Synthesis and biological testing of purine derivatives as potential ATP-competitive kinase inhibitors

Article abstract of DOI:10.1021/jm0408767

On the basis of ATP adenine, a series of adenine and purine derivatives was prepared and tested for their ability to inhibit a spectrum of disease-related kinases. There has been scant research investigating the potential of cosubstrate derived kinase inhibitors for other kinases than CDKs. Our inhibitor design combined the purine system from the original cosubstrate ATP and phenyl moieties in order to explore possible interactions with the different regions of the ATP binding site in several disease-related protein kinases. There have been a number of hits for the assayed substances, which led us to conclude that the spectrum of compounds may prove to be a valuable tool kit for the evaluation of bonding and selectivity patterns for a wide variety of kinases.

Full text of DOI:10.1021/jm0408767

710
J. Med. Chem. 2005, 48, 710-722
Synthesis and Biological Testing of Purine Derivatives as Potential
ATP-Competitive Kinase Inhibitors
Stefan A. Laufer,*,† David M. Domeyer,† Thomas R. F. Scior,‡ Wolfgang Albrecht,§ and Dominik R. J. Hauser†
Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-University Tu¨bingen, Auf der Morgenstelle 8,
72076 Tu¨bingen, Germany; Merckle GmbH, Graf-Arco-Str. 3, 89079 Ulm, Germany, and Department of Pharmacy,
Beneme´rita Universidad Auto´noma de Puebla, 14 Sur con Avienda San Claudio, C.P. 72570 Puebla, Me´xico
Received August 31, 2004
On the basis of ATP adenine, a series of adenine and purine derivatives was prepared and
tested for their ability to inhibit a spectrum of disease-related kinases. There has been scant
research investigating the potential of cosubstrate derived kinase inhibitors for other kinases
than CDKs. Our inhibitor design combined the purine system from the original cosubstrate
ATP and phenyl moieties in order to explore possible interactions with the different regions of
the ATP binding site in several disease-related protein kinases. There have been a number of
hits for the assayed substances, which led us to conclude that the spectrum of compounds may
prove to be a valuable tool kit for the evaluation of bonding and selectivity patterns for a wide
variety of kinases.
Introduction
within or close to the binding cleft that ATP does not
fully occupy. These regions, unoccupied by ATP, show
structural diversity between members of the kinase
family. This provides opportunities for the discovery or
design of selective and small molecule ATP-competitive
inhibitors.
Architecture of the ATP Binding Site. Figure 1
shows a general scheme of the important interactions
of a kinase with its cofactor ATP. The ATP binding site
is located in the center of the enzyme, which formed a
molecular hinge. This allows the opening or closure of
the ATP binding cleft in case of kinase activation.5,6
The protein kinase family offers both a challenge and
a huge opportunity for drug discovery. About 22% of the
“druggable” human genome encodes target protein
kinases.1 Approximately 30% of all marketed drugs
target G-protein-coupled receptors, about 7% address
ion channels, and roughly 4% bind to nuclear hormone
receptors. There are, however, only two drugs on the
market that address kinase targets [imatinib (Gleevec)
and gefitinib (Iressa)].2 These facts dictate a great need
for fundamental research in this field and for the
development and design of new lead structures.
Kinases regulate many different cell proliferation,
differentiation, and signaling processes by adding phos-
phate groups to target protein substrates. Reversible
protein phosphorylation is the main strategy for the
control of eukaryotic cell activities. Signal transduction
pathways maintain the balanced steady-state function-
ing of a cell. Disease arises when signal transduction
in a cell breaks down, thereby removing the tight control
that typically exists over cellular functions. Devastating
diseases such as cancer, autoimmune diseases, inflam-
mation, psoriasis, allergic reactions, neurological dis-
orders, and hormone-related diseases can result from
abnormal signal transduction.
Exploring Hydrophobic Region I. Many kinase
inhibitors target hydrophobic region I with a phenyl
moiety (Figure 2) as in the case of p38 mitogen activated
protein kinase (p38 MAP kinase) inhibitors.10 Hydro-
phobic region I is formed as a selectivity pocket, just
big enough to incorporate a 4-fluorophenyl moiety. The
pyridine nitrogen forms an H-bond with the amidic NH
of Met 109, which mimics the H-bond interaction
typically formed by the N-1 of ATP adenine.
The Purine System as a Central Scaffold in
Kinase Inhibitors. Purine derivatives have already
been reported as kinase inhibitors for some kinases,
mainly cyclin-dependent kinases (CDKs).11 Structures
such as olomoucine, a 2,6,9-substituted purine (Figure
3), were initially identified as CDK-inhibitors (IC50 ) 7
µM for CDK2) with only minor inhibitory effects on
other kinases.12 Olomoucine was used as a lead struc-
ture and was optimized in combinatorial chemistry
approaches13 to generate the more efficient analogues
related to purvalanol A (IC50 ) 0.004 µM for CDK2)
(Figure 3). The 2,6,9-substitution pattern has been
retained nearly unchanged, and most variations have
been carried out at C-2 of the purine with variations of
mainly aliphatic substituents. Substituents at N-9 have
been kept very small (methyl, ethyl, isopropyl) in order
to enable the imidazole ring of the purine to enter the
pocket formed by hydrophobic region I (see Figure 1).14
At present, 518 protein kinases3 are identified, in
which all of them the cofactor ATP (adenosine triphos-
phate) binds in a very similar way. The conservation of
structural features within the ATP binding cleft initially
indicated that specificity for ATP-site-directed inhibitors
would be difficult to achieve. Structure elucidation of
ATP complexes or the nonhydrolyzable ATP analogue
adenylyl-â,γ-imidodiphosphate (AMP-PNP)4 bound to
protein kinases has revealed that there are regions
* To whom correspondence should be addressed. Telephone: ++49
7071 2972459. Fax: ++49 7071 295037. E-mail: stefan.laufer@
uni-tuebingen.de.
† Eberhard-Karls-University Tu¨bingen.
‡ Beneme´rita Universidad Auto´noma de Puebla.
§ Merckle GmbH Ulm.
10.1021/jm0408767 CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/15/2005

Purine Derivatives as Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 711
Figure 3. Purine-based CDK inhibitors and the HSP90
inhibitor PU 3.
ler,7 the benzylamino moiety of C-6 should, however,
bind in hydrophobic region II. Therefore, it is obvious
that the purine moiety of adenine-derived ATP-competi-
tive inhibitors of CDKs does not necessarily take the
same orientation as seen for adenine of ATP.13,15
Another purine-based compound library of 8,9-disub-
stituted purine derivatives was derived from the com-
pound PU 3 (Figure 3) to provide inhibitors of the
ATPase HSP90.16,17 On binding, these inhibitors induce
a conformational change of the ATP binding region of
the HSP90 protein.17
Figure 1. Mapping important regions within the ATP binding
site of kinases (modified from Traxler7). The adenine binding
region: N-1 of adenine acts as a hydrogen-bond acceptor from
the amidic NH of the protein backbone. A proton from the
amino group at C-6 forms a hydrogen bond with the carbonyl
oxygen of the backbone. This bidentate hydrogen-bond-
donor/-acceptor system in the hinge region anchors the adenine
deep inside the binding cleft.7 The sugar pocket: The ribose
binding pocket is hydrophilic.7 Surprisingly, the ribose can be
replaced by the phenyl ring of an aniline moiety8 without
causing substantial loss to its binding ability. The phosphate
binding region: The region is surrounded by several residues
that stabilize the phosphates by chelation together with one
or two metal ions (in most cases Mg2+) and thus position the
γ-phosphate for a facilitated phosphoryl transfer.9 Hydrophobic
region I: This region is formed as a lipophilic pocket8 located
deep inside the ATP binding cleft in the direction of N-7 and
C-6 of the adenine moiety of ATP. Its expanse is controlled by
the size of the amino acid side chains surrounding this area
which is not occupied by ATP. The extent of this cavity, which
varies between different kinases, provides opportunities for
the design of selective inhibitors. Hydrophobic region II: The
region is also unoccupied by ATP. It is formed as a cleft that
opens to solvent with the lipophilic surface above and below
the level of the adenine heterocycle. The upper and lower part
of this surface provide two additional interaction possibilities,
thus gaining further affinity and selectivity for potential
inhibitors.7
Our review of the literature revealed that there has
been scant research investigating the potential of co-
substrate derived (adenine- or purine-based) kinase
inhibitors for kinases other than CDKs. Moreover, an
approach to the design of adenine-derived kinase inhibi-
tors has been urgently missed in the literature.18
As discussed above, the selectivity pocket (hydropho-
bic region I) of CDKs is too small to incorporate the
benzylamino moiety of olomoucine,14 whereas other
kinases, such as p3810,19,20 or JNK,21 are able to bind
phenyl moieties in their selectivity pocket. Therefore,
the goal of our work was to establish the optimal design
of a potential kinase inhibitor wherein the purine
system from the original cosubstrate is combined with
phenyl moieties at various positions (Figure 4). We
started our investigations with the synthesis of com-
pounds bearing only one aryl moiety at different posi-
tions of the purine system. Furthermore, these first
aryls were supplemented with second aryl moieties. The
introduction of phenyl moieties with various linkers is
necessary to exploit all possible orientations of the
purine system according to the binding properties of the
phenyl moieties in the hydrophobic regions. Thus, the
probability of finding the best inhibitor, which can
mimic the interaction observed for the adenine of ATP,
increases. In the ATP-adenine model, N-9 is linked to
the sugar moiety, thus effectively blocking any interac-
tion possible (i.e. H-bond) through this position. In
circumspect, any subsequent kinase inhibitor designed
to mimic the adenine of ATP should probably also
possess this inherent characteristic. This can be achieved
by blocking the N-9 position using small and simple
substituents (i.e. methyl). A series of about 50 deriva-
tives was synthesized and tested for inhibitory potency
on a panel of 15 disease-related kinases, including p38
MAP kinase.22 This research project demonstrates that
Figure 2. Schematic drawing of important interactions
between inhibitor SB203580 (a p38 MAP kinase inhibitor) and
the ATP binding site of p38 highlighting the selectivity
pocket.10
The hydroxyethylamino moiety of C-2 binds in the sugar
pocket.14 If the crystal structure data provided by
Schulze-Gahmen14 are carried forward to the map of the
ATP binding site introduced to the literature by Trax-

712 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Laufer et al.
Scheme 2a
a Reagents: (a) 2 N NaOH, appropriate benzyl chlorides, rt.
Scheme 3a
a Solvent: (a) n-butanol at reflux or ethylene glycol at 140 °C.
Figure 4. The ATP binding site showing the purine core
having the same orientation as the adenine of ATP. (Possible
phenyl substitutions, linkers, and interactions are highlighted
in red.)
6-Chloropurine was N-alkylated with benzyl chloride
or 4-fluorobenzyl chloride to obtain the 7- and 9-regio-
isomeric compounds 21-24. The reaction provided the
N-7 benzylated isomers as the minor product.28 Treat-
ment of compounds 21, 23, and 24 with methanolic
ammonia24 in a sealed bottle yielded the adenine
derivatives 25-27 and 6-methoxy-substituted 28 as a
side product (Scheme 4).
Scheme 1a
6-Substituted purines 29-31 having a one- or two-
carbon linker were synthesized by Wittig reaction or a
modified Wittig reaction starting from 9-tetrahydropy-
ranyl-protected 6-chloropurine29 following the proce-
dures described by Taylor30 (Scheme 5). On the other
hand, the 9-methyl-substituted styrylpurine 32 could be
obtained via Suzuki coupling of stryrylboronic acid with
6-chloro-9-methyl-9H-purine as recently described for
9-benzylpurines.31 9-Unsubstituted 6-chloropurine is
unreactive toward Suzuki coupling conditions.
The 6-phenyl-substituted purines 39 and 40 and 6,8-
diphenyl-substituted 41 were synthesized as shown in
Scheme 6. Suzuki coupling of benzeneboronic acid or
4-fluorobenzeneboronic acid with 4,6-dichloro-5-nitro-
pyrimidine32 provided the arylpyrimidines 33 or 34,
which were treated with methylamine to deliver the
4-amino-5-nitro compounds 35 or 36. Subsequent cata-
lytic reduction of the 5-nitro group afforded the diamines
37 or 38, which permitted acid-catalyzed ring closure
with the suitable ortho esters
The starting compound for the synthesis of the 6,8,9-
trisubstituted purines 44, 45, and 49-54 was 6-chloro-
9-methyl-9H-purine, which was brominated to the 8-bro-
mo derivative by the method described by Nolsoe.33 The
two different halogens at positions C-6 and C-8 of the
purine moiety allowed us to selectively introduce sub-
stituents at both positions. Substitution at position C-8
was achieved by Suzuki coupling of benzeneboronic acid
or 4-methylthiobenzeneboronic acid with the 8-bromo
derivative. The sulfur of the methylthio moiety could
be selectively oxidized to a methylsulfinyl group by
hydrogen peroxide or to a methylsulfonyl group by
m-chloroperbenzoic acid. The 8-substituted compounds
were subjected to nucleophilic substitution at the 6-po-
sition with the appropriate thiophenols (Scheme 7).
a Reagents: (a) n-butanol, triethylamine, appropriate anilines
or thiophenols, reflux (melt in the case of phenols).
systematic variation of cosubstrate-based analogues
shows promise as a way to develop selective kinase
inhibitors.
Chemistry
Purines and deazapurines substituted at C-6 (com-
pounds 1-12) were synthesized by nucleophilic substi-
tution of the chlorine of 6-chloropurine,23 6-chloro-9-
methyl-9H-purine,24 or 4-chloro-7H-pyrrolo[2,3-d]pyrim-
idine25 with the appropriate anilines, phenols, or thiophe-
nols (Scheme 1).
The 6-benzylmercaptopurines 13-16 were synthe-
sized by nucleophilic substitution of benzyl chloride or
4-fluorobenzyl chloride with 6-mercaptopurine or 6-mer-
capto-9-methylpurine24 (Scheme 2).
The 7-substituted purines 17-20 were synthesized in
a one-step reaction starting from formamidine acetate
and the appropriately substituted aminoacetonitriles26
following the Bredereck method for preparing compound
1727 (Scheme 3).

Purine Derivatives as Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 713
Scheme 4a
a Reagents: (a) DMF, K2CO3, appropriate benzyl chlorides; (b) MeOH, aqueous ammonia.
Scheme 5a
which was brominated at the 8-position by the proce-
dure described above for 42 to deliver compound 55.
Sonogashira coupling34 with but-3-yn-1-ol afforded com-
pound 56 (Scheme 8).
6,9-Substituted purines 57-60 were obtained by
nucleopilic substitution of compounds 21 or 23 with
thiophenol or 4-fluorothiophenol (Scheme 9).
Biological Results and Discussion
The inhibitory potency of the compounds was evalu-
ated with the Upstate Kinase Profiler.22 The results are
shown in Table 1 and will be discussed on the basis of
protein-ligand interactions.
a Reagents: (a) for 29 and 30: (1) benzyl(tri-n-butyl)phospho-
nium bromide or 4-fluoro-benzyl(tri-n-butyl)phosphonium bromide,
DME, n-butyllithium, -35 °C; (2) Na2CO3, H2O, rt; (3) 1 N HCl,
EtOH, rt. For 31: (1) methyltriphenylphosphonium bromide,
DME, n-butyllithium, -35 °C; (2) benzaldehyde, rt; (3) 1 N HCl,
EtOH, rt. For 32: trans-2-phenylvinylboronic acid, K2CO3, DME,
Pd(PPh3)4, 90 °C.
As reported for most kinase inhibitors,35 our com-
pounds were assumed to be ATP-competitive as well.
In this line, inhibition values are usually dependent on
the ATP concentrations in the test assay.36 From an in
vivo standpoint, the competitiveness of the potential
inhibitor is even weaker because the intracellular
concentrations of ATP are in the millimolar range.37 On
the basis of these important considerations, the ATP
concentration was maintained at higher levels than the
usual ATP concentrations. The concentrations of the
potential inhibitors were kept at a level about 3-fold
lower than that of ATP to create an especially challeng-
ing condition for the compounds.
Scheme 6a
As mentioned earlier, the adenine of ATP exhibits key
interactions when bound to kinases. Adenine in itself
without the ATP substructure would then act more like
an inhibitor, due to the fact that it would lose its ability
to transfer phosphate groups together with the kinase.
Since the central scaffold of all the potential inhibitors
are purine or adenine, it would be pertinent to also
include adenine in the assay as a comparison in judging
the values obtained. To determine the effectiveness of
an inhibitor, the cutoff value, which would be of interest,
has to be set above this particular result. Although the
compounds have not yet been optimized for a single
kinase, the result should deliver selectivity criteria and
structural clues for the substitutional pattern using
diverse phenyl substituents. Purines substituted at the
6- and 7-positions were the primary candidates, because
this allows the purine to have the same orientation as
that of adenine when ATP is bound to the protein. Their
a Reagents: (a) appropriate boronic acid, Na2CO3, toluene,
Pd(PPh3)4, 90 °C; (b) H2N-CH3 40% in H2O, reflux; (c) PtS/C, H2,
4 bar, rt; (d) appropriate ortho ester, EtOH, p-toluenesulfonic acid,
reflux.
This reaction sequence was not applicable for the
synthesis of compound 56 because of the reactivity of
the alkynyl moiety at the 8-position. Thus, 6-chloro-9-
methyl-9H-purine was first subjected to nucleophilic
substitution with thiophenol to provide compound 11,

714 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Laufer et al.
Scheme 7a
a Reagents: (a) (1) LDA, (2) BrCCl2CCl2Br, -78 °C; (b) appropriate boronic acid, K2CO3, toluene, Pd(PPh3)4, 90 °C; (c) for n ) 0, no
reaction; for n ) 1, glacial acetic acid, H2O2, rt; for n ) 2, CH2Cl2, m-chloroperbenzoic acid, reflux; (d) n-butanol, triethylamine, appropriate
thiophenols, reflux.
Scheme 8a
amino group linked to aryl moieties. This resulted in
quite unselective inhibition. Eight kinases could be
inhibited with stronger values as seen for adenine. For
these four compounds, additional methyl substitution
at the 9-position increased selectivity with four kinases
targeted at better inhibition levels in the cases of c-RAF
kinase and MAPK2 kinase. Replacement of the amino
moiety at C-6 by carbon and other heteroatoms was
done to investigate the relevance of possible alternative
linkers. As exemplified by compounds 3 and 4, the
activity decreased with the oxygen linker. For that
reason this approach was discontinued. Compounds 29
and 30 with a carbon linker at C-6 showed no activity.
This perhaps is due to the absence of H-bond interac-
tions or the reduced distance between the purine and
the aryl moiety. In compound 31, this distance was
increased by addition of one carbon atom, keeping the
linker rigid by introducing a double bond. In contrast
to the single carbon counterpart, the compound showed
activity and inhibition of the three kinases c-RAF
kinase, Lck kinase, and MAPK1 kinase. The replace-
ment of the amino moiety at C-6 of the purine with
sulfur led to compounds with activity against two or
three kinases (ROCK kinase, p38 MAP kinase, and
c-RAF kinase) in the case of compounds 7 and 8.
However, the trend of selectivity was lost in their
7-deaza analogues 9 and 10. On the basis of these
findings, subsequent investigation involved retaining,
in most compounds, the sulfur at C-6 and the nitrogen
at the 7-position.
a Reagents: (a) (1) LDA, (2) BrCCl2CCl2Br; (b) but-3-yn-1-ol,
CH2Cl2, triethylamine, PdCl2(PPh3)2, CuI, reflux.
Scheme 9a
a Reagents: (a) n-butanol, triethylamine, appropriate thiophe-
nols, reflux.
Compounds 17-20, without substituents at C-6 and
aryl substituted at N-7, did not show any activity. Their
analogues 22 and 24, with chlorine substituted at C-6,
showed similar results. This, together with the facts
discussed above, indicates the importance of heteroatom
substitution with nitrogen or sulfur at this position for
improving activity. The 6-amino-9-aryl combination of
compounds 25 and 26, where the aryls could in theory
aryl substituents should be directed to the lipophilic
pocket formed by the amino acid residues of hydrophobic
region I.
In ATP, the 6-position of adenine is substituted with
an amino group. Similarly, 1, 2, 5, and 6 contain the

Purine Derivatives as Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 715
Table 1. Inhibition Values of Kinase Activitya,b
no. c-RAFc CaMKIId GSK3âc JNK1R1c JNK2R2c JNK3d Lckc MAPK1c MAPK2c MEK1c PKAc PKBRc PKCRc ROCK-IIc SAPK2Rc
1
2
19
24
3
0
0
0
0
0
8
0
0
6
3
8
5
0
3
0
5
0
0
7
5
7
0
0
8
0
9
8
2
0
8
3
4
8
12
5
1
8
0
7
8
12
8
0
0
10
3
0
0
0
0
13
13
0
0
0
1
0
0
5
0
0
0
2
3
10
1
0
1
10
0
1
0
0
0
0
0
6
1
0
4
0
0
0
0
0
9
5
2
0
2
0
3
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
6
0
1
0
6
6
10
0
0
7
6
0
0
4
0
0
1
0
9
0
0
7
0
0
0
0
0
11
3
8
0
0
1
5
0
1
8
2
0
0
0
0
0
0
0
11
10
0
39
42
11
0
18
9
5
22
33
0
33
48
6
19
34
1
0
4
8
0
2
41
36
3
6
7
0
6
7
0
0
4
0
3
0
1
4
7
7
8
3
8
0
7
0
9
1
5
4
7
4
4
3
1
4
0
0
0
6
4
1
3
9
32
40
11
0
14
18
0
6
17
8
48
52
16
9
34
30
12
18
0
3
4
1
0
0
0
0
0
3
5
44
27
6
26
14
1
0
11
4
3
0
35
0
5
16
36
3
42
57
13
12
4
0
0
2
0
6
19
0
0
8
0
11
3
7
13
54
72
56
79
17
38
50
29
22
21
0
7
0
33
37
36
42
6
8
29
22
43
10
12
53
47
12
16
0
8
13
56
52
6
2
10
0
0
9
9
0
11
10
12
9
0
23
15
13
7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
39
40
41
44
45
49
50
51
52
53
54
55
56
57
58
59
60
61e
5
2
0
0
7
12
12
12
5
0
0
9
2
1
0
6
7
22
16
6
15
9
6
11
0
0
11
5
9
10
13
16
17
22
26
19
0
13
12
0
4
11
4
28
18
1
12
4
6
10
14
4
0
4
13
12
4
9
5
8
0
11
0
6
3
2
0
0
0
0
10
7
0
4
9
0
0
0
2
2
0
13
10
0
13
12
16
11
20
21
17
9
7
7
0
9
18
15
17
12
14
13
2
7
6
13
0
4
0
8
4
5
4
12
4
20
25
16
28
9
2
0
66
8
12
9
0
2
0
3
11
7
9
0
1
0
0
6
8
0
0
0
0
0
10
17
7
0
0
1
0
0
7
7
19
22
0
11
0
1
0
5
5
0
0
16
14
7
0
0
13
7
14
8
17
13
17
0
0
8
0
25
30
0
5
7
0
6
8
5
0
3
7
0
1
8
0
0
0
0
0
3
1
2
0
0
0
50
2
0
0
3
2
1
20
0
2
37
1
20
0
4
7
22
0
17
12
16
16
23
44
29
11
15
27
44
36
19
23
3
8
0
0
11
3
26
0
14
14
12
12
16
0
21
5
17
5
20
0
1
30
30
16
10
23
11
0
16
12
16
19
23
17
20
10
11
1
9
7
15
0
8
0
0
10
0
24
12
14
5
1
0
31
42
29
5
0
4
0
3
1
22
3
1
3
9
0
0
0
1
5
3
6
1
0
0
0
8
0
0
4
0
0
0
1
4
4
10
7
0
0
4
10
14
4
15
18
7
0
10
6
0
9
3
11
3
5
0
5
5
0
0
0
19
0
0
0
0
0
1
5
0
0
0
0
0
0
33
1
18
0
0
0
1
0
0
0
0
1
64
64
67
27
7
0
0
0
0
0
0
1
0
5
0
0
0
17
6
0
0
13
0
0
2
0
8
0
0
0
0
0
14
0
10
3
17
14
0
6
28
a Concentration of the compounds, 30 µM; concentration of ATP, 100 µM. Measured values are given as percent inhibition, and values
g25% are italic. b Abbreviations: c-RAF, Raf kinase; CaMK, calcium/calmodulin-dependent protein kinase; GSK3â, glycogen synthase
kinase 3â; JNK, c-Jun N-terminal kinase (also stress-activated protein kinase, SAPK); LCK, lymphocyte kinase; MAPK, mitogen-activated
protein kinase; MEK, MAPK kinase (also called MKK); PKA, cAMP-dependent protein kinase; PKB, protein kinase B (also called Akt);
PKC, protein kinase C; ROCK, Rho-dependent protein kinase; SAPK2R, stress-activated protein kinase 2a (also called p38 MAP kinase).
c From human. d From rat. e Compound 61 is adenine.
occupy the hydrophobic region II, showed only weak
inhibition. This was slightly improved in the 6-amino-
7-aryl or 6-methoxy-7-aryl combinations of compounds
27 and 28, where the aryls project toward the hydro-
phobic region I.
In general, most hits were found for p38 MAP kinase.
This was not particularly astonishing, because the
selectivity pocket formed by hydrophobic region I allows
the exact incorporation of phenyl moieties. The best
results for p38 MAP kinase were obtained with the
simple purines 7 and 8 and the deazapurines 9 and 10,
which have a sulfanyl linker at C-6. The size of the
sulfur linker provides greater flexibility for the bond
angles. The unsubstituted positions at N-9 (purine) or
N-7 (pyrrolopyrimidine) allow the molecule to form a
bidentate H-bond in conjunction with N-3 (purine) and
N-1 (pyrrolopyrimidine) (Figure 5). Thus, the N-9 meth-
ylated analogues 11 and 12 show weaker activity
because one hydrogen bond site is unavailable. In
analogues 1 and 2, the activity should be higher than
observed, since both N-9 (unsubstituted) and N-2 are
available for H-bonding. This was not the case. This
could be explained by an additional H-bonding site at
the amino function at C-6, which could possibly cause
unselective H-bonding patterns with the kinase. Com-
pound 10 may not necessarily take the same orientation
as the adenine in ATP because of its disposition to form
this H-bond with the enzyme backbone. This forces the
deazapurine in a different configuration needed for
activity, even though the phenylsulfanyl moiety is still

716 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Laufer et al.
Figure 5. Active site of p38 MAP kinase with compound 10
occupying the lipophilic pocket (yellow) with a 4-fluorophen-
ylsulfanyl moiety. The formation of H-bonds to the backbone
Met109 similar to those of adenine in ATP seems to be possible
(pink).
Figure 7. Overlay of p38 MAP kinase (blue) and JNK3 kinase
(orange) with compound 10. JNK3 does not form a wide
lipophilic pocket: Met 146 clashes with the 4-fluorophenyl
moiety. The highlighted amino acids were used for superposi-
tion of the protein backbones.
controlled by Met 146.4 Since Met has a bulkier side
chain than the Thr, this allows the p38 MAP kinase to
accommodate the larger phenyl moiety (Figure 7).
Compounds 51 and 52 had hits for Lck and p38 MAP
kinase. As opposed to compounds 11 and 12, these
compounds have an additional methylsulfinylphenyl
ring at C-8, which could provide the possibility for either
π-π-interactions with Tyr residues or interactions
between the sulfoxide oxygen of the methanesulfinyl
substituents and Arg residues at the phosphate binding
region.38 The 7-(4-fluorobenzyl)-substituted adenine 27
showed hits at the cutoff for three kinases at values that
have been seen for unsubstituted adenine 61 in other
kinases. The fluorobenzyl moiety possibly reaches the
area of the lipophilic pocket, whereas adenine takes a
similar orientation as in ATP but a full incorporation
does not seem likely for JNK3 kinase for the reasons
discussed above. Since the methoxy analogue 28 seems
to be more selective than adenine analogue 27, it may
serve as a template to design more potent analogues as
selective JNK3 inhibitors.
Figure 6. Active site of p38 MAP kinase with compound 59
occupying the lipophilic pocket (yellow) with a 4-fluorobenzyl
moiety. The Conolly surface is colored by the lipophilicity of
the amino acid side chains. Red, lipophilic; blue, hydrophilic.
oriented toward the lipophilic pocket, owing to the
flexibility offered by the sulfur linker.
A possible arrangement may be envisioned for com-
pound 59 in the active site of p38 MAP kinase (Figure
6). The 4-fluorobenzyl moiety is directed toward the
lipophilic pocket and the phenylsulfanyl moiety in the
area of the hydrophobic pocket II, providing additional
lipophilic interactions. H-bonding between N-1 or N-7
and the protein backbone could still be feasible. For
compounds 57-60, activity was only observed for p38
MAP kinase. A possible reason may be the size of the
phenyl substituents and their corresponding distance
to each other, which is defined by the dimension of the
purine moiety. The size of the hydrophobic regions and
the distance between them is possibly different in the
other kinases. Thus, the utilization of appropriate
substituents should exploit the distance needed to attain
additional selectivity for potential inhibitor molecules.
Surprisingly, there are only very few hits for JNK3
kinase, which is structurally closely related to p38 MAP
kinase. The size of the lipophilic pocket of p38 is
controlled by the side chain of the appropriate amino
acid. For p38 MAP kinase, the expanse of the lipophilic
pocket is controlled by Thr 106, and for JNK3, it is
Conclusion
Although the compounds do not exhibit an outstand-
ing selectivity or activity profile, we were able to show
that simple substitution of purine or adenine templates
with one or two aryl moieties bound by diverse linkers
can provide the first selectivity pattern and point out
general substitution requirements for selective kinase
inhibitors. Overall, if the panel of the compounds is
viewed as a whole, it may prove to be a valuable tool
kit for the evaluation of bonding and selectivity patterns
for a wide variety of kinases. This is especially true for
kinases, for which no potent inhibitors are presently
known. Due to its excellent H-bonding pattern, the
purine moiety should be a useful scaffold in the design
of new kinase inhibitors, if the inhibitor contains the
following attributes to gain selectivity and potency: (i)
a portion that closely mimics the ATP molecule, (ii) the
H-bonding characteristics of adenine, and (iii) portions
that protrude away from the ATP binding region and
reach into a lipophilic pocket and/or into additional
hydrophobic areas. However, other mechanisms of

Purine Derivatives as Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 717
8.37 (s, 1H), 9.78 (s, 1H, N-H), 13.18 (s, 1H, N-H); GC
(temperature program B) 11.6 min; MS m/z (%) 211 (52, M+),
210 (100), 183 (6), 156 (9), 129 (6), 105 (8), 77 (10).
achieving specificity may be possible as well. For
instance, this could be achieved through hydrophilic
interactions in the areas of the phosphate binding region
and the sugar binding region. The substitution pattern
of the purine moiety with other residues than phenyl
substituents and/or mainly lipophilic substituents will
be subject to further investigation. Some of the com-
pounds may serve as lead structures for new and more
potent kinase inhibitors on the basis of a new core
structure closely related to the adenine moiety of ATP.
(4-Fluorophenyl)(9H-purin-6-yl)amine (2). The com-
pound was prepared from 6-chloropurine23 (0.62 g, 4.0 mmol)
and 4-fluoroaniline (0.89 g, 8.0 mmol) according to general
1
procedure A to yield 2 (730 mg; 80%): mp 338 °C; H NMR δ
7.13-7.22 (m, 2H, 4-F-Ph), 7.94-8.01 (m, 2H, 4-F-Ph), 8.28
(s, 1H), 8.36 (s, 1H), 9.87 (s, 1H, N-H), 13.19 (s, 1H, N-H);
GC (temperature program B) 11.6 min; MS m/z (%) 229 (82,
M+), 228 (100), 147 (9), 95 (12), 83 (10), 75 (10).
6-Phenoxy-9H-purine (3).40 The compound was prepared
from 6-chloropurine23 (0.4 g, 2.6 mmol) and phenol (1.2 g, 12.77
mmol) according to general procedure B to yield 3 (375 mg;
Experimental Section
1
68%): mp 217 °C, H NMR δ 7.26-7.52 (m, 5H, Ph), 8.41 (s,
Molecular Models. Each structure was built with Insight
II, minimized under CVFF force field conditions, and subse-
quently docked manually into the ATP binding site of p38 MAP
kinase. The crystal structure of p38 MAP kinase with SB
203580 was used as template (PDB 1a9u).19 To take the
conformational flexibility of the protein into consideration, the
assembly of kinase and inhibitor was then minimized again
excluding all protein atoms outside a box of 12 Å around the
inhibitor from the calculation (Insight II 2000 Accelrys Inc.,
9685 Scranton Road, San Diego, CA 92121-3752).
1H), 8.48 (s, 1H), 13.60 (s, 1H, N-H); GC (temperature
program B) 10.1 min; MS m/z (%) 212 (100, M+), 211 (100),
184 (8), 158 (7), 119 (10), 94 (10), 77 (9).
6-(4-Fluorophenoxy)-9H-purine (4). The compound was
prepared from 6-chloropurine23 (0.4 g, 2.6 mmol) and 4-fluo-
rophenol (1.1 g, 9.75 mmol) according to general procedure B
to yield 4 (382 mg, 64%): mp 218 °C, 1H NMR δ 7.26-7.40
(m, 4H, 4-F-Ph), 8.43 (s, 1H), 8.53 (s, 1H), 13.60 (s, 1H, N-H);
GC (temperature program B) 9.9 min; MS m/z (%) 230 (100,
M+), 229 (70), 202 (13), 176 (9), 148 (8), 119 (23), 112 (20), 95
(12). Anal. (C11H7FN4O) C, H, N; C: calcd, 57.39; found, 57.87.
(9-Methyl-9H-purin-6-yl)phenylamine (5).41 The com-
pound was prepared from 6-chloro-9-methyl-9H-purine24 (0.43
g, 2.6 mmol) and aniline (0.9 g, 8.1 mmol) according to general
Chemistry. All reagents and solvents were of commercial
quality and used without further purification. Melting points
were determined on a Buechi Melting Point B-545 apparatus
1
and are thermodynamically corrected. H NMR spectra were
obtained on a Bruker Advance 200 at 200 MHz in DMSO-d6
unless otherwise specified. Chemical shifts are reported in
parts per million (ppm) relative to TMS as internal standard.
TLC analyses were performed on fluorescent silica gel 60
plates (Merck KGaA; Art.-Nr. 1.05554). Spots were visualized
under 254-nm UV illumination. SiO2 60 was used for purifica-
tions by column chromatography unless otherwise specified.
GC/MS analyses were carried out on a HP 6890 series GC-
system to which a HP 5973 mass selective detector was
attached. The GC was equipped with a HP-5MS capillary
column (30 m × 0.25-mm i.d.) with a 5% cross-linked phenyl-
methylsiloxane stationary phase (0.25 mm film thickness).
Helium was used as carrier gas and the following temperature
programs were employed: (A) initial isothermal period of 1.0
min at 100 °C, an increase at 10.0 °C/min to 160 °C with an
isothermal period of 10.0 min at 160 °C, and then an increase
at 15.0 °C/min to 200 °C with an isothermal period of 15.0
min at 200 °C; (B) initial isothermal period of 1.0 min at 160
°C, an increase at 10.0 °C/min to 240 °C with an isothermal
period of 5.0 min at 240 °C, and then an increase at 10.0 °C/
min to 270 °C with an isothermal period of 20.0 min at 270
°C. GC/MS analyses were performed in the single ion monitor-
ing (SIM) mode with the ionization voltage of the mass
selective detector set to 70 eV. GC data are presented as
retention times (min) and MS results as m/z and intensity (%)
relative to the base peak. Positive electrospray ionization (ESI)
mass spectra were obtained from a ThermoFinnigan TSQ
Quantum instrument (electrospray voltage, 3 kV; heated
capillary temperature, 300 °C; sheath and auxiliary gas,
nitrogen).
1
procedure A to yield 5 (430 mg; 73%): mp 169 °C; H NMR δ
3.80 (s, 3H, N9-CH3), 6.99-7.37 (m, 3H, Ph), 7.96-7.99 (m,
2H, Ph), 8.28 (s, 1H), 8.42 (s, 1H), 9.84 (s, 1H, N-H,
exchangeable); GC (temperature program B) 10.2 min; MS m/z
(%) 225 (60, M+), 224 (100), 210 (11), 197 (5), 182 (10), 156
(10), 133 (7), 77 (14).
(4-Fluorophenyl)(9-methyl-9H-purin-6-yl)amine (6). The
compound was prepared from 6-chloro-9-methyl-9H-purine24
(0.43 g, 2.6 mmol) and 4-fluoroaniline (0.58 g, 6.2 mmol)
according to general procedure A to yield 6 (474 mg; 75%): mp
182 °C; 1H NMR δ 3.80 (s, 3H, N9-CH3), 7.13-7.22 (m, 2H,
4-F-Ph), 7.94-8.01 (m, 2H, 4-F-Ph), 8.28 (s, 1H), 8.40 (s, 1H),
9.91 (s, 1H, N-H, exchangeable); GC (temperature program
B) 10.1 min; MS m/z (%) 243 (89, M+), 242 (100), 228 (11), 201
(5), 174 (10), 147 (6), 122 (8), 95 (18).
6-Phenylsulfanyl-9H-purine (7).42 The compound was
prepared from 6-chloropurine23 (0.5 g, 3.25 mmol) and thiophe-
nol (1.0 g, 9.1 mmol) according to general procedure A to yield
1
7 (290 mg; 39%): mp 245 °C; H NMR δ 7.48-7.68 (m, 5H,
Ph), 8.53 (s, 1H), 8.56 (s, 1H), 13.62 (s, 1H, N-H, exchange-
able).
6-(4-Fluorophenylsulfanyl)-9H-purine (8). The com-
pound was prepared from 6-chloropurine23 (0.5 g, 3.25 mmol)
and 4-fluorothiophenol (1.2 g, 9.4 mmol) according to general
1
procedure A to yield 8 (335 mg; 42%): mp 233 °C; H NMR δ
7.29-7.41 (m, 2H, 4-F-Ph), 7.64-7.74 (m, 2H, 4-F-Ph), 8.51
(s, 1H), 8.55 (s, 1H), 13.61 (s, 1H, N-H, exchangeable). Anal.
(C11H7FN4S) C, H, N.
4-Phenylsulfanyl-7H-pyrrolo[2,3-d]pyrimidine (9). The
compound was prepared from 4-chloro-7H-pyrrolo[2,3-d]pyrim-
idine25 (765 mg, 5 mmol) and thiophenol (1.65 g, 15 mmol)
according to general procedure A to yield 9 (624 mg; 55%): mp
159 °C; 1H NMR δ 6.03 (1H, HCdCH), 7.45-7.69 (6H, Ph and
HCdCH), 8.49 (s, 1H), 12.27 (s, 1H, NH); GC (temperature
program B) 13.9 min; MS m/z (%) 227 (50, M+), 226 (100), 199
(6), 172 (8), 145 (4), 118 (17), 109 (12), 91 (9), 77 (8). Anal.
(C12H9N3S) C, H, N.
Preparation of 6-Substituted Purines. General Pro-
cedure A. The appropriate 6-chloropurines were refluxed
together with the suitable anilines or thiophenols and triethyl-
amine (2-4 equiv) in 1-butanol for 3-8 h. The volatiles were
evaporated in vacuo, and the residue was purified by column
chromatography (CH2Cl2/EtOH 9:1).
Preparation of 6-Substituted Purines. General Pro-
cedure B. The appropriate 6-chloropurines were melted at
110 °C together with the suitable phenols for 3-8 h. The
phenols were extracted with diethyl ether, and the residue was
purified by column chromatography (CH2Cl2/EtOH 9:1).
Phenyl(9H-purin-6-yl)amine (1).39 The compound was
prepared from 6-chloropurine23 (0.62 g, 4.0 mmol) and aniline
(0.74 g, 8.0 mmol) according to general procedure A to yield 1
(642 mg; 76%): mp 278 °C; 1H NMR δ 6.98-7.09 (m, 1H, Ph),
7.28-7.41 (m, 2H, Ph), 7.95-7.99 (m, 2H, Ph), 8.28 (s, 1H),
4-(4-Fluorophenylsulfanyl)-7H-pyrrolo[2,3-d]pyrimi-
dine (10). The compound was prepared from 4-chloro-7H-
pyrrolo[2,3-d]pyrimidine25 (765 mg, 5 mmol) and 4-fluo-
rothiophenol (1.92 g, 15 mmol) according to general procedure
1
A to yield 10 (690 mg; 56%): mp 200 °C; H NMR δ 6.12 (d,
1H, 3J(H,H) ) 3.5 Hz, HCdCH), 7.31-7.41 (2H, 4-F-Ph), 7.48
(d, 1H, 3J(H,H) ) 3.5 Hz, HCdCH), 7.66-7.75 (2H, 4-F-Ph),
8.47 (s, 1H), 12.24 (s, 1H, NH); GC (temperature program B)

718 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Laufer et al.
13.9 min; MS m/z (%) 245 (43, M+), 244 (100), 217 (9), 190 (8),
127 (10), 118 (19), 95 (8), 83 (14). Anal. (C12H8FN3S) C, H, N.
9-Methyl-6-phenylsulfanyl-9H-purine (11). The com-
pound was prepared from 6-chloro-9-methyl-9H-purine24 (0.43
g, 2.6 mmol) and thiophenol (0.68 g, 6.2 mmol) according to
general procedure A to yield 11 (320 mg; 51%): mp 136 °C;
1H NMR δ 3.83 (s, 3H), 7.48-7.66 (m, 5H, Ph), 8.48 (s, 1H),
8.57 (s, 1H); GC (temperature program B) 10.8 min; MS m/z
(%) 242 (36, M+), 241 (100), 227 (9), 173 (8), 133 (5), 109 (10),
77 (8). Anal. (C12H10N4S) C, H, N.
7-(4-Fluorophenyl)-7H-purine (18). The compound was
prepared from (4-fluorophenylamino)acetonitrile (3 g, 20 mmol)
and formamidine acetate (15.61 g, 150 mmol) according to the
method described for 17 to yield 18 (1.1 g; 26%): mp 232 °C;
1H NMR δ 7.47-7.56 (m, 2H, 4-F-Ph), 7.83-7.92 (m, 2H, 4-F-
Ph), 9.08 (s, 1H), 9.20 (s, 2H); GC (temperature program B)
8.5 min; MS m/z (%) 214 (100, M+), 187 (31), 160 (38), 133
(29), 108 (18), 95 (29), 75 (20). Anal. (C11H7FN4) C, H, N.
7-Benzyl-7H-purine (19).44 A solution of benzylaminoace-
tonitrile (2.08 g, 14.2 mmol) and formamidine acetate (4.44 g,
43 mmol) in ethylene glycol (7 mL) was stirred for 5 h at 140
°C. The mixture was taken up in water and extracted with
CH2Cl2. The organic layer was dried over Na2SO4 and evapo-
rated in vacuo, and the residue was purified by column
chromatography (CH2Cl2/EtOH 9:1) to produce 19 (300 mg;
10%): mp 146 °C; 1H NMR δ 5.65 (s, 2H, CH2), 7.29-7.47 (m,
5H, Ph), 8.94 (s, 1H), 8.99 (s, 1H), 9.14 (s, 1H); GC (temper-
ature program B) 10.0 min; MS m/z (%) 210 (35, M+), 182 (4),
155 (3), 128 (4), 120 (4), 106 (3), 91 (100), 77 (20), 65 (40).
6-(4-Fluorophenylsulfanyl)-9-methyl-9H-purine (12).
The compound was prepared from 6-chloro-9-methyl-9H-
purine24 (0.43 g, 2.6 mmol) and 4-fluorothiophenol (0.8 g, 6.2
mmol) according to general procedure A to yield 12 (350 mg;
1
52%): mp 182 °C; H NMR δ 3.83 (s, 3H), 7.31-7.40 (m, 2H,
4-F-Ph), 7.65-7.72 (m, 2H, 4-F-Ph), 8.48 (s, 1H), 8.58 (s, 1H);
GC (temperature program B) 10.2 min; MS m/z (%) 260 (40,
M+), 259 (100), 245 (12), 191 (9), 127 (12), 106 (10), 83 (14).
Anal. (C12H9FN4S) C, H, N.
7-(4-Fluoro-benzyl)-7H-purine (20). The compound was
prepared from (4-fluorobenzylamino)acetonitrile (2.5 g, 15
mmol) and formamidine acetate (4.44 g, 43 mmol) according
to the method described for 19 and was purified by column
chromatography (CH2Cl2/EtOH 9:1) to yield 20 (220 mg;
6.5%): mp 138 °C; 1H NMR δ 5.61 (s, 2H, CH2), 7.17-7.26
(m, 2H, 4-F-Ph), 7.49-7.56 (m, 2H, 4-F-Ph), 8.91 (s, 1H), 8.98
(s, 1H), 9.15 (s, 1H); GC (temperature program B) 15.2 min;
MS m/z (%) 228 (79, M+), 200 (4), 173 (3), 146 (4), 126 (3), 109
(100), 83 (25), 65 (13). Anal. (C12H9FN4) C, H, N.
Preparation of 6-Benzylsulfanylpurines. General Pro-
cedure C. The appropriate benzyl chloride was added to a
solution of 6-mercaptopurine or 9-methyl-9H-purine-6-thiol24
in 2 N NaOH (10 mL) and stirred for 2 h. The mixture was
adjusted to pH 5 with glacial acetic acid, and the precipitate
was collected, washed with water and diethyl ether, and
purified by column chromatography (CH2Cl2/EtOH 9:1).
6-Benzylsulfanyl-9H-purine (13).43 The compound was
prepared from 6-mercaptopurine (0.61 g, 3.6 mmol) and benzyl
chloride (0.5 g, 3,96 mmol) according to general procedure C
to yield 13 (640 mg; 73%): mp 195 °C; 1H NMR δ 4.66 (s, 2H,
CH2), 7.24-7.50 (m, 5H, Ph), 8.47 (s, 1H), 8.75 (s, 2H), 13.52
(s, 1H, N-H, exchangeable); GC (temperature program B) 15.2
min; MS m/z (%) 242 (100, M+), 227 (3), 209 (98), 164 (20),
121 (22), 107 (39), 107 (40), 91 (90), 77 (54).
9-Benzyl-6-chloro-9H-purine (21) and 7-Benzyl-6-chloro-
9H-purine (22).28 K2CO3 (4.15 g, 30 mmol) was suspended in
a stirred solution of 6-chloropurine (1.55 g, 15 mmol) in DMF
(50 mL). To this was added benzyl chloride (1.89 g, 15 mmol)
and the mixture stirred for 20 h at room temperature. After
filtration, the solvent was evaporated in vacuo, and the isomers
were separated by column chromatography (EtOAc/hexane 7:3)
with the 9-isomer eluting prior to the 7-isomer to produce 21
(1.11 g; 46%) and 22 (140 mg; 6%). Compound 21: mp 88 °C;
1H NMR δ 5.56 (s, 2H, CH2), 7.28-7.38 (m, 5H, Ph), 8.81 (s,
1H), 8.88 (s, 1H); GC (temperature program B) 12.9 min; MS
m/z (%) 246 (24), 245 (42), 244 (78, M+), 243 (100), 209 (10),
182 (16), 167 (12), 152 (7), 128 (8), 91 (100), 65 (32). Compound
6-(4-Fluorobenzylsulfanyl)-9H-purine (14).42 The com-
pound was prepared from 6-mercaptopurine (0.61 g, 3.6 mmol)
and 4-fluorobenzyl chloride (0.57 g, 3,96 mmol) according to
general procedure C and to yield 14 (250 mg; 27%): mp 228
°C; 1H NMR δ 4.65 (s, 2H), 7.09-7.20 (m, 2H, 4-F-Ph), 7.46-
7.55 (m, 2H, 4-F-Ph), 8.46 (s, 1H), 8.74 (s, 2H), 13.55 (s, 1H,
N-H, exchangeable); GC (temperature program B) 15.0 min;
MS m/z (%) 260 (96, M+), 227 (83), 164 (22), 139 (12), 123 (40),
123 (40), 109 (100), 95 (39), 83 (26).
1
22: mp 150 °C; H NMR δ 5.76 (s, 2H, CH2), 7.17-7.41 (m,
6-Benzylsulfanyl-9-methyl-9H-purine (15).29 The com-
pound was prepared from 9-methyl-9H-purine-6-thiol24 (0.47
g, 2.8 mmol) and benzyl chloride (0.38 g, 3 mmol) according to
general procedure C and was purified by column chromatog-
raphy (CH2Cl2/EtOH 95:5) to yield 15 (430 mg; 56%): mp 133
°C; 1H NMR δ 3.82 (s, 3H), 4.66 (s, 2H), 7.24-7.49 (m, 5H,
Ph), 8.42 (s, 1H), 8.77 (s, 2H); GC (temperature program B)
13.1 min; MS m/z (%) 256 (100, M+), 241 (4), 223 (76), 208
(12), 179 (15), 134 (15), 121 (19), 91 (91), 77 (14).
5H, Ph), 8.82 (s, 1H), 9.01 (s, 1H); GC (temperature program
B) 15.1 min; MS m/z (%) 246 (20), 245 (15), 244 (62, M+), 243
(23), 209 (9), 182 (7), 153 (5), 126 (5), 91 (100), 65 (29).
6-Chloro-9-(4-fluorobenzyl)-9H-purine45 (23) and 6-Chlo-
ro-7-(4-fluorobenzyl)-7H-purine (24). The compounds were
prepared from 6-chloropurine (1.55 g, 15 mmol) and 4-fluo-
robenzyl chloride (2.16 g, 15 mmol) together with K2CO3 (4.15
g, 30 mmol) as a base according to the method described for
21 and 22. The compounds were separated by column chro-
matography (EtOAc/hexane 7:3) to yield first 23 (1.31 g; 50%)
and afterwards 24 (520 mg; 20%). Compound 23: mp 135 °C;
1H NMR δ 5.54 (s, 2H, CH2), 7.15-7.23 (m, 2H, 4-F-Ph), 7.42-
7.49 (m, 2H, 4-F-Ph), 8.80 (s, 1H), 8.86 (s, 1H); GC (temper-
ature program B) 12.9 min; MS m/z (%) 264 (28), 262 (84, M+),
227 (6), 200 (7), 167 (5), 146 (4), 109 (100), 83 (32). Compound
6-(4-Fluorobenzylsulfanyl)-9-methyl-9H-purine (16).
The compound was prepared from 9-methyl-9H-purine-6-
thiol24 (0.47 g, 2.8 mmol) and 4-fluorobenzyl chloride (0.43 g,
3 mmol) according to general procedure C and was purified
by column chromatography (CH2Cl2/EtOH 95:5) to yield 16
1
(450 mg; 58%): mp 152 °C; H NMR δ 3.82 (s, 3H), 4.65 (s,
1
24: mp 170 °C; H NMR δ 5.75 (s, 2H, CH2), 7.15-7.33 (m,
2H), 7.09-7.18 (m, 2H, 4-F-Ph), 7.47-7.54 (m, 2H, 4-F-Ph),
8.43 (s, 1H), 8.77 (s, 2H); GC (temperature program B) 12.5
min; MS m/z (%) 274 (100, M+), 259 (3), 241 (70), 226 (10),
178 (20), 134 (22), 123 (20), 109 (95), 95 (22). Anal. (C13H11-
FN4S) C, H, N.
4H, 4-F-Ph), 8.82 (s, 1H), 9.0 (s, 1H); GC (temperature
program B) 15.0 min; MS m/z (%) 264 (8), 262 (24, M+), 227
(5), 200 (6), 173 (5), 153 (4), 109 (100), 83 (32). Anal. (C12H8-
ClFN4) C, H, N.
9-Benzyl-9H-purin-6-ylamine (25).46 A solution of 9-ben-
zyl-6-chloro-9H-purine 21 (400 mg, 1.64 mmol) in 7 N metha-
nolic ammonia (40 mL) was stirred in a sealed bottle at 90 °C
for 7 h. The solvent was evaporated in vacuo and the residue
washed with diethyl ether to afford 25 (240 mg; 65%): mp 235
7-Phenyl-7H-purine (17).27 A solution of phenylaminoace-
tonitrile (1.65 g, 12.5 mmol) and formamidine acetate (6.24 g,
60 mmol) in n-butanol (13 mL) was refluxed for 5 h. The
solvent was evaporated in vacuo and the residue taken up in
water. This was extracted with CH2Cl2. The organic layer was
dried over Na2SO4 and evaporated in vacuo, and the residue
purified was by column chromatography (CH2Cl2/EtOH 9:1)
1
°C; H NMR δ 5.38 (s, 2H, CH2), 7.27-7.36 (m, 7H, phenyl,
C6-NH2 (exchangeable)), 8.17 (s, 1H), 8.28 (s, 1H); GC (tem-
perature program B) 13.3 min; MS m/z (%) 225 (44), 224 (100,
M+), 207 (7), 182 (11), 148 (13), 91 (94), 65 (52).
1
to produce 17 (710 mg; 29%): mp 186 °C; H NMR δ 7.48-
7.83 (m, 5H, Ph), 9.06 (s, 1H), 9.10 (s, 1H), 9.22 (s, 1H); GC
(temperature program B) 8.7 min; MS m/z (%) 196 (100, M+),
169 (31), 142 (31), 115 (20), 105 (10), 91 (11), 77 (30).
9-(4-Fluorobenzyl)-9H-purin-6-ylamine (26). The com-
pound was prepared from 6-chloro-9-(4-fluorobenzyl)-9H-pu-

Purine Derivatives as Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 719
rine 23 according to the method described for 25 to yield 26
(350 mg; 76%): mp 215 °C; 1H NMR δ 5.37 (s, 2H, CH2), 7.13-
7.43 (m, 6H, 4-F-Ph, C6-NH2, exchangeable), 8.17 (s, 1H), 8.28
(s, 1H); GC (temperature program B) 13.1 min; MS m/z (%)
243 (38, M+), 242 (48), 200 (7), 134 (14), 109 (100), 83 (36).
Anal. (C12H10FN5) C, H, N.
nylboronic acid (190 mg, 1.28 mmol), K2CO3 (316 mg, 2.3
mmol), DME (8 mL), and Pd(PPh3)4 (28 mg, 0.02 mmol) was
stirred under argon protected from light at 90 °C for 3 h. The
reaction mixture was filtered, the filtrate evaporated in vacuo,
and the residue purified by column chromatography (CH2Cl2/
1
EtOH 9:1) to produce 32 (32 mg; 15%): mp 167 °C; H NMR
δ 3.87 (s, 1H, N9-CH3), 7.41-7.51 (m, 3H, Ph), 7.65 (d, 1H,
3J(H,H) ) 16.2 Hz, trans), 7.77-7.82 (m, 2H, Ph), 8.42 (d, 1H,
3J(H,H) ) 16.2 Hz, trans), 8.59 (s, 1H), 8.89 (s, 1H); GC
(temperature program B) 16.9 min; MS m/z (%) 236 (25), 235
(100, M+), 221 (5), 208 (5), 193 (5), 181 (3), 167 (6), 155 (3),
140 (9), 128 (7). Anal. (C14H12N4) C, H, N.
4-Chloro-5-nitro-6-phenylpyrimidine (33). A mixture of
4,6-dichloro-5-nitropyrimidine (2 g, 10.36 mmol), benzenebo-
ronic acid (1.26 g, 10.36 mmol), Na2CO3 (3.29 g, 31 mmol), Pd-
(PPh3)4 (200 mg, 0.17 mmol), water (8 mL), and toluene (10
mL) was stirred under argon protected from light at 90 °C for
4.5 h. After the addition of water, the mixture was extracted
with CH2Cl2. The organic layer was dried (Na2SO4) and
evaporated in vacuo, and the residue was purified by column
chromatography (CH2Cl2) to afford 33 (1.54 g: 63%): mp 65
°C; 1H NMR δ 7.55-7.71 (m, 5H, Ph), 9.35 (s, 1H); GC
(temperature program A) 13.5 min; MS m/z (%) 237 (6), 253
(18, M+), 209 (28), 207 (86), 178 (18), 144 (40), 127 (100), 100
(50), 77 (85).
4-Chloro-6-(4-fluorophenyl)-5-nitropyrimidine (34). The
compound was prepared according to the procedure for 33 from
4,6-dichloro-5-nitropyrimidine (2 g, 10.36 mmol), 4-fluoroben-
zeneboronic acid (1.45 g, 10.36 mmol), Na2CO3 (3.29 g, 31
mmol), Pd(PPh3)4 (200 mg, 0.17 mmol), water (10 mL), and
toluene (25 mL) and was purified by column chromatography
(CH2Cl2) to yield 34 (1.58 g; 60%): mp 57 °C; 1H NMR (CDCl3)
δ 7.18-7.27 (m, 2H, 4-F-Ph), 7.71-7.81 (m, 2H, 4-F-Ph), 9.11
(s, 1H);); GC (temperature program A) 13.1 min; MS m/z (%)
255 (19), 253 (54, M+), 223 (21), 187 (18), 165 (23), 145 (100),
118 (25), 99 (71), 75 (44), 57 (18).
7-(4-Fluorobenzyl)-7H-purin-6-ylamine (27) and 7-(4-
Fluorobenzyl)-6-methoxy-7H-purine (28). The compounds
were prepared from 6-chloro-7-(4-fluorobenzyl)-7H-purine 24
(200 mg, 0.76 mmol) according to the method described for
25. The compounds were separated by column chromatography
(Al2O3, CH2Cl2/EtOH 9:1) yielding first 28 (80 mg; 41%) as a
previously undesired side product and subsequently 27 (20 mg;
11%). Compound 28: mp 170 °C; 1H NMR δ 4.07 (s, 3H), 5.55
(s, 2H), 7.14-7.42 (m, 4H, 4-F-Ph), 8.54 (s, 1H), 8.70 (s, 1H);
GC (temperature program B) 15.5 min; MS m/z (%) 258 (70,
M+), 243 (7), 227 (7), 201 (3), 149 (16), 109 (100), 83 (26). Anal.
1
(C12H10FN5) C, H, N. Compound 27: mp 220 °C; H NMR δ
5.69 (s, 2H, CH2), 7.05 (s, 2H, C6-NH2, exchangeable), 7.13-
7.22 (m, 4H, 4-F-Ph), 8.23 (s, 1H), 8.47 (s, 1H); GC (temper-
ature program B) 20.9 min; MS m/z (%) 243 (47, M+), 242 (30),
200 (5), 134 (11), 109 (100), 83 (27). Anal. (C13H11FN4O) C, H,
N.
6-Benzyl-9H-purine (29).30 Benzyl(tri-n-butyl)phospho-
nium bromide47 (3.42 g, 9.2 mmol) was suspended in DME (50
mL) at -35 °C. n-Butyllithium (4 g, 9.25 mmol, 15% in hexane)
was added dropwise and the mixture stirred for 1 h. A solution
of 6-chloro-9-(tetrahydro-2-pyranyl)purine29 (1 g, 4.2 mmol) in
DME (10 mL) was added. The mixture was allowed to warm
up to room temperature, at which point it was stirred for 6 h.
A solution of Na2CO3 (0.44 g, 4.2 mmol) in water (10 mL) was
added and the mixture refluxed for 3 h. The volatiles were
evaporated in vacuo, and the residue was suspended in water.
The mixture was extracted with diethyl ether, and the organic
phase dried (Na2SO4) and evaporated in vacuo. The residue
was dissolved in a mixture of ethanol (25 mL) and 1 N HCl
(10 mL) and stirred at room temperature for 4 h. The excess
acid was neutralized by addition of NaHCO3 (ca. 1 g) and the
solvent was removed in vacuo. The residue was purified by
column chromatography (CH2Cl2/EtOH 95:5) to produce 29
(390 mg; 44%): mp 150 °C; 1H NMR (CDCl3) δ 4.59 (s, 2H,
CH2), 7.19-7.45 (m, 5H, Ph), 8.24 (s, 1H), 8.98 (s, 1H), (N9-H
not visible).
6-(4-Fluorobenzyl)-9H-purine (30). The compound was
prepared from tributyl(4-fluorobenzyl)phosphonium bromide47
(3.6 g, 9.2 mmol) and 6-chloro-9-(tetrahydro-2-pyranyl)purine29
(1 g, 4.2 mmol) according to the method described for 29 and
was purified by column chromatography (CH2Cl2/EtOH 95:5)
to yield 30 (230 mg; 24%): mp 176 °C; 1H NMR (CDCl3) δ 4.55
(s, 2H, CH2), 6.92-7.01 (m, 2H, 4-F-Ph), 7.39-7.46 (m, 2H,
4-F-Ph), 8.29 (s, 1H), 8.96 (s, 1H), 12.75 (s, 1H, N9-H,
exchangeable). Anal. (C12H9FN4) C, H, N; C: calcd, 63.15;
found, 59.86.
Methyl-(5-nitro-6-phenylpyrimidin-4-yl)amine (35). A
solution of 33 (1.54 g, 6.55 mmol) and methylamine (20 mL,
40% in water) in dioxane (25 mL) was refluxed for 1 h. After
evaporation of the volatiles, the residue was dissolved in
ethanol and the solution filtered over SiO2 (CH2Cl2/EtOH 9:1).
The filtrate was evaporated in vacuo to give 35 (1.43 g; 95%):
1
3
mp 142 °C; H NMR δ 2.97 (d, 3H, CH3, J(H,H) ) 3.9 Hz),
7.50 (s, br, 5H, Ph), 8.14 (s, br, 1H, N-H, exchangeable), 8.67
(s, 1H); GC (temperature program A) 19.7 min; MS m/z (%)
230 (95, M+), 216 (46), 200 (90), 173 (39), 156 (57), 128 (85),
102 (67), 77 (100), 57 (63).
[6-(4-Fluorophenyl)-5-nitropyrimidin-4-yl]methyl-
amine (36). A solution of 34 (1.58 g, 6.25 mmol) and methyl-
amine (20 mL, 40% in water) in dioxane (25 mL) was refluxed
for 1 h. After evaporation of the volatiles, the residue was
dissolved in ethanol and the solution filtered over SiO2 (CH2-
Cl2/EtOH 9:1). The filtrate was evaporated in vacuo to give
1
6-Styryl-9H-purine (31).30 Methyltriphenylphosphonium
bromide (3.28 g, 9.2 mmol) was suspended in DME (50 mL)
at -35 °C. n-Butyllithium (4 g, 9.25 mmol, 15% in hexane)
was added dropwise and the mixture stirred for 1 h. A solution
of 6-chloro-9-(tetrahydro-2-pyranyl)purine29 (1 g, 4.2 mmol) in
DME (10 mL) was added. The mixture was allowed to warm
to room temperature, where it was stirred for 4 h. A solution
of benzaldehyde (1.78 g, 16.8 mmol) in DME (5 mL) was added
and the mixture stirred for 24 h. The precipitated phospho-
nium salt was removed by filtration and the filtrate evaporated
in vacuo. The residue was dissolved in a mixture of ethanol
(25 mL) and 1 N HCl (10 mL) and stirred at room temperature
for 4 h. Excess acid was neutralized by addition of NaHCO3
(ca. 1 g) and the solvent was removed in vacuo. The residue
was purified by column chromatography (CH2Cl2/EtOH 95:5)
35 (1.47 g; 95%): mp 114 °C; H NMR (CDCl3) δ 3.19 (d, 3H,
3
CH3, J(H,H) ) 4.9 Hz), 7.09-7.18 (m, 2H, 4-F-Ph), 7.36 (s,
br, 1H, N-H, exchangeable), 7.49-7.56 (m, 2H, 4-F), 8.66 (s,
1H); GC (temperature program A) 18.8 min; MS m/z (%) 248
(100, M+), 224 (44), 218 (72), 174 (64), 146 (71), 121 (66), 81
(57), 57 (65).
N′4′-Methyl-6-phenylpyrimidin-4,5-diamine (37). A so-
lution of 35 (1.5 g, 6.55 mmol) in a 1:1 mixture of CH2Cl2 and
ethanol (50 mL) containing Pd/C (100 mg) was stirred for 30
min under a hydrogen pressure of 4 bar. The mixture was
filtered and the solvent evaporated in vacuo to produce 37 (1.2
1
3
g; 92%): mp 161 °C; H NMR δ 2.91 (d, 3H, CH3, J(H,H) )
4.5 Hz, exchangeable), 4.52 (s, 2H, N-H2), 6.73 (d, 1H, C4-
N-H, 3J(H,H) ) 4.5 Hz, exchangeable), 7.37-7.67 (m, 5H, Ph),
8.03 (s, 1H, C2-H); GC (temperature program B) 10.7 min; MS
m/z (%) 200 (100, M+), 199 (98), 170 (20), 143 (14), 117 (15),
104 (31), 89 (22), 77 (18).
1
to produce 31 (185 mg; 20%): mp 207 °C; H NMR δ 7.37-
7.52 (m, 3H), 7.65-7.81 (m, 3H), 8.29-8.37 (m, 1H), 8.63 (s,
1H), 8.86 (s, 1H), 13.56 (s, 1H, N9-H); ESI-MS m/z (%) 323
(46, [M + 1]+), 208 (100), 154 (27), 119 (33), 115 (30), 94 (24).
6-(4-Fluorophenyl)-N′4′-methylpyrimidine-4,5-di-
amine (38). A solution of 36 (1.55 g, 6.25 mmol) in a 1:1
mixture of CH2Cl2 and ethanol (50 mL) containing Pd/C (100
mg) was stirred for 30 min under a hydrogen pressure of 4
9-Methyl-6-styryl-9H-purine (32). A mixture of 6-chloro-
9-methyl-9H-purine24 (144 mg, 0.85 mmol), trans-2-phenylvi-

720 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Laufer et al.
bar. The mixture was filtered and the solvent evaporated in
vacuo to give 38 (1.23 g; 90%): mp 169 °C; 1H NMR δ 2.90 (d,
3H, CH3, 3J(H,H) ) 4.5 Hz, exchangeable), 4.57 (s, 2H, N-H2),
6.76 (d, 1H, C4-N-H, 3J(H,H) ) 4.5 Hz, exchangeable), 7.23-
7.32 (m, 2H, 4-F-Ph), 7.65-7.72 (m, 2H, 4-F-Ph), 8.01 (s, 1H);
GC (temperature program B) 10.5 min; MS m/z (%) 218 (100,
M+), 217 (93), 188 (26), 161 (19), 139 (21), 122 (42), 107 (32),
95 (18).
(s, 3H), 7.50-7.64 (m, 8H, Ph), 7.92-7.97 (m, 2H, Ph), 8.60
(s, 1H); GC (temperature program B) 21.3 min; MS m/z (%)
318 (57, M+), 317 (100), 303 (7), 173 (8), 141 (11), 103 (15), 77
(27). Anal. (C18H14N4S) C, H, N.
6-(4-Fluorophenylsulfanyl)-9-methyl-8-phenyl-9H-pu-
rine (45). The compound was prepared from 43 (300 mg, 1.23
mmol) and 4-fluorothiophenol (400 mg, 3 mmol) according to
general procedure A and was purified by column chromatog-
raphy (CH2Cl2/EtOH 975:25) to yield 45 (310 mg; 75%): mp
157 °C; 1H NMR δ 3.90 (s, 3H), 7.32-7.40 (m, 2H, Ph), 7.61-
7.74 (m, 5H, Ph), 7.92-7.97 (m, 2H, Ph), 8.60 (s, 1H); GC
(temperature program B) 29.0 min; MS m/z (%) 336 (62), 335
(100, M+), 321 (8), 308 (7), 141 (9), 126 (4). Anal. (C18H13FN4S)
C, H, N.
9-Methyl-6-phenyl-9H-purine (39).48 A solution of 37 (600
mg, 3 mmol) and p-toluenesulfonic acid (10 mg) in a mixture
of ethanol (10 mL) and triethylorthoformate (20 mL) was
refluxed for 5 h. The volatiles were evaporated in vacuo, and
the residue was purified by column chromatography (CH2Cl2/
1
EtOH 9:1) to yield 39 (300 mg; 48%): mp 120 °C; H NMR δ
3.88 (s, 3H), 7.56-7.63 (m, 3H, Ph), 8.64 (s, 1H), 8.83-8.88
(m, 2H, Ph), 9.00 (s, 1H); GC (temperature program B) 11.7
min; MS m/z (%) 210 (100, M+), 191 (20), 183 (19), 182 (19),
141 (21), 159 (12), 114 (18), 77 (23).
6-Chloro-9-methyl-8-(4-methylsulfanylphenyl)-9H-pu-
rine (46). A mixture of 42 (500 mg, 2 mmol), 4-methylthioben-
zeneboronic acid (370 mg, 2.2 mmol), K2CO3 (345 mg, 2.5
mmol), Pd(PPh3)4 (120 mg, 0.1 mmol), and toluene (15 mL)
was stirred under argon protected light at 90 °C for 7.5 h. The
mixture was filtered, and the filtrate evaporated in vacuo. The
residue was purified by column chromatography (hexane/
EtOAc 1:1) to produce 45 (350 mg; 60%): mp 135 °C; 1H NMR
δ 2.58 (s, 3H, S-CH3), 3.95 (s, 3H, N9-CH3), 7.45-7.49 (m, 2H,
Ph), 7.45-7.49 (m, 2H, Ph), 8.78 (s, 1H); GC (temperature
program B) 32.2 min; MS m/z (%) 292 (33), 290 (100, M+), 274
(12), 253 (12), 243 (18), 149 (23).
6-(4-Fluorophenyl)-9-methyl-9H-purine (40). The com-
pound was prepared from 38 (500 mg, 2.29 mmol) according
to the method described for 39 and was purified by column
chromatography (EtOAc/hexane 7:3) to yield 40 (190 mg;
1
36%): mp 140 °C; H NMR δ 3.88 (s, 3H), 7.39-7.48 (m, 2H,
4-F-Ph), 8.64 (s, 1H), 8.89-8.95 (m, 2H), 8.98 (s, 1H); GC
(temperature program B) 11.2 min; MS m/z (%) 228 (100, M+),
223 (26), 200 (17), 188 (25), 159 (12), 132 (11), 95 (10). Anal.
(C12H9FN4) C, H, N.
6-Chloro-8-(4-methansulfinylphenyl)-9-methyl-9H-pu-
rine (47). To a solution of 46 (660 mg, 2.27 mmol) in glacial
acetic acid (6 mL) was added H2O2 (1.9 g, 30 mmol, 30% in
water) dropwise and the solution stirred for 3 h. Approximately
30 g of ice was added and the solution made basic by the
addition of ammonia (25% in water). The mixture was ex-
tracted with CH2Cl2. The organic phase was washed with
water, dried (Na2SO4), and evaporated to yield 47 (620 mg;
6-(4-Fluorophenyl)-9-methyl-8-phenyl-9H-purine (41).
The compound was prepared from 38 (600 mg, 2.75 mmol) and
orthobenzoic acid trimethyl ester (10 mL) according to the
method described for 39 and was purified by column chroma-
tography (CH2Cl2/EtOH 95:5) to yield 41 (310 mg; 37%): mp
154 °C; 1H NMR δ 3.93 (s, 3H), 7.38-7.67 (m, 5H, Ph), 7.98-
8.02 (m, 2H, 4-F-Ph), 8.92-8.99 (m, 3H, 4-F-Ph, C2-H); GC
(temperature program B) 23.1 min; MS m/z (%) 304 (100, M+),
303 (98), 289 (7), 276 (6), 247 (4), 145 (11), 118 (14), 95 (8), 77
(18). Anal. (C18H13FN4) C, H, N; C: calcd, 71.04; found, 69.57.
8-Bromo-6-chloro-9-methyl-9H-purine (42). A stirred
solution of diisopropylamine (1.70 g, 16.80 mmol) in dry THF
(15 mL) was cooled to -78 °C. n-Butyllithium (7.17 g, 16.88
mmol, 15% in hexane) was added dropwise and the mixture
stirred for 1 h. At this point, a solution of 6-chloro-9-methyl-
9H-purine24 (2.02 g, 12 mmol) in dry THF (20 mL) was added
slowly and the mixture stirred for 1 h at -78 °C. Subsequently,
a solution of dibromotetrachloroethane (7.81 g, 24 mmol) in
dry THF (20 mL) was added and the mixture stirred for 1 h
at -78 °C. A saturated aqueous solution of ammonium (10 mL)
was added. The mixture was allowed to warm to room
temperature and THF was evaporated in vacuo. Water (50 mL)
was added to the residue and extracted with CH2Cl2. The
organic phase was dried (Na2SO4) and evaporated in vacuo.
The residue was extracted with boiling hexane, which was
filtered hot. The remainder then was purified by column
chromatography (CH2Cl2/EtOAc 6:4) to afford 42 (1.96 g;
66%): mp 187 °C; 1H NMR δ 3.7 (s, 3H), 8.7 (s, 1H); GC
(temperature program B) 7.2 min; MS m/z (%) 250 (25), 249
(8), 248 (100), 247 (8, M+), 246 (70), 213 (21), 211 (23), 169
(11), 167 (33), 132 (25), 105 (35), 77 (25), 52 (12).
1
90%): mp 192 °C; H NMR δ 2.87 (s, 3H, SO-CH3), 3.98 (s,
3H, N9-CH3), 7.93-7.97 (m, 2H, Ph), 8.17-8.21 (m, 2H, Ph),
8.53 (s, 1H); GC (temperature program B) 32.4 min; MS m/z
(%) 308 (18), 306 (54, M+), 291 (100), 291 (33), 274 (20), 259
(34), 242 (19).
6-Chloro-8-(4-methanesulfonylphenyl)-9-methyl-9H-
purine (48). A solution of 46 (1.1 g, 3.8 mmol) and 3-chloro-
perbenzoic acid (2.0 g, 8.14 mmol) in CH2Cl2 (20 mL) was
refluxed for 4 h. The mixture was washed with a saturated
solution of NaHCO3 and water, dried (Na2SO4), and evaporated
1
in vacuo to yield 48 (1.16 g; 95%): mp 65 °C; H NMR δ 3.34
(s, 3H, SO-CH3), 3.97 (s, 3H, N9-CH3), 8.21 (q, 4H, Ph), 8.85
(s, 1H); GC (temperature program B) 34.0 min; MS m/z (%)
317 (51), 322 (100, M+), 321 (91), 243 (44), 207 (54), 79 (36).
9-Methyl-8-(4-methylsulfanylphenyl)-6-phenylsulfanyl-
9H-purine (49). The compound was prepared from 46 (320
mg, 1.1 mmol) and thiophenol (288 mg, 2.6 mmol) according
to general procedure A and was purified by column chroma-
tography (CH2Cl2/EtOH 975:25) to yield 49 (223 mg; 56%): mp
161 °C; 1H NMR δ 2.57 (s, 3H, S-CH3), 3.90 (s, 3H, N9-CH3),
7.44-7.52 (m, 5H, Ph), 7.63-7.68 (m, 2H, Ph), 7.87-7.91 (m,
2H, Ph), 8.58 (s, 1H); GC (temperature program B) 32.8 min;
MS m/z (%) 364 (65, M+), 363 (100), 349 (9), 348 (17), 334 (4),
316 (5), 214 (4), 181 (18), 149 (12), 77 (10). Anal. (C19H16N4S2)
C, H, N.
6-Chloro-9-methyl-8-phenyl-9H-purine (43). A mixture
of 42 (820 mg, 3.33 mmol), benzeneboronic acid (440 mg, 3.60
mmol), K2CO3 (550 mg, 4.0 mmol), Pd(PPh3)4 (120 mg, 0.1
mmol), and toluene (15 mL) was stirred under argon protected
from light at 90 °C for 7 h. The mixture was filtered, the
filtrate evaporated in vacuo, and the residue purified by
column chromatography (CH2Cl2/EtOH 975:25) to yield 43 (530
6-(4-Fluorophenylsulfanyl)-9-methyl-8-(4-methylsulfa-
nylphenyl)-9H-purine (50). The compound was prepared
from 46 (320 mg, 1.1 mmol) and 4-fluorothiophenol (333 mg,
2.6 mmol) according to general procedure A and was purified
by column chromatography (CH2Cl2/EtOH 975:25) to yield 50
1
(280 mg; 67%): mp 179 °C; H NMR δ 2.57 (s, 3H, S-CH3),
1
3.90 (s, 3H, N9-CH3), 7.31-7.48 (m, 4H, Ph), 7.67-7.74 (m,
2H, Ph), 7.86-7.91 (m, 2H, Ph), 8.58 (s, 1H). Anal. (C19H15-
FN4S2) C, H, N.
mg; 65%): mp 157 °C; H NMR (CDCl3) δ 3.99 (s, 3H), 7.56-
7.62 (m, 5H, Ph), 8.76 (s, 1H); GC (temperature program B)
8.8 min; MS m/z (%) 245 (33), 244 (82, M+), 243 (100), 207
(52), 141 (7), 103 (10), 77 (11).
8-(4-Methanesulfinylphenyl)-9-methyl-6-phenylsulfa-
nyl-9H-purine (51). The compound was prepared from 47
(300 mg, 0.98 mmol) and thiophenol (288 mg, 2.6 mmol)
according to general procedure A and was purified by column
chromatography (CH2Cl2/EtOH 95:5) to yield 51 (180 mg;
9-Methyl-8-phenyl-6-phenylsulfanyl-9H-purine (44). The
compound was prepared from 43 (350 mg, 1.43 mmol) and
thiophenol (380 mg, 6.2 mmol) according to general procedure
A and was purified by column chromatography (CH2Cl2/EtOH
975:25) to yield 44 (200 mg; 44%): mp 147 °C; 1H NMR δ 3.90
1
50%): mp 196 °C; H NMR δ 2.87 (s, 3H, SO-CH3), 3.95 (s,

Purine Derivatives as Kinase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 721
3H, N9-CH3), 7.50-7.70 (m, 5H, Ph), 7.92-7.96 (m, 2H, Ph),
8.15-8.19 (m, 2H, Ph), 8.64 (s, 1H); ESI-MS m/z (%) 381 (100,
[M + 1]+), 366 (20). Anal. (C19H16N4OS2) C, H, N.
7:3) to yield 59 (505 mg; 87%): mp 128 °C; 1H NMR δ 5.38 (s,
2H, CH2), 6.99-7.08 (m, 2H, Ph), 7.26-7.33 (m, 2H, Ph), 7.45-
7.49 (m, 3H, Ph), 7.64-7.69 (m, 2H, Ph), 7.97 (s, 1H), 8.64 (s,
1H); GC (temperature program B) 19.3 min; MS m/z (%)337
(8), 336 (28, M+), 227 (17), 110 (9), 109 (100), 83 (18), 77 (7).
Anal. (C18H13FN4S) C, H, N.
6-(4-Fluorophenylsulfanyl)-8-(4-methanesulfinylphen-
yl)-9-methyl-9H-purine (52). The compound was prepared
from 47 (300 mg, 0.98 mmol) and 4-fluorothiophenol (333 mg,
2.6 mmol) according to general procedure A and was purified
by column chromatography (CH2Cl2/EtOH 95:5) to yield 52 (74
9-(4-Fluorobenzyl)-6-(4-fluorophenylsulfanyl)-9H-pu-
rine (60). The compound was prepared from 23 (453 mg, 1.73
mmol) and 4-fluorothiophenol (570 mg, 4.46 mmol) according
to general procedure A and was purified by column chroma-
tography (EtOAc/hexane 7:3) to yield 60 (480 mg; 78%): mp
1
mg; 19%): mp 213 °C; H NMR δ 2.85 (s, 3H, SO-CH3), 3.93
(s, 3H, N9-CH3), 7.32-7.17 (m, 8H, Ph), 8.62 (s, 1H); ESI-MS
m/z (%) 399 (100, [M + 1]+), 384 (15). Anal. (C19H15FN4OS2)
C, H, N.
1
188 °C; H NMR δ 5.39 (s, 2H, CH2), 7.00-7.33 (m, 6H, Ph),
8-(4-Methanesulfonylphenyl)-9-methyl-6-phenylsulfa-
nyl-9H-purine (53). The compound was prepared from 48
(500 mg, 1.55 mmol) and thiophenol (444 mg, 4.0 mmol)
according to general procedure A and was purified by column
chromatography (CH2Cl2/EtOH 975:25) to yield 53 (260 mg;
7.60-7.67 (m, 2H, Ph), 7.98 (s, 1H), 8.64 (s, 1H); GC (temper-
ature program B) 19.3 min; MS m/z (%) 354 (45, M+), 245 (25),
109 (100), 83 (25). Anal. (C18H12F2N4S) C, H, N.
Acknowledgment. The authors would like to thank
Merckle GmbH, Blaubeuren, Germany, and Fonds der
Chemischen Industrie, Germany, for their generous
support of this work. We thank Rainer Kahlich and
Bernd Kammerer for recording the ESI-MS spectra of
compounds 31 and 51-54.
1
42%): mp 241 °C; H NMR δ 3.34 (s, 3H, SO-CH3), 3.94 (s,
3H, N9-CH3), 7.51-7.69 (m, 5H, Ph), 8.20 (q, 4H, Ph), 8.64 (s,
1H); ESI-MS m/z (%) 397 (22, [M + 1]+), 318 (100). Anal.
(C19H16N4O2S2) C, H, N.
6-(4-Fluorophenylsulfanyl)-8-(4-methanesulfonylphen-
yl)-9-methyl-9H-purine (54). The compound was prepared
from 48 (500 mg, 1.55 mmol) and 4-fluorothiophenol (512 mg,
4.0 mmol) according to general procedure A and was purified
by column chromatography (CH2Cl2/EtOH 975:25) to yield 54
(150 mg; 23%): mp 250 °C; 1H NMR δ 3.13 (s, 3H, SO-CH3),
3.99 (s, 3H, N9-CH3), 7.14-7.26 (m, 2H, 4-F-Ph), 7.62-7.69
(m, 2H, 4-F-Ph), 8.12 (q, 4H, Ph), 8.66 (s, 1H); ESI-MS m/z
(%) 415 (30, [M + 1]+), 336 (100). Anal. (C19H15FN4O2S2) C, H,
N.
Supporting Information Available: Analysis data for
the novel described test compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Hopkins, A. L.; Groom, C. R. Opinion: The druggable genome.
Nat. Rev. Drug Discov. 2002, 1, 727-730.
(2) Mueller, G. Medicinal chemistry of target family-directed mas-
terkeys. Drug Discov. Today 2003, 8, 681-691.
(3) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudar-
sanam, S. The Protein Kinase Complement of the Human
Genome. Science 2002, 298, 1912-1916, 1933.
(4) Xie, X.; Gu, Y.; Fox, T.; Coll, J. T.; Fleming, M. A.; Markland,
W.; Caron, P. R.; Wilson, K. P.; Su, M. S. S. Crystal structure of
JNK3: A kinase implicated in neuronal apoptosis. Structure
1998, 6, 983-991.
(5) Bellon, S.; Fitzgibbon, M. J.; Fox, T.; Hsiao, H. M.; Wilson, K.
P. The structure of phosphorylated p38gamma is monomeric and
reveals a conserved activation-loop conformation. Structure Fold.
Des 1999, 7, 1057-1065.
(6) Huse, M.; Kuriyan, J. The conformational plasticity of protein
kinases. Cell (Cambridge, MA) 2002, 109, 275-282.
(7) Traxler, P. Tyrosine kinase inhibitors in cancer treatment (part
II). Exp. Opin. Ther. Patents 1998, 8, 1599-1625.
(8) Traxler, P. M. Protein tyrosine kinase inhibitors in cancer
treatment. Exp. Opin. Ther. Patents 1997, 7, 571-588.
(9) Adams, J. A. Kinetic and catalytic mechanisms of protein
kinases. Chem. Rev. 2001, 101, 2271-2290.
(10) Boehm, J. C.; Adams, J. L. New inhibitors of p38 kinase. Exp.
Opin. Ther. Patents 2000, 10, 25-37.
(11) Meijer, L.; Raymond, E. Roscovitine and Other Purines as Kinase
Inhibitors. From Starfish Oocytes to Clinical Trials. Acc. Chem.
Res. 2003, 36, 417-425.
(12) Vesely, J.; Havlicek, L.; Strnad, M.; Blow, J. J.; Donella-Deana,
A.; Pinna, L.; Letham, D. S.; Kato, J. y.; Detivaud, L. Inhibition
of cyclin-dependent kinases by purine analogues. Eur. J. Bio-
chem. 1994, 224, 771-786.
(13) Chang, Y. T.; Gray, N. S.; Rosania, G. R.; Sutherlin, D. P.; Kwon,
S.; Norman, T. C.; Sarohia, R.; Leost, M.; Meijer, L.; Schultz, P.
G. Synthesis and application of functionally diverse 2,6,9-
trisubstituted purine libraries as CDK inhibitors. Chem. Biol.
1999, 6, 361-375.
(14) Schulze-Gahmen, U.; Brandsen, J.; Jones, H. D.; Morgan, D. O.;
Meijer, L. Multiple modes of ligand recognition: Crystal struc-
tures of cyclin-dependent protein kinase 2 in complex with ATP
and two inhibitors, olomoucine and isopentenyladenine. Pro-
teins: Struct., Funct., Genet. 1995, 22, 378-391.
(15) Haesslein, J. L.; Jullian, N. Recent advances in cyclin-dependent
kinase inhibition. Purine-based derivatives as anti-cancer agents.
Roles and perspectives for the future. Curr. Top. Med. Chem.
2002, 2, 1037-1050.
(16) Chiosis, G.; Lucas, B.; Shtil, A.; Huezo, H.; Rosen, N. Develop-
ment of a Purine-Scaffold Novel Class of Hsp90 Binders that
Inhibit the Proliferation of Cancer Cells and Induce the Degra-
dation of Her2 Tyrosine Kinase. Bioorg. Med. Chem. 2002, 10,
3555-3564.
8-Bromo-9-methyl-6-phenylsulfanyl-9H-purine (55). The
compound was prepared from 11 (2.42 g, 10 mmol) according
to the procedure described for 42 and was purified by column
chromatography (CH2Cl2/EtOH 95:5) to yield 55 (1.76 g;
1
55%): mp 166 °C; H NMR δ 3.82 (s, 3H), 7.44-7.67 (m, 5H,
Ph), 8.56 (s, 1H); GC (temperature program B) 12.6 min; MS
m/z (%) 322 (34), 321 (100, M+), 320 (34), 319 (96), 241 (19),
199 (7), 173 (8), 132 (10), 109 (13), 105 (12), 77 (16). Anal.
(C12H9BrN4S) C, H, N.
4-(9-Methyl-6-phenylsulfanyl-9H-purin-8-yl)but-3-yn-
1-ol (56). A mixture of 55 (482 mg, 1.5 mmol), but-3-yn-1-ol
(158 mg, 2.25 mmol), triethylamine (505 mg, 5 mmol), PdCl2-
(PPh3)2 (81 mg, 0.12 mmol), CuI (40 mg, 0.23 mmol), and CH2-
Cl2 (10 mL) was refluxed for 7 h under argon protected from
light. The mixture was filtered and the filtrate evaporated in
vacuo. The residue was purified by column chromatography
(CH2Cl2/EtOH/EtOAc 9:2:1) to afford 56 (30 mg; 6.5%): mp
1
3
231 °C (dec); H NMR δ 2.76 (t, 2H, CH2, J(H,H) ) 6.2 Hz),
3
3.69 (q, 2H, CH2, J(H,H) ) 6.2 Hz), 3.80 (s, 3H), 5.10 (t, 1H,
3
OH, J(H,H) ) 6.2 Hz), 7.51-7.64 (m, 5H, Ph), 8.58 (s, 1H).
Anal. (C16H14N4OS) C, H, N.
9-Benzyl-6-phenylsulfanyl-9H-purine (57). The com-
pound was prepared from 21 (423 mg, 1.73 mmol) and
thiophenol (490 mg, 4.46 mmol) according to general procedure
A and was purified by column chromatography (EtOAc/hexane
7:3) to yield 57 (383 mg; 70%): mp 121 °C; 1H NMR δ 5.41 (s,
2H, CH2), 7.29-7.49 (m, 8H, Ph), 7.46-7.66 (m, 2H, Ph), 7.98
(s, 1H), 8.65 (s, 1H)); GC (temperature program B) 21.1 min;
MS m/z (%) 318 (90, M+), 317 (100), 227 (44), 91 (96). Anal.
(C18H14N4S) C, H, N.
9-Benzyl-6-(4-fluorophenylsulfanyl)-9H-purine (58). The
compound was prepared from 21 (423 mg, 1.73 mmol) and
4-fluorothiophenol (570 mg, 4.46 mmol) according to general
procedure A and was purified by column chromatography
(EtOAc/hexane 7:3) to yield 58 (500 mg; 86%): mp 163 °C; 1H
NMR δ 5.42 (s, 2H, CH2), 7.12-7.37 (m, 7H, Ph), 7.60-7.67
(m, 2H, Ph), 7.98 (s, 1H), 8.65 (s, 1H); GC (temperature
program B) 19.3 min; MS m/z (%) 336 (55, M+), 335 (76), 245
(33), 91 (100). Anal. (C18H13FN4S) C, H, N.
9-(4-Fluorobenzyl)-6-phenylsulfanyl-9H-purine (59).
The compound was prepared from 23 (453 mg, 1.73 mmol) and
thiophenol (490 mg, 4.46 mmol) according to general procedure
A and was purified by column chromatography (EtOAc/hexane

722 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Laufer et al.
(17) Dymock, B.; Barril, X.; Beswick, M.; Collier, A.; Davies, N.;
Drysdale, M.; Fink, A.; Fromont, C.; Hubbard, R. E.; Massey,
A.; Surgenor, A.; Wright, L. Adenine derived inhibitors of the
molecular chaperone HSP90-SAR explained through multiple
X-ray structures. Bioorg. Med. Chem. Lett. 2004, 14, 325-328.
(18) Groom, C. R.; Hopkins, A. L. Protein kinase drugs-optimism
doesn’t wait on facts. Drug. Discov. Today 2002, 7, 801-802.
(19) Wang, Z.; Canagarajah, B. J.; Boehm, J. C.; Kassisa, S.; Cobb,
M. H.; Young, P. R.; Abdel-Meguid, S.; Adams, J. L.; Goldsmith,
E. J. Structural basis of inhibitor selectivity in MAP kinases.
Structure 1998, 6, 1117-1128.
(20) Fitzgerald, C. E.; Patel, S. B.; Becker, J. W.; Cameron, P. M.;
Zaller, D.; Pikounis, V. B.; O’Keefe, S. J.; Scapin, G. Structural
basis for p38a MAP kinase quinazolinone and pyridol-pyrimidine
inhibitor specificity. Nat. Struct. Biol. 2003, 10, 764-769.
(21) Scapin, G.; Patel, S. B.; Lisnock, J.; Becker, J. W.; LoGrasso, P.
V. The Structure of JNK3 in Complex with Small Molecule
Inhibitors: Structural Basis for Potency and Selectivity. Chem.
Biol. 2003, 10, 705-712.
(22) www.upstate.com/features/kinaseprofiler.
(23) Bader, H.; Chiang, Y. H. (Polaroid Corp.) 6-Chloropurine salts.
U.S. Patent 4,405,781, 1981.
(24) Robins, R. K.; Lin, H. H. Potential purine antagonists. IV.
Synthesis of some 9-methyl-6-substituted purines. J. Am. Chem.
Soc. 1957, 79, 490-494.
(25) Davoll, J. Pyrrolo[2,3-d]pyrimidines. J. Am. Chem. Soc., Abstr.
1960, 131-138.
(26) Cignarella, G.; Villa, S.; Barlocco, D. Synthesis of a new series
of N-substituted-3-[1-alkyl(aryl)-4-piperidyl]azetidin-2-ones as
possible muscarinic agents. J. Heterocycl. Chem. 1993, 30, 1337-
1340.
(27) Bredereck, H.; Effenberger, F.; Rainer, G. Acid amide reactions.
XXXIV. 7-Substituted purines and their cleavage into 4,5-
diaminopyrimidines. Justus Liebigs Ann. Chem. 1964, 673, 82-
87.
(28) Gundersen, L. L.; Bakkestuen, A. K.; Aasen, J.; Overas, H.; Rise,
F. 6-Halopurines in palladium-catalyzed coupling with organotin
and organozinc reagents. Tetrahedron 1994, 50, 9743-9756.
(29) Robins, R. K.; Godefroi, E. F.; Taylor, E. C.; Lewis, L. R.; Jackson,
A. Purine nucleosides. I. The synthesis of certain 6-substituted-
9-(tetrahydro-2-pyranyl)purines as models of purine deoxy-
nucleosides. J. Am. Chem. Soc. 1961, 83, 2574-2579.
(30) Taylor, E. C.; Martin, S. F. General method for alkylation and
alkenylation of heterocycles. J. Am. Chem. Soc. 1974, 96, 8095-
8101.
(34) Beers, S. A., Malloy, E.; Wachter, M. P.; Wu, W. (Ortho-McNeil
Corporation, Inc.) Preparation of substituted imidazoles useful
in the treatment of inflammatory diseases. Patent WO 9847892
A1, 1998.
(35) Cohen, P. The development therapeutic potential of protein
kinase inhibitors. Curr. Opin. Chem. Biol. 1999, 3, 459-465.
(36) Davies, S. P.; Reddy, H.; Caivano, M.; Cohen, P. Specificity and
mechanism of action of some commonly used protein kinase
inhibitors. Biochem. J. 2000, 351, 95-105.
(37) Miller, D. S.; Horowitz, S. B. Intracellular compartmentalization
of adenosine triphosphate. J. Biol. Chem. 1986, 261, 13911-
13915.
(38) Laufer, S. A.; Wagner, G. K. From Imidazoles to Pyrimidines:
New Inhibitors of Cytokine Release. J. Med. Chem. 2002, 45,
2733-2740.
(39) Nishikawa, S.; Kumazawa, Z.; Kashimura, N.; Nishikimi, Y.;
Uemura, S. Alternating dependency of cytokinin activity in the
number of methylene units in .omega.-phenylalkyl derivatives
of some purine cytokinins and 4-substituted pyrido[3,4-d]pyrim-
idine. Agric. Biol. Chem. 1986, 50, 2243-2249.
(40) Keck, J. H., Jr.; Simpson, R. A.; Wong, J. L. Reactivities and
electronic aspects of nucleic acid heterocycles. 7. Regiospecific
substituent effects in 6-substituted purines as measured by
proton magnetic resonance. J. Org. Chem. 1978, 43, 2587-2590.
(41) Thompson, R. D.; Secunda, S.; Daly, J. W.; Olsson, R. A. N6,9-
Disubstituted adenines: Potent, selective antagonists at the A1
adenosine receptor. J. Med. Chem. 1991, 34, 2877-2882.
(42) Johnston, T. P.; Holum, L. B.; Montgomery, J. A. Synthesis of
potential anticancer agents. XVI. S-Substituted derivatives of
6-mercaptopurine. J. Am. Chem. Soc. 1958, 80, 6265-6271.
(43) Elion, G. B.; Lange, W. H.; Hitchings, G. H. Condensed pyrimi-
dine systems. XVI. Purines and thiazolo[5,4-d]pyrimidines from
4-amino-5-formamido-6-mercaptopyrimidines. J. Am. Chem. Soc.
1956, 78, 2858-2863.
(44) Montgomery, J. A.; Temple, C., Jr. Synthesis of potential
anticancer agents. XXVI. The alkylation of 6-chloropurine. J.
Am. Chem. Soc. 1961, 83, 630-635.
(45) Kelley, J. L.; Krochmal, M. P.; Linn, J. A.; McLean, E. W.;
Soroko, F. E. 6-(Alkylamino)-9-benzyl-9H-purines. A new class
of anticonvulsant agents. J. Med. Chem. 1988, 31, 606-612.
(46) Wojnar, R. J., Brittain, R. J.; Bernstein, J.; Losee, K. A. (Squibb
and Sons E. R.) 9-Benzyladenine antiinflammatory agents and
their use. U.S. Patent 3,930,005, 1973.
(47) Aitken, R. A.; Drysdale, M. J.; Hill, L.; Lumbard, K. W.;
MacCallum, J. R.; Seth, S. Flash vacuum pyrolysis of stabilised
phosphorus ylides. Part 16. Model studies for the construction
of conjugated polymers. Tetrahedron 1999, 55, 11039-11050.
(48) Fuerstner, A.; Leitner, A.; Mendez, M.; Krause, H. Iron-
Catalyzed Cross-Coupling Reactions. J. Am. Chem. Soc. 2002,
124, 13856-13863.
(31) Havelkova, M.; Dvorak, D.; Hocek, M. The Suzuki-Miyaura cross-
coupling reactions of 2-, 6- or 8-halopurines with boronic acids
leading to 2-, 6- or 8-aryl- and -alkenylpurine derivatives.
Synthesis 2001, 1704-1710.
(32) Boon, W. R.; Jones, W. G. M.; Ramage, G. R. Pteridines. I. An
unambiguous synthesis of 7,8-dihydro-6-hydroxypteridines. J.
Chem. Soc. 1951, 96-102.
(33) Nolsoe, J. M. J.; Gundersen, L. L.; Rise, F. Synthesis of
8-halopurines by reaction of lithiated purines with appropriate
halogen donors. Synth. Commun. 1998, 28, 4303-4315.
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