Synthesis, bioactivity and DFT structure-activity relationship study of novel 1,2,3-thiadiazole derivatives

Article abstract of DOI:10.1007/s11164-012-0521-1

A series of novel 1,2,3-thiadiazole derivatives were designed and synthesized. Their structures were characterized by 1H NMR, MS and HRMS. The bioactivity tests indicated that compound 9b exhibits a favorable KARI inhibition rate, and some of these novel compounds also showed moderate herbicidal activity against Brassica campestris. Springer Science+Business Media B.V. 2012.

Full text of DOI:10.1007/s11164-012-0521-1

Res Chem Intermed (2012) 38:1999–2008
DOI 10.1007/s11164-012-0521-1
Synthesis, bioactivity and DFT structure–activity
relationship study of novel 1,2,3-thiadiazole derivatives
•
•
•
Xing-Hai Liu Wei-Guang Zhao Bao-Lei Wang
Zheng-Ming Li
Received: 6 January 2012 / Accepted: 26 February 2012 / Published online: 14 March 2012
Ó Springer Science+Business Media B.V. 2012
Abstract A series of novel 1,2,3-thiadiazole derivatives were designed and
1
synthesized. Their structures were characterized by H NMR, MS and HRMS. The
bioactivity tests indicated that compound 9b exhibits a favorable KARI inhibition
rate, and some of these novel compounds also showed moderate herbicidal activity
against Brassica campestris.
Keywords 1,2,3-Thiadiazole ꢀ Synthesis ꢀ KARI activity ꢀ Herbicidal activity ꢀ
DFT study
Introduction
Recently, the development of herbicides targeting at the key enzymes in the
biosynthesis pathway of branched chain amino acids has attracted extensive
attention [1]. Some reported herbicides, such as sulfonylureas [2], imidazolinones
[3], sulfonamide [4], and pyrimidinylbenzoic acids [5], can inhibit the first enzyme,
acetohydroxyacid synthase (AHAS), in the biosynthesis pathway of the branched
chain amino acids called. The success of these herbicides has stimulated numerous
research into finding efficient inhibitors for other enzymes in this pathway, such as
ketol-acid reductoisomerase (KARI; EC 1.1.1.86), which catalyzes the second key
step. Furthermore, it was noticed that KARI only exists in microorganisms and
X.-H. Liu (&)
College of Chemical Engineering and Materials Science, Zhejiang University of Technology,
Hangzhou 310014, China
e-mail: xhliu@zjut.edu.cn
W.-G. Zhao ꢀ B.-L. Wang ꢀ Z.-M. Li (&)
State Key Laboratory of Elemento-Organic Chemistry, National Pesticidal Engineering Centre,
Nankai University, Tianjin 300071, China
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X.-H. Liu et al.
CH3
P
CH3
H
C
-
-
H3C
C
H
N
C
O
CO2
IpOHA3
H3C
CO2
HOE 7042
OH
O
OH
Acetohydroxyacid
Methylhydroxyketolacid
Dihydroxyacid
R
R
R
CH2
CH2
M2+
CH2
Mg2+
H
-
-
-
H3C
C
C
O
CO2
H3C
C
O
C
CO2
H3C
C
C
CO2
R
OH
OH OH
OH
NADP+
NADPH
+ H+
H2C
-
H3C
C
O
C
CO2
O
Transition state4
H
Fig. 1 Reaction catalyzed by KARI, the known inhibitors of HOE704, IpOHA and CPD, thiadiazoles
respectively
plants, rather than in mammals, and thus it is an ideal target for non-toxic KARI-
inhibitors for potential herbicide applications.
The KARI-catalyzed reaction in the biosynthesis of branched chain amino acids
is shown in Fig. 1. The reactions include two steps [6]. Both steps require a divalent
metal ion, such as Mg2?, Mn2? or Co2?, and Mg2? is known to be the specific metal
ion for alkyl migration.
Until now, there has been many commercialized herbicide targeting at KARI,
with some reported compounds including HOE 704 [7], IpOHA [8], 1,2,3-
thiadiazoles [9, 10] and CPD derivatives [11–16] have shown certain KARI
inhibitory activities, and might be the potential KARI inhibitors (Fig. 1).
Here, we report the synthesis, structural characterizations, and bioactivity tests of
a series of novel 1,2,3-thiadiazole derivatives. Some of these compounds showed
certain herbicidal and KARI inhibitory activities. Further density functional theory
(DFT) studies showed the possible favorable functional groups of 1,2,3-thiadiazole
derivatives.
Results and discussion
Chemistry
The synthetic route of the novel 1,2,3-thiadiazole derivatives is presented in
Scheme 1. The key intermediate ethyl 4-(bromomethyl)-1,2,3-thiadiazole-5-carbox-
ylate (6) was prepared via two pathways using carbonic acid diethyl ester (1) as the
starting material. It is shown in Scheme 1. Ethyl 4-methyl-1,2,3-thiadiazole-5-
carboxylate (4) was synthesized according to the reference. To a solution of ethyl
4-methyl-1,2,3-thiadiazole-5-carboxylate (4) (0.04 mol) in CCl4 (120 mL) was
123

Synthesis, bioactivity and DFT structure
2001
O
O
N
N
O
O
N
HN
O
NH2
O
S
O
N
H
O
O
O
O
1
2
3
4
O
O
N
X
N
HN
N
O
O
S
Cl
O
O
6
5
X=Cl or Br
R1
R1
S
S
N
S
N
N
O
O
N
N
N
O
O
OH
S
S
S
O
O
O
7a-7d
8a-8b
11
R1
R1
S
S
N
S
N
S
O
O
N
N
OH
O
O
9a-9b
O
10a-10b
1 = m-Cl, p-Me, p-Cl, H
R
Scheme 1 The synthetic route of the title compounds
added NBS(7g) and a little AIBN, and the mixture was refluxed for 5 h. Afterwards,
the reaction was completed, filtrated, washed with brine, dried over Na2SO4, and
condensed under reduced pressure to afford the ethyl 4-(bromomethyl)-1,2,3-
thiadiazole-5-carboxylate (6). This intermediate was not further purified.
The combination of H2O2 and HOAc has been recognized as an efficient system
to oxidize sulfur into sulfone or sulfoxide. In our case, the sulfoxide was easily
obtained at room temperature for 12 h. The longer the reaction time is given at room
temperature, the more sulfone was obtained. When the mixture was stirred at room
temperature for 48 h, all the sulfoxide converted to sulfone. The sulfone can also be
obtained at reflux condition for short time.
Bioactivity and structure–activity relationship
To investigate the KARI inhibitory activities and herbicidal activities of the synthesized
1,2,3-thiadiazole compounds, cyclopropane-1,1-dicarboxylic acid (CPD), a potent
inhibitor of KARI in vitro, was used as the reference. The bioactivities of the selected
1,2,3-thiadiazole compounds as well as CPD are shown in Table 1. With regard to the
KARI inhibitory assay, most of the 1,2,3-thiadiazole compounds do not dissolve in the
test system, except for the compound 8, 9, 11. The KARI inhibitory activities of these
four compounds are lower than that of CPD, with the highest inhibitory activity of 60 %
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X.-H. Liu et al.
Table 1 The in vitro and in
vivo inhibition rates (%) of the
title compounds at 100 ppm
No
KARI
Brassica napus
Echinochloa
crusgalli
7a
–
30.0
25.9
27.4
31.6
44.9
0
22.0
30.0
15.0
37.0
30.1
20.8
38.4
43.0
35.0
30.0
18.0
27.7
7b
–
7c
–
7d
–
8a
0
8b
6.73
25.09
60.55
–
9a
0
9b
47.1
41.8
27.9
19.5
17.2
10a
10b
11
–
45.80
100
– indicates that the compound is
not soluble in our test system,
thus no data were obtained
CPD
of compound 9b. Based on the herbicidal activities against Brassica napus and
Echinochloa crusgalli tests, the synthesized 1,2,3-thiadiazole compounds displayed low
activities. Compounds 8a, 9b and 10a exhibited moderate inhibitory activities against B.
napus. According to the in vitro activities tests, the 1,2,3-thiadiazole compounds
containing based carboxylic acid groups as well as the electron-withdrawing groups,
such as phenyl, showed higher activity than their analogues. In addition, the sulfoxides
led to higher activity than sulfonyls. In general, it was revealed that the ketol-acid
reductoisomeraseinvitroinhibitoryactivitiesofthesenewcompoundsareratherlimited.
DFT study of structure–activity relationship
According to the frontier molecular orbital theory, HOMO and LUMO are two
crucial factors influencing the bioactivities of compounds. HOMO is usually prone
to provide electrons, while LUMO accepts electrons [16]. Based on such a concept,
the study on the frontier orbital energy can provide useful information for the
working mechanism of the bioactive compounds. DFT is a useful tool to investigate
the HOMO–LOMO orbital energies. Thus, on the selected compound 9b with the
highest KARI inhibitory activity, the DFT study was carried out using the DFT-
B3LYP/6-31G method to find the favorable functionalities as well as the possible
working mechanisms. The DFT-derived graphic results are presented in Fig. 2. The
red and green parts represent the cloud density of frontier orbitals at HOMO or
LOMO states. The total energy of the compound 9b is -1,901.67561163 a.u. The
energy gap between HOMO and LUMO of the compound 9b is rather small (0.129
a.u.) suggesting that the stability of the compound 9b is limited. As seen from Fig. 2
of compound 9b in the HOMO, electrons are mainly delocalized on the phenyl ring,
the carboxyl group as well as the sulfoxide group in compound 9b. However, when
electron transitions take place, some electrons will enter into the LUMO, then, in the
LUMO, the electrons will mainly be delocalized on the phenyl ring, carboxy group,
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2003
Fig. 2 The HOMO and LUMO of compound 9b. The red and green parts represent the cloud density of
frontier orbitals at HOMO or LOMO states
SO2 group, and the 1,2,3-thiadiazole ring. Since these polar functionalities with
favorable frontier molecular orbitals are the main positions where the interaction
with KARI takes place, the possible working mechanism between compound 9b and
the rice KARI amino acid residues is estimated to be the hydrophobic interactions.
Experimental
Materials and methods
All the reagents are analytical grade. Melting points were determined using an X-4
1
apparatus and uncorrected. H NMR spectra were measured on a Bruker AC-P500
instrument using TMS as an internal standard and CDCl3 as solvent. Mass spectra
were recorded on a Thermo Finnigan LCQ Advantage LC/mass detector apparatus.
Infrared spectra were recorded on a Nicolet MAGNA-560 spectrophotometer using
KBr tablets. HRMS data was obtained on a FTICR-MS instrument (Ionspec 7.0T).
Synthesis
Synthesis of the intermediate ethyl 4-(chloromethyl)-1,2,3-thiadiazole-
5-carboxylate (6)
Carbonic acid diethyl ester (1) (11.8 g, 0.1 mol) and hydrazine hydrate (5.6 mL,
0.095 mol, 85 %) were added into a 250-mL round-bottom flask equipped with a
condenser. The reaction mixture was heated to 50 °C and stirred for 20 min, and then
cooled to room temperature and further stirred for 30 h. Net, water, ethanol, and the
unreacted carbonic acid diethyl ester were distilled off under reduced pressure. After
drying, a white crystal (2) (9.88 g) was obtained with a yield of 95 %.
To a stirred solution of compound 2 (6.36 g, 0.06 mol) in ethanol (16.7 mL), a
solution of ethyl 4-chloro-3-oxobutanoate (9.84 g, 0.06 mol) in ethanol (3.7 mL)
was added at room temperature. The reaction mixture was stirred for 6 h. Then, the
solvent was removed in vacuo and the crude product ethyl 2-(1-chloro-4-ethoxy-4-
oxobutan-2-ylidene)hydrazinecarboxylate (3) was directly used in the next step
without further purification.
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Ethyl 2-(1-chloro-4-ethoxy-4-oxobutan-2-ylidene)hydrazinecarboxylate (3)
(15.0 g, 0.06 mol) was dissolved in dry dichloromethane (25 mL), and thionyl
chloride (25 mL) was subsequently added into the stirred reaction mixture drop
wise at 0 °C for 1 h. Next, the reaction mixture was permitted to stand for 20 h at
room temperature. The excess thionyl chloride and dichloromethane were distilled
off, and the resulting crude product was subjected to fractional distillation under
reduced pressure. An umber oil (4) was obtained with yield of 75.4 %.
Synthesis of the title compounds
General procedure for the compound 7a–7d The synthetic route was shown in
Scheme 1. To a stirred solution of 4 (1.05 g, 5.1 mmol) and K2CO3 (0.2 g,
5.6 mmol) in DMF (15 mL), a mixture of a substituted benzenethiol (5.1 mmol)
was added dropwise. The resulting mixture was stirred at room temperature
overnight. Next, the mixture was poured into water. The precipitate formed was
filtered off. The crude product was purified by chromatography over silica gel to
give 7a–7d. (PE:EA = 5:1).
General procedure for sulfoxides To a stirred solution of compound 7b–7c
(1 mmol) in HOAc (10 mL) was added H2O2 (2 mmol), then the mixture was
stirred at room temperature overnight. When the reaction reached completion
indicated by TLC, CH2Cl2 (20 mL) was added and the organic layer was separated
and washed with water. Next, the solvent was evaporated in vacuo to afford crude
compounds 8a–8c. The pure products 8a–8c were purified by chromatography over
silica gel to give 8a–8c.
General procedure for sulfones To a stirred solution of compound 7b–7c
(1 mmol) in HOAc (10 mL) was added H2O2 (2 mmol), then the mixture was
stirred at 80 °C for 1 h. During this course, a large amount of white solid was
formed. After filtration, the solid was purified by chromatography over silica gel to
give the pure 10a–10b.
General procedure for carboxylic acid To a stirred solution of ester (1 mmol) in
CH3OH (10 mL) was added NaOH (1 mmol) in CH3OH (5 mL), and the mixture
was stirred at room temperature for 24 h. Next, diluted HCl solution was added
dropwise to adjust pH to 7 and formed light yellow precipitates. After filtration, the
pure product was obtained by recrystallization from EtOH/H2O mixture.
Ethyl 4-((phenylthio)methyl)-1,2,3-thiadiazole-5-carboxylate (7a): yellow crys-
1
tal, yield 45.2 %, m.p. 28–30 °C; H NMR(CDCl3)d: 1.36(t, J = 7.14 Hz, 3H,
CH3), 4.33(q, J = 7.15 Hz, 2H, CH2), 4.80(s, 2H, SCH2), 7.20–7.24(m, 2H, Ar–H),
7.36–7.40(m, 2H, Ar–H); ESI–MS: 281.16[M?H]?; HRMS for C13H12N2O2S2:
found 303.0227, calcd. 303.0232[M?Na]?.
Ethyl 4-((p-tolylthio)methyl)-1,2,3-thiadiazole-5-carboxylate (7b): reddish-brown
1
crystal, yield 43.4 %, m.p. 36–37 °C; H NMR(CDCl3)d: 1.35(t, J = 7.13Hz, 3H,
CH3), 2.30(s, 3H, CH3), 4.30(q, J = 7.16Hz, 2H, CH2), 4.74(s, 2H, SCH2), 7.05(d,
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Synthesis, bioactivity and DFT structure
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J = 8.12Hz, 2H, Ar–H), 7.23(d, J = 8.12Hz, 2H, Ar–H); ESI-MS: 295.10[M?H]?;
HRMS for C13H14N2O2S2: found 317.0388, calcd. 317.0389 [M?Na]?.
Ethyl 4-(((4-chlorophenyl)thio)methyl)-1,2,3-thiadiazole-5-carboxylate (7c):
1
reddish-brown crystal, yield 37.6 %, m.p. 41–43 °C; H NMR(CDCl3)d: 1.37(t,
J = 7.14 Hz, 3H, CH3), 4.34(q, J = 7.12 Hz, 2H, CH2), 4.78(s, 2H, SCH2), 7.22(d,
J = 8.54 Hz, 2H, Ar–H), 7.30(d, J = 6.72 Hz, 2H, Ar–H); ESI-MS:
315.26[M?H]?; HRMS for C12H11ClN2O2S2: found 336.9846, calcd. 336.9843
[M?Na]?.
Ethyl 4-(((3-chlorophenyl)thio)methyl)-1,2,3-thiadiazole-5-carboxylate (7d):
reddish-brown oil, yield 38.9 %; 1H NMR(CDCl3)d: 1.40(t, J = 7.14 Hz, 3H,
CH3), 4.38(q, J = 7.12 Hz, 2H, CH2), 4.84(s, 2H, SCH2), 7.21(d, J = 5.26 Hz, 2H,
Ar–H), 7.28(s, 1H, Ar–H), 7.41(s, 1H, Ar–H); ESI-MS: 315.10[M?H]?; HRMS for
C12H11ClN2O2S2: found 336.9839, calcd. 336.9843 [M?Na]?.
Ethyl 4-((p-tolylsulfinyl)methyl)-1,2,3-thiadiazole-5-carboxylate (8a): yellow
1
solid, yield 31.2 %, m.p. 72–74 °C; H NMR(CDCl3)d: 1.39(t, J = 7.17 Hz, 3H,
CH3), 4.37(q, J = 7.07 Hz, 2H, CH2), 4.98(dd, J = 12.74 Hz, J = 12.74 Hz, 2H,
SCH2), 7.39(d, J = 8.51 Hz, 2H, Ar–H), 7.45(d, J = 8.60 Hz, 2H, Ar–H); ESI-MS:
620.78[2M]?, 311.15[M?H]?; HRMS for C13H14N2O3S22: found 333.0336, calcd.
333.0336 [M?Na]?.
Ethyl 4-(((4-chlorophenyl)sulfinyl)methyl)-1,2,3-thiadiazole-5-carboxylate (8b):
reddish-brown oil, yield 31.2 %; 1H NMR(CDCl3)d: 1.38(t, J = 7.14Hz, 3H, CH3),
2.40(s, 3H, CH3), 4.36(q, J = 5.24 Hz, 2H, CH2), 4.95(dd, J = 12.70 Hz,
J = 12.70 Hz, 2H, SCH2), 7.25(d, J = 8.20 Hz, 2H, Ar–H), 7.33(d, J = 8.20 Hz,
2H, Ar–H); ESI-MS: 662.69[2M]?, 331.07[M?H]?; HRMS for C12H11ClN2O3S2:
found 352.9787, calcd. 352.9792 [M?Na]?.
4-((p-tolylthio)methyl)-1,2,3-thiadiazole-5-carboxylic acid (9a): yellow solid,
1
yield 34.6 %, m.p. 139–141 °C; H NMR(CDCl3)d: 2.31(s, 3H, CH3), 4.77(s, 2H,
SCH2), 7.08(d, J = 7.98 Hz, 2H, Ar–H), 7.25(d, J = 7.98 Hz, 2H, Ar–H); ESI-MS:
530.79[2M]-, 264.92[M-H]-; HRMS for C11H10N2O2S2: found 265.0116, calcd.
265.0111[M-H]-.
4-(((4-chlorophenyl)thio)methyl)-1,2,3-thiadiazole-5-carboxylic
acid
(9b):
yellow solid, yield 35.8 %, m.p. 142–143 °C; 1H NMR(CDCl3)d: 4.79(s, 2H,
SCH2), 7.25(d, J = 8.55Hz, 2H, Ar–H), 7.33(d, J = 8.55Hz, 2H, Ar–H); FTIR
m(KBr): 3020 cm-1, 2991 cm-1, 2940 cm-1, 1720 cm-1, 1513 cm-1, 1492 cm-1
,
1415 cm-1, 1275 cm-1; ESI-MS: 570.78[2M]-, 284.79[M–H]-; HRMS for
C10H7ClN2O2S2: found 384.9567, calcd. 284.9565 [M–H]-.
Ethyl 4-(tosylmethyl)-1,2,3-thiadiazole-5-carboxylate (10a): yellow solid, yield
78.0 %, m.p. 75–78 °C; 1H NMR(CDCl3)d: 1.42(t, J = 6.80 Hz, 3H, CH3), 2.46(s,
3H, CH3), 4.41(q, J = 6.88 Hz, 2H, CH2), 5.29(s, 2H, SCH2), 7.21(d, J = 7.49 Hz,
2H, Ar–H), 7.60(d, J = 7.62 Hz, 2H, Ar–H); ESI-MS: 324.16[M–H]-,
327.17[M?H]?; HRMS for C13H14N2O3S2: found 327.0468, calcd. 327.0468
[M?H]?.
Ethyl 4-(((4-chlorophenyl)sulfonyl)methyl)-1,2,3-thiadiazole-5-carboxylate (10b):
white solid, yield 83.2 %, m.p. 114–115 °C; 1H NMR(CDCl3)d: 1.43(t, J = 7.16 Hz,
3H, CH3), 4.41(q, J =7.16 Hz, 2H, CH2), 5.32(s, 2H, SCH2), 7.51(d, J = 8.57 Hz, 2H,
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Ar–H), 7.67(d, J = 8.59 Hz, 2H, Ar–H); ESI-MS: 344.83[M-H]-; HRMS for
C12H11ClN2O3S2: found 346.9916, calcd. 346.9922 [M?H]?.
4-((p-tolylsulfinyl)methyl)-1,2,3-thiadiazole-5-carboxylic acid (11): yellow solid,
yield 31.3 %, m.p. 71–73 °C; 1H NMR(CDCl3)d: 2.42(s, 3H, CH3), 4.90(dd,
J = 12.56 Hz, J = 12.56 Hz, 2H, SCH2), 7.21(d, J = 3.77 Hz, 2H, Ar–H), 7.34(d,
J = 8.21 Hz, 2H, Ar–H); ESI-MS: 281.06[M-H]-;
Theoretical calculations
On the basis of the structure of 9b, an isolated molecule was selected as the initial
structure. DFT-B3LYP/6-31G (d,p) methods in Gaussian 03 package [17] were used
to optimize the structure of 9b. Vibration analysis showed that the optimized
structures were in accordance with the minimum points on the potential energy
surfaces. All the convergent precisions were the system default values, and all the
calculations were carried out on the Nankai Stars supercomputer at Nankai
University.
KARI assay
Cloning, expression and purification of rice KARI
The DNA sequence corresponding to mature KARI was amplified by PCR using the
oligonucleotide primers 50-aaaggatCCATGGTCGCGGCGC-30 and 50-cccAaaTTt-
gaagcttCTACG ATGACTGCCGGAG-30. In these sequences, lower case represents
mismatched bases, underlining indicates the location of introduced BamHI and
HindIII restriction sites, and italics show the Met-54 codon or the reverse
complement of the TAG stop codon. The PCR product was digested with BamHI
and HindIII and ligated into the pET-30a plasmid that had been digested with the
same enzymes. The resultant expression plasmid was used to transform Escherichia
coli BL21(DE3) cells.
A single colony of these cells was inoculated into 20 mL of LB medium
containing 50 mg/mL kanamycin. The culture was incubated overnight at 37 °C and
was used to inoculate each of two 500-mL volumes of LB medium containing
50 mg/mL kanamycin; the cultures were incubated at 37 °C with shaking. When an
OD600 of 0.8 was reached, expression was induced by adding 0.5 mM isopropyl
b-D-thiogalactoside to each culture; these were then incubated at room temperature
(*22 °C) for a further 4 h with shaking and the cells were harvested by
centrifugation.
The frozen cell pellet was thawed, suspended in ice-cold purification buffer
[20 mM Tris–HCl (pH 7.9)/500 mM NaCl] containing 5 mM imidazole and then
treated with lysozyme (10 mg/g of cells for 30 min at 0 °C). The cells were
disrupted by sonication, insoluble material was removed by centrifugation and the
supernatant was passed through a 0.45-mm filter. The cell extract was applied to a
7-mL column of HisꢀBind resin (Novagen) that had been charged by using 50 mM
NiSO4 then equilibrated with purification buffer containing 5 mM imidazole. The
loaded column was washed with 23 mL of the same buffer, followed by 30 mL of
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Synthesis, bioactivity and DFT structure
2007
purification buffer containing 25 mM imidazole, and then KARI was eluted with
30 mL of purification buffer containing 400 mM imidazole. Fractions containing
the enzyme were pooled, concentrated to 2.5 mL by ultrafiltration, and exchanged
into 20 mM Na–Hepes buffer, pH 8.0 using a Pharmacia PD-10 column. The eluate
was snap-frozen in liquid nitrogen and stored at -70 °C [18, 19].
Enzyme and protein assays
Gerwick et al. [19] reported that the inhibition of Escherichia coli KARI is time-
dependent. To characterize the steady-state inhibition constant, Escherichia coli
KARI was preincubated for 10 min with NADPH, Mg2? and the title compound,
then the reaction was initiated with hydroxypyruvate. Under these conditions, the
change in A340 was found to be linear with time.
Herbicidal activities assay
Inhibition of the root growth of rape (Brassica campestris) The evaluated
compounds were dissolved in water and emulsified if necessary. Rape seeds were
soaked in distilled water for 4 h before being placed on a filter paper in a 6-cm Petri
plate, to which 2 mL of inhibitor solution had been added in advance. Usually, 15
seeds were used on each plate. The plate was placed in a dark room and allowed to
germinate for 65 h at 28 1 °C. The lengths of ten rape roots selected from each
plate were measured, and the means were calculated. The check test was carried out
in distilled water only. The percentage of the inhibition was calculated.
Inhibition of the seedling growth of Barnyard grass (Echinochloa crusgalli) The
evaluated compounds were dissolved in water and emulsified if necessary. A total of
ten barnyard grass seeds were placed into a 50-mL cup covered with a layer of glass
beads and a piece of filter paper at the bottom, to which 5 mL of inhibitor solution
had been added in advance. The cup was placed in a bright room and allowed to
germinate for 65 h at 28 1 °C. The heights of seedlings of above-ground plant
parts from each cup were measured, and the means were calculated. The check test
was carried out in distilled water only. The percentage of the inhibition was
calculated.
Acknowledgments This work was funded by National Natural Science Foundation of China (no.
21002090), The Key Innovation Team of Science and Technology in Zhejiang Province (2010R50018)
and Scientific Research Fund of Zhejiang Education Department (Y201018479).
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