A new series of potent oxindole inhibitors of CDK2

Article abstract of DOI:10.1016/j.bmcl.2003.12.009

A novel series of oxindole-type inhibitors of CDK2 that have heteroatom substituted alkynyl moieties at their C-4 position is described. These novel 4-alkynyl-substituted inhibitors have superior potency relative to their parent compound in free enzyme and in cell based assays. The crystal structure of CDK2 in complex with one of these analogues was determined and gives insight to their increased potency. The biochemical evaluation of a representative derivative is also described.

Full text of DOI:10.1016/j.bmcl.2003.12.009

Bioorganic & Medicinal Chemistry Letters 14 (2004) 913–917
A new series of potent oxindole inhibitors of CDK2
Kin-Chun Luk, Mary Ellen Simcox, Andy Schutt, Karen Rowan, Thelma Thompson,
Yi Chen, Ursula Kammlott, Wanda DePinto, Pete Dunten and Apos Dermatakis*
Hoffmann-La Roche Inc., 340 Kingsland St., Nutley, NJ 07110-1199, USA
Received 21 October 2003; revised 26 November 2003; accepted 2 December 2003
Abstract—A novel series of oxindole-type inhibitors of CDK2 that have heteroatom substituted alkynyl moieties at their C-4
position is described. These novel 4-alkynyl-substituted inhibitors have superior potency relative to their parent compound in free
enzyme and in cell based assays. The crystal structure of CDK2 in complex with one of these analogues was determined and gives
insight to their increased potency. The biochemical evaluation of a representative derivative is also described.
# 2003 Elsevier Ltd. All rights reserved.
Cyclin-dependent kinase 2 (CDK2) is a key cell cycle
regulator. Upon complexation with its activating pro-
teins, cyclin E or cyclin A, CDK2 modulates the activity
of many cellular substrates via phosphorylation on Ser
and/or Thr residues.1 In complex with cyclin E, CDK2
plays a paramount role during the G1/S transition of
the cell cycle while in complex with cyclin A it facilitates
the progression of the S phase of the cell cycle. Recent
evidence also suggests that CDK2 may have a crucial
role in the G2 phase of the cell cycle.2 The importance
of CDK2 for cell cycle progression has led to an active
pursuit of small molecule inhibitors of this enzyme as a
possible treatment against cancer and other hyperproli-
ferative disorders.3,4
of this lead could be improved by the introduction of an
appropriate heteroatom substituted moiety at C-4.
By means of this strategy and via the incorporation of
cyclic saturated heteroatom substituted moieties at C-4,
we had previously effected the potency optimization of
screening hit 1a.5 In the case of the screening hit 1b we
decided to follow a slightly different approach for its
potency optimization. Instead of pursuing the introduc-
tion of cyclic appendages at its C-4 position we opted to
explore the use of heteroatom substituted alkynyl
moieties. Through modeling we came to the realization
that a heteroatom substituted propargyl and/or homo-
propargyl appendage at C-4 could be as beneficial for
potency as were the cyclic moieties used during the
optimization of lead 1a.
We recently disclosed a potent series of oxindole inhi-
bitors of CDK2 that is based on the screening hit 1a.5
Herein we describe a new series of oxindole type inhibi-
tors of CDK2 based on the lead 1b (Fig. 1, Table 1).
Our work toward the potency optimization of 1b started
by reviewing the CDK2-ATP co-crystal structure in
conjunction with several published crystal structures of
CDK2 in complex with oxindole-type inhibitors.6 This
review lead us to assume that lead 1b, like other
oxindoles, must bind in the CDK2 ATP pocket along the
residues Glu81–Leu83 in a donor acceptor donor motif.
In that orientation, an overlay of 1b with ATP in the
CDK2 active site led us to the conclusion that the potency
Figure 1. Screening hits 1a,b. Overlay of the core of lead 1b with ATP
in the CDK2 pocket and visual representation of its optimization
strategy. Interactions between CDK2 and the ribofuranoside
phosphate moiety of ATP have been omitted for clarity.
Keywords: CDK2; CDK Inhibitors; Oxindole.
* Corresponding author. Tel.: +1-973-235-6307; fax: +1-973-235-
2448; e-mail: apostolos.dermatakis@roche.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.009

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K.-C. Luk et al. / Bioorg. Med. Chem. Lett. 14 (2004) 913–917
The synthesis of 4-alkynyl derivatives of 1b was carried
out as described in Scheme 1.7 Via that route we pre-
pared the 5-fluoro-4-alkynyl analogues 7a–t shown in
Table 1.
lines SW480 and MDA MB 435 revealed that these
analogues were much superior to the lead compound
1b.8 In the CDK2 enzyme assay the great majority of
alkynyl oxindoles 7 had an IC50 in the single digit
nanomolar range. The cell based assays showed that
these analogues were potent inhibitors of cellular pro-
liferation, consistently better than 1b by one or two
Assay results of 4-alkynyl derivatives 7 against the
CDK2/cyclin E holoenzyme and against the cancer cell
Table 1. CDK2/Cyclin E and tumor cell growth inhibition by 4-alkynyl oxindoles 7
Compd
C-4
Substituent
CDK2/
Cyclin E
IC50 (nM)a
MDA MB 435
IC50 (nM)a
SW480
IC50 (nM)a
Compd
C-4
Substituent
CDK2/
Cyclin E
IC50 (nM)a
MDA MB 435
IC50 (nM)a
SW480
IC50 (nM)a
b
1b
H
I
847
>30,000
—
7j
95
1270
967
b
6
1000
>30,000
490
—
7k
7l
9c
300
358
210
140
7a
7b
7c
7d
7e
4c
150
130
400
110
270
7c
21
26
4c
110
7m
7n
7o
7p
18
45
3c
1070
2625
400
549
190
1185
140
130
3c
320
4c
600
290
7f
4c
300
300
80
7q
7r
6c
110
31
92
40
7g
5c
205
2c
7h
7i
8c
349
100
566
7s
7t
4c
63
44
90
21
1160
5c
110
a IC50 values were determined by a single experiment run in duplicate.
b Not tested.
c Values at assays detection limit.

K.-C. Luk et al. / Bioorg. Med. Chem. Lett. 14 (2004) 913–917
915
orders of magnitude. Analogues that were substituted
on the propargyl heteroatom by a group larger than a
methyl moiety where overall slightly less potent that
their counterparts without such groups. For instance,
analogues 7i and 7j were overall less potent than alkynyl
oxindoles 7g and 7 h. Derivatives 7m and 7n were less
potent than their respective des-methyl or methyl-sub-
stituted counterparts 7k and 7l.
pocket. Apparently it is this fourth hydrogen bond that
is responsible for the increased potency of our 4-alkynyl
derivatives. This correlates with the observation that
substitution by large groups at the propargylic hetero-
atom results in inhibitors with reduced overall potency.
The CDK2-7o co-crystal structure also revealed that the
terminal hydroxy group on the 4-alkynyl tether of 7o
makes a, seemingly unique for that particular inhibitor,
fifth hydrogen bonding interaction with residue Glu12
from the protein backbone.
The crystal structure of CDK2 complexed with deriv-
ative 7o affords a rationale for the observed potency of
our 4-alkynyl oxindoles (Fig. 2).9 That structure shows
that 7o binds in the ATP pocket of CDK2 via four
major hydrogen bonding interactions. As expected,
three of these interactions involve the oxindole-3-
methylpyrrole core of 7o and residues Glu81-Leu83.
The fourth interaction involves a hydrogen bond
between the propargylic heteroatom on the C-4 alkynyl
substituent and the Asp145 located at the ribofurano-
side phosphate binding region of the CDK2 ATP
A representative from this class, compound 7q, was
evaluated further for its effect on the phosphorylation
of retinoblastoma protein (Rb), one of the cellular
substrates of CDK2. In these experiments SW480 cancer
cells were incubated for 12 h or 24 h with 7q. This
resulted in a concentration dependent inhibition of the
Rb phosphorylation (Fig. 3).10
The cell cycle analysis11 of SW480 cells treated with 7q
showed that this 4-alkynyl-oxindole blocks cellular pro-
liferation at the G1 and G2 phase and leads to a
decrease in the percentage of cells in the S phase (Table
2). In addition, the treatment of these cells with 7q also
led to apoptosis as evident by the time- and dose-
dependent increase of the subG0 fraction of cells (Table
2) and the DNA fragmentation experiment shown in
Figure 4.12
The above biochemical effects correlate well with the
cellular effects of other known CDK2 inhibitors.13
Scheme 1. General synthesis of 4-alkynyl oxindoles 7.
Figure 3. Inhibition of Rb phosporylation by 7q.
Figure 4. Electrophoretic gel mobility of DNA isolated from
7q-treated SW480 cancer cells. Cells incubated with 7q or Taxol, as
control, for 24 h.
Figure 2. Crystal structure of inhibitor 7o complexed with CDK2.

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K.-C. Luk et al. / Bioorg. Med. Chem. Lett. 14 (2004) 913–917
Table 2. FACS cell cycle analysis of SW480 cells treated with 7q
Concentration of 7q
Lane, D. P. Curr. Med. Chem. 2000, 7, 1213. (b) Sielecki,
T. M.; Boylan, J. F.; Benfield, P. A.; Trainor, G. L. J.
Med. Chem. 2000, 43, 1.
5. Dermatakis, A.; Luk, K.-C.; DePinto, W. Bioorg. Med.
Chem. 2003, 11, 1873.
Time of
treatment
0 nM
92 nM
270 nM
6. (a) Schulze-Gahmen, U.; De Bondt, H.; Kim, S.-H. J.
Med. Chem. 1996, 39, 4540. (b) Hoessel, R.; Leclerc, S.;
Endicott, J. A.; Nobel, M. E. M.; Lawrie, A.; Tunnah, P.;
Leost, M.; Damiens, E.; Marie, D.; Marko, D.;
Niederberger, E.; Tang, W.; Eisenbrand, G.; Meijer, L. Nat.
Cell Biol. 1999, 1, 60. (c) Bramson, N. H.; Corona, J.;
Davis, S. T.; Dickerson, S. H.; Edelstein, M.; Frye, S. V.;
Gampe, R. T.; Harris, P. A.; Hassel, A.; Holmes, W. D.;
Hunter, R. N.; Lackey, K. E.; Lovejoy, B.; Luzzio, M. J.;
Montana, V.; Rocque, W. J.; Rusnak, D.; Shewchuck, L.;
Veal, J. M.; Walker, D. H.; Kuyper, L. F. J. Med. Chem.
2001, 44, 4339. (d) Huang, P.; Ramphal, J.; Rice, A.;
Tang, F.; Liang, C.; Wei, J.; McMahon, G.; Tang, C.
Proc. Am. Assoc. Cancer Res. 2001, 42, Abst. 3622.
7. For experimental details, see: (a) Chen. Y.; Dermatakis,
A.; Luk, K.-C.; Liu, J.-J. US Pat. 6,252,068 B1. (b) Chen.
Y.; Dermatakis, A.; Liu, J.-J.; Luk, K.-C. US Patent
6,303,793 B1.
4 h
G0-G1 49.25%
G2-M 18.42%
S 32.33%
G2/G1a 1.94%
%CVb 3.31%
SubG0 13.29%
G0-G1 46.55%
G2-M 14.84%
S 38.61%
G2/G1a 1.95%
%CVb 3.29%
SubG0 11.29%
G0-G1 52.41%
G2-M 14.89%
S 32.70%
G2/G1a 1.95%
%CVb 3.09%
SubG0 11.85%
G0-G1 52.49%
G2-M 18.36%
S 29.14%
G2/G1a 1.95%
%CVb 3.02%
SubG0 11.17%
G0-G1 55.84%
G2-M 15.72%
S 28.43%
G2/G1a 1.96%
%CVb 3.22%
SubG0 13.52%
G0-G1 54.68%
G2-M 15.16%
S 30.15%
G2/G1a 1.96%
%CVb 3.15%
SubG0 11.67%
G0-G1 43.90%
G2-M 21.8%
S 34.26%
G2/G1a 1.95%
%CVb 3.41%
SubG0 14.47%
G0-G1 47.74%
G2-M 38.44%
S 13.82%
G2/G1a 1.94%
%CVb 3.27%
SubG0 43.52%
G0-G1 50.00%
G2-M 35.45%
S 14.54%
G2/G1a 1.94%
%CVb 3.19%
SubG0 46.86%
16 h
24 h
a Ratio between the G2 and G1 cellular fractions.
b Coefficient of variation.
8. Assay details have been disclosed in ref 5.
9. The crystals of CDK2-7o complex were grown at 4 ꢀC by
the vapor diffusion method. CDK2 (1-298) at 12 mg/mL
was mixed with and equilibrated against 10% PEG 3350
and 0.1 M Ches pH 9. 2.5% b-mercaptoethanol was
added to reservoir after mixing of the drop. Cryoprotec-
tant was the same as the reservoir with 20% PEG 3350
and the addition of 15% ethylene glycol. X-ray data was
collected at beamline X8C at the Brookhaven National
Laboratories. Data was processed to 2.0 A with the HKL
package (Otwinowski, Z.; Minor, W. Methods Enzymol.
1997 276, 307) to an R-sym of 0.036. The structure was
refined with REFMAC (Murshudov, G. N.; Vagin, A. A.;
Dodson, E. J. Acta Crystallogr. 1997, D53, 240) to an R-
factor/Rfree of 0.234/0.273. The crystal coordinates have
been deposited in the PDB with the accession code 1R78.
10. Protocol for assessment of Rb phosphorylation in SW480
cells: SW480 (2.25Â106 cells/mL) cells were treated with
0.1% DMSO (as the control) or oxindole CDK2 inhibitor
dissolved in DMSO. Cells were treated with compound
concentrations equivalent to the IC50, IC90 & 3ÂIC90 as
determined in the MTT assay. Cells were exposed to drug
for 4 h, 16 h, and 24 h time points. For immunoblotting,
cell pellets were resuspended in PLC lysis buffer, sonicated
briefly and debris sedimented by microcentrifugation at
15,000 rpm at 4 ꢀC. The supernatant was mixed with 2X
SDS sample buffer with 5% b-ME, and equal amounts of
protein per lane loaded onto a 4–20% Tris Glycine gel.
Proteins were resolved by electrophoresis, transferred to
nitrocellulose membrane which was blocked in BSA
blocking buffer (1% BSA, 10 mM Tris, pH 7.5, 100 mM
NaCl, 0.1% Tween 20) overnight at 4 ꢀC. The membrane
was then incubated with diluted 1ꢀ antibody (NEB
Ser807/811 phospho-Rb dilution 1:1000) in BSA blocking
buffer for 1 h at room temperature. The membrane was
washed for 30 min with buffer changes every 5 min with
wash Buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.1%
Tween 20) and then incubated with 2ꢀ antibody con-
jugated to HRP (anti-mouse IgG-HRP and anti-rabbit
IgG-HRP both at 1:2000) in BLOTTO buffer (5% nonfat
milk powder, 10 mM Tris pH 7.5, 100 mM NaCl, 0.1%
Tween 20) for 1 h at room temperature. The wash proce-
dure was repeated for another 30 min after which specific
binding was detected with ECL (Amersham) folowed by
exposure to a film.
In summary, the lead oxindole 1b was transformed to a
new series of potent inhibitors of CDK2. The potency
optimization of 1b was achieved by the introduction of
propargyl and/or homopropargyl heteroatom substituted
moieties at its C-4 position.
Acknowledgements
We thank Gino Sasso, Vance Bell, Richard Szypula,
Michael Lanyi, and Theresa Burchfield for their analy-
tical and spectroscopic work. We also owe thanks to Dr.
Christine Lukacs for her help with the crystal structure
figures.
References and notes
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11. For the cell cycle analysis experiments cells were fixed in

K.-C. Luk et al. / Bioorg. Med. Chem. Lett. 14 (2004) 913–917
917
70% ethanol, centrifuged for 1 min at 3000g at 25 ꢀC,
washed once with PBS, treated with 1 mg/mL ribonu-
clease (Sigma Chemical Co.) for 15 min at 37 ꢀC and
stained with 50 mg/mL propidium iodide (Sigma Chemi-
cal Co.) for 30 min at room temperature. Flow cytometry
analyses were performed on a Becton Dickinson FaCS-
Calibur using the Becton Dickinson Cell Quest program.
12. Protocol for the cell viability-apoptosis experiments:
SW480 cells were grown in 40 mL of DMEM High
Glucose+10% heat inactivated FBS, (purchased from
GIBCO/BRL, Gaithersburg MD) at 2.5Â106 cells/150
cm2 flask for SW480 cells. After 24 h of growth at 37 ꢀC
with 5% CO2, cells were at 30% confluency. Subse-
quently, cells were treated with each drug at IC50, IC90
and 3Â IC90 for 4 h or 24 h. For the negative control cells
were treated with 40 mL of DMSO for 4 h or 24 h and for
positive control cells were treated with Taxol at the IC50,
IC90, 3Â IC90, and 0.1 mM for 4 h or 24 h. For each
sample cells were collected by transferring the growth
medium to a 50 mL centrifuge tube, collecting the 5 mL
wash with D-PBS-calcium and magnesium free (GIBCO/
BRL, Gaithersburg, MD), and collecting the trypsinized
cells (3 mL of Trypsin for 5 min at 37 ꢀC). Cells were
pelleted by centrifugation at 3000 rpm for 5 min at 4 ꢀC.
Cells were resuspended in 10 mL of D-PBS and pelleted as
above. Cells were resuspended in pre-warmed lysis buffer
(1.0 M NaCl, 50 mM Tris–HCl, pH 8.0, 0.5% SDS, and
10 mM EDTA) with Proteinase K added and lysed over-
night (16–24 h) at 37 ꢀC without shaking. DNA was
extracted with phenol:chloroform:isoamyl alcohol
(25:24:1) with vigorous vortexing for 1 min. The phases
were separated by centrifugation at 16,000g for 5 min at
room temperature. The upper aqueous phase was
removed and the extraction was repeated. The DNA was
precipitated overnight by adding 2 volumes of 100% eth-
anol to the extract, vortexed briefly, and stored at À20 ꢀC.
The DNA was pelleted at 16,000g for 5 min at room
temperature. The DNA pellet was dried and resuspended
in 30 mL of TE (10 mM Tris–HCl, pH 8.0, 1 mM EDTA).
The DNA samples were then treated with RNAse for 30
min at 37 ꢀC. The DNA was resolved by electrophoresis
(1.5% agarose, 0.5 mg/mL of ethidium bromide) in order
to assess intranucleosomal DNA cleavage. Ethidium-
stained DNA was visualized with UV light.
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