Design, Synthesis, Crystal Structure, and Fungicidal Activity of Two Fenclorim Derivatives

: Two fenclorim derivatives (compounds 6 and 7 ) were synthesized by linking active sub-structures using fenclorim as the lead compound. The chemical structures of the two compounds were conﬁrmed by NMR spectroscopy, high resolution mass spectrometry, and X-ray di ﬀ raction analysis. Their fungicidal activity against six plant fungal strains was tested. Compounds 6 and 7 both crystallized in the monoclinic system, with a P2 1 / c space group (a = 8.4842(6) Å, b = 24.457(2) Å, c = 8.9940(6) Å, V = 1855.0(2) Å 3 , Z = 4) and Cc space group (a = 10.2347(7) Å, b = 18.3224(10) Å, c = 7.2447(4) Å, V = 1357.50(14) Å 3 , Z = 4), respectively. The crystal structure of compound 6 was stabilized by C–H ··· N and C–H ··· O hydrogen bonding interactions and N–H ··· N hydrogen bonds linked the neighboring molecules of compound 7 to form a three-dimensional framework. Compound 6 displayed the most excellent activity, which is much better than that of pyrimethanil against Botrytis cinerea in vivo. Additionally, compound 6 exhibited greater in vitro activity against Pseudoperonospora cubensis compared to that of pyrimethanil. Moreover, compound 7 exhibited strong fungicidal activity against Erysiphe cichoracearum at 50 mg / L in vitro, while pyrimethanil did not. Compounds 6 and 7 could be used as new pyrimidine fungicides in the future.


Introduction
Plant diseases caused by fungi can significantly affect the growth and development of crops such as potato, soybean, and rice, and reduce the yield (20% perennial yield losses and 10% postharvest losses) of crop plants globally [1][2][3][4]. Meanwhile, fungal plant diseases also cause fresh fruit yield loss due to the shortening of storage times and secretion of fungal toxins that can damage human health [5,6]. Chemically synthesized fungicides are a major tool that producers use to protect against plant diseases. However, long-term and unreasonable application of fungicides has led to the emergence of resistance [7][8][9][10][11]. Hence, novel and efficient fungicides are necessary to solve problems arising from current fungicide resistance.
Fenclorim (4,6-dichloro-2-phenyl-pyrimidine, Figure 1a) is a commercial herbicide safener, which could alleviate the injury caused by chloroacetanilide herbicides, especially pretilachlor, without affecting their herbicide activity [12,13]. Zheng et al. [12] showed that fenclorim exhibited excellent in vivo fungicidal activity against Sclerotinia sclerotiorum, Fusarium oxysporum, Fusarium graminearum, and Thanatephorus cucumeris, and could be used as a lead compound to design novel pyrimidine-type fungicides. A fenclorim derivative, named N-(4,6-dichloropyrimidine-2-yl) benzamide, was synthesized by inserting an amide group between the phenyl ring and the pyrimidine ring in fenclorim to study the activity relationship (SAR) against fenclorim. This derivative displayed greater fungicidal activity than that of lead fenclorim and the positive control of pyrimethanil against Sclerotinia sclerotiorum and Fusarium oxysporum, with in vivo IC 50 values of 1.23 and 9.97 mg/L, of pyrimethanil against Sclerotinia sclerotiorum and Fusarium oxysporum, with in vivo IC50 values of 1. 23 and 9.97 mg/L, respectively. These results indicate that the modification of fenclorim can produce highly active fungicidal compounds and that fenclorim provides broad potential as a lead compound for screening fungicides. Azoxystrobin stably held 23-25% of the fungicide market share until 2016. Azoxystrobin is a broad-spectrum fungicide and that displays strong activity against plant fungi such as ascomycetes, deuteromycetes, and oomycetes in crop plants, vegetables, and fruits [14]. Azoxystrobin causes the mitochondrial respiration of pathogenic fungi to be hindered, by binding to the Q0 site of cytochrome bc1 enzyme complex to block electron transfer and freeze adenosine triphosphate (ATP) production [15]. Pyrimethanil, named 4,6-dimethyl-N-phenylpyrimidin-2-amine, was commercialized in 1991 and controlled plant fungi such as pear scab (Venturia pirina) and gray mold (Botrytis cinerea) in agricultural product [16]. It acts as an athogenesis inhibitor to inhibit the secretion of cell wall degrading enzymes in plant fungi [17].
The linking of active sub-structures to compounds is a common method for identifying novel pesticides [18][19][20]. Here, in order to find new fungicide candidates with high efficiency, two fenclorim derivatives (compound 6 and 7) were synthesized via the linking of active sub-structures. This was achieved by combining the (Z)-methyl 2-iodo-3-methoxyacrylate group substituted phenoxy group (red) in azoxystrobin and the aminophenyl group (pink) in pyrimethanil (Scheme 1). The chemical structures of compounds 6 and 7 were confirmed by NMR spectroscopy, high-resolution mass spectrometry (HRMS) and X-ray diffraction analysis. Their fungicidal activity against Botrytis cinerea (B. cinerea), Pseudoperonospora cubensis (P. cubensis), Erysiphe cichoracearum (E. cichoracearum), Blumeria graminis (B. graminis), Rhizoctonia solani (R. solani), and Puccinia polysora (P. polysora) were evaluated. These results provide useful guidance for designing novel fungicides using fenclorim as a lead compound.  Azoxystrobin stably held 23-25% of the fungicide market share until 2016. Azoxystrobin is a broad-spectrum fungicide and that displays strong activity against plant fungi such as ascomycetes, deuteromycetes, and oomycetes in crop plants, vegetables, and fruits [14]. Azoxystrobin causes the mitochondrial respiration of pathogenic fungi to be hindered, by binding to the Q 0 site of cytochrome bc1 enzyme complex to block electron transfer and freeze adenosine triphosphate (ATP) production [15]. Pyrimethanil, named 4,6-dimethyl-N-phenylpyrimidin-2-amine, was commercialized in 1991 and controlled plant fungi such as pear scab (Venturia pirina) and gray mold (Botrytis cinerea) in agricultural product [16]. It acts as an athogenesis inhibitor to inhibit the secretion of cell wall degrading enzymes in plant fungi [17].
The linking of active sub-structures to compounds is a common method for identifying novel pesticides [18][19][20]. Here, in order to find new fungicide candidates with high efficiency, two fenclorim derivatives (compound 6 and 7) were synthesized via the linking of active sub-structures. This was achieved by combining the (Z)-methyl 2-iodo-3-methoxyacrylate group substituted phenoxy group (red) in azoxystrobin and the aminophenyl group (pink) in pyrimethanil (Scheme 1). The chemical structures of compounds 6 and 7 were confirmed by NMR spectroscopy, high-resolution mass spectrometry (HRMS) and X-ray diffraction analysis. Their fungicidal activity against Botrytis cinerea (B. cinerea), Pseudoperonospora cubensis (P. cubensis), Erysiphe cichoracearum (E. cichoracearum), Blumeria graminis (B. graminis), Rhizoctonia solani (R. solani), and Puccinia polysora (P. polysora) were evaluated. These results provide useful guidance for designing novel fungicides using fenclorim as a lead compound.
Crystals 2020, 10, x FOR PEER REVIEW 2 of 13 of pyrimethanil against Sclerotinia sclerotiorum and Fusarium oxysporum, with in vivo IC50 values of 1.23 and 9.97 mg/L, respectively. These results indicate that the modification of fenclorim can produce highly active fungicidal compounds and that fenclorim provides broad potential as a lead compound for screening fungicides. Azoxystrobin stably held 23-25% of the fungicide market share until 2016. Azoxystrobin is a broad-spectrum fungicide and that displays strong activity against plant fungi such as ascomycetes, deuteromycetes, and oomycetes in crop plants, vegetables, and fruits [14]. Azoxystrobin causes the mitochondrial respiration of pathogenic fungi to be hindered, by binding to the Q0 site of cytochrome bc1 enzyme complex to block electron transfer and freeze adenosine triphosphate (ATP) production [15]. Pyrimethanil, named 4,6-dimethyl-N-phenylpyrimidin-2-amine, was commercialized in 1991 and controlled plant fungi such as pear scab (Venturia pirina) and gray mold (Botrytis cinerea) in agricultural product [16]. It acts as an athogenesis inhibitor to inhibit the secretion of cell wall degrading enzymes in plant fungi [17].
The linking of active sub-structures to compounds is a common method for identifying novel pesticides [18][19][20]. Here, in order to find new fungicide candidates with high efficiency, two fenclorim derivatives (compound 6 and 7) were synthesized via the linking of active sub-structures. This was achieved by combining the (Z)-methyl 2-iodo-3-methoxyacrylate group substituted phenoxy group (red) in azoxystrobin and the aminophenyl group (pink) in pyrimethanil (Scheme 1). The chemical structures of compounds 6 and 7 were confirmed by NMR spectroscopy, high-resolution mass spectrometry (HRMS) and X-ray diffraction analysis. Their fungicidal activity against Botrytis cinerea (B. cinerea), Pseudoperonospora cubensis (P. cubensis), Erysiphe cichoracearum (E. cichoracearum), Blumeria graminis (B. graminis), Rhizoctonia solani (R. solani), and Puccinia polysora (P. polysora) were evaluated. These results provide useful guidance for designing novel fungicides using fenclorim as a lead compound.

Chemicals
All chemicals used in this research, including reagents and starting materials, were obtained from the Jilin Chinese Academy of Sciences, Yanshen Technology Co., Ltd., Jilin, China. 1 H and 13 C NMR spectra were recorded using a Bruker Avance-300 spectrometer (Bruker AXS, Karlsruhe, BW, Germany) operating at 300 MHz ( 1 H) and 75 MHz ( 13 C), respectively, with chemical shifts reported in ppm (δ). Deuterated chloroform (CDCl 3 ) was used as the solvent and tetramethylsilane (TMS) was used as the internal standard. HRMS analysis data was obtained on an FTICR-MS Varian 7.0 T FTICR-MS instrument (Varian IonSpec, Lake Forest, CA, USA). Melting points were measured using a Hanon MP100 automatic melting point instrument (Jinan Hanon Instruments Co., Ltd., Jinan, Shandong, China) using an open capillary tube. X-ray crystal structures of compounds 6 and 7 were measured using a Bruker SMART APEX II X-ray single-crystal diffractometer (Bruker AXS, Karlsruhe, BW, Germany). Reagents obtained from commercial sources were used without further purification.

Synthetic Procedure
Target compounds 6 and 7 were synthesized based on methods reported in the literature [21][22][23]. The synthetic routes of target compounds 6 and 7 are described in Scheme 2; Scheme 3. atmosphere. The mixture was then stirred at this temperature for a further 12 h. The mixture was then poured into ice water (100 mL) and extracted with EtOAc (20 mL) twice. The organic extract was evaporated under vacuum and the residue was purified by silica gel column chromatography (1:7 EtOAc /hexane) to afford compound 6 (2.99 g, 79%  (7) A mixture of fenclorim 5 (1.00 g, 4.44 mmol), phenylamine (0.34 g, 3.70 mmol) and TEA (0.50 g, 4.44 mmol), was dissolved in dry N-methylpyrolidin-2-one (NMP, 20 mL) at 120 °C under nitrogen atmosphere for 24 h. The mixture was then cooled to room temperature, and a mixture of EtOAc (20 mL) and saturated sodium chloride solution (20 mL) was added. Then, this mixture was stirred for 30 min. The organic layer was separated, dried using anhydrous sodium sulfate, filtered, and concentrated under a vacuum. Next, the residue was purified using silica gel column chromatography (1:6 EtOAc/hexane) to obtain compound 7 (0.90 g, 72%

Structural Determination
Colorless single crystals of compounds 6 and 7 were obtained by slowly evaporating a methanol solution containing pure compounds 6 and 7 at room temperature. Single crystal X-ray atmosphere. The mixture was then stirred at this temperature for a further 12 h. The mixture was then poured into ice water (100 mL) and extracted with EtOAc (20 mL) twice. The organic extract was evaporated under vacuum and the residue was purified by silica gel column chromatography (1:7 EtOAc /hexane) to afford compound 6 (2.99 g, 79%  (7) A mixture of fenclorim 5 (1.00 g, 4.44 mmol), phenylamine (0.34 g, 3.70 mmol) and TEA (0.50 g, 4.44 mmol), was dissolved in dry N-methylpyrolidin-2-one (NMP, 20 mL) at 120 °C under nitrogen atmosphere for 24 h. The mixture was then cooled to room temperature, and a mixture of EtOAc (20 mL) and saturated sodium chloride solution (20 mL) was added. Then, this mixture was stirred for 30 min. The organic layer was separated, dried using anhydrous sodium sulfate, filtered, and concentrated under a vacuum. Next, the residue was purified using silica gel column chromatography (1:6 EtOAc/hexane) to obtain compound 7 (0.90 g, 72%

Structural Determination
Colorless single crystals of compounds 6 and 7 were obtained by slowly evaporating a methanol solution containing pure compounds 6 and 7 at room temperature. Single crystal X-ray • C for another 12 h and water (30 mL) was added to quench the reaction. The mixture was extracted with dichloromethane (20 mL) twice. The organic extract was washed with saturated aqueous sodium thiosulfate (30 mL) twice, saturated aqueous sodium bicarbonate (30 mL) twice, and water (30 mL) twice. The mixtures were dried using anhydrous sodium sulfate and concentrated under vacuum. The residue was further purified by silica gel column chromatography (1:6 ethyl EtOAc /hexane) to obtain 3 (white solid, 1.56 g, 75.1%).

Structural Determination
Colorless single crystals of compounds 6 and 7 were obtained by slowly evaporating a methanol solution containing pure compounds 6 and 7 at room temperature. Single crystal X-ray diffraction data of compounds 6 and 7 were obtained using a SuperNova, Dual, Cu at zero, AtlasS2 diffractometer (Agilent, CA, USA) equipped with MoKα radiation (λ = 1.54184 Å) at 100.00(10) K. The crystal dimensions of compounds 6 and 7 were 0.11 × 0.11 × 0.08 mm 3 and 0.14 × 0.13 × 0.12 mm 3 , respectively.
A total of 3608 reflections were collected by employing an ω scan mode for compound 6, 6797 of which were independent with R int = 0.1240, R sigma = 0.1384. The final R 1 was 0.0697 (I > 2σ(I)) and wR2 was 0.1869 for compound 6. A total of 4819 reflections were collected by using ω scan mode for compound 7, 1795 of which were unique with R int = 0.0317 and R sigma = 0.0245. The final R 1 was 0.0372 (I > 2σ(I)) and wR 2 was 0.0990. The structures of compounds 6 and 7 were solved using the ShelXT structure solution program by Intrinsic Phasing and refined with the ShelXL refinement package via Least Squares minimization, using Olex2 [24][25][26].
The crystal data and structure refinement details of the compounds 6 and 7 are provided in

Fungicidal Activity
The in vivo fungicidal activity (EC 50 values) of compounds 6, 7, fenclorim, and pyrimethanil against P. cubensis were tested according to methods in the reported literature [27,28]. The fungicidal activities of compounds 6, 7, fenclorim and pyrimethanil against P. cubensis, E. cichoracearum, B. graminis, R. solani, and P. polysora were tested at different concentrations in vitro using methods reported previously [5].

Synthesis and Spectroscopic Properties
The synthetic routes for compounds 6 and 7 are described in Scheme 2; Scheme 3. The chemical structures of compound 6 and 7 were characterized by NMR spectroscopy, HRMS, and X-ray diffraction analysis. For compound 6, signals corresponding to C-H protons in the phenyl ring and pyrimidine were observed at δ 7.21-8.41 and δ 6.61, respectively; signals corresponding to the C-H proton to N-H proton in imino group were observed δ 6.69. For compound 7, signals corresponding to C-H protons in methoxyl groups were observed at δ 3.54 and δ 3.67, respectively; signals corresponding to the C-H proton to C-H proton in pyrimidine ring were observed at δ 6.63, and signals corresponding to the C-H proton in the phenyl ring were at δ 7.24-8.32. In the compound 6 13 C-NMR spectra, chemical shifts of carbons that resonated at δ 107.18-170.47 were assigned to carbons in the phenyl ring and pyridine ring. The chemical shifts of carbons that resonated at δ 51.59 and 61.93, respectively, were assigned to the carbons in methoxyl groups. The signal at δ 104.46 can be assigned to carbon (C-7) in the acrylate group. In the 13 C-NMR spectra of compound 7, chemical shifts linked to aromatic rings (phenyl ring and pyrimidine ring) appeared at 100.5-164.94. All HRMS data for compounds 6 and 7 were well-matched with theoretical values calculated from their chemical formula.

Crystal Structures of Compounds 6 and 7
Compounds 6 and 7 both crystallized in the monoclinic system, with a P2 1 /c space group and Cc space group, respectively. The molecular structures of compounds 6 and 7 are described in Figure 2a,b, and selected molecular structure parameters, including bond lengths and bond angles for compound 6 and 7, are listed in Tables 2 and 3, respectively. Packing diagrams of compounds 6 and 7 are shown in Figure 3a,b respectively.

Fungicidal Activities
The in vivo fungicidal activities (EC 50 values) of compound 6, compound 7, positive control fenclorim and pyrimethanil against B. cinerea are listed in Table 6. Compound 6 displayed the greatest activity with an EC 50 value of 20.84 mg/L, which was much greater than that of compound 7 (215.45 mg/L), fenclorim (319.95 mg/L) and pyrimethanil (30.72 mg/L). This result indicates that the combination of the (Z)-methyl-2-iodo-3-methoxyacrylate group substituted phenoxyl group with fenclorim could improve the in vivo fungicidal activity of fenclorim against Botrytis cinerea. To further study the fungicidal activity of compounds 6 and 7, the in vitro inhibitory rate of compounds 6, 7, fenclorim, and pyrimethanil against P. cubensis, E. cichoracearum, B. graminis, R. Solani, and P. polysora were evaluated at different concentrations (Table 7). At 200 mg/L, compound 6 displayed the strongest fungicidal activity against P. cubensis (94.00% control) and against R. Solani (91.67% control), while compound 7 showed the strongest fungicidal activity against E. cichoracearum (91.00% control), B. graminis (90.67% control) and P. polysora (90.67% control). Furthermore, fenclorim exhibited no activity against the E. cichoracearum control and pyrimethanil only displayed good fungicidal activity against P. cubensis (89.00% control). As the concentration decreased, most of the fungicidal activity of these compounds brought to gradually. Surprisingly, compound 6 exhibited 80.33% activity of that of the control against P. cubensis, when pyrimethanil displayed only 20.67% of that of the control, when at 25 mg/L. These results revealed that the combination of active groups in commercial fungicides with the lead fenclorim could improve the in vitro fungicidal activity of fenclorim significantly.

Conclusions
In conclusion, two fenclorim derivatives (compounds 6 and 7) were synthesized by the linking of active sub-structures method. The chemical structures of the two compounds were confirmed by NMR spectroscopy, HRMS, and X-crystal diffraction, and their fungicidal activity against plant fungi were tested. Compound 6 displayed the greatest activity (EC 50 value of 20.84 mg/L), which was much greater than that of pyrimethanil (30.72 mg/L) against B. cinerea in vivo. Additionally, compound 6 at 25 mg/mL exhibited 80.33% of the control EC 50 value against P. cubensis, when pyrimethanil displayed only 20.67% control EC 50 value at 25 mg/L in vitro. Moreover, compound 7 exhibited 89% of the control EC 50 value against E. cichoracearum at 50 mg/L in vitro, while pyrimethanil only exhibited 6% of that of the control. Compounds 6 and 7 could be used further as pyrimidine fungicides in the future.