Cell Permeable Imidazole-Desferrioxamine Conjugates: Synthesis and in Vitro Evaluation

Article abstract of DOI:10.1021/acs.bioconjchem.8b00924

Desferrioxamine (DFO), a clinically approved iron chelator used for iron overload, is unable to chelate labile plasma iron (LPI) because of its limited cell permeability. Herein, alkyl chain modified imidazolium cations with varied hydrophobicities have been conjugated with DFO. The iron binding abilities and the antioxidant properties of the conjugates were found to be similar to DFO. The degree of cellular internalization was much higher in the octyl-imidazolium-DFO conjugate (IV) compared with DFO, and IV was able to chelate LPI in vitro. This opens up a new avenue in using N-alkyl imidazolium salts as a delivery vector for hydrophilic cell-impermeable drugs.

Full text of DOI:10.1021/acs.bioconjchem.8b00924

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Article
Cell permeable Imidazole-Desferrioxamine
Conjugates: Synthesis and in-vitro Evaluation
Shreya Pramanik, Saikat Chakraborty, Malavika Sivan, Birija Patro, Sucheta Chatterjee, and Dibakar Goswami
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00924 • Publication Date (Web): 14 Feb 2019
Downloaded from http://pubs.acs.org on February 17, 2019
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Cell permeable Imidazole-Desferrioxamine Conjugates: Synthesis and in-
vitro Evaluation
Shreya Pramanik,a,+ Saikat Chakraborty,b,+ Malavika Sivan,c,+ Birija S Patro,b,d Sucheta
b
b,d
Chatterjee, and Dibakar Goswami *
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aCentre for excellence in Basic Sciences, Mumbai, PIN: 400098, India
bBio-Organic Division, Bhabha Atomic Research Centre, Trombay, Mumbai, PIN: 400085,
India
cIISER, Bhopal, PIN: 462066, India,
dHomi Bhabha National Institute, Anushaktinagar, Mumbai, PIN: 400094, India
+Equal contributions
*
Corresponding author
ABSTRACT.
Desferrioxamine (DFO), a clinically approved iron chelator used for iron overload, is
unable to chelate labile plasma iron (LPI) due to its limited cell permeability. Herein, alkyl
chain modified imidazolium cations with varied hydrophobicities have been conjugated with
DFO. The iron binding abilities, and the antioxidant properties of the conjugates were found
to be similar to DFO. The degree of cellular internalisation was much higher in the octyl-
imidazolium-DFO conjugate (IV) compared to DFO, and was able to chelate LPI in-vitro.
This opens up a new avenue in using N-alkyl imidazolium salts as a delivery vector for
hydrophilic cell-impermeable drugs.
KEYWORDS. N-alkyl imidazolium salts; Desferrioxamine; hydrophobicity; Chelation;
Labile plasma iron.
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Introduction
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Iron is the most abundant metal on earth, and is a vital constituent of human body. It
plays important roles in oxygen transport, DNA synthesis and in the formation of several
biologically important enzymes. However, due to the redox toxicity of the labile iron, its
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absorption, distribution, utilization and storage in human body is tightly regulated. At the
same time, there are certain conditions (e.g. hereditary hemochromatosis, β-thalassemia etc.)
which lead to excess iron accumulation in human body. These iron pools, either accumulated
in plasma (labile plasma iron, LPI) or in cytosol (labile iron pool, LIP), generates reactive
oxygen species (ROS)via Fenton/Heber-Weiss reactions, thereby promoting oxidative stress
leading to cell death via lipid peroxidation and/or DNA damage.4
Chelation therapy, used for the treatment of iron overload and to maintain a steady
and healthy iron concentration in human body, has gained considerable attention in the last
5
few years. Three chelators viz. desferrioxamine B (DFO), deferiprone (DFP) and deferisirox
(DFX) are being regularly used in clinical practice. Apart from these, a large number of other
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chelators have also been developed and are under different phases of trial. Amongst these,
DFO, a highly specific hydroxamate based iron chelator, has been used for last 50 years with
7
a high success rate. However, its poor gastrointestinal absorptivity, and subsequent poor
bioavailability renders this drug to be administered as a parental infusion, leading to poor
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patient compatibility. DFP, despite its better bioavailability and promising results when used
in combination with DFO or DFX, has limited clinical use due to some serious side-
effects.9,10 Moreover, both bidentate (DFP) and tridentate (DFX) ligands can form partially
chelated complexes, and may be susceptible for redox recycling, which is detrimental and
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undesirable. DFO, which forms strong hexa-coordinate complex with iron, is the most
advantageous and the most preferred chelator for clinical uses.
However, the limiting cell-permeability of DFO, mainly due to its high hydrophilicity
coupled with high molecular weight, has restricted its use for chelation of LIP. The “free”
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LIP in the Fe-loaded cells has significant toxicity, and hence is an important and invincible
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target for iron chelators. To overcome this limitation, a number of DFO-conjugates e.g. with
lipophilic adamantyl groups,13 caffeine,14 peptides,4,15,16 triphenylphosphonium,17 different
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lipophilic antioxidants etc. have been synthesized and evaluatedfor membrane permeability.
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However, none of these conjugates have been approved as a chelator for clinical use, and
hence, search for an “ideal” conjugate continues.
Recently, imidazolium based compounds have shown increased antimicrobial
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acitivities. Similar kinds of salts are also being considered as a potential tool for delivering
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active pharmaceutical ingredients (API) across cells. The API can be linked to the cationic
21
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imidazolium core either as an anion or via covalent bonding. Their properties, specially
hydrophilicities of the conjugates, can be tailored using different substituents, and hence this
offers a major advantage of using imidazolium salts as delivery vectors. In this study, we
have conjugated DFO with imidazolium cations having varied alkyl substituents, thereby
possessing different degrees of hydrophobicity (Fig. 1, compounds I-IV). These conjugates
were evaluated for their iron binding abilities, their antioxidantproperties, and their cell
permeability, compared to the parent siderophore.
O
O
H
N
O
O
N
N
H
N
N
H
N
N
N
R
N
H
N
OH
O
OH
O
CH SO
3
3
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O
O
N
N
N
N
OH
N
N
OH
H
H
OH
O
OH
O
II. R = CH3
I
III. R = C H9
4
IV. R = C H
8
17
Fig. 1. Structures of imidazole-DFO conjugates (I-IV).
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Results
Synthesis of imidazole-desferrioxamine conjugates I-IV
Imidazole-DFO conjugates (I-IV) were prepared according to Scheme 1. For
synthesis of conjuagate I, imidazole (1) was reacted with ethyl bromoacetate, followed by
acid hydrolysis and subsequent conjugation to DFO using reported procedure.22,23 For
conjugate II, commercially available N-methyl imidazole (4) was similarly reacted with ethyl
bromoacetate, followed by acid hydrolysis and subsequent conjugation to DFO. Conjugates
III and IV were synthesized similarly using N-butyl imidazole (5) andN-octyl imidazole(6)
respectively.
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. R = C H
. R = C H
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iii
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HCl
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. R = CH3
. R = C H
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iii
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Br
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OH
H
OH
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10. R = CH3
1. R = C H
12. R = C H
1
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iv
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OH
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N
OH
H
OH
O
II. R = CH3
III. R = C H
4
9
IV. R = C H
8
17
o
i) Ethyl bromoacetate (for synthesis of 2) or RBr (for synthesis of 5-6) / K CO / KOH / TBAI / CH Cl / 25 C / 8h;
ii) Ethyl bromoacetate / THF / -10 C to 25 C / 6h; iii) 40% aq. HCl / reflux / 4h; iv) DFO-Mes / HOBt / DIC / DMF /
2
3
2
2
o
o
o
o
4
5 C / 1h, then DIPEA / 25 C / 8h
Scheme 1. Synthesis of conjugates I-IV.
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Purification and characterization
The crude conjugates I-IV were purified by semi preparative RP-HPLC using
Younglin RP-HPLC semi preparative system Model YL9100 consisting of a quaternary
pump (YL9110S), an UV detector (YL9120 Dual Absorbance Detector), a manual sample
injector, an automated gradient controller and a semi-preparative column (Venusil XBP C
18), at a flow rate of 2.0 mL/min (solvent A: 0.1% TFA/water and solvent B: 60%
AcCN/0.1% TFA). The absorbances were monitored at a wavelength of 254 nm, and the
gradient applied was5% B to 95% B in 90 min.
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After purification, the analysis of the purified conjugates I-IV was performed in a
®
Younglin HPLC system (analytical column Kromasil C18) with a flow rate of 1.0 mL/min
using the same solvents and gradient as mentioned above. The absorbance was monitored at a
1
wavelength of 254 nm.All the conjugates were characterized by H NMR (using Bruker AC-
200 instrument or 500 MHz Varian NMR spectrometer, as described before and also inSI),
CHN-analysis (using Elementar Vario micro cube), and FT-IR (as pellets in KBr, using a
BRUKER Tensor II spectrophotometer) spectroscopy. The retention times (R ) in analytical
t
RP-HPLC and the calculated and experimental results of CHN analyses of the purified
conjugates are tabulated in Table 1.
Table 1. Results of analytical RP-HPLC and elemental analysis for conjugates I-IV.
RP-HPLC
Elemental Analysis
Calculated (%) Experimental (%)
Conjugate
Rt
Purity
C
H
N
S
C
H
N
S
(min)
(%)
97.8
95.6
91.1
93.6
I
23.8
20.2
12.8
16.5
53.88 7.84 16.75 ----- 53.64 7.68 16.98 -----
49.34 7.51 14.39 4.12 49.06 7.48 14.75 3.74
51.20 7.86 13.65 3.91 50.81 7.74 13.96 4.17
53.41 8.27 12.78 3.66 53.12 8.62 12.95 3.64
II
III
IV
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000
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3000
2500
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-1
wave number (cm )
Fig. 2. FT-IR spectra of DFO and conjugates I-IV.
Table 2. Selected FT-IR peaks obtained in conjugates I-IV vis-à-vis DFO
-1
-1
-1
-1
-1
Vibration mode
-N-OH
DFO (cm )
I (cm ) II (cm ) III (cm ) IV (cm )
1640
1638
3491
2938
2854
1627
1464
1162
1050
1567
1648
3203
2921
2855
1634
1460
1166
1049
1567
1627
3487
2931
2859
1627
1463
1159
1051
1570
1641
3486
2935
2859
1620
1464
1165
1049
1562
-
N-H stretching
3480
-
CH stretching
2936 (asymmetric)
2
2
859 (symmetric)
1627
CO stretching
-
CH bending
1474
2
C-C stretching
N-O stretching
Imidazolium N–C–N
stretching
1162
1048
absent
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FT-IR spectra of the conjugates were recorded in KBr pellets, and compared with
-1 24
DFO (Fig. 2). It could be observed that hydroxamic group vibration (~1640 cm ) appear in
the spectra of DFO alongwith all the compounds I-IV, indicating the presence of unaffected
chelator moiety in the conjugates. Although the -C=C- stretching vibration of the
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imidazolium ring (~1640 cm ) was masked (due to overlapping N-OH vibration) in the
spectra of the conjugates I-IV, the conjugation of imidazolium group to DFO could be
-1
ascertained from the presence of imidazolium N–C–N stretching mode (~1565 cm ) in the
spectra of I-IV.26,27 The other vibrations, typically suggesting the unaltered chelating moiety
28
in the conjugates are tabulated in Table 2.
Table 3. Calculated logPOW values for DFO-mesylate and conjugates I-IV
ompound DFO-Mes
-6.39
I
II
III
IV
logPOW
-3.41
-6.82
-5.61
-4.03
2
9
The calculated (using MarvinSketch software ) logPOW (octanol/water) values for
DFO-Mes (neutral form, ammonium cation with methane sulphonate as counter anion and
conjugates I-IV (neutral form for conjugate I; neutral forms for DFO and conjugates II-IV
with either ammonium or imidazolium cation and methane sulphonate as counter anion, no
micro species with gross charges were taken into account) are tabulated in Table 3.
Conjugates II and III were calculated to be highly hydrophilic, almost comparable to DFO.
Conjugates I and IV were comparatively hydrophobic, showing promise for better cellular
uptake. Experimentally, the solubility of conjugates II and III in water (pH 7.4, > 3 mg/mL)
was much higher than those of conjugates I and IV (< 1 mg/mL). This corroborates with the
calculated logPOW values. Stock solutions of II and III in HBS, and I and IV in DMSO were
prepared. Unless otherwise mentioned, the DMSO solutions were properly diluted in HBS for
further experiments.
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Iron binding studies
The iron-binding of conjugates I-IV was checked by UV electronic spectroscopy (Fig.
3), and was compared with that of desferrioxamine (DFO). All the spectra were collected
with 10% aqueous DMSO solutions. Addition of ferrous ammonium sulfate (FAS) to DFO or
DFO-conjugates I-IV invariably formed Fe(III) complexes of the chelators, via the rapid
autooxidation of Fe(II) to Fe(III).30,31 All the solutions showed absorbance maxima around
0
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30 nm, which is typical signature of formation of ferrioxamine complex, thus indicating that
the chelator moiety was preserved in the conjugates.
The stoichiometry of iron-binding to compounds I-IV were assessed by continuous
variation (Job’s) method14,32 and compared to the parent DFO molecule.The solutions of
FAS, DFO and compound I-IV were all prepared in 10% aqueous DMSO. After addition of
instantly prepared FAS solution to the solutions of DFO and compounds I-IV, the solutions
were mixed properly, and were equilibrated for approx. 30 min. The UV-VIS spectra of the
o
solutions were recorded at 28 C. In this method total concentration of added iron and
compound I-IV were kept constant, and their relative proportions, i.e. mole fractions (Xi),
were varied. Change in absorbance of the complex at 430 nm (or in principle any parameter
which varies linearly with concentration), when plotted against the mole fraction of either
metal or ligand, is expected to show curvature plot with maxima at X = 0.5 for complexation
L
with 1:1 stoichiometry (cf. Fig. 4). Moreover, the shape of the curve provides qualitative
insight into binding equilibrium (Keq = [ML]/([M][L])); where strong binding results Keq >>
1
and curvature approaches a perfect triangle, as observed for all the four compounds. The
3
3
data were analysed according to the procedures reported by Renny et al. The stoichiometry
of iron binding to all the conjugates (I-IV) and DFO were virtually identical (Fig. 4) and
equal to 1:1 (metal:ligand) stoichiometry. Again, the imidazole conjugation to DFO did not
alter iron binding equilibrium significantly and did not add extra binding sites.
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Fig. 3. UV-VIS spectra of iron complexes of DFO and conjugates I-IV (50 μM solutions of
Fe-chelator prepared in 10% DMSO were used in each case).
Fig. 4. Job’s plots for the binding of iron to conjugates I-IV (absorbance recorded at 430
nm).
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Competition studies with calcein34
Calcein, a well-known metal-sensor,shows stoichiometric quenching of its
fluorescence in presence of iron(III).35 This property has been extensively used for
determining labile iron concentration in biological fluids. In presence of a stronger iron
chelator, the calcein-iron (Ca-Fe) complex may get disrupted, and the calcein fluorescence is
recovered. Since the fluorescence quenching of calcein is faster in presence of iron (II),36
competition studies with the proposed chelators have been conducted using aqueous Fe(II)
solution. Rapid autoxidation of Fe(II) to Fe(III) happens upon binding to calcein in ambient
air. At physiological pH, addition of different chelators (with higher Fe(III) binding affinity)
to the solution of Ca-Fe(III) led to fluorescence recovery (Fig. 5), the extent of which is
dependent on the relative affinities of the iron chelators. As evident from Fig. 5, all the
chelators efficiently competed with calcein, and their iron-binding profiles were similar to
that of DFO. This indicated the high thermodynamic stability of the iron-complexes of the
proposed chelators, indicating their possible roles in physiological iron scavenging. The
profile also indicated that the iron binding ability of the DFO was retained in their imidazole-
conjugates.
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Fig. 5. Dequenching of calcein (2 μM) fluorescence as a function of chelator concentration
(The plot represents the average of duplicate experiments repeated at least twice).
Competition studies with fluorescein-apotransferrin
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Fluorescein-labeled apotransferrin (Fl-aTf) was first used by Bruer and Cabantchik
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for the assay of non-transferrin-bound iron (NTBI). Following the reported procedure, Fl-
aTf was prepared byincubating 5-DTAF with holo-transferrin, and subsequent dialysis
against citrate (pH 5.5). This Fl-aTf undergoes fluorescence quenching upon binding iron,
and this property has been used for assessing the ability of the proposed chelators to remove
iron from the diferric transferrin. DFO, despite its higher affinity towards iron, cannot remove
iron from transferrin, mainly due to kinetic reasons. The same is also true for the proposed
chelators. The chelators, even at a 2-fold concentration of the Fl-Tf-Fe , was not able to
2
demetallise it (Fig. 6). This, in turn, is an important positive characteristic of the chelators
since these should not remove excess iron affecting its biochemical compartments.
FAS added
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Fig. 6. Recovery of fluorescence from aqueous Fl-Tf-Fe solution (2 μM) by addition of 20
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μM of chelators (The plot represents the average of duplicate experiments repeated at least
twice).
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DPPH assay
DPPH (2,2-diphenyl-1-picrylhydrazyl) is a stablefree radical at room temperature, and
gives a violet colour whendissolved in methanol. However, in presence of an antioxidant, the
radical is scavenged, and the colour is discharged. This is used to assay the antioxidant
38
property of a compound spectrophotometrically. All the chelators under study, along with
the standard antioxidant ascorbic acid, decreased the amount of DPPH in the solution (Fig.
7
). This invariably established the antioxidant properties of the chelators.
Fig. 7. DPPH radical scavenging activity of conjugates I-IV compared to DFO and ascorbic
acid (The plot represents the average of duplicate experiments repeated at least twice).
Chelation of cellular LIP
In order to assess cell permeability and chelation of intracellular LIP by compound I-
IV, a flow cytometry based fluorescence analysis of calcein acetoxymethyl ester (calcein-
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AM), a cell permeable iron sensor, was adopted. To this end, U2-OS (osteosarcoma) cells
were used as a cellular model.In this assay, cellular esterases hydrolyses calcein-AM to
calcein, which is fluorescent. Calcein fluorescence is quenched upon its binding to cellular
39
LIP, in a stoichiometric fashion. In accordance to previous report, our result showed that the
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mean fluorescence intensity (MFI), which was ~0.1 in unstained cells, increased to~100 in
CA-AM loaded cells (Fig. 8A, B). Upon FAS treatment, which enhances LIP, calcein
fluorescence was significantly reduced in the cells. This result confirmed the specificity of
the assay for measuring LIP.Addition of iron chelator, which removes iron from its complex
3
9
with calcein, increases the fluorescence emitted by the cells. The difference in the cellular
MFI (∆F) with andwithout iron chelator incubation depicts the cell permeation and LIP
4
0
chelating properties of chelators. In our experiment, DFO (100 M) caused only marginal
increase in ∆F. As shown in Fig.8A, C, compound II-III (50 and 100 M) has no or marginal
effect on ∆F. Interestingly, compound I and IV enhanced ∆F significantly, in a concentration
dependent manner. The LIP chelation efficiency of IV was higher in comparison to I.
Together, this result suggested that compound I and IV, especially IV, dequenched calcein
fluorescence most effectively by efficient penetration and chelating LIP in the cells, whereas
DFO and compound II, III did not have a significant effect on ∆F, due to their poor cell
permeating abilities. Moreover, a similar result was also observed in MCF7 (breast
carcinoma) cells (data not shown). Together, our result suggested that the LIP chelation
efficiency of IV is not limited to one cell type.
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Fig. 8. Flow cytometry measurements of LIP and its chelation by compound I-IV. U2-OS
cells were loaded with 0.2M calcein-AM for 30 min, then washed and treated with FAS (50
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
M) and ascorbic acid (200 M), for 30 min, to enhance the cellular LIP. Subsequently, cells
were washed and treated the iron chelators (50 and 100 M) for 60 min and acquired by flow
cytometry. (A-C) Mean fluorescence intensities (MFI) in histograms of unstained cells (no
calcien-AM), calcein-stained cells under different treatment were quantified by FlowJo
software. The difference in the MFI in absence and presence of chelatorrepresents ∆F, which
was used as a measure of LIP.
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Cytotoxicity and cellular uptake assay
Higher LIP chelation efficiency of compound I and IV indicated a better cellular
permeability and intracellular availability of compound I and IV, as compared to DFO.
However, higher cellular permeability of these conjugates may leads to higher cytotoxicity.
In order to assess compound I-IV and DFO induced cytotoxicity, U2-OS cells were treated
with these compounds (200 M) and cell viability was measured at 24-72 h by MTT assay.
Our results showed that compound I-IV induces no or marginal loss of cell viability during
2
4-72 h treatment (Fig. 9A).
In order to assess the mode of cellular uptake, the LIP chelating ability of compound
IV was assessed in the absence and presence of sucrose (inhibits clathrin mediated
endocytosis), nystatin (inhibits claveolae mediated endocytosis) and nocodazole (inhibits
41
macropinocytosis). Cellular uptake of compound IV was chosen for further study because
of its superior LIP chelating efficiency vis-à-vis other tested compounds in the current study.
To this end, our results showed that pre-incubation of cells with sucrose, nystatin or
nocodazole partially but significantly reduces compound IV mediated chelation of LIP in
cells (Fig. 9B). In control experiment, sucrose, nystatin or nocodazole treatment alone has no
or marginal effects on LIP status in the cells. Together, this suggested that cellular uptake of
compound IV is mediated through multiple processesvia endocytosis and macropinocytosis.
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Fig. 9. Cytotoxic and cellular uptake analysis. (A) U2-OS cells were treated with compound
I-IV or DFO (200 M) for 24-72 h. Cells viability was assessed by MTT assay. (B) U2-OS
cells were loaded with calcein-AM (0.2 M) for 30 min, then washed and treated with FAS
(50 M) and ascorbic acid (200 M), for 30 min, to enhance the cellular LIP. Further cells
were washed and incubated with medium containing either sucrose, nystatin or nocodazole
for 60 min. Subsequently, compound IV (50 M) was added for 60 min and cellular
fluorescence was acquired by flow cytometry. The difference in the MFI in absence and
presence of chelator represents ∆F, which was used as a measure of LIP.The experiments
were repeated three times. All determinations were made in 2-3 replicates and the values are
mean ± S. E. M.*p<0.05 compared to respective control in the absence of compound IV,
$p<0.05 compared to FAS + compound IV.
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Discussion
Iron overload is inevitable in many diseases including hemochromatosis, thalassemia,
myelodysplastic syndromes etc., where excess iron accumulation can occur via increased
gastrointestinal absorption or via transfusional loading. This excess iron, both extracellular
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(NTBI, non-transferrin bound iron) and intracellular (LIP) can be detrimental to human
health. Very recently, LIP level has been suggested as an alternative marker for iron overload
and related oxidative stress in β-thalassemia patients.42 This, unambiguously, shows the
importance of LIP as a target in chelation therapy. Although DFX and DFP, either used
separately or in combination with DFO, have showed considerable improvements in LIP
removal, their potential toxicity has always been a concern. Towards this, derivatives of DFO
with increased cell permeability, and unaffected chelation ability can be used as suitable
alternatives.
Earlier, imidazolium salts have been shown to possess antimicrobial properties.19,43
Their cell permeabilityincreaseswith increasing alkyl chain length, as shown in Hep-G2
4
4
cells. This property of imidazolium salts has been utilised for delivering APIs across
cells,20,21 as well as delivery reagent for siRNA. However, imidazolium salts have never
been used for delivering metal chelators across cells.
45
In this study, in a first ever attempt, we have conjugated imidazolium salts with DFO.
The synthetic strategy enabled us to tailor the hydrophobicities of the conjugates by attaching
different alkyl groups with varying chain lengths. The synthesis was straightforward, with no
practical surprises. The conjugates were obtained in moderate to good yields, and were easily
purified using RP-HPLC. All the conjugates were characterized beyond doubt.
One of our major goal was to retain all the chelation properties of the original
siderophore in the conjugates. In this study, we have shown that conjugation to imidazole did
not alter either the iron binding stoichiometry or the binding equilibria of DFO. This
invariably proved that imidazole part of the conjugate did not offer any extra binding site. In
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the DPPH-assay, the conjugates were able to scavenge DPPH radical, proving their
antioxidant capabilities. The DPPH scavenging ability of DFO has been reported earlier.14,46
The iron chelators, DFO and the conjugates I-IV, contain hydroxamate groups which can
donate a hydrogen atom to DPPH radical, resulting in the scavenging of DPPH racdical
alongwith the formation of a stable nitroxide radical.47 The scavenging abilities of the
conjugates, and thereby the antioxidant properties, were comparable with that of DFO.
Iron overload is a concern in a number of clinical conditions, as excess cellular iron
facilitates pro-oxidant reactions with oxygen or nitrogen substrates, giving rise to oxidative
0
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stress. Diseases such as hereditary hemochromatosis or thalassemia are characterized by
non-localized deposits of excess iron, however a number of conditions are known in which
iron overload is circumscribed to a specific tissue or organelle (neurodegeneration with brain
iron accumulation, hereditary X-linked sideroblastic anemia, anemia of chronic disease.49
Patients of FA typically have a decreased expression of the peptide frataxin, which assists the
assembly of iron-sulfur clusters within the cell. Clinically relevant chelators must halt this
oxidation. For this, development of cell-permeable chelators is of urgent need. In our study,
we have demonstrated that increased hydrophobicity, synergised with a charged moiety, help
the chelator to penetrate cells.
A combination of electrostatic, dispersion interactions, hydrophobic effects, and
hydrogen bonds dynamics at the interface account for the overall mechanisms of interaction
50
of an imidazolium salt with biomenbranes. The basic structures of all biomembranes are
formed by phospholipid bilayer, with hydrophilic negatively charged phosphate head and a
hydrophobic tail consisting of two fatty acid chains. The absence of an imidazolium cation in
conjugate I is responsible for its decreased cell permeability, mainly because the electrostatic
interaction of imidazolium cation with the negative charge groups in the lipid head is
diminished in conjugate I compared to that of conjugate IV, despite both having similar
hydrophobicities. Among the conjugates II, III and IV, the degree of cellular internalization
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is dependent on the alkyl chain length as reported earlier. The hydrophobic alkyl chains
have been demonstrated to internalise via hydrophobic interactions through membrane
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curvature. Recently, Durand et. al. have shown the effect of alkyl chain length in the
membrane interactions of phenolipids. According to their study, C8-alkyl chain substituted
molecules show the highest penetration depth. This corroborates with our finding that
molecule IV shows the highest penetration and chelation of LIP in cancer cells. In fact,
conjugate IV showed almost complete fluorescence recovery in our experiments, suggesting
a high degree of localisation of IV inside the cells.
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Biologically, cellular uptake of various metabolites and substances is mediated
53
primarily through two receptor dependent endocytosis process. These two processes is
regulated via clathrin and caveolae. Besides, cell also employs a non-specific and receptor
independent macropinocytosis process for internalizing various types of substances. In our
study, sucrose treatment, which inhibits clathrin-mediated endocytosis due to hypertonic
condition, partially reduced LIP chelation by compound IV in cells. Similarly, nystatin,
which inhibits lipid rafts caveolae mediated endocytosis, also partially reduced LIP chelation
by compound IV in cells. However, the extent of inhibitory effects of sucrose and nystatin
were weak, suggested an existence of alternate mode of cellular uptake for compound IV.
Apart from receptormediated endocytosis, a non-specific and receptor independent
macropinocytosis process is known to internalize various substances.41,53 Since
macropinocytosis process is dependent crucially on actin polymerization and membrane
ruffling, a microtubule and actin organization disrupting agent such as nocodazolehas been
used for inhibition ofmacropinocytosis.Intriguingly, cells treated with nocodazole
significantly reduced LIP chelation by compound IV. Together, our results suggested that
cellular uptake compound IV is initially starts with ionic and C8-chain mediated chemical
interaction with membrane phospholipids and subsequently it may be internalized through
both receptor-dependent endocytosis and receptor-independent macropinocytosis process. At
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present, it is unclear whether endocytosis and macropinocytosis processes for cellular uptake
of compound IV act cooperatively or not. It warrants further studies to unravel the molecular
mechanism of cellular internalization of compound IV.
Number of evidences has been reported till date54-57 to link iron overload to the
growth of cancer and related metastasis. Since neoplastic cells require more iron, cell-
permeable iron chelators have been projected as potential candidates for use in adjunctive
therapy in iron-overloaded cancers. Although DFO has been seen to disrupt intracellular iron
homeostasis, it acts only at a higher dose, and did not show any substantial effect at lower
doses. This is due to the high hydrophilicity of DFO, which inhibits it to chelate intracellular
iron, and therefore has not been much used as adjuvant in iron-overloaded cancers. In
contrast, compound IV has been shown to be cell-permeable and highly effective in chelating
LIP in the current study. Therefore, compound IV, as an adjuvant with chemo/radiation
therapy for iron overloaded cancer, may enhance the therapeutic outcomes.
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Conclusion
In conclusion, we have demonstrated that the conjugates I-IV retained the iron chelation
abilities of DFO, but at the same time conjugate IV showed significant enhancement in its
cell penetration efficiency. Besides its use in iron overload diseases, this property of
imidazolium cations with desired hydrophobicity can be used in drug delivery, particularly as
small molecule based vector. The possibility of using compound IV as an adjuvant in cancer
therapy and other diseases will be explored in future.
EXPERIMENTAL SECTION
General Chemistry
All chemicals (AR grade) were purchased from Sigma-Aldrich, and were used without
further purification. All solvents (AR grade) used for synthesis were purchased from SRL,
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India. Unless otherwise mentioned, HBS (NaCl 150 mM, HEPES 20 mM; pH 7.4; treated
with Chelex-100 purchased from Sigma, 1 g/100 mL) was used throughout the experiments.
-1
-1
Phosphate Buffer Saline (PBS) was prepared with 0.14 mol L NaCl; 2.6 mmol L NaH PO
2
4
-1
H O and 7.4 mmol L Na HPO .7H O, pH 7.4. For characterization, the IR spectra (KBr)
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were recorded with a BRUKER Tensor II spectrophotometer. The H and C NMR spectra
were recorded with a Bruker AC-200 (200 MHz) or a Varian 500 MHz NMR spectrometer,
and the NMR spectra were processed using either MestReNova Lite-11.0.4, ACD/1D NMR
Processor or Bruker TOPSPIN software. The elemental analyses were carried with an
Elementar Vario micro cube. The logPOW (octanol/water) of all the conjugates I-IV were
calculated using MarvinSketch 18.4.0 (ChemAxon Ltd.). UV-VIS electronic spectra were
recorded with commercial UV/VIS spectrophotometer (JASCO, V-550).
Ethylimidazol-1-yl-acetate (2)22
K CO (9.13 g, 66.09 mmol) and KOH (3.09 g, 55.09 mmol) were added
2
3
simultaneously to a solution of imidazole 1 (3.0 g, 44.07 mmol) in dry DCM (15 mL).The
suspension was stirred for 15 min. Thereafter, ethyl bromoacetate (6.09 mL, 55.09 mmol) and
o
a catalytic amount (10 mol %) of TBAI were added to it. The reaction was stirred at 25 C for
8
h. On completion (cf TLC), the reaction mixture was filtered, residue washed with CHCl3
and the filtrate was concentrated under vacuum. Purification by column chromatography
1
using 0-5% MeOH/CHCl yielded 2 (2.65 g, 39%). Yellow oil; H NMR (500 MHz, CDCl ):
3
3
δ 1.27 (t, J = 7.0 Hz, 3H), 4.22 (q, J = 7.0 Hz, 2H), 4.68 (s, 2H), 6.94 (s, 1H), 7.07 (s, 1H),
1
3
7
.49 (s, 1H). C NMR (125 MHz, CDCl ): δ 14.0, 47.9, 61.9, 120.0, 129.4, 137.9, 167.5.
3
5
.3.1H-Imidazol-1-yl-acetic acid hydrochloride (3)23
A solution of 2 (0.79 g, 5.12 mmol) in 40% aqueous HCl (10 mL) was refluxed for 4
h. After completion of reaction (cf TLC), water was removed under vacuum, and column
chromatography of the residue using 0-50% MeOH/CHCl yielded pure 3 (0.80 g, 96%).
3
o
23
o
1
White solid; m.p. 202-206 C (decom.)[Lit m.p. 206.6-207.6 C]; H NMR (500 MHz, D O):
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δ 5.18 (s, 2H), 7.52 (s, 1H), 7.54 (s, 1H), 8.82 (s, 1H); C NMR (125 MHz, D O): δ 49.7,
2
1
19.6, 123.1, 135.9, 170.0.
1H-Imidazole-1-acetic acid-DFO conjugate (I)
A suspension of 3 (0.05 g, 0.31 mmol), DFO-Mes (0.20 gm, 0.31 mmol), DIC (0.06
mL, 0.39 mmol) and HOBt (0.05 g, 0.39 mmol) in 10 ml DMF was heated under N2
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atmosphere at 45 C for 1 h. After 1 h, the reaction mixture was cooled to room temperature,
and DIPEA (0.15 mL, 0.88 mmol) was added to it. The reaction mixture was stirred for 8 h.
Solvent removal under vacuum yielded a solid residue, which was washed with EtOAc (3 x
1
10 mL) and was dissolved in water. The water extract was concentrated under vacuum. H
NMR of the crude solid confirmed the presence of the conjugate I. Purification of the crude
residue by semi-preparative RP-HPLC yielded pure I (yield 0.148 g, 72%) light yellow solid.
1H NMR (500 MHz, DMSO-d6): δ 1.17-1.28 (m, 6H), 1.31-1.41 (m, 6H), 1.42-1.51 (m, 6H),
1.97 (s, 3H), 2.27-2.35 (m, 4H), 2.58-2.67 (m, 4H), 2.89-3.09 (m, 6H), 3.42-3.48 (m, 6H),
4
.61 (s, 2H), 6.86 (s, 1H), 7.03 (s, 1H), 7.58 (s, 1H), 7.69 (broad s, 3H), 8.01-8.09 (m, 3H).
Anal. Calcd for C H N O : C, 53.88; H, 7.84; N, 16.75. Found: C, 53.64; H, 7.68; N, 16.98.
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N-butyl imidazole (5)
K CO (15.23 g, 110.17 mmol) and KOH (4.95 g, 88.14 mmol) were added to a
2
3
solution of imidazole 1(5.0 g, 73.45 mmol) in dry DCM (25 mL) and stirred for 15 minutes.
To this suspension, 1-bromo butane(11.89 ml,110.17 mmol) and a catalytic amount (10 mol
%
) of TBAI was added. The reaction was stirred for 6 h. On completion (cf TLC), the
reaction mixture was filtered, residue washed with CHCl and the filtrate was concentrated
3
under vacuum. Purification by column chromatography using 0-5% MeOH/CHCl yielded 5
3
1
(6.5 g, 71%). Yellow oil; H NMR (500 MHz, DMSO-d6): δ 0.86 (t, J = 7.5 Hz, 3H), 1.16-
1
1
.24 (m, 2H), 1.62-1.68 (m, 2H), 3.93 ((t, J = 7.5 Hz, 2H), 6.86 (s, 1H), 7.13 (s, 1H), 7.59 (s,
1
3
H); C NMR (125 MHz, DMSO-d6): δ 13.8, 19.6, 33.1, 46.1, 119.6, 128.7, 137.6. Anal.
Calcd for C H N : C, 67.70; H, 9.74; N, 22.56. Found: C, 67.77; H, 9.87; N, 22.87.
7
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N-octyl imidazole (6)
As described previously, K CO (15.23 g, 110.17 mmol) and KOH (4.95 g, 88.14
2
3
mmol) were added to a solution of imidazole 1(5.0 g, 73.45 mmol) in dry DCM (25 mL) and
stirred for 15 minutes. To this suspension, 1-bromo octane (19.03 mL, 110.17 mmol) and a
catalytic amount (10 mol%) of TBAI was added. The reaction was stirred overnight. On
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completion (cf TLC), the reaction mixture was filtered, residue washed with CHCl and the
3
filtrate was concentrated under vacuum. Purification by column chromatography using 0-5%
1
MeOH/CHCl yielded 6 (9.9 g, 75%). Yellow oil; H NMR (500 MHz, CDCl ): δ 0.87 (t, J =
3
3
7.0 Hz, 3H), 1.24-1.28 (m, 10H), 1.74-1.77 (m, 2H), 3.91 (t, J = 7.0 Hz, 2H), 6.89 (s, 1H),
13
7
.04 (s, 1H), 7.45 (s, 1H). C NMR (125 MHz, CDCl ): δ 14.0, 22.5, 26.5, 28.9, 29.0, 31.0,
3
3
1.6, 46.9, 118.7, 129.2, 137.0. Anal. Calcd for C H N : C, 73.28; H, 11.18; N, 15.54.
1
1
20
2
Found: C, 73.09; H, 11.32; N, 15.36.
1-methyl-3-(2-ethoxy-2-oxoethyl)-imidazolium bromide (7)
Ethyl bromoacetate (5.5 mL, 50.06 mmol) was added dropwise to a pre-cooled (-10
oC) solution of 4 (4.11 g, 50.06 mmol) in dry THF (25 ml). The reaction was gradually
brought to room temperature and was stirred overnight. Solvent evaporation followed by
washing of the residue with Et O (3 x 10 mL) and drying under vacuum yielded pure 7 (8.50
2
1
g, 68%). Yellow viscous oil; H NMR (200 MHz, CDCl ): δ 1.14 (t, J = 7.0 Hz, 3H), 3.95 (s,
3
3H), 4.10 (q, J = 7.2 Hz, 2H), 5.34 (s, 2H), 7.58 (d, J = 1.6 Hz, 1H), 7.67 (d, J = 1.8 Hz, 1H),
13
9
1
5
.79 (t, J = 1.8 Hz, 1H); C NMR (50 MHz, CDCl ): δ 14.0, 36.9, 50.2, 62.8, 123.2, 123.9,
3
37.9, 166.2.
.8. 1-butyl-3-(2-ethoxy-2-oxoethyl)-imidazolium bromide (8)
Ethyl bromoacetate (5.5 mL, 49.93 mmol) was added dropwise to a pre-cooled (-10
oC) solution of 5 (6.2 g, 49.93 mmol) in dry THF (25 ml). The reaction was gradually brought
to room temperature and was stirred overnight. Solvent evaporation followed by washing of
the residue with Et O (3 x 10 mL) and drying under vacuum yielded pure 8 (9.48 g, 65%).
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1
Yellow viscous oil; H NMR (500 MHz, CDCl ): δ 0.88 (t, J = 7.5 Hz, 3H), 1.22 (t, J = 7.0
3
Hz, 3H), 1.28-1.33 (m, 2H), 1.81-1.87 (m, 2H), 4.17 (q, J = 7.0 Hz, 2H), 4.26 (t, J = 7.5 Hz,
1
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2H), 5.43 (s, 2H), 7.52 (s, 1H), 7.72 (s, 1H), 10.12 (s, 1H); C NMR (125 MHz, CDCl ): δ
3
1
3.4, 14.0, 19.3, 31.9, 50.0, 50.2, 62.8, 121.7, 124.0, 137.6, 166.1.
.9. 1-octyl-3-(2-ethoxy-2-oxoethyl)-imidazolium bromide (9)
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Ethyl bromoacetate (1.3 mL, 11.48 mmol) was added dropwise to a pre-cooled (-10
oC) solution of 6 (2.07 g, 11.48 mmol) in dry THF (15 ml). The reaction was gradually
brought to room temperature and was stirred overnight. Solvent evaporation followed by
washing of the residue with Et O (3 x 10 mL) and drying under vacuum yielded pure 9 (2.19
2
1
g, 55%). Yellow viscous oil; H NMR (500 MHz, CDCl ): δ 0.83 (t, J = 7.0 Hz, 3H), 1.18-
3
1
1
.30 (m, 13H), 1.85-1.91 (m, 2H), 4.20-4.29 (m, 4H), 5.47 (s, 2H), 7.43 (s, 1H), 7.68 (s, 1H),
1
3
0.13 (s, 1H); C NMR (125 MHz, CDCl ): δ 13.9, 22.4, 26.0, 28.7, 28.8, 30.0, 31.5, 50.2,
3
62.7, 121.4, 123.9, 137.7, 166.0.
1
-methyl-3-(carboxymethyl)-imidazolium bromide (10)
A solution of 7 (8.0 g, 32.11 mmol) in 40% aqueous HCl was refluxed for 4 h. After
the reaction was complete (cf TLC), water was removed under vacuum to yield pure 10 (5.76
1
g, 81%). Yellow viscous oil; H NMR (500 MHz, D O): δ 3.88 (s, 3H), 5.06-5.13 (m, 2H),
2
1
3
7
.44-7.46 (m, 2H), 8.76 (s, 1H); C NMR (125 MHz, DMSO-d6): δ 36.1, 50.0, 123.5, 137.3,
170.0. Anal. Calcd for C H BrN O : C, 32.60; H, 4.10; N, 12.67. Found: C, 32.54; H, 4.36;
6
9
2
2
N, 12.68.
1
-butyl-3-(carboxymethyl)-imidazolium bromide (11)
A solution of 8 (9.18 g, 31.53 mmol) in 40% aqueous HCl (30 mL) was refluxed for 4
h. After the reaction was complete (cf TLC), water was removed under vacuum to yield pure
1
1
1 (7.17g, 86%). Yellow viscous oil; H NMR (500 MHz, DMSO-d6): δ 0.87 (t, J = 7.5 Hz,
3
H), 1.20-1.25 (m, 2H), 1.73-1.78 (m, 2H), 4.26 (t, J = 7.0 Hz, 2H), 5.21 and 5.34 (two s,
1
3
2H), 7.82 (s, 1H), 7.90 (s, 1H), 9.37 (s, 1H); C NMR (125 MHz, DMSO-d6): δ 13.7, 19.1,
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1.8, 49.2, 53.3, 122.6, 124.3, 137.6, 167.8. Anal. Calcd for C H BrN O : C, 41.08; H, 5.75;
9
15
2
2
N, 10.65. Found: C, 40.90; H, 6.32; N, 10.28.
1-octyl-3-(carboxymethyl)-imidazolium bromide (12)
A solution of 9 (2.0 g, 5.76 mmol) in 40% aqueous HCl was refluxed for 4 h. After
1
1
1
1
1
1
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1
1
1
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the reaction was complete (cf TLC), water was removed under vacuum to yield pure 12 (1.51
1
g, 82%). Light brown solid; H NMR (500 MHz, D O): δ 0.74 (t, J = 6.5 Hz, 3H), 1.14-1.19
2
(m, 10H), 1.76-1.79 (m, 2H), 4.13 (t, J = 7.0 Hz, 2H), 4.99 (s, 2H), 7.41 (s, 1H), 7.44 (s, 1H),
1
3
8
.75 (s, 1H); C NMR (125 MHz, D O): δ 13.4, 22.0, 25.2, 27.9, 28.1, 29.0, 30.9, 49.9, 50.1,
2
122.3, 123.6, 136.6, 170.1. Anal. Calcd for C H BrN O : C, 48.91; H, 7.26; N, 8.78. Found:
1
3
23
2
2
C, 48.44; H, 7.52; N, 8.54.
1
-methyl-3-(carboxymethyl)-imidazolium-DFO conjugate (II)
A suspension of 10 (0.155 g, 0.70 mmol), DFO-Mes (0.46 gm, 0.70 mmol), DIC (0.14
mL, 0.90 mmol) and HOBt (0.12 g, 0.90 mmol) in 10 ml DMF was heated under N2
o
atmosphere at 50 C for 1 h. After 1 h, the reaction mixture was cooled to room temperature,
and DIPEA (0.37 mL, 2.1 mmol) was added to it. The reaction mixture was stirred overnight.
Solvent removal under vacuum yielded a solid residue, which was washed with EtOAc (3 x
1
0 mL) and was dissolved in water. The water extract was concentrated under vacuum. 1H
1
NMR of the crude solid confirmed the presence of the conjugate II. H NMR of the crude
solid confirmed the presence of the conjugate II. Purification of the crude residue by semi-
1
preparative RP-HPLC yielded pure II (yield 0.371 g, 68%) light yellow solid. H NMR (200
MHz, D O): 1.20-1.41 (m, 6H), 1.53-1.57 (m, 6H), 1.64-1.68 (m, 6H), 2.12 (s, 3H), 2.47-2.50
2
(m, 4H), 2.72 (s, 3H), 2.77-2.81 (m, 4H), 3.16-3.18 (m, 4H), 3.19-3.20 (m, 1H), 3.33-3.34
(m, 1H), 3.60-3.64 (m, 6H), 3.99 (two s merged together, 3H), 5.03 (s, 2H), 7.54-7.57 (m,
1
H), 7.62 (s, 1H), 8.98 (s, 1H). Anal. Calcd for C H N O S: C, 49.34; H, 7.51; N, 14.39; S,
32 58 8 12
4
.12. Found: C, 49.06; H, 7.48; N, 14.75; S, 3.74.
1-butyl-3-(carboxymethyl)-imidazolium-DFO conjugate (III)
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A suspension of 11 (0.185 g, 0.70 mmol), DFO-Mes (0.46 gm, 0.70 mmol), DIC (0.14
mL, 0.90 mmol) and HOBt (0.12 g, 0.90 mmol) in 10 ml DMF was heated under N2
o
atmosphere at 50 C for 1 h. After 1 h, the reaction mixture was cooled to room temperature,
and DIPEA (0.37 mL, 2.10 mmol) was added to it. The reaction mixture was stirred
overnight. Solvent removal under vacuum yielded a solid residue, which was washed with
EtOAc (3 x 10 mL) and was dissolved in water. The water extract was concentrated under
vacuum. 1H NMR of the crude solid confirmed the presence of the conjugate III. Purification
of the crude residue by semi-preparative RP-HPLC yielded pure III (yield 0.380 g,
0
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66%).yellow solid. H NMR (500 MHz, DMSO-d6): δ 0.89 (t, J = 7.5 Hz, 3H), 1.20-1.26 (m,
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H), 1.35-1.38 (m, 6H), 1.48-1.50 (m, 6H), 1.75-1.78 (m, 2H), 1.95 (s, 3H), 2.25-2.30 (m,
H), 2.55-2.59 (m, 4H), 2.72-2.76 (m, 1H), 2.96-3.00 (m, 6H), 3.42-3.47 (m, 8H), 4.19-4.20
(
m, 2H), 4.84 (s, 1H), 4.95 (s, 1H), 7.67-7.76 (m, 5H), 9.11-9.15 (m, 1H), 9.62-9.67 (m, 3H).
Anal. Calcd for C H N O S: C, 51.20; H, 7.86; N, 13.65; S, 3.91. Found: C, 50.81; H,
3
5
64
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12
7
.74; N, 13.96; S, 4.17.
1
-Octyl-3-(carboxymethyl)-imidazolium-DFO conjugate (IV)
A suspension of 12 (0.115g, 0.36mmol), DFO-Mes (0.24g, 0.36 mmol), DIC (0.07ml,
0
5
.45mmol) and HOBt (0.06g, 0.45mmol) in 10 ml DMF was heated under N atmosphere at
2
o
0 C for 1 h. After 1 h, the reaction mixture was cooled to room temperature, and DIPEA
(0.19 ml, 1.08 mmol) was added to it. The reaction mixture was stirred overnight. Solvent
removal under vacuum yielded a solid residue, which was washed with EtOAc (3 x 10 mL),
water (3 x 10 mL) and MeOH (3 x 10 mL). The methanol extract was concentrated under
1
vacuum. H NMR of the crude solid confirmed the presence of the conjugate IV. Purification
of the crude residue by semi-preparative RP-HPLC yielded pure IV (yield 0.224 g, 71%).
1
yellow solid. H NMR (500 MHz, DMSO-d6): δ 0.84 (t, J = 7.0 Hz, 3H), 1.15-1.26 (m, 16H),
1
.35-1.39 (m, 6H), 1.42-1.49 (m, 6H), 1.75-1.79 (m, 2H), 1.95 (s, 3H), 2.25-2.29 (m, 4H),
2.30-2.32 (m, 2H),2.57-2.60 (m, 4H), 2.73-2.76 (m, 1H), 3.00-3.12 (m, 6H), 3.42-3.47 (m,
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Page 29 of 38
Bioconjugate Chemistry
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H), 4.18 (t, J = 7.0 Hz, 2H), 4.94 (s, 2H), 7.66-7.76 (m, 4H), 8.38 (s, 1H), 9.11 (s, 1H), 9.58-
.65 (m, 3H). Anal. Calcd for C H N O S: C, 53.41; H, 8.27; N, 12.78; S, 3.66. Found: C,
39 72
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53.12; H, 8.62; N, 12.95; S, 3.64.
Binding stoichiometry
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The solutions of FAS, DFO and conjugates I-IV (100 μM each in 10% DMSO) were
freshly prepared. FAS solution was mixed with the solutions of the conjugates I-IV (or DFO)
and were allowed to equilibrate for 30 min.The electronic spectra were recorded at 30 °C. For
Job’s plot, the electronic spectra of Fe-chelator (DFO or conjugates I-IV) solutions with
varying mole fractions (iron mole fractions ranging from 0-0.8) were recorded at 30 °C.
Competition studies with calcein
1
90 μL of 2 μM calcein (in HBS, pH 7.4) were placed in each well of a flat, transparent 96-
well microplate.Fluorescence was recorded at 37 °C on a mutliwell fluorescence plate reader
(Tecan Infinite M200, Switzerland, λexc/λem = 485/520 nm) for 10 min (till stabilization of
fluorescence). After that,10 μL of40 μM FAS (instantly prepared in water, final concentration
2
μM) was added in each well, and was allowed to react at 37 °C for 10 min. Thereafter,
fluorescence was recorded until stabilization (∼10 min). The calcein-Fe (CAFe) solutions
formed in the wells were treated with increasing concentrations (0-24 μM of the compounds
I-IV (10 μL aliquots), and fluorescence was further recorded (∼60 min).
Competition studies with fluorescein-apotransferrin
1
80 μL of 2 μM FlTf solution (in HBS, pH 7.4) was placed in each well of a flat,
transparent 96-well microplate, and the fluorescence was recorded at 37 °C on a mutliwell
fluorescence plate reader (Tecan Infinite M200, Switzerland) for 10 min (till stabilization).
After that, 10 μL of 4 μM FAS (instantly prepared in water, final concentration) was added to
each well, and fluorescence was recorded till stabilization of fluorescence quenching (∼10
min). Next, the solutions were treated with increasing concentrations (0−20 μM) of chelators
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