Synthesis and uptake of fluorescence-labeled combi-molecules by P-glycoprotein-proficient and -deficient uterine sarcoma cells MES-SA and MES-SA/DX5

Article abstract of DOI:10.1021/jm9016043

Here, we report on the first synthesis of fluorescent-labeled epidermal growth factor receptor-DNA targeting combi-molecules, and we studied the influence of P-glycoprotein status of human sarcoma MES-SA cells on their growth inhibitory effect and cellular uptake. The results showed that 6, bearing a longer spacer between the quinazoline ring and the dansyl group, was more stable and more cytotoxic than 4. In contrast to the latter, it induced significant levels of DNA damage in human tumor cells. Moreover, in contrast to doxorubicin, a drug known to be actively effluxed by P-gp, the more stable combi-molecule 6 induced almost identical levels of drug uptake and DNA damage in P-gp-proficient and -deficient cells. Likewise, in contrast to doxorubicin, 4 and 6 exerted equal levels of antiproliferative activity against the two cell types. The results in toto suggest that despite their size, the antiproliferative effects of 4 and 6 were independent of P-gp status of the cells.

Full text of DOI:10.1021/jm9016043

2104 J. Med. Chem. 2010, 53, 2104–2113
DOI: 10.1021/jm9016043
Synthesis and Uptake of Fluorescence-Labeled Combi-molecules by P-Glycoprotein-Proficient
and -Deficient Uterine Sarcoma Cells MES-SA and MES-SA/DX5
Anne-Laure Larroque-Lombard,†,§ Margarita Todorova,†,§ Nahid Golabi,† Christopher Williams,‡ and
Bertrand J. Jean-Claude*,†
†Cancer Drug Research Laboratory, Department of Medicine, Division of Medical Oncology, McGill University Health Center/Royal
Victoria Hospital, 687 Pine Avenue West Room M-719, Montreal, Quebec, H3A 1A1 Canada, and Chemical Computing Group Inc.,
‡
1010 Sherbrooke Street West, Suite 910, Montreal, Quebec H3A 2R7, Canada. §The two authors contributed equally to this paper.
Received October 30, 2009
Here, we report on the first synthesis of fluorescent-labeled epidermal growth factor receptor-DNA
targeting combi-molecules, and we studied the influence of P-glycoprotein status of human sarcoma
MES-SA cells on their growth inhibitory effect and cellular uptake. The results showed that 6, bearing a
longer spacer between the quinazoline ring and the dansyl group, was more stable and more cytotoxic
than 4. In contrast to the latter, it induced significant levels of DNA damage in human tumor cells.
Moreover, in contrast to doxorubicin, a drug known to be actively effluxed by P-gp, the more stable
combi-molecule 6 induced almost identical levels of drug uptake and DNA damage in P-gp-proficient
and -deficient cells. Likewise, in contrast to doxorubicin, 4 and 6 exerted equal levels of antiproliferative
activity against the two cell types. The results in toto suggest that despite their size, the antiproliferative
effects of 4 and 6 were independent of P-gp status of the cells.
Introduction
and MES-SA/DX5) that do not express EGFR,16 the primary
target of these combi-molecules. The use of cells deprived of
EGFR was to avoid any interference of EGFR or related
proteins with the retention or subcellular distribution of the
molecules. We have now shown elsewhere that EGFR expres-
sion influences their subcellular distribution.15 Importantly,
in contrast to the parental MES-SA cell line, the MES-SA/
DX5 has been shown to express P-gp and to be resistant to
doxorubicin and paclitaxel.17-19
The new combi-molecules reported herein were designed to
degrade into an aminoquinazoline, fluorescing in the blue,
and a dansylated DNA-damaging moiety, fluorescing in the
green, thereby facilitating the analysis of drug uptake by
fluorescence microscopy and flow cytometry. The IC50 values
for EGFR tyrosine kinase inhibition by 4 and 6 were 1.4 and
0.6 μM, respectively, and theirbinary EGFR-DNA targeting
properties are extensively discussed in a separate report.15
Here, we describe the synthesis of the two combi-molecules,
one with a short neutral ethano linker and the other with a
longer and basic N-methylethanediamine spacer. We com-
pared their differential uptake in MES-SA cells with that of
doxorubicin, by fluorescence microscopy and flow cytometry.
In contrast to the clinical anticancer drug doxorubicin, the
potency of these bulky molecules was independent of the P-gp
status of the two cell types. It should be noted that the purpose
of this study was not to comparethe potency ofthe fluorescent
molecules with that of doxorubicin, a strong DNA interca-
lator and topoisomerase II inhibitor, but was rather toanalyze
their differential uptake in comparison with that of doxo-
rubicin, a highly P-gp-dependent drug.
At the advanced stages, solid tumors express a variety of
receptors and signaling proteins that not only drive their
progression but also render them resistant to tumor drugs.
One mechanism of multidrug resistance (MDR)a is the
expression of a reflux pump [e.g., P-glycoprotein (P-gp)] that
transports drugs out of the cells,1-5 and this is responsible for
drugresistance andtreatmentfailureinapproximately90%of
cancer patients.6,7 Also, some mutations in key signaling
proteins are often responsible for chemoresistance. Several
attempts to modulate MDR led to a limited degree of success
in the clinic, and the design of agents that diffuse into the
cells regardless of their P-gp status or that selectively kill
P-gp-expressing cells8 has become a new drug development
strategy.
Recently, to circumvent problems associated with target
heterogeneity in advanced cancers, we developed novel types
of drugs capable of inhibiting refractory tumor cell growth by
blocking divergent targetssuch asthe epidermal growthfactor
receptor (EGFR) and genomic DNA. Many prototypes of
these agents termed combi-molecules have been shown to
block the growth of human cancer cells with disordered
signaling.9-14 While their ability to concomitantly damage
DNA and to induce high levels of apoptosis was demon-
strated, the influence of transport pump-mediated MDR on
the potency of these multitargeted molecules has yet to be
explored. To analyze their intracellular uptake and dependence
on MDR status, we designed fluorescence-labeled prototypes
4 (AL194) and 6 (AL237)15 and studied their differential
uptake by two human uterine sarcoma cell lines (MES-SA
Results and Discussion
*To whom correspondence should be addressed. Tel: 514-934-1934
ext 35841. Fax: 514-843-1475. E-mail: jacques.jeanclaude@staff.mcgill.ca.
aAbbreviations: P-gp, P-glycoprotein; EGFR, epidermal growth
factor receptor; MDR, multidrug resistance.
Chemistry. The synthesis of compound 4 and 6 proceeded
according to Schemes 1 and 2, respectively. Commercially
r
pubs.acs.org/jmc
Published on Web 02/12/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2105
Scheme 1. Synthesis of Compound 4a
a Reagents and conditions: (i) N-Fmoc-ethylenediamine, EtOAc, H2O, K2CO3, 0 °C. (ii) Morpholine, DMF, room temperature. (iii) Diazonium 3,
Et3N, CH3CN/Et2O, -5 °C.
Scheme 2. Synthesis of Compound 6a
a Reagents and conditions: (i) N-(2-Aminoethyl)-N-methylethanediamine, EtOAc, 0 °C. (ii) Diazonium 3, Et3N, CH3CN/Et2O, -5 °C.
available 9-fluorenylmethyl N-(2-aminoethyl)carbamate hydro-
chloride was treated with dansyl chloride in a biphasic ethyl
acetate/aqueous potassium carbonate solution to give 1
(Scheme 1). Compound 1 was deprotected with morpholine
in DMF to give the amino compound 2. In parallel, the
aminoquinazoline was diazotized in dry acetonitrile with
nitrosonium tetrafluoroborate to provide the diazonium salt
3 as previously described.14 Diazonium salt 3 was coupled
with 2 in the presence of triethylamine in situ to give the
desired compound 4. Several attempts to purify 4 by column
chromatography failed due to on-column degradation,
which we believe could be due to the conversion of 4 to a
product resulting from loss of nitrogen. Successful purifica-
tion was achieved by serial trituration of compound 4 with
methylene chloride, ether, and petroleum ether to give a pure
red-brown powder. The compound was characterized by
NMR and MS, and its purity was confirmed by elemental
analysis.
The synthesis of compound 6 proceeded in a similar
fashion. An excess of commercially available N-(2-amino-
ethyl)-N-methylethanediamine was treated with dansyl chlo-
ride to give 5 in ethyl acetate at 0 °C (Scheme 2), and this

2106 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Larroque-Lombard et al.
Table 1. Half-Lives of Compounds 4 and 6 in a Serum-Containing Medium
* Data are means of three experiments in triplicate.
product was coupled with the diazonium salt of quinazoline
3 to give 6. The purification of 6 was as difficult as that of 4.
All attempts to purify this compound by column chroma-
tography failed. Serial trituration provided a pure red-brown
powder that was analytically pure. The structure of 6 was
further confirmed by NMR and MS.
Half-Life in a Serum-Containing Medium. The sole struc-
tural difference between 4 and 6 is in the nature of the linker.
The linker in 4 is an ethylene group between the sulfonamido
moiety of the dansyl and the N3 of the triazene, leaving no
possibility for intramolecular hydrogen bonding. By con-
trast, the dansyl in 6 is separated by six bond lengths and
contains a methylamino group three bond lengths away
from the N3 of the triazene, leaving the possibility for
intramolecular hydrogen bonding. Interestingly, 4 was 5-fold
less stable than 6 with a half-life as short as 5 min (Table 1).
Perhaps, the intramolecular interaction between the N-methyl-
amino group and the triazene plays a significant stabilizing
role. We have already evoked a similar interaction for explain-
ing the stability of 7 (Scheme 2), a stable dimethyaminoethyl
triazene with a t1/2 = 108 min10 that showed significant anti-
tumor activity in vivo.20 To further elaborate on the impor-
tance of the N-methylamino group on the stability of 6,
we undertook a molecular modeling study using AM1 semi-
empirical calculation.
Figure 1. Molecular modeling using AM1 semi-empirical calcula-
˚
tion of 6. The H-N distances are given in A.
that do not express P-gp. MES-SA cells are also deprived of
EGFR expression.16 Therefore, the EGFR inhibitory acti-
vity of the quinazoline side chain would not influence the
growth inhibitory effect of compounds 4 and 6. The results
showed that 6 was 3-fold more potent than 4 (Figure 2A).
Perhaps the rapid decomposition of 4 compromised its
optimal cellular delivery. We also observed a 2-fold stronger
potency for 6 in the MDA-MB-468 breast cancer cells that
express EGFR but do not express P-gp,21,22 indicating that 6
was more potent than 4 regardless of the P-gp and the EGFR
status of the cells (Figure 2B).
Because our primary goal was to compare the cellular
penetration of the two compounds in P-gp-proficient and
-deficient cells, we tested their effects on both MES-SA (P-gp-
deficient) and its derived MES-SA/DX5 (P-gp-proficient)
cells. The latter cell line expresses high levels of P-gp and is
resistant to doxorubicin. Using the SRB assay, we showed a
20-fold difference between the sensitivity of MES-SA and
MES-SA/DX5 to doxorubicin. In contrast, no significant
difference (P > 0.05) was seen for both 4 and 6 in their
potency against the two cell lines. Combi-molecule 6 was
equally potent in both cell lines, and 4 was 1.6-fold more
potent against MES-SA/DX5 than MES-SA, indicating an
even greater potency in the cells that express P-gp.
As outlined in Figure 1, conformer 1 containing a H-bond
is computed to be ∼2 kcal/mol more stable than conformer 2,
which lacks the H-bond. Conformer 1 has the proposed
˚
H-bond (2.62 A distance) that stabilizes it. The H-bond is
absent in conformer 2, as the hydrogen and the amine
˚
nitrogen are trans to one another at a distance of 4.08 A.
Compound 4, which is not able to adopt H-bond-stabilizing
conformations, is less stable. However, in aqueous solution,
the superior stability of 6 is better rationalized in terms of the
equilibria depicted in Schemes 3 and 4. Because the central
nitrogen in 6 is protonated at physiological pH, the transi-
tion state to the formation of the doubly charged species
F should have a higher energy when compared with that for
C in Scheme 4. This may account for the slower rate of
hydrolysis for 6 when compared with 4 under physiological
conditions.
Differential Growth Inhibitory Activity. The potency of the
two dansylated compounds was compared in MES-SA cells
Cellular Uptake. To verify whether the differential res-
ponses correlated with cell penetration, we exploited the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2107
Scheme 3. Degradation Pathway of Compound 6 in Physiological Conditions
Scheme 4. Degradation Pathway of Compound 4 in Physiological Conditions
fluorescent properties of doxorubicin and those of com-
pounds 4 and 6 to analyze their intracellular content by
fluorescence microscopy and flow cytometry. We have pre-
viously used flow cytometry to analyze doxorubicin and
compound 6 internalization in human breast tumor cells.15,23
Fluorescence microscopy in the parental cell line showed a
homogeneous distribution of doxorubicin (Figure 3A, upper
panel) throughout the cell population. By contrast, in the
MES-SA/DX5 cells (Figure 3A, lower panel), the distribu-
tion was heterogeneous with a fraction of the cell popula-
tion emitting high fluorescence and another with poor to no
internalization of doxorubicin. As shown in Figure 4A, 4 and
6 penetrate the cells and decompose inside the cells to
generate its bioactive species: EGFR inhibitor, blue fluore-
scence, and a DNA-damaging species, green fluorescence.
The two colors were observed at similar intensity in both cell
types. Flow cytometric analysis permitted the quantification
of the proportion of cells that did not internalize doxorubicin
(Figure 3B) and the combi-molecules (Figure 4B). For the
more potent compound 6, at a dose range starting with the
IC50 for growth inhibition, no differential internalization
between the two cell types was seen. In contrast, 40% of the

2108 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Larroque-Lombard et al.
Figure 2. Growth inhibition of doxorubicin, 4, and 6 by SRB assay. (A) Comparison of antiproliferative activity of doxorubicin, 4, or 6 in
MES-SA and MES-SA/DX5 cells after 96 h. (B) Growth inhibition of 4 and 6 in human breast MDA-MB-468 cells. Each point represents an
average of two independent experiments performed in triplicate.
cell population could not internalize doxorubicin at this dose
range.
population when compared with MES-SA/DX5 (Figure 3C).
Cells with a higher comet tail moment than the control were
scored as damaged, and the data were presented as percent of
cells with damaged DNA. This result was consistent with the
differential level of doxorubicin uptake observed between
these two cell types.
DNA Damage. The DNA-damaging property of com-
pounds 4 and 6 was assessed by alkaline comet assay. The
visualization of DNA comet tails in both cell lines using
fluorescence microscopy showed that 6 was a strong DNA-
damaging agent, while 4 was not able to induce DNA
damage (Figure 5). This result is consistent with the stability
of the two molecules and also that of the putative dansylated
alkyldiazonium released after degradation. The rapid de-
composition of 4 seems again to limit its activity. Moreover,
6 damaged DNA in both cell lines with the same intensity,
independently of P-gp status, which is in agreement with the
growth inhibitory results, and also indicated that the combi-
molecule can penetrate and decompose inside the cells to
deliver the DNA-damaging moiety (green fluorescence,
Figure 4A). The levels of DNA damage induced by doxo-
rubicin were also analyzed in the two cell types, and a
differential response was seen with doxorubicin inducing a
2-fold greater number of DNA-damaged cells in the MES-SA
Conclusion
At the advanced stages of many cancers, drug transport is
significantly affected by several mechanisms including P-gp-
mediated drug efflux.1-5 This presents a daunting challenge to
drug development against advanced cancers. The combi-
molecules are designed to possess multiple targeting proper-
ties with the purpose of simultaneously blocking several
signaling networks in the cells. To this end, several pharma-
cophores must be appended to one single core structure.
Therefore, this leads to the branching of pharmacophores
through bulky linkers that often affect the size of the resulting
molecules and, as a result, their transport into the cells. It is
now well-known that the greater the size of an agent is, the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2109
Figure 3. Cellular fluorescence and DNA damage by doxorubicin. (A) Intracellular accumulation of doxorubicin in P-gp-positive and
-negative cells was determined by fluorescence microscopy. Cells were incubated with 25 μM doxorubicin for 2 h and washed with PBS, and
fluorescence was visualized with a Leica fluorescent microscope (40ꢀ magnification). (B) Cellular fluorescence of doxorubicin by FACS. The
levels of accumulated fluorescence for doxorubicin (0, 6.25, 12.5, 25, and 50 μM) were quantified in MES-SA and MES-SA/DX5 cells after 2 h.
(C) DNA damage induced by doxorubicin in MES-SA (0, 1.5, 6.25, 12.5, 25, and 50 μM) and MES-SA/DX5 (0, 6.25, 12.5, 25, 50, and 100 μM)
cells. Cells were exposed for 2 h to doxorubicin, and DNA damage was measured using an alkaline comet assay. Fifty cells were scored from
each dose, and the comet tail moment was calculated by comet IV software (Perceptive Instruments). Cells with a comet tail moment greater
than that in the control were scored positive and expressed as percent of cells with damaged DNA.
more likely that its transport mechanism across the cell
membrane will be influenced by efflux proteins. Here, we
discovered that at least one mechanism of drug efflux may not
prevent cell penetration by two prototypical combi-molecules
designed to block EGFR-DNA and carrying a bulky fluor-
escence-labeled moiety. Although the molecular mechanism
of such an important observation requires further elucidation,
we believe that the fact that the combi-molecules are designed
to decompose inside the cells to generate their bioactive
species might be a mean by which the rapid efflux of the intact
structure was impeded.
Compound 1, 2-Dansyl-9-fluorenylmethyl N-(2-Aminoethyl)-
carbamate. 9-Fluorenylmethyl N-(2-aminoethyl)carbamate hydro-
chloride (500 mg, 1.57 mmol) was dissolved in water saturated with
potassium carbonate and ethyl acetate (50 mL/50 mL). The mixture
was cooled to 0 °C, and a solution of dansyl chloride (3 equiv) in
ethyl acetate was added dropwise. The reaction mixture was stirred,
and the temperature was raised to room temperature. After 4 h, the
organic layer was separated, washed with brine twice and dried with
magnesium sulfate, filtered, and evaporated to provide a yellow-
green oil, which was purified by trituration in ethyl ether. A white
pure solid of compound 1 was obtained after filtration (742 mg,
92%). 1H NMR (300 MHz, DMSO-d6): δ ppm 2.75 (m, 2H), 2.80
(s, 6H), 2.95 (m, 2H), 4.21 (m, 3H), 7.25 (m, 4H), 7.37 (m, 2H), 7.59
(m, 4H), 7.85 (d, J=7.5 Hz, 1H), 7.99 (m, 1H), 8.08 (d, J=7.5 Hz,
1H), 8.24 (d, J=8.1 Hz, 1H), 8.45 (d, J=8.7 Hz, 1H).
Experimental Section
Chemistry. 1H NMR spectra and 13C NMR spectra were
recorded on a Varian 300 MHz spectrometer. Chemical shifts
are given as δ values in parts per million (ppm) and are
referenced to the residual solvent proton or carbon peak. Mass
spectrometry was performed by the McGill University Mass
Spectroscopy Center, and electrospray ionization (ESI) spectra
were performed on a Finnigan LC QDUO spectrometer. Data
are reported as m/z (intensity relative to base peak = 100).
Elemental analyses were carried out by GCL & Chemisar
Laboratories (Guelph, Ontario, Canada). The analyses were
done two times. All chemicals were purchased from Sigma-
Aldrich. Dansyl chloride was purchased from Alfa Aesar, N-(2-
aminoethyl)-N-methylethanediamine was from Wako Pure
Chemical Industries, Ltd., and mono-Fmoc-ethylenediamine
hydrochloride was from Novabiochem. The purities of all com-
pounds tested were >95% as determined by elemental analysis.
Compound 2, Dansyl-N-(2-aminoethyl)amide. The Fmoc
group was removed with morpholine/DMF 1/1 (2 mL) for 30 min
at room temperature. The reaction mixture was evaporated, and
the crude product was extracted with ethyl acetate and acidic
water phase (pH 2). The aqueous phase was alkalinized to pH 5
and extracted with ethyl acetate to eliminate the secondary
products from the aqueous layer. The pH was increased to 10,
and this aqueous layer was extracted again with ethyl acetate.
The organic layer was dried, and the magnesium sulfate was
filtered and evaporated to provide pure free amine 2 (148 mg,
1
87%). H NMR (300 MHz, DMSO-d6): δ ppm 2.45 (m, 2H),
2.75 (m, 2H), 2.81 (s, 6H), 7.24 (d, J =7.2 Hz, 1H), 7.59 (q,
J=7.5 Hz, 2H), 8.09 (d, J=7.2 Hz, 1H), 8.28 (d, J=8.4 Hz, 1H),
8.45 (d, J=8.7 Hz, 1H).
Compound 5, Dansyl-N-(2-[(2-aminoethyl)-N-methylamino]ethyl)-
amide. Dansyl chloride (1 g, 3.71 mmol) was dissolved in 20 mL

2110 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Larroque-Lombard et al.
Figure 4. (A) Cellular fluorescence of 4 and 6 by fluorescence microscopy. MES-SA and MES-SA/DX5 cells were incubated with 4 and 6 for
2 h, and imaging was performed with Leica fluorescent microscope (40ꢀ magnification). Visualization of 4 and 6 degradation species: Blue
aminoquinazoline and green dansylated species were imaged using individual filters. (B) Cellular fluorescence of 4 and 6 by FACS. The levels of
accumulated fluorescence for 4 and 6 (0, 6.25, 12.5, 25, and 50 μM) were quantified in MES-SA and MES-SA/DX5 cells by FACS, and results
were analyzed with GraphPad Prism software.
of ethyl acetate and was added dropwise to a cold solution of
N-(2-aminoethyl)-N-methylethanediamine (2.38 mL, 5 equiv) in
80 mL of ethyl acetate. The reaction mixture was stirred at 0 °C
under argon. After 2 h, the solution was extracted with neutral
water and acidic water (pH 2-3). This acidic water was alkali-
nized and was extracted with ethyl acetate two times. The
organic layer was dried with magnesium sulfate, filtered, and
evaporated to provide a yellow oil, which was purified by
crystallization in a minimum of ethyl acetate. White pure
crystals of compound 5 were obtained (1.117 g, 86%). 1H
NMR (300 MHz, DMSO-d6): δ ppm 1.92 (s, 3H), 2.13 (t, J =
6.3 Hz, 2H), 2.21 (t, J=6.7 Hz, 2H), 2.40 (t, J=6.1 Hz, 2H), 2.81
(s, 6H), 2.84 (t, J=6.9 Hz, 2H), 7.21 (d, J=7.5 Hz, 1H), 7.58 (m,
2H), 8.11 (d, J=7.2 Hz, 1H), 8.27 (d, J=8.4 Hz, 1H), 8.43 (d,
J =8.1 Hz, 1H).
Compound 4. The diazonium compound 3 was synthesized as
described in ref 14: Amino-anilinoquinazoline (50 mg, 0.184 mmol)
was dissolved in dry acetonitrile (5 mL) under argon and cooled
to -5 °C. Nitrosonium tetrafluoroborate (2 equiv) in acetoni-
trile was added directly. After 30 min at -5 °C, the resulting
clear orange solution was added dropwise to another solution of
compound 2 (1 equiv) in acetonitrile with triethylamine (2 equiv)
at 0 °C, after which the mixture was extracted with ethyl acetate
and brine. The organic layer was dried with potassium carbo-
nate and evaporated to provide a brown residue, which was
purified by serial trituration: First, the crude product was
dissolved in a minimum of methylene chloride and precipitated
with petroleum ether and after was triturated in ether/petroleum
ether 1/4 to give after filtration a red-brown solid (93 mg, 88%).
1H NMR (300 MHz, DMSO-d6): δ ppm 2.77 (s, 6H), 3.10 (m,
2H), 3.59 (m, 2H), 7.13 (d, J=8.4 Hz, 1H), 7.22 (d, J=7.5 Hz,
1H), 7.39 (m, 1H), 7.59 (m, 2H), 7.73 (d, J = 8.7 Hz, 1H), 7.86
(m, 2H), 8.13 (m, 2H), 8.27 (d, J=8.7 Hz, 1H), 8.35 (s, 1H), 8.43
(d, J=9.0 Hz, 1H), 8.58 (s, 1H), 9.89 (s, 1H), 10.59 (m, 1H). 13
C
NMR (75 MHz, DMSO-d6): δ ppm 158.13, 153.92, 152.05,
149.23, 148.90, 141.70, 136.29, 133.36, 130.71, 130.23, 129.74,
129.70, 129.50, 129.08, 128.61, 125.68, 124.28, 123.58, 121.89,
120.84, 119.71, 116.28, 115.82, 115.18, 51.72, 46.31, 45.72
(2C). ESI m/z 572.9 (MHþ with 35Cl). Anal. (C28H27ClN8O2S)
C, H, N.
Compound 6. The diazonium compound 3 was still synthe-
sized as previously described.14 Amino-anilinoquinazoline (608 mg,
2.246 mmol) was dissolved in dry acetonitrile (80 mL) under
argon and cooled to -5 °C. Nitrosonium tetrafluoroborate (2 equiv)
in acetonitrile was added directly. After 30 min at -5 °C, the
resulting clear solution was added dropwise to another solution
of compound 5 (1 equiv) in acetonitrile with triethylamine
(2 equiv) at 0 °C, after which the mixture was extracted with
ethyl acetate and brine. The organic layer was dried with
potassium carbonate and evaporated to provide a brown resi-
due, which was purified by serial trituration. The crude product
was dissolved in a minimum of methylene chloride and pre-
cipitated with petroleum ether, after which it was triturated in
ether/petroleum ether 1/4 to give after filtration a red-brown
solid (1.2 g, 85%). 1H NMR (300 MHz, DMSO-d6): δ ppm 1.99
(s, 3H), 2.32 (m, 2H), 2.52 (m, 2H), 2.77 (s, 6H), 2.90 (m, 2H),
3.53 (m, 2H), 7.12 to 7.21 (m, 2H), 7.39 (m, 1H), 7.55 (m, 2H),
7.75 to 7.97 (m, 4H), 8.12 (m, 2H), 8.30 (d, J=8.4 Hz, 1H), 8.40

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2111
Figure 5. DNA damage induced by 4 and 6 at 50 (A) and 100 μM (B). MES-SA and MES-SA/DX5 cells were exposed to each combi-molecule
for 2 h followed by assessment of drug-induced DNA damage using an alkaline comet assay. Visualization of DNA comets in both cell lines by
fluorescence microscopy (10ꢀ magnification) after staining with SYBR Gold. (C) Fifty comets were scored, and the comet tail moment was
quantified by comet IV software (Perceptive Instruments). Two independent experiments were averaged for each cell line, and data were
analyzed with GraphPad Prism software.
(m, 1H), 8.43 (s, 1H), 8.60 (s, 1H). 13C NMR (75 MHz, DMSO-
d6): δ ppm 158.10, 153.83, 152.01, 149.73, 148.78, 141.75,
136.69, 133.37, 130.72, 130.07, 129.76, 129.69, 129.56, 128.92,
128.54, 125.40, 124.25, 123.55, 121.83, 120.79, 119.77, 116.37,
115.76, 115.29, 57.04 (2C), 53.84, 46.40, 45.72 (2C), 41.51. ESI
m/z 632.1 (MHþ with 35Cl). Anal. (C31H34ClN9O2S) C, H, N.
Half-Lives. The half-lives of 4 and 6 under physiological
conditions were studied by UV spectrophotometer, Farmacia
Biotech Ultrospec 2000. The compounds were dissolved in
minimum volume of DMSO and diluted with DMEM supple-
mented with 10% FBS, and the absorbances were read at 335 nm
for 4 and 350 nm for 6 in a quartz UV cell maintained at 37 °C
with a circulating water bath. The experiment was repeated
three times for each compound. The half-life was estimated by a
one-phase exponential decay curve-fit method using the Graph-
Pad software package (GraphPad software, Inc., San Diego,
CA). The thermometer was Traceable VWR digital with (0.005 °C
accuracy.
Molecular Modeling. Molecular modeling was performed
with MOE software (version 2006.08) available from Chemical
Computing Group Inc. (Montreal, Quebec, Canada; www.
chemcomp.com).
Biology. Cell Culture. All three cell lines were purchased from
the American Type Culture Collection (ATCC, Manassas, VA).
Human uterine sarcoma cells, MES-SA, and MES-SA/DX5
(ATCC: CRL-1976 and CRL-1977, respectively) were main-
tained in McCoy 5A medium, and human breast cancer cells
MDA-MB-468 were cultured in DMEM. All media were sup-
plemented with 10% FBS, 1.5 mM L-glutamine (Wisent Inc.,
St.-Bruno, Canada), and 100 μg/mL penicillin/streptomycin
(GibcoBRL, Gaithersburg, MD). Cells were grown exponentially
at 37 °C in a humidified atmosphere of 95% air and 5% car-
bon dioxide. In all assays, cells were plated 24 h before drug
treatment.
Drug Treatment. Compounds 4 and 6 were synthesized in our
laboratory, and doxorubicin hydrochloride was purchased from
the hospital drug store (Mayne Pharma Inc., Montreal). In all
assays, molecules were dissolved in DMSO (50 mM stock solution),
and doxorubicin was dissolved in sterile water (2 mg/mL stock
solution) and subsequently diluted in McCoy 5A medium before
it was added to cells. The concentration of DMSO never
exceeded 0.1% (v/v) during treatment.
Growth Inhibition Assay. MES-SA, MES-SA/DX5, and
MDA-MB-468 cells were plated at a density of 5000 cells/well
in 96-well flat-bottomed microtiter plates (100 μL of cell/well).
Cells were allowed to attach overnight and then were treated
with different drug concentrations for 96 h. Thereafter, cells
were fixed using 50 μL of 50% trichloroacetic acid for 60 min at
4 °C, washed four times with water, stained with sulforhoda-
mine B (SRB 0.4%) for 2 h at room temperature, rinsed four
times with 1% acetic acid, and allowed to dry.24 The resulting
colored residue was dissolved in 200 μL of Tris base (10 mM,
pH 10.0), and the optical density was recorded at a wavelength
of 492 nm using a Bio-Rad microplate reader (model 2550). The
results were analyzed by GraphPad Prism (GraphPad Software,
Inc.), and the sigmoidal dose-response curve was used to
determine 50% cell growth inhibitory concentration (IC50).
Each point represents the average of at least three independent
experiments run in triplicate.
DNA Damage by Alkaline Comet Assay. The alkaline comet
assay was performed as previously described.25-27 Briefly, cells
were exposed to doxorubicin (0, 1.5, 6.25, 12.5, 25, and 50 μM),

2112 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Larroque-Lombard et al.
4, and 6 (0, 50, and 100 μM) for 2 h, harvested by trypsinization,
collected in ice-cold PBS by centrifugation at 3000 rpm for 5 min,
and then resuspended in PBS at 1 ꢀ 106 cells/mL. Cells were
mixed with low melting point agarose (0.7%) in PBS at 37 °C
(final dilution 1:10), followed by layering the agarose/cells
suspension on GelBond film (Lonza, Rockland, ME). The
agarose/cell suspension was allowed to solidify for 10 min and
then immediately placed in lysis buffer [2.5 M NaCl, 100 mM
tetra-sodium EDTA, 10 mM Tris-base, 1% (w/v) N-lauroyl-
sarcosine, 10% (v/v) DMSO, and 1% (v/v) triton X-100, pH
10.0] overnight at 4 °C. Thereafter, the gels were rinsed with
distilled water, equilibrated in alkaline electrophoresis buffer
[300 mM NaOH, 10 mM tetra-sodium EDTA, 7 mM 8-hydro-
xyquinoline, 2% (v/v) DMSO, pH 13.0] for 30 min at room
temperature, and electrophoresed at 20 V for 25 min in fresh
eletrophoresis buffer. The gels were subsequently neutralized
in 1 M ammonium acetate and then dehydrated in 100% ethanol
overnight. Comets were visualized at 10ꢀ magnification using
Leica fluorescent microscope after staining with SYBR Gold
(1:10000, Molecular Probes, Eugene, OR) for 45 min.
(5) Loo, T. W.; Clarke, D. M. Recent progress in understanding the
mechanism of P-glycoprotein-mediated drug efflux. J. Membr.
Biol. 2005, 206, 173–185.
(6) Aller, S. G.; Yu, J.; Ward, A.; Weng, Y.; Chittaboina, S.; Zhuo, R.;
Harrell, P. M.; Trinh, Y. T.; Zhang, Q.; Urbatsch, I. L.; Chang, G.
Structure of P-Glycoprotein Reveals a Molecular Basis for Poly-
Specific Drug Binding. Science (Washington, DC, U. S.) 2009, 323,
1718–1722.
(7) Perez, R. P.; Hamilton, T. C.; Ozols, R. F.; Young, R. C. Mecha-
nisms and modulation of resistance to chemotherapy in ovarian
cancer. Cancer 1993, 71, 1571–80.
(8) Turk, D.; Hall, M. D.; Chu, B. F.; Ludwig, J. A.; Fales, H. M.;
Gottesman, M. M.; Szakacs, G. Identification of Compounds
Selectively Killing Multidrug-Resistant Cancer Cells. Cancer Res.
2009, 69, 8293–8301.
(9) Brahimi, F.; Matheson, S. L.; Dudouit, F.; McNamee, J. P.; Tari,
A. M.; Jean-Claude, B. J. Inhibition of epidermal growth factor
receptor-mediated signaling by “Combi-Triazene” BJ2000, a new
probe for Combi-Targeting postulates. J. Pharmacol. Exp. Ther.
2002, 303, 238–246.
(10) Brahimi, F.; Rachid, Z.; McNamee, J. P.; Alaoui-Jamali, M. A.;
Tari, A. M.; Jean-Claude, B. J. Mechanism of action of a novel
“combi-triazene” engineered to possess a polar functional group on
the alkylating moiety: Evidence for enhancement of potency.
Biochem. Pharmacol. 2005, 70, 511–519.
(11) Brahimi, F.; Rachid, Z.; Qiu, Q.; McNamee, J. P.; Li, Y.-J.; Tari,
A. M.; Jean-Claude, B. J. Multiple mechanisms of action of ZR2002
in human breast cancer cells: A novel combi-molecule designed to
block signaling mediated by the ERB family of oncogenes and to
damage genomic DNA. Int. J. Cancer 2004, 112, 484–491.
(12) Domarkas, J.; Dudouit, F.; Williams, C.; Qiyu, Q.; Banerjee, R.;
Brahimi, F.; Jean-Claude, B. J. The Combi-Targeting Concept: Synthe-
sis of Stable Nitrosoureas Designed to Inhibit the Epidermal Growth
Factor Receptor (EGFR). J. Med. Chem. 2006, 49, 3544–3552.
(13) Banerjee, R.; Rachid, Z.; McNamee, J.; Jean-Claude, B. J. Synthe-
sis of a Prodrug Designed To Release Multiple Inhibitors of the
Epidermal Growth Factor Receptor Tyrosine Kinase and an
Alkylating Agent: A Novel Tumor Targeting Concept. J. Med.
Chem. 2003, 46, 5546–5551.
(14) Rachid, Z.; Brahimi, F.; Katsoulas, A.; Teoh, N.; Jean-Claude,
B. J. The Combi-Targeting Concept: Chemical Dissection of the
Dual Targeting Properties of a Series of “Combi-Triazenes. J. Med.
Chem. 2003, 46, 4313–4321.
(15) Todorova, M.; Larroque, A.-L.; Dauphin-Pierre, S.; Fang, Y. Q.;
Jean-Claude, B. J. Cellular imaging and signaling to AL237, a
fluorescent-labeled probe for the combi-targeting approach to
tumor targeting. Mol. Cancer Ther. 2009, accepted for publication.
(16) Yip, W. L.; Weyergang, A.; Berg, K.; Tonnesen, H. H.; Selbo, P. K.
Targeted delivery and enhanced cytotoxicity of cetuximab-saporin
by photochemical internalization in EGFR-positive cancer cells.
Mol. Pharmaceutics 2007, 4, 241–451.
(17) Harker, W. G.; MacKintosh, F. R.; Sikic, B. I. Development and
characterization of a human sarcoma cell line, MES-SA, sensitive
to multiple drugs. Cancer Res. 1983, 43, 4943–4950.
(18) Harker, W. G.; Sikic, B. I. Multidrug (pleiotropic) resistance in
doxorubicin-selected variants of the human sarcoma cell line MES-
SA. Cancer Res. 1985, 45, 4091–4096.
Fluorescence Microscopy. Fluorescent properties of mole-
cules were used to image drug transport efficiency and intracel-
lular accumulation. MES-SA and MES-SA/DX5 cells were
plated at 70% confluence in six-well plates, allowed to adhere
overnight, and treated with 0, 25, and 50 μM 4, 6, or doxo-
rubicin. After 2 h, cells were washed twice with PBS and were
directly imaged at 40ꢀ using Leica fluorescent microscope
(Leica DFC300FX camera) without fixation.
Flow Cytometry Analysis of Intracellular Drug Fluorescence.
MES-SA and MES-SA/DX5 cells were plated at 0.5ꢀ106 cells
per well in six-well plates, allowed to adhere overnight, and
incubated in the presence of 4 and 6 or doxorubicin (0, 1.25,
6.25, 12.5, 25, and 50 μM) for 2 h. Cells were collected by
trypsinization, washed twice with PBS, pelleted by centrifuga-
tion, and resuspended in 300 μL of PBS supplemented with 1%
FBS to prevent cell clumping. Intracellular fluorescence levels
were measured using a BD LSR flow cytometer (BD Bio-
sciences, San Jose, CA). For 4 and 6, fluorescence was detected
at two wavelengths for the two fluorescent products hydro-
lyzed in the cell: the aminoquinazoline [excitation at 340 nm
and emission at 451 nm (blue)] and the dansylated DNA-
damaging species [excitation at 340 nm and emission at 525 nm
(green)]. To test the transport efficiency of P-gp-proficient cells
(MES-SA/DX5) in comparison with the parental MES-SA
P-gp-deficient cells, we quantified the fluorescence levels of
doxorubicin [excitation at 480 nm and emission at 560-590 nm
(red)] and recorded the percentage of doxorubicin-positive
P-pg-expressing cells (MES-SA/DX5). FACS results were
analyzed with GraphPad Prism software.
(19) Koo, J. S.; Choi, W. C.; Rhee, Y. H.; Lee, H. J.; Lee, E. O.; Ahn, K.
S.; Bae, H. S.; Ahn, K. S.; Kang, J. M.; Choi, S. U.; Kim, M. O.; Lu,
J.; Kim, S. H. Quinoline derivative KB3-1 potentiates paclitaxel
induced cytotoxicity and cycle arrest via multidrug resistance
reversal in MES-SA/DX5 cancer cells. Life Sci 2008, 83, 700–708.
(20) Merayo, N.; Rachid, Z.; Qiu, Q.; Brahimi, F.; Jean-Claude, B. J.
The combi-targeting concept: Evidence for the formation of a novel
inhibitor in vivo. Anti-Cancer Drugs 2006, 17, 165–171.
(21) Wang, H.; Giuliano, A. E.; Cabot, M. C. Enhanced de novo
ceramide generation through activation of serine palmitoyltrans-
ferase by the P-glycoprotein antagonist SDZ PSC 833 in breast
cancer cells. Mol. Cancer Ther. 2002, 1, 719–726.
Acknowledgment. We thank the National Cancer Institute
of Canada (NCIC Grant 018475), Canadian Institutes of
Cancer Research (Grant FRN 49440), and Fonds de la
ꢀ
ꢀ
Recherche en Sante du Quebec doctoral award (M.T.) for
financial support. We gratefully acknowledge Nadim K. Saade
from the Department of Chemistry, McGill University, for
mass spectra.
(22) Liem, A. A.; Appleyard, M. V.; O’Neill, M. A.; Hupp, T. R.;
Chamberlain, M. P.; Thompson, A. M. Doxorubicin and vinorelbine
act independently via p53 expression and p38 activation respectively in
breast cancer cell lines. Br. J. Cancer 2003, 88, 1281–1284.
(23) Panasci, L.; Jean-Claude, B. J.; Vasilescu, D.; Mustafa, A.; Damian,
S.; Damian, Z.; Georges, E.; Liu, Z.; Batist, G.; Leyland-Jones, B.
Sensitization to doxorubicin resistance in breast cancer cell lines by
tamoxifen and megestrol acetate. Biochem. Pharmacol. 1996, 52,
1097–1102.
(24) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.
New colorimetric cytotoxicity assay for anticancer-drug screening.
J. Natl. Cancer Inst. 1990, 82, 1107–1112.
References
(1) Ramachandra, M.; Ambudkar, S. V.; Chen, D.; Hrycyna, C. A.;
Dey, S.; Gottesman, M. M.; Pastan, I. Human P-Glycoprotein
Exhibits Reduced Affinity for Substrates during a Catalytic Tran-
sition State. Biochemistry 1998, 37, 5010–5019.
(2) Sharom, F. J. The P-glycoprotein efflux pump: how does it trans-
port drugs? J. Membr. Biol. 1997, 160, 161–175.
(3) Szakacs, G.; Paterson, J. K.; Ludwig, J. A.; Booth-Genthe, C.;
Gottesman, M. M. Targeting multidrug resistance in cancer. Nat.
Rev. Drug Discovery 2006, 5, 219–234.
(4) Longley, D. B.; Johnston, P. G. Molecular mechanisms of drug
resistance. J. Pathol. 2005, 205, 275–292.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2113
(25) Matheson, S. L.; McNamee, J. P.; Wang, T.; Alaoui-Jamali, M. A.;
Tari, A. M.; Jean-Claude, B. J. The combi-targeting concept:
Dissection of the binary mechanism of action of the combi-triazene
SMA41 in vitro and antitumor activity in vivo. J. Pharmacol. Exp.
Ther. 2004, 311, 1163–1170.
(26) Olive, P. L.; Banath, J. P. The comet assay: A method to measure
DNA damage in individual cells. Nat. Protoc. 2006, 1, 23–29.
(27) McNamee, J. P.; McLean, J. R.; Ferrarotto, C. L.; Bellier, P. V.
Comet assay: Rapid processing of multiple samples. Mutat. Res.
2000, 466, 63–69.