Multifunctional antioxidants for the treatment of age-related diseases

Article abstract of DOI:10.1021/jm901381j

Analogues of N,N-dimethyl-4-(pyrimidin-2-yl)piperazine-1-sulfonamide possessing a free radical scavenger group (FRS), chelating groups (CHL), or both (FRS + CHL) have been synthesized. Electrospray ionization mass spectrometry studies indicate that select

Full text of DOI:10.1021/jm901381j

J. Med. Chem. 2010, 53, 1117–1127 1117
DOI: 10.1021/jm901381j
Multifunctional Antioxidants for the Treatment of Age-Related Diseases
Hongxia Jin, James Randazzo, Peng Zhang, and Peter F. Kador*
College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198
Received September 17, 2009
Analogues of N,N-dimethyl-4-(pyrimidin-2-yl)piperazine-1-sulfonamide possessing a free radical sca-
venger group (FRS), chelating groups (CHL), or both (FRS þ CHL) have been synthesized. Electro-
spray ionization mass spectrometry studies indicate that select members of this series bind ions in the
relative order of Cu1þ=Cu2þ>Fe2þ=Fe3þ > Zn2þ with no binding of Ca2þ or Mg2þ observed. In vitro
evaluation of these compounds in human lens epithelial, human retinal pigmented epithelial, and human
hippocampal astrocyte cell lines indicates that all analogues possessing the FRS group as well as the
water-soluble vitamin E analogue 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid protect
these cells against decreased cell viability and glutathione levels induced by hydrogen peroxide. In
addition, those compounds possessing CHL groups also protected these cells against hydroxyl radicals
generated by the Fenton reaction. These compounds are good candidates for the preventive treatment of
cataract, age-related macular degeneration (AMD), and Alzheimer’s dementia (AD).
Introduction
demonstrated clinical efficacy in reducing the clinical signs of
neurodegeneration or cataract.
Oxidative stress damages cells and is an accepted determi-
nant of both lifespan and health span. Oxidative stress results
from the generation of reactive oxygen species (ROSa), pri-
marily H2O2, and is enhanced by the age-related decrease of
cellular antioxidant defenses and accumulation of iron and/or
copper.1 The increased cellular presence of these redox-active
metals enhance H2O2-linked ROS by generating hydroxyl
radicals through the Fenton reaction.2,3 Age-related increases
in ROS are believed to trigger biochemical cascades that lead
to neurodegenerations such as AD,4-6 retinal degenerations
such as age-related macular degeneration (AMD),7-10 and
cataract formation.7,11 While these diseases are pathologically
different, studies suggest that they all undergo similar oxidative
damage from ROS associated with redox-active metal ions.
For example, ROS-associated Aβ in amyloid plaques, which
are characteristic of AD,4,12-15 is also present in AMD16-18
and age-related cataract.19-21
The oxidative pathways associated with neurodegenera-
tions and age-related ocular diseases have been targeted as a
potential therapeutic approach to the treatment of these age-
related diseases.5 A wide variety of antioxidants have been
examined to reduce ROS. These range from natural products
with antioxidant properties such as aged garlic extract, cur-
cumin, melatonin, resveratrol, Ginkgo biloba extract, green
tea, vitamin C, L-carnosine, vitamin E, and cannabinoids to
derivatives of lipoic acid, analogues of coenzyme Q (MitoQ),
and “thiol-delivering” glutathione mimics such as tricyclo-
decan-9-yl xanthogenate.22-29 Some of these agents have
shown limited clinical efficacy with AMD,30,31 but none have
Reducing ROS through the chelation of redox-active me-
tals is another approach that has been investigated; however,
this approach has primarily been used to treat neurodegen-
erations. Examples are illustrated in Figure 1. Of these
compounds, only the iron chelator desferrioxamine (DFO,
N0-{5-[acetyl(hydroxy)amino]pentyl}-N-[5,4- [(5-aminopentyl)-
(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxy-
succinamide) and clioquinol (5-chloro-7-iodoquinolin-8-ol)
has been investigated for both neurological32,33 and age-
related ocular diseases.34-36 Desferrioxamine is not orally
active and does not significantly cross the blood-brain
barrier.37,38 Clioquinol is an orally active antibiotic that was
extensively used as an antibiotic in Asia in the 1960s. In 1971 it
was banned in Japan after being linked to subacute myelo-
optic neuropathy (SMON) in over 10000 Japanese people. Its
chelating ability was subsequently identified during neuro-
toxicity studies39 where it has been demonstrated to decrease
Cu uptake and counteract Cu efflux activities of the amyloid
precursor protein of AD,40 disaggregate the metal ion-
induced aggregates of Aβ(1-40), and retard fibril growth
through a Zn(II) clioquinol complex formation.41 Its efficacy
has been demonstrated in vitro, in in vivo animal models, and
in several small clinical trials of AD patients.15,42,43 Because of
the promising results of clioquinol, several other 8-hydroxy-
quinoline analogues have been developed: PBT2 (structure
not disclosed to date),44,45 M-30 (5-((methyl(prop-2-ynyl)-
amino)methyl)quinolin-8-ol),46,47 VK-28 5-((4-(2-hydroxyethyl)-
piperazin-1-yl)methyl)quinolin-8-ol),46 HLA-20 (5-((4-(prop-
2-ynyl)piperazin-1-yl)methyl)quinolin-8-ol),46 deferasirox
(4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl)benzoic
acid),46-48 deferiprone (3-hydroxy-1,2-dimethylpyridin-4(1H)-
one),49 feralex (2-(3-hydroxy-2-methyl-4-oxo-3,4-dihydropyri-
din-1(2H)-yl)-N-((3R,4R,5S,6R)-2,4,5-trihydroxy-6-(hydroxyl-
methyl)-tetrahydro-2H-pyran-3-yl)acetamide,50,51 D-penacill-
amine ((S)-2-amino-3-mercapto- 3-methylbutanoic acid),52
*To whom correspondence should be addressed. Phone: 402-559-
9261. Fax: 402-559-9543. E-mail: pkador@unmc.edu.
a Abbreviations: AD, Alzheimer’s dementia; AMD, age-related ma-
cular degeneration; BSA, bovine serum albumin; CHL, chelating
groups; FRS, free radical scavenger groups; hLECs, human lens epithe-
lium cells; HA-h, human hippocampal astrocytes; ROS, reactive oxygen;
SDI, sorbitol dehydrogenase inhibitor.
r
2010 American Chemical Society
Published on Web 01/15/2010
pubs.acs.org/jmc

1118 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Jin et al.
Figure 1. Structure of antioxidant and chelators evaluated for cataract, AMD, and AD.
Figure 2. Analogues of N,N0-dimethyl-4-(pyrimidin-2-yl)piperazine-1-sulfonamide (1) synthesized and evaluated.
DP-109 (3,30-(2,20-(ethane-1,2-diylbis(oxy))-bis(2,1-phenylene))-
bis(5-(2-(octadecyloxy)ethoxy)-5-oxopentanoic acid),53 and (-)-
epigallocatechin 3-gallate (EGCG) ((2R,3R)-5,7-dihydroxy-2-(3,
4,5-trihydroxyphenyl)chroman-3-yl-3,4,5-trihydroxybenzoate).54
To date, many research efforts on the treatment of ROS-
linked complications have focused on therapeutic targets
that enhance cellular antioxidant defenses, demonstrate anti-
oxidant activity, or regulate cellular levels of transition
metal ions.43,55 Because multiple mechanisms are involved
in the development of ROS-linked disorders, drugs with at
least two mechanisms of action targeted at ROS may offer
more therapeutic benefit than those only targeting a single
mechanism. Toward this end, we have synthesized a series of
multifunctional analogues of N,N-dimethyl-4-(pyrimidin-2-yl)-
piperazine-1-sulfonamide (1) (Figure 2) possessing a FRS
group, (analogues 2, 6), CHL groups (CHL, analogues 3, 7),
or both (analogues 4, 8).56 The ring structure of the parent
compound 1 was derived from studies investigating the effect

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1119
Chemistry
of sorbitol dehydrogenase inhibitors (SDI) on sugar cataract
formation.57 FRS activity was introduced to 1 by addition of
an -OH group in the 5-position of the pyrimidine ring. This
was based on a report that 5-pyrimidinols are more effective
antioxidants than their corresponding phenols, with 2-N,N-
dimethyl-4,6-dimethyl-5-hydroxypyrimidine being 5-fold more
reactive toward alkyl radicals and essentially equally reac-
tive to peroxy radicals compared to R-tocopherol.58 Meth-
oxy groups, rather than methyl groups, were added to the
pyrimidine ring because methoxy groups stabilize the radi-
cal scavenger slightly better than the methyl groups and are
not as readily subjected to metabolic oxidation as the methyl
groups.59-61 The ability to chelate was introduced by adding
carbonyl groups directly adjacent to the amino group con-
necting the piperazine ring to the pyrimidine ring. This was
based on a report that 2-N-succinamide-1,3-pyrimidine
easily forms complexes with transition metals such as Fe3þ
Compounds 1, 5, and 6 were synthesized as outlined in
Scheme 1. N,N-Dimethylpiperazine-1-sulfonamide 10 was
obtained from commercially available piperazine 9 according
to the literature.63 Nucleophilic substitution of 2-chloropyr-
imidines 11a and 11b with N,N-dimethylpiperazine-1-sulfon-
amide 10 gave 1 and 5, respectively. Compound 6 was
obtained through directed hydroxylation of 5. The aromatic
anion of 5, which was generated in the presence of n-BuLi, was
treated with oxygen gas to give 6.64
The 5-hydroxypyrimidine analogue 2 was also initially
obtained by nucleophilic substitution of 2-chloro-5-hydroxy-
pyrimidine (15) with N,N-dimethylpiperazine-1-sulfonamide
(10) (Scheme 2). Compound 15 was obtained from commer-
cially available 2-chloro-5-nitropyrimidine (12) by first redu-
cing the nitro group to 2-chloro-5-aminoprimidine (13) by
refluxing with acetic acid in the presence of iron. Subsequent
diazotization of the amine 13 failed; however, 2-chloro-5-
hydroxypyrimidine 15 could be obtained in poor yield (33%)
by refluxing 13 with 2 M sulfuric acid.65
and Cu2þ 62
.
Scheme 1a
Because of the poor yield in obtaining 15, an alternative
approach to compound 2 was employed. As outlined in
Scheme 3, an initial nucleophilic reaction of N,N-dimethyl-
piperazine-1-sulfonamide 10 with 2-chloro-5-nitropyrimidine
12 gave the nitro product 16 which was reduced to the amine
17 by refluxing with NH4Cl and iron. Refluxing amine 17 with
2 M sulfuric acid gave 2 in 56% yield.
The piperazine-2,6-dione containing analogues were synthe-
sized by condensation of 2,20-(N,N-dimethylsulfamoylazane-
diyl)diacetic acid (19), obtained from commercially available
iminodiacetic acid (2,20-azanediyldiacetic acid) (18), with pri-
mary aromatic amines 20a-d (Scheme 4). The piperazine-
2,6-diones 3, 7, 21, and 22, respectively, were obtained by
activation of the 2,20-(N,N-dimethylsulfamoyliminodiacetic
acid (19) with acetic anhydride (step b), addition of 20a-d
(step c), and subsequent dehydration (step d).66 The benzyl
a Reagents and conditions: (a) ClSO2NMe2, K2CO3, EtOH, room
temperature; (b) TEA, THF, reflux; (c) n-BuLi, THF, O2, -78 °C to
room temperature.
Scheme 2a
a Reagents and conditions: (a) Fe, AcOH, reflux; (b) 2 M H2SO4, reflux; (c) TEA, THF, reflux.
Scheme 3a
a Reagents and conditions: (a) TEA, THF, room temperature; (b) Fe, NH4Cl, EtOH-H2O, reflux; (c) 2 M H2SO4, reflux.

1120 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Jin et al.
Scheme 4a
a Reagents and conditions: (a) ClSO2NMe2, NaOH, Na2CO3, acetone-H2O, 0 °C to room temperature; (b) Ac2O, 85 °C; (c) acetone, toluene, 80 °C;
(d) Ac2O, 80 °C; (e) Pd-C, H2, EtOAc, room temperature.
Scheme 5a
a Reagents and conditions: (a) Ac2O, 85 °C; (b) acetone, toluene, 80 °C; (c) Ac2O, 80 °C; (d) BBr3, benzene, room temperature; or TMSI, CHCl3; or
MeCN.
protecting groups of 21 and 22 were removed by hydrogeno-
lysis to give final 4and 8, respectively. Compounds 20a and 20b
were commercially available.
Scheme 6
To obtain compound 4, the synthesis outlined in Scheme 5
was initially employed. The methoxy 24 was obtained in 60%
yield in a three-step pot reaction from 19 and 23 according
to published procedures.67 However, attempts at selectively
cleaving the methoxy group of 24 failed. In the presence of
BBr3, a number of cleavage products were obtained, presum-
ably because of the instability of 2,6-dioxopiperazine in the
presence of the strong Lewis acid. Alternatively, no reaction
was observed with mild treatment of 24 with TMSI for 12 h.
Condensation of 19 with 2-amino-5-hydroxypyrimidine, 25,
the cleaved methoxy product of 23, also did not result in the
anticipated amide 27 (Scheme 6). Instead, ester 26 was formed
because the 5-hydroxyl group of 2-amino-5-hydroxypyrimi-
dine, 25, is more reactive; however, protection of the hydroxyl
group with an easily removable benzyl group gave amine 20c
which when condensed yielded compound 21. Subsequent
hydrogenolysis of 21 gave the desired compound 4.
2-Amino-5-(benzyloxy)-4,6-dimethoxypyrimidine (20d) was
obtained from commercially available 2-chloro-4,6-dimethoxy-
pyrimidine 11b (Scheme 7). Oxidation of the aromatic anion
of 11b generated in the presence of dioxygen gave the hydroxyl
28 in 44% yield. This yield was increased to 94% when
dioxygen was replaced with lithium tert-butyl peroxide
which was generated in situ from tert-butylperoxy alcohol
and n-butyllithium.68 Following benzylation of the hydroxyl
group, the 2-chlorine atom was replaced with an amino group.
Attempted substitution of the chlorine with an ammonia-
methanol solution or ammonia hydroxide was unsuccess-
ful. Refluxing chloride 29 in toluene with benzylamine resulted
in the formation of the 2-benzyllamine 30 in 33% yield and a
64% yield (according to the procedure for preparation of
compound 30) with 25% recovery of chloride 29. Replacing
toluene with dioxane increased the yield of 30 to 55% with
25% recovery of 29. Attempted selective hydrogenolysis of 30
for 2 h at room temperature and 1 atm resulted in 2-benzyl-
amino-4,6-dimethoxy-5-hydroxyprimidine in 90% yield and
2-amino-4,6-dimethoxy-5-hydroxyprimidine 31 in 10% yield.
Both benzyl groups were removed to give 31 in 98% yield when
hydrogenolysis was increased to 12 h. The benzyl group was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1121
Scheme 7a
a Reagents and conditions: (a) LDA, t-BuOOLi, THF, -78 °C; (b) BnBr, K2CO3, MeCN, room temperature; (c) BnNH2, K2CO3, dioxane, reflux;
(d) Pd-C, H2, MeOH, room temperature; (e) BnBr, K2CO3, MeOH, room temperature.
Table 1. Percent Parent Peaks Remaining at 10:1 Metal Ion Solution/
Compound Obtained by ESI-MS
% parent peaks in ion solutions
compd
Fe2þ
Fe3þ
Cu1þ
Cu2þ
Zn2þ
Ca2þ
Mg2þ
1
2
3
4
5
6
7
8
100.0
100.0
8.1
100.0
100.0
10.0
8.2
100.0
100.0
3.6
100.0
100.0
4.4
100.0
100.0
15.3
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
6.9
4.2
3.0
12.7
100.0
100.0
7.3
100.0
100.0
7.6
100.0
100.0
4.1
100.0
100.0
3.4
100.0
100.0
13.8
7.5
8.2
4.0
3.6
14.6
Figure 4. Job’s plot of compound 4 in acetate solution (1 mM, pH
6.5) at 23 °C. The final concentration of compound 4 and Fe2þ ion
was maintained constant at 0.1 mM.
Table 2. Stoichiometry of Metal Ion Binding to Compounds Obtained
from Job Plotsa
stoichiometry in ion solutions
compd
Fe2þ
0b
0b
Fe3þ
Cu1þ
Cu2þ
Zn2þ
Mg2þ Ca2þ
1
2
3
4
5
6
7
8
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
2:1.06 2.101 1.212 1:2.03 1:1.94 0b
2:1.08 2.096 1.194 1:1.99 1:2.08 0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
0b
2:1.08 2.099 1.194 1:1.99 1:1.86 0b
2:1.10 2.099 1.190 1:2.08 1:1.99 0b
a The total concentration of compound to metal ion was maintained
at 0.1 mM. b Nonintersecting lines in the Job plot.
binding was only observed with compounds 3, 4, 7, and 8 all
of which possess the required piperazine-2,6-dione groups.
These compounds bound ions with a relative binding order
of Cu1þ = Cu2þ > Fe2þ = Fe3þ > Zn2þ. No binding was
Figure 3. ESI-MS analysis of parent peak of N,N0-dimethyl-4-
(pyrimidin-2-yl)piperazine-1-sulfonamide (1) and N,N0-dimethyl-
3,5-dioxo-4-(pyrimidin-2-yl)piperazine-1-sulfonamide (3) in the
presence of increasing amounts of Fe2þ solutions.
observed with either Ca2þ or Mg2þ
.
then reintroduced to the 5-hydroxy group to give 20d under
conditions similar to those employed for the synthesis of 20c.
To obtain the stoichiometry of the chelator-ion com-
plexes, Jobs plots were prepared for compounds 1-8 with
Fe2þ, Fe3þ, Cu1þ, Cu2þ, Zn2þ, Ca2þ, and Mg2þ. The maxi-
mum absorbance for each compound as a function of the
mole fraction of the metal ion was determined and plotted to
give linear dependences at high and low molar fractions. The
stoichiometry of the formed complexes was determined by
the intersections of these lines. The intersection of these lines
corresponded to the stoichiometry of the complex formed
(Figure 4). Intersecting lines were not observed with com-
pounds 1, 2, 5, or 6 or any compounds incubated with Ca2þ
and Mg2þ; therefore, no stoichiometry could be obtained for
these compounds or ions. This was consistent with the ESI-
MS data. Binding stoichiometry for compounds 3, 4, 6,
and 7, summarized in Table 2, was determined to be 1 metal
chelator/2 metal ions for those ions with a tetrahedral/
square planar geometry (Cu1þ, Cu2þ, Zn2þ) or to be 2 metal
Chelation Studies. The relative amounts of Fe2þ, Fe3þ
,
Cu1þ, Cu2þ, Zn2þ, Ca2þ, Mg2þ ions bound by compounds
1-8 were determined by electrospray ionization mass
spectrometry (ESI-MS) by mixing each ion solution with
compound in molar ratios of 10:1, 1:1, and 1:10 and then
directly infusing each solution under constant ionization
energy. Table 1 summarizes the relative percent parent peak
observed with a 10:1 metal ion/compound solution for each
compound. Under these conditions, a relative decrease in
compound parent peak height only occurs with increased ion
binding. This is illustrated in Figure 3 where the parent peak
heights of compounds 1 and 3 are compared. Only com-
pound 3 which contains the necessary piperazine-2,6-
dione groups required for ion binding shows a decrease
in peak height with increasing concentration of Fe2þ. Ion

1122 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Jin et al.
Figure 5. In vitro MTS viability assay of HLECs subjected to ROS. Part A illustrates exposure of HLECs exposed for 2 h with 1 mM H2O2
with/without the presence of the water-soluble vitamin E analogue 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid or compounds
1-8. Part B illustrates 2 h exposure of HLECs with 1 mM Fenton’s reagent with/without the presence of the vitamin E analogue or compounds
1-8. Parts C and D illustrate similar 2 h exposure of hRPE and HA-h cells, respectively, with 1 mM Fenton’s reagent with/without the presence
of the vitamin E analogue or compounds 1-8. All studies were normalized against MTS cell staining obtained without ROS (blank control),
and the results represent the mean ( SD of three separate experiments. Significant differences (p>0.05), calculated by ANOVA, were com-
pared to control staining obtained with ROS in the absence of either the vitamin E analogue or compounds 1-8.
chelators/1 metal ion for ions having an octahedral geometry
(Fe2þ, Fe3þ). This stoichiometry was consistent with the data of
a similar chelating moiety in 2-(N-succinimidyl) pyrimidine.62
In Vitro Evaluation of Antioxidant Activity. Compounds
1-8 were evaluated for their ability to protect SRA-1
human lens epithelium cells (hLECs),69 commercially available
ARPE-19 human retinal pigmented epithelial cells (RPEs),70
and HA-h human hippocampal astrocytes (HA-hs). These
three cells types are instrumental in the development of catar-
act, AMD, and AD, respectively.
multifunctional compounds possessing both FRS and CHL
groups (compounds 4 and 8) demonstrated better protection
than 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid. Similar results were observed for the RPE and HA-h
cells. The protection afforded by compounds 1-8 against
Fenton’s reagent is summarized in Figure 5C and Figure 5D,
respectively.
In these three cell types, the rapid intracellular reduction of
cellular glutathione (GSH) is a sensitive marker of oxidative
stress. As illustrated in Figure 6A, the presence of 1 mM H2O2
rapidly resulted in a reduction of GSH levels in hLECs and a
similar reduction was also observed with compounds 1, 3, 5,
and 7, analogues not possessing the FRS group. However,
GSH levels were maintained when similar cells were treated
with either the water-soluble vitamin E analogue 6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid or compounds
2, 4, 6, and 8 which possess the FRS group. All compounds
possess the 2-amino-5-hydroxy-1,3-pyrimidine FRS group.
With Fenton generated ROS (Figure 6B), protection was also
afforded by the CHL containing compounds 3 and 7. In
addition, the multifunctional compounds possessing both
FRS and CHL groups (compounds 4 and 8) demonstrated
slightly better protection than 6-hydroxy-2,5,7,8-tetramethyl-
chroman-2-carboxylic acid. Again, similar results were ob-
served for the RPE and HA-h cells. The protection afforded
by compounds 1-8 against Fenton’s reagent is summarized in
Figure 6C and Figure 6D, respectively.
In the first set of studies, the ability of compounds 1-8 to
protect cells against ROS-induced cell death was evaluated
using a MTS Cell Viability assay. The results were compared
to the protective activity of the water-soluble vitamin E
analogue 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxy-
lic acid. All cells were exposed for 2 h of either 1.0 mM
H2O2 or Fenton reagents (1.0 mM H2O2 and 1.0 mM Fe2þ
)
with/without 1 mM compounds 1-8 or 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid. As summarized in
Figure 5A, the presence of H2O2, reduced the viability of the
hLECs to less that 50%. A similar loss of cell viability was
observed for compounds 1, 3, 5, and 7, analogues not posses-
sing the 2-amino-5-hydroxy-1,3-pyrimidine group required
for FRS activity. 6-Hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid significantly protected cell viability against
ROS, and similar protection was also observed with com-
pounds 2, 4, 6, and 8; all compounds possess the 2-amino-5-
hydroxy-1,3-pyrimidine FRS group. When ROS was gener-
ated by the Fenton reaction (Figure 5B), in addition to the
FRS containing compounds 2, 4, 6, and 8, some protection
was now also demonstrated with compounds 3 and 7 which
contain the 2,8-dioxopiperazine CHL group. Moreover, the
Experimental Section
NMR spectra were obtained with a Varian 500 MHz spectro-
meter. EI-MS utilized an Agilent 5973N MSD, and ESI-MS was
conducted with a Finnigan MAT LCQ. UV-visible spectra were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1123
Figure 6. GSH levels in in vitro cells subjected to ROS. Part A illustrates exposure of HLECs exposed for 2 h with 1 mM H2O2 with/without the
presence of the water-soluble vitamin E analogue 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid or compounds 1-8. Part B
illustrates 2 h exposure of HLECs with 1 mM Fenton’s reagent with/without the presence of the vitamin E analogue or compounds 1-8. Parts C
and D illustratesimilar 2 h exposureofhRPEand HA-h cells, respectively, with1 mM Fenton’sreagent with/withoutthe presence ofthe vitamin E
analogueorcompounds1-8. GSH levelswere expressed asnmolofGSH/mg ofprotein, withvalues normalizedtoGSH levelsincells not exposed
to H2O2 (blank control, 100%). The results represent the mean( SD of three separate experiments. Significant differences (p>0.05), calculatedby
ANOVA, were compared to control staining obtained with ROS in the absence of either the vitamin E analogue or compounds 1-8.
measuredon a MolecularDevices SpectraMax Plus384 microplate
spectrophotometer (Molecular Devices, Sunnyvale, CA). Melt-
ing points were uncorrected. Column chromatography (CC)
utilized Merck silica gel (230-400 mesh). Final compound
purities were assessed as g99% and intermediate compound
purities were assessed as g96% by reverse phase HPLC using a
Calcd for C12H21N5O4S: C, 43.49; H, 6.39; N, 21.13; S, 9.68.
Found: C, 43.60; H, 6.47; N, 21.11; S, 9.85.
4-(5-Hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N0-dimethyl-
piperazine-1-sulfonamide (6). To a solution of compound 5 (1.66 g,
5 mmol) in 40 mL of THF at -78 °C and under an Ar atmosphere
was added 0.3 mL of n-BuLi (7 mmol). After the mixture was
stirred for 3 h at -60 °C, the Ar atmosphere was replaced with
oxygen. The reaction mixture was then gradually warmed to room
temperature (RT), and stirring was continued for an additional 6
h. After adjusting the pH to 6-7 with dilute HCl, THF was
removed by evaporation and the remaining aqueous layer was
extracted with CHCl3. The CHCl3 layers were washed with brine,
dried over Na2SO4, and filtered. Solvent evaporation gave a
yellow solid, which was purified by CC with 1:1:2 CHCl3/
EtOAc/hexane to yield 530 mg (30%) of compound 6, mp
113-114 °C. The structure was confirmed by 1H NMR (CDCl3)
δ 4.21 (s, 1H), 3.95 (s, 6H), 3.76 (s, 4H), 3.29 (s, 4H), 2.86 (s, 6H);
EI-MS (m/z) 347 (Mþ). Anal. Calcd for C12H21N5O5S: C, 41.49;
H, 6.09; N, 21.16; S, 9.23. Found: C, 43.61; H, 6.13; N, 19.96; S,
9.18.
˚
250 mm ꢀ 4.6 mm C18 Luna column (5 μ100 A) with a mobile
phase of 75:25 methanol/water at a flow rate of 0.9 mL/min and
detection at 220, 254, and 280 nm. Elemental analyses were
performed by M-H-W Laboratories, Phoenix, AZ.
N,N0-Dimethylsulfamoylpiperazine (10). 1 was obtained ac-
cording to the literature.63
General Procedure for the Preparation of N,N0-Dimethyl-4-
(pyrimidin-2-yl)piperazine-1-sulfonamide (1) and 4-(4,6-dimethoxy-
pyrimidin-2-yl)-N,N0-Dimethylpiperazine-1-sulfonamide (5). Et3N
(0.73 mL, 5.2 mmol) was added to 1.0 g of N,N0-dimethylsulfa-
moylpiperazine 10 (5.2 mmol) dissolved in 20 mL of THF. 2-
Chloro-4,6-dimethoxypyrimidine 11a (0.9 g, 5.2 mmol) dissolved
in 5 mL of THF was then added, and the mixture was refluxed
with stirring for 40 h. After the mixture was cooled, THF was
removed under vacuum and the remaining yellow solid was
dissolved in 300 mL of CHCl3, washed with 0.5 N HCl, water,
and brine, dried over Na2SO4, and filtered. Removal of solvent
and CC with 1:1 CHCl3/hexane gave 1.4 g (81%) of white
compound 1, mp 157-158 °C. 1H NMR (CDCl3) δ 8.34 (s, 1H),
8.33 (s, 1H), 6.55 (t, J = 4.88 Hz, 1H), 3.92 (appt, J = 5.13 Hz,
4H), 3.31 (appt, J = 5.13 Hz, 4H), 2.86 (s, 6H); 13C NMR
(CDCl3) 161.4, 157.3, 110.5, 46.1, 43.4, 38.2; ESI-MS (m/z) 294
([M þ Na]þ). Anal. Calcd for C10H17N5O2S: C, 44.26; H, 6.31;
N, 25.81; S, 11.82. Found: C, 44.12; H, 6.42; N, 25.58; S, 12.03.
The yield of compound 5, mp 103-105 °C, was 81%. 1H NMR
(CDCl3) δ 5.41 (s, 1H), 3.86 (s, 6H), 3.89-3.86 (m, 4H), 3.28
(appt, J = 4.88 Hz, 4H), 2.86 (s, 6H); 13C NMR (CDCl3) 172.0,
160.6, 78.5, 53.5, 46.2, 43.5, 38.2; EI-MS (m/z) 331 (Mþ). Anal.
N,N-Dimethyl-4-(5-nitropyrimidin-2-yl)piperazine-1-sulfon-
amide (16). Triethylamine (7.2 mL, 51.8 mmol) was added to
a solution of 10 (10 g, 51.8 mmol) dissolved in 400 mL of THF.
2-Chloro-5-nitropyrimidine 12 (7.7 g, 48.3 mmol) dissolved in
20mLof THF was added to the stirred mixture. After the mixture
was stirred for 24 h at RT, THF was removed under vacuum and
600 mL of CHCl3 was added to the residue. The CHCl3 layer was
washed with 0.5 N HCl, water, and brine, dried over Na2SO4, and
filtered. Solvent evaporation gave 14.1 g (92%) of straw-yellow
solid 16. 1H NMR (CDCl3) δ 9.08 (s, 2H), 4.09 (appt, J = 5.13
Hz, 4H), 3.35 (appt, J = 5.13 Hz, 4H), 2.87 (s, 6H).
4-(5-Aminopyrimidin-2-yl)-N,N-dimethylpiperazine-1-sulfon-
amide (17). NH4Cl (1.43 g, 26.76 mmol) was added to a suspen-
sion of 16 (14.1 g, 44.6 mmol) in a mixture of EtOH (175 mL) and

1124 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Jin et al.
H2O (48 mL), followed by iron powder (7.5 g, 133.9 mmol). After
refluxing 1 h, the reaction mixture was filtered and concentrated to
give a brown solid which was dissolved in 800 mL of CHCl3,
washed with NaHCO3 and brine, dried over Na2SO4, and filtered.
Solvent evaporation gave a yellow solid which after CC with 100:1
the solvent followed by CC using 25:1 hexane/EtOAc gave 16.57 g
(85%) of benzyloxy 29. 1H NMR (CDCl3) δ 7.41-7.31 (m, 5H),
5.00 (s, 2H), 3.98 (s, 6H); 13C NMR (CDCl3) 163.6, 150.5, 136.4,
128.5, 128.4, 128.3, 124.4, 74.8, 54.0.
N-Benzyl-5-(benzyloxy)-4,6-dimethoxypyrimidin-2-amine (30).
A mixture of 29 (3.18 g, 11.33 mmol), BnNH2 (2.2 mL, 20 mmol),
and K2CO3 (1.66 g, 12 mmol) in dioxane (60 mL) was refluxed for
4 days. The mixture was filtered and the filtrate concentrated in
vacuo to give a yellow oil, which after CC with 25:1 to 10:1
1
CHCl3/MeOH gave 11.5 g (90%) of solid yellow 17. H NMR
(CDCl3) δ 7.98 (s, 2H), 3.76 (t, J = 4.88 Hz, 4H), 3.30 (t, J = 4.88
Hz, 4H), 2.86 (s, 6H); EI-MS (m/z) 286 (Mþ).
4-(5-Hydroxypyrimidin-2-yl)-N,N-dimethylpiperazine-1-sulfon-
amide (2). A suspension of 17 (5.76 g, 20.2 mmol) in 100 mL of
2 M H2SO4 was refluxed for 1.5 h until the suspension became
clear. After the mixture was cooled to RT, the solution pH was
adjusted to 6-7 with 10 N NaOH, and the cloudy mixture was
extracted with EtOAc. The combined organic layers were washed
with brine, dried over Na2SO4, and filtered. Solvent evaporation
gave a yellow solid, which was purified by CC using 1:1 EtOAc/
hexane to yield 3.23 g (56%) of white solid compound 2, mp
134-135 °C. 1H NMR (CDCl3) δ 8.08 (s, 2H), 3.78 (appt, J =
5.13 Hz, 4H), 3.29 (appt, J = 5.13 Hz, 4H), 2.86 (s, 6H); 13C
NMR (CDCl3) 156.9, 145.8, 143.5, 46.0, 44.3, 38.1; EI-MS (m/z)
287 (Mþ). Anal. Calcd for C10H17N5O3S: C, 41.80; H, 5.96; N,
24.37; S, 11.16. Found: C, 41.60; H, 5.98; N, 24.16; S, 11.02.
5-Hydroxy-2-aminopyrimidine (25). Under argon at RT, BBr3
(2.6 mL, 50 mmol) was added dropwise to a solution of 23
(1.25 g, 10 mmol) in 60 mL of benzene, and the mixture was
refluxed for 6 h. After the mixture stood overnight at RT, the
solvent was evaporated and the residue was carefully treated
with 10 mL of ice cold H2O. The pH of the solution was adjusted
to 6-7 with 6 N NaOH and then extracted with EtOAc. The
EtOAc layers were washed with brine, dried over Na2SO4, and
filtered. Solvent evaporation gave a yellow solid, which after CC
using 20:1 CHCl3/MeOH gave 840 mg (75%) of white solid
5-hydroxy-2-aminopyrimidine 25. 1H NMR (DMSO-d6) δ 8.90
(s, 1H), 7.86 (s, 2H), 5.91 (s, 2H); EI-MS (m/z) 111 (Mþ).
5-Benzyloxy-2-aminopyrimidine (20c). K2CO3 (276 mg, 2
mmol) was added to 222 mg of 25 (2 mmol) in 5 mL of methanol,
followed by BnBr (0.24 mL, 2 mmol). After the mixture was
stirred for 14 h at RT, the reaction was stopped by addition of
water. Methanol was evaporated. The remaining aqueous layer
was extracted with CHCl3. The combined CHCl3 layers were
washed with brine, dried over Na2SO4, and filtered. Removal of
the solvent followed by CC using 67:1 CHCl3/MeOH yielded
300 mg (75%) of white solid 20c. 1H NMR (CDCl3) δ 8.08 (s,
2H), 7.40-7.32 (m, 5H), 5.03 (s, 2H), 4.77 (br s, 2H).
1
hexane/EtOAc gave 2.54 g (64%) of white solid 30. H NMR
(CDCl3) δ 7.45-7.24 (m, 10H), 5.10 (br s, 1H), 4.85 (s, 2H), 4.55
(d, J = 5.5 Hz, 2H), 3.86 (s, 6H); 13C NMR (CDCl3) 163.5, 156.0,
139.7, 137.5, 128.5, 128.4, 128.1, 127.8, 127.6, 127.1, 117.7, 75.2,
53.7, 45.9.
2-Amino-4,6-dimethoxypyrimidin-5-ol (31). Compound 30
(2.54 g, 7.24 mmol) in 40 mL of MeOH was hydrogenated for
12 h at RT in the presence of 635 mg of 10% Pd/C catalyst. After
filtration and solvent evaporation, a white solid was obtained,
which after CC using 50:1 CHCl3/MeOH yielded 1.21 g (98%)
of 31. 1H NMR (CDCl3) δ 4.50 (s, 2H), 4.30 (s, 1H), 3.93 (s, 6H).
5-(Benzyloxy)-4,6-dimethoxypyrimidin-2-amine (20d). To 4.16 g
of 31 (24.3 mmol) in MeOH (240 mL) was added K2CO3 (3.5 g, 25
mmol), followed by BnBr (3.0 mL, 25 mmol). After the mixture
was stirred for 12 h at RT, water was added to the reaction
mixture. MeOH was evaporated, and the water layer was ex-
tracted with CHCl3. The combined CHCl3 layers were washed
with brine, dried over Na2SO4, and filtered. Removal of CHCl3
gave a yellow solid, which after purification by CC with 10:1 to 2:1
hexane/EtOAc yielded 5.14 g (81%) of 2-amine-4,6-dimethoxy-5-
1
benzyloxypyrimidine 20d. H NMR (CDCl3) δ 7.44-7.27 (m,
5H), 4.86 (s, 2H), 4.60 (s, 2H), 3.87 (s, 6H).
2,20-(N,N-Dimethylsulfamoylazanediyl)diacetic Acid (19). An
amount of 26.62 g of the iminodiacetic acid 18 (200 mmol) was
added to 16.0 g of NaOH (400 mmol) in 50 mL of H2O at 0 °C,
followed by 21.2 g of Na2CO3 (200 mmol) in 100 mL of H2O.
Then dimethylsulfamoyl chloride (200 mmol) dissolved in
50 mL of acetone was slowly added with stirring and the stirring
was continued at RT. After 48 h, the reaction mixture was
extracted with EtOAc. The pH of the aqueous layer was
adjusted to 2 with 12 N HCl and extracted again with EtOAc.
The organic layer was dried over Na2SO4 and filtered. Solvent
evaporation gave 28.75 g (60%) of 19 as a white powder which
was recrystallization from EtOAc. The structure was confirmed
by 1H NMR (DMSO-d6) δ 12.81 (br s, 2H), 3.99 (s, 4H), 2.71 (s,
6H); 13C NMR (DMSO-d6) 170.9, 49.7, 37.8.
2-Chloro-5-hydroxy-4,6-dimethoxypyrimidine (28). To a stir-
red suspension of 10.44 g 2-chloro-4,6-dimethoxypyrimidine
11b, (60 mmol) in 200 mL of THF under argon at -78 °C was
added dropwise a solution of LDA (2 M in THF/heptane/ethyl
benzene, 60 mL, 120 mmol). To a separate round-bottomed
flask at -78 °C under argon containing tert-butyl hydroper-
oxide (5.5 M in decane, 22.7 mL,125 mmol) in 150 mL of THF
was added a solution of n-BuLi (1.6 M in hexane, 78 mL, 125
mmol). Then, after being stirred for 1 h, the hydroperoxide
anion solution was added via a double-ended needle to the
aromatic anion. The temperature was gradually raised to 0 °C,
with stirring continuing an additional 3 h. The reaction was
quenched with 6 N HCl until a pH of 7 was obtained. After THF
evaporation the remaining aqueous layer was extracted with
CHCl3. The CHCl3 layers were washed with brine, dried over
Na2SO4, and filtered. Removal of solvent and subsequent
purification by CC using 100:1 CHCl3/MeOH gave 10.67 g
(93.4%) of white solid 28. 1H NMR (CDCl3) δ 4.93 (s, 1H), 4.05
(s, 6H); 13C NMR (CDCl3) 158.1, 146.7, 123.0, 55.1.
General Procedure for Preparation of N,N0-Dimethyl-3,5-di-
oxo-4-(pyrimidin-2-yl)piperazine-1-sulfonamide (3), 4-(4,6-Dime-
thoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfon-
amide (7), 4-(5-(Benzyloxy)pyrimidin-2-yl)-N,N-dimethyl-3,5-dioxo-
piperazine-1-sulfonamide (21), and 4-(5-(Benzyloxy)-4,6-dimeth-
oxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfon-
amide (22). A suspension of1.44g of diacid 19 (6 mmol) in 8.0 mL
of Ac2O was heated to 85 °C for 5 min until the solution became
clear. After excess Ac2O was removed under vacuum at 60 °C, the
resulting anhydride was obtained as a yellow oil. The resulting
anhydride, in 10 mL of toluene, was then reacted with 475 mg of
2-aminopyrimidine 20a (5 mmol) dissolved in 7 mL of acetone by
heating the mixture to 80 °C for 6 h. Solvent evaporation gave the
amide as a yellow oil. Heating amide for 4 h at 80 °C with 8 mL of
Ac2O resulted into the final piperidine-2,6-ring product as a red,
clear solution. Removal of the remaining Ac2O under vacuum
at 60 °C followed by CC with 3:1:1 hexane/EtOAc/CHCl3 gave
760 mg (50.8%) of compound 3, mp179-181 °C. 1HNMR(CDCl3)
δ 8.90 (d, J = 4.88 Hz, 2H), 7.45 (t, J = 4.88 Hz, 1H), 4.31 (s, 4H),
2.96 (s, 6H); 13C NMR (CDCl3) 167.0, 159.5, 154.8, 121.4, 49.5, 38.3;
ESI-MS (m/z) 322 ([M þ Na]þ). Anal. Calcd for C10H13-
N5O4S: C, 40.13; H, 4.38; N, 23.40; S, 10.71. Found: C, 40.40; H,
4.54; N, 23.41; S, 10.66.
2-Chloro-5-(benzyloxy)-4,6-dimethoxypyrimidine (29). K2CO3
(10.0 g, 70 mmol) was added to 28 (13.24 g, 69.5 mmol) dissolved
in 200 mL of MeCN followed by BnBr (8.3 mL, 69.5 mmol). After
the mixture was stirred for 14 h at RT, the reaction was stopped by
addition of water. MeCN was evaporated, the remaining aqueous
layer was extracted withCHCl3. The combined CHCl3 layers were
washed with brine, dried over Na2SO4, and filtered. Removal of
The white solid of compound 7, mp 200-202 °C, was obtained
in 57.9%yield. 1H NMR (CDCl3) δ 6.10 (s, 1H), 4.30(s, 4H), 3.92

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1125
(s, 6H), 2.967 (s, 6H); 13C NMR (CDCl3) 172.8, 166.7, 152.8,
90.5, 54.7, 49.4, 38.2;ESI-MS(m/z) 382([M þ Na]þ). Anal. Calcd
for C12H17N5O6S: C, 40.11; H, 4.77; N, 19.49; S, 8.92. Found: C,
40.31; H, 4.84; N, 19.28; S, 9.10.
Crude compound 21 was purified by CC with 100:1 CHCl3/
1
MeOH, and white solid of 21 was obtained in 69% yield. H
15 mM HEPES buffer (Gibco, Carlsbad, CA) containing 10%
heat-inactivated FBS and 1% penicillin/streptavidin solution.
Human hippocampal astrocytes (HA-h), obtained from the
ScienCell Research Laboratories (Carlsbad, CA), were cultured
on poly-L-lysine coated containers (2 μg/cm2) with astrocyte
medium (ScienCell, Carlsbad, CA) composed of 500 mL of basal
medium, 5 mL of astrocyte growth supplement, and 5 mL of
penicillin/streptomycin solution. All cells were cultured at 37 °C
in a humidified atmosphere of 5% CO2 and 95% air until
subconfluent (2-4 days). Cells were passaged when 80-90%
confluent by treatment with trypsin-EDTA and then plated
at a density of 1 ꢀ 104 cells onto 96-well plates or 1 ꢀ 106 cells
onto 150 mm dishes. For compound evaluation, FBS-free media
(blank and control) or FBS-free media containing either 100 μM
compounds 1-8 or 100 μM water-soluble vitamin E analogue
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)
were added to each individual well/dish. After 1 h, an additional
aliquot of FBS-free media was added to the blank well/dish,
and a similar volume of FBS-free media containing 1 mM H2O2
was added to all remaining wells/dishes. For the Fenton studies,
addition of H2O2 was immediately followed by addition of 1 mM
ammonium iron(II) sulfate hexahydrate. After 2 h of exposure, all
media were removed and the cells were washed at room tempera-
ture with PBS. All studies were conducted in triplicate.
Cell viability studies were conducted in 96-well plates using
the CellTiter 96 AQueous One Solution Cell Proliferation Assay
(MTS, Promega, Madison, WI). Briefly, after 2 h of exposure to
either H2O2 or Fenton reagents, media were removed from all
wells and each well was washed with PBS. After removal of the
PBS wash, 100 μL of blank media was added to each well
followed by addition of 20 μL of the MTS solution. The cells
were further incubated at 37 °C for 1.5 h and then spectro-
photometrically evaluated at 490 nm in a plate reader. The
results were normalized to blank control (100%) of cells not
treated with H2O2.
NMR (CDCl3) δ 8.56 (s, 2H), 7.44-7.26 (m, 5H), 5.20 (s, 2H),
4.29 (s, 4H), 2.95 (s, 6H).
Crude compound 22 was purified by CC using 100:1 CHCl3/
MeOH, and pale-yellow solid of 22 was obtained in 41% yield.
1H NMR (CDCl3) δ 7.46-7.32 (m, 5H), 5.05 (s, 2H), 4.29 (s,
4H), 3.95 (s, 6H), 2.96 (s, 6H).
General Procedure for Preparation of 4-(5-Hydroxypyrimidin-
2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (4) and 4-
(5-Hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-di-
oxopiperazine-1-sulfonamide (8). Compound21 (2.0 g, 4.9 mmol)
dissolved in 100 mL of EtOAc was hydrogenated with 500 mg of
10% Pd/C catalyst at RT for 12 h. After filtration and solvent
evaporation, compound 4 recrystallized from EtOAc to give
1
1.83 g (80%) of a white powder, mp 225-226 °C. H NMR
(DMSO-d6) δ 11.0 (s, 1H), 8.45 (s, 2H), 4.39 (s, 4H), 2.85 (s, 6H);
13C NMR (CDCl3) 168.9, 152.7, 146.9, 146.1, 50.0, 38.5; ESI-MS
(m/z) 338 ([M þ Na]þ). Anal. Calcd for C10H13N5O5S: C, 38.09;
H, 4.16; N, 22.21; S, 10.17. Found: C, 38.18; H, 4.35; N, 21.98;
S, 9.93.
Crude compound 8 was purified by CC using 80:1 CHCl3/
MeOH to yield 1.15 g (91%) of white solid 8, mp 222-224 °C.
1H NMR (CDCl3) δ 9.67 (s, 1H), 4.44 (s, 4H), 3.88 (s, 6H), 2.51
(s, 6H); 13C NMR (CDCl3) 168.1, 159.4, 140.9, 124.7, 54.7, 49.3,
37.9; ESI-MS (m/z) 398 ([M þ Na]þ). Anal. Calcd for C12H17-
N5O7S: C, 38.40; H, 4.56; N, 18.66; S, 8.54. Found: C, 38.60; H,
4.66; N, 18.49; S, 8.58.
Chelation Studies. The relative amounts of ions bound by
compounds 1-8 were determined by electrospray ionization
mass spectrometry (ESI-MS) according to the method of Baron
and Hering.71 Stock solutions (1 mM) were prepared with
doubly distilled water from ammonium iron(II) sulfate hexa-
hydrate, iron(III) nitrate nonahydrate, copper(I) chloride,
copper(II) chloride dihydrate, zinc chloride, magnesium chlor-
ide, and calcium chloride. Each ion solution was mixed with
compounds 1-8, respectively, in ratios of 10:1, 1:1, and 1:10
metal ion/compound so that a final compound concentration of
50 μM was maintained. Forty-five minutes after mixing, each
solution was directly infused into the MS under constant
ionization energy.
The stoichiometry of the complexes of compounds 1-8 with
Fe2þ, Fe3þ, Cu1þ, Cu2þ, Zn2þ, Ca2þ, and Mg2þ was determined
using the method of continuous variation (Job plots).72 Because
of their low solubility, compounds 1-8 were first dissolved in
acetonitrile (HPLC grade). These concentrated solutions were
then diluted to 0.1 mM stock solutions with 1 mM acetate
(pH 6.5). In a typical experiment (at 23 °C), several solutions of
the compound and metal ion of interest were prepared at a
constant total concentration of compound and ion but with a
different mole fraction of one component. After an equilibration
period of 45 min, the change in UV absorbance was monitored
from 210 to 310 nm. The maximum absorbance for each
compound was then recorded as a function of the mole fraction
of the metal ion. Two linear dependences were obtained, at low
and high molar fractions, which intersect at a mole fraction
value that corresponds to the stoichiometry of the complex.
Cell Culture Studies. Human lens epithelial cells (SRA-1),
obtained as a gift from Dr. Reddy,69 were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bo-
vine serum (FBS), 4 mM L-glutamine, 1.5 g/L NaHCO3, and 1%
penicillin/streptomycin solution. Human retinal pigmented
epithelial cells (ARPE-19), obtained from the American Type
Culture Collection (Manassas, VA), were cultured in a 1:1
mixture of Dulbecco’s modified Eagle’s medium and Ham’s
F12 (DMEM/F12) medium with 2.5 mM L-glutamine and
GSH measurements were conducted on cells cultured in either
100 or 150 mm dishes. After 2 h of exposure to either H2O2 or
Fenton reagents, the media were removed and the cells were
washed with PBS. The cells were removed from each dish by
scraping and homogenized in glass homogenizers. Following
centrifugation at 4 °C, protein levels in the supernatant were
measured according to Bradford.73 The cell homogenates were
then deproteinized with equal volumes of 20% trichloroacetic
acid (TCA), and GSH levels were measured at 412 nm according
to the DTNB method.74 GSH levels were expressed as nmol of
GSH/mg of protein, with values subsequently normalized to
GSH levels in cells not exposed to H2O2 (blank control, 100%).
Acknowledgment. This work was supported by National
Institutes of Health Grant EY016460.
References
(1) Brewer, G. J. Iron and copper toxicity in diseases of aging,
particularly atherosclerosis and Alzheimer’s disease. Exp. Biol.
Med. (Maywood, NJ, U. S.) 2007, 232, 323–335.
(2) Gilca, M.; Stoian, I.; Atanasiu, V.; Virgolici, B. The oxidative
hypothesis of senescence. J. Postgrad. Med. 2007, 53, 207–213.
(3) Stadtman, E. R.; Oliver, C. N.; Levine, R. L.; Fucci, L.; Rivett, A. J.
Implication of protein oxidation in protein turnover, aging, and
oxygen toxicity. Basic Life Sci. 1988, 49, 331–339.
(4) Allsop, D.; Mayes, J.; Moore, S.; Masad, A.; Tabner, B. J. Metal-
dependent generation of reactive oxygen species from amyloid
proteins implicated in neurodegenerative disease. Biochem. Soc.
Trans. 2008, 36, 1293–1298.
(5) Smith, D. G.; Cappai, R.; Barnham, K. J. The redox chemistry of
the Alzheimer’s disease amyloid beta peptide. Biochim. Biophys.
Acta 2007, 1768, 1976–1990.
(6) Valko, M.; Morris, H.; Cronin, M. T. Metals, toxicity and oxida-
tive stress. Curr. Med. Chem. 2005, 12, 1161–1208.
(7) Brennan, L. A.; Kantorow, M. Mitochondrial function and redox
control in the aging eye: role of MsrA and other repair systems in
cataract and macular degenerations. Exp. EyeRes. 2009, 88, 195–203.

1126 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Jin et al.
(8) Dunaief, J. L.; Richa, C.; Franks, E. P.; Schultze, R. L.; Aleman,
T. S.; Schenck, J. F.; Zimmerman, E. A.; Brooks, D. G. Macular
degeneration in a patient with aceruloplasminemia, a disease
associated with retinal iron overload. Ophthalmology 2005, 112,
1062–1065.
(9) Richer, S.; Rudy, D.; Statkute, L.; Karofty, K.; Frankowski, J.
Serum iron, transferrin saturation, ferritin, and dietary data in age-
related macular degeneration. Am. J. Ther. 2002, 9, 25–28.
(10) Dunaief, J. L. Iron induced oxidative damage as a potential factor
in age-related macular degeneration: the Cogan lecture. Invest.
Ophthalmol. Visual Sci. 2006, 47, 4660–4664.
(11) Garner, B.; Roberg, K.; Qian, M.; Brunk, U. T.; Eaton, J. W.;
Truscott, R. J. Redox availability of lens iron and copper: implica-
tions for HO* generation in cataract. Redox Rep. 1999, 4,
313–315.
(12) Ecroyd, H.; Carver, J. A. Crystallin proteins and amyloid fibrils.
Cell. Mol. Life Sci. 2009, 66, 62–81.
Zorov, D. B. An attempt to prevent senescence: a mitochondrial
approach. Biochim. Biophys. Acta 2009, 1787, 437–461.
(29) Gupta, S. K.; Selvan, V. K.; Agrawal, S. S.; Saxena, R. Advances in
pharmacological strategies for the prevention of cataract develop-
ment. Indian J. Ophthalmol. 2009, 57, 175–183.
(30) Evans, J. Antioxidant supplements to prevent or slow down the
progression of AMD: a systematic review and meta-analysis. Eye
(London, U. K.) 2008, 22, 751–760.
(31) Evans, J. R.; Henshaw, K. Antioxidant Vitamin and Mineral
Supplements for Preventing Age-Related Macular Degenera-
tion. In Cochrane Database Of Systematic Reviews (Online); The
Cochrane Collaboration: Oxford, U.K., 2008; CD000253.
(32) House, E.; Collingwood, J.; Khan, A.; Korchazkina, O.; Berthon,
G.; Exley, C. Aluminium, iron, zinc and copper influence the in
vitro formation of amyloid fibrils of Abeta42 in a manner which
may have consequences for metal chelation therapy in Alzheimer’s
disease. J. Alzheimer’s Dis. 2004, 6, 291–301.
(33) CNS DRug DElivey. NIH Monogram; National Institutes of Health:
Bethesda, MD.
(13) Narayanan, S.; Kamps, B.; Boelens, W. C.; Reif, B. alphaB-
crystallin competes with Alzheimer’s disease beta-amyloid peptide
for peptide-peptide interactions and induces oxidation of Abeta-
Met35. FEBS Lett. 2006, 580, 5941–5946.
(14) Lee, S.; Carson, K.; Rice-Ficht, A.; Good, T. Hsp20, a novel alpha-
Crystallin, prevents Abeta fibril formation and toxicity. Protein
Sci. 2005, 14, 593–601.
(15) Smith, D. G.; Cappai, R.; Barnham, K. J. The redox chemistry of
the Alzheimer’s disease amyloid beta peptide. Biochim. Biophys.
Acta 2007, 1768, 1976–1990.
(16) Anderson, D. H.; Talaga, K. C.; Rivest, A. J.; Barron, E.; Hage-
man, G. S.; Johnson, L. V. Characterization of beta amyloid
assemblies in drusen: the deposits associated with aging and age-
related macular degeneration. Exp. Eye Res. 2004, 78, 243–256.
(17) Bruban, J.; Glotin, A. L.; Dinet, V.; Chalour, N.; Sennlaub, F.;
Jonet, L.; An, N.; Faussat, A. M.; Mascarelli, F. Amyloid-beta-
(1-42) alters structure and function of retinal pigmented epithelial
cells. Aging Cell 2009, 8, 162–177.
(18) Wang, J.; Ohno-Matsui, K.; Yoshida, T.; Shimada, N.; Ichinose,
S.; Sato, T.; Mochizuki, M.; Morita, I. Amyloid-beta up-regulates
complement factor B in retinal pigment epithelial cells through
cytokines released from recruited macrophages/microglia: another
mechanism of complement activation in age-related macular de-
generation. J. Cell. Physiol. 2009, 220, 119–128.
(19) Surolia, I.; Sinha, S.; Sarkar, D. P.; Reddy, P. Y.; Reddy, G. B.;
Surolia, A. Concurrence of Danish dementia and cataract: insights
from the interactions of dementia associated peptides with eye lens
alpha-crystallin. PLoS One 2008, 3, No. e2927.
(20) Goldstein, L. E.; Muffat, J. A.; Cherny, R. A.; Moir, R. D.;
Ericsson, M. H.; Huang, X.; Mavros, C.; Coccia, J. A.; Faget,
K. Y.; Fitch, K. A.; Masters, C. L.; Tanzi, R. E.; Chylack, L. T., Jr.;
Bush, A. I. Cytosolic beta-amyloid deposition and supranuclear
cataracts in lenses from people with Alzheimer’s disease. Lancet
2003, 361, 1258–1265.
(34) Wong, R. W.; Richa, D. C.; Hahn, P.; Green, W. R.; Dunaief, J. L.
Iron toxicity as a potential factor in AMD. Retina 2007, 27, 997–1003.
(35) Avunduk, A. M.; Yardimci, S.; Avunduk, M. C.; Kurnaz, L.
Cataractous changes in rat lens following cigarette smoke exposure
is prevented by parenteral deferoxamine therapy. Arch. Ophthal-
mol. 1999, 117, 1368–1372.
(36) Melov, S.; Wolf, N.; Strozyk, D.; Doctrow, S. R.; Bush, A. I. Mice
transgenic for Alzheimer disease beta-amyloid develop lens catar-
acts that are rescued by antioxidant treatment. Free Radical Biol.
Med. 2005, 38, 258–261.
(37) Cuajungco, M. P.; Lees, G. J. Diverse effects of metal chelating
agents on the neuronal cytotoxicity of zinc in the hippocampus.
Brain Res. 1998, 799, 97–107.
(38) Finefrock, A. E.; Bush, A. I.; Doraiswamy, P. M. Current status of
metals as therapeutic targets in Alzheimer’s disease. J. Am. Geriatr.
Soc. 2003, 51, 1143–1148.
(39) Clifford Rose, F.; Gawel, M. Clioquinol neurotoxicity: an over-
view. Acta Neurol. Scand. Suppl. 1984, 100, 137–145.
(40) Treiber, C.; Simons, A.; Strauss, M.; Hafner, M.; Cappai, R.;
Bayer, T. A.; Multhaup, G. Clioquinol mediates copper uptake and
counteracts copper efflux activities of the amyloid precursor
protein of Alzheimer’s disease. J. Biol. Chem. 2004, 279, 51958–
51964.
(41) Raman, B.; Ban, T.; Yamaguchi, K.; Sakai, M.; Kawai, T.; Naiki,
H.; Goto, Y. Metal ion-dependent effects of clioquinol on the fibril
growth of an amyloid {beta} peptide. J. Biol. Chem. 2005, 280,
16157–16162.
(42) Jenagaratnam, L.; McShane, R. Clioquinol for the Treatment of
Alzheimer’s Disease. In Cochrane Database of Systematic Reviews
(Online); The Cochrane Collaboration: Oxford, U.K., 2006.
(43) Crouch, P. J.; Barnham, K. J.; Bush, A. I.; White, A. R. Ther-
apeutic treatments for Alzheimer’s disease based on metal bioavail-
ability. Drug News Perspect. 2006, 19, 469–474.
(44) Lannfelt, L.; Blennow, K.; Zetterberg, H.; Batsman, S.; Ames, D.;
Harrison, J.; Masters, C. L.; Targum, S.; Bush, A. I.; Murdoch, R.;
Wilson, J.; Ritchie, C. W. Safety, efficacy, and biomarker findings
of PBT2 in targeting Abeta as a modifying therapy for Alzheimer’s
disease: a phase IIa, double-blind, randomised, placebo-controlled
trial. Lancet Neurol. 2008, 7, 779–786.
(45) Adlard, P. A.; Cherny, R. A.; Finkelstein, D. I.; Gautier, E.; Robb,
E.; Cortes, M.; Volitakis, I.; Liu, X.; Smith, J. P.; Perez, K.;
Laughton, K.; Li, Q. X.; Charman, S. A.; Nicolazzo, J. A.; Wilkins,
S.; Deleva, K.; Lynch, T.; Kok, G.; Ritchie, C. W.; Tanzi, R. E.;
Cappai, R.; Masters, C. L.; Barnham, K. J.; Bush, A. I. Rapid
restoration of cognition in Alzheimer’s transgenic mice with
8-hydroxy quinoline analogs is associated with decreased intersti-
tial Abeta. Neuron 2008, 59, 43–55.
(46) Kupershmidt, L.; Weinreb, O.; Amit, T.; Mandel, S.; Carri, M. T.;
Youdim, M. B. Neuroprotective and neuritogenic activities of
novel multimodal iron-chelating drugs in motor-neuron-like
NSC-34 cells and transgenic mouse model of amyotrophic lateral
sclerosis. FASEB J. 2009, 23, 3766–3779.
(21) Meehan, S.; Berry, Y.; Luisi, B.; Dobson, C. M.; Carver, J. A.;
MacPhee, C. E. Amyloid fibril formation by lens crystallin proteins
and its implications for cataract formation. J. Biol. Chem. 2004,
279, 3413–3419.
(22) Frank, B.; Gupta, S. A review of antioxidants and Alzheimer’s
disease. Ann. Clin. Psychiatry 2005, 17, 269–286.
(23) Garcia-Arencibia, M.; Gonzalez, S.; de Lago, E.; Ramos, J. A.;
Mechoulam, R.; Fernandez-Ruiz, J. Evaluation of the neuropro-
tective effect of cannabinoids in a rat model of Parkinson’s disease:
importance of antioxidant and cannabinoid receptor-independent
properties. Brain Res. 2007, 1134, 162–170.
(24) Bolognesi, M. L.; Minarini, A.; Tumiatti, V.; Melchiorre, C. Lipoic
acid, a lead structure for multi-target-directed drugs for neurode-
generation. Mini-Rev. Med. Chem. 2006, 6, 1269–1274.
(25) Binienda, Z.; Przybyla-Zawislak, B.; Virmani, A.; Schmued, L.
L-Carnitine and neuroprotection in the animal model of mitochon-
drial dysfunction. Ann. N.Y. Acad. Sci. 2005, 1053, 174–182.
(26) Tauskela, J. S. MitoQ;a mitochondria-targeted antioxidant.
IDrugs 2007, 10, 399–412.
(27) Perluigi, M.; Joshi, G.; Sultana, R.; Calabrese, V.; De Marco, C.;
Coccia, R.; Butterfield, D. A. In vivo protection by the xanthate
tricyclodecan-9-yl-xanthogenate against amyloid beta-peptide
(1-42)-induced oxidative stress. Neuroscience 2006, 138, 1161–
1170.
(47) Weinreb, O.; Mandel, S.; Bar-Am, O.; Yogev-Falach, M.; Avra-
movich-Tirosh, Y.; Amit, T.; Youdim, M. B. Multifunctional
neuroprotective derivatives of rasagiline as anti-Alzheimer’s dis-
ease drugs. Neurotherapeutics 2009, 6, 163–174.
(28) Skulachev, V. P.; Anisimov, V. N.; Antonenko, Y. N.; Bakeeva,
L. E.; Chernyak, B. V.; Erichev, V. P.; Filenko, O. F.; Kalinina,
N. I.; Kapelko, V. I.; Kolosova, N. G.; Kopnin, B. P.; Korshunova,
G. A.; Lichinitser, M. R.; Obukhova, L. A.; Pasyukova, E. G.;
Pisarenko, O. I.; Roginsky, V. A.; Ruuge, E. K.; Senin, I. I.;
Severina, I. I.; Skulachev, M. V.; Spivak, I. M.; Tashlitsky,
V. N.; Tkachuk, V. A.; Vyssokikh, M. Y.; Yaguzhinsky, L. S.;
(48) Silver, H.; Chertkow, Y.; Weinreb, O.; Danovich, L.; Youdim, M.
Multifunctional pharmacotherapy: what can we learn from study
of selective serotonin reuptake inhibitor augmentation of anti-
psychotics in negative-symptom schizophrenia? Neurotherapeutics
2009, 6, 86–93.
(49) Forni, G. L.; Balocco, M.; Cremonesi, L.; Abbruzzese, G.; Parodi,
R. C.; Marchese, R. Regression of symptoms after selective iron

Article
chelation therapy in a case of neurodegeneration with brain iron
accumulation. Movement Disord. 2008, 23, 904–907.
(50) Kruck, T. P.; Burrow, T. E. Synthesis of feralex a novel aluminum/
iron chelating compound. J. Inorg. Biochem. 2002, 88, 19–24.
(51) Kruck, T. P.; Percy, M. E.; Lukiw, W. J. Metal sulfate-
mediated induction of pathogenic genes and repression by
phenyl butyl nitrone and feralex-G. NeuroReport 2008, 19, 245–
249.
(52) Brewer, G. J. The risks of free copper in the body and the
development of useful anticopper drugs. Curr. Opin. Clin. Nutr.
Metab. Care 2008, 11, 727–732.
(53) Lee, J. Y.; Friedman, J. E.; Angel, I.; Kozak, A.; Koh, J. Y. The
lipophilic metal chelator DP-109 reduces amyloid pathology in
brains of human beta-amyloid precursor protein transgenic mice.
Neurobiol. Aging 2004, 25, 1315–1321.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1127
(61) Pedulli, G.; Franco, L. M. Thermochemical and kinetic studies of
antioxidants. Trends Org. Chem. 2001, 9, 97–105.
(62) Zaidi, S. A.; Shahjahan, A.; Sharif, M.; Siddiqi, K. S. Transition
metal complexes of 2-(N-succinimidyl)pyrimidine. Synthesis and
reactivity. Inorg. Met.-Org. Chem. 1993, 23, 1571–1584.
(63) Fourmaintraux, E.; Depreux, P.; Lesieur, I. Synthesis of new phthal-
azinyl compounds as potential inhibitors of aldose reductase and
sorbitol dehydrogenase. Heterocycl. Commun. 1999, 33, 313–316.
(64) Parker, K. A.; Koziski, K. A. Directed hydroxylation of aromatics.
J. Org. Chem. 1987, 51, 674–676.
(65) Kizner, T. A.; Mikhaleva, M. A.; Serebryakova, E. S. Selective acid
hydrolysis of 2-substituted-5-dimethylaminomethyleneaminopyr-
imidines to 5-amino-and 5-hydroxypyrimidines. Chem. Heterocycl.
Compd. 1990, 6, 668–670.
(66) Henry, D. W. A. Facile synthesis of piperazines from primary
(54) Scott, L. E.; Orvig, C. Medicinal inorganic chemistry approaches
to passivation and removal of aberrant metal ions in disease. Chem.
Rev. 2009, 109, 4885–4910.
(55) Bush, A. I.; Tanzi, R. E. Therapeutics for Alzheimer’s disease
based on the metal hypothesis. Neurotherapeutics 2008, 5, 421–
432.
amines (1). J. Heterocycl. Chem. 1966, 3, 503–511.
(67) Priewe, H.;Gutsche, K. 5-Alkoxy-2-aminopyrimidines. DE1145622,
1963.
(68) Julia, M.; Pfeuty-Saint Jalmes, V.; Ple, K.; Verpeaux, J.-N. Hydro-
xylation of carbanions with lithium tert-butyl peroxide acting as an
oxenoid. Bull. Soc. Chim. Fr. 1996, 133, 15–24.
(56) Kador, P. F.; Jin, H. Multifunctional Compounds and Methods of
(69) Ibaraki, N.; Chen, S. C.; Lin, L. R.; Okamoto, H.; Pipas, J. M.;
Reddy, V. N. Human lens epithelial cell line. Exp. Eye Res. 1998,
67, 577–585.
(70) Dunn, K. C.; Aotaki-Keen, A. E.; Putkey, F. R.; Hjelmeland, L. M.
ARPE-19, a human retinal pigment epithelial cell line with differ-
entiated properties. Exp. Eye Res. 1996, 62, 155–169.
(71) Baron, D.; Hering, J. G. Analysis of metal-EDTA complexes by
electrospray mass. spectrometry. J. Environ. Qual. 1998, 27, 844–850.
(72) Huang, C. Y. Determination of binding stoichiometry by the
continuous variation method: the Job plot. Methods Enzymol.
1982, 87, 509–525.
(73) Bradford, M. M. A rapid and sensitive method for the quantita-
tion of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 1976, 72, 248–254.
(74) Lou, M. F.; Dickerson, J. E., Jr. Protein-thiol mixed disulfides in
human lens. Exp. Eye Res. 1992, 55, 889–896.
Use Thereof. Patent Appl. 20090105269, 2009.
(57) Kador, P. F.; Inoue, J.; Blessing, K. Anticataract activity of
analogs of a sorbitol dehydrogenase inhibitor. J. Ocul. Pharmacol.
Ther. 2004, 20, 333–344.
(58) Gino, A.; DiLabio, G. A.; Brigati, G.; Pedulli, G. F.; Valgimigli, L.
5-Pyrimidinols: novel chain-breaking antioxidants more effective
than phenols. J. Am. Chem. Soc. 2001, 123, 4625–4626.
(59) Bakalbassis, E. G.; Lithoxoidou, A. T.; Vafiadis, A. P. Theoretical
calculation of accurate absolute and relative gas- and liquid-phase
O-H bond dissociation enthalpies of 2-mono- and 2,6-disubsti-
tuted phenols, using DFT/B3LYP. J. Phys. Chem. A 2003, 107,
8594–8606.
(60) Jinno, S.; Okita, T. Synthesis and structure-activity relationships
of phaffiaol and related antioxidants. Chem. Pharm. Bull. 1998, 46,
1688–1694.