Synthesis and evaluation of simplified functionalized bongkrekic acid analogs

Article abstract of DOI:10.1016/j.tet.2018.01.018

Bongkrekic acid (BKA) is a strong inhibitor of adenine nucleotide translocase (ANT), inducing inhibition of adenosine triphosphate synthesis. We designed and synthesized simplified benzene-ring-containing BKA analogs. The key reaction is the one-pot double Sonogashira reaction, which forms the main skeleton. The analogs were efficiently synthesized in 8–10 longest linear sequence steps. This synthetic method can be applied for the preparation of other analogs having different combinations of carbon chain lengths. Furthermore, the allyloxy group on the benzene ring can be easily replaced by other functional groups. Our preliminary biological evaluation based on mitochondrial inhibitory effects revealed the high potency of the analogs bearing the same carbon chain length as that of BKA. In particular, the prefunctionalized analogs are potential ANT inhibitors.

Full text of DOI:10.1016/j.tet.2018.01.018

Accepted Manuscript
Synthesis and evaluation of simplified functionalized bongkrekic acid analogs
Satoshi Fujita, Masaki Suyama, Kenji Matsumoto, Atsushi Yamamoto, Takenori
Yamamoto, Yuka Hiroshima, Takayuki Iwata, Arihiro Kano, Yasuo Shinohara, Mitsuru
Shindo
PII:
S0040-4020(18)30040-1
10.1016/j.tet.2018.01.018
TET 29233
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 1 December 2017
Revised Date: 5 January 2018
Accepted Date: 9 January 2018
Please cite this article as: Fujita S, Suyama M, Matsumoto K, Yamamoto A, Yamamoto T, Hiroshima
Y, Iwata T, Kano A, Shinohara Y, Shindo M, Synthesis and evaluation of simplified functionalized
bongkrekic acid analogs, Tetrahedron (2018), doi: 10.1016/j.tet.2018.01.018.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
.
Synthesis and evaluation of simplified
functionalized bongkrekic acid analogs
Leave this area blank for abstract info.
Satoshi Fujita, Masaki Suyama, Kenji Matsumoto, Atsushi Yamamoto, Takenori Yamamoto, Yuka Hiroshima,
Takayuki Iwata, Arihiro Kano, Yasuo Shinohara,* and Mitsuru Shindo*

1
ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Synthesis and evaluation of simplified functionalized bongkrekic Acid analogs
Satoshi Fujita,a Masaki Suyama,a Kenji Matsumoto,e Atsushi Yamamoto,b Takenori Yamamoto,c,d Yuka
Hiroshima,c Takayuki Iwata,e Arihiro Kano,e Yasuo Shinohara,c,d,* and Mitsuru Shindoe,*
a
Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, 6-1, Kasuga-koen, Kasuga 816-8580 Japan
Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Minamitamagakicho-3500, Suzuka, Mie 513-8670, Japan
Institute for Genome Research, Tokushima University, Kuramotocho-3, Tokushima 770-8503, Japan
b
c
d Graduate School of Pharmaceutical Sciences, Tokushima University, Shomachi-1, Tokushima 770-8505, Japan
e Institute for Materials Chemistry and Engineering, Kyushu University, 6-1, Kasuga-koen, Kasuga 816-8580 Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Bongkrekic acid (BKA) is a strong inhibitor of adenine nucleotide translocase (ANT), inducing
inhibition of adenosine triphosphate synthesis. We designed and synthesized simplified
benzene-ring-containing BKA analogs. The key reaction is the one-pot double Sonogashira
reaction, which forms the main skeleton. The analogs were efficiently synthesized in 8ꢀ10
longest linear sequence steps. This synthetic method can be applied for the preparation of other
analogs having different combinations of carbon chain lengths. Furthermore, the allyloxy
group on the benzene ring can be easily replaced by other functional groups. Our preliminary
biological evaluation based on mitochondrial inhibitory effects revealed the high potency of
the analogs bearing the same carbon chain length as that of BKA. In particular, the
prefunctionalized analogs are potential ANT inhibitors.
Available online
Keywords:
Bongkrekic acid
adenine nucleotide translocator
mitochondria
Inhibitor
2009 Elsevier Ltd. All rights reserved
.
ATP
1. Introduction
the pharmacophore. Herein, we report the design and synthesis of
simplified BKA analogs bearing a benzene-ring-containing
carbon chain, and the evaluation of their ANT inhibitory activity.
Bongkrekic acid (BKA), isolated from Burkholderia gladioli
pathovar cocovenenans,1 is a strong inhibitor of adenine
nucleotide translocase (ANT) in the mitochondrial inner
membrane on the matrix side,2 inhibiting adenosine triphosphate
(ATP) synthesis and suppressing mitochondria-dependent
apoptosis (Figure 1). Recently, a cancer cell-specific death effect
was also found.3 BKA has, therefore, become an important
biological tool for research into the function of mitochondria, the
mechanism of apoptosis, and new antitumor drugs. However,
because of its limited availability,4 the efficient chemical
synthesis of BKA is required. Thus, we have developed an
efficient total synthesis of BKA in a pure form;5 however, even
this method requires more than 30 steps, and this may lead to
hesitation in the synthesis of BKA on a large scale. Because of
this supply issue, we have been developing BKA analogs by
simplifying its structure based on structure–activity relationship
studies, which indicate that tricarboxylic acids are essential
moieties for bioactivity.5a We have also synthesized highly
simplified analogs (e.g., 1) with apoptosis-suppressing activity,6
as well as inhibitory activity for mitochondrial ATP synthesis.7
Notably, the carbon chain length connecting the carboxylic acids,
is very important for the inhibitory activity. However, if
functional groups are introduced into the simplified analogs to
prepare molecular probes or more potent analogs having
additional functions, the simple alkane chain is inconvenient.
Furthermore, because the conformation of 1 is highly flexible, it
is challenging to obtain conformational information to determine
HO2C
HO2C
MeO
HO2C
HO2C
CO2H
CO2H
BKA
1
Figure 1. BKA and the simplified analog 1.
2. Results
BKA is expected to have a folded conformation induced by
intramolecular hydrogen bond(s).8 We, thus, designed
a
tricarboxylic acid (2) that contains a benzene ring in the carbon
chain of 1 (Figure 2). We anticipated that the meta-disubstitution
would be more appropriate rather than para-substitution to obtain
a more rigid folded structure.9 Furthermore, the permeability of
the cell membrane would be improved owing to higher
lipophilicity by the aromatic ring. If the benzene ring is pre-
functionalized, additional functions can be easily introduced into
the BKA analogs, as shown in 2. Based on this concept, we
decided to synthesize compound 3, which has the same total

2
Tetrahedron
ACCEPTED MANUSCRIPT
carbon number (24) including the benzene ring as analog 1, and
compound 4, which
2.1. Synthesis of 3 and 4
Compound 3 was synthesized as shown in Scheme 1. The
double Sonogashira coupling of 1-bromo-3-iodobenzene (7) with
hept-6-yn-1-ol (8)10 and, subsequently, with 5-TBSO-pentyne
(10a), afforded 11a in good yield. After complete hydrogenation
with a PtO2 catalyst, the resulting 12a was oxidized with Dessꢀ
Martin periodinane to give the corresponding aldehyde, which
was subjected to borylalkenylation by CrCl2 (Takai reaction)11 to
provide alkenylborane 14a. Suzuki-Miyaura coupling of 14a with
iodoalkene, 15,5a followed by desilylation, furnished 16a in good
yield. Finally, Jones oxidation of the diol moiety of 16a,
followed by deprotection with acid, provided 3. In the same
manner, 4 was synthesized using 10b as a starting alkyne by the
same protocol, except for the final deprotection, which proceeded
partially during oxidation.
Figure 2. BKA analogs synthesized in the present study.
contains the same (22) carbon chain length, as depicted by the
bold line in 4.

3
ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of 3 and 4.
2.2. Synthesis of 5 (1st generation synthesis)
Next, BOM-protected 2,4-diiodophenol 19 was subjected to the
Sonogashira coupling; however, this resulted in moderate yield
with poor selectivity. This product 21 was sufficiently reactive
for the Sonogashira coupling with alkyne 22. After
hydrogenation with a Pd/C catalyst, the selective deprotection of
the BOM group was carefully examined. After the screening of
the reagents and reaction conditions, we found that treatment
with B-bromocatecholborane12 at -78 ºC gave a relatively high
yield of the deprotected 25a and the doubly deprotected 25b.
After separation, 25b was subjected to selective O-allylation to
give 26. Following the procedure in the previous section, 5 was
synthesized from 26 in six steps (Scheme 2).
We then designed O-allyloxy analogs of 4, 5, and 6 because
the preliminary evaluation of the ANT-inhibitory effects suggests
that 4 was more active than 3. Furthermore, an allyloxy group
can be easily converted into other functionality. The initial
attempts on the double Sonogashira reactions of Bn- and BOM)-
protected 2-bromo-4-iodophenol 18 failed because the reactivity
of the 2-bromo group on 18 was too low for the second
Sonogashira coupling.

4
Tetrahedron
ACCEPTED MANUSCRIPT
5
OTBS
OTBDPS
6
20
22
OBOM
OBOM
OBOM
OBOM
Pd(PPh3)4
CuI, Et3N
Pd(PPh3)4
CuI, Et3N
TBDPSO
6
I
I
10% Pd/C
TBDPSO
8
toluene
rt, 12 h
30-55%
toluene
rt, 20 h
96%
toluene
rt, 20 h
95%
TBSO
I
6
19
5
5
TBSO
TBSO
21
23
24
+ 2-alkynyl product (8-24%)
+ dialkynyl product (0-22%)
O
O
B
Br
1) DMP
CH2Cl2, rt, 1 h
85%
O
OH
Allyl bromide
Cs2CO3
K2CO3
TBDPSO
8
TBDPSO
5
CH2Cl2
-78 C, 0.5 h
DMF
rt, 5 h
89%
2) 13, CrCl2, LiI
THF, rt
93%
RO
HO
6
5
26
25b (R = H: 42%)
+ 25a (R = TBS: 50%)
15
PdCl2(PPh3)2
Et3N
O
O
O
HO
HO
TBDPSO
TBDPSO
HO
TBAF
5
5
5
MeOH/THF
rt, 24 h
74%
THF
rt, 19 h
83%
O
B
5
O
5
5
CO2MOM
29
CO2MOM
28
27
O
HO2C
O
Jones reagent
acetone
6 M HCl
HO2C
HO2C
5
HO2C
THF
rt, 0.8 h
55%
0
C, 1 h
95%
5
CO2MOM
30
CO2H
5
Scheme 2. Synthesis of 5.
2.3. Synthesis of 6 (2nd generation)
With the double coupling product in hand, 33 was converted
into 36 in four steps in the same manner as described above
(Scheme 4). Although the deprotection of the acetate in 36 by
methanolysis or hydrolysis did not give the desired product,
phenol acetate-selective aminolysis with pyrrolidine successfully
afforded 38 in good yield after O-allylation.15 Finally, the
synthesis of 6 was achieved after the same transformation of 28.
Although 6 could be synthesized using the same protocol as
that of 5, we tried to improve the synthesis, due to low efficiency
of several steps (double Sonogashira coupling and deprotection
of BOM). We suspected that the BOM group was not an
appropriate protecting group and should be re-examined. As a
protecting group for phenol, an acetyl group was found to be
much better than BOM and Bn13 because the double Sonogashira
coupling was highly selective without detection of minor isomers
(Scheme 3). The one-pot double coupling reaction (from 31 to
33) was also successfully completed in 72% yield. Presumably,
the BOM group acts as a directing group for Pd inducing ortho
selectivity14 and, thus, the coupling reaction at C-2 competitively
proceeded with that at C-4. On the other hand, an acetoxy group
seemed to be worse directing group than the ether-type protecting
groups, partly because of the plausible dominant conformations
(A, B > C, D in Figure 3).

5
ACCEPTED MANUSCRIPT
Scheme 3. Stepwise and one-pot double Sonogashira coupling.
Figure 3. Possible conformations of 31.
Scheme 4. Synthesis of 6.
3. Biological evaluation
The inhibitory effects of these benzene ring-incorporated
simplified BKA analogs (3, 4, 5, 6, and 1 for comparison) on
ANT were evaluated by measuring the effects of the derivatives
on the pH change of the mitochondrial suspension accompanied
by ATP synthesis. The rationale of this experimental system is as
follows: when ATP is synthesized by mitochondrial ATP
synthase from adenosine diphosphate (ADP) and inorganic
phosphate (Pi), an increase in the pH (alkalinization) of the
incubation medium occurs.16 If certain compounds showing an
inhibitory effect on ANT are added to the mitochondrial
suspension, the alkalinization of the incubation medium should
be suppressed. Thus, the inhibitory effects of BKA analogs could
be evaluated by measuring the effects of the derivatives on the
pH change of the mitochondrial suspension accompanied by ATP
synthesis. Because the inhibitory effect of BKA on the
mitochondrial ATP synthesis is known to be stronger under
slightly acidic conditions than at neutral pH,17 the biological tests
were performed under two pH conditions: 7.4 and 6.8.
Figures 4 and 5 show the changes in the pH of the incubation
medium in the presence of BKA derivatives (1 and 10 µM),
starting at pH 6.8 and 7.4. Under more acidic conditions (pH
6.8), BKA completely inhibited ATP synthesis, even at 1 µM,
after the addition of ADP. In contrast, the BKA analogs (1, 3–6)

6
Tetrahedron
were much less potent than BKA at 1 µM. Simplified analog 1
ATP synthesis (mean ± standard deviation (SD)). The rates of
ACCEPTED MANUSCRIPT
showed high activity at 10 µM, as previously reported. Although
3 was poorly active, 4 was more potent than 3. This result
indicates the carbon chain length may be more important than the
carbon number concerning the inhibitory activity. Notably, 5 and
6 were stronger inhibitors than 3 and 4. At pH 7.4, the activity
trend seemed to be similar to that at pH 6.8, but the inhibition
was slightly less effective, as was seen in the previous results for
BKA and 1.
ATP synthesis in the absence of BKA derivatives at pH 6.8 or 7.4
were 248 ± 29 and 450 ± 42 nmol/(mg min), respectively.
In the evaluation of the inhibitory effects of BKA analogs on
ANT by measuring the pH of the incubation medium, we must
pay attention to the side effects of the BKA analogs on the
mitochondria because these side effects also influence
mitochondrial ATP synthesis. For this reason, we also
investigated membrane permeabilization effects and the
inhibitory effects on the mitochondrial electron transport system
of the BKA analogs.
The membrane permeabilization effects and inhibitory effects
on the mitochondrial electron transport system of the BKA
analogs were evaluated by measuring the rate of oxygen
consumption by the mitochondria. After the addition of a certain
amount of the individual BKA analogs, the time-dependent
changes in the oxygen concentration of the mitochondrial
suspension were monitored at 25 °C using a Clark-type oxygen
electrode (Yellow Springs Instrument, model 5331, YSI Life
Sciences, Yellow Springs, OH, USA). When the mitochondria
were suspended in the medium containing a respiratory substrate,
gradual oxygen consumption to compensate for the H+ leakage
through the mitochondrial inner membrane was observed. The
rate of oxygen consumption observed under this condition
reflects the lowest H+ permeability of the mitochondrial
membrane (shown as “control” in Figure 6). In contrast, the
addition of 100 nM SF6847, an effective protonophore,18
accelerated the mitochondrial oxygen consumption. The rate of
oxygen consumption in this state reflects the highest H+
permeability of the mitochondrial inner membrane (shown as
"SF6847" in Figure 6). Using the rate of oxygen consumption in
these two states as negative and positive controls, we examined
the permeabilization effects of the BKA analogs on the
mitochondrial inner membrane. As depicted in Figure 6, analogs
1, 5, and 6 at 10 µM showed weak acceleration effects at pH 6.8,
but the effects of all analogs were almost negligible.
Figure 4. Effects of BKA derivatives on the pH change of the
mitochondrial suspension accompanied by ATP synthesis.
Mitochondria were first incubated with BKA derivatives for 2
min, and then, 100 µM ADP was added. Time-dependent
changes in the pH of the incubation medium are shown. The
dotted lines showed the changes in the pH of the incubation
medium caused by the addition of ADP in the presence of 1%
dimethylsulfoxide (DMSO, i.e., in the absence of BKA
derivatives). The left and right traces represent the results of the
experiments at pH 6.8 and 7.4, respectively.
Figure 6. The permeabilization effects of the BKA derivatives
on the mitochondrial inner membrane. The figure represents
the effects of the BKA derivatives on the rate of mitochondrial
oxygen consumption at pH 6.8 (left) and 7.4 (right). The rates of
oxygen consumption observed in the absence of chemicals
(control) or presence of SF6847 are used as controls and shown
by the hatched columns. Those observed in the presence of BKA
and its derivatives at 1 or 10 ꢀM are shown by white and black
Figure 5. Inhibitory effects of BKA analogs on mitochondrial
ATP synthesis. Inhibitory effects of individual BKA derivatives
at 1 or 10 ꢀM (white and black columns, respectively) on the
mitochondrial ATP synthesis were conducted by measuring the
suppression of pH change caused by the addition of ADP at pH
6.8 or 7.4 (left or right graph, respectively). The results obtained
by three independent runs are shown as percentage inhibition of

7
columns, respectively. The bars represent the SD values of
individual experiments.
inhibitor than 3. This result indicates that the carbon chain
ACCEPTED MANUSCRIPT
length has a greater effect on the bioactivity than the carbon
number of the compounds. This is consistent with our previous
results concerning the apoptosis-suppressing activity. A suitable
arrangement of three carboxylic acids connected by a carbon
We also tested the inhibitory effects of BKA analogs on the
mitochondrial electron transport system. This effect was
evaluated by measuring how the rate of oxygen consumption
stimulated by SF6847 was inhibited by the BKA analogs. For
this purpose, mitochondria were pretreated with BKA, 1, or 3–6
at 1 or 10 µM for 1 min, and, then, 100 nM SF6847 was added. If
the BKA analogs had an inhibitory effect on the mitochondrial
electron transport system, then the rate of oxygen consumption
caused by the addition of SF6847 would decrease. A summary of
the results obtained with BKA and its analogs is shown in Figure
7. BKA and 1 showed moderate and relatively strong inhibitory
effects, respectively, on the mitochondrial electron transport
system at 10 µM, as was seen in the previous report.7 Analog 3
showed a very weak inhibitory effect, even at 10 µM, being
comparable to BKA. In contrast, analogs 4, 5, and, especially, 6
demonstrated weaker effects than 1 at 10 µM, in spite of showing
slightly stronger effects than BKA. These effects were almost
negligible at the 1 µM concentration. Based on these results,
although the inhibitory effects of 4, 5, and 6 on the mitochondrial
ATP synthesis were not simply attributable to the results of the
specific inhibition of the mitochondrial ADP/ATP carrier, as was
seen in the results by the simplified analog 1, the present three
analogs (4, 5, and 6) were concluded to be comparable or slightly
more potent inhibitors on the mitochondrial ADP/ATP carrier
than 1. Furthermore, these analogs had fewer side effects on the
other mitochondrial functions than 1.
chain of appropriate length (C22) and having
a folded
conformation is important for effective binding to ANT or other
target proteins. It is also interesting that the allyloxy-analogs 5
and 6 are more potent than 4. This might partially arise from the
higher lipophilicity because one of the drawbacks of BKA for
mitochondrial inhibition is its high polarity; that is, three
carboxylate moieties would reduce the cell membrane
permeability of BKA (and the analogs). On the other hand,
higher lipophilicity might cause undesirable side effects because
of non-specific binding to biomacromolecules. Analogs 3–6 did
not show the significant side effects compared to 1.
5. Conclusion
We designed and synthesized simplified, benzene-ring-
containing BKA analogs (3, 4, 5, and 6). The key reaction is the
one-pot double Sonogashira reaction forming the main skeleton.
The analogs were efficiently synthesized in 8ꢀ10 longest linear
sequence steps (total 15–17 steps), which is significantly fewer
than that of BKA (17 longest linear sequence steps, total 37
steps) and even that of the previous simplified BKA, 1 (10
longest linear sequence steps, total 19 steps). This synthetic
method can be applied for the preparation of other BKA analogs
having
a different combination of carbon chain lengths.
Furthermore, the allyloxy group on the benzene ring can be easily
replaced with other functional groups, which will bring about
new highly functionalized simplified BKA analogs.
The preliminary biological evaluation based on mitochondrial
inhibitory effects revealed the high potency of the benzene-ring-
containing BKA analogs bearing the same carbon chain length as
that of BKA. Particularly, prefunctionalized analogs 5 and 6 are
potential compounds that are relatively easily prepared and could
lead to more effective inhibitors of mitochondria. In this study,
we validated our molecular design concept for mitochondrial
inhibitors, which will contribute to mitochondrial research
including apoptosis suppression and cancer cell-specific death
effects, as well as the ADP/ATP translocating mechanism of
ANT.
6. Experimental section
6.1 General
1H NMR spectra were measured in CDCl3 solution containing
0.01% trimethylsilane (TMS, reference: 0.00 ppm) using a JEOL
JNM-ECA 600 spectrometer or JEOL JNM-AL-400 spectrometer.
13C-NMR spectra were measured in CDCl3 solution (reference:
77.0 ppm) or CDOD3 solution (reference: 49.0 ppm) using a
Figure 7. The inhibitory effects of BKA derivatives on the
mitochondrial electron transport system. The figure represents
the inhibitory effects of BKA and its derivatives on the SF6847-
stimulated oxygen consumption at pH 6.8 (left) and 7.4 (right).
The results observed in the presence of BKA and its derivatives
at 1 or 10 ꢀM are shown by 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 derivative was 174 ± 27
and 215 ± 25 natomsO/(mg min), respectively, at pH 6.8 and 7.4.
1
JEOL JNM-ECA 600 or JEOL JNM-AL-400 spectrometer. H
NMR and 13C-NMR spectra were measured at room temperature
unless otherwise noted. IR spectra were recorded on
a
SHIMADZU IRPrestige-21 FT-IR spectrophotometer using a
KBr disk or a NaCl plate. Column chromatography was
performed on silica gel (60N, spherical, pH = 7.0 ± 0.5, Kanto
Chemical Co., Inc.). Thin-layer chromatography was performed
on precoated plates (0.25 mm, silica gel, Merck 60 F254). Mass
spectra (MS) and high-resolution mass spectra (HRMS) were
measured on a JEOL JMS-700 mass spectrometer (EI and FAB),
a JEOL JMS-T100CS (ESI), and a JEOL JMS-S3000 mass
4. Discussion
The ANT inhibitory activity of the simplified, benzene ring-
containing BKA analogs (3, 4, 5 and 6) synthesized in the current
study is compared with that of BKA and the simplified analog 1
evaluated previously. From the investigation of the inhibitory
effects on the mitochondrial ATP synthesis, analogs 3 and 4 were
active, albeit less potent than 1. Notably, analog 4 was a better
spectrometer
(MALDI).
High-performance
liquid

8
Tetrahedron
chromatography (HPLC) purification was performed using a
C3 H42NaO7 513.2828; HRMS (ESI-) 513.2866 (M-H-) calcd for
ACCEPTED MANUSCRIPT
0
pump (LC-10ADvp, SHIMADZU) equipped with a photodiode
array (PDA) detector (SPD-M10Avp, SHIMADZU), a system
controller (SCL-10Avp, SHIMADZU), a degasser (DGU-12A,
SHIMADZU), and a column oven (CTO-10ACvp, SHIMADZU).
C30H41O7 513.2852.
6.6 Preparation of mitochondria from rat liver–
Mitochondria were prepared from the liver of a male Wistar rat,
as described previously.19
6.2 Spectra of 3
6.7 Inhibitory effects of BKA analogs on the mitochondrial
ATP synthesis
1H NMR (600 MHz, CDCl3) δ: 1.29–1.32 (m, 4H), 1.39–1.42
(m, 2H), 1.55–1.68 (m, 6H), 2.21 (dt, J = 6.9 Hz), 2.35 (t, J = 7.2
Hz, 2H), 2.56 (t, J = 7.8 Hz, 2H), 2.62 (t, J = 6.9 Hz, 2H), 3.36 (s,
2H), 5.71 (s, 1H), 6.22 (dt, J = 7.0, 16.1 Hz, 1H), 6.96–6.99 (m,
3H), 7.17 (t, J = 7.6 Hz, 1H), 7.45 (d, J = 16.1 Hz, 1H); 13C
NMR (150 MHz, CDCl3) δ: 23.9 (t), 28.5 (t), 28.9 (t), 29.3 (t),
30.3 (t), 31.6 (t), 33.2 (t), 33.9 (t), 35.1 (t), 36.2 (t), 40.9 (t),
117.9 (d), 125.8 (d), 126.0 (d), 126.3 (d), 128.2 (d), 128.7 (d),
141.1 (d), 141.6 (s), 142.8 (s), 148.9 (s), 170.0 (s), 176.3 (s),
180.8 (s); IR (neat): 1711, 2932, 3011 cm-1; MS (FAB) m/z 439
(M+Na+); HRMS (FAB) 439.2094 (M+Na+), calcd for
C24H32O6Na 439.2097.
To evaluate the inhibitory effects of the BKA analogs,
mitochondria were first suspended in the medium containing 200
mM sucrose, 20 mM KCl, 3 mM MgCl2, and 3 mM potassium
phosphate buffer, pH 6.8 or 7.4 to reach a protein concentration
of 0.7 mg/mL. After the addition of 10 mM succinate (plus 0.5
ꢀg/mL rotenone) as respiratory substrate, mitochondria were pre-
incubated with the BKA analogs for 2 min at 25 ºC. Then, ATP
synthesis was initiated by addition of 200 ꢀM ADP, and the time-
dependent pH change of the mitochondrial suspension was
measured using a pH meter.
6.3 Spectra of 4
6.8 Effects of BKA analogs on the permeability of
mitochondrial membrane and on the mitochondrial electron
transport system
1H NMR (600 MHz, CDCl3) δ: 1.28–1.35 (m, 12H), 1.41–1.45
(m, 2H), 1.56–1.63 (m, 4H), 2.22 (dt, J = 6.9, 6.9 Hz, 2H), 2.37
(t, J = 7.2 Hz, 2H), 2.55–2.59 (m, 4H), 3.37 (s, 2H), 5.71 (s, 1H),
6.21 (dt, J = 6.9, 15.8 Hz, 1H), 6.97–6.99 (m, 3H), 7.17 (t, J =
7.6 Hz, 1H), 7.49 (d, J = 15.8 Hz, 1H); 13C NMR (150 MHz,
CD3OD) δ: 24.5 (t), 28.4 (t), 28.54 (t), 28.56 (t), 28.85 (t), 28.93
(t), 29.0 (t), 30.9 (t), 31.3 (t), 33.3 (t), 34.0 (t), 35.8 (t), 35.9 (t),
40.5 (t), 117.9 (d), 125.6 (d), 125.8 (d), 126.4 (d), 128.1 (d),
128.6 (d), 141.3 (d), 142.5 (s), 142.6 (s), 148.7 (s), 170.6 (s),
176.2 (s), 180.7 (s); IR (neat): 1705, 2932, 3021 cm-1; MS (FAB)
m/z 481 (M+Na+); HRMS (FAB) 481.2568 (M+Na+), calcd for
C27H38O6Na 481.2566.
The effects of BKA analogs on the permeability of mitochondrial
membrane and on the mitochondrial electron transport system
were evaluated by measuring the oxygen consumption. For this,
the mitochondria were first suspended in the abovementioned
medium at 0.7 mg/mL. After addition of 10 mM succinate (plus
0.5 ꢀg/mL rotenone) as respiratory substrate, mitochondria were
pre-incubated with BKA analogs for 2 min at 25 ºC. Then, the
time-dependent changes in the oxygen concentration in the
mitochondrial suspension were measured by Clark-type oxygen
electrode (Yellow Springs Instrument, model 5331, YSI Life
Sciences)
6.4 Spectra of 5
1H NMR (600 MHz, CDCl3) δ: 1.26–1.35 (m, 10H), 1.42–1.45
(m, 2H), 1.47–1.65 (m, 6H), 2.23 (dt, J = 7.1, 7.1 Hz, 2H), 2.33 (t,
J = 6.5 Hz, 2H), 2.51 (t, J = 7.6 Hz, 2H), 2.62 (t, J = 6.9 Hz, 2H),
3.37 (s, 2H), 4.50 (d, J = 4.8 Hz, 2H), 5.24 (dt, J = 1.7, 10.7 Hz,
1H), 5.41 (dt, J = 2.1, 17.2 Hz, 1H), 5.72 (s, 1H), 6.05 (ddt, J =
4.8, 10.7, 15.6 Hz, 1H), 6.22 (dt, J = 7.1, 15.8 Hz, 1H), 6.92–6.93
(m, 2H), 7.49 (d, J = 15.8 Hz, 1H); 13C NMR (150 MHz,
CDOD3) δ: 24.5 (t), 28.36 (t), 28.41 (t), 28.57 (t), 28.60 (t), 28.7
(t), 29.5 (t), 29.7 (t), 31.2 (t), 32.7 (t), 33.4 (t), 34.4 (t), 39.7 (t),
68.2 (t), 111.1 (d), 115.0 (t), 118.2 (d), 125.9 (d), 126.3 (d), 129.5
(d), 130.5 (s), 133.7 (d), 134.2 (s), 138.5 (d), 147.5 (s), 154.2 (s),
167.9 (s), 173.0 (s), 176.1 (s); IR (neat): 1707, 2924 cm-1; MS
(EI) m/z: 514 (M+); HRMS (EI) 514.2929 (M+) calcd for
C30H42O7 514.2931.
Acknowledgments
This work was supported by a research fund from the Network
Joint Research Center for Materials and Devices, JSPS
KAKENHI Grant numbers JP16H01157 (M.S.), JP23790131
(K.M.) and 17K08274 (Y.S.).
References and notes–
1. (a) Lumbach GWM, Cox HC, Berends W. Tetrahedron. 1970;
26:5993. (b) Lumbach GWM, Cox HC, Berends W. Tetrahedron
1971; 27:1839.
2. Recent reviews: (a) Palmieri F. Mol Aspects Med. 2013; 34: 465.
(b) Clemencon B, Babot M, Trezeguet V. Mol Aspects Med. 2013;
34:485–493. See also (c) Todisco S, Di Noia MA, Onofrio A,
Parisi G, Punzi G, Redavid G, De Grassi A, Pierri CL. Biochem
Pharmacol. 2016; 100:112.
6.5 Spectra of 6
1H NMR (600 MHz, CDCl3) δ: 1.25–1.37 (m, 12H), 1.42–1.47
(m, 2H), 1.54–1.61 (m, 4H), 2.23 (dt, J = 7.1, 7.1 Hz, 2H), 2.33 (t,
J = 6.9 Hz, 2H), 2.52 (t, J = 7.2 Hz, 2H), 2.60 (t, J = 7.9 Hz, 2H),
3.37 (s, 2H), 4.51 (d, J = 4.8 Hz, 2H), 5.24 (d, J = 10.3 Hz, 1H),
5.41 (d, J = 17.2 Hz, 1H), 6.05 (ddt, J = 4.8, 10.3, 17.2 Hz, 1H),
6.22 (dt, J = 7.1, 15.8 Hz, 1H), 6.73 (d, J = 7.9 Hz, 1H), 6.91 (d,
J = 8.2 Hz, 6.93 (s, 1H), 7.49 (d, J = 15.8 Hz, 1H); 13C NMR
(150 MHz, CDCl3) δ: 24.5 (t), 28.6 (t), 28.73 (t), 28.78 (t), 29.0
(t), 29.3 (t), 29.7 (t), 30.3 (t), 31.4 (t), 33.5 (t), 34.0 (t), 35.0 (t),
40.4 (t), 68.8 (t), 111.6 (d), 116.5 (t), 118.0 (d), 126.29 (d),
126.31 (d), 130.0 (d), 131.2 (s), 133.9 (d), 134.6 (s), 141.4 (d),
148.8 (s), 154.5 (s), 171.2 (s), 176.4 (s), 180.6 (s); IR (neat):
1703, 2928, 3007 cm-1; MS (ESI+) m/z: 537 (M+Na+); MS (ESI-)
m/z: 513 (M-H-); HRMS (ESI+) 537.2877 (M+Na+) calcd for
3. Kano A, Kamita MK, Iwasaki T, Shindo M. Evergreen 2017;
4:23.
4. The cost of BKA is $433/100 µg from LKT LABS
5. (a) Matsumoto K, Suyama M, Fujita S, Moriwaki T, Sato Y, Aso
Y, Muroshita S, Matsuo H, Monda K, Okuda K. Chem. - Eur. J.
2015; 21:11590. (b) Sato Y, Aso Y, Shindo M. Tetrahedron Lett.
2009; 50:4164. For the other syntheses, see (c) Corey EJ,
Tramontano J. J. Am. Chem. Soc. 1984; 106:462. (d) Shindo M,
Sugioka T, Umaba Y, Shishido K. Tetrahedron Lett. 2004;
45:8863. (e) Kanematsu M, Shindo M, Yoshida M, Shishido K.
Synthesis (Stuttg). 2009:2893. (f) Francais A, Leyva A,
Etxebarria-Jardi G, Ley S V. Org. Lett. 2010; 12:340.
6. Okuda K, Hasui K, Abe M, Matsumoto K, Shindo M. Chem. Res.
Toxicol. 2012; 25:2253.

9
7. Yamamoto A, Hasui K, Matsuo H, Okuda K, Abe M, Matsumoto
ACCEPTED MANUSCRIPT
K, Harada K, Yoshimura Y, Yamamoto T, Ohkura K. Chem. Biol.
Drug Des. 2015; 86:1304.
8. See Supporting Information
9. Ortho-substitution may also cause several problems of steric
–
congestion between the carbon chains as well as synthetic
difficulty.
–
10. Hopf H, Kruger A. Chem. - Eur. J. 2001; 7:4378.
11. Takai K, Shinomiya N, Kaihara H, Yoshida N, Moriwake T,
Utimoto K. Synlett. 1995; 1995:963.
12. Lee K, Cha JK. J. Am. Chem. Soc. 2001; 123:5590.
13. Bates R, Gabel C, Ji J, Rama-Devi T. Tetrahedron 1995; 51:8199.
14. Riguet E, Alami M, Cahiez G. J. Organomet. Chem. 2001;
624:376.
15. Wender PA, Jesudason CD, Nakahira H, Tamura N, Tebbe AL,
Ueno Y. J. Am. Chem. Soc. 1997; 119:12976.
16. Nishimura M, Ito T, Chance B. Biochim. Biophys. Acta 1962;
59:177.
17. Kemp A, Out TA, Guiot HF, Souverijn JH. Biochim. Biophys.
Acta 1970; 223:460.
18. (a) Terada H. Biochim. Biophys. Acta 1981; 639:225. (b) Terada
H. Environ. Health Perspect. 1990; 87:213.
19. Yamamoto T, Ito M, Kageyama K, Kuwahara K, Yamashita K,
Takiguchi Y, Kitamura S, Terada H, Shinohara Y. FEBS J. 2014;
281:3933.
Supplementary Material
Supplementary data (calculated 3D structure, synthetic
procedures, biological tests) associated with this article can be
found in the online version, at http://dx.doi.org