Design, Synthesis, and 3D-QSAR Analysis of Novel 1,3,4-Oxadlazol-2(3H)-ones as Protoporphyrlnogen Oxidase Inhibitors

Article abstract of DOI:10.1021/jf9026298

Protoporphyrlnogen oxidase (PPO, EC 1.3.3.4) has been Identified as one of the most significant action targets for a large chemically diverse family of herbicides that exhibit some Interesting characteristics, such as low use rate, low toxicity to mammals, and low environmental Impact. As a continuation of research work on the development of new PPO Inhibitors, some benzothlazole analogues of oxadlargyl, an Important PPO-lnhlbltlng commercial herbicide, were designed and synthesized by ring-closing of the substltuents at the C-4 and C-5 positions. The bloassay results Indicated that the series 8, 9, and 10 have good PPO Inhibition activity with k_l values ranging from 0.25 to 18.63 μM. Most Interestingly, 9I, ethyl 2-((5-(5-fert-butyl-2-oxo-1,3,4-oxadlazol- 2(3H)-yl)-6fluorobenzothlazol-2-yl)sulfanyl) propanoate, was Identified as the most promising candidate due to Its high PPO Inhibition effect (k_l 1.42 ,μM) and broad spectrum postemergence herbicidal activity at the concentration of 37.5 g of al/ha.

Full text of DOI:10.1021/jf9026298

J. Agric. Food Chem. 2010, 58, 2643–2651 2643
DOI:10.1021/jf9026298
Design, Synthesis, and 3D-QSAR Analysis of Novel
1,3,4-Oxadiazol-2(3H)-ones as Protoporphyrinogen
Oxidase Inhibitors†
§,
§,#,
§
L
I
-L
I
J
IANG
,
Y
ING
T
C
AN
,
X
IAO-LEI
Z
HU,§ ZHI-FANG
UANG-F
W
Y
ANG
ANG
,
*
§,# YANG
,§
Z
UO
,
Q
IONG
HEN,§ ZHEN
X
I
,*,# AND
G
U
§ Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, People’s Republic of China, and #State Key
Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, People’s Republic
of China. Co-first authors.
Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) has been identified as one of the most significant
action targets for a large chemically diverse family of herbicides that exhibit some interesting
characteristics, such as low use rate, low toxicity to mammals, and low environmental impact. As a
continuation of research work on the development of new PPO inhibitors, some benzothiazole
analogues of oxadiargyl, an important PPO-inhibiting commercial herbicide, were designed and
synthesized by ring-closing of the substituents at the C-4 and C-5 positions. The bioassay results
indicated that the series 8, 9, and 10 have good PPO inhibition activity with ki values ranging from
0.25 to 18.63 μM. Most interestingly, 9l, ethyl 2-((5-(5-tert-butyl-2-oxo-1,3,4-oxadiazol-2(3H)-yl)-6-
fluorobenzothiazol-2-yl)sulfanyl) propanoate, was identified as the most promising candidate due to
its high PPO inhibition effect (ki = 1.42 μM) and broad spectrum postemergence herbicidal activity at
the concentration of 37.5 g of ai/ha.
KEYWORDS: Protoporphyrinogen oxidase; 1,3,4-oxadiazol-2(3H)-ones; benzothiazole; herbicide
INTRODUCTION
their low use rates and environmentally benign characteristics.
Since the 1970s, research on PPO inhibitors has been actively
pursued, leading to two classes of commercial products, diphenyl
ethers and N-phenyl phthalimides, the latter of which has become a
very interesting research area (13-16). Structural optimization of
the pioneering compounds of the N-phenyl phthalimides family,
chlorphthalim and oxadiazon, as shown in Figure 1, resulted in
the discovery of several commercial herbicides. Oxadiargyl, 3-[2,4-
dichloro-5-(2-propynyloxy)phenyl]-5-(1,1-dimethylethyl)-1,3,4-
oxadiazol-2-(3H)-one, is an oxadiazole-type PPO-inhibiting
herbicide introduced in 1996. Although it exhibited some
advantages over other PPO inhibitors, such as its broad spec-
trum, soil-independent, and low use rate properties, it is safe on
crops only when applied as a pre-emergence herbicide (14-16).
Therefore, it is an interesting task to discover new analogues
with suitable postemergence herbicidal activity.
Oxadiazole-type herbicides have a common structural feature
of N-2,4,5-trisubstituted phenylnitrogen. Of the phenyl substitu-
tion patterns investigated (4), the ones that led to the most active
compounds were F or Cl at C-2 and Cl at C-4, whereas the groups
at the C-5 position have an important effect not only on the
activity but also on the crop selectivity. Keep in mind that the
benzothiazole moiety is a common substructure in a large number
of compounds with a wide range of biological activities (17-21);
therefore, as a continuation of our research work on the devel-
opment of new PPO inhibitors, we are very interested in the
benzothiazole-type ring-closing analogues of oxadiargyl as
shown in Scheme 1. Herein, we report the detailed syntheses
As thelast common enzyme in thebiosynthesis pathway leading
to heme and chlorophyll synthesis, protoporphyrinogen IX oxi-
dase (PPO; EC 1.3.3.4) has been identified as one of the most
significant targets for several chemical families of herbicides such
as diphenyl ethers, phenylpyrazoles, oxadiazoles, triazolinones,
thiadiazoles, pyrimidineones, oxazolidinediones, isoxazoles, and
N-phenyl phthalimides that have been available in the commercial
market for many years (1-9). It is well-known that the oxidation
of protoporphyrinogen IX to protoporphyrin IX can take place
nonenzymatically, but the PPO-catalyzed reaction seems to be
ubiquitous in cells having aerobic metabolism (9, 10). However,
when the enzyme in plants is inhibited, the substrate protopor-
phyrinogen IX will accumulate and be exported to the cytoplasm,
where it is slowly oxidized by O2 to produce protoporphyrin
IX (11). Then, when treated plants are exposedto visible radiation,
the protoporphyrin IX, which is a highly efficient photosensitizer,
will lead to severe photochemical damage, such as lipid peroxida-
tion and cell death (12). Therefore, PPO-inhibiting herbicides are
also known as light-dependent bleaching herbicides (9).
Most of side effects, such as toxicological and environmental
impact, are associated with high use rates of herbicides (13).
Therefore, PPO-inhibiting herbicides are of great interest due to
†Part of the ECUST-Qian Pesticide Cluster.
*Corresponding authors [(Z.X.) telephone/fax þ86-22-23504782,
e-mail zhenxie@nankai.edu.cn; (G.-F.Y.) telephone þ86-27-67867800,
fax þ86-27-67867141, e-mail gfyang@mail.ccnu.edu.cn].
© 2009 American Chemical Society
Published on Web 12/02/2009
pubs.acs.org/JAFC

2644 J. Agric. Food Chem., Vol. 58, No. 5, 2010
Jiang et al.
Figure 1. Structures of some commercial PPPO inhibitors.
Scheme 1. Molecular Design of the Series 8, 9, and 10
and herbicidal activities of series 8, 9, and 10, and the results
indicated that these compounds displayed good PPO inhibition
activity and promising herbicidal activity.
reaction mixture was stirred for an additional 0.5 h. Then a solution of
pivaloyl chloride (1.74 g, 14.5 mmol) in 20 mL of CH2Cl2 was added dropwise
to the reaction system. After overnight stirring at room temperature, CH2Cl2
(100 mL) was added to the reaction mixture. The organic layer was separated
and washed successively with water (200 mL) and brine (200 mL) and then
dried over anhydrous magnesium sulfate. After evaporation of the solvent,
the oil or solid products of 3a, 3b, and 3c were obtained.
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. 1H NMR spectra were recorded on
a Varian Mercury-Plus 400 spectrometer in CDCl3 or DMSO-d6
with TMS as the internal reference. Mass spectral data were
obtained on a ThermoFisher Mass platform DSQII 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
uncorrected.
Data for 3a: yield, 92%; yellow oil; 1H NMR (400 MHz, CDCl3) δ 1.29
(s, 9H), 6.73 (d, J = 8.4 Hz, 1H), 7.10 (dd, J1 = 2.0 Hz, J2 = 8.4 Hz, 1H),
7.29 (d, J = 2.0 Hz, 1H), 7.57 (br, 1H); ESI/MS, 260.0 (Mþ).
Data for 3b: yield, 93%; mp 135-137 °C; 1H NMR (400 MHz, CDCl3)
δ 1.26 (s, 9H), 6.75 (t, J = 8.4 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 7.03 (d,
J = 11.4 Hz, 1H), 7.57 (br, 1H); ESI/MS, 266.7 (M þ Na).
Data for 3c: yield, 86%; oil; 1H NMR (400 MHz, CDCl3) δ 1.28 (s, 9H),
6.75 (dd, J1 = 5.2 Hz, J2 = 8.8 Hz, 1H), 6.91-6.96 (m, 1H), 7.22 (dd, J1 =
2.8 Hz, J2 = 7.6 Hz, 1H); ESI/MS, 290.7 (M þ H).
Preparation of (2,4-Disubstituted-phenyl)hydrazine Hydrochlo-
ride (2a-c) (22). Under a nitrogen atmosphere, a stirred solution of 2,4-
dichloroaniline (1a, 4.80 g, 30 mmol) in 30 mL of concentrated hydro-
chloric acid was cooled to -9 °C, and a solution of 2.55 g (36 mmol) of
sodium nitrite in 11 mL of water was added dropwise at a rate to keep the
reaction mixture temperature under -9 °C. The complete addition
required 20 min. The reaction mixture was stirred for an additional 1 h
at 0 to -9 °C. Then, a solution of 14.8 g (78 mmol) of stannous chloride in
30 mL of concentrated hydrochloric acid was added dropwise at a rate to
keep the reaction mixture temperature under -9 to -0 °C for 2 h. The
mixture was then vacuum filtered, and the resulting solid was allowed to
dry overnight. The solid was dissolved in hot water and gravity filtered,
and the filtrate was cooled on ice. The crystallized solid was then suction
filtered, and the product was allowed to dry overnight. 2a was obtained as
a solid in a yield of 39%, mp 191-193 °C (lit. (23) 193-194 °C). 2b and 2c
were also prepared according to this procedure.
Preparation of 5-tert-Butyl-3-(2,4-disubstituted-phenyl)-1,3,4-
oxadiazol-2(3H)-ones (4a-c) (25). Under a nitrogen atmosphere, a
solution of bis(trichloromethyl) carbonate (7.46 g, 25.37 mmol) in toluene
(50 mL) was added dropwise to a stirred solution of 3a-c (12.69 mmol),
triethylamine (2.57 g, 25.37 mmol), and toluene (260 mL) in an ice bath.
The resulting mixture was stirred overnight at room temperature under a
nitrogen atmosphere and then was refluxed for 2 h. After cooling, the
solution was washed with water (200 mL) and brine (200 mL), then dried,
filtered, and concentrated to give the desired oil or solid products.
Data for 4a: yield, 90%; oil; 1H NMR (400 MHz, CDCl3) δ 1.37 (s, 9H),
7.35 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.54 (d,
J = 2.0 Hz, 1H); ESI/MS, 286.1 (M).
Data for 4b: yield, 90%; mp, 99-101 °C; 1H NMR (400 MHz, CDCl3)
δ 1.37 (s, 9H), 7.25-7.28 (m, 2H), 7.46-7.50 (m, 1H); ESI/MS, 292.5
(M þ Na).
Data for 4c: yield, 92%; mp, 83-85 °C; 1H NMR (400 MHz, CDCl3)
δ 1.37 (s, 9H), 7.15-7.16 (m, 1H), 7.45-7.51 (m, 2H); ESI-MS, 338.4
(M þ Na).
Data for 2b: yield, 37%; mp, 209-210 °C; 1H NMR (600 MHz,
DMSO-d6) δ 7.19-7.21 (m, 1H), 7.27-7.28 (m, 1H), 7.43-7.45 (m,
1H), 8.45 (br s, 1H), 10.38 (br s, 3H).
Preparation of 5-tert-Butyl-3-(2,4-disubstituted-5-nitrophenyl)-
1,3,4-oxadiazol-2(3H)-ones (5a-c). A solution of HNO3 (1.07 g,
11.3 mmol) in concentrated H2SO4 (2.26 g, 22.6 mmol) was added
dropwise to a stirred mixture of 4a-c (11.3 mmol) and concentrated
H2SO4 (226 mL) in an ice bath. The complete addition took about 15 min.
After 3 h of stirring, the solution was slowly poured into a mixture of ice
and water (500 mL). The resulting solid was collected by filtration, washed
with water (2 L), and then dried to give to the desired solid products.
Data for 2c: yield, 36%; mp, 208.0 °C (decomposition); 1H NMR
(600 MHz, DMSO-d6) δ 7.15-7.17 (m, 1H), 7.29-7.32 (m, 1H),
7.58-7.59 (m, 1H), 7.89 (br s, 1H), 10.37 (br s, 3H).
Preparation of N0-(2,4-Disubstituted-phenyl)pivalohydrazide
(3a-c) (24). A solution of sodium hydroxide (0.82 g, 20 mmol) in
20 mL of water was added dropwise to a stirred mixture of 2a-c
(13.8 mmol), 50 mL of H2O, and 150 mL of CH2Cl2 in an ice bath. The

Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 2645
Data for 5a: yield, 90%; mp, 155-156 °C; 1H NMR (400 MHz, CDCl3)
Data for 8e: yield, 46%; mp 142-143 °C; 1H NMR (400 MHz, CDCl3)
δ1.40(s, 9H), 1.50(s, 9H), 4.08(s, 2H), 7.90(s, 1H), 7.94(s, 1H); ESI-MS,
477.1 (Mþ þ Na). Anal. Calcd for C19H22ClN3O4S2: C, 50.05; H, 4.86; N,
9.22. Found: C, 49.83; H, 4.76; N, 9.10.
δ 1.38 (s, 9H), 7.77 (s, 1H), 8.14 (s, 1H); ESI-MS, 331.9 (M þ H).
Data for 5b: yield, 83%; mp, 160-162 °C; 1H NMR (400 MHz, CDCl3)
δ 1.38 (s, 9H), 7.47 (d, J = 9.6 Hz, 1H), 8.26 (d, J = 6.8 Hz, 1H); ESI-MS,
337.1 (M þ Na).
Data for 8f: yield, 72%; mp, 137-138 °C; 1H NMR (400 MHz, CDCl3)
δ 1.39 (s, 9H), 3.80 (s, 3H), 4.18 (s, 2H), 7.90 (s, 1H), 7.97 (s, 1H); ESI-MS,
435.5 (M þ Na). Anal. Calcd for C16H16ClN3O4S2: C, 46.43; H, 3.90; N,
10.15. Found: C, 46.62; H, 4.10; N, 10.24.
Data for 5c: yield, 87%; mp, 175-176 °C; 1H NMR (400 MHz, CDCl3)
δ 1.39 (s, 9H), 7.70 (d, J = 9.6 Hz, 1H), 8.26 (d, J = 6.4 Hz, 1H); ESI-MS,
360.0 (M).
Data for 8g: yield, 61%; oil; 1H NMR (400 MHz, CDCl3) δ 1.25 (t, J =
7.2 Hz, 3H), 1.39 (s, 9H), 2.15 (m, 2H), 2.49 (t, J = 7.2 Hz, 2H), 3.41 (t,
J = 7.2 Hz, 2H), 4.14 (q, J = 7.2 Hz, 2H), 7.89 (s, 1H), 7.95 (s, 1H);
ESI-MS, 477.1 (M þ Na). Anal. Calcd for C19H22ClN3O4S2: C, 50.05; H,
4.86; N, 9.22. Found: C, 50.21; H, 4.62; N, 8.85.
Preparation of 3-(5-Amino-2,4-disubstituted-phenyl)-5-tert-butyl-
1,3,4-oxadiazol-2(3H)-ones (6a-c) (26). The powder of Fe was added
portionwise to a stirred solution of NH4Cl (0.83 g) and 5a-c (10.4 mmol)
in a mixture of EtOH/H2O (v/v, 10:1, 220 mL) at reflux temperature. After
TLC detection showing that the reaction had finished, the reactionmixture
was filtered and concentrated to dryness. The residue was dissolved in
water (200 mL) and extracted with EtOAc (200 mL); the organic phase was
washed with brine (150 mL), dried, filtered, then concentrated to give the
desired oil or solid products.
Data for 8h: yield, 59%; mp, 104-106 °C; 1H NMR (400 MHz, CDCl3)
δ 1.25 (t, J = 7.2 Hz, 3H), 1.39 (s, 9H), 2.15 (d, J = 7.2 Hz, 3H), 4.22 (q,
J = 7.2 Hz, 2H), 4.67 (q, J = 7.2 Hz, 1H), 7.90 (s, 1H), 7.96 (s, 1H);
ESI/MS, 442.05 (M þ H). Anal. Calcd for C18H20ClN3O4S2: C, 48.92; H,
4.56; N, 9.51. Found: C, 49.13; H, 4.76; N, 9.60.
Data for 8i: yield, 76%; mp, 116-117 °C; 1H NMR (400 MHz, CDCl3)
δ 1.39 (s, 9H), 4.00 (d, J = 6.0 Hz, 2H), 5.22 (d, J = 10.0 Hz, 1H), 5.37 (d,
J = 17.2 Hz, 1H), 5.96-6.03 (m, 1H), 7.89 (s, 1H), 7.97 (s, 1H); ESI/MS,
403.3 (M þ Na). Anal. Calcd for C16H16ClN3O2S2: C, 50.32; H, 4.22; N,
11.00. Found: C, 50.02; H, 4.44; N, 11.23.
Data for 6a: yield, 91%; oil; 1H NMR (400 MHz, CDCl3) δ 1.36 (s, 9H),
6.87 (s, 1H), 7.39 (s, 1H); ESI-MS, 301.6 (M).
Data for 6b: yield, 69%; oil; 1H NMR (600 MHz, CDCl3) δ 1.36
(s, 9H), 3.90 (br, 2H), 6.90 (dd, J1=7.2 Hz, J2=1.2 Hz, 1H), 7.16 (dd,
J1 = 9.6 Hz, J2 = 1.2 Hz, 1H); ESI-MS, 285.4 (M).
Data for 6c: yield, 46%; mp, 122-124 °C; 1H NMR (400 MHz, CDCl3)
δ 1.36 (s, 9H), 3.90 (br, 2H), 6.89 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 10 Hz,
1H); ESI/MS, 329.3 (M).
Data for 8j: yield, 80%; mp, 102-103 °C; 1H NMR (600 MHz, CDCl3) δ
1.20 (t, J = 7.2 Hz, 3H), 1.39 (s, 9H), 1.77 (s, 6H), 4.19 (q, J = 7.2 Hz, 2H),
7.91 (s, 1H), 7.99 (s, 1H); ESI/MS, 477.8 (M þ Na). Anal. Calcd for
C19H22ClN3O4S2: C, 50.05; H, 4.86; N, 9.22. Found: C, 49.87; H, 4.63; N, 9.12.
Data for 8k: yield, 69%; mp, 110.2-110.5 °C; 1H NMR (600 MHz,
CDCl3) δ 1.26 (d, J = 9.6 Hz, 6H), 1.39 (s, 9H), 4.13 (s, 2H), 5.09 (m, 1H),
7.90 (s, 1H), 7.94 (s, 1H); ESI/MS, 463.0 (M þ Na). Anal. Calcd
for C18H20ClN3O4S2: C, 48.92; H, 4.56; N, 9.51. Found: C, 49.13; H,
4.79; N, 9.60.
Preparation of 5-tert-Butyl-3-(6-substituted-2-mercaptobenzo-
[d]thiazol-5-yl)-1,3,4-oxadiazol-2(3H )-ones (7a-c) (27). A solution
of6a-c (2.63 g, 9.45 mmol) and potassium O-ethyl dithiocarbonate (3.03 g,
18.9 mmol) in 142 mL anhydrous DMF was heated at 120 °C for 3 h. After
TLC detection showing that the reaction had finished, the reaction mixture
was evaporated under reduced pressure to remove most of the solvent and
diluted with water (300 mL) and concentrated HCl solution until pH 3 to
induce precipitation. Stirring was continued for 10 min. The solid pre-
cipitate was collected by filtration and rinsed with water. The wet filter cake
was dried and then purified by flash column chromatography to give the
pure products 7a, 7b, and 7c in yields of 54, 60, and 53%, respectively.
Data for 7a: mp, 155-156 °C; 1H NMR (400 MHz, CDCl3) δ 1.31
(s, 9H), 7.60 (s, 1H), 8.10 (s, 1H), 14.11 (s, 1H); ESI/MS, 341.9 (M þ H).
Data for 7b: mp, 158-160 °C; 1H NMR (400 MHz, CDCl3) δ 1.31
(s, 9H), 7.52 (d, J = 6.0 Hz, 1H), 7.91 (d, J = 9.6 Hz, 1H), 13.97 (s, 1H);
ESI/MS, 387.3 (M þ H).
Data for 8l: yield, 67%; mp, 88-89 °C; 1H NMR (600 MHz, CDCl3)
δ 0.93 (t, J = 7.2 Hz, 3H), 1.39 (s, 9H), 1.67-1.68 (m, 2H), 4.14 (t, J =
6.0 Hz, 2H), 4.18 (s, 2H), 7.90 (s,1H), 7.95 (s,1H); ESI/MS, 463.0
(M þ Na). Anal. Calcd for C18H20ClN3O4S2: C, 48.92; H, 4.56; N, 9.51.
Found: C, 48.65; H, 4.36; N, 9.44.
Data for 8m: yield, 75%; mp, 96-98 °C; 1H NMR (400 MHz, CDCl3) δ
1.39 (s, 9H), 2.31 (t, J = 2.4 Hz, 1H), 4.14 (d, J = 2.4 Hz, 2H), 7.92 (s, 1H),
8.01 (s, 1H); ESI/MS, 401.8 (M þ Na). Anal. Calcd for C16H14ClN3O2S2:
C, 50.59; H, 3.71; N, 11.06. Found: C, 50.67; H, 3.90; N, 11.23.
Data for 9a: yield, 62%; mp, 113-114 °C; 1H NMR (600 MHz, CDCl3)
δ 1.39 (s, 9H), 3.80 (s, 3H), 4.18 (s, 2H), 7.60 (d, J = 9.6 Hz, 1H), 8.13
(d, J = 4.2 Hz, 1H); ESI/MS, 398.03 (M þ H). Anal. Calcd for
C16H16FN3O4S2: C, 48.35; H, 4.06; N, 10.57. Found: C, 48.57; H, 4.21;
N, 10.70.
Data for 7c: mp, 150-152 °C; 1H NMR (400 MHz, CDCl3) δ 1.31
(s, 9H), 7.60 (s, 1H), 8.23 (s, 1H), 14.10 (s, 1H); ESI/MS, 337.9 (M þ Na).
General Synthesis Procedure for the Target Series of 8, 9, and 10.
K2CO3 powder (0.33 g, 2.4 mmol) was added to a solution of 7a, 7b, and 7c
(1.2 mmol) in acetone (20 mL). After 10 min of stirring at room
temperature, halogen derivative (RX, 1.8 mmol) in acetone solution was
added dropwise to the mixture. The resulting mixture reacted for about
30 min and then was filtered and concentrated. The residue was purified
via flash chromatography to give the pure products 8, 9, and 10 in yields of
35-89%.
Data for 9b: yield, 58%; mp, 143-144 °C; 1H NMR (600 MHz, CDCl3)
δ 1.39 (s, 9H), 3.80 (s, 3H), 4.00 (s, 3H), 7.73 (d, J = 8.4 Hz, 1H), 8.13 (d,
J = 6.6 Hz, 1H); ESI/MS, 383.2 (Mþ). Anal. Calcd for C15H14FN3O4S2:
C, 46.99; H, 3.68; N, 10.96. Found: C, 47.18; H, 3.87; N, 11.14.
Data for 9c: yield, 69%; mp, 151-152 °C; 1H NMR (600 MHz, CDCl3)
δ 1.39 (s, 9H), 1.40 (t, J = 7.2 Hz, 3H), 4.46 (q, J = 7.2 Hz, 2H), 7.73 (d,
J = 9.0 Hz, 1H), 8.0 (d, J = 6.6 Hz, 1H); ESI/MS, 419.6 (M þ Na). Anal.
Calcd for C16H16FN3O4S2: C, 48.35; H, 4.06; N, 10.57. Found: C, 48.50;
H, 4.28; N, 10.80.
Data for 8a: yield, 79%; mp 117-119 °C; 1H NMR (400 MHz, CDCl3)
δ 1.39 (s, 9H), 4.09 (d, J = 7.6 Hz, 2H), 6.26 (t, J = 7.6 Hz, 1H), 7.90
(s, 1H), 7.98 (s, 1H); ESI-MS, 473.5 (Mþ þ Na). Anal. Calcd for
C16H14Cl3N3O2S2:: C, 42.63; H, 3.13; N, 9.32. Found: C, 42.89; H, 3.23;
N, 9.50.
Data for 9d: yield, 67%; mp, 89-90 °C; 1H NMR (600 MHz, CDCl3) δ
1.28 (t, J = 7.2 Hz, 3H), 1.39 (s, 9H), 4.16 (s, 2H), 4.23 (q, J = 7.2 Hz, 2H),
7.60 (d, J = 9.6 Hz, 1H), 7.97 (d, J = 6.6 Hz, 1H); ESI/MS, 433.7
(M þ Na). Anal. Calcd for C17H18FN3O4S2: C, 49.62; H, 4.41; N, 10.21.
Found: C, 49.40; H, 4.28; N, 10.10.
Data for 9e: yield, 80%; mp, 143-145 °C; 1H NMR (600 MHz, CDCl3)
δ 1.39 (s, 9H), 1.48 (s, 9H), 4.07 (s, 2H), 7.60 (d, J = 9.0 Hz, 1H), 8.0 (d,
Data for 8b: yield, 35%; mp 160-161 °C; 1H NMR (400 MHz, CDCl3)
δ 1.38-1.43 (m, 12H), 4.67 (q, J = 4.8 Hz, 2H), 8.04 (s, 1H), 8.10 (s, 1H);
ESI-MS, 436.3 (Mþ þ Na). Anal. Calcd for C16H16ClN3O4S2: C, 46.43; H,
3.90; N, 10.15. Found: C, 46.20; H, 4.05; N, 10.04.
Data for 8c: yield, 60%; mp 89-90 °C; 1H NMR (400 MHz, CDCl3)
δ 1.26 (t, J = 7.2 Hz, 3H), 1.39 (s, 9H), 2.91 (t, J = 7.2 Hz, 2H), 3.61 (t,
J = 7.2 Hz, 2H), 4.18 (q, J = 7.2 Hz, 2H), 7.89 (s, 1H), 7.96 (s, 1H);
ESI-MS, 465.4 (Mþ þ Na). Anal. Calcd for C18H20ClN3O4S2: C, 48.92; H,
4.56; N, 9.51. Found: C, 48.68; H, 4.35; N, 9.62.
J
=
6.0 Hz, 1H); ESI/MS, 460.4 (M
þ
Na). Anal. Calcd for
C19H22FN3O4S2: C, 51.92; H, 5.05; N, 9.56. Found: C, 52.03; H, 5.25;
N, 9.67.
Data for 9f: yield, 84%; oil; 1H NMR (600 MHz, CDCl3) δ 1.39 (s, 9H),
3.99 (d, J = 7.2 Hz, 2H), 5.21 (d, J = 10.2 Hz, 1H), 5.37 (d, J = 16.8 Hz,
1H), 6.01-6.02 (m, 1H), 7.59 (d, J = 9.0 Hz, 1H), 7.98 (d, J = 6.6 Hz, 1H);
ESI/MS, 387.5 (M þ Na). Anal. Calcd for C16H16FN3O2S2: C, 52.59;
H, 4.41; N, 11.50. Found: C, 52.44; H, 4.11; N, 11.26.
Data for 8d: yield, 53%; mp, 98-100 °C; 1H NMR (400 MHz, CDCl3)
δ 1.28 (t, J = 6.8 Hz, 3H), 1.39 (s, 9H), 4.17 (s, 2H), 4.24 (q, J = 6.8 Hz,
2H), 7.90 (s, 1H), 7.95 (s, 1H); ESI/MS, 427.2 (Mþ). Anal. Calcd
for C17H18ClN3O4S2: C, 47.71; H, 4.24; N, 9.82. Found: C, 47.64; H,
4.44; N, 9.94.

2646 J. Agric. Food Chem., Vol. 58, No. 5, 2010
Jiang et al.
Data for 9g: yield, 61%; oil; 1H NMR (600 MHz, CDCl3) δ 1.27 (t,
J = 6.6 Hz, 3H), 1.39 (s, 9H), 2.90 (t, J = 7.2 Hz, 2H), 3.59 (t, J = 7.2 Hz,
2H), 4.18 (q, J = 6.6 Hz, 2H), 7.59 (d, J = 9.6 Hz, 1H), 7.98 (d, J = 6.6 Hz,
1H); ESI/MS, 447.4 (M þ Na). Anal. Calcd for C18H20FN3O4S2: C, 50.81;
H, 4.74; N, 9.88. Found: C, 51.10; H, 4.64; N, 9.87.
Calcd for C18H20BrN3O4S2: C, 44.45; H, 4.14; N, 8.64. Found: C, 44.33; H,
3.95; N, 8.45.
Data for 10h: yield, 73%; oil; 1H NMR (400 MHz, CDCl3) δ 1.39 (s,
9H), 2.30 (t, J = 2.8 Hz, 1H), 4.13 (d, J = 2.8 Hz, 2H), 8.00 (s, 1H), 8.09
(s,1H); ESI/MS, 424.3 (Mþ). Anal. Calcd for C16H14BrN3O2S2: C, 45.29;
H, 3.33; N, 9.90. Found: C, 45.19; H, 3.24; N, 9.67.
Data for 9h: yield, 63%; oil; 1H NMR (600 MHz, CDCl3) δ 1.25 (t, J =
7.2 Hz, 3H), 1.39 (s, 9H), 2.16-2.18 (m, 2H), 2.50 (t, J = 7.2 Hz, 2H), 3.41
(t, J = 7.2 Hz, 2H), 4.15 (q, J = 7.2 Hz, 2H), 7.59(d, J = 9.0 Hz, 1H),
7.98(d, J = 6.6 Hz, 1H). ESI/MS: 461.1(MþNa). Anal. Calcd for
C19H22FN3O4S2: C, 51.92; H, 5.05; N, 9.56. Found: C, 51.64; H, 5.01;
N, 9.38.
1
Data for 10i: yield, 53%; oil; H NMR (400 MHz, CDCl3) δ 1.40 (s,
9H), 4.10 (d, J = 7.2 Hz, 2H), 6.27 (s, 1H), 7.99 (s, 1H), 8.10 (s,1H);
ESI/MS, 495.6 (Mþ). Anal. Calcd for C16H14BrCl2N3O2S2: C, 38.80; H,
2.85; N, 8.48. Found: C, 39.08; H, 2.97; N, 8.69.
X-ray Diffraction. Colorless blocks of 8e (0.23 mm ꢀ 0.12 mm ꢀ
0.10 mm) were counted on a quartz fiber with protection oil. Cell
dimensions and intensities were measured at 300 K on a Bruker SMART
CCD area detector diffractometer with graphite monochromated Mo KR
Data for 9i: yield, 49%; oil; 1H NMR (600 MHz, CDCl3) δ 1.20 (t, J =
6.6 Hz, 3H), 1.39 (s, 9H), 1.76 (s, 6H), 4.20 (q, J = 6.6 Hz, 2H), 7.61 (d,
J = 9.0 Hz, 1H), 7.98 (d, J = 6.6 Hz, 1H); ESI/MS, 460.9 (M þ Na). Anal.
Calcd for C19H22FN3O4S2: C, 51.92; H, 5.05; N, 9.56. Found: C, 51.62; H,
4.91; N, 9.47.
˚
radiation (λ = 0.71073 A); θmax=26.5°; 10109 measured reflections; 4538
independent reflections (Rint = 0.040) of which 3683 had I > 2σ(I). Data
were corrected for Lorentz and polarization effects and for absorption
(Tmin = 0.9160; Tmax = 0.9622). The structure was solved by direct
methods using SHELXS-97 (28); all other calculations were performed
with Bruker SAINT System and Bruker SMART programs (29).
Full-matrix least-squares refinement based on F2 using the weight of
1/[σ2(Fo 2) þ (0.0786P)2 þ 0.3001P] gave final values of R = 0.049, ωR =
0.153, and GOF(F ) = 1.106 for 386 variables and 2650 contribut-
ing reflections. Maximum shift/error = 0.000(3), and max/min resi-
Data for 9j: yield, 45%; oil; 1H NMR (600 MHz, CDCl3) δ 0.93 (t, J =
7.8 Hz, 3H), 1.39 (s, 9H), 1.68-1.69 (m, 2H), 4.14 (t, J = 6.6 Hz, 2H), 4.17
(s, 2H), 7.59 (d, J = 9.0 Hz, 1H), 7.98 (d, J = 6.6 Hz, 1H); ESI/MS, 447.3
(M þ Na). Anal. Calcd for C18H20FN3O4S2: C, 50.81; H, 4.74; N, 9.88.
Found: C, 50.93; H, 4.96; N, 9.93.
Data for 9k: yield, 62%; mp, 125-127 °C; 1H NMR (600 MHz, CDCl3)
δ 1.39 (s, 9H), 4.08 (d, J = 7.8 Hz, 2H), 6.26 (t, J = 7.8 Hz, 1H), 7.60 (d,
J = 9.0 Hz, 1H), 7.98 (d, J = 6.6 Hz, 1H); ESI/MS, 455.2 (M þ Na). Anal.
Calcd for C16H14Cl2FN3O2S2: C, 44.24; H, 3.25; N, 9.67. Found: C, 44.47;
H, 3.36; N, 9.84.
dual electron density = 1.201/-1.297 e A-3. Hydrogen atoms were
˚
observed and refined with a fixed value of their isotropic displacement
parameter.
Data for 9l: yield, 62%; mp, 82-84 °C; 1H NMR (600 MHz, CDCl3) δ
1.26 (t, J = 6.6 Hz, 3H), 1.39 (s, 9H), 1.71 (d, J = 7.2 Hz, 2H), 4.22 (q, J =
6.6 Hz, 2H), 4.67 (q, J = 7.2 Hz, 2H), 7.60 (d, J = 9.6 Hz, 1H), 7.98 (d, J =
6.6 Hz, 1H); ESI/MS, 447.0 (M þ Na). Anal. Calcd for C18H20FN3O4S2:
C, 50.81; H, 4.74; N, 9.88. Found: C, 50.59; H, 4.55; N, 9.69.
Data for 9m: yield, 64%; mp, 102-104 °C; 1H NMR (600 MHz,
CDCl3) δ 1.39 (s, 9H), 3.73 (s, 3H), 4.11 (d, J = 7.2 Hz, 2H), 6.10 (d,
J = 15.6 Hz, 2H), 7.04-7.07 (m, 1H), 7.60 (d, J = 9.0 Hz, 1H), 8.0 (d, J =
6.6 Hz, 1H); ESI/MS, 444.9 (M þ Na). Anal. Calcd for C18H18FN3O4S2:
C, 51.05; H, 4.28; N, 9.92. Found: C, 51.26; H, 4.34; N, 10.03.
Data for 9n: yield, 67%; mp, 97-99 °C; 1H NMR (600 MHz, CDCl3) δ
1.27 (d, J = 6.0 Hz, 6H), 1.39 (s, 9H), 4.12 (s, 2H), 5.08-5.10 (m, 1H), 7.60
(d, J = 7.4 Hz, 1H), 7.95 (d, J = 6.6 Hz, 1H); ESI/MS, 447.7 (M þ Na).
Anal. Calcd for C18H20FN3O4S2: C, 50.81; H, 4.74; N, 9.88. Found: C,
51.07; H, 4.82; N, 9.95.
Data for 10a: yield, 89%; mp 110-111 °C; 1H NMR (600 MHz, CDCl3) δ
1.28 (t, J = 6.6 Hz, 3H), 1.39 (s, 9H), 4.16 (s, 2H), 4.24 (q, J = 6.6 Hz, 2H),
7.95 (s, 1H), 8.07 (s, 1H); ESI/MS, 493.9 (M þ Na). Anal. Calcd for
C17H18BrN3O4S2: C, 43.22; H, 3.84; N, 8.90. Found: C, 43.43; H, 4.04; N, 8.92.
Data for 10b: yield, 88%; mp, 125-127 °C; 1H NMR (600 MHz,
CDCl3) δ 1.39 (s, 9H), 3.79 (s, 3H), 4.18 (s, 2H), 7.96 (s, 1H), 8.07 (s,1H);
ESI/MS, 479.7 (M þ Na). Anal. Calcd for C16H16BrN3O4S2: C, 41.93; H,
3.52; N, 9.17. Found: C, 42.05; H, 3.69; N, 9.33.
Enzyme Expression, Purification, and Inhibition Kinetic Analy-
sis. Recombinant human PPO was expressed by using the pTrcHis vector
and purified according to reported methods (3, 30, 31). The pTrcHis
(PPO) plasmid was transformed into Escherichia coli JM109. Transformed
cells carrying pTrcHis (PPO) were inoculated into 0.5 L of 2ꢀ YT grown
at 37 °C with 200 rpm shaking until an A600 of 0.6 was reached. The
expression of the recombinant PPO enzyme was induced by adding
isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 mM). Cells were grown
for an additional 4 h at 25 °C, harvested by centrifugation, resuspended in
50 mM Tris buffer (pH 8.0) containing 0.5 M NaCl, 1 μg/mL phenyl-
methanesulfonyl fluoride (PMSF), and 0.2% (v/v) Triton X-100, and then
disrupted by sonication. Cellular debris was removed by centrifugation at
12500 rpm. The supernatant was subjected to Ni-affinity chromatography.
Protein concentrations were determined according to the method of
Bradford with bovine serum albumin (BSA) as a standard (32). PPO
activity was assayed by fluorescence as described previously (33-36). The
product has a maximum excitation wavelength at 410 nm and a maximum
emission wavelength at 630 nm. The total volume of the reaction mixture
was 200 μL consisting of 0.1 M potassium phosphate buffer (pH 7.4),
5 mM dithiothreitol (DTT), 1 mM EDTA, 0.2 M imidazole, and 0.03%
Tween 80 (v/v). The enzymatic reaction was started by the addition
of substrate. The autoxidation rates were determined concomitantly
and were subsequently subtracted. The concentration of protoporphyr-
inogen IX was determined by the difference of the absorption of proto-
porphyrin IX before and after the complete enzyme oxidation of the
substrate monitored by UV-vis spectrophotometer at a wavelength of
410 nm. The concentration of protoporphyrin IX was calculated from
the calibration graph. In assays, the inhibitors were added from a
stock solution in dimethyl sulfoxide (DMSO) and then added 1% total
volume in each assay. The final concentration ranged from 0.005 to
250 μM.
Data for 10c:yield 75%; mp, 116-118 °C; 1H NMR (600 MHz, CDCl3) δ
1.26 (d, J = 6.0 Hz, 6H), 1.39 (s, 9H), 4.13 (s, 2H), 5.08 (m, 1H), 7.93 (s, 1H),
8.07 (s, 1H); ESI/MS, 507.7 (M þ Na). Anal. Calcd for C18H20BrN3O4S2: C,
44.45; H, 4.14; N, 8.64. Found: C, 44.19; H, 4.11; N, 8.44.
Data for 10d: yield, 61%; oil; 1H NMR (400 MHz, CDCl3) δ 1.25 (t,
J = 7.2 Hz, 3H), 1.39 (s, 9H), 2.17-2.19 (m, 2H), 2.49 (t, J = 7.2 Hz, 2H),
3.41 (t, J = 7.2 Hz, 2H), 4.13 (q, J = 7.2 Hz, 2H), 7.95 (s, 1H), 8.06 (s, 1H);
ESI/MS, 500.4 (M þ H). Anal. Calcd for C19H22BrN3O4S2: C, 45.60; H,
4.43; N, 8.40. Found: C, 45.43; H, 4.33; N, 8.32.
Data for 10e: yield, 35%; oil; 1H NMR (400 MHz, CDCl3) δ 1.25 (t,
J = 7.2 Hz, 3H), 1.39 (s, 9H), 1.71 (d, J = 7.2 Hz, 3H), 4.22 (q, J = 7.2 Hz,
2H), 4.69 (q, J = 7.2 Hz, 1H), 7.95 (s, 1H), 8.07 (s,1H); ESI/MS, 486.2
(Mþ). Anal. Calcd for C18H20BrN3O4S2: C, 44.45; H, 4.14; N, 8.64.
Found: C, 44.58; H, 4.19; N, 8.80.
The kinetic parameters were evaluated by Sigma Plot software 10.0
(SPSS, Chicago, IL). IC50 was determined by measuring PPO activity over
a range of inhibitor concentrations at a single substrate concentration.
IC50 values were calculated by fitting v versus [I] data to a single binding
site model described by eq 1
Data for 10f: yield, 79%; oil; 1H NMR (400 MHz, CDCl3) δ 1.26 (t, J =
7.2 Hz, 3H), 1.39 (s, 9H), 2.89 (t, J = 6.8 Hz, 2H), 3.59 (t, J = 6.8 Hz, 2H),
4.69 (q, J = 7.2 Hz, 2H), 7.96 (s, 1H), 8.06 (s, 1H); ESI/MS, 486.1 (Mþ).
Anal. Calcd for C18H20BrN3O4S2: C, 44.45; H, 4.14; N, 8.64. Found: C,
44.69; H, 4.25; N, 8.85.
max -min
y ¼ min þ
ð1Þ
50-x
1 þ 10logIC
where y is the percentage of maximal rate, max and min are the y values at
which the curve levels off, x is the logarithm of inhibitor concentration,
and IC50 is the inhibitor concentration that elicits 50% of the total
inhibition. The calculated Ki value is obtained by applying the following
Data for 10g: yield, 46%; oil; 1H NMR (400 MHz, CDCl3) δ 0.92 (t,
J = 7.6 Hz, 3H), 1.39 (s, 9H), 1.67-1.69 (m, 2H), 4.13 (t, J = 6.8 Hz, 2H),
4.17 (s, 2H), 7.94 (s, 1H), 8.07 (s, 1H); ESI/MS, 487.3 (M þ H). Anal.

Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 2647
relationship, which exists for competitive inhibition among Ki, Km, and
IC50 at any saturating substrate concentration (S).
Table 1. PPO Inhibition and Herbicidal Activity of Series 8, 9, and 10
postemergence,a 150 g of ai/ha
IC50
Ki
¼
ð2Þ
no.
8a
X
R
ki (μM) EC DS PA BJ AR EP
S=Km þ 1
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
CH2CHCCl2
COOCH2CH3
10.24
0.33
0.25
1.01
0.84
1.07
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
/
0
0
0
0
0
0
0
0
0
0
0
0
/
70 70 40
70 70 40
70 80 60
50 70 60
50 40 30
CoMSIA Analysis. Comparative molecular similarity indices analy-
sis (CoMSIA) (37) was applied to understand the QSAR of the com-
pounds tested in this study. The 3D structures of all compounds were built
on the basis of the crystal structure of 8e and then minimized by using
SYBYL 7.1 on a Silicon Graphics Fuel workstation. The geometries of all
molecules involved in this study were fully optimized by using the PM3
method. The lowestenergyconformationswereconsideredas the bioactive
conformations. A useful kind of net atomic charges called electrostatic
potential (ESP) fitting charges were derived from the PM3 calculated
molecular electrostatic potential distribution. The common framework of
benzothiazole and 1,3,4-oxadiazole-2(3H)-one ring was selected as the
template to superimpose all of the compounds using an atom-by-atom
least-squares fit implemented in the SYBYL FIT option in SYBYL. 8c
with the best biological activity was selected as the reference molecule.
CoMSIA similarity index descriptors AF,k for a molecule j with atoms i at
the grid point q are determined as
8b
8c
8d
8e
8f
CH2CH2COOCH2CH3
CH2COOCH2CH3
CH2COOC(CH3)3
CH2COOCH3
0
0
70 50
50
8g
8h
8i
CH2CH2CH2COOCH2CH3 2.02
0
CH(CH3)COOCH2CH3
CH2CHdCH2
1.69
3.11
1.35
2.17
0.98
8.61
3.99
0.72
1.30
1.58
1.59
15.92
0.32
50 100 70
70 70 60
60 50 50
8j
C(CH3)2COOCH2CH3
CH2COOCH(CH3)2
CH2COOCH2CH2CH3
CH2CCH
8k
8l
0
0
/
50
70
/
0
0
/
0
b
/
8m
9a
9b
9c
9d
9e
9f
CH2COOCH3
COOCH3
COOCH2CH3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
60
40
0
0
0
0
F
F
X
F
CH2COOCH2CH3
CH2COOC(CH3)3
CH2CHdCH2
50 90 50
0
2
iq
AqF kðjÞ ¼
ωprobe;kωik e-Rr
ð3Þ
F
0
0
,
i
F
60 60 40
60 50 50
50 90 70
50 60 70
50 100 90
where ωik is the actual value of the physicochemical property k (steric,
electrostatic, hydrophobic), which was evaluated using the probe atom. A
Gaussian type distance dependence was used between the grid point q and
each atom i of the molecule. The default value of 0.3 was used as the
attenuation factor R. An sp3 carbon atom with þ1.0 charge served as the
probe atom to calculate steric, electrostatic, and hydrophobic fields. Two
angstrom grid point spacing was used. The all-orientation search (AOS)
method was used for CoMSIA analysis. The AOS routine (38) optimizes
the field sampling by rotating the molecular aggregate systematically and
picking the orientation that produces the highest q2 value. Details of the
AOS routine were described previously (37, 39). Briefly, the whole
aggregate was rotated about the x, y, and z axes systematically with an
increment of 30° using the STATIC ROTATE command. For each
orientation, a conventional CoMSIA was performed as described above
and the predictive value of the model was evaluated using leave-one-out
(LOO) cross-validation with sample-distance partial least-squares
(SAMPLS). The orientation that gave the highest q2 value was selected
to produce the final model. A Sybyl Programming language (SPL) script
was written to perform the AOS routine as described (39) automatically.
Greenhouse Herbicidal Activities. The herbicidal activities of series 8,
9, and 10 against monocotyledon weeds such as Echinochloa crus-galli (EC),
Digitaria sanguinalis (DS), and Poa annua (PA) and dicotyledon weeds such
as Brassica juncea (BJ), Amaranthus retroflexus (AR), and Eclipta prostrate
(EP) were evaluated according to a previously reported procedure (40-42).
Sulfentrazone was selected as a positive control because it is one of the few
soil-active PPO inhibitors (43-45). All test compounds were formulated as
100 g/L emulsified concentrates by using DMF as solvent and Tween-80 as
emulsification reagent. The concentrated formulas 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. 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 9.5 cm
were filled with soil to a depth of 8 cm. Approximately 20 seeds of the tested
weeds were sown in the soil at the depth of 1-3 cm and grown at 15-30 °C
in a greenhouse. The air relative humidity is 50%. The diluted formulation
solutions were applied for postemergence treatment; dicotyledon weeds
were treated at the two-leaf stage, and monocotyledon weeds were treated at
the one-leaf stage, respectively. The postemergence application rate was
150 g of ai/ha. Untreated seedlings were used as the control group, and
solvent (DMF þ Tween-80) treated seedlings were used as the solvent
control group. Herbicidal activity was evaluated visually 15 days after
treatment. The results of herbicidal activities are shown in Table 1, three
replicates per treatment. Seven kinds of dicotyldon weeds, such as B. juncea,
Polygonum humifusum, Abutilon theophrasti, Cyperus iria, A. retroflexus,
Rumex acetasa, and E. prostrate were used for the further test of 8c, 8h, 9d,
9h, 9j, 9l, 9n, and 10b.
9g
9h
9i
F
CH2CH2COOCH2CH3
F
CH2CH2CH2COOCH2CH3 0.70
F
C(CH3)2COOCH2CH3
CH2COOCH2CH2CH3
CH2CHCCl2
1.54
0.70
9j
F
9k
9l
F
10.92 50 40 50 70 60 60
F
CH(CH3)COOCH2CH3
CH2CHdCHCOOCH3
CH2COOCH(CH3)2
1.42
7.48
1.17
6.96
7.46
5.97
0
0
0
0
0
0
/
50 50 75 90 95
9m
9n
10a
F
0
0
0
0
0
/
0
0
0
0
0
/
40 40 50
40 90 70
F
Br CH2COOCH2CH3
0
0
0
/
75
80
75
/
0
0
0
/
10b Br CH2COOCH3
10c Br CH2COOCH(CH3)2
10d Br CH2CH2CH2COOCH2CH3 5.83
10e
10f
Br CH(CH3)COOCH2CH3
Br CH2CH2COOCH2CH3
8.13
1.69
0
0
0
0
/
0
0
0
0
/
0
0
0
0
/
0
0
0
70 60
30
50 30
0
10g Br CH2COOCH2CH2CH3
10h Br CH2CCH
3.10
11.12
18.63
40 40 40
10i
sulfentrazone
Br CH2CHCCl2
/ /
100 100 100
/
0.72 75 70
/
a EC, Echinochloa crus-galli; DS, Digitaria sanguinalis; PA, Poa annua; BJ,
Brassica juncea; AR, Amaranthus retroflexus; EP, Eclipta prostrata. b /, not tested.
RESULTS AND DISCUSSION
Synthesis of the Title Compounds. As shown in Scheme 2, the
target series 8, 9, and 10 were prepared by a seven-step synthetic
route. The preparation of intermediates 2, 3, 4, and 5 was easily
obtained according to existing methods and resulted in good to
excellent yields. After obtaining intermediates 5, we tried to
prepare intermediate 7 directly from series 5 according to the
reported method (46). However, the reaction of intermediate 5
with sodium polysulfide and carbon disulfide was so complex that
it is difficult to isolate the desired product in good yield. However,
the use of iron powder to reduce intermediate 5 into 6, which
underwent ring-closing reaction with potassium O-ethyl carbo-
nodithioate in the DMF solution, produced the key intermediate
7 in acceptable yields (53-60%). Finally, intermediates 7a-c
reacted with various alkylation reagents to give the target series 8,
9, and 10 in yields of 35-89%. The structures of all intermediates
1
and title compounds were confirmed by elemental analyses, H
NMR, and ESI-MS spectral data. In addition, the crystal
structure of 8e was determined by X-ray diffraction analyses.
As shown in Figure 2, the dihedral angle between the benzothia-
zole ring (max deviation = 0.013 A) and 1,3,4-oxadiazol-2(3H)-
one ring is 58.69°. The crystal packing is stabilized by two
intermolecular hydrogen-bonding (C-H O) interactions.
˚
3 3 3

2648 J. Agric. Food Chem., Vol. 58, No. 5, 2010
Jiang et al.
In Vitro Activity and CoMSIA Analysis. The ki values against
human PPO of the synthesized compounds 8-10 are listed in
Table 1. Sulfentrazone, a commercial PPO inhibitor, was used as
a positive control. For the sake of clarity, 8a-m, 9a-n, and 10a-i
will be named as chlorine, fluorine, and bromine derivatives,
respectively, throughout the text. Results as shown in Table 1
indicated that 8c (ki = 0.25 μM) displayed the highest PPO
inhibitory activity, about 3 times higher than that of sulfentra-
zone (ki = 0.72 μM). Generally speaking, series 10 (X = Br)
always displayed a much lower PPO inhibitory activity than series
8 (X = Cl) and 9 (X = F) containing the same R group, for
example, 8a, 9k, and 10i (R = CH2CHCCl2), 8c, 9g, and 10f
(R = CH2CH2COOCH2CH3), 8d, 9d, and 10a (R = CH2COO-
CH2CH3). However, series 8 sometimes displayed higher activity
than series 9 containing the same R group but sometimes lower
activity than the corresponding analogues 9. For example, 8b, 8c,
8d, 8e, 8f, 8i, and 8j displayed higher activity than the correspond-
ing 9c, 9g, 9d, 9e, 9a, 9f, and 9i. In addition, Table 1 also shows
that CH2CH2COOCH2CH3 is the best group for the R position
among the investigated substituents, because 8c, 9g, and 10f
displayed the highest activity within the chlorine, fluorine, and
bromine derivatives, respectively.
To understand the substituent effects on the PPO inhibition of
these compounds, the method of CoMSIA was applied to under-
stand the quantitative structure-activity relationships. As listed
in Table 2, a predictive CoMSIA model was established with the
conventional correlation coefficient r2 = 0.933 and the cross-
validated coefficient q2 = 0.585; the contributions of steric,
electrostatic, and hydrophobic fields are 19.0, 47.5, and 33.5%,
respectively. The observed and calculated activity values for all of
the compounds are given in Table 3, and the plots of the predicted
versus the actual activity values for all of the compounds are
shown in Figure 3. In Figures 4, 5, and 6, the isocontour diagrams
of the steric, electrostatic, and hydrophobic field contributions
(“stdev*coeff”) obtained from the CoMSIA analysis are illu-
strated together with exemplary ligands. The steric field contour
map is plotted in Figure 4. The green region highlights positions
where a bulky group would be favorable for higher PPO inhibi-
tion activity. In contrast, yellow indicates positions where a
decrease in the bulk of the desired compounds is favored. As
shown in Figure 4, the CoMSIA steric contour plots indicated
that a yellow region is located around the side chain of the
benzothiazolyl group, whereas a green region is near the carboxyl
oxygen atom. This map means that the substituents at this
position should have an optimum steric effect, which supports
8c (R = CH2CH2COOC2H5, ki = 0.25 μM) being more active
than 8d (R = CH2COOC2H5, ki = 1.01 μM) and 8g (R =
CH2CH2CH2COOC2H5, ki = 2.02 μM). The electrostatic con-
tour plot is shown in Figure 5. The blue contour defines a region
where an increase in the positive charge will result in an increase in
the activity, whereas the red contour defines a region of space
where increasing electron density is favorable. As shown in
Figure 5, the target compounds bearing an electron-withdrawing
Figure 2. Crystal structure of 8e.
Scheme 2. Synthetic Route for the Title Compounds 8, 9, and 10

Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 2649
Table 2. Summary of CoMSIA Analysis
contribution (%)
2
qcv
2
rn-cv
s
F
comp steric electrostatic hydrophobic
0.585 0.933 0.142 67.706
6
19.0
47.5
33.5
Table 3. Results of the Experimental and Calculated ki Values
pkia
pkia
no.
expt
calcd
no.
exptl
calcd
Figure 5. Electrostatic map from the CoMSIA model. 8c is shown inside
the field. Blue contours (0.070 level) encompass regions where an
increase of positive charge will enhance affinity, whereas in red contoured
areas (-0.040 level) more negative charges are favorable for binding
properties.
8a
8b
8c
8d
8e
8f
4.99
6.48
6.60
6.00
6.08
5.97
5.69
5.77
5.51
5.87
5.66
6.01
5.06
5.40
6.14
5.89
5.80
5.80
5.15
6.29
6.55
6.04
6.16
6.03
5.62
5.75
5.28
5.87
5.78
5.99
5.17
5.27
6.17
6.20
5.76
5.79
9f
9g
4.80
6.49
6.15
5.81
6.15
4.96
5.85
5.13
5.93
5.16
5.13
5.22
5.23
5.09
5.77
5.51
4.95
4.73
5.12
6.42
6.15
5.83
6.03
4.86
5.63
5.02
5.87
5.24
5.10
5.25
5.19
5.34
5.67
5.63
4.92
4.63
9h
9i
9j
9k
8g
8h
8i
9l
9m
9n
8j
10a
10b
10c
10d
10e
10f
10g
10h
10i
8k
8l
8m
9a
9b
9c
9d
9e
a pki = -log ki.
Figure 6. Hydrophobic map from the CoMSIA model. 8c is shown inside
the field. Violet contours (0.010 level) encompass regions where an
increase of hydrophobic effect will enhance affinity, whereas in gray
contoured areas (-0.050 level) a more hydrophilic effect is favorable for
binding properties.
always displayed higher activity than the derivatives (8a, 8i, 8m,
9f, 9k, 10h, and 10i) bearing an alkyl group as side chain. As
shown in Figure 6, violet regions indicate areas where hydro-
phobic groups increase activity, and gray regions indicate areas
where hydrophilic groups will increase activity. Fluorine and
chlorine atoms are more hydrophilic than bromine atoms; there-
fore, series 8 and 9 are always more active than series 10. In
addition, the ester group is hydrophobic, which accounts for why
compounds containing an ester group always displayed higher
activity. In fact, the PPO inhibition of chlorine derivatives 8a-m
displayed linear correlation with the hydrophobic effects to some
extent (data not shown).
Figure 3. CoMSIA predicted as experimental pKi values.
Greenhouse Herbicidal Activities. The postemergence herbici-
dal activity of series 8, 9, and 10 was tested in the greenhouse at a
concentration of 150 g. of ai/ha; a triazolinone-type commercial
product, sulfentrazone, was selected as a positive control. As
shown in Table 1, no compound had herbicidal activity at the
concentration of 150 g of ai/ha against E. crus-galli, D. sangui-
nalis, or P. annus. On the other hand, some of the series had good
herbicidal activities against B. juncea, A. retroflexus, and E.
prostrate. The inhibition effects of 8c, 8h, 9d, 9h, 9j, 9l, 9n, and
10b against A. retroflexus are >80%. Therefore, these eight
compounds were selected for further test. As shown in Table 4,
9l was the most promising candidate. Even at the concentration of
37.5 g of ai/ha, 9l showed excellent herbicidal activity with broad
spectrum. Its inhibition effects against A. theophrasti, C. iria,
R. acetasa, and E. prostrate are >80%. At the same time, 9l
also displayed moderate herbicidal activity against B. juncea,
P. humifusum, and A. retroflexus at the concentration of 37.5 g of
ai/ha.
Figure 4. Steric map from the CoMSIA model. 8c is shown inside the field.
Sterically favored areas (0.030 level) are represented by green polyhedra.
Sterically disfavored areas (-0.004 level) are represented by yellow
polyhedra.
group at the side chain of benzothiazole ring will display higher
activity. For example, compounds containing an ester group

2650 J. Agric. Food Chem., Vol. 58, No. 5, 2010
Jiang et al.
Table 4. Herbicidal Spectrum Test of Eight Selected Compounds
(Postemergence)a
heterocyclic protoporphyrinogen oxidase inhibitors. Pest. Manag.
Sci. 2004, 60, 1178–1188.
(5) Dayan, F. E.; Green, H. M.; Weete, J. D.; Hancock, H. G.
Postemergence activity of sulfentrazone: effects of surfactants and
leaf surfaces. Weed Sci. 1996, 44, 797–803.
(6) Dayan, F. E.; Duke, S. O.; Weete, J. D.; Hancock, H. G. Selectivity
and mode of action of carfentrazone-ethyl, a novel phenyl triazoli-
none herbicide. Pestic. Sci. 1997, 51, 65–73.
compd dosage (g of ai/ha) BJ PH
AT
CI
AR
RA
EP
8c
8h
9d
9h
9j
37.5
75
60
60
60
70
70
70
0
40
50
60
50
60
80
60
70
70
50
60
60
0
0
150
(7) Shapiro, R.; DiCosimo, R.; Hennessey, S. M.; Stieglitz, B.; Campo-
piano, O.; Chiang, G. C. Discovery and development of a commer-
cial synthesis of azafenidin. Org. Process Res. Dev. 2001, 5, 593–
598.
37.5
75
0
0
0
40
60
70
0
0
0
60
60
0
30
60
0
40
70
60 100
70 100
150
(8) Dayan, F. E.; Duke, S. O.; Reddy, K. N.; Hamper, B. C.; Leschinsky,
K. L. Effects of isoxazole herbicides on protoporphyrinogen oxidase
and porphyrin physiology. J. Agric. Food Chem. 1997, 45, 967–975.
(9) Hess, F. D. Light-dependent herbicides: an overview. Weed Sci.
2000, 48, 160–170.
37.5
75
0
50
50
70
70
80
0
0
0
60
70
70
50
50
90
50
50
70
0
0
150
50
37.5
75
50
50
50
50
80
80
0
0
60
60
70
60
80
90
50
60
70
50
60
70
(10) Dayan, F. E.; Duke, S. O. Phytotoxicity of protoporphyrinogen
oxidase inhibitors: phenomenology, mode of action and mechanisms
of resistance. In Herbicide Activity: Toxicology, Biochemistry and
Molecular Biology; Roe, R. M., Burton, J. D., Kuhr, R. J., Eds.; IOS
Press: Washington, DC, 1997; pp 11-35.
(11) Lehnen, L. P.; Sherman, T. D.; Becerril, J. M.; Duke, S. O. Tissue
and cellular localization of acifluorfen-induced porphyrins in
cucumber cotyledons. Pestic. Biochem. Physiol. 1990, 37, 239–248.
(12) Duke, S. O.; Kenyon, W. H. Peroxidizing activity determined by
cellular leakage. In Target Assays for Modern Herbicides and Related
150
50
37.5
75
50
50
50
60
70
80
0
0
0
40
40
0
50
60
70
0
40
90
60
150
60 100
9l
37.5
75
70
70
75
60
70
70
80
100
90
90
60
60
95
95
80
85
95
150
100 100
90 100
€
Compounds; Boger, P., Sandmann, G., Eds.; Lewis Publishers: Boca
Raton, FL, 1993; pp 61-66.
9n
10b
37.5
75
40
50
50
40
40
60
0
0
0
50
50
60
0
85
90
50
60
70
0
0
(13) Dayan, F. E.; Duke, S. O. Herbicides: Protoporphyrinogen oxidase
inhibitors. In Encyclopedia of Agrochemicals; Plimmer, J. R., Gammon,
D. W., Ragsdale, N. N., Eds.; Wiley: New York, 2003; Vol. 2, pp 850-
863.
(14) Hwang, I. T.; Hong, K. S.; Choi, J. S.; Kim, H. R.; Jeon, D. J.; Cho,
K. Y. Protoporphyrinogen IX-oxidizing activities involved in the
mode of action of a new compound N-[4-chloro-2-fluoro-5-{3-(2-
fluorophenyl)-5-methyl-4,5-dihydroisoxazol-5-yl-methoxyl}-phenyl]-
3,4,5,6-tetrahydrophthalimide. Pestic. Biochem. Physiol. 2004, 80,
123–130.
150
70
37.5
75
0
0
0
60
70
70
0
0
0
40
40
50
50
60
80
40
50
60
0
0
0
150
a BJ, Brassica juncea; PH, Polygonum humifusum; AT, Abutilon theophrasti; CI,
Cyperus iria; AR, Amaranthus retroflexus; RA, Rumex acetasa; EP, Eclipta
prostrate.
In summary, a series of 1,3,4-oxadiazol-2(3H)-ones containing
benzothiazole substructure were designed and synthesized as
potential PPO inhibitors. The results of in vitro and greenhouse
tests indicated that series 8, 9, and 10 had good PPO inhibition
activity and herbicidal activity at the concentration of 150 g
of ai/ha. Most interestingly, 9l, ethyl 2-((5-(5-tert-butyl-2-oxo-1,3,4-
oxadiazol-2(3H)-yl)-6-fluorobenzothiazol-2-yl)sulfanyl)propanoate,
was identified as the most promising candidate due to its high PPO
inhibition effect (ki = 1.42 μM) and broad-spectrum herbicidal
activity at the concentration of 37.5 g of ai/ha. The crop selectivity
and field trial of 9l are under way.
(15) Dayan, F. E.; Meazza, G.; Bettarini, F.; Signorini, E.; Piccardi, P.;
Romagni, J. G.; Duke, S. O. Synthesis, herbicidal activity, and mode
of action of IR 5790. J. Agric. Food Chem. 2001, 49, 2302–2307.
(16) Gitsopoulos, T. K.; Froud-Williams, R. J. Effects of oxadiargyl on
direct-seeded rice and Echinochloa crus-galli under aerobic and
anaerobic conditions. Weed Res. 2004, 44, 329–334.
(17) Hutchinson, I.; Jennings, S. A.; Vishnuvajjala, B. R.; Westwell,
A. D.; Stevens, M. F. G. Antitumor benzothiazoles. 16. Synthesis
and pharmaceutical properties of antitumor 2-(4-aminophenyl)-
benzothiazole amino acid prodrugs. J. Med. Chem. 2002, 45, 744.
(18) Mortimer, C. G.; Wells, G.; Crochard, J.; Stone, E. L.; Bradshaw,
T. D.; Stevens, M. F. G.; Westwell, A. D. Antitumor benzothiazoles.
26. 2-(3,4-Dimethoxyphenyl)-5-fluoro-benzothiazole (GW 610,
NSC 721648), a simple fluorinated 2-arylbenzothiazole, shows
potent and selective inhibitory activity against lung, colon, and
breast cancer cell lines. J. Med. Chem. 2006, 49, 179.
ACKNOWLEDGMENT
We thank Dr. Jie Chen for the greenhouse test of biological
activities.
(19) Huang, W.; Zhao, P. L.; Liu, C. L.; Chen, Q.; Liu, Z. M.; Yang, G. F.
Design, synthesis and fungicidal activities of new strobilurin deriva-
tives. J. Agric. Food Chem. 2007, 55, 3004–3010.
LITERATURE CITED
(1) Duke, S. O.; Nandihalli, U. B.; Lee, H. J.; Duke, M. V. Proto-
porphyrinogen oxidase as the optimal herbicide site in the por-
phyrin pathway. Am. Chem. Soc. Symp. Ser. 1994, No. 559, 191–
204.
(2) Scalla, R.; Matringe, M. Inhibitors of protoporphyrinogen oxidase
as herbicides: diphenyl ethers and related. Rev. Weed Sci. 1994, 6,
103–132.
(3) Manelia, M. H.; Corrigalla, A. V.; Klumpb, H. H.; Davidsa, L. M.;
Kirscha, R. E.; Meissnera, P. N. Kinetic and physical characterisa-
tion of recombinant wild-type and mutant human protoporphyrino-
gen oxidases. Biochim. Biophys. Acta 2003, 1650, 10–21.
(4) Meazza, G.; Bettarini, F.; La Porta, P.; Piccardi, P.; Signorini, E;
Portoso, D.; Fornara, L. Synthesis and herbicidal activity of novel
(20) Huang, W.; Tan, Y.; Ding, M. W.; Yang, G. F. An improved
synthesis of 2-(3H)benzo- thiazolethiones under microwave irradia-
tion. Synth. Commun. 2007, 37, 369–376.
(21) Huang, W.; Yang, G. F. Microwave-sssisted, one-pot syntheses and
fungicidal activity of polyfluorinated 2-benzylthiobenzothiazoles.
Bioorg. Med. Chem. 2006, 14, 8280–8285.
(22) Dekeyser, M. A.; Canada, W.; McDonald, P. T.; Conn, M. Insecti-
cidal phenylhydrazine derivatives. U.S. Patent 5367093, 1994; Chem.
Abstr. 1994, 123, 9144.
(23) Bullock, M. W.; Hand, J. J. Synthesis of some substituted indole-3-
butyric acids. J. Am. Chem. Soc. 1956, 78, 5854–5857.
(24) Sawada, Y.; Yanai, T.; Nakagawa, H.; Tsukamoto, Y.; Yokoi, S.;
Yanagi, M.; Toya, T.; Sugizaki, H.; Kato, Y.; Shirakura, H.;

Article
J. Agric. Food Chem., Vol. 58, No. 5, 2010 2651
Watanabe, T.; Yajima, Y.; Kodama, S.; Masui, A. Synthesis and
(36) Tan, Y.; Sun, L.; Xi, Z.; Yang, G. F.; Jiang, D. Q.; Yan, X. P.; Yang,
X.; Li, H. Y. A capillary electrophoresis assay for protoporphyrino-
gen oxidase. Anal. Biochem. 2008, 383, 200–204.
(37) Klebe, G.; Abraham, U.; Mietzner, T. Molecular similarity indices in
a comparative analysis (CoMSIA) of drug molecules to correlate and
predict their biological activity. J. Med. Chem. 1994, 37, 4130–4146.
(38) Wang, R.; Gao, Y.; Liu, L.; Lai, L. All-orientation search and all-
placement search in comparative molecular field analysis. J. Mol.
Model. 1998, 4, 276–283.
(39) Gangjee, A.; Lin, X. CoMFA and CoMSIA analyses of Pneumo-
cystis carinii dihydrofolate reductase, Toxoplasma gondii dihydro-
folate deductase, and rat liver dihydrofolate reductase. J. Med.
Chem. 2005, 48, 1448–1469.
(40) Chen, C. N.; Lv, L. L.; Ji, F. Q.; Chen, Q.; Xu, H.; Niu, C. W.; Xi, Z.;
Yang, G. F. Design and synthesis of N-2,6-difluorophenyl-5-meth-
oxyl-1,2,4-triazolo[1,5-a]pyrimidine-2- sulfonamide as acetohydrox-
yacid synthase inhibitor. Bioorg. Med. Chem. 2009, 17, 3011–3017.
(41) Luo, Y. P.; Jiang, L. L.; Wang, G. D.; Chen, Q.; Yang, G. F.
Syntheses and herbicidal activities of novel triazolinone derivatives.
J. Agric. Food Chem. 2008, 56, 2118–2124.
insecticidal activity of benzoheterocyclic analogues of N0-benzoyl-
N-(tert-butyl)benzohydrazide: part 2. Introduction of substituents
on the benzene rings of the benzoheterocycle moiety. Pest Manag.
Sci. 2003, 59, 36–48.
(25) Fischer, R.; Jensen-Korte, U.; Kunisch, F.; Marhold, A.; Ooms, P.;
Schallner, O.; Santel, H. J.; Schmidt, R. R.; Krauskopf, B.; Strang,
R. H. N-Aryl-substituted nitrogencontaining heterocycles, processes
and novel intermediates for their preparation, and their use as
herbicides and plant growth regulators. U.S. Patent 6906006, 2005;
Chem. Abstr. 2005, 143, 21460.
(26) Bosch, J.; Roca, T.; Catena, J. L.; Farrerons, C.; Miquel, I. A
convergent route to 5-(arylsulfanyl)-6-sulfonamido-3-benzofura-
nones. Synthesis 2000, 721–725.
(27) Zhu, L.; Zhang, M. B.; Dai, M. A convenient synthesis of 2-
mercapto and 2-chlorobenzo-thiazoles. J. Heterocycl. Chem. 2005,
42, 727.
€
(28) Sheldrick, G. M. SHELXTL (Version 5.0); University of Gottingen:
€
Gottingen, Sweden, 2001.
(29) SMART V5.628, SAINT V6.45, and SADABS; Bruker AXS: Madi-
son, WI, 2001.
(30) Dailey, T. A.; Dailey, T. A. Human protoporphyrinogen oxidase:
expression, purification, and characterization of the cloned enzyme.
Protein Sci. 1996, 5, 98–105.
(42) Li, Y. X.; Luo, Y. P.; Xi, Z.; Niu, C. W.; He, Y. Z.; Yang, G. F.
Design and syntheses of novel phthalazin-1(2H)-one derivatives as
acetohydroxyacid synthase inhibitors. J. Agric. Food Chem. 2006, 54,
9135–9139.
(43) Grey, T. L.; Walker, R. H.; Wehtje, G. R.; Adams, J.; Dayan, F. E.;
Weete, J. D.; Hancock, H. G.; Kwon, O. Behavior of sulfentrazone
in ionic exchange resins, electrophoresis gels, and cation-saturated
soils. Weed Sci. 2000, 48, 239–247.
(44) Dayan, F. E.; Weete, J. D.; Duke, S. O.; Hancock, H. G. Soybean
(Glycine max) cultivar differences in response to sulfentrazone.
Weed Sci. 1997, 45, 634–641.
(45) Dayan, F. E.; Weete, J. D.; Hancock, H. G. Physiological basis for
differential sensitivity to sulfentrazone by sicklepod (Senna obtusifolia)
and coffee senna (Cassia occidentalis). Weed Sci. 1996, 44, 12–17.
(46) Li, Z. G.; Wang, Q. M.; Huang, J. M. Preparation of Organic
Intermediates; Chemical Industry Press: Beijing, China, 1996; p 141.
(31) Corrigall, A. V.; Siziba, K. B.; Maneli, M. H.; Shephard, E. G.;
Ziman, M.; Dailey, T. A.; Dailey, H. A.; Kirsch, R. E.; Meissner,
P. N. Purification of and kinetic studies on a cloned protoporphyrino-
gen oxidase from the aerobic bacterium Bacillus subtilis. Arch.
Biochem. Biophys. 1998, 358, 251–256.
(32) Berezovski, M.; Krylov, S. N. Using nonequilibrium capillary
electrophoresis of equilibrium mixtures for the determination of
temperature in capillary electrophoresis. Anal. Chem. 2004, 76,
7114–7117.
(33) Meissner, P. N.; Day, R. S.; Moore, M. R.; Disler, P. B.; Harley, E.
Protoporphyrinogen oxidase and porphobilinogen deaminase in
variegate porphyria. Eur. J. Clin. Invest. 1986, 16, 257–261.
(34) Shepherd, M.; Dailey, H. A. A continuous fluorimetric assay for
protoporphyrinogen oxidase by monitoring porphyrin accumula-
tion. Anal. Biochem. 2005, 344, 115–121.
Received for review July 28, 2009. Revised manuscript received
September 12, 2009. Accepted November 19, 2009. We are grateful for
financial support from the National Basic Research Program of China
(2010CB126103, 2003CB114400), the National Natural Science
Foundation of China (No. 20925206 and 20932005), and the Research
Fund for the Doctoral Program of Higher Education (No. 20060511003).
(35) Jeong, E.; Houn, T. A point mutation of valine-311 to methionine in
Bacillus subtilis protoporphyrinogen oxidase does not greatly in-
crease resistance to the diphenyl ether herbicide oxyfluorfen. Bioorg.
Chem. 2003, 31, 389–397.