Synthesis and Biological Evaluation of Novel Dispiro Compounds based on 5-Arylidenehydantoins and Isatins as Inhibitors of p53–MDM2 Protein–Protein Interaction

Article abstract of DOI:10.1007/s10593-020-02726-0

[Figure not available: see fulltext.] A series of novel hydantoin-based dispiroindolinones as potential small-molecule inhibitors of p53–MDM2 protein–protein interaction were synthesized by two methods, using 2-arylidenehydantoins as starting materials. S

Full text of DOI:10.1007/s10593-020-02726-0

DOI 10.1007/s10593-020-02726-0
Chemistry of Heterocyclic Compounds 2020, 56(6), 747–755
Synthesis and biological evaluation of novel dispiro
compounds based on 5-arylidenehydantoins and isatins
as inhibitors of p53–MDM2 protein–protein interaction
1
1
1
Anastasia Beloglazkina , Alexander Barashkin , Vladislav Polyakov ,
1
1
1
1,2
German Kotovsky , Nikita Karpov , Sofia Mefedova , Bogdan Zagribelny ,
2
3
1,4
1
Yan Ivanenkov , Marina Kalinina , Dmitry Skvortsov , Victor Tafeenko ,
1
1,5,6
1
Nikolay Zyk , Alexander Majouga , Elena Beloglazkina *
1
Lomonosov Moscow State University,
1
Build. 3 Leninskie Gory, Moscow 119991, Russia; e-mail: bel@org.chem.msu.ru
2
3
4
5
6
Moscow Institute of Physics and Technology (State University),
Institutskiy Pereulok, Dolgoprudny 141700, Moscow Region, Russia
Skolkovo Institute of Science and Technology,
Alfred Nobel St., Skolkovo 143025, Russia
Faculty of Biology and Biotechnologies, Higher School of Economics,
Vavilova St., Moscow 117312, Russia
National University of Science and Technology MISiS,
Leninsky Ave., Moscow 119049, Russia
D. Mendeleev University of Chemical Technology of Russia,
Miusskaya Sq., Moscow 125047, Russia
9
4
7
9
9
Published in Khimiya Geterotsiklicheskikh Soedinenii,
020, 56(6), 747–755
Submitted February 17, 2020
Accepted March 2, 2020
2
A series of novel hydantoin-based dispiroindolinones as potential small-molecule inhibitors of p53–MDM2 protein–protein interaction
were synthesized by two methods, using 2-arylidenehydantoins as starting materials. Some compounds demonstrate moderate
cytotoxicity against cancer cell lines with IC50 in micromolar concentration range, which is comparable to nutlin-3. Two of the
synthesized dispiroindolinones show p53-related activity in p53 reporter activation test.
Keywords: hydantoins, spirooxindole, anticancer drugs, 1,3-dipolar cycloaddition, p53–MDM2 inhibitors.
During the past few years, an increasing interest for
nonpeptide small-molecule compounds inhibiting the p53–
MDM2 protein–protein interaction of cell proteins has been
tors of p53–MDM2 interaction, p53 gene therapies, and
drugs targeting chaperones, which bind to mutant p53 and
restore its functions.5
1
observed. The p53 protein is a tumor suppressor which
It is known that MDM2 protein has two binding sites (so-
5
plays a key role in cell life cycle and apoptosis, and MDM2
called site 1 and site 2) and both sites are equally probable
2
,3
protein is its endogenous inhibitor. If this protein–protein
interaction is blocked, the released p53 can activate the
process of tumor destruction, which may be used in the
anticancer therapy. In many types of human cancer cell
lines, p53 is mutated and inactivated, which implies the
targets for small-molecule inhibitors. The structural basis
of p53–MDM2 protein–protein interaction, which was
6
established by X-ray crystallography, is primarily related
with three hydrophobic residues Phe19, Trp23, and Leu26
from the R-helix in p53 and a small, but deep pocket in
MDM2. To inhibit their interaction, it is necessary to
design a rigid core structure of small-molecule inhibitor,
which can build-in to the hydrophobic region of MDM2.
4
importance of disabling p53 function. Some strategies
have been used for the correction of p53-related regulatory
pathway dysfunction, for example, small-molecule inhibi-
0
009-3122/20/56(6)-0747©2020 Springer Science+Business Media, LLC
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Chemistry of Heterocyclic Compounds 2020, 56(6), 747–755
The anticancer compound nutlin-3,7 which currently
In this work, we synthesized a series of novel dispiro-
hydantoin-based oxindoles as potential p53–MDM2 inter-
action inhibitors and evaluated their biological activity. Con-
tinuing our project on the discovery of potential p53–MDM2
interaction inhibitors based on 2-thiohydantoins, which
are structural analogs of the nutlin-3, in the present study
we report a procedure for the synthesis of their oxo analogs
via 1,3-dipolar cycloaddition reactions of azomethine
ylides with 5-arylidenehydantoins.
undegoes clinical trials, is one of the most known inhibitors
of the p53–MDM2 protein–protein interaction; this
compound is able to bind to the p53–MDM2 pocket and
inhibit this protein interaction in nanomolar concentra-
1
4
7
tions. Treatment of cancer cells expressing wild-type p53
with nutlin-3a induced nongenotoxic stabilization of p53
protein and subsequent activation of a p53 pathway, which
was translated in vivo to a reduction in xenograft mice
8
tumor. The molecule of nutlin-3a, which definitely binds
For the synthesis of target compounds 4a–m we used two
synthetic routes shown in Scheme 1. Both methods represent
two-step procedures starting from 2-thiohydantoins 1a–m
and involve replacement of sulfur by oxygen and spiro-
cyclization, and the order of the steps may be varied. The
yields of the obtained compounds 4a–m are shown in Table 1.
In the first synthetic approach (method I, Scheme 1), a
series of dispiroindolinones 4a–m were obtained from the
corresponding thiohydantoins 1a–m using the procedure
analogous to the earlier described protocol.14 The first step
is the nucleophilic substitution of sulfur with oxygen in
thiohydantoins 1a–m, achieved by two consecutive
reactions – S-alkylation with MeI followed by hydrolysis in
refluxing aqueous HCl–EtOH mixture. The presence of
hydantoin moiety in the resulting products 2a–e,g–i,k,m
to the site 1 of MDM2 protein, may be a template for the
9
design of new p53–MDM2 inhibitor molecules.
Since the indole ring of Trp23 residue of p53 is located
deep inside a hydrophobic pocket of MDM2 and its NH
group forms a hydrogen bond with the backbone carbonyl
in MDM2, Trp23 appears to be most crucial for binding of
5
,10
p53 to MDM2. Previously,
Wang's group searched for
chemical moieties that can mimic the Trp23 interaction
with MDM2. In addition to the indole ring itself, they have
found that oxindole can perfectly mimic the side chain of
Trp23 for interaction with MDM2 (Fig. 1). The modeling
studies showed that although these compounds fit poorly
into the MDM2 cleft due to steric hindrance, the spiro-
(
oxindole-3,3'-pyrrolidine) core structure may be used as the
1
starting point for the design of a new class of MDM2 inhibi-
tors. The oxindole can closely mimic the Trp23 side chain
in p53 in both hydrogen bond formation and hydrophobic
interactions with MDM2, and the spiropyrrolidine ring
provides a rigid scaffold from which two hydrophobic groups
can be projected to mimic the side chain of Phe19 and Leu26.
The series of compounds (MI-188 and others) designed
by Wang's group11 (Fig. 1) closely mimics p53 in its
interaction with MDM2 and they should have a high
affinity for MDM2. By now, several new small-molecule
inhibitors such as RG7112 (Roche), MI-773 (Sanofi), and
DS-3032b (Daiichi Sankyo) have been developed, which
was confirmed by H NMR spectroscopy. In all cases,
1
H NMR spectra of the synthesized hydantoins 2a–e,
g–i,k,m contained signals at 11.00–12.65 ppm, which are
characteristic for NH protons in 2-arylidenehydantoins. An
alternative previously described procedure, which involves
19
treatment with chloroacetic acid in H
2
O
for replacing
sulfur with oxygen, was not successful in our case. None
of the desired products were detected in reaction mixtures
by LC-MS analysis. At the second step, hydantoins
2a–e,g–i,k,m reacted with sarcosine and isatin in refluxing
EtOH forming target dispiroindolinones 4a–e,g–i,k,m in
4–8 h. This reaction occurs according to the 1,3-dipolar
cycloaddition mechanism. [3+2] Cycloaddition reactions
are convenient and widely used for the synthesis of five-
membered heterocycles containing one to three hetero-
atoms.20
5
are at various stages of phase I clinical trials.
Previously reported small-molecule inhibitors based on
spiroindolinones have IC50 in the range 24.1 nM – 181 μM.12
Presumably, introduction of two spiro rings in the inhibitor
structure1
3–15
may increase the conformational rigidity of
For the synthesis of bromo-substituted dispiroindo-
linones 4f,j,l, we successfully used an alternative synthetic
sequence (method II, Scheme 1). Initially, spiro derivatives
the molecule and, as the result, improve its interaction with
MDM2 protein. Based on single examples of the dispiro
derivatives presented in the literature, they inhibit the
p53–MDM2 interaction with IC50 values in the range of
2
1,22
of 2-arylidenethiohydantoins 3f,j,l
were prepared, and
then they were S-alkylated and hydrolyzed to yield desired
2-arylidenehydantoin dispiro compounds 4f,j,l.
1
6–18
0
.001–0.05 μM (GI50 <0.1 mM in the MTT test).
Figure 1. The logical transition from tryptophan to oxindole fragment and further to the compounds of the MI series and the compounds
studied in this work (IC50 values are given for human prostate adenocarcinoma cell line (LNCap)).
7
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Chemistry of Heterocyclic Compounds 2020, 56(6), 747–755
Scheme 1. Synthesis of target dispirooxindoles 4a–m
ylide and dipolarophile in other approaches.23,24 Thus, exo
R1
approach of azomethine ylide to dipolarophile C=C double
bond is realized.
Method I
O
N
i
iii
The structures of compounds 4a–m were established
O
1
13
N
H
based on H and C NMR spectroscopy and mass spectro-
1
metry data. For all products, H NMR spectra showed the
R1
N
Ar
R2
R1
N
signals of indolinone NH protons at 10.60–10.85 ppm, NH
protons of 2-arylidenehydantoin moiety at 8.02–8.64 ppm,
aromatic protons as multiplets at 6.85–7.96 ppm, characte-
ristic pseudo-triplets of three CH protons of pyrrolidine
cycles at 4.09–4.61, 3.79–4.20, and 3.37–3.46 ppm, as well
O
2a–e,g–i,k,m
O
S
O
N
H
NH
Ar
Br
R1
N
Ar
HN
N
O Me
4a–m
1
a–m
O
as the signal of NCH protons at 2.08–2.19 ppm. ESI
3
S
HRMS spectra of compounds 4a–m confirmed the compo-
sition of the synthesized products. Notably, all dispiro
derivatives were isolated from reaction mixtures as single
diastereomers with (2'R*,4S*,4'S*) relative configurations.
ii
i
NH
Ar
HN
Method II
N
O Me
1
13
3
f,j,l
For compound 4d, COSY, NOESY, and Н– С HSQC
i: 1. MeI, 2% KOH in EtOH, rt, 0.5 h; 2. EtOH–HCl (1:1), , 6 h
spectra were recorded (Supplementary information file). In
1
ii: 5-Bromoisatin, sarcosine, EtOH, , 6 h
the H NMR spectrum of compound 4d, there are three
iii: Isatin, sarcosine, EtOH, , 8 h
signals of the pyrrolidine cycle protons at 4.61, 4.20, and
3
.43 ppm as characteristic pseudo-triplets (Supplementary
Table 1. Yields of target dispirooxindoles 4a–m
information file, Fig. S26). In the COSY spectrum of
compound 4d, the cross peaks are observed, indicating
pairwise interaction of protons at 4.61, 4.20, 3.43 ppm with
each other with the similar coupling constants (Supple-
mentary information file, Fig. S27). For determination
of the relative position of protons and carbon atoms,
Compound
R1
R2
Ar
4-ClC
Ph
Method
Yield, %
56
4
a
H
H
6
H
4
I
I
4
b
Ph
H
68
4
c
Ph
H
2-BrC
2-ClC
6
H
4
I
93
1
13
H– C HSQC correlation spectroscopy was used (Supplemen-
tary information file, Fig. S28). As this experiment demon-
strates, for the proton with a chemical shift of 4.61 ppm,
cross peak with the carbon atom at 44.8 ppm is observed,
and for the proton at 4.20 and 3.43 ppm, cross peak with
the carbon of 55.5 ppm is also observed. Thus, proton at
4
d
Ph
H
6
H
4
I
97
4
e
Ph
H
4-FC
4-BrC
Ph
6
H
4
I
78
4
f
Ph
Br
H
6
H
4
II
I
72
4
g
4-ClC
6
6
H
4
75
4
h
4-ClC
H
4
H
4-MeOC
6
H
4
I
82
4
.61 ppm is located at position 4 of the pyrrolidine cycle,
4
i
4-MeOC
4-EtOC
3-Cl-4-FC
6
H
4
H
4-ClC
6
6
H
4
4
I
39
and the other two protons are at position 5 of the cycle. To
see an interaction of the protons at position 5 of pyrrolidine
cycle with the sides of the diastereotope surface, NOESY
NMR spectroscopy was used (Supplementary information
file, Figs. S29–S31). When the proton at 3.43 ppm was
irradiated at its resonant frequency, the responses of the
NH protons of thiohydantoin cycle and indole fragment
were observed (Supplementary information file, Fig. S29).
Therefore, these protons are located from the same side of
the pyrrolidine cycle plane. The other two protons of the
pyrrolidine fragment have a weak correlation with each
other (Supplementary information file, Fig. S28, S29),
which means they are located on the same side of the
central cycle of the molecule. Thereby, the proton at 3.43 ppm
is located on the opposite side of the cycle from protons at
4.20 and 4.61 ppm, which differ greatly among themselves
4
j
6
H
4
Br
H
4-ClC
H
II
I
56
4
k
6
6
6
H
H
H
3
4-EtOC
4-ClC
4-MeOC
6
H
4
43
4
l
3-Cl-4-FC
3-Cl-4-FC
3
3
Br
H
6
H
4
II
I
51
4
m
6
H
4
86
The mechanism of spirocyclization reactions is
presumably similar to that for the addition of azomethine
1
5
ylides to N-substituted 5-arylidenethiohydantoins and
1
,3-oxazolones.13 It involves successive reactions of isatin
or 5-bromoisatin with sarcosine to form an iminium
intermediate and then a lactone which loses the CO
2
molecule to afford dipole (Scheme 2). This dipole regio-
and diastereoselectively attacks the C=C double bond of
5
-arylidenehydantoin. Note that the attack of dipole can
1
13
proceed from both above and below of the double bond
plane to result in a mixture of enantiomers. Possible ways
of azomethine ylide approach to dipolarophile moiety are
shown in Scheme 2.
according to H– C HSQC. Thus, using the methods of
one-dimensional and two-dimensional NMR spectroscopy,
it was possible to establish the assignment of protons to
their signals in the NMR spectrum, as well as to prove the
stereochemical structure of the obtained compound.
Based on the structure of the products, we can conclude
that cycloaddition reaction takes place through the exo-
transition state (Scheme 2), possibly due to an increase in
the transition state energy as a result of electrostatic
repulsion of the cis-located carbonyl groups of azomethine
Molecular structure of compound 4l was also confirmed
by X-ray crystallography (Supplementary information file,
Fig. 2, Table S1), which showed the formation of the
diastereomer with (2'R*,4S*,4'S*) relative configuration.
7
49

Chemistry of Heterocyclic Compounds 2020, 56(6), 747–755
Scheme 2. Possible approaches of azomethine ylide dipole to 5-arylidenehydantoin in the 1,3-dipolar cycloaddition reactions
leading to the possible formation of various diastereomeric products (the actual reaction pathway is shown in blue)
O
O
H2C
Me
O
O
N
Me
N Me
O
O +
N
H
COOH
–
CO2
N
H
N
H
N
H
HN
endo
exo
H
N
O
O
N
CH2
Me
N
CH2
Me
H
N
H
O
O
N
N
N
O
O
H
O
N
HN
Me
N
O
CH2
N
CH2
Me
exo
endo
NH
NH
N
H
N
O
Me
H
H
N
H
N
O
Me
O
N
O
O
O
N
Me
O
Me
O
N
N
N
O
N
O
N
O
N
O
NH
NH
O
O
N
O
N
O
N
N
O
O
O
O
HN
HN
HN
HN
NH
NH
N
N
Me
N
N
Me
O
O
O
O
N
H
Me
Me
N
H
exo
exo
endo
endo
The neighboring spiro-conjugated cycles are almost planar
and near perpendicular to each other. The unit cell in the
crystal of compound 4l contains two enantiomeric
molecules in P1 space group with the inversion center.
When 5-bromoisatin was used instead of unsubstituted
isatin in [3+2] cycloadditions with 5-arylidenehydantoins
2
2
a–e,g–i,k,m (R ≠ H), we could not obtain the desired
dispiro products, and only starting materials were isolated
from the reaction mixtures. This result can be rationalized
by steric hindrances between 1,3-dipole and N-substituted
arylidenehydantoin arising in the transition state that would
lead to product formation (Scheme 3). In this case, isatin
bromine atom in the transition state should be located close
1
to R substituent at the N(3) atom of hydantoin, not allowing
the reagents to approach each other. The significance of
1
steric bulk introduced by R group is supported by the fact
Figure 2. Molecular structure of compound 4l with atoms repre-
sented as thermal vibration ellipsoids of 40% probability.
1
that the plane of benzene ring of R in hydantoin molecules
7
50

Chemistry of Heterocyclic Compounds 2020, 56(6), 747–755
Scheme 3. Steric hindrances arising upon the convergence of the
Cl
NH2
i-PrO
1
,3-dipole and N-substituted arylidenehydantoin
N
OMe
NH
H
N
N
O
O
t-Bu
N
Cl
O
F
HN
N
H
4JVR
Cl
Cl
O
Nutlin-3a
4HG7
is almost perpendicular to the plane of the imidazolone ring
Figure 3. Nutlin-3a and compound 4JVR.
25
(
as we reported earlier and Scheme 3).
In this case, for the synthesis of bromo-substituted
dispiroindolinones 4f,j,l, we have successfully used an
alternative synthetic method II (Scheme 1).
4JVR protein (Fig. 4A), namely: (а) hydrogen bond
between 2-indolinone fragment and Leu54 amide carbonyl
group; (b) hydrogen bond between 2-oxo group of
hydantoin scaffold and His96 along with π-stacking
between ligand ring and His96 residue. Secondly, the score
functions of thio derivatives 3l and 3i demonstrated that
their binding to the active site is slightly less preferable
than for oxo analogs. Moreover, it should be noted that the
binding mode of hydantoins 4l and 4i significantly differs
from that of the thio analogs and does not well correlate
with reference data (Fig. 4D–F): during the docking
procedure, no key interactions were observed. This fact
could be explained by the larger size of the sulfur atom in
thiohydantoin moiety in contrast with the oxygen atom in
hydantoin analog, which makes difficult for thiohydantoins
3l and 3i to entry in the hydrophobic pocket of MDM2 and
interact with His96. In all orientations, the sulfur atom of
compounds 3l and 3i is positioned "out-of-pocket" (Fig. 4D),
while orientations performed for oxo derivatives 4l and 4i
showed "in-pocket" positioning for both oxygen atoms. The
repulsion of sulfur atom from MDM2 binding mode seemed
to be crucial for the "right" orientation that we can observe in
4JVR X-ray diffraction data. Thus, according to molecular
Thus, two alternative methods for the preparation of the
target dispiroindolinones 4a–m, differing in the sequence
of the stages of cycloaddition and replacement of the exo-
cyclic sulfur atom with oxygen have been developed. In
general, method I with the initial preparation of hydantoin
and its further introduction into spirocyclization seems to
be more convenient, allowing purification of products by
recrystallization at all stages of the synthesis. However, this
method is not applicable using 5-bromoisatin, and in this
case, the use of the method II with the initial
spirocyclization and the further replacement of the thio-
hydantoin fragment sulfur with oxygen may be rational,
although, the intermediate dispirothiohydantoin sometimes
requires purification by column chromatography.
1
8
Considering the literature data we proposed that
stability and pharmacokinetic profile of dispiro derivatives
based on 5-arylidenehydantoins would be enhanced in
comparison to their thiohydantoin analogs. On the basis of
the molecular docking data, 2-oxotetrahydro-4H-imidazol-
4
-one derivatives could be effective inhibitors of the p53–
MDM2 protein–protein interaction.
More than 50 crystallographic complexes obtained for
various small-molecule p53–MDM2 inhibitors, including
spiroindolinones, are available from PDB (Protein Data
Bank). To elucidate the possible binding affinity of the
target compounds toward MDM2, a static 3D molecular
docking study was performed using the ICM-Pro software
based on X-ray analysis data, including compounds 4HG7
(
nutlin-3a) and 4JVR, a compound structurally similar
to those investigated in this paper, as known p53 ligand
Fig. 3).
Water molecules were removed from the site, and the
(
reference compounds were docked into the constructed model
starting from 2D structures. The obtained results correlate
with the X-ray data (Fig. 4A,B). Compounds 4l and 4i were
then docked into the static pocket using an extensive range
of key force fields, particularly describing hydrophobic
interactions (Fig. 4B,C). Compounds 4l and 4i were
compared with previously described thiohydantoin analogs
Figure 4. Molecular docking of 4JVR ligand and compounds 3i,l
and 4i,l into hydrophobic MDM2 binding pocket. (A) 3D
structure of 4JVR (X-ray data) in hydrophobic MDM2 binding
10
pocket (yellow dashed line – H bond; blue dashed line –
π-stacking; green dashed circle – "in-pocket" positioning; yellow
dashed circle – "out-of-pocket" positioning); (B) The orientation
of compound 4i; (C) The orientation of compound 4l; (D) The
orientation of compound 3l with "out-of-pocket" conformation of
14
3
l and 3i; an extra docking procedure for the similar oxo
and thio derivatives was performed and analyzed.
First of all, it could be noted, that key interactions of
hydantoins 4l and 4i with MDM2 pocket (Fig. 4B,C) are
the same as presented in X-ray data for reference ligand in
2
-sulfur atom; (E) The orientation of compound 3i; (F) The
orientation of compound 3l.
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Chemistry of Heterocyclic Compounds 2020, 56(6), 747–755
docking study, dispiroindolinones based on hydantoin core
Table 2. Cytotoxicity (IC50, µM) and p53 reporter activation test
for the synthesized compounds 4a–m
should have better binding affinity to MDM2.
against different cancer cell lines
Further, the cytotoxicity of the synthesized dispiro-
indolinones 4a–m on the selected model cell lines was
investigated. This study was realized using breast cancer
MCF7, human lung carcinoma A549, human embryonic
kidney HEK293T, and lung fibroblast VA13 cell lines.
Cytotoxicity of the tested compounds was assessed using
the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
Cancer cell lines
p53 reporter
activation
test
Com-
pound
MCF7
19.3 ± 1.4 21.0 ± 1.4 12.6 ± 1.3 24.3 ± 2.0
≥100 88.6 ± 9.4 23.6 ± 1.2 ≥100
47.3 ± 3.3 39.6 ± 2.3 28.8 ± 6.2 54.2 ± 4.8
70.1 ± 5.9 55.1 ± 4.5 35.4 ± 2.3 ≥100
24.1 ± 4.1 6.6 ± 1.6 9.2 ± 0.5 11.9 ± 2.5
≥100 ≥100 ≥100 ≥100
20.0 ± 2.3 14.1 ± 1.2 15.8 ± 1.2 47.3 ± 4.6
≥100 ≥100 ≥100 ≥100
A549
HEK293T
VA13
4
a
2.5
na*
na
4b
4
c
2
6
zolium bromide) assay, all compounds were additionally
tested for the p53 activation on a transcriptional reporter.27, 28
4d
1.7
na
4
e
The results of these assays are shown in Table 2, along
with the results obtained for nutlin-3 as known inhibitor of
p53–MDM2 interaction and cisplatin as a cytotoxic drug
that does not act by the p53–MDM2 inhibition mechanism.
The most cytotoxic compound 4l demonstrates high
cytotoxicity with IC50 value in the range of 1.3–4.7 µM
against all cell lines (Table 2). However, no relevant
selectivity was observed for the most active compounds
among the cell types. Four compounds 4e,i,j,l showed
moderate selectivity against A549 cell line with IC50 value
in the range of 1.3–7.5 µM. It should be noted that
hydantoins 4j–l showed higher cytotoxicity against
HEK293T and MCF7 cell lines compared to corresponding
4f
na
4
g
2.3
na
4
h
4i
18.4 ± 5.3 7.5 ± 2.0 14.3 ± 5.0 20.8 ± 5.3
6.6 ± 0.6 4.9 ± 0.3 6.3 ± 0.6 13.3 ± 0.8
7.7 ± 0.6 11.9 ± 0.8 8.1 ± 0.6 16.2 ± 1.6
na
4
j
na
4
k
1.7
1.6
na
4
l
3.9 ± 0.3 1.3 ± 0.1 3.0 ± 0.1
4.7 ± 0.3
4m
Nutlin-322 14.9 ± 0.6 10.4 ± 0.8
58.2 ± 12.8 41.8 ± 6.4 35.9 ± 2.6 33.5 ± 2.7
N/A**
Cisplatin 7.1 ± 0.8 4.1 ± 0.6 4.3 ± 0.9
N/A
6.9
2.1
2.2 ± 0.3
14
thio analogs. For example, compound 4j has IC50 equal to
*
*
na – not active compound.
* N/A – not applicable.
6
6
.3 ± 0.6 µM at HEK293T cell line and IC50 equal to
.6 ± 0.6 µM at MCF7 cell line, while its thio analog 3j has
IC50 equal to 28.61 ± 8.68 µM and 34.9 ± 6.4 µM,
14
respectively. Compound 4l has IC50 equal to 3.0 ± 0.1 µM
at HEK293T cell line and IC50 equal to 3.9 ± 0.3 µM at
MCF7 cell line, whereas its thio analog 3l has IC50 equal to
In general, we assume that the observed data of
biological activity could be rationalized in two ways. High
cytotoxicity of compounds 4j,l with simultaneous absence
of p53 activation effect may be the result of two parallel
effects: the activation of p53 and other mechanisms that
affects the general cytotoxicity. The second possible
explanation is that weak p53 activation is a consequence of
other types of activities that cause p53 expression. Thus, a
high cytotoxicity, but the absence of p53 activation for
compounds 4j,l is more a consequence of the general toxic
effect of the molecule by a p53-independent mechanism
than the competitive processes of p53 activation.
Apparently, p53 protein is not the only target of the
cytotoxic effect of the compounds obtained. Although
according to the results of molecular docking they are
capable of fitting into the pocket of MDM2, they obviously
do not possess the ability of direct p53 activation, like
nutlin. However, the resulting hydantoin-based dispiro-
indolinones have higher toxicity than previously obtained
oxazolone analogs,13 and some ability to activate p53,
1
5.62 ± 3.34 µM and 15.52 ± 2.31 µM, respectively.14
The synthesized compounds were also tested for
28
activation of p53 in transcriptional reporter activation test
(Table 2). Nutlin, a known inhibitor of p53–MDM2
interaction was used as a positive control. We proceeded
from the assumption that the mechanisms of the cyto-
toxicity of the studied compounds may include the p53
activation. It is known that the activated p53 induces the
transcription of MDM2, which can directly interact with
transactivation domain of p53, thereby inhibiting its
transcription activity by targeting it for polyubiquitination
2
9
and further proteasome-mediated degradation. In many
cancer cells lines, including HEK293T, MCF7, and A549,
the overexpression of MDM2 is actually observed,
1
4
resulting in significant apoptosis attenuation. Nutlin-3a
causes direct activation of p53 via the block of p53–MDM2
interaction and elevates p53 activity up to five times in the
6
14
reporter assay. Treatment of the cells with cisplatin causes
which was not observed at all for thiohydantoin analogs.
DNA damage that leads to p53 activation indirectly due to
regulatory cascades: it causes about twofold p53 activation
in the reporter-based experiments.3 In our case, two
compounds 4j,l showed higher in vitro cytotoxicity against
MCF7 and A549 cell lines compared with nutlin-3a and
cisplatin. The treatment of the cells with compounds 4a,g
leads to the twofold p53 activation; it is less than for
nutlin-3a, but comparable with indirect cisplatin action,
which suggests that the investigated compounds have
In summary, a series of novel dispiroindolinones based
on 2-arylidenehydantoins were synthesized starting from
5-arylmethylene-2-hydantoins, isatins, and sarcosine using
two alternative methods. Some of the obtained dispiro-
indolinones exhibit moderate anticancer activity against
A549 cell lines in micromolar concentrations. Two dispiro-
indolinones show moderate p53 activation in p53 reporter
activation test. Cytotoxicity of the obtained dispiroindo-
linones based on 5-arylmethylene-2-hydantoins is higher
than the cytotoxicity of its previously studied thio analogs.
0
6
indirect p53-dependent mechanism of action.
7
52

Chemistry of Heterocyclic Compounds 2020, 56(6), 747–755
Experimental
4'-(2-Bromophenyl)-1'-methyl-1-phenyldispiro[imid-
azolidine-4,3'-pyrrolidine-2',3''-indoline]-2,2'',5-trione
1H and 13C NMR spectra and 2D NMR experiments
were recorded on a Bruker Avance 400 spectrometer (400
and 100 MHz, respectively) in DMSO-d
(4c). Yield 480 mg (93%, method I), white solid, mp 206–
1
208°C. H NMR spectrum, δ, ppm (J, Hz): 10.60 (1Н, s,
6
, TMS was used as
NH indolinone); 8.02 (1Н, s, NH imidazolidine); 7.64 (1H,
d, J = 7.5, H Ar); 7.51–7.27 (9H, m, H Ar); 7.03 (1H, t,
J = 8.0, H Ar); 6.85 (2H, d, J = 7.5, H Ar); 4.61 (1H, t,
J = 8.7, H pyrrolidine); 4.13 (1H, t, J = 8.7, H pyrrolidine);
internal standard. High-resolution mass spectra were recorded
on an Orbitrap Elite mass spectrometer (ESI). Melting points
were measured on a Stuart melting point apparatus SMP11 in
open capillaries and are uncorrected. TLC analysis was
carried out on silica gel plates Silufol UV254.
3.46 (1H, t, J = 8.1, H pyrrolidine); 2.14 (3H, s, CH ).
3
13
C NMR spectrum, ppm: 175.8; 173.1; 163.1; 154.2;
All reagents and solvents were reagent grade or were
144.0; 135.6; 133.0; 132.4; 131.9; 130.4; 130.3; 129.3;
128.9; 128.1; 126.9; 126.4; 124.1; 123.7; 121.8; 110.0;
25
purified by standard methods before use. Compounds 1a–m,
3
1
14
2
a–c,e–f,h, 3j,l
were prepared according to the
107.2; 77.0; 73.5; 56.4; 47.3; 34.8. Found, m/z: 517.0877
+
previously reported procedures. The synthesis of com-
[M+H] . C26
H
22BrN
4
O . Calculated, m/z: 517.0870.
3
31
14
pounds 2b,g,i–l and 3f,i,k was carried out analogously
to the previously reported procedures (described in
Supplementary information file).
4'-(2-Chlorophenyl)-1'-methyl-1-phenyldispiro[imid-
azolidine-4,3'-pyrrolidine-2',3''-indoline]-2,2'',5-trione
(4d). Yield 458 mg (97%, method I), white solid, mp 225–
1
Synthesis of compounds 4a–m (General procedure).
Method I. Sarcosine (2 mmol) and isatin (2 mmol) were
added to a stirred and refluxed solution of the respective
compound 2a–e,g–i,k,m (1 mmol) in EtOH (10 ml). The
resulted solution was refluxed for 8 h. After cooling to
room temperature, formed precipitate was filtered off and
washed with EtOH.
227°C. H NMR spectrum, δ, ppm (J, Hz): 10.63 (1Н, s,
NH indolinone); 8.15 (1Н, s, NH imidazolidine); 7.96 (1H,
d, J = 7.5, H Ar); 7.49–7.27 (9H, m, H Ar); 7.04 (1H, t,
J = 7.5, H Ar); 6.88–6.81 (1H, m, H Ar); 4.62 (1H, t,
J = 9.0, H pyrrolidine); 4.20 (1H, t, J = 9.0, H pyrrolidine);
3.44 (1H, t, J = 8.5, H pyrrolidine); 2.15 (3H, s, CH ).
3
13
C NMR spectrum, δ, ppm: 175.8; 173.3; 154.2; 144.0;
Method II. MeI (1 equiv) was added to a solution of
compound 3f,j,l (1 equiv) in 2% KOH in EtOH (10 ml),
and the reaction mixture was stirred at room temperature
for 30 min. The precipitate formed was filtered off and
dried on air. Then the resulting salt was dissolved in EtOH–
HCl, 1:1 mixture (10 ml) and refluxed for 6 h. After
cooling, the precipitate was filtered off and dried on air.
134.8; 133.8; 131.5; 131.3; 130.3; 129.1; 129.0; 128.9
(2C); 128.1; 127.6; 126.9; 126.5; 126.3; 123.7; 121.8;
109.9; 77.0; 73.5; 55.5; 44.8; 34.8. Found, m/z: 473.1375
+
[M+H] . C26
H
22ClN
4
O . Calculated, m/z: 473.1375.
3
4'-(4-Fluorophenyl)-1'-methyl-1-phenyldispiro[imid-
azolidine-4,3'-pyrrolidine-2',3''-indoline]-2,2'',5-trione
(4e). Yield 355 mg (78%, method I), white solid, mp 232–
1
4
'-(4-Chlorophenyl)-1'-methyldispiro[imidazolidine-
234°C. H NMR spectrum, δ, ppm (J, Hz): 10.61 (1Н, s,
4
,3'-pyrrolidine-2',3''-indoline]-2,2'',5-trione (4a). Yield
NH indolinone); 8.43 (1Н, s, NH imidazolidine); 7.46 (2H,
t, J = 9.5, H Ar); 7.37 (2H, d, J = 8.3, H Ar); 7.29 (2H, t,
J = 8.3, H Ar); 7.05–6.99 (2H, m, H Ar); 6.91–6.83 (5H,
m, H Ar); 4.20 (1H, t, J = 8.7, H pyrrolidine); 3.91 (1H, t,
J = 8.7, H pyrrolidine); 3.40 (1H, t, J = 7.9, H pyrrolidine);
2
22 mg (56%, method I), white solid, mp 216–218°C.
1
H NMR spectrum, δ, ppm (J, Hz): 11.72 (1Н, s, NH
indolinone); 10.68 (1Н, s, NH imidazolidine); 9.74 (1H, s,
H Ar); 7.52 (1H, s, H Ar); 7.45 (1H, d, J = 7.7, H Ar); 7.41–
13
7
.36 (4H, m, H Ar); 6.77 (1H, d, J = 7.7, H Ar); 4.16 (1H,
2.12 (3H, s, CH ). C NMR spectrum, δ, ppm: 176.3;
3
t, J = 9.5, H pyrrolidine); 3.81 (1H, t, J = 9.5,
169.5; 159.1; 155.4; 144.2; 131.5; 130.2; 129.3; 128.5;
127.0; 126.7; 126.2; 122.2; 114.1; 110.5; 76.3; 76.0; 74.8;
H pyrrolidine); 3.41 (1H, t, J = 8.9, H pyrrolidine); 2.10
13
(
3H, s, CH
3
). C NMR spectrum, δ, ppm: 176.9; 172.0;
55.5; 55.4; 54.5; 49.8; 45.4; 36.5; 35.2; 26.5. Found, m/z:
+
1
1
2
57.0; 144.1; 135.1; 132.1; 131.4; 130.3; 128.6; 126.2;
25.8; 125.0; 122.3; 110.2; 107.3; 76.8; 54.3; 45.3; 36.2;
456.1596 [M+H] . C26
H
22FN
4
O . Calculated, m/z: 456.1598.
3
5''-Bromo-4'-(4-bromophenyl)-1'-methyl-1-phenyl-
dispiro[imidazolidine-4,3'-pyrrolidine-2',3''-indoline]-
2,2'',5-trione (4f). Yield 410 mg (72%, method II), white
+
6.4. Found, m/z: 397.0991 [M+H] . C20
H
18ClN
4
O .
3
Calculated, m/z: 397.1062.
'-Methyl-1,4'-diphenyldispiro[imidazolidine-4,3'-
pyrrolidine-2',3''-indoline]-2,2'',5-trione (4b). Yield 298 mg
1
1
solid, mp 251–253°C. H NMR spectrum, δ, ppm (J, Hz):
10.87 (1H, s, H indolinone); 9.05 (1H, s, H imidazolidine);
1
1
2
(
68%, method I), white solid, mp 221–223°C. H NMR
spectrum, δ, ppm (J, Hz): 10.62 (1Н, s, NH indolinone);
.14 (1Н, s, NH imidazolidine); 7.96 (1H, d, J = 7.9,
7.50 (1H, dd, J = 8.2, J= 2.0, H Ar); 7.42 (4H, br. s,
H Ar); 7.25 (1H, d, J = 2.0, H Ar); 6.88–6.83 (4H, m,
H Ar); 6.37 (1H, s, H Ar); 6.34 (1H, s, H Ar); 3.98 (1H, t,
J = 6.9, H pyrrolidine); 3.89 (1H, t, J = 8.9, H pyrrolidine);
8
H Ar); 7.39–7.27 (9H, m, H Ar); 7.03 (1H, t,
J = 7.2, H Ar); 6.84 (1H, d, J = 8.2, H Ar); 6.80 (2H, d,
J = 7.0, H Ar); 4.25 (1H, t, J = 9.5, H pyrrolidine); 3.98
3.44 (1H, t, J = 9.2, H pyrrolidine); 2.18 (3H, s, CH ).
3
13
C NMR spectrum, δ, ppm: 175.1; 172.5; 153.6; 142.7;
(
1H, t, J = 9.5, H pyrrolidine); 3.43 (1H, t, J = 9.1,
135.0; 132.8; 132.2; 131.4; 131.2; 129.4; 128.9; 128.3;
1
3
H pyrrolidine); 2.14 (3H, s, CH
3
). C NMR spectrum,
126.6; 126.3; 120.8; 113.5; 111.9; 98.4; 77.3; 74.4; 57.1;
+
δ, ppm: 175.7; 172.6; 153.7; 143.4; 135.8; 131.3; 130.0;
49.2; 34.8. Found, m/z: 593.9904 [M+H] . C26
Calculated, m/z: 593.9902.
H
21Br
2
N
4
3
O .
1
1
4
29.8; 128.9; 128.6; 128.1; 127.4; 127.0; 126.3; 124.3;
21.6; 109.8; 77.4; 74.5; 66.4; 57.2; 50.1; 34.7. Found, m/z:
1-(4-Chlorophenyl)-1'-methyl-4'-phenyldispiro[imid-
azolidine-4,3'-pyrrolidine-2',3''-indoline]-2,2'',5-trione
+
39.1757 [M+H] . C26
H
23
N O3. Calculated, m/z: 438.1765.
4
7
53

Chemistry of Heterocyclic Compounds 2020, 56(6), 747–755
(
4g). Yield 355 mg (75%, method I), white solid, mp 211–
2,2'',5-trione (4k). Yield 230 mg (43%, method I), white
1
1
2
13°C. H NMR spectrum, δ, ppm (J, Hz): 10.62 (1Н, s,
solid, mp 219–221°C. H NMR spectrum, δ, ppm (J, Hz):
NH indolinone); 8.41 (1Н, s, NH imidazolidine); 7.70 (2H,
d, J = 7.2, H Ar); 7.55 (2H, d, J = 7.2, H Ar); 7.25–7.19
10.75 (1Н, s, NH indolinone); 8.57 (1Н, s, NH
imidazolidine); 7.48 (2H, d, J = 8.6, H Ar); 7.42 (1H, s,
H Ar); 7.35 (2H, d, J = 8.2, H Ar); 7.21 (2H, d, J = 7.7,
H Ar); 6.97–6.91 (2H, m, H Ar); 6.88 (2H, d, J = 8.2,
H Ar); 4.19 (1H, t, J = 10.4, H pyrrolidine); 4.05–4.00 (2H,
(
5H, m, H Ar); 7.09–6.85 (4H, m, H Ar); 4.25 (1H, t, J = 8.7,
H pyrrolidine); 3.97 (1H, t, J = 8.7, H pyrrolidine); 3.43
1
3
(
1H, t, J = 9.3, H pyrrolidine); 2.13 (3H, s, CH ). C NMR
3
spectrum, δ, ppm: 175.6; 172.3; 153.2; 143.4; 135.6; 132.5;
m, OCH
2
); 3.87 (1H, t, J = 10.4, H pyrrolidine); 3.42 (1H,
); 1.17 (3H, t,
1
1
3
30.1; 130.0; 129.8; 129.0; 128.6; 128.5; 127.8; 127.4;
t, J = 10.4, H pyrrolidine); 2.14 (3H, s, CH
3
13
26.8; 124.2; 121.6; 120.4; 109.8; 77.4; 74.5; 57.1; 50.0;
J = 7.4, CH ). C NMR spectrum, δ, ppm: 175.3; 169.2;
3
+
4.7. Found, m/z: 472.1304 [M+H] . C26
H
22ClN
4
O
3
.
158.4; 155.9; 154.5; 143.4; 134.5; 133.8; 133.1; 132.1;
130.7; 128.7; 128.1; 127.2; 125.8; 120.6; 120.4; 118.1;
Calculated, m/z: 472.1302.
-(4-Chlorophenyl)-4'-(4-methoxyphenyl)-1'-methyl-
dispiro[imidazolidine-4,3'-pyrrolidine-2',3''-indoline]-
,2'',5-trione (4h). Yield 412 mg (82%, method I), white
1
114.2; 112.9; 77.6; 76.3; 76.0; 74.9; 54.1; 45.3; 36.5; 35.2.
+
Found, m/z: 535.1543 [M+H] . C28
H24ClFN
4
O . Calculated,
4
2
m/z: 535.1543.
1
solid, mp 236–238°C. H NMR spectrum, δ, ppm (J, Hz):
0.64 (1Н, s, NH indolinone); 8.35 (1Н, s, NH
5''-Bromo-1-(3-chloro-4-fluorophenyl)-4'-(4-chloro-
phenyl)-1'-methyldispiro[imidazolidine-4,3'-pyrrolidine-
2',3''-indoline]-2,2'',5-trione (4l). Yield 298 mg (51%,
1
imidazolidine); 7.49 (2H, d, J = 7.4, H Ar); 7.39–7.31 (4H,
m, H Ar); 7.19 (2H, d, J = 7.4, H Ar); 7.03 (2H, d, J = 7.4,
H Ar); 6.85–6.80 (2H, m, H Ar); 4.25 (1H, t, J = 9.9,
H pyrrolidine); 3.92 (1H, t, J = 9.9, H pyrrolidine); 3.56
(
(
1
1
7
1
method II), white solid, mp 263–265°C. H NMR
spectrum, δ, ppm (J, Hz): 10.80 (1Н, s, NH indolinone);
8.64 (1Н, s, NH imidazolidine); 7.72 (1H, d, J = 8.0,
H Ar); 7.52–7.48 (4H, m, H Ar); 7.40 (2H, d, J = 9.3,
H Ar); 7.11 (1H, d, J = 8.8, H Ar); 6.95 (1H, m, H Ar);
6.83 (1H, d, J = 8.0, H Ar); 6.65 (1H, s, H Ar); 4.23 (1H, t,
J = 8.0, H pyrrolidine); 3.91 (1H, t, J = 8.0,
3H, s, OCH
3
); 3.43 (1H, t, J = 8.7, H pyrrolidine); 2.12
13
3H, s, CH ). C NMR, δ, ppm: 175.6; 172.4; 163.1;
3
59.7; 158.5; 153.8; 143.3; 132.3; 131.4; 130.9; 128.8;
28.5; 127.8; 125.3; 124.3; 124.2; 114.4; 113.9; 110.7;
+
4.5; 55.3; 55.0; 34.7. Found, m/z: 503.1481 [M+H] .
H pyrrolidine); 3.47 (1H, t, J = 9.4, H pyrrolidine); 2.15
+
C
27
4
H
24ClN
4
O
4
. Calculated, m/z: 503.1481.
(3H, s, CH
FN
3
). Found, m/z: 602.9996 [M+H] .
'-(4-Chlorophenyl)-1-(4-methoxyphenyl)-1'-methyl-
dispiro[imidazolidine-4,3'-pyrrolidine-2',3''-indoline]-
,2'',5-trione (4i). Yield 196 mg (39%, method I), white
С
26
H
19BrCl
2
4
O . Calculated, m/z: 602.9997.
3
1-(3-Chloro-4-fluorophenyl)-4'-(4-methoxyphenyl)-
2
1'-methyldispiro[imidazolidine-4,3'-pyrrolidine-2',3''-
1
solid, mp 227–229°C. H NMR spectrum, δ, ppm (J, Hz):
0.59 (1Н, s, NH indolinone); 8.22 (1Н, s, NH
indoline]-2,2'',5-trione (4m). Yield 432 mg (86%, method I),
1
1
white solid, mp 225–227°C. H NMR spectrum, δ, ppm
imidazolidine); 7.31–7.23 (8H, m, H Ar); 7.04–6.82 (2H,
m, H Ar); 6.65 (2H, d, J = 9.0, H Ar); 4.19 (1H, t, J = 9.4,
H pyrrolidine); 3.89 (1H, t, J = 9.4, H pyrrolidine); 3.88
(J, Hz): 10.65 (1Н, s, NH indolinone); 8.54 (1Н, s, NH
imidazolidine); 7.73 (1H, d, J = 9.6, H Ar); 7.51–7.44 (3H,
m, H Ar); 7.40 (2H, d, J = 7.2, H Ar); 7.32–7.27 (2H, m,
H Ar); 7.08 (1H, d, J = 7.8, H Ar); 7.01 (1H, t, J = 7.8,
H Ar); 6.85 (1H, t, J = 7.8, H Ar); 4.24 (1H, t, J = 9.0,
H pyrrolidine); 3.94 (1H, t, J = 9.0, pyrrolidine); 3.45 (1H, t,
(
3H, s, OCH ); 3.39 (1H, t, J = 8.2, H pyrrolidine); 2.08
3
13
(
3H, s, CH
3
). C NMR spectrum, δ, ppm: 169.6; 159.4;
1
1
7
55.5; 145.5; 144.1; 133.9; 133.0; 131.4; 130.8; 129.0; 128.7;
13
27.8; 126.3; 125.8; 123.5; 123.2; 114.7; 114.6; 109.7; 76.0;
J = 7.8, H pyrrolidine); 2.12 (3H, s, CH ). C NMR spectrum,
3
5.6; 75.5; 55.8; 54.3; 45.5; 36.7; 35.1. Found, m/z: 503.1481
δ, ppm: 207.0; 175.6; 172.3; 163.1; 159.8; 158.5; 157.7;
+
[M+H] . C27
4 4
H24ClN O . Calculated, m/z: 503.1481.
155.2; 153.7; 153.1; 143.4; 131.4; 131.0; 129.0; 125.2; 121.6;
5
''-Bromo-4'-(4-chlorophenyl)-1-(4-ethoxyphenyl)-
116.9; 114.4; 113.9; 110.8; 109.9; 77.4; 74.6; 57.4; 55.3; 49.2;
+
1
'-methyldispiro[imidazolidine-4,3'-pyrrolidine-2',3''-
34.7. Found, m/z: 520.1316 [M+H] . C27
H
23ClFN
4
O .
4
indoline]-2,2'',5-trione (4j). Yield 289 mg (56%, method II),
Calculated, m/z: 520.1314.
1
white solid, mp 247–259°C. H NMR spectrum, δ, ppm
Molecular docking studies of the synthesized dispiro-
indolinones 4a–m were performed using the ICM-Pro soft-
ware (official website: www.molsoft.com) and crystallo-
graphic data for proteins that were available in the PDB
(official website: www.rcsb.org). For in silico modeling, we
used crystallographic data obtained for p53–MDM2 in
complex with nutlin-3a (4HG7) and (2'S,3R,4'S,5'R)-N-
(2-aminoethyl)-6-chloro-4'-(3-chloro-2-fluorophenyl)-2'-
(2,2-dimethylpropyl)-2-oxo-1,2-dihydrospiro[indole-3,3'-
pyrrolidine]-5'-carboxamide (PDB code: 4JVR). The
predicted active conformations of the reference compounds
correlated well with the available crystallographic data of
nutlin-3a (4HG7) and 4JVR. For each compound, ≥10
different conformations were generated in ICM-Pro. All
conformations from the output were within a particular
(
J, Hz): 10.91 (1H, s, H indolinone); 9.14 (1H, s,
H imidazolidine); 7.57–7.32 (2H, m, H Ar); 7.50 (1H, d,
J = 7.1, H Ar); 7.45–7.30 (5H, m, H Ar); 7.27 (1H, s, H Ar);
6
.87–6.85 (1H, m, H Ar); 6.50 (2H, d, J = 7.1, H Ar); 4.60
(
2H, t, J = 9.0, OCH
2
); 3.90 (1H, t, J = 8.4, H pyrrolidine);
.86 (1H, t, J = 8.9, H pyrrolidine); 3.50 (1H, t, J = 9.1,
); 1.45 (3H, s, CH ).
C NMR spectrum, δ, ppm: 174.5; 169.2; 158.3; 155.0;
3
H pyrrolidine); 2.20 (3H, s, CH
3
3
1
3
1
43.2; 133.5; 133.4; 130.5; 128.6; 128.3; 127.5; 127.4; 125.3;
1
23.5; 123.0; 114.8; 114.7; 113.7; 112.4; 75.8; 75.4; 74.3;
6
3.4; 54.0; 49.1; 44.7; 36.6; 26.3. Found, m/z: 595.0742
+
[
M+H] . C28
H
25BrClN
4
4
O . Calculated, m/z: 595.0742.
1-(3-Chloro-4-fluorophenyl)-4'-(4-ethoxyphenyl)-1'-me-
thyldispiro[imidazolidine-4,3'-pyrrolidine-2',3''-indoline]-
7
54

Chemistry of Heterocyclic Compounds 2020, 56(6), 747–755
11. Zhao, Y.; Aguilar, A.; Bernard, D.; Wang, S. J. Med. Chem.
range around the minimum of the potential energy. For
assessment, we used internal ''score'' functions calculated
automatically in ICM-Pro. All scores and conformations
that were generated during modeling were thoroughly
analyzed. Of these values, the best score for the
conformation that is most closely related to the template
supramolecular interface have been selected and
represented.
2
015, 58, 1038.
1
2. Zhao, Y.; Yu, S.; Sun, W.; Liu, L.; Lu, J.; McEachern, D.;
Shargary, S.; Bernard, D.; Li, X.; Zhao, T.; Zou, P.; Sun, D.;
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1
Cytotoxicity and p53 transcriptional reporter activa-
tion tests of the synthesized dispiroindolinones 4a–m are
described in the Supplementary information file.
X-ray study of compound 4l was performed on a
STOE diffractometer with Pilatus100K detector and CuKα
radiation (λ 1.54186 Å). Crystallographic data were
deposited at the Cambridge Crystallographic Data Center
1
5. Beloglazkina, A. A.; Karpov, N. A.; Mefedova, S. R.;
Polyakov, V. S.; Skvortsov, D. A.; Kalinina, M. A.; Tafeenko, V. A.;
Majouga, A. G.; Zyk, N. V.; Beloglazkina, E. K. Russ. Chem.
Bull., Int. Ed. 2019, 1006. [Izv. Akad. Nauk, Ser. Khim. 2019,
(
deposit CCDC 1844112).
Supplementary information file containing H and 13C
1
1
006.]
1
6. Zhao, Y.; Liu, L.; Sun, W.; Lu, J.; McEachern, D.; Li, X.; Yu, S.;
Bernard, D.; Ochsenbein, P.; Ferey, V.; Carry, J.-C.;
Deschamps, J. R.; Sun, D.; Wang, S. J. Am. Chem. Soc. 2013,
NMR spectra of the synthesized compounds, COSY,
1
13
1
13
NOESY, H– C HSQC, H– C HMBC spectra of com-
pound 4d, description of the synthesis and analytical data
of the starting compounds 2d,g,i–l and 3f,i,k, HRMS data of
compounds 4b–d,h–l, X-ray data of compound 4l, as well as
description of the biological tests is available at the journal
website at http://link.springer.com/journal/10593.
1
35, 7223.
1
7. Hill, R. A.; Connolly, J. D. Nat. Prod. Rep. 2015, 32, 273.
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This study was supported by Russian Science Foundation
(
grants 19-03-00201, 19-33-90237, and 18-33-01159).
The authors are thankful to Thermo Fisher Scientific Inc.,
2
1
017, 53, 1199. [Khim. Geterotsikl. Soedin. 2017, 53,
199.]
Textronica AG group (Moscow, Russia), and personally to
Prof. Alexander Makarov for providing the Orbitrap Elite
mass spectrometer for this work. This work in part of NMR
and X-ray structural study was supported by the Lomonosov
Moscow State University Program of Development.
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