Modifying aroylhydrazone prochelators for hydrolytic stability and improved cytoprotection against oxidative stress

Article abstract of DOI:10.1016/j.bmc.2018.11.004

BSIH ((E)-N′-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isonicotinohydrazide) is a prodrug version of the metal chelator SIH ((E)-N′-(2-hydroxybenzylidene)isonicotinohydrazide) in which a boronate group prevents metal chelation until reac

Full text of DOI:10.1016/j.bmc.2018.11.004

Accepted Manuscript
Modifying aroylhydrazone prochelators for hydrolytic stability and improved
cytoprotection against oxidative stress
Qin Wang, Katherine J. Franz
PII:
S0968-0896(18)31520-7
https://doi.org/10.1016/j.bmc.2018.11.004
BMC 14608
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
29 August 2018
26 October 2018
4 November 2018
Please cite this article as: Wang, Q., Franz, K.J., Modifying aroylhydrazone prochelators for hydrolytic stability and
improved cytoprotection against oxidative stress, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/
10.1016/j.bmc.2018.11.004
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Graphical Abstract
Modifying aroylhydrazone prochelators for
hydrolytic stability and improved
Leave this area blank for abstract info.
cytoprotection against oxidative stress
Qin Wang and Katherine J. Franz*
Duke University, Department of Chemistry, 124 Science Dr., Durham, NC 27708, USA

Bioorganic & Medicinal Chemistry
Modifying aroylhydrazone prochelators for hydrolytic stability and improved
cytoprotection against oxidative stress
Qin Wang and Katherine J. Franz*
Duke University, Department of Chemistry, 124 Science Dr., Durham, NC 27708, USA
*katherine.franz@duke.edu
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
BSIH ((E)-N'-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzylidene)isonicotinohydrazide) is a prodrug version of the metal chelator SIH ((E)-N'-(2-
hydroxybenzylidene)isonicotinohydrazide) in which a boronate group prevents metal chelation
until reaction with hydrogen peroxide releases SIH, which is then available for sequestering
iron(III) and inhibiting iron-catalyzed oxidative damage. While BSIH has shown promise for
conditionally targeting iron sequestration in cells under oxidative stress, the yield of SIH is
limited by the fact that BSIH exists in cell culture media as an equilibrium mixture with its
hydrolysis products isoniazid and 2-formylphenyl boronic acid. In the current study, several
BSIH analogs were evaluated for their hydrolytic stability, reaction outcomes with H2O2, and
prochelator-to-chelator conversion efficiency. Notably, the para-methoxy derivative (p-
OMe)BSIH ((E)-N'-(5-methoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzylidene)isonicotinohydrazide) and the meta-, para- double substituted (MD)BSIH ((E)-
N'-((6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d][1,3]dioxol-5-
Available online
Keywords:
oxidative stress
iron
Fenton chemistry
chelating agent
cytoprotection
yl)methylene)isonicotinohydrazide) showed 1.3- and 1.9-fold improved hydrolytic stability
compared to BSIH, respectively, leading to a 22 and 50 % increase in chelator released.
Moreover, both prochelators were found to protect retinal pigment epithelial cells stressed with
either H2O2 or paraquat insult.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
boronate mask by hydrogen peroxide thus conscripts iron
sequestration specifically under oxidative conditions in which
Iron plays essential roles in the proper functioning of the
body, but excessive or otherwise misregulated iron is also
implicated in pathological progression of several diseases.1-3
Chelation therapy with high-affinity iron chelating agents can be
part of treatment plans for patients with systemic iron overload,
for example thalassemia-major patients who require frequent
blood transfusions.4 There are a number of diseases, however,
that present signs of iron-promoted oxidative injury but do not
involve systemic iron overload, for example neurodegenerative
and cardiovascular disorders.5-9 Several types of cancers also
exhibit significantly perturbed aspects of iron uptake, utilization
and regulation associated with cancer cell proliferation.10-12 For
these cases, conditional chelation strategies that direct iron
chelators preferentially to sites of localized need without
adversely perturbing normal metal homeostasis are desirable.11-14
Our lab has pursued a prodrug strategy for chelating agents in
which the resulting “prochelator” has negligible metal binding
affinity until a specific stimulus produces a chelation site.14
Installation of a boronate masking group in place of a phenol of a
multidentate chelating agent creates prochelators that are
responsive to hydrogen peroxide, a reactive oxygen species that
contributes to oxidative stress.15-21 The conditional removal of the
silencing redox-active iron can minimize further cell damage.
Our first prochelator BSIH ((E)-N'-(2-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzylidene)isonicotinohydrazide)
contains an aryl boronic ester in place of the phenol of the well-
studied chelator salicylaldehyde isonicotinoyl hydrazone (SIH).
The key steps involved in BSIH activation are shown in Scheme
1. BSIH is synthesized as the pinacol boronic ester, which
equilibrates in water to give the boronic acid version, BASIH
(step 1). Both BSIH and BASIH react with H2O2 in the second
Scheme 1. Key steps involved in BSIH hydrolytic degradation,
peroxide activation, and Fe3+ chelation.

step to produce SIH, which is then available in step 3 to bind
available metal ions, in the case of Fe3+ it forms a 2:1
ligand:metal complex.15 BSIH has been shown to minimize
hydroxyl radical production in vitro by inhibiting iron-catalyzed
Fenton chemistry,22 protect cells in culture from damage induced
by hydrogen peroxide insult,23 and be well tolerated by cells that
are otherwise compromised by long-term exposure to
conventional iron chelators.24 Despite these favorable properties,
the yield of SIH released from BSIH in cellular contexts is
significantly lower than predicted, requiring higher
concentrations of BSIH to protect cells against peroxide stress
compared to SIH itself.25
The low cellular BSIH-to-SIH conversion originates from the
inherent equilibrium that exists in aqueous solutions between
BSIH and its hydrolysis products isoniazid and (2-
formylphenyl)boronic acid (FBA) (Scheme 1, step 4), which
consequently reduces the amount of active SIH chelator released
upon exposure to H2O2.26 In spite of this equilibrium, BSIH itself
maintains its integrity in media and plasma, with little
degradation seen over 10 h incubation in plasma.27 SIH, on the
other hand, remains intact in buffer at pH 7.4 but degrades within
hours in cell culture media and plasma due to amino acid-
catalyzed hydrolysis reactions (Scheme 1 step 5; further
reactions likely).27,28
In an effort to minimize undesired hydrolysis, we previously
developed a later-generation prochelator based on the SIH
analogue,
HAPI
((E)-N'-(1-(2-
hydroxyphenyl)ethylidene)isonicotinohydrazide),29
which
demonstrates increased resistance to hydrolysis in plasma by
replacing the salicylaldehyde component of SIH with the
respective methyl ketone.18 Prochelator BHAPI harbors a self-
immolative linker that undergoes 1,6-benzyl elimination upon
H2O2 activation to release HAPI. While BHAPI demonstrates
superior prochelator-to-chelator conversion rates and was shown
to protect cardiomyoblast cells against oxidative injury induced
by catecholamines, high-dose or long-time incubation of either
BHAPI or HAPI was found to elicit cytotoxicity.24,30 In fact, our
original design, BSIH, was shown to have the most favorable
therapeutic ratio of low inherent toxicity to cytoprotection against
oxidative damage in comparison with several Fe chelators and
prochelators.24 As a result of the more favorable non-toxic yet
cytoprotective outcomes for BSIH compared to BHAPI, we
chose to optimize BSIH-like aroylhydrazones for improved
activity.
Chart 1. Synthetic scheme and structures of prochelators used in
this study. Structures of the corresponding chelators replace the
boronic ester or boronic acid with an OH functional group; names of
chelators mirror those of the prochelators, just without the “B”.
2. Results
2.1. Design and Synthesis
BSIH-like derivatives are readily synthesized via Schiff base
condensation reactions between hydrazide and borylated aryl
aldehyde building blocks in acidic conditions, as shown in Chart
1. This straightforward procedure provides access to a wide range
of structural variation. The compounds for the current study were
selected based on knowledge gained from prior structure-function
studies. As we previously reported, replacing the pyridine ring of
the hydrazide building block with an aryl ring diminished Fe3+
binding affinity of the resulting chelator, which in turn lowered
its efficacy to inhibit iron-promoted hydroxyl radical formation.15
The current compounds therefore retain a pyridyl ring to maintain
high Fe3+ affinity, a consequence of electronic effects, as the
pyridyl N is not directly part of the metal coordination sphere.
Substitutions on the aryl aldehyde building block, on the other
hand, had little effect on the Fe3+ affinity, but did impact the rate
of H2O2-dependent conversion from prochelator to chelator, with
electron-donating substituents favoring faster reactions than
electron-withdrawing substituents.15
The insights of BSIH hydrolysis shown in Scheme 1 provide a
foundation for tuning hydrolytic stability and cytoprotective
efficacy of BSIH analogs. In principle, new prochelators with
improved stability can be created by tweaking the
aroylhydrazone structure to shift the hydrolysis constant K
(Scheme 1 step 4) to favor the intact prochelator, which in turn
would increase the efficiency of prochelator-to-chelator
conversion upon peroxide exposure and the efficacy of
cytoprotection against oxidative stress. The ultimate goal of this
study was to identify aroylhydrazone prochelators that maintain
the favorably low inherent cytotoxicity of BSIH, yet show
improved hydrolytic stability in aqueous solutions so as to
maximize the amount of active chelator available for
cytoprotection upon cellular oxidative stress.
In the current study, several boronate-masked aroylhydrazone
prochelators were evaluated for their peroxide-activated
prochelator-to-chelator conversion efficiency, hydrolytic
stability, and cytoprotective efficacy in oxidatively stressed
retinal pigment epithelial cells.
Based on these prior observations, which were done in
methanol or water-methanol mixtures, we selected the
aroylhydrazone prochelator derivatives shown in Chart 1 for
further study in aqueous solution and to test their efficacy at
minimizing oxidative stress in cell culture. Compared to the

original BSIH structure, (p-OMe)BSIH and (m-OMe)BSNH
contain an electron-donating methoxy substituent installed at
either the para- or meta position to the boronic ester,
respectively. Nicotinic hydrazide was used for (m-OMe)BSNH
in place of the isoniazid building block for the other compounds
because the chelator version (m-OMe)SIH was not soluble above
50 M in aqueous buffers, whereas (m-OMe)BSNH and (m-
OMe)SNH can be prepared at concentrations as high as 300 M.
The methylenedioxy derivative (MD)BSIH was also prepared in
order to evaluate the effect of double substitution at both para-
and meta- positions.
Inspired by the precedence of hydrolytically stable chelators
and prochelators based on thiosemicarbazone structures,31,32
BSMTH and BSPTH embed thiosemicarbazide components to
incorporate a sulfur donor into the metal coordination sphere.
Finally, the control compound (m-OMe)BIIH repositions the
boronic ester to test the hypothesis that its placement contributes
to the hydrolytic susceptibility of the hydrazone framework and
so that reaction with H2O2 will produce a phenol that is not
positioned to provide a metal chelation site.
Figure 1. Calcein fluorescence assay with chelators and H2O2-
activated prochelators. Buffered solutions of prochelators were pre-
incubated overnight with excess H2O2 then diluted to 4 μM and
equilibrated with 2 μM calcein-Fe3+ for 2 h prior to measurement of
calcein fluorescence emission. Chelation efficiency was expressed as a
percentage of the calcein emission of the free calcein control. The grey
dashed line indicates the residual fluorescence response of 2 μM calcein
quenched by 1 equiv of Fe3+. The red dashed line indicates the ~50%
calcein emission restored by the reaction mixture of BSIH and H2O2. Data
are presented as mean ± SD, n = 3; statistical significance (t-test), * p <
0.05, compared to the BSIH group. # p < 0.05, compared to the SIH
group.
Based on the previously reported crystal structure of BSIH,
the prochelators prepared here are predicted to prefer the E
configuration about the C=N double bond, with the boronic ester
positioned anti to the imine N.22 As was the case with BSIH,
however, additional small peaks in the 1H NMR spectra of clean
samples reveal the presence of other isomers (Supp Info). The
13C NMR spectra for these prochelators appear to be missing a
peak, an observation consistent with the known line-broadening
effects of 11B on the aromatic carbon to which it is bonded.33 An
additional subtlety of these compounds is the potential for
boronic acids at the ortho position of aroylhydrazones to
dehydrate and form cyclic diazaborine monomers and anhydrated
dimers.34 While the prochelators reported here were prepared as
pinacol esters to minimize this transformation, the pinacol diol is
labile and can be lost under high vacuum or upon hydrolysis. In
all cases, the 1H NMR spectra obtained in DMSO-d6 confirm that
the compounds as prepared exist in solution as the open
aroylhydrazone form, as evidenced by the presence of a 12-H
singlet at 1.3 ppm corresponding to a pinacol coordinated to
boron, and a D2O-exchangeable NH peak ~12 ppm (Supp Info).
Given the propensity of these compounds to transform and
interconvert by the addition or removal of H2O, it is not
surprising that some of the diazaborine forms can be observed
during chromatographic analysis (see Supp Info).
out-competes calcein for Fe3+ chelation, as predicted based on
pFe values of 23 and higher for this ligand class.15,37,38
All borylated compounds in Chart 1 contain boronate masks
blocking potential metal chelation sites such that they
demonstrate negligible affinity for Fe3+ with calcein-Fe emission
remaining at the basal level (Figure S1). In contrast, reaction of
the prochelators with H2O2 should in principle convert them into
active chelators capable of chelating Fe3+ and restoring calcein
fluorescence. As indicated by the red dashed line in Figure 1,
BSIH reacted with H2O2 to incur a ~50% recovery in calcein
fluorescence. This result reinforces the conclusion that
prochelator BSIH in aqueous solution is only partially converted
to its corresponding chelator SIH for concomitant Fe3+
chelation.25-27 Among the new prochelators, (p-OMe)BSIH and
(MD)BSIH restored calcein emission to ~60% and 75% chelation
efficiency, respectively, suggesting enhanced content of active
chelator. Peroxide activation of (m-Cl)BASIH, BSPTH, and (m-
OMe)BSNH, however, resulted in approximately the same or
lower levels of calcein fluorescence compared to that of BSIH.
Incubation of the control compound, (m-OMe)BIIH with H2O2
showed no change in fluorescence compared to the calcein-Fe3+
solution, confirming that the reaction products fail to extract Fe3+,
as anticipated because the resulting phenol product is ill-
positioned to create a multidentate metal binding pocket. The
calcein emission of the methyl thiosemicarbazide-based
prochelator BSMTH after reaction with H2O2 also remained at
the basal level of the calcein-Fe3+.
2.2. Conditional Iron Chelation
A prerequisite for a prochelator is the correct transformation
into a form that binds its target metal ion under a specific
triggering condition. Here, a fluorometric competitive binding
assay was utilized to assess the efficiency of prochelator-to-
chelator conversions of the BSIH derivatives triggered by the
presence of H2O2. Calcein is a fluorescent probe with a multi-
carboxylate metal-binding site that binds Fe(III) with an affinity
and 1:1 stoichiometry that corresponds to a pFe of 20.3.35 The
term pFe (−log[Feaq3+]) is a quantitative expression that facilitates
direct comparison of chelating agents of different binding
stoichiometries by calculating the amount of uncomplexed iron
under defined conditions, typically pH 7.4 with total ligand and
iron concentrations of 10 µM and 1 µM, respectively.36 As shown
in Figure 1, the fluorescence emission of calcein was quenched
by ~70% upon complexation with 1 equivalent of Fe3+ (basal
level, gray dashed line). Addition of 2 equivalents of chelator
SIH to the calcein-Fe3+ solution resulted in an almost full
recovery of the original fluorescence, indicating that SIH readily
The conclusions from the calcein assay with peroxide-reacted
prochelators were reinforced by results of the calcein assay with
their corresponding chelator counterparts. As shown in Figure 1,
addition of SIH, (p-OMe)SIH and (m-OMe)SNH lead to an
increase in calcein fluorescence by more than 90%, indicating
that these chelators readily compete with calcein for Fe3+ binding.
Meanwhile, the S-containing chelators SMTH and SPTH failed
to fully restore calcein emission; indeed, SMTH had no effect on
the quenched signal of calcein-Fe3+ solution. The consistent
results of weak Fe3+ affinity for both thiolated chelators and
activated prochelators imply that replacement of the
aroylhydrazone chelation pocket with thiosemicarbazone scaffold
significantly reduces the binding affinity for Fe3+.

2.3. Hydrolytic Stability
prepared at 200 μM. Integration indicates a composition of 80%
intact (MD)BSIH vs. 20% hydrolysis fragments in the
equilibrated solution. These numbers are not directly comparable
with BSIH though, due to the different initial concentrations of
the prochelators.
Recognizing (p-OMe)BSIH and (MD)BSIH as the most
promising derivatives with respect to prochelator-to-chelator
conversion, these compounds were selected for further
assessment of hydrolytic stability in aqueous solutions. The
control compound (m-OMe)BIIH was included, based on our
hypothesis that the position of the boronic functionality might
influence the hydrolytic stability of the hydrazone bond.
Unlike the observations for the BSIH-based prochelators, the
hydrazone proton signal of the control compound (m-OMe)BIIH
detected in DMSO-d6 (δc = 8.38, Figure 3a) persists upon
dilution in D2O (δc = 8.17, Figure 3b). Meanwhile, no additional
peaks corresponding to potential hydrolysis products were
observed, substantiating the superior hydrolytic resistance of (m-
OMe)BIIH.
The hydrolysis equilibria of (p-OMe)BSIH and (MD)BSIH in
PBS, pH 7.4 were further monitored by UV-Vis
spectrophotometry. The spectra in Figure 4a and c show the
time-dependent changes of intact prochelators (p-OMe)BSIH and
(MD)BSIH upon dilution in PBS from corresponding stock
solutions prepared in DMSO. Similar alterations of spectral
features were also observed in the reaction mixtures of the
respective hydrolysis fragments (Figure 4 b and d). These results
reinforce the conclusion that these BSIH analogs also undergo
hydrolysis equilibria in aqueous solutions with their
corresponding hydrazide and aldehyde degradation products.
Figure 2. Hydrolysis of (p-OMe)BSIH observed by 1H NMR
spectroscopy. The aromatic regions of 1H NMR spectra of (a) (p-
OMe)BSIH (10 mM in DMSO-d6) (b) (p-OMe)BSIH (500 μM in D2O,
with 5% DMSO-d6 after 1-h equilibration) and (c) INH (500 μM in D2O,
with 5% DMSO-d6). The arrows indicate the peaks corresponding to the
aromatic protons of isoniazid (red).
According to our previous observations for BSIH hydrolysis,
intact aroylhydrazone prochelators are anticipated to establish
equilibria in aqueous solutions with corresponding degradation
fragments: hydrazides and boronate-masked salicylaldehydes. To
quantify the equilibrium ratios of intact prochelator vs.
degradation components, aqueous samples of these selected
prochelators were analyzed by NMR. Figure 2a shows the
(c)
(d)
2
1.5
1
2
INH
1 min
3 min
5 min
30 min
(MD)FBA
1
INH + (MD)FBA 1 min
INH + (MD)FBA 3 min
INH + (MD)FBA 5 min
INH + (MD)FBA 30 min
1.5
1
aromatic region of the H NMR spectra of (p-OMe)BSIH in
DMSO-d6, with the hydrazone proton from the intact prochelator
clearly visible at 9.01 ppm (peak c). Upon dilution and
equilibration in D2O for 1 h, however, the hydrazone peak
disappeared, with concomitant appearance of two extra peaks
corresponding to the aromatic protons of isoniazid (indicated by
red arrows), Figure 2b. Integration of peak a and a’ in Figure 2b
gave 85% of intact (p-OMe)BSIH vs. 15% of INH at equilibrium
in a 500-μM sample of (p-OMe)BSIH. Comparable solutions of
BSIH resulted in 80% intact chelator.26 A similar set of spectra
for (MD)BSIH are shown in Supp Info Figure S2. Because of the
lower aqueous solubility of (MD)BSIH, these samples were
0.5
0
0.5
0
250
300
350
400
450
250
300
350
400
450
Wavelength (nm)
Wavelength (nm)
Figure 4. The hydrolysis equilibria of 100 μM (a) (p-OMe)BSIH and
(c) (MD)BSIH observed by UV-Vis spectrophotometry; spectra of
reaction mixtures of 100 μM INH with100 μM of (b) (p-OMe)FBA or (d)
(MD)FBA, pH 7.4 over the course of 30 min.
The hydrolysis equilibrium constants for these two
prochelators were calculated from the spectra of equilibrated
reaction mixtures at various concentrations, which gave K values
of (1.1  0.1) × 10-5 M for (p-OMe)BSIH and (8.0  0.6) × 10-6
M for (MD)BSIH. These numbers are smaller compared to that
of BSIH [(1.5  0.1) × 10-5 M], indicating that the hydrolysis
reactions of (p-OMe)BSIH and (MD)BSIH slightly favor the
intact prochelators.
In contrast, the spectrum of the (m-OMe)BIIH control
compound remains unchanged over time in PBS buffer (Figure
S3). This observation in combination with the signal integrity in
1H NMR speciation represents a unique feature of (m-OMe)BIIH
that distinguishes it from other BSIH derivatives and implies that
installation of the boronic ester mask at a different position
prevents the hydrolysis equilibrium in aqueous buffered solutions
Figure 3. Hydrolytic stability of (m-OMe)BIIH analyzed by 1H NMR
spectroscopy. The aromatic regions of 1H NMR spectra of (a) (m-
OMe)BIIH (10 mM in DMSO-d6) and (b) (m-OMe)BIIH (500 μM in
D2O, with 5% DMSO-d6).

5d shows the spectrum of the reaction mixture of (MD)BSIH
(200 μM) with H2O2 in D2O, with byproduct INH indicated in red
arrows. Integration of a” and a’ gives ~45% formation of intact
chelator under this condition. Since the prochelator solution of
(MD)BSIH was prepared at a lower concentration, the content of
chelator formation was lower in comparison with BSIH. The
reduced equilibrium constant K obtained from data in Figure 4
and the highest prochelator-to-chelator efficiency shown in
Figure 1, however, both identify (MD)BSIH as the most
promising analog regarding improved hydrolytic stability and
maximal chelator release.
2.4. Cytotoxicity and Cytoprotection against Oxidative Stress
Having shown that selected BSIH derivatives (p-OMe)BSIH
and (MD)BSIH fulfill desirable prochelator attributes, we further
explored the structure-activity relationship of these
aroylhydrazone derivatives and investigated their ability to
protect cells subjected to oxidative damage. The cell culture
experiments were conducted in human retinal pigment epithelial
ARPE-19 cells as they suffer from iron-induced oxidative stress
during age-related macular degeneration.39,40
Figure 5. The reactions of prochelators (p-OMe)BSIH and
(MD)BSIH with H2O2 observed by 1H NMR. The aromatic regions of 1H
NMR spectra of (a) the reaction mixture of (p-OMe)BSIH (500 μM in
D2O, with 5% DMSO-d6) with H2O2 (25 mM) for 12 h (b) (p-OMe)SIH
(500 μM in D2O, with 5% DMSO-d6) (c) INH (500 μM in D2O, with 5%
DMSO-d6) (d) the reaction mixture of (MD)BSIH (200 μM in D2O, with
2% DMSO-d6) with H2O2 (10 mM) for 12 h. See main text for description
of arrows and peak labels.
Initial cytotoxicity studies revealed that all the new BSIH-
based prochelators were non-toxic at concentrations up to 100
μM for 24-h incubation (data not shown). BSIH and (MD)BSIH
remained non-toxic after 72 h incubation, however, cells were
less tolerant of elevated doses of (p-OMe)BSIH when incubation
was extended over 72 h, with a 20% reduction of cell viability at
the concentration of 100 μM (Figure 6).
observed in BSIH-like prochelators. However, this modification
also disables the metal-binding capacity of the activation product,
as the critical phenol functionality after deboronation is
inappropriately positioned for metal chelation.
Given that (p-OMe)BSIH and (MD)BSIH exhibited improved
hydrolytic stability, we speculated that the higher content of
intact prochelators in aqueous solutions would allow for
enhanced release of corresponding active chelators upon peroxide
The cytotoxicity of our self-immolative prochelator BHAPI
was also assessed for comparison, as it was previously found to
elicit toxicity in H9c2 cardiomyoblasts, with a TC50 value of 37.1
± 7.7 μM after 72 h.24 A relatively moderate toxic effect was
observed in ARPE-19 cells exposed to BHAPI, with a 25%
viability reduction at the highest concentration tested (100 μM).
1
activation. Figure 5a shows the H NMR spectra of a 500-μM
sample of (p-OMe)BSIH in D2O after reaction with excess H2O2.
The intact chelator (p-OMe)SIH as well as the activation
byproducts INH (indicated in red arrows) and (p-
OMe)salicylaldehyde (indicated in green arrows) were clearly
identified. Integration of the aromatic peak a derived from (p-
OMe)SIH and peak a’ from INH indicates ~67% of intact
chelator vs. ~33% of byproduct formation, which is significantly
improved compared to the 51% of SIH generated from peroxide
activation of BSIH at the same concentration.26 Likewise, Figure
Figure 7 shows the efficacy of the new aroylhydrazone
prochelators to protect cells from peroxide damage. To avoid
direct reaction of prochelators with H2O2 in the treatment
medium, cells were first preincubated with the test compound
prior to exposure to a toxic 200-μM bolus of H2O2. After 19 h,
cells were found to be significantly protected from death by most
of the prochelators tested, with the exceptions of (m-OMe)BIIH
and BSMTH. The two prochelators with improved hydrolytic
stability, (p-OMe)BSIH and (MD)BSIH, demonstrated the most
potent cytoprotective activity, being effective at 25 μM. Similar
dose-dependent protection was also observed in peroxide-
stressed cells exposed to BHAPI.18 Meanwhile, the thiolated
prochelator BSPTH demonstrated comparable protective potency
to BSIH, being effective at concentrations between 25 and 50
μM. In contrast, the lack of cytoprotection observed in the cases
of BSMTH and the (m-OMe)BIIH control is likely due to the
non-chelating property of their corresponding activation
products.
To further explore the role of iron chelation in the mode of
protection for aroylhydrazone prochelators, we also tested the
cytoprotective effects of corresponding chelators against
oxidative stress. As shown in Figure S4, the thiosemicarbazone
SMTH that lacks affinity for Fe3+ had no protective effect on
H2O2-stressed ARPE-19 cells, while other chelators with
adequate metal binding affinity protected cells from the oxidative
damage induced by H2O2, being effective at 5 μM.
Figure 6. Inherent cytotoxicity of selected aroylhydrazone
prochelators after 72-h incubation in ARPE-19 retinal pigment epithelial
cells. Error bars represent standard deviations from triplicate runs (n = 3).
In addition to stressing cells with exogenous H2O2 added in
bolus form, cells were also subjected to paraquat, a viologen

Figure 7. Cytoprotective effects of aroylhydrazone prochelators from
oxidative damage induced by H2O2 in ARPE-19 cells. ARPE-19 cells
were pre-incubated with various concentrations of aroylhydrazone
prochelators for 5 h prior to exposure to H2O2. Cell viability was
measured 19 h after peroxide treatment and expressed as percentage of
the untreated control group (100%). Error bars represent standard
deviations from triplicate runs (n = 3).
Figure 8. Cytoprotective effects of aroylhydrazone prochelators
against oxidative damage induced by 10 mM paraquat in ARPE-19
cells. Cell viability was measured after 48 h and expressed as
percentage of the untreated control group (100%). Error bars represent
standard deviations from triplicate runs (n = 3).
3. Discussion
herbicide that is a known environmental risk factor for
Parkinson’s disease. Upon reduction in mitochondria, paraquat
elicits overproduction of superoxide with concomitant release of
H2O2 and liberation of Fe from Fe-S clusters.41,42 The
cytoprotective effect of aroylhydrazone prochelators was tested
in ARPE-19 cells stressed with 10 mM paraquat for 48 h, the
timepoint at which cell death by paraquat exposure was observed
under these conditions. BHAPI was previously shown to be
protective from paraquat-induced cell death, with a 40% viability
restoration at 100 μM.18 Figure 8 shows that the cytoprotection
of (MD)BSIH tracked very closely with BHAPI to give a dose-
dependent recovery of cell viability. (p-OMe)BSIH also
displayed adequate protective effects on paraquat-stressed cells,
with 100 μM prochelators capable of restoring viability to ~30%
of the untreated cells. BSIH and other prochelators, however,
failed to protect cells from paraquat damage. The observation
that the most stable prochelators are the only ones that protect
cells against paraquat exposure highlights the importance of
prolonged prochelator stability for maximal cellular protection.
Structural modification to the hydrazide and aryl aldehyde
building blocks of the parent BSIH compound tunes its chemical
properties and biological activity. Changing the electronic nature
of the aldehyde-containing ring has substantive effects on the
hydrolytic stability of the aroylhydrazone prochelators. The
electron-donating p-OMe derivative is more stable with a higher
content of intact prochelator present in aqueous solutions
compared to the parent compound. The m- and p- double
substitution with electron-donating methylenedioxy functionality
affords prochelator (MD)BSIH with even greater hydrolytic
stability and a hydrolysis equilibrium constant K ~2-fold lower
than the value of parent BSIH. We reason that the electron-
donating functionalities installed on the aldehyde-containing ring
increase the electron density of the hydrazone carbon, reducing
the susceptibility of C=N to the nucleophilic attack by water.
Consistent with this reasoning, the prochelator with m-Cl
substitution that is electron withdrawing with respect to the
hydrazone carbon was less effective in the peroxide-triggered
calcein assay, regardless of its chelator’s similar Fe affinity.15
This result suggests the electron withdrawing substituent
aggravates hydrazone hydrolysis.
To determine whether the lack of cytoprotection by BSIH,
BSMTH and BSPTH was due to insufficient prochelator-to-
chelator conversion or inability of the corresponding chelator to
protect cells against paraquat damage, the cytoprotective effects
of corresponding chelators were assessed in paraquat-stressed
ARPE-19 cells. As shown in Figure S5, SIH, (p-OMe)SIH,
HAPI and SPTH all demonstrated ~40% cytoprotection against
paraquat-induced cell death, whereas the non-chelating SMTH
failed to protect cells. Combined, these results substantiate the
importance of iron chelation in the protective activity against this
model of oxidative stress.
Additionally, modifications on the aldehyde-containing ring
were previously found to significantly influence the kinetics of
prochelator-to-chelator conversion activated by H2O2.15 In
moving from the electron-withdrawing m-Cl to the electron-
donating p-OMe compound, a nearly 5-fold increase in the H2O2-
dependent conversion rates was observed. The accelerated
kinetics of peroxide activation was further shown to associate
with increased inhibition of metal-promoted ROS formation in
vitro.15 The current study shows that improved hydrolytic
stability is another benefit of the electron-donating derivatives (p-
OMe)BSIH and (MD)BSIH, which ultimately leads to higher
yields of active chelator upon peroxide activation and enhanced
cytoprotective efficacy against cellular oxidative damage.

The exception to these structure-activity relationships is the
control compounds, (m-OMe)BIIH. Moving the boronic ester
functionality to the meta-position of the aldehyde/hydrazone
group yields a hydrolytically stable aroylhydrazone framework.
Despite the desired structural integrity of this compound,
selective deborylation initiated by H2O2 generates a product with
a hobbled metal chelation site. Therefore, we surmise that the
aqueous susceptibility of other BSIH-derivatized prochelators
likely derives from the electron-deficient boronic ester
functionality installed at the ortho-position to the hydrazone
bond. While this ortho-borylation is necessary for peroxide-
triggered metal binding, the electron-withdrawing effect of boron
reduces the electron density of the adjacent hydrazone carbon
sufficiently to make it susceptible to nucleophilic attack by water.
and used without further purification; (5-chloro-2-
formylphenyl)boronic acid and (2-formy-4-methoxy-
lphenyl)boronic acid were purchased from Matrix Scientific;
(4,5-dimethoxymethylene-2-formylphenyl)boronic acid and (2-
formyl-5-methoxyphenyl)boronic acid were purchased from
Frontier Scientific. Other reagents were purchased from Sigma-
Aldrich. All solvents were reagent grade and all aqueous
solutions were prepared from nanopure water. Compound stock
solutions (10 mM) were prepared in DMSO and stored at -20 °C.
1H NMR and 13C NMR spectroscopy was performed on a Varian
Inova 400 or 500 MHz spectrometer. High-resolution mass
spectra (HRMS) were recorded on an Agilent G6224 LCMS-
TOF system. Purity of all final compounds was determined by
high performance liquid chromatography (HPLC) to be 95% or
higher. The instrument was an Agilent 1100 Series LCMS
apparatus with a Supelco Ascentis C18 (50  1.0 mm) column.
Replacing the aroyl oxygen donor with a sulfur donor to
create an O,N,S coordination sphere was unfavorable in our
system, as it significantly decreased the Fe(III) affinity of the
SPTH and SMTH chelators compared to SIH, as evidenced by
the lack of competition with calcein under the conditions tested.
While BSPTH retained modest cellular protection against
peroxide exposure, BSMTH was ineffective. This difference in
activity likely arises from electronic differences between the
phenyl vs. methyl substituents of BSPTH vs BSMTH, something
that has been observed in disulfide-bridged prochelators built on
a similar scaffold.31,43 Interestingly, the disulfide prochelators
introduced by Tomat and coworkers are reductively-activated and
create S,N,S or S,N,O iron chelators with interesting
antiproliferative activity against cancer cells.12,43 The boronate
prochelators discussed here, on the other hand, are oxidatively
activated and create O,N,O coordination spheres. The
combination of these two approaches reveals the opportunity
available to tune prochelators for desirable biological
applications by adjusting their coordination sphere, activation
mechanism and stability.
UV-Vis spectra were recorded on
a Cary 50 UV-Vis
spectrophotometer. Absorbance or fluorescence readings were
measured using clear, flat-bottom 96-well plates and a Perkin
Elmer Victor 3V multi-label counter maintained at 25 °C.
5.2. Synthesis
5.2.1. (p-OMe)BSIH
(E)-N'-(5-methoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzylidene)isonicotinohydrazide)
A portion of 4-methoxy-2-formylphenylboronic acid (1mmol,
0.180 g) was added to a nearly saturated solution of isoniazid (1
mmol, 0.137 g) in 0.1 M sodium acetate buffer, pH 4.5. The
reaction mixture was stirred over an oil bath at 100 °C for 4 min.
The white insoluble product was collected via vacuum filtration,
washed with diethyl ether, and dried in vacuo to give an off-
white
isonicotinoylhydrazineylidene)methyl)-4-methoxyphenyl)boronic
acid ((p-OMe)BASIH) in 88% yield. Portions of (p-
powder
corresponding
to
(E)-(2-((2-
4. Conclusions
OMe)BASIH (0.5 mmol, 0.150 g) and pinacol (0.5 mmol, 0.059
g) were then dissolved in DMF and added to a round bottom
flask. The reaction stirred for 21 h at rt then dried via rotary
The structure-activity relationships of the new aroylhydrazone
derivatives studied here reveal that the potency of cytoprotection
against oxidative stress correlates with prochelator-to-chelator
conversion efficiency as well as the Fe-chelating capacity of the
resulting chelators. In particular, the electron-donating
substituted prochelators (p-OMe)BSIH and (MD)BSIH with
improved hydrolytic stability provided the best cytoprotection to
cells stressed with either H2O2 or paraquat. The overall trends in
cytoprotection observed for this set of aroylhydrazone
compounds suggest that the major source of their protective
effects derives from preferential Fe chelation in response to
oxidative stress. These advances are important steps in
developing this promising class of conditionally active iron
chelators to potentially mitigate iron-associated oxidative damage
in vivo.
1
evaporation to give an off-white powder in 82% yield. H NMR
(DMSO-d6, 500 MHz) δ (ppm): 12.24 (s, 1H), 9.01 (s, 1H), 8.79
(d, J = 5.6 Hz, 2H), 7.82 (d, J = 5.6 Hz, 2H), 7.71 (d, J = 8.4 Hz,
1H), 7.56 (d, J = 2.6 Hz, 1H), 7.04 (dd, J = 8.3, 2.6 Hz, 2H), 3.84
(s, 3H), 1.32 (s, 12H). 13C NMR (DMSO-d6, 125 MHz) δ (ppm):
162.24, 161.50, 150.28, 149.20, 141.54, 140.76, 137.82, 121.78,
115.80, 109.55, 83.72, 55.21, 24.61. HR-MS (ESI) (m/z): [M +
H]+ calcd for ([C20H24BN3O4] + H)+, 382.1933, found 382.1933.
5.2.2. (MD)BSIH
(E)-N'-((6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzo[d][1,3]dioxol-5-yl)methylene)isonicotinohydrazide
A portion of 2-formyl-4,5-methylenedioxyphenylboronic acid
(1 mmol, 0.194 g) and pinacol (1 mmol, 0.118 g) were dissolved
in DMF and added to a round bottom flask. The reaction stirred
for 21 h at rt then dried by rotary evaporation to give an off-white
5. Experimental Section
powder
corresponding
to
6-(4,4,5,5-tetramethyl-1,3,2-
(MD-
5.1. Materials and Instrumentation
dioxaborolan-2-yl)benzo[d][1,3]dioxole-5-carbaldehyde
BSIH ((E)-N'-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzylidene)isonicotinohydrazide),22 (m-Cl)BASIH) (E)-(5-
chloro-2-((2-
FBApin) in 80% yield. A portion of isoniazid (0.5 mmol, 0.068g)
was dissolved in 1 mL methanol and added to a solution of MD-
FBApin (0.5 mmol, 0.138 g) dissolved in 2 mL of methanol. The
reaction was stirred for 2 h over an oil bath at 100 °C and cooled
over ice. The liquid phase was then collected via vacuum
filtration and dried by rotary evaporation. A yellow powder was
isonicotinoylhydrazineylidene)methyl)phenyl)boronic acid,15 and
SIH ((E)-N'-(2-hydroxybenzylidene)isonicotinohydrazide)44 were
prepared as reported in the literature. The starting materials
isoniazid and (2-formylphenyl)boronic acid pinacol ester were
purchased in reagent grade from Thermo Fisher Scientific Inc
1
isolated in 45% yield. H NMR (DMSO-d6, 500 MHz) δ (ppm):
12.15 (s, 1H), 8.95 (s, 1H), 8.79 (d, J = 5.2 Hz, 2H), 7.80 (d, J =

5.2 Hz, 2H), 7.52 (s, 1H), 7.14 (s, 1H), 6.12 (s, 2H), 1.33 (s, 12
H). 13C NMR (DMSO-d6, 125 MHz) δ (ppm): 162.07, 150.24,
150.19, 148.83, 148.61, 140.83, 135.40, 121.73, 113.76, 104.71,
101.75, 83.97, 24.54. HR-MS (ESI) (m/z): [M + H]+ calcd for
([C20H22BN3O5] + H)+, 396.1725, found 396.1720.
methoxyphenylboronic acid. A white powder was isolated in
68% yield. H NMR (DMSO-d6, 500 MHz) δ (ppm): 11.95 (s,
1
1H), 8.78 (d, J = 5.5 Hz, 2H), 8.39 (s, 1H), 8.05 (s, 1H), 7.81 (d, J
= 5.6 Hz, 2H), 7.74 (d, J = 8.7 Hz, 1H), 7.07 (d, J = 8.6 Hz, 2H),
3.81 (s, 3H), 1.30 (s, 12H). 13C NMR (DMSO-d6, 125 MHz) δ
(ppm): 165.54, 161.46, 150.31, 148.74, 140.58, 134.70, 133.20,
125.76, 121.50, 110.97, 83.35, 55.60, 24.64. HR-MS (ESI) (m/z):
[M + H]+ calcd for ([C20H24BN3O4] + H)+, 382.1933, found
382.1929.
5.2.3. (m-OMe)BSNH
(E)-N'-(4-methoxy-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzylidene)nicotinohydrazide
5.2.7. (p-OMe)SIH
(E)-N'-(2-hydroxy-5-
A similar procedure to the one described above for (MD)BSIH
methoxybenzylidene)isonicotinohydrazide
was
followed,
but
replacing
2-formyl-4,5-
methylenedioxyphenylboronic
acid with
2-formyl-5-
A similar procedure to the one described for SIH was
followed, but replacing salicylaldehyde with 2-hydroxy-5-
methoxybenzaldehyde. A yellow powder was isolated in 87%
methoxyphenylboronic acid. An off-white powder was isolated in
1
9% yield. H NMR (DMSO-d6, 400 MHz) δ (ppm): 12.02 (s,
1H), 9.04 (d, J = 1.9 Hz, 1H), 8.87 (s, 1H), 8.76 (dd, J = 4.7, 1.5
Hz, 1H), 8.23 (dt, J = 8.0, 1.9 Hz, 1H), 8.00 (d, J = 8.7 Hz, 1H),
7.57 (dd, J = 7.8, 4.7 Hz, 1H), 7.19 (d, J = 2.7 Hz, 1H), 7.15 (dd,
J = 8.7, 2.8 Hz, 1H), 3.82 (s, 3H). 13C NMR (DMSO-d6, 125
MHz) δ (ppm): 161.96, 159.84, 152.11, 148.74, 135.61, 131.98,
129.60, 127.58, 123.59, 119.52, 117.32, 84.09, 55.29, 24.61. HR-
MS (ESI) (m/z): [M + H]+ calcd for ([C20H24BN3O4] + H)+,
382.1933, found 382.1932.
1
yield. H NMR (DMSO-d6, 500 MHz) δ (ppm): 12.26 (s, 1H),
10.50 (s, 1H), 8.80 (d, J = 4.9 Hz, 2H), 8.67 (s, 1H), 7.84 (d, J =
4.9 Hz, 2H), 7.17 (d, J = 2.4 Hz, 1H), 6.93 (dd, J = 8.7, 2.6 Hz,
2H), 6.87 (d, J = 8.7 Hz, 1H), 3.73 (s, 3H). 13C NMR (DMSO-d6,
125 MHz) δ (ppm): 161.36, 152.17, 151.52, 150.36, 148.18,
140.07, 121.51, 118.98, 118.66, 117.34, 111.70, 55.51. HR-MS
(ESI) (m/z): [M + H]+ calcd for ([C14H13N3O3] + H)+, 272.1030,
found 272.1029.
5.2.8. (m-OMe)SNH
5.2.4. BSMTH
(E)-N'-(2-hydroxy-4-methoxybenzylidene)nicotinohydrazide
(E)-N-methyl-2-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzylidene)hydrazine-1-carbothioamide
Equimolar quantities of nicotinic hydrazide (1 mmol, 0.137 g)
and 2-hydroxy-4-methoxybenzaldehyde (1 mmol, 0.152 g), both
in methanol were added to a 25-mL round-bottom flask equipped
with a stir bar. The reaction mixture was stirred over an oil bath
at 80 °C for 4 min. The precipitate was collected via vacuum
filtration and washed with water to give a white powder in 85%
One equivalent of (2-formylphenyl)boronic acid pinacol ester
(1 mmol, 0.232 g) was added to a 2-mL solution of 4-methyl-3-
thiosemicarbazide (1 mmol, 0.105 g) in ethanol and heated to 60
°C. After stirring for 10 min, 1 mL of chilled diethyl ether was
added and the reaction was chilled over ice. A white precipitate
was collected via vacuum filtration, washed with diethyl ether
and dried in vacuo to give a white powder in 81% yield. 1H NMR
(DMSO-d6, 400 MHz) δ (ppm): 11.74 (s, 1H), 8.68 (s, 1H), 8.48
(d, J = 4.5 Hz, 1H), 8.29 (d, J = 7.9 Hz, 1H), 7.69 (d, J = 7.3 Hz,
1H), 7.50 (t, J = 7.5 Hz, 1H), 7.38 (t, J = 7.3 Hz, 1H), 3.02 (s,
3H), 1.33 (s, 12H). 13C NMR (DMSO-d6, 125 MHz) δ (ppm):
177.81, 142.47, 139.69, 135.46, 130.90, 128.60, 125.23, 83.94,
30.82, 24.54. HR-MS (ESI) (m/z): [M + H]+ calcd for
([C15H22BN3O2S] + H)+, 320.1599, found 320.1598.
1
yield. H NMR (DMSO-d6, 500 MHz) δ (ppm): 12.12 (s, 1H),
11.48 (s, 1H), 9.07 - 9.05 (m, 1H), 8.75 (d, J = 4.8 Hz, 1H), 8.54
(s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.56 (dd, J = 8.0, 4.8 Hz, 1H),
7.46 (d, J = 8.5 Hz, 1H), 6.52 (dd, J = 8.5, 2.3 Hz, 1H), 6.49 (d, J
= 2.3 Hz, 1H), 3.75 (s, 3H). 13C NMR (DMSO-d6, 125 MHz) δ
(ppm): 162.24, 161.18, 159.43, 152.37, 149.12, 148.57, 135.39,
131.05, 128.77, 123.64, 111.71, 106.57, 101.17, 55.34. HR-MS
(ESI) (m/z): [M + H]+ calcd for ([C14H13N3O3] + H)+, 272.1030,
found 272.1031.
5.2.9. SMTH
5.2.5. BSPTH
(E)-N-phenyl-2-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzylidene)hydrazine-1-carbothioamide
(E)-2-(2-hydroxybenzylidene)-N-methylhydrazine-1-
carbothioamide
Equimolar quantities of 4-methyl-3-thiosemicarbazide (1
mmol, 0.105 g) and salicylaldehyde (1 mmol, 0.122 g), both in
methanol were added to a 25-mL round-bottom flask equipped
with a stir bar. The reaction mixture was stirred over an oil bath
at 80 °C for 4 min. The precipitate was collected via vacuum
filtration and washed with water to give a white powder in 72%
A similar procedure to the one described above for BSMTH
was followed, but replacing 4-methyl-3-thiosemicarbazide with
4-phenylthiosemicarbazide. A white powder was isolated in 75%
1
yield. H NMR (DMSO-d6, 500 MHz) δ (ppm): 12.10 (s, 1H),
10.08 (s, 1H), 8.79 (s, 1H), 8.42 (d, J = 7.9 Hz, 1H), 7.71 (d, J =
7.4 Hz, 1H), 7.57 (d, J = 7.9 Hz, 2H), 7.51 (t, J = 7.6 Hz, 1H),
7.41 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.6 Hz, 2H), 7.20 (t, J = 7.3
Hz, 1H), 1.35 (s, 12 H). 13C NMR (DMSO-d6, 125 MHz) δ
(ppm): 176.09, 143.69, 139.38, 139.08, 135.44, 130.95, 128.90,
128.01, 125.85, 125.25, 83.98, 24.56. HR-MS (ESI) (m/z): [M +
H]+ calcd for ([C20H24BN3O2S] + H)+, 382.1755, found 382.1756.
1
yield. H NMR (DMSO-d6, 500 MHz) δ (ppm): 11.41 (s, 1H),
9.88 (s, 1H), 8.42 (s, 1H), 8.36 (s, 1H), 7.93 (d, J = 7.5 Hz, 1H),
7.21 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.82 (t, J = 7.4
Hz, 1H), 3.00 (s, 3H). 13C NMR (DMSO-d6, 125 MHz) δ (ppm):
177.52, 156.31, 138.92, 130.94, 126.57, 120.48, 119.17, 116.03,
30.83. HR-MS (ESI) (m/z): [M + H]+ calcd for ([C9H11N3OS] +
H)+, 210.0696, found 210.0696.
5.2.6. (m-OMe)BIIH
(E)-N'-(4-methoxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzylidene)isonicotinohydrazide
5.2.10. SPTH
(E)-2-(2-hydroxybenzylidene)-N-phenylhydrazine-1-
carbothioamide
A similar procedure to the one described above for (p-
A similar procedure to the one described above for SMTH
was followed, but replacing 4-methyl-3-thiosemicarbazide with
OMe)BSIH was followed, but replacing 4-methoxy-2-
formylphenylboronic
acid
with
5-formyl-2-

4-phenylthiosemicarbazide. A white powder was isolated in 82%
yield. H NMR (DMSO-d6, 500 MHz) δ (ppm): 11.76 (s, 1H),
To determine the hydrolysis equilibrium constant K of (p-
OMe)BSIH and (MD)BSIH, the UV-Vis absorption spectra of
equilibrated (p-OMe)BSIH or (MD)BSIH solutions in PBS
buffer at various initial concentrations were recorded. The
absorbance values at 263 nm and 321 nm from each spectrum
was used to calculated the equilibrated concentrations of the
intact prochelator and hydrolysis products INH and FBA based
on Eq. 1, where ε represents the extinction coefficient at the
indicated wavelength and [INH] or [FBA] refers to the
equilibrated concentrations of isoniazid and substituted formyl
phenyl boronic acid components. Briefly, the extinction
coefficients for each prochelator, FBA and INH were determined
from the UV-Vis spectra of solutions of each component at
various concentrations in DMSO or PBS, pH 7.4. The absorbance
at 263 nm or 321 nm vs. concentrations provides a linear plot
where the slope is the corresponding extinction coefficient, ε. If
[prochelator] was assumed to be x, [INH] = [FBA] = ci – x, where
ci represents the initial concentration of BASIH added to the PBS
solution. The readings of absorbance at 263 nm or 321 nm from
the equilibrated UV-Vis spectra and the ε values were plugged
into Eq. 1 to solve for x, which afforded the compositions of the
equilibrated solution mixtures.
1
10.04 (s, 1H), 8.49 (s, 1H), 8.08 (d, J = 8.1 Hz, 1H), 7.57 (d, J =
7.7 Hz, 2H), 7.36 (t, J = 7.6 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H),
7.19 (t, J = 7.3 Hz, 1H), 6.88 (d, J = 8.1 Hz, 1H), 6.84 (t, J = 7.5
Hz, 1H). 13C NMR (DMSO-d6, 125 MHz) δ (ppm): 175.70,
156.59, 139.14, 131.31, 128.02, 127.93, 127.05, 125.67, 125.15,
120.25, 119.22, 116.03. HR-MS (ESI) (m/z): [M + H]+ calcd for
([C14H13N3OS] + H)+, 272.0852, found 272.0852.
5.3. Calcein Fluorescence Assay for Prochelator-to-Chelator
Conversion Efficiency
The relative Fe3+ binding affinity of chelators and peroxide-
activated prochelators was assessed by a calcein fluorometric
assay. The fluorescence probe calcein binds Fe2+ or Fe3+ to form
1:1 Fe-calcein complexes, which readily oxidize to the Fe3+ state
in aerobic environments.29,45 Experiments were performed at 25
°C using black-walled 96-well plates with clear bottoms in a
Wallac Victor 1420 plate reader equipped with F485 and F535
incident and emission filters, respectively. The fluorescence
emission of calcein is quenched when coordinated to Fe3+. Stocks
of prochelators and chelators were prepared immediately before
use at 100 μM concentrations in PBS buffer at pH 7.4. The
reaction mixtures of prochechlators with H2O2 were generated by
incubating a 100 μM working solution of prochelators with 5
mM H2O2 for 12 h. The Fe(calcein) stock solution was prepared
by equilibrating 4 μM ferric ammonium citrate and 4 μM calcein
(from a 1 mM stock in 1M NaHCO3) in PBS buffer for 4 h to
ensure formation of the ferric calcein complex. In each well,
Fe3+-calcein at a final concentration of 2 μM was combined with
4 μM of chelators, prochelators, or reaction mixtures of
prochelator with H2O2 in PBS buffer. Emission values from
triplicate wells were averaged for each condition. Wells
containing only calcein were used to determine the 100%
chelation efficiency.
A = εprochelator [prochelator] + εINH [INH] + εFBA [FBA] (1)
The equilibrium constants K at each initial concentration of
BSIH were further calculated according to Eq. 2 and averaged to
give the final equilibrium constant of (p-OMe)BSIH and
(MD)BSIH.
[INH][FBA]
(2)
K=
[prochelator]
5.6. Cell Culture
All cell culture reagents, including minimal essential medium
(MEM), Dulbecco’s modified eagle medium (DMEM), F12
Ham’s nutrient mix (F12), fetal bovine serum (FBS), pencillin–
streptomycin (pen-strep), l-glutamine, and trypsin–EDTA were
purchased from Gibco. The spontaneously immortalized human
retinal pigment epithelial cell line ARPE-19 was purchased from
American Type Culture Collection. The cells were grown in 1:1
DMEM and F12 medium supplemented with FBS (10%), pen-
strep (1%), and glutamine (1%). Cells were cultured until
confluent in 24-well Falcon plates and incubated at 37 °C in a
fully humidified atmosphere containing 5% CO2. CellTiter-Blue
Cell Viability Assay was obtained from Promega. Cell viability
assays were performed on a Perkin Elmer Victor3 1420 plate
reader.
5.4. NMR Characterization of Hydrolytic Stability and Activation
Products of Aroylhydrazone Prochelators
Stock solutions of prochelators (10 mM) in DMSO-d6 were
1
prepared for H-NMR investigations of hydrolytic stability and
reaction with H2O2. Portions of prochelator stock solution were
diluted to 500 μM in D2O with 5% DMSO-d6, or 200 μM with
2% DMSO-d6 in the (MD)BSIH case, and equilibrated for 1 h
prior to NMR analysis. The peroxide activation of the
prochelators was triggered by addition of concentrated H2O2 to
give a final concentration of 25 mM. Products were identified
after 24 h of equilibration by comparison of their 1H NMR
spectra to authentic samples. The percentage of chelator was
calculated taking the ratio of integrated peak areas corresponding
to pyridine Ha from the intact chelator vs. the sum of integrated
peak areas for Ha of the chelator plus Ha’ of INH.
For cytotoxicity assays, the growth medium was removed
when cells reached confluence. Cells were then washed three
times with MEM and treated with various prochelators at final
concentrations ranging from 5–100 M in MEM with up to 1%
DMSO. After 72 h of incubation, cell viability was determined
by the CellTiter-Blue assay. For cytoprotection against H2O2,
washed cells were pre-treated for 5 h with various prochelators or
chelators at the indicated concentrations prior to addition of H2O2
(200 μM final concentration). The cells were further incubated
for 19 h before determination of viability. For cytoprotection
against paraquat, washed cells were co-incubated with 10 mM
paraquat and various prochelators or chelators for 48 h prior to
determination of viability. The conditions compared in each
experiment were: positive control (cells treated only with MEM),
negative control (cells treated with MEM containing H2O2 or
paraquat), and cells treated with H2O2 or paraquat and
5.5. UV-Vis Spectrophotometry for Determination of Hydrolysis
Equilibrium Constants of Aroylhydrazone Prochelators
UV-Vis absorption spectra were collected on a Cary 50 UV-
Vis spectrophotometer using quartz cuvettes with 1-cm
pathlength. For experiments tracking spectral changes of
prochelator hydrolysis, the compositions of the prochelator
solution in PBS buffer at pH 7.4 were monitored by collecting
UV-Vis absorption spectra at 1 min intervals over 30 min.

14. Wang, Q; Franz, KJ. Stimulus-Responsive Prochelators for
Manipulating Cellular Metals. Acc. Chem. Res. 2016; 49: 2468-
2477.
compounds at a range of concentrations. Each condition was run
in triplicate and variability was determined as the standard
deviation of the results.
15. Charkoudian, LK; Pham, DM; Kwon, AM; Vangeloff, AD;
Franz, KJ. Modifications of boronic ester pro-chelators
triggered by hydrogen peroxide tune reactivity to inhibit metal-
promoted oxidative stress. Dalton Trans. 2007: 5031-5042.
16. Dickens, MG; Franz, KJ. A prochelator activated by hydrogen
Acknowledgments
We thank the National Institutes of Health (RO1-GM084176)
for supporting this work. We thank Ms. Abigail C. Jackson for
help with NMR spectra, and Dr. George Dubay and Dr. Peter
Silinski of the Duke Chemistry Instrumentation Facility for
assisting with LC-MS analysis.
peroxide prevents metal-induced amyloid
ChemBioChem 2010; 11: 59-62.
β aggregation.
17. Leed, MG; Wolkow, N; Pham, DM; Daniel, CL; Dunaief, JL;
Franz, KJ. Prochelators triggered by hydrogen peroxide provide
hexadentate iron coordination to impede oxidative stress. J.
Inorg. Biochem. 2011; 105: 1161-1172.
Supplementary Material
18. Kielar, F; Helsel, ME; Wang, Q; Franz, KJ. Prochelator BHAPI
protects cells against paraquat-induced damage by ROS-
triggered iron chelation. Metallomics 2012; 4: 899-909.
19. Kielar, F; Wang, Q; Boyle, PD; Franz, KJ. A boronate
prochelator built on a triazole framework for peroxide-triggered
tridentate metal binding. Inorg. Chim. Acta 2012; 393: 294-303.
20. Thiele, NA; Abboud, KA; Sloan, KB. Novel double prodrugs of
the iron chelator N,N′-bis(2-hydroxybenzyl)ethylenediamine-
N,N′-diacetic acid (HBED): Synthesis, characterization, and
investigation of activation by chemical hydrolysis and
oxidation. Eur. J. Med. Chem. 2016; 118: 193-207.
21. Major Jourden, J; Cohen, S. Hydrogen Peroxide Activated
Matrix Metalloproteinase Inhibitors: A Prodrug Approach.
Angew. Chem. Int. Ed. 2010; 49: 6795-6797.
22. Charkoudian, LK; Pham, DM; Franz, KJ. A pro-chelator
triggered by hydrogen peroxide inhibits iron-promoted hydroxyl
radical formation. J. Am. Chem. Soc. 2006; 128: 12424-12425.
23. Charkoudian, LK; Dentchev, T; Lukinova, N; Wolkow, N;
Dunaief, JL; Franz, KJ. Iron prochelator BSIH protects retinal
pigment epithelial cells against cell death induced by hydrogen
peroxide. J. Inorg. Biochem. 2008; 102: 2130-2135.
24. Jansova, H; Machacek, M; Wang, Q; Haskova, P; Jirkovska, A;
Potuckova, E; Kielar, F; Franz, KJ; Simunek, T. Comparison of
various iron chelators and prochelators as protective agents
against cardiomyocyte oxidative injury. Free Radical Biol.
Med. 2014; 74: 210-221.
25. Jansova, H; Bures, J; Machacek, M; Haskova, P; Jirkovska, A;
Roh, J; Wang, Q; Franz, KJ; Kovarikova, P; Simunek, T.
Characterization of cytoprotective and toxic properties of iron
chelator SIH, prochelator BSIH and their degradation products.
Toxicology 2016; 350: 15-24.
1
Supplemental figures S1–S5 and H, 13C NMR and LC-MS
spectral data for new compounds. This file is available free of
charge.
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