Design, synthesis, and bioevaluation of novel strobilurin derivatives

Article abstract of DOI:10.1002/cjoc.201200607

Strobilurins are one of the most important natural products with fungicidal activities and well known for their novel action mode, broad fungicidal spectrum, lower toxicity against mammalian cells, and environmentally benign characteristics. Design and syntheses of strobilurin analogues therefore have attracted great attention in the field of agrochemistry. Previously, we successfully developed a new molecular design method of pharmacophore-linked fragment virtual screening (PFVS) and discovered a lead compound (E)-methyl-2-(2-(((3-(imino-(phenyl)methyl)phenyl)thio)methyl)phenyl) -3-methoxyacrylate (1). To discover new strobilurin analogues with higher fungicidal activity, the structural modification of compound 1 was carried out guided by bioisosterism. A series of benzophenone derivatives 2a-2j were synthesized, among which compound 2j with a K_i value of 1.89 nmol/L was identified as the most promising inhibitor of porcine cytochrome bc ₁ complex, 157-fold improved binding affinity compared to the commercially available bc₁ inhibitor Azoxystrobin (AZ). In addition, most of the new compounds displayed excellent fungicidal activity against Sphaerotheca fuliginea at the concentration of 200 μmol/L. The present work indicates that strobilurin analogues containing benzophenone side chains may be the ideal leads for future fungicide discovery.

Full text of DOI:10.1002/cjoc.201200607

FULL PAPER
DOI: 10.1002/cjoc. 201200607
Design, Synthesis, and Bioevaluation of Novel Strobilurin
Derivatives†
Zhu, Xiaolei(朱晓磊)
Wang, Fu(王福)
Li, Hui(李慧)
Yang, Wenchao(杨文超)
Chen, Qiong(陈琼)
Yang, Guangfu*(杨光富)
Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China
Normal University, Wuhan, Hubei 430079, China
Strobilurins are one of the most important natural products with fungicidal activities and well known for their
novel action mode, broad fungicidal spectrum, lower toxicity against mammalian cells, and environmentally benign
characteristics. Design and syntheses of strobilurin analogues therefore have attracted great attention in the field of
agrochemistry. Previously, we successfully developed a new molecular design method of pharmacophore-linked
fragment virtual screening (PFVS) and discovered a lead compound (E)-methyl-2-(2-(((3-(imino-(phenyl)-
methyl)phenyl)thio)methyl)phenyl)-3-methoxyacrylate (1). To discover new strobilurin analogues with higher fun-
gicidal activity, the structural modification of compound 1 was carried out guided by bioisosterism. A series of
benzophenone derivatives 2a—2j were synthesized, among which compound 2j with a Ki value of 1.89 nmol/L was
identified as the most promising inhibitor of porcine cytochrome bc1 complex, 157-fold improved binding affinity
compared to the commercially available bc1 inhibitor Azoxystrobin (AZ). In addition, most of the new compounds
displayed excellent fungicidal activity against Sphaerotheca fuliginea at the concentration of 200 μmol/L. The pre-
sent work indicates that strobilurin analogues containing benzophenone side chains may be the ideal leads for future
fungicide discovery.
Keywords strobilurin, cytochrome bc1 complex, benzophenone, bioisosterism
Introduction
gicides has led to the rapid development of resistance in
a range of important plant pathogens.[8,9] Developing
novel strobilurin analogues may overcome this problem
and has attracted much attention from agricultural
chemists in recent years.
Cytochrome bc1 complex (EC 1.10.2.2, bc1), also
known as Complex III, is an essential component of the
cellular respiratory chain or the photosynthetic appara-
tus in photosynthetic bacteria. It catalyzes the electron
transfer from quinol to a soluble cytochrome c (cyt c)
and couples this electron transfer to the translocation of
protons across the membrane, generating the proton
gradient required for ATP biosynthesis.[1-4] Inhibition of
the function of bc1 complex would block the electron
transfer and cause serious problem even death to the
organism, which makes inhibitors targeting bc1 complex
become a significant area in fungicides discovery.
N
N
O
O
O
O
CN
O
O
O
N
O
O
Azoxystrobin
Kresoxim-methyl
Figure 1 Structures of commercialized products azoxystrobin
Among the existing bc1 complex inhibitors specifi-
cally binding to the Qo site, strobilurins derivatives have
attracted the greatest attention due to the broad spectrum
and efficient activity. Since the first commercialized
products azoxystrobin and kresoxim-methyl (Figure 1)
were launched in 1996, thousands of analogues with
diverse structure[5-7] have been synthesized and over 10
strobilurin fungicides are available for field application.
However, the widespread application of strobilurin fun-
and kresoxim-methyl.
Recently, we developed a new molecular design
method of pharmacophore-linked fragment virtual
screening (PFVS),[10] which produced a new lead com-
pound (E)-methyl-2-(2-(((3-(imino(phenyl)-methyl)-
phenyl)thio)methyl)phenyl)-3-methoxyacrylate (1) as
shown in Figure 2. Unfortunately, although compound 1
was successfully synthesized, it was found to be unstable
*
E-mail: gfyang@mail.ccnu.edu.cn
Received June 24, 2012; accepted August 6, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc.201200607 or from the author.
Dedicated to the 80th Anniversary of Chinese Chemical Society.
†
Chin. J. Chem. 2012, 30, 1999—2008
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1999

Zhu et al.
FULL PAPER
O
NH
S
S
bioisosterim substitution
O
OCH3
O
OCH3
OCH3
OCH3
1
2a
O
OCH3
O
cyclization
O
X
O
O
OCH3
R
OCH3
OCH3
2j
—
2b 2i
(X = O, S, NH, or NCH3)
Figure 2 Structural optimization of lead compound 1.
Scheme 1 Synthetic route for the newly designed compounds 2a—2j
O
XH
O
O
R
X
O
4
R
O
a
2a 2h X = S, O, NH
—
Br
O
+
O
O
O
OH
O
O
3
5
O
N
O
a
O
O
2j
O
O
H
N
b
O
O
O
O
O
2c
2i
Reagent and conditions: (a) K2CO3, acetone, reflux; (b) K2CO3, CH3I, acetone, reflux
due to the existence of the imino. We also found that its
N-methylated derivatives were unstable. Because oxy-
gen is the classical bioisosterim of NH, we therefore
designed compound 2a, whose Ki value against porcine
cytochrome bc1 complex was determined to be 13.95
nmol/L. This result prompted us to study the potential of
the benzophenone-containing strobilurin derivatives as
inhibitors of cytochrome bc1 complex. In fact, as a
critical intermediate in the biosynthetic pathway of flu-
triafol,[11] benzophenone has demonstrated a diverse
array of pharmacological activities, such as antifungal
activities. However, to the best of our knowledge, no
benzophenone-containing strobilurin analogues have
been reported so far. Herein, nine benzophenonecon-
taining strobilurin derivatives with different linking
modes between benzophenone and the pharmacophore
of β-methoxyacrylate were synthesized and assayed. Fi-
nally, a fluorenone-containing strobilurin derivative 2j
was designed and synthesized by the cyclization strat-
egy. Very interestingly, compound 2j with a Ki value of
1.89 nmol/L was identified as the most promising in-
hibitor of porcine cytochrome bc1 complex. Further in
vivo evaluation results revealed that compounds 2b, 2g
and 2j showed good fungicidal activity against
Sphaerotheca fuliginea at the concentration of 200
μmol/L.
Experimental
Unless otherwise noted, all chemical reagents were
commercially available and treated with standard
methods before use. Silica gel column chromatography
2000
www.cjc.wiley-vch.de
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 1999—2008

Design, Synthesis, and Bioevaluation of Novel Strobilurin Derivatives
(CC): silica gel (200—300 mesh; Qingdao Makall
Group Co., Ltd., Qingdao, China). Solvents were dried
in a routine way and redistilled. 1H and 13C spectra were
recorded in CDCl3 or d6-DMSO on a Varian Mercury
600 or 400 spectrometer and resonances (δ) are given
relative to tetramethylsilane (TMS). The following ab-
breviations were used to designate chemical shift muti-
plicities: s ＝ singlet, d ＝ doublet, t ＝ triplet, m ＝
multiplet, br＝broad. High Resolution Mass Spectra
(HRMS) were acquired in positive mode on a WATERS
MALDI SYNAPT G2 HDMS (MA, USA). Melting
points were taken on a Buchi B-545 melting point ap-
paratus and uncorrected. Intermediates 4 and 5 were
purchased from Sigma-Aldrich.
3-methoxyacrylate (2b) Yield 77%. White solid, m.p.
80—81 ℃. 1H NMR (600 MHz, CDCl3) δ: 3.67 (s, 3H,
COOCH3), 3.79 (s, 3H, ＝CH-OCH3), 5.02 (s, 2H,
CH2), 7.13 (d, J＝6.6 Hz, 1H, Ar-H), 7.18 (d, J＝7.2 Hz,
1H, Ar-H), 7.32—7.35 (m, 5H, Ar-H), 7.44—7.46 (m,
2H, Ar-H), 7.53 (d, J＝6.6 Hz, 1H, Ar-H), 7.56—7.57
(m, 2H, ＝CH-OCH3 and Ar-H), 7.77 (d, J＝7.8 Hz,
2H, Ar-H); 13C NMR (100 MHz, d6-DMSO) δ: 195.2,
166.8, 160.5, 158.1, 138.3, 136.8, 135.4, 132.5, 132.0,
131.1, 129.6, 129.4, 128.4, 127.4, 122.1, 119.1, 115.2,
108.7, 67.7, 61.7, 51.1; HRMS (MALDI) calcd for
C25H22O5 [M＋Na]＋ 425.1359, found 425.1340.
(E)-Methyl-2-(2-(((3-benzoylphenyl)amino)-
methyl)phenyl)-3-methoxyacrylate (2c) Yield 75%.
1
Yellow oil. H NMR (600 MHz, d6-DMSO) δ: 3.59 (s,
General procedure for the synthesis of target com-
pounds 2a—2h and 2j
3H, COOCH3), 3.79 (s, 3H, ＝CH-OCH3), 4.09 (d, J＝
5.4 Hz, 2H, CH2), 6.52—6.54 (m, 1H, Ar-H), 6.72 (d,
J＝7.8 Hz, 1H, Ar-H), 6.82 (d, J＝7.2 Hz, 1H, Ar-H),
6.88 (s, 1H, NH), 7.05 (d, J＝7.2 Hz, 1H, Ar-H),
7.17—7.22 (m, 2H, Ar-H), 7.23—7.26 (m, 1H, Ar-H),
7.33 (d, J＝7.8 Hz, 1H, Ar-H), 7.48—7.51 (m, 2H,
Ar-H), 7.61—7.64 (m, 1H, Ar-H), 7.66—7.67 (m,
3H, ＝CH-OCH3 and Ar-H); 13C NMR (100 MHz,
d6-DMSO) δ: 196.1, 167.0, 160.6, 148.6, 138.0, 137.6,
137.3, 132.3, 131.7, 130.9, 129.4, 128.9, 128.3, 127.4,
126.2, 126.1, 117.2, 115.9, 112.7, 108.9, 61.7, 51.2,
44.0; HRMS (MALDI) calcd for C25H23NO4 [M＋Na]＋
424.1519, found 424.1520.
To a mixture of anhydrous K2CO3 (0.82 g, 6.0 mmol)
in dry acetone (20 mL), 5.0 mmol of intermediate 4 or
2-hydroxy-9H-fluoren-9-one (5) was added, then the
resulted mixture was stirred and refluxed for 0.5 h. Then,
the intermediate of (E)-methyl-2-(2-(bromomethyl)-
phenyl)-3-methoxyacrylate 3 (1.42 g, 5.0 mmol) was
added into the solution, the reaction mixture was stirred
for a further 5—8 h under reflux. The resulting mixture
was cooled to room temperature and filtered off by suc-
tion, and the solvent was evaporated to give the crude
product, followed by chromatography purification on
silica using a mixture of petroleum ether and ethyl ace-
tate (10∶1) as eluent to give the target compounds
2a—2h and 2j in yields of 65%—81%.
(E)-Methyl-2-(2-((2-benzoylphenoxy)ethyl)phenyl)-
1
3-methoxyacrylate (2d) Yield 70%. Yellow oil. H
NMR (600 MHz, CDCl3) δ: 3.68 (s, 3H, COOCH3),
3.79 (s, 3H, ＝CH-OCH3), 4.89 (s, 2H, CH2), 6.89 (d,
J＝8.4 Hz, 2H, Ar-H), 7.02—7.05 (m, 1H, Ar-H),
7.07—7.11 (m, 2H, Ar-H), 7.19—7.21 (m, 1H, Ar-H),
7.37—7.40 (m, 1H, Ar-H), 7.41—7.46 (m, 3H, Ar-H),
7.55—7.57 (m, 2H, Ar-H and ＝CH-OCH3), 7.84 (d,
J＝7.8 Hz, 2H, Ar-H); 13C NMR (100 MHz, d6-DMSO)
δ: 195.8, 166.7, 161.0, 155.6, 137.5, 135.3, 133.1, 132.0,
130.8, 130.7, 129.1, 128.9, 128.7, 128.6, 127.1, 126.8,
125.8, 120.7, 112.9, 108.1, 67.2, 61.8, 51.2; HRMS
(MALDI) calcd for C25H22O5 [M＋Na]＋ 425.1359,
found 425.1733.
General procedure for the synthesis of target com-
pounds (2i)
To a mixture of 5.0 mmol compound 2c in dry ace-
tone (20 mL), anhydrous K2CO3 (1.38 g, 10 mmol) and
CH3I (1.42 g, 10 mmol) were added respectively, stirred
and refluxed for 2 h. The resulting mixture was cooled
to room temperature and filtered off by suction, and the
solvent was evaporated to give the crude product, fol-
lowed by chromatography purification on silica using a
mixture of petroleum ether and ethyl acetate (6∶1) as
eluent to give the target compounds 2i.
(E)-Methyl-2-(2-((3-benzoylphenoxy)methyl)-
phenyl)-3-methoxyacrylate (2e) Yield 77%. Yellow
(E)-Methyl-2-(2-(((3-benzoylphenyl)thio)methyl)-
phenyl)-3-methoxyacrylate (2a) Yield 81%. Yellow
1
oil. H NMR (600 MHz, d6-DMSO) δ: 3.62 (s, 3H,
1
oil. H NMR (600 MHz, d6-DMSO) δ: 3.65 (s, 3H,
COOCH3), 3.82 (s, 3H, ＝CH-OCH3), 5.03 (s, 2H,
CH2), 7.06 (d, J＝8.4 Hz, 2H, Ar-H), 7.15 (d, J＝5.4 Hz,
1H, Ar-H), 7.30—7.33 (m, 2H, Ar-H), 7.49—7.50 (m,
1H, Ar-H), 7.52—7.54 (m, 2H, Ar-H), 7.62—7.64 (m,
1H, Ar-H), 7.67—7.69 (m, 3H, Ar-H and ＝CH-OCH3),
7.73 (d, J＝8.4 Hz, 2H, Ar-H); 13C NMR (100 MHz,
d6-DMSO) δ: 194.4, 167.0, 162.0, 160.8, 137.7, 135.2,
132.2, 132.1, 131.2, 129.5, 129.3, 128.4, 127.7, 127.67,
COOCH3), 3.78 (s, 3H, ＝CH-OCH3), 4.04 (s, 2H,
CH2), 7.13 (d, J＝7.2 Hz, 1H, Ar-H), 7.21—7.23 (m,
1H, Ar-H), 7.25—7.27 (m, 1H, Ar-H), 7.31—7.35 (m,
2H, Ar-H), 7.45—7.47 (m, 3H, Ar-H), 7.57—7.59 (m,
3H, Ar-H), 7.66 (s, 1H, ＝CH-OCH3), 7.71—7.72 (m,
2H, Ar-H); 13C NMR (100 MHz, d6-DMSO) δ: 195.1,
167.0, 160.6, 137.6, 137.2, 136.7, 135.8, 133.0, 132.6,
132.4, 131.4, 129.6, 129.3, 129.0, 128.9, 128.4, 127.4,
127.1, 126.9, 109.1, 61.6, 51.1, 36.5; HRMS (MALDI)
calcd for C25H22O4S [M ＋ Na] ＋ 441.1131, found
441.1107.
127.62, 114.5, 108.7, 67.9, 61.9, 51.3; HRMS (MALDI)
＋
calcd for C25H22O5 [M ＋ Na]
425.1359, found
425.1373.
(E)-Methyl-2-(2-((3-(2-fluorobenzoyl)phenoxy)-
methyl)phenyl)-3-methoxyacrylate (2f) Yield 69%.
(E)-Methyl-2-(2-((3-benzoylphenoxy)methyl)phenyl)-
Chin. J. Chem. 2012, 30, 1999—2008
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2001

Zhu et al.
FULL PAPER
Yellow oil. H NMR (600 MHz, CDCl3) δ: 3.70 (s, 3H,
1
7.46—7.47 (m, 1H, Ar-H), 7.53—7.56 (m, 2H, Ar-H),
7.66—7.68 (m, 3H, Ar-H and ＝CH-OCH3); 13C NMR
(100 MHz, d6-DMSO) δ: 192.7, 166.8, 160.7, 159.5,
144.2, 136.4, 135.4, 134.9, 133.3, 131.9, 131.1, 128.1,
127.5, 127.4, 127.2, 123.8, 122.2, 121.0, 120.2, 110.0,
108.6, 67.9, 61.8, 51.2; HRMS (MALDI) calcd for
C25H20O5 [M＋Na]＋ 423.1203, found 423.1321.
COOCH3), 3.82 (s, 3H, ＝CH-OCH3), 5.05 (s, 2H,
CH2), 6.93—6.95 (m, 2H, Ar-H), 7.13—7.16 (m, 1H,
Ar-H), 7.18—7.19 (m, 1H, Ar-H), 7.23—7.24 (m, 1H,
Ar-H), 7.33—7.35 (m, 2H, Ar-H), 7.48—7.51 (m, 3H,
Ar-H), 7.59 (s, 1H, ＝CH-OCH3), 7.78—7.80 (m, 2H,
Ar-H); 13C NMR (100 MHz, d6-DMSO) δ: 191.0, 166.9,
162.8, 160.8, 160.1, 157.6, 135.1, 132.3, 131.9, 131.2,
130.1, 130.0, 129.5, 127.7, 127.6, 124.7, 124.6, 116.2,
116.0, 114.8, 108.6, 67.9, 61.8, 51.3; HRMS (MALDI)
calcd for C25H21FO5 [M ＋ Na] ＋ 443.1265, found
443.1278.
Enzyme inhibition kinetic activity
The preparation of succinate-cytochrome c reductase
(SCR, mixture of respiratory complex II and bc1 com-
plex) from porcine heart was essentially same as re-
ported.[12] The activity of SCR was measured by moni-
toring the increase of cytochrome c at 550 nm, by using
(E)-Methyl-2-(2-((3-(3-chlorobenzoyl)phenoxy)-
methyl)phenyl)-3-methoxyacrylate (2g) Yield 65%.
－
－
1
1
1
the extinction coefficient of 18.5 mmol•L •cm . The
succinate-ubiquinone reductase (complex II) activity
was measured by monitoring the decrease of 2,6-di-
chlorophenolindophenol (DCIP) at 600 nm, by using the
Yellow oil. H NMR (600 MHz, CDCl3) δ: 3.71 (s, 3H,
COOCH3), 3.83 (s, 3H, ＝CH-OCH3), 5.06 (s, 2H,
CH2), 6.96 (d, J＝8.8 Hz, 2H, Ar-H), 7.19—7.20 (m,
1H, Ar-H), 7.33—7.35 (m, 2H, Ar-H), 7.44 (d, J＝8.4
Hz, 2H, Ar-H), 7.51—7.53 (m, 1H, Ar-H), 7.60 (s, 1H,
＝CH-OCH3), 7.70 (d, J＝8.4 Hz, 2H, Ar-H), 7.75 (d,
J＝8.4 Hz, 2H, Ar-H); 13C NMR (100 MHz, d6-DMSO)
δ: 193.1, 166.8, 162.1, 160.7, 136.9, 136.3, 135.1, 132.1,
132.0, 131.1, 130.0, 129.1, 128.5, 127.6, 127.5, 114.5,
108.6, 67.8, 61.8, 51.2; HRMS (MALDI) calcd for
C25H21ClO5 [M＋Na]＋ 459.0970, found 459.0971.
(E)-Methyl-2-(2-(((3-benzoylphenyl)amino)methyl)-
phenyl)-3-methoxyacrylate (2h) Yield 67%. Yellow
－
1
－
1
extinction coefficient of 21 mmol•L •cm . The reac-
tion mixture may be scaled down to 1.8 mL with final
concentrations of PBS (pH 7.4), 100 mmol/L; EDTA,
0.3 mmol/L; succinate, 20 mmol/L; oxidized cyto-
chrome c, 60 μmol/L (or DCIP, 53 μmol•L); and an ap-
propriate amounts of enzyme.[13]
The ubiquinol-cytochrome c reductase (bc1 complex)
activity in catalyzing the oxidation of DBH2 by cyto-
chrome c was assayed in 100 mmol/L PBS (pH 6.5), 2
mmol/L EDTA, 750 μmol/L lauryl maltoside (n-dodecyl-
β-D-maltoside), 20—120 μmol/L DBH2, 100 μmol/L
oxidized cytochrome c, and an appropriate amount of
SCR.[14] The preparation of DBH2 from DB was carried
out according to the procedure described in previous
publications,[15,16] and the concentration of DBH2 was
determined by measuring the absorbance difference
between 288 and 320 nm using an extinction coefficient
1
oil. H NMR (600 MHz, d6-DMSO) δ: 3.70 (s, 3H,
COOCH3), 3.83 (s, 3H, ＝CH-OCH3), 4.24 (s, 2H,
CH2), 6.56 (d, J＝8.4 Hz, 2H, Ar-H), 7.16—7.17 (m,
1H, Ar-H), 7.32—7.33 (m, 2H, Ar-H), 7.41—7.45 (m,
3H, Ar-H), 7.50—7.53 (m, 1H, Ar-H), 7.57 (s, 1H,
＝CH-OCH3), 7.70—7.73 (m, 4H, Ar-H); 13C NMR
(100 MHz, d6-DMSO) δ: 193.4, 167.0, 160.8, 152.8,
139.0, 137.7, 132.4, 131.9, 131.1, 128.8, 128.2, 127.6,
126.5, 126.2, 124.0, 111.1, 108.9, 61.8, 51.3, 43.7;
HRMS (MALDI) calcd for C25H23NO4 [M ＋ Na] ＋
423.1519, found 423.1895.
－
－
1
of 4.14 mmol•L •cm for the calculation.[17,18] The
nonionic detergent lauryl maltoside was used to de-
crease the interfering nonenzymatic activity,[8,17,18]
though it was expected to affect the Km value of DBH2
to bc1 complex. For each reaction, the nonenzymatic
rate for cytochrome c reduction was followed for at
least 100 s before enzyme was added to initiate the re-
action.
For the inhibitory kinetic studies, the reaction was
carried out in the presence of varying concentrations of
the inhibitor. All of the reactions were initiated by the
addition of enzyme and monitored continuously by fol-
lowing the absorbance change at certain wavelengths on
a Perkin-Elmer Lambda 45 spectrophotometer equipped
with a magnetic stirrer at 23 ℃.
Determination of Michaelis-Menten parameters Km
and Vmax was performed by nonlinear regression. The
inhibition type was assayed by the Lineweaver-Burk
plot, and the kinetic parameters were evaluated by
Sigma Plot software 9.0 for the classical inhibitors.
For slow, tight-binding inhibitors, the reaction
mechanism could be considered as shown in Scheme 2,
where E, S, and I represent enzyme, substrate, and in-
1
(E)-Methyl-2-(2-(((3-benzoylphenyl)(methyl)-
amino)methyl)phenyl)-3-methoxyacrylate
(2i)
Yield 68%. Yellow oil. 1H NMR (600 MHz, d6-DMSO)
δ: 2.93 (s, 3H, N-CH3), 3.49 (s, 3H, COOCH3), 3.70 (s,
3H, ＝CH-OCH3), 4.28 (s, 2H, CH2), 6.78—6.81 (m,
2H, Ar-H), 6.88 (s, 1H, Ar-H), 6.96—6.99 (m, 2H,
Ar-H), 7.12—7.20 (m, 3H, Ar-H), 7.37—7.39 (m, 2H,
Ar-H), 7.51—7.54 (m, 2H, Ar-H and ＝CH-OCH3),
7.59 — 7.60 (m, 2H, Ar-H); 13C NMR (100 MHz,
d6-DMSO) δ: 196.2, 166.9, 160.5, 148.9, 137.7, 137.2,
136.9, 132.4, 131.8, 131.3, 129.5, 129.0, 128.4, 127.5,
126.4, 125.6, 117.4, 115.7, 111.9, 108.8, 61.8, 53.6, 51.2,
38.7; HRMS (MALDI) calcd for C26H25NO4 [M＋Na]＋
438.1676, found 438.1680.
(E)-Methyl-3-methoxy-2-(2-(((9-oxo-9H-fluoren-
2-yl)oxy)methyl)phenyl)acrylate (2j)
Red solid, m.p. 132—133 ℃. H NMR (600 MHz,
d6-DMSO) δ: 3.63 (s, 3H, COOCH3), 3.85 (s, 3H,
＝CH-OCH3), 5.01 (s, 2H, CH2), 7.01 (s, 1H, Ar-H),
7.10—7.13 (m, 2H, Ar-H), 7.26—7.33 (m, 3H, Ar-H),
Yield 80%.
1
2002
www.cjc.wiley-vch.de
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 1999—2008

Design, Synthesis, and Bioevaluation of Novel Strobilurin Derivatives
hibitor, respectively. The progress curves at different
inhibitor concentrations can be described by Eq. 1.[14]
Uncompetitive:
k＋' 0[S]
A＝
k－' 0[S]
Km∗＋[S]
Scheme 2 Kinetic model used for the present study
B＝
(4)
Km＋[S]
I
+
I
+
k1
k2
ES
E + P
S + E
k-1
Computational methods
'
k+0
k-1
k+0
'
k-1
The three dimensional (3D) structure of the porcine
bc1 was obtained from our previous work.[14] The
AutoDock 4.0 program was applied to dock these ben-
zophenone-based strobilurins into the Qo site of bc1
complex. The parameters used in molecular docking
were the same as our previous work used.[14] All the
complex structures derived from molecular docking
were used as starting structures for further energy
minimizations using the Sander module of the Amber8
program before the final binding structures were ob-
tained. The atomic charges used for these inhibitors
were the restrained electrostatic potential (RESP)
charges, determined by using the standard RESP pro-
cedure implemented in the Antechamber module of the
Amber8 program following the electronic structure and
electrostatic potential calculations at the HF/6-31G*
level. First, the ligand was minimized with the protein
fixed. Then, the backbone atoms of the protein were
fixed and the other atoms were relaxed to be minimized.
The final minimization was performed with both the
ligand and protein relaxed. In each step, the energy
minimization was executed by using the steepest de-
scent method for the first 2000 cycles and the conju-
gated gradient method for the subsequent 3000 cycles
*
k1
S + EI
EIS
*
k-1
v0－vs
obst
[P]＝vst＋
(1－e－k
)
(1)
kobs
The data were analyzed using the nonlinear regres-
sion program of Sigma Plot 9.0 to give the individual
parameters for each progress curve; v0 (initial velocity),
vs (steady-state velocity), kobs (apparent first-order rate
constant for the transition from v0 to vs) according to Eq.
1. Usually, kobs constant:
kobs＝A[I]0＋B
k＋0 Km＋k＋' 0[S]
A＝
Km＋[S]
k－0 Km∗＋k－' 0[S]
B＝
Km∗＋[S]
－
－
1
1
with a convergence criterion of 0.1 kcal•mol •Å .
Finally, the 20 ps MD simulation was carried out for
each complex structure. For temperature regulation, the
Langevin thermostat was used to maintain at 300 K. The
atomic coordinates were saved per ps. The last snapshot
of the MD simulation was minimized to a convergence
We have Km and K∗ as the Michaelis-Menten con-
m
stants:
k－1＋k2
Km＝
－
－
1
1
k1
criterion of 0.1 kcal•mol •Å . The enthalpy was cal-
culated using MM/PBSA method and entropy was cal-
culated as our recent publications described.[19,20] The re-
sult was shown in Table 1.
k－∗ 1
Km∗＝
k1∗
Evaluation of fungicidal activity
Like the classical inhibitors, the slow, tight-binding
inhibitors can also be classified as competitive, non-
competitive, and uncompetitive on the basis of similar
considerations. Experimentally, the type of inhibition
can be ascertained by studying the effect of [S] on the
apparent rate constants A and B:
The in vivo fungicidal activities of compounds 2a—
2j against cucumber Sphaerotheca fuliginea were tested
according to the procedure described previously.[20] The
results are listed in Table 2, in which the inhibition per-
centage was calculated by averaging the values obtained
in three independent experiments. Azoxystrobin, a
commercial fungicide, was used as control.
Competitive:
k＋0 Km
A＝
B＝k－0
Results and Discussion
(2)
(3)
Km＋[S]
Noncompetitive:
k ,
Synthetic chemistry
The synthetic route for the target compounds is out-
lined in Scheme 1. The key intermediate (E)-methyl-2-
A
＝
B k
＝
0
＋
－
0
Chin. J. Chem. 2012, 30, 1999—2008
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2003

Zhu et al.
FULL PAPER
Table 1 Inhibitory activities of compounds 2a—2j against porcine SCR with cytochrome c as substrate
Redisue interaction energy/
－
∆G/(kcal•mol )
1
∆H/
－T∆S/
No.
Structure
Ki/(nmol•L－
)
－
(kcal•mol )
1
1
－
(kcal•mol ) (kcal•mol )
－
1
1
cal.
exp.
F274
F128
O
O
S
13.95
－50.00
－51.63
—
19.94
19.14
—
－30.06 －10.74
－32.49 －11.60
－7.130
－7.345
2a
O
O
O
O
O
O
O
3.28
－10.472
－6.872
2b
2c
O
NH
＞1000
—
—
—
—
O
O
O
O
O
O
2d
＞1000
—
—
—
—
—
—
O
O
O
O
12.49
16.87
－49.93
－46.70
－49.21
—
19.40
17.59
17.70
—
－30.53 －10.82
－29.11 －10.63
－31.51 －10.92
－7.629
－6.042
－8.377
—
－5.258
－8.175
－5.555
—
2e
2f
O
O
O
O
O
O
O
O
F
Cl
O
10.35
2g
2h
O
O
O
O
NH
＞1000
—
—
O
O
O
O
2004
www.cjc.wiley-vch.de
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 1999—2008

Design, Synthesis, and Bioevaluation of Novel Strobilurin Derivatives
Continued
Redisue interaction energy/
－
∆G/(kcal•mol )
1
∆H/
－T∆S/
No.
Structure
Ki/(nmol•L－
)
－
(kcal•mol )
1
1
－
(kcal•mol ) (kcal•mol )
－
1
1
cal.
exp.
F274
F128
O
N
2i
＞1000
—
—
—
—
—
—
O
O
O
O
O
1.89
－50.90
－43.42
18.25
19.42
－32.65 －11.93
－23.00 －8.92
－9.14
－8.96
－6.44
－4.29
2j
O
O
O
AZ
297.60
—
Table 2 In vivo fungicidal activities of compounds 2a—2j and
effect on the activity.
AZ
Table 1 also summarizes the Ki values against the
porcine bc1 complex for the inhibition by the synthe-
sized compounds and also by the commercial compound
azoxystrobin that was used as the control. Interestingly,
the calculated binding free energies correlated reasona-
bly well with the binding free energies derived from the
corresponding experimental Ki values with a correlation
coefficient of r2＝0.89, confirming the reliability of our
computational models. As shown in Table 1, compound
2j distinguishes itself as the most promising candidate
with the highest potency (Ki＝1.89 nmol/L), with about
157-fold improved binding affinity compared to AZ.
Figure 3 showed the simulated binding model of
compound 2j, from which we can conclude that the
pharmacophore of this new inhibitor bound in the fash-
ion of typical MOA inhibitors. It bounded with Phe128,
Tyr131, Phe274, and Glu271, formed a H-bond between
the methoxy of the methoxyacrylate and the backbone N
of Glu271. Very importantly, the π-π interactions be-
tween Phe274 and the fluorenone ring of compound 2j
were improved significantly compared with that of
azoxystrobin, which accounts for the higher potency of
compound 2j.
200 mg/L (Inhibition rate, %)
No.
2a
2b
2c
S. fuliginea
No.
2g
2h
2i
S. fuliginea
0
93
0
96
0
0
2d
2e
0
2j
80
85
56
43
AZ
2f
(2-(bromomethyl)phenyl)-3-methoxyacrylate (3) was
prepared according to the existing method.[6] Then,
compound 3 reacted with various benzophenones 4 or
2-hydroxy-9H-fluoren-9-one (5) to afford the target
compounds 2a—2h and 2j in yields of 65%—81%.
Compound 2i was prepared by the N-methylation of 2c
in a yield of 68%. The structures of all the target com-
pounds were characterized by H NMR, 13C NMR and
1
HRMS spectra.
Cytochrome bc1 inhibition activity
As shown in Table 1, six compounds (2a, 2b, 2e, 2f,
2g, and 2j) were found to exhibit higher (from 21-fold
to 157-fold) inhibitory activities against the porcine cy-
tochrome bc1 complex than the control of azoxystrobin,
whereas compounds 2c, 2d, 2h, and 2i showed very low
inhibition effects against porcine cytochrome bc1 com-
plex. In addition, the O-bridged derivatives 2b (Ki＝
3.28 nmol/L) is more active than its corresponding
S-bridged derivative 2a (Ki＝13.95 nmol/L) and the
NH-bridged derivative 2c (Ki＞1000 nmol/L). Besides,
compounds 2e, 2f and 2g showed comparable potency
as compound 2a, indicating that substitution on the
benzophenone as pharmacophore has not significant
Enzymatic kinetics
As shown in Figure 4A illustrating progress curves,
compound 2j exhibits slow-binding behavior to porcine
bc1 complex. Over a time period in which the uninhibi-
ted enzyme displays a simple linear progress curve like
line 1 in Figure 4A, the data in the presence of 2j will
display a quasi-linear relationship with time in the early
part of the curve, converting later to a slower linear re-
lationship between product and time. Increasing the
concentration of 2j led to the decrease of the
steady-state rate (vs) in the progress curve and the pro-
gress curves obtained using various concentrations of 2j
Chin. J. Chem. 2012, 30, 1999—2008
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2005

Zhu et al.
FULL PAPER
the values of A and B determined from Figure 4B are
equal to the true association and dissociation rate con-
stants k＋ and k－ according to Eq. 3, respectively, and
0
0
the inhibition constant for 2j can be calculated as Ki＝
k－ /k＋ ＝(1.8987±0.0432) nmol/L.
0
0
In order to study the effect of substrate ubiquinol on
the inhibition kinetics of 2j, the bc1 complex activity of
SCR was further measured directly by using decylu-
biquinol (DBH2) or cytochrome c as substrates in the
absence and in the presence of 2j. In the presence of 2j,
with succinate used as substrate, the progress curves
exhibited a similar curvilinear (Figure 6A and 6B).
However, when the experiments were performed at a
fixed concentration of 2j, the kobs value decreased with
increasing concentration of DBH2, which is different
from what is observed in Figure 5B. To further elucidate
the inhibitory mechanism, we carried out six sets of in-
hibitory experiments in the presence of varying concen-
trations of DBH2 and 2j. Similarly, from these progress
curves of product formation at fixed concentrations of
DBH2, the values of v0, vs and kobs can be determined as
described previously. Plots of kobs against the inhibitor
concentration give a straight line (insets of Figure 6A
and 6B), from which the values of A (slope) and B (in-
tercept) at indicated DBH2 concentrations were obtained.
Figures 6C and 6D show the dependence of the A and B
values on the DBH2 concentration. Clearly, A deceased
with increasing the concentration of DBH2, while B was
not influenced, suggesting that 2j is a competitive in-
hibitor with respect to substrate ubiquinol. Thus, the
microscopic inhibition rate constants k＋0＝(0.3713±
Figure 3 Simulated binding model of compound 2j (gray) in
bc1 complex (light gray).
were fitted to Eq. 1 to determine v0, vs and kobs. The kobs
values were respectively 0.0298, 0.0333, 0.0469, 0.0944
and 0.1740 s－1 when the concentrations of 2j were re-
spectively 1, 2.5, 5, 10 and 20 nmol/L. The plot for kobs
versus [I] is shown in Figure 4B. kobs is proportional to
the inhibitor concentration, the slope corresponds to the
－
1
－
1
apparent rate constants A＝(0.0079±0.0004) s •nmol
and the intercept corresponds to the apparent rate con-
－
1
stants B＝(0.015±0.004) s .
To distinguish the mode of inhibition of a time-de-
pendent inhibitor, it is convenient to analyze the effect
of various substrate concentrations on kobs at fixed in-
hibitor concentration. A competitive-type inhibitor will
display a decrease of kobs with increasing substrate con-
centration. In contrast, with uncompetitive inhibitors the
value of kobs will increase with increasing substrate
concentration, while kobs is independent of substrate
concentration for noncompetitive-type inhibition. So we
monitored the time courses of the bc1 complex-cata-
lyzed reaction in the presence of different concentra-
tions of cytochrome c and at a fixed concentration of 2j
(Figure 5A). The values of kobs can be obtained by fit-
ting the progress curves according to the substrate reac-
tion kinetic theory and are plotted against the concentra-
tion of cytochrome c in Figure 5B. The data clearly re-
vealed that kobs was independent of the concentration of
substrate cytochrome c, indicating that 2j is a noncom-
petitive inhibitor with respect to cytochrome c. Thus,
－
－
1
0.0259) s •nmol•L and k－ ＝(0.0018±0.0003) s－
1
1
0
can be obtained by fitting Eq. 2 to the experimental data,
and the inhibition constant Ki＝k－ /k＋ ＝(4.848±0.316)
0
0
nmol/L can be derived. The Ki value determined is
about 2.55-fold higher than that measured from the suc-
cinate-cytochrome c reductase activity inhibition, which
is mainly due to the presence of lauryl maltoside since
increasing the concentration of lauryl maltoside, the kobs
value will be decreased.
Fungicidal activity
The in vivo fungicidal activities of all the new com-
pounds against S. fuliginea were screened. As listed in
Figure 4 (A) Inhibitory kinetics of bc1 complex by 2j. Each reaction mixture contains 100 mmol/L PBS (pH 7.4), 0.3 mmol/L EDTA,
20 mmol/L succinate, 100 μmol/L cytochrome c, 0.1 nmol/L SCR, and a certain amount of 2j (1, 0 nmol/L; 2, 1 nmol/L; 3, 2.5 nmol/L; 4,
5 nmol/L; 5, 10 nmol/L and 6, 20 nmol/L). (B) Plot of kobs against concentration of 2j.
2006
www.cjc.wiley-vch.de
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 1999—2008

Design, Synthesis, and Bioevaluation of Novel Strobilurin Derivatives
Figure 5 (A) Effect of cytochrome c concentration on the inhibition of bc1 complex by 2j. The assays were carried out in the presence
of 10 nmol/L 2j and various concentrations of cytochrome c (1, 12 μmol/L; 2, 20 μmol/L; 3, 28 μmol/L; 4, 44 μmol/L and 5, 60 μmol/L).
Each reaction was initiated by adding 0.1 nmol/L SCR. (B) Plot of kobs against concentration of cytochrome c.
Figure 6 (A, B) Inhibitory kinetics of porcine bc1 complex by 2j. Each reaction mixture contains 100 mmol/L PBS (pH 6.5), 2 mmol/L
EDTA, 750 μmol/L lauryl maltoside, 100 μmol/L oxidized cytochrome c, 0.05 nmol/L SCR, a certain amount of DBH2 (A, 20 μmol/L; B,
120 μmol/L), and 2j (1, 10 nmol/L; 2, 20 nmol/L and 3, 30 nmol/L). Experimental data are shown as dots and theoretical values as lines.
Insets: Plots of kobs against concentration of 2j. (C) Plot of the apparent rate constant A against concentration of DBH2. Inset: Plot of 1/A
against concentration of DBH2. (D) Plot of the apparent rate constant B against concentration of DBH2.
Table 2, some compounds exhibited significant fungi-
cidal activity against S. fuliginea at the concentration of
200 mg/L, and overall, the order of the fungicidal activ-
ity is m-benzophenone derivative ＞ p-benzophenone
derivative ＞o-benzophenone derivative. Among them,
compound 2j, a m-benzophenone derivative, displayed
the same level of fungicidal activity as azoxystrobin.
Most importantly, compounds 2b and 2g showed higher
fungicidal activity than azoxystrobin, which indicates
that benzophenone-containing derivatives worth further
investigation.
Conclusions
In summary, we have successfully designed and
synthesized a series of benzophenone/fluorenone-con-
taining derivatives 2a—2j as inhibitors of cytochrome
bc1 complex. Very promisingly, compound 2j with a Ki
Chin. J. Chem. 2012, 30, 1999—2008
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2007

Zhu et al.
FULL PAPER
L.; Yu, L.; Xia, D. Biochemistry 2002, 41, 11692.
[3] Berry, E. A.; Huang, L. FEBS Lett. 2003, 555, 13.
[4] Esser, L.; Quinn, B.; Li, Y. F.; Zhang, M.; Elberry, M.; Yu, L.; Yu,
C. A.; Xia, D. J. Mol. Biol. 2004, 341, 281.
[5] Beautement, K.; Clough, J. M.; de Fraine, P. J.; Godfrey, C. R. A. J.
Pestic Sci. 1991, 31, 499.
[6] Zhao, P. L.; Liu, C. L.; Huang, W.; Wang, Y. Z.; Yang, G. F. J. Ag-
ric. Food Chem. 2007, 55, 5697.
[7] Huang, W.; Zhao, P. L.; Liu, C. L.; Chen, Q.; Liu, Z. M.; Yang, G. F.
J. Agric. Food Chem. 2007, 55, 3004.
[8] Fisher, N.; Meunier, B. Pest Manag. Sci. 2005, 61, 973.
[9] Gisi, U.; Sierotzki, H.; Cook, A.; McCaffery, A. Pest Manag. Sci.
2002, 58, 859.
[10] Hao, G.-F.; Wang, F.; Li, H.; Zhu, X.-L.; Yang, W.-C.; Huang, L.-S.;
Wu, J.-W.; Berry, E. A.; Yang, G.-F. J. Am. Chem. Soc. 2012, 134,
11168.
value of 1.89 nmol/L was found to display about
157-fold improved binding affinity compared to the
commercial inhibitor azoxystrobin. The further inhibi-
tory kinetics studies revealed that compound 2j is a non-
competitive inhibitor with respect to substrate cyto-
chrome c, but is a competitive inhibitor with respect to
substrate ubiquinol. The present work demonstrated that
strobilurin analogues containing benzophenone or fluo-
renone side chain could be used as new leads for future
fungicide discovery. Further structural optimization and
in vivo fungicidal activities about benzophenone-based
strobilurin derivatives are well under way.
Acknowledgement
[11] Worthington, P. A. J. Pestic. Sci. 1991, 31, 457.
[12] Yu, L.; Yu, C. J. Biol. Chem.1982, 257, 2016.
[13] King, T. E. Methods Enzymol. 1967, 10, 216.
[14] Zhao, P. L.; Wang, L.; Zhu, X. L.; Huang, X.; Zhan, C. G.; Wu, J.
W.; Yang, G. F. J. Am. Chem. Soc. 2010, 132, 185.
[15] Rieske, J. S. Methods Enzymol. 1967, 10, 239.
[16] Luo, C.; Long, J.; Liu, J. Clin. Chim. Acta 2008, 395(1—2), 38.
[17] Fisher, N.; Bourges, I.; Hill, P.; Brasseur, G.; Meunier, B. Eur. J.
Biochem. 2004, 271, 1292.
[18] Fisher, N.; Brown, A. C.; Sexton, G.; Cook, A.; Windass, J. Eur. J.
Biochem. 2004, 271, 2264.
[19] Pan, Y.; Gao, D.; Zhan, C. G. J. Am. Chem. Soc. 2008, 130, 5140.
[20] Zhu, X. L.; Yang, W. C.; Yu, N. X.; Yang, S. G.; Yang, G. F. J. Mol.
Model. 2011, 17, 495.
The research was supported in part by the National
Basic Research Program of China (No. 2010CB126103),
the NSFC (Nos. 20902034, 20925206 and 21102052)
and the National Key Technologies R&D Program (No.
2011BAE06B05).
References
[1] Kim, H.; Xia, D.; Yu, C. A.; Xia, J. Z.; Kachurin, A. M.; Zhang, L.;
Yu, L.; Deisenhofer, J. Proc. Natl. Acad. Sci. U. S. A. 1998, 95,
8026.
[2] Gao, X.; Wen, X.; Yu, C. A.; Esser, L.; Tsao, S.; Quinn, B.; Zhang,
(Pan, B.; Qin, X.)
2008
www.cjc.wiley-vch.de
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 1999—2008