Synthesis and evaluation of new potent inhibitors of CK1 and CDK5, two kinases involved in Alzheimer's disease

Article abstract of DOI:10.1007/s00044-012-0334-1

Cyclin-dependent kinase 5 (CDK5) and Casein kinase 1 (CK1) are both involved in the hyperphosphorylation of the Tau protein and in the amyloid-β production, the two major hallmarks of Alzheimer's disease. In the present paper, we describe the synthesis and biological evaluation of new series of 2,6,9-trisubstituted purines derived from DRF53, a dual specificity inhibitor of the kinase activity of CDK5 (IC₅₀ = 80 nM) and CK1 (IC₅₀ = 10 nM), and are able to prevent in a dose-dependent manner the CK1-dependent production of amyloid-β in a cell model. Several molecules (e.g., 6e, 6g, 7c) displayed potent kinase inhibitory activities against CDK5 and CK1 (IC₅₀ values ranging from 20 to 50 nM) among which a selective inhibitor of CK1 has been identified (5a, IC₅₀ = 60 nM). In addition, some compounds exhibit sub-micromolar activities against DYRK1A (dual specificity, tyrosine phosphorylation regulated kinase 1A), a kinase involved in Down syndrome and Alzheimer's disease (6g, IC₅₀ = 510 nM).

Full text of DOI:10.1007/s00044-012-0334-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0334-1
ORIGINAL RESEARCH
Synthesis and evaluation of new potent inhibitors of CK1
and CDK5, two kinases involved in Alzheimer’s disease
•
Luc Demange Olivier Lozach Yoan Ferandin
•
•
•
•
Nha Thu Hoang Laurent Meijer Herve Galons
´
Received: 4 April 2012 / Accepted: 6 November 2012
Ó Springer Science+Business Media New York 2012
Abstract Cyclin-dependent kinase 5 (CDK5) and Casein
kinase 1 (CK1) are both involved in the hyperphosphory-
lation of the Tau protein and in the amyloid-b production,
the two major hallmarks of Alzheimer’s disease. In the
present paper, we describe the synthesis and biological
evaluation of new series of 2,6,9-trisubstituted purines
derived from DRF53, a dual specificity inhibitor of the
kinase activity of CDK5 (IC50 = 80 nM) and CK1
(IC50 = 10 nM), and are able to prevent in a dose-depen-
dent manner the CK1-dependent production of amyloid-b
in a cell model. Several molecules (e.g., 6e, 6g, 7c) dis-
played potent kinase inhibitory activities against CDK5
and CK1 (IC50 values ranging from 20 to 50 nM) among
which a selective inhibitor of CK1 has been identified (5a,
IC50 = 60 nM). In addition, some compounds exhibit sub-
micromolar activities against DYRK1A (dual specificity,
tyrosine phosphorylation regulated kinase 1A), a kinase
involved in Down syndrome and Alzheimer’s disease
(6g, IC50 = 510 nM).
Keywords Alzheimer’s disease Á Down syndrome Á
CDK5 Á CK1 Á Kinase Á Roscovitine
Introduction
Alzheimer’s disease (AD) is characterized by a progressive
and irreversible alteration of memory, a loss of the language
skill, and a general deregulation of the cerebral activity. This
is the leading cause of dementia among the elderly, and its
economical impact is continually growing due to the increase
of life expectancy. To date, very few drugs, including
inhibitors of acetylcholinesterase and a NMDA receptor
antagonist (Gravitz, 2011), are clinically used to temporarily
restrain the effects of AD, but there is no treatment to prevent
or to cure this pathology at the moment.
The two major hallmarks of AD are the hyperphosph-
orylation of the Tau protein (responsible for the formation of
neurofibrillary tangles of paired helical filaments and for the
¨
cellular death) (Gotz et al., 2012) and the amyloid-b pro-
duction (responsible for the formation of extracellular
¨
amyloid plaques) (Ittner and Gotz, 2011). It has been sug-
gested that the deregulation of several multi-function protein
kinases (PKs) including Cyclin-dependent kinases 1 and 5
(CDK1, CDK5) (Cruz and Tsai 2004, 2006; Sadleir and
Vassar, 2012; Camins et al., 2006), Casein Kinase (CK1)
(Flajolet et al., 2007), Glycogen Synthase Kinase-3 (GSK3)
(Huang and Klein, 2006; Martin et al., 2011; Dominguez
et al., 2012), and the dual specificity tyrosine phosphoryla-
tion kinase 1A (DYRK1A) (Wegiel et al., 2011), might be
involved in those hallmarks. Thus, these PKs form a prom-
ising new class of potential targets in AD and their inhibition
might be promising against this pathology.
L. Demange (&) Á N. T. Hoang Á H. Galons
Laboratoire de Chimie et Biochimie Pharmacologiques et
´
Toxicologiques (LCBPT), UMR 8601 CNRS, Universite Paris
Descartes, Sorbonne Paris Cite, UFR Biomedicale des Saints
´
´
`
`
Peres, 45 rue des Saints Peres, and UFR de Pharmacie, 4, avenue
de l’Observatoire, 75270 Paris cedex 06, France
e-mail: lucdemange@yahoo.ca
O. Lozach Á Y. Ferandin Á L. Meijer (&)
CNRS, USR3151, ‘Protein Phosphorylation & Human Disease’
Group, Station Biologique, 29680 Roscoff, Bretagne, France
e-mail: meijer@manros-therapeutics.com
L. Meijer
ManRos Therapeutics, Hotel de Recherche, Centre de Perharidy,
29680 Roscoff, France
We previously reported the synthesis and the biological
evaluation of new families of 2,6,9-trisubstituted purines
ˆ
123

Med Chem Res
selective inhibitor
dual inhibitor
HN
N
N
N
HN
N
HN
N
N
N
HN
N
N
N
N
N
N
N
N
N
N
N
N
N
HN
HN
HN
N
Cl
OH
OH
OH
(R )-roscovitine
5a
DRF 53
7c
CDK1/cyclinB
CDK5/p25
CK1
0.35 µM
0.20 µM
2.3 µM
-
0.22 µM
0.08 µM
0.01 µM
10.0 µM
1.3 µM
0.05 µM
0.06 µM
0.06 µM
Fig. 1 IC50 comparison between previously reported kinase inhibitors (roscovitine and DRF53) and two selected molecules from the present
study. See legend of Table 1 for details; - not tested
(Oumata et al., 2008; Bettayeb et al., 2008; Demange et al.,
2008, 2012), structurally related to Roscovitine, an
advanced CDK inhibitor currently in clinical trials phase 2
or 2b against different kinds of cancers and in phase 1
against renal diseases including glomerulonephritis (Meijer
et al., 2006). Among these new series of molecules, DRF53
(Fig. 1), is one of the more potent inhibitors of CDK5
kinase activity (IC50 = 80 nM) and CK1 kinase activity
(IC50 = 10 nM). Moreover, this molecule is able to pre-
vent, in a dose-dependent manner, the CK1-dependent
production of amyloid-b in a cell model (Oumata et al.,
2008), and consequently might have applications to prevent
the appearance and development of AD’s hallmarks. We
explored in the present study the potencies of new deriv-
atives of the DRF53 family bearing a 6-aminobiarylic core
including a pyrimidine ring in terms of inhibition and
selectivity on relevant kinases (CDK1, CDK5, GSK-3,
CK1, and DYRK1A).
coupling reaction using 2-bromopyrimidine or 5-bromopy-
rimidine to give products 4a–b and 5a–b in high yield. Both
steps occur with a good regioselectivity, and without the
formation of secondary materials. Finally, the substitution of
the 2-chlorine of the purine ring by amino alcohols with an
excess of triethylamine in DMSO at 150 °C led to an average
yield of 35 % depending on the amino alcohol nucleophi-
licity to the expected 2,6,9-trisubstituted purine families
6a–k and 7a–k.
The structures of the newly synthesized trisubstituted
1
purines were confirmed by spectral data: H NMR, 13C
1
NMR, and mass spectra analysis. The NMR H spectra
were recorded in CDCl3; these spectra are divided into two
parts. The aliphatic protons correspond to the C2 amino–
alcohol, and to the N9 isopropyl, that appears as a classical
doublet (d range between 1.47 and 1.63 ppm) coupled with
a heptuplet (d range between 4.21 and 4.67 ppm) with
3J coupling constant range between 6.3 and 8.0 Hz. Among
the aromatic protons, the singlet at 7.88 ppm is character-
istic from the purine H8, and taking advantage of multi-
plicity, integration, and chemical displacement, the other
aromatic protons might be easily attributed to the phenyl or
pyrimidinyl ring. 13C NMR experiments confirm the scaf-
fold of the structures and the ion peaks of the mass spec-
trum are in agreement with the molecular formulas of all
compounds. Lastly, we determined the melting point for
each fully solid new molecule (see ‘‘Experimental proce-
dures’’ section for details).
Result and discussion
Chemistry
The new families of 2,6,9-trisubstituted purines were easily
synthesized from the commercial 2,6-dichloropurine as
previously described (Scheme 1) (Oumata et al., 2008).
Briefly, the regiospecific 9-alkylation of the starting material
was achieved in 70 % yield using isopropylbromide in
DMSO at 18 °C (in order to avoid regioselectivity problems,
the temperature should be carefully respected), and the
resulting 2,6-dichloro-9-isopropylpurine 1 was allowed to
react with appropriate boranylaniline to afford in high yield
the corresponding stable purines 2 and 3. These compounds
are converted into a biarylic core through a Suzuki cross-
Biological assay
The inhibition of CDK5/p25, GSK-3a/b, and CK1d/e ser-
ine threonine kinase (STK) activity was determined in the
presence of a range of concentrations of newly synthesized
products using an assay with [c–33P]-ATP as described in
123

Med Chem Res
O
O
B
O
B
O
HN
N
HN
Cl
Cl
N
N
N
N
N
N
N
N
N
N
N
N
b
a
N
Cl
Cl
N
Cl
N
Cl
H
3
2
1
c
N
N
N
N
N
N
HN
N
HN
HN
HN
N
N
N
N
N
N
N
N
N
N
N
N
R
N
R
Cl
N
N
N
Cl
N
N
N
H
4a
H
5a
7a-g
6a-d
N
d
N
N
N
HN
HN
N
N
HN
HN
N
N
N
N
N
N
N
N
N
N
N
N
Cl
N
N
R
Cl
N
R
N
N
N
4b
N
N
5b
H
H
6e-k
7h-k
Scheme 1 Reagents and conditions (a) 2-bromopropane, K2CO3, DMSO 15–18 °C, 5 days; (b) Ar–NH2, NEt3, BuOH, 100 °C, 1 day;
(c) 2-bromopyrimidine or 5-bromopyrimidine, Na2CO3, Pd[P(C6H5)3]4, H2O, dioxane, 8 h.; (d) RNH2, NEt3, DMSO, 150 °C, 2–5 days
the ‘‘Experimental procedures’’ section. In order to evalu-
ate the selectivity of the compounds, their inhibition of
kinase activity was controlled under the same conditions
with other purified kinases (DYRK1A, CDK1/cyclin B,
CDK2/cyclin A). IC50 values were determined from dose–
response curves. Globally, all newly synthesized purines
are micromolar or sub-micromolar inhibitors of the cata-
lytic activity of CDK5 and CK1.
DRF 53 (Table 1) or purine bioisosteres such as pyrazol-
o[1,5-a]-1,3,5-triazine (IC50 range between 70 and 320
nM) (Popowycz et al., 2009). Moreover, their activities are
very similar to the CDK5-specific inhibitors described by
others with different scaffolds including pyrazolopyrimi-
dine ring (IC50 = 30 nM) (Heathcore et al., 2010),
2,4-diaminothiazoles scaffold (IC50 range between 15 nM
and 1 lM) (Laha et al., 2011), and a cyclohexyl-thiophene
moiety linked with triazole (IC50 range between 35 nM and
1 lM) (Shiradkar et al., 2011).
The most active compounds against the kinase activity
of CDK5 reported in this paper are 6g, 7c, 7j, and 6e (IC50
range between 20 nM and 60 nM). Several newly synthe-
sized purines are more potent than Roscovitine or its
optimized derivative Purvalanol A (IC50 = 75 nM) and
exhibit IC50 values similar to our reference compound
Interestingly, the structure of the biarylic core seems to
be very important in terms of selectivity for CK1. In par-
ticular, compounds bearing the phenyl-3-(2-pyrimidinyl)
moiety (compounds 5a, 7a–7g) are very efficient against
123

Med Chem Res
Table 1 Biological evaluation of compounds bearing 6-Phenyl-4-(2-pyrimidinyl) and 6-Phenyl-4-(5-pyrimidinyl)
Compd. Scaffold
R
CDK1 CDK2 CDK5 GSK 3a/b DYRK1A CK1
6a
6b
6c
6d
(R)-1-hydroxy-but-2-yl
0.18
0.5
–
0.10
0.27
0.083
1.0
25
[10
7.1
9
–
1
(S)-1-hydroxy-but-2-yl
0.35
0.083
0.6
0.65
0.5
0.8
1.7
1.8
2.3
N
(R)-1-hydroxy-3-methylbut-2-yl
(S)-1-hydroxy-3-methylbut-2-yl
N
–
HN
N
N
N
N
R
NH
4b
6e
6f
Cl
–
1.1
2.1
[10
21
3.2
–
0.41
0.6
(R)-1-hydroxy-but-2-yl
(S)-1-hydroxy-but-2-yl
0.04
0.07
–
0.06
0.2
N
0.2.
0.03
0.61
0.16
0.33
0.2
100
10
1.2
0.51
1.1
1.1
1.2
2.3
1.3
N
6g
6h
6i
(R)-1-hydroxy-3-methylbut-2-yl 0.017
0.02
0.48
0.54
0.19
0.11
0.34
1.8
(S)-1-hydroxy-3-methylbut-2-yl
(S)-1-hydroxy-4-methylpent-2-
1,3-dihydroxyprop-2-yl
0.27
[10
13
HN
N
–
–
–
2.2
N
N
N
6j
35
0.64
0.5
R
6k
1-hydroxy-2-methylprop-2-yl
11
NH
Purines were tested at various concentrations on CDK1/cyclin B, CDK2/cyclin A, CDK5/p25, GSK-3a/ß, DYRK1A, and CK1d/e, as described in
the ‘‘Experimental procedures’’ section. IC50 values, calculated from the dose–response curves, are reported in lM. IC50 values are reported in
lM. IC50 value reported as [10 indicates that the compound did not display any inhibitory activity at the highest concentration tested (10 lM)
- not tested
this kinase (Table 2). In this family, compound 7c is a dual
inhibitor of CDK5 and CK1 (CDK 5 : IC50 = 50 nM;
CK1 : IC50 = 60 nM).
moiety. These stabilizing interactions (Fig. 2) might also
take place in the case of the strongest CK1 inhibitors
reported in this paper (compounds 5a or 7a–g, see
Table 2).
Products with a chlorine atom in the 6 position of the
purine ring are also highly selective for CK1, and the
presence of this halogen is not detrimental in terms of
inhibition as shown in the case of compound 5a (see
Table 2, CDK 5 : IC50 = 1900 nM; CK1 : IC50 = 60 nM)
which exhibits an IC50 value very close to that of DRF 53.
In either case, the substitution of chlorine by an amino
alcohol moiety does not improve the potency of the mol-
ecule against CK1—moreover, this substitution might be
detrimental (compare IC50 values for compounds 4b, and
6e–k, Table 1). Those results are very surprising because
previously reported 2,6,9-trisubstituted purines acting as
CK1 inhibitors have an amino alcohol in the 6 position
(DRF 53, see structure in Fig. 1, IC50 = 10 nM) (Oumata
et al., 2008).
In the past decade, several CK1 nM inhibitors (isolated
from natural sources) such as hymenialdisine (CDK
5:IC50 = 30 nM; CK1:IC50 = 30 nM), which is an pyr-
role-imidazole alkaloid extracted from marine sponge
(Meijer et al., 2000), or synthetic compounds such as the
potent 2,4-diaryl-imidazole family described by Peifer and
coworkers (IC50 values against CK1 kinase activity ranging
from 4 to 1500 nM) (Peifer et al., 2009) have been reported
(for a recent review, see Perez et al., 2011). More recently,
few pyrimidinyl pyrrolopyridinones (IC50 range between 3
nM and 800 nM) (Huang et al., 2012) and two polyhet-
erocyclic molecules, including both thiazole and benz-
imidazole rings (IC50 range between 40 and 100 nM)
(Bischof et al., 2012), have been reported as selective and
potent CK1 inhibitors, but their activities against CDKs
and precisely against CDK5 are still unknown. Moreover,
all these reported inhibitors have generally complex
structures and their preparation involves long multi-step
syntheses (see Papeo et al., 2005 and Mangu et al., 2010 in
We previously studied the docking of DRF53 in the
ATP-binding domain of the CK1 catalytic site (Oumata
et al., 2008) and suggested the presence of an H-bond
between the side chain of Arg 16 and the nitrogen in the 2
position of the pyridyl group belonging to the biarylic
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Med Chem Res
Table 2 Biological evaluation of compounds bearing 6-Phenyl-3-(2-pyrimidinyl) and 6-Phenyl-3-(5-pyrimidinyl)
Compd. Scaffold
R
CDK1 CDK2 CDK5 GSK 3a/b DYRK1A CK1
5a
7a
7b
7c
7d
Cl
10
0.3
0.73
–
1.9
1.3
0.2
0.5
0.05
0.9
0.11
0.62
2
[10
11
[10
–
0.06
0.06
0.1
(R)-1-hydroxy-but-2-yl
(S)-1-hydroxy-but-2-yl
(R)-1-hydroxy-3-methylbut-2-yl
(S)-1-hydroxy-3-methylbut-2-yl
1-hydroxy-2-methylprop-2-yl
1,3-dihydroxyprop-2-yl
(S)-1-hydroxy-4-methylpent-2-yl
0.32
0.074
0.7
–
17
2.1
2
N
HN
N
4.3
11
0.06
0.18
0.05
0.07
0.31
N
N
N
–
2
N
7e
7f
–
9.9
10
4.7
7
R
NH
–
–
7g
–
–
1.5
0.71
5b
7h
7i
Cl
–
–
1
[10
7.3
3
–
–
1
3
0.38
0.3
(R)-1-hydroxy-but-2-yl
(S)-1-hydroxy-but-2-yl
(R)-1-hydroxy-3-methylbut-2-yl
1-hydroxy-2-methylprop-2-yl
0.13
0.3
–
–
0.07
0.3
–
20
0.2
HN
N
N
7j
0.051
0.11
0.052
0.11
6.9
0.41
0.32
N
7k
–
[10
N
N
R
N
NH
See legend of Table 1 for details. All values are reported in lM
the case of hymenialdisine derivatives); consequently, the
subsequent pharmacomodulations, in order to improve
activity or selectivity, might be very challenging. By
contrast, the purine scaffold that is described here is
chemically accessible, stable, and adjustable. Purines are
widely used in medicinal chemistry due to their very
favorable biological properties (Legraverend and Grierson,
2006) and their use has been previously suggested in the
case of AD, for example to reduce the activity of
c-secretase and therefore the formation of amyloid-b pep-
tide and the amyloid plaques (Rivkin et al., 2010).
Lastly, we evaluated the effects of our new purines’
derivatives on DYRK1A. This serine/threonine kinase has
critical roles in human development and its deregulation is
suggested in multiple neurodegenerative pathologies. Thus,
its gene is located on chromosome 21, within the Down
syndrome (DS) critical region, and its over expression is
involved in the phosphorylation abnormalities of protein
tau observed in DS and AD (Frost et al., 2011). In addition,
DYRK1A has been reported to be involved in the neuro-
fibrillary tangles formation in AD (Wegiel et al., 2008;
2011). Altogether, these results pointed out that this kinase
might be a very promising therapeutic target against these
pathologies. With the significant exceptions of the analogs
of Lamellarin D including in their structure a complex
chromeno[3,4-b]indole skeleton (Neagoie et al., 2012), the
substituted bromo-indolic scaffold of meridianins deriva-
tives (Giraud et al., 2011), and few polysubstituted quin-
azolines (Rosenthal et al., 2011), there are very few
sub-micromolar DYRK1A inhibitors reported in the recent
literature (Becker and Sippl, 2011; Wang et al., 2012;
Debdab et al., 2011). Moreover, none of these molecules
have reached the stage of clinical evaluation. Among our
newly synthesized purines, several compounds such as 6b,
6d, and 7g exhibit significant inhibition of DYRK1A
(IC50 range between 0.5 and 0.7 lM). Moreover, 7g seems
to be quite selective for this kinase (see Table 2). To the
best of our knowledge, these molecules are the first purines
described as DYRK1A inhibitors. Therefore, they might be
H
N
Arg 16
H
N
N
H
H
Leu 88
O
N
H
N
N
N
N
N
N
H
N
R
Leu 88
Fig. 2 Suggestion of H-bonding between compounds 7a–g and key
amino acids from the ATP-binding pocket of CK1. This figure takes
advantage of our previously reported modeling of DRK53 in ATP
domain of the CK1 catalytic site (Oumata et al., 2008)
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Med Chem Res
General procedure for the Suzuki cross-coupling reaction
considered as potentially promising for development of
new DYRK1A kinase selective inhibitors.
To a solution of 2 or 3 (0.38 g, 1.0 mmol) in dioxane
(5 mL) and Na2CO3 1 M (5 mL) under N2 atmosphere,
Pd(PPh3)4 (58 mg, 0.05 mmol) was added. After 5 min. of
stirring, 2-bromopyrimidine or 5-bromopyrimidine
(238 mg, 1.5 mmol) was added. The mixture was heated
for 1 day at 100 °C, then concentrated in vacuo. The
resulting material is diluted in CH2Cl2, then washed with
water and brine. The organic layer was dried and evapo-
rated. The crude material was purified by chromatography
on silica gel, using AcOEt/Cyclohexane as the solvent.
Conclusion
We prepared in good yields new series of 6–aminobiarylic
purines structurally related to roscovitine and DRF 53
(Oumata et al., 2008) using a convergent synthesis proce-
dure that includes a Suzuki cross-coupling reaction as the
key step. Thus, we identified very potent inhibitors of the
kinase activity of CDK5 and CK1 (IC50 ranging below
50 nM). The strong binding of CK1 by these inhibitors is
reliable to a potential H-bond involving the Arg-16 of the
ATP binding site, as previously suggested by molecular
modeling in the case of DRF 53. Interestingly, some newly
synthesized molecules are promising inhibitors of
DYRK1A, another serine/threonine kinase involved in
several neuronal pathologies including AD. Although these
molecules endowed several atoms considered as H-bond
donors and acceptors involved in the inhibitor binding to
the ATP kinase pocket, a recent review (Ghose et al., 2012)
reveals that such kind of a compound might be considered
for the development of new CNS pathology targeted drugs.
Thus, further studies will allow us to determine whether the
most potent compounds are able to reduce the production
of amyloid-b in a cell model in order to identify their
potency to be used against Alzheimer’s disease.
2-Chloro-6-[4-(2-pyrimidinyl)phenylamino]-9-iso-pro-
pylpurine (4a) 1H NMR(CDCl3):
d 1.63 (d, 6H,
J = 6.8 Hz, CH(CH3)2), 4.88 (hept, 1H, J = 6.8 Hz,
CH(CH3)2), 7.16 (t, 1H, J = 4.8 Hz, Hpyrimidyl), 7.92 (s,
1H, 8-H), 7.92 (d, 2H, J = 9.2 Hz, Hphenyl), 8.49 (d, 2H,
J = 8.8 Hz, Hphenyl), 8.79 (d, 2H, J = 4.8 Hz, Hpyrimidyl).
2-Chloro-6-[4-(5-pyrimidinyl)phenylamino]-9-iso-pro-
pylpurine (4b) m.p. 264–268 °C. 1H NMR(CDCl3): d
1.63 (d, 6H, J = 9 Hz, (CH3)2CHNH), 4.89 (hept, 1H,
J = 9 Hz, (CH3)2CHNH), 7.64 (d, 1H, J = 9 Hz, Hphenyl),
7.90 (s, 1H, NH), 7.92 (s, 1H, 8-H), 7.98 (d, 1H, J = 9 Hz,
H
phenyl), 8.98 (s, 2H, Hpyrimidinyl), 9.20 (s, 1H, Hpyrimidinyl).
13C NMR (DMSO): d 22.0, 47.0, 119.3, 121.3, 127.0,
128.1, 132.8, 139.8, 140.6, 150.4, 151.9, 152.1, 154.2,
156.7. MS (ES?) m/z 366 (M?H)?, 388 (M?Na)?.
2-Chloro-6-[3-(2-pyrimidinyl)phenylamino]-9-iso-pro-
1
pylpurine (5a) m.p. [ 270 °C. H NMR(CDCl3): d 1.62
Experimental procedures
Chemistry
(d, 6H, J = 9 Hz, (CH3)2CHNH), 4.88 (hept, 1H,
J = 9 Hz, (CH3)2CHNH), 5.30 (s, 1H, NH), 7.23 (t, 1H,
J = 6 Hz, Hpyrimidinyl), 7.56 (t, 1H, J = 9 Hz, Hphenyl),
7.89 (s, 1H, 8-H), 8.22 (d, 1H, J = 8 Hz, Hphenyl), 8.26 (d,
1H, J = 8 Hz, Hphenyl), 8.60 (bs, 1H, Hphenyl), 8.84 (d, 1H,
J = 6 Hz, Hpyrimidinyl). 13C NMR(DMSO): d 22.1, 47.1,
119.2, 120.1, 121.0, 123.0, 123.9, 129.0, 137.8, 139.3,
140.6, 150.5, 152.1, 152.5, 157.8, 163.3. MS (ES?) m/z 366
(M?H)?, 388 (M?Na)?.
General procedures
Chemical reagents and solvents were purchased from
Sigma-Aldrich, Fluka, and Carlo Erba. Reactions were
monitored by TLC using Merk silica gel 60F-254 thin layer
plates. Column chromatographies were performed on SDS
Chromagel 60 ACC 40–63 lM. Melting points were
¨
determined on a Kofler hot-stage (Reichert) and were
2-Chloro-6-[3-(5-pyrimidinyl)phenylamino]-9-iso-pro-
pylpurine (5b) m.p. 188–190 °C. 1H NMR(CDCl3): d
1.63 (d, 6H, J = 6.9 Hz, (CH3)2CHNH), 4.88 (hept, 1H,
J = 6.9 Hz, (CH3)2CHNH), 7.35 (d, 1H, J = 7.8 Hz,
uncorrected. NMR spectra were recorded on Brucker
Avance 400 MHz (100 MHz for 13C NMR) at 300 K.
Chemical shifts were reported as d values (ppm) indirectly
referenced to the solvent signal or to tetramethylsilane
(TMS) as the internal standard. Data are reported in the
conventional form. Mass spectra were recorded on a ZQ
2000 Waters using a Z-spray (ESI–MS).
Hphenyl), 7.54 (t, 1H, J = 7.8 Hz, Hphenyl, 7.77 (d, 1H,
J = 8.6 Hz, Hphenyl), 7.90-7.96 (m, 2H, 8-H ? NH), 8.23
(s, 1H, Hphenyl), 9.03 (s, 2H, Hpyrimidyl), 9.24 (s, 1H,
pyrimidyl). 13C NMR(DMSO): d 22.0, 47.0, 119.1, 119.4,
H
121.3, 121.6, 129.5, 133.2, 133.8, 139.8, 140.5, 150.4,
151.9, 152.2, 154.5, 157.3. MS (ES?) m/z 366 (M?H)?,
388 (M?Na)?.
Synthesis and structures of compounds 1, 2, and 3 have
been previously reported in the literature and are not
described in this section.
123

Med Chem Res
General procedure for the N2 amination
(S)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[4-(2-pyrimid-
inyl)phenylamino]-9-iso-propylpurine (6d) Yield 47 %,
m.p. 129–135 °C. 1H NMR(CDCl3): d 0.98 (d, 6H,
J = 6.9 Hz, (CH3)2CHCH), 1.47 (d, 6H, J = 6.6 Hz,
NCH(CH3)2), 1.91–2.02 (m, 1H, CHCH(CH3)2), 3.68 (dd,
1H, J = 10.3 Hz and J’ = 7.8 Hz, CH2OH), 3.79–3.93 (m,
2H, CH2OH ? CHNH), 4.52 (hept, 1H, J = 6.6 Hz,
(CH3)2CHNH), 5.00 (1H, bd, J = 6.4 Hz, NH), 7.07 (t,
1H, J = 4.8 Hz, Hpyrimidinyl), 7.52 (s,1H, 8-H), 7.82 (d, 2H,
J = 8.6 Hz, Hphenyl), 7.91 (s, 1H, NH), 8.35 (d, 2H,
J = 8.6 Hz, Hphenyl), 8.70 (d, 2H, J = 4.8 Hz, Hpyrimidinyl).
13C NMR: d 18.9, 19.4, 22.4, 30.0, 46.4, 59.5, 65.0, 115.0,
118.4, 119.2, 128.8, 131.5, 135.0, 141.9, 150.7, 151.8,
157.1, 159.8, 164.4. MS (ES?) m/z 433 (M?H)?, 455
(M?Na)?.
To a solution of 4 or 5 (0.2 g, 0.5 mmol) and the selected
amino alcohol (2.5 mmol) in DMSO (1 mL), NEt3 (0.5 mL)
was added. The mixture was heated at 150 °C for 1–5 days.
After cooling to r.t., 20 mL of CH2Cl2 was added, and the
resulting solution was extracted three times with water and
brine. The organic layer was dried and evaporated. The crude
material was purified by chromatography on silica gel, using
AcOEt/Cyclohexane/NEt3 as the solvent.
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[4-(2-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (6a) Yield 39 %. 1H
NMR(CDCl3): d 0.99 (t, 3H, J = 8 Hz, CH3CH2), 1.48 (d,
6H, J = 6.8 Hz, CH(CH3)2), 1.52–1.67 (m, 2H, CH3CH2),
3.57-3.66 (m, 1H, CH2OH), 3.77–3.85 (m, 1H, CH2OH),
3.90-3.99 (m, 1H, CHNH), 4.55 (hept, 1H, J = 6.8 Hz,
NCH(CH3)2), 4.97–5.03 (m, 1H, NH), 7.07 (t, 1H,
J = 5.0 Hz, Hpyrimidinyl), 7.53 (s,1H, 8-H), 7.84 (d, 2H,
J = 8.5 Hz, Hphenyl), 8.36 (d, 2H, J = 8.5 Hz, Hphenyl),
8.71 (d, 2H, J = 5.0 Hz, Hpyrimidinyl). 13C NMR: d 10.9,
22.4, 22.5, 24.9, 46.6, 56.1, 63.7, 115.2, 118.5, 119.4,
129.0, 131.9, 135.3, 141.6, 152.0, 157.1, 164.4. MS (ES?)
m/z 419 (M?H)?, 441 (M?Na)?.
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[4-(5-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (6e) Yield 40 %, m.p.
1
112–118 °C. H NMR(CDCl3): d 1.06 (t, 3H, J = 7,2 Hz,
CH3CH2), 1.57 (d, 6H, J = 6,8 Hz, NCH(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, J = 6,8 Hz,
CH(CH3)2), 7.64 (d, 1H, J = 8.8 Hz, Hphenyl), 7.86 (s, 1H,
8-H), 7.99 (d, 2H, J = 8.8 Hz, Hphenyl), 8.97 (s, 2H,
H
pyrimidinyl), 9.2 (s, 1H, pyrimidinyl). 13C NMR: d 10.9, 22.4,
22.5, 24.9, 46.7, 55.9, 67.1, 115.1, 120.4, 127.3, 128.0,
133.9, 135.3, 140.2, 150.8, 152.0, 154.4, 156.9, 159.6, MS
(ES?) m/z 419 (M?H)?, 441 (M?Na)?, 457 (M?K)?.
(S)-2-(1-Hydroxy-but-2-ylamino)-6-[4-(2-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (6b) Yield 43 %. 1H
NMR(CDCl3): d 1.07 (t, 3H, J = 7.2 Hz, CH3CH2), 1.59
(d, 6H, J = 6.8 Hz, (CH3)2CHN), 1.63-1.67 (m, 2H,
CH3CH2), 3.70–3.85 (m, 2H, CH2OH), 3.96–4.02 (m, 1H,
CHNH), 4.21 (hept, 1H, J = 6.8 Hz, (CH3)2CHN), 7.15 (t,
1H, J = 4.8 Hz, Hpyrimidinyl), 7.62 (s,1H, 8-H), 7.92 (d, 2H,
J = 8.4 Hz, Hphenyl), 8.45 (d, 2H, J = 8.4 Hz, Hphenyl),
8.78 (d, 2H, J = 4.8 Hz, Hpyrimidinyl). 13C NMR: d 10.8,
22.3, 22.5, 24.9, 46.5, 56.1, 63.6, 115.2, 118.5, 119.3,
129.0, 131.8, 135.3, 141.6, 152.0, 157.2, 164.4. MS (ES?)
m/z 419 (M?H)?, 441 (M?Na)?.
(S)-2-(1-Hydroxy-but-2-ylamino)-6-[4-(5-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (6f) Yield 29 %, m.p.
1
110–115 °C. H NMR(CDCl3): d 0.96 (t, 3H, J = 7,2 Hz,
CH3CH2),1.63 (d, 6H, J = 6.8 Hz, NCH(CH3)2),
1.63–1.67 (m, 2H, CH3CH2), 3.70–3.85 (m, 2H, CH2OH),
3.96-4.02 (m, 1H, CHNH), 4.82 (hept, 1H, J = 6.8 Hz,
CH(CH3)2), 7.64 (d, 1H, J = 8.4 Hz, Hphenyl), 7.86 (s, 1H,
8-H), 7.99 (d, 2H, J = 8.4 Hz, Hphenyl), 8.97 (s, 2H,
H
pyrimidinyl), 9.2 (s, 1H, Hpyrimidinyl). 13C NMR: d 10.9,
22.3, 22.4, 24.8, 46.7, 55.8, 66.6, 115.2, 120.4, 127.1,
127.8, 133.8, 135.2, 151.9, 154.3, 156.7, 159.5. MS (ES?)
m/z 419 (M?H)?, 441 (M?Na)?, 457 (M?K)?.
(R)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[4-(2-pyri-
midinyl)phenylamino]-9-iso-propylpurine
(6c) Yield
1
38 %, m.p. 128–133 °C. H NMR(CDCl3): d 0.99 (d, 6H,
J = 6.5 Hz, (CH3)2CHCH), 1.47 (d, 6H, J = 6.6 Hz,
NCH(CH3)2), 1.91–2.02 (m, 1H, CHCH(CH3)2), 3.69 (dd,
1H, J = 10.8 Hz and J’ = 8.0 Hz, CH2OH), 3.78–3.92 (m,
2H, CH2OH ? CHNH), 4.52 (hept, 1H, J = 6.4 Hz,
(CH3)2CHNH), 5.00 (1H, bd, J = 7.1 Hz, NH), 7.07 (t,
1H, J = 4.5 Hz, Hpyrimidinyl), 7.52 (s,1H, 8-H), 7.82 (d, 2H,
J = 7.8 Hz, Hphenyl), 7.91 (s, 1H, NH), 8.36 (d, 2H,
J = 7.8 Hz, Hphenyl), 8.71 (d, 2H, J = 4.5 Hz, Hpyrimidinyl).
13C NMR: d 18.9, 19.4, 22.4, 30.0, 46.4, 59.5, 65.1, 115.1,
118.4, 119.2, 128.8, 131.7, 135.1, 141.9, 150.0, 151.8,
157.1, 159.8, 164.4. MS (ES?) m/z 433 (M?H)?, 455
(M?Na)?.
(R)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[4-(5-pyri-
midinyl)phenylamino]-9-iso-propylpurine
(6g) Yield
39 %. 1H NMR(CDCl3): d 0.99 (d, 6H, J = 6.8 Hz,
CH3CH),1.50 (d, 6H, J = 6.6 Hz, NCH(CH3)2), 1.93–2.03
(m, 1H, (CH3)2CH), 3.68 (dd, 1H, J = 10.8 Hz and
J’ = 7.7 Hz, CH2OH), 3.82–3.90 (m, 2H, CH2OH ?
CHNH), 4.57 (hept, 1H, J = 6.6 Hz, CH(CH3)2), 5.03 (1H,
bd, J = 7.4 Hz, NH), 7.51 (d, 2H, J = 8.1 Hz, Hphenyl),
7.54 (s, 1H, 8-H), 7.81 (bs, 1H, NH), 7.88 (d, 2H,
J = 8.1 Hz, Hphenyl), 8.89 (s, 2H, Hpyrimidinyl), 9.11 (s, 1H,
123

Med Chem Res
Hpyrimidinyl). 13C NMR: d 19.0, 19.4, 22.4, 22.5, 30.0, 46.6,
59.6, 65.3, 115.1, 120.3, 127.2, 127.9, 133.8, 135.3, 140.3,
150.8, 151.9, 154.4, 156.9, 159.8. MS (ES?) m/z 433
(M?H)?, 455 (M?Na)?.
8-H), 7.81 (bs, 1H, NH), 7.90 (d, 2H, J = 8.3 Hz, Hphenyl),
8.96 (s, 2H, Hpyrimidinyl), 9.18 (s, 1H, Hpyrimidinyl). 13C
NMR: d 22.3, 24.9, 47.0, 55.3, 71.5, 114.9, 120.6, 127.2,
128.2, 133.7, 135.3, 139.9, 149.9, 152.2, 154.3, 156.8,
158.2.
(S)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[4-(5-pyrimid-
inyl)phenylamino]-9-iso-propylpurine (6h) Yield 41 %.
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[3-(2-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (7a) Yield 31 %, m.p.
117–124 °C. 1H NMR(CDCl3): 1.00 (t, 3H, J = 8 Hz,
CH3CH2), 1.51 (m, 6H, CH(CH3)2), 1.58–1.67 (m, 2H,
CH3CH2), 3.62–3.80(m, 2H, CH2OH), 4.00–4.08 (m, 1H,
CHNH), 4.59 (hept, 1H, J = 6.8 Hz, CH(CH3)2),
7.14–7.20 (m, 1H, Hpyrmidinyl), 7.43 (t, 1H, J = 7 Hz,
1H NMR(CDCl3):
d
0.99 (d, 3H, J = 6.8 Hz,
CH3CH2),1.51 (d, 6H, J = 6.8 Hz, NCH(CH3)2),
1.90–2.03 (m, 1H, (CH3)2CH), 3.60–3.71 (m, 1H, CH2OH),
3.82–3.90 (m, 2H, CH2OH ? CHNH), 4.59 (hept, 1H,
J = 6.8 Hz, CH(CH3)2), 5.06 (1H, bd, J = 7.6 Hz, NH),
7.52 (d, 2H, J = 8.6 Hz, Hphenyl), 7.57 (s, 1H, 8-H), 7.93
(d, 2H, J = 8.6 Hz, Hphenyl), 8.40 (bs, 1H, NH) 8.90
(s, 2H, Hpyrimidinyl), 9.10 (s, 1H, Hpyrimidinyl). 13C NMR: d
19.0, 19.4, 22.4, 22.5, 30.0, 46.6, 59.6, 65.4, 115.1, 120.3,
127.3, 128.0, 133.8, 135.3, 140.3, 152.0, 154.4, 156.9,
159.8. MS (ES?) m/z 433 (M?H)?, 455 (M?Na)?.
H
phenyl), 7.55 (s, 1H, 8-H), 7.58 (m, 1H, Hphenyl), 7.67–7.74
(m, 1H, Hphenyl) 8.09 (d, 1H, J = 7 Hz, Hphenyl), 8.76 (d,
2H, J = 8 Hz, Hpyrimidinyl). 13C NMR: d 10.9, 22.5, 22.6,
25.0, 46.4, 56.0, 68.1, 119.2, 119.4, 122.1, 122.8, 128.7,
129.1, 130.9, 132.4, 135.1, 138.1, 139.6, 152.2, 157.2,
159.6, 164.6. 167.7. MS (ES?) m/z 419 (M?H)?, 441
(M?Na)?.
(S)-2-(1-Hydroxy-4-methylpent-2-ylamino)-6-[4-(5-
pyrimidinyl)phenylamino]-9-iso-propylpurine (6i) Yield
49 %. 1H NMR(CDCl3):
d
0.83–1.02 (m, 6H,
(S)-2-(1-Hydroxy-but-2-ylamino)-6-[3-(2-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (7b) Yield 35 %, m.p.
(CH3)2CHCH2), 1.36–1.48 (m, 2H, CHCH2CH), 1.57 (d,
6H, J = 6.5 Hz, (CH3)2CH), 1.71–1.84 (m, 1H,
(CH3)2CHCH2), 3.84 (d, 1H, J = 11.2 Hz, CH2OH),
4.07–4.25 (m, 2H, CH2OH ? CHNH), 4.64 (hept, 1H,
J = 6.5 Hz, CH(CH3)2), 5.01 (1H, bd, J = 7.1 Hz, NH),
7.56 (d, 2H, J = 8.5 Hz, Hphenyl), 7.61 (s, 1H, 8-H), 7.85
(bs, 1H, NH), 7.93 (d, 2H, J = 8.5 Hz, Hphenyl), 8.95
(s, 2H, Hpyrimidinyl), 9.17 (s, 1H, Hpyrimidinyl). 13C NMR: d
22.3, 22.4, 23.1, 24.8, 41.0, 46.5, 52.0, 67.2, 114.9, 120.3,
127.1, 127.8, 133.8, 135.2, 140.3, 150.9, 151.9, 154.3,
156.7, 159.4, MS (ES?) m/z 447 (M?H)?, 469 (M?Na)?.
1
118–125 °C. H NMR(CDCl3): d 1.01 (t, 3H, J = 7,2 Hz,
CH3CH2), 1.57 (d, 6H, J = 6,8 Hz, CH(CH3)2), 1.63-1.67
(m, 2H, CH3CH2), 3.70–3.85(m, 2H, CH2OH), 3.96–4.02
(m, 1H, CHNH), 4.63 (hept, 1H, J = 6.8 Hz, CH(CH3)2),
7.23 (t, 1H, J = 4.8 Hz,
J = 7.6 Hz, Hphenyl), 7.61 (s, 1H, 8-H), 7.68 (m, 1H,
phenyl), 7.72–7.82 (m, 1H, Hphenyl 8.15 (d, 1H,
Hpyrmidinyl), 7.48 (t, 1H,
H
)
J = 7.6 Hz, Hphenyl), 8.83 (d, 2H, J = 4.8 Hz, Hpyrimidinyl).
13C NMR: d 10.8, 22.5, 22.6, 25.0, 46.4, 55.9, 66.9, 115.0,
119.2, 119.4, 122.1, 122.7, 129.1, 135.1, 138.1, 139.7,
150.8, 152.2, 157.2, 159.6, 164.5. MS (ES?) m/z 441
(M?Na)?.
1,3-(Dihydroxyprop-2-yl)-6-[4-(5-pyrimidinyl)phenyla-
1H
mino]-9-iso-propylpurine
(6j) Yield
25 %.
NMR(CDCl3): d 1.58 (d, 6 H, J = 6.6 Hz (CH3)2CH),
3.70–3.90 (m, 7H, (CH2OH)2 ? (CH2OH)2 ? CH
(CH2OH)2), 4.66 (hept, 1H, J = 6.6 Hz, CH(CH3)2), 5.80
(bd, 1H, J = 6.0 Hz, NH), 7.58 (d, 2H, J = 8.1 Hz,
(R)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[3-(2-pyri-
(7c) Yield
midinyl)phenylamino]-9-iso-propylpurine
1
23 %, m.p. 115–122 °C. H NMR(CDCl3): d 1.03 (d, 3H,
J = 6.7 Hz, CH3CH), 1.05 (d, 3H, J = 6.7 Hz, CH3CH),
1.55 (d, 3H, J = 6.8 Hz, CH3CHN), 1.56 (d, 3H,
J = 6.8 Hz, CH3CHN), 2.03–2.14 (m, 1H, (CH3)2CH CH),
3.76–3.82 (m, 2H, CH2OH), 4.06–4.16 (m, 1H,
CHCH2OH), 4.65 (hept, 1H, J = 6.7 Hz, (CH3)3CH),
5.01–5.10 (m, 1H, NH), 7.22 (t, 1H, J = 4.8 Hz, Hpyrimidinyl),
7.47 (t, 1H, J = 7.9 Hz, Hphenyl), 7.60 (s, 1H, 8-H), 7.70–7.82
(m, 1H, Hphenyl), 7.82–7.87 (m, 1H, Hphenyl), 8.15 (d, 1H,
J = 7.9 Hz, Hphenyl)), 8.82 (d, 2H, J = 4.8 Hz, Hpyrimidinyl),
9.00–9.20 (m, 1H, NH). 13C NMR: d 19.6, 22.5, 22.6, 29.6,
46.3, 59.3, 114.9, 119.2, 119.4, 122.1, 122.6, 129.0, 135.0,
138.1, 139.8, 152.2, 157.2, 159.8, 164.5. MS (ES?) m/z 433
(M?H)?, 455 (M?Na)?.
H
phenyl), 7.64 (s, 1H, 8-H), 8.03 (d, 2H, J = 8.1 Hz,
H
phenyl), 8.19 (s, 2H, Hpyrimidinyl), 8.33 (s, 1H, Hpyrimidinyl).
13C NMR(DMSO): 22.3, 46.5, 55.1, 60.5, 114.4, 120.6,
127.0, 127.4, 133.4, 136.9, 141.5, 151.6, 152.1, 154.4,
156.8, 158.8. MS (ES?) m/z 443 (M?Na)?.
2-(1-Hydroxy-2-methylprop-2-yl)-6-[4-(5-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (6k) Yield 29 %, m.p.
1
235–238 °C. H NMR(CDCl3): d 1.44 (s, 6H, C(CH3)2),
1.60 (d, 6H, J = 6.5 Hz, (CH3)2CH), 3.75 (bs, 2H,
CH2OH), 4.62 (hept, 1H, J = 6.5 Hz, (CH3)2CH), 5.16 (bs,
1H, NH), 7.58 (d, 2H, J = 8.3 Hz, Hphenyl), 7.62 (s, 1H,
123

Med Chem Res
(S)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[3-(2-pyrimid-
inyl)phenylamino]-9-iso-propylpurine (7d) Yield 22 %,
J = 7.8 Hz, Hphenyl), 7.53 (s, 1H, 8-H), 7.71–7.74 (m, 1H,
phenyl), 7.99 (d, 1H, J = 8.3 Hz, Hphenyl), 8.09 (d, 1H,
H
1
m.p. 126–135 °C. H NMR(CDCl3): d 1.01–1.07 (m, 6H,
J = 8.2 Hz, Hphenyl)), 8.55 (bs, 1H, NH), 8.75 (d, 2H,
J = 4.5 Hz, Hpyrimidinyl). 13C NMR: d 22.4, 25.1, 46.8,
55.3, 72.0, 115.0, 119.1, 119.9, 122.8, 123.0, 129.1, 135.1,
138.2, 139.2, 149.6, 152.6, 157.1, 158.3, 164.3. MS (ES?)
m/z 419 (M?H)?, 441 (M?Na)?.
(CH3)2CH), 1.52–1.58 (m, 6H, 1.55 (d, 3H, J = 6.8 Hz,
CH(CH3)2), 2.03–2.13 (m, 1H, (CH3)2CH CH), 3.77–3.83
(m, 2H, CH2OH), 4.02–4.16 (m, 1H, CHCH2OH), 4.64
(hept, 1H, J = 6.7 Hz, (CH3)3CH), 5.02–5.12 (m, 1H,
NH), 7.22 (t, 1H, J = 4.7 Hz, Hpyrimidinyl), 7.47 (t, 1H,
J = 7.8 Hz, Hphenyl), 7.60 (s, 1H, 8-H), 7.71–7.80 (m, 1H,
(R)-2-(1-Hydroxy-but-2-ylamino)-6-[3-(5-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (7h) Yield 30 %, m.p.
H
phenyl), 7.82–7.88 (m, 1H, Hphenyl), 8.15 (d, 1H,
1
J = 7.6 Hz, Hphenyl)), 8.82 (d, 2H, J = 4.8 Hz, Hpyrimidi-
nyl), 8.98–9.12 (m, 1H, NH). 13C NMR: d 19.6, 22.5, 22.6,
29.6, 46.3, 59.3, 114.9, 119.2, 119.4, 122.0, 122.7, 129.0,
135.1, 138.0, 139.7, 149.0, 152.1, 157.2, 159.8, 164.5. MS
(ES?) m/z 433 (M?H)?, 455 (M?Na)?.
116–122 °C. H NMR(CDCl3): d 1.03 (t, 3H, J = 8 Hz,
CH3CH2), 1.58 (d, 6H, J = 8 Hz, CH(CH3)2), 1.62–1.74
(m, 2H, CH3CH2), 3.64–3.77 (m, 1H, CH2OH), 3.86 (dd,
1H, J = 12 Hz and J’ = 4 Hz, CH2OH), 3.95–4.04 (m,
1H, CHNH), 4.65 (hept, 1H, J = 8 Hz, CH(CH3)2), 4.99
(d, 1H, J = 5 Hz, NH), 7.28–7.30 (m, 1H, Hphenyl), 7.49 (t,
1H, J = 9 Hz, Hphenyl), 7.62 (s, 1H, 8-H), 7.70–7.78 (m,
2H, Hphenyl), 8.15 (bs, 1H, NH), 9.00 (s, 2H, Hpyrimidinyl),
9.22 (s, 1H, Hpyrimidinyl). 13C NMR: d 10.9, 22.4, 22.5, 24.8,
46.6, 56.0, 67.1, 115.0, 118.2, 120.2, 121.3, 129.9, 134.3,
134.9, 135.4, 140.2, 152.1, 154.9, 157.4, 159.6. MS (ES?)
m/z 441 (M?Na)?.
(S)-2-(1-Hydroxy-4-methylpent-2-ylamino)-6-[3-(2-
pyrimidinyl)phenylamino]-9-iso-propylpurine (7g) Yield
1
27 %, m.p. 114–120 °C. H NMR(CDCl3): d 0.84 (d, 3H,
J = 6.5 Hz, (CH3)2CHCH2), 0.89 (d, 3H, J = 6.4 Hz,
(CH3)2CHCH2), 1.34–1.44 (m, 2H, CHCH2CH), 1.49 (d,
6H, J = 6.8 Hz, (CH3)2CH), 1.68–1.81 (m, 1H,
(CH3)2CHCH2), 3.56–3.63 (m, 1H, CH2OH), 3.66–3.76
(m, 1H, CH2OH), 4.19–4.30 (m, 1H, CHNH), 4.59 (hept,
1H, J = 6.8 Hz, CH(CH3)2), 7.16 (t, 1H, J = 4.9 Hz,
(S)-2-(1-Hydroxy-but-2-ylamino)-6-[3-(5-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (7i) Yield 31 %, m.p.
1
H
pyrimidinyl), 7.40 (t, 1H, J = 8.1 Hz, Hphenyl), 7.54 (s, 1H,
8-H), 7.60–7.71 (m, 1H, Hphenyl), 7.73–7.80 (m, 1H,
phenyl), 8.09 (d, 1H, J = 7.7 Hz, Hphenyl)), 8.76 (d, 2H,
120–127 °C. H NMR(CDCl3): d 0.95 (t, 3H, J = 8 Hz,
CH3CH2), 1.50 (d, 6H, J = 8 Hz, CH(CH3)2), 1.57–1.67
(m, 2H, CH3CH2), 3.60 (dd, 1H, J = 12 Hz and
J’ = 8 Hz, CH2OH), 3.79 (dd, 1H, J = 12 Hz and
J’ = 4 Hz, CH2OH), 3.88–3.97 (m, 1H, CHNH), 4.58
(hept, 1H, J = 8 Hz, CH(CH3)2), 4.93 (d, 1H, J = 8 Hz,
NH), 7.20–7.22 (m, 1H, Hphenyl), 7.42 (t, 1H, J = 9 Hz,
H
J = 4.8 Hz, Hpyrimidinyl), 8.94–9.06 (m, 1H, NH). 13C
NMR: d 22.2, 22.4, 22.5, 23.3, 24.8, 41.2, 46.3, 52.3, 67.4,
114.8, 119.1, 119.4, 122.1, 122.7, 129.0, 135.0, 138.0,
139.7, 152.2, 157.2, 159.4, 164.6. MS (ES?) m/z 447
(M?H)?, 469 (M?Na)?.
H
phenyl), 7.55 (s, 1H, 8-H), 7.78–7.82 (bs, 1H, Hphenyl), 8.09
(bs, 1H, NH), 8.93 (s, 2H, Hpyrimidinyl), 9.15 (s, 1H,
pyrimidinyl). 13C NMR: d 10.9, 22.4, 22.5, 24.7, 46.7, 56.0,
1,3-(Dihydroxyprop-2-yl)-6-[3-(2-pyrimidinyl)phenyla-
H
mino]-9-iso-propylpurine
136–144 °C. 1H NMR(DMSO):
J = 6.3 Hz, (CH3)2CH),
(7f) Yield
30 %,
m.p.
H,
67.2, 115.1, 118.2, 120.2, 121.3, 129.9, 134.3, 134.9,
135.4, 140.2, 152.1, 154.9, 157.4, 159.6. MS (ES?) m/z 419
(M?H)?, 441 (M?Na)?, 457 (M?K)?.
d
1.46 (d, 6
3.55–3.68
(m,
5H,
(CH2OH)2 ? CH(CH2OH)2), 4.56 (hept, 1H, J = 6.3 Hz,
CH(CH3)2), 5.74–5.84 (m, 1H, NH), 7.22–7.27 (m, 1H,
(R)-2-(1-Hydroxy-3-methylbut-2-ylamino)-6-[3-(5-pyri-
midinyl)phenylamino]-9-iso-propylpurine
H
pyrimidinyl), 7.33 (t, 1H, J = 7.6 Hz, Hphenyl), 7.71 (s, 1H,
(7j) Yield
1
8-H), 7.87–8.00 (m, 3H, Hphenyl), 8.73–8.80 (d, 2H,
J = 4.5 Hz, Hpyrimidinyl). 13C NMR(DMSO): d 22.1, 40.3,
54.5, 60.6, 113.7, 119.7, 121.6, 121.7, 128.7, 136.0, 137.6,
140.3, 150.9, 151.8, 157.4, 158.3, 163.6. MS (ES?) m/z 421
(M?H)?, 443 (M?Na)?.
23 %. H NMR(CDCl3): d 0.98–1.03 (m, 6H, CH3CH),
1.57 (d, 6H, J = 6.6 Hz, NCH(CH3)2), 1.95–2.03 (m, 1H,
(CH3)2CH), 3.68–3.76 (m, 1H, CH2OH), 3.83–3.90 (m, 2H,
CH2OH ? CHNH), 4.67 (hept, 1H, J = 6.6 Hz,
CH(CH3)2), 5.13 (1H, bd, J = 7.8 Hz, NH), 7.27–7.30 (m,
1H, Hphenyl), 7.50 (t, 1H, J = 8.8 Hz, Hphenyl), 7.64 (s, 1H,
8-H), 7.84–7.91 (m, 1H, Hphenyl), 8.17–8.30 (m, 2H,
Hphenyl ? NH), 9.02 (s, 2H, Hpyrimidinyl), 9.22 (s, 1H,
2-(1-Hydroxy-2-methylprop-2-yl)-6-[3-(2-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (7e) Yield 20 %, m.p.
1
H
pyrimidinyl). 13C NMR: d 18.9, 19.4, 22.4, 22.5, 29.9, 46.6,
159–162 °C. H NMR(CDCl3): d 1.35 (s, 6H, C(CH3)2),
1.51 (d, 6H, J = 6.5 Hz, (CH3)2CH), 3.65–3.71 (m, 2H,
CH2OH), 4.35 (hept, 1H, J = 6.5 Hz, (CH3)2CH), 5.11 (bs,
1H, NH), 7.14 (t, 1H, J = 4.5 Hz, Hpyrimidinyl), 7.26 (t, 1H,
59.7, 65.4, 118.2, 120.2, 121.2, 129.9, 134.3, 134.8, 135.3,
139.5, 140.2, 152.1, 154.9, 157.4, 159.8. MS (ES?) m/z 433
(M?H)?, 455 (M?Na)?.
123

Med Chem Res
2-(1-Hydroxy-2-methylprop-2-yl)-6-[3-(5-pyrimidinyl)phe-
nylamino]-9-iso-propylpurine (7k) Yield 20 %. 1H
NMR(CDCl3): d 1.41 (s, 6H, C(CH3)2), 1.59 (d, 6H,
J = 7.1 Hz, (CH3)2CH), 3.73 (bs, 2H, CH2OH), 4.62 (hept,
1H, J = 7.1 Hz, (CH3)2CH), 5.09–5.13 (bs, 1H, NH),
7.25–7.30 (m, 1H, Hphenyl), 7.50 (t, 1H, J = 7.5 Hz,
ml) in a final volume of 30 ll. After 30 min of incubation
at 30 °C, 25lL aliquots of supernatant were spotted onto
2.5 cm 9 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 phos-
phoric acid/liter of water. The wet filter was counted in the
presence of 1 mL of ACS (Amersham) scintillation fluid.
GSK-3a/b (porcine brain, native) was assayed, as
described for CDK1, but in Buffer A and using a GSK-3-
specific substrate (GS-1: YRRAAVPPSPSLSRHSSPHQ-
SpEDEEE) (pS stands for phosphorylated serine) (Bach
et al., 2005).
H
phenyl), 7.63 (s, 1H, 8-H), 7.82 (d, 2H, J = 8.5 Hz,
phenyl), 7.95–7.98 (bs, 1H, Hphenyl), 7.99–8.02 (bs, 1H,
H
NH), 8.98 (s, 2H, Hpyrimidinyl), 9.22 (s, 1H, Hpyrimidinyl). 13
C
NMR: d 22.4, 24.8, 25.1, 47.0, 55.4, 72.0, 114.9, 118.6,
119.0, 120.7, 121.7, 130.0, 134.2, 134.9, 135.3, 139.8,
149.8, 152.4, 154.9, 157.4, 158.3. MS (ES?) m/z 419
(M?H)?, 441 (M?Na)?.
CK1d/e (porcine brain, native) was assayed in threefold
diluted buffer C as described for CDK1, but using 25 lM
CKS peptide (RRKHAAIGpSAYSITA), a CK1-specific
substrate (Reinhardt et al., 2007).
Biology–protein kinase assays
DYRK1A (human, recombinant, expressed in E. coli as a
GST fusion protein) was purified by affinity chromatog-
raphy on glutathione-agarose and assayed in buffer A
(? 0.5 mg BSA/ml) with using Woodtide (KKISGRL-
SPIMTEQ) (1.5 lg/assay) as a substrate.
Biochemical reagents
Sodium orthovanadate, EGTA, EDTA, Mops, b-glycero-
phosphate, phenylphosphate, sodium fluoride, dithiothreitol
(DTT), glutathione–agarose, glutathione, bovine serum
albumin (BSA), nitrophenylphosphate, leupeptine, aproti-
nine, pepstatin, soybean trypsin inhibitor, benzamidine,
and histone H1 (type III-S) were obtained from sigma
Chemicals. [c-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 (YRRAAVPPSPSLSRHSSPHQp-
SEDEEE) was obtained from GenScript Corporation.
Acknowledgments This research was supported by grants from the
´
‘‘Canceropole Grand-Ouest’’ (LM), the ‘‘Institut National du Cancer’’
(INCa ‘‘Cancer Detection d’innovations 2006’’) (LM), the ‘‘Associ-
´
`
ation France-Alzheimer Finistere’’ (LM), and the ‘‘Ligue Nationale
contre le Cancer (Grand-Ouest)’’ (LM).
Pr. Christiane Garbay is gratefully acknowledged for critical
reading of the manuscript.
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