Meriolins (3-(pyrimidin-4-yl)-7-azaindoles): Synthesis, kinase inhibitory activity, cellular effects, and structure of a CDK2/Cyclin A/meriolin complex

Article abstract of DOI:10.1021/jm700940h

We report the synthesis and biological characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins), a chemical hybrid between the natural products meridianins and variolins, derived from marine organisms. Meriolins display potent inhibitory activities toward cyclin-dependent kinases (CDKs) and, to a lesser extent, other kinases (GSK-3, DYRK1A). The crystal structures of 1e (meriolin 5) and variolin B (Bettayeb, K.; Tirado, O. M.; Marionneau-Lambert, S.; Ferandin, Y.; Lozach, O.; Morris, J.; Mateo-Lozano, S.; Drückes, P.; Sch?chtele, C.; Kubbutat, M.; Liger, F.; Marquet, B.; Joseph, B.; Echalier, A.; Endicott, J.; Notario, V.; Meijer, L. Cancer Res. 2007, 67, 8325-8334) in complex with CDK2/cyclin A reveal that the two inhibitors are orientated in very different ways inside the ATP-binding pocket of the kinase. A structure-activity relationship provides further insight into the molecular mechanism of action of this family of kinase inhibitors. Meriolins are also potent antiproliferative and proapoptotic agents in cells cultured either as monolayers or in spheroids. Proapoptotic efficacy of meriolins correlates best with their CDK2 and CDK9 inhibitory activity. Meriolins thus constitute a promising class of pharmacological agents to be further evaluated against the numerous human diseases that imply abnormal regulation of CDKs including cancers, neurodegenerative disorders, and polycystic kidney disease.

Full text of DOI:10.1021/jm700940h

J. Med. Chem. 2008, 51, 737–751
737
Meriolins (3-(Pyrimidin-4-yl)-7-azaindoles): Synthesis, Kinase Inhibitory Activity, Cellular
Effects, and Structure of a CDK2/Cyclin A/Meriolin Complex†
,§
Aude Echalier,‡,§ Karima Bettayeb, Yoan Ferandin, Olivier Lozach, Monique Cle´ment,¶ Annie Valette,× François Liger,∞
Bernard Marquet,∞ Jonathan C. Morris,# Jane A. Endicott,‡ Benoît Joseph,*,∞ and Laurent Meijer*,
Laboratory of Molecular Biophysics and Department of Biochemistry, The Rex Richards Building, UniVersity of Oxford, South Parks Road, Oxford,
OX1 3QU, U.K., C.N.R.S., Cell Cycle Group & UPS2682, Station Biologique, B.P. 74, 29682 Roscoff Cedex, Bretagne, France, Département de
Recherches en Cancérologie, UniVersité de Nantes, Inserm U692, 9 Quai Moncousu, 44093 Nantes Cedex 01, France, LBCMCP, UMR-CNRS 5088,
IFR 109, UniVersité Paul Sabatier Bat 4R3B1, 118 Route de Narbonne, 31062 Toulouse Cedex, France, Laboratoire de Chimie Organique 1, Institut
de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR-CNRS 5246, UniVersité de Lyon, UniVersité Claude BernardsLyon 1, Bâtiment
Curien, 43 BouleVard du 11 NoVembre 1918, 69622 Villeurbanne Cedex, France, and Department of Chemistry, School of Chemistry and Physics,
UniVersity of Adelaide, Adelaide SA 5005, Australia
ReceiVed July 31, 2007
We report the synthesis and biological characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins), a
chemical hybrid between the natural products meridianins and variolins, derived from marine organisms.
Meriolins display potent inhibitory activities toward cyclin-dependent kinases (CDKs) and, to a lesser extent,
other kinases (GSK-3, DYRK1A). The crystal structures of 1e (meriolin 5) and variolin B (Bettayeb, K.;
Tirado, O. M.; Marionneau-Lambert, S.; Ferandin, Y.; Lozach, O.; Morris, J.; Mateo-Lozano, S.; Drückes,
P.; Schächtele, C.; Kubbutat, M.; Liger, F.; Marquet, B.; Joseph, B.; Echalier, A.; Endicott, J.; Notario, V.;
Meijer, L. Cancer Res. 2007, 67, 8325-8334) in complex with CDK2/cyclin A reveal that the two inhibitors
are orientated in very different ways inside the ATP-binding pocket of the kinase. A structure-activity
relationship provides further insight into the molecular mechanism of action of this family of kinase inhibitors.
Meriolins are also potent antiproliferative and proapoptotic agents in cells cultured either as monolayers or
in spheroids. Proapoptotic efficacy of meriolins correlates best with their CDK2 and CDK9 inhibitory activity.
Meriolins thus constitute a promising class of pharmacological agents to be further evaluated against the
numerous human diseases that imply abnormal regulation of CDKs including cancers, neurodegenerative
disorders, and polycystic kidney disease.
Introduction
of CDK activities has been demonstrated in viral infections,16,17
proliferative renal diseases18–21 including polycystic kidney
disease,22 inflammatory responses,23 and pain signaling.3,24
Taken together, these observations in many therapeutic areas
constitute strong support for the search for and optimization of
selective small molecular weight inhibitors of CDKs. Over 120
pharmacological inhibitors of CDKs have been described
(reviews in 25–29). Among these, a few compounds have
already reached clinical phase evaluation (Figure 1), mostly
against cancer and renal disease. All CDK inhibitors identified
so far act by competing with ATP for binding at the catalytic
site of their kinase targets.30 Selectivity studies show that
although some kinase inhibitors are rather unselective, many
display a definite specificity profile.31
Essentially all physiological processes and most human
diseases involve protein phosphorylation. Among the 518
kinases present in man, cyclin-dependent kinases (CDKsa) stand
out as some of the best studied kinases because of their key
functions in cell cycle regulation,1 neuronal cell physiology,2
pain signaling,3 apoptosis,4 transcription, RNA splicing,5,6 and
insulin secretion.7 Numerous abnormalities in CDK activity and
regulation have been described in cancers.8 CDK5 is abnormally
up-regulated in neurodegenerative disorders such as Alzhe-
imer’s,9 Parkinson’s,10,11 and Nieman-Pick’s12 diseases, in
ischemia,13,14 and in traumatic brain injury.15 Direct involvement
†
The structures of pT160 CDK2/cyclin A/meriolin 3, pT160 CDK2/
Meridianins, a family of 3-(2-aminopyrimidin-4-yl)indoles,
were recently identified as kinase inhibitors.32 Meridianins were
first extracted from the Ascidian Aplidium meridianum33 and
later chemically synthesized by various groups.34–36 Meridianins
share some structural analogy with another family of marine
natural products known as variolins, initially extracted from the
Antarctic sponge Kirkpatrickia Variolosa.37,38 The synthesis of
variolins has been reported recently.39–43 Variolin B and
deoxyvariolin B (PM01218) are cytotoxic to human cancer cell
lines.38,44,45 They are currently under investigation as antitumor
drugs by PharmaMar. As this work was in progress, variolin B
and deoxyvariolin B were reported to inhibit CDKs.45
cyclin A/meriolin 5, and pT160 CDK2/cyclin A/variolin B have been
deposited in the PDB with accession codes 3BHT, 3BHU, and 3BHV,
respectively.
* To whom correspondence should be addressed. For B.J. (chemistry):
phone, +33 (0)4 72 44 81 35; fax, +33 (0)4 72 44 81 35; e-mail,
benoit.joseph@univ-lyon1.fr. For L.M. (biochemistry and cell biology):
phone, +33 (0)2 98 29 23 39; fax, +33 (0)2 98 29 25 26; e-mail, meijer@sb-
roscoff.fr.
‡
University of Oxford.
These authors contributed equally.
§
C.N.R.S., Cell Cycle Group & UPS2682.
Université de Nantes.
Université Paul Sabatier.
Université de Lyon.
¶
×
∞
#
University of Adelaide.
In this article we report on the synthesis and biological
characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins),
a chemical hybrid between meridianins and variolins. Meriolins
display potent inhibitory activities toward CDKs (especially
a Abbreviations: CDK, cyclin-dependent kinase; CK1, casein kinase 1;
FCS, fetal calf serum; GSK-3, glycogen synthase kinase-3; LDH, lactate
dehydrogenase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymetho-
xyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PBS, phosphate buffered saline.
10.1021/jm700940h CCC: $40.75
2008 American Chemical Society
Published on Web 01/31/2008

738 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Echalier et al.
Figure 1. Seven CDK inhibitors currently in clinical trials as anticancer drugs: (A) flavopiridol (Alvocidib) (Sanofi-Aventis);78 (B) R-roscovitine
(CYC202, Seliciclib) (Cyclacel Pharmaceuticals);79,80 (C) R547 (Ro-4584820) (Hoffmann-LaRoche Inc.);81,82 (D) SNS-032 (BMS-387032) (Bristol-
Myers Squibb);83 (E) PD-0332991 (Pfizer);84 (F) AZD5438 (AstraZeneca);85 (G) AG-024322 (Pfizer).29
Scheme 1a
Figure 2. Structure of meriolins and related compounds. Meridianins
originate from the Ascidian Aplidium meridianum,33 while variolins
were initially extracted from the Antarctic sponge Kirkpatrickia
Variolosa.37,38
CDK9) and other kinases (GSK-3, CK1, DYRK1A). Meriolins
are also potent antiproliferative and proapoptotic agents in cell
cultures. The crystal structures of 1e (meriolin 5) and variolin
B in complex with CDK2/cyclin A reveal that the two inhibitors
are orientated in very different ways inside the ATP-binding
pocket of the kinase. A structure-activity relationship provides
some further insight into the molecular mechanism of action of
this family of kinase inhibitors. Proapoptotic efficacy of
meriolins correlates best with their CDK2 and CDK9 inhibitory
activity.
a Reagents and conditions: (i) Ac2O, TFA, reflux, 8 h, 3a ) 85%, 3b )
55%, 3c ) 99%, 3d ) 84%, 3e ) 80%; (ii) NaH, PhSO2Cl, 0 °C to room
temp, 4 h, 4a ) 93%, 4b ) 90%, 4c ) 88%, 4d ) 68%, 4e ) 70%; (iii)
I2, KOH, DMF, room temp, 2.5 h, 82%; (iv) NaH, PhSO2Cl, 0 °C to room
temp, 4 h, 68%; (v) (a) n-tributyl(1-ethoxyvinyl)stannane, Pd(PPh3)4, LiCl,
DMF, 80 °C, 18 h; (b) 10% HCl, 89%; (vi) Me2SO4, K2CO3, acetone, reflux,
5.5 h, 81%.
Results and Discussion
The chemical structure resemblance between the two natural
products meridianins and variolin B prompted us to synthesize
a hybrid structure, which we named meriolins (Figure 2).
Chemistry. The synthetic approach of meriolins was based
on the previous preparation of 3-[(2-amino)pyrimidin-4-yl]-7-
azaindole 1 from commercially available 7-azaindole.46 4-Chloro-
7-azaindole 2a, 4-methoxy-7-azaindole 2b, and 6-bromo-7-
azaindole 2c were commercially available. Starting materials
2d, 2e, and 2f were obtained by Mitsunobu reaction between
4-hydroxy-7-azaindole (see Experimental Section)47 and ap-
propriate alcohols. Acylation of 2a-e in the presence of acetic
anhydride and trifluoroacetic acid afforded the derivatives 3a-e
(Scheme 1).48 N-Benzenesulfonylation of 3a-e was readily
carried out in the presence of benzenesulfonyl chloride and
sodium hydride to give derivatives 4a-e. 3-Acetyl-1-benzene-
sulfonyl-4-(1-methylethyl)-7-azaindole 4f was prepared by an

Kinase Inhibitory 3-(Pyrimidin-4-yl)-7-azaindoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 739
Scheme 3a
Scheme 2a
a Reagents and conditions: (i) 48% HBr/AcOH, reflux, 2 h, 8 ) 90%; 9
) 92%; 11 ) 92%; (ii) CuCl, formamide, 170 °C, 18 h, 26%; (iii) 2-methyl-
2-thiopseudourea sulfate, K2CO3, 2-methoxyethanol, 100–110 °C, 5 days,
30%.
a Reagents and conditions: (i) DMF-DMA, DMF, 90 °C, 8 h; (ii)
guanidine·HCl, K2CO3, 2-methoxyethanol, 100–110 °C, 36 h.
The difference electron density map for the CDK2/cyclin
A/meriolin 5 complex after cycles of rigid body refinement
showed strong density at the CDK2 ATP binding site. The
quality of the data and the resulting electron density maps
resulted in the unambiguous positioning of the inhibitor in the
electron density map (Figure 3).
Meriolin 5 is bound within the kinase ATP binding cleft, and
with the exception of the glycine-rich loop (residues 12–20)
that will be described in detail below, the overall conformation
of CDK2/cyclin A bound to meriolin 5 is essentially identical
to that bound to meriolin 3 (described in ref 66). The binding
modes of the two inhibitors are also identical. The pyrrolopy-
ridine central scaffold of meriolins 5 and 3 and the pyrrolopy-
rimidine system fused to the pyridine ring of variolin B (as
described in ref 66) are sandwiched between the two lobes of
CDK2. The ring systems pack against the side chain of Leu134
and of Ala31. This is a common feature of many protein kinase
inhibitors containing planar ring scaffolds.
As described for ATP and for almost all ATP-competitive
kinase inhibitors, the meriolins and variolins also interact with
CDK2 via hydrogen bonds with the hinge region of the protein,
namely, with the Glu81 main chain carbonyl group and the
Leu83 main chain amide nitrogen (Figures 3 and 4). Interest-
ingly, despite sharing a large proportion of their scaffold,
meriolin 5 and variolin B interact with the CDK2 hinge via
different groups. These interactions occur with the pyrrole-
pyridine core of meriolin 5 and through the pyridine-amine ring
of variolin B (Figures 3 and 4, respectively). As a result, the
two inhibitors explore different regions of the CDK2 ATP site.
The different orientations of the meriolin 5 and variolin B
scaffolds result in the meriolin 5 pyrrole ring facing the gatekeeper
residue Phe80 (Figure 3). However, in the variolin B structure this
ring faces out of the ATP site toward the selectivity surface (Figure
4). It is likely that the pyridine-amine group of variolin B is too
bulky to be accommodated in the space facing the gatekeeper, and
as a result, the inhibitor is rotated 180° compared to the binding
alternative way to avoid the O-dealkylation of 7-azaindole 2f
during the first acetylation step in acidic conditions (Scheme
1). Iodination of 4-(1-methylethyl)-7-azaindole 2f was first
realized to give 3-iodo derivative 5. N-Benzenesulfonation of
5 was then performed to reach 6. Stille reaction between 6 and
tri-n-tributyl(1-ethoxyvinyl)stannane afforded the desired deriva-
tive 4f.49 N-Methylation of compound 3b was also performed
to give derivative 4g. Reaction of 4a-g with dimethylformamide
dimethylacetal in DMF afforded enaminones 7a-g (Scheme
2). Final formation of pyrimidine nucleus occurred by heating
7a-g in the presence of guanidine ·HCl and anhydrous K2CO3
in 2-methoxyethanol to afford final derivatives 1a-g in fair
yields. Under these reaction conditions, compound 7a gave 1a
(51%) and 1h (31%) via the displacement of the chlorine atom
by the alcohol. O-Demethylation of 1b and 1g was done in
acidic conditions (48% HBr/AcOH) to reach 4-hydroxy deriva-
tives 8 and 9 in good yields. Derivatives 10 and 11 were
prepared from 2b (Scheme 3). The latter compound was treated
with CuCl and formamide to give compound 10 in 26% yield.50
O-Demethylation of 10 afforded 11 in 92% yield. Compound
12 was prepared in 30% yield by heating 7b and 2-methyl-2-
pseudsothiourea sulfate.51
Cocrystallization of Meriolin and Variolin with CDK2/
Cyclin A. We report the structure of CDK2/cyclin A in complex
with meriolin 5 (compound 1e) and compare it to that of the
structures of complexes of CDK2/cyclin A bound to meriolin
3 (compound 1b) and to variolin B. CDK2 adopts a fully active
conformation upon association with one of its cyclin partners
(cyclin A or E) and phosphorylation of Thr160 in the CDK2
activation loop. CDK2 displays a classical protein kinase fold
with the N-terminal lobe (composed of a twisted antiparallel
ꢀ-sheet) linked by a short hinge to the C-terminal domain (which
is almost exclusively R-helical).30

740 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Echalier et al.
Figure 3. Cocrystal structure of meriolin 5 (1e) bound to CDK2/cyclin A. (A) (2Fo - Fc)Rcalc electron density for meriolin 5 calculated at the end
of refinement using map coefficients output from REFMAC with resolution between 20.0 and 2.3 Å. The map is contoured at a level of 0.27 e Å-3
corresponding to 1.0 times the rms deviation of the map from its mean value. CDK2 residues surrounding the meriolin 5 molecule are drawn in ball
and stick mode. Meriolin 5 carbon atoms are drawn in magenta and those of CDK2 in yellow. Oxygen atoms are colored red, and nitrogen atoms
are blue. Dotted lines represent hydrogen bonds. (B) Schematic diagram of the meriolin 5 binding mode. Arrows indicate hydrogen bonds and the
distance (Å) between the donor and acceptor atoms.
Figure 4. Cocrystal structure of variolin B bound to CDK2/cyclin A. (A) (2Fo - Fc)Rcalc electron density for variolin B calculated at the end of
refinement using map coefficients output from REFMAC with resolution between 20 and 2.1 Å. The map is contoured at a level of 0.27 e Å-3
corresponding to 1.0 times the rms deviation of the map from its mean value. CDK2 residues surrounding the variolin B molecule are drawn in ball
and stick mode. Variolin B carbon atoms are drawn in cyan, and other color symbols are as in Figure 3. (B) Schematic diagram of the variolin B
binding mode. Arrows indicate hydrogen bonds and the distance (Å) between the donor and acceptor atoms.
mode of meriolin 5. Meriolin 5 additionally interacts via its
pyrimidine-amine group with the side chains of Glu51 and Lys33
at the back of the ATP cleft (Figure 3), whereas variolin B only
interacts with the side chain of Lys33 via the hydroxyl group of
its pyridine ring.
The glycine-rich loop is the only CDK2 structural motif that
differs between the structures discussed in this report. This loop
undergoes rearrangements upon activation and on inhibitor
binding, the extent of which is dependent on the ligand bound
at the active site.86–88 It is inherently flexible and upon activation

Kinase Inhibitory 3-(Pyrimidin-4-yl)-7-azaindoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 741
Figure 5. Comparison of the binding modes of meriolin 5 (1e) and other CDK inhibitors within the CDK2 ATP-binding pocket. The crystal
structure of CDK2/cyclin A in complex with meriolin 5 (PDB code 3BHT) was superimposed on that of CDK2/cyclin A-variolin B (PDB code
3BHV) (A), CDK2/cyclin A-roscovitine (PDB code, in preparation) (B), CDK2/cyclin A-indirubin-3′-oxime-5-sulfonate (PDB code 1E9H) (C),
CDK2-SU9516 (PDB code 1PF8) (D), CDK2/cyclin A-NU2058 (PDB 1H1P) (E), CDK2-aloisine A (M. Noble, personal communication) (F).
CDK2 residues (taken from the meriolin 5 complex structure) surrounding the inhibitors are drawn in ball and stick mode. Meriolin 5 carbon atoms
are drawn in magenta and those of CDK2 in yellow. Dotted lines represent hydrogen bonds, and red stars represent water molecules.
moves away from the active site to expose the ribose binding
site to solvent. The loop retains its flexibility in fully activated
CDK2, and different orientations have been described in several
pThr160-CDK2/cyclin A/ATP-competitive inhibitor complex
structures. For example, NU2058 bound to CDK2/cyclin A
promotes interactions between the side chains of Tyr15 and
Glu51 and between the Thr14 side chain and the Asp145
carboxylate group.86 The same interactions are observed in one
of the two copies of CDK2 within the asymmetric unit of the
CDK2/cyclin A/meriolin 5 crystal. However, in the variolin
B-bound structure, the glycine-rich loop is located slightly
further away from the ATP cleft and is more flexible such that
Tyr15 in one copy has weak density and a dual conformation
was modeled. The size and the orientation of variolin B in the
active site are likely to cause this conformational change. In
contrast with the meriolin 5 structure in which the pyrimidine-
amine group is located at the back of the ATP cleft, the
pyrimidine-amine group in the variolin B structure packs against
the glycine-rich loop and bulges slightly out of the cleft (Figure
4). Indeed, this side of variolin B is decorated with water
molecules bridging it to the glycine-rich loop. It is anticipated
that, despite being numerous, the interactions of variolin B with
the CDK2 glycine-rich loop are not very strong because this
region of the protein displays signs of flexibility. In contrast,
meriolin 5 seems to better stabilize the glycine-rich region.
However, even between these two closely related compounds
differences in the orientation of Tyr15 can be seen. The
molecular basis of these two different conformations is not clear
but further outlines the inherent flexibility of this region.
We next compared the CDK2/cyclin A/meriolin 5 structure
with other inhibitors that have been cocrystallized with CDK2
(Figure 5), namely, variolin B,66 roscovitine,52 indirubin 3′-
oxime-5-sulfonate,53 SU9516,54 NU2058,55 and aloisine A.56
All these inhibitors are ATP-competitive and form at least two
hydrogen bonds with the hinge region of CDK2. A third
hydrogen bond, with the carbonyl group of Leu83, is observed
with roscovitine (Figure 5B), indirubin 3′-oxime-5-sulfonate
(Figure 5C), SU9516 (Figure 5D), NU2058 (Figure 5E), and
aloisine A (Figure 5F). An appropriate substitution on meriolin
might provide this additional hydrogen bond to Leu83 to
enhance binding. Among the inhibitors described here, only
meriolins 5 and 3, variolin B, and indirubin 3′-oxime-5-sulfonate
(through the sulfonate substituent) make a hydrogen bond
with the side chain of Lys33 and only the meriolins interact

742 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Echalier et al.
Table 1. CDK2/Cyclin A (1b, Meriolin 3), CDK2/Cyclin A (1e, Meriolin 5), and CDK2/Cyclin A (Variolin B) Cocrystal Structures: Statistics of the
Data Sets Used and of the Refined Structures
CDK2/cyclin A
(meriolin 3, 1b)
CDK2/cyclin A
(meriolin 5, 1e)
CDK2/cyclin A
(variolin B)
cell dimension (Å)
maximal resolution (Å)
observations
74.1, 133.8, 147.8
2.00
347 777
95 411 (95.5)
0.051
74.1, 134.0, 147.8
2.30
209 608
65 608 (99.3)
0.087
74.2, 134.0, 147.9
2.10
345 484
86 384 (99.7)
0.068
unique reflections, completeness (%)
a
Rmerge
mean I/σ(I)
16.3
2.11–2.00
77.0
4.8
0.225
11.1
2.42-2.30
98.3
3.0
0.439
13.1
2.21–2.10
98.9
3.8
0.386
highest resolution bin (Å)
completeness (%)
(mean I)/(mean σ(I))
Rmerge
resolution range (Å)
20.00–2.00
0.181
0.220
8.4
2.3
20.00–2.30
0.194
0.240
10.4
4.2
20.00–2.10
0.182
0.229
36.7
42.1
b
Rconv
c
Rfree
mean protein temperature factors (Å)b
mean ligand temperature factors (Å)b
PDB code
3BHT
3BHU
3BHV
a Rmerge ) (∑h∑jIh,j - I jh)/(∑h∑jIh,j), where Ih,j is the intensity of the jth observation of unique reflection h. b Rconv ) (∑h||Fo,h| - |Fc,h||)/(∑h|Fo,h|), where
Fo,h and Fc,h are the observed and calculated structure factor amplitudes for reflection h. c Rfree is equivalent to Rconv but is calculated using a 5% disjoint set
of reflections excluded from the least-squares refinement stages.
Table 2. Effects of Variolin B and 1, 1a-h, 8–12 (Meriolins 1–14) on Seven Protein Kinasesa
compd
variolin B
CDK1/cyclin B
CDK2/cyclin A
CDK5/p25
CDK9/cyclin T
GSK-3R/ꢀ
CK1
DYRK1A
0.06
0.78
0.057
0.17
0.01
0.007
0.008
35.0
1.20
25.0
0.24
2.20
1.80
0.9
0.08
0.09
0.018
0.011
0.007
0.003
0.0051
>10
1.8
>10
0.06
1.3
2.1
0.7
0.09
0.51
0.050
0.17
0.005
0.003
0.003
14.0
5.50
73.0
0.23
0.68
2.30
0.7
0.026
0.026
0.018
0.006
0.007
0.0056
0.0056
5.30
1.2
>10
0.05
1.00
1.10
0.25
0.22
0.07
0.63
0.40
0.23
0.03
0.025
0.021
63.0
4.60
>100
2.00
30.0
7.00
1.8
0.005
0.2
0.05
0.2
0.1
0.2
0.14
100.0
2.3
>10
3.0
0.08
0.13
0.035
0.029
0.032
0.037
0.040
>10
1.2
>10
0.13
0.3
1.0
0.9
1
8
meriolin 1
meriolin 2
meriolin 3
meriolin 4
meriolin 5
meriolin 6
meriolin 7
meriolin 8
meriolin 9
meriolin 10
meriolin 11
meriolin 12
meriolin 13
meriolin 14
1b
1d
1e
1f
1h
9
1g
1a
1c
12
11
10
1.3
0.6
0.9
0.6
1.3
0.8
1.3
1.1
0.23
a Variolin B and meriolins were tested at various concentrations in seven kinase assays, as described in the Experimental Section. IC50 values were
calculated from the dose–response curves and are reported in micromolar.
with the Glu51 side chain. A more detailed comparative analysis
of these cocrystal structures is provided in the Supporting
Information.
3ꢀ.57 This albeit limited SAR study, complemented with the
crystal structure, provides clear-cut information on the interac-
tions of meriolins with kinases.
Kinase Inhibitory Activities and Structure-Activity
Relationship. All synthesized meriolins 1–14, along with
variolin B as a reference, were first tested on seven purified
protein kinases, CDK1/cyclin B, CDK2/cyclin A, CDK5/p25,
CDK9/cyclin T, GSK-3R/ꢀ, CK1δ/ꢀ, and DYRK1A (Table 2).
Meriolins 1–6 are potent inhibitors of all seven kinases. As
previously observed for many CDK inhibitors, meriolins and
variolin B are also high to low nanomolar inhibitors of GSK-
Meriolin 5 binds to the CDK2 hinge region via hydrogen
bonds involving the two nitrogens within the pyrrolo[2,3-
b]pyridine ring. These positions are therefore anticipated to
tolerate little variation. This is confirmed to be the case for all
protein kinases tested in this study by the loss of potency of
meriolins upon addition of a methyl group at the position R3
(compare meriolins 2 and 8, and compare meriolins 3 and 9).

Kinase Inhibitory 3-(Pyrimidin-4-yl)-7-azaindoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 743
Table 3. Effects of Variolin B and 1, 1a-h, 8–12 (Meriolins 1–14) on
Position R2 on meriolin 5 faces the main chain carbonyl group
of Leu83. Addition of a bromide atom at this position (compare
meriolins 1 and 11) leads to a drop in inhibitory activity for
almost all protein kinases tested, but this effect is particularly
pronounced against CDK9 and GSK-3. CDK1, CDK2, and
CDK5 are less affected by the bromide addition. This result
might reflect a potential difference between the ATP binding
sites of the different CDKs that might be exploited to develop
more discriminating inhibitors.
the Survival of Various Cell Linesa
compd
SH-SY5Y HEK293
GBM
KMS-11 LS 174T
variolin B
meriolin 1
meriolin 2
0.24
0.67
0.41
1.92
2.20
0.96
0.24
0.22
0.33 ( 0.12 0.08 ( 0.04 0.88
1.38 ( 0.21 1.27 ( 0.27 1.50
1.21 ( 0.39 0.60 ( 0.10 1.06
0.14 ( 0.10 0.12 ( 0.02 0.13
0.13 ( 0.09 0.06 ( 0.03 0.10
1
8
1b meriolin 3
1d meriolin 4
1e meriolin 5
1f meriolin 6
1h meriolin 7
meriolin 8
1g meriolin 9
1a meriolin 10 1.92
1c meriolin 11 43
12 meriolin 12 >100
11 meriolin 13 28.0
10 meriolin 14 10.2
0.073
0.081
0.026
0.038
>100
>30
0.085 0.08 ( 0.03 0.04 ( 0.01 0.047
0.089 0.08 ( 0.02 0.04 ( 0.02 0.060
>100 >20
>20
>30
>30
>30
>10
>30
>30
23.5
10.0
Third, the replacement of the amino group by -SCH3
(compare meriolins 12 and 3) or by -H (compare meriolins 13
and 2, and compare meriolins 14 and 3) also leads to a reduction
of inhibitory activity. This is likely to result from the loss of a
key hydrogen bond between the amine group on the pyrimidine
ring with CDK2 Glu51 (Figure 3).
9
>30
>30
10.8
>20
>20
>30
>20
>20
10.0 ( 5
7.5 ( 3.5
>15
>20
>15
10.0 ( 4
>100 >20
66
84
35.5
>20
>20
10.0 ( 6
Fourth, we investigated the effects of substitution at position
R1. The effects of the substitutions could be rationalized by
reference to the structures of CDK2/cyclin A/meriolin 3 (ref
66) and /meriolin 5 (this study). The inhibitory activity of the
meriolins was particularly sensitive to modifications at this
position. Increasing the size of aliphatic groups added at R1
(meriolins 2-6) resulted in an increase in affinity. Groups at
R1 interact with the glycine-rich loop region, and the propyl
group of meriolin 5 is observed to pack more extensively against
the glycine-rich loop than the smaller meriolin 3 methyl group.
The most potent inhibitors in this series were meriolins 5 and
6, which exhibited IC50 values in the low nanomolar range for
CDKs (Table 2), 20 nM for GSK-3, 40 nM for DYRK1A, and
submicromolar values for CK1.
a Variolin B and meriolins were tested at various concentrations for their
effects on neuroblastoma SH-SY5Y cells, HEK293 cells, glioma GBM cells,
myeloma KMS-11, and colorectal adenocarcinoma LS 174T cells. Cell
survival was estimated 48 h after the addition of each compound using the
MTS reduction assay (SH-SY5Y, HEK293, LS 174T) or neutral red (GBM,
KMS-11). IC50 values were calculated from the dose–response curves and
are reported in micromolar (average ( SE of three independent measure-
ments performed in triplicate).
merase II on Ser2, its CDK9-specific site, was most sensitive
to the same meriolins (Figure 6C).
We next analyzed the level of Mcl-1, a key survival factor
frequently expressed in tumor cells (reviews in refs 62 and 63),
in SH-SY5Y cells exposed to all the synthesized meriolins
(Figure 6D). Several meriolins triggered a complete disappear-
ance of Mcl-1. An excellent correlation was observed with
inhibition of CDK9 (Tables 2 and 3) with the exception of
meriolin 10.
We also assayed the activity of caspases in SH-SY5Y cells
exposed to two concentrations of each meriolin (Figure 7).
Results show solid induction of caspase activity by the most
potent meriolins as detected in vitro (purified kinases) and in
vivo (cell death induction, inhibition of CDK substrate phos-
phorylation, and induction of Mcl-1).
As this work was in progress, 3-(N-substituted 2-aminopy-
rimidin-4-yl)-7-azaindoles were reported as potent CDK1 in-
hibitors.58 These molecules share the central core of meriolins
but have bulky substitutions on the pyrimidin-2-ylamine group
that would appear to be incompatible with the mode of binding
of the meriolins to CDK2. However, an unambiguous assign-
ment of the binding mode must await determination of a CDK2
cocomplex structure with Dr. Huang’s molecules.
Cellular Effects of Meriolins. All synthesized meriolins 1–14
were next tested for their effect on cell survival of five cell
lines: neuroblastoma SH-SY5Y cells, HEK293 cells, glioma
GBM cells, multiple myeloma KMS-11, and colorectal adeno-
carcinoma LS 174T cells (Table 3). Cell survival was estimated
48 h after the addition of each compound using the MTS
reduction assay (SH-SY5Y, HEK293, LS 174T) or neutral red
(GBM, KMS-11). IC50 values were calculated from the dos-
e–response curves. Some meriolins were particularly potent at
compromising cell survival. Although an overall correlation was
observed between kinase inhibition of the six enzymes with cell
death induction, an excellent correlation was observed with
inhibition of CDK9 (Tables 2 and 3) with the exception of
meriolin 10.
Finally we tested the effects of a 5 day treatment with meriolin
3 on the proliferation of HCT116, a human colon cancer cell
line grown in 3D spheroids, using a method adapted from Del
Duca et al.64 This method allows the preparation of individual-
ized spheroids that are homogeneous in terms of size distribu-
tion. Each spheroid was prepared from 500 HCT116 cells. Four
days after the beginning of the culture, spheroids were treated
with various concentrations of meriolin 3 for a 5 day period.
The size of the spheroids was then measured under the
microscope and compared to the spheroids’ size at the beginning
of the treatment (Figure 8A). While the control spheroids
reached a 1300 µm diameter, the meriolin 3 treated spheroids
only reached a diameter of 720 µm. Meriolin 3 prevented
HCT116 cell proliferation in a concentration-dependent manner
(Figure 8B), with an IC50 value of 0.063 ( 0.015 µM. Spheroid
culture shows chemoresistance to a wide range of agents
compared to conventional monolayers.65 Meriolin 3 showed
equal or even better activity in HCT116 spheroids than in the
respective monolayer cell culture (IC50 ) 0.94 µM),66 indicating
that this compound has the potential to circumvent multicellular
drug resistance and, as such, may show promising activity
against solid tumors.
We next tested the effects of all these compounds on the
phosphorylation of several CDK substrates in neuroblastoma
SH-SY5Y cells (Figure 6). Some meriolins prevented the
phosphorylation of retinoblastoma protein (CDK2/CDK4 sites)59
(Figure 6A), protein phosphatase 1R (CDK1 site)60 (Figure 6B),
and RNA polymerase II (CDK9 site)61 (Figure 6C). The
meriolins that were initially detected as being most active at
inhibiting purified kinases (Table 2) and at inducing cell death
(Table 3) were also found to be the most active at inhibiting
site-specific phosphorylation of CDK substrates in cell culture.
In agreement with the extreme sensitivity of CDK9 to several
meriolins in vitro, the in vivo phosphorylation of RNA poly-
Conclusion
Meriolins represent a hybrid structure between the natural
products meridianins and variolins and constitute a potent kinase

744 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Echalier et al.
Figure 6. Effects of meriolins 1–14 (1, 1a-h, 8–12) and variolin B on the phosphorylation at CDK-specific sites of retinoblastoma protein Rb,
protein phosphatase PP1R and RNA polymerase II, and Mcl-1 levels. Cells were exposed for 24 h to variolin B or meriolins 1–14 (0.1 and/or 1 µM
final concentration) or vehicle (DMSO). Proteins were then resolved by SDS-PAGE followed by Western blotting with antibodies phospho-Rb
(A), phospho-Thr320 PP1R (B), phospho-Ser2 RNA polymerase II (C), and Mcl-1 (D).
to use. All reactions requiring anhydrous conditions were conducted
in a flame-dried apparatus. Melting points were determined using
a Büchi capillary instrument and are uncorrected. IR spectra were
recorded on a Perkin-Elmer 681 infrared spectrophotometer. 1H
NMR and 13C spectra were recorded on a Bruker Avance 300 or
500 MHz spectrometer. Chemical shifts are reported in ppm (δ)
relative to tetramethylsilane as an internal standard. Mass spectra
were recorded with a Perkin-Elmer SCIEX API spectrometer.
Elemental analyses were performed on a Thermoquest Flash 1112
series EA analyzer. Thin layer chromatography (TLC) analyses were
conducted on aluminum sheets silica gel, Merck 60F254. The spots
were visualized using an ultraviolet light. Flash chromatography
was carried out on silica gel 60 (40–63 µm, Merck) using the
indicated solvents. The light petroleum ether refers to the fraction
boiling at 40–60 °C. Compounds 2a-c were commercially avail-
Figure 7. Effects of all meriolins 1–14 (1, 1a-h, 8–12), and variolin
B on the activity of caspases. Cells were exposed for 24 h to variolin
B or meriolins 1–14 (0.1 or 1 µM final concentration) or vehicle
(DMSO). DEVDase activity was measured as arbitrary fluorescence
units. Every point is the mean ( SE of three independent determinations.
able. 4-Hydroxy-7-azaindole has been prepared according to the
previously reported method.47
Variolin B. Variolin B was synthesized in eight steps as
described previously.43 The trifluoroacetate salt was neutralized by
sonication with concentrated aqueous NH3/MeOH to give the free
base. Concentration in vacuo (35 °C, 0.03 mm/Hg to remove
ammonium trifluoroacetate) gave variolin B as a brown-orange
solid. Mp 320 °C (dec) (lit.37 45 °C, dec). IR (KBr): ν 3085, 1670,
inhibitory scaffold. Their cocrystallization with CDK2/cyclin
A and the determination of SARs for a small series of analogues
provide some interesting clues in order to understand their
molecular mechanism of action and to improve their effects.
The strong antiproliferative and proapoptotic effects of meriolins
in tumor cells cultured under various conditions (in monolayers,
in spheroids, and as xenografts in nude mice66) provide a sound
rationale for their further optimization and evaluation of their
antitumor properties. Meriolins also provide promising phar-
macological inhibitors of CDKs involved in noncancer patholo-
gies such as polycystic kidney disease, various neurodegener-
ative diseases, stroke, inflammation, and some viral infections.
1
1572, 1477, 1458, 1298 cm-1. H NMR (500 MHz, DMSO- d6):
δ 6.81 (d, 1H, J ) 5.6 Hz, H-3), 6.97 (s, 2H, NH2), 7.14 (d, 1H,
J ) 5.6 Hz, H-5′), 7.23 (d, 1H, J ) 6.7 Hz, H-6), 7.63 (d, 1H, J
) 6.7 Hz, H-7), 8.17 (d, 1H, J ) 5.6 Hz, H-2), 8.27 (d, 1H, J )
5.6 Hz, H-6′), 8.45 (broad s, 1H, NH), 9.79 (broad s, 1H, NH),
16.05 (s, 1H, OH). 13C NMR (75 MHz, DMSO- d6): δ 99.6 (C-5),
100.3 (C-6), 106.0 (C-5′), 107.5 (C-3), 111.2 (C-4a), 137.1 (C-
5a), 143.1 (C-2), 144.6 (C-7), 144.9 (C-10a), 150.3 (C-9), 158.3
(C-4′), 159.8 (C-4), 160.0 (C-6′), 161.4 (C-2′). HRMS (ES): calcd
for C14H12N7O (MH+) 294.1103, found 294.1096.
Deoxyvariolin B. This material was prepared using the synthesis
described by Morris and co-workers43 and afforded deoxyvariolin
B as a yellow microcrystalline solid. Mp 220–240 °C (dec) (lit.41
160–162 °C). IR (KBr): ν 3304, 3142–2849 (series of weak bands),
Experimental Section
1
1647, 1574, 1514, 1472, 1456, 1269 cm-1. H NMR (500 MHz,
Chemistry. General Procedures. Commercial reagents (Fluka,
Aldrich) were used without purification. Solvents were distilled prior
DMSO-d6): δ 6.51 (s, 2H, NH2), 7.05 (d, 1H, J ) 5.4 Hz, H-5′),

Kinase Inhibitory 3-(Pyrimidin-4-yl)-7-azaindoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 745
Figure 8. Effects of meriolin 3 (1b) on the survival of HCT116 cells grown in spheroids. After 4 days of culture in spheroids, HCT116 cells were
exposed to various concentrations of meriolin 3 for 5 days. (A) Photograph of control and meriolin 3 (1 µM) treated spheroids (scale, 100 µm). (B)
Spheroid volumes were estimated and compared to those of the initial day spheroid volume. A ratio of 1 (dashed line) indicates complete inhibition
of cell growth.
7.57 (dd, 1H, J ) 4.8, 8.2 Hz, H-3), 7.62 (d, 1H, J ) 6.3 Hz, H-7),
7.67 (d, 1H, J ) 6.3 Hz, H-6), 8.22 (d, 1H, J ) 5.4 Hz, H-6′), 8.44
(dd, 1H, J ) 1.5, 4.8 Hz, H-2), 8.50 (broad s, 1H, NH), 8.90 (dd,
1H, J ) 1.5, 8.2 Hz, H-4), 9.40 (broad s, 1H, 9-NH). 13C NMR
(75 MHz, DMSO-d6): δ 99.6 (C-5), 101.9 (C-6), 107.1 (C-5′), 121.0
(C-3), 121.7 (C-4a), 129.4 (C-4), 138.3 (C-5a), 140.3 (C-2), 143.0
(C-10a), 144.0 (C-7), 149.9 (C-9), 158.2 (C-6′), 161.6 (C-4′), 163.6
(C-2′). MS (EI): m/z (rel intensity) 277 (100), 236 (54). HRMS
(EI): **calcd for C14H11N7 (M+) 277.1076, found 277.1072.
reflux for 8 h. After the mixture was cooled, saturated aqueous
Na2CO3 was added to neutralize the medium (pH 7–8). After
addition of EtOAc, stirring, and decantation, phases were separated.
The organic phase was dried over MgSO4 and evaporated. The
crude residue was purified by column chromatography (EtOAc) to
give 3a (98 mg, 85%). Mp >210 °C (MeOH). IR (KBr): ν 3196,
1
3088, 3012, 2872, 1656, 1565, 1455, 1391, 1289, 817 cm-1. H
NMR (300 MHz, CDCl3): δ 2.63 (s, 3H, CH3), 7.41 (d, 1H, J )
5.6 Hz, H-5), 8.12 (s, 1H, H-2), 8.27 (d, 1H, J ) 5.6 Hz, H-6). MS
(IS): m/z 195 (M + H+). Anal. (C9H7ClN2O) C, H, N.
3-[(2-Amino)pyrimidin-4-yl]-1 H-pyrrolo[2,3-b]pyridine (1).
Meriolin 1. This compound was prepared using the synthesis
3-Acetyl-4-methoxy-1H-pyrrolo[2,3-b]pyridine (3b). Accord-
ing to the same procedure, compound 3b was prepared in 55%
yield from 4-methoxy-7-azaindole 2b. Mp >210 °C (MeOH). IR
(KBr): ν 3198, 3122, 2841, 1640, 1583, 1463, 1400, 1311, 1100,
1
described by Fresneda and Molina.46 Mp >210 °C (MeOH). H
NMR data (300 MHz, DMSO-d6) were identical to those described
previously.46
4-Ethoxy-1H-pyrrolo[2,3-b]pyridine (2d). Under N2 atmo-
sphere, diethyl azodicarboxylate (520 µL, 3.3 mmol) was added
dropwise to a solution of triphenylphosphine (1.04 g, 3.96 mmol)
in dry THF (13 mL). This mixture was added to a solution of
4-hydroxy-7-azaindole (222 mg, 1.65 mmol) and EtOH (115 µL,
1.98 mmol) in dry THF (41 mL). The final mixture was stirred for
2 h at room temperature. The solvent was evaporated. The crude
residue was purified by column chromatography (CH2Cl2/MeOH,
98:2) to give 2d (214 mg, 80%). Mp 176–178 °C (Et2O). 1H NMR
(300 MHz, CDCl3): δ 1.52 (t, 3H, J ) 7.1 Hz, CH3), 4.26 (q, 2H,
J ) 7.1 Hz, CH2), 6.53 (d, 1H, J ) 5.6 Hz, H-5), 6.59 (broad s,
1H, H-3), 7.18 (broad s, 1H, H-2), 8.18 (d, 1H, J ) 5.6 Hz, H-6),
9.64 (broad s, 1H, NH). MS (IS): m/z 163 (M + H+). Anal.
(C9H10N2O) C, H, N.
1
848 cm-1. H NMR (300 MHz, DMSO-d6): δ 2.50 (s, 3H, CH3),
3.92 (s, 3H, CH3), 6.79 (d, 1H, J ) 5.6 Hz, H-5), 8.11 (s, 1H,
H-2), 8.17 (d, 1H, J ) 5.6 Hz, H-6), 12.36 (broad s, 1H, NH). MS
(IS): m/z 191 (M + H+). Anal. (C10H10N2O2) C, H, N.
3-Acetyl-6-bromo-1H-pyrrolo[2,3-b]pyridine (3c). According
to the same procedure, compound 3c was prepared in 99% yield
from 6-bromo-7-azaindole 2c. Mp >210 °C (MeOH). IR (KBr): ν
3122, 3035, 2985, 2893, 1651, 1573, 1512, 1418, 1313, 1275, 1102,
1
943, 918, 818 cm-1. H NMR (300 MHz, DMSO-d6): δ 2.46 (s,
3H, CH3), 7.42 (d, 1H, J ) 8.2 Hz, H-5), 8.38 (d, 1H, J ) 8.2 Hz,
H-4) 8.49 (d, 1H, J ) 3.0 Hz, H-2), 12.68 (broad s, 1H, NH). MS
(IS): m/z 241 (81Br, M + H+), 239 (79Br, M + H+). Anal.
(C9H7BrN2O) C, H, N.
4-Propoxy-1H-pyrrolo[2,3-b]pyridine (2e). According to the
same procedure, compound 2e was prepared in 78% yield from
4-hydroxy-7-azaindole and PrOH. Mp 189–191 °C (Et2O). 1H NMR
(300 MHz, CDCl3): δ 1.10 (t, 3H, J ) 7.4 Hz, CH3), 1.88–1.99
(m, 2H, CH2), 4.20 (t, 2H, J ) 6.6 Hz, CH2), 6.59 (d, 1H, J ) 5.8
Hz, H-5), 6.63 (d, 1H, J ) 3.6 Hz, H-3), 7.21 (d, 1H, J ) 3.6 Hz,
H-2), 8.15 (d, 1H, J ) 5.8 Hz, H-6), 10.34 (broad s, 1H, NH). MS
(IS): m/z 177 (M + H+). Anal. (C10H12N2O) C, H, N.
4-(1-Methylethoxy)-1H-pyrrolo[2,3-b]pyridine (2f). According
to the same procedure, compound 2f was prepared in 76% yield
from 4-hydroxy-7-azaindole and i-PrOH. Mp 182–184 °C (Et2O).
1H NMR (300 MHz, CDCl3): δ 1.44 (d, 6H, J ) 6.0 Hz, 2 CH3),
4.82 (hept, 1H, J ) 6.0 Hz, CH), 6.52 (d, 1H, J ) 5.7 Hz, H-5),
6.57 (d, 1H, J ) 3.6 Hz, H-3), 7.19 (d, 1H, J ) 3.6 Hz, H-2), 8.17
(d, 1H, J ) 5.7 Hz, H-6), 10.70 (broad s, 1H, NH). MS (IS): m/z
177 (M + H+). Anal. (C10H12N2O) C, H, N.
3-Acetyl-4-ethoxy-1H-pyrrolo[2,3-b]pyridine (3d). According
to the same procedure, compound 3d was prepared in 84% yield
from 4-ethoxy-7-azaindole 2d. Mp >210 °C (MeOH). IR (KBr): ν
1
3198, 3119, 2837, 1643, 1580, 1463, 1309, 1086, 844 cm-1. H
NMR (300 MHz, DMSO-d6): δ 1.43 (t, 3H, J ) 7.2 Hz, CH3),
2.54 (s, 3H, CH3), 4.22 (q, 2H, J ) 7.2 Hz, CH2), 6.78 (d, 1H, J
) 5.7 Hz, H-5), 8.04 (s, 1H, H-2), 8.14 (d, 1H, J ) 5.7 Hz, H-6),
12.33 (broad s, 1H, NH). MS (IS): m/z 205 (M + H+). Anal.
(C11H12N2O2) C, H, N.
3-Acetyl-4-propoxy-1H-pyrrolo[2,3-b]pyridine (3e). According
to the same procedure, compound 3e was prepared in 80% yield
from 4-propoxy-7-azaindole 2e. Mp >210 °C (MeOH). IR (KBr):
ν 3198, 3124, 2879, 1640, 1583, 1464, 1401, 1292, 1091, 852 cm-1
.
1H NMR (300 MHz, DMSO-d6): δ 1.07 (t, 3H, J ) 7.3 Hz, CH3),
1.77–1.88 (m, 2H, CH2), 2.53 (s, 3H, CH3), 4.11 (t, 2H, J ) 6.3
Hz, CH2), 6.78 (d, 1H, J ) 5.6 Hz, H-5), 8.08 (s, 1H, H-2), 8.14
(d, 1H, J ) 5.6 Hz, H-6), 12.32 (broad s, 1H, NH). MS (IS): m/z
219 (M + H+). Anal. (C12H14N2O2) C, H, N.
3-Acetyl-4-chloro-1H-pyrrolo[2,3-b]pyridine (3a). A solution
of acetic anhydride (0.13 mL, 1.8 mmol), 4-chloro-7-azaindole 2a
(90 mg, 0.59 mmol), and trifluoroacetic acid (5 mL) was heated at

746 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Echalier et al.
3-Acetyl-1-benzenesulfonyl-4-chloro-1H-pyrrolo[2,3-b]pyri-
dine (4a). At 0 °C, sodium hydride (26 mg, 0.64 mmol, 60% in
oil) was added portionwise to a solution of 3a (125 mg, 0.64 mmol)
in dry THF (20 mL). The solution was stirred for 45 min at 0 °C,
and a solution of benzenesulfonyl chloride (0.11 mL, 0.83 mmol)
was added to the reaction mixture. The final solution was stirred at
room temperature for 4 h. Addition of water was performed at 0
°C, and then solvent was removed. The residue was partitioned
between H2O and EtOAc (50 mL, 1:1) and extracted. The organic
phase was dried over MgSO4 and evaporated. The crude solid was
purified by column chromatography (petroleum ether/EtOAc, 8:2)
to give 4a (200 mg, 93%). Mp 160–162 °C (CH2Cl2/pentane). IR
(KBr): ν 3152, 3020, 2943, 1686, 1382, 1199, 1185, 1092, 995,
746 cm-1. 1H NMR (300 MHz, CDCl3): δ 2.63 (s, 3H, CH3), 7.30
(d, 1H, J ) 5.2 Hz, H-5), 7.54 (broad t, 2H, J ) 7.4 Hz, Harom),
7.66 (broad t, 1H, J ) 7.4 Hz, Harom), 8.25 (broad d, 2H, J ) 7.4
Hz, Harom), 8.32 (d, 1H, J ) 5.2 Hz, H-6), 8.34 (s, 1H, H-2). MS
(IS): m/z 335 (M + H+). Anal. (C15H11ClN2O3S) C, H, N.
H-5), 7.26 (s, 1H, H-2) 8.14 (d, 1H, J ) 5.8 Hz, H-6). MS (IS):
m/z 303 (M + H+). Anal. (C10H11IN2O) C, H, N.
1-Benzenesulfonyl-3-iodo-4-(1-methylethoxy)-1H-pyrrolo[2,3-
b]pyridine (6). Following the procedure described for the prepara-
tion of 4a, compound 6 was obtained in 68% yield from 5. Mp
151–153 °C (CH2Cl2/pentane). IR (KBr): ν 3137, 2979, 1588, 1572,
1487, 1372, 1293, 1189, 1172, 1114, 819, 740 cm-1 1H NMR
.
(300 MHz, CDCl3): δ 1.42 (d, 6H, J ) 6.2 Hz, CH3), 4.71 (hept,
1H, J ) 6.2 Hz, CH), 6.59 (d, 1H, J ) 5.7 Hz, H-5), 7.48 (broad
t, 2H, J ) 7.9 Hz, Harom), 7.58 (broad t, 1H, J ) 7.9 Hz, Harom),
7.69 (s, 1H, H-2), 8.17 (broad d, 2H, J ) 7.9 Hz, Harom), 8.27 (d,
1H, J ) 5.7 Hz, H-6). MS (IS): m/z 443 (M + H+). Anal.
(C16H15IN2O3S) C, H, N.
3-Acetyl-1-(1-benzenesulfonyl)-4-(1-methylethoxy)-1H-pyr-
rolo[2,3-b]pyridine (4f). To a stirred mixture of 6 (67 mg, 0.15
mmol), freshly prepared Pd(PPh3)4 (18 mg, 0.015 mmol), and LiCl
(16 mg, 0.38 mmol) in dry DMF (3 mL) was added n-tributyl(1-
ethoxyvinyl)stannane (77 µL, 0.23 mmol). The solution was stirred
at 80 °C for 18 h. After the mixture was cooled, 5% HCl solution
(10 mL) was added and the solution was stirred for 20 min at room
temperature. The solvent was evaporated in vacuo. The residue was
partitioned between saturated aqueous Na2CO3 solution and EtOAc
(pH 7–8) and extracted. The organic phase was washed with KF
solution, dried over MgSO4, and evaporated. The crude solid was
purified by column chromatography (petroleum ether/EtOAc, 7:3)
to give 4f (48 mg, 89%). Mp 118–120 °C (CH2Cl2/petroleum ether).
IR (KBr): ν 3127, 2981, 1686, 1586, 1528, 1496, 1376, 1300, 1172,
1121, 817, 748 cm-1. 1H NMR (300 MHz, CDCl3): δ 1.43 (d, 6H,
J ) 6.0 Hz, CH3), 2.65 (s, 3H, CH3), 4.78 (hept, 1H, J ) 6.0 Hz,
CH), 6.69 (d, 1H, J ) 6.0 Hz, H-5), 7.51 (broad t, 2H, J ) 7.9 Hz,
3-Acetyl-1-benzenesulfonyl-4-methoxy-1H-pyrrolo[2,3-b]py-
ridine (4b). According to the same procedure, compound 4b was
prepared in 90% yield from 3b. Mp 156–158 °C (CH2Cl2/pentane).
IR (KBr): ν 3149, 3015, 2943, 1662, 1574, 1511, 1374, 1293, 1196,
1
1177, 1111, 750 cm-1. H NMR (300 MHz, CDCl3): δ 2.63 (s,
3H, CH3), 3.98 (s, 3H, CH3), 6.73 (d, 1H, J ) 5.6 Hz, H-5), 7.51
(broad t, 2H, J ) 7.4 Hz, Harom), 7.62 (broad t, 1H, J ) 7.4 Hz,
H
arom), 8.21 (s, 1H, Harom), 8.24 (broad d, 2H, J ) 7.4 Hz, Harom),
8.34 (d, 1H, J ) 5.7 Hz, H-6). MS (IS): m/z 331 (M + H+). Anal.
(C16H14N2O4S) C, H, N.
3-Acetyl-1-benzenesulfonyl-6-bromo-1H-pyrrolo[2,3-b]pyri-
dine (4c). According to the same procedure, compound 4c was
prepared in 88% yield from 3c. Mp 172–174 °C (CH2Cl2/pentane).
IR (KBr): ν 3141, 3087, 1673, 1585, 1529, 1440, 1387, 1207, 1183,
H
arom), 7.61 (broad t, 1H, J ) 7.9 Hz, Harom), 8.17 (s, 1H, H-2),
8.23 (broad d, 2H, J ) 7.9 Hz, Harom), 8.29 (d, 1H, J ) 6.0 Hz,
H-6). MS (IS): m/z 359 (M + H+). Anal. (C18H18N2O4S) C, H, N.
3-Acetyl-4-methoxy-1-methyl-1H-pyrrolo[2,3-b]pyridine (4g).
Under an inert atmosphere, a solution of 3b (80 mg, 0.42 mmol),
dimethyl sulfate (0.07 mL, 0.74 mmol), and K2CO3 (82 mg, 0.59
mmol) in dry acetone (15 mL) was refluxed for 5.5 h. After the
mixture was cooled, solvent was evaporated. The residue was
partitioned between H2O and EtOAc (30 mL, 1:1) and extracted.
The organic phase was dried over MgSO4 and evaporated. The
crude residue was purified by column chromatography (EtOAc) to
give 4g (70 mg, 81%). Mp 79–81 °C (CH2Cl2/hexane). IR (KBr):
ν 3123, 2984, 1649, 1590, 1573, 1521, 1448, 1335, 1313, 1285,
1
1098, 1087, 974, 929, 758 cm-1. H NMR (300 MHz, CDCl3): δ
2.56 (s, 3H, CH3), 7.44 (d, 1H, J ) 8.3 Hz, H-5), 7.58 (broad t,
2H, J ) 7.9 Hz, Harom), 7.68 (broad t, 1H, J ) 7.9 Hz, Harom), 8.29
(s, 1H, Harom), 8.31 (broad d, 2H, J ) 7.9 Hz, Harom), 8.42 (d, 1H,
J ) 8.3 Hz, H-4). MS (IS): m/z 381 (81Br, M + H+), 379 (79Br, M
+ H+). Anal. (C15H11BrN2O3S) C, H, N.
3-Acetyl-1-benzenesulfonyl-4-ethoxy-1H-pyrrolo[2,3-b]pyri-
dine (4d). According to the same procedure, compound 4d was
prepared in 68% yield from 3d. Mp 151–153 °C (CH2Cl2/pentane).
IR (KBr): ν 3154, 3062, 2981, 1660, 1571, 1508, 1376, 1291, 1197,
1
1179, 1102, 751 cm-1. H NMR (300 MHz, CDCl3): δ 1.52 (t,
1239, 1199, 1083, 1054, 961, 800 cm-1 1H NMR (300 MHz,
.
3H, J ) 7.2 Hz, CH3), 2.65 (s, 3H, CH3), 4.21 (q, 2H, J ) 7.2 Hz,
CH2), 6.70 (d, 1H, J ) 5.7 Hz, H-5), 7.51 (broad t, 2H, J ) 7.4
Hz, Harom), 7.61 (broad t, 1H, J ) 7.4 Hz, Harom), 8.20 (s, 1H, H-2),
8.23 (broad d, 2H, J ) 7.4 Hz, Harom), 8.31 (d, 1H, J ) 5.7 Hz,
H-6). MS (IS): m/z 345 (M + H+). Anal. (C17H16N2O4S) C, H, N.
3-Acetyl-1-benzenesulfonyl-4-propoxy-1H-pyrrolo[2,3-b]py-
ridine (4e). According to the same procedure, compound 4e was
prepared in 70% yield from 3e. Mp 119–121 °C (CH2Cl2/pentane).
IR (KBr): ν 3137, 2967, 1676, 1583, 1520, 1496, 1374, 1301, 1189,
CDCl3): δ 2.61 (s, 3H, CH3), 3.85 (s, 3H, CH3), 3.99 (s, 3H, CH3),
6.64 (d, 1H, J ) 5.5 Hz, H-5), 7.76 (s, 1H, H-2), 8.23 (d, 1H, J )
5.5 Hz, H-6). MS (IS): m/z 205 (M + H+). Anal. (C11H12N2O2) C,
H, N.
1-(1-Benzenesulfonyl-4-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)-
3-dimethylaminopropenone (7a). Under an inert atmosphere, a
solution of 4a (450 mg, 1.34 mmol) and DMF-DMA (1.07 mL,
8.0 mmol) in dry DMF (15 mL) was stirred at 90 °C for 8 h. After
the mixture was cooled, the solvent was evaporated under reduced
pressure. The residue was partitioned between H2O and EtOAc (50
mL, 1:1) and extracted. The organic phase was dried over MgSO4
and evaporated. The crude solid was purified by column chroma-
tography (EtOAc) to give 7a (310 mg, 60%). Mp 139–141 °C
(MeOH). IR (KBr): ν 3132, 3021, 2902, 1646, 1562, 1526, 1381,
1
1171, 1115, 748 cm-1. H NMR (300 MHz, CDCl3): δ 1.09 (t,
3H, J ) 7.3 Hz, CH3), 1.87–1.96 (m, 2H, CH2), 2.64 (s, 3H, CH3),
4.09 (t, 2H, J ) 6.6 Hz, CH2), 6.70 (d, 1H, J ) 5.7 Hz, H-5), 7.51
(broad t, 2H, J ) 7.4 Hz, Harom), 7.61 (broad t, 1H, J ) 7.4 Hz,
H
arom), 8.19 (s, 1H, H-2), 8.23 (broad d, 2H, J ) 7.4 Hz, Harom),
8.31 (d, 1H, J ) 5.7 Hz, H-6). MS (IS): m/z 359 (M + H+) Anal.
(C18H18N2O4S) C, H, N.
1
1169, 989, 885, 728 cm-1. H NMR (300 MHz, CDCl3): δ 2.93
(broad s, 3H, CH3), 3,15 (broad s, 3H, CH3), 5.49 (d, 1H, J ) 12.6
Hz, dCH), 7.23 (d, 1H, J ) 5.3 Hz, H-5), 7.49 (t, 2H, J ) 7.4 Hz,
3-Iodo-4-(1-methylethoxy)-1H-pyrrolo[2,3-b]pyridine (5). At
room temperature and under an inert atmosphere, a solution of
iodine (74 mg, 0.29 mmol) in dry DMF (0.5 mL) was added
dropwise to a solution of 4-(1-methylethoxy)-1H-pyrrolo[2,3-
b]pyridine 2f (50 mg, 0.28 mmol) and potassium hydroxide (88
mg, 1.56 mmol) in dry DMF (0.5 mL). The solution was stirred
for 2.5 h at room temperature. The solvent was evaporated, and
the crude residue was taken up in water. The suspension obtained
was filtered to afford 5 (70 mg, 82%). Mp 181–183 °C (MeOH).
IR (KBr): ν 3198, 3116, 2977, 1586, 1509, 1316, 1107, 922, 802
H
arom), 7.47–7.65 (m, 2H, Harom + dCH), 8.00 (s, 1H, H-2), 8.19
(d, 2H, J ) 7.7 Hz, Harom), 8.29 (d, 1H, J ) 5.3 Hz, H-6). MS
(IS): m/z 390 (M + H+). Anal. (C18H16ClN3O3S) C, H, N.
1-(1-Benzenesulfonyl-4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-
yl)-3-dimethylaminopropenone (7b). According to the same
procedure, compound 7b was obtained in 78% yield from 4b. Mp
210–212 °C (MeOH). IR (KBr): ν 3144, 2950, 1632, 1578, 1544,
1414, 1371, 1294, 1193, 1166, 888, 798 cm-1. 1H NMR (300 MHz,
CDCl3): δ 2.91 (broad s, 3H, CH3), 3.12 (broad s, 3H, CH3), 3.94
(s, 3H, CH3), 5.62 (d, 1H, J ) 12.6 Hz, dCH), 6.68 (d, 1H, J )
1
cm-1. H NMR (300 MHz, CDCl3): δ 1.49 (d, 6H, J ) 6.0 Hz,
CH3), 4.80 (hept, 1H, J ) 6.0 Hz, CH), 6.55 (d, 1H, J ) 5.8 Hz,

Kinase Inhibitory 3-(Pyrimidin-4-yl)-7-azaindoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 747
5.7 Hz, H-5), 7.47 (t, 2H, J ) 7.4 Hz, Harom), 7.57 (t, 1H, J ) 7.4
Hz, Harom), 7.65 (broad d, 1H, J ) 12.6 Hz, dCH), 7.96 (s, 1H,
H-2), 8.18 (d, 2H, J ) 7.4 Hz, Harom), 8.31 (d, 1H, J ) 5.7 Hz,
H-6). MS (IS): m/z 386 (M + H+). Anal. (C19H19N3O4S) C, H, N.
1-(1-Benzenesulfonyl-6-bromo-1H-pyrrolo[2,3-b]pyridin-3-
yl)-3-dimethylaminopropenone (7c). According to the same
procedure, compound 7c was obtained in 62% yield from 4c. Mp
181–183 °C (MeOH). IR (KBr): ν 3142, 3070, 2912, 1643, 1569,
tography (CH2Cl2/MeOH, 95:5) to afford 1a (45 mg, 51%) and
1 h (32 mg, 31%). Mp >210 °C (MeOH). IR (KBr): ν3310,
3135, 3021, 2852, 1659, 1563, 1515, 1459, 1398, 1311 cm-1
.
1H NMR (300 MHz, DMSO-d6): δ 6.48 (broad s, 2H, NH2),
6.85 (d, 1H, J ) 5.3 Hz, H-5), 7.26 (d, 1H, J ) 5.3 Hz, H-5′),
7.94 (s, 1H, H-2), 8.20 (d, 1H, J ) 5.3 Hz, H-6), 8.22 (d, 1H,
J ) 5.3 Hz, H-6′), 12.49 (broad s, 1H, NH), MS (IS): m/z 246
(M + H+). Anal. (C11H8ClN5) C, H, N.
1555, 1526, 1391, 1279, 1172, 1085, 1034, 964, 889, 817 cm-1
.
3-[(2-Amino)pyrimidin-4-yl]-4-(2-methoxyethoxy)-1H-pyrro-
lo[2,3-b]pyridine (1h). Meriolin 7. Compound 1h was obtained
in 31% yield from 7a. Mp 172–174 °C (MeOH). IR (KBr): ν 3314,
1H NMR (300 MHz, CDCl3): δ 2.89 (broad s, 3H, CH3), 3.10 (broad
s, 3H, CH3), 5.52 (d, 1H, J ) 12.2 Hz, dCH), 7.34 (d, 1H, J ) 8.2
Hz, H-5), 7.49 (t, 2H, J ) 7.5 Hz, Harom), 7.58 (t, 1H, J ) 7.5 Hz,
1
3182, 3097, 2924, 1640, 1575, 1465, 1401, 1326 cm-1. H NMR
H
arom), 7.72 (d, 1H, J ) 12.2 Hz, dCH), 8.15 (s, 1H, H-2), 8.26
(300 MHz, DMSO-d6): δ 3.70 (t, 2H, J ) 5.1 Hz, CH2), 3.99 (s,
3H, CH3), 4.46 (t, 2H, J ) 5.1 Hz, CH2), 6.37 (broad s, 2H, NH2),
6.84 (d, 1H, J ) 5.6 Hz, H-5), 7.28 (d, 1H, J ) 5.3 Hz, H-5′), 8.03
(s, 1H, H-2), 8.18 (d, 1H, J ) 5.3 Hz, H-6′), 8.21 (d, 1H, J ) 5.6
Hz, H-6), 12.49 (broad s, 1H, NH). MS (IS): m/z 286 (M + H+).
Anal. (C14H15N5O2) C, H, N.
3-[(2-Amino)pyrimidin-4-yl]-4-methoxy-1H-pyrrolo[2,3-b]py-
ridine (1b). Meriolin 3. According to the same procedure,
compound 1b was obtained in 75% yield from 7b. Mp >210 °C
(MeOH). IR (KBr): ν 3326, 3148, 3014, 1638, 1574, 1503, 1453,
1310, 1097 cm-1. 1H NMR (300 MHz, DMSO-d6): δ 3.97 (s, 3H,
CH3), 6.32 (broad s, 2H, NH2), 6.79 (d, 1H, J ) 5.5 Hz, H-5),
7.27 (d, 1H, J ) 5.3 Hz, H-5′), 7.92 (s, 1H, H-2), 8.16 (d, 1H, J )
5.5 Hz, H-6), 8.17 (d, 1H, J ) 5.3 Hz, H-6′), 12.13 (broad s, 1H,
NH). MS (IS): m/z 242 (M + H+). Anal. (C12H11N5O) C,
H, N.
(d, 2H, J ) 7.5 Hz, Harom), 8.44 (d, 1H, J ) 8.2 Hz, H-4). MS
(IS): m/z 436 (81Br, M + H+), 434 (79Br, M + H+). Anal.
(C18H16BrN3O3S) C, H, N.
1-(1-Benzenesulfonyl-4-ethoxy-1H-pyrrolo[2,3-b]pyridin-3-
yl)-3-dimethylaminopropenone (7d). According to the same
procedure, compound 7d was obtained in 68% yield from 4d. Mp
187–189 °C (MeOH). IR (KBr): ν 3146, 2985, 1636, 1576, 1536,
1420, 1368, 1293, 1279, 1166, 1105, 893, 787 cm-1 1H NMR
.
(300 MHz, CDCl3): δ 1.44 (t, 3H, J ) 7.2 Hz, CH3), 2.90 (broad
s, 3H, CH3), 3.11 (broad s, 3H, CH3), 4.18 (q, 2H, J ) 7.2 Hz,
CH2), 5.60 (d, 1H, J ) 12.6 Hz, dCH), 6.65 (d, 1H, J ) 5.6 Hz,
H-5), 7.46 (t, 2H, J ) 7.1 Hz, Harom), 7.55–7.63 (m, 2H, Harom
+
dCH), 7.94 (s, 1H, H-2), 8.18 (d, 2H, J ) 7.7 Hz, Harom), 8.29 (d,
1H, J ) 5.6 Hz, H-6). MS (IS): m/z 400 (M + H+). Anal.
(C20H21N3O4S) C, H, N.
1-(1-Benzenesulfonyl-4-propoxy-1H-pyrrolo[2,3-b]pyridin-3-
yl)-3-dimethylaminopropenone (7e). According to the same
procedure, compound 7e was obtained in 69% yield from 4e. Mp
139–141 °C (CH2Cl2/pentane). IR (KBr): ν 3140, 2932, 1638, 1575,
3-[(2-Amino)pyrimidin-4-yl]-6-bromo-1H-pyrrolo[2,3-b]pyri-
dine (1c). Meriolin 11. According to the same procedure, com-
pound 1c was obtained in 68% yield from 7c. Mp >210 °C
(MeOH). IR (KBr): ν 3303, 3130, 2886, 1658, 1575, 1555, 1509,
1454, 1421, 1308, 1271, 1114 cm-1. 1H NMR (300 MHz, DMSO-
d6): δ 6.54 (broad s, 2H, NH2), 7.05 (d, 1H, J ) 5.3 Hz, H-5′),
7.36 (d, 1H, J ) 8.3 Hz, H-5), 8.15 (d, 1H, J ) 5.3 Hz, H-6′), 8.38
(s, 1H, H-2), 8.87 (d, 1H, J ) 8.3 Hz, H-4), 12.42 (broad s, 1H,
NH). MS (IS): m/z 292 (Br81, M + H+), 290 (79Br, M + H+).
Anal. (C11H8BrN5) C, H, N.
1551, 1370, 1295, 1280, 1190, 1170, 1110, 1083, 897, 737 cm-1
.
1H NMR (300 MHz, CDCl3): δ 1.02 (t, 3H, J ) 7.3 Hz, CH3),
1.77–1.89 (m, 2H, CH2), 2.90 (broad s, 3H, CH3), 3.10 (broad s,
3H, CH3), 4.05 (q, 2H, J ) 6.4 Hz, CH2), 5.59 (d, 1H, J ) 12.6
Hz, dCH), 6.64 (d, 1H, J ) 5.6 Hz, H-5), 7.46 (t, 2H, J ) 7.4 Hz,
H
arom), 7.56 (broad t, 1H, J ) 7.4 Hz, Harom), 7.62 (broad d, 1H, J
) 12.6 Hz, dCH), 7.91 (s, 1H, H-2), 8.18 (d, 2H, J ) 7.4 Hz,
3-[(2-Amino)pyrimidin-4-yl]-4-ethoxy-1H-pyrrolo[2,3-b]pyri-
dine (1d). Meriolin 4. According to the same procedure, compound
1d was obtained in 63% yield from 7d. Mp >210 °C (MeOH). IR
(KBr): ν 3338, 3167, 1653, 1578, 1570, 1500, 1439, 1410, 1390,
H
arom), 8.28 (d, 1H, J ) 5.6 Hz, H-6). MS (IS): m/z 414 (M +
H+). Anal. (C21H23N3O4S) C, H, N.
1-(1-Benzenesulfonyl)-4-(1-methylethoxy)-1H-pyrrolo[2,3-
b]pyridin-3-yl)-3-dimethylaminopropenone (7f). According to the
same procedure, compound 7f was obtained in 69% yield from 4f.
Mp 150–152 °C (MeOH). IR (KBr): ν 3150, 2925, 1638, 1552,
1
1310, 1092, 1014 cm-1. H NMR (300 MHz, DMSO-d6): δ 1.45
(t, 3H, J ) 7.2 Hz, CH3), 4.26 (q, 2H, J ) 7.2 Hz, CH2), 6.33
(broad s, 2H, NH2), 6.75 (d, 1H, J ) 5.5 Hz, H-5), 7.36 (d, 1H, J
) 5.3 Hz, H-5′), 7.90 (s, 1H, H-2), 8.13 (d, 1H, J ) 5.5 Hz, H-6),
8.17 (d, 1H, J ) 5.3 Hz, H-6′), 12.09 (broad s, 1H, NH). MS (IS):
m/z 256 (M + H+). Anal. (C13H13N5O) C, H, N.
1
1495, 1373, 1290, 1190, 1168, 1110, 895 cm-1. H NMR (300
MHz, CDCl3): δ 1.37 (d, 6H, J ) 6.0 Hz, CH3), 2.95 (broad s, 3H,
CH3), 3.14 (broad s, 3H, CH3), 4.72 (hept, 1H, J ) 6.0 Hz, CH),
5.68 (broad d, 1H, J ) 12.5 Hz, dCH), 6.65 (d, 1H, J ) 5.7 Hz,
H-5), 7.47 (t, 2H, J ) 7.5 Hz, Harom), 7.57 (t, 1H, J ) 7.5 Hz,
3-[(2-Amino)pyrimidin-4-yl]-4-propoxy-1H-pyrrolo[2,3-b]py-
ridine (1e). Meriolin 5. According to the same procedure, compound
1e was obtained in 60% yield from 7e. Mp >210 °C (MeOH). IR
(KBr): ν 3327, 3158, 2937, 1649, 1566, 1500, 1451, 1409, 1309, 1091
H
arom), 7.73–7.79 (m, 1H, dCH), 7.98 (s, 1H, H-2), 8.20 (d, 2H, J
) 7.5 Hz, Harom), 8.28 (d, 1H, J ) 5.7 Hz, H-6). MS (IS): m/z 414
(M + H+). Anal. (C21H23N3O4S) C, H, N.
cm-1. H NMR (300 MHz, DMSO-d6): δ 1.01 (t, 3H, J ) 7.3 Hz,
1
CH3), 1.81–1.90 (m, 2H, CH2), 4.16 (t, 2H, J ) 6.2 Hz, CH2), 6.32
(broad s, 2H, NH2), 6.76 (d, 1H, J ) 5.5 Hz, H-5), 7.34 (d, 1H, J )
5.3 Hz, H-5′), 7.90 (s, 1H, H-2), 8.14 (d, 1H, J ) 5.5 Hz, H-6), 8.16
(d, 1H, J ) 5.3 Hz, H-6′), 12.07 (broad s, 1H, NH). MS (IS): m/z 270
(M + H+) Anal. (C14H15N5O) C, H, N.
3-[(2-Amino)pyrimidin-4-yl]-4-(1-methylethoxy)-1H-pyrro-
lo[2,3-b]pyridine (1f). Meriolin 6. According to the same proce-
dure, compound 1f was obtained in 60% yield from 7f. Mp >210
°C (MeOH). IR (KBr): ν 3323, 3168, 2921, 1655, 1570, 1510, 1462,
1305, 1105 cm-1. 1H NMR (300 MHz, DMSO-d6): δ 1.39 (d, 6H,
J ) 6.0 Hz, CH3), 4.91 (hept, 1H, J ) 6.0 Hz, CH), 6.30 (broad s,
2H, NH2), 6.77 (d, 1H, J ) 5.6 Hz, H-5), 7.34 (d, 1H, J ) 5.3 Hz,
H-5′), 7.87 (s, 1H, H-2), 8.11 (d, 1H, J ) 5.6 Hz, H-6), 8.17 (d,
1H, J ) 5.3 Hz, H-6′), 12.03 (broad s, 1H, NH). MS (IS): m/z 270
(M + H+). Anal. (C14H15N5O) C, H, N.
1-(4-Methoxy-1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-di-
methylaminopropenone (7g). According to the same procedure,
compound 7g was obtained in 82% yield from 4g. Mp 138–140
°C (MeOH). IR (KBr): ν 3116, 3014, 2906, 1631, 1531, 1503, 1418,
1355, 1282, 1232, 1184, 1072, 887, 789 cm-1. 1H NMR (300 MHz,
CDCl3): δ 3.00 (broad s, 6H, CH3), 3.87 (s, 3H, CH3), 4.00 (s, 3H,
CH3), 6.08 (d, 1H, J ) 12.6 Hz, dCH), 6.63 (d, 1H, J ) 5.6 Hz,
H-5), 7.74 (d, 1H, J ) 12.6 Hz, dCH), 7.76 (s, 1H, H-2), 8.23 (d,
1H, J ) 5.6 Hz, H-6). MS (IS): m/z 260 (M + H+). Anal.
(C14H17N3O2) C, H, N.
3-[(2-Amino)pyrimidin-4-yl]-4-chloro-1H-pyrrolo[2,3-b]pyri-
dine (1a). Meriolin 10. A solution of 7a (140 mg, 0.36 mmol),
guanidine · HCl (52 mg, 0.54 mmol), and K2CO3 (106 mg, 0.76
mmol) in 2-methoxyethanol (5 mL) was heated at 100–110 °C
for 36 h. After the mixture was cooled, the solution was poured
into H2O. The final solution was extracted by EtOAc twice. The
organic phase was dried over MgSO4 and evaporated under
reduced pressure. The residue was purified by column chroma-
3-[(2-Amino)pyrimidin-4-yl]-4-methoxy-1-methyl-1H-pyrro-
lo[2,3-b]pyridine (1g). Meriolin 9. According to the same proce-
dure, compound 1g was obtained in 92% yield from 7g. Mp >210

748 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Echalier et al.
°C (MeOH). IR (KBr): ν 3347, 3312, 3177, 2985, 1640, 1599, 1572,
to 13 mg/mL in 40 mM HEPES, pH 7.0, 200 mM NaCl, 0.01%
monothioglycerol. The protein solution was incubated 20 min on
ice with meriolin 5 (0.8 mM), meriolin 3 (1.8 mM), or variolin B
(1.8 mM) before setting up hanging drop crystallization trials. The
reservoir solution contained 0.6–0.8 M KCl, 0.9–1.2 M (NH4)2SO4,
and 100 mM HEPES, pH 7.0. Orthorhombic crystals grew within
3 weeks at 4 °C. Crystals were briefly soaked in 8 M sodium
formate before being frozen in liquid nitrogen.
1
1522, 1458, 1327, 1304, 1284, 1201, 1074 cm-1. H NMR (300
MHz, CDCl3): δ 3.91 (s, 3H, CH3), 4.02 (s, 3H, CH3), 4.93 (broad
s, 2H, NH2), 6.65 (d, 1H, J ) 5.5 Hz, H-5), 7.45 (d, 1H, J ) 5.3
Hz, H-5′), 7.92 (s, 1H, H-2), 8.26 (d, 1H, J ) 5.3 Hz, H-6′), 8.27
(d, 1H, J ) 5.5 Hz, H-6). MS (IS): m/z 256 (M + H+). Anal.
(C13H13N5O) C, H, N.
3-[(2-Amino)pyrimidin-4-yl]-4-hydroxy-1H-pyrrolo[2,3-b]py-
ridine (8). Meriolin 2. A solution of 1b (50 mg, 0.20 mmol) in
48% HBr/AcOH (6 mL) was refluxed for 2 h. After the mixture
was cooled, solvent was evaporated. The residue was dissolved in
EtOAc (10 mL), and then the solution was neutralized by addition
of saturated aqueous Na2CO3 (pH 7–8). The phases were separated,
and the aqueous phase was washed by EtOAc twice (2 × 10 mL).
The combined organic phases were dried over MgSO4 and
evaporated under reduced pressure. The solid obtained was
recrystallized from methanol to afford 8 (43 mg, 90%). Mp >210
°C (MeOH). IR (KBr): ν 3478, 3303, 3082, 2827, 1643, 1581, 1461,
X-ray Crystallography Data Collection and Processing,
Structure Solution, and Structure Refinement. Data were col-
lected on a single crystal of each CDK2/cyclin A/inhibitor complex
at 100 K at either the Elettra beamline X-ray diffraction (for
meriolin 5) or the ESRF ID14-EH-2 (for meriolin 3 and variolin
B). Data processing and integration were carried out using
MOSFLM and SCALA.68 The structures were solved by molecular
replacement with MOLREP using a well refined structure of CDK2/
cyclin A3 (unpublished results) as the search model. Two CDK2/
cyclin A dimers were found in the asymmetric unit. Within each
electron density map, strong electron density corresponding to the
bound inhibitor was seen at the ATP site after rigid body refinement.
The structure refinement to 2.30 Å for meriolin 5, 2.0 Å for meriolin
3, and 2.10 Å for variolin B and the inhibitor model building were
performed using the CCP4 software suite.68 Alternate cycles of
rebuilding in Coot69 and refinement in Refmac70 were carried out
to obtain the final models. Data collection and refinement statistics
are presented in Table 1. The structures of CDK2/cyclin A/meriolin
3, CDK2/cyclin A/meriolin 5, and CDK2/cyclin A/variolin B have
been deposited in the PDB with accession codes 3BHT, 3BHU,
and 3BHV, respectively.
Protein Kinase Assays. Biochemical Reagents. Sodium ortho-
vanadate, EGTA, EDTA, Mops, ꢀ-glycerophosphate, phenyl phos-
phate, sodium fluoride, dithiothreitol (DTT), glutathione-agarose,
glutathione, bovine serum albumin (BSA), nitrophenyl phosphate,
leupeptin, aprotinin, pepstatin, soybean trypsin inhibitor, benzami-
dine, and histone H1 (type III-S) were obtained from Sigma
Chemicals. [γ-33P]ATP was obtained from Amersham. The GSK-
3-specific substrate GS-1 (YRRAAVPPSPSLSRHSSPHQSpEDEEE)
and the CK1-specific peptide substrate (RRKHAAIGSpAYSITA)71
were synthesized by the Peptide Synthesis Unit, Millegen, Prologue
Biotech, Labege, France.
1
1392, 1287, 1225, 1156 cm-1. H NMR (300 MHz, DMSO-d6 +
D2O): δ 6.51 (d, 1H, J ) 5.5 Hz, H-5), 7.15 (d, 1H, J ) 5.5 Hz,
H-5′), 7.99 (d, 1H, J ) 5.5 Hz, H-6), 8.17 (d, 1H, J ) 5.5 Hz,
H-6′), 8.24 (s, 1H, H-2). MS (IS): m/z 228 (M + H+). Anal.
(C11H9N5O) C, H, N.
3-[(2-Amino)pyrimidin-4-yl]-4-hydroxy-1-methyl-1H-pyrro-
lo[2,3-b]pyridine (9). Meriolin 8. According to the same procedure,
compound 9 was obtained in 92% yield from 1g. Mp >210 °C
(MeOH). IR (KBr): ν 3440, 3310, 3170, 2929, 1647, 1573, 1542,
1
1497, 1326, 1290, 1223, 1186, 1173 cm-1. H NMR (300 MHz,
DMSO-d6 + D2O): δ 3.78 (s, 3H, CH3), 6.56 (d, 1H, J ) 5.5 Hz,
H-5), 7.08 (d, 1H, J ) 5.5 Hz, H-5′), 8.04 (d, 1H, J ) 5.5 Hz,
H-6), 8.16 (d, 1H, J ) 5.5 Hz, H-6′), 8.28 (s, 1H, H-2). MS (IS):
m/z 242 (M + H+). Anal. (C12H11N5O) C, H, N.
3-Pyrimidin-4-yl-4-methoxy-1H-pyrrolo[2,3-b]pyridine (10).
Meriolin 14. A solution of 4-methoxy-7-azaindole 2b (100 mg,
0.67 mmol), CuCl (27 mg, 0.27 mmol), and formamide (300 µL)
was heated at 170 °C for 18 h. After the mixture was cooled, water
was added to the solution (pH 9) and extraction was performed
with EtOAc. The organic phase was dried over MgSO4 and
evaporated. The crude residue was purified by column chromatog-
raphy (EtOAc/MeOH, 9:1) to afford 10 (40 mg, 26%). Mp >210
°C (MeOH). IR (KBr): ν 3083, 3021, 2923, 2851, 1575, 1544, 1354,
Buffers. Homogenization buffer consisted of 60 mM ꢀ-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 phenylphosphate, 10 µg of leupeptin/
mL, 10 µg of aprotinin/mL, 10 µg of soybean trypsin inhibitor/
mL, and 100 µM benzamidine.
1
1299, 1093 cm-1. H NMR (300 MHz, CD3OD + D2O): δ 4.09
(s, 3H, CH3), 6.88 (d, 1H, J ) 5.6 Hz, H-5), 8.12 (s, 1H, H-2),
8.20–8.22 (m, 2H, H-5′, H-6), 8.65 (d, 1H, J ) 5.3 Hz, H-6′), 8.99
(s, 1H, H-2′). MS (IS): m/z 227 (M + H+). Anal. (C12H10N4O) C,
H, N.
Buffer A consisted of 10 mM MgCl2, 1 mM EGTA, 1 mM DTT,
25 mM Tris-HCl, pH 7.5, and 50 µg of heparin/mL.
Buffer C consisted of homogenization buffer but 5 mM EGTA,
no NaF, and no protease inhibitors.
3-(Pyrimidin-4-yl)-4-hydroxy-1H-pyrrolo[2,3-b]pyridine (11).
Meriolin 13. According to the same procedure described for the
preparation of 8, compound 11 was obtained in 92% yield from
10. Mp >210 °C (MeOH). IR (KBr): ν 3429, 3078, 2923, 2851,
1
1636, 1592, 1554, 1463, 1406, 1308, 1290, 1157, 1011 cm-1. H
Kinase Preparations and Assays. Kinase activities were assayed
in buffer A or C 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 30 min incubation. The activities
are usually expressed as percent of the maximal activity, i.e., in
the absence of inhibitors. Controls were performed with appropriate
dilutions of dimethyl sulfoxide.
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.72 The kinase
activity was assayed in buffer C, with 1 mg of histone H1/mL, in
the presence of 15 µM [γ-33P]ATP (3000 Ci/mmol, 10 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 Whatman P81
phosphocellulose paper filters, 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 ACS (Amersham) scintillation fluid.
NMR (300 MHz, DMSO-d6): δ 6.53 (d, 1H, J ) 5.1 Hz, H-5),
8.05 (d, 1H, J ) 5.1 Hz, H-6), 8.12 (d, 1H, J ) 5.6 Hz, H-5′), 8.58
(s, 1H, H-2), 8.71 (d, 1H, J ) 5.6 Hz, H-6′), 9.10 (s, 1H, H-2′),
12.42 (broad s, 1H, NH), 14.24 (s, 1H, OH). MS (IS): m/z 213 (M
+ H+). Anal. (C11H8N4O) C, H, N.
3-[(2-Methylsulfanyl)pyrimidin-4-yl]-4-methoxy-1H-pyrrolo[2,3-
b]pyridine (12). Meriolin 12. According to the same procedure
described for the preparation of 1a, compound 12 was obtained in
30% yield from 7b and 2-methyl-2-thiopseudourea sulfate (time
reaction ) 5 days). Mp >210 °C. IR (KBr): ν 3134, 3083, 2900,
1
2840, 1585, 1556, 1468, 1415, 1363, 1306, 1202, 1095 cm-1. H
NMR (300 MHz, DMSO-d6): δ 2.55 (s, 3H, CH3), 4.01 (s, 3H,
CH3), 6.84 (d, 1H, J ) 5.5 Hz, H-5), 7.84 (d, 1H, J ) 5.5 Hz,
H-5′), 8.18 (s, 1H, H-2), 8.21 (d, 1H, J ) 5.5 Hz, H-6), 8.50 (d,
1H, J ) 5.5 Hz, H-6′), 12.38 (broad s, 1H, NH). MS (IS): m/z 273
(M + H+). Anal. (C13H12N4OS) C, H, N.
Crystallography. Expression, Purification, and Cocrystalli-
zation of Human CDK2/Cyclin A with Meriolins 3 (1b) and 5
(1e) and Variolin B. Thr160-phosphorylated CDK2/cyclin A3
complex was purified as described previously67 and concentrated
CDK2/cyclin A (human, recombinant, expressed in insect cells)
was assayed as described for CDK1/cyclin B.

Kinase Inhibitory 3-(Pyrimidin-4-yl)-7-azaindoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 749
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 purified by
affinity chromatography on glutathione-agarose (vectors kindly
provided by Dr. L. H. Tsai) (p25 is a truncated version of p35, the
35 kDa CDK5 activator). Its activity was assayed with histone H1
in buffer C as described for CDK1/cyclin B.
were suspended on the lid of agar-coated 24-well dishes containing
1 mL of culture media. After the 72 h period required for cell
aggregation, the spheroids were transferred to the culture medium.
Spheroids were treated with different concentrations of meriolin
3, 4 days after the start of the culture and for a 5 day period. The
size of three spheroids per experimental condition was determined
at day 0 (V0) and day 5 (V5) by measuring two orthogonal diameters
(d1 and d2) using an inverted microscope fitted with a calibrated
eyepiece reticule. Volumes were determined according to the
formula V ) (4/3)πr3 where r ) (1/2)(d1d2)1/2. Each experiment
was repeated three times.
CDK9/cyclin T (human, recombinant, expressed in insect cells)
was assayed as described for CDK1/cyclin B but using a pRB
fragment (aa773–928) (3.5 µg/assay) as a substrate.
GSK-3R/ꢀ was purified from porcine brain by affinity chroma-
1
tography on immobilized axin.73 It was assayed, following a /100
Acknowledgment. We are grateful to Dr. J. Boix for the
SH-SY5Y cell line, to Tristan Gallenne for GBM cells, and to
the beamline scientists at the ESRF (ID14-2) and at beamline
X-ray diffraction Elettra for providing excellent facilities for
crystal data collection. This research was supported by grants
from the EEC (Grant FP6-2002, Life Sciences & Health, PRO-
KINASE Research Project) (L.M., J.A.E.), “Cancéropole Grand-
Ouest” (L.M.), and the Australian Research Council (J.C.M).
K.B. was supported by a fellowship from the “Ministère de la
Recherche”.
dilution in 1 mg of BSA/mL of 10 mM DTT, with 5 µL of 4 µM
GS-1 peptide substrate, in buffer A, in the presence of 15 µM
[γ-33P]ATP (3000 Ci/mmol, 10 mCi/mL) in a final volume of 30
µL. After 30 min of incubation at 30 °C, 25 µL aliquots of
supernatant were processed as described above.
CK1δ/ꢀ was purified from porcine brain by affinity chromatog-
raphy on an immobilized axin fragment.74 It was assayed as
described for CDK1 but using a CK1-specific peptide substrate.
DYRK1A (rat, recombinant, expressed in E. coli as a GST fusion
protein) was purified by affinity chromatography on glutathione-
agarose and assayed as described for CDK1/cyclin B.
Cell Biology. Antibodies and Chemicals. CelTiter 96 kit
containing the MTS reagent was purchased from Promega (Madi-
son, WI). The protease inhibitor cocktail was from Roche, and fetal
calf serum (FCS) was from Invitrogen. Unless otherwise stated,
the nonlisted reagents were from Sigma.
Supporting Information Available: Results from elemental
analysis and detailed comparative analysis of the cocrystal struc-
tures. This material is available free of charge via the Internet at
http://pubs.acs.org.
Cell Lines and Culture Conditions. SH-SY5Y human neuro-
blastoma cell line was grown in DMEM with L-glutamine medium
from Invitrogen (Cergy Pontoise, France), antibiotics, and 10%
volume of FCS from Invitrogen.
HEK293 cells were grown in MEM with Glutamax medium from
Invitrogen, antibiotics, and 10% volume of FCS.
References
(1) Malumbres, M.; Barbacid, M. Mammalian cyclin-dependent kinases.
Trends Biochem. Sci. 2005, 30, 630–641.
(2) Camins, A.; Verdaguer, E.; Folch, J.; Canudas, A. M.; Pallas, M. The
role of CDK5/P25 formation/inhibition in neurodegeneration. Drug
News Perspect. 2006, 19, 453–460.
(3) Pareek, T. K.; Keller, J.; Kesavapany, S.; Pant, H. C.; Iadarola, M. J.;
Brady, R. O.; Kulkarni, A. B. Cyclin-dependent kinase 5 activity
regulates pain signaling. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 791–
796.
(4) Borgne, A.; Versteege, I.; Mahé, M.; Studeny, A.; Léonce, S.; Naime,
I.; Rodriguez, M.; Hickman, J. A.; Meijer, L.; Golsteyn, R. M. Analysis
of cyclin B1 and CDK activity during apoptosis induced by camp-
tothecin treatment. Oncogene 2006, 25, 7361–7372.
(5) Garriga, J.; Grana, X. Cellular control of gene expression by T-type
cyclin/CDK9 complexes. Gene 2004, 337, 15–23.
(6) Loyer, P.; Trembley, J. H.; Katona, R.; Kidd, V. J.; Lahti, J. M. Role
of CDK/cyclin complexes in transcription and RNA splicing. Cell.
Signalling 2005, 17, 1033–1051.
(7) Wei, F. Y.; Nagashima, K.; Ohshima, T.; Saheki, Y.; Lu, Y. F.;
Matsushita, M.; Yamada, Y.; Mikoshiba, K.; Seino, Y.; Matsui, H.;
Tomizawa, K. Cdk5-dependent regulation of glucose-stimulated insulin
secretion. Nat. Med. 2005, 11, 1104–1108.
(8) Vermeulen, K.; Van Bockstaele, D. R.; Berneman, Z. N. The cell cycle:
a review of regulation, deregulation and therapeutic targets in cancer.
Cell Proliferation 2003, 36, 131–149.
(9) Cruz, J. C.; Tsai, L.-H. Cdk5 deregulation in the pathogenesis of
Alzheimer’s disease. Trends Mol. Med. 2004, 10, 452–458.
(10) Smith, P. D.; Crocker, S. J.; Jackson-Lewis, V.; Jordan-Sciutto, K. L.;
Hayley, S.; Mount, M. P.; O’Hare, M. J.; Callaghan, S.; Slack, R. S.;
Przedborski, S.; Anisman, H.; Park, D. S. Cyclin-dependent kinase 5
is a mediator of dopaminergic neuron loss in a mouse model of
Parkinson’s disease. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 13650–
13655.
(11) Smith, P. D.; O’Hare, M. J.; Park, D. S. CDKs: taking on a role as
mediators of dopaminergic loss in Parkinson’s disease. Trends Mol.
Med. 2004, 10, 445–451.
(12) Zhang, M.; Li, J.; Chakrabarty, P.; Bu, B.; Vincent, I. Cyclin-dependent
kinase inhibitors attenuate protein hyperphosphorylation, cytoskeletal
lesion formation, and motor defects in Niemann-Pick type C mice.
Am. J. Pathol. 2004, 165, 843–852.
(13) Wang, J.; Liu, S.; Fu, Y.; Wang, J. H.; Lu, Y. Cdk5 activation induces
hippocampal CA1 cell death by directly phosphorylating NMDA
receptors. Nat. Neurosci. 2003, 6, 1039–1047.
(14) Rashidian, J.; Iyirhiaro, G.; Aleyasin, H.; Rios, M.; Vincent, I.;
Callaghan, S.; Bland, R. J.; Slack, R. S.; During, M. J.; Park, D. S.
Multiple cyclin-dependent kinases signals are critical mediators of
ischemia/hypoxic neuronal death in vitro and in vivo. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 14080–14085.
KMS-11, a multiple myeloma adherent cell line,75 and GBM, a
multiform glioblastoma primary culture,76 were grown in cultured
in RPMI 1640 medium supplemented with 5% and 10% fetal calf
serum, respectively, 2 mM glutamine, antibiotics (100 IU/mL
penicillin and 100 µg/mL streptomycin), and 10 µΜ ꢀ-mercapto-
ethanol (Life Technologies). Cells were subcultured at confluency
after dispersal with 0.025% trypsin in 0.02% EDTA. For experi-
ments cells were used at 70-80% confluence.
General culture conditions were an atmosphere of 5% CO2 and
a temperature of 37 °C.
Culture dishes and other plastic disposable tools were supplied
by Corning (Corning, NY). Drug treatments were performed on
exponentially growing cultures at the indicated time and concentra-
tions. Control experiments were also carried out using appropriate
dilutions of DMSO.
Cell Viability Assessment. Cell viability was determined by
measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
boxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS).
The procedure has been previously described in detail.77
Alternatively, cell viability was assessed by the neutral red uptake
assay (KMS-11 and GBM cells). The 20 000 cells were plated in
96-well culture microtiter plates (Falcon) and incubated at 37 °C
in 5% CO2. After 24 h drugs were added in fresh medium, and
after 48 h cells were loaded for 3 h with 50 µg of neutral red/mL.
This weakly cationic dye penetrates cell membranes by nonionic
diffusion and binds intracellularly to anionic carboxylic and/or
phosphate groups of the lysosomal matrix. Thereafter, the medium
was removed, cells were fixed for 5 min with a mixture of 1%
formaldehyde and 1% CaCl2, and the dye was extracted with 0.2
mL of 1% acetic acid in 50% ethanol. Plates were left overnight at
4 °C, and absorbance was recorded at 570 nm (Multiskan EX,
Thermo-Electron Corporation) and corrected with a blank. Experi-
ments were performed at least in triplicate, four wells being used
per cell line.
Preparation and Analysis of Cell Spheroids. We have adapted
the hanging-drop method64 to produce parental HCT116 spheroids
of homogeneous diameter. The 25 µL drops containing 500 cells

750 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4
Echalier et al.
(15) Di Giovanni, S.; Movsesyan, V.; Ahmed, F.; Cernak, I.; Schinelli, S.;
Stoica, B.; Faden, A. I. Cell cycle inhibition provides neuroprotection
and reduces glial proliferation and scar formation after traumatic brain
injury. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8333–8335.
(16) Schang, L. M. Effects of pharmacological cyclin-dependent kinase
inhibitors on viral transcription and replication. Biochim. Biophys. Acta
2004, 1697, 197–209.
(17) Pumfery, A.; de la Fuente, C.; Berro, R.; Nekhai, S.; Kashanchi, F.;
Chao, S. H. Potential use of pharmacological cyclin-dependent kinase
inhibitors as anti-HIV therapeutics. Curr. Pharm. Des. 2006, 12, 1949–
1961.
(18) Nelson, P. J.; Shankland, S. J. Therapeutics in renal disease: the road
ahead for antiproliferative targets. Nephron Exp. Nephrol. 2005, 103,
e6–e15.
(19) Gherardi, D.; D’Agati, V.; Tearina Hu, T.-H.; Barnett, A.; Gianella-
Borradori, A.; Gelman, I. H.; Nelson, P. J. Reversal of collapsing
glomerulopathy in mice with the cyclin-dependent kinase inhibitor
CYC202. J. Am. Soc. Nephrol. 2004, 15, 1212–1222.
(20) Griffin, S. V.; Krofft, R. D.; Pippin, J. W.; Shankland, S. J. Limitation
of podocyte proliferation improves renal function in experimental
crescentic glomerulonephritis. Kidney Int. 2005, 67, 977–986.
(21) Price, P. M.; Yu, F.; Kaldis, P.; Aleem, E.; Nowak, G.; Safirstein,
R. L.; Megeyesi, J. Dependence of cisplatin-induced cell death in vitro
and in vivo on cyclin-dependent kinase 2. J. Am. Soc. Nephrol. 2006,
17, 2434–2442.
(22) Bukanov, N. O.; Smith, L. A.; Klinger, K. W.; Ledbetter, S. R.;
Ibraghimov-Beskrovnaya, O. Long-lasting arrest of murine polycystic
kidney disease with CDK inhibitor Roscovitine. Nature 2006, 444,
949–952.
(23) Rossi, A. G.; Sawatzky, D. A.; Walker, A.; Ward, C.; Sheldrake, T. A.;
Riley, N. A.; Caldicott, A.; Martinez-Losa, M.; Walker, T. R.; Duffin,
R.; Gray, M.; Crescenzi, E.; Martin, M. C.; Brady, H. J.; Savill, J. S.;
Dransfield, I.; Haslett, C. Cyclin-dependent kinase inhibitors enhance
the resolution of inflammation by promoting inflammatory cell
apoptosis. Nat. Med. 2006, 12, 1056–1064.
(24) Pareek, T. K.; Keller, J.; Kesavapany, S.; Agarwal, N.; Kuner, R.;
Pant, H. C.; Iadarola, M. J.; Brady, R. O.; Kulkarni, A. B. Cyclin-
dependent kinase 5 modulates nociceptive signaling through direct
phosphorylation of transient receptor potential vanilloid 1. Proc. Natl.
Acad. Sci. U.S.A. 2007, 104, 660–665.
N(3′)-methyltetrahydrovariolin B. Tetrahedron 1994, 50, 3993–4000.
(39) Anderson, R. J.; Morris, J. C. Total synthesis of variolin B. Tetrahedron
Lett. 2001, 42, 8697–8699.
(40) Molina, P.; Fresneda, P. M.; Delgado, S. Carbodiimide-mediated
preparation of the tricyclic pyrido[3′,2′:4,5]pyrrolo[1,2-c]pyrimidine
ring system and its application to the synthesis of the potent antitumoral
marine alkaloid variolin B and analog. J. Org. Chem. 2003, 68, 489–
499.
(41) Ahaidar, A.; Fernandez, D.; Danelon, G.; Cuevas, C.; Manzanares,
I.; Albericio, F.; Joule, J. A.; Alvarez, M. Total syntheses of variolin
B and deoxyvariolin B. J. Org. Chem. 2003, 68, 10020–10029.
(42) Mendiola, J.; Baeza, A.; Alvarez-Builla, J.; Vaquero, J. J. Reaction
of bromomethylazoles and tosylmethyl isocyanide. A novel hetero-
cyclization method for the synthesis of the core of marine alkaloids
variolins and related azolopyrimidines. J. Org. Chem. 2004, 69, 4974–
4983.
(43) Anderson, R. J.; Hill, J. B.; Morris, J. Concise total syntheses of
variolin B and deoxyvariolin B. J. Org. Chem. 2005, 70, 6204–6212.
(44) Erba, E.; Balconi, G.; Faretta, M.; Bergamaschi, D.; Guidi, G.; Jimeno,
J.; Faircloth, G.; D’Incalci, M. Cell cycle phase perturbation and
apoptosis induced by variolin B, a novel antitumor agent of marine
origin. Proc. Am. Assoc. Cancer Res. 1996, 37, 28.
(45) Simone, M.; Erba, E.; Damia, G.; Vikhanskaya, F.; Di Francesco,
A. M.; Riccardi, R.; Bailly, C.; Cuevas, C.; Fernandez Sousa-Faro,
J. M.; D’Incalci, M. Variolin B and its derivate deoxy-variolin B: new
marine natural compounds with cyclin-dependent kinase inhibitor
activity. Eur. J. Cancer. 2005, 41, 2366–2377.
(46) Fresneda, P. M.; Molina, P.; Bleda, J. A. Synthesis of the indole
alkaloids meriadinins from the tunicate aplidium meridianum. Tetra-
hedron 2001, 57, 2355–2363.
(47) Girgis, N. S.; Larson, S. B.; Robins, R. K.; Cottam, H. B. The synthesis
of 5-azaindoles by substitution-rearrangement of 7-azaindoles upon
treatment with certain primary amines. J. Heterocycl. Chem. 1989,
26, 317–325.
(48) Zhang, Z.; Yang, Z.; Wong, H.; Zhu, J.; Meanwell, N. A.; Kadow,
J. F.; Wang, T. An effective procedure for the acylation of azaindoles
at C-3. J. Org. Chem. 2002, 67, 6226–6227.
(49) Kwon, H. B.; McKee, B. H.; Stille, J. K. Palladium-catalyzed coupling
reactions of (1-ethoxyvinyl)trimethylstannane with vinyl and aryl
triflate. J. Org. Chem. 1990, 55, 3114–3118.
(25) Knockaert, M.; Greengard, P.; Meijer, L. Pharmacological inhibitors
of cyclin-dependent kinases. Trends Pharmacol. Sci. 2002, 123, 417–
425.
(26) Fischer, P. M.; Gianella-Borradori, A. Recent progress in the discovery
and development of cyclin-dependent kinase inhibitors. Expert Opin.
InVest. Drugs 2005, 14, 457–477.
(27) Shapiro, G. I. Cyclin-dependent kinase pathways as targets for cancer
treatment. J. Clin. Oncol. 2006, 24, 1770–1783.
(50) Helland, I.; Lejon, T. Synthesis of 4-phenylpyrimidine from acetophe-
none and formamide. Heterocycles 1999, 51, 611–615.
(51) Fernandez, D.; Ahaidar, A.; Danelon, G.; Cironi, P.; Marfil, M.; Perez,
O.; Cuevas, C.; Albericio, F.; Joule, J. A.; Alvarez, M. Synthesis of
polyheterocyclic nitrogen-containing marine natural products.
Monatsch. Chem. 2004, 135, 615–627.
(52) Azevedo, W. F.; Leclerc, S.; Meijer, L.; Havlicek, L.; Strnad, M.;
Kim, S. H. Inhibition of cyclin-dependent kinases by purine analogues:
crystal structure of human cdk2 complexed with roscovitine. Eur.
J. Biochem. 1997, 243, 518–526.
(28) Misra, R. N. Clinical progress of selective cyclin-dependent kinase
(CDK) inhibitors. Drugs Future 2006, 31, 43–52.
(29) CDK Inhibitors of Cyclin-Dependent Kinases as Anti-Tumor Agents;
Smith, P. J., Yue, E., Eds; Monographs on Enzyme Inhibitors, Vol.
2, CRC Press: Boca Raton, FL, 2006.
(30) Noble, M. E.; Endicott, J. A.; Johnson, L. N. Protein kinase inhibitors:
insights into drug design from structure. Science 2004, 303, 1800–
1805.
(31) Bach, S.; Blondel, M.; Meijer, L. Evaluation of CDK Inhibitors’
Selectivity: From Affinity Chromatography to Yeast Genetics. In CDK
Inhibitors and Their Potential as Anti-Tumor Agents; Yue, E, Smith,
P. J., Eds; Monographs on Enzyme Inhibitors, Vol. 2; CRC Press:
Boca Raton, FL, 2006; pp 103–119.
(32) Gompel, M.; Leost, M.; Bal de Kier Joffe, E.; Puricelli, L.; Hernandez
Franco, L.; Palermo, J.; Meijer, L. Meridianins, a new family of protein
kinase inhibitors isolated from the Ascidian Aplidium meridianum.
Bioorg. Med. Chem. Lett. 2004, 14, 1703–1707.
(53) Hoessel, R.; Leclerc, S.; Endicott, J.; Noble, M.; Lawrie, A.; Tunnah,
P.; Leost, M.; Damiens, E.; Marie, D.; Marko, D.; Niederberger, E.;
Tang, W.; Eisenbrand, G.; Meijer, L. Indirubin, the active constituent
of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases.
Nat. Cell Biol. 1999, 1, 60–67.
(54) Moshinsky, D. J.; Bellamacina, C. R.; Boisvert, D. C.; Huang, P.;
Hui, T.; Jancarik, J.; Kim, S. H.; Rice, A. G. SU9516: biochemical
analysis of cdk inhibition and crystal structure in complex with cdk2.
Biochem. Biophys. Res. Commun. 2003, 310, 1026–1031.
(55) Davies, T. G.; Bentley, J.; Arris, C. E.; Boyle, F. T.; Curtin, N. J.;
Endicott, J. A.; Gibson, A. E.; Golding, B. T.; Griffin, R. J.; Hardcastle,
I. R.; Jewsbury, P.; Johnson, L. N.; Mesguiche, V.; Newell, D. R.;
Noble, M. E.; Tucker, J. A.; Wang, L.; Whitfield, H. J. Structure-
based design of a potent purine-based cyclin-dependent kinase
inhibitor. Nat. Struct. Biol. 2002, 9, 745–749.
(33) Franco, L. H.; Bal de Kier Joffe, E.; Puricelli, L.; Tatian, M.; Seldes,
A. M.; Palermo, J. A. Indole alkaloids from the tunicate Aplidium
meridianum. J. Nat. Prod. 1998, 61, 1130–1132.
(56) Mettey, Y.; Gompel, M.; Thomas, V.; Garnier, M.; Leost, M.; Ceballos-
Picot, I.; Noble, M.; Endicott, J.; Vierfond, J.-M.; Meijer, L. Aloïsines,
a new family of CDK/GSK-3 inhibitors. SAR study, crystal structure
in complex with CDK2, enzyme selectivity, and cellular effects.
J. Med. Chem. 2003, 46, 222–236.
(57) Leclerc, S.; Garnier, M.; Hoessel, R.; Marko, D.; Bibb, J. A.; Snyder,
G. L.; Greengard, P.; Biernat, J.; Mandelkow, E.-M.; Eisenbrand, G.;
Meijer, L. Indirubins inhibit glycogen synthase kinase-3ꢀ and CDK5/
p25, two kinases involved in abnormal tau phosphorylation in
Alzheimer’s disease. A property common to most CDK inhibitors?
J. Biol. Chem. 2001, 276, 251–260.
(34) Fresneda, P. M.; Molina, P.; Delgado, S.; Bleda, J. A. Synthetic studies
towards the 2-aminopyrimidine alkaloids variolins and meridianins
from marine origin. Tetrahedron Lett. 2000, 41, 4777–4780.
(35) Jiang, B.; Yang, C.-G. Synthesis of indolylpyrimidines via cross-
coupling of indolylboronic acid with chloropyrimidines: facile syn-
thesis of meridianin D. Heterocycles 2000, 53, 1489–1498.
(36) Franco, L. H.; Palermo, J. A. Synthesis of 2-(pyrimidin-4-yl)indoles.
Chem. Pharm. Bull. 2003, 51, 975–977.
(37) Perry, N. B.; Ettouati, L.; Litaudon, M.; Blunt, J. W.; Munro, M. H. G.;
Parkin, S.; Hope, H. Alkaloids from the Antarctic sponge Kirkpatrickia
Varialosa. Part 1: variolin B, a new antitumour and antiviral compound.
Tetrahedron 1994, 50, 3987–3992.
(38) Trimurtulu, G.; Faulkner, D. J.; Perry, N. B.; Ettouati, L.; Litaudon,
M.; Blunt, J. W.; Munro, M. H. G.; Jameson, G. B. Alkaloids from
the Antarctic sponge Kirkpatrickia Varialosa. Part 2: variolin A and
(58) Huang, S.; Li, R.; Connolly, P. J.; Emanuel, S.; Middleton, S. A.
Synthesis of 2-amino-4-(7-azaindol-3-yl)pyrimidines as cyclin de-
pendent kinase 1 (CDK1) inhibitors. Bioorg. Med. Chem. Lett. 2006,
16, 4818–4821.
(59) Zarkowska, T.; Mittnacht, S. Differential phosphorylation of the
retinoblastoma protein by G1/S cyclin-dependent kinases. J. Biol.
Chem. 1997, 272, 12738–12746.

Kinase Inhibitory 3-(Pyrimidin-4-yl)-7-azaindoles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 4 751
(60) Kwon, Y. G.; Lee, S. Y.; Choi, Y.; Greengard, P.; Nairn, A. C. Cell
cycle-dependent phosphorylation of mammalian protein phosphatase
1 by cdc2 kinase. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2168–2173.
(61) Ahn, S. H.; Kim, M.; Buratowski, S. Phosphorylation of serine 2 within
the RNA polymerase II C-terminal domain couples transcription and
3′ end processing. Mol. Cell 2004, 13, 67–76.
(62) Mandelin, A. M.; Pope, R. M. Myeloid cell leukemia-1 as a therapeutic
target. Expert Opin. Ther. Targets 2007, 11, 363–373.
(63) Yang-Yen, H. F. Mcl-1: a highly regulated cell death and survival
controller. J. Biomed. Sci. 2006, 13, 201–204.
(78) Christian, B. A.; Grever, M. R.; Byrd, J. C.; Lin, T. S. Flavopiridol in
the treatment of chronic lymphocytic leukemia. Curr. Opin. Oncol.
2007, 19, 573–578.
(79) Meijer, L.; Raymond, E. Roscovitine and other purines as kinase
inhibitors. From starfish oocytes to clinical trials. Acc. Chem. Res.
2003, 36, 417–25.
(80) Meijer, L.; Bettayeb, K.; Galons, H. Roscovitine (CYC202, Seliciclib).
In CDK Inhibitors and Their Potential as Anti-Tumor Agents; Yue,
E., Smith, P. J., Eds.; Monographs on Enzyme Inhibitor, Vol. 2; CRC
Press: Boca Raton, FL, 2006; pp 187–226.
(81) Chu, X. J.; DePinto, W.; Bartkovitz, D.; So, S.-S.; Vu, B. T.; Packman,
K.; Lukacs, C.; Ding, Q.; Jiang, N.; Wang, K.; Goelzer, P.; Yin, X.;
Smith, M. A.; Higgins, B. X.; Chen, Y.; Xiang, Q.; Moliterni, J.;
Kaplan, G.; Graves, B.; Lovey, A.; Fotouhi, N. Discovery of [4-amino-
2-(1-methanesulfonylpiperidin-4-ylamino)pyrimidin-5-yl](2,3-difluoro-
6-methoxyphenyl)methanone (R547), a potent and selective cyclin-
dependent kinase inhibitor with significant in vivo antitumor activity.
J. Med. Chem. 2006, 49, 6549–6560.
(82) DePinto, W.; Chu, X.-J.; Yin, X.; Smith, M.; Packman, K.; Goelzer,
P.; Lovey, A.; Chen, Y.; Qian, H.; Hamid, R.; Xiang, Q.; Tovar, C.;
Blain, R.; Nevins, T.; Higgins, B.; Luistro, L.; Kolinsky, K.; Felix,
B.; Hussain, S.; Heimbrook, D. In vitro and in vivo activity of R547:
a potent and selective cyclin-dependent kinase inhibitor currently in
phase I clinical trials. Mol. Cancer Ther. 2006, 5, 2644–2658.
(83) Misra, R. N.; Xiao, H.-Y.; Kim, K. S.; Lu, S.; Han, W.-C.; Barbosa,
S. A.; Hunt, J. T.; Rawlins, D. B.; Shan, W.; Ahmed, S. Z.; Qian, L.;
Chen, B.-C.; Zhao, R.; Bednarz, M. S.; Kellar, K. A.; Mulheron, J. G.;
Batorsky, R.; Roongta, U.; Kamath, A.; Marathe, P.; Ranadive, S. A.;
Sack, J. S.; Tokarski, J. S.; Pavletich, N. P.; Lee, F. Y. F.; Webster,
K. R.; Kimball, S. D. N-(Cycloalkylamino)acyl-2-aminothiazole
inhibitors of cyclin-dependent kinase 2. N-[5-[[[5-(1,1-Dimethylethyl)-
2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS-
387032), a highly efficacious and selective antitumor agent. J. Med.
Chem. 2004, 47, 1719–1728.
(84) Baughn, L. B.; Di Liberto, M.; Wu, K.; Toogood, P. L.; Louie, T.;
Gottschalk, R.; Niesvizky, R.; Cho, H.; Ely, S.; Moore, M. A. S.;
Chen-Kiang, S. A novel orally active small molecule potently induces
G1 arrest in primary myeloma cells and prevents tumor growth by
specific inhibition of cyclin-dependent kinase 4/6. Cancer Res. 2006,
66, 7661–7667.
(85) Camidge, D. R.; Smethurst, D.; Growcott, J.; Barrass, N. C.; Foster,
J. R.; Febbraro, S.; Swaisland, H.; Hughes, A. A first-in-man phase I
tolerability and pharmacokinetic study of the cyclin-dependent kinase-
inhibitor AZD5438 in healthy male volunteers. Cancer Chemother.
Pharmacol. 2007, 60, 391–398.
(64) Del Duca, D.; Werbowetski, T.; Del Maestro, R. F. Spheroid
preparation from hanging drops: characterization of a model of brain
tumor invasion. J. Neurooncol. 2004, 67, 295–303.
(65) Desosize, D.; Jardillier, J. Multicellular resistance: a paradigm for
clinical resistance. Crit. ReV. Oncol. Hematol. 2000, 36, 193–207.
(66) Bettayeb, K.; Tirado, O. M.; Marionneau-Lambert, S.; Ferandin, Y.;
Lozach, O.; Morris, J.; Mateo-Lozano, S.; Drückes, P.; Schächtele,
C.; Kubbutat, M.; Liger, F.; Marquet, B.; Joseph, B.; Echalier, A.;
Endicott, J.; Notario, V.; Meijer, L. Meriolins, a new class of cell
death-inducing kinase inhibitors with inhibitors with enhanced selec-
tivity for cyclin-dependent kinases. Cancer Res. 2007, 67, 8325–8334.
(67) Brown, N. R.; Noble, M. E.; Endicott, J. A.; Johnson, L. N. The
structural basis for specificity of substrate and recruitment peptides
for cyclin-dependent kinases. Nat. Cell Biol. 1999, 1, 438–443.
(68) Collaborative Computational Project, Number 4. The CCP4 suite:
programs for protein crystallography. Acta Crystallogr. 1994, D50,
760–763.
(69) Emsley, P.; Cowtan, K. Coot: model-building tools for molecular
graphics. Acta Crystallogr. 2004, D60, 2126–2132.
(70) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method. Acta
Crystallogr. 1997, D53, 240–255.
(71) Marin, O.; Meggio, F.; Pinna, L. A. Design and synthesis of two new
peptide substrates for the specific and sensitive monitoring of casein
kinases 1 and 2. Biochem. Biophys. Res. Commun. 1984, 198, 898–
905.
(72) Meijer, L.; Borgne, A.; Mulner, O.; Chong, J. P. J.; Blow, J. J.; Inagaki,
N.; Inagaki, M.; Delcros, J. G.; Moulinoux, J. P. Biochemical and
cellular effects of roscovitine, a potent and selective inhibitor of the
cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur. J. Biochem. 1997,
243, 527–536.
(73) Primot, A.; Baratte, B.; Gompel, M.; Borgne, A.; Liabeuf, S.; Romette,
J. L.; Costantini, F.; Meijer, L. Purification of GSK-3 by affinity
chromatography on immobilised axin. Protein Expression Purif. 2000,
20, 394–404.
(74) Reinhardt, J.; Ferandin, Y.; Meijer, L. Purification of native, active
casein kinase 1 (CK1) by affinity chromatography on immobilised
axin fragment. Protein Expression Purif. 2007, 54, 101–109.
(75) Namba, M.; Ohtsuki, T.; Mori, M.; Togawa, A.; Wada, H.; Sugihara,
T.; Yawata, Y.; Kimoto, T. Establishment of five human myeloma
cell lines. In Vitro Cell DeV. Biol. 1989, 25, 723–729.
(76) Cartron, P.-F.; Juin, P.; Oliver, L.; Martin, S.; Meflah, K.; Vallette,
F. M. Nonredundant role of Bax and Bak in Bid-mediated apoptosis.
Mol. Cell. Biol. 2003, 23, 4701–4712.
(77) Ribas, J.; Boix, J. Cell differentiation, caspase inhibition, and
macromolecular synthesis blockage, but not BCL-2 or BCL-XL
proteins, protect SH-SY5Y cells from apoptosis triggered by two CDK
inhibitory drugs. Exp. Cell Res. 2004, 295, 9–24.
(86) Davies, T. G.; Tunnah, P.; Meijer, L.; Marko, D.; Eisenbrand, G.;
Endicott, J. A.; Noble, M. Inhibitor binding to active and inactive
CDK2. The crystal structure of CDK2-cyclin-A/indirubin-5-sulphonate.
Structure 2001, 9, 389–397.
(87) Davies, T. G.; Pratt, D. J.; Endicott, J. A.; Johnson, L. N.; Noble,
M. E. M. Structure-based design of cyclin-dependent kinase inhibitors.
Pharmacol. Ther. 2002, 93, 125–133.
(88) Kontopidis, G.; McInnes, C.; Pandalaneni, S. R.; McNae, I.; Gibson,
D.; Mezna, M.; Thomas, M.; Wood, G.; Wang, S.; Walkinshaw, M.;
Fischer, P. M. Differential binding of inhibitors to active and inactive
CDK2 provides insights for drug design. Chem. Biol. 2006, 13,
201–211.
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