In vitrostudies of deferasirox derivatives as potential organelle-targeting traceable anti-cancer therapeutics

Article abstract of DOI:10.1039/d0cc08156f

We report here strategic functionalization of the FDA approved chelator deferasirox (1) in an effort to produce organelle-targeting iron chelators with enhanced activity against A549 lung cancer cells. Derivative 8 was found to have improved antiproliferative activity relative to 1. Fluorescent cell imaging revealed that compound 8 preferentially localises within the lysosome.

Full text of DOI:10.1039/d0cc08156f

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In vitro studies of deferasirox derivatives as
potential organelle-targeting traceable
anti-cancer therapeutics†
Cite this: Chem. Commun., 2021,
57, 5678
Received 16th December 2020,
Accepted 4th May 2021
a
Axel Steinbrueck,
Sajal Sen,
Jonathan L. Sessler
Adam C. Sedgwick,a Hai-Hao Han,b Michael Y. Zhao,a
Dan-Ying Huang,a Yi Zang,c Jia Li,*c Xiao-Peng He*b and
a
a
DOI: 10.1039/d0cc08156f
*
rsc.li/chemcomm
We report here strategic functionalization of the FDA approved
To address these challenges, our efforts have focused on the
chelator deferasirox (1) in an effort to produce organelle-targeting development of chelators that show enhanced cytotoxicity and
iron chelators with enhanced activity against A549 lung cancer produce a steep, non-plateauing dose–response curve, a pro-
cells. Derivative 8 was found to have improved antiproliferative perty that is considered favourable for chemotherapeutics. This
activity relative to 1. Fluorescent cell imaging revealed that com- is because small, clinically achievable increases in the concen-
pound 8 preferentially localises within the lysosome.
tration of a drug above its IC50 value can translate into a
disproportionally larger fractional killing of cancer cells.17,18
A design feature of 1 that limits its efficacy as an anticancer
Over the past decades, iron chelators have gained increasing
attention as potential primary or adjuvant cancer therapies.1–4 agent is the terminal carboxylic acid (highlighted in blue in
The depletion of iron from rapidly proliferating cancer cells has Scheme 1), which imparts an overall negative charge on this
been shown to result in effective growth inhibition and to ligand at physiological pH, thereby disfavouring the effective
ultimately induce apoptotic cell death.5–7 Within this arena, passage of 1 through lipid cell membranes.19 To ameliorate the
the clinical iron sequestration agent deferasirox (1, Scheme 1) above-mentioned drawback of 1 we have investigated the
has shown encouraging initial anticancer activity in vitro and strategic derivatization of the carboxylic acid via introduction
in vivo for example against inter alia AML,8 triply negative of organelle targeting groups designed to improve cellular
breast cancer,9 and lung cancer.10,11 Despite this promise, the uptake while guiding preferential localization within the lyso-
treatment of solid tumours with 1 remains challenging and new some or mitochondria.20,21 These latter organelles constitute
chelators are required that are taken up into cancerous cells
efficiently and which display enhanced cytotoxicity.12,13 Only a
few reports are currently available in the literature that discuss
the derivatization of 1 to produce anticancer agents, and the
majority of derivatives synthesized in such a context show only
moderate cytotoxicity in vitro with concentrations 4100 mM
required to achieve baseline eradication of cancer cells.14–16
a Department of Chemistry, University of Texas at Austin, 105 E 24th street A5300,
Austin, TX 78712-1224, USA. E-mail: sessler@cm.utexas.edu
b Key Laboratory for Advanced Materials and Joint International Research
Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize
Scientist Joint Research Center, Frontiers Center for Materiobiology and Dynamic
Chemistry, School of Chemistry and Molecular Engineering, East China University
of Science and Technology, 130 Meilong Rd., Shanghai 200237, China.
Scheme 1 (a) Structures of 1 and its complex with Fe3+. The donor set is
highlighted in red and the carboxylate moiety is highlighted in blue. (b)
E-mail: xphe@ecust.edu.cn
c National Center for Drug Screening, State Key Laboratory of Drug Research,
Conditions: (i) Methanol, reflux, 48 h. (ii) Urea (4 equiv.), imidazole
(2 equiv.), microwave 150 W, 170 1C, 20 min. (iii) EDC (2 equiv.), TEA
(2 equiv.), NHS (cat.), amine (3 equiv.), CH2Cl2, r.t., 16 h. (iv) Same as (iii) with
N-BOC ethylenediamine, then TFA, r.t., 16 h.
Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, P. R. China. E-mail: jli@simm.ac.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc08156f
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Table 1 IC50 and Hill slope (HS) values for 1–10 against the A549 human
lung cancer cell line after 72 h and 24 h incubation. Experiments were
performed in triplicate
appealing targets due to their key role in mediating intracel-
lular iron metabolism and their reported sensitivity to iron
chelation therapy, which is thought to be mediated via the
induction of oxidative stress caused by ROS generation in these
organelles, for example, in the cases of the potent iron chela-
tors salinomycin and Dp44mT, respectively.6,20–23
We have now examined the antiproliferative activity of
several new derivatives of 1 using the A549 human lung cancer
cell line and as detailed below identified derivative 8 as having
an enhanced therapeutic activity compared to 1. In contrast to
deferasirox, 8 proved fluorescent in aqueous environments,
which allowed its subcellular localisation in the lysosome to
be followed by fluorescent microscopy.
Compound IC50 (72 h)
HS (72 h) IC50 (24 h)
HS (24 h)
Ox-Pt
1
2
3
4
5
6
0.5 Æ 0.1 mM 1.2 Æ 0.2
450 mM
450 mM
N/A
N/A
8.5 Æ 2.0 mM 1.0 Æ 0.2
2.5 Æ 0.7 mM 0.9 Æ 0.2
8.3 Æ 2.2 mM 0.8 Æ 0.2
3.6 Æ 1.6 mM 0.6 Æ 0.2
6.0 Æ 0.6 mM 1.9 Æ 0.3
21.0 Æ 3.0 mM 1.3 Æ 0.2
450 mM
450 mM
N/A
N/A
29.0 Æ 1.4 mM 2.9 Æ 0.6
450 mM
N/A
450 mM N/A
7
8
9
10
3.8 Æ 0.5 mM 1.0 Æ 0.1
3.7 Æ 0.3 mM 2.7 Æ 0.6
7.7 Æ 0.8 mM 1.7 Æ 0.3
23.3 Æ 1.5 mM 1.8 Æ 0.2
12.3 Æ 1.3 mM 3.5 Æ 1.0
12.6 Æ 0.8 mM 2.7 Æ 0.4
450 mM
N/A
450 mM
N/A
First, guided by the premise that they would benefit from
improved passive diffusion through lipid membranes and thus
faster internalization with respect to 1, derivatives with neutral study showed activities that were either improved relative to 1
sidechains were prepared (compounds 2–4, Scheme 1b). Next, or similar. The exception was 6, which proved inactive. Redu-
we synthesized derivatives containing hydrophilic amine and cing the exposure time to 24 h decreased the apparent activity
ammonium side chains (6–9, Scheme 1b) as these function- of the charge neutral derivatives 2–4, as well as Ox-Pt by over an
alities have been reported to promote preferential localisation order of magnitude. This finding leads us to suggest that
in the lysosome.24 Finally, we prepared one derivative several cell cycles are required for these compounds to exert a
(5, Scheme 1b) with a cationic triphenylphosphonium moiety, cytotoxic effect. Interestingly, the activity of the derivatives with
a hydrophobic subunit reported to drive localisation toward the lysosomal targeting amine groups 7, 8 and 9, were less
mitochondria.20,25 Detailed synthetic procedures for all com- impacted by shorter exposure times with 8 (IC50 = 12.3 mM)
pounds prepared in this study, as well as their respective and 9 (IC50 = 12.6 mM) exhibiting the highest activity under
characterization by 1H- and 13C-NMR spectroscopy and high these conditions. Compounds 8 and 9 furthermore produced
resolution mass spectrometry, are available in the ESI.† All new the highest HS parameters after both 72 h and 24 h exposures,
derivatives were found to be stable under physiological condi- while 1, Ox-Pt, and the derivatives with neutral side chains (i.e.,
tions at 37 1C over the course of several days (cf. ESI†). The 2–4) produced rather shallow dose–response curves (Table 1)
lipophilicity of 1–9 was quantified by determining their dis- and required concentrations Z100 mM to achieve baseline
tribution coefficients (log D) at pH 7.2 and at pH 4.5 (to mimic eradication of A549 cancer cells. The determined IC50 values
lysosomal pH) (cf. Table S1 in the ESI†). In accord with the of the potent derivatives 2 and 8, as well as 5 bearing a
design expectations, the lipophilicity of the carboxylic acid 1 mitochondria directing group, were verified via crystal violet
increased at lower pH while the amine derivatives became more staining as a secondary measure of activity. In all cases, IC50
hydrophilic in more acidic environments.
values close to those determined via MTT assay were obtained
The antiproliferative activity of compounds 1–9 and control (cf. Fig. S6 in the ESI†).29
10 was evaluated in A549 lung cancer cells, a cell line with a
Comparison studies for derivatives 2 and 8, as well as the
well-established sensitivity to iron imbalance.11,26 The clinical parent chelator 1, in HCT116 colon cancer cells and L929 non-
chemotherapeutic oxaliplatin (Ox-Pt) with a known activity cancerous mouse fibroblast cells showed consistent activity for
profile in A549 cells was included as a positive control.27,28 each chelator, respectively, across all three cell lines (cf. Fig. S5
For each compound, cellular proliferation profiles were pro- in the ESI† for proliferation profiles and Table S2 for IC50
duced via a standard MTT assay for exposure times of 72 h and values). These results were taken as evidence that the cell type
24 h, respectively (cf. Fig. S1 and S2 in the ESI†). From these did not significantly affect the activity of the present iron
proliferation profiles, averaged IC50 values and Hill slope (HS) chelators. The combination of favourable cytotoxicity and HS
parameters were determined via nonlinear regression analysis. seen at 72 h in the case of 8 led us to prepare its diether
The results are summarised in Table 1. IC50 values represent a analogue 10 via methylation of the phenol moieties (cf. ESI†
commonly reported metric of toxicity, while HS parameters Scheme S1). This derivative was expected to display a reduced
provide insight into the shape of the proliferation profile, metal binding affinity thus serving as a negative control for 8.
wherein higher HS values are desirable as they correspond to In fact, derivative 10 exerted no appreciable antiproliferative
a steeper dose–response curve. HS values have attracted inter- activity against A549 cells under conditions identical to those
est in recent years as this parameter was found to exhibit used to test compounds 1–9 as shown in Fig. 1. Additionally,
greater consistency when compared across different cell lines when cells were supplemented with 50 mM FeCl3, both 1 and 8
than the IC50 value.17
no longer produced any observable cytotoxicity after 72 h of
The determined IC50 values of 1 and Ox-Pt are in good exposure (cf. Fig. S3 in the ESI†). This finding is taken as
agreement with previous literature reports for an exposure time evidence that intracellular iron chelation plays a key role in
of 72 h.11,27 At 72 h incubation time, the new derivatives of this mediating their in vitro antiproliferative activity.
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a mitochondrial localisation effect in the case of simple defer-
asirox derivatives and that other factors may govern the intra-
cellular distribution of this class of chelator.
Our recent efforts have focused on overcoming the observed
unwanted cytotoxicity towards healthy cells. We found, for
instance, that deferasirox derivatives can be effectively encap-
sulating into the ubiquitous blood protein human serum
albumin (HSA), an established tumour localizing agent.30,31
The parent ligand 1, as well as the potent derivatives 2 and 8,
were found to bind to HSA as inferred from Stern–Volmer
Fig. 1 Selected proliferation profiles of 1, 8, 10 and oxaliplatin (Ox-Pt) in
A549 cells. The high HS parameter of 8 translates to sharp decline in cell
viability with increasing drug concentration. Derivative 8 also achieves
baseline eradication of cancer cells at the lowest concentration of all analyses (cf. Fig. S6–S8 for the titration data and Table S3 for
evaluated drugs. See text for discussion.
the Stern–Volmer quenching constants, ESI†). The resulting
HSA-complexes of 2 and 8, respectively, showed a 2- and 3-fold
In contrast to deferasirox 1, derivatives 2 and 8 at concen-
trations of 50 mM give rise to distinct fluorescence emission
bands centred 510 nm and 480 nm, respectively, in PBS
(cf. Fig. S9–S11, ESI†). This allowed their subcellular location
to be explored with the results shown in Fig. 2. As confirmed by
comparisons with Lysotrackers red, derivative 8 was found to
localise in the lysosome as determined by fluorescent cell
microscopy using A549 cells. In contrast, no discernible orga-
nelle targeting was seen in the case of 2, a chelator that lacks a
recognized organelle targeting unit. The enhanced cytotoxicity
and localization in the lysosome seen for 8 proved reproducible
in HeLa cells (cf. Fig. S12 in the ESI†).
No detectable increase in ROS production was seen in A549
cells after incubation with the potent derivative 8 as evidenced
by confocal microscopy (cf. Fig. S13 in the ESI†). These results
lead us to suggest that the enhanced therapeutic efficacy seen
in the case of 8 is due, at least in part, to its intracellular
localization and that the cytotoxicity of this derivative is exerted
via an ROS-independent mechanism. This sets 8 apart from
other lysosome directed iron chelators, such as salinomycin.21
We further utilized the fluorescent properties of the
triphenylphosphonium-bearing derivative 5 to trace success-
fully this chelator inside A549 cells (cf. ESI† Fig. S13). However,
no colocalization with Mitotrackers red was observed for 5,
which may in part explain the low activity of this derivative.
Notably, the inherent fluorescent properties of this derivative
leads us to conclude that introduction of a well-established
mitochondria directing group20,25 does not necessarily produce
increase in cytotoxicity in A549 cells relative to the uncom-
plexed chelators (cf. Fig. S5 for proliferation profiles and Table
S2 in the ESI† for IC50 values). Encouraged by these results, our
future efforts will focus on exploring the tumour targeted
delivery of deferasirox derivatives.
In conclusion, we report the synthesis of eight new derivatives of
deferasirox including examples with neutral, cationic and amine-
containing side chains. These derivatives were evaluated for their
antiproliferative activity in A549 cells after incubation times of 24 h
and 72 h. The derivatives that contained lysosome targeting moi-
eties, such as 8, were found to exert notable cytotoxicity after 24 h
exposure time and also showed steeper dose–response curves with
respect to the parent chelator 1. Derivative 8, as well as the majority
of the new compounds reported here, proved fluorescent in aqueous
media (Fig. S4–S6, ESI†), allowing their subcellular localisation to be
tracked inside live cells. It was found that the chelator 8, but not the
control system 2 lacking a localising functionality, localised well to
the lysosome. The ability to produce an antiproliferative response as
well as providing for fluorescence-based tracking, are considered
attractive features of the present systems and serve to underscore the
versatility of the deferasirox platform in terms of potential iron
chelation-based approaches to anticancer drug discovery. More
broadly, the present results highlight the benefits that can accrue
by optimizing the drug-like properties and targeting features of
chelators displaying therapeutic potential.
The initial work in Austin was supported by the National
Institutes of Health (grant CA 68682 to J. L. S.). Subsequent
studies were supported by the Robert A. Welch Foundation (grant
F-0018 to J. L. S.). X.-P. H. thank the NSFC (no. 91853201), the
Shanghai Municipal Science and Technology Major Project
(2018SHZDZX03), the National Key Sci-Tech Special Projects of
Infection Diseases of China (2018ZX10732202) and the Interna-
tional Cooperation Program of Shanghai Science and Technology
Committee (17520750100). H.-H. H. would like to thank the China
Postdoctoral Science Foundation (No. 2020M681196) for support.
Conflicts of interest
Fig. 2 Confocal microscopy imaging of 2 and 8 (20 mM) in A549 cells.
Colocalization was observed between 8 and Lysotrackers red. Blue
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
channel: Ex/Em
580–620 nm.
= 405/440–480 nm. Red channel: Ex/Em = 559/
5680
|
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14 S. Salehi, A. S. Saljooghi and A. Shiri, Eur. J. Pharmacol., 2016, 781,
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