Conjugates of desferrioxamine and aromatic amines improve markers of iron-dependent neurotoxicity

Article abstract of DOI:10.1007/s10534-020-00277-7

Abstract: Alzheimer’s Disease (AD) is a complex neurodegenerative disorder associated in some instances with dyshomeostasis of redox-active metal ions, such as copper and iron. In this work, we investigated whether the conjugation of various aromatic amines would improve the pharmacological efficacy of the iron chelator desferrioxamine (DFO). Conjugates of DFO with aniline (DFOANI), benzosulfanylamide (DFOBAN), 2-naphthalenamine (DFONAF) and 6-quinolinamine (DFOQUN) were obtained and their properties examined. DFOQUN had good chelating activity, promoted a significant increase in the inhibition of β-amyloid peptide aggregation when compared to DFO, and also inhibited acetylcholinesterase (AChE) activity both in vitro and in vivo (Caenorhabditis elegans). These data indicate that the covalent conjugation of a strong iron chelator to an AChE inhibitor offers a powerful approach for the amelioration of iron-induced neurotoxicity symptoms. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10534-020-00277-7

Biometals (2021) 34:259–275
https://doi.org/10.1007/s10534-020-00277-7(0123456789(). -, volV
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Conjugates of desferrioxamine and aromatic amines
improve markers of iron-dependent neurotoxicity
.
Rodrigo R. V. Carvalho Tanara V. Peres Cleber W. Liria M. Teresa Machini
.
.
.
.
Michael Aschner Breno P. Esposito
´
Received: 27 May 2020 / Accepted: 27 November 2020 / Published online: 3 January 2021
Ó The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021
Abstract Alzheimer’s Disease (AD) is a complex
neurodegenerative disorder associated in some
instances with dyshomeostasis of redox-active metal
ions, such as copper and iron. In this work, we
investigated whether the conjugation of various aro-
matic amines would improve the pharmacological
efficacy of the iron chelator desferrioxamine (DFO).
Conjugates of DFO with aniline (DFOANI), benzo-
had good chelating activity, promoted a significant
increase in the inhibition of b-amyloid peptide aggre-
gation when compared to DFO, and also inhibited
acetylcholinesterase (AChE) activity both in vitro and
in vivo (Caenorhabditis elegans). These data indicate
that the covalent conjugation of a strong iron chelator
to an AChE inhibitor offers a powerful approach for
the amelioration of iron-induced neurotoxicity
symptoms.
sulfanylamide
(DFOBAN),
2-naphthalenamine
(DFONAF) and 6-quinolinamine (DFOQUN) were
obtained and their properties examined. DFOQUN
Supplementary information The online version of this
article (https://doi.org/10.1007/s10534-020-00277-7) contains
supplementary material, which is available to authorized users.
´
R. R. V. Carvalho (&) Á B. P. Esposito
Department of Fundamental Chemistry, Institute of
Chemistry, University of Sa˜o Paulo, Sa˜o Paulo,
SP, Brazil
e-mail: rodrigovictor65@hotmail.com
T. V. Peres Á M. Aschner
Department of Molecular Pharmacology, Albert Einstein
College of Medicine, Bronx, NY, USA
C. W. Liria Á M. T. Machini
Department of Biochemistry, Institute of Chemistry,
University of Sa˜o Paulo, Sa˜o Paulo, SP, Brazil
123

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Biometals (2021) 34:259–275
Graphic abstract
Quinoline
Desferrioxamine
DFOQUN Conjugate
Anti-AChE
Anti-Aβ
Iron Chelator
Keywords Desferrioxamine Á Iron Á Alzheimer’s
disease Á Acetylcholinesterase Á Beta amyloid Á
Caenorhabditis elegans
reactive iron and generation of reactive oxygen
species (ROS) that will damage organs, such as
pancreas, heart, liver and brain (Cabantchik et al.
2013). Iron overload is a common complication of
diseases such as hereditary hemochromatosis and
thalassemias. The risks of complications associated
with excess iron in neurodegenerative disorders may
be increased due the oxidation of lipids, proteins and
enzymes, as well as destruction of RNA and DNA
(Hider et al. 2011). Hence, oxidative stress may
disturb iron homeostasis and damage neuronal cells
Introduction
Iron (Fe), the fourth most abundant element in the
earth’s crust, has many roles in biological systems,
mostly derived from its capacity to mediate the traffic
of electrons in redox reactions and to form reversible
complexes with molecular oxygen that are crucial to
biochemical processes, such as ATP or DNA synthesis
and oxygen transport (Ward et al. 2015). However,
precisely due to its ability to catalyze electron transfer
reactions in an oxygenated environment, whenever in
excess and/or outside its assigned biochemical com-
partments, iron will behave as a potent toxin (Breuer
et al. 2000; Cabantchik et al. 2013; Knutson 2019).
Indeed, excess of this metal in the bloodstream leads,
for example, to labile plasma iron (LPI) formation,
which enhances the levels of intracellular pools of
´
´
(Rodrıguez-Rodrıguez et al. 2012). The brain is
particularly vulnerable to damage caused by oxidative
stress due to its high oxygen consumption, low
concentration of antioxidants and its propensity to
´ ´
accumulate metals over time (Rodrıguez-Rodrıguez
et al. 2012).
These factors may be relevant to the development
of Alzheimer’s Disease (AD). AD is a complex
neurodegenerative disorder associated with memory
loss and decline of cognitive functions, promoting
dementia in the elderly. It is a fatal, incurable disorder,
which affects over 36 million people in the world
123

Biometals (2021) 34:259–275
261
´ ´
(Rodrıguez-Rodrıguez et al. 2012). Due to population
In this study, we coupled the aromatic amines
aniline, 2-naphthalenamine, 6-quinolinamine (ana-
logue of the AChE inhibitor tacrine) and benzosul-
fanilamide to obtain four new DFO conjugates (Fig. 1)
and evaluated their effects on biochemical markers
relevant to AD therapy. These molecules were
selected due their ability to introduce p–p stacking
interactions with AD targets similar to other drugs
(Chen et al. 2018a, b; Martinez and Iverson 2012;
Piemontese et al. 2018; Riwar et al. 2017; Thakuria
et al. 2019). As molecule designers, our rationale is
always to prepare a single molecule with dual activity,
rather than relying on the administration of two
different treatments. This approach decreases the risks
and complications inherent to polytherapies (Palleria
et al. 2013), while developing synthetic strategies to
impart new functions to established chelators. In
addition, a succinic spacing group was introduced
between the chelator and the aromatic amine moieties
in our conjugates, thus making it impossible to
adequately compare the effects of the final product
and their constituting parts alone.
aging, it is estimated that this figure will increase to 66
´
million by 2030 and 115 million by 2050 (Rodrıguez-
´
Rodrıguez et al. 2012). In the search for a cure,
different targets have been identified and studied.
Inhibition of the production and deposition of b-
amyloid (Ab) peptide, which causes the accumulation
of neurotoxic senile plaques in the brain (Hayne et al.
2014), and inhibition of acetylcholinesterase (AChE)
(Berg et al. 2011; Tiiman et al. 2013), the enzyme
responsible for the rapid conversion of acetylcholine
to choline and acetic acid required in the termination
of impulse transmission, have been the main
approaches to anti-AD therapy. Only five drugs have
been approved by the US Food and Drug administra-
tion (FDA) for the treatment of AD symptoms: tacrine,
donepezil, rivastigmine, galantamine (cholinesterase
inhibitors) and memantine (NMDA receptor antago-
´
nist) (Colovic et al. 2013; Schneider 2000).
There is ample evidence linking increased iron
brain levels and Ab aggregation. Both Fe(II) and
Fe(III) can form hexacoordinated complexes with Ab
through Asp, Glu and His residues (Kozlowski et al.
2012). Conversely, chelation therapy disrupts metal-
Ab interaction (Keri et al. 2013, 2016; Quintanova
et al. 2015b). One of the most specific iron chelators is
desferrioxamine (DFO), a siderophore secreted by the
microorganism Streptomyces pilosus and employed in
the treatment of iron overload disorders (Kurth et al.
2016). DFO forms a kinetically inert, strong hexaden-
tate complex with excess iron, which is readily
excreted (Kurth et al. 2016). However, DFO is highly
hydrophilic, with poor cell permeability that makes its
traffic through the blood–brain barrier (BBB) unlikely
(Heli et al. 2011; Pramanik et al. 2019; Raines et al.
2015; Sheth 2014).
Experimental section
Chemicals
All chemicals were at least 99% pure and used without
further purification. Desferrioxamine mesylate (DFO)
´
was obtained from Cristalia (Brazil). Ferrous ammo-
nium sulfate (FAS), aniline, calcein, benzosulfany-
lamide, 6-quinolinamine, 2-naphthalenamine and
other chemicals were purchased from Sigma Aldrich
(USA), unless otherwise noted. Trifluoroacetic acid,
analytical TLC plates pre-coated with silica gel and
chromatographic grade solvents used for RP-HPLC
and LC–MS were from Merck (USA). Dihydrorho-
damine chloride was obtained from Biotium (USA).
The multifactorial nature and complexity of AD has
challenged scientists searching for new anti-AD drugs.
To this end, the strategy of multi-target-directed-
ligands (MTDL) has been explored to develop new
anti-AD candidates. MTDL is based on conjugation of
one or more molecules with other pharmacophoric
moieties in order to enhance their pharmacological
proprieties (Santos et al. 2016). Our research group
used this strategy to create new DFO conjugates such
as DFCAF and DFO-TAT peptide, which are DFO
derivatives with enhanced lipophilicity and cell per-
meability (Alta et al. 2014; Goswami et al. 2014).
Instruments
Elemental analysis was performed in a CHN 2400
(Perkin Elmer, Waltham, USA). Melting points were
determined in a Kofler Apparatus (Fisher Scientific,
Loughbourough, United Kingdom). NMR spectra
were recorded in a Varian Inova (Oklahoma, USA)
or Bruker DRX Spectrometer (Bruker, Billerica, USA)
operating at 300 MHz and 500 MHz, respectively (for
123

262
Biometals (2021) 34:259–275
Fig. 1 Structures of DFO and the DFO conjugates prepared in this study
1H NMR), or 75 MHz and 128 MHz, respectively (for
13C NMR). NMR spectra were calibrated using
DMSO–d6 solvent signals (dH = 2.50 and dC = 39.52)
and reported in ppm. Mass spectra of carboxylic acids
were recorded in an Amazon Speed ETD—Bruker
(Germany). The capillary voltage of the electrospray
ionization source (ESI) was set to 4.5 kV. The
capillary temperature was set at 200 °C. Nebulizer
pressure was set to 12 psi. Fluorescence and electronic
absorbance measurements were conducted in the
microplate readers SpectraMax M4 (Molecular
Devices, Sunnyvale, USA) or FluoStar Optima
(BMG, Ortenberg, Germany). Chromatographic
purification by preparative reverse-phase HPLC was
carried in a RP-HPLC system from Waters (Millford,
MA, USA) composed of pump (Waters Delta 600
pump), pump controller (Waters 600 Controller), one
UV detector (Waters 2487 Dual k Absorbance detec-
tor), a manual injector sample (Rheodyne 7725), a
recorder (Kipp & Zonen Servogor 124 recorder) and a
Shimadzu Corporation RP-HPLC system (Kyoto,
Japan, flow 0.8 mL/min) composed of a DGU model
degasser 20A3, two pumps model LC-20AD, one
Rheodyne 8125 injector, one column oven model
CTO-20A (T = 32 °C), one C18 column (0.46 cm 9
˚
25.00 cm, 5 lm particle diameter, 300 A pore diam-
eter; Grace-Vydac, Hesperia, CA, USA) and a UV/Vis
detector model SPD-20A. This RP-HPLC system is
coupled to a Bruker Daltonics (Fahrenheitstrasse,
Germany) AmaZon X mass spectrometer with elec-
trospray ionization source, positive ESI mode and an
ion trap analyzer. The capillary voltage was 4500 V
and the end-plate offset voltage was 500 V (for
positive mode) and 220 °C. For spectrum analysis
the HyStar 3.2 software was used.
Synthesis of carboxylic acids
2a, 2b and 4: The syntheses were based on previously
described methods (Balakrishna et al. 2014). Distilled
water (5 mL) was mixed with the appropriate amine
(aniline or benzosulfanylmide to obtain 2a or 2b;
˚
Vydac C18 column (10 lm, 300 A, 2.2 9 25 cm). The
characterization by LC–MS was carried out with a
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Biometals (2021) 34:259–275
263
1 mmol) and stirred constantly. Three portions of
succinic anhydride (1 mmol, 1 mmol and 0.6 mmol)
were added to the mixture with 1 min intervals
between them. The reaction mixture was stirred for
10 min at room temperature and under open air. The
white precipitate was washed with deionized water
and dried under vacuum for 24 h to achieve the desired
product. To obtain 4, distilled water (50 mL) was
mixed with 2-naphtylamine (10 mmol) and succinic
anhydride (10 mmol). The reaction mixture was
stirred for 15 min at room temperature and under
open air. The brown precipitate was washed with
concentrated acetic acid, distilled water and dried
under vacuum for 24 h to achieve the desired product.
The reactions were monitored by TLC using chloro-
form/ acetic acid/methanol (85:10:5) eluent.
128.75, 127.87, 127.66, 126.82, 124.88, 120.25,
115.27, 31.12, 29.23. ESIMS (m/z): 244 (M ? H)?
4-oxo-4-(quinolin-6-ylamino)butanoic acid (6):
Yield: 82% m.p: 230–232 °C Rf: 0.30 1H-NMR
(300 MHz, DMSO-d6): d (ppm) 12.14 (s, 1H), 10.33
(s, 1H), 8.75 (dd, J1 = 4.2, J2 = 1.7 Hz, 1H); 8.38 (d,
J = 2.3 Hz, 1H), 8.24 (dd, J = 8.3, 1.7 Hz, 1H), 7.94
(d, J = 9.1 Hz, 1H); 7.76 (dd, J1 = 9.1, J2 = 2.4 Hz,
1H), 7.45 (dd, J = 8.3, 4.2 Hz, 1H), 2.69–2.50 (m,
4H). 13C RMN (75 MHz, DMSO-d6): d (ppm) 174.32,
171.09, 149.29, 144.96, 137.58, 135.89, 129.87,
128.80, 123.62, 122.19, 115.04, 31.55, 29.20. ESIMS
(m/z): 245 (M ? H)?
General procedure for the synthesis of DFO
conjugates
6: The synthesis was based on a previous report
(Sinha and Shrivastava 2013). 6-quinolinamine
(2 mmol) and succinic anhydride (2 mmol) were
mixed in 15 mL toluene and stirred constantly in
reflux under nitrogen. After 4 h, the yellow solid was
washed with CHCl3/MeOH (95:5) and dried under
vacuum for 24 h to achieve the desired product. The
reaction was monitored by TLC using chloroform/
acetic acid/methanol (85:10:5) eluent.
DFO conjugates were prepared based on previous
reports (Liu et al. 2010). In DMF (15 mL), 1 mmol of
the carboxylic acids were mixed with DFO (1 mmol),
EDC (1.5 mmol) and HOBt (1.5 mmol). The mixture
was stirred constantly and heated to 50 °C for 5 min.
After the reagents dissolved, DIPEA (2 mmol) was
added to the solution and the mixture was stirred for
16 h at room temperature under nitrogen. The solid
was washed with diethyl ether, distilled water and
methanol. The reactions were monitored by TLC using
chloroform/methanol (80:20) eluent.
4-oxo-4-(phenylamino)butanoic acid (2a): Yield:
70%. m.p: 144–146 °C. Rf: 0.43. 1H-NMR
(300 MHz, DMSO-d6): d (ppm) 12.10 (s, 1H), 9.92
(s, 1H), 7.61–7.51 (m, 2H), 7.33–7.20 (m, 2H),
7.06–6.94 (m, 1H), 2.61–2.47 (m, 4H). 13C RMN
(75 MHz, DMSO-d6): d (ppm) 174.25, 170.48,
139.72, 129.09, 123.33, 119.32, 31.47, 29.24. ESIMS
(m/z): 192 (M-H)-.
Purification and characterization of DFO
conjugates
Purification by preparative reverse-phase HPLC was
carried in a RP-HPLC with two eluents: A (trifluo-
roacetic acid 0.1% in water) and B (60% acetonitrile,
0.09% trifluoroacetic acid in water). A sample of
250 mg of crude conjugate was injected and the
elution sequence was 20% B for 15 min, 20–60% B in
60 min, 60–95% B in 15 min, 95% B for 5 min. The
fractions were analyzed by direct infusion mass
spectrometry and those containing the desired conju-
gate were pooled and lyophilized. Analysis of the
purified conjugate, at the concentration 1 mg/mL in
DMSO, was carried out in a LC–MS system using the
same eluents described above. The gradient applied
was 5 to 95% B in 30 min, the capillary voltage was
4500 V and the end plate offset voltage was 500 V
(for positive mode).
4-oxo-4-((4-sulfamoylphenyl)amino)butanoic
acid (2b): Yield: 0.50% m.p: 204–205 °C Rf: 0.10.
1H-NMR (300 MHz, DMSO-d6): d (ppm) 12.14 (s,
1H), 10.29 (s, 1H), 7.72 (s, 4H), 7.22 (s, 2H),
2.65–2.47 (m, 4H). 13C RMN (75 MHz, DMSO-d6):
d (ppm) 174.19, 171.17, 142.58, 138.47, 127.12,
118.84, 31.55, 29.07 ESIMS (m/z): 273 (M ? H)?
4-(naphthalen-2-ylamino)-4-oxobutanoic acid
(4): Yield: 35% m.p: 194–195 °C Rf: 0.50 1H-
NMR (300 MHz, DMSO-d6): d (ppm) 12.17 (s, 1H),
10.18 (s, 1H), 8.30 (d, J = 2.1 Hz, 1H), 7.88–7.73 (m,
3H), 7.56 (dd, J1 = 8.9, J2 = 2.1 Hz, 1H); 7,41 (dddd,
J1 = 6.9, J2 = 8.0, J3 = 6.8, J4 = 1.3 Hz, 2H),
2.69–2.50 (m, 4H) 13C RMN (75 MHz, DMSO-d6):
d (ppm) 174.34, 170.86, 137.28, 133.90, 130.03,
123

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Biometals (2021) 34:259–275
DFOANI: Yield: 26.7% m.p: 180–182 8C. Rf:
0.52. H-NMR (500 MHz; DMSO) d(ppm) 9.92 (s;
ESIMS (m/z): 786 (M ? H)?. Elemental Formula
C39H59N7O10 Calculated (%): C 59.6 H 7.6 N 12.5
Found (%): C 59.9 H 7.9 N 12.0 Experimental
Formula: C39H59N7O10. 0.1CH3OH.
1
1H), 9.66 (s; 1H), 9.61 (s; 2H), 7.84 (t; J = 5.4 Hz;
1H); 7.78 (t; J = 5.3 Hz; 2H), 7.57 (d; J = 7.7 Hz;
2H); 7.27 (t; J = 7.9 Hz; 2H), 7.01 (t; J = 7.4 Hz; 1H),
3.45 (t; J = 6.4 Hz; 6H), 3.05–2.96 (m; 6H), 2.58 (t;
J = 7.1 Hz; 3H), 2.54 (dd; J = 9.4; 5.1 Hz; 3H), 2.39
(t; J = 7.3 Hz; 2H), 2.27 (t; J = 7.2 Hz; 4H), 1.97 (d;
J = 5.0 Hz; 3H), 1.51 (dd; J = 22.7; 16.1 Hz; 6H);
1.37 (d; J = 6.8 Hz; 6H), 1.22 (d; J = 6.7 Hz; 6H). 13C
RMN (126 MHz., DMSO) d (ppm) 172.44, 171.78,
171.49, 170.96, 139.80, 129.11, 123.33, 119.37, 47.55,
47.25, 38.90, 32.23, 30.80, 30.37, 29.28, 28.03, 26.49,
23.96, 20.82. ESIMS (m/z): 736 (M ? H)?. Elemen-
tal Formula C35H57N7O10, Calculated (%): C 57.1,
H 7.8, N 13.3, Found (%): C 55.6, H 7.9, N 12.9,
Experimental Formula: C35H57N7O10.0.4CH3-
OH.0.95H2O. e [DFOANI(Fe)] complex (kmax-
DFOQUN: Yield: 24.3% m.p: 143–145 8C. Rf: 0.5
1H-NMR (500 MHz; DMSO) d(ppm) 10.38 (s, 1H),
9.63 (s, 2H), 8.85 (dd, J = 4.4, 1.5 Hz, 1H), 8.52–8.37
(m, 2H), 8.01 (d, J = 9.1 Hz, 1H), 7.85 (dd, J = 9.1,
2.3 82 Hz, 2H), 7.77 (t, J = 5.4 Hz, 2H), 7.60 (dd,
J = 8.4, 4.5 Hz, 1H), 3.01 (dq, J = 13.2, 6.7 Hz, 8H),
2.67–2.53 (m, 8H), 2.42 (q, J = 6.9 Hz, 4H), 2.26 (t,
J = 7.1 Hz, 5H), 1.95 (s, 3H), 1.54–1.44 (m, 6H),
1.41–1.33 (m, 6H), 1.26–1.12 (m, 6H).13C RMN
(126 MHz., DMSO) d (ppm) 172.43, 171.79, 171.67,
171.42, 147.89, 138.31, 129.11, 128.42, 128.00,
124.84, 122.32, 114.99, 47.53, 47.24, 38.89, 32.25,
30.64, 30.39, 29.28, 28.02, 26.48, 23.94, 20.79.
ESIMS (m/z): 787 (M ? H)?. Elemental Formula
C38H58N8O10 Calculated (%): C 58.0 H 7.4 N 14.2
Found (%): C 58.3 H 7.5 N 14.1 Experimental
Formula: C38H58N8O10 e [DFOQUN(Fe)] complex
= 430 nm): 2037.3 L mol-1 cm-1
.
DFOBAN: Yield: 24.9% m.p: 184–185 8C. Rf:
0.0.25 1H-NMR (500 MHz; DMSO) d(ppm) 10.28 (s,
1H), 9.66 (s, 1H), 9.61 (s, 2H), 7.85 (t, J = 5.5 Hz,
1H), 7.77 (t, J = 5.3 Hz, 2H), 7.74 (d, J = 9.5 Hz, 4H),
7.23 (s, 2H), 3.45 (t, J = 7.0 Hz, 6H), 3.06–2.95 (m,
6H), 2.63–2.53 (m, 6H), 2.43–2.36 (m, 2H), 2.27 (t,
J = 7.1 Hz, 4H), 1.96 (s, 3H), 1.55–1.44 (m, 6H),
1.42–1.34 (m, 6H), 1.22 (d, J = 6.8 Hz, 6H). 13C
RMN (126 MHz., DMSO) d (ppm) 172.44, 171.61,
171.37, 142.68, 138.44, 127.13, 118.84, 47.55, 47.25,
32.21, 30.51, 30.37, 29.28, 28.03, 26.50, 23.96, 20.82.
ESIMS (m/z): 815 (M ? H)?. Elemental Formula
C35H58N8O12S Calculated (%): C 51.8, H 7.2, N 13.8
Found (%): C 50.8, H 7.2, N 13.2 Experimental
(kmax = 430 nm): 1231.8 L mol-1 cm-1
.
Calcein competition studies
The ability of the new chelators to scavenge iron was
studied by competition against the fluorescent chelator
´
calcein, according to published protocols (Esposito
et al. 2002a). Aliquots of 190 lL of ferric calcein
(CAFe; 2 lM) in HBS/Chelex (Hepes Buffered
Saline; hepes 20 mM, NaCl 150 mM, Chelex 1 g/
100 mL; pH 7.4) were added to transparent 96-well
microplates. Next, 10 lL of conjugates (final concen-
trations: 0–15 lM) in DMSO were added to each well.
Fluorescence was recorded after 2 h incubation at
37 °C in a BMG FluoStar Optima microplate reader
(kexc/kem = 485/520 nm). Two independent assays
were performed, each one in duplicate.
Formula: C35H58N8O12S.0.55CH3OH.0.45H2O
e
[DFOBAN(Fe)] complex (kmax = 430 nm): 1642.7
L mol-1 cm-1
.
DFONAF: Yield: 43.8% m.p: 180–182 Rf: 0.6
1H-NMR (500 MHz; DMSO) d(ppm) 10.14 (s, 1H),
9.65 (s, 1H), 9.61 (s, 2H), 8.29 (s, 1H), 7.89–7.75 (m,
6H), 7.59–7.54 (m, 1H), 7.49–7.42 (m, 1H), 7.41–7.35
(m, 1H), 3.45 (t, J = 7.0 Hz, 6H), 3.01 (td, J = 13.2,
6.6 Hz, 6H), 2.64–2.52 (m, 6H), 2.43 (t, J = 7.2 Hz,
2H), 2.27 (t, J = 6.6 Hz, 4H), 1.97 (s, 3H), 1.49 (d,
J = 6.3 Hz, 6H), 1.39 (dq, J = 14.8, 7.5 Hz, 6H), 1.22
(d, J = 6.2 Hz, 6H). 13C RMN (126 MHz., DMSO) d
(ppm) 172.44, 171.78, 171.49, 171.28, 137.38, 133.93,
130.05, 128.74, 127.89, 127.68, 126.82, 124.87,
120.33, 115.28, 47.55, 47.25, 38.92, 38.89, 32.29,
30.79, 30.37, 29.28, 28.03, 26.50, 23.96, 20.82.
DHR antioxidant activity
The ability of the new chelators to block iron-
dependent oxidation of DHR was studied according
´
to previous methods (Esposito et al. 2003). Aliquots of
10 lL of ferric nitrilotriacetate (Fe(NTA)) were added
to transparent 96 wells microplates followed by 10 lL
of the conjugates (0–300 lM) in DMSO. Next, 180 lL
of fluorogenic mixture (DHR 50 lM and ascorbic acid
40 lM in HBS/Chelex) were transferred to the wells.
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Biometals (2021) 34:259–275
265
Final concentrations of Fe(NTA) and conjugates were
2 lM and 0–15 lM, respectively, with 5% final
DMSO. The fluorescence was measured in a BMG
Fluostar Optima (kexc/kem = 485/520 nm) at 37 8C
for 1 h. Two independent assays were performed, each
one in duplicate.
aggregation of the peptide, which in this test would be
equivalent to releasing the probe into the aqueous
medium and quenching its fluorescence. According to
the methodology (Guzior et al. 2015), the Ab1–42
fragment (Sigma Aldrich A9810) was solubilized in
DMSO to obtain a 100 lM stock solution and then
diluted in PBS pH 7.4 to 5 lM (containing 5%
DMSO). Tacrine, DFO and DFOQUN were dissolved
in DMSO (15 mM) and diluted in PBS (100 lM).
Finally, aliquots of 20 lL Ab1–42 (5 lM), 20 lL
conjugate (100 lM) and 4 lL FeSO4 (50 lM) were
transferred to a 96-well black microplate. Each well
was filled with PBS, at a final volume of 100 lL. The
microplate was incubated in the dark under constant
shaking at room temperature for 24 h. After incuba-
tion, 2 lL of ThT solution (44.4 lM in PBS) was
transferred to each well. The microplate was inserted
into a Spectra Max M4 reader and the fluorescence
was recorded (kexc/kem = 446/490 nm). Final concen-
trations of Ab1–42, compounds, FeSO4 and ThT were
1, 20, 2 and 1 lM, respectively, and final DMSO
percentage equal to 1%. The aggregation inhibition
percentage was calculated according to the following
the equation (Hiremathad et al. 2016):
DPPH anti-oxidant activity
Aliquots of 10 lL of DFO conjugates (0–2 mM)
dissolved in DMSO were added to 96-well micro-
plates, followed by addition of 190 lL of DPPH
(0.1 mM in methanol) (Molyneux 2004). Final con-
centrations of conjugates were 0–100 lM and final
DMSO percentage was 5%. The microplates were
incubated for 30 min at room temperature in the dark
and the absorbance (k = 520 nm) was measure in a
SpectraMax M4 microplate reader. Antioxidant activ-
ity was calculated according to the equation:
ðAbs DPPH À Abs SampleÞ
AAð%Þ ¼
 100
ðAbs DPPHÞ
where AA(%) is the percentage of antioxidant activity.
Two independent assays were performed, each one in
duplicate.
Ið%Þ ¼ ð1 À Fi=FoÞ Â 100
where Fi Fluorescence of treated Ab1- 42, Fo Fluores-
cence of Ab1–42, I (%) percentage of inhibition. Three
independent assays were performed, each in triplicate.
Fe(II) speciation calculation
CHEAQS NEXTÒ is a computational software used
for chemical equilibrium calculations in aqueous
systems, widely used in geological and environmental
studies (Walaszek et al. 2018). The interest in
calculating Fe(II) speciation was to estimate the levels
of bioavailable iron that can affect worm mortality.
Speciation calculation was performed considering
ionic strength of 0.09 M, pE of 0.9128 (54 mV),
FeSO4 6.2 mM (at ‘ LC50) and H? 1.1 lM (free
activity), allowing for the formation of insoluble
species (Xie et al. 2009).
C. elegans strain and maintenance
N2 nematodes were acquired from the Caenorhabditis
Genetics Center (CGC—University Minnesota, Twin
Cities, MN, USA). Worms were cultivated at 20 °C in
standard culture conditions in plastic petri dishes
containing agar 8P (Bianchi and Driscoll 2006).
Synchronized populations at the L1 larval stage were
obtained by isolation of eggs of gravid hermaphrodites
using bleach solution as previously described (1%
NaOCl, 0.25 M NaOH) (Kenyon 1988). Eggs were
hatched in NGM plates without food. For all the
experiments, synchronized worms were washed from
the hatching plates, treated and then transferred to
NGM plates containing nematode growth medium
and E. coli OP50 strain as food (Brenner 1974).
Inhibition of Ab1–42 aggregation
To investigate the inhibition of Ab1–42 aggregation by
the DFO conjugates in the presence and absence of Fe,
an assay was performed using the fluorescent probe
Thioflavin-T (ThT). When ThT binds to the peptide
structure, the probe fluoresces. Any anti-Alzheimer
drug candidate would be expected to disrupt the
123

266
Biometals (2021) 34:259–275
Lethality assay
normalized to protein content, quantified by the
Bradford method (Hart 2006; Sashidhara et al. 2014b).
The worms were treated with various concentrations
of FeSO4 to determine the LC50. After synchroniza-
tion, 2500 worms in the L1 stage were treated with Fe
(0–100 mM) for 1 h in NaCl (0.5%) solution at 20 °C.
Then, 30–50 worms were washed and transferred to
NGM plates with bacteria. After 48 h, the number of
living worms was counted. Three independent assays
were performed each in triplicate.
AChE activity (in vitro assay)
Ten lL of conjugate solutions (0–300 lM) dissolved
in HBS (DMSO 6.7%) were transferred to 96-well
black microplates. Next, 10 lL FeSO4 (300 lM), 10
lL acetylcholine (300 lM) and 70 lL HBS buffer
were added to each well, and received 100 lL of the
reagent mixture (400 lM amplex red and 1 U/mL
AChE, without acetylcholine) of the assay kit
described above. The final concentration of the
conjugates was 0–15 lM and the final DMSO per-
centage was 0.1%. The microplate was incubated at
room temperature in the dark for 30 min and fluores-
cence was measured using BMG Fluostar Optima
(kexc/kem = 544/590 nm). Three independent assays
were performed, each in triplicate.
Survival assay
We found the LC50 to be equal to 12.5 mM. In order to
have the least possible number of worms dying while
observing toxicity, we used an iron concentration of
6.2 mM (1/2 LC50) in all subsequent assays. The same
procedures for lethality assays were employed, but
after the addition of iron, we co-administered DFO.
We studied both the effects of DFO concentration and
DMSO percentage. Three DFO concentrations were
studied (1: 2.5 lM with 0.015% DMSO; 2: 5 lM with
0.03% DMSO; 3: 10 lM with 0.06% DMSO).
Survival rate was improved under condition 2.
Accordingly, survival and subsequent assays were
carried out at the L1 stage with DFO and other
chelators under the following conditions: Fe
(6.2 mM), chelator (5 lM) and DMSO (0.03%).
DMSO was not toxic to C. elegans up to a 10% level
Body bend assay
The behavior of the nematode reflects the activity of
its central nervous system, which depends on several
factors such as external stimulus, neuronal structure
and changes in the animal environment (Hart 2006). In
addition to its effects on cognitive function, acetyl-
choline acts on neuromuscular junctions in the CNS.
In C. elegans, acetylcholine functions to increase
locomotion secondary to muscle contraction (Gjorg-
jieva et al 2014). Reactive oxygen species generated
by iron damage the cholinergic system, interrupting
the communication between motor neurons and mus-
cle cells in the worm. The number of body bends was
selected for analysis of locomotion. Worms were
transferred to NGM plates and the number of body
bends was counted with a stereomicroscope. Two
types of treatment were used after exposure of the L1
worms to Fe and chelators. In treatment 1, worms were
transferred to 60 mm Petri dishes without food and
body curvature counted for a period of 20 s. In
treatment 2, worms were transferred to 60 mm Petri
dishes with food, and after a 2 h period, again
transferred to plates without food for counting the
curvatures for the same period. Analyses were carried
out in at least ten worms per group, in triplicate.
Assays were repeated independently three times.
´
(Esposito et al. 2020).
AChE activity in C. elegans (in vivo assay)
After treating the worms with Fe and conjugates (in
suspensions of 20,000 worms/500 lL in Eppendorf
tubes), we quantified the activity of AChE in wild-type
(N2) worms. Worms were washed with NaCl (0.5%)
to remove bacteria. Samples were frozen 4 consecu-
tive times in acetone/dry ice bath and sonicated three
times on ice at 15 s intervals using 40% amplitude.
Then, samples were centrifuged at 14,000 rpm for
30 min to remove insoluble residues. The supernatant
was assayed for AChE levels with the Amplex Red
ACh/AChE assay kit (Invitrogen A12217) according
to the manufacturer’s instructions. Absorbance was
measured in a BMG Fluostar Optima reader (kexc
/
kem = 544/590 nm). Three independent experiments
were performed in triplicate and final fluorescence was
123

Biometals (2021) 34:259–275
log P calculation
267
The coupling of DFO to carboxylic acids to prepare
DFO-aniline (DFOANI), DFO-benzosulfanylamide
(DFOBAN), DFO-2-naphthalenamine (DFO-NAF)
and DFO-6-quinolinamine (DFOQUN) was per-
formed (Scheme 1) according to described methods
(Liu et al. 2010). Coupling was assisted by 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC) and
benzotriazol-1-ol (HOBt), which forms a tetrahedral
intermediate that is a good leaving group. The crude
products containing the desired compounds, as veri-
fied by RP-HPLC and mass spectrometry analyses,
were purified by RP-HPLC to a limit of 250 mg per
run. Difficulties in dissolving the products in the
solvents used for this type of chromatography plus
addition of DMSO to overcome such problems led to
product losses and to relatively low purification yields.
The conjugates were not sufficiently soluble in water
or methanol to enable purification using flash chro-
matography and were thereafter purified using prepar-
ative RP-HPLC, with the following yields: DFOANI
(27%), DFOBAN (25%), DFONAF (44%) and
Lipophilicity is the tendency of a compound be
dissolved preferentially in an apolar phase over an
aqueous phase. This information is usually presented
as partition coefficient (P). The log P value of the
conjugates was calculated by the Marvin Sketch
16.6.27.0 software (https://chemaxon.com/pro-ducts/
marvin). The standard software method was used for
the calculations, based on the contribution of all atoms
to generate the log P. The concentrations of Na?, Cl-
and K? cations were defined as 0.1 M (Arndt et al.
2017).
Statistical analysis
In the case of biological assays, statistical analyses
were performed using the Graph Pad Prism 6.0
software
(https://www.graphpad.com/scientific-
software/prism/) with one-way ANOVA followed by
Tukey’s post hoc test. Results were expressed as
Means Standard Deviation (SD) and differences
were considered significant when p \ 0.05. n = 2 or 3
was used as effects were statistically significant even
with few replicates, as occurs with other drug tested in
C. elegans (e.g., Sashidhara et al. 2014a, b; Chand
et al. 2018; Maqbool et al. 2020).
DFOQUN (24%). Techniques such as H NMR, 13C
1
NMR, UV–Vis and Mass Spectrometry were used to
characterize both carboxylic acids precursors and the
conjugates (see Supporting Information).
Calculation of lipophilicity of the conjugates were
obtained by Marvin SketchTM. log P of DFONAF
(0.05), DFOANI (-0.94) and DFOQUN (-0.78) were
higher than water-soluble DFO (- 2.02). This means
that the addition of aromatic rings to DFO enhanced
the hydrophobicity of the conjugates. Indeed, these
conjugates were soluble only in DMSO. However,
when a benzene ring has polar groups, such as in
DFOBAN, hydrophilicity increased (log P = - 2.34).
Results and discussion
Synthesis, purification and lipophilicity
of the conjugates
Succinylation of the aromatic amines in water and
open air was adapted from a previously described
method (Balakrishna et al. 2014) to obtain the
carboxylic acid derivatives of aniline (2a; yield:
70%), benzosulfanylamide (2b; yield: 50%) and
2-naphthalenamine (4; yield: 35%). Increasing reac-
tion time and/or succinic anhydride concentration did
not increase the yield of 4 (data not shown), probably
due to resonance effects over the extended ring system
that decreased the reactivity of the aromatic amine.
The method above did not work for the preparation of
the carboxylic derivative of 6-quinolinamine (6); this
product was obtained in high yield (82%) only when
toluene was used under reflux.
Iron binding and antioxidant studies
Calcein is a fluorescent probe with high affinity for
Fe(III) (log KML = 24). Upon iron binding, the
complex ferric calcein (CAFe) if formed, and its
fluorescence is stoichiometrically (1:1) quenched.
Being a stronger iron chelator than calcein, DFO
(log KML = 30.6) scavenges iron and regenerates
calcein fluorescence in a concentration-dependent
´
manner (Esposito et al. 2002b).
The DFO conjugates under study, at physiological
pH, displayed different binding patterns (Fig. 2a),
suggesting that in some cases, the conjugation of
aromatic rings to the DFO backbone altered the
123

268
Biometals (2021) 34:259–275
Scheme 1 Schematic representation of the DFO conjugates
syntheses. Reagents and conditions: a succinic anhydride, H2O,
10 min, b succinic anhydride, H2O, 15 min, c succinic
anhydride, reflux, Toluen, N2(g), 4 h, d DFO, EDC, HOBT,
DIPEA, DMF, N2(g), RT, 16 h
coordination environment. The conjugate DFONAF
did not display any fluorescence recovery, which may
be a reflex of additional solubility problems (Ihnat
et al. 2002). DFONAF was therefore excluded from
further consideration as a MTDL chelator for brain
iron. DFOANI is one ring smaller than DFONAF, and
in this case we observed better fluorescence recovery,
indicating that the metal has access to the otherwise
unmodified tris(hydroxamate) binding site. DFOBAN
presented also a high fluorescence recovery, possibly
because the polar benzosulfanylamide group posi-
tively assists to the metal binding and at the same time
does not hamper coordination (Ihnat et al. 2002).
DFOQUN showed a binding pattern almost identical
to that of DFO, indicating that the quinoline ring did
neither attach to the tris(hydroxamate) binding site nor
hamper metal approximation.
as the ferric nitrilotriacetate, FeNTA) to participate in
ROS-generating reactions mediated by ascorbate in a
physiological buffer (HBS). The generated ROS will
oxidize DHR, unless a suitable iron chelator is present
´
in the medium (Esposito et al. 2003). Successful
clinical chelators such as DFO can inhibit DHR
oxidation (Fig. 2b), and any DFO-derived candidate
drug should maintain the same ability. Except for
DFONAF, this was verified for all conjugates, sug-
gesting that they could potentially mitigate neurode-
generation promoted by iron-induced ROS generation
(Chaston and Richardson 2003).
Finally, tests with the free radical 2,2-diphenyl-1-
picrylhydrazyl (DPPH) (Molyneux 2004) showed that
hydrogen atoms from all DFO conjugates are available
for DPPH, as all of them behave as antioxidants
(Fig. 3). Therefore, they can all confer antioxidant
protection irrespective of their iron chelation efficacy.
Iron-dependent oxidation processes occur in phys-
iological conditions, and a very convenient and
relevant form to assess antioxidants is the inspection
of the effect of chelators in the dihydrorhodamine
(DHR)/ascorbate system. This antioxidant test is
based on the ability of redox-active iron species (such
123

Biometals (2021) 34:259–275
269
100
Lethality and survival assay in wild type C.
elegans
(a)
80
60
40
20
0
Several iron compounds (such as iron chelates, zero-
valent iron nanoparticles or iron salts) have been
recognized to be neurotoxic in C. elegans. Ferrous
sulfate at concentrations up to 2 mM has been found to
negatively impact worm behavioral responses and
neuronal viability (Fagundez et al. 2015; Hu et al.
2008; Soares et al. 2018). Therefore, the efficacy of the
DFO conjugates in mitigating iron-induced damage
was examined. First, we constructed a dose–response
curve (Fig. 4a) to determine the optimal level for iron
exposure. We selected a dose corresponding to half of
the LC50, 6.2 mM, which lead to lethality in * 27%
of the worms, and thus allowed us to test whether the
effects of Fe might be reversible. Also, this concen-
tration of free iron did not decrease the pH of the
solution to toxic levels (higher FeSO4 concentrations
acidify the solution and increase toxicity). Under the
aqueous, saline conditions employed, chemical speci-
ation simulations (Supplementary Material) indicated
that at 6.2 mM total iron, most of the metal would be
in insoluble (hydro)oxide forms and only 0.9 lM
would be soluble, and therefore bioavailable. This
information guided us in the selection of DFO
concentration (5 lM) that might revert iron-induced
toxicity without being toxic themselves (Fig. 4b). Our
results revealed that among the DFO conjugates, only
DFOQUN performed the same as DFO in attenuating
Fe-induced toxicity in the C. elegans model (Fig. 4c).
DFOQUN by itself was not toxic. Interestingly, some
authors described quinolines as promoters of the
expression of superoxide dismutase, an important
antioxidant enzyme (Salgueiro et al. 2014, 2017) and
since DFOQUN is based on a quinoline, that could be
an additional therapeutic effect. According to the
positive results obtained, only DFOQUN was selected
for subsequent testing in behavioral and mechanistic
assays.
0
2
4
6
8
10
12
14
16
Concentration (μM)
(b)
300
250
200
150
100
50
0
0
2
4
6
8
10
12
14
16
Concentration (μM)
Fig. 2 a CAFe (2 lM) was treated with increasing concentra-
tions of the conjugates (incubation time: 120 min). The
experiment was repeated twice in duplicates. b Antioxidant
activity of DFO conjugates against ascorbate oxidation cat-
alyzed by iron. Fe(NTA) = 2 lM, Ascorbate = 40 lM, DHR =
50 lM. The experiment was repeated twice in duplicates
70
60
50
40
30
20
10
0
In vitro and in vivo AChE activity
Owing to the involvement of acetylcholinesterase
(AChE) in the termination of signal transmission in the
post-synaptic membranes, AChE inhibitors promote
the accumulation of choline, and increase the duration
of its effects, which are of interest for the treatment of
AD symptoms (Lazarevic-Pasti et al. 2017). In this
0
20
40
60
80
100
Concentration (μM)
Fig. 3 Antioxidant activity of DFO conjugates (in DMSO)
against 0.1 mM DPPH in methanol
123

270
Biometals (2021) 34:259–275
Fig. 4 Iron toxicity and
chelator activity in the N2 C.
elegans strain. a Lethality
induced by FeSO4
**
100
**
***
100
80
60
40
20
0
**
80
60
40
**
(LC50 = 13.6 mM).
***
b Survival of worms treated
with 5 lM DFO. c Survival
of worms treated with 5 lM
DFOQUN. For both b and c:
[Fe] = 6.2 mM; vehicle:
0.03% DMSO. All values
are means SD of n = 3
experiments performed in
triplicate. Statistical
analysis was carried out by
means of one-way ANOVA,
followed by Tukey’s post
hoc test. *p \ 0.05,
20
0
e
l
c
e
ol
O
3.1
100
0
12.5
25
6.2
50
Fe
F
tr
i
n
DF
h
O +
Co
Concentration (mM)
Ve
DF
*
(b)
(a)
*
***
***
100
80
60
40
20
0
**
***
**p \ 0.01, ***p \ 0.001;
NS not significant
l
e
e
F
e
ro
ont
F
M)
cl
hi
µ
+
5
(
C
Ve
M)
N
QU
µ
5
(
O
DF
N
QU
O
F
D
(c)
context, conjugation of an AChE inhibitor moiety
(quinoline, inspired by tacrine) to an iron chelator
could potentiate the effect of both compounds. Thus,
in this study, AChE inhibition by the chelators was
studied in vitro (AChE from Electrophorus electricus)
and in C. elegans in vivo (Fig. 5). The results showed
that DFOQUN significantly increased AChE inhibi-
tion compared to DFO (Fig. 5a; p \ 0.05). Most
likely, the quinoline moiety of DFOQUN blocks the
docking of acetylcholine in the catalytic active site
through p-p staking interactions with residues such as
phenylalanine and tryptophan (Chand et al. 2018;
McKenna et al. 1997). Furthermore, in the presence of
iron, the enzyme inhibition by DFO or DFOQUN was
enhanced, suggesting that the complexes may have
structural characteristics that may further contribute to
their inhibitory effects. This is an intriguing possibility
for the development of metal-based AChE inhibitors
for the treatment of AD.
chelators inhibited the enzyme more robustly
(Fig. 5c). The similarity of effect between DFO and
DFOQUN might be the result of metabolic deactiva-
tion, which should be confirmed in future metabolo-
mics studies.
Finally, low concentrations of DMSO alone can
increase peroxidase activity (by up to 10%), promot-
ing resorufin production (Rammler 1967), which may
account for the increased levels of AChE observed in
the vehicle.
In vitro inhibition of Ab1–42 aggregation
The inhibitory capacity of DFO conjugates against Ab
aggregation was studied with the thioflavin-T (ThT)
assay. ThT is a dye that fluoresces upon binding to Ab
fibrils; therefore, anti-Ab aggregation agents lead to
reduced ThT fluorescence (Hayne et al. 2014). As
expected, free iron increased Ab aggregation (Fig. 6).
DFOQUN promoted greater inhibition of aggregation
compared with DFO (p \ 0.01), likely due to the
presence of a lipophilic quinoline moiety in its
No difference in AChE inhibition was observed in
C. elegans for both chelators at the concentrations
studied (Fig. 5b). However, in the presence of iron the
123

Biometals (2021) 34:259–275
271
N S
***
150
150
100
50
**
NS
NS
NS
**
*
100
50
0
*
*
N S
*
** * ** *
** *
** *
** *
Fe
DFO
0
Vehicle
Control
Tacrine
DFOQUN
DFO + Fe
N
Tacrin(ea+ F)e
DFO
QU
DFOQUN + Fe
O
DF
Tacrine
(b)
(c)
Fig. 5 Effect of the test compounds on AChE activity in the
presence and absence of Fe(II). a in vitro test with AChE from E.
tacrine] = [Fe(II)] = 5 lM; vehicle = 0.03% DMSO; 15 min
incubation. The values are means SD of n = 3 experiments
performed in triplicate. Statistical analysis was carried out by
means of one-way ANOVA, followed by Tukey’s post hoc test.
*p \ 0.05, **p \ 0.01, ***p \ 0.001. NS not significant
electricus
after
60 min
incubation;
[chelators] = [
tacrine] = [Fe(II)] = 15 lM; vehicle = 0.1% DMSO. Tests
with C. elegans were conducted in the absence b and presence
(c)
of
6.2 mM
iron(II),
and
[chelators] = [
Inhibition of Aβ1-42 aggregation(%):
Aβ1-42 + DFO = 8.6 0.6
Aβ1-42 + DFO + Fe = 13.4 1.5
Aβ1-42 + DFOQUN = 11.5 0.8
Aβ1-42 + DFOQUN + Fe = 13.4 1.4
Aβ1-42 + Tacrine = 15.4 0.7
Aβ1-42 + Tacrine + Fe = 14.3 1.4
Fig. 6 Normalized fluorescence of thioflavin T probe. The
values are means SD of n = 3 experiments performed in
triplicate. Statistical analysis was carried out by means of one-
way ANOVA, followed by Tukey’s post hoc test. *p \ 0.05,
**p \ 0.01, ***p \ 0.001, NS = Not Significant. DFO, DFO-
QUN and tacrine = 20 lM; Ab1-42 = 1 lM; Fe(II) = 2 lM;
DMSO percentage = 1%
structure. Indeed, tacrine, which was used as a positive
control, also displayed inhibitory capacity (11.5%) of
peptide aggregation. This suggests that the conforma-
tion adopted by DFOQUN can disrupt b-sheet
123

272
Biometals (2021) 34:259–275
***
**
50
40
30
20
10
0
N S
N S
30
20
10
0
***
NS
***
***
***
***
N S
NS
***
N S
*
***
***
***
***
l
r
e
l
t
Fe
c
C
i
Fe
Ctrl
h
V e
Vehicle
DFO + Fe
DFO + Fe
Tacrine + Fe
Tacrine + Fe
DFOQUN + Fe
DFOQUN + Fe
(c)
(a)
NS
***
40
***
50
NS
***
***
***
***
***
30
20
10
0
40
30
20
10
0
***
***
***
l
r
e
l
t
C
ne
ic
Ctrl
DFO
DFO
e h
V
Vehicle
Tacrine
Tacri
DFOQUN
DFOQUN
(d)
(b)
Fig. 7 Body bend assay in the N2 strain of C. elegans under
starvation (a, b) or regular feeding (c, d), in the presence (a, c) or
absence (b, d) of 6.2 mM iron. DFOQUN = DFO = Tacrine =
5 lM. Vehicle = 0.03% DMSO. The values are means SD
of n = 3 experiments performed in triplicate. Statistical analysis
was carried out by means of one-way ANOVA, followed by
Tukey’s post hoc test. *p \ 0.05, **p \ 0.01, ***p \ 0.001,
NS not significant
interactions of Ab (Hiremathad et al. 2016; Quin-
tanova et al. 2015a). Since the chelators are efficient
iron scavengers, co-administration of iron with the
chelators also did not lead to fibril aggregation.
DFO is a highly hydrophilic molecule, however, as
is the case for other chelators, its metal complexes are
more lipophilic due to the lower exposure of donor
atoms to the solvent. Increased lipophilicity hampers
the aggregation of Ab, likely accounting for the fact
that FeDFO has a greater anti-aggregation activity
than DFO alone.
altering their functions (Manalo and Medina 2020). As
a defense mechanism, neurons will increase lys-7
expression (involved in defense responses in other
organisms) and sod-5 expression (an ortholog of
human superoxide dismutase 1; Manalo and Medina
2020; Wang et al. 2010). This process, in turn, will
promote stress in the worms, increasing their locomo-
tion frequency (see Fig. 7) as reflected by increased
number of body bends for worms treated with vehicle
when compared to saline controls.
Our results showed that under starvation (Fig. 7a,
b) there was a significant recovery of motility loss
caused by iron, when either chelators or tacrine
(p \ 0.001) were present at the same time (however,
the locomotion levels were never the same as those for
vehicle control, suggesting that simple DMSO distur-
bance was not the main effect). In the absence of iron-
Body bend assay
Body bend assay in C. elegans was performed to
assess locomotion deficits (Rand 2007). It is notewor-
thy that DMSO affects mechanoreceptor neurons,
123

Biometals (2021) 34:259–275
273
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induced stress, motility was higher and without
significant differences between treatments. Under
normal feeding (Fig. 7c, d) DFOQUN, but not DFO,
promoted an increase in worm motility disturbed by
iron (again, absent recovery to the levels of vehicle
control). In this condition, again there was no signif-
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tacrine, DFO and DFOQUN in the absence of iron-
induced stress. Since acethylcoline plays an important
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(2016/03709-9 and 2018/19684-0 to BPE and 2015/14360-4 to
MTM) and CAPES (Brazilian agencies). MA was supported in
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Funding FAPESP 15/14360-4, 16/03709-9 and 18/19684-0;
CAPES 88881.131918/2016-01; NIEHS R01ES07331 and
R01ES10563.
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