**1. 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 [1–4]. 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 [7–11]. 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<sup>50</sup> 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. of pyrimethanil against *Sclerotinia sclerotiorum* and *Fusarium oxysporum*, with in vivo IC<sup>50</sup> 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. *Crystals* **2020**, *10*, x FOR PEER REVIEW 2 of 13 of pyrimethanil against *Sclerotinia sclerotiorum* and *Fusarium oxysporum*, with in vivo IC<sup>50</sup> values of

1.23 and 9.97 mg/L, respectively. These results indicate that the modification of fenclorim can

**Figure 1.** Chemical structure of fenclorim (**a**), azoxystrobin (**b**), and pyrimethanil (**c**). **Figure 1.** Chemical structure of fenclorim (**a**), azoxystrobin (**b**), and pyrimethanil (**c**). (a) (b) (c)

Azoxystrobin (Figure 1b) and pyrimethanil (Figure 1c) are commercial pyrimidine fungicides. Azoxystrobin, named methyl (*E*)-2-{[6-(2-cyanophenoxy)-4-pyrimidinyl]oxy}-alpha-(methoxy methylene)benzeneacetate, belongs to the strobilurin fungicides, and was commercialized in 1996. 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<sup>0</sup> 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]. Azoxystrobin (Figure 1b) and pyrimethanil (Figure 1c) are commercial pyrimidine fungicides. Azoxystrobin, named methyl (*E*)-2-{[6-(2-cyanophenoxy)-4-pyrimidinyl]oxy}-alpha-(methoxy methylene)benzeneacetate, belongs to the strobilurin fungicides, and was commercialized in 1996. 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<sup>0</sup> 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]. **Figure 1.** Chemical structure of fenclorim (**a**), azoxystrobin (**b**), and pyrimethanil (**c**). Azoxystrobin (Figure 1b) and pyrimethanil (Figure 1c) are commercial pyrimidine fungicides. Azoxystrobin, named methyl (*E*)-2-{[6-(2-cyanophenoxy)-4-pyrimidinyl]oxy}-alpha-(methoxy methylene)benzeneacetate, belongs to the strobilurin fungicides, and was commercialized in 1996. 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<sup>0</sup> 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

The linking of active sub-structures to compounds is a common method for identifying novel pesticides [18–20]. 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 *s*pectrometry (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. The linking of active sub-structures to compounds is a common method for identifying novel pesticides [18–20]. 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. 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 [18–20]. 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 *s*pectrometry (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.

**Scheme 1.** Design strategies for compounds **6** and **7**. **Scheme 1.** Design strategies for compounds **6** and **7**. **Scheme 1.** Design strategies for compounds **6** and **7**.

#### **2. Materials and Methods** (1:7 EtOAc /hexane) to afford compound **6** (2.99 g, 79%). Compound **6** was a white solid with the following characteristics: m.p. 121–122 °C; 1H NMR (300 MHz, CDCl3) *δ* [ppm]: 3.54 (s, 3H, OCH3), (1:7 EtOAc /hexane) to afford compound **6** (2.99 g, 79%). Compound **6** was a white solid with the

#### *2.1. Chemicals* 3.67 (s, 3H, OCH3), 6.63 (s, 1H, PyH), 7.24–7.26 (m, 2H, ArH), 7.32–7.35 (m, 2H, ArH), 7.39–7.44 (m, 3.67 (s, 3H, OCH3), 6.63 (s, 1H, PyH), 7.24–7.26 (m, 2H, ArH), 7.32–7.35 (m, 2H, ArH), 7.39–7.44 (m,

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. <sup>1</sup>H and <sup>13</sup>C NMR spectra were recorded using a Bruker Avance-300 spectrometer (Bruker AXS, Karlsruhe, BW, Germany) operating at 300 MHz (1H) and 75 MHz (13C), respectively, with chemical shifts reported in ppm (δ). Deuterated chloroform (CDCl3) 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. 4H, ArH+CH), 8.28–8.32 (m, 2H, ArH); 13C NMR (75 MHz, CDCl3) *δ* [ppm]:170.5, 167.4, 165.0, 162.0, 161.0, 150.3, 135.7, 132.7, 131.6, 129.1, 128.7, 128.5, 126.1, 125.9, 122.0, 107.2, 104.5, 61.8, 50.8 HRMS (ESI+) *m*/*z*: 397.0950 ([M+H]<sup>+</sup> ); found: 397.0946. 2.2.5. 6-chloro-N-2-diphenylpyrimidin-4-amine (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%). Compound **7** was a white 4H, ArH+CH), 8.28–8.32 (m, 2H, ArH); <sup>13</sup>C NMR (75 MHz, CDCl3) *δ* [ppm]:170.5, 167.4, 165.0, 162.0, 161.0, 150.3, 135.7, 132.7, 131.6, 129.1, 128.7, 128.5, 126.1, 125.9, 122.0, 107.2, 104.5, 61.8, 50.8 HRMS (ESI+) *m*/*z*: 397.0950 ([M+H]<sup>+</sup> ); found: 397.0946. 2.2.5. 6-chloro-N-2-diphenylpyrimidin-4-amine (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

*Crystals* **2020**, *10*, x FOR PEER REVIEW 4 of 13

*Crystals* **2020**, *10*, x FOR PEER REVIEW 4 of 13

atmosphere. The mixture was then stirred at this temperature for a further 12 h. The mixture was

was evaporated under vacuum and the residue was purified by silica gel column chromatography

was evaporated under vacuum and the residue was purified by silica gel column chromatography

following characteristics: m.p. 121–122 °C; 1H NMR (300 MHz, CDCl3) *δ* [ppm]: 3.54 (s, 3H, OCH3),

#### *2.2. Synthetic Procedure* solid with the following characteristics: m.p. 95-96 °C; 1H NMR (300 MHz, CDCl3) *δ* [ppm]: 6.61 (s, chromatography (1:6 EtOAc/hexane) to obtain compound **7** (0.90 g, 72%). Compound **7** was a white solid with the following characteristics: m.p. 95-96 °C; 1H NMR (300 MHz, CDCl3) *δ* [ppm]: 6.61 (s,

122.6, 100.5; HRMS (ESI+) *m*/*z*: 282.0790 ([M+H]<sup>+</sup>

Target compounds 6 and 7 were synthesized based on methods reported in the literature [21–23]. The synthetic routes of target compounds **6** and **7** are described in Scheme 2; Scheme 3. 1H, PyH), 6.92 (s, 1H, NH), 7.21–7.27 (m, 1H, ArH), 7.36–7.50 (m, 7H, ArH), 8.37–8.41 (m, 2H, ArH); <sup>13</sup>C NMR (75 MHz, CDCl3) *δ* [ppm]: 164.9, 162.1, 160.9, 137.5, 136.5, 131.1, 129.5, 128.4, 128.3, 125.4, 122.6, 100.5; HRMS (ESI+) *m*/*z*: 282.0790 ([M+H]<sup>+</sup> ); found: 282.0793. 1H, PyH), 6.92 (s, 1H, NH), 7.21–7.27 (m, 1H, ArH), 7.36–7.50 (m, 7H, ArH), 8.37–8.41 (m, 2H, ArH); <sup>13</sup>C NMR (75 MHz, CDCl3) *δ* [ppm]: 164.9, 162.1, 160.9, 137.5, 136.5, 131.1, 129.5, 128.4, 128.3, 125.4,

); found: 282.0793.

**Scheme 2.** The synthetic route of target compound **6**. **Scheme 2.** The synthetic route of target compound **6**. **Scheme 2.** The synthetic route of target compound **6**.

**Scheme 3.** The synthetic route of target compound **7**. **Scheme 3.** The synthetic route of target compound **7**. **Scheme 3.** The synthetic route of target compound **7**.

*2.3. Structural Determination 2.3. Structural Determination* 2.2.1. Synthesis of (Z)-methyl 2-iodo-3-methoxyacrylate (**2**)

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 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 A mixture of **1** (1.00 g, 8.61 mmol), *N*-iodosuccinimide (NIS, 2.32 g, 10.33 mmol), glacial acetic acid (0.98 mL, 17.22 mmol), and dichloromethane (15 mL) was stirred at 20 ◦C for 24 h. Afterwards, triethylamine (TEA, 4.2 mL, 30 mmol) was added dropwise. The reaction mixture was then stirred at

20 ◦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%).
