Supporting Information
Development of a Cyclin Dependent Kinase Inhibitor Devoid of ABC
Transporter-Dependent Drug Resistance
Authors: Maciej Kaliszczak†, Hetal Patel†, Sebastian H. B. Kroll§, Laurence Carroll†,
Graham Smith†, Sean Delaney†, Dean A. Heathcote†, Alexander Bondke§, Matthew J.
Fuchter §, R. Charles Coombes†, Anthony G. M. Barrett§, Simak Ali†, and Eric O.
Aboagye†*
†
Department of Surgery and Cancer, Imperial College London, Hammersmith
Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom. §Department
of Chemistry, Imperial College London, London SW7 2AZ, United Kingdom,
The following files are included as supplementary information:
Supplementary Materials and Methods
Supplementary Figures

Figure S1. Cross resistance of BS-194 in an ovarian cancer cell line model
resistant to doxorubicin.

Figure S2. Chemical structure of a novel series of pyrazolo[1,5-a]pyrimidine
derivatives.

Figure S3. Impact of MRP1 inhibition on the growth inhibitory effect of
pyrazolo[1,5-a]pyrimidine derivatives in A549 cells.

Figure S4. Assessment of ICEC-0782 toxicity.

Figure S5. CDK2 inhibition following treatment with ICEC-0782 in MCF7
xenograft model.

Figure S6. Treatment with ICEC-0782 does not induce expression of ABCG2
in MCF7 xenograft model.
Tables

Table S1. Inhibition of recombinant CDK7 by pyrazolo[1,5-a]pyrimidine
derivatives.

Table S2. Physico-chemical features of pyrazolo[1,5-a]pyrimidine derivatives.

Table S3. Metabolic stability of a novel series of pyrazolo[1,5-a]pyrimidine
derivatives in human and mouse microsomes and S9 fractions.

Table S4. Growth inhibitory effect of pyrazolo[1,5-a]pyrimidine derivatives in
HCT-116 colon, MCF7 breast and A549 lung cancer cell lines.
Supplementary Materials and Methods
Synthesis and characterisation of ICEC-0782
Pyrazolopyrimidines Xa – z were synthesised from dichloride A (Williamson et al,
2005) by sequential substitution on the C-7 position with benzylamine or ortho-fluoro
benzylamine, N-Boc protection and subsequent palladium catalysed C-5 amination
with side chains S1- 5 and global deprotection according to our recent reports (Ali et
al, 2009; Heathcote et al, 2010). Masking the terminal amine in ICEC-0229 as methyl
carbamate was achieved by reacting the amine with dimethyl dicarbonate to give
prodrug ICEC-0782 (Scheme X).
F
F
R1
Cl
N N
Cl
A
N N
N N
HN
R2
N
NH
[a] - [d]
N
NH
NH
HN
N
Xa - z
NH2
ICEC0229
N N
[e]
HN
N
HN
O
O
ICEC0782
Scheme X: Reaction sequence to pyrazolopyrimidines Xa - z.
Reagents and conditions: [a] R1NH2, EtOH, reflux, 16 h; [f] Boc2O, DMAP, THF, 20
°C, 20 h; [c] S, Pd2dba3, rac-BINAP, NaOtBu, PhMe, 95 ºC, 16 h; [d] HCl, MeOH,
20 °C, 3 h; [e] (MeCO2)2, cat. DMAP, THF, 20 ºC, 2 h, 64%.
δH (400 MHz, CDCl3) 1.30 (3H, s), 1.32 (3H, s), 1.55-1.65 (8H, m), 2.05-2.13 (3H,
m), 3.68 (3H, s), 4.62 (2H, d, J = 6.1 Hz), 5.05 (1H, s), 7.12 (1H, t, J = 8.8 Hz), 7.17
(1H, t, J = 7.7), 7.33-7.39 (2H, m), 7.67 (1H, s); δC (100 MHz, CDCl3) 23.2, 29.7,
30.7, 30.9, 31.1, 31.70, 31.73, 39.8, 112.5, 114.2, 115.7 (d, J = 19.6), 124.8, 129.4,
129.5, 160.6 (d, J = 246.3); δF (377 MHz; CDCl3) -118.8; m/z (ESI) ([M+H+]+) 455.
1.0 1H nmr.esp
0.9
0.8
Normalized Intensity
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
9
8
7
6
5
Chemical Shift (ppm)
160
140
120
4
3
2
100
80
Chemical Shift (ppm)
60
40
1
0.065 13C nmr.esp
0.060
0.055
0.050
Normalized Intensity
0.045
0.040
0.035
0.030
0.025
0.020
0.015
0.010
0.005
180
20
0
1.0 19F nmr.esp
0.9
0.8
Normalized Intensity
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
100
50
0
-50
-100
Chemical Shift (ppm)
-150
-200
-250
-300
HPLC-UV detection.
The HPLC-UV system for analysis of drug concentrations comprised of a Waters 600
connected to Waters 2487 dual λ absorbance detector and a Waters 717 plus
autosampler (Sunnyvale, CA). Data were acquired and processed with Empower Pro
version 5.0 software (Waters, Dionex, Sunnyvale, CA, USA). Compounds were
separated on a LC-ABZ alkylamide column (50 x 4.6 mm I.D.; 5 µm particle size;
Sigma) protected by a C18 guard cartridge (Phenomenex, Torrance, CA, USA). The
HPLC method used gradient elution; mobile phase solvent A comprised of 10 mM
ammonium acetate solution and mobile phase B was acetonitrile for all compounds
apart from BS-194, BS-195, ICEC-0067 and ICEC-0167; for those compounds,
solvent A was 0.1% formic acid solution and solvent B was methanol. The initial
mobile phase composition of 90% solvent A and 10% solvent B was maintained for
0.5 min. Between 0.5 and 6.5 min the percentage of mobile phase B was increased to
90%, kept constant for 5.5 min and then back to the initial mobile phase composition
within 0.2 min, with a total run time of 15 min. The column eluted at a flow rate of 1
ml.min-1 and a temperature of 23°C.
Pharmacokinetic profiling. ICEC-0782 and ICEC-0229 were prepared in 10%
DMSO, 10% solutol HS15 (BASF, Wyandotte, MI, USA) in PBS and administered at
10 mg.kg-1 to female BALB/c mice (Harlan, UK) by oral gavage (p.o.),
intraperitoneally (i.p.) or intravenously (i.v.). All animal experiments were done by
licensed investigators in accordance with the United Kingdom Home Office Guidance
on the Operation of the Animal (Scientific Procedures) Act 1986 (HMSO, London,
United Kingdom, 1990) and within guidelines set out by the United Kingdom
National Cancer Research Institute Committee on Welfare of Animals in Cancer
Research (Workman et al). Three animals were used per time point. Blood samples
were recovered by cardiac puncture under general anaesthesia and the plasma isolated
by centrifugation. Analyte detection and quantification were performed under contract
by Cyprotex Discovery Ltd (Macclesfield, UK). Snap frozen samples were
precipitated with methanol, centrifuged for 30 min at 4°C and the supernatant was
diluted with 200 µL of HPLC grade water prior to LC-MS/MS analysis. The
chromatographic system comprised of an Agilent HP 1100 binary LC pump (Agilent
Technologies, UK, Ltd., Stockport, Cheshire, UK) and CTC autosampler (Presearch
Ltd, Hitchin, Herts, UK). Separation was effected on an Atlantis C18 3 μm column
(10 x 2.1 mm; Waters Ltd, Elstree, Herts, UK) maintained at 40°C and running a
solvent gradient of 10 mM ammonium acetate in deionised water (eluent A) and 10
mM ammonium acetate in methanol (eluent B). Solvent composition was maintained
at 100% A for 0.1 min following injection of each sample. A linear gradient was then
employed reaching 5% eluent A at 1.5 min (held for 0.3 min), and 100% eluent A
from 1.85 min until the end of the run. The flow rate was 0.5 mL.min-1. Test
compound was quantified from a five point standard curve prepared by spiking
plasma with varying concentrations of test compound dissolved in DMSO over a final
concentration range of 3-3000 ng.mL-1 (final DMSO concentration 1%) and treated in
an identical manner to the test samples as described above. The data were plotted
using GraphPad prism software and the AUC determined by the trapezoid rule.
Supplementary Figure legends
Supplementary Figure S1. Cross resistance of BS-194 in an ovarian cancer cell
line model resistant to doxorubicin. Growth inhibitory effect of BS-181 and BS-194
in A2780AD doxorubicin resistant cells compared to the parental cell line A2780.
A2780 and A2780AD were pre-treated or not with ABCB1 inhibitor verapamil (ver) at
10 µM for 1 h prior to compound exposure. The results are mean ± SEM of 6
replicates representative of 3 independent experiments.
Supplementary Figure S2. Chemical structure of pyrazolo[1,5-a]pyrimidine
derivatives.
Supplementary Figure S3. Impact of MRP1 inhibition on the growth inhibitory
effect of pyrazolo[1,5-a]pyrimidine derivatives in A549 cells. Results are expressed
as ratio between the GI50 when A549 cells where pre-treated or not with 50 μM of
MK-571. Results are means ± SEM of 3 experiments performed in triplicate.
Supplementary Figure S4. Assessment of ICEC-0782 toxicity. Treatment with
ICEC-0782 does not lead to significant toxicity as shown by body weight
measurements in nu/nu-BALB/c athymic nude mice treated at 15 mg.kg-1 BID (i.e, 30
mg.kg-1.day-1) via the oral route of injection.
Supplementary Figure S5. CDK2 inhibition following treatment with ICEC-0782
in MCF7 xenograft model. Effect of ICEC-0782 on phosphorylation of Rb at
ser807/811 and at threonine 821 as determined by immunohistochemistry. MCF7
tumors were excised after 14 days of oral treatment at 15 mg.kg-1 BID. Positive cells
have brown staining. Phosphorylation labeling index was calculated from 10 random
fields of view per slice (2 slices per tumor, and 3 tumors per group) (P < 0.0001). Bar
is 100 µm.
Supplementary Figure S6. Treatment with ICEC-0782 does not induce
expression of ABCG2 in MCF7 xenograft model. ABCG2 expression in MCF7
xenografts excised after 14 days of treatment with ICEC-0782 as determined by
immunohistochemistry. Results are expressed as a percentage of positive cells (n = 3
tumors per group). Bar is 50 µm.
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Supplementary Figure S4
Supplementary Figure S5
Supplementary Figure S6
Supplementary Table S1. Inhibition of recombinant CDK7 by pyrazolo[1,5a]pyrimidine derivatives.
n.d. not determined.
Supplementary Table S2. Physico-chemical features of pyrazolo[1,5a]pyrimidine derivatives.
* Compounds associated with an active efflux by the ABC transporters ABCG2 and ABCB1
Supplementary Table S3. Metabolic stability of pyrazolo[1,5-a]pyrimidine
derivatives in human and mouse microsomes and S9 fractions.
n.d. not determined.
Supplementary Table S4. Growth inhibitory effect of pyrazolo[1,5-a]pyrimidine
derivatives in HCT-116 colon, MCF7 breast and A549 lung cancer cell lines.
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