N-4-iodophenyl-N′-2-chloroethylurea, a novel potential anticancer agent with colon-specific accumulation: Radioiodination and comparative in vivo biodistribution profiles

Article abstract of DOI:10.1007/s10637-009-9222-z

In a search for more selective anticancer drugs, we have designed nitrogen mustard and nitrosourea conjugates leading to a series of N-4-aryl-N′-2- chloroethylureas (CEUs). The iodinated derivative N-4-iodophenyl-N′-2- chloroethylurea (4-ICEU) has demonstrated significant antineoplastic and antiangiogenic potency in preclinical evaluations. In this study, 4-ICEU was radiolabelled with [¹²⁵I]iodide in order to carry out a comparative study of its in vivo behavior profile. 4-[¹²⁵I]-ICEU was synthesized by direct electrophilic radioiodination with 80% radiochemical yield and 97% radiopurity. Three different routes of administration (intraperitoneal (ip), intravenous (iv) and intratumoral (it)) were tested in mice bearing subcutaneously implanted CT-26 murine colon carcinoma. The results clearly established that 4-ICEU was more stable to biotransformation than previously studied CEUs congeners. It was readily bioavailable and reached the CT-26 colorectal tumor regardless of the route of administration. Additionally, the colon mucosa was an important target tissue where 4-ICEU accumulated and remained largely untransformed. In conclusion, these results justify further investigations for developing 4-ICEU as a new chemotherapeutic agent for colorectal cancer.

Full text of DOI:10.1007/s10637-009-9222-z

Invest New Drugs (2010) 28:124–131
DOI 10.1007/s10637-009-9222-z
PRECLINICAL STUDIES
N-4-iodophenyl-N′-2-chloroethylurea, a novel
potential anticancer agent with colon-specific
accumulation: radioiodination and comparative
in vivo biodistribution profiles
Emmanuelle Mounetou & Elisabeth Miot-Noirault &
René C. Gaudreault & J. Claude Madelmont
Received: 19 December 2008 /Accepted: 23 January 2009 /Published online: 10 February 2009
#
Springer Science + Business Media, LLC 2009
Summary In a search for more selective anticancer drugs,
we have designed nitrogen mustard and nitrosourea
conjugates leading to a series of N-4-aryl-N′-2-chloroethy-
lureas (CEUs). The iodinated derivative N-4-iodophenyl-
N′-2-chloroethylurea (4-ICEU) has demonstrated significant
antineoplastic and antiangiogenic potency in preclinical
evaluations. In this study, 4-ICEU was radiolabelled with
developing 4-ICEU as a new chemotherapeutic agent for
colorectal cancer.
.
.
Keywords Chloroethylurea Anticancer agent
.
Colon specificity Radioiodination
[
125I]iodide in order to carry out a comparative study of its
Introduction
in vivo behavior profile. 4-[125I]-ICEU was synthesized by
direct electrophilic radioiodination with 80% radiochemical
yield and 97% radiopurity. Three different routes of
administration (intraperitoneal (ip), intravenous (iv) and
intratumoral (it)) were tested in mice bearing subcutane-
ously implanted CT-26 murine colon carcinoma. The
results clearly established that 4-ICEU was more stable to
biotransformation than previously studied CEUs congeners.
It was readily bioavailable and reached the CT-26 colorectal
tumor regardless of the route of administration. Addition-
ally, the colon mucosa was an important target tissue where
4-ICEU accumulated and remained largely untransformed.
In conclusion, these results justify further investigations for
N-Aryl-N′-(2-chloroethyl)ureas (CEUs) are a new class of
soft alkylating agents exhibiting potent antiangiogenic and
antitumoral activities. We designed these compounds as
conjugates of the aromatic ring of nitrogen mustards
(e.g. chlorambucil) as the biofunctional moiety and the
2-chloroethylurea group of aliphatic nitrosoureas (e.g. car-
mustine) as the pharmacophore (Fig. 1) [1–3]. Our goal was
to develop new drugs offering both a higher therapeutic
index and a lower capacity to induce tumor chemoresistance
in response to two majors challenges in modern cancer
chemotherapy [4–6]. We previously reported that CEUs are
not only more cytotoxic than the parent drugs (i.e.
chlorambucil and carmustine) against a wide panel of tumor
cell lines but also remain active in cell lines that have
developed resistance to several antineoplastic agents [7–10].
In addition, CEUs do not exhibit mutagenicity [9]. Their
main target is microtubules which are crucial components of
the cytoskeleton in cancer cells and are involved in mitosis
[11, 12]. CEUs act as antimitotic agents, preventing
microtubule assembly through β-tubulin alkylation via
covalent binding to the 198 glutamic acid residue leading
to the arrest of cell cycle progression t in G2/M phase, and to
apoptosis[13–15].
:
:
E. Mounetou (*) E. Miot-Noirault J. C. Madelmont
INSERM UMR 484, EA 4231, BP 184,
63005 Clermont-Ferrand, France
e-mail: emmanuelle.mounetou@inserm.fr
:
:
E. Mounetou E. Miot-Noirault J. C. Madelmont
Université Clermont-Ferrand 1,
63001 Clermont-Ferrand, France
R. C. Gaudreault
Centre de Recherche, CHUQ, Hôpital Saint-François d’Assise,
10, rue de l’Espinay,
Preclinical evaluations carried out with the first lead
compound of the series, i.e. N-(4-tert-butyl)phenyl-N′-
Québec, QC G1L3L5, Canada

Invest New Drugs (2010) 28:124–131
125
Fig. 1 Chemical structures
of N-(4-tert-butyl)phenyl-N′-
(2-chloroethyl)urea (4-tBCEU)
and N-4-iodophenyl-N′-
H
H
shifts (δ) are reported in parts per million relative to the
internal tetramethylsilane standard. Infrared (IR) spectra
were recorded on a Bruker Vector 22 FTIR spectrometer.
Melting points (mp), uncorrected, were determined on
an electrothermal melting point apparatus. Elemental
analyses were performed by the CNRS Service Central
d’Analyses (Vernaison, France). Electrospray ionization
mass spectrometry (ESI-MS) was performed on a Brüker
ESQUIRE-LC mass spectrometer. Chemicals were supplied
by Sigma-Aldrich Chimie SARL (St Quentin Fallavier,
France). Sodium [125I] iodide as no carrier added in
solution reductant-free aqueous sodium hydroxide was
purchased from Amersham Biosciences UK (Little Chal-
font, England). Analytical thin layer chromatography
(TLC) was conducted on precoated silica gel plates (SDS,
60 F254, 0.25 mm thick) with both detection by ultraviolet
light at 254 nm and visualization by iodine. Radioanalysis
of TLC plates was carried out by scanning with an Ambis
400 detector (B. Braun Scientec). High pressure liquid
chromatography (HPLC) analysis were performed on a HP
1100 System with a UV/visible detector (Hewlett Packard,
Les Ulis, France) equipped with a reverse phase Lichrocart
column (Lichrosphere® RP18-e-, 5 μm, 125×4 mm,
Hewlett Packard, Les Ulis, France) and connected to a
500TR flow scintillation analyser (Packard Instrument SA,
Rungis, France). The flow rate was 1 mL/min with a
gradient mobile phase starting from an acetonitrile/water
mixture:30/70 (v/v) to 100/0 at 25 min. Specific activity
was evaluated using an automated Packard 5530 gamma
counter (EGG Instruments, Evry, France).
N
N
Cl
O
(2-chloroethyl)urea (4-ICEU)
4-tBCEU
H
N
H
N
Cl
O
I
4-ICEU
(2-chloroethyl)urea (4-tBCEU; Fig. 1), showed that although
its antineoplastic activity on animal models was limited
mainly due to metabolic inactivation via hydroxylation of the
tert-butyl group, 4-tBCEU exhibited a particular tropism for
the gastrointestinal tract organs, notably the colon [16, 17].
This observation prompted us to carry out further inves-
tigations to assess the potential of CEUs for colorectal cancer
treatment. We recently focused our efforts on the iodinated
bioisostere N-4-iodophenyl-N′-(2-chloroethyl)urea (4-ICEU;
Fig. 1) which exhibits not only improved antiproliferative
and antiangiogenic activity in various cancer cell lines but
also increased antitumoral activity and life-span in murine
colon carcinoma [14, 17, 18]. We hypothesized that
replacing the tert-butyl group with the bioisosteric iodine
atom should increase in vivo stability. In addition, the
presence of an iodine-substituted phenyl ring allowed its
radioiodination. Indeed, the radioiodinated 4-ICEU would be
a useful tool for confirming that the colon is a relevant target
organ. 125I radioisotope was chosen for its appropriate range
of energy decay (35 keV) and half-life time (T1/2=60 d)
compatible with exploring in vivo tissue distribution via
quantitative whole-body autoradiography [19, 20].
As part of the preclinical development of 4-ICEU as a
potential anticancer agent, we studied the radiosynthesis of
4-[125I]-ICEU and comparative pharmacokinetic profiles in
CT-26 murine colon carcinoma-bearing mice with particu-
lar emphasis on in vivo stability and organ targeting. Three
different administration routes were tested, i.e. intraperito-
neal (ip), intravenous (iv) and intratumoral (it). These
crucial data on the bioavailability of the drug candidate
substantiated the anticipated colon tropism, and confirmed
that the colon is a relevant target for future development of
4-ICEU in anticancer therapy.
The standard N-4-iodophenyl-N′-2-chloroethylurea
(4-ICEU) and the precursor N-phenyl-N′-2-chloroethylurea
were synthesized as previously described [7].
Synthesis of N-4-[125I]iodophenyl-N′-2-chloroethylurea
(4-[125I]ICEU)
To a 1 mL conical reaction vial fitted with a Teflon
lined stopper containing a suspension of N-phenyl-N′-2-
chloroethylurea (3 mg, 5 μmol) in a 0.25 M phosphoric
acid aqueous solution (100 μL) was added sodium [125I]
iodide (37 MBq, 1 mCi) as a 0.15 M sodium iodide
aqueous solution (50 μL) and a 0.15 M chloramine-T
aqueous solution (50 μL). The mixture was magnetically
stirred at 80°C for 20 min and the reaction was quenched
by the addition of a 80 μM aqueous solution of sodium
metabisulfite (50 μL). After cooling at 4°C, the vial was
centrifuged at 3,000 rpm for 5 min. The precipitate was
isolated after removal of the supernatant and several
washings with water to afford 4-[125I]ICEU in 80%
radiochemical yield. The radioiodinated product comigrated
with the unlabeled standard (Rf=0.25, eluent: ether/cyclo-
hexane: 50/50, v/v). Radiochemical and chemical purity
Experimental
Chemistry
General
Proton nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker AM-400 spectrometer. Chemical

126
Invest New Drugs (2010) 28:124–131
was >97% as determined by HPLC analysis (one peak at
12.1 min). Analytical data for the unlabeled compound
obtained with non-radioactive sodium iodide: mp 194–
196°C; 1H-NMR (DMSO-d6) δ 3.40–3.44, 3.60–3.66 (2 m,
4H, CH2CH2Cl), 6.42 (broad s, 1H, NHCH2, exchanges
with D2O), 7.21–7.54 (dd, 4H, Ar–H), 8.75 (broad s, 1H,
ArNH, exchanges with D2O); 13C-NMR (DMSO-d6) 41.96
(NCH2), 45.09 (CH2Cl), 84.62, 120.80, 137.97, 140.94
(Ar–C), 155.62 (CO); ΙR (KBr) ν 3320 (NH), 1635 (C=O)
cm−1. MS (ESI): m/z 323.90 ([M+], C9H10ClIN2O, calcd
323.95). Anal. (C9H10ClIN2O) C, H, N.
methanol by magnetic stirring for 1.5 h at room tempera-
ture. The extracts were combined and centrifuged at
2,000×g for 10 min at 4°C. The supernatant volume was
measured, aliquots taken and radioactivity determined. The
recovery of radioactivity from extraction procedures was in
the range of 80–85%. After evaporation of the extracts
under reduced pressure of the extracts, the dry residues
were dissolved in the HPLC mobile phase (acetonitrile/
water:30/70 (v/v)) and filtered through Dynagard syringe
filters (Spectrum Microgon, Laguna Hills, CA, USA).
HPLC analysis was performed using the aforementioned
HPLC conditions.
Pharmacokinetics studies
Excretion of the radioactivity
Six-week-old male BALB/c mice (Charles River Company,
Lyon, France) were injected subcutaneously in the right
flank with 2.5×105 mouse colon carcinoma CT-26 cells
obtained from Dr. I.J. Fidler (MD Anderson Cancer center,
USA). After 10 days, the studies were performed after iv, ip
or it injection of 4-[125I]-ICEU (185 kBq i.e. 10 mg kg−1,
740 kBq i.e. 47 mg kg−1 and, 666 kBq i.e. 36 mg kg−1
respectively).
Animals (8) were housed in metabolic cages (Iffa-Credo,
L’arbresle, France) enabling separate collection of feces and
urine. Urine and feces were collected 24, 48, 72 h after
administration. Radioactivity of urine and dried feces
samples was directly measured using an automated Packard
5530 gamma counter (EGG Instruments, Evry, France).
Blood kinetics and tissue distribution
Results
Animals were sacrificed by CO2 inhalation at various time
intervals after 4-[125I]-ICEU administration, and rapidly
frozen by immersion in liquid nitrogen. They were then
embedded in a 2% gel of carboxymethyl cellulose, frozen
in liquid nitrogen and were sagittally sectioned at −22°C
with a Reichert-Jung Cryopolycut cryomicrotome (Heidel-
berg, Germany). For each mouse, eight body sections
selected at different levels were taken using no. 810 Scotch
band tape (3 M, Saint Paul, MN, USA), dried for 48 h
at −22°C and analyzed with an Ambis 4000 detector
(B. Braun Sciencetec), which allows visualization and
quantification of the radioactivity distribution in whole-
body sections. The results were expressed as the percentage
of the injected dose of radioactivity per gram of tissue
(% ID/g). For the quantitation of radioactivity uptake within
the colon mucosa, mice were sacrificed by CO2 inhalation
at various time intervals after administration. Colon was
dissected out. Its content was eliminated and the mucosa
washed. Radioactivity of mucosa aliquots was measured
using an automated Packard 5530 gamma counter (EGG
Instruments, Evry, France). The results were corrected for
radioactive decay and expressed as the percentage of the
injected dose of radioactivity per gram of tissue (% ID/g).
Chemistry
As high specific activity was not required, the most
straightforward method for radioiodination of small mole-
cules, i.e. iodine exchange, was first assessed [19, 20]. To
that end, the reaction of 4-ICEU with sodium [125I]-iodide
was carried out at 135°C in citrate buffer in the presence of
a catalytic amount of copper sulfate (Table 1, entry 1) [21].
However, radio-TLC analysis of the medium did not
confirmed the formation of 4-[125I]-ICEU. Another strategy
using the well-described oxidative iododestannylation
reaction was therefore devised [19]. Using the tributyltin
group allows the regiospecific radioiodination of aryl
rings in high radiochemical yield [22]. The widely used
tri-n-butylstannylaryl precursor is prepared by tetrakis
(triphenylphosphine)palladium-mediated stannylation of
the aryl iodide in the presence of hexabutylditin in refluxing
toluene [23]. However, this procedure yielded only 30%
of the desired stannane, even after 24 h of reaction.
Finally, the direct electrophilic iodination of N-phenyl-N′-
2-chloroethylurea by sodium [125I]-iodide in the presence
of chloramine-T as oxidizing agent was attempted in
aqueous medium under various pH conditions (Table 1,
entries 2–4). Reaction time and temperature were also
optimized. The iodination was initially carried out at the mg
scale using non-radioactive sodium iodide to assess the
regiospecificity of the reaction. The 1H-NMR spectrum
exhibited a doublet of doublets at 7.21–7.54 ppm that
In vivo stability
Plasma, urine, rehydrated feces and organs homogenized in
a Potter were sampled and extracted three times with

Invest New Drugs (2010) 28:124–131
Table 1 Radiosynthesis of 4-[125I]-ICEU
127
H
N
H
N
H
N
H
N
Na125
I
Cl
Cl
O
O
X
125I
4-[125I]-ICEU
Entry
X
Method
Conditions
Yielda (%)
1
2
3
4
127I
H
H
H
Isotopic exchange
CuSO4/citrate buffer, 135°C/1 h
0
50
65
80
Electrophilic iodination
Electrophilic iodination
Electrophilic iodination
Chloramine-T/H2O/phosphate buffer, 80°C/30 min
Chloramine-T/H2O/H3PO4, 60°C/20 min
Chloramine-T/H2O/H3PO4, 80°C/20 min
a Radiochemical yield determined by radio-TLC analysis
confirmed the para-substitution of the aromatic ring and
was in agreement with our previously reported NMR data
on 4-ICEU [7]. In all the experiments, the labelled
compound was identified by comparing its chromatograph-
ic behavior (TLC, HPLC) with that of the authentic non-
radioactive standard prepared as previously described [7].
Finally, the reaction conducted in phosphoric acid at 80°C
for 20 min gave optimal yield (Table 1, entry 4). After
purification, 4-[125I]-ICEU was obtained in 80% radio-
chemical yield and, 97% radiopurity, with a specific activity
of 1.5 TBq mmol−1.
Blood radioactivity concentrations in of it-route-injected
mice were lower than in ip- or iv-treated mice but were still
significant (e.g., 1.84 0.27%ID/g at 2 h pi) even at early
time-points, allowing a rapid whole-body distribution of the
radioactivity (Table 2).
Tissue distribution Tables 2, 3 and 4 show the quantitative
biodistribution of total radioactivity at time points from
15 min to 24 h pi in representative tissues of mice treated
with 4-[125I]-ICEU. These data were determined by quanti-
tative whole-body autoradiography. However, γ-counting of
tissue aliquots was used as a complementary method,
especially for the evaluation of radioactivity levels in the
colon, since it allowed isolation of the colon mucosa, thus
avoiding contamination by its contents.
Pharmacokinetic studies
Mice subcutaneously implanted with CT-26 murine colon
carcinoma received an iv, ip or it injection of 4-[125I]-ICEU
(185 kBq, i.e. 10 mg kg−1; 740 kBq, i.e. 47 mg kg−1 and,
666 kBq, i.e. 36 mg kg−1 respectively).
Following ip administration of 4-[125I]-ICEU, radioac-
tivity was widely distributed into most tissues. All tissues
showed high uptake rapidly achieved 30 min pi. The
Blood kinetics Radioactivity levels in the blood of mice
treated with either administered ip, iv or it 4-[125I]-ICEU are
shown in Fig. 2. In mice injected by the iv route, blood
radioactivity kinetics displayed an initial stable phase with
levels of around 4–4.5%ID/g up to 2 h post injection (pi)
followed by a sudden and rapid clearance between 2 and 3 h
pi and a final stabilization phase at around 1–2%ID/g until
6 h pi. Radioactivity was totally eliminated at 24 h after
administration. In mice treated ip route, radioactivity concen-
tration rapidly reached values of around 4%ID/g and
remained relatively constant, with a maximum of 5.30
0.93%ID/g at 3 h pi. From four hours pi, the level of radio-
activity decreased slowly, reaching 1.5 0.5%ID/g after 24 h.
Whereas blood radioactivity levels were initially similar
in ip- and iv-treated mice, they decreased more rapidly over
time in iv-treated mice as illustrated at the 3 h time-point
(1.10 0.50%ID/g vs. 5.30 0.93%ID/g respectively).
7
ip
6
iv
5
4
3
2
1
0
16
0
4
8
12
20
24
time (h)
Fig. 2 Blood kinetics of total radioactivity following administration
of 4-[125I]-ICEU by ip or iv route in BALB/c mice implanted with
murine CT-26 colon carcinoma. Values are expressed as the
percentage of the injected dose per gram of blood and correspond to
the mean standard deviation for two animals for each time-point

128
Invest New Drugs (2010) 28:124–131
Table 2 Biodistribution data for 4-[125I]-ICEU after intraperitoneal (ip) administration to BALB/c mice implanted with murine CT-26 colon
carcinoma
Tissue
Percentage of injected dose per gram of tissue (%ID/g)
15 min
30 min
1 h
3 h
2.72 0.30
6 h
12 h
24 h
b
b
Brain
Lung
Thyroid
Liver
Kidney
Spleen
Stomach
Colona
Tumor
Muscle
1.13 0.44
2.53 0.20
2.12 0.23
5.38 2.32
14.92 2.00
7.18 1.97
3.62 1.89
8.03 2.06
6.09 0.43
12.96 1.43
2.61 0.53
1.91 0.59
2.21 0.52
4.73 0.99
16.05 2.80
5.94 0.89
4.61 1.21
3.46 1.09
9.13 1.09
10.51c
0.28 0.25
3.15 0.93
17.55 9.73
2.51 0.68
3.31 0.2
1.77 0.02
18.92 7.57
9.85c
–
–
–
–
–
b
b
5.01 0.15
17.66 4.79
6.68 0.56
4.72 0.82
6.06 0.02
14.71 1.97
12.42 3.63
3.27 0.32
2.32 0.39
b
c
–
31.07 14.90
1.47 0.25
3.85 0.31
4.58 1.85
2.44 0.59
b
b
–
–
–
b
b
–
1.12 0.28
15.34 2.54
6.32c
2.40 0.30
0.71 0.5
2.52 0.02
31.16c
1.29 0.19
1.29 0.02
5.67c
2.98 0.71
2.57 0.61
2.64 0.54
1.98 0.51
2.40 0.46
0.66 0.02
b
–
Values are the mean standard deviation for the quantification of eight whole-body sections from 11 animals
a Quantified using a γ counter after collection of the organ, elimination of the contents, and washings (see experimental section)
b Could not be quantified
c Quantified in one animal
radioactivity remained stable or increased slightly up to 3 h
pi. It was cleared rapidly until 6 h pi and more gradually
thereafter. The highest concentrations were found in the
colon mucosa (12.96 1.43%ID/g), spleen, lung and liver
(Table 2). The lowest radioactivity levels were observed in
the brain and muscles. At 24 h pi, most of the radioactivity
has been eliminated from the tissues, except for the
stomach, liver and colon mucosa. In contrast, tumor uptake
did not vary significantly over the time frame studied, and
remained at around 2–3%ID/g globally, with. tumor (3.27
0.32%ID/g), kidney and heart exhibiting intermediates
values. Moreover, as generally observed with molecules
directly radiolabelled with iodine, radioactivity accumulat-
ed in the thyroid and stomach.
After iv administration, tissues radioactivity kinetics
generally followed four phases: rapid distribution in a large
number of tissues, followed by stabilization at peak levels
between 1 and 2 h, fast elimination between 2 and 3 h, and
a final more gradual elimination up to 24 h (Table 3). The
highest activities were detected in well-perfused tissues,
particularly the lung (15.2 3.00% ID/g at 1 h pi) and the
colon mucosa (18.90 2.60%ID/g at 1 h pi). At late time-
points, high retention was still observed in these organs
compared with the other tissues. High levels of radioactiv-
ity (8–10%ID/g) were also found in liver at 1 h pi, kidney
and stomach at 2 h pi. Tumor and spleen levels of
radioactivity rapidly became substantial (2.5–3.5%ID/g)
and remained constant until 2 h post-administration.
Table 3 Biodistribution data for 4-[125I]-ICEU after intravenous (iv) administration to BALB/c mice implanted with murine CT-26 colon
carcinoma
Tissue
Percentage of injected dose per gram of tissue (%ID/g)
30 min
1 h
2 h
3 h
6 h
24 h
b
Brain
Lung
Liver
Kidney
Spleen
Stomach
Colona
Tumor
Muscle
1.10 0.50
14.50 2.20
7.20 1.70
6.10 1.40
2.40 0.50
4.30 1.20
5.70 1.50
2.70 0.40
1.50 0.60
1.90 0.8
15.2 3.0
2.10 0.60
0.20 0.06
2.80 0.70
1.40 0.90
1.60 0.60
0.70 0.30
2.70 0.40
3.00 1.80
0.40 0.10
0.30 0.10
0.10 0.06
5.00 1.30
1.80 0.30
1.50 0.40
1.30 0.50
–
12.60 2.50
7.80 2.10
8.40 1.00
4.10 0.60
9.20 1.90
13.6 3.40
3.00 0.60
2.10 0.20
0.80 0.20
0.40 0.20
0.10 0.02
0.30 0.20
10.9 3.20
7.40 0.60
3.90 0.80
3.00 0.50
18.9 2.60
3.60 0.40
2.30 1.30
b
b
–
–
4.00 1.60
0.80 0.20
0.40 0.10
0.10 0.01
0.10 0.05
b
–
Values are the mean standard deviation for the quantification of eight whole-body sections from eight animals
a Quantified using a γ counter after collection of the organ, elimination of the contents, and washings (see experimental section)
b Could not be quantified

Invest New Drugs (2010) 28:124–131
129
Table 4 Biodistribution data for 4-[125I]-ICEU after intratumoral (it)
administration to BALB/c mice implanted with murine CT-26 colon
carcinoma
remained significantly present in the tumor and colon at
later time-points accounting for 15–20% of total radioac-
tivity (i.e. 6 h pi for the iv route). Finally, metabolization
in the colon was generally slower than in the tumor.
Tissue/fluid Percentage of injected dose per gram of tissue (%ID/g)
1 h
2 h
3 h
Excretion of radioactivity The radioactivity in urine and
feces of mice given ip 4-[125I]-ICEU was gradually and
mainly eliminated in urine (>50% in 48 h). Fecal excretion
was low (25% in 48 h). Finally, over 80% of the
administrated dose was excreted within 72 h, with about
10% remaining in the carcasses.
HPLC analysis of fecal extracts and urine samples
collected over 48 h indicated that intact 4-[125I]-ICEU
accounted for only 10% of total radioactivity.
Blood
Lung
1.81 0.66
7.01 1.34
1.84 0.27
4.09 0.89
10.96b
1.83 0.39
1.88 0.43
3.25b
29.36 10.20
40.87 12.50
0.36 0.11
1.31 0.73
2.94 0.73
a
a
Thyroid
Liver
Kidney
Stomach
Colon
–
–
3.83 1.05
2.00 0.56
0.46 0.01
7.90 1.90
29.4 15.70
1.28 0.63
1.48 0.15
1.32 0.15
a
–
23.15 4.40
19.00 7.10
0.50 0.45
Tumor
Muscle
Values are the mean standard deviation for the quantification of eight
whole-body sections from two animals
a Could not be quantified
b Quantified in one animal
A
ip administration
blood
colon
tumor
100
80
60
40
20
0
Conversely, brain and muscles displayed the lowest uptakes
at any given time.
Following it administration of 4-[125I]-ICEU, more than
50%ID/g was distributed in all the organs examined at 2 h
pi (Table 4). The peak radioactivity concentrations were
observed in the lung, liver and, kidney at 1 h pi and in the
stomach, thyroid, colon at 2 h pi. There was a slow
elimination after this time-point. However, a particularly
high accumulation of radioactivity in the colon was noted
over the period examined (23.15 4.40%ID/g at 3 h pi).
1h
2h
3h
6h
Time
iv administration
B
100
80
60
40
20
0
blood
colon
tumor
In vivo stability The blood and target organs extracts
collected from mice treated with 4-[125I]-ICEU were
analyzed by HPLC to determine the percentage of 4-
[
125I]-ICEU in total radioactivity. The data obtained are
presented in Fig. 3. The chromatographic profiles of the
samples analyzed were very similar. Under the used HPLC
conditions, 4-[125I]-ICEU eluted at 12.0 min. The metabo-
lites detected were more polar, with retention times of
around 3.0 min and 8.0 min, and are still unidentified.
Comparing the administration routes, the stability of
4-[125I]-ICEU was in the order it > ip > iv. For example,
the percentages of unchanged ICEU in the liver at 1 h pi
were 91%, 45% and 12% following it, ip and iv
administration respectively. The highest rate of biotrans-
formation was observed in the blood, and the percentage
of 4-[125I]-ICEU was invariably below 5% from 3 h after
ip or iv administration. The fraction of 4-[125I]-ICEU was
higher in the tumor and colon, and declined more slowly
than in blood at all time-points. In these organs, the levels
of the intact form were in the 40–60% range at 1 h pi and
decreased to 20–30% at 2–3 h pi. However, 4-[125I]-ICEU
1h
2h
3h
6h
Time
it administration
C
100
80
60
40
20
0
blood
colon
tumor
1h
2h
Time
3h
6h
Fig. 3 Stability of 4-[125I]-ICEU after administration to BALB/c mice
implanted with murine CT-26 colon carcinoma. a ip route; b iv route;
c it route

130
Invest New Drugs (2010) 28:124–131
Discussion
tration. Radioactivity levels were significantly higher in
colon mucosa than in non-target organs (brain, muscles)
or blood. Colon/blood ratios ranged between 2 and 3,
and metabolization was slower in the colon mucosa than
in blood, independently of the route of administration
used. The drug showed stability and retention in the
colon, particularly after ip administration (20% of
unchanged form remained at 6 h pi). Mice whole-body
autoradiograms corroborated the biodistribution profile
reported above and clearly showed a dense signal
corresponding to the intestinal tract, more specifically
the colon (Fig. 4). The substantial concentration of
radioactivity detected in the colon at 2–3 h post it injection
also confirmed the colon-specific localization and accu-
mulation. These results are in agreement with the
particular tropism for the gastrointestinal tract previously
found for the first lead compound, 4-tBCEU [16].
The pharmacokinetics data presented here indicates that 4-
ICEU is rapidly and widely distributed to most organs and
tissues, whatever the route of administration. Additionally,
blood and tissues kinetics displayed highly similar pat-
terns. The peak concentrations were reached at 2 h pi for it-
or iv-treated mice. For ip-treated mice, peak concentration
were reached at 3 h pi, after which. elimination then
occurred gradually over 72 h pi. Excretion was mainly
urinary. As the biostability evaluation indicated that, 4-
[
125I]-ICEU was stable in vivo up to 3 h pi, whatever the
route of administration used, the radioactivity detected
until that time can be considered as related to 4-[125I]-
ICEU. The HPLC analysis of blood and target organs
demonstrated that the biotransformation of 4-ICEU was
slow and not as extensive as for its previously studied
congener 4-tBCEU [16]. These results confirmed that 4-
ICEU possesses promising properties for future therapeutic
development.
Tumor uptake was confirmed by tumor/blood ratios in
the range of 0.6–0.9 before significant metabolization
occurred at 2 and 6 h pi for ip and iv injection, respectively.
Moreover, metabolization rate was generally slower in the
tumor than in blood. These data are in accordance with our
previous results [17–18]. The antitumor activity of it-
administered 4-ICEU was reproduced in ip administered,
with high tolerance and efficacy (around 60% tumor growth
inhibition and 45% life-span increase). Representative
slices showing the localization of 4-[125I]-ICEU in the
tumor are presented in Fig. 1. The highest levels of
radioactivity were generally found in blood-rich tissues
(lung, spleen) and in the metabolization and elimination
organs (liver, kidney). As is usual for iodinated compounds,
the thyroid and stomach displayed higher concentrations of
radioactivity at later time-points. Conversely, the brain and
muscles showed no accumulation of radioactivity at any
time-points, whatever the route of administration.
In conclusion, 4-[125I]-ICEU was obtained in high
radiochemical purity and high yield by direct electrophilic
iodination of N-phenyl-N′-2-chloroethylurea. Radiola-
belled 4-ICEU proved a useful tool for assessing the in
vivo behavior of the drug in mice bearing subcutaneous
CT-26 colon carcinoma, consequently validating its
potential for the treatment of colorectal cancers. Bio-
pharmaceutical profiles for the three routes of adminis-
tration tested showed that: (1) compared with a tert-butyl
group, an iodine atom at the 4-position of the phenyl ring
enhanced the biostability of the CEU, (2) 4-ICEU was
biodistributed into the CT-26 colon carcinoma tumor, and
(3) 4-ICEU accumulated specifically in the colon mucosa.
Taken together, these properties combined with the
previously demonstrated antineoplastic activity [14–17]
clearly suggest that radiolabelled 4-ICEU is a candidate
for colorectal cancer therapy, offering potential for
developing new selective therapies for this resilient
cancer [24]. Since the data presented here appear of
utmost importance, further investigation on both the
antitumor efficacy and biodistribution profiles of 4-ICEU
will be performed in mice bearing orthotopically trans-
planted CT-26 colon carcinoma tumors [25].
There was important accumulation of radioactivity in
the colon mucosa, irrespective of the route of adminis-
Fig. 4 Representative two-
intestines
intestines
tumor
dimensional whole-body sagittal
section autoradiograms of
BALB/c mice implanted with
murine CT-26 colon carcinoma
at 2 h post-ip administration of
4-[125I]-ICEU

Invest New Drugs (2010) 28:124–131
131
disrupting microtubules that are unaffected by cell adhesion-
mediated drug resistance. Cancer Res 64:4654–4663 doi:10.1158/
0008-5472.CAN-03-3715
References
1. Rajski SR, Williams RM (1998) Cross-linking agents as antitumor
drugs. Chem Rev 98:2723–2731 doi:10.1021/cr9800199
2. Weiss RB, Issel BF (1982) The nitrosoureas: carmustine (BCNU)
and lomustine (CCNU). Cancer Treat Rev 9:313–330 doi:10.1016/
S0305–7372(82)80043–1
3. Bank B (1992) Studies of chlorambucil-DNA adducts. Biochem
Pharmacol 44:571–575 doi:10.1016/0006–2952(92)90451-N
4. Eckardt S (2002) Recent progress in the development of
anticancer agents. Curr Med Chem Anticancer Agent 2:419–439
5. Palumbo M (2004) Anticancer agents: towards the future. Curr
Med Chem Anticancer Agents 4:425–427
6. Cozzi P, Mongelli N, Suarato A (2004) Recent anticancer
cytotoxic agents. Curr Med Chem Anticancer Agents 4:93–121
7. Mounetou E, Legault J, Lacroix J, Gaudreault RC (2001)
Antimitotic antitumor agents: synthesis, structure activity relation-
ships and biological characterization of N-Aryl-N′-(2-chloroethyl)
ureas as new selective alkylating agents. J Med Chem 44:694–702
doi:10.1021/jm0010264
8. Mounetou E, Legault J, Lacroix J, Gaudreault RC (2003) A new
generation of N-Aryl-N′-(1-alkyl-2-chloroethyl)ureas as microtu-
bule disrupters: synthesis, antiproliferative activity, and β-tubulin
alkylation kinetics. J Med Chem 64:5055–5063 doi:10.1021/
jm030908a
15. Bouchon B, Chambon C, Mounetou E, Papon J, Miot-Noirault E,
Gaudreault RC, Madelmont JC, Degoul F (2005) Alkylation of β-
tubulin on GLU 198 by a microtubule disrupter. Mol Pharmacol
68:1415–1422 doi:10.1124/mol.105.015586
16. Maurizis JC, Rap M, El Mostafa A, Gaudreault RC, Veyre A,
Madelmont JC (1998) Disposition and metabolism of a novel
antineoplastic agent, 4-tert-butyl-[3-(2-chloroethyl)ureido]ben-
zene, in mice. Drug Metab Dispos 26:146–151
17. Miot-Noirault E, Legault J, Cachin F, Mounetou E, Degoul F,
Gaudreault R, Moins N, Madelmont JC (2004) Antineoplastic
potency of arylchloroethylurea derivatives in murine colon
carcinoma. Invest New Drugs 22:369–378 doi:10.1023/B:
DRUG.0000036679.12112.4c
18. Borel M, Degoul F, Communal Y, Mounetou E, Bouchon B,
Gaudreault R, Madelmont JC, Miot-Noirault E (2007) N-
(4-Iodophenyl)-N′-(2-chloroethyl)urea (ICEU) as a microtubule
disrupter: in vitro and in vivo profiling of antitumoral activity on
CT-26 murine colon carcinoma cell line cultured and grafted to
mice. Br J Cancer 96:1684–1691 doi:10.1038/sj.bjc.6603778
19. Volkert WA, Hoffman TJ (1999) Therapeutic radiopharmaceut-
icals. Chem Rev 99:2269–2292 doi:10.1021/cr9804386
20. Baldwin RM (1986) Chemistry of radioiodine. Appl Radiat Isot
37:817–821
9. Lacroix J, Gaudreault R, Pagé M, Joly P (1998) In vitro and in
vivo activity of 1-aryl-3-(2-chloroethyl)urea derivatives as new
antineoplastic agents. Anticancer Res 8:595–598
10. Gaudreault R, Alaoui-Jamali M, Batist G, Béchard P, Lacroix J,
Poyet P (1994) Lack of cross-resistance to a new cytotoxic
arylchloroethylurea in various drug-resistant tumor cells. Cancer
Chemother Pharmacol 33:489–492 doi:10.1007/BF00686506
11. Jordan MA (2002) Mechanism of action of antitumor drugs that
interact with microtubules and tubulin. Curr Med Chem Antican-
cer Agents 2:1–17
21. Moreau MF, Michelot J, Papon J, Bayle M, Labarre P, Madelmont
JC, Parry D, Boire JY, Moins N, Seguin H, Veyre A, Mauclaire L
(1995) Synthesis, radiolabeling and preliminary evaluation in
mice of some (N-diethylaminoethyl)-4-iodobenzamide derivatives
as melanoma imaging agents. Nucl Med Biol 22:737–747
doi:10.1016/0969-8051(95)00020-X
22. Wilbur DS (1992) Radio halogenation of proteins: an overview of
radionuclides, labeling methods, and reagents for conjugate
labeling. Bioconjug Chem 3:433–470 doi:10.1021/bc00018a001
23. Azizian H, Eaborn C, Pidcock AJ (1989) Synthesis of organo-
trialkylstannanes. J Organomet Chem 215:49–58 doi:10.1016/
S0022-328X(00)84615-X
24. Van Custem E, Verslype C, Demedts I (2002) The treatment of
advanced colon cancer: where are we now and where do we go?
Best Pract Res Clin Gastroenterol 16:319–330 doi:10.1053/
bega.2002.0288
25. Wilmanns C, Fan D, O’Brian CA, Bucana CD, Fidler IJ (1992)
Orthotopic and ectopic organ environments differentially influ-
ence the sensitivity of murine colon carcinoma cells to doxoru-
bicin and 5-fluorouracil. Int J Cancer 52:98–104 doi:10.1002/
ijc.2910520118
12. Islam MN, Iskander MN (2004) Microtubulin binding sites as
target for developing anticancer agents. Mini Rev Med Chem
4:1077–1104
13. Legault J, Gaulin JF, Mounetou E, Bolduc S, Lacroix J, Poyet P,
Gaudreault R (2000) Microtubule disruption induced in vivo by
alkylation of β-tubulin by 1-aryl-3-(2-chloroethyl)urea, a novel class
of antineoplastic soft alkylating agents. Cancer Res 60:985–992
14. Petitclerc E, Deschenes R, Cote MF, Marquis C, Janvier R,
Lacroix J, Miot-Noirault E, Legault J, Mounetou E, Madelmont
JC, Gaudreault R (2004) Antiangiogenic and antitumoral activity
of phenyl chloroethylureas: a class of soft alkylating agents