Ametoctradin is a Potent Qo Site Inhibitor of the Mitochondrial Respiration Complex III

Article abstract of DOI:10.1021/acs.jafc.5b00228

Ametoctradin is a new Oomycete-specific fungicide under development by BASF. It is a potent inhibitor of the bc₁ complex in mitochondrial respiration. However, its detailed action mechanism remains unknown. In the present work, the binding mode of ametoctradin was first uncovered by integrating molecular docking, MD simulations, and MM/PBSA calculations, which showed that ametoctradin should be a Q_o site inhibitor of bc₁ complex. Subsequently, a series of new 1,2,4-triazolo[1,5-a]pyrimidine derivatives were designed and synthesized to further understand the substituent effects on the 5- and 6-position of 1,2,4-triazolo[1,5-a]pyrimidine. The calculated binding free energies (ΔG_cal) of newly synthesized analogues as Q_o site inhibitors correlated very well (R² = 0.96) with their experimental binding free energies (ΔG_exp). Two compounds (4a and 4c) with higher inhibitory activity against porcine SQR than ametoctradin were successfully identified. The structural and mechanistic insights obtained from the present study will provide a valuable clue for future designing of a new promising bc₁ inhibitor.

Full text of DOI:10.1021/acs.jafc.5b00228

Article
pubs.acs.org/JAFC
Ametoctradin is a Potent Qo Site Inhibitor of the Mitochondrial
Respiration Complex III
Xiaolei Zhu,† Mengmeng Zhang,† Jingjing Liu,† Jingming Ge,† and Guangfu Yang*,†,‡
†Key Laboratory of Pesticide and Chemical Biology, College of Chemistry, Ministry of Education, Central China Normal University,
Wuhan 430079, P.R. China
‡Collaborative Innovation Center of Chemical Science and Engineering, Tianjing 30071, P.R.China
S
* Supporting Information
ABSTRACT: Ametoctradin is a new Oomycete-specific fungicide under development by BASF. It is a potent inhibitor of the bc1
complex in mitochondrial respiration. However, its detailed action mechanism remains unknown. In the present work, the
binding mode of ametoctradin was first uncovered by integrating molecular docking, MD simulations, and MM/PBSA
calculations, which showed that ametoctradin should be a Qo site inhibitor of bc1 complex. Subsequently, a series of new 1,2,4-
triazolo[1,5-a]pyrimidine derivatives were designed and synthesized to further understand the substituent effects on the 5- and 6-
position of 1,2,4-triazolo[1,5-a]pyrimidine. The calculated binding free energies (ΔGcal) of newly synthesized analogues as Qo
site inhibitors correlated very well (R2 = 0.96) with their experimental binding free energies (ΔGexp). Two compounds (4a and
4c) with higher inhibitory activity against porcine SQR than ametoctradin were successfully identified. The structural and
mechanistic insights obtained from the present study will provide a valuable clue for future designing of a new promising bc1
inhibitor.
KEYWORDS: cytochrome bc1 complex, ametoctradin, molecular docking, molecular dynamics
that ametoctradin is an inhibitor of mitochondrial respiration
bc1 complex with a new chemical scaffold, the pyrimidylamines.
Field trials indicated that ametoctradin is a highly selective and
effective fungicide that can be used in preventive spray
applications against late blight and downy mildews in a wide
range of special crops. It has an excellent toxicological and
ecotoxicological profile and is highly suitable for use in
integrated crop management programs. Since its discovery in
2004, an increasing interest in ametoctradin was not from its
high potency but from its having no cross-resistance to the
existing Qo inhibitors (e.g., strobilurins). However, it remains
unclear whether ametoctradin is a Qo site inhibitor or a Qi site
inhibitor of the bc1 complex.
In order to uncover the detailed interaction mechanism
between ametoctradin and bc1 complex, a series of 5,6-
disubstituted-7-amino-1,2,4-triazolo[1,5-a]pyrimidines 4a ∼ z
(Figure 1) were designed and synthesized. Herein, we first
described the synthetic chemistry, inhibition effects against
porcine bc1 complex, and the structure−activity relationships of
these newly synthesized compounds. Then, we carried out
molecular docking of these compounds based on Qo and Qi
sites, respectively, and followed by molecular dynamics (MD)
simulations and the molecular mechanics Poisson−Boltzmann
surface area (MM/PBSA) calculations. We believe that the
detailed molecular mechanism of ametoctradin uncovered by
the present study would put forward the structure-based design
INTRODUCTION
■
Fungicides have become an integral part of efficient food
production. In agrochemical research, the discovery of new
fungicides with broad-spectrum fungal control and excellent
crop selectivity is still remaining as a challenge. The
cytochrome bc1 complex (EC 1.10.2.2, bc1, also known as
complex III) is an essential component in the respiratory chain;
it catalyzes electron transfer from quinol to cyt c1 and
concomitantly translocates protons across membranes.1−4
The bc1 complex has a diheme cytochrome b, an iron−sulfur
protein (ISP) with a Rieske-type Fe2S2 cluster, and cyt c1 that
undergoes reduction and oxidation during the turnover of the
enzyme.5 The bc1 complex has been found in the plasma
membrane of bacteria and in the inner mitochondrial
membrane of eukaryotes. Due to its critical roles in life
processes, the bc1 complex has been identified as a promising
action target for agricultural fungicides. So far, two separate
catalytic sites, a quinone reduction site near the negative side of
the membrane (Qi) and a quinol oxidation site close to the
positive side of the membrane (Qo), have been identified and
confirmed by X-ray crystallographic studies.6,7 The detailed
action mechanism of a number of specific Qo and Qi sites
inhibitors of cytochrome bc1 complex have been studied
intensively.8,9 Among them, antimycin A, funiculosin, ilicicolin
H, and dichlorophenyl dimethyl urea are well-known Qi site
inhibitors, whereas the Qo site inhibitors include azoxystrobin,
pyraclostrobin, kresoxim-methyl, stigmatellin, various hydrox-
yquinones and myxothiazol.10−14 Besides, some inhibitors, such
as NQNO and ascochlorin, can bind both at Qo and Qi sites
simultaneously.15
Received: January 15, 2015
Revised: March 16, 2015
Accepted: March 16, 2015
Ametoctradin (Initium)16,17 is an innovative new fungicide
under development by BASF. The existing research has proved
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jafc.5b00228
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
Figure 1. Design of the title compounds.
and discovery of novel ametoctradin analogues and fungicides
targeting the bc1 complex.
reactive temperature was heated to 160 °C under reflux for 12−
16 h as monitored by TLC detection. The mixture was cooled
to room temperature, and the product was precipitated in the
form of colorless crystals. Water (5 mL) was added to the
reaction mixture with pH equal to 8−9 adjusted by adding 20%
triethylamine. Finally, the precipitate was purified by flash
chromatography on silica gel to provide the desired product.
Due to the complexity of reaction, the yield of some compound
is lower than 10%. Our purpose was to obtain a certain
compound to assay its activity. So, we did not to optimize the
reaction condition to increase the yield.
MATERIALS AND METHODS
■
General Methods. Unless otherwise noted, all chemical
reagents were commercially available from Acros Organics and/
or Sigma-Aldrich and treated with standard methods before
1
used. H NMR spectra were recorded in CDCl3 or DMSO-d6
solution on a varian Mercury 600 or 400 MHz spectrometer.
Chemical shifts were recorded in parts per million (ppm), with
TMS as the internal reference. The following abbreviations
were used to designate chemical shift multiplicities: s = singlet,
d = doublet, t = triplet, m = multiplet, br = broad; MS spectra
were determined with a Tracems2000 organic mass spec-
trometer, and signals were given in m/z. Melting points were
measured on a Buchi B-545 melting point apparatus. Element
analyses (EA) were measured on a Vario ELIII CHNSO
elemental analyzer. The silica gel (200−300 mesh) for flash
column chromatography was purchased from QingDao Marine
Chemical Factory in China.
General Procedure for the Preparation of Intermedi-
ate 2.18−20 Under a nitrogen atmosphere, THF (tetrahy-
drofuran, 30 mL) and anhydrous acetonitrile (25 mmol) was
mixed and stirred for 5 min at −78 °C. BuLi (2.M) was added
into the mixture and stirred for 1 h at −78 °C. Then, different
substituent benzyl bromide was added dropwise slowly and
then the mixture was stirred for 20 min and monitored by TLC
detection. After 20 min, the reaction was complete. The
resulting suspension was then diluted with water (15 mL) and
extracted with ethyl acetate (2 × 25 mL). The combined
organic extracts were washed with brine (2 × 25 mL), dried
with Na2SO4, and concentrated, and the residue was purified by
flash chromatography on silica gel to provide the desired
product.
General Procedure for the Preparation of Intermedi-
ate 3.18−20 A mixture of solid potassium methoxide (1.75g)
and o-xylene (2.7 mL) was stirred and heated with reflux. When
the mixture changed to light yellow, as monitored by HPLC
detection, only 10% of 2 (total 10 mmol) and ester (total 20
mmol) were added in 5 min and then stirred for 15 min. The
complete addition required another 10 min. The reaction
mixture was refluxed 2.5 h and was allowed to warm room
temperature. Water (3.3 mL) was added to the reaction mixture
with pH equal to 5−6 adjusted by adding 20% hydrochloric
acid. Then it was extracted with ethyl acetate (3 × 15 mL). The
combined organic extracts were washed with brine 15 mL and
dried with Na2SO4 to yield 2-propionyldecanonitrile.
5-Ethyl-6-octyl-1,2,4-triazolo[1,5-a]pyrimidin-7-amine
1
(ametoctradin). Yield: 76%; mp 197.7−198.7 °C; H NMR
(600 MHz, d6-DMSO) δ 8.33 (s, 1H), 7.71 (s, 2H), 2.74 (q, J =
7.2 Hz, 2H), 2.63−2.56 (m, 2H), 1.41 (d, J = 13.8 Hz, 4H),
1.33−1.19 (m, 11H), 0.86 (t, J = 6.6 Hz, 3H); MS: m/z =
275.33 (M+).
6-Octyl-5-(p-tolyl)-1,2,4-triazolo[1,5-a]pyrimidin-7-
1
amine (4a). Yield: 10%; mp 156−158 °C; H NMR (600
MHz, d6-DMSO): δ 8.44 (s, 1H), 8.01 (s, 2H), 7.34 (d, J = 7.2
Hz, 2H), 7.28 (d, J = 7.2 Hz, 2H), 2.56 (s, 2H), 2.38 (s, 3H),
1.33 (s, 2H), 1.25 (d, J = 12.0 Hz, 2H), 1.23−1.16 (m, 2H),
1.08 (dd, J = 42.6, 6.0 Hz, 6H), 0.83 (t, J = 7.2 Hz, 3H); MS:
m/z = 337.27 (M+). Anal. Calcd for C20H27N5: C, 71.18; H,
8.06; N, 20.75. Found: C, 71.22; H, 8.102; N, 20.50.
6-Octyl-5-(3-(trifluoromethyl)phenyl)-1,2,4-triazolo-
[1,5-a]pyrimidin-7-amine (4b). Yield: 11%; mp 130−131
°C; 1H NMR (600 MHz, CDCl3): δ 8.42 (s, 1H), 7.78 (s, 1H),
7.73 (dd, J = 15.6, 7.2 Hz, 2H), 7.62 (t, J = 7.8 Hz, 1H), 6.00 (s,
2H), 2.60−2.55 (m, 2H), 1.58−1.46 (m, 2H), 1.23 (dd, J =
25.2, 19.8 Hz, 10H), 0.86 (t, J = 67.2 Hz, 3H). Anal. Calcd for
C20H24F3N5: C, 61.37; H, 6.18; N, 17.89. Found: C,61.35; H,
6.070; N, 18.01.
5-(3-Chlorophenyl)-6-octyl-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4c). Yield: 7%; mp 154−155 °C; H
NMR (600 MHz, d6-DMSO): δ 8.47 (s, 1H), 8.12 (s, 2H),
7.58−7.47 (m, 3H), 7.42 (d, J = 7.2 Hz, 1H), 2.53 (d, J = 7.8
Hz, 1H), 1.39- 1.28 (m, 2H), 1.21 (dd, J = 13.8, 7.2 Hz, 2H),
1.17- 0.99 (m, 8H), 0.83 (t, J = 7.2 Hz, 3H); MS: m/z = 357.26
(M+). Anal. Calcd for C19H24ClN5: C, 63.77; H, 6.76; N, 19.57.
Found: C, 63.61; H, 6.783; N, 19.31.
5-Ethyl-6-propyl-1,2,4-triazolo[1,5-a]pyrimidin-7-
1
amine (4d). Yield: 64%; mp 179−181 °C; H NMR (400
MHz, d6-DMSO): δ 8.34 (s, 1H), 7.74 (s, 2H), 2.75 (d, J = 7.2
Hz, 2H), 2.58 (s, 2H), 1.45 (s, 2H), 1.23 (t, J = 7.2 Hz, 3H),
0.98 (t, J = 6.8 Hz, 3H); MS: m/z = 205.06 (M+). Anal. Calcd
for C10H15N5: C, 58.51; H, 7.37; N, 34.12. Found: C, 58.32; H,
7.379; N, 34.22.
General Procedure for the Preparation of the Title
Compounds 4a ∼ z. A mixture of 3 (3 mmol), 1,2,4-triazol-3-
amine (3 mmol), and o-xylene (2.5 mL) was quickly stirred,
and then ClSO3H (0.6 mmol) was slowly added dropwise. The
6-Benzyl-5-phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7-
1
amine (4e). Yield: 33%; mp 249−251 °C; H NMR (400
MHz, d6-DMSO): δ 8.49 (s, 1H), 8.05 (s, 2H), 7.64−7.27 (m,
B
DOI: 10.1021/acs.jafc.5b00228
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
5H), 7.27−7.19 (m, 2H), 7.18−7.09 (m, 1H), 6.96 (d, J = 7.2
Hz, 2H), 4.01 (s, 2H); MS: m/z = 300.09 (M+). Anal. Calcd
for C18H15N5: C, 71.74; H, 5.02; N, 23.24. Found: C, 71.80; H,
5.169; N, 23.07.
(M+). Anal. Calcd for C14H14IN5: C, 44.34; H, 3.72; N, 18.47.
Found: C, 44.59; H, 3.550; N, 18.50.
6-(2-Chlorobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4n). Yield: 59%; mp 225−227 °C; H
6-Benzyl-5-(3,3,3-trifluoropropyl)-1,2,4-triazolo[1,5-
NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.50
(d, J = 7.8 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 7.19 (t, J = 7.8 Hz,
1H), 6.75 (d, J = 7.8 Hz, 1H), 4.09 (s, 2H), 2.55−2.46 (m,
5H), 1.08 (t, J = 7.2 Hz, 3H); MS: m/z = 287.01 (M+). Anal.
Calcd for C14H14ClN5: C, 58.44; H, 4.90; N, 24.34. Found: C,
58.17; H, 4.710; N, 24.21.
1
a]pyrimidin-7-amine (4f). Yield: 39%; mp 212−214 °C; H
NMR (600 MHz, d6-DMSO): δ 8.44 (s, 1H), 8.06 (s, 2H), 7.29
(t, J = 7.2 Hz, 2H), 7.20 (t, J = 7.2 Hz, 1H), 7.12 (d, J = 7.2 Hz,
2H), 4.11 (s, 2H), 2.93−2.80 (m, 2H), 2.61 (dd, J = 16.2, 10.8
Hz, 2H); MS: m/z = 321.08 (M+). Anal. Calcd for
C15H14F3N5: C, 56.07; H, 4.39; N, 21.80. Found: C, 56.24;
H, 4.514; N, 21.65.
5-Ethyl-6-(4-(trifluoromethoxy)benzyl)-1,2,4-triazolo-
[1,5-a]pyrimidin-7-amine (4o). Yield: 69%; mp 212−213
1
6-(4-Bromobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]-
°C; H NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s,
1
pyrimidin-7-amine (4g). Yield: 53%; mp 239−241 °C; H
2H), 7.25 (dd, J = 23.4, 8.4 Hz, 4H), 4.11 (s, 2H), 2.63 (q, J =
7.2 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H); MS: m/z = 337.02 (M+).
Anal. Calcd for C15H14F3N5O: C, 53.41; H, 4.18; N, 20.76.
Found: C, 53.53; H, 4.336; N, 20.56.
NMR (600 MHz, d6-DMSO): δ 8.39 (s, 1H), 7.88 (s, 2H), 7.46
(d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 4.05 (s, 2H), 2.61
(q, J = 7.2 Hz, 2H), 1.09 (t, J = 7.2 Hz, 3H); MS: m/z = 332.96
(M+). Anal. Calcd for C14H14BrN5: C, 50.62; H, 4.25; N, 21.08.
Found: C, 50.73; H, 4.103; N, 21.99.
6-(3-Chlorobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4p). Yield: 80%; mp 217−218 °C; H
5-Ethyl-6-(4-fluorobenzyl)-1,2,4-triazolo[1,5-a]-
NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.30
(t, J = 7.8 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.20 (s, 1H), 7.05
(d, J = 7.8 Hz, 1H), 4.10 (s, 2H), 2.63 (q, J = 7.2 Hz, 2H), 1.10
(t, J = 7.2 Hz, 3H); MS: m/z = 287.02 (M+). Anal. Calcd for
C14H14ClN5: C, 58.44; H, 4.90; N, 24.34.
1
pyrimidin-7-amine (4h). Yield: 85%; mp 216−218 °C; H
NMR (600 MHz, d6-DMSO): δ 8.39 (s, 1H), 7.87 (s, 2H), 7.15
(t, J = 7.2 Hz, 2H), 7.09 (t, J = 9.0 Hz, 2H), 4.06 (s, 2H), 2.63
(q, J = 7.2 Hz, 2H), 1.09 (t, J = 7.2 Hz, 3H); MS: m/z = 271.06
(M+). Anal. Calcd for C14H14FN5: C, 61.98; H, 5.20; N, 25.81.
Found: C, 62.15; H, 5.269; N, 25.56.
6-(3,4-Dichlorobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4q). Yield: 48%; mp 256−257 °C; H
6-(3-Bromobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]-
NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.52
(d, J = 8.4 Hz, 2H), 7.41 (s, 1H), 7.06 (d, J = 7.8 Hz, 1H),4.08
(s, 2H), 2.62 (d, J = 7.8 Hz, 2H), 1.11 (t, J = 7.2 Hz, 3H); MS:
m/z = 321.19 (M+). Anal. Calcd for C14H13Cl2N5: C, 52.19; H,
4.07; N, 21.74. Found: C, 52.23; H, 4.251; N, 21.49.
1
pyrimidin-7-amine (4i). Yield: 52%; mp 211−212 °C; H
NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.39
(d, J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.09
(d, J = 7.8 Hz, 1H), 4.09 (s, 2H), 2.63 (q, J = 7.2 Hz, 2H), 1.10
(t, J = 7.2 Hz, 3H); MS: m/z = 332.93 (M+). Anal. Calcd for
C14H14BrN5: C, 50.62; H, 4.25; N, 21.08. Found: C, 50.73; H,
4.103; N, 20.99.
5-Benzyl-6-phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7-
1
amine (4r). Yield: 17%; mp 251−252 °C; H NMR (600
MHz, d6-DMSO): δ 8.45 (s, 1H), 7.47 (ddd, J = 24.0, 15.6, 7.8
Hz, 4H), 7.33 (d, J = 4.2 Hz, 1H), 7.25 (d, J = 6.6 Hz, 2H),
7.17 (t, J = 7.2 Hz, 2H), 7.13 (d, J = 7.2 Hz, 1H), 6.94 (d, J =
7.2 Hz, 2H), 3.78 (s, 2H); MS: m/z = 300.07 (M+). Anal.
Calcd for C18H15N5: C, 71.74; H, 5.02; N, 23.24. Found: C,
71.64; H, 5.270; N, 23.13.
5-Ethyl-6-(2-fluorobenzyl)-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4j). Yield: 66%; mp 209−211 °C; H
NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.89 (s, 2H), 7.26
(dd, J = 13.2, 6.6 Hz, 1H), 7.20 (t, J = 9.0 Hz, 1H), 7.05 (t, J =
7.2 Hz, 1H), 6.84 (t, J = 7.8 Hz, 1H), 4.06 (s, 2H), 4.47 (q, J =
7.2 Hz, 2H), 1.08 (t, J = 7.2 Hz, 3H); MS: m/z = 271.03 (M+).
Anal. Calcd for C14H14FN5: C, 61.98; H, 5.20; N, 25.81. Found:
C, 61.82; H, 5.347; N, 25.80.
5-Benzyl-6-(4-fluorophenyl)-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4s). Yield: 19%; mp 248−250 °C; H
NMR (600 MHz, d6-DMSO): δ 8.44 (s, 1H), 7.48 (s, 2H),
7.31−7.24 (m, 4H), 7.18 (t, J = 7.2 Hz, 2H), 7.14 (d, J = 7.2
Hz, 1H), 6.94 (d, J = 7.2 Hz, 2H), 3.78 (s, 2H); MS: m/z =
318.07 (M+). Anal. Calcd for C18H14FN5: C, 67.70; H, 4.42; N,
21.93. Found: C, 67.74; H, 4.250; N, 21.88.
6-(2-Bromobenzyl)-5-ethyl-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4k). Yield: 59%; mp 228−239 °C; H
NMR (600 MHz, d6-DMSO): δ 8.40 (s, 1H), 7.90 (s, 2H), 7.67
(d, J = 7.8 Hz, 1H), 7.23 (t, J = 7.2 Hz, 1H), 7.17 (t, J = 7.8 Hz,
1H), 6.73 (d, J = 7.8 Hz, 1H), 4.06 (s, 2H), 2.48 (d, J = 7.5 Hz,
1H), 1.08 (t, J = 7.2 Hz, 3H); MS: m/z = 332.99 (M+). Anal.
Calcd for C14H14BrN5: C, 50.62; H, 4.25; N, 21.08. Found: C,
50.75; H, 4.234; N, 21.29.
5-Benzyl-6-(4-chlorophenyl)-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4t). Yield: 5%; mp 262−263 °C; H
NMR (600 MHz, d6-DMSO): δ 8.44 (s, 1H), 7.52 (d, J = 8.4
Hz, 4H), 7.25 (d, J = 8.4 Hz, 2H), 7.18 (t, J = 7.2 Hz, 2H), 7.14
(d, J = 7.2 Hz, 1H), 6.95 (d, J = 7.2 Hz, 2H), 3.78 (s, 2H); MS:
m/z = 334.01 (M+). Anal. Calcd for C18H14ClN5: C, 64.38; H,
4.20; N, 20.86. Found: C, 64.17; H, 4.308; N, 20.67.
5-Ethyl-6-(3-fluorobenzyl)-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4l). Yield: 65%; mp 226−228 °C; H
NMR (600 MHz, d6-DMSO): δ 8.39 (s, 1H), 7.88 (s, 2H), 7.31
(dd, J = 14.4, 7.8 Hz, 1H), 7.02 (t, J = 7.8 Hz, 1H), 6.95 (t, J =
10.2 Hz, 2H), 4.10 (s, 2H), 2.63 (q, J = 7.2 Hz, 2H), 1.10 (t, J =
7.2 Hz, 3H); MS: m/z = 271.08 (M+). Anal. Calcd for
C14H14FN5: C, 61.98; H, 5.20; N, 25.81. Found: C, 61.92; H,
5.467; N, 25.71.
6-Benzyl-5-ethyl-1,2,4-triazolo[1,5-a]pyrimidin-7-
1
amine (4u). Yield: 80%; mp 229−230 °C; H NMR (400
MHz, CDCl3) δ 8.40 (s, 1H), 7.88 (s, 2H), 7.27 (d, J = 7.2 Hz,
2H), 7.19 (d, J = 6.4 Hz, 1H), 7.12 (d, J = 6.4 Hz, 2H), 4.08 (s,
2H), 2.80−2.57 (m, 2H), 1.09 (t, J = 7.2 Hz, 3H); MS: m/z =
253.09 (M+). Anal. Calcd for C14H15N5: C, 66.38; H, 5.97; N,
27.65. Found: C, 66.41; H, 5.842; N, 27.60.
5-Ethyl-6-(3-iodobenzyl)-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4m). Yield: 87%; mp 214−215 °C; H
NMR (600 MHz, d6-DMSO): δ 8.39 (s, 1H), 7.88 (s, 2H), 7.55
(d, J = 15.6 Hz, 2H), 7.08 (s, 2H), 4.06 (s, 2H), 2.62 (dd, J =
13.8, 6.5 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H); MS: m/z = 378.92
5-Ethyl-6-phenyl-1,2,4-triazolo[1,5-a]pyrimidin-7-
1
amine (4v). Yield: 60%; mp 216−218 °C; H NMR (400
MHz, CDCl3) δ 8.43 (s, 1H), 7.66−7.50 (m, 2H), 7.47 (d, J =
C
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max −min
7.2 Hz, 1H), 7.35 (s, 2H), 7.29 (s, 2H), 2.40 (dd, J = 14.8, 7.2
Hz, 2H), 1.06 (t, J = 7.6 Hz, 3H); MS: m/z = 238.09 (M+).
Anal. Calcd for C13H13N5: C, 65.25; H, 5.48; N, 29.27. Found:
C, 65.25; H, 5.48; N, 29.27.
y = min +
1 + 10x−logIC
50
(1)
Sigma Plot software 9.0 was used to determine all kinetic
constants.
5-Ethyl-6-(4-fluorophenyl)-1,2,4-triazolo[1,5-a]-
1
Computational Methods and Binding-Free-Energy
Calculations. Molecular Docking. The three-dimensional
(3D) structure of bc1 complex was obtained from PDB
database (PDB ID: 1PPJ, from bovine). The AutoDock 4.2
program22 was applied to dock ametoctradin and its derivates
into the Qo and or Qi site of the bc1 complex. The Gasteiger
charges were used for these inhibitors. To select the best set of
docking parameters and to test the reliability of the docking
results, the antimycin and stigmatellin were first docked into
the Qi and Qo site, respectively. In the docking process, a
conformational search was performed for the antimycin/
stigmatellin molecule using the Solis and Wets local search
method, and the Lamarckian genetic algorithm (LGA)23,24 was
applied for the conformational search of the binding complex of
antimycin/stigmatellin with bc1 complex. Among a series of
docking parameters, the grid size was set to be 40 × 40 × 40,
and the used grid space was the default value of 0.375 Å. The
interaction energy that resulted from probing the cyt b with the
antimycin/stigmatellin molecule was assessed by the standard
AutoDock scoring function. Among a set of 100 candidates of
the docked complex structures, the best one was first selected
according to the interaction energy and was then compared
with the conformation of antimycin/stigmatellin extracted from
the crystal structure. By tuning the docking parameters, the
final antimycin/stigmatellin-bc1 complex was obtained, based
on the smallest root-mean-square deviation (rmsd) of the
docked conformation of antimycin/stigmatellin from its
conformation in the crystal structure. The RMSD between
the selected conformation and previously noted in the X-ray
crystal structure is 0.84 for stigmatellin and 0.89 for antimycin
(data not shown), confirming the reliability of docking
parameters. The same docking parameters were adopted to
perform molecular docking of other compounds.
Molecular Dynamics Simulations. 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 achieved. The Amber ff99 force field was used
for amino acid and General Amber force field was used for
ligands. For temperature regulation, the Langevin thermostat
was used to maintain a temperature of 300 K. The bcc charges
were used as the atomic charges for ligands. There are two
stages for energy minimization. First, the ligand was minimized
with the protein fixed. Second, the backbone atoms of the
protein were fixed, and other atoms were relaxed. The final
minimization was performed with both the ligand and the
protein relaxed. In each step, the energy minimization was
executed by using the steepest descent method for the first
1000 cycles, and the conjugated gradient method for the
subsequent 3000 cycles, with a convergence criterion of 0.1 kcal
mol−1 Å−1. The atomic coordinates were saved every
picosecond. Then, an additional 10 ns MD simulation was
performed for ametoctradin-Qo and ametoctradin-Qi complex.
In order to reduce the computational cost, only 20 ps MD
simulations were performed for 12 newly synthesized
analogues.
pyrimidin-7-amine (4w). Yield: 57%; mp 224−225 °C; H
NMR (400 MHz, d6-DMSO): δ 8.43 (s, 1H), 7.35 (dd, J =
20.4, 8.0 Hz, 5H), 2.40 (dd, J = 14.8, 7.2 Hz, 2H), 1.07 (t, J =
7.6 Hz, 3H); MS: m/z = 256.07 (M+). Anal. Calcd for
C13H12FN5: C, 60.69; H, 4.70; N, 27.22. Found: C, 60.72; H,
4.822; N, 27.41.
6-(4-Chlorophenyl)-5-ethyl-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4x). Yield: 47%; mp 235−236 °C; H
NMR (400 MHz, d6-DMSO): δ 8.42 (s, 1H), 7.57 (d, J = 8.0
Hz, 2H), 7.43 (s, 2H), 7.36 (d, J = 8.0 Hz, 2H), 2.40 (dd, J =
15.2, 7.6 Hz, 2H), 1.06 (t, J = 7.6 Hz, 3H); MS: m/z = 272.05
(M+). Anal. Calcd for C13H12ClN5: C, 57.04; H, 4.42; N, 25.59.
Found: C, 57.01; H, 4.527; N, 25.31.
6-(4-Bromophenyl)-5-ethyl-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4y). Yield: 39%; mp 242−244 °C; H
NMR (400 MHz, d6-DMSO): δ 8.42 (s, 1H), 7.70 (d, J = 8.0
Hz, 2H), 7.44 (s, 2H), 7.30 (d, J = 8.0 Hz, 2H), 2.47−2.23 (m,
2H), 1.07 (t, J = 7.6 Hz, 3H); MS: m/z = 316.01 (M+). Anal.
Calcd for C13H12BrN5: C, 49.07; H, 3.80; N, 22.01. Found: C,
49.01; H, 3.926; N, 22.09.
6-Phenyl-5-(trifluoromethyl)-1,2,4-triazolo[1,5-a]-
1
pyrimidin-7-amine (4z). Yield: 11%; mp 279−280 °C; H
NMR (400 MHz, d6-DMSO): δ 8.67 (s, 1H), 8.05 (s, 2H), 7.50
(d, J = 5.2 Hz, 3H), 7.36 (d, J = 7.2 Hz, 2H); MS: m/z =
279.03 (M+). Anal. Calcd for C12H8F3N5: C, 51.62; H, 2.89; N,
25.08. Found: C, 51.48; H, 3.128; N, 25.35.
Enzyme Assays. The preparation of succinate−cytochrome
c reductase (SCR, mixture of respiratory complex II and bc1
complex) from porcine heart was essentially as reported.21 The
activity of SCR was measured by monitoring the increase in
absorbance of cytochrome c1 at 550 nm, by using the extinction
coefficient of 18.5 mM−1 cm−1. The succinate−ubiquinone
reductase (complex II) activity was measured by monitoring
the decrease in absorbance of 2,6-dichlorophenolindophenol
(DCIP) at 600 nm, by using the extinction coefficient of 21
mM−1 cm−1. The reaction mixture may be scaled down to 1.8
mL with final concentrations of PBS (pH 7.4), 100 mM;
EDTA, 0.3 mM; succinate, 20 mM; oxidized cytochrome c1, 60
uM (or DCIP, 53 uM); and appropriate amounts of enzyme to
start the reaction. The ubiquinol−cytochrome c1 reductase (bc1
complex) activity in catalyzing the oxidation of DBH2 by
cytochrome c1 was assayed in 100 mM PBS (pH 7.4), 0.3 mM
EDTA, 750 uM lauryl maltoside (n-dodecyl-β-D-maltoside),
20−120 uM DBH2, 100 uM oxidized cytochrome c1, and an
appropriate amount of SCR. The preparation of DBH2 from
DB was carried out according to the procedure described in
previous publications, and the concentration of DBH2 was
determined by measuring the absorbance difference between
288 and 320 nm by using an extinction coefficient of 4.14
mM−1 cm−1 for the calculation. For the steady-state studies, the
reaction was carried out in the absence or presence of various
concentrations of the inhibitor.21
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.
MM/PBSA Calculation. For ametoctradin-Qo and ametoc-
tradin-Qi, 100 snapshots of the simulated structure within the
D
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Scheme 1. Synthetic Route of the Title Compounds: (a) BunLi THF −78 °C Ice Bath; (b) CH3OK o-Xylene Reflux Stirred; (c)
ClSO3H o-Xylene Stirred Reflux
Figure 2. X-ray crystal structure of ametoctradin.
stable MD trajectory were selected and minimized to perform
the MM/PBSA calculations (see below). For newly synthesized
analogues, the last snapshot of the MD simulation was
minimized to a convergence criterion of 0.1 kcal mol−1 Å−1.
Then, the ΔH calculation was performed on the minimized
complex, as described elsewhere.25,26
In this method, the binding free energy of the protein−ligand
complex (ΔGbind) is obtained from the difference between the
free energies of the protein−ligand complex (ΔGcpx), the
unbound receptor (△Grec), and the free ligand (△Glig) as
follows:
molecule and surrounding solvent were set to 1 and 80, and
the probe radius of the solvent was set to 1.4 Å.
The entropy contribution to the binding free energy can be
divided into two parts: the solvation free entropy (ΔSsolv) and
the conformational free entropy (ΔSconf).
ΔSsol = ΔSsolv + ΔSconf
(7)
The solvation free entropy is gained by the tendency of water
molecules to minimize their contacts with hydrophobic groups
in protein. The conformational free entropy is related to the
change of the number of rotatable bonds during the binding
process. The detailed computational procedure used to evaluate
the entropy contribution (−TΔS) to the binding free energy
was as previously described.26
ΔGbind = △Gcpx − △Grec − △Glig
(2)
The binding free energy (ΔGbind) can be evaluated as the
sum of the changes in the molecular mechanical (MM) gas-
phase binding energy (ΔEMM), solvation free energy (ΔGsol),
and entropic contribution (−TΔS). The ΔEMM was evaluated
as a sum of electrostatic energy (ΔEele) and van der Waals
interaction energy (ΔEvdw).
RESULTS AND DISCUSSION
■
Synthetic Chemistry of the Title Compounds. As
shown in Scheme 1, the target compounds 4a ∼ z were
smoothly prepared by a three-step synthetic route using
bromide compound as starting materials. Compound 2,
resulting from the reaction of bromide compound with
acetonitrile, underwent an elimination reaction with substituent
ester to afford the key intermediates β-ketonitriles (3). By
performing the cyclization reaction with 1,2,4-triazol-3-amine,
compound 3 was successfully transformed into the correspond-
ing substituted 1,2,4-triazolo[1,5-a]pyrimidine derivates 4a ∼ z.
The structures of all synthesized compounds were
characterized by element analyses, 1H NMR, and GC-MS
techniques. The crystal structure of ametoctradin was
determined by X-ray diffraction analysis (CCDC 981965). As
shown in Figure 2, the crystal structure showed that
ametoctradin had an extended conformation.
ΔGbind = ΔEMM + ΔGsol − TΔS
(3)
ΔEMM = ΔEele + ΔEvdw
(4)
The solvation free energy ΔGsol was composed of two parts:
ΔGsol = ΔGPB + ΔGnp
(5)
ΔGnp = γSASA + β
(6)
The electrostatic contribution to the solvation free energy
(ΔGPB) is evaluated by Poisson−Boltzmann (PB) methods.
The nonpolar solvation energy, ΔGnp, can be estimated by an
empirical relation of ΔGnp = γSASA + β, where SASA and is
defined as the solvent-accessible surface area, and the solvation
parameters γ and β were set to 0.00542 kcal·mol−1 Å−2 and 0.92
kcal·mol−1, respectively. The dielectric constant for the
Inhibition Effect against bc1 Complex of Compounds
4a ∼ z. 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.27 Complex II (also
E
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Table 1. Percent Inhibition and IC50 Value of the Title Compounds against Porcine SCR
no.
R1
R2
% inhibition (100 μM)
IC50 (μM)
a
a
a
4a
CH3(CH2)6CH2
CH3(CH2)6CH2
CH3(CH2)6CH2
4-CH3−Ph
3-CF3−Ph
3-Cl-Ph
82%
0.12 0.01
36.33 1.23
0.15 0.01
4b
75.5%
77.6%
24.1%
<10%
12.6%
68.9%
23.0%
72.5%
29.3%
57.5%
16.7%
84.1%
48.2%
94.3%
65.0%
92.8%
<10%
16.3%
12.3%
<10%
36.7%
14.2%
70.0%
76.4%
37.9%
100%
4c
a
b
4d
CH3CH2CH2
CH3CH2
Ph
/
4e
PhCH2
/
4f
PhCH2
CF3CH2CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
PhCH2
/
4g
4-Br-PhCH2
4-F-PhCH2
3-Br-PhCH2
2-F-PhCH2
2-Br-PhCH2
3-F-PhCH2
3-I-PhCH2
2-Cl-PhCH2
24.89 1.32
4h
/
4i
26.82 1.44
4j
/
4k
82.62 1.35
4l
/
4m
10.90 1.27
4n
/
4o
4-CF3O-PhCH2
3-Cl-PhCH2
5.56 1.13
4p
42.27 1.24
4q
34-Cl2−PhCH2
4.00 1.14
a
4r
Ph
/
a
4s
4-F-Ph
PhCH2
/
a
4t
4-Cl-Ph
PhCH2
/
4u
PhCH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CH3CH2
CF3
/
a
4v
Ph
/
a
4w
4-F-Ph
/
a
4x
4-Cl-Ph
33.20 1.30
25.14 1.26
/
a
4y
4z
4-Br-Ph
a
Ph
a
ametoctradin
CH3(CH2)6CH2
CH3CH2
0.30 0.02
a
b
Meaning that the substituted 2 could be purchased. Not tested.
known as succinate-ubiquinone oxidoreductase, SQR) first
passes electrons from succinate to ubiquinone, and then the
cytochrome bc1 complex passes electrons from reduced
ubiquinone to cytochrome c1. The activity of complex II in
SCR was selectively determined using succinate and dichlor-
ophenolindophenol (DCIP) as substrates, and the activity of
only the cytochrome bc1 complex in SCR was determined using
decylubiquinol (DBH2) and cytochrome c1 as substrates,
whereas the overall activity of SCR (both complex II and bc1
complex) was determined using succinate and cytochrome c1 as
substrates.28,29
inhibitors, which would lead to different binding free energy of
a ligand toward the protein and would be discussed in the
following section.
Binding Mode of Ametoctradin. Although ametoctradin
has been known as inhibitor of cytochrome bc1 complex for
many years, its detailed binding mode is still unknown so far.
Many existing studies have shown that molecular docking is a
very powerful technique to predict the binding mode of
ligands;30−34 therefore, the AutoDock4.2 program was
integrated with the molecular dynamic simulations and MM/
PBSA calculation to uncover the detailed binding mode of
ametoctradin.
The results indicated that all of the compounds exhibited no
effect on the activity of complex II (data not shown) but
markedly inhibited the activities of the bc1 complex and SCR.
As shown in Table 1, some compounds showed very high
percent inhibition, such as 4a (82%), 4q (92.8%), 4o (94.3%),
4m (84.1%), against porcine SCR. To reduce the time and
workload of the biological assay, the IC50 values of some
compounds were not determined when its percent inhibition
was less than 50%. Among the 26 newly synthesized
compounds, 4a was identified as the most potent inhibitor
with a IC50 value of 0.12 μM against SCR from porcine heart
mitochondria (Table 1), which indicated that the potency of 4a
against porcine SCR is nearly twice as much as ametoctradin
(0.3 μM). In addition, when R1 was −CH2(CH2)6CH3 and or
substituent −CH2Ph, the inhibitor always displayed much
higher potency against porcine SCR, such as compound 4a, 4c,
4m, 4o, and 4q. Remarkably, 2-substituent and or 3-substituent
benzyl derivatives in R1 displayed lower antifungal activity
compared with the 4-substituent. These results indicated that
the substituent on R1 will affect the bioactive conformation of
Before docking, sequence alignment of cytochrome bc1
complex from different species was performed (the data were
included in the Supporting Information). The bovine
cytochrome b (1PPJ)35 showed about 50.92% sequence
identity with yeast (3CX5),36 74.67% with chicken (1BCC),11
49.08% with Rhodobacter capsulates (1ZRT),37 and 48.02% with
Paracoccus denitrificans (2YIU).38 In addition, the residues
around the Qo-site region are highly conserved. Up to now,
most crystal structures of inhibitor-bound bc1 complex are from
vertebrate heart, such as bovine and chicken. Previously, we
used the bovine bc1 complex (PDB ID: 1PPJ) as the receptor
model to study the binding mode of some new Qo-site
inhibitors, which successfully guided the design of a series of
new subnanomolar, even picomolar, Qo site inhibitors of bc1
complex.39,40 Hence, the bovine PDB crystal structure (1PPJ)
was selected herein as receptor model.
As mentioned before, there are two binding sites in the
cytochromore bc1 complex, a quinone reduction site near the
negative side of the membrane (Qi site) and a quinol oxidation
F
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Figure 3. Binding energy of ametoctradin with bc1 complex in Qo (A) and Qi (B) site. The red and blue histogram means the right and wrong
binding mode, respectively. The figure was directly obtained from AutoDock cluster analysis with default parameters. Based on our knowledge, the
1,2,4-triazolo[1,5-a]pyrimidine ring in ametoctradin should locate into the Q0/i-site, and then this binding mode cluster was defined as right. On the
contrary, it was defined as wrong when 1,2,4-triazolo[1,5-a]pyrimidine ring pointed to other position in Qo/i-site, such as the entrance of Qo/i-site.
Table 2. Binding Free Energy of Ametoctradin in the Qo and Qi site (kcal/mol)
a
a
a
a
a
AutoDock binding energy
ΔEele
ΔEVDW
ΔEnp
ΔEpol
ΔHPB
ametoctradin-Qi
ametoctradin-Qo
−6.0
−7.0
−15.49
−18.27
−38.97
−46.23
−5.16
−5.10
38.50
45.15
−21.11
−24.46
a
Obtained from 10 ns MD simulation.
site close to the positive side of the membrane (Qo site). Then,
we, respectively, docked ametoctradin into the Qo and Qi sites,
and obtained two ametoctradin-bound complexes, ametoctra-
din−Qo and ametoctradin−Qi complex (Figure 1S, Supporting
Information). As shown in Figure 3, the Qo site binding energy
of ametoctradin (about −7.0 kcal/mol) was more favorable
than the Qi site binding energy (about −6.0 kcal/mol), showing
that ametoctradin might be a Qo site inhibitor. Then, we
performed 10 ns molecular dynamics (MD) simulations for
ametoctradin−Qo and ametoctradin−Qi complex (Figure 2S,
Supporting Information), and the 100 snapshots obtained from
the last 1 ns of each complex were used to calculate the binding
free energy using MM/PBSA module. As shown in Table 2, the
binding energies (ΔH) of ametoctradin−Qo and ametoctra-
din−Qi are −24.46 and −21.11 kcal/mol, respectively. It is
necessary to point out that, if we prolonged the simulation time
(e.g., 12 ns), ametoctradin would leave from the ametoctradin−
Qi complex, whereas the ametoctradin−Qo complex remains
very stable (data not shown). On the basis of these results, we
proposed that ametoctradin should bind into the Qo site of bc1
complex.41
It is very interesting to compare the MD-simulated
ametoctradin−Qo complex with the existing crystal structures
of Qo site inhibitor-bound bc1 complex. According to the
reported crystal structures of Qo site inhibitor-bound bc1
complex,4 the E271 and F274 would always form an H-bond
and hydrophobic interaction with the inhibitor, indicating that
both residues played an important role in the binding of Qo site
inhibitors.39,40,42 For example, stigmatellin forms an H-bond
with E271 and H161 and hydrophobic interaction with F274
(PDB code: 1PPJ)35 (Figure 4A). As shown in Figure 4B, the
amine group of ametoctradin also formed an H-bond with
E271, and the aliphatic chain of ametoctradin formed a
hydrophobic interaction with F274, F128, I146, L149, and
L294 when binding into the Qo site. As we knew, the long
Figure 4. (A) Binding mode of stigmatellin in the Qo site. The blue
line represents the H-bond distance between stigmatellin and residue.
(B) Binding mode of ametoctradin in the Qo site. The red line
represents the H-bond distance between ametoctradin and residue.
(C) The overlay of ametoctradin and stigmatellin in the Qo site. For
clarity, only the key residues were shown. The blue line represents the
H-bond distance between stigmatellin and the residue. (D) The
overlay of ametoctradin and 4a in the Qo site.
aliphatic substituent in antimycin and stigmatellin adopted an
extended conformation in the crystal complex structure (PDB
code: 1PPJ). Therefore, it was reasonable that the octyl group
in ametoctradin also took on an extended conformation, and
G
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then 1,2,4-triazolo[1,5-a]pyrimidine ring in ametoctradin
formed a hydrophobic interaction with P270. However, the
distance between ametoctradin and H161 is 4.0 Å, which could
account for the reason why ametoctradin (IC50 = 0.3 μM) has
lower binding affinity than stigmatellin (IC50 = 2.4 nM for bc1
complex from bovine heart) (Figure 4C,D).43,44
distance between Gly143 in wide type bc1 complex and
ametoctradin was about 4.2 Å, while the closest distance
between A143 in mutant bc1 complex and ametoctradin
decreased to 3.7 Å (Figure 5B). As a result, the binding free
energy of ametoctradin upon G143A mutant and wild type bc1
complex were −25.61 kcal/mol and −21.68 kcal/mol,
respectively, which means G143A mutation increased the
binding affinity of ametoctradin. These results showed that the
established binding mode of ametoctradin should be
reasonable.
Binding-Free-Energy Calculation. In order to further
confirm the binding mode of ametoctradin and understand the
structure−activity relationships of ametoctradin analogues at
the atomic level, we carried out molecular docking for 12 newly
synthesized analogues with determined IC50 values and
calculated their binding free energies using MM/PBSA method
for the resulted analogue−Qo and analogue−Qi complex (Table
3), respectively.
As we know,45 the G143A mutation showed a nearly killing
effect upon all of strobilurin inhibitors, whereas ametoctradin
did not show cross-resistance to strobilurin fungicides.16,17 The
resistance mechanism for G143A mutation was that residue
A143 would create steric hindrance between the methoxyacry-
lamide moiety of inhibitor and cytochrome b.44,46 Schiffer et
al.47,48 had pointed out that the residue mutation would lead to
less effect upon ligand activity with longer distance between
ligand and residue. Therefore, we monitored the distance
between ametoctradin with G143. As shown in Figure 5A, the
For the analogue−Qo complex, the correlation coefficients
(R2) between the experimental binding free energies (ΔGexp
,
derived from the experimental binding affinities) and the
calculated energy terms are 0.20 for enthalpy contribution
(ΔH) and 0.64 for entropy compensation (−TΔS). Although
these individual correlations are very low, the overall calculated
binding free energies (ΔGcal) correlated very well (R2 = 0.96)
with the experimental binding free energies (ΔGexp) (Figure 6).
Figure 5. (A) Binding mode overlay between ametoctradin (yellow
sticks) and azoxystrobin (PDB code 1SQB, green sticks). (B) The
binding mode overlay of ametoctradin with wide type bc1 complex
(yellow stick) and G143A mutation (magenta stick). For clarity, only
Gly143 or Ala143 was shown as a stick.
closest distance between Gly143 and ametoctradin was about
4.2 Å, while it decreased to 3.6 Å with azoxystrobin (1SQB).
The binding free energy of azoxystrobin upon G143A mutant
and wild type bc1 complex were, respectively, −23.59 kcal/mol
and −26.74 kcal/mol (IC50 = 21 nM toward wild type bc1
complex from yeast; IC50 = 1394 nM toward G143A mutant bc1
form yeast44), which accounted for the resistance mechanism of
G143A mutation to azoxystrobin. On the contrary, the closest
Figure 6. Correlation between the ΔGcal and ΔGexp
.
Table 3. Individual Energy Terms of the Calculated Free Energy of Title Compounds (kcal/mol)
Qo
Qi
a
no.
ΔGexp
ΔH
−TΔS
ΔGcal
ΔH
−TΔS
ΔGcal
4a
−31.98
−28.24
−31.15
−31.94
−30.45
−29.76
−33.06
−35.45
−29.97
−30.67
−29.59
−30.34
−32.94
0.20
8.59
11.67
9.10
−23.39
−16.57
−22.05
−17.06
−16.58
−14.69
−18.69
−19.58
−15.91
−18.66
−16.23
−16.70
−21.68
0.96
−30.20
−29.31
−30.38
−23.97
−23.26
−24.76
−22.80
−30.73
−21.63
−21.94
−25.88
−26.78
−29.32
0.06
8.05
11.35
8.37
5.83
4.00
4.03
3.83
10.93
5.60
5.66
7.91
10.69
9.27
0.21
−22.15
−17.96
−22.01
−18.14
−19.26
−20.73
−18.97
−19.80
−16.03
−16.28
−17.97
−18.14
−20.05
−9.41
−6.03
−9.27
−6.26
−6.21
−5.55
−6.74
−7.10
−5.94
−7.34
−6.09
−6.25
−8.86
1.00
4b
4c
4g
14.88
13.87
15.07
14.37
15.89
14.06
12.01
13.36
13.64
11.26
0.64
4i
4k
4m
4o
4p
4q
4x
4y
ametoctradin
b
R2
0.0047
a
b
ΔGexp = −RT × lnIC50. The correlation between ΔGexp and the individual energy terms of the calculated free energy
H
DOI: 10.1021/acs.jafc.5b00228
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
However, for the analogue−Qi complex, when the compounds
were docked into the Qi site, the corresponding R2 values
between ΔGexp and the calculated energy terms were decreased
to 0.06 for ΔH, 0.21 for −TΔS, and 0.0047 for ΔGcal,
respectively. Therefore, these molecular docking and binding
free energy calculation results further confirmed that
ametoctradin and its analogues should binding to the Qo site
of bc1 complex.
describes the RMSD change during MD simulations for
ametoctradin-Qo/Qi. Table 1S shows the individual energy
terms of the calculated free energy of title compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: gfyang@mail.ccnu.edu.cn. Tel.: 86-27-67867800. Fax:
86-27-67867141.
■
It is very interesting to analyze the contribution of the
individual energy terms. Although the enthalpy contribution
(ΔH) always reflects the direct interaction between ligand and
receptor, and plays an important role in the binding of ligand,
the binding free energy arises from a more complex interplay
between the enthalpy (ΔH) and entropy (−TΔS) terms. For
the composition of enthalpy (ΔH) term, the ΔEVDW (ranging
from −43.83 to 49.27 kcal/mol) and the ΔEpol (ranging from
31.25 to 45.10 kcal/mol) are two largest components of the
binding free energy for each complex (Table 1S, Supporting
Funding
The research was supported in part by the National Key
Technologies R&D Program (2011BAE06B05) and the
National Natural Science Foundation of China (No.
21472065).
Notes
The authors declare no competing financial interest.
Information). For the analogues bearing R1
=
REFERENCES
■
−CH2(CH2)6CH3, their entropy term(−TΔS, ranging from
8.59 to 11.67 kcal/mol) is lower than that of other compounds,
but their binding affinity is higher than that of other
(1) Kim, H.; Xia, D.; Yu, C. A.; Xia, J. Z.; Kachurin, A. M.; Zhang, L.;
Yu, L.; Deisenhofer, J. Inhibitor binding changes domain mobility in
the iron-sulfur protein of the mitochondrial bc1 complex from bovine
heart. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 8026−8033.
(2) Gao, X. G.; Wen, X. L.; Yu, G. A.; Esser, L.; Tsao, S.; Quinn, B.;
Zhang, L.; Yu, L. D.; Xia, D. The crystal structure of mitochondrial
cytochrome bc1 in complex with famoxadone: the role of aromatic-
aromatic interaction in inhibition. Biochemistry. 2002, 41, 11692−
11702.
compounds, such as 4a (IC50 = 0.12 μM) and 4c (IC50
=
0.15 μM). These result indicated that the R1 group has an
important role in the entropy compensation, which is a critical
aspect for the binding of ametoctradin and its analogues.
The mode of ametoctradin−Qo complex is shown in Figure
4B. As shown in Figure 4B, the octyl (R1 group) of
ametoctradin pointed to the outside of the Qo pocket, whereas
its ethyl (R2) group inserted inside the Qo pocket. As a result,
when R2 was a bulky group, such as substituted phenyl (4-
CH3−Ph, 3-CF3−Ph, and 3-Cl-Ph), the compound (4a, 4b, and
4c) would move up about 3.0 Å compared to ametoctradin
(Figure 4D) due to steric clash. It should be pointed out that
this movement will lead to an entropy compensation, which is
favorable to the binding of inhibitors.
(3) Berry, E. A.; Huang, L. S. Observations concerning the quinol
oxidation site of the cytochrome bc1 complex. FEBS Lett. 2003, 555,
13−20.
(4) Esser, L.; Quinn, B.; Li, Y. F.; Zhang, M. Q.; Elberry, M.; Yu, L.
D.; Yu, C. A.; Xia, D. Crystallographic studies of quinol oxidation site
inhibitors: a modified classification of inhibitors for the cytochrome bc1
complex. J. Mol. Biol. 2004, 341, 281−302.
(5) Wang, F.; Hui, L.; Wang, L.; Yang, W. C.; Wu, J. W.; Yang, G. F.
Design, syntheses, and kinetic evaluation of 3-(phenylamino)-
oxazolidine-2,4-diones as potent cytochrome bc1 complex inhibitors.
Bioorg. Med. Chem. 2011, 19, 4608−4615.
(6) Berry, E. A.; Huang, L. S. Conformationally linked interaction in
the cytochrome bc1 complex between inhibitors of the Qo site and the
Rieske iron-sulfur protein. Biochim. Biophys. Acta 2011, 1807, 1349−
1363.
(7) Huang, L. S.; Shen, J. T.; Wang, A. C.; Berry, E. A.
Crystallographic studies of the binding of ligands to the dicarboxylate
site of Complex II, and the identity of the ligand in the ″oxaloacetate-
inhibited″ state. Biochim. Biophys. Acta 2006, 1757, 1073−1083.
(8) Mitchell, P. Possible molecular mechanisms of the protonmotive
function of cytochrome systems. J. Theor. Biol. 1976, 62, 267−327.
(9) Berry, E. A.; Huang, L. S.; Lee, D. W.; Daldal, F.; Nagai, K.;
Minagawa, N. Ascochlorin is a novel, specific inhibitor of the
mitochondrial cytochrome bc1 complex. Biochim. Biophys. Acta 2010,
1797, 360−370.
(10) Gutierrez-Cirlos, E. B.; Merbitz-Zahradnik, T.; Trumpower, B.
L. Inhibition of the yeast cytochrome bc1 complex by ilicicolin H, a
novel inhibitor that acts at the QN site of the bc1 complex. J. Biol. Chem.
2004, 279, 8708−8714.
(11) Zhang, Z.; Huang, L.; Shulmeister, V. M.; Chi, Y. I.; Kim, K. K.;
Hung, L. W.; Crofts, A. R.; Berry, E. A.; Kim, S. H. Electron transfer by
domain movement in cytochrome bc1. Nature. 1998, 392, 677−684.
(12) Iwata, S.; Lee, J. W.; Okada, K.; Lee, J. K.; Iwata, M.; Rasmussen,
B.; Link, T. A.; Ramaswamy, S.; Jap, B. K. Complete structure of the
11-subunit bovine mitochondrial cytochrome bc1 complex. Science.
1998, 281, 64−71.
(13) Esser, L.; Yu, C. A.; Xia, D. Structural Basis of Resistance to
Anti-Cytochrome bc1 complex Inhibitors: Implication for Drug
Improvement. Curr. Pharm. Design. 2014, 20, 704−724.
In summary, a series of novel 1,2,4-triazolo[1,5-a]pyrimidine
derivates 4a ∼ z were designed and synthesized using
ametoctradin as a lead compound. The in vitro assay indicated
that compound 4a, 6-octyl-5-p-tolyl-1,2,4-triazolo[1,5-a]-
pyrimidine-7-amine, is a new inhibitor of bc1 complex with
higher binding affinity (IC50 = 0.12 μM) against porcine bc1
complex than ametoctradin (IC50 = 0.30 μM). In addition, the
results of molecular docking, molecular dynamics simulations,
and MM/PBSA calculations strongly supported that ametoc-
tradin and its analogues should bind to the Qo site rather than
the Qi site of cytochrome bc1 complex. Inside the Qo pocket,
ametoctradin and its analogues formed an H-bond with E721
and hydrophobic interactions with F274, F128, I146, L149, and
L294.
On the basis of the results obtained from the present study,
the following guidance for the future inhibitor design could be
proposed: (1) 1,2,4-triazolo[1,5-a]pyrimidine should be
substituted to form an H-bond with H161, a key residue for
the binding of inhibitor; (2) Introduction of a bulky and
hydrophobic group onto R2 position is favorable to the binding
due to their contribution to the entropy compensation.
ASSOCIATED CONTENT
* Supporting Information
■
S
Supplemental cluster results showed the sequence alignment of
bc1 complex from different species. Figure 1S describes the
binding mode of ametoctradin in the Qi site, and Figure 2S
I
DOI: 10.1021/acs.jafc.5b00228
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
Article
(14) Berry, E. A.; Guergova-Kuras, M.; Huang, L.; Crofts, A. R.
Structure and function of cytochrome bc1 complexes. Annu. Rev.
Biochem. 2000, 69, 1005−1075.
Deletion in Protoporphrinogen Oxidase through Computational
Modeling. J. Phys. Chem. B 2009, 113, 4865−4875.
(33) Hao, G. F.; Yang, G. F.; Zhan, C. G. Computational Mutation
Scanning and Drug Resistance Mechanisms of HIV-1 Protease
Inhibitors. J. Phys. Chem. B 2010, 114, 9663−9676.
(15) Gao, X.; Wen, X.; Esser, L.; Quinn, B.; Yu, L.; Yu, C. A.; Xia, D.
Structural basis for the quinone reduction in the bc1 complex: a
comparative analysis of crystal structures of mitochondrial cytochrome
bc1 with bound substrate and inhibitors at the Qi site. Biochemistry.
2003, 42, 9067−9080.
(34) Chen, H.; Zhang, Y.; Ye, C.; Feng, T. T.; Han, J. G. Insight into
the binding modes and inhibition mechanisms of adamantyl-based 1,3-
disubstituted urea inhibitors in the active site of the human soluble
epoxide hydrolase. J. Biomol. Struct. Dyn. 2014, 32, 1231−1247.
(35) Huang, L. S.; Cobessi, D.; Tung, E. Y.; Berry, E. A. Binding of
the Respiratory Chain Inhibitor Antimycin to the Mitochondrial bc1
Complex: A New Crystal Structure Reveals an Altered Intramolecular
Hydrogen-bonding Pattern. J. Mol. Biol. 2005, 351, 573−597.
(36) Solmaz, S. R.; Hunte, C. Structure of complex III with bound
cytochrome c in reduced state and definition of a minimal core
interface for electron transfer. J. Biol. Chem. 2008, 283, 17542−17549.
(37) Berry, E. A.; Huang, L. S.; Saechao, L. K.; Pon, N. G.; Valkova-
Valchanova, M.; Daldal, F. X-Ray Structure of Rhodobacter Capsulatus
Cytochrome bc1: Comparison with its Mitochondrial and Chloroplast
Counterparts. Photosynth. Res. 2004, 81, 251−275.
(38) Kleinschroth, T.; Castellani, M.; Trinh, C. H.; Morgner, N.;
Brutschy, B.; Ludwig, B.; Hunte, C. X-ray structure of the dimeric
cytochrome bc1 complex from the soil bacterium Paracoccus
denitrificans at 2.7-Å resolution. Biochim. Biophys. Acta 2011, 1807,
1606−1615.
(39) 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. Computational Discovery of
Picomolar Qo Site Inhibitors of Cytochrome bc1 Complex. J. Am.
Chem. Soc. 2012, 134, 11168−11176.
(40) Zhao, P. L.; Wang, L.; Zhu, X. L.; Huang, X.; Zhan, C. G.; Wu, J.
W.; Yang, G. F. Subnanomolar Inhibitor of Cytochrome bc1 Complex
Designed by Optimizing Interaction with Conformationally Flexible
Residues. J. Am. Chem. Soc. 2010, 132, 185−194.
(41) Zhu, X. L.; Zhang, M. M.; Yang, G. F. Synthesis and fungicidal
activity of novel ametoctradin derivates, 248th American Chemical
Society National Meeting & Exposition, San Francisco, California,
August 10−14, 2014 (http://abstracts.acs.org/chem/248nm/
program/view.php?obj_id=261551&terms).
(42) Xiao, Y. M.; Esser, L.; Zhou, F.; Li, C.; Zhou, L. H.; Yu, C. A.;
Qin, Z. H.; Xia, D. Studies on Inhibition of Respiratory Cytochrome
bc1 Complex by the Fungicide Pyrimorph Suggest a Novel Inhibitory
Mechanism. PLoS One 2014, 9, e93765.
(43) Biagini, G. A.; Fisher, N.; Berry, N.; Stocks, P. A.; Meunier, B.;
Williams, D. P.; Bonar-Law, R.; Bray, P. G.; Owen, A.; O’Neill, P. M.;
Ward, S. A. Acridinediones: Selective and Potent Inhibitors of the
Malaria Parasite Mitochondrial bc1 Complex. Mol. Pharmacol. 2008,
73, 1347−55.
́
(16) Gold, R.; Scherer, M.; Rether, J.; Speakman, J.; Nave, B.; Levy,
T.; Storer, R.; Marris. D. Initium® -An Innovative Fungicide of a New
Chemical Class for the Control of Oomycetes, Proceedings of BCPC
Congress, Glasgow, U.K., November 9−11, 2009.
(17) Chai, B. S.; Fu, C. X.; Sun, X. F.; Li, Q.; Li, Z. N.; Li, Z. N.
Synthesis of Ametoctradin and Its Bioactivity. Agrochemicals. 2012, 51,
645−674.
(18) Grammenos, W.; Gewehr, M.; Mueller, B.; Blettner, C.; Blasco,
J. T. I. 5, 6-Dialkyl-7-Aminotriazolopyrimidines, their Preparation and
their Use for Controlling Harmful Fungi, and Compositions
Comprising these Compounds. U.S. Patent No. US20080119493,
2005.
(19) Dietz, J.; Grotz, T.; Grammenos, W.; Muller, B.; Lohmann, J. K.;
Renner, J.; Ulmschneider, S.; Tormo, I. B. J. Use of Substituted
Triazolopyrimidines for Controlling Phytopathogenic Harmful Fungi.
Patent No. WO 2007113136, 2007.
(20) Vogelbacher, U. J.; Otto, P.; Rack, M. Method for the
production of beta-ketonitriles. U.S. Patent No. US20100105903,
2008.
(21) King, T. E. Preparations of succinate-cyto-chrome reductase and
the cytochrome bc1 particle, and reconstitution of succinate-
cytochrome c reductase. Methods Enzymol. 1967, 10, 216−225.
(22) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew,
R. K.; Goodsell, D. S.; Olson, A. J. Autodock4 and AutoDockTools4:
automated docking with selective receptor flexiblity. J. Comput. Chem.
2009, 30, 2785−2791.
(23) Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart,
W. E.; Belew, R. K.; Olson, A. J. Automated docking using a
lamarckian genetic algorithm and an empirical binding free energy
function. J. Comput. Chem. 1998, 19, 1639−1662.
(24) Sitkoff, D.; Sharp, K. A.; Honig, B. Accurate calculation of
hydration free energies using macroscopic solvent models. J. Phys.
Chem. 1994, 98, 1978−1988.
(25) Connolly, M. L. Analytical molecular surface calculation. J. Appl.
Crystallogr. 1983, 16, 548−558.
(26) Pan, Y. M.; Gao, D. Q.; Zhan, C. G. Modeling the catalysis of
anti-cocaine catalytic antibody: competing reaction pathways and free
energy barrier. J. Am. Chem. Soc. 2008, 130, 5140−5149.
(27) Chen, J.; Chen, C. L.; Alevriadou, B. R.; Zweier, J. L.; Chen, Y.
R. Excess NO predisposes mitochondrial succinate−cytochrome c
reductase to produce hydroxyl radical. Biochim. Biophys. Acta 2011,
1807, 491−502.
(28) Zhu, X. L.; Xiong, L.; Li, H.; Song, X. Y.; Liu, J. J.; Yang, G. F.
Computational and Experimental Insight into the Molecular
Mechanism of Carboxamide Inhibitors of Succinate-Ubquinone
Oxidoreductase. ChemMedChem. 2014, 9, 1512−1521.
(29) Li, H.; Zhu, X. L.; Yang, W. C.; Yang, G. F. Comparative
Kinetics of Qi Site Inhibitors of Cytochrome bc1 Complex: Picomolar
Antimycin and Micromolar Cyazofamid. Chem. Biol. Drug Des. 2013,
83, 71−80.
(30) Zhu, X. L.; Yang, W. C.; Yu, N. X.; Yang, S. G.; Yang, G. F.
Computational Simulations of Structural Role of the Active-site
W374C Mutation of Acetyl-Coenzyme-A Carboxylase: Multi-drug
Resistance Mechanism. J. Mol. Model. 2011, 17, 495−503.
(31) Zhu, X. L.; Hao, G. F.; Zhan, C. G.; Yang, G. F. Computational
Simulations of the Interactions between Acetyl-Coenzyme-A Carbox-
ylase and Clodinafop: Resistance Mechanism Due to Active and
Nonactive Site Mutations. J. Chem. Inf. Model. 2009, 49, 1936−1943.
(32) Hao, G. F.; Zhu, X. L.; Ji, F. Q.; Zhang, L.; Yang, G. F.; Zhan, C.
G. Understanding Mechanism of Drug Resistance Due to a Codon
(44) Fisher, N.; Meunier, B. Re-examination of inhibitor resistance
conferred by Qo-site mutations in cytochrome b using yeast as a model
system. Pest Manag. Sci. 2005, 61, 973−978.
(45) Mutations associated with Qo-I-resistance (2014). http://www.
frac.info.
(46) Gisi, U.; Sierotzki, H.; Cook, A.; McCaffery, A. Mechanisms
influencing the evolution of resistance to Qo inhibitor fungicides. Pest
Manag. Sci. 2002, 58, 859−867.
̈
(47) Ozen, A.; Haliloglu, T.; Schiffer, C. A. HIV-1 Protease and
Substrate Coevolution Validates the Substrate Envelope as the
Substrate Recognition Pattern. J. Chem. Theory Comput. 2012, 8,
703−714.
(48) Altman, M. D.; Ali, A.; Reddy, G. S.; Nalam, M. N.; Anjum, S.
G.; Cao, H.; Chellappan, S.; Kairys, V.; Fernandes, M. X.; Gilson, M.
K.; Schiffer, C. A.; Rana, T. M.; Tidor, B. HIV-1 protease inhibitors
from inverse design in the substrate envelope exhibit subnanomolar
binding to drug-resistant variants. J. Am. Chem. Soc. 2008, 130, 6099−
6113.
J
DOI: 10.1021/acs.jafc.5b00228
J. Agric. Food Chem. XXXX, XXX, XXX−XXX