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Beloglazkina et al.
of the starting compound (TLC monitoring). A white precipitate
formed after cooling was filtered off and dried in air. If necessary,
the resulting product was purified by column chromatography
(the eluent was chloroform—methanol, 20 : 1).
causes a 5—8-fold increase in the p53 activation (compared
to control) in the transcriptional reporter test. The mech-
anism of cisplatin action is based on a DNA damage, which
leads to the indirect activation of p53 through regulatory
channels and to a two-fold increase in the p53 activation
in the transcriptional reporter test. As Table 1 shows,
compound 11 provides the p53 activation comparable with
that of cisplatin, which may suggest an indirect p53-de-
pendent mechanism of activation.
Comparing the data on the cytotoxicity of dispiroox-
azolones with that of dispirothioxoimidazolinones,27 one
can conclude that a change in the nature of heterocycle in
the starting 1,3-dipolarophile results in the compounds
exhibiting selective activity against studied cell cultures
only in certain cases.
Thus, in the present work we synthesized a series of
new N-unsubstituted dispiroindolinones by the regioselec-
tive 1,3-dipolar cycloaddition of azomethine ylides to the
corresponding N-unsubstituted heterocyclic derivatives
(5-arylidene thiohydantoins, hydantoins, and thiazolidine-
2,4-diones), evaluated the cytotoxicity of the obtained
compounds, and measured the p53 activation in the tran-
scriptional reporter test. The obtained data suggest that
the hydantoin derivative 9 is the most promising for further
modification and study as an inhibitor of the р53—MDM2
interaction, since this compound exhibited the highest
selectivity against human A549 lung carcinoma cell line
and moderate value of the p53 activation.
5´´-Bromo-4´-(4-chlorophenyl)-1´-methyl-2-thioxodispiro-
[imidazolidine-4,3´-pyrrolidine-2´,3´´-indoline]-2´´,5-dione (7) was
prepared from thiohydantoin 1 (0.3 g, 0.5 mmol) and 5-bromo-
isatin (0.22 g, 1.0 mmol). The yield was 0.61 g (42%), white
crystalline powder, m.p. 167 °C. 1H NMR, δ: 2.08 (s, 3 Н,
NCH3); 3.39 (t, 1 H, CH2, aliph., J = 8.9 Hz); 3.79 (t, 1 H, CH2,
aliph., J = 9.5 Hz); 4.15 (t, 1 H, CH, aliph., J = 9.2 Hz). 6.77
(d, 1 H, CH, arom., J = 8.4 Hz); 7.37—7.47 (m, 5 H, CH, arom.);
7.50 (d, 1 H, CH, arom., J = 7.4 Hz); 9.11 (s, 1 H, N(1)H, imid-
azolidine); 11.61 (s, 1 H, NH, indolinone). HRMS (ESI), m/z:
490.9946 [M + H]+. Calculated for C20H17BrClN4O2S: 490.9944.
5´´-Bromo-4´-(4-fluorophenyl)-1´-methyl-2-thioxodispiro-
[imidazolidine-4,3´-pyrrolidine-2´,3´´-indoline]-2´´,5-dione (8) was
prepared from thiohydantoin 2 (0.2 g, 0.15 mmol), sarcosine
(0.15 g, 0.267 mmol), and 5-bromoisatin (0.67 g, 0.3 mmol). The
yield was 0.4 g (98%), white crystalline powder, m.p. 186 °C.
1H NMR, δ: 2.10 (s, 3 H, NCH3); 3.41 (t, 1 H, CH2, aliph.,
J = 9.5 Hz); 3.80 (t, 1 H, CH2, aliph., J = 9.5 Hz); 4.17 (t, 1 H,
CH, aliph., J = 9.2 Hz); 6.77 (d, 1 H, CH, arom., J = 8.2 Hz);
7.15 (t, 2 H, CH, arom., J = 8.7 Hz); 7.36—7.53 (m, 4 H,
CH, arom.); 9.71 (s, 1 H, N(1)H, imidazolidine); 10.73 (s, 1 H,
N(3)H, imidazolidine); 11.61 (s, 1 H, NH, indolinone). HRMS
(ESI), m/z: 475.0242 [M + H]+. Calculated for C20H17BrFN4O2S:
475.0240.
5´´-Bromo-4´-(4-chlorophenyl)-1´-methyl-2-oxodispiro[imid-
azolidine-4,3´-pyrrolidine-2´,3´´-indoline]-2´´,5-dione (9) was
prepared from hydantoin 5 (0.2 g, 0.5 mmol), sarcosine (0.09 g,
1.0 mmol), and 5-bromoisatin (0.22 g, 1.0 mmol). The yield was
0.33 g (56%), white crystalline powder, m.p. 185 °C. 1H NMR,
δ: 2.07 (s, 3 H, NCH3); 3.49 (t, 1 H, CH2, aliph., J = 9.6 Hz);
3.78 (t, 1 H, CH2, aliph., J = 9.3 Hz); 4.15 (t, 1 H, CH, aliph.,
J = 9.4 Hz); 6.56 (s, 1 H, CH2, arom.); 6.77 (d, 1 H, CH, arom.,
J = 8.2 Hz); 7.37 (s, 2 H, CH, arom.); 7.44 (d, 2 H, CH, arom.,
J = 7.7 Hz); 7.64 (d, 1 H, CH, arom., J = 7.9 Hz); 10.79 (s, 1 H,
NH, imidazolidine); 11.70 (s, 1 H, NH, indolinone). HRMS
(ESI), m/z: 475.0176 [M + H]+. Calculated for C20H17BrClN4O3:
474.0173.
1´-Methyl-4´-(4-fluorophenyl)-2-oxodispiro[imidazolidine-
4,3´-pyrrolidine-2´,3´´-indoline]-2´´,5-dione (10) was prepared from
hydantoin 6 (0.2 g, 0.5 mmol), sarcosine (0.09 g, 1.0 mmol), and
isatin (0.19 g, 1.3 mmol). The yield was 0.31 g (42%), white
crystalline powder, m.p. 197 °C. 1H NMR, δ: 2.07 (s, 3 H,
NCH3); 3.37 (t, 1 H, CH2, aliph., J = 9.6 Hz); 3.80 (t, 1 H, CH2,
aliph., J = 9.4 Hz); 4.12 (t, 1 H, CH, aliph., J = 9.4 Hz); 6.80
(d, 1 H, CH, arom., J = 7.7 Hz); 6.98 (t, 1 H, CH, arom.,
J = 7.6 Hz). 7.21—7.27 (m, 1 H, CH, arom.); 7.33 (d, 1 H, CH,
arom., J = 7.6 Hz); 7.41 (d, 1 H, CH, arom., J = 8.5 Hz); 7.47
(s, 1 H, CH, arom.); 10.56 (s, 1 H, NH, imidazolidine); 11.33
(s, 1 H, NH, indolinone). HRMS (ESI), m/z: 381.1366 [M + H]+.
Calculated for C20H17FN4O3: 381.1363.
5´´-Bromo-4´-(4-chlorophenyl)-1´-methyl-2-oxodispiro[thia-
imidazolidine-4,3´-pyrrolidine-2´,3´´-indoline]-2´´,5-dione (11) was
prepared from (Z)-5-(4-chlorobenzylidene)thiazolidine-2,4-
dione (0.5 g, 0.8 mmol), sarcosine (0.26 g, 1.6 mmol), and
5-bromoisatin (0.51 g, 1.6 mmol). The yield was 0.54 g (52%),
white crystalline powder, m.p. 217 °C. 1H NMR, δ: 2.06 (s, 3 H,
NCH3); 3.48 (t, 1 H, CH2, aliph., J = 8.3 Hz); 3.77 (t, 1 H, CH2,
Experimental
The course of the reaction and the purity of compounds were
monitored by thin-layer chromatography (TLC) on Silufol-
UV254 plates with fixed silica gel layer. Melting points were
determined in a unit with open capillary. Uncorrected melting
points are given. 1H NMR spectra were recorded on a Bruker
Avance instrument (400 MHz) in DMSO-d6. Chemical shifts
are given in the δ scale relative to hexamethyldisiloxane as the
internal standard. High-resolution electrospray ionization mass
spectra were recorded on a Bruker microTOF II instrument
(positive ion mode, the capillary voltage of 4500 V). The mass
scan range was m/z 50—3000 Da, the calibration was external or
internal (Electrospray Calibration Solution, Fluka). Compounds
were injected as solutions in acetonitrile; the flow rate was
3 μL min–1. The nebulizer gas was nitrogen (4 L min–1) and the
interface temperature was 180 °C.
All starting compounds, viz., thiohydantoins 1 and 2, hydan-
toins 3 and 4, thiazolidines 5 and 6, were prepared according to
the procedures described earlier.21,24
Synthesis of dispiro derivatives based on N-unsubstituted
heterocycles (general procedure). A round-bottom flask equipped
with a reflux condenser was charged with the corresponding
unsubstituted 2-thiohydantoin 1 or 2, hydantoin 3 or 4, thiazolid-
ine 5 or 6 (1 mmol, 1 equiv.) and 95% EtOH (10 mL). The
mixture was heated to reflux and sarcosine (2 mmol, 2 equiv.)
and isatin (2 mmol, 2 equiv.) were added portionwise. The mix-
ture was refluxed with stirring for about 5 h until disappearance