Synthesis and fungicidal activity of novel chloro-containing 1-Aryl-3-oxypyrazoles with an oximino ester or oximino amide moiety

Article abstract of DOI:10.3390/molecules19068140

Six novel chloro-containing 1-aryl-3-oxypyrazoles TMa-TMf with an oximino ester or an oximino amide moiety were prepared by the reaction of 1-aryl-1H-pyrazol-3-ols with benzyl bromide. Their structures were characterized by ¹H-NMR, ¹³C-NMR, IR, MS, and elemental analysis. A preliminary in vitro bioassay indicated that compounds TMa, TMe and TMf displayed excellent fungicidal activity against Rhizoctonia solani and could be used as potential lead compounds for further development of novel fungicides.

Full text of DOI:10.3390/molecules19068140

Molecules 2014, 19, 8140-8150; doi:10.3390/molecules19068140
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Fungicidal Activity of Novel Chloro-Containing
1-Aryl-3-oxypyrazoles with an Oximino Ester or Oximino
Amide Moiety
Yuanyuan Liu 1,*, Yi Li 2,*, Nanqing Chen 3, Kunzhi Lv 3, Chao Zhou 1, Xiaohui Xiong 2 and
Fangshi Li 3
1
Department of Chemical and Pharmaceutical Engineering, Southeast University ChengXian
College, Nanjing 210088, China
2
College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211816, China
3
College of Science, Nanjing Tech University, Nanjing 211816, China
* Authors to whom correspondence should be addressed;
E-Mails: liuyuanyuan1985419@163.com (Y.L.); liynj2012@njtech.edu.cn (Y.L.);
Tel./Fax: +86-25-5866-2849 (Y.L.); +86-25-5813-9432 (Y.L.).
Received: 25 April 2014; in revised form: 9 June 2014 / Accepted: 12 June 2014 /
Published: 17 June 2014
Abstract: Six novel chloro-containing 1-aryl-3-oxypyrazoles TMa–TMf with an oximino
ester or an oximino amide moiety were prepared by the reaction of 1-aryl-1H-pyrazol-3-ols
with benzyl bromide. Their structures were characterized by 1H-NMR, 13C-NMR, IR, MS,
and elemental analysis. A preliminary in vitro bioassay indicated that compounds TMa,
TMe and TMf displayed excellent fungicidal activity against Rhizoctonia solani and could
be used as potential lead compounds for further development of novel fungicides.
Keywords: synthesis; fungicidal activity; chloro-containing 1-aryl-3-oxypyrazoles;
structure-activity relationships
1. Introduction
Since the discovery of the strobilurin fungicide azoxystrobin by Syngenta scientists [1], this novel
fungicide class has occupied an important position in the agrochemical market due to its lower
mammalian toxicity, wider spectrum, and higher bioactivity [2–4]. Generally, the chemical structure of
strobilurin fungicides can be divided into three parts: (A) an (E)-β-methyl methoxyiminoacetate or an

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isosteric (E)-β-methyl methoxyacrylate moiety as pharmacophore; (B) an aromatic bridge moiety; and (C)
a side chain. Previous researchers have mainly focused on maintaining the pharmacophore but
changing the side chain, and some new strobilurins such as trifloxystrobin, SYP-1620 and pyraclostrobin
(Figure 1) have been commercialized [5–8]. Our group has also devoted considerable effort to the
development of novel fungicides. Bioactive (alkyl)oxyacetate, thiazolidine-2-thione, (O-acetyl)gluco-
pyranosyl or benzoyl moieties were introduced into the 1-aryl-3-oxypyrazole structure of
pyraclostrobin to replace its methoxycarbamate pharmacophore which could not be prepared in an
environmentally friendly way, and some novel aryloxypyrazoles with good fungicidal activity were
reported [9–11].
Figure 1. Design strategy for the target products TM.
Furthermore, several biological studies have also indicated the 4-chloro-3-oxypyrazole moiety as a
bioactive structure, and side chain modifications such as introduction of chlorine can indeed lead to a
better fungicidal efficacy [12,13]. In continuation of our studies on fungicidal aryloxypyrazoles, the
chlorine and oximino ester or oximino amide pharmacophore of strobilurins were introduced into the
1-aryl-3-oxypyrazole structure of pyraclostrobin according to the principle of active parts combination,
and new target products TM were designed (Figure 1).
In this paper, the synthesis of six novel chloro-containing 1-aryl-3-oxypyrazoles TMa–TMf with an
oximino ester or an oximino amide moiety is reported. Meanwhile, their fungicidal activity has been
investigated with the aim of understanding the structure-activity relationships and developing novel
fungicides. Preliminary in vitro bioassay data indicated that some compounds showed excellent
fungicidal activity against Rhizoctonia solani, especially TMa, TMe and TMf, which displayed a
higher activity than pyraclostrobin at a dosage of 0.1 μg/mL.
2. Results and Discussion
2.1. Chemistry
Intermediate 4-chloro-1-aryl-1H-pyrazol-3-ols IIa–IId were synthesized from arylhydrazines via
addition-cyclization, oxidation and chlorination (Scheme 1) [14,15]. A previous report by Li and Yang

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described that the benzyl bromide (E)-methyl 2-(2-(bromomethyl)phenyl)-2-(methoxyimino)acetate V
could be prepared from 1-(o-tolyl)ethanone via a serial of reactions including oxidation, esterification,
oximation and bromination [16]. However, due to the fact the oxidation was carried out under
heterogeneous conditions, the yield was low, so in our procedure, we optimized the reaction conditions,
and the oxidation was carried out by phase-transfer catalysis with tetrabutylammonium bromide
(Bu4N+Br−) under alkaline conditions, which gave 2-oxo-2-(o-tolyl)acetic acid III in 80% yield
(Scheme 2). In the following step, the esterification of III was carried out in methanol, using
concentrated sulfuric acid as catalyst. The compound obtained was directly used for oximation without
further purification. The bromination of oximino ester IV was carried out in a molar ratio of IV to
N-bromosuccinimide (NBS) 1:1.1 equiv. in CCl4 as solvent, and with benzoyl peroxide (BPO) as catalyst.
Scheme1. Synthesis of intermediate 4-chloro-1-aryl-1H-pyrazol-3-ols IIa–IId.
Scheme 2. Synthesis of intermediate benzyl bromide V.
In our previous studies, we have prepared several ethyl 2-(1,5-diaryl-1H-pyrazol-3-yloxy)acetates
by the substitution reaction of 1,5-diaryl-1H-pyrazol-3-ols with ethyl 2-bromoacetate in acetone, using
potassium carbonate (K2CO3) as acid-binding agent [11]. Motivated by this reaction, in our procedure,
hydroxypyrazoles Ib and IIa–IId were allowed to react with benzyl bromide V in a 1:1.1 molar equiv.
ratio in boiling acetone in the presence of K2CO3, which afforded the target products TMa–TMe in
80%–82% yield as sole isolable products (Scheme 3). The reaction process for TMf was carried out in
a molar ratio of TMe to methylamine in anhydrous methanol (30%) 1:3 equiv. at reflux temperature
until TMe was completely consumed as judged by TLC. The crude product was purified via column
chromatography to give the pure TMf in 90% isolated yield (Scheme 3).
1
The structures of TMa–TMf were confirmed by their NMR spectra. In the H-NMR spectra, as a
result of the deshielding effect of aryl and chloro groups, the CH of the pyrazole ring in TMa–TMd
appeared at low field (δ 7.68–7.81 ppm) as a singlet, whereas the corresponding CH proton in
TMe–TMf appeared at δ 7.65 ppm and δ 5.85 ppm, respectively, as two doublets with coupling
constants around 2.5 Hz. The aromatic protons of TMa–TMf resonated in the range of δ 7.79–7.19 ppm
in 1H-NMR spectra, and the 13C-NMR signals were observed around δ 146.6–114.3 ppm. The chemical

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shifts of MeO H-atoms in TMa–TMe appeared as two singlets around δ 4.06 ppm and δ 3.84 ppm,
respectively, whereas one MeO proton signal in TMf was absent and replaced by the Me proton at δ
13
2.89 ppm. All compounds exhibited a carbonyl (C=O) C signal at the lowest field in the region of
δ 163.9–163.3 ppm.
Scheme 3. Synthesis of target products TMa–TMf.
2.2. Fungicidal Activity
Products TMa–TMf were screened for fungicidal activity against Rhizoctonia solani at the dosages
of 10, 1, and 0.1 μg/mL, respectively. As can be seen in Tables 1 and 2, most compounds have good
fungicidal activity at 10 μg/mL, especially TMa, TMe and TMf, with 100% antifungal activity. When
the concentration was reduced to 0.1 μg/mL, TMe, TMa and TMf also had 99%, 83% and 71%
inhibition rates, which were better than those of the reference compound pyraclostrobin. Compound
TMe had an optimal EC90 lower than 0.1 μg/mL, which was better than TMa (EC90 = 0.20 μg/mL) and
TMf (EC90 = 0.32 μg/mL). The 4-chloropyrazole containing a Cl on the phenyl ring (TMb) displayed
higher activity than that with an electron-donating CF3O (TMc) or electron-withdrawing CF3 (TMd)
group. According to the different positions of chlorine, the sequence of fungicidal activity is
Cl-substituted phenyl ring > Cl-substituted pyrazole ring. For example, the structure only containing a
Cl on the phenyl ring (TMe) showed better fungicidal activity than that on the pyrazole ring (TMa).
However, with increasing number of the Cl groups, the fungicidal activity was decreased, as seen in
the comparison of TMa and TMe vs. TMb, which might because the larger molecular volume was
unfavourable for the intracellular uptake and transport in the fungus. In terms of the pharmacophore,
the compound with an oximino ester moiety (TMe) had better fungicidal activity than that with an
oximino amide (TMf) or a methoxycarbamate moiety (pyraclostrobin). The present work indicated that
TMa, TMe and TMf could be used as potential lead compounds for further studies of novel fungicides.

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Table 1. Antifungal activity of tested compounds (% inhibition).
Rhizoctonia solani a
Compounds
TMa
Structure
10 μg/mL
1 μg/mL
0.1 μg/mL
+++
+++
+++
TMb
+++
+++
+
TMe
TMf
+++
+++
+++
+++
+++
++
TMc
++
+
+
TMd
++
++
+++
+
++
+
Pyraclostrobin
a Activity is expressed in four categories: (−) < 20%, (+) 21%–50%, (++) 51%–80%, and (+++) 81%–100%.
Table 2. Antifungal activity and EC90 values of compounds TMa, TMe and TMf.
concn (μg/mL) a
Compounds
EC90 (μg mL−1)
10
1
0.1
TMa
TMe
TMf
100.00
100.00
100.00
98.23
100.00
94.70
83.40
98.94
71.40
0.20
<0.1
0.32
a 100.00 = total kill.
3. Experimental
3.1. General Information
Melting points were measured on an X-4 microscope electrothermal apparatus (Taike, Beijing,
China) and were uncorrected. NMR spectra were obtained in CDCl3 or DMSO-d6 on a Brucker NMR
1
13
spectrometer operating at 400 MHz for H and 100 MHz for C, with TMS as an internal standard.
Elemental analyses were performed on a Flash EA-1112 elemental analyzer.

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3.2. Synthesis and Characterization
Intermediates 1-aryl-1H-pyrazol-3-ols Ia–Id were prepared according to the reported method, and
the spectral data matched those previously reported [14].
3.2.1. General Procedure for the Synthesis of IIa–IId
A mixture of SOCl2 (5 mL), Ia–Id (1.0 mmol), and a catalytic amount of DMF (0.1 mmol) were
heated under reflux for about 4 h (monitored by TLC), then excess SOCl2 was evaporated and H2O
(200 mL) was added with good stirring. The precipitate that formed was filtered off, washed with H2O, and
then purified by a silica-gel column chromatography (petroleum ether/EtOAc = 5:1) to afford IIa–IId.
4-Chloro-1-phenyl-1H-pyrazol-3-ol (IIa). Yield 80%; White solid; mp 183–184 °C; 1H-NMR (DMSO-d6)
δ 11.00 (s, 1H, OH), 8.54 (s, 1H, CH), 7.69–7.21 (m, 5H, Ar-H). Anal. calcd for C9H7ClN2O: C 55.54,
H 3.63, N 14.39; found C 55.65, H 3.62, N 14.34.
4-Chloro-1-(4-chlorophenyl)-1H-pyrazol-3-ol (IIb). Yield 85%; White solid; mp 204–205 °C;
1H-NMR (CDCl3) δ 7.69 (s, 1H, CH), 7.46–7.39 (m, 4H, Ar-H). Anal. calcd for C9H6Cl2N2O: C 47.19,
H 2.64, N 12.23; found C 47.12, H 2.64, N 12.26.
4-Chloro-1-(4-(trifluoromethoxy)phenyl)-1H-pyrazol-3-ol (IIc). Yield 78%; White solid; mp
1
172–173 °C; H-NMR (CDCl3) δ 7.62 (s, 1H, CH), 7.40 (d, J = 8.8 Hz, 2H, Ar-H), 7.25 (d,
J = 8.8 Hz, 2H, Ar-H). Anal. calcd for C10H6ClF3N2O2: C 43.11, H 2.17, N 10.05; found C 43.33, H
2.13, N 10.12.
4-Chloro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-ol (IId).Yield 76%; White solid; mp 210–211 °C;
1H-NMR (CDCl3) δ 7.74 (s, 1H, CH), 7.66–7.46 (m, 4H, Ar-H). Anal. calcd for C10H6ClF3N2O: C
45.73, H 2.30, N 10.67; found C 45.55, H 2.26, N 10.75.
3.2.2. General Procedure for the Synthesis of III
A mixture of 1-(o-tolyl)ethanone (10 g, 74.6 mmol), H2O (150 mL), NaOH (2 g, 50 mmol) and
Bu4N+Br− (0.1 g, 0.295 mmol) was stirred at 0 °C, then KMnO4 (23 g, 0.146 mol) was added gradually.
The mixture was stirred at 30 °C until the color of the solution remained unchanged. The black
precipitate was filtered off, and the filtrate was neutralized by NaHSO3 solution, acidified with HCl
(36.5%) and filtered. The filtrate was extracted with EtOAc, and then the solvent was removed under
reduced pressure to afford 2-oxo-2-(o-tolyl)acetic acid (III). Yield 80%; White oil; 1H-NMR (CDCl3) δ
7.83–7.25 (m, 4H, Ar-H), 2.52 (s, 3H, CH3). Anal. calcd for C9H8O3: C 65.85, H 4.91; found C 65.62,
H 4.93.
3.2.3. General Procedure for the Synthesis of IV
A mixture of III (5 g, 0.03 mol), H2SO4 (1 mL), and MeOH (100 mL) was heated to reflux until the
starting material had been completely consumed as judged by TLC. The mixture was neutralized by
NaHCO3, and CH3ONH2·HCl (3 g, 0.036 mol) was added. The reaction was monitored by TLC, then

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excess MeOH was removed under reduced pressure. The residue was neutralized by NaHCO3,
extracted by EtOAc, dried, filtered, and evaporated under reduced pressure. It was then purified by a
silica-gel column chromatography (petroleum ether/EtOAc = 8:1) to afford (E)-methyl 2-(methoxy-
1
imino)-2-(o-tolyl)acetate (IV). Yield 70%; White solid; mp 63–64 °C; H-NMR (CDCl3) δ 7.31–7.10
(m, 4H, Ar-H), 4.05 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 2.19 (s, 3H, CH3). Anal. calcd for C11H13NO3:
C 63.76, H 6.32, N 6.76; found C 63.98, H 6.29, N 6.74.
3.2.4. General Procedure for the Synthesis of V
To the solution of IV (5 g, 24.0 mmol) in CCl4 (150 mL) was added NBS (4.7 g, 26.4 mmol) and
BPO (1 g, 4.13 mmol). The reaction mixture was heated to reflux until the starting material had been
completely consumed as judged by TLC. The precipitate was filtered off, and the solvent was
evaporated under reduced pressure. The residue was purified by a silica-gel column chromatography
(petroleum ether/EtOAc = 15:1) to give (E)-methyl 2-(2-(bromomethyl)phenyl)-2-(methoxyimino)-
1
acetate (V). Yield 85%; Yellow oil; H-NMR (CDCl3) δ 7.47-7.13 (m, 4H, Ar-H), 4.32 (s, 2H, CH2),
4.04 (s, 3H, OCH3), 3.85 (s, 3H, OCH3). Anal. calcd for C11H12BrNO3: C 46.18, H 4.23, N 4.90; found
C 46.32, H 4.21, N 4.88.
3.2.5. General Procedure for the Synthesis of TMa–TMe
To a solution of Ib and IIa–IId (1.0 mmol) in acetone (30 mL) was added K2CO3 (1.5 mmol). The
mixture was refluxed for 15 min and V (1.05 mmol) was added slowly. The mixture was refluxed for
about 4 h, then K2CO3 was filtered off. The solvent was evaporated under reduced pressure. The
residue was purified by a silica-gel column chromatography (petroleum ether/EtOAc = 10:1) to afford
products TMa–TMe.
(E)-Methyl 2-(2-{[(4-chloro-1-phenyl-1H-pyrazol-3-yl)oxy]methyl}phenyl)-2-(methoxyimino)acetate
1
(TMa). Yield 80%; White solid; mp 104–105 °C; H-NMR (CDCl3) δ 7.73 (s, 1H, CH), 7.63 (d,
J = 7.4 Hz, 1H, oximino-ArH), 7.53–7.19 (m, 8H, ArH), 5.25 (s, 2H, CH2), 4.04 (s, 3H, MeO), 3.84 (s,
13
3H, MeO). C-NMR (CDCl3) δ 163.3, 158.9, 149.4, 139.7, 134.8, 130.1, 129.5, 129.4, 128.9, 128.5,
128.1, 125.7, 125.6, 117.6, 98.3, 69.5, 63.9, 53.1. IR (KBr): 3147, 2949, 2895, 1734, 1598, 1557, 1511,
1463, 1438, 1406, 1356, 1323, 1223, 1122, 1063, 1012, 984, 947, 895, 862, 795, 756, 684. MS (ESI):
calcd for [M+H]+ 400.11, [M+Na]+ 422.09; found [M+H]+ 400.11, [M+Na]+ 422.09. Anal. calcd for
C20H18ClN3O4: C 60.08, H 4.54, N 10.51; found C 59.83, H 4.58, N 10.57.
(E)-Methyl 2-(2-{[(4-chloro-1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy]methyl}phenyl)-2-(methoxyimino)
acetate (TMb). Yield 82%; White solid; mp 147–148 °C; 1H-NMR (CDCl3) δ 7.68 (s, 1H, CH), 7.61
(d, J = 7.4 Hz, 1H, oximino-ArH), 7.44 (d, J = 8.0 Hz, 2H, N-ArH), 7.40 (d, J = 8.0 Hz, 2H, N-ArH),
7.33 (m, 2H, oximino-ArH), 7.21 (dd, J = 1.6, 7.4 Hz, 1H, oximino-ArH), 5.23 (s, 2H, CH2), 4.03 (s,
3H, MeO), 3.84 (s, 3H, MeO). 13C-NMR (CDCl3) δ 163.3, 159.0, 149.4, 138.2, 134.7, 131.0, 130.1,
129.5, 128.9, 128.5, 128.2, 125.6, 118.6, 98.8, 69.5, 63.9, 53.1. IR (KBr): 3140, 2940, 1721, 1594,
1547, 1495, 1437, 1355, 1327, 1253, 1217, 1114, 1063, 1016, 959, 829, 785, 723, 689. MS (ESI):

Molecules 2014, 19
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calcd for [M+H]+ 434.07, [M+Na]+ 456.05; found [M+H]+ 434.07, [M+Na]+ 456.05. Anal. calcd for
C20H17Cl2N3O4: C 55.31, H 3.95, N 9.68; found C 55.52, H 3.91, N 9.62.
(E)-Methyl
2-(2-{[(4-chloro-1-(4-(trifluoromethoxy)phenyl)-1H-pyrazol-3-yl)oxy]methyl}phenyl)-2-
(methoxyimino)acetate (TMc). Yield 75%; White solid; mp 149–150 °C; 1H-NMR (CDCl3) δ 7.71 (s,
1H, CH), 7.62 (d, J = 7.2 Hz, 1H, oximino-ArH), 7.52 (d, J = 8.4 Hz, 2H, N-ArH), 7.42 (m, 2H,
oximino-ArH), 7.25 (d, J = 8.4 Hz, 2H, N-ArH), 7.21 (dd, J = 1.6, 7.4 Hz, 1H, oximino-ArH), 5.24 (s,
2H, CH2), 4.04 (s, 3H, MeO), 3.84 (s, 3H, MeO). 13C-NMR (CDCl3) δ 163.3, 159.2, 149.4, 146.6,
138.2, 134.7, 130.1, 129.5, 128.9, 128.5, 128.2, 125.7, 122.2, 118.6, 99.0, 69.6, 63.9, 53.0. IR (KBr):
3144, 2946, 1722, 1607, 1558, 1507, 1441, 1393, 1370, 1298, 1261, 1208, 1146, 1115, 1063, 1017,
957, 842, 808, 759, 728, 685. MS (ESI): calcd for [M+H]+ 484.09, [M+Na]+ 506.07; found [M+H]+
484.09, [M+Na]+ 506.06. Anal. calcd for C21H17ClF3N3O5: C 52.13, H 3.54, N 8.68; found C 52.35,
H 3.60, N 8.61.
(E)-Methyl
2-(2-{[(4-chloro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-yl)oxy]methyl}phenyl)-2-
(methoxyimino)acetate (TMd). Yield 83%; White solid; mp 119–120 °C; 1H-NMR (CDCl3) δ 7.81 (s,
1H, CH), 7.79 (s, 1H, N-ArH), 7.68 (d, J = 8.0 Hz, 1H, N-ArH), 7.62 (d, J = 7.4 Hz, 1H,
oximino-ArH), 7.51 (t, J = 7.6 Hz, 1H, N-ArH), 7.42 (m, 3H, oximino-ArH), 7.21 (t, J = 7.6 Hz, 1H,
N-ArH), 5.26 (s, 2H, CH2), 4.05 (s, 3H, MeO), 3.85 (s, 3H, MeO). 13C-NMR (CDCl3) δ 163.3, 159.3,
149.4, 140.0, 134.5, 130.2, 130.1, 129.5, 129.0, 128.5, 128.2, 125.6, 122.1, 122.0, 120.1, 114.3, 99.6,
69.7, 63.9, 53.1. IR (KBr): 3140, 2946, 1730, 1598, 1562, 1518, 1458, 1406, 1358, 1328, 1306, 1226,
1169, 1122, 1067, 1014, 948, 889, 794, 758, 692. MS (ESI): calcd for [M+H]+ 468.09, [M+Na]+
490.08; found [M+H]+ 468.10, [M+Na]+ 490.08. Anal. calcd for C21H17ClF3N3O4: C 53.91, H 3.66, N
8.98; found C 54.12, H 3.60, N 8.92.
(E)-Methyl 2-(2-(((1-(4-chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)phenyl)-2-(methoxyimino)acetate
1
(TMe). Yield 82%; White solid; mp 131–132 °C; H-NMR (CDCl3) δ 7.66 (d, J = 2.4 Hz, 1H, CH),
7.60 (d, J = 7.2 Hz, 1H, oximino-ArH), 7.52 (d, J = 9.0 Hz, 2H, N-ArH), 7.42 (m, 2H, oximino-ArH),
7.36 (d, J = 9.0 Hz, 2H, N-ArH), 7.20 (dd, J = 1.8, 7.2 Hz, 1H, oximino-ArH), 5.85 (d, J = 2.4 Hz, 1H,
13
CH), 5.18 (s, 2H, CH2), 4.08 (s, 3H, OCH3), 3.84 (s, 3H, OCH3). C-NMR (CDCl3) δ 163.7, 163.0,
149.1, 138.3, 134.8, 130.1, 129.4, 129.1, 129.0, 128.1, 127.5, 127.3, 119.2, 118.5, 94.2, 68.7, 63.4,
52.6. IR (KBr): 3142, 2997, 2945, 1740, 1589, 1542, 1475, 1357, 1306, 1225, 1069, 1016, 978, 935, 895,
845, 784, 749. MS (ESI): calcd for [M+H]+ 400.11, [M+Na]+ 422.09; found [M+H]+ 400.10, [M+Na]+
422.09. Anal. calcd for C20H18ClN3O4: C 60.08, H 4.54, N 10.51; found C 59.85, H 4.59, N 10.58.
3.2.6. General Procedure for the Synthesis of TMf
Compound TMe (0.4 g, 1.0 mmol) was dissolved in dried MeOH (40 mL), and then CH3NH2 in
EtOH (0.31 g, 30%) was added slowly. The mixture was heated to reflux until the starting material had
been completely consumed as judged by TLC analysis. The solvent was evaporated under reduced pressure,
and the residue was purified by a silica-gel column chromatography (petroleum ether/EtOAc = 3:1) to
afford TMf.

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(E)-2-(2-(((1-(4-Chlorophenyl)-1H-pyrazol-3-yl)oxy)methyl)phenyl)-2-(methoxyimino)-N-methylacetami
de (TMf). Yield 90%; Yellow oil; 1H-NMR (CDCl3) δ 7.65 (d, J = 2.7 Hz, 1H, CH), 7.58 (d, J = 6.9 Hz,
1H, oximino-ArH), 7.51 (d, J = 9.0 Hz, 2H, N-ArH), 7.43 (m, 2H, oximino-ArH), 7.37 (d, J = 9.0 Hz,
2H, N-ArH), 7.22 (dd, J = 1.8, 6.9 Hz, 1H, oximino-ArH), 6.74 (s, 1H, NH), 5.85 (d,
J = 2.7 Hz, 1H, CH), 5.17 (s, 2H, CH2), 3.95 (s, 3H, OCH3), 2.89 (d, J = 4.8 Hz, 3H, CH3). 13C-NMR
(CDCl3) δ 163.9, 162.6, 150.8, 138.3, 135.0, 130.1, 129.0, 128.9, 128.3, 128.0, 127.4, 127.3, 127.2,
118.5, 94.2, 68.9, 62.9, 25.9. IR (KBr): IR (KBr): 3406, 3147, 2937, 1682, 1595, 1545, 1484, 1456,
1417, 1358, 1301, 1267, 1237, 1166, 1094, 1027, 981, 932, 884, 830, 783, 751. MS (ESI): calcd for
[M+H]+ 399.12, [M+Na]+ 421.10; found [M+H]+ 399.12, [M+Na]+ 421.11. Anal. calcd for C20H19ClN4O3:
C 60.23, H 4.80, N 14.05; found C 60.02, H 4.75, N 14.13.
3.3. Fungicidal Activity Assays
Fungicidal activity of TMa–TMf against Rhizoctonia solani was evaluated according to the
literature procedures [10,17]. Compounds TMa–TMf were dissolved in acetone at a concentration of
10 ppm, 1 ppm and 0.1 ppm, respectively, and then added to a sterile agarized Czapek-Dox medium at
45 °C. The control sample contained only one equivalent of acetone. The media were poured onto
8-cm Petri dishes (10 mL for each dish) and after 2 days inoculated with 5-mm PDA discs of
overgrown mycelium. The medium was inoculated by a prick of laboratory needle containing fungus
spores. The Petri dishes were incubated at r.t. in the dark. After 4 days, the diameters of the inoculation
of the cultures were measured. The percentage inhibition of fungal growth was determined by
comparison between the development of fungi colonies on media containing compounds and on the
control. Three replicates of each test were carried out. Comparative studies involving pyraclostrobin
were carried out under the same conditions using solutions in acetone. For the most active compounds,
concentrations that would give 90% growth inhibition (EC90) were calculated by the statistical method
of linear regression between the activity and the logarithm of the concentration.
4. Conclusions
In summary, six novel chloro-containing 1-aryl-3-oxypyrazoles TMa–TMf with an oximino ester
or an oximino amide moiety were synthesized by the reaction of 1-aryl-1H-pyrazol-3-ols with benzyl
bromide. Their fungicidal activity were tested in vitro against Rhizoctonia solani, and most compounds
displayed good fungicidal activity, especially TMa, with an optimal EC90 lower than 0.1 μg/mL, TMe
and TMf with EC90s of 0.20 and 0.32 μg/mL, respectively, which were better than those of
pyraclostrobin. In terms of the different positions of chlorine, the sequence of fungicidal activity is
Cl-substituted phenyl > Cl-substituted pyrazole. The fungicidal activity was however decreased with
increasing chlorine substitution. The present work indicated that TMa, TMe and TMf could be used as
potential lead compounds for further studies of novel fungicides.
Acknowledgments
The authors gratefully acknowledge the financial supports of the Science Foundation for Youth
Scholars of Jiangsu Province (BK20130749) and the Youth Foundation of Southeast University
ChengXian College (7303600001) of China.

Molecules 2014, 19
8149
Author Contributions
LY, LY, CN, LK, ZC, XX and LF designed research; LY, LY, CN, LK, ZC, XX and LF performed
research and analyzed the data; LY, LY, XX and LF wrote the paper. All authors read and approved
the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the intermediates and target products are available from the authors.
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