Synthesis and biological activity of olomoucine II.

Article abstract of DOI:10.1016/S0960-894X(02)00693-5

Based on our previous experiences with synthesis of purines, novel 2,6,9-trisubstituted purine derivatives were prepared and assayed for the ability to inhibit CDK1/cyclin B kinase. One of newly synthesized compounds designated as olomoucine II, 6-[(2-hydroxybenzyl)amino]-2-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpurine, displays 10 times higher inhibitory activity than roscovitine, potent and specific CDK1 inhibitor. Olomoucine II in vitro cytotoxic activity exceeds purvalanol A, the most potent CDK inhibitor, as it kills the CEM cells with IC(50) value of 3.0 microM.

Full text of DOI:10.1016/S0960-894X(02)00693-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3283–3286
Synthesis and Biological Activity of Olomoucine II
Vladimır Krystof,a Rene Lenobel,a Libor Havlıcek,b Marek Kuzmac and
Miroslav Strnada,*
a
ˇ
Laboratory of Growth Regulators, Palacky University and Institute of Experimental Botany, Slechtitelu 11, 783 71 Olomouc,
´
˚
Czech Republic
Isotope Laboratory, Institute of Experimental Botany, Czech Academy of Sciences, Vıdenˇska 1083, 14220 Prague 4, Czech Republic
b
´
´
Institute of Microbiology, Czech Academy of Sciences, Vıdenˇska 1083, 14220 Prague 4, Czech Republic
c
´
´
Received 24 July 2002; accepted 19 August 2002
Abstract—Based on our previous experiences with synthesis of purines, novel 2,6,9-trisubstituted purine derivatives were prepared
and assayed for the ability to inhibit CDK1/cyclin B kinase. One of newly synthesized compounds designated as olomoucine II, 6-
[(2-hydroxybenzyl)amino]-2-{[1-(hydroxymethyl)propyl]amino}-9-isopropylpurine, displays 10 times higher inhibitory activity than
roscovitine, potent and specific CDK1 inhibitor. Olomoucine II in vitro cytotoxic activity exceeds purvalanol A, the most potent
CDK inhibitor, as it kills the CEM cells with IC50 value of 3.0 mM.
# 2002 Elsevier Science Ltd. All rights reserved.
A number of reports during the past decade witness the
crucial role of cyclin dependent kinases (CDK) in cell
division and proliferation. Their discovery as enzymes
driving progress through cell division was followed clo-
sely with revelation of frequent alterations of cell cycle
regulatory genes in many primary tumours and cancer
cells. Acting in the centre of all particular processes
during cell division, CDK’s became promising targets
for specific inhibitors able to block cell division at
defined points.1 The potential use of these substances in
the therapy of cancer and other proliferative diseases
initiated an intensive search for chemical CDK inhibitors.
Crystal structures of CDK2–purine complexes revealed
inhibitors fixed in the ATP binding pocket, occupying
approximately the same region, but demonstrating dif-
ferent orientation with respect to the protein.5 Detailed
analysis of molecular and cellular effects of purine CDK
inhibitors, supported by co-crystal analysis, has moti-
vated several scientific teams to continue with the
synthesis and biological testing of 2,6,9-trisubstituted
purines.6 The research contributed to the identification
of purvalanol A, the compound with about 100-fold
increase of inhibitory potency over roscovitine.7
Comparing the previously published purine inhibitors
of CDK1, the most potent compound purvalanol B
bears a 3-chlorophenylamino substituent at position 6
in contrast to the parental benzylamino moiety of cyto-
kinins, from which purine inhibitors have been origin-
ally developed. Our initial experiments suggested that
the hydroxy substituent on the benzyl ring enhances
slightly the ability of purines to block CDK1 activity.2
That idea has been recently confirmed by Legraverend
and co-workers, who have found different substituents
at benzyl ring (Cl, OCH3) to be favourable.8 We show
here that hydroxy substitutions of the benzyl ring
modifies very positively the affinity of purine derivatives
to CDK1. Hence prepared derivatives of bohemine and
other trisubstituted purines bearing various hydro-
xylated benzylamino groups at position C-6 are about
10 times more active than unmodified parental
Systematic screening of purine derivatives of cytokinin
origin leading to the discovery of 2,6,9-trisubstituted
purines revealed a large number of highly active CDK
inhibitors.2 One of the first CDK specific inhibitors
olomoucine, 2-(2-hydroxyethylamino)-6-benzylamino-
9-methylpurine, was found to block selectively CDK1,
CDK2and CDK5 kinases at micromolar concentra-
tions.2 Its structure modifications led to the development
of roscovitine, 2-{[(1-hydroxymethyl) propyl]amino}-6-
benzylamino-9-isopropylpurine,3 a novel substance with
enhanced inhibitory activity towards CDK1, increased
selectivity and antimitotic activity.4
*Corresponding author. Tel.: +420-68-563-4850; fax: +420-68-522-
1357; e-mail: strnad@aix.upol.cz
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00693-5

3284
V. Krysˇtof et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3283–3286
Table 1. CDK1/cyclin B inhibitory activity of 2,6,9-trisubstituted purines
Compd
Substitution
IC50 (mM)a
C-2C-6
N-9
Olomoucine
Bohemine
B23-Hydroxypr-oHpyldarmoxinyobenzylamino
2-Hydroxyethylamino
3-Hydroxypropylamino
Benzylamino
Benzylamino
Methyl
Isopropyl
7b
1.1b
2 Isopropyl
0.1
B3
B4
3-Hydroxypropylamino
3-Hydroxypropylamino
3-Hydroxybenzylamino
4-Hydroxybenzylamino
Benzylamino
2-Hydroxybenzylamino
3-Hydroxybenzylamino
4-Hydroxybenzylamino
3-Chloroanilino
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
Isopropyl
1.0
2.6
0.45b
0.02
0.2
Roscovitine
Olomoucine II
R3
R4
Purvalanol A
[1-(Hydroxymethyl)propyl]amino
[1-(Hydroxymethyl)propyl]amino
[1-(Hydroxymethyl)propyl]amino
[1-(Hydroxymethyl)propyl]amino
[1-(Hydroxymethyl)-2-methylpropyl]amino
0.3
0.05b
P-2m[1e-t(hHylypdrro-oHpxyylmd]aremotxhinyyobl)e-n2zylamino2
Isopropyl
0.08
P3
P4
[1-(Hydroxymethyl)-2-methylpropyl]amino
[1-(Hydroxymethyl)-2-methylpropyl]amino
3-Hydroxybenzylamino
4-Hydroxybenzylamino
Isopropyl
Isopropyl
0.12
0.15
aValues are means of three experiments.
bOlomoucine, bohemine, roscovitine and purvalanol A IC50 values measured in our laboratory.
compounds. Together with synthesis of the described
purine derivatives, their ability to block recombinant
CDK1/cyclin B kinase activity and their in vitro
cytotoxic properties are also presented.
Compounds P2and P3 were prepared as described
previously.9,11
Results and Discussion
All newly synthesized trisubstituted purines were tested
in a CDK1/cyclin B kinase inhibition assay as described
previously,9 with enzyme purified on NiNTA column
(according to Qiagen manual). Resulting data presented
in Table 1, including IC50 values of olomoucine, bohe-
mine, roscovitine and purvalanol A for comparison,
demonstrate that hydroxybenzylamino moiety at posi-
tion C-6 increase the activity of almost all derivatives.
The 6-benzylamino group is thought to be responsible
for the specificity of purine CDK inhibitors. Addition of
a hydroxyl group at the C-6 benzylamino substituent of
bohemine and other related analogues improved CDK1
inhibition. Now it becomes clear that the activity grows
with the addition of one hydroxyl group. The most sig-
nificant increase of CDK1 inhibition is obvious when
decorating benzyl positions 2and 3, respectively. In
contrast to that, 6-(4-hydroxybenzylamino) derivatives
display only slight change in activity.
Chemistry
The straightforward two-step synthesis of 2,6,9-trisub-
stituted purines started from 2,6-dichloro-9-iso-
propylpurine,9 which has to be used as a parental
compound to avoid alkylation of phenolic OH during
introduction of N-isopropylgroup via classic alkylation.
The starting compounds were reacted with appropriate
hydroxybenzylamines in butan-1-ol in the presence of
triethylamine (112 ꢀC, 2h). Resulting -c2hloro-6-
(hydroxybenzyl)amino-9-isopropylpurines were further
coupled with 3-aminopropan-1-ol (160 ꢀC, 2h), ( R/S)-2-
aminobutan-1-ol (160 ꢀC, 3 h) or (R/S)-2-amino-3-
methylbutan-1-ol (160 ꢀC, 20 h), respectively. The pro-
ducts were purified by column chromatography. Data
characterizing prepared purine derivatives are given in
ref 10.
When the compounds were assayed for in vitro anti-
tumour activity against various cancer cell lines,12 we
obtained data showing that drug activity rises remark-
ably with enhancement of its CDK1 inhibitory potency
(Table 2). These results suggest that the CDK1 inhibi-
tion may be at least partly responsible for the
antiproliferative activity of the compounds.13 2-Hydro-
xybenzylamino compounds were again the most active
from the tested series. Cytotoxicity values exceeded their
respective 6-benzylaminopurines more than 2-fold.
Table 2. In vitro cytotoxicity of selected trisubstituted purines
Compd
IC50 (mM)a
K562CEM HOS
MCF7
G361
160
58
17.2
26
Olomoucine
Bohemine
B2
B3
B4
Roscovitine
Olomoucine II
R3
R4
Purvalanol A
132
28
>167
113
24
62149
27
45
13.3
26
12.4
14.7
12.7
11.1
5.3
13.7
7.8
10.7
10.7
17.6
18.2
18.23324
3.0
9.9
11.0
7.4
56
57
40
11.1
26
24
9.0
26
23
Olomoucine, the first cytokinin CDK inhibitor, has only
weak cytotoxic activity on cancer cell lines in compar-
ison to olomoucine II, being effective in low micromolar
concentrations. On the other hand, olomoucine II is
more potent in vitro against tumour cells than purvala-
nol A, the most active CDK1 inhibitor. The results
provide valuable information for the design of CDK1
inhibiting compounds with enhanced cytotoxic effect.
6.3
11.0
13.0
22
6.3
19.4
11.1
24
P25.7
P3
P4
10.9
4.4
8.3
7.8
6.7
5.3
14.8
9.3
16.1
14.8
12.0
12.0
12.0
11.1
aValues are means of three experiments.

V. Krysˇtof et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3283–3286
3285
Acknowledgements
110–122 ꢀC; yield 80%. MS: 319 (25), 317 (62, C15H16N5OCl,
ꢁ0.6) 276 (12), 274 (32), 213 (12), 211 (40), 171 (31), 169 (100),
134 (28), 122 (60, C7H8NO, ꢁ1.8), 107 (60, C7H7O, ꢁ1.5), 77
(31). 1H NMR (300 MHz, CDCl3): 1.638 (6H, d, J=6.6 Hz,
(CH3)2CH), 4.682(2H, bd, J=4.8 Hz, CH2NH), 4.913 (1H,
sept, J=6.7 Hz, CH(CH3)2), 6.685 (2H, d, J=8.2Hz, H–Ar),
7.129 (2H, d, J=8.5 Hz, H–Ar), 8.173 (1H, bs, OH), 8.686
(1H, s, HC8). Anal. (C15H16ClN5O) C, H, N. 6-(2-hydro-
xybenzylamino)-2-[(3-hydroxypropyl)amino]-9-isopropylpurine
(B2): Column chromatography stepwise 0.5, 1, 2% MeOH in
CHCl3; amorphous glass-like product; yield 70%. MS: 356
(57, M+), 325 (6), 312 (8), 298 (7), 250 (69), 219 (65), 206 (71),
205 (100), 177 (37), 163 (72), 150 (37), 134 (50), 108 (38), 106
(28), 78 (53). 1H NMR (300 MHz, CDCl3): 1.520 (6H, d,
J=6.6 Hz, (CH3)2CH), 1.782(H2 , m, CH 2CH2CH2), 3.660
(4H, m, CH2CH2CH2), 4.54–4.60 (2H, bs, CH2Ph), 4.575 (1H,
sept, J=6.6, CH(CH3)2), 5.11 (1H, bt, exch H), 6.53 (1H, exch
H), 6.82–6.93 (2H, m, H-Ar), 7.17–7.27 (2H, m, H–Ar), 7.520
(1H, s, HC8). Anal. (C18H24N6O2) C, H, N. 6-(2-hydro-
xybenzylamino)-2-{[1(R/S)-(hydroxymethyl)propyl]amino}-9-
isopropylpurine (R2, olomoucine II): Column chromatography
stepwise 1, 2, 3, 4% MeOH in CHCl3; amorphous glass-like
product; mp 110–114 ꢀC; yield 76%. MS ESI+: 371.3 (100,
M+H+), 372(20). 1H NMR (300 MHz, CDCl3): 1.055 (3H, t,
J=7.1, CH3CH2), 1.532and 1.538 (6H, 2 ꢂ d, J=6.6 Hz,
CH(CH3)2), 1.67 (2H, m, CH2CH3), 3.70 (1H, m, CHHOH),
3.84 (1H, dd, J=2.9, J=11 Hz, CHHOH), 3.985 (1H, m,
NHC*H), 4.597 (1H, sept, J=6.6 Hz, (CH3)2CH), 4.60 (2H,
bs, CH2Ph), 5.57 (1H, bs, NH), 6.78–6.94 (2H, m, H-Ar),
7.12–7.20 (2H, m, H-Ar), 7.567 (1H, s, HC8). Anal.
(C19H26N6O2) C, H, N. 6-(3-hydroxybenzylamino)-2-[(3-
hydroxypropyl)amino]-9-isopropylpurine (B3): Column chro-
matography stepwise 1, 2, 3, 4% MeOH in CHCl3; mp 134–
136 ꢀC; yield 77%. MS: 356 (100, C18H24N6O2, ꢁ0.1, M+),
325 (43), 312 (42), 311 (43), 298 (24), 191 (17), 122 (24), 107
(62). 1H NMR(400 MHz, CDCl3): 1.562(6H, d, J=6.8 Hz,
CH(CH3)2), 1.747 (2H, m, CH2CH2CH2), 3.65 (4H, m,
CH2CH2CH2, 4.59 (2H, bd, CH2Ph), 4.66 (1H, sept,
J=6.8 Hz, (CH3)2CH), 6.4 (1H, bs, exch H), 6.73–6.82(3H, m,
H–Ar), 7.06–7.12(1H, m, H–Ar), 7.265 (1H, s, HC 8), 7.605
(1H, s, phenolic H). The proton 2D-COSY spectrum was used
for the assignment of signals. Anal. (C18H24N6O2) C, H, N. 6-
(3-hydroxybenzylamino)-2-{[1(R/S)-(hydroxymethyl)propyl]amino}-
9-isopropylpurine (R3): Column chromatography stepwise 1, 2,
3, 4% MeOH in CHCl3; crystallized from EtOAc; mp 150–
152 ꢀC; yield 81%. MS: 370 (25, C19H26N6O2, 0.7, M+), 352
(13), 339 (100, C18H23N6O, +0.4), 323 (20), 309 (7), 298 (9),
The authors thank D. P. Lane (University of Dundee)
for baculoviruses and K. Novakova, O. Hustakova and
E. Friesnerova for technical assistance. This work was
supported by grants from the Grant Agency of the
Czech Republic (No. 301/02/0475) and from the Minis-
try of Education, Youth and Sports (MSMT
153100008).
References and Notes
1. (a) Garrett, M. D.; Fattaey, A. Curr. Opin. Genet. Dev.
1999, 9, 104. (b) Gray, N.; Detivaud, L.; Doerig, C.; Meijer, L.
Curr. Med. Chem. 1999, 9, 859.
2. Vesely, J.; Havlıcek, L.; Strnad, M.; Blow, J. J.; Donella-
Deana, A.; Pinna, L.; Letham, D. S.; Kato, J.; Detivaud, L.;
LeClerc, S.; Meijer, L. Eur. J. Biochem. 1994, 224, 771.
3. Havlıcek, L.; Hanus, J.; Vesely, J.; Leclerc, S.; Meijer, L.;
Shaw, G.; Strnad, M. J. Med. Chem. 1997, 40, 408.
4. Meijer, L.; Borgne, A.; Mulner, O.; Chong, J. P.; Blow, J. J.;
Inagaki, N.; Inagaki, M.; Delcros, J. G.; Moulinoux, J. P. Eur.
J. Biochem. 1997, 243, 527.
5. De Azevedo, W. F.; LeClerc, S.; Meijer, L.; Havlıcek, L.;
Strnad, M.; Kim, S. H. Eur. J. Biochem. 1997, 243, 518.
6. (a) Schow, S. R.; Mackman, R. L.; Blum, C. L.; Brooks, E.;
Horsma, A. G.; Joly, A.; Kerwar, S. S.; Lee, G.; Shiffman, D.;
Nelson, N. G.; Wang, X.; Wick, M. M.; Zhang, X.; Lum, R. T.
Bioorg. Med. Chem. Lett. 1997, 7, 2697. (b) Legraverend, M.;
Ludwig, O.; Bisagni, E.; LeClerc, S.; Meijer, L.; Giocanti, N.;
Sadri, R.; Favaudon, V. Bioorg. Med. Chem. 1999, 7, 1281. (c)
Imbach, P.; Capraro, H. G.; Furet, P.; Mett, H.; Meyer, T.;
Zimmermann, J. Bioorg. Med. Chem. Lett. 1999, 9, 91. (d)
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. Chem. Biol. 1999, 6, 361.
7. 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.
8. Legraverend, M.; Tunnah, P.; Noble, M.; Ducrot, P.; Lud-
wig, O.; Grierson, D. S.; Leost, M.; Meijer, L.; Endicott, J. J.
Med. Chem. 2000, 43, 1282.
9. Otyepka, M.; Krystof, V.; Havlıcek, L.; Siglerova, V.;
Strnad, M.; Koca, J. J. Med. Chem. 2000, 43, 2506.
1
217 (7), 134 (8), 122 (6), 107 (33), 43 (13), 41 (11). H NMR
10. Data for prepared compounds: 2-chloro-6-(2-hydro-
xybenzylamino)-9-isopropylpurine: Column chromatography
stepwise 1, 2, 3, 4% MeOH in CHCl3; crystallized from Et2O;
mp 170–171 ꢀC; yield 83%. MS ESI+: 318.3 (100, M+H+),
319 (18), 320 (25). 1H NMR (300 MHz, CDCl3): 1.636 (6H, d,
J=7.0 Hz, (CH3)2CH), 4.682(2H, bd, J=5.3 Hz, CH2NH),
4.916 (1H, sept, J=6.8, CH(CH3)2), 6.866 (1H, ddd, J=6.7,
J=6.7, J=1.1 Hz, H-50), 6.966 (1H, dd, J=8.2, J=1.1 Hz, H-
30), 7.198 (1H, ddd, J=8.0, J=8.1, J=1.6 Hz, H-40), 7.280
(1H, dd, J=7.5, J=1.7 Hz, H-60) 8.44 (1H, bs, OH), 8.592
(1H, s, HC8). Anal. (C15H16ClN5O) C, H, N. 2-chloro-6-(3-
hydroxybenzylamino)-9-isopropylpurine: The title compound
crystallized from 1-butanol after reaction and cooling; recrys-
tallization from CHCl3 gave mp 217–218 ꢀC; yield 92%. MS
ESI+: 318.3 (100, M+H+), 319 (18), 320 (25). 1H NMR
(300 MHz, CDCl3): 1.61 (6H, d, J=6.7 Hz, (CH3)2CH), 4.72
(2H, bs, CH2NH), 4.83 (1H, sept, J=6.7 Hz, CH(CH3)2),
6.70–7.20 (4H, m, H–Ar), 7.92 (1H, s, HC8). Anal.
(C15H16ClN5O) C, H, N. 2-chloro-6-(4-hydroxybenzylamino)-
9-isopropylpurine: Column chromatography stepwise 1, 2, 3,
4% MeOH in CHCl3; crystallized from benzene; mp (dec)
(400 MHz, CDCl3): 1.032(3H, t, J=7.5 Hz, CH3CH2), 1.50–
1.70 (3H, m, CH2OH+CH2CH3), 1.555 (6H, d, J=6.8 Hz,
CH(CH3)2), 3.637 (1H, dd, J=7.8, J=10.7 Hz, CHHOH),
3.830 (1H, dd, J=2.8, 10.7 Hz, CHHOH), 3.95 (1H, m,
NHC*H), 4.59 (2H, bd, CH2Ph), 4.64 (1H, sept, J=6.8 Hz,
(CH3)2CH), 4.937 (1H, d, J=6.4 Hz, exch H), 6.4 (1H, bs,
NH), 6.746 (1H, ddd, J=1.1, 2.5, 8.1 Hz, H–Ar), 6.848 (1H,
ddd, J=1.0, 1.7, 7.5 Hz, H–Ar), 6.871 (1H, m, H–Ar), 7.131
(1H, dd, J=7.5, 8.1 Hz, H–Ar), 7.579 (1H, s, HC8). Anal.
(C19H26N6O2) C, H, N. 6-(4-hydroxybenzylamino)-2-[(3-
hydroxypropyl)amino]-9-isopropylpurine (B4): Column chro-
matography stepwise 1, 2, 3, 4% MeOH in CHCl3; crystallized
from EtOAc–Et2O; mp 169–170 ꢀC; yield 79%. MS: 356 (43,
C18H24N6O2, +0.3, M+), 250 (46, C11H18N6O, +1.5), 219
(62, C10H15N6O, +0.4), 164 (45), 163 (100), 150 (55), 134 (73),
122 (91), 108 (48), 107 (65), 78 (48), 77 (47), 51 (44), 43 (72).
1H NMR (200 MHz, CDCl3): 1.509 (6H, d, J=6.8 Hz,
CH(CH3)2), 1.70 (2H, m, CH2CH2CH2), 3.6 (4H, m,
CH2CH2CH2, 4.6 (3H, m, CH2Ph +CH(CH3)2), 6.71 (2H, d,
J=7.5 Hz, H–Ar), 7.10 (2H, d, J=7.5 Hz, H–Ar), 7.512(1H,
s, HC8). Anal. (C18H24N6O2) C, H, N. 6-(4-hydro-

3286
V. Krysˇtof et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3283–3286
xybenzylamino)-2-{[1(R/S)-(hydroxymethyl)propyl]amino}-9-
isopropylpurine (R4): Column chromatography stepwise 1, 2,
3% MeOH in CHCl3; crystallized from CHCl3–Et2O; mp 135–
(300 MHz, CDCl3): 0.980 (3H, d, J=6.9 Hz, (CH3)2CHC*),
1.003 (3H, d, J=6.9 Hz, (CH3)2CHC*), 1,547 (3H, d,
J=6.9 Hz, (CH3)2CHN9), 1.558 (3H, d, J=6.9 Hz,
(CH3)2CHN9), 2.023 (1H, m, (CH3)2CHC*), 3.660–3.700 (2H,
m, CH2OH), 3.90–3.98 (1H, m, CH2C*H), 4.54–4.68 (3H, m,
CH2Ph+N9CH(CH3)2), 6.728 (2H, d, J=8.5 Hz, H–Ar), 7.193
(2H, d, J=8.5 Hz, H–Ar), 7.760 (1H, s, HC8). The proton 2D-
COSY experiments were used for the assignment of signals.
Anal. (C20H28N6O2) C, H, N.
11. Havlıcek, L.; Hajduch, M.; Strnad, M. US Patent
6221873, 1998.
12. For experimental details, see: Travnıcek, Z.; Malon, M.;
Sindelar, Z.; Dolezal, K.; Rolcık, J.; Krystof, V.; Strnad, M.;
Marek, J. J. Inorg. Biochem. 2001, 84, 2 3.
140 ꢀC; yield 70%. MS: 370 (M+, 11), 352(M
ꢁH2O, 11),
+
340 (13), 339.1942(C 18H23N6O, ꢁ0.9, 40), 246.1605
(C12H18N6, ꢁ1.2, 100), 233 (72), 217 (59), 204 (40), 175 (30).
1H NMR (300 MHz, CDCl3): 1.02(3H, t,
J=7.4 Hz,
CH3CH2), 1.54 (6H, d, J=6.8 Hz, CH(CH3)2), 1.60 (2H, m,
CH2CH3), 3.66 (1H, dd, J=10.9, 7.7 Hz, CHHOH), 3.83 (1H,
dd, J=10.9, 2.7 Hz, CHHOH), 3.99 (1H, m, C8HNH), 4.63
(3H, bs+sept, J=6.8 Hz, CH2NH+CH(CH3)2), 6.63 (2H, d,
J=7.8 Hz, H–Ar), 6.92(2H, d, J=7.8 Hz, H–Ar), 7.57 (1H, s,
HC8). Anal. (C19H26N6O2) C, H, N. 6-(4-hydro-
xybenzylamino)-2-{[1(R/S)-(hydroxymethyl)}-2-(methyl)propy-
l]amino}-9-isopropylpurine (P4): Column chromatography
stepwise 0, 0.5, 1.0, 2.0% MeOH in CHCl3; the syrup-like
product; crystallized from CHCl3–Et2O; mp 176–178; yield
40%. MS ESI+: 385 (100, M+H+), 386 (20). 1H NMR
13. Vermeulen, K.; Strnad, M.; Krystof, V.; Havlıcek, L.; Van
der Aa, A.; Lenjou, M.; Nijs, G.; Rodrigus, I.; Stockman, B.;
Van Onckelen, H.; Van Bockstaele, D. R.; Berneman, Z. N.
Leukemia 2002, 16, 299.