Synthesis and inhibitory activity of ubiquinone-acetogenin hybrid inhibitor with bovine mitochondrial complex I

Article abstract of DOI:10.1016/S0960-894X(03)00439-6

To elucidate the inhibitory action of acetogenins, the most potent inhibitors of mitochondrial complex I, we synthesized an acetogenin analogue which possesses a ubiquinone ring (i.e., the physiological substrate of complex I) in place of the α,β-unsatura

Full text of DOI:10.1016/S0960-894X(03)00439-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2385–2388
Synthesis and Inhibitory Activity of Ubiquinone–Acetogenin
Hybrid Inhibitor with Bovine Mitochondrial ComplexI
Hiromi Yabunaka, Masato Abe, Atsushi Kenmochi, Takeshi Hamada, Takaaki Nishioka
and Hideto Miyoshi*
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received 7 March 2003;accepted 13 April 2003
Abstract—To elucidate the inhibitory action of acetogenins, the most potent inhibitors of mitochondrial complex I, we synthesized
an acetogenin analogue which possesses a ubiquinone ring (i.e., the physiological substrate of complex I) in place of the a,b-unsat-
urated g-lactone ring of natural acetogenins, and named it Q-acetogenin. Our results indicate that the g-lactone ring of acetogenins
is completely substitutable with the ubiquinone ring. This fact is discussed in light of the inhibitory action of acetogenins.
# 2003 Elsevier Science Ltd. All rights reserved.
Acetogenins have very potent and diverse biological
effects such as antitumor, antimalarial, pesticidal and
antifeedant activities.1,2 The inhibitory effects of aceto-
genins on mitochondrial NADH-ubiquinone oxido-
reductase (complex I) are of particular note as the
diverse biological activities are thought to be attribu-
table to this effect.1,2 In fact, some acetogenins, such as
bullatacin (=rolliniastatin-2, Fig. 1) and rolliniastatin-1,
are the most potent inhibitors of this enzyme identified
to date.3À6 Although the acetogenins are thought to act
at the terminal electron transfer step of complex I,5À6
there is still no hard experimental evidence to verify
whether the inhibitors bind to the ubiquinone reduction
site. Additionally, there are few structural similarities
between the acetogenins and ordinary complex I inhibi-
tors such as piericidin A and rotenone. Thus, consider-
ing the unusual structural characteristics as well as the
very strong inhibitory potency of acetogenins, a detailed
analysis of the inhibitory actions of these inhibitors is
important to elucidate the structural and functional
features of the terminal electron transfer step of com-
plex I.
ural acetogenins, is substitutable with a ubiquinone ring
(i.e., 2,3-dimethoxy-5-methyl-1,4-benzoquinone) using
mucocin and squamocin D as the mother compounds.7
Taking into consideration the fact that ubiquinone is a
physiological substrate of complex I, this finding is very
interesting to elucidate the mode of action of aceto-
genins. Unfortunately, mucocin and squamocin D are
about 300- and 80-fold less potent, respectively, than
the most potent acetogenins like bullatacin.8 Moreover,
it seems somewhat confusing that mucocin showed
rather less activity than the quinone-mucocin hybrid
inhibitor. Therefore, it remains to be verified whether
the ubiquinone ring is generally substitutable for the g-
lactone ring. In the present study, we synthesized an
acetogenin analogue which possesses a ubiquinone ring
in place of the a,b-unsaturated g-lactone ring, named Q-
acetogenin (Fig. 1), using the most potent acetogenin
(compound 1) synthesized in our laboratory as the
mother compound. The inhibitory action of Q-aceto-
genin was examined with bovine heart mitochondrial
complex I.
Recently, Hoppen et al. indicated that the a,b-unsatu-
rated g-lactone ring of acetogenins, one of the most
common structural features of a large number of nat-
Synthesis
The synthetic procedures are outlined in Scheme 1. The
vinyl iodide 5 was prepared by methylation of the
reduced form of quinone 4, which was synthesized by
reacting 3 and (E, Z)-1,9-diiodo-1-nonene and a sequen-
tial retro-Diels–Alder reaction.9 Synthetic procedures of
compound 3 were described in ref 9.
*Corresponding author. Tel.: +81-75-753-6119;fax: +81-75-753-
6408;e-mail: miyoshi@kais.kyoto-u.ac.jp
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00439-6

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H. Yabunaka et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2385–2388
The key intermediate 6 was synthesized as described
previously.10 Treatment of 6 with 0.5 equiv TsCl (repe-
ated twice) and sequential MOM ether protection
afforded 7. Desilylation of 7 with TBAF, and the open-
ing of epoxide 8 with 1-lithium-1-nonyne in the presence
of BF3 etherate11 provided 9. After acetyl protection,
deprotection of the MOM ether and sequential treat-
ment with TsCl afforded 11. Hydrolysis of the acetyl
groups gave epoxide 12. The opening of epoxide 12 with
lithium (trimethylsilyl)acetylide in the presence of BF3
Bioactivity
The inhibition of complex I activity was determined by
NADH oxidase assay using bovine heart sub-
mitochondrial particles.16 Previous studies indicated
that the inhibitory potency of compound 1 is compar-
able to that of bullatacin, one of the most potent natural
acetogenins.17,18 The inhibitory potencies of 1 and Q-
acetogenin in terms of IC50, that is, the molar concen-
tration needed to halve the control NADH oxidase
activity, were 0.9 and 1.2 nM, respectively, in the pre-
sent study. Complete inhibition by Q-acetogenin was
attained at about 4 nM. We confirmed that Q-acet-
ogenin has no effect on the enzyme activities down-
stream of complex I. Thus, the ubiquinone ring is
almost completely substitutable for the g-lactone ring.
This result supports not only the result of the earlier
work by Hoppen et al.,7 but also our previous proposal
that the a,b-unsaturated g-lactone is not an essential
structural factor required to elicit potent activity of
acetogenins (Table 1).15,18
1
etherate provided 13, H and 13C NMR data and the
optical rotation of 13 were identical to those reported.12
Pd(0)-catalyzed coupling13 of alkyne 13 with vinyl
iodide 4 gave the corresponding eneyne in poor yield.
Therefore, vinyl iodide 5 converted from 4 was used as a
coupling partner to obtain eneyne 14.7 Catalytic hydro-
genation of 14 with Wilkinson’s catalyst and sequential
oxidation by CAN afforded Q-acetogenin.14
Compounds 1, 2 and DB were the same samples as
those used previously.15
The IC50 value of the precursor of Q-acetogenin (com-
pound 15) was 280 nM, indicating a significant con-
tribution of the ubiquinone ring to the inhibition.
Considering the structural similarity between a,b-unsa-
turated g-lactone and ubiquinone rings, the presence of
a conjugated carbonyl group may be required for this
moiety.
We previously showed that acetogenin acts as a potent
inhibitor only when the g-lactone and the THF ring
moieties are directly linked by an alkyl spacer.15 To
confirm whether this is also the case for Q-acetogenin,
we examined the inhibition by combined use of two
substructures (i.e., compound 2 and DB) at various
molar ratios, from 0.01 to 100. Under the experimental
conditions, the IC50 of compound 2 was 4.5 mM.19 No
synergistic enhancement was observed, indicating that the
ubiquinone ring and the THF ring moieties must be
directly linked by an alkyl spacer to elicit potent inhibition.
Discussion
Recent photoaffinity labeling studies using pyridaben20
and fenpyroximate21 indicated that PSST, ND5, and IP
49 kDa subunits contribute to the inhibitor binding
domain in bovine heart mitochondrial complex I.
Nevertheless, the binding subunit(s) of acetogenin,
which is significantly different from the two inhibitors
structurally, is still unknown. The present study along
with the earlier work7 indicated that the ubiquinone
ring is almost completely substitutable for the g-lactone
Table 1. Inhibitory potencies of test compounds
Compd
IC50 (nM)
Bullatacin
0.9
0.9
1.2
Compound 1
Q-acetogenin
Compound 2
Compound 15
4500
280
Figure 1. Structures of acetogenins and substructures examined in this
study.

H. Yabunaka et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2385–2388
2387
Scheme 1. (a) KOBu-t, THF/DMF (9:1) at À20 ꢀC, and 1,9-diiodo-1-nonene, À20 ꢀC to 0 ꢀC, 3 h, 65%;(b) toluene, 90 ꢀC, 2 h, 96%;(c) (i)
Na2S2O4, n-hexane/H2O (1:1), rt, (ii) CH3I, K2CO3, acetone, 40 ꢀC, 16 h, 75%;(d) (i) TsCl (0.5 equiv), DMAP, Et N, CH2Cl2, rt, (repeated twice,
3
ꢀ
.
55%), (ii) MOMCl, (i-Pr)2NEt, 97%;(e) TBAF, THF, rt, 88%;(f) 1-nonyne, n-BuLi, BF3 Et2O, THF, À78 C, 0.5 h, 69%;(g) AcCl, DMAP,
CH2Cl2, 0 ꢀC to rt, 96%;(h) (i) BF 3 Et2O, Me2S, À20 C, 1.5 h, 94%, (ii) TsCl, DMAP, Et3N, CH2Cl2, 55 C, 3 h, 95%;(i) KOH, MeOH, rt, 0.5 h,
ꢀ
ꢀ
.
ꢀ
.
92%;(j) (i) lithium (trimethylsilyl)acetylide, BF 3 Et2O, À78 C, 15 min, (ii) K2CO3, MeOH, rt, 5 h, 69%;(k) (Ph 3P)4Pd, CuI, Et3N, rt, 5 h, 72%, (e)
H2, (Ph3P)3RhCl, benzene, rt, 1 day, 91%;(m) Ce(NH 4)2(NO3)6, pyridine-2,6-dicarboxylic acid, CH3CN/H2O (1:1), 0 ꢀC, 4 h, 86%.
ring of natural acetogenins. This result is important to
elucidating the inhibitory action of acetogenins since
ubiquinone is the physiological substrate of complex I.
Actually, the so-called DB (n-decylbenzoquinone) has
been widely used in complex I assays as an electron
accepting substrate. Therefore, at first sight, Q-aceto-
genin and consequently natural acetogenins seem to
bind to the ubiquinone reduction site of the enzyme.
acetogenins. Although we cannot exclude the possibility
that the ubiquinone ring of the hybrid inhibitors accepts
electrons from complex I, this effect would not be
responsible for their inhibitory action since the hybrid
inhibitors elicited inhibition at concentrations far lower
than the concentration of NADH, an electron donor,
under the experimental conditions. The mode of action
of acetogenins including their active conformation
remains to be elucidated. Nevertheless, the fact that Q-
acetogenin is a good mimic of potent acetogenins may
open a new experimental approach to the study of
ligand-complex I interaction, for example, the redox-
reaction induced FTIR spectroscopic technique used for
other electron-transfer enzymes in combination with [1-
or 4-13C1]-ubiquinones.23,24
However, this possibility can be ruled out for the fol-
lowing two reasons. Firstly, a Lineweaver–Burk plot for
NADH-DB (or Q1) oxidoreductase activity in the pre-
sence of Q-acetogenin or compound 1 suggested that the
inhibition by these inhibitors is noncompetitive against
DB and Q1 (data not shown, cf. ref 17). Secondly, since
the Km of DB and the Kd of potent acetogenin differ by
three orders of magnitude,22 it is unlikely that the ubi-
quinone moiety of Q-acetogenin binds tightly to the
ubiquinone reduction site. Therefore, the ubiquinone
ring of quinone-acetogenin hybrid inhibitors synthe-
sized here and earlier7 may serve merely as a substitute
of the a,b-unsaturated g-lactone ring of natural
Acknowledgements
We thank Dr. Kazuhiro Irie (Kyoto Univ.) for his help
with the measurement of HRMS spectra.

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15. Kuwabara, K.;Takada, M.;Iwata, J.;Tatsumoto, K.;
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Tetrahedron
1
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2.41 (d, J=4.1 Hz, 2H), 2.01 (s, 3H), 2.00–1.92 (m, 4H), 1.74–
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29.9, 29.7, 29.6, 29.5, 29.4, 29.3, 29.0, 28.8, 28.4, 26.4, 25.7,
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