-8), 151.5 (CH-2), 152.0 (C-4); HRMS (EI) molecular
ion calculated for C17H19N5 293.16405, found 293.16393.
4.3.6. (9-Naphthalen-1-yl-9H-purin-6-yl)-phenylamine
(9). White powder; H NMR (DMSO, 400 MHz) d 7.06 (t,
1
J¼7 Hz, 1H, CH-40), 7.34 (m, 3H, CH-arom), 7.54 (t,
J¼7 Hz, 1H, CH-arom), 7.64 (t, J¼7 Hz, 1H, CH-arom),
7.72 (m, 2H, CH-arom), 7.99 (d, J¼8 Hz, 2H, CH-20), 8.13
(d, J¼8 Hz, 1H, CH-arom), 8.19 (d, J¼8 Hz, 1H, CH-
arom), 8.31 (s, 1H, CH-2), 8.61 (s, 1H, CH-8), 10.07 (s,
1H, NH); 13C NMR (DMSO, 100 MHz) d 119.5 (C-arom),
121.0 (C-arom), 122.4 (C-arom), 122.7 (C-arom), 125.6
(C-arom), 125.9 (C-arom), 126.9 (C-arom), 127.6 (C-arom),
128.3 (C-arom), 128.4 (C-arom), 129.4 (C-arom), 129.7 (C-
arom), 130.7 (C-arom), 133.7 (C-arom), 139.6 (C-arom),
142.5 (C-arom), 151.2 (CH-8), 152.4 (CH-2), 152.6 (C-4);
HRMS (EI) molecular ion calculated for C21H15N5
337.13275, found 337.13349.
4.4. Kinase inhibition assays in vitro
Glutathione S-transferase (GST) fused v-Src proteins were
expressed in Escherichia coli and purified on glutathione
beads as described previously.4 In the v-Src kinase assay,
various concentrations of inhibitor were incubated with
50 mM Tris (pH 8.0), 10 mM MgCl2, 1.6 mM glutathione,
1 mg/mL BSA, 0.1 mg/mL peptide substrate (IY-
GEFKKK), 3.3% DMSO, and 11 nM (2 mCi) [g-32P]ATP
(6000 Ci/mmol, NEN), and v-Src kinase in a total volume
of 30 mL for 30 min. Reaction mixtures (27 mL) were spot-
ted onto a phosphocellulose disk, and washed with 0.5%
H3PO4. The transfer of 32P was measured by standard scin-
tillation counting. IC50 values were defined to be the con-
centration of inhibitor at which the radioactivity counts
remaining on the phosphocellulose disk were inhibited
by 50%.
4.3.3. Phenyl-(9-phenyl-9H-purin-6-yl)-amine (6). White
powder; IR (thin film) 3048, 1623, 1584, 1500, 1475,
1
1373, 751, 691 cmꢂ1; H NMR (DMSO, 400 MHz) d 7.05
(t, J¼8 Hz, 1H, CH-40), 7.34 (t, J¼8 Hz, 2H, CH-30), 7.46
(t, J¼8 Hz, 1H, CH-400), 7.60 (t, J¼8 Hz, 2H, CH-300), 7.92
(d, J¼8 Hz, 2H, CH-20), 7.97 (d, J¼8 Hz, 2H, CH-200),
8.46 (s, 1H, CH-2), 8.76 (s, 1H, CH-8), 9.99 (s, 1H, NH);
13C NMR (DMSO, 100 MHz) d 120.3 (C-arom), 120.9 (C-
arom), 122.8 (C-arom), 123.2 (C-arom), 127.7 (C-arom),
128.4 (C-arom), 129.5 (C-arom), 134.9 (C-arom), 139.6
(C-arom), 140.6 (C-arom), 149.3 (CH-8), 152.4 (CH-2),
152.6 (C-4); HRMS (EI) molecular ion calculated for
C17H13N5 287.11710, found 287.11692.
4.3.4. Phenyl-(9-p-tolyl-9H-purin-6-yl)-amine (7). White
powder; IR (thin film) 3048, 1623, 1585, 1516, 1497, 1475,
1373, 750 cmꢂ1; 1H NMR (DMSO, 400 MHz) d 2.38 (s, 3H,
CH3), 7.04 (t, J¼8 Hz, 1H, CH-40), 7.33 (t, J¼8 Hz, 2H, CH-
30), 7.39 (d, J¼8 Hz, 2H, CH-300), 7.78 (d, J¼8 Hz, 2H, CH-
20), 7.97 (d, J¼8 Hz, 2H, CH-200), 8.44 (s, 1H, CH-2), 8.71 (s,
1H, CH-8), 9.97 (s, 1H, NH); 13C NMR (DMSO, 100 MHz)
d 20.6 (CH3), 120.3 (C-arom), 120.9 (C-arom), 122.7
(C-arom), 123.0 (C-arom), 128.4 (C-arom), 129.9 (C-
arom), 132.4 (C-arom), 137.2 (C-arom), 139.6 (C-arom),
140.6 (C-arom), 149.3 (CH-8), 152.3 (CH-2), 152.5 (C-4);
HRMS (EI) molecular ion calculated for C18H15N5
301.13275, found 301.13294.
6ꢀHis tagged CDK2 and cyclin E were expressed in SF9
cells and purified on Ni-NTA beads as described previously.5
In the CDK2 kinase assay, various concentrations of inhibi-
tor were incubated with 20 mM Tris (pH 7.4), 100 mM
NaCl, 10 mM MgCl2, 1 mg/mL BSA, 0.1 mg/mL peptide
substrate (RGGKSPRKGNSSKKK), 3.3% DMSO, and
11 nM (2 mCi) [g-32P]ATP (6000 Ci/mmol, NEN), and
CDK2/ClnE complex in a total volume of 30 mL for
30 min. The other procedures of the CDK2 kinase assay
are the same as those of the v-Src assay.
4.5. Halo assay for cdc28-as1 inhibition
4.3.5. (9-Naphthalen-2-yl-9H-purin-6-yl)-phenylamine
(8). White powder; IR (thin film) 1619, 1583, 1497, 1477,
The cdc28-as1 yeast was created as previously described.5
Approximately 2ꢀ105 cells isolated during log phase growth

5838
C. Zhang, K. M. Shokat / Tetrahedron 63 (2007) 5832–5838
P. G.; Rose, M. D.; Wood, J. L.; Morgan, D. O.; Shokat,
K. M. Nature 2000, 407, 395–401.
6. Adams, J.; Huang, P.; Patrick, D. Curr. Opin. Chem. Biol. 2002,
6, 486–492.
7. Zhang, C.; Kenski, D. M.; Paulson, J. L.; Bonshtien, A.; Sessa,
G.; Cross, J. V.; Templeton, D. J.; Shokat, K. M. Nat. Methods
2005, 2, 435–441.
(OD660z0.5) in liquid YPD were spread evenly on
each plate before four circular cellulose disks (0.6 cm dia-
meter) were gently laid on top of the plate. 10 mL of each
inhibitor solution (in DMSO at 2 mM) was subsequently
added to one cellulose disk. The images of the plates were
taken on an Alpha Innotech Imaging Center after 30-h incu-
bation at 30 ꢁC.
8. Chen, X.; Ye, H.; Kuruvilla, R.; Ramanan, N.; Scangos, K. W.;
Zhang, C.; Johnson, N. M.; England, P. M.; Shokat, K. M.;
Ginty, D. D. Neuron 2005, 46, 13–21.
Acknowledgements
9. Ventura, J. J.; Hubner, A.; Zhang, C.; Flavell, R. A.; Shokat,
K. M.; Davis, R. J. Mol. Cells 2006, 21, 701–710.
10. Hanke, J. H.; Gardner, J. P.; Dow, R. L.; Changelian, P. S.;
Brissette, W. H.; Weringer, E. J.; Pollok, B. A.; Connelly,
P. A. J. Biol. Chem. 1996, 271, 695–701.
We thank J. D. Blethrow for kindly providing the CDK2 pro-
teins and NIH for funding (AI44009).
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