Searching for Dual Inhibitors of the MDM2-p53 and MDMX-p53 Protein-Protein Interaction by a Scaffold-Hopping Approach

Article abstract of DOI:10.1111/cbdd.12474

Two libraries of substituted benzimidazoles were designed using a 'scaffold-hopping' approach based on reported MDM2-p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X-ray structure. Benzimidazole libraries were prepared using an efficient solution-phase approach and screened for inhibition of the MDM2-p53 and MDMX-p53 protein-protein interactions. Key examples showed inhibitory activity against both targets.

Full text of DOI:10.1111/cbdd.12474

Received Date : 17-Jun-2014
Revised Date : 18-Sep-2014
Accepted Date : 15-Oct-2014
Article type : Research Article
Searching for dual inhibitors of the MDM2-p53 and
MDMX-p53 protein-protein interaction by a
scaffold-hopping approach
Andrey Zaytsev,1 Barry Dodd,1 Matteo Magnani,3 Chiara Ghiron,3 Bernard T. Golding,1 Roger
J. Griffin,1 Junfeng Liu,2 Xiaohong Lu,2 Iolanda Micco,3 David R. Newell,2 Alessandro Padova,3
Graeme Robertson,3 John Lunec,2 Ian R. Hardcastle1*
1 Newcastle Cancer Centre, Northern Institute for Cancer Research and School of Chemistry,
Bedson Building, Newcastle University, Newcastle, NE1 7RU, UK
2 Newcastle Cancer Centre, Northern Institute for Cancer Research, Paul O’Gorman Building,
Medical School, Framlington Place, Newcastle University, Newcastle, NE2 4HH, UK
3Siena Biotech S.p.A., Strada del Petriccio e Belriguardo, 35, Siena, 53100, Italy
Corresponding author mail id: i.r.hardcastle@newcastle.ac.uk
ABSTRACT
Two libraries of substituted benzimidazoles were designed using a ‘scaffold-hopping’ approach
based on reported MDM2-p53 inhibitors. Substituents were chosen following library enumeration
and docking into an MDM2 X-ray structure. Benzimidazole libraries were prepared using an efficient
solution-phase approach, and screened for inhibition of the MDM2-p53 and MDMX-p53 protein-
protein interactions. Key examples showed inhibitory activity against both targets.
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lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12474
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INTRODUCTION
The design of small molecule inhibitors of protein-protein interactions has received
considerable interest in recent years.(1-4) In particular, the interaction of p53 with the regulatory
proteins MDM2 and MDMX have provided a focus for efforts because of their importance in a
number of cancers, and the prospect that inhibition of these interactions may provide new
therapies. Overexpression of the regulatory proteins MDM2 and MDMX has been demonstrated to
silence the tumor suppressor function of p53. MDM2 amplification occurs in around 11% of all
tumors, and is most common in hepatocellular carcinoma (44%), osteosarcomas (20%), and soft
tissue sarcomas (31%). Similarly, MDMX (MDM4) occurs in approximately 10-25% of tumors, e.g.
brain (11%), breast (5-40%), and sarcomas (17%).(5-8)
Expression of MDM2 is p53-dependent, and forms a regulatory feedback loop with p53 by
binding to the p53 transactivation domain. Acting as an E3-ligase, MDM2 promotes proteasomal
degradation of p53.(9-12) MDMX expression is not p53-dependent and MDMX levels remain
constant. The protein inhibits p53 transcriptional activity, but does not act as an E3 ligase
independently of MDM2.(13) Thus, MDM2 and MDMX both regulate p53, but their functions are
different and non-redundant.(14)
The X-ray crystal structure of MDM2 bound to a p53-derived peptide reveals the α-helical
peptide bound into a deep groove on the surface of the protein, and key interactions are formed
with three hydrophobic residues of p53 (Figure 1).(15) Inhibitors of MDM2 with diverse chemotypes
have been reported.(16, 17) X-ray crystal structures of a range of potent MDM2-p53 inhibitors have
been published, such as Nutlin-3 (1), spirooxindoles, e.g. MI-63 (2) and pyrrolidones (3).(18-21)
Inhibitors show cellular activity and in vivo antitumor activity.(18, 22) Recently, potent MDM2-p53
inhibitors have entered clinical trials, including RG7112,(23, 24) RG7388(25) and MI-773(26) (Chart
1).
The p53 transactivation loop binds in a cleft on the surface of MDMX similar to that
identified for MDM2 but with a closed conformation for Tyr99.(27, 28). Steric hindrance of Met53 in
the Leu23 pocket for MDMX may be responsible for the lack of potency against MDMX for MDM2-
p53 inhibitors such as the Nutlins and spirooxindoles.(29) A high degree of flexibility has been
measured in computational studies of MDMX, and significant conformational differences, owing to
the induced fit of ligands are observed in crystal structures.(30-32) Reports of small-molecule
MDMX-p53 inhibitors have not been as numerous as for MDM2.(21) The selective MDMX inhibitor
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5-oxo-pyrazolylidene SJ-172550 (4) was identified in an MDMX high-throughput assay.(33) The 3-
imidazolylindole 5 is a mixed MDM2- and MDMX-p53 inhibitor with modest potency against
MDMX.(29) Recently, indolylhydantoins, e.g. (6) have been reported as MDM2-p53 and MDMX-p53
inhibitors.(34)
In this paper, we describe the application of a scaffold-hopping approach to design new
inhibitor chemotypes for MDM2 and MDMX(35, 36) based on heterocycle replacement, and derived
from two MDM2-p53 inhibitors with significantly different core structures. In particular, starting
from cis-imidazoline (1),(18) and a class of oxindoles (7),(37, 38) benzimidazole derivatives were
designed and evaluated through docking studies. The benzimidazole heterocycle was chosen as a
‘privileged structure’ in medicinal chemistry that is readily synthetically accessible.(39) The synthesis
of small libraries yielded mixed MDM2-p53 and MDMX-p53 inhibitors with promising potency.
MATERIALS AND METHODS
MDM2-p53 ELISA assay. Compounds were assayed for MDM2-p53 inhibitory activity using ELISA
assays in a 96-well format using the published method.(40)
MDMX-p53 ELISA assay. MDMX-p53 inhibitory activity was determined using an analogous method
incorporating a pCMV-XL5-MDMX cDNA construct (OriGene Technologies) for the in vitro coupled T7
transcription and rabbit reticulocyte lysate translation of MDMX, and a rabbit anti-MDMX antigen
affinity-purified polyclonal antibody (Bethyl Laboratories Inc, via UK supplier Cambridge Bioscience,
UK, Cat No. A300-287 A).(41)
Docking experiments. Docking calculations were performed with the GOLD software.(42) Ligands
were docked within the p53 binding site of MDM2, using the crystal structure of human MDM2 in
complex with the small molecule inhibitor Nutlin-2 (PDB code: 1RV1).(18) A single protein chain was
selected from the unit cell. The binding site was defined by hydrophobic fitting points calculated on
the target for a 8Å radius around the co-crystallized ligand. GoldScore was used as a fitness function,
while default genetic algorithm parameter settings were applied. 30 poses were generated for each
ligand, early termination being allowed when the top 10 solutions were within 1.5Å R.M.S.D.
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Chemical Synthesis.
General Procedure A – Synthesis of compounds (14).
A mixture of the appropriate aldehyde (1.2 eq.), sodium dithionite (86% purity; 1.98 g, 9.78 mmol, 3
eq.) and 13 (1.16 g, 3.26 mmol, 1.0 eq.) in methanol (12 mL) and water (3 mL) was heated in a
microwave reactor for 10 minutes at 100 oC. The sample was diluted with ethyl acetate (20 mL) and
washed with water (20 mL) and brine (20 mL). The organic layer was dried (MgSO4), and evaporated.
Chromatography (silica; ethyl acetate, petrol) gave compound 14.
General Procedure B. Compound 14 was dissolved in DCM (2 mL) and TFA (3 mL) and the mixture
stirred at rt for 1 hour, then evaporated. The residue was diluted with DCM (20 mL) and neutralised
with sodium carbonate (sat.; 20 mL). The organic layer was dried (MgSO4) to give 15 which was used
without further purification.
General procedure C. Either an aliquot of a solution of 15 in dry DCM (1 mL, 0.049 M), or weighed 15
in dry DCM (1 mL) was treated with the named isocyanate (1.5 eq.). The mixture was quenched with
NH2-silica and filtered through a plug of NH2-silica. Evaporation gave 8 without further purification.
(S)-Ethyl 3-(3-(5-chloro-2-(3-methoxybenzyl)-1H-benzo[d]imidazol-1-yl)piperidine-1-
carboxamido)benzoate 8{3,7}.
General procedure C: 15{3} (1 mL, 49.3 µmol) and ethyl 3-isothiocyanatobenzoate (12 µl, 76 µmol).
8{3,7} (26 mg, 96%) as a white solid; HPLC purity (as area %) > 98; 1H NMR (300MHz, CDCl3) 1.38 (1H,
t, J 7.2, CH2CH3), 1.54 (1H, qt, J 3.6 and 13.1), 1.72 (1H, app.dd), 1.87 (1H, app.d), 2.20 (1H, qd, J 4.0
and 12.7), 2.80 (1H, td, J 2.2 and 13.1), 3.38(1H, app.t), 3.63 (3H, s, OCH3), 3.77 (1H, app. dd), 4.21
(1H, app.d), 4.30 (2H, s, CH2), 4.36 (2H, q, J 7.2), 6.54 (1H, br.d), 6.65-6.69 (1H, m, Ar), 6.77 (2H, d, J
8.3, Ar), 7.17 (1H, app.t, Ar), 7.20 (1H, m, Ar), 7.32-7.39 (2H, m, Ar), 7.67-7.81 (4H, m, Ar); 13C NMR
(100MHz, CDCl3) 14.3, 24.8, 28.6, 35.0, 44.1, 47.2, 53.5, 55.2, 61.1, 112.2, 112.5, 114.3, 119.8, 120.5,
121.0, 122.7, 124.4, 124.7, 127.7, 128.9, 129.9, 131.0, 132.1, 137.5, 138.9, 144.0, 154.2, 154.7,
160.0, 166.3.
(S)-Ethyl 3-(3-(5-chloro-2-(4-methoxybenzyl)-1H-benzo[d]imidazol-1-yl)piperidine-1-
carboxamido)benzoate 8{4,7}.
General procedure C: 15{4} (1 mL, 49 µmol) and 3-isocyanatobenzoate (12 µl, 76 µmol). 8{4,7} (23
1
mg, 85%), white solid; HPLC purity (as area %) > 98; H NMR (400MHz, CDCl3) 1.33 (3H, t, J 4.1,
CH2CH3), 1.48 (1H, qt, J 4.1 and 13.3), 1.62 (1H, app.d), 1.82 (1H, app.d), 2.17 (1H, qd, J 4.1 and 12.8),
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2.78 (1H, td, J 2.3 and 13.3), 3.36 (1H, app.t), 3.55 (3H, s, OCH3), 3.82 (1H, dd, J 4.1 and 12.8), 4.15
(1H, d, J 16.0), 4.17 (1H, app.d), 4.23 (1H, d, J 16.0), 4.25-4.33 (1H, m), 4.30 (2H, q, J 7.3, CH2CH3),
6.70-6.73 (3H, app.d), 7.04 (2H, d, J 8.7, Ar), 7.15 (1H, dd, J 1.8 and 8.7), 7.28-7.33 (2H, m, Ar), 7.59-
7.62 (1H, m, Ar), 7.63-7.70 (2H, m, Ar), 7.81 (1H, app.d, Ar); 13C NMR (100MHz, CDCl3) 14.3, 24.9,
28.6, 34.1, 44.2, 47.2, 53.4, 55.0, 61.0, 112.2, 114.2, 119.7, 121.1, 124.4, 124.8, 127.6, 127.7, 128.9,
129.4, 131.0, 132.2, 139.0, 144.0, 154.7, 154.9, 158.7, 166.3; MS (ESI+) m/z = 547.3 [M+H]+.
(S)-N-Benzyl-3-(2-benzyl-5-chloro-1H-benzo[d]imidazol-1-yl)piperidine-1-carbothioamide 16.
General procedure C: 15{1} (16 mg, 49.2 µmol) and benzyl isothiocyanate (7.2 µl, 54.1 µmol). 16 (15
1
mg, 64%) as a white solid; HPLC purity (as area %) > 93; H NMR (500 MHz, CDCl3) 1.43 (1H, qt, J 4.1
and 13.3), 1.55 (1H, app.d), 1.77-1.80 (1H, m), 2.25 (1H, qd, J 12.8 and 4.4), 3.03 (1H, td, J 13.4 and
2.4), 3.50 (1H, app.t), 4.35-4.46 (4H, m), 4.67-4.71 (1H,m), 4.74 (1H, dd, J 14.6 and 4.6), 4.97 (1H, dd,
J 14.6 and 5.4), 5.65 (1H, app.t, NH), 7.15-7.22 (6H, m, Ar), 7.30-7.36 (6H, m, Ar), 7.73 (1H, d, J 2.0,
Ar); 13C NMR (125 MHz, CDCl3) 24.5, 28.4, 35.0, 48.0, 50.4, 51.3, 52.7, 112.0, 119.9, 122.7, 127.2,
127.7, 127.8, 127.9, 128.4, 128.8, 128.9, 132.1, 136.2, 137.8, 144.1, 154.5, 183.2. LC-MS (ESI+) m/z =
475.3 [M+H]+
.
(S)- 2-Benzyl-1-(1-(benzylsulfonyl)piperidin-3-yl)-5-chloro-1H-benzo[d]imidazole 17.
Phenylmethanesulfonyl chloride (12 mg, 60.9 µmol, 1.1 eq) was added to a solution of 15 (18 mg,
55.4 µmol, 1eq) and triethylamine (11.6 µl, 83.1 µmol, 1.5 eq) in DCM (1mL). The mixture was stirred
at RT for 1h, then quenched with water (3mL). The organic layer was separated, dried (MgSO4), and
evaporated. Chromatography (5% DCM, methanol) gave 17 (25 mg, 94%) as a white solid; HPLC
purity (as area %) > 96; 1H NMR (500 MHz, CDCl3) 1.34-1.41 (2H, m), 1.64-1.68 (1H, m), 1.94 (1H, qd,
J 12.6 and 4.2), 2.54 (1H, td, J 12.6 and 2.6), 3.01 (1H, app.t), 3.54 (1H, dd, J 12.3 and 4.6), 3.61-3.64
(1H, m), 4.13 (1H, d, J 14.1), 4.17 (1H, d, J 14.1), 4.19 (1H, d, J 15.9), 4.24 (1H; tt, J 12.0 and 4.1), 4.40
(1H, d, J 15.9), 7.11 (1H, d, J 8.7, Ar), 7.14 (1H, dd, J 8.7 and 1.8, Ar), 7.18-7.23 (3H, m, Ar), 7.27-7.30
(3H, m, Ar), 7.32-7.37 (4H, m, Ar), 7.72 (1H, d, J 1.8, Ar); LC-MS (ESI+) m/z = 480.3 [M+H]+
.
General Procedure E: Benzimidazole Intermediate Synthesis (20)
A mixture 18 (0.3g, 1.41 mmol), sodium dithionite (0.67g, 3.84 mmol) and 19 (1.0 eq.) in methanol
o
(16 mL) and water (4 mL) was heated by microwave for 10 mins at 100 C, then diluted with ethyl
acetate (20 mL), washed with water (20 mL) and brine (20 mL), dried (MgSO4) and evaporatated.
Recrystallisation from ethyl acetate, petrol gave 20.
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General Procedure F: Boc deprotection (21)
A mixture of 20 (0.40 g, 0.94 mmol ), TFA (0.6 mL) and DCM (2 mL) was stirred at rt for 4.5 h, then
concentrated and diluted with methanol (4 mL). K2CO3 (0.2 g) was added and the suspension was
stirred at rt, then filtered, and concentrated. Chromatography (5% DCM, MeOH) gave 21.
Intermediates 21{2},21{3}, 21{4} were used directly in the subsequent step without purification.
General Procedure G: Benzimidazole Synthesis (9)
21 (0.01 g, 0.028 mmol) was added to a solution of the appropriate isocyanate (1.2 eq.) in DCM (2
mL) and the mixture was stirred at rt for 1 h, then concentrated. Chromatography (NH-silica; 2%
MeOH, DCM) gave 9 as glassy solids.
N-(4-Acetylphenyl)-4-(6-chloro-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-2-yl)piperidine-1-
carboxamide 9{2,2}
General Procedure G: 21{2} (0.011 g, 0.029mmol), 4-acetylphenylisocyanate (0.005 g, 0.032 mmol).
9{2,2} 81%;
HPLC purity (as area %) > 99; 1H NMR (400 MHz ,CDCl3) 1.88 (m, 2H, CH2CH), 2.09 (m, 2H,
CH2NCO), 2.49, (s, 3H, COCH3) 2.99 (m, 2H, CH2NCO), 3.07(m, 1H, CH2CH), 4.17 (m, 2H, CH2CH2NCO),
5.31 (s, 2H, CH2Ar), 6.49 (s, 1H, N-H), 6.85 (m, 1H, Ar-H), 7.06 (s, 1H, Ar-H), 7.14 (d, 2H, J 8.37 Hz, Ar-
H) 7.17 (d, 1H, J 1.82 Hz, Ar-H), 7.39 (d, 3H, J 7.91 Hz Ar-H), 7.38 (d, 2H, J 8.85 Hz, Ar-H), 7.81 (d, 1H, J
8.56 Hz, Ar-H);
LC-MS (ESI+) m/z = 519 [M+H]+.
4-(6-Chloro-1-(3-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)-N-(3,4,5-trimethoxyphenyl)piperidine-1-
carboxamide 9{2,3}
General Procedure G: 21{2} (0.05 g, 0.14 mmol), 3,4,5-trimethoxyphenylisocyanate (0.035 g, 0.16
mmol). 9{2,3} 73 %; HPLC purity (as area %) > 92; 1H NMR (400 MHz ,CDCl3) 1.82 (m, br, 2H, CH2CH2-
NH), 2.10 (m, 2H, CH2CH2-NH), 2.99 (m, 3H, CH2-CH2-NH, CH-CH2), 3.73 (s, 3H, Ar-OMe), 3.77 (s, 6H,
Ar-(OMe)2), 4.12 (m, 2H, CH2-N), 5.27 (s, 2H, CH2-N), 6.31 (s, 1H, N-H), 6.59 (s, 2H, Ar-H), 6.72 (d,1H, J
7.21, Ar-H), 7.01 (s, 1H, Ar-H), 7.13 (m, 3H, Ar-H), 7.62 (d, 1H, 8.33 Hz, Ar-H); LC-MS (ESI+) m/z 569
[M+H]+;
Ethyl-4-(4-(6-chloro-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-2-yl)piperidine-1-carboxamido)
benzoate 9{2,8}
General Procedure G: 21{2} (0.011 g, 0.029 mmol), ethyl 4-isocyanobenzoate (0.006 g, 0.032 mmol).
9{2,8} 80%;
HPLC purity (as area %) > 98; 1H NMR (400 MHz ,CDCl3) 1.40 (t, 3H, 7.11 Hz, OCH2CH3),
1.89 (m, 2H, CH2CH), 2.05 (m, 2H, CH2NCO), 3.04 (m, 2H, CH2NCO), 3.08 (m, 1H, CH2CH), 4.19 (m, 2H,
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CH2CH2NCO), 4.37 (q, 2H, J 7.12 Hz OCH2CH3), 5.35 (s, 2H, CH2Ar), 6.58 (s, 1H, N-H), 6.85 (m, 1H, Ar-
H), 7.06 (s, 1H, Ar-H), 7.21 (d, 1H, J 1.82 Hz, Ar-H), 7.38 (m, 3H, Ar-H), 7.43 (d, 2H, J 8.83 Hz, Ar-H),
7.69 (d, 1H, J 8.56 Hz, Ar-H), 7.97 (d, 2H, J 8.77 Hz, Ar-H; LC-MS (ESI+) m/z = 549 [M+H]+.
N-(4-Acetylphenyl)-4-(6-chloro-1-(4-methoxybenzyl)-1H-benzo[d]imidazol-2-yl)piperidine-1-
carboxamide 9{4,2}
General Procedure G: 21{4} (0.097 g, 0.27 mmol), 4-acetylphenylisocyanate (0.074 g, 0.46 mmol).
1
Chromatography (50% EtOAc, petrol), 9{4,2} 55 %
;
HPLC purity (as area %) > 96; H NMR (400 MHz
,CDCl3) 1.82 (m, 2H, CH2CH2-NH), 2.05 (m, 2H, CH2CH2-NH), 2.48(s, 3H, Ar-COCH3) 2.94 (m, 3H, CH2-
CH2-NH and CH-CH2), 3.71 (s, 3H, Ar-OMe), 4.12 (m, br 2H, CH2-N), 5.22 (s, 2H, CH2-N), 6.62 (s, 1H, N-
H), 6.79 (d, 2H, J 8.7, Ar-H), 6.84 (d, 2H, J 8.7, Ar-H), 7.18 (m, 2H, Ar-H), 7.39 (d, 2H, J 8.8, Ar-H), 7.6
(dd, 1H, J 8.1, 1.04, Ar-H), 7.89 (d, 2H, J 8.8, Ar-H).; LC-MS (ESI+) m/z 517 [M+H]+.
RESULTS
Design
The search for MDM2–p53 inhibitors has revealed several classes of small molecules able to bind
MDM2 at the p53 binding site and restore p53 activity.(21) Reflecting the hydrophobic nature of the
MDM2-p53 interaction, all of these compounds are characterized by a central scaffold that directs
hydrophobic substituents towards the three MDM2 sub-pockets, thus mimicking the key amino acid
side-chains of p53.
In an attempt to identify novel chemotypes acting as an anchor point for hydrophobic substituents,
our attention was focused on compounds 1 and 7, taken as representative of two potent and
structurally different classes of MDM2 inhibitors. The scaffold-hopping approach initially matched
the two nitrogens of the Nutlin imidazoline ring 1 with the two nitrogens in the benzimidazole
scaffold (Figure 2, red). Overlay of the Nutlin chlorophenyl rings with the benzimidazole gave two
possible orientations of the chloro group in the scaffold (8 and 9). Positions 1 and 2 of the
benzimidazole ring were used to append the hydrophobic piperidinyl amide substituents judged to
be necessary for activity, resulting in two different substitution patterns, represented by general
structures 8 and 9 (Figure 2).
Docking experiments showed two separate binding modes for the enantiomers of 8{1,6}. In the case
of the (R)-enantiomer the chlorobenzimidazole occupies the Phe19 pocket and the aryl urea fills the
Leu26 pocket, whereas for the (S)-enantiomer the positions are reversed (Figures 3a and 3b,
respectively). The docked pose for 9{1,6} shows the arylurea occupying the Leu26 pocket and the
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benzyl group positioned within the Phe19 pocket (Figure 3c). Interestingly, the Trp23 subpocket is
unoccupied for all of the binding modes generated. This pocket is known to be important from the
published X-ray structures of small-molecule MDM2-p53 inhibitors. The other subpockets (Phe19
and Leu26) were occupied by the hydrophobic moieties of the ligands, thus rendering both libraries
8 and 9 worthy of exploration. For the alternative benzimidazoles 8, the (S)-enantiomers appeared
to be more promising than (R)-enantiomers and therefore were prioritized for synthesis.
Synthesis
The rapid synthesis of benzimidazole libraries has been described recently using a number of
approaches, including solid-phase synthesis,(43, 44) and solution-phase methods.(45-48) In this
case, the required N1-substitution was introduced via an SNAr reaction with an o-fluoronitrobenzene,
followed by in-situ nitro-reduction, using sodium dithionite under microwave heating, and
cyclisation with the desired aldehyde to give the the appropriately substituted benzimidazoles 8 and
20.(49)
The synthesis of benzimidazoles 8 bearing substituted aryl groups required the use of suitably
substituted arylacetaldehydes (10). These were prepared from the respective benzaldehydes using a
Wittig reaction giving the methyl enol ethers (11), which were subjected to acidic hydrolysis (Scheme
1). The SNAr reaction of (S)-12 and 4-chloro-1-fluoro-2-nitrobenzene gave nitroaniline (13) in good
yield. Reductive cyclisation of 13 with sodium dithionite in the presence of arylacetaldehydes 10,
under microwave heating, provided benzimidazoles 14{1-4} cleanly and in good yields. Deprotection
with TFA gave piperidines 15{1-4} which were reacted in parallel with a series of isocyanates to give
the final (S)-benzimidazoles 8{1-4,1-10} (Scheme 2). Single examples of the thiourea 16 and
sulfonamide 17 derivatives were also prepared (Scheme 3).
The synthesis of 2-(piperidin-4-yl)-benzimidazoles 9 required the Boc-protected isonipecotic
aldehyde (18), prepared according to a literature procedure (Scheme 4).(50)
Reaction of 4-chloro-1-fluoro-2-nitrobenzene with the required benzylamine under microwave
heating gave nitroanilines (19). Reductive cyclisation of 19 with sodium dithionite in the presence of
aldehydes 14, under microwave heating gave the benzimidazoles 20{1-4} in good yields.
Deprotection with TFA gave piperidines 21{1-4} which were reacted in parallel with a series of
isocyanates giving the final benzimidazoles 9{1-4,1-10} (Scheme 5).
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Biological evaluation
We have previously demonstrated that the MDM2 and MDMX ELISA assays show good sensitivity
over a wide range of IC50 values. In particular, compounds with low potency and poor predicted
solubility have given reliable results.(40, 41) Compounds from libraries 8 and 9 were assayed against
MDM2 and MDMX in parallel. The results are displayed in Tables 1 and 2. Overall, both series
displayed limited MDM2 inhibition with only 8{4,7}, and 9{2,8} showing sub-100 μM activity (MDM2
LE = 0.14 and 0.15, respectively). In contrast, 27/40 examples of series 8 were sub-100 μM inhibitors
of MDMX, and two examples showed sub-50 μM activity, i.e. 8{3,4} and 8{4,7} (MDMX LE = 0.15).
Series 9 also had a greater number of sub-100 μM MDMX inhibitors (10/30), and four examples
showed sub-50 μM activity, i.e. 9{2,2}, 9{2,3},9{2,8} and 9{4,2} (MDMX LE = 0.15 - 0.16).
Interestingly, the unsubstituted thiourea derivative 16 and the sulfonamide 17 showed improved
MDMX inhibitory activity when compared with the equivalent urea 15{1,6}.
DISCUSSION
Recently, analysis of the contribution to binding of each portion of Nutlin-3 (1) has been reported, by
the synthesis and analysis of fragments of the lead compound.(51) Systematic removal of the groups
accessing each of the three sub-pockets of MDM2 allowed an assessment of their relative
contribution to binding, and showed that removal of the 4-chlorophenyl group accessing the Trp23
pocket resulted in the most significant loss of activity. For example, 22 showed a Kd for MDM2 of 1
mM (LE = 0.10). The activity of libraries 8 and 9 are consistent with the loss of potency associated
with leaving the Trp23 sub-pocket vacant as predicted in the docked binding modes of both series.
Interestingly, the results for MDMX suggest that the Trp23 sub-pocket may play a somewhat less
significant role in the overall binding affinity, as libraries 8 and 9 showed improved activity.
CONCLUSIONS
Libraries of substituted benzimidazoles based on scaffold-hopping from structures 1 and 7 have
been docked into MDM2 and evaluated. The efficient synthesis of two libraries (8 and 9) has been
achieved by microwave-assisted reductive cyclisation. Both libraries were assayed for inhibition of
the MDM2-p53 and MDMX-p53 protein-protein interaction. Members of each library showed sub-
100 μM activity for MDM2 or MDMX and several mixed MDM2-MDMX inhibitors were identified
with modest potency.
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SUPPORTING INFORMATION
Synthetic and analytical details for compounds 8, 13, 14, 15, 16, 19, 20, 21.
ACKNOWLEDGEMENTS
This work was supported by Cancer Research UK and the European Union FP6 (DePPICT, grant
number LSHC-CT-2007-037834). Carlo Bawn and Karen Haggerty for expert technical assistance. Use
of the EPSRC National Mass Spectrometry Service at the University of Wales (Swansea) is gratefully
acknowledged.
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TABLE CAPTIONS
Table 1. MDM2-p53 and MDMX-p53 inhibitory activity of compound libraries 13 and 8.
Table 2. MDM2-p53 and MDMX-p53 inhibitory activity of compound libraries 20 and 9.
FIGURE CAPTIONS
Chart 1: MDM2-p53 clinical trials candidates with disclosed structures.
Figure 1: Structure of the p53-MDM2 complex (1RV1) (MDM2 white, p53 peptide yellow).(15)
Figure 2: Schematic for scaffold hopping and docking approach.
Figure 3: Putative binding modes for compounds: a) 8 (R)-enantiomers; b) 8 (S)-enantiomers; c) 9.
SCHEME CAPTIONS
Scheme 1: Synthesis of substituted phenylacetaldehydes 10.a
a Reagents and Conditions: a) MeOCH2PPh3Br, KOt-Bu, THF, rt; b) HCO2H, DCM, rt.
Scheme 2: Synthesis of benzimidazole library 8.a
a Reagents and Conditions: a) DMF, Na2CO3, 70 oC; b) ArCH2CHO, 13, Na2S2O4, MeOH, H2O, MW, 100
oC; c)i) TFA, DCM, rt; ii) K2CO3, MeOH, rt; d) R’NCO, DCM, rt.
Scheme 3: Synthesis of benzimidazoles 16 and 17.a
a Reagents and Conditions: a) R’NCS, DCM, rt; b) PhCH2SO2Cl, Et3N, DCM, rt.
Scheme 4: Synthesis of piperidine 18.a
a Reagents and Conditions: a) Boc2O, NaOH, dioxane, rt; b) BH3-THF, THF, rt; c) PCC, NaOAc, 4Å
molecular sieves, DCM, rt.
Scheme 5: Synthesis of benzimidazole library 9.a
a Reagents and Conditions: a) ArNH2, K2CO3, EtOH, rt; b) 19, Na2S2O4, MeOH, H2O, MW, 100 oC; c)
i)TFA, DCM, rt; ii) K2CO3, rt; d) R’NCO, DCM, rt.
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Table 1.
Compound
MDM2 IC50
(μM)
MDMX IC50
(μM)
Compound
MDM2 IC50
(μM)
MDMX IC50
(μM)
13{1}
13{2}
13{3}
>500
290 ± 38
>500
248
104 ± 23
>500
8{3,1}
8{3,2}
8{3,3}
8{3,4}
8{3,5}
8{3,6}
8{3,7}
8{3,8}
8{3,9}
8{3,10}
8{4,1}
8{4,2}
8{4,3}
8{4,4}
8{4,5}
8{4,6}
8{4,7}
8{4,8}
8{4,9}
8{4,10}
16
170
-
64 ± 20
-
121 ± 40
120 ± 37
213 ± 158
191
146 ± 63
>500
92 ± 18
43 ± 15
74 ± 12
187
77 ± 20
116
13{4}
211
130
8{1,1}
8{1,2}
8{1,3}
8{1,4}
8{1,5}
8{1,6}
8{1,7}
8{1,8}
8{1,9}
8{1,10}
8{2,1}
8{2,2}
8{2,3}
8{2,4}
8{2,5}
8{2,6}
8{2,7}
8{2,8}
8{2,9}
8{2,10}
183 ± 20
208 ± 33
202 ± 24
176
202 ± 51
218
203 ± 80
196 ± 61
>500
70 ± 19
105 ± 54
54 ± 14
117
92 ± 51
100
61 ± 30
69 ± 5
156
257 ± 146
177
131 ± 48
100
-
-
201 ± 96
115
95 ± 13
207
257
151
169
86
166 ± 55
188 ± 102
188 ± 33
72 ± 20
202 ± 157
198 ± 75
131 ± 45
144 ± 44
212 ± 1
78 ± 16
173 ± 121
109 ± 39
43 ± 24
76 ± 24
99 ± 26
64 ± 13
49 ± 14
59 ± 36
134 ± 86
148 ± 61
201 ± 128
114 ± 38
125 ± 35
154 ± 81
153 ± 112
165 ± 30
103 ± 36
81 ± 33
60 ± 10
154 ± 79
80 ± 13
83 ± 29
57 ± 14
50 ± 6
73 ± 14
63 ± 35
17
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Table 2.
Compound
MDM2 IC50
(μM)
253
MDMX IC50
(μM)
>500
217
>500
>500
>500
-
154 ± 70
94 ± 37
98 ± 62
151
Compound
MDM2 IC50
MDMX IC50
(μM)
-
212
(μM)
-
101
20{1}
20{2}
20{3}
20{4}
21{1}
9{2,9}
9{2,10}
9{3,1}
9{3,2}
9{3,3}
9{3,4}
9{3,5}
9{3,6}
9{3,7}
9{3,8}
9{3,9}
9{3,10}
9{4,1}
9{4,2}
9{4,3}
9{4,4}
9{4,5}
9{4,6}
9{4,7}
9{4,8}
9{4,9}
9{4,10}
248
>500
>500
282
-
-
201
103
182 ± 21
291 ± 117
183
269 ± 39
>500
149 ± 61
218
81 ± 20
252 ± 136
>500
200 ± 100
>500
94 ± 38
123
9{1,1}
9{1,2}
9{1,3}
9{1,4}
9{1,5}
9{1,6}
9{1,7}
9{1,8}
9{1,9}
9{1,10}
9{2,1}
9{2,2}
9{2,3}
9{2,4}
9{2,5}
9{2,6}
9{2,7}
9{2,8}
-
307 ± 132
256 ± 51
141 ± 22
>500
>500
>500
151
>500
204 ± 69
-
231
194
194
218
201
>500
314 ± 65
>500
-
>500
49 ± 22
145
>500
111 ± 63
-
46 ± 11
45
-
-
130 ± 65
243
156 ± 40
-
-
>500
-
180 ± 15
-
245
-
73 ± 16
-
184
113 ± 74
-
-
-
161 ± 68
88 ± 43
68 ± 29
31 ± 10
>500
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Cl
Cl
O
N
N
O
N
HN
O
O
Nutlin-3 (1)
H
N
N
O
O
Cl
F
NH
O
N
Cl
H
MI-63 (2)
HO
O
N
CO2H
Cl
Cl
(3)
N
N
O
O
O
O
Cl
O
SJ-172550 (4)
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N
N
H
N
N
O
Cl
Cl
N
N
(5)
OH
OH
O
N
NH
O
F
F
HN
Cl
O
N
H
RO-5963 (6)
H
Cl
R
N
O
O
N
N
H
(7)
Cl
OCH3
Cl
N
N
O
N
HN
OCF3
8{3,4}
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OCH3
Cl
N
N
O
O
N
OEt
HN
8{4,7}
O
N
N
N
O
HN
Cl
Cl
CH3
9{2,2}
O
OCH3
OCH3
OCH3
N
N
N
HN
Cl
Cl
9{2,3}
O
N
N
N
O
HN
Cl
Cl
OEt
9{2,8}
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O
N
N
N
O
HN
Cl
CH3
H3CO
9{4,2}
Phe19
pocket
Leu23
pocket
O
O
N
N
N
N
S
O
O
22
Cl
Cl
H
O
O
N
S
O O
N
Cl
H
O
N
F
N
Cl
O
F
N
N
OH
NH
CO2H
O
NH
CN
O
O
N
Cl
H
Cl
F
RG7112
RG7388
MI-773
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OCH3
Cl
OCH3
Ar =
{1}
{2}
{3}
{2}
{4}
OCH3
OCH3
OCH3
O
CF3
R' =
NO2
OCF3
{5}
{1}
{3}
{8}
{4}
{9}
O
O
OEt
F
OEt
{6}
{7}
{10}
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