Synthesis, in vitro cytotoxicity and radiosensitizing activity of novel 3-[(2,4-dinitrophenylamino)alkyl] derivatives of 5-fluorouracil

Article abstract of DOI:10.1111/cbdd.12211

Previously, it was reported that 3[3-(2,4-dinitrophenylamino)-propyl]-5- fluorouracil 8c unlike its components 5-fluorouracil (5-FU) 6 and 2,4-dinitroaniline 2 in HT-29 cells under aerobic conditions had no cytotoxicity but showed radiosensitizing activity. In this study several analogues of 8c differing in the number of linking methylene groups were prepared and tested for in vitro cytotoxicity and radiosensitizing activity under both aerobic and hypoxic conditions. Tethered compound 8a was prepared in one pot by the reaction of 5-FU 6 with paraformaldehyde and 2,4-dinitroaniline 2 in the presence of the concentrated hydrochloric acid, and compounds 8b-f were prepared by the reaction of N-(bromoalkyl) - 2,4-dinitrobenzeneamines 5b-f with 1-(t-butoxycarbonyl)-5-fluorouracil 7 followed by hydrolysis of the protecting group. The cytotoxicity of the tested compounds were measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and propidium iodide (PI)-digitonin assays and values of sensitization enhancement ratio (SER) as a measure of the radiosensitizing activity were measured from radiation survival curves in the absence and presence of each sensitizer for 37% survival respectively. Results showed that tethered compounds 8a-f induced time- and concentration-dependent cytotoxicity under hypoxia but had no significant effect under aerobic conditions. These compounds also showed selective and concentration-dependent radiocytotoxicity under hypoxic conditions. Several novel 3[3-(2,4-dinitro-phenylamino)-alkyl]-5-fluorouracil resulting from linkage of 2,4-dinitrophenylamine through 1-6 methylene units with 5-fluorouracil were prepared and tested for in vitro cytotoxicity in HT-29 cell line under aerobic and hypoxic conditions with and without radiation. These compounds induced time- and concentration-dependent cytotoxicity which were higher under hypoxic than aerobic conditions, and showed selective radiosensitizing activities under hypoxic conditions.

Full text of DOI:10.1111/cbdd.12211

Chem Biol Drug Des 2014; 83: 183–190
Research Article
Synthesis, In Vitro Cytotoxicity and Radiosensitizing
Activity of Novel 3-[(2,4-Dinitrophenylamino)Alkyl]
Derivatives of 5-Fluorouracil
Ali Khalaj1,2,*, Khosrou Abdi1,3, Seyed Nasser
Received 30 June 2013, revised 10 August 2013 and
accepted for publication 12 August 2013
Ostad4, Mohammad Reza Khoshayand5, Navid
Lamei2 and Hasan Ali Nedaie6
1Department of Medicinal Chemistry, Faculty of Pharmacy,
Dual functional agents consisting of an electron affinic
Tehran University of Medical Sciences,Tehran, Iran
2Drug Design & Development Research Center, Tehran
University of Medical Sciences,Tehran, Iran
nitroaromatic or nitro-heterocyclic moiety as radiosensitizer
and a cytotoxic group (1) such as nitrophenyl mustards
(2), and 2-nitroimidazole derivatives containing haloalkyl-
amines and aziridines (2–4) are known as hypoxia selective
cytotoxins and as radiosensitizers in hypoxic tumor cells.
These compounds are known as second generation anti-
cancer agents, which have shown to improve the mode of
treatment by radiotherapy (5).
3Department of Radiopharmacy, Faculty of Pharmacy,
Tehran University of Medical Sciences,Tehran, Iran
4Department of Toxicology and Pharmacology, Faculty of
Pharmacy,Tehran University of Medical Sciences,Tehran, Iran
5Department of Drug and Food Control, Faculty of
Pharmacy,Tehran University of Medical Sciences,Tehran,Iran
6Department of Radiotherapy, Oncology Cancer Institute,
Tehran University of Medical Sciences,Tehran, Iran
*Corresponding author: Ali Khalaj, akhalaj@tums.ac.ir
Results of our previous investigations on in vitro aerobic
cytotoxicity of 3[3-(2,4-dinitrophenylamino)-propyl]-5-fluoro-
uracil 8c in HT-29 cell line with and without radiation
showed that this compound unlike its components has no
cytotoxicity but is radiosensitizer (6). Also in another report,
it was shown that 2,4-dinitrophenyl derivatives of 5-FU 6
(7) (Figure 1) compared to 5-FU 6 had minimal or no cyto-
toxicity under both aerobic and hypoxic conditions, and
their SER values were lower under aerobic and higher
under hypoxic conditions than 1.6 which is the minimum
value required for an in vitro radiosensitizing activity (8).
Previously, it was reported that 3[3-(2,4-dinitrophenylami-
no)-propyl]-5-fluorouracil 8c unlike its components 5-fluo-
rouracil (5-FU) 6 and 2,4-dinitroaniline 2 in HT-29 cells
under aerobic conditions had no cytotoxicity but showed
radiosensitizing activity. In this study several analogues of
8c differing in the number of linking methylene groups
were prepared and tested for in vitro cytotoxicity and ra-
diosensitizing activity under both aerobic and hypoxic
conditions. Tethered compound 8a was prepared in one
pot by the reaction of 5-FU 6 with paraformaldehyde and
2,4-dinitroaniline 2 in the presence of the concentrated
hydrochloric acid, and compounds 8b–f were prepared by
the reaction of N-(bromoalkyl) - 2,4-dinitrobenzeneamines
5b–f with 1-(t-butoxycarbonyl)-5-fluorouracil 7 followed by
hydrolysis of the protecting group. The cytotoxicity of the
tested compounds were measured by 3-(4,5-Dim-
ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),
and propidium iodide (PI)-digitonin assays and values of
sensitization enhancement ratio (SER) as a measure of the
radiosensitizing activity were measured from radiation
survival curves in the absence and presence of each sen-
sitizer for 37% survival respectively. Results showed that
tethered compounds 8a–f induced time- and concentra-
tion-dependent cytotoxicity under hypoxia but had no
significant effect under aerobic conditions. These com-
pounds also showed selective and concentration-depen-
dent radiocytotoxicity under hypoxic conditions.
In continuation of our investigations on this type of hybrid
compounds and in the search for more selective and
potent hypoxia cytotoxins and hypoxic cell radiosensitizers,
several analogues of 8c differing in the length of linker
chain were prepared, and their in vitro cytotoxicities under
both aerobic and hypoxic conditions with and without radi-
ation were investigated.
Material and Methods
Chemistry
5-fluorouracil 6, di-t-butoxydicarbonate (Boc)2O, 2,4-dinitro-
fluorobenzene 1, 2,4-dinitroaniline 2, triethylamine, sodium
hydride, and other chemicals and solvents not listed
here were from Merck Chemical Co. (Germany). Thin
layer chromatography was carried out using Kieselgel 60,
230–400 mesh (Merck, Germany) to monitor the progress
of reactions. Melting points were determined on a MEL-270
Key words: 2,4-dinitrophenylamine, 5-fluorouracil, HT-29 cell
line, Radiosensitizing, Tethered compounds
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12211
183

Khalaj et al.
Sibata melting point apparatus and are uncorrected.
1HNMR spectra were recorded on 500 MHz a Bruker
(Germany), using DMSO-d6 or CDCl3 as solvent. Chemical
shifts (d) are reported in ppm relative to TMS as internal
standard. Mass spectra were obtained on a Finningan TSQ-
70 instrument. Infrared spectra were recorded on a Nicolet
Magna IR-550 spectrometer. Elemental analyses for C, H,
and N were performed using a Heracus CHN–O-rapid ele-
mental analyzer, and results are within Æ0.4% of the theo-
retical values. All compounds were characterized by the
above techniques. Analytical and spectroscopic data for the
known compounds were consistent with reported literature
values, and data only for new compounds are presented.
Figure 1: Structures of 2, 4-dinitrophenyl derivatives of 5-
fluorouracil.
Syntheses
Tethered compound 8a with one methylene unit in the
linker chain was prepared in one pot in 33% yield by the
reaction of 5-fluorouracil 6 with paraformaldehyde and
2,4-dinitroaniline 2 in the presence of one drop of the
concentrated hydrochloric acid in a sealed tube.
Tethered compounds 8b–f were prepared by the reaction
of N-(bromoalkyl)-2,4-dinitrobenzeneamines 5b–f with 1-(t-
butoxycarbonyl)-5-fluorouracil 7 (9) in DMF in the presence
of sodium hydride followed by removal of 1-(t-butoxycar-
bonyl) protecting group by potassium carbonate in metha-
nol (Scheme 1).
N-(bromoalkyl) - 2,4-dinitrobenzeneamines 5c–e had been
prepared in this laboratory through ultrasound irradiation of
the reaction of 2,4-dinitroaniline 2 with dibromoalkanes
4c–e in acetonitrile in the presence of sodium hydride (10),
and the novel derivatives 5b,f were prepared in high yields
by the reaction of hydrobromide salts of 2,4-dinitrofluoro-
benzene 1 with the corresponding bromoalkylamines 3b,f
in 80% ethanol in the presence of triethylamine. Bro-
moalkylamines 3b,f were prepared by the Gabriel synthe-
sis through N-alkylation of potassium phthalimide with the
corresponding dibromoalkanes 4b,f followed by acid
hydrolysis of the phthalyl group (11).
Scheme 1: Reagents and conditions (i):80% EtOH, Et3N, r.t.
30 min; (ii):CH3CN, NaH, 40 °C, ultrasound; (iii):HCl, 180 °C,
Sealed tube; (iv):DMF, NaH, 80 °C; (v):CH3OH, K2CO3.
Synthesis of N-(bromoalky)-2,4-
dinitrobenzeneamines (5b, f)
H6-phenyl), 3.83 (s, 4H, CH2); IR (KBr, cmax,): 3360, 3108,
2924, 1623, 1521, 1335/cm; Mass (m/e): 289 (40%), 291
(34%), 196 (100%), 167 (40%).
A solution of 2,4-dinitrofluorobenzene 1 (1.5 mmol) and
hydrobromide salt of the corresponding bromoalkylamines
3b,f (1.5 mmol) and triethylamine (1 g, 10 mmol) in 80%
EtOH (10 mL) was stirred at room temperature for 30 min,
and the residues after evaporation of the solvent were
crystallized from 50% EtOH to give N-(bromoalky)-2,4-
dinitrobenzeneamines 5b, f.
N-(6-bromohexyl)-2,4-dinitrobenzeneamine (5f)
Yield 90%, mp. 108–110 °C. 1HNMR (CDCl3) d: 9.16 (d,
J = 2.5 Hz, 1H, H3-phenyl), 8.56 (bs, 1H, NH), 8.31 (dd,
J = 9.5, 2.5 Hz, 1H, H5-phenyl), 6.92 (d, J = 9.5 Hz, 1H,
H6-phenyl), 3.44 (m, 4H, NHCH2 & CH2Br), 1.89 (m, 2H,
CH2), 1.82 (m, 2H, CH2), 1.54 (m, 4H, CH2-CH2); IR (KBr,
N-(2-bromoethyl)-2,4-dinitrobenzeneamine (5b)
Yield 98%, Mp. 83.9–84.5 °C. 1HNMR (CDCl3) d: 9.2 (d,
J = 2.5 Hz, 1H, H3-phenyl), 8.6 (bs, 1H, NH), 8.3 (dd,
J = 9.5, 2.5 Hz, 1H, H5-phenyl), 6.99 (d, J = 9.5 Hz, 1H,
cmax,): 3310, 3020, 2944, 1774, 1659, 1520, 1352/cm;
Mass (m/e): 345 (30%), 347 (28%), 266 (70%), 196(100%),
149 (80%).
184
Chem Biol Drug Des 2014; 83: 183–190

2,4-Dinitrophenylamin Derivatives of 5-Fluorouracil
Synthesis of 5-flouro-3-[(2,4-dinitrophenylamino)
J = 6 Hz, 2H, CH2-N), 3.71 (m, 2H, CH2-NH); IR (KBr,
alkyl)]-pyrimidine-2,4-(1H)-diones 8a–f
cmax,): 3352, 3073, 2953, 1696, 1654, 1610, 1547, 1326/
cm; Mass (m/e) 339 (10%), 338(9%), 210(40%), 196
(100%), 130 (95%) Anal. Calcd. for C12H10FN5O6: C,
42.49; H, 2.97; N, 20.64. Found: C, 42.56; H, 2.89; N,
20.53.
3-[(2,4-dinitrophenylamino)methyl]-5-
fluoropyrimidine-2,4(1H,3H)-dione (8a)
A mixture of 5-FU 6 (0.13 g, 1 mmol), 2,4-dinitroaniline 2
(0.183 g, 1 mmol), paraformaldehyde (0.06 g, 2 mmol)
and one drop of concentrated hydrochloric acid in a
tightly closed hydrogenation bomb was heated with stirring
at 180˚C for 2 h. The mixture after cooling to room
temperature was subjected to column chromatography
(EtOAc-Hexan 6.5:5.5) to give 8a (0.104 g, 32%). mp.
187–190 °C. 1H NMR (DMSO-d6) d: 9.07 (t, J = 7.05 Hz,
1H, NH), 8.85 (d, J = 2.5 Hz, 1H, H3-phenyl), 8.40 (dd,
J = 9.5, 2.5 Hz, 1H, H5-phenyl), 7.93 (d, J = 5.3 Hz, 1H,
CH=CF), 7.64 (d, J = 9.5, 1H, H6-phenyl), 5.57 (d,
J = 7.05 Hz, 2H, CH2); IR (KBr, cmax,): 3370, 3200, 2928,
1739, 1660, 1526, 1332/cm; Mass (m/e): 325 (20%), 306
(70%), 196 (100%), 183 (95%), 167 (80%), 130 (83%), 91
(90%).
5-Flouro-3-[(3-(2,4-dinitrophenylamino)propyl)]-
pyrimidine-2,4-(1H)-dione (8c)
Yield 29%, mp. 182–186 °C. 1HNMR (DMSO-d6) d: 9.16
(bs, 1H, NH), 8.77 (d, J = 2.5 Hz, 1H, H3-phenyl), 8.31
(dd, J = 9.5, 2.5 Hz, 1H, H5-phenyl), 7.21 (d, J = 5 Hz,
CH=CF), 6.92 (d, J = 9.5 Hz, 1H, H6-phenyl), 3.84
(t, J = 6.5 Hz, 2H, CH2-N), 3.51 (m, 2H, CH2-NH), 2.12
(m, 2H, CH2); IR (KBr, cmax,): 3350, 3073, 2950, 1695,
1654, 1618, 1551, 1325/cm; Mass (m/e) 353 (15%), 224
(40%), 196 (100%), 166 (30%), 130(87%). Anal.Calcd. for
C13H12FN5O6: C, 44.20; H, 3.42; N; 19.82. Found: C,
44.35; H, 3.34; N, 19.69.
Anal. Calcd. for C11H8FN5O6: C, 40.63; H, 2.48; N, 21.53.
Found: C, 40.59; H, 2.39; N, 21.76.
5-Flouro-3-[(4-(2,4-dinitrophenylamino)butyl)]-
pyrimidine-2,4-(1H)-dione (8d)
Yield 40%, mp. 205–207 °C. 1HNMR (DMSO-d6) d: 11.10
(bs, 1H, NH), 8.89 (bs, 1H, NH), 8.86 (d, J = 2.5 Hz, 1H,
H3-phenyl), 8.25 (dd, J = 9.4, 2.5 Hz, 1H, H5-phenyl),
7.84 (d, J = 5 Hz, CH=CF), 7.26 (d, J = 9.4 Hz, 1H, H6-
phenyl), 3.82 (t, J = 6.5 Hz, 2H, CH2-N), 3.52 (m, 2H,
CH2-NH), 1.63 (m, 4H, CH2); IR (KBr, cmax,): 3350, 3073,
2943, 1690, 1657, 1555, 1329/cm; Mass: (m/e) 367
(10%), 236 (15%), 196 (100%), 149 (50%), 129 (45%).
Anal. Calcd. for C14H14FN5O6: C, 45.78; H, 3.84; N,
19.07. Found: C, 45.69; H, 3.76; N, 19.12.
General procedure for the synthesis of 5-flouro-
3-[(3-(2,4-dinitrophenylamino)alkyl)]-pyrimidine-
2,4-(1H)-diones (8b–f)
To a stirred solution of 4 g (1.73 mmol) of 1-(t-butoxycar-
bonyl)-5-fluorouracil 7 (9) in 10 mL of anhydrous DMF in a
round bottom flask which was kept in an ice bath was
added within 10 min 30 mg (1.73 mmol) of 60% sodium
hydride and after stirring for 30 min at 0 °C, the mixture
was then treated with 1.73 mmol of N-(bromoalkyl)-2,4-
dinitrobenzeneamines 5b–f and stirred at room tempera-
ture for 30 min and then at 80 °C for 18 h. The resulting
mixture was then cooled to room temperature, diluted with
the same volume of water, acidified with concentrated
hydrochloric acid, and extracted with ethyl acetate
(3 9 20 mL). The organic layer was washed with water
(5 9 10 mL), dehydrated using anhydrous sodium sulfate,
and evaporated under vacuum. The residue after evapora-
tion of the solvent was dissolved in MeOH (20 mL), treated
with 2.83 g potassium carbonate, and stirred at room
temperature for 2 h. The precipitate after evaporation of
the organic solvent was washed with cold water
(2 9 5 mL) and subjected to flash chromatography using
chloroform-CH3OH (9:1), and then, after evaporation of the
solvent, the residue was crystallized from chloroform to
give the corresponding products 8b–f.
5-Flouro-3-[(5-(2,4-dinitrophenylamino)pentyl)]-
pyrimidine-2,4-(1H)-dione (8e)
Yield 44%, mp. 215–218 °C. 1HNMR (DMSO-d6) d: 11.19
(bs, 1H, NH), 8.95 (d, J = 2.6 Hz, 1H, H3-phenyl), 8.36
(bs, 1H, NH), 7.86 (dd, J = 9.5, 2.6 Hz, 1H, H5-phenyl),
7.18 (d, J = 4.8 Hz, CH=CF), 6.56 (d, J = 9.5 Hz, 1H,
H6-phenyl), 3.57 (t, J = 7.5 Hz, 2H, CH2-N), 3.20 (m, 2H,
CH2-NH), 1.64 (m, 4H, CH2), 1.31 (m, 2H, CH2); IR (KBr,
cmax,): 3330, 3073, 2939, 1692, 1664, 1551, 1330/cm;
Mass (m/e) 381 (30%), 335 (25%), 252 (44%), 196 (85%),
129 (100%). Anal. Calcd. for C15H16FN5O6: C, 47.25; H,
4.23; N, 18.37. Found: C, 47.35; H, 4.15; N 18.42.
5-Flouro-3-[(6-(2,4-dinitrophenylamino)hexyl)]-
pyrimidine-2,4-(1H)-dione (8f)
5-Flouro-3-[(2-(2,4-dinitrophenylamino)ethyl)]-
pyrimidine-2,4-(1H)-dione (8b)
Yield 22%, mp. 175–179 °C. 1HNMR (DMSO-d6) d: 8.98
(bs, 1H, NH), 8.8 (d, J = 2.5 Hz, 1H, H3-phenyl), 8.27 (dd,
J = 9.5, 2.5 Hz, 1H, H5-phenyl), 7.83 (d, J = 5 Hz,
CH = CF), 7.35 (d, J = 9.5 Hz, 1H, H6-phenyl), 4.05 (t,
Yield 41%, mp. 219–223 °C. 1HNMR (DMSO-d6) d:11.31
(bs, 1H, NH), 8.88 (d, J = 2.5 Hz, 1H, H3-phenyl), 8.40
(bs, 1H, NH), 8.07 (dd, J = 9.4, 2.5 Hz, 1H, H5-phenyl),
7.21 (d, J = 5 Hz, CH = CF), 6.76 (d, J = 9.5 Hz, 1H, H6-
phenyl), 3.48 (t, J = 7.5 Hz, 2H, CH2-N), 3.22 (m, 2H,
CH2-NH), 1.57 (m, 2H, CH2), 1.50 (m, 2H, CH2), 1.29 (m,
Chem Biol Drug Des 2014; 83: 183–190
185

Khalaj et al.
2H, CH2), 1.20 (m, 2H, CH2); IR (KBr, cmax,): 3343, 3065,
2939, 1692, 1664, 1551, 1330/cm; Mass (m/e): 396
(35%), 350 (30%), 229(40%), 196 (55%), 166 (35%), 130
(100%). Anal. Calcd. for C16H18FN5O6: C, 48.61; H, 4.81;
N, 17.71. Found: C, 48.52; H, 4.75; N, 17.64.
DMSO, and its absorbance was read in Elisa reader at
550 nm and a background wavelength of 690 nm.
Survival was scored by the ratio of the absorbance of the
treated cells to untreated cells with the tested compounds
(control) and is expressed as percentage of the cell sur-
vival (12). IC50 values were determined from cell survival
curves in which the percentages of cells that survived
were plotted as a function of the concentration of the
tested compounds (Table 1).
Biology
Cytotoxicity
Cytotoxicity of 2,4-dinitroaniline 2, 5-fluorouracil 6 and
tethered compounds 8a–f in HT-29 cell line under both
aerobic and hypoxic conditions were determined by mea-
surement of the percent of the surviving cells by MTT (12)
and propidium iodide (PI)-digitonin assays (13,14).
PI-Digitonin assay
20 lL of a 1.53 mmol solution of propidium iodide (PI) in
PBS for a final concentration of 30 lmol was added to
each well, and cell cultures were incubated for 60 min at
37 °C, and then, the initial fluorescence intensity from the
dead cells was measured in a multiwell plate reader, (Bio-
Tek, Synergy HT, USA) with 530-nm excitation and 645-
nm emission filters. Then, digitonin (6 9 10À7 mol) was
added to each well to permeabilize all cells and label all
nuclei with PI and after 30 min incubation, the fluore-
scence intensity was measured again to obtain a value
corresponding to the total number of cells (13). The per-
centage of dead cells was calculated as the proportion of
fluorescence intensity of the dead cells to that of total cells
multiplied by 100, and percentage of the cell viability was
calculated by subtracting the percentage of dead cells
from 100% (14). IC50 values were determined from cell
survival curves in which the percentages of cells that sur-
vived were plotted as a function of the concentration of
the tested compounds (Table 1).
HT-29 cells (human colon cancer cells) from Pasteur Insti-
tute (Tehran, Iran), RPMI 1640 from Gibco (UK), fetal
bovine serum (FBS), penicillin/streptomycin 1009, Trypsin-
EDTA 10 9 and trypan blue from Biosera (UK), MTT and
PI from Sigma (USA) and digitonin from Merck (Germany)
were used in this study.
Cell culture
Studies were carried out on HT-29 cells, originally derived
from human colorectal carcinoma obtained from Pasteur
Institute of Iran. Cells were grown as an attached mono-
layer in RPMI 1640 supplemented with 10% fetal bovine
serum, penicillin (50 unit/mL), and streptomycin (50 lg/mL).
Cells were routinely grown in tissue culture flasks and kept
in a humidified atmosphere of 5% CO2 and 95% air at
37 °C (15).
Cells were seeded at the density of 104 cells/well in 96-
well culture dishes and incubated for 4 h. Stock solutions
of the tested compounds in DMSO at concentrations of
50 mmol which were prepared prior to the experiments
diluted with culture media and at concentrations of 6.25–
400 lmol/mL added to each well of plates. Cells in the
control wells were treated with the same volume of med-
ium which contained DMSO in concentration equal to
those which were used in incubations treated with the
tested compound. Cell incubations were kept at 37 °C
under either air by purging with 95% nitrogen and 5%
CO2 or hypoxia in sealed GasPack jar by using Anoximat
(Netherland) and an anaerobic chamber GasPak Jar
(Merck, Germany) and GasPak plus (Merck, Germany) (16)
for 3 or 48 h. After removal of the supernatant of the cul-
ture medium, wells were washed with phosphate buffer
saline (PBS), and the numbers of live cells were deter-
mined by MTT (12) and PI-digitonin (13,14) assays.
Radiosensitizing evaluation
HT-29 cells under hypoxic or aerobic conditions were
incubated with different concentrations of the tested
compounds at 37 °C for 3 h and then exposed to diffe-
rent doses of radiation at 1.953 Gy/min dose rate using
a
60Co source. Radiosensitization of the tested com-
pounds were determined through measurement of the
cell growth inhibition using clonogenic assay (17). Results
are presented in terms of sensitization enhancement ratio
(SER) in Table 2 which were calculated from the equation
SER= Do untreated cells/Do treated cells. Do values rep-
resent radiation doses that lead to 37% cell survival and
were obtained from the survival curves of irradiation in
the presence and absence of the tested compounds
(18).
Clonogenic forming assay and cell survival curves
The medium of the cell cultures after treatment with the
tested compounds and/or irradiation was first separated to
remove the tested compounds and washed with PBS.
Cells were seeded in 6-well plates at concentration of
5 9 103 cells per well and maintained at 37 °C for
14 days. The cells were then fixed in methanol, colonies
MTT Assay
25 lL of the reagent (5 mg/mL in PBS) was added to each
well, and cell cultures were incubated for 4 h at 37 °C, and
then, the precipitated formazan was dissolved in 100 lL of
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2,4-Dinitrophenylamin Derivatives of 5-Fluorouracil
Table 1: IC50values for the cell viabilities of 2,4-dinitroaniline 2, 5-
tested compounds were assessed from cell survival curves
in which the percentage of cells that survived after 48 h
incubation was plotted as a function of their concentrations.
Similar to results of our investigations on 2,4-dinitrophenyl
derivatives of 5-FU (7) and as it can be found from the data
in Table 2, the IC50 values of tethered compounds 8a–f
determined by MTT and PI-digitonin assays were not differ-
ent significantly.
a
Fluorouracil 6 and tethered compounds 8a–f determined by
b
MTT and PI-digitonin assays on human colon cancer (HT-29)
cells under aerobic and hypoxic conditions
IC50 (lmol)
MTTa
PIb
Compound
Aerobic
Hypoxic
Aerobic
Hypoxic
The IC50 values of the tethered compounds 8a–f under
both aerobic and hypoxic conditions were significantly
higher than those of 2,4-dinitroaniline 2 and 5-fluorouracil
6 (p < 0.05), which is in agreement with results of other
investigations that N-acylation or alkylation of 5-fluorouracil
with groups that are not labile to hydrolysis reduces its
cytotoxicity (19).
2
93 Æ 9
89 Æ 7
88 Æ 7
84 Æ 5
6
134 Æ 12
176 Æ 15
210 Æ 16
216 Æ 18
223 Æ 20
220 Æ 19
225 Æ 18
103 Æ 10
116 Æ 9
141 Æ 10
185 Æ 12
224 Æ 15
221 Æ 16
229 Æ 18
228 Æ 21
232 Æ 16
110 Æ 11
122 Æ 12
130 Æ 9
8a
8b
8c
8d
8e
8f
124 Æ 11
126 Æ 10
135 Æ 11
142 Æ 13
145 Æ 12
133 Æ 11
141 Æ 13
150 Æ 14
149 Æ 14
a3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
bPropidium Iodide.
While tethered compounds 8a–f under hypoxic conditions
as are shown for compounds 8b,d reduced cell viabilities
in a concentration-dependent manner, and these com-
pounds under aerobic conditions comparatively were not
cytotoxic and as are shown for the same compounds at
concentrations up to 50 lmol reduced the cell viabilities to
<10% (Figure 3). Consistent with these results, the IC50
values of tethered compounds 8a–f under hypoxic relative
to aerobic conditions were significantly lower (p < 0.05),
indicating that these compounds have selective-hypoxia
cytotoxicity.
that were formed stained with crystal violet, and those
containing more than 50 cells were scored. The number of
colonies of treated and untreated dishes with the tested
compounds was compared and expressed as the per-
centage of survival by clonogenic assay (17).
Results and Discussion
The cytotoxicity of the tethered compounds 8a–f under
both aerobic and hypoxic conditions as are shown for com-
pounds 8b,d (Figure 2) was time dependent, and the per-
centage of the cell survival at 3 h after treatment of
incubations of HT-29 cells with different concentrations of
these compounds was similar but after 48 h (Figure 3) was
significantly different (p < 0.05). The IC50 values of the
The radiosensitization effects of compounds 8a–f under
aerobic conditions at various concentrations as are
shown for compounds 8b,d (Figure 4A) were not differ-
ent significantly but under hypoxic conditions as are
shown for the same compounds (Figure 4B) increased
significantly (p < 0.05) with increases in their concentra-
tions.
Figure 2: Clonogenic survival of incubation of HT-29 cell line with different concentrations (6.25, 12.5, 25, 50, 100 lmol) of compounds
8b and 8d after 3 h under aerobic (▲) and hypoxic (●) conditions. Data points were obtained from three independent experiments, and
the standard deviations are given for each point (mean Æ SD, each set of experiments was performed in triplicates).
Chem Biol Drug Des 2014; 83: 183–190
187

Khalaj et al.
Figure 3: Clonogenic cell survival of incubation of HT-29 cell line with different concentrations (6.25, 12.5, 25, 50, 100 lmol) of compounds
8b and 8d after 48 h under aerobic (▲) and hypoxic (●) conditions, († = p < 0.05). Data points were obtained from three independent
experiments, and the standard deviations are given for each point (mean Æ SD, each set of experiments was performed in triplicates).
Table 2: Sensitization Enhancement Ratio (SER) values of 2,4-dinitroaniline 2, 5-flourouracil 6 and tethered compounds 8a–f under aero-
bic and hypoxic conditions
SER
Concentration (lmol)
20
50
100
Compound No
Aerobic
Hypoxic
Aerobic
Hypoxic
Aerobic
Hypoxic
2
1.07 Æ 0.10
1.05 Æ 0.11
1.12 Æ 0.10
1.10 Æ 0.11
1.09 Æ 0.10
1.05 Æ 0.08
1.06 Æ 0.09
1.04 Æ 0.10
1.03 Æ 0.10
1.07 Æ 0.08
1.42 Æ 0.13
1.33 Æ 0.12
1.09 Æ 0.10
1.32 Æ 0.11
1.31 Æ 0.11
1.25 Æ 0.12
1.12 Æ 0.09
1.12 Æ 0.10
1.28 Æ 0.12
1.25 Æ 0.12
1.26 Æ 0.12
1.21 Æ 0.10
1.09 Æ 0.09
1.12 Æ 0.10
1.18 Æ 0.11
1.32 Æ 0.12
2.05 Æ 0.17
1.98 Æ 0.16
1.95 Æ 0.17
1.90 Æ 0.14
1.86 Æ 0.16
1.80 Æ 0.17
1.16 Æ 0.10
1.25 Æ 0.12
1.47 Æ 0.15
1.43 Æ 0.13
1.40 Æ 0.13
1.34 Æ 0.12
1.20 Æ 0.11
1.19 Æ 0.11
1.20 Æ 0.11
1.20 Æ 0.12
2.05 Æ 0.17
2.81 Æ 0.25
2.66 Æ 0.21
2.52 Æ 0.20
2.42 Æ 0.20
2.46 Æ 0.22
6
8a
8b
8c
8d
8e
8f
Additionally, the SER values of the tethered compounds
8a–f at all concentrations were higher under hypoxic
compared to aerobic conditions and at concentrations of
50 and 100 lmol were lower under aerobic and higher
under hypoxic conditions than 1.6, which is the minimum
required value for an in vitro radiosensitizing activity (8),
indicating that compounds are hypoxia -selective radio-
sensitizer.
hypothesized to produce reactive species upon reduc-
tion that have the potential to act in
manner.
a synergistic
Conclusion
While the IC50 values of 5-FU 6 under aerobic and hypoxic
conditions against HT-29 cell line were similar, and this
drug at all concentrations had no radiosensitizing activity
(SER: 1.04–1.16 < 1.6), and its 2,4-dinitrophenylamine
derivatives showed high cytotoxicity and radiosensitizing
activity under hypoxic compared to aerobic conditions. As
in radiotherapy of tumors, it is desirable to use com-
pounds that sensitize hypoxic cells to the effects of radia-
tion selectively, further exploration of these compounds
which showed concentration-dependent enhanced radio-
cytotoxicity might result in 5-fluorouracil derivatives with
Among the tethered compounds, 8a with one methylene
unit in the linker chain had the lowest IC50 value under
both aerobic and hypoxic conditions, and corresponding
values for other tethered compounds increased
significantly (p < 0.05) with increase in the linker length
only under hypoxic conditions. The highest cytotoxicity
of 8a may be attributed to its structural similarities
with o- and p-nitrobenzyloxycarbonyl (20) and 2,4-
dinitrophenyl derivatives of 5-FU 6 (7) which have been
188
Chem Biol Drug Des 2014; 83: 183–190

2,4-Dinitrophenylamin Derivatives of 5-Fluorouracil
A
B
Figure 4: Clonogenic cell survival of HT-29 cells upon exposure to different doses of Gamma-radiation (0, 2, 4, 8 and 12 Gy) after
incubation with different concentrations (0, 20, 50 and 100 lmol) of tested compounds 8b and 8d under aerobic (A) and hypoxic (B)
conditions. Data points were obtained from three independent experiments, and the standard deviations are given for each point
(mean Æ SD, each set of experiments was performed in triplicates).
little or no cytotoxicity on normal tissues. To achieve this
goal future, investigation is directed to mononitro or other
phenylamine derivative of 5-Fu which would be reduced
anaerobically to cytotoxic agents. The modes of binding of
compounds 8b,c with bovine serum albumin (BSA), the
binding constants of the resulting complexes, and the
effects of these compounds on the stability and conforma-
tions of BSA determined by FT-IR, UV-Vis spectroscopic
methods as well as molecular modeling have been
reported recently (21).
samples t-test was used to investigate the mean differ-
ences between irradiated and unirradiated cells treated with
the tested compounds and for comparing more than two
groups, Holm–Sidak multiple comparison test followed by
one-way analysis of variance (one-way ANOVA) was performed
and p value smaller than 0.05 was considered statistically
significant.
Acknowledgment
The authors thank the deputy of research of Tehran Uni-
versity of Medical Sciences, for the financial support of this
investigation.
Statistical Analyses
For the cell survival and cell death assays, each experi-
ment was repeated three times, and results are
mean Æ SD of three independent experiments. Statistical
analysis was performed using Sigmaplot 12 (SYSTAT soft-
ware Inc.) for window, and descriptive statistics are shown
as arithmetic mean Æ standard deviation. Independent
Conflict of Interest
The authors declare that they have no competing inter-
ests.
Chem Biol Drug Des 2014; 83: 183–190
189

Khalaj et al.
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