新型4(3<i>H</i>)-喹唑啉酮席夫碱衍生物的合成及抗烟草花叶病毒活性

引用本文

王翔, 蒋向辉, 汤承浩, 韦国兰 . 新型4(3H)-喹唑啉酮席夫碱衍生物的合成及抗烟草花叶病毒活性[J]. 农药学学报, 2016, 18(6): 686-696,doi: 10.16801/j.issn.1008-7303.2016.0096 复制到剪切板

WANG Xiang, JIANG Xianghui, TANG Chenghao, WEI Guolan . Synthesis and anti-TMV activity of novel 4(3H)-quinazolinone derivatives containing a Schiff base moiety[J]. Chinese Journal of Pesticide Science, 2016, 18(6): 686-696, doi:10.16801/j.issn.1008-7303.2016.0096 复制到剪切板

新型4(3H)-喹唑啉酮席夫碱衍生物的合成及抗烟草花叶病毒活性

王翔¹, 蒋向辉^1,2, 汤承浩^1,2, 韦国兰^1,2

1. 凯里学院, 黔东南民族药综合利用工程技术研究中心, 贵州凯里 556011;
2. 凯里学院, 化学与材料工程学院, 贵州凯里 556011

摘要: 合成了一系列含席夫碱的4（3H）-喹唑啉酮衍生物，并采用半叶枯斑法测试了目标化合物抗烟草花叶病毒活性。结果表明：目标化合物在500 μg/mL下对烟草花叶病毒的治疗活性为40.4%~53.1%，保护活性为51.2%~72.6%，钝化活性为46.0%~90.7%，其中，化合物3d的保护活性较好，有效抑制中浓度（EC₅₀）值为203.89 μg/mL，略优于商业化抗病毒剂宁南霉素（EC₅₀值214.90 μg/mL）。初步构效关系结果表明：在苯环的R¹及R²位上引入吸电子基团有利于活性的提高。本研究结果表明，含席夫碱的4（3H）-喹唑啉酮衍生物能较好地控制烟草花叶病毒。

关键词: 4(3H)-喹唑啉酮席夫碱合成抗烟草花叶病毒活性

Synthesis and anti-TMV activity of novel 4(3H)-quinazolinone derivatives containing a Schiff base moiety

WANG Xiang¹, JIANG Xianghui^1,2, TANG Chenghao^1,2, WEI Guolan^1,2

1. Qiandongnan Engineering and Technology Research Center for Comprehensive Utilization of National Medicine, Kaili University, Kaili 556011, Guizhou Province, China;
2. College of Chemistry and Materials Engineering, Kaili University, Kaili 556011, Guizhou Province, China

Received: Sep 16, 2016 Accepted: Oct 31, 2016

Fond project: Guizhou Province Natural Science United Fund (No. JLKK [2013]03 and LH [2015]7740) and Guizhou Province Education Quality Promotion Engineering Project (No. KY [2014]228)

^* Wang Xiang,author for correspondence,female,professor,the research interest is focused on pesticide science,medicinal chemistryand organic synthesis,E-mail:wangx0828@sina.com

Abstract: A series of 4(3H)-quinazolinone derivatives containing a Schiff base moiety were synthesized and their antiviral activity against tobacco mosaic virus (TMV) was evaluated using halfleaf method in vivo. Bioactivity results exhibited that the target compounds 3a-3x under 500 μg/mL displayed moderate to good anti-TMV activities with the curative activity, protection activity, and inactivation activity values of 40.4%-53.1%, 51.2%-72.6%, and 46.0%-90.7%, respectively. Especially, compound 3d showed excellent protection activity against TMV, with a half-maximal effective concentration (EC₅₀) value of 203.89 μg/mL, which was even superior to that of ningnanmycin (214.90 μg/mL). SAR analysis showed that compounds with electron-withdrawing groups at R¹ and R² positions displayed better anti-TMV activity. These results indicated that the series of 4(3H)-quinazolinone derivatives containing a Schiff base moiety can effectively control TMV.

Key words: 4(H)-quinazolinone Schiff base synthesis anti-TMV activity

Tobacco mosaic virus (TMV) is a positive-sense and single stranded RNA virus which could infect 9 families and at least 125 species of plants including tobacco, tomato, pepper, cucumbers, and a number of ornamental flowers. TMV diseases could cause a huge loss in agricultural production worldwide every year^[1]. Ningnanmycin, isolated from Strepcomces noursei var. xichangensis, was found to be more effective than existing products in the treatment of TMV. However, using ningnanmycin in field trials is not suitable because of its unsatisfactory curative rates and high control costs^[2]. Therefore, the search for new antiviral agents remains an important task in pesticide science^[3].

4 (3H) -Quinazolinone derivatives have a variety of biological activities including antibacterial^[4-7], antifungal^[6-9], antiviral^[10-13], anti-inflammatory^{[5, 14-15]}, and anticancer^[16-17] activities. In the past decade, a series of novel 4 (3H) -quinazolinone derivatives were synthesized and displayed excellent anti-TMV activity^[10-13]. Meanwhile, Schiff base, which exhibited a broad-spectrum biological activities such as antibacterial^{[4, 6]}, antifungal^[4], antiviral^[18-19], herbicide^[20], and insecticide^{[18, 21]} activities, could be used as an ideal lead structure for the development of agrochemicals and medicines. In our previous work, a series of 4 (3H) -quinazolinone derivatives containing a Schiff base moiety had been synthesized, and their antibacterial and antifungal activities have been evaluated^{[4, 6]}.

To continue our efforts on developing highly antiviral activity and also in continuation of our earlier research, a series of 4 (3H) -quinazolinone derivatives containing a Schiff base moiety were designed and synthesized. In this paper, we would like to report their synthesis and anti-TMV activity. The synthetic route of the target compounds 3a-3x is shown in Scheme 1.

Scheme1 Synthetic route of the target compounds 3a-3x.

1 Materials and Methods 1.1 General procedure

Unless noted, all solvents and reagents were freshly distilled or purified according to standard procedures. The IR spectra were recorded on a VECTOR 22 spectrometer (Bruker, Germany) in KBr disk. NMR date was performed in solvent CDCl₃ on a JEOL-ECX 500 NMR spectrometer (JEOL, Japan) at room temperature spectrometer operating at 500 and 125 MHz, respectively, using TMS as an internal standard. Elemental analysis was performed on an Elementar Vario-III CHN analyzer (Elementar, Germany) . Mass spectral studies were conducted on an Agilent 5973 organic mass spectrometer (Agilent, USA) . X-ray data for 3t were collected using a Bruker Smart Apex CCD area detector diffractometer (Bruker, Germany) with Mo-Ka radiation. Analytical TLC was performed on silica gel GF₂₅₄. Flash column chromatography was carried out on silica gel 200-300 mesh.

1.2 General procedure for the preparation of the target compounds 3a-3x

As shown in Scheme 1, the target compounds 3a-3x were synthesized using o-aminobenzoic acid as the starting material according to the previously reported methods^{[4, 6]}. The physical characteristics, IR, ¹H NMR, ¹³C NMR, MS, and elemental analysis data for all the target compounds 3a-3x are shown below.

(E) -3- (2- ( (2-fluorobenzylidene) amino) ethyl) -2-anilino-4 (3H) -quinazolinone (3a) . White solid. IR (KBr) , ν/cm^-1: 3 460 (N-H, Qu-ring-NH-Ar) , 3 059 (Ar-C-H) , 1 674 (C=O) , 1 610 (C=N) , 1 473-1 585 (C=C and N-H, benzene and Qu-ring and bending of N-H) , 1 444 (C-H) , 1 278 (C-N) , 1 172-1 253 (C-F) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.83 (s, 1H, Qu-ring-NH-Ar) , 8.68 (s, 1H, -N=CH-) , 8.19 (d, 1H, J = 8.05 Hz, Ar-H) , 7.90 (t, 1H, J = 6.87 Hz, Ar-H) , 7.43-7.62 (m, 5H, Ar-H) , 7.03-7.30 (m, 6H, Ar-H and Qu-H) , 4.63 (s, 2H, =N-CH₂-Ar) , 4.12 (s, 2H, Qu-ring-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.58, 158.56, 148.75, 148.48, 139.33, 134.36, 133.56, 133.49, 128.89, 128.03, 126.82, 125.76, 124.49, 123.76, 123.05, 120.50, 118.33, 116.38, 116.22, 60.17, 45.21; MS (ESI) , m/z: 387.3 ([M+H]⁺) , 409.3 ([M+Na]⁺) ; Anal. Calcd. for C₂₃H₁₉FN₄O: C 71.49%, H 4.96%, N 14.50%; found: C 71.62%, H 5.04%, N 14.64%.

(E) -3- (2- ( (2-chlorobenzylidene) amino) ethyl) -2-anilino-4 (3H) -quinazolinone (3b) . White solid. IR (KBr) , ν/cm^-1: 3 263 (N-H, Qu-ring-NH-Ar) , 3 062 (Ar-C-H) , 1 674 (C=O) , 1 610 (C=N) , 1 587-1 473 (C=C and N-H, benzene and Qu-ring and bending of N-H) , 1 444 (C-H) , 1 303 (C-N) , 754-551 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.90 (s, 1H, Qu-ring-NH-Ar) , 8.82 (s, 1H, -N=CH-) , 8.19 (d, 1H, J = 8.05 Hz, Ar-H) , 7.97 (d, 1H, J = 7.40 Hz, Ar-H) , 7.62-7.23 (m, 10H, Ar-H and Qu-H) , 7.03 (t, 1H, J = 7.15 Hz, Ar-H) , 4.63 (s, 2H, =N-CH₂-Ar) , 4.15 (s, 2H, Qu-ring-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.59, 161.85, 148.74, 148.49, 139.29, 135.71, 134.42, 132.69, 132.25, 130.28, 128.88, 128.54, 127.12, 126.83, 125.76, 123.76, 123.04, 120.50, 118.32, 59.87, 45.21; MS (ESI) , m/z: 403.2 ([M+H]⁺) , 425.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₃H₁₉ClN₄O: C 68.57%, H 4.75%, N 13.91%; found: C 68.53%, H 4.50%, N 13.84%.

(E) -3- (2- (2-fluorobenzylideneamino) ethyl) -2- (4- (trifluoromethoxy) anilino) -4 (3H) -quinazolinone (3c) . White solid. IR (KBr) , ν/cm^-1: 3 248 (N-H, Qu-ring-NH-Ar) , 1 670 (C=O) , 1 589 (C=N) , 1 566-1 473 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 273 (C-N) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.99 (s, 1H, Qu-ring-NH-Ar) , 8.67 (s, 1H, -N=CH-) , 8.19 (d, 1H, J = 6.90 Hz, Qu-H) , 7.84 (t, 1H, J = 6.85 Hz, Qu-H) , 7.62 (t, 1H, J = 7.2 Hz, Ar-H) , 7.54-7.43 (m, 4H, Ar-H and Qu-H) , 7.27-7.25 (m, 1H, Ar-H) , 7.19-7.09 (m, 4H, Ar-H) , 4.63 (s, 2H, =N-CH₂-) , 4.13 (s, 2H, -N-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.44, 163.38, 161.36, 158.88, 148.58, 148.18, 144.32, 138.12, 134.56, 133.74, 133.67, 127.98, 126.91, 125.71, 124.52, 124.07, 121.78, 121.75, 121.39, 118.43, 116.53, 116.37, 60.11, 45.19; MS (ESI) , m/z: 471.3 ([M+H]⁺) , 493.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₁₈F₄N₄O₂: C 61.26%, H 3.99%, N 12.08%; found: C 61.28%, H 3.86%, N 11.91%.

(E) -3- (2- (2-chlorobenzylideneamino) ethyl) -2- (4- (trifluoromethoxy) anilino) -4 (3H) -quinazolinone (3d) . White solid. IR (KBr) , ν/cm^-1: 3 238 (N-H, Qu-ring-NH-Ar) , 1 676 (C=O) , 1 591 (C=N) , 1 566-1 436 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 282 (C-N) , 763 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 9.08 (s, 1H, Qu-ring-NH-Ar) , 8.83 (s, 1H, -N=CH-) , 8.20 (d, 1H, J = 6.90 Hz, Qu-H) , 7.91 (d, 1H, J = 8.05 Hz, Qu-H) , 7.63 (t, 1H, J = 7.75 Hz, Qu-H) , 7.47-7.42 (m, 5H, Qu-H and Ar-H) , 7.28-7.25 (m, 2H, Ar-H) , 7.12 (d, 2H, J = 8.60 Hz, Ar-H) , 4.63 (s, 2H, =N-CH₂-) , 4.14 (s, 2H, N-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.36, 161.97, 148.45, 148.08, 144.21, 137.96, 135.65, 134.46, 132.76, 132.14, 130.30, 128.30, 127.01, 126.81, 125.60, 123.98, 121.63, 121.53, 121.28, 118.32, 59.69, 45.08; MS (ESI) , m/z: 487.2 ([M+H]⁺) , 509.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₁₈ClF₃N₄O₂: C 59.31%, H 3.98%, N 11.53%; found: C 59.21%, H 3.73%, N 11.51%.

(E) -3- (2- (4-methylbenzylideneamino) ethyl) -2- (4- (trifluoromethoxy) anilino) -4 (3H) -quinazolinone (3e) . White solid. IR (KBr) , ν/cm^-1: 3 271 and 3 209 (N-H, Qu-ring-NH-Ar) , 3 024-2 895 (C-H) , 1 672 (C=O) , 1 597 (C=N) , 1 575-1 429 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 265 (C-N) ; ¹H NMR (500 MHz, CDCl₃) , δ: 9.15 (s, 1H, Qu-ring-NH-Ar) , 8.34 (s, 1H, Qu-H) , 8.19 (d, 1H, J =8.05 Hz, -N=CH-) , 7.61-7.43 (m, 6H, Qu-H and Ar-H) , 7.27-7.14 (m, 5H, Ar-H) , 4.61 (s, 2H, =N-CH₂-) , 4.07 (s, 2H, N-CH₂-) , 2.39 (s, 3H, -CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 164.93, 163.49, 148.75, 148.25, 144.31, 142.57, 138.21, 134.50, 132.26, 129.66, 128.54, 126.88, 125.69, 123.99, 121.74, 121.52, 119.64, 118.42, 59.78, 45.34, 21.69; MS (ESI) , m/z: 467.3 ([M+H]⁺) , 489.3 ([M+Na]⁺) ; Anal. Calcd. for C₂₅H₂₁F₃N₄O₂: C 64.22%, H 4.20%, N 12.20%; found: C 64.37%, H 4.54%, N 12.01%.

(E) -3- (2- (3-nitrobenzylideneamino) ethyl) -2- (4- (trifluoromethoxy) anilino) -4 (3H) -quinazolinone (3f) . Yellow solid. IR (KBr) , ν/cm^-1:3 298 (N-H, Qu-ring-NH-Ar) , 2 879 (C-H) , 1 666 (C=O) , 1 612 (C=N) , 1 539 (N=O) , 1 506-1 436 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 251 (C-N) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.65 (s, 1H, Qu-ring-NH-Ar) , 8.51 (s, 1H, Ar-H) , 8.45 (s, 1H, -N=CH-) , 8.32 (d, 1H, J = 8.00 Hz, Ar-H) , 8.18 (d, 1H, J = 7.45 Hz, Qu-H) , 7.94 (d, 2H, J = 8.00 Hz, Ar-H) , 7.63-7.57 (m, 4H, Qu-H and Ar-H) , 7.42 (d, 1H, J = 8.00 Hz, Ar-H) , 7.26 (t, 1H, J = 7.42 Hz, Ar-H) , 7.18 (d, 2H, J = 8.55 Hz, Ar-H) , 4.67 (s, 2H, =N-CH₂-) , 4.18 (s, 2H, -N-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.33, 162.74, 148.72, 148.19, 148.09, 144.56, 140.50, 137.79, 136.34, 134.65, 134.40, 130.22, 126.84, 126.22, 125.73, 124.18, 122.45, 121.96, 121.32, 118.35, 60.05, 44.85; MS (ESI) , m/z: 498.3 ([M+H]⁺) , 520.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₁₈F₃N₅O₄: C 57.66%, H 3.67%, N 14.54%; found: C 57.95%, H 3.65%, N 14.08%.

(E) -3- (2- (3-chloro-4-nitrobenzylideneamino) ethyl) -2- (4- (trifluoromethoxy) anilino) -4 (3H) -quinazolinone (3g) . Yellow solid. IR (KBr) , ν/cm^-1: 3 296 (N-H, Qu-ring-NH-Ar) , 2 893 (C-H) , 1 672 (C=O) , 1 593 (C=N) , 1 539 (N=O) , 1 506-1 440 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 265 (C-N) , 777 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.37 (d, 2H, J = 1.70 Hz, Qu-ring-NH-Ar and Ar-H) , 8.28 (d, 1H, J = 1.70 Hz, -N=CH-) , 8.17 (d, 1H, J = 8.00 Hz, Qu-H) , 7.74-7.54 (m, 5H, Qu-H and Ar-H) , 7.43 (d, 1H, J = 8.05 Hz, Ar-H) , 7.28-7.19 (m, 3H, Ar-H) , 4.65 (s, 2H, =N-CH₂-) , 4.16 (s, 2H, -N-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.29, 161.66, 148.43, 148.07, 144.63, 137.68, 134.70, 134.58, 132.71, 132.57, 130.22, 126.83, 125.74, 124.34, 124.21, 122.02, 121.64, 121.35, 119.60, 118.29, 60.00, 44.71; MS (ESI) , m/z: 532.2 ([M+H]⁺) , 554.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₁₇ClF₃N₅O₄: C 53.93%, H 3.15%, N 13.52%; found: C 54.20%, H 3.22%, N 13.17%.

(E) -3- (2- ( (2-fluorobenzylidene) amino) ethyl) -2- (4-methylanilino) -4 (3H) -quinazolinone (3h) . White solid. IR (KBr) , ν/cm^-1: 3 253 (N-H, Qu-ring-NH-Ar) , 3 037 (Ar-C-H) , 1 674 (C=O) , 1 610 (C=N) , 1 473-1 585 (C=C and N-H, benzene and Qu-ring and bending of N-H) , 1 456 (C-H) , 1 309 (C-N) , 1 024-1 384 (C-F) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.70 (s, 1H, Qu-ring-NH-Ar) , 8.66 (s, 1H, -N=CH-) , 8.17 (d, 1H, J = 6.90 Hz, Ar-H) , 7.89 (d, 1H, J = 5.70 Hz, Ar-H) , 7.07-7.58 (m, 10H, Ar-H and Qu-H) , 4.61 (s, 2H, =N-CH₂-Ar) , 4.11 (s, 2H, Qu-ring-CH₂-) , 2.31 (s, 3H, Ar-CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.61, 161.37, 158.42, 149.05, 148.64, 136.65, 134.35, 133.45, 132.72, 129.41, 128.03, 126.81, 125.71, 124.48, 123.54, 122.85, 120.87, 118.20, 116.20, 60.23, 45.16, 20.94; MS (ESI) , m/z: 401.3 ([M+H]⁺) , 423.3 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₂₁FN₄O: C 71.98%, H 5.29%, N 13.99%; found: C 72.00%, H 5.58%, N 13.93%.

(E) -3- (2- ( (3-nitrobenzylidene) amino) ethyl) -2- (4-methylanilino) -4 (3H) -quinazolinone (3i) . Yellow crystal. IR (KBr) , ν/cm^-1: 3 292 (N-H, Qu-ring-NH-Ar) , 2 914 (Ar-C-H) , 1 662 (C=O) , 1 612 (C=N) , 1 587-1 473 (C=C, N=O and N-H, benzene and Qu-ring and bending of N-H and -NO₂ of N=O) , 1 436 (C-H) , 1 352 (N=O) , 1 284-1 033 (C-N) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.67 (s, 1H, Qu-ring-NH-Ar) , 8.42 (s, 1H, -N=CH-) , 8.31 (d, 1H, J = 9.70 Hz, Ar-H) , 8.24 (s, 1H, Ar-H) , 8.16 (d, 1H, J = 6.30 Hz, Ar-H) , 7.96 (d, 1H, J = 7.45 Hz, Ar-H) , 7.59 (t, 2H, J = 7.72 Hz, Ar-H) , 7.40 (d, 3H, J = 8.00 Hz, Qu-H) , 7.24 (t, 1H, J = 8.00 Hz, Qu-H) , 7.12 (d, 2H, J = 8.55 Hz, Ar-H) , 4.66 (s, 2H, =N-CH₂-Ar) , 4.17 (s, 2H, Qu-ring-CH₂-) , 2.31 (s, 3H, Ar-CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.53, 162.44, 148.67, 148.64, 148.55, 136.44, 136.30, 134.47, 134.45, 133.06, 130.11, 129.59, 126.75, 126.08, 125.74, 123.67, 122.65, 120.72, 118.11, 60.20, 44.81, 20.92; MS (ESI) , m/z: 428.3 ([M+H]⁺) , 450.3 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₂₁N₅O₃: C 67.44%, H 4.95%, N 16.38%; found: C 67.06%, H 4.97%, N 16.78%.

(E) -3- (2- (3-chloro-4-nitrobenzylideneamino) ethyl) -2- (3-methylanilino) -4 (3H) -quinazolinone (3j) . Yellow solid. IR (KBr) , ν/cm^-1: 3 444 and 3 246 (N-H, Qu-ring-NH-Ar) , 3 049 and 2 897 (C-H) , 1 670 (C=O) , 1 602 (C=N) , 1 558 (N=O) , 1 525-1 473 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 361-1 033 (C-N) , 823-765 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.35 (s, 1H, Qu-ring-NH-Ar and Ar-H) , 8.27 (d, 1H, J = 1.75 Hz, -N=CH-) , 8.14 (s, 2H, Qu-H) , 7.78 (d, 1H, J = 6.85 Hz, Qu-H) , 7.63-7.55 (m, 2H, Qu-H and Ar-H) , 7.42-7.14 (m, 5H, Ar-H) , 6.90 (d, 1H, J = 7.45 Hz, Ar-H) , 4.63 (s, 2H, =N-CH₂-) , 4.15 (s, 2H, -N-CH₂-) , 2.31 (s, 3H, Ar-CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.44, 161.41, 148.42, 148.35, 138.19, 137.88, 134.71, 134.54, 132.60, 132.52, 129.99, 130.08, 129.01, 126.77, 125.79, 124.74, 124.44, 123.84, 121.06, 118.14, 117.60, 60.14, 44.70, 21.60; MS (ESI) , m/z: 462.3 ([M+H]⁺) , 484.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₂₀ClN₅O₃: C 67.44%, H 4.95%, N 16.38%; found: C 67.00%, H 4.97%, N 16.86%.

(E) -3- (2- ( (3-chloro-4-nitrobenzylidene) amino) ethyl) -2- (4-chloroanilino) -4 (3H) -quinazolinone (3k) . Yellow crystal. IR (KBr) , ν/cm^-1: 3 265 (N-H, Qu-ring-NH-Ar) , 3 057 (Ar-C-H) , 1 674 (C=O) , 1 606 (C=N) , 1 581-1 471 (C=C, N=O and N-H, benzene and Qu-ring and bending of N-H and -NO₂ of N=O) , 1 442 (C-H) , 1 357 (N=O) , 1 284-1 024 (C-N) , 813-758 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.36 (s, 1H, Qu-ring-NH-Ar) , 8.31 (s, 1H, -N=CH-) , 8.28 (s, 1H, Ar-H) , 8.16 (d, 1H, J = 7.45 Hz, Ar-H) , 7.74-7.24 (m, 9H, Qu-H and Ar-H) , 4.64 (s, 2H, =N-CH₂-Ar) , 4.15 (s, 2H, Qu-ring-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.32, 161.61, 148.41, 148.12, 148.07, 137.53, 134.67, 134.58, 132.72, 132.56, 130.23, 129.13, 128.35, 126.82, 125.74, 124.41, 124.14, 121.62, 118.24, 60.03, 44.72; MS (ESI) , m/z: 482.2 ([M+H]⁺) , 504.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₃H₁₇Cl₂N₅O₃: C 57.27%, H 3.55%, N 14.52%; found: C 57.35%, H 3.58%, N 14.25%.

(E) -3- (2- ( (3-nitrobenzylidene) amino) ethyl) -2- (4-chloroanilino) -4 (3H) -quinazolinone (3l) . Light yellow crystal. IR (KBr) , ν/cm^-1: 3 271 (N-H, Qu-ring-NH-Ar) , 2 875 (Ar-C-H) , 1 672 (C=O) , 1 606 (C=N) , 1 587-1 475 (C=C, N=O and N-H, benzene and Qu-ring and bending of N-H and -NO₂ of N=O) , 1 435 (C-H) , 1 350 (N=O) , 1 303-1 022 (C-N) , 813-758 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.65 (s, 1H, Qu-ring-NH-Ar) , 8.44 (s, 2H, -N=CH- and Ar-H) , 8.32 (d, 1H, J = 8.55 Hz, Ar-H) , 8.17 (d, 1H, J = 6.90 Hz, Ar-H) , 7.94 (d, 1H, J = 8.00 Hz, Ar-H) , 7.62 (dd, 2H, ⁴J = 8.60 Hz, ³J = 8.00 Hz, Ar-H) , 7.51 (d, 2H, J = 8.60 Hz, Ar-H) , 7.41 (d, 1H, J = 8.00 Hz, Qu-H) , 7.41 (dd, 3H, ⁴J = 2.30 Hz, ³J = 8.00 Hz, Ar-H and Qu-H) , 4.66 (s, 2H, =N-CH₂-Ar) , 4.17 (s, 2H, Qu-ring-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.37, 162.70, 148.70, 148.18, 148.14, 137.63, 136.34, 134.64, 134.44, 130.22, 129.05, 128.15, 126.82, 126.19, 125.72, 124.12, 122.47, 121.60, 118.29, 60.06, 44.85; MS (ESI) , m/z: 448.3 ([M+H]⁺) , 470.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₃H₁₈ClN₅O₃: C 61.68%, H 4.05%, N 15.64%; found: C 61.29%, H 4.23%, N 15.39%.

(E) -3- (2- ( (3-nitrobenzylidene) amino) ethyl) -2-anilino-4 (3H) -quinazolinone (3m) . Yellow solid. IR (KBr) , ν/cm^-1: 3 282 (N-H, Qu-ring-NH-Ar) , 1 674 (C=O) , 1 610 (C=N) , 1 475-1 589 (C=C and N-H, benzene and Qu-ring and bending of N-H) , 1 440 (C-H) , 1 350 (C-N) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.67 (s, 1H, Qu-ring-NH-Ar) , 8.44 (s, 1H, -N=CH-) , 8.36 (s, 1H, Ar-H) , 8.25 (d, 1H, J = 7.45 Hz, Ar-H) , 8.16 (d, 1H, J = 8.05 Hz, Ar-H) , 7.95 (d, 1H, J = 7.45 Hz, Ar-H) , 7.61-7.54 (m, 4H, Ar-H and Qu-H) , 7.42 (d, 1H, J = 7.80 Hz, Ar-H) , 7.32 (t, 2H, J = 8.00 Hz, Ar-H) , 7.25-7.23 (m, 1H, Ar-H) , 7.07 (t, 1H, J = 7.55 Hz, Ar-H) , 4.68 (s, 2H, =N-CH₂-Ar) , 4.20 (s, 2H, Qu-ring-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.49, 162.56, 148.69, 148.39, 148.37, 138.98, 136.41, 134.54, 134.39, 130.13, 129.11, 126.77, 126.12, 125.78, 123.88, 123.36, 122.68, 1120.40, 118.23, 60.14, 44.87; MS (ESI) , m/z: 414.3 ([M+H]⁺) , 436.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₃H₁₉N₅O₃: C 66.82%, H 4.63%, N 16.94%; found: C 66.95%, H 4.41%, N 16.93%.

(E) -3- (2- (4-methylbenzylideneamino) ethyl) -2-anilino-4 (3H) -quinazolinone (3n) . White solid. IR (KBr) , ν/cm^-1: 3 250 and 3 188 (N-H, Qu-ring-NH-Ar) , 3 061-2 887 (C-H) , 1 670 (C=O) , 1 606 (C=N) , 1 583-1 473 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 446 (C-H) , 1 257 (C-N) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.99 (s, 1H, Qu-ring-NH-Ar) , 8.32 (s, 1H, Qu-H) , 8.18 (d, 1H, J =8.05 Hz, -N=CH-) , 7.97-6.93 (m, 12H, Qu-H and Ar-H) , 4.60 (s, 2H, =N-CH₂-) , 4.07 (s, 2H, -N-CH₂-) , 2.37 (s, 3H, -CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 164.77, 163.62, 148.92, 148.54, 142.34, 139.43, 134.36, 132.35, 129.61, 128.89, 128.61, 126.80, 125.76, 123.68, 122.97, 121.34, 118.34, 59.85, 45.38, 21.69; MS (ESI) , m/z: 383.2 ([M+H]⁺) , 405.1 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₂₂N₄O: C 75.37%, H 5.80%, N 14.65%; found: C 74.92%, H 5.40%, N 15.00%.

(E) -3- (2- (3-chloro-4-nitrobenzylideneamino) ethyl) -2-anilino-4 (3H) -quinazolinone (3o) . Yellow solid. IR (KBr) , ν/cm^-1: 3 286 (N-H, Qu-ring-NH-Ar) , 3 066 (C-H) , 1 670 (C=O) , 1 608 (C=N) , 1 533 (N=O) , 1 589-1 475 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 444 (C-H) , 1 348 (C-O) , 1 253 (C-N) , 765 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.35 (s, 1H, Qu-ring-NH-Ar) , 8.28 (s, 1H, -N=CH-) , 8.25 (s, 1H, Qu-H) , 8.16 (d, 1H, J = 7.80 Hz, Ar-H) , 7.60 (d, 1H, J = 8.00 Hz, Ar-H) , 7.72-7.06 (m, 9H, Qu-H and Ar-H) , 4.65 (s, 2H, =N-CH₂-) , 4.16 (s, 2H, -N-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.43, 161.46, 148.37, 148.24, 138.89, 134.65, 134.57, 132.62, 132.56, 130.08, 129.18, 126.78, 125.78, 124.57, 123.90, 123.53, 120.39, 118.17, 60.08, 44.72; MS (ESI) , m/z: 448.1 ([M+H]⁺) , 470.1 ([M+Na]⁺) ; Anal. Calcd. for C₂₃H₁₈ClN₅O₃: C 61.68%, H 4.05%, N 15.64%; found: C 61.17%, H 3.84%, N 15.90%.

(E) -3- (2- (2-chlorobenzylideneamino) ethyl) -2- (4-methylanilino) -4 (3H) -quinazolinone (3p) . White solid. IR (KBr) , ν/cm^-1: 3 263 (N-H, Qu-ring-NH-Ar) , 1 674 (C=O) , 1 589 (C=N) , 1 568-1 475 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 253 (C-N) , 759 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.82 (s, 1H, Qu-ring-NH-Ar) , 8.78 (s, 1H, -N=CH-) , 8.17 (d, 1H, J = 8.00 Hz, Qu-H) , 7.97 (d, 1H, J = 8.00 Hz, Qu-H) , 7.60 (t, 1H, J = 7.45 Hz, Qu-H) , 7.42-7.06 (m, 9H, Qu-H and Ar-H) , 4.62 (s, 2H, =N-CH₂-) , 4.14 (s, 2H, -N-CH₂-) , 2.31 (s, 3H, Ar-CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.64, 161.73, 149.05, 148.64, 136.59, 135.71, 134.36, 132.73, 132.66, 132.26, 130.27, 129.39, 128.56, 127.11, 126.81, 125.72, 123.55, 120.88, 118.18, 59.92, 45.16, 20.92; MS (ESI) , m/z: 417.3 ([M+H]⁺) , 439.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₂₁ClN₄O: C 69.14%, H 5.08%, N 13.44%; found: C 69.13%, H 5.19%, N 13.45%.

(E) -3- (2- (4-methylbenzylideneamino) ethyl) -2- (4-methylanilino) -4 (3H) -quinazolinone (3q) . White solid. IR (KBr) , ν/cm^-1: 3 281 and 3 209 (N-H, Qu-ring-NH-Ar) , 3 028-2 995 (C-H) , 1 668 (C=O) , 1 608 (C=N) , 1 581-1 573 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 255 (C-N) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.85 (s, 1H, Qu-ring-NH-Ar) , 8.31 (s, 1H, Qu-H) , 8.16 (d, 1H, J = 8.05 Hz, -N=CH-) , 7.64-7.57 (m, 3H, Qu-H and Ar-H) , 7.41-7.09 (m, 8H, Ar-H) , 4.59 (s, 2H, =N-CH₂-) , 4.06 (s, 2H, -N-CH₂-) , 2.38 (s, 3H, Ar-CH₃) , 2.32 (s, 3H, Ar-CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 164.66, 163.66, 149.23, 148.71, 142.30, 136.74, 134.31, 132.64, 132.37, 129.59, 129.40, 128.61, 126.78, 125.73, 123.46, 120.98, 118.20, 59.91, 45.32, 21.70, 20.96; MS (ESI) , m/z: 397.3 ([M+H]⁺) , 419.3 ([M+Na]⁺) ; Anal. Calcd. for C₂₅H₂₄N₄O: C 75.73%, H 6.10%, N 14.13%; found: C 75.53%, H 5.97%, N 14.06%.

(E) -3- (2- (3-chloro-4-nitrobenzylideneamino) ethyl) -2- (4-methylanilino) -4 (3H) -quinazolinone (3r) . Yellow solid. IR (KBr) , ν/cm^-1: 3 282 (N-H, Qu-ring-NH-Ar) , 3 064 and 3 032 (C-H) , 1 672 (C=O) , 1 585 (C=N) , 1 533 (N=O) , 1 516-1 471 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 253 (C-N) , 763 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.33 (s, 1H, Qu-ring-NH-Ar and Ar-H) , 8.27 (s, 1H, -N=CH-) , 8.13 (s, 2H, Qu-H) , 7.73-7.12 (m, 9H, Qu-H and Ar-H) , 4.63 (s, 2H, =N-CH₂-) , 4.14 (s, 2H, -N-CH₂-) , 2.33 (s, 3H, Ar-CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.46, 161.32, 148.53, 148.36, 136.19, 134.67, 134.51, 133.32, 132.59, 129.99, 129.66, 126.75, 125.73, 124.52, 123.68, 120.79, 118.03, 60.13, 44.65, 20.95; MS (ESI) , m/z: 462.3 ([M+H]⁺) , 484.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₂₀ClN₅O₃: C 62.41%, H 4.36%, N 15.16%; found: C 62.48%, H 4.50%, N 15.35%.

(E) -3- (2- ( (2-fluorobenzylidene) amino) ethyl) -2- (3-methylanilino) -4 (3H) -quinazolinone (3s) . White solid. IR (KBr) , ν/cm^-1: 3 263 (N-H, Qu-ring-NH-Ar) , 1 672 (C=O) , 1 589 (C=N) , 1 566-1 473 (C=C and N-H, benzene and Qu-ring and bending of N-H) , 1 436 (C-H) , 1 296 (C-N) , 767-754 (C-F) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.75 (s, 1H, Qu-ring-NH-Ar) , 8.68 (s, 1H, -N=CH-) , 8.18 (d, 1H, J = 6.85 Hz, Ar-H) , 7.93 (s, 1H, Ar-H) , 7.59-7.11 (m, 9H, Ar-H and Qu-H) , 6.86 (d, 1H, J = 6.30 Hz, Ar-H) , 4.62 (s, 2H, =N-CH₂-Ar) , 4.12 (s, 2H, Qu-ring-CH₂-) , 2.25 (s, 3H, Ar-CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.61, 158.53, 148.92, 148.54, 139.11, 138.68, 134.40, 133.55, 133.48, 128.77, 128.11, 126.81, 125.77, 124.48, 123.96, 123.70, 121.28, 118.26, 118.06, 116.38, 116.22, 60.28, 45.21, 21.52; MS (ESI) , m/z: 401.3 ([M+H]⁺) , 423.3 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₂₁FN₄O: C 71.98%, H 5.29%, N 13.99%; found: C 71.63%, H 5.17%, N 14.35%.

(E) -3- (2- (2, 5-dimethoxybenzylideneamino) ethyl) -2- (3-methylanilino) -4 (3H) -quinazolinone (3t) . White solid. IR (KBr) , ν/cm^-1: 3 263 (N-H, Qu-ring-NH-Ar) , 3 047 (Ar-C-H) , 1 670 (C=O) , 1 593 (C=N) , 1 586-1 473 (C=C and N-H, benzene and Qu-ring and bend of N-H) , 1 301 (C-N) , 1 166 (Ar-C-O) , 1 089 (-OCH₃) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.97 (s, 1H, Qu-ring-NH-Ar) , 8.78 (s, 1H, -N=CH-) , 8.18 (d, 1H, J = 8.00 Hz, Qu-H) , 7.60 (t, 1H, J = 7.45 Hz, Ar-H) , 7.43 (s, 3H, Qu-H) , 7.24-6.99 (m, 4H, Ar-H) , 6.87 (t, 2H, J = 9.45 Hz, Ar-H) , 4.60 (s, 2H, =N-CH₂-) , 4.09 (s, 2H, -N-CH₂-) , 3.79 (s, 3H, 2-OCH₃) , 3.61 (s, 3H, 5-OCH₃) , 2.22 (s, 3H, Ar-CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.68, 160.94, 153.78, 153.74, 153.64, 149.13, 148.65, 139.12, 138.72, 134.28, 128.70, 126.80, 125.75, 123.93, 123.85, 123.51, 121.49, 119.65, 118.25, 112.77, 111.05, 60.52, 56.08, 55.63, 45.49, 21.31; MS (ESI) , m/z: 443.2 ([M+H]⁺) , 465.1 ([M+Na]⁺) ; Anal. Calcd. for C₂₆H₂₆N₄O₃: C 70.57%, H 5.92%, N 12.66%; found: C 70.15%, H 5.47%, N 12.99%.

(E) -3- (2- ( (3-nitrobenzylidene) amino) ethyl) -2- (3-methylanilino) -4 (3H) -quinazolinone (3u) . Yellow crystal. IR (KBr) , ν/cm^-1: 3 288 (N-H, Qu-ring-NH-Ar) , 3 074 and 2 875 (Ar-C-H) , 1674 (C=O) , 1 598 (C=N) , 1 567-1 475 (C=C, N=O and N-H, benzene and Qu-ring and bending of N-H and -NO₂ of N=O) , 1 436 (C-H) , 1 344 (N=O) , 1 303-1 029 (C-N) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.64 (s, 1H, Qu-ring-NH-Ar) , 8.43 (s, 1H, -N=CH-) , 8.31 (d, 1H, J = 8.00 Hz, Qu-H) , 8.26 (s, 1H, Qu-H) , 8.17 (d, 1H, J = 8.05 Hz, Ar-H) , 7.99 (d, 1H, J = 8.00 Hz, Ar-H) , 7.60 (dd, 2H, ⁴J = 6.85 Hz, ³J = 8.05 Hz, Ar-H) , 7.42-7.19 (m, 5H, Ar-H) , 6.87 (d, 1H, J = 7.45 Hz, Ar-H) , 4.65 (s, 2H, =N-CH₂-Ar) , 4.17 (s, 2H, Qu-ring-CH₂-) , 2.28 (s, 3H, Ar-CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.50, 162.53, 148.55, 148.49, 148.45, 138.93, 138.84, 136.48, 134.49, 134.35, 130.09, 128.93, 126.76, 126.08, 125.79, 124.27, 123.81, 122.81, 121.03, 118.19, 117.82, 60.22, 44.84, 21.58; MS (ESI) , m/z: 428.3 ([M+H]⁺) , 450.3 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₂₁N₅O₃: C 62.41%, H 4.36%, N 15.16%; found: C 62.12%, H 4.58%, N 15.36%.

(E) -3- (2- ( (2-chlorobenzylidene) amino) ethyl) -2- (4-chloroanilino) -4 (3H) -quinazolinone (3v) . White solid. IR (KBr) , ν/cm^-1: 3 236 (N-H, Qu-ring-NH-Ar) , 3 061 and 2 966 (Ar-C-H) , 1 668 (C=O) , 1 604 (C=N) , 1 585-1 473 (C=C and N-H, benzene and Qu-ring and bending of N-H) , 1 435 (C-H) , 1 301-1 089 (C-N) , 812-696 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 9.02 (s, 1H, Qu-ring-NH-Ar) , 8.82 (s, 1H, -N=CH-) , 8.19 (d, 1H, J = 8.05 Hz, Ar-H) , 7.90 (d, 1H, J = 7.45 Hz, Qu-H) , 7.63 (t, 1H, J = 7.45 Hz, Qu-H) , 7.41-7.38 (m, 5H, Qu-H and Ar-H) , 7.26-7.20 (m, 4H, Ar-H) , 4.62 (s, 2H, =N-CH₂-Ar) , 4.13 (s, 2H, Qu-ring-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.48, 162.01, 148.54, 148.24, 137.93, 135.74, 134.54, 132.82, 132.24, 130.38, 128.82, 128.43, 127.82, 126.89, 125.70, 123.99, 121.71, 118.35, 59.08, 45.17; MS (ESI) , m/z: 437.2 ([M+H]⁺) , 459.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₃H₁₈C_l2N₄O: C 63.17%, H 4.15%, N 12.81%; found: C 63.37%, H 4.17%, N 12.84%.

(E) -3- (2- ( (2-fluorobenzylidene) amino) ethyl) -2- (4-chloroanilino) -4 (3H) -quinazolinone (3w) . White solid. IR (KBr) , ν/cm^-1: 3 246 (N-H, Qu-ring-NH-Ar) , 3 035 (Ar-C-H) , 1 674 (C=O) , 1 610 (C=N) , 1 587-1 473 (C=C and N-H, benzene and Qu-ring and bending of N-H) , 1 406 (C-H) , 1 278-1 089 (C-N) , 813-758 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 8.93 (s, 1H, Qu-ring-NH-Ar) , 8.66 (s, 1H, -N=CH-) , 8.18 (d, 1H, J = 7.45 Hz, Ar-H) , 7.83 (s, 1H, Qu-H) , 7.62 (t, 1H, J = 7.45 Hz, Qu-H) , 7.43 (d, 4H, J = 7.45 Hz, Qu-H and Ar-H) , 7.25-7.09 (m, 5H, Ar-H) , 4.61 (s, 2H, =N-CH₂-Ar) , 4.11 (s, 2H, Qu-ring-CH₂-) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 163.45, 161.36, 158.78, 148.56, 148.24, 137.97, 134.52, 133.71, 128.83, 127.99, 127.85, 126.89, 125.70, 124.54, 124.00, 122.79, 122.70, 121.73, 118.36, 116.50, 116.33, 60.12, 45.18; MS (ESI) , m/z: 421.3 ([M+H]⁺) , 443.2 ([M+Na]⁺) ; Anal. Calcd. for C₂₃H₁₈ClFN₄O: C 65.64%, H 4.31%, N 13.31%; found: C 65.84%, H 4.45%, N 13.25%.

(E) -3- (2- (4-methylbenzylideneamino) ethyl) -2- (4-chloroanilino) -4 (3H) -quinazolinone (3x) . White solid. IR (KBr) , ν/cm^-1: 3 224 (N-H, Qu-ring-NH-Ar) , 3 026 (Ar-C-H) , 1 672 (C=O) , 1 606 (C=N) , 1 585-1 473 (C=C and N-H, benzene and Qu-ring and bending of N-H) , 1 406 (C-H) , 1 301-1 012 (C-N) , 812-761 (C-Cl) ; ¹H NMR (500 MHz, CDCl₃) , δ: 9.11 (s, 1H, Qu-ring-NH-Ar) , 8.34 (s, 1H, -N=CH-) , 8.17 (s, 1H, Ar-H) , 7.61-7.20 (m, 11H, Qu-H and Ar-H) , 4.59 (s, 2H, =N-CH₂-Ar) , 4.06 (s, 2H, Qu-ring-CH₂-) , 2.40 (s, 3H, Ar-CH₃) ; ¹³C NMR (125 MHz, CDCl₃) , δ: 164.87, 163.50, 148.72, 148.30, 142.52, 138.07, 134.46, 132.26, 129.66, 128.81, 128.54, 127.76, 126.85, 125.69, 123.90, 121.82, 118.35, 59.78, 45.33, 21.70; MS (ESI) , m/z: 417.3 ([M+H]⁺) , 439.3 ([M+Na]⁺) ; Anal. Calcd. for C₂₄H₂₁ClN₄O: C 69.14%, H 5.08%, N 13.44%; found: C 68.94%, H 4.71%, N 13.04%.

1.3 Purification of TMV

Nicotiana tabacum cv. (N. tabacum cv.) K326 was provided by Tobacco Research Institude of Chinese Academy of Agricultural Sciences and TMV was purchased from Wuhan Institute of Virology, CAS. TMV was propagated and maintained in the leaves of N. tabacum cv. K326 and purified by the Gooding method^[22]. The concentration of virus was determined by an ultraviolet spectrophotometer at 260 nm.

Virus concentration (mg/mL) = (A₂₆₀ × dilution ratio) /E^0.1%_{1 cm}^{260 nm}, where E represents the extinction coefficient for TMV, E^0.1%_{1 cm}^{260 nm} is 3.1.

1.4 Curative activity of the target compounds against TMV in vivo

Growing leaves of Nicotiana tabacum L. of the same age were selected. TMV, with the concentration of 6 × 10^-3 mg/mL, was dipped and inoculated in the whole leaves that had been previously scattered with silicon carbide with a brush. The leaves were washed with water and dried after inoculation. The compound solution was smeared on the left side of the leaf and the distilled water was smeared on the right side as a control. Then local lesion numbers were counted and recorded 3-4 days after inoculation. Three replicates were conducted for each compound.

1.5 Protection activity of the target compounds against TMV in vivo

The compound solution was smeared on the left side of growing N. tabacum leaves and the distilled water served as the control was smeared on the right side. The leaves that had been previously scattered with silicon carbide were inoculated with TMV with the concentration of 6 × 10^-3 mg/mL, and then washed with water after 12 h inoculation. About 3-4 days after inoculation, the number of local lesions was counted. Three replicates for each compound were performed.

1.6 Inactivation activity of the target compounds against TMV in vivo

The virus was inhibited when it was mixed with the same volume of the target compound solution for 30 min. The right side of the N. tabacum leaves was then inoculated with the solvent, and the virus mixture was used as the control. All of the leaves had been previously scattered with silicon carbide. The number of local lesions was recorded 3-4 d after inoculation. Three replicates were used for each compound.

1.7 Data analysis

The inhibition rate of the target compound was calculated according to the following formula.

Inhibition rate/% = [ (means average of local lesion number of control (not treated with compound) -means average of local lesion number smeared with compound) / means average of local lesion number of control (not treated with compound) ] × 100

According to the previous bioassays, the results of inhibition rate (expressed by EC₅₀) of the target compounds against TMV were also evaluated at five double-declining concentrations (e.g., 200, 100, 50, 25 and 12.5 μg/mL) and calculated with SPSS 17.0 software to obtain their corresponding EC₅₀ values. The experiments were repeated three times for each compound.

2 Results and Discussion 2.1 Chemistry

In this study, using o-aminobenzoic acid as the starting material, as shown in Scheme 1, the target compounds 3a-3x were synthesized with the good yields of 69%-98%. And the structures of the target compounds 3a-3x were characterized by IR, ¹H NMR, ¹³C NMR, elemental analysis and X-ray diffraction crystallography. The crystal structure data collection and handling of the synthesized compound 3t are shown in Table 1.

Table 1 Crystal structure data collection and handling of the synthesized compound 3t

2.2 Analytical spectral data of the target compounds 3a-3x

The IR spectral data of compounds 3a-3x showed characteristic absorption bands at 3 640-3 188 cm^-1, which were assigned to the N-H of Qu-NH-Ar. The absorption bands of the C=O and C=N groups of the skeletal stretching frequency appeared at 1 676-1 662 cm^-1 and 1 610-1 589 cm^-1, respectively. A singlet varying from 9.15-8.16 in the ¹H NMR spectra belonged to Qu-NH-Ar and -N=CH-. The chemical shifts from 164.93-158.53 in the ¹³C NMR spectra confirmed the existence of C=O and C=N, respectively. All the final products were confirmed by MS according to their molecular formulae. All the spectra exhibited parent peaks because of molecular ions (M + 1) . The structure of compound 3t was also confirmed by X-ray diffraction (Fig. 1) .

Fig. 1 The crystal structure of 3t

2.3 Antiviral activity

The results of antiviral activity against TMV of 3a-3x were determined (Table 2) according to the methods described previously^[22]. The commercial antiviral agent of ningnanmycin, served as a positive control, was evaluated at the same conditions. The preliminary biological activity results revealed that the target compounds 3a-3x exhibited moderate to good anti-TMV activity at 500 μg/mL. Meanwhile, compounds 3a-3x exhibited better curative activity (40.4%-53.1%) against TMV which were slightly lower than that of Ningnanmycin (56.4%) . The results in Table 2 showed that compounds 3c, 3m, 3q and 3v showed potent inactivation activity against TMV, with the values of 89.4%, 89.7%, 90.5% and 90.7%, respectively, which were similar to that of Ningnanmycin (91.3%) . Especially, compounds 3a, 3d, 3n, 3p, 3r, 3s and 3v exhibited excellent protection activity, with inhibition rates of 69.5%, 71.8%, 69.4%, 71.8%, 72.6%, 71.1% and 71.4%, respectively, which were equal to or even superior to that of Ningnanmycin (69.8%) .

Table 2 Antiviral activity against TMV of the target compounds 3a-3w at 500 μg/mL in vivo

The EC₅₀ values of some synthesized compounds were determined (Table 3) . As indicated in Table 3, compounds 3a, 3d, 3n, 3p, 3r, 3s, 3v and 3x exhibited potent protection activity against TMV, with the EC₅₀ values of 216.0, 203.9, 268.4, 277.5, 276.0, 307.7, 292.6 and 288.8 µg/mL, respectively. Particularly, compound 3d showed the best protection activity against TMV, with the EC₅₀ value of 203.9 µg/mL, which was even better than that of Ningnanmycin (214.9 µg/mL) . Moreover, compound 3a showed significant protection activity against TMV, with the EC₅₀ value of 216.2 µg/mL, which was similar to that of ningnanmycin.

Table 3 EC₅₀ values for protection activity of the testing compounds against TMV in vivo.

2.4 The structure-activity relationship analyses

As an extension of this approach, the structure-activity relationships (SAR) were discussed on the basis of the activity values in Tables 2 and Table 3. First, as shown in Table 2, when R¹ and R² were 4-CH₃ and 2-F, respectively, the corresponding compound 3h possessed better curative activity (55.1%) against TMV at 500 μg/mL, which was better than the other compounds. Second, when R¹ and R² substituent groups were 4-Cl and 2-Cl, respectively, the corresponding compound 3v exhibited better inactivation activity (90.7%) against TMV. Especially, Tables 2 and Table 3 revealed that when R¹ and R² were 4-OCF₃ and 2-Cl, respectively, the corresponding compound 3d exhibited excellent protection activity (EC₅₀ = 203.9 µg/mL) against TMV.

3 Conclusions

In conclusion, a series of 4 (3H) -quinazolinone derivatives containing a Schiff base moiety were synthesized and those compounds were characterized by IR, ¹H NMR, ¹³C NMR, MS, elemental analysis, and X-ray diffraction crystallography and their antiviral activity against TMV in vivo was evaluated. Bioassay results demonstrated that the above mentioned compounds presented moderate to good anti-TMV activity in vivo. Particularly, compound 3d demonstrated the best protection activity against TMV in vivo, with the EC₅₀ value of 203.9 µg/mL, which was even better than that of ningnanmycin. SAR analysis showed that when R¹ and R² substituent are electron-withdrawing groups, the corresponding compounds displayed better anti-TMV activity. This work demonstrated that 4 (3H) -quinazolinone derivatives containing a Schiff base moiety can be used to develop potential antiviral agents for plants.

Conflicts of Interest: The authors declare no conflict of interest.

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农药学学报 2016, Vol. 18 Issue (6): 686-696	PDF