An optimized pyrimidinol multifunctional radical quencher

Article abstract of DOI:10.1021/ml400130z

A series of aza analogues (4-9) of the experimental neuroprotective drug idebenone (1) have been prepared and evaluated for their ability to attenuate oxidative stress induced by glutathione depletion and to compensate for the decrease in oxidative phosphorylation efficiency in cultured Friedreich's ataxia (FRDA) fibroblasts and lymphocytes and also coenzyme Q₁₀-deficient lymphocytes. Modification of the redox core of the previously reported 3 improved its antioxidant and cytoprotective properties. Compounds 4-9, having the same redox core, exhibited a range of antioxidant activities, reflecting side chain differences. Compounds having side chains extending 14-16 atoms from the pyrimidinol ring (6, 7, and 9) were potent antioxidants. They were superior to idebenone and more active than 3, 4, 5, and 8. Optimized analogue 7 and its acetate (7a) are of interest in defining potential therapeutic agents capable of blocking oxidative stress, maintaining mitochondrial membrane integrity, and augmenting ATP levels. Compounds with such properties may find utility in treating mitochondrial and neurodegenerative diseases such as FRDA and Alzheimer's disease.

Full text of DOI:10.1021/ml400130z

Letter
pubs.acs.org/acsmedchemlett
An Optimized Pyrimidinol Multifunctional Radical Quencher
Omar M. Khdour, Pablo M. Arce, Basab Roy, and Sidney M. Hecht*
Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe,
Arizona 85287, United States
S
* Supporting Information
ABSTRACT: A series of aza analogues (4−9) of the experimental
neuroprotective drug idebenone (1) have been prepared and evaluated
for their ability to attenuate oxidative stress induced by glutathione
depletion and to compensate for the decrease in oxidative phosphorylation
efficiency in cultured Friedreich’s ataxia (FRDA) fibroblasts and
lymphocytes and also coenzyme Q10-deficient lymphocytes. Modification
of the redox core of the previously reported 3 improved its antioxidant and cytoprotective properties. Compounds 4−9, having
the same redox core, exhibited a range of antioxidant activities, reflecting side chain differences. Compounds having side chains
extending 14−16 atoms from the pyrimidinol ring (6, 7, and 9) were potent antioxidants. They were superior to idebenone and
more active than 3, 4, 5, and 8. Optimized analogue 7 and its acetate (7a) are of interest in defining potential therapeutic agents
capable of blocking oxidative stress, maintaining mitochondrial membrane integrity, and augmenting ATP levels. Compounds
with such properties may find utility in treating mitochondrial and neurodegenerative diseases such as FRDA and Alzheimer’s
disease.
KEYWORDS: Mitochondrial dysfunction, electron transport chain, lipid peroxidation, cytoprotection, adenosine triphosphate
itochondrial dysfunction is linked to numerous neuro-
degenerative diseases including Alzheimer’s disease,
M
Parkinson’s disease, Huntington’s disease, and Friedreich’s
ataxia.1−6 Mitochondria are vitally important organelles
involved in many essential cellular functions, notably energy
metabolism. They produce 90% of our cellular ATP through
oxidative phosphorylation within the mitochondrial respiratory
chain.7−11 Within mitochondria, the primary site of reactive
oxygen species (ROS) generation is the electron transport
chain;12,13 normally, mitochondria have an extensive network of
antioxidant and detoxification systems, ensuring that lipid
peroxidation and levels of ROS are kept at physiologically
acceptable levels.14,15 Defects in the mitochondrial respiratory
chain can undoubtedly lead to increased electron leakage and
consequently to increased ROS production, causing progressive
oxidative damage and ultimately cell death.2−5,10,16
Figure 1. Structures of compounds synthesized and studied.
The antioxidant and bioenergetic effects of coenzyme Q
(CoQ10) are well-known,17,18 but its clinical utility is limited by
its extreme hydrophobicity, which results in low bioavaibil-
ity.19,20 To facilitate the delivery of such molecules to the
mitochondria of cells, we have designed antioxidants bearing
smaller lipophilic side chains and having pyrimidinol21 or
pyridinol22−26 redox cores, based on earlier studies.27−30 We
demonstrated that an aza analogue of idebenone (3, Figure 1),
having the 1,4-benzoquinone core replaced with a pyrimidinol
core, retained the ability to function within the mitochondria.21
Because these aza analogues function at a number of levels to
suppress damage that would otherwise be caused by electron
leakage from the electron transport chain, we have denoted
them multifunctional radical quenchers.21 Recently, we
described the importance of side chain optimization to improve
antioxidant activity and the ability to maintain ATP levels in
cellular mitochondrial disease models in comparison with 3.26
These encouraging results suggested that further optimization
of the redox core and the side chain might afford multifunc-
tional radical quenchers exhibiting improved potency and
efficacy.
Presently, we describe the preparation and characterization of
six new pyrimidinol derivatives (4−9). These compounds have
redox cores different than that in previously reported 3;21 the
methyl group ortho to the phenolic OH has been replaced with
a methoxyl group. They also have modified side chains of
varying lengths lacking the hydroxyl group present in
Received: April 5, 2013
Accepted: May 28, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml400130z | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
idebenone and 3 (Figure 1). Compounds 1−9 were evaluated
for their ability to suppress lipid peroxidation, maintain
mitochondrial membrane potential, confer cytoprotection to
cultured cells under induced oxidative stress, and support
electron transport through the respiratory chain as judged by an
increase in ATP levels.
The preparation of 1 and 3 have been described
previously.21,31 Decylubiquinone (2) was commercially avail-
able. The syntheses of the 2-(N,N-dimethylamino)-4-alkyl-6-
methoxypyrimidin-5-ols (4, 5, and 7) were accomplished using
the strategy exemplified in Scheme 1 for 7 and its acetate 7a.
Scheme 1. Route Employed for the Synthesis of 7 and Its
Acetate (7a)
These were prepared in four and five steps, respectively. First,
commercially available 2-amino-6-methoxy-4-methylpyrimidine
was treated with methyl iodide in the presence of sodium
hydride to afford 10 in 63% yield. Then, 10 was monoalkylated
on the C-4 methyl group by treatment with n-BuLi in the
presence of pentadecyl bromide, affording 11 in 62% yield.
Aminopyrimidine 11 was then brominated at position 5 to
obtain 5-bromo-2-(N,N-dimethylamino)-4-hexadecyl-6-me-
thoxypyrimidine (12) in 95% yield. Finally, 7 was obtained in
55% yield by treating 12 successively with n-BuLi in the
presence of N,N,N′,N′-tetramethylenediamine (TMEDA), then
with trimethyl borate, and finally with hydrogen peroxide.
Compound 7 was O-acetylated in 80% yield to obtain 7a.
Compounds 4 and 5 were prepared in the same fashion,
using the appropriate alkyl bromides for the alkylation of
intermediate 10 (Scheme S1, Supporting Information). For the
preparation of 6, 8, and 9, a slightly different strategy was
employed (Scheme S2, Supporting Information). Specifically,
the alkylation of 10 was carried out with allyl bromide,
affording 2-(N,N-dimethylamino)-4-(3-butenyl)-6-methoxypyr-
imidine in 50% yield. Following conversion of this compound
to key intermediate 5-benzyloxy-2-(N,N-dimethylamino)-4-(3-
butenyl)-6-methoxypyrimidine, the side chains of 6, 8, and 9
were introduced by cross-metathesis reactions using the
appropriate terminal alkenes and Grubbs’ second generation
catalyst.32 The characterization of all new compounds included
high-resolution mass spectrometry.
The antioxidant and bioenergetics properties of the newly
prepared pyrimidinol analogues were tested and analyzed in
selected biochemical and biological assays. The test compounds
were dissolved in DMSO and added to the assay medium at
final DMSO concentrations <0.5%. The ability of the
pyrimidinol analogues to quench lipid peroxidation was studied
in FRDA lymphocytes that had been depleted of glutathione by
treatment with diethyl maleate (DEM). The fluorescent lipid
peroxidation-sensitive fatty acid-conjugated dye (C-11-BODI-
PY581/591) probe was used as described previously.22,24,25 Figure
2 shows representative C11-BODIPY581/591-green (oxidized)
FACS (fluorescence-activated cell sorting) histogram overlays
Figure 2. Lipid peroxidation in FRDA lymphocyte cells depleted of
glutathione was detected by utilizing the oxidation-sensitive fatty acid
probe C11-BODIPY581/591 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-
bora-3a,4a-diaza-s-indacene-3-propionic acid) using FACS. Increased
C11-BODIPY-green fluorescence (oxidized form), a measure of
intracellular lipid peroxidation, was determined by increasing the
geometric mean fluorescence intensity (GMFI) of C11-BODIPY-green
relative to the untreated control. A bar graph representing the
percentage of lipid peroxidation scavenging activity is shown. Data are
expressed as the mean SEM (n = 3).
B
dx.doi.org/10.1021/ml400130z | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
of FRDA lymphocyte cells stained with C11-BODIPY581/591-red
(reduced) and analyzed using the FL1-H channel, as described
in the Supporting Information. DEM treatment caused the C11-
BODIPY581/591-green fluorescence to shift right on the x-axis of
the FACS histogram, indicating increased membrane perox-
idation as a result of glutathione depletion. Optimization of the
redox core by replacing the methyl group ortho to the phenolic
OH (3) with a better electron donating (methoxyl) group, as in
4, significantly increased its ability to quench lipid peroxidation
(Figure 2). These results are in agreement with the ability of
more electron donating groups to lower the bond dissociation
energy of the O−H bond, as described by Valgimigli et al.33 We
have recently reported the importance of side chain length on
the interaction of coenzyme Q analogues with the mitochon-
drial respiratory chain to achieve improved antioxidant
activity.26 In the light of these findings, we prepared analogues
having different side chain lengths attached to the modified
redox core. As shown (Figure 2), the optimal length for
antioxidant activity was between 14 and 16 atoms. The
analogue with an octadecyl side chain (8) had significantly
reduced ability to quench lipid peroxidation in comparison to
14 and 16 atom side chains (6, 7, and 9).
The ability of the pyrimidinol analogues to preserve
mitochondrial membrane integrity following treatment of
FRDA lymphocytes with diethyl maleate (DEM) was assessed
by FACS analysis of mitochondrial membrane potential (Δψm)
using the cationic fluorescent probe tetramethylrhodamine
methyl ester (TMRM), as described previously.24,25 Represen-
tative flow cytometric two-dimensional color density dot plot
analyses of the mitochondrial membrane potential measure-
ments in the presence and absence of selected compounds are
shown (upper panel, Figure 3). The percentage of TMRM
stained cells with intact mitochondrial membrane potential
appears in the top right quadrant of individual treatments.
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone
(FCCP), a commonly used uncoupler of oxidative phosphor-
ylation in mitochondria, was employed as positive control to
dissipate the chemiosmotic proton gradient, which results in
lowering of TMRM fluorescence as a result of the
depolarization of mitochondrial inner membrane potential.
The bar graph in the lower panel of Figure 3 summarizes the
percentage of the cells with intact Δψm of the flow cytometric
profiles. It shows clearly that 6, 7, and 9 had the greatest
potency. Compound 4, having the idebenone side chain, was
slightly better than compound 3 in this assay. Idebenone was
less active than 3 or 4 (Figure 3). Decylubiquinone (2) and 5
afforded significant protection of mitochondrial membrane
integrity, as shown in Figure 3. Compound 8, having an 18
carbon side chain, was as effective as 3 and 4.
The ability of the compounds to protect FRDA skin
fibroblasts from oxidative damage-induced death was measured
as described previously with some modification.34,35 The cells
were subjected to L-buthionine (S,R)-sulfoximine (BSO), an
inhibitor of de novo glutathione (GSH) biosynthesis.36 The
half-maximal effective concentrations (EC50) were determined
(Table 1). Oxidative damage-induced death of FRDA
fibroblasts was blocked by exogenous antioxidants.34,35
Compound 6, 7, and 9 were by far the most efficient, having
EC50 values of 22 7, 25 4, and 32 4 nM, respectively
(Table 1). Again, in this assay the optimal side chain length was
between 14 and 16 atoms. Compounds having a side chain with
less than 14 or more than 16 atoms were less protective in this
assay. Compounds 3 and 4, having the idebenone side chain,
Figure 3. Representative flow cytometric two-dimensional color
density dot plot analyses of the ability of 1−9 to maintain
mitochondrial membrane potential (Δψm) in DEM-treated FRDA
lymphocyte cells stained with 250 nM TMRM and analyzed using the
FL2-H channel as described in the Supporting Information. The
percentage of cells with intact Δψm is indicated in the top right
quadrant of captions. Representative examples from at least three
independent experiments are shown. A total of 10,000 events were
recorded for each sample and analyzed with the CellQuest software
(BD Biosciences). A bar graph representing the percentage of the cells
with intact Δψm is shown. Data are expressed as means SEM of
three independent experiments run in duplicate.
C
dx.doi.org/10.1021/ml400130z | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
heart mitochondria.26 Our earlier study suggested that
compounds having lipophilic side chains exhibit lesser
inhibition of mitochondrial respiratory chain complexes and
correspondingly permit more effective maintenance of cellular
ATP levels.26 In that study, two pyrimidinol analogues having
lipophilic side chains were shown to be able to increase ATP
levels in CoQ10-deficient lymphocytes, albeit not in Friedreich’s
ataxia lymphocytes, when used at low concentration.
Table 1. Effect of Pyrimidinol Antioxidants on the Cellular
Viability of FRDA Fibroblasts Treated with BSO
compd
EC50 (nM)
551 23
idebenone (1)
decylubiquinone (2)
56
4
3
456 22
345 21
4
5
68
22
25
8
7
4
In the present study, 1−9 were evaluated for their ability to
enhance ATP levels in CoQ10 deficient lymphocytes and in
FRDA lymphocytes. As noted above, idebenone (1) and
decylubiquinone (2) simply diminished ATP levels in a
concentration-dependent fashion. Compound 3, which was
included in the earlier study,26 had the same effect as did 4.
Compound 5, having a 6-methoxyl substituent on the redox
core and a 4-decyl substituent noted to be suboptimal for the
assays described above, also failed to increase ATP levels.
Compound 8, whose side chain was found to be suboptimal in
the assays described above, was found to be without effect on
ATP levels in either of the lymphocyte cell lines studied. In
comparison, 6, 7, 7a, and 9 all effected a statistically significant
increase in ATP levels when tested at 5 μM concentration in
both CoQ10-deficient lymphocytes and FRDA lymphocytes.
The most favorable response was noted for 7 in CoQ10-
deficient lymphocytes, namely, 118 2% of control. The cell
line used for this experiment (GM17932, Coriell Cell
Repository) has been shown previously to have an ATP level
that is about 20% lower than normal human lymphocytes,26 so
7 effected essentially full restoration of ATP levels.
6
7
8
266 19
32
>50000
9
4
pyrimidinol redox core
were both more efficient than idebenone, but less effective than
decylubiquinone and 5 (Table 1).
In this study, we used a nutrient-sensitized screening strategy
to identify CoQ10 analogues that function within the
mitochondrial respiratory chain by measuring the total ATP
level in galactose-containing media.37 Total cellular ATP was
measured in cultured FRDA and CoQ10-deficient cell lines as
described previously.26 The cells were grown on glucose-free
media supplemented with galactose. Since cells grown in
galactose rely mostly on oxidative phosphorylation (OXPHOS)
to produce their ATP, they become more sensitive to
mitochondria respiratory chain inhibitors than cells grown in
glucose medium.37,38 Compound 7 and its acetate (7a)
increased ATP levels in the CoQ10-deficient lymphocytes
above the basal level when used at 5 and 10 μM concentrations
and slightly lowered ATP levels in both cell lines at 25 μM
(Table 2). Idebenone strongly diminished ATP levels in a
concentration-dependent fashion in the CoQ10 deficient
lymphocytes, as well as in Friedreich’s ataxia lymphocytes
(Table 2). In the presence of 25 μM idebenone, ATP
concentrations were essentially completely depleted. Decylubi-
quinone (2), which differs from idebenone only by the absence
of the side chain OH group, showed an ATP level reduced by
37% at 25 μM concentration compared to the control. There
was no increase in ATP levels at the lower concentrations (5
and 10 μM) beyond the basal level. The reduction in ATP
levels in idebenone-treated cells likely reflects the ability of
idebenone to inhibit complex I activity in the mitochondrial
electron transport chain, as we have shown previously in bovine
Given the important roles of CoQ10 in mitochondria,17,18 we
assessed the effects of idebenone (1), 4, and 7 on FRDA
fibroblasts in which CoQ10 had been depleted pharmacologi-
cally. Treatment of cultured mammalian cells with 4-nitro-
benzoate, which competitively inhibits 4-hydroxybenzoate:po-
lyprenyltransferase (COQ2),39 was reported to decrease CoQ10
biosynthesis compared to control.39−41 Analogous treatment of
FRDA fibroblasts with 1 mM 4-nitrobenzoate depleted CoQ10
levels by 29% compared to control (Figure S1a, Supporting
Information) and reduced ATP levels by 20% compared to
control (Figure S1b, Supporting Information). Co-treatment of
FRDA fibroblasts with 1 mM 4-nitrobenzoate and 5 μM
compound 7 restored ATP, essentially to normal levels (Figure
S2, Supporting Information), while 4 having the idebenone side
chain was less effective at 5 μM concentration and ineffective at
Table 2. Total ATP Concentration in CoQ10 Deficient Lymphocytes and FRDA Lymphocytes Following Incubation with
Compounds 1−9 for 48 h
total ATP level (% of control)
CoQ10 deficient lymphocytes
Friedreich’s ataxia lymphocytes
compd
5 μM
100
10 μM
25 μM
5 μM
100
10 μM
25 μM
untreated control
100
100
100
100
idebenone (1)
80
3
3
5
46
86
86
7
7
2
2
6
3
2
1
73
7
4
5
4
7
46
4
2
4
5
2
4
4
3
1
decylubiquinone (2)
100
99
6
2
63
46
54
58
65
90
96
96
64
4
4
4
6
5
8
6
4
5
93
76
63
28
46
58
55
80
92
102
72
6
2
5
5
3
4
4
3
77
46
4
102
96
95
83
80
66
5
94
87
6
105
118
116
98
3
2
4
97
106
109
98
109
108
109
101
107
4
5
5
2
3
97
7
9
4
96
7a
8
101
102
103
4
3
3
3
3
5
3
9
105
2
97
3
D
dx.doi.org/10.1021/ml400130z | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(8) McBride, H. M.; Neuspiel, M.; Wasiak, S. Mitochondria: more
than just a power house. Curr. Biol. 2006, 16, R551−R560.
(9) Bras, M.; Queenan, B.; Susin, S. A. Programmed cell death via
mitochondria: different modes of dying. Biochemistry 2005, 70, 231−
239.
10 μM concentration. Idebenone itself diminished ATP levels
with or without 4-nitrobenzoate coadministration. It has been
reported that addition of decylubiquinone (2) to 4-nitro-
benzoate treated cells significantly increased respiration beyond
that of the treated control.39
(10) Orrenius, S.; Gogvadze, V.; Zhivotovsky, B. Mitochondrial
oxidative stress: implications for cell death. Annu. Rev. Pharmacol.
Toxicol. 2007, 47, 143−183.
In summary, new pyrimidinol derivatives, have been
synthesized and characterized biochemically. The results
indicate that careful optimization of the redox core and side
chain can afford multifunctional radical quenchers capable of
potently suppressing ROS and lipid peroxidation, preserving
mitochondrial membrane potential, affording cytoprotection
against induced oxidative stress, and maintaining normal ATP
levels. The greatly improved cytoprotective activities of the
present compounds capable of enhancing ATP production, in
comparison with prototype analogue 3, suggests that optimal
activity results from multiple properties of the compounds in
suppressing lipid peroxidation and ROS, in addition to
enhancing energy production, consistent with our earlier
mechanistic hypothesis.21 While the limited number of
analogues studied here does not completely define the side
chain and redox core optimal for improving antioxidant and
bioenergetic properties, it does suggest that further structural
optimization may afford compounds suitable for therapeutic
intervention in mitochondrial and neurodegenerative diseases
associated with increased levels of ROS and reduced energy
production.
(11) Graier, W. F.; Frieden, M.; Malli, R. Mitochondria and Ca2+
signaling: old guests, new functions. Eur. J. Physiol. 2007, 455, 375−
396.
(12) Turrens, J. F. Mitochondrial formation of reactive oxygen
species. J. Physiol. 2003, 552, 335−344.
(13) Murphy, M. P. How mitochondria produce reactive oxygen
species. Biochem. J. 2009, 417, 1−13.
(14) Mates, J. M.; Perez-Gomez, C.; Nunez de Castro, I. Antioxidant
enzymes and human diseases. Clin. Biochem. 1999, 32, 595−603.
(15) Fridovich, I. Fundamental aspects of reactive oxygen species, or
what’s the matter with oxygen? Ann. N.Y. Acad. Sci. 1999, 893, 13−18.
(16) Lenaz, G. Role of mitochondria in oxidative stress and ageing.
Biochim. Biophys. Acta 1998, 1366, 53−67.
(17) Ernster, L.; Dallner, G. Biochemical, physiological and medical
aspects of ubiquinone function. Biochim. Biophys. Acta 1995, 1271,
195−204.
(18) Nohl, H.; Kozlov, A. V.; Staniek, K.; Gille, L. The multiple
functions of coenzyme Q. Bioorg. Chem. 2001, 29, 1−13.
(19) Bhagavan, H. N.; Chopra, R. K. Plasma coenzyme Q10 response
to oral ingestion of coenzyme Q10 formulations. Mitochondrion 2007,
7, S78−S88.
(20) CoQ10 can be rendered water soluble by formulation. See, e.g.,
Borowy-Borowski, H.; Sikorska-Walker, M.; Walker, P. R. Water
soluble compositions of bioactive lipophilic compounds. U. S. Patent
6,632,443, October 14, 2003.
(21) Arce, P. M.; Khdour, O. M.; Goldschmidt, R.; Armstrong, J. S.;
Hecht, S. M. A strategy for suppressing redox stress within
mitochondria. ACS Med. Chem. Lett. 2011, 2, 608−613.
(22) Khdour, O. M.; Lu, J.; Hecht, S. M. An acetate prodrug of a
pyridinol-based vitamin E analogue. Pharm. Res. 2011, 28, 2896−2909.
(23) Lu, J.; Khdour, O. M.; Armstrong, J. S.; Hecht, S. M. Design,
synthesis, and evaluation of an α-tocopherol analogue as a
mitochondrial antioxidant. Bioorg. Med. Chem. 2010, 18, 7628−7638.
(24) Cai, X.; Khdour, O. M.; Jaruvangsanti, J.; Hecht, S. M.
Simplified bicyclic pyridinol analogues protect mitochondrial function.
Bioorg. Med. Chem. 2012, 20, 3584−3595.
(25) Arce, P. M.; Goldschmidt, R.; Khdour, O. M.; Madathil, M. M.;
Jaruvangsanti, J.; Dey, S.; Fash, D. M.; Armstrong, J. S.; Hecht, S. M.
Analysis of the structural and mechanistic factors in antioxidants that
preserve mitochondrial function and confer cytoprotection. Bioorg.
Med. Chem. 2012, 20, 5188−5201.
ASSOCIATED CONTENT
* Supporting Information
■
S
Procedures and characterization for all new compounds,
procedures for biochemical assays, and effects of 1, 4, and 7
on ATP levels in 4-nitrobenzoate-treated human fibroblasts.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(S.M.H.) Tel: 480-965-6625. Fax: 480-965-0038. E-mail: sid.
hecht@asu.edu.
■
Funding
This work was supported by a research grant from the
Friedreich’s Ataxia Research Alliance (FARA).
Notes
The authors declare no competing financial interest.
(26) Goldschmidt, R.; Arce, P. M.; Khdour, O. M.; Collin, V. C.;
Dey, S.; Jaruvangsanti, J.; Fash, D. M.; Hecht, S. M. Effects of
cytoprotective antioxidants on lymphocytes from representative
mitochondrial neurodegenerative diseases. Bioorg. Med. Chem. 2013,
21, 969−978.
(27) Pratt, D. 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.
(28) Wijtmans, M.; Pratt, D. A.; Valgimigli, L.; DiLabio, G. A.;
Pedulli, G. F.; Porter, N. A. 6-Amino-3-pyridinols: towards diffusion-
controlled chain-breaking antioxidants. Angew. Chem., Int. Ed. 2003,
42, 4370−4373.
REFERENCES
■
(1) Markesbery, W. R.; Carney, J. M. Oxidative alterations in
Alzheimer’s disease. Brain Pathol. 1999, 9, 133−146.
(2) Calabrese, V.; Lodi, R.; Tonon, C.; D’Agata, V.; Sapienza, M.;
Scapagnini, G.; Mangiameli, A.; Pennisi, G.; Stella, A. M.; Butterfield,
D. A. Oxidative stress, mitochondrial dysfunction and cellular stress
response in Friedreich’s ataxia. J. Neurol. Sci. 2005, 233, 145−162.
(3) Lin, M. T.; Beal, M. F. Mitochondrial dysfunction and oxidative
stress in neurodegenerative diseases. Nature 2006, 443, 787−795.
(4) DiMauro, S.; Schon, E. A. Mitochondrial disorders in the nervous
system. Annu. Rev. Neurosci. 2008, 31, 91−123.
(5) Armstrong, J. S.; Khdour, O. M.; Hecht, S. M. Does oxidative
stress contribute to the pathology of Friedreich’s ataxia? A radical
question. FASEB J. 2010, 24, 2152−2163.
(29) Nam, T.-G.; Rector, C. L.; Kim, H.-Y.; Sonnen, A. F.-P.; Meyer,
R.; Nau, W. M.; Atkinson, J.; Rintoul, J.; Pratt, D. A.; Porter, N. A.
Tetrahydro-1,8-naphthyridinol analogues of α-tocopherol as antiox-
idants in lipid membranes and low-density lipoproteins. J. Am. Chem.
Soc. 2007, 129, 10211−10219.
(6) Browne, S. E.; Ferrante, R. J.; Beal, M. F. Oxidative stress in
Huntington’s disease. Brain Pathol. 1999, 9, 147−163.
(7) Newmeyer, D. D.; Ferguson-Miller, S. Mitochondria: Releasing
power for life and unleashing the machineries of death. Cell 2003, 112,
481−490.
(30) Serwa, R.; Nam, T.-G.; Valgimigli, L.; Culbertson, S.; Rector, C.
L.; Jeong, B.-S.; Pratt, D. A.; Porter, N. A. Preparation and
E
dx.doi.org/10.1021/ml400130z | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
investigation of vitamin B6-derived aminopyridinol antioxidants.
Chem.Eur. J. 2010, 16, 14106−14114.
(31) Duveau, D. Y.; Arce, P. M.; Schoenfeld, R. A.; Raghav, N.;
Cortopassi, G. A.; Hecht, S. M. Synthesis and characterization of
mitoQ and idebenone analogues as mediators of oxygen consumption
in mitochondria. Bioorg. Med. Chem. 2010, 18, 6429−6441.
(32) Marsh, G. P.; Parsons, P. J.; McCarthy, C.; Corniquet, X. G. An
efficient synthesis of nitroalkenes by alkene cross metathesis: facile
access to small ring systems. Org. Lett. 2007, 9, 2613−2616.
(33) Valgimigli, L.; Brigati, G.; Pedulli, G. F.; DiLabio, G. A.;
Mastragostino, M.; Arbizzani, C.; Pratt, D. A. The effect of ring
nitrogen atoms on the homolytic reactivity of phenolic compounds:
understanding the radical-scavenging ability of 5-pyrimidinols.
Chem.Eur. J. 2003, 9, 4997−5010.
(34) Jauslin, M. L.; Wirth, T.; Meier, T.; Schoumacher, F. A cellular
model for Friedreich ataxia reveals small-molecule glutathione
peroxidase mimetics as novel treatment strategy. Hum. Mol. Genet.
2002, 11, 3055−3063.
(35) Jauslin, M. L.; Vertuani, S.; Durini, E.; Buzzoni, L.; Ciliberti, N.;
Verdecchia, S.; Palozza, P.; Meier, T.; Manfredini, S. Protective effects
of Fe-Aox29, a novel antioxidant derived from a molecular
combination of idebenone and vitamin E, in immortalized fibroblasts
and fibroblasts from patients with Friedreich ataxia. Mol. Cell. Biochem.
2007, 302, 79−85.
(36) Griffith, O. W.; Meister, A. Potent and specific inhibition of
glutathione synthesis by buthionine sulfoximine (S-n-butyl homo-
cysteine sulfoximine). J. Biol. Chem. 1979, 254, 7558−7560.
(37) Aguer, C.; Gambarotta, D.; Mailloux, R. J.; Moffat, C.; Dent, R.;
McPherson, R.; Harper, M. E. Galactose enhances oxidative
metabolism and reveals mitochondrial dysfunction in human primary
muscle cells. PLoS One 2011, 6, e28536.
(38) Robinson, B. H.; Petrova-Benedict, R.; Buncic, J. R.; Wallace, D.
C. Nonviability of cells with oxidative defects in galactose medium: a
screening test for affected patient fibroblasts. Biochem. Med. Metab.
Biol. 1992, 48, 122−126.
(39) Forsman, U.; Sjoberg, M.; Turunen, M.; Sindelar, P. J. 4-
Nitrobenzoate inhibits coenzyme Q biosynthesis in mammalian cell
culture. Nat. Chem. Biol. 2010, 6, 515−517.
(40) Quinizii, C.; Naini, A.; Salviati, L.; Trevisson, E.; Navas, P.;
DiMauro, S.; Hirano, M. A mutation in para-hydroxybenzoate-
polyprenyl transferase (COQ2) causes primary coenzyme Q10
deficiency. Am. J. Hum. Genet. 2006, 78, 345−349.
(41) Quinizii, C. M.; Tadesse, S.; Naini, A.; Hirano, M. Effects of
inhibiting CoQ10 biosynthesis with 4-nitrobenzoate in human
fibroblasts. PLoS One 2011, 7, e30606.
F
dx.doi.org/10.1021/ml400130z | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX