A 5-azacyclic structure substituted berberine derivative with antibacterial and insecticidal activity, and a preparation method and application thereof
By introducing a 5-aza-heterocyclic structure into the molluscum alkaloid molecule, a novel molluscum alkaloid derivative was synthesized, solving the problem of poor antibacterial effect of existing derivatives. This resulted in highly efficient inhibition of peach aphids, plant pathogenic fungi, and bacteria, enriching the applications of molluscum alkaloid derivatives.
Patent Information
- Application Number
- CN202310414858.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-18
- Publication Date
- 2026-02-27
- Estimated Expiration
- 2043-04-18
AI Technical Summary
Existing derivatives of purslane are not particularly effective in inhibiting bacteria, and nitrogen heterocyclic structures are rarely reported in purslane molecules. There is a lack of compounds with novel structures and good antibacterial and insecticidal activities.
A derivative of purslane with a 5-nitrogen heterocyclic structure was synthesized by introducing a nitrogen heterocyclic structure into the purslane molecule. Specifically, the compound can have mono- or poly-substituted positions at 2, 3, 4, and 5. R1 is a halogen, hydroxyl, amino, or other groups. The derivative was prepared by a synthetic reaction.
The obtained purslane derivative showed excellent insecticidal activity against peach aphids at a concentration of 500 μg/mL, with an LC50 below 10 μg/mL; at a concentration of 50 μg/mL, it showed antibacterial activity of more than 70% against ten plant pathogenic fungi, and also had antibacterial activity against bacteria such as Escherichia coli.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of drug research, and particularly relates to a 5-azacyclic structure substituted sanguinarine derivative with antibacterial and insecticidal activity and a preparation method and application thereof. BACKGROUND
[0002] There are multiple ways for new drug development, such as random screening, similar synthesis, natural product model and biological rational design, among which, the development of new pesticides and medicines by using biological active natural products as a lead for structural modification shows great potential and broad application prospect. Sanguinarine is extracted from the rich Macleaya cordata resources in Wuling Mountain area. The sanguinarine parent drug and sanguinarine botanical insecticide developed therefrom have the effects of stomach poison, contact killing and nerve paralysis, and can be used for preventing and treating bean aphids, crucifer vegetable caterpillars and aphids. In addition, studies have shown that the alkaloids of Macleaya cordata have excellent disease prevention effect on multiple plant pathogenic fungi under laboratory conditions; and the sanguinarine alkaloids have good prevention and treatment effect on rice bacterial leaf blight and wheat smut. It can be seen that sanguinarine is a good lead compound.
[0003] In addition, in the existing pesticide and medicine varieties, there are generally multiple nitrogen structures, such as triazole and sulfonylurea pesticide varieties. The excellent activity of these multiple nitrogen pesticide varieties makes the multiple nitrogen structure more and more valued by researchers. In addition, it can be known from computational chemistry and biochemistry that when a nitrogen-containing compound is combined with a receptor, the nitrogen atom in the molecule is easy to form a hydrogen bond with the amino acid residue on the receptor protein, and the hydrogen bond is one of the most important forces between the drug molecule and the receptor. Whether the hydrogen bond is formed or not and the number of hydrogen bonds directly affect the binding ability between the drug molecule and the receptor, and have an important influence on the biological activity of the compound. Therefore, the continuous research on azacyclic ring, multiple nitrogen chain and multiple azacyclic ring structure is still a hot spot in the research and development of new drugs. In addition, there is no report on introducing an azacyclic ring structure into the sanguinarine molecule.
[0004] The current sanguinarine derivative related to Macleaya cordata is still not outstanding in the antibacterial effect, and there is a great space for progress. The azacyclic ring structure is not uncommon in the structure of a drug molecule, but the introduction of an azacyclic ring structure into the sanguinarine molecule is rarely reported in the research of agricultural chemicals or drugs.
[0005] Therefore, it is the focus of the current research to find a sanguinarine derivative with novel structure and good antibacterial effect. SUMMARY
[0006] In order to solve the above technical problems in the prior art, the present application provides a 5-azacyclic structure substituted sanguinarine derivative with antibacterial and insecticidal activity, which is as follows:
[0007] A 5-azacyclic structure substituted berberine derivative with antibacterial and insecticidal activity, which is represented by the following general formula (I):
[0008]
[0009] Further, in the general formula I, R1 can be single or multiple substituted at 2, 3, 4, 5 of the heterocyclic ring.
[0010] Further, in the general formula (I), R1 is not H.
[0011] Further, in the general formula (I), R1 is halogen, hydroxyl, amino, trifluoromethyl, C1-C5 alkyl, C1-C5 alkoxy, C1-C5 fluoroalkyl or C1-C5 fluoroalkoxy, aryl (such as phenyl, pyridyl, imidazolyl, oxazolyl, thiazolyl, triazolyl), aryloxy (such as phenoxy, pyridyloxy) and the like.
[0012] Further, in the general formula (I), R1 is halogen, hydroxyl, amino, trifluoromethyl, C1-C5 alkyl, C1-C5 alkoxy, C1-C5 fluoroalkyl or C1-C5 fluoroalkoxy, aryl (such as phenyl, pyridyl, imidazolyl, oxazolyl, thiazolyl, triazolyl), aryloxy (such as phenoxy, pyridyloxy) and the like.
[0013] Further, in the general formula (I), R1 is halogen, hydroxyl, amino, trifluoromethyl, C1-C5 alkyl, C1-C5 alkoxy, C1-C5 fluoroalkyl or C1-C5 fluoroalkoxy, aryl (such as phenyl, pyridyl, imidazolyl, oxazolyl, thiazolyl, triazolyl), aryloxy (such as phenoxy, pyridyloxy) and the like.
[0014] Further, in the general formula (I), X and Y can be hydrogen or nitrogen, i.e. the azacyclic structure formed can be pyrrole, pyrazole, imidazole, triazole and the like.
[0015] The preparation method of the 5-azacyclic structure substituted berberine derivative with antibacterial and insecticidal activity is synthesized by the following reaction:
[0016]
[0017] The application of the 5-azacyclic structure substituted berberine derivative with antibacterial and insecticidal activity in insecticidal, bacteriostatic and antibacterial aspects, especially in the bacteriostatic activity of bacteria and fungi.
[0018] The berberine derivative in the present application is exemplified by the table, including the compounds listed in the table, but not limited to the compounds in the table.
[0019]
[0020]
[0021] The compound I of the present application shows excellent insecticidal activity on Myzus persicae. At a concentration of 500 g / mL, the compound I shows excellent insecticidal activity on Myzus persicae, and the corrected mortality of most compounds is above 85%. In the LC50 determination, the LC50 of some compounds is below 10 g / mL, and the compound still shows excellent insecticidal activity on Myzus persicae.
[0022] The compound 1 of the present application shows better fungistatic activity on ten plant pathogenic fungi. At a concentration of 50 g / mL, the compound I shows certain fungistatic activity on ten pathogenic fungi. Some sanguinarine derivatives such as I-2, I-6, I-7, I-10 show better fungistatic activity, and the inhibition rate on several tested pathogenic fungi is above 70% at a concentration of 50 μg / mL, and the inhibition rate on individual pathogenic fungi reaches above 90%.
[0023] The compound 1 of the present application also shows certain fungistatic activity on bacteria such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Most compounds show fungistatic activity on Staphylococcus aureus and Escherichia coli, wherein I-2 shows strong fungistatic activity on Escherichia coli and Pseudomonas aeruginosa, and I-5 shows strong fungistatic activity on Staphylococcus aureus. It is worth further studying.
[0024] Compared with the prior art, the technical effects of the present application are embodied in:
[0025] (1) The present application obtains a sanguinarine derivative with novel structure and better antibacterial and insecticidal activity, which has a nitrogen-containing ring structure at the 5-position, and enriches the application of sanguinarine derivatives in the field of agricultural chemicals and medicines.
[0026] (2) The sanguinarine derivative of the present application has good insecticidal activity. At a concentration of 500 g / mL, it shows excellent insecticidal activity on Myzus persicae, and the corrected mortality of most compounds is above 85%. In the LC50 determination, the LC50 of some compounds is below 10 g / mL, and the compound still shows excellent insecticidal activity on Myzus persicae.
[0027] (3) The sanguinarine derivative of the present application shows better fungistatic activity on ten plant pathogenic fungi, and shows certain fungistatic activity on ten pathogenic fungi at a concentration of 50 g / mL. At a concentration of 50 μg / mL, the sanguinarine derivative of the present application shows an inhibition rate of above 70% on several tested pathogenic fungi, and the inhibition rate on individual pathogenic fungi reaches above 90%.
[0028] (4) The sanguinarine derivative of the present application also shows certain fungistatic activity on bacteria such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, and it is worth further studying. BRIEF DESCRIPTION OF DRAWINGS
[0029] Figure 1 is a structural diagram of the 5-azacyclic structure substituted sanguinarine derivative with antibacterial and insecticidal activity of the present application.
[0030] Figure 2 is a structural diagram of the 5-azacyclic structure substituted sanguinarine derivative with antibacterial and insecticidal activity and its derivative synthesis process. DETAILED DESCRIPTION
[0031] The technical solutions of the present application will be further limited in combination with specific embodiments, but the scope of protection is not limited to the description.
[0032] Example 1
[0033] Example 1: Preparation of compound I-1
[0034] (1) Treatment of solvent. Tetrahydrofuran as affinity reaction solvent must be removed water, remove a small amount of oxide in the solvent. Method: 200 ml of analytical pure tetrahydrofuran slowly add 2.1 g of lithium aluminum hydride, during the solution to prevent splash. In constant temperature oil bath, 77 ℃ under reflux for 2 hours, change the reflux device for distillation device, dry tetrahydrofuran is evaporated, after temperature stabilization, collect 63-64 ℃ component, collect the middle part.
[0035] (2) Reaction step. 2-methyl 1H-pyrrole (1.1 eq) is dissolved in 25 mL of dry tetrahydrofuran solvent, placed in a three-necked flask, one of which is connected with N2 balloon three-way valve, the other two are inserted into the appropriate hollow plug. Using three-way valve access vacuum pump, replace the air in the three-necked flask several times, so that the whole three-necked flask environment is under nitrogen protection. The three-necked flask is placed in a constant temperature oil bath, the temperature is controlled between 23-30 ℃, under nitrogen protection, NaH (1.4 eq) is added under stirring, after stirring at room temperature for half an hour, sanguinarine hydrochloride (1 eq) is added, the stirring speed is increased and stirred vigorously for 24 hours, during which the reaction progress can be detected by TLC.
[0036] (3) Post-processing: after the reaction is basically complete, 75 ml of water is slowly added under stirring to extinguish the reaction, then 50 ml of ethyl acetate is used for extraction three times, the combined extract is dried with anhydrous magnesium sulfate, concentrated, and used as column chromatography raw material. A 300 mesh silica gel is used to pack a chromatography column, ethyl acetate: petroleum ether is used as the chromatography system, and gradient elution is used to pass through the column to separate the target compound. White powder 0.40 g, yield 33%. 1H NMR (CDC13, 500 MHz) δ 10.40 (s, 1H), 7.82 (s, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.23 (s, 1H), 7.00 (d, J = 8.1 Hz, 1H), 6.11 (dd, J = 15.0, 14.1 Hz, 4H), 5.35 (s, 1H), 5.26 (s, 1H), 4.99 (s, 1H), 2.66 (s, 3H), 2.01 (s, 3H). 13 CNMR (CDC13, 75 MHz) δ 148.26, 147.65, 147.19, 145.41, 140.57, 130.90, 129.71, 127.27, 127.25, 126.24, 123.97, 123.96, 120.45, 116.82, 114.80, 107.99, 106.20, 104.82, 104.42, 101.90, 101.58, 101.02, 55.30, 42.92, 13.30; HRMS calcd for C 25 H 20 N2O4(M+H) + 412.1423, found 413.1509.
[0037] Example 2: Preparation of compound I-2
[0038] (1) Reaction step. The treatment of reaction solvent tetrahydrofuran must be anhydrous and free of oxidant, same as example 1. Dissolve 1H-4-methylimidazole (1.1 eq) in 25 mL dry tetrahydrofuran solvent, put into a three-necked flask, one of which is connected to a three-way valve with a N2balloon, and the other two are inserted into suitable hollow plugs. Use the three-way valve to connect to the vacuum pump, replace the air in the three-necked flask several times by repeatedly pumping, and make the whole three-necked flask environment under nitrogen protection. Put the three-necked flask into a constant temperature oil bath, control the temperature between 23-30°C, under nitrogen protection, add NaH (1.4 eq) under stirring, stir for half an hour at room temperature, then add sanguinarine hydrochloride (1 eq), increase the stirring speed and stir vigorously for 24 hours, during which time the reaction progress can be detected by TLC.
[0039] (2) Post-treatment: after the reaction is basically complete, slowly add 75 mL water under stirring to quench the reaction, then extract with 50 mL*3 ethyl acetate, dry with anhydrous magnesium sulfate, concentrate, then recrystallize once using chloroform: petroleum ether = 1:4 as the recrystallization system, continue to recrystallize the obtained solid sample 1-2 times using ethyl acetate: petroleum ether = 1:3 as the recrystallization solvent, and separate to obtain the purified target product. White powder 0.27 g, yield 26%.1 HNMR (CDC13, 300 MHz) δ 7.84 (d, J = 8.7 Hz, 1H), 7.63 - 7.53 (m, 3H), 7.35 - 7.22 (m, 2H), 7.17 (d, J = 8.2 Hz, 1H), 6.59 (s, 1H), 6.50 (s, 1H), 6.24 (d, J = 3.9 Hz, 1H), 6.20 - 6.09 (m, 4H), 2.80 (s, 3H), 1.86 (s, 3H). 13 C NMR (CDC13, 75 MHz) δ 148.72, 148.03, 147.86, 145.87, 137.87, 136.81, 135.12, 131.23, 126.41, 125.50, 124.79, 122.78, 120.39, 117.41, 114.40, 110.28, 110.05, 104.81, 102.70, 101.87, 100.22, 67.06, 41.87, 13.99. HRMS calcd for C 24 H 19 N3O4 (M+H) + 413.1367, found 414.1402.
[0040] In summary, the post-treatment method mainly has two methods of column chromatography and recrystallization, depending on the separation difficulty of each target compound. Some target compounds are extremely difficult to separate and purify, and preparative chromatography can also be used for separation. The treatment method needs to be tried several times to obtain pure target compounds.
[0041] Example 3: Insecticidal activity determination of I series compounds
[0042] (1) Test materials
[0043] Test agents: I-1 ~ I-10.
[0044] Test pests: Myzus persicae, collected from field population.
[0045] (2) Test treatment and method
[0046] The virulence determination method is as follows: first, the raw material is prepared into a mother liquor (2% by mass) with N,N-dimethylformamide, and then diluted to the determination concentration with a Triton X-100 aqueous solution (0.05% by mass). The aphid-carrying leaves selected in advance are immersed in the liquid medicine by immersion method, and after being shaken gently for 10 s, the excess liquid is absorbed with a water-absorbing paper. After counting, the leaves are placed in a culture dish, which contains a wet water-absorbing paper for moisture retention, and covered with a preservative film. The culture dish is placed in a 25-1C incubator for recovery. A water solution without the medicine is used as a blank control. The results are checked under a table magnifying lens after 48 h. The insect body is gently touched with a brush tip. Insects that do not react and cannot normally crawl are considered dead. The concentration of the medicine for the general screening is 500 μg / mL, and the concentration gradient for the determination of LC50 is set to 1, 5, 10, 20, 80, 200, 400 μg / mL. Each gradient is determined in triplicate. The test results are statistically analyzed by Probit software to calculate the LC50 value. The insecticidal activity data of the target compounds are shown in Tables 1 and 2.
[0047] (3) Test results
[0048] Table 1 Insecticidal activity of I series compounds against Myzus persicae at a concentration of 500 μg / mL
[0049]
[0050] Table 2 LC50 value and 95% confidence limit of target compound I against Myzus persicae 50
[0051] Number LC 50 Values and 95% confidence limits (pg / mL) Number LC 50 Values and 95% confidence limits (pg / mL) I-1 15.4(10.4～24.3) I-9 98.1(71.7～139.6) I-2 9.4(5.4～16.8) I-10 101.5(58.0～217.9) I-3 256.1(94.4～953.7) I-4 8.0(4.8～13.7) I-5 92.4(47.1～188.9) I-6 101.38(57.8～214.8) I-7 51.0(36.9～78.9) I-8 174.1(106.6～317.8)
[0052] Example 4: Determination of antibacterial activity of I series compounds against plant pathogenic fungi
[0053] (1) Experimental method: The test compound is dissolved and prepared into a high-concentration mother liquor of 20,000 μg / mL with dimethylformamide. The test medicine is added to sterilized and cooled PDA medium to a concentration of 50 μg / mL, and mixed. A blank control without the medicine is set, and each treatment is repeated three times. Carbendazim is used as a control medicine for the determination of antibacterial activity.
[0054] Colony diameter method. A fresh mycelium block of the test fungus is inoculated in the center of the medium plate containing the medicine, with the mycelium facing downward. In a constant-temperature incubator, the colony diameter of the treatment is measured by the cross method when the colonies of the blank control cover more than 2 / 3 of the culture dish. The average of three repetitions represents the size of the colony.
[0055] (2) Treatment method:
[0056] Inhibition rate of mycelial growth of various pathogenic fungi by medicine treatment (see the following formula)
[0057] Mycelial growth inhibition rate / % = (blank mycelium diameter - mycelium diameter of sample) / (blank mycelium diameter - culture dish diameter)
[0058] (3) The test results are as follows:
[0059] Table 3 Mycelial growth inhibition rate of various pathogenic fungi treated by the sample
[0060]
[0061]
[0062] Example 5: Determination of antibacterial activity of the compounds in series I against bacteria
[0063] (1) Test strains: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa.
[0064] (2) Experimental method and treatment: The culture medium is liquid and solid broth protein peptone medium. A small amount of bacterial coating is taken from each fresh strain slope and inoculated into nutrient broth after activation, and then cultured in a 37°C incubator for 24 hours to obtain the original bacterial solution. The original bacterial solution is then prepared into a bacterial suspension containing 104-105 CFU / ml using sterilized saline. The concentration of the ten samples tested is 100 μg / mL. The experimental method uses a simple filter paper method: filter paper with a thickness of 1.5 mm is punched into 6 mm diameter paper pieces using a punch machine, and the filter paper pieces are sterilized, dried, and placed in solutions of the same concentration and different samples for 2 hours. 0.2 ml of each test bacterial solution is spread on the solid culture medium to obtain a bacterial plate, and the filter paper pieces soaked in various concentrations are attached to the bacterial plate using sterile forceps, with 5 pieces placed equidistantly on each plate. Three pieces are soaked in the sample, one piece is soaked in saline, and one piece is soaked in 400,000 units of penicillin, which is placed in the middle. Each treatment is repeated three times, and each treated culture dish is placed in a 37°C incubator and incubated for 24 hours. The size of the inhibition zone diameter is measured. The width of the inhibition ring (W) is calculated. w>3mm strong inhibition, l-2.5mm inhibition, W<0.9mm weak or no inhibition. W: (D-d) / 2
[0065] In the formula: W - inhibition ring width;
[0066] D - total diameter of sample and inhibition ring;
[0067] d - sample diameter.
[0068] (3) The determination results: the antibacterial effect (mm) of each sample on three kinds of pathogenic bacteria, repeated three times and averaged.
[0069] Table 4: Inhibition effect (mm) of each sample on three pathogenic bacteria
[0070]
[0071] From the above, the 5-azacycle-substituted bloodroot alkaloid derivative (I) has good insecticidal, bacteriostatic and antibacterial activities, and can be well applied in the field of agricultural chemicals and the field of antibacterial drugs.
[0072] Finally, it should be pointed out that the above examples are only more representative examples of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, but can also have many variations. All variations that can be directly derived or inferred by those skilled in the art from the content disclosed herein should be considered as falling within the scope of protection of the present application.
Claims
1. A 5-azo-substituted hematoxylin derivative with antibacterial and insecticidal activity, characterized in that, It is any of the following structures:
2. The application of the 5-azo-substituted hematoxylin derivative with antibacterial and insecticidal activity as described in claim 1 in the preparation of insecticides or bacteriostatic agents.