Bongkrekic Acid Analogue, Lacking One of the Carboxylic Groups of its Parent Compound, Shows Moderate but pH-insensitive Inhibitory Effects on the Mitochondrial ADP/ATP Carrier

Article abstract of DOI:10.1111/cbdd.12594

Bongkrekic acid, isolated from Burkholderia cocovenenans, is known to specifically inhibit the mitochondrial ADP/ATP carrier. However, the manner of its interaction with the carrier remains elusive. In this study, we tested the inhibitory effects of 17 bo

Full text of DOI:10.1111/cbdd.12594

Chem Biol Drug Des 2015; 86: 1304–1322
Research Article
Bongkrekic Acid Analogue, Lacking One of the
Carboxylic Groups of its Parent Compound, Shows
Moderate but pH-insensitive Inhibitory Effects on the
Mitochondrial ADP/ATP Carrier
1
2
Atsushi Yamamoto , Keisuke Hasui ,
acid for its pH-dependent action. A direct inhibitory
effect of KH-7 on the mitochondrial ADP/ATP carrier
was also clearly demonstrated.
2
3
Hiroshi Matsuo , Katsuhiro Okuda ,
Masato Abe , Kenji Matsumoto , Kazuki
Harada , Yuya Yoshimura , Takenori
Yamamoto , Kazuto Ohkura ,
3
3
4
,5
4,5
4
,5
1
Key words: ADP/ATP carrier, bongkrekic acid, mitochondria,
mitochondrial solute carrier (SLC25a)
3
,
4,5,
Mitsuru Shindo * and Yasuo Shinohara
*
Abbreviations: BKA, bongkrekic acid; CATR, carboxyatracty-
loside.
1
Faculty of Pharmaceutical Sciences, Suzuka University of
Medical Science, Minamitamagakicho-3500, Suzuka, Mie
5
13-8670, Japan
Interdisciplinary Graduate School of Engineering
Received 12 April 2015, revised 18 May 2015 and accepted
for publication 27 May 2015
2
Sciences, Kyushu University, 6–1 Kasuga-koen, Kasuga
8
16-8580, Japan
3
Institute for Materials Chemistry and Engineering, Kyushu
University, Kasugakoen-6, Kasuga, Fukuoka 816-8580,
Japan
Most of the cellular ATP is synthesized by the process of
oxidative phosphorylation in the mitochondria. During this
process, energy of nutrient molecules is first converted into
4
Institute for Genome Research, Tokushima University,
Kuramotocho-3, Tokushima 770-8503, Japan
+
an electrochemical gradient of H across the inner mito-
5
Faculty of Pharmaceutical Sciences, Tokushima
University, Shomachi-1, Tokushima 770-8505, Japan
chondrial membrane. Then, using the electrochemical gra-
+
dient of H across this membrane as a driving force, ATP
*Corresponding authors: Mitsuru Shindo,
is synthesized by F F -ATP synthase. Therefore, to enable
0
1
shindo@cm.kyushu-u.ac.jp; Yasuo Shinohara,
yshinoha@genome.tokushima-u.ac.jp
effective energy conversion, permeability of the inner mito-
chondrial membrane must be kept very low. However, var-
ious molecules such as those involved in the process of
ATP synthesis or in the TCA cycle or b-oxidation must be
conveyed across the inner mitochondrial membrane. The
transport of various metabolites and ions across this inner
membrane is known to be catalyzed by transporters spe-
cific for each individual metabolite. These transporters are
thought to have arisen from a common ancestral gene,
because they show structural similarities such as 6-times
membrane spanning topology, and so they are referred to
as the mitochondrial solute carrier family, SLC25a.
Bongkrekic acid, isolated from Burkholderia cocove-
nenans, is known to specifically inhibit the mitochon-
drial ADP/ATP carrier. However, the manner of its
interaction with the carrier remains elusive. In this
study, we tested the inhibitory effects of 17 bongkrekic
acid analogues, derived from the intermediates
obtained during its total synthesis, on the mitochon-
drial ATP/ATP carrier. Rough screening of these chem-
icals, performed by measuring their inhibitory effects
on the mitochondrial ATP synthesis, revealed that 4 of
them, KH-1, KH-7, KH-16, and KH-17, had moderate
inhibitory effects. Further characterization of the
actions of these 4 analogues on mitochondrial function
showed that KH-16 had moderate; KH-1 and KH-17,
weak; and KH-7, negligible side effects of both per-
meabilization of the mitochondrial inner membrane and
inhibition of the electron transport, indicating that only
KH-7 had a specific inhibitory effect on the mitochon-
drial ADP/ATP carrier. Although the parental bon-
gkrekic acid showed a strong pH dependency of its
action, the inhibitory effect of KH-7 was almost insen-
sitive to the pH of the reaction medium, indicating the
importance of the 3 carboxyl groups of bongkrekic
The ADP/ATP carrier has been the most extensively stud-
ied member of this solute carrier family [for recent review,
see refs. (1–4)]. It was identified in 1964/65 (5–7), and its
primary structure was determined in 1982 (8). Two dec-
ades later, in 2002, its crystal structure was revealed (9).
Despite extensive studies, the catalytic mechanism of the
ADP/ATP carrier still remains elusive. It is thought that the
ADP/ATP carrier catalyzes the transports of ADP and ATP
by changing its conformation between that facing the
cytosolic side (c-state) and that facing the matrix side (m-
state). Key molecules for understanding the catalytic
1
304
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12594

pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier
A
mechanism of this carrier are two specific inhibitors of it,
that is, carboxyatractyloside (CATR) and bongkrekic acid
B
(BKA), isolated as toxins from Atractylis gummifera and
Burkholderia cocovenenans, respectively (10). These two
inhibitors bind to the ADP/ATP carrier from the cytosolic
side and matrix side, respectively, and fix the carrier in the
c-state and m-state, respectively. Therefore, studies on
the interaction between the ADP/ATP carrier and these
inhibitors are expected to be effective for understanding
the molecular mechanism of this nucleotide exchange.
The detailed manner of interaction of the ADP/ATP carrier
with CATR has been clarified by structural analysis of their
co-crystal (9); however, that with BKA remains elusive.
C
For a better understanding of the interaction of the ADP/
ATP carrier with BKA, studies on the actions of BKA
analogues should be helpful. Thus, in this study, we exam-
ined the effects of a variety of BKA analogues, derived from
the intermediates obtained during the process of BKA syn-
thesis, on mitochondrial functions and the ADP/ATP carrier.
Figure 1: General structure of the bongkrekic acid (BKA)
analogues used in the present study (A) and chemical structure of
KH-7 (B) and parental BKA (C) For the structural properties of the
BKA analogues, see the text. For the substituents R , R , X, or
chain length n of individual analogues, see Table 1.
1
2
Table 1: Structural features of the bongkrekic acid analogues
used in the present study
Materials and Methods
1
2
Chemicals and reagents
Code
R
R
X
n
Alias or reference
Authentic BKA was provided by Prof. Hans J. Duine (Delft
t
3
0
KH-1
KH-2
KH-3
KH-4
KH-5
KH-6
KH-7
KH-8
KH-9
KH-10 CH
KH-11 CH
KH-12 CH
KH-13 CH
KH-14 CH
H
Si( Bu)Ph
H,H
H,H
H,H
H,H
H,H
H,H
6
6
6
8
8
8
18a
21a
17a
18b
21b
17b
2
2
2
2
2
2
2
2
2
University, The Netherlands). [2,8- H] adenosine 5 -diphos-
t
3
CH
CH
H
CH
CH OCH
H
CH
CH
2
CH
OCH
2
CN Si( Bu)Ph
phate (code NET241), hereafter referred to just as [ H]ADP,
t
2
3
Si( Bu)Ph
was purchased from PerkinElmer, Inc., Waltham, MA, USA
t
Si( Bu)Ph
t
2
CH
2
CN Si( Bu)Ph
t
The BKA analogues, compounds KH-1~14, summarized in
Figure 1A and Table 1, were prepared as indicated below
Si( Bu)Ph
2
3
t
Si( Bu)Ph
H,H 10 18c
H,H 10 21c
H,H 10 17c
H,H 10 27a
H,H 10 27b
H,H 10 27c
H,H 10 28
O
O
O
O
t
(see also ref. 11 and Schemes 1 and 2). The method
2
2
2
2
2
2
2
CH
2
CN Si( Bu)Ph
t
OCH
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
3
Si( Bu)Ph
for synthesis of KH-15, 16, and 17 was already reported
previously (12).
t
Si( Bu)Me
2
SiPh
CPh
H
CH
H
3
3
Materials for chemical synthesis
2
OCH
3
10 35
1
13
The H- and C-NMR spectra were recorded using a JNM
LA-400 spectrometer, JEOL Ltd., Tokyo, Japan (400 and
KH-15
KH-16 CH
KH-17
H
10 Ref. (12)
16 Ref. (12)
16 Ref. (12)
2
OCH
3
H
H
H
100 MHz). The IR spectra were recorded on a Shimadzu FT/
IR-8300 spectrometer (Shimadzu Co., Kyoto, Japan) using a
KBr disk or a NaCl cell. Mass spectra were obtained on a
JEOL JMS-700. Column chromatography was performed
on silica gel (Kanto Chemical Co., Tokyo, Japan). Thin-layer
chromatography was performed on precoated plates
This table summarizes the structural properties of the 17 BKA
analogues (see Figure 1A for their general structure). The symbols
with ‘KH-xx’ represent the names of the individual chemicals; and
1
2
structures or atoms shown in the columns ‘R ’, ‘R ’, and ‘X’ are
the substituents of the individual chemicals at these positions. The
numbers shown in column ‘n’ represent the number of repeated
alkyl chain. The actual structure of one compound, KH-7, of
(0.25 mm, silica gel 60 F254, Merck & Co, Inc., Kenilworth,
NJ, USA.). Reaction mixtures were stirred magnetically.
1
2
t
2
which R , R , X, and n are H, Si( Bu)Ph , H2, and 10, respec-
tively, is shown in Figure 1B. The codes or reference No. shown
in the column ‘Alias or reference’ indicate the alias of individual
chemicals used in the process of their synthesis (see Schemes 1
and 2) or the reference No. for a study in which the procedures
for the synthesis of these analogues are described.
8
-((tert-butyldiphenylsilyl)oxy)octan-1-ol (13a)
To a solution of 1.8-octanediol (10 g, 68.4 mmol) in CH
230 mL) were added imidazole (5.60 g, 82.1 mmol) and
TBDPSCl (13.1 g, 47.8 mmol). The reaction mixture was
stirred at RT for 12 h and added sat, NaHCO aq,
extracted with CH Cl and washed with brine, dried over
2
Cl
2
(
3
1
colorless oil (9.81 g, 37%): H-NMR (400 MHz in CDCl ) d:
2
2,
3
MgSO
4
. The crude product was purified by silica gel
1.05 (s, 9H), 1.20–1.29 (m, 7H), 1.52–1.59 (m, 5H), 3.61–
3.67 (m, 4H), 7.26–7.44 (m, 6H), 7.68 (m, 4H); C-NMR
13
column chromatography (Hexane/EtOAc = 4/1) to give
Chem Biol Drug Des 2015; 86: 1304–1322
1305

Yamamoto et al.
Scheme 1: Outline of the procedure used for the synthesis of KH-1~9.
(
3
100 MHz, CDCl
2.5, 32.7, 62.8, 63.9, 127.5, 129.4, 134.0, 135.5; IR
3
) d: 19.1, 25.6, 25.7, 25.8, 26.8, 29.3,
reaction mixture stirred at ꢀ78 °C for 20 min, and NEt
(13.5 mL, 96.2 mmol) was added at ꢀ78 °C. The mixture
was stirred at RT for 1 h and quenched with sat, NaHCO
aq., extracted with CH Cl and washed with brine, dried
3
ꢀ
1
(Neat): 3342, 2930, 2850, 1589 cm
.
3
2
2,
over MgSO
4
. The crude product was purified by silica gel
8
-((tert-butyldiphenylsilyl)oxy)octanal (14a)
column chromatography (Hexane/EtOAc = 5:1) to give a
yellow oil. (4.32 g, 87%): H-NMR (400 MHz in CDCl ) d:
3
1.03 (s, 9H), 1.23–1.30 (m, 8H),1.52–1.61 (m, 2H), 2.41
(m, 2H), 3.65 (t, J = 9.4 Hz, 2H), 7.35–7.42 (m, 6H), 7.65–
7.68 (m, 4H), 9.76 (t, J = 2.8 Hz, 1H).
1
To a solution of oxalyl chloride (2.70 mL, 31.2 mmol) in
CH Cl (70 mL) was added DMSO (2.77 mL, 39.0 mmol)
2
2
at ꢀ78 °C. and stirred for 20 min, and alcohol (5.0 g,
1
3.0 mmol) in CH
2
Cl
2
(10 mL) was added at ꢀ78 °C. The
1
306
Chem Biol Drug Des 2015; 86: 1304–1322

pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier
Scheme 2: Outline of the procedure used for the synthesis of KH-10~14.
(
E)-tert-butyldiphenyl((9-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)non-8-en-1-yl)oxy)silane (15a)
To a suspension of CrCl (5.77 g, 47.0 mmol) and LiI
4.20 g, 31.4 mmol) in THF (20 mL) were added pinacol
borane (2.48 g, 11.8 mmol) in THF (10 mL) and aldehyde
(3.00 g, 7.84 mmol) in THF (10 mL). The mixture was stir-
2
red at RT for 3 h, and added sat.NaHCO
3
aq, extracted
(
with EtOAc, and washed with brine, dried over MgSO .
4
Chem Biol Drug Des 2015; 86: 1304–1322
1307

Yamamoto et al.
1
3
The crude product was purified by silica gel column chro-
(m, 6H), 7.56 (d, J = 16.4 Hz, 1H), 7.67 (m, 4H); C-
NMR (100 MHz, CDCl ) d:19.1, 25.4, 26.8, 28.6, 28.8,
matography (Hexane/EtOAc = 10/1) to give pale yellow oil
3
1
(
2.37 g, 60%): H-NMR (400 MHz in CDCl
3
) d: 1.04 (s,
28.9, 32.2, 33.4, 40.0, 57.6, 64.0, 90.1, 118.1, 126.3,
127.6, 129.5, 133.9, 135.6, 140.8, 147.7, 165.0; IR (Neat):
9
H), 1.27–1.39(m, 20H), 1.53–1.56 (m, 2H), 2.13 (q,
ꢀ1
J = 7.2 Hz, 4H), 3.64 (t, J = 6.4 Hz, 6H), 5.42 (d,
J = 18.4 Hz, 1H), 6.63 (td, J = 6.4, 18 Hz, 1H), 7.36–7.42
3244, 2930, 2856, 1716, 1695, 1633, 1602 cm .
1
3
(
m, 6H), 7.66–7.68 (m, 4H); C-NMR (100 MHz, CDCl
d:19.1, 24.7, 24.8, 25.7, 26.8, 28.2, 29.4, 29.3, 32.5,
5.8, 63.9, 82.8, 127.5, 129.4, 134.0, 135.5, 154.7; IR
3
)
(Z)-3-((E)-9-((tert-butyldiphenylsilyl)oxy)non-1-en-1-
3
yl)pent-2-enedioic acid (KH-1, 18a)
ꢀ1
(Neat): 2933, 2852, 1641 cm
.
To a solution of ester (10 mg, 0.018 mmol) in Et
2
O
(1.7 mL) was added MgBr2 (33 g, 0.18 mmol). The reac-
tion mixture was stirred at RT for 20 min, and added 3 M
HCl and H O, extracted with EtOAc and washed with
(2Z,4E)-methoxymethyl-12-((tert-butyldiphenylsilyl)
2
,
oxy)-3-(2-hydroxyethyl)dodeca-2,4-dienoate (16a)
To a suspension of Pd(PPh Cl (225 mg, 0.32 mmol) in
MeOH (4.0 mL) were added Segment (540 mg,
.89 mmol) and boronic ester (800 mg, 1.58 mmol) at RT.
The mixture stirred at RT for 10 min, and NEt3 (1.55 mL,
1.0 mmol) was added. The reaction mixture was stirred
brine, dried over MgSO
4
. The crude product was purified
3
)
2
2
by silica gel column chromatography (CHCl
3
/MeOH=/19/1)
to give colorless solid (5.7 mg, 62%): H-NMR (400 MHz in
CDCl ) d: 1.04 (s, 9H), 1.23–1.28(m, 6H), 1.41 (brs, 2H),
1
A
1
3
1.52–1.54 (m, 2H), 2.21 (q, J = 7.6 Hz, 2H), 3.38 (s,2H),
3.65 (t, J = 13.6 Hz, 2H), 5.73 (s,1H), 6.22 (td, J = 7.2,
16.0 Hz, 1H), 7.36–7.42 (m, 6H), 7.51 (d, J = 15.6 Hz,
1
at RT for 6 h, and evaporated. The crude product was
purified by silica gel column chromatography (Hexane/
13
3
1H), 7.67 (m, 4H); C-NMR (100 MHz, CDCl ) d:20.0,
1
EtOAc = 2/1) to give a yellow oil (626 mg, 74%): H-NMR
26.8, 27.3, 29.9, 30.1, 30.2, 33.5, 34.3, 41.2, 65.0,
119.8, 127.9, 128.7, 130.8, 135.1, 136.7, 140.3, 149.1,
169.3, 174.4; IR (Neat): 3508, 2929, 2856, 1697, 1631,
(
400 MHz in CDCl ) d: 1.05 (s, 9H), 1.25–1.28(m, 4H),
3
1
.35–1.42 (m, 2H), 1.53–1.58 (m, 4H), 2.21 (q,
ꢀ
1
J = 7.6 Hz, 2H), 2.63 (t, J = 6.4 Hz, 2H), 3.48 (s, 3H),
1604 cm
.
3
2
7
4
2
1
1
1
.65 (t, J = 6.6 Hz, 2H), 3.79 (q, J = 6.8 Hz, 2H), 5.28 (s,
H), 5.69 (s, 1H),6.22 (td, J = 7.6 Hz, 16.4 Hz, 1H), 7.36–
.44 (m, 6H), 7.53 (d, J = 16.4 Hz, 1H), 7.66–7.68 (m,
10-(tert-butyldiphenylsilyloxy)decan-1-ol (13b)
To a solution of 1.10-decanediol (10 g, 58.0 mmol) in
1
3
H); C-NMR (100 MHz, CDCl
3
) d:19.1, 25.6, 26.8, 28.9,
9.0, 29.2, 32.4, 33.4, 37.5, 57.5, 61.8, 63.9, 89.8,
15.7, 126.5, 127.5, 129.4, 134.0, 135.5, 140.0, 153.1,
65.4; IR (Neat): 3421, 2930, 2858, 1716, 1635,
CH Cl2 (290 mL) were added imidazole (4.73 g,
2
69.6 mmol) and TBDPSCl (9.56 g, 34.8 mmol). The reac-
tion mixture was stirred at RT for 40 min, and added sat,
ꢀ
1
597 cm
.
NaHCO
3
aq, extracted with CH
2
Cl2, and washed with
brine, dried over MgSO
4
. The crude product was purified
by silica gel column chromatography (Hexane/EtOAc=4/1)
1
(3Z,4E)-12-((tert-butyldiphenylsilyl)oxy)-3-(2-
to give colorless oil (5.39 g, 23%): H-NMR (400 MHz in
(
methoxymethoxy)-2-oxoethylidene)dodec-4-enoic
CDCl ) d: 1.04 (s, 9H), 1.26 (brs, 16H), 3.62–3.3.67 (m,
4H), 7.37–7.7.42 (m, 6H), 7.65–7.68 (m, 4H); C-NMR
3
13
acid (KH-3, 17a)
To a solution of alcohol (200 mg, 0.371 mmol) in CH Cl
(100 MHz, CDCl ) d: 19.1, 25.7, 26.8, 29.3, 29.4, 32.5,
2
2
3
(
14.8 mL) was added DMP (314 mg, 0.742 mmol).
The reaction mixture was stirred at RT for 0.5 h,
2
and added sat. Na aq., extracted with CH Cl2,
62.9, 63.9, 127.5, 129.4, 134.1, 135.5; IR (Neat): 3340,
ꢀ1
2929, cm
.
2
S
2
O
3
washed with brine, dried over MgSO
product.
4
and gave a crude
10-((tert-butyldiphenylsilyl)oxy)decanal (14b)
To a solution of oxalyl chloride (2.60 mL, 30.2 mmol) in
To a solution of the crude product in tBuOH/THF/2-
methyl-2-butene = 3/1/1 (7.6 mL) were added NaClO
43 mg, 0.47 mmol) and NaH PO ・2H (103 mg,
.66 mmol) in H O (7.6 mL). The reaction mixture was stir-
CH Cl2 (74 mL) was added DMSO (2.68 mL, 37.8 mmol)
2
2
at ꢀ78 °C. and stirred for 20 min, and alcohol (5.22 g,
(
0
2
4
2
O
12.6 mmol) in CH
2
Cl
2
(10 mL) was added at ꢀ78 °C. The
2
reaction mixture stirred at ꢀ78 °C for 20 min, and NEt
3
red at RT for 0.5 h, and extracted with EtOAc, and
(7.47 mL, 53.2 mmol) was added at ꢀ78 °C. The mixture
washed with brine, dried over MgSO . The crude product
was stirred at RT for 1 h, and quenched with sat, NaHCO3
4
was purified by silica gel column chromatography (Hex-
ane/EtOAc = 2/1) to give colorless oil (180 mg, 87%): H-
2
aq., extracted with CH Cl2, and washed with brine, dried
1
over MgSO . The crude product was purified by silica gel
4
NMR (400 MHz in CDCl
3
) d: 1.04 (s, 9H), 1.24–1.28(m,
column chromatography (Hexane/EtOAc =4:1) to give a
1
6
H), 1.42 (brs, 2H), 1.53–1.54 (m, 2H), 2.22 (q,
yellow oil. (4.27 g, 83%): H-NMR (400 MHz in CDCl
3
) d:
J = 7.2 Hz, 2H), 3.38 (s, 2H), 3.48 (s, 3H), 3.65 (t,
J = 6.6 Hz, 2H), 3.65 (t, J = 6.8 Hz, 2H), 5.29 (s, 1H),
1.04 (s, 9H), 1.26 (brs, 12H), 1.52–1.62 (m, 3H), 2.41 (m,
2H), 3.64 (t, J = 6.3 Hz, 2H), 7.35–7.7.42 (m, 6H), 7.65–
7.68 (m, 4H), 9.76 (t, J = 2.8 Hz, 1H).
5
.74 (s, 2H),6.22 (td, J = 7.2 Hz, 16.4 Hz, 1H), 7.36–7.44
1
308
Chem Biol Drug Des 2015; 86: 1304–1322

pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier
with brine, dried over MgSO . The crude product was
(
1
(
E)-tert-butyldiphenyl(13-(4,4,5,5-tetramethyl-
,3,2-dioxaborolan-2-yl)tridec-12-enyloxy)silane
15b)
To a suspension of CrCl2 (4.60 g, 37.4 mmol) and LiI
4.56 g, 34.1 mmol) in THF (50 mL) were added pinacol
borane (3.58 g, 17.0 mmol) in THF (10 mL) and aldehyde
3.50 g, 8.52 mmol) in THF (15 mL). The mixture was stir-
red at RT for 14 h, and added sat.NaHCO aq, extracted
with EtOAc, and washed with brine, dried over MgSO
4
purified by silica gel column chromatography (Hexane/
1
EtOAc=2/1) to give colorless oil (17 mg, 55%): H-NMR
(400 MHz in CDCl ) d: 1.04 (s, 9H), 1.26–1.28 (m, 10H),
3
(
1.43 (brs, 2H), 1.51–1.56 (m, 2H), 2.22 (q, J = 6.8 Hz,
2H), 3.38 (s, 2H), 3.48 (s, 3H), 3.65 (t, J = 6.6 Hz, 2H),
5.28 (s, 2H), 5.74 (s, 1H), 6.20 (td, J = 6.8 Hz, 16.8 Hz,
1H), 7.35–7.44 (m, 6H), 7.56 (d, J = 16 Hz, 1H), 7.68 (m,
(
3
13
4
.
3
4H); C-NMR (100 MHz, CD OD) d: 20.0, 26.8, 27.4,
The crude product was purified by silica gel column chro-
29.9, 30.2, 30.3, 30.4, 30.6, 33.6, 34.4, 57.7, 65.0, 90.9,
118.6, 127.7, 128.7, 130.8, 135.1, 136.6, 141.3, 150.6,
166.6, 174.1; IR (Neat): 3512, 2929, 1716, 1633,
matography (Hexane/EtOAc=10/1) to give yellow oil
1
(
2.03 g, 44%): H-NMR (400 MHz in CDCl
3
) d: 1.04 (s,
ꢀ
1
+
9
2
1
H), 1.24–1.57(m, 26H), 2.13 (m, 2H), 3.64 (t, J = 6.4 Hz,
1602 cm ; Mass (FAB) m/z 581 (M +1); HRMS calcd for
Si: 581.3298 found 581.3285.
H), 5.42 (d, J = 18.1 Hz, 1H), 6.63 (td, J = 6.5, 17.8 Hz,
48 49 6
C H O
13
H), 7.35–7.44 (m, 6H), 7.65–7.68 (m, 4H);
C-NMR
(
100 MHz, CDCl ) d: 19.1, 24.7, 25.6, 26.8, 28.1, 29.0,
3
2
1
9.1, 32.5, 35.7, 63.8, 82.8, 127.5, 129.4, 134.0, 135.5,
(Z)-3-((E)-11-((tert-butyldiphenylsilyl)oxy)undec-1-
en-1-yl)pent-2-enedioic acid (KH-4, 18b)
ꢀ1
54.6; IR (Neat): 2929, 2858, 1637, cm
.
2
To a solution of ester (84 mg, 0.14 mmol) in Et O (11 mL)
was added MgBr2 (258 g, 0.14 mmol). The reaction mix-
ture was stirred at RT for 20 min, and added 3M HCl and
(
2Z,4E)-methoxymethyl-14-((tert-butyldiphenylsilyl)
oxy)-3-(2-hydroxyethyl)tetradeca-2,4-dienoate
16b)
To a suspension of Pd(PPh
MeOH (3.3 mL) were added Segment
.74 mmol) and boronic ester (800 g, 1.45 mmol) at RT.
The mixture stirred at RT for 10 min, and NEt3 (1.44 mL,
0.2 mmol) was added. The reaction mixture was stirred
H
2
O, extracted with EtOAc
,
and washed with brine, dried
(
over MgSO . The crude product was purified by silica gel
4
3
)
2
Cl
2
(204 mg, 0.29 mmol) in
(497 mg,
3
column chromatography (CHCl /MeOH=/19/1) to give col-
1
A
orless solid (37.7 mg, 50%): H-NMR (400 MHz in CDCl )
3
1
d: 1.04 (s, 9H), 1.21–1.41 (m, 10H), 1.41 (brs, 2H), 1.53–
1.56 (m, 2H), 2.21 (q, J = 6.8 Hz, 2H), 3.37 (s,2H), 3.65
(t, J = 6.6 Hz, 2H), 5.72 (s,1H), 6.22 (dt, J = 7.2, 16.0 Hz,
1H), 7.36–7.42 (m, 6H), 7.50 (d, J = 16.4 Hz, 1H), 7.67
1
at RT for 3 h, and evaporated. The crude product was
purified by silica gel column chromatography (Hexane/
EtOAc=2/1) to give a yellow oil (414 mg, 50%): H-NMR
13
(m, 4H); C-NMR (100 MHz, CD OD) d: 19.2, 25.7, 26.9,
3
1
28.8, 29.2, 29.3, 29.4, 32.5, 33.5, 40.3, 64.0, 117.9,
126.3, 127.5, 129.4, 134.1, 135.6, 141.5, 148.9, 171.2,
176.2; IR (Neat): 3497, 2926, 2852, 1699, 1631,
(400 MHz in CDCl ) d: 1.04 (s, 9H), 1.16–1.60 (m, 14H),
3
2
3
5
7
4
2
8
1
1
.20 (q, J = 6.8 Hz, 2H), 2.63 (t, J = 6.4 Hz, 2H), 3.42 (s,
H), 3.65 (t, J = 6.5 Hz, 2H), 3.79 (q, J = 6.8 Hz, 2H),
.28 (s, 1H), 5.69 (s, 2H),6.22 (td, J = 6.8, 16.0 Hz, 1H),
.30–7.44 (m, 6H), 7.48 (d, J = 16.2 Hz, 1H), 7.67 (m,
ꢀ
1
1606 cm
.
1
3
H); C-NMR (100 MHz, CDCl ) d:19.1, 25.7, 26.8, 28.9,
12-(tert-butyldiphenylsilyloxy)dodecan-1-ol (13c)
To a solution of 1.12-dodecanediol (5.00 g, 24.7 mmol)
3
9.1, 29.2, 29.3, 29.4, 32.5, 33.4, 37.5, 57.5, 61.8, 63.9,
9.8, 115.7, 126.5, 127.5, 129.4, 134.0, 135.5, 140.0,
53.1, 165.4; IR (Neat): 3481, 2929, 2856, 1718,
in CH Cl2 were added imidazole (2.01 g, 29.6 mmol)
2
and TBDPSCl (4.75 g, 17.3 mmol). The reaction mixture
ꢀ
1
134 cm
.
was stirred at RT for 2 h, and added sat, NaHCO
extracted with CH Cl2, and washed with brine, dried
over MgSO . The crude product was purified by silica
gel column chromatography (Hexane/EtOAc=3/1) to give
3
aq,
2
4
(3Z,4E)-16-(tert-butyldiphenylsilyloxy)-3-(2-
1
(
methoxymethoxy)-2-oxoethylidene)hexadec-4-
3
colorless oil (3.83 g, 35%): H-NMR (400 MHz in CDCl )
enoic acid (KH-6, 17b)
d: 1.05 (s, 9H), 1.25–1.31(m, 18H), 1.53–1.57 (m, 6H),
3.65 (t, J = 6.6 Hz, 4H), 7.36–7.42 (m, 6H), 7.67 (m,
4H): C-NMR (100 MHz, CDCl ) d:19.2, 25.7, 26.8,
3
2 2
To a solution of alcohol (30 mg, 0.053 mmol) in CH Cl
13
1
.1 mL) was added DMP (45 mg, 0.11 mmol). The reac-
tion mixture was stirred at RT for 1 h, and added sat.
Na aq., extracted with CH Cl2, washed with brine,
29.3, 29.4, 29.5, 29.6, 32.5, 32.7, 63.0, 64.0, 127.5,
129.4, 134.1, 135.5; IR (Neat): 3336, 2928, 2854,
2
S
2
O
3
2
ꢀ
1
dried over MgSO and gave a crude product.
1589 cm .
4
To a solution of the crude product in tBuOH/THF/2-
methyl-2-butene = 3/1/1 (1.1 mL) were added NaClO
2
12-(tert-butyldiphenylsilyloxy)dodecanal (14c)
(24 mg, 0.27 mmol) and NaH
2
PO
4
2
・2H O
(58 mg,
To a solution of oxalyl chloride (0.95 mL, 11.0 mmol) in
0
.37 mmol) in H O (1.1 mL). The reaction mixture was stir-
CH Cl2 (30 mL) was added DMSO (0.97 mL, 13.7 mmol)
2
2
red at RT for 1 h, and extracted with EtOAc, and washed
at ꢀ78 °C. and stirred for 20 min, and alcohol 13c
Chem Biol Drug Des 2015; 86: 1304–1322
1309

Yamamoto et al.
(
2.02 g, 4.58 mmol) in CH
2
Cl
78 °C. The reaction mixture stirred at ꢀ78 °C for
0 min, and NEt (4.83 mL, 34.4 mmol) was added at
78 °C. The mixture was stirred at RT for 1 h, and
aq., extracted with CH
2
(10 mL) was added at
(3Z,4E)-16-(tert-butyldiphenylsilyloxy)-3-(2-
(methoxymethoxy)-2-oxoethylidene)hexadec-4-
enoic acid (KH-9, 17c)
ꢀ
2
3
ꢀ
To a solution of alcohol 16c (200 mg, 0.336 mmol) in
quenched with sat, NaHCO
3
2
Cl2,
CH
The reaction mixture was stirred at RT for 1 h, and added
sat. Na aq., extracted with CH Cl2, washed with
brine, dried over MgSO and gave a crude product.
2 2
Cl (6.7 mL) were added DMP (570 mg, 1.34 mmol).
and washed with brine, dried over MgSO . The crude
product was purified by silica gel column chromatography
4
2
S
2
O
3
2
(
Hexane/EtOAc =4: 1) to give a yellow oil. (1.86 g, 93%):
4
1
3
H-NMR (400 MHz in CDCl ) d: 1.07 (s, 9H), 1.25–1.29
(m, 15H), 1.52–1.64 (m, 7H), 2.42 (t, J = 8.0 Hz, 2H), 3.65
(t, J = 6.6 Hz, 2H), 7.36–7.44 (m, 6H), 7.67 (m, 4H), 9.76
(t, J = 4.0 Hz, 1H).
To a solution of the crude product in tBuOH/THF/2-
methyl-2-butene =3/1/1 (6.7 mL) were added NaClO2
(152 mg, 1.69 mmol) and NaH
2
PO
4
・2H
2
O
(368 mg,
2
.36 mmol) in H O (6.7 mL). The reaction mixture was
2
stirred at RT for 1 h, and extracted with EtOAc, and
washed with brine, dried over MgSO . The crude product
was purified by silica gel column chromatography
(
1
(
E)-tert-butyldiphenyl(13-(4,4,5,5-tetramethyl-
,3,2-dioxaborolan-2-yl)tridec-12-enyloxy)silane
15c)
To a suspension of CrCl2 (4.05 g, 33.0 mmol) and LiI
2.94 g, 22.0 mmol) in THF (50 mL) were added pinacol
4
(Hexane/EtOAc=2/1) to give colorless oil (173 mg,
1
86%): H-NMR (400 MHz in CDCl3) d: 1.04 (s, 9H),
(
1.24–1.31(m, 12H), 1.43 (brs, 2H), 1.53–1.58 (m,4H), 2.20
(q, J = 7.2 Hz, 2H), 3.38 (s, 2H), 3.48 (s, 3H), 3.65 (t,
J = 6.6 Hz, 2H), 5.28 (s, 2H), 5.74 (s, 1H), 6.20
(dt, J = 6.8 Hz, 16 Hz, 1H), 7.36–7.42 (m, 6H), 7.56 (d,
borane 11 (2.20 g, 11.0 mmol) in THF (5.0 mL) and alde-
hyde 14c (2.41 g, 5.50 mmol) in THF (5.0 mL). The mix-
ture was stirred at RT for 3 h, and added sat.NaHCO
extracted with EtOAc, and washed with brine, dried over
MgSO . The crude product was purified by silica gel col-
umn chromatography (Hexane/EtOAc=10/1) to give yellow
oil (1.87 g, 60%): H-NMR (400 MHz in CDCl
9
J = 6.8 Hz, 4H), 3.65 (t, J = 6.6 Hz, 6H), 5.42 (d,
J = 18.4 Hz, 1H), 6.63 (td, J = 6.8, 18.0 Hz, 1H), 7.36–
7
3
aq,
13
J = 16.4 Hz, 1H), 7.67 (m, 4H);
C-NMR (100 MHz,
4
CD OD) d: 20.0, 26.9, 27.4, 29.9, 30.2, 30.3, 30.5, 30.6,
3
30.7, 33.6, 34.4, 41.1, 57.7, 65.0, 90.9, 118.6, 127.8,
128.7, 130.8, 135.1, 136.7, 141.3, 150.6, 166.7, 174.0;
1
3
) d: 1.04 (s,
ꢀ
1
H), 1.26–1.42(m, 28H), 1.52–1.59 (m, 2H), 2.14 (q,
IR (Neat): 3292, 2933, 2854, 1710, 1635, 1602 cm ;
+
Mass (FAB) m/z 631 (M +Na); HRMS calcd for
52NaO Si: 631.3431 found 631.3427.
C
36
H
6
13
.43 (m, 6H), 7.66–7.68 (m, 4H) 01-193; C-NMR
(
100 MHz, CDCl ) d:19.2, 24.7, 25.7, 26.8, 28.1, 29.2,
3
2
1
1
9.3, 29.4, 29.5, 29.6, 32.5, 35.8, 63.9, 82.9, 127.5,
29.4, 134.1, 135.5, 154.8; IR (Neat): 2978, 2928,
(Z)-3-((E)-13-((tert-butyldiphenylsilyl)oxy)tridec-1-
en-1-yl)pent-2-enedioic acid (KH-7, 18c)
ꢀ
1
637 cm
.
To a solution of ester 17c (56 mg, 0.092 mmol) in Et
2
O
(
7.0 mL) was added MgBr (102 mg, 0.55 mmol). The
2
reaction mixture was stirred at RT for 20 min, and added
3M HCl and H O, extracted with EtOAc and washed with
(
2Z,4E)-methoxymethyl-16-(tert-
butyldiphenylsilyloxy)-3-(2-hydroxyethyl)hexadeca-
,4-dienoate (16c)
To a suspension of Pd(PPh
MeOH (20 mL) were added Segment
.96 mmol) and boronic ester 15c (1.00 g, 1.78 mmol) at
RT. The mixture stirred at RT for 10 min, and NEt
1.76 mL, 12.5 mmol) was added. The reaction mixture
2
,
brine, dried over MgSO
4
. The crude product was purified
2
by silica gel column chromatography (CHCl /MeOH=/19/1)
3
1
3
)
2
Cl
2
(250 mg, 0.36 mmol) in
(560 mg,
to give colorless solid (30 mg, 58%): H-NMR (400 MHz in
A
3
CDCl ) d: 1.04 (s, 9H), 1.24 (brs, 14H), 1.41–1.43 (m, 2H),
1
1.51–1.61 (m, 2H), 2.22 (q, J = 7.6 Hz, 2H), 3.38 (s,2H),
3.65 (t, J = 6.6 Hz, 2H), 5.73 (s,1H), 6.22 (dt, J = 7.2,
16.0 Hz, 1H), 7.35–7.43 (m, 6H), 7.51 (d, J = 16.4 Hz,
3
(
13
was stirred at RT for 3 h, and evaporated. The crude
product was purified by silica gel column chromatography
3
1H), 7.66–7.68 (m, 1H); C-NMR (100 MHz, CD OD):
19.2, 25.8, 26.9, 28.9, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7,
33.5, 40.2, 64.1, 117.8, 126.3, 127.6, 129.4, 134.1,
135.6, 141.5, 148.9, 170.9, 175.9; IR (Neat): 3349, 2926,
(
Hexane/EtOAc=2/1) to give a yellow oil (781 mg, 73%):
1
H-NMR (400 MHz in CDCl
m, 9H), 1.39–1.42 (m,3H), 1.53–1.58 (m, 6H), 2.22 (q,
J = 7.6 Hz, 2H), 2.63 (t, J = 6.6 Hz, 2H), 3.48 (s, 3H),
.65 (t, J = 7.0 Hz, 2H), 3.78 (q, J = 6.6 Hz, 2H), 5.28
s, 2H), 5.68 (s, 1H),6.22 (dt, J = 6.8 Hz, 16.4 Hz, 1H),
3
) d: 1.05 (s, 9H), 1.20–1.28
ꢀ
1
(
2852, 1697, 1631, 1606 cm
.
3
(
(2Z,4E)-2-cyanoethyl 12-((tert-butyldiphenylsilyl)
7
4
2
6
1
1
.36–7.44 (m, 6H), 7.53 (d, J = 16.0 Hz, 1H), 7.67 (m,
oxy)-3-(2-hydroxyethyl)dodeca-2,4-dienoate (20a)
1
3
H);
C-NMR (100 MHz, CDCl
3
) d:19.1, 25.7, 26.8,
3 2 2
To a suspension of Pd(PPh ) Cl (166 mg, 0.24 mmol) in
8.9, 29.2, 29.3, 29.4, 29.5, 32.5, 33.5, 37.5, 57.5,
1.8, 64.0, 89.8, 115.7, 126.5, 127.5, 129.4, 134.1,
35.5, 140.1, 153.1, 165.4; IR (Neat): 3481, 2929, 2856,
MeOH (6.0 mL) were added cyanoethylester (417 mg,
1.41 mmol) and boronic ester (600 g, 1.18 mmol) at RT.
3
The mixture stirred at RT for 10 min, and NEt (1.15 mL,
ꢀ
1
718 cm
.
8.19 mmol) was added. The reaction mixture was stirred
1
310
Chem Biol Drug Des 2015; 86: 1304–1322

pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier
at RT for 3 h, and evaporated. The crude product was
purified by silica gel column chromatography (Hexane/
EtOAc=2/1) to give a yellow oil (328 mg, 51%): H-NMR
12-(tert-butyldimethylsilyloxy)dodecanal (24a)
To a solution of oxalyl chloride (0.39 mL, 4.56 mmol) in
1
CH
at ꢀ78 °C. and stirred for 20 min, and alcohol 22a
(600 mg, 1.90 mmol) in CH Cl (5.0 mL) was added at
ꢀ78 °C. The reaction mixture stirred at ꢀ78 °C for
20 min, and NEt (1.88 mL, 13.3 mmol) was added at
ꢀ78 °C. The mixture was stirred at RT for 1 h, and
quenched with sat, NaHCO aq., extracted with CH
and washed with brine, dried over MgSO . The crude
2 2
Cl (15 mL) was added DMSO (0.40 mL, 5.70 mmol)
(
400 MHz in CDCl ) d: 1.05 (s,9H), 1.24–1.59 (m, 10H),
3
2.22 (q, J = 7.6 Hz, 2H), 2.63 (t, J = 6.6 Hz, 2H), 2.72 (t,
2
2
J = 6.4 Hz, 2H), 3.65 (t, J = 6.6 Hz, 2H), 3.79 (q,
J = 6.0 Hz, 2H), 4.31 (t, J = 6.4 Hz, 2H), 5.68 (s, 1H),
3
6
.25 (td, J = 6.8, 16.0 Hz, 1H), 7.35–7.44 (m, 6H), 7.49
13
(
(
3
1
d, J = 16.0 Hz, 1H), 7.66–7.68 (m, 4H);
100 MHz, CDCl ) d:18.0, 19.2, 25.6, 26.8, 28.9, 29.2,
2.4, 33.5, 37.5, 58.1, 61.8, 63.8, 63.9, 114.9, 117.0,
26.3, 127.5, 129.4, 134.1, 135.5, 140.5, 153.7, 165.2;
C-NMR
3
2
Cl2,
3
4
product was purified by silica gel column chromatography
(Hexane/EtOAc =4: 1) to give a yellow oil. (524 g, 88%):
ꢀ
1
1
IR (Neat): 3419, 2930, 2857, 2252, 1716 cm
.
H-NMR (400 MHz in CDCl ) d: 0.05 (s, 9H), 0.89 (s, 9H),
3
1
3
.27 (brs, 14H), 1.55–1.63 (m, 4H), 2.40–2.43 (m, 2H),
.60 (t, J = 6.0 Hz, 2H), 9.76 (m, 1H).
(3Z,4E)-14-((tert-butyldiphenylsilyl)oxy)-3-(2-(2-
cyanoethoxy)-2-oxoethylidene)tetradec-4-enoic
acid (KH-2, 21a)
(E)-tert-butyldimethyl(13-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)tridec-12-enyloxy)silane
(25a)
2 2
To a solution of alcohol (100 mg, 0.183 mmol) in CH Cl
(
7.3 mL) was added DMP (233 mg, 0.549 mmol). The
reaction mixture was stirred at RT for 0.5 h, and added
sat. Na aq., extracted with CH Cl2, washed with
To a suspension of CrCl
2
(1.22 g, 9.96 mmol) and LiI
2
S
2
O
3
2
(888 mg, 6.64 mmol) in THF (9.0) were added pinacol
borane 11 (525 mg, 2.49 mmol) in THF (4.0 mL) and
aldehyde 24a (524 mg, 1.66 mmol) in THF (4.0 mL). The
mixture was stirred at RT for 2 h, and added sat.NaH-
brine, dried over MgSO and gave a crude product.
4
To a solution of the crude product in tBuOH/THF/2-
methyl-2-butene =3/1/1 (4.0 mL) were added NaClO
2
3
CO aq, extracted with EtOAc, and washed with brine,
(
1
91 mg, 1.01 mmol) and NaH PO ꢁ2H O (229 mg,
dried over MgSO . The crude product was purified by
2
4
2
4
.47 mmol) in H
2
O (4.0 mL). The reaction mixture was stir-
silica gel column chromatography (Hexane/EtOAc=10/1)
1
red at RT for 0.5 h, and extracted with EtOAc, and
to give yellow oil (524 g, 70%): H-NMR (400 MHz in
washed with brine, dried over MgSO . The crude product
CDCl ) d: 0.05 (s, 6H), 0.89 (s, 9H), 1.24–1.52 (m,
4
3
was purified by silica gel column chromatography (Hex-
ane/EtOAc=2/1) to give colorless oil (94 mg, 91%): H-
30H), 2.14 (q, J = 7.2 Hz, 2H), 3.59 (t, J = 6.6 Hz, 2H),
5.42 (d, J = 18.0 Hz, 1H), 6.63 (td, J = 6.4, 18.4 Hz,
1
13
NMR (400 MHz in CDCl
6
2
3
) d: 1.04 (s, 9H), 1.24–1.28 (m,
H), 1.42 (brs, 2H), 1.52–1.54 (m, 2H),2.23 (q, J = 7.2 Hz,
H), 2.73 (t, J = 6.2 Hz, 2H), 3.39 (s, 2H), 3.65 (t,
1H);
3
C-NMR (100 MHz, CDCl ) d: 18.0, 24.4, 24.5,
25.4, 25.6, 27.8, 28.9, 29.0, 29.1, 29.2, 29.3, 29.4,
32.5, 35.5, 62.9, 82.5, 154.4; IR (Neat): 2928, 2854,
ꢀ
1
J = 6.0 Hz, 2H), 4.32 (t, J = 6.6 Hz, 2H), 5.74 (s,1H), 6.22
1639 cm .
(
td, J = 7.2, 15.6 Hz, 1H), 7.36–7.44 (m, 6H), 7.52 (d,
13
J = 16.4 Hz, 1H), 7.68 (m, 4H);
C-NMR (100 MHz,
CD OD) d: 18.4, 20.1, 26.8, 27.4, 29.9, 30.1, 35.5, 41.1,
(2Z,4E)-methoxymethyl-16-(tert-
butyldimethylsilyloxy)-3-(2-hydroxyethyl)hexadeca-
2,4-dienoate (26a)
3
5
1
9.9, 64.9, 118.4, 127.7, 128.7, 130.8, 135.1, 136.7,
41.5.
To a suspension of Pd(PPh
MeOH (1.0 mL) were added Segment
3
)
2
Cl
2
(32 mg, 0.046 mmol) in
(72 mg,
A
1
2-(tert-butyldimethylsilyloxy)dodecan-1-ol (22a)
0.25 mmol) in MeOH (1.8 mL) and boronic ester 25a
(101 mg, 0.23 mmol) in MeOH (1.8 mL) at RT. The mix-
ture stirred at RT for 10 min, and NEt3 (0.23 mL,
1.61 mmol) was added. The reaction mixture was stirred
at RT for 3 h, and evaporated. The crude product was
purified by silica gel column chromatography (Hexane/
To a solution of 1.12-dodecanediol (5.00 g, 24.7 mmol) in
CH Cl2 were added imidazole (2.01 g, 29.6 mmol) and
TBSCl (2.70 g, 17.3 mmol). The reaction mixture was stir-
red at RT for 2 h, and added sat, NaHCO
with CH Cl2, and washed with brine, dried over MgSO .
The crude product was purified by silica gel column chro-
2
3
aq, extracted
2
4
1
EtOAc=2/1) to give a yellow oil (80 mg, 74%): H-NMR
matography (Hexane/EtOAc=4/1) to give colorless oil
(400 MHz in CDCl ) d: 0.05 (s, 6H), 0.89 (s, 9H), 1.27 (s,
3
1
(1.38 g, 18%): H-NMR (400 MHz in CDCl
3
) d: 0.05 (s,
15H), 1.40–1.54 (m, H), 2.22 (q, J = 6.8 Hz, 2H), 2.64 (t,
J = 6.6 Hz, 2H), 3.48 (s, 3H), 3.60 (t, J = 6.8 Hz, 2H),
3.78 (q, J = 5.6 Hz, 2H), 5.28 (s, 2H), 5.68 (s, 1H), 6.23
(td, J = 6.8, 16.4 Hz, 1H), 7.53 (d, J = 16.0 Hz, 1H); IR
6
3
1
3
1
3
H), 0.89 (s, 9H), 1.22 (brs, 14H), 1.46–1.51 (m, 2H),
.53–3.59 (m, 4H); C-NMR (100 MHz, CDCl ) d: 18.0,
3
13
9.2, 25.7, 26.8, 29.0, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6,
2.6, 33.5, 37.5, 58.1, 61.9, 64.0, 114.9, 116.9, 126.3,
27.5, 129.4, 134.1, 135.5, 140.7, 153.7, 165.2; IR (Neat):
ꢀ1
(Neat): 3398, 2928, 2854, 1716, 1635, 1597 cm ; Mass
+
(EI) m/z 470 (M ); HRMS calcd for C26
found 470.3432.
H
50
O
5
Si: 470.3428
ꢀ
1
335, 2928, 2854 cm
.
Chem Biol Drug Des 2015; 86: 1304–1322
1311

Yamamoto et al.
1
(3Z,4E)-16-(tert-butyldiphenylsilyloxy)-3-(2-
3
H-NMR (400 MHz in CDCl ) d: 1.22–1.29 (m, 16H), 1.60–
(methoxymethoxy)-2-oxoethylidene)hexadec-4-
1.64 (m, 4H), 2.41 (t, J = 7.4 Hz, 2H), 3.78 (t, J = 6.4 Hz,
enoic acid (KH-10, 27a)
2H), 7.40 (m, 9H), 7.62 (m, 6H), 9.76 (s, 1H).
To a solution of alcohol 26a (80 mg, 0.17 mmol) in
CH
The reaction mixture was stirred at RT for 0.5 h, and
added sat. Na aq., extracted with CH Cl2, washed
and gave a crude prod-
2 2
Cl (3.4 mL) was added DMP (288 mg, 0.68 mmol).
(E)-triphenyl(13-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)tridec-12-enyloxy)silane (25b)
2
S
2
O
3
2
with brine, dried over MgSO
uct.
4
2
To a suspension of CrCl (1.23 g 10.0 mmol) and LiI
(892 mg, 6.68 mmol) in THF (9.0 mL) were added pinacol
borane 11 (528 mg, 2.51 mmol) in THF (4.0 mL) and alde-
hyde 24b (767 mg, 1.67 mmol) in THF (4.0 mL). The mix-
To a solution of the crude product in tBuOH/THF/2-
methyl-2-butene = 3/1/1 (3.4 mL) were added NaClO
2
ture was stirred at RT for 3 h, and added sat.NaHCO
extracted with EtOAc, and washed with brine, dried over
MgSO . The crude product was purified by silica gel col-
3
,
(
78 mg, 0.866 mmol) and NaH PO ・2H O (187 mg,
2
4
2
1
.20 mmol) in H
2
O (3.4 mL). The reaction mixture was
4
stirred at RT for 1.5 h, and extracted with EtOAc, and
umn chromatography (Hexane/EtOAc=10/1) to give yellow
1
washed with brine, dried over MgSO . The crude product
oil (672 mg, 69%): H-NMR (400 MHz in CDCl ) d1.21–
4
3
was purified by silica gel column chromatography (Hex-
ane/EtOAc=2/1) to give colorless oil (50.7 mg, 62%): H-
1.40 (m, 30H), 1.54–1.61 (m, 2H), 2.14 (q, J = 6.8 Hz,
2H), 3.78 (t, J = 6.6 Hz, 2H), 5.42 (d, J = 18.0 Hz, 1H),
6.63 (td, J = 6.4, 18.4 Hz, 1H), 7.35–7.45 (m, 9H), 7.61–
1
NMR (400 MHz in CDCl
3
) d: 0.05 (s, 6H), 0.88 (s, 9H),
13
1
2
5
.24–1.52 (m, 22 H), 2.22 (q, J = 7.2 Hz, 2H), 3.37 (s,
7.63 (m, 6H): C-NMR (100 MHz, CDCl ) d:25.7, 28.9,
3
H), 3.48 (s, 3H), 3.61 (t, J = 6.6 Hz, 2H), 5.29 (s, 2H),
.74 (s, 1H), 6.21 (td, J = 7.2, 16.0 Hz, 1H), 7.56 (d,
29.2, 29.3, 29.4, 29.5, 32.5, 33.5, 37.5, 57.5, 61.9, 64.0,
89.9, 115.7, 126.5, 127.8, 129.9, 134.4, 135.3, 140.1,
1
3
ꢀ1
J = 16.0 Hz, 1H); C-NMR (100 MHz, CDCl ) d: 18.2,
153.1, 165.4; IR (Neat): 2926, 2854, 1637 cm
.
3
2
5
5.6, 28.3, 29.1, 29.2, 29.3, 29.4, 29.5, 32.7, 33.3, 37.4,
7.3, 61.6, 63.2. 89.7, 115.5, 126.4, 139.8, 153.2, 165.4;
ꢀ
1
IR (Neat): 3325, 2928, 2854, 1716, 1635 1602 cm
Mass (EI) m/z 484 (M ); HRMS calcd for C H O Si:
;
(2Z,4E)-methoxymethyl 3-(2-hydroxyethyl)-16-
((triphenylsilyl)oxy)hexadeca-2,4-dienoate (26b)
+
26 48 6
4
84.3220 found 484.3218.
To a suspension of Pd(PPh
MeOH (7.0 mL) were added Segment
.53 mmol) and boronic ester 25b (750 mg, 1.28 mmol)
at RT. The mixture stirred at RT for 10 min, and NEt
3
(1.26 mL, 8.96 mmol) was added. The reaction mixture
was stirred at RT for 3 h, and evaporated. The crude
product was purified by silica gel column chromatography
3
)
2
Cl
2
(180 mg, 0.256 mmol) in
A
(440 mg,
1
1
2-(triphenylsilyloxy)dodecan-1-ol (22b)
To a solution of 1.12-dodecanediol (5.00 g, 24.7 mmol) in
CH Cl were added imidazole (2.01 g, 29.6 mmol) and tri-
phenylsilyl chloride (5.01 g, 17.3 mmol).The reaction mix-
ture was stirred at RT for 1 h, and added H O, extracted
with CH Cl2, and washed with brine, dried over MgSO
2
2
2
(Hexane/EtOAc=2/1) to give a yellow oil (316 mg, 40%):
1
2
4
.
3
H-NMR (400 MHz in CDCl ) d: 1.22–1.28 (m, 14H), 1.43
The crude product was purified by silica gel column chro-
(brs, 2H), 1.54–1.62 (m, 2H), 2.22 (q, J = 6.8 Hz, 2H),
2.63 (t, J = 6.8 Hz, 2H), 3.48 (s, 3H), 3.76–3.81 (m, 4H),
5.27 (s, 2H), 5.68 (s, 1H), 6.22 (td, J = 6.8, 16.4 Hz, 1H),
matography (Hexane/EtOAc=4/1) to give colorless oil
1
(
2.87 g, 25%): H-NMR (400 MHz in CDCl ) d: 1.23–1.29
3
(
7
m, 20H), 3.64 (brs, 2H), 3.78 (t, J = 9.6 Hz, 2H), 7.35–
7.35–7.45 (m, 9H), 7.53 (d, J = 16.0 Hz, 1H), 7.61–7.63
(m, 6H); C-NMR (100 MHz, CDCl ) d: 25.7, 28.9, 29.2,
3
13
13
.43 (m, 9H), 7.60–7.64 (m, 6H) C-NMR (100 MHz,
) d:25.7, 29.2, 29.4, 29.6, 32.5, 32.7, 63.0, 63.9,
27.8, 130.0, 134.4, 135.3; IR (Neat): 3312, 2924,
CDCl
1
2
3
29.3, 29.4, 29.5, 29.6, 32.4, 33.4, 37.6, 57.4, 61.8, 63.9,
89.8, 115.6, 126.5, 127.7, 129.8, 134.4, 135.3, 140.0,
153.1, 165.4; IR (Neat): 3489, 2928, 2854, 1718, 1635,
ꢀ
1
879 cm
.
ꢀ
1
1
597 cm .
1
2-(triphenylsilyloxy)dodecanal (24b)
To a solution of oxalyl chloride (0.45 mL, 5.21 mmol) in
CH Cl (15 mL) was added DMSO (0.46 mL, 6.51 mmol)
(3Z,4E)-3-(2-(methoxymethoxy)-2-oxoethylidene)-
16-(triphenylsilyloxy)hexadec-4-enoic acid (KH-11,
27b)
2
2
at ꢀ78 °C and stirred for 20 min, and alcohol 22b
(
1.00 g, 2.17 mmol) in CH Cl (5.0 mL) was added at
To a solution of alcohol 26b (100 mg, 0.161 mmol) in
2
2
ꢀ
78 °C. The reaction mixture stirred at ꢀ78 °C for
CH
The reaction mixture was stirred at RT for 1 h, and added
sat. Na aq., extracted with CH Cl2, and washed with
brine, dried over MgSO and gave a crude product.
2 2
Cl (3.5 mL) was added DMP (273 mg, 0.644 mmol).
2
0 min, and NEt3 (2.13 mL, 15.2 mmol) was added at
ꢀ
78 °C. The mixture was stirred at RT for 1 h, and
2
S
2
O
3
2
quenched with sat. NaHCO aq., extracted with CH
3
2
Cl2,
4
and washed with brine, dried over MgSO . The crude
4
product was purified by silica gel column chromatography
To a solution of the crude product in tBuOH/THF/2-
methyl-2-butene =3/1/1 (3.24 mL) were added NaClO2
(Hexane/EtOAc =4: 1) to give a yellow oil. (767 mg, 77%):
1
312
Chem Biol Drug Des 2015; 86: 1304–1322

pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier
(
1
73 mg, 0.81 mmol) and NaH
2
PO
4
・2H
2
O
(177 mg,
at ꢀ78 °C and stirred for 20 min, and alcohol 22c (1.00 g,
.13 mmol) in H O. The reaction mixture was stirred at RT
2
2.25 mmol) in CH
2
Cl
2
(5.0 mL) was added at ꢀ78 °C. The
for 1 h, and extracted with EtOAc, and washed with brine,
dried over MgSO . The crude product was purified by sil-
ica gel column chromatography (Hexane/EtOAc=2/1) to
give yellow oil (62 mg, 61%): H-NMR (400 MHz in CDCl )
reaction mixture stirred at ꢀ78 °C for 20 min, and NEt
(2.13 mL, 15.2 mmol) was added at ꢀ78 °C. The mixture
was stirred at RT for 1 h, and quenched with sat, NaHCO
aq., extracted with CH Cl and washed with brine, dried
3
4
3
1
3
2
2,
d: 1.22–1.26 (m, 14H), 1.43 (brs, 2H), 1.54–1.62 (m, 2H),
over MgSO
4
. The crude product was purified by silica gel
2
.21 (q, J = 6.8 Hz, 2H), 3.36 (s, 2H), 3.47 (s, 3H), 3.79 (t,
column chromatography (Hexane/EtOAc = 4: 1) to give a
yellow oil. (847 mg, 85%): H-NMR (400 MHz in CDCl ) d:
3
1.25–1.29 (m, 14H), 1.61–1.64 (m, 2H), 2.41 (t,
J = 4.8 Hz, 2H), 3.78 (t, J = 6.4 Hz, 2H), 7.20–7.24 (m,
3H), 7.27–7.31 (m, 6H), 7.44 (m, 6H),9.76 (s, 1H).
1
J = 6.6 Hz, 2H), 5.27 (s, 2H), 5.72 (s, 1H), 6.19 (td,
J = 6.8, 16.4 Hz, 1H), 7.35–7.44 (m, 9H), 7.56
13
(
(
2
1
d, J = 16.0 Hz, 1H), 7.61–7.63 (m, 6H);
100 MHz, CDCl ) d: 25.7, 28.8, 29.2, 29.3, 29.4, 29.5,
9.6, 32.5, 33.4, 40.0, 57.6, 64.0, 90.1, 118.0, 126.3,
27.8, 129.9, 134.4, 135.4, 140.8, 147.6, 165.1, 176.1;
C-NMR
3
ꢀ
1
IR (Neat):, 3228, 2928, 2854, 1717, 1635, 1602 cm
.
(E)-4,4,5,5-tetramethyl-2-(13-(trityloxy)tridec-1-
enyl)-1,3,2-dioxaborolane (25c)
2
To a suspension of CrCl (1.41 g, 11.46 mmol) and LiI
(
3Z,4E)-16-hydroxy-3-(2-(methoxymethoxy)-2-
(1.02 g, 7.64 mmol) in THF (10 mL) were added pinacol
borane 11 (603 mg, 2.86 mmol) in THF (5.0 mL) and alde-
hyde 24c (846 mg, 1.91 mmol) in THF (5.0 mL). The mix-
oxoethylidene)hexadec-4-enoic acid (KH-13, 28)
To a solution of silyl ether 27b (10 mg, 0.016 mmol) in
THF (1.0 mL) was added TBAF (1M in THF, 32 L,
ture was stirred at RT for 3 h, and added sat.NaHCO
3
0
0
.032 mmol) at 0 °C. The reaction mixture was stirred at
aq., extracted with EtOAc, and washed with brine, dried
°C for 10 min, and quenched with sat. NH Cl aq.,
over MgSO . The crude product was purified by silica gel
4
4
extracted with EtOAc, and washed with brine, dried over
column chromatography (Hexane/EtOAc = 10/1) to give
1
MgSO . The crude product was purified by silica gel col-
yellow oil (708 g, 65%): H-NMR (400 MHz in CDCl ) d:
4
3
umn chromatography (CHCl
oil (4.3 mg, 73%): H-NMR (400 MHz in CDCl ) d: 1.27
3
/MeOH=19/1) to give yellow
1.23–1.42 (m, 28H), 1.57–1.65 (m, 2H), 2.06 (q,
J = 6.8 Hz, 2H), 2.98 (t, J = 6.8 Hz, 2H), 3.03
(t, J = 6.8 Hz, 2H), 5.38 (d, J = 18.4 Hz, 1H), 6.58 (td,
J = 7.2, 17.2 Hz, 1H), 7.07-7.10 (m, 3H), 7.27–7.43 (m,
1
3
(
(
brs, 14H), 1.43 (brs, 2H), 1.54–1.56 (m, 2H), 2.23
q, J = 6.8 Hz, 2H), 3.37 (s, 2H), 3.48 (s, 3H), 3.65 (t,
13
J = 6.4 Hz, 2H), 5.8 (s, 2H), 5.74 (s, 1H), 6.20
6H), 7.36–7.39 (m, 6H); C-NMR (100 MHz, CDCl ) d:
3
13
(td, J = 7.2, 16.0 Hz, 1H), 7.56 (d, J = 16.0 Hz, 1H); C-
25.2, 26.7, 28.6, 29.6, 29.7, 29.8, 29.9, 30.0, 30.1, 30.4,
36.2, 57.2, 64.0, 83.3, 86.6, 126.6, 127.1, 128.1, 128.7,
129.1, 129.8, 144.9, 155.2; IR (Neat):, 2926, 2854, 1637,
NMR (100 MHz, CDCl ) d: 25.5, 28.7, 29.0, 29.1, 29.2,
3
2
1
2
9.3, 29.4, 29.5, 32.5, 33.4, 40.1, 57.6, 63.0, 90.0,
17.9, 126.3, 140.8, 148.0, 165.1, 174.8; IR (Neat): 3417,
ꢀ
1
1597 cm
.
ꢀ
1
924, 2852, 1639 cm
.
(2Z,4E)-methoxymethyl 3-(2-hydroxyethyl)-16-
1
2-(trityloxy)dodecan-1-ol (22c)
To a solution of 1.12-dodecanediol (5.00 g, 24.7 mmol) in
CH Cl were added DMAP (150 mg, 1.24 mmol) and
NEt3 (5.21 mL, 37.1 mmol) and trityl chloride (6.89 g,
(trityloxy)hexadeca-2,4-dienoate (26c)
To a suspension of Pd(PPh ) Cl (167 mg, 0.238 mmol) in
3
2
2
2
2
MeOH (6.0 mL) were added Segment
1.53 mmol) in MeOH (3.0 mL) and boronic ester 25c
A
(440 mg,
2
1
4.7 mmol). The reaction mixture was stirred at RT for
h, and added H O, extracted with CH Cl2, and washed
(750 mg, 1.28 mmol) in MeOH (3.0 mL) at RT. The mix-
2
2
ture stirred at RT for 10 min, and NEt
3
(1.26 mL,
with brine, dried over MgSO . The crude product was
8.96 mmol) was added. The reaction mixture was stirred
at RT for 3 h, and evaporated. The crude product was
purified by silica gel column chromatography (Hexane/
4
purified by silica gel column chromatography (Hexane/
1
EtOAc=4/1) to give colorless oil (4.84 g, 44%): H-NMR
1
(400 MHz in CDCl
3
) d: 1.19–1.34 (m, 18H), 1.53–1.65 (m,
EtOAc=2/1) to give a yellow oil (113 mg, 16%): H-NMR
4
7
H), 3.04 (t, J = 6.8 Hz, 2H), 3.64 (q, J = 5.6 Hz, 2H),
3
(400 MHz in CDCl ) d: 1.24–1.43 (m, 14H), 1.58–1.63 (m,
13
.20–7.24 (m, 3H), 7.27–7.31 (m, 6H), 7.44 (m, 6H); C-
) d:25.7, 26.1, 29.3, 29.4, 29.5,
9.6, 29.9, 32.6, 62.6, 63.5, 86.1, 126.6, 127.5, 128.5,
4H), 2.15 (q, J = 7.2 Hz, 2H), 2.63 (t, J = 6.4 Hz, 2H),
3.03 (t, J = 6.6 Hz, 2H), 3.48 (s, 3H), 3.78 (q, J = 6.4 Hz,
2H), 5.27 (s, 2H), 5.68 (s, 1H), 6.22 (dt, J = 7.6, 15.6 Hz,
1H), 7.20–7.24 (m, 3H), 7.27–7.31 (m, 6H), 7.44 (m, 6H),
NMR (100 MHz, CDCl
2
1
3
ꢀ
1
44.4; IR (Neat): 3346, 2926, 2852 cm
.
13
7
.53 (d, J = 16.4 Hz, 1H); C-NMR (100 MHz, CDCl ) d:
3
2
6.2, 28.9, 29.2, 29.4, 29.5, 29.6, 30.0, 33.5, 37.5, 86.2,
1
2-(trityloxy)dodecanal (24c)
To a solution of oxalyl chloride (0.49 mL, 5.40 mmol) in
CH Cl (18 mL) was added DMSO (0.48 mL, 6.75 mmol)
89.9, 115.7, 126.5, 126.7, 127.7, 128.7, 140.2, 144.5,
153.1, 165.4; IR (Neat): 3481, 2926, 1716, 1636,
1597 cm .
ꢀ
1
2
2
Chem Biol Drug Des 2015; 86: 1304–1322
1313

Yamamoto et al.
(
1
3Z,4E)-3-(2-(methoxymethoxy)-2-oxoethylidene)-
6-(trityloxy)hexadec-4-enoic acid (KH-12 27c)
To a solution of the crude product in tBuOH/THF/2-
methyl-2-butene = 3/1/1 (3.4 mL) were added NaClO
(80 mg, 0.880 mmol) and NaH PO ・2H (192 mg,
2
To a solution of alcohol 26c (112 mg, 0.187 mmol) in
2
4
2
O
CH Cl (4.0 mL) was added DMP (317 mg, 0.748 mmol).
1.23 mmol) in H O (3.4 mL). The reaction mixture was stir-
2
2
2
The reaction mixture was stirred at RT for 1 h, and added
sat. Na S O aq., extracted with CH Cl washed with
red at RT for 1 h, and extracted with EtOAc, and washed
with brine, dried over MgSO . The crude product was
2
2
3
2
2,
4
brine, dried over MgSO
4
and gave a crude product.
purified by silica gel column chromatography (Hexane/
EtOAc=2/1) to give colorless oil (98 mg, 96%): H-NMR
1
To a solution of the crude product in tBuOH/THF/2-methyl-
3
(400 MHz in CDCl ) d: 1.04 (s, 9H), 1.26 (s, 10H), 1.43
2
0
H
-butene = 3/1/1 (3.8 mL) were added NaClO
2
(85 mg,
(brs, 2H), 1.51–1.57 (m, 2H), 2.23 (q, J = 6.8 Hz, 2H),
2.72 (t, J = 6.6 Hz, 2H), 3.38 (s, 2H), 3.65 (t, J = 6.6 Hz,
2H), 4.32 (t, J = 6.2 Hz, 2H), 5.73 (s,1H), 6.22 (td,
J = 7.6, 16.0 Hz, 1H), 7.36–7.44 (m, 6H), 7.52 (d,
.94 mmol) and NaH PO ・2H O (206 mg, 1.32 mmol) in
2
4
2
2
O. The reaction mixture was stirred at RT for 1 h, and
extracted with EtOAc, and washed with brine, dried over
MgSO . The crude product was purified by silica gel col-
umn chromatography (Hexane/EtOAc=2/1) to give yellow oil
13
4
J = 16.0 Hz, 1H), 7.67 (m, 4H);
C-NMR (100 MHz,
CD OD) d:18.4, 20.0, 26.8, 27.4, 30.0, 30.2, 30.3, 30.4,
3
1
(
(
38 mg, 37%): H-NMR (400 MHz in CDCl ) d: 1.24–1.65
m, 18H), 2.21 (q, J = 7.2 Hz, 2H), 3.03 (t, J = 6.6 Hz, 2H),
30.6, 33.6, 34.4, 41.1, 59.9, 65.0, 118.1, 127.7, 128.7,
130.8, 135.1, 136.7, 141.5, 150.7, 166.1, 174.1; IR (Neat):
3
ꢀ1
3
.38 (s,2H), 3.46 (s, 3H), 5.28 (s, 2H), 5.74 (s, 1H), 6.19 (dt,
3498, 2930, 2857, 2254, 1716, 1633, 1602 cm .
J = 7.2, 16.0 Hz, 1H), 7.20–7.24 (m, 3H), 7.27–7.30 (m,
13
6
H), 7.44 (m, 6H), 7.56 (d, J = 16.4 Hz, 1H); C-NMR
(
100 MHz, CDCl ) d: 26.2, 28.8, 29.2, 29.4, 29.5, 29.6,
3
(2Z,4E)-2-cyanoethyl-16-((tert-butyldiphenylsilyl)
oxy)-3-(2-hydroxyethyl)hexadeca-2,4-dienoate
(20c)
3
1
6
0.0, 33.4, 40.0, 57.6, 63.7, 86.2, 90.0, 118.1, 126.3,
26.8, 127.6, 128.7, 140.9, 144.5, 147.6, 165.1, 175.9 03-
ꢀ
1
4; IR (Neat): 3244, 2928, 2854, 1717, 1636, 1600 cm
.
To a suspension of Pd(PPh
MeOH (6.0 mL) were added cyanoethylester 19 (400 mg,
.37 mmol) and boronic ester 15c (600 g, 1.06 mmol) at
3 2 2
) Cl (189 mg, 0.27 mmol) in
1
(
2Z,4E)-2-cyanoethyl-14-((tert-butyldiphenylsilyl)
oxy)-3-(2-hydroxyethyl)tetradeca-2,4-dienoate
20b)
To a suspension of Pd(PPh ) Cl (154 mg, 0.22 mmol) in
RT. The mixture stirred at RT for 10 min, and NEt3
(1.04 mL, 7.42 mmol) was added. The reaction mixture
was stirred at RT for 3 h, and concentrated. The crude
product was purified by silica gel column chromatography
(
3
2
2
MeOH (2.0 mL) were added cyanoethylester (385 mg,
.31 mmol) and boronic ester (600 g, 1.09 mmol) at RT.
The mixture stirred at RT for 10 min, and NEt (1.10 mL,
1.0 mmol) was added. The reaction mixture was stirred
(Hexane/EtOAc=2/1) to give
42%): H-NMR (400 MHz in CDCl ) d: 1.05 (s, 9H), 1.25–
1.28 (m, 14H), 1.44–1.46 (m, 2H), 1.52–1.59 (m, 2H), 2.23
(q, J = 6.8 Hz, 2H), 2.63 (t, J = 6.6 Hz, 2H), 2.72 (t,
J = 6.4 Hz, 2H), 3.65 (t, J = 6.6 Hz, 2H), 3.79 (q,
a
yellow oil (255 mg,
1
1
3
3
1
at RT for 3 h, and evaporated. The crude product was
purified by silica gel column chromatography (Hexane/
J = 6.0 Hz, 2H), 4.31 (t, J = 6.6 Hz, 2H), 5.68 (s, 1H),
6.25 (td, J = 6.8, 16.0 Hz, 1H);
1
13
EtOAc=2/1) to give a yellow oil (316 mg, 50%): H-NMR
C-NMR (100 MHz,
(
1
400 MHz in CDCl
.52–1.57 (m, 2), 2.23 (q, J = 7.6 Hz, 2H), 2.63 (t,
J = 6.6 Hz, 2H), 2.72 (t, J = 6.2 Hz, 2H), 3.65
t, J = 6.6 Hz, 2H), 3.79 (q, J = 6.4 Hz, 2H), 4.31 (t,
J = 6.4 Hz, 2H), 5.68 (s, 1H), 6.25 (td, J = 6.8, 16.0 Hz,
3
) d: 1.05 (s,9H), 1.24–1.44 (m, 12H),
CDCl ) d: 18.0, 19.2, 25.7, 26.8, 29.0, 29.1, 29.2, 29.3,
3
29.4, 29.5, 29.6, 32.6, 33.5, 37.5, 58.1, 61.9, 64.0,
114.9, 116.9, 126.3, 127.5, 129.4, 134.1, 135.5, 140.7,
153.7, 165.2; IR (Neat): 3431, 2928, 2854, 2254, 1714,
(
ꢀ
1
+
1633 cm ; Mass (FAB) m/z 603 (M ); HRMS calcd for
37 53 4
C H O NSi: 603.3744 found 603.3749.
1
7
2
6
1
1
H), 7.35–7.44 (m, 6H), 7.49 (d, J = 16.0 Hz, 1H), 7.66–
1
3
.68 (m, 4H); C-NMR (100 MHz, CDCl ) d:18.0, 19.2,
3
5.7, 26.9, 28.8, 29.2, 29.3, 29.5, 32.5, 33.5, 39.8, 58.3,
4.0, 117.2, 126.1, 127.6, 129.5, 134.1, 135.6, 141.5,
48.2, 164.9; IR (Neat): 3419, 2930, 2857, 2252,
(3Z,4E)-14-((tert-butyldiphenylsilyl)oxy)-3-(2-(2-
cyanoethoxy)-2-oxoethylidene)tetradec-4-enoic
acid (KH-8, 21c)
ꢀ
1
716 cm
.
2 2
To a solution of alcohol 20c (65 mg, 0.11 mmol) in CH Cl
(
2.2 mL) was added DMP (140 mg, 0.33 mmol). The reac-
tion mixture was stirred at RT for 1 h, and added sat.
Na aq., extracted with CH Cl2, washed with brine,
(3Z,4E)-14-((tert-butyldiphenylsilyl)oxy)-3-(2-(2-
cyanoethoxy)-2-oxoethylidene)tetradec-4-enoic
acid (KH-5, 21b)
2
S
2
O
3
2
dried over MgSO and gave a crude product.
4
2 2
To a solution of alcohol (100 mg, 0.173 mmol) in CH Cl
(
7.0 mL) was added DMP (220 mg, 0.519 mmol). The
reaction mixture was stirred at RT for 1 h, and added sat.
Na aq., extracted with CH Cl2, washed with brine,
dried over MgSO and gave a crude product.
To a solution of the crude product in tBuOH/THF/2-
methyl-2-butene = 3/1/1 (0.90 mL) were added NaClO2
2
S
2
O
3
2
(41 mg, 0.46 mmol) and NaH
2
PO
4
2
・2H O
(99 mg,
0.64 mmol) in H O (0.90 mL). The reaction mixture was
4
2
1
314
Chem Biol Drug Des 2015; 86: 1304–1322

pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier
stirred at RT for 1 h, and extracted with EtOAc, and
washed with brine, dried over MgSO . The crude product
and MOMCl (7.04 g, 54.5 mmol) at 0 °C. The reaction
4
mixture was stirred at RT for 2 h, and added sat, NaHCO
aq, extracted with CH Cl2, and washed with brine, dried
3
was purified by silica gel column chromatography (Hex-
2
1
ane/EtOAc=2/1) to give colorless oil. (45 mg, 66%): H-
over MgSO . The crude product was purified by silica gel
4
NMR (400 MHz in CDCl
4H), 1.44 (brs, 2H), 1.52–1.57 (m, 2H), 2.23 (q,
J = 7.6 Hz, 2H), 2.72 (t, J = 6.4 Hz, 2H), 3.38 (s, 2H),
.65 (t, J = 6.6 Hz, 2H), 4.32 (t, J = 6.2 Hz, 2H), 5.73
s,1H), 6.22 (td, J = 6.8, 16.8 Hz, 1H), 7.36–7.44 (m, 6H),
3
) d: 1.04 (s, 9H), 1.24–1.28 (s,
column chromatography (Hexane/EtOAc=2/1) to give col-
1
1
orless oil (5.42 g, 99%): H-NMR (400 MHz in CDCl ) d:
3
1.24–1.28 (m, 16H), 1.56–1.66 (m, 6H), 2.35 (t,
J = 7.6 Hz, 2H), 3.46 (s, 3H), 3.63 (t, J = 6.6 Hz, 2H),
3
(
13
5.23 (s, 2H); C-NMR (100 MHz, CDCl
3
) d: 24.7, 25.7,
13
7.52 (d, J = 16.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 4H); C-
29.0, 29.2, 29.3, 29.4, 29.5, 32.7, 34.2, 57.5, 62.9, 90.1,
ꢀ
1
NMR (100 MHz, CD OD) d: 18.4, 20.0, 26.8, 27.4, 29.9,
173.4; IR (Neat): 3460, 2926, 2854, 1747 cm ; Mass (EI)
3
+
3
1
1
1
0.2, 30.3, 30.5, 30.6, 33.6, 34.4, 41.1, 59.9, 65.0,
18.1, 118.7, 127.8, 128.7, 130.8, 135.1, 136.6, 141.5,
50.8, 166.7, 174.2.; IR (Neat): 3466, 2928, 2857, 2254,
29 4
m/z 261 (M +1); HRMS calcd for C14H O : 261.1998
found 261.2073.
ꢀ
1
+
739 cm ; Mass (FAB) m/z 618 (M + 1); HRMS calcd
for C H O Si: 618.3615 found 618.3620.
Methoxymethyl 12-oxododecanoate (32)
3
7 52 5
To a solution of oxalyl chloride (0.24 mL, 2.76 mmol) in
CH Cl2 (10 mL) was added DMSO (0.24 mL, 3.45 mmol)
2
1
(
2-((tert-butyldiphenylsilyl)oxy)dodecanoic acid
29)
To a solution of aldehyde 14c in tBuOH/THF/2-methyl-2-
butene = 3/1/1 (60 mL) and H O (60 mL) were added Na-
ClO2 (11.3 g, 125 mmol) and NaH PO ・2H O (19.5 g,
at ꢀ78 °C. and stirred for 20 min, and alcohol 31
(300 mg, 1.15 mmol) in CH Cl (6.0 mL) was added at
2
2
ꢀ78 °C. The reaction mixture stirred at ꢀ78 °C for
20 min, and NEt (1.21 mL, 8.63 mmol) was added at
ꢀ78 °C. The mixture was stirred at RT for 1 h, and
2
quenched with sat, NaHCO aq., extracted with CH Cl2,
2
3
2
4
2
125 mmol). The reaction mixture was stirred at RT for
3
1.5 h, and extracted with EtOAc, and washed with brine,
and washed with brine, dried over MgSO . The crude
4
dried over MgSO
4
. The crude product was purified by sil-
product was purified by silica gel column chromatography
ica gel column chromatography (Hexane/EtOAc=2/1) to
give colorless oil (4.97 g, 87%): H-NMR (400 MHz in
(Hexane/EtOAc = 4:1) to give a yellow oil. (295 mg, 99%):
H-NMR (400 MHz in CDCl ) d: 1.28 (s, 13H), 1.59–1.64
3
1
1
CDCl
3
) d: 1.05 (s, 9H), 1.24–1.31 (m, 14H), 1.53–1.63 (m,
(m, 3H), 2.35 (t, J = 7.4 Hz, 2H), 2.42 (t, J = 7.4 Hz, 2H),
4
7
H), 2.35 (t, J = 7.4 Hz, 2H), 3.65 (t, J = 6.6 Hz, 2H),
.37–7.42 (m, 6H), 7.67 (m, 2H); C-NMR (100 MHz,
3.46 (s, 3H), 5.23 (s, 2H), 9.77 (s, 1H).
13
CDCl ) d: 19.2, 24.7, 25.7, 26.9, 29.0, 29.2, 29.3, 29.4,
3
2
1
9.5, 29.6, 32.5, 34.1, 64.0, 127.5, 129.4, 134.1, 135.6,
(E)-methoxymethyl 13-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)tridec-12-enoate (33)
ꢀ
1
80.3; IR (Neat): 3134, 2928, 2854, 1708, 1589 cm
.
To a suspension of CrCl
2
(840 mg 6.84 mmol) and LiI
(610 mg, 4.56 mmol) in THF (4.0 mL) were added pinacol
Methoxymethyl 12-((tert-butyldiphenylsilyl)oxy)
dodecanoate (30)
borane 11 (480 mg, 2.28 mmol) in THF (3.0 mL) and alde-
hyde 32 (295 mg, 1.14 mmol) in THF (3.0 mL). The mix-
ture was stirred at RT for 2.5 h, and added sat.NaHCO3,
extracted with EtOAc, and washed with brine, dried over
To a solution of carboxylic acid 29 (4.97 g, 10.9 mmol) in
CH Cl were added diisopropylethylamine (5.27 g,
5.4 mmol) and MOMCl (7.04 g, 54.5 mmol) at 0 °C. The
reaction mixture was stirred at RT for 1.5 h, and added
sat, NaHCO aq, extracted with CH Cl2, and washed with
2
2
6
MgSO
4
. The crude product was purified by silica gel col-
umn chromatography (Hexane/EtOAc=10/1) to give green
1
3
2
3
oil (140 mg, 32%): H-NMR (400 MHz in CDCl ) d: 1.24–
brine, dried over MgSO . The crude product was purified
1.40 (m, 26H), 1.64–1.66 (m, 2H), 2.14 (q, J = 6.8 Hz,
2H), 2.35 (t, J = 7.4 Hz, 2H), 3.46 (s, 3H), 5.23 (s, 2H),
5.42 (d, J = 18.4 Hz, 1H), 6.63 (td, J = 6.4, 18.4 Hz, 1H);
4
by silica gel column chromatography (Hexane/EtOAc=2/1)
1
to give colorless oil (5.42 g, 99%): H-NMR (400 MHz in
13
CDCl
H), 2.36 (t, J = 7.6 Hz, 2H), 3.46 (s, 3H), 3.65 (t,
J = 6.6 Hz, 2H), 5.23 (s, 2H), 7.36–7.42 (m, 6H), 7.69–
3
) d: 1.05 (s, 9H), 1.25–1.28 (m, 14H), 1.54–1.55 (m,
3
C-NMR (100 MHz, CDCl ) d: 24.7, 28.1, 29.0, 29.1,
4
29.2, 29.3, 29.4, 29.5, 34.3, 35.8, 57.5, 82.9, 90.1,
ꢀ
1
154.7, 173.3; IR (Neat): 2928, 2854, 1745, 1637 cm
;
:
1
3
+
7
2
9
2
.71 (m, 2H); C-NMR (100 MHz, CDCl
3
) d: 19.1, 24.7,
Mass (FAB) m/z 383 (M +1); HRMS calcd for C21H40BO
5
5.7, 26.8, 29.0, 29.2, 29.3, 29.5, 32.5, 34.2, 57.4, 63.9,
0.0, 127.5, 127.6, 129.4, 134.1, 135.2, 135.5; IR (Neat):
929, 2856, 1739 cm
383.2969 found 383.2973.
ꢀ
1
.
(
2Z,4E)-bis(methoxymethyl) 3-(2-hydroxyethyl)
hexadeca-2,4-dienedioate (34)
To a suspension of Pd(PPh ) Cl (67 mg, 0.0952 mmol)
Methoxymethyl 12-hydroxydodecanoate (31)
3
2
2
To a solution of ester 30 (4.97 g, 10.9 mmol) in CH
2
Cl
2
in MeOH (1.0 mL) were added Segment A (148 mg,
were added diisopropylethylamine (5.27 g, 65.4 mmol)
0.523 mmol)
and
boronic
ester
33
(182 mg,
Chem Biol Drug Des 2015; 86: 1304–1322
1315

Yamamoto et al.
Figure 2: Experimental procedure to evaluate inhibitory effects of bongkrekic acid (BKA) analogues on mitochondrial ATP synthesis. The
experimental system used to evaluate the effects of BKA analogues on the mitochondrial ATP synthesis is shown. Broken line indicates
the changes in the pH of the incubation medium without the addition of ADP and BKA analogues (negative control), and dotted line
indicates the changes in the pH of the incubation medium caused by the addition of ADP in the absence of BKA analogues (positive
control). The left and right traces represent the typical results of the experiments at pH 7.4 and 6.8, respectively.
1
1
73.4; IR (Neat): 3444, 2926, 2854, 1732, 1716, 1635,
597 cm ; Mass (EI) m/z 414 (M ); HRMS calcd for
ꢀ
1
+
C H O : 414.2618 found 414.2612.
22 38 7
(
(
3Z,4E)-16-(methoxymethoxy)-3-(2-
methoxymethoxy)-2-oxoethylidene)-16-
oxohexadec-4-enoic acid (KH-14, 35)
2 2
To a solution of alcohol 34 (100 mg, 0.24 mmol) in CH Cl
(
5.0 mL) was added DMP (407 mg, 0.96 mmol). The reac-
tion mixture was stirred at RT for 1 h, and added sat.
Na aq., extracted with CH Cl2, washed with brine,
dried over MgSO and gave a crude product.
2
S
2
O
3
2
4
Figure 3: Inhibitory effects of bongkrekic acid (BKA) analogues
on mitochondrial ATP synthesis. Inhibitory effects of individual
BKA analogues at 1 lM (white column) or 10 lM (black column)
on mitochondrial ATP synthesis were examined by measuring the
suppression of the pH change caused by the addition of ADP at
pH 6.8. The results obtained by 3 independent runs are shown as
To a solution of the crude product in tBuOH/THF/2-
methyl-2-butene =3/1/1 (5.0 mL) were added NaClO
(109 mg, 1.20 mmol) and NaH PO ・2H O (262 mg,
2
2
4
2
1
.68 mmol) in H
red at RT for 1 h, and extracted with EtOAc, and washed
with brine, dried over MgSO . The crude product was
2
O (5.0 mL). The reaction mixture was stir-
4
%
inhibition of ATP synthesis (mean ꢂ SD). The rate of the ATP
purified by silica gel column chromatography (Hexane/
synthesis in the absence of BKA analogues (i.e., positive control
1
EtOAc=2/1) to give colorless oil (38 mg, 37%): H-NMR
in Figure 1B) was 297 ꢂ 46 nmol/mg/min.
(400 MHz in CDCl
3
) d: 1.24–1.30 (m, 14H), 1.43 (brs, 2H),
1
.63–1.66 (m, 2H), 2.22 (q, J = 7.6 Hz, 2H), 2.36 (t,
0
1
.476 mmol) at RT. The mixture stirred at RT for
0 min, and NEt (1.76 mL, 12.5 mmol) was added. The
J = 7.4 Hz, 2H), 3.38 (s, 2H), 3.47 (s, 3H), 3.48 (s, 3H),
5.24 (s, 2H), 5.28 (s, 2H), 5.74 (s, 1H), 6.21 (td, J = 7.6,
3
13
reaction mixture was stirred at RT for 3 h, and evapo-
rated. The crude product was purified by silica gel col-
15.6 Hz, 1H), 7.56 (d, J = 16.0 Hz, 1H);
C-NMR
(100 MHz, CD OD) d: 25.9, 29.9, 30.1, 30.2, 30.3, 30.4,
3
umn chromatography (Hexane/EtOAc = 2/1) to give a
30.5, 30.6, 34.4, 35.1, 41.0, 57.7, 90.9, 91.2, 118.6,
127.8, 141.4, 150.7, 166.7, 174.1, 174.9; IR (Neat): 3083,
1
yellow oil (195 mg, 98%): H-NMR (400 MHz in CDCl
3
)
ꢀ
1
d: 1.27 (brs, 12H), 1.43 (brs, 2H), 1.63–1.66 (m, 2H),
.21 (q, J = 7.6 Hz, 2H), 2.35 (t, J = 7.8 Hz, 2H), 2.64
t, J = 6.6 Hz, 2H), 3.46 (s, 3H), 3.48 (s, 3H), 3.79 (q,
J = 6.4 Hz, 2H), 5.23 (s, 2H), 5.28 (s, 2H), 5.69 (s, 1H),
2933, 2928, 2854, 1737, 1717, 1635 cm ; Mass (FAB)
+
2
(
37 8
m/z 429 (M +1); HRMS calcd for C22H O : 429.2488
found 429.2498.
6
1
2
6
.22 (td, J = 7.2, 16.0 Hz, 1H), 7.53 (d, J = 16.4 Hz,
1
3
H); C-NMR (100 MHz, CD OD) d: 24.8, 28.9, 29.0,
Preparation of mitochondria from rat liver
Mitochondria were prepared from the liver of 8-week-old
male Wistar rats by the differential centrifugation procedure
3
9.1, 29.2, 29.3, 29.4, 29.5, 33.4, 34.3, 37.5, 57.5,
1.9, 89.7, 90.1, 115.7, 126.5, 140.1, 153.1, 165.4,
1
316
Chem Biol Drug Des 2015; 86: 1304–1322

pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier
A
B
Figure 4: Evaluation of the permeabilization effects of the four bongkrekic acid (BKA) analogues on the mitochondrial inner membrane.
Panel A presents the experimental system used for evaluation of permeabilization of the mitochondrial inner membrane. The very slow
oxygen consumption observed in the absence of added chemicals (broken line) and the rapid oxygen consumption caused by the
addition of SF6847 (dotted line) indicated the mitochondrial inner membrane showing lowest and highest permeability, respectively. Panel
B shows the effects of the BKA analogues on the rate of mitochondrial oxygen consumption. The rates of oxygen consumption observed
in the absence of chemicals or in the presence of SF6847 are shown as hatched columns. Those observed in the presence of BKA or its
analogues at 1 and 10 lM are shown with white and black columns, respectively. The bars represent the SD values of individual
experiments.
A
B
Figure 5: Evaluation of the inhibitory effects of bongkrekic acid (BKA) analogues on the mitochondrial electron transport system. Panel A
outlines the experiment used to evaluate the inhibitory effects of BKA on the mitochondrial electron transport system. The solid line shows
the result obtained in the absence of BKA; and the broken line, that in the presence of 10 lM BKA. Panel B shows the inhibitory effect of
BKA and its analogues on the SF6847-stimulated oxygen consumption. The results observed in the presence of BKA and its analogues at
1
or 10 lM are shown with white and black columns, respectively. The bars represent the SD values of individual experiments. The
maximum rate of oxygen consumption observed in the absence of BKA or its analogue (trace ‘control’ in panel A) was 141 ꢂ 17
natomsO/mg/min.
reported previously (13). Final mitochondrial pellets were
suspended in aliquots of medium containing 250 mM
sucrose and 2 mM Tris-Cl at pH 7.4. The resulting suspen-
sion was used as a stock solution of mitochondria and
kept on ice during the experiments. Its protein concentra-
tion was determined by the biuret method using bovine
serum albumin as a standard.
Evaluation of the inhibitory effects of BKA
analogues on mitochondrial ATP synthesis
The velocity of ATP synthesis in the mitochondrial suspen-
sion was measured by calculating the change in the pH of
the incubation medium (13,14). Briefly, mitochondria were
suspended in the appropriate amount of medium (3 mM
potassium phosphate buffer, pH 7.4 or 6.8, containing
Chem Biol Drug Des 2015; 86: 1304–1322
1317

Yamamoto et al.
Figure 6: Comparison of the pH
dependency of the inhibitory effects of
bongkrekic acid (BKA) and KH-7 on ATP
synthesis. Inhibitory effects on the
mitochondrial ATP synthesis of BKA and
KH-7 at various concentrations in three
incubation media of pH 6.3, 6.8, and 7.4
were examined by measuring the pH of the
incubation medium as stated in Figure 1.
Rates of ATP synthesis in the absence of
BKA or KH-7 (i.e., control) at pH 6.3, 6.8,
and 7.4 were 174 ꢂ 22, 297 ꢂ 46, and
384 ꢂ 36 nmol/mg/min, respectively.
electron transport system were determined by measuring
the rate of oxygen consumption by mitochondria. For this
purpose, mitochondria were suspended in the above-men-
tioned medium (pH 6.8), supplemented with 5 mM succi-
nate (plus 1.25 lg/mL rotenone) as
a
respiratory
substrate. After the addition of a certain amount of individ-
ual BKA analogues, time-dependent changes in the oxy-
gen concentration of the mitochondrial suspension were
monitored at 25 °C by a Clark-type oxygen electrode (Yel-
low Springs Instrument, model 5331, YSI Life Sciences,
Yellow Springs, OH, USA).
Evaluation of the inhibitory effects of KH-7 on the
mitochondrial ADP uptake
To evaluate the direct action of KH-7 on the mitochondrial
3
ADP/ATP carrier, we measured the uptake of [ H]ADP into
mitochondria as described previously (15). Briefly, mito-
chondria (1 mg protein) were suspended in the above-
mentioned medium (0.5 mL, pH 7.4, not supplemented
with the respiratory substrate) and incubated at 25 °C for
2 min in the presence or absence of certain chemicals
(BKA, CATR or KH-7). Then, a 10-lL aliquot of 2 mM ADP
Figure 7: Direct inhibitory effects of KH-7 on the mitochondrial
ADP/ATP carrier. For evaluation of the direct effects of KH-7 on
the mitochondrial ADP/ATP carrier, uptake of ADP was measured
by using [ H]ADP as a tracer. ADP uptakes observed in the
absence (none) and presence of an inhibitor (CATR or BKA at
3
3
1
0 lM) were used as positive and negative controls, respectively.
solution containing [ H]ADP (specific radioactivity of
For details of experiments, see the Methods section.
1
1
85 MBq/mmol ADP) was added. After an incubation for
0 seconds, the reaction was terminated by the addition of
2
00 mM sucrose, 20 mM KCl, 3 mM MgCl ) to give a pro-
a sufficient amount of CATR to make its final concentration
of 10 lM; and mitochondria were then pelleted by centrifu-
gation at 5500 9 g for 1 min. After complete removal of the
supernatant, the mitochondrial pellet was solubilized with
200 lL of 1% SDS solution. The radioactivity in 50 lL of
the solubilized mitochondrial solution was counted in an
Aloka liquid scintillation counter, model LSC-3500, Hitachi
Aloka Medical, Ltd., Tokyo, Japan.
2
tein concentration of 0.7 mg/mL. After the addition of
0 mM succinate (plus 1.25 lg/mL rotenone) as a respira-
1
tory substrate, ATP synthesis was started by the addition
of 200 lM ADP. The incubation time-dependent change in
the pH of the mitochondrial suspension was monitored by
pH electrode model PCE108CW (Tokokagaku, Tokyo,
Japan) and recorded at 25 °C. The changes in pH were
calibrated by the addition of 100 lM oxalic acid.
Results
Evaluation of the membrane permeabilization
effects of BKA analogues, and inhibitory effects of
these analogues on the mitochondrial electron
transport system
The membrane permeabilization effects of the BKA ana-
logues, and their inhibitory effects on the mitochondrial
Bongkrekic acid analogues used in the present
study
Bongkrekic acid has 3 carboxylic acids connected by a
branched chain of partially unsaturated fatty acid. This
structure suggested to us that the anionic functional
1
318
Chem Biol Drug Des 2015; 86: 1304–1322

pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier
groups such as carboxylate would be essential for the
binding of BKA to biomolecules and that the fatty acid
chain would control the spatial configuration of these func-
tional groups. Hence, we designed a general structure for
the BKA analogues used in the present study (Figure 1A),
one consisting of two parts, left and right. As shown in
Table 1, the left part contained carboxylate(s) and/or an
As shown in Figure 3, the inhibitory effects of the BKA
analogues on mitochondrial ATP synthesis were much
more moderate than that effect of BKA, and no analogues
showed perfect inhibition at 1 lM, as seen with BKA. How-
ever, the actions of 4 of the analogues, that is, KH-1, KH-
7, KH-16 and KH-17, were relatively remarkable; and
these compounds at 10 lM completely suppressed the
alkalinization of the incubation medium. Therefore, we
focused on these four BKA analogues in subsequent
experiments.
1
ester (R ) of acid-labile methoxymethyl ester or base-labile
cyanoethyl ester, a (Z,E)-conjugated diene, and a lipophilic
fatty acid chain of various length (n = 6, 8, 10, and 16);
and the right part, a functional group of hydrophilic car-
boxy (COOH), methoxymethyl ester (methoxymethyloxycar-
bonyl group), hydroxy (OH) group, highly hydrophilic
As stated above, an increase in the pH of the incubation
medium reflects inhibition of ATP synthesis. In the case of
BKA at low concentrations, its inhibitory effect on ATP syn-
thesis could be simply attributable to its inhibitory action on
the ADP/ATP carrier, because it does not show remarkable
side effects on other mitochondrial functions. However,
when we evaluate the effects of new chemicals on mito-
chondrial ATP synthesis, we must pay adequate attention
to their possible side effects that may influence this synthe-
sis. Thus, we next tested whether these analogues had
any (i) permeabilization effects on the mitochondrial inner
membrane, or (ii) inhibitory effects on the mitochondrial
electron transport system, because these actions are major
side effects that would influence mitochondrial ATP synthe-
sis. These effects could be evaluated by measuring the
oxygen consumption by mitochondria.
t
t
(
lipophilic) trialkylsiloxy groups (Si BuPh
2 2 3
, Si BuPh , SiPh ),
or alkyl ether (CPh ). We prepared a total of 17 BKA ana-
3
logues and examined their effects on the mitochondrial
ADP/ATP carrier.
Inhibitory effects of these 17 BKA analogues on
mitochondrial ATP synthesis
First, we roughly tested the inhibitory effects of these 17 BKA
analogues on the mitochondrial ADP/ATP carrier by measur-
ing their effects on the ATP synthesis in isolated mitochon-
dria. If a certain one of these chemicals showed an inhibitory
effect on the mitochondrial ADP/ATP carrier, import of ADP
into the mitochondrial matrix would be suppressed; and,
+
hence, ATP synthesis would be inhibited. Because H is
consumed during ATP synthesis, as shown below,
Side effects of BKA analogues affecting ATP
synthesis
ADP þ Pi þ Hþ ! =ATP þ H2O
We first evaluated the permeabilization effects of BKA ana-
logues on the mitochondrial inner membrane. As shown in
Figure 4A, when mitochondria were suspended in the med-
ium containing a respiratory substrate (trace ‘control’), grad-
ATP synthesis in mitochondria can be determined by mea-
suring the pH of the incubation medium (14). In addition, the
inhibitory effect of BKA, the parental compound of the
chemicals used, on the mitochondrial ATP synthesis is
known to be sensitive to the pH of the incubation medium
+
ual oxygen consumption, to compensate for the H leakage
through the mitochondrial inner membrane, was observed.
The addition of 100 nM SF6847, a most effective protono-
phore (17,18), remarkably accelerated the mitochondrial
oxygen consumption. Using these two conditions as the
(16). Thus, the actions of the test chemicals were evaluated
under two pH conditions, those of 7.4 and 6.8. As shown in
Figure 2, the addition of ADP to the mitochondria sus-
pended in medium containing inorganic phosphate (Pi)
caused alkalinization of the incubation medium, thus reflect-
ing ATP synthesis, regardless of the differences in pH of the
incubation medium. In the medium of pH 7.4, BKA at 1 lM
had a partial inhibitory effect on the alkalinization of the incu-
bation medium, but that at 10 lM suppressed it perfectly.
On the contrary, in the medium of pH 6.8, BKA was effec-
tive in suppressing the alkalinization of the incubation med-
ium even at 1 lM. As these results supported the reported
pH-dependent action of BKA on mitochondrial ATP synthe-
sis, that is, stronger effects at a slightly acidic pH than at
neutral pH (16), this experimental system was concluded to
be suitable for evaluation of the inhibitory effects of BKA
analogues on mitochondrial ATP synthesis; and so we
adopted it for evaluating the action of the 17 BKA ana-
logues. Because the inhibitory effect of BKA on the mito-
chondrial ATP synthesis was more remarkable at pH 6.8
than at pH 7.4, we tested the analogue action at pH 6.8.
+
membrane states showing the lowest and the highest H
permeabilities, respectively, we examined the permeabiliza-
tion effects of the 4 BKA analogues on the mitochondrial
inner membrane. As shown in Figure 4B, KH-16 at 10 lM
markedly increased the rate of mitochondrial oxygen con-
sumption, and KH-1 and KH-17 showed weak acceleration
effects when tested at the same concentration. KH-7 also
slightly accelerated the mitochondrial oxygen consumption
at 10 lM, but this effect was almost negligible.
We next evaluated the inhibitory effects of BKA analogues
on the mitochondrial electron transport system. This
effect was evaluated by measuring how the rate of oxy-
gen consumption stimulated by SF6847 would be inhib-
ited by the individual BKA analogues. For this purpose,
mitochondria were pretreated with BKA or with KH-1,
KH-7, KH-16 or KH-17 at 1 or 10 lM for 1 min, and then
100 nM SF6847 was added. If BKA analogues had an
Chem Biol Drug Des 2015; 86: 1304–1322
1319

Yamamoto et al.
inhibitory effect on the mitochondrial electron transport
system, then the rate of oxygen consumption effected by
the addition of SF6847 would be decreased. A typical
result obtained with 10 lM BKA is shown in Figure 5A;
and a summary of the results obtained with the 4 BKA
analogues, in Figure 5B. BKA itself showed moderate
inhibitory effects on the mitochondrial electron transport
system at 10 lM BKA. KH-7 also showed moderate
inhibitory effects at 10 lM, almost to the same extent as
observed with BKA; but the other 3 BKA analogues, KH-
simply evaluate its direct action on the carrier, as demon-
strated in the last part of the Results section. However, it
is also very important to know whether it shows remark-
able side effects on other mitochondrial functions aside
from the ADP/ATP carrier, especially when we test the
compound on the whole mitochondria rather than on the
isolated ADP/ATP carrier. Therefore, in the present study,
we first examined the effects of BKA analogues on mito-
chondrial ATP synthesis and on other properties of mito-
chondria. Among the 17 analogues tested, 4 of them, that
is, KH-1, KH-7, KH-16, and KH-17, showed inhibitory
effects on mitochondrial ATP synthesis. Of these, the
inhibitory effect of KH-16 on mitochondrial ATP synthesis
could hardly be attributed to inhibition of ADP/ATP carrier,
because it showed remarkable side effects on the mito-
chondrial membrane permeability and respiratory chain.
When we focused on the remaining 3 analogues (KH-1,
KH-7, and KH-17), it became evident that all of them had
a common left part of their structure, the part consisting
of two carboxyl groups. This moiety may have been
essential for their inhibitory effects on ADP/ATP carrier,
because substitution of R1 position of KH-7 with MOM
group (i.e., KH-9) caused a loss of the inhibitory effects of
KH-7.
1
, KH-16, and KH-17, showed much stronger effects
than BKA. However, these effects were almost negligible
at the 1 lM concentration. Based on these results, the
inhibitory effects of KH-1, KH-16, and KH-17 on the
mitochondrial ATP synthesis were concluded to be not
simply attributable to the results of the specific inhibition
of mitochondrial ADP/ATP carrier; but KH-7 was con-
cluded to specifically inhibit mitochondrial ADP/ATP car-
rier without showing remarkable side effects on the other
mitochondrial functions.
pH dependency of the inhibitory effect of BKA and
its analogues on mitochondrial ATP synthesis
We next examined the pH dependency of the inhibitory
effect of BKA and KH-7 on mitochondrial ATP synthesis.
As shown in Figure 6 (see also Figure 2, left traces), BKA
showed a strong pH-dependent inhibitory effect on the
ATP synthesis, and it showed strong inhibition, with it
being stronger at a slightly acidic pH than at pH 7.4. On
the contrary, KH-7 showed a weak and opposite pH
dependency; that is, it was most effective at neutral pH
Interestingly, moreover, KH-17 had 3 carboxyl groups, like
the parental BKA. It should be also emphasized that the
length of the alkyl chain connecting left and right parts of
KH-17 was essentially the same as that of the parental
BKA, as schematically shown in our recent paper (12).
Therefore, the presence of a third carboxyl group at right
part of the molecule, even at a distance from the two car-
boxyl groups in the left part, similar to that of BKA, was a
‘non-sufficient condition’ for strong inhibitory effects
against ADP/ATP carrier. When we compared the struc-
tures of BKA and KH-17, BKA had 3 additional methyl
groups and one OMe group. Inclusion of these side chains
would be expected to have made the molecule much
more hydrophobic. If we assume that the higher hydro-
phobicity of BKA, especially at the right side of the mole-
cule, was necessary for the stronger inhibitory effect on
the ADP/ATP carrier, the strongest inhibitory effect of HK-
7 among the 17 BKA analogues observed in the present
study becomes explainable, because it has the same left
side structure as that of BKA, and has a sterically bulky
hydrophobic group, the tert-butyl diphenylsilyl (TBDPS)
group, as the right part of the structure. However, it
should be mentioned that other analogues sharing the
same right and left parts of the molecule as those of KH-
7, but having different alkyl chain lengths, such as KH-1
and KH-4, showed weak or no inhibitory effects on the
mitochondrial ADP/ATP carrier. A possible explanation for
the different activities of these analogues is that the pres-
ence of this TBDPS group at an appropriate distance from
the left part of the structure containing the two carboxyl
groups was important for the inhibitory action on the ADP/
ATP carrier. To validate these interpretations, further struc-
ture–activity relationship studies, using analogues lacking
(7.4) and rather weaker at the slightly acidic pH. Therefore,
KH-7 was concluded to have lost its parental pH depen-
dency. Possibly, the third carboxyl group present in the
parental BKA but lacking in KH-7 was responsible for the
pH dependency of the inhibitory action of BKA.
Evaluation of the direct inhibitory effects of KH-7
on the mitochondrial ADP/ATP carrier
Finally, we tested the direct action of KH-7 on the mitochon-
3
drial ADP/ATP carrier by measuring the uptake of [ H]ADP.
KH-7 at lower concentrations such as 1–10 lM was not
remarkably effective, but clear suppression of ADP uptake
was observed at 20 or 50 lM, as shown in Figure 7. It
should be emphasized that the ADP uptake via the ADP/
ATP carrier observed in the present experimental condition
was not influenced by the factors such as an increase in the
+
H permeability caused by SF6847. Therefore, a moderate
but specific inhibitory effect of KH-7 on the mitochondrial
ADP/ATP carrier was clearly demonstrated.
Discussion
To examine whether a certain compound has an inhibitory
effect on the mitochondrial ADP/ATP carrier, we can
1
320
Chem Biol Drug Des 2015; 86: 1304–1322

pH-resistant Inhibitor of Mitochondrial ADP/ATP Carrier
the TBDPS group as a negative control, would seem to be
required.
Food Industry. We thank Prof. A. Kano (Kyushu University)
for helpful discussions.
One of the remarkable characteristics observed for KH-7
was its pH-independent inhibitory action toward the ADP/
ATP carrier. We would like to consider why KH-7 showed
such a unique property. As clearly mentioned in the past
study (19), for the inhibition of the ADP/ATP carrier, BKA
must be translocated to the matrix side of the mitochon-
drial inner membrane; and for efficient translocation
across the mitochondrial inner membrane, the carboxyl
groups of the BKA molecule should not be dissociated
Conflict of Interest
The authors have no financial/commercial conflicts of inter-
est to be declared.
References
(
i.e., only the neutral form of BKA can be easily translo-
1. Palmieri F. (2013) The mitochondrial transporter family
SLC25: identification, properties and physiopathology.
Mol Aspect Med;34:465–484.
2. Cl eꢀ mencßon B., Babot M., Tr eꢀ z eꢀ guet V. (2013) The
mitochondrial ADP/ATP carrier (SLC25 family): patho-
logical implications of its dysfunction. Mol Aspect
Med;34:485–493.
3. Klingenberg M. (2008) The ADP and ATP transport in
mitochondria and its carrier. Biochim Biophys
Acta;1778:1978–2021.
cated to the matrix side of the mitochondrial inner mem-
brane). Therefore, loss of pH dependence in the
inhibitory action on the ADP/ATP carrier observed with
KH-7 could be attributable to the absence of the car-
boxyl group in its right part. In addition, lack of this third
carboxyl group in its right part may also have significantly
weakened the inhibitory action of KH-7 toward the ADP/
ATP carrier. To examine these possibilities, a further
study on the structure/activity relationship of BKA ana-
logues, such as that examining the BKA molecule just
lacking its right carboxyl group or that evaluating the KH-
4. Nury H., Dahout-Gonzalez C., Tr eꢀ z eꢀ guet V., Lauquin
G.J., Brandolin G., Pebay-Peyroula E. (2006) Relations
between structure and function of the mitochondrial
ADP/ATP carrier. Annu Rev Biochem;75:713–741.
7
derivative having the right carboxyl group, would seem
to be necessary.
5
. Bruni A., Luciani S., Contessa A.R. (1964) Inhibition by
atractyloside of the binding of adenine nucleotides of
rat liver mitochondria. Nature;201:1219–1220.
In the present study, we successfully identified KH-7 as
a new analogue of BKA. Although the parental BKA
molecule is hard to synthesize and shows strong pH
dependence in its action, synthesis of KH-7 was
much easier (12); and this analogue did not show strong
pH dependence. Although KH-7 showed a much weaker
inhibitory effect on the mitochondrial ADP/ATP carrier
than BKA, its action was specific for the carrier.
Thus, KH-7 could be a nice tool to understand the
catalytic mechanism of the mitochondrial ADP/ATP
carrier.
6. Du eꢀ e E.D., Vignais P.V. (1965) Exchange between
extra- and intramitochondrial adenine nucleotides. Bio-
chim Biophys Acta;107:184–188.
7. Pfaff E., Klingenberg M., Heldt H.W. (1965) Unspecific
permeation and specific exchange of adenine nucleo-
tides in liver mitochondria. Biochim Biophys
Acta;104:312–315.
8. Aquila H., Misra D., Eulitz M., Klingenberg M. (1982)
Complete amino acid sequence of the ADP/ATP carrier
from beef heart mitochondria. Hoppe-Seyler’s J Physiol
Chem;363:345–349.
9. Pebay-Peyroula E., Dahout-Gonzalez C., Kahn R.,
Tr eꢀ z eꢀ guet V., Lauquin G.J.M., Brandolin G. (2003)
Structure of mitochondrial ADP/ATP carrier in complex
with carboxyatractyloside. Nature;426:39–44.
Presently, we only focused on the inhibitory effects of
BKA analogues on its primary target protein, that is, the
mitochondrial ADP/ATP carrier. However, BKA is also
thought to work as an inhibitor of mitochondrion-depen-
dent apoptosis, because it largely suppresses the mito-
chondrial permeability transition (20), which causes the
release of mitochondrial cytochrome c (a trigger of apop-
tosis), into cytosol. Thus, studies on BKA analogues are
important for possible development of therapeutics
for the treatment of certain mitochondrion-dependent
diseases.
10. Stubbs M. (1981) Inhibitors of the adenine nucleotide
translocase. In: Ereci nꢀ ska M., Wilson D.F., editors.
Inhibitors of Mitochondrial Functions. Oxford: Perg-
amon Press; p. 283–304.
11. Sato Y., Aso Y., Shindo M. (2009) Efficient synthesis of
bongkrekic acid. Three-component convergent strat-
egy. Tetrahedron Lett;50:4164–4166.
1
2. Okuda K., Hasui K., Abe M., Matsumoto K., Shindo
M. (2012) Molecular design, synthesis, and evaluation
of novel potent apoptosis inhibitors inspired from bon-
gkrekic acid. Chem Res Toxicol;25:2253–2260.
Acknowledgments
This study was supported by a research fund from the
Network Joint Research Center for Materials and Devices,
and by a grant from Science and Technology Research
Promotion Program for Agriculture, Forestry, Fisheries, and
13. Terada H., Shima O., Yoshida K., Shinohara Y. (1990)
Effects of the local anesthetic bupivacaine on oxidative
phosphorylation in mitochondria. Change from
Chem Biol Drug Des 2015; 86: 1304–1322
1321

Yamamoto et al.
decoupling to uncoupling by formation of a leakage type
ion pathway specific for H in cooperation with hydro-
inhibition of oxidative phosphorylation by bongkrekic
acid. Biochim Biophys Acta;223:460–462.
+
phobic anions. J Biol Chem;265:7837–7842.
17. Terada H. (1981) The interaction of highly active un-
couplers with mitochondria. Biochim Biophys
Acta;639:225–242.
18. Terada H. (1990) Uncouplers of oxidative phosphoryla-
tion. Environ Health Perspect;87:213–218.
1
4. Nishimura M., Ito T., Chance B. (1962) Studies on
bacterial photophosphorylation. III. A sensitive and
rapid method of determination of photophosphoryla-
tion. Biochim Biophys Acta;59:177–182.
1
5. Shinohara Y., Nagamune H., Terada H. (1987) The
hydrophobic cationic cyanine dye inhibits oxidative
phosphorylation by inhibiting ADP transport, not by
electrophoretic transfer, into mitochondria. Biochem
Biophys Res Commun;148:1081–1086.
19. Klingenberg M., Appel M., Babel W., Aquila H. (1983)
The binding of bongkrekate to mitochondria. Eur J Bio-
chem;131:647–654.
20. Zoccarato F., Rugolo M., Siliprandi D., Siliprandi N.
2
+
(1981) Correlated effluxes of adenine nucleotides, Mg
2+
1
6. Kemp A. Jr, Out T.A., Guiot H.F., Souverijn J.H. (1970)
The effect of adenine nucleotides and pH on the
and Ca induced in rat-liver mitochondria by external
2+
Ca and phosphate. Eur J Biochem;114:195–199.
1
322
Chem Biol Drug Des 2015; 86: 1304–1322