Pyrazolo[3,4-b]quinoxalines. A new class of cyclin-dependent kinases inhibitors

Article abstract of DOI:10.1016/S0968-0896(02)00069-X

Protein kinases are involved in most physiological processes and in numerous diseases. Therefore, inhibitors of protein kinases have therefore a wide therapeutic potential. While screening for inhibitors of cyclin-depent kinases (CDK's) and glycogen synthase kinase-3 (GSK-3), we identified pyrazolo[3,4-b]quinoxalines as sub-micromolar inhibitors of CDK1/cyclin B. A preliminary structure-activity relationship study suggests that this family of compounds can be optimized to inhibit CDK's and GSK-3. Compounds were tested for their anti-proliferative activity and the results show that several of them displayed a significant inhibitory effect on CDK1/cyclin B. The most active compound (1) was also tested against the brain kinases CDK5/p25 and GSK-3, and proved to be a good inhibitor of both of them. On the contrary, none of the compounds showed any activity in the CDC25 phosphatase assay. As an additional approach, affinity chromatography on immobilized pyrazolo[3,4-b]quinoxalines will be used to identify the intracellular targets of this family of compounds.

Full text of DOI:10.1016/S0968-0896(02)00069-X

Bioorganic & Medicinal Chemistry 10 (2002) 2177–2184
Pyrazolo[3,4-b]quinoxalines. A New Class of
Cyclin-Dependent Kinases Inhibitors
Miguel A. Ortega, Marıa E. Montoya, Belen Zarranz, Andre
´
´ ´
s Jaso,a
a
b
a
a
c
c
a,
Ignacio Aldana, Sophie Leclerc, Laurent Meijer and Antonio Monge *
aUnidad en Investigacio´n y Desarrollo de Medicamentos, Centro de Investigacio´n en Farmacobiologı´a Aplicada (CIFA),
Universidad de Navarra, E-31080 Pamplona, Spain
b
Facultad de Farmacia y Bioquı´mica, Universidad Nacional Mayor de San Marcos, Lima, Peru
cCell Cycle Laboratory, CNRS, Station Biologique, BP 74, 29682 Roscoff Cedex, Bretagne, France
Received 6 November 2001; revised 28 January 2002; accepted 8 February 2002
Abstract—Protein kinases are involved in most physiological processes and in numerous diseases. Therefore, inhibitors of protein
kinases have therefore a wide therapeutic potential. While screening for inhibitors of cyclin-depent kinases (CDK’s) and glycogen
synthase kinase-3 (GSK-3), we identified pyrazolo[3,4-b]quinoxalines as sub-micromolar inhibitors of CDK1/cyclin B. A pre-
liminary structure–activity relationship study suggests that this family of compounds can be optimized to inhibit CDK’s and GSK-3.
Compounds were tested for their anti-proliferative activity and the results show that several of them displayed a significant inhibi-
tory effect on CDK1/cyclin B. The most active compound (1) was also tested against the brain kinases CDK5/p25 and GSK-3, and
proved to be a good inhibitor of both of them. On the contrary, none of the compounds showed any activity in the CDC25 phos-
phatase assay. As an additional approach, affinity chromatography on immobilized pyrazolo[3,4-b]quinoxalines will be used to
identify the intracellular targets of this family of compounds. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
ing binding to cyclins, which is a broad class of positive
regulatory CDK-binding proteins. In addition to this,
there are other different ways by which these proteins
are regulated: subunit production, complex formation,
(de)phosphorylation, intracellular translocations and
interaction with various natural protein inhibitors.
Recently, deregulations of CDK’s have been proven in
Most anticancer agents which have been approved for
clinical use are molecules that damage deoxyribonucleic
acid (DNA), block DNA synthesis by inhibiting the
nucleic acid precursors biosynthesis, alter tubulin
polymerization/depolymerization or disrupt hormonal
1
6
stimulation of cell growth. However, there has been a
human primary tumors and in tumor cell lines and
therefore, CDK’s constitute attractive targets for the
recent shift of emphasis towards novel mechanistic tar-
gets that have directly arisen from the in-depth study of
the underlying genetic changes related to the cancerous
state. Thus, for instance, the identification of oncogenes
such as ras which are activated in tumors, has suggested
many pathways and specific molecular entities as
rational targets for anticancer drug discovery.2,3
3
,7À21
In addition, the
development of antitumor drugs.
involvement of CDK5 in Alzheimer’s disease provides a
1
1
new potential application for CDK inhibitors.
Among the chemical agents that act selectively as CDK
inhibitors are: a lactone (butyrolactone I), flavonoids
(flavopiridol), indirubins, hymenialdisine and several
More recently, medicinal chemistry research has been
focused on the proteins that drive and control cell cycle
progression. Among them, the cyclin-dependent kinases
purine derivatives (see Fig. 1). Butyrolactone I has
shown antiproliferative activity for colon and pancreatic
2
2
carcinoma cell lines. Flavopiridol is the first CDK
inhibitor that has entered clinical trials as an anticancer
(
of serine/threonine kinases which rule the transmission
CDK’s) play an essential role. The CDK’s are a group
23
agent. The class of purine-related derivatives comprises
25
4
,5
24
26
between successive stages of the cell cycle. The activity
of CDK’s is regulated by multiple mechanisms, includ-
olomoucine,
purine-derived compounds as well as flavopiridol have been
roscovitine
and purvalanols.
These
23,27
shown to bind to the ATP-binding pocket of CDK’s.
For several years our group has been investigating new
quinoxaline and quinoxaline 1,4-di-N-oxide derivatives
*Corresponding author. Tel.: +34-948-425653; fax: +34-945-425652;
e-mail: amonge@unav.es
0
968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00069-X

2178
M. A. Ortega et al. / Bioorg. Med. Chem. 10 (2002) 2177–2184
Figure 1. CDK inhibitors.
that have been shown to possess antitumoral proper-
8,29
reaction with hydrazine hydrate under reflux. Reaction
of derivatives 1 and 6 with copper(II) chloride and tert-
butylnitrite in acetonitrile at 80 C under nitrogen
atmosphere yielded compounds 7 and 8. Attempts to
synthesize the 3-(dialkylaminoalkylamino) derivatives
from the corresponding 3-chloro-1H-pyrazolo[3,4-b]
quinoxalines (7 and 8) and N,N - dialkylaminoalkyl-
amines always proved to be unsuccessful (Scheme 1);
either the starting compounds or unmanageable mixtures
were obtained in each case.
ties.2
As a continuation of this research, novel pyr-
ꢀ
azolo[3,4-b]quinoxalines have been synthesized and
evaluated as potential CDK inhibitors. These chemical
structures were chosen due to their structural similarity
with some purine derivatives, such as olomoucine and
roscovitine. The biological evaluation of the new com-
pounds would provide information regarding how the
quinoxaline-fused pyrazol ring may modify the anti-
tumoral properties of previously synthesized quinoxalines
and quinoxaline 1,4-di-N-oxides. In addition, with the
attachment of different aliphatic amines to the pyrazole
ring we attempted to achieve a structural similarity
with well-known antitumor drugs.
To obtain derivatives 9–16, the corresponding 1H-
pyrazolo[3,4-b]quinoxalines (1–6) were dissolved in
chloroform and reacted with the appropriate N,N-di-
alkylaminoalkyl chloride in the presence of potassium
carbonate. Finally, compound 17 was obtained from 1 by
reaction with isonicotinic acid chloride in dichloro-
methane, in the presence of triethylamine which facilitates
the condensation process.
Results
Chemistry
The compounds reported in this paper were prepared
according to Scheme 1. The synthesis of compounds 1
With the aim of studying the influence of the substituents
in positions 6 and 7 of the different pyrazolo[3,4-b]quin-
oxalines, several groups were chosen for these positions.
In addition, monosubstitution as well as disubstitution
were assessed. Thus, the trifluoromethyl group and
chlorine were selected as withdrawing groups whereas
3
0
and 7 has already been reported; however, in this
paper, a new synthetic route for compound 7 is being
described. Compounds 1–6 were formed from the
3
1
corresponding 3-chloroquinoxaline-2-carbonitrile by
ꢀ
Scheme 1. Synthesis of compounds 1–17: (A) hydrazine hydrate, reflux; (B) copper(II) chloride, tert-butylnitrite, acetonitrile, 80 C; (C) N,N-dia-
lkylaminoalkyl chloride or isonicotinic acid chloride, in the presence of potassium carbonate or triethylamine, respectively; (D) N,N-dialkylamino-
alkylamine, potassium carbonate, chloroform.

M. A. Ortega et al. / Bioorg. Med. Chem. 10 (2002) 2177–2184
2179
methyl and methoxy were chosen as electrodonating
substitutions.
None of the compounds showed any activity in the
CDC25 phosphatase assay. However, several compounds
displayed a significant inhibitory effect on CDK1/cyclin B
(Table 1, Fig. 2). The most active compound was further
tested against the brain kinases CDK5/p25 and GSK-3.
As expected from the close relationship between CDK1
and CDK5, compound 1 was a good inhibitor of CDK5
(IC : 0.4 mM). As recently suggested, many CDK inhi-
As is well known, in principle, isomer-position mixtures
of the 3-aminoquinoxaline-2-carbonitrile 1,4-di-N-oxide
derivatives can be formed from the corresponding mono-
3
2
substituted benzofuroxanes and thus, the obtainment of
isomer mixtures of the 3-aminoquinoxaline-2-carboni-
trile, 3-chloroquinoxaline-2-carbonitrile and 1H-pyr-
azolo[3,4-b]quinoxaline derivatives would be expected.
However, in the case of the compound that contains a
methoxy group (5) and those derived from 5(6)-chlor-
obenzofuroxan (6, 8, 10, 13 and 15), one of the isomers
was obtained in a very low proportion and discarded
during the purification processes; according to previous
research carried out in our lab and literature refer-
5
0
bitors are also excellent inhibitors of the glycogen syn-
3
5
thase kinase-3 (GSK-3). This indeed turned out to be
the case for compound 1 (IC : 1 mM) (Table 1, Fig. 2).
5
0
Discussion
We here describe a new class of kinase inhibitors with
some selectivity towards cyclin-dependent kinases and
glycogen synthase kinase-3. The most active compound
acts with an IC50 of 0.6 and 1 mM on CDK1 and GSK-3.
It is clear that the synthesis of more analogues is
required to reach a good structure–activity relationship.
Nevertheless, the first analogues which have been syn-
thesized clearly show that even minor modifications lead
to significant changes in kinase inhibitory activity. It is
also evident that more work is required to reach a good
view on the selectivity of this class of compounds. The
initial results presented here confirm the hypothesis that
3
2
ences, we assume that this corresponds to the 7-chloro
isomer and, therefore, the isomer reported here would
correspond to the 6-chloro. Finally, regarding compounds
2
and 4, we were unable to separate the isomers and, thus,
they were actually evaluated as the corresponding isomer–
position mixture (A and B, in Experimental).
Pharmacology
We then tested the effects of the synthesized pyrazolo
3,4-b]quinoxalines on two purified cell cycle regulators,
[
3
5
the CDK1/cyclin B kinase and the CDC25 phosphatase.
CDK1/cyclin B which regulates the G2/M transition was
purified from M phase starfish oocytes by affinity chro-
most CDK inhibitors are also inhibitors of GSK-3. To
investigate the selectivity of pyrazolo[3,4-b]quinox-
alines, we need to test a large panel of kinases as pre-
viously done with purines, hymenialdisine, indirubins.
Alternatively, and this is the approach we will favor in
the future, we would like to use affinity chromatography
on immobilized pyrazolo[3,4-b]quinoxalines as a strategy
to identify the intracellular targets of this family of
compounds. This approach has been successfully used
CKShs1
matography on p9
2
-sepharose and assayed with
3
histone H1 and [ P]-ATP (15 mM, final concentration)
3
3
as substrates. The CDC25 phosphatase regulates the
phosphorylation of CDK1 and is responsible for its
activation at the G2/M transition. It was expressed as a
recombinant fusion protein in Escherichia coli and
3
4
36
37
assayed with p-nitrophenylphosphate as a substrate.
with purvalanol and flavopiridol.
Table 1. Effect of various compounds on the enzymatic activity of the CDK1/cyclin B kinase and the CDC25 phosphatase, assayed as described in
the Experimental procedure section
Compound
R
1
R
3
R
6
R
7
CDK1/cyclin B IC50 (mM)
CDC25 IC50 (mM)
1
2
3
4
5
6
7
8
9
H
H
H
H
H
NH
NH
NH
NH
NH
NH
Cl
2
2
2
2
2
2
H
CF /H
H
H/CF
3
CH
H/CH
H
H
H
H
H
H
CH
H
H
H
H
0.6
4.5
21
3.5
4.8
4.56
22
15
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3
CH
CH /H
3
3
3
3
OCH
Cl
3
H
H
H
Cl
H
Cl
H
N(CH
N(CH
N(CH
N(CH
N(CH
N(CH
N(CH
N(CH
Cl
(CH
2
)
3
3
)
2
HCl
NH
NH
NH
NH
NH
NH
NH
NH
NH
2
2
2
2
2
2
2
2
2
10
11
12
13
14
15
16
17
(CH
(CH
(CH
(CH
(CH
(CH
(CH
2
2
2
2
)
3
)
3
)
2
)
2
3
3
3
3
)
)
)
)
2
2
2
2
CH
H
Cl
H
Cl
CH
H
3
3
3
2
)
2
)
2
)
2
2
2
2
CH
2
CH
2
CH
3
)
3
)
3
)
2
2
2
CH
H
3
Pyridyl-4-carbonyl

2180
M. A. Ortega et al. / Bioorg. Med. Chem. 10 (2002) 2177–2184
targets to identify novel therapeutic agents. From the
lead compound, more potent analogues can certainly be
generated. The kinase selectivity of pyrazolo[3,4-b]
quinoxalines remains to be investigated.
Experimental
Synthesis
Melting points were determined with a Mettler
FP82+FP80 apparatus (Greifense, Switzerland) and
1
have not been corrected. The H NMR spectra were
recorded on a Bruker AC-200E (Rheinstetten, Germany)
using TMS as the internal standard. The IR spectra were
performed on a Perkin-Elmer 1600 FTIR (Norwalk, CT,
USA) in KBr pellets. Elemental microanalyses were
obtained on a Elemental Analyzer (Carlo Erba 1106,
Milan, Italy) from vacuum-dried samples; results of
analyses indicated by the symbols of the elements or
functions are shown for each compound. The mass
spectra were recorded on a Hewlett-Packard 5988-A
instrument (Palo Alto, CA, USA) at 70 eV.
Silicagel 60 (0.040–0.063 mm) 1.09385.2500 (Merck
KGaA, 64271 Darmstadt, Germany) was used for col-
umn chromatography and Alugram SILG/UV
2
54
(Layer: 0.2 mm) (Macherey-Nagel GmbH & Co. KG.
Postfach 101352. D-52313 Duren, Germany) was used
¨
for Thin Layer Chromatography. HPLC conditions:
Column Nova Pack C18 60 A 4 mm (3.9Â150 mm);
mobile phase: methanol/water 80/20; flux: 1 mL/min.
Chemicals were purchased from E. Merck (Darmstadt,
Germany), Scharlau (F.E.R.O.S.A., Barcelona, Espan
Panreac Quımica S.A. (Montcada i Reixac, Barcelona,
Espana), Signa-Aldrich Quımica, S.A., (Alcobendas,
Madrid, Espana), Acros Organics (Janssen Pharma-
ceuticalaan 3a, 2440 Geel, Belgie) and Lancaster
Bischheim-Strasbourg, France).
˜
a),
´
˜
´
˜
¨
(
General procedure for compounds 2–6
These compounds were synthesized by following the
3
0
procedure used for compound 1. Yields: 20–90%.
3-Amino-6(7)-trifluoromethyl-1H-pyrazolo[3,4-b]quinox-
aline (2). From 3-chloro-6(7)-trifluoromethylquinox-
aline-2-carbonitrile and hydrazine hydrate. Mp 251–
ꢀ
À1
2
54 C. IR (KBr) n 3414, 3326, 1560, 1326, 1124 cm
;
1
H NMR (DMSO-d ) d 6.35 (s, 2H, NH ), 7.90 (d, 1H,
6
2
H (A) J =9.0 Hz), 8.02–8.28 (m, 2H, H (A)+H (B)),
8
12.54 (s, 1H, NH) ppm (A: 7-CF isomer; B: 6-CF iso-
3 3
7
7À8
.35–8.37 (m, 2H, H (B)+H (B)), 8.47 (s, 1H, H (A)),
8
6
5 8 5
Figure 2. Compound 1 inhibits CDK1/cyclin B, CDK5/p25 and GSK-3.
Dose–response curves.
mer). MS: m/z (percentage of relative abundance): 253
Á
+
(M , 100), 211 (15), 197 (23), 171 (47), 144 (8), 75 (20).
Anal. (C H F N ) C, H, N; C: calcd: 47.43; found:
Conclusion
1
0
6
3
5
46.98. H: calcd: 2.38; found: 2.46. N: calcd: 27.66;
found: 27.29. HPLC: R =2.79 min (A+B).
We have established a rapid and efficient protocol for
the synthesis of pyrazolo[3,4-b]quinoxalines. Here, we
show that this class of compounds are inhibitors of
CDK’s and GSK-3, two enzymes involved in important
physiological processes and possibly good screening
t
3-Amino-6,7-dimethyl-1H-pyrazolo[3,4-b]quinoxaline (3).
From 3-chloro-6,7-dimethylquinoxaline-2-carbonitrile and
ꢀ
hydrazine hydrate. Mp 297 C. IR (KBr) n 3324, 1648,

M. A. Ortega et al. / Bioorg. Med. Chem. 10 (2002) 2177–2184
2181
À1
1
1
595, 1559 cm ; H NMR (DMSO-d ) d 2.46 (s, 6H,
20, the compound was separated and recrystallized
from n-hexane/dichloromethane (yellow solid). Yields:
5–20%.
6
2CH ), 6.01 (s, 2H, NH ), 7.76 (s, 1H, H ), 7.88 (s, 1H,
H ), 12.08 (s, 1H, NH) ppm. MS: m/z (percentage of
relative abundance): 213 (M , 100), 198 (13), 170 (9),
43 (3), 130 (4), 116 (5), 103 (6). Anal. (C H N ) C, H,
N. C (%): calcd: 61.95; found: 61.85. H (%): calcd: 5.19;
found: 5.29. N (%): calcd: 32.84; found: 32.67.
3
2
8
5
+
Á
1
3- Chloro -1H - pyrazolo[3,4 -b]quinoxaline (7). From 3-
amino-1H-pyrazolo[3,4-b]quinoxaline, copper(II) chlor-
1
1
11
5
ꢀ
ide and tert-butyl nitrite. Mp 225 C (decomposition).
À1
1
IR (KBr) n 3127, 3039, 1625, 1595 cm ; H NMR
(DMSO-d ) d 7.83–8.00 (m, 2H, H +H ), 8.16 (d, 1H,
3-Amino-6(7)-methyl-1H-pyrazolo[3,4-b]quinoxaline (4).
From 3-chloro-6(7)-methylquinoxaline-2-carbonitrile and
hydrazine hydrate. Mp 276–277 C. IR (KBr) n 3398,
323, 1646, 1560 cm ; H NMR (DMSO-d ) d 2.51 (s,
3H, CH ), 6.09 (s, 2H, NH ), 7.52 (dd, 1H, H (B),
J6 =8.2 Hz, J =1.5 Hz), 7.67 (dd, 1H, H (A),
J7 =8.8 Hz, J =1.5 Hz) 7.77–8.02 (m, 3H, H (A)+
7À5
H (A)+H (B)), 8.04 (d, 1H, H (B) J =8.7 Hz), 12.16
s, 1H, NH) ppm (A: 7-CH isomer; B: 6-CH isomer)
6
6
7
H , J =8.5 Hz), 8.29 (d, 1H, H , J =8.4 Hz), 14.30
8
8À7
5
5À6
(s, 1H, NH) ppm. MS: m/z (percentage of relative
ꢀ
À1
1
+Á
3
abundance): 204 (M , 100), 169 (16), 143 (28), 116 (21),
90 (17). Anal. (C H ClN ) C, H, N. C (%): calcd: 52.84;
6
3
2
6
9
5
4
found: 52.90. H (%): calcd: 2.44; found: 2.40. N (%):
calcd: 27.38; found: 27.59.
À5
À8
8
6À8
7
5
8
5
5À6
(
3,6-Dichloro-1H-pyrazolo[3,4-b]quinoxaline (8). From 3-
amino-6-chloro-1H-pyrazolo[3,4-b]quinoxaline, copper
3
3
+
Á
,
MS: m/z (percentage of relative abundance): 199 (M
00), 170 (2), 156 (13), 143 (5), 116 (9), 895 (6), 77 (3).
Anal. (C H N ) C, H, N. C (%): calcd: 60.20; found:
ꢀ
1
(II) chloride and tert-butyl nitrite. Mp 224–225 C. IR
À1
1
(KBr) n 3140, 1623, 1560, 873 cm ; H NMR (DMSO-
d ) d 7.96 (dd, 1H, H , J =2.7 Hz, J =9.0 Hz), 8.17
(d, 1H, H , J =9.1 Hz), 8.36 (d, 1H, H , J =2.3
5À7
Hz), 14.29 (s, 1H, NH) ppm. MS: m/z (percentage of
relative abundance): 238 (M ,100), 203 (15), 177 (36),
150 (29), 88 (27). Anal. (C H Cl N ) C, H, N. C (%):
1
0
9
5
6
3
0.18. H (%): calcd: 4.55; found: 4.65. N (%): calcd:
5.15; found: 35.24. HPLC: R =2.81 min (A+B).
6
7
7À5
7À8
t
8
8À7
5
+
Á
3-Amino-6-methoxy-1H-pyrazolo[3,4-b]quinoxaline (5).
From 3-chloro-7-methoxyquinoxaline-2-carbonitrile and
9
4
2
4
ꢀ
hydrazine hydrate. Mp 250 C. IR (KBr), n 3408, 1636,
calcd: 45.21; found: 44.79. H (%): calcd: 1.68; found:
1.69. N (%): calcd: 23.43; found: 23.08.
À1
1
1
OCH ), 5.95 (s, 2H, NH ), 7.44–7.52 (m, 2H, H +H ),
602, 1224 cm ; H NMR (DMSO-d ) d 3.93 (s, 3H,
6
3
2
6
8
7
.90 (d, 1H, H , J =9.1 Hz), 12.11 (s, 1H, NH) ppm .
5
5À6
General procedure for compounds 9–16
+
Á
,
MS: m/z (percentage of relative abundance): 215 (M
00), 200 (31), 172 (26), 143 (1), 130 (12), 103 (4). Anal.
C H N O) C, H, N. C (%): calcd: 55.80; found: 55.90.
1
(
A mixture of the 6 and/or 7-substituted 3-amino-1H-
pyrazolo[3,4-b]quinoxaline (2.25 mmol) and the corre-
sponding N,N-dialkylaminoalkylamine (14.50 mmol) in
the presence of potassium carbonate (0.5 g) was stirred
in dry chloroform (30 mL) for 10 days and kept from
light and moisture. The mixture was filtered off, and the
residue obtained was washed with dry chloroform. The
solvent was removed under low pressure and the red
solid obtained was purified by flash chromatography
using a mixture of dichloromethane/methanol as the
mobile phase. Once the solvent was removed, the new
solid was dissolved in methanol and a small amount of
active carbon was added. After filtering the suspension
and removing the solvent, a final red solid was obtained.
Yields: 12–79%.
1
0
9
5
H (%): calcd: 4.21; found: 4.30. N (%): calcd: 32.54;
found: 32.85. HPLC: R =2.51 min.
t
3-Amino-6-chloro-1H-pyrazolo[3,4-b]quinoxaline (6).
From 3,7-dichloroquinoxaline-2-carbonitrile and hydra-
zine hydrate. Mp >280 C. IR (KBr) n 3388, 3322,
ꢀ
À1
1
1
645, 1618, 1560 cm ; H NMR (DMSO-d ) d 6.20 (s,
6
2
H , J =9.2 Hz), 8.16 (s, 1H, H ), 12.36 (s, 1H, NH)
H, NH ), 7.82 (d, 1H, H , J =9.0 Hz), 8.01 (d, 1H,
2
6
6À5
5
5À6
ppm. MS: m/z (percentage of relative abundance): 219
8
+
Á
(
Anal. (C H ClN ) C, H, N. C (%): calcd: 49.21; found:
M
, 100), 190 (2), 164 (7), 150 (9), 136 (8), 110 (4).
9
6
5
4
3
9.02. H (%): calcd: 2.75; found: 2.78. N (%): calcd:
1.88; found: 31.52. HPLC: R =2.63 min.
t
3
-Amino-1-(3-dimethylamino-1-propyl)pyrazolo[3,4-b]
quinoxaline hydrochloride (9). From 3-amino-1H-pyr-
azolo[3,4-b]quinoxaline and 3-dimethylamino-1-propyl-
amine. This compound was prepared as hydrochloride
salt as follows: The red solid obtained from the column
was dissolved in dichloromethane and washed with a
solution of NaOH 2 N (3Â100 mL) and water (3Â100
mL). It was dried with anhydrous sodium sulfate and
the solvent was removed under low pressure. The new
residue was dissolved in ethyl acetate and three drops of
hydrochloric acid 35% were added. The red precipitate
which appeared was isolated by filtration. The solid was
dissolved in methanol and a small amount of active
carbon was added in order to remove the impurities.
After filtering and removing the solvent, a red solid was
General procedure for compounds 7 and 8
The corresponding 3-amino-1H-pyrazolo[3,4-b]quinox-
aline (2.70 mmol) in acetonitrile was added to a stirred
suspension of copper II) chloride (0.62 g; 4.61 mmol) in
ꢀ
acetonitrile under reflux (80–85 C) and under nitrogen
atmosphere. A first fraction of tert-butyl nitrite (0.25
mL) was then added. The same amount was added
every 15 min, up to a total of five times. The mixture
was allowed to cool and filtered off; the solvent was
removed under low pressure. The residue was then
washed with plenty of dichloromethane, and the new
solution was dried by removing the solvent under low
pressure. The dark solid obtained was purified by flash
chromatography using a mixture of dichloromethane/
ethyl acetate as the mobile phase. With the fraction 80/
ꢀ
obtained. Mp 238–240 C. IR (KBr) n 3361, 3303, 2945,
À1
1
2609, 1637 cm ; H NMR (DMSO-d ) d 2.20–2.28 (m,
6

2182
M. A. Ortega et al. / Bioorg. Med. Chem. 10 (2002) 2177–2184
2
H, CH ), 2.69 (s, 6H, 2CH ), 3.10 (t, 2H, CH ), 4.31 (t,
2
110 (0.2), 58 (100). Anal. (C H ClN ) C, H, N. C (%):
13 15 6
3
2
2
Hz), 7.84 (t, 1H, H , J =7.3 Hz), 8.01 (d, 1H, H , J
H, CH ), 6.41 (s, 2H, NH ), 7.68 (t, 1H, H , J =7.0
=
calcd: 53.70; found: 53.65. H (%): calcd: 5.20; found:
5.20. N (%): calcd: 28.90; found: 28.87.
2
2
6
6À7
8
7
7À6
8À7
8
HCl) ppm. MS: m/z (percentage of relative abundance):
.3 Hz), 8.16 (d, 1H, H , J =8.3 Hz), 10.61 (bs, 1H,
5
5À6
3-Amino-1-(2-diethylamino-1-ethyl)pyrazolo[3,4-b]quin-
oxaline (14). From 3-amino-1H-pyrazolo[3,4-b]quinox-
+
Á
2
(
70 (M , 40), 198 (34), 171 (5), 129 (12), 102 (15), 84
ꢀ
.
.
90), 58 (100). Anal. (C H N HCl 0.25H O) C, H, N.
aline and 2-diethylamino-1-ethylamine. Mp 102–104 C.
À1
1
4
18
6
2
1
C (%): calcd: 54.01; found: 54.36. H (%): calcd: 6.31;
found: 6.28. N (%): calcd: 26.99; found: 26.76.
IR (KBr) n 3314, 2973, 1642, 1572, 752 cm ; H NMR
(CDCl ) d 0.94 (t, 6H, 2CH ), 2.59 (c, 4H, 2CH ), 2.96
3
3
2
(
t, 2H, CH ), 4.40 (t, 2H, CH ), 4.68 (s, 2H, NH ), 7.49
2 2 2
3
-Amino-6-chloro-1-(3-dimethylamino-1-propyl)pyrazolo
3,4-b]quinoxaline (10). From 3-amino-6-chloro-1H-pyr-
(t, 1H, H , J =6.6 Hz), 7.64 (t, 1H, H , J =6.9 Hz),
6 7
6À7
7À6
7.93 (d, 1H, H , J =8.4 Hz), 8.02 (d, 1H, H , J
[
azolo[3,4-b]quinoxaline and 3-dimethylamino-1-propyl-
amine. Mp 238–240 C. IR (KBr) n 3366, 1647, 1567,
=
8
8À7
5
5À6
8.4 Hz) ppm. MS: m/z (percentage of relative abundance):
ꢀ
+Á
284 (M , 5), 198 (4), 144 (2), 129 (4), 102 (7), 86 (100),
.
58 (5). Anal. (C H N H O) C, H, N. C (%): calcd:
À1
1
8
81cm ; H NMR (CDCl ) d 2.09 (m, 2H, CH ), 2.22
3
2
15 20
6
2
(
s, 6H, 2CH ), 2.35 (t, 2H, CH ) 4.40 (t, 2H, CH ), 4.75
59.58; found: 59.83. H (%): calcd: 6.66; found: 7.11. N
(%): calcd: 27.79; found: 27.71.
3
2
2
(
Hz), 7.98 (d, 1H, H , J =9.1 Hz), 8.13 (d, 1H, H ,
s, 2H, NH ), 7.68 (dd, 1H, H , J =9.2 Hz, J =2.0
2
7
7À8
7À5
8
8À7
J5 =1.7 Hz) ppm. MS: m/z (percentage of relative
5
3-Amino-6-chloro-1-(2-diethylamino-1-ethyl)pyrazolo[3,4-
b] quinoxaline (15). From 3-amino-6-chloro-1H-pyrazolo
[3,4-b]quinoxaline and 2-diethylamino-1-ethylamine.
À7
abundance): 304 (M , 3), 232 (25), 205 (4), 178 (3), 163
+
Á
(
(
10), 136 (11), 110 (1), 84 (95), 58 (100). Anal.
C H ClN ) C, H, N. C (%): calcd: 55.17; found:
ꢀ
À1
Mp 152–154 C. IR (KBr) n 3319, 1648, 1570, 829 cm
;
1
4
17
6
1
5
2
4.76. H (%): calcd: 5.62; found: 5.65. N (%): calcd:
7.57; found: 27.27.
H NMR (CDCl ) d 1.06 (t, 6H, 2CH ), 2.72 (c, 4H,
3
3
2CH ), 3.08 (t, 2H, CH ), 4.52 (t, 2H, CH ), 4.73 (s, 2H,
NH ), 7.69 (dd, 1H, H , J =9.1 Hz, J =2.2 Hz),
2
2
2
2
7
7À8
7À5
3-Amino-6,7-dimethyl-1-(3-dimethylamino-1-propyl)pyra-
zolo[3,4-b]quinoxaline (11). From 3-amino-6,7-dimethyl-
7.98 (d, 1H, H , J =9.1 Hz), 8.13 (s, 1H, H ) ppm.
8 8À7 5
MS: m/z (percentage of relative abundance): 318 (M
+
Á
,
1
H-pyrazolo[3,4-b]quinoxaline and 3-dimethylamino-1-
ꢀ
1), 232 (4), 205 (2), 178 (2), 163 (3), 136 (4), 86 (100), 58
(10). Anal. (C H ClN ) C, H, N. C (%): calcd: 56.51;
found: 56.23. H (%): calcd: 6.00; found: 6.08. N (%):
calcd: 26.36; found: 25.98.
propylamine. Mp 150–152 C. IR (KBr) n 3329, 1641,
1
5
19
6
À1
1
1
2
2
570, 861 cm ; H NMR (CDCl ) d 2.08 (q, 2H, CH ),
3
2
.16 (s, 6H, 2CH ), 2.34 (t, 2H, CH ), 2.47 (d, 6H,
CH ), 4.39 (t, 2H, CH ), 4.64 (s, 2H, NH ), 7.80 (s, 1H,
3
2
3
2
2
H ), 7.87 (s, 1H, H ) ppm. MS: m/z (percentage of rela-
8
tive abundance): 298 (M , 5), 240 (32), 226 (41), 214
3-Amino-6,7-dimethyl-1-(2-diethylamino-1-ethyl)pyra-
zolo[3,4-b]quinoxaline (16). From 3-amino-6,7-dime-
thyl-1H-pyrazolo[3,4-b]quinoxaline and 2-diethylamino-
5
+
Á
(
(
10), 199 (5), 157 (10), 130 (3), 103 (10), 84 (77), 58
100). Anal. (C H N ) C, H, N. C (%): calcd: 64.40;
ꢀ
1-ethylamine. Mp 136–137 C. IR (KBr) n 3313, 1646,
1
6
22
6
À1
1
found: 63.99. H (%): calcd: 7.43; found: 7.41. N (%):
calcd: 28.16; found: 28.03.
1573, 865 cm ; H NMR (CDCl ) d 0.97 (t, 6H, 2CH ),
3
3
2.46 (s, 3H, CH ), 2.48 (s, 3H, CH ), 2.58 (c, 4H, 2CH ),
2.93 (t, 2H, CH ), 4.41 (t, 2H, CH ), 4.63 (s, 2H, NH ),
3
3
2
2 2 2
3-Amino-1-(2-dimethylamino-1-ethyl)pyrazolo[3,4-b]quin-
oxaline (12). From 3-amino-1H-pyrazolo[3,4-b]quinox-
7.78 (s, 1H, H ), 7.85 (s, 1H, H ) ppm. MS: m/z (per-
8 5
+
Á
centage of relative abundance): 312 (M , 4), 226 (4),
213 (3), 199 (1), 157 (3), 130 (1), 103 (3), 86 (100), 58 (8).
Anal. (C H N ) C, H, N. C (%): calcd: 65.35; found:
aline and 2-dimethylamino-1-ethylamine. Mp 174–
ꢀ
À1
1
1
78 C. IR (KBr) n 3294, 1651, 1572, 756 cm ; H
1
7
24
6
NMR (CDCl ) d 2.25 (s, 6H, 2CH ), 2.79 (t, 2H, CH ),
65.65. H (%): calcd: 7.74; found: 8.11. N (%): calcd:
26.89; found: 26.52.
3
3
2
4
J6 =7.3 Hz), 7.67 (t, 1H, H , J =6.9 Hz), 7.96 (d,
.40 (t, 2H, CH ), 4.67 (s, 2H, NH ), 7.52 (t, 1H, H ,
2 2 6
À7
7
7À6
H, H , J =8.5 Hz), 8.05 (d, 1H, H , J =8.4 Hz)
1
ppm. MS: m/z (percentage of relative abundance): 256
Procedure for 3-amino-1-(pyridyl-4-carbonyl)pyrazolo
[3,4-b]quinoxaline (17). A mixture of 3-amino-1H-pyr-
azolo[3,4-b]quinoxaline (0.2 g; 1.08 mmol), isonicotinic
acid chloride (0.23 g; 1.65 mmol) and triethylamine (0.5
mL) in dichloromethane (20 mL) was kept from light
and moisture, and stirred for 6 h. After filtering and
washing the residue with dichloromethane, a yellow
5
5À6
8
8À7
+
Á
(
(
M
, 2), 198 (2), 144 (1), 129 (2), 102 (6), 76 (1), 58
100). Anal. (C H N ) C, H, N. C (%): calcd: 60.91;
1
3
16
6
found: 61.11. H (%): calcd: 6.29; found: 6.31. N (%):
calcd: 32.72; found: 32.41.
ꢀ
3
-Amino-6-chloro-1-(2-dimethylamino-1-ethyl)pyrazolo
3,4-b]quinoxaline (13). From 3-amino-6-chloro-1H-pyr-
solid was obtained. Mp 246–248 C. IR (KBr) n 3362,
À1
1
[
azolo[3,4-b]quinoxaline and 2-dimethylamino-1-ethyl-
amine. Mp 188–191 C. IR (KBr) n 3291, 1654, 1570,
1699, 1638, 1560 cm ; H NMR (CDCl ) d 7.15 (s, 2H,
3
NH ), 7.79 (d, 2H, 2CH pyridine, J=5.3 Hz), 7.88–8.05
(m, 2H, H +H ), 8.17 (d, 1H, H , J =8.1 Hz), 8.28 (d,
6 7 5
2
ꢀ
5À6
À1
1
1
391, 868 cm
CH ), 2.89 (t, 2H, CH ), 4.47 (t, 2H, CH ), 4.70 (s, 2H,
;
H NMR (CDCl ) d 2.34 (s, 6H,
1H, H , J =8.3 Hz), 8.84 (d, 2H, 2CH pyridine, J=5.5
8
3
8À7
2
NH ), 7.67 (dd, 1H, H , J =9.3 Hz, J =2.1 Hz),
Hz) ppm. MS: m/z (percentage of relative abundance):
Á
290 (M , 84), 261 (2), 106 (100), 78 (40). Anal. (C H
15 10
3
2
2
+
2
7
7À8
.97 (d, 1H, H , J =9.3 Hz), 8.12 (d, 1H, H , J =1.5
7À5
7
Hz) ppm. MS: m/z (percentage of relative abundance):
2
N O) C, H, N. C (%): calcd: 62.06; found: 62.40. H
6
(%): calcd: 3.47; found: 3.55. N (%): calcd: 28.95;
found: 29.39.
8
8À7
5
5À7
+
Á
90 (M , 1), 232 (1), 205 (1), 178 (1), 163 (1), 135 (2),

M. A. Ortega et al. / Bioorg. Med. Chem. 10 (2002) 2177–2184
2183
Kinase assays
CDK5/p25 was reconstituted by mixing equal amounts
of recombinant mammalian CDK5 and p25 expressed
in E. coli as Glutathione-S-transferase (GST) fusion
proteins, and purified by affinity chromatography on
glutathione-agarose (vectors kindly provided by Dr. J.
H. Wang) (p25 is a truncated version of the p35, the 35
kDa CDK5 activator). Its activity was assayed in buffer
C as described for CDK1/cyclin B.
Biochemical reagents. Sodium ortho-vanadate, EGTA,
EDTA, RNAse A, Mops, b-glycerophosphate, phenyl-
phosphate, sodium fluoride, glutathione-agarose, dithio-
threitol (DTT), bovine serum albumin (BSA), nitrophenyl
phosphate, leupeptin, aprotinin, pepstatin, soybean tryp-
sin inhibitor, benzamidine, histone H1 (type III-S) were
3
2
obtained from Sigma Chemicals. [g- P]-ATP (PB 168)
was obtained from Amersham.
GSK-3b was expressed in and purified from insect Sf9
3
8
cells. It was assayed, following a 1/100 dilution in 1
mg BSA/mL10 mM DTT, with 5 mL , 40mM GS-1
peptide as a substrate, in buffer A, in the presence of 15
The GS-1 peptide (YRRAAVPPSPSLSRHSSPHQSpE
DEEE) was synthesized by the Peptide Synthesis Unit,
Institute of Biomolecular Sciences, University of
Southampton, Southampton SO16 7PX, UK.
3
2
mM [g- P] ATP (3,000 Ci/mmol; 1 mCi/mL) in a final
ꢀ
volume of 30 mL. After 30 min incubation at 30 C, 25-
mLaliquots of supernatant were spotted onto P81
phosphocellulose papers and treated as described above.
Buffers. Homogenization buffer. 60 mM b-glycerophos-
phate, 15 mM p-nitrophenylphosphate, 25 mM Mops
(
pH 7.2), 15 mM EGTA, 15 mM MgC l , 1 mM DTT,
2
1
mM sodium vanadate, 1 mM NaF, 1 mM phenyl-
phosphate, 10 mg leupeptin/mL, 10 mg aprotinin/mL,
0 mg soybean trypsin inhibitor/mLand 100 mM benz-
Acknowledgements
1
The authors are thankful to the Departamento de
Industria del Gobierno de Navarra for a grant given to
amidine.
Bele
grants from the ‘Association pour la Recherche sur le
Cancer’ (ARC 5343) (to L.M.) and the ‘Conseil Regio-
´
n Zarranz. This research was also supported by
Buffer A. 10 mM MgCl , 1 mM EGTA, 1 mM DTT,
2
´
2
5 mM Tris–HCl pH 7.5, 50 mg heparin/mL.
nal de Bretagne’ (to L.M.). The Iberoamerican Program
CYTED is also gratefully acknowledged.
Buffer C. Homogenization buffer but 5 mM EGTA,
no NaF and no protease inhibitors.
References and Notes
Tris-buffered saline-Tween-20 (TBST). 50 mM Tris
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