Essential structural factors of acetogenins, potent inhibitors of mitochondrial complex I.

Article abstract of DOI:10.1016/S0960-894X(02)00374-8

To elucidate the role of the hydrophobic alkyl tail of acetogenins in the inhibitory action, we synthesized an acetogenin derivative possessing the shortest tail (i.e., methyl group) and examined its inhibitory activity against bovine heart mitochondrial

Full text of DOI:10.1016/S0960-894X(02)00374-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2089–2092
Essential Structural Factors of Acetogenins, Potent Inhibitors of
Mitochondrial Complex I
Tomoko Motoyama, Hiromi Yabunaka and Hideto Miyoshi*
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received 11 April 2002; accepted 16May 2002
Abstract—To elucidate the role of the hydrophobic alkyl tail of acetogenins in the inhibitory action, we synthesized an acetogenin
derivative possessing the shortest tail (i.e., methyl group) and examined its inhibitory activity against bovine heart mitochondrial
complex I. Our results indicated that the alkyl tail, which is one of the common structural features of natural acetogenins, is not an
essential structural factor required for the potent inhibition. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
groups such as hydroxy group in the spacer, and ste-
reochemistry around the THF and the g-lactone ring
moieties are not essential structural factors for the
potent activity; (ii) acetogenin acts as a potent inhibitor
only when the g-lactone and the THF ring moieties are
directly linked by an alkyl spacer; and (iii) the optimal
length of the spacer is about 13 carbon atoms. On the
basis of these results, we proposed that the g-lactone
ring is not the only reactive species interacting directly
with the enzyme and that both ring moieties act in a
cooperative manner on the enzyme with the support of
some specific conformation of the spacer. On the other
hand, there have been no studies concerning the role of
the long alkyl tail, although the importance of hydro-
phobic interactions of the tail with the enzyme (or its
environment) was suggested.6 We synthesized an acet-
ogenin derivative (1) possessing the shortest tail (i.e.,
methyl group) and compared its inhibitory activity for
bovine complex I with that of its mother compound 2.
Acetogenins have very potent and diverse biological
effects including cytotoxic, antitumor, antimalarial,
pesticidal and antifeedant activities.1 The inhibitory
effects of acetogenins on mitochondrial NADH-ubiqui-
none oxidoreductase (complex I) are of particular note
as the diverse biological activities are thought to be
attributable to this effect.1 In fact, some of these com-
pounds, such as bullatacin (Fig. 1) and rolliniastatin-1,
are the most potent inhibitors of this enzyme identified
to date.2,3 Although the acetogenins act at the terminal
electron transfer step of complex I similarly to the
ordinary complex I inhibitors,2,3 there are few structural
similarities between the acetogenins and ordinary com-
plex I inhibitors such as piericidin A and rotenone.
Thus, considering their unusual structural character-
istics as well as the very strong inhibitory potency of
acetogenins, detailed analysis of the inhibitory actions
of these inhibitors is important to elucidate the struc-
tural and functional features of the terminal electron
transfer step of complex I. As the first step toward this
purpose, identification of the crucial structural factors
of acetogenins required for their potent inhibitory
effects would be very useful.3,4
Chemistry
The synthetic procedures are outlined in Scheme 1. The
key intermediate 8 was synthesized according to the
procedures reported by Hoye and Ye,7 except that 3 was
prepared by reacting tert-butyl acetate and trans-1,4-
dibromo-2-butene.8 Reduction of 3 with DIBAL-H and
Wittig extension of the resultant dialdehyde gave 4. This
was reduced with DIBAL-H followed by double Sharp-
less asymmetric epoxidation to afford 5. Silylation with
TBDPSCl and sequential Sharpless asymmetric dihy-
droxylation gave 6, which was immediately treated with
In previous structure/activity studies using a series of
natural and synthetic acetogenins,5 we showed that: (i)
number of THF rings, the presence of polar functional
*Corresponding author. Tel.: +81-75-753-6119; fax: +81-75-753-
6408; e-mail: miyoshi@kais.kyoto-u.ac.jp
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00374-8

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T. Motoyama et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2089–2092
trifluoroacetic acid to produce 7. Treatment with TsCl
afforded 8, the 1H NMR data and an optical rotation of
which were completely identical to those reported.7
After brief treatment of 8 with TBAF (0.5 molar equiv,
20 min), the reaction mixture was treated with K2CO3 to
produce a mixture of two useful products, 9 and 10 (43
and 17%, respectively, based upon recovered starting
1
material).9 The H and 13C NMR data and an optical
rotation of 10 also completely matched the reported
data.7
Opening of epoxide 9 by catalytic hydrogenation with
Pd/C selectively yielded the secondary alcohol 11.10
Treatment of 11 with excess TBAF produced epoxide
12. Opening of epoxide 12 with lithium (trimethylsilyl)
acetylide in the presence of boron trifluoride etherate11
and desilylation provided 13. Pd(0)-catalyzed coupling12
of alkyne 13 with vinyl iodide 14, which was prepared
by the reported method,13 gave the eneyne 15. Catalytic
hydrogenation of 15 using Wilkinson’s catalyst and
sequential thermal elimination of the sulfide moiety
were performed according to the reported method7 to
afford 1.14
Compound 2 was the same sample as that synthesized
previously.5c
Bioactivity
The inhibition of complex I activity was determined by
NADH oxidase assay using bovine heart sub-
mitochondrial particles.15 Previous studies indicated
that the inhibitory potency of synthetic acetogenin 2 is
comparable to that of bullatacin, one of the most potent
natural acetogenins.5b,5c The potency of 2 in terms of
IC50 value, that is the molar concentration needed to
halve the control NADH oxidase activity, was 0.9 nM in
Figure 1. Structures of bullatacin and synthetic acetogenins examined
in this study.
Scheme 1. (a) LDA, THF, ꢀ40 ꢁC, 1 h, 83%; (b) (i) DIBAL-H (2.0 molar equiv), CH2Cl2, ꢀ78 ꢁC, 1 h; (ii) PH3P¼CHCOOEt, CH2Cl2, rt, 9 h, 65%;
(c) (i) DIBAL-H, CH2Cl2, ꢀ78 ꢁC, 2 h; (ii) L-(+)-DET, Ti(i-PrO)4, t-BuOOH, CH2Cl2, ꢀ20 ꢁC, 1 day, 58%; (d) (i) TBDPSCl, DMAP, Et3N,
CH2Cl2, rt, 16h; (ii) AD-mix- b, MeSO2NH2, t-BuOH-H2O, 0 ꢁC, 16h 68%; (e) TFA, CH Cl2, rt, 1.5 h 82%; (f) TsCl, DMAP, Et3N, CH2Cl2, 55 ꢁC,
24 h, 91%; (g) (i) TBAF (0.5 molar equiv), THF, rt, 20 min; (ii) K2CO3, MeOH, rt, 16h; (h) H 2, Pd/C, MeOH, 4 days, 95%; (i) TBAF, THF, rt, 5 h,
2
ꢁ
.
85%; (j) (i) lithium (trimethylsilyl)acetylide, BF3 Et2O, ꢀ78 C, 15 min; (ii) K2CO3, MeOH, rt, 16h, 62%; (k) (Ph 3P)2PdCl2, CuI, Et3N, rt, 24 h,
72%; (l) (i) H2, (Ph3P)3RhCl, benzene, rt, 1 day; (ii) m-CPBA, CH2Cl2, 0 ꢁC, 30 min; (iii) toluene, 100 ꢁC, 2 h, 32%.

T. Motoyama et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2089–2092
2091
the present study. Under the same experimental condi-
tions, IC50 of 1 was 3.1 nM, and complete inhibition was
attained at about 10 nM. Thus, irrespective of drastic
shorting of the tail length, compound 1 retained suffi-
ciently potent inhibitory activity, indicating that large
hydrophobicity of the tail is not essentially important
for the activity. This result is consistent with the obser-
vations that the presence of polar hydroxy group(s) in
the tail of some natural acetogenins is not unfavorable
for the activity.2a,5a
not support the model proposed by Shimada et al. The
active conformation of acetogenins in complex I
remains to be elucidated. However, we cannot necessa-
rily exclude the validity of their model for partitioning of
acetogenins into the liposomal membrane. In the lipo-
somal membrane, the average location of THF and g-
lactone ring moieties could be primarily determined by
their hydrophobicity.16
Acknowledgements
We thank Dr. Kazuhiro Irie for his help in measure-
ment of HRMS spectra.
Discussion
In general, the activities of potent biologically active
compounds are markedly diminished (by several orders
of magnitude) by structural modification of essential
structural unit(s). In this sense, essential structural fac-
tors of the THF and the g-lactone ring moieties of
acetogenins are not necessarily obvious, except for the
important role of the alkyl spacer connecting both ring
moieties, as described in the introductory section. In
addition to the THF ring with flanking hydroxy groups
and the g-lactone ring, the presence of a long alkyl tail is
also a common structural feature of a large number of
natural acetogenins.1a Our results indicated that the
hydrophobic tail is preferable for the activity, but is not
an essential structural factor. Thus, regardless of the
very potent activity, crucial structural features of acet-
ogenins are quite ambiguous. This unusual character-
istic may be related to the high degree of flexibility of
the alkyl spacer that links the functionally important
THF and g-lactone ring moieties. The present study
provided useful insight into design strategies of novel
acetogenin mimics for further wide structural modifi-
cations, which would enable development of novel
agents for pharmaceutical and agrochemical use.
References and Notes
1. (a) Zeng, L.; Ye, Q.; Oberlies, N. H.; Shi, G.; Gu, Z.-M.;
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1
Based on the results of H NMR and differential scan-
ning calorimetry studies of acetogenins in liposomal
membrane, Shimada et al. proposed a model of active
conformation of these inhibitors, wherein the alkyl tail
and THF ring with flanking hydroxy groups work as
hydrophobic and hydrophilic anchors, respectively, in
the mitochondrial membrane, and only the g-lactone
interacts directly with the target site of complex I by
lateral diffusion in the membrane.6 As a prerequisite for
the conformational model of Shimada et al., they noted
a significant contribution of the intermolecular hydro-
gen bonds between the hydroxy groups in the adjacent
THF rings and the oxygen atoms in the glycerol back-
bone of the phospholipid when the THF rings moiety
acts as hydrophilic anchor at the liposomal membrane
surface. If the hydrogen bond-donating ability of the
hydroxy groups is important for complex I inhibition,
acetylation of one or both hydroxy groups would result
in a marked decrease in the inhibitory potency, but this
was not the case.5b Furthermore, if the g-lactone ring
can be regarded as the only reactive species directly
interacting to the enzyme, chemical modification of this
moiety would result in drastic decrease in the inhibitory
potency, but this was also not the case.5b,5c Thus, the
present results along with the previous studies5b,5c do
6. Shimada, H.; Grutzner, J. B.; Kozlowski, J. F.; McLaugh-
lin, J. L. Biochemistry 1998, 37, 854.
7. Hoye, T. R.; Ye, Z. J. Am. Chem. Soc. 1996, 118, 1801.
8. Li, P.; Yang, J.; Zhao, K. J. Org. Chem. 1999, 64, 2259.
9. Compound 9: 1H NMR (300 MHz, CDCl3) d 7.74 (d,
J=8.2 Hz, 2H), 7.61 (brd, J=6.0 Hz, 4H), 7.44–7.30 (m, 6H),
7.23 (d, J=8.2 Hz, 2H), 4.65 (ddd, J=4.6, 4.6 and 4.6 Hz, 1H),
4.28 (m, 1H), 3.92–3.80 (m, 3H), 3.69 (dd, J=12.2 and 4.5 Hz,
1H), 3.59 (m, 1H), 2.94 (ddd, J=4.2, 4.2 and 2.3 Hz, 1H), 2.72
(dd, J=6.2 and 3.1 Hz, 1H), 2.64 (m, 1H), 2.39 (s, 3H), 2.12–
1.60 (m, 8H), 1.02 (s, 9H). MS (ESI) m/z 659 [M+Na]+.
1
Compound 10: H NMR (300 MHz, CDCl3) d 3.97–3.89 (m,
4H), 2.96(ddd, J=4.2, 4.2 and 2.3 Hz, 2H), 2.76–2.70 (m, 4H),
2.18–2.05 (m, 2H), 2.03–1.92 (m, 2H), 1.88–1.75 (m, 4H). 13C
NMR (75 MHz, CDCl3) d 82.1, 78.8, 54.2, 44.1, 28.8, 27.9. MS
23
(ESI) m/z 249 [M+Na]+. ½aꢂD =ꢀ15.9 (c 3.34, ethyl acetate).
10. Uckun, F. M.; Mao, C.; Vassilev, A. O.; Navara, C. S.;
Narla, R. K. S.; Jan, S. T. Bioorg. Med. Chem. Lett. 2000, 10,
1015.
11. Yamaguchi, M.; Hirano, I. Tetrahedron Lett. 1983, 24,
391.
12. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron
Lett. 1975, 16, 4467.
13. (a) Hoye, T. R.; Hanson, P. R.; Kovelesky, A. C.; Ocain,
T. D.; Zhuang, Z. J. Am. Chem. Soc. 1991, 113, 9369. (b)

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T. Motoyama et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2089–2092
Makabe, H.; Tanaka, A.; Oritani, T. J. Chem. Soc., Perkin
Trans. 1 1994, 1975.
15. Bovine heart submitochondrial particles were prepared by
the method of Matsuno-Yagi and Hatefi (J. Biol. Chem. 1985,
260, 14424) and stored in a buffer containing 0.25 M sucrose
and 10 mM Tris–HCl (pH 7.4) at ꢀ82 ꢁC. The NADH oxidase
activity in the particles was followed spectrometrically with a
Shimadzu UV-3000 (340 nm, e=6.2 mMꢀ1 cmꢀ1) at 30 ꢁC.
The reaction medium (2.5 mL) contained 0.25 M sucrose,
1 mM MgCl2 and 50 mM phosphate buffer (pH 7.4). The final
mitochondrial protein concentration was 30 mg of protein/mL.
The reaction was started by adding 50 mM NADH after equi-
libration of the particles with inhibitor for 5 min. The IC50
values were averaged from two independent experiments.
16. Diamond, J. M.; Katz, Y. J. Membr. Biol. 1974, 17, 121.
14. Colorless oil. 1H NMR (300 MHz, CDCl3) d 6.98 (m, 1H),
5.00 (dq, J=1.5, 7.0 Hz, 1H), 3.83–3.90 (m, 3H), 3.78 (dt,
J=5.0, 7.5 Hz, 1H), 3.57 (dq, J=6.3, 6.3 Hz, 1H), 3.37 (m,
1H), 2.79 (br, s, 2H), 2.28 (m, 2H), 1.96–2.04 (m, 4H), 1.43–
1.80 (m, 8H), 1.21–1.43 (m, 18H), 1.41 (d, J=7.0 Hz, 3H), 1.13
(d, J=6.3 Hz, 3H). 13C NMR (75 MHz, CDCl3) d 173.9,
148.9, 134.4, 84.5, 83.2, 81.8, 81.7, 77.5, 77.0, 76.6, 74.1,
70.5, 33.5, 29.7, 29.6, 29.5, 29.3, 29.2, 29.0, 28.4, 27.4, 25.7,
24
25.2, 19.2, 18.8. ½aꢂD =+6.8 (c 0.40, ethyl acetate). HRMS
(FAB) for C28H49O6 (M+H)+ calcd 481.3529, found
481.3522.