Synthesis and characterization of Δlac-acetogenins that potently inhibit mitochondrial complex I

Article abstract of DOI:10.1016/j.bmc.2008.10.071

Four Δlac-acetogenins have been prepared and characterized as inhibitors of the mitochondrial respiratory chain, to define the effects of unsaturation within the alkyl substituents. In keeping with earlier reports, the presence of acetylenic functionaliti

Full text of DOI:10.1016/j.bmc.2008.10.071

Bioorganic & Medicinal Chemistry 17 (2009) 2204–2209
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of
mitochondrial complex I
Dlac-acetogenins that potently inhibit
*
Jean-Charles Chapuis, Omar Khdour, Xiaoqing Cai, Jun Lu, Sidney M. Hecht
Center for BioEnergetics, The Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Four
Dlac-acetogenins have been prepared and characterized as inhibitors of the mitochondrial respira-
Received 17 April 2008
Revised 3 August 2008
Accepted 31 October 2008
Available online 5 November 2008
tory chain, to define the effects of unsaturation within the alkyl substituents. In keeping with earlier
reports, the presence of acetylenic functionalities within the alkyl substituents slightly diminished their
potency of inhibition of NADH oxidase activity, which measures the overall transfer of electrons from
NADH to oxygen through mitochondrial complexes I, III, and IV. In contrast, both of the acetylenic
D
lac-acetogenins were far more active in a NADH–ubiquinone Q1 oxidoreductase assay that measures
Keywords:
complex I function per se.
Annonaceous acetogenins
Mitochondrial complex I
NADH–ubiquinone oxidoreductase
NADH oxidase
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
In recent studies of the structural requirements for complex I
inhibition, the alkyl substituents of acetogenins and
Dlac-acetoge-
Members of the family Annonaceae produce a very interesting
class of natural product known as acetogenins.1 Many compounds
in this structural class exhibit potent biological activities such as
anti-tumor, anti-malarial, and pesticidal activities.2 These diverse
biological activities have been attributed to the compelling inhibi-
tory effects of these compounds on mitochondrial complex I
(NADH–ubiquinone oxidoreductase) in mammalian and insect
mitochondrial electron transport systems.3 They have also been re-
ported as potent inhibitors of NADH oxidase of the plasma mem-
brane of cancer cells.4
nins were altered in length and by introduction of single and multi-
ple double and triple bonds within the alkyl substituent. While
dramatic effects were observed with varying chain length, the ef-
fects of unsaturation were less clear, producing little change in
some cases and a pronounced diminution of activity in others.6,7
As part of an effort to characterize the function of the mitochon-
drial respiratory chain, we have prepared four
Dlac-acetogenins
(1–4) (Fig. 1), two of which contain alkynes within the alkyl sub-
stituents. We have characterized these compounds in a series of
biological assays including (i) complex I assay measured by
NADH–ubiquinone oxidoreductase, (ii) complexes I, III, and IV as-
say measured by NADH oxidase, (iii) evaluation of the conse-
quences of the compounds in inducing a shift to glycolysis, and
(iv) evaluation of the cytotoxicities of all four compounds in sev-
eral mammalian cell lines.
We found that acetylenic compounds 1 and 3, not previously
studied as complex I inhibitors, strongly inhibit complex I and
actually exhibit significantly stronger activities than the reported
inhibitors 28 and 4.9 Presently, we detail the preparation, biochem-
Many of the naturally occurring acetogenins contain
a,b-unsat-
urated -methylbutyrolactone substituents at the terminus of a
c
linear alkyl substituent, as exemplified by bullatacin, trilobin, and
trilobacin (Fig. 1). The intrinsic electrophilic nature of such func-
tionalities may be capable of mediating enhanced potency through
alkylation of their target proteins, albeit with the potential for loss
of selectivity of action. Bullatacin is one of the most potent inhib-
itors of bovine heart complex I NADH–ubiquinone oxidoreductase,
having activity reported at low nanomolar concentration.
Recently, structurally related compounds lacking the
urated -methylbutyrolactone ring have been described. These
lac-acetogenins appear to act at a site on complex I different than
a
,b-unsat-
ical, and biological characterization of these interesting new
acetogenin complex I inhibitors.
Dlac-
c
D
the prototype acetogenins, or the more traditional mitochondrial
2. Results and discussion
complex I inhibitors such as rotenone and piericidin.5
Inhibitors 1 and 2 were prepared as described previously.9
Known9 compounds 3 and 4 were prepared analogously but using
a new route for a key synthetic intermediate (8), as outlined in
* Corresponding author. Tel.: +1 480 965 6625; fax: +1 480 965 0038.
E-mail address: sidney.hecht@asu.edu (S.M. Hecht).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.10.071

J.-C. Chapuis et al. / Bioorg. Med. Chem. 17 (2009) 2204–2209
2205
OH
R1
R2
O
O
HO
HO
O
O
O
O
O
O
HO
HO
trilobin R1 = OH, R2 = H
trilobacin R1 = H, R2 = OH
bullatacin
HO
HO
O
O
O
O
HO
HO
2
1
HO
HO
O
O
O
O
O
O
O
O
HO
HO
4
3
Figure 1.
Dlac-acetogenins 1–4 and structurally related natural compounds.
Scheme 1. n-Butylphenoxypropionaldehyde 6 was prepared read-
ily in two steps as shown in the Scheme, and then converted to 8
via dibromobutenyl ether 7. Nucleophilic opening of the epoxide
moieties in 9 then afforded inhibitor 3 in 99% yield as a colorless
oil. Catalytic hydrogenation of 3 afforded 4 in 87% yield.
Both the NADH oxidase and NADH–ubiquinone oxidoreductase
activities of compounds 1–4 were measured. NADH oxidase activ-
ity reflects forward electron transfer in the respiratory chain,
wherein NADH is oxidized by endogenous coenzyme Q10 with
transfer of electrons through complexes III and IV to O2. It was
found that the bovine heart submitochondrial particles (SMPs)
functioned significantly more robustly in Hepes buffer,10 than the
reported5,6,8,9 phosphate buffer. Accordingly, the assay was run in
Hepes buffer. As shown in Table 1, acetylenic derivatives 1 and 3
had IC50 values of 30.1 and 38.8 nM in this assay, while the corre-
sponding saturated derivatives 2 and 4 gave values of 12.3 and
19.6 nM, respectively. Thus, the presence of the triple bonds in 1
and 3 diminished the NADH oxidase activity of these compounds
to some extent.11
Inhibition of complex I formation was also measured by mea-
suring NADH–ubiquinone oxidoreductase activity in the presence
of exogenous ubiquinone Q1, as well as complex III inhibitor anti-
mycin A and complex IV inhibitor cyanide. As shown in Table 1,
acetylenic compound 1 was significantly more active in this assay
than acetylenic compound 3 (48 vs 1700 nM IC50 values, respec-
tively). Compounds 2 and 4 exhibited little inhibitory activity in
this assay system.12
optimal chain length and balance of hydrophobicity of the two al-
kyl substituents, as well as the steric nature of the alkyl substitu-
ents, were important factors for respiratory chain inhibition.8,14
In another study, these workers found that local flexibility in a spe-
cific region of one of the alkyl chains (studied by introducing dou-
ble and triple bonds) was not obviously important for the
inhibitory activity of the acetogenins.6
According to the Warburg effect, which is defined by the observa-
tion that cancer cells are intrinsically more glycolytic than normal
cells, tumor cells depend less on mitochondrial function for ATP pro-
duction and more on glycolysis than their normal counterparts.15
Further, it has been reported that more generally when cells lack
oxygen, they have a propensity to switch to anaerobic metabolism,
resulting in an increase in glycolysis.16 The Pasteur effect, defined
as the inhibition of glycolysis by respiration, is based on such a prin-
ciple.17 When cells are subjected to decreased mitochondrial func-
tion due to metabolic stress, glycolysis is stimulated in order to
maintain relatively constant ATP synthesis. Under such conditions,
pyruvate is reduced to lactate to regenerate oxidized pyridine nucle-
otides. Although not as efficient as aerobic respiration, two ATP and
two lactate molecules are formed per glucose metabolized. The out-
come is increased lactate production sufficient to compensate for
inhibition of respiration. We have used this principle to evaluate
known anti-mitochondrial toxins, such as antimycin A, as well as
the
Dlac-acetogenins that are the focus of this study.
Figure 2 shows the titration of semi-confluent MCF-7 cells ex-
posed to varying concentrations of compounds 1–4 for 3 h. Acety-
lenic compounds 1 and 3 stimulated significantly more lactate
production than 2 and 4. In order to compare these compounds,
Previous studies by Miyoshi and co-workers have focused on
the alkyl substituents of
Dlac-acetogenins and indicated that an

2206
J.-C. Chapuis et al. / Bioorg. Med. Chem. 17 (2009) 2204–2209
O
O
Br
CBr4, PPh3
CH2Cl2, O o
AcOH/H2O
K2CO3/acetone
45 o
C
C
O
OH
O
O
O
91%
66% (two steps)
O
H
6
5
Br
Br
n-BuLi, THF
60%
O
O
8
7
n-C4H9
n-C4H9
O
HO
HO
O
O
1) n-BuLi, THF
2) BF3.OEt2, THF
3) 8, THF
O
O
H2, Pd/C
EtOH
O
O
O
87%
99%
O
n-C4H9
O
O
HO
HO
O
n-C4H9
4
3
9
Scheme 1.
cytotoxicity of synthetic hybrid acetogenins toward a panel of 39
human cancer cell lines.20 In the present study, additional cell
lines, such as two human glioblastoma lines (Table 3), were found
to be largely unaffected by these compounds as well. For the cell
lines tested, 1 was generally more potently cytotoxic than 2–4.
One exception was Lewis lung carcinoma which was inhibited
strongly by all of the compounds, especially by compounds 2–4.
Liu et al.21 recently reported micromolar inhibition of HT-29 cells
by a modified bullatacin derivative, but obtained nanomolar activ-
ities against cancerous human liver cells and a lack of response in
normal hepatocytes. Whether these effects are all due to action at
the locus of mitochondrial complex I is unclear at present.
Table 1
Inhibitory activities toward mitochondrial complex Ia of the described
acetogenins
Dlac-
b
Compound
IC50
NADH oxidase (nM)
NADH–ubiquinone (Q1)
oxidoreductase (lM)
1
2
3
4
30.1 0.8
12.3 0.3
38.8 1.3
19.6 1.9
0.048 0.002
10.4 0.3
1.7 0.2
>15
a
Test performed on bovine heart submitochondrial particles (SMP).
The molar concentration required for half-maximal inhibition of the control.
b
Differences in the mode of ATP production between normal and
cancer cells, that is, the extent of use of glycolysis versus the mito-
chondrial respiratory chain, have been suggested as a basis for
selective cancer chemotherapy.22,23 In addition to their lack of an
Values are means SD of three independent experiments. NADH–ubiquinone oxi-
doreductase was determined with ubiquinone 1 (Q1) as the ubiquinone analogue.
Control activity was approximately 0.92
l
mol minꢀ1 mgꢀ1 for NADH oxidase and
0.75
l
mol minꢀ1 mgꢀ1 for NADH–ubiquinone oxidoreductase.
electrophilic
a,b-unsaturated c-methylbutyrolactone constituent,
the lac-acetogenins are of interest as mitochondrial inhibitors
D
the concentration required to obtain half of the maximal lactate
production was calculated. Table 2 presents that value for each
of compounds 1–4 compared to antimycin A under identical condi-
tions. Compounds 2 and 4 were found to be less able to induce a
shift to glycolysis than the two acetylenic compounds 1 and 3,
the latter of which had activity in the nanmolar range. Increasing
the incubation time from 3 to 6 h did not significantly change
due to the ability of at least some of these compounds (e.g., 4) to
inhibit mitochondrial complex I function without strongly induc-
ing superoxide production.24 In the present study, we have demon-
strated that introduction of an alkyne moiety within the aliphatic
substituent of such compounds can result in more potent inhibi-
tion of NADH–ubiquinone oxidoreductase activity, and a signifi-
cantly enhanced ability to induce a shift to glycolysis. This design
feature may find utility in implementing therapeutic strategies
that exploit differences in ATP production between normal and
cancer cells.
the values measured (0.44, 4.4, 0.32, and 18.8
respectively).
lM for 1–4,
Also determined were the cytotoxicities of 1–4 toward cultured
mammalian cells. Compounds 1–4 were not strongly cytotoxic. The
results were confirmed by SRB assay18 which gave similar values
(data not shown). In both assays, compound 1 was found to be
the most active. ‘Normal’ human breast cells (MCF-10A) were
found slightly more sensitive to compound 1, although the differ-
ence was not large. Natural acetogenins, such as bullatacin and
derivatives have been previously reported19 not to be as cytotoxic
toward MCF-7 cells as compared with A549 (lung) or HT-29 (colon)
cells. Other workers have recently reported a lack of significant
3. Experimental
3.1. Chemistry
Chemicals and solvents were of reagent grade and were used
without further purification. Anhydrous grade solvents were pur-
chased from VWR. All reactions involving air or moisture sensitive

J.-C. Chapuis et al. / Bioorg. Med. Chem. 17 (2009) 2204–2209
2207
200.0
180.0
160.0
140.0
120.0
100.0
80.0
60.0
40.0
20.0
0.0
0
0.08
0.4
2
10
50
Concentration (µg/mL)
Figure 2. Lactate production by MCF-7 cells exposed to compound 1 ( ), 2 ( ), 3 ( ), or 4 ( ) for 3 h.
heated at reflux overnight. The cooled reaction mixture was fil-
tered and concentrated to dryness. The residue was dissolved in
20 mL of water and the aqueous mixture was adjusted to a pH be-
tween 2 and 3 using 1.2 N HCl. The solution was extracted with
ethyl acetate and the combined organic phase was washed with
brine, dried over anhydrous Na2SO4, and concentrated under
diminished pressure to yield a crude oil. The residue was purified
by flash chromatography on a silica gel column (25.4 ꢁ 2.6 cm).
Elution with 1:20 ethyl ether/hexanes gave 5 as a colorless oil:
yield 1.4 g (91%); silica gel TLC Rf 0.42 (1:5 ethyl ether/hexanes);
1H NMR (CDCl3) d 0.90 (t, 3H, J = 7.2 Hz), 1.32 (m, 2H), 1.53 (m,
2H), 2.12 (m, 2H), 2.53 (t, 2H, J = 8.1 Hz), 3.86 (t, 2H, J = 2.1 Hz),
3.97 (t, 2H, J = 2.7 Hz), 4.08 (t, 2H, J = 4.8 Hz), 5.01 (t, 1H,
J = 4.8 Hz), 6.80 (d, 2H, J = 8.4 Hz), and 7.06 (d, 2H, J = 8.4 Hz); 13C
NMR (CDCl3) d 13.95, 22.28, 33.89, 33.91, 34.72, 63.62, 64.90,
102.13, 114.31, 129.21, 135.07, and 156.84; mass spectrum (EI),
m/z 250.1568 (M+) (C15H22O3 requires m/z 250.1569).
Table 2
Lactate production by MCF-7 human breast cancer cells incubated for 3 h in the
presence of compounds 1, 2, 3, or 4
Compound
Concentration (
lM)
Incremental lactate production (lM)
1
2
3
4
0.38
6.6
0.44
18.2
<0.15
1080
616
1280
572
Antimycin A
1520
reagents or intermediates were performed under an argon atmo-
sphere. Flash chromatography was carried out using Silicycle
200–400 mesh silica gel. Analytical TLC was carried out using
0.25 mm EM silica gel 60 F250 plates that were visualized by irradi-
ation (254 nm) or by staining with p-anisaldehyde stain. 1H and 13
C
NMR spectra were obtained using Gemini 300, Inova 400, Inova
500 and VNMRS 800 MHz Varian instruments. Chemical shifts
were reported in parts per million (ppm, d) referenced to the resid-
ual 1H resonance of the solvent (CDCl3, 7.26 ppm).13C spectra were
referenced to the residual 13C resonance of the solvent (CDCl3,
77.0 ppm). Splitting patterns were designated as follows: s, singlet;
br, broad; d, doublet; dd, doublet of doublets; t, triplet; q, quartet;
m, multiplet. High resolution mass spectra of 4, 5, 7, and 8 were ob-
tained at the Michigan State University-NIH Mass Spectrometry
Facility. For compound 3, the high resolution mass spectrum was
obtained at the Ohio State University Mass Spectrometry Facility.
3.1.2. 3-(4-n-Butylphenoxy)propionaldehyde (6)
A solution containing 200 mg (1.60 mmol) of 5 in 5 mL of 4:1
acetic acid/water was stirred at 45 °C for 5 h. The cooled reaction
mixture was adjusted to a pH between 6 and 7 with satd aq NaH-
CO3. The aqueous mixture was then extracted with ethyl acetate
and the combined organic phase was washed with brine and dried
over anhydrous MgSO4. Concentration under diminished pressure
gave a crude oil. Crude 6 was used for the next step without further
purification; silica gel TLC Rf 0.38 (1:5 ethyl ether/hexanes).
3.1.1. 2-(2-(4-n-Butylphenoxy)ethyl)-1,3-dioxolane (5)
3.1.3. 1-Butyl-4-(4,4-dibromobut-3-enyloxy)benzene (7)
To a dried flask were added 419 mg (1.26 mmol) of CBr4 and
2 mL of anhydrous CH2Cl2 at 0 °C. A solution containing 662 mg
(2.52 mmol) of PPh3 in 2 mL of anhydrous CH2Cl2 was then added
dropwise. The reaction mixture was stirred at 0 °C for 10 min, and
To a suspension of 3.31 g (4.00 mmol) of K2CO3 in 20 mL of ace-
tone was added 900 mg (6.00 mmol) of 4-n-butylphenol at 70 °C.
After stirring for 1 h, 2.17 g (12.0 mmol) of 2-(2-bromoethyl)-1,3-
dioxolane was added to the mixture. The reaction mixture was
Table 3
Human and mouse cell line inhibition values (IC50) in l
M for compounds 1–4 determined by MTT assaya
Compound
MCF-7
MCF-10A
CRL-2365
CRL-2366
A549
3LL
5.2 0.6
0.99 0.06
0.48 0.14
0.64 0.06
1
2
3
4
7.6 2.2
>20
>16
2.0 0.2
>20
>16
20.3 2.5
>20
14.6 1.4
>16
>20.9
>20
>16
13.2 1.0
>20
>16
>16
>16
>16
>16
a
Cell type: MCF-7 (breast carcinoma); MCF-10A (‘normal’ human breast); CRL-2365 (brain glioblastoma); CRL-2366 (DNA-repair deficient brain glioblastoma); A549 (lung
carcinoma); 3LL (mouse Lewis lung carcinoma).

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J.-C. Chapuis et al. / Bioorg. Med. Chem. 17 (2009) 2204–2209
a solution containing 130 mg (0.63 mmol) of 6 in 3 mL of anhy-
drous CH2Cl2 was added slowly. The reaction mixture was stirred
for 1.5 h and then 3 mL of water was added. The mixture was ex-
tracted with portions of CH2Cl2 and the combined organic phase
was washed with brine and dried over anhydrous MgSO4, then
concentrated under diminished pressure. Twenty milliliters of
ether was added to the residue and the resulting suspension was
filtered to remove triphenylphosphine oxide. The solid was washed
again with ether. The combined filtrate was concentrated and the
residue was purified by flash chromatography on a silica gel col-
umn (25 ꢁ 2.6 cm). Elution with 1:25 ether/hexanes gave 7 as a
colorless oil: yield 130 mg (66% over two steps); silica gel TLC Rf
0.85 (1:5 ethyl ether/hexanes); 1H NMR (CDCl3) d 0.92 (t, 3H,
J = 7.2 Hz), 1.33 (m, 2H), 1.53 (m, 2H), 2.55 (m, 4H), 3.99 (t, 2H,
J = 6.6 Hz), 6.58 (t, 1H, J = 7.2 Hz), 6.80 (d, 2H, J = 8.7 Hz), and 7.09
(d, 2H, J = 8.1 Hz); 13C NMR (CDCl3) d 14.11, 22.44, 33.39, 34.03,
34.88, 65.46, 90.93, 114.56, 129.46, 135.03, 135.60 and 156.69;
mass spectrum (EI), m/z 359.9715 (M+) (C14H18O79Br2 requires m/
z 359.9724).
in 1 mL of ethanol was stirred overnight under a H2 atm at room
temperature. The catalyst was removed by filtration and the result-
ing mixture was concentrated and purified by flash chromatogra-
phy on a silica gel column (20 ꢁ 1.7 cm). Elution with 1:2 ethyl
ether/hexanes gave 4 as a colorless oil: yield 12.2 mg (87%); silica
gel TLC Rf 0.45 (1:3 hexane/ethyl acetate); 1H NMR (CDCl3) d 0.91
(t, 6H, J = 7.5 Hz), 1.34–1.69 (m, 24H), 1.76–1.79 (m, 4H), 1.97–
1.99 (m, 4H), 2.48 (br, 2H), 2.54 (t, 4H), 3.40 (m, 2H), 3.83–3.87
(m, 4H), 3.92 (t, 4H, J = 6.5 Hz), 6. 80 (d, 4H, J = 8.5 Hz), and 7.07
(d, 4H, J = 8.0 Hz); 13C NMR (CDCl3) d 13.94, 22.29, 25.42, 26.15,
28.35, 28.94, 29.31, 33.37, 33.89, 34.72, 67.87, 73.94, 81.76,
83.12, 114.27, 129.18, 134.82 and 157.11; mass spectrum (ESI),
m/z 639.4628 (M+H)+ (C40H63O6 requires m/z 639.4625); ½a D25
ꢃ
+0.025° (c 0.75, CH2Cl2).
3.2. Biological evaluation
3.2.1. Lactate assay
Using an adaptation of the method of Everse,26 mitochondrial
toxins were assessed in MCF-7 human breast carcinoma cells. Cells
were plated at 2 ꢁ 105 cells per well in tissue culture treated 12-
well plates and incubated for 24 h (resulting in about 50% cell con-
fluence). The wells were rinsed with a pre-warmed solution of
Krebs–Ringer buffer (Sigma) and the compounds were then added
at the appropriate concentrations in 0.5 mL of buffer and incu-
bated for 6 h in comparison with controls containing the same
amount of buffer. The supernatant was recovered and the variation
3.1.4. 1-(But-3-ynyloxy)-4-n-butylbenzene (8)
A solution of 0.46 mL of n-BuLi in hexane (2.5 M in hexane,
1.15 mmol) was added dropwise to a stirred solution containing
190 mg (0.53 mmol) of 7 in 20 mL of anhydrous THF at ꢀ78 °C.
After 45 min, the reaction mixture was slowly warmed to 0 °C.
The reaction was quenched with satd NH4Cl after 1 h. The reaction
mixture was extracted with ethyl acetate and the organic phase
was washed with brine, and dried over anhydrous MgSO4, then
concentrated under diminished pressure. The residue was purified
by flash chromatography on a silica gel column (25 ꢁ 2.6 cm). Elu-
tion with 1:60 ethyl ether/hexanes gave 8 as a colorless oil: yield
60 mg (60%); silica gel TLC Rf 0.77 (1:20 ethyl ether/hexanes); 1H
NMR (CDCl3) d 0.96 (t, 3H, J = 5.4 Hz), 1.36 (m, 2H), 1.57 (m, 2H),
2.06 (d, 1H, J = 2.7 Hz), 2.58 (t, 2H, J = 5.7 Hz), 2.68 (m, 2H), 4.11
(t, 2H, J = 5.4 Hz), 6.86 (d, 2H, J = 6.3 Hz), and 7.12 (d, 2H,
J = 6.6 Hz); 13C NMR (CDCl3) d 13.98, 19.59, 22.32, 33.90, 34.76,
66.08, 69.83, 80.57, 114.51, 129.32, 135.53, and 156.45; mass spec-
trum (EI), m/z 202.1360 (M+) (C14H18O requires m/z 202.1358).
in absorbance (
tion ( g/mL) was calculated from the following formula: [lacta-
te] = [( OD363) (10f)(90)]/9.1 where f is the dilution factor used
DOD) was measured at 363 nm. Lactate concentra-
l
D
to bring the lactate concentration within the measurable range
of the lactate titration curve, 90 represents the molecular weight
of lactic acid and 9.1 is the mM extinction coefficient of the reac-
tion at 363 nm. IC50 values were calculated as the dose which pro-
duced a 50% inhibition of the maximum lactate production
measured.
3.2.2. MTT assay
In vitro inhibition of human cancer cell growth was assessed
3.1.5. (1R,10R)-1,10-((2R,20R,5R,50R)-Octahydro-2,20-bifuran-5,50-
diyl)-bis-(6-(4-n-butylphenoxy)hex-3-yn-1-ol) (3)
using the standard MTT assay as previously described.27
Compound 3 was prepared according to the method of Ichimaru
et al.7 To a solution of 143 mg (0.71 mmol) of 8 in 2 mL of anhy-
drous THF was added 1.28 mL of a solution of n-BuLi in hexane
(2.5 M in hexane, 0.71 mmol) at ꢀ78 °C. After 30 min, 101 mg
(0.71 mmol) of BF3ꢂEt2O was added and the reaction mixture was
stirred for another 30 min. A solution of 7 mg (0.03 mmol) of 925
in 1.0 mL of THF was then added and the reaction mixture was stir-
red for 1.5 h. The reaction was quenched by the addition of sat
NH4Cl. The reaction mixture was extracted with ether. The com-
bined organic phase was washed with brine and dried over anhy-
drous MgSO4, then concentrated under diminished pressure to
give a crude oil. The residue was purified by flash chromatography
on a silica gel column (20 ꢁ 1.7 cm). Elution with 1:2 ethyl ether/
hexanes gave 3 as a colorless oil: yield 19.5 mg (99%); silica gel
TLC Rf 0.40 (3:1 ethyl acetate/hexanes); 1H NMR (CDCl3) d 0.90
(t, 6H, J = 7.2 Hz), 1.32 (m, 4H), 1.55 (m, 4H), 1.85 (m, 4H), 1.91
(m, 4H), 2.40 (t, 4H, J = 3.9 Hz), 2.51 (t, 4H), 2.59–2.64 (m, 6H),
3.58 (br, 2H), 3.86 (m, 2H), 4.01 (m, 6H), 6.79 (d, 4H, J = 8.4 Hz)
and 7.06 (d, 4H, J = 8.1 Hz); mass spectrum (ESI), m/z 653.3821
(M+Na)+ (C40H54O6 requires m/z 653.3818).
3.2.3. Mitochondrial complex I activity
Beef heart mitochondria were obtained by a large-scale proce-
dure.28 Inverted submitochondrial particles (SMP) were prepared
by the method of Matsuno-Yagi and Hatefi,29 and stored in a buffer
containing 0.25 M sucrose and 10 mM Tris–HCl (pH 7.4) at ꢀ80 °C.
Inhibitory effects of compounds 1 and 3 on bovine heart mitochon-
drial complex I (NADH oxidase and NADH:ubiquinone oxidoreduc-
tase) were evaluated by modification of a method described
previously.8 Stock solutions (2 mg/mL in dimethylsulfoxide) of
compounds 1 and 3 (Fig. 1) were prepared and kept in the dark
at ꢀ80 °C. Maximal dimethyl sulfoxide concentration never ex-
ceeded 2% and had no influence on the control enzymatic activity.
Beef heart SMP were diluted to 0.5 mg/mL, and treated with
300
were assayed at 30 °C and monitored spectrophotometrically with
l
V NADH to activate complex I.30 The enzymatic activities
a
e
Molecular
Devices
SPECTRA
Max-M5
(340 nm,
= 6.22 mMꢀ1 cmꢀ1). NADH oxidase activity was determined in a
reaction medium (2.5 mL) containing 50 mM Hepes, pH 7.5, con-
taining 5 mM MgCl2. The final mitochondrial protein concentration
was 30 lg. The reaction was initiated by adding 50 lM NADH after
the pre-equilibration of SMP with inhibitor for 5 min. The initial
rates were calculated from the linear portion of the traces. The
inhibition of NADH-Q1 oxidoreductase activity was also deter-
mined under the same experimental conditions except that the
reaction medium (2.5 mL) contained 0.25 M sucrose, 1 mM MgCl2,
3.1.6. (1R,10R)-1,10-((2R,20R,5R,50R)-Octahydro-2,20-bifuran-5,50-
diyl)-bis-(6-(4-n-butylphenoxy)hexan-1-ol) (4)
Compound 4 was prepared according to the method of Ichimaru
et al.9 A mixture of 13.8 mg (0.02 mmol) of 3 and 3 mg of 10% Pd/C

J.-C. Chapuis et al. / Bioorg. Med. Chem. 17 (2009) 2204–2209
2209
inhibitor, was subtracted when calculating the IC50 values. Residual NADH
oxidase activities were 12% and 18% for compounds 1 and 3, respectively.
Residual NADH-Q1 oxidoreductase activities were 18% and 25% for compounds
1 and 3, respectively.
2 lM antimycin A, 2 mM KCN, 50 lM ubiquinone Q1, and 50 mM
phosphate buffer, pH 7.4.
13. Duval, R. A.; Lewin, G.; Peris, E.; Chahboune, N.; Garofano, A.; Droese, S.; Cortes,
D.; Brandt, U.; Hocquemiller, R. Biochemistry 2006, 45, 2721.
14. Chimaru, N.; Abe, M.; Murai, M.; Senoh, M.; Nishioka, T.; Miyoshi, H. Bioorg.
Med. Chem. Lett. 2006, 16, 3555.
15. Warburg, O.; Wind, F.; Negalein, E. J. Physiol. 1927, 8, 519.
16. Brown, J. Metab. Clin. Exp. 1962, 11, 1098.
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,
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compounds
2 and 4 gave values of 7.5 0.3 nM and 0.83 0.04 nM,
respectively.9
12. Residual enzymatic activity,13 defined as NADH oxidase or NADH-Q1
oxidoreductase activity not inhibited by
a high (1 lM) concentration of