Novel protoporphyrinogen oxidase inhibitors: 3H-pyrazolo[3,4-d][1,2,3] triazin-4-one derivatives

Article abstract of DOI:10.1021/jf801774k

A series of 3H-pyrazolo[3,4-d][1,2,3]triazin-4-one derivatives were synthesized as candidate herbicides by diazotization of different 5(3)-amino-N-phenyl-1H-pyrazole-4-carboxamide derivatives prepared by the reaction of substituted 5(3)-amino-pyrazole-4-carbonyl chloride with a substituted aniline. Their structures were identified by ¹H NMR and elemental analyses. The isomers D and E were isolated, and their structures were identified by two-dimensional NMR analyses (heteronuclear single quantum coherence and heteronuclear multiple-bond correlation) and single-crystal X-ray diffraction analysis. The bioassay results showed that some of the title compounds exhibited both excellent herbicidal activity at a dose of 93.75 g/ha and strong inhibition against protoporphyrinogen oxidase activity in vitro. The structure-activity relationship showed that D16 possessed the highest activities both in vivo and in vitro when the N-substituted group of the pyrazole ring was allyl and the N-substituted group of benzooxazinone was propargyl.

Full text of DOI:10.1021/jf801774k

J. Agric. Food Chem. 2008, 56, 9535–9542 9535
Novel Protoporphyrinogen Oxidase Inhibitors:
3H-Pyrazolo[3,4-d][1,2,3]triazin-4-one Derivatives
HUA-BIN LI, YOU-QUAN ZHU, XIAO-WEI SONG, FANG-ZHONG HU, BIN LIU,
YONG-HONG LI, ZI-XIA NIU, PEI LIU, ZHI-HONG WANG, HAI-BIN SONG,
XIAO-MAO ZOU,* AND HUA-ZHENG YANG*
State Key Laboratory of Elemento-organic Chemistry, Institute of Elemento-organic Chemistry,
Nankai University, Tianjin 300071, People’s Republic of China
A series of 3H-pyrazolo[3,4-d][1,2,3]triazin-4-one derivatives were synthesized as candidate herbicides
by diazotization of different 5(3)-amino-N-phenyl-1H-pyrazole-4-carboxamide derivatives prepared
by the reaction of substituted 5(3)-amino-pyrazole-4-carbonyl chloride with a substituted aniline. Their
1
structures were identified by H NMR and elemental analyses. The isomers D and E were isolated,
and their structures were identified by two-dimensional NMR analyses (heteronuclear single quantum
coherence and heteronuclear multiple-bond correlation) and single-crystal X-ray diffraction analysis.
The bioassay results showed that some of the title compounds exhibited both excellent herbicidal
activity at a dose of 93.75 g/ha and strong inhibition against protoporphyrinogen oxidase activity in
vitro. The structure-activity relationship showed that D16 possessed the highest activities both in
vivo and in vitro when the N-substituted group of the pyrazole ring was allyl and the N-substituted
group of benzooxazinone was propargyl.
KEYWORDS: 3H-Pyrazolo[3,4-d][1,2,3]triazin-4-one; PPO inhibitor; SAR; herbicidal activity
INTRODUCTION
herbicidal activities, such as flumioxazin (2) (Figure 1). In our
previous work (9), the imidazotetrazinone moiety of mitozolo-
mide A and temozolomide B with antineoplastic activity (Figure
1) (10, 11) was modified into pyrazolotetrazinone C, and some
of these compounds provided more than 80% control of Brassica
campestris at 10 µg/mL. To further improve their herbicidal
activity and find valuable lead compounds, according to the
bioisosteric principle, novel 3H-pyrazolo[3,4-d][1,2,3]triazin-
4-one derivatives D and E were designed (Figure 1). In this
paper, their synthesis, herbicidal activities in vivo, and inhibition
activities in vitro against PPO have been described.
The protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4) is the
last enzyme in the common tetrapyrrole biosynthesis pathway
before the pathway branches toward chlorophyll and heme
synthesis. The enzyme is the target of many classes of herbicides
including diphenyl ether, cyclic imides, and thiadiazolidines.
The application of PPO-inhibiting herbicides to plant leads to
the peroxidative destruction of cellular membrane and bleaching
of tissues in the presence of light. In contrast to other herbicides,
PPO inhibitors have some general characteristics: (i) PPO
inhibitors give long-lasting control for up to 30 days after the
application. (ii) PPO inhibitors are effective on currently difficult
to control weeds. (iii) PPO inhibitors are more rapid than many
other herbicides, causing necrosis within 24 h and death in 2-5
days. (iv) PPO inhibitors in combination with other herbicides
can provide one-shot weed control with a wide window of
application. For these characteristics, PPO inhibitors constitute
a kind of important herbicide (1). Generally, commercial cyclic
imides possess the following structural features: (i) a heterocycle
structure with one or more nitrogen atoms; (ii) a polysubstituted
benzene ring that links with the nitrogen atom of the heterocycle
ring (2-8). It was noticed that when the polysubstituted benzene
ring was replaced by a benzo-heterocycle ring, the corresponding
compounds also possessed excellent PPO inhibitory activity and
MATERIALS AND METHODS
Synthetic Procedures. 1H NMR spectra were obtained on 300 MHz
(a Bruker AV300 spectrometer), 400 MHz (Varian Mercury Plus400
spectrometer), or 600 MHz (a Bruker AV600) in CDCl3 solution with
tetramethylsilane as the internal standard. Chemical shift values (δ)
are given in ppm. Elemental analyses were determined on a Yanaca
CHN Corder MT-3 elemental analyzer. Melting points were taken on
a Thomas-Hoover melting point apparatus and are uncorrected. Yields
were not optimized. Solvents were dried according to standard methods
and distilled prior to use.
General Synthetic Procedure for F and H. Compounds F and H
were synthesized as the literature described (12-14).
General Synthetic Procedure for D1-13 (15, 16). After the mixture
of F (10 mmol) in THF-MeOH (50 mL, V:V ) 1:1) with 2.5 N NaOH
(25 mL) was heated at 60 °C for 4 h, the solvent was removed under
reduced pressure, and the residue was acidified with 6 N HCl at 0 °C. A
gray solid was precipitated, filtered, and washed with water. Then, the
* To whom correspondence should be addressed. Tel: +86-22-
23503799. Fax: +86-22-23503627. E-mail: nk_yanghz@126.com or
zyq8165@nankai.edu.cn.
10.1021/jf801774k CCC: $40.75  2008 American Chemical Society
Published on Web 09/23/2008

9536 J. Agric. Food Chem., Vol. 56, No. 20, 2008
Li et al.
Figure 1. Chemical structures of A-E and flumioxazin.
Scheme 1
Scheme 2
dry solid was stirred with SOCl2 at room temperature for 3 h, and the
corresponding acid chloride was obtained by vacuo to remove the excess
of SOCl2. The substituted aniline (10 mmol) in CH2Cl2 (50 mL) and
pyridine (5 mL) was mixed with the acid chloride at 0 °C and stirred at
room temperature overnight and concentrated, and the residue was washed
with 0.5 N HCl, saturated NaHCO3 solution, and brine and was dried in
vacuo to obtain G used for next step without further purification.
The solution of G (1 mmol) in 10 mL of 6 N HCl and 1 mL of
MeOH was stirred for 10 h and then cooled down below 0 °C, sodium
nitrite (3 mmol) in 2 mL of water was added dropwise and stirred for
2.5 h, and then, ice water (10 mL) was added and extracted with ethyl
acetate (15 mL × 3). The organic layer was washed with brine and
dried over anhydrous Na2SO4. The solvent was removed under reduced
pressure, and the residue was purified by flash column chromatography
on silica gel using ethyl acetate-petroleum ether as an eluent to afford
the products D1-13 (Scheme 1).
General Synthetic Procedure for D14-19 and E1-6. NaH (60%,
11 mmol) was added slowly to a stirred solution of H (10 mmol) in 50
mL of dry dimethyl formamide (DMF) at 0 °C. After 0.5 h, the
appropriate alkyl halide (11 mmol) was added dropwise and stirred
for 4 h at room temperature. Then, the mixture was slowly poured into
ice water (200 mL) and extracted with ethyl acetate. The organic layer
was washed with brine and dried over anhydrous Na2SO4. The solvent
was removed under reduced pressure to give a mixture of F and I (17),
and the mixture was directly used for the preparation of G and J without
isolation according to the procedure of Scheme 1.
General Synthetic Procedure for D20-28. A solution of D3 (0.68
mmol) in 10 mL of DMF was cooled down below 0 °C, and 0.5 mL
of NaOH (1.4 mmol) solution was added slowly. The cooling bath was
removed after addition, and the solution was stirred at room temperature
for 2 h. The reaction mixture was poured slowly into 50 mL of ice
water, the pH was adjusted to 6, and the mixture was extracted with
ethyl acetate. The organic layer was washed with brine and dried over
anhydrous Na2SO4. The solvent was removed in vacuo, and the residue
was purified by flash column chromatography on silica gel, using ethyl
acetate-petroleum ether as the eluent to afford the pure target product
D20 (18).
Different halides were reacted with D20 (0.34 mmol) and K2CO3
(0.35 mmol) in 10 mL of DMF to afford the pure target products
D21-28, respectively (Scheme 3). The melting points, yields, and
elemental analyses of compounds D1-28 and E1-6 are listed in Table
1
1-3, and the data of their H NMR are listed in Table 4.
Bioassays. For comparative purposes, the herbicidal activities of the
title compounds D, E, and flumioxazin {2-(7-fluoro-3-oxo-4-(prop-2-
ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-4,5,6, 7-tetrahydro-2H-
isoindole-1,3-dione} were evaluated using a previously reported
procedure (19). All treatments were triplicated.
Inhibitory Activity in Vitro against PPO. The pI50 values against
PPO of compounds D and E were assayed according to the procedure
reported (20-25). Etioplasts were prepared from the etiolated leaves
of corn. Seven day old leaves of dark-grown corn seedlings were
homogenized with an FSH-2A Variable High Speed Homogenizer for
20 s at 15000 rpm using a fresh weight to volume ratio of 1:5.
Homogenization buffer consisted of 50 mM Hepes (pH 7.8 at 25 °C),
500 mM sucrouse, 1 mM EDTA, 1 mM MgCl2, 1 mM dithiothreitol
(DTT), and 0.2% bovine serum albumin (BSA). Homogenate was
filtered through two layers of fabric, and crude cell debris was removed
by centrifugation at 800g for 2 min at 4 °C. Etioplasts were collected
As polarities of G and J are very similar, the mixture of these
compounds was used to prepare the target products D14-19 and E1-6
according to the procedure of Scheme 1. Their structures were identified
by two-dimensional (2D) NMR analyses [heteronuclear single quantum
coherence (HSQC) and heteronuclear multiple-bond correlation (HMBC)]
and single-crystal X-ray diffraction analysis (Scheme 2).

3H-Pyrazolo[3,4-d][1,2,3]triazin-4-one Derivatives
J. Agric. Food Chem., Vol. 56, No. 20, 2008 9537
Scheme 3
Table 1. List of Compounds D and E
no.
R1
R2
R3
R4
no.
R1
R2
R3
R4
D1
D2
D3
D4
D5
D6
D7
D8
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
allyl
allyl
allyl
allyl
H
H
H
CF3
CH3
CH3
H
H
H
CF3
CF3
CH3
CH3
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
OCH2C′CH
OCH2CH)CH2
OCO2CH2CH3
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
E1
n-C3H7
n-C3H7
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
allyl
allyl
allyl
allyl
n-C3H7
n-C3H7
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R3R4dOCH2CONCH2C′CH
R3R4dOCH2CONCH2CHdCH2
OH
OCH3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
OCH2C′CH
OCH2C′CH
OC2H5
O(n-C4H9)
O(n-C5H11)
OCH2CH)CH2
R3R4dOCH2CONCH2C′CH
R3R4dOCH2CONCH2CHdCH2
R3R4dOCH2CON(n-C3H7)
R3R4dOCH2CONCH2C′CH
R3R4dOCH2CONCH2CHdCH2
R3R4dOCH2CONCH2C′CH
R3R4dOCH2CONCH2CHdCH2
OCH2C′CH
OCH2CHdCHCl(E)a
OCH2C(Cl)dCH2
OCH2CN
D9
D10
D11
D12
D13
D14
D15
D16
D17
OCH2OC2H4OCH3
OCH2C′CH
OCH2CHdCH2
E2
E3
E4
E5
Cl
Cl
R3R4dOCH2CONCH2C′CH
R3R4dOCH2CONCH2CHdCH2
R3R4dOCH2CONCH2C′CH
R3R4dOCH2CONCH2CHdCH2
OCH2CHdCH2
R3R4dOCH2CONCH2C′CH
R3R4dOCH2CONCH2CHdCH2
E6
a It showed the configuration of the alkenyl substituent.
Table 2. Melting Points and Yields of Compounds D and E
no.
mp (°C)
yield (%)
no.
mp (°C)
yield (%)
no.
mp (°C)
yield (%)
D1
D2
D3
D4
D5
D6
D7
D8
215-216
191-193
182-184
144-145
192-194
123-124
245-246
235-237
208-210
261-263
238-239
235-237
27.8a
34.2a
48.9a
29.0a
16.5a
18.0a
47.7a
60.9a
52.2a
27.2a
20.5a
19.5a
D13
D14
D15
D16
D17
D18
D19
D20
D21
D22
D23
D24
212-213
147-149
149-151
193-195
136-137
173-175
124-126
235-237
220-222
204-205
162-163
144-145
17.6a
20.0b
9.9b
D25
D26
D27
D28
E1
E2
E3
E4
E5
182-184
155-157
137-139
115-117
169-170
132-134
189-191
162-163
215-217
196-198
97.3
91.9
71.7
16.2b
11.5b
18.4b
19.1b
99.7
67.8
30.0b
14.9b
24.3b
17.2b
27.6b
28.6b
D9
79.4
99.5
84.1
89.0
D10
D11
D12
E6
a Yield calculated from starting material F. b Yield calculated from starting material H.
Table 3. Elemental Analysis Data of Compounds D and E
elemental analysis (%, calcd)
elemental analysis (%, calcd)
H
no.
C
H
N
no.
C
N
D1
D2
D3
D4
D5
D6
D7
D8
50.21 (50.39)
49.93 (50.09)
45.72 (45.73)
44.79 (44.85)
51.91 (51.81)
51.31 (51.51)
54.39 (54.24)
53.71 (53.93)
53.62 (53.63)
48.46 (48.35)
48.28 (48.12)
55.29 (55.44)
54.99 (55.13)
53.41 (53.42)
53.22 (53.12)
56.70 (56.84)
56.64 (56.54)
2.92 (2.72)
3.39 (3.30)
2.99 (3.02)
2.19 (2.01)
3.31 (3.19)
3.97 (3.75)
3.17 (3.13)
3.52 (3.68)
4.07 (4.22)
2.62 (2.39)
2.72 (2.85)
3.77 (3.56)
4.11 (4.08)
3.21 (3.08)
3.90 (3.62)
3.56 (3.45)
4.07 (3.95)
20.77 (20.99)
20.82 (20.86)
18.99 (19.05)
17.29 (17.43)
20.01 (20.14)
20.11 (20.02)
23.48 (23.72)
23.62 (23.59)
23.21 (23.45)
19.96 (19.90)
19.64 (19.81)
22.60 (22.82)
22.60 (22.69)
19.22 (19.47)
19.10 (19.36)
21.92 (22.10)
21.70 (21.98)
D18
D19
D20
D21
D22
D23
D24
D25
D26
D27
D28
E1
56.35 (56.54)
56.04 (56.25)
44.40 (44.69)
46.28 (46.54)
48.00 (48.23)
51.19 (51.22)
52.40 (52.54)
45.16 (45.43)
45.18 (45.43)
46.42 (46.65)
46.70 (46.95)
53.25 (53.42)
53.08 (53.12)
56.54 (56.84)
56.50 (56.54)
56.58 (56.54)
56.21 (56.25)
4.09 (3.95)
4.59 (4.46)
2.37 (2.39)
3.07 (2.93)
3.43 (3.43)
4.26 (4.30)
4.65 (4.68)
2.95 (2.72)
2.84 (2.72)
2.48 (2.41)
3.96 (3.94)
3.20 (3.08)
3.40 (3.62)
3.62 (3.45)
4.08 (3.95)
3.93 (3.95)
4.31 (4.46)
22.17 (21.98)
21.81 (21.86)
23.48 (23.69)
22.41 (22.61)
21.40 (21.63)
19.81 (19.91)
19.01 (19.15)
18.92 (18.92)
18.68 (18.92)
25.06 (25.11)
18.38 (18.25)
19.31 (19.47)
19.47 (19.36)
21.86 (22.10)
22.09 (21.98)
22.09 (21.98)
21.92 (21.86)
D9
D10
D11
D12
D13
D14
D15
D16
D17
E2
E3
E4
E5
E6
from this supernatant by centrifugation at 17000g for 6 min at 4 °C.
The extracts were resuspended in assay buffer (50 mM Tris, 2 mM
EDTA, and 20% glycerol, pH 7.3, at 25 °C) and stored at -80 °C
until use.
Protoporphyrinogen IX (protogen IX) was prepared according to
Jacobs (23) with the following modifications. Protoporphyrin IX (Proto
IX) stock solution (0.5 mM in 20% ethanol containing 10 mM KOH)
was reduced to protogen IX with approximately one-eighth volume of

9538 J. Agric. Food Chem., Vol. 56, No. 20, 2008
Table 4. 1H NMR of Compounds D and E
Li et al.
no.
δ (ppm)
D1 (400 MHz)
2.57 (t, 1H, J ) 2.2 Hz, ’CH), 4.31 (s, 3H, NCH3), 4.79 (d, 2H, J ) 2.2 Hz,
OCH2), 7.21 (d, 1H, J ) 6.3 Hz, Ph), 7.38 (d, 1H, J ) 8.9 Hz, Ph), 8.25 (s,
1H, pyrazole-H)
D2 (300 MHz)
D3 (300 MHz)
4.31 (s, 3H, NCH3), 4.61 (d, 2H, J ) 5.1 Hz, OCH2), 5.32-5.50 (m, 2H,
dCH2), 5.99-6.15 (m, 1H, CH), 7.02 (d, 1H, J ) 6.4 Hz, Ph), 7.37 (d, 1H,
J ) 9.0 Hz, Ph), 8.24 (s, 1H, pyrazole-H)
1.41 (t, 3H, J ) 7.1 Hz, CH3), 4.31 (s, 3H, NCH3), 4.37 (q, 2H, J ) 7.1 Hz,
OCH2), 7.44 (d, 1H, J ) 9.0 Hz, Ph), 7.46 (d, 1H, J ) 6.7 Hz, Ph), 8.24 (s,
1H, pyrazole-H)
D4 (400 MHz)
D5 (400 MHz)
2.57 (t, 1H, J ) 2.2 Hz, ’CH), 4.36 (s, 3H, NCH3), 4.79 (d, 2H, J ) 2.2 Hz,
OCH2), 7.20 (d, 1H, J ) 6.3 Hz, Ph), 7.39 (d, 1H, J ) 8.9 Hz, Ph)
2.57 (t, 1H, J ) 1.9 Hz, ’CH), 2.65 (s, 3H, CCH3), 4.21 (s, 3H, NCH3), 4.78 (d,
2H, J ) 1.9 Hz, OCH2), 7.19 (d, 1H, J ) 6.3 Hz, Ph), 7.37 (d, 1H, J ) 9.0
Hz, Ph)
D6 (400 MHz)
D7 (300 MHz)
D8 (300 MHz)
D9 (400 MHz)
D10 (400 MHz)
D11 (400 MHz)
D12 (400 MHz)
D13 (400 MHz)
D14 (400 MHz)
2.65 (s, 3H, CCH3), 4.21 (s, 3H, NCH3), 4.60 (d, 2H, J ) 5.1 Hz, OCH2),
5.32-5.48 (m, 2H, dCH2), 6.00-6.09 (m, 1H, CH), 7.00 (d, 1H, J ) 6.4
Hz, Ph), 7.37 (d, 1H, J ) 9.0 Hz, Ph)
2.28 (t, 1H, J ) 2.4 Hz, ’CH), 4.32 (s, 3H, NCH3), 4.70 (d, 2H, J ) 2.4 Hz,
OCH2), 4.74 (s, 2H, OCH2), 6.99 (d, 1H, J ) 9.7 Hz, Ph), 7.29 (d, 1H, J )
6.7 Hz, Ph), 8.25 (s, 1H, pyrazole-H)
4.31 (s, 3H, NCH3), 4.54-4.56 (m, 2H, NCH2), 4.74 (s, 2H, OCH2), 5.18-5.27
(m, 2H, dCH2), 5.78-5.90 (m, 1H, CH), 6.97 (d, 1H, J ) 9.7 Hz, Ph), 7.04
(d, 1H, J ) 6.8 Hz, Ph), 8.24 (s, 1H, pyrazole-H)
0.95 (t, 3H, J ) 7.4 Hz, CH3), 1.65-1.74 (m, 2H, CH2), 3.87 (t, 2H, J ) 7.4
Hz, NCH2), 4.31 (s, 3H, NCH3), 4.69 (s, 2H, OCH2), 6.96 (d, 1H, J ) 9.7 Hz,
Ph), 7.02 (d, 1H, J ) 6.8 Hz, Ph), 8.25 (s, 1H, pyrazole-H)
2.28 (t, 1H, J ) 2.4 Hz, ’CH), 4.36 (s, 3H, NCH3), 4.69 (d, 2H, J ) 2.3 Hz,
OCH2), 4.74 (s, 2H, OCH2), 6.98 (d, 1H, J ) 9.7 Hz, Ph), 7.28 (d, 1H, J )
6.7 Hz, Ph)
4.35 (s, 3H, NCH3), 4.54-4.55 (m, 2H, NCH2), 4.74 (s, 2H, OCH2), 5.18-5.27
(m, 2H, dCH2), 5.78-5.88 (m, 1H, CH), 6.96 (d, 1H, J ) 9.7 Hz, Ph), 7.03
(d, 1H, J ) 6.8 Hz, Ph)
2.27 (t, 1H, J ) 2.4 Hz, ’CH), 2.65 (s, 3H, CCH3), 4.21 (s, 3H, NCH3), 4.68 (d,
2H, J ) 2.4 Hz, CH2), 4.73 (s, 2H, OCH2), 6.97 (d, 1H, J ) 9.6 Hz, Ph),
7.27 (d, 1H, J ) 7.8 Hz, Ph)
2.65 (s, 3H, CCH3), 4.21 (s, 3H, NCH3), 4.54-4.55 (m, 2H, NCH2), 4.73 (s,
2H, OCH2), 5.18-5.26 (m, 2H, dCH2), 5.79-5.88 (m, 1H, CH), 6.95 (d, 1H,
J ) 9.7 Hz, Ph), 7.03 (d, 1H, J ) 6.8 Hz, Ph)
2.57 (t, 1H, J ) 2.1 Hz, ’CH), 4.78 (d, 2H, J ) 2.1 Hz, OCH2), 5.25 (d, 2H, J
) 5.9 Hz, NCH2), 5.34-5.39 (m, 2H, dCH2), 6.07-6.17 (m, 1H, CH), 7.21
(d, 1H, J ) 6.3 Hz, Ph), 7.38 (d, 1H, J ) 8.9 Hz, Ph), 8.28 (s, 1H,
pyrazole-H)
D15 (400 MHz)
D16 (400 MHz)
4.60-4.61 (m, 2H, OCH2), 5.24-5.26 (m, 2H, NCH2), 5.32-5.48 (m, 4H,
2dCH2), 6.00-6.15 (m, 2H, 2CH), 7.01 (d, 1H, J ) 6.3 Hz, Ph), 7.37 (d,
1H, J ) 8.6 Hz, Ph), 8.27 (s, 1H, pyrazole-H)
2.28 (t, 1H, J ) 2.4 Hz, ’CH), 4.69 (d, 2H, J ) 2.4 Hz, NCH2), 4.74 (s, 2H,
OCH2), 5.26 (d, 2H, J ) 6.0 Hz, NCH2), 5.35-5.40 (m, 2H, dCH2),
6.08-6.18 (m, 1H, CH), 6.98 (d, 1H, J ) 9.7 Hz, Ph), 7.29 (d, 1H, J ) 6.7
Hz, Ph), 8.28 (s, 1H, pyrazole-H)
D17 (400 MHz)
D18 (400 MHz)
D19 (400 MHz)
4.54-4.55 (m, 2H, NCH2), 4.74 (s, 2H, OCH2), 5.18-5.26 (m, 4H, 2CH2),
5.34-5.39 (m, 2H, dCH2), 5.79-5.88 (m, 1H, CH), 6.07-6.17 (m, 1H, CH),
6.96 (d, 1H, J ) 9.7 Hz, Ph), 7.04 (d, 1H, J ) 6.8 Hz, Ph), 8.27 (s, 1H,
pyrazole-H)
1.00 (t, 3H, J ) 7.4 Hz, CH3), 2.04-2.12 (m, 2H, CH2), 2.27 (t, 1H, J ) 1.9
Hz, ’CH), 4.61 (t, 2H, J ) 7.2 Hz, NCH2), 4.69 (d, 2H, J ) 1.9 Hz, NCH2),
4.73 (s, 2H, OCH2), 6.98 (d, 1H, J ) 9.6 Hz, Ph), 7.29 (d, 1H, J ) 6.7 Hz,
Ph), 8.25 (s, 1H, pyrazole-H)
1.00 (t, 3H, J ) 7.4 Hz, CH3), 2.04-2.10 (m, 2H, CH2), 4.54-4.55 (m, 2H,
NCH2), 4.60 (t, 2H, J ) 7.1 Hz, NCH2), 4.74 (s, 2H, OCH2), 5.19-5.26 (m,
2H, dCH2), 5.80-5.88 (m, 1H, CH), 6.96 (d, 1H, J ) 9.8 Hz, Ph), 7.04 (d,
1H, J ) 6.7 Hz, Ph), 8.24 (s, 1H, pyrazole-H)
D20 (300 MHz)
D21 (400 MHz)
D22 (400 MHz)
4.31 (s, 3H, NCH3), 5.60 (s, 1H, OH), 7.16 (d, 1H, J ) 6.5 Hz, Ph), 7.33 (d,
1H, J ) 8.8 Hz, Ph), 8.25 (s, 1H, pyrazole-H)
3.91 (s, 3H, OCH3), 4.31 (s, 3H, NCH3), 7.00 (d, 1H, J ) 6.3 Hz, Ph), 7.37 (d,
1H, J ) 8.9 Hz, Ph), 8.25 (s, 1H, pyrazole-H)
1.48 (t, 3H, J ) 6.9 Hz, CH3), 4.09 (q, 2H, J ) 6.9 Hz, OCH2), 4.30 (s, 3H,
NCH3), 6.99 (d, 1H, J ) 6.3 Hz, Ph), 7.36 (d, 1H, J ) 8.9 Hz, Ph), 8.24 (s,
1H, pyrazole-H)
D23 (400 MHz)
0.98 (t, 3H, J ) 7.4 Hz, CH3), 1.49-1.57 (m, 2H, CH2), 1.79-1.86 (m, 2H,
CH2), 4.02 (t, 2H, J ) 6.4 Hz, OCH2), 4.30 (s, 3H, NCH3), 6.99 (d, 1H, J )
6.4 Hz, Ph), 7.35 (d, 1H, J ) 9.0 Hz, Ph), 8.24 (s, 1H, pyrazole-H)

3H-Pyrazolo[3,4-d][1,2,3]triazin-4-one Derivatives
Table 4. Continued
J. Agric. Food Chem., Vol. 56, No. 20, 2008 9539
no.
δ (ppm)
D24 (400 MHz)
0.93 (t, 3H, J ) 7.2 Hz, CH3), 1.36-1.50 (m, 4H, 2CH2), 1.81-1.88 (m, 2H,
CH2), 4.01 (t, 2H, J ) 6.5 Hz, OCH2), 4.30 (s, 3H, NCH3), 6.99 (d, 1H, J )
6.4 Hz, Ph), 7.35 (d, 1H, J ) 9.0 Hz, Ph), 8.24 (s, 1H, pyrazole-H)
D25 (300 MHz)
D26 (400 MHz)
4.31 (s, 3H, NCH3), 4.58 (dd, 2H, J1 ) 1.4 Hz, J2 ) 5.8 Hz, OCH2),
6.14-6.22 (m, 1H, CH), 6.44-6.49 (m, 1H, CH), 7.01 (d, 1H, J ) 6.4 Hz,
Ph), 7.39 (d, 1H, J ) 8.9 Hz, Ph), 8.25 (s, 1H, pyrazole-H)
4.31 (s, 3H, NCH3), 4.64 (s, 2H, OCH2), 5.49-5.50 (m, 1H, )CH2), 5.65-5.66
(m, 1H, )CH2), 7.02 (d, 1H, J)6.3 Hz, Ph), 7.39 (d, 1H, J)8.9 Hz, Ph),
8.25 (s, 1H, pyrazole-H)
D27 (300 MHz)
D28 (300 MHz)
4.32 (s, 3H, NCH3), 4.86 (s, 2H, OCH2), 7.25 (d, 1H, J ) 5.4 Hz, Ph), 7.44 (d,
1H, J ) 8.9 Hz, Ph), 8.26 (s, 1H, pyrazole-H)
3.35 (s, 3H, OCH3), 3.55 (t, 2H, J ) 3.3 Hz, OCH2), 3.88 (t, 2H, J ) 3.0 Hz,
OCH2), 4.31 (s, 3H, NCH3), 5.35 (s, 2H, OCH2), 7.36 (d, 1H, J ) 9.0 Hz,
Ph), 7.41 (d, 1H, J ) 6.6 Hz, Ph), 8.24 (s, 1H, pyrazole-H)
2.57 (t, 1H, J ) 2.4 Hz, ’CH), 4.79 (d, 2H, J ) 2.4 Hz, OCH2), 5.05 (d, 2H, J
E1 (400 MHz)
) 6.3 Hz, NCH2), 5.43-5.50 (m, 2H, dCH2), 6.07-6.17 (m, 1H, CH), 7.23
(d, 1H, J ) 6.4 Hz, Ph), 7.36 (d, 1H, J ) 9.0 Hz, Ph), 8.26 (s, 1H,
pyrazole-H)
E2 (400 MHz)
E3 (400 MHz)
4.60-4.62 (m, 2H, OCH2), 5.04-5.06 (m, 2H, NCH2), 5.31-5.50 (m, 4H,
2dCH2), 6.00-6.17 (m, 2H, 2CH), 7.03 (d, 1H, J ) 6.4 Hz, Ph), 7.35 (d,
1H, J ) 9.0 Hz, Ph), 8.26 (s, 1H, pyrazole-H)
2.27 (t, 1H, J ) 1.8 Hz, ’CH), 4.69 (d, 2H, J ) 1.8 Hz, NCH2), 4.72 (s, 2H,
OCH2), 5.06 (d, 2H, J ) 5.6 Hz, NCH2), 5.43-5.50 (m, 2H, dCH2),
6.07-6.17 (m, 1H, CH), 6.96 (d, 1H, J ) 9.6 Hz, Ph), 7.30 (d, 1H, J)6.7
Hz, Ph), 8.27 (s, 1H, pyrazole-H)
E4 (400 MHz)
E5 (400 MHz)
E6 (400 MHz)
4.54-4.56 (m, 2H, NCH2), 4.72 (s, 2H, OCH2), 5.05 (d, 2H, J ) 6.3 Hz,
NCH2), 5.18-5.26 (m, 2H, dCH2), 5.42-5.50 (m, 2H, dCH2), 5.79-5.88
(m, 1H, CH), 6.07-6.17 (m, 1H, CH), 6.94 (d, 1H, J ) 9.7 Hz, Ph), 7.06 (d,
1H, J ) 6.8 Hz, Ph), 8.26 (s, 1H, pyrazole -H)
1.00 (t, 3H, J ) 7.4 Hz, CH3), 2.04-2.13 (m, 2H, CH2), 2.27 (t, 1H, J ) 2.4
Hz, ’CH), 4.40 (t, 2H, J ) 7.0 Hz, NCH2), 4.69 (d, 2H, J ) 2.1 Hz, NCH2),
4.72 (s, 2H, OCH2), 6.96 (d, 1H, J ) 9.7 Hz, Ph), 7.30 (d, 1H, J ) 6.8 Hz,
Ph), 8.23 (s, 1H, pyrazole-H)
1.00 (t, 3H, J ) 7.4 Hz, CH3), 2.05-2.11 (m, 2H, CH2), 4.40 (t, 2H, J ) 6.9
Hz, NCH2), 4.54-4.55 (m, 2H, NCH2), 4.72 (s, 2H, OCH2), 5.18-5.26 (m,
2H, dCH2), 5.79-5.87 (m, 1H, CH), 6.94 (d, 1H, J ) 9.6 Hz, Ph), 7.06 (d,
1H, J ) 6.8 Hz, Ph), 8.22 (s, 1H, pyrazole-H)
freshly ground sodium amalgam. Residual amalgam and porphyrin
aggregates were removed by filtration. The resulting colorless filtrate
was adjusted to pH 8 with 10% HCl and diluted with triplication volume
of assay buffer, consisting of 100 mM Tris (pH 7.5) with 1 mM EDTA
and 4 mM DTT. The resulting preparation was stable for at least 1
week at -80 °C. All operations were conducted under dim light.
The assay of PPO inhibitory activity was carried out as follows.
The reaction mixture (1 mL) contained 100 mM Tris (pH 7.5), 1 mM
EDTA, 4 mM DTT, 2-5 µM protogen IX, and 0.3-0.6 mg of
etioplasts. The reaction was initiated by the addition of protogen IX
with or without test compound. After incubation for 30 min at 30 °C
in darkness, the mixture was transferred to 2.0 mL of 100 mM Tris
(pH 7.8) buffer that contained 1 mM EDTA, 5 mM DTT, and 1% (V/
V) Tween 80, and then, fluorescence at 630 nm (with excitation at 410
nm) was immediately measured with a fluorescence spectrophotometer
(960MC Shanghai Lengguang Corp.). Heat-denatured etioplasts were
used in the control experiments. Data were fit to a one-site competition
model (26) using GraphPad Prism versions 4.03 for Windows (D.
Radushev, Graph Pad Software Inc. Trial), and the pI50 values were
obtained (Table 5).
Treatment. The emulsions of purified compounds were prepared by
dissolving them in 100 µL of N,N-dimethylformamide with the addition
of a little Tween 20 and proper water. There were three replicates for
each treatment. The mixture of the same amount of water, N,N-
dimethylformamide, and Tween 20 was used as the control.
Pre-emergence. Sandy clay (100 g) in a plastic box (11 cm × 7.5
cm × 6 cm) was wetted with water. Fifteen sprouting seeds of the
weed under test were planted in fine earth (0.6 cm depth) in the
glasshouse and sprayed with the test compound solution.
emergence test. For both methods, the fresh weights were determined
15 days later, and the percentage inhibition relative to the controls was
calculated. The herbicidal activity is summarized in Tables 6 and 7.
RESULTS AND DISCUSSION
Synthesis and Structure Characterization. The intermediate
pyrazole derivatives F and H were synthesized as the literature
described (12-14). Compound F was hydrolyzed and then
converted to its acid chloride, which reacted directly with
substituted aniline in CH2Cl2 in the presence of pyridine to
afford G. Suggs reported that the cyclization temperature of G
to D1-13 was below -25 °C (16); however, a higher temper-
ature (90 ( 5 °C) and stronger acid atmosphere were more
Table 5. pI50 of Compounds D and E against PPO in Corn
no. pI50 SE no. pI50 SE
no.
pI50
SE
D1 7.12 0.08 D13 6.28 0.07 D25
D2 6.33 0.11 D14 7.94 0.07 D26
D3 6.31 0.04 D15 7.19 0.01 D27
D4 5.71 0.05 D16 8.02 0.01 D28
D5 6.49 0.02 D17 7.78 0.01 E1
D6 6.10 0.04 D18 7.87 0.03 E2
D7 7.85 0.03 D19 7.75 0.05 E3
D8 7.34 0.06 D20 6.23 0.06 E4
D9 7.37 0.11 D21 6.12 0.03 E5
D10 5.74 0.07 D22 6.53 0.01 E6
6.55
6.58
6.64
6.09
4.51
<4.50
5.27
4.83
5.00
5.04
0.07
0.02
0.04
0.01
0.06
0.02
0.05
0.01
0.03
D11 5.41 0.10 D23 6.61 0.03 Flumioxazin 8.49 [reference value 0.03
(27): 8.50]
D12 6.63 0.07 D24 6.32 0.07
Postemergence. Seedlings (one leaf and one stem) of the weed were
sprayed with the test compounds at the same rate as used for the pre-

9540 J. Agric. Food Chem., Vol. 56, No. 20, 2008
Li et al.
Table 6. Herbicical Activity of Compounds (Percent Inhibition) (Rate )
Table 7. Herbicical Activity of Compounds (Percent Inhibition)a
B. campestris A. retroflexus E. crus-galli D. sanguinalis
1500g/ha)a
B. campestris
A. retroflexus
E. crus-galli
D. sanguinalis
no.
rate (g/ha) pre
post pre
100
post pre post pre
post
no.
pre
post
pre
post
pre
0
post
20.8
pre
post
D7
D9
750
375
187.5
93.75
750
97.1 -
-
100
-
100
100
80.0
26.8
87.8
65.4
14.2
3.9
-
95.6 -
77.7 -
47.3 -
79.9 -
57.1 -
38.5 -
32.2 -
96.3 -
66.0 -
60.2 -
95.3 -
93.2 -
89.5 -
83.8 -
41.0 41.5
27.9 12.2
21.3 7.3
34.4 100
94.8 -
48.4 -
47.1 -
91.2 -
69.6 -
28.4 -
0
0
0
0
-
-
-
-
-
-
-
D1
D2
D3
D4
D5
D6
D7
D8
12.7
7.4
0
9.2
1.2
0
0
10.6
0
15.5
0
0
0
7.7
5.5
0
0
0
0
19.6
17.9
0
11.0 28.0
19.8 37.7
21.6
9.5
32.7
0
0
0
2.4
0
6.7
0
375
38.3
0
100
0
94.1
8.3
0
187.5
93.75
750
7.8
98.8
17.5
100
0
0
44.4
34.6
7.4
30.5
7.5
0
45.6 100
14.7
71.8 100
17.2 21.0
16.6
39.2
27.0
47.5
0
-
58.4
9.1
D14
D15
D16
0
0
0
7.4
3.4
0
17.1 17.4 19.4
20.9
20.5 100
20.8
12.0
0
0
18.5
10.3
30.6
20.3
54.1
0
24.4
375
11.2 13.0
7.3 6.5
0
0
D9
94.8 46.9
187.5
750
375
187.5
750
375
D10
D11
D12
D13
D14
D15
0
1.2
3.0
0
15.8
22.3
0
8.2
13.6
0
0
38.3
16.4 2.9
11.9 0
9.0 0
16.9 22.0
4.3 11.1
0
0
0
27.9 85.4 10.0 18.0
1.6 12.2 6.5 2.4
0
0
0
0
30.0
50.0
87.0
0
0
88.6
57.0
47.7
37.4
10.5
100
92.3
100
90.7
0
0
0
0
100 100 100 100
100 100 100 83.5 97.3 43.5 100 4.2
98.9 51.6 100 16.7
41.3
23.1
32.1
31.2
33.9
52.8
9.2
20.6
0
60.1 100
30.1 32.3
187.5
93.75 100 18.5 100
100 72.9 100 5.8
0
63.7 27.8 77.6
0
0
-
-
D16 100
D17 100
100
100
100
100
100
100
100
85.2
96.5 89.3
95.4 91.5
86.4 20.1
3.5 13.2
53.4 7.0
34.0 -
25.1 -
44.9
2.4
0
45
22.5
66.7 -
22.3 -
86.2 -
76.6 -
D18 98.1
D19 100
90.7 100
97.5 100
99.6
100
25.1
D17
750
375
187.5
93.75
45
22.5
750
375
187.5
750
375
187.5
750
375
187.5
750
375
187.5
750
375
100 100 100 85.1 89.2 49.9 67.4 4.2
100 95.3 100 53.7 47.4 23.2 51.0
93.3 55.6 100 30.6 34.4 14.2 26.5
D20
D21
D22
D23
D24
D25
D26
D27
D28
E1
0
0
29.8
4.8
13.9
0
27.9
28.3
21.5
16.1
6.1
23.7
0
0
0
-
-
40.5
0
0
0
0
11.0
37.5
70.6
16.6
23.2
14.0
54.1
11.3 1.0
12.4 20.2
0
60.4 19.6 100 19.0 10.0 0.2
7.4 -
5.4 -
20.4
17.1
4.9
0
0
0
0
40.7 -
12.7 -
82.8 -
3.4 -
1.1 15.3
9.7 11.1
15.9 31.1
17.5 32.6
5.6 43.6
40.9 20.3
39.6 35.8
0
0
2.9
0
1.4
0
0
0
D18
D19
E1
82.1 70.3 100 87.8 87.0 61.0 93.5 16.7
0
67.9 64.6
63.4 35.4
100 78.9 100 100
82.8 45.1
26.9 12.6
37.3 36.6
25.4 18.9
16.4 2.9
73.8 41.5 80.1 19.0 91.3 5.6
37.7 14.6 43.7 0.5 80.4
0
0
31.3
28.0
40.5 100
51.8 74.9
2.3
2.3
79.6
18.0
80.7
90.0
51.6
78.6
0
0
0
62.8 7.3
55.0 2.3
67.2 48.8 42.9 1.5
71.7 33.3
67.4 25.0
37.0 8.3
47.8 13.9
6.5 8.3
2.2 0
8.7 44.4
4.3 33.3
65.8
50.7
100
100
75.1
88.7
70.3 100
60.8 100
70.9 100
95.6 100
27.9 100
50.3 100
100
100
100
100
100
100
36.1
60.7
32.7
0
43.8
0
77.0 100
E2
E3
E4
E5
100
100
38.7
30.8
93.4 100
90.2 100
41.0 22.0
19.5 9.3
0
0
0.5
0
37.8 40.9
60.4 11.2
E6
E2
23.9 32.6 100 100
100 100
100 36.6
22.1 18.0
18.4 16.1
a Post, postemergence; pre, pre-emergence; and -, not measured.
13.4 8.6
3.0 6.3
0
14.1
95.7 23.6 67.4
100 38.9 100 48.8 49.1 17.6 24.5
0
22.2
0
0
0
0
0
0
0
0
E3
100 50.4 100 100
helpful to salify G to afford D1, D2, D4, D10, and D11. To
make the substituent R4 more representative, D21-28 were
synthesized via the intermediate D20 as shown in Scheme 3.
Approximately a 1:1 mixture of the F and I was obtained by
alkylation of H.
187.5
93.75
750
90.8 0
26.0 0
100 88.5 100 100
100 40.5 13.3 14.7 16.3
100 30.6 3.5 12.1 6.1
89.7 18.1 75.5
100 60.8 100 83.5 27.9 17.2 40.8
E4
375
187.5
93.75
750
95.9 12.3 100 45.5 18.2 2.8
16.3
6.1
Because the polarities of F and I are very similar, the mixture
was directly used for the next step without further isolation.
For the same reason, the mixture of G and J obtained was
reacted with hydrogen chloride and NaNO2 (Scheme 1). After
general workup, a two-component mixture was obtained, and
the target products were isolated by flash column chromatog-
raphy on silica gel. A combination of 2D NMR analyses (HSQC
and HMBC) of D16 and E3 not only confirmed the position of
allyl group but also allowed complete hydrogen and carbon
assignments. Some of the major long-range correlations (J2 and
J3) observed in the HMBC contour plots of compounds D16
and E3 were outlined in the structure (Figure 2). Tables 1-3
summarized the chemical structures, physical constants, yields,
and elemental analysis data of the new compounds D and E.
1H NMR data were listed in Table 4. To further validate their
structures, their starting material of the mixture of F and I (R1
) allyl; R2 ) H) (Scheme 2) was hydrolyzed, and 1-allyl-3-
amino-1H-pyrazole-4-carboxylic acid was obtained by crystal-
51.2 10.2 100 28.9
55.2 19.4 100 100
45.5 18.9 100 100
16.4 11.4 100 56.1
69.4 33.7 100 100
8.2 0
E5
E6
16.9 25.9 32.6 22.2
5.6 11.2 26.1 16.7
375
187.5
750
375
0 10.2
35.9 5.4
0
13.9
0
0
54.3
39.1
4.3
55.2 29.1 100 85.4 16.0 2.4
13.4 0 27.9 7.3 10.8 0
187.5
flumioxazin 187.5
0
100
93.75 100
-
-
-
100
-
-
-
-
100
100
71.4 -
40.9 -
-
-
100
100
100
-
-
-
-
100
100
100
45
22.5
100
92.6 -
85.4
a Post, postemergence; pre, pre-emergence; -, not measured.
1
lizing with ethanol. Its structure was determined by H NMR
and X-ray analysis (Figure 3). Then, it was reacted with SOCl2,
arylamine, HCl, and NaNO2, respectively, according to Scheme
1, and the final product was confirmed to be E3.
Figure 2. Selected long-range HMBC correlations found in the corre-
sponding contour plots of compounds D16 and E3.
Structure-Activity Relationship. As shown in ref 1, many
commercial heterocycle PPO inhibitors always contain a
2-fluoro-4-chloro-5-alkoxybenzene ring, and the polysubstituted
benzene ring was crucial for their herbicidal activities. In our

3H-Pyrazolo[3,4-d][1,2,3]triazin-4-one Derivatives
J. Agric. Food Chem., Vol. 56, No. 20, 2008 9541
selectivity at 187.5 g/ha in pre-emergence treatments against
dicotyledon weeds.
Compound D’s herbicidal activities correlated well with their
PPO inhibitory activity. Although the pI50 values of the isomers
D and E differed greatly, E1-6 showed nearly as good
herbicidal activity as D14-19. This suggested that the R1
position has important influence on the combination of the target
compounds with PPO, and compounds E may share other type
of mode of action against the weeds besides PPO. In addition,
the pI50 value of D16 increased slightly as compared to D18
and D7. However, the herbicidal activity increased greatly.
Compound D16 showed the highest herbicidal activity. There-
fore, the changing R2 group from methyl group to allyl or propyl
group can increase the herbicidal activity of the target com-
pounds greatly.
Figure 3. Crystal strucure of 1-allyl-3-amino-1H-pyrazole-4-carboxylic acid.
In this paper, the synthesis and herbicidal evaluation of a
series of 3H-pyrazolo[3,4-d][1,2,3]triazin-4-one derivatives as
a novel class of PPO inhibitors were described. Their herbicidal
activities were optimized. When the R3R4 moiety of the title
compounds is 4-(prop-2-ynyl)-2H-1,4-oxazin-3(4H)-one, the R2
moiety is an H atom, and when the R1 moiety is a methyl, allyl,
or propyl group, they have better herbicidal activities. The
bioassay data show that some of them, for example, D16, D17,
and E3, have promising herbicidal activities. They are chosen
as lead compounds, and further investigation on lead optimiza-
tion, herbicidal activity, and crop selectivity in vivo for the
compounds is underway in our group.
Figure 4. Chemical structure of D16.
previous paper (9), compound C (R1 ) CH3) exhibited better
herbicidal activity when the imidazotetrazinone moiety was
modified into pyrazolotetrazinone. On the basis of the above,
the imidazotetrazinone moiety was modified into pyrazolotri-
azinone, and a series of compounds was designed and synthe-
sized. As shown in Table 5, the pI50 was affected greatly by
the variance of R1, R2, R3, and R4. When R3 was chloro (D1-6,
D14-15, D20-28, and E1-2), only D14 showed better
inhibitory activity (pI50 ) 7.94). When the benzene ring was
modified into benzoxazinone (R1 ) CH3, CH2CHdCH2, or
n-C3H7; R2 ) H, CH3, or CF3) and R1 was methyl, allyl, or
propyl group and R2 was hydrogen (D7-9, D16-19), their pI50
values increased greatly. Especially, compound D16 (Figure
4) showed similar inhibitory activity against PPO with flumi-
oxazin (pI50 ) 8.49). When R2 was CH3 or CF3 (D10-13), the
pI50 values changed slightly. When the position of R1 was
changed, their inhibitory activity against PPO decreased sharply
(E1-6). The result showed that (i) the inhibitory activity in
vitro was related with the position of R1, while allyl and propyl
groups are more suitable than the methyl group for R1, and (ii)
that the H atom is more satisfactory for increasing pI50 value
than trifluoromethyl group and methyl group on R2.
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Received for review June 11, 2008. Revised manuscript received August
5, 2008. Accepted August 5, 2008. We gratefully acknowledge the
support of this work by the National Key Project for Basic Research
(2003CB114400), the National Natural Science Foundation of China
(20572054; 20772066), and the Research Foundation for the Doctoral
Program of Higher Education (20050055042).
JF801774K