Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction

Article abstract of DOI:10.1021/acs.jmedchem.0c02188

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

Full text of DOI:10.1021/acs.jmedchem.0c02188

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Structure-Based Design of Potent and Orally Active Isoindolinone
Inhibitors of MDM2-p53 Protein−Protein Interaction
Gianni Chessari,* Ian R. Hardcastle,* Jong Sook Ahn, Burcu Anil, Elizabeth Anscombe, Ruth H. Bawn,
Luke D. Bevan, Timothy J. Blackburn, Ildiko Buck, Celine Cano, Benoit Carbain, Juan Castro, Ben Cons,
Sarah J. Cully, Jane A. Endicott, Lynsey Fazal, Bernard T. Golding, Roger J. Griffin, Karen Haggerty,
Suzannah J. Harnor, Keisha Hearn, Stephen Hobson, Rhian S. Holvey, Steven Howard,
Claire E. Jennings, Christopher N. Johnson, John Lunec, Duncan C. Miller, David R. Newell,
Martin E. M. Noble, Judith Reeks, Charlotte H. Revill, Christiane Riedinger, Jeffrey D. St. Denis,
́
Emiliano Tamanini, Huw Thomas, Neil T. Thompson, Mladen Vinkovic, Stephen R. Wedge,
Pamela A. Williams, Nicola E. Wilsher, Bian Zhang, and Yan Zhao
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ABSTRACT: Inhibition of murine double minute 2 (MDM2)-p53
protein−protein interaction with small molecules has been shown to
reactivate p53 and inhibit tumor growth. Here, we describe rational,
structure-guided, design of novel isoindolinone-based MDM2 inhibitors.
MDM2 X-ray crystallography, quantum mechanics ligand-based design,
and metabolite identification all contributed toward the discovery of
potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with
representative compounds inducing cytostasis in an SJSA-1 osteosarco-
ma xenograft model following once-daily oral administration.
into potent MDM2-p53 inhibitors with in vivo antitumor
activity.
INTRODUCTION
■
The transcription factor p53 acts as a molecular signaling
node that determines a cell’s fate in response to stress,
including DNA damage.1−3 Cellular stress pathways activate
RESULTS AND DISCUSSION
■
p53 by phosphorylation giving rise to a conformational switch
to a transcriptionally activated protein that activates >150
genes, leading to a variety of cellular responses including DNA
repair and apoptosis.4 The response to p53 activation is
dependent on the stress, cell type, and tissue.5 Mutations of
p53 resulting in its inactivation are found in approximately
50% of tumors.6 The murine double minute 2 (MDM2)
protein regulates p53 by binding its transactivation domain
resulting in the degradation of p53. In around 7% of tumors
MDM2 overexpression inactivates p53, leading to oncogenic
transformation.7,8 Inhibition of the MDM2-p53 protein−
protein interaction with peptides and small-molecules has
been shown to reactivate p53 and inhibit tumor growth.9−12 A
number of MDM2-p53 inhibitors have entered clinical trials
that is ALRN-6924,13 AMG 232,14,15 APG-155,16 BI
907828,17 CGM-097,16 milademetan DS-3032b,17 siremadlin
(HDM201),18 RG7112,19−21 idasanutlin (RG7388),22 and
SAR405838.23
First Isoindolinone-MDM2 X-ray Crystal Structure. In
an effort to determine the binding mode of isoindolinone
molecules with MDM2, surface-entropy reduced mutants were
used to improve the crystallization characteristics of the
protein.28 Importantly, introduction of the E69A and K70A
surface mutations into MDM2 enabled the first isoindolinone
X-ray co-crystal structure to be solved, with compound 1 as
the ligand (Figure 1A). The structure shows that the
compound occupies the three subpockets involved in the
recognition of the residues Phe19, Trp23, and Leu26 of the
transactivation domain of p53.29 The isoindolinone central
motif binds to the Phe pocket, making van der Waals contacts
with Val93 and forming an edge-to-face aromatic interaction
with Tyr67. The C3-chlorophenyl ring occupies the Trp
Received: December 18, 2020
Published: March 24, 2021
Previously, we have reported an isoindolinone series with
MDM2-p53 inhibitory activity.24−27 In this paper, we describe
the structure based optimization of early isoindolinone leads
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Figure 1. X-ray crystal structures of compounds 1 (A), 4 (B), and 5 (C) bound to MDM2. Key hydrogen bond interactions between the ligands
and the protein residues are highlighted with red dashed lines represented. Coordinates for MDM2 complexes with compounds 1 and 5 have
been deposited in the Protein Data Bank (PDB) under accession codes 7BJ0 and 7BMG, respectively. The coordinates of the MDM2 complexes
with compound 4 are available as a part of the Supporting Information. (D) Electrostatic potential surface of the 4-Cl-phenyl and the 4-
ethynylbenzyl side chains of 5 in the bound conformation. Negative areas are colored red, and positive areas are colored blue. The red circle
highlights a potential electrostatic repulsion between the π clouds of the two aromatic rings. (E) Solution conformation of 5 as observed by
NMR. Through space ROESY correlations in the NMR spectrum are indicated with arrows.
pocket as seen in many other inhibitor structures.12 The ether
side chain branching off the C3 position binds to a shallow
subpocket located on the top of the α2 helix near Phe55,
Gly58, and Gln59, with the hydroxy group forming H-bond
with the carbonyl backbone of Phe55. The Leu pocket is filled
by the 4-nitrobenzyl group which is engaged in a stacking
interaction with His96. Replacement of the nitro group with
simple lipophilic groups proved to be effective, with 2 and 3
having similar potency to 1 (see Table 1). The determination
of this X-ray crystal structure was key to define the subsequent
lead optimization strategy. The structure confirmed the
binding mode for this class of compounds and showed that
the isoindolinone scaffold offers good vectors to explore the
canonical MDM2 pockets.
Preliminary Isoindolinone SAR. Our analysis of Nutlin-
3a MDM2 crystal structures (PDB 4HG7) suggested that
there may be protein flexibility in the Phe pocket.28 In
particular, the sidechain of Tyr67 can adopt an OUT
conformation which increases the size of the Phe pocket.
We started to explore this induced fit with our series by
studying a compound with a tert-butyl side chain at the C6
position of the isoindolinone scaffold. This substitution in
compound 4 did not show any advantage in terms of potency;
however, in the X-ray co-crystal structure of 4 with MDM2,
we observed that the 6-t-butyl group caused the desired Tyr67
movement (Figure 1B). The replacement of a methyl of the t-
butyl group with a hydroxyl group maintained the Try67
OUT conformation while forming a favorable H-bond with
Gln72 (Figure 1C). The substitution was also advantageous as
it introduced a less lipophilic and more drug-like substituent
into the molecule. The dimethylcarbinols 5 and 6 exhibited
improved MDM2 inhibitory activity, and importantly, they
were the first isoindolinones with submicromolar cell growth
inhibition activity in the TP53 wild-type SJSA-1 cell line
(Table 1).
Evaluation of Metabolic Stability of Isoindolinone 6.
Encouraged by the good progress made in terms of binding
and cellular activity and selectivity against the TP53 mutant
cell line SN40R1, we decided to investigate the pharmacoki-
netic (PK) profile of 6. The in vitro ADME properties of 6
were characterized by high intrinsic clearance, leading to a
short half-life when incubated in mouse and human
microsomes (<5 and 9.5 min, respectively) and very high
plasma protein binding of >99% in both mouse and human
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Table 1. Structure Activity Relationships for Isoindolinones 1−11
a
Data are expressed as geometric mean (n ≥ 2).
a
Table 2. DMPK Properties of Key Isoindolinones
b
c
d
e
f
g
PPB m/h
(%)
LM t1/2 m/h
CYP3A4 t1/2
CYP3A4 IC50
Cl m
Cl NHP
% F
g
f
Cmp
(min)
(min)
(μM)
(mL/min/kg)
% F m
6.5
(mL/min/kg)
NHP
6
99.9/99.8
99.0/n.a.
99.9/99.6
99.8/99.7
98.3/99.2
90.6/91.4
-/-
<5/9.5
40/>360
16/>70
12/17
<5/5.7
27/52
23/18
25/51
>70/>360
<5
25
7 (rac)
8
9
13
14
9.0
7.7
21
11
12.1
34
31
25
28
46
44
92
83
<5
<5
14
6.5
>360
>360
1.2
46%@8 μM
11
16
17
9
31
5.6
66
19
21
75%@10 μM
97.4/98.7
96.6/97.7
0.2
0.6
23
7.1
150
a
b
c
Data are expressed as mean (n ≥ 2). Plasma protein binding (PPB) in male BALB/c mouse (m) and human (h) plasma, n = 1−4. Half-life (t1/2
)
d
in male CD1 mouse (m) and mixed gender human (h) liver microsomes (LM), n = 1−3. Half-life (t1/2) in human supersomes expressing
CYP3A4, n = 1. Inhibition of CYP3A4 using midazolam as the probe substrate, where an IC50 could not be determined the concentration that
e
f
resulted in inhibition nearest to 50% is given, n = 1. Plasma clearance (Cl) and oral bioavailability (F) determined from sparse sampling in mouse,
n = 3 per timepoint, combined to generate a composite profile. Data from serial sampling nonhuman primate (NHP), n = 3.
g
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Figure 2. Metabolism of 6 following incubation with mouse liver microsomes for 5 min. Representative chromatograms (A) 6 multiple reaction
monitoring (MRM) 526 > 424; retention time 2.30 min, (B) acyl-iminium ion MRM 424 > 125; retention times 2.13 and 2.30 min, (C) 7 MRM
■
●
540−125; retention time 2.12 min. (D) Disappearance of 6 ( ) and 7 ( ) over time following incubation in mouse liver microsomes (n = 2)
▲
and formation of 7 ( ) detected in 6 incubations; peak areas normalized to peak area of product at time zero.
Scheme 1. Acid Catalyzed Hydrolysis of Compound 6
plasma (Table 2). The high plasma protein binding resulted in
moderate in vivo clearance in mice (25 mL/min/kg) with low
oral bioavailability of 6.5%.
7, it demonstrated that reducing the clearance through
modification of the C3 substituent could lead to compounds
with better oral bioavailability in the mouse.
Following incubation of 6 in vitro in mouse microsomes, in
addition to parent compound, detection of metabolites by
liquid chromatography−mass spectrometry (LC−MS) was
facilitated by the formation of an acyl iminium ion. It was
hypothesized that the metabolites arose from either
metabolism of the C3 side chain or direct glucuronide
conjugation of the hydroxy group. Further LC−MS analysis
revealed the major parent-related product corresponded to a
[M + H]+ ion of m/z 540, a m/z increase of +14 mass units in
molecular weight, compared with 6. The metabolite was
putatively assigned as the carboxylic acid derivative arising
from oxidation of the C3 sidechain. A minor peak with a [M +
H]+ ion consistent with an acyl-glucuronide conjugate of the
putative carboxylic acid (+190 mass units) was also detected
in the 6 incubations.
Carboxylic acid 7, the putative metabolite of 6, was
synthesized and proved to be 10-fold less potent than the
parent and reduced cellular activity. Compound 7 was
incubated in mouse microsomes alongside 6, and it was
confirmed that the metabolite generated in the 6 samples was
the carboxylic acid 7 (Figure 2). Semi-quantitation of the
metabolite formation indicated that 7 was the major
metabolite of 6. Thus, the methylene adjacent to the hydroxyl
group on the C3 sidechain appeared to be a hot spot for
metabolic oxidation.
Deuteration was considered as an alternative approach to
modulate the metabolism of the series. Selective replacement
of hydrogen with deuterium leads to increased bond strength
which in turn can increase the metabolic stability of the
molecule.30,31 We considered that the incorporation of
deuterium into the C3 hydroxymethylcyclopropyl chain
adjacent to the terminal hydroxyl group would reduce the
rate of oxidation, potentially giving a more favorable PK
profile. Compound 9 was prepared using d4-dihydroxyme-
thylcyclopropane to introduce the C3 sidechain. Comparison
with the nondeuterated matched pair 6 indicated that the
modification of the side chain did not affect the potency or
cellular activity (Table 1). The deuteration did, however,
improve the mouse microsome half-life for 9 (Table 2)
showing increased metabolic stability as a result of deuterium
replacement in the C3 chain. A lower plasma clearance (7.7
mL/min/kg) and higher oral bioavailability (25%) were also
observed in mouse, compared with the nondeuterated 6,
further highlighting the merit of stabilizing the C3 substituent.
Evaluation of Chemical Stability of Isoindolinone 6.
Before conducting further optimization, we sought to
investigate the chemical stability of this class of compounds.
The alkoxy-isoindolinone scaffold, on which this series is
based, is known to generate acyl-iminium ions, particularly
under acidic conditions (Scheme 1).32 Furthermore, the
synthesis of the compounds themselves relies on acyl-iminium
ion chemistry. In a simple test of chemical stability, 6 was
incubated in dilute aqueous HCl (pH 1, RT) and showed
∼40% hydrolysis after 1 day, with almost complete hydrolysis
after 5 days. We hypothesized that introduction of an
electron-withdrawing group, such as fluorine, into the
Compound 8, with two methyl groups on the α-carbon to
the hydroxy, was more stable than 6 in mouse and human
microsomes (16 and >70 min half-life, respectively).
Consequently, plasma clearance of 8 in mice was reduced to
9 mL/min/kg, and oral bioavailability was increased to 31%.
Although, the potency of 8 was not improved compared with
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Table 3. Structure Activity Relationships for Isoindolinones 12−19
a
Data are expressed as geometric mean (n ≥ 2). For percent inhibitions, the average of the percentages at the reported concentration is provided.
isoindolinone motif would destabilize the acyl-iminium ion
intermediate and therefore reduce the rate of hydrolysis.
Incubation of the fluorinated-analogue 11 under identical
conditions (pH 1) showed <10% hydrolysis even after 5 days.
Based on the improved chemical stability and 2-fold
improvement in potency shown by 11, the fluorinated
isoindolinone scaffold was adopted for subsequent optimiza-
tion of the series.
SAR in the Leu Pocket. Following the encouraging PK
profile of 9 and the improved chemical stability of 11, we
decided to use the scaffold in Table 3 as a starting point for
the next round of optimization with the aim of further
improving potency and also lowering lipophilicity (all
compounds in Table 1 have ClogP > 5).
top of each other (Figure 1C). An analysis of the electrostatic
potential surface (ESP) of bound conformation of 5 suggested
a potential electrostatic repulsion between the π clouds of the
two aromatic rings (Figure 1D). This observation prompted
an investigation to understand whether the folded con-
formation seen in the X-ray crystal structure was maintained
1
by 5 in the unbound state. The H NMR spectrum of 5 in
DMSO-d6 showed unusual chemical shifts for the C3
hydroxymethylcyclopropylmethylene side chain (data in the
NMR section of the Supporting Information document).
Thus, the hydrogens of the methylene linker and the
cyclopropyl are shielded indicating that they are lying above
1
one or more of the aromatic rings. H−1H through space
correlations observed in the ROESY spectrum show that one
of the diastereotopic methylene linker hydrogens is located
above the phenyl ring of the isoindolinone and the other is
above the phenylacetylene. Similarly, the cyclopropyl hydro-
gens are located above phenylacetylene. The observations in
The X-ray crystal structure of 5 bound to MDM2 shows
that 4-Cl-phenyl and the 4-ethynylbenzyl side chains bind to
the Trp and Leu pockets, respectively, adopting a folded
conformation where the two aromatic rings stack one on the
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Figure 3. (A) Thermodynamic cycle used to estimate the strength of the aromatic interaction highlighted with the red dashed line. Other
potential noncovalent interactions are highlighted with green dashed lines. (B) Calculated values of the aromatic interaction (Eπ−π) between the
chlorophenyl and R groups. (C,D) Electrostatic potential surface relative to two compounds in Figure 3B. Negative areas are colored red, and
positive areas are colored blue.
solution are compatible with a major contribution from a
conformation in which ethynylbenzyl and chlorophenyl are
pointing away from each other, rather than stacked as
observed in bound conformation (see Figure 1E). Stabiliza-
tion of the ligand conformation in the bound state is a secure
way of increasing binding affinity, and it works best when the
bound molecule is not in its lowest energy conformation.33,34
In order to optimize the interaction between the phenyl rings
in the bound conformation, the thermodynamic cycle in
Figure 3A was designed. This method allows the interaction of
interest (red dashed line, Figure 3A) to be dissected out of the
array of other noncovalent interactions (e.g. green dashed
lines, Figure 3A).35 The energy of each molecule in the cycle
was calculated at the ωB97XD/cc-pVTZ level of theory,
which is well suited to investigate aromatic stacking
interactions.36 The method is described in more details in
the Computational Chemistry section in the Supporting
Information. The interaction energy between the aromatic
rings (Eπ−π) was calculated using the equation in Figure 3A.
The results in Figure 3B indicate that the aromatic
interactions in the bound conformation could be improved
with the introduction of electron withdrawing groups at the
para position of the benzyl ring and also by introducing
electron deficient six membered aromatic rings such as
pyridines or pyrimidines. Figure 3D shows the improved
electrostatic interactions associated with replacing the benzyl
ring with an electron deficient chloropyridine.
polar aza-aryl rings are also tolerated. Compounds 13, 14, 15,
and 17 show low nM potency in the ELISA assay and sub μM
cellular activity in the SJSA-1 TP53WT cell line. Importantly,
these gains in potency were obtained with reduction in
lipophilicity. Compounds 14 and 15 have ChromlogD values
below 4, and they both achieve a good selectivity profile
against the SN40R1 TP53mut cell line. X-ray crystal structures
of 13 and 14 bound to MDM2 were obtained (see Figure
4A,B) showing that the introduction of polar aromatic rings in
the Leu pocket does not alter the binding mode previously
observed for 5. The enantiomers of 13 and 14 were also
tested in vitro, both showing an approximately 100-fold
decrease in potency in the ELISA assay and μM cellular
activity in the SJSA-1 cell line. A small molecule crystal
structure of compound 18 (a close analogue of 11) was
obtained showing that the molecule adopts a folded
conformation with the chloropyridinyl side chain stacking
against the chlorophenyl ring as suggested by the modeling
studies (see Figure 4C).
Although 13 was potent with good cellular activity (Table
3) and had oral bioavailability of 28% in the mouse (Table 2),
it was also identified as a CYP3A4 substrate (half-life < 5
min). CYP3A (CYP3A4 and CYP3A5) is the predominant
CYP isozyme expressed in the human gastrointestinal (GI)
tract, and intestinal metabolism can limit human oral
bioavailability of compounds that are predominantly metab-
olized by CYP3A4.37 Species differences in CYP isoforms,
specificity, and expression can mean that oral bioavailability is
less affected by CYP3A intestinal metabolism in mice.38
Compound 14, the cyanopyridine analogue of compound 13,
was similarly active in SJSA-1 cells (Table 3) and was also
orally bioavailable in mouse (46%). The cyanopyridine,
Following these modeling studies, an array of compounds
was synthesized with the aim of optimizing the folded bound
conformation while improving protein−ligand interactions in
the Leu pocket. Historically, this pocket has been targeted
with lipophilic aryl groups, but the data in Table 3 shows that
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Figure 4. X-ray crystal structures of compounds 13 (A), 14 (B), and 23 (D) bound to MDM2. Key hydrogen bond interactions between the
ligands and the protein residues are highlighted with red dashed lines. Coordinates for MDM2 complexes with compounds 13, 14, and 23 have
been deposited in the PDB under accession codes 7BJ6, 7BIV, and 7BIR, respectively. (C) Small-molecule X-ray crystal structure of compound
18 (CCDC/FIZ Karlsruhe database deposition number 2059877), showing two molecules in the asymmetric unit.
however, displayed a longer half-life in CYP3A4 incubations
(14 min) compared with the chloropyridine (<5 min).
Compound 14 was, therefore, preferentially progressed into
a nonhuman primate (NHP) PK study to evaluate a
nonrodent species which better represents human intestinal
metabolism. CYP3A8 (NHP isoform of CYP3A4) and
CYP3A5 are highly expressed in the NHP intestine, although
the first-pass effect can overestimate human.37 Plasma
clearance of 14 in NHP was low at 11 mL/min/kg, with a
good oral bioavailability of 31% (Table 2). Compound 19, the
nondeuterated analogue of 14, was also relatively stable in
mouse microsomes (23 and 27 min half-life, respectively) and
had similar oral bioavailability in mouse (44%), suggesting
that the impact of the C3 metabolism may be reduced for the
cyanopyridine. The CYP3A4 half-life of 19, however, was
shorter at 6.5 min. In NHP, although clearance of 19 (16 mL/
min/kg) was comparable to 14, oral bioavailability was
reduced to 5.6%, showing that deuteration of the C3 sidechain
minimized intestinal CYP3A4 metabolism and maximized oral
bioavailability.
identify C3-side chains which combine good potency with
stability to CYP3A4 metabolism. A selection of the most
interesting compounds identified is reported in Table 4.
Reduction of the C3-side chain to a methyl group
exemplified by compound 20 resulted in a 13-fold reduction
in potency, emphasizing the importance of the side chain
interactions with the protein. In comparison, the cyclo-
propylalcohol 21 resulted in a smaller reduction of in vitro
MDM2 potency and cellular activity. Compound 22, the
methyl ether of 21, has a similar in vitro MDM2 potency,
demonstrating that a hydrogen bond donor is not essential at
this position. Compound 21 exhibited submicromolar
inhibition in the CYP3A4 inhibition assay, which may
contribute to the low mouse and human microsomal clearance
and good oral bioavailability observed in both mouse and
NHP (see Table 2). The cyclopropylcarboxamide 23 also had
comparable MDM2 and cellular potency and selectivity to 21.
The X-ray structure of 23 (Figure 4D) shows that the
cyclopropyl group of the C3 ether chain adopts a similar
orientation to the hydroxymethylcyclopropyl examples (e.g.
compound 6), and an H-bond is formed between the amide
carbonyl and the NH2 of Gln59. The PK profile of 23 was
investigated (Table 2) and reduced plasma protein binding of
up to 97.7% in human plasma was observed, with good
SAR in the C3 Region. The identification of the 3-
substituent as a main site of metabolism for the isoindolinone
series prompted further investigation at this position. A range
of compounds with diverse C3-substitutions were prepared to
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Table 4. Structure Activity Relationships for Isoindolinones 20−32
a
Data are expressed as geometric mean (n ≥ 2). For percent inhibitions, the average of the percentages at the reported concentration is provided.
stability in vitro in the microsomal and CYP3A4 phenotyping
assays. Consequently, the plasma clearance of 23 in mouse
and NHP was low (7.1 and 9.0 mL/min/kg, respectively), and
oral bioavailability was good in both species, consistent with
the long CYP3A4 half-life (>360 min). A range of analogues,
based on amide 23, were prepared. The N-methyl analogue 24
showed reduced potency, whereas the α-methyl amide 26
retained cell-free potency but was 3-fold less potent in the
cellular assay. The cyclopropylamine 27 showed a similar
profile to 26, whereas the acetamide derivative 28 was less
potent. Cyanocyclopropyl derivative 29 and the methylsulfone
analogue 30 showed good potency and cellular activity. The
tetrahydrofuran (THF) derivatives 31 and 32 were also potent
with the (S)-isomer showing good cellular potency.
In Vivo Antitumor Activity of Lead Compounds.
Isoindolinones 14 and 23 were selected to investigate target
engagement in vivo. Both compounds are characterized by
good in vitro cellular activity and selectivity and good overall
PKs properties (see Table 2 and Figure 5A,B). Pharmacody-
namic studies were performed in the mouse SJSA-1
osteosarcoma xenograft model in athymic mice to confirm
p53 activation in vivo. Following a single oral dose of 50 mg/
kg of 14, a maximal 3.7-fold increase in p53 expression was
observed at 3 h compared to the vehicle control group.
Consistent with p53 activation, the increase in p53 levels was
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Figure 5. In vivo characterization of compounds 14 (A,C,E) and 23 (B,D,F) at 50 mg/kg in female CD-1 nude mice bearing SJSA-1
subcutaneous xenografts. (A,B) PK profiles in plasma and tumor. (C,D) p53 and MDM2 pharmacodynamics profile in tumor. (E,F) In vivo
efficacy.
followed by an increase in the expression of MDM2, a
transcriptional target of p53. MDM2 levels were at their
highest at 6 h with a 10.3-fold increase in expression
compared to the vehicle control (Figure 5C). A single oral
dose of 50 mg/kg of 23 showed a maximal increase of 4.2-fold
in p53 expression at 3 h and 13.2-fold increase in MDM2 at 6
h (Figure 5D).
The antitumor activities of 14 and 23 were also investigated
in the SJSA-1 xenograft model, which has been widely used to
assess the in vivo efficacy of different inhibitors of MDM2-p53
interaction.13−23 Daily oral administration of 14 at 50 mg/kg
for 7 days exerted a significant antitumor effect with the
maximal response T/C of 21% (P = 0.0022 vs vehicle
treatment). 1 out of 10 mice showed a complete regression
(Figure 5E). Similarly, daily oral administration of 23 at 50
mg/kg for 7 days significantly inhibited tumor growth with the
maximal response T/C of 20% observed on day 7 (P < 0.0001
vs vehicle treatment). Partial regression was observed in 2 out
of 10 mice and complete regression in 1 out of 10 mice,
indicating that 23 confers sustained in vivo efficacy (Figure
5F). In all studies, the compounds were well tolerated and
neither excessive bodyweight loss nor significant adverse
effects were observed.
Chemistry. We have described previously the synthesis of
substituted isoindolinones from substituted phthalic anhy-
drides;24,27 however, the separation of mixtures of re-
gioisomers was undesirable. A regioselective approach to the
benzoylbenzoic acid key intermediates was adopted following
the method described by Jacq et al. (Scheme 2).38,39
Condensation of the appropriate salicylaldehyde (33a−f)
with 4-chlorobenzhydrazide gave the hydrazides (34a−f).
Lead tetraacetate mediated rearrangement of the hydrazides
34a−f gave the required benzoylbenzaldehydes 35a−f,
followed by Pinnick oxidation to give the benzoylbenzoic
acid 36a−f. Treatment of each benzoic acid with thionyl
chloride gave the respective chloroisobenzofuran which was
then treated in situ with the relevant benzylamine to give the
3-hydroxyisoindolinone 37a−l. Etherification at the C-3
position could be achieved by chlorination of isoindolinone
37a−l with thionyl chloride and reaction with the relevant
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Scheme 2. Reagents and Conditions: (a) 4-Chlorobenzhydrazide, AcOH, rt, 20 min; (b) Pb(OAc)4, THF, 0 °C, 1−2 h; (c)
Sodium Chlorite, Sulfamic Acid, MeCN, H2O, rt, 1−2 h; (d) (i) SOCl2, N,N-dimethylformamide (DMF), THF, rt, 4 h; (ii)
Arylmethylamine, N,N-Diisopropylethylamine (DIPEA), THF, rt, 18 h; (e) (i) SOCl2, DMF, THF, rt, 4 h; (ii) ROH, K2CO3,
THF, rt, 18 h; or (f) ROH, InBr3, DCE, 80 °C; (g) TBAF, THF
Scheme 3. Reagents and Conditions: (a) (i) SOCl2, DMF, THF, rt, 4 h; (ii) ROH, K2CO3, THF, rt, 18 h; (b) MeMgBr,
ZnCl2, THF, 0 °C; (c) TBAF, THF
alcohol as described previously;40 however, in some cases this
sequence gave unreliable yields. Lewis acid catalyzed ether-
ification reactions have been reported,41,42 and a screen of
conditions demonstrated that reaction of isoindolinones
(37b,f,k,l) with the required alcohols under indium bromide
catalysis gave reliable yields of the desired ethers (2, 41j−n).
4-Ethynyl examples (3a, 4a, and 5a) were prepared using 4-
TIPS-ethynylbenzylamine, and deprotection with tetrabuty-
lammonium fluoride (TBAF) gave the desired products 3−5
(Schemes 2 and 3).
bromide to the 6-acetyl isoindolinones 40a−d in the presence
of either ZnCl2 or LaCl3 (Scheme 3). Alternatively, the 6-
carbinol was prepared from 6-bromoisoindolinone 41a−n by
palladium-catalyzed coupling with isopropenylboronic acid
pinacol ester to give the 6-isopropenylisoindolinones
(42a,b,d,f) (Scheme 4). These 6-isopropenyl derivatives
were then converted directly to the 6-carbinols (10−12, 17,
23) by hydroxymercuration with mercuric acetate, followed by
treatment with perchloric acid and reduction with alkaline
borohydride. Alternatively, Stille coupling of tributyl(1-
ethoxyvinyl)tin with 6-bromo derivatives (41c−f,j−m),
followed by acidic hydrolysis of the resulting enol ether,
Introduction of the 6-carbinol at the isoindolinone 6-
position was achieved by addition of methyl magnesium
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Scheme 4. Reagents and Conditions: (a) Isopropenylboronic Acid Pinacol Ester, Pd(dppf)Cl2, NaOH, N,N-
Dicyclohexylmethylamine, THF, 120 °C; (b) (i) Hg(OAc)2, H2O; (ii) HClO4; (iii) NaOH, NaBH4, 0 °C; (c) Tributyl(1-
ethoxyvinyl)tin, Pd(PPh3)4, LiCl, Dioxane/Toluene (1:1), 110 °C; (d) MeMgBr, ZnCl2, THF, 0 °C
provided an alternative route to the 6-acetyl isoindolinones
(43a−i). Treatment of the 6-acetylisoindolinones with methyl
magnesium bromide in the presence of zinc chloride gave the
6-carbinol isoindolinones (13−16, 18−22, 24−32). Unless
stated otherwise, all final compounds were purified by
preparative chiral HPLC and isolated as single enantiomers.
The tetra-deuterated cyclopropyl side chain was prepared
by reduction of dimethyl cyclopropane-1,1-dicarboxylate with
LiAlD4 in THF (S1 Supporting Information). The carboxylic
acid 7 was prepared by oxidation of 6 with ruthenium(III)
chloride and sodium periodate.
EXPERIMENTAL SECTION
■
General Chemistry. All solvents employed were commercially
available anhydrous grade, and reagents were used as received unless
otherwise noted. Hereafter, petrol denotes the petroleum ether
fraction boiling at 40−60 °C. Flash column chromatography was
performed on a Biotage SP4 system prepacked silica columns
included: Biotage KP-Sil, KP-NH, KP-C18, Agilent Si50 or 35 and
NH, SiliCycle Siliasep, and GraceResolv 40 and NH. Chiral semi-
preparative HPLC was carried out using a Varian Prostar instrument
equipped with a Daicel Chiralpak AD-H250 × 10 mm column
eluting with typically heptane/2-propanol (50:50) and 0.2%
diethanolamine (DEA), at a flow rate of 4.0 mL/min, monitoring
by UV at λ = 254 nm. NMR spectra were recorded on a Bruker
AV400 (AVANCE 400 MHz) spectrometer. LC−MS were
conducted in dimethylsulfoxide (DMSO) or methanol solvents
using a Waters Acquity UPLC system with PDA and ELSD. Samples
were eluted on Acquity UPLC BEH C18, 1.7 μm, 2.1 × 50 mm, with
a flow rate of 0.6 mL/min using 5−95% 0.1% formic acid/MeCN,
overall times were either 2 or 3 min. Analytical HPLC was carried
out using either Waters XTerra RP18 5 μm, 150 × 4.6 mm, or
Waters XSELECT CSH C18 3.5 μm, 4.6 × 100 mm with a flow rate
of 1 mL/min using 0.1% formic acid/MeCN or 0.1% ammonia/
MeCN 5−100% with the highest purity quoted. Optical rotation
measurements were carried out using an Optical Activity Ltd. polAAr
3001 automatic polarimeter and were performed in, and blanked to,
ethanol. Compounds were assigned (R)- or (S)-directly on the basis
of small-molecule X-ray or ligand-bound protein crystallography or
indirectly on the basis of chiral HPLC retention time and MDM2-
p53 inhibitory activity. HRMS were calculated for 35Cl and 79Br
where applicable. HRMS were provided by the ESPRC National
Mass Spectrometry Service, University of Wales, Swansea. NMR
spectra were recorded on a Bruker AVANCE III 500 plus (1H at 500
MHz, 13C at 125 MHz) or on a Bruker Spectrospin AC 300E (1H at
300 MHz, 13C at 75 MHz). Chemical shifts (δ) are reported as parts
per million (ppm) relative to the deuterated solvent. All compounds
were purified to >95% purity by analytical HPLC and 1H NMR,
unless stated otherwise.
CONCLUSIONS
■
In summary, we have identified potent, selective, and orally
efficacious inhibitors of the p53-MDM2 protein−protein
interaction. The application of structure-aided and ligand-
based design, together with metabolite identification, was used
to optimize binding affinity for MDM2 and oral bioavail-
ability, respectively. MDM2 X-ray crystallography was
extensively used to understand ligand binding modes and in
particular to explore the induced fit resulting from the
movement of Tyr67. Furthermore, quantum mechanics
analysis and small molecule NMR were used to aid the
design of more potent compounds which display a
preorganized, low energy, conformation consistent with that
seen in the protein bound state. Finally, there were challenges
of chemical and metabolic stability to overcome in the series.
Modulation of the electronic character of the isoindolinone
system by incorporation of fluorine greatly reduced the
propensity for C3 side chain elimination and application of
the deuterium isotope effect to these molecules proved a
successful strategy for reducing CYP3A4-mediated oxidation
of metabolically labile sites. Of the compounds described, 14
and 23 showed the best balance of properties (potency,
selectivity, and oral bioavailability) and were used to
demonstrate in vivo pharmacodynamic effects and antitumor
efficacy in the SJSA-1 xenograft model at doses which were
well tolerated.
6-[5-Bromo-1-(4-chlorophenyl)-7-fluoro-1-hydroxy-3-oxo-
1,3-dihydro-isoindol-2-ylmethyl]pyridine-3-carbonitrile (37e).
To a solution of 5-bromo-2-(4-chlorobenzoyl)-3-fluorobenzoic acid
(see Supporting Information) 36a (20 g, 56.02 mmol) in THF (200
mL) was added dropwise SOCl2 (8.17 mL, 112.04 mmol) in THF
(200 mL) and anhydrous DMF (1 mL). The reaction mixture was
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stirred at r.t. for 4 h before being concentrated in vacuo. The residue
was dissolved in THF (200 mL), and 6-aminomethyl-pyridine-3-
carbonitrile (16.7 g, 61.62 mmol, 76% w/w) and DIPEA (32.1 mL,
184.87 mmol) were added. The reaction mixture was stirred at r.t. for
18 h. The mixture was diluted with ethyl acetate (EtOAc) (500 mL)
and washed with water (500) and brine (500 mL). The EtOAc layer
was concentrated in vacuo, and then, heptane was added to induce
crystallization. The resulting solid was isolated by vacuum filtration
and washed with EtOAc and heptane to give 37e 18.94 g (72%) of
6-Bromo-3-(4-chlorophenyl)-2-(5-chloropyridin-2-ylmeth-
yl)-4-fluoro-3-hydroxy-2,3-dihydroisoindol-1-one (37f). To a
solution of 5-bromo-2-(4-chlorobenzoyl)-3-fluorobenzoic acid (see
Supporting Information) (10.7 g, 30.0 mmol) in THF (100 mL) was
added DMF (0.1 mL) and SOCl2 (4.4 mL, 60.0 mmol). The
resulting solution was stirred for 16 h under N2 and then
concentrated in vacuo. The residue was dissolved in THF (100
mL), cooled to 0 °C under N2, (5-chloropyridine-2-yl)methaneamine
dihydrochloride (6.9 g, 32.0 mmol) and DIPEA (16.7 mL, 96.0
mmol) were added, and the reaction mixture was stirred at room
temperature for 4 h. Water (150 mL) was added, and the product
was extracted with EtOAc (2 × 150 mL). The combined organic
layers were washed with brine (200 mL) and dried over MgSO4. The
solvent was removed in vacuo, and the residue was triturated with
EtOAc/petrol (1:1, 100 mL) to afford 37f as an off-white solid (9.56
1
the title compound. H NMR (400 MHz, DMSO-d6): 8.80 (1H, d),
8.14 (1H, dd), 7.88−7.80 (2H, m), 7.64 (1H, s), 7.38 (1H, d),
7.35−7.26 (4H, m), 4.62 (1H, d), 4.46 (1H, d), 3.33 (2H, s). LCMS
(ESI−) m/z: 471 [M − H]−.
6-{[5-Bromo-1-(4-chlorophenyl)-7-fluoro-1-({1-
[hydroxydideuteromethyl]cyclopropyl}dideuteromethoxy)-3-
oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carboni-
trile (41e). To a solution [5-bromo-1-(4-chlorophenyl)-7-fluoro-1-
hydroxy-3-oxo-1,3-dihydro-isoindol-2-ylmethyl]-nicotinonitrile 37e
(1.9 g, 4.0 mmol) in DCE (40 mL) were added {1-
[hydroxydideuteromethyl]cyclopropyl}dideuteromethanol (1.97 g,
18.7 mmol) and InBr3 (2.13 g, 6.0 mmol), and the reaction mixture
was stirred for 3 h under nitrogen at 90 °C. The reaction mixture was
cooled and washed with water (2 × 150 mL). The organic phase was
dried over MgSO4, concentrated in vacuo, and purified by
chromatography (silica, EtOAc/petrol, 0−60%) which gave the title
compound as a white solid (0.9 g, 40%). 1H NMR (400 MHz,
DMSO-d6): 8.77 (1H, dd), 8.11 (1H, dd), 7.96−7.91 (1H, m),
7.91−7.82 (1H, m), 7.40−7.30 (1H, m), 7.28 (4H, s), 4.61 (1H, d),
4.55−4.45 (1H, m), 3.36 (2H, s). LCMS (ESI−) m/z: 454 [M −
D4C5O2]−.
1
g, 66%). H NMR (400 MHz, DMSO-d6): 8.37 (1H, d), 7.84 (1H,
d), 7.80 (1H, dd), 7.73 (1H, dd), 7.61 (1H, s), 7.35−7.26 (4H, m),
7.22 (1H, d), 4.54 (1H, d), 4.39 (1H, d). LCMS (ESI+) m/z: 483
[M + H]+.
1-[5-Bromo-1-(4-chlorophenyl)-2-(5-chloropyridin-2-yl-
methyl)-7-fluoro-3-oxo-2,3-dihydro-1H-isoindol-1-
yloxymethyl]cyclopropanecarboxylic Acid Amide (41d). To
bromide 37f (0.962 g, 2.0 mmol) in THF (20 mL) was added
dropwise thionyl chloride (0.29 mL, 4.0 mmol) and catalytic
anhydrous DMF. The mixture was stirred at r.t. for 4 h and before
being concentrated in vacuo. The residue was dissolved in a solution
of 1-hydroxymethylcyclopropanecarboxylic acid amide (0.46 g, 4
mmol) in THF (10 mL). K2CO3 (0.552 g, 4.0 mmol) was added,
and then, the reaction mixture was stirred at r.t. for 18 h. The
mixture was diluted with EtOAc (20 mL), washed with water (3 ×
20 mL) and brine (20 mL), dried (MgSO4), and then concentrated
in vacuo. Chromatography (silica; EtOAc/petrol 0−50%) gave 41d as
6-{[5-Acetyl-1-(4-chlorophenyl)-7-fluoro-1-({1-
[hydroxydideuteromethyl]cyclopropyl}dideuteromethoxy)-3-
oxo-2,3-dihydro-1H-isoindol-2-yl]methyl}pyridine-3-carboni-
trile (43e). The bromide 41e (5.3 g, 9.8 mmol) was dissolved in a
mixture of toluene (50 mL) and 1,4-dioxane (50 mL). LiCl (1.3 g,
29.3 mmol). Tributyl(1-ethoxyvinyl)tin (3.3 mL, 9.8 mmol) and
Pd(PPh3)4 (580 mg, 0.48 mmol) were added, and the reaction
mixture was heated to 110 °C for 1.5 h. After cooling to room
temperature, the reaction mixture was diluted with dichloromethane
(DCM) and washed with saturated NaHCO3(aq). The organic phase
was dried over MgSO4 and concentrated in vacuo and purified by
SiO2 chromatography (0−100% EtOAc/petrol). The isolated
product was dissolved in 1,4-dioxane (50 mL), and 2 M HCl (30
mL) was added, and the reaction was stirred for 30 min. The
reaction was quenched with saturated NaHCO3(aq) and then
extracted with EtOAc. The combined organic extracts were dried
over MgSO4 and concentrated in vacuo to give the title compound
(3.8 g, 77%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): 8.78
(1H, m), 8.22 (1H, m), 8.12 (1H, dd), 7.99 (1H, d), 7.37 (1H, d),
7.29 (4H, s), 4.64 (1H, d), 4.54 (1H, d), 4.41 (1H, s), 3.32, (2H, s),
2.70 (3H, s). LCMS (ESI+) m/z: 522 (M − H+).
1
a white solid (0.81 g, 70%). H NMR (400 MHz, DMSO-d6): 8.34
(1H, d), 7.91 (1H, d), 7.86 (1H, dd), 7.73 (1H, dd), 7.35−7.17 (5H,
m), 6.94 (2H, m), 4.55−4.44 (2H, m), 3.52 (1H, d), 3.30 (2H, s),
3.10 (1H, d), 1.05−0.87 (2H, m), 0.61−0.50 (2H, m). LCMS
(ESI−) m/z: 461 [M − C5H9NO2]−.
1-(4-Chlorophenyl)-2-(5-chloropyridin-2-ylmethyl)-7-fluo-
ro-3-oxo-2,3-dihydro-1H-isoindol-1-yloxymethyl-6-(prop-1-
en-2-yl)cyclopropanecarboxylic Acid Amide (42d). Bromide
41d (0.8 g, 1.40 mmol) was dissolved in THF (15 mL) and
isopropenylboronic acid pinacol ester (0.4 mL, 2.1 mmol), NaOH
(0.06 g, 1.40 mmol) and N,N-dicyclohexylmethylamine (0.3 mL,
1.40 mmol) were added, and the solution was degassed for 15 min.
Pd(dppf)Cl2 (0.05 mg, 0.14 mmol) was added, and the reaction was
heated to reflux for 2 h, cooled to room temperature, filtered through
Celite, diluted with EtOAc (50 mL), and washed with 2 M HCl (30
mL) and brine (30 mL). The organic phase was dried over MgSO4,
concentrated in vacuo, and purified by chromatography (silica,
EtOAc/petrol, 0−80%) to give 42d as an orange solid (0.64 g, 85%).
1H NMR (400 MHz, DMSO-d6): 8.35 (1H, d), 8.32 (1H, s), 7.80
6 - { [ ( 1 R ) - 1 - ( 4 - C h l o r o p h e n y l ) - 7 - fl u o r o - 1 - ( { 1 -
[hydroxydideuteromethyl]cyclopropyl}dideuteromethoxy)-5-
(2-hydroxypropan-2-yl)-3-oxo-2,3-dihydro-1H-isoindol-2-yl]-
methyl}pyridine-3-carbonitrile (14). MeMgCl (9.7 mL, 29 mmol,
3 M in THF) and ZnCl2 (2.9 mL, 1.45 moll, 0.5 M in THF) were
dissolved in THF (50 mL) and stirred for 10 min and then cooled to
0 °C. To the cooled solution was then added a solution of ketone
(43e) (3.8 g, 7.3 mmol in THF). The solution was stirred at 0 °C for
30 min after which a further 2.4 mL of 3 M MeMgCl was added.
After stirring for a further 10 min, the mixture was quenched by
addition of sat. aq NH4Cl. The mixture was extracted with DCM,
and then, the organic layer was dried (MgSO4) and concentrated in
vacuo. Purification on SiO2 (0−70% EtOAc/hexanes) gave the title
compound (1.65 g) as a light orange solid. Separation by chiral
preparative LCMS Chiralpak ADH with a mobile phase of heptane/
2-propanol (50:50) and 0.2% DEA gave 14 (0.83 g) as a colorless
solid. 1H NMR (400 MHz, DMSO-d6): 8.77 (1H, d), 8.10 (1H, dd),
7.81 (1H, d), 7.53 (1H, d), 7.39−7.21 (5H, m), 5.38 (1H, s), 4.55
(2H, q), 4.39 (1H, s), 1.49 (6H, s), 0.40−0.30 (2H, m), 0.26−0.09
(2H, m); LCMS (ESI−) m/z: 538 [M − H]−.
(1H, d), 7.73 (1H, dd), 7.63 (1H, dd), 7.30 (2H, d), 7.27−7.17 (3H,
m), 7.03 (1H, s), 6.88 (1H, s), 5.69 (1H, s), 5.31 (1H, s), 4.48 (2H,
s), 3.52 (1H, d), 3.08 (1H, d), 2.18 (4H, s), 1.03−0.94 (1H, m),
0.94−0.83 (1H, m), 0.59−0.47 (2H, m). LCMS (ESI+) m/z: 540 [M
+ H]+.
1-({[(1R)-1-(4-Chlorophenyl)-2-[(5-chloropyridin-2-yl)-
methyl]-7-fluoro-5-(2-hydroxypropan-2-yl)-3-oxo-2,3-dihy-
dro-1H-isoindol-1-yl]oxy}methyl)cyclopropane-1-carboxa-
mide (23). Hg(OAc)2 (0.6 g, 1.90 mmol) and water (1.4 mL) were
stirred together for 5 min. THF (3 mL) was added, followed by a
solution of 42d (0.64 g, 1.19 mmol) in THF (1.5 mL). The solution
was stirred at r.t. for 2 h, followed by the addition of perchloric acid
(60%, 32 μL). After 3 h, 2 M NaOH (1.67 mL) was added followed
by NaBH4 (0.09 g, 2.38 mmol). The mixture was stirred at r.t. for 16
h, then filtered through Celite followed by filtration through a
polymer-supported thiol cartridge, and the solvent removed in vacuo.
Chromatography (silica, EtOAc/petrol, 30−100%) followed by
purification by preparative chiral HPLC gave 23 as a colorless
1
solid (0.1 g). H NMR (400 MHz, DMSO-d6): 8.35 (1H, d), 7.80
(1H, s), 7.73 (1H, dd), 7.53 (1H, d), 7.31 (2H, d), 7.23 (3H, dd),
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7.03 (1H, d), 6.85 (1H, s), 5.38 (1H, s), 4.47 (2H, s), 3.47 (1H, d),
3.08 (1H, d), 1.48 (6H, s), 0.99−0.87 (2H, m), 0.59−0.44 (2H, m).
LCMS (ESI+) m/z: 558 [M + H]+.
with Scala. The integration of the data using a monoclinic cell
yielded a total of 51587 reflections to a maximum Θ angle of 34.23°
(0.622 Å resolution), of which 18390 were independent (redundancy
2.8), completeness = 75.0% (Rmeas = 0.065, Rpim = 0.026).
Crystallography. Three clones were used to support the
structural biology of MDM2 described in this paper, namely
clone1 (GST-3C-17-109 E69A K70A), clone2 (GST-3C-17-125
E69A K70A), and clone3 (GST-3C 17−125 K51A).DNA encoding
residues 17−109 or 17−125 of the MDM2 gene were cloned into a
pGEX-6P-1 vector (GE Healthcare), and point mutations introduced
by QuikChange mutagenesis (Agilent). All three constructs were
expressed and purified using the same protocol. MDM2 was
expressed in Escherichia. coli ArticExpress (DE3) or BL21 (DE3)
pLysS cells using Terrific Broth or Luria−Bertani media, respectively.
The E. coli cultures were incubated at 37 °C, 200 rpm in baffled
flasks until reaching an optical density at 600 nm (OD600) of ∼1.0.
The temperature was reduced to 18 °C, and then, expression was
induced by the addition of isopropyl β-d-1-thiogalactopyranoside to a
final concentration of 0.1 mM. Cell cultures were then left to grow
overnight and harvested the next day by centrifugation.
Cells from 4 to 6 L cultures were resuspended in buffer A (25 mM
HEPES-NaOH, pH 7.4, 100 mM NaCl, 5 mM dithiothreitol)
supplemented with protease inhibitors (Calbiochem #539134), 2 μg/
mL DNase I (Sigma-Aldrich), and 1 mM MgCl2 (final concen-
trations.) The resuspended cells were sonicated on ice and then
clarified by centrifugation (60,000g, 45 min, 4 °C). The supernatant
was then tumbled with 5 mL of GST 4B resin (GE Healthcare) for
1−2 h or O/N, before being poured into a low-pressure column,
washed with buffer A, and then eluted in buffer A supplemented with
20 mM reduced glutathione (pH adjusted to 7.4). The GST fusion
protein was removed with PreScission 3C protease at 4 °C. The
cleaved protein was applied to a Superdex75 26/60 or Superdex200
26/60 column (GE Healthcare) equilibrated in buffer A. Purified
MDM2 was pooled and then frozen or used for co-crystallization.
The co-complex structures were generated by co-crystallization;
crystallization trials were setup using all constructs, and crystals were
obtained with at least one construct. A total of 3 mg of protein in
approximately 15 mL of buffer was placed in a 15 mL tube and 12
μL of 50 mM compound dissolved in 100% DMSO-d6 was added.
The protein-compound mix was tumbled overnight at 4 °C and then
concentrated (Amicon Ultracel-3) to approximately 200 μL. The
protein-compound complex was then used in crystallization trials
against the Qiagen AmSO4 Suite using protein/precipitant ratios of
1:1 or 2:1. The trays were incubated at 4 °C, and crystals were
usually obtained in several conditions after 2−5 days. The crystals
were briefly cryo-protected with reservoir solution supplemented
with 20% glycerol, before being flash cooled in liquid nitrogen. Data
were collected using in-house or synchrotron (ESRF or diamond) X-
ray sources.
X-ray diffraction data were reduced and used in molecular
replacement to solve the structures using the CCP443 and Global
Phasing software suites.44 Ligand placement was performed using
AutoSolve45 and the protein-compound structure refined using
Refmac.46 Data collection and refinement statistics are presented in
Table SX. The coordinates were deposited in the PDB under the
accession codes listed below.
NMR Studies on Isoindolinone 5. A sample of 5 was dissolved
in DMSO-d6 (160 μL) and transferred to a 2.5 mm NMR capillary
(New Era). NMR data (1H, COSY, HSQC, HMBC, and ROESY)
were acquired at 300 K on a Bruker AVANCE III 500 MHz
spectrometer equipped with a 5 mm TXI cryoprobe. Data processing
was performed with MestReNova 14 (Mestre Lab Research, Santiago
de Compostela, Spain). Further details are supplied in the
Supporting Information.
Determination of the Single Crystal Structure of Iso-
indolinone 18. A colorless single crystal of 18 was used for the X-
ray crystallographic analysis. The X-ray intensity data were measured
at 100(2) K at ESRF ID23.1 beamline using a 0.70000 Å wavelength
radiation and Dectris Pilatus 6M detector.
A total of 1800 frames were collected with a scan width of 0.2° in
φ. The frames were integrated with XDS software package and scaled
The unit cell constants were determined to be a = 14.1815(10) Å,
b = 11.4100(10) Å, c = 16.4315(10) Å, α = 90°, β = 108.590(10)°, γ
= 90°, and volume = 2520.1(3) Å3 and belonged to space group P21.
The structure was solved using direct methods implemented in
SHELXS-97 and refined using SHEXL-97. Two molecules of
compound 18 were found in the asymmetric unit. The final
anisotropic full-matrix least-squares refinement on F2 with 653
variables converged at wR2 = 11.93% for all 17401 reflections and R1
= 3.93% for 17,132 reflections greater than 2σ(I). The goodness-of-
fit was 1.058, and the Flack parameter was calculated to be 0.06(2).
The largest peak on the final difference electron density map was
0.51 e−/Å3, and the largest hole was −0.49 e−/Å3 with a root-mean-
square deviation of 0.11 e−/Å3. On the basis of the final model, the
calculated density was 1.390 g/cm3 and F(000) = 1104 e−.
ELISA Methods. MDM2 was expressed in vitro using the TNT
T7 coupled reticulocyte lysate system (Promega) and a plasmid
DNA preparation containing the MDM2 DNA sequence. The
reaction was incubated at 30 °C for 2 h, and the resulting expression
was diluted 1/70 in 12 mM Na2HPO4, 150 mM NaCl, pH 7.5 for its
immediate use or stored at −20 °C.
The ELISA was performed in 96-well streptavidin coated plates
(streptavidin FlashPlate PLUS SMP103A, PerkinElmer). 200 μL per
well of 1 μg/mL IP3 peptide (Biotin-aminohexanoyl-MPRFMDY-
WEGLN, Alta Biosciences) in 12 mM Na2HPO4, 150 mM NaCl, pH
7.5 was added to each well, and the plate was incubated for 1 h. The
plate was washed with 12 mM Na2HPO4, 150 mM NaCl, 0.05% (v/
v) Tween-20, pH 7.5.
Compounds were preincubated with the in vitro expressed MDM2
in 12 mM Na2HPO4, 150 mM NaCl, pH 7.5 for 20 min 200 μL of
the compound-MDM2 mix was then transferred to the IP3 treated
streptavidin plate and incubated at 4 °C for 90 min. After washing
the plate with 12 mM Na2HPO4, 150 mM NaCl, 0.05% (v/v)
Tween-20, pH 7.5, the plate was incubated at room temperature for
1 h with 200 μL of primary mouse Ab-5 monoclonal anti-MDM2
antibody (Ab-5 mouse 4B2C1.11, Calbiochem) at 500 ng/mL in 50
mM Tris−HCl, 150 mM NaCl, 0.05% (v/v) Tween-20, pH 7.5. The
plate was washed with 200 μL of 50 mM Tris−HCl, 150 mM NaCl,
0.05% (v/v) Tween-20, pH 7.5 and incubated with 200 μL of
polyclonal goat-anti-mouse horseradish peroxidase (HRP) conju-
gated secondary antibody (Millipore) at 1 ng/mL in 50 mM Tris−
HCl, 150 mM NaCl, 0.05% (v/v) Tween-20, pH 7.5 for 45 min. The
plate was washed with 50 mM Tris−HCl, 150 mM NaCl, 0.05% (v/
v) Tween-20, pH 7.5. HRP activity was measured in a TopCount
NXT luminescence reader (PerkinElmer) after adding 100 μL of a
1:1 (v/v) mixture of enhanced chemiluminescence reagents 1 and 2
(Amersham Biosciences). IC50 curves were generated using the four-
parameter logistic curve fit.
Sulforhodamine-B Cell Proliferation Assays. The TP53 wild-
type human osteosarcoma SJSA-1 (American Type Culture
Collection, Virginia USA) and its TP53 mutant derivative
SN40R247 cell lines were used to assess the antiproliferative effects
of different compounds. All cell cultures were grown in RPMI 1640
medium (Gibco, Paisley, UK) supplemented with 10% fetal calf
serum and routinely tested and confirmed negative for mycoplasma
infection. 100 μL of 3 × 104/mL and 2 × 104/mL SJSA-1 and
SN40R2 cells, respectively, was 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 concentrations of compounds and
incubated for a further 72 h to allow cell growth before adding 25 μL
of 50% trichloroacetic acid (TCA) to fix the cells for 1 h at 4 °C.
The TCA was washed off with distilled water and 100 μL of
sulforhodamine-B (SRB) dye (0.4% w/v in 1% acetic acid) (Sigma-
Aldrich, Poole, Dorset) 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. The SRB
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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 = 570 nm was measured in each well using a
FluoStar Omega Plate reader. The GI50 was calculated by nonlinear
regression analysis of the data using the GraphPad Prism software.
Plasma Protein Binding. Test compounds were incubated in
duplicates at 1 μM with mouse (male BALB/c) and human (single
donor) plasma which was diluted 1 in 10 with Dulbecco’s phosphate
buffered saline (PBS). Equilibrium dialysis was carried out using a
HTDialysis plate, molecular weight cut off 12,000−14,000 Da.
Spiked and blank PBS and 10% plasma were placed appropriately
either side of the dialysis membrane and then incubated on a shaking
platform at 37 °C for 48 h. Samples (20 μL) were removed, and the
matrix matched at 48 h only.
In Vitro Intrinsic Clearance in Mouse and Human Micro-
somes. Test compounds were incubated in duplicates at 1 μM with
mouse (male CD-1) or human (mixed gender 150 donor pool) liver
microsomes at 37 °C for 40 min at a protein concentration 0.25 mg/
mL, in the presence of a NADPH regenerating system, UDPGA, and
alamethacin. Diclofenac was included alongside test compounds as a
positive control reference compound. Incubations were sampled (50
μL) at 0, 5, 10, 20, and 40 min. Negative control incubations, in the
absence of NADP and UDPGA, were performed and sampled at 0
and 40 min only.
Metabolite Identification. Selected sample extracts from the in
vitro mouse microsome intrinsic clearance assay, using the standard
conditions or incubated at a higher test compound concentration of
10 μM, were further analyzed for metabolite identification by LC/
MS. Metabolites were detected by MRM of the common iminium
ion (m/z 424) formed in the MS source during electrospray
ionization and comparison with negative control samples. Molecular
ions of metabolites were identified by “parents of m/z 424” scans.
CYP3A4 Phenotyping. Test compounds were incubated in
duplicates at 1 μM with human Supersomes expressing CYP3A4 at
37 °C for 40 min at a protein concentration of 20 pmol/mL, in the
presence of NADPH. Midazolam was included alongside test
compounds as a positive control reference compound. Incubations
were sampled (50 μL) at 0, 5, 10, 20, and 40 min. Negative control
incubations, in the absence of NADPH, were performed and sampled
at 0 and 40 min.
CYP3A4 Inhibition. CYP3A4 inhibition studies were performed
at Cyprotex (Manchester UK). Test compounds were incubated at
37 °C in human liver microsomes, at 6 final assay concentrations, up
to 25 μM, in the presence of 2.5 μM of the probe substrate
midazolam. Protein content was 0.1 mg/mL, and NAPDH was added
at a final concentration of 1 mM. A specific positive control CYP3A4
inhibitor, ketoconazole, was screened alongside the test compounds.
Negative control incubations, in the absence of inhibitors, were also
performed. All incubations were sampled after 5 min and analyzed
for the metabolite 1-hydroxymidazolam by LC−MS.
test compounds were formulated for IV dosing in 10% NMP, 90%
PEG400 (dose volume 0.2 mL/kg) or 10% NMP, 20% PEG200, and
saline (dose volume 1 mL/kg). Oral NHP doses were formulated in
5% DMSO, 95% hydroxypropyl-β-cyclodextrin (20% aq, w/v), dose
volume of 5 mL/kg. Blood samples were collected at appropriate
times, up to 24 h, following test compound administration into tubes
containing K2EDTA as the anticoagulant. Whole blood was
centrifuged to obtain plasma and stored at −20 °C or below prior
to bioanalysis.
The mean composite (mouse) or individual (NHP) plasma
compound concentration−time profiles were plotted, and PK
parameters were obtained using WinNonLin (Certara USA, Inc.)
noncompartmental analysis software.
Bioanalysis. All in vitro and in vivo samples were extracted by
protein precipitation with acetonitrile containing the internal
standard (1:3 v/v). For quantitative studies, calibration standards
and quality controls were prepared in the blank matrix and extracted
under the same conditions. Test compound bioanalysis of samples
was typically performed using a Xevo TQ-S tandem quadrupole mass
spectrometer coupled to an Aquity i-Class autosampler and UPLC
system (Waters, UK) or a Qtrap 4000 hybrid triple quadrupole/
linear ion trap mass spectrometer (AB Sciex, UK) coupled to a
Agilent 1200 HPLC system (Agilent, UK). Test compound and the
internal standard were ionized using positive mode (ESI+) electro-
spray ionization. Analytes were detected using MRM. Compounds
typically ran on a gradient HPLC method over 6 min with a 10 mM
ammonium acetate (aq) and acetonitrile mobile phase at a flow rate
of 0.5 mL/min. Separation was typically achieved using an Acquity
BEH C18 Phenyl 1.7 μm 50 × 2.1 mm column maintained at 40 °C,
connected to a BEH C18 vanguard precolumn.
Derivation of Chromatographic LogD (Chrom logD).
Chromatographic hydrophobicity index (CHI) values were measured
using the reversed phase HPLC column (PolymerX RP-1,
Phenomenex, UK) with a fast acetonitrile gradient at a starting
mobile phase of pH = 7.4. CHI values are derived directly from the
gradient retention times by using a calibration line obtained for
standard compounds. The CHI value approximates to the volume %
organic concentration when the compound elutes. CHI is linearly
transformed into Chrom LogD (pH 7.4) by least-squares fitting of
experimental CHI values to calculated ClogP values for over 20,000
research compounds using the following formula: Chrom LogD (pH
7.4) = 0.0857CHI-2.00. The average error of the assay is 3 CHI
unit or 0.25 Chrom LogD (pH 7.4).
In Vivo Mouse PK and Pharmacodynamic Studies. The care
and treatment of animals were in accordance with the United
Kingdom Coordinating Committee for Cancer Research guidelines
and with the United Kingdom Animals (Scientific Procedures) Act
1986 law. The study protocols were approved by Animal Welfare
Ethical Review Boards at the University of Cambridge and Newcastle
University (Newcastle upon Tyne, UK). SJSA-1 subcutaneous
xenografts were established by injecting 1 × 107 cells in 50 μL of
RPMI media mixed 1:1 with Matrigel into the right hind flank of
female CD-1 nude mice. Tumor-bearing mice were treated with 14
or 23 administrated orally at a dose volume of 10 mL/kg. Doses were
given as mg free base equivalent per kg body weight. The compounds
were formulated in 5% ethanol and 95% hydroxypropyl methyl
cellulose (1% w/v aq). Blood samples were then collected into tubes
containing potassium EDTA at indicated times post dose. Samples
were centrifuged, and the resultant plasma was separated and stored
at −20 °C until bioanalysis. At terminal time points, animals were
sacrificed and tumors were excised immediately and flash-frozen in
liquid nitrogen. Tumors were stored at −80 °C until analyzed.
For assessment of pharmacodynamic markers, tumor lysates were
prepared by grinding the frozen tumor tissue to a fine powder with a
mortar/pestle under liquid nitrogen and then adding ice-cold Meso
Scale Discovery (MSD, Maryland, USA) lysis buffer, containing
protease inhibitors [complete mini tablet (Roche), 50 mM NaF, and
1 mM Na3V04], to the ground-up tumor powder. Samples were
vortexed and left on ice for at least 30 min. Lysates were cleared by
centrifugation, and samples of the supernatant were removed for
Animal Husbandry and Handling. Mouse studies were
performed according to the Animal (Scientific Procedures) Act
(1986) law. Mice were allowed access to food and water ad libitum.
NHP studies were performed at WuXi AppTec (Suzhou, China) in
accordance with the Wuxi IACUC standard animal procedures along
with the IACUC guidelines that are in compliance with the Animal
Welfare Act, the Guide for the Care and Use of Laboratory Animals,
the Office of Laboratory Animal Welfare (OLAW), and following
local ethical reviews. NHPs were housed individually in stainless steel
mesh cages in a controlled environment (temperature 18 to 29 °C,
relative humidity 30 to 70%) and fed twice daily.
In Vivo PK Studies. Test compounds were administered by
intravenous (IV) bolus or oral gavage to male BALB/c mice (n = 3
per timepoint) or male cynomolgus monkeys (n = 3). The IV dose
was 0.5 mg/kg (mice) or 0.2 mg/kg (NHP), and the oral dose was 5
mg/kg. For mouse studies, test compounds were formulated in 10%
NMP, 20% PEG400 or PEG200, 70% 15 mM sodium bicarbonate
(pH 8.4) or saline (IV) and 10% NMP, 90% 15 mM sodium
bicarbonate (pH 8.4) or water (PO) and administered at dose
volumes of 5 mL/kg and 10 mL/kg, respectively. For NHP studies,
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protein determination by BCA assay (Pierce). Levels of p53 and
MDM2 were measured by MSD analyses using the single-plex MSD
kits following the manufacturer’s instructions. The plates were read
using the Sector Imager 2400 plate reader.
For PK bioanalysis, compound levels in plasma and tumor samples
were measured as described previously.
In Vivo Efficacy Studies. For tumor growth inhibition studies,
tumor-bearing animals were randomized into groups of 10 with the
average tumor volume of 100 mm3. Tumors were measured every 2−
3 days externally by using a pair of digital calipers and volumes
calculated by applying the formula for ellipsoid. Statistical analyses
were performed using GraphPad Prism version 8. The effects of
treatments were compared using one-way ANOVA and two-way
ANOVA with Dunnett multiple comparison test against vehicle
control. Differences were deemed statistically significant when P <
0.05.
Luke D. Bevan − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
Timothy J. Blackburn − Cancer Research UK Newcastle
Drug Discovery Unit, Newcastle University Centre for
Cancer, Chemistry, School of Natural and Environmental
Sciences, Newcastle University, Newcastle upon Tyne NE1
7RU, U.K.
Ildiko Buck − Astex Pharmaceuticals, Cambridge CB4 0QA,
U.K.
Celine Cano − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU,
U.K.; orcid.org/0000-0002-2032-2272
Benoit Carbain − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU, U.K.
Juan Castro − Astex Pharmaceuticals, Cambridge CB4 0QA,
U.K.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02188.
■
sı
Ben Cons − Astex Pharmaceuticals, Cambridge CB4 0QA,
U.K.
Molecular formula string (CSV)
Model of the X-ray crystal structure of MDM2 with
Sarah J. Cully − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU, U.K.
Jane A. Endicott − Cancer Research UK Newcastle Drug
Discovery Unit, Translational and Clinical Research
Institute, Faculty of Medical Sciences, Newcastle University,
Newcastle upon Tyne NE2 4HH, U.K.
compound 4 (CIF)
Structure factors of the X-ray crystal structure of
MDM2 with compound 4 (CIF)
Additional experimental details for compounds not
included in the main section and analytical data for all
compounds and intermediates; NMR studies on 5; X-
ray structure tables; and computational chemistry
(PDF)
Lynsey Fazal − Astex Pharmaceuticals, Cambridge CB4 0QA,
U.K.
Accession Codes
Bernard T. Golding − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU, U.K.
Roger J. Griffin − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU, U.K.
Karen Haggerty − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU, U.K.
Suzannah J. Harnor − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU,
U.K.; orcid.org/0000-0003-1646-593X
Coordinates for MDM2 complexes with compounds 1, 5, 13,
14, and 23 have been deposited in the Protein Data Bank
(PDB) under accession codes 7BJ0, 7BMG, 7BJ6, 7BIV, and
7BIR, respectively. Coordinates for the small molecule crystal
structure of compound 18 have been deposited in the
CCDC/FIZ Karlsruhe database under the deposition number
2059877.
AUTHOR INFORMATION
Corresponding Authors
■
Gianni Chessari − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.; orcid.org/0000-0002-8151-7778;
Email: Gianni.chessari@astx.com
Ian R. Hardcastle − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU,
U.K.; orcid.org/0000-0001-7495-3769;
Keisha Hearn − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
Email: ian.hardcastle@newcastle.ac.uk
Stephen Hobson − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU, U.K.
Rhian S. Holvey − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
Steven Howard − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
Claire E. Jennings − Cancer Research UK Newcastle Drug
Discovery Unit, Translational and Clinical Research
Institute, Faculty of Medical Sciences, Newcastle University,
Newcastle upon Tyne NE2 4HH, U.K.
Authors
Jong Sook Ahn − Astex Pharmaceuticals, Cambridge CB4
0QA, U.K.
Burcu Anil − Department of Biochemistry, University of
Oxford, Oxford OX1 3QU, U.K.
Elizabeth Anscombe − Department of Biochemistry,
University of Oxford, Oxford OX1 3QU, U.K.
Ruth H. Bawn − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU, U.K.
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Christopher N. Johnson − Astex Pharmaceuticals,
Cambridge CB4 0QA, U.K.; orcid.org/0000-0001-8618-
9729
John Lunec − Cancer Research UK Newcastle Drug Discovery
Unit, Translational and Clinical Research Institute, Faculty
of Medical Sciences, Newcastle University, Newcastle upon
Tyne NE2 4HH, U.K.
Duncan C. Miller − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU,
U.K.; orcid.org/0000-0001-6846-2007
ACKNOWLEDGMENTS
■
We acknowledge valuable scientific discussions with col-
leagues, including Christopher W. Murray, David C. Rees, and
Andrew J. Woodhead. The authors thank Stuart Whibley for
provision of high-resolution mass spectrometry data and Anne
Cleasby for crystallographic support. The authors also
gratefully acknowledge support for a proportion of this work
from a Cancer Research UK (CRUK) Programme grant
(C2215/A21421). Use of the EPSRC Mass Spectrometry
Service at the University of Wales (Swansea) is gratefully
acknowledged.
David R. Newell − Cancer Research UK Newcastle Drug
Discovery Unit, Translational and Clinical Research
Institute, Faculty of Medical Sciences, Newcastle University,
Newcastle upon Tyne NE2 4HH, U.K.
Martin E. M. Noble − Cancer Research UK Newcastle Drug
Discovery Unit, Translational and Clinical Research
Institute, Faculty of Medical Sciences, Newcastle University,
Newcastle upon Tyne NE2 4HH, U.K.
ABBREVIATIONS
■
ADME, absorption distribution metabolism excretion;
CYP3A4, cytochrome P450 3A4; DCE, dichloroethane;
DCM, dichloromethane; DMF, N,N-dimethylformamide;
DMSO, dimethylsulfoxide; EtOAc, ethyl acetate; MDM2,
murine double minute 2; QM, quantum mechanics; ROESY,
rotating frame Overhauser effect spectroscopy; THF, tetrahy-
drofuran
Judith Reeks − Astex Pharmaceuticals, Cambridge CB4 0QA,
U.K.
Charlotte H. Revill − Cancer Research UK Newcastle Drug
Discovery Unit, Newcastle University Centre for Cancer,
Chemistry, School of Natural and Environmental Sciences,
Newcastle University, Newcastle upon Tyne NE1 7RU, U.K.
Christiane Riedinger − Department of Biochemistry,
University of Oxford, Oxford OX1 3QU, U.K.
Jeffrey D. St. Denis − Astex Pharmaceuticals, Cambridge
CB4 0QA, U.K.
Emiliano Tamanini − Astex Pharmaceuticals, Cambridge
CB4 0QA, U.K.; orcid.org/0000-0002-2776-011X
Huw Thomas − Cancer Research UK Newcastle Drug
Discovery Unit, Translational and Clinical Research
Institute, Faculty of Medical Sciences, Newcastle University,
Newcastle upon Tyne NE2 4HH, U.K.
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