Isoindolinone inhibitors of the murine double minute 2 (MFM2)-p53 protein-protein interaction: Structure-activity studies leading to improved potency

Article abstract of DOI:10.1021/jm1011929

Inhibition of the MDM2-p53 interaction has been shown to produce an antitumor effect, especially in MDM2 amplified tumors. The isoindolinone scaffold has proved to be versatile for the discovery of MDM2-p53 antagonists. Optimization of previously reported inhibitors, for example, NU8231 (7) and NU8165 (49), was guided by MDM2 NMR titrations, which indicated key areas of the binding interaction to be explored. Variation of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification of 3-(4-chlorophenyl)-3-((1- (hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhibitor (IC₅₀ = 0.23 A± 0.01 μM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (+)-R-enantiomer (74a; IC₅₀ = 0.17 A± 0.02 μM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for wild-type p53 cell lines in growth inhibition assays.

Full text of DOI:10.1021/jm1011929

ARTICLE
pubs.acs.org/jmc
Isoindolinone Inhibitors of the Murine Double Minute 2 (MDM2)-p53
Protein-Protein Interaction: Structure-Activity Studies Leading to
Improved Potency
Ian R. Hardcastle,*,† Junfeng Liu,‡ Eric Valeur,† Anna Watson,† Shafiq U. Ahmed,‡ Timothy J. Blackburn,†
Karim Bennaceur,‡ William Clegg,† Catherine Drummond,‡ Jane A. Endicott,§ Bernard T. Golding,†
Roger J. Griffin,† Jan Gruber,§ Karen Haggerty,† Ross W. Harrington,† Claire Hutton,‡ Stuart Kemp,†
Xiaohong Lu,‡ James M. McDonnell,§ David R. Newell,‡ Martin E. M. Noble,§ Sara L. Payne,†
Charlotte H. Revill,† Christiane Riedinger,§ Qing Xu,‡ and John Lunec*,‡
†Newcastle Cancer Centre, Northern Institute for Cancer Research and School of Chemistry, Bedson Building, Newcastle University,
Newcastle, NE1 7RU, United Kingdom
‡Newcastle Cancer Centre, Northern Institute for Cancer Research, Paul O’Gorman Building, Medical School, Framlington Place,
Newcastle University, Newcastle, NE2 4HH, United Kingdom
§Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU,
United Kingdom
S Supporting Information
b
ABSTRACT: Inhibition of the MDM2-p53 interaction has been
shown to produce an antitumor effect, especially in MDM2
amplified tumors. The isoindolinone scaffold has proved to be ver-
satile for the discovery of MDM2-p53 antagonists. Optimization of
previously reported inhibitors, for example, NU8231 (7) and
NU8165 (49), was guided by MDM2 NMR titrations, which
indicated key areas of the binding interaction to be explored. Variation
of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification
of 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-
2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhi-
bitor (IC50 = 0.23 ( 0.01 μM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (þ)-
R-enantiomer (74a; IC50 = 0.17 ( 0.02 μM). The cellular activity of key compounds has been examined in cell lines with defined p53 and
MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for
wild-type p53 cell lines in growth inhibition assays.
’ INTRODUCTION
Inactivation of p53 by a range of mechanisms is a frequent
causal event in the development and progression of cancer.
These include inactivation by mutation, targeting by oncogenic
viruses, and, in a significant proportion of cases, amplification of
the MDM2 gene, resulting in overexpression or increased acti-
vation of the MDM2 protein. MDM2 amplification has been
reported to occur in approximately 11% of all tumors and is
found at higher levels in certain tumor types, for example,
hepatocellular carcinoma (44%), osteosarcomas (20%), and soft
tissue sarcomas (31%).4,5 Normally, transcriptional activation of
MDM2 by activated p53 results in increased MDM2 protein
levels, forming a negative feedback loop.6,7 MDM2 binds to the
p53 transactivation domain, blocking p53-dependent transcrip-
tional activity. MDM2 also functions as an E3 ligase, which
The response of the tumor suppressor protein p53 to cellular
stresses, such as hypoxia, DNA damage, and oncogenic activa-
tion, is to act as a signaling node in the diverse pathways that
become activated. Phosphorylation of p53, resulting from activa-
tion of kinases including ATM, CHK1 and 2, and DNA-PK,
results in a stabilized and transcriptionally active form of the
protein.1 The resulting transcription of a range of genes is
responsible for diverse functions, such as apoptosis, survival,
cell-cycle arrest, DNA repair, angiogenesis, invasion, and auto-
regulation, giving rise to the observed cellular effect.1,2 The
eventual fate of a cell, that is, apoptosis, cell-cycle arrest, or
senescence, following p53 activation is thought to be determined
by its genetic background. For tumor cells, the apoptotic pathway
may be favored due to the loss of tumor suppressor proteins and
associated cell cycle checkpoint controls, coupled with oncogenic
stress.3
Received: September 13, 2010
Published: February 11, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Substituted Isoindolinones 48-103a
a Reagents and conditions: (a) SOCl2, DMF, THF; (b) R1NH2, i-Pr2EtN, or Et3N, THF; (c) SOCl2, DMF, THF; (d) R2OH, K2CO3, or i-Pr2EtN, THF.
have been reported.10-12 The most widely studied series to date are
the Nutlins, for example, Nutlin-3 (1),13 and the spirooxindoles, for
example, MI-147 and MI-219 (2, 3);14,15 both series have demons-
trated cellular activity consistent with MDM2-p53 inhibition and have
shown in vivo antitumor activity.13,16 Other small-molecule inhibitors
include benzodiazepinediones (4),17,18 2-methyl-7-(phenyl(phenyl-
amino)methyl)quinolin-8-ol (5),19 and chromenotriazolopyrimi-
dines (6).20 Previously, we have reported isoindolinones, for example,
NU8231 (6) and NU8165 (49) with low micromolar MDM2-p53
inhibitory potency and cellular activity.21 In this paper, we describe the
application of NMR-based structural biology and SAR studies to
inform binding models, leading to MDM2-p53 inhibitory isoindoli-
nones with significantly improved potency and cellular activity over
the parentcompounds.
Scheme 2. Synthesis of Sulfoxide 29a
a Reagents and conditions: (a) mCPBA, DCM.
Scheme 3. Synthesis of Benzoic Acids 34 and 91a
a Reagents and conditions: (a) (CH3)3SiOK, THF.
Scheme 4. Synthesis of Pyridine N-Oxides 40-42 and 100-
102a
’ CHEMISTRY
Substituted isoindolinones were prepared using the method
described previously.21,22 The appropriate benzoylbenzoic acids
(8a-d) were converted into the corresponding ψ-acid chlorides
(9a-d), which were condensed with the R1-primary amines to give
the 4-hydroxyisoindolinones (10-47). The 4-hydroxy com-
pounds (10-47) were converted into their respective chlorides
and subsequently reacted with the appropriate alcohol in the
presence of base (Et3N or DIPEA) to give the final substituted
isoindolinones (48-104) as racemates (Scheme 1). The 4-methyl-
sulfoxide (29) was prepared from the 4-methylthio derivative (28)
by oxidation with mCPBA (Scheme 2). The 4-carboxylic acids (34
and 92) were prepared by cleavage of the corresponding methyl
esters (34 and 91) by saponification with potassium trimethylsila-
nolate in THF (Scheme 3). The 2-, 3-, and 4-pyridine N-oxides
(40-42 and 100-102) were prepared by the reaction of the
parent pyridines with mCPBA in DCM (Scheme 4). The 4-tri-
fluoromethylsulfone derivative (104) was prepared from the
3-hydroxy precursor (45) directly using boron trifluoride etherate
to activate the 3-hydroxyl group, followed by reaction with the
a Reagents and conditions: (a) mCPBA, DCM.
ubiquitylates the MDM2-p53 complex and has been shown to
export the complex from the nucleus and target it for proteaso-
mal destruction.8,9
The tractability of the MDM2-p53 interaction as a drug target
has been demonstrated and a number of classes of potent inhibitors
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Scheme 5. Synthesis of Trifluoromethylsulfone 104a
a Reagents and conditions: (a) (i) BF3 Et2O, DCM, 0 °C; (ii) 1,1-
3
bis(hydroxymethyl)cyclopropane, DCM, 0 °C.
Scheme 6. Synthesis of 4-(4-(Ammoniomethyl)phenyl)-4H-
1,2,4-triazol-4-ium Chloride 107a
Figure 2. Structure of (R)-NU8165 bound to MDM2 deduced from
15N-13C HSQC NMR studies: (A) GOLD docked structure; (B)
binding mode cartoon.
petrol by vapor diffusion, enabling a high-resolution X-ray crystal
structure to be obtained that established the absolute stereo-
chemistry as (S) (Figure 1). Suitable crystals were not obtained
for the (þ)-enantiomer 74a.
a Reagents and conditions:(a) (i) SOCl2, 5 °C to r.t.; (ii) NH2NH2 H2O,
3
r.t.; (b) toluene, reflux; (c) H2, 10% Pd/C, MeOH, aq. HCl.
’ RESULTS AND DISCUSSION
Structure-Based Design. In our previous studies, we de-
monstrated the limitations of computer docking approaches in
guiding the design of novel isoindolinone ligands for the p53-
binding site of MDM2.21 These studies were hampered by the
large number of low energy binding modes available. As a result,
we sought to gain structural information with key inhibitors
bound to MDM2 through X-ray crystallography and NMR
experiments. Unfortunately, to date, suitable cocrystals of iso-
indolinones bound to MDM2 have not been obtained. By
contrast, NMR has provided useful structural information,
despite the relatively low affinities of the ligands employed.24
Initial NMR experiments using 6 (IC50 = 16 μM) showed a
significant chemical shift change in the Leu54 region attributed
to an interaction with the hydroxypropoxy substituent
(Figure 2). Reasoning that additional potency may be gained
by introducing rigidity to the 3-alkoxy substituent, the cis-
cyclopentyl derivative 50a was proposed (Figure 3). The binding
mode model for 6 also suggested that the substitution of the N-
benzyl moiety could introduce additional favorable interactions
and, hence, improve potency.
Structure-Activity Relationships for Inhibition of the
MDM2-p53 Interaction. 3-Hydroxy Isoindolinones. To under-
stand the relative contributions to potency of the substituents on
the isoindolinone scaffold, the 3-hydroxyisoindolinones were
assayed for their MDM2-p53 inhibitory activity (Table 1). The
introduction of small, lipophilic substituents at the 4-position of
the N-benzyl group resulted in significant gains in potency, for
example, the 4-chloro compound (13) is 17 times more potent
than the parent compound (11). Similarly, the introduction of a
4-nitro substituent (20) resulted in a 30-fold improvement in
potency over 11. Retaining the 4-nitrobenzyl substituent and
varying the 3-aryl substituent demonstrated that the 4-chloro or
4-bromo substituents confer the greatest potency (20 and 21)
Figure 1. X-ray structure of 74b, indicating the (S)-enantiomer.
relevant diol (Scheme 5). The 4-(1,2,4-triazolyl)benzylamine
precursor was prepared by the reaction of dimethylaminomethy-
lene-N,N-dimethylformohydrazonamide (105)23 and 4-aminoben-
zonitrile, followed by reduction of the nitrile (106) to the
methylamine (107; Scheme 6).
Compound 74 was resolved by HPLC using a chiral column.
The (-)-enantiomer 74b was crystallized from EtOAc and
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Table 1. 3-Hydroxyisoindolinone SAR: Variation of the 2-N-
Benzyl Substituent
Figure 3. Modeled structure of the trans-(R)-3-((1R,3R)-3-hydroxycy-
clopentyloxy analogue of NU8165.
compared with the unsubstituted 3-phenyl (18), whereas the
4-fluoro compound (19) was less active. Switching the nitro
group to the 3-position (22) resulted in a dramatic loss of
activity. The 1-naphthyl (26) and 3-bromo (27) derivatives were
similarly nonpotent, indicating a lack of tolerance for steric bulk
at this position. Homologation of 11 to the phenethyl derivative
(23) resulted in a 10-fold gain in potency, whereas, the 4-nitro-
and 4-anilino-substituted phenethyl derivatives (25 and 26) did
not gain additional potency. A range of additional 4-substituents
was investigated, including classical and nonclassical isosteres of
the nitro group (29-42). Of these, only the methyl 4-benzoate
(33) retained significant activity.
3-Alkoxy Isoindolinones. A series of 3-alkoxyisoindolinones
was assayed for MDM2-p53 inhibitory activity (Table 2). As
predicted by the NMR-based binding model, the 3-hydroxycy-
clopentoxy derivative (50), a mixture of cis- and trans-isomers,
showed a 5-fold improvement in potency compared with the
acyclic derivative (6), demonstrating the positive effect of
conformational restriction of the alkoxy side chain. Gratifyingly,
inclusion of the 4-nitrobenzyl group, observed to be beneficial in
the 3-OH series, had an additive effect on potency (51).
However, refinement of the stereochemistry to the cis-derivative
52 produced only a modest improvement in potency over the cis/
trans mixture. The desired trans-diol was not commercially
available, and attempts to epimerize the cis-derivative 52 met
with failure. In contrast to the 3-hydroxy series, inclusion of a
4-cyanobenzyl group (53) did not improve potency significantly
over 50. Further conformational restriction was introduced with
the unsaturated cis-3-hydroxycyclopent-2-enyloxy group. Intro-
duction of 4-chloro or 4-methyl substituents resulted in modest
gains in potency (55 and 56), and the 4-nitro substituent gave a
similar gain in potency (57), although this compound was 5-fold
less potent than 52. Interestingly, the acyclic unsaturated Z-4-
hydroxybut-2-enyloxy derivative 58 was equipotent with the
trans-cyclopentoxy derivative 50.
a n = 1.
of available starting materials, and efforts to epimerize final
trans-compounds met with failure. As an alternative, the acyclic
derivatives were revisited with the inclusion of selected favorable
4-substituted benzyl groups identified in the 3-OH series. Interest-
ingly, in the 4-nitro series, the hydroxypropoxy and hydroxybutoxy
compounds 59 and 60 were equipotent with the trans-cyclic deri-
vative 52. The 4-cyanohydroxybutoxy compound 61 also exhibited
similar potency to the corresponding trans-cyclic derivative 53.
Some stereospecificity of inhibition was observed with the 4-
chloro-R-methylbenzylamine derivatives, the (R)-isomer 62 was
2-fold more potent than the (S)-isomer 63, although 62 was 2-fold
less potent than the 4-nitro derivative 61. Homologation to the
4-nitrophenethylamino derivative 64 resulted in a 7-fold loss in
activity. The modest drop in activity on replacement of 4-chlor-
ophenyl by 4-bromophenyl was also seen in the hydroxypropoxy
and hydroxybutoxy compounds 65 and 66.
The SAR around the 3-alkoxy substituent was explored further,
retaining the 4-nitrobenzyl group as optimal (Table 3). A range of
suitable diols was introduced to probe the spatial and steric
requirements at this position, and all the compounds prepared
inhibited the MDM2-p53 interaction with submicromolar potency.
The trans-1,4-cyclohexanedimethanol derivative 68 was nearly
2-fold more active than 1,4-phenylenedimethanol derivative 67,
which was the least potent in the series. The 1,3-phenylenedi-
methanol derivative 69 displayed improved potency compared
with 67. The trans-1,4-cyclooctane diol, trans-1,4-cyclohexane diol,
The synthesis of the cis-3-hydroxycyclopentoxyisoindolinones,
predicted to be favored by the model, was complicated by the lack
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Table 2. Isoindolinone SAR: Variation of the 2-N-Benzyl and
3-Alkoxy Substituent
Table 3. 2-N-4-Nitrobenzylisoindolinone SAR: Variation of
the 3-Alkoxy Substituent
* Ref 21.
and trans-1,4-cyclohex-2-ene diol derivatives 70, 75, and 76 showed
similar activity to the 1,4-butanediol derivative 60, whereas the but-
2-yne-1,4-diol and the cis-1,2-cyclohexanedimethanol derivatives
71 and 72 were somewhat less potent. Steric restriction of the 1,
3-propanediol chain with 2,2-dimethyl, 2-cyclopropanyl, or 2-
methylene groups (73, 74, and 75) maintained the potency of
the parent 59, with the 2-(1,1-cyclopropane)dimethanol derivative
74 showing the best activity in this series.
The possible pharmacological liabilities associated with the nitro
group present in 74 prompted the evaluation of a number of
alternatives including classical and nonclassical isosteres (Table 4).
Replacement with simple lipophilic substituents (78-86) resulted
in a 5-10-fold loss in potency, with the more potent being the
cyano, bromo, and iodo derivatives (78, 81, and 82). The introduc-
tion of more sterically demanding, or meta-substituents, resulted in
dramatic losses in potency, for example, trifluoromethyl (89) and
the 1-naphthalene derivative (87). Isosteric and pseudoisosteric
replacements for the nitro group were also inactive, for example,
carboxyl (92), carboxamide (94), and acetyl (95). The 2-, 3- and
4-pyridyl isomers (97-99) lacked significant potency, as did their
respective pyridine-N-oxides (100-102). We considered that the
1,2,4-triazolyl group could act as a nitro group bioisostere, as it has
similar shape and electronic characteristics. Disappointingly, the
4-(1,2,4-triazolyl) derivative (103) was inactive, possibly indicating
a lack of steric tolerance for the larger heterocycle. An aromatic nitro
group was replaced successfully by a trifluoromethylsulfone group
in the development of the Bcl-XL inhibitor ABT-737.25 In our case,
however, the same replacement (104) resulted in a complete loss of
activity. These results suggest that the nitro group’s interactions
with the protein are mostly lipophilic and strongly directional and
that its electron withdrawing character is also important.
The enantiomers of 74, the most potent compound identified
in this study, were separated by chiral HPLC and showed that
potent MDM2-p53 activity resided with the (þ)-R-enantiomer
74a (IC50 = 0.171 ( 0.015 μM). By contrast, the S-enantiomer
74b was weakly active (IC50 = 1.30 ( 0.11 μM). The binding
mode for 74a as deduced from NMR experiments and GOLD
docking is similar to the original model for 6 (Figure 4).24 The
2-(1,1-cyclopropane)dimethanol interacts with the surface of the
region close to Leu54 of MDM2, a region not occupied by
Nutlin-2 or the benzodiazepine 3, as seen in X-ray structures
(1rv1 and 1t4e). The 4-chlorophenyl group occupies the Ile99
pocket, also occupied by halophenyl groups in the Nutlin-2 and
3. The deep pocket filled by Trp23 of p53 is occupied by the
isoindolinone scaffold. The 4-nitrophenyl group occupies the
Gln54 pocket, which is occupied by the piperidinone moiety of
Nutlin-2 and the iodoaryl portion of 3. The SAR for the
4-substituted phenyl group is not readily explained by this model
and further structural studies are ongoing.
Cell Biology. Potent compounds identified in the cell-free
ELISA were selected for investigation in intact cells. SJSA-1 cells
(MDM2 amplified, p53wt) were treated with increasing
concentrations of 68, 70-74, 74a, and 74b using Nutlin-3 as a
positive control. Cells were lysed at a 6 h time point and protein
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Table 4. 3-(1-Hydroxymethylcyclopropylmethoxy)
isoindolinone SAR: Variation of the 2-N-Benzyl Substituent
Figure 5. Cellular activity of 70, 73, and 74 for the p53 pathway
activation detected by Western blotting (SJSA-1 cells, 4 h treatment).
* n = 1.
Figure 6. Cellular activity of 71, 68, and 72 for the p53 pathway
activation detected by Western blotting (SJSA-1 cells, 4 h treatment).
Figure 4
extracts analyzed by Western blotting, probing for MDM2, p53 and
p21 to test for p53 pathway activation, and either actin or R-tubulin as
protein loading controls (Figures 5-7). Compounds 68, 70-74,
74a, and 74b produced a concentration-dependent induction of
MDM2, p53, and p21 over the 1-20 μM dose range, with 72,68,and
74 showing greater induction of p53 and p21 at the 10 μM point.
Nutlin-3 showed comparable effects over a 1-10 μM dose range.
The enantiomers of 74, 74a, and 74b, and Nutlin-3 were compared
over a 1-20 μM dose range (Figure 7). The potent enantiomer 74a
showed strong induction of MDM2, p53, and p21, whereas the less
potent enantiomer 74b showed only weak activity at the highest
concentration (20 μM). The lack of cellular activity of 74b offers
strong evidence that the cellular activation of p53 transcription by the
isoindolinone inhibitors is mediated by their inhibition of MDM2-
p53 binding.
Figure 7. Cellular activity of 74, 74a, and 74b for the p53 pathway
activation detected by Western blotting (SJSA-1 cells, 4 h treatment).
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Figure 8. Comparison of concentration dependent growth inhibition of 74a and Nutlin-3 using an SRB-based growth inhibition assay in a panel of cell
lines with differing MDM2 and p53 status.
Table 5. Growth Inhibitory Activity of 74 and Enantiomers in Comparison to Nutlin-3, as Determined by SRB Assay
SRB GI50a (μM)
compound
MDM2-p53 IC50 (μM)
SJSA-1
LS
NGPb
HCT116(þ/þ)
HCT116(-/-)
SAOS
74
0.23 ( 0.01
0.17 ( 0.02
1.30 ( 0.11
0.061 ( 0.021
8.3 ( 1.1 (n = 4)
5.2 ( 1.5
6.9 ( 1.3 (n = 4)
6.1 ( 1.8
6.9
3.4
13
15 ( 0.3
20 ( 2
21 ( 1
13 ( 3
20 ( 1
24 ( 2
24 ( 1
34 ( 4
8.9 ( 2.1
11 ( 3
74a
74b
20 ( 3
153 ( 4
14 ( 1
Nutlin-3
2.6 ( 0.1
2.3 ( 0.3
2.1
18 ( 0.2
a Values are n = 3 unless otherwise stated. b n = 1.
The growth-inhibitory and cytotoxic effects of 74a and Nutlin-
3 were compared in a range of cell lines using the SRB assay
(Figure 8). Three MDM2 amplified cell lines were used, all wild
type for p53 (SJSA-1 osteosarcoma, NGP and LS neu-
roblastoma), and three MDM2 nonamplified cell lines, an
isogenic pair of wild type p53 and null colorectal cancer cell
lines (HCT116 p53(þ/þ) and p53(-/-) colon), and a p53
null osteosarcoma cell line (SaOS-2). The results for both
compounds show that MDM2 amplified cell lines were more
sensitive to the growth inhibitory effects of MDM2-p53 inhibi-
tion. A comparison of GI50 values showed that 74a was 2-3-fold
less growth inhibitory than Nutlin-3 in the three MDM2-
amplified cell lines consistent with the 2.5-fold lower potency
versus MDM2-p53 of 74a (Table 5). The less-active enantiomer
74b was 6-10-fold less growth inhibitory than Nutlin-3 but is
approximately 20-fold less potent versus MDM2-p53, suggesting
that some nonspecific growth inhibitory activity is present.
Nutlin-3 showed the greatest difference in activity in the paired
p53(þ/þ) and (-/-) HCT-116 cell lines and had the lowest
potency in the p53 null SAOS line. These results suggest that the
initial growth inhibitory activity of the MDM2-p53 inhibitors
tested resulted from MDM2-mediated p53 activation but that, at
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higher concentrations, non-p53 related off-target effects resulted
in additional growth inhibitory activity.
the required amine (1.1 equiv) and DIPEA (1.1 equiv) or Et3N (1.1
equiv) were added, under N2. The resulting mixture was stirred over-
night at rt and then concentrated in vacuo. The residue was diluted with
EtOAc (50 mL) and filtered, and the filtrate washed with water (3 ꢀ 25
mL) and brine (1 ꢀ 25 mL). The organic layer was dried (MgSO4 or
Na2SO4) and concentrated in vacuo to afford the required 3-hydro-
xysioindolinone as a solid, which was recrystallized (EtOAc, petrol) or
purified by chromatography.
General Procedure B: 3-Alkoxy Isoindolinones. To a solu-
tion of the appropriate isoindolinone (1.37 mmol, 1 equiv) in THF (10
mL) under N2 was added thionyl chloride (2 equiv) and 3 drops of DMF,
and the resulting mixture was stirred for 4 h at rt and then concentrated in
vacuo. The residue was diluted with THF (10 mL) and the required
alcohol (2 equiv), potassium carbonate (2 equiv) or DIPEA (1.1 equiv)
were added, and the resulting mixture was stirred overnight at rt and then
concentrated in vacuo. The residue was diluted with EtOAc (50 mL) and
washed with water (3 ꢀ 25 mL) and brine (1 ꢀ 25 mL). The organic layer
was dried (MgSO4) and concentrated in vacuo to afford an oil that was
purified by chromatography (silica; EtOAc/petrol).
’ CONCLUSIONS
The isoindolinone series of MDM2-p53 inhibitors has been
optimized for potency and cellular activity. The addition of a
4-nitro substituent to the N-benzyl moiety of 6 resulted in a
greater than 30-fold improvement in potency (59, IC50 = 0.45 (
0.01 μM). A further gain was achieved by introducing conforma-
tional restriction to the isoindolinone 3-hydroxypropoxy side-
chain in the form of a 2-cyclopropyl group (74, IC50 = 0.23 (
0.01 μM). As expected, only one enantiomer (R)-74a (IC50
=
0.17 ( 0.02 μM) was responsible for the inhibitory potency of
the racemate. The cellular activation of p53 of 74a was similar to
Nutlin-3, albeit at a lower potency. Similarly, the profile of
growth inhibitory activity in cell lines with defined p53 and
MDM2 status was similar to Nutlin-3, however, p53-indepen-
dent, off-target effects were seen at lower concentrations than for
Nutlin-3. We consider that the isoindolinones such as 74a are
promising leads for the development of MDM2-p53 targeted
cancer therapeutics. Further studies are ongoing to address the
potency, selectivity, and drug-like properties for this class of
inhibitors.
E-3-(4-Chlorophenyl)-3-(4-hydroxybut-2-enyloxy)-2-(4-
nitrobenzyl)-2,3-dihydroisoindol-1-one (58). General Proce-
dure B: 20 (400 mg, 1.0 mmol), thionyl chloride (214 μL, 2.75 mmol),
cis-butenediol (445 mg, 5.0 mmol), and K2CO3 (380 mg, 2.75 mmol)
gave 58 as a yellow oil (272 mg, 58%): 1H NMR (300 MHz, CDCl3) δ
1.70 (br s, 1H, OH), 3.32 (dd, 1H, J = 12.1, 6.3 Hz, OCH), 3.45 (dd, 1H,
J = 12.1, 6.3 Hz, OCH0), 3.79 (d, 2H, J = 6.6 Hz, HOCH2), 4.25 (d, 1H, J
= 15.0 Hz, N-CH), 4.64 (d, 1H, J = 15.0 Hz, N-CH0), 5.26-5.35 (m,
1H, OCH2CH), 5.53-5.62 (m, 1H, OCH2CH0), 7.12-7.23 (m, 5H,
Ar-H), 7.36-7.39 (m, 2H, Ar-H), 7.51-7.57 (m, 2H, Ar-H), 7.91-
7.93 (m, 1H, C(O)dCdCH), 8.00-8.04 (m, 2H, Ar-H); 13C NMR
(75 MHz, CDCl3) δ 42.57, 58.48, 59.20, 95.0, 123.4, 123.9, 126.8, 128.0,
128.7, 130.0, 130.2, 131.4, 132.0, 133.1, 135.0, 136.9, 144.6, 145.1, 147.5,
168.2; (ESIþ) m/z = 465 [M þ H]þ; CHN.
’ EXPERIMENTAL SECTION
General Methods. Reagents were purchased from fine chemicals
vendors, and used as received unless otherwise stated. Solvents were
purified and stored according to standard procedures. Petrol refers to
that fraction in the boiling range 40-60 °C. THF refers to anhydrous
tetrahydrofuran, either by distillation from sodium benzophenone, or
from commercial sources. Melting points were obtained on a Stuart
Scientific SMP3 apparatus and are uncorrected. Thin layer chromatog-
raphy was performed using silica gel plates (Kieselgel 60F254; 0.2 mm),
and visualized with UV light or potassium permanganate. Chromatog-
raphy was conducted under medium pressure in glass columns or using a
Biotage SP4 instrument in prepacked columns (FLASHþ Silica col-
umns (40-63 μm, 60 Å)). Chiral semipreparative HPLC was conducted
using a Varian Prostar instrument equipped with a Daicel Chiralpak AD-
H 250 ꢀ 10 mm column eluting with hexane, ethanol (4:1), at a flow rate
of 3.5 mL/min, monitoring by UV at λ = 270 nm. Proton (1H) and
carbon (13C) nuclear magnetic resonance (NMR) spectra were re-
corded on a Bruker Spectrospin AC 300E (1H at 300 MHz, 13C at 75
MHz), a Jeol JNM-LA500 spectrometer (1H at 500 MHz, 13C at 125
MHz), or a Bruker Avance II 500 (1H at 500 MHz, 13C at 125 MHz)
employing the solvent as internal standard. IR spectra were recorded on
a Bio-Rad FTS 3000MX diamond ATR. Liquid chromatography-mass
spectrometry (LCMS) was carried out on a Micromass Platform
instrument operating in positive and negative ion electrospray mode,
employing a 50 ꢀ 4.6 mm C18 column (Waters Symmetry or Waters
Atlantis) 5 or 12 min gradient elution with 0.05% formic acid in
methanol (10-90%). All compounds gave g95% purity by 1H NMR,
HPLC, or LCMS, unless stated otherwise. Elemental analyses were
performed by The School of Pharmacy, Analytical Facility, University of
London, WC1N 1AX. Accurate masses were measured using a Finnigan
MAR 95 XP or a Finnigan MAR 900 XLT at the EPSRC National Mass
Spectrometry Service Centre (Chemistry Department, University of
Wales, Swansea, Wales, SA2 8PP).
3-(4-Chlorophenyl)-3-(3-hydroxypropoxy)-2-(4-nitrobenzyl)-
2,3-dihydroisoindol-1-one (59). General Procedure B: 20 (400 mg,
1.0 mmol), 1,3-propanediol (365 μL, 5.1 mmol), thionyl chloride (214 μL,
2.75 mmol), and potassium carbonate (380 mg, 2.75 mmol). Chromatog-
raphy (silica; 40% EtOAc, petrol) gave 59 as a yellow solid (342 mg, 75%):
1
mp 141-143 °C; IR (cm-1) 3397, 1682 cm-1; H NMR (300 MHz,
CDCl3) δ 1.49-1.60 (m, 2H, OCH2CH2), 1.65 (br s, 1H, OH), 2.94 (m,
2H, OCH2), 3.62 (t, 2H, J = 6.0 Hz, HOCH2), 4.33 (d, 1H, J = 15.0 Hz, N-
CH), 4.63 (d, 1H, J=15.0Hz,N-CH0),7.14-7.20 (m, 5 H, Ar-H), 7.36-
7.40 (m, 2H, Ar-H), 7.52-7.58 (m, 2H, Ar-H), 7.92-7.94 (m, 1H,
C(O)dCdCH), 8.02-8.06 (m, 2H, Ar-H); 13CNMR(75MHz, CDCl3)
δ 32.1, 42.4, 60.0, 60.5, 94.9, 123.1, 123.3, 123.9, 127.9, 128.7, 129.9, 130.1,
131.5, 133.0, 134.9, 137.1, 144.7, 145.2, 147.4, 168.2; LCMS (ESIþ) m/z453
[M þ H]þ. CHN.
3-(4-Chlorophenyl)-3-(4-hydroxybutoxy)-2-(4-nitrobenzyl)-
2,3-dihydroisoindol-1-one (60). General Procedure B: 20 (400 mg,
1.0 mmol), 1,4-butanediol (455 mg, 5.0 mmol), thionyl chloride (214 μL,
2.75 mmol), and potassium carbonate (380 mg, 2.75 mmol). Chromatog-
raphy (silica; 40% EtOAc, petrol) gave 60 as a yellow oily solid (320 mg,
68%). IR (cm-1) 3440, 2940, 1697, 1519; 1H NMR (300 MHz, CDCl3) δ
1.21-1.51 (m, 4H, OCH2CH2CH2), 1.62 (br s, 1H, OH), 2.77 (t, 2H, J =
5.1 Hz, OCH2), 3.52 (t, 2H, J = 5.7 Hz, HOCH2), 4.25 (d, 1H, J = 15.0 Hz,
N-CH), 4.62 (d, 1H, J = 15.0 Hz, N-CH0), 7.08-7.22 (m, 5 H, Ar-H),
7.39-7.35 (m, 2H, Ar-H), 7.49-7.55 (m, 2H, Ar-H), 7.88-7.94 (m,
1H, C(O)dC=CH), 8.04-8.01 (m, 2H, Ar-H); 13C NMR (75 MHz,
CDCl3) δ 25.7, 29.4, 42.4, 62.3, 62.9, 94.8, 123.1, 123.3, 123.8, 128.0, 128.7,
130.0, 130.0, 131.4, 133.0, 134.8, 137.2, 144.8, 145.3, 147.4, 168.2; (ES)
m/z: [M]þ. CHN.
General Procedure A: 3-Hydroxy Compounds. To a solu-
tion of the appropriate benzoic acid (1 equiv) in THF (10 mL), under
N2, was added thionyl chloride (2 equiv) and 3 drops of anhydrous
DMF, and the resulting mixture was stirred for 4 h at rt and then
concentrated in vacuo. The residue was diluted with THF (10 mL), and
3-(4-Bromophenyl)-3-(4-hydroxybutoxy)-2-(4-nitrobenzyl)
isoindolin-1-one (66). General Procedure B: 21 (0.50 g, 1.1 mmol),
thionyl chloride (214 μL, 2.75 mmol), 1,4-butanediol (0.20 mL, 2.3
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Journal of Medicinal Chemistry
ARTICLE
mmol), and K2CO3 (526 mg, 5.0 mmol). Chromatography (Biotage SP4,
[R] = -20.10° (at 24.8 °C, wavelength = 589 nm, tube length = 0.25
dm, concentration = 0.398 g/100 mL).
silica; 10-50% EtOAc, petrol) gave 66 as a pale yellow oil (0.50 g, 85%).
1
IR (cm-1) 3397, 2925, 2872, 1687, 1605, 1519, 1467; H NMR (300
3-(4-Chlorophenyl)-3-(4-hydroxycyclohexyloxy)-2-(4-nitro-
benzyl)-2,3-dihydroisoindol-1-one (75). General Procedure B: 20
(400 mg, 1.0 mmol), thionyl chloride (214 μL, 2.75 mmol), cis/trans-1,4-
cyclohexanediol (586 mg, 5.0 mmol), and K2CO3 (526 mg, 5.0 mmol)
gave 75 as a white solid (338 mg, 68%); mp 88-91 °C; IR (cm-1) 3412,
MHz, CDCl3) δ 1.36-1.45 (m, 4H, (CH2)2), 2.76 (t, 1H, J = 6.1 Hz,
OCH2), 3.49 (t, 2H, J = 6.2 Hz, HOCH2), 4.25 (d, 1H, J = 15.0 Hz, N-
CH), 4.60 (d, 1H, J = 15.0 Hz, N-CH0), 7.08-7.13 (m, 3 H, Ar-H),
7.29-7.36 (m, 4H, Ar-H), 7.48-7.51 (m, 2H, Ar-H), 7.87-7.89 (m,
1H, C(O)dCdCH), 7.97-8.00 (m, 2H, Ar-H); 13C NMR (75 MHz,
CDCl3) δ 26.0, 29.7, 42.7, 62.5, 63.2, 95.1, 123.2, 123.5, 123.6, 124.1,
128.6, 130.3, 130.3, 131.8, 131.9, 133.3, 138.1, 145.1, 145.6, 147.7, 168.5;
CHN.
3-(4-Chlorophenyl)-3-(5-hydroxycyclooctyloxy)-2-(4-nitro-
benzyl)-2,3-dihydroisoindol-1-one (70). General Procedure B:
20 (400 mg, 1.0 mmol), thionyl chloride (214 μL, 2.75 mmol), cis-1,5-
cyclooctanediol (728 mg, 5.0 mmol), and K2CO3 (380 mg, 2.75 mmol)
gave 70 as a yellow solid (342 mg, 65%); mp 69-72 °C; IR (cm-1) 3440,
2928, 2360, 1707, 1520; 1H NMR (300 MHz, CDCl3) δ 1.29-1.83 (m,
12H, CH) 3.18-3.24 (m, 1H, OCH), 3.52-3.59 (m, 1H, HOCH), 4.20
(d, 1H, J = 15.3 Hz, N-CH), 4.86 (d, 1H, J = 15.3 Hz, N-CH0), 7.18-
7.03 (m, 7 H, Ar-H), 7.60-7.52 (m, 2H, Ar-H), 7.95-7.91 (m, 3H,
Ar-H and C(O)dCdCH); 13C NMR (75 MHz, CDCl3) δ 20.1, 20.3,
33.9, 34.3, 35.9, 36.5, 42.8, 71.2, 73.6, 94.5, 123.1, 123.6, 124.1, 128.1,
128.3, 129.7, 130.2, 131.8, 132.6, 134.8, 137.6, 144.9, 145.9, 147.2, 168.4;
CHN.
3-(4-Chlorophenyl)-3-(3-hydroxy-2,2-dimethylpropoxy)-
2-(4-nitrobenzyl)-2,3-dihydroisoindol-1-one (73). General Proce-
dure B: 20 (400 mg, 1.0 mmol), thionyl chloride (214 μL, 2.75 mmol), and
neopentyl glycol K2CO3 (526 mg, 5.0 mmol) gave 73 as an off-white solid
(267 mg, 55%); mp 91-94 °C; IR (cm-1) 3425, 3077, 2959, 2871, 2160,
1689, 1606, 1519, 1468; 1H NMR (300 MHz, CDCl3) δ 0.83 (d, 6H, J = 3.9
Hz, (CH3)2), 1.71 (br s, 1H, OH), 2.63 (d, 1H, J = 8.7 Hz, OCH), 2.78 (d,
1H, J = 8.7 Hz, OCH0), 3.37-3.42 (m, 2H, HOCH2), 4.44 (d, J = 15.3 Hz,
1H, N-CH), 4.58 (d, J = 15.3 Hz, 1H, N-CH0), 7.12-7.15 (m, 5 H, Ar-
H), 7.28-7.32 (m, 2H, Ar-H), 7.55-7.58 (m, 2H, Ar-H), 7.93-7.96 (m,
1H, C(O)dCdCH), 7.98-8.02 (m, 2H, Ar-H); 13C NMR (75 MHz,
CDCl3) δ 21.8, 36.4, 42.4, 69.0, 69.6, 94.5, 123.2, 123.2, 123.8, 127.9, 128.6,
129.9, 130.2, 131.6, 133.1, 134.9, 137.3, 144.6, 145.2, 147.3, 168.3; MS
(ESIþ) m/z 481 [M þ H]þ. CHN.
1
3076, 2935, 2861, 2159, 1694, 1606, 1519, 1490, 1468, 1424; H NMR
(300 MHz, CDCl3) mixture of diastereoisomers: δ 1.26-1.86 (m, 11H,
OH and Cy-H), 3.09-3.26 (m, 1H, OCH), 3.62-3.70 (m, 1H, HOCH-),
4.78 and 4.24 (d: AB, J = 15.0 Hz, 2H, N-CH2), 7.02-7.18 (m, 7H, Ar-
H), 7.51-7.56 (m, 2H, Ar-H), 7.90-7.93 (m, 3H, O2N-C-CH and
C(O)dCdCH); 13C NMR (75 MHz, CDCl3) mixture of diastereoi-
somers δ 29.0, 29.2, 30.3, 30.7, 31.0, 32.3, 32.5, 42.7, 67.7, 68.6, 69.2, 71.4,
94.3, 94.3, 123.1, 123.8, 128.2, 128.2, 128.3, 128.3, 129.7, 130.2, 130.2,
131.6, 132.7, 134.7, 137.7, 144.8, 146.0, 147.2, 168.3; MS (ESIþ) m/z =
493 [M þ H]þ; CHN.
3-(4-Chlorophenyl)-3-(4-hydroxycyclohex-2-enyloxy)-2-(4-
nitrobenzyl)-2,3-dihydroisoindol-1-one (76). General Procedure B:
20 (400 mg, 1.01 mmol), thionyl chloride (214 μL, 2.75 mmol) and trans-1,4-
cyclohex-2-enediol (576 mg, 5.0 mmol), K2CO3 (526 mg, 5.0 mmol) gave 76
as a white solid (263 mg, 53%): mp 81-83 °C; IR (cm-1) 3077, 2935, 2860,
1
2159, 2026, 1699, 1606, 1519, 1490, 1468, 1424; H NMR (300 MHz,
CDCl3) mixture of diastereoisomers: δ1.14-2.12 (m, 4H, CH2), 3.67-3.77
(m, 2H, (OCH-)2), 4.19-4.34 (m, 4H, (HOCH)2 and (N-CH)2), 4.80
(d, 1H, J = 15 Hz, N-CH0), 4.87 (d, 1H, J = 15 Hz, N-CH0), 5.28-5.40
(m, 2H, (CH)2), 5.69-5.83 (m, 2H, (CH)2), 7.03-7.14 (m, 8H, Ar-H),
7.17-7.23 (m, 6H, Ar-H), 7.55-7.61 (m, 4H, Ar-H), 7.9-7.99 (m, 3H,
Ar-H and C(O)dCdCH); 13C NMR (75 MHz, CDCl3) mixture of
diastereoisomers: δ 28.6, 30.5, 30.9, 43.1, 65.8, 65.9, 68.3, 68.4, 94.8, 94.9,
123.5, 123.5, 124.2, 124.3, 128.6, 128.6, 128.65, 128.67, 123.0, 130.1, 130.2,
130.6, 130.7, 131.9, 133.2, 134.1, 134.6, 135.2, 137.6, 137.7, 145.0, 146.1,
147.6, 145.1, 168.6; MS (ESIþ) m/z = 491 [M þ H]þ. CHN.
Determination of Inhibition of the MDM2-p53 Interaction
Using a Binding Assay (ELISA). Assays were carried out as
described previously.21
Cell-Based Assays. Western Blot Analysis for p53 Activation in
Intact Cells. Western blot analysis of p53, MDM2, p21WAF1 and actin
proteins in cells treated with the MDM2-p53 antagonists was carried out as
described previously.21 Detection of R-tubulin on Western blots with the
Clone DM1A monoclonal antibody (Sigma-Aldrich, Dorset, U.K.) at
1:2000 dilution was used as an additional protein loading control.
Cellular Growth Inhibition (GI50). The concentrations of com-
pounds required for inhibition of cell growth by 50% (GI50) were
determined for a range of cell lines of differing MDM2 and p53 gene
status. The MDM2 amplified cell lines tested were SJSA-1 osteosarcoma,
as well as LS and NGP neuroblastoma. The MDM2 nonamplified cell
lines comprised SaOS-2 (p53 null osteosarcoma) and an isogenic
matched pair of p53 wild-type and deleted colorectal carcinoma cell
lines (HCT116 þ/þ and HCT116 -/-). All cell cultures were grown
in RPMI 1640 medium (Gibco, Paisley, U.K.) supplemented with 10%
fetal calf serum and routinely tested and confirmed negative for
mycoplasma infection. The growth of cells and their inhibition were
measured using the sulphorhodamine B (SRB) method, as previously
outlined.26,27 Cells were seeded into 96-well tissue culture plates and
incubated at 37 °C in a 5% CO2 humidified incubator for 24 h, after
which the medium was replaced with 100 μL of test medium containing
a range of MDM2-p53 antagonist concentrations and incubated for a
further 72 h to allow cell growth before adding 25 μL of 50% tri-
chloroacetic acid (TCA) to fix the cells for 1 h at 4 °C. The TCA was
washed off with distilled water and 100 μL of SRB dye (0.4% w/v in 1%
acetic acid; Sigma-Aldrich, Poole, Dorset) was added to each well of the
plate. Following incubation with the SRB dye at room temperature for
30 min, the plates were washed with 1% acetic acid and left to dry over-
3-(4-Chlorophenyl)-3-(1-hydroxymethylcyclopropylmethoxy)-
2-(4-nitrobenzyl)-2,3-dihydroisoindol-1-one (74). General Proce-
dure B: 20 (400 mg, 1.0 mmol), thionyl chloride (214 μL, 2.75 mmol), 1,1-
bis(hydroxymethyl)cyclopropane (0.2 mL, 2.0 mmol), and K2CO3 (526 mg,
5.0 mmol). Chromatography (Biotage SP4, silica; 10-50% EtOAc, petrol)
gave 74 as a cream solid (442 mg, 92%), mp 148-149 °C. IR (cm-1) 3471,
3076, 2878, 1705, 1599, 1514 cm-1; 1H NMR (300 MHz, CDCl3) 0.12-0.22
(m, 2H, CH2) 0.40-0.43 (m, 2H, CH2), 2.81 (s, 2H, OCH2), 3.43-3.51 (m,
2H, HOCH2) 4.49(s, 2H, N-CH2), 7.12-7.19 (m, 5 H, Ar-H), 7.32-7.29
(m, 2H, Ar-H), 7.55-7.52 (m, 2H, Ar-H), 7.89-7.92 (m, 1H, C-
(O)dCdCH), 7.98-8.01 (m, 2H, Ar-H); 13C NMR (75 MHz, CDCl3),
8.9, 8.9, 22.7, 42.8, 67.8, 95.0, 123.5, 123.6, 124.1, 128.3, 129.0, 130.2, 130.5,
131.9, 133.5, 135.3, 137.5, 144.9, 145.5, 168.5; HRMS (C26H23ClN2O5) Calcd,
478.1290; obsd, 478.1291; CHN.
Separation of enantiomers was achieved by chiral preparative HPLC
(Daicel Chiralpak AD-H 250 x10 mm; Hexane/Ethanol (4:1) 3.5 mL/
min)
(þ)-R-3-(4-Chlorophenyl)-3-(1-hydroxymethylcyclopropyl-
methoxy)-2-(4-nitrobenzyl)-2,3-dihydroisoindol-1-one (74a).
Yellow solid, Rt = 16.5 min. Optical rotation: specific rotation
o
[R] = þ22.66 (at 24.8 °C, wavelength = 589 nm, tube length = 0.25
dm, concentration = 0.406 g/100 mL).
(-)-S-3-(4-Chlorophenyl)-3-(1-hydroxymethylcyclopropyl-
methoxy)-2-(4-nitrobenzyl)-2,3-dihydroisoindol-1-one (74b).
Off-white solid, Rt = 22.5 min. Optical rotation: specific rotation
1241
dx.doi.org/10.1021/jm1011929 |J. Med. Chem. 2011, 54, 1233–1243

Journal of Medicinal Chemistry
ARTICLE
night. The SRB stained protein, which is a measure of the number of cells
in a well, was then resuspended in 100 μL of 10 mM Tris-HCl (pH 10.5)
and the absorbance at λ = 590 nm was measured in each well using a
Spectra Max Pro 400 plate reader. The GI50 was calculated by nonlinear
regression analysis of the data using Prism v4.0 statistical software.
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Liu, E. A. In vivo activation of the p53 pathway by small-molecule
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’ ASSOCIATED CONTENT
S
Supporting Information. Additional experimental details
b
for compounds not included in the main section and analytical data
for all compounds and intermediates. X-ray structure tables. Table
of combustion analysis data. Additional Western blot analysis for 6,
7, 47, 50, and 53-55. Additional SRB growth inhibitory data for
74, 74a, and 74b. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel.: þ44 191 222 6645 (I.R.H.); þ44 191 246 4420 (J.L.). Fax:
þ44 191 8591 (I.R.H.); þ44 191 4301 (J.L.). E-mail: i.r.hard-
castle@ncl.ac.uk (I.R.H.); john.lunec@ncl.ac.uk (J.L.).
’ ACKNOWLEDGMENT
The authors thank Cancer Research UK and the Wellcome
Trust for funding. Use of the EPSRC Mass Spectrometry Service
at the University of Wales (Swansea) is gratefully acknowledged.
’ ABBREVIATIONS USED
ATM, ataxia telangiectasia mutated; Bcl-XL, B-cell lymphoma-
extra large; CHK1 and 2, checkpoint kinase 1 and 2; DCM,
dichloromethane; DIPEA, diisopropylethyl amine; DNA-PK,
DNA-dependent protein kinase; mCPBA, meta-chloroperben-
zoic acid; MDM2, murine double minute 2; SRB, sulforhoda-
mine B; THF, tetrahydrofuran
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