Synthesis and herbicidal evaluation of triketone-containing quinazoline-2,4-diones

Article abstract of DOI:10.1021/jf5048089

Exploring novel 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) inhibitors is one of the most promising research directions in herbicide discovery. To discover new triketone herbicides with broad-spectrum weed control as well as excellent crop selectivity, a series of (total 52) novel triketone-containing quinazoline-2,4-dione derivatives were synthesized and further bioevaluated. The greenhouse testing indicated that many of the newly synthesized compounds showed better or excellent herbicidal activity against broadleaf and monocotyledonous weeds at the dosages of 37.5-150 g of active ingredient (ai)/ha. The structure and activity relationship in this study indicated that the triketone-containing quinazoline-2,4-dione motif has possessed great impact on herbicide activity and may be used for further optimization. Among the new compounds, III-b and VI-a-VI-d displayed a broader spectrum of weed control than mesotrione. In addition, the compound III-b also demonstrated comparatively superior crop selectivity to mesotrione, thus possessing great potential for weed control in the field.

Full text of DOI:10.1021/jf5048089

Article
pubs.acs.org/JAFC
Synthesis and Herbicidal Evaluation of Triketone-Containing
Quinazoline-2,4-diones
Da-Wei Wang,† Hong-Yan Lin,† Run-Jie Cao,† Sheng-Gang Yang,† Qiong Chen,† Ge-Fei Hao,†
Wen-Chao Yang,*,† and Guang-Fu Yang*,†,‡
†Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal
University, Wuhan, Hubei 430079, People’s Republic of China
‡Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, People’s Republic of China
S
* Supporting Information
ABSTRACT: Exploring novel 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) inhibitors is one of the most
promising research directions in herbicide discovery. To discover new triketone herbicides with broad-spectrum weed control as
well as excellent crop selectivity, a series of (total 52) novel triketone-containing quinazoline-2,4-dione derivatives were
synthesized and further bioevaluated. The greenhouse testing indicated that many of the newly synthesized compounds showed
better or excellent herbicidal activity against broadleaf and monocotyledonous weeds at the dosages of 37.5−150 g of active
ingredient (ai)/ha. The structure and activity relationship in this study indicated that the triketone-containing quinazoline-2,4-
dione motif has possessed great impact on herbicide activity and may be used for further optimization. Among the new
compounds, III-b and VI-a−VI-d displayed a broader spectrum of weed control than mesotrione. In addition, the compound III-
b also demonstrated comparatively superior crop selectivity to mesotrione, thus possessing great potential for weed control in the
field.
KEYWORDS: 4-hydroxyphenylpyruvate dioxygenase, crop selectivity, herbicidal activity, quinazoline-2,4-dione, synthesis
well as some annual monocotyledon weeds. More importantly,
mesotrione could control some of the resistant biotypes, for
example, glyphostate-resistant, acetyl coenzyme carboxylase
(ACCase) and acetohydroxy acid synthase (AHAS) resistant
weeds.14 In 2009, the market value of mesotrione was about
$485 million, and now, it is one of the top five best-selling
herbicides worldwide. However, there are still some limitations
of mesotrione, such as toxicity to wheat, soybean, rape, cotton,
and other crops, because of its poor selectivity. In addition,
some grass weeds, for example, Setaria faberii, are not sensitive
to mesotrione. Therefore, it is necessary to discover a novel
triketone herbicide with improved crop selectivity and broader
spectrum of weed control (especially against grass weeds).
In our previous work,15 we have designed and synthesized a
series of novel triketone-containing quinazoline-2,4-dione
derivatives with a variety of substitutes at the N-1 position of
the quinazoline-2,4-dione ring. According to our data of
herbicidal activity and crop selectivity, the methyl group turned
to be the optimum substitute at the N-1 position. Moreover,
the lead compound I-f (Table 1) not only showed a broad
spectrum of weed control but also possessed better selectivity
for maize and wheat at the dosage of 150 g of active ingredient
(ai)/ha. In this ongoing study, we performed thorough
modification on the triketone-containing quinazoline-2,4-
dione motif and synthesized a series of novel triketone-
INTRODUCTION
■
In agrochemical research, the discovery of new herbicides with
broad-spectrum weed control and excellent crop selectivity is
still remaining as a challenge. 4-Hydroxyphenylpyruvate
dioxygenase (EC 1.13.11.27, HPPD), catalyzing the conversion
of 4-hydroxyphenyl pyruvic acid (HPPA) into homogentisic
acid (HGA), belongs to the family of non-heme Fe(II)-
containing enzymes.1,2 As an important enzyme in regulating
the biosynthesis of tocopherols and plastoquinone in plants,
HPPD is an important target for herbicide discovery. HPPD-
inhibiting-based herbicides can block photosynthesis, which
resulted in unique bleaching symptoms in sunlight and finally
caused necrosis and death of treated plants.3−6 HPPD
inhibitors exhibit many advantages, such as excellent crop
selectivity, low application rate, low toxicity, broad-spectrum
weed control, and benign environmental effects.7−11
Thus far, HPPD inhibitors that are widely used in the field as
herbicides can be classified into three categories: triketones,
pyrazoles (diketonitrile), and isoxazoles. Among the commer-
cialized HPPD herbicides, triketone derivatives are the most
deeply studied, owing to their structural diversity. The story of
discovering triketone herbicides can be traced back to the
1970s; scientists in California noticed that a few plants grew
under Callistemon citrinus, and the natural product leptosper-
mone was accordingly extracted.7,8 Further studies lead to the
discovery of the first triketone herbicide sulcotrione by Stauffer
in 1991. About 10 years later, the second triketone herbicide
mesotrione was successfully launched into the market, which is
more potent compared to sulcotrione.12,13 It can effectively
control all of the major broadleaved weeds in a corn field as
Received: October 4, 2014
Revised: November 17, 2014
Accepted: November 18, 2014
Published: November 18, 2014
© 2014 American Chemical Society
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Journal of Agricultural and Food Chemistry
Article
Table 1. Chemical Structures, Postemergence Herbicidal Activities (Inhibition Rating of 0−100) of Compounds I, and Their
a
Inhibitory Activities against AtHPPD
b
b
b
b
b
b
c
compound
R1
R2
dose (g of ai/ha)
EC
SF
DS
AR
EP
AJ
Ki (nM)
I-a
CH3
CH3
CH3
4-Cl
150
75
90
70
100
60
90
60
50
70
40
0
90
60
85
50
0
90
50
0
21
27
23
2
5
1
37.5
150
75
50
50
60
I-b
I-c
3-Cl
2-Cl
80
100
20
100
70
80
30
30
80
40
30
75
95
100
80
20
20
85
70
60
75
70
100
100
50
37.5
150
75
40
0
60
100
90
100
90
100
90
40
75
75
100
95
85
0
80
75
37.5
150
150
150
75
60
60
60
I-d
I-e
I-f
CH3
CH3
CH3
4-CH3
3-CH3
2-CH3
75
75
100
100
100
100
42
39
32
1
6
1
70
100
100
100
90
100
100
95
87.5
37.5
150
150
75
97.5
0
82.5
0
97.5
I-g
I-h
H
2-CH3
2-CH3
0
10
15
100
100
100
100
100
627 42
CH2CH3
100
100
100
100
100
100
100
100
5
1
97.5
60
60
50
30
0
100
90
95
80
50
90
30
20
0
100
95
37.5
150
75
97.5
I-i
CH2CH2CH3
2-CH3
87.5
80
70
50
0
90
87.5
65
20
10
10
10
50
10
20
0
97.5
15
2
87.5
70
35
0
37.5
150
150
150
150
150
150
150
75
87.5
I-j
CH2CCH
2-CH3
2-CH3
2-CH3
2-CH3
2-CH3
2-CH3
90
50
90
0
33
19
1
1
2
9
8
I-k
I-l
CH2CH2CH2CH3
CH2CH(CH3)2
CH2C6H5
0
0
0
20
0
17
I-m
I-n
I-o
0
0
355
139
CH2−2-F−C6H4
CH2−3-OCH3−C6H4
50
50
85
75
30
30
25
95
60
30
0
90
80
0
0
0
141 55
13
mesotrione
100
100
100
100
100
100
100
1
100
100
37.5
0
a
b
For the method for the preparation of compounds I, see ref 15. Abbreviations: EC, Echinochloa crus-galli; SF, Setaria faberii; DS, Digitaria
c
sanguinalis; AR, Amaranthus retroflexu; EP, Eclipta prostrata; AJ, Abutilon juncea. Inhibition constant of the enzymatic reaction.
Figure 1. Design of title compounds (II−VI).
containing quinazoline-2,4-dione derivatives possessing a
methyl group at the N-1 position of the quinazoline-2,4-
dione ring (Figure 1). The biological test indicated that most
newly synthesized compounds showed excellent herbicidal
activity against broadleaf and monocotyledonous weeds at the
dosages of 37.5−150 g of ai/ha. Notably, some compounds
showed significantly improved crop selectivity compared to
mesotrione. Herein, we have revealed the detailed synthesis,
herbicidal activity, and structure−activity relationship (SAR) of
compounds II−VI as potent herbicide candidates.
MATERIALS AND METHODS
■
The experimental details and analytical data for all of the intermediates
and compounds II−VI are shown in the Supporting Information.
X-ray Diffraction. Compound IV-d was recrystallized from a
mixture of chloroform and n-hexane to afford a suitable single crystal.
Light brown crystals of IV-d (0.12 × 0.10 × 0.10 mm) were mounted
on a quartz fiber with protection oil. Cell dimensions and intensities
were measured at 296 K on a Bruker SMART APEX DUO area
detector diffractometer with graphite monochromated Mo Kα
radiation (λ = 0.710 73 Å), with θmax = 25.49, 16 744 measured
reflections, and 4817 independent reflections (Rint = 0.0253). The data
sets were integrated and reduced using SAINT Plus Programme.16
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Journal of Agricultural and Food Chemistry
Article
Table 2. Chemical Structures, Postemergent Herbicidal Activity (Inhibition Rating of 0−100) of Compounds II, and Their
Inhibitory Activities against AtHPPD
a
a
a
a
a
a
compound
R2
dose (g of ai/ha)
EC
SF
DS
AR
EP
AJ
Ki (nM)
II-a
II-b
H
150
150
75
65
80
70
50
85
60
50
85
70
60
50
100
40
30
80
40
0
65
80
60
40
75
60
30
75
60
20
70
50
20
30
70
30
0
100
80
70
80
50
30
80
50
30
95
60
0
80
85
50
0
51
53
9
3
2-F
75
37.5
150
75
70
II-c
II-d
II-e
3-F
4-F
2-Br
85
100
30
0
28
29
27
4
1
2
80
37.5
150
75
70
100
20
20
90
100
70
100
40
20
100
90
65
85
100
65
50
37.5
150
75
60
87.5
75
100
100
100
95
82.5
70
60
50
82.5
37.5
150
150
75
45
75
45
II-f
3-Br
4-Br
85
16
17
1
4
II-g
85
85
100
100
100
100
100
100
55
60
100
95
37.5
150
75
30
45
II-h
2-OCH3
100
100
92.5
100
100
90
100
100
100
60
75
95
90
65
50
60
85
85
75
60
40
95
85
80
100
82.5
97.5
68
5
75
60
67.5
80
85
80
75
75
60
85
65
50
80
82.5
85
0
90
85
37.5
150
150
150
75
97.5
95
II-i
II-j
II-k
3-OCH3
4-OCH3
2-CF3
92.5
85
25
100
60
17
15
46
4
3
5
50
95
95
100
95
100
100
90
95
97.5
37.5
150
150
150
75
87.5
85
92.5
90
90
55
II-l
3-CF3
80
97.5
65
24
47
1
2
2
II-m
II-n
4-CF3
85
30
100
100
95
2-OCF3
85
80
97.5
95
77.5
70
80
50
37.5
150
150
150
75
50
90
30
II-o
II-p
II-q
4-OCF3
4-NO2
2-C2H5
77.5
50
50
95
50
12
18
29
1
4
1
10
90
60
85
82.5
90
100
100
90
87.5
80
95
90
37.5
150
75
87.5
97.5
87.5
50
0
60
II-r
2-iPr
80
90
80
60
50
70
50
70
65
55
20
82.5
60
50
60
97.5
29
3
82.5
70
92.5
75
55
80
95
85
80
65
80
70
30
70
95
60
30
95
80
60
37.5
150
150
150
75
90
II-s
II-t
II-u
2,6-di-iPr
2,6-di-Cl
60
20
100
85
35
106
47
3
2
5
80
40
80
2,6-di-CH3
100
100
100
100
100
100
100
66
92.5
87.5
82.5
85
100
100
37.5
150
150
75
97.5
II-v
2,4-di-Cl
92.5
87.5
100
85
13
53
3
1
II-w
2-CH3-5-Cl
60
80
55
80
20
0
90
100
100
90
85
65
37.5
150
150
75
70
30
II-x
3,5-di-Cl
80
100
100
100
100
17
13
2
1
mesotrione
85
100
100
100
100
75
100
100
37.5
30
0
a
Abbreviations: EC, Echinochloa crus-galli; SF, Setaria faberii; DS, Digitaria sanguinalis; AR, Amaranthus retroflexu; EP, Eclipta prostrata; AJ, Abutilon
juncea.
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Journal of Agricultural and Food Chemistry
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Table 3. Chemical Structures, Postemergent Herbicidal Activity (Inhibition Rating of 0−100) of Compounds III−V, and Their
Inhibitory Activities against AtHPPD
a
a
a
a
a
a
compound
R2
R3
dose (g of ai/ha)
EC
SF
DS
AR
EP
AJ
Ki (nM)
III-a
4-Br
5-CH3
150
75
80
70
50
40
30
100
95
90
45
100
50
40
80
50
30
95
50
40
35
50
50
30
30
50
60
60
40
10
50
80
75
50
95
85
80
80
90
80
95
90
95
90
80
95
92.5
85
80
90
70
50
85
70
60
90
50
40
82.5
92.5
87.5
80
25
1
37.5
150
75
60
65
82.5
100
III-b
2,4-di-Cl
5-CH3
100
100
95
100
95
100
15
4
95
90
100
100
100
100
60
37.5
150
150
75
92.5
85
IV-a
IV-b
H
5,5-di-CH3
5,5-di-CH3
77.5
90
70
50
85
50
40
80
50
40
75
70
20
0
95
54
20
5
3
2-F
90
100
70
50
37.5
150
75
50
60
40
IV-c
IV-d
3-F
4-F
5,5-di-CH3
5,5-di-CH3
95
85
85
56
5
9
50
70
30
37.5
150
75
50
60
30
95
100
60
100
50
100
30
37.5
150
150
75
30
50
40
IV-e
IV-f
2-Br
3-Br
5,5-di-CH3
5,5-di-CH3
95
95
100
100
100
100
100
50
245 21
85
100
100
100
100
95
100
29
3
65
100
100
37.5
150
150
150
150
150
150
150
150
150
150
150
75
20
IV-g
IV-h
IV-i
4-Br
5,5-di-CH3
5,5-di-CH3
5,5-di-CH3
5,5-di-CH3
5,5-di-CH3
5,5-di-CH3
5,5-di-CH3
5,5-di-CH3
5,5-di-CH3
5,5-di-CH3
5,5-di-CH3
60
85
60
80
50
40
65
0
92.5
85
87.5
24
4
2-OCH3
3-OCH3
4-OCH3
3-CF3
85
287 21
103 12
118 19
55
80
75
55
IV-j
87.5
82.5
60
90
67.5
80
100
IV-k
IV-l
90
92.5
48
42
24
58
23
1
9
4
5
3
4-CF3
80
55
60
100
100
100
IV-m
IV-n
IV-o
IV-p
IV-q
4-OCF3
4-NO2
90
95
80
25
80
80
2-CH2CH3
2-iPr
80
55
92.5
97.5
90
85
87.5
75
25
80
75
95
85
75
30
60
100
100
95
70
50
90
97.5
249 37
180 10
2,6-di-Cl
87.5
85
97.5
80
95
90
82.5
37.5
150
75
70
70
100
80
70
80
IV-r
2,6-di-CH3
5,5-di-CH3
50
87.5
60
100
85
262
8
87.5
82.5
75
37.5
150
150
150
150
150
75
82.5
30
40
85
45
20
0
75
50
80
IV-s
IV-t
IV-u
V
2,4-di-Cl
2-CH3-5-Cl
3,5-di-Cl
2,6-di-Cl
5,5-di-CH3
5,5-di-CH3
5,5-di-CH3
6,6-di-CH3
20
30
50
100
65
362 39
138 11
82.5
90
95
80
100
80
0
39
49
13
3
5
1
60
97.5
80
100
100
100
100
mesotrione
95
100
100
100
100
60
100
100
37.5
0
30
a
Abbreviations: EC, Echinochloa crus-galli; SF, Setaria faberii; DS, Digitaria sanguinalis; AR, Amaranthus retroflexu; EP, Eclipta prostrata; AJ, Abutilon
juncea.
Data were corrected for Lorentz and polarization effects and
absorption (Tmax = 0.9613, and Tmin = 0.9538). The structure was
solved by a direct method using SHELXS97 and refined with
SHELXL970.17 Full-matrix least-squares refinement based on F2 using
0.506 e Å−3. Hydrogen atoms were observed and placed at their ideal
positions with a fixed value of their isotropic displacement parameter.
Crystallographic data for compound IV-d has been deposited with
the Cambridge Crystallographic Data Centre as a supplementary
publication with the deposition number 977355. These data can be
obtained free of charge from http://www.ccdc.cam.ac.uk/.
Herbicidal Activities. The postemergent herbicidal activities of
compounds I−VI, against Echinochloa crus-galli (EC), Setaria faberii
(SF), Digitaria sanguinalis (DS), Amaranthus retroflexus (AR), Eclipta
2
the weight of 1/[σ2(F0 ) + (0.1322P)2 + 0.6124P] gave final values of
R1 = 0.0628, ωR2 = 0.2008, and GOF(F) = 1.116 for 403 variables, 403
parameters, and 4817 contributing reflections. Maximum shift/error =
0.002, and maximum/minimum residual electron density = 0.679/−
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dx.doi.org/10.1021/jf5048089 | J. Agric. Food Chem. 2014, 62, 11786−11796

Journal of Agricultural and Food Chemistry
Article
Table 4. Chemical Structures, Postemergent Herbicidal Activity (Inhibition Rating of 0−100) of Compounds III−V, and Their
Inhibitory Activities against AtHPPD
a
a
a
a
a
a
R2
R4
dose (g of ai/ha)
EC
SF
DS
AR
EP
AJ
Ki (nM)
VI-a
2-CH3
CH3
150
75
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
95
100
100
100
100
100
95
100
100
100
100
100
95
100
100
16
28
10
9
1
4
1
1
1
37.5
150
75
97.5
VI-b
2-OCH3
2-CF3
CH3
CH3
CH3
100
100
37.5
150
75
97.5
100
92.5
100
97.5
97.5
VI-c
100
95
100
95
100
90
97.5
90
37.5
150
75
95
100
100
95
20
0
90
90
90
90
VI-d
2,6-di-CH3
100
100
100
100
95
100
100
95
100
95
100
100
100
95
37.5
150
75
87.5
97.5
mesotrione
85
100
100
100
100
100
100
100
100
100
13
75
60
37.5
30
0
30
a
Abbreviations: EC, Echinochloa crus-galli; SF, Setaria faberii; DS, Digitaria sanguinalis; AR, Amaranthus retroflexu; EP, Eclipta prostrata; AJ, Abutilon
juncea.
prostrata (EP), and Abutilon juncea (AJ) were evaluated according to
the above-mentioned procedure;18−20 the commercially available
triketone herbicide mesotrione was selected as a positive control. All
test compounds were formulated as 100 g/L emulsified concentrates
using N,N-dimethylformamide (DMF) as the solvent and Tween-80 as
the emulsification reagent. The concentrates were diluted with water
to the required concentration and applied to pot-grown plants in a
greenhouse. Clay soil was used with pH 6.5, 1.6% organic matter,
37.3% clay particles, and cation-exchange capacity (CEC) of 12.1 mol/
kg. The rate of application (g of ai/ha) was calculated by the total
amount of active ingredient in the formulation divided by the surface
area of the pot. Plastic pots with a diameter of 9 cm were filled with
soil to a depth of 8 cm. Approximately 20 seeds of selected weeds were
sown in soil at the depth of 1−3 cm and grown at the temperature of
15−30 °C in a greenhouse. Atmospheric relative humidity was 50%.
The diluted formulation solutions were applied as a postemergence
treatment. Broadleaf weeds were treated at the 2-leaf stage, and
monocotyledon weeds were treated at the 1-leaf stage. The
postemergence application rate was 150 g of ai/ha. Untreated
seedlings were used as the control group, and the solvent (DMF +
Tween-80)-treated seedlings were used as the solvent control group.
Herbicidal activity was evaluated visually after 15 days from post-
treatment. The results of herbicidal activities are shown in Tables 1−4,
with three replicates per treatment.
Crop Selectivity. The conventional rice, soybean, cotton, wheat,
rape, and maize were planted separately in pots (diameter = 12 cm)
containing selected soil and grown in a greenhouse at 20−25 °C.21
After the (crop) plants had reached the 4-leaf stage, new herbicides
were applied (spraying) at the dosage of 150 g of ai/ha. The visual
injury and growth rate of the individual plants were observed at regular
intervals. After 15 days, the final results of crop safety were determined
(Table 5), using three replicates per treatment.
Plasmid Construction, Protein Expression and Extraction,
HPPD Purification, Activity Assays, and Kinetic Inhibition
Studies. Arabidopsis thaliana HPPD (AtHPPD) was constructed by
polymerase chain reaction (PCR) using cDNA of HPPD in pMD19-T
Simple (Hangzhou BIOSCI Biotechnology Company) as the template.
The primers, used here were 5′-CATGCCATGGGCCAC-
CAAAACGCCGC-3′ (NcoI) and 5′-CGCGGATCCT-
CAGTGGTGGTGGTGGTGGTGTCCCACTAACTGTTT-3′
(BamHI). PCR conditions were 35 cycles at 94 °C for 30 s, 55 °C for
Table 5. Crop Selectivity (Injury Rating of 0−100) of
Selected Compounds (Postemergence, 150 g of ai/ha)
compound
soybean
rape
cotton
maize
rice
wheat
II-h
20
45
0
100
85
20
25
30
0
40
0
50
20
30
35
II-k
II-u
55
0
20
10
III-b
VI-a
65
45
0
20
10
100
100
90
100
100
100
100
100
100
86
90
90
70
15
50
30
70
10
100
100
100
100
50
90
VI-b
VI-c
60
100
60
VI-d
mesotrione
100
55
40
30 s, and 68 °C for 1.5 min. The amplion was introduced into the
expression vector pET-15b and subsequently transformed into
Escherichia coli BL21(DE3). The DNA sequences of the positive
clones were confirmed by DNA sequencing with Shanghai SANGON
Company.
Recombinant AtHPPD was overexpressed in E. coli BL21(DE3)
cells with pET-15b-HPPD plasmid. Recombinant HGD from human
was overexpressed in E. coli BL21(DE3) cells with pET-28a-HGD
plasmid. The cells were grown at 37 °C in Luria−Bertani broth
supplemented with 50 μg/mL kanamycin (pET-28a plasmid) or 100
μg/mL antimycin (pET-15b plasmid) according to the previous
publication.22 Expression of the AtHPPD plasmid was incubated at 37
°C for 12 h. HGD was induced by 0.5 mM isopropyl-β-D-
thiogalactopyranoside (IPTG) when bacterial growth reached A600 of
0.6, and then the cells were incubated for another 40 h at 15 °C. After
harvesting by centrifugation (5000g for 30 min), the pellet was
resuspended in buffer [20 mM N-2-hydroxyethylpiperazine-N′-2-
ethanesulfonic acid (HEPES) and 20 mM NaCl at pH 7.0] and
washed twice, followed by sonication using a cell disruptor. A crude
cell-free supernatant was obtained by centrifugation at 20000g for 30
min. The over-expression and the relative molecular weight of
recombinant AtHPPD were validated by both sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) stained with
Coomassie Brilliant Blue-R250 and Simple Western analysis using
Simon instrumentation (ProteinSimple, CA)
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Table 6. Binding Free Energies (kcal/mol) of Compounds II-k and VI-c
a
b
compound
ΔEELE
ΔEVDW
ΔEGAS
ΔGSOL
ΔEMM
−TΔS
ΔGcal
ΔGexp
II-k
−128.75 (8.15)
−138.94 (7.44)
−33.57 (3.46)
−31.98 (3.81)
−162.31 (8.11)
115.98 (7.74)
124.09 (7.11)
−46.33 (4.91)
−46.82 (5.31)
34.42 (2.65)
37.72 (3.01)
−11.91
−9.16
−10.93
−10.02
VI-c
−170.91 (6.57)
a
b
The results were determined by the MM/PBSA calculations. The experimental values of ΔGexp were derived from the reported experimental Ki
values.
a
Scheme 1. Synthetic Route for the Title Compounds II−V
a
Reagents and conditions: (a) KOH, KMnO4, HCl; (b) CH3OH, H2SO4, reflux; (c) H2, 10% Pd/C; (d) (un)substituted phenyl isocyanates,
pyridine, 100 °C; (e) Cs2CO3, CH3I, DMF, rt; (f) HOAc, H2SO4, H2O, 100 °C; (g) SOCl2, THF, reflux; (h) substituted 1,3-cyclohexanediones,
Et3N, CHCl3, 0 °C; (i) acetone cyanohydrin, Et3N, CH2Cl2, rt.
AtHPPD was purified in two chromatographic steps. The crude cell-
free supernatant was loaded onto a nickel−nitrilotriacetic acid (Ni−
NTA) column (Qiagen, Canada), equilibrated with 20 mM HEPES at
pH 7.0. Then, HPPD was eluted with 20 mM HEPES at pH 7.0, 150
mM NaCl, and 250 mM imidazole. The fractions containing HPPD
were concentrated, and the buffer was exchanged for 20 mM HEPES
at pH 7.0 by ultrafiltration in Ultrafree filter devices (Millipore, MA).
For further purification of the recombinant HPPD, anion-exchange
chromatography was carried out on Q resin (Amersham-Pharmacia
Biotech, Germany) in 20 mM HEPES at pH 7.0. Elution of the
recombinant HPPD was carried out in a linear gradient from 0 to 250
mM NaCl. Again, the HPPD-containing fractions were collected, and
the buffer was exchanged to 20 mM HEPES at pH 7.0 by
ultrafiltration.
Our coupled enzyme assays for the in vitro activity and inhibition of
HPPD were measured by a modification of methods previously
reported in the literature.23 Assays were performed in 96-well plates at
30 °C using an ultraviolet/visible plate reader to monitor the
formation of maleylacetoacetate at 318 nm (ε330 = 13 500 M−1 cm−1).
The reaction mixture in a total assay volume of 200 μL contained
appropriate amounts of HPPA, 100 μM FeSO4, 2 mM sodium
ascorbate, 20 mM HEPES buffer (pH 7.0), HPPD, and HGD. Before
assays were conducted, all reaction components were pre-equilibrated
at 30 °C for at least 10 min. The amount of HGD activity was
predetermined to be in a large excess of the HPPD activity to ensure
that the reaction was tightly coupled (the Km of HGD for HGA was 25
μM). Each experiment was repeated at least 3 times, and the values
were averaged. HPPD inhibitors were dissolved in dimethyl sulfoxide
(DMSO) for stock solution and diluted to various concentrations with
reaction buffer just before use. The inhibition constant (Ki), the
indication of the potency of an inhibitor, was obtained from the Dixon
plot of plotting 1/v against the concentration of the inhibitor at certain
concentrations of substrate. Bovine serum albumin is usually added up
to 0.5% of the total reaction volume for coating of the target enzyme
during the incubation. In our assay, no obvious effect from bovine
serum albumin on the activity of the compounds has been found,
which indicated that the new compounds selectively inhibited the
target enzyme but did not interact with bovine serum albumin.
Computational Methods. The crystal structure of AtHPPD was
taken from the Protein Data Bank (PDB ID 1TFZ). Compounds were
constructed and optimized using SYBYL 7.0 (Tripos, Inc.), and
Gasteiger−Huckel charges were calculated for them. Docking
calculations were performed on the two molecules using Auto-
Dock4.0.24 The protein and ligand structures were prepared with
AutoDock Tools. A total of 256 runs were launched for each molecule.
Each docked structure was scored by the built-in scoring function and
clustered by 0.8 Å of root-mean-square deviation (RMSD) criterions.
The best binding modes were determined by docking scores and also
the comparison to available complex crystal structure of HPPD and
commercial inhibitors (Streptomyces avermitilis HPPD complexed with
inhibitor NTBC). Standard Amber ff99 force field parameters were
assigned to protein, and general AMBER force field (gaff) was assigned
to ligands. The partial atomic charges of ligands were calculated using
the AM1-BCC method, and the system was solvated in an octahedral
box of TIP3P water with the crystallographic water molecules kept.
The edge of the box was at least 8 Å from the solute, and appropriate
counterions were added to the system to preserve neutrality. In each
step, energy minimization was first performed using the steepest
descent algorithm for 1000 steps and then the conjugated gradient
algorithm for another 2000 steps.
The MD simulation was performed under periodic boundary
conditions using the Sander module of the AMBER9 program.25 The
stable MD trajectory was used to perform the binding free energy
(ΔGbind) calculation using a modified MM/PBSA method (Table 6). A
total of 100 snapshots were taken from the last 500 ps trajectory with
an interval of 5 ps to analyze the binding energy, and at the same time,
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a
Scheme 2. Synthetic Route for the Title Compounds VI
a
Reagents and conditions: (a) ICl, CH3COOH, rt; (b) substituted phenyl isocyanates, pyridine, 100 °C; (c) Cs2CO3, CH3I, DMF, rt; (d) CuCN,
DMF, reflux; (e) H2SO4, CH3COOH, H2O, 100 °C; (f) SOCl2, THF, reflux; (g) 1,3-cyclohexanediones, Et3N, CHCl3, 0 °C; (h) acetone
cyanohydrin, Et3N, CH2Cl2, rt.
Figure 2. Crystal structure of compound IV-d.
the counterions and water molecules (water related to the crucial
hydrogen bond was not included) were stripped.
and H2SO4) as the hydrolysis reagents, the hydrolysis of
intermediates 6a−6x could smoothly proceed with yields of
78−98%. Subsequently, intermediates 7a−7y reacted with
SOCl2 in tetrahydrofuran (THF) to obtain the corresponding
acid chlorides that were very unstable. Thus, the acid chlorides
were used directly in the next step without further isolation.
After the reaction of the acid chlorides with substituted-1,3-
cyclohexanediones, the key enol esters (8a−8x, 9a and 9b,
10a−10v, and 11) were generated. Finally, using acetone
cyanohydrin as the Fries rearrangement catalyst, the target
compounds II−V were obtained in yields of 64−94%.
Similarly, the title compounds VI can be prepared, in an 8-
step synthetic route using 2-amino-6-methylbenzoic acid as the
starting material, after an iodination reaction with ICl in acetic
acid; the key intermediate 6-amino-3-iodo-2-methylbenzoic
acid (13) was obtained in a yield of 73%. Intermediates 14a−
14d and 15a−15d were obtained using the same methods as
the preparation of intermediates 6a−6x. Then, compounds
15a−15d reacted with 2 equiv of CuCN in DMF, and the
intermediates 16a−16d were obtained in yields of 69−85%.
Because the quinazoline-2,4-dione rings of intermediates 16a−
16d were also very sensitive to strong base, the reaction could
not be proceeded in aqueous base solution. Using mixed acids
(H2SO4 and HOAc) as the reactant, the hydrolysis of the cyano
group in the intermediates 16a−16d proceeded smoothly.
Using the same method as the synthesis of previously enol
RESULTS AND DISCUSSION
■
Chemistry. Dependent upon the substituent at R4,
compounds II−VI were synthesized by two different synthetic
routes. When the substituent at R4 is H, the detailed synthetic
routes of compounds II−V are outlined in Scheme 1. When R4
is methyl, the detailed synthetic routes of title compounds VI
are outlined in Scheme 2. As seen in Scheme 1, the target
compounds II−V can be prepared in 10-step synthetic routes
using 5-methyl-2-nitrobenzoic acid as the starting material.
After oxidation, esterification, and reduction reactions, the key
intermediate dimethyl 4-aminoisophthalate (4) was obtained in
a yield of 83%. According to the reported methods,26,27 the key
intermediate 4 reacted with various (un)substituted phenyl
isocyanates in pyridine to afford quinazoline-2,4-dione
intermediates 5a−5x in good yields. Then, the intermediates
5a−5x reacted with CH3I to afford the corresponding
intermediates 6a−6x in yields of 81−99%. It was found that
the intermediates 6a−6x were very unstable in strong base
solution, such as aqueous concentrated NaOH and KOH
solution, because of the decomposition of the quinazoline-2,4-
dione rings of 6a−6x caused by base. However, to our delight,
the quinazoline-2,4-dione rings of intermediates 6a−6x were
very stable in acid solution. Finally, using mixed acids (HOAc
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esters, the intermediates 18a−18d could be prepared from
intermediates 17a−17d in yields of 62−80%. Subsequent Fries
rearrangement reaction using acetone cyanohydrin as the
catalyst afforded compounds VI in yields of 71−78%.
methyl-substituted analogues (I-f > I-e and I-d), the methoxyl-
substituted analogues (II-h > II-i and II-j), and the
trifluoromethyl-substituted analogues (II-k > II-m and II-l).
Notably, compounds with multiple substitutions on the phenyl
ring showed a decreased herbicidal activity (II-s−II-x), even
through some of them with substituents at the ortho positions.
It was also found that compounds with ortho substitiutions of
electron-donating groups (I-f, CH3; II-h, OCH3) displayed
improved herbicidal activity compared to that of the
compounds with electron-withdrawing groups (I-c, Cl; II-b,
F; II-e, Br; II-k, CF3; and II-n, OCF3). The similar structure−
activity trends could also be observed for the compounds with
substituents at the para positions, although there may be a few
exceptions.
The SAR results suggested that substituents in the ortho
positions of compounds can usually improve their herbicidal
activity. Inspired by this, we then synthesized compounds II-q−
II-s with different sizes of substituents on ortho positions to
examine the steric effects on herbicidal activity. As shown in
Table 2, the steric factors had a significant impact on herbicidal
activity. Compounds II-q (R2 = 2-C2H5) and II-r (R2 = 2-iPr)
showed over 80% control in six deferent weed tests at the rate
of 150 g of ai/ha. However, compound II-s (R2 = 2,6-di-iPr)
with more sterically bulky substituents in its ortho positions
displayed decreased effects and was almost inactive toward S.
faberii and A. juncea. A possible explanation for compound II-s
with lower activity is that the large subsitutuents would prevent
making bonds with plant HPPD.28
The structures of all of the synthetic triketone-containing
1
compounds were confirmed by H and 13C nuclear magnetic
resonance (NMR) and high-resolution mass spectrometry
(HRMS) spectral data. In addition, the crystal structure of
compound IV-d was further determined by X-ray diffraction
analysis (Figure 2).
Herbicidal Activity and SAR. The postemergence
herbicidal activities of all of the target compounds were
evaluated against monocotyledon weeds (E. crus-galli, S. faberii,
and D. sanguinalis) and broadleaf weeds (A. retroflexus, E.
prostrata, and A. juncea) in the greenhouse environment.
Mesotrione was used as the positive control, and the results are
shown in Tables 1−4. As anticipated, the treated weeds
developed unique bleaching symptoms, as typical HPPD
herbicides. The herbicidal activity evaluation indicated that
most of the synthetic compounds showed between “good” to
“excellent” herbicidal activity against the test weeds. Among
them, compounds III-b and VI-a−VI-d showed an even
broader spectrum of weed control than mesotrione, because of
their superior control efficacy against the monocotyledon
weeds to mesotrione. To summarize systematically the SAR of
triketone-containing quinazoline-2,4-diones, some of our
previously synthesized compounds were also added to the
results depicted in Table 1.15
Previously, we have found that compound I-f was worth
considering for further optimization; therefore, a series of
substituents with diversity were introduced at the N-1 position
of the quinazoline-2,4-dione ring. As shown in Table 1,
compound I-h with an ethyl group at R1 displayed almost
equipotent herbicidal activity as compound I-f. However,
compound I-i with n-Pr at R1 displayed reduced herbicidal
activity compared to compound I-h. It seems that too small or
sterically bulky substituents at R1 are detrimental to the activity
of target compounds; for example, compounds I-g (R1 = H), I-
k (R1 = n-Bu), and I-m (R1 = CH2C6H5) are almost inactive
against six tested weeds. A possible reason for the poor
herbicidal activity of these compounds may be due to their
uneasily access into the weeds or rapid degradation in the
weeds. However, when we placed an electron-withdrawing (I-n,
F) or electron-donating (I-o, OCH3) group on the benzyl
group of I-m, the herbicidal activity usually displayed enhanced
effects (I-n and I-o > I-m). Therefore, the SAR at this position
can be summarized as follows: CH3 and CH2CH3 > n-Pr >
CH2CCH > CH2−2-F−C6H4, CH2−3-OCH3−C6H4, iBu, n-
Bu, H, and CH2C6H5.
On the basis of the above SAR results, we kept the methyl
group at R1 and synthesized a series of novel compounds with
different R2 substitutions to explore further their herbicidal
activity (Scheme 1). A variety of substituents were introduced
at R2, including electron-withdrawing (F, Br, OCF3, CF3, and
NO2), electron-donating (OCH3), sterically small (H), or bulky
(2-iPr and 2.6-di-iPr) groups, to evaluate the substituent effect
of the substituent at this position on herbicidal activity. As
depicted in Tables 1 and 2, when a single group was introduced
at R2, in most cases, compounds with substituents at the ortho
positions would have increased activity and broader spectrum
weed control than those with equivalent substitutions on para
and meta positions. For example, the above rule is applied to
the chloro-substituted analogues (I-c > I-a and I-b), the
To examine the effect of R3 substitution on herbicidal
activity, we then synthesized another set of compounds (III−
V). As in Table 3, compounds III-a and III-b, with a methyl on
the 5 position of the 1,3-cyclohexanedione ring, displayed an
increased herbicidal effect compared to their non-substituted
analogues II-g and II-v, respectively. Furthermore, compound
III-b exhibited over 85% control in six deferent weed tests,
even at a rate as low as 37.5 g of ai/ha, demonstrating broader
spectrum of weed control than mesotrione. However, when
another methyl group was also introduced to the 5 position, the
herbicidal activity (IV-g and IV-s) was impaired in most cases.
Because of our curiosity, two methyl groups were introduced at
6 positions, with compound V displaying an improved
herbicidal activity compared to its parent compound II-t.
One possible reason for this phenomenon is that introducing
two methyl groups on 6 posotions would block the metabolism
of weeds in vivo.7
We found that most of the commercial HPPD herbicides
have substituents in the ortho positions of their benzoyl
groups.7,8,10 It is assumed that placing a substitutent in the
ortho position would be favorable for increasing their herbicidal
activity. In addition, introducing a methyl group in the ortho
position has been reported, to be able to enhance the herbicidal
activity.9,13 Owning to the structure feature of the triketone-
containing quinazoline-2,4-dione motif, we intended to
introduce a methyl group in the 5 position of the quinazo-
line-2,4-dione ring (ortho position). To understand whether
placing a methyl group in the 5 position is beneficial for
herbicidal activity or not, four representative compounds I-f, II-
h, II-k, and II-u with a promising and broad spectrum of weed
control were selected for further optimization. The synthetic
methods for these methyl-substituted analogues (VI-a−VI-d)
are outlined in Scheme 2, the herbicidal activity results are
shown in Table 4. Surprisingly, compounds VI-a−VI-d, with a
methyl group in the ortho positions, showed significantly
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Figure 3. Simulated binding models of four representative compounds (A) II-r, (B) II-s, (C) II-k, and (D) VI-c in the active site of AtHPPD, with
panels A and B shown as translucent surfaces and panels C and D shown as directly binding models. Fe(II) is depicted as the aquamarine sphere; the
structures of inhibitors are indicated in yellow sticks; and the key residues surrounding the active site are displayed in light blue.
improved herbicidal activity compared to their corresponding
parent compounds I-f, II-h, II-k, and II-u, respectively. All four
methyl-substituted analogues displayed strong inhibition
(>85%) and broad spectrum for weed control, even at a
dosage as low as 37.5 g of ai/ha. Especially, compound VI-a
displayed almost 100% inhibition in both tests against
monocotyledon and dicotyledon weeds, with its herbicidal
potency against dicotyledon weeds (A. retroflexus, E. prostrata,
and A. juncea) being almost equipotent to mesotrione and its
potency against monocotyledon weeds (E. crus-galli, S. faberii,
and D. sanguinalis) being superior to mesotrione.
Crop Selectivity. Crop selectivity is one of the vital
concerns in pesticide discovery. Some representative com-
pounds (II-h, II-k, II-u, III-b, and VI-a−VI-d) were chosen for
crop selectivity evaluation. The results are listed in Table 5. It is
very interesting that compound III-b was safe for three crops of
cotton, maize, and wheat after postemergence application at a
rate of 150 g of ai/ha, whereas mesotrione was not selective for
cotton (70% injury) and wheat (40% injury) at the
concentration of 150 g of ai/ha. This result indicated that
there is a great potential for III-b to be used in cotton, maize,
and wheat fields to control weeds. Furthermore, II-u was safe
for maize and wheat, suggesting that II-u has potential to
develop as a effective herbicide for controlling weeds in maize
and wheat fields. In addition, II-h was safe for rape at the rate of
150 g of ai/ha, whereas mesotrione displayed 100% injury effect
against it at the same conditions, indicating that II-h might has
potential to develop as a new herbicide in a rape field.
Moreover, II-k was safe for maize at the rate of 150 g of ai/ha,
which indicated that II-k can be developed as a herbicide for
maize field. Although compounds VI-a−VI-d displayed
excellent herbicidal activity, they were not safe for the six
selected crops. A possible explanation of compounds VI-a−VI-
d to improve their herbicidal activity while decreasing crop
selectivity is that the methyl group at R4 would block
metabolism of plants at this site.13
the Ki values of compounds II−VI against AtHPPD are shown
in Tables 2−4. Mesotrione was used as the positive control.
Notably, most of newly synthesized herbicides displayed a
strong AtHPPD inhibitory activity, and some of them showed
even better potency than mesotrione. Compound VI-d (Ki = 9
nM) displayed the highest HPPD inhibitory activity, showing
slightly more potency than mesotrione (Ki = 13 nM).
Furthermore, compounds VI-c (Ki = 10 nM), II-o (Ki = 12
nM), and II-v (Ki = 13 nM) also showed excellent inhibitory
activity, which are comparable to mesotrione.
On the basis of the in vitro activity of those compounds,
some SARs can be drawn. As shown in Table 2, when the right
side of the benzene ring is single-substituted, compounds with
para substitutions generally have improved activity compared
to those with the same substituents in meta and ortho positions.
With regard to the multi-substituted analogues, the positions of
substituents can also affect the HPPD inhibitory activity.
Compounds with substituents at 2,4 and 3,5 positions would
have enhanced activity compared to those at 2,6 positions (II-v
and II-x > II-t). It seemed that the electronic factors did not
significantly affect the activity; for example, compounds with
either para substitutions of strongly electron-donating (II-j,
OCH3) groups or electron-withdrawing (II-p, NO2) groups
showed almost equal activity (II-j = II-p). In addition, the steric
factors of substituents also have a significant impact on the
inhibitory activity. Compounds with sterically bulky groups on
the ortho position were found detrimental to activity (II-r > II-
s). This was consistent with the herbicide inhibition activity. To
understand the structural basis, we investigated the binding
modes of the new compounds using compounds II-r and II-s as
two representatives. As shown in panels A and B of Figure 3,
the two compounds can form a bidentate association with the
active site ferrous ion via its 5′ and 7′ oxygens and the
quinazoline-2,4-dione ring can form π−π stacking interaction
with the conserved Phe360 and Phe403. In addition, the 2-
isopropyl-substituted benzene ring on the quinazoline-2,4-
dione moiety of compound II-r embedded deep into the
hydrophobic cavity and occupied a major part of this pocket.
HPPD Inhibition and SAR. For further understanding, the
structural basis for these compounds as HPPD inhibitors and
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However, the 2,6-diisopropyl-substituted benzene ring of
compound II-s occupied beyond the hydrophobic pocket,
showing a strong steric repulsion effect.
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It is worth mentioning that, when a methyl group was
introduced to the 5 position of the quinazoline-2,4-dione ring,
the inhibition of compounds VI-a−VI-d against AtHPPD has
improved significantly compared to their parent compounds I-f,
II-h, II-k, and II-u, respectively (Table 4). To investigate why
the methyl group at the R4 position significantly improved the
activity of the target compounds, compounds II-k and VI-c
were chosen as the representative inhibitors for computational
modeling. The energy component analysis showed that the
difference mainly came from the electrostatic interaction,
solvation, and entropy effects (Table 5). Compound II-k had
an electrostatic interaction of −128.75 kcal/mol and a solvation
penalty of 115.98 kcal/mol, which result in an enthalpy change
of −46.33 kcal/mol. Compound VI-c had a lower electrostatic
interaction of −138.94 kcal/mol and a higher solvation penalty
of 124.09 kcal/mol, which result in a similar enthalpy change of
−46.82 kcal/mol. However, a higher entropy penalty of VI-c
(37.72 kcal/mol compared to 34.42 kcal/mol for II-k) resulted
in a lower binding affinity (−9.16 kcal/mol compared to
−11.91 kcal/mol for II-k), which demonstrated that the
introduction of the methyl group at the 5 position of the
quinazoline-2,4-dione ring can help reduce the entropy penalty
and be advantageous for activity improvement.
In summary, a series of novel triketone-containing quinazo-
line-2,4-diones were designed and subsequently optimized on
the basis of a systemic SAR study, leading to the discovery of
several novel HPPD inhibitors with the excellent in vitro
inhibitory potency and greenhouse herbicidal activity. Specif-
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found with a potential of displaying a broader spectrum of weed
control than mesotrione. More importantly, compound III-b
was very safe for three crops of cotton, maize, and wheat at the
rate of 150 g of ai/ha, showing great applicability in the field.
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compound II-u was safe for maize and wheat. The promising
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dione motif is well worth further optimization. Further
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ASSOCIATED CONTENT
* Supporting Information
■
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Modern Crop Protection Compounds, 2nd ed.; Kramer, W., Schirmer, U.,
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Experimental details and analytical data for all of the
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AUTHOR INFORMATION
Corresponding Authors
■
*Telephone: +86-27-67867800. Fax: +86-27-67867141. E-mail:
tomyang@mail.ccnu.edu.cn.
*Telephone: +86-27-67867800. Fax: +86-27-67867141. E-mail:
gfyang@mail.ccnu.edu.cn.
(17) Sheldrick, G. M. SHELXS97; University of Gottingen:
Gottingen, Germany, 1997.
Funding
The authors are very grateful to the National Key Technologies
Research and Development Program of China
(2011BAE06B05) for the financial support for this work.
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Notes
The authors declare no competing financial interest.
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