Syntheses and In Vitro Anticancer Properties of Novel Radiosensitizers

Article abstract of DOI:10.1111/j.1747-0285.2012.01442.x

Series of 4-(ethylsulfonyl)-1-halogen-2-nitrobenzene (3a-e) and 1-(4-halogen-3-nitrophenyl) propan-1-one (5a-d) analogs designed as novel radiosensitizers using bromonitropropiophenone and bromonitrobenzonitrile as lead compounds were synthesized. The anticancer activities of the compounds were evaluated in vitro using human prostate cancer (DU-145) and breast cancer (MCF-7) cell lines and the MTT assay. From the series, six compounds (3b-e, 5b-c) exhibited potent growth inhibitory effects against both cell lines. The most active, compound 3d, is an iodosulfone and is significantly more potent than the lead compound 5c at 10μm. Compounds were then compared with doxorubicin, a clinically used anticancer compound for breast and prostate cancers. Our most active compound 3d is more effective than doxorubicin at the dose level of 10μm at 3days after radiation, cell viabilities of 18%, 13% compared to 87%, 94% against MCF-7, and 15%, 20% compared to 60%, 75% against DU-145 without and with radiation, respectively. At 10μm, compound 5c had no effects as compared to control, whereas compound 3d reduced DU-145 cell viability to 16% and that of MCF-7 cells to 9% even at 5days after radiation. These results are very encouraging. Future studies include testing the compounds in vivo with and without radiation. Our most active compound 3d is significantly more effective than doxorubicin or lead compound 5c at the dose level of 10μm, without and with radiation; and in MCF-7 and DU-145 cell lines. These results are very encouraging. Future studies include testing the compounds in vivo without and with radiation.

Full text of DOI:10.1111/j.1747-0285.2012.01442.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01442.x
Chem Biol Drug Des 2012; 80: 853–861
Research Article
Syntheses and In Vitro Anticancer Properties of
Novel Radiosensitizers
Zeynep Ates-Alagoz1,2, Natalia Coleman1,
has been suggested that abnormalities of structure and function in
tumor vessels can lead to decreased oxygen delivery to tumor tissue
(2), which can become hypoxic and resistant to radiation therapy and
chemotherapy (3,4). An increase in radiation dosage can combat these
Marlena Martin1, Aaron Wan1 and Adeboye
Adejare1,*
1Department of Pharmaceutical Sciences, Philadelphia College of
radioresistant hypoxic tumor cells. However, this increase can cause
damage to normal surrounding stroma and presents increased risk for
radiation-induced secondary cancers (5). Radiation doses >60 Gy
appear to exponentially increase the risk for secondary bone sarco-
mas (6). Tumor hypoxia can be exploited for selective anticancer drug
treatment using hypoxic cell cytotoxins or hypoxic cell radiosensitizers
(7).
Pharmacy, University of the Sciences in Philadelphia, Philadelphia,
PA 19104, USA
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Ankara University, 06100 Tandogan, Ankara, Turkey
*Corresponding author: Adeboye Adejare, a.adejar@usciences.edu
Series of 4-(ethylsulfonyl)-1-halogen-2-nitrobenzene
(3a–e) and 1-(4-halogen-3-nitrophenyl) propan-1-one
(5a–d) analogs designed as novel radiosensitizers
Radiosensitizers are intended to enhance tumor cell killing while
having much less effects on normal tissues (8). Hypoxic cell radio-
sensitizers can have oxygen-mimicking effects on hypoxic tumor
cells. This leads to repairs in the radiation-induced damaged DNA
or other biomolecules (9). In vitro and in vivo studies have demon-
strated that many chemotherapeutic agents, given at subcytotoxic
doses, are capable of radiosensitizing activity in tumoral but not in
normal cells. When used in chest radiotherapy, synergistic response
is observed with these drugs. It is assumed that radiosensitization
increases the lethal damage to the tumor caused by radiotherapy
and decreases the ability of cancer cells to repair the damage,
which suggests that the maximum synergistic effects can be
obtained when the treatments are given in tandem (10).
using
bromonitropropiophenone
and
bro-
monitrobenzonitrile as lead compounds were syn-
thesized. The anticancer activities of the
compounds were evaluated in vitro using human
prostate cancer (DU-145) and breast cancer (MCF-
7) cell lines and the MTT assay. From the series,
six compounds (3b–e, 5b–c) exhibited potent
growth inhibitory effects against both cell lines.
The most active, compound 3d, is an iodosulfone
and is significantly more potent than the lead
compound 5c at 10 lM. Compounds were then
compared with doxorubicin, a clinically used anti-
cancer compound for breast and prostate cancers.
Our most active compound 3d is more effective
than doxorubicin at the dose level of 10 lM at
3 days after radiation, cell viabilities of 18%, 13%
compared to 87%, 94% against MCF-7, and 15%,
20% compared to 60%, 75% against DU-145 with-
out and with radiation, respectively. At 10 lM,
compound 5c had no effects as compared to con-
trol, whereas compound 3d reduced DU-145 cell
viability to 16% and that of MCF-7 cells to 9%
even at 5 days after radiation. These results are
very encouraging. Future studies include testing
the compounds in vivo with and without radiation.
Radiosensitizer capabilities of compound 5c (Figure 1) have been
reported (11). It exhibited the highest average radiosensitivity factor
(RSF) of 4.62 among 870 compounds and was selected for further
investigation (11). Compound 5c radiosensitizes U251 glioma tumor
cells, HT-29 colorectal cancer cells and A549 lung tumor cells and
does not radiosensitize normal cells.
Key words: anticancer, bromonitropropiophenone, ethylsulfonyl
nitrobenzene, MTT assay, radiosensitizers
Received 2 April 2012, revised 23 May 2012 and accepted for publica-
tion 19 June 2012
The growth and survival of cells in solid tumors are dependent on
adequate supply of oxygen and nutrients that diffuse from blood ves-
sels and are consumed by tumor cells (1). Oxygen consumption in
tumor tissue is increased by a high level of tumor cell proliferation. It
Figure 1: Structure of compound 5c.
853

Ates-Alagoz et al.
the presence of tellurium, rongalite, and 1 M aqueous sodium
hydroxide (12,13) gave ethylsulfonyl derivatives (2a–d). This was
followed by reaction with conc. H2SO4 and potassium nitrate to
give target novel nitro compounds (14). Syntheses of carbonyl deriv-
atives (5a–d) were carried out as shown in Scheme 2. A mixture
of HNO3 and H2SO4 was added to commercially available aryl ke-
tones (4a–c) at )15 ꢀC to give (4-halogen-3-nitrophenyl) propan-1-
one derivatives (15,16). Yields were not optimized. Compounds 3e
and 5d are commercially available and were purchased from
Sigma–Aldrich company.
1-(Ethylsulfonyl)-benzene (2a)
A reddish solution of sodium telluride, prepared by heating a mixture
of powdered tellurium (10 mmol, 1.28 g), rongalite (50 mmol,
7.71 g), and 1 M aqueous sodium hydroxide (25 mL), was added
dropwise to a stirred solution of benzenesulfonyl chloride (1a,
10 mmol, 1.94 g) and triethylbenzylammonium chloride (TEBAC)
(0.1 mmol, 0.23 g) in THF (30 mL) at room temperature under nitro-
gen. An instantaneous reaction occurred and the color of the reaction
mixture changed to deep black. After 5 min while stirring, iodoe-
thane (50 mmol, 4 mL) in THF (3 mL) was added and the resulting
mixture was kept at 90 ꢀC for 5 h. After cooling, the solvent was
removed under reduced pressure and the residue was treated with
aqueous ammonium chloride and benzene. Organic phase was sepa-
rated, dried over sodium sulfate, and the solvent evaporated. The
residue was purified by column chromatography (hexane ⁄ ethylace-
Scheme 1: General synthetic scheme to compounds 3(a–e).
1
tate 2:1) to give a white solid, m.p. 43–44 ꢀC, (0.6 g, 35% yield). H
NMR (CDCl3): 1.3 (t, 3H, CH3), 3.13 (q, 2H, CH2), 7.59 (m, 2H, Ar-H),
7.68 (m, 1H, Ar-H), 7.93 (m, 2H, Ar-H); MS (ESI, +ion): 171 M+.
Scheme 2: General synthetic scheme to compounds 5(a–d).
In view of the above and the need for new compounds with better
anticancer properties, we designed and synthesized a new series of
sulfonyl and carbonyl analogs of compound 5c possessing m-nitro
and p-halogen substituted benzene moiety (Schemes 1 and 2) to
examine their in vitro anticancer properties. Abilities of these
compounds to act as radiosensitizers and in vivo studies are being
pursued.
1-(Ethylsulfonyl)-4-chlorobenzene (2b)
Compound 2b was prepared in analogous manner to compound
2a, starting from p-chlorobenzene-1-sulfonyl chloride (1b, 10 mmol,
3.02 g) to give white oily compound (0.36 g, 18% yield). 1H NMR
(400 MHz, CDCl3) d ppm 7.86 (d, J = 8.53 Hz, 2H), 7.56 (d,
J = 8.56 Hz, 2H), 3.13 (q, J = 7.43 Hz, 2H), 1.29 (t, J = 7.44 Hz,
3H); MS (ESI, +ion): 205 M+ (100.0%), 207 (36.5%).
Materials and Methods
1-(Ethylsulfonyl)-4-fluorobenzene (2c)
Chemistry
Compound 2c was prepared in analogous manner to compound 2a,
starting from p-fluorobenzene-1-sulfonyl chloride (1c, 10 mmol,
3.02 g). The residue was purified by column chromatography (hex-
ane ⁄ ethylacetate 1:1) to give a white solid. HPLC indicated 94% pur-
Melting points were determined with a Mel-Temp electrothermal
apparatus and are uncorrected. H NMR spectra were recorded with
1
a 400 MHz Bruker NMR spectrophotometer with TMS as internal
standard and CDCl3 as solvent. Mass spectra were obtained with a
Varian 1200 Triple Quadruple instrument using electrospray ionization
(ESI) technique. Column chromatography was conducted using Merck
silica gel, grade 9385, 230–400 mesh, 60 ꢀ. HPLC was conducted
using C18 column, elution solution of water ⁄ acetonitrile ⁄ formic acid
and Hitachi Elite LaChrom instrument with UV detection.
1
ity, m.p. 39–40 ꢀC, (0.30 g, 16% yield). H NMR (CDCl3): 1.6 (t, 3H,
CH3), 3.23 (q, 2H, CH2), 7.27 (m, 2H, Ar-H), 7.94 (m, 2H, Ar-H); MS (ESI,
+ion): 189 M+. Anal. calcd. for C8H9FO2S: C, 51.05; H, 4.82; F, 10.09;
S, 17.04 and found C, 51.07; H, 4.67; F, 10.36; S,16.85.
1-(Ethylsulfonyl)-4-iodobenzene (2d)
Compound 2d was prepared in analogous manner to compound
2a, starting from pipsyl chloride (1d, 10 mmol, 3.02 g). A white
solid was obtained. HPLC indicated 100% purity, m.p. 78–79 ꢀC,
Syntheses
Syntheses of the sulfonyl derivatives (3a–d) were carried out start-
ing from commercially available aryl sulfonyl chlorides (1a–d,
Scheme 1). Alkylation of the sulfonyl chlorides with iodoethane in
1
(0.60 g, 20% yield). H NMR (CDCl3): 1.28 (t, 3H, CH3), 3.12 (q, 2H,
CH2), 7.63 (d, 2H, Ar-H), 7.96 (d, 2H, Ar-H); MS (ESI, +ion): 296 M+.
854
Chem Biol Drug Des 2012; 80: 853–861

Anticancer Properties of Novel Radiosensitizers
Anal. calcd. for C8H9IO2S. )0.2 C6H14 )0.1 H2O: C, 35.06; H, 3.83;
I, 40.26; S, 10.17 and found C, 35.04; H, 3.41; I, 40.32; S, 9.74.
was added dropwise with stirring at )15 ꢀC. After the addition
was complete, the reaction mixture was stirred at )15 ꢀC for an
additional 15 min and then poured into ice. The resulting mixture
was extracted with ether and washed with 5% NaHCO3 and water,
and the solvent was removed on a rotary evaporator. The residue
was purified by column chromatography (CH2Cl2 ⁄ hexane 30:70) to
give a light yellow solid, m.p. 50–52 ꢀC, (0.265 g, 10% yield). 1H
NMR (400 MHz, CDCl3): d ppm 8.45 (d, J = 2.01 Hz, 1H), 8.11 (dd,
J = 8.39 Hz, 1H), 7.69 (d, J = 8.39 Hz, 1H), 3.04 (q, J = 7.17 Hz,
2H), 1.32–1.24 (m, 3H); MS (ESI, +ion): 214 M+. Anal. calcd. for
C9H8ClNO3: C, 50.60; H, 3.77; Cl, 16.60; N, 6.56 and found C, 50.66;
H, 3.64; Cl, 16.57; N, 6.37.
1-(Ethylsulfonyl)-3-nitrobenzene (3a)
To a solution of 1-(ethylsulfonyl)-benzene (2a, 1.35 mmol, 0.230 g)
in sulfuric acid (1.4 mL) was added potassium nitrate (0.264 g) at
80 ꢀC. The mixture was stirred at 90 ꢀC for 2 h. Ice water (5 mL)
was added, and the mixture was extracted with ethyl acetate
(25 mL) and washed with water (20 mL) and brine (10 mL). The
organic extracts were combined and dried over sodium sulfate and
concentrated. The residue was purified by column chromatography
(hexane ⁄ ethylacetate 2:1) to give a light yellow solid, m.p. 98–
100 ꢀC, (0.22 g, 75% yield). 1H NMR (400 MHz, DMSO) d ppm
8.62–8.55 (m, 2H), 8.37–8.31 (m, 1H), 8.03–7.95 (m, 1H), 3.47 (q,
J = 7.33 Hz, 2H), 1.14 (t, J = 7.33 Hz, 3H); MS (ESI, -ion): 214 M).
Anal. calcd. for C8H9NO4S )0.01 C6H14: C, 44.80; H, 4.26; N, 6.48;
S, 14.83 and found C, 45.14; H, 4.25; N, 6.41; S, 14.78.
1-(4-fluoro-3-nitrophenyl)propan-1-one (5b)
Compound 5b was prepared in analogous manner to compound
5a, starting from 1-(4-fluorophenyl)propan-1-one (4b, 0.9 mL,
6.4 mmol) and concentrated nitric acid (0.5 mL) and concentrated
sulfuric acid (1.5 mL). The residue was purified by column chroma-
tography (ethylacetate ⁄ hexane 10:90) to give a light yellow solid.
1
4-(Ethylsulfonyl)-1-chloro-2-nitrobenzene (3b)
Compound 3b was prepared in analogous manner to compound 3a,
starting from 1-(ethylsulfonyl)-4-chlorobenzene (2b, 2.0 mmol, 0.42 g)
m.p. 25 ꢀC, (0.190 g, 15% yield). H NMR (400 MHz, CDCl3): d ppm
8.67 (dd, J = 7.13 Hz, 1H), 8.28 (ddd, J = 8.69, 2.26 Hz, 1H), 7.47–
7.38 (m, 1H), 3.03 (qd, J = 14.69, 7.23 Hz, 2H), 1.32–1.22 (m, 3H);
MS (ESI, +ion): 198 M+. Anal. calcd. for C9H8FNO3 )0.1 H2O: C,
54.32; H, 4.15; N, 7.04 and found C, 54.23; H, 4.02; N, 7.26.
1
to give a light yellow solid, m.p. 96–97 ꢀC (0.35 g, 63% yield). H
NMR (400 MHz, CDCl3) d ppm 8.42 (d, J = 2.03 Hz, 1H), 8.06 (dd,
J = 8.39 Hz, 1H), 7.83 (t, J = 6.45 Hz, 1H), 3.21 (q, J = 7.44 Hz, 2H),
1.36 (t, J = 7.44 Hz, 3H); MS (ESI, +ion): 249.99 M+ (100.00%), 251.00
(36.5). Anal. calcd. for C8H8ClNO4S: C, 38.48; H, 3.23; N, 5.61; Cl, 14.20;
S, 12.84 and found C, 38.79; H, 3.23; N, 5.25; Cl, 14.41; S, 12.64.
1-(4-bromo-3-nitrophenyl)propan-1-one (5c)
Compound 5c was prepared in analogous manner to compound 5a,
starting from 1-(4-bromophenyl)propan-1-one (4c, 1.36 g, 6.4 mmol)
and conc. nitric acid (0.5 mL) and conc. sulfuric acid (1.5 mL). The
residue was purified by column chromatography (CH2Cl2 ⁄ hexane
30:70) to give a light yellow solid, m.p. 62–64 ꢀC, (0.330 g, 20%
4-(Ethylsulfonyl)-1-fluoro-2-nitrobenzene (3c)
Compound 3c was prepared in analogous manner to compound 3a,
starting from 1-(ethylsulfonyl)-4-fluorobenzene (2c, 1.36 mmol,
0.257 g) to give a light yellow solid. HPLC indicated 98% purity,
1
yield). H NMR (400 MHz, CDCl3): d ppm 8.40 (d, J = 2.00 Hz, 1H),
8.02 (td, J = 5.58, 3.68 Hz, 1H), 7.89 (dd, J = 8.79 Hz, 1H), 3.10–
2.98 (m, 2H), 1.33–1.23 (m, 3H); MS (ESI, +ion): 259 M+. Anal.
calcd. for C9H8BrNO3: C, 41.89; H, 3.12; Br, 30.96; N, 5.43 and
found C, 42.12; H, 3.06; Br, 31.09; N, 5.48.
1
m.p. 128–131 ꢀC, (0.21 g, 66% yield). H NMR (CDCl3): d ppm 1.35
(t, 3H, CH3), 3.21 (q, 2H, CH2), 7.55 (t, 1H, Ar-H), 8.21 (m, 1H, Ar-H),
8.64 (d, 1H, Ar-H); MS (ESI, -ion): 229 M). Anal. calcd. for
C8H8FNO4S: C, 41.20; H, 3.46; N, 6.01; F, 8.15; S, 13.75 and found
C, 41.31; H, 3.35; N, 5.85; F, 8.17; S, 13.53.
Biology
We tested the effects of novel synthesized compounds (3a–e, 5a–
d) on cell proliferation in vitro using two different cell lines by
MTT, a cell viability assay.
4-(Ethylsulfonyl)-1-iodo-2-nitrobenzene (3d)
Compound 3d was prepared in analogous manner to compound
3a, starting from 1-(ethylsulfonyl)-4-iodobenzene (2d, 0.34 mmol,
0.10 g) to give a light yellow solid. HPLC indicated 98% purity, m.p.
1
124–126 ꢀC, (0.052 g, 45% yield). H NMR (CDCl3): d ppm 1.35 (t,
Cell culture
3H, CH3), 3.22 (q, 2H, CH2), 7.77 (dd, 1H, Ar-H), 8.31 (d, 1H, Ar-H),
8.34 (d, 1H, Ar-H); MS (ESI, -ion): 338.9 M). Anal. calcd. for
C8H8INO4S: C, 28.17; H, 2.36; N, 4.11; I, 37.20; S, 9.40 and found
C, 28.25; H, 2.25; N, 3.99; I, 37.39; S, 9.18.
Human prostate cancer (DU-145, ATCC) and human breast cancer
(MCF-7, ATCC) cell lines were used in the study. The cells were
routinely propagated using Eagle's minimum essential medium
(EMEM, ATCC), supplemented with 10% fetal bovine serum (ATCC)
and 1% penicillin ⁄ streptomycin (Gibco) in 100 cm2 Petri dishes
(Corning) at 37 ꢀC in 5% CO2.
1-(4-chloro-3-nitrophenyl)propan-1-one (5a)
A flask charged with 7 mL concentrated sulfuric acid was cooled to
)20 ꢀC and to this was added 1-(4-chlorophenyl)propan-1-one (4a,
2.16 g, 12.8 mmol) dropwise with stirring. Then, a mixture of con-
centrated nitric acid (1 mL) and concentrated sulfuric acid (3 mL)
Treatment
The nine compounds (3a–e, 5a–d) were obtained as solids. Stock
solutions (100 mM) were prepared in dimethylsulphoxide (DMSO).
Chem Biol Drug Des 2012; 80: 853–861
855

Ates-Alagoz et al.
The treatment concentrations were 100 and 10 lM with 0.1%
DMSO in media and 100 and 10 lM with 0.5% DMSO for com-
pounds 3d, 5c, and 5d because of compound solubility issues.
Cells were allowed to attach overnight prior to treatment. H2O2 and
100% DMSO served as positive controls, 0.1% DMSO and 0.5%
DMSO served as vehicle controls. Working concentrations were pre-
pared immediately prior to treatment by dilution into medium.
Results and Discussion
Compounds belonging to two classes, 4-(ethylsulfonyl)-1-halogen-2-
nitrobenzene (3a–e) and 1-(4-halogen-3-nitrophenyl)propan-1-one
(5a–d), were synthesized or obtained from a commercial source.
Yields were not optimized but several milligram quantities of each
compound were obtained. Structure and purity were confirmed
using several techniques, including melting point, NMR, MS, and
elemental analysis. Each target compound was tested on human
prostate (DU-145) and breast (MCF-7) cancer cell lines and com-
pared with a reference compound, doxorubicin, a clinically used
anticancer compound for breast and prostate cancers (20,21), using
MTT assay. In this assay, the number of surviving cells is directly
proportional to the level of formazan product formed because of
reduction by mitochondrial dehydrogenases in living cells. To begin
with, the inhibition effects of synthesized compounds on carcinoma
cells were tested at 100 lM concentration for 24 h (Figure 2). The
sulfonyl derivatives (3b–e) showed cytotoxic effects at 100 lM by
decreasing cell viability to 8–13%. In contrast, compound 3a that
has no halogen substituent exhibited cell proliferation activity. The
presence of a halogen substituent on the aromatic ring is implied
to be critical for inhibition of cell viability by this class of com-
pounds.
The MTT assay
The MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbro-
mide) assay is a well documented and widely used cell viability
assay. MTT is incorporated into the cell by endocytosis and is
reduced by mitochondrial enzymes in living cells to a blue-colored
formazan precipitate. The absorption of dissolved formazan in the
visible region correlates with the number of intact alive cells (17).
Cells were plated at a concentration of 13 000–30 000 cells ⁄ well
on a 96-well round-bottom plate. MTT assay (ATCC) was performed
according to manufacturer's instructions. The following determinants
were optimized for each cell line: plating cell concentration, incuba-
tion time with MTT reagent, and incubation time with detergent
reagent. Absorbance was recorded at 570 nm by a microtiter plate
reader (VICTOR; PerkinElmer). The number of surviving cells is
directly proportional to the level of the formazan product created.
This experiment was repeated on three separate occasions, and
results are presented as the mean absorbance € SD.
For the carbonyl compounds (5a–d), again, the one without halo-
gen (5d) exerted cell proliferation activities like compound 3a,
exhibiting 113% and 108% on DU-145 and MCF-7, respectively, as
compared to control. For the halogen containing ones, the trend is
Radiosensitizing activity
All compounds were evaluated for their in vitro anticancer activity in
combination with c-radiation. This study was conducted to evaluate
the ability of these compounds to enhance the cell killing effect of c-
radiation. Treatment was performed with paired 96-well plates: one
plate was exposed to 4 Gy, and the other plate was a non-irradiated
control. One hour after radiation, the media was replaced with fresh
media alone (18). The MTT assay was used to evaluate cell survival at
different time-points: 0, 1, 3, and 5 days after radiation. Irradiation
was performed at the Department of Oncology, Thomas Jefferson Uni-
versity, using Pantak XRAD 320 cabinet X-ray machine.
Figure 2: The inhibition effects of synthesized compounds on
carcinoma cells (as % of untreated control) at 100 lM concentration
(24 h, n = 3, *p < 0.05).
Clonogenic assay
The clonogenic survival assay was performed as previously
described (18). Optimal cell densities were determined for each cell
line. Control flasks for the DU-145 cell line contained 1000 cells;
control flasks for the MCF-7 cell line contained 5000 cells. Treat-
ment flasks had cell densities 10 times as many cells. Cells were
then incubated and allowed to attach overnight. The following day,
cells were treated with 10 lM concentration of compounds 3c or
3d for 24 h. One of two treated flasks was exposed to ionizing
radiation (IR) at a dose of 4 Gy. One hour later, media was changed
in all flasks. Flasks were then incubated for 14 days at 37 ꢀC in an
atmosphere of 5% CO2. On day 14, the cells were stained with
0.25% crystal violet staining solution. Media was removed, and
cells were incubated with 3 mL of staining solution for 10 min.
Flasks were then rinsed and left to air dry. Colonies were counted,
and survival was assessed using a cell efficiency rate (no. of
colonies ⁄ cell density) (19).
Table 1: Relative cell viability (as % of untreated control) of
MCF-7 and DU-145 cells, exposed for 24 h at 100 lM concentration
of novel synthesized compounds (n = 3)
Compound
Conc. (lM)
MCF-7
DU-145
Control
3a
100
100
100
100
100
100
100
100
100
100
100
103.94 € 6.88
9.49 € 0.82
7.78 € 0.27
7.39 € 0.89
8.55 € 0.12
102.12 € 3.19
7.82 € 0.98
9.18 € 1.82
94.15 € 7.57
107.5 € 8.49
12.63 € 1.61
10.56 € 0.91
10.2 € 1.62
11.41 € 1.07
99.15 € 17.33
14.4 € 5.29
15.6 € 2.70
113.34 € 3.89
3b
3c
3d
3e
5a
5b
5c
5d
856
Chem Biol Drug Des 2012; 80: 853–861

Anticancer Properties of Novel Radiosensitizers
not clear although they all exhibited inhibition of cell proliferation.
Relative viability data of DU-145 and MCF-7 cells exposed for 24 h
at 100 lM concentrations of target compounds are summarized in
Table 1.
against DU-145 and MCF-7 cell lines. To confirm the inhibitory effects
of the novel compounds from this study, we compared their activities
with a widely used antineoplastic agent, doxorubicin (Figures 3–6).
All compounds did not show significant cytotoxic effects at 10 lM
concentration on both cell lines with and without radiation for 24 h
and 1 day after radiation (Tables 2 and 3). Tables 4 and 5 represent
relative cell viability (as % of untreated control) of MCF-7 and DU-145
Compounds (3a–e, 5a–d) were further tested at concentration of
10 lM, exposed for 24, 48, 96, and 144 h with or without radiation
B
A
Figure 3: The inhibition effects of synthesized compounds on (A) MCF-7 and (B) DU-145 carcinoma cells at 10 lM concentration (24-h
treatment; n = 3, 0 day after radiation).
A
B
Figure 4: The inhibition effects of synthesized compounds on (A) MCF-7 and (B) DU-145 carcinoma cells at 10 lM concentration (48-h
treatment; n = 3, 1 day after radiation).
A
B
Figure 5: The inhibition effects of synthesized compounds on (A) MCF-7 and (B) DU-145 carcinoma cells at 10 lM concentration (96-h
treatment; n = 3, 3 day after radiation).
Chem Biol Drug Des 2012; 80: 853–861
857

Ates-Alagoz et al.
A
B
Figure 6: The inhibition effects of synthesized compounds on (A) MCF-7 and (B) DU-145 carcinoma cells at 10 lM concentration (144-h
treatment; n = 3, 5 day after radiation).
Table 2: Relative cell viability
Without radiation
(MCF-7)
With radiation
(MCF-7)
Without radiation
(DU-145)
With radiation
(DU-145)
(as % of untreated control) of
MCF-7 and DU-145 cells, exposed
for 24 h at 10 lM concentration of
novel synthesized compounds with
or without radiation (n = 3)
Compound
Control
Doxorubicin
3a
100
100
100
100
98.048 € 3.94
91.65 € 1.15
91.25 € 0.84
87.46 € 1.49
87.12 € 3.09
91.03 € 0.39
87.15 € 1.89
92.72 € 0.60
95.60 € 1.43
97.87 € 1.88
97.42 € 1.06
89.82 € 2.65
87.22 € 2.32
86.99 € 0.65
90.39 € 1.78
86.27 € 5.38
90.39 € 1.25
90.08 € 3.64
100.02 € 10.4
89.65 € 3.32
86.91 € 3.03
76.38 € 3.23
86.97 € 1.67
87.84 € 2.98
84.06 € 5.32
75.13 € 5.89
81.18 € 4.47
95.52 € 6.87
98.34 € 5.23
107.44 € 5.27
95.20 € 7.09
89.81 € 4.18
76.26 € 8.07
81.44 € 8.15
84.03 € 1.14
93.84 € 6.20
90.32 € 7.37
94.86 € 9.95
97.13 € 10.97
101.69 € 5.00
3b
3c
3d
3e
5a
5b
5c
5d
Table 3: Relative cell viability
(% of untreated control) of MCF-7
and DU-145 cells, exposed for 48 h
at 10 lM concentration of novel
synthesized compounds with (1 day
after radiation) or without radiation
(n = 3)
Without radiation
(MCF-7)
With radiation
(MCF-7)
Without radiation
(DU-145)
With radiation
(DU-145)
Compound
Control
Doxorubicin
3a
100
100
100
100.81 € 4.66
95.88 € 2.13
82.95 € 5.49
78.5243.48
69.38 € 5.83
86.74210.33
95.0731.89
100
94.56 € 0.13
92.60 € 1.08
95.70 € 3.22
93.68 € 3.10
86.30 € 1.59
87.35 € 1.83
94.24 € 1.82
90.47 € 1.82
89.65 € 1.50
95.87 € 1.08
92.59 € 2.87
93.02 € 1.34
92.60 € 1.40
86.98 € 0.94
93.90 € 4.76
90.36 € 2.98
94.32 € 1.82
93.63 € 4.12
99.98 € 1.07
103.99 € 3.17
86.58 € 2.40
96.85 € 2.86
92.62 € 4.21
86.52 € 3.17
82.65 € 10.32
91.90 € 4.24
92.88 € 2.00
96.95 € 3.07
102.91 € 1.07
111.24 € 2.43
3b
3c
3d
3e
5a
5b
99.36 € 11.21
96.7644.65
103.98 € 4.47
5c
5d
cells exposed for 96 and 144 h at 10 lM concentration of novel
synthesized compounds with and without radiation (n = 3).
radiation, compound 3d had outstanding activity with radiation
against MCF-7 cell line with 9% cell viability and compounds 3c–3e
were more active than doxorubicin, 37%, 16% and 23% cell viabilities
resulted compared to 71%, against DU-145 with radiation.
Our most active compound 3d is more active than doxorubicin at the
dose level of 10 lM for 3 days after radiation, cell viabilities of 18%,
13% compared to 87%, 94% against MCF-7 cells, and 15%, 20%
compared to 60%, 75% against DU-145 cells without and with
radiation (Table 4). Compounds 3b–3e showed more considerable
cytotoxic effects against DU-145 cells compared against MCF-7 cells
by decreasing cell viability to 15–44% (Table 4). The 5th day after
Preliminary clonogenic survival assays were performed for com-
pounds 3c and 3d using MCF-7 and DU-145 cell lines. Effects of
10 lM of compounds 3c and 3d on MCF-7 and DU-145 colony for-
mation are depicted in Tables 6 and 7, respectively. The activity
trends for both compounds in both cell lines seem to be similar.
858
Chem Biol Drug Des 2012; 80: 853–861

Anticancer Properties of Novel Radiosensitizers
Table 4: Relative cell viability
(as % of untreated control) of
MCF-7 and DU-145 cells, exposed
for 96 h at 10 lM concentration of
novel synthesized compounds with
(3 day after radiation) or without
radiation (n = 3)
Without radiation
(MCF-7)
With radiation
(MCF-7)
Without radiation
(DU-145)
With radiation
(DU-145)
Compound
Control
Doxorubicin
3a
100
100
100
100
87.62 € 1.26
89.69 € 2.60
91.03 € 5.54
91.83 € 5.17
18.29 € 2.96
95.76 € 1.57
97.02 € 1.04
93.96 € 3.27
106.83 € 2.35
108.48 € 1.41
94.27 € 1.37
87.76 € 1.68
87.61 € 4.31
76.36 € 4.33
13.42 € 2.07
83.25 € 6.03
91.69 € 0.29
90.88 € 1.24
91.36 € 1.21
94.93 € 2.53
60.20 € 4.76
110.65 € 3.94
35.13 € 3.69
18.88 € 1.28
15.02 € 0.08
20.62 € 0.45
72.46 € 4.13
74.82 € 1.35
126.71 € 8.38
123.80 € 6.05
75.36 € 4.45
115.70 € 1.82
43.84 € 4.16
22.45 € 1.12
20.54 € 0.63
25.29 € 8.30
80.43 € 1.71
86.38 € 1.71
127.57 € 0.82
129.54 € 3.09
3b
3c
3d
3e
5a
5b
5c
5d
Table 5: Relative cell viability
(as % of untreated control) of
MCF-7 and DU-145 cells, exposed
for 144 h at 10 lM concentration
of novel synthesized compounds
with (5 days after radiation) or
without radiation (n = 3)
Without radiation
(MCF-7)
With radiation
(MCF-7)
Without radiation
(DU-145)
With radiation
(DU-145)
Compound
Control
Doxorubicin
3a
100
100
100
100
92.57 € 3.19
86.72 € 1.56
93.28 € 1.10
94.79 € 1.58
14.57 € 4.93
98.22 € 3.73
84.98 € 2.56
86.07 € 1.01
96.80 € 3.78
94.35 € 3.40
95.47 € 2.03
98.48317 € 2.21
96.19 € 3.36
72.89 € 7.32
8.62 € 2.39
88.62 € 1.40
87.11798 € 3.37
92.47 € 5.69
95.34 € 2.3
75.45 € 2.85
86.55 € 2.13
69.011 € 4.21
59.36 € 7.04
12.92 € 0.49
38.11 € 2.64
93.84 € 4.08
91.89 € 1.38
97.01 € 1.23
112.73 € 0.42
70.58 € 0.91
108.79 € 0.54
72.28 € 3.79
36.96 € 8.16
16.26 € 0.87
23.03 € 2.13
81.44 € 2.02
86.14 € 1.65
95.73 € 0.06
115.94 € 2.85
3b
3c
3d
3e
5a
5b
5c
5d
93.63 € 0.79
Table 6: Effects of 10 lM treatment of compounds 3c and 3d
on MCF-7 colony formation. Mean € SD of at least two experi-
ments. The plating efficiency and survival fractions were calculated
Table 7: Effects of 10 lM treatment of compounds 3c and 3d
on DU-145 colony formation. Mean € SD of at least two experi-
ments. The plating efficiency and survival fractions were calculated
Mean number
of colonies
Plating
efficiency (%)
Survival
fraction
Mean number
of colonies
Plating
efficiency (%)
Survival
fraction
Experiment
Experiment
Control
149 € 9.89
131 € 26.16
15 € 0.70
0
0
2.98
0.262
0.03
0
N ⁄ A
N ⁄ A
0.01
N ⁄ A
0
Control
161 € 12.50
178 € 8.48
114 € 27.57
0
0
16.1
1.78
1.14
0
N ⁄ A
N ⁄ A
0.01
N ⁄ A
0
3c on MCF-7 cells: Non-IR
3c on MCF-7 cells: IR
3d on MCF-7 cells: Non- IR
3d on MCF-7 cells: IR
3c on DU-145 cells: Non-IR
3c on DU-145 cells: IR
3d on DU-145 cells: Non- IR
3d on DU-145 cells: IR
0
0
Compared with control, compound 3c had more survival decrease
with radiation than without radiation (Figure 7, Tables 6 and 7). Col-
ony formation of tumor cells disappeared totally using our most
active compound 3d, with and without radiation (Figure 8, Tables 6
and 7). Based on the results of these studies, we believe that com-
pound 3d is deserving of further studies for prostate and breast
cancers. The mechanism(s) through which this molecule elicits its
effects, dose–response studies as well its effects on normal cell
lines is subject of further investigations. In vivo studies of the com-
pound with and without radiation are also desired future studies.
obtaining effective antiproliferative compounds. Initial radiosensitiz-
ing studies of compound 5c have been reported (11). The goal of
this study was to synthesize a series of compounds that have been
designed as radiosensitizers and test their effects against cancer
cell lines. When used with radiotherapy, these compounds are
expected to have synergistic responses and thereby increase their
values. The combination of radiosensitization and radiotherapy to
target hypoxic tumors can be a powerful anticancer therapeutic.
Our data show that compound 3d is a very potent antiproliferative
compound, more so than the lead compound 5c, especially at the
lower concentration of 10 lM (Tables 4 and 5). At that concentra-
tion, compound 5c had no effects as compared to control, whereas
compound 3d reduced DU-145 cell viability to 16% and that of
MCF-7 cells to 9% even 5 days after radiation. The clonogenic
assays confirm potent activities of compound 3d, reducing colony
formation for both cell lines to non-detectable levels.
Conclusions and Future Directions
Taking together, data presented suggest that introduction of halo-
gens to both sulfonyl and carbonyl classes has great potential in
Chem Biol Drug Des 2012; 80: 853–861
859

Ates-Alagoz et al.
carbanions is shown to involve a coupling reaction between the
electrogenerated radical and the parent anionic species, both on
preparative and kinetic grounds (26). The halogen moieties act as
electron 'sinks' on irradiation, the carbon-halogen bond breaking on
electron attachment to liberate free halide and form a carbon-cen-
tered free radical. This species can add oxygen to form a peroxyl
radical and carry out strand-breaking reactions as DNA base ⁄ hy-
droxyl radical adduct (23). We believe these may be possible mech-
anisms of action for our compounds based on their aromatic nitro,
sulfonyl and halogen groups, which are capable of generating free
radicals. The free radicals can then lead to cell death.
In conclusion, the in vitro evaluation of cytotoxicity of 4-(ethylsulfo-
nyl)-1-halogen-2-nitrobenzene (3a–e) and 1-(4-halogen-3-nitrophe-
nyl)propan-1-one (5a–d) derivatives revealed the high potential of
compound 3d as a cytotoxic compound. Further studies are needed
to evaluate this compound as a new cytotoxic agent with therapeu-
tic potentials.
Figure 7: The inhibitory effects of (A) control; (B) compound 3c
without radiation; and (C) compound 3c with radiation on colony
formation in MCF-7.
Acknowledgment
We acknowledge US patent application no. 12 ⁄ 677,478 and PCT
application no. PCT ⁄ US08 ⁄ 76208 (Constantinos Koumenis, Brian E
Lally, Steven Kridel, Gary D Kao, and Adeboye Adejare, inventors)
from which this work was derived. We would also like to thank Dr
Phyllis Wachsberger and Barbara Andersen of Department of Radia-
tion Oncology, Jefferson Medical College, Thomas Jefferson Univer-
sity, Philadelphia, PA, USA, for helping us with use of the Pantak
XRAD 320 cabinet X-ray machine.
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861