Proapoptotic modification of substituted isoindolinones as MDM2-p53 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.10.049

A series of novel amino acid ester derivatives of 2,3-substituted isoindolinones was synthesized and evaluated for p53-mediated apoptotic activity. The rationale for augmentation of the target activity of 2,3-substituted isoindolinones was based on the introduction of new fragments in the structure of the inhibitor that would provide additional binding sites in the hydrophobic cavity of MDM2. To select for the anticipated modifications we employed molecular docking. Synthesized molecules were evaluated for their ability to induce apoptosis in two cancer cell lines and their derivatives with different status of p53 (colorectal HCT116 and osteosarcoma U2OS cells) by Annexin V staining. The target activity was estimated using high-content imaging system Operetta. Valine and phenylglycine ester derivatives were identified as potentially active MDM2-p53 inhibitors.

Full text of DOI:10.1016/j.bmcl.2017.10.049

Accepted Manuscript
Proapoptotic modification of substituted isoindolinones as MDM2-p53 inhibi-
tors
Tatyana A. Grigoreva, Daria S. Novikova, Alexey V. Petukhov, Maxim A.
Gureev, Alexander V. Garabadzhiu, Gerry Melino, Nickolai A. Barlev,
Vyacheslav G. Tribulovich
PII:
S0960-894X(17)31043-0
https://doi.org/10.1016/j.bmcl.2017.10.049
BMCL 25378
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
6 September 2017
18 October 2017
19 October 2017
Please cite this article as: Grigoreva, T.A., Novikova, D.S., Petukhov, A.V., Gureev, M.A., Garabadzhiu, A.V.,
Melino, G., Barlev, N.A., Tribulovich, V.G., Proapoptotic modification of substituted isoindolinones as MDM2-
p53 inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.10.049
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Proapoptotic modification of substituted
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Tatyana A. Grigoreva, Daria S. Novikova, Alexey V. Petukhov, Maxim A. Gureev, Alexander V. Garabadzhiu,
Gerry Melino, Nickolai A. Barlev and Vyacheslav G. Tribulovich

Bioorganic & Medicinal Chemistry Letters
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Proapoptotic modification of substituted isoindolinones as MDM2-p53 inhibitors
a, ∗
Tatyana A. Grigoreva , Daria S. Novikova , Alexey V. Petukhov , Maxim A. Gureev , Alexander V.
a
a
a,b
a
Garabadzhiu , Gerry Melino , Nickolai A. Barlev and Vyacheslav G. Tribulovich
c
d
a
a
St. Petersburg State Institute of Technology (Technical University), St. Petersburg 190013, Russia
b
I.M. Sechenov First Moscow State Medical University, Moscow 119991, Russia
c
University of Rome Tor Vergata, Rome 00173, Italy
d
Institute of Cytology, Russian Academy of Sciences, St. Petersburg 194064, Russia
ARTICLE INFO
ABSTRACT
Article history:
Received
A series of novel amino acid ester derivatives of 2,3-substituted isoindolinones was synthesized
and evaluated for p53-mediated apoptotic activity. The rationale for augmentation of the target
activity of 2,3-substituted isoindolinones was based on the introduction of new fragments in the
structure of the inhibitor that would provide additional binding sites in the hydrophobic cavity of
MDM2. To select for the anticipated modifications we employed molecular docking.
Synthesized molecules were evaluated for their ability to induce apoptosis in two cancer cell
lines and their derivatives with different status of p53 (colorectal HCT116 and osteosarcoma
U2OS cells) by Annexin V staining. The target activity was estimated using high-content
imaging system Operetta. Valine and phenylglycine ester derivatives were identified as
potentially active MDM2-p53 inhibitors.
Revised
Accepted
Available online
Keywords:
p53
MDM2
protein-protein interaction
computer modelling
apoptosis
2
009 Elsevier Ltd. All rights reserved.
The p53 protein, 'the guardian of the genome', is a tumor
suppressor that regulates the most important processes in the cell
including DNA repair, cell cycle arrest and apoptosis. p53 exerts
its functions as a transcription factor, whose activity and stability
are regulated mostly at the post-transcriptional level by covalent
terminus of the MDM2 protein that implies strict conformational
constrains on the interactor (Fig. 1), rational design can be used
to predict small molecules that mimic this interaction.
6
,7
1
modifications in response to various forms of stress. In the
absence of stress, p53 is maintained at a basal level by its
2
negative regulatory protein MDM2 (murine double minute 2).
The MDM2 protein is an E3 ubiquitin ligase, which promotes the
3
degradation of p53 through ubiquitin-dependent proteolysis. The
p53-MDM2 negative feedback loop plays crucial role in the
regulation of the p53 activity under physiological conditions. It is
therefore not surprising that MDM2 overexpression suppresses
the pro-apoptotic function of p53 and hence promotes
4
uncontrollable proliferation of cancer cells.
In general, the ubiquitin-dependent protein degradation system
is a well-known target for anticancer agents. Of note, several
proteasome inhibitors, including bortezomib, have been approved
5
by FDA and are currently being used in clinics. However, all
Figure 1. An α-helix of p53 in the hydrophobic cavity of MDM2 (PDB
these drugs have severe side effects. Therefore, small molecules
that control specific E3 ligases with high substrate selectivity
provide an attractive alternative approach to the use of
proteasome inhibitors. From this perspective, MDM2 represents
a suitable target for anticancer drug development. Also, since the
α-helix of p53 binds into a relatively deep cleft in the amino-
8
YCR). Three critical amino acids of the p53-MDM2 interaction are denoted
1
with different colors: Phe19 (blue), Trp23 (green), Leu26 (red).
Details of the MDM2-p53 interaction have been extensively
7
studied by X-ray crystallography and NMR spectroscopy. Three
critical amino acid residues Phe19, Trp23, and Leu26 within the
—
——
∗
Corresponding author. Tel.: +7-812-494-9266, +7-911-982-5418; e-mail: rozentatiana@gmail.com

Figure 2. MDM2 inhibitors. Canonical binding to three hydrophobic pockets (Phe19, Trp23, Leu26) is shown for Nutlin-3a; additional binding of carboxyl
group with His96 of MDM2 protein is shown for AM8553; additional π-stacking interaction of phenyl fragment with His96 imidazole moiety of MDM2 protein
is shown for RG7388.
transactivation domain (TAD) of the p53 protein have been
identified. These residues of p53 interact with three hydrophobic
cavities in the MDM2 molecule, called Phe19, Trp23, and Leu26
pockets, respectively. Notably, there are co-crystal structures of
8
MDM2 not only with the α-helix of the p53 protein, but also
number of prospects for additional binding to the hydrophobic
cavity of the protein. Two cis-imidazolines, Nutlin-3a and its
analog modified with hydrophobic terminal CF
3
group,
7
,9
(CF ˗Nutlin), showed significantly higher activity compared to
3
14
the parental compound, seemed the most interesting; a
comparative analysis of their interactions with MDM2 showed
similar mode of posing, which corresponds to the classical
pharmacophore hypothesis of three-point binding along with a
with a number of MDM2 inhibitors, such as compounds of cis-
imidazoline and spiro-oxindole series that were originally
designed to mimic the p53 α-helix and have the same modes of
7
,10
binding to MDM2.
new binding point for CF
Fig. 4).
3
˗Nutlin in the region 56–59 of MDM2
(
Thus, the most widely used pharmacophore model stipulates
the presence of three centers for binding of a small molecule
11
compound to the lipophilic cavity of MDM2. However, the
presence of additional binding sites is suggested to enhance the
activity of MDM2 inhibitors, such as spiro-oxindoles and
pyrrolidones (Fig. 2). These compounds are currently being
evaluated in preclinical trials as highly active MDM2-p53
protein-protein interaction (PPI) inhibitors. For these compounds
the binding to the His96 residue or amino acid residues of the
highly mobile region 1–18 of MDM2 is assumed in addition to
1
2
the classical three-point interaction.
Thus, using computer modeling we sought to study the effect
of introducing an additional binding fragment into the molecule
of a well-known MDM2-p53 inhibitor on the formation of the
binding interface in the cavity of MDM2.
Substituted isoindolinones proposed by the group of Prof.
6
Hardcastle were chosen as templates for subsequent
3
Figure 4. Nutlin-3a (green) and its modified analog CF ˗Nutlin (orange) in
the lipophilic cavity of MDM2. The triad of canonical binding sites is
indicated in dashed yellow circles for both substrates. Additional binding
modifications. Isoindolinones are inferior to the most active
compounds in efficiency, but their structures allow relatively
easy modifications of the molecules to introduce the additional
binding point (Fig. 3).
3
sites for CF -Nutlin are shown in dashed red.
Figure 3. Binding mode of substituted isoindolinones.
To date, the number of small molecule compounds possessing
different ability to inhibit the MDM2-p53 protein-protein
1
3
interaction is accounted for several hundred. Thus, there is the
possibility of computer analysis of a sufficient large chemical
space consisting of compounds belonging to various chemical
classes. Virtual screening of known MDM2 inhibitors showed a
Figure 5. The substituted isoindolinone 4a (green) and its optimized
analog 7a (orange) in the lipophilic cavity of MDM2. The triad of canonical
binding sites is indicated in dashed yellow circles for both substrates, and the
new binding point for 7a is indicated in dashed red.

Thus, the idea of the terminal hydroxyl group modification of
compound 4 by a fragment capable of hydrophobic binding
seemed advantageous. However, an excessive increase in the
lipophilicity of a small molecule compound may lead to a
significant decrease in its solubility. In this regard, we decided to
modify the hydroxyl group through esterification with lipophilic
amino acids, which increased the calculated value of logP by an
acceptable value.
(Table 1). Analysis of the calculated data and ligand binding
poses in the MDM2 cavity showed that the role of new fragments
was to form additional interactions with the 56–59 residues of
MDM2 as in the case of cis-imidazolines (Fig. 4,5). The presence
of a flexible linker between two ether bonds (Fig. 3) provided an
optimal position for both the indole moiety in the ‘canonical’
pockets and the amino acid fragment in the N-terminal region of
MDM2.
6
,17
We performed a comparative molecular docking using three
different PDB models: 1) truncated MDM2 co-crystallized with
Compounds 1-4 were synthesized as described previously.
Compound 2 was obtained by cyclization of benzoylbenzoic acid
(1), and the resulting lactone without isolation was condensed
with the appropriate amines to obtain N-substituted-3-
hydroxyisoindolinones (3). 3-Cl-derivatives were obtained by the
1
Nutlin-3a (17–125 a.a., PDB: 4HG7) ; 2) an MDM2 model
5
containing the N-terminal region co-crystallized with Pip-1
7
inhibitor (1–125 a.a., PDB: 2LZG) , and 3) the same region of
1
6
MDM2 without a ligand (1–118 a.a., PDB: 1Z1M) . These
procedures allowed us deduce the contribution of modifying
groups to the binding with MDM2. The results of the docking
study are shown in Table 1.
2
addition of SOCl , and their further etherification with
appropriate diols in the presence of TEA produced isoindolinone
ethers (4). Acetates (5) were obtained by the reaction with acetyl
chloride in the presence of TEA. Protected amino acid esters (6)
were synthesized by the Steglich esterification with DDC as a
coupling reagent and 4-DMAP as a catalyst. Thermal de-
protection of protected esters (6) was used to obtain final esters
An increase in the calculated target activity (GoldScore) was
clearly observed when these were compared between the original
compound, acetate, α-aminoacetate, and α-amino acid esters
18
19
(
7,8) (Scheme 1).
Table 1. Results of substituted isoindolinone docking to the active site of MDM2.
Structure
GoldScore
1Z1M
Compound
1
2
R
R
4HG7
54.2
2LZG
55.5
4
4
a
–H
–Cl
–H
–Cl
–H
–Cl
–H
–Cl
–H
–Cl
–H
–Cl
–H
–Cl
–H
–Cl
52.2
54.7
53.6
53.9
59.0
61.1
56.1
60.7
64.0
65.7
65.4
59.9
63.2
67.3
60.0
59.9
58.1
b
54.5
53.7
55.3
59.5
60.6
56.6
59.2
65.8
70.1
63.2
61.5
71.8
72.5
66.1
66.3
60.6
56.1
54.5
54.8
65.6
69.1
64.4
67.0
73.1
64.3
66.2
64.4
71.5
70.4
65.3
70.3
56.9
4
c
4d
5a
5b
5
c
5d
7a
7b
7
c
7d
8a
8b
8
c
8
d
Nutlin-3a

Scheme 1. Reagents and conditions: (a) SOCl
2
, DMF, THF; (b) appropriate benzylamine, TEA, THF; (c) SOCl
2
, DMF, appropriate diol, TEA; (d) AcCl,
TEA, THF; (e) N-Boc-protected L-amino acid, CH
2
Cl , DCC, 4-DMAP; (f) silica, 120ºC.
2
The starting structure 4 comprises a quaternary carbon atom,
which is asymmetrically substituted and, therefore, stereogenic.
Thus, such compounds form pairs of enantiomers, which are
divided into diastereomeric pairs after the formation of esters
with optically active amino acids. Notwithstanding, we studied
the pro-apoptotic activity of only racemic mixtures, which were
synthesized under achiral conditions.
Western blot analysis (Fig. 6) confirmed an increase in the
+/+
protein levels of p53 and its target MDM2 in HCT116 p53
cells after 24 hour exposure of cells with the studied compounds
at a concentration of 10µM. In case of the modified compound
8d, expression levels of the p53 and MDM2 proteins were
increased compared to the levels induced by the original
compound 4d.
20
All the resulting isoindolinones were subsequently tested for
their ability to inhibit cell growth as well as to induce apoptosis.
These experiments were performed using human colon cancer
Table 2. Growth inhibition of HCT116 cells with different
р53 status by studied compounds and Nutlin-3a.
Mean GI ± SE(µM)
5
0
+
/+
-/-
Compound
+
/+
-/-
cells HCT116 with different р53 status (p53 and p53 ) and the
reporter U2OS cell line (wild-type p53) that express EGFP
HCT116 p53
9±9
HCT116 p53
90±10
7
11a
4a
plasmid under control of p53
.
4
b
60±8
71±9
50±7
50±6
46±5
38±4
35±4
20±3
16±2
20±2
11±2
14±3
12±2
15±3
8±2
68±9
82±10
65±8
80±9
68±7
70±8
70±8
65±7
60±6
65±6
40±4
43±5
45±4
44±4
38±4
27±4
Inhibitory effects of the compounds on proliferation of cancer
cells were evaluated using the Operetta HCS system (Perkin
Elmer). Following the treatment with the compounds cells were
examined with Invitrogen Click-iT Plus EdU Alexa Fluor 647
Imaging Kit, which allows quantitative measurement of new
DNA synthesis based on incorporation of the nucleoside analog
EdU (5-ethynyl-2'-deoxyuridine) into DNA. Thus, proliferating
cells should accumulate EdU-Alexa Fluor 647 in the nuclei,
which provide fluorescence in red spectrum. In contrast, non-
proliferating cells do not incorporate the dye and therefore can be
visualized only in the UV-spectrum after staining with Hoechst
4
c
4
5
5
d
a
b
5c
5
d
7a
+
/+
-/-
3
3342. Specifically, HCT116 p53 and HCT116 p53 cells
7
b
were treated with compounds for 48 h, and then were analyzed
using the Operetta system. The compounds were used in the
range of 2–100 µM, in 3–4 replicates for each concentration.
Results of the experiment are shown in Table 2. The GI50 values
7c
7
8
8
d
a
b
-
/-
+/+
in the case of p53 cells were higher than those for the p53
cells for all the studied compounds thereby strongly suggesting
that the compounds cause the inhibition of proliferation in a p53-
-
dependent manner. The GI50p53 / GI50p53 ratio was in the
/-
+/+
8
c
range of 1.1–1.3 for the unmodified compounds 4а-4d, which
corresponds to the previously obtained data. For the modified
1
7b
8d
+
/+
compounds 7а-8d a significant decrease in GI50p53 and an
Nutlin-3a
8±2
-
/-
+/+
increase in the GI50p53 / GI50p53 ratio up to 4.75 for 8d were
observed. Collectively, these results point not only to an increase
in the inhibitory activity, but also an improved specificity of the
proliferation inhibition mechanism.

compounds using automated microscopy (e.g. Operetta, Perkin
Elmer).
To study the effect of synthesized compounds we used the
Operetta system. The U2OS p53-EGFP cells were incubated with
compounds for 24 h. The total cell number in each well (Hoechst
3
3342 staining), as well as the number of cells with intense green
fluorescence was then calculated. The proportion of fluorescent
cells to the total number of cells was a criterion for comparing
the ability of the compounds to activate p53.
The known MDM2 inhibitor, Nutlin-3a, and DMSO solution
were used as positive and negative controls, respectively. As
shown in Table 3, the effect of compounds on activation of
EGFP-positive cells was dose-dependent and correlated with the
ability of the known compounds to inhibit MDM2-p53 PPI.
Figure 6. Western blot analysis of HCT116 cells with different р53 status
treated with compounds 8d and 4d.
The treatment of these reporter cells with valine and
phenylglycine esters (7,8) resulted in significant increase in the
percentage of EGFP-positive cells (Table 3).
To confirm the direct involvement of the studied compounds
in the MDM2-p53 PPI inhibition, we conducted an in vitro study
of compounds 4d and 8d using a ImmunoSet p53/MDM2
complex ELISA kit. Compounds were studied at a concentration
of 10µM, Nutlin-3a at a concentration 10µM and DMSO solution
were used as controls. The percentage of the MDM2-p53 PPI
inhibition was 27% for Nutlin-3a, 8% and 26% for compounds
The apoptotic effect of the compounds was confirmed using
21
Annexin V-FITC assay. U2OS cells were treated with the
studied compounds at a concentration of 10 µM for 24 h to
examine the apoptotic effect. The results obtained, as shown in
Fig. 7, suggest that compound 8d is a significantly more potent
pro-apoptotic agent than the parental 4d.
4
d and 8d, respectively.
Table 3. p53 activation by the studied compounds and
Nutlin˗3a in U2OS p53-EGFP cells.
%
of EGFP-positive cells
Compound
5
µM
.1
10 µM
4.0
1
4
4
a
b
1.3
3.8
3.1
1.8
2.1
4.0
4.1
5.8
7.0
7.8
14
4.2
5.0
5.1
4.4
4.7
10
10
16
18
18
33
15
14
17
38
31
4
c
4d
5a
5b
5
c
5d
7a
7b
Figure 7. Proapoptotic effect of 4d, 8d and Nutlin-3a on U2OS cells.
7
c
In sum, the results of this study demonstrate that the correct
extension of the pharmacophore hypothesis can enhance the
specificity and activity of well-known compounds. In our case,
modification of the compounds based on formalized computer
modeling resulted in ten-fold increase in the pro-apoptotic
activity of isoindolinones esterified with amino acids compared
with the original compounds. The biochemical analysis showed
that the newly synthesized compounds, like the original ones,
potently induce apoptosis through the p53 signaling pathway.
The latter was likely activated via inhibition of the MDM2-p53
interaction.
7d
8a
8b
6.1
7.1
8.1
15
8
c
8
d
Nutlin-3a
14
To prove the effect of the studied compounds on the
transcription activity of p53, we used U2OS p53-EGFP reporter
cell line that responds to an increase in p53 activity by
augmented expression of EGFP, whose expression is under
control of p53 response elements, resulting in green fluorescence
of the activated cells. This approach allows high content
screening of sufficiently large libraries of small molecule
The accurate positioning of the lateral substituents of the
modified compounds within the binding cavity of MDM2, which
represents four-point binding mode of PPI inhibitors, could serve
as the basis for further modifications and successful design of
new scaffolds.

Acknowledgments
M. E. M.; Newell, D. R.; Calvert, A. H. WO 2006024837 A1; (d)
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Valeur, E.; Watson, A. F.; Noble, M. US 2011224274 A1.
8. Nadimpally, K. C.; Thalluri, K.; Palakurthy, N. B.; Saha, A.;
Mandal, B. Tetrahedron Lett. 2011, 52, 2579.
This work was financially supported by the Russian Science
Foundation (project №. 16-13-10358).
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1
7. Synthetic procedures for 2,3-substituted isoindolinones (4) also
can be found in: (a) Hardcastle, I. R.; Ahmed, S. U.; Atkins, H.;
Calvert, A. H.; Curtin, N. J.; Farnie, G.; Golding, B. T.; Griffin, R.
J.; Guyenne, S.; Hutton, C.; Kallblad, P.; Kemp, S. J.; Kitching,
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Clegg, W.; Drummond, C.; Endicott, J. A.; Golding, B. T.;
Griffin, R. J.; Gruber, J.; Haggerty, K.; Harrington, R. W.; Hutton,
C.; Kemp, S.; Lu, X.; McDonnell, J. M.; Newell, D. R.; Noble, M.
E.; Payne, S. L.; Revill, C. H.; Riedinger, C.; Xu, Q.; Lunec, J. J.
Med. Chem. 2011, 54, 1233; (c) Willems, H. M. G.; Kallblad, P.;
Hardcastel, I. R.; Griffin, R. J.; Golding, B. T.; Lunec, J.; Nobel,

•
2,3-Substituted isoindolinone-based inhibitors
were successfully modified.
Additional binding to MDM2 contributes to the
effect of new compounds.
New modification results in a tenfold increase in
proapoptotic activity.
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