Synthesis of carbon-14 labeled pyraoxystrobin, a novel fungicide

Article abstract of DOI:10.1002/jlcr.1929

SYP-3343, (E)-2-(2-((3-(4-chlorophenyl)-1-methyl-1H-pyrazole-5-yloxy) methyl)) phenyl)-3-methoxyacrylate, is a novel fungicide with broad-spectrum and high activity against fungi. In this paper, radioactive pyraoxystrobin, labeled in the pyrazole ring sys

Full text of DOI:10.1002/jlcr.1929

Research Article
Received 10 June 2011,
Revised 28 July 2011,
Accepted 31 July 2011
Published online 22 September 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1929
Synthesis of carbon-14 labeled
pyraoxystrobin, a novel fungicide
a
a
a
b
*
Xun-yue Liu, Qing-fu Ye, Deng-lei Kan, Zheng Zhang, and
a**
Xing-cheng Ding
SYP-3343, (E)-2-(2-((3-(4-chlorophenyl)-1-methyl-1H-pyrazole-5-yloxy)methyl)) phenyl)-3-methoxyacrylate, is a novel fungi-
cide with broad-spectrum and high activity against fungi. In this paper, radioactive pyraoxystrobin, labeled in the pyrazole
ring system was achieved from 14C labeled 4-chlorobenzoic acid through substitution, cyclization, and condensation, in an
overall chemical and radiochemical yield of 44%. The chemical and radiochemical purity of the title compound was 98.5%
and 98.2%, respectively. This labeled SYP-3343 was used as a radiotracer for metabolism, toxicology, mode of action, and
other environmental studies.
Keywords: pyraoxystrobin; radiosynthesis; 14C-labeled; SYP-3343
which may be attractive sites for labeling. We selected the
pyrazole ring for labeling, because potential precursors were
readily available. Both C-14 labeled 4-chloroacetophenone
and 4-chlorobenzoic acid were considered as possible start-
Introduction
SYP-3343, (E)-2-(2-((3-(4-chlorophenyl)-1-methyl-1H-pyrazole-5-
yloxy) methyl)) phenyl)-3-methoxyacrylate (Figure 1), is a novel
pyraoxystrobin fungicide that was independently developed
by the ShenYang Research Institute of Chemical Industry in
China. It is highly effective and has broad-spectrum activity
against Pseudoperonospora cubensis, Blumeria graminis, Erysiphe
cichoracearum, Plasmopara viticola, and rice blast. It is widely
ing materials; however, the yields in the synthesis of 6 from
4-chloroacetophenone were significantly lower than when
4-chlorobenzoic acid was used. The radiolabeled compound, [14C]-
pyrazole ring SYP-3343, was synthesized from 4-chlorobenzoic-
[carbonyl-14C] acid according to the method shown in Scheme 1.
used to control many species of acariens on various crops such
In the preparation of 1, 3, and 6, it was necessary to vigorously
as rice, vegetables, and teas.1–8 SYP-3343 has low toxicity to
eliminate water from the reactions or the yields were significantly
mammals. Its acute oral lethal dose 50% is 1000 mg/kg in male
decreased. Reaction of carbonyldiimidazole with 4-chlorobenzoic-
[carbonyl-14C] acid (2) and subsequent reaction with the magnesium
rats and 1022 mg/kg in female rats. It does not cause skin irrita-
tion in rabbits.9 It obtained temporary registration in China in
salt of mono-ethyl malonate (1) yielded ethyl 3-(4-chlorophenyl-
3-[14C]-oxopropanoate (3) in 98% yield. Reaction of 3 with
2009 for cucumber downy mildew with a use rate of 80–100 g
active ingredient per hectare.10
N-methylhydrazine yielded 3-(4-chlorophenyl)-1-methyl-1H-
[3-14C]-pyrazol-5-ol (4) in 87% yield. Subsequent reaction of
Insecticides, fungicides, and some herbicides directly enter
into the soils after treatment.11 Pesticide residues in soils, the
4 and (E)-methyl 2-(2-(chloromethyl) phenyl)-3-methoxyacrylate
(5) yielded 6 in 68% crude yield. The last step of the reaction pro-
dynamics of degradation, and the effects of pesticides on inver-
tebrates, microbiology, and plants are important issues.12–14
duced several by-products that required semi-preparative HPLC
Pesticides degrade differently in different soils under differing
to remove, resulting in a 51% yield for the last step and 44% overall
yield from 4-chlorobenzoic-[carbonyl-14C] acid (2). HPLC-mass
conditions. When pesticide degradation is studied under differ-
ent environmental conditions, a mathematical model can be
established combining actual rainfall and temperature, to predict
spectrometry (MS), MS (electron impact [EI]), and 1H-NMR analysis
of 6 were consistent with those obtained for unlabeled SYP-3343.
the dynamics of degradation and the effects on the environ-
The chemical and radiochemical purities were over 98%; the
ment.15–17 At the present time, insufficient data are available
on the toxicology of SYP-3343, and therefore, radiolabeled
aInstitute of Nuclear Agricultural Science, Zhejiang University, Hangzhou 310029,
Zhejiang Province, China
SYP-3343 is necessary to further elucidate the precise mechan-
ism of action and support ongoing studies on the environmental
behavior and fate of SYP-3343.18,19 In this paper, the synthesis of
pyrazole ring labeled [14C]-SYP-3343 is described.
bDepartment of Eye Science, Zhejiang University, Hangzhou 310009, Zhejiang
Province, China
*Correspondence to: Zheng Zhang, Eye Center of Affiliated Second Hospital,
Zhejiang University 88 Jiefang RoadHangzhou, 310009, Zhejiang, Province, China.
E-mail: zhangzhengzju@yahoo.com.cn
Results and discussion
In the design of a procedure for the preparation of a radiolabeled
compound for use in environmental fate studies, it is important
to locate the C-14 label in a metabolically stable position. In
**Correspondence to: Xing-cheng Ding, Institute of Nuclear-Agricultural Science,
Zhejiang University, 268 Kaixuan Road, Hangzhou, 310029, Zhejiang, Province, China.
SYP-3343, there are two benzene rings and a pyrazole ring, E-mail: dingxch@zju.edu.cn
J. Label Compd. Radiopharm 2011, 54 780–782
Copyright © 2011 John Wiley & Sons, Ltd.

X.-Y. Liu et al.
Cl
Clara, CA, USA). HPLC-MS was conducted on an Agilent 1100
LC/MS instrument (Agilent Technologies). HPLC analysis and
semi-preparative purifications were completed on a Waters
2695 series system (Waters Co., Milford, MA, USA) consisting
of an automated gradient pump, photodiode array detector
with Inertsil ODS-3 column (4.6 Â 250 mm, GL Science Co., Tokyo,
Japan), and Hypersil semi-preparative column (8 mm  300 mm,
Dalian Elite Co., Liaoning, China). TLC was run on GF254 plates, and
O
O
N
OCH3
N
CH3
OCH3
Figure 1. SYP-3343.
specific activity was 5.04 mCi/mmol as determined by HPLC, radio- radio-TLC plates were scanned on a Fujifilm BAS-1800II laser-based
thin layer chromatography (TLC), and HPLC-LSC (Liquid Scientilla- fluorescence and radioisotope imaging system (Fujifilm Global,
tion Counting) methods.
Tokyo Japan). Radioactivity was determined with a WinSpectral-
1414 liquid scintillation spectrometer (Wallac, Turku, Finland).
Experimental
Magnesium ethyl 2-hydroperoxyacrylate (1)
General
Magnesium ethanolate (3.43 g, 30 mmol) and 2-(ethylperoxy)
acrylic acid (7.93 g, 60 mmol) were mixed in anhydrous THF
and stirred for 2 h at room temperature. The reaction mixture
was concentrated in vacuo, to yield (1) as a gray solid (8.07 g,
94% yield).
[1-14C]4-Chlorobenzoic acid (15 mCi, 5.1 mCi/mmol) (2) was pur-
chased from American Radiolabeled Chemicals Inc., (St. Louis,
MO, USA). Paraformaldehyde, 2,5-diphenyloxazole (PPO, scintilla-
tion grade, 99%), and 1,4-bis(5-phenyloxazol-2-yl) benzene
(POPOP, scintillation grade) were obtained from Acros Organics
(Geel, Belgium). Methanol for HPLC was of chromatographic
Ethyl 3-(4-chlorophenyl)-[3-14C]-oxopropanoate (3)
grade from SK Chemicals (Seoul, Korea), and ultrapure water A THF (dried by Na) solution (2 mL) of 4-chlorobenzoic-[carbonyl-
was prepared on a Milli-Q academic instrument (Millipore, 14C] acid (3.0 mmol, 5.1 mCi/mmol) was stirred under nitrogen.
Alsace, France). The standard of SYP-3343 and the related inter- N, N-carbonyldiimidazole (535 mg, 3.3 mmol) was added to the reac-
mediates (>99%) were provided by the ShenYang Research tion mixture, and stirring was continued for 2 h. THF (5 mL) was
Institute of Chemical Industry in China. Scintillation cocktail was transferred by using a syringe into the reaction mixture, and stirring
prepared as follows: PPO (7.0 g) and POPOP (0.5 g) were dis- was continued for an additional 4 h at room temperature. The reac-
solved in a mixture of xylene (650 mL) and 2-methoxyethanol tion mixture was diluted in t-butyl methyl ether (30 mL) and washed
(350 mL). All other regents were of analytical grade and were with water (30 mL, three times). The organic solution was washed
commercially available and were used without further purifica- with brine, dried over anhydrous Na2SO4, and evaporated to
1
tion. 1H-NMR spectra were measured on a Bruker DRX 300 spec- afford (3) (665 mg, 98%). H-NMR (300 MHz, CDCl3), d(ppm): 7.89
trometer (Bruker, Fallanden, Switzerland), and MS was performed (d, J= 8.7 Hz, 2H, ArH), 7.45 (d, J= 8.7 Hz, 2H, ArH), 4.21
on an Agilent 5973 instrument (Agilent Technologies, Santa (q, J= 7.1 Hz, 2H, CH2), 3.97 (s, 2H, CH2), 1.25 (t, J= 7.1 Hz, 3H, CH3).
a
Mg(OC2H5)2 2C2H5OOCCH2COOH
Mg(OOCCH2COOC2H5)2
94%
1
Cl
Cl
Cl
b, 1
c
*
O
OEt
98%
*
87%
OH
N
N
*
O
O
O
CH3
2
4
3
Cl
d, 4
Cl
O
*
51%
N
O
OCH3
N
O
OCH3
CH3
OCH3
OCH3
5
6
* = 14
C
a, Na, THF, 20-25oC; b, N,N-Carbonyldiimidazole (CDI); c, 40% methylhydrazine in MeOH,
HOAc, 90-100oC; d, DMF, anhydrous K2CO3, 100-110oC
Scheme 1. Synthesis of [14C] SYP-3343.
J. Label Compd. Radiopharm 2011, 54 780–782
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

X.-Y. Liu et al.
3-(4-Chlorophenyl)-1-methyl-1H-[14C]-pyrazol-5-yloxy)methyl (4)
Acknowledgements
To a flask was added methanol (10 mL), acetic acid (1.0 mL), and
(3) (665 mg, 2.9 mmol) in portions together with 40% methyl
hydrazine (407 mg, 3.53 mmol). The mixture was refluxed for
8 h at 90–100ꢀC. The solution was cooled and then was extracted
with dichloromethane and washed with water. The organic
extracts were combined and washed with brine, dried over anhy-
drous Na2SO4, and evaporated to afford (4) (532 mg, 87%).
This work was financially supported by the Natural Science Foun-
dation of China (J20080934), Doctoral Fund of Ministry of Educa-
tion of China (J20081244), Special Fund for Agro-scientific
Research in the Public Interest (201103007), and the Project
of Science Technology Department of Zhejiang Province
(2010R50033). The authors would like to express appreciation
to Dr Miao Li and Professor Changling Liu of ShenYang Research
1H-NMR (500 MHz, CDCl3), d(ppm): 7.60 (d, J = 8.5 Hz, 2H, ArH), Institute of Chemical Industry of China for their kind cooperation
and for providing the raw materials.
7.40 (d, J = 8.5 Hz, 2H, ArH), 3.58 (s, 2H, CH2), 3.41 (s, 3H, NCH3).
REFERENCES
[1] L. Miao, C. L. Liu, J. C. Yang, J. B. Zhang, Z. N. Li, H. Zhang, Z. M. Li, J.
Agri. Food Chem. 2010, 58, 2664–2667.
(E)-2-(2-((3-(4-Chlorophenyl)-1-methyl-1H-14C-pyrazole-5-
yloxy) methyl))phenyl)-3-methoxyacrylate (6)
Compound (4) (532 mg, 2.6 mmol) was added to a solution of
Dimethyl Formamide (DMF) (10 mL), (E)-methyl 2-(2-chloromethyl)
phenyl)-3-methoxyacrylate (5) (675 mg, 2.8 mmol), and anhy-
drous K2CO3 (530 mg, 3.8 mmol) and warmed to 110ꢀC for 4 h. The
reaction was checked by using the TLC trace method, and the mix-
ture was diluted in ethyl acetate (30 mL) and washed with water
(30 mL, three times) and brine, and dried over anhydrous Na2SO4.
The solution was filtered, and the mixture was dissolved in a solu-
tion of petroleum ether (PE) and Dichloromethane (DCM) (volume
ratio 1:1). Silica gel (2.0 g, 100–200 mesh) was added to the solution
and mixed for flash chromatography (PE/Ethyl Acetate (EA) 5:1 by
volume). Fractions containing the product were combined, and
solvent was removed to give impure (6). The impure products were
subjected to semi-preparative HPLC (column: Diamonsil C18, 10mm,
250 Â 10.0 mm; inject volume: 200 mL; methanol : water 80:20, by
volume; flow rate: 4.5 mL/min; detection UV: 254 nm; column tem-
perature: 30ꢀC). Fractions between 7.610 and 8.750 min were col-
lected and concentrated to afford (6) (538 mg, 51%). Chemical
[2] W. Z. Li, Agrochem 2008, 47(7), 489–491.
[3] Z. J. Fan, H. K. Zhang, L. Ma,X. F. Liu, J. Y. Chen, C. L. Liu, J. Sichuan
Norm. Uni. (Natu. Sci.) 2008, 31(3), 358–360.
[4] L. Wang, B. J. Li, W. S. Xiang, Y. X. Shi, C. L. Liu, Agrochem 2008, 47(5),
378–380.
[5] L. Wand, Y. X. Shi, B. J. Li, Chinese J. Pestic. Sci. 2008, 10(4), 417–422.
[6] Y. P. Liu, H. B. Sun, F. J. Zeng, J. M. Liu, Y. L. Xu, S. W. Wang, Guang-
dong Agri. Sci. 2010, 9, 46–47.
[7] H. K. Zhang, Z. J. Fan, L. Ma, Proceedings of 13th Annual Meeting, Chinese
Chemical Society, Pesticide Committee, Jiangxi 2005, 306–312.
[8] J. C. Jia, Z. J. Fan, L. Ma, Proceedings of 13th Annual Meeting,
Chinese Chemical Society, Pesticide Committee, Jiangxi, 2005, 600–611.
[9] N. G. Si, L. Chen, J. L. Liu, Proceedings of 12th Annual Meeting,
Chinese Chemical Society, Pesticide Committee, Xi’an, 2005, 419–427.
[10] M. Li, L. Li, C. L. Liu, Z. M. Li, Z. N. Li, N. G. Shi, Proceedings of 7th
Chinese Annual Meeting for Innovative Pesticide, 2009, 242–246.
[11] C. X. Wu, X. Lu, W. Mu, Chinese J. Pestic. Sci 2002, 4(1), 45–51.
[12] Z. B. Zhang, D. F. Fan, C. F. Qian, China Agri. Pres, Beijing, 1989, 111–128.
[13] D. Ambrost, P. C. Kearney, J. A. Macchia, J. Agri. Food Chem. 1977,
25(4), 868–872.
[14] T. L. Mervosh, G. K. Sims, E. W. Stoller, J. Agri. Food Chem., 1995, 43(3),
537–543.
purity: 98.5%; radiochemical purity: 98.2%; and specific activity: [15] Y. J. Wang, Acta Phytophy. Sin 1994, 26(4), 363–366.
5.04 are determined by using HPLC,20 radio-TLC, and HLPC-LSC
[16] X. M. Ou, Y. J. Wang, G. H. Qiao, Proceedings (abstract) of 15th Inter-
methods.21 HPLC-MS (ESI) m/z: 413.2(M + H+), 414.2(M + H+ + 1),
national Plant Protection Congress, Beijing: Foreign Languages
Press, 2004, 170.
[17] X. M. Ou, H. Pei, X. G. Wang, D. F. Fan, J. Safe. Envir 2006, 6(4), 31–34.
[18] Z. M. Yang, Q. F. Ye, L. Lu, J. Label. Compd. Radiopharm. 2008, 51,
435.1(M + Na+). MS(EI, 70 eV) m/z(%): 412(M+, 56), 145.1(100).
1H-NMR (300 MHz, CDCl3), d(ppm): 7.64 (d, J= 7.0 Hz, 2H, ArH),
182–186.
7.61 (s, 1H, =CH-), 7.55–7.52 (m, 1H, ArH), 7.39–7.36 (m, 2H, ArH),
7.32 (d, J =7.0 Hz, 2H, ArH), 7.22–7.19 (m, 1H, ArH), 5.73 (s, 1H,
Het-H), 5.03 (s, 2H, CH2), 3.83 (s, 3H, NCH3), 3.70 (s, 3H, OCH3), 3.68
(s, 3H, OCH3). The data were consistent with those in the pre-
vious studies.1–3
[19] Z. M. Yang, Z. Q. Ji, Q. F. Ye, W. J. Wu, J. Label. Compd. Radiopharm.
2008, 51, 109–112.
[20] Z. M. Yang, Q. F. Ye, Q. H. Tang, L. Lu, J. Nucl.Agri.Sci. 2006, 20, 423–428.
[21] Z. M. Yang, Q. F. Ye, Q. H. Tang, L. Lu, Z. Y. Chen, Acta Chim. Sin. 2005,
63, 1999–2003.
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J. Label Compd. Radiopharm 2011, 54 780–782