Synthesis of 1,2,3-thiadiazole and thiazole-based strobilurins as potent fungicide candidates

Article abstract of DOI:10.1021/acs.jafc.6b05128

Strobilurin fungicides play a crucial role in protecting plants against different pathogens and securing food supplies. A series of 1,2,3-thiadiazole and thiazole-based strobilurins were rationally designed, synthesized, characterized, and tested against various fungi. Introduction of 1,2,3-thiadiazole greatly improved the fungicidal activity of the target molecules. Compounds 8a, 8c, 8d, and 10i exhibited a relatively broad spectrum of fungicidal activity. Compound 8a showed excellent activities against Gibberella zeae, Sclerotinia sclerotiorum, and Rhizoctonia cerealis with median effective concentrations (EC₅₀) of 2.68, 0.44, and 0.01 μg/mL, respectively; it was much more active than positive controls enestroburin, kresoxim-methyl, and azoxystrobin with EC₅₀ between 0.06 and 15.12 μg/mL. Comparable or better fungicidal efficacy of compound 8a compared with azoxystrobin and trifloxystrobin against Sphaerotheca fuliginea and Pseudoperonspera cubensis was validated in cucumber fields at the same application dosages. Therefore, compound 8a is a promising fungicidal candidate worthy of further development.

Full text of DOI:10.1021/acs.jafc.6b05128

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Article
Synthesis of 1,2,3-Thiadizole and Thiazole Based
Strobilurins as Potent Fungicide Candidates
Lai Chen, Yu-Jie Zhu, Zhijin Fan, Xiao-Feng Guo, Zhi-Ming Zhang, Jing-Hua Xu, Ying-
Qi Song, Yury Yurievich Morzherin, Nataliya P. Belskaya, and Vasiliy A. Bakulev
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05128 • Publication Date (Web): 05 Jan 2017
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Synthesis of 1,2,3ꢀThiadiazole and Thiazole Based
Strobilurins as Potent Fungicide Candidates
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Lai Chen, † YuꢀJie Zhu, † ZhiꢀJin Fan,*, †, ‡ XiaoꢀFeng Guo, † ZhiꢀMing Zhang, †
JingꢀHua Xu, † YingꢀQi Song, † Morzherin Y. Yurievich, § Nataliya P. Belskaya, §
Vasiliy A. Bakulev*, §
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† State Key Laboratory of ElementoꢀOrganic Chemistry, Nankai University, Tianjin 300071,
P. R. China
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‡ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai
University, Tianjin 300071, P. R. China.
§ The Ural Federal University Named after the First President of Russia B. N. Yeltsin, Yeltsin
UrFU 620002, Ekaterinburg, Russia
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* Address correspondence to this author at State Key Laboratory of ElementoꢀOrganic
Chemistry, Nankai University, No. 94, Weijin Road, Nankai District, Tianjin 300071,
P. R. China (telephone +86ꢀ13920714666; Fax:+86 22ꢀ23503620; eꢀmail:
fanzj@nankai.edu.cn, or v.a.bakulev@urfu.ru)
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Strobilurin fungicides play a crucial role in protecting plants against different
pathogens and securing food supplies. A series of 1,2,3ꢀthiadiazole and thiazole
based strobilurins were rationally designed, synthesized, characterized and tested
against various fungi. Introduction of 1,2,3ꢀthiadiazole greatly improved the
fungicidal activity of the target molecules. Compounds 8a, 8c, 8d and 10i exhibited
a relative broadꢀspectrum of fungicidal activity. Compound 8a showed excellent
activities against Gibberella zeae, Sclerotinia sclerotiorum and Rhizoctonia cerealis
with the median effective concentration (EC50) of 2.68, 0.44 and 0.01 ꢁg/mL
respectively; it was much more active than positive controls enestroburin,
kresoximꢀmethyl and azoxystrobin with EC50 between 0.06 and 15.12 ꢁg/mL.
Comparable or better fungicidal efficacy of compound 8a than azoxystrobin and
trifloxystrobin against Sphaerotheca fuliginea and Pseudoperonspera cubensis was
validated in the cucumber fields at the same application dosages. Therefore,
compound 8a was a promising fungicidal candidate worthy of further development.
Keyword: Fungicide, strobilurin, 1,2,3ꢀthiadiazole, thiazole, antifungal activity.
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Plant pathogens can not only cause dramatic crop yield losses, but also greatly
affect the food security.1 One extreme example is that estimated annually economic
loss for American corn growers was over one billion US dollars directly from
southern corn leaf blight which is caused by Cochliobolus heterostrophus, anamorph
Bipolaris maydis.2 Some plant pathogens such as the scab fungus could produce
mycotoxins called polyketides which contaminate grain based foods, causing a serious
food safety and economic problem in the United States.3 Therefore, effective
measures for managing plant pathogens are in great demand.
Fungicides are an effective tool for plant disease management,4, 5 especially, new
fungicides are always welcome for farmers to phytopathogen managements. As the
second biggest classes of fungicides,6 being developed based on the lead compound
first isolated from fermentations of Stroblurus tenacellus in 1977 by block electron
transfer between cytochrome b and cytochrome c1,7 strobilurin fungicides have been
widely used against major plant pathogens.8 Majority of strobilurin fungicides
possesses a similar scaffold called βꢀmethoxyacrylate. Over a dozen strobilurin
fungicides have been launched and more are possible in the future.9ꢀ18
Heterocyclic rings as a substructure is one of the important measures for novel
pesticide discovery including fungicides development. Approximately 70% of
pharmaceuticals and agrochemicals contain at least one heterocyclic ring.19
Heterocyclic rings functionalities differ from each other in biologically active
compounds,20 and work as scaffolds in active ingredients, prodrugs, tools for
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fineꢀtuning physicochemical properties and isosteric replacements of functional
groups, alicyclic rings or other heterocyclic rings.21 Thiadiazole and thiazole
derivatives possess properties that include environmental compatibility and broad
spectrum of bioactivities such as antifungal,22 antimicrobial,23 systemic acquired
resistance24 and antitumor activities.25
This study was aimed at developing novel strobilurin fungicides by employing
the active scaffold of strobilurin fungicides and active 1,2,3ꢀthiadiazole, thiazole or
thiazole’s derivatives as a substructures to the target molecules (Figure 1). A series
of novel 1,2,3ꢀthiadiazole and thiazole based strobilurins were rationally designed,
synthesized and characterized. The active compound was evaluated as fungicide
candidate against Sphaerotheca fuliginea and Pseudoperonspora cubensis in the
cucumber fields.
75 Materials and Methods
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Equipment and materials: Melting points of synthesized compounds were
taken on an Xꢀ4 melting point apparatus and were uncorrected. 1H and 13C NMR
spectra were obtained on a Bruker Avance 400 MHZ spectrometer at 400 MHz and
100MHz in deuteroꢀchloroform (CDCl3) and tetramethylsilane (TMS) as an internal
standard. High resolution mass spectra (HRMS) were determined on a 7.0T
FTICRꢀMS instrument. Elemental analyses were performed on a Vario EL III
elemental analysis instrument. Crystal structure was recorded by a Bruker SMART
1000CCD diffraction meter. All solvents were of analytical grade.
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General Synthetic Procedure for Compounds 3a-c (Figure 2). Compounds
1a and 1b were commercially available. Compound 1c was synthesized according to
the published procedures.26 Compound 2 was prepared from the corresponding acid
1.27 A solution of methyl magnesium bromide in ethyl ether (Et2O) (3 mol/L, 6.22
mL, 18.66 mmol) was added dropwise to a solution of 2 (12.44 mmol) in anhydrous
tetrahydrofuran (THF) (45 mL) at ꢀ30 °C under N2. The mixture was then stirred at
ꢀ30 °C for 1 h and at room temperature for another 1 h. When the reaction was
complete as monitored with thin layer chromatography (TLC), it was quenched with
the sat. aq ammonium chloride (NH4Cl) solution (50 mL). After removal of Et2O
and THF under vacuo, the aqueous phase was extracted with ethyl acetate (3×50 mL).
The combined organic layers were washed with water (50 mL), saturated brine (50
mL), and dried over anhydrous sodium sulfate. After filtration, the solvent was
evaporated. The residue was then purified on a silica gel column (203 mm x 26
mm)eluted with a mixture of ethyl acetate and petroleum ether (60ꢀ90 °C fraction) at a
ratio of 1:5 (v/v) to obtain compounds 3a-c in a 90ꢀ93% yield (Figure 2).
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General Synthetic Procedure for Compounds 6a-c (Figure 2). Compound 4a
and 4b were commercially available. Compound 4c was synthesized as reference
description.26 Compound 5 was synthesized by reduction of compound 4.28
DessꢀMartin periodinane (12.00 mmol) was added to a solution of compound 5 (10.87
mmol) in dichloromethane in an ice bath. The reaction mixture was stirred at room
temperature overnight. After filtration, the solvent was evaporated. The residue
was then purified on a silica gel column eluted with a mixture of ethyl acetate and
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petroleum ether (60ꢀ90 °C fraction) at a ratio of 1:4(v/v) to obtain compounds 6a-c in
a 50ꢀ80% yield.
General Synthetic Procedure for Compounds 8a-f (Figure 3). Compound 7 was
prepared according to Strazzolini and Pavsler.29 A solution of compound 7 (1.12
mmol) in 15 mL ethanol was added to the compound 3 or 6 (1.02 mmol) in ethanol
(15 mL), followed by addition of a catalytic amount of hydrochloric acid (2 mol/L,
0.1 mL. The reaction mixture was stirred at room temperature overnight. After
removal of ethanol under vacuum, the residue was purified by recrystallization in
ethanol or silica gel column chromatography eluted with a mixture of ethyl acetate
and petroleum ether (60ꢀ90 °C fraction) at a ratio of 1:9ꢀ1:4 (v/v) to obtain the
corresponding compound 8 (Figure 3). The yields, physical properties and HRMS or
elemental analyses of the target compounds 8a-f were as follows:
Data for 8a: yellow solid; yield, 48%; m.p.: 70ꢀ72 oC; Anal. calcd for
C16H18N4O4S: C, 53.03; H, 5.01, N, 15.46. Found C, 53.29; H, 4.86, N, 15.40.
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Data for 8b: yellow solid; yield, 67%; m.p.: 98ꢀ100 C; HRMS (m/z) calcd for
C15H16N4O4S: (M+H)+: 349.0892, found: 349.0969.
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Data for 8c: colorless crystal; yield, 53%; m.p.: 82ꢀ83 C; Anal. calcd for
C16H18N4O4S: C, 53.03; H,5.01, N, 15.46. Found C, 53.02; H, 4.99; N, 15.60.
Data for 8d: white solid; yield, 70%; m.p.: 82ꢀ84 oC; Anal. calcd for
C15H16N4O4S: C, 51.71; H, 4.63; N, 16.08. Found C, 51.83; H, 4.58; N, 16.23.
Data for 8e: white solid; yield, 48%; m.p.: 91ꢀ93 oC; Anal. calcd for
C26H34N4O6S: C, 58.85; H, 6.46; N, 10.56. Found C, 58.25; H, 6.26; N, 10.16.
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Data for 8f: yellow oil; yield, 47%; HRMS (m/z) calcd for C25H32N4O6S (M+H)+:
517.2043, found: 517.2114.
General Synthetic Procedure for Compounds 10a-o. Compound 9 was
prepared according to the report of Domagala et al.30 Compounds 9 (0.75 mmol),
1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide hydrochloride (EDCI) (0.17 g, 0.90
mmol) and 1ꢀhydroxybenzotriazole (HOBt) (0.11 g, 0.77 mmol) were dissolved in
dichloromethane (25 mL) in an ice bath and was slowly warmed up to room
temperature and stirred for another 45 min. A solution of amine in dichloromethane
(25 mL) was then added followed by addition of trimethylamine (Et3N) (0.09 g, 0.90
mmol), the reaction mixture was stirred at room temperature overnight, the organic
layer was successively washed with water (2×30 mL), saturated brine (40 mL), and
dried over magnesium sulfate and concentrated in vacuo. Compound 10aꢀo in the
residue were then purified on a silica gel eluted with a mixture of ethyl acetate and
petroleum ether (60ꢀ90 °C fraction) at a ratio of 1:1ꢀ1:6(v/v) (Figure 4). The yields,
physical properties and HRMS, elemental analyses of the target compounds 10a-o
were as follows:
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Data for 10a: yellow solid; yield, 83%; m.p.: 70ꢀ72 C; HRMS (m/z) calcd for
C17H19F2N5O3S (M+H)+: 412.1177, found: 412.1255.
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Data for 10b: yellow powder; yield, 89%; m.p.: 110ꢀ112 C; HRMS (m/z) calcd
for C18H19N5O3S (M+H)+: 386.1209, found: 386.1285.
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Data for 10c: yellow powder; yield, 69%; m.p.: 83ꢀ85 C; Anal. calcd for
C18H21N5O3S: C, 55.80; H, 5.46, N, 18.08. Found C, 55.83; H, 5.53; N, 18.23.
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Data for 10d: colorless crystal; yield, 68%; m.p.: 71ꢀ73 C; HRMS (m/z) calcd
for C16H19N5O3S (M+H)+: 362.1209, found: 362.1289.
Data for 10e: yellow oil; yield, 68%; HRMS (m/z) calcd for C20H27N5O3S2
(M+H)+: 450.1555, found: 350.1635.
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Data for 10f: white solid; yield, 57%; m.p.: 98ꢀ101 C; HRMS (m/z) calcd for
C17H19F2N5O3S (M+H)+: 412.1177, found: 412.1257.
Data for 10g: colorless crystal; yield, 85%; m.p.: 131ꢀ133 oC; HRMS (m/z) calcd
for C18H19N5O3S (M+H)+: 386.1209, found: 386.1287.
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Data for 10h: yellow crystal; yield, 90%; m.p.: 125ꢀ127 C; Anal. calcd for
C18H21N5O3S: C, 55.80; H,5.46, N, 18.08. Found C, 55.39; H, 5.53; N, 18.09.
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Data for 10i: white powder; yield, 82%; m.p.: 123ꢀ124 C; Anal. calcd for
C16H19N5O3S: C, 53.17; H, 5.30, N, 19.38. Found C, 52.47; H, 5.43; N, 19.67.
Data for 10j: yellow oil; yield, 78%; HRMS (m/z) calcd for C20H27N5O3S2
(M+H)+: 450.1555, found: 450.1632.
Data for 10k: yellow oil; yield, 87%; HRMS (m/z) calcd for C27H35F2N 5O5S
(M+H)+: 580.2327, found: 580.2393.
Data for 10l: yellow oil; yield, 91%; Anal. calcd for C28H35N5O5S: C, 60.74; H,
6.37; N, 12.65. Found C, 60.12; H, 6.18; N, 12.10.
Data for 10m: yellow oil; yield, 91%; HRMS (m/z) calcd for C28H37N O5S
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(M+H)+: 556.2515, found: 556.2594.
Data for 10n: yellow solid; yield, 64%; m.p.: 101ꢀ103 oC; HRMS (m/z) calcd for
C26H35N5O5S (M+H)+: 530.2359, found: 530.2442.
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Data for 10o: yellow oil; yield, 96%; HRMS (m/z) calcd for C30H43N5O5S2
(M+H)+: 618.2706, found: 618.2791.
Crystal Structure Determination for Compound 8b
The crystal of compound 8b was obtained from a mixture of dichloromethane
and petroleum ether for structure validation (Figure 5). Xꢀray intensity data of
compound 8b were recorded on a Bruker SMART 1000CCD diffraction meter
equipped with graphiteꢀmonochromatic Mo Kα radiation (λ = 0.71073 Ǻ). A total of
9395 measured reflections; 3936 unique (Rint = 0.0308) in the range of 1.96°≤ θ ≤
27.96°(h, ꢀ10 to 10; k, ꢀ13 to 13; l, ꢀ15 to 15), of which 2775 had |Fo| > 2|Fo|. Direct
methods were used for the structure solving by the SHELXSꢀ97 program. The
structure were refined anisotropically by fullꢀmatrix leastꢀsquares for all nonꢀH atoms
2
to give the final R=0.0359 and wR=0.1010 (w=1/[σ2((Fo )+(0.0651P)2+0.0000P],
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2
where P=(Fo +2Fc )/3), (ꢂ/σ)max=0.982 and S=1.029. The hydrogen atoms were
located according to theoretical models.
Fungicidal Activity
The fungicidal activities against Alternaria solani (A. s), Botrytis cinerea (B. c),
Cercospora arachidicola (C. a), Gibberella zeae (G. z), Phytophthora infestans (Mont)
de Bary (P. i), Physalospora piricola (P. p), Pellicularia sasakii (P. s), Sclerotinia
sclerotiorum (S. s) and Rhizoctonia cerealis (R. c) were detected in vitro at 50ꢁg/mL
according to a reported method by using azoxystrobin as a positive control.31 For
any active compound with growth inhibition over 90% at 50 ꢁg/mL, their median
effective concentration (EC50) were determined according to the reference by using
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enestroburin, kresoximꢀmethyl and azoxystrobin as positive controls.32
The preventive activities of the target compounds against Pseudoperonospora
cubensis, Erysiphe graminis, Puccinia sorghi Schw, Colletotrichum lagenarium were
conducted at 400 ꢁg/mL by fungal spores inoculation.9 The test solution was diluted
by mother solution with distilled water (containing 0.1% Tween 80), the mother
solution was prepared by dissolving each target compound (0.0111 g) in 0.5mL DMF.
Control plants were sprayed with water solution containing the same concentration of
DMF and Tween 80 in the test solutions. The percentage of disease control was
compared with the control plants by checking complete disease control as 100 and no
disease control as 0. Azoxystrobin was used as a positive control.
Evaluation of Fungicidal Activity of Compound 8a in the Field
Due to high potency in laboratory and greenhouse, the fungicidal activity of
compound 8a was further evaluated in the field. An emulsifiable concentrate (8.88%)
formulation was prepared. Its field efficacy against S. fuliginea and P. cubensis on
cucumbers was conducted in Wuqing County, Tianjin, China. Azoxystrobin (250g/L,
suspension concentrate), trifloxystrobin (50% water dispersible granule) and
pyraclostrobin (350 g/L, emulsifiable concentrate) were used as positive controls.
An application rates of compound 8a and positive standards were 37.5 and 75 g ai/ha
for S. fuliginea and P. cubensis, respectively. Disease index (DI) was determined by
the evaluated as Eqn. 1:
DI=Σ(A×B)×100/(C×9)
(Eqn. 1)
Where A: number of disease leaves; B: corresponding grade of A; C: total
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number of investigation leaves.
Preventative efficacy was evaluated as Eqn. 2
Efficacy (%)=[1ꢀCK0×PT1/(CK1×PT0)]×100%
(Eqn. 2)
Where CK0: DI of control group before water application; PT0: DI of treatment
group before compound application; CK1: DI of control group after water application;
PT1: DI of treatment group after compound application. Data analysis was
conducted by Duncan's Multiple Range Test (DMRT).
224 Results and Discussion
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Chemistry
Compounds 3 and 6 were obtained from the corresponding acid 1 and ester 4,
respectively (Figure 2). Intermediate 2 was prepared with yield 90ꢀ98% by reaction
between compound 1 and N,Oꢀdimethylhydroxylamine hydrochloride.27 This
intermediate 2 was reacted with methyl magnesium bromide in anhydrous THF to
give compound 3 in 90ꢀ93% yield. After reduction of compound 4 by sodium
borohydride and subsequent DessꢀMartin oxidation, compound 6 was obtained in
50ꢀ80% yield.
The compounds 8 were obtained by the reaction between the intermediate 7 and
the compounds 3 or 6 catalyzed by hydrochloric acid (Figure 3 ).29 The compounds
10a-10o were prepared by a condensation reaction between the hydrolytic product 9
of compounds 8a, 8c or 8e and amine R3NH2 with a yield ranging from 64ꢀ96%
1
(Figure 4). All structures were confirmed by H NMR, 13C NMR, HRꢀMS or
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elemental analysis. Therefore, to develop new strobilurin fungicides by integrating
the active scaffold of strobilurin and 1,2,3ꢀthiadiazoles or thiazoles into one molecule,
a series of compounds were successfully designed, synthesized and well
characterized.
Fungicidal activity
The in vitro antifungal activities were assessed at 50 ꢁg/mL and results are listed
in Table 1. The Eꢀisomer of compound 8a (E-8a) exhibited slightly better fungicidal
activities against A. solani, G. zeae, P. piricola, B. cinerea, S. sclerotiorum and R.
cerealis than compound 8a (Z-8a). Because the difference between E-8a and Z-8a
was not significant, and the two isomers might be interconverted under biological
conditions, we used mixtures in all the bioassays in the same way as commercialized
positive controls. Compounds 8 series with a 1,2,3ꢀthiadiazole ring at R1 showed
good activity against G. zeae, B. cinerea, S. sclerotiorum, R. cerealis and P. infestans.
Compound E-8a exhibited a similar inhibition to azoxystrobin with 100% activity
against G. zeae and S. sclerotiorum. Compound 8c exhibited similar 100%
inhibition against G. zeae, R. cerealis and P. infestans to azoxystrobin, and it showed
better efficacy against B. cinerea with 100% of growth inhibition than azoxystrobin
(79%). Compound 8d had similar activity against S. sclerotiorum, R. cerealis and P.
infestans with 100% growth inhibition to azoxystrobin. In addition, compounds 10b,
10c, 10d, 10g and 10i also showed the similar activity against PI with 100% growth
inhibition to azoxystrobin, and compound 10i exhibited the same activity against G.
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zeae, R. cerealis and P. infestans as azoxystrobin, having totally inhibited all fungal
growth.
Table 2 showed EC50 values of compounds 8 and 10 series. Being much better
than the positive controls such as enestroburin, kresoximꢀmethyl and azoxystrobin,
the compound 8a showed excellent activities against G. zeae, S. sclerotiorum and R.
cerealis with EC50 values of 2.68, 0.44 and 0.01 ꢁg/mL respectively, and compound
8a also had a similar EC90 against G. zeae as azoxystrobin, and much better activity
against S. sclerotiorum with EC90 more than 10 times than that of the positive controls
such as enestroburin, kresoximꢀmethyl and azoxystrobin. Compound 8c had an EC90
against B. cinerea greater than 20 times than that of the three positive controls such as
enestroburin, kresoximꢀmethyl and azoxystrobin.
The in vivo protective activity of all target compounds against P. cubensis, E.
graminis, P. sorghi Schw, and C. lagenarium at 400 ꢁg/mL were evaluated (Table 3).
It showed that only 8a and 8c displayed better activity against P. cubensis. than the
positive control azoxystrobin.
This indicated that the introduction of a
1,2,3ꢀthiadiazole ring and methyl group on the side chain at R1 and R2 respectively
could increase activities against P. cubensis. Compounds 10a, 10f and 10n
exhibited 100% growth inhibition against C. lagenarium, which were higher than
azoxystrobin (80%). Compounds 8a, 8c-e, 10d, 10e and 10i exhibited the same level
of activity against E. graminis as azoxystrobin.
Subsequently, compounds 8a and 8c with good in vivo fungicidal activity were
chosen for further experiments in the greenhouse at the concentration of 50 ꢁg/mL,
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12.5 ꢁg/mL, 3.13 ꢁg/mL, 0.78 ꢁg/mL and 0.20 ꢁg/mL. Compound 8c exhibited
lower activity against P. cubensis, P. sorghi Schw and C. lagenarium, however, it
showed the same activity against E. graminis as trifloxystrobin and azoxystrobin.
Compound 8a exhibited 100% inhibition activity against E. graminis even at 0.20
ꢁg/mL, which was equal to azoxystrobin (100% inhibition) and better than
trifloxystrobin (95% inhibition) and enestroburin (30% inhibition). Compound 8a
exhibited 60% inhibition against P. cubensis at 50 ꢁg/mL, whereas trifloxystrobin
only showed 20% inhibition, and enestroburin had no inhibition under the same
conditions. Therefore, compound 8a exhibited high in vivo fungicidal activity and
warranted further development.
These results indicated that the introduction of a 1,2,3ꢀthiadiazole ring and a
methyl group on the side chain at R1 and R2 respectively could increase fungicidal
activities.
Evaluation of Fungicidal Activity of Compound 8a under the Field Condition
The compound 8a was chosen for field trials in 2015 against S. fuliginea and P.
cubensis on cucumbers (Table 5). Compound 8a showed significant better efficacy
against S. fuliginea (p<0.01) than azoxystrobin and trifloxystrobin at an application
dosage of 37.5g ai/ha. At an application rate of 75 g ai/ha, compound 8a showed
significant better efficacy (p<0.05) against P. cubensis than trifloxystrobin and similar
efficacy to azoxystrobin. The results indicated that, the compound 8a could be a
fungicide candidate against S. fuliginea and P. cubensis for further development on
the basis of this field experiment.
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The bioassay results described above indicated that as an active substructure,
1,2,3ꢀthiadiazole could significantly improve the antifungal activity of the target
strobilurin molecules. Compound 8a displayed comparable or better field efficacy
than the commercial fungicides and warranted further studies as a fungicide
candidate.
ABBREVIATIONS USED
DMAP: 4ꢀdimethylaminopyridine; EC50: median effective concentration; EDCI:
1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide
hydrochloride;
HOBt:
1ꢀhydroxylbenzotriazole; TLC: thin layer chromatography; TMS: tetramethylsilane;
THF: tetrahydrofuran; DMRT: Duncan's Multiple Range Test.
ACKNOWLEDGMENT
This work was supported in part by the National Natural Science Foundation of China
(Grant No. 21372132), the International Science & Technology Cooperation Program
of China (Grant No. 2014DFR41030), the Tianjin Natural Science Foundation (No.:
14JCYBJC20400) and the "111" Project of Ministry of Education of China (Project
No. B06005). V. A. Bakulev thanks the Ministry of Education and Science of the
Russian Federation (State task 4.1626.2014/K).
SUPPORTING INFORMATION
1H NMR, 13C NMR data and spectra for the target compounds; Crystal data of the
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compound 8b. This material is available free of charge via the Internet at http:
//pubs.acs.org.
327
328
329
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Table 1. In Vitro Fungicidal Activity of Compounds 8a-f and 10a-o.
Fungicidal activity (%) at 50 ꢁg/mL
Compd.
A. sa
49
37
28
48
34
42
45
8
C. a
79
79
36
68
60
54
54
12
12
44
44
20
12
20
20
44
20
13
33
33
58
13
68
G. z
100
95
P. p
47
33
40
58
33
53
53
33
30
51
44
47
9
B. c
88
69
32
100
74
75
73
32
37
53
58
32
32
32
32
42
47
55
46
43
77
41
79
S. s
100
96
R. c
93
P. s
7
P. i
80
E-8a
Z-8a
8b
84
7
80
55
80
73
100
53
41
40
60
47
73
67
33
40
33
60
47
53
65
29
65
6
66
14
78
68
55
42
3
71
8c
100
82
100
100
78
100
100
63
8d
8e
52
8f
39
78
54
10a
55
22
43
10b
10c
8
100
64
85
62
41
90
8
100
100
100
81
28
34
15
8
80
10d
10e
100
45
76
80
10f
45
22
8
33
10g
8
55
33
47
47
2
56
24
8
100
24
10h
10i
8
45
51
34
15
9
100
45
100
61
78
14
30
45
30
55
15
84
100
33
10j
10k
10l
21
36
36
56
52
21
65
50
17
27
27
42
5
58
74
46
10m
10n
10o
52
68
29
48
70
54
21
38
17
Azoxystrobin
44
100
100
100
100
a
A. s: Alternaria solani; B. c: Botrytis cinerea; C. a: Cercospora arachidicola; G.z:
Gibberella zeae; P. i: Phytophthora infestans (Mont) de Bary; P. p: Physalospora piricola; P. s:
Pellicularia sasakii; S. s: Sclerotinia sclerotiorum; R. c: Rhizoctonia cerealis.
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Table 2. EC50 and EC90 of Compounds 8 and 10 Series.
EC50 (ꢁg/mL) and EC90 (ꢁg/mL)
Compd.
B. ca
G. z
P. i
P. s
R. c
S. s
EC50 EC90
EC50 EC90 EC50 EC90 EC50 EC90 EC50 EC90
EC50 EC90
0.44 6.89
c
ndb
nd
0.77 4.18
2.68 18.02 nd
nd
nd
nd
nd
nd
nd
nd
0.01
0.27
17.75
nd
>
8a
1.37 40.14 1.41 4.89 nd
>
nd
nd
>
8c
8d
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
>
8.76 70.17 nd
>
53.84
nd
3.34
nd
7.38
>
>
>
>
>
>
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
>
10b
nd
>
16.66
1.88
nd
nd
10c
1.40
nd
6.36 99.30 nd
nd
10d
nd
>
13.92
0.41
nd
nd
nd
>
nd
nd
10g
10i
1.40
nd
0.07 16.82 nd
0.12 32.54 2.17
1.73 126.90 13.87
>
99.84
18.40
Enestroburin
Kresoxim-methyl
Azoxystrobin
19.05 96.35 8.77 24.10 0.71 12.08 10.71
15.11 101.36 6.92 18.08 0.40 7.58 3.68
>
4.43 145.97 15.12 81.89
0.06 4.04
>
>
>
a B. c: Botrytis cinerea; G. z: Gibberella zeae; P. i: Phytophthora infestans (Mont) de Bary; P.
b
s: Pellicularia sasakii; R. c: Rhizoctonia cerealis; S. s: Sclerotinia sclerotiorum. nd: not
determined; c >: data more than 200 ꢁg/mL;
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Table 3. In Vivo Fungicidal Activity of Compounds 8a-f and 10a-o.
Fungicidal activity (%) at 400 ꢁg/mL
Compd.
P. cubensis E. graminis P. sorghi Schw C. lagenarium
100
0
100
0
100
0
85
0
8a
8b
90
0
80
80
0
30
0
0
100
100
100
60
0
85
30
80
70
80
85
60
85
60
0
30
60
0
0
50
0
70
80
75
85
100
80
80
85
80
100
80
80
80
0
8c
8d
8e
8f
10a
10b
10c
10d
10e
0
0
100
100
30
0
70
100
0
30
0
0
0
75
0
10f
10g
10h
10i
10j
10k
10l
0
60
65
75
85
0
60
40
85
0
60
80
100
0
0
80
0
10m
10n
10o
Azoxystrobin
0
0
100
0
80
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Table 4. Greenhouse Fungicidal Activity Validation Studies In Vivo
.
Fungicidal activity (%)
C(ꢁg/mL) P. cubensis E. graminis P. sorghi Schw C. lagenarium
Compd.
0.2
0.78
3.13
12.5
50
0
0
0
15
60
0
100
100
100
100
100
95
0
20
55
100
100
10
40
55
60
85
98
0
8a
0.2
8c
0.78
3.13
12.5
50
0
0
0
0
0
0
0
0
20
0
0
0
0
0
0
0
0
100
100
100
100
95
100
100
100
100
30
65
95
98
100
98
25
40
75
98
95
98
100
100
100
30
0
0
0
30
65
85
98
98
100
65
85
95
98
100
100
100
100
100
100
0.2
Trifloxystrobin
Enestroburin
Azoxystrobin
0.78
3.13
12.5
50
0.2
0.78
3.13
12.5
50
75
98
100
100
100
100
100
100
100
0.2
0.78
3.13
12.5
50
100
100
100
100
0
0
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Table 5. Fungicidal Efficacy of Compound 8a in the Cucumber Field.
DD c
Rate
(g ai/ha)
37.5
37.5
37.5
ndf
Base After
DIa DIb
Field
Efficacy(%)
76.61±3.63
70.19±2.91
68.02±5.58
nd
Disease
Compd.
5%
ad
1%
ce
de
8a EC
1.83 8.23
1.66 9.59
1.64 10.04
1.07 20.63
3.70 5.78
3.73 7.24
3.57 5.78
3.58 24.96
S.
Azoxystrobin SC
Trifloxystrobin WG
CK
bd
fuliginea
b
d
nd
a
nd
cde
d
8a EC
75
77.52±2.15
72.02±1.61
77.02±1.77
nd
Trifloxystrobin WG
Pyraclostrobin EC
CK
75
b
P.
75
a
cd
nd
cubensis
nd
nd
aBase DI, disease index base. bAfter DI, disease index after compound application. cDD,
distinct difference. da and b: distinct difference at 95% of confidence limits; ec, d and cd:
distinct difference at 99% of confidence limits. fnd, not determined.
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FIGURE CAPTIONS
Figure 1. Design of the title compounds
Figure 2. General synthetic procedure for compounds 3a-c and 6a-c.
Figure 3. General synthetic procedure for compounds 8a-f.
Figure 4. General synthetic procedure for compounds 10a-o.
Figure 5. Crystal structure for compound 8b by X-ray diffraction determination.
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Journal of Agricultural and Food Chemistry
Figure graphics
CH3
N
S
N
N
Boc
N
S
Hetero aryl
β-methoxyacrylate
β-methoxyacrylate
N
Hetero aryl
N
Ph
O
O
R1
CH3
O
O
N
N
R3
β-methoxyacrylate:
H3CO
N
H3CO
OCH3
H
1
1
Figure 1
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aReagents and conditions: (i) Et3N, CH3NHOCH3ñHCl, EDCI, DMAP, dry CH2Cl2,
overnight; (ii) CH3MgBr, dry THF, 0 °C to r.t.; (iii) NaBH4, CH3OH;(iv) Dess-Martin
periodinane, CH2Cl2, overnight.
Figure 2
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Figure 3
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