Synthesis and mitochondrial complex I inhibition of dihydroxy-cohibin A, non-THF annonaceous acetogenin analogue

Article abstract of DOI:10.1016/j.bmcl.2003.11.057

To elucidate the inhibitory action of acetogenins, we synthesized an acetogenin derivative which possesses tetraol in place of the tetrahydrofuran ring and examined its inhibitory activity against bovine heart mitochondrial complex I. Our results indicate that these hydroxy groups are an essential structural factor though it is not effective as bis-THF hydroxy groups combination.

Full text of DOI:10.1016/j.bmcl.2003.11.057

Bioorganic & Medicinal Chemistry Letters 14 (2004) 629–632
Synthesis and mitochondrial complex I inhibition of
dihydroxy-cohibin A, non-THF annonaceous
acetogenin analogue
Hiroyuki Konno,a,* Naoki Hiura,a Hidefumi Makabe,b Masato Abec
and Hideto Miyoshic,*
aDepartment of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minamijosanjima-cho,
Tokushima 770-8506, Japan
bIntegrated Department of Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, 8304 Minamiminowa,
Kamiina, Nagano 399-4598, Japan
cDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received 3 October 2003; accepted 18 November 2003
Abstract—To elucidate the inhibitory action of acetogenins, we synthesized an acetogenin derivative which possesses tetraol in
place of the tetrahydrofuran ring and examined its inhibitory activity against bovine heart mitochondrial complex I. Our results
indicate that these hydroxy groups are an essential structural factor though it is not effective as bis-THF hydroxy groups combi-
nation.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
In previous structure–activity relationships using nat-
Among the rapidly growing family of the Annonaceous
acetogenin,1,2 these are endemic to certain plants of the
Annonaceae and have been shown to possess a broad
spectrum of biological activities involving cytotoxic, anti-
tumor, pesticidal, antifeedant, and immunosuppressive
properties. Although action of their compounds is
thought the inhibition of the complex I (NADH-ubiqui-
none oxidoreductase) in mammalian and insect mito-
chondrial electron transport systems3,4 and of the NADH
oxidase found in the plasma membranes of cancer cells,5,6
there is still no hard experimental evidence to verify
whether the inhibitors bind to the ubiquinone reduction
site. Although bullatacin (2),4 bis-THF acetogenin iso-
lated from Annona bullata, is actually one of the most
potent inhibitors of this enzyme identified to date, there
are few structural similarities between the acetogenin
and ordinary complex I inhibitors such as piericidin A
and rotenone. Thus, studying their essential structural
sites as well as inhibitory property of acetogenin, struc-
tural–activity relationshipis important to elucidate the
terminal electron transfer system of complex I.
ural and synthetic acetogenin, these are shown follow-
ing: (i) acetogenin acts as a potent inhibitor only when
the g-lactone and THF ring moieties are directly linked
by an alkyl spacer (13 carbon atoms is better); (ii)
number of THF rings, the presence of polar functional
groups such as hydroxy group in the spacer, and ste-
reochemistry around the THF are not essential struc-
tural factors for the potent activity.4 On the other hand,
Shimada et al. proposed a model of active conformation
of acetogenins, wherein THF ring with flanking
hydroxy groups work as hydrophilic anchor in mito-
chondrial membrane (Fig. 1).7 There are few studies of
structural simplification of THF ring based on natural
acetogenin without catechol and ether linked aceto-
genins.6 Taking into consideration the fact that hydro-
philicity of the place of THF site is essential, this finding
is very interesting to elucidate the mode of action of
acetongenin.
In the present study, we synthesized an acetogenin analo-
gue which possesses tetraol in place of the THF ring,
named dihydroxy-cohibin A (1), non-THF acetogenin
(Fig. 2). There are minor groupof non-THF ring
compounds which can be assumed to be degradation
products and intermediary precursors in the biosynth-
esis onto acetogenins. Cohibin A has been isolated by
Keywords: Annonaceous acetogenin; Biological activity; Complex I
inhibitor; Stereoselective synthesis; Aetogenin analogue.
* Corresponding author. Tel.: +81-88-656-9213; fax: +81-88-656-
9213; e-mail: konno@bio.tokushima-u.ac.jp
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.057

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H. Konno et al. / Bioorg. Med. Chem. Lett. 14 (2004) 629–632
Figure 1. Model of the active conformation of acetogenins interacting with complex I in the mitochondrial membrane proposed by Shimada et al.7
catacin 3. After protecting the hydroxy group of 3 as a
MOM ether, the partial reduction of the resulting lac-
tone with DIBAL afforded acetal, which was then sub-
mitted to a careful Horner–Emmons reaction at ꢀ78 ꢁC
to give the chain-extented a,b-E-unsaturated ester 4.
Protection of the hydroxy groupof 4 as an EE ether and
subsequent Sharpless asymmetric dihydroxylation using
AD-mix b furnished dihydroxy ester, which, after pro-
tecting the hydroxy groupwith ethyl vinyl ether, was
reduced with LiAlH4 to yield 5. A three stepsequence of
reactions involving treatment with p-TsCl, hydrolysis of
the EE groupand oxirane ring formation with KOH
provided dihydroxy epoxide 6, which was proved to
have a 92% diastereomeric excess by 1H NMR analysis.
Fortunately, the undesired diastereomer could be
removed from 6 by column chromatography. Protection
of the hydroxy groupof 6 as a MOM ether, a coupling
reaction with lithum TMS-acetylide in the presence of
.
BF3 Et2O and deprotection of TMS group with KF
afforded 7. The g-lactone moiety 8 was prepared by
application of the method that had been reported earlier
by our group.12 A Pd-mediated cross-coupling reaction
between 7 and 8 with Pd(PPh3)4, CuI, and pyrrolidine
without any solvent yielded enyne 9.13 Catalytic hydro-
genation of 9 with Wilkinson’s catalyst gave 10.
Sequential thermal elimination of the sulfide moiety
were performed according to the reported method to
afford 11. Finally deprotection of MOM group of 11
Figure 2. Structure of cohibin A, synthetic dihydroxy-cohibin A (1)
and bullatacin (2).
.
Cave groupfrom the roots of the tropical fruit, Annona
muricata.8 Dihydroxy-cohibin A (1), acetogenin analo-
gue that possessed dihydroxy function in the place of
olefin based on cohibin A, was designed to be increase
the hydrophilicity. Stereochemistry of dihydroxy-cohi-
bin A (1) was led to cohibin A and previous publica-
tions.9,10 The inhibitory action of dihydroxy-cohibin A
(1) and 11 was examined with bovine heart mitochon-
drial complex I.
with BF3 Et2O in Me2S gave dihydroxy-cohibin A (1).
3. Bioactivity
The inhibition of complex I activity was determined by
NADH oxidase assay using bovine heart sub-
mitochondrial particles.14 The potency of bullatacin (2),
one of the most potent natural acetogenins, in terms of
IC50 value was 0.8 nM in the present study. Under the
same experimental conditions, IC50 of 1 and 11 were 20
and 4100 nM, respectively. Thus, free hydroxy groups
between the spacer and the tail are essentially important
for the activity though it is not effective as bis-THF
hydroxy groups combination. This was supported by
the marked loss of inhibitory activity of 11, which has
lonely free hydroxy groupwith tri-MOM rpotected
hydroxy groups located in the middle of the inhibitor.
2. Chemistry
The synthetic procedures are outlined in Scheme 1. The
key intermeditate 6 of enantiomer form was synthesized
earlier by us9 and all stereoisomers of muricatacin 3
could be easily obtained in an enantiomerically pure
form.11 We commenced synthesis using (+)-muri-

H. Konno et al. / Bioorg. Med. Chem. Lett. 14 (2004) 629–632
631
Scheme 1. (a) (i) MOMCl, iPr2NEt, CH2Cl2, 65%; (ii) DIBAL-H, CH2Cl2, 95%; (iii) (EtO)2P(O)CH2CO2Et, NaH, THF, 75%; (b) (i)
EtOCH¼CH2, PPTS, CH2Cl2, 87%; (ii) AD-mix b, MeSO2NH2, tBuOH–H2O, 92%; (iii) EtOCH¼CH2, PPTS, CH2Cl2, 81%; (iv) LiAlH4, THF,
.
99%; (c) (i) pTsCl, pyridine; (ii) PPTS, MeOH; (iii) KOH, 50%; (d) (i) MOMCl, iPr2NEt, CH2Cl2; (ii) trimethylsilyl acetylene, nBuLi, BF3 Et2O,
THF; (iii) KF, MeOH, 15%; (e) Pd(PPh3)4, CuI, pyrrolidine, 58%; (f) H2, Rh(PPh3)3Cl, benzene, 97%; (g) (i) mCPBA, NaHCO3, CH2Cl2; (ii)
.
toluene reflux, 73%; (h) BF3 Et2O, Me2S, 56%.
4. Discussion
acetogenin. Taking into account our hypothesis the
synthesis and active conformation of complex I, but
simplified analogues design is underway.
The present study indicated that free hydroxy groups in
the place of THF ring are preferable for the activity but
is not effective as bullatacin type structure. The present
results do not support Shimada’s model from hydro-
philic property of tetraol unit and previous publica-
tions.4 If the hydrogen bond-donating ability of the
hydroxy groups as hydrophilic anchor at the liposomal
membrane surface is important for Complex I inhibi-
tion, potent activity of tetraol acetogenin would have
shown to the intermolecular hydrogen bonds between
the hydroxy groups and oxygen in phospholipid. Mole-
cular structure of hydrophilic site, gylcerol backbone of
the phospholipid, can be regarded high degree of flex-
ibility in the liposomal membrane. Structure of hydro-
philic anchor at the liposomal membrane surface also
can permit to be flexible structure. However bullatacin
type structure contained rigid bis THF unit remains to
be the most potent inhibitor. We propose that both g-
lactone ring and THF ring act in cooperative manner on
the enzyme with the support of some specific con-
formation of the spacer.15 Therefore, we guess that
bioactivity of dihydroxy-cohibin A (1) are diminish (20
times-fold) by the high degree of flexibility of the tetraol
unit and more rigid structure contained with free
hydroxy groups need to increase in activity. Thus, in
order to examine the potential of these compounds as
novel agents for pharmaceutical and agrochemical use,
it seems worthwhile to design and study more simple
analogue incorporating key elements of Annonaceous
Acknowledgements
We thank Prof. Hitoshi Hori (University of Tokush-
ima), and Prof. Hiromasa Kiyota (Tohoku University)
for useful discussion. This work was supported by the
Agricultural Chemical Research Foundation and Mit-
subishi Chemical Corporation Fund.
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