Design, syntheses, and kinetic evaluation of 3-(phenylamino)oxazolidine-2, 4-diones as potent cytochrome bc1 complex inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.06.008

The cytochrome bc₁ complex (EC 1.10.2.2, bc₁) is one of the most promising targets for new drugs and agricultural fungicides. Among the existing bc₁ complex inhibitors specifically binding to the Q_o site, oxazolidinedione derivatives have attracted great attention. With the aim to understand the substituent effects of oxazolidinedione derivatives on the inhibition activity against the bc₁ complex, a series of new oxazolidinedione derivatives were designed, synthesized, and biologically evaluated. The further inhibitory kinetics studies against porcine succinate-cytochrome c reductase (SCR) revealed that the representative compound 8d and famoxadone are both non-competitive inhibitors with respect to the substrate cytochrome c, but competitive inhibitors with respect to substrate decylubiquinol (DBH₂). In addition, compound 8d and famoxadone showed, respectively, 35-fold and 15-fold greater inhibitory activity against the porcine SCR than the porcine bc₁ complex, indicating that these two inhibitors not only inhibited the activity of the bc₁ complex, but possibly affect the interaction between the complex II and the bc ₁ complex. To our knowledge, this is the first report that famoxadone and its analogs have effects on the interaction between the complex II and the bc₁ complex.

Full text of DOI:10.1016/j.bmc.2011.06.008

Bioorganic & Medicinal Chemistry 19 (2011) 4608–4615
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, syntheses, and kinetic evaluation of
3-(phenylamino)oxazolidine-2,4-diones as potent cytochrome bc1 complex
inhibitors
Fu Wang a,1, Hui Li a,1, Le Wang b, Wen-Chao Yang a, Jia-Wei Wu b, , Guang-Fu Yang a,
⇑
⇑
a Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, PR China
b MOE Key Laboratory of Bioinformatics, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The cytochrome bc1 complex (EC 1.10.2.2, bc1) is one of the most promising targets for new drugs and
agricultural fungicides. Among the existing bc1 complex inhibitors specifically binding to the Qo site, oxa-
zolidinedione derivatives have attracted great attention. With the aim to understand the substituent
effects of oxazolidinedione derivatives on the inhibition activity against the bc1 complex, a series of
new oxazolidinedione derivatives were designed, synthesized, and biologically evaluated. The further
inhibitory kinetics studies against porcine succinate–cytochrome c reductase (SCR) revealed that the rep-
resentative compound 8d and famoxadone are both non-competitive inhibitors with respect to the sub-
strate cytochrome c, but competitive inhibitors with respect to substrate decylubiquinol (DBH2). In
addition, compound 8d and famoxadone showed, respectively, 35-fold and 15-fold greater inhibitory
activity against the porcine SCR than the porcine bc1 complex, indicating that these two inhibitors not
only inhibited the activity of the bc1 complex, but possibly affect the interaction between the complex
II and the bc1 complex. To our knowledge, this is the first report that famoxadone and its analogs have
effects on the interaction between the complex II and the bc1 complex.
Received 4 May 2011
Revised 2 June 2011
Accepted 2 June 2011
Available online 12 June 2011
Keywords:
Oxazolidinedione
Cytochrome bc1 complex
Famoxadone
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
introduced into the agricultural fungicide market, including meth-
oxyacrylate, oxazolidinedione and imidazolinone derivatives.5
The cytochrome bc1 complex (EC 1.10.2.2, bc1) is an essential
component of the cellular respiratory chain and the photosynthetic
apparatus in photosynthetic bacteria. The function of the bc1 com-
plex is to catalyze the electron transfer from quinol to a soluble
cytochrome c (cyt c) and couple this electron transfer to the trans-
location of protons across the membrane.1–4 The bc1 complex has a
diheme cytochrome b, an iron–sulfur protein (ISP) with a Rieske-
type Fe2S2 cluster, and cytochrome c1 that undergo reduction and
oxidation during the turnover of the enzyme. Due to its crucial role
in the life cycle, inhibition of the bc1 complex has become an
important area for the discovery of fungicides useful in controlling
crop diseases. So far, two separate catalytic sites of the bc1 complex
have been identified and have been confirmed by X-ray crystallo-
graphic studies: the quinol oxidation site (Qo site) and the quinone
reduction site (Qi site). A number of inhibitors specifically binding
to the Qo site of the bc1 complex, termed QoI fungicides, have been
Based on specific binding interactions and conformational
changes observed in both cyt b and the ISP domain of the bc1 com-
plex, Qo inhibitors can be further divided into two subgroups.4 Sub-
group I includes azoxystrobin (AZ) and any methoxyacrylate-type
inhibitors, while subgroup II contains stigmatellin, famoxadone,
and UHDBT. The ISP domain is still mobile after the binding of sub-
group I inhibitors, but becomes fixed after the binding of subgroup
II inhibitors. Stigmatellin formed a hydrogen bond with ISP, so it is
very easy to understand why it holds the ISP in a fixed position.4
However, unlike stigmatellin, famoxadone do not make a hydrogen
bond (or any direct contact of any kind) with the ISP, and thus it is
somewhat of a mystery how famoxadone fix the ISP. Therefore, it
has been proposed that the mechanism by which inhibitors control
the position of the ISP is likely to be related to mechanism of
enforcement of the bifurcated reaction at Qo site.2,4,6
Famoxadone,
5-methyl-5-(4-phenoxyphenyl)-3-(phenylami-
no)-2,4-oxazolidinedione, is a new agricultural fungicide discov-
ered by the collaboration between DuPont and the Geffken
research group at University of Bonn, Germany.7 It is a member
of a new class of oxazolidinedione fungicides which belong to
the bc1 complex QoI family. However, site mutations at the bc1
complex have resulted in an explosive increase in resistance
⇑
Corresponding authors.
E-mail addresses: jiaweiwu@mail.tsinghua.edu.cn (J.-W. Wu), gfyang@mail.
ccnu.edu.cn (G.-F. Yang).
1
Co-first authors.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.008

F. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4608–4615
4609
associated with QoI fungicides.8–10 The resistance of the bc1 com-
plex to famoxadone developed much slower than resistance to
the methoxyacrylate(MOA)-type inhibitors like azoxystrobin and
kresoxim-methyl.10–12 Therefore, the design and syntheses of oxa-
zolidinedione derivatives have attracted much attention from the
synthetic chemists with the aim of discovering novel fungicides
with both high potency and low risk of resistance.13,14
and only one product was obtained. Then, the THF solution of
the Grignard reagent 2 reacted with a cooled (ꢀ78 °C) solution of
ethyl pyruvate to afford the key intermediate of ethyl 2-(6-bromo-
pyridin-3-yl)-2-hydroxyl-propanoate 3. Finally, compound 3 was
reacted with 1,10-carbonyldiimidazole (CDI) to form the intermedi-
ate acylimidazole. Without isolation, the obtained acylimidazole
was subjected to a one-pot reaction with various substituted phen-
ylhydrazines and acetic acid (HOAc) to give the target compounds
4 with isolated yields of 25–69%. The phenylhydrazine bearing an
electron-withdrawing group, for example, CF3, NO2, always re-
sulted in low yields of products, while those phenylhydrazines
bearing electron-donating groups, for example, 4-OCH3, 2,4-
(CH3)2, always resulted in relative high yields of products. In addi-
tion, acetic acid is critical to the one-pot reaction. If acetic acid was
omitted, no product was obtained. As shown in Scheme 2, com-
pounds 8a–k was prepared according to a similar procedure as
the preparation of compounds 4a–k in yields of 36–57%. The
Oxazolidinedione-type QoI fungicides have the common struc-
tural feature of a 3,5,5-trisubstituted oxazolidinedione (Fig. 1).
Studies of the structure–activity relationship indicated that the
R1 and R3 groups were sensitive to structural variation, whereas
compounds with a wide variety of R2 groups were fungicidally ac-
tive. The optimal group at the R2 position was the phenoxyphenyl
group. However, the earlier structure–activity relationships were
established according to results of in vivo fungicidal activity.7
Although the crystal structure of mitochondrial cytochrome bc1
in complex with famoxadone has been determined,15 the substitu-
ent effects of oxazolidinedione derivatives on the inhibitory activ-
ity against the bc1 complex remain unclear. Therefore, to
understand the detailed mechanism of the interaction between
the oxazolidinedione-type inhibitors and the bc1 complex, we de-
signed and synthesized a series of oxazolidinedione derivatives
4a–k (R1 = CH3; R2 = 6-bromopyridin-3-yl; R3 = various substituted
phenylamino) and 8a–k (R1 = CH3; R2 = phenoxyphenyl; R3 = vari-
ous substituted phenylamino). Herein, we present the results of
the inhibition activities against the porcine bc1 complex and dis-
cuss the structure-activity relationships of these newly synthe-
sized compounds. Moreover, the X-ray diffraction of bc1 complex
with bound famoxadone15 indicated that it belongs the family of
Qo site inhibitors, but famoxadone has been reported as a non-
competitive inhibitor of bc1 complex with respect to the substrate
QH2.16 This earlier kinetic result does not seem to be feasible be-
cause inhibitors binding to the same pocket as the substrate should
be competitive. We speculated that this earlier study was likely not
to take the non-enzymatic oxidation of QH2 into consideration, a
process that our previous work showed has significant influence
on the kinetic behavior.17 Therefore, we undertook a detailed
investigation of the inhibitory kinetics of famoxadone and the rep-
resentative compound (8d) against the porcine bc1 complex with
respect to the substrates of cytochrome c and DBH2. The results
showed clearly that famoxadone and compound 8d are both
non-competitive inhibitors with respect to substrate cytochrome
c, but are competitive inhibitors with respect to substrate DBH2.
difference is that 1-bromo-4-phenoxybenzene
smoothly with magnesium to afford Grignard reagent 6, whereas
2,5-dibromopyridine needs to undergo halogen-magnesium
5 can react
1
exchange. The structures of all intermediates and title compounds
were confirmed by 1H NMR and HRMS spectral data.
2.2. Inhibition activities of compounds 4a–k and 8a–k against
porcine succinate–cytochrome c reductase (SCR)
SCR is the mixture of respiratory complex II and the bc1 complex
(complex III), which was also deemed to form complex II–complex
III supercomplexes.18 Complex II (SQR) firstly passes electrons
from succinate to ubiquinone, and then the cytochrome bc1 com-
plex passes electrons from reduced ubiquinone to cytochrome c.
The activity of complex II in SCR was selectively determined using
succinate and dichlorophenolindophenol (DCIP) as substrates, and
the activity of only the cytochrome bc1 complex in SCR was deter-
mined using decylubiquinol (DBH2) and cytochrome c as sub-
strates, whereas the overall activity of SCR (both complex II and
bc1 complex) was determined using succinate and cytochrome c
as substrates.
The results indicated that all of the compounds exhibited no ef-
fect on the activity of complex II (data not shown), but markedly
inhibited the activities of the bc1 complex and SCR. The IC50 values
of compounds 4a–k and 8a–k against SCR from porcine heart mito-
chondria (in Table 1) show that although the potency of the newly
synthesized compounds was not superior to famoxadone, some
interesting structure–activity relationships are evident. Com-
pounds 4a–k always showed very low inhibition activities against
SCR, suggesting that the phenoxyphenyl group is critical to main-
tain high potency. The X-ray diffraction of bc1 complex with bound
famoxadone indicated that the van der Waals interactions between
the phenoxyphenyl group and hydrophobic residues M124, F128,
I146, P270, Y273, F274, Y278, and I298, made great contributions
to the binding of famoxadone.15 The 2-bromopyridyl group
(C log P = 1.59) is more hydrophilic than the phenoxyphenyl group
(C log P = 4.24), resulting in a notable reduction in the van der
Waals interactions between the 2-bromopyridyl group and the
above hydrophobic residues. The greater hydrophobic interactions
also account for the higher inhibitory activity of compounds 8a–k
as compared to compounds 4a–k. However, the substituents on
the phenylamino group have great effects on the inhibition activ-
ity. According to the results of X-ray diffraction analysis,15 the phe-
nylamino group of famoxadone was located deep inside the Qo
pocket and interacted with hydrophobic residues M138, G142,
V145, I146, I268, P270, and Y278, of which V145, I268, and Y278
are part of the iron–sulfur protein (ISP) docking crater. Therefore,
a hydrophobic group (4-Br, 4-Cl and 4-CH3) at this site is favorable,
whereas an electron-withdrawing group (4-COOH, 4-CN and
2. Results and discussion
2.1. Synthetic chemistry of the title compounds
The synthetic route for compound 4a–k is shown in Scheme 1.
Due to the strong electron-withdrawing ability of the nitrogen
atom, it is very difficult for 2,5-dibromopyridine 1 to react with
magnesium to afford a Grignard reagent. Therefore, 2,5-dibromo-
pyridine was firstly converted to the Grignard reagent of
(6-bromopyridin-3-yl)-magnesium chloride 2 via a procedure of
halogen-magnesium exchange. By controlling the conditions, the
reaction took place selectively at position 5 of the pyridine ring
O
R1
R2
O
O
O
N
N
N
H
R3
O
O
O
3,5,5-Trisubstitutedoxazolidinone
Famoxadone
Figure 1. Chemical structures of famoxadone and 3,5,5-trisubstituted
oxazolidinedione.

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F. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4608–4615
O
N
OH
R
O
Br
O
MgCl
NH
c, d, e
a
b
O
one-pot
Br
N
O
Br
N
Br
N
Br
N
3
2
1
4
Scheme 1. Synthesis of 5-(6-bromopyridin-3-yl)-5-methyl-3-(phenylamino)oxazolidine-2,4-diones 4a–k. Reagents and conditions: a, i-PrCl, Mg, I2, THF, reflux; b, ethyl
pyruvate, ꢀ78 °C, THF; c, CDI, 25 °C, CH2Cl2; d, H2O, HOAc, rt; e, substituted phenylhydrazine, CH2Cl2, rt.
O
O
OH
N
Br
NH
MgBr
O
a
b
c, d, e
O
O
O
one-pot
O
O
O
R
6
8
5
7
Scheme 2. Synthesis of 5-methyl-5-(4-phenoxyphenyl)-3-(phenylamino)oxazolidine-2,4-diones 8a–k. Reagents and conditions: a, Mg, I2, THF, reflux; b, ethyl pyruvate,
ꢀ78 °C, THF; c, CDI, 25 °C, CH2Cl2; d, H2O, HOAc, rt; e, substituted phenylhydrazine, CH2Cl2, rt.
Table 1
Furthermore, we examined the effect of 8d on the reactions of
the SCR pathway catalyzed by the bc1 complex. We determined
The inhibitory activities of compounds 4a–k and 8a–k against porcine SCR
the kinetics of the bc1 complex activity with respect to the sub-
strate ubiquinol by using the artificial electron donor decylubiqui-
nol (DBH2) and cytochrome c as substrates in the absence and
presence of 8d. As shown in Figure 2B, 8d competitively inhibited
that portion of SCR activity catalyzed by the bc1 complex in a com-
petitive manner with respect to the substrate DBH2. These results
indicate that 8d binds to the same hydrophobic binding pocket
a
a
as the ubiquinol-binding site. It is consistent with some X-ray
structures of bc1 complex in which this type of inhibitor binds to
the Qo site of the bc1 complex.4 A similar result was obtained for
the commercial fungicide famoxadone. As shown in Figure 2C
and D, famoxadone is a non-competitive inhibitor (Ki = 3.62 nM)
with respect to the substrate of cytochrome c, but is a competitive
inhibitor (Ki = 51.43 nM) with respect to the substrate of DBH2. It is
well known that most of the quinol analogs are readily autooxi-
dized, thus producing both superoxide and hydrogen peroxide
which can ultimately lead to reduction of cytochrome c, resulting
in an exchange of electrons indirectly with cytochrome c in solu-
tion.19 Therefore, it is very important to minimize the nonenzy-
matic oxidation of the quinol substrate when assaying the bc1
complex activity.19 In contrast to earlier studies,16 we added the
nonionic detergent lauryl maltoside to prevent the nonenzymatic
oxidation of DBH2. The nonenzymatic rate for cytochrome c reduc-
tion in our experiment was followed for at least 100 s before the
enzyme was added to initiate the reaction for each study. In our
data analysis, we subtracted the rate of the nonenzymatic activity
from the overall reaction rate. In earlier investigations performed
by other workers,15 reduced quinone was added into the enzyme
solution to start the reaction. As a result, the nonenzymatic and
enzymatic reactions could not be distinguished. Famoxadone can
only inhibit the enzyme catalyzed transfer of electrons to cyto-
chrome c and the contribution of the nonenzymatic reaction led
other investigators to conclude that famoxadone is a non-compet-
itive inhibitor of the bc1 complex with respect to the substrate QH2,
an analog of DBH2. In addition, Figure 2E and F clearly indicated
that azoxystrobin is a non-competitive inhibitor (Ki = 295.10 nM)
with respect to the substrate of cytochrome c, but is a competitive
inhibitor (Ki = 204.20 nM) with respect to the substrate of DBH2.
We compared the inhibitory activities of three inhibitors
against porcine SCR and the bc1 complex (Figure 2 and Table 2).
Compound 8d and famoxadone exhibited, respectively, 35-fold
No.
R
IC50 (nM)
No.
R
IC50 (nM)
4a
4b
4c
4d
4e
4f
4g
4h
4i
H
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
8a
8b
8c
8d
8e
8f
8g
8h
3-COOH
2-Br-4-F
2,6-(CH3)2
4-Br
4-CF3
4-CN
4-CH3
4-Cl
4-OCH3
2,4-(CH3)2
4-COOH
24.04
59.98
1516.92
9.44
63.26
275.17
23.76
18.79
57.92
15.93
5312.08
3.62
2-Br-4-F
2,6-(CH3)2
4-Br
4-CF3
4-CN
4-CH3
4-Cl
4-OCH3
2,4-(CH3)2
4-NO2
8i
8j
8k
4j
4k
Famoxadone
a
Determined with the substrate of cytochrome c.
4-CF3) would be unfavorable. For example, compound 8d (R = 4-
Br) exhibited relatively high inhibition, while compound 8k
(R = 4-COOH) showed 563-times lower activity than compound
8d (Table 1). In addition, the conformation of the phenylamino
group is very important for the binding. If the substituents affected
the conformation of the phenylamino group, the inhibitory activity
will greatly reduced. For example, compound 8c is 419-times less
potent than famoxadone due to its 2,6-dimethyl substitution
pattern.
2.3. Inhibitory kinetics of 8d and famoxadone
In order to understand the mechanism by which these com-
pounds inhibit this electron transport complex, we further studied
the inhibitory kinetics of 8d against SCR and also selectively exam-
ined the effects on only the bc1 complex. SCR catalyzed the overall
electron transfer from succinate to the water-soluble electron
acceptor cyt. c. As shown in Figure 2A, double-reciprocal plots
showed non-competitive inhibition of 8d with respect to cyt. c.

F. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4608–4615
4611
B
A
1200
2000
1600
1200
800
400
0
Km = 78.0287 7.8149 µM
Vmax = 0.0574 0.0058 µM /sec
Ki = 328.9037 31.0051 nM
Km = 4.4501 0.2467 µM
Vmax = 0.0081 0.0002 µM /sec
Ki = 9.4355 0.3473 nM
6
5
4
1000
800
600
400
200
0
5
4
3
2
1
v
3
2
1
02
0.00
.02
.04
.06
-.4 -.2 0.0 .2
.4
-1
.6
.8 1.0
-1
1/[DBH ] (µM
)
1/[cyt.c] (µM
)
2
C
D
1600
1400
1200
1000
800
600
400
200
0
700
600
500
400
300
200
100
0
Km = 4.4678 0.2172 µM
5
Km = 66.2108 5.8387 µM
Vmax = 0.0598 0.0021 µM/sec
Ki = 51.4339 3.3391 nM
Vmax = 0.0081 0.0002 µM /sec
Ki = 3.6195 0.1390 nM
6
4
5
4
3
2
1
v
3
2
1
.02
0.00
.02
.04
.06
-.4 -.2 0.0 .2
.4
-1
.6
.8 1.0
-1
1/[DBH ] (µM
)
2
1/[cyt.c] (µM
)
F
E
1800
1500
1200
900
600
300
0
Km = 4.3700 0.2401 µM
Vmax = 0.0099 0.0002 µM /sec
Ki = 295.0677 9.7350 nM
1800
1500
1200
900
600
300
0
Km = 72.1025 10.3968 µM
Vmax = 0.0528 0.0033 µM /sec
Ki = 204.1721 20.1691 nM
5
4
6
5
3
2
1
v
v
4
3
2
1
6
-.4 -.2 0.0 .2 .4 .6 .8 1.0
02
0.00
.02
.04
.06
-1
-1
1/[DBH ] (µM
)
1/[cyt.c] (µM
)
2
Figure 2. Kinetic analysis of inhibition by 8d (A and B), famoxadone (C and D) and azoxystrobin (E and F). The inhibition of porcine SCR by (A) 8d (1, 0 nM; 2, 1 nM; 3, 5 nM; 4,
10 nM and 5, 15 nM), (C) famoxadone (1, 0 nM; 2, 0.5 nM; 3, 1 nM; 4, 2.5 nM and 5, 5 nM) and (E) azoxystrobin (1, 0 nM; 2, 100 nM; 3, 200 nM; 4, 400 nM and 5, 600 nM).
Each reaction mixture contains 100 mM PBS (pH 7.4), 0.3 mM EDTA, 20 mM succinate, 0.1 nM enzyme, 1.16–16.02 lM cytochrome c and the indicated amount of 8d,
famoxadone or azoxystrobin. By assuming non-competitive inhibition, Km (cyt. c), Vmax, and Ki were estimated. The inhibition of porcine bc1 complex by (B) 8d (1, 0 nM; 2,
500 nM; 3, 1000 nM; 4, 2000 nM; 5, 3000 nM; and 6, 5000 nM), (D) famoxadone (1, 0 nM; 2, 50 nM; 3, 100 nM; 4, 200 nM; 5, 300 nM; and 6, 500 nM) and (F) azoxystrobin (1,
0 nM; 2, 100 nM; 3, 500 nM; 4, 1000 nM; 5, 3000 nM; and 6, 5000 nM). Each reaction mixture contains 100 mM PBS (pH 7.4), 0.3 mM EDTA, 750
lM lauryl maltoside, 100
lM
oxidized cytochrome c, 0.1 nM SCR, 20–200
Ki were also estimated.
lM DBH2 and the indicated amount of 8d, famoxadone or azoxystrobin. By assuming competitive inhibition, Km (DBH2), Vmax, and
Table 2
The inhibition effect of some inhibitors against porcine SCR and bc1 complex
Inhibitor
SCR (Succinate–cyt.c system 23 °C)
IC50 (nM) Inhibition type (with cyt.c) Kia (nM)
bc1 complex (DBH2–cyt.c system 23 °C)
IC50 without LMa (nM) IC50 with LMb (nM) Inhibition type (with DBH2) Kib (nM)
a
8d
9.03
4.78
Non-competitive
Non-competitive
Non-competitive
9.44 0.35 337.13
3.62 0.14 48.21
295.10 9.74 434.71
1049.32
142.02
527.12
Competitive
Competitive
Competitive
328.9 31.0
51.43 3.34
204.2 20.2
Famoxadone
Azoxystrobin 291.30
a
Reaction buffer: 100 mM PBS (pH 7.4), 0.3 mM EDTA.
Reaction buffer: 100 mM PBS (pH 7.4), 0.3 mM EDTA, 750
b
l
M lauryl maltoside (n-dodecyl-b-
D
-maltoside).

4612
F. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4608–4615
A120
B 120
C 120
100
80
60
40
20
0
100
80
60
40
20
100
80
60
40
20
0
0
-10
-9
-8
-7
-6
-5
-9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0
-10
-8
-6
-4
-2
Inhibitor Concentration (log Molarity)
Inhibitor Concentration (log Molarity)
Inhibitor Concentration (log Molarity)
Figure 3. The inhibition of SCR and QCR activities of porcine SCR by 8d (A), famoxadone (B) and azoxystrobin (C). Open circles (SCR activity): each reaction mixture contains
100 mM PBS (pH 7.4), 0.3 mM EDTA, 20 mM succinate, 0.1 nM enzyme, 60 M cytochrome c and the indicated amount of inhibitors. Solid circles (QCR activity): each reaction
mixture contains 100 mM PBS (pH 7.4), 0.3 mM EDTA, 100 M DBH2, 0.1 nM enzyme, 100 M cytochrome c and the indicated amount of inhibitors.
l
l
l
and 15-fold greater inhibition against the porcine SCR than against
the porcine bc1 complex, as indicated by the Ki values. We also
found that compound 8d and famoxadone did not show inhibitory
report showed no evidence for association of complex-II with
anything by blue native-polyacrylamide gel electrophoresis,23 but
a further analysis of the exothermic enthalpy change of thermo-
denaturation of a protein-phospholipid vesicle containing both
complex II and complex III also suggested the presence of a specific
interaction between complexes II and III.24 However, no reports
have indicated that a small molecule can interfere with the
interaction between complex II and III. Interestingly, in contrast
to compound 8d and famoxadone, azoxystrobin (AZ) inhibited
the SCR and the bc1 complex with similar Ki values (Figure 2E, 2F
and Table 2). The above results indicated that although all three
compounds belong to the family of Qo-specific inhibitors, 3-(phe-
nylamino)-oxazolidine-2,4-dione derivatives (such as famoxadone
and compound 8d) and methoxyacrylate-type derivatives (such as
AZ) should have different mechanisms of biological action. This
phenomenon might also account for why famoxadone showed
lower prevalence of resistance than methoxyacrylate-type
Qo-specific inhibitors.
It has been established that the ISP domain has a dynamic role
in the electron transfer between cytochrome b and c1 by function-
ing as a tethered, but flexible shuttle between the two redox cen-
ters. The ISP is held in a fixed position close to cytochrome b when
famoxadone is bound, whereas it becomes mobilized when AZ is
bound. The flexibility of the ISP domain is required for electron
flow in the high-potential chain. Although famoxadone and AZ oc-
cupy overlapping regions in the Qo pocket, they do exhibit different
characteristics according to their different binding modes with the
ISP domain, which was further confirmed by our experimental re-
sults. We therefore speculate that oxazolidinedione derivatives
such as famoxadone and our novel compound 8d possibly block
the interaction between complexes II and III by fixing the ISP
domain.
effects against complex II at concentrations as high as 20 lM (data
not shown). Furthermore, we also measured the IC50 values of the
three inhibitors with respect to the bc1 complex in the absence and
presence of the nonionic detergent lauryl maltoside. The differ-
ences in IC50 values were retained even when the same reaction
buffer was used both in the succinate–cyt. c system and DBH2–
cyt. c system. Figure 3 shows that the inhibition ability of com-
pound 8d and famoxadone decreased more than ten times while
that for azoxystrobin decreased only by half when measuring bc1
complex activity with DBH2 and cyt. c as substrates. The reason
for the inhibition decline may be attributed to the use of DBH2.
When DBH2 was used as substrate to measure the bc1 complex
activity, the Vmax of the bc1 complex was seven-fold higher than
that of SCR. Thus, we had to increase the concentration of inhibi-
tors to achieve the same degree of inhibition. For example, when
we determined the Ki values of famoxadone in these two systems,
we set the inhibitor concentrations of 0–5 nM for measuring SCR
activity but concentrations ranging from 0 to 500 nM were re-
quired for inhibiting the bc1 complex activity. Of course, both the
competitive effect of substrate in the high quinol concentration
and the effect of detergent will lead to high IC50 values, but difficul-
ties in doing kinetic experiments with water-insoluble substrates
and inhibitors, makes it difficult to prove. Furthermore, a recent re-
port20 has showed that decylubiquinone (DB) can increase the
activities of complex I/III and complex II/III, attenuate reductions
in oxygen consumption at high concentrations of the complex III
inhibitor, and induce increases in mitochondrial function in the
nerve terminal during complex I or III inhibition. Our experimental
results also indicated that DBH2 had some similar features as DB.
It is well known that the rate-limiting step will control the over-
all reaction process when considering the overall rate of an enzyme
sequence. The difference in rates between SCR and bc1 suggest that
the rate limiting step is before the bc1 complex. This rate-limiting
step may be most sensitive to these inhibitors, as indicated by the
lower Ki values when the SCR is measured. Therefore, we proposed
that these two inhibitors not only inhibited the activity of the bc1
complex, but also might affect the interaction between the com-
plex II and the bc1 complex. In fact, earlier biophysical investiga-
tions have suggested the existence of a complex II-complex III
pro-supercomplex, although to date this supercomplex has not
been isolated.21,22 When we prepared the protein from porcine
heart, we found it is easier for co-purification of complex II–
complex III (SCR) with excellent quality and activity. Another
3. Conclusion
In summary, a series of new oxazolidinedione derivatives
were designed and synthesized as potent inhibitors against the
cytochrome bc1 complex. Based on the results of the inhibition
activities against porcine bc1 complex, the structure–activity
relationships of these newly synthesized compounds were dis-
cussed. Detailed investigation of the effect of the inhibitors on
the steady state kinetics revealed that the representative com-
pound 8d and famoxadone are both non-competitive inhibitors
with respect to the substrate cytochrome c, but competitive
inhibitors with respect to the substrate DBH2. In addition, com-

F. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4608–4615
4613
pound 8d and famoxadone showed, respectively, 35-fold and 15-
fold greater inhibitory activity against the porcine SCR than
against the porcine bc1 complex, indicating that these two inhib-
itors not only inhibited the activity of the bc1 complex, but pos-
sibly interfered with the interaction between the complex II and
the bc1 complex. To our knowledge, this is the first report that
famoxadone and its analogs might affect the interaction between
the complex II and the bc1 complex.
Then, the organic layer was separated and the aqueous layer was
extracted twice with methylene chloride. The combined organic
phases were dried with MgSO4 and evaporated at reduced pressure
to obtain the crude product, which was then purified by flash col-
umn chromatography to give the pure products in yields of 25–
69%.
4.3.1. Data for 4a
White solid, yield 68%, mp 144–145 °C. 1H NMR(600 MHz,
CDCl3):
d 2.01(s, 3H, Het–CH3), 6.09 (s, 1H, NH), 6.74 (d,
4. Materials and methods
J = 7.8 Hz, 1H, ArH), 7.04 (t, J = 7.2 Hz, 1H, ArH), 7.26–7.29 (m,
2H, ArH), 7.59 (d, J = 8.4 Hz, 1H, Py-H), 7.81 (dd, J = 3.6 and
8.4 Hz, 1H, Py-H), 8.65 (d, J = 7.8 Hz, 1H, Py-H); HRMS(MALDI):
Calcd for C15H12BrN3O3 [M+H]+ 362.0153. Found: 362.0140.
4.1. Reagents and equipment
Unless otherwise noted, all chemical reagents were commer-
cially available and treated with standard methods before use. Sil-
ica gel column chromatography (CC): silica gel (200–300 mesh);
Qingdao Makall Group Co., Ltd; Qingdao; China). Solvents were
dried in a routine way and redistilled. 1H spectra were recorded
in CDCl3 or DMSO-d6 on a Varian Mercury 600 or 400 spectrometer
and resonances (d) are given in ppm relative to tetramethylsilane
(TMS). The following abbreviations were used to designate chemi-
cal shift mutiplicities: s = singlet, d = doublet, t = triplet, m = multi-
plet, br = broad. High resolution mass spectra (HRMS) were
acquired in positive mode on a WATERS MALDI SYNAPT G2
HDMS(MA, USA) or an Agilent 6520 Accurate-Mass Q-TOF liquid
chromatography/mass spectrometry (LC/MS)(USA). Melting points
were taken on a Buchi B-545 melting point apparatus and are
uncorrected.
4.3.2. Data for 4b
White solid, yield, 61%, mp 143–144 °C. 1H NMR(600 MHz,
CDCl3): d 2.01 (s, 3H, Het–CH3), 6.49 (dd, J = 4.8 and 9.6 Hz, 1H,
ArH), 6.54 (s, 1H, NH), 6.95 (s, 1H, ArH), 7.30 (dd, J = 2.4 and
7.8 Hz, 1H, ArH), 7.59 (d, J = 8.4 Hz, 1H, Py-H), 7.80 (dd, J = 2.4 Hz
and J = 8.4 Hz, 1H, Py-H), 8.64 (d, J = 2.4 Hz, 1H, Py-H); HRMS(MAL-
DI): Calcd for C15H10Br2FN3O3 [M+H]+ 457.9149. Found: 457.9151.
4.3.3. Data for 4c
White solid, yield, 53%, mp 134–135 °C. 1H NMR(600 MHz,
CDCl3): d 2.33 (s, 3H, Het–CH3), 2.42 (s, 6H, Ar-2ꢁCH3), 6.07 (s,
1H, NH), 6.77 (s, 1H, ArH), 6.92 (s, 1H, ArH), 7.00 (s, 1H, ArH),
7.03(s, 1H, Py-H), 7.20 (s, 1H, Py-H), 8.09(s, 1H, Py-H); HRMS(MAL-
DI): Calcd for C17H16BrN3O3 [M+Na]+ 412.0154. Found: 412.0273.
4.2. Synthesis of the intermediate 325–27
4.3.4. Data for 4d
White solid, yield, 25%, mp 177–178 °C. 1H NMR (600 MHz,
Under
a nitrogen atmosphere, i-propyl chloride (780 mg,
CDCl3):
d 2.00(s, 3H, Het–CH3), 6.08 (s, 1H, NH), 6.64 (t,
10 mmol) was added dropwise to a mixture of Mg (288 mg,
12 mmol) and a small amount of iodine in anhydrous THF
(10 mL). After refluxing for 2 h, 2,5-dibromopyridine (2.36 g,
10 mmol) was added slowly. Then, the resulting mixture was stir-
red for 2.5 h at 70 °C, and cooled to room temperature to afford the
intermediate of Grignard reagent 2, which was dissolved in THF
and added dropwise to a cooled (ꢀ78 °C) solution of ethyl pyruvate
(1.16 g, 10 mmol). After completion of the addition, the reaction
temperature was slowly allowed to warm to 20 °C and kept over-
night. The reaction mixture was poured into an ice solution of
HCl and then extracted with methylene chloride (3 ꢁ 40 mL). The
methylene chloride extract was washed with brine (30 mL) and
dried with MgSO4. Evaporation of methylene chloride at reduced
pressure afforded the crude product. After purification by flash col-
umn chromatography, the intermediate 3 (1.64 g) was obtained as
a pale yellow oil in a yield of 60%. 1H NMR (600 MHz, CDCl3): d 1.28
(t, J = 6.9 Hz, 1H, CH3), 1.78 (s, 3H, CH3), 3.99 (s, 1H, OH), 4.23–4.30
(m, 2H, CH2), 7.47 (d, J = 8.4 Hz, 1H, Py-H), 7.79 (d, J = 8.4 Hz, 1H,
Py-H), 8.59 (s, 1H, Py-H).
J = 8.2 Hz, 2H, ArH), 7.38 (d, J = 8.8 Hz, 2H, ArH), 7.59 (d,
J = 8.0 Hz, 1H, Py-H), 7.78 (dd, J = 2.4 and 8.0 Hz, 1H, Py-H), 8.64
(d, J = 2.4 Hz, 1H, Py-H); HRMS(MALDI): Calcd for C15H11Br2N3O3
[M+H]+ 439.9227. Found: 439.9245.
4.3.5. Data for 4e
White solid, yield, 58%, mp 151–152 °C. 1H NMR(600 MHz,
CDCl3): d 2.03 (s, 3H, Het–CH3), 6.34 (s, 1H, NH), 6.76 (d,
J = 9.0 Hz, 2H, ArH), 7.53 (d, J = 8.4 Hz, 2H, ArH), 7.60 (d,
J = 9.0 Hz, 1H, Py-H), 7.81 (dd, J = 2.4 and 8.4 Hz, 1H, Py-H), 8.65
(d, J = 2.4 Hz, 1H, Py-H); HRMS(MALDI): Calcd for C16H11BrF3N3O3
[M+H]+ 430.0008. Found: 430.0014.
4.3.6. Data for 4f
White solid, yield, 69%, mp 183–184 °C. 1H NMR(400 MHz,
CDCl3): d 2.04 (s, 3H, Het–CH3), 6.37 (s, 1H, NH), 6.73 (d,
J = 8.4 Hz, 2H, ArH), 7.57–7.62 (m, 3H, ArH and Py-H), 7.81 (dd,
J = 2.4 and 8.4 Hz, 1H, Py-H), 8.65 (d, J = 2.4 Hz, 1H, Py-H);
HRMS(MALDI): Calcd for C16H11BrN4O3 [M+H]+ 387.0101. Found:
387.0093.
4.3. General procedures for the preparation of target
compounds 4a–k7,28–30
4.3.7. Data for 4g
White solid, yield, 48%, mp 162–163 °C. 1H NMR(600 MHz,
CDCl3): d 1.99 (s, 3H, Het–CH3), 2.28 (s, 3H, Ar-CH3), 5.97 (s, 1H,
NH), 6.69 (d, J = 8.4 Hz, 2H, ArH), 7.07 (d, J = 8.4 Hz, 2H, ArH),
7.57 (d, J = 8.4 Hz, 1H, Py-H), 7.79 (dd, J = 2.4 and 9.0 Hz, 1H, Py-
H), 8.64 (d, J = 3.0 Hz, 1H, Py-H); HRMS(MALDI): Calcd for
A mixture of ethyl 2-(6-bromopyridin-3-yl)-2-hydroxypropan-
oate (3, 4 mmol), 1,10-carbonyldiimidazole (CDI) (5 mmol) and
dry dichloromethane (15 mL) was stirred at 25 °C for 24 h. When
the reaction was completed, as monitored by TLC detection, 8 mL
of water was then added and the mixture was stirred for a further
15 min. Then, acetic acid (3 mL) and the substituted phenylhydr-
azine (5.32 mmol) were then added, and the obtained mixture
was stirred at 25 °C for 26 h. After the reaction was completed,
as evidenced by TLC detection, 30 mL of water was added and
the pH value of the solution was adjusted to about 2 with HCl.
C
16H14BrN3O3 [M+H]+ 376.0272. Found: 376.0297.
4.3.8. Data for 4h
White solid, yield, 51%, mp 160–161 °C. 1H NMR(400 MHz,
CDCl3):
d 2.00 (s, 3H, Het–CH3), 6.10 (s, 1H, NH), 6.69 (t,
J = 4.2 Hz, 2H, ArH), 7.24 (t, J = 4.2 Hz, 2H, ArH), 7.59 (d,

4614
F. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4608–4615
J = 8.4 Hz, 1H, Py-H), 7.78 (t, J = 5.6 Hz, 1H, Py-H), 8.633 (d,
J = 2.4 Hz, 1H, Py-H); HRMS(MALDI): Calcd for C15H11BrClN3O3
[M+H]+ 395.9752. Found: 395.9751.
NH); HRMS(MALDI): Calcd for C23H18N2O6 [M+Na]+ 441.0118.
Found: 441.1063.
4.5.2. Data for 8b
4.3.9. Data for 4i
White solid, yield, 36%, mp 144–145 °C. 1H NMR(600 MHz,
CDCl3): d 2.00 (s, 3H, Het–CH3), 6.48 (s, 1H, NH), 6.50 (dd,
J = 5.4 Hz, J = 9.0 Hz, 1H, ArH), 6.93 (t, J = 8.4 Hz, 1H, ArH), 7.04 (t,
J = 6.6 Hz, 4H, ArH), 7.17 (t, J = 7.2 Hz, 1H, ArH), 7.29 (d, J = 7.8 Hz,
1H, ArH), 7.38 (t, J = 7.5 Hz, 2H, ArH), 7.53 (d, J = 8.4 Hz, 2H, ArH);
HRMS(ESI): Calcd for C22H16BrFN2O4 [M+K]+ 508.9964. Found:
508.9915.
White solid, yield, 49%, mp 129–130 °C. 1H NMR(400 MHz,
CDCl3): d 1.97 (s, 3H, Het–CH3), 3.76 (s, 3H, Ar–OCH3), 5.95 (s,
1H, NH), 6.80–6.86 (m, 4H, ArH), 7.57 (d, J = 8.8 Hz, 1H, Py-H),
7.79 (t, J = 3.0 Hz, 1H, Py-H), 8.62 (d, J = 2.4 Hz, 1H, Py-H);
HRMS(MALDI): Calcd for C16H14BrN3O4 [M+H]+ 392.0268. Found:
392.0246.
4.3.10. Data for 4j
4.5.3. Data for 8c
White solid, yield, 52%, mp 142–143 °C. 1H NMR(400 MHz,
CDCl3): d 2.00 (s, 3H, Het–CH3), 2.24 (s, 3H, Ar-CH3), 2.30 (s, 3H,
Ar-CH3), 5.92 (s, 1H, NH), 6.40 (d, J = 8.4 Hz, 1H, ArH), 6.89 (d,
J = 8.8 Hz, 1H, ArH), 6.96 (s, 1H, ArH), 7.57 (d, J = 8.4 Hz, 1H, Py-
H), 7.81 (t, J = 4.4 Hz, 1H, Py-H), 8.64 (d, J = 2.8 Hz, 1H, Py-H);
HRMS(MALDI): Calcd for C17H16BrN3O3 [M+H]+ 390.0444. Found:
390.0453.
White solid, yield, 44%, mp 100–101 °C. 1H NMR(600 MHz,
CDCl3):d 1.91 (s, 3H, Het–CH3), 2.26 (s, 6H, Ar-2ꢁCH3), 5.85 (s,
1H, NH), 6.94 (t, J = 7.5 Hz, 1H, ArH), 6.98–7.02 (m, 6H, ArH), 7.16
(t, J = 7.5 Hz, 1H, ArH), 7.37 (t, J = 8.1 Hz, 2H, ArH), 7.50 (d,
J = 9.0 Hz, 2H, ArH); HRMS(ESI): Calcd for C24H22N2O4 [M+K]+
441.1259. Found: 441.1217.
4.5.4. Data for 8d
4.3.11. Data for 4k
White solid, yield, 57%, mp 174–175 °C. 1H NMR(600 MHz,
CDCl3): d 1.99 (s, 3H, Het–CH3), 6.03 (s, 1H, NH), 6.64 (d,
J = 8.4 Hz, 2H, ArH), 7.04 (t, J = 4.2 Hz, 4H, ArH), 7.17 (s, 1H, ArH),
7.38 (dd, J = 8.4 and 15.0 Hz, 4H, ArH), 7.52 (d, J = 9.0 Hz, 2H,
ArH); HRMS(ESI): Calcd for
Found: 453.0450.
White solid, yield, 42%, mp 92–93 °C. 1H NMR(400 MHz, CDCl3):
d 2.05 (s, 3H, Het–CH3), 6.44 (s, 1H, NH), 6.74 (d, J = 8.4 Hz, 2H,
ArH), 7.62 (d, J = 8.8 Hz, 1H, Py-H), 7.80 (t, J = 5.6 Hz, 1H, Py-H),
8.19(d, J = 9.6 Hz, 2H, ArH), 8.66 (d, J = 2.4 Hz, 1H, Py-H);
HRMS(MALDI): Calcd for C15H11BrN4O5 [M+H]+ 406.9988. Found:
406.9991.
C
22H17BrN2O4 [M+H]+ 453.1701.
4.5.5. Data for 8e
4.4. Synthesis of intermediate 726,27
White solid, yield, 47%, mp 166–167 °C. 1H NMR(400 MHz,
CDCl3): d 2.01 (s, 3H, Het–CH3), 6.25 (s, 1H, NH), 6.76 (d,
J = 8.4 Hz, 2H, ArH), 7.04–7.07 (m, 4H, ArH), 7.18 (t, J = 7.2 Hz, 1H,
ArH), 7.39 (t, J = 8.0 Hz, 2H, ArH), 7.50–7.55 (m, 4H, ArH); HRMS(E-
SI): Calcd for C23H17F3N2O4 [M+Na]+ 465.1072. Found: 465.1038.
A
THF solution (15 mL) of 1-bromo-4-phenoxybenzene
(2.48 g, 10 mmol) was added dropwise to a mixture of Mg
(288 mg, 12 mmol) and a small amount of iodine in anhydrous
THF (10 mL). After refluxing for 1 h, the Grignard reagent of
(4-phenoxyphenyl)magnesium bromide was obtained, which
was dissolved in THF and added dropwise to a cooled (ꢀ78 °C)
solution of ethyl pyruvate (1.16 g, 10 mmol). After completion
of the addition, the reaction temperature was slowly allowed
to rise to 20 °C and kept overnight. The reaction mixture was
poured into an ice solution of HCl and then extracted with
methylene chloride (3 ꢁ 40 mL). The methylene chloride extract
was washed with brine (30 mL) and dried with MgSO4. Evapora-
tion of methylene chloride at reduced pressure afforded the
crude product. After purification by flash column chromatogra-
phy, the intermediate 7 (1.49 g) was obtained as a pale yellow
oil in a yield of 52%. 1H NMR (600 MHz, CDCl3): d 1.27 (t,
J = 7.2 Hz, 1H, CH3), 1.77 (s, 3H, CH3), 3.78 (s, 1H, OH), 4.22–
4.27 (m, 2H, CH2), 6.97 (d, J = 9.0 Hz, 2H, ArH), 7.01 (d,
J = 7.8 Hz, 2H, ArH), 7.11 (t, J = 7.5 Hz, 1H, ArH), 7.34 (t,
J = 7.8 Hz, 2H, ArH), 7.51 (d, J = 8.4 Hz, 2H, ArH).
4.5.6. Data for 8f
Yellow oil, yield, 53%. 1H NMR(600 MHz, CDCl3): d 2.05 (s, 3H,
Het–CH3), 6.34 (s, 1H, NH), 6.72 (d, J = 7.8 Hz, 2H, ArH), 7.05 (t,
J = 7.8 Hz, 4H, ArH), 7.19 (d, J = 7.8 Hz, 1H, ArH), 7.39 (t,
J = 8.1 Hz, 2H, ArH), 7.54 (t, J = 6.3 Hz, 4H, ArH); HRMS(MALDI):
Calcd for C23H17N3O4 [M+K]+ 438.0842. Found: 438.0856.
4.5.7. Data for 8g
White solid, yield, 41%, mp 158–159 °C. 1H NMR(600 MHz,
CDCl3): d 1.98 (s, 3H, Het–CH3), 2.27 (s, 3H, Ar-CH3), 5.97 (s, 1H,
NH), 6.69 (d, J = 8.4 Hz, 2H, ArH), 7.05 (dd, J = 8.4 and 12.6 Hz,
6H, ArH), 7.17 (d, J = 7.2 Hz, 1H, ArH), 7.37 (t, J = 7.8 Hz, 2H, ArH),
7.53 (d, J = 9.0 Hz, 2H, ArH); HRMS(MALDI): Calcd for C23H20N2O4
[M+Na]+ 411.1454. Found: 411.1321.
4.5.8. Data for 8h
White solid, yield, 43%, mp 149–150 °C. 1H NMR(600 MHz,
CDCl3): d 1.99 (s, 3H, Het–CH3), 6.03 (s, 1H, NH), 6.69 (d,
J = 8.4 Hz, 2H, ArH), 7.05 (t, J = 4.5 Hz, 4H, ArH), 7.17 (s, 1H, ArH),
7.22 (d, J = 8.4 Hz, 2H, ArH), 7.38 (t, J = 7.8 Hz, 2H, ArH), 7.52 (d,
J = 9.0 Hz, 2H, ArH); HRMS(ESI): Calcd for C22H17ClN2O4 [M+K]+
447.0519. Found: 447.0514.
4.5. General procedures for the preparation of target
compounds 8a–k
According to the similar procedure as for the preparation of
compounds 4a–k, compounds 8a–k was prepared in yields of
36–57%.
4.5.9. Data for 8i
4.5.1. Data for 8a
White solid, yield, 45%, mp 157–158 °C. 1H NMR(600 MHz,
CDCl3): d 1.96 (s, 3H, Het–CH3), 3.76 (s, 3H, Ar–OCH3), 5.90 (s,
1H, NH), 6.82 (dd, J = 9.0 and 20.4 Hz, 4H, ArH), 7.03 (d,
J = 8.4 Hz, 4H, ArH), 7.16 (t, J = 7.5 Hz, 1H, ArH), 7.37 (t, J = 8.1 Hz,
2H, ArH), 7.52(d, J = 8.4 Hz, 2H, ArH); HRMS(MALDI): Calcd for
White solid, yield, 49%, mp 148–149 °C. 1H NMR(400 MHz,
DMSO):
d 2.03 (s, 3H, Het–CH3), 6.75 (d, J = 7.8 Hz, 2H,
ArH),7.07 (d, J = 8.4 Hz, 2H, ArH), 7.11 (d, J = 8.4 Hz, 2H, ArH),
7.20 (s, 1H, ArH), 7.26 (d, J = 8.4 Hz, 2H, ArH), 7.43 (d,
J = 7.5 Hz, 2H, ArH), 7.55 (d, J = 7.8 Hz, 2H, ArH), 8.84 (s, 1H,
C
23H20N2O5 [M+Na]+ 427.1271. Found: 427.1270.

F. Wang et al. / Bioorg. Med. Chem. 19 (2011) 4608–4615
4615
Vmax½Sꢂ
4.5.10. Data for 8j
ꢀ
ꢀ
ꢁ
m
m
¼
¼
ð2Þ
White solid, yield, 37%, mp 174–175 °C. 1H NMR(600 MHz,
CDCl3): d 1.99 (s, 3H, Het–CH3), 2.24 (s, 3H, Ar-CH3), 2.30 (s, 3H,
Ar-CH3), 5.90 (s, 1H, NH), 6.43 (d, J = 8.4 Hz, 1H, ArH), 6.88 (s,
J = 7.8 Hz, 1H, ArH), 6.95(s, 1H, ArH), 7.04 (t, J = 9.0 Hz, 4H, ArH),
7.17 (d, J = 7.2 Hz, 1H, ArH), 7.37 (t, J = 7.8 Hz, 2H, ArH), 7.54 (d,
½Iꢂ
1 þ K Km þ ½Sꢂ
I
Vmax½Sꢂ
ꢁ
ð3Þ
½Iꢂ
1 þ K ðKm þ ½SꢂÞ
I
J = 8.4 Hz, 2H, ArH); HRMS(MALDI): Calcd for
C24H22N2O4
[M+Na]+ 425.1491. Found: 425.1477.
Acknowledgments
4.5.11. Data for 8k
White solid, yield, 35%, mp 220–221 °C. 1H NMR(400 MHz,
CDCl3): d 2.03(s, 3H, Het–CH3), 6.42 (s, 1H, NH), 6.68 (d, J = 8.4 Hz,
2H, ArH), 7.06 (dd, J = 3.6 and 8.4 Hz, 4H, ArH), 7.18 (s, 1H, ArH),
7.38 (d, J = 7.6 Hz, 2H, ArH), 7.96 (d, J = 8.4 Hz, 2H, ArH); HRMS(ESI):
Calcd for C23H18N2O6 [M+K]+ 457.0806. Found: 457.0802.
The research was supported in part by the National Basic
Research Program of China (No. 2010CB126103) and the NSFC
(Nos. 20925206, 20932005, and 20872045). The authors also
acknowledge Dr. Z. X. Wang for critical discussions and reading
of the manuscript.
References and notes
4.6. Enzyme assays
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3. Berry, E. A.; Huang, L. S. FEBS Lett. 2003, 555, 13.
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6. Berry, E. A.; Huang, L. S. Biochim. Biophys. Acta 2011. doi:10.1016/
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8. Meunier, B.; Fisher, N. FEMS 2008, 8, 183.
9. Wenz, T.; Covian, R.; Hellwig, P.; MacMillan, F.; Meunier, B.; Trumpower, B. L.;
Hunte, C. J. Biol. Chem. 2007, 282, 3977.
10. Jordan, D. B.; Livingston, R. S.; Bisaha, J. J.; Duncan, K. E.; Pember, S. O.;
Picollelli, M. A.; Schwartz, R. S.; Sternberg, J. A.; Tang, X. S. Pest. Sci. 1999, 55,
105.
11. Sierotzki, H.; Parisi, S.; Steinfeld, U.; Tenzer, I.; Poirey, S.; Gisi, U. Pest Manag.
Sci. 2000, 56, 833.
12. Pasche, J. S.; Piche, L. M.; Gudmestad, N. C. Plant Dis. 2005, 89,
269.
The preparation of succinate–cytochrome c reductase (SCR,
mixture of respiratory complex II and bc1 complex) from porcine
heart was essentially as reported.31 The activity of SCR was mea-
sured by monitoring the increase of cytochrome c at 550 nm, by
using the extinction coefficient of 18.5 mMꢀ1 cmꢀ1. The succi-
nate–ubiquinone reductase (complex II) activity was measured
by monitoring the decrease of 2,6-dichlorophenolindophenol
(DCIP) at 600 nm, by using the extinction coefficient of 21 mMꢀ1
cmꢀ1. The reaction mixture may be scaled down to 1.8 mL with fi-
nal concentrations of PBS (pH 7.4), 100 mM; EDTA, 0.3 mM; succi-
nate, 20 mM; oxidized cytochrome c, 60
lM (or DCIP, 53 lM); and
appropriate amounts of enzyme to start the reaction.32
The ubiquinol–cytochrome c reductase (bc1 complex) activity in
catalyzing the oxidation of DBH2 by cytochrome c was assayed in
100 mM PBS (pH 7.4), 0.3 mM EDTA, 750
lM lauryl maltoside
(n-dodecyl-b- -maltoside), 20–120 M DBH2, 100 l
D
l
M oxidized
cytochrome c, and an appropriate amount of SCR.17 The preparation
of DBH2 from DB was carried out according to the procedure
described in previous publications,33,34 and the concentration of
DBH2 was determined by measuring the absorbance difference be-
tween 288 and 320 nm using an extinction coefficient of
4.14 mMꢀ1 cmꢀ1 for the calculation.35,36 The nonionic detergent
lauryl maltoside was used to decrease the interfering nonenzymatic
oxidative activity,19,35–37 though it was expected to affect the Km va-
lue of the bc1 complex for DBH2.19 For each reaction, the nonenzy-
matic rate for cytochrome c reduction was followed for at least
100 s before enzyme was added to initiate the reaction.17
For the steady state studies, the reaction was carried out in the
absence or presence of various concentrations of the inhibitor. To
obtain the Ki values, all reactions were initiated by the addition
of enzyme and monitored continuously by following the absor-
bance change at certain wavelengths on a Perkin–Elmer Lambda
45 spectrophotometer equipped with a magnetic stirrer at 23 °C.
13. Jordan, D. B.; Livingstone, R. S.; Bisaha, J. J.; Duncan, K. E.; Pember, S. O.;
Picollelli, M. A.; Schwartz, R. S.; Sternberg, J. A.; Tang, X. S. Pest. Sci. 1999, 55,
197.
14. Huggett, M.J.; Whittingham, W.G.; Buchanan, K.I. PCT Int. Patent Appl. WO/
1997/038995, 1997.
15. Gao, X. G.; Wen, X. L.; Yu, C. A.; Esser, L.; Tsao, S.; Quinn, B.; Zhang, L.; Yu, L.;
Xia, D. Biochemistry 2002, 41, 11692.
16. Pember, S. O.; Fleck, L. C.; Moberg, W. K.; Walker, M. P. Arch. Biochem. Biophys.
2005, 435, 280.
17. Zhao, P. L.; Wang, L.; Zhu, X. L.; Huang, X. Q.; Zhan, C. G.; Wu, J. W.; Yang, G. F. J.
Am. Chem. Soc. 2010, 132, 185.
18. Chen, J. F.; Chen, C. L.; Alevriadou, B. R.; Zweier, J. L.; Chen, Y. R. Biochim.
Biophys. Acta 2011. doi:10.1016/j.bbabio.2011.03.001.
19. Chretien, D.; Slama, A.; Brière, J.; Munnich, A.; Rotig, A.; Rustin, P. Curr. Med.
Chem. 2004, 11, 233.
20. Telford, J. E.; Kilbride, S. M.; Davey, G. P. J. Biol. Chem. 2010, 285, 8639.
21. Yu, A.; Yu, L.; King, T. E. J. Biol. Chem. 1974, 249, 4905.
22. Yu, A.; Yu, L. Biochim. Biophys. Acta 1980, 591, 409.
23. Schagger, H.; Pfeiffer, K. J. Biol. Chem. 2001, 276, 37861.
24. Gwak, S. H.; Yu, L.; Yu, C. A. Biochemistry 1986, 25, 7675.
25. Trécourt, F.; Breton, G.; Bonnet, V.; Mongin, F.; Marsais, F.; Quéguiner, G.
Tetrahedron 2000, 56, 1349.
26. Johnnie, L.; Leazer, J.; Raymond, C. Org. Syn. 2005, 82, 115.
27. Ghosh, S.; Pardo, S. N.; Salomon, R. G. J. Org. Chem. 1982, 47, 4692.
28. Geffken, D.; Rayner, D. R. U.S. Patent 4957933, 1990.
29. Geffken, D.; Rayner, D. R. U.S. Patent 5223523, 1993.
30. Sternberg, J. A.; Sun, K. M.; Toji, M.; Witterholt, V. G. U.S. Patent 5552554,
1996.
31. Yu, L.; Yu, C. A. J. Biol. Chem. 1982, 257, 2016.
32. King, T. E. Methods Enzymol. 1967, 10, 216.
33. Rieske, J. S. Methods Enzymol. 1967, 10, 239.
4.7. Data analysis
The concentrations at 50% inhibition (absolute IC50 values) for
experiments with SCR were obtained from a nonlinear regression
of the activity data according to a four parameter logistic model.
The absolute IC50 was calculated according to Eq. 1.
34. Luo, C.; Long, J.; Liu, J. Clin. Chim. Acta 2008, 395, 38.
35. Fisher, N.; Bourges, I.; Hill, P.; Brasseur, G.; Meunier, B. Eur.J. Biochem. 2004,
271, 1292.
36. Fisher, N.; Brown, A. C.; Sexton, G.; Cook, A.; Windass, J.; Meunier, B. Eur. J.
Biochem. 2004, 271, 2264.
max ꢀ min
y ¼ min þ
ð1Þ
1 þ 10log IC
50ꢀx
The inhibition type was determined by Lineweaver–Burk plots,
and computer fitted to the appropriate equations like Eqs. 2 and 3.
Sigma Plot software 9.0 was used to determine all kinetic
constants.
37. Fisher, N.; Meunier, B. Pest Manag. Sci. 2005, 61, 973.