Molecular design, synthesis, and evaluation of novel potent apoptosis inhibitors inspired from bongkrekic acid

Article abstract of DOI:10.1021/tx300315h

Bongkrekic acid (BKA) is an inhibitor of adenine nucleotide translocase (ANT). Since inhibition of ANT is connected to the inhibition of cytochrome c release from mitochondria, which then results in the suppression of apoptosis, it has been used as a tool for the mechanistic investigation of apoptosis. BKA consists of a long carbon chain with two asymmetric centers, a nonconjugated olefin, two conjugated dienes, three methyl groups, a methoxyl group, and three carboxylic acids. This complicated chemical structure has caused difficulties in synthesis, supply, and biochemical mechanistic investigations. In this study, we designed and synthesized more simple tricarboxylic acids that were inspired by the molecular structure of BKA. Their cytotoxicity and apoptosis-preventing activity in HeLa cells and the effect on the mitochondrial inner membrane potential (Δψm) in HL-60 cells were then evaluated. All tested tricarboxylic acid derivatives including BKA showed little toxicity against HeLa cells. BKA and two of the synthesized derivatives significantly suppressed staurosporine (STS)-induced reductions in cell viability. Furthermore, STS-induced Δψm collapse was significantly restored by pretreatment with BKA and a tricarboxylic acid derivative. Other derivatives, in which one of three carboxylic acids was esterified, exhibited potent toxicity, especially a derivative bearing a carbon chain of the same length as that of BKA. In conclusion, we have developed a new lead compound as an apoptosis inhibitor bearing three carboxylic acids connected with the proper length of a long carbon chain.

Full text of DOI:10.1021/tx300315h

Article
pubs.acs.org/crt
Molecular Design, Synthesis, and Evaluation of Novel Potent
Apoptosis Inhibitors Inspired from Bongkrekic Acid
Katsuhiro Okuda,† Keisuke Hasui,‡ Masato Abe,† Kenji Matsumoto,† and Mitsuru Shindo*,†
†Institute for Materials Chemistry and Engineering, Kyushu University, 6-1 Kasuga-koen, Kasuga, Fukuoka 816-8580, Japan
‡Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, 6-1 Kasuga-koen, Kasuga, Fukuoka 816-8580, Japan
S
* Supporting Information
ABSTRACT: Bongkrekic acid (BKA) is an inhibitor of
adenine nucleotide translocase (ANT). Since inhibition of
ANT is connected to the inhibition of cytochrome c release
from mitochondria, which then results in the suppression of
apoptosis, it has been used as a tool for the mechanistic
investigation of apoptosis. BKA consists of a long carbon chain
with two asymmetric centers, a nonconjugated olefin, two conjugated dienes, three methyl groups, a methoxyl group, and three
carboxylic acids. This complicated chemical structure has caused difficulties in synthesis, supply, and biochemical mechanistic
investigations. In this study, we designed and synthesized more simple tricarboxylic acids that were inspired by the molecular
structure of BKA. Their cytotoxicity and apoptosis-preventing activity in HeLa cells and the effect on the mitochondrial inner
membrane potential (ΔΨm) in HL-60 cells were then evaluated. All tested tricarboxylic acid derivatives including BKA showed
little toxicity against HeLa cells. BKA and two of the synthesized derivatives significantly suppressed staurosporine (STS)-
induced reductions in cell viability. Furthermore, STS-induced ΔΨm collapse was significantly restored by pretreatment with
BKA and a tricarboxylic acid derivative. Other derivatives, in which one of three carboxylic acids was esterified, exhibited potent
toxicity, especially a derivative bearing a carbon chain of the same length as that of BKA. In conclusion, we have developed a new
lead compound as an apoptosis inhibitor bearing three carboxylic acids connected with the proper length of a long carbon chain.
nonsynaptosomal mitochondria,13 glucose-induced electrical
INTRODUCTION
Bongkrekic acid (BKA, Scheme 1) is a poisonous antibiotic
produced by Burkholderia Cocovenenans. Its name comes from
■
activity in pancreatic beta-cells through the stimulation of ATP-
sensitive potassium channel activity,14 mitochondrial Ca2+
efflux, and Mg2+, K+, and adenine nucleotide release, which
was promoted by a chelating agent EGTA and stimulated by
Scheme 1. Chemical Structures of BKA and Its Simplified
Analogue 10h
menadione in rat liver mitochondria.15 Furthermore, BKA
reduced the uncoupling of the mitochondrial oxidative
phosphorylation induced by KATP channel openers16 and
prevented losses in the mitochondrial inner membrane
potential (ΔΨm) induced by H2O2 in cerebellar granule
neurons.17 Recently, it was also reported that BKA inhibits
mitochondrial membrane-bound glutathione transferase
(mtMGST1) in rat livers through membrane components.
This inhibition was observed only in mtMGST1 on the inner
an Indonesian equivalent of tofu called tempeh bongkrek. BKA
mitochondrial membrane, although mtMGST1 resides in both
was isolated in 1934,1 and its structure was elucidated between
inner and outer mitochondrial membranes.18
1970 and 1973.2−4 BKA was found to be an inhibitor of
These particular effects on mitochondria have been shown to
adenine nucleotide translocase (ANT), which mediates ADP/
suppress apoptosis.19,20 BKA prevented a number of phenom-
ATP exchange in mitochondria.5,6 Further investigations have
ena linked to apoptosis, such as depletion of reduced
found that BKA inhibits the mitochondrial permeability
glutathione, generation of reactive oxygen species, translocation
of NFκB, exposure of phosphatidylserine residues on the outer
transition pore (MPTP) via binding to ANT and fixes its
conformation in the m-state, where its binding site faces the
mitochondrial matrix. In an opposite manner, a different ANT
inhibitor, atractyloside (ATR), and its derivative carboxya-
plasma membrane, cytoplasmic vacuolization, chromatin
condensation, and oligonucleosomal DNA fragmentation.21 It
also protected multidrug-resistant tumor cells against apoptosis
tractyloside (CATR), induced MPTP opening by changing the
caused by 1,4-anthraquinone.22 BKA protected MCF-7 cells
ANT conformation to its c-state where its binding site faced the
cytosol side.5,7−11
BKA inhibited chromatin condensation induced by valino-
Received: July 9, 2012
Published: September 21, 2012
mycin in BV-2, mouse microglia cells,12 K+ efflux in brain
© 2012 American Chemical Society
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Japan. Cells were cultured in RPMI-1640 supplemented 10% (v/v)
heat-inactivated FBS, 100 units/mL penicillin G, and 100 μg/mL
streptomycin. Cells were kept at 37 °C in 5% CO2 and 95% humidified
air. Cells were suspended in complete RPMI-1640 medium at a
density of 4.0 × 105 cells/tube and preincubated for 1 h at 37 °C in the
presence or absence of tricarboxylic acid derivatives. To investigate the
ability to prevent against reductions in ΔΨm, 2 μM STS was added 1
h after preincubation of the test compound, and incubation was
continued for another 3 h. Thirty minutes before finishing incubation,
the fluorescent dye JC-128 was loaded for cells at a final concentration
of 0.5 μM. In the case of CCCP exposure, it was added at the same
time as JC-1 addition. Cells were centrifuged (900g, 3 min) and
washed with PBS at room temperature to remove the drugs and
unincorporated free dye. The fluorescence ratio, red aggregates/green
monomers linked to the ΔΨm, of resuspended cells in PBS was
immediately detected at 515 nm excitation and 595/535 nm emission
using an RF-1500 spectrofluorophotometer (Shimadzu Co., Kyoto,
Japan).
Statistical Analysis. Results were expressed as the mean SD.
Statistical analysis was performed using a one-way ANOVA followed
by the Tukey−Kramer test to assess the significance of differences in
mean values between drug treatment groups and a suitable reference
group. Significance was accepted when the P value was less than 0.05.
Chemical Synthesis. 3-((tert-Butyldiphenylsilyl)oxy)propan-1-ol
(1a). Imidazole (2.15 g, 31.5 mmol) was added to a solution of 1,3-
propanediol (2.0 g, 26.3 mmol) in CH2Cl2 (130 mL). The reaction
mixture was stirred at room temperature for 35 min, and TBDPSCl
(4.82 mL, 18.4 mmol) was added. The reaction mixture was stirred at
room temperature for 10 min, and then the saturated aqueous solution
of NaHCO3 was added. The aqueous layer was extracted with CH2Cl2,
and the organic layer was washed with brine, dried over MgSO4,
filtered, and concentrated in vacuo. The crude product was purified by
silica gel column chromatography (hexane/EtOAc = 3/1) to give a
colorless oil (3.44 g, 42%). 1H NMR (400 MHz in CDCl3) δ: 1.06 (s,
9H), 1.76−1.81 (m, 2H), 3.81−3.85 (m, 4H), 7.35−7.42 (m, 6H),
7.66−7.68 (m, 4H).
against the proliferation inhibition effect of the chemotherapy
drug Tamoxifen23 and reduced ischemic-induced neuronal
death.24
As a result, BKA has become a general tool for the
elucidation of apoptosis mechanisms, appearing in hundreds of
publications in spite of being an expensive and valuable reagent.
To date, we have established an efficient synthetic method for
BKA, which has gradually improved overall yield and reduced
the steps in the longest linear sequence.25−27 If more easily
available potent apoptosis inhibitors could be designed and
synthesized, bioactive compounds of this category would be
useful for biochemical mechanistic investigations. In this study,
we designed and synthesized simpler tricarboxylic acids as novel
apoptosis inhibitors, inspired by the molecular structure of
BKA. (Scheme 1), and their cytotoxicity and ability to prevent
staurosporine (STS)-induced apoptosis in HeLa cells were
evaluated. Furthermore, some of them were nominated for
estimations of the effect on ΔΨm reduced by STS in HL-60
cells.
MATERIALS AND METHODS
■
Materials for Biological Assays. All new compounds were
synthesized with the method based on BKA synthesis.26 Representa-
tive methods for the compounds and their spectra data are described
below, and the others are in Supporting Information. DMEM, RPMI-
1640, STS, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP)
were purchased from Wako Pure Chemical Industries (Osaka, Japan).
WST-1 and 1-methoxy-5-methylphenazinium methylsulfate (1-me-
thoxy-PMS) were from Dojindo Molecular Technologies, Inc.
(Kumamoto, Japan). JC-1 was from Enzo Life Sciences, Inc.
(Farmingdale, NY). Other chemicals used were of the highest quality
commercially available.
Materials for Chemical Synthesis. The melting points were
1
measured on a Yanako MP-S3. The H- and 13C NMR spectra were
3-((tert-Butyldiphenylsilyl)oxy)propanal (2a). Me2SO (1.48 mL,
20.9 mmol) in CH2Cl2 (4 mL) was added to a solution of oxalyl
chloride (1.48 mL, 16.7 mmol) in CH2Cl2 (60 mL) at −78 °C and was
stirred for 20 min, and alcohol 1a (2.18 g, 6.93 mmol) in CH2Cl2 (6
mL) was added at −78 °C under an argon atmosphere. The reaction
mixture was stirred at −78 °C for 20 min, and triethylamine (6.86 mL,
48.8 mmol) was added at −78 °C. The reaction mixture was stirred at
room temperature for 30 min, and the saturated aqueous solution of
NaHCO3 was then added. The aqueous layer was extracted with
CH2Cl2, and the organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo. The crude product was
purified by silica gel column chromatography (hexane/EtOAc = 4/1)
to give a pale yellow oil (1.84 g, 89%). 1H NMR (400 MHz in CDCl3)
δ: 1.04 (s, 9H), 2.59 (td, J = 2.0, 6.0 Hz, 2H), 4.02 (t, J = 6.0 Hz, 2H),
7.36−7.44 (m, 6H), 7.65−7.67 (m, 4H), 9.80 (t, J = 2.0 Hz, 1H).
(12-Hydroxydodecyl)triphenylphosphonium bromide (12f). PPh3
(5.12 g, 19.5 mmol) was added to a solution of 12-bromododecan-1-ol
11f (5.18 g, 19.5 mmol) in acetonitrile (73 mL). The reaction mixture
was stirred under reflux for 90 h and evaporated. The crude product
was purified by silica gel column chromatography (MeOH/CHCl3 =
1/9) and recrystallization (EtOH/EtOAc) to give a white powder
(9.63 g, 94%). 1H NMR (400 MHz in CDCl3) δ: 1.20−1.28 (m, 14H),
1.51−1.63 (m, 6H), 3.62 (t, J = 6.8 Hz, 2H), 3.76 (brs, 2H), 7.71−
7.87 (m, 15H).
recorded using a JEOL JNM AL-400 spectrometer (400 and 100
MHz). The IR spectra were recorded on a Shimadzu FT/IR-8300
spectrometer 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.). Thin-layer chromatog-
raphy was performed on precoated plates (0.25 mm, silica gel Merck
60 F254). Reaction mixtures were stirred magnetically.
WST-1 Assay. HeLa human cervical cancer cells were cultured to
subconfluency in DMEM supplemented 10% (v/v) heat-inactivated
FBS, 100 units/mL penicillin G, and 100 μg/mL streptomycin. Cells
were kept at 37 °C in 5% CO2 and 95% humidified air. Cells
suspended in culture medium were seeded on 96-well microtiter
plastic culture plates at a density of 2.0 × 104 cells/100 μL/well. Media
were exchanged to serum free DMEM on the next day, and incubation
was continued for another 24 h. Test compounds dissolved in
dimethylsulfoxide and diluted with the culture medium were added.
To investigate the ability to prevent against apoptosis, staurosporine
(STS) was added 2 h after the test compound had been added, and
incubation was continued for another 22 h. Cell viability was assayed
according to the WST-1 method of the modified MTT assay. This
colorimetric assay measures the metabolic activity of viable cells based
on cleavage of the tetrazolium salt WST-1 to water-soluble formazan
by mitochondrial dehydrogenase in living cells. The assay was
performed by incubation with 0.26 mM WST-1 and 0.01 mM 1-
methoxy-PMS as an electron carrier in serum free DMEM for 0.5 h.
After thorough shaking, the formazan produced by metabolically active
cells in each sample was measured at a wavelength of 415 nm, with 620
nm as a reference, using a Microplate Reader Sunrise remote (Tecan
(Z)-15-((tert-Butyldiphenylsilyl)oxy)pentadec-12-en-1-ol (13g).
nBuLi (2.6 M in hexane, 5.50 mL, 14.3 mmol) was added to a
suspension of phosphonium salts 12f (3.47 g, 7.15 mmol) in THF (40
mL) at 0 °C under an argon atmosphere. The reaction mixture was
stirred under 0 °C for 10 min, and aldehyde 2a (1.49 g, 4.77 mmol) in
THF (8 mL) was added at 0 °C. The mixture was stirred at 0 °C for
10 min, and NH4Cl was added and extracted with EtOAc, and the
organic layer was washed with brine and dried over MgSO4. The crude
product was purified by silica gel column chromatography (hexane/
Group Ltd., Mannedorf, Switzerland). Absorbance readings were
̈
normalized against those of control wells with medium alone.
Measurement of Mitochondrial Membrane Potentials. The
HL-60 promyelocytic leukemia cell line (RCB0041) was provided by
RIKEN BRC through the National Bio-Resource Project of MEXT,
1
EtOAc = 4/1) to give a colorless oil (1.83 g, 80%). H NMR (400
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Scheme 2. Synthesis of Tricarboxylic Acid Derivatives 10 Bearing Different Carbon Chain Lengths (n = 4, 7, 10, 13, 16, 19, and
22)
MHz in CDCl3) δ: 1.05 (s, 9H), 1.27 (brs, 16H), 1.53−1.58 (m, 4H),
1.96 (q, J = 6.8 Hz, 2H), 2.31 (q, J = 17.2 Hz, 2H), 3.61−3.68 (m,
4H), 5.33−5.46 (m, 2H), 7.35−7.43 (m, 6H), 7.66−7.68 (m, 4H). 13C
NMR (100 MHz, CDCl3) δ: 19.2, 25.7, 26.9, 27.3, 29.2, 29.3, 29.4,
29.5, 29.6, 29.7, 30.8, 32.8, 63.1, 63.7, 125.5, 127.6, 127.7, 131.9, 134.0,
135.6. IR (Neat): 3354, 2928, 2855 cm−1; mass (FAB) m/z 481 [M +
H]+. HRMS calcd for C31H49O2Si: 481.3502; found, 481.3495.
15-((tert-Butyldiphenylsilyl)oxy)pentadecan-1-ol (1g). Alcohol
13g (0.98 g, 2.04 mmol) in MeOH (5 mL) was added to a
suspension of Pd/C (10%, 109 mg, 0.10 mmol) in MeOH (15 mL)
under H2. The reaction mixture was stirred at room temperature for 2
h, filtered, and concentrated to afford a colorless oil (892 mg, 91%).
1HNMR (400 MHz in CDCl3) δ: 1.04 (s, 9H), 1.25 (brs, 22H), 1.56
(m, 4H), 3.64−3.67 (m, 4H), 7.35−7.41 (m, 6H), 7.66−7.68 (m, 4H).
13C NMR (100 MHz, CDCl3) δ: 19.2, 25.7, 25.8, 26.9, 29.4, 29.4, 29.6,
29.7, 32.6, 32.8, 63.1, 64.0, 127.5, 127.7, 129.4, 129.8, 134.2, 135.5.
15-((tert-Butyldiphenylsilyl)oxy)pentadecanal (2g). Me2SO (0.41
mL, 5.71 mmol) in CH2Cl2 (1 mL) was added to a solution of oxalyl
chloride (0.40 mL, 4.57 mmol) in CH2Cl2 (16 mL) at −78 °C and
stirred for 20 min, and then alcohol 1g (919 mg, 1.90 mmol) in
CH2Cl2 (3 mL) was added at −78 °C under argon atmosphere. The
reaction mixture was stirred at −78 °C for 20 min, and triethylamine
(1.87 mL, 13.3 mmol) was added at −78 °C. The reaction mixture was
stirred at room temperature for 30 min, and then the saturated
aqueous solution of NaHCO3 was added. The aqueous layer was
extracted with CH2Cl2, and the organic layer was washed with brine,
dried over MgSO4, filtered, and concentrated in vacuo. The crude
product was purified by silica gel column chromatography (hexane/
(100 MHz, CDCl3) δ: 19.2, 22.1, 25.8, 26.9, 29.2, 29.3, 29.4, 29.4,
29.6, 29.6, 29.6, 29.6, 32.6, 43.9, 64.2, 127.5, 129.4, 134.2, 135.6, 202.9.
IR (Neat): 2928, 2855, 1728 cm−1; mass (FAB) m/z 423 [M −
C4H9]+. HRMS calcd for C27H39O2Si: 423.2719; found, 423.2718.
(E)-tert-Butyldiphenyl((16-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)hexadec-15-en-1-yl)oxy)silane (4g). Aldehyde 2g (770 mg,
1.60 mmol) in THF (2 mL), pinacol borate 329 (470 mg, 2.24 mmol)
in THF (2 mL), and TMSCl (1.22 mL, 9.61 mmol) were added to a
suspension of CrCl2 (200 mg, 1.60 mmol), LiI (1.29 g, 9.61 mmol),
and Mn (260 mg, 6.41 mmol) in THF (12 mL) under a nitrogen
atmosphere. The mixture was stirred at room temperature for 16.5 h,
and then the saturated aqueous solution of NaHCO3 was added. The
aqueous layer was extracted with EtOAc, and the organic layer was
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo. The crude product was purified by silica gel column
chromatography (hexane/EtOAc = 20/1) to give a colorless oil
1
(624 mg, 65%). H NMR (400 MHz in CDCl3) δ: 1.04 (s, 9H), 1.24
(s, 20H), 1.36−1.41 (m, 2H), 1.52−1.59 (m, 2H), 2.14 (q, J = 6.8 Hz,
2H), 3.65 (t, J = 6.8 Hz, 2H), 5.42 (d, J = 18.4 Hz, 1H), 6.63 (dt, J =
6.8, 18.4 Hz, 1H), 7.35−7.43 (m, 6H), 7.66−7.68 (m, 4H). 13C NMR
(100 MHz, CDCl3) δ: 19.2, 24.8, 25.8, 26.9, 28.2, 29.2, 29.4, 29.5,
29.6, 29.6, 29.7, 32.6, 35.8, 64.0, 83.0, 127.5, 129.4, 134.2, 135.6, 154.8.
IR (Neat): 2926, 2855, 1638 cm−1; mass (FAB) m/z 589 [M − CH3]+.
HRMS calcd for C37H58BO3Si: 589.4248; found, 589.4265.
(2Z,4E)-Methoxymethyl 19-((tert-Butyldiphenylsilyl)oxy)-3-(2-
hydroxyethyl)nonadeca-2,4-dienoate (7g). Segment A 626 (341
mg, 1.19 mmol) in MeOH (3 mL) and boronic ester 4g (594 mg, 0.99
mmol) in THF (10 mL) were added to a suspension of Pd(PPh3)2Cl2
(139 mg, 0.20 mmol) in MeOH (7 mL) at room temperature. The
mixture was stirred at room temperature for 10 min, and triethylamine
(0.98 mL, 6.94 mmol) was added. The reaction mixture was stirred at
room temperature for 40 h, filtered, and concentrated in vacuo. The
1
EtOAc = 4/1) to give a colorless oil (797 mg, 87%). H NMR (400
MHz in CDCl3) δ: 1.05 (s, 9H), 1.20−1.33 (m, 20H), 1.52−1.64 (m,
4H), 2.41 (td, J = 2.0, 6.8 Hz, 2H), 3.65 (t, J = 6.8 Hz, 2H), 7.35−7.43
(m, 6H), 7.66−7.68 (m, 4H), 9.76 (t, J = 2.0 Hz, 1H)). 13C NMR
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Scheme 3. Synthesis of tert-Butyldiphenylsilylated Long Chain Alkane Diol 2
crude product was purified by silica gel column chromatography
(hexane/EtOAc = 3/1) to give a yellow oil (463 mg, 73%). H NMR
concentrated in vacuo. The crude product was purified by
recrystallization (CH3CN) to give a colorless powder (51 mg, 95%);
mp 129−133 °C. 1H NMR (400 MHz in CD3OD) δ: 1.29 (brs, 18H),
1.41−1.45 (m, 2H), 1.57−1.61 (m, 2H), 2.19 (q, J = 6.8 Hz, 2H), 2.27
(t, J = 7.2 Hz, 2H), 3.34 (s, 2H), 5.70 (s, 1H), 6.20 (dt, J = 6.8, 16.8
Hz, 1H), 7.49 (d, J = 15.6 Hz, 1H). 13C NMR (100 MHz, CD3OD) δ:
26.1, 30.0, 30.2, 30.4, 30.6, 30.6, 30.7, 30.7, 30.7, 34.3, 35.0, 41.1,
119.8, 127.9, 140.3, 149.1, 169.4, 174.3, 177.7 . IR (KBr): 2920, 2851,
1703, 1632, 1603 cm−1. EA calcd for C21H34O6: C 65.94%, H 8.96%;
found, C 65.69%, H 8.99%.
1
(400 MHz in CDCl3) δ: 1.04 (s, 9H), 1.24−1.27 (m, 20H), 1.40−1.43
(m, 2H), 1.53−1.56 (m, 2H), 2.21 (q, J = 6.8 Hz, 2H), 2.63 (t, J = 6.8
Hz, 2H), 3.48 (s, 3H), 3.65 (t, J = 6.4 Hz, 2H), 3.78 (q, J = 6.4 Hz,
2H), 5.27 (s, 2H), 5.68 (s, 1H), 6.22 (dt, J = 6.8, 16.4 Hz, 1H), 7.34−
7.41 (m, 6H), 7.52 (d, J = 16.8 Hz, 1H), 7.66−7.68 (m, 4H); 13C
NMR (100 MHz, CDCl3) δ: 19.2, 25.8, 26.9, 29.0, 29.3, 29.4, 29.6,
29.6, 29.7, 32.6, 33.5, 37.5, 57.6, 61.9, 64.0, 89.9, 115.8, 126.5, 127.5,
129.4, 134.2, 135.6, 140.2, 153.1, 165.5. IR (Neat): 3418, 2928, 2855,
1715, 1699, 1633 cm−1; mass (FAB) m/z 575 [M − C2H3O2]+. HRMS
calcd for C37H55O3Si: 575.3920; found, 575.3920.
(2Z,4E)-Methoxymethyl 19-hydroxy-3-(2-hydroxyethyl)-
nonadeca-2,4-dienoate (8g). TBAF (1 M in THF, 1.38 mL, 1.38
mmol) was added to a solution of 7g (440 mg, 0.69 mmol) in THF (6
mL) at room temperature. The reaction mixture was stirred at room
temperature for 16 h and quenched with the saturated aqueous
solution of NH4Cl. The aqueous layer was extracted with EtOAc, and
the organic layer was washed with brine, dried over MgSO4, filtered,
and concentrated in vacuo. The crude product was purified by silica gel
column chromatography (hexane/EtOAc = 1/1) to give a colorless
powder (227 mg, 83%); mp 48−52 °C. 1H NMR (400 MHz in
CDCl3) δ: 1.25−1.31 (m, 20H), 1.42−1.45 (m, 2H), 1.53−1.58 (m,
2H), 2.22 (q, J = 6.8 Hz, 2H), 2.63 (t, J = 6.4 Hz, 2H), 3.48 (s, 3H),
3.64 (t, J = 6.4 Hz, 2H), 3.78 (t, J = 6.8 Hz, 2H), 5.28 (s, 2H), 5.68 (s,
1H), 6.23 (dt, J = 6.8, 16.4 Hz, 1H), 7.52 (d, J = 16.4 Hz, 1H). 13C
NMR (100 MHz, CDCl3) δ: 25.7. 28.9, 29.3, 29.4, 29.4, 29.5, 29.5,
29.6, 29.6, 32.8, 33.5, 37.5, 57.5, 61.9, 63.0, 89.9, 115.7, 126.5, 140.1,
153.1, 165.4. IR (KBr): 3423, 2928, 2849, 1695, 1629, 1591 cm−1. EA
calcd for C23H42O5: C 69.31%, H 10.62%; found, C 68.96%, H
10.52%.
RESULTS
■
Synthesis of Tricarboxylic Acids. We have reported a
convergent strategy for the total synthesis of BKA.26 A series of
tricarboxylic acids bearing a fatty acid chain were synthesized by
the modified convergent strategy (Scheme 2). A conjugated
double-bond was formed via Suzuki−Miyaura coupling
between Segment A (6) and pinacol borate bearing various
lengths of protected or unprotected fatty alcohols 4 and 5.
Suzuki−Miyaura coupling prior to deprotection (4→7→8)
afforded the target diols 8 in higher yield than vice versa (4→
5→8). Oxidation of the diols 8 was tried with two distinct
methods via 2 step-oxidation by Dess−Martin oxidation,
followed by Pinnick oxidation, and via straightforward oxidation
with Jones reagent to provide the carboxylic acids 9 at a similar
level of yield. Several noncommercially available 1,n-diols 1
having a long carbon chain (n = more than 10) were
synthesized by the Wittig reaction, followed by catalytic
hydrogenation (Scheme 3).
(3Z,4E)-3-(2-(Methoxymethoxy)-2-oxoethylidene)nonadec-4-ene-
dioic acid (9g). Alcohol 8g (208 mg, 0.52 mmol) in acetone (2 mL)
was added dropwise to a solution of Jones reagent (2.5 M in acetone,
1.67 mL, 4.18 mmol) in acetone (8.5 mL) at 0 °C. The reaction
mixture was stirred at 0 °C for 30 min and quenched with iPrOH, and
H2O was added. The aqueous layer was extracted with Et2O, and the
organic layer was washed with brine, dried over MgSO4, filtered, and
concentrated in vacuo. The crude product was purified by
recrystallization (EtOAc/hexane) to give a colorless powder (88.7
Biological Assays for Tricarboxylic Acid Derivatives
10. To evaluate the cytotoxicity of BKA and tricarboxylic acid
derivatives 10, the WST-1 assay was conducted after 22 h
exposure to each compound in HeLa cells. As shown in Figure
1, all tested compounds including BKA showed little toxicity
against HeLa cells. The most toxic compound among these was
10g (n = 13), but 85% of cells were still alive even at a
concentration of 200 μM.
1
mg, 40%); mp 99−101 °C. H NMR (400 MHz in CDCl3) δ: 1.26−
The apoptosis preventing activities of BKA and derivatives
10 were estimated by the WST-1 assay under the conditions of
2 h of pretreatment of test compounds followed by 22 h of
exposure to 100 nM of STS, which is well-known as an
apoptosis inducer (Figure 2).30,31 Under these conditions, STS
reduced cell viability at most 74%, and cell viability significantly
recovered by pretreatment with 200 μM BKA. As for
tricarboxylic acid derivatives, 10f (n = 10) and 10g (n = 13)
significantly suppressed STS-induced reductions in cell viability.
The other compounds (10b, 10d, 10i, and 10j) did not show
substantial recovery. Apoptosis inducible ability of STS was
confirmed by modified TdT-mediated dUTP nick-end labeling
1.30 (m, 18H), 1.41−1.43 (m, 2H), 1.60−1.64 (m, 2H), 2.23 (q, J =
7.2 Hz, 2H), 2.36 (t, J = 6.8 Hz, 2H), 3.38 (s, 2H), 3.48 (s, 3H), 5.29
(s, 2H), 5.74 (s, 1H), 6.21 (dt, J = 6.8, 16.8 Hz, 1H), 7.55 (d, J = 16.8
Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 24.4, 28.5, 28.8, 28.8, 28.9,
29.0, 29.1, 29.2, 29.3, 29.4, 33.4, 33.9, 40.4, 57.6, 90.1, 118.1, 126.3,
141.1, 147.8, 165.1, 176.6, 180.5. IR (KBr): 2922, 2849, 1701, 1634,
1603 cm−1. EA calcd for C23H38O7: C 64.76%, H 8.98%; found, C
64.79%, H 8.94%.
(2Z,4E)-3-(Carboxymethyl)nonadeca-2,4-dienedioic acid (10g).
HCl (6 M, 2.8 mL, 16.7 mmol) was added to a solution of ester 9g
(60 mg, 0.14 mmol) in THF (2.8 mL) at room temperature. The
reaction mixture was stirred at room temperature for 1 h, and H2O was
then added, extracted with Et2O, dried over MgSO4, filtered, and
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Figure 3. Effect of BKA and tricarboxylic acid derivatives 10 on
reductions in ΔΨm caused by STS in HL-60 cells. Cultured HL-60
cells were preincubated with these compounds for 1 h. ΔΨm collapse
was induced in cells by exposure to 2 μM STS for 3 h together with
the test compound. Values were evaluated using the ΔΨm sensitive
fluorescence dye JC-1. Each value represents the mean SD of three
experiments. The significance of differences was assessed using the
Tukey−Kramer test. *P < 0.05 was significantly different from the
reference group.
Figure 1. Cytotoxicity of BKA and tricarboxylic acid derivatives 10 in
HeLa cells. Cultured HeLa cells were exposed to these compounds for
22 h. Cell viability was determined by the WST-1 assay. Each value
represents the mean SD of three (control n = 6) experiments.
Biological Assays for MOM Esters 9. Cytotoxicity and
the ability to prevent apoptosis of MOM esters were also
evaluated (Figure 4). These compounds are not only synthetic
Figure 2. Effect of BKA and tricarboxylic acid derivatives 10 on cell
viability in HeLa cells. Cultured HeLa cells were preincubated with
these compounds for 2 h. Apoptosis was induced in cells by exposure
to 100 nM STS for 22 h together with the test compound. Cell
viability was determined by the WST-1 assay. Each value represents
the mean SD of three experiments. The significance of differences
was assessed using the Tukey−Kramer test. *P < 0.05 was significantly
different from the reference group.
Figure 4. Cytotoxicity of mono-MOM ester derivatives 9 in HeLa
cells. Cultured HeLa cells were exposed to these compounds for 22 h.
Cell viability was determined by the WST-1 assay. Each value
represents the mean SD of three (control n = 6) experiments.
(TUNEL) assay, and then DNA fragmentation was observed in
a dose-dependent manner (Figure S1, Supporting Informa-
tion).
Since some of the tricarboxylic acid derivatives possessed
BKA-like activity, further assessment was conducted to confirm
their availabilities. Since it has been reported that BKA is
capable of preventing disruptions in the inner mitochondrial
transmembrane potential (ΔΨm),20 the ability to prevent
disruptions in the ΔΨm induced by STS in HL-60 cells was
evaluated using the derivatives 10 (Figure 3). The fluorescence
ratio of the ΔΨm sensitive dye JC-1 represents the relative
ΔΨm in cells.32 The STS-induced ΔΨm collapse was
significantly restored by pretreatment with BKA as reported.17
In the case of the tricarboxylic acid derivatives, ΔΨm tended to
increase as carbon chains got shorter, but only 10d (n = 7)
exhibited significant ΔΨm recovery. In contrast, 10j (n = 22)
strongly promoted reductions in ΔΨm. CCCP (carbonyl
cyanide m-chlorophenylhydrazone) was used as a positive
control and completely induced uncoupling on mitochondria.
When the cell viabilities of these cells were measured right after
the JC-1 assay using trypan blue, more than 90% of cells were
still alive even in only STS- or CCCP-treated groups (data not
shown); however, apoptotic changes in morphology were
observed in STS treated cells (Figure S2, Supporting
Information).
intermediates but also more hydrophobic derivatives than the
corresponding tricarboxylic acid. Compound 9j (n = 22) was
not tested because we were not able to purify it enough due to
its detergent-like properties. As shown in Figure 4, 9h (n = 16)
exhibited potent toxicity in HeLa cells, which stood out among
the other derivatives. Compound 9i (n = 19) also somewhat
exhibited toxicity, however, which was weaker than that of 9h,
although the derivatives became more toxic as their carbon
chain length got longer.
The only compound that showed a slight ability to prevent
STS-induced apoptosis was 9d (n = 7) (Figure 5). Compounds
9h and 9i magnified reductions in cell viability, and the other
derivatives had no effect on cells.
DISCUSSION
■
BKA is well-known as a selective inhibitor for ANT that
mediates ADP/ATP exchange in the inner mitochondrial
membrane.5,6 Since the inhibition of ANT could be linked to
oxidative phosphorylation impairments, BKA treatment may
cause cell death. However, it did not exhibit potent cytotoxicity
in HeLa and HL-60 cells. This paradoxical phenomenon may
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able to magnify the activity of these derivatives. A simple
structure is helpful for such a delivery system and for the design
of further derivatives.
Once the MOM ester moiety of derivatives 9 is hydrolyzed
by intracellular esterase or under acidic conditions like the
intermembrane space of mitochondria, it becomes an active
compound, tricarboxylic acid. As such, it had been considered
as the easier membrane permeable precursor of tricarboxylic
acid derivatives. However, some of the cytotoxicities and
preventing activities of 9f and 9g were weaker than those of the
corresponding tricarboxylic acids 10f and 10g. It should be
considered that the carbon chain may be shortened by β-
oxidation in the mitochondrial matrix and that the ester moiety
would not be hydrolyzed by the intracellular esterase.
Only 10d could significantly suppress reductions in ΔΨm,
and 10b, 10f, and 10g tended to recover the potential to some
degree. The optimal carbon chain length for apoptosis
prevention was n = 10 and for ΔΨm collapse inhibition was
n = 7. This variety may come from differences in cells having
distinct sensitivities, metabolic abilities, and amounts of protein
expression. Furthermore, it could be considered that the
apoptosis preventing mechanism of these derivatives including
BKA were not only ANT inhibition but also the inhibition or
activation of other target molecules.
In conclusion, our results indicate that the BKA carbon
chain, which has an olefin, two conjugated olefins, three methyl
groups, and a methoxyl group, can be simplified to a saturated
carbon hydrate chain but that the chain length is critical to its
activity. According to our previous unpublished data, the
carboxylic acid group at the unbranched terminal is essential for
making the compound nontoxic. It is highly possible that ANT
and/or the target of these derivatives recognize the three
carboxylic acid moieties and then bind to some of them. We
have successfully achieved the design and synthesis in more
easily available potent apoptosis inhibitors, and these
compounds would be useful for biochemical mechanistic
investigations. Since there are several points that need to be
uncovered about ANT as a component of MPTP, a novel
reagent effect to ANT will play an important role as an
apoptosis research tool. Further structure−activity relationship
studies are ongoing, and investigations may elucidate a
pharmacophore of BKA.
Figure 5. Effect of mono-MOM ester derivatives 9 on cell viability in
HeLa cells. Cultured HeLa cells were preincubated with these
compounds for 2 h. Apoptosis was induced in cells by exposure to
100 nM STS for 22 h together with the test compound. Cell viability
was determined by the WST-1 assay. Each value represents the mean
SD of three experiments. The significance of differences was
assessed using the Tukey−Kramer test. *P < 0.05 was significantly
different from the reference group.
happen because these kinds of cancer cells use aerobic
glycolysis with reduced mitochondrial oxidative phosphoryla-
tion for glucose metabolism.33 Under such ATP supplied
conditions, BKA prevents MPTP opening and cytochrome c
release from mitochondria, thus suppressing apoptosis
induction.24 There are several apoptotic pathways without
mitochondrial uncoupling, for instance, a pathway through
caspase 8 activation. We considered that this is one of the
reasons why BKA could not completely rescue cells from STS-
induced apoptosis.
Synthesized tricarboxylic acids and their MOM esters were
applied to the WST-1 assay to estimate their cytotoxicity, and
only MOM esters 9h and 9i, bearing a long carbon chain, were
found to be positioned as toxic compounds. Conformational
searches using the MMFF force field (CONFLEX ver. 5;
CONFLEX Corporation, Tokyo, Japan) indicated that BKA
would adopt cyclic conformations, rather than linear ones,
partially due to hydrogen bonding with two carboxylic acid
moieties. In the case of the tricarboxylic acids synthesized
herein, cyclic conformations may be possible when their carbon
chain is long enough (n = around 13 or more). Although it is
not clear whether cyclic conformations are essential for binding
to target proteins, three carboxylic acid moieties and
appropriate spatial positioning among these moieties would
be crucial for the inhibition of apoptosis.
The most effective apoptosis inhibitor among these
derivatives was 10f, and 10g was as potent as 10f. The
MOM ester 9d also showed a slight inhibitory activity.
Contrary to our expectations, the tricarboxylic acid 10h,
which was designed and synthesized as a BKA equivalent
bearing the same carbon chain length, did not exhibit significant
activity. However, the fact that a compound with a shorter
carbon chain possesses higher activity is desirable for further
investigation.
Usually cationic and lipophilic compounds selectively localize
on mitochondria because of the high negative transmembrane
electrical potential maintained by functional mitochondria.34
An anionic compound like carboxylic acid would not reach the
inner mitochondrial membrane where ANT localizes and works
except by passive diffusion. Thus, an efficacious carrier for the
delivery of anionic compounds into mitochondria35 may be
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, DNA fragmentation caused by STS
addition in HeLa cells, and apoptotic changes in morphology in
HL-60 cells exposed with STS. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +81-92-583-7802. Fax: +81-92-583-7875. E-mail:
shindo@cm.kyushu-u.ac.jp.
Funding
This work was supported by the Program for Promotion of
Basic and Applied Research for Innovations in the Bio-oriented
Industry (BRAIN) from Bio-oriented Technology Research
Advancement Institution, a Grant-in-Aid for Exploratory
Research (22659023) (to M.S.), and a Grant-in-Aid for
Young Scientists (B) (23790131) (to K.M.) from the Ministry
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(13) Brustovetsky, T., Shalbuyeva, N., and Brustovetsky, N. (2005)
Lack of manifestations of diazoxide/5-hydroxydecanoate-sensitive
KATP channel in rat brain nonsynaptosomal mitochondria. J. Physiol.
568, 47−59.
(14) Kiranadi, B., Bangham, J. A., and Smith, P. A. (1991) Inhibition
of electrical activity in mouse pancreatic beta-cells by the ATP/ADP
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(15) Toninello, A., Salvi, M., Schweizer, M., and Richter, C. (2004)
Menadione induces a low conductance state of the mitochondrial
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37, 1073−1080.
of Education, Culture, Sports, Science and Technology of
Japan.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Professor Atsushi Maruyama (Institute for Materials
Chemistry and Engineering, Kyushu University) for kindly
providing HeLa cells.
(16) Kopustinskiene, D. M., Toleikis, A., and Saris, N.-E. L. (2003)
Adenine nucleotide translocase mediates the K(ATP)-channel-open-
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ABBREVIATIONS
■
BKA, bongkrekic acid; ANT, adenine nucleotide translocase;
MPTP, mitochondrial permeability transition pore; ΔΨm,
mitochondrial inner membrane potential; STS, staurosporine;
CCCP, carbonyl cyanide m-chlorophenyl hydrazone; 1-
methoxy-PMS, 1-methoxy-5-methylphenazinium methylsulfate;
TBDPSCl, tert-butyldiphenylchlorosilane; TMSCl, trimethyl-
chlorosilane; TBAF, tetrabutyl ammonium fluoride; PPh3,
triphenylphosphine; nBuLi, n-butyl lithium; HRMS, high
resolution mass spectrometry; EA, elemental analysis
(17) Teshima, Y., Akao, M., Li, R. A., Chong, T. H., Baumgartner, W.,
́
Johnston, M. V., and Marban, E. (2003) Mitochondrial ATP-sensitive
potassium channel activation protects cerebellar granule neurons from
apoptosis induced by oxidative stress. Stroke 34, 1796−1802.
(18) Ulziikhishig, E., Lee, K. K., Hossain, Q. S., Higa, Y., Imaizumi,
N., and Aniya, Y. (2010) Inhibition of mitochondrial membrane
bound-glutathione transferase by mitochondrial permeability transition
inhibitors including cyclosporin A. Life Sci. 86, 726−732.
́
(19) Zamzami, N., Susin, S. A., Marchetti, P., Hirsch, T., Gomez-
Monterrey, I., Castedo, M., and Kroemer, G. (1996) Mitochondrial
control of nuclear apoptosis. J. Exp. Med. 183, 1533−1544.
(20) Zamzami, N., Marchetti, P., Castedo, M., Hirsch, T., Susin, S. A.,
Masse, B., and Kroemer, G. (1996) Inhibitors of permeability
transition interfere with the disruption of the mitochondrial trans-
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Haeffner, A., Hirsch, F., Geuskens, M., and Kroemer, G. (1996)
Mitochondrial permeability transition is a central coordinating event of
apoptosis. J. Exp. Med. 184, 1155−1160.
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