Synthesis and biological evaluations of pyrazolo[3,4-d]pyrimidines as cyclin-dependent kinase 2 inhibitors

Article abstract of DOI:10.1016/S0223-5234(03)00065-5

A series of 1,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines 15-19, 30-38 capable of selectively inhibiting CDK2 activity were synthesized by derivatization at C-4, C-6 and N-1 with various amines and lower alkyl groups. For above synthetic compounds, biological evaluation was carried out and structure-activity relationship was examined. In our series, 4-anilino compounds exhibited better CDK2 inhibitory activity and antitumor activity compared to 4-benzyl compounds. The compounds 33a,b having a 3-fluoroaniline group at C-4 showed comparable or superior CDK2 inhibitory activity to those of olomoucine and roscovitine as reference compounds. In general, the unsubstituted compounds (30a,b, 33a,b, 36a,b) at N-1 possessed higher potency than the substituted compounds (32a,b, 34a,b) for the CDK2 inhibitory activity. As for EGFR inhibitory activity, most compounds didnot have a significant activity. The compounds 32a,b exhibited potent cell growth inhibitory activity against human cancer cell lines, but their CDK2 inhibitory activities were slightly poorer than olomoucine.

Full text of DOI:10.1016/S0223-5234(03)00065-5

European Journal of Medicinal Chemistry 38 (2003) 525ꢀ532
/
www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological evaluations of pyrazolo[3,4-d]pyrimidines as
cyclin-dependent kinase 2 inhibitors
Dong Chan Kim a, Yeo Ran Lee a, Beom-Seok Yang a, Kye Jung Shin a,
Dong Jin Kim a, Bong Young Chung b,*, Kyung Ho Yoo a,*
a Medicinal Chemistry Research Center, Korea Institute of Science and Technology, Seoul 130-650, Republic of Korea
b Department of Chemistry, Korea University, Seoul 136-701, Republic of Korea
Received 2 September 2002; received in revised form 31 October 2002; accepted 10 January 2003
Abstract
A series of 1,4,6-trisubstituted pyrazolo[3,4-d]pyrimidines 15ꢀ
synthesized by derivatization at C-4, C-6 and N-1 with various amines and lower alkyl groups. For above synthetic compounds,
biological evaluation was carried out and structureꢀactivity relationship was examined. In our series, 4-anilino compounds
/
19, 30ꢀ38 capable of selectively inhibiting CDK2 activity were
/
/
exhibited better CDK2 inhibitory activity and antitumor activity compared to 4-benzyl compounds. The compounds 33a,b having a
3-fluoroaniline group at C-4 showed comparable or superior CDK2 inhibitory activity to those of olomoucine and roscovitine as
reference compounds. In general, the unsubstituted compounds (30a,b, 33a,b, 36a,b) at N-1 possessed higher potency than the
substituted compounds (32a,b, 34a,b) for the CDK2 inhibitory activity. As for EGFR inhibitory activity, most compounds didnot
have a significant activity. The compounds 32a,b exhibited potent cell growth inhibitory activity against human cancer cell lines, but
their CDK2 inhibitory activities were slightly poorer than olomoucine.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: CDK; Kinase inhibitors; Pyrazolo[3,4-d]pyrimidines; CDK2 inhibitory activity; Cell growth inhibitory activity
1. Introduction
on the protein kinases. Cyclin-dependent kinases
(CDKs) have recently raised considerable interest as
It is becoming clear that many types of cancer are the
result of abnormal signal-transducing proteins which
lead to continual production of the signal for cell
division. The mutated genes that encode these defective
signaling proteins are oncogenes. They were originally
discovered in tumor-causing viruses. A variety of human
tumor cells have been shown to produce their own
growth factors [1]. The central role of tyrosine phos-
phorylation in cell proliferative signaling mechanisms
provides another target for chemotherapy. It suggests
that a specific tyrosine kinase inhibitor would have
significant therapeutic potential as an antitumor agent
the principal regulators of the cell division cycle [16ꢀ
Olomoucine [23ꢀ26] and roscovitine [27,28] (Fig. 1), the
representative compounds of purine derivatives, exhibit
moderate inhibitory activities (IC50ꢁ7 and 0.5 mM) but
good selectivity for CDK1/cyclin B1, CDK2/cyclin A,
CDK2/cyclin E, CDK5/p25, and CDK7/cyclin H of
CDKs over other CDKs or other kinases.
In this work, we carried out the chemical modification
by the introduction of pyrazole ring instead of imidazole
ring on purine to give a specific binding mode different
/
22].
/
/
from the olomoucine analogues [6,29ꢀ
we synthesized 1,4,6-trisubstituted pyrazolo[3,4-d]pyri-
midines 15ꢀ19, 30ꢀ38 (Fig. 1) by introducing a variety
/31]. To this end,
[2ꢀ
Currently, 2,4,9-trisubstituted purines [5ꢀ
quinazolines [12ꢀ15] exhibit a potent inhibitory effect
/
4].
/
/
/11] and
of the substituent groups at C-4, C-6 and N-1 of
pyrazolopyrimidine ring for a potent and selective
inhibitor of the CDK2 by competitive binding at the
ATP site, and evaluated for their CDK2, EGFR
inhibitory activities and cell growth inhibitory activities.
/
* Corresponding auther.
E-mail address: khyoo@kist.re.kr (K.H. Yoo).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00065-5

526
D.C. Kim et al. / European Journal of Medicinal Chemistry 38 (2003) 525ꢀ532
/
Fig. 1. Structure of olomoucine, roscovitine, and pyrazolo[3,4,d]pyrimidines.
2. Chemistry
line yielded 4-chloro-6-methylmercaptopyrazolo[3,4-
d]pyrimidine (6) in 92% yield.
1,4,6-Trisubstituted pyrazolopyrimidines 15ꢀ
a variety of the substituent groups at N-1, C-4, and C-6
positions were prepared as shown in Fig. 3.
Treatment of 6 with benzylamines 7 in the presence of
Hunig’s base in n-butanol resulted in 4-(4?-substituted
benzylamino)-6-methylmercaptopyrazolo[3,4-d]pyrimi-
dines (8) by nucleophilic displacement of the chlorine
atom, respectively. N-1 substituted pyrazolo[3,4-d]pyr-
imidines 10, 11 were prepared by N-alkylation of 8 with
the appropriate alcohols 9 using Mitsunobu condition
4-Chloro-6-methylmercaptopyrazolo[3,4-d]pyrimi-
dine (6) [32], as a key intermediate for the synthesis of
title compounds, was synthesized starting from ethox-
ymethylenemalononitrile (1) by the sequence of reac-
tions shown in Fig. 2.
This synthesis was performed in a process that the
pyrazole ring was first formed and the pyrimidine ring
formation was followed. In pyrazolopyrimidine ring
system, the chloro substituent as a leaving group was
introduced at C-4 which was the most reactive site to
nucleophilic attack. And the methylmercapto group was
employed at C-6 as a precursor to introduce later the
methylsulfonyl group which is five times more reactive
than the chloro substituent [33].
Ethoxymethylenemalononitrile (1) was cyclized by
nucleophilic substitution of hydrazine to give 3-amino-
4-cyanopyrazole (2) in 84% yield, which was converted
to 3-amino-4-pyrazolecarboxamide hydrosulfate (3) by
hydrolysis of the nitrile group with 95% sulfuric acid in
88% yield. Subsequent fusion of 3 with thiourea
provided 4-hydroxy-6-mercaptopyrazolo[3,4-d]pyrimi-
dine (4) in moderate yield. 4 was alkylated with
iodomethane and sodium hydroxide at 5 8C to afford
4-hydroxy-6-methylmercaptopyrazolo[3,4-d]pyrimidine
(5) in quantitative yield. Chlorination of 5 with phos-
phorus oxychloride in the presence of N,N-dimethylani-
/19 with
[34] in 86ꢀ96% yields. Then, in order to introduce the
/
amino group at C-6 position of 8, 10, 11, oxidation of
methylmercapto groups using m-CPBA and followed by
nucleophilic displacement of the resulting activated
methylsulfonyl groups with hydroxylamines 14 led to
the corresponding 1,4,6-trisubstituted pyrazolo[3,4-
d]pyrimidines 15ꢀ18. Compounds 18 bearing 4-nitro-
/
benzyl moiety at C-4 were carefully reduced with Pd/C
in methanol to yield 19 of 4-aminobenzyl moiety.
On the other hand, compounds 30ꢀ38 were prepared
/
from 6 (Fig. 4), by means of reactions such as amination
at C-4 by anilines 20, alkylation at N-1 using Mitsunobu
condition, oxidation of methylmercapto groups with m-
CPBA, and nucleophilic substitution of activated sul-
fones with hydroxylamines 29 in a mixture of n-BuOH
and DMSO.
Fig. 2. Reagents and reaction conditions: (i) H2NNH2×
/H2O (84%); (ii) 95% H2SO4 (88%); (iii) thiourea (70%); (iv) CH3I, NaOH (99%); (v) POCl3,
PhNMe2 (92%).

D.C. Kim et al. / European Journal of Medicinal Chemistry 38 (2003) 525ꢀ
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527
Fig. 3. Reagents and reaction conditions: (i) Hunig’s base, n-BuOH; (ii) PPh3, DEAD, THFꢀ/CH2Cl2 (1:1); (iii) mCPBA, CH2Cl2, 0 8C; (iv) n-
BuOHꢀDMSO (4:1); 90 8C, 4 h; (v) Pd/C, MeOH.
/
Fig. 4. Reagents and reaction conditions: (i) Hunig’s base, n-BuOH; (ii) PPh3, DEAD, THFꢀ/CH2Cl2 (1:1); (iii) mCPBA, CH2Cl2, 0 8C; (iv) n-
BuOHꢀDMSO (4:1), 90 8C, 4 h.
/

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D.C. Kim et al. / European Journal of Medicinal Chemistry 38 (2003) 525ꢀ532
/
Table 1
Physical properties and spectral data of the selected intermediates
Compound M.p.
(8C)
1H-NMR (solvent: d, ppm)
13C-NMR (d, ppm)
8a
212ꢀ
214
/
DMSO-d6: 8.77 (t, 1H, Jꢁ
4.71 (d, 2H, Jꢁ5.7 Hz), 2.44 (s, 3H)
DMSO-d6: 13.32 (s, 1H), 8.92 (t, 1H), 8.21 (d, 2H, Jꢁ
1H), 7.60 (d, 2H, Jꢁ8.6 Hz), 4.82 (d, 2H, Jꢁ5.7 Hz), 2.39 (s, 3H) 43.6, 14.4
95 DMSO-d6: 7.71 (s, 1H), 7.36ꢀ
2.58 (s, 3H)
CDCl3: 7.75 (s, 1H), 7.38ꢀ
(d, 2H, Jꢁ5.5 Hz), 2.60 (s, 3H), 1.54 (d, 6H, Jꢁ
DMSO-d6: 9.43 (s, 1H), 8.30 (s, 1H), 7.40ꢀ
/
5.7 Hz), 8.09 (s, 1H), 7.37ꢀ
/
7.23 (m, 5H), 168.9, 156.0, 153.9, 139.9, 133.3, 129.1, 128.2, 127.7,
/
98.5, 43.9, 14.3
8b
208ꢀ
/
/
8.6 Hz), 8.06 (s, 169.0, 156.1, 148.4, 147.3, 133.4, 129.1, 124.1, 98.6,
210
/
/
10a
10b
12a
12b
12c
21a
21b
94ꢀ
/
/7.31 (m, 5H), 4.81 (d, 2H), 3.94 (s, 3H), 167.8, 154.8, 152.9, 138.7, 131.2, 127.9, 127.1, 126.5,
97.6, 42.8, 32.8, 13.1
166ꢀ
/
/
7.28 (m, 5H), 5.09 (m, 1H, Jꢁ
/6.6 Hz), 4.83 169.0, 156.1, 153.5, 138.1, 133.5, 129.3, 128.2, 127.7,
167
/
/6.6 Hz)
98.7, 49.1, 45.5, 22.3, 14.6
205ꢀ
/
/7.27 (m, 5H), 4.78 (s, 2H), 163.6, 157.5, 154.5, 139.3, 133.6, 129.3, 128.7, 128.1,
208
3.29 (s, 3H)
101.3, 46.7, 39.8
110ꢀ
/
DMSO-d6: 9.46 (s, 1H), 8.25 (s, 1H), 7.41ꢀ
/
7.27 (m, 5H), 4.78 (d, 2H, 163.7, 157.5, 152.8, 139.4, 133.2, 129.5, 128.8, 128.4,
101.7, 44.9, 40.1, 35.0
115
Jꢁ
CDCl3: 7.98 (s, 1H), 7.37ꢀ
(d, 2H, Jꢁ5.6 Hz), 3.31 (s, 3H), 1.54 (d, 6H, Jꢁ
/
5.1 Hz), 3.95 (s, 3H), 3.30 (s, 3H)
122ꢀ
/
/
7.27 (m, 5H), 5.19 (m, 1H, Jꢁ
/
6.6 Hz), 4.87 162.7, 157.3, 151.7, 137.9, 131.5, 129.1, 128.4, 127.3,
101.9, 49.8, 45.6, 39.5, 22.4
124
/
/6.6 Hz)
ꢀ/
250 DMSO-d6: 10.33 (s, 1H), 8.29 (s, 1H), 8.31ꢀ
/
7.29 (m, 4H), 2.54 (s, 3H) 168.0, 155.4, 153.0, 140.7, 132.3, 130.5, 125.7, 123.1,
121.3, 119.3, 98.4, 13.6
242ꢀ
/
DMSO-d6: 10.19 (s, 1H), 8.22 (s, 1H), 7.93ꢀ
/
6.87 (m, 4H), 2.51 (s, 3H) 168.9, 164.4, 161.2, 156.2, 153.7, 141.7, 141.5, 133.2,
131.0, 130.8, 117.0, 110.4, 110.1, 108.4, 108.0, 99.1,
14.4
247
21c
23a
23b
26a
26b
26c
218ꢀ
222
/
DMSO-d6: 10.04 (s, 1H), 8.21 (s, 1H), 7.60ꢀ
2.54 (s, 3H)
/
6.70 (m, 4H), 3.80 (s, 3H), 169.2, 160.5, 156.4, 154.2, 141.3, 133.7, 130.5, 114.1,
109.9, 107.7, 99.3, 56.0. 14.7
141ꢀ
/
DMSO-d6: 10.22 (s, 1H), 8.19 (s, 1H), 8.30ꢀ
/
7.27 (m, 4H), 3.89 (s, 3H), 168.3. 153.6, 152.9, 140.6, 131.6, 130.6, 125.7, 123.2,
121.4, 119.3, 98.8, 33.4, 13.8
144
2.57 (s, 3H)
110ꢀ
/
CDCl3: 7.89 (s, 1H), 7.49ꢀ
(s, 3H), 1.52 (d, 6H, Jꢁ6.7 Hz)
DMSO-d6: 7.90 (s, 1H), 8.12ꢀ7.15 (m, 4H), 3.30 (s, 3H)
/7.26 (m, 4H), 5.08 (m, 1H, Jꢁ
/
6.7 Hz), 2.64 167.7, 154.0, 153.4, 139.1, 132.0, 131.0, 129.4, 127.1,
123.2, 122.5, 98.6, 49.4, 22.3, 14.7
115
/
208ꢀ
/
/
163.0, 155.0, 153.0, 141.0, 139.5, 134.0, 127.6, 124.6,
122.3, 120.7, 102.4, 40.2
210
ꢀ/
260 DMSO-d6: 10.73 (s, 1H), 8.34ꢀ
/
7.33 (m, 5H), 3.99 (s, 3H), 3.41 (s, 3H) 163.0, 155.0, 153.0, 140.8, 133.2, 131.8, 127.7, 124.6,
122.5, 120.8, 102.6, 40.2, 35.1
200ꢀ
/
CDCl3: 8.39 (s, 1H), 7.36ꢀ/6.80 (m, 4H), 5.06 (m, 1H, Jꢁ
/
6.7 Hz), 3.33 164.9, 162.2, 161.6, 152.2, 139.2, 132.4, 130.8, 119.3,
113.1, 110.7, 101.9, 49.9, 39.7, 22.3
204
(s, 3H), 1.42 (d, 6H, Jꢁ6.7 Hz)
/
Physical properties and spectral data of the selected
compounds are given Tables 1 and 2.
corresponding 4-benzyl analogues 15ꢀ19. The com-
/
pounds 33a,b having a 3-fluoroaniline group at C-4
were as active as roscovitine and much more active than
olomoucine as reference compounds, whereas they
possessed poor cell division inhibitory activity. The
unsubstituted compounds (30, 33, 36) at N-1 exhibited
higher potency compared to the substituted compounds
(31, 32, 34, 35, 37, 38) for the CDK2 inhibitory activity.
As a nitrogen atom as a hydrogen bond acceptor in
pyrazolopyrimidine scaffold is missing at C-3 position
equivalent to N-7 position of olomoucine, pyrazolopyr-
imidine ring system has to flip for hydrogen bonding
with the protein hinge region. To this reason, the
unsubstituted compounds at N-1 are more active owing
to the possibility of an additional hydrogen bonding by
ring flip (Fig. 5).
In the correlation of the CDK2 inhibitory activity
with the substituent size at C-6 of pyrazolopyrimidine
skeleton, there was no significant difference between
mono-ethanolamine and branched di-ethanolamine
compounds. Most compounds did not show any sig-
nificant EGFR inhibitory activity, indicating good
selectivity in the situation of protein kinase inhibitors.
3. Biological results and discussion
CDK2 and EGFR inhibitory activities of pyra-
zolo[3,4-d]pyrimidines 15ꢀ
/
19, 30ꢀ38 prepared above
/
were shown in Table 3, together with those of olomou-
cine, roscovitine and PYK2104 as reference compounds.
CDK2 is one of CDK family protein which has a proline
directed serine/threonine kinase activity, phosphorylat-
ing serine or threonine residue ahead of proline, whereas
EGFR belongs to receptor tyrosine kinase family.
Compounds were further evaluated for their cell division
inhibitory activities against three human tumor cell
lines. These tumor cells divide at least twice for 2-day
period of the cell division inhibition test in the absence
of inhibitory compounds and the kinase activity of
CDK2/cyclin A is indispensable for the division of these
cells.
In general, 4-anilino compounds 30ꢀ/38 showed better
CDK2 and cell division inhibitory activities than the

D.C. Kim et al. / European Journal of Medicinal Chemistry 38 (2003) 525ꢀ
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529
Table 2
Physical properties and spectral data of the selected pyrazolopyrimidines
Compound M.p.
(8C)
1H-NMR (solvent: d, ppm)
13C-NMR (d, ppm)/FABHRMS m/z
15a
15b
16a
230ꢀ
234
/
DMSO-d6: 12.53 (s, 1H), 8.21 (s, 1H), 7.85 (s, 1H), 7.34ꢀ
7.23 (m, 5H), 6.39 (s, 1H), 4.67 (d, 2H), 3.51ꢀ3.31 (m, 4H) 44.5, 43.7
DMSO-d6: 8.32 (s, 1H), 7.83 (s, 1H), 7.37ꢀ7.23 (m, 5H),
4.62 (m, 3H), 3.60ꢀ3.46 (m, 8H)
95 CD3OD: 7.80 (s, 1H), 7.37ꢀ
(s, 3H), 3.70 (t, 2H, Jꢁ5.4 Hz), 3.54 (t, 2H, Jꢁ
3.57 (t, 2H)
CD3OD: 7.79 (s, 1H), 7.36ꢀ
(s, 11H)
CDCl3: 8.18 (d, 2H), 7.64 (s, 1H), 7.49 (d, 2H), 5.78 (bs,
/
162.4, 158.2, 157.3, 140.8, 133.1, 129.1, 128.3, 127.6, 96.0, 61.2,
/
242ꢀ
/
/
161.3, 158.1, 156.9, 141.0, 133.1, 129.1, 128.3, 127.6, 95.1, 60.4,
52.5, 44.6
245
/
94ꢀ
/
/
7.20 (m, 5H), 4.72 (d, 2H), 3.77 162.5, 157.4, 155.5, 142.1, 139.6, 131.5, 128.3, 127.4, 126.9, 61.6,
/
/
5.5 Hz) 49.2, 46.2, 32.2
16b
18a
130ꢀ
/
/
7.23(m, 5H), 4.70 (d, 2H), 3.77 161.9, 157.1, 155.9, 140.0, 131.5, 128.4, 127.4, 127.0, 95.6, 61.8,
135
52.4, 44.1, 32.1
127ꢀ
/
162.5, 157.4, 155.8, 147.8, 146.7, 130.7, 128.4, 124.3, 96.4, 64.9,
130
1H), 5.41 (t, 1H), 4.85 (d, 2H), 3.84 (s, 3H), 3.82 (t, 2H), 45.4, 44.6, 33.9
3.59 (t, 2H)
18b
30a
30b
155ꢀ
157
/
CDCl3: 8.19 (d, 2H), 7.68 (s, 1H), 7.50 (d, 2H), 5.87 (bs,
1H), 5.40 (t, 1H), 4.84 (m, 2H), 4.84 (m, 1H), 3.82 (t, 2H), 48.4, 44.9, 44.1, 21.8
3.59 (t, 2H), 1.49 (d, 6H)
161.9, 157.0, 154.2, 147.2, 146.3, 130.3, 128.0, 123.8, 96.3, 64.5,
ꢀ/
260 DMSO-d6: 7.85 (s, 1H), 8.22ꢀ
/
7.09 (m, 4H), 3.66 (t, 2H),
162.0, 158.5, 154.9, 142.8, 141.3, 133.3, 131.2, 125.7, 121.4, 119.7,
96.6, 61.0, 44.7/Calc. for C13H13BrN6O (Mꢂ
H)ꢂ 349.0334,
Found 349.0412
7.10 (m, 4H), 3.77 (m, 4H), 160.8, 158.3, 154.2, 142.2, 133.0, 131.2, 125.4, 122.9, 122.1, 119.2,
95.7, 60.3, 52.4/Calc. for C15H17BrN6O2 (Mꢂ
H)ꢂ 393.0596,
Found 393.0667
3.47 (t, 2H)
/
254ꢀ
/
DMSO-d6: 7.82 (s, 1H), 8.21ꢀ
3.49 (m, 4H)
/
257
/
31a
31b
33a
173ꢀ
174
/
DMSO-d6: 9.67 (s, 1H), 8.00ꢀ
3.74 (s, 3H), 3.59 (q, 2H), 3.43 (q, 2H)
DMSO-d6: 9.78 (s, 1H), 8.32ꢀ7.19 (m, 5H), 4.82 (s, 2H),
3.75ꢀ3.72 (m, 11H)
/
6.94 (m, 5H), 4.70 (s, 1H),
161.9, 156.5, 154.8, 142.5, 132.2, 131.4, 125.7, 123.1, 122.3, 119.6,
96.8, 60.9, 44.8, 33.8
218ꢀ
/
/
160.9 156.5, 154.4, 142.3, 132.2, 131.4, 125.6, 123.1, 122.3, 119.3,
96.2, 60.8, 52.7, 33.6
220
/
ꢀ/
260 DMSO-d6: 12.79 (s, 1H), 9.69 (s, 1H), 8.14ꢀ
/
6.79 (m, 5H), 164.7, 161.9, 161.5, 158.3, 154.8, 142.8, 142.7, 133.2, 130.9, 130.7,
116.3, 109.4, 109.2, 107.9, 107.5, 96.5, 60.8, 44.6/Calc. for
4.73 (t, 2H), 3.60ꢀ3.37 (m, 4H)
/
C13H13FrN6O (Mꢂ
H)ꢂ 289.1135, Found 289.1201
164.5, 161.3, 161.0, 158.4, 154.4, 142.6, 142.4, 133.2, 131.0, 130.9,
/
33b
ꢀ/
260 DMSO-d6: 12.82 (s, 1H), 9.80 (s, 1H), 8.05 (s, 1H), 7.95ꢀ
/
6.81 (m, 4H), 4.01 (s, 1H), 3.71ꢀ3.64 (m, 8H)
/
116.4, 109.6, 109.3, 107.7, 107.4, 95.9, 60.5, 52.7/Calc. for
15H17FN6O2 (Mꢂ
H)ꢂ 333.1397, Found 333.1475
C
/
34a
34b
36a
176ꢀ
179
/
CD3OD: 7.96ꢀ
4H)
DMSO-d6: 9.81 (s, 1H), 8.03 (s, 1H), 7.90ꢀ
4.80 (s, 2H), 3.84 (m, 3H), 3.80ꢀ3.57 (m, 8H)
/
6.79 (m, 5H), 3.81 (s, 3H), 3.79ꢀ
/
3.60 (m,
164.4, 161.9, 161.5, 156.5, 154.9, 142.7, 142.6, 132.2, 130.9, 130.8,
116.5, 109.6, 109.3, 107.9, 107.6, 96.8, 60.9, 44.7, 33.7
164.6, 161.4, 160.9, 156.5, 154.5, 142.5, 142.3, 132.2, 131.0, 130.9,
116.4, 109.7, 109.4, 107.8, 107.4, 96.2, 60.0, 52.5, 33.6
213ꢀ
/
/6.81 (m, 4H),
215
/
254ꢀ
/
DMSO-d6: 9.48 (s, 1H), 8.02 (s, 1H), 7.60-6.59 (m, 5H), 4.72 161.1, 159.4, 157.34, 154.2, 141.2, 132.4, 129.2, 112.5, 108.0,
(s, 1H), 3.77 (s, 3H), 3.55 (t, 2H), 3.36 (s, 2H) 105.9, 95.6, 60.0, 55.0, 43.7/Calc. for C14H16N6O2 (Mꢂ
H)ꢂ
301.1335, Found 301.1413
161.2, 160.4, 158.3, 154.6, 141.9, 133.2, 130.2, 113.3, 109.1, 106.5,
95.9, 60.2, 56.0, 52.3/Calc. for C16H20N6O3 (Mꢂ
H)ꢂ 345.1597,
Found 345.1675
6.67 (m, 4H), 3.83 (s, 3H), 3.81 162.0, 160.3, 156.5, 155.1, 142.0, 132.3, 130.1, 113.5, 109.1, 106.8,
256
/
36b
ꢀ/
260 DMSO-d6: 9.51 (s, 1H), 8.01 (s, 1H), 7.48ꢀ
/6.58 (m, 4H),
4.73 (s, 2H), 3.75 (s, 3H), 3.74ꢀ3.62 (m, 8H)
/
/
37a
37b
184ꢀ
/
CD3OD: 7.89 (s, 1H), 7.53ꢀ
(s, 3H), 3.77 (t, 2H), 3.61 (t, 2H)
CDCl3: 9.59 (s, 1H), 8.04 (1H, s), 7.51ꢀ
(brs, 2H), 3.78ꢀ3.67 (m, 14H)
/
188
96.8, 60.9, 55.9, 44.7, 33.7
171ꢀ
/
/
6.60 (m, 4H), 4.80 161.1, 160.4, 156.4, 154.7, 141.8, 132.3, 130.2, 113.3, 109.2, 106.4,
96.2, 60.2, 55.9, 52.3 33.6
175
/
The compounds 32a,b exhibited potent cell division
inhibitory activity against the target organisms but poor
CDK2 inhibitory activity.
It seems that there is not a good correlationship
between in vitro CDK2 inhibitory activity and in vivo
cell growth inhibition activity among some tested
compounds. For example, comparing compound 30b
with 32a and 32b, 30b is more potent than 32a and 32b
for CDK2 inhibition. However, the degree of in vivo cell
growth inhibition is reversed between them. Currently it
is not clear for the reason. It may be due to different
abilities to internalize inside cells, or it may be resulted
from different inhibitory activities against some other
cellular targets within cells besides CDK2 that might be
critical for cell growth.
The results demonstrate that structural variations at
C-4, C-6, and N-1 of pyrazolo[3,4-d]pyrimidines might
be led to potent inhibitors of the CDKs, and these
informations will be helpful for designing new inhibi-
tors.

530
D.C. Kim et al. / European Journal of Medicinal Chemistry 38 (2003) 525ꢀ532
/
Table 3
CDK2, EGFR, and cell division inhibitory activities of pyrazolo[3,4-
rected. IR spectra were measured on a PerkinꢀElmer
/
16F PC FT-IR spectrophotometer. H- and 13C-NMR
spectra were recorded on a 300 MHz Bruker NMR
1
d]pyrimidines 15ꢀ
/
19, 30ꢀ38
/
1
spectrometer (300 MHz for H and 75.5 MHz for 13C)
IC50 (mM)
GI50 (mM)
using tetramethylsilane as an internal standard. Mass
spectra were obtained on a JEOL SX-102. Microanaly-
tical data were obtained by using a EA 1108 Fisons
Instruments. Column chromatography was carried out
Compound
CDK2 EGFR A431 a SNU638 b HCT116 c
15a
15b
29.5
37.2
93.3
55.0
26.56
20.77
100 28.98
4.50
93.63
ꢀ/
16a
16b
ꢀ
/
100
ꢀ
/
100 42.67
24.28
37.08
25.28
25.17
26.11
24.92
23.45
96.75
38.20
ꢀ100
/
using silica gel (230ꢀ400 mesh). Solvents and liquid
reagents were transferred using hypodermic syringes. All
other reagents and solvents used were reagent grade.
/
ꢀ
/
100 77.6
53.44
57.60
47.68
48.52
77.71
17a
17b
ꢀ
/
100
ꢀ
/
100 35.60
100 21.88
100 23.36
100 32.02
100 58.88
100 55.27
ꢀ
/
100
ꢀ
/
18a
18b
17.8
46.8
ꢀ
/
4.1.1. General procedure for the synthesis of 4-substituted
amino-6-methylmercaptopyrazolo[3,4-d]pyrimidines 8
and 21
ꢀ
/
19a
19b
ꢀ
/
100
ꢀ
/
ꢀ
/
100
100
100
100
100
ꢀ
/
100
ꢀ
/
ꢀ
/
30a
30b
8.1
ꢀ/
100
36.3
100 47.9
100
ꢀ
ꢀ/
/
100 69.22
100 38.88
ꢀ
/
To a solution of 4-chloro-6-methylmercaptopyra-
zolo[3,4-d]pyrimidine (6, 1.00 g, 5.1 mmol) in n-BuOH
(80 mL) were added the appropiate amine (7, 20, 5.1
mmol) and diisopropylethylamine (1.60 mL, 9.2 mmol),
and the mixture was stirred at 50 8C for 24 h. The
mixture was concentrated in vacuo to give the residue,
which was diluted with EtOAc and H2O. The organic
layer was dried over anhydrous Na2SO4, and concen-
trated in vacuo. The residue was purified by silica gel
3.8
ꢀ
/
31a
31b
ꢀ
/
31.86
18.16
6.20
4.28
4.10
ꢀ/
ꢀ
/
ꢀ
/
100 2.94
100 2.19
100 2.58
16.61
6.46
5.06
32a
32b
28.2
29.5
0.5
ꢀ
/
ꢀ
/
33a
33b
66.1
28.85
100 54.33
ꢀ/100
ꢀ/100
0.9
ꢀ
/
ꢀ/100
ꢀ/100
34a
34b
21.4
36.3
49.0
47.9
3.1
ꢀ
/
100 16.29
3.94
15.84
2.56
7.57
7.95
28.24
8.89
29.5
35a
35b
ꢀ
/
100 7.80
19.28
10.88
38.19
ꢀ
/
100 6.25
column chromatography (n-hexaneꢀ
/
EtOAcꢁ1.5:1) to
/
36a
36b
ꢀ
/
100
ꢀ
ꢀ/
/
100 17.07
100 100
give the title compound.
10.2
ꢀ/
100
ꢀ/
ꢀ/100
37a
37b
ꢀ
/
100
57.5
100
ꢀ
/
100 19.14
100 12.94
100 13.08
100 4.43
16.60
21.73
10.49
7.55
90.44
17.19
21.44
12.56
12.05
4.1.2. General procedure for the synthesis of 1-alkyl-4-
substituted amino-6-methylmercaptopyrazolo[3,4-
ꢀ
/
38a
38b
ꢀ
/
ꢀ
/
66.0
7.0
ꢀ
/
7.92
16.62
d]pyrimidines 10, 11 and 23ꢀ25
/
Olomoucine
Roscovitine
PYK2104
Doxorubicine
ꢀ/
ꢀ/
To a solution of triphenylphosphine (1.30 g, 5.0
mmol) in a mixture of THF (2.50 mL) and CH2Cl2
(2.50 mL) was added diethyl azodicarboxylate (0.39 mL,
2.5 mmol) in portions, and the mixture was stirred at
0 8C for 1 h. To a solution of compound 8, 21 (2.0
mmol) in a mixture of THF (2.5 mL) and CH2Cl2 (2.5
mL) was added the appropriate alcohol 9, 22 (2.0
mmol), followed by the above-prepared solution using
cannula at 0 8C, and the mixture was stirred at room
temperature (r.t.) for 3 h. When the reaction was
completed, the mixture was concentrated in vacuo.
The residue was purified by silica gel column chromato-
0.5
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
0.0008
ꢀ/
0.18
0.13
a
A431, human vulvar epidermoid carcinoma cell line.
SNU638, human stomach epitherial carcinoma cell line.
HCT116, human colon epitherial carcinoma cell line.
b
c
graphy (n-hexaneꢀ
/
EtOAcꢁ2:1) to afford the title
/
compound.
4.1.3. General procedure for the synthesis of 1-alkyl-4-
substituted amino-6-methylsulfonylpyrazolo[3,4-
Fig. 5. Comparison of the binding modes of olomoucine with
compound 33a.
d]pyrimidines 12,13, and 26ꢀ
To a stirred solution of the appropriate compound 10,
11, 23ꢀ25 (1.4 mmol) in CH2Cl2 (100 mL) was added
70% solution of 3-chloroperbenzoic acid (0.74 g, 3.0
mmol) at 0 8C for 3 h. After being stirred at r.t. for 3 h,
the mixture was concentrated in vacuo to give the
residue, which was diluted with EtOAc and saturated
aqueous NaHCO3 solution. The organic layer was
washed with saturated aqueous NaHCO3 solution,
/
28
/
4. Experimental
4.1. Chemistry
Melting points were determined on a Thomasꢀ
/
Ho-
over capillary melting point apparatus and are uncor-

D.C. Kim et al. / European Journal of Medicinal Chemistry 38 (2003) 525ꢀ
/532
531
H2O and brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by silica
4.2.2. Cell growth inhibition assay
Human cancer cell lines, A431 (cervical cancer cell
line), SNU638 (stomach cancer cell line), and HCT116
(colon cancer cell line) were obtained from Cell Bank in
Medical College of Seoul National University, Seoul,
Korea. Cells were grown in RPMI 1640 medium
containing 10% fetal bovine serum at 37 8C and 5%
CO2. For cell division inhibition assay, 1000 cells were
plated on 96 well plate and next day, test compounds as
well as olomucine and doxorubicin (Sigma, USA) as
reference compounds were treated to the cells at various
concentrations. Cells were allowed to grow further for
two days in the presence of compounds, then fixed by
adding equal volume of 4% formalin (Sigma) for 30 min.
Fixed cells were washed with tap water five times and
stained in 0.1% sulforhodamine B (Sigma) for 30 min.
Subsequently cells were washed with 1% acetic acid for
four times and the dyes attached to cells were eluted by
gel column chromatography (n-hexaneꢀ
/
EtOAcꢁ2:1)
/
to give the title compound.
4.1.4. General procedure for the synthesis of 1-alkyl-4-
substituted amino-6-substituted
hydroxylaminopyrazolo[3,4-d]pyrimidines 15ꢀ
30ꢀ38
To a stirred solution of the appropriate compound 12,
13, 26ꢀ28 (1.0 mmol) in n-BuOH (16 mL) and DMSO
/
19 and
/
/
(4 mL) was added the corresponding ethanolamine 14,
29 (0.18 mL). After being stirred at 90 8C for 12 h, the
mixture was concentrated in vacuo to give the residue,
which was diluted with EtOAc. The organic layer was
washed with H2O, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by silica
gel column chromatography (CH2Cl2ꢀ
/
MeOHꢁ20:1) to
/
give the title compound.
adding 100 mL of 0.1 M Trisꢀ/HCl (pH 8.0) and shaking
for 10 min. The absorbance was measured at 520 nm
wavelength using microplate reader (Molecular Dy-
namics). The absorbance is proportional to cell number
in each well. Measurements were done triplicate and
averaged. The absorbance from cells at the time of
compound treatment was designated as 0% and the
absorbance from cells after two days growth with no
compound treatment was assigned as 100%. The GI50
value was defined as the inhibitor concentration which
gives 50% cell growth inhibition during 2 days period of
compound treatment.
4.2. Microbiology methods
4.2.1. Enzymatic activity inhibition assay
The inhibition studies of cell cycle dependent kinase 2
were performed for the synthesized compounds along
with olomoucine and roscovitine as reference com-
pounds. Olomoucine was purchased from Sigma
(USA) and synthesized roscovitine was kindly provided
by Oh et al. [7]. CDK2/cyclin A enzyme was purified
from infected sf21 insect cells. For baculoviral over-
expressions of proteins, we subcloned human CDK2 c-
DNA tagged by hexa-histidine on its N-terminal and
human cyclin A c-DNA into pBacPak 8 expression
vector (Clon Tech, USA), respectively, and baculovirus
which carries each gene was generated using baculovirus
generating kit from Clon Tech. CDK2/cyclin A enzyme
was purified using Ni2ꢂ-affinity resin (Novagen, USA)
from sf21 insect cell culture into which CDK2 and cyclin
A carrying baculoviruses were cotransfected. Enzyme
Acknowledgements
We are grateful to the Korea Institute of Science and
Technology for financial support.
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/
HCl
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reaction was continued for 10 min in the presence of
inhibitors and stopped by adding 10 mL of 30%
phosphoric acid. The stopped mixtures were spotted
onto P81 paper (Whatman, USA) and were washed with
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