Synthesis and biological activities of N-(3-Carboxylpropyl)-5-amino-2- hydroxy-3-tridecyl-1,4-benzoquinone and analogues

Article abstract of DOI:10.1021/np3007099

The synthesis of benzoquinone natural product 2 and three analogues is described. The synthesized compounds were tested for their ability to protect Friedreich's ataxia (FRDA) lymphocytes from induced oxidative stress. One of the analogues (3) conferred c

Full text of DOI:10.1021/np3007099

Article
pubs.acs.org/jnp
Synthesis and Biological Activities of N‑(3-Carboxylpropyl)-5-amino-
2-hydroxy-3-tridecyl-1,4-benzoquinone and Analogues
Manikandadas M. Madathil,†,‡ Omar M. Khdour,† Jennifer Jaruvangsanti,†,‡ and Sidney M. Hecht*,†,‡
‡
†Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe,
Arizona 85287-2904, United States
S
* Supporting Information
ABSTRACT: The synthesis of benzoquinone natural product 2 and three
analogues is described. The synthesized compounds were tested for their
ability to protect Friedreich’s ataxia (FRDA) lymphocytes from induced
oxidative stress. One of the analogues (3) conferred cytoprotection in a
dose-dependent manner in FRDA lymphocytes at micromolar concen-
trations. The biological assays suggest that the modification of the 2-hydroxy and N-(3-carboxypropyl) groups in the natural
product can improve its antioxidant activity and significantly enhance its ability to protect mitochondrial function under
conditions of oxidative stress.
itochondria are intracellular organelles that play a critical
role in a number of metabolic pathways.1 A primary
Herein we describe the synthesis of natural product 2 and
analogues 1, 3, and 4 (Figure 1). The synthesized compounds
1−4 were studied for their ability to confer cytoprotection to
FRDA lymphocytes placed under conditions of induced
oxidative stress. Compound 3 showed promising antioxidant
activity and maintained cell viability in a dose-dependent
manner (Table 1) at micromolar concentrations in oxidatively
stressed FRDA lymphocytes. Compound 3 was also shown to
M
function of the mitochondria is the generation of sufficient ATP
to meet the energy requirements of the cell.1 Mitochondrial
damage can play a critical role in the etiology and progression
of several neurodegenerative diseases, including Friedreich’s
ataxia (FRDA).2 In normal cells, the generation of reactive
oxygen species (ROS) and free radicals in the mitochondria is
controlled by cellular antioxidant enzymes. In diseased cells,
disruptions in the mitochondrial electron transport chain can
cause enhanced oxidative stress and damage, challenging the
intrinsic cellular antioxidant capacity.2
Natural products have been an important source for the
development of small-molecule therapeutic agents for the
treatment of many diseases, including neurodegenerative
diseases.3 Quinones such as coenzyme Q10 (CoQ10) are an
important class of natural products and play a key role in
cellular respiration.4 The pharmacological and toxicological
properties of quinones have been well documented.5 Natural
product 2, first isolated from Embelia ribes Burm. (Myrsina-
ceae), which is a species used in traditional Chinese medicine,
has been synthesized previously, but its biological activity has
not been well studied.6,7
Compound 2 has a structure similar to the redox-active
quinone core of the natural product geldanamycin. Geldana-
mycin is a benzoquinone ansamycin that has been widely
studied for its antiproliferative activities against a wide panel of
human tumor cell lines.8 Interestingly, it has been shown
experimentally that an analogue of geldanamycin (17-AAG)
undergoes reduction in normal epithelial cells under physio-
logical conditions and that this reduction significantly enhances
its affinity for the cellular protein target Hsp90.9 Given the
structural similarity between the redox cores of 2 and
geldanamycin, it seemed likely that 2 and its structural
analogues would also undergo reduction in situ to afford the
corresponding hydroquinones under physiological conditions,
potentially enabling them to protect cells from oxidative stress.
Figure 1. Chemical structures of 5-[N-(3-carboxypropyl)amino]-2-
hydroxy-3-tridecyl-1,4-benzoquinone (2), three derivatives (1, 3 and
4) and geldanamycin.
Received: October 10, 2012
Published: November 28, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
2209
dx.doi.org/10.1021/np3007099 | J. Nat. Prod. 2012, 75, 2209−2215

Journal of Natural Products
Article
The synthesis of 2 was attempted by treatment of 8 with
sodium bicarbonate and γ-aminobutyric acid, which would
involve conjugate addition of the amino acid to the vinylogous
methyl ester.7,12 The reaction did not proceed as expected,
possibly due to γ-butyrolactam formation. Therefore, 8 was
treated with γ-aminobutyric acid tert-butyl ester hydrochloride
salt in the presence of a large excess of sodium bicarbonate to
afford tert-butyl ester 1 in 45% yield. Finally the tert-butyl ester
was cleaved by treatment with trifluoroacetic acid in the
presence of anisole;13 compound 2 precipitated from methanol
as a red amorphous solid in 88% yield. Alternatively, tert-butyl
ester 1 was methylated with dimethyl sulfate in dry acetone to
yield 3 in 91% yield, which, upon treatment with trifluoroacetic
acid in the presence of anisole, afforded acid 4 in 76% yield.
Biochemical and Biological Evaluation. Cytoprotection.
The ability of the test compounds to confer cytoprotection was
evaluated in cultured Friedreich’s ataxia lymphocytes. These
cells were treated with diethyl maleate to induce oxidative stress
through depletion of glutathione. As shown in Table 1,
compound 3 was the most efficient, exhibiting 84%
cytoprotection at 2.5 μM concentration. The cytoprotection
afforded by this compound increased at higher doses up to 5
μM concentration. Benzoquinone analogue 4 afforded greater
cyoprotection to FRDA lymphocytes at 5 μM concentration
than did the tert-butyl ester 1 (74% vs 50%). The natural
product 2 afforded the least protection when tested at this
concentration.
Table 1. Cytoprotection of Cultured FRDA Cells from the
Effects of Oxidative Stress
a
viable cells (%)
compound
5 μM
2.5 μM
0.25 μM
0.125 μM
1
2
3
4
50 2.9
36 7.3
93 4.0
74 5.5
84 5.0
58 4.4
48 5.6
a
The viability of untreated cells was defined as 100%; cells treated with
DEM alone had 18 10% viability.
suppress ROS production and lipid peroxidation and to
maintain mitochondrial membrane potential in cultured
FRDA lymphocytes that had been subjected to oxidative stress.
These findings are consistent with the possible use of this class
of compounds as therapeutic agents for the management of
oxidative stress.
RESULTS AND DISCUSSION
■
The synthesis of 2 (Scheme 1) began with the n-butyllithium-
mediated alkylation of compound 510,11 with purified 1-
bromotridecane to yield 6 in 73% yield.11 The alkylated
tetramethoxybenzene 6 was then subjected to oxidation with
cerium(IV) ammonium nitrate to give a crude mixture
containing quinones 7 and 8, which underwent selective
perchloric acid-catalyzed demethylation to afford hydroxyqui-
none 8 in 54% yield over two steps.11 Regioselective
demethylation took place with the removal of the more
hindered methoxy group.11
Inhibition of Lipid Peroxidation. The ability of compound 2
and its analogues to quench lipid peroxidation was evaluated in
FRDA lymphocytes. These cells were placed under oxidative
stress by depleting them of glutathione (GSH) using diethyl
Scheme 1. Synthesis of Natural Product 2 and Analogues
2210
dx.doi.org/10.1021/np3007099 | J. Nat. Prod. 2012, 75, 2209−2215

Journal of Natural Products
Article
maleate (DEM).14−16 The extent of lipid peroxidation was
quantified using the fatty acid sensitive fluorescent reporter C11-
BODIPY581/591 (Molecular Probes).17,18 Upon oxidation of the
phenylbutadiene moiety of the fluorophore, the red-emitting
form of the dye (595 nm) is converted into a green-emitting
form (520 nm). Increased C11-BODIPY581/591-green (oxidized)
fluorescence, a measure of peroxyl radical production, was
determined by flow cytometric analysis, which is expressed as
percent scavenging activity (Table 2). The results in Table 2
in the stressed cells at 5 μM concentration, while the natural
product 2 was only very weakly active at the same
concentration. Compounds 1 and 4 were less effective than
compound 3.
Preservation of Mitochondrial Membrane Potential
(Δψm). The ability of the test compounds to preserve
mitochondrial membrane potential under conditions of
oxidative stress was studied. Assessment of Δψm is an important
indicator of cellular function during stress-induced cell death.
Changes in mitochondrial membrane potential were measured
using two different fluorescent dyes, tetramethylrhodamine
methyl ester (TMRM) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-
tetraethylbenzimidazolocarbocyanine iodide (JC-1). TMRM is
a potentiometric, cell-permeable fluorescent indicator that
accumulates in the highly negatively charged interior of the
mitochondrial inner membrane in a Nernstian manner.19 The
fluorescence signal of TMRM can be directly correlated to Δψm
across the inner mitochondrial membrane. Therefore, the
accumulation of dye in the mitochondria and the intensity of
the signal are direct functions of mitochondrial potential. Loss
of mitochondrial membrane potential is indicated by a
reduction in TMRM red fluorescence. The detection of
mitochondrial depolarization using TMRM was accomplished
by flow cytometry. Figure 3 illustrates representative two-
dimensional density dot plots of TMRM-stained lymphocyte
cells showing the percentage of cells with intact Δψm (TMRM
fluorescence in top right quadrant) vs the percentage of cells
with reduced Δψm (TMRM fluorescence in bottom left and
right quadrants). Figure 3 (bottom panel) shows a bar graph of
the percentage (mean SE) of FRDA lymphocytes with intact
Δψm. The results show that treatment with 5 mM DEM
decreased the percentage of cells with TMRM fluorescence in
the top right quadrant, indicating that DEM treatment caused
depolarization of Δψm. Compound 3 preserved mitochondrial
membrane polarization the most effectively when used at 5 μM
concentration (85%), while compound 2 was the least effective
(27%). Intermediate values were determined for compounds 1
and 4 (40% and 60%, respectively).
These results were further confirmed using JC-1 dye in
primary FRDA fibroblasts treated with buthionine sulfoximine
(BSO) (Figure 4). BSO was used in this cellular model to
induce an oxidative insult by inhibiting de novo glutathione
synthesis.20 JC-1 is a lipophilic, cationic dye that can selectively
enter into mitochondria and reversibly change color from green
to red as the membrane potential increases by forming
aggregates.21 The dye exhibits red fluorescence when it
aggregates in the matrix of healthy energized mitochondria,
whereas it has green fluorescence in cells with depolarized Δψm.
In untreated FRDA cells and cells treated with 5 μM
compound 3 in addition to 1 mM BSO, the JC-1 dye was
mainly in the aggregated state (red−orange) (Figure 4A and
D), indicating that compound 3 preserved the mitochondrial
membrane potential in spite of the presence of BSO. Treatment
with 1 mM BSO prevented JC-1 mitochondrial accumulation in
the cells, resulting in a pronounced green fluorescence due to
complete loss of mitochondrial membrane potential (Figure
4C). A significant mitochondrial membrane depolarization was
observed with natural product 2 in BSO-treated cells (Figure
4E). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone
(FCCP), a commonly used uncoupler of oxidative phosphor-
ylation in mitochondria, was employed to dissipate the
chemiosmotic proton gradient (ΔμH+). The pronounced
green fluorescence resulting from FCCP treatment reflects
Table 2. Suppression of Lipid Peroxidation by Compounds
1−4 in Cultured FRDA Lymphocytes Treated with Diethyl
a
Maleate
scavenging activity (%)
compound
5 μM
100
10 μM
100
b
untreated control
c
treated control
0
0
1
2
3
4
26 6.7
8.0 6.6
72 1.8
41 7.2
37 1.4
24 7.4
86 1.8
51 5.0
a
Values have been calculated as [(100 − % mean)/(100 − % mean of
b
c
the untreated control)] × 100. No DEM treatment. DEM treatment.
show that analogue 3 was very effective in suppressing lipid
peroxidation at 5 and 10 μM concentrations (72% and 86%
suppression of lipid peroxidation), while the natural product 2
was much less active (24% suppression at 10 μM concen-
tration). Compounds 1 and 4 also exhibited concentration-
dependent suppression of lipid peroxidation, affording 37% and
51% suppression, respectively, at 10 μM concentration.
Suppression of Reactive Oxygen Species. The ability of
compounds 1−4 to suppress ROS production was determined
in FRDA lymphocytes and CEM leukemia lymphocytes in the
presence and absence of the test compounds by monitoring the
fluorescence of the ROS-sensitive dye dichlorofluorescein
(DCF). Increased DCF fluorescence, a measure of intracellular
oxidation and ROS production, was determined by flow
cytometry. These cells were placed under oxidative stress by
depleting them of GSH using DEM.14−16 The results in Figure
2 show that analogue 3 was very effective in suppressing ROS
Figure 2. Flow cytometric analysis of CEM leukemia lymphocytes
(gray bars) and FRDA lymphocytes (black bars) stained with
dichlorodihydrofluorescein diacetate (DCFH-DA) for 20 min,
following pretreatment with the test compounds at 5 μM
concentration for 16 h, and subsequent treatment with diethyl maleate
(DEM) for 40 or 80 min to induce the production of ROS in CEM
and FRDA lymphocytes, respectively. Data shown represent the mean
SEM of two different experiments run as duplicates.
2211
dx.doi.org/10.1021/np3007099 | J. Nat. Prod. 2012, 75, 2209−2215

Journal of Natural Products
Article
Figure 3. Effect of nitrogen-containing 1,4-benzoquinone derivatives on preservation of mitochondrial membrane potential of cultured FRDA cells
following treatment with the test compounds. Representative flow cytometric two-dimensional color density dot plot analyses of mitochondrial
membrane potential Δψm in FRDA lymphocytes stained with TMRM and analyzed using the FL2-H channel as described in the Experimental
Section. The cells were washed twice in phosphate-buffered saline and suspended in phosphate-buffered saline containing 20 mM glucose. The
percentage of cells with intact Δψm is indicated in the top right quadrant of the panels. In each analysis, 10 000 events were recorded. Data are
expressed as means SEM of three independent experiments run in duplicate. The bar graph shows the percentage of cells with intact Δψm
calculated using CellQuest software.
chemicals were all ACS reagent grade and were used without further
purification, except for 1-bromotridecane, which was purified by silica
gel flash column chromatography prior to use. The reactions were
carried out under an atmosphere of argon. Flash column
chromatography was carried out using silica gel (Silicycle R10030B,
60 particle size, 230−400 mesh), applying a low-pressure stream of
nitrogen. Analytical thin-layer chromatographic separations were
carried out on glass plates coated with silica gel (60 particle size
F254, SiliCycle TLG-R10011B-323). The TLC chromatograms were
developed by immersing the plates in 2.5% potassium permanganate in
ethanol or 2% anisaldehyde + 5% sulfuric acid + 1.5% glacial acetic
acid in ethanol, followed by heating, or else visualized by UV
irradiation (254 nm). Melting points were recorded on a MelTemp
apparatus and are uncorrected. Tetrahydrofuran was distilled from
sodium/benzophenone ketyl and dichloromethane from calcium
the depolarization of mitochondrial inner membrane potential
(Figure 4B). These data indicate that compound 3 is able to
prevent oxidative stress induced collapse of Δψm, an event
associated with the disruption of mitochondrial function that
occurs prior to cell death.
Recently, we have described three structural series of
compounds that are capable of protecting cells with suboptimal
mitochondrial function resulting from the effects of induced
oxidative stress.22 These compounds suppress lipid peroxida-
tion and reactive oxygen species and confer cytoprotection to
cultured mammalian cells under oxidative stress. The present
study extends these findings to a fourth structural class,
providing an additional pharmacophore of potential utility for
the design of therapeutic agents for the treatment of
mitochondrial disorders.
1
hydride. H and 13C NMR spectra were recorded on a Gemini 300
or Varian Inova 400 or on a Varian Inova 500 spectrometer, using
CDCl3 as solvent and internal standard, unless otherwise indicated. 1H
NMR chemical shifts were reported relative to residual CDCl3 at 7.26
ppm or to residual DMSO-d6 at 2.50 ppm; 13C NMR shifts were
reported relative to the central line of CDCl3 at 77.16 ppm or to
residual DMSO-d6 at 39.51 ppm. Splitting patterns are designated as s,
singlet; d, doublet; dd, doublet of doublets; m, multiplet; q, quartet;
quin, quintet; br, broad. High-resolution mass spectrometric data was
obtained at the Michigan State Mass Spectrometry Facility or at the
Arizona State University CLAS High Resolution Mass Spectrometry
Facility.
In conclusion, we have prepared natural product 2 and three
structural analogues and tested them for their ability to prevent
ROS-induced damage of cellular lipid membranes, maintain
mitochondrial membrane potential, and confer cytoprotection
to FRDA lymphocytes despite severe induced oxidative stress.
EXPERIMENTAL SECTION
General Experimental Procedures. All the chemicals were
purchased from Sigma Aldrich and Chem-Impex International. The
■
2212
dx.doi.org/10.1021/np3007099 | J. Nat. Prod. 2012, 75, 2209−2215

Journal of Natural Products
Article
solution of this mixture in 2.6 mL of dichloromethane was added 1.1
mL (13 mmol) of 70% perchloric acid dropwise at 0 °C. The reaction
mixture was stirred at 0 °C for 9 h, diluted with 10 mL of
dichloromethane, then washed successively with distilled water and
brine, and dried (MgSO4). The solvent was concentrated under
diminished pressure to afford a crude residue. The residue was applied
to a silica gel column (7 × 2 cm). Elution with 1:4 ethyl acetate−
hexanes gave 8 as a yellow-orange solid: yield 48 mg (54%); mp 90−
92 °C; lit.23 mp 90−91 °C; silica gel TLC Rf 0.58 (1:1 ethyl acetate−
1
hexanes); H NMR (CDCl3) δ 0.85 (3H, t, J = 6.8 Hz), 1.17−1.33
(20H, m), 1.39−1.49 (2H, m), 2.41 (2H, t, J = 8 Hz), 3.84 (3H, s),
5.82 (1H, s), and 7.32 (1H, s); 13C NMR (CDCl3) δ 14.2, 22.7, 22.8,
28.1, 29.48, 29.54, 29.68, 29.69, 29.77, 29.78, 29.79, 29.80, 32.0, 56.9,
102.3, 119.4, 151.7, 161.2, 181.8, and 183.0.
4-(4-Hydroxy-3,6-dioxo-5-tridecylcyclohexa-1,4-
dienylamino)butyric Acid tert-butyl Ester (1) (ref 7). To a
solution of 42 mg (0.13 mmol) of 2-hydroxy-5-methoxy-3-tridecyl-1,4-
benzoquinone (8) and 1.0 g (13 mmol) of sodium bicarbonate in 9.7
mL of ethanol was added 39 mg (0.19 mmol) of γ-aminobutyric acid
tert-butyl ester hydrochloride salt. The reaction mixture was stirred at
45 °C for 27 h under an argon atmosphere. The reaction mixture was
then diluted with 5 mL of water and extracted with seven 2 mL
portions of dichloromethane. The combined organic layer was washed
successively with water and brine and then dried (Na2SO4). The
solvent was concentrated under diminished pressure to afford a crude
residue. The residue was applied to a silica gel column (5 × 2 cm).
Elution with dichloromethane gave 1 as a dark red solid: yield 27 mg
(45%); mp 96−97 °C; lit.7 mp 82−85 °C; silica gel TLC Rf 0.38
Figure 4. Representative fluorescence microscopy images of JC-1-
stained primary FRDA fibroblasts were examined under a Zeiss
fluorescent microscope. Red indicates JC-1 aggregates, which are
formed in the mitochondria when a sufficiently high membrane
potential is reached. When the Δψm collapses as a result of glutathione
depletion, the reagent (JC-1) no longer accumulates inside the
mitochondria, and instead, it is distributed throughout the cell in the
monomeric form, which fluoresces green. Hoechst 33342 was used to
identify all nuclei. (A) Untreated primary FRDA fibroblasts, (B)
FRDA fibroblasts treated for 2 h with a 25 μM concentration of the
uncoupler FCCP, (C) FRDA fibroblasts treated for 24 h with 1 mM
BSO, (D) FRDA fibroblasts pretreated for 12 h with 5 μM compound
3 and then treated for 24 h with 1 mM BSO, and (E) FRDA
fibroblasts pretreated for 12 h with 5 μM compound 2 and then
treated for 24 h with 1 mM BSO.
1
(dichloromethane); H NMR (CDCl3) δ 0.86 (3H, t, J = 6.5 Hz),
1.20−1.32 (20H, m), 1.38−1.46 (11H, m), 1.94 (2H, quin, J = 6.9
Hz), 2.31 (2H, t, J = 7.0 Hz), 2.34−2.40 (2H, m), 3.21 (2H, dd, J =
12.9 and 6.6 Hz), 5.35 (1H, s), 6.58 (1H, s), and 8.10 (1H, br s); 13C
NMR (CDCl3) δ 14.3, 22.79, 22.84, 23.5, 28.23, 28.24, 29.5, 29.6,
29.73, 29.75, 29.81, 29.83, 29.84, 32.1, 32.8, 42.4, 81.2, 91.9, 115.9,
149.9, 155.1, 172.1, 179.0, and 182.6; mass spectrum (LCT
electrospray), m/z 486.3181 (M + Na)+ (C27H45NO5Na requires m/
z 486.3195).
Chemistry. Compounds 5 and 6 were synthesized according to
literature procedures.10,11
5-[N-(3-Carboxylpropyl)amino]-2-hydroxy-3-tridecyl-1,4-
benzoquinone (2). To a solution containing 28 mg (60 μmol) of 1
in 0.4 mL of dichloromethane was added 6.5 μL (60 μmol) of anisole
and 400 μL (5.4 mmol) of trifluoroacetic acid. The reaction mixture
was stirred for 24 h at room temperature under an argon atmosphere.
The reaction mixture was concentrated under diminished pressure,
and the excess trifluoroacetic acid removed by co-evaporation three
times with cyclohexane to afford a crude residue. The residue was
precipitated from methanol to give 2 as a red, amorphous solid: yield
2,3,5,6-Tetramethoxyphenyl-1-tridecylbenzene (6) (ref 11).
To a solution containing 1.0 g (5.0 mmol) of 1,2,4,5-tetramethox-
ybenzene (5)11 and 87 μL (90 mg, 0.5 mmol) of hexamethylphos-
phoramide in 25 mL of dry THF was added 3.4 mL (1.6 M in hexanes,
5.5 mmol) of n-butyllithium dropwise at −40 °C over a period of 5
min. The reaction mixture was warmed to 0 °C over a period of 2 h;
then 1.4 mL (1.4 g, 5.5 mmol) of purified 1-bromotridecane was
added, and the reaction mixture was stirred at room temperature under
argon for 15 h. The reaction mixture was quenched with 20 mL of
saturated NH4Cl and extracted with five 10 mL portions of ether. The
organic layer was washed with distilled water and brine, then dried
(MgSO4). The solvent was concentrated under diminished pressure to
afford a crude residue. The residue was applied to a silica gel column
(6 × 3 cm). Elution with 1:9 ethyl acetate−hexanes afforded 6 as a
colorless solid: yield 1.4 g (73%); mp 31−32 °C; lit.11 mp 31−32 °C.
A 0.20 g (20%) amount of unreacted 1,2,4,5-tetramethoxybenzene (5)
21 mg (88%); mp 194−195 °C; lit.7 mp 177−180 °C; H NMR
1
(DMSO-d6) δ 0.85 (3H, t, J = 6.8 Hz), 1.15−1.42 (22H, m), 1.74 (2H,
quin, J = 14.4 and 7.2 Hz), 2.26 (4H, q, J = 6.9 Hz), 3.14 (2H, dd, J =
13.8 and 6.7 Hz), 5.32 (1H, s), 7.78 (1H, t, J = 6.2 Hz), 10.49 (1H, br
s), and 12.2 (1H, br s); 13C NMR (DMSO-d6) δ 14.0, 22.1, 22.2, 22.8,
27.6, 28.8, 28.9, 29.0, 29.02, 29.06, 29.08, 29.10, 30.9, 31.3, 41.4, 91.8,
115.6, 149.3, 156.7, 174.2, 178.5, and 182.5; mass spectrum (LCT
electrospray), m/z 430.2564 (M + Na)+ (C23H37NO5Na requires m/z
430.2569).
1
was recovered: silica gel TLC Rf 0.45 (1:1 ethyl ether−hexanes); H
4-(4-Methoxy-3,6-dioxo-5-tridecylcyclohexa-1,4-
dienylamino)butyric Acid tert-Butyl Ester (3). To a solution
containing 22 mg (47 μmol) of 1 and 0.25 g (1.8 mmol) of potassium
carbonate in 1.2 mL of dry acetone was added 23 μL (0.23 mmol) of
dimethyl sulfate. The reaction mixture was heated at reflux overnight,
allowed to cool to room temperature, and concentrated under
diminished pressure. The crude mixture was redissolved in 10 mL of
dichloromethane and washed with 5 mL of 1 N HCl, and the aqueous
layer was extracted with three 10 mL portions of dichloromethane.
The combined organic layer was dried (MgSO4) and concentrated
under diminished pressure. The residue was purified by flash column
chromatography on a silica gel column (24 × 2 cm). Elution with 1:5
ethyl acetate−hexane gave 3 as a bright red, amorphous solid: yield 21
NMR (CDCl3) δ 0.87 (3H, t, J = 6.8 Hz), 1.14−1.46 (20H, m), 1.47−
1.58 (2H, m), 2.61 (2H, dd, J = 8.8 and 6.9 Hz), 3.76 (6H, s), 3.82
(6H, s), and 6.40 (1H, s); 13C NMR (CDCl3) δ 14.1, 22.7, 24.7, 29.4,
29.5, 29.6, 29.70, 29.75, 29.76, 30.0, 30.8, 32.0, 56.2, 60.4, 60.9, 96.7,
131.1, 141.1, and 148.8.
2-Hydroxy-5-methoxy-3-tridecyl-1,4-benzoquinone (8) (ref
11). To a solution containing 0.10 g (0.26 mmol) of 2,3,5,6-
tetramethoxyphenyl-1-tridecylbenzene (6) in 2.6 mL of acetonitrile
was added dropwise a solution containing 0.28 g (0.52 mmol) of
cerium(IV) ammonium nitrate in 2.6 mL of 7:3 acetonitrile−water at
−7 °C (salt−ice bath) over a period of 30 min. The reaction mixture
was stirred at room temperature for 3 h and diluted with 10 mL of
ether. The organic layer was washed successively with distilled water
and brine, then dried (MgSO4). The solvent was concentrated under
diminished pressure to afford a mixture of quinones 7 and 8. To a
1
mg (91%); silica gel TLC Rf 0.60 (1:2 ethyl acetate−hexanes); H
NMR (CDCl3) δ 0.87 (3H, t, J = 6.8 Hz), 1.16−1.42 (22H, m), 1.45
2213
dx.doi.org/10.1021/np3007099 | J. Nat. Prod. 2012, 75, 2209−2215

Journal of Natural Products
Article
(9H, s), 1.82−2.03 (2H, quin, J = 9 Hz), 2.31 (2H, t, J = 7.2 Hz),
2.35−2.39 (2H, m), 3.14 (2H, dd, J = 13.0 and 6.8 Hz), 4.10 (3H, s),
5.28 (1H, s), and 5.94 (1H, t, J = 5.6 Hz); 13C NMR (CDCl3) δ 14.3,
22.8, 23.1, 23.6, 28.20, 28.24, 28.8, 29.5, 29.6, 29.7, 29.81, 29.83, 32.1,
32.9, 42.1, 61.8, 81.1, 96.1, 127.6, 146.9, 158.5, 172.18, 172.20, 181.8,
and 183.9; mass spectrum (APCI), m/z 478.3532 (M + H)+
(C28H48NO5 requires m/z 478.3532).
Calibur). Briefly, 1 mL of cells (5 × 105 cells) was plated in a 24-well
plate, treated with the test compounds at 5 μM concentration, and
incubated at 37 °C for 16 h in a humidified atmosphere containing 5%
CO2 in air. Cells were treated with 5 mM diethyl maleate (97%,
Sigma-Aldrich) for 40 or 80 min to induce oxidative stress by
glutathione depletion in the treated cells. The cells were collected by
centrifugation at 300g for 3 min and then washed with PBS
(Invitrogen). Cells were resuspended in PBS containing 20 mM
glucose and incubated at 37 °C in the dark for 20 min with 10 μM
DCFH-DA. Cells were collected by centrifugation at 300g for 3 min
and then washed with PBS. The samples were analyzed immediately
by flow cytometry (BD FACS Calibur) using a 488 nm excitation laser
and FL1-H channel 538 nm emission filter. In each analysis, 10 000
events were recorded. The results obtained were verified in two
independent experiments.
5-[N-(3-Carboxypropyl)amino]-2-methoxy-3-tridecyl-1,4-
benzoquinone (4). To a solution containing 9.0 mg (19 μmol) of 3
in 0.12 mL of dichloromethane were added 2.0 μL (19 μmol) of
anisole and 0.13 mL (1.7 mmol) of trifluoroacetic acid. The reaction
mixture was stirred at room temperature for 24 h under an atmosphere
of argon. The reaction mixture was co-evaporated with six 5 mL
portions of cyclohexane, and the solvent was concentrated under
diminished pressure to afford a crude residue. The residue was purified
by flash column chromatography on a silica gel column (22 × 2 cm).
Elution with 100:1 chloroform−methanol gave 4 as a red, amorphous
solid: yield 6.0 mg (76%); silica gel TLC Rf 0.32 (1:1 ethyl acetate−
Assessment of Mitochondrial Membrane Potential. Mitochon-
drial membrane potential was measured using two different fluorescent
dyes, TMRM and JC-1. Δψm was determined as previously described
by staining FRDA lymphocyte cells with TMRM (Molecular Probes)
and analyzing fluorescence emission by flow cytometry in detection
channel 2 (FL2-H).16 Briefly, FRDA lymphocytes were pretreated with
or without the test compounds for 16 h. The cells were then treated
with 5 mM DEM for 140 min, collected by centrifugation at 300g for 3
min, and then washed twice with phosphate-buffered saline. The cells
were resuspended in PBS containing 20% glucose and incubated with
250 nM TMRM at 37 °C in the dark for 15 min. Cells were collected
by centrifugation at 300g for 3 min and were then washed with
phosphate-buffered saline. The samples were analyzed immediately by
flow cytometry using a 488 nm excitation laser and the FL2-H channel.
The results obtained were verified in three independent experiments.
FCCP, a mitochondrial uncoupler, was used to produce a negative
control to dissipate Δψm. In each analysis, 10 000 events were
recorded. We qualitatively examined the mitochondrial membrane
potential using JC-1 dye in primary FRDA fibroblasts GM04078
(Coriell Institute) after treatment with 1 mM buthionine sulfoxime, in
the presence and absence of the tested compounds (5 μM
concentration). JC-1 is a cationic dye that is accumulated in
mitochondria according to membrane potential. In polarized
mitochondria, it accumulates in aggregated form and appears as red
punctate staining, whereas in cells having depolarized mitochondria,
JC-1 diffuses throughout the cell and appears as green diffused
monomeric staining. Briefly, FRDA fibroblasts (2 × 105 cells/mL)
were seeded in coverslips (Corning) in six-well plates. The plates were
incubated at 37 °C overnight in a humidified atmosphere of 5% CO2
in air to allow attachment of the cells to the coverslips. The following
day, cells were treated with the test compounds and incubated for an
additional 12 h before treatment with 1 mM BSO. Δψm was assessed
after 24 h using JC-1 Mitochondrial Membrane Potential Detection
Kit (Biotium, Inc.) following the manufacturer’s protocol. Glass
coverslips were rinsed with phosphate-buffered saline and mounted
onto slides, and images were recorded and analyzed with a Zeiss
AxioCam MRm and AxioVision 3.1 software (Carl Zeiss) on a Zeiss
Axiovert 200 M inverted microscope, equipped with a 40× oil
immersion objective.
1
hexanes); H NMR (CDCl3) δ 0.88 (3H, t, J = 6.9 Hz), 1.22−1.41
(22H, m), 1.98 (2H, quin, J = 6.9 Hz), 2.33−2.40 (2H, m), 2.47 (2H,
t, J = 6.9 Hz), 3.20 (2H, q, J = 6.6 Hz), 4.11 (3H, s), 5.29 (1H, s), and
5.97 (1H, s); 13C NMR (CDCl3) δ 14.3, 18.5, 22.8, 23.1, 23.2, 28.8,
29.5, 29.6, 29.7, 29.81, 29.84, 31.3, 32.1, 42.0, 51.0, 58.6, 61.8, 96.2,
127.7, 146.9, 158.5, 176.6, 181.8, and 184.0; mass spectrum (APCI),
m/z 422.2898 (M + H)+ (C24H40NO5 requires 422.2906).
Biological Evaluation. Cytoprotection. Cell viability was
determined by the use of a trypan blue exclusion assay22c in
Friedreich’s ataxia lymphocyte cell line GM15850 (Coriell Institute).
This technique was used to assess the cytoprotective effects of the
tested compounds in cultured cells treated with DEM to induce cell
death by GSH depletion. The viability of DEM-treated FRDA cells was
determined by their ability to exclude the dye trypan blue. Viable cells
exclude trypan blue, whereas nonviable cells take up the dye and are
stained blue. Briefly, FRDA lymphocytes were grown in RPMI 1640
medium (Gibco) supplemented with 15% fetal calf serum, 2 mM
glutamine (HyClone), and 1% penicillin−streptomycin mix (Cellgro).
Cells were seeded at a density of 5 × 105 cells/mL and treated with
different concentrations of the indicated compounds. Cells were
incubated at 37 °C in a humidified atmosphere of 5% CO2 in air for 17
h. After preincubation, the cells were treated with 5 mM DEM and
maintained for an additional 6 h. Cell viability was determined after
staining cells with 0.4% trypan blue by the use of a hemacytometer. At
least 500 cells were counted in each experimental group. At the time of
assay, <20% of DEM-treated cells were viable (trypan blue negative),
whereas in the non-DEM-treated control, >90% cells were viable. Cell
viability was expressed as the percentage of control. Data are expressed
as means SEM (n = 3).
Lipid Peroxidation Assay. A quantitative FACS analysis of lipid
peroxidation of FRDA lymphocytes, which had been treated with
diethyl maleate following incubation in the presence and absence of
the test compounds, was measured as described previously.16 Briefly,
FRDA lymphocytes (5 × 105 cell/mL) were treated with the test
compounds at final concentrations of 5 and 10 μM and incubated at
37 °C for 16 h in a humidified atmosphere containing 5% CO2 in air.
Cells were treated with 1 μM C11-BODIPY581/591 in phenol red-free
RPMI-1640 medium and incubated at 37 °C in the dark for 30 min.
Oxidative stress was induced with 5 mM DEM in phenol red-free
RPMI-1640 medium for 2 h. Treated cells were collected by
centrifugation at 300g for 3 min and then washed with phosphate-
buffered saline (PBS). Cells were resuspended in PBS and analyzed by
FACS (FACS Calibur flow cytometer, Becton Dickinson) to monitor
the change in intensity of the C11-BODIPY581/591 green (oxidized)
fluorescence signal. In each analysis, 10 000 events were recorded.
Results obtained were verified by running duplicates and repeating the
assays in three independent experiments. Results are expressed as
percent scavenging activity.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds 1−4, 6, and 8. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: sid.hecht@asu.edu. Phone: 1-480-965-6625. Fax: 1-
480-965-0038.
Scavenging of Reactive Oxygen Species. Changes in cellular ROS
in CEM leukemia or FRDA lymphocytes were measured by the ROS
reactive fluorescent indicator 2,7-dichlorodihydrofluorescein diacetate
(DCFH-DA) (Molecular Probes) using flow cytometry (BD FACS
Notes
The authors declare no competing financial interest.
2214
dx.doi.org/10.1021/np3007099 | J. Nat. Prod. 2012, 75, 2209−2215

Journal of Natural Products
Article
ACKNOWLEDGMENTS
This work was supported in part by a grant from the
Friedreich’s Ataxia Research Alliance.
■
REFERENCES
■
(1) (a) Bras, M.; Queenan, B.; Susin, S. A. Biochemistry (Moscow)
2005, 70, 231−239. (b) McBride, H. M.; Neuspiel, M.; Wasiak, S.
Curr. Biol. 2006, 16, R551−560.
(2) (a) Markesbery, W. R.; Carney, J. M. Brain Pathol. 1999, 9, 133−
146. (b) Barnham, K. J.; Masters, C. L.; Bush, A. I. Nat. Rev. Drug
Discovery 2004, 3, 205−214. (c) Calabrese, V.; Lodi, R.; Tonon, C.;
D’Agata, V.; Sapienza, M.; Scapagnini, G.; Mangiameli, A.; Pennisi, G.;
Stella, A. M. G.; Butterfield, D. A. J. Neurol. Sci. 2005, 233, 145−162.
(d) Lin, M. T.; Beal, M. F. Nature 2006, 443, 787−795. (e) DiMauro,
S.; Schon, E. A. Annu. Rev. Neurosci. 2008, 31, 91−123. (f) Armstrong,
J. S.; Khdour, O. M.; Hecht, S. M. FASEB J. 2010, 24, 2152−2163.
(3) Joyner, P. M.; Cichewicz, R. H. Nat. Prod. Rep. 2011, 28, 26−47.
(4) Ernster, L.; Dallner, G. BBA-Mol. Basis Dis. 1995, 1271, 195−204.
(5) Monks, T. J.; Jones, D. C. Curr. Drug Metab. 2002, 3, 425−438.
(6) Lin, P.; Li, S.; Wang, S.; Yang, Y.; Shi, J. J. Nat. Prod. 2006, 69,
1629−1632.
(7) McErlean, C. S. P.; Moody, C. J. J. Org. Chem. 2007, 72, 10298−
10301.
(8) Whitesell, L.; Shifrin, S. D.; Schwab, G.; Neckers, L. M. Cancer
Res. 1992, 52, 1721−1728.
(9) Maroney, A. C.; Marugan, J. J.; Mezzasalma, T. M.; Barnakov, A.
N.; Garrabrant, T. A.; Weaner, L. E.; Jones, W. J.; Barnakova, L. A.;
Koblish, H. K.; Todd, M. J.; Masucci, J. A.; Deckman, I. C.; Galemmo,
R. A., Jr.; Johnson, D. L. Biochemistry 2006, 45, 5678−5685.
(10) Matsumoto, M.; Kobayashi, K.; Hotta, Y. J. Org. Chem. 1984, 49,
4740−4741.
(11) Poigny, S.; Guyot, M.; Samadi, M. Tetrahedron 1998, 54,
14791−14802.
(12) Stahl, P.; Kissau, L.; Mazitschek, R.; Huwe, A.; Furet, P.;
Giannis, A.; Waldmann, H. J. Am. Chem. Soc. 2001, 123, 11586−
11593.
(13) Chauvel, E. N.; Coric, P.; Llorens-Cortes, C.; Wilk, S.; Roques,
B. P.; Fournie-Zaluski, M.-C. J. Med. Chem. 1994, 37, 1339−1346.
(14) Armstrong, J. S.; Jones, D. P. FASEB J. 2002, 16, 1263−1265.
(15) Armstrong, J. S.; Whiteman, M.; Rose, P.; Jones, D. P. J. Biol.
Chem. 2003, 278, 49079−49084.
(16) Khdour, O. M.; Lu, J.; Hecht, S. M. Pharm. Res. 2011, 28, 2896−
2909.
(17) Pap, E. H. W.; Drummen, G. P. C.; Winter, V. J.; Kooij, T. W.
A.; Rijken, P.; Wirtz, K. W. A.; Op den Kamp, J. A. F.; Hage, W. J.;
Post, J. A. FEBS Lett. 1999, 453, 278−282.
(18) Drummen, G. P. C.; van Liebergen, L. C. M.; Op den Kamp, J.
A. F.; Post, J. A. Free Radical Biol. Med. 2002, 33, 473−490.
(19) Ehrenberg, B.; Montana, V.; Wei, M. D.; Wuskell, J. P.; Loew, L.
M. Biophys. J. 1988, 53, 785−794.
(20) Griffith, O. W.; Meister, A. J. Biol. Chem. 1979, 254, 7558−7560.
(21) Smiley, S. T.; Reers, M.; Mottola-Hartshorn, C.; Lin, M.; Chen,
A.; Smith, T. W.; Steele, G. D., Jr.; Chen, L. B. Proc. Natl. Acad. Sci. U.
S. A. 1991, 88, 3671−3675.
(22) (a) Lu, J.; Khdour, O. M.; Armstrong, J. S.; Hecht, S. M. Bioorg.
Med. Chem. 2010, 18, 7628−7638. (b) Arce, P. M.; Khdour, O. M.;
Goldschmidt, R.; Armstrong, J. S.; Hecht, S. M. ACS Med. Chem. Lett.
2011, 2, 608−613. (c) Arce, P. M.; Goldschmidt, R.; Khdour, O. M.;
Madathil, M. M.; Jaruvangsanti, J.; Dey, S.; Fash, D. M.; Armstrong, J.
S.; Hecht, S. M. Bioorg. Med. Chem. 2012, 20, 5188−5201.
(23) Wong, S.-M.; Pezzuto, J. M.; Fong, H. H. S.; Farnsworth, N. R. J.
Pharm. Sci. 1985, 74, 1114−1116.
2215
dx.doi.org/10.1021/np3007099 | J. Nat. Prod. 2012, 75, 2209−2215