Discovery of a new insecticide lead by optimizing a target-diverse scaffold: Tetrazolinone derivatives

Article abstract of DOI:10.1016/j.bmc.2006.12.002

In order to discover lead compounds with novel action mechanism, a series of tetrazolinone derivatives bearing structurally diverse substituents, 1-aryl-4-substituted-1,4-di-hydro-5H-tetrazol-5-ones 2, 1-((5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)-4-(sub

Full text of DOI:10.1016/j.bmc.2006.12.002

Bioorganic & Medicinal Chemistry 15 (2007) 1716–1724
Discovery of a new insecticide lead by optimizing a
target-diverse scaffold: Tetrazolinone derivatives
Yan-Ping Luo and Guang-Fu Yang*
Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, PR China
Received 12 November 2006; revised 1 December 2006; accepted 2 December 2006
Available online 6 December 2006
Abstract—In order to discover lead compounds with novel action mechanism, a series of tetrazolinone derivatives bearing structurally
diverse substituents, 1-aryl-4-substituted-1,4-di-hydro-5H-tetrazol-5-ones 2, 1-((5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)-4-(substi-
tuted)- phenyl-1H-tetrazol-5(4H)-ones 5, and 1-((5-(alkylthio)-1,3,4-thiadiazol-2-yl)methyl)-4- (substituted)phenyl-1H-tetrazol-
5(4H)-ones 7, were designed and synthesized in good yields by a multiple-step synthetic procedure. The results of greenhouse in vivo test
indicated that all the target compounds did not displayed herbicidal activity, however, some of them exhibited excellent in vivo insec-
ticidal activity against Tetranychus cinnabarinus at the concentration of 250 mg Lꢀ1. To our knowledge, this is the first report about the
insecticidal activity of tetrazolinone derivatives, which indicated that the tetrazolinone scaffold could be identified as a novel insecticidal
lead structure. The present work demonstrated that optimizing a target-diverse scaffold is an effective way to discover new lead
compounds with new action mechanism or biological activity.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
able substituent into 4-position of this scaffold, it is
possible to discover new lead compounds with novel
action mechanism or new biological activity.
The discovery of new lead structure is one of the most
important topics of research in medicinal and pesticide
chemistry. Among various biologically heterocyclic scaf-
folds, tetrazolinone derivatives attracted considerable
attentions from pesticide chemists in last decade due
to their promising herbicidal activity.1 For example,
fentrazamide,2 a commercial herbicide as shown in
Figure 1, was highly effective against many weeds by
inhibiting the cell division. Additionally, 1-aryl-4-
(3-fluoropropyl)-tetrazolinones (compound 1b in
Fig. 1) were developed as a new and highly active family
of protoporphyrinogen oxidase-inhibiting herbicides.3
We noticed that fentrazamide and compound 1b have
the same scaffold, but different substituent at 4-position
resulted in these two compounds having different action
mechanism. This observation indicated that tetrazoli-
none might be regarded as a ‘target-diverse’ scaffold
and R group at 4-position might have dramatically
influenced the action mechanism. If introducing a suit-
1,3,4-Thiadiazole and 1,3,4-oxadiazole derivatives
have been approved to be important scaffolds with
broad-spectrum biological activities for many years.
For example, some 1,3,4-thiadiazole and 1,3,4-oxadia-
zole derivatives displayed antiproliferative,4 bacterio-
static,5 hypoglycemic,6 antibacterial,7 and anti-
inflammatory activity.8,9 In addition, broad-spectrum
insecticidal,10 herbicidal,11 and fungicidal activities12
were also found to be associated with 1,3,4-thiadiazole
and 1,3,4–oxadiazole derivatives.
As a part of our extensive program to discover lead com-
pound with novel action target, we designed and synthe-
sized the title compounds, 1-((5-(alkylthio)-1,3,4-
oxadiazol-2-yl)methyl)-4-(substituted)phenyl-1H-tetra-
zol-5(4H)-ones 5, and 1-((5-(alkylthio)-1,3,4-thiadiazol-
2-yl)-methyl)-4-(substituted)phenyl-1H-tetrazol-5(4H)-
ones 7, by introducing various substituents including
1,3,4-thiadiazole and 1,3,4-oxadiazole moieties into the
4-position of tetrazolinone scaffold. The preliminary
in vitro bioassay against six kinds of weed indicated
that some derivatives displayed good inhibition activity
against the stem or root growth, however, further
Keywords: Tetrazolinone; 1,3,4-Thiadiazole; 1,3,4-Oxadiazole; Insecti-
cidal activity; Lead compound.
*
Corresponding author. Tel.: +86 27 67867706; fax: +86 27
67867141; e-mail: gfyang@mail.ccnu.edu.cn
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.002

Y.-P. Luo, G.-F. Yang / Bioorg. Med. Chem. 15 (2007) 1716–1724
1717
F
R1
O
O
Cl
O
O
Cl
N
N
F
N
N
R3
N
N
R
N
N
N
N
N
N
N
HN
SO2C2H5
R4
1
1b
Fentrazamide (1a)
Figure 1. General structures of herbicidal tetrazolinones.
greenhouse in vivo test indicated that all these compounds
displayed no herbicidal activity. Interestingly, some com-
pounds were found to exhibit good in vivo insecticidal
activities against Tetranychus cinnabarinus at the dosage
of 250 mg Lꢀ1. To our knowledge, this is the first report
about the insecticidal activity of tetrazolinone derivatives.
by the nucleophilic substitution reaction of 4 and 6
with alkyl halides or substituted benzyl halides, respec-
tively. The structures of all of the title compounds
were confirmed by 1H NMR, MS, and elemental
analyses.
2.2. Bioactivity
2. Results and discussion
2.1. Chemistry
The inhibition activities of the title compounds against
the stem and root growth of some weeds, such as wheat
(Triticum aestivum), barnyard grass (Echinochloa crus-
galli), jowar (Sorghum bicolor), rape (Brassica campes-
tris), radish (Raphanus sativus), and cucumber
(Cucumis sativus), were determined using the reported
method.14 The results as shown in Table 1 indicated that
some of the tested compounds displayed good inhibition
activities against the stem or root growth of tested weeds
at the concentration of 100 mg Lꢀ1. For example, com-
pounds 2c and 7l displayed 80% inhibition against both
root and stem growth of wheat, compound 7c displayed
80% inhibition against both root and stem growth of
barnyard grass, compounds 2g and 7h displayed over
80% inhibition activities against both root and stem
growth of rape, compounds 2c and 4 displayed over
80% inhibition activities against both root and stem
growth of radish. Additionally, compound 2e displayed
95% inhibition activities against the root growth of jo-
war. It should be noted that compound 7 seems to dis-
play broader spectrum activities than other
compounds. However, unfortunately, the further green-
house experiment indicated that all these compounds
did not exhibit herbicidal activity at the dosage of
450 g ai/ha.
The target compounds 5a–d and 7a–o were synthesized
by a five-step procedure. First, the starting material,
1-substituted phenyl-1H-tetrazol-5(4H)-ones, was pre-
pared by the reaction of substituted phenylisocyanate
with sodium azide in N,N-dimethylformamide (DMF)
according to the existing method.13 Treatment of tetraz-
olinones 1 with alkyl halides or substituted benzyl
halides in the presence of K2CO3 in DMF solution at
room temperature afforded 1-aryl-4-substituted-1,4-
dihydro-5H-tetrazolin-5-ones 2a–o in good yields
(Scheme 1).
Refluxing
compound
2e
ðR ¼ CH2COOC2H5;
W ¼ 4-ClÞ with hydrazine hydrate in ethanol for 4 h
afforded 2-(4-(4-chlorophenyl)-5-oxo-4,5-dihydrotetra-
zol-1-yl)acetohydrazide 3. It should be noted that
treatment of compound 3 with carbon disulfide under
different reaction conditions afforded different prod-
ucts. Under refluxing condition in the presence of
potassium hydroxide, 5-mercapto-1,3,4-oxadiazoles 4
were obtained. However, 5-mercapto-1,3,4-thiadiazoles
6 were obtained when the reaction was carried out at
room temperature (Scheme 2). After having two key
intermediates 4 and 6 in hand, the target compounds
1-((5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)-4-(substi-
tuted)phenyl-1H-tetrazol-5(4H)-ones 5, and 1-((5-
(alkylthio)-1,3,4-thiadiazol-2-yl)methyl)-4-(substituted)-
phenyl-1H-tetrazol-5(4H)-ones7, were prepared easily
The insecticide activity results of all target compounds
against Aphis medicagini, Nilaparvata lugen, Mythima
separata and Tetranychus cinnabarinus are shown in
Table 2, two commercial products, Dichlorvos and Pyri-
dabea, were used as controls. As shown in Table 2, some
compounds displayed excellent insecticide activities
O
O
RX / K2CO3, DMF
rt, 4-6h
N
NH
N
N
R
W
W
N
N
N
N
1
2
Scheme 1. Syntheses of 1-aryl-4-substituted-1,4-dihydro-5H-tetrazol-5-ones.

1718
Y.-P. Luo, G.-F. Yang / Bioorg. Med. Chem. 15 (2007) 1716–1724
O
O
S
S
S
R2
SH
e
N
N
N
N
W
W
N
N
N
N
N
N
N
N
7a-o
6
d
O
O
OC2H5
NHNH2
a
N
N
N
N
W
W
N
N
N
N
O
O
2e
3
b
O
O
O
O
S
R1
c
SH
N
N
N
N
N
N
N
N
W
W
N
N
N
N
5a-d
4
Scheme 2. Syntheses of 1-((5-(alkylthio)-1,3,4-oxa(thia)diazol-2-yl)methyl)-4-(substituted)-phenyl-1H-tetrazol-5(4H)-ones. Reagents and conditions:
(a)NH2NH2ÆH2O, EtOH, reflux, 4 h; (b) CS2, KOH, EtOH, reflux, 24 h, HOAc ; (c) R1Br, DMF, K2CO3, rt, 6–8 h; (d) CS2, KOH, EtOH, rt, 24 h;
H2SO4, NaOH, HCl; (e) R2 X, DMF, K2CO3, rt, 4–8 h.
against T. cinnabarinus or A. medicagini, M. separata.
For example, at the concentration of 250 mg Lꢀ1, com-
pounds 2a, 2b, 2e, 2o, 5a, and 7g showed excellent insec-
ticide activity against T. cinnabarinus, and compound 7b
showed excellent insecticide activity against A. medica-
gini, compound 2c showed excellent insecticide activity
against M. separata. However, all the title compounds
had no or very low insecticide activity against N. legen.
Additionally, we can find from Table 2 that the insecti-
cide activities of the target compounds were influenced
by the substituents on 4-position of tetrazolinone. For
example, some compounds bearing methyl, ethyl, allyl,
ester group, and chromenone-3-methene displayed good
insecticide activity, while compounds bearing benzyl
always showed no or lower insecticide activity. To our
knowledge, this is the first report about the insecticide
activity of tetrazolinone derivatives. Further studies on
structural optimization, structure-insecticide activity
relationships, and insecticidal mode of action about this
scaffold are under way.
scaffold displaying insecticide activity implied a poten-
tial new action mechanism. In a word, the present
work demonstrated that optimizing a target-diverse
scaffold is an effective way to discover new lead com-
pounds with new action mechanism or biological
activity.
4. Experimental
4.1. Materials
All chemical reagents were commercially available and
treated with standard methods before use. Solvents were
dried in a routine way and redistilled. Nilaparvata lugen
(N. lugen), Tetranychus cinnabarinus (T. cinnabarinus),
Aphis medicagini (A. medicagini), Mythima separata
(M. separata), and wheat (Triticum aestivum), barnyard
grass (Echinochloa crusgalli), jowar (Sorghum bicolor),
rape (Brassica campestris), radish (Raphanus sativus),
cucumber (Cucumis sativus) were provided through the
courtesy of the Center for Bioassay, Zhejiang Chemical
Industry Research Institute.
3. Conclusion
In conclusion, by introducing various substituents into
the 4-position of tetrazolinone derivatives, the scaffold
of tetrazolinone, which originally displayed herbicidal
activities, was identified as a novel insecticide lead
structure for the first time. Previously, it has been
identified that the herbicidal tetrazolinone derivatives
killed weeds by inhibiting the cell division or the activ-
ity of protoporphyrinogen oxidase. The mechanism of
insect cell division is different greatly from that of
weed and there is no protoporphyrinogen oxidase in
the body of insects. Therefore, the original herbicidal
4.2. Analysis and instruments
Melting points are uncorrected and determined with
Electro thermal digital melting point apparatus. MS
spectra were determined using a Finnigan Trace MS
organic mass spectrometry, and signals was given in
1
m=z. H NMR are recorded in CDCl3 or DMSO on a
Varian Mercury 400 MHz spectrometer and resonance
are given in ppm (d) relative to TMS. Elementary anal-
yses (EA) were performed on a Vario EL III elementary
analysis instrument.

Y.-P. Luo, G.-F. Yang / Bioorg. Med. Chem. 15 (2007) 1716–1724
1719
Table 1. The results of inhibition activities of the title compounds against stem and root growth (100 mg Lꢀ1
)
Compound
W
R (R1 or R2)
T. aestivum
E. crusgalli
S. bicolor
B. campestris
R. sativus
C. sativus
Stem Root Stem Root Stem Root Stem
Root Stem Root Stem root
2a
2,4-Cl2,
5-OCH3
2-Cl
CH3
50
70
30
30
30
70
0
0
0
0
60
60
2b
2c
2d
2e
2f
CH2@CHCH2
CH2@CHCH2
CH2CO2C2H5
50
80
30
50
80
30
80
0
0
50
0
0
0
0
60
20
40
0
0
60
20
95
0
60
50
70
0
0
50
85
0
0
80
40
50
0
0
80
80
70
0
60
30
0
0
40
0
4-Cl
4-Cl
0
4-Cl
4-Cl
CH(CH3)CO2C2H5 70
0
0
0
0
2-CH3-C6H5CH2
3-CH3-C6H5CH2
4-CH3-C6H5CH2
2-F-C6H5CH2
30
0
0
0
0
0
0
0
2g
2h
2i
4-Cl
4-Cl
0
0
0
0
0
90
0
80
0
70
0
80
0
0
0
0
0
0
0
0
0
0
0
4-Cl
4-Cl
0
0
0
0
0
0
0
0
0
0
0
0
2j
3-F-C6H5CH2
40
0
40
0
0
70
0
0
0
0
0
0
0
30
0
50
0
2k
2l
4-Cl
4-Cl
3-Cl-C6H5CH2
4-Br-C6H5CH2
3-CH3O-C6H5CH2
0
0
0
0
0
0
0
30
0
0
0
0
0
0
0
0
0
0
0
0
2m
4-Cl
0
0
0
0
0
0
0
0
0
0
0
O
CH2
2n
2o
2-Cl
4-Cl
30
30
0
0
0
0
0
0
30
0
50
0
30
0
50
0
30
0
50
0
0
0
0
0
O
O
CH2
O
4
/
0
0
0
0
0
0
0
0
0
0
60
0
70
70
60
60
0
80
80
50
80
0
80
60
0
90
60
50
0
0
50
0
0
60
0
5a
5b
5c
5d
6
4-Cl
4-Cl
4-Cl
4-Cl
CH2@CHCH2
CH2CO2C2H5
CH(CH3)CO2C2H5
C8H17
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
/
CH3
70
50
40
70
40
30
0
70
60
50
70
40
0
70
0
70
30
60
80
70
0
60
40
60
0
60
60
70
80
50
60
50
30
80
60
60
0
70
70
60
60
40
40
0
70
0
70
50
40
70
0
80
50
50
70
30
40
40
70
80
40
60
40
80
50
50
60
60
30
70
0
60
40
0
7a
7b
7c
7d
7e
7f
7g
7h
7i
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
C2H5
C3C7
70
80
30
0
60
70
50
70
40
70
85
60
60
50
80
40
0
50
0
C4H9
C6H13
40
40
0
30
0
0
0
C8H17
CH2CO2C2H5
30
50
70
50
60
60
80
40
60
0
40
0
0
30
50
60
0
60
CH(CH3)CO2C2H5 50
0
50
70
0
50
80
40
50
30
70
0
60
50
40
40
40
60
50
30
30
60
0
40
70
70
70
70
70
0
50
60
60
60
60
50
0
CH2@CHCH2
C6H5CH2
40
60
60
80
70
70
7j
70
30
70
40
50
60
0
60
0
7k
7l
2-CH3-C6H4CH2
2-F-C6H4CH2
3-F-C6H4CH2
3-Cl-C6H4CH2
70
40
40
70
30
40
70
30
0
7m
7n
0
CO2CH3
7o
4-Cl
40
0
0
30
0
60
30
30
30
60
30
30
O
CH2
4.2.1. General procedure for the preparation of 1-aryl-4-
substituted-1,4-dihydro-5H-tetrazol-5-ones (2). A mixture
of 1-aryl-1H-tetrazol-5(4H)-one (1) (0.1 mol), the appro-
priate alkyl halide or substituted benzyl halide (0.11 mol),
and anhydrate potassium carbonated (0.2 mol) in DMF
(25 mL) was stirred for 4–6 h according to thin-layer
chromatographic (TLC) analysis monitored, and then
ice water was added to the reaction mixture to form pre-
cipitate, then filtered, washed with water, dried, and
recrystallized from ethanol to afford compound 2:
Compound 2b: Yield: 87%; oil (lit.,3 oil); 1H NMR
(400 MHz, CDCl3): d 7.43–7.60 (m, 4H), 5.99–6.06 (m,
2H), 5.37–5.41 (m, 1H), 4.65–4.67 (m, 2H); EIMS (probe)
70 eV, m=z (rel. int.): 237 [M]+ (38), 152 (100), 124 (46), 89
(54). Anal. Calcd for C10H9ClN4O: C, 50.75; H, 3.83; N,
23.67. Found: C, 50.60; H, 3.95; N, 23.71.
Compound 2c: Yield: 94%; mp 63–64 °C (lit.,3 62–
63 °C); 1H NMR (400 MHz, CDCl3): d 7.77 (dd,
J ¼ 8:8 Hz, 4H), 5.94–6.04 (m, 2H), 5.37–5.64 (m,
1H), 4.63–4.65 (m, 2H); 13C NMR (100 MHz, CDCl3):
d 148.39, 139.22, 133.07, 129.81, 129.37, 120.01,
114.14, 47.12; EIMS (probe) 70 eV, m=z (rel. int.): 237
[M]+ (100), 152 (99), 124 (63), 89 (38).
Compound 2a: Yield: 47%; mp 155–157 °C (lit.,3 158–
1
159 °C); H NMR (400 MHz, CDCl3): d 7.45 (m, 2H),
3.91 (s, 3H), 3.72 (s, 3H).

1720
Y.-P. Luo, G.-F. Yang / Bioorg. Med. Chem. 15 (2007) 1716–1724
70 eV, m=z (rel. int.): 301 [M]+ (100), 152 (63), 124
(51), 88 (81). Anal. Calcd for C15H13ClN4O: C, 59.91;
H, 4.36; N, 18.63. Found: C, 59.90; H, 4.31; N, 18.67.
Table 2. The results of insecticide activities of the title compounds
Compound
250 mg Lꢀ1
500 mg Lꢀ1
T. cinnabarinus A. medicagini N. legen M. separata
Compound 2g: Yield: 87%; mp 90–91 °C; 1H NMR
(400 MHz, CDCl3): d 7.69 (dd, J ¼ 8:8 Hz, 4H), 7.16–
7.27 (m, 4H), 5.13 (s, 2H), 2.36 (s, 3H); EIMS (probe)
70 eV, m=z (rel. int.): 301 [M]+ (100), 152 (95), 124
(41), 104 (45), 89 (55). Anal. Calcd for C15H13ClN4O:
C, 59.91; H, 4.36; N, 18.63. Found: C, 60.16; H, 4.29;
N, 18.49.
2a
2b
100.0
98.6
4.3
0.0
1.7
3.3
8.3
0.0
21.7
8.3
3.3
5.0
0.0
0.0
13.3
3.3
13.3
1.7
5.0
0.0
8.3
3.3
5.7
82.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
5.7
0.0
18.6
0.0
2.9
2.9
—
10.0
23.3
10.0
13.3
16.7
10.0
0.0
0.0
0.0
100.0
0.0
0.0
0.0
0.0
0.0
60.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
13.3
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
100.0
2c
2d
0.0
2e
97.1
2.9
2f
2g
4.3
0.0
2h
2i
10.0
13.3
0.0
10.0
5.7
1
2j
2k
Compound 2h: Yield: 93%; mp 130–133 °C; H NMR
0.0
0.0
6.7
6.7
(400 MHz, CDCl3): d 7.68 (dd, J ¼ 8:8 Hz, 4H), 7.27
(dd, J ¼ 8 Hz, 4H), 5.14 (s, 2H), 2.35 (s, 3H); EIMS
(probe) 70 eV, m=z (rel. int.): 301 [M]+ (100), 152 (84),
124 (55), 89 (81). Anal. Calcd for C15H13ClN4O: C,
59.91; H, 4.36; N, 18.63. Found: C, 60.05; H, 4.46; N,
18.43.
2l
2m
0.0
2.9
0.0
6.7
2n
2o
100.0
96.8
2.9
10.0
6.7
5a
5b
16.7
10.0
13.3
0.0
5c
5d
0.0
2.9
1
Compound 2i: Yield: 91%; mp 101–103 °C; H NMR
7a
7b
0.0
5.7
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 7.10–
7.45 (m, 4H); 5.26 (s, 2H); EIMS (probe) 70 eV, m=z
(rel. int.): 305 [M]+ (0.5), 152 (53), 124 (44), 108 (100),
88 (28). Anal. Calcd for C14H10ClFN4O: C, 55.18; H,
3.31; N, 18.39. Found: C, 55.30; H, 3.35; N, 18.25.
3.3
6.7
7c
7d
0.0
5.7
0.0
7e
4.3
2.9
10.0
6.7
7f
7g
80.0
0.0
3.3
6.7
1
Compound 2j: Yield: 88%; mp 106–107 °C; H NMR
7h
7i
(400 MHz, CDCl3): d 7.69 (dd, J ¼ 8:8 Hz, 4H), 7.06–
7.37 (m, 4H), 5.17 (s, 2H); 13C NMR (100 MHz,
CDCl3): d 164.12, 161.66, 148.59, 136.20, 133.37,
133.09, 130.71, 130.63, 129.54, 124.08, 120.21, 115.92,
115.71, 115.61, 115.39, 48.17; EIMS (probe) 70 eV,
m=z (rel. int.): 305 [M]+ (100), 152 (66), 124 (26), 108
(79), 88 (40). Anal. Calcd for C14H10ClFN4O: C,
55.18; H, 3.31; N, 18.39. Found: C, 55.09; H, 3.54; N,
18.25.
72.9
2.9
0.0
0.0
7j
7k
0.0
0.0
0.0
7l
7m
10.0
10.0
6.7
2.9
5.7
7n
7o
0.0
0.0
100.0
Dichlorvos
(500 mg Lꢀ1
Pyridaben
(50 mg Lꢀ1
—
)
100.0
—
—
—
)
1
Compound 2k: Yield: 94%; mp 135–137 °C; H NMR
(400 MHz, CDCl3): d 7.69 (dd, J ¼ 8:8 Hz, 4H), 7.33–
7.44 (m, 4H), 5.15 (s, 2H); EIMS (probe) 70 eV, m=z
(rel. int.): 321 [M]+ (45), 152 (100), 124 (53), 88 (17),
Anal. Calcd for C14H10Cl2N4O: C, 52.36; H, 3.14; N,
17.45. Found: C, 52.47; H, 3.11; N, 17.38.
1
Compound 2d: Yield: 90%; mp 115–116 °C, H NMR
(400 MHz, CDCl3): d 7.47 (dd, J ¼ 8:8 Hz, 4H), 4.81
(s, 2H), 4.30 (q, J ¼ 7:2 Hz, 2H), 1.32 (t, J ¼ 7:2 Hz,
3H); 13C NMR (100 MHz, CDCl3): d 165.68, 148.69,
133.39, 133.02, 129.54, 120.24, 62.52, 45.56, 14.03;
EIMS (probe) 70 eV, m=z (rel. int.): 283 [M]+ (2),
155(29), 153(100), 125(18). Anal. Calcd for
C11H11ClN4O3: C, 46.74; H, 3.92; N, 19.82. Found: C,
46.72; H, 3.95; N, 19.43.
1
Compound 2l: Yield: 98%; mp 149–150 °C; H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 7.44
(dd, J ¼ 8:0 Hz, 4H), 5.13 (s, 2H); 13C NMR (100 MHz,
CDCl3): d 148.53, 133.33, 133.04, 132.84, 132.17,
130.21, 129.51, 122.99, 120.16, 48.14; EIMS (probe)
70 eV, m=z (rel. int.): 366 [M]+ (100), 152 (52), 124 (63),
89 (96). Anal. Calcd for C14H10BrClN4O: C, 45.99; H,
2.76; N, 15.32. Found: C, 45.86; H, 2.87; N, 15.32.
1
Compound 2e: Yield: 95%; mp 108–110 °C, H NMR
(400 MHz, CDCl3): d 7.71 (dd, J ¼ 8:8 Hz, 4H), 5.05
(q, J ¼ 7:2 Hz, 1H), 4.26 (q, J ¼ 7:2 Hz, 2H), 1.89 (d,
J ¼ 7:2 Hz, 3H), 1.30 (t, J ¼ 7:2 Hz, 3H); EIMS (probe)
70 eV, m=z (rel. int.): 297 [M]+ (11), 155 (62), 153 (100),
125 (31), 90 (16). Anal. Calcd for C12H13ClN4O3: C,
48.58; H, 4.42; N, 18.88. Found: C, 48.44; H, 4.26; N,
19.07.
1
Compound 2m: Yield: 82%; mp 112–113 °C; H NMR
(400 MHz, CDCl3): d 7.69 (dd, J ¼ 8:8 Hz, 4H), 6.88–
7.32 (m, 4H), 5.14 (s, 2H), 3.82 (s, 3H); 13C NMR
(100 MHz, CDCl3): d 159.45, 148.39, 136.28, 133.09,
132.03, 129.91, 129.52, 121.09, 119.93, 113.65, 113.53,
55.08, 47.77; EIMS (probe) 70 eV, m=z (rel. int.): 317
[M]+ (100), 152 (48), 124 (41), 107 (12), 89 (54). Anal.
Calcd for C15H13ClN4O2: C, 56.88; H, 4.14; N, 17.69.
Found: C, 56.69; H, 4.08; N, 17.67.
1
Compound 2f: Yield: 97%; mp 124–125 °C; H NMR
(400 MHz, CDCl3): d 7.69 (dd, J ¼ 8:8 Hz, 4H), 7.22–
7.39 (m, 4H), 5.19 (s, 2H), 2.49 (s, 3H); EIMS (probe)

Y.-P. Luo, G.-F. Yang / Bioorg. Med. Chem. 15 (2007) 1716–1724
1721
1
Compound 2n: Yield: 62%; mp 133–135 °C; H NMR
(400 MHz, CDCl3): d 8.17 (s, 1H), 7.41–8.27 (m, 8H),
5.14 (s, 2H); EIMS (probe) 70 eV, m=z (rel. int.): 355
[M]+ (3), 291 (28), 277 (43), 172 (54), 146 (100), 125
(23), 89 (32); Anal. Calcd for C17H11ClN4O3: C, 57.56,
H, 3.13, N, 15.79; Found: C, 57.63; H, 3.36; N, 15.37.
6.00 (m, 1H), 5.42 (s, 2H), 5.20–5.40 (m, 2H), 3.87–
3.89 (m, 2H); 13C NMR (100 MHz, CDCl3): d 165.81,
160.47, 147.98, 133.51, 132.71, 131.15, 129.51, 120.16,
120.00, 39.10, 35.04; EIMS (probe) 70 eV, m=z (rel.
int.): 351 [M]+ (29), 235 (17), 208 (23), 152 (100), 125
(61), 111 (29), 90 (50). Anal. Calcd for C13H11ClN6O2S:
C, 44.51; H, 3.16; N, 23.96; S, 9.14. Found: C, 44.79; H,
3.41; N, 23.64; S, 9.12.
1
Compound 2o: Yield: 88%; mp:195–197 °C; H NMR
(400 MHz, CDCl3): d 8.20 (s, 1H), 7.44–8.24 (m, 8H),
5.11 (s, 2H); EIMS (probe) 70 eV, m=z (rel. int.): 355
[M]+ (100), 292 (49), 172 (36), 146 (59), 125 (58), 89
(56). Anal. Calcd for C17H11ClN4O3: C, 57.56, H,
3.13, N, 15.79. Found: C, 57.83; H, 3.27; N, 15.60.
Compound 5b: Yield: 42%; mp 68–69 °C; 1H NMR
(400 MHz, CDCl3): d 7.69 (dd, J ¼ 8:8 Hz, 4H), 5.42 (s,
2H), 4.23 (q, J ¼ 7:2 Hz, 2H), 4.06 (s, 2H), 1.28 (t,
J ¼ 7:2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d
166.89, 164.90, 160.72, 147.85, 133.30, 132.60, 129.36,
120.05, 62.23, 38.95, 34.10, 13.82; EIMS (probe) 70 eV,
m=z (rel. int.): 397 [M]+ (6), 153 (100), 125 (25), 90 (10).
Anal. Calcd for C14H13ClN6O4S: C, 42.38; H, 3.30; N,
21.18; S, 8.08. Found: C, 42.83; H, 3.46; N, 21.12; S, 8.47.
4.2.2. Preparation of 2-(4-(4-chlorophenyl)-5-oxo-4,5-
dihydrotetrazol-1-yl)acetohydrazide (3). Hydrazine hy-
drate (0.5 mol) was added into a solution of ethyl 2-(2-
(4chlorophenyl)-5-oxo-4,5-dihydrotetrazol-1-yl)acetate
(2e) (0.1 mol) in ethanol (100 mL). The reaction mix-
ture was heated under reflux for 4 h, concentrated in
vacuum, cooled, and diluted with water. The precipi-
tate obtained was filtered, washed with ice-cold water,
dried, and recrystallized from ethanol, Yield: 88%;
mp 235–237 °C; 1H NMR (400 MHz, DMSO-d6): d
9.47 (s, 1H), 7.66 (dd, J ¼ 8:8 Hz, 4H), 4.69 (s, 2H),
4.39 (s, 2H); EIMS (probe) 70 eV, m=z (rel. int.): 268
[M]+ (10), 155 (31), 153 (100), 127 (10), 125 (27), 115
(16). Anal. Calcd for C9H9ClN6O2: C, 40.24; H, 3.38;
N, 31.28. Found: C, 40.23; H, 3.27; N, 30.95.
Compound 5c: Yield: 61%; mp 55–57 °C; 1H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 5.43
(s, 2H), 4.43 (q, J ¼ 8:4 Hz, 1H), 4.19 (q, J ¼ 7:2 Hz,
2H), 1.70 (d, J ¼ 8:4 Hz, 3H), 1.27 (t, J ¼ 7:2 Hz, 3H);
EIMS (probe) 70 eV, m=z (rel. int.): 411 [M]+ (13), 153
(100), 125 (26), 90 (13). Anal. Calcd for
C15H15ClN6O4S: C, 43.85; H, 3.68; N, 20.46; S, 7.80.
Found: C, 44.09; H, 3.80; N, 20.18; S, 8.17.
Compound 5d: Yield: 87%; mp 58–60 °C; 1H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 5.41
(s, 2H), 3.25 (t, J ¼ 7:2 Hz, 2H), 1.26–1.81 (m, 12H),
0.88 (t, J ¼ 7:2 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 166.72, 160.19, 148.01, 133.50, 132.74, 129.51, 120.16,
39.12, 32.54, 31.61, 28.96, 28.94, 28.82, 28.44, 22.48,
13.96; EIMS (probe) 70 eV, m=z (rel. int.): 423 [M]+
(7), 376 (17), 352 (15), 153 (100), 125 (37), 111 (15), 90
(21). Anal. Calcd for C18H23ClN6O2S: C, 51.12; H,
5.48; N, 19.87; S, 7.58. Found: C, 51.12; H, 5.31; N,
19.59; S, 7.88.
4.2.3. Preparation of 1-(4-chlorophenyl)-4-(2-mercapto-
1,3,4-oxadiazol-5-yl)-methylene-1H- tetrazol-5(4H)-one
(4). To a vigorously stirred solution of 0.012 mol of
potassium hydroxide and 80 mL of anhydrous ethanol,
0.01 mol of compound 3 was added. Then, a solution
of 1 g of carbon disulfide in 20 mL of anhydrous ethanol
was added dropwise, and the resulted reaction mixture
was refluxed for 24 h. The solvent was evaporated in
vacuum and the residue was dissolved in 50 mL of
water, then acidified to pH ꢁ5–6 with glacial acetic acid,
filtered off, dried, and purified by recrystallization from
a DMF/water solution (5:1). Yield: 36%; mp 153–
155 °C; 1HNMR (400 MHz, DMSO-d6): d 14.65 (s,
1H), 7.79 (dd, J ¼ 8:8 Hz, 4H), 5.47 (s, 2H); EIMS
(probe) 70 eV, m=z (rel. int.): 311 [M]+ (14), 153 (43),
125 (100), 111 (30), 90 (74). Anal. Calcd for
C10H7ClN6O2S: C, 38.65; H, 2.27; N, 27.05; S, 10.32.
Found: C, 38.73; H, 2.35; N, 26.74; S, 10.65.
4.2.5. Preparation of 1-(4-chlorophenyl)-4-(2-mercapto-
1,3,4-thiadiazol-5-yl)-methylene-1H-tetrazol-5(4H)-one (6).
To a vigorously stirred solution mixture of 0.011 mol of
potassium hydroxide, 0.01 mol of the compound 3, and
100 mL of anhydrous ethanol, a solution of 7.5 g of car-
bon disulfide in 20 mL of anhydrous ethanol was added
dropwise. Then, the reaction mixture was stirred at
room temperature for 24 h and filtered to yield a white
solid, which was used for the next reaction with further
purification. So, 0.005 mol of above solid was added
into 100 mL of concentrated sulfuric acid, the resulted
mixture was stirred for 24 h at room temperature and
then poured into 100 mL of ice water. The precipitate
was filtered and dissolved with sodium hydroxide solu-
tion (25%), which was acidified with concentrated
hydrochloride acid and filtered to give the crude prod-
uct, dried, and recrystallized from a DMF/water solu-
tion (5:1). Yield: 84%; mp 180–182 °C; 1H NMR
(DMSO-d6): d 14.68 (s, 1H), 7.66–7.90 (m, 4H), 5.49(s,
2H); EIMS (probe) 70 eV, m=z (rel. int.): 327 [M]+
(20), 153 (90), 125(100), 111 (30), 90 (68), 75 (39). Anal.
Calcd for C10H7ClN6OS2: C, 36.75; H, 2.16; N, 25.72.
Found: C, 36.81; H, 2.11; N, 25.71.
4.2.4. General procedure for the preparation of 1-((5-
(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)-4-(substituted)-
phenyl-1H-tetrazol-5(4H)-ones (5). To a stirring solu-
tion of mercaptooxadiazole 4 (1 mmol) in DMF
(5 mL), the appropriate alkyl halide (allyl halide or ethyl
acetate halide) (1.1 mmol) and anhydrate potassium car-
bonated (2 mmol) were added gradually. The reaction
mixture was stirred at room temperature for 4–8 h.
Then, the reaction mixture was poured into 100 mL of
ice water. The precipitate was filteredoff, washed with
water, dried, and recrystallized from ethanol.
Compound 5a: Yield: 65%; mp 65–67 °C; 1H NMR
(400 MHz, CDCl3): d 7.69 (dd, J ¼ 8:8 Hz, 4H), 5.93–

1722
Y.-P. Luo, G.-F. Yang / Bioorg. Med. Chem. 15 (2007) 1716–1724
4.2.6. General procedure for the preparation of 1-(2-
alkylthio-4H-1,3,4-thiadiazol-5-yl)-methylene-4-(4-chloro-
phenyl)-1H- tetrazol-5(4H)-one(7). To a stirring solution
of mercaptothiadiazole (6) (1 mmol) in DMF (5 mL),
the appropriate alkyl halide (allyl halide or ethyl acetate
halide) (1.1 mmol) and anhydrate potassium carbonated
(2 mmol) was added gradually. The reaction mixture
was stirred at room temperature for 4–8 h according to
TLC monitored. Then, the reaction mixture was poured
into 100 mL ice water. The precipitate was filtered-off,
washed with water, dried and recrystallized from
ethanol.
Compound 7f: Yield: 52%; mp 85–86 °C; 1H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 5.59
(s, 2H), 3.32 (t, J ¼ 7:2 Hz, 2H), 1.27–1.81 (m, 12H),
0.87 (t, J ¼ 7:2 Hz, 3H); EIMS (probe) 70 eV, m=z (rel.
int.): 439 [M]+ (10), 391 (62), 172 (43), 152 (100), 124
(53), 110 (27), 89 (36). Anal. Calcd for C18H23ClN6OS2:
C, 49.25; H, 5.28; N, 19.14; S, 14.61. Found: C, 48.92;
H, 4.93; N, 18.89; S, 14.74.
Compound 7g: Yield: 48%; mp 75–77 °C; 1H NMR
(400 MHz, CDCl3): d 7.69 (dd, J ¼ 8:8 Hz, 4H), 5.60
(s, 2H), 4.23 (q, J ¼ 7:2 Hz, 2H), 4.15 (s, 2H), 1.28 (t,
J ¼ 7:2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d
167.51, 166.78, 161.68, 148.10, 133.56, 132.77, 129.57,
120.22, 62.24, 43.09, 35.39, 14.02; EIMS (probe) 70 eV,
m=z (rel. int.): 413 [M]+ (5), 185 (20), 172 (4), 153
(100), 124 (38), 110 (14), 89 (27). Anal. Calcd for
C14H13ClN6O3S2: C, 40.73; H, 3.17; N, 20.35; S, 15.53.
Found: C, 40.84; H, 2.97; N, 20.22; S, 15.42.
1
Compound 7a: Yield: 61%; mp128–130 °C; H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 5.59
(s, 2H), 2.79 (s, 3H); 13C NMR (100 MHz, CDCl3): d
169.81, 160.81, 148.14, 133.61, 132.79, 129.59, 120.22,
43.18, 16.43; EIMS (probe) 70 eV, m=z (rel. int.): 341
[M]+ (60), 187 (73), 153 (100), 125 (70), 111 (21), 90
(88). Anal. Calcd for C11H9ClN6OS2: C, 38.77; H,
2.66; N, 24.66; S, 18.82. Found: C, 39.14; H, 2.54; N,
24.29; S, 18.42.
Compound 7h: Yield: 73%; mp 63–65 °C; 1H NMR
(400 MHz, CDCl3): d 7.68 (dd, J ¼ 8:8 Hz, 4H), 5.61
(s, 2H), 4.60 (q, J ¼ 7:2 Hz, 2H), 4.21 (q, J ¼ 7:2 Hz,
1H), 1.68 (d, J ¼ 7:2 Hz, 3H), 1.26 (t, J ¼ 7:2 Hz, 3H);
EIMS (probe) 70 eV, m=z (rel. int.): 427 [M]+ (95), 353
(23), 172 (17), 153 (100), 124 (35), 111 (23), 89 (28).
Anal. Calcd for C15H15ClN6O3S2: C, 42.20; H, 3.54;
N, 19.69; S, 15.02. Found: C, 42.62; H, 3.55; N, 19.67;
S, 14.97.
Compound 7b: Yield: 71%; mp 93–94 °C; 1H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 5.59
(s, 2H), 3.35 (q, J ¼ 7:2 Hz, 2H), 1.47 (t, J ¼ 7:2 Hz,
3H); EIMS (probe) 70 eV, m=z (rel. int.): 355 [M]+
(10), 202 (13), 153 (100), 125 (70), 90 (23). Anal. Calcd
for C12H11ClN6OS2: C, 40.62; H, 3.12; N, 23.68; S,
18.07. Found: C, 40.75; H, 2.93; N, 23.55; S, 18.17.
Compound 7i: Yield: 58%; mp 63–65 °C; 1H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 5.94–
6.60 (m, 1H), 5.59 (s, 2H), 5.20–5.39 (m, 2H), 3.95–
3.97 (m, 2H); 13C NMR (100 MHz, CDCl3): d 167.98,
161.19, 148.11, 133.58, 132.78, 131.49, 129.57, 120.20,
119.87, 43.15, 36.67; EIMS (probe) 70 eV, m=z (rel.
int.): 367 [M]+ (16), 351 (90), 152 (100), 124 (52), 110
(20), 89 (49). Anal. Calcd for C13H11ClN6OS2: C,
42.56; H, 3.02; N, 22.91; S, 17.48. Found: C, 42.51; H,
2.80; N, 22.70; S, 17.31.
Compound 7c: Yield: 76%; mp 83–85 °C; 1H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 5.59
(s, 2H), 3.31 (t, J ¼ 7:2 Hz, 2H), 1.83 (m, 2H), 1.05 (t,
J ¼ 7:2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d
169.17, 160.77, 148.15, 133.60, 132.81, 129.59, 120.23,
43.18, 36.08, 22.44, 13.02; EIMS (probe) 70 eV, m=z
(rel. int.): 369 [M]+ (6), 326 (16), 172 (22), 153 (100),
125 (54), 90 (29). Anal. Calcd for C13H13ClN6OS2: C,
42.33; H, 3.55; N, 22.78; S, 17.39. Found: C, 42.43; H,
3.29; N, 22.63; S, 17.44.
Compound 7j: Yield: 74%; mp 98–100 °C; 1H NMR
(400 MHz, CDCl3): d 7.49–7.92 (m, 4H), 7.31–7.42 (m,
5H), 5.59 (s, 2H), 4.57 (s, 2H); EIMS (probe) 70 eV,
m=z (rel. int.): 417 [M]+ (100), 263 (20), 206 (14), 176
(47), 125 (9), 112 (23). Anal. Calcd for C17H13ClN6OS2:
C, 48.98; H, 3.14; N, 20.16; S, 15.38. Found: C, 49.43;
H, 3.09; N, 20.02; S, 15.24.
Compound 7d: Yield: 71%; mp 62–63 °C; 1H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 5.59
(s, 2H), 3.33 (t, J ¼ 7:2 Hz, 2H), 1.44–1.80 (m, 4H),
0.94 (t, J ¼ 7:2 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 169.21, 160.74, 148.15, 133.61, 132.81, 129.59, 120.22,
43.17, 33.91, 30.95, 21.78, 13.46; EIMS (probe) 70 eV,
m=z (rel. int.): 383 [M]+ (21), 335 (83), 173 (73), 153
(100), 125 (65), 111 (22), 89 (51). Anal. Calcd for
C14H15ClN6OS2: C, 43.92; H, 3.95; N, 21.95; S, 16.75.
Found: C, 43.84; H, 3.72; N, 21.79;S, 16.68.
Compound 7k: Yield: 68%; mp 77–78 °C; 1H NMR
(400 MHz, CDCl3): d 7.15–7.92 (m, 8H), 5.60 (s, 2H),
4.61 (s, 2H), 2.42 (s, 3H); EIMS (probe) 70 eV, m=z
(rel. int.): 431 [M]+ (93), 397 (25), 243 (14), 162 (37),
125 (18), 105 (100). Anal. Calcd for C18H15ClN6OS2:
C, 50.17; H, 3.51; N, 19.50; S, 14.88. Found: C, 50.37;
H, 3.40; N, 19.49; S, 15.19.
Compound 7e: Yield: 75%; mp 68–70 °C; 1H NMR
(400 MHz, CDCl3): d 7.70 (dd, J ¼ 8:8 Hz, 4H), 5.59
(s, 2H), 3.33 (t, J ¼ 7:2 Hz, 2H), 1.30–1.81 (m, 8H),
0.88 (t, J ¼ 7:2 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d 169.21, 160.74, 148.15, 133.59, 132.80, 129.58, 120.20,
43.18, 34.22, 31.12, 28.90, 28.28, 22.39, 13.91; EIMS
(probe) 70 eV, m=z (rel. int.): 411 [M]+ (100), 363 (84),
172 (28), 153 (89), 125 (68), 111 (27), 90 (47). Anal.
Calcd for C16H19ClN6OS2: C, 46.76; H, 4.66; N, 20.45;
S, 15.61. Found: C, 46.90; H, 4.59; N, 20.37; S, 15.36.
Compound 7l: Yield: 74%; mp 90–91 °C; 1H NMR
(400 MHz, CDCl3): d 7.06–7.92 (m, 8H), 5.59 (s, 2H),
4.61 (s, 2H); 13C NMR (100 MHz, CDCl3): d 167.69,
162.08, 161.35, 159.61, 148.01, 133.44, 132.71, 131.36,
131.33, 129.88, 129.80, 129.47, 124.18, 124.14, 122.91,
122.77, 120.09, 115.56, 115.35, 43.01, 31.14; EIMS

Y.-P. Luo, G.-F. Yang / Bioorg. Med. Chem. 15 (2007) 1716–1724
1723
(probe) 70 eV, m=z (rel. int.): 435 [M]+ (100), 281 (37),
165 (24), 152 (20), 125 (10), 109 (55). Anal. Calcd for
C17H12ClFN6OS2: C, 46.95; H, 2.78; N, 19.32; S,
14.75. Found: C, 47.06; H, 2.71; N, 19.17; S, 14.93.
M. separata, and A. medicagini. These insects were
reared in a room maintained at 25 ( 1) °C, 60 ( 5)% rel-
ative humidity, and 14 h light photoperiod. Stock solu-
tions of each test compound were prepared in DMF at
a concentration of 1.0 g Lꢀ1, and then diluted to the re-
quired test concentrations with water containing TW-80.
Groups of 10 insects of each species were transferred to
glass Petri dishes and sprayed with test solutions using a
Potter sprayer. After air-drying, they were kept in a
room for normal cultivation. The mortality was deter-
mined by the number and size of live larvae in the treat-
ed bottles relative to that in the untreated controls in
72 h. In the case of N. lugen, rice seedlings (second
semester) were dipped in the test solution for 5 s, air-
dried, and then placed in a large test tube. Each test rube
contained 20 seedlings. Twenty insects (fifth instar) were
introduced into the tube, and the mouth of the tube was
covered with white cheesecloth. The tube was kept at
room temperature, and the number of live and dead in-
sects counted after 72 h. In a control experiment, carried
out under the same conditions, 1 mL DMF was applied
on each insect. All experiments and the respective con-
trols were carried out in three replicates and the data
were subjected to probit analysis. The results of insecti-
cidal activities are listed in Table 2.
Compound 7m: Yield: 71%; mp 87–88 °C; 1H NMR
(400 MHz, CDCl3): d 6.98–7.92 (m, 8H), 5.59 (s, 2H),
4.55 (s, 2H); 13C NMR (100 MHz, CDCl3): d 167.58,
163.94, 161.39, 148.12, 137.97, 133.63, 132.78, 130.26,
130.18, 129.60, 124.86, 124.84, 120.22, 116.19, 115.97,
115.08, 114.88, 43.13, 37.36; EIMS (probe) 70 eV, m=z
(rel. int.): 435 [M]+ (100), 281 (43), 166 (54), 153 (30),
125 (12), 109 (41). Anal. Calcd for C17H12ClFN6OS2:
C, 46.95; H, 2.78; N, 19.32; S, 14.75. Found: C, 46.95;
H, 2.93; N, 18.92; S, 14.77.
Compound 7n: Yield: 62%; mp 90–91 °C; 1H NMR
(400 MHz, CDCl3): d 7.23–7.92 (m, 8H), 5.59 (s, 2H),
4.53 (s, 2H); 13C NMR (100 MHz, CDCl3): d 167.46,
161.40, 148.07, 137.58, 134.39, 133.56, 132.75, 129.91,
129.55, 129.13, 128.11, 127.34, 120.16, 43.09, 37.22;
EIMS (probe) 70 eV, m=z (rel. int.): 451 [M]+ (100),
297 (45), 182 (84), 156 (64), 125 (96), 91 (63). Anal.
Calcd for C17H12Cl2N6OS2: C, 45.24; H, 2.68; N,
18.62; S, 14.21. Found: C, 45.36; H, 2.87; N, 18.24; S,
14.42.
1
Compound 7o: Yield: 36%; mp 144–146 °C; H NMR
Acknowledgments
(400 MHz, CDCl3): d 7.15–7.92 (m, 8H), 5.58 (s, 2H),
4.50 (s, 3H), 3.84 (s, 3H), 3.69 (s, 3H); 13C NMR
(100 MHz, CDCl3): d 168.84, 167.63, 161.01, 160.37,
148.10, 134.41, 133.55, 132.84, 132.79, 131.38, 130.04,
129.55, 128.22, 127.95, 120.22, 109.76, 62.00, 51.66,
43.09, 36.44; EIMS (probe) 70 eV, m=z (rel. int.): 531
[M]+ (5), 454 (100), 153 (54), 125 (36), 111 (14), 89
(27). Anal. Calcd for C22H19ClN6O4S2: C, 49.76; H,
3.61; N, 15.83; S, 12.08. Found: C, 49.95; H, 3.64; N,
15.59; S, 12.36.
We thank Dr. Jie Chen for the test of biological activity.
We also thank the financial support from the National
Key Project for Basic Research (2003CB114400),
National NSFC (Nos. 20572030, 20432010, and
20528201), Key project of Ministry of Education (Nos.
103116 and 104205), Foundation for Ph.D.
(20060511003), and Program for Excellent Research
Group of Hubei Province (No. 2004ABC002).
4.3. Biological assays
References and notes
4.3.1. Herbicidal tests. Stock solutions of each test com-
pound were prepared in DMF at a concentration of
1.0 g Lꢀ1, and then diluted to the required test concentra-
tions with water containing TW-80. The biological tests
were carried out in Petri dish (id = 9 cm) containing two
pieces of filter paper and 10 pregerminated seeds of wheat
(barnyard grass, jowar, rape, radish or cucumber), and
then 9 mL of solution (100 mg Lꢀ1) was added to each
Petri dish containing seeds. In a control experiment, car-
ried out underthe same conditions, 9 mL ofthe same solu-
tion without compounds was applied on each Petri dish.
The cultivations were kept at 28 1 °C, 70–80% relative
humidity (RH), a photoperiod of 16:8 (L:D) h, and with
exposure to light of 3000 LX for 5 days. The root and stem
growth was measured for the 10 seeds in each Petri dish
and averaged. All experiments and the respective controls
were carried out in three replicates and the data were
subjected to probit analysis. The results of herbicidal
activities are listed in Table 1.
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