Imidazole piperazines: SAR and development of a potent class of cyclin-dependent kinase inhibitors with a novel binding mode

Article abstract of DOI:10.1016/j.bmcl.2008.06.027

A piperazine series of cyclin-dependent kinase (CDK) inhibitors have been identified. The compounds exhibit excellent physiochemical properties and a novel binding mode, whereby a bridging interaction via a water molecule with Asp 86 of CDK2, leads to sel

Full text of DOI:10.1016/j.bmcl.2008.06.027

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4442–4446
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Imidazole piperazines: SAR and development of a potent class
of cyclin-dependent kinase inhibitors with a novel binding mode
a
a
a
a
M. Raymond V. Finlay a, , David G. Acton , David M. Andrews , Andrew J. Barker , Michael Dennis ,
Eric Fisher a, Mark A. Graham a, Clive P. Green a, David W. Heaton a, Galith Karoutchi a, Sarah A. Loddick a,
Rémy Morgentin c, Andrew Roberts a, Julie A. Tucker b, Hazel M. Weir a
*
a Cancer and Infection Research Area, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
b Lead Generation Discovery Enabling Capabilities and Sciences, AstraZeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
c
AstraZeneca Research Unit, Chemin de Vrilly, Z.I. la Pompelle BP 1050, 51689 Reims Cedex 2, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A piperazine series of cyclin-dependent kinase (CDK) inhibitors have been identified. The compounds
exhibit excellent physiochemical properties and a novel binding mode, whereby a bridging interaction
via a water molecule with Asp 86 of CDK2, leads to selectivity for the CDK family of enzymes over other
kinases. Piperazines 2e and 2i were subsequently shown to inhibit tumour growth when dosed orally in a
nude mouse xenograft study. Additional chemical series that exploit this unexpected interaction with Asp
86 are also described.
Received 29 April 2008
Revised 6 June 2008
Accepted 6 June 2008
Available online 12 June 2008
Keywords:
CDK
Ó 2008 Elsevier Ltd. All rights reserved.
Kinase
Inhibitor
Cancer
In a previous letter,1 we described the development of a series
of novel imidazole-substituted CDK inhibitors. To further explore
SAR within this area, we decided to examine alternative substitu-
ents to the previously described sulfonamides and sulfones at the
4-position of the aniline ring (Table 1).
Table 1
Structures and in vitro activity for imidazoles 1 and 2
X
N
X
N
N
N
N
N
N
N
N
N
N
N
Whilst these changes led to the identification of a number of po-
tent CDK2 inhibitors (i.e., 1a and 1e), these compounds demon-
H
H
strated
a number of suboptimal properties during initial
screening. These included cytochrome P450 inhibition, high levels
of serum protein-binding and affinity for the hERG ion channel
(data not shown). Acquired long-QT syndrome causes significant
cardiac side effects and represents a major problem in clinical
studies of drug candidates. One of the reasons for development
of arrhythmias related to long QT is inhibition of the human
ether-a-go-go-related-gene (hERG) potassium channel.2 Therefore,
early identification of hERG affinity is becoming increasingly
important. We thus chose to profile the compounds early in the
cascade using a high-throughput patch-clamp hERG assay.3 In
addition, P450 inhibition by a CDK inhibitor raises the concern of
possible drug interactions resulting from abrogation of the P450
pathway(s) of metabolism, and causing toxicity due to elevated
exposures to other drugs metabolized by these pathways.
Compound
X
CDK2 IC50
(
l
M)
MCF-7 proliferation
IC50 (lM)
1a
1b
1c
1d
1e
1f
CN
<0.003
0.024
0.008
0.07
0.068
—
0.100
—
0.045
0.072
0.145
NMe2
OMe
F
0.003
Cl
<0.003
<0.003
0.032
SO2NHCH2CH2OMe
SO2CH3
COCH3
2a
2b
<0.003
Most of the properties described above could be attributed to
the relatively high lipophilicity of these compounds. The less lipo-
philic piperazine series (2), however, appeared to be potent and
devoid of the above issues, and we elected to further explore this
class of inhibitor.
* Corresponding author. Tel.: +44 1625 510379.
E-mail address: ray.finlay@astrazeneca.com (M. Raymond V. Finlay).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.027

M. R. V. Finlay et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4442–4446
4443
amide. This could undergo conjugate addition with both sodium
methoxide and dimethylamine to give 2c and 2d, respectively
(Scheme 2).
Methyl sulfonamide 2a was prepared in a slightly different way
as shown in Scheme 3. Reaction of guanidine with aminoprope-
none 7 gave the amino pyrimidine 9. Buchwald coupling4 with bro-
mo compound 11 (derived from sulfonylation of commercial
piperazine 10) then provided the required compound 2a.
Although the piperazine sulfonamides displayed good cell and
enzyme potency6 (Table 2), the aqueous solubility of the neutral
O
O
N
F
N
N
(a), (b)
+
O
N
N
O
H
H2N
3
4
5
(c)
O
N
N
sulfonamides was less than 20 lM, possibly driven by their moder-
NH
ate lipophilicity (LogD between 2 and 3), and we elected to explore
the piperazinyl amide series to see if this property could be
modulated.
Mindful of the solubility properties of the sulfonamides, a
number of basic and hydrophilic amides were targeted. The lat-
ter were prepared by coupling with activated carboxylic acids
(e.g., glycolic acid or S-lactic acid) leading to amides 2e5 and
2f (Scheme 2).
Basic amides (2g and 2h) were accessed by reaction with
chloro-acetyl chloride followed by displacement with dimethyl-
amine or diethylamine. We were pleased to observe that once
more the compounds displayed good levels of enzyme and cell po-
tency (Table 2), and in general manifested acceptable physical
properties.
H2N
N
6
H
H
N
(d), (e)
N
N
O
N
N
N
N
N
N
H
N
7
8
Scheme 1. Synthesis of piperazine intermediate 8. Reagents and conditions:
(a) K2CO3, NMP, 120 °C, 2 h, 80%; (b) H2 gas, 10% Pd/C, EtOH, 99%; (c) NCNH2,
HCl, Dioxane, EtOH, 90 °C, 30 h, 70%; (d) 2-Methoxyethanol, 110 °C, 75%; (e) concd
HCl, iso-propanol, 28%.
H
SO2R
N
N
N
N
N
N
N
N
N
N
Clearly, a reduction in LogD benefits aqueous solubility (cf. 2a
and 2e) although in the case of 2e (LogD = 1.7 and solubility = 160)
and 2f (LogD = 2.2 and solubility = 370), we anticipate that the
presence of the chiral methyl group assists solubility (despite the
lipophilicity increase), due to disruption of crystal packing. Moving
from sulfonamide to amide derivatives also enhanced the hERG
properties of the compounds (again, cf. 2a, 2c and 2e). This finding
was in accordance with the general trend for the piperazine series,
that increased LogD led to a greater affinity for the hERG ion chan-
nel (Fig. 1).
N
N
H
N
N
(a), (b)
H
8
2c R = (CH2)2NMe2
2d R = (CH2)2OMe
(c) or (d)
O
R
N
N
N
N
N
N
N
H
Whilst introduction of a basic side chain in the amide series sig-
nificantly increased solubility (cf. 2e and 2g), the compounds dem-
onstrated low levels of exposure following oral dosing in rats and
were not pursued further. In a related series of basic inhibitors, ef-
flux had been shown to be the cause (data not shown). In contrast,
the hydroxylated amides showed low levels of serum protein bind-
ing, and excellent oral exposure. To more fully understand the rea-
sons behind the observed enzyme potency, we undertook
structural studies to investigate the binding mode of the pipera-
zines when bound to CDK2. The crystal structure of CDK2 com-
plexed with 2e is shown in Figure 2.
The hydrogen bonding interactions in the hinge region of CDK2
between Leu83 NH and the pyrimidine N, and between Leu 83 O
and the aniline NH are preserved. However, a number of additional
key features are visible. The tertiary amine nitrogen of the pipera-
zine overlays on the sulfur atom of the corresponding sulfonamide
analogues,1 and interacts via a water molecule with the backbone
2e-2h
Scheme 2. Synthesis of piperazine amides and sulfonamides 2. Reagents: (a)
ClSO2CH2CH2Cl, Et3N, CH2Cl2, 31%; (b) NMe2, THF, 80%, or NaOMe, Methanol, 28%;
(c) Carboxylic acid, HATU, DIPEA, DMF, 20–95%; (d) i—chloroacetyl chloride
i-Pr2EtN, CH2Cl2, 87%; ii—amine, THF, 80–90%.
The piperazines were prepared using a modification to the route
described by us previously1 (Scheme 1).
Thus, 4-fluoro-nitrobenzene (3) was coupled with 1-acetyl-
piperazine (4) under basic conditions. Following reduction of the
nitro group, the resulting aniline 5 was reacted with cyanamide
to produce guanidine 6 as the bicarbonate salt. Cyclisation with
the known aminopropenone 7 followed by hydrolysis gave the
piperazine 8. Further reaction with 2-chloro-1-ethanesulfonyl
chloride followed by in situ elimination gave the vinyl sulfon-
O O
NH
S
N
N
(b)
(a)
O O
N
S
N
Br
N
Br
N
(c)
10
7
11
N
N
N
H
N
N
2a
N
N
NH2
N
9
Scheme 3. Synthesis of piperazine 2a. Reagents and conditions: (a) Guanidine hydrochloride, NaOMe, Butanol, reflux, 40%; (b) MeSO2Cl, CH2Cl2, 80%; (c) Pd2(DBA)3, (2-(di-
tertbutylphosphino)biphenyl, NaOtBu, 1,4-dioxane, 15%.

4444
M. R. V. Finlay et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4442–4446
Table 2
Structures and in vitro activity for piperazines 2 and morpholines 12
X
N
O
N
Y
N
Y
N
N
N
N
N
N
N
N
N
H
H
N
Compound
X
Y
CDK2 IC50
(l
M)
MCF-7 IC50
(l
M)
% Free (Rat)
hERG Mean
Solubility pH
LogD pH 7.4
Oral DMPK AUC
M h)18
IC50
(l
M)
7.4 (l
M)
(l
2a
2c
2d
2e
2f
2g
2h
2i
12a
12b
SO2Me
H
H
H
H
H
H
H
F
<0.003
0.003
<0.003
0.017
0.017
0.016
-
0.006
0.009
0.008
0.145
<0.039
0.093
0.17
8
5.5
18
9
—
2.6
2.5
2.8
1.7
2.2
3.3
—
2.2
2.9
3.0
0.08
—
—
0.4
0.16
—
0.0
0.17
0.16
0.19
SO2(CH2)2NMe2
SO2(CH2)2OMe
COCH2OH
COCH(S-CH3)OH
COCH2NMe2
COCH2NEt2
COCH2OH
—
—
—
22
—
40
—
—
—
—
8.4
>24
>24
>32
—
18
—
11
14
160
370
>3400
>3700
160
0.22
<0.027
<0.022
0.075
0.29
H
F
56
120
—
0.11
5.3
5.2
5.1
5
4.9
4.8
4.7
4.6
4.5
4.4
1.6
1.8
2
2.2
2.4
2.6
2.8
3
3.2
3.4
Log D
Figure 1. LogD versus IonWorks hERG pIC50; red, piperazine amides, blue,
piperazine sulfonamides.
Figure 2. Crystal structure of CDK2 complexed with 2e.7 Selected nearby protein
residues are shown. Hydrogen bonding interactions with the protein are indicated
as dashed red lines. The figure was prepared using Bobscript and Raster3D.8,9
and side chain of Asp 86. This same interaction was observed in the
crystal structure of an analogous imidazole sulfonamide com-
plexed with CDK2, and helps to rationalise the selectivity that
the piperazine series displays for this enzyme compared to other
kinases. This is in accord with our previous observations that inter-
actions with Asp86 confer an increase in CDK selectivity, and has
been supported by selectivity testing in kinase screens (data not
shown).16 To explore further the nature and generality of this
water-mediated contact, we elected to prepare further analogues
that could potentially exploit a similar binding mode. Towards this
goal, we targeted the morpholine 12a (Table 2), prepared in an
analogous fashion to intermediate 8. Morpholine 12a displayed cell
activity and good physiochemical properties, in combination with
higher in vivo clearance, higher lipophilicity and lower CDK po-
tency than displayed by the piperazinyl amides. In an attempt to
further investigate the SAR of the piperazinyl series, we next
looked at substitution at the 5-position of the central pyrimidine
ring. Whilst some substituents (not shown) proved beneficial from
a potency and DMPK point of view, the effect on physical proper-
ties was, on the whole, significant with a reduction of aqueous sol-
ubility and an increase in plasma protein binding due to increased
O
O
(a)
N
N
N
N
N
N
F
7
13
(b) X = O
(c) X = NCOCH3
X
F
N
N
N
X
N
N
H
N
N
NH
N
H
H2N
12b X = O
2i X = NCOCH2OH
Scheme 4. Synthesis of 5-fluoro piperazines and morpholines 2i and 12b. Reagents
and conditions: (a) Selectfluor, MeCN, 52%; (b) 2-methoxyethanol, 110 °C, 85%;
(c) i—2-methoxyethanol, 110 °C, 83%; ii—IPA, concd HCl, 85 °C, 91%; iii—acetoxy-
acetyl chloride, Et3N, CH2Cl2, RT, then 20% NH3 in MeOH, RT, O/N 81% over two
steps.

M. R. V. Finlay et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4442–4446
4445
Table 3
Structures and in vitro activity for azetidine 14, pyrrolidine 15 and proline amide 16
H
N
O
H
N
F
N
F
N
F
N
N
N
N
O
N
N
N
N
N
N
N
N
N
N
N
N
O
H
H
H
N
Compound
CDK2 IC50
(l
M)
MCF-7 IC50
(l
M)
% Free (Rat)
Solubility (
l
M) pH 7.4
LogD pH 7.4
14
15
16
0.0005
>0.0003
0.046
—
0.14
—
5
—
—
120
9
—
2.8
3.3
—
Table 4
Structures and DMPK properties for piperazines 2e, 2i and morpholine 12b
O
O
OH
OH
N
N
N
O
N
F
N
F
N
N
N
N
N
N
N
N
N
N
N
N
H
H
H
N
N
N
Compound
Rat Vdss (L/Kg)
Rat Clp (ml/min/kg)
Rat bioavailability (%)
Dog Vdss (L/Kg)
Dog Clp (ml/min/Kg)
Dog bioavailability (%)
2e
2i
12b
2.5
1.0
1.3
15
16
26
59
67
34
3.4
3.5
2.9
19
24
140
79
31
3
%inhibition [ SW620 ]
LogD. Fluorination, in contrast, provides excellent gains in potency
without the impact displayed by more lipophilic moieties. Fluori-
nation of aminopropenone 7 was achieved using SelectFluor17 in
acetonitrile ( Scheme 4) to give the product 13 as a golden crystal-
line solid.
vehicle
95.00
75.00
55.00
35.00
15.00
-5.00
2e @ 7.5 mg/kg p.o.
2i @ 12.5 mg/kg p.o.
Cyclisation as described above with a suitable guanidine deliv-
ered the fluorinated pyrimidine that could be further elaborated in
an analogous fashion to the hydrogen pyrimidines (vide supra).
The beneficial potency effect of the fluorinated compounds can
be rationalised by the electronegative fluorine atom acidifying
the aniline NH, and thereby increasing its hydrogen-bond donor
strength to the hinge region of the protein. Gratifyingly, applica-
tion of this SAR learning to the morpholine scaffold 12a also led
to noticeably more potent CDK inhibitors (e.g., 12b, Table 2). A
crystal structure of 12b in complex with CDK2 confirmed the bind-
ing mode (data not shown), including retention of the water med-
iated contact. The results obtained for azetidine (14), pyrrolidine
(15) and proline amide (16) templates (Table 3) are also consistent
with the fluoro SAR leading to increased potency, and the water-
mediated nitrogen contact giving CDK selectivity.
Encouraged by the evidence of oral exposure from rat cassette
work, pleasing anti-proliferative cell potency and good physical
properties of compounds 2e, 2i and 12b, we next elected to under-
take discrete rat and dog PK studies with these compounds. The re-
sults from this work are shown below in Table 4.
Whilst the two piperazine compounds 2e and 2i showed oral
exposure in both rat and dog, it was apparent that the bioavailabil-
ity of morpholine compound 12b in the dog was limited by in-
creased clearance relative to the rat. In view of these data,
compounds 2e and 2i were subsequently evaluated for their ability
to inhibit the growth of human SW620 xenografts in nude mice.
Pleasingly, both compounds were shown to be active in this dis-
ease model with 2e showing 80% tumour growth inhibition when
dosed orally once a day at 7.5 mg/kg for 28 days. Compound 2i
showed 89% tumour growth inhibition when dosed orally once a
day at 12.5 mg/kg for 28 days ( Fig. 3).
5
10
15
20
25
30
day post implant
Figure 3. Inhibition of SW620 tumour xenografts in the nude mouse for
compounds 2e and 2i. Compounds were dosed orally once a day for 28 days.
In summary, we have described a series of CDK inhibitors with a
novel binding mode that exploits an unexpected interaction with
Asp86, in an analogous but subtly different way to that displayed
by the previously described sulfonamides. Two of these com-
pounds have also been shown to inhibit in vivo tumour growth
in clinically relevant disease models.
Acknowledgments
We acknowledge the excellent technical expertise of the follow-
ing scientists: Kate F. Byth, Catherine Geh, Claire A. Brassington,
Sandra E. Oakes, Malcolm Anderson, Stefan Gerhardt, Jon Read, Ju-
dith Stanway, Richard Fitzpatrick, Mike Walker, Sarah Kearney,
Karoline Pitts and Sandra McGregor.
References and notes
1. Anderson, M.; Andrews, D. M.; Barker, A. J.; Brassington, C. A.; Breed. J.; Byth,
K. F.; Culshaw, J. D.; Finlay, M. R. V.; Fisher, E.; Gingell, H. H. J.; Green, C. P.;
Heaton, D. W.; Nash, I. A.; Newcombe, N. J.; Oakes, S. E.; Pauptit, R. A.; Roberts,
A.; Stanway, J. J.; Thomas, A. P.; Tucker, J. A.; Weir, H. M. Bioorg. Med. Chem.
Lett., submitted for publication.

4446
M. R. V. Finlay et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4442–4446
2. Thai, K.-M.; Ecker, G. F. Bioorg. Med. Chem. 2008, 16, 4107.
7. Protein and crystals were obtained according to established procedures.10,11
Diffraction data were collected on beamline PX14.2 at the SRS, Daresbury, at
100 K. Crystals were soaked in 5 mM compound 2e overnight in mother liquor
containing 10% DMSO. Data processing, data reduction and structure solution
by molecular replacement were carried out using programs from the CCP4
suite.12 Compound 2e was modelled into the electron density using QUANTA.13
The protein–compound complex model was refined using Refmac5,14 and the
final structure15 has been deposited in the Protein Data Bank with the
deposition code 2vv9 together with structure factors and detailed
experimental conditions.
3. Schroeder, K.; Neagle, B.; Trezise, D. J.; Worley, J. J. Biomol. Screening 2003, 8, 50.
4. For recent reviews, see: (a) Yang, B. H.; Buchwald, S. L. J. Organomet. Chem. 1999,
576, 125; (b) Hartwig, J. F. Angew. Chem., Int. Ed. Engl. 1998, 37, 2046; (c) Wolfe, J.
P.; Wagaw, S.; Marcoux, J.-F.; Buchwald, S. L. Acc. Chem. Res. 1998, 31, 805.
5. NMR data for compound 2e : dH (300 MHz, DMSO-d6) 1.38 (6H, d, J = 8.0 Hz),
2.46 (3H, s), 3.05 (4H, s), 3.48–3.60 (4H, m), 4.12 (2H d, J = 5.4 Hz), 4.57 (1H t,
J = 5.5 Hz), 5.64–5.73 (1H, m), 6.91 (2H d, J = 8.9 Hz), 6.94 (1H, s), 7.38 (1H, s),
7.46 (2H d, J = 8.9 Hz), 8.30 (1H, d, J = 5.2 Hz), 9.16 (1H, s).
6. Inhibition of proliferation in whole cells was assayed using the Roche BrdU
incorporation Elisa kit (Roche 1-647-229). MCF-7 cells were seeded into 96-well
plates at 8 Â 103 cells/well in DMEM (D6546) supplemented with 10% FCS and
1% Glutamine and allowed to adhere overnight. They were then treated with
inhibitors for 48 h at 37 °C. Ten microlitres of BrdU labelling reagent were added
to each well and plates were re-incubated at 37 °C for 2 h.Cells were fixed with
8. Esnouf, R. M. J. Mol. Graph. Model. 1997, 15, 132.
9. Merritt, E. A.; Murphy, M. E. P. Acta Crystallogr. 1994, D50, 869.
10. Lawrie, A. M.; Noble, M. E.; Tunnah, P.; Brown, N. R.; Johnson, L. N.; Endicott, J.
A. Nat. Struct. Biol. 1997, 4, 796.
11. Legraverend, M.; Tunnah, P.; Noble, M.; Ducrot, P.; Ludwig, O.; Grierson, D. S.;
Leost, M.; Meijer, L.; Endicott, J. J. Med. Chem. 2000, 43, 1282.
12. CCP4, Acta Crystallogr. 1994, D50, 760.
50 ll of FixDenat for 45 min, washed with wash buffer and then exposed to 75 ll
of AntiBrdU POD for 90 min at RT.
Cells were washed with wash buffer and 100
l
l of TMB substrate was added to
13. Quanta2000, Accelrys.
each well and plates were shaken vigorously for 10 min at RT. The reaction was
stopped by the addition of 25 l of 1 M H2SO4. Absorbance was then measured
14. Refmac version 5.1.17, Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta
Crystallogr. 1997, D53, 240.
l
on a 96-well plate reader at 450 nm. The IC50 values for CDK inhibitors were
determined by performing dose–response curves with individual compounds
and determining the concentration of inhibitor producing fifty percent decrease
in maximal signal. Cdk2/Cyclin E enzyme inhibition was screened using Spatial
15. Crystallographic statistics for the CDK2-compound 2e complex are as follows:
space group P212121, unit cell 53.2, 71.1, 71.9 Å, resolution 1.90 Å, 21,456
reflections from 57,306 observations give 97.3% completeness with Rmerge of
4.0% and mean I/r(I) of 16.0. The final model containing 2206 protein, 152
Proximity Assay radiometric technology (Amersham RPNQ0019 Protein
A
water and 32 compound atoms has an R-factor of 19.1% (Rfree using 5% of the
data 22.7%). Mean temperature factors for the protein and the ligand are 37.9
and 33.4 Å2, respectively.
coated beads). The ability of Cdk2/E inhibitors to inhibit phosphorylation of
the Retinoblastoma protein (gene region 792–928 expressed in a GST expression
system and purified from Escherichia coli) by partially purified non-GST tagged
CDK2/E (from a baculoviral insect cell lysate) was assessed. CDK2/E enzyme
activity was titrated on a batch to batch basis. CDK2/E inhibitors were dissolved
in 100% DMSO to 10 mM and diluted down in 5% DMSO to give a final
16. Analysis of kinase protein sequences reveals only 34 kinases (including CDKs)
having an Asp residue at this position. In a panel of 43 kinases, when tested at
10
compound 2i showed >90% inhibition against six enzymes when tested at
10 M against a panel of 24 kinases.
lM, compound 2e showed >90% inhibition against 10 enzymes. Similarly,
concentration range of 10 to 0.003
Ten microlitres of inhibitor were mixed with 25
(XS), 400 nM cold ATP, +0.145 rmuCi [
33P]ATP Redivue (Amersham AH9968).
Rb was stored at À80 °C in a buffer containing 50 mM Hepes, 10 mM MnCl2,
1 mM DTT, 100 M NaF, 100 M NaVan, 10 mM NaGlycPhos, 5 g/ml Aprotinin,
2.5 g/ml Leupetin and 100 M PMSF. Max wells had 10 l of 5% DMSO and Min
wells 10 l of 10 M final concentration Roscovitine (Calbiochem 557360).
The reaction was started by the addition of 20 l Cdk2/E (in a buffer containing
1 mg/ml BSA, 50 mM Hepes, 10 mM MnCl2, 1 mM DTT, 100 M NaVan, 100
NaF and 10 mM NaGlycPhos). Reaction volume was 55 l. Plates were incubated
at RT for 60 min. The reaction was stopped by the addition of 150 l STOP
l
M.
l
l
l of GST-Rb at 1.25
lg/well
17. Nyffeler, P. T.; Duron, S. G.; Burkart, M. D.; Vincent, S. P.; Wong, C.-H. Angew.
Chem., Int. Ed. 2005, 44, 192.
c
18. A cocktail of six compounds was formulated in propylene glycol using a
combination of vortex mixing; sonication and high speed shear mixing. This
formulation consisted of five test compounds (1 mg/ml) and a QC compound
(0.5 mg/ml). The formulation was dosed (2 ml/kg) to two male rats (170–
250 g) which had been fasted for 616 h. The dose for the test compounds was
2 mg/kg and for the QC was 1 mg/kg. Serial blood samples were taken from
rats at 0.5, 1, 2 and 4 h post-dose via the tail vein and a terminal sample was
taken at 6 h post-dose. The blood samples were centrifuged and plasma was
removed for analysis. The two plasma samples for a given time point were
combined prior to analysis. A single set of six calibration standards containing
all six compounds covering the concentration range (0.3 ng/ml–3 ug/ml) were
prepared by spiking blank plasma. The samples and standards were extracted
by precipitation with two volumes of acetonitrile followed by centrifugation.
The resulting supernatant was then diluted with water (10-fold). The results
l
l
l
l
l
l
l
l
l
l
lM
l
l
solution containing 50 mM Hepes, 1:500 dilution of Anti-GST Ab (Molecular
Probes A-5800), 0.5 M EDTA and Protein A-coated SPA beads (reconstituted in
25 ml of 50 mM Hepes). After 2 h incubation at RT, the plates were centrifuged at
2500 rpm for 5 min and then read on a TopCount NXT Microplate Scintillation
Counter.
The IC50 values for Cdk2 inhibitors were determined by performing dose–
response curves with individual compounds and determining the concentration
of inhibitor producing 50% decrease in maximal signal (diluent control).
obtained were used to determine the Cmax
for a given compound. All reported data are normalised to a dose of 1
(
l
M), AUC0–6h
(
l
M h) and tmax (h)
M/kg.
l