Synthesis and characterization of mitoQ and idebenone analogues as mediators of oxygen consumption in mitochondria

Article abstract of DOI:10.1016/j.bmc.2010.06.104

Analogues of mitoQ and idebenone were synthesized to define the structural elements that support oxygen consumption in the mitochondrial respiratory chain. Eight analogues were prepared and fully characterized, then evaluated for their ability to support oxygen consumption in the mitochondrial respiratory chain. While oxygen consumption was strongly inhibited by mitoQ analogues 2-4 in a chain length-dependent manner, modification of idebenone by replacement of the quinone methoxy groups by methyl groups (analogues 6-8) reduced, but did not eliminate, oxygen consumption. Idebenone analogues 6-8 also displayed significant cytoprotective properties toward cultured mammalian cells in which glutathione had been depleted by treatment with diethyl maleate.

Full text of DOI:10.1016/j.bmc.2010.06.104

Bioorganic & Medicinal Chemistry 18 (2010) 6429–6441
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of mitoQ and idebenone analogues as
mediators of oxygen consumption in mitochondria
Damien Y. Duveau a, Pablo M. Arce a, Robert A. Schoenfeld b, Nidhi Raghav a, Gino A. Cortopassi b,
,
*
Sidney M. Hecht a,
*
a Center for BioEnergetics, Biodesign Institute, and Department of Chemistry, 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:
Analogues of mitoQ and idebenone were synthesized to define the structural elements that support oxy-
gen consumption in the mitochondrial respiratory chain. Eight analogues were prepared and fully char-
acterized, then evaluated for their ability to support oxygen consumption in the mitochondrial
respiratory chain. While oxygen consumption was strongly inhibited by mitoQ analogues 2–4 in a chain
length-dependent manner, modification of idebenone by replacement of the quinone methoxy groups by
methyl groups (analogues 6–8) reduced, but did not eliminate, oxygen consumption. Idebenone ana-
logues 6–8 also displayed significant cytoprotective properties toward cultured mammalian cells in
which glutathione had been depleted by treatment with diethyl maleate.
Received 4 May 2010
Revised 29 June 2010
Accepted 30 June 2010
Available online 4 August 2010
Keywords:
Mitochondrial respiration
Oxygen consumption
Idebenone
Ó 2010 Elsevier Ltd. All rights reserved.
Ubiquinones
Cytoprotection
1. Introduction
agents. Such compounds should ideally accumulate within mito-
chondria, and mediate electron transport through all or defined
Mitochondrial dysfunction is thought to contribute to a number
of human degenerative diseases, many of them lacking specific
treatments.1 Because of their central role in energy production
mitochondria, and particularly the mitochondrial respiratory
chain, are important potential drug targets.2 The study and reme-
diation of impaired electron flow through the complexes of the
mitochondrial respiratory chain has been hampered by the diffi-
culty in delivering redox-active molecules selectively to mitochon-
dria in vivo. Coenzyme Q10 (Fig. 1) is one of the electron carriers
used in the respiratory chain, but its bioavailability as an exoge-
nous agent might be expected to be low due to its extreme hydro-
phobicity. In addition to transporting electrons between complexes
I/II and III of the respiratory chain, the hydroquinone moiety of
coenzyme Q has been shown to possess antioxidant properties.
The multiple functions associated with coenzyme Q highlights
the need for quinones that can be used to provide better insight
into the fate of electrons circulating within the mitochondrial inner
membrane and potentially facilitate the design of therapeutic
parts of the respiratory chain.
A family of mitochondria targeted ubiquinones denoted mitoQs
has been developed recently;3,4 they feature a ubiquinone moiety
attached to alkyl chains of varying length, which terminate with
a triphenylphosphonium cation. This latter functional group en-
ables such lipophilic cations to accumulate within the mitochon-
dria as a consequence of the large mitochondrial membrane
potential. A second family of ubiquinones was developed in the
present study based on the idebenone scaffold as a coenzyme Q
surrogate. Although its side chain is less lipophilic than that of
coenzyme Q, it partitions non-specifically into phospholipid bilay-
ers, but does not accumulate efficiently in mitochondria.
As part of an effort to define the structural elements in coen-
zyme Q10 essential to support mitochondrial respiration, eight
ubiquinones (1–8) were synthesized (Fig. 1), namely mitoQ3 (1),
mitoQ5 (2), mitoQ10 (3), mitoQ15 (4), idebenone (5), and three ana-
logues of idebenone (6–8). Compounds 6–8 include methyl groups
and methoxy groups at varying positions on the quinone ring. The
syntheses and characterization of these compounds are described
herein. Analogues 1–8 were then evaluated for their ability to sup-
port oxygen consumption in the mitochondrial respiratory chain,
and also for their ability to confer protection from oxidative stress
to cultured CEM cells treated with diethyl maleate.
* Corresponding author. Tel.: +1 480 965 6625; fax: +1 480 965 0038 (S.M.H.);
tel.: +1 530 754 9665; fax: +1 530 754 9342 (G.A.C.).
E-mail addresses: gcortopassi@ucdavis.edu (G.A. Cortopassi), sidney.hecht@
asu.edu (S.M. Hecht).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.104

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D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
O
O
O
O
O
R1
R2
MeO
MeO
MeO
MeO
OH
PPh3
n
Br
H
10
O
10
mitoQ3 (1)
mitoQ5 (2)
mitoQ10 (3)
mitoQ15 (4)
coenzyme Q
R1 = R2 = MeO-
R1 = R2 = Me-
R1 = MeO-, R2 = Me-
n = 3
n = 5
n = 10
n = 15
idebenone (5)
6
7
R1 = Me-, R2 = MeO-
8
Figure 1. Coenzyme Q, and structurally related ubiquinones.
mediates by column chromatography. This synthetic strategy al-
lowed facile regioselective functionalization with the allyl
substituent, and the mild conditions employed in this step should
allow introduction of substituents bearing different functional
groups. This should permit the convenient syntheses of coenzyme
Q analogues. Finally, reactive quinone 12 was reduced using so-
dium dithionite in 1:1 THF/water, in the presence of a catalytic
amount of n-BuN4Br, and maintained as the hydroquinone by the
use of a reducing agent through phase transfer catalysis. These
conditions were compatible with permethylation of the hydroqui-
none using dimethyl sulfate and sodium hydroxide, and yielded 13
in 81% yield over two steps. This one-pot procedure afforded 13 in
much higher yield, as compared to the reduction of quinone 12
with NaBH4 or LiAlH4, followed by methylation of the obtained
crude hydroquinone, even when strictly anaerobic conditions were
employed in the latter case.
Hydroboration of 13 using 9-borabicyclo[3.3.1]nonane (9-BBN)
in THF (Scheme 2), followed by oxidation using hydrogen peroxide/
aqueous sodium hydroxide led to primary alcohol 14 in 79% yield.
Alcohol 14 was converted to the corresponding mesylate 15, using
MsCl and triethylamine in CH2Cl2, followed by treatment with so-
dium bromide in DMF at 23 °C to afford bromide 16. Finally, treat-
ment of 16 with CAN in 1:1 acetonitrile/water led to the
chemoselective oxidative cleavage of the two para-methoxy
groups to afford quinone 17 in 95% yield.8 Slow addition of 4-bro-
mo-1-butene to a dilute (0.01 M) solution containing 13 and
Grubb’s second generation catalyst9 (1,3-bis-(2,4,6-trimethyl-
phenyl)-2-(imidazolidene)(dichlorophenylmethylene)(tricy-
clohexylphosphine)ruthenium) in CH2Cl2 at reflux led to the
product of olefin cross-metathesis (18) in 64% yield. While ring
closing metathesis and polymeric olefin metathesis have been
studied extensively, olefin cross-metathesis has been relatively
underrepresented in the area of olefin metathesis.9 The present re-
2. Results and discussion
2.1. Synthesis of mitoQ analogues 1–4
While all four of the mitoQ derivatives described here have
been reported previously,5,6 a variety of alkylation chemistries
were employed for introduction of side chain precursors, and for
their subsequent conversion to triphenylphosphonium derivatives,
the latter of which were also prepared as several different salt
derivatives. Variations of this approach were employed in the pres-
ent study for the syntheses of mitoQs 3 and 4 to obtain the prod-
ucts more conveniently, in an improved state of purity, and with
a uniform bromide counterion to facilitate direct comparison of
biochemical properties. One important variation involved reduc-
tion of the intermediate quinones to the hydroquinones prior to
introduction of the triphenylphosphonium moiety; this afforded
the (reoxidized) final products in an improved state of purity.
For mitoQs 1 and 2, new synthetic strategies were employed to
facilitate side chain introduction and to enhance the potential flex-
ibility of the introduced substituent as a precursor to a variety of
functionalized side chains. The syntheses of mitoQ3 (1) and mitoQ5
(2) involved the use of allyl arene 13 (Scheme 1) as a common syn-
thetic intermediate.
Treatment of coenzyme Q0 (9) with freshly distilled cyclopenta-
diene in acetic acid at 23 °C led to Diels–Alder cycloadduct 10 in
94% yield.7 The angular proton in position 7 was removed che-
moselectively using potassium tert-butoxide in THF at 0 °C, and
the resulting anion was treated with allyl bromide to provide 11
in 80% yield. Compound 11 was isolated as a racemate, but having
exclusively a cis-orientation of methyl and allyl groups. Heating 11
in toluene at reflux led to cleavage of the cyclopentadiene moiety,
providing quinone 12 in quantitative yield. This synthetic sequence
was carried out on a gram scale without purifying any of the inter-
O
O
O
O
a
c
b
MeO
MeO
MeO
MeO
MeO
MeO
O
O
9
10
11
O
O
OMe
d
MeO
MeO
MeO
MeO
OMe
12
13
Scheme 1. Synthesis of intermediate 13. Reagents: (a) cyclopentadiene, AcOH, 23 °C, 94%; (b) t-BuOK, allyl bromide, THF, 0 °C, 80%; (c) toluene, 110 °C, 98%; (d) (i) Na2S2O4, n-
Bu4NBr, 1:1 THF/H2O; (ii) (MeO)2SO2, NaOH, 1:1 THF/H2O, 81%.

D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
6431
OMe
OMe
a
b
MeO
MeO
MeO
MeO
OH
OMe
OMe
13
14
OMe
OMe
O
c
d
MeO
MeO
MeO
MeO
MeO
MeO
Br
3
OMs
Br
3
3
OMe
OMe
O
16
15
17
OMe
OMe
e
d
MeO
MeO
MeO
MeO
Br
OMe
OMe
18
13
O
O
O
f
MeO
MeO
MeO
MeO
Br
Br
5
O
19
20
Scheme 2. Syntheses of quinones 17 and 20. Reagents and conditions: (a) (i) 9-BBN, THF, 0 °C, then 60 °C; (ii) aq NaOH, aq H2O2, 0 °C, 79%; (b) MsCl, Et3N, DMAP, CH2Cl2,
23 °C, 97%; (c) NaBr, DMF, 23 °C, 65%; (d) CAN, 1:1 CH3CN–H2O, 0 °C, then 23 °C, 95% (17), 71% (19); (e) 4-bromo-1-butene, Grubbs’ second generation catalyst, CH2Cl2, 40 °C,
64%; (f) H2 (1 atm), Pd/C, MeOH, 23 °C, 99%.
O
O
a
MeO
MeO
MeO
MeO
Br
10
O
O
9
21
O
O
O
O
O
b
c
d
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
OH
15
Br
15
OMs
15
O
O
O
24
9
22
23
Scheme 3. Syntheses of quinones 21 and 24. Reagents and conditions: (a) (i) 11-bromoundecanoic acid, SOCl2, 79 °C, then pyridine, aq H2O2, Et2O, 23 °C; (ii) 5, AcOH, 118 °C,
22%; (b) 16-hydroxyhexadecanoic acid, AgNO3, K2S2O8, 1:1 CH3CN/H2O, 75 °C, 21%; (c) MsCl, Et3N, DMAP, CH2Cl2, 23 °C, 97%; (d) NaBr, acetone, 56 °C, 66%.
port is to date the only example of olefin cross-metathesis em-
ployed in the synthesis of redox active molecules. Treatment of
18 with CAN in 1:1 acetonitrile/water led to oxidative cleavage
of the two para-methoxy groups to afford quinone 19 in 71% yield.
The quinone and the double bond were then reduced concomi-
tantly by catalytic hydrogenation at atmospheric pressure (Pd/C
in MeOH) to yield a hydroquinone, which air-oxidized spontane-
ously10 to quinone bromide 20 in 99% yield over two steps.
Treatment of 11-bromoundecanoic acid (Scheme 3) with thio-
nyl chloride at reflux led to the corresponding acyl chloride, which
was then immediately treated with aqueous hydrogen peroxide in
the presence of pyridine in diethyl ether to afford 11-bromoun-
decanoic peroxo-anhydride.11
The crude solid so obtained was immediately stirred with coen-
zyme Q0 (9) in acetic acid at reflux to afford quinone bromide 21 in
a modest 22% yield. Similarly, treatment of 15-hydroxyhexadeca-
noic acid and of 9 with silver nitrate and potassium peroxosulfate
in 1:1 acetonitrile/water12 at 75 °C led to quinone 22 in 21% yield.
Conversion of 22 to the corresponding mesylate 23 using MsCl and
triethylamine in CH2Cl2, followed by treatment with sodium

6432
D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
the reaction, upon washing with ether–CH2Cl2, did not lead to the
O
O
O
a
isolation of any product having a satisfactory level of purity. In com-
parison, purification by column chromatography using CH2Cl2–
EtOH enabled efficient separation of the product from unreacted
starting material. Consistent with their structures, the mitoQs do
not readily crystallize; under anhydrous conditions, they form
foams that collapse into gums when exposed to the air.
MeO
MeO
MeO
MeO
Br
PPh3
Br
n
n
O
mitoQ3 (1)
mitoQ5 (2)
mitoQ10 (3)
mitoQ15 (4)
17
20
n = 3
n = 5
n = 10 21
n = 15 24
2.2. Synthesis of idebenone (5) and idebenone analogues 6–8
The syntheses of analogues 6–8 commenced with the prepara-
tion of their corresponding quinone precursors 26, 30, and 35
(Scheme 5). Thus oxidation of 2,3,5-trimethylphenol (25) using a
mixture of iodine, sulfuric acid, and aqueous hydrogen peroxide13
at 23 °C afforded 2,3,5-trimethyl-p-benzoquinone (26) in 94%
yield. Treatment of 2,5-dimethyl-p-benzoquinone with acetic
anhydride and boron trifluoride-etherate14 at 40 °C afforded
1,2,4-triacetoxy-3,6-dimethylbenzene (28) in 92% yield; 28 was
then treated with sodium hydroxide and dimethyl sulfate in meth-
anol14 at 23 °C to provide 3,6-dimethyl-1,2,4-trimethoxybenzene
(29) in 82% yield. Finally, 29 was oxidized using phenyliodine diac-
etate (PIDA)15 to obtain 3,6-dimethyl-2-methoxy-p-benzoquinone
(30) in 65% yield. Jones oxidation of 2,6-dimethylphenol (31) pro-
vided 2,6-dimethyl-p-benzoquinone (32) in 94% yield.16 The qui-
none so obtained was then subjected to the same synthetic
procedures used for the preparation of 30 from 27, in order to pro-
vide quinone 35.
Commercially available 11-bromoundecanoic acid (Scheme 6)
was treated with the sodium salt of benzyl alcohol in DMF at
75 °C to afford the O-benzylated derivative 36 in 60% yield, or with
aqueous potassium hydroxide at reflux to afford 37 in 96% yield.
Treatment of 9 and of 26 with 36, silver nitrate, and potassium per-
sulfate, in acetonitrile/water12 in the dark at 75 °C afforded 38 and
39 in 17% and 37% yields, respectively. Cleavage of the benzyl ether
protecting group by catalytic hydrogenation using palladium-on-
carbon in methanol led to idebenone (5) and to idebenone ana-
logue 6 in 82% and 51% yields, respectively. Similarly, treatment
of 30 and of 35 with 37, silver nitrate, and potassium persulfate,
Scheme 4. Syntheses of mitoQs 1–4. Reagents and conditions: (a) (i) Na2S2O4, 1:1
EtOAc/H2O; (ii) PPh3, EtOH, sealed tube, 85 °C, (iii) air, Pd/C, MeOH, 23 °C, 55–92%.
bromide in acetone at reflux led to quinone 24 in 64% yield over
two steps.
Quinones 17, 20, 21, and 24 (Scheme 4) were reduced using
aqueous sodium dithionite. The crude hydroquinones so obtained
were then treated immediately with a stoichiometric amount of tri-
phenylphosphine in ethanol in a sealed tube in the dark at 85 °C to
afford, after purification by silica gel column chromatography,
mitoQ3 (1), mitoQ5 (2), mitoQ10 (3), and mitoQ15 (4) in 55–92%
yields. While mitoQ5 (2) was obtained as the quinone, 1 was ob-
tained as a mixture containing the quinone (20%) and the corre-
sponding hydroquinone (80%); mitoQ10 (3) and mitoQ15 (4) were
obtained as a mixture of quinone (40%) and hydroquinone (60%).
Stirring these mixtures in methanol in the presence of a catalytic
amount palladium-on-carbon under aerobic conditions led to the
pure quinones in each case. This last step proceeded in higher yields
when the quinone was first reduced to the hydroquinones with
Na2S2O4. In any case, the small scale at which the reaction was
run prevented the use of solvent-less conditions.6 The use of non-
polar aprotic solvents such as toluene did not favor the formation
of the final mitoQ, nor did polar aprotic solvents such as
acetonitrile. The conversion worked best using ethanol, a solvent
in which both reagents and products were soluble. Contrary to
the reported procedure,6 purification of the reaction mixture and
removal of the excess PPh3, and of the Ph3PO generated during
OH
O
a
OH
O
25
26
O
OMe
O
O
OAc
OMe
OMe
OAc
c
b
d
O
OMe
OAc
27
28
29
30
OMe
OAc
O
O
OH
O
b
e
c
d
OMe
OMe
OAc
OMe
OAc
O
31
32
33
34
35
Scheme 5. Syntheses of quinones 26, 30, and 35. Reagents and conditions: (a) I2, H2O2, H2SO4, MeOH, 23 °C, 94%; (b) Ac2O, BF3ÁOEt2, 40 °C, 92% (for 28) and 82% (for 33); (c)
Me2SO4, aq NaOH, MeOH, 23 °C, 82% (for 29) and 24% (for 34); (d) PIDA, 9:1 H2O/MeOH, 23 °C, 65% (for 30) and 66% (for 35); (e) Jones reagent, 1:1 Et2O/H2O, 23 °C, 94%.

D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
6433
O
O
a
b
BnO
HO
11-bromoundecanoic acid
OH
OH
10
10
36
37
O
R1
O
c
OR3
HO
OR3
R2
10
O
10
R3 = Bn
R3 = H
36
37
R1 = R 2 = MeO, R3 = Bn
R1 = R 2 = Me, R3 = Bn
R1 = R 2 = MeO, R3 = H
38
d
39
5
d
6
7
8
R
1 = R2 = Me, R3 = H
R1 = MeO, R2 = Me, R3 = H
R1 = Me,R2 = MeO, R3 = H
Scheme 6. Syntheses of idebenone (analogues) 5–8. Reagents and conditions: (a) BnOH, NaH, DMF, 75 °C, 60%; (b) aq KOH, 100 °C, 96%; (c) 9, 26, 30, or 35, AgNO3, K2S2O8, 1:1
CH3CN/H2O, 75 °C, 10–37%; (d) H2, Pd/C, MeOH, 23 °C, then air, Pd/C, MeOH, 23 °C, 51–82%.
in acetonitrile/water12 in the dark at 75 °C afforded 7 and 8 in 15%
and 10% yields, respectively.
none + idebenone stimulated mitochondrial O2 consumption about
50% above the basal level. This was consistent for each of the ideb-
enone analogues as well, and all four compounds stimulated max-
imal O2 consumption about threefold. While the errors associated
with this measurement do not permit a more detailed analysis of
those structural elements within the quinone moiety conducive
to mitochondrial oxygen consumption, they do suggest that some
variation in substituents on the quinone moiety can be tolerated,
and thus support the concept that exogenously supplied coenzyme
Q analogues may envisioned as mechanistically and therapeuti-
cally relevant probes.
2.3. Oxygen consumption assay
C2C12 cells pretreated with 25 nM rotenone were further trea-
ted with 10
lM compounds 1–8, either in the presence or absence
of 5 M FCCP (carbonylcyanide p-trifluoromethoxyphenylhydraz-
l
one). Oxygen consumption was monitored for 2 h and quantified.
As shown in Figure 2, FCCP activated mitochondrial oxygen con-
sumption under basal conditions, and mitoQ3 did not inhibit O2
consumption, but mitoQ5, mitoQ10, and mitoQ15 each inhibited
O2 consumption; this inhibition increased with increasing side
chain length. Thus coenzyme Q analogues having the triphenyl-
phosphonium functionality within their side chain function poorly
in supporting mitochondrial O2 consumption. In contrast, rote-
2.4. Cytoprotective effects of idebenone analogues
The promising effects noted with idebenone (5) and its ana-
logues (6–8) in supporting mitochondrial oxygen consumption
Figure 2. Effect of compounds 1–8 in supporting mitochondrial O2 consumption. C2C12 cells were pretreated with 25 nM rotenone to simulate a partial blockage of
mitochondrial Complex I, after which 10 M compounds 1–8, either without or with 5 M FCCP (to maximally stimulate mitochondrial oxygen consumption), were added.
l
l
Oxygen consumption was monitored for 2 h and quantified by calculating M/T (see Section 4.2.1). Compounds were tested in triplicate. Error bars represent median absolute
deviation. In the left part of the figure, one sees that under basal conditions, FCCP activated mitochondrial oxygen consumption, and that mitoQ3 did not inhibit O2
consumption, but that mitoQ5, mitoQ10 and mitoQ15 each inhibited O2 consumption, with inhibition increasing with increased side chain length. In the right part of the figure,
one sees that rotenone + idebenone stimulated mitochondrial O2 consumption about 50% above the basal level This was consistent for each of the idebenone analogues (6–8)
as well, and all of the idebenone analogues stimulated maximal O2 consumption by approximately threefold.

6434
D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
Table 1
110 °C prior to use. Tetrahydrofuran was distilled from sodium/
benzophenone ketyl, dichloromethane was distilled from calcium
hydride, benzene, and toluene were distilled from sodium, and tri-
ethylamine and diisopropylamine were distilled from potassium
hydroxide. All other solvents were of analytical grade and were
used without any further purification. Flash column chromatogra-
phy was carried out using silica gel (Silicycle R10030B, 60 Å parti-
cle size, 230–400 mesh), applying a low-pressure stream of
nitrogen. Analytical thin layer chromatographic separations were
carried on glass plates coated with silica gel (60 Å particle size,
Cytoprotective effects of compounds 5–8 on cultured CEM cells treated with diethyl
maleatea
Compound
Viable cells (% of control)
0.5
0.1
l
M
lM
2.5 lM
5
6
7
8
24.6 1.2
34.8 3.4
22.9 0.8
8.27 0.7
35.8 3.3
77.3 2.8
74.7 7.7
21.2 1.0
86.2 4.1
96.3 3.7
82.3 3.7
77.1 1.4
a
CEM leukemia cells were pretreated with compounds 5–8 at the concentrations
indicated for 18 h. The cells were then treated with 5 mM diethyl maleate for 4 h
and subsequently assayed for viability using trypan blue.
250 lm thickness, F-254, Silicycle). The TLC chromatograms were
developed using iodine vapor, or by immersing the plates either
in 2.5% phosphomolybdic acid in ethanol, or in 2.0% anisaldehyde
in ethanol/sulfuric acid/acetic acid, followed by heating (heat
gun). 1H NMR chemical shifts were reported relative to residual
CHCl3 at 7.26 ppm or DMSO at 3.31 ppm; 13C NMR chemical shifts
were reported relative to the central line of CDCl3 at 77.0 ppm or of
DMSO-d6 at 39.5 ppm, and 31P NMR shifts were reported relative to
the central line of H3PO4 at 0.00 ppm.
prompted us to assay these compounds for their ability to protect
cultured mammalian cells from the effects of oxidative stress.
Accordingly, cultured CEM leukemia cells were treated with
5 mM diethyl maleate to deplete cellular glutathione, thus expos-
ing the cells to the effects of oxidative stress. Pretreatment of the
cells with compounds 5–8 prior to diethyl maleate treatment actu-
ally did confer protection to the cells, as summarized in Table 1. As
shown in the table, compound 6, containing two methyl groups in
place of the methoxyl groups in idebenone, was actually the most
effective, conferring virtually complete protection against deple-
4.1.1. 4,5-Dimethoxy-2-methyltricyclo[6.2.1.02,7]undeca-4,
9-diene-3,6-dione (10)7
A stirred solution containing 2.00 g (11.0 mmol) of 2,3-dime-
thoxy-5-methyl-1,4-benzoquinone (9) and 1.24 mL (16.5 mmol)
of freshly distilled cyclopentadiene in 6.6 mL of glacial acetic acid
was stirred at 23 °C for 18 h. The reaction solution was chilled by
means of an ice-water bath and was then treated with saturated
aq NaOH until a basic pH was reached. The product was extracted
with five 20-mL portions of ethyl acetate. The combined organic
layer was washed successively with 20 mL of distilled water, and
20 mL of brine, and was then dried (MgSO4), and concentrated un-
der diminished pressure to provide 4,5-dimethoxy-2-methyltricy-
clo[6.2.1.02,7]undeca-4,9-diene-3,6-dione (10) as a dark yellow oil:
yield 2.56 g (94%); silica gel TLC Rf 0.20 (3:2 hexanes/ethyl ace-
tate); 1H NMR (300 MHz, CDCl3) d 1.45 (s, 3H), 1.45–1.65 (m, 2H,
AB system), 2.80 (d, 1H, J = 3.85 Hz), 3.06 (br m, 1H), 3.40 (br m,
1H), 3.93 (s, 3H), 3.95 (s, 3H), 6.00 (dd, 1H, J = 5.5, 3.8 Hz), and
6.14 (dd, 1H, J = 5.5, 2.8 Hz); 13C NMR (125 MHz, CDCl3) d 26.8,
44.5, 46.6, 49.1, 53.7, 57.3, 58.8, 60.9, 134.6, 137.3, 138.27,
138.48, 150.63 and 150.69.
tion of glutathione when employed at a concentration of 2.5 lM.
3. Conclusion
The syntheses of four mitoQs (1–4), as well as idebenone (5)
and three idebenone analogues (6–8) have been accomplished.
While the side chains of mitoQs 3–4 and of idebenone analogues
5–8 were obtained upon radical alkylation of their quinone precur-
sors, Diels–Alder cycloadduct 10 allowed the facile functionaliza-
tion of 9, leading to the precursors of mitoQ3 (1) and mitoQ5 (2).
This strategy should be readily applicable to modification of the re-
dox core of coenzyme Q and the introduction of a variety of side
chains. Olefin cross-metathesis was used successfully to elongate
the side chain of intermediate 13, en route to mitoQ5 (2). While mi-
toQ analogue 1 did not materially increase or decrease basal or
maximal mitochondrial O2 consumption in the presence of rote-
none, 2, 3, and 4 decreased basal and maximal mitochondrial O2
consumption in order of increasing side chain length. By contrast,
idebenone (5) and each of its analogues 6–8 promoted basal mito-
chondrial O2 consumption by about 50%, and maximal mitochon-
drial O2 consumption by about 300%, that is, much more than in
the presence of rotenone itself. The method by which idebenone
and its analogues increase mitochondrial O2 consumption could
be either through uncoupling mitochondria, or through supporting
maximal electron transport activity. Since rotenone + FCCP only
produced a 50% increase in O2 over rotenone alone, whereas ideb-
enone and analogues + FCCP produced a 300% increase, the latter
hypothesis (i.e., facilitation of maximal electron transport activity)
is better supported. However, further studies of absolute mito-
chondrial productivity (i.e., ATP synthesis) are necessary to confirm
this idea. The ability of the idebenone analogues to protect diethyl
maleate-treated CEM cells from oxidative stress is also quite
encouraging, especially in view of the enhanced protection relative
to idebenone obtained with compound 6.
4.1.2. 2-Allyl-4,5-dimethoxy-7-methyltricyclo[6.2.1.02,7]-
undeca-4,9-diene-3,6-dione (11)
To a stirred solution containing 1.00 g (4.04 mmol) of 4,5-dime-
thoxy-2-methyltricyclo[6.2.1.02,7]-undeca-4,9-diene-3,6-dione
(10) in 6 mL of tetrahydrofuran at 0 °C, was added portionwise
0.72 g (6.06 mmol) of potassium tert-butoxide. The reaction mix-
ture turned from yellow to dark red and was stirred at 0 °C for
30 min. A solution containing 0.53 mL (6.12 mmol) of allyl bromide
in 6 mL of tetrahydrofuran was added dropwise to the reaction
suspension at 0 °C. The reaction mixture was stirred at 0 °C for
1.5 h, and was then treated with 8 mL of distilled water. The prod-
uct was extracted with five 10-mL portions of ether. The combined
organic layer was washed with 10 mL of distilled water, then with
10 mL of brine, and was then dried (MgSO4) and concentrated un-
der diminished pressure to provide 2-allyl-4,5-dimethoxy-7-meth-
yltricyclo[6.2.1.02,7]-undeca-4,9-diene-3,6-dione (11) as a yellow
oil: yield 0.94 g (80%); silica gel TLC Rf 0.33 (2:1 hexanes/ethyl ace-
tate); 1H NMR (500 MHz, CDCl3) d 1.46 (m, 1H), 1.49 (s, 3H), 1.75
(d, 1H, J = 9.8 Hz), 2.54 (m, 1H, AB system), 2.66 (m, 1H, AB sys-
tem), 3.01 (s, 1H), 3.10 (s, 1H), 3.88 (s, 3H), 3.89 (s, 3H), 5.04 (m,
2H), 5.77 (m, 1H), and 6.04 (s, 2H); 13C NMR (125 MHz, CDCl3) d
23.8, 41.8, 43.4, 52.7, 54.4, 56.5, 59.3, 60.4, 60.5, 95.0, 118.9,
134.2, 137.7, 138.1, 149.5, 150.6 and 177.0; mass spectrum (LCT
electrospray), m/z 311.1260 (M+Na)+ (C17H20O4Na requires
311.1259).
4. Experimental
4.1. Chemistry
Reactions were carried out under an atmosphere of argon un-
less specified otherwise. The glassware was dried in an oven at

D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
6435
4.1.3. 2-Allyl-3-methyl-5,6-dimethoxy-1,4-benzoquinone (12)
A solution containing 0.91 g (3.17 mmol) of 2-allyl-4,5-
dimethoxy-7-methyltricyclo[6.2.1.02,7]-undeca-4,9-diene-3,6-
dione (11) in 16 mL of toluene was stirred at reflux for 5 h, during
which time the reaction solution turned from yellow to dark or-
ange. The solvent was concentrated under diminished pressure
to provide 2-allyl-3-methyl-5,6-dimethoxy-1,4-benzoquinone
(12) as a red oil: yield 0.69 g (98%); silica gel TLC Rf 0.38 (2:1 hex-
anes/ethyl acetate); 1H NMR (500 MHz, CDCl3) d 2.02 (s, 3H), 3.23
(d, 2H, J = 7.1 Hz), 3.99 (s, 6H), 5.04 (m, 2H), and 5.75 (m, 1H); 13C
NMR (125 MHz, CDCl3) d 12.2, 15.7, 18.2, 30.0, 30.5, 61.4, 116.8,
133.2, 140.0, 144.5, 183.7 and 184.6; mass spectrum (LCT electro-
spray), m/z 223.0962 (M+H)+ (C12H15O4 requires 223.0970).
and 148.3; mass spectrum (LCT electrospray), m/z 271.1539
(M+H)+ (C14H23O5 requires 271.1545).
4.1.6. 1-(3-Methanesulfoxypropyl)-2,3,4,5-tetramethoxy-6-
methylbenzene (15)
To a solution containing 221 mg (0.817 mmol) of 1-(3-hydroxy-
propyl)-2,3,4,5-tetramethoxy-6-methylbenzene
(3.27 mmol) of triethylamine, and 2.0 mg (0.016 mmol) of dimeth-
ylaminopyridine in 5.5 mL of dichloromethane was added 200
(14),
500 lL
lL
(1.72 mmol) of methanesulfonyl chloride. The reaction solution
was stirred at 23 °C for 4.5 h. The reaction mixture was transferred
into a separatory funnel containing 20 mL of distilled water. The
layers were separated and the aqueous layer was extracted with
three 15-mL portions of ether. The combined organic layer was
washed with 15 mL of brine, and then dried (MgSO4) and concen-
trated under diminished pressure. The crude product was applied
to a column of silica gel (13 Â 3 cm); elution with 2:1 hexanes/ethyl
acetate afforded 1-(3-methanesulfoxy)-2,3,4,5-tetramethoxy-6-
methylbenzene (15) as a clear colorless oil: yield 275 mg (97%); sil-
ica gel TLC Rf 0.15 (2:1 hexanes/ethyl acetate); 1H NMR (500 MHz,
CDCl3) d 1.93 (dt, 2H, J = 15.9, 6.3 Hz), 2.17 (s, 3H), 2.70 (t, 2H,
J = 7.7 Hz), 3.03 (s, 3H), 3.78 (s, 3H), 3.82 (s, 3H), 3.89 (s, 3H), 3.91
(s, 3H), and 4.27 (t, 2H, J = 6.3 Hz); 13C NMR (125 MHz, CDCl3) d
11.6, 22.9, 29.4, 37.4, 60.1, 60.6, 61.0, 70.0, 125.0, 127.7, 144.7,
145.3, 147.7, and 147.9; mass spectrum (LCT electrospray), m/z
371.1147 (M+Na)+ (C15H24O7SNa requires 371.1140).
4.1.4. 1-Allyl-2,3,4,5-tetramethoxy-6-methylbenzene (13)
To a vigorously stirred solution at 23 °C containing 0.474 g
(2.13 mmol) of 2-allyl-3-methyl-5,6-dimethoxy-1,4-benzoquinone
(12) and 0.069 g (0.214 mmol) of n-tetrabutylammonium bromide
in 42 mL of 1:1 tetrahydrofuran/water was added portionwise
4.37 g (21.3 mmol) of sodium dithionite. The reaction mixture
was stirred vigorously at 23 °C for 0.5 h, during which time the
reaction solution turned from orange to light yellow. The reaction
mixture was then chilled by means of an ice-water bath, then
1.28 g (32.0 mmol) of solid sodium hydroxide was added portion-
wise, followed immediately by 4.00 mL (42.3 mmol) of dimethyl
sulfate. The cooling bath was removed and the reaction mixture
was stirred at 23 °C for 2.5 h. The product was extracted with five
30-mL portions of ether. The combined organic layer was washed
successively with 30 mL of distilled water, then with 30 mL of
brine and was dried (MgSO4) and concentrated under diminished
pressure. The crude product was applied to a silica gel column
(12 Â 4 cm); elution with 7:1 hexanes/ethyl acetate provided 1-al-
lyl-2,3,4,5-tetramethoxy-6-methylbenzene (13) as a colorless oil:
yield 0.438 g (81%); silica gel TLC Rf 0.63 (2:1 hexanes/ethyl ace-
tate); 1H NMR (500 MHz, CDCl3) d 2.12 (s, 3H), 3.39 (d, 2H,
J = 5.9 Hz), 3.76 (s, 3H), 3.78 (s, 3H), 3.88 (s, 3H), 3.89 (s, 3H),
4.92 (d, 1H, J = 16.6 Hz), 5.00 (d, 1H, J = 9.8 Hz), and 5.92 (m, 1H);
13C NMR (125 MHz, CDCl3) d 11.9, 31.2, 61.0, 61.3, 61.4, 61.5,
115.0, 125.8, 127.0, 136.7, 144.8, 145.4, 148.0, and 148.0; mass
spectrum (LCT electrospray), m/z 275.1249 (M+Na)+ (C14H20O4Na
requires 275.1259).
4.1.7. 1-(3-Bromopropyl)-2,3,4,5-tetramethoxy-6-methylben-
zene (16)
A solution containing 230 mg (0.660 mmol) of 1-(3-methane-
sulfoxypropyl)-2,3,4,5-tetramethoxy-6-methylbenzene (15) in
3 mL of dimethylformamide was treated with 682 mg (6.60 mmol)
of sodium bromide at 23 °C for 24 h. The reaction mixture was par-
titioned between distilled water (5 mL) and ethyl acetate (5 mL).
The aqueous layer was further extracted with six 5-mL portions
of ethyl acetate. The combined organic layer was washed with
one 10-mL portion of 3% aq HCl, then with two 10-mL portions
of distilled water and 10 mL of brine. The organic phase was dried
(MgSO4) and concentrated under diminished pressure. The result-
ing oil was applied to a silica gel column (13 Â 3 cm); elution with
4:1 hexanes/ethyl acetate afforded 1-(3-bromopropyl)-2,3,4,5-tet-
ramethoxy-6-methylbenzene (16) as a colorless oil: yield 199 mg
(90%); silica gel TLC Rf 0.32 (1:1 hexanes/ethyl acetate); 1H NMR
(400 MHz, CDCl3) d 2.02 (m, 2H), 2.18 (s, 3H), 2.72 (dd, 2H,
J = 8.8, 6.8 Hz), 3.47 (t, 2H, J = 6.8 Hz), 3.78 (s, 3H), 3.83 (s, 3H),
3.89 (s, 3H), and 3.91 (s, 3H); 13C NMR (100 MHz, CDCl3) d 11.7,
25.7, 33.2, 33.9, 60.6, 60.98, 61.01, 61.1, 125.0, 128.1, 144.6,
145.1, 145.2, and 147.8; mass spectrum (LCT electrospray), m/z
355.0525 (M+Na)+ (C14H21O4BrNa requires 355.0521).
4.1.5. 1-(3-Hydroxypropyl)-2,3,4,5-tetramethoxy-6-
methylbenzene (14)17
To a stirred solution containing 307 mg (1.22 mmol) of 1-allyl-
2,3,4,5-tetramethoxy-6-methylbenzene (13) in 1.8 mL of tetrahy-
drofuran at 0 °C was added dropwise 3.00 mL of a 1.0 M 9-BBN
solution in tetrahydrofuran. The reaction mixture was stirred at
23 °C for 16 h, then at reflux for 2 h. The reaction solution was then
treated successively at 0 °C with 2.0 mL (6.0 mmol) of 3.0 M aq so-
dium hydroxide and then with 2.0 mL (5.83 mmol) of 30% aq
hydrogen peroxide. The reaction mixture was stirred at 0–2 °C
for 0.5 h. The reaction mixture was diluted with 20 mL of distilled
water and the product was extracted with four 20-mL portions of
ether. The combined organic layer was washed with 20 mL of
brine, dried (Na2SO4) and then concentrated under diminished
pressure. The crude oil was applied to a silica gel column
(12 Â 3 cm); step-gradient elution with 4:1?2:1 hexanes/ethyl
acetate provided 1-(3-hydroxypropyl)-2,3,4,5-tetramethoxy-6-
methylbenzene (14) as a colorless oil: yield 261 mg (79%); silica
gel TLC Rf 0.08 (2:1 hexanes/ethyl acetate); 1H NMR (500 MHz,
CDCl3) d 1.71 (quint, 2H, J = 5.9 Hz), 2.15 (s, 3H), 2.69 (t, 2H,
J = 7.3 Hz), 3.52 (t, 2H, J = 5.9 Hz), 3.76 (s, 3H), 3.82 (s, 3H), 3.87
(s, 3H), and 3.89 (s, 3H); 13C NMR (125 MHz, CDCl3) d 11.8, 22.7,
30.0, 32.3, 60.9, 61.3, 61.5, 61.7, 125.3, 128.4, 144.5, 145.2, 147.9,
4.1.8. 2-(5-Bromopropyl)-3-methyl-5,6-dimethoxy-1,4-benzoqui-
none (17)
To a stirred solution containing 199 mg (0.597 mmol) of 1-(3-
bromopropyl)-2,3,4,5-tetramethoxy-6-methylbenzene (16) in
6 mL of 1:1 acetonitrile/water was added a solution containing
833 mg (1.49 mmol) of cerium (IV) ammonium nitrate (CAN) in
6 mL of 1:1 acetonitrile/water. The cooling bath was removed,
and the reaction solution was stirred at 23 °C for 1 h. The reaction
mixture was partitioned between 10 mL of distilled water and
10 mL of ethyl acetate. The aqueous phase was extracted with five
10-mL portions of ethyl acetate. The combined organic layer was
washed with 5 mL of brine, then dried (NaSO4) and concentrated
under diminished pressure. The resulting oil was applied to a silica
gel column (13 Â 3 cm); elution with 2:1 hexanes/ethyl acetate
afforded 2-(5-bromopropyl)-3-methyl-5,6-dimethoxy-1,4-benzo-
quinone (17) as a yellow oil: yield 164 mg (95%); silica gel TLC Rf

6436
D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
0.36 (2:1 hexanes/ethyl acetate); 1H NMR (400 MHz, CDCl3) d 1.96
(m, 2H), 2.05 (s, 3H), 2.62 (t, 2H, J = 7.6 Hz), 3.43 (t, 2H, J = 6.8 Hz),
and 3.98 (s, 6H); 13C NMR (100 MHz, CDCl3) d 12.0, 25.2, 31.5, 33.1,
61.2, 139.7, 141.1, 144.3, 144.4, 184.0, and 184.4; mass spectrum
(APCI), m/z 303.0233 (M+H)+ (C12H16O4Br requires 303.0232).
acetate); 1H NMR (300 MHz, CDCl3) d 0.89 (m, 2H), 1.34 (m, 6H),
2.01 (s, 3H), 2.45 (m, 2H, J = 7.4 Hz), and 3.98 (s, 6H); 13C NMR
(125 MHz, CDCl3) d 12.1, 14.2, 22.7, 26.6, 28.6, 32.2, 61.4, 138.9,
143.3, 144.5, 144.6, 184.4, and 185.0; mass spectrum (APCI), m/z
331.0548 (M+H)+ (C14H20O4Br requires 331.0545).
4.1.9. 1-(5-Bromo-2-pentenyl)-2,3,4,5-tetramethoxy-6-
methylbenzene (18)
4.1.12. 2-(10-Bromodecyl)-3-methyl-5,6-dimethoxy-1,
4-benzoquinone (21)6
A solution containing 115 mg (0.453 mmol) of 1-allyl-2,3,4,5-
tetramethoxy-6-methylbenzene (13) and 230 lL (2.27 mmol) of
A solution containing 3.00 g (11.3 mmol) of 11-bromoundeca-
noic acid in 1.50 mL (20.6 mmol) of thionyl chloride was stirred
at reflux for 20 min. Excess thionyl chloride was removed by distil-
lation under diminished pressure; the residue was diluted with
10 mL of ether and the resulting light yellow clear solution was
stirred at 0 °C. It was treated with 1.40 mL of 30% aq hydrogen per-
oxide at 0 °C, followed by the slow addition at 0 °C (over the course
of 15 min) of a solution containing 1 mL of pyridine and 1 mL of
ether. As the pyridine was added, a white precipitate formed. Once
the addition was complete, the resulting suspension was stirred at
room temperature for 1.25 h. The entire reaction mixture was then
transferred into a separatory funnel containing 150 mL of ether
and 50 mL of distilled water. The layers were separated and the or-
ganic layer was washed successively with two 50-mL portions of
distilled water, 50 mL of 10% aq HCl, 50 mL of distilled water,
two 50-mL portions of 0.5 M aq sodium bicarbonate, 50 mL of dis-
tilled water, and then with 50 mL of brine. The organic phase was
dried (MgSO4) and concentrated under diminished pressure to af-
ford the crude peroxo-anhydride as a white solid which was used
immediately for the coupling step with 2,3-dimethoxy-5-methyl-
1,4-benzoquinone (9): yield 2.98 g (94%).
4-bromo-1-butene was stirred in 45 mL of dichloromethane in
the presence of 19.4 mg (0.023 mmol) of benzylidene [1,3-
bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene] dichloro (tricy-
clohexylphosphine) ruthenium (Grubbs’ second generation
catalyst9) at reflux for 6 h. The solvent was concentrated under
diminished pressure and the crude residue was applied to a silica
gel column (14 Â 3 cm); elution with 6:1 hexanes/ethyl acetate
afforded 1-(5-bromo-2-pentenyl)-2,3,4,5-tetramethoxy-6-methyl-
benzene (18) as a light amber oil: yield 105 mg (64%); silica gel
TLC Rf 0.51 (2:1 hexanes/ethyl acetate); 1H NMR (500 MHz, CDCl3)
d 2.12 (s, 3H), 2.52 (q, 2H, J = 6.8 Hz), 3.32 (m, 4H), 3.76 (s, 3H), 3.78
(s, 3H), 3.88 (s, 3H), 3.89 (s, 3H), 5.30 (m, 1H), and 5.59 (m, 1H); 13
C
NMR (125 MHz, CDCl3) d 11.9, 30.0, 33.0, 36.1, 60.9, 61.27, 61.33,
61.5, 125.7, 127.19, 127.22, 131.6, 144.8, 145.4, 147.85, and
147.96; mass spectrum (LCT electrospray), m/z 381.0688 (M+Na)+
(C16H23O4BrNa requires 381.0677).
4.1.10. 2-(5-Bromo-2-pentenyl)-3-methyl-5,6-dimethoxy-1,4-
benzoquinone (19)
A solution containing 1.29 g (7.08 mmol) of 2,3-dimethoxy-5-
methyl-1,4-benzoquinone (9) and 2.98 g (10.6 mmol) of the freshly
prepared peroxo-anhydride in 42 mL of glacial acetic acid was stir-
red at reflux for 20 h. The resulting red solution was allowed to
cool to room temperature and was then transferred into a separa-
tory funnel containing 300 mL of ether. The solution was washed
successively with two 100-mL portions of distilled water, two
100-mL portions of 10% aq HCl, 100 mL of distilled water, two
100-mL portions of 0.5 M aq sodium bicarbonate, 100 mL of dis-
tilled water, and then with two 100-mL portions of brine. The dried
organic layer (MgSO4) was concentrated under diminished pres-
sure and the resulting red solid was applied to a silca gel column
(15 Â 4 cm); elution with 20:1 dichloromethane/diethyl ether
afforded 2-(10-bromodecyl)-3-methyl-5,6-dimethoxy-1,4-benzo-
quinone (21) as a red oil: yield 0.62 g (22%); silica gel TLC Rf 0.72
(2:1 hexanes/ethyl acetate); 1H NMR (500 MHz, CDCl3) d 1.25–
1.40 (m, 14H), 1.82 (dt, 2H, J = 14.7, 6.8 Hz), 1.98 (s, 3H), 2.42 (t,
2H, J = 6.8 Hz), 3.37 (t, 2H, J = 6.8 Hz), 3.95 (s, 3H), and 3.96 (s,
3H); 13C NMR (125 MHz, CDCl3) d 12.2, 26.7, 28.4, 28.9, 29.0,
29.5, 29.6, 29.7, 30.1, 33.1, 34.3, 61.4, 138.8, 143.2, 144.4, 184.2,
and 184.7.
To a stirred solution at 0 °C containing 133 mg (0.369 mmol)
1-(5-bromo-pentenyl)-2,3,4,5-tetramethoxy-6-methylben-
of
zene (18) in 3.7 mL of 1:1 acetonitrile/water was added a solution
containing 514 mg (0.923 mmol) of cerium (IV) ammonium nitrate
in 3.7 mL of 1:1 acetonitrile/water. The reaction mixture was stirred
at 23 °C for 2.5 h. The reaction mixture was partitioned between
10 mL of distilled water and 10 mL of ethyl acetate. The aqueous
layer was extracted with three 5-mL portions of ethyl acetate. The
combined organic layer was washed with 5 mL of brine, then dried
(Na2SO4) and concentrated under diminished pressure. The result-
ing crude oil was applied to a silica gel column (14 Â 3 cm); elution
with 2:1 hexanes/ethyl acetate afforded 2-(5-bromo-2-pentenyl)-
3-methyl-5,6-dimethoxy-1,4-benzoquinone (19) as a red oil: yield
86.5 mg (71%); silica gel TLC Rf 0.09 (4:1 hexanes/ether; 1H NMR
(300 MHz, CDCl3) d 1.99 (s, 3H), 2.50 (q, 2H, J = 5.5 Hz), 3.16 (d,
2H, J = 3.3 Hz), 3.31 (t, 2H, J = 6.8 Hz), 3.95 (s, 6H), and 5.42 (m,
2H); 13C NMR (100 MHz, CDCl3) d 12.0, 29.2, 29.8, 32.4, 35.2, 53.4,
61.2, 127.8, 129.1, 184.4, and 185.0; mass spectrum (LCT electro-
spray), m/z 351.0208 (M+Na)+ (C14H17O4BrNa requires 351.0214).
4.1.11. 2-(5-Bromopentyl)-3-methyl-5,6-dimethoxy-1,4-benzo-
quinone (20)17
4.1.13. 2-(15-Hydroxypentadecyl)-3-methyl-5,6-dimethoxy-1,
4-benzoquinone (22)6
A solution containing 85 mg (0.26 mmol) of 2-(5-bromo-2-pen-
tenyl)-3-methyl-5,6-dimethoxy-1,4-benzoquinone (19) in 2.6 mL
of methanol was stirred in the presence of 9 mg of 10% palla-
dium-on-carbon under hydrogen (1 atm) at 23 °C for 3 h, during
which time the reaction mixture turned from yellow to colorless.
The catalyst was removed by filtration through a short pad of silica
gel and the filtrate was stirred at 23 °C under aerobic conditions for
0.5 h, and was then concentrated under diminished pressure to
To a stirred suspension containing 546 mg (3.00 mmol) of 2,3-
dimethoxy-5-methyl-1,4-benzoquinone (9), 816 mg (3.00 mmol)
of 16-hydroxyhexadecanoic acid, and 464 mg (2.73 mmol) of silver
nitrate in 60 mL of 1:1 acetonitrile/water at 75 °C, was added
slowly over the course of 2 h a solution containing 900 mg
(3.33 mmol) of potassium persulfate in 35 mL of distilled water.
When the addition was complete, the reaction mixture was stirred
at 75 °C for 1 h, then was allowed to cool to room temperature, and
the precipitated solid was filtered under vacuum. The filtrate was
transferred to a separatory funnel and the product was extracted
with five 50-mL portions of ether. The combined organic layer
was washed successively with two 100-mL portions of distilled
water, two 100-mL portions of 1.0 M aq sodium bicarbonate,
afford
a
mixture containing ꢀ80% of 2-(5-bromopentyl)-3-
methyl-5,6-dimethoxy-1,4-benzoquinone (20), and ꢀ20% of the
corresponding hydroquinone as an orange oil: yield 84 mg (99%);
treatment of an aerated methanolic solution of the hydroqui-
none/quinone mixture with 10% palladium-on-carbon provided
the quinone quantitatively; silica gel TLC Rf 0.35 (1:1 hexanes/ethyl

D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
6437
100 mL of distilled water and a 50-mL portion of brine. The dried
(Na2SO4) organic phase was then concentrated under diminished
pressure. The crude solid was applied to a silica gel column
(12 Â 3 cm); elution with 10:1 dichloromethane/ether afforded 2-
(15-hydroxypentadecyl)-3-methyl-5,6-dimethoxy-1,4-benzoqui-
none (22) as a dark yellow solid: yield 260 mg (21%); silica gel TLC
Rf 0.10 (20:1 dichloromethane/ether); mp: 63–64 °C; 1H NMR
formed in the presence of a solution containing 1.10 g (5.37 mmol)
of sodium dithionite in 5.4 mL of distilled water. The layers were
separated and the aqueous layer was extracted with 2.7 mL of
ethyl acetate. The combined organic layer was dried (MgSO4),
and was then concentrated under diminished pressure. The crude
hydroquinone so obtained was quickly dissolved in 2.7 mL of 95%
ethanol and the resulting light yellow solution was stirred in the
presence of 72.4 mg (0.276 mmol) of triphenylphosphine in a
sealed tube in the dark at 85 °C for 72 h. The solvent was concen-
trated under diminished pressure and the resulting yellow foam
was applied to a silica gel column (12 Â 3 cm); elution with 9:1
dichloromethane/ethanol afforded 2-(5-triphenylphosphonium-
propyl)-3-methyl-5,6-dimethoxy-1,4-benzoquinone bromide (1)
as a yellow foam and as a mixture of quinone and hydroquinone
(Q:QH2 ꢀ20:80): yield 126 mg (82%); treatment of an aerated
methanolic solution of the hydroquinone/quinone mixture with
10% palladium-on-carbon provided the quinone quantitatively; sil-
ica gel TLC Rf 0.22 (9:1 dichloromethane/ethanol); 1H NMR
(400 MHz, CDCl3) d 1.87 (m, 2H), 2.16 (s, 3H), 2.94 (dd, 2H,
J = 7.6, 7.4 Hz), 3.76 (m, 2H), 3.94 (s, 3H), 3.95 (s, 3H), and 7.66–
7.89 (m, 15H); 13C NMR (100 MHz, CDCl3) d 11.5, 21.8, 22.1, 26.7,
60.9, 61.0, 117.3, 118.0, 118.7, 120.6, 130.5, 133.7, 135.0, 137.9,
184.2, and 184.4; 31P NMR (162 MHz, CDCl3) d 24.3; mass spec-
trum (LCT electrospray), m/z 485.1883 (MÀBr)+ (C30H30O4P re-
quires 485.1882).
(500 MHz, CDCl3)
d 1.23–1.36 (m, 24H), 1.54 (quint, 2H,
J = 6.8 Hz), 1.99 (s, 3H), 2.42 (t, 2H, J = 7.8 Hz), 3.62 (t, 2H,
J = 6.8 Hz), 3.96 (s, 3H), and 3.97 (s, 3H); 13C NMR (125 MHz, CDCl3)
d 12.2, 26.0, 26.7, 29.0, 29.68, 29.73, 29.80, 29.88, 29.90, 29.93,
30.1, 33.1, 61.4, 63.4, 138.8, 143.3, 144.4, 184.2, and 184.8; mass
spectrum (LCT electrospray), m/z 431.2772 (M+Na)+ (C24H40O5Na
requires 431.2773).
4.1.14. 2-(15-Methanesulfoxypentadecyl)-3-methyl-5,
6-dimethoxy-1,4-benzoquinone (23)
To a stirred solution at 23 °C containing 91.8 mg (0.225 mmol)
of 2-(15-hydroxypentadecyl)-3-methyl-5,6-dimethoxy-1,4-benzo-
quinone (22), 70.0
(0.025 mmol) of dimethylaminopyridine in 0.5 mL of dichloro-
methane, was added 21.0 L (0.271 mmol) of methanesulfonyl
lL (0.502 mmol) of triethylamine, and 3.1 mg
l
chloride. After stirring at 23 °C for 1 h, the reaction mixture was di-
luted with 5 mL of ether and was poured into a separatory funnel
containing 5 mL of distilled water. The layers were separated and
the aqueous layer was extracted with two 3-mL portions of ether.
The combined organic layer was washed with two 3-mL portions
0.5 M aq sodium bicarbonate, then with 3 mL of brine. The dried
(MgSO4) organic layer was then concentrated under diminished
pressure to afford 2-(15-methanesulfoxypentadecyl)-3-methyl-
5,6-dimethoxy-1,4-benzoquinone (23) as a yellow solid: yield
107 mg (97%); silica gel TLC Rf 0.40 (20:1 dichloromethane/ether);
mp: 42–44 °C; 1H NMR (500 MHz, CDCl3) d 1.23–1.38 (m, 24H),
1.72 (quint, 2H, J = 6.8 Hz), 1.98 (s, 3H), 2.42 (t, 2H, J = 6.8 Hz),
2.98 (s, 3H), 3.96 (s, 6H), and 4.20 (t, 2H, J = 6.8 Hz); 13C NMR
(125 MHz, CDCl3) d 12.2, 25.7, 26.7, 29.0, 29.3, 29.4, 29.66, 29.70,
29.79, 29.88, 29.90, 30.1, 37.6, 61.4, 70.4, 138.8, 143.2, 144.0,
144.4, 184.2, and 184.8; mass spectrum (LCT electrospray), m/z
509.2555 (M+Na)+ (C25H42O7SNa requires 509.2549).
4.1.17. 2-(5-Triphenylphosphoniumpentyl)-3-methyl-5,
6-dimethoxy-1,4-benzoquinone bromide (MitoQ5, 2)
A solution containing 47 mg (0.14 mmol) of 2-(5-bromopentyl)-
3-methyl-5,6-dimethoxy-1,4-dihydroxybenzene (20) in 2.8 mL of
ethyl acetate was shaken thoroughly in a separatory funnel in
the presence of a solution containing 582 mg (2.84 mmol) of so-
dium dithionite in 5.6 mL of distilled water. The layers were sepa-
rated and the aqueous layer was extracted with 2.8 mL of ethyl
acetate. The combined organic layer was dried (MgSO4) and was
then concentrated under diminished pressure. The crude hydro-
quinone was quickly dissolved in 1.4 mL of 95% ethanol and the
resulting light yellow solution was stirred in the presence of
38 mg (0.14 mmol) of triphenylphosphine in a sealed tube in the
dark at 85 °C for 40 h. The solvent was concentrated under dimin-
ished pressure and the resulting yellow foam was applied to a sil-
ica gel column (12 Â 3 cm); elution with 9:1 dichloromethane/
ethanol afforded 2-(5-triphenylphosphoniumpentyl)-3-methyl-
5,6-dimethoxy-1,4-benzoquinone (2) as a yellow foam: yield
46 mg (55%); silica gel TLC Rf 0.22 (9:1 dichloromethane/ethanol);
1H NMR (400 MHz, CDCl3) d 1.40 (m, 2H), 1.65 (m, 2H), 1.71 (m,
2H), 1.96 (s, 3H), 2.36 (dd, 2H, J = 8.2, 8.0), 3.80 (m, 2H), 3.95 (s,
6H), and 7.66–7.85 (m, 15H); 13C NMR (100 MHz, CDCl3) d 12.0,
22.1, 22.6, 25.8, 27.8, 29.9, 61.11, 61.13, 117.9, 118.6, 130.4,
133.7, 134.9, 139.3, 142.1, 144.2, 184.2, and 184.5; 31P NMR
(162 MHz, CDCl3) d 24.56; mass spectrum (LCT electrospray), m/z
513.2198 (MÀBr)+ (C32H34O4P requires 513.2195).
4.1.15. 2-(15-Bromopentadecyl)-3-methyl-5,6-dimethoxy-1,
4-benzoquinone (24)
A solution containing 58.0 mg (0.119 mmol) of 2-(15-methane-
sulfoxypentadecyl)-3-methyl-5,6-dimethoxy-1,4-benzoquinone
(23) and 123 mg (1.19 mmol) of finely ground sodium bromide in
1.2 mL of acetone was stirred at reflux for 6.5 h. The acetone was
concentrated under diminished pressure; the crude residue was
suspended in a minimum amount of dichloromethane and the
supernatant was applied to a silica gel column (13 Â 2 cm). Elution
with 4:1 hexanes/ethyl acetate afforded 2-(15-bromopentadecyl)-
3-methyl-5,6-dimethoxy-1,4-benzoquinone (24) as a yellow solid:
yield 37.0 mg (66%) as well as essentially all unreacted starting
material: yield 18.7 mg (32%); silica gel TLC Rf 0.53 (4:1 hexanes/
ethyl acetate); mp: 43–44 °C; 1H NMR (500 MHz, CDCl3) d 1.23–
1.42 (m, 24H), 1.83 (quint, 2H, J = 6.8 Hz), 1.98 (s, 3H), 2.42 (t,
2H, J = 6.8 Hz), 3.38 (t, 2H, J = 6.8 Hz), and 3.96 (s, 6H); 13C NMR
(125 MHz, CDCl3) d 12.0, 26.5, 28.2, 28.8, 29.45, 29.50, 29.58,
29.59, 29.7, 29.8, 29.9, 32.9, 34.1, 61.2, 138.6, 143.0, 183.9, and
184.6; mass spectrum (LCT electrospray), m/z 493.1924 (M+Na)+
(C24H39O4BrNa requires 493.1929).
4.1.18. 2-(10-Triphenylphosphoniumdecyl)-3-methyl-5,
6-dimethoxy-1,4-benzoquinone bromide (MitoQ10, 3)
A solution containing 97.7 mg (0.243 mmol) of 2-(10-bromode-
cyl)-3-methyl-5,6-dimethoxy-1,4-benzoquinone (21) in 5 mL of
ethyl acetate was thoroughly shaken in a separatory funnel in
the presence of a solution containing 499 mg (2.44 mmol) of so-
dium dithionite in 5 mL of distilled water. The layers were sepa-
rated and the organic layer was shaken a second time with a
solution containing 499 mg (2.44 mmol) of sodium dithionite in
5 mL of distilled water. The combined aqueous layer was extracted
with 5 mL of ethyl acetate. The combined organic layer was dried
(MgSO4) and concentrated under diminished pressure. The crude
hydroquinone was quickly dissolved in 2.4 mL of 95% ethanol
4.1.16. 2-(5-Triphenylphosphoniumpropyl)-3-methyl-5,6-
dimethoxy-1,4-benzoquinone bromide (MitoQ3, 1)
A solution containing 81.8 mg (0.266 mmol) of 2-(5-bromopro-
pyl)-3-methyl-5,6-dimethoxy-1,4-dihydroxybenzene
(17)
in
2.7 mL of ethyl acetate was thoroughly shaken in a separatory

6438
D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
and the resulting light yellow solution was stirred in the presence
of 64.0 mg (0.244 mmol) of triphenylphosphine in a sealed tube in
the dark at 85 °C for 60 h. The solvent was concentrated under
diminished pressure and the resulting crude yellow oil was applied
to a silica gel column (13 Â 2 cm); elution with 9:1 dichlorometh-
4.1.21. 1,2,4-Triacetoxy-3,6-dimethylbenzene (28)14
To stirred solution at 23 °C containing 1.00 g (7.34 mmol) of
2,5-dimethyl-p-benzoquinone (27) in 8.0 mL of acetic anhydride
was added 400 lL (3.13 mmol) of boron trifluoride etherate. The
reaction mixture was stirred at 40 °C for 48 h and was then poured
into 100 mL of water. The formed precipitate was collected by fil-
tration, and was then dried under diminished pressure to afford
1,2,4-triacetoxy-3,6-dimethylbenzene (28) as a colorless solid:
yield 1.89 g (92%); mp: 97–98 °C; silica gel TLC Rf 0.5 (1:1 hex-
anes/ethyl acetate); 1H NMR (400 MHz, CDCl3) d 1.95 (s, 3H),
2.14 (s, 3H), 2.29 (s, 9H), and 6.84 (s, 1H); 13C NMR (100 MHz,
CDCl3) d 10.1, 16.0, 20.2, 20.7, 20.8, 121.4, 122.5, 129.5, 139.2,
141.8, 146.7, 167.8, 167.9, and 168.8; mass spectrum (EI), m/z
280.0927 (M)+ (C14H16O6 requires 280.0947).
ane/ethanol
afforded
2-(10-triphenylphosphoniumdecyl)-3-
methyl-5,6-dimethoxy-1,4-benzoquinone bromide (3) as a yellow
foam and as a mixture of quinone and hydroquinone (Q:QH2
ꢀ40:60): yield 149 mg (92%); treatment of an aerated methanolic
solution of the hydroquinone/quinone mixture with 10% palla-
dium-on-carbon provided the quinone quantitatively; 1H NMR
(400 MHz, CDCl3) d 1.21–1.62 (m, 16H), 2.13 (s, 3H), 2.56 (dd,
2H, J = 8.2, 8.0 Hz), 3.82 (m, 2H), 3.98 (s, 6H), and 7.68–7.88 (m,
15H); 13C NMR (100 MHz, CDCl3) d 11.9, 15.3, 22.7, 23.0, 26.4,
28.7, 29.2, 29.4, 29.7, 30.4, 31.6, 60.8, 61.2, 118.2, 118.9, 130.5,
133.8, 134.9, 138.7, 143.0, 144.3, 184.2, and 184.7; 31P NMR
(162 MHz, CDCl3) d 24.7; mass spectrum (LCT electrospray), m/z
583.2975 (MÀBr)+ (C37H44O4P requires 583.2977).
4.1.22. 3,6-Dimethyl-1,2,4-trimethoxybenzene (29)14
To a stirred solution at 23 °C containing 1.84 g (6.74 mmol) of
1,2,4-triacetoxy-3,6-dimethylbenzene (28) in 6.0 mL of methanol
were added 5.0 mL (52.3 mmol) of dimethyl sulfate followed by
the slow addition of a solution containing 2.17 g (54.3 mmol) of so-
dium hydroxide in 2.5 mL of water. The reaction mixture was stir-
red at 23 °C for 16 h. The reaction mixture was poured into 60 mL
of water and was then extracted with 80 mL of 1:1 diethyl ether/
hexanes and then with 60 mL of hexanes. The combined organic
layer was washed with 80 mL of water and then with 80 mL of
brine. The solution was dried (MgSO4) and was then concentrated
under diminished pressure. The residue was applied to a silica gel
column (10 Â 6 cm); elution with 3:1 hexanes/ethyl acetate affor-
ded 3,6-dimethyl-1,2,4-trimethoxybenzene (29) as a colorless oil;
yield 1.08 g (82%); silica gel TLC Rf 0.62 (4:1 hexanes/diethyl
ether); 1H NMR (400 MHz, CDCl3) d 2.12 (s, 3H), 2.27 (s, 3H), 3.78
(s, 3H), 3.79 (s, 3H), 3.83 (s, 3H), and 6.43 (s, 1H); 13C NMR
(100 MHz, CDCl3) d 8.7, 16.0, 55.7, 60.3, 60.3, 107.5, 118.0, 128.5,
145.3, 151.8, and 153.8; mass spectrum (EI), m/z 196.1094 (M)+
(C11H16O3 requires 196.1100).
4.1.19. 2-(15-Triphenylphosphoniumpentadecyl)-3-methyl-5,
6-dimethoxy-1,4-benzoquinone bromide (MitoQ15, 4)
A solution containing 66.8 mg (0.142 mmol) of 24 in 2.8 mL of
ethyl acetate was thoroughly shaken in a separatory funnel in
the presence of a solution containing 581 mg (2.83 mmol) of so-
dium dithionite in 2.8 mL of distilled water. The layers were sepa-
rated and the aqueous layer was extracted with 3 mL of ethyl
acetate. The combined organic layer was washed with 5 mL of
brine and then dried (MgSO4) and concentrated under diminished
pressure. The crude hydroquinone was quickly dissolved in 1.4 mL
of 95% ethanol and the resulting yellow solution was treated with
37.2 mg (0.142 mmol) of triphenylphosphine in a sealed tube in
the dark at 85 °C for 63 h. The solvent was concentrated under
diminished pressure and the resulting crude yellow oil was applied
to a silica gel column (13 Â 2 cm); elution with 9:1 dichlorometh-
ane/ethanol afforded 2-(15-triphenylphosphoniumpentadecyl)-3-
methyl-5,6-dimethoxy-1,4-benzoquinone bromide (4) as a yellow
foam and as a mixture of quinone and hydroquinone (Q:QH2
ꢀ40:60): yield 57.0 mg (55%); treatment of an aerated methanolic
solution of the hydroquinone/quinone mixture with 10% palla-
dium-on-carbon provided the quinone quantitatively; silica gel
TLC Rf 0.30 (9:1 dichloromethane/ethanol); 1H NMR (400 MHz,
CDCl3) d 1.15–1.42 (m, 22H), 1.58 (m, 4H), 2.10 (s, 3H), 2.54 (dd,
2H, J = 8.2, 8.0 Hz), 3.68 (m, 2H), 3.85 (s, 6H), and 7.66–7.82 (m,
15H); 13C NMR (100 MHz, CDCl3) d 11.8, 22.47, 22.53, 22.6, 22.9,
26.25, 26.30, 28.6, 29.1, 29.27, 29.39, 29.46, 29.49, 29.53, 29.8,
30.4, 60.7, 61.1, 117.9, 118.6, 130.5, 133.6, 135.0, 136.6, 138.6,
139.9, 144.2, 184.1, and 184.6; 31P NMR (162 MHz, CDCl3) d 24.5;
mass spectrum (LCT electrospray), m/z 653.3754 (MÀBr)+
(C42H54O4P requires 653.3760).
4.1.23. 3,6-Dimethyl-2-methoxy-p-benzoquinone (30)
To a stirred solution containing 900 mg (4.85 mmol) of 3,6-di-
methyl-1,2,4-trimethoxybenzene (29) in 30 mL of 9:1 water/meth-
anol at 23 °C was added 2.21 g (6.86 mmol) of phenyliodine
diacetate (PIDA). The reaction mixture was stirred at 40 °C for
16 h, and then poured into 100 mL of water. The product was ex-
tracted with 150 mL of ether. The organic layer was washed with
100 mL of water, then with 100 mL of satd aq sodium bicarbonate
solution and 100 mL of brine, and was then dried (MgSO4) and con-
centrated under diminished pressure. The residue was applied to a
silica gel column (10 Â 6 cm); elution with 3:1 hexanes/ethyl ace-
tate afforded 3,6-dimethyl-2-methoxy-p-benzoquinone (30) as a
yellow solid: yield: 543 mg (65%); mp: 58–59 °C; silica gel TLC Rf
0.46 (4:1 hexanes/ether); 1H NMR (400 MHz, CDCl3) d 1.93 (s,
3H), 2.03 (s, 3H), 3.98 (s, 3H), and 6.51 (s, 1H); 13C NMR
(100 MHz, CDCl3) d 8.8, 15.7, 60.8, 128.8, 133.1, 143.7, 155.5,
183.6, and 188.5; mass spectrum (EI), m/z 166.0632 (M)+
(C9H10O3 requires 166.0630).
4.1.20. Trimethyl-p-benzoquinone (26)13
To a stirred solution containing 1.00 g (6.57 mmol) of trimethyl-
p-hydroquinone (25) in 20 mL of methanol at 23 °C were added
83.3 mg (0.33 mmol) of iodine followed by 329
lL (2.90 mmol) of
4.1.24. 2,6-Dimethyl-p-benzoquinone (32)16
30% aq hydrogen peroxide and 329 L (0.93 mmol) of sulfuric acid.
l
The reaction mixture was stirred at 23 °C for 3 h and was then di-
luted with 150 mL of ether. The organic layer was washed with
three 75-mL portions of water, then with one 75-mL portion of
satd aq sodium thiosulfate and then with 75 mL of brine. The or-
ganic layer was dried (MgSO4) and concentrated under diminished
pressure to afford trimethyl-p-benzoquinone (26) as yellow crys-
tals: yield 930 mg (94%); mp: 29–30 °C; silica gel TLC Rf 0.65 (3:1
hexanes/ethyl acetate); 1H NMR (400 MHz, CDCl3) d 2.03 (m, 9H)
and 6.56 (s, 1H); 13C NMR (CDCl3) d 12.0, 12.3, 15.9, 133.0, 140.7,
140.9, 145.3, 187.5, and 187.9.
To a stirred solution at 23 °C containing 2.00 g (16.4 mmol) of
2,6-dimethylphenol (31) in 20 mL of diethyl ether was added drop-
wise a solution containing 11.0 g (36.9 mmol) of sodium dichro-
mate-dihydrate and 7 mL of sulfuric acid in 20 mL of water
(Jones reagent). The reaction mixture was stirred at 23 °C for
16 h, and was then poured into 150 mL of water. The product
was extracted using three 150-mL portions of ether. The combined
organic layer was washed with 150 mL of water, then with 150 mL
of brine, and was then dried (MgSO4) and concentrated under
diminished pressure to afford 2,6-dimethyl-p-benzoquinone (32)

D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
6439
as a yellow solid; yield 2.10 g (94%); mp: 65–66 °C; silica gel TLC Rf
0.60 (5:1 hexanes/ethyl acetate); 1H NMR (400 MHz, CDCl3) d 2.05
(s, 6H) and 6.55 (s, 2H); 13C NMR (100 MHz, CDCl3) d 16.0, 17.0,
133.3, 133.3, 145.8, 145.8, 187.6, and 188.2.
510 mg (4.71 mmol) of anhydrous benzyl alcohol followed by
1.00 g (3.77 mmol) of 11-bromoundecanoic acid. The reaction mix-
ture was stirred at 75 °C for 3 h, and was then poured into 100 mL
of ether. The formed precipitate was collected by filtration. The so-
lid was then dissolved in 70 mL of water and the pH was adjusted
to ꢀ1. The product was extracted with 75 mL of dichloromethane.
The organic layer was washed with two 60-mL portions of water,
then with 60 mL of brine, and was then dried (MgSO4) and concen-
trated under diminished pressure to afford 11-benzyloxyundeca-
noic acid (36) as a wax: yield 660 mg (60%); silica gel TLC Rf 0.15
(4:1 hexanes/ethyl acetate); 1H NMR (400 MHz, CDCl3) d 1.28 (m,
12H), 1.62 (m, 4H), 2.34 (t, 2H, J = 7.6 Hz), 3.46 (t, 2H, J = 6.8 Hz),
4.50 (s, 2H), 6.98 (m, 1H), and 7.03 (m, 4H); 13C NMR (100 MHz,
CDCl3) d 24.7, 26.1, 29.0, 29.2, 29.3, 29.4, 29.5, 29.7, 34.0, 70.5,
72.8, 127.4, 127.6, 127.6, 128.3, 128.3, 138.7, and 179.7; mass
spectrum (APCI), m/z 293.2106 (M+H)+ (C18H29O3 requires
293.2117).
4.1.25. 1,2,4-Triacetoxy-3,5-dimethylbenzene (33)14
To a stirred solution containing 1.10 g (8.08 mmol) of 2,6-di-
methyl-p-benzoquinone (32) in 8.0 mL of acetic anhydride at
23 °C was added 360 lL (3.23 mmol) of boron trifluoride-etherate.
The reaction mixture was stirred at 40 °C for 48 h. The reaction
mixture was poured into 100 mL of water and the product was ex-
tracted with two 75-mL portions of ethyl acetate. The combined
organic layer was washed with 60 mL of water, then with 60 mL
of satd aq sodium bicarbonate and 60 mL of brine, and was dried
(MgSO4) and concentrated under diminished pressure. The residue
was applied to a silica gel column (12 Â 3 cm); elution with 3:1
hexanes/ethyl acetate afforded 1,2,4-triacetoxy-3,5-dimethylben-
zene (33) as a colorless solid: yield 1.86 g (82%); mp: 94–95 °C; sil-
ica gel TLC Rf 0.25 (3:1 hexanes/ethyl acetate); 1H NMR (400 MHz,
CDCl3) d 1.97 (s, 3H), 2.13 (s, 3H), 2.25 (s, 3H), 2.33 (s, 3H), 2.99 (s,
3H), and 6.92 (s, 1H); 13C NMR (100 MHz, CDCl3) d 10.5, 16.3, 20.2,
20.4, 20.6, 121.9, 125.1, 128.7, 139.2, 139.9, 145.8, 167.8, 168.3,
and 168.3; mass spectrum (APCI), m/z 281.1031 (M+H)+
(C14H17O6 requires 281.1025).
4.1.29. 11-Hydroxyundecanoic acid (37)
To 70 mL of 2 M aq potassium hydroxide solution was added
1.60 g (6.08 mmol) of 11-bromoundecanoic acid. The reaction mix-
ture was stirred at 100 °C for 16 h, and was then cooled to 23 °C.
The pH was adjusted to ꢀ1 by addition of concd HCl. The formed
precipitate was collected by filtration and was then dried under
diminished pressure to afford 11-hydroxyundecanoic acid (37) as
a colorless solid: yield 1.18 g (96%); mp: 65–66 °C; 1H NMR
(400 MHz, CDCl3) d 1.28 (m, 12H), 1.62 (m, 4H), 2.34 (t, 2H,
J = 7.6 Hz), and 3.63 (t, 2H, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3)
d 24.6, 25.6, 29.0, 29.1, 29.3, 29.3, 29.4, 32.7, 34.0, 63.0, and
179.3; mass spectrum (APCI), m/z 203.1651 (M+H)+ (C11H23O3 re-
quires 203.1647).
4.1.26. 3,5-Dimethyl-1,2,4-trimethoxybenzene (34)14
To a stirred solution containing 1.77 g (6.31 mmol) of 1,2,4-tri-
acetoxy-3,5-dimethylbenzene (33) in 5.0 mL of methanol at 23 °C
was added 5.0 mL (52.3 mmol) of dimethyl sulfate followed by
the slow addition of a solution containing 5.25 g (56.3 mmol) of so-
dium hydroxide in 6 mL of water. The reaction mixture was stirred
at 23 °C for 16 h, and was then poured into 60 mL of water. The
product was extracted with 80 mL of 1:1 ethyl acetate/hexanes
and then with 60 mL of hexanes. The combined organic layer
was washed with 80 mL of water, then with 80 mL of brine, and
was dried (MgSO4) and concentrated under diminished pressure.
The residue was applied to a silica gel column (12 Â 3 cm); elution
with 3:1 hexanes/ethyl ether afforded 3,5-dimethyl-1,2,4-trime-
thoxybenzene (34) as a colorless oil: yield 292 mg (24%); silica
gel TLC Rf 0.5 (9:1 hexanes/ether); 1H NMR (400 MHz, CDCl3) d
2.21 (s, 3H), 2.52 (s, 3H), 3.66 (s, 3H), 3.77 (s, 3H), 3.81 (s, 3H),
and 6.56 (s, 1H); 13C NMR (100 MHz, CDCl3) d 9.4, 16.0, 55.9,
59.9, 60.3, 111.5, 125.2, 125.7, 145.8, 148.9, and 150.6; mass spec-
trum (EI), m/z 196.1094 (M)+ (C11H16O3 requires 196.1100).
4.1.30. 2,3-Dimethoxy-6-methyl-5-benzyloxydecyl-p-benzo-
quinone (38)
To a stirred solution containing 900 mg (3.07 mmol) of 11-ben-
zyloxyundecanoic acid (36) and 500 mg (2.74 mmol) of coenzyme
Q0 (9) in 50 mL of acetonitrile at 23 °C was added 464 mg
(2.74 mmol) of silver nitrate. The reaction mixture was heated to
75 °C, then 900 mg (3.32 mmol) of potassium persulfate in 50 mL
of water was added very slowly. The reaction mixture was stirred
at 75 °C for 4 h and was then cooled to 23 °C, and poured into
25 mL of water. The product was extracted with 50 mL of ethyl
acetate. The organic layer was washed with 25 mL of satd aq so-
dium bicarbonate, then with 25 mL of brine, and was then dried
(MgSO4), and concentrated under diminished pressure. The residue
was applied to a silica gel column (12 Â 4 cm); elution with 3:1
hexanes/ethyl acetate afforded 2,3-dimethoxy-6-methyl-5-ben-
zyloxydecyl-p-benzoquinone (38) as an orange oil: yield 200 mg
(17%); silica gel TLC Rf 0.75 (4:1 hexanes/ethyl acetate); 1H NMR
(400 MHz, CDCl3) d 1.34 (m, 14H), 1.60 (quint, 2H, J = 5.2 Hz),
2.04 (s, 3H), 2.44 (t, 2H, J = 8.4 Hz), 3.46 (t, 2H, J = 6.4 Hz), 3.98 (s,
6H), 4.50 (s, 2H), 7.27 (m, 1H), and 7.34 (m, 4H); 13C NMR
(100 MHz, CDCl3) d 11.9, 26.2, 26.4, 28.7, 29.3, 29.4, 29.4, 29.5,
29.8, 29.8, 61.1, 61.1, 70.5, 72.8, 127.44, 127.6, 127.6, 128.3,
128.3, 138.6, 138.7, 143.1, 144.29, 144.30, 184.1, and 184.7; mass
spectrum (APCI), m/z 429.2637 (M+H)+ (C26H37O5 requires
429.2641).
4.1.27. 3,5-Dimethyl-2-methoxy-p-benzoquinone (35)
To a stirred solution containing 500 mg (2.55 mmol) of 3,5-di-
methyl-1,2,4-trimethoxybenzene (34) in 15 mL of 9:1 water/meth-
anol at 23 °C was added 1.22 g (3.78 mmol) of PIDA. The reaction
mixture was stirred at 40 °C for 16 h, and was then poured into
50 mL of water. The product was extracted with 150 mL of ether.
The organic layer was washed with 100 mL of water, then with
100 mL of satd aq sodium bicarbonate, and with 100 mL of brine,
and was dried (MgSO4) and concentrated under diminished pres-
sure. The residue was applied to a silica gel column (12 Â 3 cm);
elution with 3:1 hexanes/diethyl ether afforded 3,5-dimethyl-2-
methoxy-p-benzoquinone (35) as a yellow solid: yield 282 mg
(66%); mp: 55–56 °C; silica gel TLC Rf 0.47 (4:1 hexanes/diethyl
ether); 1H NMR (400 MHz, CDCl3) d 1.95 (s, 3H), 2.03 (s, 3H), 4.01
(s, 3H), and 6.43 (s, 1H); 13C NMR (100 MHz, CDCl3) d 8.8, 15.7,
60.8, 128.8, 131.4, 145.7, 155.5, 183.6, and 188.5; mass spectrum
(EI), m/z 166.0625 (M)+ (C9H10O3 requires 166.0630).
4.1.31. Idebenone (5)18
To a stirred solution containing 200 mg (0.467 mmol) of 2,3-
dimethoxy-6-methyl-5-benzyloxydecyl-p-benzoquinone (38) in
5 mL of anhydrous methanol at 23 °C was added 15 mg of 10 %
Pd/C in one portion. The reaction mixture was stirred at 23 °C un-
der an atmosphere of hydrogen for 24 h. Air was then bubbled
through the reaction mixture at 23 °C for 24 h. The suspension
was filtered through Celite and the filtrate was concentrated under
4.1.28. 11-Benzyloxyundecanoic acid (36)
To a stirred solution containing 330 mg (8.25 mmol) of sodium
hydride in 15 mL of anhydrous DMF at 23 °C was added dropwise

6440
D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
diminished pressure to afford idebenone (5) as an orange solid:
yield 130 mg (82%); mp: 46–47 °C; 1H NMR (400 MHz, CDCl3) d
1.34 (m, 14H), 1.60 (quint, 2H, J = 7.6 Hz), 2.04 (s, 3H), 2.44 (t,
2H, J = 8.0 Hz), 3.63 (t, 2H, J = 6.8 Hz), and 3.99 (s, 6H); 13C NMR
(100 MHz, CDCl3) d 11.9, 25.7, 26.4, 28.7, 29.3, 29.3, 29.4, 29.5,
29.8, 32.7, 61.1, 61.1, 63.0, 138.6, 143.1, 144.3, 144.3, 184.1, and
184.7.
was applied to a silica gel column (10 Â 3 cm); elution with 19:1
dichloromethane/ethyl ether afforded 3,6-dimethyl-2-methoxy-5-
hydroxydecyl-p-benzoquinone (7) as an orange solid: yield
25 mg (15%); mp: 58–59 °C; silica gel TLC Rf 0.32 (3:1 hexanes/
ethyl acetate); 1H NMR (400 MHz, CDCl3) d 1.28 (m, 14H), 1.59
(quint, 2H, J = 7.0 Hz), 1.93 (s, 3H), 2.01 (s, 3H), 2.44 (t, 2H,
J = 7.0 Hz), 3.62 (t, 2H, J = 6.5 Hz), and 3.95 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 8.8, 12.2, 26.16, 26.22, 28.7, 29.4, 29.4, 29.5,
29.8, 29.8, 60.9, 63.0, 72.8, 128.6, 140.3, 143.1, 155.4, 183.4, and
188.7; mass spectrum (APCI), m/z 322.2217 (M+H)+ (C19H31O4 re-
quires 322.2222).
4.1.32. 2,3,6-Trimethyl-5-benzyloxydecyl-p-benzoquinone (39)
To a stirred solution containing 600 mg (3.11 mmol) of 11-ben-
zyloxyundecanoic acid (36) and 450 mg (3.00 mmol) of trimethyl-
p-benzoquinone (26) in 50 mL of acetonitrile at 23 °C was added
509 mg (3.00 mmol) of silver nitrate. The reaction mixture was
heated to 75 °C and then 981 mg (3.63 mmol) of potassium persul-
fate in 50 mL of water was added very slowly. The reaction mixture
was stirred at 75 °C for 3 h, was then allowed to cool to 23 °C, and
was poured into 25 mL of water. The product was extracted with
50 mL of ethyl acetate. The organic layer was washed with 25 mL
of water, then with 25 mL of satd aq sodium bicarbonate, and
25 mL of brine, then dried (MgSO4) and concentrated under dimin-
ished pressure. The residue was purified by chromatography on a
silica gel column (13 Â 4 cm); elution with 3:1 hexanes/ethyl ace-
tate afforded 2,3,6-trimethyl-5-benzyloxydecyl-p-benzoquinone
(39) as an orange oil: yield 330 mg (37%); silica gel TLC Rf 0.75
(3:1 hexanes/ethyl acetate); 1H NMR (400 MHz, CDCl3) d 1.26 (m,
14H), 1.59 (quint, 2H, J = 7.2 Hz), 2.00 (m, 9H), 2.43 (t, 2H,
J = 8.0 Hz), 3.44 (t, 2H, J = 6.4 Hz), 4.50 (s, 2H), 7.25 (m, 1H), and
7.31 (m, 4H); 13C NMR (100 MHz, CDCl3) d 12.1, 12.3, 12.3, 26.2,
26.6, 28.8, 29.34, 29.4, 29.4, 29.5, 29.8, 29.9, 70.5, 72.8, 127.4,
127.6, 127.6, 128.3, 128.3, 138.7, 140.0, 140.3, 140.3, 144.5,
187.1, and 187.8; mass spectrum (EI), m/z 296.2676 (M)+
(C26H36O3 requires 396.2665).
4.1.35. 3,5-Dimethyl-2-methoxy-6-hydroxydecyl-p-
benzoquinone (8)
To a stirred solution at 23 °C containing 364 mg (1.79 mmol) of
11-hydroxyundecanoic acid (37) and 282 mg (1.69 mmol) of 3,5-
dimethyl-2-methoxy-p-benzoquinone (35) in 10 mL of acetonitrile
at 23 °C was added 288 mg (1.69 mmol) of silver nitrate. The reac-
tion mixture was heated to 75 °C, then 552 mg (2.04 mmol) of
potassium persulfate in 10 mL of water was added dropwise. The
reaction mixture was stirred at 75 °C for 4 h, then allowed to cool
to 23 °C, and then poured into 50 mL of water. The mixture was ex-
tracted with 75 mL of dichloromethane. The organic layer was
washed with 75 mL of water, then with 75 mL satd aq sodium
bicarbonate, and 75 mL of brine, and was then dried (MgSO4) and
concentrated under diminished pressure. The residue was applied
to a silica gel column (12 Â 3 cm); elution with 19:1 dichlorometh-
ane/ether afforded 3,5-dimethyl-2-methoxy-6-hydroxydecyl-p-
benzoquinone (8) as an orange wax: yield 56 mg (10%); silica gel
TLC Rf 0.33 (3:1 hexanes/ethyl acetate); 1H NMR (400 MHz, CDCl3)
d 1.28 (m, 14H), 1.59 (quint, 2H, J = 7.2 Hz), 1.93 (s, 3H), 2.01 (s,
3H), 2.43 (t, 2H, J = 8.0 Hz), 3.62 (t, 2H, J = 6.8 Hz), and 3.95 (s,
3H); 13C NMR (100 MHz, CDCl3) d 8.8, 12.2, 26.2, 26.2, 28.7, 29.4,
29.4, 29.5, 29.8, 29.8, 60.9, 63.0, 72.8, 128.6, 140.3, 143.1, 155.4,
183.4, and 188.7; mass spectrum (APCI), m/z 323.2229 (M+H)+
(C19H31O4 requires 323.2222).
4.1.33. 2,3,6-Trimethyl-5-hydroxydecyl-p-benzoquinone (6)
To a stirred solution containing 330 mg (0.832 mmol) of 2,3,6-
trimethyl-5-benzyloxydecyl-p-benzoquinone (39) in 6 mL of anhy-
drous methanol at 23 °C was added 20 mg of 10% Pd/C. The
reaction mixture was stirred at 23 °C under a hydrogen atmo-
sphere for 24 h, then air was bubbled through the reaction mixture
at 23 °C for 24 h. The suspension was filtered through Celite and
the filtrate was concentrated under diminished pressure. The resi-
due was applied to a silica gel column (13 Â 4 cm); elution with
3:1 hexanes/ethyl acetate afforded 2,3,6-trimethyl-5-hydroxyde-
cyl-p-benzoquinone (6) as a yellow solid: yield 130 mg (51%);
mp: 61–62 °C; silica gel TLC Rf 0.4 (3:1 hexanes/ethyl acetate);
1H NMR (400 MHz, CDCl3) d 1.26 (m, 14H), 1.53 (quint, 2H,
J = 6.4 Hz), 2.00 (m, 9H), 2.43 (t, 2H, J = 8.0 Hz) and 3.61 (t, 2H,
J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) d 12.1, 12.3, 12.3, 25.7,
26.6, 28.8, 29.31, 29.34, 29.4, 29.5, 29.8, 32.7, 63.0, 140.0, 140.3,
140.4, 144.5, 187.2, and 187.9; mass spectrum (APCI), m/z
307.2270 (M+H)+ (C19H31O3 requires 307.2273).
4.2. Biological experiments
4.2.1. Measurement of oxygen consumption
C2C12 cells were grown in DMEM supplemented with 10% fetal
bovine serum and 2 mM glutamine. Mitochondrial O2 consumption
was performed essentially as described previously19 with minor
modifications. Briefly, C2C12 cells were trypsinized and resus-
pended in phenol red-free growth medium, treated with 25 nM
rotenone and plated at 200,000 cells/well in a 96-well BD Oxygen
Biosensor plate. Ten micromolar compounds 1–8 were added and
fluorescence was monitored for 2 h in a PerSeptive Biosystems
Cytofluor Series 4000 plate reader (ex 485 nm; em 620 nm). Oxy-
gen consumption was quantified by calculating M/T, that is, the
slope at 50% maximum divided by the time to reach 50%
maximum.
4.1.34. 3,6-Dimethyl-2-methoxy-5-hydroxydecyl-p-benzo- quinone
(7)
4.2.2. Measurement of cytoprotective effects of idebenone
analogues
To a stirred solution containing 110 mg (0.54 mmol) of 11-
hydroxyundecanoic acid (37) and 85.0 mg (0.51 mmol) of 2,5-di-
methyl-3-methoxy-p-benzoquinone (30) in 2.5 mL of acetonitrile
at 23 °C was added 87.0 mg (0.51 mmol) of silver nitrate. The reac-
tion mixture was heated to 75 °C, then 167 mg (0.61 mmol) of
potassium persulfate in 2.5 mL of water was added very slowly.
The reaction mixture was stirred at 75 °C for 4 h, and was then al-
lowed to cool to 23 °C, and poured into 50 mL of water. The prod-
uct was extracted with 75 mL of dichloromethane. The organic
layer was washed with 75 mL of water, then with 75 mL of satd
aq sodium bicarbonate, and 75 mL of brine, and was then dried
(MgSO4) and concentrated under diminished pressure. The residue
CEM cells were grown in RPMI medium 1640 (Gibco, Grand Is-
land, NY) supplemented with 10% fetal bovine serum (Hyclone,
South Logan, Utah) and 1% penicillin-streptomycin solution (Cell-
gro, Manassas, VA). In a 12-well cell culture plate 5 Â 105 cells
per well were seeded and treated with the idebenone analogues
for 18 h. Cells were then treated with 5 mM DEM (diethyl maleate)
at 37 °C for 4 h in a humidified atmosphere of 5% CO2 in air. The
viability of the cells was measured by staining with 0.4% trypan
blue. Viable cells exclude the dye and non-viable cells take up
the dye. Cell viability is calculated as percentage of positive control

D. Y. Duveau et al. / Bioorg. Med. Chem. 18 (2010) 6429–6441
6441
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Acknowledgments
This work was supported at Arizona State University by a grant
from the Friedreich’s Ataxia Research Alliance. R.A.S. was sup-
ported by NIH Grant R01 AG16719.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.104. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
16. Liotta, D.; Arbiser, J.; Short, J. W.; Saindane, M. J. Org. Chem. 1983, 48, 2932.
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