Aloisines, a new family of CDK/GSK-3 inhibitors. SAR study, crystal structure in complex with CDK2, enzyme selectivity, and cellular effects

Article abstract of DOI:10.1021/jm020319p

Cyclin-dependent kinases (CDKs) regulate the cell cycle, apoptosis, neuronal functions, transcription, and exocytosis. The observation of CDK deregulations in various pathological situations suggests that CDK inhibitors may have a therapeutic value. In th

Full text of DOI:10.1021/jm020319p

222
J . Med. Chem. 2003, 46, 222-236
Aloisin es, a New F a m ily of CDK/GSK-3 In h ibitor s. SAR Stu d y, Cr ysta l Str u ctu r e
in Com p lex w ith CDK2, En zym e Selectivity, a n d Cellu la r Effects
Yvette Mettey,† Marie Gompel,‡ Virginie Thomas,‡ Matthieu Garnier,‡ Maryse Leost,‡ Ire`ne Ceballos-Picot,§
Martin Noble, J ane Endicott, J ean-michel Vierfond,† and Laurent Meijer*,‡
Faculte´ de Me´decine et de Pharmacie, 34 rue du J ardin des Plantes, B.P. 199, 86005 Poitiers Cedex, France, Cell Cycle Group,
Station Biologique, C.N.R.S., B.P. 74, 29682 Roscoff Cedex, Bretagne, France, INSERM U 383, Ge´ne´tique, Chromosomes et
Cancer, Hoˆpital Necker, 75015 Paris, France, and Laboratory of Molecular Biophysics, Department of Biochemistry, University
of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom
Received J uly 29, 2002
Cyclin-dependent kinases (CDKs) regulate the cell cycle, apoptosis, neuronal functions,
transcription, and exocytosis. The observation of CDK deregulations in various pathological
situations suggests that CDK inhibitors may have a therapeutic value. In this article, we report
on the identification of 6-phenyl[5H]pyrrolo[2,3-b]pyrazines (aloisines) as a novel potent CDK
inhibitory scaffold. A selectivity study performed on 26 kinases shows that aloisine A is highly
selective for CDK1/cyclin B, CDK2/cyclin A-E, CDK5/p25, and GSK-3R/â; the two latter
enzymes have been implicated in Alzheimer’s disease. Kinetic studies, as well as the resolution
of a CDK2-aloisine cocrystal structure, demonstrate that aloisines act by competitive inhibition
of ATP binding to the catalytic subunit of the kinase. As observed with all inhibitors reported
so far, aloisine interacts with the ATP-binding pocket through two hydrogen bonds with
backbone nitrogen and oxygen atoms of Leu 83. Aloisine inhibits cell proliferation by arresting
cells in both G1 and G2.
Sch em e 1
In tr od u ction
Protein kinases catalyze the phosphorylation of serine,
threonine, and tyrosine residues of proteins, using
adenosine triphosphate (ATP) or guanosine 5′-triphos-
phate (GTP) as the phosphate donor. Protein phospho-
rylation is considered one of the main post-translational
mechanisms used by cells to finely tune their metabolic
and regulatory pathways. Protein kinases (800+ in the
human genome) and their counterparts, protein phos-
phatases, appear to be involved in most human diseases.
This is the reason screening for potent and selective
inhibitors of protein kinases has intensified over the
past few years (reviewed in refs 1-4). In our laboratory,
we have focused our efforts on two families of kinases:
cyclin-dependent kinases (CDKs) and glycogen synthase
kinase-3 (GSK-3). CDKs are involved in controlling the
cell cycle, apoptosis, neuronal functions and neurode-
generation, transcription, and exocytosis (reviewed in
refs 5-11). GSK-3, an essential element of the WNT
signaling pathway, is involved in multiple physiological
processes including cell cycle regulation, in which it
controls the levels of cyclin D1 and â-catenin, dorso-
ventral patterning during development, insulin action
on glycogen synthesis, axonal outgrowth, HIV-1 Tat-
mediated neurotoxicity, and phosphorylation of tau, a
characteristic of Alzheimer’s disease (reviewed in refs
12-17). Potential applications of CDK/GSK-3 inhibitors
are being evaluated against cancers, neurodegenerative
disorders such as Alzheimer’s disease, proliferation of
protozoan parasites, and viral infections (HIV, cytome-
galovirus, and herpes virus) (reviewed in ref 18).
CDK inhibitors include the purines olomoucine,19
roscovitine,20,21 purvalanols,22,23 CVT-313,24 C2-alkyl-
ynated purines,25 H717,26 and NU2058,27 piperidine-
substituted purines,28 toyocamycin,29 flavopiridol,30 in-
dirubins,31,32 paullones,33-35 γ-butyrolactone,36 hymenial-
disine,37 indenopyrazoles,38 the pyrimidines NU602727
and CGP60474,39 pyridopyrimidine,40 the aminopyri-
midine PNU 112455A,41 oxindoles,42-44 PD0183812,45
cinnamaldehydes,46 quinazolines,47,48 fasclaplysin,49
SU9516,50 benzocarbazoles,51 and aminothiazole52 (re-
viewed in refs 18, 53-59). GSK-3 inhibitors include
indirubins,35 paullones,35 maleimides,60,61 and lithium.62
In this paper, we report on the identification of
aloisines, a new family of kinase inhibitors selective for
CDK1/2/5 and GSK-3R/â. These 6-phenyl[5H]pyrrolo-
[2,3-b]pyrazines act in the submicromolar range by
competing with ATP for binding to the kinase active
site, as revealed by a CDK2-aloisine crystal structure
which is presented here. Aloisine binds to the kinase
ATP-binding pocket by two hydrogen bonds to the CDK2
backbone and numerous hydrophobic interactions. A
comparison with the binding of other CDK inhibitors is
provided. A structure/activity relationship study has
been performed with 50 aloisine analogues. Finally, we
show that aloisines display antiproliferative activity.
* To whom correspondence should be addressed. E-mail: meijer@
sb-roscoff.fr. Tel.: 33 (0)2.98.29.23.39. Fax: 33 (0)2.98.29.23.42.
† Faculte´ de Me´decine et de Pharmacie.
‡ C.N.R.S.
Ch em istr y
A series of 6-aryl[5H]pyrrolo[2,3-b]pyrazines were
§ Hoˆpital Necker.
University of Oxford.
prepared by reaction of methylpyrazines with aromatic
10.1021/jm020319p CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/18/2002

Aloisines Inhibit CDKs and GSK-3
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 223
Ta ble 1. Structure, Yields, and Physical Characteristics of 6-Aryl[5H]pyrrolo[2,3-b]pyrazines
compound
R2
R3
Ar
yield (%)
mp (°C)
formula
1 (RP19)
2 (RP6)
3 (RP128)
4 (RP13)
5 (RP7)
6 (RP17)
7 (RP12)
8 (RP18)
9 (RP124)
H
H
H
H
H
H
H
2-furyl
6
8
232.6
C
C
C
C
C
C
C
C
C
10H7N3O
10H7N3S
10H7N3S
11H8N4
H
H
H
H
H
2-thienyl
3-thienyl
2-pyridyl
phenyl
260.3
6
230 dec
233.1
8
37
17
44
26
41
216.0a
216.4
12H9N3
1-naphthyl
phenyl
phenyl
16H11N3
16H11N3
13H11N3
15H12N3Cl
-C4H4-
260 decb
261.8
H
H
CH3
H
1-(4-chlorophenyl)cyclopropyl
189.7
a
b
Lit.64 mp 215-216 °C. Lit.64 mp 233 °C dec.
Ta ble 2. Structure, Yields, and Physical Characteristics of Substituted 6-Phenyl[5H]pyrrolo[2,3-b]pyrazines
compound
R5
R7
X
yield (%)
mp (°C)
formula
10 (RP9)
H
H
2-OCH3
2-OH
3-OCH3
3-OH
4-OCH3
4-OH
3,5-OCH3
3,4,5-OCH3
4-F
4-Cl
3,5-Cl
4-Br
4-CF3
4-CN
4-CH3
2-dioxolyl
4-N(CH3)2
4-OCH3
4-OH
3,4-OCH3
4-Cl
OSO2N(CH3)2
OCH3
OH
4-OCH3
4-OCH3
4-OCH3
4-Cl
4-OCH3
4-OH
4-Cl
4-OCH3
4-OCH3
4-OH
27
22a
55
40a
48
30a
50
50
52
49
20
24
31
6
156.9
250 dec
195.7
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
13H11N3O
12H12N3O2Br
13H11N3O
12H10N3OBr
13H11N3O
12H12N3O2Br
14H13N3O2
15H15N3O3
12H8N3F
12H8N3Cl
12H7N3Cl2
12H8N3Br
13H8N3F3
13H8N4
11 (RP109)
12 (RP10)
13 (RP134)
14 (RP11)
15 (RP26)
16 (RP21)
17 (RP16)
18 (RP76)
19 (RP14)
20 (RP15)
21 (RP77)
22 (RP8)
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
258 dec
256.1b
255 dec
216.7
H
H
H
H
231.7
H
244 dec
250 decc
252 dec
256 dec
238 dec
340 dec
265.4
H
H
H
H
23 (RP20)
24 (RP78)
25 (RP122)
26 (RP129)
27 (RP95)
28 (RP96)
29 (RP123)
30 (RP80)
31 (RP125)
32 (RP127)
33 (RP132)
34 (RP110)
35 (RP126)
36 (RP102)
37 (RP90)
38 (RP106)
39 (RP107)
40 (RP108)
41 (RP111)
42 (RP104)
43 (RP112)
44 (RP92)
45 (RP91)
46 (RP98)
47 (RP99)
48 (RP22)
H
H
49
43
5
13H11N3
15H13N3O2
14H14N4
H
265.6
271 dec
221.6
262 dec
230.2
260 dec
235.1
188.5
244 dec
193.8
H
CH3
CH3
H
47
41a
51
44
72
26
50a
36
10
2
15
32
66a
70
50
30
54a
24
20
1
14H13N3O
13H12N3OBr
15H15N3O2
13H10N3Cl
15H16N4SO3
16H17N3O
15H16N3OBr
16H15N3O
16H16N3OCl
16H17N3O
15H14N3Cl
17H19N3O
16H17N3O
16H16N3Cl
20H25N3O
17H17N3O
16H18N3O2Br
19H15N3
CH3
CH3
(CH2)2CH3
(CH2)2CH3
CH2sCHdCH2
(CH2)2CH2Cl
CH(CH3)2
CH(CH3)2
(CH2)3CH3
(CH2)3CH3
(CH2)3CH3
(CH2)6CH3
CH2-C3H5
CH2-C3H5
CH2-C6H5
CH2-C6H5
CH2-C6H11
CH2-C6H11
H
178 dec
204.8
208.6
183.8
281.4
200.0
132.5
193.9
260 dec
209.8
H
Cl
4-OCH3
4-Cl
H
266.3
19H14N3Cl
20H23N3O
19H20N3Cl
13H11N3
220.3
4
54
203.5
72.3
a
b
Calculated from methoxy compound. Lit.64 mp 238-240 °C. c Lit.64 mp 250 °C dec.
nitriles, using our previously described procedure63
(Scheme 1). Compounds 1-9 were obtained in moderate
or poor yields, together with 1-amino-1-aryl-2-(2-pyrazi-
nyl)ethene. Yields and physical characteristics of the
products 1-9 are recorded in Table 1. From biological
results, we similarly prepared a series of substituted
6-phenyl[5H]pyrrolo[2,3-b]pyrazines from methyl- or
alkylpyrazines and appropriate substituted benzoni-
triles. Alkylpyrazines were obtained by reaction of
pyrazinylmethyllithium with bromoalkanes, and ben-
zonitriles were commercially available products. Dem-
ethylation of methoxy compounds was achieved by
refluxing in 47% hydrobromidic acid at 126 °C. The time
required for demethylation varied from 3 to 20 h. The
yields were moderate. These results are presented in
Table 2.
We also prepared 6-[1-(4-chlorophenyl)1-cyclopropyl]-
7-methyl[5H]pyrrolo[2,3-b]pyrazine (49, RP 130) and
2-(4-methoxyphenyl)[1H]pyrrolo[2,3-b]pyridine64 (50, RP
97) from 2-picoline and p-methoxybenzonitrile (Table 3).

224 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Mettey et al.
F igu r e 1. Aloisine A and B inhibit CDK1/cyclin B, CDK5/p25, and GSK-3â. Kinases were assayed as described in the Experimental
Section in the presence of increasing concentrations of aloisines A and B. Activity is presented as percentage of maximal activity,
i.e., measured in the absence of inhibitors.
Ta ble 3.
the backbone oxygen of Glu81, but instead it accepts
and donates a hydrogen bond respectively from the
backbone nitrogen and oxygen atoms of Leu83 (Figure
4).65,66 This hydrogen-bonding pattern has previously
been observed in the structures of monomeric CDK2 in
complex with olomoucine,66 roscovitine,21 purvalanol
B,22 OL567,25 and H717.26 The CDK2 ATP-binding site
is tolerant of a number of positions for the planar
heterocyclic ring systems which are a characteristic of
the CDK inhibitors identified to date (reviewed in ref
54). The position of the aloisine B fused ring system
within the CDK2 ATP-binding site most closely re-
sembles that of indirubin-5-sulfonate31 and oxindole-3.43
However, being smaller than indirubin-5-sulfonate and
in a different orientation to oxindole-3, aloisine B does
not fill the back of the ATP-binding cleft and form an
equivalent edge-to-ring stacking interaction with the
side chain of Phe80 (Figure 5).
In the apo-CDK2 structure,65 and in other monomeric
CDK2-inhibitor complex structures,21,22,27,31,37,66-68 two
regions of CDK2 have been reported to be flexible. These
regions have been excluded from some CDK2-inhibitor
models and constitute the loop connecting strand â3 to
helix RC, and the “activation segment” containing
Thr160, which is phosphorylated in the fully active
CDK2-cyclin A complex.69 The CDK2-aloisine B com-
plex also is flexible in the first of these regions.
Interpretable electron density confirms that the confor-
mation of the activation segment resembles that seen
in the monomeric CDK2-ATP complex, although this
electron density is rather weak (<0.15 e- Å-3) for
residues 155-162. Thus, aloisine B binding to CDK2
does not significantly decrease the temperature factors
of this segment, as has been reported for certain
compounds in a series of guanine-based inhibitors.27
This might be because the aloisine B isopropyl group
that binds in the ATP ribose site is much smaller than
the substituents of the O6-substituted guanine series
and does not contact CDK2 in this region (Figures 5A
and 6A).
compound
yield (%)
mp (°C)
formula
49 (RP130)
50 (RP97)
41
20
223.9
C
C
16H14N3Cl
14H12N2O
206.2a
a
Lit.64 mp 205-206 °C.
Resu lts a n d Discu ssion
Aloisin es, a F a m ily of CDK1/2 a n d GSK-3 In h ibi-
tor s. Two decades ago, we proposed an original method
for the one-pot synthesis of [5H]pyrrolo[2,3-b]pyra-
zines.63 While screening for new protein kinase inhibi-
tors, we discovered that some of these diazaindoles were
potent inhibitors of CDKs and GSK-3â (Figure 1; Tables
4 and 5). In the presence of 15 µM ATP, the compounds
were found to inhibit CDKs and GSK-3 in the submi-
cromolar range (Figure 1). Given the key function of
both CDK5 and GSK-3 in the hyperphosphorylation of
tau in Alzheimer’s disease, we named this family of
compounds “aloisines”, following the first name of Dr.
Alzheimer, Alois. Aloisine represents the unsubstituted
6-phenyl[5H]pyrrolo[2,3-b]pyrazine scaffold. Two aloi-
sines, aloisine A (39, RP107) and aloisine B (37, RP90),
were selected for further studies to investigate their
molecular mechanism of action, cocrystallization with
CDK2, selectivity, and cellular effects (see below).
Aloisin e Is a Com p etitive In h ibitor of ATP Bin d -
in g. To investigate the mechanism of aloisine’s action,
kinetic experiments were performed by varying both
ATP levels and aloisine A concentrations (Figure 2).
These experiments were performed with CDK1/cyclin
B, CDK5/p25, and GSK-3â. Double-reciprocal plotting
of the data demonstrates that aloisine A acts as a
competitive inhibitor for ATP. These results are in
complete agreement with the localization of aloisine B
within the ATP-binding pocket of CDK2 (see below).
Aloisin e-CDK2 Cocr yst a l St r u ct u r e, Over a ll
Con for m a tion of P r otein a n d Liga n d , a n d Com -
p a r ison w ith Oth er CDK2-In h ibitor Str u ctu r es.
Aloisine B was chosen for soaking into monomeric CDK2
crystals (Table 6). Aloisine B occupies the CDK2 ATP-
binding site and makes two hydrogen bonds to the
CDK2 backbone within the hinge sequence that links
the two lobes of the kinase (Figure 3). Unlike the
natural ligand, ATP, aloisine B does not interact with
The aloisine B 4-chlorophenyl group points out of the
ATP-binding site cleft toward the surface of the CDK2
C-terminal domain (Figure 3). A superposition of the
structures of the CDK2-aloisine B and CDK2-oxin-

Aloisines Inhibit CDKs and GSK-3
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 225
Ta ble 4. Structure/Activity Relationship of Aloisinesa
substituent
IC50 (µM)
compound
a
b
c
d
7
CDK1/cyclin B
CDK5/p25
GSK-3
5 (RP7)
5.00
20.00
2.50
13.00
2.50
2.00
1.20
4.00
23.00
3.00
2.30
3.30
6.50
3.20
10 (RP9)
OCH3
OH
11 (RP109)
12 (RP10)
OCH3
OH
10.00
13 (RP134)
14 (RP11)
OCH3
OH
4.00
1.00
>100.00
>100.00
1.00
1.10
1.20
60.00
85.00
1.90
15 (RP26)
16 (RP21)
OCH3
OCH3
OCH3
OCH3
100.00
100.00
2.30
17 (RP16)
OCH3
F
Cl
18 (RP76)
19 (RP14)
1.80
20 (RP15)
Cl
Cl
>100.00
>100.00
>100.00
>100.00
10.00
>100.00
6.00
7.20
2.60
4.80
12.00
0.46
2.00
0.40
2.50
0.50
0.60
0.92
>10.00
1.10
6.80
0.50
1.70
0.75
5.90
6.80
1.00
8.00
0.52
1.80
0.65
3.00
21 (RP77)
Br
4.00
22 (RP8)
CF3
6.00
24 (RP78)
CH3
3.00
23 (RP20)
CN
3.00
13.00
26 (RP129)
27 (RP95)
N(CH3)2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
20.00
0.30
>100.00
CH3
CH3
0.80
29 (RP123)
32 (RP127)
35 (RP126)
36 (RP102)
34 (RP110)
38 (RP106)
41 (RP111)
42 (RP104)
46 (RP98)
OCH3
1.10
1.00
(CH2)2CH3
(CH2)3Cl
CH(CH3)2
CH2sCHdCH2
(CH2)3CH3
(CH2)6CH3
CH2C3H5
CH2C6H11
CH3
0.40
0.50
1.30
3.00
1.00
2.00
1.00
2.00
0.70
1.50
7.00
>100.00
>100.00
>100.00
0.90
1.00
5.00
31 (RP125)
30 (RP80)
OsSO2sN (CH3)2
0.70
Cl
Cl
Cl
Cl
CH3
0.40
5.00
13.00
>100.00
>100.00
>100.00
>100.00
0.20
1.20
0.20
>100.00
37 (RP90) (aloisine B)
40 (RP108)
45 (RP91)
CH(CH3)2
(CH2)3CH3
CH2C6H5
CH2C6H5
CH2C6H11
CH3
0.85
0.20
40.00
2.00
44 (RP92)
47 (RP99)
Cl
10.00
0.25
25.00
0.15
50.00
28 (RP96)
OH
OH
OH
OH
33 (RP132)
39 (RP107) (aloisine A)
43 (RP112)
(CH2)2CH3
(CH2)3CH3
CH2C3H5
a
Enzyme activities were assayed as described in the Experimental Section, in the presence of increasing concentrations of aloisines.
IC50’s were calculated from the dose-response curves.
dole-3 complexes (Figure 6B) shows that the positions
of the aloisine B 4-chlorophenyl and the oxindole-3
benzylsulfonamide groups are overlaid so that the
aloisine B chlorine atom occupies the same position as
the sulfur atom of the oxindole-3 sulfonamide group.
However, the chlorine atom cannot mimic the sulfona-
mide interactions with the backbone nitrogen and the
side chain of Asp86 through one of its oxygens and its
amine group, respectively. The purvalanol B 3-chloro-
4-carboxyanilino group also probes this region of CDK2.22
In this case, the chlorine atom has a polar interaction
with the side chain of Asp86, and the carboxyl group is
in van der Waals contact with the side chain of Lys89.
As the anilino group of purvalanol B is not constrained
to be coplanar with its purine ring, it adopts a confor-
mation rather different from that of aloisine B and
oxindole-3. This conformation allows it to exploit the
potential stacking interaction of its aromatic ring with
the planar peptide bond between His84 and Gln85.
Because it is conjugated with its heterocyclic ring
system, the phenyl ring of aloisine B cannot adopt this
conformation.
as a few related compounds. The IC50 values of all
compounds against CDK1/cyclin B, CDK5/p25, and
GSK-3â were determined from dose-response curves
and are presented in Tables 4 and 5. This modest
structure/activity study constitutes only the first steps
of a larger study that should be continued on the basis
of the data given by the aloisine B-CDK2 cocrystal
structure and its comparison with other inhibitor-
CDK2 structures. Nevertheless, these preliminary data
provide some key SAR features.
First, as the three nitrogen atoms of the phenylpyr-
rolo[2,3-b]pyrazine skeleton are engaged in hydrogen
bonds with the Leu83 and Lys33 residues of CDK2 (see
above, and Figures 4 and 5), substitutions on these three
positions (i.e., 1, 4, and 5) are to be excluded. Further-
more, replacement of any of the nitrogen atoms in this
skeleton by a carbon abolishes the inhibitory activity
(Tables 4 and 5). Replacement of the nitrogen atom in
position 1 by a carbon (50), or substitution of the
nitrogen atom in position 5 by a methyl group (48),
results in a dramatic decrease in the inhibitory activity.
Thus, substitutions on other positions (a-e, 7) were
undertaken to orientate the synthesis of derivatives
toward improved CDK and GSK-3 inhibitory activity.
Aloisin es: Str u ctu r e/Activity Rela tion sh ip . A
variety of aloisine derivatives were synthesized, as well

226 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Mettey et al.
Ta ble 5. Structure/Activity Relationship of Aloisine-Related Compoundsa
a
Enzyme activities were assayed as described in the Experimental Section, in the presence of increasing concentrations of aloisines.
IC50’s were calculated from the dose-response curves and are presented in micromolar.
We first focused on position c, without any other
additional substitution. If we consider the aloisine
B-CDK2 cocrystal structure and admit that the inhibi-
tor has the same position in the ATP-binding pocket,
regardless of what moiety is in position c, none of the
substituents tested in this position is within a distance
of 4 Å from the side-chain CO2H or backbone NH of
Asp86. This distance is too large to allow the formation
of hydrogen bonds. It would be interesting to test larger
substituents which could get close enough to these
functions to provide hydrogen bonds. Indeed, from the
comparison with the oxindole-CDK2 complex structure,
in which the SO2NH2 group sits very close to that of
the aloisine B chlorine, one can imagine some interest-
ing substitutions at position a. The SO2NH2 moiety
makes two hydrogen bonds with CDK2: one of the O
atoms accepts a hydrogen bond from the backbone NH
of Asp86, and the NH2 group donates to the side-chain

Aloisines Inhibit CDKs and GSK-3
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 227
F igu r e 2. Aloisine A acts by competing with ATP. Double reciprocal plots of kinetic data from assays of CDK1/cyclin B (A),
CDK5/p25 (B), and GSK-3â (C). Kinase activities at different concentrations of aloisine A (indicated in micromolar). Enzyme
activities were assayed as described in the Experimental Section. ATP concentrations in the reaction mixture varied from 0.1 to
0.25 mM (CDK1 and CDK5) or from 0.015 to 0.15 mM (GSK-3â). The concentrations of histone H1 (A, B) and GS-1 (C) were kept
constant at 0.7 mg/mL and 6.7 µM, respectively.
Ta ble 6. CDK2-Aloisine B Cocrystal Structure: Statistics of
the Dataset Used and of the Refined Structure
cell dimensions (Å)
maximal resolution (Å)
observations
53.0, 71.3, 71.9
1.9
79 169
21 476 (97.9)
0.081
7.6
unique reflections (completeness, %)
a
Rmerge
mean I/σ(I)
highest resolution bin (Å)
completeness (%)
mean I/mean σ(I)
Rmerge
2.00-1.90
97.9
2.1
0.36
protein atoms
residues
other atoms
2340
1-36, 44-298
188 water; 19 aloisine B
72.55-1.90
0.17
resolution range (Å)
b
Rconv
c
Rfree
0.23
mean protein temperature factors (Å)b 23.8
mean ligand temperature factors (Å)b 34.8
a
Rmerge ) [∑h∑jIh,j - hIh]/[∑h∑jIh,j], where Ih,j is the intensity of
b
the jth observation of unique reflection h. Rconv ) [∑h||Foh| -
|Fch||]/[∑h|Foh|], where Foh and Fch are the observed and calculated
c
structure factor amplitudes for reflection h. Rfree is equivalent
to Rconv but is calculated using a 5% disjoint set of reflections
excluded from the least-squares refinement stages.
CO2H of Asp86. Thus, groups such as CONH2, CO2H,
or SO3H, with both donating and accepting capability,
might interact potently with the Asp86 residue. Among
the moieties tested so far, the OH group turned out to
be the best (followed by OCH3 and Cl, respectively).
Much of the work was then dedicated to substituting
position 7 with either -OH, -OCH3, or -Cl in position
c. From this study, two major facts emerged. First, the
introduction of bulky groups in position 7 seems to be
detrimental for the CDK and GSK-3 inhibitory activity.
Second, small aliphatic saturated alkyl chains [(CH2)n,
n ) 1-4] give the best results among the groups tested.
Moreover, the aloisine-related molecules did not show
good results, except for the thienyl-pyrrolo[2,3-b]pyra-
zine compounds, which approximately equaled their
aloisine homologues’ performances on CDK1, CDK5,
and GSK-3 (Table 5).
F igu r e 3. Binding of aloisine B to CDK2. CDK2 is drawn in
ribbon representation and color-ramped from blue to red,
starting at the N-terminus. The N-terminal lobe is dominated
by a five-stranded antiparallel â-sheet, and the C-terminal lobe
is predominantly R-helical. Aloisine B is drawn in ball-and-
stick mode bound at the ATP-binding site, which lies in the
cleft between the two domains. Aloisine B carbon atoms are
colored cyan, nitrogen atoms blue, and the chlorine atom
yellow.
tone H1, casein, myelin basic protein, and peptides),
with 15 µM ATP and in the presence of increasing
concentrations of aloisine A. IC50 values were estimated
from the dose-response curves and are presented in
Table 7. Most kinases tested were inhibited poorly or
not at all (IC50 > 10 µM). However, two families of
kinases, GSK-3R/â and CDKs, were strongly sensitive
to aloisine A (IC50’s of 0.65 and 0.15 µM, respectively)
(Figure 1; Table 7). Among the CDKs, CDK1, CDK2,
Aloisin es: Kin a se Selectivity. Aloisine A, the most
active aloisine so far, was tested for selectivity on 26
highly purified kinases (Table 7). Kinase activities were
assayed with appropriate substrates (for example, his-

228 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Mettey et al.
F igu r e 4. Stereoview showing the interactions between aloisine B and the CDK2 ATP-binding site. Residues that lie within 4
Å of the bound aloisine B molecule are drawn in ball-and-stick mode. Aloisine B carbon atoms are drawn in cyan and those of
CDK2 in green. Oxygen atoms are colored red, nitrogen atoms blue, and the chlorine atom yellow. Dotted lines represent hydrogen
bonds (d(OfN) or d(N-O) < 3.4 Å) between aloisine B and the backbone nitrogen and oxygen atoms of Leu83. The figure also
includes (2Fo - Fc)Rcalc electron density for aloisine B, calculated at the end of refinement using map coefficients output from
REFMAC with resolution between 20 and 1.9 Å. The map is contoured at a level of 0.19 e- Å-3, corresponding to 1.0 times the
rms deviation of the map from its mean value.
F igu r e 5. Molecular surface of the CDK2 ATP-binding site in the CDK2-aloisine B complex. The Connelly molecular surface
was calculated within the program Aesop using a probe radius of 1.4 Å and is shown in gold. Secondary structural elements are
colored blue. Atoms of the CDK2 structure seen through the surface representation are rendered in ball-and-stick mode and
colored green, and carbon atoms of aloisine B are drawn in cyan. (A) View looking into the active site with the N-terminal domain
above. Aloisine B binds into the adenine-binding pocket but does not contact residues in the ribose-phosphate binding site. (B)
View looking down onto the surface of the CDK2 C-terminal domain in the ATP-binding pocket from the N-terminal domain. The
hydrogen bonds between the backbone oxygen and nitrogen atoms of Leu83 and aloisine B are drawn as dotted lines. There is a
third hydrogen bond between aloisine B and the ꢀ-amino group of Lys33.
and CDK5, but not CDK4, were inhibited by aloisine
A. This is reminiscent of other CDK inhibitors, such as
purines,22,70 hymenialdisine,37 paullones,33,34 and in-
dirubins,31 which inhibit CDK1/2/5 but have much less
or no effect on CDK4/6. Although aloisines appear to
be remarkably specific to CDKs and GSK-3, the actual

Aloisines Inhibit CDKs and GSK-3
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 229
F igu r e 6. Superposition of aloisine B and ATP (A) and aloisine B and oxindole-3 (B) bound to CDK2. The CDK2-aloisine B
complex is shown in each case with CDK2 carbon atoms in green, aloisine B carbon atoms in cyan, and ATP (A) or oxindole-3 (B)
carbon atoms colored magenta, respectively. The structures were superimposed on the basis of all protein atoms. In each figure,
the fold of CDK2 is rendered in ribbon mode, and the residues identified in Figure 3 are drawn in ball-and-stick representation.
Ta ble 7. Kinase Inhibition Selectivity of Aloisine Aa
Cellu la r Effects of Aloisin es. The cellular effects
of aloisine A were evaluated on undifferentiated human
teratocarcinoma cells (NT2) and differentiated postmi-
totic neurons (hNT). First, NT2 cells were treated or
not with 10 µM aloisine A and counted at different
intervals over time. Results show that aloisine A
completely blocks the proliferation of dividing cells, as
the number of aloisine A-treated NT2 cells remains
essentially constant, while control cells, exposed to
vehicle (0.1% DMSO), double every 24 h (Figure 7A).
The time course also shows that very few cells actually
die in the presence of aloisine A. This lack of toxicity is
further supported by the reversibility of aloisine A’s
effect (Figure 7B). The proliferation inhibitory activity
of aloisine A was dose-dependent, with an IC50 of 7 µM
(Figure 7C). A slightly higher IC50 value (10.5 µM) was
found for hNT viability (Figure 7D).
The effect of aloisine A on the cell cycle distribution
was investigated for NT2 cells by flow cytometry.
Unsynchronized cells (Figure 7E) were exposed to 20
µM aloisine A for 40 h (Figure 7F). The proliferation
arrest induced by aloisine A in exponentially growing
cells was clearly accompanied by an accumulation of the
G2/M phase. No signs of apoptosis were detectable,
confirming the lack of apparent toxicity observed before.
We next investigated the effects of aloisine A on NT2
cells synchronized either in G0/G1 by serum deprivation
or in G2/M by nocodazole treatment (Figure 8). Serum
deprivation for 24 h led to a significant increase of cells
in G0/G1 (Figure 8A). Cells were then re-exposed to a
a
Enzyme activities were assayed as described in the Experi-
serum-enriched media for 40 h in the absence (Figure
8B) or presence (Figure 8C) of 20 µM aloisine A. Aloisine
A-treated cells remained essentially in G0/G1, with a
small additional accumulation of G2/M cells, most
probably derived from the initial S phase subpopulation
mental Section, in the presence of increasing concentrations of
aloisine A. IC50’s were calculated from the dose-response curves
and are presented in micromolar.
spectrum of their intracellular targets remains to be
identified. For this purpose, we are currently designing
an immobilized aloisine matrix to purify aloisine-
binding proteins by the affinity chromatography method
described for purines71 and paullones.72
(Figure 8C). Control cells redistributed in a classical cell
cycle pattern (Figure 8B). Nocodazole treatment for 24
h led to a massive accumulation of cells in G2/M (Figure
8D). Cells were then washed to remove nocodazole and
incubated for 40 h in the absence (Figure 8E) or

230 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Mettey et al.
F igu r e 7. Reversible inhibition of exponential cell growth by aloisine A. (A) Exponentially growing NT2 cells were treated either
with 10 µM aloisine A ([) or 0.1% DMSO (]). Cells were harvested by trypsinization at the indicated times and counted. (B) Cell
growth arrest induced by aloisine A is reversible. Exponentially growing NT2 cells were treated with 10 µM aloisine A for 24 h
(arrow). They were then washed with PBS and maintained in the presence of either 10 µM aloisine A ([) or 0.1% DMSO (]).
Cells were harvested by trypsinization at the indicated times and counted. (C) Dose-dependent antiproliferative effects of aloisine
A. NT2 cells were exposed to several concentrations of aloisine A for 40 h. Thereafter, cells were incubated with MTT, and the
antiproliferative effect of aloisine A was evaluated through the measurement of the inhibition of cellular reduction of MTT to
MTT formazan, a reflection of the density of living cells. Cell viability is expressed as percentage of the 0.1% DMSO-treated
control. The values are given as the mean ( SD (n ) 8). (D) Antiproliferative effect of aloisine A on hNT cells. On day 5 after the
second replate, hNT cells were exposed to several concentrations of aloisine A for 40 h. Thereafter, cells were incubated with
MTT, and the toxic effect of aloisine A was evaluated as described above (n ) 4). (E) Cell cycle distribution of unsynchronized
NT2 cells treated for 40 h with 0.1% DMSO. The same profile was observed for untreated cells. (F) Cell cycle distribution of
unsynchronized NT2 cells treated for 40 h with 20 µM aloisine, leading to an accumulation of G2/M arrested cells.
presence (Figure 8F) of 20 µM aloisine A. Forty hours
after nocodazole withdrawal, control cells redistributed
in the various cell cycle phases (Figure 8E). In contrast,
the majority of cells exposed to aloisine A after nocoda-
zole treatment remained in G2/M (Figure 8F), preclud-
ing the increase in G1/G0 seen in control cells. A small
sub-G2 peak may indicate a minor onset of apoptotic
cell death.
Con clu sion
In conclusion, we have identified 6-phenyl[5H]pyrrolo-
[2,3-b]pyrazines (aloisines) as a new scaffold structure
for protein kinase inhibition, active at submicromolar
concentrations on cyclin-dependent kinases 1, 2, and 5.
The excellent selectivity of these compounds for CDKs
and for glycogen synthase kinase-3, two families of
therapeutically relevant targets, is encouraging. The
cocrystal structure of aloisine with CDK2 and the early
structure/activity relationship studies provide some
interesting clues to improve the potency of aloisines.
Altogether, these data indicate that aloisine A has
antiproliferative properties and that it is able to block
both the exit from G0/G1 and the exit from G2/M,
suggesting the existence of several intracellular targets.
A G1 arrest correlates with aloisine A’s high potency
against CDK2/cyclin E. The inability to enter S phase
might also result from inhibition of GSK-3, a kinase
known to be involved in cyclin D1 degradation.73 The
G2/M arrest correlates well with the potency of aloisine
A against CDK1/cyclin B.
Exp er im en ta l Section
Abbr evia tion s. BSA, bovine serum albumin; CDK, cyclin-
dependent kinase; DTT, dithiothreitol; GSK-3, glycogen syn-
thase kinase-3; MTT, (4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide; PBS, phosphate-buffered saline; TBS,
Tris-buffered saline.

Aloisines Inhibit CDKs and GSK-3
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 231
F igu r e 8. Comparison of the effects of aloisine A on G0/G1 (A-C) and G2/M (D-F) synchronized cells. The cell cycle phase
distribution was analyzed by flow cytometry following propidium iodide staining. (A-C) NT2 cells were synchronized by serum
deprivation for 24 h (A) and then cultured for 40 h in fresh medium without (B) or with 20 µM aloisine A (C). (D-F). NT2 cells
were synchronized by nocodazole treatment (0.2 µg/mL) for 24 h (D) and then cultured for 40 h in fresh medium without (E) or
with 20 µM aloisine A (F).
Ch em istr y. Melting points were measured in open capillary
tubes on an Electrothermal 9200 apparatus and are uncor-
rected. IR spectra were taken in KBr on an ATI Mattson
Genesis series FTIR. 1H NMR spectra were recorded on a
Varian EM 360 A spectrometer (60 MHz), and chemical shifts
(ppm) are reported relative to tetramethylsilane. Signals are
designated as follows: bs, broad singlet; s, singlet; d, doublet;
dd, doublet of doublets; t, triplet; m, multiplet. Mass spectra
were determined on an LKB 209 (EI at 70 eV). Elemental
microanalyses are indicated by the symbol of the elements,
and the results were within (0.4% of the theoretical values
unless otherwise stated; they were performed on a Perkin-
Elmer 240 apparatus.
was added, and the solution was stirred for 30 min at -40 °C
and further (1-20 h) at 20 °C, and then hydrolyzed with a
10% aqueous solution of NH4Cl. The organic layer was dried
over Na2SO4 and concentrated under vacuum. The crude
product was chromatographed on silica gel and eluted with
methylene chloride and then ethyl acetate. If necessary, the
product was crystallized from ethanol or a methylene chloride-
ethanol mixture.
6-(2-F u r yl)[5H]p yr r olo[2,3-b]p yr a zin e (1, RP 19): mp
232.6 °C; IR 3157, 3143, 3102 cm-1; 1H NMR (60 MHz, DMSO-
d6) δ 6.50-6.70 (m, 1H), 6.80 (s, 1H), 7.05 (d, 1H, J ) 3 Hz),
7.80 (bs, 1H), 8.20 and 8.35 (2d, 1H each, J ) 3 Hz), 12.45 (bs,
1H). Anal. (C10H7N3O) C, H, N.
All experiments involving butyllithium or sodium hydride
were carried out in dried apparatus under an atmosphere of
dry, oxygen-free nitrogen. Tetrahydrofuran (THF) was distilled
from benzophenone-sodium. Diisopropylamine and methyl
heterocycles were distilled and stored over barium oxide.
Butyllithium (1.6 M solution in hexane) was supplied by Acros
and was assayed by titration against diphenylacetic acid.
Alkyl- and aralkylpyrazines were prepared according to usual
procedures. Grace silica gel 60 A, 20-45 µm, was employed
for column chromatography. 2-Phenylindole was purchased
from Aldrich and used as received.
Gen er a l Meth od for th e Syn th esis of Aloisin es. Diiso-
propylamine (2.23 g; 0.022 mol) in THF (50 mL) was cooled to
0 °C, and butyllithium (0.022 mol) was added dropwise. After
being stirred for 30 min at 0 °C, the solution was cooled to
-40 °C before addition of the heterocycle (0.02 mol) in THF
(20 mL). After 30 min, the nitrile (0.01 mol) in THF (20 mL)
6-(2-Th ien yl)[5H]p yr r olo[2,3-b]p yr a zin e (2, RP 6): mp
260.3 °C; IR 3208, 3150, 3068 cm-1; 1H NMR (60 MHz, DMSO-
d6) δ 6.95 (bs, 1H), 7.15-7.45 (m, 1H), 7.75 (s, 1H), 7.85 (bd,
1H), 8.25 and 8.45 (2d, 1H each, J ) 2.8 Hz), 12.85 (bs, 1H).
Anal. (C10H7N3S) C, H, N.
6-(3-Th ien yl)[5H ]]p yr r olo[2,3-b]p yr a zin e (3, R P 128):
1
mp 230 °C dec; IR 3095, 3050, 3000 cm-1; H NMR (60 MHz,
DMSO-d6) δ 6.95 (s,1H), 7.60-7.75 (m, 2H), 8.10-8.20 (m, 2H),
8.30 (d, 1H, J ) 2.6 Hz), 12.30 (bs, 1H). Anal. (C10H7N3S) C,
H, N.
6-(2-P yr id yl)[5H]p yr r olo[2,3-b]p yr a zin e (4, RP 13): mp
233.1 °C; IR 3100, 3059 cm-1; 1H NMR (60 MHz, DMSO-d6) δ
7.30-7.60 (m, 2H), 7.95-8.20 (m, 2H), 8.30 and 8.45 (2d, 1H
each, J ) 2.8 Hz), 8.75 (d, 1H, J ) 5 Hz), 12.65 (bs, 1H). Anal.
(C11H8N4) C, H, N.
6-P h en yl[5H]p yr r olo[2,3-b]p yr a zin e (5, RP 7): mp 216
1
°C (lit.63 mp 215-216 °C); IR 3135, 3050 cm-1; H NMR (60

232 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Mettey et al.
MHz, DMSO-d6) δ 7.00 (s, 1H), 7.55 (m, 3H), 7.90 (m, 2H),
8.25 and 8.50 (2d, 1H each, J ) 3 Hz), 11.90 (bs, 1H); MS m/ e
209 (M+, 100). Anal. (C12H9N3) C, H, N.
DMSO-d6) δ 7.45 (s, 1H), 7.90-8.65 (m, 6H), 12.85 (bs, 1H).
Anal. (C13H8N4) C, H, N.
6-(4-Methylphenyl)[5H]pyrrolo[2,3-b]pyrazine (24,RP 78):
mp 265.4 °C; IR 3150, 3120, 2940, 2920 cm-1 1H NMR (60
;
6-(1-Na p h t h yl)[5H ]p yr r olo[2,3-b]p yr a zin e (6, R P 17):
mp 216.4 °C; IR 3214, 3110, 3048 cm-1 1H NMR (60 MHz,
;
MHz, DMSO-d6) δ 2.40 (s, 3H), 7.05 (s, 1H), 7.30 and 7.90 (2d,
2H each, J ) 8 Hz), 8.15 and 8.30 (2d, 1H each, J ) 2.4 Hz),
12.45 (bs, 1H). Anal. (C13H11N3) C, H, N.
DMSO-d6) δ 6.95 (s, 1H), 7.50-7.85 (m, 4H), 7.95-8.40 (m,
4H), 8.50 (d, 1H, J ) 2.5 Hz), 12.05 (bs, 1H). Anal. (C16H11N3)
C, H, N.
6-[4-(2-Dioxolyl)-ph en yl][5H]pyr r olo[2,3-b]pyr azin e (25,
RP 122): mp 265.6 °C; IR 3120, 2980, 2889 cm-1; H NMR (60
MHz, DMSO-d6) δ 3.95 (s, 4H), 5.75 (s, 1H), 7.15 (s, 1H), 7.50
and 8.05, (2d, 2H each, J ) 7 Hz), 8.20 and 8.35 (2d, 1H each,
J ) 3 Hz), 12.45 (bs, 1H). Anal. (C15H13N3O2) C, H, N.
6-(4-Dim et h yla m in op h en yl)[5H ]p yr r olo[2,3-b]p yr a -
zin e (26, RP 129): mp 271 °C dec; IR 3211, 3157, 2900, 2818
6-P h en yl[5H]p yr r olo[2,3-b]qu in oxa lin e (7, RP 12): mp
1
260 °C dec (lit.63 mp 233 °C dec); IR 3150 cm-1; H NMR (60
MHz, DMSO-d6) δ 7.30 (s, 1H), 7.60 (m, 5H), 8.10 (m, 4H),
12.40 (bs, 1H). Anal. (C16H11N3) C, H, N.
3-Meth yl-6-ph en yl[5H]pyr r olo[2,3-b]pyr azin e (8, RP 18):
1
mp 261.8 °C; IR 3104, 3030, 2985, 2915, 2878, 2801 cm-1; H
1
cm-1; H NMR (60 MHz, DMSO-d6) δ 3.15 (s, 6H), 6.80-6.90
NMR (60 MHz, DMSO-d6) δ 2.55 (s, 3H), 7.15 (s, 1H), 7.35-
7.60 (m, 3H), 7.75-8.10 (m, 2H), 8.40 (s, 1H), 12.30 (bs, 1H);
MS m/ e 209 (M+, 100). Anal. (C13H11N3) C, H, N.
(m, 3H), 7.85 (d, 2H, J ) 8.Hz), 8.05 and 8.20 (2d, 1H each, J
) 3 Hz), 12.15 (bs, 1H). Anal. (C14H14N4) C, H, N.
6-(4-Meth oxyp h en yl)-7-m eth yl[5H]p yr r olo[2,3-b]p yr a -
zin e (27, RP 95): mp 221.6 °C; IR 3142, 3043, 2955, 2844
cm-1; 1H NMR (60 MHz, DMSO-d6) δ 2.40 (s, 3H), 3.80 (s, 3H),
7.10 and 7.70 (2d, 2H each, J ) 7 Hz), 8.15 and 8.30 (2d, 1H
each, J ) 2.6 Hz), 12.00 (bs, 1H). Anal. (C14H13N3O) C, H, N.
6-(3,4-Meth oxyph en yl)-7-m eth yl[5H]pyr r olo[2,3-b]pyr a-
zin e (29, RP 123): mp 230.2 °C; IR 3102, 2963, 2920, 2850
cm-1; 1H NMR (60 MHz, DMSO-d6) δ 2.55 (s, 3H), 3.80 (s, 3H),
3.85 (s, 3H), 7.00-7.40 (m, 3H), 8.15 and 8.30 (2d, 1H each, J
) 3 Hz), 12 (bs, 1H). Anal. (C15H15N3O2) C, H, N.
6-[1-(4-Ch lor op h en yl)-1-cyclop r op yl][5H]p yr r olo[2,3-
b]p yr a zin e (9, RP 124): mp 189.7 °C; IR 3210, 3125, 3049,
1
3000, 2940, 2850 cm-1; H NMR (60 MHz, DMSO-d6) δ 1.50
(d, 4H), 6.15 (s, 1H), 7.30 (s, 4H), 8.10 and 8.25 (2d, 1H each,
J ) 3 Hz), 12.00 (bs, 1H). Anal. (C15H12N3Cl) C, H, N.
6-(2-Met h oxyp h en yl)[5H ]p yr r olo[2,3-b]p yr a zin e (10,
RP 9): mp 156.9 °C; IR 3080, 3051, 2925, 2887, 2830 cm-1; 1H
NMR (60 MHz, DMSO-d6) δ 3.90 (s, 3H), 6.95-7.35 (m, 4H),
7.80-8.10 (m, 1H), 8.20 and 8.35 (2d, 1H each, J ) 2.5 Hz),
11.90 (bs, 1H). Anal. (C13H11N3O) C, H, N.
6-(3-Met h oxyp h en yl)[5H ]p yr r olo[2,3-b]p yr a zin e (12,
RP 10): mp 195.7 °C; IR 3123, 2968, 2921, 2836 cm-1; 1H NMR
(60 MHz, DMSO-d6) δ 3.95 (s, 3H), 6.90-7.80 (m, 5H), 8.25
and 8.40 (2d, 1H each, J ) 2.5 Hz), 12.55 (bs, 1H). Anal.
(C13H11N3O) C, H, N.
6-(4-Met h oxyp h en yl)[5H ]p yr r olo[2,3-b]p yr a zin e (14,
RP 11): mp 256.1 °C (lit.63 mp 238-240 °C dec); IR 3143, 3035,
2959, 2857 cm-1; 1H NMR (60 MHz, DMSO-d6) δ 3.80 (s, 3H),
6.95-7.10 (m, 3H), 8.00 (d, 2H, J ) 8 Hz), 8.15 and 8.35 (2d,
1H each, J ) 2.6 Hz), 12.35 (bs, 1H). Anal. (C13H11N3O) C, H,
N.
6-(4-Ch lor op h en yl)-7-m et h yl[5H ]p yr r olo[2,3-b]p yr a -
zin e (30, RP 80): mp 260 °C dec; IR 3148, 2920, 2853 cm-1
;
1H NMR (60 MHz, DMSO-d6) δ 2.40 (s, 3H), 7.40-7.85 (m,
4H), 8.15 and 8.30 (2d, 1H each, J ) 2.5 Hz), 12.00 (bs, 1H).
Anal. (C13H10N3Cl) C, H, N.
6-(4-Dim eth ylam in osu lfam oyloxyph en yl)-7-m eth yl[5H]-
p yr r olo[2,3-b]p yr a zin e (31, RP 125): mp 235.1 °C; IR 3140,
1
3045, 2970, 2925, 2880 cm-1; H NMR (60 MHz, DMSO-d6) δ
2.45 (s, 3H), 2.95 (s, 6H), 7.45 and 7.90 (2d, 2H each, J ) 8
Hz), 8.30-8.50 (m, 2H), 12.25 (bs, 1H). Anal. (C15H16N4SO3)
C, H, N.
6-(4-Meth oxyp h en yl)-7-p r op yl[5H]p yr r olo[2,3-b]p yr a -
zin e (32, RP 127): mp 188.5 °C; IR 3215, 3158, 3055, 2958,
2934, 2866, 2836 cm-1; 1H NMR (60 MHz, DMSO-d6) δ 0.9 (t,
3H, J ) 7 Hz), 1.70 (m, 2H), 2.80 (t, 2H, J ) 7 Hz), 3.80 (s,
3H), 7.05 and 7.65 (2d, 2H each, J ) 8 Hz), 8.15 and 8.30 (2d,
1H each, J ) 3 Hz), 12.00 (bs, 1H). Anal. (C16H17N3O) C, H,
N.
6-(3,5-Dim eth oxyph en yl)[5H]pyr r olo[2,3-b]pyr azin e (16,
RP 21): mp 216.7 °C; IR 3150, 2950, 2880 cm-1; 1H NMR (60
MHz, DMSO-d6) δ 3.90 (s, 6H), 6.55 (s, 1H), 7.20 (m, 3H), 8.15
and 8.35 (2d, 1H each, J ) 2.5 Hz), 12.40 (bs, 1H). Anal.
(C14H13N3O2) C, H, N.
6-(3,4,5-Tr im e t h oxyp h e n yl)[5H ]p yr r olo[2,3-b]p yr a -
zin e (17, RP 16): mp 231.7 °C; IR 3098, 2964, 2939, 2834
cm-1; 1H NMR (60 MHz, DMSO-d6) δ 3.75 (s, 3H), 3.95 (s, 6H),
7.25 (s, 1H), 7.40 (bs, 2H), 8.25 and 8.40 (2d, 1H each, J ) 2
Hz), 12.45 (bs, 1H). Anal. (C15H15N3O3) C, H, N.
7-Allyl-6-(4-m et h oxyp h en yl)[5H ]p yr r olo[2,3-b]p yr a -
zin e (34, RP 110): mp 193.8 °C; IR 3135, 3063, 2962, 2934,
1
2878, 2838 cm-1; H NMR (60 MHz, CDCl3) δ 3.65-3.85 (m,
2H), 3.90 (s, 3H), 4.80-5.20 (m, 2H), 5.75-6.45 (m, 1H), 7.10
and 7.75 (2d, 2H each, J ) 8.2 Hz), 8.05 and 8.40 (2d, 1H each,
J ) 2.4 Hz), 11.85 (bs, 1H). Anal. (C16H15N3O) C, H, N.
7-(3-Ch lor op r op yl)-6-(4-m et h oxyp h en yl)[5H ]p yr r olo-
[2,3-b]p yr a zin e (35, RP 126): mp 178 °C dec; IR 3220, 3159,
3050, 3000, 2835 cm-1; 1H NMR (60 MHz, DMSO-d6) δ 2.00-
2.60 (m, 2H), 3.00 (m, 2H), 3.60 (t, 2H, J ) 6 Hz), 3.80 (s, 3H),
7.10 and 7.70 (2d, 2H each, J ) 8 Hz). 8.15 and 8.35 (2d, 1H
each, J ) 3 Hz), 12.00 (bs, 1H). Anal. (C16H16N3OCl) C, H, N.
7-Isopr opyl-6-(4-m eth oxyph en yl)[5H]pyr r olo[2,3-b]pyr a-
zin e (36, RP 102): mp 204.8 °C; IR 3135, 3050, 2957, 2924,
2859 cm-1; H NMR (60 MHz, DMSO-d6) δ 1.30 (d, 6H), 3.50
(m, 1H), 3.80 (s, 3H), 7.10 and 7.55 (2d, 2H each, J ) 8 Hz),
8.15 and 8.35 (2d, 1H each, J ) 2.4 Hz), 11.75 (bs, 1H). Anal.
(C16H17N3O) C, H, N.
6-(4-Flu or oph en yl)[5H]pyr r olo[2,3-b]pyr azin e (18,RP 76):
mp 244 °C dec; IR 3149 cm-1; 1H NMR (60 MHz, DMSO-d6) δ
7.05-7.50 (m, 3H), 7.65-8.10 (m, 2H), 8.20 and 8.35 (2d, 1H
each, J ) 2.4 Hz),12.45 (bs, 1H). Anal. (C12H8N3F) C, H, N.
6-(4-Chlor ophen yl)[5H]pyr r olo[2,3-b]pyr azin e (19,RP 14):
1
mp 250 °C dec (lit.63 mp 250 °C dec); IR 3300 cm-1; H NMR
(60 MHz, DMSO-d6) δ 7.20 (s, 1H), 7.55 and 8.05 (2d, 2H each,
J ) 8. Hz), 8.20 and 8.35 (2d, 1H each, J ) 2.4 Hz), 12.45 (bs,
1H). Anal. (C12H8N3Cl) C, H, N.
6-(3,5-Dich lor op h en yl)[5H]p yr r olo[2,3-b]p yr a zin e (20,
1
RP 15): mp 252 °C dec; IR 3216, 3164, 3114 cm-1; H NMR
(60 MHz, DMSO-d6) δ 7.35 (s, 1H), 7.75 (m, 1H), 8.15 (d, 2H),
8.25 and 8.40 (2d, 1H each, J ) 2.2 Hz), 12.40 (bs, 1H). Anal.
(C12H7N3Cl2) C, H, N.
6-(4-Br om oph en yl)[5H]pyr r olo[2,3-b]pyr azin e (21,RP 77):
mp 256 °C dec; IR 3211, 3109 cm-1; 1H NMR (60 MHz, DMSO-
d6) δ 7.20 (s, 1H), 7.70 and 8.00 (2d, 2H each, J ) 8.2 Hz),
8.20 and 8.35 (2d, 1H each, J ) 2.5 Hz), 12.45 (bs, 1H). Anal.
(C12H8N3Br) C, H, N.
6-(4-Ch lor op h en yl)-7-isop r op yl[5H]p yr r olo[2,3-b]p yr a -
zin e (37, RP 90): mp 208.6 °C; IR 3130, 3051, 2977, 2925,
2869 cm-1 1H NMR (60 MHz, CDCl3) δ 1.50 (d, 6H, J ) 6
;
Hz), 3.25 (m, 1H), 7.60 (s, 4H), 8.20 and 8.35 (2d, 1H each, J
) 2.5 Hz), 12.00 (bs, 1H). Anal. (C15H14N3Cl) C, H, N.
7-n -Bu tyl-6-(4-m eth oxyp h en yl)[5H]p yr r olo[2,3-b]p yr a -
zin e (38, RP 106): mp 183.8 °C; IR 3143, 3050, 2956, 2934,
6-(4-Tr iflu or om et h ylp h en yl)[5H ]p yr r olo[2,3-b]p yr a -
1
zin e (22, RP 8): mp 238 °C dec; IR 3164 cm-1; H NMR (60
1
2870 cm-1; H NMR (60 MHz, DMSO-d6) δ 1.00 (t, 3H, J )
MHz, DMSO-d6) δ 7.35 (s, 1H), 7.85 (d, 2H, J ) 8.2 Hz), 8.10-
8.50 (m, 4H), 12.70 (bs, 1H). Anal. (C13H8N3F3) C, H, N.
6-(4-Cyan oph en yl)[5H]pyr r olo[2,3-b]pyr azin e (23,RP 20):
7.2 Hz), 1.60 (m, 4H), 3.00 (t, 2H, J ) 7.6 Hz), 3.90 (s, 3H),
7.10 and 7.70 (2d, 2H each, J ) 8 Hz), 8.00 and 8.30 (2d, 1H
each, J ) 2.6 Hz), 11.75 (bs, 1H). Anal. (C17H19N3O) C, H, N.
1
mp 340 °C dec; IR 3464, 3056, 2205 cm-1; H NMR (60 MHz,

Aloisines Inhibit CDKs and GSK-3
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 233
7-n -Bu t yl-6-(4-ch lor op h en yl)[5H ]p yr r olo[2,3-b]p yr a -
zin e (40, RP 108): mp 200 °C; IR 3161, 3048, 2954, 2924, 2856
7.50 (m, 7H), 8.65 (bs, 2H), 13.45 (bs, 1H). Anal. (C12H9N3O,
HBr) C, H, N.
1
cm-1; H NMR (60 MHz, DMSO-d6) δ 0.90 (t, 3H, J ) 6 Hz),
6-(4-H yd r oxyp h en yl)[5H ]p yr r olo[2,3-b]p yr a zin e h y-
d r obr om id e (15, RP 26): mp 255 °C dec; IR 3448, 3176, 3060
cm-1; 1H NMR (60 MHz, DMSO-d6) δ 6.65-6.85 (m, 4H), 7.70
and 7.85 (2d, 2H each, J ) 8.2 Hz), 8.15 and 8.25 (2d, 1H each,
J ) 3 Hz), 9.65 (s, 1H), 12.10 (bs, 1H). Anal. (C12H9N3O, HBr,
H2O) C, H, N.
1.20-2.00 (m, 4H), 2.95 (t, 2H, J ) 7.2 Hz), 7.65 (s, 4H), 8.25
and 8.40 (2d, 1H each, J ) 2.5 Hz), 12.05 (bs, 1H). Anal.
(C16H16N3Cl) C, H, N.
7-n -Heptyl-6-(4-m eth oxyph en yl)[5H]pyr r olo[2,3-b]pyr a-
zin e (41, RP 111): mp 132.5 °C; IR 3142, 3064, 2955, 2925,
1
2850 cm-1; H NMR (60 MHz, CDCl3) δ 0.90-2.00 (m, 13H),
6-(4-Hyd r oxyp h en yl)-7-m eth yl[5H]p yr r olo[2,3-b]p yr a -
zin e h yd r obr om id e (28, RP 96): mp 262 °C dec; IR 3465,
3.05 (t, 2H, J ) 7.2 Hz), 3.90 (s, 3H), 7.05 and 7.70 (2d, 2H
each, J ) 8.2 Hz), 8.00 and 8.40 (2d, 1H each, J ) 3 Hz), 12.05
(bs, 1H). Anal. (C20H25N3O) C, H, N.
1
3143, 3090, 2796, 2759 cm-1; H NMR (60 MHz, DMSO-d6) δ
2.45 (s, 3H), 7.00 and 7.70 (2d, 2H each, J ) 8.2 Hz), 8.50 (bs,
2H), 9.80 (s, 2H), 13.00 (bs, 1H). Anal. (C13H11N3O, HBr) C,
H, N.
6-(4-Meth oxyph en yl)-7-m eth ylcyclopr opyl[5H]pyr r olo-
[2,3-b]p yr a zin e (42, RP 104): mp 193.9 °C; IR 3142, 3080,
1
3046, 3000, 2931, 2820 cm-1; H NMR (60 MHz, DMSO-d6) δ
6-(4-Hyd r oxyp h en yl)-7-p r op yl[5H]p yr r olo[2,3-b]p yr a -
zin e h yd r obr om id e (33, RP 132): mp 244 °C dec; IR 3187,
0.20-0.50 (m, 4H), 1.00-1.40 (m, 1H), 2.90 (d, 2H, J ) 6 Hz),
3.85 (s, 3H), 7.15 and 7.75 (2d, 2H each, J ) 8.2 Hz), 8.20 and
8.40 (2d, 1H each, J ) 2.6 Hz), 12.20 (bs, 1H). Anal. (C17H17N3O)
C, H, N.
1
3100, 2965, 2873, 2798 cm-1; H NMR (60 MHz, DMSO-d6) δ
0.85 (t, 3H, J ) 7 Hz), 1.35-1.90 (m, 2H), 3.10-2.75 (m, 2H),
7.20 and 7.65 (2d, 2H each, J ) 8.2 Hz), 8.50 (s, 2H), 9.8 (s,
2H), 13.1 (s, 1H). Anal. (C15H15N3O, HBr) C, H, N.
7-Ben zyl-6-ph en yl[5H]pyr r olo[2,3-b]pyr azin e (44, RP 92):
1
mp 209.8 °C; IR 3144, 3056, 3024, 2929, 2871 cm-1; H NMR
7-n -Bu tyl-6-(4-h yd r oxyp h en yl)[5H]p yr r olo[2,3-b]p yr a -
zin e (39, RP 107): mp 281.4 °C; IR 3134, 3100, 2946, 2924,
(60 MHz, DMSO-d6) δ 4.30 (s, 2H), 7.20 (s, 6H), 7.75-8.25 (m,
4H), 8.30 and 8.40 (2d, 1H each, J ) 3 Hz), 12.25 (bs, 1H).
Anal. (C19H15N3) C, H, N.
1
2867 cm-1; H NMR (60 MHz, DMSO-d6) δ 0.90 (t, 3H, J ) 7
Hz), 1.20-1.90 (m 4H), 2.90 (t, 2H, J ) 7.5 Hz), 6.95 and 7.60
(2d, 2H each, J ) 7 Hz), 8.15 and 8.30 (2d, 1H each, J ) 2.6
Hz), 9.80 (bs, 1H), 11.80 (bs, 1H). Anal. (C16H17N3O) C, H, N.
6-(4-Hydr oxyph en yl)-7-m eth ylcyclopr opyl[5H]pyr r olo-
[2,3-b]p yr a zin e h yd r obr om id e (43, RP 112): mp 260 °C dec;
7-Ben zyl-6-(4-ch lor op h en yl)[5H ]p yr r olo[2,3-b]p yr a -
zin e (45, RP 91): mp 266.3 °C; IR 3138, 3050, 3025, 2928,
1
2858 cm-1; H NMR (60 MHz, DMSO-d6) δ 4.25 (s, 2H), 7.15
(s, 5H), 7.55 (s, 4H), 8.20 and 8.35 (2d, 1H each, J ) 3 Hz),
1
IR 3482, 3335, 3064, 2983 cm-1 ; H NMR (60 MHz, DMSO-
12.25 (bs, 1H). Anal. (C19H14N3Cl) C, H, N.
d6) δ 1.60 (d, 4H, J ) 6 Hz), 2.00-2.60 (m, 3H), 2.90-3.60 (m,
4H), 7.05 and 7.90 (2d, 2H each, J ) 8.2 Hz), 8.55-8.80 (m,
2H), 13.45 (bs, 1H). Anal. (C16H15N3O, HBr, H2O) C, H, N.
Bioch em istr y. (a ) Bioch em ica l Rea gen ts. Sodium ortho-
vanadate, EGTA, EDTA, Mops, â-glycerophosphate, phenyl
phosphate, sodium fluoride, dithiothreitol (DTT), glutathione
agarose, glutathione, bovine serum albumin (BSA), nitrophe-
nyl phosphate, leupeptin, aprotinin, pepstatin, soybean trypsin
inhibitor, benzamidine, and histone H1 (type III-S) were
obtained from Sigma Chemicals. [γ-32P]ATP (PB 168) was
obtained from Amersham. The GS-1 peptide (YRRAAVPP-
SPSLSRHSSPHQSpEDEEE) was synthesized by the Peptide
Synthesis Unit, Institute of Biomolecular Sciences, University
of Southampton, Southampton SO16 7PX, U.K.
(b) Bu ffer s. 1. Hom ogen iza tion bu ffer : 60 mM â-glyc-
erophosphate, 15 mM p-nitrophenyl phosphate, 25 mM Mops
(pH 7.2), 15 mM EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM
sodium vanadate, 1 mM NaF, 1 mM phenyl phosphate, 10 µg
of leupeptin/mL, 10 µg of aprotinin/mL, 10 µg of soybean
trypsin inhibitor/mL, and 100 µM benzamidine.
6-(4-Meth oxyp h en yl)-7-m eth ylcycloh exyl[5H]p yr r olo-
[2,3-b]p yr a zin e (46, RP 98): mp 220.3 °C; IR 3434, 3135,
1
2921, 2850 cm-1; H NMR (60 MHz, DMSO-d6) δ 0.85-1.80
(m, 11H), 2.80 (d, 2H, J ) 6.5 Hz), 3.80 (s, 3H), 7.15 and 7.70
(2d, 2H each, J ) 8.2 Hz), 8.15 and 8.35 (2d, 1H each, J ) 2.5
Hz), 11.90 (bs, 1H). Anal. (C20H23N3O) C, H, N.
6-(4-Ch lor op h en yl)-7-m et h ylcycloh exyl[5H ]p yr r olo-
[2,3-b]p yr a zin e (47, RP 99): mp 203.5 °C; IR 3142, 3048,
1
2928, 2847 cm-1; H NMR (60 MHz, DMSO-d6) δ 0.80-1.75
(m, 11H), 2.80 (d, 2H, J ) 6.5 Hz), 7.65 (s, 4H), 8.20 and 8.40
(2d, 1H each, J ) 2.4 Hz), 12.10 (bs, 1H). Anal. (C19H20N3Cl)
C, H, N.
5-Met h yl-6-p h en ylp yr r olo[2,3-b]p yr a zin e (48, R P 22):
mp 72.3 °C; IR 3100, 3051, 2948 cm-1 1H NMR (60 MHz,
;
DMSO-d6) δ 3.75 (s, 3H), 6.70 (s, 1H), 7.45 (s, 5H), 8.20 and
8.40 (2d, 1H each, J ) 2.4 Hz). Anal. (C13H11N3) C, H, N.
6-[1-(4-Ch lor op h en yl)-1-cyclop r op yl]-7-m et h yl[5H ]-
p yr r olo[2,3-b]p yr a zin e (49, RP 130): mp 223.9 °C; IR 3135,
1
3055, 2921, 2857, 2780 cm-1; H NMR (60 MHz, DMSO-d6) δ
1.40 (s, 4H), 2.30 (s, 3H), 7.30 (bs, 4H), 8.20 and 8.35 (2d, 1H
2. Bu ffer A: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25
mM Tris-HCl (pH 7.5), 50 µg of heparin/mL.
each, J ) 2.8 Hz), 11.95 (bs, 1H). Anal. (C16H14N3Cl) C, H, N.
2-(4-Met h oxyp h en yl)[1H ]p yr r olo[2,3-b]p yr id in e (50,
RP 97): mp 206.2 °C; IR 3143, 2930, 2846, cm-1; 1H NMR (60
MHz, DMSO-d6) δ 3.45 (s, 3H), 6.75 (s, 1H), 6.90-7.15 (m, 3H),
7.70-7.95 (m, 3H), 8.15 (d, 1H, J ) 4 Hz), 11.95 (s, 1H). Anal.
(C14H12N2O) C, H, N.
Gen er a l Meth od for th e Dem eth yla tion of Meth oxy-
Su bstitu ted 6-P h en yl[5H]p yr r olo[2,3-b]p yr a zin es. First,
hydrobromidic acid was redistilled over a trace of 50% hypo-
phosphorous acid: 1 g for each 100 g of 48% hydrobromidic
acid. Methoxy compound (0.003 mol) was heated with hydro-
bromidic acid (20 mL). After removal of the aqueous forerun,
the temperature reached 126 °C. The time required for
demethylation varied from 3 to 10 h. The excess hydrobromidic
acid was removed under reduced pressure, and the crude
product was crystallized from ethanol.
3. Bu ffer C: same as homogenization buffer but with 5 mM
EGTA, no NaF, and no protease inhibitors.
4. Tr is-bu ffer ed sa lin e-Tw een -20 (TBST): 50 mM Tris
(pH 7.4), 150 mM NaCl, 0.1% Tween-20.
5. Hyp oton ic lysis bu ffer (HLB): 50 mM Tris-HCl (pH
7.4), 120 mM NaCl, 10% glycerol, 1% Nonidet-P40, 5 mM DTT,
1 mM EGTA, 20 mM NaF, 1 mM orthovanadate, 5 µM
microcystin, 100 µg/mL each of leupeptin, aprotinin, and
pepstatin.
(c) Kin a se P r ep a r a tion s a n d Assa ys. Kinases activities
were assayed in buffer A or C (unless otherwise stated), at 30
°C, at a final ATP concentration of 15 µM. Blank values were
subtracted and activities calculated as picomoles of phosphate
incorporated for a 10-min incubation. The activities are usually
expressed in percent of the maximal activity, i.e., in the
absence of inhibitors. Controls were performed with appropri-
ate dilutions of dimethyl sulfoxide. In a few cases, phospho-
rylation of the substrate was assessed by autoradiography
after SDS-PAGE.
6-(2-H yd r oxyp h en yl)[5H ]p yr r olo[2,3-b]p yr a zin e h y-
d r obr om id e (11, R P 109): mp 250 °C dec; IR 3419, 3354,
1
3090, 2710, 2641 cm-1; H NMR (60 MHz, DMSO-d6) δ 5.80
(s, 3H), 6.80-7.30 (m, 4H), 7.70-8.00 (m, 1H), 8.40 (bs, 3H),
12.85 (bs, 1H). Anal. (C12H9N3O, HBr, H2O) C, H, N.
GSK-3â was either purified from porcine brain or expressed
in and purified from insect Sf9 cells.32 It was assayed, following
a 1/100 dilution in 1 mg of BSA/mL of 10 mM DTT, with 5 µL
of 40 µM GS-1 peptide as a substrate, in buffer A, in the
6-(3-H yd r oxyp h en yl)[5H ]p yr r olo[2,3-b]p yr a zin e h y-
d r obr om id e (13, R P 134): mp 258 °C dec; IR 3448, 3137,
3085, 2700, 2630 cm-1; 1H NMR (60 MHz, DMSO-d6) δ 6.70-

234 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2
Mettey et al.
presence of 15 µM [γ-32P]ATP (3000 Ci/mmol; 1 mCi/mL) in a
final volume of 30 µl. After 30 min of incubation at 30 °C, 25-
µL aliquots of supernatant were spotted onto 2.5- × 3-cm pieces
of Whatman P81 phosphocellulose paper, and 20 s later, the
filters were washed five times (for at least 5 min each time)
in a solution of 10 mL of phosphoric acid/L of water. The wet
filters were counted in the presence of 1 mL of ACS (Amer-
sham) scintillation fluid.
improvement in the value of Rfree. Statistics of the final models
are given in Table 6.
The analysis of the surface area involved in the CDK2-
aloisine B complex was carried out using the program Aesop
(M.N., unpublished). Atomic radii were taken from a database
of unified atom values, and a solvent probe radius of 1.4 Å
was used.
Cell Biology. (a ) Rea gen ts. Penicillin, streptomycin, no-
codazole, insulin, transferrin, progesterone, putrescine, sodium
selenite, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), RNAse A, and propidium iodide were pur-
chased from Sigma.
CDK1/cyclin B was extracted in homogenization buffer from
M phase starfish (Marthasterias glacialis) oocytes and purified
by affinity chromatography on p9CKShs1-sepharose beads, from
which it was eluted by free p9CKShs1 as previously described.32,35
The kinase activity was assayed in buffer C, with 1 mg of
histone H1/mL, in the presence of 15 µM [γ-32P]ATP (3000 Ci/
mmol; 1 mCi/mL) in a final volume of 30 µL. After 10 min of
incubation at 30 °C, 25-µL aliquots of supernatant were
spotted onto P81 phosphocellulose papers and treated as
described above.
(b) Cell Cu ltu r es. Clonal human NT2 teratocarcinoma cells
were obtained from Stratagene (La J olla, CA) and grown in
Dulbecco’s Modified Eagle Medium Nutrient Mixture F-12
with 2 mM l-glutamine (BIO WHITTAKER) supplemented
with 5% FCS and containing penicillin (20 IU/mL) and
streptomycin (20 µg/mL) at 37 °C, in a humidified atmosphere
containing 5% CO2 in air.
CDK5/p25 was reconstituted by mixing equal amounts of
recombinant mammalian CDK5 and p25 expressed in E. coli
as GST (glutathione-S-transferase) fusion proteins and puri-
fied by affinity chromatography on glutathione agarose (vectors
kindly provided by Dr. J . H. Wang) (p25 is a truncated version
of p35, the 35 kDa CDK5 activator). Its activity was assayed
in buffer C as described for CDK1/cyclin B.
(c) NT2 Differ en tia tion . Differentiation of hNT cells from
NT2 cells was induced according to the method of Pleasure et
al.,82 modified by Soulie´ et al. (submitted). Briefly, after the
second replating, cells were cultured in serum-free medium
with
a combination of mitotic inhibitors (1 µM cytosine
arabinoside, 10 µM fluorodeoxyuridine, and 10 µM uridine)
and a mixture of salt and hormones (25 µg of insulin/mL, 100
µg of transferrin/mL, 20 nM progesterone, 60 µM putrescine,
and 30 nM sodium selenite) for 5 days before treatment.
Other kinases were expressed, purified, and assayed as
described previously.20,37
Cr ysta llogr a p h y. (a ) Exp r ession , P u r ifica tion , a n d
Cr ysta lliza tion of Hu m a n CDK2. Human CDK2 was ex-
pressed from a recombinant baculovirus in Sf9 insect cells and
purified according to slightly modified published method.74,75
Monomeric unphosphorylated CDK2 crystals were grown as
previously described.68
(d ) Tr ea tm en t w ith Aloisin e. Exponentially growing cells
were incubated for 24 h with aloisine A (stock solution
dissolved in dimethyl sulfoxide). Nocodazole treatment of cells
was performed at a concentration of 0.04 µg of nocodazole/mL
of medium for 24 h. Following the nocodazole treatment, cells
were washed twice with fresh medium and cultured with or
without aloisine A for 24 h. To perform serum deprivation,
cells were maintained in serum-free medium for 40 h. Follow-
ing serum deprivation, cells were washed twice and cultured
in fresh serum-containing medium with or without aloisine A
for 40 h.
(b) X-r a y Cr ysta llogr a p h y Da ta Collection a n d P r o-
cessin g. The CDK2-aloisine B dataset was collected from a
monomeric CDK2 crystal soaked for 60 h in 1 mM aloisine B
in 1× mother liquor solution (50 mM ammonium acetate, 10%
PEG3350, 15 mM NaCl, 100 mM HEPES, pH 7.4) plus 5%
DMSO. Data were collected on beamline X-RAY DIFFRAC-
TION at the Elettra Light Source at 100K after the crystal
had been transferred briefly to cryoprotectant (mother liquor
adjusted to contain 20% glycerol). The images were integrated
with the MOSFLM package,76 and reflections were subse-
quently scaled and merged using SCALA.77 Subsequent data
reduction and structure refinement were pursued through
programs of the CCP4 suite.77 Statistics of the datasets used
in the structure determination are given in Table 6.
(e) Cell Via bility Assa y. To quantify the toxicity of aloisine
A on NT2 cells and hNT human neurons, we measured the
inhibition of cellular reduction of MTT to MTT formazan
according to Saille´ et al.83 Following aloisine A exposure, cells
were incubated with 0.5 mg of MTT/mL of fresh medium at
37 °C for 1 h. The formazan products were dissolved in DMSO
and quantified by measurement of the absorbance at 562 nm.
(f) Cell Cycle An a lysis by F low Cytom etr y. Cells were
trypsinized, collected by centrifugation, and fixed in cold 70%
ethanol for at least 4 h. Fixed cells were washed in PBS,
incubated with 10 µg of RNAse A/mL, and stained with 25 µg
of propidium iodide/mL for 1 h at 37 °C. The stained cells were
then analyzed for cell cycle distribution on a FACSort flow
cytometer (Becton Dickinson). Cell cycle analyzes were per-
formed as described84 using multiCYCLE (P Rabanovitch).
(c) Str u ctu r e Solu tion a n d Refin em en t. The starting
model for CDK2 refinement was a 1.3-Å resolution model of
CDK2 in complex with an O6-substituted guanine derivative
(unpublished data). The positioning of this search model in
our isomorphous crystals was confirmed by molecular replace-
ment using AMoRE.78 Refinement was begun by carrying out
rigid body refinement of the structure using REFMAC79 and
using sequentially higher resolution data. As the resolution
of the data included was increased from 3.0 to 1.9 Å, increasing
numbers of rigid bodies were used, so that initially the whole
molecule was treated as a single rigid body, and finally
individual secondary structural elements were allowed to
refine independently. Following rigid body refinement, the (Fo
- Fc)Rcalc maps included readily interpretable electron density
for the bound inhibitor. The structure was then compared with
2Fo - Fc electron density maps using the program “O”,80 and
minor structural changes were introduced. Following the first
round of model building, a model of aloisine B generated within
the CCP4 molecular sketcher77 was built into the electron
density map and included in subsequent refinement steps.
Refinement was then pursued with alternating cycles of
interactive model building and maximum likelihood refine-
ment using REFMAC. Toward the end of the refinement, water
molecules were added using the program ARP.81 Structure
refinement was completed when addition of further waters,
or the rebuilding of the disordered loops, resulted in no
Ack n ow led gm en t. We thank the beamline scien-
tists at the Elettra Light source, beamline X-RAY
DIFFRACTION, for excellent facilities. We thank David
Morgan for his generous gift of the AcCDK2 baculovirus,
J . Wang for the CDK5 and p25 clones, J ohn Sinclair
and Elspeth Garman for assistance during data collec-
tion, and Alison Lawrie for cell culture. At the LMB,
the authors thank I. Taylor, R. Bryan, and G. Coates.
This research was supported by the Royal Society and
the BBSRC. This research was also supported by grants
from the “Association pour la Recherche sur le Cancer”
(ARC 5343 and ARC5732) (L.M.), the “Conseil Re´gional
de Bretagne” (L.M.), and a grant (“Mole´cules & Cibles
The´rapeutiques”) from the “Ministere de la Recherche/
INSERM/CNRS”.

Aloisines Inhibit CDKs and GSK-3
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 2 235
Newell, D. R.; Noble, M. E. M.; Sausville, E. A.; Schultz, R.; Yu,
W. Identification of novel purine and pyrimidine cycline-depend-
ent kinase inhibitors with distinct molecular interactions and
tumor cell growth inhibition profiles. J . Med. Chem. 2000, 43,
2797-2804.
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