Roscovitine-derived, dual-specificity inhibitors of cyclin-dependent kinases and casein kinases 1

Article abstract of DOI:10.1021/jm800109e

Cyclin-dependent kinases (CDKs) and casein kinases 1 (CK1) are involved in the two key molecular features of Alzheimer's disease, production of amyloid-β peptides (extracellular plaques) and hyper-phosphorylation of Tau (intracellular neurofibrillary tangles). A series of 2,6,9-trisubstituted purines, structurally related to the CDK inhibitor roscovitine, have been synthesized. They mainly differ by the substituent on the C-6 position. These compounds were screened for kinase inhibitory activities and antiproliferative effects. Several biaryl derivatives displayed potent inhibition of both CDKs and CK1. In particular, derivative 13a was a potent inhibitor of CDK1/cyclin B (IC₅₀: 220 nM), CDK5/p25 (IC₅₀: 80 nM), and CK1 (IC ₅₀: 14 nM). Modeling of these molecules into the ATP-binding pocket of CK1δ provided a rationale for the increased selectivity toward this kinase. 13a was able to prevent the CK1-dependent production of amyloid-β in a cell model. CDK/CK1 dual-specificity inhibitors may have important applications in Alzheimer's disease and cancers.

Full text of DOI:10.1021/jm800109e

J. Med. Chem. 2008, 51, 5229–5242
5229
Roscovitine-Derived, Dual-Specificity Inhibitors of Cyclin-Dependent Kinases and Casein
Kinases 1
Nassima Oumata,† Karima Bettayeb,‡ Yoan Ferandin,‡ Luc Demange,† Angela Lopez-Giral,† Marie-Lore`ne Goddard,†
Vassilios Myrianthopoulos,§ Emmanuel Mikros,§ Marc Flajolet,| Paul Greengard,| Laurent Meijer,*,‡ and Herve´ Galons*,†
Laboratoire de Chimie Organique 2, INSERM U 648, UniVersite´ Paris-Descartes, 4 aVenue de l’ObserVatoire, 75270 Paris cedex 06, France,
CNRS, Protein Phosphorylation & Human Disease Group, Station Biologique, B.P. 74, 29682 Roscoff cedex, Bretagne, France, Department of
Pharmacy, UniVersity of Athens, GR-15771 Athens, Greece, Laboratory of Molecular & Cellular Neuroscience, The Rockefeller UniVersity,
1230 York AVenue, New York, New York 10065-6399
ReceiVed February 2, 2008
Cyclin-dependent kinases (CDKs) and casein kinases 1 (CK1) are involved in the two key molecular features
of Alzheimer’s disease, production of amyloid-ꢀ peptides (extracellular plaques) and hyper-phosphorylation
of Tau (intracellular neurofibrillary tangles). A series of 2,6,9-trisubstituted purines, structurally related to
the CDK inhibitor roscovitine, have been synthesized. They mainly differ by the substituent on the C-6
position. These compounds were screened for kinase inhibitory activities and antiproliferative effects. Several
biaryl derivatives displayed potent inhibition of both CDKs and CK1. In particular, derivative 13a was a
potent inhibitor of CDK1/cyclin B (IC50: 220 nM), CDK5/p25 (IC50: 80 nM), and CK1 (IC50: 14 nM).
Modeling of these molecules into the ATP-binding pocket of CK1δ provided a rationale for the increased
selectivity toward this kinase. 13a was able to prevent the CK1-dependent production of amyloid-ꢀ in a cell
model. CDK/CK1 dual-specificity inhibitors may have important applications in Alzheimer’s disease and
cancers.
Introduction
human diseases (reviewed in ref 11) provides a growing impetus
for the discovery of selective CK1 inhibitors.12
Protein phosphorylation is catalyzed by protein kinases. The
human kinome comprises 518 kinase genes: 40 atypical and
478 classical protein kinases. These latter consist of 338 serine/
threonine kinases, 90 tyrosine kinases, and 50 sequences that
lack a functional catalytic site.1–4 Abnormalities in protein
phosphorylation have been observed in numerous major human
diseases, strongly encouraging the search for pharmacological
inhibitors of protein kinases.5–7
Our laboratory has focused its efforts on the search for,
optimization, and characterization of inhibitors of several
disease-relevant kinases, such as cyclin-dependent kinases
(CDKs), glycogen synthase kinases-3 (GSK-3),a and casein
kinases 1 (CK1). Inhibitors of CDKs have a great potential in
the treatment of pathological conditions such as solid tumors,
leukemia, neurodegenerative disorders (Alzheimer’s and Par-
kinson’s diseases, stroke, traumatic brain injury), renal diseases
(polycystic kidney disease, glomerulonephritis), pain, inflam-
mation, diabetes, and some viral diseases (reviewed in refs 8,
9). Inhibitors of GSK-3 are being studied for their potential use
in Alzheimer’s disease, manic depression, diabetes, etc., (re-
viewed in ref 10). CK1s are less studied as therapeutic targets,
but their implication in multiple physiological events and some
Purines represent a large family of biologically active
molecules and constitute the scaffold of a wide variety of
promising drugs, including kinase inhibitors (reviewed in refs
13–15). Among the large diversity of purines that have been
synthesized, 2,6,9-trisubstituted purines have generated consid-
erable interest as protein kinase inhibitors (reviewed in ref 16).
The most advanced molecule, roscovitine (or CYC202 or
Seliciclib),17–19 developed by Cyclacel Pharmaceuticals, is
currently in phase 2b clinical trials against non-small cell lung
cancer, in phase 2 against nasopharyngeal cancer, in phase 1
against various renal diseases (glomerulonephritis), and in
preclinical evaluation against polycystic kidney disease,20
Alzheimer’s disease (in preparation), stroke,21 and inflamma-
tion.22 The metabolism of roscovitine, which is orally available,
is well established. The main metabolic pathways are the
oxidation of the alcohol of the C-2 side chain into a carboxylate
group, the conjugation of a glucose or glucuronic acid residue
on this alcohol, and the loss of the isopropyl group on N9.23,24
Interestingly, the benzyl group has not been reported to be
affected by metabolism. Although frequently considered as
rather selective for some CDKs (CDK1, CDK2, CDK5, CDK7,
CDK9), roscovitine interacts with several other targets, although
with a lower affinity (DYRK1A, CK1, pyridoxal kinase).25–28
All of this information first suggests that the trisubstituted
purine motive of roscovitine remains an attractive scaffold to
develop new derivatives with reasonable chances of generating
useful bioactivity associated with moderate toxicity. Inhibitors
should be selected more on the basis of their inhibitory profile
on several key relevant kinases implicated in a particular
pathology rather than on potent inhibition of a single target.
For instance, in Alzheimer’s disease, hyperphosphorylation of
Tau is carried out by a small set of kinases, CDK5, GSK3, and
CK1 being the most important ones (reviewed in refs 29, 30).
In addition, amyloid-ꢀ formation from its ꢀ-amyloid precursor
* To whom correspondence should be addressed. For biochemistry: L.M.
phone +33.(0)2.98.29.23.39; fax, +33.(0)2.98.29.25.26; E-mail, meijer@
sb-roscoff.fr. For chemistry: H.G., phone +33.(0)1.53.73.96.84; fax,
+33.(0)1.43.29.05.92; E-mail, herve.galons@univ-paris5.fr.
†
Laboratoire de Chimie Organique 2, INSERM U 648, Universite´ Paris-
Descartes.
‡
CNRS, Protein Phosphorylation & Human Disease Group, Station
Biologique.
§
Department of Pharmacy, University of Athens.
Laboratory of Molecular & Cellular Neuroscience, The Rockefeller
|
University.
a Abbreviations: Aꢀ, amyloid ꢀ peptide; ꢀ-APP, ꢀ-amyloid precursor
protein; CDK, cyclin-dependent kinase; CK1, casein kinase 1; FCS, fetal
calf serum; GSK-3, glycogen synthase kinase-3; MTS, 3-(4,5-dimethylthi-
azol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H -tetrazolium.
10.1021/jm800109e CCC: $40.75
2008 American Chemical Society
Published on Web 08/13/2008

5230 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Oumata et al.
Scheme 1a
afforded the expected molecules. However, we failed to achieve
the nucleophilic substitution of the 6-chlorine with anilines
bearing a pyridyl group (Scheme 2). Reaction of intermediate
2 with 4-bromoaniline afforded the corresponding amine 9.
Suzuki coupling of boronic ester with bromoderivative 9 did
not lead to the expected biaryl but to compound 12. Reaction
of boranylanilines with 2 afforded the corresponding amines
10, which subsequently reacted with bromopyridines to afford
the expected biaryls 11. Finally, reaction with amino-alcohols
afforded the trisubstituted purines 13.
The synthesis of a large number of 2,6,9-trisubstituted purines,
including combinatorial methods, has been previously des-
cribed.46–50 Generally, the synthesis of 2,6,9-trisubstituted
purines start from commercially available 2,6-dichloropurine 1
and most authors start with the substitution on C6-position. We
found easier to achieve first the preparation of the key
intermediate 2. Regiospecific N9 alkylation was achieved by
precise control of the reaction temperature.
Biological Evaluation: Purified Protein Kinases. All syn-
thesized compounds were evaluated for their potential inhibitory
effects on purified kinases (CDK1/cyclin B, CDK5/p25, GSK-
3R/ꢀ, CK1δ/ε) as described in the Experimental Section. IC50
values were determined from dose-response curves and com-
pared to those of the reference molecules, (R)-roscovitine 8a
and (S)-roscovitine 8b (Tables 1, 2, and 3).
All three 6-aryl-substituted purines (6a, 6b, 6c) were com-
pletely inactive, confirming the essential hydrogen bond donat-
ing N6 present in all active purines.18,19,52 All other compounds
showed submicromolar activities on CDK1 and CDK5. The
substituent in C2-position had generally limited influence. The
most potent inhibitors were the biarylanilines series 13 with
the pyridine nucleus meta to the amine. In contrast, all
roscovitine analogues remained either inactive or poorly active
on GSK-3 (IC50 in the 2-50 µM range), despite the observation
that many CDK inhibitors are also excellent GSK-3 inhibitors.53
IC50 values for CDK1 and CDK5 were very similar, a likely
consequence of the close similarity of both kinases, especially
in their ATP-binding site.
a Reagents and conditions: (a) 2-bromopropane, K2CO3, DMSO 15-18
°C; (b) boronate, Na2CO3, Pd[P(C6H5)3]4, H2O, dioxane, 100 °C; (c) ArNH2
or ArCH2NH2, NEt3, BuOH 100 °C; (d) RNH2, NBu3, DMSO, 165 °C.
Results showed the appearance of significant CK1 inhibitory
activity among many derivatives or analogues of roscovitine
(Tables 1, 2, and 3), especially compounds 13a-13f. The most
potent CK1 inhibitors shared a biaryl substituent on N6. (R)-
DRF053 (13a) was the most potent inhibitor of CK1 (IC50: 0.014
µM), compared to (R)-roscovitine (8a) (IC50: 2.3 µM). In fact,
13a appears to be one of the most potent known inhibitor of
CK1.12
protein (ꢀ-APP) also involves CDK5,31 GSK-3,32,33 and CK1.34
Thus CDK1,35–37 CDK5,38,39 GSK-3,32,40–42 and CK134,43–45 all
appear to be involved in the two diagnostic features of
Alzheimer’s disease, amyloid-ꢀ production (responsible for
formation of amyloid plaques) and Tau hyperphosphorylation
(responsible for the formation of neurofibrillary tangles of paired
helical filaments). Multitarget inhibitors acting on this selection
of kinases could therefore become of great therapeutic value.
The aim of the present study was to synthesize and evaluate a
series of trisubstituted purines that mainly differed by the C-6
substituent. The prepared compounds were evaluated on the four
main kinases involved in Alzheimer’s disease, CDK1, CDK5,
GSK-3, and CK1. This study uncovered CDK/CK1 dual specificity
inhibitors, which should be evaluated for their potential therapeutic
applications, especially in the Alzheimer’s disease field.
Modeling of the CK1/Purine Interactions. The conversion
of (R)-roscovitine (8a) to C&R8 (8m) and then to DRF053 (13a)
(Figure 1) showed a gradual improvement of biological activity
toward CK1 (Table 1–3). Enhanced affinity of 13a was also
demonstrated for CDK1 and CDK5, however to a lesser degree.
To provide a structural rationale for this biological data, we
tried to locate and evaluate differences in the interaction mode
of these analogues with the two groups of kinases. A sequence
alignment of CDK1, CDK2, CDK5, and various isoforms of
CK1 (Figure 2) showed that there are several important
variations of residues located in the vicinity of the ATP binding
pocket between the CDKs and the CK1 isoforms. These
variations are located either at the ribose binding site or at the
Results and Discussion
Chemistry. Most of the trisubstituted purine derivatives
described herein were obtained following a three steps synthetic
route outlined in Scheme 1. Starting from commercially
available 2,6-dichloropurine 1, alkylation of N9 with 2-bro-
mopropane provided 2,6-dichloro-9-isopropylpurine 2. Displace-
ment of the 6-chloro was accomplished either by Suzuki cross-
coupling or by nucleophilic substitution with appropriate amines.
In the last step, nucleophilic substitution of the 2-chlorine
glycine loop. More specifically, at the ribose site Asp86CDK2
,
an important residue that in many cases interacts directly either
with the natural substrate (ATP-CDK2; 1B38) or with inhibitors
(roscovitine-CDK5; 1UNL) is replaced by a serine (Ser88CK1-
δ
). Moreover, Lys89CDK2 is replaced by an aspartate in the CK1

CDK/CK1 Dual-Specificity Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5231
Scheme 2a
a Reagents and conditions: (a) ArNH2, NEt3, BuOH 100 °C; (b) boronate, Na2CO3, Pd[P(C6H5)3]4, H2O, dioxane, 100 °C; (c) ArBr, Na2CO3, Pd[P(C6H5)3]4,
H2O, dioxane, 100 °C; (d) RNH2, NBu3, DMSO 165 °C.
isoforms (Asp91CK1-δ). Two additional residue variations appear
at the glycine loop: Glu8CDK2 is replaced by a basic residue,
arginine in CK1-R, -δ (Arg13CK1-δ), and -ε isoforms and lysine
in CK1-γ1, -γ2, and -γ3 isoforms. Lys20CDK2 is replaced by a
leucine in CK1 (Leu25CK1-δ).
Arg13CK1-δ in CK1 explains the fact that 8m does not
demonstrate an improved affinity for CDK1 and CDK5.
However, the increased binding affinity of 13a toward both CK1
and CDKs with respect to 8m could not be interpreted by
docking calculations but only by considering the alteration of
N6 stereoelectronic properties. The conversion of the 4-pyridi-
nylbenzyl substitution of 8m N6 to a 3-pyridinylphenyl in 13a
increases the hydrogen bonding potential of N6. This is caused
by the increased polarization of the N-H bond resulting from
the enhancement of the sp2 character of the N6 of 13a, which
carries two aromatic substitutions compared to analogue 8m.
An ab initio single-point energy calculation of 13a and 8m at
the B3LYP/6-31+G* level validated the aforementioned hy-
pothesis. The gap between the negative partial charge of nitrogen
and the positive charge of hydrogen is 1.14e for 13a and 0.58e
for 8m. The stronger hydrogen bonding potential of 13a N6H
is consistent with experimental data showing that N-methyla-
niline exhibits half of the hydrogen bond acidity compared to
diphenylamine.51 To reproduce this experimental result theoreti-
cally in our system and further examine the influence of
substitutions on the hydrogen bonding potential of secondary
amines, we have performed ab initio interaction energy calcula-
tions. The second-order Møller-Plesset perturbation theory
method was chosen in order to include electron correlation
effects and obtain accurate results. Interaction energy calcula-
tions of the complexes of diphenylamine-CK1 and
N-methylaniline-CK1 were performed. The results obtained at
the MP2/6-31+G* level showed that the interaction energy was
1.07 kcal/mol more favorable in the case of the diphenylamine-
receptor complex. The experimental ∆∆G between 13a and 8m,
which is in the range of 1.45-1.97 kcal/mol, is in very good
agreement with the above calculated values, considering the
approximations for the ab initio calculations (see Experimental
Section). These results suggest that the improved affinity of 13a,
where N6 carries two aromatic substituents, compared to that
of 8a, with only one aromatic substituent, could be mainly
Docking of 13a in CK1-δ resulted in two distinct binding
modes. In both of them, the usually observed adenine type
interaction between the ligand and the receptor was sustained
with purine N6 donating and N7 receiving a hydrogen bond
from the receptor backbone of the binding cavity (Figure
3A-B). However, two possible orientations of the 3-pyridi-
nylphenylamino moiety were obtained. The first binding mode
is characterized by a mixed hydrogen bond stacking interaction
of pyridine with the side chain of Arg16 of the glycine loop.
The pyridine nitrogen forms a hydrogen bond with the guanidine
moiety, while the phenyl ring is oriented parallel to Arg
guanidinium, stabilized through a π-π interaction (Figure 3B).
In the second binding mode, the phenylamino N6-C1′ bond
demonstrates a ∼60° rotation, which orients the 3-pyridinylphe-
nylamino group toward the C-terminal lobe where it forms
hybrophobic interactions with Pro87CK1-δ and stacking with
Phe95CK1-δ. Docking of 8m in CK1-δ (Figure 3C) resulted in
two binding poses that were similar to the structures, which
resulted in the case of 13a-CK1-δ complex.
The favorable interaction of pyridine with Arg13CK1-δ
proposed by the first binding mode of both 13a and 8m, which
does not exist in the case of roscovitine 8a (Figure 3D), could
provide an explanation of the improved affinity of 3- and
4-pyridinyl substituted analogues with respect to CK1. In the
case of CDKs, the corresponding residue of Arg13CK1-δ is a
conserved acidic residue (Glu8CDK1, 2, 5) with no capacity to
act either as a hydrogen bond donor or as a stacking partner for
pyridine. A 2-aromatic substituted pyridine (calculated pKa 4.25)
can accommodate mainly π-π aromatic stacking and cation-π
interactions or hydrogen bonds where pyridine nitrogen serves
as hydrogen bond acceptor (Figure 3B-C). The absence of an
interaction in the CDKs similar to that between pyridine and

5232 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Oumata et al.
Table 1. Effects of Purines 6a-8h on Four Protein Kinases and on the Survival of SH-SY5Y and HEK293 Cellsa
compd
Ar
R
CDK1
CDK5
GSK-3R/ꢀ
CK1
SH-SY5Y
HEK293
6a
6b
6c
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
phenyl
3-pyridyl
4-pyridyl
phenyl-2 -phenyl
phenyl-3-phenyl
phenyl-4-phenyl
phenyl-3-phenyl
phenyl-4-phenyl
phenyl-3-phenyl
phenyl
phenyl
2-pyridyl
2-pyridyl
3-pyridyl
3-pyridyl
4-pyridyl
4-pyridyl
phenyl-3-phenyl
phenyl-3-(2-pyridyl)
phenyl-3-(3-pyridyl)
phenyl-3-(4-pyridyl)
phenyl-4-(2-pyridyl)
phenyl-4-(2-pyridyl)
phenyl-4-(2-pyridyl)
phenyl-4-(3-pyridyl)
phenyl-4-(3-pyridyl)
phenyl-4-(4-pyridyl)
phenyl-4-phenyl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-3-methylbut-2-yl
(R)-1-hydroxy-3-methylbut-2-yl
1-hydroxy-2-methylprop-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(S)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(S)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(S)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(S)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-3-methylbut-2-yl
1-hydroxy-2-methylprop-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-3-methylbut-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
>100
>100
>100
29
>100
>100
>100
17
0.4
0.1
>100
>100
>100
>100
>100
90
>100
50
>10
>10
-
14.5
>33
>10
>10
>10
>10
>100
3.3
>10
58
12
10
>50
>50
>10
>50
0.05
0.08
0.6
0.048
0.6
2.3
6.0
7
13
2.2
4.1
2.0
2.2
1.1
>100
>100
>100
29
37.6
26.8
11
28.6
11.6
17.5
-
165.1
-
7.7
-
64.8
-
2.4
3.3
7.4
6.5
0.43
0.76
0.94
1.25
0.74
1.69
0.75
>100
>100
>100
6.6
-
-
15.6
-
16.8
46.7
-
190
-
20.5
-
1.1
0.23
0.05
0.28
0.09
0.35
0.55
0.74
1.15
0.13
0.13
0.33
0.39
3.2
0.25
0.12
1.6
0.13
0.08
0.21
1.9
0.02
0.08
0.05
0.20
0.25
1.00
1.70
0.16
0.14
0.23
0.41
2.60
0.2
0.08
1.6
0.13
0.08
0.21
2.0
73.7
-
2.5
2.8.
5.2
20
0.9
0.9
1.2
0.6
8m
8n
8o
8p
8q
8r
8s
0.56
0.90
-
0.6
16
84
6
35.5
>100
0.68
0.73
0.7
1.1
0.8
2.2
0.9
2.3
3.0
0.9
2.2
2.52
0.76
1.54
1.33
a Purines were tested at various concentrations on CDK1/cyclin B, CDK5/p25, GSK-3R/ꢀ, and CK1δ/ε, as described in Experimental Section. IC50
values, calculated from the dose-response curves, are reported in µM. The compounds were tested at various concentrations for their effects on SH-SY5Y
and HEK293 cell survival after 48 h incubation as estimated using the MTS reduction assay. IC50 values, calculated from the dose-response curves, are
reported in µM. -: not tested. When IC50 values are reported as >10, 33, or 100 µM, this indicates that the compound did not display any inhibitory activity
at the highest concentration tested (10, 33, or 100 µM). Some compounds were essentially inactive at 10 µM (indicated as >10 µM), they were not tested
at higher concentrations. Some displayed a small but significant inhibition at this concentration and were thus tested up to 100 µM, when solubility was not
an issue. 8a, (R)-roscovitine; 8b, (S)-roscovitine; 8m, (R)-C&R8.
attributed to the enthalpic gain offered by the hydrogen bond
formed between N6 and the protein backbone carbonyl.
The dual CDK-CK1 specificity character gained by ana-
logues 13a and 8m with respect to roscovitine can be considered
as a combined result of the favorable interactions provided by
the 3- or 4-pyridinyl substitution toward CK1 and the enhanced
hydrogen bonding potential of N6 carrying two aromatic
substituents toward both CDK and CK1 receptors.
Biological Evaluation: Cell Proliferation Assays. All
compounds were tested for their antiproliferative effects. Human
neuroblastoma SH-SY5Y and human embryonic kidney cells
were exposed for 48 h to various concentrations of each
compound. Viability was then estimated by the MTS reduction
assay as described in the Experimental Section. The three 6-aryl-
substituted purines (6a, 6b, 6c) and compounds 8c and 12a were
completely inactive. All other compounds displayed significant
cell death inducing activity, the IC50 values ranging from 0.43
to 65 µM.
line, IC50 values were 0.43 and 0.76 µM, respectively), were
far from being the most potent kinase inhibitors. Further, another
interesting aspect of the SAR studies was the observation that
the replacement of an heterobiaryl moiety in 8m and 8n by a
biphenyl in 8s reduced the inhibitory potency on CDK1 and
CDK2 (IC50 >2 µM). Nevertheless, a potent antiproliferative
effect was observed with this compound.
Altogether, the synthesized compounds can be divided in three
groups in terms of biological activity (kinase and cell survival):
(1) Inactive on both kinases and cell proliferation: compounds
6a-6c.
(2) Active on both kinases and cell proliferation: the
biarylmethylamine series (compounds 8i-8s) demonstrated high
antiproliferative action, which contrasted with variable enzyme
inhibition. No clear correlation could be established between
kinase inhibition and antiproliferative effects. Different cellular
properties (cell permeability, intracellular stability, intracellular
distribution, or targets) may account for this lack of direct
correlation. The 8i-8s series of molecules could be developed
as potential anticancer agents. As an illustration, we have
investigated in detail the cellular effects of the most active
compound, 8m (C&R8).54
Structure-activity relationships based on CDKs and CK1
inhibition were difficult to correlate with the antiproliferative
effects determined on both cell lines. For example, biaryl-
methyamines compounds (e.g., compounds 8m and 8n), which
displayed the highest antiproliferative effects (on SH-SY5Y cell

CDK/CK1 Dual-Specificity Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5233
Table 2. Effects of Purines 12 on Four Protein Kinases and on the
at purine position 6 that can interact favorably with an arginine
(or alternatively a lysine) located in the glycine loop of CK1R,
γ, δ, and ε isoforms takes advantage of the conserved variation
of this residue in the CDKs and inhibitors like 8m gain 1 order
of magnitude in potency with respect to CK1 compared to
roscovitine. Further improvement of potency but in a common
trend in both families of kinases is achieved by the transforma-
tion of the biarylmethylamine substituent of purine position 6
to a biarylamine. The amine group attached at position 6 acts
as a hydrogen bond donor that stabilizes the protein-inhibitor
complex. The bond formed in the case of the biarylamines is
stronger than that formed in the case of the biarylmethylamine
analogues. The stronger bonding potential of biarylamines like
13a was confirmed with a high level ab initio interaction energy
calculation.
Survival of SH-SY5Y and HEK293 Cellsa
compd
Ar
CDK1 CDK5 GSK-3R/ꢀ CK1 SH-SY5Y HEK293
12a
12b
3-pyridyl 0.41 0.73
4-pyridyl 0.59 0.6
>100
>100
0.091 >100
0.083 60
-
-
a Purines were tested at various concentrations on CDK1/cyclin B, CDK5/
p25, GSK-3R/ꢀ, and CK1δ/ε, as described in Experimental Section. IC50
values, calculated from the dose-response curves, are reported in µM. The
compounds were tested at various concentrations for their effects on SH-
SY5Y and HEK293 cell survival after 48 h incubation as estimated using
the MTS reduction assay. IC50 values, calculated from the dose-response
curves, are reported in µM. -: not tested. When IC50 values are reported
as >10, 33, or 100 µM, this indicates that the compound did not display
any inhibitory activity at the highest concentration tested (10, 33, or 100
µM). Some compounds were essentially inactive at 10 µM (indicated as
>10 µM), they were not tested at higher concentrations. Some displayed a
small but significant inhibition at this concentration and were thus tested
up to 100 µM, when solubility was not an issue.
As both CK1 and CDK families of kinases are involved in
the development of Alzheimer’s disease, these modifications
on the 2,6,9-trisubstituted purines deserve to be further explored
in terms of both pharmacological tools for the study of
neurodegeneration pathways in cellular and animal models and
possibly for potential therapeutic applications.
Experimental Section
Chemistry. General Methods. Melting points were determined
on a Kofler hot-stage (Reichert) and are uncorrected. NMR spectra
were recorded on Bruker Avance 400 MHz (100 MHz for 13C
NMR). Chemical shifts are given in ppm downfield of tetrameth-
ylsilane (TMS) used as an internal standard. Infrared spectra were
recorded on a FTIR Schimadzu 8300. Reactions were monitored
by TLC using Merk silica gel 60F-254 thin layer plates. Column
chromatography was carried out on SDS Chromagel 60 ACC,
40-63 µm. The HPLC analyses were carried out on a system
consisting of a Waters 600 system controller, a Jones Chromatog-
raphy heater-chiller oven, and a Waters 994 photodiode array
detector. Microanalysis (C, H, N) of compounds 6, 7, 8, and 13
agreed ((0.4) with calculated values. Biarylamines were prepared
by Suzuki-Myaura coupling.24 Results of compounds microanaly-
sis are presented in the Supporting Information.
2,6-Dichloro-9-iso-propylpurine (2). To a solution of 2,6-
dichloro-9H-purine 1 (9.00 g, 47.6 mmol) in DMSO (50 mL) at
15 °C was added K2CO3 (19.74 g, 142.8 mmol) and 2-bromopro-
pane (21.85 mL, 238 mmol). After 8 h stirring at 15-18 °C, water
was added and the solution was extracted with AcOEt. The organic
layers were assembled, washed with H2O (3 × 20 mL), dried
(Na2SO4), concentrated, and chromatographed on a silica gel column
(Cyclohexane/AcOEt 1:9) to yield a white solid (7.80 g, 71%); mp
149-152 °C. 1H NMR (CDCl3): δ 1.65 (d, 6H, J ) 6.8 Hz,
CH(CH3)2), 4.91 (hept, 1H, J ) 6.8 Hz, CH(CH3)2), 8.17 (s, 1H,
H-8).
General Procedure for the Synthesis of 6-Aryl-2-chloropu-
rines (3). To solution of 2 (2.31 g, 10 mmol) in 40 mL dioxane
under argon were added Pd[P(C6H5)3]4 (0.575 g, 0.5 mmol). After
5 min stirring under N2, 1 M Na2CO3 aqueous solution (20 mL)
and pinacolboronate derivatives (11 mmol) were added. The
reaction mixture was refluxed under N2 for 6 h and then cooled to
room temperature. The mixture was concentrated in vacuo to half
of its initial volume and extracted with AcOEt (3 × 15 mL). The
combined organic extracts were washed with H2O and brine and
then dried (Na2SO4). The solvent was removed in vacuo. Com-
pounds 3 crystallized upon trituration with cyclohexane.
(3) Active on kinases but poor antiproliferative activity: the
biarylamine type compounds (series 13) were highly potent
CDK/CK1 inhibitors but displayed low antiproliferative effect.
As a general rule, CK1 inhibition inversely correlated with
antiproliferative activity. This series of molecules could be
developed as protective agents, especially against neurodegen-
erative diseases, as illustrated below with compound 13a.
Biological Evaluation: Inhibition of Amyloid-ꢀ Production.
Recently, CK1 was demonstrated to regulate the production of
amyloid-ꢀ (Aꢀ) formation and CK1 inhibitors to prevent Aꢀ
production.34 To test whether our molecules could act as CK1
inhibitors in a cellular system, we exposed N2A cells stably
expressing amyloid-ꢀ precursor protein (APP) (N2A-APP695
cells) to various concentrations of the selection of three
compounds, (R)-roscovitine (8a), (R)-C&R8 (8m), and (R)-
DRF053 (13a), for a 3 h period. Cell supernatants were then
collected and A-ꢀ40 levels measured by an ELISA assay.
Results show a dose-dependent inhibitory effect on A-ꢀ40
production for the three compounds, (R)-DRF053 (13a) being
the most efficient (Figure 4). Although these results might
suggest that the three compounds are able to inhibit CK1 in a
cellular context, leading to reduced production of amyloid-ꢀ
from the amyloid-ꢀ precursor protein, this needs to be formally
demonstrated and there are alternative explanations, including
an effect through other targets (kinases and non-kinases). The
use of chemically different CK1 inhibitors, specific siRNAs,
and compounds that do not affect other kinases than CK1, will
shed some light on the link between CK1 and amyloid-ꢀ
production.
Conclusion
2-Chloro-6-phenyl-9-iso-propylpurine (3a). Yield 87%, mp
112-113 °C. 1H NMR (CDCl3): δ 1.66 (d, 6H, J ) 6.8 Hz,
CH(CH3)2), 4.97 (hept, 1H, CH(CH3)2), 7.54-7.67 (m, 3H, Hphenyl),
7. 75-7.90 (m, 2H, Hphenyl), 8.17 (s, 1H, H-8).
2-Chloro-6-(3-pyridyl)-9-iso-propylpurine (3b). Yield 53%, mp
163-164 °C. 1H NMR (CDCl3): δ 1.66 (d, 6H, J ) 6.8 Hz,
CH(CH3)2), 4.97 (hept, 1H, CH(CH3)2), 7.30 (m, 1H, Hpyridyl), 7.90
(m, 2H, Hpyridyl), 8.22 (s, 1H, H-8), 8.65 (d, 1H, Hpyridyl).
In this study, the introduction of a biarylamine or a biaryl-
methylamine on the 6 position of the purine system led to potent
inhibitors of CDKs. Unexpectedly, some compounds displayed
potent inhibition of CK1 as well. Docking calculations suggested
that potency enhancement toward the CDKs and CK1 result
from interactions between the protein binding pocket and two
separate parts of the inhibitor molecule. Introducing a substituent

5234 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Oumata et al.
Table 3. Effects of Purines 13 on Four Protein Kinases and on the Survival of SH-SY5Y and HEK293 Cellsa
compd
Ar
R
CDK1
CDK5
GSK-3R/ꢀ
CK1
SH-SY5Y
HEK293
13a
13b
13c
13d
13e
13f
13g
13h
13i
phenyl-3-(2-pyridyl)
phenyl-3-(2-pyridyl)
phenyl-3-(2-pyridyl)
phenyl-3-(3-pyridyl)
phenyl-3-(3-pyridyl)
phenyl-3-(4-pyridyl)
phenyl-4-(2-pyridyl)
phenyl-4-(2-pyridyl)
phenyl-4-(3-pyridyl)
phenyl-4-(3-pyridyl)
phenyl-4-(4-pyridyl)
phenyl-4-(4-pyridyl)
(R)- 1-hydroxy-but-2-yl
0.22
0.18
0.48
0.18
0.05
0.18
0.05
0.06
0.18
0.18
0.02
0.03
0.08
0.08
0.16
0.05
0.01
0.05
0.03
0.05
0.02
0.02
0.02
0.02
4.1
5.1
4.9
60
13
20
40
90
7.5
8.0
1.9
12
0.014
0.031
0.020
0.05
0.08
0.11
0.13
0.4
0.22
0.63
0.23
1.2
17.2
16.8
16.0
15.4
7.5
15.8
10
17.5
6.5
–
–
–
–
9.2
11.6
10.4
14.6
7.4
8.3
8
(R)-1-hydroxy-3-methylbut-2-yl
1-hydroxy-2-methylprop-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-3-methylbut-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-3-methylbut-2-yl
(R)-1-hydroxy-but-2-yl
13j
13k
13l
(R)-1-hydroxy-3-methylbut-2-yl
(R)-1-hydroxy-but-2-yl
(R)-1-hydroxy-3-methylbut-2-yl
8.9
7
6.6
6.3
a Purines were tested at various concentrations on CDK1/cyclin B, CDK5/p25, GSK-3R/ꢀ, and CK1δ/ε, as described in Experimental Section. IC50
values, calculated from the dose-response curves, are reported in µM. The compounds were tested at various concentrations for their effects on
SH-SY5Y and HEK293 cell survival after 48 h incubation as estimated using the MTS reduction assay. IC50 values, calculated from the dose-response
curves, are reported in µM. -: not tested. When IC50 values are reported as >10, 33, or 100 µM, this indicates that the compound did not display any
inhibitory activity at the highest concentration tested (10, 33, or 100 µM). Some compounds were essentially inactive at 10 µM (indicated as >10
µM), they were not tested at higher concentrations. Some displayed a small but significant inhibition at this concentration and were thus tested up to
100 µM, when solubility was not an issue.
2-Chloro-6-(4-phenylphenylamino)-9-iso-propylpurine (4c).
1
Yield 65%, mp 123-126 °C. H NMR (CDCl3): δ 1.54 (d, 6H, J
) 6 Hz, CH(CH3)2), 4.82 (hept, 1H, CH(CH3)2), 7.25-7.82(m, 10H,
H
phenyl), 8.10(s, 1H, H-8).
2-Chloro-6-[(2-pyridyl)methylamino]-9-iso-propylpurine (5a).
1
Yield 45%, mp 67-69 °C. H NMR (CDCl3): δ 1.49 (d, 6H, J )
6.6 Hz, CH(CH3)2), 4.55 (hept, 1H, CH(CH3)2), 4.88 (bs, 2H,
NHCH2), 6.53(bs, 1H, NH), 7.10 (dd, 1H, J ) 7.6 Hz and J′ ) 5.0
Hz, Hpyridyl), 7.29 (d, 1H, Hpyridyl), 7.47 (s, 1H, H-8), 7.54 (td, 1H,
J ) 7.6 Hz and J′ ) 1.8 Hz, Hpyridyl), 8.48 (dd, 1H, Hpyridyl).
2-Chloro-6-[(3-pyridyl)methylamino]-9-iso-propylpurine (5b).
Yield 56%, mp 184-186 °C. H NMR (CDCl3): δ 1.58 (d, 6H, J
) 6.8 Hz, CH(CH3)2), 4.83 (hept, 1H, CH(CH3)2), 4.85-4.90 (m,
2H, NHCH2), 6.22 (bs, 1H, NH), 7.24-7.29 (m, 1H, Hpyridyl) 7.73
(m, 1H, Hpyridyl), 7.78 (s, 1H, H-8), 8.54 (m, 1H, Hpyridyl), 8.66 (bs,
1H, Hpyridyl).
Figure 1. Structure of (R)-roscovitine (8a), (R)-C&R8 (8m), and
DRF053 (13a). IC50 values for CDK5/p25 and CK1 are indicated under
each structure in µM.
1
2-Chloro-6-(4-pyridyl)-9-iso-propylpurine (3c). Yield 76%, mp
145-148 °C. 1H NMR (CDCl3): δ 1.66 (d, 6H, J ) 6.8 Hz,
CH(CH3)2), 4.97 (hept, 1H, CH(CH3)2), 8.22 (s, 1H, H-8), 8.63
(bd, 2H, J ) 5.5 Hz, Hpyridyl), 8.82 (bd, 2H, Hpyridyl).
2-Chloro-6-[(4-pyridyl)methylamino]-9-iso-propylpurine (5c).
1
Yield 77%, mp 120-122 °C. H NMR (CDCl3): δ 1.58 (d, 6H, J
) 6.8 Hz, CH(CH3)2), 4.84 (hept, 1H, CH(CH3)2), 4.85-4.91 (m,
2H, NHCH2), 6.25 (bs, 1H, NHCH2), 7.29 (d, 2H, Hpyridyl), 7.80 (s,
1H, H-8), 8.56 (d, 2H, J ) 5.6 Hz, Hpyridyl).
General Procedure for the Synthesis of 6-Amino-2-chloropu-
rines (Preparation of Compounds 4, 5, 9, 10). To a solution of 2
(2.31 g, 10 mM) in 15 mL n-BuOH was added the primary amine
(12 mM) and NEt3 (2.20 mL, 16 mM). After 2 h heating at 100
°C, n-BuOH was evaporated in vacuo. To the residue, 10 mL water
were added and this mixture was extracted with AcOEt (3 × 20
mL). The combined organic extracts were dried (Na2SO4) and the
solvent was removed in vacuo. The residue was chromatographied
on a silica gel column using (CH2Cl2/AcOEt, various ratio) as
eluent.
2-Chloro-6-[3-(phenyl)phenylmethylamino]-9-iso-propylpu-
rine (5d). Yield 45%, mp 105-107 °C. 1H NMR (CDCl3): δ 1.35
(d, 6H, J ) 6.7 Hz, CH(CH3)2), 4.60 (hept, 1H, CH(CH3)2), 4.80
(bs, 2H, NHCH2), 7.26-7.49 (m, 10H, Hphenyl), 7.82 (s, 1H, H-8).
2-Chloro-6-[4-(phenyl)phenylmethylamino]-9-iso-propylpu-
1
rine (5e). Yield 32%, mp 98-101 °C. H NMR (CDCl3): δ 1.57
(d, 6H, J ) 6.7 Hz, CH(CH3)2) 4.64 (hept, 1H, J ) 6.8 Hz,
CH(CH3)2), 4.82 (bs, 2H, NHCH2), 6.65 (brs, 1H, NH); 7.30-7.34
(m, 1H, Hphenyl), 7.44 (t, 2H, J ) 8 Hz, Hphenyl), 7.52-7.63 (m, 5H,
Hphenyl + H-8), 7.81-7.84 (d, 2H, J ) 8.8 Hz, Hphenyl).
2-Chloro-6-(2-phenylphenylamino)-9-iso-propylpurine (4a).
1
Yield 56%, mp 68 °C. H NMR (CDCl3): δ 1.66 (d, 6H, J ) 6.8
Hz, CH(CH3)2), 4.97 (hept, 1H, CH(CH3)2), 6.90-7.47(m, 10H,
H
phenyl), 8.22(s, 1H, H-8).
2-Chloro-6-[3-(2-pyridyl)phenylmethylamino]-9-iso-propylpu-
rine (5f). Yield 44%, mp 120-122 °C. H NMR (CDCl3): δ 1.34
(d, 6H, J ) 6.8 Hz, CH(CH3)2), 4.78 (hept, 1H, CH(CH3)2); 4.86
(bs, 2H, NHCH2), 5.80 (brs, 1H, NH); 7.23 (m, 2H, Hpyridyl), 7.80
2-Chloro-6-(3-phenylphenylamino)-9-iso-propylpurine (4b).
1
1
Yield 75%, mp 117-119 °C. H NMR (CDCl3): δ 1.66 (d, 6H, J
) 6.8 Hz, CH(CH3)2), 4.97 (hept, 1H, CH(CH3)2), 6.90-7.35 (m,
10H, Hphenyl), 8.22 (s, 1H, H-8).

CDK/CK1 Dual-Specificity Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5235
Figure 2. An alignment of the sequences of CDK1, 2, and 5 and five isoforms of CK1. Identical residues are enclosed in red-filled boxes
and similar residues in blue-bordered boxes. The red arrow indicates Arg13CK1-δ. This position is occupied by a basic residue, arginine or
lysine, in all CK1 isoforms, while in the CDKs, it is a glutamate. A favorable interaction between this residue and the pyridinyl ring of
C&R8 (8m) and 13a in CK1, and its absence in the CDKs could account for the specificity gain of the latter two analogues with respect to
roscovitine for CK1.
(dd, 1H, Hpyridyl), 7.38-7.65 (m, 5H, Hphenyl + H-8), 8.63 (d, 1H,
J ) 2.5 Hz, Hpyridyl).
1H, Hphenyl), 7.86 (dd, 1H, J ) 1.6 and J′ ) 8 Hz, Hpyridyl), 8.59
(dd, 1H, J ) 1.6 Hz and J′ ) 4.8 Hz, Hpyridyl), 8.85 (d, 1H, J ) 2.4
Hz, Hpyridyl).
2-Chloro-6-[3-(3-pyridyl)phenylmethylamino]-9-iso-propylpu-
rine (5g). Yield 65%, mp 115-117 °C. 1H NMR (CDCl3): δ 1.55
(d, 6H, J ) 6.8 Hz, CH(CH3)2), 4.81 (hept, 1H, CH(CH3)2), 4.90
(d, 2H, NHCH2), 6.66 (bs, 1H, NH), 7.30 (dd, 1H, J ) 2.8, 8 Hz,
2-Chloro-6-[3-(4-pyridyl)phenylmethylamino]-9-iso-propylpu-
rine (5h). Yield 48%, mp 145-148 °C. 1H NMR (CDCl3): δ
1.58 (d, 6H, J ) 6.8 Hz, CH(CH3)2), 4.79 (hept, 1H, CH(CH3)2),
4.85 (bs, 2H, NHCH2), 6.59 (bs, 1H, NH), 7.20-7.49 (d, 2H, J
Hpyridyl), 7.41-7.52 (m, 3H, Hphenyl), 7.61 (s, 1H, H-8), 7.69 (s,

5236 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Oumata et al.
Figure 3. (A) Overall structure of CK1-δ complexed with analogue 13a (magenta). The kinase is depicted as a ribbon and colored according
to its secondary structure (red: R helix; cyan: beta-sheet; gray: random coil). (B) Analogue 13a docked in the ATP-binding pocket of CK1-δ.
The hydrogen bonds formed between the ligand and the kinase backbone as well as with the side chain of Arg13 are depicted as yellow
dashed lines. The gatekeeper residue Met82 near the hydrophobic cavity of the pocket is also visible. (C) Analogue 8m docked in the
ATP-binding pocket of CK1-δ. Same representation as in (B). (D) (R)-Roscovitine 8a docked in the ATP-binding pocket of CK1-δ. While
the two hydrogen bonds formed with the receptor backbone are present, Arg13 does not interact with the ligand and adopts a different
orientation.
) 8.4 Hz, Hphenyl) 7.65(dd, 2H, pyridyl), 7.76 (s, 1H, 8-H), 7.98
(d, 2H, J ) 8.4 Hz, Hphenyl), 8.79(dd, 2H, Hpyridyl).
Amination by Nucleophilic Substitution of the 2 Chlorine.
Preparation of 6, 7, 8, and 13. A mixture of compound 3, 4, 5, or
11, (2 mmol), NBu3 (10 mmol), amino-alcohol (10 mmol), and
DMSO 0.5 mL was heated at 165 °C for 3 h. After cooling to rt,
5 mL of H2O was added and the mixture was extracted with AcOEt
(3 × 10 mL). The residue was chromatographied using AcOEt-
EtOH-NEt3 as eluent.
2-Chloro-6-[4-(2-pyridyl)phenylmethylamino]-9-iso-propylpu-
rine (5i). Yield 56%, mp 176-179 °C. 1H NMR (CDCl3): δ 1.58
(d, 6H, J ) 6.8 Hz, CH(CH3)2), 4.79 (hept, 1H, CH(CH3)2), 4.85
(bs, 2H, NHCH2), 6.59 (bs, 1H, NHCH2), 7.20-7.23 (m, 1H,
H
H
pyridyl), 7.49 (d, 2H, J ) 8 Hz, Hphenyl), 7.73-7.71 (m, 2H,
pyridyl), 7.79 (s, 1H, H-8), 7.98 (d, 2H, Hphenyl), 8.71 (d, 1H, J
(R)-2-(1-Hydroxy-but-2-ylamino)-6-phenyl-9-iso-propylpurine
1
) 4.8 Hz, Hpyridyl).
(6a). Yield 56%, mp 100-104 °C. H NMR (CDCl3): δ 1.01 (t,
3H, J ) 7.4 Hz, CH3CH2), 1.53 (d, 6H, J ) 6.6 Hz, CH(CH3)2),
1.71 (m, 2H, CH3CH2), 3.62 (dd, 1H, J ) 10,3 Hz and J′ ) 7.5
Hz, CH2OH), 3.75 (dd, 1H, J ) 10.3 Hz and J′ ) 2.4 Hz, CH2OH),
4.60 (hept, 1H, CH(CH3)2), 7.48-7.66(m, 3H, Hphenyl), 7. 75-7.92
(m, 2H, Hphenyl), 8.22 (s, 1H, H-8).
2-Chloro-6-[4-(3-pyridyl)phenylmethylamino]-9-iso-propylpu-
1
rine (5j). Yield 68%, mp 158-160 °C. H NMR (CDCl3): δ 1.56
(d, 6H, J ) 7.2 Hz, CH(CH3)2), 4.84 (hept, 1H, CH(CH3)2), 4.88
(bs, 2H, NHCH2), 7.37 (dd, 1H, J ) 2.8 Hz and J′ ) 7.6 Hz,
H
H
pyridyl), 7.52 (d, 2H, J ) 8.4 Hz, Hphenyl), 7.57 (d, 2H, J ) 8.4 Hz,
phenyl), 7.80 (s, 1H, H-8), 7.82 (dt, 1H, J ) 1.6 Hz and J′ ) 8.4
(R)-2-(1-Hydroxy-but-2-ylamino)-6-(3-pyridyl)-9-iso-propylpu-
1
Hz, Hpyridyl), 8.60 (d, 1H, J ) 3.6 Hz, Hpyridyl), 8.84 (d, 1H, J )
rine (6b). Yield 42%, mp 98-100 °C. H NMR (CDCl3): δ 0.99
1.6, Hpyridyl).
(t, 3H, J ) 7.4 Hz, CH3CH2), 1.54 (d, 6H, J ) 6.9 Hz, CH(CH3)2),
1.72 (m, 2H, CH3CH2), 3.66 (dd, 1H, J ) 6.6 Hz and J′ ) 10.8
Hz, CH2OH), 3.83 (dd, 1H, J ) 3.2 Hz and J′ ) 10.8 Hz, CH2OH),
4.02 (m, 1H, CHNH), 4.66 (hept, 1H, CH(CH3)2), 5.20 (d, 1H, J
) 6.5 Hz, CHNH), 7.35 (dd, 1H, J ) 4.8 Hz and J′ ) 8.1 Hz,
2-Chloro-6-[4-(4-pyridyl)phenylmethylamino]-9-iso-propylpu-
rine (5k). Yield 56%, mp 127-132 °C. 1H NMR (CDCl3): δ 1.53
(d, 6H, J ) 7 Hz, CH(CH3)2), 4.84 (hept, 1H, CH(CH3)2), 4.88
(bs, 2H, NHCH2), 7.52 (d, 2H, J ) 8.4 Hz, Hphenyl), 7.57 (d, 2H,
phenyl), 7.80 (s, 1H, H-8), 7.62 (dd, 2H, Hpyridyl), 8.82 (dd, 2H,
pyridyl).
H
H
H
pyridyl), 7.78 (s, 1H, H-8), 8.64 (d, 1H, J ) 4.5 Hz, Hpyridyl), 8.88
(d, 1H, J ) 7.8 Hz, Hpyridyl), 9.73 (s, 1H, Hpyridyl). 13C NMR: δ 9.8,

CDK/CK1 Dual-Specificity Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5237
Figure 4. Dose-dependent inhibition of amyloid-ꢀ production in N2A-APP695 cells by (R)-roscovitine (8a), (R)-C&R8 (8m), and (R)-DRF053
(13a). N2A cells stably expressing APP-695 were incubated in the absence or presence of various concentrations of (R)-roscovitine (8a),
(R)-C&R8 (8m), and (R)-DRF053 (13a). Cell supernatants were collected after 3 h of incubation and subjected to Aꢀ40 ELISA assays. Data
from at least three experiments (mean ( SEM) were compared (GraphPad Prism v4.0) with the control conditions (DMSO). (*: P < 0.05;
**: P < 0.01; ***: P < 0.001; one-tailed Student’s t test, 95% significance level).
21.3, 21.4, 23.9, 45.7, 54.8, 65.6, 122.3, 124.8, 130.8, 135.8, 138.5,
149.6, 150, 152, 152.8, 158.4.
3.72-3.77 (m, 1H, CH2OH), 3.87-3.93 (m, 2H, CH2OH and
CHNH), 4.64 (hept, 1H, NCH(CH3)2), 5.01 (d, 1H, J ) 5.1 Hz,
CHNH), 7.30-7.34 (m, 1H, Hphenyl), 7.44 (t, 2H, J ) 8 Hz, Hphenyl),
7.59-7.61 (m, 5H, 8-H + Hphenyl), 7.67 (s, 1H, NHphenyl), 7.81-7.84
(d, 2H, J ) 8.8 Hz, Hphenyl).
2-(1-Hydroxy-2-methylprop-2-ylamino)-6-[3-(phenyl)-pheny-
lamino]-9-iso-propylpurine (7e). Yield 27%, mp 80-85 °C. 1H
NMR (CDCl3): δ 1.40 (s, 6H, (HOCH2CCH3)2), 1.59 (d, 6H, J )
6.8 Hz, NHCH(CH3)2), 3.72 (d, 2H, J ) 6 Hz, CH2OH), 4.61 (hept,
1H, NCH(CH3)2), 7.14-7.75 (m, 10H, Hphenyl). 13C NMR: δ 22.4
(×2), 25.1 (×2), 46.9, 55.4, 72.3, 115.0, 119.2, 119.5, 122.3, 127.2
(×2), 127.3 (×2), 128.6, 129.2, 135.2, 139.0, 141.0, 142.0, 149.6,
152.6, 158.4.
(R)-2-(1-Hydroxy-but-2-ylamino)-6-(4-pyridyl)-9-iso-propylpu-
rine (6c). Yield 58%, mp 136-138 °C. 1H NMR (CDCl3): δ 0.99
(t, 3H, J ) 7.4 Hz, CH3CH2), 1.54 (d, 6H, J ) 6.8 Hz, CH(CH3)2),
1.77-1.31 (m, 2H, CH3CH2), 3.70 (m, 1H, CH2OH), 3.83 (dd, 1H,
J ) 3.4 Hz and J′ ) 10.9 Hz, CH2OH), 3.99-4.09 (m, 1H, CHNH),
4.68 (hept, 1H, CH(CH3)2), 5.20 (d, 1H, J ) 3.4 Hz, CHNH), 7.81
(s, 1H, H-8), 8.43 (d, 2H, J ) 2.8 Hz, Hpyridyl), 8.70 (d, 2H, J )
2.8 Hz, Hpyridyl). 13C NMR: δ 9.8, 21.3, 21.4, 23.8, 45.8, 54.8, 65.5,
122.2 (2×), 125.3, 138.9, 142.2, 149.2 (2×), 151.6, 153.3, 158.4.
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[2-(phenyl)-phenylamino]-
1
9-iso-propylpurine (7a). Yield 65%, mp 134-138 °C. H NMR
(CDCl3): δ 0.94 (t, 3H, J ) 7.2 Hz, CH3CH2), 1.43 (d, 6H, J )
6.8 Hz, CH(CH3)2), 1.48-1.60 (m, 2H, CH3CH2), 3.54 (dd, 1H, J
) 3.2 Hz and J′ ) 10.4 Hz, CH2OH), 3.71 (dd, 1H, J ) 2.4 and
J′ ) 10 Hz, CH2OH), 3.81-3.85 (m, 1H, CHNH), 4.51 (hept, 1H,
CH(CH3)2), 5.1 (d, 1H, J ) 6 Hz, CHNH), 7.10 (t, 1H, J ) 7.6
Hz, Hphenyl), 7.23-7.40 (m, 8H, H-8 + Hphenyl), 7.54 (s, 1H,
NHphenyl), 8.25 (d, 1H, J ) 8.4 Hz, Hphenyl). 13C NMR: δ 10.9,
22.4, 22.5, 24.8, 46.5, 56.0, 67.4, 115.1, 123.0, 123.9, 127.7, 127.8,
128.8 (×2), 129.3, 130.4, 133.6, 135.1 (×2), 135.4, 138.4, 150.5,
152.6, 159.6.
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[3-(phenyl)phenylamino)-9-
iso-propylpurine (7b). Yield 78%, mp 100-105 °C. 1H NMR
(CDCl3): δ 1.02 (t, 3H, J ) 7.6 Hz, CH3-CH2), 1.56 (d, 6H, J )
6.8 Hz, CH(CH3)2), 1.59-1.69 (m, 2H, CH3CH2), 3.65 (dd, 1H, J
) 7.2 Hz and J′ ) 10 Hz, CH2OH), 3.85 (d, 1H, J ) 10 Hz,
CH2OH), 3.95-4.03 (m, 1H, CH-NH), 4.63 (hept, 1H, CH(CH3)2),
(R)-2-(1-Hydroxybut-2-ylamino)-6-[4-(phenyl)-phenylamino]-9-
iso-propylpurine (7f). Yield 42%, mp 76-79 °C. 1H NMR (CDCl3):
δ 1.06 (t, 3H, J ) 7.4 Hz, CH3CH2), 1.57 (d, 6H, J ) 6.8 Hz,
CH(CH3)2), 1.62-1.72 (m, 2H, CH3CH2), 3.69-3.72 (m, 1H,
CH2OH), 3.83-3.89 (m, 1H, CH2OH), 3.93-4.00 (m, 1H, CHNH),
4.64 (hept, 1H, CH(CH3)2), 5.00 (bs, 1H, CHNH), 7.30-7.34 (m,
1H, Hphenyl), 7.42-7.46 (m, 2H, Hphenyl), 7.59-7.75 (m, 5H, 8-H
+ Hphenyl), 7.81-7.84 (m, 2H, Hphenyl). 13C NMR: δ 10.9, 22.5,
22.6, 24.9, 46.7, 56.2, 67.7, 115.3, 120.2 (×2), 126.8 (×2), 126.9,
127.5 (×2), 128.7 (×2), 135.2, 135.8, 138.3, 140.7, 152.3, 153.3,
159.7.
(R)-2-(1-Hydroxybut-2-ylamino)-6-[(2-pyridyl)-methylamino]-9-
iso-propylpurine (8c). Yield 44%, mp 109-113 °C. 1H NMR
(CDCl3): δ 0.94 (t, 3H, J ) 7.3 Hz, CH3CH2), 1.47 (d, 6H, J )
6.6 Hz, CH(CH3)2), 1.55 (m, 2H, CH3CH2), 3.60 (dd, 1H, J ) 10.8
Hz and J′ ) 7.1 Hz, CH2OH), 3.75 (dd, 1H, J ) 10.8 Hz and J′ )
3.0 Hz, CH2OH), 3.85 (m, 1H, CHNH), 4.55 (hept, 1H, CH(CH3)2),
4.83 (bs, 2H, NHCH2), 5.08 (d, 1H, J ) 6.3 Hz, CHNH), 7.10 (dd,
1H, J ) 7.6 Hz and J′ ) 5.0 Hz, Hpyridyl), 7.25 (bs, 1H, NHCH2),
7.29 (d, 1H, J ) 7.6 Hz, Hpyridyl), 7.47 (s, 1H, 8-H), 7.54 (td, 1H,
J ) 7.6 Hz and J′ ) 1.8 Hz, Hpyridyl), 8.46 (dd, 1H, J ) 5.0 Hz and
J′ ) 1.8 Hz, Hpyridyl). 13C NMR: δ 10.9, 22.4, 22.5, 24.8, 45.5,
46.3, 55.9, 67.2, 114.5, 121.6, 122.0, 134.5, 136.6, 148.9, 154.7,
158.2, 159.9.
4.98 (d, 1H, J ) 6.4 Hz, CHNH), 7.14-7.75 (m, 10H, Hphenyl
+
H-8). 13C NMR: δ 10.9, 22.5, 24.9, 46.7, 56.1, 67.5, 115.1, 118.8,
121.9, 127.2, 127.4, 128.7, 129.2, 135.2, 139.4, 141.1, 142.0, 152.4
(×2), 159.7.
(R)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[3-(phenyl)pheny-
lamino]-9-iso-propylpurine (7c). Yield 56%, mp 90-105 °C. H
1
NMR (CDCl3): δ 1.01 (d, 6H, J ) 6.4 Hz, CHCH(CH3)2), 1.56 (d,
6H, J ) 6.8 Hz, NCH(CH3)2), 2.00 (hept, 1H, J ) 6.4 Hz,
CHCH(CH3)2), 3.71 (dd, 1H, J ) 7.2 Hz and J′ ) 11 Hz, CH2OH),
3.87 (d, 1H, J ) 11 Hz, CH2OH), 4.09-4.24 (m, 1H, CHNH),
4.64 (hept, 1H, NCH(CH3)2), 5.01 (d, 1H, J ) 6.4 Hz, CHNH),
7.14-7.75 (m, 10H, Hphenyl). 13C NMR: δ 18.7, 19.5, 22.5 (×2),
30.0, 46.5, 59.8, 65.8, 115.2, 118.8, 121.8, 127.2 (×2), 127.4 (×3),
128.7, 129.2, 135.1, 139.5, 141.1, 141.9, 152.3 (×2), 160.0.
(R)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[4-(phenyl)pheny-
lamino]-9-iso-propylpurine (7d). Yield 45%, mp 115-118 °C. 1H
NMR (CDCl3): δ 1.05 (d, 6H, J ) 6.8 Hz, CHCH(CH3)2), 1.57 (d,
6H, J ) 6.8 Hz, NCH(CH3)2), 2.00-2.10 (m, 1H, CHCH(CH3)2),
(S)-2(1-Hydroxybut-2-ylamino)-6-[(2-pyridyl)methylamino]-9-
iso-propylpurine (8d). Yield 54%, mp 117-120 °C. 1H NMR
(CDCl3) δ 0.95 (t, 3H, J ) 7.3 Hz, CH3CH2), 1.47 (d, 6H, J ) 6.6
Hz, CH(CH3)2), 1.57 (m, 2H, CH3CH2), 3.58 (dd, 1H, J ) 10.8 Hz
and J′ ) 7.1 Hz, CH2OH), 3.75 (dd, 1H, J ) 10.8 Hz and J′ ) 3.0
Hz, CH2OH), 3.85 (m, 1H, CHNH), 4.55 (hept, 1H, CH(CH3)2),
4.83 (bs, 2H, NHCH2), 5.08 (d, 1H, J ) 6.3 Hz, CHNH), 7.10 (dd,
1H, J ) 7.6 Hz and J′ ) 5.0 Hz, Hpyridyl), 7.25 (bs, 1H, NHCH2),
7.29 (d, 1H, J ) 7.6 Hz, Hpyridyl), 7.47 (s, 1H, 8-H), 7.54 (td, 1H,

5238 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Oumata et al.
J ) 7.6 Hz and J′ ) 1.8 Hz, Hpyridyl), 8.48 (dd, 1H, J ) 5.0 Hz and
J′ ) 1.8 Hz, Hpyridyl).
7.50 (s, 1H, 8-H), 7.53 (d, 2H, J ) 8 Hz, Hphenyl), 7.86 (dt, 1H, J
) 2 Hz and J′ ) 7.6 Hz, Hpyridyl), 8.58 (dd, 1H, J ) 1.6 Hz and J′
) 8.6 Hz, Hpyridyl), 8.82 (d, 1H, J ) 2 Hz, Hpyridyl).
(R)-2-(1-Hydroxybut-2-ylamino)-6-[(3-pyridyl)methylamino]-9-
iso-propylpurine (8e). Yield 55%, mp 117-120 °C. 1H NMR
(CDCl3) δ 0.99 (t, 3H, J ) 7.5 Hz, CH3CH2), 1.47 (d, 6H, J ) 6.8
Hz, CH(CH3)2), 1.61 (m, 2H, CH3CH2), 3.60 (dd, 1H, J ) 10.6 Hz
and J′ ) 7.3 Hz, CH2OH), 3.77 (dd, 1H, J ) 10.7 Hz and J′ ) 2.9
Hz, CH2OH), 3.88 (m, 1H, CHNH), 4.59 (hept, 1H, CH(CH3)2),
4.73 (m, 2H, NHCH2), 4.96 (d, 1H, J ) 6.6 Hz, CHNH), 6.65 (bs,
1H, NHCH2), 7,21 (dd, 1H, J ) 7.8 Hz and J′ ) 4.8 Hz, Hpyridyl),
7.46 (s, 1H, 8-H), 7.66 (m, 1H, Hpyridyl), 8.47 (dd, 1H, J ) 4.8 Hz
(R)-2-(1-Hydroxybut-2-ylamino)-6-[3-(4-pyridyl)phenylmethy-
1
lamino]-9-iso-propylpurine (8l). Yield 78%, mp 144-146 °C. H
NMR (CDCl3): δ 0.98 (t, 3H, J ) 6.8 Hz, CH3CH2), 1.60 (d, 6H,
J ) 6.8 Hz, CH(CH3)2), 1.90-2.00 (m, 2H, CH2CH3), 3.70 (dd,
1H, J ) 2.4 Hz and J′ ) 10.0 Hz, CH2OH), 3.75-3.85 (m, 2H,
CHNH + CH2OH), 4.62 (hept, 1H, CH(CH3)2), 4.80 (bs, 2H,
NHCH2), 5.15 (d, 1H, NHCH), 6.22 (bs, 1H, NH),7.45-7.63(m,
5H, Hphenyl + 8-H), 8.45 (d, 2H, J ) 2.8 Hz, Hpyridyl), 8.68 (d, 2H,
J ) 2.8 Hz, Hpyridyl).
and J′ ) 1.5 Hz, Hpyridyl) 8.60 (d, 1H, J ) 1.8 Hz, Hpyridyl).
13C
NMR: δ 11.1, 22.6, 22.7, 25.4, 42.4, 47.9, 55.8, 65.3, 114.6, 125.2,
136.6, 137.5, 137.9, 148.5, 149.4, 152.0, 155.9, 161.0.
(R)-2-(1-Hydroxybut-2-ylamino)-6-[4-(2-pyridyl)phenylmethy-
1
lamino]-9-iso-propylpurine (8m). Yield 66%, mp 89-93 °C. H
(S)-2-(1-Hydroxybut-2-ylamino)-6-[(3-pyridyl)methylamino]-9-
iso-propylpurine (8f). Yield 70%, mp 115-116 °C. 1H NMR
(CDCl3): δ 0.99 (t, 3H, J ) 7.5 Hz, CH3CH2), 1.50 (d, 6H, J )
6.5 Hz, CH(CH3)2), 1.64 (m, 2H, CH3CH2), 3.62 (dd, 1H, J ) 10.0
Hz and J′ ) 7.3 Hz, CH2OH), 3.77 (dd, 1H, J ) 10.0 Hz and J′ )
2.9 Hz, CH2OH), 3.92 (m, 1H, CHNH), 4.59 (hept, 1H, CH(CH3)2),
4.75 (m, 2H, NHCH2), 5.21 (d, 1H, J ) 6.6 Hz, CHNH), 6.63 (bs,
1H, NHCH2), 7,22 (dd, 1H, J ) 7.8 Hz and J′ ) 4.8 Hz, Hpyridyl),
7.41 (s, 1H, 8-H), 7.63 (m, 1H, Hpyridyl), 8.45 (dd, 1H, J ) 4.8 Hz
and J′ ) 1.5 Hz, Hpyridyl) 8.61 (d, 1H, J ) 1.8 Hz, Hpyridyl).
(R)-2-(1-Hydroxybut-2-ylamino)-6-[(4-pyridyl)methylamino]-9-
iso-propylpurine (8g). Yield 67%, mp 130-132 °C. 1H NMR
(CDCl3): δ 0.97 (t, 3H, J ) 7.4 Hz, CH3CH2), 1.53 (d, 6H, J )
6.7 Hz, -CH(CH3)2), 1.61 (m, 2H, CH3CH2), 3.60 (dd, 1H, J )
10.8 Hz and J′ ) 7.5 Hz, -CH2OH), 3.78 (dd, 1H, J ) 10.8 Hz
and J′ ) 2.4 Hz, CH2OH), 3.85 (m, 1H, CHNH), 4.60 (hept, 1H,
CH(CH3)2), 4.77 (m, 2H, NHCH2), 4.92 (d, 1H, J ) 6.1 Hz,
CHNH), 6.55 (bs, 1H, NHCH2), 7.26 (d, 2H, J ) 5.8 Hz, Hpyridyl),
7.51 (s, 1H, 8-H), 8.51 (d, 2H, J ) 2.8 Hz, Hpyridyl). 13C NMR: δ
10.5, 22.0, 22.1, 24.5, 42.7, 46.1, 55.7, 67.5, 114.1, 121.8 (×2),
134.5, 148.1, 149.5 (×2), 154.3 (×2), 159.4.
NMR (CDCl3): δ 0.95 (t, 3H, J ) 7.2 Hz, CH3CH2), 1.47 (d, 6H,
J ) 6.8 Hz, CH(CH3)2), 1.50-1.60 (m, 2H, CH3CH2), 3.56 (dd,
1H, J ) 2.8 Hz and J′ ) 10.8 Hz, CH2OH), 3.75 (dd, 1H, J ) 2.4
Hz and J′ ) 10.8 Hz, CH2OH), 3.79-3.85 (m, 1H, CHNH), 4.54
(hept, 1H, CH(CH3)2), 4.76 (bs, 2H, NHCH2), 4.90 (bs, 1H, CHNH),
6.10 (bs, 1H, NHCH2), 7.16 (td, 1H, J ) 1.6 and J′ ) 5.4 Hz,
Hpyridyl), 7.41 (d, 2H, J ) 8.4 Hz, Hphenyl), 7.45 (s, 1H, 8-H),
7.63-7.70 (m, 2H, Hpyridyl), 7.89 (d, 2H, J ) 8.4 Hz, Hphenyl), 8.62
(d, 1H, J ) 4.8 Hz, Hpyridyl). 13C NMR (CDCl3): δ 10.5, 22.3, 22.4,
24.1, 38.0, 45.9, 55.0, 66.8, 113.6, 120.0, 120.9, 125.5, 126.8 (×2),
127.7, 130.0, 134.1, 135.9, 137.7, 140.0, 149.1, 154.1, 156.8, 159.1.
(R)-2-(1-Hydroxy-3-methylbut-2-yl)-6-[4-(2-pyridyl)phenylme-
thylamino]-9-iso-propylpurine (8n). Yield 76%, mp 128-135 °C.
1H NMR (CDCl3): δ 0.94 (d, 6H, J ) 6.8 Hz, CHCH(CH3)2), 1.47
(d, 6H, J ) 6.8 Hz, CH(CH3)2), 1.67-1.80 (m, 1H, CHCH(CH3)2,
3.60-3.65 (m, 1H, CH2OH), 3.76-3.79 (m, 2H, CHNH and
CH2OH), 4.55 (hept, 1H, CH(CH3)2), 4.78 (bs, 2H, NHCH2), 6.10
(bs, 1H, NHCH2), 7.14-7.18 (m, 1H, Hpyridyl), 7.41 (d, 2H, J )
8.2 Hz, Hphenyl), 7.48 (s, 1H, H-8), 7.63-7.70 (m, 2H, Hpyridyl), 7.89
(d, 2H, J ) 8.2 Hz, Hphenyl), 8.61 (d, 1H, J ) 5.2 Hz, Hpyridyl). 13
C
NMR: δ 15.2, 18.9, 19.4, 22.5, 22.6, 30.1, 46.4, 59.9, 65.8, 114.6,
120.4, 122.0, 127,1 (×2), 128.0 (×2), 134.5 (×2), 134.7, 138.4,
139.7, 149.6, 154.8, 157.1, 160.2.
(S)-2-(1-Hydroxybut-2-ylamino)-6-[(4-pyridyl)methylamino]-9-
iso-propylpurine (8h). Yield 55%, mp 128-130 °C. 1H NMR
(CDCl3): δ 0.97 (t, 3H, J ) 7.4 Hz, CH3CH2), 1.53 (d, 6H, J )
6.7 Hz, CH(CH3)2), 1.61 (m, 2H, CH3CH2), 3.60 (dd, 1H, J ) 10.8
Hz and J′ ) 7.5 Hz, CH2OH), 3.78 (dd, 1H, J ) 10.8 Hz and J′ )
2.4 Hz, CH2OH), 3.85 (m, 1H, CHNH), 4.60 (hept, 1H, CH(CH3)2),
4.77 (m, 2H, NHCH2), 4.92 (d, 1H, J ) 6.1 Hz, CHNH), 6.55 (bs,
1H, NHCH2), 7.26 (d, 2H, J ) 5.8 Hz, Hpyridyl), 7.51 (s, 1H, 8-H),
8.51 (d, 2H, J ) 2.8 Hz, Hpyridyl).
2-(1-Hydroxy-2-methylprop-2-yl)-6-[4-(2-pyridyl)phenylmethy-
lamino]-9-iso-propylpurine (8o). Yield 65%, mp 110-112 °C. 1H
NMR (CDCl3): δ 1.42 (s, 6H, C(CH3)2), 1.56 (d, 6H, J ) 6.8 Hz,
NCH(CH3)2), 2.5 (bs, 1H, CH2OH), 3.74 (s, 2H, CH2OH), 4.59
(hept, 1H, NCH(CH3)2), 4.81 (bs, 2H, NHCH2), 6.15 (bs, 1H,
NHCH2), 7.14-7.18 (m, 1H, Hpyridyl), 7.40 (d, 2H, J ) 8.0 Hz,
Hphenyl), 7.52 (s, 1H, 8-H), 7.65-7.72 (m, 2H, Hpyridyl), 7.93 (d,
2H, J ) 8.0 Hz, Hphenyl), 8.65 (d, 1H, J ) 5.0 Hz, Hpyridyl).
(R)- 2-(1-Hydroxybut-2-ylamino)-6-[4-(3-pyridyl)phenylmethy-
lamino]-9-iso-propylpurine (8p). Yield 62%, mp 104-106 °C. 1H
NMR (CDCl3): δ 0.97(t, 3H, J ) 7.5 Hz, CH3CH2), 1.48 (d, 6H,
J ) 6.8 Hz, CH(CH3)2), 1.61 (m, 2H, CH3CH2), 3.62 (dd, 1H, J )
10.6 Hz and J′ ) 7.3 Hz, CH2OH), 3.77 (dd, 1H, J ) 10.7 Hz and
J′ ) 2.9 Hz, CH2OH) 4.60 (hept, 1H, CH(CH3)2), 4.80 (bs, 2H,
NHCH2), 5.03 (brt, 1H, J ) 6.8 Hz, NHCH2), 7.36 (dd, 1H, J )
3.2 and J ) 7.4 Hz, Hpyridyl), 7.48 (d, 2H, J ) 8 Hz, Hphenyl), 7.50
(s, 1H, 8-H), 7.53 (d, 2H, J ) 8 Hz, Hphenyl), 7.86 (dt, 1H, J ) 2
Hz and J′ ) 7.6 Hz, Hpyridyl), 8.58 (dd, 1H, J ) 1.6 Hz and J′ )
4.6 Hz, Hpyridyl), 8.85 (d, 1H, J ) 2 Hz, Hpyridyl).
(R)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[4-(3-pyridyl)phe-
nylmethylamino]-9-iso-propylpurine (8q). Yield 89%, mp 83-85
°C. 1H NMR (CDCl3): δ 0.99 (d, 6H, J ) 6.8 Hz, CHCH(CH3)2),
1.60 (d, 6H, J ) 6.8 Hz, NCH(CH3)2), 1.90-2.00 (m, 1H,
CHCH(CH3)2, 3.67 (dd, 1H, J ) 2.4 Hz and J′ ) 10.4 Hz,
-CH2OH), 3.78-3.87 (m, 2H, CHNH + CH2OH), 4.60 (hept, 1H,
CH(CH3)2), 4.83 (bs, 2H, NHCH2), 4.99 (d, 1H, J ) 6.8 Hz,
NHCH), 6.25 (bs, 1H, NHCH2), 7.36 (dd, 1H, J ) 3.2 Hz and J′
) 7.4 Hz, Hpyridyl), 7.48 (d, 2H, J ) 8 Hz, Hphenyl), 7.50 (s, 1H,
8-H), 7.53 (d, 2H, J ) 8 Hz, Hphenyl), 7.86 (dt, 1H, J ) 2 Hz and
J′ ) 7.6 Hz, Hpyridyl), 8.58 (dd, 1H, J ) 1.6 Hz and J′ ) 8.6 Hz,
(R)-2-(1-Hydroxybut-2-ylamino)-6-[3-(phenyl)phenylmethylamino]-
9-iso-propylpurine (8i). Yield 42%, mp 100-102 °C. 1H NMR
(CDCl3): δ 0.91 (t, 3H, J ) 7.3 Hz, CH3CH2), 1.40 (d, 6H, J )
6.7 Hz, CH(CH3)2), 1.45-1.56 (m, 2H, CH3CH2), 3.54 (m, 1H,
CH2OH), 3.78 (d, 1H, J ) 10.6 Hz, CH2OH), 3.79-3.82 (m, 1H,
CHNH), 4.50 (hept, 1H, CH(CH3)2), 4.74 (bs, 2H, NHCH2), 4.87
(d, 1H, J ) 5,7 Hz, CHNH), 6.48 (bs, 1H, NHCH2), 7.23-7.52
(m, 10H, H-8). 13C NMR (CDCl3): δ 10.2, 21.4, 21.5, 23.9, 43,4,
45.4, 55.2, 67.2, 113.6, 118.5, 125.1, 125.6, 126.1 (2×), 126.3,
127.7 (2×), 128, 133.5 (2×), 138.5, 139.9, 140.5, 153.9, 159.
(R)-2-(1-Hydroxybut-2-ylamino)-6-[3-(2-pyridyl)phenylmethy-
1
lamino]-9-iso-propylpurine (8j). Yield 32%, mp 163-165 °C. H
NMR (CDCl3): δ 0.91 (t, 3H, J ) 7.3 Hz, CH3CH2), 1.40 (d, 6H,
J ) 6.7 Hz, CH(CH3)2), 1.45-1.56 (m, 2H, CH3CH2), 3.54 (m,
1H, CH2OH), 3.78 (d, 1H, J ) 10.6 Hz, CH2OH), 3.79-3.82 (m,
1H, CHNH), 4.50 (hept, 1H, CH(CH3)2), 4.74 (bs, 2H, NHCH2),
4.87 (brs, 1H, NH) 6.48 (bs, 1H, NH), 7.32-7.62 (m, 6H, Hphenyl
+ Hpyridyl); 7.76 (s, 1H, 8-H), 8.66 (d, 1H, J ) 4.5 Hz, Hpyridyl),
8.85 (d, 1H J ) 2.8 Hz Hpyridyl).
(R)-2-(1-Hydroxybut-2-ylamino)-6-[3-(3-pyridyl)phenylmethy-
lamino]-9-iso-propylpurine (8k). Yield 67%, mp 123-126 °C. 1H
NMR (CDCl3): δ 0.99 (t, 3H, J ) 6.8 Hz, CH3CH2), 1.60 (d, 6H,
J ) 6.8 Hz, CH(CH3)2), 1.90-2.00 (m, 2H, CH2CH3)2, 3.67 (dd,
1H, J ) 2.4 Hz and J′ ) 10.4 Hz, CH2OH), 3.78-3.87 (m, 2H,
CHNH and CH2OH), 4.60 (hept, 1H, CH(CH3)2), 4.83 (bs, 2H,
NHCH2), 4.99 (d, 1H, NHCH), 6.25 (bs, 1H, NH), 7.36 (dd, 1H, J
) 3.2 Hz and J′ ) 7.4 Hz, Hpyridyl), 7.48 (d, 2H, J ) 8 Hz, Hphenyl),
H
pyridyl), 8.82 (d, 1H, J ) 2 Hz, Hpyridyl).
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[4-(4-pyridyl)phenylmethy-
lamino]-9-iso-propylpurine (8r). Yield 56%, mp 70-72 °C. 1H
NMR (CDCl3): δ 0.97 (t, 3H, J ) 7.5 Hz, CH3CH2), 1.49 (d, 6H,

CDK/CK1 Dual-Specificity Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5239
J ) 6.8 Hz, CH(CH3)2), 1.61 (m, 2H, CH3CH2), 1.55-1.71 (m,
2H, CH3CH2), 3.57 (dd, 1H, J ) 7.8 Hz and J′ ) 10.7 Hz, CH2OH),
3.75 (dd, 1H, J ) 2.4 Hz, J′ ) 10.7 Hz, CH2OH), 3.79-3.82 (m,
1H, CHNH), 4.56 (hept, 1H, CH(CH3)2), 4.71 (bs, 2H, NHCH2),
7.41 (d, 2H, J ) 6 Hz, Hpyridyl), 7.48-7.75 (m 5H, Hphenyl + H-8),
8.60 (d, 2H, Hpyridyl).
2-(3-Pyridyl)-6-(4-bromophenylamino)-9-iso-propylpurine (12a).
Yield 26%, mp 122-125 °C. H NMR (CDCl3): δ 1.68 (d, 6H,
1
J ) 6.4 Hz, CH(CH3)2), 4.96 (hept, 1H, CH(CH3)2), 7.37-7.45
(m, 1H, Hpyridyl), 7.54 (d, 2H, J ) 8.4 Hz, Hphenyl), 7.81 (d, 2H,
J ) 8.4 Hz, Hphenyl), 7.94 (s, 1H, 8-H), 8.62-8.75 (m, 2H,
H
pyridyl), 9.70 (s, 1H, Hpyridyl). 13C NMR: δ 22.5, 47.9, 115.5,
120.1, 127.7, 123.2, 123.4, 132.0, 139.1, 149.9, 144.8, 151.4,
156.2.
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[4-(phenyl)phenylmethy-
lamino]-9-iso-propylpurine (8s). Yield 67%, mp 117-120 °C. 1H
NMR (CDCl3): δ 0.97 (t, 3H, J ) 7.4 Hz, CH3CH2), 1.47 (d, 6H,
J ) 6.8 Hz, CH(CH3)2), 1.55-1.71 (m, 2H, CH3CH2), 3.58 (dd,
1H, J ) 7.8 Hz and J′ ) 10.8 Hz, CH2OH), 3.75 (dd, 1H, J ) 2.4
Hz, J′ ) 10.8 Hz, CH2OH), 3.79-3.82 (m, 1H, CHNH), 4.55 (hept,
1H, CH(CH3)2), 4.74 (bs, 2H, NHCH2), 4.82-4.84 (m, 1H, CHNH),
6.04 (bs, 1H, NHCH2), 7.24-7.56 (m, 10H, 8-H). 13C NMR: δ
10.9, 22.5, 22.6, 25.0, 38.7, 46.4, 56.4, 68.6, 113.6, 118.5, 125.1,
125.6 (×2), 127, 127.2, 127.3, 128.1, 128.7, 134.6 (2×), 137.8,
140.3, 140.8, 154.8, 159.9.
2-(4-Pyridyl)-6-(4-bromophenylamino)-9-iso-propylpurine (12b).
1
Yield 20%, mp 200-215 °C. H NMR (CDCl3): δ 1.63 (d, 6H, J
) 6.4 Hz, CH(CH3)2), 4.90 (hept, 1H, CH(CH3)2), 7.48 (d, 2H, J
) 8.6 Hz, Hphenyl), 7.75 (d, 2H, J ) 8.6 Hz, Hphenyl), 7.90 (s, 1H,
H-8), 8.25 (d, 2H, J ) 4.8 Hz, Hpyridyl), 8.69 (d, 2H, J ) 4.8 Hz,
H Hpyridyl). 13C NMR: δ 22.6, 47.4, 115.3, 119.6, 120.1, 121.2,
121.8, 128.0, 130.3, 131.7, 137.7, 139.3, 145.4, 149.9, 151.1, 155.9.
(R)-2-(1-Hydroxybut-2-ylamino)-6-[3-(2-pyridyl)phenylamino]-
9-isopropylpurine (13a). Yield 79%, mp 77-85 °C. 1H NMR
(CDCl3): δ 0.98 (t, 3H, J ) 7.2 Hz, CH3CH2), 1.49 (d, 6H, J )
6.8 Hz, CH(CH3)2), 1.67-1.79 (m, 2H, CH3CH2), 3.64 (m, 1H,
CH2OH), 3.74 (dd, 1H, J ) 2.4 Hz and J′ ) 10,6 Hz, CH2OH),
3.96-4.07 (m, 1H, CHNH), 4.58 (hept, 1H, CH(CH3)2), 4.94 (m,
1H, CHNH), 7.38 (t, 1H, J ) 7.2 Hz, Hphenyl), 7.50-7.54 (m, 2H,
Hphenyl + Hpyridyl) 7.68-7.71 (m, 3H, Hphenyl + Hpyridyl) 7.86 (s, 1H,
H-8), 8.62 (d, 1H, J ) 4.3 Hz, Hpyridyl). 13C NMR: δ 10.8, 22.5,
22.6, 24.9, 46.4, 55.9, 66.7, 114.9, 118.6, 120.3, 121.0, 121.4, 122.2,
129.1, 135.1, 136.9, 139.8, 140.1, 149.4, 152.2, 157.3, 159.6.
(R)-2(1-Hydroxy-3-methyl-but-2-ylamino)-6-[3-(2-pyridyl)phe-
nylamino]-9-isopropylpurine (13b). Yield 50%, mp 95-110 °C.
1H NMR (CDCl3): δ 0.94 (d, 6H, J ) 6.8 Hz, CHCH(CH3)2), 1.49
(d, 6H, J ) 6.8 Hz, NCH(CH3)2), 1.95-2.06 (m, 1H, CHCH(CH3)2,
3.62-3.77 (m, 2H, CH2OH), 3.91-3.99 (m, 1H, CHNH), 4.58
(hept, 1H, NCH(CH3)2), 4.91 (m, 1H, CHNH), 7.36 (t, 1H, J )
7.2 Hz, Hphenyl), 7.50-7.73 (m, 6H, Hphenyl, 8-H), 8.62 (d, 1H J )
4.2 Hz, Hpyridyl). 13C NMR: δ 18.4, 19.7, 22.5, 22.6, 29.5, 46.4,
59.6, 65.2, 115.1, 118.6, 120.3, 121.0, 121.3, 122.3, 129.2, 135.0,
136.9, 139.7, 140.1, 149.5, 152.3, 157.4, 159.9.
2-Chloro-6-(4-bromophenylamino)-9-iso-propylpurine (9). Yield
1
71%, mp 172 °C. H NMR (CDCl3): δ 1.54 (d, 6H, J ) 6.8 Hz,
CH(CH3)2), 4.79 (hept, 1H, CH(CH3)2), 7.42 (d, 2H, J ) 8.8 Hz,
Hphenyl), 7.62 (d, 2H, J ) 8.8 Hz, Hphenyl), 7.82 (s, 1H, 8-H).
2-Chloro-6-[3-(tetramethyldioxaborolanyl)phenylamino]-9-iso-
propylpurine (10a). Yield 65%, mp 213 °C. 1H NMR (CDCl3): δ
1.37 (s, 12 H, (CH3)2COB), 1.61 (d, 6H, J ) 7.0 Hz, CH(CH3)2),
4.85 (hept, 1H, CH(CH3)2), 7.44 (t, 1H, J ) 7.3 Hz, Hphenyl), 7.57
(d, 1H, ) 7.3 Hz, Hphenyl), 7.72 (s, 1H, NH), 7.82 (s, 1H, Hphenyl),
7.87 (s, 1H, 8-H), 8.27 (d, 1H, J ) 7.3 Hz, Hphenyl).
2-Chloro-6-[4-(tetramethyldioxaborolanyl)phenylamino]-9-iso-
propylpurine (10g). Yield ) 59%, mp 168 °C. 1H NMR (CDCl3):
δ 1.34 (s, 12 H, (CH3)2COB), 1.61 (d, 6H, J ) 6.8 Hz, CH(CH3)2),
4.87 (hept, 1H, CH(CH3)2), 7.78-7.91 (m, 4H, Hphenyl), 8.07 (s,
1H, H-8).
2-Chloro-6-[3-(2-pyridyl)phenylamino]-9-iso-propylpurine (11a).
1
Yield 76%, mp 142-148 °C. H NMR (CDCl3): δ 1.61 (d, 6H, J
) 6.7 Hz, CH(CH3)2), 4.85 (hept, 1H, CH(CH3)2), 7.53 (t, 1H, J )
7.5 Hz, Hphenyl), 7.67-7.75 (m, 3H, Hphenyl + Hpyridyl), 7.77-7.85
(m, 2H, Hphenyl + Hpyridyl), 7.91-7.95 (m, 1H, anilin or pyridin),
8.35 (bs, 1H, Hpyridyl), 8.69 (d, 1H, J ) 4.1 Hz, Hpyridyl).
2-(1-Hydroxy-2-methylprop-2-ylamino)-6-[3-(2-pyridyl)pheny-
lamino]-9-iso-propylpurine (13c). Yield 48%, mp 103-109 °C. 1H
NMR (CDCl3): δ 1.40 (s, 6H, C(CH3)2), 1.56 (d, 6H, J ) 6.8 Hz,
NCH(CH3)2), 2.54(bs, 1H, CH2OH), 3.74 (s, 2H, CH2OH), 4.59
(hept, 1H, NCH(CH3)2), 6.81 (bs, 1H, NH), 7.24 (td, 1H, J ) 1.6
and J′ ) 6.8 Hz, Hpyridyl), 7.45 (t, 1H, J ) 8 Hz, Hphenyl), 7.59 (s,
1H, NHPhenyl), 7.65 (d, 1H, J ) 3.2 Hz, Hphenyl), 7.70-7.77 (m,
2H, Hpyridyl) 7.90 (d, 1H, J ) 8.4 Hz, Hphenyl), 7.99 (s, 1H, H-8),
8.23 (s, 1H, Hpyridyl) 8.69 (d, 1H, J ) 4.8 Hz, Hpyridyl). 13C NMR:
δ 22.4 (×2), 25.1, 46.9, 55.4 (×2), 72.1, 115.0, 118.9, 120.7, 120.9,
121.8, 122.2, 129.2, 135.2, 136.7, 139.3, 140.1, 149.5, 149.6, 152.6,
157.1, 158.4.
2-Chloro-6-[3-(3-pyridyl)phenylamino]-9-iso-propylpurine (11d).
1
Yield 66%, mp 85-88 °C. H NMR (CDCl3): δ 1.62 (d, 6H, J )
6.6 Hz, CH(CH3)2), 4.86 (hept, 1H, CH(CH3)2), 7.36 (d, 1H, J )
7.6 Hz, Hpyridyl), 7.40 (dd, 1H, J ) 4.2 Hz and J′ ) 7.6 Hz, Hpyridyl),
7.50 (t, 1H, J ) 8.0 Hz, Hphenyl), 7.79 (d, 1H, J ) 8.0 Hz, Hphenyl),
7.90 (s, 1H, 8-H), 7.95 (d, 1H, J ) 8.0 Hz, Hphenyl), 8.07 (s, 1H,
H-8), 8.62 (d, 1H, J ) 4.2 Hz, Hpyridyl), 8.91 (s, 1H, Hpyridyl).
2-Chloro-6-[3-(4-pyridyl)phenylamino]-9-iso-propylpurine (11f).
1
Yield 45%, mp 160-165 °C. H NMR (CDCl3): δ 1.55 (d, 6H,
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[3-(3-pyridyl)phenylamino]-
9-iso-propylpurine (13d). Yield 60%, mp 82-89 °C. 1H NMR
(CDCl3): δ 0.94 (t, 3H, J ) 6.8 Hz, CH3CH2), 1.49 (d, 6H, J )
6.8 Hz, CH(CH3)2), 1.53-1.66 (m, 2H, CH3CH2), 3.61 (dd, 1H, J
) 7.2 Hz and J′ ) 10.4 Hz, CH2OH), 3.78 (dd, 1H, J ) 2.4 Hz
and J′ ) 10.4 Hz, CH2OH), 3.88-3.98 (m, 1H, CHNH), 4.56 (hept,
1H, J ) 6.8 Hz, CH(CH3)2), 4.94 (d, 1H, J ) 6 Hz, CHNH), 7.29
(dd, 1H, J ) 4 Hz and J ) 7.6 Hz, Hpyridyl), 7.37 (t, 1H, J ) 7.6
Hz, Hpyridyl), 7.53 (s, 1H, H-8), 7.62-7.70 (m, 2H, Hphenyl and
CH(CH3)2), 4.81 (sept, 1H, J ) 6.8 Hz, CH(CH3)2), 7.35 (d, 1H, J
) 7.6 Hz, Hphenyl), 7.44 (t, 1H, J ) 7.6 Hz, Hphenyl), 7.52 (d, 2H, J
) 4 Hz, Hpyridyl), 7.74 (d, 1H, J ) 7.6 Hz, Hphenyl), 7.85 (s, 1H,
H
phenyl), 7.99 (s, 1H, 8-H), 8.63 (d, 2H, J ) 4 Hz, Hpyridyl).
2-Chloro-6-[4-(2-pyridyl)phenylamino]-9-iso-propylpurine (11g).
Yield 57%, mp 170 °C. 1H NMR (CDCl3): δ 1.62 (d, 6H,
CH(CH3)2), 4.86 (hept, 1H, CH(CH3)2), 7.70-7.91 (m, 5H, Hphenyl
,
Hpyridyl and 8-H), 7.92 (d, 1H, J ) 8.4 Hz, Hpyridyl), 8.07 (d, 2H, J
) 8.8 Hz, Hphenyl), 8.70 (d, 1H, J ) 4 Hz, Hpyridyl).
H
pyridyl), 7.82 (d, 1H, J ) 7.6 Hz, Hphenyl), 7.99 (s, 1H, Hphenyl),
8.52 (d, 1H, J ) 4 Hz, Hpyridyl), 8.81 (s, 1H, Hpyridyl). 13C NMR: δ
10.8, 22.6, 24.8, 46.7, 56.0, 67.2, 115.1, 118.5, 119.5, 121.6, 123.5,
129.5, 134.5, 135.3, 136.5, 138.5, 139.8, 148.3, 148.5, 152.3, 159.6.
(R)-2(1-Hydroxy-3-methyl-but-2-ylamino)-6-[3-(3-pyridyl)phe-
nylamino]-9-iso-propylpurine (13e). Yield 51%, mp 90-97 °C. 1H
NMR (CDCl3): δ 0.93 (d, 6H, J ) 6.8 Hz, CH-CH(CH3)2), 1.48
(d, 6H, J ) 6.8 Hz, NCH(CH3)2), 1.95 (hept, 1H, J ) 6.8 Hz,
CHCH(CH3)2, 3.68 (dd, 1H, J ) 7.6 Hz, J′ ) 10,8 Hz, CH2OH),
3.84 (dd, 1H, J ) 2.6 Hz and J′ ) 10.8 Hz, CH2OH), 3.84-3.90
(m, 1H, CHNH), 4.55 (hept, 1H, J ) 6.8 Hz, NCH(CH3)2), 4.91
(d, 1H, J ) 6.6 Hz, CHNH), 7.30 (dd, 1H, J ) 4.8 Hz and J ) 7,6
Hz, Hpyridyl), 7.38 (t, 1H, J ) 7.6 Hz, Hphenyl), 7.53 (s, 1H, 8-H),
7.61-7.70 (m, 2H, Hphenyl), 7.84 (d, 1H, J ) 7.6 Hz, Hpyridyl), 8.00
2-Chloro-6-[4-(3-pyridyl)phenylamino]-9-iso-propylpurine (11h).
1
Yield 52%, mp 204-207 °C. H NMR (CDCl3) δ 1.56 (d, 6H, J
) 6.4 Hz, CH(CH3)2), 4.82 (hept, 1H, CH(CH3)2), 7.30 (dd, 1H, J
) 4.8 Hz and J′ ) 8 Hz, Hpyridyl), 7.57 (d, 2H, J ) 8.8 Hz, Hphenyl),
7.82 (dt, 1H, J ) 1.6 Hz and J′ ) 8 Hz, Hpyridyl), 7.86 (d, 2H, J )
8.8 Hz, Hphenyl), 7.94 (s, 1H, 8-H), 8.51 (dd, 1H, J ) 1.6 Hz and
J′) 4.8 Hz, Hphenyl), 8.80 (d, 1H, J ) 1.6 Hz, Hpyridyl).
2-Chloro-6-[4-(4-pyridyl)phenylamino]-9-iso-propylpurine (11k).
Yield 70%, yellow oil. 1H NMR (CDCl3): δ 1.56 (d, 6H, J )
6.5 Hz, CH(CH3)2), 4.85 (hept, 1H, CH(CH3)2), 7.10-7.20 (m,
2H, Hphenyl), 7.40 (d, 2H, J ) 7.7 Hz, Hphenyl), 7.54 (d, 2H, J )
5.0 Hz, Hpyridyl), 7.75 (s, 1H, 8-H), 8.00 (d, 2H, J ) 5.0 Hz,
H
pyridyl).

5240 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Oumata et al.
(s, 1H, Hphenyl), 8.53 (d, 1H, J ) 4 Hz, Hpyridyl), 8.82 (s, 1H, Hpyridyl).
13C NMR: δ 18.8, 19.4, 22.5, 29.9, 46.6, 59.7, 65.6, 115.1, 118.5,
119.5, 121.6, 123.6, 129.6, 134.5, 135.4, 136.6, 138.6, 140.0, 148.3,
148.7, 152.2, 159.9.
7.89-7.95 (m, 2H, Hpyridyl), 8.60-8.65 (m, 2H, Hpyridyl). 13C NMR:
δ 11.0, 22.8, 25.4, 47.1, 53.5, 56.3, 120.4, 121.5, 127.6, 128.6,
129.4, 132.4, 135.7, 140.6, 148.2, 150.6, 152.2, 159.7.
(R)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[4-(4-pyridyl)phe-
nylamino]-9-iso-propylpurine (13l). Yield 41%, mp 121-123 °C.
1H NMR (CDCl3): δ 0.99 (d, 6H, J ) 7.9 Hz, CHCH(CH3)2), 1.51
(d, 6H, J ) 7.9 Hz, NCH(CH3)2), 1.97-1.99 (m, 1H, CHCH(CH3)2,
3.61-3.71 (m, 2H, CH2OH), 3.82-3.85 (m, 1H, CHNH), 4.55-4.62
(m, 1H, NCH(CH3)2), 4.99-5.01 (m, 1H, CHNH), 7.44 (d, 2H, J
) 4.1 Hz, Hphenyl), 7.55 (s, 1H, 8-H), 7.59 (d, 2H, J ) 4.1 Hz,
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[3-(4-pyridyl)phenylamino]-
9-iso-propylpurine (13f). Yield 88%, mp 85-97 °C. 1H NMR
(CDCl3): δ 0.96 (t, 3H, J ) 7.2 Hz, CH3CH2), 1.50 (d, 6H, J )
6.8 Hz, CH(CH3)2), 1.52-1.66 (m, 2H, CH3CH2), 3.60 (dd, 1H, J
) 7.2 Hz, J′ ) 10.6 Hz, CH2OH), 3.79 (dd, 1H, J ) 2.4 Hz and
J′ ) 10.6 Hz, CH2OH), 3.89-3.96 (m, 1H, CHNH), 4.58 (hept,
1H, CH(CH3)2), 4.92 (d, 1H, J ) 6.4 Hz, CHNH), 7.26 (d, 1H, J
) 7.6 Hz, Hphenyl), 7.39 (t, 1H, J ) 7.6 Hz, Hphenyl), 7.46 (d, 2H, J
) 6 Hz, Hpyridyl), 7.55 (s, 1H, H), 7.69 (s, 1H, Hphenyl), 7.73 (d, 1H,
J ) 7.6 Hz, Hphenyl), 8.60 (d, 2H, J ) 6 Hz, Hpyridyl). 13C NMR: δ
10.8, 22.3, 25.1, 46.7, 56.3, 67.2, 115.3, 118.5, 120.6, 121.5, 121.6,
129.4, 135.4, 138.8, 140.1, 148.5, 150.0, 152.1, 159.4.
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[4-(2-pyridyl)phenylamino]-
9-iso-propylpurine (13g). Yield 76%, yellow oil. 1H NMR (CDCl3):
δ 0.98 (t, 3H, J ) 7.2 Hz, CH3CH2), 1.46 (d, 6H, J ) 6.8 Hz,
CH(CH3)2), 1.52-1.68 (m, 2H, CH3CH2), 3.63 (dd, 1H, J ) 6.8
Hz and J′ ) 10,8 Hz, CH2OH), 3.81 (dd, 1H, J ) 2.8 Hz, J′ )
10,8 Hz, CH2OH), 3.90-3.98 (m, 1H, CHNH), 4.53 (hept, 1H, J
) 6.8 Hz, CH(CH3)2), 5.05 (d, 1H, J ) 6.8 Hz, CHNH), 7.09-7.14
(m, 1H, Hpyridyl), 7.51 (s, 1H, H-8), 7.60-7.68 (m, 2H, Hpyridyl),
7.80 (d, 2H, J ) 8.4 Hz, Hphenyl), 7.90 (d, 2H, J ) 8.4 Hz, Hphenyl),
8.59 (d, 1H, J ) 4.8 Hz, Hpyridyl). 13C NMR: δ 10.4, 21.9, 24.5,
45.8, 55.4, 65.8, 114.3, 119.5, 119.6, 121.1, 127.0, 132.7, 134.7,
136.4, 140.1, 148.7, 150.5, 151.4, 159.7, 158.8.
(R)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[4-(2-pyridyl)phe-
nylamino]-9-iso-propylpurine (13h). Yield 87%, mp 78-82 °C. 1H
NMR (CDCl3): δ 0.98 (d, 6H, J ) 7 Hz, CHCH(CH3)2), 1.48 (d,
6H, J ) 6.8 Hz, NCH(CH3)2), 1.97 (m, 1H, CHCH(CH3)2), 3.68
(dd, 1H, J ) 7,6 Hz, J′ ) 11 Hz, CH2OH), 3.84 (dd, 1H, J ) 2.6
Hz and J′ ) 11 Hz, CH2OH), 3.86-3.88 (m, 1H, CHNH), 4.54
(hept, 1H, J ) 6.8 Hz, NCH(CH3)2), 5.03 (d, 1H, J ) 6.8 Hz,
CHNH), 7.10-7.15 (m, 1H, Hpyridyl), 7.53 (s, 1H, 8-H), 7.64-7.68
(m, 2H, Hpyridyl), 7.80 (d, 2H, J ) 8.4 Hz, Hphenyl), 7.93 (d, 2H, J
) 8.4 Hz, Hphenyl), 8.60 (d, 1H, J ) 4.6 Hz, Hpyridyl). 13C NMR: δ
18.7, 19.8, 21.7, 29.5, 46.4, 51.8, 59.4, 114.2, 119.2, 119.6, 121.1,
126.9, 133.0, 134.5, 136.2, 139.5, 148.9, 151.4, 156.2, 159.2.
(R)-2-(1-Hydroxybut-2-ylamino)-6-[4-(3-pyridyl)phenylamino]-
9-iso-propylpurine (13i). Yield 79%, mp 85-105 °C. 1H NMR
(CDCl3): δ 1.06 (t, 3H, J ) 7.2 Hz, CH3CH2), 1.57 (d, 6H, J )
6.8 Hz, CH(CH3)2), 1.60-1.70 (m, 2H, CH3CH2), 3.70 (dd, 1H, J
) 6.8 Hz and J′ ) 11 Hz, CH2OH), 3.88 (d, 1H, J ) 11 Hz,
CH2OH), 3.96-4.02 (m, 1H, CHNH), 4.65 (hept, 1H, CH(CH3)2),
5.04 (d, 1H, J ) 8 Hz, CHNH), 7.35 (dd, 1H, J ) 4.8 Hz, J′ ) 7.6
Hz, Hpyridyl), 7.56-7.62 (m, 3H, Hphenyl + Hpyridyl), 7.68 (s, 1H, 8-H),
7.88 (d, 2H, J ) 8.4 Hz, Hphenyl), 8.57 (d, 1H, J ) 4.8 Hz, Hpyridyl),
8.86 (s, 1H, Hpyridyl). 13C NMR: δ 10.9, 22.5, 24.9, 46.7, 56.0, 67.3,
115.2, 120.3, 123.6, 127.4, 132.0, 133.8, 135.3, 136.2, 139.3, 148.0,
152.1, 159.7.
H
H
phenyl), 7.84 (d, 2H, J ) 6.2 Hz, Hpyridyl), 8.57 (d, 2H, J ) 6.2 Hz,
pyridyl). 13C NMR: δ 19.0, 19.5, 22.4, 30.0, 46.6, 59.7, 64.0, 115.1,
119.9, 121.0, 127.3, 131.9, 135.0, 140.3, 147.7, 150.0, 152.0, 160.0.
Docking on CK1. The multiple sequence alignment of the
proteins was created using ClustalW 1.82 (http://www.ch.embnet.
org/software/ClustalW.html). The crystal structure of CK1-δ (1CKJ)
with sequence numbering based on SwissProt entry P48730 was
used for all CK1 calculations. The ligands were placed in the
receptor binding cavity and were minimized with the two hydrogen
bonds formed between the receptor backbone and the purine ring
constrained at their crystallographic distances and angles.55 Then
a Monte Carlo local search of 1000 steps as implemented in
Macromodel v.956 was performed with all residues in a sphere of
6 Å around the ligand free to move and all remaining residues
frozen. In all calculations the TNCG minimizer, the AMBER* force
field and the GB/SA implicit solvent model were used. Prior to
docking, the three ligand molecules were subjected to a 1000-step
conformational search. An ab initio single point calculation of the
global minimum conformation was performed at the DFT level
using B3LYP method and the 6-31+G* basis set of JAGUAR v.4
for partial electrostatic atomic charge calculation. The pKa calcula-
tions were performed with Pallas v.3 software (Pallas 3.1.1.2,
CompuDrug Chemistry Ltd.). Alignment depictions were created
with ESPript.57 For the interaction energy calculations, the local
MP2 method as implemented in JAGUAR v.458 and the 6-31+G*
basis set were used. The LMP2 method is designed to avoid basis
set superposition error, so the BSSE was calculated only for the
HF energies using the Boys-Bernardi counterpoise method.59
Interaction energy calculations were performed by transforming the
ligand of the minimized 13a-CK1 complex in diphenylamine or
N-methylaniline, subtracting all protein atoms except the backbone
carbonyl acting as hydrogen bond acceptor and its amide nitrogen,
adding hydrogens to fill valences and performing single point
calculations. Experimental ∆∆G was calculated from IC50 values
13a
using the equation ∆∆G ) ∆G13a - ∆GC&R8 ) RT ln(IC50
IC50C&R8), approximating IC50 ∼ Kd.
/
Protein Kinase Assays. Biochemical Reagents. Sodium ortho-
vanadate, EGTA, EDTA, Mops, ꢀ-glycerophosphate, phenylphos-
phate, sodium fluoride, dithiothreitol (DTT), glutathione-agarose,
glutathione, bovine serum albumin (BSA), nitrophenylphosphate,
leupeptin, aprotinin, pepstatin, soybean trypsin inhibitor, benzami-
dine, and histone H1 (type III-S) were obtained from Sigma
Chemicals. [γ-33P]-ATP was obtained from Amersham. The CK-S
peptide (RRKHAAIGpSAYSITA) (pS stands for phosphorylated
serine) was purchased from Millegen, and the GS-1 peptide
(YRRAAVPPSPSLSRHSSPHQpSEDEEE) was obtained from Gen-
Script Corporation.
(R)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[4-(3-pyridyl)phe-
nylamino]-9-iso-propylpurine (13j). Yield 84%, mp 116-120 °C.
1H NMR (CDCl3) δ 1.05 (d, 6H, J ) 6.8 Hz, CHCH(CH3)2), 1.57
(d, 6H, J ) 6.8 Hz, NCH(CH3)2), 2.01 (hept, 1H, J ) 7 Hz,
CHCH(CH3)2), 3.75 (dd, 1H, J ) 8.0 Hz and J′ ) 10.4 Hz,
CH2OH), 3.90 (d, 1H, J ) 10.4 Hz, CH2OH), 4.09-4.17 (m, 1H,
CHNH), 4.64 (hept, 1H, NCH(CH3)2), 5.08 (d, 1H, J ) 6 Hz,
CHNH), 7.35 (dd, 1H, J ) 4.8 Hz, J′ ) 7.6 Hz, Hpyridyl), 7.56-7.62
(m, 3H, Hphenyl + Hpyridyl), 7.68 (s, 1H, 8-H), 7.88 (d, 2H, J ) 8.4
Hz, Hphenyl), 8.57 (d, 1H, J ) 4.8 Hz, Hpyridyl), 8.86 (s, 1H, Hpyridyl).
13C NMR: δ 19.0, 19.2, 22.3, 30.1, 46.7, 59.7, 65.2, 115.2, 120.1,
123.7, 127.5, 132.2, 133.7, 135.0, 136.3, 139.2, 147.9, 152.0, 159.9.
(R)-2-(1-Hydroxybut-2-ylamino)-6-[4-(4-pyridyl)phenylamino]-
Buffers. Buffer A: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT,
25 mM Tris-HCl pH 7.5, 50 µg heparin/mL. Buffer C: 60 mM
ꢀ-glycerophosphate, 15 mM p-nitrophenylphosphate, 25 mM Mops
(pH 7.2), 5 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM sodium
vanadate, 1 mM phenylphosphate.
Kinase Preparations and Assays. Kinase activities were assayed
in buffer A or C, at 30 °C, at a final ATP concentration of 15 µM.
Blank values were subtracted and activities expressed in % of the
maximal activity, i.e., in the absence of inhibitors. Controls were
performed with appropriate dilutions of DMSO.
CDK1/cyclin B (M phase starfish oocytes, native) and CDK5/
p25 (human, recombinant) were prepared as previously described.54
Their kinase activity was assayed in buffer C, with 1 mg histone
H1/mL, in the presence of 15 µM [γ-33P] ATP (3000 Ci/mmol; 10
1
9-iso-propylpurine (13k). Yield 37%, mp 126-130 °C. H NMR
(CDCl3): δ 0.94-0.99 (m, 3H, CH3CH2), 1.44-1.52 (m, 6H,
CH(CH3)2), 1.65-1.80 (m, 2H, CH3CH2), 3.66-3.75 (m, 1H,
CH2OH), 3.82-3.90 (m, 1H, CH2OH), 3.97-4.05 (m, 1H, CHNH),
4.58-4.65 (m, 1H, CH(CH3)2), 5.00-5.05 (m, 1H, CHNH),
7.43-7.47 (m, 2H, Hphenyl), 7.53-7.65 (m, 3H, Hphenyl + 8-H),

CDK/CK1 Dual-Specificity Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5241
mCi/mL) in a final volume of 30 µL. After 30 min incubation at
30 °C, 25 µL aliquots of supernatant were spotted onto 2.5 cm ×
3 cm pieces of Whatman P81 phosphocellulose paper, and, 20 s
later, the filters were washed five times (for at least 5 min each
time) in a solution of 10 mL of phosphoric acid/liter of water. The
wet filters were counted in the presence of 1 mL of ACS
(Amersham) scintillation fluid. GSK-3R/ꢀ (porcine brain, native)
was assayed as described for CDK1 but in buffer A and using GS-
1, a GSK-3 specific substrate.27 CK1 (porcine brain, native) was
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Health, PRO-KINASE Research Project) (L.M.), “Cance´ropole
Grand-Ouest” (L.M.), the “Ligue Nationale contre le Cancer”
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“Institut National du Cancer” (INCa) and the “Ministe`re de la
Recherche”. This work was supported by National Institutes of
Health Grant AG09464 (PG and MF).
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