Synthesis and Fungicidal Activity of Novel Chloro-Containing 1-Aryl-3-oxypyrazoles with an Oximino Ester or Oximino Amide Moiety

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.


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][3][4]. Generally, the chemical structure of strobilurin fungicides can be divided into three parts: (A) an (E)-β-methyl methoxyiminoacetate or an OPEN ACCESS 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][6][7][8]. Our group has also devoted considerable effort to the development of novel fungicides. Bioactive (alkyl)oxyacetate, thiazolidine-2-thione, (O-acetyl)glucopyranosyl 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][10][11]. 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.

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 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 (Bu 4 N + 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 CCl 4 as solvent, and with benzoyl peroxide (BPO) as catalyst. 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 (K 2 CO 3 ) 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 K 2 CO 3 , 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).
The structures of TMa-TMf were confirmed by their NMR spectra. In the 1 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 1 H-NMR spectra, and the 13 C-NMR signals were observed around δ 146.6-114.3 ppm. The chemical 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 2.89 ppm. All compounds exhibited a carbonyl (C=O) 13 C signal at the lowest field in the region of δ 163.9-163.3 ppm. Scheme 3. Synthesis of target products TMa-TMf.

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 EC 90 lower than 0.1 μg/mL, which was better than TMa (EC 90 = 0.20 μg/mL) and TMf (EC 90 = 0.32 μg/mL). The 4-chloropyrazole containing a Cl on the phenyl ring (TMb) displayed higher activity than that with an electron-donating CF 3 O (TMc) or electron-withdrawing CF 3 (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.

General Information
Melting points were measured on an X-4 microscope electrothermal apparatus (Taike, Beijing, China) and were uncorrected. NMR spectra were obtained in CDCl 3 or DMSO-d 6 on a Brucker NMR spectrometer operating at 400 MHz for 1 H and 100 MHz for 13 C, with TMS as an internal standard. Elemental analyses were performed on a Flash EA-1112 elemental analyzer.

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].

General Procedure for the Synthesis of IIa-IId
A mixture of SOCl 2 (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 SOCl 2 was evaporated and H 2 O (200 mL) was added with good stirring. The precipitate that formed was filtered off, washed with H 2 O, and then purified by a silica-gel column chromatography (petroleum ether/EtOAc = 5:1) to afford IIa-IId.

General Procedure for the Synthesis of IV
A mixture of III (5 g, 0.03 mol), H 2 SO 4 (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 NaHCO 3 , and CH 3 ONH 2 ·HCl (3 g, 0.036 mol) was added. The reaction was monitored by TLC, then excess MeOH was removed under reduced pressure. The residue was neutralized by NaHCO 3 , 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-(methoxyimino)-2-(o-tolyl)acetate (IV

General Procedure for the Synthesis of V
To the solution of IV (5 g, 24.0 mmol) in CCl 4 (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)acetate (V

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 (EC 90 ) were calculated by the statistical method of linear regression between the activity and the logarithm of the concentration.

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 EC 90 lower than 0.1 μg/mL, TMe and TMf with EC 90 s 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.