Effects of alkyl side chain modification of coenzyme Q10 on mitochondrial respiratory chain function and cytoprotection

Article abstract of DOI:10.1016/j.bmc.2013.01.075

The effect of the alkyl side chain length of coenzyme Q₁₀ on mitochondrial respiratory chain function has been investigated by the use of synthetic ubiquinone derivatives. Three analogues (3, 4 and 6) were identified that exhibited significantl

Full text of DOI:10.1016/j.bmc.2013.01.075

Bioorganic & Medicinal Chemistry 21 (2013) 2346–2354
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effects of alkyl side chain modification of coenzyme Q10 on
mitochondrial respiratory chain function and cytoprotection
David M. Fash a, Omar M. Khdour a, Sunil J. Sahdeo b, Ruth Goldschmidt a, Jennifer Jaruvangsanti a,
Sriloy Dey a, Pablo M. Arce a, Valérie C. Collin a, Gino A. Cortopassi b, , Sidney M. Hecht a,
⇑
⇑
a Center for BioEnergetics, Biodesign Institute, and Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA
b Department of Molecular Biosciences, University of California, Davis, CA 95616, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The effect of the alkyl side chain length of coenzyme Q10 on mitochondrial respiratory chain function has
been investigated by the use of synthetic ubiquinone derivatives. Three analogues (3, 4 and 6) were iden-
tified that exhibited significantly improved effects on mitochondrial oxygen consumption and mitochon-
drial membrane potential, and also conferred significant cytoprotection on cultured mammalian cells in
which glutathione had been depleted by treatment with diethyl maleate. The analogues also exhibited
lesser inhibition of the electron transport chain than idebenone. The results obtained provide guidance
for the design of CoQ10 analogues with improved activity compared to that of idebenone (1), the latter
of which is undergoing evaluation in the clinic as a therapeutic agent.
Received 21 December 2012
Revised 22 January 2013
Accepted 31 January 2013
Available online 13 February 2013
Keywords:
Coenzyme Q10
Electron transport chain
Oxygen consumption
Reactive oxygen species
Cytoprotection
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
antioxidant systems and exposing cellular macromolecules to oxi-
dative damage.10–15
Mitochondria are key organelles which play important roles in
the life and death of the cell. They participate in essential cellular
functions including energy metabolism, and regulate a number of
signaling cascades, including apoptosis, calcium homeostasis and
programmed cell death.1–3 Mitochondria are the primary intracel-
lular site of oxygen consumption and the major source of reactive
oxygen species (ROS), mostly originating from intrinsic limits on
the efficiency of the mitochondrial respiratory chain.4–6
Coenzyme Q10 is a naturally occurring, fat-soluble quinone that
is synthesized endogenously and also assimilated from the diet and
localized mainly in the mitochondrial membranes.16 CoQ10 acts as
an electron carrier in the mitochondrial respiratory chain by trans-
ferring electrons from complexes I and II to complex III.17 It also
transfers protons to the mitochondrial inter-membrane space, con-
tributing to maintenance of membrane potential and to the biosyn-
thesis of ATP.18 Further, reduced CoQ10 is a powerful antioxidant
that prevents oxidative damage by free radicals, including the oxi-
dation of lipids within the mitochondrial membrane.17,19
Functional CoQ10 deficiency can result from impairment in
CoQ10 biosynthesis or enhanced oxidative stress. The result will
be less cellular ATP, diminished conversion of CoQ10 to its re-
duced form and reduced protection against oxidative stress. Re-
duced mitochondrial respiratory chain activities and ATP
synthesis, and increased oxidative stress in CoQ10-deficient cells
have been found to correlate with the severity of primary
CoQ10 deficiency.20,21 Also, a decreased ratio of reduced to oxi-
dized CoQ10 has been found in the platelets of individuals with
Parkinson’s disease.21,22
In mitochondria, superoxide (O2Åꢀ) is generated at a number of
sites within the electron transport chain, linked to bioenergetic
function.4,5 However, non-optimal electron transfer at any point
in the respiratory chain can have a major impact on mitochondrial
coupling (ATP synthesis) and the production of reactive oxygen
species. Cells have developed a number of efficient ROS scavenging
systems, including antioxidant enzymes, the glutathione redox cy-
cle with its associated constitutive enzymes, and the pool of coen-
zyme Q10 (CoQ10). Accordingly, cells can readily deal with normal
levels of ROS produced within the mitochondria.7–9 However, im-
paired oxidative phosphorylation can lead to significantly in-
creased production of ROS, further challenging the endogenous
Coenzyme Q10 has a long history as an experimental therapeutic
agent; clinical trials of CoQ10 in mitochondrial and neurodegener-
ative diseases have produced mixed results.23,24 The effectiveness
of CoQ10 in mitochondria in such studies may be hampered by its
extreme hydrophobicity, which results in low bioavailability. It
⇑
Corresponding authors. Tel.: +1 530 754 9665; fax: +1 530 754 9342 (G.A.C.);
tel.: +1 480 965 6625; fax: +1 480 965 0038 (S.M.H.).
E-mail addresses: gcortopassi@ucdavis.edu (G.A. Cortopassi), sidney.hecht@asu.
edu (S.M. Hecht).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.075

D. M. Fash et al. / Bioorg. Med. Chem. 21 (2013) 2346–2354
2347
Grubbs' 2nd generation catalyst,
3,3-dimethyl-1-butene, CH2Cl2
H2, Pd/C, CH3OH
86%
HO
HO
4
4
77%
7
8
HO
p-TsCl, TEA, DMAP, CH2Cl2
89%
NaI, acetone
98%
TsO
6
6
9
10
O
O
O
H3CO
H3CO
H3CO
H3CO
t-BuOK, DMF
84%
I
+
6
O
11
12
13
O
O
CH3O
CH3O
toluene, 95 °C
100%
5
Scheme 1. Route employed for the synthesis of compound 5.
has been shown in rats that only 3% of orally administered CoQ10
could be absorbed.25 This has resulted in a number of strategies
to develop mitochondrially accessible CoQ10 analogues for sup-
pressing redox stress within mitochondria, and otherwise enhanc-
ing mitochondrial function.26–29
The syntheses and characterization of several compounds of
this type are described herein. Compounds 1–6 were evaluated
for their effects within the mitochondrial electron transport chain,
for their ability to support oxygen consumption and maintain
mitochondrial membrane potential (Dwm), and also for their abil-
One of the best studied CoQ10 analogue is idebenone (1), which
has been evaluated in a number of clinical trials. In an earlier study,
we demonstrated that idebenone analogues having one or both
OCH3 groups replaced with CH3 groups retained the ability to sup-
port O2 consumption within the mitochondrial respiratory chain.30
In the present study, structural variations in CoQ10 were limited to
the alkyl side chain at the 6-position of the 1,4-benzoquinone ring.
A number of CoQ10 analogues with modified side chains have been
prepared and evaluated to identify compounds that can support
electron transport through the respiratory chain and protect cells
against oxidative stress.
ity to confer cytoprotection to cultured CEM cells from oxidative
stress induced by diethyl maleate treatment.
2. Results and discussion
2.1. Compound design and synthesis
Compounds 1–4 were synthesized in analogy with procedures
reported previously.30–32 To investigate the tolerance for steric
bulk at the side chain terminus, compound 5 (Scheme 1) was pre-
pared. Cross metathesis using Grubbs’ 2nd generation catalyst33
p-TsCl, TEA, DMAP, CH2Cl2
NaI, acetone
OH
6
OTs
6
95%
90%
14
O
O
H3CO
H3CO
H3CO
t-BuOK, DMF
82%
toluene, 95 °C
98%
+
I
6
H3CO
O
O
6
O
O
15
12
16
O
O
1. Grubbs' 2nd generation catalyst,
vinylcyclohexane, CH2Cl2
2. H2, Pd/C, CH3OH
CH3O
CH3O
CH3O
CH3O
6
6
59%
17
6
Scheme 2. Route employed for the synthesis of compound 6.

2348
D. M. Fash et al. / Bioorg. Med. Chem. 21 (2013) 2346–2354
O
CH3O
CH3O
H
10
ClogP 20.15
coenzyme Q10
O
O
O
O
CH3O
CH3O
CH3O
OH CH3O
N3
O
2
ClogP 3.22
ClogP 5.49
ClogP 7.86
1 (idebenone)
O
O
CH3O
CH3O
CH3O
CH3O
O
O
ClogP 5.21
3 (decylubiquinone)
4
O
O
CH3O
CH3O
CH3O
CH3O
O
O
ClogP 6.01
ClogP 7.86
5
6
Figure 1. Coenzyme Q10, and structurally related ubiquinones (1–6).
Table 1
Table 3
Inhibitory effects of CoQ10 analogues (1–6) on bovine heart mitochondrial NADH
oxidase activity
Effects of CoQ10 analogues (1–6) on mitochondrial oxygen consumption in RGC-5
cellsa
Compound
NADH oxidase activitya (%)
Compound
Fold change over vehicle
1.5 h 24 h
10 10 lM
1
l
M
5
lM
10 lM
1 (Idebenone)
2
3 (Decylubiquinone)
4
5
6
58.3 0.8
89.8 1.2
93.5 2.6
85.2 4.0
68.3 5.4
88.3 0.8
20.3 1.0
75.0 1.8
83.3 1.3
66.3 4.0
50.6 3.8
81.9 1.1
13.4 0.8
64.0 1.2
61.5 0.9
59.7 1.3
36.2 4.2
78.7 1.5
3
l
M
lM
3
lM
1 (Idebenone)
2
3 (Decylubiquinone)
4
5
6
1.12 0.04
1.01 0.03
1.19 0.02
1.14 0.02
1.08 0.03
1.17 0.06
0.99 0.06
0.97 0.01
1.2 0.04
1.15 0.02
1.05 0.03
1.21 0.06
0.98 0.17
1.68 0.13
0.99 0.52
1.49 0.11
1.55 0.07
1.06 0.22
0.98 0.17
1.01 0.19
1.08 0.11
1.52 0.48
1.16 0.45
1.51 0.55
a
Relative to untreated control. The specific activity of the enzyme was
323 8.2 nmol NADH/min/mg of protein.
a
Oxygen consumption values are normalized as fold change from vehicle (0.1%
DMSO) fluorescence values at each time point.
Table 2
The inhibitory effects of CoQ10 analogues (1–6) on bovine heart mitochondrial
complex I
Table 4
Suppression of reactive oxygen species (ROS) by CoQ10 analogues (1–6) in cultured
CEM leukemia cells treated with DEM
a
Compound
IC50
(lM)
Imax (%)
Compound
ROS scavenging activitya (%)
2.5 lM
1 (Idebenone)
2
3 (Decylubiquinone)
4
5
6
5.8 0.7
>30
>30
>30
14.7 2.1
14.9 0.4
75.0 1.6
1
l
M
Untreated control
Treated control
1 (Idebenone)
2
100
0
100
0
69.0 1.2
55.4 2.8
45 6.0
71 3.3
80 2.2
100 2.1
88 2.0
100 1.9
65 5.5
87 2.2
95 3.2
100 3.1
100 2.7
100 2.9
a
3 (Decylubiquinone)
Maximum inhibition achievable.
4
5
6
was used to couple compound 734 with excess 3,3-dimethyl-1-bu-
tene to give alkene 8 in good yield. Intermediate 8 was reduced
with Pd/C under a hydrogen atmosphere to give the saturated
derivative 9. tert-Butyl alcohol 9 was then converted to its tosylate
10 in 89% yield. Displacement of the tosylate group with sodium
iodide gave the final side chain precursor 11 in nearly quantitative
yield. Alkylation of quinone 1235 using potassium tert-butoxide at
low temperature afforded intermediate 13 in 84% yield. A retro
a
Results are expressed as % of scavenging activity of ROS measured by DCF
median fluorescence intensity.
Diels–Alder reaction gave the desired saturated quinone 5 in quan-
titative yield.
Cyclohexyldecyl analogue 6 was easily obtained from the cross
metathesis reaction of intermediate 17 with Grubbs’ 2nd genera-
tion catalyst33 in the presence of vinylcyclohexane using the

D. M. Fash et al. / Bioorg. Med. Chem. 21 (2013) 2346–2354
2349
Figure 2. Effect of CoQ10 analogues (1–6) on the mitochondrial membrane potential of DEM-treated CEM leukemia cells. Representative flow cytometric two-dimensional
color density dot plot analyses of mitochondrial membrane potential in CEM cells stained with TMRM and analyzed using the FL2-H channel as described in Experimental
Section. The cells were washed in phosphate buffered saline, and then suspended in phosphate buffered saline containing 20 mM glucose. The percentage of cells with intact
D
w
m is indicated in the top right quadrant of captions. 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 wm calculated using CellQuest software. The data are also summarized in the bar graph.
D
general alkene coupling procedure (Scheme 2). Compound 17 was
prepared from the commercially available 9-decen-1-ol and inter-
mediate 12.35 Tosylation of the alcohol led to intermediate 14 in
95% yield. Compound 14 underwent a Finkelstein reaction with so-
dium iodide to give 15 in 90% yield. The alkenyl iodide 15 was sub-
sequently used to alkylate quinone 1235 in the presence of
potassium tert-butoxide to give the desired intermediate 16, which
was heated at 95 °C in toluene to give the quinone 17 in nearly
quantitative yield. As noted above, 6 was then obtained in 59%
yield via a cross metathesis reaction with vinylcyclohexane and
subsequent reduction of the resulting alkene.
2.2. Biochemical and biological evaluation
2.2.1. Mitochondrial electron transport chain function
The effect of the alkyl side chain of the CoQ10 analogues (Fig. 1)
in their electron transfer properties in the mitochondrial respira-
tory chain was studied. The alkyl side chains varied in chain length,

2350
D. M. Fash et al. / Bioorg. Med. Chem. 21 (2013) 2346–2354
branching and polarity. The inhibitory effects of the test com-
pounds on bovine heart mitochondrial complex I alone, or on com-
plexes I, III and IV together were evaluated using submitochondrial
particles (SMP). Both NADH:ubiquinone oxidoreductase activity
(complex I) and NADH oxidase activity (complexes I, III and IV)
were measured spectrophotometrically. NADH:ubiquinone oxido-
reductase activity was measured using coenzyme Q1 as a substrate.
The results are presented in Tables 1 and 2, and show that com-
pounds 2, 3 (decylubiquinone) and 4 were not strongly inhibitory
to respiratory chain function. In comparison, compound 1 (idebe-
none) having a polar terminal hydroxyl group strongly inhibited
both NADH oxidase and NADH:ubiquinone oxidoreductase activi-
ties. The branched chain analogues (compounds 5 and 6) were
slightly more inhibitory to complex I than linear analogues 2–4. Gi-
ven that the analogues are capable of promoting enhanced oxygen
consumption under some conditions (vide infra), presumably via
electron transport to complex IV, it seems possible that some of
CoQ10 analogues to suppress ROS. Cellular ROS production was
monitored using 20,70-dichlorodihydrofluorescein diacetate
(DCFH-DA), a membrane permeable and oxidant-sensitive fluores-
cent dye.36 Once this dye is taken up by cells, DCFH-DA is hydro-
lyzed by cellular esterases to the non-fluorescent DCFH; this
traps the dye in the cell. In the presence of ROS including hydrogen
peroxide, DCFH is oxidized to the highly fluorescent dichlorofluo-
rescein (DCF). Intracellular DCF fluorescence is used as an index
of cellular ROS. The results are presented in Table 4, and show that
analogues 3, 4, 5 and 6 were the most potent in suppressing ROS.
All of the compounds tested were better ROS scavengers than ideb-
enone. It is logical to think that the oxidized forms of compounds
1–6 suppressed ROS by acting as prooxidants, that is, by accepting
electrons from ROS. We have shown previously that both the oxi-
dized and reduced forms of idebenone had some activity in this as-
say, presumably due to their intracellular interconversion.29
the analogues may be acting as competitive substrates to CoQ10
,
2.2.4. Preservation of mitochondrial membrane potential
and that the apparent inhibition simply reflects that competition.
(Dwm)
Mitochondrial membrane potential collapse is an event that fol-
2.2.2. Oxygen consumption
lows ROS-induced mitochondrial damage and precedes cell death.
Compounds 1–6 were assessed for their ability to preserve mito-
chondrial membrane potential following induced oxidative stress
Changes in mitochondrial oxygen consumption were deter-
mined in RGC-5 cells using oxygen biosensor plates after 1.5 or
24 h following treatment with compounds 1–6. The results showed
in CEM leukemia lymphocytes.
Dwm was assessed by the extent
that compounds 1, 3, 4, 5 and 6 (3
oxygen consumption between 1.1- and 1.2-fold after 1.5 h (Ta-
ble 3). Interestingly, at 10 M concentration, only compounds 3,
4 and 6 exhibited analogous increases. The results from the 24-h
studies were more variable; only compounds 2, 4 and 5 signifi-
l
M concentration) increased
of retention of the cationic fluorescent probe tetramethylrhod-
amine methyl ester (TMRM), which preferentially accumulates in
the mitochondria due to the negative membrane potential across
the inner mitochondrial membrane, in accordance with the Nernst
potential.37 The red fluorescent signal in mitochondria decreases
l
cantly increased oxygen consumption when tested at 3
l
M con-
when Dwm collapses, and the detection of mitochondrial depolar-
centration, and did so by approximately 1.5-fold. At 10
l
M
ization using TMRM was accomplished by flow cytometry. Figure 2
illustrates representative two-dimensional density dot plots of
TMRM-stained lymphocyte cells showing the percentage of cells
concentration, compounds 4–6 may have been active, but the error
bars were large. Overall, the results indicate that some CoQ10 ana-
logues can significantly potentiate mitochondrial oxygen con-
with intact
D
w
m (TMRM fluorescence in top right quadrant) versus
wm (TMRM fluorescence in
sumption at 3
lM concentration after 1.5 and 24 h. Since the
the percentage of cells with reduced D
compounds suppress ROS (vide infra), increased oxygen consump-
tion cannot be due to enhanced formation of superoxide. Logically,
the enhanced oxygen consumption must be due to greater reduc-
tion of oxygen to water in complex IV. The putatively enhanced
flow of electrons into complex IV suggested that the analogues
might increase ATP production. This was found not to be the case
(data not shown), presumably due to the concurrent inhibitory ef-
fects on NADH oxidase activity (Table 1).
bottom left and right quadrants). Figure 2 (bottom panel) shows
a bar graph of the percentage (means SEM) of CEM lymphocytes
with intact
centage of cells with TMRM fluorescence in the top right quadrant,
indicating that DEM treatment caused depolarization of
These data indicate that compounds 3, 4 and 6 were able to
block oxidative stress-induced collapse of wm completely, and
Dwm. Treatment with 5 mM DEM decreased the per-
D
wm.
D
clearly better than idebenone. Compounds 2 and 5 had activity
intermediate between idebenone and the best compounds. Treat-
ment with compounds 4 and 5 in the absence of DEM treatment
had no effect on mitochondrial membrane potential. Also run as
a control was FCCP (carbonyl cyanide p-(trifluoromethyl) phen-
2.2.3. Suppression of reactive oxygen species
The ability of these short side chain CoQ10 analogues to sup-
press ROS induced by depletion of glutathione with diethyl male-
ate (DEM) was evaluated in CEM leukemia lymphocyte cells.
DEM treatment depletes glutathione stoichiometrically by direct
chemical reaction, thus exposing the cells to oxidative stress. This
facilitated the observation of ROS, as well as the ability of the
ylhydrazone),
a potent uncoupler of mitochondrial oxidative
phosphorylation.
2.2.5. Cytoprotection
The ability of the test compounds (1–6) to confer cytoprotection
was evaluated in cultured CEM leukemia cells that had been trea-
ted with diethyl maleate to induce oxidative stress through deple-
tion of glutathione. As shown in Table 5, compounds 3, 4 and 6
were the most effective, even at the low concentrations. Idebenone
was the least protective of the compounds, and compounds 2 and 5
exhibited intermediate effects.
Table 5
Cytoprotective effects of CoQ10 analogues (1–6) on the viability of cultured CEM
leukemia cells treated with DEMa
Compound
Viable cells (%)
0.1
lM
0.5
l
M
1
lM
5 lM
1 (Idebenone)
2
3 (Decylubiquinone)
4
5
6
25 1.2
11 0.9
69 7.4
82 5.2
38 3.3
21 2.0
62 2.8
87 2.1
43 4.1
83 3.2
71 1.2
72 1.7
79 5.3
90 3.8
72 2.4
87 2.8
74 5.4
92 2.1
87 6.4
94 2.4
93 7.0
93 1.6
3. Conclusions
Six structural analogues of CoQ10 were evaluated for their abil-
ity to support respiratory chain function. Analogues 2–6 were
found to be superior to idebenone (1) in their abilities to suppress
ROS and maintain mitochondrial membrane potential in CEM
9
0.8
76 4.4
a
The viability of untreated cells was defined as 100%; cells treated with DEM
alone had 18 10% viability.

D. M. Fash et al. / Bioorg. Med. Chem. 21 (2013) 2346–2354
2351
leukemia cells that had been treated with diethyl maleate (DEM) to
deplete cellular glutathione. These compounds were also less
inhibitory to respiratory chain function than idebenone, and were
generally more effective than idebenone in affording cytoprotec-
tion to DEM-treated CEM leukemia cells. All of the analogues were
capable of enhancing mitochondrial oxygen consumption in RGC-5
cells under at least some conditions, with analogues 4–6 having
the most robust effects. Compounds 4 and 6 had especially favor-
able properties overall, illustrating the importance of an optimized
side chain on the properties of exogenously supplied CoQ10 ana-
logues. Since these analogues are significantly less hydrophobic
than CoQ10 itself, their bioavailability following exogenous admin-
istration should be more favorable. Clearly, careful optimization of
benzoquinones and aza analogues capable of analogous behavior,
has the potential to afford compounds of interest for therapeutic
applications.
concentrated under diminished pressure and the residue was puri-
fied by flash chromatography on a silica gel column (20 ꢁ 2 cm).
Elution with 5:1 hexanes/ethyl ether gave 9 as a colorless oil: yield
0.21 g (86%); silica gel TLC Rf 0.29 (3:1 hexanes/ethyl ether); 1H
NMR (CDCl3) d 0.84 (s, 9H), 1.13–1.23 (m, 12H), 1.54 (m, 2H),
1.96 (br s, 1H) and 3.60 (t, 2H, J = 6.8 Hz); 13C NMR (CDCl3) d
24.5, 25.7, 29.4, 29.7, 30.2, 30.5, 32.7, 44.2 and 62.9; high resolu-
tion mass spectrum could not be obtained due to decomposition
of the product.
4.1.3. 9,9-Dimethyldecyl-4-methylbenzenesulfonate (10)
To a solution of 0.21 g (1.14 mmol) of alcohol 9 in 5 mL of
CH2Cl2 was added 0.26 g (1.37 mmol) of p-toluenesulfonyl chlo-
ride, 14.0 mg (0.11 mmol) of DMAP, and 0.32 mL (2.3 mmol) of tri-
ethylamine. The reaction mixture was stirred for 4 h, then
quenched by the addition of 50 mL of H2O. The mixture was ex-
tracted with four 20-mL portions of CH2Cl2. The combined organic
layer was washed with brine, dried over MgSO4, and then concen-
trated under diminished pressure. The residue was purified by
flash chromatography on a silica gel column (40 ꢁ 4 cm). Elution
with 5:1 hexanes/ethyl ether gave 10 as a colorless oil: yield
0.35 g (89%); silica gel TLC Rf 0.63 (3:1 hexanes/ethyl ether); 1H
NMR (CDCl3) d 0.84 (s, 9H), 1.13–1.29 (m, 12H), 1.62 (m, 2H),
2.44 (s, 3H), 4.00 (t, 2H, J = 6.4 Hz), 7.33 (d, 2H, J = 7.6 Hz) and
7.77 (d, 2H, J = 7.6 Hz); 13C NMR (CDCl3) d 21.6, 24.4, 25.3, 28.7,
28.9, 29.4, 44.2, 70.6, 127.8, 129.7, 133.1 and 144.5; mass spectrum
(APCI), m/z 341.2159 (M+H)+ (C19H33O3S requires m/z 341.2150).
4. Experimental section
4.1. Chemistry
1H NMR spectra were recorded on a Varian Inova 400 MHz,
using chloroform-d or methanol-d4 as solvents. 1H NMR chemical
shifts were reported relative to residual chloroform at 7.26 ppm
or to residual methanol at 3.31 ppm. 13C NMR chemical shifts were
reported relative to residual chloroform at 77.1 ppm or to residual
methanol at 49.0 ppm. All solvents were of analytical grade and
were used without further purification. All chemicals were pur-
chased from Sigma–Aldrich Chemical Co. and were used without
further purification. Reactions were carried out under an argon
atmosphere. Column chromatography was carried out using silica
gel (60 Å particle size, 230–240 mesh, Silicycle). Analytical thin
layer chromatography separations were carried out on glass plates
coated with silica gel (60 Å particle size, F-254, E. Merck). The TLC
chromatograms were visualized by UV irradiation or by immersing
the plates in 2.5% potassium permanganate in ethanol, or 2% anis-
aldehyde + 5% sulfuric acid + 1.5% glacial acetic acid in ethanol,
both applications being followed by heating of the TLC plates.
Melting points were recorded on a MelTemp apparatus and are
uncorrected. High resolution mass spectra were obtained in the
Arizona State University CLAS High Resolution Mass Spectrometry
Laboratory. Decylubiquinone was purchased from Sigma.
4.1.4. 1-Iodo-9,9-dimethyldecane (11)
To a solution of 0.35 g (1.02 mmol) of 10 in 50 mL of acetone
was added 0.457 g (3.05 mmol) of sodium iodide. The reaction
mixture was stirred at 80 °C for 16 h, and then filtered through a
Celite pad. The pad was washed with acetone and the filtrate
was concentrated under diminished pressure. The residue was dis-
solved in 50 mL of ethyl ether and washed with three 20-mL por-
tions of 10% aq sodium thiosulfate. The organic layer was washed
with brine, dried over MgSO4, and then concentrated under dimin-
ished pressure to give 11 as a colorless oil: yield 0.346 g (98%); sil-
ica gel TLC Rf 0.95 (3:1 hexanes/ethyl ether); 1H NMR (CDCl3) d 0.85
(s, 9H), 1.16–1.38 (m, 12H), 1.82 (m, 2H) and 3.18 (t, 2H,
J = 7.2 Hz); 13C NMR (CDCl3) d 7.2, 24.5, 28.6, 29.4, 29.5, 30.3,
30.5, 33.6 and 44.2; mass spectrum (APCI), m/z 296.0997 (M)+
(C12H25I requires m/z 296.1001).
4.1.1. 9,9-Dimethyldec-7-en-1-ol (8)
4.1.5. 2-(9,9-Dimethyldecyl)-4,5-dimethoxy-7-
To a solution of 0.100 g (0.780 mmol) of alkene 734 and 12.0 mL
(93.1 mmol) of 3,3-dimethyl-1-butene in 50 mL of CH2Cl2 was
added 33 mg (0.039 mmol) of Grubbs’ 2nd generation catalyst.33
The reaction mixture was stirred at 40 °C for 10 h. The reaction
mixture was concentrated under diminished pressure and the res-
idue was purified by flash chromatography on silica gel column
(20 ꢁ 2 cm). Elution with 2:1 hexanes/ethyl ether gave 13 as a col-
orless oil: yield 0.110 g (77%); silica gel TLC Rf 0.43 (1:1 hexanes/
ethyl ether); 1H NMR (CDCl3) d 0.97 (s, 9H), 1.31–1.35 (m, 6H),
1.55 (m, 2H), 1.75 (br s, 1H), 1.95 (m, 2H), 3.61 (t, 2H, J = 6.8 Hz),
5.29 (m, 1H) and 5.41 (d, 1H, J = 16.0 Hz); 13C NMR (CDCl3) d
25.6, 28.8, 29.6, 29.8, 32.5, 32.7, 62.9, 124.5 and 141.5; mass spec-
trum (APCI), m/z 184.1823 (M)+ (C12H24O requires m/z 184.1827).
methyltricyclo[6.2.1.02,7]undeca-4,9-diene-3,6-dione (13)
To a solution of 63.0 mg (0.25 mmol) of 1235 in 4 mL of DMF at
ꢀ40 °C was added 30.0 mg (0.25 mmol) of potassium tert-butox-
ide, followed by a solution of 68 mg (0.23 mmol) of iodide 11 in
2 mL of DMF. The reaction mixture was stirred at ꢀ40 °C for 1 h,
then allowed to warm to room temperature. The reaction mixture
was stirred at room temperature for 3 h, then quenched by the
addition of 50 mL of 10% aq LiCl. The mixture was extracted with
four 20-mL portions of ethyl ether. The combined organic layer
was washed with 50 mL of 10% aq sodium thiosulfate, dried over
MgSO4, and then concentrated under diminished pressure. The res-
idue was purified by flash chromatography on a silica gel column
(20 ꢁ 2 cm). Elution with 6:1 hexanes/ethyl acetate gave 13 as a
yellow oil: yield 80 mg (84%); silica gel TLC Rf 0.87 (3:1 hexanes/
ethyl acetate); 1H NMR (CDCl3) d 0.84 (s, 9H), 1.12–1.24 (m,
14H), 1.42 (m, 1H), 1.46 (s, 3H), 1.72 (m, 2H), 1.90 (m, 1H), 2.98
(s, 1H), 3.08 (s, 1H), 3.89 (s, 3H), 3.92 (s, 3H) and 6.04 (s, 2H);
13C NMR (CDCl3) d 23.2, 24.5, 26.3, 29.3, 29.4, 29.6, 30.2, 30.4,
30.5, 37.2, 43.3, 44.2, 52.6, 54.2, 56.2, 59.5, 60.1, 60.2, 137.1,
138.1, 149.1, 150.5, 198.2 and 198.8; high resolution mass spec-
trum could not be obtained due to decomposition of the product.
4.1.2. 9,9-Dimethyldecan-1-ol (9)
To a solution of 0.30 g (1.77 mmol) of alkene 8 in 20 mL of
methanol was added a catalytic amount of Pd/C. Hydrogen was
bubbled through the stirred reaction mixture for 15 min, then
the reaction mixture was maintained under a hydrogen atmo-
sphere for 4 h. The reaction mixture was filtered through a Celite
pad and the pad was washed with methanol. The filtrate was

2352
D. M. Fash et al. / Bioorg. Med. Chem. 21 (2013) 2346–2354
4.1.6. 2-(9,9-Dimethyldecyl)-5,6-dimethoxy-3-
methylcyclohexa-2,5-diene-1,4-dione (5)
tion with 6:1 hexanes/ethyl acetate gave 16 as a yellow oil: yield
0.27 g (82%); silica gel TLC Rf 0.75 (3:1 hexanes/ethyl acetate); 1H
NMR (CDCl3) d 1.21–1.33 (m, 12H), 1.40 (m, 2H), 1.43 (s, 3H),
1.67 (m, 2H), 1.86 (m, 1H), 1.97 (m, 1H), 2.94 (s, 1H), 3.04 (s,
1H), 3.86 (s, 3H), 3.89 (s, 3H), 4.87 (d, 1H, J = 10.0 Hz), 4.93 (d,
1H, J = 17.2 Hz), 5.75 (m, 1H) and 6.00 (s, 2H); 13C NMR (CDCl3) d
23.1, 26.2, 28.8, 28.9, 29.2, 29.3, 30.3, 33.7, 37.1, 43.2, 52.5, 54.1,
56.1, 59.3, 60.1, 114.1, 137.0, 138.1, 139.0, 149.0, 150.4, 198.1
and 198.7; mass spectrum (APCI), m/z 387.2524 (M+H)+
(C24H35O4 requires m/z 387.2535).
A solution of 0.08 g (0.19 mmol) of 17 in 20 mL of toluene was
stirred at 95 °C for 12 h. The reaction mixture was concentrated
under diminished pressure and the residue was purified by flash
chromatography on a silica gel column (20 ꢁ 2 cm). Elution with
3:1 hexanes/ethyl acetate gave 5 as an orange oil: yield 0.068 g
(100%); silica gel TLC Rf 0.93 (3:1 hexanes/ethyl acetate); 1H
NMR (CDCl3) d 0.84 (s, 9H), 1.31–1.35 (m, 14H), 2.00 (s, 3H), 2.42
(t, 2H, J = 7.6 Hz) and 3.98 (s, 6H); 13C NMR (CDCl3) d 11.9, 24.5,
26.4, 28.7, 29.4, 29.6, 29.8, 30.3, 30.6, 44.2, 61.1, 138.6, 143.1,
144.2, 144.3, 184.1 and 184.7; mass spectrum (APCI), m/z
351.2527 (M+H)+ (C21H35O4 requires m/z 351.2535).
4.1.10. 2-(9-Decenyl)-5,6-dimethoxy-3-methylcyclohexa-2,5-
diene-1,4-dione (17)
A solution of 0.270 g (0.699 mmol) of 16 in 20 mL of toluene
was stirred at 95 °C for 12 h. The reaction mixture was concen-
trated under diminished pressure and the residue was purified
by flash chromatography on a silica gel column (20 ꢁ 2 cm). Elu-
tion with 6:1 hexanes/ethyl acetate gave 17 as a yellow oil: yield
0.22 g (98%); silica gel TLC Rf 0.47 (3:1 hexanes/ethyl acetate); 1H
NMR (CDCl3) d 1.25–1.33 (m, 12H), 1.98 (s, 3H), 2.01 (t, 2H,
J = 6.8 Hz), 2.41 (t, 2H, J = 6.8 Hz), 3.96 (s, 6H), 4.89 (d, 1H,
J = 10.4 Hz), 4.95 (d, 1H, J = 17.2 Hz) and 5.77 (m, 1H); 13C NMR
(CDCl3) d 11.8, 26.3, 28.6, 28.8, 29.0, 29.2, 29.3, 29.7, 33.7, 61.1,
114.1, 138.6, 139.1, 143.0, 144.2, 184.0 and 184.6; mass spectrum
(APCI), m/z 321.2066 (M+H)+ (C19H29O4 requires m/z 321.2066).
4.1.7. Dec-9-enyl-4-methylbenzenesulfonate (14)
To a solution of 0.22 g (1.40 mmol) of 9-decen-1-ol in 10 mL of
CH2Cl2 was added 0.32 g (1.68 mmol) of p-toluenesulfonyl
chloride, 17.0 mg (0.14 mmol) of DMAP, and 0.39 mL (2.8 mmol)
of triethylamine. The reaction mixture was stirred at room temper-
ature overnight, then quenched with 100 mL of H2O. The mixture
was extracted with four 50-mL portions of CH2Cl2. The combined
organic layer was washed with brine, dried over MgSO4, and then
concentrated under diminished pressure. The residue was purified
by flash chromatography on a silica gel column (40 ꢁ 4 cm). Elu-
tion with 3:1 hexanes/ethyl ether gave 14 as a colorless oil: yield
0.41 g (95%); silica gel TLC Rf 0.56 (3:1 hexanes/ethyl ether); 1H
NMR (CDCl3) d 1.16–1.30 (m, 10H), 1.56 (m, 2H), 1.96 (m, 2H),
2.37 (s, 3H), 3.95 (t, 2H, J = 6.4 Hz), 4.91 (d, 1H, J = 11.2 Hz), 4.93
(d, 1H, J = 17.2 Hz), 5.72 (m, 1H), 7.28 (d, 2H, J = 8.0 Hz) and 7.73
(d, 2H, J = 8.0 Hz); 13C NMR (CDCl3) d 21.4, 25.1, 28.6, 28.7, 29.0,
33.5, 70.5, 114.0, 127.6, 129.6, 133.0, 138.8 and 144.4; mass spec-
trum (APCI), m/z 311.1672 (M+H)+ (C17H27O3S requires m/z
311.1681).
4.1.11. 2-(10-Cyclohexyldecyl)-5,6-dimethoxy-3-
methylcyclohexa-2,5-diene-1,4-dione (6)
To a solution of 0.05 g (0.16 mmol) of 17 and 3.0 mL (21 mmol)
of vinyl cyclohexane in 5 mL of CH2Cl2 was added 7 mg (8 lmol) of
Grubbs’ 2nd generation catalyst.33 The reaction mixture was stir-
red at 40 °C for 8 h, then concentrated under diminished pressure.
The residue was dissolved in 20 mL of CH3OH and to this solution
was added a catalytic amount of Pd/C. Hydrogen was bubbled
through the stirred reaction mixture for 30 min, then the reaction
mixture was maintained under a hydrogen atmosphere overnight.
The reaction mixture was filtered through a Celite pad and the pad
was washed with methanol. The solution was concentrated under
diminished pressure and the residue was purified by flash chroma-
tography on a silica gel column (20 ꢁ 2 cm). Elution with 6:1 hex-
anes/ethyl acetate gave 6 as a yellow oil: yield 37 mg (59%); silica
gel TLC Rf 0.71 (3:1 hexanes/ethyl acetate); 1H NMR (CDCl3) d 1.14–
1.37 (m, 25H), 1.66 (m, 4H), 2.00 (s, 3H), 2.44 (t, 2H, J = 7.6 Hz) and
3.98 (s, 6H); 13C NMR (CDCl3) d 11.9, 14.1, 22.3, 25.9, 26.4, 26.8,
28.7, 29.4, 29.5, 29.6, 29.7, 29.8, 30.0, 31.9, 33.4, 37.5, 37.7, 61.1,
138.6, 143.1, 144.2, 144.3, 184.1 and 184.7; mass spectrum (APCI),
m/z 405.2996 (M+H)+ (C25H41O4 requires m/z 405.3005).
4.1.8. 10-Iododec-1-ene (15)
To a solution of 0.70 g (2.25 mmol) of tosylate 14 in 40 mL of
acetone was added 0.37 g (2.48 mmol) of sodium iodide. The reac-
tion mixture was stirred at 80 °C for 5 h, then filtered through a
Celite pad. The pad was washed with acetone and the filtrate
was concentrated under diminished pressure. The residue was dis-
solved in 50 mL of ethyl ether and washed with three 20-mL por-
tions of 10% aq sodium thiosulfate. The organic layer was washed
with brine, dried over MgSO4, and then concentrated under dimin-
ished pressure. The residue was purified by flash chromatography
on a silica gel column (40 ꢁ 4 cm). Elution with 6:1 hexanes/ethyl
ether gave 15 as a colorless oil: yield 0.541 g (90%); silica gel TLC Rf
0.94 (3:1 hexanes/ethyl ether); 1H NMR (CDCl3) d 1.29–1.38 (m,
10H), 1.82 (m, 2H), 2.03 (m, 2H), 3.18 (t, 2H, J = 7.2 Hz), 4.92 (d,
1H, J = 10.0 Hz), 4.99 (d, 1H, J = 16.8 Hz) and 5.80 (m, 1H); 13C
NMR (CDCl3) d 7.2, 28.5, 28.8, 29.0, 29.2, 30.4, 33.5, 33.7, 114.2
4.2. Biochemical and biological evaluation of the CoQ10
analogues
and 139.0; mass spectrum (APCI), m/z 267.0629 (M+H)+ (C10H20
requires m/z 267.0610).
I
4.2.1. Cell culture growth conditions
4.1.9. 2-(9-Decenyl)-4,5-dimethoxy-7-
CCRF-CEM leukemia lymphocytes (ATCC, CRL-119) were cul-
tured in RPMI (Gibco) with 10% fetal bovine serum (Fisher Scien-
tific), 2 mM glutamine (HyClone) and 1% penicillin–streptomycin
mix (Cellgro). Cells were maintained in log phase at a concentra-
tion between 1 ꢁ 105 and 1 ꢁ 106 cells/mL. RGC5 cells (ATCC
PTA-6600) were grown in phenol-red free growth media (DMEM
supplemented with 10% FBS).
methyltricyclo[6.2.1.02,7]undeca-4,9-diene-3,6-dione (16)
To a solution of 0.212 g (0.854 mmol) of 1235 in 8 mL of DMF at
ꢀ40 °C was added 0.111 g (0.939 mmol) of potassium tert-butox-
ide, followed by a solution of 0.227 g (0.854 mmol) of iodide 15
in 2 mL of DMF. The reaction mixture was stirred at ꢀ40 °C for
3 h, then allowed to warm to room temperature. The reaction mix-
ture was stirred at room temperature for 2 h, then quenched with
50 mL of 10% aq LiCl. The mixture was extracted with four 20-mL
portions of ethyl ether. The combined organic layer was washed
with 50 mL of 10% aq sodium thiosulfate, dried over MgSO4, and
concentrated under diminished pressure. The residue was purified
by column chromatography on a silica gel column (20 ꢁ 2 cm). Elu-
4.2.2. NADH oxidase activity
The inhibitory effects of the test compounds on bovine heart
mitochondrial complexes I, III and IV were evaluated. Beef heart
mitochondria were obtained by a large-scale procedure.38 Submi-
tochondrial particles (SMP) were prepared by the method of

D. M. Fash et al. / Bioorg. Med. Chem. 21 (2013) 2346–2354
2353
Matsuno-Yagi and Hatefi,39 and stored in a buffer containing
0.25 M sucrose and 10 mM Tris–HCl, pH 7.4, at ꢀ80 °C. Bovine
heart SMPs were diluted to 0.5 mg/mL. Activity was assayed at
30 °C and monitored spectrophotometrically using a Beckman
(Molecular Probes) and analyzing fluorescence emission by flow
cytometry in detection channel 2 (FL2-H). Briefly, CEM leukemia
cells (5 ꢁ 105 cells) were pre-treated with or without the test com-
pounds for 16 h. The cells were treated with 5 mM DEM for
120 min, collected by centrifugation at 300ꢁg for 3 min and then
washed with phosphate buffered saline. The cells were resus-
pended in PBS containing 20% glucose and incubated at 37 °C in
the dark for 15 min with 250 nM TMRM. Cells were collected by
centrifugation at 300ꢁg for 3 min and were then washed with
phosphate buffered saline. The samples were analyzed immedi-
ately by flow cytometry using a 488 nm excitation laser and the
FL2-H channel. The results obtained were verified in three inde-
pendent experiments. FCCP, a mitochondrial uncoupler, was used
to produce a negative control. In each analysis, 10,000 events were
recorded.
Coulter DU-530 (340 nm, e
6.22 mMꢀ1 cmꢀ1). NADH oxidase activ-
ity was determined in a reaction medium (2.5 mL total volume)
containing 50 mM Hepes, pH 7.5, and 5 mM MgCl2. The final mito-
chondrial protein concentration was 30 lg/mL. After the pre-equil-
ibration of SMP with inhibitor for 5 min, the initial rates were
calculated from the linear portion of the traces.
4.2.3. NADH:ubiquinone oxidoreductase activity
The inhibition of NADH-Q oxidoreductase activity was deter-
mined using the same experimental conditions described previ-
ously.40,41 Twelve micrograms of SMPs were incubated at 39 °C
for 5 min with the test compound in 1 mL of 50 mM phosphate
buffer, pH 7.4, containing 0.25 M sucrose, 1 mM MgCl2, 2
mycin A and 2 mM KCN. The reaction was initiated by the addition
of 50 M NADH and 50 M coenzyme Q1. Enzymatic activity, mea-
sured by the decrease in NADH absorbance, was monitored at
340 nm.
l
M anti-
4.2.7. Cytoprotection (trypan blue exclusion assay)
Cell viability was determined by the use of a hemocytometer-
based trypan blue exclusion assay in CEM leukemia cells. The via-
bility of DEM-treated cells was determined by their ability to ex-
clude the dye trypan blue. Viable cells exclude trypan blue,
whereas non-viable cells take up the dye and are stained blue.
l
l
4.2.4. Oxygen consumption assay
Briefly, CEM leukemia cells were seeded at a density of
Before initiating the assay, cell density was determined using
the Vi-Cell counter (Beckman Coulter), and 70,000 RGC-5 cells
per well were aliquoted into 384-well oxygen biosensor plates
5 ꢁ 105 cells/mL and treated with different concentrations of the
test compounds. Cells were incubated at 37 °C in a humidified
atmosphere of 5% CO2 in air for 17 h. Oxidative stress was then in-
duced by incubation with 5 mM DEM for 6 h, followed by evalua-
tion of cytoprotection. Cell viability was determined by the use
of 0.4% trypan blue. Cytoprotection by the test compounds was as-
sessed with respect to the untreated controls. Cells not treated
with DEM had >90% cell viability whereas DEM treatment reduced
cell viability to <20%. The ability of the test compounds to protect
the cells against the effects of DEM was determined. Cell viability
was expressed as the percentage of untreated control. Data are ex-
pressed as means S.E.M. (n = 3).
(BD Biosciences) in 90 lL of phenol-red free growth media (DMEM
supplemented with 10% fetal bovine serum), and the cells were al-
lowed to attach for 20-30 min. Ten microliters of test compounds
(10 mM stock in DMSO) or vehicle were dispensed into wells after
an intermediate dilution in PBS, giving a final DMSO concentration
of 0.1%. Test compounds were all tested at 3 and 10
concentrations. There were 6 negative control (DMSO vehicle only)
and 6 positive control (5 M FCCP) wells on each plate. Plates were
lM final assay
l
incubated at 37 °C under 5% CO2 for 1.5 or 24 h and then fluores-
cence was measured (485 nm excitation; 630 nm emission) (BMG
Labtech, Germany). The statistical significance of the data was
determined with a two-tailed Student t test (p-value <0.05).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.075.
4.2.5. Suppression of reactive oxygen species (ROS)29,41,42
Intracellular ROS production was measured using the oxidant
sensitive fluorescent probe 2,7-dichlorodihydrofluorescein diace-
tate (DCFH-DA) (Molecular Probes). One milliliter of CEM leukemia
lymphocyte cells (5 ꢁ 105 cells) were plated in a 24-well plate,
treated with the test compounds 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 (DEM) (97%, Sigma–Aldrich)
for 1 h, collected by centrifugation at 300ꢁg for 3 min and then
washed with phosphate buffered saline (PBS) (Invitrogen). Cells
were resuspended in PBS containing 20 mM glucose and incubated
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