English

• 1. INTRODUCTION

  Epidemic crops and trees diseases could strongly reduce the crop production and enormous losses each year. Researches revealed that more than 19, 000 types of fungi and oomycetes caused nearly 200 plant diseases, which accounted for nearly 10% reduction of the major crops^{[1, 2]}. This phenomenon had become a severe issue drawing increasing concerns all over the world. In addition, pathogenic oomycetes are virulent and hemibiotrophic pathogens which often cause great damage on some important vegetables^[3]. For example, Phytophthora infestans (P. infestans) and Phytophthora capsici (P. capsici) can cause destructive effects on the productivity of potato and pepper, respectively^[4-6]. In addition, forest fungi have led to huge losses in the forest production, such as pecan and torreya. In the past decades, the application of fungicides was still an effective way to control these pathogenic fungi and oomycetes^{[7, 8]}. However, the widespread use and misuse of fungicides have caused rapidly increasing resistance in these pathogens^[9]. Therefore, it is very urgent to develop novel, effective and environment friendly fungicides to control plant pathogenic fungi and oomycetes.

  The application of heterocyclic group as active substructure was an important approach for fungicide design. Especially, nitrogen containing heterocycles are the key pharmacophores in many bioactive molecules^{[10, 11]}. Among them, pyrazole is an important five-membered heterocycle^[12] and has been used in many synthetic agrochemicals owing to its diverse activities such as insecticidal^[13], antifungal^[14-17] and nematocidal activities^[18-21]. A number of pyrazole derivatives were commercialized as fungicides with broad antifungal spectrum, including sedaxane, bixafen, isopyrazam and so on^[22]. Moreover, the carboxamide moiety represents an important pharmacophore presented in many commercial pesticides, which displayed notable antibacterial, antifungal, herbicidal and insecticidal activities^[23-27].

  Based on these findings, novel pyrazole-benzene carboxamide derivatives 2a~2d were synthesized via two steps and their structures were confirmed by NMR, HRMS spectra and X-ray diffraction. The in vitro anti-fungal/anti-oomycete activities toward two forest fungi, two crop fungi and two crop oomycetes were studied, and the in vivo antifungal bioassay of compound 2b was conducted as well.

  2. EXPERIMENTAL

  2.1 Materials and methods

  Melting points were measured by an OptiMelt melting point apparatus (USA) and uncorrected. NMR spectra of the title compounds were gathered through a Bruker AV-600 spectrometer (MA, USA). The crystal structure of compound 2d was measured on an Agilent Super Nova Dual Atlas CCD diffractometer (USA). HRMS data were collected via a Waters G2-xs instrument (USA). The reagents and chemicals (AR) were bought from Juyou Chemical Co., Ltd. (China).

  2.2 Synthesis procedure of intermediate 1

  A mixture of 4-(1H-pyrazol-1-yl)benzonitrile (0.338 g, 2.0 mmol), NaOH (0.160 g, 4.0 mmol) and EtOH/H₂O (25 mL, v/v = 1:1) was refluxed in 105 ℃ for 16 hours. Then water (30 mL) was added to the product, which was extracted with EtOAc (30 mL). The water phase was acidified by HCl (6.0 mol/L) to pH = 5. The precipitate formed was filtered and dried to give compound 1. White solid, yield 65%, m.p. 269~271 ℃. ¹H NMR (600 MHz, DMSO-d₆) δ 13.13 (s, 1H), 8.65 (d, J = 2.3 Hz, 1H), 8.10 (d, J = 8.5 Hz, 2H), 8.02 (d, J = 8.6 Hz, 2H), 7.85 (s, 1H), 6.63 (s, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.71, 142.73, 141.88, 130.94, 128.24, 128.15, 117.92, 108.63.

  2.3 Synthesis procedure of the title compounds 2a~2d

  4-Phenoxyaniline (1.0 mmol), EDCI (1.2 mmol) and DMAP (0.2 mmol) were added separately into the solution of compound 1 (1.2 mmol) in dichloromethane (10 mL). The mixture was then stirred at 25 ℃ for 2~5 h. After completion, the organic phase was filtered. The solid formed was washed with petroleum ether and desiccated to afford compounds 2a~2d.

  Compound 2a: white solid, yield 70%, m.p. 179~181 ℃. ¹H NMR (600 MHz, DMSO-d₆): δ 10.33 (d, J = 6.9 Hz, 1H), 8.65 (d, J = 2.5 Hz, 1H), 8.11 (d, J = 7.0 Hz, 2H), 8.02 (d, J = 8.8 Hz, 2H), 7.84~7.80 (m, 3H), 7.39 (d, J = 8.5, 7.4 Hz, 2H), 7.12 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 8.9 Hz, 2H), 7.00 (d, J = 7.7 Hz, 2H), 6.61 (dd, J = 2.3, 1.9 Hz, 1H). ¹³C NMR (150 MHz, DMSO-d₆): δ 165.15, 164.44, 157.31, 152.15, 141.71, 132.03, 130.00, 129.29, 128.19, 123.06, 122.20, 119.25, 118.31, 117.97, 117.71, 108.50. ESI-HRMS: m/z calcd. for C₂₂H₁₈N₃O₂ [M+H]⁺ 356.1399; found 356.1393.

  Compound 2b: white solid, yield 73%, m.p. 243~245 ℃. ¹H NMR (600 MHz, DMSO-d₆): δ 10.83 (s, 1H), 7.98 (dd, J₁ = 7.8 Hz, J₂ = 1.5 Hz, 2H), 7.84 (d, J = 9.0 Hz, 2H), 7.60~7.55 (m, 4H), 7.50 (s, 1H), 7.44~7.41 (m, 2H), 7.10~7.08 (m, 2H), 7.03~7.02 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆): δ 164.46, 156.33, 151.69, 141.80, 141.69, 135.40, 132.00, 129.80, 129.28, 128.16, 126.72, 122.19, 119.54, 119.47, 117.71, 108.47. ESI-HRMS: m/z calcd. for C₂₂H₁₇ClN₃O₂ [M+H]⁺ 390.1009; found 390.1013.

  Compound 2c: white solid, yield 89%, m.p. 189~190 ℃. ¹H NMR (600 MHz, DMSO-d₆): δ 10.32 (s, 1H), 8.65 (d, J = 2.5 Hz, 1H), 8.10 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 8.8 Hz, 2H), 7.82 (d, J = 1.5 Hz, 1H), 7.80 (m, 2H), 7.60 (dd, J = 8.0, 1.5 Hz, 1H), 7.36 (td, J = 8.2, 1.6 Hz, 1H), 7.20 (td, J = 7.8, 1.2 Hz, 1H), 7.06 (dd, J = 8.2, 1.4 Hz, 1H), 7.00 (m, 2H), 6.61 (t, J = 2.3 Hz, 1H). ¹³C NMR (150 MHz, DMSO-d₆): δ 164.65, 161.01, 154.87, 152.12, 141.69, 130.70, 129.21, 128.77, 128.14, 125.02, 122.16, 122.05, 120.34, 118.10, 117.70, 108.46. ESI-HRMS: m/z calcd. for C₂₂H₁₇ClN₃O₂ [M+H]⁺ 390.1009; found 390.1011.

  Compound 2d: white solid, yield 80%, m.p. 213~215 ℃. ¹H NMR (600 MHz, DMSO-d₆): δ 10.27 (s, 1H), 8.65 (d, J = 2.5 Hz, 1H), 8.10 (d, J = 8.7 Hz, 2H), 8.01 (d, J = 8.7 Hz, 2H), 7.82 (d, J = 1.6 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.00~6.95 (m, 6H), 6.61 (t, J = 2.1 Hz, 1H), 3.75 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆): δ 165.01, 164.79, 155.87, 154.20, 150.53, 142.23, 142.17, 134.66, 129.68, 128.63, 122.60, 120.62, 118.28, 118.17, 115.52, 108.95, 55.89. ESI-HRMS: m/z calcd. for C₂₃H₂₀N₃O₃ [M+H]⁺ 386.1505; found 386.1512.

  2.4 Crystal structure determination

  Suitable crystal of compound 2d was cultured from CH₂Cl₂ at RT. A colorless prism of 2d (0.14mm × 0.12mm × 0.1mm) was measured on a fiberglas at a random orientation. The data were gathered on a Dual Atlas CCD diffractometer with graphite-monochromated CuKα radiation (λ = 1.54184 Å) through an ω scan mode within the range of 3.141≤θ≤66.591° (–49≤h≤50, –5≤k≤6, –5≤l≤9) at 169.99(10) K. Totally 5791 reflections were gathered, in which 3169 were independent (R_int = 0.0534) and 2657 were observed with I > 2σ(I). The structure was solved by SHELXT-2018/2 and refined through full-matrix least-squares procedure on F² with SHELXL-2018/3^[28]. All non-hydrogen atoms were refined anisotropically using the observed reflections with I > 2σ(I). All H atoms were generated geometrically and refined in terms of the riding model. The final refinement got R = 0.0763, wR = 0.2136 (w = 1/[σ²(F_o²) + (0.1185P)² + 1.4089P], where P = (F_o² + 2F_c²)/3), S = 1.087, (Δ/σ)_max = 0.001, (Δρ)_max = 0.371 and (Δρ)_min = −0.371 e/ų.

  2.5 In vitro anti-fungal/anti-oomycete activity of compounds 2a~2d

  The antifungal bioassay was screened at 50 mg/L according to the previous report^{[29, 30]}. Forest fungi, Botryosphaeria dothidea (B. dothidea); Fusarium solani (F. solani); crop fungi, Botrytis cinerea (B. cinerea), Gibberella zeae (G. zeae); crop oomycete: Phytophthora infestans (P. infestans) and Phytophthora capsici (P. capsici) were obtained from the Agricultural Culture Collection of China (ACCC). Fluopicolide and boscalid were used as positive control.

  2.6 In vivo bioassay on tomato fruits

  The in vivo bioassay procedure of compound 2b was conducted according to the previous report^[31]. Boscalid was co-assayed as positive control.

  3. RESULTS AND DISCUSSION

  3.1 Synthesis

  As illustrated in Scheme 1, compounds 2a~2d were prepared by treating the key intermediate (1) with corresponding 4-phenoxyaniline via amidation reaction with high yields ranging in 70~90%. NMR and HRMS spectra were utilized to confirm the structures. For instance, the molecular formula of compound 2d could be determined as C₂₃H₁₉N₃O₃ through its HR-MS spectrum (m/z calcd. for C₂₃H₂₀N₃O₃: 386.1505 [M+H]⁺; found: 386.1512). In the ¹H-NMR data of 2d, the appearance of singlet at δ 10.27 ppm belongs to the proton of the amide group (N(3)). Two doublets at δ 8.65 and 7.82 ppm and a triplet at δ 6.61 ppm can be attributed to three heterocyclic protons in the pyrazole ring at C(10), C(8) and C(9), respectively. Two doublets at δ 8.10 and 8.01 ppm can also be attributed to four aromatic protons of benzene ring at C(2)/C(6) and C(3)/C(5), respectively. The doublet at 7.75 ppm (2H) and six protons at 7.00~6.95 ppm are assigned to eight aromatic protons in the 4-phenoxyphenyl moiety. In addition, the singlet at δ 3.75 ppm belongs to three protons of the methoxyl group (C(23)). The ¹³C-NMR spectrum of compound 2d exhibits 17 well resolved resonances corresponding to 23 carbon atoms. Among them, the absorption peak at δ 55.89 ppm is due to the methylene carbon (C(23)), and three peaks at δ 142.17, 128.63 and 108.95 ppm result from the carbons in the pyrazole ring (C(8), C(10), C(9)). Meanwhile, four peaks at δ 142.23, 134.66, 129.68(2C) and 115.52(2C) ppm are attributed to the six carbons at the benzene ring (C(1)~C(6)), while the peak at δ 165.01 ppm is the signal of amide carboxyl carbon (C(7)). In brief, the signals appearing in the NMR spectra of compound 2d are in good accordance with its structure. Furthermore, the structures of compounds 2a~2c could also be characterized by their spectral data in a similar manner.

  Scheme 1

  

  Scheme 1.  Synthesis route of compounds 2a~2d

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  3.2 Crystal structure description

  The crystal molecular structure of compound 2d is illustrated in Fig. 1. The bond lengths and bond angles within the pyrazole and benzene rings agreed well with the normal values (Table 1). The dihedral angle between the pyrazole heterocycle and its adjacent benzene ring (C(1)~C(6)) is 14.22(18)°, indicating that the two rings are not completely coplanar and thus not well conjugated, which was possibly owning to the steric hindrance between the two rings. Accordingly, the bond length of N(1)–C(4) is 1.413(4) Å, similar to the value of typical N–C bond (1.471 Å). However, the bond distance of N(3)–C(7) is 1.361(4) Å, shorter than the isolated N–C bond (1.471 Å) but longer than N=C bond (1.273 Å), which is due to the p-π conjugation effect between N(3) and the carbonyl group. The torsion angles C(2)–C(1)–C(7)–N(3) and C(7)– N(3)–C(11)–C(12) are −27.2(4)° and −35.2(4)°, respectively, indicating that the amide plane is not coplanar with both of its adjacent benzene rings (C(1)~C(6) and C(11)~C(16)). Thus, the dihedral angle between these two phenyl rings is 60.41(7)°. In addition, owing to the existence of ether group (O(2)), the dihedral angle between the two phenyl rings (C(11)~C(16)) and (C(17)~C(22)) is 76.91(7)°.

  Figure 1

  

  Figure 1.  X-ray crystal structure of compound 2d

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  Table 1

  

  Table 1.  Selected Bond Lengths (Å) and Bond Angles (°)

  DownLoad: CSV

  Bond	Dist.		Bond	Dist.		Bond	Dist.
  N(1)–C(4)	1.413(4)		C(1)–C(7)	1.496(4)		O(2)–C(17)	1.388(3)
  N(1)–C(10)	1.357(4)		N(3)–C(7)	1.361(4)		C(17)–C(18)	1.386(4)
  N(2)–C(8)	1.325(4)		N(3)–C(11)	1.411(4)		C(19)–C(20)	1.393(4)
  C(8)–C(9)	1.394 (5)		C(11)–C(12)	1.401(4)		O(3)–C(20)	1.375(3)
  O(1)–C(7)	1.229(4)		C(13)–C(14)	1.377(4)		O(3)–C(23)	1.423(4)
  Angle	(°)		Angle	(°)		Angle	(°)
  N(1)–C(10)–C(9)	107.4(3)		C(16)–C(11)–N(3)	118.2(2)		C(18)–C(17)–O(2)	116.4(3)
  N(2)–C(8)–C(9)	111.8(3)		C(13)–C(14)–O(2)	121.0(3)		O(3)–C(20)–C(19)	115.1(2)
  C(10)–N(1)–N(2)	111.1(3)		C(17)–O(2)–C(14)	119.7(2)		C(20)–O(3)–C(23)	117.2(2)
  N(2)–N(1)–C(4)–C(3)	11.7(4)		C(2)–C(1)–C(7)–N(3)	−27.2(4)		C(17)–O(2)–C(14)–C(13)	−67.8(4)
  C(6)–C(1)–C(7)–O(1)	−24.1(4)		C(7)–N(3)–C(11)–C(12)	−35.2(4)		C(23)–O(3)–C(20)–C(21)	−10.2(4)

  The molecular packing diagram of compound 2d is presented in Fig. 2. There are two orientations of molecules presented in the crystal structure. The molecules are connected by two types of hydrogen bonds (N(3)–H(3)···O(1) and C(10)– H(10)···N(2)) (Table 2), forming chains along the b axis. In addition, the two orientations of molecules pack alternately along the c axis via Van de Waals interactions, forming a layer of molecules. Along the a axis, different layers of compounds pack through a head-to-head and tail-to-tail manner to form a three-dimensional network.

  Table 2

  

  Table 2.  Hydrogen Bond Lengths (Å) and Bond Angles (°) of Compound 2d

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  D–H···A	D–H	H···A	D···A	∠DHA
  N(3)–H(3)···O(1)a	0.88	2.37	3.212(3)	161
  C(10)–H(10)···N(2)b	0.95	2.56	3.361(4)	143
  Symmetry codes: (a) x, y+1, z; (b) x, y–1, z

  Figure 2

  

  Figure 2.  Molecular packing of compound 2d

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  3.3 In vitro anti-fungal/anti-oomycete activity

  As presented in Table 3, compounds 2a~2d displayed different degree of antifungal activity against the tested fungi. The primary bioassay showed that 2a~2d exhibited mild activities against two oomycetes P. capsici and P. infestans, and very low inhibition against two fungal strains G. zeae and F. solani. On the contrary, these compounds showed notable inhibitions to B. cinerea and B. dothidea. Compound 2b displayed 67.1% inhibition against B. cinerea, which was near the value of boscalid (75.3% inhibition). Meanwhile, compound 2c exhibited 51.6% inhibition rate against forest fungi B. dothidea, which was approached to fluopicolide (59.5% inhibition). Based on such results, compound 2b was selected to further evaluate its fungicidal potency against B. cinerea (EC₅₀ = 1.61 mg/L), which was equivalent to that of boscalid (EC₅₀ = 1.41 mg/L) (Table 4).

  Table 3

  

  Table 3.  In vitro Anti-fungal/anti-oomycete Results of 2a~2d at 50 mg/L

  DownLoad: CSV

  Compound	Inhibition rate (%)a
  	Crop fungi		Crop oomycete		Forest fungi
  	B. cinerea	G. zeae	P. infestans	P. capsici	B. dothidea	F. solani
  2a	48.3 ± 2.8	9.3 ± 4.9	7.9 ± 0.6	8.3 ± 0.1	23.7 ± 3.3	8.3 ± 1.7
  2b	67.1 ± 1.0	< 5.0	20.8 ± 5.9	23.9 ± 1.0	43.1 ± 3.2	< 5.0
  2c	54.3 ± 1.9	17.6 ± 4.0	12.8 ± 3.8	12.2 ± 0.1	51.6 ± 0.9	< 5.0
  2d	< 5.0	14.8 ± 3.2	10.8 ± 3.5	33.0 ± 0.1	28.4 ± 3.3	< 5.0
  Fluopicolide	14.6 ± 1.1	19.8 ± 2.8	40.3 ± 0.6	28.3 ± 1.7	59.5 ± 0.9	0
  Boscalid	75.3 ± 2.0	91.7 ± 1.4	37.5 ± 3.9	98.0 ± 0.5	90.3 ± 0.5	0
  a Values are the mean ± standard deviation (SD) of three replicates.

  Table 4

  

  Table 4.  EC₅₀ of Compound 2b and Boscalid against B. Cinerea

  DownLoad: CSV

  Compound	EC50 (mg/L)	Regression equation	95% Cl	R2
  2b	1.61	y = 3.93x – 0.81	1.34~1.96	0.999
  Boscalid	1.41	y = 2.52x – 0.38	0.81~3.10	0.988

  As for compounds 2a~2d, most of them demonstrate fair to notable anti-fungal/anti-oomycete activities toward B. cinerea and B. dothidea, with inhibition rates ranging from 0 to 70%. In general, 2b and 2c with electron-withdrawing -Cl group on the diphenyl core presented better antifungal effects than 2a or 2d with -H or electron-donating -OCH₃ group, which demonstrate that the electron-withdrawing groups on the phenyl moiety, such as -Cl group, could be more beneficial to the anti-fungal/anti-oomycete activity of these derivatives than the electron-donating groups.

  3.4 In vivo antifungal activity of compound 2b against B. cinerea

  The results presented in Fig. 3 and Table 5 indicate that compound 2b displays considerable protective effects against B. cinerea at two dosages. Especially, 2b exhibits potent protective effects (85.3%) at 200 mg/L, which is equipotent to boscalid (88.6%). Meanwhile, it also displays good curative effect (77.1% and 61.1%) toward B. cinerea at the dosages of 200 and 100 mg/L, respectively, which is better than that of boscalid (41.1% and 40.0%). These results indicate that the selected compound 2b shows good curative effects to tomato fruits infected by B. cinerea.

  Figure 3

  

  Figure 3.  In vivo antifungal activity of compounds 2b

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  Table 5

  

  Table 5.  In vivo Antifungal Activity of Compounds 2b

  DownLoad: CSV

  Compound	Concentration (mg/L)	Protective activity (%)a		Curative activity (%)a
  		Diameter lesion length (cm)	Inhibition (%)	Diameter lesion length (cm)	Inhibition (%)
  2b	200	0.725 ± 0.040	85.3	0.9 ± 0.1	77.1
  	100	1.05 ± 0.07	64.1	1.18 ± 0.04	61.1
  Boscalid	200	0.675 ± 0.040	88.6	1.53 ± 0.04	41.1
  	100	1.03 ± 0.04	65.4	1.55 ± 0.07	40.0
  Negative control	-	2.03 ± 0.04	-	2.25 ± 0.04	-
  a Values are the mean ± standard deviation (SD) of nine replicates.

  4. CONCLUSION

  In this study, four novel pyrazole-benzene carboxamide derivatives were synthesized, characterized and screened for their anti-fungal/anti-oomycete activity. A potential bioactive compound 2b is found with considerable antifungal activity against B. cinerea. This work provides new insights into the novel fungicides investigation. The in-depth structural modification and antifungal mechanism studies will be carried out in the future.

  
  
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  Scheme 1  Synthesis route of compounds 2a~2d

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  Figure 1  X-ray crystal structure of compound 2d

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  Figure 2  Molecular packing of compound 2d

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  Figure 3  In vivo antifungal activity of compounds 2b

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  Table 1.  Selected Bond Lengths (Å) and Bond Angles (°)

  Bond	Dist.		Bond	Dist.		Bond	Dist.
  N(1)–C(4)	1.413(4)		C(1)–C(7)	1.496(4)		O(2)–C(17)	1.388(3)
  N(1)–C(10)	1.357(4)		N(3)–C(7)	1.361(4)		C(17)–C(18)	1.386(4)
  N(2)–C(8)	1.325(4)		N(3)–C(11)	1.411(4)		C(19)–C(20)	1.393(4)
  C(8)–C(9)	1.394 (5)		C(11)–C(12)	1.401(4)		O(3)–C(20)	1.375(3)
  O(1)–C(7)	1.229(4)		C(13)–C(14)	1.377(4)		O(3)–C(23)	1.423(4)
  Angle	(°)		Angle	(°)		Angle	(°)
  N(1)–C(10)–C(9)	107.4(3)		C(16)–C(11)–N(3)	118.2(2)		C(18)–C(17)–O(2)	116.4(3)
  N(2)–C(8)–C(9)	111.8(3)		C(13)–C(14)–O(2)	121.0(3)		O(3)–C(20)–C(19)	115.1(2)
  C(10)–N(1)–N(2)	111.1(3)		C(17)–O(2)–C(14)	119.7(2)		C(20)–O(3)–C(23)	117.2(2)
  N(2)–N(1)–C(4)–C(3)	11.7(4)		C(2)–C(1)–C(7)–N(3)	−27.2(4)		C(17)–O(2)–C(14)–C(13)	−67.8(4)
  C(6)–C(1)–C(7)–O(1)	−24.1(4)		C(7)–N(3)–C(11)–C(12)	−35.2(4)		C(23)–O(3)–C(20)–C(21)	−10.2(4)

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  Table 2.  Hydrogen Bond Lengths (Å) and Bond Angles (°) of Compound 2d

  D–H···A	D–H	H···A	D···A	∠DHA
  N(3)–H(3)···O(1)a	0.88	2.37	3.212(3)	161
  C(10)–H(10)···N(2)b	0.95	2.56	3.361(4)	143
  Symmetry codes: (a) x, y+1, z; (b) x, y–1, z

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  Table 3.  In vitro Anti-fungal/anti-oomycete Results of 2a~2d at 50 mg/L

  Compound	Inhibition rate (%)a
  	Crop fungi		Crop oomycete		Forest fungi
  	B. cinerea	G. zeae	P. infestans	P. capsici	B. dothidea	F. solani
  2a	48.3 ± 2.8	9.3 ± 4.9	7.9 ± 0.6	8.3 ± 0.1	23.7 ± 3.3	8.3 ± 1.7
  2b	67.1 ± 1.0	< 5.0	20.8 ± 5.9	23.9 ± 1.0	43.1 ± 3.2	< 5.0
  2c	54.3 ± 1.9	17.6 ± 4.0	12.8 ± 3.8	12.2 ± 0.1	51.6 ± 0.9	< 5.0
  2d	< 5.0	14.8 ± 3.2	10.8 ± 3.5	33.0 ± 0.1	28.4 ± 3.3	< 5.0
  Fluopicolide	14.6 ± 1.1	19.8 ± 2.8	40.3 ± 0.6	28.3 ± 1.7	59.5 ± 0.9	0
  Boscalid	75.3 ± 2.0	91.7 ± 1.4	37.5 ± 3.9	98.0 ± 0.5	90.3 ± 0.5	0
  a Values are the mean ± standard deviation (SD) of three replicates.

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  Table 4.  EC₅₀ of Compound 2b and Boscalid against B. Cinerea

  Compound	EC50 (mg/L)	Regression equation	95% Cl	R2
  2b	1.61	y = 3.93x – 0.81	1.34~1.96	0.999
  Boscalid	1.41	y = 2.52x – 0.38	0.81~3.10	0.988

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  Table 5.  In vivo Antifungal Activity of Compounds 2b

  Compound	Concentration (mg/L)	Protective activity (%)a		Curative activity (%)a
  		Diameter lesion length (cm)	Inhibition (%)	Diameter lesion length (cm)	Inhibition (%)
  2b	200	0.725 ± 0.040	85.3	0.9 ± 0.1	77.1
  	100	1.05 ± 0.07	64.1	1.18 ± 0.04	61.1
  Boscalid	200	0.675 ± 0.040	88.6	1.53 ± 0.04	41.1
  	100	1.03 ± 0.04	65.4	1.55 ± 0.07	40.0
  Negative control	-	2.03 ± 0.04	-	2.25 ± 0.04	-
  a Values are the mean ± standard deviation (SD) of nine replicates.

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