English

• 1.   Introduction

  Succinate dehydrogenase (SDH, also known as complex II), which catalyzes the oxidation of succinate to fumarate in the mitochondrial matrix, is the only enzyme complex simultaneously involved in the respiration chain and tricarboxylic acid cycle.^[1-3] Due to its crucial role in life processes, SDH has been particularly considered as a promising target for agrochemical discovery. Succinate dehydrogenase inhibitor (SDHI) fungicides are an important category of fungicides. SDHIs have been widely used to control destructive plant pathogenic fungi. They exert their fungicidal activities by disrupting the mitochondrial tricarboxylic acid cycle and respiration chain.^[4] Moreover, they have no cross-resistance with other commercial fungicides because of their unique mode of action.^[4] Therefore, SDHIs have been extensively investigated in the world.^[5-8] Eighteen SDHIs have been successfully developed and employed since the first SDHI carboxin was used in 1966. Meanwhile, the researches and innovations of SDHIs have been the frontier of the fungicidal field and novel SDHIs are ceaselessly reported.^[9-12] These SDHIs have the following common features in molecular structure: (a) amide scaffolds, (b) halogen atoms, methyl groups, methoxy groups or (c) heterocyclic rings, such as furan rings, thiophene rings, pyrazole rings, pyridine rings (Figure 1).

  Figure 1

  

  Figure 1.  Representatives of succinate dehydrogenase inhibitor fungicides

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  Pyrimidine derivatives are an important class of compounds. They have been found in a variety of natural products, such as nucleic acids and thiamine (vitamin B1), which are important to the research and development of pharmaceutical and agrochemical compounds. In the meantime, pyrimidine derivatives possess diverse and important biological properties, including antibacterial, ^[13-15] anticancer, ^[16-19] antiviral, ^[20-21] anti-inflammatory, ^[22-24] analgesic, ^{[22, 24]} antimalarial, ^[25-26] anti-tuberculosis, ^[27-28] anticonvulsant^[29-30] and antioxidant.^[22] In addition, pyrimidine derivatives are important fungicides in the agrochemical field. Dozens of fungicides containing pyrimidine rings have been successfully developed and employed since the first pyrimidine fungicide ethirimol was used in 1968 (Figure 2). Currently, structural modifications on pyrimidine rings are still ongoing to discover more bioactive compounds.

  Figure 2

  

  Figure 2.  Representatives of commercial pyrimidine derivative fungicides

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  As mentioned above, in the present study, thirty-six new pyrimidine analogues were designed in accordance with the principle of splicing bioactive substructures and the following modifications of pyrimidine rings: (a) substituting the hydrogen atom at position-2 with the methyl group, pyrazole ring or amide moiety, (b) substituting the hydrogen atom at position-4 with the furan ring/5-methyl furan ring or thiophene ring/5-bromothiophen ring and (c) substituting the hydrogen atom at position-6 with the phenyl ring substituted by the fluorine atom, chlorine atom, bromine atom, methyl group or methoxy group (Scheme 1). Meanwhile, the designed compounds were synthesized and their antifungal activities were evaluated in the laboratory to discover novel SDHIs with simple structures and excellent performances. Molecular docking studies were conducted to detect the probable interactions between the active compounds and SDH.

  Scheme 1

  

  Scheme 1.  Design of target compounds

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  2.   Results and discussion

  2.1   Chemistry

  The syntheses and chemical structures of the target compounds are shown in Scheme 2. The syntheses of the designed compounds were performed according to the methods reported previously and modified appropriately.^[31-32] Intermediate 1 was obtained by Claisen-Schmidt condensation reaction and the characterizing data of these compounds conformed to the literatures.^[33-36] The targetcompounds (2a~2l, 3a~3l and 4a~4l) were synthesized by Michael addition between the α, β-unsaturated C＝C double bond of intermediate 1 and acetimidamide, pyrazole-1-carboximidamide or guanyl urea in the presence of base, which was followed by a cyclization reaction. All the synthesized compounds were purified by crystallization or silica gel chromatography. Their purity was checked by thin layer chromatography (TLC). The structures of all the synthesized compounds were confirmed by IR, ¹H NMR, ¹³C NMR and HRMS. The results showed that their spectrum data were in agreement with the proposed structures.

  Scheme 2

  

  Scheme 2.  Synthesis of target compounds

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  2.2   Antifungal activity

  Compared with the efficient fungicide fluopyram, all the synthesized compounds were submitted to laboratorial assay using R. solani, F. graminearum, H. maydis, S. sclerotiorum and B. cinerea as targets. The results are listed in Table 1. Most of the target compounds displayed remarkable antifungal activities at 20 mg/L. Among them, 2a, 2c, 2f, 3d, 3j, 4b and 4j possessed excellent antifungal activities and their inhibitory rate reached 100% at 20 mg/L against S. sclerotiorum. Moreover, compounds 2c and 3d showed better activities than fluopyram against R. solani and F. graminearum at 20 mg/L.

  Table 1

  

  Table 1.  Antifungal activity of target compounds at 20 mg/L

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  Compound	Inhibition ratea/%
  	R. solani	F. graminearum	H. maydis	S. sclerotiorum	B. cinerea
  2a	51.8±2.2	30.1±0.9	88.2±3.5	100.0±0.0	76.5±3.5
  2b	39.0±1.3	26.6±1.1	50.3±2.0	80.1±3.8	43.8±1.9
  2c	75.6±2.9	68.2±3.3	91.6±4.1	100.0±0.0	83.2±2.8
  2d	58.3±2.6	40.1±1.9	83.4±3.8	98.6±1.1	73.3±3.2
  2e	56.7±2.0	43.4±1.3	81.7±3.4	96.8±2.8	78.2±3.1
  2f	61.9±2.4	45.5±2.2	86.7±3.8	100.0±0.0	80.5±3.5
  2g	32.8±1.3	26.4±0.9	51.9±2.1	64.3±2.1	40.3±1.9
  2h	30.5±0.7	21.3±0.3	55.0±2.3	68.5±2.2	44.7±1.8
  2i	44.5±0.5	30.5±0.9	67.5±2.8	73.3±3.3	51.8±2.2
  2j	41.2±1.3	38.9±1.0	65.1±2.7	68.0±2.8	55.4±1.9
  2k	51.1±1.2	36.5±1.6	84.3±4.0	94.7±2.4	77.9±2.8
  2l	50.3±1.7	38.1±1.8	70.3±3.1	81.2±4.0	58.4±2.1
  3a	60.3±2.2	48.5±2.1	84.7±3.6	94.9±3.8	74.5±3.3
  3b	54.1±1.9	44.2±2.0	82.5±3.6	97.6±2.0	77.0±2.5
  3c	40.7±1.7	33.2±1.3	68.2±2.7	82.7±2.6	59.2±2.5
  3d	74.1±2.3	67.9±2.5	90.3±3.4	100.0±0.0	81.5±3.2
  3e	40.8±2.1	36.4±1.5	67.2±2.6	75.6±3.2	54.3±2.0
  3f	32.0±0.8	23.5±1.0	56.4±2.3	66.0±3.1	47.6±1.4
  3g	30.9±0.9	20.2±0.2	53.8±2.1	68.0±2.7	47.9±2.5
  3h	35.2±1.6	22.1±0.3	56.4±2.4	68.7±3.1	50.5±1.6
  3i	60.0±2.2	42.6±1.9	83.5±3.7	97.9±1.7	75.3±2.9
  3j	57.0±2.3	49.3±1.5	88.6±4.2	100.0±0.0	76.1±2.4
  3k	46.3±1.5	37.6±1.4	61.3±2.0	78.2±3.1	55.7±2.8
  3l	45.7±1.9	30.6±1.0	67.0±2.6	84.3±3.9	58.1±2.0
  4a	51.4±1.3	42.4±1.7	83.2±3.9	97.2±1.9	75.1±2.1
  4b	60.9±2.9	48.2±2.2	87.6±4.0	100.0±0.0	74.3±3.3
  4c	32.6±0.7	25.3±1.3	51.9±2.6	65.3±2.3	43.5±2.0
  4d	39.1±1.3	28.6±0.4	61.4±3.0	73.8±2.3	58.0±1.8
  4e	52.7±1.7	41.1±1.8	81.9±3.5	94.1±3.1	60.2±2.4
  4f	32.0±1.0	20.9±1.1	50.6±2.3	61.3±2.8	41.6±1.0
  4g	33.2±1.4	23.6±0.6	52.8±2.2	60.2±2.5	43.2±1.3
  4h	31.7±1.5	26.9±0.9	45.1±1.8	55.1±1.3	38.4±1.4
  4i	50.8±1.7	45.6±1.2	77.8±3.3	95.7±2.1	65.7±1.2
  4j	60.6±2.4	45.3±1.6	87.6±4.3	100.0±0.0	76.9±2.2
  4k	41.6±1.5	35.4±0.6	60.3±2.7	71.2±1.0	52.3±2.4
  4l	51.5±2.3	41.8±1.5	78.9±3.1	92.3±3.3	58.2±2.2
  Fluopyram	71.4±1.9	62.9±1.0	100.0±0.0	100.0±0.0	100.0±0.0
  a Data are the mean±standard deviation (SD) of three replicates.

  As shown in Table 1, all the synthesized compounds showed inhibitory activities against the above five plant pathogenic fungi in the following order: S. sclerotiorum > H. maydis > B. cinerea > R. solani > F. graminearum. If compounds 2c, 3b, 4a and 4b were compared with compounds 2e, 3e, 4c and 4e, respectively, it could be found that when the hydrogen atom at position-5 of the furan ring was substituted by a methyl group, the antifungal activity of the corresponding compounds decreased, which needs to be further studied. Likewise, when compounds 2c, 3b, 3c, 4a and 4b were compared with compounds 2h, 3g, 3h, 4f and 4h, respectively, it was noticeable that when the furan ring of these compounds was changed into a thiophene ring, the antifungal activities of the corresponding compounds also decreased. However, if compounds 2h, 2i, 3f, 3g, 3h, 4f, 4g and 4h were contrasted with compounds 2k, 2l, 3i, 3k, 3l, 4i, 4k and 4l, it could be found that when the hydrogen atom at position-5 of the thiophene ring was replaced by a bromine atom, the antifungal activities of the corresponding compounds increased, whose causes also deserve to be further investigated.

  The EC₅₀ values of the compounds with excellent effects against S. sclerotiorum were further determined to investigate their antifungal activities. The results are displayed in Table 2 and Figure 3. The EC₅₀ values of seventeen compounds (2a, 2c~2f, 2k, 3a, 3b, 3d, 3i, 3j, 4a, 4b, 4e, 4i, 4j and 4l) ranged from 0.072~1.230 mg/L. Among them, the EC₅₀ values of compounds 2c and 3d reached 0.072 and 0.077 mg/L, respectively, whereas the EC₅₀ value of fluopyram was 0.244 mg/L, indicating that they possessed better inhibitory activities than fluopyram. Similarly, in accordance with the EC₅₀ values of compounds 2a, 2f, 3j, 4b and 4j, it can be known that their inhibitory activities were close to fluopyram.

  Table 2

  

  Table 2.  EC₅₀ values of the compounds with excellent effects against S. sclerotiorum

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  Compound	EC50a/(mg·L-1)
  2a	0.258±0.024
  2c	0.072±0.010
  2d	0.632±0.081
  2e	0.768±0.091
  2f	0.272±0.027
  2k	1.003±0.102
  3a	0.984±0.110
  3b	0.711±0.082
  3d	0.077±0.010
  3i	0.704±0.081
  3j	0.299±0.030
  4a	0.740±0.079
  4b	0.267±0.026
  4e	1.106±0.161
  4i	0.807±0.091
  4j	0.327±0.032
  4l	1.230±0.186
  Fluopyram	0.244±0.019
  a Data are the mean±standard deviation (SD) of three replicates.

  Figure 3

  

  Figure 3.  Comparison of EC₅₀ values among compounds with excellent effects against S. sclerotiorum

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  Although definite structure-activity relationships could not be found through the above-mentioned findings, some interesting results obtained can be used for designing and synthesizing more analogues to further study their quantitative structure-activity relationship so that more bioactive compounds or lead compounds may be discovered. For example, compounds 2c and 3d might be further used as lead compounds because they exhibited excellent inhibitory activities and had simple structures.

  2.3   Inhibitory activity against enzyme

  The assay of the inhibitory activity against SDH was performed to investigate whether SDH is a potential target enzyme of the synthesized compounds. Using fluopyram as the comparative standard, the inhibitory activities of compounds 2c and 3d were tested against the SDH from the mitochondria of S. sclerotiorum. The results are listed in Table 3. The IC₅₀ values of compounds 2c and 3d were 0.115, 0.121 mg/L, respectively, which revealed that they possessed better inhibitory activity than fluopyram (IC₅₀＝0.356 mg/L). Therefore, the findings implied that SDH may be probably one action target of these compounds.

  Table 3

  

  Table 3.  Inhibitory activities of compounds 2c and 3d against SDH

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  Compound	IC50a/(mg·L-1)
  2c	0.115±0.011
  3d	0.121±0.012
  Fluopyram	0.356±0.040
  a Data are the mean±standard deviation (SD) of three replicates.

  2.4   Homology modelling and molecular docking analysis

  The theoretical binding modes of compounds 2c, 3d and fluopyram were simulated to investigate whether SDH is a potential target enzyme of the target compounds. Their theoretical binding modes to SDH are displayed in Figure 4. Fluopyram fitted in the gap composed of subunits B, C and D (Figure 4A). The 2-trifluoromethylphenyl group of fluopyram was located in the hydrophobic pocket surrounded by residues B/Pro-226, B/Trp-230 and B/Ile-275, whereas the 3-chloro-5-(trifluoromethyl) pyridine ring of fluopyram stretched into the hydrophobic pocket that consisted of residues C/Trp-81, C/Ile-82 and C/Ile-89, forming strong hydrophobic binding. In addition, the 2-trifluoro- methylphenyl group of fluopyram formed a cation-π interaction with residue C/Arg-88.

  Figure 4

  

  Figure 4.  Theoretical binding modes of fluopyram (A), compound 2c (B), fluopyram and compound 2c (C), compound 3d (D), fluopyram and compound 3d (E) to SDH

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  The binding mode of compound 2c to SDH is illustrated in Figure 4B. Compound 2c also fitted in the gap composed of subunits B, C and D. It was located in the hydro phobic pocket surrounded by residues B/Pro-226, B/Trp- 230, B/Ile-275, C/Leu-72, C/Trp-81 and C/Ile-82, forming strong hydrophobic binding. Like fluopyram, the furan ring of compound 2c also formed a cation-π interaction with residue C/Arg-88. Besides, compound 2c and residue B/Trp-230 formed one hydrogen bond with a length of 3.1 Å.

  As mentioned above, compound 2c and fluopyram shared similar binding modes (Figure 4C). The main difference was that compound 2c formed an extra hydrogen bond with residue B/Trp-230 when compared with fluopyram, which might make compound 2c more active than fluopyram against SDH. Meanwhile, the estimated binding energy of compound 2c and fluopyram was -32.2 and -28.9 kJ/mol, respectively, which implied compound 2c had stronger affinity to SDH than fluopyram.

  The binding mode of compound 3d to SDH is outlined in Figure 4D. Compound 3d also fitted in the gap composed of subunits B, C and D. It was located in the hydrophobic pocket surrounded by residues B/Trp-230, B/Ile- 275, C/Leu-72, C/Trp-81, C/Ile-82 and C/Ile-89, forming strong hydrophobic binding. The 4-chlorophenyl group of compound 3d formed a π-π stacking interaction with residue C/Trp-81. Like fluopyram, the pyrazole ring of compound 3d also formed a cation-π interaction with residue C/Arg-88. In addition, compound 3d and residue B/Trp- 230 formed one hydrogen bond with a length of 3.2 Å.

  In a word, compound 3d and fluopyram also had similar binding modes (Figure 4E). The main difference was also that compound 3d formed an extra hydrogen bond with residue B/Trp-230 when compared with fluopyram, which might make compound 3d more active than fluopyram against SDH. Furthermore, the estimated binding energy of compound 3d and fluopyram was -31.8 and -28.9 kJ/mol, respectively, which signified compound 3d had stronger affinity to SDH than fluopyram.

  The above molecular docking results displayed that compounds 2c and 3d might be potential inhibitors of SDH. Meanwhile, the different binding energy between SDH and compounds 2c, 3d or fluopyram revealed that compounds 2c and 3d possessed stronger affinity to SDH than fluopyram, which was in agreement with the results of the antifungal activity assay. These results rendered rational explanations of the interactions between SDH and compounds 2c, 3d or fluopyram and provided some valuable information for the further research and development of SDHI fungicides.

  3.   Conclusions

  Thirty-six pyrimidine derivatives were designed, synthesized and tested for their antifungal activities against the above five phytopathogenic fungi. The seven compounds (2a, 2c, 2f, 3d, 3j, 4b and 4j) with excellent antifungal activity were discovered against S. sclerotiorum. They have simple structures and are easily synthesized. In particular, compounds 2c and 3d exhibited potent activities against S. sclerotiorum and their antifungal activities even surpassed fluopyram. They can be further used as lead compounds to design and synthesize more analogues for the discovery of more active compounds. The results of molecular docking studies may be conducive to further exploring the possible fungicidal mechanism of the above compounds and the interactions between similar fungicidal compounds and SDH.

  4.   Experimental section

  4.1   General

  IR spectra were recorded on a Nicolet 380 spectrometer. NMR spectra (¹H NMR and ¹³C NMR) were taken on an AVANCE NEO 400 spectrometer using deuterated dimethyl sulfoxide (DMSO-d₆) as the solvent. HRMS data were taken on an LTQ Orbitrap Elite mass spectrometer. Melting points were determined using an X-4B micro-melting point apparatus and were not corrected. All chemicals and solvents were purchased from commercial sources unless specified otherwise. R. solani, F. graminearum, H. maydis, S. sclerotiorum and B. cinerea were obtained from the Chinese Academy of Agricultural Sciences. They were preserved at 4 ℃.

  4.2   Synthesis of target compounds

  4.2.1   Synthesis of intermediate 1

  As shown in Scheme 1, in accordance with the methods reported previously, ^[33-36] a mixture of selected aldehyde (0.01 mol), substituted acetophenone (0.01 mol) and anhydrous ethanol (20 mL) was stirred in an ice bath. 10% NaOH anhydrous ethanol (5 mL) was added to the mixture. The reaction was kept at 0~5 ℃ for 3~4 h. The course of the reaction was monitored by thin-layer chromatography (TLC). After completion, distilled water was added to the reaction mixture. The pH value of the mixture was adjusted to about 7 by 10% HCl solution. The solid that precipitated was filtered and washed with distilled water. The obtained crude product was recrystallized from anhydrous ethanol to afford intermediate 1.

  4.2.2   Synthesis of target compounds 2a~2l

  As shown in Scheme 1, a mixture of intermediate 1 (0.005 mol), anhydrous ethanol (10 mL) and 10% NaOH anhydrous ethanol (5 mL) was stirred at room temperature. The solution of acetimidamide hydrochloride (0.01 mol) and anhydrous ethanol (10 mL) was slowly added to the above mixture. The reaction was kept at 65~70 ℃ for 4~5 h. The process of the reaction was monitored by TLC. After completion, the reaction mixture was neutralized with HCl (10%) till the solution became neutral. The solvent was removed under reduced pressure. The obtained residue was purified by recrystallization from anhydrous ethanol to obtain compounds 2a~2l.

  4-(4-Fluorophenyl)-6-(furan-2-yl)-2-methylpyrimidine (2a): White powder, yield 81%. m.p. 124~125 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.37~8.32 (m, 2H), 8.11 (s, 1H), 8.01 (dd, J＝1.6, 0.8 Hz, 1H), 7.50 (dd, J＝3.6, 0.4 Hz, 1H), 7.40 (t, J＝8.8 Hz, 2H), 6.77 (dd, J＝3.2, 1.6 Hz, 1H), 2.69 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.2, 164.5 (d, J＝247.0 Hz), 163.0, 156.1, 151.8, 146.6, 133.2 (d, J＝3.0 Hz), 130.0 (d, J＝9.0 Hz), 116.3 (d, J＝21.0 Hz), 113.5, 113.3, 107.6, 26.5; IR (KBr) v_max: 1603, 1565, 1536, 1509, 1381, 1102, 842 cm^-1; HRMS (ESI) calcd for C₁₅H₁₂N₂OF [M+H]⁺ 255.0928, found 255.0924.

  4-(3, 4-Dichlorophenyl)-6-(furan-2-yl)-2-methylpyrimidine (2b): Yellow powder, yield 89%. m.p. 139~140 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.53 (d, J＝2.0 Hz, 1H), 8.28 (dd, J＝8.4, 1.6 Hz, 1H), 8.23 (s, 1H), 7.84 (d, J＝8.4 Hz, 1H), 7.54 (d, J＝3.2 Hz, 1H), 6.78 (dd, J＝3.2, 1.6 Hz, 1H), 2.71 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.3, 161.5, 156.5, 151.6, 146.8, 137.3, 134.2, 132.4, 131.6, 129.3, 127.6, 113.9, 113.3, 108.2, 26.4; IR (KBr) v_max: 1605, 1549, 1523, 1379, 1121, 819 cm^-1; HRMS (ESI) calcd for C₁₅H₁₁Cl₂N₂O [M+H]⁺ 305.0243, found 305.0237.

  4-(Furan-2-yl)-2-methyl-6-(p-tolyl)pyrimidine (2c): Yellow powder, yield 85%. m.p. 86~87 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.18 (d, J＝8.4 Hz, 2H), 8.07 (s, 1H), 8.00 (d, J＝0.8 Hz, 1H), 7.48 (d, J＝2.8 Hz, 1H), 7.37 (d, J＝8.0 Hz, 2H), 6.77 (dd, J＝3.2, 1.6 Hz, 1H), 2.69 (s, 3H), 2.40 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.1, 164.0, 156.0, 151.9, 146.5, 141.5, 134.0, 130.0, 127.5, 113.3, 113.2, 107.3, 26.5, 21.5; IR (KBr) v_max: 1605, 1522, 1383, 1106, 829 cm^-1; HRMS (ESI) calcd for C₁₆H₁₅N₂O [M+H]⁺ 251.1179, found 251.1179.

  4-(4-Chlorophenyl)-2-methyl-6-(5-methylfuran-2-yl)-pyrimidine (2d): Yellow powder, yield 77%. m.p. 132~133 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.28 (d, J＝8.4 Hz, 2H), 8.04 (s, 1H), 7.62 (d, J＝8.8 Hz, 2H), 7.41 (d, J＝3.2 Hz, 1H), 6.40 (d, J＝2.8 Hz, 1H), 2.68 (s, 3H), 2.43 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.2, 162.5, 156.2, 156.1, 150.2, 136.3, 135.6, 129.4, 129.3, 115.0, 109.8, 107.2, 26.5, 14.2; IR (KBr) v_max: 1606, 1573, 1517, 1381, 1095, 836 cm^-1; HRMS (ESI) calcd for C₁₆H₁₄N₂OCl [M+H]⁺ 285.0789, found 285.0785.

  2-Methyl-4-(5-methylfuran-2-yl)-6-(p-tolyl)pyrimidine (2e): Brown powder, yield 88%. m.p. 143~144 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.16 (d, J＝8.0 Hz, 2H), 7.98 (s, 1H), 7.39~7.36 (m, 3H), 6.39 (d, J＝2.8 Hz, 1H) 2.67 (s, 3H), 2.44 (s, 3H), 2.41 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.0, 163.7, 156.0, 155.9, 150.4, 141.4, 134.0, 130.0, 127.4, 114.6, 110.0, 106.7, 26.5, 21.5, 14.2; IR (KBr) v_max: 1604, 1515, 1382, 1117, 814 cm^-1; HRMS (ESI) calcd for C₁₇H₁₇N₂O [M+H]⁺ 265.1335, found 265.1338.

  4-(4-Methoxyphenyl)-2-methyl-6-(5-methylfuran-2-yl)-pyrimidine (2f): Yellow powder, yield 80%. m.p. 107~108 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.23 (d, J＝8.8 Hz, 2H), 7.94 (s, 1H), 7.37 (d, J＝3.2 Hz, 1H), 7.10 (d, J＝8.8 Hz, 2H), 6.39 (d, J＝2.8 Hz, 1H), 3.86 (s, 3H), 2.65 (s, 3H), 2.43 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.9, 163.4, 162.1, 155.8, 150.4, 129.1, 127.5, 114.7, 114.5, 109.7, 106.2, 55.9, 26.5, 14.2; IR (KBr) v_max: 2923, 1629, 1573, 1515, 1381, 1108 cm^-1; HRMS (ESI) calcd for C₁₇H₁₇N₂O₂ [M+H]⁺ 281.1285, found 281.1280.

  4-(4-Chlorophenyl)-2-methyl-6-(thiophen-2-yl)pyrimidine (2g): White powder, yield 83%. m.p. 86~87 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.42 (s, 1H), 8.36 (d, J＝8.4 Hz, 2H), 8.30 (dd, J＝3.6, 1.2 Hz, 1H), 7.85 (dd, J＝5.2, 1.2 Hz, 1H), 7.65 (d, J＝8.8 Hz, 2H), 7.29 (dd, J＝4.8, 3.6 Hz, 1H), 2.68 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.1, 162.7, 160.0, 142.7, 136.4, 135.6, 131.6, 129.6, 129.4, 129.3, 108.2, 26.4; IR (KBr) v_max: 2923, 1629, 1515, 1381, 1097, 817 cm^-1; HRMS (ESI) calcd for C₁₅H₁₂- N₂SCl [M+H]⁺ 287.0404, found 287.0410.

  2-Methyl-4-(thiophen-2-yl)-6-(p-tolyl)pyrimidine (2h): White crystal, yield 80%. m.p. 103~104 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.34 (s, 1H), 8.28 (d, J＝3.6 Hz, 1H), 8.23 (d, J＝8.0 Hz, 2H), 7.83 (d, J＝4.8 Hz, 1H), 7.38 (d, J＝8.0 Hz, 2H), 7.29 (t, J＝4.0 Hz, 1H), 2.67 (s, 3H), 2.41 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.0, 163.8, 159.7, 142.9, 141.4, 134.0, 131.3, 129.9, 129.2, 127.6, 125.4, 107.7, 26.4, 21.5; IR (KBr) v_max: 1630, 1523, 1383, 1107 cm^-1; HRMS (ESI) calcd for C₁₆H₁₅N₂S [M+H]⁺ 267.0950, found 267.0945.

  4-(4-Methoxyphenyl)-2-methyl-6-(thiophen-2-yl)pyrimidine (2i): Yellow powder, yield 86%. m.p. 167~168 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.31~8.26 (m, 4H), 7.82 (d, J＝4.8 Hz, 2H), 7.28 (t, J＝4.0 Hz, 1H), 7.11 (d, J＝8.8 Hz, 2H), 3.86 (s, 3H), 2.66 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.9, 163.5, 162.2, 159.5, 143.0, 131.1, 129.3, 129.2, 129.1, 114.7, 107.2, 55.9, 26.4; IR (KBr) v_max: 1603, 1565, 1536, 1509, 1381, 1102, 842 cm^-1; HRMS (ESI) calcd for C₁₆H₁₅N₂OS [M+H]⁺ 283.0940, found 283.0970.

  4-(4-Bromophenyl)-6-(5-bromothiophen-2-yl)-2-methyl-pyrimidine (2j): White powder, yield 77%. m.p. 161~162 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.48 (s, 2H), 8.42 (s, 2H), 8.31 (d, J＝8.0 Hz, 1H), 8.15 (d, J＝4.0 Hz, 1H), 7.77 (d, J＝8.0 Hz, 1H), 7.53 (t, J＝8.0 Hz, 1H), 7.43 (d, J＝4.0 Hz, 1H), 2.66 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.2, 162.4, 159.1, 144.2, 138.9, 131.5, 130.3, 130.1, 126.6, 122.9, 117.7, 108.0, 26.3; IR (KBr) v_max: 1574, 1525, 1389, 1103, 798 cm^-1; HRMS (ESI) calcd for C₁₅H₁₁N₂SBr₂ [M+H]⁺ 408.9004, found 408.9000.

  4-(5-Bromothiophen-2-yl)-2-methyl-6-(p-tolyl)pyrimidine (2k): Light yellow powder, yield 85%. m.p. 167~168 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.35 (s, 1H), 8.22 (d, J＝8.0 Hz, 2H), 8.13 (d, J＝4.0 Hz, 1H), 7.42 (d, J＝4.0 Hz, 1H), 7.38 (d, J＝8.0 Hz, 2H), 2.66 (s, 3H), 2.40 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 168.0, 164.1, 158.7, 144.5, 141.6, 133.8, 132.7, 130.0, 129.9, 127.6, 117.4, 107.3, 26.3, 21.5; IR (KBr) v_max: 1574, 1523, 1379, 1121, 822, 785 cm^-1; HRMS (ESI) calcd for C₁₆H₁₄N₂SBr [M+H]⁺ 345.0056, found 345.0046.

  4-(5-Bromothiophen-2-yl)-6-(4-methoxyphenyl)-2-methylpyrimidine (2l): Light yellow powder, yield 82%. m.p. 139~140 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.31 (s, 1H), 8.29 (d, J＝8.8 Hz, 2H), 8.12 (d, J＝4.0 Hz, 1H), 7.42 (d, J＝4.0 Hz, 1H), 7.11 (d, J＝8.4 Hz, 2H), 3.87 (s, 3H), 2.65 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 167.9, 163.7, 162.3, 158.5, 144.6, 132.6, 129.7, 129.3, 128.9, 117.2, 114.7, 106.7, 55.9, 26.3; IR (KBr) v_max: 1605, 1574, 1517, 1390, 1253, 1114, 837, 801 cm^-1; HRMS (ESI) calcd for C₁₆H₁₄N₂OSBr [M+H]⁺ 361.0005, found 361.0004.

  4.2.3   Synthesis of target compounds 3a~3l

  As shown in Scheme 1, a mixture of intermediate 1 (0.005 mol), anhydrous methanol (10 mL) and NaOH (0.02 mol) was stirred at room temperature. The solution of pyrazole-1-carboximidamide hydrochloride (0.01 mol) and anhydrous methanol (10 mL) was slowly added to the above mixture. The reaction was kept at 70~75 ℃ for 5~6 h. The process of the reaction was monitored by TLC. After completion, the reaction mixture was poured into ice water and neutralized by HCl solution (10%) till the solution became neutral and extracted with ethyl acetate (10 mL×3). The organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. The obtained residue was purified by silica gel (200~300 mesh) chromatography using petroleum ether (b.p. 60~90 ℃)/ethyl acetate (V:V＝1:2) as the eluting system to give compounds 3a~3l.

  4-(4-Bromophenyl)-6-(furan-2-yl)-2-(1H-pyrazol-1-yl)- pyrimidine (3a): Brown powder, yield 85%. m.p. 100~101 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.89 (s, 1H), 8.37 (d, J＝8.4 Hz, 2H), 8.26 (s, 1H), 8.10 (s, 1H), 7.91 (s, 1H), 7.82 (d, J＝8.0 Hz, 2H), 7.70 (d, J＝2.4 Hz, 1H), 6.85 (s, 1H), 6.66 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.8, 157.9, 156.3, 151.2, 147.4, 143.7, 135.2, 132.5, 130.4, 129.9, 126.0, 115.2, 113.6, 109.2, 108.2; IR (KBr) v_max: 1601, 1517, 1456, 1397, 1256, 1175, 835, 759 cm^-1; HRMS (ESI) calcd for C₁₇H₁₂N₄OBr [M+H]⁺ 367.0189, found 367.0183.

  4-(Furan-2-yl)-2-(1H-pyrazol-1-yl)-6-(p-tolyl)pyrimidine (3b): Black powder, yield 85%. m.p. 71~72 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.88 (s, 1H), 8.36 (d, J＝8.4 Hz, 2H), 8.26 (s, 1H), 8.09 (s, 1H), 7.91 (s, 1H), 7.81 (d, J＝8.0 Hz, 2H), 7.69 (d, J＝2.4 Hz, 1H), 6.84 (s, 1H), 6.65 (s, 1H), 2.42 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 161.9, 154.9, 153.3, 148.2, 144.4, 140.7, 132.2, 129.5, 127.4, 126.9, 123.0, 112.2, 110.6, 106.2, 105.2, 23.0; IR (KBr) v_max: 1585, 1521, 1455, 1396, 1366, 1077, 824, 769 cm^-1; HRMS (ESI) calcd for C₁₈H₁₅N₄O [M+H]⁺ 303.1246, found 303.1248.

  4-(Furan-2-yl)-6-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)pyrimidine (3c): Yellow powder, yield 80%. m.p. 114~115 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.88 (s, 1H), 8.39 (d, J＝8.4 Hz, 2H), 8.16 (s, 1H), 8.08 (s, 1H), 7.90 (s, 1H), 7.65 (d, J＝3.2 Hz, 1H), 7.15 (d, J＝8.4 Hz, 2H), 6.83 (s, 1H), 6.65 (s, 1H), 3.89 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.2, 157.2, 155.6, 150.5, 146.7, 143.0, 134.5, 131.8, 129.7, 129.2, 125.3, 114.5, 112.9, 108.5, 107.5, 53.4; IR (KBr) v_max: 1605, 1576, 1516, 1452, 1253, 1177, 1028, 829 cm^-1; HRMS (ESI) calcd for C₁₈H₁₅N₄O₂ [M+H]⁺ 319.1195, found 319.1179.

  4-(4-Chlorophenyl)-6-(5-methylfuran-2-yl)-2-(1H-pyrazol-1-yl)pyrimidine (3d): White powder, yield 88%. m.p. 110~111 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.88 (d, J＝2.4 Hz, 1H), 8.48 (dd, J＝8.8, 5.6 Hz, 2H), 8.14 (s, 1H), 7.9 (s, 1H), 7.59 (d, J＝3.6 Hz, 1H), 7.44 (t, J＝8.4 Hz, 2H), 6.65~6.64 (m, 1H), 6.48 (d, J＝3.2 Hz, 1H), 2.48 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 159.7, 156.3, 154.5, 147.0, 146.0, 142.6, 135.6, 135.1, 131.5, 129.7, 129.6, 109.4, 107.6, 106.1, 104.6, 14.5; IR (KBr) v_max: 1582, 1521, 1458, 1395, 1365, 1200, 942, 819, 796 cm^-1; HRMS (ESI) calcd for C₁₈H₁₄N₄OCl [M+H]⁺ 337.0856, found 337.0825.

  4-(5-Methylfuran-2-yl)-2-(1H-pyrazol-1-yl)-6-(p-tolyl)pyrimidine (3e): Black powder, yield 83%. m.p. 123~124 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.73 (s, 1H), 8.21 (d, J＝8.4 Hz, 2H), 8.11 (s, 1H), 7.94 (s, 1H), 7.76 (s, 1H), 7.66 (d, J＝8.0 Hz, 2H), 7.54 (d, J＝2.4 Hz, 1H), 6.69 (s, 1H), 2.36 (s, 3H), 2.25 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 159.7, 156.3, 154.5, 147.0, 146.0, 142.6, 135.6, 135.1, 131.5, 129.7, 129.6, 109.4, 107.6, 106.1, 104.6, 24.1, 14.5; IR (KBr) v_max: 1582, 1522, 1458, 1396, 1365, 1182, 1038, 942, 819, 797 cm^-1; HRMS (ESI) calcd for C₁₉H₁₇N₄O [M+H]⁺ 317.1402, found 317.1475.

  4-(4-Chlorophenyl)-2-(1H-pyrazol-1-yl)-6-(thiophen-2-yl)pyrimidine (3f): Yellow powder, yield 87%. m.p. 217~218 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.89 (d, J＝2.4 Hz, 1H), 8.48 (dd, J＝8.8, 5.6 Hz, 2H), 8.16 (s, 1H), 7.91 (s, 1H), 7.60 (d, J＝3.6 Hz, 1H), 7.45 (t, J＝8.4 Hz, 2H), 7.27 (s, 1H), 6.65 (s, 1H), 6.48 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ:160.4, 153.9, 152.0, 146.2, 136.7, 135.8, 135.3, 132.2, 131.7, 129.9, 129.8, 129.0, 121.5, 109.6, 107.8; IR (KBr) v_max: 1604, 1576, 1516, 1454, 1390, 1358, 1253, 1178, 1027, 828 cm^-1; HRMS (ESI) calcd for C₁₇H₁₂N₄SCl [M+H]⁺ 339.0471, found 339.0458.

  2-(1H-pyrazol-1-yl)-4-(thiophen-2-yl)-6-(p-tolyl)pyrimi-dine (3g): Black powder, yield 86%. m.p. 235~236 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.88 (s, 1H), 8.36 (d, J＝8.4 Hz, 2H), 8.26 (s, 1H), 8.09 (s, 1H), 7.91 (s, 1H), 7.81 (d, J＝8.0 Hz, 2H), 7.69 (s, 1H), 6.84 (s, 1H), 6.65 (s, 1H), 2.42 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 160.7, 154.2, 152.3, 146.5, 142.2, 137.0, 136.8, 132.5, 132.0, 131.0, 129.3, 129.0, 121.8, 110.0, 108.1, 21.7; IR (KBr) v_max: 1601, 1516, 1455, 1397, 1357, 1255, 1173, 1098, 834, 758 cm^-1; HRMS (ESI) calcd for C₁₈H₁₅N₄S [M+H]⁺ 319.1017, found 319.1054.

  4-(4-Methoxyphenyl)-2-(1H-pyrazol-1-yl)-6-(thiophen-2-yl)pyrimidine (3h): Grey powder, yield 88%. m.p. 125~126 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.83 (s, 1H), 8.34 (d, J＝6.8 Hz, 2H), 8.11 (s, 1H), 8.03 (s, 1H), 7.85 (s, 1H), 7.60 (s, 1H), 7.10 (d, J＝5.6 Hz, 2H), 6.78 (s, 1H), 6.60 (s, 1H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 163.4, 161.0, 154.6, 152.7, 146.9, 132.9, 132.4, 132.0, 129.7, 128.9, 122.2, 115.0, 110.3, 108.5, 105.9, 56.9; IR (KBr) v_max: 2961, 2925, 1600, 1561, 1524, 1487, 1453, 1398, 1261, 1078, 1041, 1010, 950, 820, 757 cm^-1; HRMS (ESI) calcd for C₁₈H₁₅N₄OS [M+H]⁺ 335.0956, found 335.0920.

  4-(5-Bromothiophen-2-yl)-6-(4-chlorophenyl)-2-(1H-pyrazol-1-yl)pyrimidine (3i): Light yellow powder, yield 79%. m.p. 188~189 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.81 (d, J＝2.4 Hz, 1H), 8.52~8.49 (m, 3H), 8.21 (d, J＝4.0 Hz, 1H), 7.91 (d, J＝0.8 Hz, 1H), 7.47~7.42 (m, 3H), 6.64 (dd, J＝2.4, 1.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.9, 160.7, 155.9, 143.8, 143.5, 132.9, 131.2, 130.6, 130.5, 130.2, 118.6, 116.5, 116.3, 109.2, 108.0; IR (KBr) v_max: 1580, 1522, 1456, 1395, 1367, 1226, 944, 837, 801, 761 cm^-1; HRMS (ESI) calcd for C₁₇H₁₁- N₄SClBr [M+H]⁺ 416.9576, found 416.9566.

  4-(4-Bromophenyl)-6-(5-bromothiophen-2-yl)-2-(1H-pyrazol-1-yl)pyrimidine (3j): Light yellow powder, yield 86%. m.p. 179~180 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.87 (d, J＝2.4 Hz, 1H), 8.47 (dd, J＝8.8, 5.6 Hz, 2H), 8.14 (s, 1H), 7.90 (s, 1H), 7.58 (d, J＝3.4 Hz, 1H), 7.44 (t, J＝8.4 Hz, 2H), 6.65~6.64 (m, 1H), 6.47 (d, J＝3.2 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.8, 163.5, 160.6, 155.8, 143.7, 143.4, 132.8, 132.3, 131.1, 130.5, 130.4, 130.2, 118.5, 109.1, 107.9; IR (KBr) v_max: 1585, 1521, 1456, 1446, 1393, 1366, 1011, 943, 824, 768 cm^-1; HRMS (ESI) calcd for C₁₇H₁₁Br₂N₄S [M+H]⁺ 460.9066, found 460.9072.

  4-(5-Bromothiophen-2-yl)-2-(1H-pyrazol-1-yl)-6-(p-tolyl)pyrimidine (3k): White powder, yield 89%. m.p. 182~183 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.79 (d, J＝2.4 Hz, 1H), 8.44 (s, 1H), 8.33 (d, J＝8.0 Hz, 2H), 8.22 (d, J＝4.0 Hz, 1H), 7.91 (s, 1H), 7.46 (d, J＝4.0 Hz, 1H), 7.40 (d, J＝8.0 Hz, 2H), 6.64 (t, J＝2.0 Hz, 1H), 2.42 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 165.9, 160.5, 155.9, 143.7, 142.4, 133.1, 132.9, 131.0, 130.2, 130.0, 127.9, 118.4, 109.2, 107.7, 21.6; IR (KBr) v_max: 1584, 1523, 1459, 1397, 1365, 1182, 1038, 941, 819, 797, 774, 754 cm^-1; HRMS (ESI) calcd for C₁₈H₁₄N₄SBr [M+H]⁺ 397.0117, found 397.0111.

  4-(5-Bromothiophen-2-yl)-6-(4-methoxyphenyl)-2-(1H-pyrazol-1-yl)pyrimidine (3l): Yellow powder, yield 79%. m.p. 157~158 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 8.78 (d, J＝2.4 Hz, 1H), 8.49~8.45 (m, 3H), 8.18 (d, J＝4.0 Hz, 1H), 7.88 (s, 1H), 7.44~7.39 (m, 3H), 6.61 (dd, J＝2.4, 1.6 Hz, 1H), 3.84 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 165.9, 160.4, 155.8, 143.6, 142.3, 133.0, 132.8, 130.9, 130.1, 129.9, 127.8, 118.3, 109.1, 107.6, 57.1; IR (KBr) v_max: 2916, 1584, 1523, 1459, 1396, 1365, 1200, 1038, 971, 818, 797, 774 cm^-1; HRMS (ESI) calcd for C₁₈H₁₄N₄OSBr [M+H]⁺ 413.0071, found 413.0070.

  4.2.4   Synthesis of target compounds 4a~4l

  As shown in Scheme 1, a mixture of intermediate 1 (0.005 mol), dimethylformamide (DFM, 10 mL) and K₂CO₃ (0.025 mol) was stirred at room temperature. The solution of guanyl urea sulfate (0.01 mol) and DFM (10 mL) was slowly added to the above mixture. The reaction was kept at 55~60 ℃ for 5~6 h. The course of the reaction was monitored by TLC. After completion, the reaction mixture was poured into ice water and neutralized with HCl solution (10%) till the solution became neutral and extracted with dichloromethane (DCM, 10 mL×3). The organic phase was dried with anhydrous Na₂SO₄ and concentrated under reduced pressure. The obtained residue was purified by silica gel (200~300 mesh) chromatography using petroleum ether (b.p. 60~90 ℃)/ethyl acetate (V:V＝1:1) as the eluting system to give compounds 4a~4l.

  1-(4-(4-Chlorophenyl)-6-(furan-2-yl)pyrimidin-2-yl)urea (4a): Light yellow powder, yield 80%. m.p. 207~208 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.64 (s, 1H), 8.64 (s, 1H), 8.28 (dd, J＝8.4, 5.6 Hz, 2H), 8.03 (s, 1H), 7.90 (s, 1H), 7.55 (d, J＝3.2 Hz, 1H), 7.41 (t, J＝8.8 Hz, 2H), 7.18 (s, 1H), 6.79 (dd, J＝3.2, 1.6 Hz, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.2, 159.0, 156.1, 155.2, 151.1, 146.9, 130.2, 130.1, 116.5, 116.3, 114.3, 113.4, 104.1; IR (KBr) v_max: 3399, 2928, 1694, 1603, 1573, 1538, 1510, 1394, 1227, 834 cm^-1; HRMS (ESI) calcd for C₁₅H₁₁N₄O₂ClNa [M+Na]⁺ 337.0464, found 337.0458.

  1-(4-(Furan-2-yl)-6-(4-methoxyphenyl)pyrimidin-2-yl)urea (4b): Light yellow powder, yield 80%. m.p. 214~215 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.56 (s, 1H), 8.71 (s, 1H), 8.19 (d, J＝8.8 Hz, 2H), 8.02 (d, J＝0.8 Hz, 1H), 7.84 (s, 1H), 7.52 (d, J＝3.6 Hz, 1H), 7.16 (s, 1H), 7.12 (d, J＝8.8 Hz, 2H), 6.78 (dd, J＝3.6, 1.6 Hz, 1H), 3.86 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.8, 162.5, 159.0, 155.8, 155.3, 151.3, 146.7, 129.3, 128.6, 114.8, 113.9, 113.3, 103.4, 55.9; IR (KBr) v_max: 3380, 1698, 1600, 1538, 1512, 1478, 1399, 1346, 1241, 1176, 835 cm^-1; HRMS (ESI) calcd for C₁₆H₁₅N₄O₃ [M+Na]⁺ 333.0959, found 333.0939.

  1-(4-(4-Chlorophenyl)-6-(5-methylfuran-2-yl)pyrimidin-2-yl)urea (4c): White powder, yield 86%. m.p. 103~104 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.57 (s, 1H), 8.72 (s, 1H), 8.20 (d, J＝8.8 Hz, 2H), 8.03 (s, 1H), 7.85 (s, 1H), 7.53 (s, 1H), 7.13 (d, J＝8.8 Hz, 2H), 6.80 (s, 1H), 2.43 (s, 3H), ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.3, 162.0, 155.3, 150.7, 138.2, 133.4, 132.2, 131.4, 128.9, 127.3, 114.1, 109.4, 100.1, 14.1; IR (KBr) v_max: 3372, 3130, 2922, 2853, 1693, 1585, 1535, 1430, 1396, 1351, 819 cm^-1; HRMS (ESI) calcd for C₁₆H₁₄N₄O₂Cl [M+H]⁺ 322.0805, found 322.0836.

  1-(4-(5-Methylfuran-2-yl)-6-(p-tolyl)pyrimidin-2-yl)urea (4d): White powder, yield 88%. m.p. 108~109 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.57 (s, 1H), 8.72 (s, 1H), 8.20 (d, J＝8.8 Hz, 2H), 8.04 (s, 1H), 7.85 (s, 1H), 7.53 (d, J＝3.6 Hz, 1H), 7.13 (d, J＝8.8 Hz, 2H), 6.80 (dd, J＝3.6, 1.6 Hz, 1H), 2.44 (s, 3H), 2.41 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 165.8, 163.5, 160.0, 156.8, 156.3, 152.3, 147.7, 130.3, 129.6, 115.8, 114.9, 114.3, 104.4, 21.2, 14.0; IR (KBr) v_max: 3329, 3133, 2964, 1692, 1584, 1530, 1394, 1367, 1239, 817, 788 cm^-1; HRMS (ESI) calcd for C₁₇H₁₇N₄O₂ [M+H]⁺ 309.1351, found 309.1343.

  1-(4-(4-Methoxyphenyl)-6-(5-methylfuran-2-yl)pyrimidin-2-yl)urea (4e): Grey powder, yield 74%. m.p. 178~179 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.56 (s, 1H), 8.71 (s, 1H), 8.19 (d, J＝8.8 Hz, 2H), 8.03 (s, 1H), 7.84 (s, 1H), 7.52 (d, J＝3.6 Hz, 1H), 7.16 (s, 1H), 7.12 (d, J＝8.8 Hz, 2H), 3.87 (s, 3H), 2.40 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 162.8, 161.3, 155.5, 154.9, 154.8, 153.3, 142.8, 131.4, 128.3, 114.4, 107.9, 106.4, 105.0, 56.3, 14.8; IR (KBr) v_max: 3374, 2922, 2853, 1693, 1585, 1535, 1429, 1396, 1351, 1097, 819 cm^-1; HRMS (ESI) calcd for C₁₇H₁₇N₄O₃ [M+H]⁺ 347.1117, found 347.1106.

  1-(4-(4-Chlorophenyl)-6-(thiophen-2-yl)pyrimidin-2-yl)urea (4f): Green powder, yield 80%. m.p. 134~135 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.64 (s, 1H), 8.49 (s, 1H), 8.33 (dd, J＝8.8, 5.6 Hz, 2H), 8.29 (dd, J＝3.6, 0.8 Hz, 1H), 8.15 (s, 1H), 7.88 (dd, J＝5.2, 0.8 Hz, 1H), 7.43 (t, J＝8.8 Hz, 2H), 7.31 (dd, J＝5.2, 4.0 Hz, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.2, 160.0, 158.8, 155.2, 142.1, 131.7, 130.2, 130.1, 130.0, 129.5, 116.5, 116.3, 104.4; IR (KBr) v_max: 3327, 1693, 1588, 1536, 1395, 1230, 1096, 828 cm^-1; HRMS (ESI) calcd for C₁₅H₁₁N₄OSClNa [M+Na]⁺ 353.0235, found 353.0232.

  1-(4-(Thiophen-2-yl)-6-(p-tolyl)pyrimidin-2-yl)urea (4g): Light yellow powder, yield 79%. m.p. 201~202 ℃; IR (KBr) v_max: 3331, 1692, 1585, 1531, 1394, 1343, 1238, 1099, 818; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.56 (s, 1H), 8.55 (s, 1H), 8.28 (d, J＝3.6 Hz, 1H), 8.16 (d, J＝8.0 Hz, 2H), 8.11 (s, 1H), 7.86 (d, J＝4.8 Hz, 1H), 7.39 (d, J＝8.0 Hz, 2H), 7.30 (dd, J＝4.8, 4.0 Hz, 2H), 2.41 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 165.1, 159.9, 158.8, 155.2, 142.2, 141.9, 133.7, 131.5, 130.0, 129.9, 129.5, 127.6, 104.2, 21.5 cm^-1; HRMS (ESI) calcd for C₁₆H₁₄N₄OSNa [M+Na]⁺ 333.0781, found 333.0759.

  1-(4-(4-Methoxyphenyl)-6-(thiophen-2-yl)pyrimidin-2-yl)urea (4h): White powder, yield 87%. m.p. 98~99 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.62 (s, 1H), 8.47 (s, 1H), 8.31 (dd, J＝8.8, 5.6 Hz, 2H), 8.26 (dd, J＝3.6, 0.8 Hz, 1H), 8.13 (s, 1H), 7.85 (dd, J＝5.2, 0.8 Hz, 1H), 7.41 (t, J＝8.8 Hz, 2H), 7.29 (dd, J＝5.2, 4.0 Hz, 2H), 3.88 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 165.3, 163.0, 159.5, 156.3, 155.8, 151.8, 147.3, 129.8, 129.1, 115.3, 114.5, 113.8, 103.9, 56.4; IR (KBr) v_max: 3328, 2964, 1693, 1588, 1536, 1395, 1230, 1097, 828 cm^-1; HRMS (ESI) calcd for C₁₆H₁₅N₄O₂S [M+H]⁺ 349.0731, found 349.0759.

  1-(4-(5-Bromothiophen-2-yl)-6-(4-chlorophenyl)pyrimidin-2-yl)urea (4i): Black powder, yield 83%. m.p. 196~197 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.67 (s, 1H), 8.35~8.28 (m, 3H), 8.15 (s, 1H), 8.13 (d, J＝4.0 Hz, 1H), 7.47~7.42 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.3, 159.1, 158.8, 155.0, 143.7, 133.0, 130.7, 130.3, 130.2, 117.7, 116.5, 116.3, 104.1; IR (KBr) v_max: 3418, 1605, 1577, 1549, 1510, 1446, 1410, 1364, 1223, 1103, 974, 798 cm^-1; HRMS (ESI) calcd for C₁₅H₁₁ON₄SBrCl [M+H]⁺ 430.9347, found 430.9381.

  1-(4-(4-Bromophenyl)-6-(5-bromothiophen-2-yl)pyrimidin-2-yl)urea (4j): White powder, yield 84%. m.p. 157~158 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.67 (s, 1H), 8.32~8.29 (m, 3H), 8.15 (s, 1H), 8.13 (d, J＝4.0 Hz, 1H), 7.47~7.42 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.7, 159.5, 158.4, 154.8, 141.8, 141.5, 133.2, 131.1, 129.6, 129.4, 129.0, 127.2, 103.8; IR (KBr) v_max: 3419, 1605, 1577, 1548, 1510, 1446, 1410, 1225, 843, 799 cm^-1; HRMS (ESI) calcd for C₁₅H₁₀N₄OSBr₂Na [M+Na]⁺ 474.8834, found 474.8838.

  1-(4-(5-Bromothiophen-2-yl)-6-(p-tolyl)pyrimidin-2-yl)urea (4k): Grey powder, yield 86%. m.p. 245~246 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.60 (s, 1H), 8.41 (s, 1H), 8.14 (d, J＝2.0 Hz, 2H), 8.11 (d, J＝7.6 Hz, 2H), 7.44 (d, J＝4.0 Hz, 1H), 7.39 (d, J＝8.0 Hz, 2H), 7.22 (s, 1H), 2.41 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 165.2, 159.0, 158.8, 155.1, 143.9, 142.1, 133.5, 132.9, 130.5, 130.0, 127.6, 117.5, 103.8, 21.5; IR (KBr) v_max: 3379, 1693, 1585, 1535, 1396, 1351, 1097, 818 cm^-1; HRMS (ESI) calcd for C₁₆H₁₃N₄OSBrNa [M+Na]⁺ 410.9886, found 410.9888.

  1-(4-(5-Bromothiophen-2-yl)-6-(4-methoxyphenyl)-pyrimidin-2-yl)urea (4l): Yellow powder, yield 81%. m.p. 163~164 ℃; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.67 (s, 1H), 8.32~8.29 (m, 3H), 8.15 (s, 1H), 8.13 (d, J＝4.0 Hz, 1H), 7.47~7.42 (m, 4H), 3.83 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ: 164.2, 159.0, 156.1, 155.2, 151.1, 146.9, 130.1, 130.0, 116.5, 116.3, 114.3, 113.3, 104.1, 55.1; IR (KBr) v_max: 3326, 2963, 1693, 1588, 1536, 1395, 1346, 1262, 1230, 1096, 828 cm^-1; HRMS (ESI) calcd for C₁₆H₁₄N₄O₂SBr [M+H]⁺ 426.9836, found 426.9863.

  4.3   Antifungal assay

  All the synthesized compounds were screened for their antifungal activities against R. solani, F. graminearum, H. maydis, S. sclerotiorum and B. cinerea by the mycelium growth rate method.^[6] Each tested compound was dissolved in DMSO (1 mL) and diluted with 1% Tween-80 solution into 10 mL to prepare the solution with a concentration of 1000 mg/L. The diluted solution (1 mL) was added to the sterile potato dextrose agar (PDA, 49 mL). Then, mycelion dishes of 5 mm diameter were cut from the culture medium, picked up with a sterilized inoculating needle, and inoculated in the center of the PDA plates (9 cm in diameter). The inoculated plates were incubated at (28±1) ℃. The compounds were tested at a concentration of 20 mg/L for primary screening. The inhibitory activities of the compounds with excellent effects were further tested at five different concentrations by the two-fold dilution method based on the concentration of 20 mg/L. Fluopyram (purity 99%) was employed as the positive control, whereas the corresponding solution without the tested compound and fluopyram was used as the blank control. Each treatment was performed three times. When the diameter of the colony used as the blank control reached 5~6 cm, the diameter of each colony was measured by the cross method. The corrected inhibitory rates were calculated using the formula I＝[(C-T)/(C-0.5)]×100%, where I represents the inhibition rate, C represents the average diameter of mycelia for the blank control and T represents the average diameter of mycelia for the treated PDA. The corresponding EC₅₀ values were calculated using the IBM SPSS Statistics 25.0 software.

  4.4   Assay of enzyme inhibition activity

  4.4.1   Isolation of SDH from S. sclerotiorum

  Mitochondria from S. sclerotiorum were isolated according to a previously reported method.^[37] Cultures were inoculated at 0.05 OD_{600 nm} and grown on a reciprocal shaker (180 r/min, 25 ℃) for 5 d in Sabouraud maltose broth (SMB) medium. Cells were harvested by vacuum filtration and disrupted in liquid nitrogen using a mortar and pestle. The resultant powder was resuspended to 10% (w/v) in extraction buffer [10 mmol/L KH₂PO₄, pH 7.2, 10 mmol/L KCl, 10 mmol/L MgCl₂, 0.5 mol/L sucrose, 0.2 mmol/L ethylene diamine tetraacetic acid (EDTA), 2 mmol/L phenylmethanesulfonyl fluoride (PMSF)]. The extract was clarified by centrifugation (5000 g, 4 ℃ for 10 min, 2 times), and intact mitochondria were then pelleted at 10000 g for 20 min at 4 ℃ and resuspended in the same buffer. Mitochondrial suspensions were brought to a concentration of 10 mg/mL and stored at -80 ℃ until use. SDH activity was found to remain stable for months.

  4.4.2   Succinate: ubiquinone/2, 6-dichloro-4-[(4-hydro-xyphenyl)imino]-2, 5-cyclohexadien-1-one (DCPIP) ac- tivity inhibition

  Mitochondrial suspensions were diluted 1/20 in extraction buffer and preactivated at 30 ℃ for 30 min in the presence of 10 mmol/L succinate. Succinate: ubiquinone/ DCPIP activity inhibition measurements were performed by adding 10 μL of preactivated mitochondria to 200 μL of assay buffer (50 mmol/L phosphate-sodium, pH 7.2, 250 mmol/L sucrose, 3 mmol/L NaN₃, 10 mmol/L succinate) supplemented with 140 μmol/L dichlorophenolindophenol (DCIP) and 1 mmol/L 2, 3-dimethoxy-5-methyl-1, 4-benzoquinone (Q₀). Inhibitor concentrations ranged between 4.4 and 150 μmol/L, with uniform 2×dilution factor steps (six inhibitor concentrations+DMSO control). A total of 96 well plates were pre-equilibrated at reaction temperature (30 ℃) for 10 min before the reactions were started by the addition of 10 μL of preactivated S. sclerotiorum mitochondrion suspension. DCPIP reduction was conducted at 30 ℃ and monitored at 595 nm. Calculated absorbance slopes (OD/h) were used for IC₅₀ calculations using the IBM SPSS Statistics 25.0 software.

  4.5   Homology modeling and molecular docking

  4.5.1   Homology modeling

  The NCBI protein database (https://www.ncbi.nlm.nih.gov/protein) was used to search the SDH amino acid sequence of S. sclerotiorum. The employed hypothetical protein sequences were XP_001591238, XP_001594577, XP_001597467 and XP_001593251, which were reported by Birren. The SDH from porcine heart (PDB ID: 1ZOY) was applied as the template, and the three-dimensional (3D) structure of the SDH was obtain by SWISS-MODEL, a fully automated protein structure homology-modelling server (http://swissmodel.expasy.org/).

  4.5.2   Molecular docking

  Molecular docking studies were performed to investigate the binding modes of compounds 2c, 3d and fluopyram to SDH using Autodock vina 1.1.2. The 3D structure of compounds 2c, 3d and fluopyram was drawn by ChemBioDraw Ultra 14.0 and ChemBio3D Ultra 14.0 softwares. The AutoDock Tools 1.5.6 package was employed to generate the docking input files. The search grid of SDH was identified as center_x: 86.459, center_y: 65.6, and center_z: 85.537 with dimensions size_x: 15, size_y: 15, and size_z: 15. The value of exhaustiveness was set to 20. For Vina docking, the default parameters were used if it was not mentioned. The best-scoring pose as judged by the Vina docking score was chosen and visually analyzed using PyMoL 1.7.6 software (http://www.pymol.org/).

  Supporting Information ¹H NMR, ¹³C NMR, IR, HRMS spectra of 2a~2l, 3a~3l and 4a~4l. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn/.

  
  
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  Figure 1  Representatives of succinate dehydrogenase inhibitor fungicides

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  Figure 2  Representatives of commercial pyrimidine derivative fungicides

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  Scheme 1  Design of target compounds

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  Scheme 2  Synthesis of target compounds

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  Figure 3  Comparison of EC₅₀ values among compounds with excellent effects against S. sclerotiorum

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  Figure 4  Theoretical binding modes of fluopyram (A), compound 2c (B), fluopyram and compound 2c (C), compound 3d (D), fluopyram and compound 3d (E) to SDH

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  Table 1.  Antifungal activity of target compounds at 20 mg/L

  Compound	Inhibition ratea/%
  	R. solani	F. graminearum	H. maydis	S. sclerotiorum	B. cinerea
  2a	51.8±2.2	30.1±0.9	88.2±3.5	100.0±0.0	76.5±3.5
  2b	39.0±1.3	26.6±1.1	50.3±2.0	80.1±3.8	43.8±1.9
  2c	75.6±2.9	68.2±3.3	91.6±4.1	100.0±0.0	83.2±2.8
  2d	58.3±2.6	40.1±1.9	83.4±3.8	98.6±1.1	73.3±3.2
  2e	56.7±2.0	43.4±1.3	81.7±3.4	96.8±2.8	78.2±3.1
  2f	61.9±2.4	45.5±2.2	86.7±3.8	100.0±0.0	80.5±3.5
  2g	32.8±1.3	26.4±0.9	51.9±2.1	64.3±2.1	40.3±1.9
  2h	30.5±0.7	21.3±0.3	55.0±2.3	68.5±2.2	44.7±1.8
  2i	44.5±0.5	30.5±0.9	67.5±2.8	73.3±3.3	51.8±2.2
  2j	41.2±1.3	38.9±1.0	65.1±2.7	68.0±2.8	55.4±1.9
  2k	51.1±1.2	36.5±1.6	84.3±4.0	94.7±2.4	77.9±2.8
  2l	50.3±1.7	38.1±1.8	70.3±3.1	81.2±4.0	58.4±2.1
  3a	60.3±2.2	48.5±2.1	84.7±3.6	94.9±3.8	74.5±3.3
  3b	54.1±1.9	44.2±2.0	82.5±3.6	97.6±2.0	77.0±2.5
  3c	40.7±1.7	33.2±1.3	68.2±2.7	82.7±2.6	59.2±2.5
  3d	74.1±2.3	67.9±2.5	90.3±3.4	100.0±0.0	81.5±3.2
  3e	40.8±2.1	36.4±1.5	67.2±2.6	75.6±3.2	54.3±2.0
  3f	32.0±0.8	23.5±1.0	56.4±2.3	66.0±3.1	47.6±1.4
  3g	30.9±0.9	20.2±0.2	53.8±2.1	68.0±2.7	47.9±2.5
  3h	35.2±1.6	22.1±0.3	56.4±2.4	68.7±3.1	50.5±1.6
  3i	60.0±2.2	42.6±1.9	83.5±3.7	97.9±1.7	75.3±2.9
  3j	57.0±2.3	49.3±1.5	88.6±4.2	100.0±0.0	76.1±2.4
  3k	46.3±1.5	37.6±1.4	61.3±2.0	78.2±3.1	55.7±2.8
  3l	45.7±1.9	30.6±1.0	67.0±2.6	84.3±3.9	58.1±2.0
  4a	51.4±1.3	42.4±1.7	83.2±3.9	97.2±1.9	75.1±2.1
  4b	60.9±2.9	48.2±2.2	87.6±4.0	100.0±0.0	74.3±3.3
  4c	32.6±0.7	25.3±1.3	51.9±2.6	65.3±2.3	43.5±2.0
  4d	39.1±1.3	28.6±0.4	61.4±3.0	73.8±2.3	58.0±1.8
  4e	52.7±1.7	41.1±1.8	81.9±3.5	94.1±3.1	60.2±2.4
  4f	32.0±1.0	20.9±1.1	50.6±2.3	61.3±2.8	41.6±1.0
  4g	33.2±1.4	23.6±0.6	52.8±2.2	60.2±2.5	43.2±1.3
  4h	31.7±1.5	26.9±0.9	45.1±1.8	55.1±1.3	38.4±1.4
  4i	50.8±1.7	45.6±1.2	77.8±3.3	95.7±2.1	65.7±1.2
  4j	60.6±2.4	45.3±1.6	87.6±4.3	100.0±0.0	76.9±2.2
  4k	41.6±1.5	35.4±0.6	60.3±2.7	71.2±1.0	52.3±2.4
  4l	51.5±2.3	41.8±1.5	78.9±3.1	92.3±3.3	58.2±2.2
  Fluopyram	71.4±1.9	62.9±1.0	100.0±0.0	100.0±0.0	100.0±0.0
  a Data are the mean±standard deviation (SD) of three replicates.

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  Table 2.  EC₅₀ values of the compounds with excellent effects against S. sclerotiorum

  Compound	EC50a/(mg·L-1)
  2a	0.258±0.024
  2c	0.072±0.010
  2d	0.632±0.081
  2e	0.768±0.091
  2f	0.272±0.027
  2k	1.003±0.102
  3a	0.984±0.110
  3b	0.711±0.082
  3d	0.077±0.010
  3i	0.704±0.081
  3j	0.299±0.030
  4a	0.740±0.079
  4b	0.267±0.026
  4e	1.106±0.161
  4i	0.807±0.091
  4j	0.327±0.032
  4l	1.230±0.186
  Fluopyram	0.244±0.019
  a Data are the mean±standard deviation (SD) of three replicates.

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  Table 3.  Inhibitory activities of compounds 2c and 3d against SDH

  Compound	IC50a/(mg·L-1)
  2c	0.115±0.011
  3d	0.121±0.012
  Fluopyram	0.356±0.040
  a Data are the mean±standard deviation (SD) of three replicates.

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