新型含氟吡唑酰肼类化合物的设计合成与生物活性研究

doi:10.6023/cjoc202112021

摘要/Abstract

漆酶在微生物体内具有重要的生理功能, 是一种潜在的作用靶标. 为了寻找具有漆酶高抑制活性的化合物, 以4-氯肉桂醛缩氨基硫脲(PMDD-5Y)为先导化合物, 利用活性亚结构拼接原理, 设计并合成了15个未见文献报道的含二氟甲基吡唑的肉桂醛酰肼类衍生物, 其结构均经过¹H NMR、¹³C NMR和HRMS确证. 漆酶抑制活性测试结果表明, 所制备的化合物均具有良好的活性, 其中N'-((Z)-3-(3,4-二氯苯基)-3-苯基烯丙亚基)-3-(二氟甲基)-1-甲基-1H-吡唑-4-甲酰肼(5l)和N'-((Z)-3-(2-氯苯基)-3-(4-氯苯基)烯丙亚基)-3-(二氟甲基)-1-甲基-1H-吡唑-4-甲酰肼(5m)的IC₅₀值分别为0.069和0.063 mmol/L, 明显优于先导化合物PMDD-5Y (IC₅₀=0.553 mmol/L)和对照药剂半胱氨酸(IC₅₀=0.298 mmol/L). 离体抑菌活性测试结果表明, 在50 μg/mL的浓度下, 具有较好漆酶抑制活性的化合物对苹果腐烂病菌(V. mali)、小麦赤霉病菌(F. graminearum)和稻瘟病菌(M. grisea)有一定的抑菌活性. 对稻瘟病菌(M. grisea)的活体实验表明, 化合物5l在200 μg/mL时的治疗活性为78.55%, 表现出一定的活体防治效果. 进一步通过分子对接研究发现, 高漆酶抑制活性化合物5l与漆酶蛋白有较好的蛋白结合模式.

关键词: 漆酶, 漆酶抑制剂, 含氟吡唑片段, 分子对接, 抑菌活性

Laccase is a potential target and has important physiological functions in micro organisms. In order to find compounds with high laccase inhibitory activity, 15 cinnamaldehyde hydrazide derivatives containing difluoromethylpyrazole were designed and synthesized by using the principle of active substructure splicing with 4-chlorocinnamaldehyde thiosemicarbazone (PMDD-5Y) as the lead compound. The structures of the compounds were confirmed by ¹H NMR, ¹³C NMR and HRMS. The results of laccase inhibitory activity test showed that all the prepared compounds have good activity, and N'- ((Z)-3-(3,4-dichlorophenyl)-3-phenylallylidene)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carbohydrazide (5l) and N'-((Z)- 3-(2-chlorophenyl)-3-(4-chlorophenyl)allylidene)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carbohydrazide (5m) had the half-maximal inhibiting concentration (IC₅₀) values of 0.069 and 0.063 mmol/L, respectively, which were significantly better than that of lead compound PMDD-5Y (IC₅₀=0.553 mmol/L) and positive control cysteine (IC₅₀=0.298 mmol/L). The in vitro bioassay showed that the compounds with good laccase inhibitory activity showed certain fungicidal activity against V. mali, F. graminearum and M. grisea at a concentration of 50 μg/mL. In vivo trials against M. grisea demonstrated that compound 5l was effective with curative activity of 78.55% at a concentration of 200 μg/mL, which showed a certain control efficiency in vivo. Further molecular docking study revealed that compound 5l with high laccase inhibitory activity had a good protein binding mode with laccase protein.

Key words: laccase, laccase inhibitor, fluoropyrazole fragment, molecular docking, fungicidal activity

引用此文

王长凯, 孙腾达, 张学博, 杨新玲, 路星星, 徐欢, 石发胜, 张莉, 凌云. 新型含氟吡唑酰肼类化合物的设计合成与生物活性研究[J]. 有机化学, 2022, 42(5): 1527-1536.

Changkai Wang, Tengda Sun, Xuebo Zhang, Xinling Yang, Xingxing Lu, Huan Xu, Fasheng Shi, Li Zhang, Yun Ling. Design, Synthesis and Bioactivity of Novel Fluoropyrazole Hydrazides[J]. Chinese Journal of Organic Chemistry, 2022, 42(5): 1527-1536.

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Compd.	R¹	R²	IC₅₀/(mmol•L^–1)	95% confidence interval	Regression equation	r
5a	H	H	0.094	0.083~0.110	y=3.00x–4.65	0.98
5b	H	2-CH₃	0.071	0.063~0.081	y=4.85x–7.02	0.97
5c	H	3-CH₃	0.073	0.065~0.087	y=5.77x–8.43	0.99
5d	H	4-CH₃	0.067	0.060~0.079	y=4.22x–6.00	0.93
5e	4-CH₃	4-CH₃	0.111	0.098~0.144	y=5.59x–9.26	0.98
5f	4-OCH₃	4-OCH₃	0.136	0.107~0.259	y=5.23x–9.36	0.99
5g	H	2-F	0.089	0.079~0.103	y=4.31x–6.66	0.96
5h	H	2-Cl	0.086	0.078~0.097	y=6.86x–10.66	0.99
5i	H	2-Br	0.136	0.098~0.592	y=3.13x–5.64	0.98
5j	H	4-Cl	0.089	0.081~0.103	y=7.11x–11.13	0.98
5k	4-Cl	4-Cl	0.089	0.077~0.115	y=3.99x–6.39	0.99
5l	H	3,4-Cl₂	0.069	0.063~0.076	y=4.89x–7.29	0.99
5m	4-Cl	2-Cl	0.063	0.058~0.069	y=4.57x–6.64	0.98
5n	H	4-NO₂	0.091	0.083~0.102	y=7.22x–11.39	0.99
5o	H	2-Ph	0.066	0.058~0.080	y=4.06x–6.00	0.98
PMDD-5Y			0.553	0.491~0.610	y=5.74x–12.18	0.97
半胱氨酸			0.298	0.218~0.483	y=2.77x–4.23	0.98

Compd.	R¹	R²	IC₅₀/(mmol•L^–1)	95% confidence interval	Regression equation	r
5a	H	H	0.094	0.083~0.110	y=3.00x–4.65	0.98
5b	H	2-CH₃	0.071	0.063~0.081	y=4.85x–7.02	0.97
5c	H	3-CH₃	0.073	0.065~0.087	y=5.77x–8.43	0.99
5d	H	4-CH₃	0.067	0.060~0.079	y=4.22x–6.00	0.93
5e	4-CH₃	4-CH₃	0.111	0.098~0.144	y=5.59x–9.26	0.98
5f	4-OCH₃	4-OCH₃	0.136	0.107~0.259	y=5.23x–9.36	0.99
5g	H	2-F	0.089	0.079~0.103	y=4.31x–6.66	0.96
5h	H	2-Cl	0.086	0.078~0.097	y=6.86x–10.66	0.99
5i	H	2-Br	0.136	0.098~0.592	y=3.13x–5.64	0.98
5j	H	4-Cl	0.089	0.081~0.103	y=7.11x–11.13	0.98
5k	4-Cl	4-Cl	0.089	0.077~0.115	y=3.99x–6.39	0.99
5l	H	3,4-Cl₂	0.069	0.063~0.076	y=4.89x–7.29	0.99
5m	4-Cl	2-Cl	0.063	0.058~0.069	y=4.57x–6.64	0.98
5n	H	4-NO₂	0.091	0.083~0.102	y=7.22x–11.39	0.99
5o	H	2-Ph	0.066	0.058~0.080	y=4.06x–6.00	0.98
PMDD-5Y			0.553	0.491~0.610	y=5.74x–12.18	0.97
半胱氨酸			0.298	0.218~0.483	y=2.77x–4.23	0.98

Treatment	Curative activity			Protective activity
Treatment	Morbidity/%	Disease index/%	Control efficiency/%	Morbidity/%	Disease index/%	Control efficiency/%
5l	100	8.73	78.55	100	16.13	60.36
PMDD-5Y	100	11.21	72.45	100	16.97	58.29
IPT^a	100	11.83	70.93	100	12.82	68.49
CK	100	40.69		100	40.69

Treatment	Curative activity			Protective activity
Treatment	Morbidity/%	Disease index/%	Control efficiency/%	Morbidity/%	Disease index/%	Control efficiency/%
5l	100	8.73	78.55	100	16.13	60.36
PMDD-5Y	100	11.21	72.45	100	16.97	58.29
IPT^a	100	11.83	70.93	100	12.82	68.49
CK	100	40.69		100	40.69