Novel substituted (Z)-5-((N-benzyl-1H-indol-3-yl)methylene)imidazolidine-2,4-diones and 5-((N-benzyl-1H-indol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-triones as potent radio-sensitizing agents

Article abstract of DOI:10.1016/j.bmcl.2009.11.082

A series of (Z)-5-((N-benzyl-1H-indol-3-yl)methylene)imidazolidine-2,4-dione (9a-9m) and 5-((N-benzyl-1H-indol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (10a-10i) derivatives that incorporate a variety of aromatic substituents in both the indole and N-benzyl moieties have been synthesized. These analogs were evaluated for their radiosensitization activity against the HT-29 cell line. Three analogs, 10a, 10b, and 10c were identified as the most potent radiosensitizing agents.

Full text of DOI:10.1016/j.bmcl.2009.11.082

Bioorganic & Medicinal Chemistry Letters 20 (2010) 600–602
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel substituted (Z)-5-((N-benzyl-1H-indol-3-yl)methylene)imidazolidine-
2,4-diones and 5-((N-benzyl-1H-indol-3-yl)methylene)pyrimidine-
2,4,6(1H,3H,5H)-triones as potent radio-sensitizing agents
Y. Thirupathi Reddy a, , Konjeti R. Sekhar b, , Nidhish Sasi b, P. Narsimha Reddy a,
Michael L. Freeman b, Peter A. Crooks a,
*
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA
b Department of Radiation Oncology/Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of (Z)-5-((N-benzyl-1H-indol-3-yl)methylene)imidazolidine-2,4-dione (9a–9m) and 5-((N-ben-
zyl-1H-indol-3-yl)methylene)pyrimidine-2,4,6(1H,3H,5H)-trione (10a–10i) derivatives that incorporate
a variety of aromatic substituents in both the indole and N-benzyl moieties have been synthesized. These
analogs were evaluated for their radiosensitization activity against the HT-29 cell line. Three analogs,
10a, 10b, and 10c were identified as the most potent radiosensitizing agents.
Received 14 October 2009
Revised 16 November 2009
Accepted 17 November 2009
Available online 22 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Aplysinopsin analogs
Radiosensitizing agents
Indolyl barbiturates
Indolyl hydantoins
Rectal cancer is one of the most frequent cancers in Western
countries, and over 40,000 Americans were diagnosed with rectal
cancer in 2008.1 Radiation therapy has been used widely as adju-
vant therapy with or without chemotherapy to improve treatment
outcomes. Postoperatively administered radio-chemotherapy pro-
vides improved local control and overall survival in locally ad-
vanced disease. Debulking of tumors using ionizing radiation
provides another opportunity to decrease recurrence events due
to an improved ability to obtain radial margins.2 The colorectal
adenocarcinoma cells (HT-29) used in the present study express
the cancer stem cell markers CD44 and EpCAM,3 and are extremely
resistant to radiation.4 Failure to control primary disease leads to
progression to devastating invasive disease, which is associated
with an increased risk of metastasis and cancer mortality.5,6 Unfor-
tunately, in cases of aggressive disease, ionizing radiation does not
always provide local-regional control. Hence, there is an urgent
need to develop radiosensitizers.
hypothesis.9–12 Chromosomal aberrations induced by ionizing
radiation are a consequence of misrepaired or unrepaired DNA
double strand breaks, which contribute significantly to radiation-
induced cell death.13 Hyperthermia suppresses the rejoining of
DNA double strand breaks induced by ionizing radiation, but does
not affect the formation of radiation-induced breaks.14 Thus, heat
shock enhances the formation of radiation-induced chromosomal
aberration by inhibiting the repair of DNA double strand breaks.
Although many recent clinical studies have demonstrated the ther-
apeutic benefit of adjuvant hyperthermia in treating aggressive
cancers such as chest wall, cervical, bladder, and head and neck,16
technical problems in heat delivery has severely hampered the
clinical use of hyperthermia.10,15
In order to overcome the problem of inadequate thermal dosing,
we have used an unbiased compound discovery program that em-
ployed a forward chemical genetics screen to identify small mole-
cule indole-based compounds that increase (Ptwo-fold) thermal
sensitivity and thermal radiosensitization of tumor cells at the clin-
ically achievable temperature of 41 °C. We have used indomethacin
as a structural platform to design novel thermal-sensitizers.
Previously, (Z)-2-(N-benzyl-indol-3-ylmethylene)quinuclidin-3-one,
(Z)-( )-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol, (Z)-2-(N-
benzenesulfonylindol-3-ylmethylene)quinuclidin-3-one, (Z)-( )-2-
(N-benzenesulfonylindol-3-yl-methylene)qui- nuclidin-3-ol, and
(Z)-( )-2-(N-(4-chlorobenzyl) indol-3-ylmethylene)quinuclidin-3-ol
(1–5, respectively; Fig. 1) were identified as potent thermal-sensi-
Therapeutic hyperthermia (temperatures greater than 42 °C) is
one of the most potent radiation sensitizers identified to date.7
Cytotoxicity exerted by heat shock is believed to be the conse-
quence of changes in protein conformation, which includes protein
unfolding and aggregation.8 Several studies have confirmed this
* Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257 7585.
E-mail address: pcrooks@email.uky.edu (P.A. Crooks).
These authors equally contributed to this Letter.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.082

Y. T. Reddy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 600–602
601
Figure 1. Chemical structures of the potent thermal radiosensitizing agents (1–5)
and potent aplysinopsin anti-cancer agents (6).
tizers that could lower the threshold needed for thermal sensitivity
to radiation treatment.17,18
Scheme 1. Reagents and conditions: (a) substituted benzyl halide, 50% NaOH aq,
triethylbenzylammonium chloride, DCM, rt; (b) NH4OAc, AcOH, microwave irradi-
ation, 40–60 s; (c) MeOH, rt, 4–6 h.
The indole nucleus plays an important role in the activity of
these compounds, as lack of an indole moiety produced inactive
compounds.17 Also, absence of the N-benzyl group also results in
inactive or less active compounds. N-Benzenesulfonyl derivatives
(3 and 4) were found to be less potent than N-benzyl or N-4-chlo-
robenzyl derivatives (2 and 5).
Aplysinopsin analogs (6; Fig. 1) have structural similarities to
compounds 1–5, and are reported to be potent cytotoxic agents
against various cancer cells. These observations prompted us to de-
sign, synthesize, and evaluate the radiosensitization activity of a
series of (Z)-5-((N-benzyl-1H-indol-3-yl)methylene)imidazol-
idine-2,4-diones (9a–9m) and 5-((N-benzyl-1H-indol-3-yl)methy-
lene)pyrimidine-2,4,6(1H,3H,5H)triones (10a–10i), and also to
undertake a more detailed investigation of the structure–activity
relationships of these analogs that incorporate a variety of different
aromatic substituents in both the N-benzyl moiety and the indole
moiety. The synthetic routes to these two groups of analogs are
illustrated in Scheme 1.
The appropriate substituted N-benzylindole-3-carboxaldehyde
8 was prepared in 85–90% yield by the reaction of the correspond-
ing indole-3-carboxaldehyde 7 with various substituted benzyl ha-
lides under phase-transfer catalytic (PTC) conditions utilizing
triethylbenzylammonium chloride and a 50% w/v aqueous NaOH
solution in dichloromethane.
The substituted (Z)-5-((N-benzyl-1H-indol-3-yl)methylene)imi-
dazolidine-2,4-dione derivatives (9a–9m) were prepared in 80–
85% yield by the condensation of 8 with hydantoin in the presence
of NH4OAc in acetic acid under microwave irradiation conditions.
The condensation of 8 with barbituric acid in methanol at room
temperature afforded the corresponding 5-((N-benzyl-1H-indol-
and then incubated at 37 °C for 90 min. Analog was then washed off,
and cells were incubated in fresh medium for 14 days. Viable cells
formed colonies that were stained with crystal violet and counted.
The plating efficiency was defined as the ratio of the number of col-
onies counted divided by the initial number of cells exposed to vehi-
cle control (DMSO) for 2 h. Vehicle treatment (2 h) was not toxic to
cells, as survival was not significantly different from plating effi-
ciency measured in the absence of vehicle (p>0.05 Student’s t-test)
and was arbitrarily set at 1.0. All analogs were tested at 25 lM.
The percentage of surviving cells following exposure to analog alone
was 70% or greater. Induction of heat shock protein 70 (Hsp70) and
activation of heat shock factor 1 (Hsf1) was studied with analog 10a;
these results will be published elsewhere. The surviving fraction of
cells irradiated in the absence or presence of analog (2 h/37 °C)
was calculated from the ratio of the number of colonies counted di-
vided by the initial number of cells treated. The surviving fraction is
corrected for both plating efficiency and analog toxicity. Radiosensi-
tization is defined as a decrease in survival following exposure to
radiation plus compound that exceeds 25%.
(Z)-5-((N-Benzyl-1H-indol-3-yl)methylene)imidazolid-ine-2,4-
dione derivatives (9a–9m, Table 1) that incorporate a variety of
aromatic substituents in both the indole and N-benzyl moieties
did not show any radiosensitization activity against the HT-29 cell
line at 25
The
l
M concentration.
5-((N-benzyl-1H-indol-3-yl)methylene)pyrimidine-2,4,
6(1H,3H,5H)trione derivatives (10a–10i, Table 2) exhibited greater
radiosensitization activity over the corresponding (Z)-5-((N-ben-
zyl-1H-indol-3-yl)methyl-lene)imidazolidine-2,4-dione deriva-
tives. Introduction of a variety of aromatic substituents into the
indole moiety of 5-((N-benzyl-1H-indol-3-yl)methylene)pyrimi-
dine-2,4,6(1H,3H,5H)trione derivatives (10e, 10f, and 10g) did not
increase radiosensitization activity compared to solvent control
(p<0.05 Student’s t-test). However, introduction of electron with-
drawing groups, such as 4-CN, 4-NO2, or 4-COOCH3 into the N-ben-
zyl moiety (i.e., compounds 10a, 10b, and 10c) significantly
increased radiosensitization activity compared to solvent control
(p<0.05 Student’s t-test).
3-yl)methylene)pyrimidine-2,4,6-(1H,3H,5H)-trione
derivatives
(10a–10i) in 85–90% yield. All the synthesized compounds were
characterized by 1H NMR and 13C NMR spectrometry and HRMS
analysis.19 The geometry of the double bond in the substituted
(Z)-5-((N-benzyl-1H-indol-3-yl)-methylene)imidazolidine-2,4-
dione derivatives (9a–9m) was established from X-ray crystallo-
graphic data.20,21
Colony formation assays of tumor cells were used to assess the
potency of newly synthesized analogs.17 HT-29 cells growing expo-
nentially in T25 flasks (n = 4) were exposed to DMSO/analog for
30 min, irradiated (4 Gy) at room temperature using a Cs137 source,

602
Y. T. Reddy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 600–602
Table 1
incorporating an electron withdrawing substituent such as –CN,
–NO2, or –COOCH3 at the 4-position of the N-benzyl group, exhibit
potent radiosensitizing properties. The present study demonstrates
for the first time that novel indole derivatives that mimic heat
shock can be designed and synthesized.
Relative survival of cultured HT-29 cells and the relative potency of substituted (Z)-5-
((N-benzyl-1H-indol-3-yl)methylene)-imidazolidine-2,4-diones determined follow-
ing exposure to 25
lM concentration of analog and 4 Gy radiation
Analog
Relative survivala of HT-29 cells
Relative potencyb
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
1.53
1.30
1.14
1.44
1.00
1.18
0.91
1.38
1.29
1.18
1.06
1.38
1.06
1.00
0.65
0.77
0.88
0.69
1.00
0.85
1.10
0.72
0.77
0.85
0.94
0.72
0.94
1.00
Acknowledgements
This research was supported by NIH/National Cancer Institute
Grant PO1 CA104457 and Pilot Project support from P50 CA95103.
References and notes
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a
Relative survival represents survival following radiation plus analog divided by
that produced by irradiation alone.
b
Relative potency is defined as the reciprocal of relative survival value.
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Table 2
Relative survival of cultured HT-29 cells and the relative potency of substituted 5-((N-
benzyl-1H-indol-3-yl)methylene) pyrimidine-2,4,6(1H,3H,5H)trione analogs deter-
mined following exposure to 25 lM concentration and 4Gy radiation
Analog
Relative survivala of HT-29 cells
Relative potencyb
10ª
10b
10c
10d
10e
10f
10g
10h
10i
0.23c
0.64c
0.44c
1.35
1.38
1.73
0.91
1.18
1.14
1.00
4.35c
1.56c
2.27c
0.74
0.72
0.58
1.10
0.85
0.88
1.00
12. Nguyen, V. T.; Morange, M.; Bensaude, O. J. Biol. Chem. 1989, 264, 10487.
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Vehicle
a
Relative survival represents survival following radiation plus analog divided by
19. Analytical data and yields for four of the most active compounds: (9a): 1H NMR
(DMSO-d6): d 5.56 (s, 2H), 6.73 (s, 1H), 7.13–7.23 (m, 2H), 7.44–7.46 (d, 2H),
7.51–7.53 (d, 1H), 7.56 (m, 3H), 8.30 (s, 1H), 10.15 (br s, 1H), 11.07 (br s, 1H);
13C NMR (DMSO-d6): d 50.0, 101.4, 109.2, 111.0, 111.2, 119.2, 119.3, 121.3,
123.4, 124.9, 128.1, 128.7, 130.6, 133.2, 136.2, 143.7, 155.9, 165.8. HRMS (EI+):
m/z found 342.1117, calcd C20H14N4O2 (EI+) 342.1116; Yield: 93%: (10a): 1H
NMR (DMSO-d6): d 5.81 (s, 2H), 7.33–7.36 (m, 2H), 7.43–7.45 (d, 2H), 7.61–7.64
(m, 1H), 7.82–7.84 (d, 2H), 7.90–7.93 (m, 1H), 8.68 (s, 1H), 9.63 (s, 1H), 11.07
(br s, 1H), 11.15 (br s, 1H); 13C NMR (DMSO-d6): d 49.7, 109.4, 110.5, 110.8,
111.8, 117.9, 118.4, 123.0, 123.8, 128.0, 129.6, 132.6, 136.1, 141.7, 141.8, 142.6,
159.2, 162.9, 164.1. HRMS (EI+): m/z found 370.1066, calcd C21H14N4O3 (EI+)
370.1067; Yield: 95%: (10b): d 3.82 (s, 3H), 5.79 (s, 2H), 7.33–7.36 (m, 2H),
7.40–7.42 (d, 2H), 7.61–7.63 (m, 1H), 7.91–7.93 (m,1H), 7.94–7.95 (d, 2H), 8.68
(s, 1H), 9.62 (s, 1H), 11.06 (br s, 1H), 11.14 (br s, 1H); 13C NMR (DMSO-d6): d
49.9, 52.1, 109.2, 110.7, 111.8, 117.8, 122.9, 123.7, 127.4, 129.0, 129.5, 129.6,
136.2, 141.4, 141.7, 142.6, 150.1, 162.8, 164.1, 165.5. HRMS (EI+): m/z found
403.1168, calcd C22H17N3O5 (EI+) 403.1167; Yield: 94%: (10c): 1H NMR (DMSO-
d6): d 5.81 (s, 2H), 7.33–7.36 (m, 2H), 7.43–7.45 (d, 2H), 7.61–7.64 (m, 1H),
7.82–7.84 (d, 2H), 7.90–7.93 (m, 1H), 8.68 (s, 1H), 9.63 (s, 1H), 11.07 (br s, 1H),
11.15 (bs, 1H); 13C NMR (DMSO-d6): d 49.7, 109.4, 110.5, 110.8, 111.8, 117.9,
118.4, 123.0, 123.8, 128.0, 129.6, 132.6, 136.1, 141.7, 141.8, 142.6, 159.2, 162.9,
164.1. HRMS (EI+): m/z found 390.0964, calcd C20H14N4O5 (EI+) 390.0965;
Yield: 93%:
that produced by irradiation alone.
b
Relative potency is defined as the reciprocal of relative survival value.
These analogs produced significant radiosensitization (p<0.05 Student’s t-test).
c
The goal of the present study was to develop analogs that mimic
heat shock which could be used as radiosensitizers at physiological
temperatures. As discussed above, our initial indole analogs (1–5)
exhibited thermal sensitization only when the cells were heated
at 41 °C. In the present study, the second generation indole deriv-
atives exhibited radio-sensitization at physiological temperature,
37 °C. Compound 10a was studied in detail to demonstrate its abil-
ity to induce Hsp70 protein expression and to activate Hsf1. Also,
several other cancer cell lines, including pancreatic cancer (Panc1),
lung cancer (H460), and breast cancer (MCF-7) cell lines were
radiosensitized in the presence of compound 10a (unpublished
data). These results also provide evidence to support the hypothe-
sis that radiosensitization by heat shock is independent of cancer
cell type.
20. Penthala, N. R.; Yerramreddy, T. R.; Parkin, S.; Crooks, P. A. Acta Crystallogr., Sect.
E 2008, 64, 2122.
21. Penthala, N. R.; Yerramreddy, T. R.; Parkin, S.; Crooks, P. A. Acta Crystallogr., Sect.
E 2009, E65, 62.
In conclusion, novel compounds containing an N-benzylindole
nucleus linked to a barbituric acid moiety via a double bond, and