In situ spectroeletrochemistry and cytotoxic activities of natural ubiquinone analogues

Article abstract of DOI:10.1016/j.tet.2011.06.026

Quinones are a group of potent antineoplastic agents. Here we described effective and facile routes to synthesize a series of ubiquinone analogues (UQAs). These unique compounds have been investigated by electrochemistry and in situ UV-vis spectroelectrochemistry to explore their electron-transfer processes and radical properties in aprotic media. The structure-activities relationships of inhibiting cancer cell proliferation of UQAs were examined in murine melanoma B16F10 cells using a 72 h continuous exposure MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Our results revealed that UQAs had improved antiproliferative activity and displayed better inhibitory effects than natural ubiquinone 10. The cytotoxic activities of UQAs were correlated to the semiubiquinone radicals, which were confirmed by in situ electron spin resonance (ESR). In the cytotoxicity test, 6-vinylubiquinone 5 and 6-(4′-fluorophenyl) ubiquinone 7 that possess half maximal inhibitory concentration value (IC₅₀) of 6.1 μM and 6.2 μM. This would make them as valuable candidates for future pharmacological studies.

Full text of DOI:10.1016/j.tet.2011.06.026

Tetrahedron 67 (2011) 5990e6000
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
In situ spectroeletrochemistry and cytotoxic activities of natural ubiquinone
analogues
,
b,
*
Wei Ma a, Hao Zhou a, Yi-Lun Ying a, Da-Wei Li a, Guo-Rong Chen a, Yi-Tao Long a , Hong-Yuan Chen
*
a Shanghai Key Laboratory of Functional Materials Chemistry and Department of Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237,
PR China
b Department of Chemistry, Nanjing University, 22 Hankou Road, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 April 2011
Received in revised form 2 June 2011
Accepted 10 June 2011
Available online 16 June 2011
Quinones are a group of potent antineoplastic agents. Here we described effective and facile routes to
synthesize a series of ubiquinone analogues (UQAs). These unique compounds have been investigated by
electrochemistry and in situ UVevis spectroelectrochemistry to explore their electron-transfer processes
and radical properties in aprotic media. The structureeactivities relationships of inhibiting cancer cell
proliferation of UQAs were examined in murine melanoma B16F10 cells using a 72 h continuous ex-
posure MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Our results revealed
that UQAs had improved antiproliferative activity and displayed better inhibitory effects than natural
ubiquinone 10. The cytotoxic activities of UQAs were correlated to the semiubiquinone radicals, which
were confirmed by in situ electron spin resonance (ESR). In the cytotoxicity test, 6-vinylubiquinone 5 and
6-(40-fluorophenyl) ubiquinone 7 that possess half maximal inhibitory concentration value (IC50) of
Keywords:
Ubiquinone/coenzyme Q
Electrochemistry
ESR
In situ spectroelectrochemistry
Cytotoxicity
6.1
m
M and 6.2
m
M. This would make them as valuable candidates for future pharmacological studies.
Ó 2011 Published by Elsevier Ltd.
1. Introduction
ubiquinol exist inside hydrophobic cell membranes. It is advanta-
geous to study their electrochemical properties in trace water en-
The ubiquinones, also known as coenzyme Qn, constitute es-
sential cellular components of all phylogenetic branches of life. The
various kinds of ubiquinones are distinguished by their number of
isoprenoid units, which are composed of the redox active ubiq-
uinonyl ring with a tail of isoprenoid units (n¼1e12) in different
homologue forms occurring in nature.1e3 Ubiquinone 10 is the
predominant form found in humans and most mammals, which is
located predominantly at the hydrophobic core of the phospholipid
bilayer of the inner membrane of the mitochondria. As an essential
cofactor in the electron transport chain, it shuttles the electrons
between complex I (or complex II) and complex III.4,5 In the pho-
tosynthetic reaction centers, ubiquinone serves as a mobile elec-
tron and proton carrier transferring electrons and protons from the
reaction centers to other components of the bioenergetic cycle.6,7
In addition to the enzymological importance, the pharmaceu-
tical activities and the nutritional importance of ubiquinone 10
have been revealed to indicate that ubiquinone serves versatile
functions in living systems.2,8 Most of such biological functions are
associated with the redox activity of ubiquinone. The ubiquinones
and their intermediates semiubiquinones, as well as reduced forms
vironment, such as aprotic organic solvents, which mimic the
nonpolar environment in a living cell where the electrochemical
properties differ from that observed in aqueous systems.9 In buffer
solution, the redox chemistry of ubiquinone shows a well-
documented two electron, two proton transfer process, giving the
ubiquinol as the final product.9,10 However, in dry aprotic media,
the ubiquinone is reduced to form the semiubiquinone radical
anion (Qꢀꢁ) and in a second step accepts another electron to pro-
duce the ubiquinone dianion (Q2ꢁ).9,10 The electrochemical re-
duction mechanism of ubiquinone in different solvents is shown in
Scheme 1a and b. In aprotic solvents, the monoanion and dianions
could undergo strong hydrogen-bonding with trace water.11 The
details for electrochemical reduction mechanism of ubiquinone in
unbuffered solution and aprotic solvents with trace water can be
seen in Supplementary data.
Although the synthesis and their biological applications of co-
enzyme Q10 in recent years are diverse and well established, there
are very few examples of systemic ubiquinone analogues (UQAs)
studies for electron transfer and biological application.12ꢁ16 For
example, Yu and co-workers demonstrated that when ubiquinone
acts as an electron acceptor for succinate-ubiquinone reductase, an
alkyl side chain of six or more carbons gives maximum activity;
whereas an alkyl side chain of 10 or more carbons gives maximum
activity when ubiquinone is an electron donor for ubiquinol/
* Corresponding authors. Tel./fax: þ86 21 642 50032; e-mail addresses: ytlong@
ecust.edu.cn (Y.-T. Long), hychen@netra.nju.edu.cn (H.-Y. Chen).
0040-4020/$ e see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.tet.2011.06.026

W. Ma et al. / Tetrahedron 67 (2011) 5990e6000
5991
2. Results and discussion
2.1. Electrochemical characteristics
The electrochemical measurements of the UQAs were carried
out by using cyclic voltammetry (CV) and differential pulse vol-
tammetry (DPV) techniques. Fig. 1 illustrates the cyclic voltam-
metric responses at glassy carbon (GC) electrode in 75 mM
tetrabutylammonium perchlorate (TBAP) CH3CN solution contain-
ing 0.5 mM of UQAs at various scan rates. The scanning range of
potential is between ꢁ2.2 and 0.2 V versus Ag/AgCl. The initial scan
started from w0.2 V versus Ag/AgCl. The values of formal potential
for all UQAs during the redox process, for the first step producing
the semiubiquinone radical (Qꢀꢁ/Ec1) and then further reducing to
the dianion (Q2ꢁ/Ec2) at more negative potentials (corrected for iR
drop in the cell) at GC electrode versus Ag/AgCl are given in Table 1.
As shown in Fig. 1, the redox peak potentials for different UQAs
are substantially different under the same conditions. As expected,
several trends are clear from the CVs in Fig. 1. First, all UQAs reveal
two couples of redox peaks, which correspond to two successive
single electron-transfer processes to give mono- and dianionic
species8,9 in aprotic CH3CN solutions, as shown in Scheme 1b. Ca-
thodic to anodic peak-to-peak separations were typically
65e70 mV for the first process and about 80e100 mV for the sec-
ond process at a scan rate of 100 mV sꢁ1. With increasing the scan
rate, peak-to-peak separations for the two redox processes di-
verged slightly, which gives a clear indication of reversible for first
redox process and quasi-reversible for the second process in this
system.23 The second trend in the CVs is the ratio of cathodic peak
current to the anodic peak current for the first redox process is
close to unity for all UQAs, throughout the scan rate range from 20
to 200 mV sꢁ1, which further confirms that it is a reversible process.
However, the ratio of peak currents for the second redox process
was found to be 0.85e0.95 at scan rate of 100 mV sꢁ1, indicating
quasi-reversibility. Third, it can be seen that the height of the sec-
ond peak is less than that of the first peak in the CVs of all UQAs,
suggesting chemical reactions may occur in the second reduction
steps of UQAs. Among several mechanisms considered in attemp-
ted simulations, the assumption involving adsorbed species been
discarded, reduction of a dimer formed by the reaction between
two semiubiquinones, and the complexation reaction between
ubiquinones and their dianions has been proposed.9,24 Similar
phenomenon also exists in other quinones.9,23,24 However, it is
important to note that even though the suggested complexation or
chemical reaction affected the peak currents significantly, it turned
out to have no effect on the reduction potentials.24 The increase of
scan rate promoted an increase of current peak in both oxidation
and reduction reactions. The anodic peak and cathodic peak shift to
more positive potential and more negative potential, respectively,
as the scan rate increased from 20 to 200 mV sꢁ1 in Fig. 1. The
detailed description of the relationship between peak currents and
scan rates is shown in a plot (see inset in Fig. 1). Both anodic and
cathodic peak currents vary approximately linearly with the square
root of the scan rate over the range of 20e200 mV sꢁ1, which
suggests that the process is predominantly diffusion controlled.25
However, all zero intercept and the negligible change in shape of
voltammograms at higher scan rates are indications for no complex
mass transport conditions involving trapped solution and linear
diffusion.
Scheme 1. Chemical structures of synthesized UQAs and their electrochemical re-
duction mechanism in different solvents.
cytochrome c reductase.12 On the other hand, the electron-transfer
activity of ubiquinone, to serve as an electron/proton carrier, differs
significantly by the effects of side chain variations and the ar-
rangement of the substituents on the ubiquinonyl ring.16 It is im-
portant to note that a modification in the direct substitution pattern
on the ubiquinonyl ring impacts its capability to accept electrons
and, thus, its capacity to mediate biological reactions.
A number of studies have examined the protective effects of
UQAs as antioxidants,2,8 which hints at the prospect of employing
UQAs as drugs. But little work has been done on the systematic
design of UQAs as anticancer drugs targeted toward inhibiting the
tumor cell growth. Here we present a preliminary study that the
relationship between structure and anticancer activities of UQAs.
We developed facile methods to synthesize a series of UQAs that
contain the following substituents in the 6-position: hydrogen,
halide, alkyl, vinyl, and aryl, as shown in Scheme 1c. The alkyl
substituted compounds 6-methylubiquinone
3
and 6-
ethylubiquinone 4 were synthesized starting from commercially
available ubiquinone 0. The alkyl residue was introduced by a rad-
ical Hundsdiecker decarboxylation of acetic acid/propanoic acid
using silver nitrate and peroxydisulfate in the degassed acetoni-
trile/water mixture.17 The alkyl substituted ubiquinones were
obtained in only one step, although the yield was low (20%). The
synthesis of 6-vinylubiquinone 5 was carried out by a Wittig cou-
pling-reaction.18 In addition, the three aromatic substituted ubi-
quinones 6, 7, 8 were obtained by palladium-mediated Suzuki
coupling-reactions.19,20 The target products were obtained by two
steps using 6-bromoubiquinone 221 and the corresponding boronic
acids with high yields. The 6-(10-bromodecyl) ubiquinone 9 was
synthesized using 11-bromoundecanoic acid and ubiquinone 0.22
The electrochemical study of UQAs was performed in aprotic
organic media as well as the full characterization of the electro-
chemically generated semiubiquinone radical anionic species by
in situ UVevis and electron spin resonance (ESR) spectroelec-
trochemistry. Furthermore, we also studied the bioactivity of UQAs
against tumor cells to develop key structureepropertyeactivity
relationships on the basis of their intrinsic chemical interests. 10
UQAs (1e10) were studied against murine melanoma B16F10 cells
by the standard microplate colorimetric MTT assay. These com-
pounds were designed to obtain structureebioactivity relation-
ships arising from the effect of substituent group at 6-position on
cytotoxicity. These cytotoxic data provide a valuable insight to se-
lect potential candidates for further development of UQAs as an-
ticancer drugs in vitro.
Further redox information was obtained by carrying out DPV
under the same conditions, which is a more sensitive electro-
chemical technique. As shown in Fig. 2, UQAs exhibit the expected
two reduction peaks, also attributing to two-step single-electron
redox process. The different ubiquinones show variation of the
peak potential values in the same experimental condition, which
could be explained by the electronic effects of the substituents. It is

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W. Ma et al. / Tetrahedron 67 (2011) 5990e6000
Fig. 1. Cyclic voltammetric curves of 0.5 mM UQAs obtained at GC electrode (w3 mm in diameter) in distilled CH3CN containing 75 mM TBAP at different scan rates of 20, 40, 60, 80,
100, 150, and 200 mV sꢁ1. The inset shows the linear increasing of the redox peak current depends on increasing the square root of the scan rate.
expected that the substitutional change on the ubiquinonyl ring,
either by an electron-withdrawing group or an electron-donating
group, has a dramatic impact on the redox properties of UQAs, as
the corresponding redox potentials shifted positively or negatively
by hundreds of millivolts. The substitutional differences on UQAs
have obvious effect on electron density at the ubiquinonyl ring and
thus influence their electron affinity. Compound 2, having the
electron-withdrawing group (bromo) on the ubiquinonyl ring, and
3 with electron-donating group (methyl) were investigated in
Fig. 2. The reduction of 3 was more difficult than that of 1 as evi-
denced by the fact that the reduction potential of 3 appeared at
ꢁ0.771 and ꢁ1.480 V, respectively, which are more negative than
those for 1, i.e., ꢁ0.650 and ꢁ1.327 V, respectively. The reduction
potential of 2 was ꢁ0.524 and ꢁ1.266 V, which illustrates that the
reduction of 2 was more favorable than that of 1. The potential
variation of 3 and 4 is hardly distinguishable for second peak, but 3
had a slightly more positive reduction potential than 4 for the first
electron-transfer step. It shows that electron-donating effects of

W. Ma et al. / Tetrahedron 67 (2011) 5990e6000
5993
M)
Table 1
Cathodic and anodic peak potentials (V), UVevis absorbance (nm) and cytotoxicity (
m
M) of ubiquinone analogues
UVevis absorbance/nm
Ubiquinone analogues
Formal potential/V
Cytotoxicity (IC50, m
Qc
Qꢀꢁd
Q2ꢁe
a
a
b
b
Ea1
Ea2
Ec1
Ec2
Ubiquinone 0 (1)
6-Bromoubiquinone (2)
6-Methylubiquinone (3)
6-Ethyl ubiquinone (4)
6-Vinylubiquinone (5)
6-Phenylubiquinone (6)
6-(40-Fluorophenyl) ubiquinone (7)
6-(40-Methoxyphenyl) ubiquinone (8)
6-(10-Bromodecyl)-ubiquinone (9)
Ubiquinone 10 (10)
ꢁ0.580
ꢁ0.476
ꢁ0.708
ꢁ0.723
ꢁ0.640
ꢁ0.648
ꢁ0.628
ꢁ0.660
ꢁ0.639
ꢁ0.699
ꢁ1.230
ꢁ1.172
ꢁ1.384
ꢁ1.388
ꢁ1.288
ꢁ1.316
ꢁ1.280
ꢁ1.328
ꢁ1.351
ꢁ1.380
ꢁ0.650
ꢁ0.524
ꢁ0.771
ꢁ0.786
ꢁ0.713
ꢁ0.717
ꢁ0.697
ꢁ0.726
ꢁ0.716
ꢁ0.765
ꢁ1.327
ꢁ1.266
ꢁ1.480
ꢁ1.412
ꢁ1.372
ꢁ1.406
ꢁ1.376
ꢁ1.426
ꢁ1.448
ꢁ1.452
263, 400
288, 425
273, 409
275, 425
302, 415
320, 412, 439
319, 416, 442
320, 417, 442
321, 417, 443
303, 357, 454
320, 420, 444
318, 423, 443
320, 421
320
324
320
321
366
325
327
328
322
317
24.8ꢂ3.1
15.9ꢂ2.3
20.4ꢂ2.4
21.9ꢂ2.7
6.1ꢂ1.1
12.8ꢂ1.1
6.2ꢂ1.0
10.3ꢂ1.1
48.3ꢂ3.3
>100
265, 312, 401
263, 314, 406
267, 351, 450
275, 417
321, 417, 422
321, 413, 440
273, 419
a
Anodic peak potential versus Ag/AgCl at scan rate of 100 mV sꢁ1
.
b
c
Cathodic peak potential versus Ag/AgCl at scan rate of 100 mV sꢁ1
UVevis absorbance peak value for ubiquinone material.
UVevis absorbance peak value of semiubiquinone.
.
d
e
UVevis absorbance peak value of ubiquinonyl dianion.
methyl and ethyl on ubiquinonyl ring was almost identical. Based
on the previous work, we knew that conjugated system can pro-
mote the transfer ability of electron.26 To validate this possibility,
we investigated the voltammetric behaviors of 5, the ubiquinone
with the rigid alkyl side chains. The formal reduction potentials of 5
were ꢁ0.713 and ꢁ1.372 V, respectively, while corresponding po-
tentials for 4 were ꢁ0.786 and ꢁ1.412 V. Consequently, it demon-
strates that the electron-transfer activity of ubiquinone with a rigid
alkyl side chain would be more favorable than ubiquinone with
flexible alkyl side chains. The reduction potentials of 6-aryl ubi-
quinones were investigated in order to characterize the change in
redox behavior associated with substitutional change on the aro-
matic ring (not directly on ubiquinonyl ring). Furthermore, the
expected electrochemical behaviors were observed with 6, 7, and 8
in Fig. 2.
Generally, the inductive effect is great when the substituent is
located closely and vice versa. The slight difference of reduction
potentials (ꢁ0.717 and ꢁ1.406 V for 6, ꢁ0.697 and ꢁ1.376 V for 7,
ꢁ0.726 and ꢁ1.426 V for 8) arising from changes of substituent
effects on aromatic ring, are qualitatively smaller than the effects of
groups attached directly to ubiquinonyl ring. The variations of the
potential could be correlated to the electron withdrawing or do-
nating character of substitutional change on the ubiquinonyl ring.
Therefore, the ubiquinones with electron-withdrawing groups ex-
hibit substantially positive potential shifts, while ubiquinones with
electron-donating group possess more negative potential values.27
As clearly shown in Fig. 2, the height of second reduction peak with
different degree is less than that of the first peak for the ten UQAs.
This also suggests a chemical reaction, which could be correlated
with electronic effects of the substituent, may occur in the second
electrochemical step of UQAs.
electrochemical cell. As the reduction of UQAs with constant po-
tential, a substantial change in the UVevis spectra was observed.
Fig. 3 shows the characteristic spectra of UQAs before and after
electrochemical reduction.
Before reduction, UQAs exhibit the characteristic bands as shown
in black line of Fig. 3. The UVevis spectra of three aromatic
substituted ubiquinones 6, 7, and 8 displayed long wavelength ab-
sorption bands with comparable intensity at 312, 314, and 350 nm,
respectively. The absorption bands can be assigned to pe
p* transi-
tions of the aryl-substituent.30 The stronger absorptions at shorter
wavelengths and the relative weak absorptions at the longer
wavelength are an indication of the
pe
p* and nep* electronic
transitions of the ubiquinonyl rings30 at 265 nm and 415 nm, 263 nm
and 410 nm, 267 nm and 430 nm, respectively. As can be seen from
the Fig. 3, 5 displayed a strong absorption band at 302 nm. It was the
overlapped result of the
pe
p* transition of the conjugated vinyl
group and the
pe
p* transition of the ubiquinonyl ring. Absorption
spectral studies show that the effect of overlapped peak led to a red
shift together with a wider absorption band than others. The relative
weak absorption at 415 nm is assigned to the nep
* electronic tran-
sition of the ubiquinonyl ring.30 However, the UQAs with non-
aromatic substitutions 1, 2, 3, 4, 9, and 10 do not display the ab-
sorption bands at 310e350 nm, which are assigned to the
pe
p*
transitions of the aromatic ring in black line of Fig. 3. The UVevis
spectra of these UQAs illustrate strong diagnostic absorption bands
at 260e300 nm, which correspond to the
pe
p* transition in the
ubiquinonyl systems. The spectra also contain relative weak and
broad absorption bands at 400e425 nm which are an indication of
the nep
* electronic transitions of the ubiquinonyl rings.
With the application of a first reduction potential of Ec1 in Table
1, one-electron participated in the reduction of UQAs. As shown in
red line of Fig. 3, after the first reduction step, the characteristic
absorption bands at 260e290 nm disappear and were replaced by
a sharp and intense absorbance at around 320 nm, which are at-
In this study, the oxidation potentials for reduced product of
UQAs also followed the harmony with the electronic nature of the
substituent and remained the same as observed for the two-step
single-electron electrochemical process.28,29
tributed to the
pe
p* absorption spectra of ubiquinonyl ring con-
verting to aromatic ring.27 In particular, the strong absorption band
2.2. In situ UVevis spectroelectrochemistry
at 302 nm of 5 is still kept except appearance of new peak at
355 nm, which correspond to the
pep
* transition of the conjugated
The information of the semiubiquinone anions of UQAs in
aprotic solvent has not been fully characterized by electrochemical
performance. In order to further investigate the deeper changes in
radical intermediates (semiubiquinone) and final reduced species
(dianion) as indicated by their electrochemical properties, we ex-
plored the corresponding effects of UQAs on their electronic tran-
sitions by in situ UVevis spectroelectrochemical experiments. The
UVevis spectra were recorded during the reduction of UQAs in
CH3CN containing TBAP in an optically transparent thin layer
vinyl group and the
pe
p* absorption spectra of ubiquinonyl ring
converting to aromatic ring. In red line of Fig. 3, the new wide
absorption bands appear at 410e450 nm are assigned to the nep*
absorption spectra of semiquinone radicals. This indicates that
ubiquinones were converted to the semiubiquinone radicals.27
With the application of a more negative potential at Ec2 in the
Table 1, semiubiquinone anions were reduced and accepted another
electron (two electron overall). The band of the semiubiquinone
radicals disappears and the corresponding dianionic characteristic

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W. Ma et al. / Tetrahedron 67 (2011) 5990e6000
On the basis of the electrochemical and spectroscopic results in
Figs. 1 and 3, a mechanism can be proposed for the generations of
the reduced species in aprotic CH3CN solution. The electron-
transfer steps could occur consecutively with the constant poten-
tial reduction. After applying a first reduction potential of Ec1, the
initial one-electron reduction of Q (oxidation state of UQAs) pro-
duced Qꢀꢁ (semiubiquinones), which could be further reduced by
accepting another electron to form Q2ꢁ (dianion) immediately ap-
plied a second reduction potential of Ec2. The spectroscopic ex-
periments indicate that the state of UQAs is influenced by the
different reduction potentials, and thus the UVevis spectra display
different extremes. It is confirmed that the substitutional change on
the ubiquinonyl ring, such as aromatic ring or vinyl bond, has
a dramatic impact on characteristic bands, as the corresponding
absorption peak value or shape. The assignment of all absorption
bands from UVevis spectra of UQAs with their reduced in-
termediate and final species at various potentials are summarized
in Table 1.
2.3. Cytotoxicity test
The redox properties of ubiquinone play critical roles in their
therapeutic and toxicological properties. In its reduced form
(ubiquinol), ubiquinone may function as a class of potentially in-
teresting drug of antioxidant and free radical scavengers.2,16 How-
ever, recent demonstrations show that ubiquinones may provide
another possibility of indirect antioxidant function.31 Many qui-
nones possess cytotoxic properties that made them useful anti-
cancer and antibacterial drugs. Adriamycin is a good case in point as
an effective antitumor therapeutical agent.32 Consequently, there is
a high prospect of development of ubiquinone based cytotoxic
agents. With this anticipation our designed UQAs were subjected to
cytotoxicity testing to determine if a structureeactivity relation-
ship could be identified.
In vitro experiments to determine cytotoxicity were performed
on a series of synthesized UQAs using a 72 h continuous exposure
MTT assay33 against B16F10 murine melanoma cell line. Results
were expressed as a percentage of viability of cells grown in the
absence of drug, but also with 3% DMSO medium. Cytotoxicity
(IC50) was defined as the concentrations of UQAs (1e10) corre-
sponding to 50% tumor cell viability of the untreated control in the
absence of an inhibitor and was calculated from three independent
experiments. All IC50 values are mM values, for B16F10 cell were
shown in Table 1. In this study, the compound was thought to be
ineffective against the growth of murine melanoma cells when IC50
value >100 mM. As shown in cytotoxicity assays (Fig. 4), the cyto-
toxic effect of the UQAs is directly related to their structural char-
acteristics of substituent including chain length, functional groups,
aryl ring, and degree of saturation. The result of cytotoxicity test
showed that UQAs decreased the viability and proliferation of tu-
mor cells in a dose-dependent manner, but at different sensitivities.
All UQAs were active at micromole concentration levels to induce
the death of tumor B16F10 cells, but the cytotoxicity was com-
pletely lost when ubiquinone 10 10 was used. Fig. 4 clearly dem-
onstrates that synthesized UQAs can effectively inhibit tumor
growth under experimental conditions with an IC50 value.
In the cytotoxicity test, 5 and 7 are most active, and possess IC50
Fig. 2. Differential pulse voltammetry (DPV) curves of 0.5 mM UQAs obtained at GC
electrode in CH3CN containing 75 mM TBAP during reduction process, the potential is
from 0.2 to ꢁ2.5 V with a pulse amplitude of 50 mV and a sample pulse width of
100 ms. Arrow indicates initial scan direction.
value of 6.1 and 6.2
with and without the substituents which belong to
m
M, respectively. Comparison of the compounds
electron
p
conjugated system with ubiquinonyl ring (entries 5e8, Table 1)
indicate that the presence of the conjugated system led to a marked
increase in cytotoxicity. For instance, a vinyl group presented at the
6-position of ubiquinonyl ring, activity increased in the inhibition
of B16F10 tumor cell growth, compared with ethyl groups in the
absorbance bands of UQAs located at about 320 nm were found,27 as
shown in green line of Fig. 3. The absorbance of 6-vinylubiquinone 5
was observed at 366 nm. Although similar in shape of absorption
bands of other UQAs, it differed significantly regarding its wave-
length, which is attributed to an interference of vinyl bond.
corresponding position (IC50 values of 4 and 5 was 21.9 and 6.1
mM,
respectively). The same trend can be observed for compounds 6

W. Ma et al. / Tetrahedron 67 (2011) 5990e6000
5995
Fig. 3. In situ UVevis spectroelectrochemistry obtained in an optically transparent thin layer electrochemical cell during the constant potential reduction of 0.2 mM UQAs in aprotic
CH3CN contained 30 mM TBAP (black line: the UVevis spectra of oxidation state of ubiquinone; red line: the UVevis spectra of semiubiquinone were obtained after applying a first
reduction potential of ER1; green line: the UVevis spectra of ubiquinonyl dianion were obtained after applying a second reduction potential of ER2).
(IC50¼12.8
m
M), 7 (IC50¼6.2
m
M) and 8 (IC50¼10.3
m
M), which show
ring, cytotoxicity against murine melanoma B16F10 cells appeared
less difference. This was also particularly evident in many proper-
ties of electrochemistry and in situ UVevis spectroelectrochemistry
higher activity on cytotoxicity. This result discloses that compounds
containing aromatic rings were more active than compounds
containing aliphatic chains against the growth of murine mela-
noma cells. In other words, the compounds with conjugated system
were more active than compounds with non-conjugated system.
Moreover, as the compounds 6, 7, and 8 have a slight difference in
the structure, which of them contain a subset similar to aromatic
shown in the Figs. 1 and 2. Compounds 1 (IC50¼24.8
m
M), 2
M), and 9
M) exhibit mild to moderate activity. The observation
(IC50¼15.9
(IC50¼48.3
m
M), 3 (IC50¼20.4
m
M), 4 (IC50¼21.9
m
m
indicates that the compounds with longer chain show less activity
to inhibit the growth of murine melanoma B16F10 cells than the

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W. Ma et al. / Tetrahedron 67 (2011) 5990e6000
for detecting paramagnetic species, ESR is very sensitive to the
structure of the substituent even in much closed conformation. The
nuclei in the substituents of UQAs split the ESR lines and affected
the behavior of their radicals, resulting in the characteristic ESR
spectra. The substitutional effects on the electronic structures of
the semiubiquinone radical anion species are obvious. Different ESR
signals for four UQAs may also be attributed to the substitutional
change leading to localization of the charge and then spin onto the
ubiquinone moiety. With applying the potential close to the first
reduction of UQAs, the intensity of semiubiquinone radical anion
species for 2, 5, 6, and 10 has been progressively increased, as
shown in Fig. 5.
Fig. 4. IC50 values of UQAs (1e10) toward B16F10 murine melanoma cell were de-
termined by MTT assay over an exposure of 72 h.
corresponding those of shorter side chain. In the Fig. 4, we clearly
observed that 2 showed higher anticancer activity than 9. A similar
relationship can be observed if compounds 3 and 4 were consid-
ered, although there was only a negligible variation in structures.
It will be important to have data concerning the toxicity of these
molecules against normal cells to have an idea about the safety
window as anticancer agent. We demonstrated a high toxicity of
UQAs against murine melanoma cells, while at the same concen-
trations they were low toxicity for normal cell (human umbilical
vein endothelial cells). The details as described in Supplementary
data. In this study, the results of cytotoxicity test revealed that
synthesized UQAs (1e9) were capable of inducing a decrease of
tumor B16F10 cell growth and have greater effects against tumor
cell growth than natural product 10. The major differences were
observed for UQAs with different substituents indicates that the
presence of the conjugated system with ubiquinonyl ring (for ex-
ample, aryl ring or vinyl bond) led to a striking enhancement in
cytotoxicity. Further studies using other model compounds are
required to better understand structureeactivity relationships, but
these are the first step to investigate such properties of ubiquinone.
The experimental result of cytotoxicity test highlights that a series
of ubiquinone compounds are worthy of the further investigation, it
would provide us the information to get better understanding of
relationship between the side chain effects of UQAs and the bi-
ological activities.
Fig. 5. Increase in the intensity of ESR spectra of four UQAs (2, 5, 6, and 10) with
electrolysis time.
To obtain further information about semiubiquinone radicals
and cytotoxicity, relative radical intensity of ESR signals with decay
time was used. When the intensity of semiubiquione radical
maintained nearly a constant value with increase of electrolysis
time, the applied potential was removed. In the Fig. 6, it was ob-
served that the ESR signal gradually decreased with decay time.
This indicates that semiubiquinone radicals were converted to
ubiquinones and their disproportionated species.37 The rate of
variation of ESR signal intensity as a function of decay time for four
UQAs was significantly different. In the Fig. 6, the rate of radical
decay of 10 was much faster than others, which showed that more
radical anions quenched per unit time. It is interesting to note that
the more cytotoxic active compound 5 and 6 has the longer decay
2.4. In situ ESR spectroelectrochemistry
Anticancer experimental therapeutics show that quinone un-
dergo enzymatic reduction via one or two electrons to give the
corresponding semiquinone radical or hydroquinone anion as part
of their mechanism of cell death induction.34e36 To investigate the
possible link between radical and cytotoxicity of ubiquinone com-
pounds, we chose four typical ubiquinone compounds, 5, which
was the most active, 2, 6, which exhibited mild activity and 10,
which was inactive against the growth of murine melanoma cell
line.
In situ ESR spectroelectrochemical experiments were carried out
for these ubiquinone compounds. The radical intermediate anions
of UQAs (semiubiquinones) characterized by ESR were prepared
in situ by electrochemical reduction in CH3CN. At the applied po-
tential corresponding to the first reduction peak obtained from the
cyclic voltammetric experiments, the corresponding ESR signals
were observed and recorded with the time. The investigated UQAs
formed stable paramagnetic intermediates after the first reduction
step with well-resolved ESR signals in CH3CN. The ESR spectra of
the radical anion of UQAs show the variation of the ESR signal in-
tensity with the electrolysis time in Fig. 5. As a versatile technique
Fig. 6. The relative intensity variation of semiubiquinone radicals of UQAs (2, 5, 6, and
10) with decay time.

W. Ma et al. / Tetrahedron 67 (2011) 5990e6000
5997
time for semiubiquinone radical decay, indicating that the radical
4.2. Electrochemistry
anion of 5 and 6 was more stable. The trend of radical decay of
UQAs 2 and 10 are also in good agreement with cytotoxic study. The
cytotoxic activities of present compounds against the growth of
murine melanoma cell line versus their intensity variation of
semiubiquinone radical anions showed a linear relationship with
decay time. Such a correlation suggests the cytotoxic activities
depend upon the stability of semiubiquinone radical. This area
deserves further research, but our reported results only hint the
relationship between cytotoxic activity and semiubiquinone radical
anion.
HPLC grade CH3CN was initially dried by distilling over CaH2
before use. All solutions for electrochemistry were dried by placing
the solvent, electrolyte, and UQAs inside a 25 mL vacuum syringe
(Yikang Medical Instrument Group Co. Ltd, Jiangxi, China) con-
ꢀ
taining 3 A molecular sieves (that were dried under vacuum at
240 ꢃC for 12 h) and storing the syringe under a dry nitrogen at-
mosphere for at least 48 h. During the measurement, a dry nitrogen
purge maintains an oxygen and moisture free environment. A
standard jacketed three-electrode cell was used for electrochem-
istry. A Pt electrode and a Ag/AgCl electrode were used as counter
electrode and reference electrode, respectively. The measurement
of approximate 0.5 mM UQAs was carried out at glassy carbon (GC)
electrode (w3 mm diameter) in CH3CN test solution. Immediately
before use, the working electrode was polished with alumina sus-
pension and rinsed with ultrapure water, then rinsed with acetone
and dried with N2. The electrolyte solution was 75 mM TBAP in
CH3CN test solution. Accurate potentials were obtained using fer-
rocenium/ferrocene as an internal standard. The temperature was
controlled by using 25 ꢃC circulating water bath through the outer
cell jacket. Cyclic voltammetries were recorded at from 20 to
200 mV sꢁ1. The measurements were performed at CHI 660 elec-
trochemical workstation (Shanghai Chenhua Co., Ltd., China).
3. Conclusions
In conclusion, the effective and facile synthetic routes for UQAs
have allowed the full investigation of electrochemical, UVevis, ESR
spectroelectrochemical properties and the biological activities of
these compounds. The degree of electron transfer in aprotic media
was estimated from CV and DPV. This study shows that UQAs un-
dergo two sequential single-electron reductions to give the mono-
and dianion at the surface of GC electrode in distilled aprotic media.
As seen from Figs.1 and 2, different substituents on the ubiquinonyl
ring of 6-position produce significant effect on the electron-transfer
activity of UQAs. It is also demonstrated that the UVevis spectra of
a series of UQAs accurately predict electron-transfer reactions as
well as the effect of structural changes on spectra.
Quinones are a kind of important additions to the repertoire of
compound as a strategy for the treatment of cancer and have po-
tentials as anticancer agents. Given the documented elevation in
many tumor types, especially those cancers with few treatment
options, such as melanoma carcinoma, the continued exploration of
the therapeutic utility is critical. Transcripts profiling from IC50
value of growth suppress on murine melanoma B16F10 cells in-
dicates that synthesized UQAs induce tumor cell death. In the cy-
4.3. In situ UVevis spectroelectrochemistry
The UVevis spectrum was recorded during reduction of UQAs in
an optically transparent thin layer electrochemical cell (optical path
length is 0.4ꢂ0.1 mm) via an Ocean Optics DT-minutesi-2 halogen
recourse and USB2000þ spectrometer. The cell featured a platinum
net working electrode and salt bridge to a platinum wire counter
electrode and Ag/AgCl reference electrode. Electrochemistry during
the UVevis spectral measurement was controlled by CHI 1232A
electrochemical workstation (Shanghai Chenhua Co., Ltd., China).
UQAs solutions were prepared by dissolving UQAs in aprotic CH3CN
solution containing TBAP. The prepared solutions were de-
oxygenated for 30 min prior to each experiment and the cell was
kept under a nitrogen atmosphere throughout the experiment.
totoxicity test, 6-vinylubiquinone
ubiquinone 7 are the most active, possessing IC50 value of 6.1 and
6.2 M, respectively and IC50 value of ubiquinone 10 is >100 M. To
5
and 6-(40-fluorophenyl)
m
m
our astonishing, synthesized UQAs have a more potent ability to
inhibit tumor cell growth than natural product ubiquinone 10.
Results of these studies may help to develop insights in this
emerging area of interest in order to further explore the relation-
ship between the side chain of UQAs and the biological activities. In
an effort to define their mechanism of action, we evaluated cyto-
toxic activities by in situ ESR. The ability of ubiquinone compounds
induced to death of cancer cell is relative with the decay time of
semiubiquinone radical. It is noteworthy that more cytotoxic active
compounds 5 and 6 have longer decay time of semiubiquinone
radical.
4.4. Cytotoxicity test using MTT assay
Cytotoxicity of UQAs on murine melanoma B16F10 cells and
human umbilical vein endothelial cells was determined using the
standard microplate colorimetric MTT assay. MTT is a yellow tetra-
zolium salt: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide. The MTT test is a colorimetric assay, that is, used to
quantify spectrophotometrically the amount of living cells, due to
the reduction of MTT to purple formazan by the mitochondria of the
living cell. Experiments of the cell viabilities of different samples
were performed on the cell cultures for 14e16 days. The murine
melanoma B16 cells in log phase were trypsinized (0.01%) and
seeded in 96-well plates at a density of 2.0ꢄ103 cells/well. The cells
were incubated at 37 ꢃC under 5% CO2 for 24 h. Murine melanoma
B16 cells were cultured for 72 h under regular growth conditions
with samples of six different concentrations, respectively. Briefly, the
cultures were incubated with medium for 3 h at 37 ꢃC after addition
of 20 mL MTT (5 mg/mL). The supernatant was removed and di-
methyl sulfoxide (DMSO) was added to each well to dissolve the
formazan crystals. Afterward, the solution from each well was
transferred to a 96-well plate. The absorbance values were read on
a Molecular Devices Spectra MAX 340 at 550 nm with background
subtraction at 690 nm. The IC50 value was calculated at 50% of the
cell growth using at least two separate replicates.
Notably, it is believed that our route of the structureebioactivity
relationship can be used to select potential candidates for further
development of UQAs as anticancer drugs.
4. Experimental section
4.1. Chemicals
HPLC grade acetonitrile (CH3CN) and other analytical grade re-
agents were purchased from SigmaeAldrich. Tetrabutylammonium
perchlorate (98%) (TBAP) were purchased from Aldrich; Coenzyme
Q0 were of the best available grade (>98%) from Sigma; N2
(99.998%, prepurified) was obtained from Cryogenic Gases (Detroit,
MI). All chemical reagents for synthesis were of analytical grade,
obtained from commercial suppliers, and used without further
purification unless otherwise noted. All electrodes for electro-
chemical experiments were purchased from Shanghai Chenhua Co.,
Ltd., China.

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W. Ma et al. / Tetrahedron 67 (2011) 5990e6000
4.5. In situ electron spin resonance (ESR)
7.05e7.15(m, 2H, ArH), 7.40e7.50 (m, 3H, ArH) ppm; MS (EI): 258.1,
spectroelectrochemistry
found: 258.09.
ESR measurements were performed in the X-band region with
a Bruker EMX-8/2.7 spectrometer at room temperature. The poten-
tial during the ESR measurements was controlled with CHI 1232A
electrochemical workstation. Silver wire was used as the working
electrode and the length of it matched the height of the ESR cavity.
An alloy wire electrode and a Ag/AgCl electrode served as the
counter electrode and the reference electrode, respectively. ESR scan
parameters used the following: microwave frequency 9.87 GHz,
modulated frequency 100 kHz, modulated amplitude 0.15 G, time
constant 81.92 s, conversion time 163.84 s. UQAs solutions were
used for in situ ESR spectroelectrochemical experiments. These so-
lutions were prepared by dissolving the desired ubiquinone com-
pounds in aprotic CH3CN containing TBAP. The prepared solutions
were deoxygenated for 30 min prior to each experiment and the cell
was kept under a nitrogen atmosphere throughout the experiment.
4.6.4. 6-(40-Fluorophenyl)ubiquinone (7). 1H NMR (500.0 MHz,
CDCl3, 298 K): 3.95 (s, 3H, eOCH3), 4.05 (s, 3H, eOCH3), 1.95 (s, 3H,
eCH3), 7.08e7.15 (m, 4H, ArH) ppm; 13C NMR (125.7 MHz, CDCl3,
298 K): 184.5 (C]O),184.1 (C]O),164.3 (C, AreF),145.3 (C, Ar),145.1
(C, Ar), 144.8 (C, Ar), 144.5 (C, Ar), 140.3 (C, Ar), 140.1 (C, Ar), 132.3 (C,
Ar), 127.5 (C, Ar),115.3 (C, Ar), 61.2 (2ꢄ eOCH3), 12.9(eCH3) ppm;
HRMS (ESI): calcd for C15H14O4F [MþH]þ 277.0870, found 277.0870.
4.6.5. 6-(40-Methoxyphenyl)ubiquinone (8). 1H NMR (500.0 MHz,
CDCl3, 298 K): 3.95 (s, 3H, eOCH3), 4.05 (s, 3H, eOCH3), 1.95 (s, 3H,
eCH3), 6.95(m, 2H, ArH), 7.05(m, 2H, ArH), 3.85 (s, 3H, eArOCH3)
ppm (Scheme 3).
4.6. Synthesis of ubiquinone analogues (UQAs)
THF was distilled freshly from benzophenone/sodium ketyl un-
der a nitrogen atmosphere. CH3CN was degassed in the ultrasonic
cleaner with degas function. 1H and 13C NMR spectra were recorded
on a Bruker FT-500 MHz spectrometer at room temperature. Mass
spectra (EI) were recorded on an MA1212 instrument using standard
conditions. All reactions were monitored by thin layer chromatog-
raphy (TLC) using silica-coated plates and visualizing under UV light.
Light petroleum of the distillation range 60e90 ꢃC was used. Evap-
oration of solvents was performed at reduced pressure, using a ro-
tary evaporator. Column chromatographic experiments were
performed with Silica Gel (300e400 mesh) (Scheme 2).
Scheme 3. Synthesis of 6-methylubiquinone 3 and 6-ethylubiquinone 4.
4.6.6. 6-Ethylubiquinone (4). To a solution of coenzyme Q0
1
(1.00 g, 5.4 mmol) in degassed CH3CN (120 mL) at 25 ꢃC were added
AgNO3 (0.25 g, 1.5 mmol) and propanoic acid (0.48 g, 6.5 mmol).
After the mixture was heated to 65 ꢃC, (NH4)2S2O8 (2.80 g,
12.5 mmol) in 10 mL degassed H2O was added dropwisely over 30
min17 (see Scheme 3). After 3 h, the reaction mixture was cooled to
25 ꢃC and was extracted with EtOAc (3ꢄ150 mL), and the organic
layers were washed with brine (150 mL), dried (MgSO4), and con-
centrated. The crude product was purified by silica chromatography
to give 4 (20%). 1H NMR (500.0 MHz, CDCl3, 298 K): 3.99 (s, 3H,
eOCH3), 4.00 (s, 3H, eOCH3), 2.02 (s, 3H, eCH3), 1.05 (t, 3H,
eCH2CH3), 2.45 (q, 2H, eCH2CH3) ppm.
Scheme 2. Synthesis of 6-bromoubiquinone 2 and 6-arylubiquinone 6, 7, 8.
4.6.7. 6-Methylubiquinone (3). The same method for synthesizing
4. 1H NMR (500.0 MHz, CDCl3, 298 K): 3.99 (s, 6H, 2ꢄ eOCH3), 2.02
(s, 6H, 2ꢄ eCH3) ppm; MS (EI): 196.1, found: 196.07 (Scheme 4).
4.6.1. 6-Bromoubiquinone (2). To a stirred solution of coenzyme Q0
1 (10.60 g, 58.0 mmol) in 120 mL of carbon tetrachloride was added
dropwisely bromine (10.52 g, 68.0 mmol) at room temperature21
(see Scheme 2). The reaction mixture was stirred for 4 h, and
then treated with water, dried with magnesium sulfate, and
evaporated. The reaction mixture was purified by column chro-
matography to give 2 (81%) as red needle crystals. 1H NMR
(500.0 MHz, CDCl3, 298 K): 3.95 (s, 3H, eOCH3), 4.05 (s, 3H, eOCH3),
2.22 (s, 3H, eCH3) ppm.
4.6.8. 2,3,4,5-Tetramethoxytoluene (5a). To a solution of coenzyme
Q0 1 (4.00 g, 21.7 mmol) in methanol (30 mL) at 0 ꢃC was dropwise
added a solution of KBH4 (5.85 g, 108.5 mmol) in methanol (30 mL).
After 10 min, the reaction was quenched by the addition of EtOAc
and then 5% aqueous HCl38,39 (see Scheme 4). The mixture was
extracted with EtOAc (3ꢄ50 mL) and the organic layer was washed
successively with water and brine, dried (MgSO4), and evaporated
at reduced pressure. The crude hydroquinone (4.20 g) was dis-
solved in EtOH (20 mL) and to this solution at room temperature
was added a solution of NaOH (2.20 g in 6 mL H2O) and dimethyl
sulfate (5.30 mL, 56.0 mmol) with cooling in a ice water bath in six
portions simultaneously. After 45 min, 5% aqueous HCl was added
and the mixture was extracted with EtOAc (3ꢄ50 mL). The organic
layer was washed successively with water and brine, dried
(MgSO4), and evaporated to give 5a (74%) as a light yellow liquid.40
1H NMR (500.0 MHz, CDCl3, 298 K): 3.99 (s, 12H, 4ꢄ eOCH3), 2.25
(s, 3H, eCH3), 6.45(s, 1H, ArH) ppm.
4.6.2. 6-Arylubiquinone (6e8). To a mixture of 2 (1.0 mmol), bo-
ronic acid (0.13 g, 1.0 mmol), Pd(PPh3)4 (3.0 mol%) were added
CHCl3 (8.5 mL) and aqueous K2CO3 (1.5 mL, 2 mmol) under argon
atmosphere19,20 (see Scheme 2). The reaction mixture was refluxed
overnight under 60 ꢃC. After cooling to room temperature, ice
cooled water (10 mL) was added and then the reaction mixture was
extracted with CH2Cl2 (3ꢄ10 mL). The organic layer was washed
with brine, dried (Na2SO4), filtered, and concentrated in vacuum.
The residue was purified by column chromatography to afford the
corresponding compound (6e8) as dark purple oily substances.
4.6.9. 2,3,4,5-Tetramethoxy-6-methylbenzaldehyde (5b). To a stirred
solution of 5a (4.24 g, 20 mmol) in CH2Cl2 (30 mL) was added
dichloromethyl methyl ether (6.89 g, 60 mmol) at 0 ꢃC followed by
addition of TiCl4 (11.38 g, 60 mmol)41 (see Scheme 4). The resulting
4.6.3. 6-Phenylubiquinone (6). 1H NMR (500.0 MHz, CDCl3, 298 K):
3.95 (s, 3H, eOCH3), 4.05 (s, 3H, eOCH3), 1.95 (s, 3H, eCH3),

W. Ma et al. / Tetrahedron 67 (2011) 5990e6000
5999
Scheme 4. Synthesis of 6-vinylubiquinone 5.
mixture was stirred for 4 h at ambient temperature and then
poured into ice water. After stirring vigorously for 10 min, the or-
ganic layer was separated. It was washed with water, dried, and
evaporated. The residue was chromatographed on silica gel to give
5b (90%) as a light yellow liquid. 1H NMR (500.0 MHz, CDCl3, 298 K):
3.99 (s,12H, 4ꢄ eOCH3), 2.25 (s, 3H, eCH3),10.45 (s, H, eCHO) ppm.
4.6.12. 6-(10-Bromodecyl)ubiquinone
(9). 11-Bromoundecanoic
acid (4.00 g,15.1 mmol) and SOCl2 (1.6 mL, 21.5 mmol) were heated
at 90 ꢃC for 15 min. Excess SOCl2 was removed by distillation under
reduced pressure and the residue was dissolved in diethyl ether
(20 mL) and cooled to 0 ꢃC. Hydrogen peroxide (30%, 1.8 mL) was
added, followed by dropwise addition of pyridine (1.4 mL) over
45 min, then diethyl ether (10 mL) was added.22 After 1 h at room
temperature, the product was diluted with diethyl ether (150 mL),
washed with H2O (2ꢄ70 mL), 1.2 M HCl (2ꢄ70 mL), H2O (70 mL),
0.5 M NaHCO3 (2ꢄ70 mL), and H2O (70 mL). After drying over
NaSO4, the solvent was removed under reduced pressure, giving
white solid 11-bromoundecanoic peroxide as crude, which was
used in the next step without delay. The crude product (3.51 g,
12.5 mmol), coenzyme Q0 (1.31 g, 7.19 mmol), and acetic acid
(60 mL) was synthesized by stirring for 20 h at 100 ꢃC22 (see
Scheme 5). After cooling to room temperature, the reaction mix-
ture was diluted with diethyl ether (300 mL), washed with H2O
(3ꢄ200 mL), 1 M HCl (3ꢄ250 mL), 0.5 M NaHCO3 (3ꢄ250 mL), and
H2O (3ꢄ200 mL), and dried over NaSO4. Removal of the solvent
under reduced pressure obtained a reddish solid. The residue was
purified by silica gel column chromatography to afford 9 as a red oil
(1.07g, 37%). 1H NMR (500 MHz, CDCl3, 298 K): 4.00 (s, 6H, 2ꢄ
eOCH3), 3.43 (t, 2H, eCH2Br), 2.45 (t, 2H, AreCH2e), 2.05 (s, 3H,
eCH3), 1.87 (s, 2H, eCH2eCH2Br), 1.25e1.47 (m, 14H, e(CH2)7e)
ppm; MS (EI): calculated 400.2/402.2; found 400.12/402.12.
4.6.10. 2,3,4,5-Tetramethoxy-6-methylstyrene (5c). To a mixture of
triphenylmethylphosphonium bromide (5.71 g, 16 mmol) and
sodium amide (0.78 g, 20 mmol) was added dry THF (50 mL) and
stirred for 12 h at room temperature in the atmosphere of argon. The
yellow supernatant liquid was added through a syringe to the solu-
tion of 5b (3.07 g,12.8 mmol) in dry THF (10 mL) under the protection
of argon18 (see Scheme 4). The reaction mixture was stirred for 24 h at
room temperature and then quenched with 2% aqueous HCl and
extracted with EtOAc (3ꢄ30 mL). The organic extracts were washed
with water, dried, and evaporated. The residuewaschromatographed
on silica gel to give 5c (75%) as a white solid. 1H NMR (500.0 MHz,
CDCl3, 298 K): 3.95 (s, 6H, 2ꢄ eOCH3), 3.80 (s, 6H, 2ꢄ eOCH3), 2.25
(s, 3H, eCH3), 5.50 (m, 2H, ]CH2), 6.70 (m, 1H, eCH]) ppm.
4.6.11. 6-Vinylubiquinone (5). To a solution of the compound 5c
(0.02 g, 0.8 mmol) in THF (14 mL), silver(II) oxide (0.49 mg) and 6 N
HNO3 (1 mL) were added. After being stirred for 5 min at 0 ꢃC,
silver(II) oxide (2.00 g) and 6 N HNO3 (2 mL) were further added42
(see Scheme 4). The reaction mixture was stirred at room tem-
perature for 30 min and was purified by column chromatography to
give 5 (77%) as red needle crystals. 1H NMR (500.0 MHz, CDCl3,
298 K): 3.99 (s, 6H, 2ꢄ eOCH3), 2.25 (s, 3H, eCH3), 5.80 (m, 2H, ]
CH2), 6.60 (m, 1H, eCH]) ppm; 13C NMR (125.7 MHz, CDCl3,
298 K): 184.7 (C]O), 184.2 (C]O), 144.3 (C, Ar), 143.1 (C, Ar), 141.1
(C, Ar), 148.7 (C, Ar), 133.6 (eCH]), 121.6 (]CH2), 61.2 (2ꢄ eOCH3),
12.9 (2ꢄ eCH3) ppm (Scheme 5).
Acknowledgements
We are grateful for help from Drs. Jia Li and Jing-Ya Li of the
Chinese National Center for Drug Screening, Shanghai Institute of
Materia Medica, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences. This research was supported by the Ministry
of Health (2009ZX 10004-301), the open research fund from Key
Lab of Analytical Chemistry for Life Science of Nanjing University,
the Major Research plan of the Natural Science Foundation of China
(Grant No. 91027035) and the Fundamental Research Funds for the
Central Universities (Grant No. WK1013002). Y.T.L. is supported by
The Program for Professor of Special Appointment (Eastern Scholar)
at Shanghai Institutions of Higher Learning.
Supplementary data
These data include electrochemical reduction mechanism of
ubiquinone; cytotoxicity of UQAs toward normal cell; NMR and
Scheme 5. Synthesis of 6-(10-bromodecyl)ubiquinone 9.

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Supplementary data related to this article can be found online at
doi:10.1016/j.tet.2011.06.026.
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