Synthesis, bioactivities and structure activity relationship of N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N'-phenyl Ureas

Article abstract of DOI:10.1002/cjoc.201200268

Twenty nine novel N-4-methyl-1,2,3-thiadiazole-5-carbonyl-N'-phenyl ureas were designed and synthesized, and their structures were confirmed by proton nuclear magnetic resonance (¹H NMR), infra red spectroscopy (IR) and high-resolution mass spectroscopy (HRMS). Compounds V-9, V-11, V-12, V-15, V-19, V-21, V-22 and V-24 exhibit excellent activity against Culex pipiens pallens. Compounds V-12 and V-22 present good insecticidal activity against Plutella xylostella L. Their median lethal concentrations (LC₅₀) are 164.15 and 89.69 mg·L^-1, respectively. Compound V-11 also has potential wide spectrum of fungicide activity. Its median effective concentrations (EC₅₀) detected from 3.82 iμg·mL ^-1 against Physalospora piricola to 31.60 iμg· mL^-1 against Cercospora arachidicola. Compounds V-15 and V-24 show outstanding induction activities as same as positive controls TDL and ningnanmycin, furthermore V-24 has the highest induction activity of 41.85%i±4.43%. To elucidate the structure activity relationship in these compounds, a 3D-QSAR model has been built. The established model showed a reliable predicting ability with q² values of 0.643 and r ² values of 0.982. 29 novel N-4-methyl-1,2,3-thiadiazole-5-carbonyl- N'-phenyl ureas were designed and synthesized. Target compounds exhibit insecticidal activities. Bioassays indicated that compounds V-24 and V-22 exhibit outstanding insecticidal activity against Culex pipiens pallens and Plutella xylostella, respectively. Compound V-11 also has a broad-spectrum of fungicide activity against all the fungi tested. Five compounds show good SAR-inducing activity as well. These results establish basis for both disease and insect control in agricultural practice and novel pesticide development. Structure-activity relationship studies reveal that, with the contour maps based on steric and electrostatic CoMFA coefficients, we can conclude the relationship between the structures and bioactivity and continue the further design of highly active compounds. Copyright

Full text of DOI:10.1002/cjoc.201200268

FULL PAPER
DOI: 10.1002/cjoc.201200268
Synthesis, Bioactivities and Structure Activity Relationship of
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-phenyl Ureas
Guo, Dandana(国丹丹)
Zhao, Huia(赵晖) Zhang, Weib(张薇)
Wu, Qingjunc(吴青君) Zhang, Youjunc(张友军)
Wang, Zhiwena(王治文)
Cheng, Jiagaob(程家高)
Fan, Qianc(范谦)
Fan, Zhijin*,a(范志金)
Yang, Jiaqiangc(杨家强)
a State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, China
b Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Tech-
nology, Shanghai 200237, China
c Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China
d Sichuan Functional Heterocyclic Compounds Engineering Technology Research Center, Mianyang, Sichuan
621000, China
Twenty nine novel N-4-methyl-1,2,3-thiadiazole-5-carbonyl-N'-phenyl ureas were designed and synthesized,
and their structures were confirmed by proton nuclear magnetic resonance (1H NMR), infra red spectroscopy (IR)
and high-resolution mass spectroscopy (HRMS). Compounds V-9, V-11, V-12, V-15, V-19, V-21, V-22 and V-24
exhibit excellent activity against Culex pipiens pallens. Compounds V-12 and V-22 present good insecticidal activ-
ity against Plutella xylostella L. Their median lethal concentrations (LC50) are 164.15 and 89.69 mg•L―1, respec-
tively. Compound V-11 also has potential wide spectrum of fungicide activity. Its median effective concentrations
－
(EC50) detected from 3.82 μg•mL－ against Physalospora piricola to 31.60 μg•mL against Cercospora arachidi-
cola. Compounds V-15 and V-24 show outstanding induction activities as same as positive controls TDL and ning-
nanmycin, furthermore V-24 has the highest induction activity of 41.85%±4.43%. To elucidate the structure activ-
ity relationship in these compounds, a 3D-QSAR model has been built. The established model showed a reliable
predicting ability with q2 values of 0.643 and r2 values of 0.982.
1
1
Keywords 4-methyl-1,2,3-thiadiazole derivatives, benzoylurea pesticides, insecticidal activity, 3D QSAR model
Introduction
azole derivatives.[8] Tiadinil (TDL, N-(3-chloro-4-meth-
ylphenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide)
has been commercialized as an important elicitor for
SAR, and has been applied in rice field.[9] Its metabolite
4-methyl-1,2,3-thiadiazole-5-carboxylic acid shows a
good SAR-inducing activity as well.[10] Considering
insecticidal activity of these compounds, we focused our
studies on the environment benign benzoylureas pesti-
cides (BUPs) lead. During the last three decades, BUPs
played an important role in agricultural pest control with
its unique mode of action on a target site which is absent
in plants and mammals,[11] therefore, their insecticidal
activity was highly selective, and non-toxic to ecologi-
cal environment. In order to design compounds with
both insecticidal activity and SAR, 1,2,3-thiadiazole
group has been introduced into the lead of BUPs.
Most of viral diseases of plants are transmitted
through the infection of insects.[1] Insect-plant disease
associations, which connect insect, pathogen and plant
inseparably, lead to greater risk of economic loss.[2] This
requires designation of compounds which can not only
control pests but also enhance the defense level of
plants. Therefore, these compounds can protect plants
from the injury caused by pests, succeeding fungi and
virus simultaneously, which means they can show in-
secticidal activities as well as systemic acquired disease
resistance. Systemic acquired resistance (SAR) refers to
a distinct signal transduction pathway that plays an im-
portant role in the ability of plants to defend themselves
against pathogens.[3]
Derivatives of 1,2,3-thiadiazole have versatile bio-
logical activities such as anti-Human Immunodeficiency
Virus (HIV),[4] antitumor,[5] antibacterial,[6] and anti-
allergic[7] activities. One of the most significant proper-
ties is that SAR can be induced by some 1,2,3-thiadi-
A 3D-QSAR model has been established by CoMFA
so as to interpret the relationship between their struc-
tures and activities. Comparative molecular field analy-
sis (CoMFA) is one of the most popular QSAR methods,
*
E-mail: fanzj@nankai.edu.cn; Tel.: 0086-022-23499464
Received March 19, 2012; accepted September 4, 2012.
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cjoc. 201200268 or from the author.
2522
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Chin. J. Chem. 2012, 30, 2522—2532

Synthesis, Bioactivities and Structure Activity Relationship
Synthesis of 4-methyl-1,2,3-thiadiazole-5-carboxylic
acid chloride (Compound I)
which use interactive graphics and statistical techniques
for correlating biological properties. In CoMFA, the
bioactivities of molecules are correlated with steric and
electrostatic potential energies in terms of Lennare-
Jones and Coulombic potentials, respectively. The per-
formance of the standard CoMFA procedure requires the
specification of both conformation and alignments of
molecules.[12] The synthetic routes were shown in
Scheme 1. All the structures of the target compounds
A mixture of 4-methyl-1,2,3-thiadiazole-5-carbox-
ylic acid (10 g 0.067 mol) in thionyl dichloride (25 mL)
was refluxed at 80 ℃ for 6 h. After cooled to room
temperature, the thionyl dichloride was removed under
reduced pressure. The 4-methyl-1,2,3-thiadiazole-5-
carboxylic acid chloride was obtained with the yield of
85% as a light-yellowish oil and used without purifica-
tion.
1
have been confirmed by H NMR, and high-resolution
mass spectroscopy. Their bioactivities have been evalu-
ated systemically.
Synthesis of 4-methyl-1,2,3-thiadiazole-5-formamide
(Compound II)
Scheme 1 General synthetic route of the title compounds V-1—
V-29
The mixture of aqueous solution of ammonia (25%,
30 mL), triethylamine (5 mL) and THF (50 mL) was
cooled with an ice-water bath, 4-methyl-1,2,3-thiadia-
zole-5-carboxylic acid chloride (10 g 0.062 mol) was
slowly added drop-wise to the cooled mixture. After-
wards, the mixture was stirred in an ice-water bath for
another 1 h, and then the mixture was stirred at room
temperature overnight. After that, the mixture was
poured into the separating funnel, organic layer was
collected and the aqueous phase was extracted by THF
(15 mL×3). The combined organic layers were washed
with saturated brine, and then dried over anhydrous so-
dium sulfate. After filtration, the organic layer was con-
centrated under reduced pressure to give a white solid
4-methyl-1,2,3-thiadiazole-5-carboxamide with a yield
of 90%, which was used without purification.
O
Cl
CH3
C
CH3
.
N
N
N
Cl
NH3 H2O (25%)
Cl
O
NH2
N
THF/TEA
S
S
C
O
Reflux
O
I
II
CH3
HN
CH3
N
R NH2
IV
R
NCO
HN
C
O
N
C
O
N
N
S
C
O
S
III
V
R＝4-nitro, 2-nitro, 3-nitro, 2,4-dinitro, 2-chloro, 4-chloro, 3-chloro,
2,5-dichloro, 3,5-dichloro, 3,4-dichloro, 2,4,5-trichloro, 2,4-difluoro-
3,5-dichloro, 4-bromo, 2-fluoro, 2-fluoro-4-hydroxyl, 3-fluoro-4-
methyl, 2-methyl-4-chloro, 3-chloro-4-methyl, 2-trifluoromethyl-5-
chloro, 2-carboxy, 4-trifluoromethyl, 2-chloro-3,5-ditrifluoromethyl,
4-trifluoromethoxyl, 2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxyl),
2,4-dimethyl, 4-ethyl, 4-carbonyl, 2-hydroxyl, 3,5-dichloro-4-hy-
droxyl
Synthesis of 4-methyl-1,2,3-thiadiazole-5-carbonyl
isocyanate (Compound III)
A mixture of 4-methyl-1,2,3-thiadiazole-5-carbox-
amide (2 g 0.014 mol) and oxalyl chloride (3.55 g 0.028
mol) in 1,2-dichloroethane (20 mL) was stirred at room
temperature for 30 min, subsequently, the mixture was
heated at 80 ℃ for 10 h. After cooling to room tem-
perature, the mixture was concentrated under reduced
pressure to give a brown oil as 4-methyl-1,2,3-thiadia-
zole-5-carbonyl isocyanate with a yield of 80% for fur-
ther reaction without purification.
Experimental
Equipments and materials
Melting points of all compounds were determined on
an X-4 binocular microscope (Gongyi Tech. Instrument
Co., Henan, China) and the thermometer was not cor-
rected. Proton NMR spectra were obtained using a
Bruker AVANCE-400 MHZ spectrometer and chemical
shift values (δ) were reported with deutero-dimethyl
sulfoxide (DMSO-d6) as solvent and tetramethylsilane
(TMS) as the internal standard. High resolution mass
spectrometry (HRMS) data were obtained on a Varian
7.0T FTICR-MS instrument. IR was recorded on a
Bruker Vector 22 FTIR spectrometer using a KBr pellet
press. All solvents were analytical reagent grade and
were dried in advance and distilled before use. Column
chromatography purification was carried out using silica
gel.
Synthesis of target compound N-4-methyl-1,2,3-
thiadiazole-5-carbonyl-N'-phenyl ureas V (1—29)
A solution of 4-methyl-1,2,3-thiadiazole-5-carbonyl
isocyanate (0.59 g 3.5 mmol) in 1,2-dichloroethane (5
mL) was added drop-wise to a solution of aromatic
amine (3.5 mmol) in 1,2-dichloroethane (20 mL) at
room temperature, and the mixture was allowed to stand
for 8 h. Then part of the solvent was removed under
reduced pressure, and the precipitated solid was filtered
to give a solid as crude product. After recrystallization
from DMF/ethanol, a pure solid was obtained.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(4-ni-
trophenyl) urea (V-1) White solid, yield 58%, m.p.
178—180 ℃; 1HNMR (DMSO-d6, 400 MHz) δ: 2.89 (s,
3H, CH3), 7.92 (d, J＝12.4 Hz, 2H, Ar-H), 8.32 (d, J＝
12.4 Hz, 2H, Ar-H), 10.71 (s, 1H, NH), 11.68 (s, 1H,
General synthesis
4-Methyl-1,2,3-thiadiazole-5-carboxylic acid was
synthesized according to the reference [13].
Chin. J. Chem. 2012, 30, 2522—2532
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www.cjc.wiley-vch.de
2523

Guo et al.
FULL PAPER
NH); IR (KBr) ν: 3350, 3131, 2945, 1705, 1598, 1513
cm － 1; HRMS calcd for C11H9N5O4S (M ＋ ＋Na):
330.0267, found 330.0268.
10.97 (s, 1H, NH), 11.86 (s, 1H, NH); IR (KBr) ν: 3221,
3124, 1709, 1592, 814 cm － 1; HRMS calcd for
C11H8Cl2N4O2S (M－－H): 328.9672, found 328.9684.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(3,5-
dichlorophenyl) urea (V-9) White solid, yield 72%,
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2-ni-
trophenyl) urea (V-2) White solid, yield, 39%, m.p.
1
1
158—160 ℃; H NMR (DMSO-d6, 400 MHz) δ: 2.84
m.p. 194—197 ℃; H NMR (DMSO-d6, 400 MHz) δ:
(s, 3H, CH3), 7.41 (t, J＝15.6 Hz, 1H, Ar-H), 7.80 (t,
J＝15.6 Hz, 1H, Ar-H), 8.18 (d, J＝8.0 Hz, 1H, Ar-H),
8.44 (d, J＝8.4 Hz, 1H, Ar-H), 11.77 (s, 1H, NH), 11.84
(s, 1H, NH); IR (KBr) ν: 3556, 3413, 3127, 1697, 1551,
1492 cm ; HRMS calcd for C11H9N5O4S (M －H):
306.0302, found 306.0304.
2.82 (s, 3H, CH3), 7.34 (s, 1H, Ar-H), 7.71 (s, 2H, Ar-H),
10.43 (s, 1H, NH), 11.60 (s, 1H, NH); IR (KBr) ν: 3219,
3124, 1703, 1590, 757 cm － 1; HRMS calcd for
C11H8Cl2N4O2S (M－－H): 328.9672, found 328.9680.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(3,4-
dichlorophenyl) urea (V-10) White solid, yield 84%,
－
－
1
1
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N'-(3-ni-
trophenyl) urea (V-3) White solid, yield 65%, m.p.
m.p. 155—157 ℃; H NMR (DMSO-d6, 400 MHz) δ:
2.82 (s, 3H, CH3), 7.54 (d, J＝8.8 Hz, Ar-H), 7.64 (d,
J＝8.8 Hz, 2H, Ar-H), 7.98 (s, 1H, Ar, 1H -H), 10.41 (s,
1H, NH), 11.56 (s, 1H, NH); IR (KBr) ν: 3223, 3128,
1
171—173 ℃; H NMR (DMSO-d6, 400 MHz) δ: 2.84
(s, 3H, CH3), 7.66(t, J＝8.2 Hz, 1H, Ar-H), 7.96 (m, 2H,
Ar-H), 8.64 (s, 1H, Ar-H), 10.54 (s, 1H, NH), 11.60 (s,
1H, NH); IR (KBr) ν: 3261, 3104, 2961, 1699, 1597,
－
1
1696, 1583, 759 cm ; HRMS calcd for C11H8Cl2N4O2S
(M－－H): 328.9672, found328.9666.
－
－
1
1545 cm ; HRMS calcd for C11H9N5O4S (M －H):
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2,4,
5-trichlorophenyl) urea (V-11) White solid, yield
306.0302, found 306.0296.
1
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2,4-
dinitrophenyl) urea (V-4) Yellow solid, yield 81%,
23%, m.p. 122—124 ℃; H NMR (DMSO-d6, 400
MHz) δ: 2.88 (s, 3H, CH3), 8.05 (s, 1H, Ar-H), 8.59 (s,
1
m.p. 164—166 ℃; H NMR (DMSO-d6, 400 MHz) δ:
1H, Ar-H), 11.03 (s, 1H, NH), 11.98 (s, 1H, NH); IR
－
1
2.84 (s, 3H, CH3), 8.61 (d, J＝9.2 Hz, 1H, Ar-H), 8.79
(d, J＝9.2 Hz, 1H, Ar-H), 8.90 (s, 1H, Ar-H), 12.02 (s,
1H, NH), 12.30 (s, 1H, NH); IR (KBr) ν: 3138, 3077,
2975, 1698, 1599, 1494 cm － 1; HRMS calcd for
C11H8N6O6S (M－－H) 351.0153, found 351.0157.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2-
chlorophenyl) urea (V-5) White solid, yield 70%,
(KBr) ν: 3220, 3131, 1692, 1574, 689 cm ; HRMS
calcd for C11H7Cl3N4O2S (M－－H): 362.9283, found
362.9286.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2,4-
difluoro-3,5-dichlorophenyl) urea (V-12)
White
solid, yield 92%, m.p. 187—189 ℃; 1H NMR (DMSO-
d6, 400 MHz) δ: 2.82 (s, 3H, CH3), 8.25 (s, 1H, Ar-H),
10.56 (s, 1H, NH), 11.92 (s, 1H, NH); IR (KBr) ν: 3232,
1
m.p. 171—173 ℃; H NMR (DMSO-d6, 400 MHz) δ:
－
1
2.88 (s, 3H, CH3), 7.23 (t, J＝7.6 Hz, 1H, Ar-H), 7.45 (t,
J＝8.0 Hz, 1H, Ar-H), 7.62 (d, J＝8.0 Hz, 1H, Ar-H),
8.33 (d, J＝8.4 Hz, Ar-H), 10.90 (s, 1H, NH), 11.79 (s,
1H, NH); IR (KBr) ν: 3230, 3126, 2952, 1699, 1589,
3129, 1696, 1603, 1232, 757 cm ; HRMS calcd for
C11H6Cl2F2N4O2S (M－－H): 364.9484, found 364.9480.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(4-
bromophenyl) urea (V-13) White solid, yield 78%,
－
－
1
1
767 cm ; HRMS calcd for C11H9ClN4O2S (M －H):
m.p. 195—197 ℃; H NMR (DMSO-d6, 400 MHz) δ:
295.0062, found 295.0054.
2.82 (s, 3H, CH3), 7.55—7.56 (m, 4H, Ar-H), 10.29 (s,
1H, NH), 11.49 (s, 1H, NH); IR (KBr) ν: 3216, 3117,
2943, 1590, 1679, 500 cm － 1; HRMS calcd for
C11H9BrN4O2S (M－－H): 338.9557, found 338.9551.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2-
fluorophenyl) urea (V-14) White solid, yield 86%,
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(4-
chlorophenyl) urea (V-6) White solid, yield 73%,
1
m.p. 173—175 ℃; H NMR (DMSO-d6, 400 MHz) δ:
2.83 (s, 3H, CH3), 7.42 (d, J＝8.0 Hz, 1H, Ar-H), 7.62
(d, J＝7.6 Hz, 1H, Ar-H), 10.28 (s, 1H, NH), 11.48 (s,
1H, NH); IR (KBr) ν: 3220, 3123, 2946, 1699, 1594,
1
m.p. 229—231 ℃; H NMR (DMSO-d6, 400 MHz) δ:
2.84 (s, 3H, CH3), 7.73 (s, 2H, Ar-H), 7.92 (d, 2H, J＝
－
－
1
757 cm ; HRMS calcd for C11H9ClN4O2S (M －H):
295.0062, found 295.0054.
7.2 Hz, Ar-H), 11.58 (s, 1H, NH), 11.90 (s, 1H, NH); IR
－
1
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(3-
chlorophenyl) urea (V-7) White solid, yield 72%,
(KBr) ν: 3206, 3120, 2947, 1716, 1620, 1229 cm ;
HRMS calcd for C11H9FN4O2S (M－－H): 279.0357,
found 279.0353.
1
m.p. 166—168 ℃; H NMR (DMSO-d6, 400 MHz) δ:
2.83 (s, 3H, CH3), 7.19 (d, J＝8.0 Hz, 1H, Ar-H), 7.43
(m, 2H, Ar-H), 7.81 (s, 1H, Ar-H), 10.32 (s, 1H, NH),
11.52 (s, 1H, NH); IR (KBr) ν: 3391, 3128, 1702, 1595,
753 cm ; HRMS calcd for C11H9ClN4O2S (M －H):
295.0062, found 295.0056.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2-
fluoro-4-hydroxylphenyl) urea (V-15) Light yellow
solid, yield 83%, m.p. 234 —236 ℃; 1H NMR
(DMSO-d6, 400 MHz) δ: 2.81 (s, 3H, CH3), 6.72—6.62
(m, 2H, Ar-H), 7.74 (s, 1H, Ar-H), 9.88 (s, 1H, OH),
10.13 (s, 1H, NH), 11.56 (s, 1H, NH); IR (KBr) ν: 3212,
3120, 1701, 1613, 1236, 1204 cm ; HRMS calcd for
C11H9FN4O3S (M－－H): 295.0307, found 295.0303.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(3-
fluoro-4-methylphenyl) urea (V-16) White solid,
－
－
1
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2,5-
dichlorophenyl) urea (V-8) White solid, yield 26%,
－
1
1
m.p. 186—190 ℃; H NMR (DMSO-d6, 400 MHz) δ:
2.82 (s, 3H, CH3), 7.24—7.25 (m, 1H, Ar-H), 7.62 (d,
J＝8.6 Hz, 1H, Ar-H), 8.37 (d, J＝2.4 Hz, 1H, Ar-H),
2524
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 2522—2532

Synthesis, Bioactivities and Structure Activity Relationship
1
yield 85%, m.p. 191—195 ℃; H NMR (DMSO-d6,
trifluoromethoxylphenyl) urea (V-23): White solid,
yield 75%, m.p. 144—146 ℃; H NMR (DMSO-d6,
1
400 MHz) δ: 2.82 (s, 3H, CH3), 7.23—7.26 (m, 2H,
Ar-H), 7.50—7.53 (m, 1H, Ar-H), 10.28 (s, 1H, NH),
11.48 (s, 1H, NH); IR (KBr) ν: 3222, 3103, 2952, 1704,
400 MHz) δ: 2.83 (s, 3H, CH3), 7.38 (d, J＝8.4 Hz, 2H,
Ar-H), 7.71 (d, J＝8.4 Hz, 2H, Ar-H), 10.33 (s, 1H,
NH), 11.50 (s, 1H, NH); IR (KBr) ν: 3230, 3124, 2952,
1715, 1611, 1280, 1221 cm － 1; HRMS calcd for
C12H9F3N4O3S (M－－H): 345.0275, found 345.0270.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-[2,5-
dichloro-4-(1,1,2,3,3,3-hexafluoropropoxyl)phenyl]
urea (V-24) White solid, yield 49%, m.p. 155—157
℃; 1H NMR (DMSO-d6, 400 MHz) δ: 2.83 (s, 3H, CH3),
6.61—6.45 (m, 1H, CHF), 7.80 (s, 1H, Ar-H), 8.55 (s,
－
1
1602, 1229, 1674, 1415, 1311 cm ; HRMS calcd for
C12H11FN4O2S (M－－H): 293.0514, found 293.0515.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2-
methyl-4-chlorophenyl) urea (V-17) White solid,
1
yield 43%, m.p. 194—196 ℃; H NMR (DMSO-d6,
400 MHz) δ: 2.20 (s, 3H, Ar-CH3), 2.83 (s, 1H, CH3),
7.29 (d, J＝8.7 Hz, 1H, Ar-H), 7.38 (s, 1H, Ar-H), 7.96
(d, J＝8.9 Hz, 1H, Ar-H), 10.23 (s, 1H, NH), 11.65 (s,
1H, NH); IR (KBr) ν: 3221, 3130, 2937, 1699, 1586,
1H, Ar-H), 11.00 (s, 1H, NH), 11.92 (s, 1H, NH); IR
－
－
1
(KBr) ν: 3422, 1701, 1588, 1222, 1211, 760 cm－
;
1
757 cm ; HRMS calcd for C12H11ClN4O2S (M －H):
HRMS calcd for C14H8Cl2F6N4O3S (M－－H): 494.9526,
found 494.9526.
309.0218, found 309.0215.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(3-
chloro-4-methylphenyl) urea (V-18) White solid,
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2,4-
dimethylphenyl) urea (V-25) White solid, yield 57%,
1
yield 70%, m.p. 166—168 ℃; H NMR (DMSO-d6,
1
400 MHz) δ: 2.29 (s, 3H, CH3), 2.82 (s, 3H, CH3), 7.31
—7.38 (m, 2H, Ar-H), 7.79 (s, 1H, Ar-H), 10.26 (s, 1H,
NH), 11.51 (s, 1H, NH); IR (KBr) ν: 3209, 3118, 2951,
m.p. 176—178 ℃; H NMR (DMSO-d6, 400 MHz) δ:
2.26 (s, 6H, CH3), 2.83 (s, 3H, CH3), 7.03 (d, J＝8.4 Hz,
1H, Ar-H), 7.08 (s, 1H, Ar-H), 7.78 (d, J＝8.4 Hz, 1H,
Ar-H), 10.14 (s, 1H, NH), 11.54 (s, 1H, NH); IR (KBr)
－
1
1713, 1603, 752 cm ; HRMS calcd for C12H11ClN4O2S
(M－－H): 309.0218, found 309.0217.
－
1
ν: 3218, 3131, 2935, 1700, 1557 cm ; HRMS calcd for
C13H14N4O2S (M－－H): 289.0765, found 289.0768.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(4-
ethylphenyl) urea (V-26) White solid, yield 68%, m.p.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2-
trifluoromethyl-5-chlorophenyl) urea (V-19) White
solid, yield 50%, m.p. 190 —192 ℃; 1H NMR
(DMSO-d6, 400 MHz) δ: 2.83 (s, 3H, CH3), 7.54 (d, J＝
8.3 Hz, 1H, Ar-H), 7.84 (d, J＝8.2 Hz, 1H, Ar-H), 8.66
(s, 1H, Ar-H), 11.09 (s, 1H, NH), 11.92 (s, 1H, NH); IR
(KBr) ν: 3202, 3137, 2940, 1708, 1607 cm ; HRMS
calcd for C12H8ClF3N4O2S (M－－H): 362.9936, found
362.9941.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N'-(2-
carboxyphenyl) urea (V-20) White solid, yield 91%,
m.p. 197—199 ℃; H NMR (DMSO-d6, 400 MHz) δ:
2.82 (s, 3H, CH3), 7.25—7.19 (m, 1H, Ar-H), 7.67—
7.59 (m, 1H, Ar-H), 7.98 (d, J＝8.0 Hz, 1H, Ar-H), 8.46
(d, J＝8.0 Hz, 1H, Ar-H), 11.48 (s, 1H, NH), 12.16 (s,
1H, NH); IR (KBr) ν: 3222, 3125, 1681, 1584 cm ;
HRMS calcd for C12H10N4O4S (M－－H): 305.0350,
found 305.0349.
1
169—161 ℃; H NMR (DMSO-d6, 400 MHz) δ: 1.18
(t, J＝14.8 Hz, 3H, CH3), 2.59 (q, J＝6.8, 7.6 Hz, 2H,
CH2), 2.83 (s, 3H, CH3), 7.20 (d, J＝7.6 Hz, 2H, Ar-H),
7.47 (d, J＝8.0 Hz, 2H, Ar-H), 10.18 (s, 1H, NH), 11.41
(s, 1H, NH); IR (KBr) ν: 3224, 3111, 2965, 1711, 1599
cm － 1; HRMS calcd for C13H14N4O2S (M － －H):
289.0765, found 289.0763.
－
1
1
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(4-
formylphenyl) urea (V-27) Red solid, yield 95%, m.p.
over 240 ℃; 1H NMR (DMSO-d6, 400 MHz) δ: 2.83 (s,
3H, CH3), 7.80 (d, J＝8.4 Hz, 2H, Ar-H), 7.92 (d, J＝
8.8 Hz, 2H, Ar-H), 9.92 (s, 1H, CHO), 10.57 (s, 1H,
NH), 11.58 (s, 1H, NH); IR (KBr) ν: 3222, 3114, 1709,
－
1
－
－
1
1688, 1547 cm ; HRMS calcd for C12H10N4O3S (M －
H): 289.0401, found 289.0395.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N'-(4-
trifluoromethylphenyl) urea (V-21) White solid,
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2-
hydroxylphenyl) urea (V-28) White solid, yield 93%,
1
yield 92%, m.p. 168—170 ℃; H NMR (DMSO-d6,
1
m.p. 186—188 ℃; H NMR (DMSO-d6, 400 MHz) δ:
400 MHz) δ: 2.83 (s, 3H, CH3), 7.73 (s, 2H, Ar-H), 7.79
2.88 (s, 3H, CH3), 6.99—6.83 (m, 3H, Ar-H), 8.16 (d,
J＝8.0 Hz, 1H, Ar-H), 10.27 (s, 1H, OH), 10.67 (s, 1H,
NH), 11.54 (s, 1H, NH); IR (KBr) ν: 3278, 3131, 1688,
(s, 2H, Ar-H), 10.52 (s, 1H, NH), 11.58 (s, 1H, NH); IR
－
1
(KBr) ν: 3227, 3132, 2944, 1680, 1606, 1318 cm ;
HRMS calcd for C12H9F3N4O2S (M－－H): 329.0326,
found 329.0326.
－
－
1
1547, 1212 cm ; HRMS calcd for C11H10N4O3S (M －
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(2-
chloro-3,5-ditrifluoromethylphenyl) urea (V-22)
White solid, yield 70%, m.p. 162—164 ℃; H NMR
H): 277.0401, found 277.0397.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(3,5-
dichloro-4-hydroxylphenyl) urea (V-29) White solid,
yield 77%, m.p. 237—239 ℃; 1HNMR (DMSO-d6, 400
MHz) δ: 2.82 (s, 3H, CH3), 7.64 (s, 2H, Ar-H), 10.00 (s,
1H, OH), 10.16 (s, 1H, NH), 11.50 (s, 1H, NH); IR
(KBr) ν: 3234, 1695, 1551, 1215, 754 cm ; HRMS
calcd for C11H8Cl2N4O3S (M－－H): 344.9621, found
344.9615.
1
(DMSO-d6, 400 MHz) δ: 2.84 (s, 3H, CH3), 7.95 (d, J＝
1.6 Hz, 2H, Ar-H), 8.93 (d, J＝1.6 Hz, 2H, Ar-H), 11.26
(s, 1H, NH), 12.00 (s, 1H, NH); IR (KBr) ν: 3245, 3142,
－
1
－
1
3113, 1709, 1602, 1223, 759 cm ; HRMS calcd for
C13H7ClF6N4O2S (M－－H): 430.9810, found 430.9816.
N-4-Methyl-1,2,3-thiadiazole-5-carbonyl-N′-(4-
Chin. J. Chem. 2012, 30, 2522—2532
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2525

Guo et al.
FULL PAPER
Biological assay
the same compound by five to seven different concen-
trations; the median effective concentration (EC50) was
calculated by the linear regression of logarithm of the
concentration with probability of the corresponding
growth inhibition by Excel.[15]
Biological activities of the target compounds in-
cluding fungicide and insecticidal activities were evalu-
ated systematically according to the standard operation
practice (SOP) as described in Ref. [8] and the follow-
ing procedures.
Systemic acquired resistance screening
Larvicidal activity against C. pipiens pallens
Protective effects of the target compounds against
tobacco mosaic virus (TMV) in vivo The healthy
fresh tobacco leaves growing at 6 leaves age were se-
lected for the tests. The compound solution was smeared
on the whole leaves, and then the leaves were dried in
the green house. After 12 h, the TMV at a concentration
of 5.88×10－2 μg/mL was inoculated on the upper three
leaves using the conventional juice robbing method, and
the solvent was smeared on the lower three leaves as a
control. The local lesion numbers were then recorded 2
—3 days after inoculation. For each compound, three
repetitions were conducted. All compounds tested were
conducted at concentrations 100 μg/mL respectively.
For the inactivation effect of the target compounds
against TMV in vivo, the TMV virus with a concentra-
tion of 5.88×10－2 μg/mL was inhibited by mixing with
the target compound solution at the same volume for 30
min, then the mixture was inoculated on the upper three
leaves using the conventional juice robbing method, and
the solvent was smeared on the lower three leaves as a
control. For the curative effect of target compounds on
Each compound of 2 mg was weighed into a penicil-
lin bottle; 10 mL of acetone was added to dissolve the
compound to prepare 200 μg/mL of mother solution.
The working solution of 5 μg/mL was prepared by di-
luting 1 mL of mother solution with 29 mL of water and
10 mL of feeding solution into a 100 mL beaker. The
working solutions of 2 or 1 μg/mL were prepared by
diluting the solution of 5 μg/mL successively. Ten 4th
instar larvae of C. pipiens pallens were transferred into
the beaker by double distilled water. Thereafter, the
beakers with mosquito larvae were put into the standard
conditioned rooms for further cultivation with tempera-
ture of 25 ℃ and humidity of 80%. After 24 h or 96 h
of cultivation, the mortalities of the larvae were calcu-
lated relative to the corresponding CK (control check),
which was prepared by 1 mL of acetone for substituting
1 mL of mother solution, the observation was conducted
until all the larvae metamorphosized into pupation or
died. Chlorfluazuron was used as a positive control.
TMV in vivo, TMV at a concentration of 5.88×10－
2
Toxicity against P. xylostella
μg/mL was inoculated on the whole leaves using the
conventional juice robbing method, after the leaves
were dried in the green house, the compound solution
was smeared on the upper three leaves, and the solvent
was smeared on the lower three leaves as control. Sys-
temic acquired resistance of the target compounds was
detected using tobacco against TMV system as de-
scribed in reference.[8,16] After 7 days of induction of the
target compounds to tobacco plant, TMV at a concentra-
The toxicities of the target compounds against P.
xylostella were tested by the leaf dip method using the
reported procedure.[14] Fresh cabbage leaves were
dipped into the 200 µg/mL test water solution for 10 s
which was prepared with a 5% of acetone to help the
compounds to dissolve. After air-drying for evaporating
off the acetone and water, the treated leaves were cut
into small pieces and placed in the Petri dishes with 9
cm diameter. Ten individuals of 2nd instars larvae of P.
xylostella L. were then transferred into the 10 cm di-
ameter of Petri dish. The Petri dishes were finally fas-
tened with rubbers and placed in the standard cultivation
room for 96 or 120 h at 25 ℃ with 80% of humidity.
The percentage of mortalities was evaluated relative to
the corresponding CK which uses water only. The in-
sects which had no reaction when touched by brush pen
were regarded as dead insect.
tion of 5.88×10－ μg/mL was inoculated on the newly
2
grown leaves using the conventional juice robbing
method. The induction activity was evaluated using the
antivirus inhibition ratio which was calculated by the
average number of the viral inflammations on the in-
oculated leaves with the corresponding control accord-
ingly. TDL and ningnanmycin were chosen as positive
control and negative control respectively, and all com-
pounds tested were conducted at concentrations of 100
µg/mL respectively. The activity of protection, inactiva-
tion, curative effects and induction activity against TMV
were calculated by the average number of the viral in-
flammations on the inoculated leaves with the corre-
sponding control according to Equation 1:
Fungicide screening
Preliminary screening was conducted by fungus
growth inhibition method according to Ref. [8]. Fungi
used in this studies included Alternaria solani (AS),
Botrytis cinerea (BC), Cercospora. arachidicola (CA),
Gibberella zeae (GZ), Phytophthora infestans (Mont) de
Bary (PI), Physalospora piricola (PP), Pellicularia-
sasakii (Shirai) (PS), Sclerotinia sclerotiorum (SS), and
Rhizoctonia cerealis (RC). Precision toxicity studies
were conducted according to the above methods by de-
termination of growth inhibition of specific fungi using
CK－A
CK
Y＝
×100
(1)
where Y is the antivirus inhibition ratio (protection, in-
activation, curative effects and induction activity in vivo)
(%), CK is the average number of viral inflammations
2526
www.cjc.wiley-vch.de
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 2522—2532

Synthesis, Bioactivities and Structure Activity Relationship
on the control leaves in vivo, A is the average number of
viral inflammations on the target compound treated
leaves in vivo.
of the tested compounds.[12,19,20] The compounds for
CoMFA studies were listed in Table 1. Each structure
was optimized by molecular mechanics method based
on the TRIPOS force field and Gasteiger and Hückel
charges. The CoMFA steric and electrostatic interaction
fields were calculated at each lattice intersection on a
regularly spaced grid of 2.0 Å. The grid pattern was
generated automatically by the SYBYL/CoMFA routine,
and an sp3 carbon atom with a van der Waals radius of
1.52 Å and a＋1.0 charge was used as the probe to cal-
culate the steric (Lennard-Jones 6—12 potential) field
energies and electrostatic (Coulombic potential) fields
with a distance-dependent dielectric at each lattice point.
Values of the steric and electrostatic fields were trun-
cated at ±30.0 kcal/mol. In our study, with standard
options for scaling of variables, a standard Leave-One-
Out (LOO) cross-validation was finally carried out with
the optimal number of components 4 (noncross-vali-
dated conventional analysis), then models with the
highest r2 and lowest PRESS was obtained.[12,19,20]
Extraction and activity determination of phenyla-
lanine ammonialyase (PAL)
The PAL extraction and assay protocols were con-
ducted according to the description of Zhang et al.[17]
5
g of tobacco leaves were cut and homogenized in 10 mL
of 25 mmol•L－1 of boric acid buffer solutions (pH＝8.0
containing 5 mmol•L － of mercaptoethanol and 1
1
mmol•L－1 of EDTA) with 0.5 g of PVP and Quartz sand
washed and clinched (purity more than 99.8%). The
homogenate was filtered through 8 layers of cheesecloth
and centrifuged at 10000 g for 15 min at 0—4 ℃, the
supernatant fluid was used as crude enzyme of PAL for
immediate specific activity determination. Adding 1 mL
of PAL crude enzyme and 2 mL of double distilled wa-
ter to 1 mL solution of 20 mmol•L－ of phenylalanine,
1
the mixture was then heated to (30±1) ℃ for 30 min
in the dark, after cooling to room temperature by water,
the absorbance (OD) of the mixture was measured at
290 nm. One enzyme unit (U) was calculated by in-
creasing of 0.01 of OD value, specific activity was cal-
culated according to the corresponding content of pro-
Results and Discussion
Synthesis
As shown in Scheme 1, 4-methyl-1,2,3-thiadia-
zole-5-carboxylic acid chloride was prepared from 4-
methyl-1,2,3-thiadiazole-5-carboxylic acid. Then 4-
methyl-1,2,3-thiadiazole-5-carboxylic acid chloride was
reacted with ammonia to provide 4-methyl-1,2,3-
thiadiazole-5-carboxamide. In the next step, 4-methyl-
1,2,3-thiadiazole-5-carbonyl isocyanate was synthesized
by the reaction of 4-methyl-1,2,3-thiadia-zole-5-car-
boxamide and oxalyl chloride with good yield. Finally,
the target compounds V-1—V-29 were produced by the
combination of 4-methyl-1,2,3-thiadiazole 5-carbonyl
isocyanate and various kinds of aromatic amine succes-
sively. Our group found some interesting phenomenon
during these processes. First, controlling of temperature
of oxalyl chloride and 4-methyl-1,2,3-thiadiazole-
5-carboxamide during the refluxing was critical, when
the temperature exceeded 120 ℃, 4-methyl-1,2,3-thia-
－
1
teins and was demonstrated as U•[(mg of protein) •
－
1
min ]. Protein had been measured by the method of
Coomassie blue according to the description of Brad-
ford et al.[18]
3D-QSAR models
In this study, a 3D-QSAR analysis was performed by
using CoMFA method within SYBYL 7.0 (Tripos, St.
Louis, MO, USA) molecular modeling software. Total
17 compounds including V-2, V-4, V-5, V-6, V-7, V-8,
V-10, V-13, V-14, V-16, V-17, V-18, V-20, V-23, V-27,
V-28 and V-29 were chosen for CoMFA analysis, which
showed insecticidal activities against Culex pipiens
pallens and their activities were within 100%. The ac-
tivity data were transformed and expressed in terms of A
by the formula A＝lg{a/[100－a]Mw}, where a is the
percentage of inhibition and Mw is the molecular weight
Table 1 Activities [A] of molecules used for 3D-QSAR models
lg A
lg A
Pre CoMFA
－2.41
－2.31
－1.99
－3.09
－3.49
－3.05
－3.33
－3.11
No V-no
Activity/%
No V-no
Activity/%
Exp
Pre CoMFA
－2.86
－2.70
－2.85
－2.65
－2.91
－3.15
－2.39
－2.75
－3.37
Res
Exp
Res
0.12
1
2
3
4
5
6
7
8
9
V-2
V-4
30
40
30
40
30
20
60
40
10
－2.86
－2.72
－2.84
－2.65
－2.84
－3.12
－2.34
－2.71
－3.40
0.00
10 V-16
60
60
70
20
10
20
10
20
－2.29
－2.32
－2.12
－3.10
－3.49
－3.07
－3.40
－3.14
－0.02 11 V-17
0.01
V-5
0.01
0.00
12 V-18
13 V-20
14 V-23
15 V-27
16 V-28
17 V-29
－0.13
0.01
V-6
V-7
0.07
0.00
V-8
0.03
－0.02
－0.07
－003
V-10
V-13
V-14
0.05
0.04
－0.02
Chin. J. Chem. 2012, 30, 2522—2532
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2527

Guo et al.
FULL PAPER
diazole-5-carbonyl isocyanate could be formed in much
lower yield. Because the carbonyl isocyanate is unstable,
it was important to control the temperature of the reac-
tion and use fresh material.[21] Second, we observed that
the reaction occurred as expected even when the aro-
matic ring contained a hydroxyl group, as in V-15, V-20,
V-28 and V-29. Third, it was found that the reaction
would not proceed at or below room temperature when
the aromatic ring contained a nitro group, as in V-1 to
V-4, but proceeded to completion by refluxing in
1,2-dicholroethane.
were chosen for structure-activity relationship studies.
The results of insecticidal activities of target compounds
against C. pipiens pallens are listed in Tables 2 and 3.
Preliminary screenings were tested at the concentration
of 5 μg•mL－1 for insecticidal activity against C. pipiens
pallens. As the results show, the compounds V-9, V-11,
V-12, V-15, V-19, V-21, V-22 and V-24 exhibited ex-
cellent activity, which suggests that introducing halogen
atoms and CF3, OCF2CHFCF3, OCF2CHF2 groups into
the target molecules had improved the insecticidal ac-
tivity against C. pipiens pallens. At a lower test concen-
tration, the mortality of C. pipiens pallens also de-
creased accordingly, with the best active compound,
Biological activity
－
1
Insecticidal Activity against Culex pipiens pallens
Different types of substitution in aromatic amine moiety
V-24, giving 50% control at 0.1 μg•mL . None of the
test compounds reached the activity of the commercial
Table 2 Insecticidal activities of the compounds V-1—V-29
Plutella xylostella L.
R
Concentration/
Culex pipiens pallens
Compd.
Concentration/
Insecticidal activity/%
Insecticidal activity/%
(μg•mL－
200
200
400
200
400
400
400
200
200
400
200
400
200
400
400
200
200
200
400
400
400
400
400
)
(μg•mL－
)
1
1
V-1
V-2
4-nitro
0
0
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
0
2-nitro
30
V-3
3-nitro
10.3
0
40
V-4
2,4-dinitro
2-chloro
40
V-5
5.0
7.7
38.5
0
30
V-6
4-chloro
40
V-7
3-chloro
30
V-8
2,5-dichloro
3,5-dichloro
3,4-dichloro
2,4,5-trichloro
20
V-9
5.0
7.7
3.5
97.5
0
100
60
V-10
V-11
V-12
V-13
V-14
V-15
V-16
V-17
V-18
V-19
V-20
V-21
V-22
V-23
100
100
40
2,4-difluoro-3,5-dichloro
4-bromo
2-fluoro
2.6
5.0
5.0
2.5
0
10
2-fluoro -4-hydroxyl
3-fluoro -4-methyl
2-methyl-4-chloro
3-chloro-4-methyl
2-trifluoromethyl-5-chloro
2-carboxy
100
60
60
70
10.3
5.0
27.5
100
59.4
100
20
4-trifluoromethyl
2-chloro-3,5-ditrifluoromethyl
100
100
10
4-trifluoromethoxyl
2,5-dichloro-4-(1,1,2,3,3,3-hexa-
fluoropropoxyl)
V-24
400
75
5
100
V-25
V-26
V-27
V-28
V-29
2,4-dimethyl
400
400
400
400
10.3
17.9
12.8
15
5
5
5
5
5
20
20
20
10
20
4-ethyl
4-formyl
2-hydroxyl
3,5-dichloro-4-hydroxyl
400
200
400
19.0
72.4
100
Positive control Chlorfluazuron
5
100
2528
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 2522—2532

Synthesis, Bioactivities and Structure Activity Relationship
Table 3 Insecticidal active against Culex pipiens pallens at
V-12 and V-22 were shown in Table 4 with the LC50
value of 164.15 and 89.69 μg•mL － 1, respectively.
Therefore, substitutes on the phenyl ring played a vital
role of the insecticidal activity of the target compounds
against P. xylostella.
lower concentration of compounds V-9, V-11, V-12, V-15, V-19,
V-22 and V-24 (Those compounds exhibit 100% at 5 μg•mL－
)
1
Compd.
V-9
Concentration/(µg•mL－
)
Death rate/%
1
2
2
1
40
100
40
V-11
Table 4 Precision toxicological determination of compounds
V-12 and V-22 against Plutella xylostella L. (96 h, %)
V-12
2
1
100
40
LC50 (95% confidence
Compd.
Slope±SD Chi-square
limits)/(µg•mL－
)
1
V-15
V-19
2
2
60
50
164.15 (127.10—211.99) 2.94±0.48
89.69 (52.50—153.24) 3.95±0.86
7.23
0.16
0.19
V-12
V-22
V-22
1
100
20
Chlorfluazuron
1.38 (0.14—13.43)
0.76±0.24
0.5
V-24
0.25
0.1
100
50
Fungicide activity
Though the main structure of target compounds was
the derivatives of a insecticide lead, derivatives of N-4-
methyl-1,2,3-thiadiazole-5-carbonyl-N'-aromatic ureas
also showed excellent fungicide activity. The fungicide
activities of the title compounds against the representa-
tive typical fungi often occurring in the Chinese
agro-ecosystem such as AS, BC, CA, GZ, PI, PP, PS,
Chlorfluazuron
2
1
100
100
100
100
100
100
100
0.5
0.25
0.1
0.05
0.025
SS and RC detected at 50 μg•mL－ were shown in Ta-
1
ble 5. The results indicated that some compounds had
good fungicide activity, compounds V-2, V-16 inhibited
more than 60% of the growth against AS; compounds
V-1, V-2, V-9, V-11, V-13, V-16, V-22 and V-25 inhib-
ited more than 50% of the growth to CA; compounds
V-9, V-11, V-13, V-16 and V-22 inhibited more than
50% of the growth against GZ; compounds V-2, V-11,
V-13, V-14, V-22 and V-25 inhibited more than 50% of
the growth against PP; compounds V-2, V-4, V-11,
V-16, V-20, V-23, V-25, and V-28 inhibited more than
50% of the growth against BC; compounds V-7, V-11,
V-13, V-14, V-17, V-19, V-20, V-22, V-24, V-27 and
V-28 inhibited more than 50% of the growth against RC;
compounds V-1, V-11 and V-20 inhibited more than
50% of the growth against PS; compounds V-9, V-11,
V-13, V-25, V-28 and V-29 inhibited more than 70% of
the growth against SS; compounds V-13 inhibited more
than 80% of the growth against PI. Compound V-11
was a broad-spectrum of fungicide activity against all of
the fungi tested with growth inhibition from 50% to
100%. Precision toxicity of compound V-11 was studied
further, the EC50 values were detected as from 3.82
standard, chlorfluazuron, which gave 100% control at
0.025 μg•mL－1 (Table 3).
Insecticidal activity against Plutella xylostella
The results of insecticidal against larval P. xylostella
were listed in Table 2. We have tested some of these
compounds at 200 μg•mL－1 at first, however, the insec-
ticidal activities against Plutella xylostella of these
compounds are unfavorable and hard to compare with
each other. Thereafter, we tested the rest of the com-
－
1
pounds at 400 μg•mL . On the basis of these results, to
examine the electronic effect of substitute on the phenyl
ring, the electron-donating substitutes CH3, C2H5 and
electron-withdrawing substitutes Cl, F, Br, CF3, CHO,
OCF3, NO2 were introduced into the target compounds.
The bioassay results demonstrated that, compounds with
CF3, OCF3 presented optimum insecticidal activity
against P. xylostella, while the rest of compounds with
electron-withdrawing substitutes showed lower insecti-
cidal activity than those with electron-donating substi-
tutes. Further results of compounds V-1, V-2, V-3 and
V-5, V-6, V-7 indicated that, the introduction of Cl and
NO2 into meta-position of the aromatic ring had ac-
ceptable biological activity, however, para-substituted
and ortho-substituted derivatives showed no insecticidal
activity. Moreover, as can be seen from Scheme 1 and
Table 2, V-12, V-22 and V-24 had more than 2 sub-
stitutes in the phenyl ring; they were the most active
compounds with insecticidal activity over 75% against
μg•mL－ against Physalospora piricola to 31.60 μg•
1
mL－1 against Cercospora arachidicola, and these were
equal to 14.31 μmol•L－ against P. piricola to 86.43
1
μmol•L－1 against C. arachidicola among the four tested
fungi (Table 6). These results indicated that compound
V-11 was a fungicide lead with good selectivity and
deserved for further modification. Halogen-substitution
in aromatic derivatives improved the fungicide activity
of the title compounds.
－
1
P. xylostella at 400 μg•mL . Further insecticidal preci-
sion toxicological determination results of compounds
Chin. J. Chem. 2012, 30, 2522—2532
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2529

Guo et al.
FULL PAPER
Table 5 The antifungal activities of compounds V-1—V-29 in
nanmycin, among the compounds with best insecticide
activity, compound V-24 had good half leaf activity as
that of ningnanmycin did; the five compounds had
lower protective activity, curative activity and inactiva-
tion activity, but three compounds including V-15 and
V-24 had good induction activity as that of both TDL
and ningnanmycin did, compound V-24 had highest
induction activity of 41.85%±4.43%. All these were
validated by PAL activity determination, all these three
compounds with good induction acidity improved their
PAL activity as compared with TDL did. These results
suggested that N-4-methyl-1,2,3-thiadiazole-5-carbonyl-
N'-aromatic ureas possessed good insecticide activity
and also showed good systemic acquired resistance, it is
valuable for both disease control caused by fungi or vi-
rus and insects damage which can also spread the fungi
invasion and virus attacking.
vitro at 50 μg•mL－
1 a
Compd.
V-1
AS CA GZ PP BC RC PS SS PI
33 77 26 42 27 37 61 32 14
V-2
61 62 33 54 65 48 48
0
0
14
28
V-3
0
14
0
0
0
9
0
32 10 37
56 11
32 10 27
0
0
3
V-4
10 38
0
14 16
24
V-5
0
0
0
7
0
V-6
14
21
29 29 45 44 29 16
11 19 53 50 12
V-7
0
V-8
28 31 13
39 62 54
8
4
23 31 21
0
14
0
V-9
15 21 33 100
V-10
V-11
V-12
V-13
V-14
V-15
V-16
V-17
V-18
V-19
V-20
V-21
V-22
V-23
V-24
V-25
V-26
V-27
V-28
15 26 13 35 23 45
50 77 72 83 100 96 90 100
49 23 13 10 24 33
0
3
4
9
2
60 18
3D-QSAR model analysis
33 54 97 100 23 94 30 100 82
23 31 27 50 47 57 39 50 14
To obtain more information about relationship be-
tween structure and insecticidal activities against Culex
pipiens pallens, we need to choose some compounds to
do 3D-QSAR analysis. Because these 17 compounds
(V-2, V-4, V-5, V-6, V-7, V-8, V-10, V-13, V-14, V-16,
V-17, V-18, V-20, V-23, V-27, V-28, V-29) present a
good statistical correlation of their data of predicted
activities (red line) and experimental activities (black
dots) in Figure 1, we choose them. Under Tripos force
field, the best CoMFA model was achieved with opti-
mal components 3, which showed a good q2 (cross-
validate r2)＝0.643, r2＝0.982 and F (variance ratio)＝
71.771. The proportion of steric and electrostatic con-
tribution was 60.8% and 39.2% respectively. Further-
more, the CoMFA steric and electrostatic fields were
plotted as 3D colored contour maps, which were helpful
to identify important regions changing in steric and
electrostatic fields. In order to explain the field contri-
butions of different properties, N-4-methyl-1,2,3-
thiadiazole-5-carbonyl-N′-[3,5-dichloro-4-(1,1,2,2-
tetrafluoroethoxyl)phenyl] urea whose value of insecti-
cidal activity against Culex pipiens pallens is 100%, and
crystal structure (CCDC 888301) has been obtained dis-
plays as a template molecule. The steric and electro-
static contour map from CoMFA analysis was presented
in Figure 2. The yellow regions indicated that increasing
the steric bulk would decrease the activity. The green
regions illustrated that increasing the steric bulk might
enhance the activity. Similarly, the red regions indicated
where increasing electronegative substitutions could
enhance the activity, while the blue regions showed
where increasing electropositive substitutions might
enhance the activity.
8
14
9
8
16 25
4
57
3
9
7
67 62 54 42 58 46 34 32
20 12 38 34 33 70 27 34
10 44
26
17 17 22 31 50 65 67 57 14
17 27 41 27 47 21
17 50 68 67 29 76 43 57 11
0
0
22
0
0
14 13
7
16 29 41 50 23 30 17
6
6
8
0
7
0
45
5
33 28
0
16
7
10 26 19 30 42 50
5
10
5
0
57
14
0
0
0
6
3
77 59 49 14 73 24
29 37 12 16
0
0
10 16
35 37
10 26
42 50 55 33 35 15
45 54 55 11 71 11
V-29
45 38 40
5
81 22
Chlorfluazuron
(Negative control)
Azoxystrobin
5
7
0
4
11 22
3
14
8
60 78 86 100 100 92 77 95 90
(Positive control)
a AS, Alternaria solani; BC, Botrytis cinerea; CA, Cercospora
arachidicola; GZ, Gibberellazeae; PI, Phytophthora infestans
(Mont) de Bary; PP, Physalospora piricola; PS, Pellicularia sa-
sakii (Shirai) ; SS, Sclerotinia sclerotiorum; RC, Rhizoctonia
cerealis.
Systemic acquired resistance for tobacco against to-
bacco mosaic virus
The results of anti-TMV activity determination were
listed in Table 7. Bioassay indicated that ningnanmycin
presented good anti-TMV activity including all test
modes, TDL only had good inactivation and induction
activities, these results accorded with the reported re-
sults.[10,22] While, the negative control chlorfluazuron
only had good inactivation activity and certain degree of
protective activity which was lower than that of ning-
Figure 2 depicted a green region on the side of ben-
zene ring, indicating that a larger bulky group in this
region favored the activity of the compound. In contrast,
a smaller steric group in the lower left yellow region
might decrease activity of the compound. A blue region
and a red region were shown on the two sides of ben-
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 2522—2532

Synthesis, Bioactivities and Structure Activity Relationship
Table 6 EC50 determination of compound V-11 against four fungi
1
1
Fungus
Regression equation
y＝0.7548x＋3.8680
y＝1.2042x＋4.2986
y＝1.8112x＋3.1698
y＝2.2147x＋1.9125
R2
EC50/(μg•mL－
)
EC50/(μg•mL－
86.43
)
Cercospora arachidicola
Physalospora piricola
Rhizoctonia cerealis
Pellicularia sasakii
0.9496
0.9374
0.9829
0.9481
31.60
3.82
14.31
10.24
27.90
24.78
67.83
Table 7 Activity against TMV of the target compoundsa
PAL specific
Activity/
Conc./
Half leaf±SD/ Protection±SD/ Curative±SD/ Inactivation±SD/ Induction±SD/
Compd.
(μg•mL－
－
1
%
%
%
%
%
U•[(mg of protein) •
1
)
min－
2.05
1.72
1.89
2.73
1.10
2.21
ND
]
1
V-15
V-22
100
100
100
100
100
100
100
—
24.23±3.89
20.87±2.33
26.19±2.06
32.95±3.61
15.15±2.63
15.95±2.01
33.50±4.08
—
16.23±4.23
28.07±2.01
14.91±5.47
26.75±4.02
38.16±2.63
14.04±3.31
48.41±3.77
—
27.11±3.36
24.45±2.04
10.22±2.77
20.00±2.67
11.11±2.04
17.78±2.04
46.99±2.11
—
21.09±2.57
24.15±3.28
27.55±2.70
24.83±3.28
40.48±3.59
45.24±4.82
41.32±1.94
—
32.62±3.25
19.86±3.25
21.99±3.25
41.85±4.43
23.40±2.13
38.30±2.13
39.80±7.42
—
V-23
V-24
Chlorfluazuron
TDL
Ningnanmycin
CK
1.95
a ND: not detected; positive control: TDL and Ningnanmycin; negative control: Chlorfluazuron
Figure 1 Experimental versus predicted [A] for CoMFA model.
zene ring, respectively. The red region of benzene indi-
cated increasing the electronegative substitutions might
promote the activity. For example, compounds V-16
and V-18, containing 3-F and 3-Cl substitution respec-
tively, presented an increasing activity in turn. As a re-
sult, we designed compounds V-19, V-22 and V-24. We
use Cl, CF3 etc. to increase the electronegativity of the
benzene red region, and use bigger substitutions in the
green region, like V-24. As can be seen in Table 2, these
three compounds exhibit excellent insecticidal activity
Figure 2 (a) Alignment of the 17 compounds; (b) contour maps
for CoMFA model in steric and electrostatic fields. The sterically
favored areas are in green; sterically unflavored areas are in yel-
low. Positive-charge favored areas are in blue; negative-charge
favored areas are in red.
against Culex pipiens pallens as we predicted based on
CoMFA analysis.
Conclusions
In summary, 29 novel N-4-methyl-1,2,3-thiadiazole-
5-carbonyl-N'-phenyl ureas were designed and synthe-
sized. Target compounds exhibit insecticidal activities
against C. pipiens pallens and P. xylostella L. Bioassays
indicated compounds V-24 and V-22 exhibit out-
Chin. J. Chem. 2012, 30, 2522—2532
© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
2531

Guo et al.
FULL PAPER
Zurcher, E. Viruses of Plants, CAB International, Cambridge,
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standing insecticidal activity against Culex pipiens
pallens and Plutella xylostella, respectively. Compound
V-11 also had a broad-spectrum of fungicide against all
of the fungi. Five compounds show good SAR-inducing
activity as well. These compounds establish basis for
both disease and insect control in agricultural practice
and novel pesticide development. Structure-activity re-
lationship studies reveal that, improvement of insecti-
cidal activity requires a reasonable combination of sub-
stituent on the phenyl ring, and the type and position of
substituent on the phenyl ring are critical. With the
contour maps based on steric and electrostatic CoMFA
coefficients, we can conclude the relationship between
the structures and bioactivity and continue the further
design of highly active compounds.
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Abrreviations Used
DMF: N,N-dimethylformamide; DMSO-d6: deutero-
dimethyl sulfoxide; EC50: median effective concentra-
tions; HIV: Human Immunodeficiency Virus; 1H NMR:
Hydrogen Nuclear Magnetic Resonance; HRMS:
High-Resolution Mass Spectrometry; IR: infra red spec-
troscopy; LC50: median lethal concentrations; PAL:
phenylalanine ammonialyase; PXL: Plutella xylostella
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Research (No. 2010CB126105), the National Key
Technology Research and Development Program (No.
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© 2012 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chin. J. Chem. 2012, 30, 2522—2532