Pyrazolo[4,3-d]pyrimidines as new generation of cyclin-dependent kinase inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00631-0

A search among analogues of anti-CDK purines led to the identification of substituted pyrazolo[4,3-d]pyrimidines as novel inhibitors of CDK1/cyclin B. Some of these derivatives also show antiproliferative activity on cancer cell line K-562, thus may find

Full text of DOI:10.1016/S0960-894X(03)00631-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2989–2992
Pyrazolo[4,3-d]pyrimidines as New Generation of
Cyclin-Dependent Kinase Inhibitors
Daniela Moravcova,a,y Vladimır Krystof,b,y Libor Havlıcek,a Jirı Moravec,a
Rene Lenobelb and Miroslav Strnadb,*
a
´
Isotope Laboratory, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Vıdenˇska 1083,
´
14220 Prague 4, Czech Republic
ˇ
Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany, Slechtitelu 11,
b
´
783 71 Olomouc, Czech Republic
˚
Received 25 February 2003; accepted 11 March 2003
Abstract—A search among analogues of anti-CDK purines led to the identification of substituted pyrazolo[4,3-d]pyrimidines as
novel inhibitors of CDK1/cyclin B. Some of these derivatives also show antiproliferative activity on cancer cell line K-562, thus may
find an application as anticancer agents.
# 2003 Elsevier Ltd. All rights reserved.
While searching for new groups of CDK inhibitors we
found that substituted pyrazolo[4,3-d]pyrimidines,
studied in early 1970s by plant physiologists as cyto-
kinin antagonists with antiproliferative effects on plant
cells,1 also potently inhibit human CDK1. In contrast to
C2, C6, N9-trisubstituted purine inhibitors,2 the pyr-
azolo[4,3-d]pyrimidines demonstrate the comparable
inhibitory effect with only two side chains at positions 3
and 7, respectively. Hence, we prepared several 3,7-dis-
ubstituted pyrazolo[4,3-d]pyrimidines and compared
their CDK1 inhibitory properties and antiproliferative
activity against cancer cell lines with corresponding 6,9-
disubstituted purines. Novel compounds showed sig-
nificantly higher biological activities.
stituted pyrazole III was then cyclised with formamidin
acetate in the presence of Et3N to 7-hydroxy-3-iso-
propylpyrazolo[4,3-d]pyrimidine (IV). The 7-hydroxy
derivative IV was chlorinated with a mixture of SOCl2-
DMF to 7-chloro derivative V, which did not have to be
isolated as a pure compound and thus can be immediately
used for nucleophilic substitution with different amines
to obtain selected 7-substituted-3-isopropylpyrazolo[4,3-
d]pyrimidines (VI). In total, 11 new pyrazolo[4,3-
d]pyrimidine derivatives have been prepared by two
different methods.4 Method A: The substitution of
Cl–C7 was achieved in CHCl3 by reaction of V with 5
equivalents of the appropriate amine at 60 ꢀC for 1 h.
Method B: Five equivalents of the selected amines in
3 mL of ethanol and 3 equiv of N-ethyldiisopropylamine
were added to the CHCl3 solution of V. The mixture
was stirred at 60 ꢀC for 1 h. The crude products were
purified by column chromatography in both cases.
Chemistry of Pyrazolo[4,3-d]pyrimidines
A series of new 3,7-disubstituted pyrazolo[4,3-d]pyr-
imidines was synthesised using a general synthetic route
outlined in Figure 1. The starting material,3 3-isopropyl-
4-nitropyrazolecarboxylic acid (I), was esterified in
methanol saturated with HCl. The resulting nitroester II
was reduced with hydrogen on RaNi under atmospheric
pressure to the corresponding aminoester III. Sub-
Synthesis of 6,9-Disubstituted Purines
The straightforward synthesis of 9-isopropyl-6-substitu-
ted purines started from 6-chloro-9-isopropylpurine.5
The 6-chloro atom was displaced by refluxing with a
primary amine in butan-1-ol in the presence of N-ethyl-
diisopropylamine at 112ꢀC for 1 h. Compounds obtained
by this method were also purified by column chromato-
graphy and data characterising the prepared purines are
*Corresponding author. Tel. +420-58-563-4850; fax: +420-58-563-
4870; e-mail: strnad@aix.upol.cz
yBoth authors contributed equally to this work.
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00631-0

´
D. Moravcova et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2989–2992
2990
Figure 1. Synthesis of 3,7-disubstituted pyrazolo[4,3-d]pyrimidines.
given in ref 4 Compounds VIIa and VIIc were prepared
as described previously.6,7
independent reports by Gray et al.2c which showed that
trisubstituted purines possessing a substituted arylamine
at C6 were strongly reactive. Relative to the parent
compound purvalanol A, good levels of activity were
also observed for the equivalent pyrazolo[4,3-d]pyr-
imidine analogue VIk. Cytotoxicity of new derivatives
usually increased in relation to the improved CDK
inhibitory activity. The pyrazolo[4,3-d]pyrimidine deri-
vatives were again much more potent than appropriate
6,9-disubstituted purine analogues VIIa–k (Table 1).
Results and Discussion
As a part of our ongoing study on structure–activity
relationships of purine CDK inhibitors, we prepared
and tested disubstituted pyrazolo[4,3-d]pyrimidines for
their ability to block the activity of CDK1/cyclin B and
proliferation of myeloid leukemia cell line K-562
according to a published method.8 The results are sum-
marised in Table 1.
To summarise the data, the best results were obtained
with o-hydroxy or m-chloro groups on the benzylamino
and aminophenyl substituents, respectively. These
results confirm that the capacity of new pyrazolo[4,3-
d]pyrimidines can be modulated through variation of
the C7 position aromatic ring substituents. Further-
more, their increased CDK inhibitory activity and anti-
proliferative capacity brings further potential to the
area of development of novel CDK inhibitors.
All synthesised pyrazolo[4,3-d]pyrimidine derivatives
strongly inhibited CDK1 kinase at concentrations
approximately 2–10 times lower than the respective 6,9-
disubstituted purines. In the present study, the first
modification of VIa to be examined was introduction of
o-Br (VIb), which however led to a big loss of activity.
Introduction of an o-MeO group to give the analogue
VIc resulted in a slight loss in activity, whereas the
hydroxybenzyl derivatives VId–f were usually more
potent. The most active substance VId, bearing a 2-
hydroxybenzylamino moiety at C7 like the trisub-
stituted purine olomoucine II,8 demonstrated the impact
and the power of hydroxylated aromatic substituents.
Introduction of a second MeO group to 3-hydroxy
group on the aromatic ring gave 3-hydroxy-4-methoxy-
benzyl analogue VIg with a minimal gain in activity.
Interestingly, certain losses of activity were observed
after substitution of the C7 position with effective cyto-
kinin or anticytokinin side chains like furfuryl (VIh),
pentyl (VIi) and isopent-2-en-(1-yl) (VIj). The prepara-
tion of compound VIk was undertaken on the basis of
Acknowledgements
We thank D. P. Lane for baculoviruses, K. Novakova,
and E. Friesnerova for technical assistance, and L. H.
Jones for English corrections. The work was supported
by Czech Grant Agency (303/02/0875) and Grant
Agency of the ASCR (KJB4038301).
References and Notes
1. Hecht, S. M.; Bock, R. M.; Schmitz, R. Y.; Skoog, F.;
Leonard, N. J. Proc. Natl. Acad. Sci. U.S.A. 1978, 68, 2608.
2. (a) Vesely, J.; Havlıcek, L.; Strnad, M.; Blow, J. J.;
Donella-Deana, A.; Pinna, L.; Letham, D. S.; Kato, J.; Deti-
vaud, L.; LeClerc, S.; Meijer, L. Eur. J. Biochem. 1994, 224,
771. (b) Havlıcek, L.; Hanus, J.; Vesely, J.; LeClerc, S.; Meijer,
L.; Shaw, G.; Strnad, M. J. Med. Chem. 1997, 40, 408. (c)
Gray, N. S.; Wodicka, L.; Thunnissen, A. M.; Norman, T. C.;
Kwon, S.; Espinoza, F. H.; Morgan, D. O.; Barnes, G.;
LeClerc, S.; Meijer, L.; Kim, S. H.; Lockhart, D. J.; Schultz,
P. G. Science 1998, 281, 533.
Table 1. CDK1/cyclin B inhibitory and in vitro antiproliferative
activities of 6,9-disubstituted purines and corresponding 3,7-di-
substituted pyrazolo[4,3-d]pyrimidines. IC50 values (given in mmol/dm3)
are means of three experiments (olomoucine added for comparison)
Pyrazolo[4,3-d] pyrimidine
IC50
Purine
IC50
CDK1 K562CDK1 K562
3. Baraldi, P. G.; Cacciari, B.; Leoni, A.; Recanatini, M.;
Roberti, M.; Rossi, M. Farmaco 1991, 46, 1337.
49
VIa
VIb
VIc
VId
VIe
VIf
VIg
VIh
VIi
1.266
11
VIIa
VIIb
VIIc
VIId
VIIe
VIIf
VIIg
VIIh
3.6
2
3.8 >167
4.4 125
3.1 >167
4.0 >167
4.5
9.1 >167
6.9
17
5.9
113
3
29
46
54
88
112
26
4. Data for prepared compounds: (II): 5-Isopropyl-4-nitro-
pyra-zole-3-carboxylic acid was added to a 4.5 M solution of
HCl in absolute methanol. The reaction mixture was heated at
60 ꢀC for 7 h and evaporated to dryness. The title compound
was crystallised from ethyl acetate; yield 91%; mp=78–80 ꢀC.
MS ESI+: 214.1 (100, M+H+). 1H NMR (300 MHz,
CDCl3): 1.39 d (6H, J=7.1 Hz); 3.64 sept (1H, J=7.1 Hz),
3.98 s (3H). Anal. (C8H11N3O4) C, H, N. Methyl 4-amino-5-
isopropylpyrazol-3-carboxylate (III): To a solution of methyl 5-
isopropyl-4-nitropyrazol-3-carboxylate (4.34 g, 24 mmol) in
2.3
0.44
1.7
1.8
1.1
2.5 >167
1.261
4.5
0.9
104
VIIi
VIIj
144
>167
54
VIj
VIk
152
18.4
VIIk
Olomoucine
7
163

´
D. Moravcova et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2989–2992
2991
20 mL ethanol and 5 mL water was added 1 g RaNi (an activ-
ity W5). The mixture was stirred under hydrogen atmosphere
(760 torr) for 12h. The RaNi was filtered off and the filtrate
was evaporated in vacuo. The residue crystals were washed
with cold ethyl acetate; yield 95%; mp=122–123 ꢀC. MS
ESI+: 184.1 (100, M+H+). 1H NMR (400 MHz, CDCl3):
1.31 d (6H; J=6.9 Hz), 2.93 sept (1H; J=6.9 Hz), 3.9 s (3H).
Anal. (C8H13N3O2) C, H, N. 7-Hydroxy-3-isopropylpyrazolo[4,3-
(20:0.6:0.1); mp 220–221 ꢀC; yield 48%. MS ESI+: 274.3 (100,
M+H+). 1H NMR (300 MHz, DMSO-d6): 1.380 d (6H,
J=7.14 Hz, ðCH3Þ2CH); 3.340 sept (1H, J=7.14 Hz,
CHðCH3Þ2); 4.678 s (2H, CH2NH), 6.631–7.173 m (4H, Ar–
H), 8.221 s (1H, H–C5). Anal. (C15H17N5O) C, H, N. 7-(4-
Hydroxybenzyl)amino-3-isopropylpyrazolo[4,3-d]pyrimidine (VIf):
Method B. Column chromat.: CHCl3/methanol/AcOH
(20:1:0.1); mp 234–236 ꢀC; yield 49%. MS ESI+: 274.3 (100,
d]pyrimidine (IV):
A
mixture of amino ester III (1.5 g,
M+H+). 1H NMR (300 MHz, MeOD): 1.420
d (6H,
8.42 mmol), formamidine acetate (2.47 g, 24 mmol) and tri-
ethylamine (5ꢀ.25 mL) in 32 mL of 2-ethoxyethanol was heated
for 2h at 90 C under argon atmosphere. The excess of tri-
ethylamine was evaporated from 2-ethoxyethanol (Cellosolve)
solution in vacuo, crystallised product was filtered off and
washed with CHCl3. An analytical sample was obtained by re-
crystallisation from ethanol. Yield 96%; mp=302–304 ꢀC. MS
ESI+: 178.3 (100, M+H+). 1H NMR (300 MHz, CD3OD):
1.41 d (6H, J=7.15 Hz), 3.40 sept (1H, J=7.15 Hz), 7.82s
(1H). 13C NMR (400 MHz, DMSO-d6+AcOD): 21.912,
25.985, 141.85, 172.17. Anal. (C8H10N4O) C, H, N. 7-Chloro-
3-isopropylpyrazolo[4,3-d]pyrimidine (V): 7-hydroxy-3-iso-
propylpyrazolo[[4,3-d]]pyrimidine IV (200 mg, 1.122 mmol)
was dissolved in the mixture of 0.81 mL (11 mmol) SOCl2,
0.12mL (1.56 mmol) of dimethylformamide and 5 mL CHCl 3.
This mixture was heated under reflux for 3 h. The solution was
evaporated to dryness in vacuo and the residue was dissolved
in CHCl3. This solution was extracted twice with a small por-
tions of water and combined chloroform extract was dried by
Na2SO4. Column chromat. 1.5% MeOH in CHCl3; mp 84–
J=6.9 Hz, ðCH3Þ2CH); 3.449 sept (1H, J=6.9 Hz,
CHðCH3Þ2); 4.683 s (2H, CH2NH); 6.780 d (2H; J=8.3 Hz,
Ar–H), 7.247 d (2H; J=8.3 Hz, Ar–H); 8.261 bs (1H, H–C5).
Anal. (C15H17N5O) C, H, N. 7-(3-Hydroxy-4-methoxybenzyl)-
amino-3-isopropylpyrazolo[4,3-d]pyrimidine (VIg): Method B.
Column chromat.: CHCl3/methanol/aq NH4OH (94:6:0.2);
crystallised from a mixture CHCl3/Et2O; mp 197–199 ꢀC; yield
1
62%. MS ESI+: 314.3 (100, M+H+). H NMR (300 MHz,
CH3OD): 1.427 d (6H, J=7.2Hz, ðCH3Þ2CH); 3.446 hept
(1H, J=7.2Hz, CHðCH3Þ2); 3.833 s (3H, –O–CH3); 4.670 bs
(2H, CH2NH), 6.820–6.904 m (3H, Ar–H), 8.244 s (1H, H–
C5). Anal. (C16H19N5O2) C, H, N. 7-Furfurylamino-3-iso-
propylpyrazolo[4,3-d]pyrimidine (VIh): Method A. Column
chromat.: CHCl3/MeOH/aq NH4OH (98:2:0.2); mp 179–
182 ꢀC; yield 43%. MS ESI+: 258.3 (100, M+H+). 1H NMR
(500 MHz, MeOD): 1.422 d (6H, J=7.0 Hz, ðCH3Þ2CH); 3.455
sept (1H, J=7.0 Hz, CHðCH3Þ2); 4.802s (2H, CH2NH); 6.373
s (2H, furfuryl-(30+40)); 7.468 s (1H, furfuryl-(50)); 8.273 s
(1H, H–C5). Anal. (C13H15N5O) C, H, N. 7-Pentylamino-3-
isopropylpyrazolo[4,3-d]pyrimidine (VIi): Method A. Column
chromat.: 1% MeOH in CHCl3; mp 73–75 ꢀC; yield 52%. MS
ESI+: 248.2 (100, M+H+). 1H NMR (500 MHz, MeOD):
0.933 t (3H, J=7.0 Hz, –ðCH2Þ4ÀCH3); 1.374–1.388 m (4H,
86 ꢀC; yield 62%. MS ESI+: 197.2 (100, M+H+). H NMR
1
(300 MHz, CDCl3): 1.513 d (6H, J=7.2Hz, ðCH3Þ2ÀCH);
3.591 sept (1H, J=7.2Hz, CHðCH3Þ2); 7.925 bs (1H, ¼NH);
8.878 s (1H, H–C5). Anal. (C8H9N4Cl) C, H, N, Cl. 7-Benzy-
lamino-3-isopropylpyrazolo[4,3-d]pyrimidine (VIa): Method A.
Column chromat.: 1.5% MeOH in CHCl3; mp 153–154 ꢀC;
yield 82%. MS ESI+: 268.3 (100, M+H+). 1H NMR
(400 MHz, CDCl3): 1.403 d (6H, J=7.0 Hz, ðCH3Þ2CH); 3.407
sept (1H, J=7.0 Hz, CHðCH3Þ2); 4.791 s (2H, CH2NH), 6.530
bs (1H, C7–NHÀ), 7.214–7.284 m (5H, H–Ar), 8.405 s (1H, H–
C5). Anal. (C15H17N5) C, H, N. 7-(2-Brombenzyl)amino-3-iso-
propylpyrazolo[4,3-d]py-rimidine (VIb): Method B. Column
chromatography 1.5% MeOH in CHCl3; crystallised from
Et2O; mp 194–196 ꢀC; yield 42%. MS ESI+: 246.2 (100,
M+H+). 1H NMR (300 MHz, CH3OD): 1.435 d (6H,
J=6.9 Hz, ðCH3Þ2CH); 3.468 hept (1H, J=6.9 Hz,
CHðCH3Þ2); 4.893 s (2H, CH2NH); 7.203 t (1H, J=7.2Hz,
À
Á
– CH2 ÀCH3); 1.418 d (6H, J=6.9 Hz, ðCH3Þ2CH); 1.715
À
Á
2
pent (2H, J=7.0 Hz, –CH2– CH2 ÀCH3), 3.447 hept (1H,
J=6.9 Hz, CHðCH3Þ ); 3.583t (2H,2 J=7.0 Hz, –NH–CH2À);
2
8.207 s (1H, H–C5). Anal. (C13H21N5) C, H, N. 7-(Isopent-2-en-
1-ylamino)-3-isopropylpyrazolo[4,3-d] pyrimidine (VIj): Method
B. Column chromat.: CHCl3/methanol/aq NH4OH (98:2:1);
syrup; yield 48%. MS ESI+: 246.5 (100, M+H+). H NMR
1
(400 MHz, CDCl3): 1.449 d (6H, J=7.0 Hz, ðCH3Þ2CH); 1.647
d (6H, J=1.3 Hz, ¼C–ðCH3Þ2), 3.467 sept (1H, J=7.0 Hz,
CHðCH3Þ2); 4.178 d (2H, J=6.8 Hz, –CH2–CH¼), 5.250 m
(1H, –CH2–CH¼), 6.252 s (1H, –NH–C7), 8.430 s (1H, H–C5).
COSY [1.45 d (6H, J=6.96 Hz); 3.47 sept (1H, J=6.96 Hz)],
COSY [1.65 d (6H, J=1.28 Hz); 4.18 d (2H, J=1.28 Hz); 5.25
m (1H, J=1.28 Hz)], COSY [4.18 d (2H); 6.25 s (1H)]. Anal.
0
0
Ar–H4 ); 7.322 t (1H, J=7.2Hz, Ar–H5 ); 7.451 m (1H, Ar–
(C13H19N5)
C,
H,
N.
7-(3-Chloroanilino)-3-iso-
0
0
H6 ); 7.618 d (1H, J=7.2Hz, Ar–H3 ); 8.238 bs (1H, H–
C5). Anal. (C15H16BrN5) C, H, N, Br. 7-(4-Methoxybenzyl)-
amino-3-isopropylpyrazolo[4,3-d]pyrimidine (VIc): Method A.
Column chromat.: 2% MeOH in CHCl3; crystallised from
Et2O; mp 143–144 ꢀC; yield 42%. MS ESI+: 298.3 (100,
propylpyrazolo[4,3-d]pyrimi-dine (VIk): Method A. The pro-
duct was precipitated when the reaction mixture was cooled at
room temperature. The colourless crystals were washed with
Et2O; the analytical sample was obtained by re-crystallisation
from mixture EtOH/Et2O. Yield 58%; mp=213–216 ꢀC. MS
ESI+: 288.5 (100, M+H+). 1H NMR (400 MHz, DMSO-d6):
M+H+). 1H NMR (300 MHz, CDCl3): 1.358
J=6.9 Hz, ðCH3Þ2CH); 3.457 sept (1H, J=6.9 Hz,
J=3.6 Hz,
d (6H,
1.400
d
(6H, J=6.9 Hz, ðCH3Þ2CH); 3.482sept (1H,
CHðCH3Þ2); 3.693 s (3H, OCH3), 4.862d (H2 ,
J=6.90Hz, CHðCH3Þ2); 7.299 dd (1H, J=7.8 Hz, J=1.7 Hz,
0
CH2NH); 6.721 d (2H, J=8.8 Hz, Ar–H); 7.295 d (2H,
J=8.8 Hz, Ar–H); 8.340 s (1H, H–C5). Anal. (C16H19N5O) C,
H, N. 7-(2-Hydroxybenzyl)amino-3-isopropylpyrazolo[4,3-
d]pyrimidine (VId): Method B. Column chromat.: CHCl3/
methanol/AcOH (20:0.4:0.1); mp 214–217 ꢀC; yield 40%. MS
Ar–H6 ), 7.488 dd (1H, J=8.0 Hz, J=8.0 Hz, Ar–H5 ), 7.862
0
dd (1H, J=7.8 Hz, J=1.7 Hz, Ar–H3 ), 8.202 bs (1H, H–C5);
20
.
.
8.750 s (1H, Ar–H ). Anal. (C14H14N5Cl HCl H2O) C, H, N,
Cl. 6-(2-Bromobenzyl)amino-9-isopropylpurine (VIIb): Column
chromat. in CHCl3; crystallised from Et2O; mp 112–114 ꢀC;
yield 79%. MS ESI+: 346.2(100, M+H +). 1H NMR
(300 MHz, CDCl3): 1.629 d (6H, J=6.9 Hz, ðCH3Þ2CH); 3.870
hept (1H, J=6.6 Hz, CHðCH3Þ2); 5.46 bs (2H, CH2NH);
7.124–7.580 m (4H, Ar–H); 7.938 bs (1H, H–C8); 8.320 bs (1H,
H–C2). Anal. (C15H16N5Br) C, H, N, Br. 6-(2-Hydroxy-
benzyl)amino-9-isopropylpurine (VIId): Column chromat. 2%
MeOH in CHCl3; crystallised from a mixture EtOH/Et2O; mp
ESI+: 274.3(100, M+H+). H NMR (400 MHz, DMSO-d6):
1
1.358
d
(6H, J=7.0 Hz, ðCH3Þ2CH); 3.297 sept (1H,
J=7.0 Hz, CHðCH3Þ2); 4.644 s (2H, CH2NH), 6.775 ddd (1H,
0
J=8.1 Hz, J=7.2Hz, J=1.3 Hz, ArÀH5 ), 6.861 d (1H,
0
J=8.0 Hz, Ar–H30 ), 7.119 ddd (1H, J=7.7 Hz, J=1.7 Hz,
J=1.4 Hz, Ar–H4 ), 7.259 dd (1H, J=7.5 Hz, J=1.3 Hz, Ar–
0
H6 ), 8.232 s (1H, H–C5). Anal. (C15H17N5O) C, H, N. 7-(3-
Hydroxybenzyl)amino - 3 - isopropylpyrazolo[4,3 - d]pyrimidine
(VIe): Method B. Column chromat.: CHCl3/methanol/AcOH
162–163 ꢀC; yield 85%. MS ESI+: 284.2 (100, M+H+). H
1
NMR (300 MHz, MeOD): 1.603
d
(6H, J=6.7 Hz,

´
D. Moravcova et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2989–2992
2992
0
8
ðCH3Þ2CH); 4.715 bs (2H, CH2NH); 4.814 sept (1H,
7.362s (1H, furfuryl-(5 )); 7.882bs (1H, H–C ); 8.192bs (1H,
J=6.7 Hz, CHðCH3Þ2); 6.758–6.828 m (2H, Ar–H); 7.109 dd
H–C2). Anal. (C13H15N5O) C, H, N. 6-Pentylamino-9-iso-
propylpurine (VIIi): Column chromat. 1% MeOH in CHCl3;
crystallized from CHCl3; mp 48–50 ꢀC; yield 78%. MS ESI+:
248.2 (100, M+H+). 1H NMR (300 MHz, CDCl3): 0.926 (3H,
t, J=6.9 Hz), 1.408 (4H, m, J=6.9 Hz) 1.635 (6H, d,
J=6.9 Hz), 1.709 (2H, m, J=6.9 Hz), 4.609 (2H, t, J=7.1 Hz)
4.863 (1H, sept, J=6.9 Hz), 7.885 (1H, s), 8.192(1H, s). Anal.
(C13H21N5) C, H, N. 6-(Isopent-2-en-1-ylamino)-9-iso-
propylpurine (VIIj): Column chromat. 0.6% MeOH in CHCl3;
crystallised from Et2O; mp 99–100 ꢀC; yield 89%. MS ESI+:
246.2 (100, M+H+). 1H NMR (300 MHz, CDCl3): 1.631 d
(6H, J=6.9 Hz, ðCH3Þ2CH); 1.764 s (6H, CH¼C–ðCH3Þ );
0
(1H, J=7.6 Hz, J=7.6 Hz, Ar–H4 ); 7.256 (1H, J=7.4 Hz, Ar–
0
H6 ), 8.159 bs (1H, H–C8), 8.270 s (1H, H–C2). Anal.
(C15H17N5O) C, H, N. 6-(3-Hydroxybenzyl)amino-9-iso-
propylpurine (VIIe): Column chromat. 3% MeOH in CHCl3;
crystallised from a mixture EtOH/Et2O; mp 170–171 ꢀC; yield
1
61%. MS ESI+: 284.2 (100, M+H+). H NMR (300 MHz,
MeOD): 1.613 d (6H, J=6.6 Hz, ðCH3Þ2CH), 4.756 bs (2H,
CH2NH), 4.824 sept (1H, J=6.60Hz, CHðCH3Þ2), 6.737 dd
(1H, J=8.0 Hz, J=2.2 Hz, Ar–H3 ), 6.804–6.843 m (2H, Ar–
0
H), 7.122 dd (1H, J=8.0 Hz, J=8.0 Hz, Ar–H5 ), 8.164 s (1H,
H–C8), 8.248s (1H, H–C2). Anal. (C15H17N5O) C, H, N. 6-(4-
Hydroxybenzyl)amino-9-isopropylpurine (VIIf): Column chro-
mat. 3.5% MeOH in CHCl3; crystallised from a mixture
EtOH/Et2O; mp 162–163 ꢀC; yield 85%. MS ESI+: 284.2
(100, M+H+). 1H NMR (300 MHz, MeOD): 1.610 d (6H,
J=6.9 Hz, ðCH3Þ2CH); 4.70 bs (2H, CH2NH); 4.819 sept (1H,
J=6.9 Hz, CHðCH3Þ2); 6.737 d (2H, d, J=8.4 Hz, Ar–H);
7.208 d (2H, J=8.4 Hz, Ar–H); 8.154 s (1H, H–C8), 8.252 s
(1H, H–C2). Anal. (C15H17N5O) C, H, N. 6-(3-Hydroxy-4-
methoxybenzyl)amino-9-isopropylpu-rine (VIIg): Column chro-
mat.: 3.5% MeOH in CHCl3; crystallised from a mixture
CHCl3/Et2O; mp 163–164 ꢀC; yield 61%. MS ESI+: 314.3
4.245 bs (1H, –CH¼); 4.860 sept (1H, J=6.9 Hz, CHðCH3Þ2),
2
5.385 t (2H, J=7.0 Hz, CH2CH¼); 7.84 bs (1H, H–C8), 8.36
bs (1H, H–C2). Anal. (C13H19N5) C, H, N. 6-(3-Chlor-
oanilino)-9-isopropylpurine (VIIk): Column chromat. CHCl3;
crystallised from Et2O; mp 72–74 ꢀC; yield 33%. MS ESI+:
288.1 (100, M+H+). 1H NMR (300MHz, MeOD): 1.638 d (6H,
J=6.6 Hz, ðCH3Þ2CH); 4.921 sept (1H, J=6.6 Hz, CHðCH3Þ2);
0
7.111 dd (1H, J=8.0 Hz, J=1.9 Hz, Ar–H6 ); 7.300 dd (1H,
0
J=8.0 Hz, J=8.0 Hz, Ar–H5 ); 7.597 dd (1H, J=8.1 Hz,
0
0
J=2.0 Hz, Ar–H4 ); 7.975 bs (1H, Ar–H2 ); 8.425 bs (1H, H–
C8), 8.514 s (1H, H–C2). Anal. (C14H14N5Cl) C, H, N,Cl.
5. Kelley, J. L.; Bullock, R. M.; Krochmal, M. P.; McLean,
E. W.; Linn, J. A.; Durcan, M. J.; Cooper, B. R. J. Med.
Chem. 1997, 40, 3207.
1
(100, M+H+). H NMR (300 MHz, CH3OD): 1.601 d (6H,
J=6.9 Hz, ðCH3Þ2ÀCH); 3.821 s (3H, –O–CH3); 4.69 bs (2H,
CH2NH), 4.821 hept (1H, J=6.9 Hz, CHðCH3Þ2); 6.800–6.882
m (3H, Ar–H), 8.166 s (1H, H–C8); 8.261 s (1H, H–C2). Anal.
(C16H19N5O2) C, H, N. 6-Furfurylamino-9-isopropylpurine
(VIIh): Column chromat. 2.5% MeOH in CHCl3; crystallised
from Et2O; mp 128–130 ꢀC; yield 68%. MS ESI+: 258.2 (100,
6. Otyepka, M.; Krystof, V.; Havlıcek, L.; Siglerova, V.;
Strnad, M.; Koca, J. J. Med. Chem. 2000, 43, 2506.
7. Franek, F.; Havlıcek, L.; Siglerova, V.; Strnad, M.; Ecks-
chlager, T.; Weigl, E. Collect. Czech. Chem. Commun. 2002,
67, 257.
M+H+). 1H NMR (300 MHz, CDCl3): 1.631
d (6H,
J=6.9 Hz, ðCH3Þ2CH); 4.866 sept (1H, J=6.9 Hz, CHðCH3Þ );
8. Krystof, V.; Lenobel, R.; Havlıcek, L.; Kuzma, M.; Strnad,
M. Bioorg. Med. Chem. Lett. 2002, 12, 3283.
4.934 bs (2H, CH2NH); 6.308–6.339 m (2H, furfuryl-(30+402));