Design and synthesis of 1-(benzothiazol-5-yl)-1H-1,2,4-triazol-5-ones as protoporphyrinogen oxidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.03.056

Protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4) is the action target for several structurally diverse herbicides. A series of novel 4-(difluoromethyl)-1- (6-halo-2-substituted-benzothiazol-5-yl)-3-methyl-1H-1,2,4-triazol-5(4H)-ones 2a-z were designed and synthesized via the ring-closure of two ortho-substituents. The in vitro bioassay results indicated that the 26 newly synthesized compounds exhibited good PPO inhibition effects with K_i values ranging from 0.06 to 17.79 μM. Compound 2e, ethyl 2-{[5-(4- (difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl) -6-fluorobenzo-thiazol-2-yl]thio}acetate, was the most potent inhibitor with K_i value of 0.06 μM against mtPPO, comparable to (K_i = 0.03 μM) sulfentrazone. Further green house assays showed that compound 2f (K_i = 0.24 μM, mtPPO), ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5- oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzothiazol-2-yl]thio} propanoate, showed the most promising post-emergence herbicidal activity with broad spectrum even at concentrations as low as 37.5 g ai/ha. Soybean exhibited tolerance to compound 2f at the dosages of 150 g ai/ha, whereas they are susceptible to sulfentrazone even at 75 g ai/ha. Thus, compound 2f might be a potential candidate as a new herbicide for soybean fields.

Full text of DOI:10.1016/j.bmc.2013.03.056

Bioorganic & Medicinal Chemistry 21 (2013) 3245–3255
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of 1-(benzothiazol-5-yl)-1H-1,2,4-triazol-
5-ones as protoporphyrinogen oxidase inhibitors
Zuo Yang a, , Yang Sheng-Gang a, , Luo Yan-Ping a, Tan Ying a,b, Hao Ge-Fei a, Wu Qiong-You a, Xi Zhen b,
,
⇑
Yang Guang-Fu a,
⇑
a Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, PR China
b State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4) is the action target for several structurally diverse herbi-
cides. A series of novel 4-(difluoromethyl)-1-(6-halo-2-substituted-benzothiazol-5-yl)-3-methyl-1H-
1,2,4-triazol-5(4H)-ones 2a–z were designed and synthesized via the ring-closure of two ortho-substitu-
ents. The in vitro bioassay results indicated that the 26 newly synthesized compounds exhibited good
Received 11 February 2013
Revised 7 March 2013
Accepted 9 March 2013
Available online 30 March 2013
PPO inhibition effects with Ki values ranging from 0.06 to 17.79
romethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzo-thiazol-2-yl]thio}acetate,
was the most potent inhibitor with Ki value of 0.06 M against mtPPO, comparable to (Ki = 0.03 M) sulf-
entrazone. Further green house assays showed that compound 2f (Ki = 0.24 M, mtPPO), ethyl 2-{[5-(4-
lM. Compound 2e, ethyl 2-{[5-(4-(difluo-
Keywords:
l
l
Molecular design
Protoporphyrinogen oxidase
1,2,4-Triazol-5-one
Benzothiazole
l
(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzothiazol-2-
yl]thio}propanoate, showed the most promising post-emergence herbicidal activity with broad spectrum
even at concentrations as low as 37.5 g ai/ha. Soybean exhibited tolerance to compound 2f at the dosages
of 150 g ai/ha, whereas they are susceptible to sulfentrazone even at 75 g ai/ha. Thus, compound 2f might
be a potential candidate as a new herbicide for soybean fields.
Herbicide
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
triazolinone derivatives have entered the market as agricultural
herbicides. As shown in Figure 1, for example, sulfentrazone, devel-
Protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4), the last com-
mon enzyme in the tetrapyrrole biosynthetic pathway leading
to chlorophyll in plant and heme in animal,1–6 has been identi-
fied as one of the most significant targets for herbicide discov-
ery. The PPO-inhibiting commercial herbicides include several
structurally diverse groups,7 such as diphenylethers, phenylpy-
razoles, oxadiazoles, triazolinones, thiadiazoles, pyrimidinones,
oxazolidinediones, isoxazoles, and N-phenyl phthalimides. Unfor-
tunately, many products that were initially seen to have great
oped by the FMC Corporation in the 1990s,14 is the first commer-
cial herbicide of this class for weed control in a number of crops,
such as soybean and sugarcane. It provides pre-emergence control
of several broadleaf weeds, as well as a number of selected grass
weeds, for the soybean markets. A few years later, carfentrazone-
ethyl15 and azafenidin16 were subsequently developed by FMC
and DuPont, respectively, as commercial products. Bencarbazone
developed by Bayer and Arysta LifeScience17 is a relatively new
triazolinone herbicide for the post-emergence control of broadleaf
weeds in cereals and corn. It provides good control of bedstraw,
velvetleaf, redroot pigweed, common lambsquarter, and speedwell
at application rates of 20–30 g ai/haꢀ1. Bencarbazone has many of
the features associated with Protox herbicides, particularly those of
the Protox herbicide sulfentrazone. The most striking chemical fea-
ture of bencarbazone is the replacement of the phenyl 4-chloro
group with a thioamide group. Although the above-mentioned
phenyl triazolinone herbicides showed ultrahigh herbicidal activ-
ity, they displayed no safety to crops. When spraying these herbi-
cides for weed control, the addition of safe agents is necessary.
Therefore, the design of new phenyl triazolinone derivatives with
both high herbicidal activity and crop safety is an interesting syn-
thetic goal.
potential were not successful and only
a few compounds
achieved any significant market share. While it was relatively
easy to identify chemistries with good PPO-inhibiting herbicidal
activity, it was much more difficult to demonstrate clear crop
selectivity, with either pre-emergent or post-emergent
application.7
Among the above mentioned chemical families, phenyl triazoli-
none derivatives have attracted considerable attention in the field
of pesticide chemistry in recent decades.8–13 To date, four phenyl
⇑
Corresponding authors. Tel.: +86 27 67867800; fax: +86 27 67867141.
E-mail address: gfyang@mail.ccnu.edu.cn (Y. Guang-Fu).
Co-first authors.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.056

3246
Z. Yang et al. / Bioorg. Med. Chem. 21 (2013) 3245–3255
F
F
O
Cl
O
Cl
F
F
O
F
O
H2N
Cl
O
N
N
N
F
N
F
Cl
N
N
F
Cl
N
N
N
N
S
N
N
O
S
HN
SO2CH3
O
O
Cl
OC2H5
Sulfentrazone
Carfentrazone-ethyl
Bencarbazone
Azafenidin
Figure 1. Structure of phenyl triazolinone-type PPO-inhibiting herbicides.
The existing triazolinone-type herbicides always possess a com-
mon structural feature: the (2,4,5-trisubstituted)-phenyl group. Of
the investigated phenyl substitution patterns, most active com-
pounds were modified with F or Cl at C-2, and Cl (e.g., sulfentraz-
one, carfentrazone-ethyl and azafenidin) or CSNH2 (e.g.,
bencarbazone) at C-4, while a variety of other groups were found
to be acceptable at C-5.8–17 In addition, the benzothiazole deriva-
tives are known to possess a wide range of biological activities
including antitumor, antiviral, antimicrobial, and antiglutamate
properties.18–23 Some of the benzothiazole derivatives have also
been widely used in agriculture. For example, as shown in Figure 2,
bentaluron, chlobenthiazone and TCMTB have been used as com-
mercial fungicides for many years.22,23 Previously, we have also de-
signed a series of N-(benzothiazol-5-yl)-4,5,6,7-tetrahydro-1H-
isoindole-1,3(2H)-diones and N-(benzothiazol-5-yl)isoindoline-
1,3-diones by the ring-closing strategy of substituents at C-4 and
C-5.24 Most interestingly, we discovered compound 1 with broad
spectrum herbicidal activity at the concentration of 37.5 g ai/ha.
Very promisingly, compound 1 exhibited high safety to wheat even
at the dosage of 300 g ai/ha, indicating that the introduction of the
benzothiazole moiety might be an effective way to improve the
crop selectivity of PPO-inhibiting herbicides. Therefore, as a contin-
uation of our research work on the development of new PPO inhib-
itors,11–13,24–31 we were very interested in the design and synthesis
of 1-(benzothiazol-5-yl)-1H-1,2,4-triazol-5-ones 2a–z (Fig. 3).
Herein, we report the detailed synthetic chemistry, PPO inhibition
effects, and herbicidal activities of compounds 2a–z, and the re-
sults indicated that some compounds displayed good PPO inhibi-
tion effects, promising herbicidal activity, and excellent crop
safety.
2. Materials and methods
All chemical reagents were commercially available and treated
with standard methods before use. Solvents were dried in a routine
way and redistilled before use. 1H NMR spectra were recorded on a
VARIAN Mercury-Plus 600 or 400 spectrometer in CDCl3 or DMSO-
d6 with TMS as the internal reference. Mass spectral data were ob-
tained on a FINNIGAN Mass platform TRACEMS 2000 by electro-
spray ionization (ESI-MS). Elemental analyses were performed on
a Vario EL III elemental analysis instrument. Melting points were
taken on
a Buchi B-545 melting point apparatus and were
uncorrected.
2.1. Preparation of pyruvic acid, 2,4-disubstitutedhydrazone (3)
Under a nitrogen atmosphere, a stirred solution of 2,4-disubsti-
tuted aniline (0.05 mol) in 50 mL of concentrated HCl was cooled
to ꢀ9 °C, and a solution of sodium nitrite (4.25 g, 0.05 mol) in
18 mL of water was added dropwise at a rate to keep the reaction
mixture temperature under ꢀ9 °C. The complete addition required
30 min. The reaction mixture was stirred for an additional 1 h at
ꢀ9 to 0 °C. Then,
a solution of stannous chloride (24.65 g,
0.13 mol) in 70 mL of concentrated HCl was added dropwise at a
rate to maintain the reaction mixture temperature under ꢀ9 to
0 °C. The complete addition required 40 min. The reaction mixture
stirred at ꢀ9 to 0 °C for an additional 30 min, then was allowed to
warm to ambient temperature where it was stirred for 2 h. Water
(40 mL) was added to the reaction mixture, followed by addition of
a solution of pyruvic acid (4.4 g, 0.05 mol) in water (40 mL) over a
5 min period. The reaction mixture was stirred for an additional
30 min and the precipitate was then filtered, washed with water
and dried to give of pyruvic acid, 2,4-disubstituted hydrone.
Cl
O
N
S
SCN
N
N
S
N
H
N
H
S
O
S
Chlobenthiazone
Bentaluron
TCMTB
Figure 2. Chemical structures of some typical benzothiazole derivatives.
O
F
N
S
O
X
O
N
S
O
O
F
O
N
N
F
S
Ring-closing strategy
1
N
N
R
S
Cl
F
2
Cl
S
N
N
a~z
X = F, Cl, Br
R, various substituents
N
F
O
N
O
CH3
H
Sulfentrazone
Figure 3. Design strategy of the title compounds.

Z. Yang et al. / Bioorg. Med. Chem. 21 (2013) 3245–3255
3247
2.1.1. 2-(2-(4-Bromo-2-fluorophenyl)hydrazono)propanoic acid
(3a)
with methylene chloride, and the combined extracts were washed
with water; the organic layer was dried with sodium sulfate, filtered,
and concentrated under reduced pressure to a residue. The residue
was purified by column chromatography on silica gel using 20:1
petroleum ether/acetone as an eluent. The appropriate fractions
were combined and concentrated under reduced pressure to yield
intermediates.
Yield, 88%; mp 176–178 °C [lit.32 172–173 °C]; 1H NMR
(600 MHz, CDCl3), d 2.08 (s, 3H, CH3), 7.35 (d, 1H, J = 9 Hz, ArH),
7.52 (d, 1H, J = 10.8 Hz, ArH), 7.61 (t, 1H, J = 9 Hz, ArH), 9.27 (s,
1H, NH), 12.25 (s, 1H, COOH); EI-MS, m/z 273.9 (M)+.
2.1.2. 2-(2-(2,4-Dichlorophenyl)hydrazono)propanoic acid (3b)
Yield, 88%; mp 188–190 °C [lit.32 193–194 °C]; 1H NMR
(600 MHz, CDCl3), d 2.11 (s, 3H, CH3), 7.39 (d, 1H, J = 9 Hz, ArH),
7.53 (d, 1H, J = 8.4 Hz, ArH), 7.64 (q, 1H, J = 6.6 Hz, ArH), 8.65 (s,
1H, NH), 12.40 (s, 1H, COOH); EI-MS, m/z 245.9 (M)+.
2.3.1. 1-(4-Bromo-2-fluorophenyl)-4-difluoromethyl-3-methyl-
1H-1,2,4-triazol-5-one (5a)
Yield, 91%; mp 143–145 °C; [lit.32 148–151 °C]; 1H NMR
(400 MHz, CDCl3), d 2.48 (s, 3H, CH3), 7.05 (t, 1H, J = 58 Hz, CHF2),
7.38–7.44 (m, 3H, ArH). EI-MS, m/z 320.9 (M)+.
2.1.3. 2-(2-(2-Bromo-4-fluorophenyl)hydrazono)propanoic acid
(3c)
2.3.2. 1-(2,4-Dichlorophenyl)-4-difluoromethyl-3-methyl-1H-
1,2,4-triazol-5-one (5b)
Yield, 88%; mp 153–155 °C; 1H NMR (600 MHz, CDCl3), d 2.10 (s,
3H, CH3), 7.27 (t, 1H, J = 9 Hz, ArH), 7.57 (d, 1H, J = 9 Hz, ArH), 7.67
(q, 1H, J = 5.4 Hz, ArH), 8.43 (s, 1H, NH), 12.34 (s, 1H, COOH); EI-MS,
m/z 273.9 (M)+.
Yield, 83%; mp 111–113 °C [lit.32 108–110 °C]; 1H NMR
(400 MHz, CDCl3), d 2.48 (s, 3H, CH3), 7.05 (t, 1H, J = 58 Hz, CHF2),
7.26–7.55 (m, 3H, ArH); EI-MS, m/z 292.9 (M)+.
2.2. Preparation of 1-(2,4-disubstituted phenyl)-3-methyl-4H-
1,2,4-triazol-5-one (4)
2.3.3. 1-(2-Bromo-4-fluorophenyl)-4-difluoromethyl-3-methyl-
1H-1,2,4-triazol-5-one (5c)
Yield, 75%; mp 99–101 °C; 1H NMR(400 MHz, CDCl3), d 2.48 (s,
3H, CH3), 7.07 (t, 1H, J = 58 Hz, CHF2), 7.14–7.47 (m, 3H, ArH). EI-
MS, m/z) 320.9 (M)+.
To
a
stirred suspension of 2,4-disubstitutedhydrazone
(0.05 mol) in 30 mL of toluene was added triethylamine (5.1 g,
0.05 mol). The reaction mixture became homogeneous and diphen-
ylphosphoryl azide (13.75 g, 0.05 mol) was added. The reaction
mixture was heated to reflux for 2 h. Then it was allowed to cool
to ambient temperature and extracted with 50 mL of aqueous
1 N NaOH. The extract was neutralized with concentrated HCl
and the precipitate was collected by filtration, washed with water
and dried to give 1-(2,4-disubstituted phenyl)-3-methyl-4H-1,2,4-
triazol-5-one.
2.4. Preparation of 1-(2,4-disubstituted-5-nitrophenyl)-3-
methyl-4-difluoromethane-1,2,4-triazol-5-one (6)
To a stirred solution of 1-(2,4-disubstituted phenyl)-3-methyl-
4-difluoromethyl-1,2,4-triazol-5-one (0.01 mol) in 10 mL of con-
centrated H2SO4 was slowly added 68% nitric acid (0.9 g); the tem-
perature was maintained at 25 °C for 60 min, and then the mixture
was poured into ice water. The resulting precipitate was collected
by filtration, washed with water and dried to give 1-(2,4-disubsti-
tuted-5-nitrophenyl)-3-methyl-4-difluoro-methane-1,2,4-triazol-
5-one.
2.2.1. 1-(4-Bromo-2-fluorophenyl)-3-methyl-1H-1,2,4-triazol-
5(4H)-one (4a)
Yield, 88%; mp 204–206 °C [lit.32 201–203 °C]; 1H NMR
(400 MHz, CDCl3), d 2.28 (s, 3H, CH3), 7.22–7.44 (m, 3H, ArH),
11.72 (s, 1H, NH); EI-MS, m/z 270.9 (M)+.
2.4.1. 1-(4-Bromo-2-fluoro-5-nitrophenyl)-4-difluoromethyl-3-
methyl-1H-1,2,4-triazol-5-one (6a)
2.2.2. 1-(2,4-Dichlorophenyl)-3-methyl-1H-1,2,4-triazol-5(4H)-
one (4b)
Yield, 79%; mp 108–110 °C [lit.32 99–105 °C]. 1H NMR
(600 MHz, CDCl3), d 2.50 (s, 3H, CH3), 7.05 (t, 1H, J = 58.2 Hz,
CHF2), 7.65 (d, 1H, J = 9.6 Hz, ArH), 8.21 (d, 1H, J = 6.6 Hz, ArH).
EI-MS, m/z 365.9 (M)+.
Yield, 92%; mp 179–181 °C [lit.32 174–175 °C]; 1H NMR
(400 MHz, CDCl3), d 2.27 (s, 3H, CH3), 7.19–7.56 (m, 3H, ArH),
11.85 (s, 1H, NH). EI-MS, m/z 242.9 (M)+.
2.4.2. 1-(2,4-Dichloro-5-nitrophenyl)-4-difluoromethyl-3-
methyl-1H-1,2,4-triazol-5-one (6b)
2.2.3. 1-(2-Bromo-4-fluorophenyl)-3-methyl-1H-1,2,4-triazol-
5(4H)-one (4c)
Yield, 94%; mp 103–105 °C [lit.32 95–97 °C]. 1H NMR (600 MHz,
CDCl3), d 2.50 (s, 3H, CH3), 7.05 (t, 1H, J = 58.2 Hz, CHF2), 7.60 (s, 1H,
ArH), 8.10 (s, 1H, ArH). EI-MS, m/z 337.9 (M)+.
Yield, 85%; mp 179–181 °C; 1H NMR (400 MHz, CDCl3), d 2.28 (s,
3H, CH3), 7.13–7.48 (m, 3H, ArH), 11.70 (s, 1H, NH); EI-MS, m/z 270.9
(M)+.
2.4.3. 1-(2-Bromo-4-fluoro-5-nitrophenyl)-4-difluoromethyl-3-
methyl-1H-1,2,4-triazol-5-one (6c)
2.3. Preparation of 1-(2,4-disubstituted phenyl)-3-methyl-4-
difluoromethyl-1,2,4-triazol-5-one (5)
Yield, 70%; mp 80–82 °C; 1H NMR (600 MHz, CDCl3), d 2.50 (s,
3H, CH3), 7.05 (t, 1H, J = 58.2 Hz, CHF2), 7.71 (d, 1H, J = 9.6 Hz,
ArH), 8.21 (d, 1H, J = 6.6 Hz, ArH). EI-MS, m/z 365.9 (M)+.
To a stirred solution of 1-(2,4-disubstituted phenyl)-3-methyl-
4H-1,2,4-triazol-5-one (0.01 mol) in THF (50 mL), powdered potas-
sium hydroxide (1.12 g, 0.02 mol) and tetrabutylammonium bro-
mide (0.32 g, 1 mmol) were added subsequently, and CHF2Cl was
bubbled through the reaction mixture at reflux temperature. After
12 h, an additional portion of powdered potassium hydroxide
(0.56 g, 0.01 mol) was added to the reaction mixture, and CHF2Cl
continued to be bubbled through the reaction mixture. The progress
of the reaction was monitored by TLC and when it was complete, the
reaction mixture was concentrated under reduced pressure and
water (100 mL) was added. The resulting solution was extracted
2.5. Preparation of 1-(5-amino-2,4-disubstitutedphenyl)-3-
methyl-4-difluoromethane-1,2,4-triazol-5-one (7)
1-(2,4-Disubstituted-5-nitrophenyl)-3-methyl-4-difluorome-
thane-1,2,4-triazol-5-one (0.01 mmol) and ammonium chloride
(1.06 g, 0.02 mol) were dissolved in a mixture of ethanol (25 mL)
and water (3 mL). The reaction mixture was heated to reflux and
powered iron (1.12 g, 0.02 mol) was added at reflux temperature.
The resulting mixture was refluxed for 4 h and filtered through dia-

3248
Z. Yang et al. / Bioorg. Med. Chem. 21 (2013) 3245–3255
tomaceous earth, and the filtrate was concentrated under reduced
pressure. The residue was extracted with ethyl acetate, and the com-
bined extracts were washed with brine; the organic layer was dried
with sodium sulfate, filtered, and concentrated under reduced pres-
sure to a residue.
2.7. General procedure for the synthesis of the title compounds
(2a–z)
To a stirred solution of 1 mmol of intermediate 6 in acetone
(20 mL) was added potassium carbonate (1.2 mmol). The mixture
was stirred for 10 min before the addition of the corresponding ha-
lide derivative (1 mmol). The progressof the reactionwas monitored
by TLC and when it was complete, the reaction mixture was filtered
and concentrated. The residue was purifiedby column chromatogra-
phy on silica gel using 10:1 petroleum ether/acetone as an eluent.
2.5.1. 1-(5-Amino-4-bromo-2-fluorophenyl)-4-difluoromethyl-
3-methyl-1H-1,2,4-triazol-5-one (7a)
Yield, 79%; mp 121–123 °C [lit.32 117–119 °C]. 1H NMR
(600 MHz, CDCl3), d 2.46 (s, 3H, CH3), 4.05 (s, 2H, NH2), 6.87 (d,
1H, J = 6.6 Hz, ArH), 7.10 (t, 1H, J = 58.2 Hz, CHF2), 7.33 (d, 1H,
J = 9.6 Hz, ArH). EI-MS, m/z 335.9 (M)+.
2.7.1. 1-[6-Fluoro-2-(methylthio)benzothiazol-5-yl]-3-methyl-
4-difluoromethyl-1H-1,2,4-triazol-5(4H)-one (2a)
White solid. Yield 85%; mp 129–131 °C; 1H NMR (400 MHz,
CDCl3): d 2.50 (s, 3H, CH3), 2.80 (s, 3H, CH3), 7.09 (t, 1H,
J = 58 Hz, CHF2), 7.61 (d, 1H, J = 9.6 Hz, ArH), 7.98 (d, 1H,
J = 6.4 Hz, ArH); EI-MS: 346.2 (M)+; Anal. Calcd for C12H9F3N4OS2:
C, 41.61; H, 2.62 N, 16.18; S, 18.52. Found: C, 41.37; H, 2.28; N,
16.08; S, 18.69.
2.5.2. 1-(5-Amino-2,4-dichlorophenyl)-4-difluoromethyl-3-
methyl-1H-1,2,4-triazol-5-one (7b)
Yield, 94%; mp 137–139 °C [lit.32 133–135 °C]. 1H NMR
(600 MHz, CDCl3), d 2.46 (s, 3H, CH3), 4.19 (s, 2H, NH2), 6.82 (s,
1H, ArH), 7.05 (t, 1H, J = 58.2 Hz, CHF2), 7.40 (s, 1H, ArH). EI-MS,
m/z 307.9 (M)+.
2.7.2. 1-[6-Fluoro-2-(propylthio)benzothiazol-5-yl]-3-methyl-4-
difluoromethyl-1H-1,2,4-triazol-5(4H)-one (2b)
2.5.3. 1-(5-Amino-2-bromo-4-fluorophenyl)-4-difluoromethyl-
3-methyl-1H-1,2,4-triazol-5-one (7c)
White solid. Yield 75%; mp 103–105 °C; 1H NMR (400 MHz,
CDCl3): d 1.08 (t, 3H, J = 7.2 Hz, CH3), 1.83–1.89 (m, 2H, CH2),
2.50 (s, 3H, CH3), 3.33 (t, 2H, J = 7.2 Hz, CH2), 7.08 (t, 1H,
J = 58 Hz, CHF2), 7.60 (d, 1H, J = 9.6 Hz, ArH), 7.98 (d, 1H,
J = 6.4 Hz, ArH); EI-MS m/z: 374.3 (M)+; Anal. Calcd for
Yield, 70%; mp 89–91 °C; 1H NMR (600 MHz, CDCl3), d 2.46 (s,
3H, CH3), 3.82 (s, 2H, NH2), 6.84 (d, 1H, J = 8.4 Hz, ArH), 7.29 (t,
1H, J = 58.2 Hz, CHF2), 7.40 (d, 1H, J = 10.8 Hz, ArH). EI-MS, m/z
335.9 (M)+.
C
14H13F3N4OS2: C, 44.91; H, 3.50; N, 14.96; S, 17.13. Found: C,
2.6. Preparation of 1-(6-substituted-2-mercaptobenzothiazol-5-
yl)-3-methyl-4-(difluoromethyl)-1H-1,2,4-triazol-5(4H)-one (8)
44.71; H, 3.54; N, 14.82; S, 17.18.
2.7.3. 1-[2-(Allylthio)-6-fluorobenzothiazol-5-yl]-3-methyl-4-
difluoromethyl-1H-1,2,4-triazol-5(4H)-one (2c)
A solution of 1-(6-substituted-2-mercapto-benzothiazol-5-yl)-
3-methyl-4-(difluoromethyl)-1H-1,2,4-triazol-5(4H)-one
White solid. Yield 76%; mp 79–81 °C; 1H NMR (400 MHz,
CDCl3): d 2.50 (s, 3H, CH3), 4.00 (d, 2H, J = 7.2 Hz, CH2), 5.30 (dd,
2H, J = 16.8, 10.4 Hz, @CH2), 5.98–6.04 (m, 1H, CH), 7.08 (t, 1H,
J = 58 Hz, CHF2), 7.60 (d, 1H, J = 9.6 Hz, ArH), 7.98 (d, 1H,
J = 6.4 Hz, ArH); EI-MS: 433.2 (M)+; Anal. Calcd for C14H11F3N4OS2:
C, 45.15; H, 2.98; N, 15.31; S, 17.22. Found: C, 38.64; H, 2.54; N,
12.77; S, 14.72.
(0.01 mol) and potassium O-ethyl dithiocarbonate (3.2 g, 0.02 mol)
in 30 mL DMF was heated at 120 °C for 3 h. The reaction mixture
was diluted with 100 mL of water and acidified with concentrated
HCl solution to pH 3 to induce precipitation. Stirring was continued
for 10 min and the precipitate was collected by filtration. The fil-
tration residue was washed with water and dried to give 1-(6-
substituted-2-mercaptobenzothiazol-5-yl)-3-methyl-4-(difluoro-
methyl)-1H-1,2,4-triazol-5(4H)-one.
2.7.4. 1-[2-(Butylthio)-6-fluorobenzothiazol-5-yl]-3-methyl-4-
difluoromethyl-1H-1,2,4-triazol-5(4H)-one (2d)
2.6.1. 1-(6-Fluoro-2-mercaptobenzothiazol-5-yl)-3-methyl-4-
difluoromethyl-1H-1,2,4-triazol-5(4H)-one (8a)
White solid. Yield 78%; mp 67–68 °C; 1H NMR (400 MHz, CDCl3):
d 0.97 (t, 3H, J = 7.2 Hz, CH3), 1.47–1.82 (m, 4H, CH2), 2.50 (s, 3H,
CH3), 3.35 (t, 2H, J = 7.2 Hz, CH2), 7.08 (t, 1H, J = 58 Hz, CHF2), 7.61
(d, 1H, J = 9.6 Hz, ArH), 8.00 (d, 1H, J = 6.4 Hz, ArH); EI-MS: 388.4
(M)+; Anal. Calcd for C15H15F3N4OS2: C, 46.38; H, 3.89; N, 14.42; S,
16.51. Found: C, 46.43; H, 4.02; N, 14.11; S, 16.17.
White solid. Yield 73%; mp 248–250 °C; 1H NMR (400 MHz,
DMSO-d6): d 2.50 (s, 3H, CH3), 7.52 (t, 1H, J = 58 Hz, CHF2), 7.49 (d,
1H, J = 10.0 Hz, ArH), 7.90 (d, 1H, J = 6.4 Hz, ArH), 13.99 (s, 1H, SH);
EI-MS m/z: 332.0 (M)+; Anal. Calcd for C11H7F3N4OS2: C, 39.76; H,
2.12; N, 16.86; S, 19.30. Found: C, 39.56; H, 2.16; N, 17.00; S, 19.42.
2.7.5. Ethyl 1-{5-[4-difluoromethyl-3-methyl-5-oxo-4,5-
dihydro-1,2,4-triazol-1-yl]-6-fluoro benzothiazol-2-
ylthio}acetate (2e)
2.6.2. 1-(6-Chloro-2-mercaptobenzothiozol-5-yl)-3-methyl-4-
difluoromethyl-1H-1,2,4-triazol-5(4H)-one (8b)
White solid. Yield 68%; mp 201–203 °C; 1H NMR (400 MHz,
CDCl3): d 2.52 (s, 3H, CH3), 7.11 (t, 1H, J = 58 Hz, CHF2), 7.25 (s,
1H, ArH), 7.54 (s, 1H, ArH), 11.03 (s, 1H, SH); EI-MS m/z: 348.1
(M)+; Anal. Calcd for C11H7ClF2N4OS2: C, 37.88; H, 2.02; N, 16.06;
S, 18.39. Found: C, 37.57; H, 1.96; N, 16.30; S, 18.85.
White solid. Yield 84%; mp 91–93 °C; 1H NMR (400 MHz,
CDCl3): d 1.29 (t, 3H, J = 7.2 Hz, CH3), 2.50 (s, 3H, CH3), 4.16 (s,
2H, CH2), 4.24 (q, 2H, J = 7.2 Hz, CH2), 7.08 (t, 1H, J = 58 Hz, CHF2),
7.62 (d, 1H, J = 9.2, 6.4 Hz, ArH), 7.95 (d, 1H, J = 9.2, 6.4 Hz, ArH);
EI-MS: 418.4 (M)+; Anal. Calcd for C15H13F3N4O3S2: C, 43.06; H,
3.13; N, 13.39; S, 15.33. Found: C, 43.10; H, 3.19; N, 13.38; S, 15.46.
2.6.3. 1-(6-Bromo-2-mercaptobenzothiazol-5-yl)-3-methyl-4-
difluoromethyl-1H-1,2,4-triazol-5(4H)-one (8c)
2.7.6. Ethyl 1-{5-[4-difluoromethyl-3-methyl-5-oxo-4,5-
dihydro-1,2,4-triazol-1-yl]-6-fluoro benzothiazol-2-
ylthio}propanoate (2f)
White solid. Yield 48%; mp 254–256 °C; 1H NMR (400 MHz,
CDCl3): d 2.52 (s, 3H, CH3), 7.12 (t, 1H, J = 58 Hz, CHF2), 7.15
(s, 1H, ArH), 7.69 (s, 1H, ArH), 11.05 (s, 1H, SH); EI-MS m/z:
391.9 (M)+; Anal. Calcd for C11H7BrF2N4OS2: C, 33.60; H, 1.79;
N, 14.25; S, 16.31. Found: C, 33.94; H, 2.11; N, 13.72; S,
15.24.
White solid. Yield 66%; mp 60–61; 1H NMR (400 MHz, CDCl3): d
1.27 (t, 3H, J = 7.2 Hz, CH3), 1.71 (d, 3H, J = 7.2 Hz, CHCH3), 2.50 (s,
3H, CH3), 4.23 (q, 2H, J = 7.2 Hz, OCH2), 4.67 (m, 1H, J = 7.2 Hz, CH),
7.09 (t, 1H, J = 58 Hz, CHF2), 7.62 (d, 1H, J = 9.2, 6.4 Hz, ArH), 7.95

Z. Yang et al. / Bioorg. Med. Chem. 21 (2013) 3245–3255
3249
(d, 1H, J = 9.2, 6.4 Hz, ArH); EI-MS: 432.2 (M)+; Anal. Calcd for
16H15F3N4O3S2: C, 44.44; H, 3.50; N, 12.96; S, 14.83. Found: C,
44.45; H, 3.23; N, 12.92; S, 14.75.
J = 58 Hz, CHF2), 7.90 (d, 2H, J = 6.8 Hz, ArH); EI-MS: 364.3
(M+H)+; Anal. Calcd for C12H9ClF2N4OS2: C, 39.73; H, 2.50; N,
15.44; S, 17.68. Found: C, 39.41; H, 2.44; N, 15.45; S, 17.43.
C
2.7.7. S-5-[4-Difluoromethyl-3-methyl-5-oxo-4,5-dihydro-1,2,4-
triazol-1-yl]-6-fluorobenzothiazol-2-yl O-methyl
carbonothioate (2g)
2.7.14. 1-[6-Chloro-2-(propylthio)benzothiazol-5-yl]-3-methyl-
4-difluoromethyl-1H-1,2,4-triazol-5(4H)-one (2n)
White solid. Yield 54%; mp 88–90 °C; 1H NMR (400 MHz,
CDCl3): d 1.07 (t, 3H, J = 7.2 Hz, CH3), 1.83–1.89 (m, 2H, CH2),
2.50 (s, 3H, CH3), 3.33 (t, 2H, J = 7.2 Hz, CH2), 7.08 (t, 1H,
J = 58 Hz, CHF2), 7.89 (d, 2H, J = 6.8 Hz, ArH); EI-MS: 392.4
(M+H)+; Anal. Calcd for C14H13ClF2N4OS2: C, 43.02; H, 3.35; N,
14.33; S, 16.41. Found: C, 43.17; H, 3.29; N, 14.19; S, 16.39.
White solid. Yield 96%; mp 138–140 °C; 1H NMR (400 MHz,
CDCl3): d 2.51 (s, 3H, CH3), 4.01 (s, 3H, OCH3), 7.09 (t, 1H,
J = 58 Hz, CHF2), 7.75 (d, 1H, J = 9.2 Hz, ArH), 8.10 (d, 1H,
J = 6.4 Hz, ArH); EI-MS: 390.2 (M)+; Anal. Calcd for C13H9F3N4O3S2:
C, 40.00; H, 2.32; N, 14.35; S, 16.43. Found: C, 40.11; H, 2.44; N,
14.11; S, 16.46.
2.7.15. 1-[2-(Allylthio)-6-chlorobenzothiazol-5-yl]-3-methyl-4-
difluoromethyl-1H-1,2,4-triazol-5(4H)-one (2o)
2.7.8. S-5-[4-Difluoromethyl-3-methyl-5-oxo-4,5-dihydro-1,2,4-
triazol-1-yl]-6-fluorobenzothiazol-2-yl O-ethyl carbonothioate
(2h)
White solid. Yield 75%; mp 119–121 °C; 1H NMR (400 MHz,
CDCl3): d 2.50 (s, 3H, CH3), 4.00 (d, 2H, J = 7.2 Hz, CH2), 5.30 (dd,
2H, J = 16.8, 10.0 Hz, @CH2), 5.97–6.04 (m, 1H, CH), 7.08 (t, 1H,
J = 58 Hz, CHF2), 7.91 (d, 2H, J = 9.6 Hz, ArH); EI-MS: 389.2 (M)+;
Anal. Calcd for C14H11ClF2N4OS2: C, 43.24; H, 2.85; N, 14.41; S,
16.49. Found: C, 43.42; H, 2.85; N, 14.56; S, 16.51.
White solid. Yield 90%; mp 149–151 °C; 1H NMR (400 MHz,
CDCl3): d 1.41 (t, 3H, J = 7.2 Hz, CH3), 2.51 (s, 3H, CH3), 4.47 (q,
2H, J = 7.2 Hz, CH2), 7.09 (t, 1H, J = 58 Hz, CHF2), 7.75 (d, 1H,
J = 9.2 Hz, ArH), 8.09 (d, 1H, J = 6.4 Hz, ArH); EI-MS: 404.3 (M)+;
Anal. Calcd for C14H11F3N4O3S2: C, 41.58; H, 2.74; N, 13.85; S,
15.86. Found: C, 41.30; H, 2.96; N, 13.88; S, 15.99.
2.7.16. 1-[2-(Butylthio)-6-chlorobenzothiazol-5-yl]-3-methyl-4-
difluoromethyl-1H-1,2,4-triazol-5(4H)-one (2p)
2.7.9. Methyl 2-[5-(4-difluoromethyl-3-methyl-5-oxo-4,5-
dihydro-1,2,4-triazol-1-yl)-6-fluoro benzothiazol-2-
ylthio]acetate (2i)
White solid. Yield 78%; mp 104–106 °C; 1H NMR (400 MHz,
CDCl3): d 0.97 (t, 3H, J = 7.2 Hz, CH3), 1.48–1.85 (m, 4H, CH2),
2.50 (s, 3H, CH3), 3.36 (t, 2H, J = 7.2 Hz, CH2), 7.08 (t, 1H,
J = 58 Hz, CHF2), 7.90 (d, 2H, J = 8.4 Hz, ArH); EI-MS: 404.4
(MꢀH)+; Anal. Calcd for C15H15ClF2N4OS2: C, 44.50; H, 3.73; N,
13.84; S, 15.84. Found: C, 44.05; H, 3.65; N, 13.98; S, 16.59.
White solid. Yield 92%; mp 98–100 °C; 1H NMR (400 MHz,
CDCl3): d 2.50 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 4.12 (s, 2H, CH2),
7.09 (t, 1H, J = 58 Hz, CHF2), 7.61 (d, 1H, J = 9.2 Hz, ArH), 7.95 (d,
1H, J = 6.4 Hz, ArH); EI-MS: 404.6 (M)+; Anal. Calcd for
C
14H11F3N4O3S2: C, 41.58; H, 2.74; N, 13.85; S, 15.86. Found: C,
2.7.17. S-6-Chloro-5-[4-difluoromethyl-3-methyl-5-oxo-4,5-
dihydro-1,2,4-triazol-1-yl]benzothiazol-2-yl O-methyl
carbonothioate (2q)
41.47; H, 2.88; N, 13.92; S, 15.61.
2.7.10. 3-{5-[4-Difluoromethyl-3-methyl-5-oxo-4,5-dihydro-
1,2,4-triazol-1-yl]-6-fluorobenzothiazol-2-ylthio}propanoic
acid (2j)
White solid. Yield 97%; mp 165–167 °C; 1H NMR (400 MHz,
CDCl3): d 2.52 (s, 3H, CH3), 4.02 (s, 3H, OCH3), 7.09 (t, 1H,
J = 58 Hz, CHF2), 8.06 (s, 2H, ArH); EI-MS: 408.4 (M+H)+; Anal. Calcd
for C13H9ClF2N4O3S2: C, 38.38; H, 2.23; N, 13.77; S, 15.76. Found: C,
38.56; H, 2.50; N, 13.78; S, 15.57.
White solid. Yield 84%; mp 161–163 °C; 1H NMR (400 MHz,
DMSO-d6): d 2.39 (s, 3H, CH3), 2.82 (t, 2H, J = 6.8 Hz, CH2), 3.53 (t,
2H, J = 6.8 Hz, CH2), 7.55 (t, 1H, J = 58 Hz, CHF2), 8.06 (d, 1H,
J = 6.8 Hz, ArH), 8.20 (d, 1H, J = 6.0 Hz, ArH), 12.53 (s, 1H, OH); EI-
MS: 404.4 (M)+; Anal. Calcd for C14H11F3N4O3S2: C, 41.58; H,
2.74; N, 13.85; S, 15.86. Found: C, 41.47; H, 2.76; N, 13.67; S, 15.58.
2.7.18. S-6-Chloro-5-[4-difluoromethyl-3-methyl-5-oxo-4,5-
dihydro-1,2,4-triazol-1-yl]benzothiazol-2-yl O-ethyl
carbonothioate (2r)
White solid. Yield 95%; mp 196–198 °C; 1H NMR (400 MHz,
CDCl3): d 1.42 (t, 3H, J = 7.2 Hz, CH3), 2.51 (s, 3H, CH3), 4.48 (q,
2H, J = 7.2 Hz, CH2), 7.09 (t, 1H, J = 58 Hz, CHF2), 8.06 (s, 2H,
ArH); EI-MS: 420.7 (M)+; Anal. Calcd for C14H11ClF2N4O3S2: C,
39.96; H, 2.63; N, 13.31; S, 15.24. Found: C, 40.06; H, 2.83; N,
13.13; S, 15.09.
2.7.11. 2-[5-(4-Difluoromethyl-3-methyl-5-oxo-4,5-dihydro-
1,2,4-triazol-1-yl)-6-fluorobenzothiazol-2-ylthio]propanoic
acid (2k)
White solid. Yield 69%; mp 161–163 °C; 1H NMR (400 MHz,
DMSO-d6): d 1.64 (d, 3H, J = 7.2 Hz, CH3), 2.40 (s, 3H, CH3), 4.63
(q, H, J = 7.2 Hz, CH), 7.55 (t, 1H, J = 58 Hz, CHF2), 8.06–8.90 (m,
2H, ArH), 13.23 (s, 1H, CO2H); EI-MS: 404.0 (M)+; Anal. Calcd for
2.7.19. Methyl 1-{6-chloro-5-[4-difluoromethyl-3-methyl-5-
oxo-4,5-dihydro-1,2,4-triazol-1-yl] benzothiazol-2-
ylthio}acetate (2s)
C14H11F3N4O3S2: C, 41.58; H, 2.74; N, 13.85; S, 15.86. Found: C,
41.30; H, 2.88; N, 13.80; S, 15.61.
White solid. Yield 89%; mp 104–106 °C; 1H NMR (400 MHz,
CDCl3): d 2.51 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 4.18 (s, 2H, CH2),
7.09 (t, 1H, J = 58 Hz, CHF2), 7.91 (s, 2H, ArH); EI-MS: 420.4
(MꢀH)+; Anal. Calcd for C14H11ClF2N4O3S2: C, 39.96; H, 2.63; N,
13.31; S, 15.24. Found: C, 39.78; H, 2.83; N, 13.23; S, 15.34.
2.7.12. 2-[5-(4-Difluoromethyl-3-methyl-5-oxo-4,5-dihydro-
1,2,4-triazol-1-yl)-6-fluorobenzothiazol-2-ylthio]acetic acid (2l)
White solid. Yield 77%; mp 169–171 °C; 1H NMR (400 MHz,
DMSO-d6): d 2.42 (s, 3H, CH3), 4.27 (s, 3H, CH3), 7.54 (t, 1H,
J = 58 Hz, CHF2), 8.03–8.22 (m, 2H, ArH), 13.11 (s, 1H, CO2H); EI-
MS: 389.9 (M)+; Anal. Calcd for C13H9F3N4O3S2: C, 40.00; H, 2.32;
N, 14.35; S, 16.43. Found: C, 40.02; H, 2.57; N, 14.09; S, 16.42.
2.7.20. Ethyl 1-{6-chloro-5-[4-difluoromethyl-3-methyl-5-oxo-
4,5-dihydro-1,2,4-triazol-1-yl] benzothiazol-2-
ylthio}propanoate (2t)
2.7.13. 1-[6-Chloro-2-(methylthio)benzothiazol-5-yl]-3-methyl-
4-difluoromethyl-1H-1,2,4-triazol-5(4H)-one (2m)
White solid. Yield 66%; mp 65–67 °C; 1H NMR (400 MHz,
CDCl3): d 1.27 (t, 3H, J = 7.2 Hz, CH3), 1.71 (d, 3H, J = 7.2 Hz, CHCH3),
2.51 (s, 3H, CH3), 4.23 (q, 2H, J = 7.2 Hz, OCH2), 4.69 (q, 1H,
J = 7.2 Hz, CH), 7.09 (t, 1H, J = 58 Hz, CHF2), 7.91 (s, 2H, ArH); EI-
White solid. Yield 77%; mp 172–174 °C; 1H NMR (400 MHz,
CDCl3): d 2.50 (s, 3H, CH3), 2.80 (s, 3H, CH3), 7.08 (t, 1H,

3250
Z. Yang et al. / Bioorg. Med. Chem. 21 (2013) 3245–3255
MS: 450.2 (M+H)+; Anal. Calcd for C16H15ClF2N4O3S2: C, 42.81; H,
3.37; N, 12.48; S, 14.29. Found: C, 42.97; H, 3.30; N, 12.35; S, 14.45.
a maximum excitation wavelength at 410 nm and a maximum
emission wavelength at 630 nm.36–39 Thus, we monitored the for-
mation of protoporphyrin IX at room temperature using a fluores-
cence detector with the excitation and emission wavelengths set at
410 and 631 nm, respectively. The concentration of protoporphy-
rinogen IX was determined according to the absorption of proto-
porphyrin IX and the concentration of protoporphyrin IX was
calculated from the calibration graph. In each assay, the stock di-
methyl sulfoxide (DMSO) solution (1% total volume) of inhibitor
was added into the reaction system. The final inhibitor concentra-
2.7.21. Ethyl 1-{6-chloro-5-[4-difluoromethyl-3-methyl-5-oxo-
4,5-dihydro-1,2,4-triazol-1-yl] benzothiazol-2-ylthio}acetate
(2u)
White solid. Yield 69%; mp 86–87 °C; 1H NMR (400 MHz,
CDCl3): d 1.29 (t, 3H, J = 7.2 Hz, CH3), 2.50 (s, 3H, CH3), 4.16 (s,
2H, CH2), 4.24 (q, 2H, J = 7.2 Hz, CH2), 7.08 (t, 1H, J = 58 Hz, CHF2),
7.91 (s, 2H, ArH); EI-MS: 436.2 (M+H)+; Anal. Calcd for
C15H13ClF2N4O3S2: C, 41.43; H, 3.01; N, 12.88; S, 14.75. Found: C,
tion ranged from 0.005 to 250 lM. The enzymatic reaction rate was
41.39; H, 2.99; N, 12.91; S, 14.81.
measured in a reagent buffer of 100 mM potassium phosphate (pH
7.5), 5 mM DTT, 1 mM EDTA, Tween 80 (0.03%, v/v), 200 mM imid-
2.7.22. 3-{6-Chloro-5-[4-difluoromethyl-3-methyl-5-oxo-4,5-
dihydro-1,2,4-triazol-1-yl]benzothiazol-2-ylthio}propanoic
acid (2v)
azole, 5 lM FAD, and approximately 0–40 lg of protein. The ki-
netic parameters were evaluated by Sigma Plot software 10.0
(SPSS, Chicago, USA). The IC50 value was determined by measuring
PPO activity over a range of inhibitor concentrations at a fixed sub-
White solid. Yield 84%; mp 150–151 °C; 1H NMR (400 MHz,
DMSO-d6): d 2.43 (s, 3H, CH3), 2.82 (t, 2H, J = 6.4 Hz, CH2), 3.54 (t,
2H, J = 6.4 Hz, CH2), 7.56 (t, 1H, J = 58 Hz, CHF2), 8.14 (s, 1H, ArH),
8.43 (s, 1H, ArH), 12.54 (s, 1H, OH); EI-MS: 420.4 (MꢀH)+; Anal.
Calcd for C14H11ClF2N4O3S2: C, 39.96; H, 2.63; N, 13.31; S, 15.24.
Found: C, 40.04; H, 2.89; N, 13.05; S, 15.26.
strate concentration. IC50 values were calculated by fitting
[I] data to a single binding site model described by Eq. 1
v
versus
max ꢀ min
Y ¼ min þ
50ꢀx
ð1Þ
1 þ 10log IC
where y is the percentage of maximal rate, with max and min being
the y values at which the curve levels off, x is the logarithm of inhib-
itor concentration, and IC50 is the concentration of inhibitor that
caused 50% of the total inhibition. Because the inhibitors belong
to the class of competitive inhibitors, the calculated Ki value (Ta-
ble 1) can be obtained by applying the following relationship among
Ki, Km, and IC50 at any saturating substrate concentration (S) by the
following Eq. 2:
2.7.23. 2-[6-Chloro-5-(4-difluoromethyl-3-methyl-5-oxo-4,5-
dihydro-1,2,4-triazol-1-yl)benzothiazol-2-ylthio]acetic acid
(2w)
White solid. Yield 81%; mp 127–129 °C; 1H NMR (400 MHz,
DMSO-d6): d 2.42 (s, 3H, CH3), 4.27 (s, 3H, CH3), 7.55 (t, 1H,
J = 58 Hz, CHF2), 8.03-8.23 (m, 2H, ArH), 13.15 (s, 1H, CO2H); EI-
MS: 408.4 (M+H)+; Anal. Calcd for C13H9ClF2N4O3S2: C, 38.38; H,
2.23; N, 13.77; S, 15.76. Found: C, 38.47; H, 2.39; N, 13.68; S, 15.74.
IC50
S=Km þ 1
Ki ¼
ð2Þ
2.7.24. 2-[6-Chloro-5-(4-difluoromethyl-3-methyl-5-oxo-4,5-
dihydro-1,2,4-triazol-1-yl)benzothiazol-2-ylthio]propanoic acid
(2x)
2.9. Computational simulations and binding free energy
calculations
White solid. Yield 73%; mp 116–118 °C; 1H NMR (400 MHz,
DMSO-d6): d 1.64 (d, 3H, J = 7.2 Hz, CH3), 2.41 (s, 3H, CH3), 4.62
(q, 1H, J = 7.2 Hz, CH), 7.55 (t, 1H, J = 58 Hz, CHF2), 8.06–8.90 (m,
2H, ArH), 13.24 (s, 1H, CO2H); EI-MS: 420.5 (M)+; Anal. Calcd for
The software of SYBYL 7.0 was used to construct the three-
dimensional chemical structures of all compounds. Then, the opti-
mized geometries were used as the starting structures for the fol-
lowing docking study. The structures of mtPPO and ligand were
prepared with AutoDock Tools40 and docking calculations were
performed with the program of AutoDock4.0.41 A total of 256 runs
were performed for each molecule. Most of the parameters for the
docking calculation were set to the default values recommended
by the program. To obtain the binding free energies of the ligands,
a modified molecular mechanics/Poisson-Boltzmann surface area
(MM/PBSA) described previously25,42,43 was performed for each
docked structure. Before the MM/PBSA calculation, the complex
structure was refined with integration of the steepest descent
and the conjugated gradient algorithm by using the Amber9 pack-
age.44 The AM1-BCC method implemented in the Antechamber
module of the Amber9 program was used to determine the partial
atomic charges of the ligands.44 During the energy minimization
process, first only the ligand was kept free, then the ligand and
the side-chains of the residues were kept free. Finally, all atoms
of the system were kept free and refined to a convergence of
0.01 kcal/(mol Å). The refined structure was used in the final bind-
ing energy calculation.
C14H11ClF2N4O3S2: C, 39.96; H, 2.63; N, 13.31; S, 15.24. Found: C,
40.03; H, 2.77; N, 13.35; S, 15.26.
2.7.25. Ethyl 1-{6-bromo-5-[4-difluoromethyl-3-methyl-5-oxo-
4,5-dihydro-1,2,4-triazol-1-yl] benzothiazol-2-ylthio}acetate
(2y)
White solid. Yield 79%; mp 95–97 °C; 1H NMR (400 MHz,
CDCl3): d 1.29 (t, 3H, J = 7.2 Hz, CH3), 2.51 (s, 3H, CH3), 4.16 (s,
2H, SCH2), 4.24 (q, 1H, J = 7.2 Hz, CH2), 7.09 (t, 1H, J = 58 Hz,
CHF2), 7.90 (s, 1H, ArH), 8.08 (s, 1H, ArH); EI-MS: 480.3 (M+H)+;
Anal. Calcd for C15H13BrF2N4O3S2: C, 37.59; H, 2.73; N, 11.69; S,
13.38. Found: C, 37.79; H, 2.43; N, 11.75; S, 13.65.
2.7.26. 1-[2-(Allylthio)-6-bromobenzothiazol-5-yl]-3-methyl-4-
difluoromethyl-1H-1,2,4-triazol-5(4H)-one (2z)
White solid. Yield 80%; mp 138–140 °C; 1H NMR (400 MHz,
CDCl3): d 2.51 (s, 3H, CH3), 4.00 (d, 2H, J = 7.2 Hz, CH2), 5.30 (dd,
2H, J = 10.8 Hz, @CH2), 5.95-6.04 (m, 1H, CH), 7.09 (t, 1H,
J = 58 Hz, CHF2), 7.92 (s, 1H, ArH), 8.07(s, 1H, ArH); EI-MS: 434.4
(M+H)+; Anal. Calcd for C14H11BrF2N4OS2: C, 38.81; H, 2.56; N,
12.93; S, 14.80. Found: C, 38.75; H, 2.30; N, 12.95; S, 15.13.
2.10. Greenhouse herbicidal activities
2.8. Evaluation of PPO inhibition kinetics
The herbicidal activities of compounds 2a–z against monocoty-
ledon weeds such as Echinochloa crus-galli (EC), Digitaria sanguinal-
is (DS), and Setaria faberi (SF), and dicotyledon weeds such as
The recombinant tobacco PPO enzyme (mtPPO) was expressed
according to the established protocol33–35 and purified as described
in our previous papers.36 The product of the enzymatic reaction has

Z. Yang et al. / Bioorg. Med. Chem. 21 (2013) 3245–3255
3251
Table 1
The Ki values (lM) of compounds 2a–z against mtPPO, and their calculated binding free energies (DGcal, kcal/mol)
X
O
CHF2
N
N
S
N
R
N
S
a
No.
X
R
D
EELE
D
EVDW
D
EGAS
D
GSOL
D
GPB
ꢀT
D
S
D
Gcal
D
Gexp
Ki
0.51
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
F
F
F
F
F
F
F
F
F
F
F
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
CH3
ꢀ22.93
ꢀ21.53
ꢀ24.17
ꢀ27.04
ꢀ33.71
ꢀ30.99
ꢀ24.99
ꢀ23.94
ꢀ31.66
ꢀ30.37
ꢀ29.43
ꢀ28.63
ꢀ20.61
ꢀ24.30
ꢀ23.78
ꢀ25.19
/
ꢀ48.12
ꢀ50.49
ꢀ49.25
ꢀ52.08
ꢀ52.73
ꢀ54.55
ꢀ48.71
ꢀ51.03
ꢀ53.89
ꢀ49.22
ꢀ52.09
ꢀ49.29
ꢀ48.6
ꢀ71.05
ꢀ72.02
ꢀ73.42
ꢀ79.12
ꢀ86.44
ꢀ85.54
ꢀ73.69
ꢀ74.97
ꢀ85.55
ꢀ79.59
ꢀ81.52
ꢀ77.92
ꢀ69.21
ꢀ75.39
ꢀ73.96
ꢀ80.22
/
44.81
44.39
45.28
50.87
56.35
55.19
46.02
46.44
57.31
50.82
53.51
49.96
44.61
47.61
46.9
ꢀ26.24
ꢀ27.63
ꢀ28.14
ꢀ28.25
ꢀ30.09
ꢀ30.35
ꢀ27.68
ꢀ28.53
ꢀ28.24
ꢀ28.77
ꢀ28.01
ꢀ27.96
ꢀ24.6
13.184
14.532
14.448
14.560
15.701
16.477
14.233
15.535
15.409
16.310
15.430
15.488
14.154
15.442
15.126
15.296
/
ꢀ13.06
ꢀ13.10
ꢀ13.69
ꢀ13.69
ꢀ14.39
ꢀ13.87
ꢀ13.45
ꢀ13.00
ꢀ12.83
ꢀ12.46
ꢀ12.58
ꢀ12.47
ꢀ10.45
ꢀ12.34
ꢀ11.93
ꢀ12.07
/
ꢀ8.60
ꢀ8.91
ꢀ9.15
ꢀ9.12
ꢀ9.87
ꢀ9.04
ꢀ8.93
ꢀ8.52
ꢀ8.15
ꢀ7.89
ꢀ8.08
ꢀ7.95
ꢀ6.64
ꢀ7.78
ꢀ7.52
ꢀ7.55
/
CH2CH2CH3
CH2CH@CH2
CH2CH2CH2CH3
CH2COOCH2CH3
CH(CH3)COOCH2CH3
COOCH3
COOCH2CH3
CH2COOCH3
CH2CH2COOH
CH(CH3)COOH
CH2COOH
0.30
0.20
0.21
0.06
0.24
0.29
0.58
1.08
1.67
1.21
1.52
13.86
2.01
3.13
2.97
CH3
CH2CH2CH3
CH2CH@CH2
CH2CH2CH2CH3
COOCH3
COOCH2CH3
CH2COOCH3
CH(CH3)COOCH2CH3
CH2COOCH2CH3
CH2CH2COOH
CH2COOH
ꢀ51.09
ꢀ50.18
ꢀ55.03
/
ꢀ27.78
ꢀ27.06
ꢀ27.37
/
52.85
/
/
b
/
/
/
/
/
/
/
/
/
2s
2t
ꢀ30.74
ꢀ30.04
ꢀ30.17
ꢀ27.17
ꢀ30.87
ꢀ29.44
ꢀ29.6
ꢀ53.05
ꢀ55.95
ꢀ54.6
ꢀ83.8
57.73
58.28
55.15
50.13
54.59
54.47
56.01
47.11
ꢀ26.06
ꢀ27.71
ꢀ29.62
ꢀ28.76
ꢀ27.83
ꢀ28.15
ꢀ28.84
ꢀ27.93
15.717
15.928
16.755
18.753
17.366
16.701
17.639
17.213
ꢀ10.34
ꢀ11.78
ꢀ12.87
ꢀ10.01
ꢀ10.46
ꢀ11.45
ꢀ11.20
ꢀ10.72
ꢀ6.58
ꢀ7.47
ꢀ8.01
ꢀ6.49
ꢀ6.81
ꢀ7.06
ꢀ6.96
ꢀ6.68
15.18
3.39
1.37
17.79
10.26
6.73
8.00
12.78
0.03
ꢀ85.99
ꢀ84.77
ꢀ78.88
ꢀ82.42
ꢀ82.62
ꢀ84.85
ꢀ75.04
2u
2v
2w
2x
2y
2z
ꢀ51.72
ꢀ51.55
ꢀ53.18
ꢀ55.25
ꢀ49.92
CH(CH3)COOH
CH2COOCH2CH3
CH2CH@CH2
ꢀ25.12
Sulfentrazone
a
D
Gexp = ꢀRT ln Ki.
b
Not tested.
Brassica juncea (BJ), Amaranthus retroflexus (AR), and Chenopodium
album (CA) were evaluated according to the previously reported
procedure.28,45–47 Sulfentrazone was selected as a positive control.
All test compounds were formulated as 100 g/L emulsified concen-
trates by using DMF as solvent and Tween-80 as emulsification re-
agent. The concentrated formulae were diluted with water to the
required concentration and applied to pot-grown plants in a green-
house. The soil used was a clay soil, pH 6.5%, 1.6% organic matter,
37.3% clay particles, and CEC 12.1 mol/kg. The rate of application
(g 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 9.5 cm were filled with soil to a depth of
O
X
X
X
N
c
b
a
NH
Y
NH
O
Y
N
Y
Y
NH2
N
HO
4
3
O
X
O
O
N
X
X
CHF2
CHF2
CHF2
N
d
f
e
N
N
N
N
Y
N
Y
N
N
H2N
O2N
6
7
5
O
X
O
X
CHF2
CHF2
N
g
N
N
S
N
S
N
N
N
R
HS
N
S
8
2
Scheme 1. Synthetic route for the title compounds 2a–z. Reagents and conditions: (a) NaNO2, HCl; SnCl2 HCl; CH3COCO2H, H2O; (b) (PhO)2PON3, toluene, reflux; (c) CHClF2
KOH, THF, reflux; (d) H2SO4, HNO3; (e) Fe, NH4Cl, EtOH, reflux; (f) C2H5OCS2K, DMF, reflux; (g) RX, K2CO3, acetone.

3252
Z. Yang et al. / Bioorg. Med. Chem. 21 (2013) 3245–3255
8 cm. Approximately 20 seeds of the tested weeds were sown in
the soil at the depth of 1–3 cm and grown at the temperature of
15–30 °C in a greenhouse. The air relative humidity was about
50%. The diluted formulation solutions were applied for post-emer-
gence treatment. Dicotyledon weeds were treated at the 2-leaf
stage and monocotyledon weeds were treated at the 1-leaf stage.
The post-emergence application rate was 150 g ai/ha. Untreated
seedlings were used as the control group and the solvent
(DMF + Tween-80) treated seedlings were used as the solvent con-
trol group. Herbicidal activity was evaluated visually at 15 days
post treatment. The results of herbicidal activities are reported
based on three replicates per treatment. An additional 17 weeds,
including Veronica didyma, Malachium aquaticum, Ludwigia pro-
strata, Cerastium arvense, Eleusine indica, Amaranthus spinosus,
Boehmeria nivea, Aster subulatus, Capsella bursa-pastoris, Ammannia
baccifera, Solanum nigrum, Polygonum humifusum, Radix phytolac-
cae, Sonchus asper, Physalis alkekengi, Cassia tora, and Commelina
communis, were used for further screening of the herbicidal
spectrum.
uation for crop safety of compound 2f was conducted by visual
observation in 30 days after treatment on the 0–100 scale.
3. Results and discussion
3.1. Synthesis of the title compounds
As shown in Scheme 1, the target compounds 2a–z were
smoothly prepared by a multiple step synthetic route using 2,4-
disubstitutedanilines as starting materials. Compound 3, resulting
from the reaction of 2,4-disubstitutedaniline with 2-oxopropanoic
acid, underwent a cyclization reaction with diphenyl phosphoraz-
idate to afford the key intermediates 1-(2,4-disubstitutedphenyl)-
3-methyl-1H-1,2,4-triazol-5-ones (4). Then, compound 4 was re-
acted with chlorodifluoromethane in the presence of potassium
hydroxide to afford another important intermediate 4-(difluoro-
methyl)-1-(2,4-disubstitutedphenyl)-3-methyl-1H-1,2,4-triazol-5-
ones (5) in yields of 75–91%, which underwent nitration and sub-
sequent reduction reactions to produce 1-(5-amino-2,4-disubsti-
tuted-phenyl)-4-(difluoro-methyl)-3-methyl-1H-1,2,4-triazol-5-
ones (7). By performing the cyclization reaction with potassium O-
ethyl carbonodithioate, compound 7 was successfully transformed
into the corresponding 4-(difluoro-methyl)-1-(2-mercapto-6-halo-
benzothiazol-5-yl)-3-methyl-1H-1,2,4-triazol-5-one (8). Finally,
the intermediates 8a–c were reacted with various alkylation re-
agents to give the target compounds 2a–z in yields of 54–96%.
The structures of all intermediates and title compounds were con-
firmed by elemental analyses and by 1H NMR and ESI-MS spectral
data.
2.11. Crop selectivity11,48
The conventional rice, soybean, cotton, wheat, rape and maize
were respectively planted in plots (diameter = 12 cm) containing
test soil and grown in a greenhouse at 20–25 °C. After the plants
had reached the 4-leaf stage, the spraying treatment was con-
ducted at different dosages by diluting the formulation of com-
pound 2f with water. The visual injury and growth state of the
individual plants were observed at regular intervals. The final eval-
3.2. PPO inhibition activity
For the sake of clarity, 2a–l, 2m–x and 2y–z will be termed
respectively as fluorine, chlorine and bromine derivatives through
the text. The fluorine series compound 2a–l always displayed high-
er mtPPO-inhibiting activity (as judged by lower Ki values), than
the corresponding chlorine compounds 2m–x and bromine com-
pounds 2y–z. Among the 26 newly synthesized compounds, 2e
was identified as the most potent inhibitor with a Ki value of
0.06
l
M against mtPPO, which indicated that the potency of 2e
M) of the control
against mtPPO is about half of that (Ki = 0.03
l
compound, sulfentrazone. In addition, within the fluorine or chlo-
rine series, compounds (e.g., 2e and 2u) bearing the R group of
CH2COOC2H5 always displayed the highest potency against mtPPO.
On the other hand, the R group of the fluorine derivatives displayed
slightly different structure–activity relationships than the chlorine
derivatives. For example, for the fluorine series, the potency order
is 2e (R = CH2COOC2H5) ꢁ 2b (R = CH2CH2CH3)–2c (R = CH2CH
@CH2)–2d (R = CH2CH2CH2CH3)–2f (R = CH(CH3)COOC2H5)–2g
Figure 4. Correlation between the calculated and experimental binding free
energies.
(R = COOCH3) > 2a
(R = CH3)–2h
(R = COOC2H5) > 2i
(R =
Figure 5. Computational simulated binding models of compounds 2e (A) and 2d (B).

Z. Yang et al. / Bioorg. Med. Chem. 21 (2013) 3245–3255
3253
Table 2
CH2COOCH3), 2k (R = CH(CH3)COOH) > 2j (R = CH2CH2COOH), 2l
(R = CH2COOH). In contrast, the potency order of the
chlorine series is 2u (R = CH2COOC2H5) > 2n (R = CH2CH2CH3) >
Herbicidal activities of compound 2 (post-emergency)
a
Compound
Dosage (g ai/
ha)
EC
DS
SF
BJ
AR
CA
2p
(R = CH2CH2CH2CH3)–2o
(R = CH2CH@CH2)–2t
(R =
(R =
b
CH(CH3)COOC2H5) > 2v
(R = CH(CH3)COOH) > 2w
2a
2b
2c
2d
2e
150
150
150
150
37.5
75
150
37.5
75
150
37.5
75
150
37.5
75
150
37.5
75
150
37.5
75
150
37.5
75
150
37.5
75
150
150
150
150
150
150
150
37.5
75
150
150
150
37.5
75
150
37.5
75
150
37.5
75
+
ꢀ
+
ꢀ
+++
++
+
++
+++
+++
ꢀ
++
+++
++
ꢀ
+++
++
+++
ꢀ
++
++
+++
ꢀ
++
ꢀ
ꢀ
CH2COOH) > 2m (R = CH3) > 2v (R = CH2CH2COOH). These results
indicated that the substituents (e.g., F, Cl, and Br) on position-2 will
affect the bioactive conformation of inhibitors, which has been dis-
cussed previously.25
To understand the structure–activity relationships at the atomic
level, we performed the docking and binding free energies calcula-
tions for all of the newly synthesized compounds. As summarized
ꢀ
++
+++
+++
ꢀ
+++
+++
+++
++
++
+
+
ꢀ
++++ ++++ ++++ ++++ ++
+++
+++
2f
2g
2h
2i
+++
+++
++++ ++++ +++
++++ ++++ +++
++++ ++++ ++++ ++++ ++++ ++++
in Table 1, the calculated binding free energies (
ꢀ10.01 to ꢀ14.39 kcal/mol, while the experimental binding free
energies (
DGcal) range from
+
+
++
++
++
ꢀ
ꢀ
ꢀ
ꢀ
+
++++
ꢀ
+
+
++
ꢀ
ꢀ
+
++
++
ꢀ
++++ ++++ ++++
D
Gexp) range from ꢀ6.49 to ꢀ9.87 kcal/mol. Obviously,
ꢀ
ꢀ
ꢀ
ꢀ
the MM/PBSA calculations systematically overestimated the abso-
lute binding affinities of the ligands toward PPO. However, the rel-
ative order of the calculated binding affinities of the compounds
were still meaningful because a good linear correlation between
ꢀ
ꢀ
ꢀ
ꢀ
++
+
ꢀ
++++ ++++ ++++
ꢀ
++
++
++
++++
ꢀ
ꢀ
++++ ++++ ++++
++++ ++++ ++++ ++++ ++++ ++++
D
Gcal and DGexp was obtained with a correlation coefficient of
2j
ꢀ
ꢀ
ꢀ
++
++
+++
r2 = 0.96 (Fig. 4), confirming that the reliability of the computa-
tional models constructed in this study.
ꢀ
+
+
++++ ++++ ++++
++++ ++++ ++++
++++ ++++
+
+
ꢀ
As shown in Table 1, compound 2e is the most potent inhibitor
with the Ki value of 0.06 lM against mtPPO. The simulated binding
2k
2l
++
++
ꢀ
++
++
+
ꢀ
++++ ++++ ++++
++++ ++++ ++++ ++++ ++++ ++++
model of 2e (Fig. 5A), showed that the 1,2,4-triazol-5-one ring was
sandwiched between the residues of F392 and G175 and formed
hydrophobic interactions with these two residues. The benzothia-
zole moiety was located between the residues of L356 and L372. In
addition, the carbonyl oxygen of the side chain of the benzothia-
zole ring formed two strong hydrogen bonds with residue R98,
which contributed greatly to the potency of PPO inhibitors.
When the side chains of benzothiazole of the inhibitors are ali-
phatic chain, such as CH3, CH2CH2CH3, CH2CH2CH2CH3, and
CH2CH@CH2, the inhibitor should displayed much lower affinity
due to the loss of hydrogen-bonding interactions with residue
R98. Surprisingly, compounds 2a, 2b, 2c, and 2d still displayed rel-
atively higher potency than compounds bearing an ester group as
the side chain, such as 2i, 2j, 2k, and 2l. In order to understand the
molecular basis of such a difference, we compared the binding
model of compound 2d with that of compound 2e. As shown in Fig-
ure 5B, the simulated binding model of compound 2d is totally dif-
ferent from that of 2e (Fig. 5A). The benzothiazole moiety of 2d was
located between the residues of L356 and L372, while the butyl
side chain, not the 1,2,4-triazol-5-one ring, interacted with F392.
Very interestingly, instead of the ester group observed in 2e, the
carbonyl oxygen of the 1,2,4-triazol-5-one formed two strong
hydrogen bonds with R98, which accounts for why 2d displayed
only a 3.5-fold lower potency than 2e.
+
+
ꢀ
+
+
ꢀ
++++ +++
++++ +++
ꢀ
+++
++++ ++++ ++++ ++++ ++++ ++++
2m
2n
2o
2p
2q
2r
ꢀ
ꢀ
++
ꢀ
ꢀ
ꢀ
+
+
ꢀ
ꢀ
++
ꢀ
ꢀ
ꢀ
+
++
+
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
++++ ++
++
+
++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2s
++
+++
++
++++
+
+
++++ ++++
++++ ++++ ++++ ++++ ++++ ++
+++ +++ +++ ++++ ++++ ++
++++ ++++ +++
2t
2u
2v
++++ +++
+
++
++++ ++++ ++++
++
++++ ++++ ++++
ꢀ
ꢀ
ꢀ
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
++
++
ꢀ
ꢀ
ꢀ
2w
2x
2y
2z
ꢀ
+
+++
ꢀ
++++ ++++ ++++ ++++ ++++ ++++
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
++
++++
ꢀ
ꢀ
ꢀ
+++
++
150
37.5
75
150
150
++++ ++++ ++++ ++++ ++++ ++++
+
++
+
ꢀ
+
+
+++
+++
++++ ++++
++++ ++++
++
+++
ꢀ
++
+++
++
++
+++
++++ ++++ ++++
+
+++
++
+++
+
++
Sulfentrazone 37.5
+++
++++
ꢀ
75
+
++++ ++++ ++++
From the energy component analysis, we concluded that the en-
ergy difference between 2e and 2d mainly came from the electro-
static effects, the solvation effects, and the entropy penalty.
Compound 2e had a stronger electrostatic energy (ꢀ33.71 kcal/
mol), higher solvation effect (56.35 kcal/mol) and entropy penalty
(15.70 kcal/mol) than did compound 2d, whose corresponding val-
ues are ꢀ27.04, 50.87 and 14.56 kcal/mol, respectively. Hence, the
replacement of the ester group side chain of the benzothiazole
150
++++ ++
++++ ++++ ++++ ++++
a
EC for Echinochloa crus-galli, DS for Digitaria sanguinalis, SF for Setaria faberii, BJ
for Brassica juncea, AR for Amaranthus retroflexus and CA for Chenopodium album.
Rating system for the growth inhibition percentage: ++++, =90%; +++, 80–89%;
++, 60–79%; +, 50–59%; ꢀ, <50%.
b
Table 3
Crop selectivity of compound 2f and sulfentrazone
Compound
Dosage (g ai/ha)
Rice
Cotton
Soybean
Maize
Rape
Wheat
2f
75
/
/
0
/
/
/
150
75
150
50
/
70
70
/
100
0
10
20
30
/
70
100
/
100
80
/
20
Sulfentrazone

3254
Z. Yang et al. / Bioorg. Med. Chem. 21 (2013) 3245–3255
moiety with an aliphatic group may result in a totally different
binding model and lower binding affinity.
150 g ai/ha, whereas they were susceptible to sulfentrazone even
at the concentration of 75 g ai/ha. These results indicated that
compound 2f might be a potential candidate for development as
a new herbicide for soybean fields.
3.3. Green house herbicidal activities
The post-emergence herbicidal activities of compounds
2
Acknowledgments
against monocotyledon weeds, such as E. crus-galli, D. sanguinalis,
and S. faberi, and dicotyledon weeds, such as B. juncea, A. retroflex-
us, and C. album, were tested in the green house at the concentra-
tion of 150 g ai/ha; sulfentrazone was selected as a control. As
shown in Table 2, most of compounds were found to display prom-
ising herbicidal activities with broad spectrum at the concentra-
tion of 150 g ai/ha. Very promisingly, compounds 2f, 2i, 2k, 2l,
2w and 2x exhibited over 90% control against the six tested weeds,
and compounds 2e, 2s, and 2y showed over 80% control against
five of the six tested weeds. In addition, compounds 2g, 2h, 2j
and 2v exhibited over 90% control against the tested dicotyledon
weeds, but did not show herbicidal activity against the tested
monocotyledon weeds. Therefore, the above twelve compounds
with promising herbicidal activities were selected for further test-
ing at lower dosages. As shown in Table 2, at the concentration of
75 g ai/ha, compounds 2e, 2i, 2j, 2k, 2l, 2s, 2w, and 2y still exhib-
ited over 80% control against dicotyledon weeds such as B. juncea,
A. retroflexus, and C. album, but did not show significant herbicidal
activity against the tested monocotyledon weeds. Even at the con-
centration of 37.5 g ai/ha, compounds 2e and 2y still showed
promising herbicidal activity against dicotyledon weeds such as
B. juncea, A. retroflexus, and C. album. In addition, compound 2f
showed excellent herbicidal activity even at the concentration of
37.5 g ai/ha. For the weeds such as D. sanguinalis, S. faberi, and B.
juncea, 2f showed much higher herbicidal activity than sulfentraz-
one at the concentration of 37.5 g ai/ha. On the contrary, 2f showed
comparable herbicidal activity with sulfentrazone against E. crus-
galli, A. retroflexus and C. album at the concentration of 37.5 g ai/
ha. The results of further herbicidal spectrum tests indicated that
at the concentration of 75 g ai/ha 2f showed excellent post-emer-
gence herbicidal activity and exhibited total control against V. didy-
ma, C. arvense, E. indica, A. spinosus, B. nivea, A. subulatus, C. bursa-
pastoris, S. nigrum, P. humifusum, and S. asper, and showed over 90%
control against M. aquaticum, L. prostrata, A. baccifera, R. phytolac-
cae, P. alkekengi, C. tora, and C. communis. As a control, sulfentraz-
one showed over 90% control against M. aquaticum, C. arvense, and
C. tora, and total control against other species at the same concen-
tration. These results indicated that compound 2f showed compa-
rable herbicidal activity with sulfentrazone.
We thank Dr. Jie Chen for the greenhouse testing of biological
activities. The research was supported in part by the National Basic
Research Program of China (No. 2010CB126103), the NSFC (No.
21002038, 20902034, and 20925206), and the National Key Tech-
nologies R&D Program (2011BAE06B05).
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