Essential structural features of acetogenins: Role of hydroxy groups adjacent to the bis-THF rings

Article abstract of DOI:10.1016/j.bmcl.2003.11.021

The presence of two hydroxy groups adjacent to the THF ring(s) is a common structural feature of natural acetogenins. To elucidate the role of each hydroxy group in the inhibitory action of acetogenins, we synthesized three acetogenin analogues which lack

Full text of DOI:10.1016/j.bmcl.2003.11.021

Bioorganic & Medicinal Chemistry Letters 14 (2004) 779–782
Essential structural features of acetogenins: role of hydroxy
groups adjacent to the bis-THF rings
Masato Abe, Atsushi Kenmochi, Naoya Ichimaru, 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 6 October 2003; accepted 6 November 2003
Abstract—The presence of two hydroxy groups adjacent to the THF ring(s) is a common structural feature of natural acetogenins.
To elucidate the role of each hydroxy group in the inhibitory action of acetogenins, we synthesized three acetogenin analogues
which lack either or both of the hydroxy groups, and investigated their inhibitory activities with bovine heart mitochondrial com-
plex I. Our results indicate that the presence of either of the two hydroxy groups sufficiently sustains a potent inhibitory effect.
# 2003Elsevier Ltd. All rights reserved.
Acetogenins have diverse biological effects such as anti-
tumor, antimalarial, pesticidal and antifeedant activ-
ities.1,2 The inhibitory effects of acetogenins on
hydroxylated THF ring(s) are not essential structural
factors for potent activity; (ii) natural g-lactone ring
itself is not crucial for the activity, and can be sub-
stitutable with an ubiquinone ring; and (iii) acetogenin
acts as a strong inhibitor only when the g-lactone and
the THF ring moieties are directly linked by an alkyl
spacer, the optimal length of which is about 13carbon
atoms. Thus, except for the important role of the alkyl
spacer, essential structural factors including the active
conformation of acetogenins remain to be elucidated.
mitochondrial
NADH-ubiquinone
oxidoreductase
(complex I) are of particular note as the diverse biolo-
gical activities are thought to be attributable to this
effect.1,2 Actually some acetogenins, such as bullatacin
(=rolliniastatin-2, Fig. 1) and rolliniastatin-1, are the
most potent inhibitors of this enzyme identified to
date.3À6 Acetogenins are thought to act at the terminal
electron transfer step of complex I,5,6 but there is still no
hard experimental evidence to verify whether the inhi-
bitors bind to the ubiquinone reduction site. Consider-
ing the unusual structural characteristics as well as the
very strong inhibitory effect of acetogenins, a detailed
study on the inhibitory action of these inhibitors is nee-
ded to elucidate the structural and functional features of
the terminal electron transfer step of complex I. To this
end, identification of the crucial structural factors of
acetogenins required for their potent inhibition would
be very useful.
The presence of two OH groups adjacent to the THF
ring(s) is a common structural feature of a large number
of natural acetogenins. Some reports suggested an impor-
tant role for the OH group in the cytotoxicity of acet-
ogenins,12,13 whereas the interpretation of results of
cytotoxic assays is somewhat complicated since one has
to take into consideration factors such as membrane
transport and metabolism. We previously showed
that acetylation of both OH groups of a potent syn-
thetic acetogenin (compound 1 in this study) resulted in
a rather slight, just 6-fold, decrease in the inhibitory
potency with mitochondrial complex I.9 Although this
result was quite unexpected, it suggests that high polar-
ity (or hydrophilicity) around the THF ring moiety,
rather than the hydrogen bond-donating ability of the
OH groups, may be required for the inhibitor to adopt
an active conformation.14 To elucidate the role of each
OH group, selective deoxygenation is necessary. In this
study, we synthesized three acetogenin analogues which
lack either or both of the two hydroxy groups located
In previous structure–activity studies using a series of
natural and synthetic acetogenins with mitochondrial
complex I,7À11 we showed that: (i) the presence of polar
functional group(s) like an OH group in the spacer,
number of THF rings and stereochemistry around the
* Corresponding author. Tel.: +81-75-753-6119; fax: +81-75-753-
6408; e-mail: miyoshi@kais.kyoto-u.ac.jp
0960-894X/$ - see front matter # 2003Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.021

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M. Abe et al. / Bioorg. Med. Chem. Lett. 14 (2004) 779–782
1-lithium-1-nonyne in the presence of BF3 etherate15
afforded 6. Hydrogenation of 6 with Pd/C and sequential
selective protection of the primary alcohol by TBDPSCl
gave 7. Deoxygenation of the free hydroxy group in 7
was carried out by the Barton two-step procedure,16 that
is conversion to the corresponding thiocarbonate 8 and
then treatment with n-Bu3SnH to produce 9.
After deprotection of the MOM ether of 9, tosylation of
a free hydroxy group and sequential desilylation gave
the epoxide 10. Opening of the epoxide with lithium
(trimethylsilyl)acetylide in the presence of BF3 etherate
and sequential desilylation afforded 11. Pd(0)-catalyzed
coupling17 of alkyne 11 with vinyl iodide 12, which was
prepared as described,18 gave the eneyne 13. Since
selective hydrogenation of 13 with Wilkinson’s catalyst
resulted in an appreciable reduction of the butenolide
double bond as pointed out by Marshall and Chen,19
hydrogenation was carried out with diimide, generated
in situ from tosylhydrazine,20 to obtain 2.21
Initially, we tried to synthesize compound 3 by a
similar sequence of reactions starting from 5, as
shown in Scheme 2. However, the radical deoxygena-
tion of the thiocarbonate in 14 resulted in a very poor
yield of the product. Several modifications of the
reaction conditions were investigated, but attempts to
improve the yield were unsuccessful. We therefore
examined an alternate approach (Scheme 2). After
acetylation of both hydroxy groups of 15, which was
obtained by catalytic hydrogenation of 6 with Pd/C,
deprotection of MOM ether and sequential tosylation
afforded 16. Hydrolysis of the acetyl group gave epoxide
17. The opening of epoxide 17 with lithium (trimethyl-
silyl)acetylide in the presence of BF3 etherate and
sequential desilylation provided 18. Cross-coupling of 18
with the spacer moiety 12 and hydrogenation of the
resultant eneyne by the above method afforded 19.
Deoxygenation of a free hydroxy group in 19 by the
Barton method and sequential deprotection of the MOM
ether afforded compound 3.21
Figure 1. Structures of acetogenin analogues examined in this study.
on the sides of bis-THF rings, and investigated their
inhibitory activities with bovine heart mitochondrial
complex I.
1. Synthesis
The synthesis of compound 2 is outlined in Scheme 1.
The key intermediate 5 was synthesized as described
previously.10,11 The opening of epoxide
5 with
ꢀ
.
Scheme 1. (a) 1-Nonyne, n-BuLi, BF3 Et2O, THF, À78 C, 0.5 h, 77%; (b) (i) H2, Pd/C, EtOH, (ii) TBDPSCl (1.2 equiv), DMAP, CH2Cl2, rt, 92%;
(c) thiocarbonyldiimidazole, DMAP, CH2Cl2, rt, 93%; (d) n-Bu3SnH, AIBN, dry toluene, rt, 2 h, 65%; (e) (i) TMSBr, CH2Cl2, À30 ꢀC, 1 h, (ii) TsCl,
ꢀ
.
DMAP, Et3N, CH2Cl2, rt, (iii) TBAF, THF, rt, 65%; (f) lithium (trimethylsilyl)acetylide, BF3 Et2O, THF, À78 C, 15 min, (ii) K2CO3, MeOH, rt, 5
h, 69%; (g) (Ph3P)4Pd, CuI, Et3N, rt, 5 h, 72%; (h) TsNHNH2, NaOAc, DME/H2O (1:1), reflux, 85%.

M. Abe et al. / Bioorg. Med. Chem. Lett. 14 (2004) 779–782
781
ꢀ
ꢀ
.
Scheme 2. (a) n-Bu3SnH, AIBN, dry toluene, rt; (b) (i) AcCl, DMAP, CH2Cl2, 0 C to rt, (ii) BF3 Et2O, Me2S, À20 C, 2 h; (iii) TsCl, DMAP, Et3N,
ꢀ
.
CH2Cl2, rt, 80%; (c) KOH, MeOH, rt, 2 h, 95%; (d) lithium (trimethylsilyl)acetylide, BF3 Et2O, THF, À78 C, 15 min; (ii) K2CO3, MeOH, rt, 5 h,
75%; (e) (i) compound 12, (Ph3P)4Pd, CuI, Et3N, rt, 5 h; (ii) TsNHNH2, NaOAc, DME/H2O (1:1), reflux, 62%; (f) (i) thiocarbonyldiimidazole,
DMAP, CH2Cl2, rt; (ii) n-Bu3SnH, AIBN, dry toluene, rt, 2 h; (iii) 4% AcCl in MeOH, CH2Cl2, rt, 1 h, 54%.
Compound 4 was synthesized by deoxygenation of a
free hydroxy group in compound 2 by the Barton
method in 53% yield (two steps).21
moiety may explain why both the number of THF rings
and the stereochemistry surrounding the hydroxylated
THF ring moiety affect little the inhibitory potency.
Furthermore, from a molecular viewpoint, the impor-
tance of high polarity may be associated with the fact
that the putative target subunit of acetogenin in bovine
complex I (PSST subunit23) is hydrophilic and contains
no transmembrane helices according to prediction of
secondary structure.24À26
2. Bioactivity
The inhibition of complex I activity was determined by
NADH oxidase assay using bovine heart sub-
mitochondrial particles (SMP).7 Previous studies indi-
cated that the inhibitory potency of compound 1, a
standard compound in this study, is comparable to that
of bullatacin, one of the most potent natural acet-
ogenins.7,9 The inhibitory potency of compound 1 in
terms of the IC50, that is the molar concentration
needed to halve the control NADH oxidase activity, was
0.75 (Æ0.08) with the present SMP preparations. The
IC50 values of compounds 2 and 3 were 3.1 (Æ0.4) and
2.7 (Æ0.3) nM, respectively. The IC50 value of com-
pound 4 was 85 (Æ9) nM, and maximum inhibition was
saturated at about 85% even at 2 mM.22 These results
indicate that a lack of both OH groups resulted in a
drastic decrease in inhibitory potency, but the presence
of either is enough to retain fairly strong activity. This is
consistent with the observation that a mono-acetyl
derivative of compound 1, wherein the OH group loca-
ted on the side of the spacer is acetylated, elicits fairly
potent activity, the IC50 being 2.1 nM.9 Our results for
the first time revealed that the contributions of the two
OH groups toward the inhibitory action are equivalent.
We previously showed that the IC50 value of diepomur-
icanin (Fig. 1), which also has no free OH group, is 2.8
mM.9 Compared to diepomuricanin, compound 4
appeared to be a much more potent inhibitor. This is
probably due to the relatively fixed spatial position (i.e.,
lower flexibility) of the two ether oxygen atoms of
compound 4 which favors an active conformation. The
present study not only is helpful in elucidating the active
conformation of acetogenins, but also provides useful
guiding principles for wide structural modification of
acetogenins with the aim of developing practical agents.
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4H), 3.38 (m, 1H), 2.49 (s, 1H), 2.26 (m, 2H), 2.08–1.96
(m, 4H), 1.65–1.23(m, 46H), 1.41 (d, J=7.0 Hz, 3H), 0.88
(t, J=6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3) d 173.9,
148.9, 134.4, 83.0, 82.1, 81.2, 80.0, 74.1, 35.8, 33.4, 32.1,
31.9, 29.8, 29.6, 29.5, 29.4, 29.2, 28.9, 28.8, 28.4, 27.4,
26.2, 25.7, 25.2, 22.7, 19.2, 14.1. [a]2D3=+20.0 (c 0.46,
EtOH). ESI-MS (m/z) 613.5 [M+Na]+. Compound 3:
colorless oil. 1H NMR (300 MHz, CDCl3) d 6.98 (m, 1H),
5.00 (dq, J=1.5, 7.0 Hz, 1H), 3.95–3.80 (m, 4H), 3.38 (m,
1H), 2.49 (s, 1H), 2.26 (m, 2H), 2.08–1.96 (m, 4H), 1.65–
1.23(m, 46H), 1.41 (d, J=7.0 Hz, 3H), 0.88 (t, J=6.9 Hz,
3H). 13C NMR (75 MHz, CDCl3) d 173.9, 148.9, 134.4,
83.0, 82.1, 81.2, 80.0, 74.1, 35.8, 33.4, 32.1, 31.9, 29.8, 29.6,
29.5, 29.4, 29.2, 28.9, 28.8, 28.4, 27.4, 26.2, 25.7, 25.2, 22.7,
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1.85–1.23(m, 48H), 1.41 (d, J=7.0 Hz, 3H), 0.88 (t, J=6.9
Hz, 3H). 13C NMR (75 MHz, CDCl3) d 173.9, 148.9, 134.4,
81.4, 79.8, 35.8, 32.1, 31.9, 29.8, 29.6, 29.5, 29.3, 29.2, 28.5,
27.4, 26.2, 25.2, 22.7, 19.2, 14.1. [a]2D3=+9.5 (c 0.11,
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