Synthesis and optimization of new 3,6-disubstitutedindole derivatives and their evaluation as anticancer agents targeting the MDM2/MDMx complex

Article abstract of DOI:10.1248/cpb.c15-00608

Twelve derivatives of the general formula 3-substituted-6-chloroindoles were synthesized and tested for their growth inhibitory effects versus p53^+/+ colorectal cancer HCT116 and its p53 knockout isogenic cells; colorectal cancer cell p53^-

Full text of DOI:10.1248/cpb.c15-00608

34
Chem. Pharm. Bull. 64, 34–41 (2016)
Vol. 64, No. 1
Regular Article
Synthesis and Optimization of New 3,6-Disubstitutedindole Derivatives
and Their Evaluation as Anticancer Agents Targeting the MDM2/MDMx
Complex
Mohamed Salah Rezk,a,b Mohammad Abdel-Halim,a Adam Keeton,c Derek Franklin,d,e
Matthias Bauer,f Frank Michael Boeckler,f Matthias Engel,b Rolf Wolfgang Hartmann,b
,a
Yanping Zhang,d,e Gary Anthony Piazza,c and Ashraf Hassan Abadi*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo;
b
Cairo 11835, Egypt: Department of Pharmaceutical and Medicinal Chemistry, Saarland University; Saarbrücken
c
66123, Germany: Department of Oncologic Sciences and Pharmacology, Drug Discovery Research Center,
d
Mitchell Cancer Institute, University of South Alabama; Mobile, AL 36604, U.S.A.: Department of Radiation
Oncology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill; NC 27514,
e
U.S.A.: Department of Pharmacology, University of North Carolina at Chapel Hill; NC 27599–7365, U.S.A.: and
f Department of Pharmaceutical & Medicinal Chemistry, Institute of Pharmaceutical Sciences, Eberhard-Karls-
University Tuebingen; Auf der Morgenstelle 8, Tuebingen 72076, Germany.
Received August 5, 2015; accepted October 30, 2015
Twelve derivatives of the general formula 3-substituted-6-chloroindoles were synthesized and tested for
their growth inhibitory effects versus p53+/+ colorectal cancer HCT116 and its p53 knockout isogenic cells;
colorectal cancer cell p53ꢀ/ꢀ SW480; the lung cancer cell line p53ꢀ/ꢀ H1299; mouse embryonic fibroblasts
(MEF) p53+/+ and its p53 knockout isogenic cells. The compounds were also evaluated for their ability to
induce p53 nuclear translocation and binding to murine double minute 2 (MDM2) and murine double minute
4 (MDM4). Of these, compound 5a was the most active in inhibiting the growth of cells, with selectivity to-
wards the p53+/+ cell lines, and it showed stronger binding to MDM4 rather than MDM2. The activity profile
of compound 5a is strongly similar to that of Nutlin-3.
Key words indole derivative; p53; murine double minute 4 (MDM4); murine double minute 2 (MDM2)
In nearly all human cancers inactivation of tumor suppres- overexpressing cells and lead to selective apoptosis or cell
sor p53 can be among the underlying causes of the uncon- cycle arrest in cancer cells.10–14)
trolled cell proliferation, that is why the reactivation of the
p53 protein is considered one of the attractive approaches for of p53–MDM2 interaction. The crystal structure showed that
cancer treatment that is currently targeted.1)
the interaction occurred between a small but deep hydropho-
X-Ray crystallography has established the structural basis
p53 plays a crucial role in the prevention of cancer develop- bic cleft in the MDM2 and a short α helix formed by residues
ment through acting as “the guardian of the genome” by ac- 13–29 of p53 in which the three hydrophobic amino acids
tivation of the transcription of a number of genes that are in- (Phe19, Trp23, Leu26) play a crucial role.15) Based on these in-
duced in response to various stress signals and are responsible teractions various classes of small-molecule MDM2 inhibitors
for the regulation of the cell cycle check points, DNA repair, have been reported. Nutlins, cis-imidazoline analogues, are
and finally induction of apoptosis.2–5)
the prototypes of this class of inhibitor.10,16,17) Subsequently,
Two major structurally related oncoproteins: murine double several spiro-oxindole derivatives e.g. MI-219 and 1,4-benzo-
minute 2 (MDM2) and 4 (MDM4/x) regulate the activity of diazepine-2,5-diones e.g. TDP222669, were introduced (Fig.
p53 through three parallel mechanisms: (1) MDM2/x binds to 1). More recently, indolyl-hydantoin derivatives (e.g. RO-2443)
the transactivation domain (TAD) of p53 resulting in the inhi- were reported to potently block p53 binding with both MDM2
bition of p53-responsive gene expression, (2) MDM2/x exports and MDMx by inhibitor-driven homo- and/or heterodimeriza-
p53 from the nucleus where it can no longer activate tran- tion of MDM2 and MDMx proteins.18,19)
scription, and (3) by ubiquiltylating the MDM2–p53 complex
In this report, we explore the scope and limitations of in-
causing it to undergo proteosomal degradation.6–8) MDMx is dolylhydantoin compounds by synthesizing twelve novel small
clearly involved in the first two mechanisms through forma- organic in the context of compound RO-2443 reported by
tion of heterodimers with MDM2.9)
Graves et al.19) The new molecules possess a 6-chloroindole
In approximately half of known cancer cases, p53 activity ring, the core hydantoin ring is kept or changed to the struc-
has been nullified by mutations and other genomic alterations turally related thiohydantoin or thiazolidinedione. The termi-
in the DNA-binding domain of p53.1) In most of remaining nal nitrogen was substituted by different halogen-substituted
cancer cases, the p53 retains its wild-type form, but it be- benzyl derivatives or 3,4-difluorobenzoyl substituent. The
comes functionally inactive by the effect of the overexpressed compounds were tested for their effects on cell lines and mod-
MDM2/x oncoprotein.6) Non-peptide small-molecules which els with different expression state to p53 and for p53 nuclear
inhibit the MDM2/x–p53 protein–protein interaction (PPI) can translocation as shown hereunder.
restore the normal activity of the wild-type p53 in MDM2/x-
*To whom correspondence should be addressed. e-mail: ashraf.abadi@guc.edu.eg
© 2016 The Pharmaceutical Society of Japan

Vol. 64, No. 1 (2016)
Chem. Pharm. Bull.
35
Fig. 1. Representative Examples of Known MDM2 and MDM4 Antagonists
i) POCl3, s: DMF, 1.5h, 0°C; 2h, room temp. ii) Hydantoin (4a) or thiohydantoin (4b) or thiazolidinedione (4c), NaHCO3, s: H2O or ethanol, 70°C, pH=7;
HOCH2CH2NH2, 90°C; overnight, reflux. iii) Substituted benzyl chlorides, K2CO3, C: KI, s: DMF, 45min, reflux.
Chart 1
designed compounds.
Results and Discussion
The synthetic protocol of the target compounds is depicted
As a preliminary assay, all the compounds were tested for
in Charts 1 and 2. In brief, the 6-chlorindole-3-carboxalde- their potential growth inhibitory activity against the wild type
hyde was prepared by reaction of 6-chloroindole with di- (p53+/+) HCT116 human colorectal carcinoma cell line. The
methylformamide and phosphorus oxychloride, the arylidenes resulting activities of the compounds are shown in Table 1.
(4a–c) were formed by reaction of the aldehyde with the All the tested compounds showed anticancer activity against
commercially available hydantoin, thiahydantoin or thazol- HCT116 with IC50 values ranging from 2.83 to 24µM, where
idinedione, followed by arylation by the appropriate benzyl 5a was the most potent compound with an IC50 value of
halide, Chart 1. To prepare the saturated, flexible analogue 2.83µM and 5g the most potent thiohydantoin derivative with
of 5d (compound 7), intermediate 4a was reduced using ex- an IC50 value of 5.10µM. The thiazolidinedione derivative 5J
cess sodium borohydride followed by benzylation using the was the least active among all tested compounds. In most of
3,4-difluorobenzyl chloride. Also, compound 4a was benzo- cases, the hydantoin derivatives were more active than their
ylated to obtain the oxo-analogue of 5d (compound 8), Chart thiohydantoin and thiazolidinedione congeners e.g. 5a, d and e
2. All the synthesized compounds were sufficiently character- versus 5f, h and i. In addition the hydantoins were more active
1
ized by H-, 13C-NMR as well as LC-MS. The analytical data than their thiazolidinedione derivative 5c versus j. This indi-
were consistent with the provided molecular structures of the cates the importance of the nature of the core ring on growth

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Chem. Pharm. Bull.
Vol. 64, No. 1 (2016)
i) NaBH4, CoCl2, CH3COOH, s: iPrOH, s: DMF, overnight, rt. ii) 3,4-Difuorobenzyl chlorides or 3,4-difluorobenzoyl chloride, K2CO3, C: KI, s: DMF, 45min, reflux.
Chart 2
Table 1. The Growth Inhibitory Activity of the Synthesized Compounds
Table 2. The Activity of the Most Potent Compounds against Different
against Wild Type HCT116 Cell Line
Cell Lines
Compound
IC50 HCT116 (µM)
Compound
IC50 HCT116 (µM)
IC50 (µM)
Compound
5a
5b
5c
5d
5e
5f
2.8
3.7
5.5
5.0
7.8
7.4
5.1
5g
5.1
9.3
HCT116
SW480
H1299
5h
5a
5b
2.8
3.7
5.0
5.1
2.0
6.7
n.d
9.2
11.3
n.d
5i
13.2
19.8
24.0
12.2
2.0
5j
5d
13.0
5.7
7
8
5g
16.6
22.2
Nutlin-3
16.0
5g
Nutlin-3
inhibition of cancerous cell line HCT116.
growth. For compound 5a, this effect apparently reached a
In our attempt to test the selectivity across various cancer plateau between 5.56 and 16.67µM, which might be attributed
cell lines with various expression levels of p53; the most to the poor solubility and high lipophilicity of the compound;
potent compounds 5a, b, d, g were further tested against p53- however, compound 5g showed its strongest effect at 50µM.
mutated cell lines; human adenocarcinoma strain (SW480) The highest increase in nuclear p53 was observed after the
and p-53 null human non-small cell lung carcinoma cell line treatment with 5a at 16.67µM, which is consistent with re-
(H1299). The results in Table 2 showed moderate selectivity of sults obtained from cellular growth assays suggesting that the
the tested compounds towards the HCT116 cell line relative to dominant mode of action for compound 5a might be through
SW480 and H1299 cell lines with selectivity index (SI) of 2.36 inhibiting p53 stability. Western blotting data supported these
and 3.26 for compound 5a. Nutlin-3 was the most selective findings and further showed that p53 accumulation induced by
compound with SI of 8.00 and 11.11, respectively.
5a was accompanied by up-regulation of MDM2 and p21 pro-
In order to elucidate the p53-dependent mode of action, teins in wild type MEF and HCT116 cells (data represented in
the most potent and selective compound (5a) was further Fig. 3), all these data confirm that the mode of action of 5a is
tested against (p53−/−) HCT116, (p53−/−) mouse embryonic p53-dependent through inhibition of the protein–protein inter-
fibroblasts (MEF), wild type (p53+/+) HCT116 and wild type action between p53 and its oncoproteins MDM2/x.
(p53+/+) MEF. These isogenic cell lines are commonly used
In an attempt to further explore the mode of action of 5a;
to show the p53/MDM2-dependent mode of action.20) Indeed, an assessment of the binding affinity characteristics of the
compound 5a and Nutlin-3 potently inhibited the growth of compound towards MDM2 was examined via competitive
HCT116 and MEF wild type p53 cell lines but showed greatly fluorescence polarization assay described by Vogel et al.21)
reduced activity in the same cell lines with p53 deletion as Kd values were calculated from IC50 values using equations
shown in Fig. 2, supporting the hypothesis that the cell growth described by Nikolovska-Coleska et al.22) Nutlin-3 was used as
inhibitory activity of both 5a and Nutlin-3 is p53-dependent.
control, and as expected, Nutlin-3 disrupted these complexes
Additionally, a p53 translocation assay was performed using with Kd values for MDM2 and MDM4 that were similar to
immunocytochemical staining and high content screening. Se- growth inhibitory IC50 values for HCT116 and SW480 cells,
lection of the tested compounds 5a, b, g were based on activ- respectively.
ity against wild type (p53+/+) HCT116 cell line using Nutlin-3
In contrast, 5a displayed approximately 14-fold difference
as a positive control. From the results shown in Table 3, it’s between Kd values and growth inhibitory IC50 for the same
clear that compounds 5a and g induced a dose-dependent in- pairs (MDM2 vs. HCT116 and MDM4 vs. SW480). This is
crease in the nuclear p53 at lower concentrations which inhibit consistent with the expected mechanism for this and a similar

Vol. 64, No. 1 (2016)
Chem. Pharm. Bull.
37
Fig. 2. Compound 5a Exhibits p53 Dependent Inhibition of Cell Proliferation in Both HCT116 (A) and MEF Cells (B)
Cell proliferation in the presence of DMSO, 10µM Nutlin-3, or 10µM compound 5a were completed using the BioRad TC10 Automated Cell Counter. Curves are the
combination of multiple experiments MEF (n=4) and HCT116 (n=2). Relative growth was calculated as current cell number divided by Day 0 cell number. Error bars
represent S.E.M.
Table 3. p53 Translocation Assay
Fold change in nuclear p53 staining vs. DMSO control
Compound conc.
50.00µM
16.67µM
5.56µM
1.85µM
5a
5b
4.24 1.63
1.10 0.25
5.56 0.87
3.85 0.40
1.64 0.24
1.48 0.28
1.17 0.23
0.92 0.15
5g
4.87 2.39
2.06 0.41
1.21 0.23
1.06 0.43
Nutlin-3
13.96 0.1 (30µM)
8.77 0.98 (10µM)
3.87 0.17 (3.3µM)
1.93 0.24 (1.1µM)
scaffold,19) which was shown to affect p53 stability through
Experimental
MDM2 and MDMx homo- and heterodimerization rather than
Chemistry General Solvents and reagents were ob-
direct binding to MDM2 (results shown in Table 4). However, tained from commercial suppliers and used as received. A
1
we also cannot completely rule out that affinity to other tar- Bruker DRX 300 spectrometer was used to obtain H- and
gets could contribute to the observed cellular profile e.g. while 13C-NMR spectra. The chemical shifts are referenced to the
5g and 5a have very similar IC50 in anti-proliferation assays, residual protonated solvent signals or tetramethylsilane (TMS)
5g barely affects the expression of MDM2 and p21.
was used as a reference. Electrospray LC-MS analysis was
In summary, compound 5a showed considerable growth performed using a TSQ quantum (Thermo Electron Corpo-
inhibitory activity, while biochemical and biological assays ration) instrument prepared with a triple quadrupole mass
showed that 5a disrupts the MDM2/x–p53 interaction and ac- detector (ThermoFinnigan) and an electrospray ionization
tivates the p53 pathway in cells with wild-type p53 leading to (ESI) source. All samples were inserted using an autosampler
cell cycle arrest in both normal and tumor cells.
In conclusion, indolylhydantoin seems to be a promising The MS detection was determined using a source collision
scaffold for MDM2/MDMx inhibition. induced dissociation (CID) of 10V and carried out at a spray
(Surveyor, ThermoFinnigan) by an injection volume of 10µL.

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Chem. Pharm. Bull.
Vol. 64, No. 1 (2016)
General Procedure of Knoevenagel Condensation
Imidazolidine-2,4-dione or its appropriate sulfur-isostere
(10.0mmol) was dissolved in 10mL of water or ethanol at
70°C with stirring. After complete dissolution, the pH of the
mixture was adjusted to 7.0 with saturated NaHCO3 solution.
Ethanolamine (0.9mL) was added to the reaction mixture,
and the temperature was increased to 90°C by use of an oil
bath. To this, an equimolar quantity of 6-chloro-1H-indole-
3-carbaldehyde (3) (10.0mmol) solution in 10mL of alcohol
was added dropwise with continuous stirring. The temperature
was raised to 120°C and kept under reflux at that temperature
overnight. The mixture was cooled, the precipitate was filtered
and washed with water in order to remove the soluble impuri-
ties, then left to dry.
5-((6-Chloro-1H-indol-3-yl)methylene)imidazolidine-2,4-
dione (4a)
Synthesized according to the general procedure of Kno-
evenagel condensation using imidazolidine-2,4-dione and
6-chloro-1H-indole-3-carbaldehyde (3); yellowish white solid;
yield: 1.60g (61%); 1H-NMR (300MHz, dimethyl sulfoxide
(DMSO)) δ: 12.29 (s, 1H), 11.97 (s, 1H), 10.49 (s, 1H), 8.19 (s,
1H), 7.82 (d, J=8.6Hz, 1H), 7.48 (d, J=1.9Hz, 1H), 7.13 (dd,
J=8.5, 1.9Hz, 1H), 6.88 (s, 1H); 13C-NMR (75MHz, DMSO)
δ: 164.49, 153.42, 136.18, 128.09, 127.06, 125.67, 122.56,
120.52, 119.73, 111.52, 108.46, 103.10; MS ESI: m/z=385.71
(M)+; purity: 97%.
Fig. 3. Western Blotting Analysis for MDM2, p53 and p21 Proteins in
Wild Type and (p53−/−) MEF (A) and HCT116 Cells (B) When Treated
with 10µM Nutlin-3, 10 µM 5a or 10µM 5g and Incubated for 24h
5-((6-Chloro-1H-indol-3-yl)methylene)-2-thioxoimidazolidin-4-
one (4b)
Synthesized according to the general procedure of Kno-
evenagel condensation using 2-thioxoimidazolidin-4-oneand
6-chloro-1H-indole-3-carbaldehyde (3); redsolid; yield: 1.35g
Table 4. Fluorescence Polarization Assay
1
(49%); H-NMR (300MHz, DMSO) δ: 12.79 (s, 1H), 11.94 (s,
MDM2
MDMx
1H), 11.59 (s, 1H), 8.40 (s, 1H), 8.20 (d, J=8.5Hz, 1H), 7.52
(d, J=1.9Hz, 1H), 7.13 (dd, J=8.5, 1.9Hz, 1H), 7.10 (s, 1H);
13C-NMR (75MHz, DMSO) δ: 169.90, 158.62, 136.79, 135.19,
132.96, 127.05, 125.36, 121.19, 120.73, 115.72, 111.78, 111.24.
5-((6-Chloro-1H-indol-3-yl)methylene)thiazolidine-2,4-dione
(4c)
IC50 (µM) S.E. (µM) Kd (µM) IC50 (µM) S.E. (µM) Kd (µM)
5a
160
2.0
5.5
6.5
41.2
0.5
330
14.0
8.7
90.3
14.8
Nutlin-3
54.2
voltage of 4.2kV, a nitrogen sheath gas pressure of 4.0×105 Pa,
Synthesized according to the general procedure of Knoeve-
a capillary temperature of 400°C, a capillary voltage of 35V nagel condensation using thiazolidine-2,4-dione and 6-chlo-
and an auxiliary gas pressure of 1.0×105 Pa. The stationary ro-1H-indole-3-carbaldehyde (3); yellow solid; yield: 1.25g
1
phase used was an RP C18 NUCLEODUR 100-3 (125×3mm) (45%); H-NMR (300MHz, DMSO) δ: 12.44 (s, 1H), 12.23
column (Macherey–Nagel). The solvent system consisted of (s, 1H), 8.18 (s, 1H), 7.93 (d, J=8.6Hz, 1H), 7.83 (s, 1H), 7.28
water containing 0.1% trifluoroacetic acid (TFA) (A) and 0.1% (dd, J=8.0, 1.1Hz, 1H), 7.19 (dd, J=8.5, 1.4Hz, 1H), 13C-NMR
TFA in acetonitrile (B). HPLC method: flow rate 400µL/min. (75MHz, DMSO) δ: 167.56, 165.77, 137.28, 131.63, 128.26,
The percentage of B started at an initial of 5%, was increased 126.35, 126.02, 121.91, 120.60, 114.97, 112.56, 111.01.
up to 100% during 16min, kept at 100% for 2min, and flushed
back to 5% in 2min.
General Procedure of Benzylation To a suspension
of 5-((6-chloro-1H-indol-3-yl)methylene)imidazolidine-2,4-dione
General Procedure of Vilsmeier–Haack Reaction A so- or its appropriate sulfur-isostere (4a–c) (10mmol), K2CO3
lution of 6-chloroindole (30mmol) in N,N-dimethylformamide (40 mmol) and KI (0.6mmol) in dry DMF (20mL), the ap-
(DMF) (5mL) was added dropwise to a solution of phospho- propriate benzyl chloride was added (12mmol). The resulting
rus oxychloride (44mmol) in DMF (32mL) which had been mixture was refluxed for 45min. The reaction was stopped by
stirred for 1.5h at 0°C. The reaction mixture was warmed adding water, and then the mixture was extracted with EtOAc
to room temperature and stirred for 2h. The solution with (3×20mL). The combined organic phases were washed with
the precipitate formed was poured on ice and basified with brine, dried over anhydrous Mg2SO4, filtered and concen-
solid KOH and left overnight. The precipitate was filtered and trated. The crude residue was then purified by column chro-
washed with a mixture of 10% ethylacetate in petroleum ben- matography on silica gel.
zene several times, then left to dry to give the aldehyde.
6-Chloro-1H-indole-3-carbaldehyde (3)
5-((6-Chloro-1H-indol-3-yl)methylene)-3-(4-chlorobenzyl)-
imidazolidine-2,4-dione (5a)
Synthesized according to the general procedure of Vils-
The title compound was prepared by reaction of
meier–Haack reaction; light brick red solid; yield 4.04g (75%). 5-((6-chloro-1H-indol-3-yl)methylene)imidazolidine-2,4-dione

Vol. 64, No. 1 (2016)
Chem. Pharm. Bull.
39
(4a) and 1-chloro-4-(chloromethyl)benzene according to the 2H), 7.48 (d, J=1.7Hz, 1H), 7.13 (dd, J=8.5, 1.8Hz, 1H), 6.89
general procedure for benzylation; yellowish white solid; (s, 1H), 4.78 (s, 2H); 13C-NMR (75MHz, DMSO) δ: 163.63,
1
yield: 1.55g (40%); H-NMR (300MHz, DMSO) δ: 11.97 (s, 154.40, 141.44, 136.19, 128.08, 128.07, 127.08, 125.99, 125.67,
1H), 10.49 (s, 1H), 8.19 (s, 1H), 7.82 (d, J=8.6Hz, 1H), 7.48 125.50, 122.39, 122.53, 120.53, 119.73, 111.53, 108.46, 103.14,
(d, J=1.9Hz, 1H), 7.44–7.38 (m, 2H), 7.37–7.29 (m, 2H), 7.13 40.83; MS (ESI): m/z=419.68 (M)+; purity: 96%.
(dd, J=8.5, 1.9Hz, 1H), 6.88 (s, 1H), 4.68 (s, 2H); 13C-NMR
5-((6-Chloro-1H-indol-3-yl)methylene)-3-(4-chlorobenzyl)-2-
(75MHz, DMSO) δ: 163.59, 154.42, 136.18, 135.75, 132.08, thioxoimidazolidin-4-one (5f)
129.33, 128.54, 128.07, 127.06, 125.67, 122.56, 120.52, 119.73,
The title compound was prepared by reaction of
111.52, 108.46, 103.00, 40.58; MS (ESI): m/z=387.39 (M+H)+; 5-((6-chloro-1H-indol-3-yl)methylene)-2-thioxoimidazolidin-4-
purity: 98.2%.
one (4b) and 1-chloro-4-(chloromethyl)benzene according to
5-((6-Chloro-1H-indol-3-yl)methylene)-3-(3-chlorobenzyl)- the general procedure for benzylation; orange solid; yield:
1
imidazolidine-2,4-dione (5b)
1.52g (38%); H-NMR (300MHz, DMSO) δ: 11.94 (s, 1H),
The title compound was prepared by reaction of 11.59 (s, 1H), 8.40 (s, 1H), 8.20 (d, J=8.5Hz, 1H), 7.59–7.53
5-((6-chloro-1H-indol-3-yl)methylene)imidazolidine-2,4-dione (m, 2H), 7.52 (d, J=1.9Hz, 1H), 7.46–7.36 (m, 2H), 7.13
(4a) and 1-chloro-3-(chloromethyl)benzene according to the (dd, J=8.5,1.9Hz, 1H), 7.10 (s, 1H), 4.59 (s, 2H); 13C-NMR
general procedure for benzylation; yellowish white solid; (75MHz, DMSO) δ: 169.91, 158.72, 136.77, 136.62, 135.19,
1
yield: 1.69g (44%); H-NMR (300MHz, DMSO) δ: 11.97 (s, 132.96, 132.00, 130.73, 128.48, 127.05, 125.36, 121.19, 120.73,
1H), 10.50 (s, 1H), 8.19 (s, 1H), 7.83 (d, J=8.5Hz, 1H), 7.48 115.72, 111.78, 111.24, 32.47; MS (ESI): m/z=403.68 (M+H)+;
(d, J=1.8Hz, 1H), 7.43–7.32 (m, 3H), 7.31–7.23 (m, 1H), 7.13 purity: 95%.
(dd, J=8.5, 1.8Hz, 1H), 6.89 (s, 1H), 4.69 (s, 2H); 13C-NMR
5-((6-Chloro-1H-indol-3-yl)methylene)-3-(3,4-dichlorobenzyl)-2-
(75MHz, DMSO) δ: 163.62, 154.40, 139.21, 136.18, 133.13, thioxoimidazolidin-4-one (5g)
130.53, 128.10, 127.47, 127.28, 127.07, 126.06, 125.68, 122.53,
The title compound was prepared by reaction of
120.53, 119.74, 111.53, 108.47, 103.11, 40.67; MS (ESI): 5-((6-chloro-1H-indol-3-yl)methylene)-2-thioxoimidazolidin-4-
m/z=387.68 (M+H)+; purity: 96%.
one (4b) and 1,2-dichloro-4-(chloromethyl)benzene according
5-((6-Chloro-1H-indol-3-yl)methylene)-3-(3,4-dichlorobenzyl)- to the general procedure for benzylation; dark yellow solid;
imidazolidine-2,4-dione (5c)
yield: 1.70g (39%); 1H-NMR (300MHz, DMSO) δ: 11.95
The title compound was prepared by reaction of (s, 1H), 11.62 (s, 1H), 8.41 (s, 1H), 8.19 (d, J=8.5Hz, 1H),
5-((6-chloro-1H-indol-3-yl)methylene)imidazolidine-2,4-dione 7.84 (d, J=1.9Hz, 1H), 7.63–7.53 (m, 2H), 7.52 (d, J=1.9Hz,
(4a) and 1,2-dichloro-4-(chloromethyl)benzene according to 1H), 7.15 (dd, J=8.5, 1.9Hz, 1H), 7.08 (s, 1H), 4.59 (s, 2H);
the general procedure for benzylation; yellowish white solid; 13C-NMR (75MHz, DMSO) δ: 169.88, 158.50, 139.01, 136.83,
1
yield: 2.00g (49%); H-NMR (300MHz, DMSO) δ: 11.97 (s, 135.10, 132.92, 130.94, 130.88, 130.64, 129.95, 129.20, 127.08,
1H), 10.50 (s, 1H), 8.19 (s, 1H), 7.82 (d, J=8.6Hz, 1H), 7.61 (d, 125.34, 121.20, 120.77, 115.90, 111.79, 111.21, 31.91; MS (ESI):
J=8.3Hz, 1H), 7.58 (d, J=2.0Hz, 1H), 7.48 (d, J=1.9Hz, 1H), m/z=437.90 (M+H)+; purity: 98%.
7.29 (dd, J=8.3, 2.0Hz, 1H), 7.13 (dd, J=8.5, 1.9Hz, 1H), 6.88
5-((6-Chloro-1H-indol-3-yl)methylene)-3-(3,4-difluorobenzyl)-2-
(s, 1H), 4.69 (s, 2H); 13C-NMR (75MHz, DMSO) δ: 163.60, thioxoimidazolidin-4-one (5h)
154.34, 137.85, 136.18, 131.09, 130.80, 130.13, 129.54, 128.11,
The title compound was prepared by reaction of
127.76, 127.07, 125.67, 122.53, 120.53, 119.73, 111.53, 108.46, 5-((6-chloro-1H-indol-3-yl)methylene)-2-thioxoimidazolidin-4-
103.15, 40.69; MS (ESI): m/z=419.50 (M−H)+; purity: 96.9%.
one (4b) and 4-(chloromethyl)-1,2-difluorobenzene accord-
5-((6-Chloro-1H-indol-3-yl)methylene)-3-(3,4-difluorobenzyl)- ing to the general procedure for benzylation; orange solid;
imidazolidine-2,4-dione (5d)
yield: 1.60g (40%); 1H-NMR (300MHz, DMSO) δ: 11.95
The title compound was prepared by reaction of (s, 1H), 11.61 (s, 1H), 8.41 (s, 1H), 8.21 (d, J=8.6Hz, 1H),
5-((6-chloro-1H-indol-3-yl)methylene)imidazolidine-2,4-dione 7.67–7.57 (m, 1H), 7.52 (d, J=1.9Hz, 1H), 7.44–7.33 (m,
(4a) and 4-(chloromethyl)-1,2-difluorobenzene according to 2H), 7.16 (dd, J=8.5, 1.9Hz, 1H), 7.09 (s, 1H), 4.59 (s, 2H);
the general procedure for benzylation; faint yellow solid; 13C-NMR (75MHz, DMSO) δ: 169.91, 158.60, 151.40, 147.38,
1
yield: 1.74g (45%); H-NMR (300MHz, DMSO) δ: 11.97 (s, 136.82, 135.42, 135.13, 132.94, 127.08, 125.80, 125.35, 121.22,
1H), 10.49 (s, 1H), 8.19 (s, 1H), 7.82 (d, J=8.5Hz, 1H), 7.48 120.75, 117.73, 117.73, 115.82, 111.79, 111.24, 32.11; MS (ESI):
(d, J=1.8Hz, 1H), 7.46–7.33 (m, 2H), 7.13 (dd, J=8.5, 1.9Hz, m/z=403.60 (M)+; purity: 96%.
2H), 6.88 (s, 1H), 4.68 (s, 2H); 13C-NMR (75MHz, DMSO) δ:
5-((6-Chloro-1H-indol-3-yl)methylene)-2-thioxo-3-(4-
163.60, 162.32, 154.38, 150.87, 136.18, 134.45, 128.08, 127.07, (trifluoromethyl)benzyl)imidazolidin-4-one (5i)
125.67, 124.25, 122.58, 120.52, 119.73, 117.64, 116.67, 111.53,
The title compound was prepared by reaction of
108.47, 103.05, 40.58; MS (ESI): m/z=387.77 (M)+; purity: 5-((6-chloro-1H-indol-3-yl)methylene)-2-thioxoimidazolidin-4-
98.5%.
one (4b) and 1-(chloromethyl)-4-(2,2,2-trifluoroethyl)benzene
5-((6-Chloro-1H-indol-3-yl)methylene)-3-(4-(trifluoromethyl)- according to the general procedure for benzylation; brick
1
benzyl)imidazolidine-2,4-dione (5e)
red solid; yield: 1.74g (40%); H-NMR (300MHz, DMSO) δ:
The title compound was prepared by reaction of 11.95 (s, 1H), 11.63 (s, 1H), 8.39 (d, J=2.7Hz, 1H), 8.19 (d,
5-((6-chloro-1H-indol-3-yl)methylene)imidazolidine-2,4- J=8.6Hz, 1H), 7.80–7.68 (m, 4H), 7.52 (d, J=1.8Hz, 1H), 7.14
dione (4a) and 1-(chloromethyl)-4-(2,2,2-trifluoroethyl)- (dd, J=8.5, 1.9Hz, 1H), 7.06 (s, 1H), 4.69 (s, 2H); 13C-NMR
benzeneaccording to the generalprocedure for benzylation; (75MHz, DMSO) δ: 169.90, 158.56, 142.61, 136.78, 135.13,
yellow solid; yield: 2.31g (55%); 1H-NMR (300MHz, 133.01, 129.63, 127.92, 127.06, 125.99, 125.34, 122.39, 121.20,
DMSO-d6) δ: 11.98 (s, 1H), 10.53 (s, 1H), 8.20 (s, 1H), 7.83 120.73, 115.86, 111.78, 111.22, 32.58; MS (ESI): m/z=435.67
(d, J=8.5Hz, 1H), 7.72 (d, J=8.2Hz, 2H), 7.52 (d, J=8.1Hz, (M)+; purity: 96%.

40
Chem. Pharm. Bull.
Vol. 64, No. 1 (2016)
5-((6-Chloro-1H-indol-3-yl)methylene)-3-(3,4-dichlorobenzyl)- MS (ESI): m/z=401.71 (M)+; purity: 98%.
thiazolidine-2,4-dione (5j)
Cell Culture Cancer cell lines cultured included wild-
The title compound was prepared by reaction of type p53 cell line (HCT-116), and p53 null cell line (H1299)
5-((6-chloro-1H-indol-3-yl)methylene)thiazolidine-2,4-dione which were obtained from the American Type Culture Col-
(4c) and 1,2-dichloro-4-(chloromethyl)benzene according to lection (ATCC). Both cell lines were cultured in a 37°C
the general procedure for benzylation; yellow solid; yield: humidified incubator with 5% CO2 with the same medium
1.22g (28%); 1H-NMR (300MHz, DMSO-d6) δ: 12.23 (s, (RPMI-1640 supplemented with 5% fetal bovine serum), and
1H), 8.18 (s, 1H), 7.93 (d, J=8.6Hz, 1H), 7.83 (s, 1H), 7.60 (d, passaged twice weekly. Only cultures exhibiting greater than
J=8.3Hz, 2H), 7.53 (d, J=8.0, 1H), 7.28 (dd, J=8.0, 1.1Hz, 95% viability were used in growth inhibition experiments (de-
1H), 7.19 (dd, J=8.5, 1.4Hz, 1H), 4.82 (s, 2H); 13C-NMR termined by trypan blue exclusion).
(75MHz, DMSO) δ: 167.56, 165.77, 137.28, 137.12, 131.63,
Growth Inhibition Assay Cells were seeded in 96-well
131.34, 130.93, 130.52, 130.32, 128.45, 128.26, 126.35, 126.02, tissue culture-treated assay plates at a density of 1.5×104 cells/
121.91, 120.60, 114.97, 112.56, 111.01, 43.86; MS (ESI): cm2, then allowed to attach overnight before addition of ex-
m/z=437.62 (M)+; purity: 97%.
perimental compounds. Compounds were dissolved in DMSO,
5-((6-Chloro-1H-indol-3-yl)methyl)imidazolidine-2,4-dione then diluted to the final concentration indicated so as to
(6)
Synthesized by
ensure DMSO concentration less than 0.2%. After treatment
solution of with either a single screening concentration or a titration se-
a
reaction between
a
5-((6-chloro-1H-indol-3-yl)methylene)imidazolidine-2,4-dione ries of concentrations of compounds, cells were incubated for
(4a) (2mmol) in a mixed solvent of DMF–isopropanol (1:2, an additional 72h. Relative cell growth was determined by ad-
60mL), CoCl2 (12mmol), acetic acid (90mmol) and NaBH4 dition of PromegaCellTiterGlo luciferase-based assay of ATP
(120mmol) portion wise. The mixture was stirred at room content. The resultant luminescence was measured, and each
temperature overnight and then diluted with ethyl acetate data set was analyzed using DMSO (vehicle control) as a base-
(100mL). The mixture was washed sequentially with saturated line value for growth inhibition. GraphPad Prism software
NaHCO3 (50mL), 1N HCl (50mL), saturated NaCl (50mL) and was used to develop dose–response curves and IC50 values for
then dried over anhydrous MgSO4, filtered and concentrated. active compounds.
The crude residue was then purified by column chromatogra-
Immunofluorescent Labeling p53 Cells were seeded
1
phy on silica gel; white powder; yield: 0.13g (25%); H-NMR in 96-well tissue culture-treated assay plates at a density of
(300MHz, DMSO) δ: 11.11 (s, 1H), 11.01 (s, 1H), 8.37 (s, 1.5×104 cells/cm2, then allowed to attach overnight before ad-
1H), 7.51 (d, J=8.5Hz, 1H), 7.36 (d, J=1.8Hz, 1H), 6.92 (dd, dition of experimental compounds.
J=8.5, 1.9Hz, 1H), 6.35–6.27 (m, 1H), 4.46 (t, J=4.1Hz, 1H),
Compound Treatment: Compounds were dissolved in
3.18 (dd, J=14.9, 4.3Hz, 1H), 3.06 (dd, J=14.9, 4.3Hz, 1H); DMSO, then diluted to the final concentration indicated so as
13C-NMR (75MHz, DMSO) δ: 174.02, 156.51, 136.16, 127.27, to ensure DMSO concentration less than 0.2%. Experimental
125.59, 122.93, 120.09, 118.68, 110.85, 107.58, 57.18, 25.96.
5-((6-Chloro-1H-indol-3-yl)methyl)-3-(3,4-difluorobenzyl)- ing equal concentration of DMSO vehicle among all tested
imidazolidine-2,4-dione (7) compound concentrations. After 20h treatment period, cells
compounds were added to cells in equal volume, maintain-
The title compound was prepared by reaction of were fixed with 4% formaldehyde (Sigma-Aldrich) for 20min
5-((6-chloro-1H-indol-3-yl)methyl)-2-thioxoimidazolidin-4-one at room temperature.
(6) and 4-(chloromethyl)-1,2-difluorobenzene according to the
Fixative was removed, and samples washed thrice in phos-
general procedure for benzylation; white solid; yield: 1.55g phate buffered saline (PBS). Nonspecific binding sites were
1
(40%); H-NMR (300MHz, DMSO) δ: 11.01 (s, 1H), 8.37 (s, blocked and cells permeabilized by 60min incubation with
1H), 7.51 (d, J=8.5Hz, 1H), 7.36 (d, J=1.8Hz, 1H), 7.11 (d, 5% fetal bovine serum and 0.3% Triton X-100 in 0.9% PBS.
J=2.3Hz, 1H), 7.04 (dt, J=10.8, 8.5Hz, 1H), 6.92 (dd, J=8.5, Samples were incubated overnight at 4°C with anti-p53 rab-
1.9Hz, 1H), 6.76 (ddd, J=11.3, 7.8, 2.0Hz, 1H), 6.35–6.27 bit monoclonal antibody (Cell Signaling Technology) diluted
(m, 1H), 4.46 (t, J=4.1Hz, 1H), 4.34 (d, J=15.7Hz, 1H), 4.22 1:1600 in antibody dilution buffer (1% bovine serum albumin
(d, J=15.7Hz, 1H), 3.18 (dd, J=14.9, 4.3Hz, 1H), 3.06 (dd, and 0.3% Triton X-100 in PBS). Samples were washed thrice
J=14.9, 4.3Hz, 1H); 13C-NMR (75MHz, DMSO) δ: 173.62, in PBS. Samples were then incubated 2h at room temperature
156.27, 151.07, 147.10, 136.16, 133.95, 127.27, 125.90, 125.59, with Alexa Fluor 488 anti rabbit polyclonal antibody conjugate
122.93, 120.09, 118.68, 116.90, 115.68, 110.85, 107.58, 57.18, (Life Technology) diluted 1:500 in antibody dilution buffer,
41.12, 25.96; MS (ESI): m/z=389.71 (M)+; purity: 97.6%.
5-((6-Chloro-1H-indol-3-yl)methylene)-3-(3,4-difluorobenzoyl)- (Sigma-Aldrich; 1µg/mL) for 30min at room temperature.
imidazolidine-2,4-dione (8)
Imaging Assay: Samples were imaged using the Molecu-
then washed thrice in PBS. Nuclei were stained with DAPI
The title compound was prepared by reaction of lar Devices ImageXpressMicroXL high content screening
5-((6-chloro-1H-indol-3-yl)methylene)imidazolidine-2,4-dione instrument fitted with a 20× ELWD Nikon objective lens.
(4a) and 3,4-difluorobenzoyl chloride according to the gen- DAPI and AlexaFluor 488 fluorophores were visualized using
eral procedure for benzylation; dark yellow solid; yield: 3.00g 405ex/450em and 488ex/525em filter sets, respectively. Four
1
(75%); H-NMR (300MHz, DMSO) δ: 11.33 (s, 1H), 10.58 (s, identically located fields from within each well were captured
1H), 8.36 (d, J=1.8Hz, 1H), 8.14 (s, 1H), 8.10–7.99 (m, 2H), in each color channel. MetaXpress image analysis software
7.78 (dd, J=8.8, 4.5Hz, 2H), 7.52 (dd, J=8.5, 1.9Hz, 1H), 6.64 was used to segment images and measure p53-associated fluo-
(s, 1H); 13C-NMR (75MHz, DMSO) δ: 166.70, 165.43, 155.96, rescent intensity within the nuclear and cytoplasmic region of
152.54, 149.74, 142.56, 136.04, 131.14, 130.69, 129.01, 128.83, each cell. Average intensity values, cell number, and nuclear
127.90, 125.15, 121.06, 119.78, 118.65, 116.11, 114.52, 97.39; to cytoplasmic intensity ratio for each well are reported.

Vol. 64, No. 1 (2016)
Chem. Pharm. Bull.
41
7) Tang Y., Zhao W., Chen Y., Zhao Y., Gu W., Cell, 133, 612–626
(2008).
Western Blotting After lysing cells in 0.5% NP-40
buffer, the lysates were resolved on a 12.5% polyacrylamide
gel and then transferred to a 0.2-µm nitrocellulose membrane.
Membranes were blocked for at least 30min in PBS block-
ing buffer with 0.1% Tween-20 (PBST) and 5% non-fat dried
milk. Membranes were incubated for 2h to overnight with
the appropriate primary antibody, incubated for 1–2h in sec-
ondary horseradish peroxidase (HRP)-conjugated antibody,
and exposed with Supersignal West Pico or Dura reagent
(Pierce). Mouse monoclonal MDM2 (2A10, Calbiochem), p53
(NCL-505, Leica), actin (MAB1501, Chemicon International),
and goat polyclonal p21 (C-19; Santa Cruz) antibodies were
purchased commercially.
Fluorescence Polarization Assay The assay was per-
formed as previously described by Vogel et al.21) Briefly, the
association of a FAM labeled peptide (derived from the amino
terminus of p53 from recombinant MDM2 (AA16–116) or
MDM4 (AA2–125) was measured by anisotropy. Dissociation
constants were determined by direct competition of the tested
compounds.
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Acknowledgments The authors are grateful to the Alex-
ander von Humboldt Foundation, Bonn, Germany for partial
sponsoring of this work through a “Research groups linkage
Program” for Prof. Dr. Ashraf Abadi, German University in
Cairo, Egypt and Prof. Dr. Frank Boeckler, University of Tue-
bingen, Germany.
Conflict of Interest The authors declare no conflict of
interest.
20) Shangary S., Qin D., McEachern D., Liu M., Miller R. S., Qiu S.,
Nikolovska-Coleska Z., Ding K., Wang G., Chen J., Bernard D.,
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