Subnanomolar Inhibitor of Cytochrome bc1
A R T I C L E S
dCH-OCH3), 5.07 (s, 2H, CH2), 5.82 (s, 1H, HetH), 6.87 (t, J )
8.0 Hz, 1H, ArH), 6.99 (d, J ) 8.0 Hz, 1H, ArH), 7.15-7.23 (m,
3H, ArH), 7.38-7.43 (m, 2H, ArH), 7.53 (dd, J ) 3.6 Hz, J ) 4.8
Hz, 1H, ArH), 7.63 (s, 1H, dCH-OCH3); EI-MS m/z (relative
intensity) 395 ([M + 1]+, 17), 394 (M+, 100), 362 (19), 330 (14),
205 (60), 204 (42), 188 (13), 145 (83), 131 (23), 117 (38), 115
(25), 102 (51). Anal. Calcd for C22H22N2O5: C, 66.99; H, 5.62; N,
7.10. Found: C, 67.19; H, 5.38; N, 7.21.
7.60 (s, 1H, dCH-OCH3), 7.71 (s, 1H, ArH); EI-MS m/z (relative
intensity) 423 ([M + 1]+, 8), 422 (M+, 9), 217 (13), 206 (18), 205
(91), 173 (14), 146 (13), 145 (100), 131 (19), 130 (20), 116 (24),
103 (12). Anal. Calcd for C24H26N2O5: C, 68.23; H, 6.20; N, 6.63.
Found: C, 68.43; H, 5.99; N, 6.65.
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Data for 6c: yield, 82%; mp 83-85 °C; H NMR (400 MHz,
CDCl3) δ 3.65 (s, 3H, NCH3), 3.67 (s, 3H, COOCH3), 3.70 (s, 3H,
dCH-OCH3), 4.91 (s, 2H, Ph-OCH2), 5.15 (s, 2H, Het-OCH2),
6.13 (s, 1H, HetH), 6.99-7.04 (m, 2H, ArH), 7.18 (dd, J ) 4.0
Hz, J ) 5.2 Hz, 1H, ArH), 7.31-7.39 (m, 6H, ArH), 7.43-7.48
(m, 3H, ArH), 7.51 (s, 1H, dCH-OCH3), 7.94 (d, J ) 7.6 Hz,
1H, ArH); EI-MS m/z (relative intensity) 484 (M+, 9), 280 (13),
204 (100), 177 (21), 174 (14), 173 (23), 144 (98), 131 (32), 117
(16), 114 (54), 103 (37), 101 (35). Anal. Calcd for C29H28N2O5: C,
71.88; H, 5.82; N, 5.78. Found: C,71.72; H, 5.62; N, 5.62.
Data for 5b: yield, 84%; mp 143-145 °C; 1H NMR (400 MHz,
CDCl3) δ 2.29 (s, 3H, Ar-CH3), 3.68 (s, 3H, NCH3), 3.70 (s, 3H,
COOCH3), 3.84 (s, 3H, dCH-OCH3), 5.06 (s, 2H, CH2), 5.81 (s,
1H, HetH), 6.88 (d, J ) 8.0 Hz, 1H, ArH), 6.98 (d, J ) 8.4 Hz,
1H, ArH), 7.20-7.24 (m, 2H, ArH), 7.37-7.41 (m, 2H, ArH), 7.52
(dd, J ) 3.2 Hz, J ) 4.8 Hz, 1H, ArH), 7.62 (s, 1H, dCH-OCH3);
EI-MS m/z (relative intensity) 409 ([M + 1]+, 6), 408 (M+, 44),
205 (23), 204 (24), 175 (10), 146 (11), 145 (100), 131 (28), 103
(34). Anal. Calcd for C23H24N2O5: C, 67.63; H, 5.92; N, 6.86.
Found: C, 67.36; H, 5.83; N, 6.90.
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Data for 6d: yield, 81%; mp 76-77 °C; H NMR (400 MHz,
CDCl3) δ 3.61 (s, 3H, NCH3), 3.75 (s, 3H, COOCH3), 3.87 (s, 3H,
dCH-OCH3), 5.39 (s, 2H, CH2), 5.76 (s, 1H, HetH), 7.11-7.19
(m, 2H, ArH), 7.28-7.33 (m, 3H, ArH), 7.34-7.40 (m, 3H, ArH),
7.61 (d, J ) 7.2 Hz, 1H, ArH), 7.69 (s, 1H, dCH-OCH3), 7.87
(d, J ) 7.8 Hz, 1H, ArH), 7.99 (d, J ) 7.8 Hz, 1H, ArH), 8.23 (t,
J ) 7.2 Hz, 1H, ArH); EI-MS m/z (relative intensity) 517 ([M +
1]+, 6), 516 (M+, 8), 484 (20), 456 (24), 147 (14), 145 (43), 144
(23), 131 (17), 123 (100), 115 (22), 103 (23), 102 (14). Anal. Calcd
for C29H25FN2O6: C, 67.43; H, 4.88; N, 5.42. Found: C, 67.15; H,
4.69; N, 5.12.
Data for 5c: yield, 81%; mp 189-191 °C;1H NMR (600 MHz,
CDCl3) δ 3.68 (s, 3H, NCH3), 3.73 (s, 3H, COOCH3), 3.88 (s, 3H,
dCH-OCH3), 5.06 (s, 2H, CH2), 5.73 (s, 1H, HetH), 6.88 (d, J )
8.8 Hz, 1H, ArH), 7.08 (dd, J ) 1.4 Hz, J ) 9.0 Hz, 1H, ArH),
7.21 (dd, J ) 3.6 Hz,J ) 5.2 Hz, 1H, ArH), 7.33 (d, J ) 2.4 Hz,
1H, ArH), 7.37 (dd, J ) 3.2 Hz, J ) 5.6 Hz, 2H, ArH), 7.50 (dd,
J ) 3.6 Hz, J ) 5.2 Hz, 1H, ArH), 7.64 (s, 1H, dCH-OCH3);
EI-MS m/z(relative intensity) 428 (M+, 2), 204 (36), 189 (14), 160
(32), 144 (100), 128 (29), 115 (26), 101 (29), 91 (34). Anal. Calcd
for C22H21ClN2O5: C, 61.61; H, 4.94; N, 6.53. Found: C, 61.35; H,
5.15; N, 6.59.
X-ray Diffraction. A colorless block of 5b (0.20 mm × 0.20
mm × 0.10 mm) was mounted on a quartz fiber. Cell dimensions
and intensities were measured at 299 K on a Bruker SMART CCD
area detector diffractometer with graphite-monochromated Mo KR
radiation (λ ) 0.71073 Å); θmax ) 22.13; 6204 measured reflections;
3658 independent reflections (Rint ) 0.0229), of which 2701 had
|Fo| > 2|Fo|. Data were corrected for Lorentz and polarization effects
and for absorption (Tmin ) 0.9819; Tmax ) 0.9909). The structure
was solved by direct methods using SHELXS-97;37 all other
calculations were performed with Bruker SAINT System and Bruker
SMART programs.38 Full-matrix least-squares refinement based on
F2 using the weight of 1/[σ2(Fo 2) + (0.0934P)2 + 0.1767P] gave
final values of R ) 0.0570, ωR ) 0.1576, and GOF(F) ) 1.055
for 289 variables and 1634 contributing reflections. Maximum shift/
error ) 0.000(3), max/min residual electron density ) 0.209/-0.169
e Å-3. Hydrogen atoms were observed and refined with a fixed
value of their isotropic displacement parameter.
Data for 5d: yield, 67%; mp 150-152 °C;1H NMR (600 MHz,
CDCl3) δ 3.66 (s, 3H, NCH3), 3.86 (s, 3H, COOCH3), 4.08 (s, 3H,
dN-OCH3), 5.07 (s, 2H, CH2), 5.82 (s, 1H, HetH), 6.86 (t, J )
4.4 Hz, 1H, ArH), 6.98 (d,J ) 7.8 Hz, 1H, ArH), 7.15-7.25 (m,
2H, ArH), 7.40-7.54 (m, 4H, ArH); EI-MS m/z (relative intensity)
395 (M+, 42), 206 (10), 205 (20), 189 (28), 160 (41), 146
(31),131(96), 118 (37), 116 (100), 115 (19), 89 (27). Anal. Calcd
for C21H21N3O5: C, 63.79; H, 5.35; N, 10.63. Found: C, 63.52; H,
5.58; N, 10.44.
General Procedure for the Synthesis of Target Compounds
6. A reaction system with 1.0 mmol of compounds 5a,b and 0.16 g
(1.2 mmol) of anhydrous K2CO3 or 0.12 g (1.2 mmol) of
triethylamine in dry N,N-dimethylformamide (DMF) (6 mL) was
stirred at room temperature for 0.5 h, followed by the addition of
1.1 mmol of halogenated compounds or substituted benzoyl
chloride. The reaction was continued for 2-24 h. The resulting
mixture was poured into 20 mL of water, extracted with ethyl
acetate (10 mL × 3), washed with saturated sodium chloride (10
mL × 3), dried with anhydrous magnesium sulfate, and filtered
off with suction. The solvent was evaporated to give the crude,
which was purified by chromatography on silica using a mixture
of petroleum ether and acetone (20:1) as eluent to give the target
compounds 6a-d in yields of 46-82%.
3. Inhibitory Kinetics of Porcine bc1 Complex. The preparation
of succinate-cytochrome c reductase (SCR, mixture of respiratory
complex II and bc1 complex) from porcine heart was essentially as
reported.39 The activity of SCR in catalyzing the oxidation of
succinate by cytochrome c was determined in 1.8 mL of reaction
mixture containing 100 mM PBS (pH 7.4), 0.3 mM EDTA, 20 mM
succinate, 60 µM oxidized cytochrome c, and an appropriate amount
of SCR at 23 °C. The enzymatic assay for succinate-ubiquinone
reductase (complex II)-catalyzed oxidation of succinate by 2,6-
dichloroindophenol (DCIP) was carried out in essentially the same
reaction mixture, except for the replacement of the oxidized
cytochrome c with 53 µM DCIP. The reaction was initiated by the
addition of enzyme and monitored continuously by following the
absorbance change at certain wavelengths on a Perkin-Elmer
Lambda 45 spectrophotometer equipped with a magnetic stirrer.
Data for 6a: yield, 78%; mp 117-119 °C; 1H NMR (400 MHz,
CDCl3) δ 3.68 (s, 3H, NCH3), 3.70 (s, 3H, COOCH3), 3.73 (s, 3H,
dCH-OCH3), 3.86 (s, 3H, Ar-OCH3), 5.06 (s, 2H, CH2), 5.81
(s, 1H, HetH), 6.85 (t, J ) 8.0 Hz, 1H, ArH), 6.98 (d, J ) 7.2 Hz,
1H, ArH), 7.07 (dd, J ) 3.6 Hz, J ) 5.6 Hz, 1H, ArH), 7.16-7.24
(m, 2H, ArH), 7.38-7.42 (m, 2H, ArH), 7.54 (dd, J ) 3.6 Hz, J )
5.6 Hz, 1H, ArH), 7.64 (s, 1H, dCH-OCH3); EI-MS m/z (relative
intensity) 410 ([M + 1]+, 3), 408 (M+, 2), 394 (100), 378 (10),
376 (31), 375 (83), 361 (17), 318 (13), 161 (16), 145 (26), 118
(21), 115 (14), 102 (29). Anal. Calcd for C23H24N2O5: C, 67.63; H,
5.92; N, 6.86. Found: C, 67.83; H, 6.01; N, 6.76.
The extinction coefficients used were 18.5 mM-1 cm-1 for A
550
red-ox
600
for cytochrome c reduction and 21 mM-1 cm-1 for A
for DCIP
red-ox
reduction. For the inhibitory kinetic studies, the reaction was carried
out in the presence of varying concentrations of the inhibitor.
The ubiquinol-cytochrome c reductase (bc1 complex) activity
in catalyzing the oxidation of DBH2 by cytochrome c was assayed
in 100 mM PBS (pH 6.5), 2 mM EDTA, 750 µM lauryl maltoside
(n-dodecyl-ꢀ-D-maltoside), 20-115 µM DBH2, 100 µM oxidized
Data for 6b: yield, 46%; mp 152-153 °C; 1H NMR (400 MHz,
CDCl3) δ 2.32 (s, 3H, Ar-CH3), 3.70 (s, 3H, NCH3), 3.77 (s, 3H,
COOCH3), 3.84 (s, 3H, dCH-OCH3), 3.87 (s, 3H, Ar-OCH3),
5.06 (s, 2H, CH2), 6.08 (s, 1H, HetH), 6.85 (d, J ) 8.4 Hz, 1H,
ArH), 7.09 (t, J ) 7.2 Hz, 1H, ArH), 7.21 (d, J ) 7.8 Hz, 1H,
ArH), 7.36-7.40 (m, 2H, ArH), 7.53 (t, J ) 7.2 Hz, 1H, ArH),
(37) Sheldrick, G. M. SHELXTL, Version 5.0; University of Go¨ttingen:
Go¨ttingen, Germany, 2001.
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J. AM. CHEM. SOC. VOL. 132, NO. 1, 2010 189