Function of the alkyl side chains of Δlac-acetogenins in the inhibitory effect on mitochondrial complex I (NADH-ubiquinone oxidoreductase)

Article abstract of DOI:10.1016/j.bmcl.2006.03.082

We synthesized a series of Δlac-acetogenins in which the two alkyl side chains were systematically modified, and examined their inhibitory effect on bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase). The results revealed that the physicochemical properties of the side chains, such as the balance of hydrophobicity and the width (or bulkiness) of the chains, are important structural factors for a potent inhibitory effect of amphiphilic Δlac-acetogenins. This is probably because such properties decide the precise location of the hydrophilic bis-THF ring moiety in the enzyme embedded in the inner mitochondrial membrane.

Full text of DOI:10.1016/j.bmcl.2006.03.082

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3555–3558
Function of the alkyl side chains of Dlac-acetogenins in the
inhibitory effect on mitochondrial complex I
(NADH-ubiquinone oxidoreductase)
Naoya Ichimaru, Masato Abe, Masatoshi Murai, Mai Senoh,
Takaaki Nishioka and Hideto Miyoshi*
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
Received 21 February 2006; revised 6 March 2006; accepted 24 March 2006
Available online 18 April 2006
Abstract—We synthesized a series of Dlac-acetogenins in which the two alkyl side chains were systematically modified, and exam-
ined their inhibitory effect on bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase). The results revealed that
the physicochemical properties of the side chains, such as the balance of hydrophobicity and the width (or bulkiness) of the chains,
are important structural factors for a potent inhibitory effect of amphiphilic Dlac-acetogenins. This is probably because such prop-
erties decide the precise location of the hydrophilic bis-THF ring moiety in the enzyme embedded in the inner mitochondrial
membrane.
Ó 2006 Elsevier Ltd. All rights reserved.
Acetogenins isolated from the plant family Annonaceae
have potent and diverse biological effects such as antitu-
mor, antimalarial, and insecticidal activities.1–3 Their
inhibitory effect on mitochondrial NADH-ubiquinone
oxidoreductase (complex I) is of particular importance
since the diverse biological activities of acetogenins are
thought to be attributable to this effect.2 Their unique
structural features and multiple chiral centers, especially
more than four in the THF portion, make acetogenins
challenging synthetic targets.4 On the other hand, struc-
tural simplification, while maintaining all the essential
functionalities of acetogenins may facilitate the task of
synthesizing a variety of acetogenin mimics which may
be used not only as possible chemotherapeutic agents,
but also as molecular probes to investigate the function-
al features of mitochondrial complex I,5,6 one of the
largest known membrane protein complexes.
a,b-unsaturated c-lactone ring.7,8 Some Dlac-acetoge-
nins had strong inhibitory effect on bovine heart mito-
chondrial complex I at the nanomolar level despite
lacking a c-lactone ring which is a structural feature
common to a large number of natural acetogenins.1–3
Interestingly, several lines of evidence, for example,
competition tests using a fluorescent ligand and the
effect on superoxide production from complex I, re-
vealed that the binding site of Dlac-acetogenins is differ-
ent from that of natural acetogenins as well as ordinary
complex I inhibitors such as rotenone and piericidin
A.7,8 Thus, Dlac-acetogenins were shown to be a new
type of inhibitor acting at the terminal electron transfer
step of complex I. Accordingly, a detailed analysis of
their inhibitory action would provide valuable insights
into the functional features of complex I.
To elucidate the mode of inhibition of Dlac-acetogenins,
identification of the crucial structural factors required
for the inhibition is highly helpful. A previous struc-
ture–activity study concerning Dlac-acetogenins indicat-
ed that the number of carbon atoms (i.e.,
hydrophobicity) of the alkyl side chains remarkably
affects the inhibitory potency (see compounds 1–4,
Fig. 1).7 The optimal length of the side chains for the
inhibition is about 11 carbons, as revealed for 3. A com-
parison of the inhibitory effects of compounds 3 and 5–7
We recently synthesized new acetogenin mimics named
Dlac-acetogenins, that consist of the hydroxylated adja-
cent bis-THF ring and two alkyl side chains without a
Keywords: Complex I; Respiratory inhibitor; Acetogenin; Structure–
activity relationship.
*
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 Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.082

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N. Ichimaru et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3555–3558
Figure 1. Structures of Dlac-acetogenin derivatives examined in this study. The molar concentrations in parentheses are the IC50 values determined in
the previous study.7
suggested that the balance of hydrophobicity of the two
side chains attached to the C2-symmetric bis-THF por-
tion is an important structural factor of these inhibitors.
However, since compounds 3 and 5–7 largely differ in
the total number of carbon atoms, it remains to be
established whether the difference in inhibitory effect is
actually due to an imbalance of hydrophobicity or a dif-
ference in the hydrophobicity of the entire molecule. The
solution of this question is unavoidable not only to
know the function of the side chains, but also to predict
the function and location of the hydrophilic bis-THF
ring moiety. Therefore, to solve this important question,
we newly synthesized compounds 8 and 9 that possess
two side chains of different lengths, but maintain a total
of 22 carbons as is the case of a potent derivative 3. In
addition, to further elucidate the function of the side
chains, we also synthesized a series of derivatives and
evaluated their inhibitory effects on bovine heart mito-
chondrial complex I.
To synthesize 12 and 13, we initially examined the
simultaneous opening of the diepoxide i-89 with the
corresponding lithium acetylide in the presence of
Et2AlCl, as shown in Scheme 1B, but the yield of
the product was very poor. We attempted to improve
this reaction step under various conditions; for
instance, BF3ÆEt2O14 was used in place of Et2AlCl at
several molar ratios and temperatures. All cases, how-
ever, resulted in unsatisfactory results. Therefore, we
synthesized 12 and 13 by the same procedures used
for 9.12 For the synthesis of 13, 3-ethyl-1-heptyne
was prepared by a Corey–Fuchs reaction from com-
mercially available 2-ethyl-1-hexanal.
The inhibition of complex I activity was determined
by NADH oxidase assay using bovine heart submito-
chondrial particles.8 Previous study indicated that the
inhibitory effect of 3 (C11–C11) is comparable to that
of bullatacin,7 one of the most potent natural acetog-
enins.15,16 The IC50, that is, the molar concentration
needed to halve the control NADH oxidase activity,
of 3 was 1.7 ( 0.08) nM with the present submito-
chondrial particles preparation. The IC50 values of
compounds 8 and 9 were 7.5 ( 0.39) and 34 ( 6.6)
nM, respectively. Taking into account the identical
total number of carbon atoms on the side chains of
3 (C11–C11), 8 (C13–C9), and 9 (C15–C7), it is obvious
that the greater the loss of the balance, the weaker
the inhibitory effect becomes. Similar results were
obtained for other compound sets, 10 (C15–C2,
IC50 = 410 23 nM) versus 5 (C11–C2, IC50 = 280
30 nM) and 9 (C15–C7, IC50 = 34 6.6 nM) versus 7
(C11–C7, IC50 = 3.2 0.30 nM), though the total num-
ber of carbons of the former is larger than that of
the latter in these pairs. These results indicate that
the balance in hydrophobicity of the two side chains
is an important structural factor for a potent inhibi-
tory effect. It is thus likely that the balance decides
the precise location of the hydrophilic bis-THF ring
moiety, which may be located at or close to the
membrane surface area of the enzyme embedded in
the inner mitochondrial membrane.
The synthetic procedures of compounds 8–11 are out-
lined in Scheme 1A, taking 9 as an example. The key
intermediate i-1 was synthesized as described previous-
ly.9,10 Treatment of i-1 with 0.5 mol equiv of TsCl
(repeated twice) and sequential MOM ether protection
of the secondary hydroxy group afforded i-2. Desilyla-
tion of i-2 with TBAF, and the opening of the epoxide
i-3 with the lithium acetylide, derived from 1-tridecyne,
in the presence of Et2AlCl11 provided i-4 (72% in two
steps). After acetyl protection of both hydroxy groups,
deprotection of the MOM ether and sequential treat-
ment with TsCl afforded i-5. Hydrolysis of the acetyl
groups gave epoxide i-6 (80%). The opening of the epox-
ide i-6 with the lithium acetylide, derived from 1-pen-
tyne, in the presence of Et2AlCl provided i-7 (78%).
Hydrogenation of i-7 with 10% Pd/C afforded 9.12 To
obtain compound 10, the final mono-epoxide i-6 was
opened by catalytic hydrogenation with 10% Pd/C.12
For the synthesis of 11, 3-n-propyl-1-hexyne was synthe-
sized by a Corey–Fuchs reaction13 from 2-n-propyl-1-
pentanal, which was prepared by Swern oxidation of
commercially available 2-n-propyl-1-pentanol.

N. Ichimaru et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3555–3558
3557
Scheme 1. Reagents and conditions: (a) i—TsCl (0.5 equiv), 4-DMAP, Et3N, CH2Cl2, rt, 12 h, 55% (repeated twice); ii—MOMCl, (i-Pr)2NEt,
CH2Cl2, rt, 15 h, 98%; (b) TBAF, THF, 50 °C, 2 h, 88%; (c) 1-tridecyne (10 equiv), n-BuLi (10 equiv), Et2AlCl (10 equiv), toluene, 0 °C, 1 h, 82%; (d)
i—AcCl, 4-DMAP, CH2Cl2, 35 °C, 2 h, 96%; ii—BF3ÆEt2O, Me2S, ꢀ20 °C, 1 h, 96%; iii—TsCl, 4-DMAP, Et3N, CH2Cl2, 35 °C, 12 h, 92%; (e)
K2CO3, MeOH, rt, 2 h, 80%; (f) 1-pentyne (10 equiv), n-BuLi (10 equiv), Et2AlCl (10 equiv), toluene, 0 °C, 1 h, 78%; (g) H2, 10% Pd/C, EtOH, rt,
12 h, 94%.
Next, to examine the effect of the shape of the side chain
on the activity, we synthesized compound 11 possessing
a branched side chain of 11 carbons, which is one of the
bulkiest structures that can be synthesized by the proce-
dures used for the preceding analogues. The total num-
ber of carbon atoms of the side chains in this derivative
is identical to that of 3. The IC50 value of 11 was 27
( 2.1) nM, indicating that the branched structure is
unfavorable for the inhibition. The replacement of both
side chains of 3 with the branched chain of 11 carbons
is not available, this feature may be closely related to
the specific nature of the putative binding site in com-
plex I embedded in the inner mitochondrial membrane.
Acknowledgment
This work was supported in part by a Grant-in-Aid for
Scientific Research from the Japan Society for the
Promotion of Science (Grant No. 17380073 to H.M.).
resulted in
a
drastic decrease in activity [12,
IC50 = 1500 190 nM]. On the basis of this result, we
next synthesized compound 13 possessing an ethyl
branch in both chains in anticipation of the recovery
of inhibitory potency. Expectedly, 13 (IC50 = 870
20 nM) significantly recovered the inhibitory effect com-
pared to 12. It is therefore clear that expansion of the
width of the side chain is remarkably unfavorable for
the inhibitory action. This is probably because the side
chains directly interact with the hydrophobic domain
of complex I rather than merely partitioning into the
lipid membrane phase, whereupon the enzyme recogniz-
es the molecular shape of the side chains in a strict sense.
References and notes
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In conclusion, the precise location of the hydrophilic
bis-THF ring moiety, which is decided by the physico-
chemical properties of the side chains, is critical to the
inhibitory effect of amphiphilic Dlac-acetogenins.
Although a high-resolution 3D structure for complex I

3558
N. Ichimaru et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3555–3558
9, 282; (d) Huang, G.-R.; Jiang, S.; Wu, Y.-L.; Jin, Y.;
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3H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d 84.44, 83.10, 81.75, 81.72, 73.97, 70.41, 33.42,
31.86, 29.68 (2C), 29.63 (2C), 29.58 (2C), 29.55 (2C),
29.30, 28.95, 28.92, 28.30, 28.26, 25.61, 22.63, 18.60, 14.06;
ESI-MS (m/z) 413.4 [M+H]+. For compound 11: colorless
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19
oil; ½aꢁD +28.3 (c 0.12, EtOH); 1H NMR (400 MHz,
CDCl3) d 3.90–3.81 (m, 4H), 3.41–3.37 (m, 2H), 2.44 (br s,
2H), 1.98–1.95 (m, 4H), 1.72–1.61 (m, 4H), 1.54–1.47 (m,
2H), 1.45–1.36 (m, 4H), 1.39–1.37 (m, 1H), 1.33–1.16 (m,
26H), 0.88 (t, J = 6.8 Hz, 3H), 0.87 (t, J = 7.0 Hz, 6H); 13
C
NMR (100 MHz, CDCl3) d 83.18, 83.15, 81.78 (2C), 74.12,
74.07, 36.99, 36.06, 36.01, 33.95, 33.79, 33.49, 31.92 (2C),
29.74, 29.63 (2C), 29.34, 28.97 (2C), 28.37 (2C), 25.67,
22.79, 22.69, 19.82, 19.79, 14.53 (2C), 14.12; ESI-MS (m/z)
483.6 [M+H]+. For compound 12: colorless oil;
19
½aꢁD +27.0 (c 0.20, EtOH); 1H NMR (400 MHz, CDCl3)
d 3.90–3.80 (m, 4H), 3.42–3.36 (m, 2H), 2.52 (br s,
2H), 2.02–1.90 (m, 4H), 1.72–1.60 (m, 4H), 1.54–1.46
(m, 2H), 1.45–1.18 (m, 28H), 0.87 (t, J = 7.0 Hz, 12H);
13C NMR (100 MHz, CDCl3) d 83.20 (2C), 81.79 (2C),
74.10 (2C), 36.98 (2C), 36.05 (2C), 36.00 (2C), 33.94
(2C), 33.78 (2C), 28.99 (2C), 28.37 (2C), 22.79 (2C),
19.81 (2C), 19.78 (2C), 14.54 (4C); ESI-MS (m/z) 483.4
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18
12. The data for compound 8: colorless oil; ½aꢁD +1.3 (c 0.15,
EtOH); 1H NMR (400 MHz, CDCl3) d 3.89–3.81 (m, 4H),
3.39–3.38 (m, 2H), 2.47 (br s, 2H), 1.99–1.94 (m, 4H),
1.69–1.63 (m, 4H), 1.57–1.48 (m, 2H), 1.43–1.25 (m, 34H),
0.87 (t, J = 6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) d
83.14 (2C), 81.77 (2C), 74.06 (2C), 33.49 (2C), 31.93,
31.89, 29.74 (2C), 29.67, 29.65, 29.62, 29.57, 29.36 (2C),
29.30 (2C), 28.97 (2C), 28.37 (2C), 25.67 (2C), 22.69,
22.68, 14.12 (2C); ESI-MS (m/z) 483.4 [M+H]+. For
20
[M+H]+. For compound 13: colorless oil; ½aꢁD +25.2 (c
0.19, EtOH); 1H NMR (400 MHz, CDCl3) d 3.94–3.90
(m, 4H), 3.44–3.35 (m, 2H), 2.53 (br s, 2H), 2.02–1.91
(m, 4H), 1.78–1.60 (m, 4H), 1.58–1.51 (m, 2H), 1.51–
1.10 (m, 28H), 0.88 (t, J = 6.9 Hz, 6H), 0.83 (t,
J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3) d 83.19
(2C), 81.79 (2C), 74.09 (2C), 38.87, 38.83, 33.93 (2C),
33.32 (2C), 32.87, 32.83, 29.00 (2C), 28.99 (2C), 28.37
(2C), 25.86, 25.79, 23.15 (2C), 22.84 (2C), 14.18 (2C),
10.89, 10.84; ESI-MS (m/z) 483.6 [M+H]+.
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18
1
compound 9: colorless oil; ½aꢁD +10.9 (c 0.11, EtOH); H
NMR (400 MHz, CDCl3) d 3.90–3.81 (m, 4H), 3.41–3.36
(m, 2H), 2.44 (br s, 2H), 1.99–1.94 (m, 4H), 1.70–1.63 (m,
4H), 1.58–1.47 (m, 2H), 1.41–1.25 (m, 34H), 0.87 (t,
J = 6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) d 83.14
(2C), 81.77 (2C), 74.06 (2C), 33.49 (2C), 31.93 (2C), 31.82
(2C), 29.70 (2C), 29.40 (2C), 29.36 (2C), 28.97 (2C), 28.36
(2C), 25.67 (2C), 25.62 (2C), 22.70, 22.63, 14.12, 14.09;
ESI-MS (m/z) 483.6 [M+H]+. For compound 10: colorless
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19
oil; ½aꢁD +15.4 (c 0.13, EtOH); 1H NMR (400 MHz,
CDCl3) d 3.89–3.81 (m, 3H), 3.81–3.75 (dt, J = 6.4, 7.5 Hz,
1H), 3.61–3.54 (dt, J = 6.7, 13.4 Hz, 1H), 3.40–3.38 (m,
1H), 2.80 (br s, 1H), 2.62 (br s, 1H), 2.00–1.94 (m, 4H),
1.67–1.47 (m, 4H), 1.41–1.25 (m, 26H), 1.13 (d, J = 6.3 Hz,