Critical role of a methyl group on the γ-lactone ring of annonaceous acetogenins in the potent inhibition of mitochondrial complex i

Article abstract of DOI:10.1016/j.bmcl.2013.01.018

C34-epi and C34-epi-C35-trifluoro analogues of solamin, a mono-THF annonaceous acetogenin, were synthesized. Their inhibitory activity, along with previously synthesized analogues (C35-fluoro, C35-difluoro, and C35-trifluorosolamins), against bovine mitochondrial NADH-ubiquinone oxidoreductase (complex I) was determined. The present study revealed that the methyl group on the γ-lactone moiety is critical to the potent inhibition of complex I by natural acetogenins.

Full text of DOI:10.1016/j.bmcl.2013.01.018

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1217–1219
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Bioorganic & Medicinal Chemistry Letters
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Critical role of a methyl group on the
c-lactone ring of annonaceous
acetogenins in the potent inhibition of mitochondrial complex I
b
c
c
c
a
Naoto Kojima a, , Masato Abe , Yuki Suga , Kazufumi Ohtsuki , Tetsuaki Tanaka , Hiroki Iwasaki ,
⇑
Masayuki Yamashita a, Hideto Miyoshi b,
⇑
a Kyoto Pharmaceutical University, 1 Shichono-cho, Yamashina-ku, Kyoto 607-8412, Japan
b Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
c Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
C34-epi and C34-epi-C35-trifluoro analogues of solamin, a mono-THF annonaceous acetogenin, were syn-
thesized. Their inhibitory activity, along with previously synthesized analogues (C35-fluoro, C35-difluoro,
and C35-trifluorosolamins), against bovine mitochondrial NADH–ubiquinone oxidoreductase (complex I)
Received 1 December 2012
Revised 28 December 2012
Accepted 4 January 2013
Available online 12 January 2013
was determined. The present study revealed that the methyl group on the
the potent inhibition of complex I by natural acetogenins.
c-lactone moiety is critical to
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Acetogenin
Respiratory complex I
Structure–activity relationship
More than 500 annonaceous acetogenins have been isolated
from the plant family Annonaceae since the discovery of uvaricin
in 1982.1–3 Acetogenins have very potent and diverse biological ef-
fects such as antitumor, antimalarial, and pesticidal activities.2,3
Annonacin, the major acetogenin in the tropical plant Annona muri-
cata, is highly toxic to cultured neurons and may play a role in
some neurodegeneration in humans.4,5 The inhibitory effect of ace-
togenins on mitochondrial NADH–ubiquinone oxidoreductase
(complex I) is of particular importance since their diverse biologi-
cal activities are thought to be attributable to this effect.
the THF ring(s) sufficiently sustains the potent activity,15 and (iii)
a long alkyl tail is preferable, but not essential since even a methyl
derivative elicited strong inhibition at the nanomolar level.16 Thus,
the crucial structural factors of acetogenins are ambiguous, sug-
gesting that complex I recognizes each of the multiple functional
groups of the inhibitors in a fairly loose way. It is however note-
worthy that acetogenins act as strong inhibitors only when the
c
-lactone and THF moieties are directly linked by a long alkyl
spacer:17 the optimal length of the spacer for exhibiting the inhibi-
tion is approximately 13 carbon atoms.18,19
The chemical structure of most natural acetogenins is charac-
Kojima et al. synthesized three C35-fluorinated solamin ana-
logues (C35-fluoro, C35-difluoro, and C35-trifluorosolamins 1–3,
Fig. 2) and investigated their growth inhibitory activities against
human cancer cell lines.20,21 Interestingly, the activity decreased
as the number of fluorine atoms increased, though it remained to
be clarified whether the decrease is due to a reduction of the activ-
ity at the target enzyme level (i.e., complex I) or to other factors
such as differences in metabolic stability. If the former is a cause,
terized by four segments: (i) an
a,b-unsaturated c-lactone ring,
(ii) one to three tetrahydrofuran (THF) rings with flanking OH
group(s), (iii) a long alkyl tail, and (iv) an alkyl spacer linking the
two pharmacophores (i.e.,
c-lactone and THF moieties) (Fig. 1).
On the basis of structure–activity relationship studies carried out
by ourselves and other groups, the roles of each segment in the
inhibitory action against bovine complex I have been elucidated
as follows: (i) a natural c-lactone ring itself is not crucial for the
activity and is substitutable with other structures,6–10 (ii) neither
the number of THF rings nor the stereochemistry around the THF
ring moiety with the flanking hydroxy group is an essential fac-
tor,11–14 and the presence of either of two OH groups adjacent to
⇑
Corresponding authors. Tel.: +81 75 595 4672; fax: +81 75 595 4775 (N.K.); tel.:
+81 75 753 6119; fax: +81 75 753 6408 (H.M.).
E-mail addresses: kojima@mb.kyoto-phu.ac.jp (N. Kojima), miyoshi@kais.kyoto-
u.ac.jp (H. Miyoshi).
Figure 1. Representative structure of natural acetogenins.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.018

1218
N. Kojima et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1217–1219
methyl 13-tetradecynoate followed by the construction of
a,b-
unsaturated
c
-lactone moiety.25 When synthesizing C34-epi-sol-
amin 4, a more efficient method was examined (Scheme 2). Direct
asymmetric alkynylation26 of 12 with the alkyne 13,27 which has
a
c-lactone moiety, proceeded smoothly to give propargyl
alcohol 14 in good yield with high diastereoselectivity (95:5).
Hydrogenation of 14 with Pd–C in EtOAc afforded a saturated
alcohol 15 in 64% yield. Oxidation of 15 followed by thermal
elimination of the resulting sulfoxide afforded a crude product
including alcohol 16. Deprotection of TBS ether was proceeded
by purification of the reaction mixture by column chromatogra-
phy over silica gel giving C34-epi-solamin 4.
Figure 2. C35-fluorinated solamins and their epimer.
it means that the c-lactone moiety is strictly recognized by the en-
zyme, and hence a critical structural factor required for the inhibi-
tion, but we did not examine the inhibition of complex I in that
report. To further elucidate the inhibitory action of acetogenins,
we here synthesized C34-epi- and C34-epi-C35-trifluorosolamins,
and determined the inhibitory activity of all fluorinated analogues,
C34-epi-solamin, and solamin against bovine heart mitochondrial
complex I.
We determined the inhibitory activity of all fluorinated ana-
logues (1–3, 5), C34-epi-solamin 4, and solamin against complex
I using bovine heart submitochondrial particles (Table 1). The
activity decreased in the following order: C35-fluoro > C35-difluor-
o > C35-trifluorosolamin. This tendency is consistent with that of
the growth inhibitory activity against human cancer cell lines.20
A structural ‘bulkiness’ of fluorine, which often replaces hydrogen
in organic molecules but the size and stereoelectronic influences of
the two atoms are quite different,28,29 may disturb the intermolec-
To clarify the effect of stereochemistry of the methyl group in
the c
-lactone on the biological activity,22 we planned the synthesis
of C34-epi-solamin 4 and C34-epi-C35-trifluorosolamin 5. Deriva-
tive 5 was synthesized using the same procedure as that for C35-
trifluorosolamin 3,20 as shown in Scheme 1. A commercially avail-
able (S)-3,3,3-trifluoro-1,2-epoxypropane 6 was converted to io-
dide 7 by reported procedures. The Sonogashira coupling of 7
and the THF-ring fragment 8 followed by hydrogenation with
Wilkinson’s catalyst gave sulfide 10. Oxidation of 10, followed by
ular interaction between the c-lactone ring and the enzyme. It is
worth noting that both C34-epi- and C34-epi-C35-trifluorosola-
mins remarkably lost the activity; the loss due to epimerization
was greater for the former. These results unambiguously indicate
that both the bulkiness and the stereochemistry of C34–CH3 are
very important structural factors for interacting with the enzyme:
in other words, the
enzyme.
c-lactone moiety is strictly recognized by the
thermal elimination of the resulting sulfoxide, afforded
a,b-unsat-
urated -lactone 11. The synthesis of C34-epi-C35-trifluorosolamin
5 was completed via deprotection of TBS ether with acidic
c
The present results are inconsistent with our previous work. We
had performed a structure–activity relationship study concerning
conditions.
the
c-lactone moiety using bis-THF acetogenin derivatives (not
We previously reported the total synthesis of solamin by
mono-THF derivatives), indicating that a natural
c
-lactone ring it-
asymmetric alkynylation of
a-tetrahydrofuranyl aldehyde 12,
self is not crucial for the activity and can be substituted with other
which was stereoselectively synthesized by our method,23,24 with
structures.6 We concluded therefore that the enzyme might not
Scheme 1. Reagents and conditions: (a) Pd(PPh3)2Cl2, CuI, Et3N, rt, 87%; (b) Rh(PPh3)3Cl, H2 (1 atm), benzene–MeOH (1:1), rt, 55%; (c) mCPBA, CH2Cl2, 0 °C; (d) toluene, 60–
65 °C, 57% in two steps; (e) 48% HF aq, MeCN–THF (1:2), rt, 93%.
Scheme 2. Reagents and conditions: (a) Zn(OTf)2, (1R,2S)-N-methylephedrine, i-Pr2NEt, toluene, rt, 81%, dr = 95:5; (b) Pd–C, H2 (3 atm), EtOAc, rt, 64%; (c) mCPBA, CH2Cl2,
0 °C; (d) toluene, 80 °C then silicagel column chromatography, 68% in two steps.

N. Kojima et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1217–1219
1219
Table 1
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6. Takada, M.; Kuwabara, K.; Nakato, H.; Tanaka, A.; Iwamura, H.; Miyoshi, H.
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Inhibition of bovine heart mitochondrial complex I by solamin analogues
Compounds
IC50 (nM)
Natural solamin (C35-CH3)
C35-Fluorosolamin 1
C35-Difluorosolamin 2
C35-Trifluorosolamin 3
C34-epi-Solamin 4
2.1 0.20
2.9 0.22
31 1.9
310 28
270 20
570 49
C34-epi-C35-Trifluorosolamin 5
8. Torm, J. R.; Estornell, E.; Gallardo, T.; Gonzalez, M. C.; Cave, A.; Granell, S.;
Cortes, D.; Zafra-Polo, M. C. Bioorg. Med. Chem. Lett. 2001, 11, 681.
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The IC50 values, which is the molar concentration (nM) needed to reduce the control
NADH oxidase activity (0.63–0.75 mol NADH/min/mg of protein) in bovine heart
submitochondrial particles by half. Values are means SD of three independent
experiments.
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Nishioka, T.; Miyoshi, H. Biochemistry 2008, 47, 6260.
20. Kojima, N.; Suga, Y.; Hayashi, H.; Yamori, T.; Yoshimitsu, T.; Tanaka, T. Bioorg.
Med. Chem. Lett. 2011, 21, 5745.
21. For a recent review of the synthesis of acetogenin analogues, see: Kojima, N.;
Tanaka, T. Molecules 2009, 14, 3621.
22. Brown and co-workers reported the total synthesis of cis-solamin and their
C35-methyl group epimer, and their cytotoxicity against B16F10 murine
melanoma cell line: Cecil, A. R. L.; Hu, Y.; Vicent, M. J.; Duncan, R.; Brown, R. C.
D. J. Org. Chem. 2004, 69, 3368.
23. For a systematic synthesis of poly-THF cores, see: (a) Maezaki, N.; Kojima, N.;
Tanaka, T. Synlett 2006, 993; (b) Kojima, N. Yakugaku Zasshi 2004, 124, 673; (c)
Kojima, N.; Maezaki, N.; Tominaga, H.; Yanai, M.; Urabe, D.; Tanaka, T. Chem.
Eur. J. 2004, 10, 672; (d) Kojima, N.; Maezaki, N.; Tominaga, H.; Asai, M.; Yanai,
M.; Tanaka, T. Chem. Eur. J. 2003, 9, 4980; (e) Maezaki, N.; Kojima, N.;
Tominaga, H.; Yanai, M.; Tanaka, T. Org. Lett. 2003, 5, 1411; (f) Maezaki, N.;
Kojima, N.; Asai, M.; Tominaga, H.; Tanaka, T. Org. Lett. 2002, 4, 2977.
24. For the application of our developed methodology to total synthesis of
annonaceous acetogenins and their analogues, see: (a) Kojima, N.; Morioka, T.;
Yano, M.; Suga, Y.; Maezaki, N.; Tanaka, T. Heterocycles 2009, 79, 387; (b)
Kojima, N.; Hayashi, H.; Suzuki, S.; Tominaga, H.; Maezaki, N.; Tanaka, T.;
Yamori, T. Bioorg. Med. Chem. Lett. 2008, 18, 6451; (c) Kojima, N.; Fushimi, T.;
Maezaki, N.; Tanaka, T.; Yamori, T. Bioorg. Med. Chem. Lett. 2008, 18, 1637; (d)
Maezaki, N.; Urabe, D.; Yano, M.; Tominaga, H.; Morioka, T.; Kojima, N.; Tanaka,
T. Heterocycles 2007, 73, 159; (e) Tominaga, H.; Maezaki, N.; Yanai, M.; Kojima,
N.; Urabe, D.; Ueki, R.; Tanaka, T. Eur. J. Org. Chem. 2006, 1422; (f) Maezaki, N.;
Tominaga, H.; Kojima, N.; Yanai, M.; Urabe, D.; Ueki, R.; Tanaka, T.; Yamori, T.
Chem. Eur. J. 2005, 11, 6237; (g) Maezaki, N.; Tominaga, H.; Kojima, N.; Yanai,
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recognize this moiety in a strict sense. To further elucidate the
inhibitory mechanism of acetogenins as well as important struc-
tural factors required for the inhibitory action, a discussion of pos-
sible causes of the discrepancy is needed.
There is a point to be made before the discrepancy is discussed
however. We produced
c
‘D
lac-acetogenins’ by deleting the
-lactone ring from natural bis-THF acetogenins.30–32 It is worth
noting that
Dlac-acetogenins elicit an inhibitory effect on complex
I as strongly as natural acetogenins do, whereas the binding site of
the inhibitors differs from that of natural acetogenins;31,32 in other
words, deletion of the
to a different type of complex I inhibitor. By contrast, deletion of
the -lactone ring from mono-THF acetogenins results in an almost
complete loss of the activity.30 Thus,
lac-acetogenin-like inhibi-
c-lactone ring converts natural acetogenins
c
D
tory behavior occurs only in the case of bis-THF derivatives. This
complicates the profile of structure–activity relationship for natu-
ral bis-THF acetogenins: a decrease in the inhibitory activity due to
structural modifications of the
c-lactone moiety is apparently
masked by the inhibitory activity elicited as
Dlac-acetogenin since
the two separate events cannot be distinguished.33 To overcome
this problem and to examine the effects of structural modifications
of the
togenins are better than bis-THF derivatives as control compounds.
Altogether, the present study unambiguously indicates that the
c-lactone moiety on the inhibitory activity, mono-THF ace-
c-
lactone moiety, which binds to the region spanning the fourth to
fifth transmembrane helices (Val144-Glu192) in the ND1 subunit
of bovine complex I,34 is strictly recognized by the enzyme, and
hence a critical structural factor of natural acetogenins required
for the potent inhibition.
Acknowledgments
This study was supported in part by a Grant-in-Aid for Young
Scientists (B) [Grant 23790130 to N.K.], a Grant-in-Aid for Scientific
Research (B) [Grant 22390021 to T.T., Grant 23380064 to H.M.]
from the Japan Society for the Promotion of Sciences (JSPS), and
the MEXT (the Ministry of Education, Culture, Sports, Science and
Technology, Japan)-Supported Program for the Strategic Research
Foundation at Private Universities, 2008–2012. We are indebted
to Ms. Natsuki Sawada of our laboratory for her experimental
contributions.
25. Kojima, N.; Morioka, T.; Urabe, D.; Yano, M.; Suga, Y.; Maezaki, N.; Ohashi-
Kobayashi, A.; Fujimoto, Y.; Maeda, M.; Yamori, T.; Yoshimitsu, T.; Tanaka, T.
Bioorg. Med. Chem. 2010, 18, 8630.
26. Frantz, D. E.; Fässler, R.; Carreira, E. M. J. Am. Chem. Soc. 2000, 122, 1806.
27. The alkyne 13 was prepared using the same procedure as that for their
enantiomer in Ref. 25.
28. Müller, K.; Faeh, C.; Diederich, F. Science 1881, 2007, 317.
29. Hagmann, W. K. J. Med. Chem. 2008, 51, 4359.
30. Ichimaru, N.; Murai, M.; Abe, M.; Hamada, T.; Yamada, Y.; Makino, S.; Nishioka,
T.; Makabe, H.; Makino, A.; Kobayashi, T.; Miyoshi, H. Biochemistry 2005, 44,
816.
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