A Double Prodrug with Improved Membrane Permeability over the Parent Chelator HBED Provides Superior Cytoprotection against Hydrogen Peroxide

Article abstract of DOI:10.1002/cmdc.201600197

The clinical use of N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED) has been hindered by its lack of bioavailability. N,N′-bis(2-boronic pinacol ester benzyl)ethylenediamine-N,N′-diacetic acid methyl, ethyl, and isopropyl esters 7 a–c, respectively, and their dimesylate salts 8 a–c, are double prodrugs that mask the two phenolate and two carboxylate donors of HBED as boronic esters and carboxylate esters, respectively. Their activation by chemical hydrolysis and oxidation, their passive diffusivity, and their cytoprotective capabilities have been investigated here. 8 a–c hydrolyzed in minimum essential medium at 37 °C with half-lives of 0.69, 0.81, and 2.28 h, respectively. The intermediate formed, 9 [N,N′-bis(2-boronic acid benzyl)ethylenediamine-N,N′-diacetic acid], then underwent oxidative deboronation by H₂O₂to give HBED (k=1.82 m^?1min^?1). Solubility measurements in mineral oil and in phosphate buffer indicated that 7 a had a better balance between lipid and aqueous solubilities than did HBED. 7 a was also able to passively diffuse across a lipid-like silicone membrane (log flux=?0.36), whereas HBED-HCl was not. 8 c provided better protection to retinal cells than did HBED against a lethal dose of H₂O₂(84 % vs. 28 % protection, respectively, at 44 μm). These results suggest that the double prodrugs have better membrane permeability than does HBED, and therefore could be therapeutically useful for improving the delivery of HBED.

Full text of DOI:10.1002/cmdc.201600197

DOI: 10.1002/cmdc.201600197
Communications
A Double Prodrug with Improved Membrane Permeability
over the Parent Chelator HBED Provides Superior
Cytoprotection against Hydrogen Peroxide
Nikki A. Thiele* and Kenneth B. Sloan[a]
The clinical use of N,N’-bis(2-hydroxybenzyl)ethylenediamine-
N,N’-diacetic acid (HBED) has been hindered by its lack of bio-
availability. N,N’-bis(2-boronic pinacol ester benzyl)ethylenedia-
mine-N,N’-diacetic acid methyl, ethyl, and isopropyl esters 7a–
c, respectively, and their dimesylate salts 8a–c, are double pro-
drugs that mask the two phenolate and two carboxylate
donors of HBED as boronic esters and carboxylate esters, re-
spectively. Their activation by chemical hydrolysis and oxida-
tion, their passive diffusivity, and their cytoprotective capabili-
ties have been investigated here. 8a–c hydrolyzed in minimum
essential medium at 378C with half-lives of 0.69, 0.81, and
2.28 h, respectively. The intermediate formed, 9 [N,N’-bis(2-bor-
onic acid benzyl)ethylenediamine-N,N’-diacetic acid], then un-
derwent oxidative deboronation by H2O2 to give HBED (k=
1.82mꢀ1 minꢀ1). Solubility measurements in mineral oil and in
phosphate buffer indicated that 7a had a better balance be-
tween lipid and aqueous solubilities than did HBED. 7a was
also able to passively diffuse across a lipid-like silicone mem-
brane (log flux=ꢀ0.36), whereas HBED-HCl was not. 8c pro-
vided better protection to retinal cells than did HBED against
a lethal dose of H2O2 (84% vs. 28% protection, respectively, at
44 mm). These results suggest that the double prodrugs have
better membrane permeability than does HBED, and therefore
could be therapeutically useful for improving the delivery of
HBED.
of novel HBED double prodrugs of the general form 7 that
mask the two phenolate and two carboxylate donors of HBED
as boronic acids/esters and simple alkyl carboxylate esters, re-
spectively (Scheme 1).[7] Three of the double prodrugs were
confirmed to undergo activation at physiological pH by chemi-
cally mediated hydrolysis to give intermediate 9 (Scheme 1),
which is itself a prodrug because of its weak affinity for Fe3+
and Cu2+. Subsequent oxidation of 9 by H2O2 generated the
active parent chelator HBED (Scheme 1). These double pro-
drugs may be useful therapeutic agents for diseases where
focal iron accumulation and oxidative stress are implicated in
disease progression (e.g., Parkinson’s disease and other neuro-
degenerative diseases, skin photo-aging, and age-related mac-
ular degeneration). In the present work, we describe the rates
of activation of the double prodrugs in cell culture medium,
which more closely mimics physiological conditions; the pas-
sive diffusivity of a double prodrug across a lipid-like mem-
brane; and the protection afforded by the double prodrugs to
retinal pigment epithelial cells exposed to a lethal dose of
H2O2.
As previously described,[7] double prodrugs 7a–c were syn-
thesized as their dimesylate salts 8a–c over four steps
(Scheme 2). Briefly, 3 was synthesized via reductive amination
and then condensed with pinacol to give 5. The bis acetic acid
esters were installed onto 5 using a simple N-alkylation reac-
tion, which gave 7a–c as oils. Conversion of 7a–c to their cor-
responding dimesylate salts afforded 8a–c as pure solids. In
studies requiring 7a, 8a was simply re-converted to its free
base using diisopropylethylamine (see Supporting Information).
We have previously shown that the double prodrugs 8a–
c convert to the parent iron chelator, HBED, by chemical hy-
drolysis and H2O2 oxidation in N-methylmorpholine and phos-
phate buffers at pH 7.4 with methanol added as a cosolvent.[7]
Here, before investigating the effectiveness of the double pro-
drugs in cell culture, we wanted to verify that the conversion
to HBED occurs in cell culture medium. Consecutive hydrolysis
and H2O2 oxidation of the double prodrugs was monitored by
UV in minimum essential medium (MEM) at 378C. 8a, 8b, and
8c hydrolyzed with pseudo first-order rate constants of
N,N’-bis(2-hydroxybenzyl)ethylenediamine-N,N’-diacetic
acid
(HBED), a hexadentate iron chelator, was investigated some
time ago as an orally administered alternative to desferrioxa-
mine, which is given by subcutaneous infusion, for the treat-
ment of transfusional iron overload.[1] Unfortunately, HBED was
found to be relatively ineffective at removing excess iron from
iron-loaded primates[2] and from humans with b-thalassemia,[3]
probably because it is not well absorbed from the gastrointes-
tinal tract. In an effort to improve oral bioavailability, simple
alkyl ester prodrugs of HBED were prepared,[1b,4] but these too
failed to be effective in iron-loaded primates[2,5] because they
were unable to chemically[4] or enzymatically[6] hydrolyze to
give the active chelator in vivo. Recently, we described a series
0.0167ꢁ0.003 minꢀ1
,
0.0143ꢁ0.0019 minꢀ1
,
and 0.0051ꢁ
0.0014 minꢀ1, respectively, which correspond to half-lives of
0.69, 0.81, and 2.28 h. The final spectrum obtained from each
prodrug’s hydrolysis matched that of an authentic sample of 9
(Figure S1). The rate-limiting step for the hydrolysis of 8a–
c most likely is the hydrolysis of the carboxylate esters, since
pinacol ester hydrolysis is very rapid.[7] Thus, the more sterically
hindered the carboxylate esters were (IPr> Et> Me), the
[a] Dr. N. A. Thiele, Dr. K. B. Sloan
Department of Medicinal Chemistry, University of Florida
Gainesville, FL 32610 (USA)
E-mail: nikkithiele@yahoo.com
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600197.
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Scheme 1. Double prodrugs of the general form 7 undergo activation by nonenzymatic hydrolysis to form 9, which then is oxidatively deboronated by H2O2
to generate the parent chelator HBED.
Scheme 2. Synthesis of HBED double prodrugs as their dimesylate salts 8a–c. a) MeOH, RT, 2 h, then NaBH4, 08C!RT, 1.5 h, 87%; b) pinacol, toluene, reflux,
22 h; c) alkyl bromoacetate, diisopropylethylamine, dry ACN, relux, o/n; d) methanesulfonic acid, dry ether, RT, 2 h, 59–81% over three steps.
longer were the macro half-lives of the double prodrugs (8c>
8b>8a). Interestingly, these half-lives in MEM are substantially
shorter than those obtained for 8a–c in pH 7.4 phosphate
buffer containing 50% MeOH (half-lives of 3.8, 5.9, and 26.3 h,
respectively),[7] probably due to catalysis by amino acids such
as glutamine present in high concentrations in MEM.[8]
bases of 8b and 8c, were not available in sufficient quantities
to determine their lipid solubilities. However, double prodrug
7a, the free base of 8a, was found to possess a high solubility
in mineral oil, a lipid-like solvent: 115ꢁ3.5 mm. In contrast, the
parent chelator, HBED, was insoluble as its monohydrochloride
salt in mineral oil. This lack of lipid solubility was anticipated
since HBED lacks oral availability due to its polar functional
groups and to its ionization at physiological pH. Aqueous solu-
bility is also important for passive diffusivity. HBED-HCl dis-
played substantial aqueous solubility (>10 mm) at pH 7.4 in
phosphate buffer, while the double prodrugs 8a, 8b, and 8c
had more moderate aqueous solubilities of 618 mm, 171 mm,
and 54 mm, respectively.[7] Due to the rapid hydrolysis of the
boronic pinacol esters of 8a–c, the aqueous solubilities of
these prodrugs are probably reflective of the corresponding
boronic acid species present in solution. Taken together, these
solubility studies suggest that the membrane permeability of
the double prodrugs may be improved compared to that of
HBED because they possess both good lipid and good aque-
ous solubilities,[9] while HBED lacks lipid solubility.
To a solution of 9 formed from the hydrolysis of 8a in MEM
was then added 7.5-30 mm of H2O2 (50–200ꢁ excess). UV spec-
tra obtained during a representative oxidation reaction are
given in Figure S2. The final spectrum for each oxidation reac-
tion matched that of an authentic sample of HBED. A second-
order rate constant (k) of 1.82mꢀ1 minꢀ1 for the macro oxida-
tion of 9 to HBED was obtained from the slope of the linear fit
of the plot of kobs versus H2O2 concentration. The results of the
serial hydrolysis and oxidation study provides proof of activa-
tion of the double prodrugs in MEM to the parent chelator
HBED.
In order to access the interior of a cell to express its activity,
a new drug or the prodrug of that new drug must be able to
passively diffuse through the cell membrane. One physico-
chemical property that may facilitate passive diffusion is ade-
quate lipid solubility. Double prodrugs 7b and 7c, the free
Passive diffusion studies of 7a and HBED-HCl using Franz
diffusion cells equipped with silicone membranes further sup-
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port the improved membrane permeability of the double pro-
drugs. Silicone membranes, which are lipid-like, have been vali-
dated as a surrogate for flux across a biological membrane:
namely, the skin.[10] Mineral oil was used as the donor vehicle
in which the compounds were dissolved so that 7a would
remain intact in the donor phase throughout the experiment.
Use of a protic solvent would most likely have led to its de-
composition. The receptor chambers contained phosphate
buffer. All receptor phase samples taken from diffusion cells to
which 7a was applied were allowed to hydrolyze to 9 before
analysis because 7a and 9 do not have sufficiently different UV
spectra to enable one to calculate the amount of each in
a mixed sample. The amount of 9 in receptor phase samples
presumably corresponds to the amount of 7a that was able to
diffuse intact through the silicone membranes before subse-
quently hydrolyzing in the receptor chambers. 7a showed ex-
cellent diffusion across the silicone membranes, having a maxi-
mum flux of 0.43ꢁ0.04 mmolcm2hꢀ1 (log flux=ꢀ0.36). By con-
trast, no flux was observed for HBED-HCl through the silicone
membranes from a mineral oil vehicle. Once again, this sug-
gests that the double prodrugs may have better membrane
permeability than the parent chelator. While a better compari-
son could have been made if the flux of HBED as the free base
had been investigated, it was not readily available.
Figure 1. H2O2 kill curve for ARPE-19 cells. After cells were grown to 100%
confluence in growth medium (DMEM:F12 with 10% FBS), the medium was
removed and MEM was applied. After 15 h, various concentrations of H2O2
were added to the wells, and the plates were incubated for another 8 h. Cell
viability was determined by an MTT assay, and is reported as the average of
triplicate wells ꢁ1 standard deviation for each concentration of H2O2 ap-
plied.
given no prodrug or HBED (p <0.001). Additionally, while nei-
ther 8a, 8b, nor 9 showed enhanced protection over that of
HBED at any equivalent dose, 8c significantly out-protected
HBED at concentrations of 13, 20, 30, and 44 mm (p<0.001). At
44 mm, 8c provided 3-fold higher protection to cells compared
to HBED. 8c was also not toxic to cells at this dose (Figure S3).
From these cytoprotection studies, several points can be
highlighted. First, despite the indication that 8a and 8b may
have improved membrane permeability from the results of the
solubility and diffusion cell studies, they did not perform as
well as 8c. 8c may provide better protection to cells than 8a
or 8b because it remains intact the longest— its carboxylate
esters are the slowest to hydrolyze—in MEM, which facilitates
its entry into cells. Second, 8c has enhanced membrane per-
meability compared to HBED. Presumably, 44 mm of 8c was
converted by hydrolysis and oxidation to 44 mm HBED. If 8c
and HBED have similar membrane permeability, 8c would have
provided protection similar to that of HBED, or even less pro-
tection because the prodrugs are not able to instantly protect
the cells upon exposure to H2O2: they first must be oxidized.
Therefore, the superior protection by 8c compared with HBED
further supports the superior membrane permeability of 8c.
Indeed, 8a (as the free base 7a), the methyl ester double pro-
drug, is much more lipophilic than HBED, so 8c, as the isopro-
pyl ester double prodrug, is even more lipophilic: partition co-
efficients increase monolithically with the sequential addition
of CH2 groups.[13] The third point that can be made is that the
primary mode of protection of 8c is not consumption of H2O2
during its unmasking. The oxidation of 44 mm of 8c (presuma-
bly present as 9 after the 15 h pretreatment period) by H2O2
will consume a total of 88 mm of H2O2, leaving 412 mm of the
500 mm dose of H2O2 to challenge the cells. According to
Figure 1, exposure to a similar amount of H2O2 (417 mm) de-
creased cell viability by ~71%. But in the presence of 44 mm of
8c, cell viability was only decreased by 22% (not shown).
Therefore, while cytoprotection by 8c may have been slightly
improved by the consumption of H2O2, it is more likely that
Attention was then turned to investigating the abilities of
the double prodrugs 8a–c and prodrug 9 to undergo activa-
tion in vitro to afford cytoprotection against H2O2-induced
death, compared with the cytoprotective ability of the parent
chelator, HBED. H2O2 has been found to oxidize ferritin, the
major iron storage protein, leading to a release of iron that
can catalyze free radical damage.[11] Protection by the prodrugs
will require their entry into cells and their timely activation to
HBED so that the freed iron can be sequestered. A spontane-
ously-arising human retinal pigment epithelial cell line (ARPE-
19) was chosen for its relevance to age-related macular degen-
eration. This incurable disease is the most common cause of
vision loss for people over the age of 50 and increased labile
iron is implicated in its pathogenesis.[12]
Confluent cells were pretreated with 8a–c, 9, or HBED (as its
HCl salt) in MEM for 15 h so that all compounds would have
sufficient time to enter the cells if they are membrane permea-
ble, and so that the double prodrugs would also have time to
hydrolyze. The cells were then treated for 8 h with a lethal
dose of 500 mm of H2O2, which decreased cell viability by ap-
proximately 79% in
a preliminary kill curve experiment
(Figure 1). Percent protection afforded by the compounds was
calculated from the results of an MTT cell viability assay (see
Supporting Information). The results of these cytoprotection
studies are given in Figure 2. Cytoprotection was dose depen-
dent for all compounds tested. At their highest concentrations
of 150 mm, 8a, 8b, 9, and HBED all provided moderate protec-
tion to cells from the lethal dose of H2O2 relative to cells ex-
posed to H2O2 but given no prodrug or HBED (62–68% protec-
tion, p<0.001 by t-test). In contrast, despite applying it at
lower concentrations due to solubility limitations, 8c gave the
highest cytoprotection: at only 44 mm, 8c afforded 84% pro-
tection against H2O2 relative to cells exposed to H2O2 but
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Figure 2. Protection of ARPE-19 cells against 500 mm H2O2 by double prodrugs 8a–c, prodrug 9, and HBED. After cells were grown to 100% confluence in
growth medium (DMEM:F12 with 10% FBS), the medium was removed and the cells were pretreated with various concentrations of prodrug or HBED in
MEM for 15 h. This was followed by an 8 h challenge with 500 mm H2O2. Percent protection was calculated from the results of an MTT cell viability assay and
is reported as the mean ꢁ1 standard deviation (n=6) for each concentration of drug applied.
the primary mechanism of protection by 8c against H2O2-in-
duced cell death is from chelation of free iron upon the activa-
tion of 8c to HBED.
Keywords: boronic acids · chelation therapy
peroxide · iron · prodrugs
· hydrogen
In summary, we have shown here that selected double pro-
drugs of HBED can be activated under physiologically relevant
conditions in vitro to give HBED, the parent iron chelator, and
can ultimately provide cells with protection against oxidative
stress promoted by the application of exogenous H2O2. The
studies suggest that at least one double prodrug, 8c, has im-
proved membrane permeability and cytoprotective capacity
over HBED. Therefore, double prodrugs of this kind may prove
useful in improving the oral bioavailability of HBED and its de-
livery to specific tissues where an iron chelator is needed to
curtail Fenton chemistry. This has implications for the treat-
ment of a broad range of diseases that are promoted by oxida-
tive stress.
[1] a) F. L’Eplattenier, I. Murase, A. E. Martell, J. Am. Chem. Soc. 1967, 89,
837–843; b) R. W. Grady, A. Jacobs in Development of Iron Chelators for
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Badman), Elsevier/North Holland, Inc., New York, NY, 1981, pp. 133–
164; c) R. W. Grady, C. Hershko, Ann. N. Y. Acad. Sci. 1990, 612, 361–368.
[2] H. H. Peter, R. J. Bergeron, R. R. Streiff, J. Wiegand in The Development of
Iron Chelators for Clinical Use (Eds.: R. J. Bergeron, G. M. Brittenham),
CRC Press, Boca Raton, FL 1994, pp. 373–394.
[3] R. W. Grady, A. D. Salbe, M. W. Hilgartner, P. J. Giardina in Progress in Iron
Research (Eds.: C. Hershko, A. M. Konain, P. Aisen), Plenum Press, NY
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[4] C. G. Pitt, Y. Bao, J. Thompson, M. C. Wani, H. Rosenkrantz, J. Metterville,
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Supporting Information
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[8] J. L. Buss, P. Ponka, Biochim. Biophys. Acta Gen. Subj. 2003, 1619, 177–
186.
The Supporting Information for this article contains the com-
plete Experimental Section for the work described, which in-
cludes synthesis, solubility determination, activation studies,
diffusion cell studies, and biological assay protocols.
[9] K. B. Sloan in Prodrugs: Topical and Ocular Drug Delivery (Ed.: K. B.
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Abbreviations
HBED (N,N’-bis(2-hydroxybenzyl)ethylenediamine-N,N’-diacetic
acid); MEM (minimum essential medium).
[12] P. Hahn, A. H. Milam, J. L. Dunaief, Arch. Ophthalmol. 2003, 121, 1099–
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[13] H. D. Beall, J. J. Getz, K. B. Sloan, Int. J. Pharm. 1993, 93, 37–47.
Acknowledgements
The authors would like to thank Dr. Stephan C. Jahn (University
of Florida) for providing training and advice related to the cell
culture studies.
Received: April 13, 2016
Revised: June 20, 2016
Published online on && &&, 0000
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COMMUNICATIONS
Double your prodrug, double your
fun! N,N’-bis(2-hydroxybenzyl)ethylene-
diamine-N,N’-diacetic acid (HBED), an
iron chelator, has poor oral bioavailabil-
ity that limits its clinical use. Here,
N. A. Thiele,* K. B. Sloan
&& – &&
A Double Prodrug with Improved
Membrane Permeability over the
Parent Chelator HBED Provides
Superior Cytoprotection against
Hydrogen Peroxide
double prodrugs of HBED that are acti-
vated by hydrolysis and oxidation were
studied. Solubility, diffusivity, and cyto-
protection studies all suggest that the
double prodrugs may have improved
membrane permeability over HBED that
could lead to better delivery in vivo.
ChemMedChem 2016, 11, 1 – 5
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These are not the final page numbers! ÞÞ