Design, synthesis, and biological activities of arylmethylamine substituted chlorotriazine and methylthiotriazine compounds

Article abstract of DOI:10.1021/jf203383s

Heterocyclic rings were introduced into the core structure of s-triazine to design and synthesize a series of novel triazines containing arylmethylamino moieties. These compounds were characterized by using spectroscopic methods and elemental analysis. Their herbicidal, insecticidal, fungicidal, and antitumor activities were evaluated. Most of these compounds exhibited good herbicidal activity, especially against the dicotyledonous weeds, and compound F8 was almost at the same level as the control compound atrazine. Their structure-activity relationships were discussed. At the same time, some triazines had interesting fungicidal and insecticidal activities, of which F4 exhibited 100% efficacy against Puccinia triticina even at 20 ppm, and F5 showed Lepidopteran-specific activity in both leaf-piece and artificial diet assays. Moreover, these compounds showed antitumor activities against leukemia HL-60 cell line and lung adenocarcinoma A-549 cell line.

Full text of DOI:10.1021/jf203383s

ARTICLE
pubs.acs.org/JAFC
Design, Synthesis, and Biological Activities of Arylmethylamine
Substituted Chlorotriazine and Methylthiotriazine Compounds
Huaping Zhao,† Yuxiu Liu,*,† Zhipeng Cui,† David Beattie,‡ Yucheng Gu,*,‡ and Qingmin Wang*,†
†State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University,
Tianjin 300071, People’s Republic of China
‡Syngenta Jealott’s Hill International Research Centre, Bracknell, Berks, RG42 6EY, U.K.
ABSTRACT: Heterocyclic rings were introduced into the core structure of s-triazine to design and synthesize a series of novel
triazines containing arylmethylamino moieties. These compounds were characterized by using spectroscopic methods and
elemental analysis. Their herbicidal, insecticidal, fungicidal, and antitumor activities were evaluated. Most of these compounds
exhibited good herbicidal activity, especially against the dicotyledonous weeds, and compound F8 was almost at the same level as the
control compound atrazine. Their structureꢀactivity relationships were discussed. At the same time, some triazines had interesting
fungicidal and insecticidal activities, of which F4 exhibited 100% efficacy against Puccinia triticina even at 20 ppm, and F5 showed
Lepidopteran-specific activity in both leaf-piece and artificial diet assays. Moreover, these compounds showed antitumor activities
against leukemia HL-60 cell line and lung adenocarcinoma A-549 cell line.
KEYWORDS: s-triazine, heterocyclic ring, herbicidal activity, insecticidal activity, fungicidal activity, antitumor activity, structure-
ꢀactivity relationships
’ INTRODUCTION
substituted cyanoacrylates and benzylaminotriazines have com-
mon benzylamino moieties which were assumed to bind to the
very same site.5,6 In our previous research on cyanoacrylates,
when benzylamino group was alternated by different heterocyclic
methylamino groups, their herbicidal activity changed to a large
extent, and some of them exhibited enhanced herbicidal activity.7
We wonder if a similar effect could be found in the triazine
system. Literature review suggested that substituted benzylami-
notriazines or heterocyclic methylamino substituted triazines
had not been thoroughly investigated. Thus, some representative
arylmethylamino-containing chlorotriazines and methylthiotria-
zines were synthesized to investigate the influence of heterocyclic
methylamino moieties of triazines on their herbicidal activity
(Figure 1).
Many herbicidal triazines are also reported to have antifungal
activity.8 In recent years, versatile bioactivities of s-triazine deriva-
tives have been widely reported and aroused the study enthu-
siasm ofscientistsfromallovertheworld.9 Therefore, the synthesized
triazine compounds were also investigated for their potential as
fungicidal, insecticidal, or antitumor agents.
s-Triazines are a group of herbicides, with atrazine registered
in 1958 as the biggest product of its class. They can displace
plastoquinone from the QB binding niche of the D-1 protein of
photosystem II in the photosynthetic electron transport chain of
plants, therefore inhibiting electron transport in the photosynth-
esis of the plant and resulting in the wilt of leaves and then the
death of the plant.1 Due to their excellent herbicidal activities,
tens of s-triazine herbicides were commercialized, and many of
them are still in use. The simple alkyl-substituted s-triazines
atrazine and ametryne are such representative pesticides. Nowa-
days, new s-triazines compounds are still in progress. In the late
1990s, triaziflam, a diaminotriazine herbicide bearing a dimethyl-
phenoxyethyl group at one of the nitrogen atoms, was developed
by a Japanese company, Idemitsu Kosan.2 More recently, indazi-
flam, with a dihydroindene moiety, was developed by Bayer in
2008 and will be launched to the market in the next few months.3
Herein we report the synthesis of arylmethylamino-containing
chlorotriazines and methylthiotriazines (F) and their biological
activities.
Benzylaminotriazines and (α-substituted)benzylaminotriazines
had also been synthesized and evaluated as herbicide in 1990s.4
Some of these compounds exhibited potent phytotoxicity against
weeds and high selectivity between rice and weeds, but had
different phytotoxic properties to atrazine. However, none of the
benzylaminotriazines had been commercialized due to the high
dosage required to establish control in the field.
’ MATERIALS AND METHODS
Instruments. 1H nuclear magnetic resonance (NMR) spectra were
obtained at 400 MHz using a Bruker AV400 spectrometer in CDCl3 or
Received: June 24, 2011
Accepted: October 4, 2011
As PSII electron transport inhibitor and therefore binding
with the same binding niche of the D1 protein, benzylamino-
Revised:
September 28, 2011
Published: October 04, 2011
r
2011 American Chemical Society
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Figure 1. Design of arylmethylamino-containing chlorotriazines and methylthiotriazines.
mp 179ꢀ180 °C [lit. 172.5ꢀ174 °C11]. 1H NMR (400 M, CDCl3) δ:
1.15ꢀ1.26 (m, 9H), 3.30ꢀ3.49 (m, 2H), 3.99ꢀ4.28 (m, 1H), 5.03ꢀ5.20
(m, 1H), 5.32ꢀ5.61 (m, 1H).
Scheme 1
Synthesis of 2-Chloro-4-isopropylamino-6-(p-fluorobenzylamino)-
1,3,5-triazine (F2). To a solution of 1 (2.07 g, 10 mmol) in toluene
(3 mL) was added a solution of sodium hydroxide (0.40 g, 10 mmol) in
water (2 mL). Then, the mixture was held constant at 15 °C, and a
solution of p-fluorobenzyl amine (0.56 g, 10 mmol) in toluene (3 mL)
was added slowly. After addition was completed, the mixture was stirred
at room temperature for 5 h. Then, toluene was removed under reduced
pressure, and water (50 mL) was added. The solid was filtered, washed
with water abundantly, and recrystallized from methanol to afford F2
1
as a white solid (2.27 g, 76.8%); mp 135ꢀ137 °C. H NMR (400 M,
CDCl3) δ: 1.12ꢀ1.25 (m, 6H), 4.02ꢀ4.24 (m, 1H), 4.50ꢀ4.60 (m, 2H),
5.08ꢀ5.33 (m, 1H), 5.43ꢀ6.67 (m, 1H), 6.97ꢀ7.06 (m, 2H), 7.22ꢀ7.32
(m, 2H). Anal. Calcd for C13H15ClFN5: C, 52.80; H, 5.11; N, 23.68.
Found: C, 52.69; H, 4.97; N, 23.50.
d6-DMSO solution with tetramethylsilane as the internal standard.
Chemical-shift values (δ) are given in parts per million (ppm). Elemental
analyses were determined on a Yanaca CHN Corder MT-3 elemental
analyzer. The melting points were determined on an X-4 binocular
microscope melting point apparatus (Beijing Tech Instruments Co.,
Beijing, China) and are uncorrected. Yields were not optimized.
General Synthesis. The reagents were of analytical grade or chemi-
cally pure. All anhydrous solvents were dried and purified by standard
techniques prior to use.
Synthetic Procedure for the Target Compounds F1ꢀF11
(Schemes 1 and 2). Synthesis of 2,4-Dichloro-6-isopropylamino-
1,3,5-triazine (1). A solution of cyanuric chloride (11.06 g, 60 mmol) in
tetrahydrofuran (60 mL) was cooled to ꢀ10 °C by an iceꢀsalt bath, and
then isopropylamine (7.08 g, 120 mmol) in tetrahydrofuran (20 mL)
was added slowly and the mixture was stirred at room temperature for 30
min. Then, the mixture was concentrated under reduced pressure,
followed by addition of water (70 mL). The aqueous phase was extracted
by ethyl acetate twice (70 mL ꢁ 2). The combined organic solution was
washed with saturated brine (70 mL), then dried over anhydrous magne-
sium sulfate, and filtered. The filtrate was concentrated under reduced
pressure. The residue was purified by flash-column chromatography on
silica gel using a mixture of petroleum ether and ethyl acetate (10:1) as
the eluent to afford 1 as a white solid (12.09 g, 97.4%); mp 42ꢀ44 °C
[lit. 45 °C10]. 1H NMR (400 M, CDCl3) δ: 1.25 (d, 3JHH = 6.4 Hz, 6H),
4.16ꢀ4.28 (m, 1H), 5.70 (s, 1H).
The target compounds F3ꢀF6 were prepared by following the same
procedure as for compound F2. The corresponding starting materials
2-tetrahydrofuranmethylamine, 2-chloro-5-pyridylmethylamine,7c 3-phen-
yl-1,2-oxazole-5-methylamine,7g and2-bromo-5-thiazolylmethylamine7b were
commercial available or prepared according to our previous paper.
2-Chloro-4-isopropylamino-6-(2-tetrahydrofuranmethylamino)-
1,3,5-triazine (F3): yield, 87.8%; an oil.8b 1H NMR (400 M, CDCl3) δ:
1.15ꢀ1.24 (m, 6H), 1.53ꢀ1.67 (m, 1H), 1.86ꢀ2.03 (m, 3H), 3.29ꢀ3.45
(m, 1H), 3.48ꢀ3.61 (m, 1H), 3.68ꢀ3.81 (m, 1H), 3.84ꢀ3.93 (m, 1H),
3.97ꢀ4.08 (m, 1H), 4.10ꢀ4.27 (m, 1H), 5.04ꢀ5.53 (m, 1H), 5.59ꢀ6.21
(m, 1H).
2-Chloro-4-isopropylamino-6-(2-chloro-5-pyridylmethylamino)-
1
1,3,5-triazine (F4): yield, 76.6%; a white solid; mp 137ꢀ138 °C. H
NMR (400 M, CDCl3) δ: 1.13ꢀ1.23 (m, 6H), 3.90ꢀ4.26 (m, 1H),
4.54ꢀ4.65 (m, 2H), 5.06ꢀ5.42 (m, 1H), 5.55ꢀ5.75 (m, 1H), 7.26ꢀ7.34
(m, 1H), 7.58ꢀ7.69 (m, 1H), 8.36ꢀ8.40 (m, 1H). Anal. Calcd for
C12H14Cl2N6: C, 46.02; H, 4.51; N, 26.83. Found: C, 45.88; H, 4.43;
N, 26.70.
2-Chloro-4-isopropylamino-6-(3-phenyl-1,2-oxazole-5-methylamino)-
1
1,3,5-triazine (F5): yield, 80.8%; a white solid; mp 173ꢀ174 °C. H
NMR (400 M, CDCl3) δ: 1.15ꢀ1.23 (m, 6H), 4.05ꢀ4.25 (m, 1H),
4.71ꢀ4.82 (m, 2H), 5.15ꢀ5.42 (m, 1H), 5.64ꢀ7.26 (m, 1H), 6.45ꢀ6.55
(m, 1H), 7.40ꢀ7.48 (m, 3H), 7.73ꢀ7.79 (m, 2H). Anal. Calcd for
C16H17ClN6O: C, 55.73; H, 4.97; N, 24.37. Found: C, 55.65; H, 4.90; N,
24.16. MS (ESI) for C16H18ClN6O (M+1): 345.
Synthesis of Atrazine (F1). To a solution of 1 (1.92 g, 9.3 mmol) in
toluene (4.96 g, 53.9 mmol) was added a solution of sodium hydroxide
(0.37 g, 9.3 mmol) in water (1.5 mL). Then, the mixture was maintained
at 15 °C, and ethylamine (0.42 g, 9.3 mmol) was added slowly. After the
completion of addition, the mixture was stirred at room temperature for
15 min. Then, toluene was removed under reduced pressure, and water
(40 mL) was added. The mixture was filtered to afford F1 as a white solid
(1.67 g, 83.5%). Further recrystallization from a mixture of petroleum
ether (60ꢀ90 °C) and dichloromethane afforded a white solid;
2-Chloro-4-isopropylamino-6-(2-bromo-5-thiazolylmethylamino)-
1
1,3,5-triazine (F6): yield, 66.9%; a white solid; mp 201ꢀ202 °C. H
NMR (400 M, CDCl3) δ: 1.06ꢀ1.15 (m, 6H), 3.91ꢀ4.18 (m, 1H),
4.49ꢀ4.58 (m, 2H), 7.55ꢀ7.61 (m, 1H), 7.66ꢀ8.00 (m, 1H), 8.06ꢀ8.44
(m, 1H). Anal. Calcd for C10H12BrClN6S: C, 33.03; H, 3.33; N, 23.11.
Found: C, 32.87; H, 3.29; N, 22.91.
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Scheme 2
Synthesis of Ametryne (F7). To a solution of F1 (0.68 g, 3 mmol) in
tetrahydrofuran (12mL) wasaddedasolutionof sodiummethylmercaptan
(2.10 g, 30 mmol) in water (8.5 mL). Then, the mixture was refluxed for
24 h, and tetrahydrofuran was removed under reduced pressure. The
residue was added water (50 mL) and extracted by dichloromethane
(50 mL ꢁ 4). The combined organic extract was washed with saturated
brine (50 mL), then dried over anhydrous magnesium sulfate, and
filtered. The filtrate was concentrated under reduced pressure. The
residue was purified by recrystallization from a mixture of methanol and
water to afford F7 as a white solid (0.45 g, 65.3%); mp 84ꢀ86 °C [lit.
86ꢀ88 °C12].
Table 1. Herbicidal Activities of Compounds F1ꢀF11 (1.5
kg/ha, Percent Inhibition, %)
preemergence treatment
postemergence treatment
rape
barnyard grass
rape
barnyard grass
F1 (atrazine)
100
100
100
100
20.1
5.2
100
2.1
100
100
100
100
42.4
10.0
100
100
100
100
100
100
F2
F3
F4
F5
F6
75.7
41.3
68.6
17.7
10.4
100
46.4
68.9
4.3
The target compounds F8ꢀF11 were prepared by following the same
procedure as for compound F7.
11.3
100
21.4
88.6
37.6
74.6
2-Isopropylamino-4-(p-fluorobenzylamino)-6-methylthio-1,3,5-tria-
zine (F8): yield, 66.5%; a white solid; mp 105ꢀ106 °C. 1H NMR (400
M, CDCl3) δ: 1.13ꢀ1.24 (m, 6H), 2.39ꢀ2.48 (m, 3H), 4.00ꢀ4.28
(m, 1H), 4.46ꢀ4.63 (s, 1H), 4.70ꢀ5.15 (m, 1H), 5.20ꢀ5.68 (m, 1H),
6.96ꢀ7.04 (m, 2H), 7.21ꢀ7.32 (m, 2H). Anal. Calcd for C14H18FN5S: C,
54.70; H, 5.90; N, 22.78. Found: C, 54.51; H, 5.82; N, 22.72.
2-Isopropylamino-4-(2-chloro-5-pyridylmethylamino)-6-methylthio-
F7 (ametryne) 100
F8
100
100
33.1
87.1
100
F9
88.6
14.3
92.0
F10
F11
1
1,3,5-triazine (F9): yield, 68.2%; a white solid; mp 163ꢀ164 °C. H
750 L/ha spray volume. The dosage (activity ingredient) for each com-
pound corresponded to 1.5 kg/ha. Compounds were sprayed immediately
after seed planting (preemergence treatment) or after the expansion of the
first true leaf (postemergence treatment). The mixture of same amount of
water, N,N-dimethylformamide, and Tween 20 was sprayed as the control.
Each treatment was triplicated. The fresh weight of the above ground tissues
was measured 10 days after treatment. The inhibition percent was used to
describe the control efficiency of the compounds. The activity numbers in
Table 1 represent the percent displaying herbicidal damage as compared to
the control, where complete control of the target is 100 and no control is 0.
Screening Conducted at Syngenta. The compounds were screened
for herbicidal, insecticidal, and fungicidal activity in a variety of laboratory
and glasshouse-based assays.
Glasshouse Herbicide Screening. The glasshouse herbicidal screen-
ing performed by Syngenta followed similar methods to those at Nankai
University, using the dicotyledonous weed species Amaranthus retro-
flexus and Stellaria media and the monocotyledonous species Lolium
perenne and Digitaria sanguinalis as plant material. The compounds were
formulated and applied at 1000 g/ha to preemergent and postemergent
plants, which were then grown in the glasshouse for 12 days. Assess-
ments were made of % herbicidal effects, and test samples were given a
score between 0 and 100, with 100 as complete control of the target and
0 as no effect. Glyphosate was included in the test as a positive control
compound. The results of these tests are presented in Table 2.
96-Well Plate Herbicide Screening. Compounds F2ꢀF11 and atra-
zine were tested for herbicidal activity against Arabidopsis thaliana at 10
ppm and Poa annua at 32 ppm, with two replicates of each treatment.
NMR (400 M, CDCl3) δ: 1.06ꢀ1.28 (s, 6H), 2.37ꢀ2.46 (m, 3H),
3.95ꢀ4.25 (m, 1H), 4.50ꢀ4.60 (m, 2H), 4.70ꢀ5.10 (m, 1H), 5.45ꢀ6.79
(m, 1H), 7.23ꢀ7.29 (m, 1H), 7.57ꢀ7.67 (m, 1H), 8.36 (s, 1H). Anal.
Calcd for C13H17ClN6S: C, 48.07; H, 5.28; N, 25.87. Found: C, 48.12;
H, 5.21; N, 25.74.
2-Isopropylamino-4-(3-phenyl-1,2-oxazole-5-methylamino)-6-me-
thylthio-1,3,5-triazine (F10): yield, 77.6%; awhite solid; mp139ꢀ140 °C.
1H NMR (400 M, CDCl3) δ: 1.18 (s, 6H), 2.40ꢀ2.48 (m, 3H), 4.06ꢀ4.25
(m, 1H), 4.70ꢀ4.80 (m, 2H), 4.82ꢀ5.02 (m, 1H), 5.40ꢀ6.20 (m, 1H),
6.46 (s, 1H), 7.41ꢀ7.48 (m, 3H), 7.74ꢀ7.79 (m, 2H). Anal. Calcd for
C17H20N6OS: C, 57.28; H, 5.66; N, 23.58. Found: C, 57.23; H, 5.63; N,
23.44. MS (ESI) for C17H21N6OS (M+1): 357.
2-Isopropylamino-4-(2-bromo-5-thiazolylmethylamino)-6-methylthio-
1
1,3,5-triazine (F11): yield, 61.6%; a yellow solid; mp 123ꢀ124 °C. H
NMR (400 M, CDCl3) δ: 1.22 (s, 6H), 2.35ꢀ2.49 (m, 3H), 4.00ꢀ4.28
(m, 1H), 4.55ꢀ4.75 (m, 2H), 4.85ꢀ5.09 (m, 1H), 5.35ꢀ6.05 (m, 1H),
7.45ꢀ7.52 (m, 1H). Anal. Calcd for C11H15BrN6S2: C, 35.20; H, 4.03;
N, 22.39. Found: C, 35.42; H, 4.24; N, 22.51.
Biological Assay. Herbicide Screening Conducted at Nankai Uni-
versity. The glasshouse herbicidal activities of compounds F2ꢀF11 and
atrazine were evaluated using a previously reported procedure in Nankai
University.7a One dicotyledonous crop, rape (Brassica napus L.), and one
monocotyledonous weed, barnyard grass (Echinochloa crus-galli), were used
to test the herbicidal activities of compounds. Purified compounds were
dissolved in 100 μL of N,N-dimethylformamide with the addition of a little
Tween 20 and then were sprayed using a laboratory belt sprayer delivering a
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Table 2. Herbicidal Activities of Selected Compounds (Mean Scores/Percent Inhibition, %)a
96-well plate tests
postemergence glasshouse
preemergence glasshouse
AT
PA
AR
LP
SM
DA
AR
LP
SM
DA
F1 (atrazine)
99
99
99
99
99
99
99
ꢀ
0
100
100
100
100
100
70
100
50
100
90
70
0
100
100
100
100
100
100
ꢀ
80
30
70
90
50
10
ꢀ
90
90
80
90
90
90
ꢀ
0
F2
0
70
0
F4
0
80
100
100
100
100
ꢀ
0
F7 (ametryne)
F8
0
100
80
100
80
90
ꢀ
90
0
0
F9
0
50
0
b
norflurazon
glyphosate
99
ꢀ
ꢀ
90
ꢀ
80
ꢀ
0
80
90
90
0
0
a For 96-well plate tests, AT at 10 ppm and PA at 32 ppm, results are mean assessment scores. For glasshouse screening, 1.0 kg/ha. AT: Arabidopsis
thaliana. PA: Poa annua. AR: Amaranthus retroflexus. LP: Lolium perenne. SM: Stellaria media. DA: Digitaria sanguinalis. Results are percent inhibition of
growth. b “ꢀ” means not tested.
Table 3. Fungicidal Activities of Compounds F1ꢀF10 (Mean Assessment Score across Replicates)
Pythium dissimilea,b Alternaria solania,b Fusarium graminearuma,b Botrytis cinereaa,b Septoria triticic,d Phytophthora infestansc,e Uromyces viciae-fabaec,d
F1
F2
F4
F5
F6
F7
F8
F9
F10
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
18
99
99
69
0
0
0
0
0
0
0
0
0
0
NCHf
99
0
99
0
0
NCH
77
0
49
0
0
0
0
27
0
0
NCH
NCH
NCH
99
27
0
0
55
77
0
0
NCH
a Tests conducted in artificial media. b 2 ppm. c Tests conducted on leaf pieces. d 100 ppm. e 200 ppm. f “NCH” indicates that no assessment was possible
due to herbicidal activity on the leaf piece.
Test plates were incubated for seven days in a controlled environment
cabinet before assessment. The plates were then assessed, and each
treatment replicate was scored as either 0 (for no observable effect) or 99
(where an herbicidal effect was observed). Norflurazon and glyphosate
was tested alongside the compounds as a positive control. The data
presented in Table 2 are the mean scores of the two replicates.
96-Well Plate Fungicide Screening. Compounds F1ꢀF11 were
evaluated in mycelial growth tests in artificial media against Pythium
dissimile, Alternaria solani, Fusarium graminearum (Gibberella zeae), and
Botrytis cinerea (Botryotinia fuckeliana), at a rate of 2 ppm. The com-
pounds were also evaluated in leaf-piece assays, at rates of 100 ppm for
Septoria tritici on wheat and Uromyces viciae-fabae on bean, and 200
ppm and 60 ppm for Phytophthora infestans on tomato. Each assay
contained two replicates for each rate, except the Septoria tritici assay,
which contained three replicates. Chemicals were applied to leaf pieces
prior to inoculation with spores of the pathogen, or incorporated into
the growth medium for the artificial media assays. The plates were stored
in controlled environment cabinets for between four and fourteen days,
depending on the assay, after which mycelia growth or disease inhibition
was assessed. Each well was scored using a three-banded system, with
complete inhibition of mycelia growth or disease symptoms scored as
99, partial inhibition as 55, and no inhibition as 0. Azoxystrobin and
prochloraz were included in the test as positive control compounds. The
biological data presented in Table 3 are the mean scores for each
treatment across replicates.
applied in a similar fashion to the 96-well tests, with the exception of a
curative test against Puccinia triticina on wheat, in which the formulated
compound was applied to leaf pieces after they had been inoculated with
pathogen spores. The pathogens tested in this expanded screen were
Phytophthora infestans on tomato, Plasmopara viticola on vine, Puccinia
triticina on wheat (with both preventative and curative applications),
Septoria nodorum (Phaeosphaeria nodorum) on wheat, Pyrenophora teres
on barley, and Alternaria solani on tomato. The compounds were applied
at rates of 200, 60, and 20 ppm. Assessments were made between three
and nine days after inoculation, depending on the assay. Treatments
were scored as percentage inhibition of disease development relative to
untreated controls. The biological data presented in Table 4 are the
mean scores for each treatment across replicates.
96-Well Plate Insecticide Screening. The compounds were tested for
activity against an aphid species and the Lepidoptera Heliothis virescens in
leaf-disk assays at 1000 ppm. The compounds were also evaluated at a
rate of 500 ppm on Plutella xylostella in an artificial diet assay. Thiamethox-
am and indoxacarb were included as positive control compounds. Each
test contained three replicate treatments. The assay plates were stored in
controlled environment cabinets for five to nine days (depending on the
species). Mortality was then assessed relative to untreated control wells,
with wells showing significant levels of mortality scored as 99, and wells
without significant mortality scored as 0. The data presented in Table 5
are the mean scores of the three replicates.
Antitumor Activity Assays. The bioassay was tested at the National
Center for Drug Screening (Shanghai, P. R. China). The antitumor
activities of compounds F2ꢀ11 on leukemia HL-60 cell line and lung
adenocarcinoma A-549 cell line have been measured by using MTT
Additional Fungicide Screening. Compounds F2, F4, and F10 were
also screened on a broader range of fungal pathogens on leaf pieces, and
against additional species in artificial media. The compounds were
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Table 4. Fungicidal Activities of Compounds F2, F4 and F10
(Percent Inhibition, %)a
Table 6. Antitumor Activities of Compounds F1ꢀF10
(Percent Inhibition, %)
leaf-piece assays
lung adenocarcinoma A-549 cell
leukemia HL-60 cell
test rates (ppm) PI PV BG PTP PTC SN PT AS
10ꢀ4 M
10ꢀ5 M
10ꢀ4 M
10ꢀ5 M
F2
200
60
0
0
0
0
0
0
0
0
0
50 70 100
20 20 20
100
100
20
70
20
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
F1
F2
F4
F5
F6
F7
F8
F9
F10
40.6
33.5
55.4
44.3
35.7
39.6
27.3
29.1
27.0
2.8
5.8
55.5
52.6
70.2
64.4
56.5
52.9
58.8
63.2
57.2
6.4
13.7
1.7
20
0
0
0
0
0
0
0
k
0
17.0
12.0
11.5
5.1
200
60
90 100
100
100
100
100
100
70
pt
pt
0
14.4
10.5
0
F4
0
0
70
0
20
200
60
70 100
70 70
pt
70
0
2.7
6.6
F10
4.9
9.6
20
0
0
2.3
14.1
a pt: phytotoxic effect observed on leaf piece. k: test well not assessed due
to contamination. PI: Phytophthora infestans (tomato-preventative). PV:
Plasmopara viticola (grapevine-preventative). BG: Blumeria graminis f. sp.
tritici (wheat-preventative). PTP: Puccinia triticina (wheat-preventative).
PTC: Puccinia triticina (wheat-curative). SN: Stagonospora nodorum (wheat-
preventative). PT: Pyrenothora teres (barley-preventative). AS: Alternaria
solani (tomato-preventative).
F2ꢀF11 were also complex, but their structures and purity were
definitely ensured by MS and elemental analysis.
Herbicidal Activities. The glasshouse screening of 4-fluoro-
benzylamino and representative arylmethylamino-substituted tria-
zines (F2ꢀF6 and F8ꢀF11) were evaluated and compared with
known atrazine (F1) and ametryne (F7) (Table 1). As shown in
the table, most of the compounds exhibited good herbicidal
activities against dicotyledonous species (rape) both in post-
emergence treatment and in preemergence treatment. From the
activities against barnyard grass in both treatments we could
obviously find that the compounds containing the methylthio
moiety (F7ꢀF11) gave higher herbicidal activities than the
corresponding chloro analogues (F1ꢀF6). Compounds with
different aryl ring exhibited different level of inhibitory activity. In
general, compounds bearing an isoxazole or thiazole ring, such as
F5 and F6, displayed lower activity on all of the four screening
modes, while 4-fluorobenzylamino-substituted triazines F2 and
F8 and 2-chloropyridyl-substituted compounds F4 and F9 gave
relatively higher activities, but still lower than commercial
compounds F1 and F7, which gave 100% inhibition during all
four screening modes.
Compounds F2, F4, F8, and F9 were further screened at
Syngenta in order to better characterize their herbicidal spec-
trum. The 96-well plate test and glasshouse screen were con-
ducted, and the data are listed in Table 2. F1, F7, norflurazon, and
glyphosate were added as control compounds. In the plate tests,
the four compounds together with F1 and F7 were active only
against the dicotyledonous species AT (Arabidopsis thaliana),
but not the monocotyledonous species PA (Poa annua). In the
glasshouse herbicide screening, a similar result could be con-
cluded since almost all the compounds exhibited a little higher
activity against dicotyledonous species (AR and SM) than
monocotyledonous species (LP and DA). Overall, the herbicide
screening data suggest that while the compounds are not inactive
against monocot species, there is a degree of selectivity toward
activity on dicots. The data also showed that all the compounds,
including control compounds, exhibited the same or a little
higher activity in postemergence treatment than in preemeergence
treatment against the same species. Among the four compounds,
F8 gave a level of overall activity similar to the commercial
herbicide atrazine and higher than glyphosate.
Table 5. Insecticidal Activities of Compounds F1ꢀF10
(Mean Assessment Scores)
aphid speciesa
Heliothis virescensa
Plutella xylostellab
F1
F2
F4
F5
F6
F7
F8
F9
F10
33
0
0
0
0
0
0
0
0
0
99
0
99
0
0
0
0
0
0
0
0
33
0
0
0
0
0
a Leaf-disk assays. 1000 ppm. b Artificial diet assay. 500 ppm.
(methyl-thiazolyl-tetrazolium) and SRB (sulfur rhodamine B) methods
respectively after incubation of cancer cell with various concentrations of
the reagents for 72 h at 37 °C according to the reported method.13 The
antitumor activity data are presented in Table 6.
’ RESULTS AND DISCUSSION
Synthesis. Known compounds 1, F1 (atrazine), and F7
(ametryne) were synthesized according to published procedures
(Scheme 1). Because of the existance of both monomer and dimer
of triazine, and because each has a different set of chemical shifts,
there are three sets of proton signals in the 1H NMR spectrum,
and the overlap of part of the signals of the monomer and the
dimer makes their spectrum complex. Similar phenomena were
found and studied by Carper in 2002.14 Compound 1 reacted
with different aromatic or heterocyclic methylamines, for example,
4-fluorophenylmethylamine, 2-tetrahydrofuranmethylamine, 2-chloro-
5-pyridylmethylamine, 3-phenyl-1,2-oxazole-5-methylamine, and
2-bromo-5-thiazolylmethylamine gave corresponding target mol-
ecules F2ꢀF6, which were converted to corresponding methylthio-
substituted triazines F8ꢀF11 by reacting with sodium thiometh-
Fungicidal Activities. Compounds F1ꢀF10 were evaluated
in a series of in vitro fungicidal tests, against a range of phytopatho-
genic species. The resulting data (Table 3) revealed that several
compounds displayed a degree of fungicidal activity. Compounds
1
oxide (Scheme 2). Just like atrazine, the H NMR spectra of
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Journal of Agricultural and Food Chemistry
ARTICLE
F2 and F5 showed activity against B. cinerea, S. tritici and U. viciae-
fabae, and compound F10 was active against B. cinerea and
U. viciae-fabae, but has high phytotoxicity to the leaf pieces in the
S. tritici assay for a result to be recorded. Similarly, F4 was active
against S. tritici, but has high phytotoxicity on the leaf pieces of
the U. viciae-fabae assay for a result to be recorded, as did several
other compounds. No obvious structureꢀactivity relationship
was found.
Compounds F2, F4, and F10 were also screened on more
detailed in vitro fungicide tests against a broader range of species
(Table 4). All three compounds showed activity on the leaf-piece
assays, particularly against Puccinia triticina (both as preventative
and curative applications) and Blumeria graminis. Compound F4
gave 100% control of P. triticina at the lowest rate tested, but
phytotoxic effects from this compound were recorded on leaf
pieces in the Septoria nodorum assay, as well as for compound F10
at the highest rate. None of the compounds displayed any activity
on additional tests in artificial media (data not shown), and this,
coupled with the activity from these compounds in the herbicide
screens, suggests that the disease control observed is an artifact of
phytotoxic activity rather than evidence of intrinsic fungicidal
properties of the compounds (although this would not explain
the better fungicidal control in the curative applications for P.
triticina compared to the preventative).
Funding Sources
This work was supported by the National Key Project for Basic
Research (2010CB126106) and the National Natural Science
Foundation of China (20972080), the Syngenta PhD (postgraduate)
Programme, and the National Key Technology Research and
Development Program (2011BAE06B05-3).
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’ AUTHOR INFORMATION
Corresponding Author
*Tel: +86-(0)22-23499842. Fax: +86-(0)22-23499842. E-mail:
wang98h@263.net; wangqm@nankai.edu.cn.
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Journal of Agricultural and Food Chemistry
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