Double-hydrogenated nopyl quaternary ammonium salt with antibacterial and anticancer effects and application thereof
阅读说明：本技术 一种具有抑菌、抗癌作用的双氢化诺卜基季铵盐及其应用 (Double-hydrogenated nopyl quaternary ammonium salt with antibacterial and anticancer effects and application thereof ) 是由 王宗德 张丽 冯雪贞 肖转泉 范国荣 陈尚钘 廖圣良 罗海 杨宇玲 张骥 贺璐 于 2021-10-16 设计创作，主要内容包括：本发明适用于天然产物化学合成和医药领域,提供了一种具有抑菌、抗癌作用的双氢化诺卜基季铵盐及其应用,本发明用氢化诺卜基的叔胺和含氢化诺卜基的卤代烃在弱极性溶剂下搅拌反应,反应后产物经抽滤、洗涤和真空干燥,分别合成得到6种双氢化诺卜基季铵盐化合物。并且这6种双氢化诺卜基季铵盐化合物对10种植物病原菌和4种有害细菌具有优良的抑制作用,同时还具有显著的抗癌活性。本发明原料丰富,合成方作简单,反应条件温和,纯化后处理操作简单。相比于传统的抗菌、抗癌药剂,这类化合物具有结构简单,环境污染小,安全无毒等优点,具有良好的应用前景。本发明为植物来源的抗菌、抗癌药物的应用提供了药用基础。(The invention is suitable for the field of natural product chemical synthesis and medicine, and provides a double-hydrogenated nopyl quaternary ammonium salt with bacteriostatic and anticancer effects and application thereof. And the 6 double-hydrogenated nopyl quaternary ammonium salt compounds have excellent inhibition effect on 10 plant pathogenic bacteria and 4 harmful bacteria, and simultaneously have obvious anticancer activity. The invention has the advantages of rich raw materials, simple synthesis method, mild reaction conditions and simple operation of purification post-treatment. Compared with the traditional antibacterial and anticancer medicaments, the compounds have the advantages of simple structure, small environmental pollution, safety, no toxicity and the like, and have good application prospect. The invention provides a medicinal basis for the application of antibacterial and anticancer drugs from plant sources.)
1. A double hydrogenated nopyl quaternary ammonium salt with antibacterial and anticancer effects is characterized in that the double hydrogenated nopyl quaternary ammonium salt consists of two hydrogenated nopyl groups, and the structural formula of the double hydrogenated nopyl quaternary ammonium salt is as shown in formula (1):
。
2. a pharmaceutical composition comprising at least one dihydrogenated quaternary ammonium salt compound having a structure of formula (1) according to claim 1 as an active ingredient.
3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is a solid preparation or a liquid preparation, or a pharmaceutically acceptable salt or solvate thereof, a pharmaceutic adjuvant and a pharmaceutic carrier.
4. The application of the bihydrogenated nopyl quaternary ammonium salt with bacteriostatic and anticancer effects is characterized in that the novel antibacterial and anticancer derivative represented by the formula (1), the mixture of the derivative or the pharmaceutically acceptable salt or the solvation drug thereof is applied to treatment or prevention of cancer, induction of programmed cell death, prevention of programmed cell death and tissue death caused by other factors.
5. The application of the bihydrogenated nopyl quaternary ammonium salt with bacteriostatic and anticancer effects is characterized in that the novel antibacterial and anticancer derivative represented by the formula (1), a mixture of the derivative or a pharmaceutically acceptable salt or a solvated medicament thereof is applied to treatment or prevention of infection of phytopathogen.
6. The application of the bihydrogenated nopyl quaternary ammonium salt with bacteriostatic and anticancer effects is characterized in that the novel antibacterial and anticancer derivative represented by the formula (1), a mixture of the derivative or a pharmaceutically acceptable salt or a solvated medicament thereof is applied to treatment or prevention of bacterial infection.
Technical Field
The invention belongs to the fields of natural product chemical synthesis and medicines, and particularly relates to a double-hydrogenated nopyl quaternary ammonium salt with antibacterial and anticancer effects and application thereof.
Background
Microbial infections represent a significant threat to human health and are a major cause of quality and yield loss in agricultural products. Chemical antibacterial agents are the traditional methods for preventing and treating microbial infectious diseases. However, the use of a single chemical antimicrobial agent can result in serious microbial resistance and can have a negative impact on the environment and public health. Therefore, in order to cope with the increasing drug resistance, new, environmentally friendly, highly effective, low-toxic antibacterial agents are continuously developed.
Cancer is a leading cause of disease death worldwide and has become one of the major causes of serious impact on the average life span of people worldwide. Current conventional approaches to cancer treatment include surgery, cytotoxic chemotherapy, radiation therapy, targeted biological therapy, immunotherapy, and the like. Research shows that the plant-derived metabolite can effectively inhibit the proliferation of cancer cells, stimulate autophagy and induce apoptosis, and can also play a synergistic role with traditional anti-cancer drugs, so that the incidence rate and the recurrence risk of cancer are reduced. At present, research on the development of plant-derived metabolites or the structural modification of plant-derived metabolites as anticancer drugs has become a hot research focus of current anticancer drugs.
The plant is natural treasury of antibacterial and anticancer active substances, and the metabolite derived from plant is natural component, has bioactivity of resisting cancer, bacteria, inflammation and virus, and has low adverse side effect, and can be used for developing plant bactericide and anticancer medicine. The plant-derived metabolite has good biocompatibility, high biodegradation speed, small cross resistance or no cross drug resistance, diversified chemical structures and is beneficial to being applied to the prevention and treatment of cancer cell and microbial infection diseases. Pine resources are very rich in China, and a large amount of renewable resource turpentine oil with low price can be obtained by distillation. The beta-pinene is one of the main components of turpentine, has wide pharmacological activity and important research and development values. The synthesis of derivatives with biological activity by chemical modification of beta-pinene structure has become one of the research hotspots in forestry chemistry and organic synthesis chemistry.
The quaternary ammonium salt is a kind of cationic surfactant, and the compound containing quaternary ammonium salt skeleton has various biological activities including antibiosis, antifungal, antiparasitic, antiviral, etc., and is widely concerned by pesticides and medicine. Therefore, the patent researches the synthesis of the compound with the quaternary ammonium salt framework by taking the beta-pinene as the raw material, and researches the activity of the compound containing the beta-pinene and the quaternary ammonium salt framework in the aspects of bacteriostasis and anticancer.
Disclosure of Invention
The embodiment of the invention aims to provide a bihydrogenated nopyl quaternary ammonium salt with bacteriostatic and anticancer effects, which is prepared from a hydronopyl tertiary amine compound (formula (2): N, N-dimethylhydronopyl amine, N-hydronopyl piperidine, N-hydronopyl morpholine) and a hydronopyl halohydrocarbon (formula (3): hydronopyl chloride, hydronopyl bromide, hydronopyl iodide) as a starting material, heating in a weak polar solvent to react to generate a quaternary ammonium salt compound containing two hydronopyl groups, namely dimethyl dihydrogennium chloride, dimethyl dihydrogennium iodide, N-dihydrogennium piperidine iodide, N-dihydrogennium morpholine chloride, N-dihydrogennium morpholine bromide and N, N-dihydrogennium morpholine iodide. The reaction process has simple apparatus, simple operation and high product yield. The chemical reaction formula of the synthesis process is as follows:
the invention also aims to disclose 6 synthesized double hydrogenated nopyl quaternary ammonium salt compounds for inhibiting the following 4 bacteria and 2 cancer cells.
The embodiment of the invention is realized in such a way that the double-hydrogenated nopyl quaternary ammonium salt with the bacteriostatic and anticancer effects consists of two hydrogenated nopyl quaternary ammonium salts, and the structural formula of the double-hydrogenated nopyl quaternary ammonium salt is shown as a formula (1):
。
a pharmaceutical composition comprising at least one dihydrogenated nopyl quaternary ammonium salt compound having the structure of formula (1) in claim 1 as an active ingredient.
In a further technical scheme, the pharmaceutical composition is a solid preparation or a liquid preparation, or pharmaceutically acceptable salt or solvate thereof, a pharmaceutic adjuvant and a pharmaceutic carrier.
The application of the novel antibacterial and anticancer derivative represented by the formula (1), the mixture of the derivative or the pharmaceutically acceptable salt or the solvate of the derivative in treating or preventing cancer, inducing programmed cell death, preventing programmed cell death and tissue death caused by other factors is disclosed.
In a further technical scheme, the novel antibacterial and anticancer derivative represented by the formula (1), a mixture of the derivative or a pharmaceutically acceptable salt or a solvated medicament thereof is applied to treatment or prevention of infection in phytopathogen.
In a further technical scheme, the novel antibacterial and anticancer derivatives represented by the formula (1), the mixture of the derivatives thereof or the pharmaceutically acceptable salt or the solvated medicament thereof are applied to treatment or prevention of bacterial infection.
The embodiment of the invention provides a bihydrogenated nopyl quaternary ammonium salt with bacteriostatic and anticancer effects and an application thereof, wherein a hypha growth rate method is adopted to determine that 6 quaternary ammonium salt compounds inhibit the activity of 10 plant pathogenic bacteria, and the fungi: watermelon wilt (Fusarium oxysporum f.sp.niveum), Phytophthora nicotianae (Phytophthora nicotianae var. nicotianae), loquat anthracnose (Colletotrichum acutatum), rice sheath blight (Rhizoctonia solani), Coriolus versicolor (Coriolus versicolor), Fusarium verticillium (Fusarium verticillioides), conidiobolus pinus (Diplodia pinea), cotton rot fungus (pora variola), Fusarium esculentum (Fusarium oxysporum), camellia anthracnose (Colletotrichum gloosporium). The inhibition activity of 6 quaternary ammonium salt compounds on 4 harmful bacteria is determined by adopting a resazurin microplate method, wherein the inhibition activity of the bacteria: escherichia coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa), and Bacillus subtilis (Bacillus subtilis). The inhibitory activity of 6 quaternary ammonium compounds on the growth of 2 cancer cells was determined using the cell counting kit (CCK-8) assay, cancer cells: human breast cancer cells (MCT-7), human colon cancer cells (HCT-116). The experimental results show that: most of the compounds show moderate to excellent in vitro bacteriostatic activity against 10 plant pathogens and 4 bacteria. Particularly, when the concentration of the compounds dimethyl bihydrogenated nopyl ammonium chloride, dimethyl bihydrogenated nopyl ammonium iodide and N, N-bihydrogenated nopyl piperidine iodide is 50 mg/mL, the inhibition rate of the compounds on tobacco phytophthora parasitica, rice sheath blight bacteria, pine blight conidioides, cotton pythium aphanidermatum and fuscoporia esculenta is 82.90-100.00%, and the inhibition effect is close to or even better than that of positive control. In addition, the compounds dimethyl double-hydrogenated nopyl ammonium chloride and dimethyl double-hydrogenated nopyl ammonium iodide have stronger activity on four bacteria, and the MIC values of the compounds are within the range of 0.625-80 mu g/mL. Besides, most of the compounds show excellent inhibitory activity in vitro on human breast cancer cells (MCT-7) and human colon cancer cells (HCT-116), and particularly, the compounds dimethyl ammonium dihydrogen norpyl iodide, N-dihydrogen norpyl iodide piperidine and N, N-dihydrogen norpyl morpholine iodide have the inhibition rate of more than 97% on human breast cancer cells (MCT-7) and human colon cancer cells (HCT-116) and the inhibition rate of 97.94% -100% on 50 mu M concentration.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the following examples further illustrate the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Specific implementations of the present invention are described in detail below with reference to specific embodiments.
Example 1:
the general synthesis method comprises the following steps: adding 0.05 mol of N, N-dimethyl norbomylamine hydride and 0.05 mol of norbomyl chloride into a 150 mL conical flask containing 30 mL of ethyl acetate, heating, stirring and heating for reflux by magnetic heating, stopping after reacting for 2-3 d, removing the ethyl acetate by using a rotary evaporator, cooling, adding petroleum ether, washing for 2-3 times, performing suction filtration, and performing vacuum drying to obtain dimethyl double-hydrogenated norbomylammonium chloride, wherein the structural formula is as follows:
white solid, yield: 80%, m.p.: 222.1-227.5 ℃;1H-NMR (CDCl3, 400 MHz) δH: 3.438 (t, J=4.8Hz, 4H, 2 11-CH2), 3.416(s, 6H, 2 α-CH3), 2.321(m, 2H, 2 2-CH), 1.943~1.820(m, 2H, 12(10-CH2, 7-CH, 1-CH, 5-CH, 3-CH)), 1.709(m, 4H, 2 4-CH2), 1.428(m, 2H, 2 3-CH), 1.184(s, 6H 2 9-CH3), 1.001(s, 6H, 2 8-CH3), 0.866(d, J=9.8Hz, 2H, 2 7-CH); 13C-NMR (CDCl3, 100 Hz) δc 62.11(2 C-11), 51.50(2C-α), 46.27(2C-2), 40.88(2C-5), 38.43(2 C-6), 38.18(2C-1), 33.20(2 C-10), 30.03(2C-7), 27.78(2C-9), 25.91(2C-4), 23.18(2 C-8), 21.96(2 C-3);1R, ymax(cm-1): 3429;IR (KBr) v (cm -1)3446.78, 2981.91, 2935.72, 2899.66, 2866.20 1469.88, 1366.31; LC-MS, C24H44NCl, m/z 346.34(M+-Cl).
example 2:
the general synthesis method comprises the following steps: other experimental procedures and conditions were the same as in example 1, and the reactants were N, N-dimethylhydronopyl amine and hydronopyl iodide. Vacuum drying to obtain dimethyl ammonium dihydrogen Norphtyl iodide with a structural formula as follows:
white solid, yield: 82%, m.p.: 224.5 to 230.8 ℃; 1H-NMR (CDCl3, 400 MHz) delta H: 3.407(m, 4H, 2)11-CH2), 3.286 (s, 6H, 2 α-CH3), 2.243(m, 2H, 2 2-CH), 1.959~1661,((m, 16H, 2 (7-CH, 10-CH2, 5-CH, 1-CH, 4-CH, 3-CH)), 1.348(m, 2H, 2 3-CH), 1.107(s, 6H, 2 9-CH3), 0.934(s, 6H, 2 8-CH), 0.788 (d, J=10Hz, 2H, 2 7-CH);13C-NMR (CDCl3, 100 Hz) δc : 62.82(2 C-11), 51.79(2 C-α), 46.26(2C-2), 41.01(2C-5), 38.56(2C-6), 38.25 ( 2 С-1), 33.31(2C-10), 30.14(2C-7), 27.91(2C-9), 26.05(2C-4), 23.36(2C-8), 22.15(2C-3)；IR (KBr) v (cm -1) 2996.90, 2984.47, 2929.30, 2888.99, 2865.64, 1469.01, 1448.93, 1362.64; LC-MS, C24H44NI, m/z 346.34(M+-I).
Example 3:
the general synthesis method comprises the following steps: other experimental methods and conditions were the same as in the examples, and the reactants were N-hydronopylpiperidine and hydronopyl iodide. Vacuum drying to obtain N, N-double hydrogenated nopyl piperidine iodide with a structural formula as follows:
white solid, yield: 84%, m.p.: 252.1-258.9 ℃; 1H-NMR (CDCl3, 400 MHz) delta H: 3.712 (m.4H, 2)α-CH2), 3.381(m, 4H, 2 11-CH2), 2.321(m, 2H, 2 2-CH), 2.061~1.694((m, 22H, 2(7- CH, 10-CH2, 5-CH, 1-CH, 4-CH2, 3-CH), 2 β-CH2, γ-CH2), 1.418 (m, 2H, 2 3-CH), 1.181(s, 6H, 2 9-CH3), 0.999(s, 6H, 2 8-CH3), 0.878(d, J=9.6Hz, 2H, 2 7-CH); 13C-NMR (CDCl3, 100 Hz) δc: 59.20(2C-11), 57.31(2C-α), 46.46( 2C-2), 40.99(2C-5), 38.55(2C-6), 38.44(2C-1), 33.33(2C-10), 29.11(2C-7), 27.90(2C-9), 26.01(2C-4), 23.35(2C-8), 22.25(2C-3), 20.45(C-γ), 19.98 (2C-β); IR (KBr) v (cm -1) 2978.50, 2936.90, 2899.96, 2865.87, 1468.43, 1382.56, 1366.37; LC-MS, C27H48NI, m/z 386.37 (M+-I).
Example 4:
the general synthesis method comprises the following steps: other experimental procedures and conditions were the same as in the example, and N-hydronopyl morpholine and hydronopyl chloride were reacted. Vacuum drying to obtain N, N-dihydronopyl morpholine chloride with the structural formula as follows:
white solid, yield: 81%, m.p.: 240.2-246.9 ℃; 1H-NMR (CDCl3, 400 MHz) Δ H: 4.312(t, J =12Hz, 4H, 2β-CH2), 3.958(d, J= 10.8Hz, 4H, 2 11=CH2), 3.411(d, J=11.6Hz, 4H, 2 α-CH2), 2.339(m, 2H, 2 2-CH), 1.992~1.746 ((m, 16H, 2(7-CH, 10-CH2, 5-CH, 1-CH, 4-CH2, 3-CH)), 1.464(m, 2H, 2 3-CH), 1.178(s, 6H, 2 9-CH3), 1.028(s, 6H, 2 8-CH3), 0.868(d, J=9.6Hz, 2H, 2 7-CH); 13C-NMR (CDCl3, 100 Hz) δc : 63.10 (2C-β), 56.55 (2C-11), 51.25(2C-α), 45.46(2C-2), 40.71(2C-5), 38.44(2C-1), 38.17(2C-6), 32.86(2C-10), 29.97(2C-7), 27.48(2C-9), 25.63(2C-4), 22.72( 2C-8), 21.51(2C-3); IR (KBr) v (cm -1) 2974.57, 2909.85, 2867.29, 2530.74, 2439.84, 1452.12, 1406.70, 1406.70, 1126.11, 1090.08; LC-MS, C26H46NOCl, m/z388.35(M+-Cl).
Example 5:
the general synthesis method comprises the following steps: other experimental procedures and conditions were the same as in the example, N-hydronopyl morpholine and hydronopyl bromide were reacted. Vacuum drying to obtain N, N-double-hydrogenated nopyl morpholine bromide, wherein the structural formula is as follows:
white solid, yield: 80%, m.p.: 246.8 to 250.7 ℃; 1H-NMR (CDCl3, 400 MHz) delta H: 4.357(t, J =11.2Hz, 4H, 2β-CH2), 3.975(d, J=12.8 Hz, 4H, 2 11-CH2), 3.452(d, J=11.6 Hz, 4H, 2 α-CH2), 2.334( m, 2H. 2 2-CH), 2.006~1.807 (m, 16H, 2(7-CH2, 10-CH2, 5-CH2, 1-CH2, 4-CH2, 3-CH)), 1.469(m, 2H, 2 3-CH), 1.163(s, 6H, 2 9-CH3), 1.015(s, 6H, 2 8-CH3), 0.849 (d, J=9.6Hz, 2H, 2 7-CH), 13C-NMR (CDCl3, 100 Hz) δc : 62.96(2C-β), 56.53(2C-11), 51.19(2C-α), 45.44(2C-2), 40.69(2C-5), 38.36 (2C-1), 38.16 (2C-6), 32.85(2C-10), 29.93(2C-7), 27.46(2C-9), 25.61(2C-4), 22.78(2C-8), 21.46(2C-3); IR (KBr) v (cm -1) 2973.91, 2909.09, 2641.53, 2593.82, 2558.96, 2470.55, 1468.60, 1442.80, 1126.62, 1090.98; LC-MS, C26H46NOBr m/z 388.35(M+-Br).
Example 6:
the general synthesis method comprises the following steps: other experimental methods and conditions were the same as in the examples, and the reactants were N-hydronopyl morpholine and hydronopyl iodide. Vacuum drying to obtain N, N-double-hydrogenated nopyl morpholine iodide, wherein the structural formula is as follows:
white solid, yield: 82%, m.p.: 265.4-271.6 ℃; 1H-NMR (CDCl3, 400 MHz) delta H: 4.049(s; 4H, 2 beta-CH)2), 3.712(s, 4H, 2 α-CH2), 3.602~ 3.474 (m, 4н, 2 11-CH2). 2.305(m,2H, 2-CH), 2.084~1.739[m, 16H, 2(7-CH, 10-CH, 5-CH, 1-CH, 4-CH, 3-CH)], 1.393(m, 2H, 2 3-CH),1.158(s, 6H,2 9-CH3),0.988(s, 6H, 2 8-CH3),0.861(d, J=10Hz, 2H, 2 7-CH).13C-NMR (CDCl3, 100 Hz) δc: 60.09(2C-β), 58.07(2C-α), 57.94(2C-11), 45.88(2C-2), 40.49(2C-5), 38.04 (2C-6), 37.83(2C-10), 32.82 (2C-10), 28.67(2C-7), 27.39(2C-9), 25.53(2C-4), 22.90(2C-8), 21.69(2C-3).IR (KBr) v (cm -1) 2981.81, 2936.92, 2904.26, 2869.57, 1128.74, 906.29; LC-MS, C26H46NOI: m/z 388.35 (M+-I).
Application example 1:
fungi inhibition experiment: the antifungal activity of the quaternary ammonium salt of dihydronopyl on 10 plant pathogenic fungi was determined by a hyphal growth inhibition method. The quaternary ammonium salt of norbo-di-hydride was dissolved in dimethyl sulfoxide (DMSO), and 200. mu.L of the solution was added to 50 mL of sterilized Potato Dextrose Agar (PDA) to obtain a PDA-containing plate having a concentration of 50. mu.g/mL. Inoculating the hypha cake (0.5 cm) of plant pathogenic bacteria on PDA plate, and culturing at 25 deg.C. 3 replicates per sample were used to measure the diameter (cm) of the cake by the cross method. The blank control is DMSO (0.4%, v/v), and the positive control is the fungicide chlorothalonil. The EC50 value was the concentration required to inhibit 50% of mycelium growth. The inhibition rate was calculated as follows:
the inhibition rate (%), at a concentration of 50. mu.g/mL, of the compound of Table 1, for the growth of hyphae of a plant pathogenic bacterium
Remarking: a: dimethyl di-hydrogenated nopyl ammonium chloride, b: dimethyl bis-hydrogenated nopyl ammonium iodide, c: n, N-dihydronopyl piperidine iodide, d: n, N-dihydronopyl morpholine chloride, e: n, N-dihydronopyl morpholine bromide, f: and N, N-dihydronopyl morpholine iodide; a: fusarium oxysporum f.sp.niveum), B: phytophthora nicotianae (Phytophthora nicotianae var. nicotianae), C: loquat anthracnose pathogen (Colletotrichum acutatum), D: rhizoctonia solani (Rhizoctonia solani), E: coriolus versicolor (Coriolus versicolor), F: fusarium verticillium (Fusarium verticillioides), G: pine wilt bacterium (Diplodia pinea), H: fuscoporia gossypii (Poria vaporaria), I: fusarium aesculi (Fusicoccumasculi), J: camellia anthracis (Colletotrichum gloeosprioides).
The results of the test on the activity of 6 double-hydrogenated nopyl quaternary ammonium salt compounds for inhibiting 10 plant pathogenic bacteria in the table 1 show that most of the compounds have moderate to excellent in-vitro bacteriostatic activity on the 10 plant pathogenic bacteria. When the concentration is 50 mg/mL, the compound N, N-dihydronopyl morpholine chloride (D) has better inhibition effects on rice sheath blight bacteria (D), Fusarium aesculosum (I) and oil tea anthracnose bacteria (J), and the inhibition rates are 66.96%, 55.73% and 60.78% respectively; the N, N-double-hydrogenated nopyl morpholine bromide (e) has a good inhibition effect on fusarium verticillium (F), and the inhibition rate is 51.22%; n, N-dihydronopyl morpholine iodide (F) shows good inhibition effects on rhizoctonia solani (D), fusarium verticillium (F) and Fusarium aestivum (I), and the inhibition effects are respectively 80.59%, 63.05% and 51.75%. In addition, the inhibition rate of the compounds dimethyl double hydrogenated nopyl ammonium chloride (a), dimethyl double hydrogenated nopyl ammonium iodide (b) and N, N-double hydrogenated nopyl piperidine iodide (c) on 10 plant pathogenic bacteria is more than 50%, and the inhibition rate of the compound N, N-double hydrogenated nopyl piperidine iodide (c) on coriolus versicolor (E) is 42.61%. The inhibition rate of the compounds dimethyl double hydrogenated nopyl ammonium chloride (a), dimethyl double hydrogenated nopyl ammonium iodide (B) and N, N-double hydrogenated nopyl piperidine iodide (c) on tobacco phytophthora parasitica (B), rice sheath blight bacteria (D), pine blight bacteria (G), cotton pythium aphanidermatum (H) and sporotrichum esculentum (I) is 82.90-100.00%, and the inhibition effect is close to or even superior to that of positive control. At a concentration of 50 mg/mL, chlorothalonil has relatively weak inhibition effects on watermelon wilt pathogenic bacteria (A) and loquat anthracnose pathogenic bacteria (C), but the inhibition effects of the compounds dimethyl double-hydrogenated nopyl ammonium chloride (a), dimethyl double-hydrogenated nopyl ammonium iodide (b) and N, N-double-hydrogenated nopyl piperidine iodide (C) on the two plant pathogenic bacteria are better than the inhibition effects of chlorothalonil. Particularly, the inhibition effect of the dimethyl ammonium dihydrogen chloride (a) on the fusarium oxysporum f.sp.sp.sp.cubense (A) is obviously higher than that of chlorothalonil, and the inhibition rate is 1.61 times of that of the chlorothalonil. This indicates that dimethyl bis-hydrogenated nopyl ammonium chloride (a), dimethyl bis-hydrogenated nopyl ammonium iodide (b) and N, N-bis-hydrogenated nopyl piperidine iodide (c) have broad-spectrum phytogerm-inhibiting activity.
Application example 2:
bacteria inhibition experiment: adopting a resazurin microplate method: the Minimum Inhibitory Concentration (MIC) is determined by a broth double dilution method, 100 mu L of LB culture medium is added into a sterile 96-well culture plate, a compound to be tested is added into the 1 st well, and gradient dilution is carried out on 1-11 wells to prepare 0.625 mu g/mL, 1.25 mu g/mL, 2.5 mu g/mL, 5 mu g/mL, 10 mu g/mL, 20 mu g/mL, 40 mu g/mL, 80 mu g/mL, 160 mu g/mL, 320 mu g/mL and 640 mu g/mL of the monad quaternary ammonium salt solution containing the hydrogenated nopyl, and the 12 th well is used as a negative control. After adding 50. mu.L of the bacterial suspension into each well, adding 50. mu.L of the resazurin indicator solution, and culturing in a constant temperature incubator at 37 ℃ for 12 hours, observing the color of each well, wherein the color changes from blue to red to show bacterial growth, and the color remains blue and does not change, which shows that no bacterial growth exists. Finally, 96-well plates were photographed using a fully automated colony counter (Shineso) and MIC values were recorded. The experiment uses 0.9% physiological saline as a blank control group and chloramphenicol as a positive control group.
TABLE 2 minimum inhibitory concentration of the Compounds against bacteria (μ g/mL)
Remarking: a: dimethyl di-hydrogenated nopyl ammonium chloride, b: dimethyl bis-hydrogenated nopyl ammonium iodide, c: n, N-dihydronopyl piperidine iodide, d: n, N-dihydronopyl morpholine chloride, e: n, N-dihydronopyl morpholine bromide, f: and N, N-dihydronopyl morpholine iodide; escherichia coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa), and Bacillus subtilis (Bacillus subtilis).
The results of the measurement of the activity of 6 types of quaternary ammonium salt compounds having nopyl double hydride against 4 types of harmful bacteria in Table 2 show that: most of the quaternary ammonium salt compounds with double hydrogenated nopyl show moderate to excellent bacteriostatic activity. The compounds dimethyl double-hydrogenated nopyl ammonium chloride (a) and dimethyl double-hydrogenated nopyl ammonium iodide (b) have stronger activity on four bacteria, and the MIC value is in the range of 0.625-80 mu g/mL. Particularly, the compound dimethyl double-hydrogenated nopyl ammonium chloride (a) has the best bacteriostatic effect on escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and bacillus subtilis, and MIC values are respectively 2.5 mu g/mL, 0.625 mu g/mL, 1.25 mu g/mL and 1.25 mu g/mL; moreover, the bacteriostatic activity of the complex is superior to that of positive control chloramphenicol (MIC: 5 mug/mL) on escherichia coli, and the bacteriostatic activity on staphylococcus aureus and bacillus subtilis is close to that of the positive control chloramphenicol (MIC: 0.625 mug/mL and 0.625 mug/mL).
Application example 3:
anti-cancer experiments: cytotoxicity of the compounds on two cancer cells was determined by cell counting kit (CCK-8) assay, MCF-7 and HCT-116 cell lines were cultured in DMEM and McCOY' S5A medium, respectively. Taking HCT-116 and MCF-7 cells in a logarithmic growth phase, counting the cells, adjusting the cell concentration, inoculating the cells into a 96-well plate according to 4 multiplied by 103 per well, and culturing the cells in a constant temperature incubator at 37 ℃ for overnight at 5% CO 2. Wherein the blank group is added with 100 mu L/hole culture medium; the test groups were added with 100. mu.L/well of 50. mu.M compound diluent, and the positive control was sorafenib. The culture was carried out for 24 hours according to the above grouping treatment. The medium was removed. Each well was washed three times with PBS, and a medium containing 10% CCK-8, 5% CO2, was added at 100. mu.L/well, and incubated at 37 ℃ for 2 hours in a constant temperature incubator. And detecting the absorbance value at 450 nm by using a microplate reader.
Table 3 inhibition ratio of Compound on two kinds of cancer cells at 50. mu.M concentration (%)
Remarking: a: dimethyl di-hydrogenated nopyl ammonium chloride, b: dimethyl bis-hydrogenated nopyl ammonium iodide, c: n, N-dihydronopyl piperidine iodide, d: n, N-dihydronopyl morpholine chloride, e: n, N-dihydronopyl morpholine bromide, f: and N, N-dihydronopyl morpholine iodide; human colon cancer cell line (HCT-116), human breast cancer cell (MCF-7).
As shown in the results of the 6 bi-hydrogenated nopyl quaternary ammonium salt compounds in Table 3 against cancer in human breast cancer cells (MCT-7) and human colon cancer cells (HCT-116), most of the compounds exhibited excellent inhibitory activity against 2 cancer cells in vitro. Under the concentration of 50 mu M, the inhibition rates of the compounds dimethyl double-hydrogenated nopyl ammonium chloride (a), dimethyl double-hydrogenated nopyl ammonium iodide (b), N-double-hydrogenated nopyl piperidine iodide (c) and N, N-double-hydrogenated nopyl morpholine iodide (f) on human breast cancer cells (MCT-7) and human colon cancer cells (HCT-116) are all more than 97 percent, and the inhibition rates are 97.94 to 100 percent; to show that at this concentration, dimethyl bis-hydrogenated nopyl ammonium iodide (b), N-bis-hydrogenated nopyl piperidine iodide (c) and N, N-bis-hydrogenated nopyl morpholine iodide (f) inhibited the growth of human breast cancer cells (MCT-7) and human colon cancer cells (HCT-116) and even caused apoptosis; and the inhibition effect is better than that of the positive control sorafenib.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
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