Design and synthesis of N-hydroxyalkyl substituted deferiprone: a kind of iron chelating agents for Parkinson's disease chelation therapy strategy

Article abstract of DOI:10.1007/s00775-021-01863-x

The blood–brain barrier (BBB) permeability of molecules needs to meet stringent requirements of Lipinski’s rule, which pose a difficulty for the rational design of efficient chelating agents for Parkinson's disease chelation therapy. Therefore, the iron c

Full text of DOI:10.1007/s00775-021-01863-x

JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
https://doi.org/10.1007/s00775-021-01863-x
ORIGINAL PAPER
Design and synthesis of N‑hydroxyalkyl substituted deferiprone:
a kind of iron chelating agents for Parkinson’s disease chelation
therapy strategy
Qingchun Zhang1 · Shufan Feng1 · Yulian Zhao2 · Bo Jin1 · Rufang Peng1
Received: 13 October 2020 / Accepted: 16 March 2021 / Published online: 8 May 2021
© Society for Biological Inorganic Chemistry (SBIC) 2021
Abstract
The blood–brain barrier (BBB) permeability of molecules needs to meet stringent requirements of Lipinski’s rule, which
pose a difculty for the rational design of efcient chelating agents for Parkinson’s disease chelation therapy. Therefore,
the iron chelators employed N-aliphatic alcohols modifcation of deferiprone were reasonably designed in this work. The
chelators not only meet Lipinski’s rule for BBB permeability, but also ensure the iron afnity. The results of solution ther-
modynamics demonstrated that the pFe3+ value of N-hydroxyalkyl substituted deferiprone is between 19.20 and 19.36,
which is comparable to that of clinical deferiprone. The results of 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays
indicated that the N-hydroxyalkyl substituted deferiprone also possesses similar radical scavenging ability in comparison to
deferiprone. Meanwhile, the Cell Counting Kit-8 assays of neuron-like rat pheochromocytoma cell-line demonstrated that
the N-hydroxyalkyl substituted deferiprone exhibits extremely low cytotoxicity and excellent H2O2-induced oxidative stress
protection efect. These results indicated that N-hydroxyalkyl substituted deferiprone has potential application prospects as
chelating agents for Parkinson’s disease chelation therapy strategy.
Graphic abstract
Extended author information available on the last page of the article
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JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
Keywords Hydroxypyridinone · Chelating agents · Deferiprone · Cytotoxicity · Solution thermodynamics · Parkinson’s
disease
H-bond donors, or possess more than 10 H-bond acceptors,
the BBB permeability of the drugs does not increase in pro-
portion to lipid solubility, then the drugs are probably poor
BBB-penetrating molecules [12]. The BBB limits the brain
penetration of essentially 100% of large-molecular drugs and
more than 98% of small-molecular drugs [13, 14]. Consider-
ing that the number of individuals with a CNS condition will
grow with an aging population, few satisfactory iron chela-
tors become particularly problematic. Therefore, we urgently
needed to develop iron chelators for PD chelation therapy.
In the past, chelation therapy has long been successfully
used in treating beta-thalassemia, and then interest in the
use of chelators as a therapeutic strategy in neurodegenera-
tive disorders. But so far, only three iron chelators, defer-
iprone, deferasirox, and desferrioxamine (Fig. 1a), have
been approved for clinical use in the chelation treatment of
beta-thalassemia. Among them, only deferiprone has oral
activity and BBB permeability, which have been employed
in clinical studies for PD chelation therapy [15]. However,
deferiprone is rapidly metabolized into non-chelating defer-
iprone 3-O-glucuronide by UDP-glucuronosyltransferases
(UGTs, Fig. 1b) [16, 17], which leads to relatively high
doses of deferiprone (75−100 mg kg−1) being adopted in
Introduction
Iron is an essential cofactor for many proteins that are
involved in the normal function of neuronal tissue, such
as the non-haem iron enzyme tyrosine hydroxylase, which
is required for dopamine synthesis. However, it has also
become apparent that iron progressively accumulates in the
brain with age, and that the redox cycle of ferric/ferrous
valence states contributes to the enhanced generation of
H2O2, hydroxyl radicals, and other reactive oxygen species
(ROSs), which can attack nigral dopaminergic neurons and
induce neurodegeneration [1–5], and thereby induce neuro-
degenerative Parkinson’s disease (PD). Therefore, the devel-
opment of iron chelators that can mobilize iron out of the
brain and eliminate the ROSs is generally considered to be a
promising therapeutic strategy for disease mitigation [6–11].
However, the most important factor limiting the devel-
opment of new iron chelators for the central nervous sys-
tem (CNS) is the blood–brain barrier (BBB). The chelators
cross the BBB via lipid-mediated free difusion. According
to Lipinski’s rule, if the molecular weight of the drugs is
greater than 500 Da and the calculated logarithm of partition
coefcient (log P) is greater than 5, or possess more than 5
(a)
(b)
(c)
O
H
N
OH
OH
N
O
OH
N
O
OH
H2N
N
CH3
N
H
N
H
4
4
5
HN N
N
CH3
CH3
O
O
O
O
O
BA
Deferasirox
Deferiprone
Desferrioxamine
HOOC
O
O
N
O
HO
OH
O
HO
Metabolized by UGTs
OH
H3C
N
CH3
CH3
CH3
Deferipron 3-O-glucuronide
Deferiprone
O
O
N
O
O
O
N
O
OH
F
OH
F
OH
OH
OH
OH
N
N
N
F
N
CH3
N
CH3
N
CH3
CH3
CH3
CH3
CH3
CH3
External modification of HOPO
Internal modification of HOPO
Fig. 1 a The molecular structures of three clinical use iron chelators. b The glucuronidation reaction of deferiprone. c The external and internal
modifcation of HOPO
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JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
469
the clinic. In the case of considering Linpinski’s rule [12]
for BBB permeability, there have been several attempts to
modify the deferiprone structure for optimizing the rate of
metabolism. Such as internal and external modifcation of
hydroxypyridinone (HOPO) ring (Fig. 1c). However, the
introduction of additional nitrogen in or fuorine on HOPO
ring [18, 19] signifcantly reduced iron afnity in compari-
son with deferiprone [19, 20]. Fortunately, the N-substituted
modifcation of deferiprone has little efect on iron afnity
[21–24]. Meanwhile, aliphatic alcohols could be glucuro-
nidated by UGTs [25] and expected to reduce the rate of
metabolism to non-chelating deferiprone 3-O-glucuronide.
Therefore, the iron chelators employed N-aliphatic alcohols
modifcation of deferiprone were reasonably designed in
this work, which not only meet Lipinski’s rule for BBB per-
meability and ensure iron afnity, but also may reduce the
rate of deferiprone metabolism to non-chelating deferiprone
3-O-glucuronide. In consideration of the importance of Ca2+
and Zn2+ in organisms, the afnity of N-hydroxyalkyl substi-
tuted deferiprone with Ca2+ and Zn2+ also be studied.
To the best of our knowledge, the radical scavenging
activity and thermodynamic stability consequences of Fe3+,
Ca2+ and Zn2+ chelation by synthetic N-hydroxyalkyl sub-
stituted deferiprone chelators are as yet unexplored. Herein,
the detailed characterization of the solution thermodynamic
stability with Fe3+, Ca2+ and Zn2+, the radical scavenging
activity, the cytotoxicity, and the H2O2-induced oxidative
stress protective efect of N-hydroxyalkyl substituted defer-
iprone are also discussed.
O
HO
NH2
O
n
BnBr, KOH
OBn
CH3
OH
3a-c
NaOH
O
2
O
1
CH3
O
O
Pd/C, H2
OH
OBn
CH3
a n = 2
b n = 3
c n = 4
N
CH3
N
n
n
OH
OH
5a-c (L1-3H)
4a-c
Scheme 1 Synthesis of N-hydroxyalkyl substituted deferiprone chela-
tors. Metal-binding atoms are shown in red, and aliphatic alcohol that
can be glucuronidated by UGTs is shown in blue
1
The H NMR signals of the HOPO protons of chelators
L1−3H in D2O were identifed as two doublets at 7.65 ppm
(d, J=7.2 Hz, 1H, Py-H) and 6.52 ppm (d, J=7.2 Hz, 1H,
Py-H). The results indicated that the structures of chelators
L1−3H were as we expected.
Solution thermodynamics
In the presence of dissolved metal ions (Ma+) and proto-
nated chelators (LHh, where L is a ligand with h removable
protons), a pH-dependent metal–ligand complex with the
general formula MmLlHh forms. The relative amount of each
species in solution was determined by Eq. 1, whose rear-
rangement provides the standard formation constant notation
of log βmlh (Eq. 2). The log βmlh value describes a cumulative
formation constant, and a stepwise formation constant (log
K) can be calculated from log βmlh value by using Eq. 3.
When addressing protonation constants, the stepwise forma-
Results and discussion
Synthesis and characterization
The synthesis of N-hydroxyalkyl substituted deferiprone
chelators 5a − c (also designated as L1−3H, due to their
neutral form involved one dissociable proton) was shown in
Scheme 1. 3-benzyloxy-2-methyl-4-pyrone 2 was prepared
according to a universal hydroxyl protection method by
using benzyl bromide. Alkanolamine 3a−c and 2 were con-
densed using NaOH to obtain the desired pyridones 4a−c
with up to 82% yield [26]. Deprotection of the hydroxyl
groups under typical catalytic hydrogenation conditions with
the removal of the benzyl group gave the target product che-
lators L1−3H up to 90% yield.
H
tion constants are commonly reported as log Kh .
[MmLlHh] =ꢀmlh[M]m[L]l[H]h
(1)
(2)
(
[MmLlHh]
)
log ꢀmlh = log
[M]m[L]l[H]h
(
)
(
)
[LHh]
ꢀ01h
log K01h = log
= log
(3)
[LHh−1][H]
ꢀ01(h−1)
The chelators L1−3H were fully characterized by nuclear
magnetic resonance (NMR), Fourier-transform infrared
(FTIR) spectroscopy, and mass spectra (MS). The FTIR
spectra of chelators L1−3H showed characteristic peaks
at about 3304 cm−1 for ν(O − H) of the HOPO hydroxy
group, 1622 cm−1 for ν(C = O) of HOPO carbonyl, and
1562 cm−1, 1499 cm−1 for pyridone ring skeleton vibration.
Protonation of chelators L1−3
H
Before studying the complexation of metal ions by the inves-
tigated chelators L1−3H, the log KhH value of chelators L1−3
should be determined. The log KhH value of chelators L1−3
H
H
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470
JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
Fig. 2 Incremental spectropho-
tometric titration of chelator
L1H a by 0.1 M HNO3, b
by 0.1 M KOH. Conditions:
[L1H]=0.2 mM, I=0.1 M KCl,
T=298 K
H
Table 1 Summary of log Kh value of chelators L1−3H and defer-
iprone
H
H
Chelator
log K1
log K2
L1Ha
10.16 (3)
10.01 (4)
9.98 (2)
9.82
3.29 (2)
3.34 (2)
3.22 (3)
3.66
L2Ha
L3Ha
Deferiproneb
aValue was determined from incremental spectrophotometric titra-
tions at [L1−3H]=0.2 mM, I=0.1 M KCl, T=298 K. bValue was
obtained from Ref. [30] at [deferiprone]=0.32 mM, I=0.1 M KCl,
T=298 K
Fig. 3 Representative speciation diagram of chelator L1H calculated
at concentration of 10 μM
L1−3H is ascribed to the protonation of carbonylic oxygen of
the HOPO moiety (Scheme 2). The value is slightly lower
than that of deferiprone [30] (Table 1), which is attributed to
the intermolecular hydrogen bond between aliphatic alcohol
and carbonyl that makes carbonyl difcult to protonate.
was calculated by program HypSpec [27], ftting incremental
spectrophotometric titration data. In their neutral form, the
initial intensity of peaks at 280 nm gradually decreases with
a decrease in pH in the range 5.6 − 2.1, which was attrib-
uted to the protonation of the carbonyl group. And the initial
maximum UV–visible absorption peaks at 280 nm redshift
to 310 nm with an increase in pH in the range 5.7− 11.2,
which was attributed to the deprotonation of the hydroxyl of
HOPO (Fig. 2 and Figures S1−S2). The log KhH value was
used to calculate the speciation diagram by program HySS
[28] and three diferent species obtained between pH 2.0 and
12.0 (Fig. 3 and Figure S3). To determine the accuracy of
the results, the log KhH value of chelators L1−3H was con-
frmed by program HyperQuad [29] ftting potentiometric
titration curves (Figure S4). The curves of free chelators
L1−3H all show a break at m=0 (mol base/mol chelator) and
low pH value (pH<9.0), which is indicative of the deproto-
nation of the hydroxyl of HOPO. The log KhH value extrapo-
lated from both methods was in excellent agreement (Fig-
Iron(III) complexation
The afnities of chelators L1−3H with Fe3+ were deter-
mined by incremental spectrophotometric titrations using
a 1:3 Fe3+ to chelator ratio to ensure the formation of
mononuclear complexes. Compared with the free chela-
tors, the UV-visible of complexes Fe3+ − L1−3H have new
absorption peaks at wavelengths greater than 400 nm.
The absorption peaks at about 580 nm blue-shift to about
460 nm with an increase in pH in the range 0.4 − 10.1,
were attributed to the ligand to metal charge transfer
(LMCT) peaks of complexes Fe3+ − L1−3H (Fig. 4 and
Figure S6 − S7). In the UV–visible spectra, the LMCT
peaks can be assigned to three spectrophotometrically
distinguishable species of complexes, which were used
to determine the log βmlh value. The Fe3+ − L1−3H com-
ure S5). The determined log KhH value of chelators L1−3
H
is listed in Table 1. The log K1H value of chelators L1−3H,
corresponding to the frst protonation of the stronger basic
oxygen atoms of the HOPO anion moiety (Scheme 2), is in
good agreement with the reported value of log K1H =9.82 for
deferiprone [30] (Table 1). The log K2H value of chelators
plexes routinely form [FeL1−3 2−
] , which are generated at
pH 1.0. The LMCT peaks at 580 nm gradually increase
and blue-shift to about 500 nm with an increase in pH in
the range of about 1.0 − 5.5, which indicated the depro-
tonation of another free chelator and complexation with
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JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
471
Scheme 2 Protonation process
of chelators L1−3
H
Fig. 4 Incremental spectropho-
tometric titration of the complex
L1H−Fe3+ a by 0.1 M KOH,
b by 0.1 M HNO3. Conditions:
[L1H]=3 × [Fe3+]=0.2 mM,
I=0.1 M KCl, T=298 K
replaced water molecules coordinated with Fe3+ with an
increase in pH value (Scheme 3).
The determined thermodynamic parameters of
Fe3+ − L1−3H complexes and corresponding complexes of
deferiprone are listed in Table 2. Because log βmlh value is
species-dependent, a species-independent metric is needed
to compare afnities of various chelators with Fe3+. In this
regard, pM is the metric employed, where pM= −log[Mfree].
“Mfree” refers to solvated metal ions free of complexation by
chelators or hydroxides, with a high pM value corresponding
to low concentrations of free metal ions in solution. In this
study, pM value is calculated using standard conditions of
[M]=1 μM and [L1−3H]=10 μM at a typical pH of 7.4. The
pFe3+ value of the chelators L1−3H is signifcantly higher
than that of nitrogen internal modifcation of HOPO [19]
and fuorine external modifcation of HOPO [19, 20], which
are attributed to the more electronegative nitrogen atom
replacing carbon atom in HOPO moiety and the negative
inductive efect of fuorine on HOPO moiety, respectively.
In comparison to deferiprone, the pFe3+ value of chelators
L1−3H is slightly lower (Table 2) [30], which is attributed
Fig. 5 Representative speciation diagram of complex Fe3+ −L1H cal-
culated at [Fe3+]=1 μM and [L1H]=10 μM
[FeL1−3 2− continuing to generate [Fe(L1−3)2]−. Subse-
]
quently, the LMCT peaks at 500 nm further gradually
increase and blue-shift to about 460 nm with an increase
in pH in the range of about 5.5 − 7.0, which indicated the
complete deprotonation of free chelators L1−3H and com-
plexation with [Fe(L1−3)2]− to generate [Fe(L1−3)3] (Fig. 5
and Figure S8). The complexation processes are proposed
that complete deprotonation chelators L1−3H gradually
to the longer N-hydroxyalkyl substituent of chelators L1−3
H
leading to greater steric hindrance.
n
Scheme 3 Proposed compl-
exation processes of chelators
L1−3H; the charge numbers are
omitted for clarity
HO
N
n
CH3
H3C
OH
HO
N
n
O
Fe
O
N
O
O
O
O
CH3
OH
CH3
OH
H3C
n
H2O
+L-
-L-
+L-
-L-
n
O
N
OH2
OH2
O
N
O
O
O
Fe
Fe
O
OH2
H2O
H2O
CH3
N
n
OH
[FeL]2-
[FeL2]-
FeL3
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JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
Table 2 Summary of the
Chelator
log ß110
log ß120
log ß130
pFe3+ e
thermodynamic parameters of
complexes Fe3+ −L1−3H and
comparison with corresponding
complexes of deferiprone
L1Ha
14.83 (3)
14.58 (3)
14.50 (4)
9.6
10.2
/
27.18 (4)
27.10 (3)
27.07 (5)
17.2
19.3
/
37.08 (2)
36.62 (3)
36.44 (3)
23.2
26.8
/
19.36 (3)
19.35 (3)
19.20 (4)
14.8
17.2
18.5
L2Ha
L3Ha
3-hydroxy-1-methylpyridazin-4-oneb
5-hydroxy-1-methylpyridazin-4-oneb
5-fuoro-3-hydroxy-1-methyl-4(1H)-pyridonec
5-fuoro-3-hydroxy-1,2-dimethyl-4(1H)-pyridonec
deferiproned
/
/
/
18.5
20.67
15.01
27.30
37.43
aValue was determined from incremental spectrophotometric titrations at [L1−3H]=3×[Fe3+]=0.2 mM,
I=0.1 M KCl, T=298 K. bValue was obtained from Ref. [19]. cValue was obtained from Ref. [20]. dValue
e
was obtained from Ref. [30] at [deferiprone]=3×[Fe3+]=0.75 mM, I=0.1 M KCl, T=298 K. pFe3+ is
the negative logarithm of the free Fe3+ concentration in equilibrium with complexed and free chelator at a
fxed pH of 7.4 with 1 µM total Fe3+ concentration and 10 μM total chelator concentration
Calcium(II) and Zinc(II) complexation
[26], 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′′′-tetraacetic
acid (DOTA) [31], and diethylenetriaminepentaacetic acid
(DTPA) [32] are listed in Table 3. The pZn2+ value of chela-
tors L1−3H is signifcantly lower than those of the traditional
chelators DOTA and DTPA, and also similar to deferiprone
[26] and other HOPO chaletors [33–35], indicating that che-
lators L1−3H exhibited the poor afnity with Zn2+.
The afnities of chelators L1−3H with Ca2+ or Zn2+ were
also determined by incremental spectrophotometric titrations
using a 1:1 metal cation to chelator ratio.
The initial maximum UV − visible absorption peaks of
complexes Ca2+ −L1−3H at 280 nm red-shift to 310 nm and
the solution becomes cloudy with an increase in pH in the
range about 5.7−11.2, which was attributed to the depro-
tonation of the hydroxyl of HOPO (Figure S9− S11) and
the hydrolysis of Ca2+. The initial pH values of the solu-
tion of complexes Ca2+ − L1−3H compared to that of free
chelators L1−3H are almost unchanged. Meanwhile, the
curves of absorbance at 280 nm and 310 nm of free chela-
tors L1−3H and complexes Ca2+ −L1−3H as a function of pH
were almost overlapping (Figure S12), and the potentiomet-
ric titration curves of free chelators L1−3H and complexes
Ca2+ −L1−3H were also almost overlapping (Figure S4). The
results indicated that the free chelators L1−3H have been
completely deprotonated, but not coordinated with Ca2+ to
form complexes. Hence, the afnity of chelators L1−3H with
Ca2+ was negligible.
The results of the above-mentioned solution thermody-
namics demonstrated that the chelators L1−3H have almost
no chelating ability to important Ca2+ and Zn2+ in vivo, but
have strong chelating ability to Fe3+, the pFe3+ value up to
19.36, which indicated that the chelators L1−3H exhibited
selective chelating ability to Fe3+.
Table 3 Summary of the
Chelator
log ß110 pZn2+ e
thermodynamic parameters of
complexes Zn2+ −L1−3H and
comparison with corresponding
complexes of deferiprone,
DOTA, and DTPA
L1Ha
L2Ha
L3Ha
7.08 (2) 6.08 (3)
7.06 (3) 6.11 (4)
7.02 (3) 6.10 (3)
Deferiproneb 7.19
6.20
17.90
14.80
DOTAc
DTPAd
21.10
17.45
The initial maximum UV − visible absorption peaks of
complexes Zn2+ −L1−3H at 280 nm also red-shift to 310 nm
with an increase in pH in the range of about 5.2−10.2, which
was attributed to the deprotonation of the hydroxyl of HOPO
(Figure S13 − S15). The initial pH values of the solution
of complexes Zn2+ −L1−3H compared to that of free chela-
tors L1−3H, have a signifcant decrease. The results indi-
cated that the free chelators L1−3H have a certain afnity for
Zn2+. Incremental spectrophotometric titrations were used
to determine the log βmlh value of complexes Zn2+ −L1−3H,
and variations in the UV − visible absorbance at varying
pH were modeled by the formation of one distinguish-
able species between about pH 6.0 and 10.0 (Figure S16).
The determined thermodynamic parameters of complexes
Zn2+ −L1−3H and corresponding complexes of deferiprone
aValue
from
was
determined
spectro-
incremental
photometric
titrations
at
[L1−3H]=[Zn2+]=0.2
mM,
I=0.1
M KCl, T=298 K.
bValue was obtained from
Ref. [26] at I=0.1 M KNO3,
c
T=298 K. Value was obtained
from Ref. [31]. dValue was
obtained from Ref. [32]. pZn2+
e
is the negative logarithm of the
free Zn2+ concentration in equi-
librium with complexed and
free chelator at a fxed pH of 7.4
with 1 µM total Zn2+ concentra-
tion and 10 μM total chelator
concentration
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JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
473
phenolic hydroxyl leading to lower EC50 value. The results
were as expected.
Free radical scavenging activity
2,2-diphenyl-1-picrylhydrazyl (DPPH•) as stable free
radical [36] has been widely used to monitor the free radi-
cal scavenging ability of antioxidants [37–39]. The assays
were carried out in methanol, and the results are expressed
as EC50, which represents the antioxidant concentration
required to decrease the initial DPPH• concentration by
50%. A low EC50 value indicates a high radical scaveng-
ing ability. The kinetics of the DPPH• reaction indicated
that chelators L1−3H can be classifed as slow reaction
[40], and the reaction reached steady-state at least 3 h.
The EC50 value of chelators L1−3H was obtained from the
curves of the percentage of remaining DPPH• as a func-
tion of concentration n, where n is the molar ratio of anti-
oxidant to DPPH•. The EC50 value of chelators L1−3H is
between 2.00 and 2.11, which is slightly more than 1.86
of deferiprone (Figure S17 and Table S1). In polar metha-
nol, the reaction between DPPH• and phenolic hydroxyl
analogs dominated by an electron transfer mechanism. The
process of electron transfer from phenolic hydroxyl ana-
logs to DPPH• is very fast [41]. Therefore, the number
of phenolic hydroxyl analogs determines the free radical
scavenging ability [42]; the more the phenolic hydroxyl
analogs, the better the performance [43]. The chelators
L1−3H, deferiprone, and butylated hydroxyanisole [44]
possess analogue monophenolic structure, which leads
them to approximate EC50 values (Table S1). In compari-
son with chelators L1−3H, the catechol possesses more
Cytotoxicity and H2O2‑induced oxidative stress
protective efect
Cytotoxicity and H2O2-induced oxidative stress protec-
tion efect of chelators L1−3H and deferiprone were evalu-
ated by using neuron-like rat pheochromocytoma cell-line
(PC12) in vitro, which was widely used to study neuro-
degenerative diseases [45]. The PC12 cells were incu-
bated with the chelators L1−3H and deferiprone for 48 h
under various concentrations, and then the Cell Counting
Kit-8 (CCK-8) assays were performed to examine cyto-
toxicity. In comparison with a control group, the relative
viability of PC12 cells incubation with chelators L1−3
H
increased slightly at all concentrations. Meanwhile, the
relative viability of chelators L1−3H was comparable to
that of clinical deferiprone (Fig. 6). The results concluded
that the chelators L1−3H are non-toxic to PC12 cells. The
PC12 cells were incubated with 100 µM H2O2 and vari-
ous concentrations of chelators L1−3H and deferiprone
for 48 h, and then the CCK-8 assays were performed to
examine the H2O2-induced oxidative stress protection
efect. In comparison with the control group incubated
with only 100 µM H2O2, the relative viability increased
signifcantly with the addition of chelators L1−3H, which
increased with the increase of concentration of chelators
L1−3H (Fig. 7a−c). The results indicated that the chelators
Fig. 6 The relative viability of
PC12 cell incubation with a
chelator L1H, b chelator L2H, c
chelator L3H, and d deferiprone
for 48 h under various concen-
trations. Value is reported as the
mean SD of three independent
experiments
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JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
Fig. 7 The relative viability
of PC12 cells incubation with
100 µM H2O2 and a chela-
tor L1H, b chelator L2H, c
chelator L3H, d deferiprone for
48 h under various concentra-
tions. Value is reported as the
mean SD of three independent
experiments
L1−3H protected PC12 cells against H2O2-induced oxida-
tive stress, and the antioxidative efect was signifcant.
The PC12 cells’ incubation with 100 µM H2O2 and vari-
ous concentrations of deferiprone was also tested, and the
relative viability was increased to about 110 and 97% at
lower concentration 25 and 50 µM, respectively (Fig. 7d).
But as the concentration increased, the relative viability
rate quickly decreased to 38% at concentration of 200 µM
(Fig. 7d), and no protective efect on H2O2-induced oxida-
tive stress was observed, which indicated a maximum dose
threshold to the protective benefts of deferiprone.
Table 4 Parameters of iron chelators for evaluating permeability
according to Lipinski’s rule
Chelator
Molecu- H bond H bond
log P
lar weight donors
acceptors
Lipinski’s rule threshold 500
5
2
2
2
1
10
4
4
4
3
5
L1Ha
169
183
197
139
− 1.1
− 0.89
− 0.75
− 0.77
L2Ha
L3Ha
Deferipronea
aThe log P value was obtained from Ref. [46] and determined
by hand-shake method at n-octanol and water system, pH=7.4,
T=298 K
BBB permeability
Conclusions
The chelators cross the BBB via lipid-mediated free dif-
fusion in pharmacologically signifcant amounts, which is
mainly controlled by molecular weight, lipophilicity, and
net charge. These general parameters have been used by
Lipinski [12] to predict BBB permeability. If the molecu-
lar weight of the drugs is lower than 500 Da and calculated
log P is lower than 5, and the drugs possess less than 5
H-bond donors as well as less than 10 H-bond acceptors,
the drugs are probably BBB-penetrating molecules. The
four parameters of chelators L1−3H are slightly higher than
deferiprone, but in full compliance with Linpinski’s rule
(Table 4), indicated that chelators L1−3H may have good
permeability similar to deferiprone.
A series of N-hydroxyalkyl substituted deferiprone chela-
tors L1−3H, which could be used for PD chelation therapy,
have been synthesized. The results of solution thermody-
namics demonstrated that the chelators L1−3H have almost
no chelating ability to important Ca2+ and Zn2+, but have
strong chelating ability to Fe3+, the pFe3+ value as high as
19.36. It was concluded that the chelators L1−3H exhibited
selective chelating ability to Fe3+. The results of DPPH•
scavenging assays indicated that the chelators L1−3H also
exhibit similar radical scavenging ability in comparison
to deferiprone. Meanwhile, the results of CCK-8 assays
indicated that the chelators L1−3H possessed extremely low
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JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
475
cytotoxicity and excellent H2O2-induced oxidative stress
protection efect on PC12 cells. These results indicated
that the chelators L1−3H have potential application pros-
pects as chelating agents for Parkinson’s disease chelation
therapy strategy.
Spectrophotometric titrations
Spectrophotometric titrations were also carried out with
INESA ZDJ-4B automated titrator. The solution was main-
tained at 298 K by an external thermostated water bath. All
analyte solutions were prepared in a 50.0 mL volumetric
fask. The measurements were conducted with a chelator to
metal ratio of 3:1 for Fe3+ complexes and a chelator to metal
ratio of 1:1 for Ca2+ and Zn2+ complexes by careful addition
of the chelator stock solution and corresponding metal ion
stock solution. Measurements of analyte solutions with dif-
ferent pHs were made on a Thermo Scientifc Evolution 201
UV − vis spectrophotometer using a 10.0-mm quartz fow
cell. The scan range of spectra was typically 200−360 nm,
except the Fe3+ complexes were 300−800 nm. The stability
constants and spectral deconvolution were refned using the
least-squares ftting program HypSpec [27].
Experimental section
Chelators and metal cations stock solutions
Aqueous stock solutions of chelators L1−3H were freshly
prepared by direct dissolution of a weighed amount of
chelators in 0.1 M KCl aqueous solution before each set
of experiments. A Fe3+ stock solution was prepared by
dissolving a weighed amount of corresponding ferric
nitrate (Fe(NO3)3·9H2O, 99.99% metals basis) in ultrapure
water and calibration by EDTA method to obtain 0.2 M
stock solution. A Ca2+ and Zn2+ stock solution was pre-
pared by dissolving a weighed amount of correspond-
ing calcium chloride (CaCl2·2H2O, 99.9% metals basis)
and (ZnCl2·2H2O, 99.9% metals basis) in ultrapure water
to obtain 0.2 M stock solution. All solid reagents were
weighed on a Sartorius BT25S analytical balance that was
accurate to 0.01 mg. All titration solutions were prepared
using distilled water from Ulupure ULUP-IV ultra water
system and degassed by ultrasonic device.
Titration data treatment
The potentiometric titration and spectrophotometric titration
data were, respectively, analyzed by the programs Hyper-
quad [29] and HypSpec [27], utilizing nonlinear least-
squares ftting to refne stability constants and protonation
constants. The pM value and speciation diagrams were cal-
culated by the program HySS [28] with protonation con-
stants of chelators (Table 1), formation constants of metal
complexes (Tables 2 and 3), metal cation hydrolysis stability
constants, and water solubility product constant (Table S2).
Errors were determined as t*s/(n)1/2 at the 95% probability
level, where n is the number of samples, s/(n)1/2 is the stand-
ard deviation, and t* is the distribution over n−1 degree of
freedom. The errors quoted in Tables 1, 2 and 3 were used
to account for error propagations, which are the standard
deviations of the overall thermodynamic parameters given
directly by the programs Hyperquad [29] and HypSpec [27]
for the input potentiometric titration data and batch spectro-
photometric titration data, which include the experimental
points of all the titration data.
Potentiometric titrations
Potentiometric titrations were carried out with INESA
ZDJ-4B automated titrator. The solution was maintained at
298 K by an external thermostated water bath. The analyte
solution was bubbled with N2 gas in each set of experi-
ments and measurement performed in 0.1 M KCl elec-
trolyte to correct for the ionic strength. The pH electrode
(INESA, equipped with a Metrohm combination electrode
in saturated KCl) was calibrated to measure p[H] (hydro-
gen ion concentration) by standardized pH bufer (Hamil-
ton). All analyte solutions were prepared in a 50.0 mL vol-
umetric fask. The potential measurements were conducted
with solution of a chelator to metal ratio of 3:1 for Fe3+
complexes and a chelator to metal ratio of 1:1 for Ca2+ and
Zn2+ complexes by careful addition of the chelator stock
solution, corresponding metal ion stock solution and 50.0
μL standardized 0.1 M HNO3 solution. Titrations of free
chelators and metal complexes used standardized 0.1 M
KOH as the titrant (Aladdin). The potentiometric titrations
data were used in the Hyperquad refnements [29], and
titrations were repeated a minimum of three times.
DPPH• scavenging assays
An aliquot of methanol (50.0 µL) and diferent aliquots of
stock methanol solutions of 2.5 mM chelators were added
to a 2.4-mL methanol solution of 0.06 mM DPPH•, and the
volume was adjusted to a fnal value of 3.0 mL with metha-
nol. Absorbances at 517 nm were measured on a Thermo
Scientifc Evolution 201 UV−vis spectrophotometer after
the solution in a dark environment for 3 h until the reaction
reached a steady state. Five diferent concentrations were
measured for each assay, which was repeated a minimum of
three times. Then the EC50 value was plotted to obtain from
the percentage of remaining.
1 3

476
JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
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Cells and culture conditions
The PC12 cells (iCell Bioscience Inc, Shanghai) were
cultured in F-12 K medium containing 15.0 vol% heat-
inactivated horse serum (Yuanyue Bio-Technology Co.,
Ltd, Shanghai) and 5.0 vol% heat-inactivated fetal bovine
serum (Invigentech, Xi’an), under 5% CO2 at 37 °C. The
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Acknowledgments The authors would like to thank Dr. Heyan Huang
(Analytical and Testing Center, Southwest University of Science and
Technology) for nuclear paramagnetic resonance measurements.
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Author contributions QZ and RP conceived and designed the experi-
ments; QZ, SF and YZ performed the experiments; QZ, and BJ ana-
lyzed the data; QZ and RP wrote and edited the manuscript.
Funding The research was funded by the National Natural Science
Foundation of China (grant number 51972278), the Project of State
Key Laboratory of Environment-friendly Energy Materials, Southwest
University of Science and Technology (grant number 19fksy04), and
the Natural Science Foundation of Southwest University of Science
and Technology (grant number 18zx7136).
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Declarations
Conflict of interest The authors declare that they have no confict of
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JBIC Journal of Biological Inorganic Chemistry (2021) 26:467–478
Authors and Afliations
Qingchun Zhang1 · Shufan Feng1 · Yulian Zhao2 · Bo Jin1 · Rufang Peng1
2
* Qingchun Zhang
School of Life Science and Engineering, Southwest
zhangqingchun@swust.edu.cn
University of Science and Technology, Mianyang 621010,
China
* Rufang Peng
pengrufang@swust.edu.cn
1
State Key Laboratory of Environment-Friendly Energy
Materials, School of Materials Science and Engineering,
Southwest University of Science and Technology,
Mianyang 621010, China
1 3