Bile acid-based polyaminocarboxylate conjugates as targeted antitumor agents

Article abstract of DOI:10.1039/b823000e

Bile acid-polyaminocarboxylate conjugates containing NE3TA, a potential iron chelator displayed significant cytotoxicities in both HeLa and HT29 colon cancer cells, and cholic acid-NE3TA attached to an organic fluorophore was shown to enter the HT29 cance

Full text of DOI:10.1039/b823000e

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Bile acid-based polyaminocarboxylate conjugates as targeted
antitumor agentsw
Hyun-Soon Chong,* Hyun A. Song, Xiang Ma, Sooyoun Lim, Xiang Sun
and Santosh B. Mhaske
Received (in Austin, TX, USA) 22nd December 2008, Accepted 19th March 2009
First published as an Advance Article on the web 1st May 2009
DOI: 10.1039/b823000e
Bile acid–polyaminocarboxylate conjugates containing NE3TA,
a potential iron chelator displayed significant cytotoxicities in
both HeLa and HT29 colon cancer cells, and cholic acid–NE3TA
attached to an organic fluorophore was shown to enter the HT29
cancer cells.
thalassaemic patients indicates that the chelator possesses an
iron-mobilizing capability comparable to DFO.21
Previously, we synthesized and evaluated the polyamino-
carboxylate chelators in the NETA and NE3TA series
(NETA, NE3TA, and NE3TA-Bn, Fig. 1) as potential
iron-depleting antitumor agents.22 NETA, NE3TA, and
NE3TA-Bn showed greatly increased inhibitory activity in
HeLa and HT29 colon cancer cells as compared to the
clinically available agents DFO and DTPA. The chelators
were evaluated for their cytotoxicities in the cancer cells and
non-cancerous human lung fibroblast cells (MRC-5). The
chelators displayed much greater inhibitory activity in the
cancer cells as compared to the normal cells, while no or
slightly differential cytotoxicity between the cancer cells and
the normal cells was observed with DFO and DTPA.22
As an ongoing effort to develop targeted iron-depleting
antitumor agents, we designed bile acid-based NE3TA
conjugates. Amphiphilic bile acid including cholic acid (CA),
deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA)
is known to form helical globular aggregates with a favorable
pharmacokinetic profile and proven to be a useful shuttle
system for selective delivery of various drugs.23–25 The experi-
mental studies demonstrate that highly hydrophobic bile acid
such as deoxycholic acid and chenodeoxycholic acid enter
colon cancer cells which over-produce bile acid transporter,
carriers, and receptor proteins.26–30
Many reports suggest that high levels of iron accumulated in
animals and humans is associated with both the initiation and
progression of cancers.1–3 It is known that cancer cells require
more iron than normal cells and are sensitive to iron depletion
due to enhanced production of iron-related proteins such as
the iron-containing enzyme ribonucleotide reductase (RR) and
transferrin receptor (TfR).4 Iron depletion using iron chelators
has emerged as a promising antitumor therapeutic strategy.5–7
Iron chelators have been shown to target TfR, RR, or other
proteins involved in iron uptake that are over-expressed in
various cancers including HeLa and colon cancers and
colorectal liver metastases, resulting in cellular iron depletion
and potent antitumor activities.8–11
Among the iron chelators being evaluated in clinical
and preclinical settings are Triapine (3-aminopyridine-2-
carboxaldehyde thiosemicarbazone), DFO (desferoxamine),
and DTPA (diethylenetriaminepentaacetic acid). Triapine
(Fig. 1), a tridentate iron-depleting anticancer agent, effec-
tively inhibits the enzyme RR.12,13 A number of phase I and II
clinical studies of Triapine in patients with various cancers
have been conducted.5,7 A naturally-occurring hexadentate
siderophore DFO (Fig. 1) was the first iron chelator that
received FDA approval for use in the treatment of iron
overload that results from chronic transfusions in patients
with b-thalassemia major. DFO was found to induce apoptotic
cell death and display inhibitory and anti-proliferative activity
on tumor cells including leukemia, bladder carcinoma, neuro-
blastoma and hepatocelluar carcinoma.5,14,15 Two clinical
trials involving leukemia patients produced encouraging
therapeutic efficacy profiles.16,17 A polyaminocarboxylate acyclic
chelator DTPA (Fig. 1) was shown to display inhibitory
activity against human neuroblastoma and ovarian carcinoma
cells.18–20 The clinical study of DTPA in the iron-overloaded
In this communication, we report the synthesis and evalua-
tion of bile acid-based polyaminocarboxylates as antitumor
agents for targeted iron-depleting therapy. Although a number
of iron chelators have been prepared and evaluated
as antitumor agents, their use in targeted iron-depletion
anticancer therapy has been little explored. The bile
acid–NE3TA conjugates reported herein are the first examples
of targeted iron-depleting antitumor agents evaluated on
tumor cells.
Chemistry Division, Biological, Chemical, and Physical Sciences
Department, Illinois Institute of Technology, Chicago, IL, USA.
E-mail: chong@iit.edu; Fax: +1 312-567-3494;
Tel: +1 312-567-3235
w Electronic supplementary information (ESI) available: Experimental
section including synthetic procedures and characterization and
relaxivity data. See DOI: 10.1039/b823000e
Fig. 1 Iron chelators for antitumor therapy in preclinical and clinical
use.
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is the most potent inhibitor against both HeLa and HT29 cells.
The respective IC50 values of 6.3 Æ 0.1 mM and 7.5 Æ 0.3 mM
in HeLa and HT29 cancer cells were observed with
DCA–NE3TA. It should be noted that conjugation of NE3TA
to DCA is well tolerated and has little impact on cytotoxicity
of the parent chelator NE3TA. The HeLa cells were approxi-
mately 40-fold more sensitive to DCA–NE3TA than DTPA.
CA–NE3TA and CDCA–NE3TA possess similar cytotoxicity
in HeLa cells (24.0 Æ 2.7 mM vs. 24.6 Æ 1.0 mM), while
CA–NE3TA (8.3 Æ 2.4 mM) possesses a two-fold increased
cytotoxicity in HT29 cancer cells compared to CDCA–NE3TA
(15.5 Æ 3.2 mM).
TEM images of CDCA–NE3TA and CA–NETA indicate that
the hydrophobic bile acid conjugated to NE3TA forms discrete
semi-square micelles on the nanometre scale (B15–60 nm,
Fig. 2). Self-assembly of the conjugate results from the presence
of a hydrophobic cholic acid moiety surrounded by hydrophilic
NE3TA ligand.
Scheme 1 Synthesis of bile acid-based polyaminocarboxylates.
The synthesis of the novel bile acid-based chelators
CA–NETA, CA–NE3TA, DCA–NE3TA, CDCA–NE3TA,
and CA–NETA is shown in Scheme 1. CA–NE3TA was
prepared by a known procedure as reported previously.31
Bile acids (CA, DCA, and CDCA) were preactivated with
2-mercaptothiazoline to provide bile acid analogues 1–3,
respectively. The desired bile acid–NE3TA conjugates 6–9
were prepared by the reaction of the activated bile acid
analogues 1,31 2, or 3 with the bifunctional ligand NE3TA
(4)32 or NETA analogue (5)32 containing the amino group.
tert-Butyl groups in 6–9 were removed by 4 M HCl in
1,4-dioxane to provide bile acid conjugated chelators
CA–NETA, CA–NE3TA, CDCA–NE3TA, and DCA–NE3TA,
respectively.
With inhibitory efficacy observed with CA–NE3TA in
HT29 cancer cells, we wanted to visualize cellular uptake of
the conjugate using fluorescence imaging. NE3TA was
successfully conjugated to an organic fluorescent moiety NBD
(4-nitrobenzo-2-oxa-1,3-diazole) to produce NBD–CA–NE3TA
(Scheme 2).
The key step of the synthesis of NBD–CA–NE3TA is the
reaction of bifunctional ligand 4 containing an amino group
with preactivated aza cholic acid analogue 11. Aza cholic acid
1035 was prepared starting from the readily available cholic
acid according to the literature procedure.36 10 was pre-
activated with 2-mercaptothiazoline to produce 11. A coupling
reaction of compound 11 with 432 provided compound 12 in
52% yield. tert-Butyl groups in 12 were removed by treatment
with 4 M HCl in 1,4-dioxane to provide compound 13 in 96%
yield. The azide group in 13 was reduced to an amino
group using hydrogenation to provide compound 14, which
was further reacted with NBD-Cl to provide the fluorescent
bile acid-based NE3TA conjugate NBD–CA–NE3TA.
NBD–CA–NE3TA has the respective excitation and emission
wavelength of 446 nm and 535 nm (ESIw, Fig. S3).
Fluorescence and phase contrast images of control HT29
cells or HT29 cells incubated with NBD–CA–NE3TA (50 mM)
were obtained using a confocal microscope with a band-pass
filter set at 446/20 nm (excitation) and 535/30 nm (emission).
Confocal microscopic images shown in Fig. 3 indicate that
NBD–CA–NE3TA does accumulate in the HT29 cancer cells,
as evidenced by the green fluorescence that appeared in the
cells. It was noted that the control cancer cells also emit
auto-fluorescence at the wavelength of excitation, as shown
in Fig. 3.
The cytotoxicity of the bile-acid based NE3TA conjugates
CA–NE3TA, CDCA–NE3TA, and DCA–NE3TA using MTS
assay33 was evaluated using the HeLa and HT29 cell lines.
Inspection of the cytotoxicity data indicates that all bile
acid–NE3TA conjugates possess higher inhibitory activity
against the HeLa and HT29 cancer cells compared to the
clinically available chelators DFO and DTPA over the entire
concentration range evaluated (ESIw, Fig. S1 and S2). This
result is significant as there are reports that structural
modification to a chelator for introduction of a functional
unit could significantly affect its complexation with metal.22,34
IC50 values of bile acid–chelator conjugates are shown in
Table 1. Among all the compounds evaluated, DCA–NE3TA
Table 1 IC50 values of bile acid-based ligands in HeLa and HT29
cancer cells
IC50/mM
Ligand
HeLa
HT29
NE3TAb
5.7 Æ 0.3
4.7 Æ 0.3
8.3 Æ 2.4
7.5 Æ 0.3
15.5 Æ 3.2
36.1 Æ 1.4
39.1 Æ 4.0
CA-NE3TA
DCA-NE3TA
CDCA-NE3TA
DFOa
24.0 Æ 2.7
6.3 Æ 0.1
24.6 Æ 1.0
450
DTPAa
264.5 Æ 36.2
a
The chelators were incubated with the cells for 72 h, and the cell
b
viability was determined using MTS assay. The data were cited for
Fig. 2 TEM image unstained of CDCA–NE3TA (10 mM aqueous
comparison22
.
solution) and CA–NETA (1 mM aqueous solution).
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the fluorescence images. We thank Dr Sankar Bhuniya for
preparation of compound 3 and Mr Haisung Lee for preparing
the samples for TEM measurements.
Notes and references
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In summary, the bile acid–NE3TA conjugates (CA–NE3TA,
CDCA–NE3TA, and DCA–NE3TA) were synthesized and
evaluated for their anti-proliferative activities in the HeLa
and HT29 cancer cell lines. All bile acid–NE3TA conjugates
studied herein were much stronger inhibitors than the clini-
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was identified as the most potent antitumor agent, and
exhibited cytotoxic activity against the cancer cells comparable
to the non-functionalized parent chelator NE3TA. The cyto-
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