A benzothiazole compound and a preparation method and application thereof

Abstract

The application discloses a benzothiazole compound and a preparation method and application thereof, and belongs to the technical field of phytochemistry. A novel benzothiazole compound is found from a strain of fungus Tennessee Aspergillus 1022LEF, named 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol, with a molecular formula of C9H9NO2S, and a preparation method of the compound and insecticidal activity of the compound on Spodoptera exigua are reported for the first time, and the compound has the potential to develop into a novel microbial natural product pesticide.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of phytochemistry, and more particularly to a benzothiazole compound and a preparation method and application thereof. BACKGROUND

[0002] Spodoptera frugiperda is an important pest that damages food crops worldwide, with records of damage to 353 plant species. Spodoptera frugiperda is native to the Americas, with extremely strong migration and serious damage, and has characteristics such as wide host range, strong reproductive capacity, and strong adaptability, and is listed as one of the world's top ten plant pests by the Food and Agriculture Organization of the United Nations.

[0003] The control measures for Spodoptera frugiperda mainly include chemical control measures (such as organophosphorus, carbamate and pyrethroid pesticides) and biological control measures (natural enemy insect control, physical and chemical induction control, biological pesticide control and transgenic prevention). Chemical pesticides have the characteristics of high efficiency, rapid effect and convenience, and have played an important role in the control of Spodoptera frugiperda. However, due to the long-term abuse of chemical pesticides, Spodoptera frugiperda has developed resistance to pesticides and has a high resistance, and the control effect has been significantly weakened. Biological control has the characteristics of high efficiency, safety and good environmental compatibility, and has become the preferred control measure. Among them, microbial natural product pesticides have the advantages of novel structure, significant activity, "drug-like" and high, and have become a new hotspot of biological pesticide research.

[0004] Benzothiazole compounds have good biological activity and low toxicity, so the discovery, preparation, design and biological activity research of this kind of compounds are of great significance to the development of innovative drugs and biological pesticides. Since the 1960s, the American Beckman Company developed the fungicide benzothiocyano, which has produced many commercial agents containing benzothiazole groups, such as the fungicide benzothiazole, the herbicide benzothiazole, the herbicide benzothiazole, the herbicide benzothiazole and the herbicide benzothiazole. Chinese patent CN109897031B discloses a benzothiazole derivative containing a coumarin group and its preparation method and application. The derivative has obvious toxic effect on Plutella xylostella and Aphis craccivora, and can be used for controlling agricultural pests.

[0005] In summary, how to provide a new type of benzothiazole compound is a problem that those skilled in the art need to solve. SUMMARY

[0006] Therefore, the application provides a benzothiazole compound and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned purpose, the application adopts the following technical scheme:

[0008] The first object of the application is to provide a novel benzothiazole compound.

[0009] A benzothiazole compound, which is named as 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol, has a molecular formula of C9H9NO2S, and a molecular structure formula as follows:

[0010]

[0011] The second object of the application is to provide the application of the above-mentioned benzothiazole compound in the control of Spodoptera exigua, which provides a new compound entity for the biological control of Spodoptera exigua.

[0012] The application of the above-mentioned compound in the control of Spodoptera exigua.

[0013] A Spodoptera exigua insecticide comprising the above-mentioned compound.

[0014] The third object of the application is to provide a preparation method of the above-mentioned benzothiazole compound.

[0015] The preparation method of the above-mentioned compound is to ferment and culture Tennessee Aspergillus 1022LEF, and the fermentation liquor can be obtained after separation and purification;

[0016] The Tennessee Aspergillus 1022LEF has been preserved by the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC No.21469, a classification name of Aspergillus tennesseensis, a Latin name of Aspergillus tennesseensis, a preservation date of March 16, 2021, and a preservation place of Institute of Microbiology, Chinese Academy of Sciences, No.3, Yikhina West Road, Beijing City, Chaoyang District.

[0017] Further, the specific steps are as follows:

[0018] (1) Tennessee Aspergillus 1022LEF is sequentially fermented in PDA plate medium and rice medium to obtain fermentation liquor;

[0019] (2) The fermentation liquor is extracted with ethyl acetate and concentrated to obtain a crude extract extract;

[0020] (3) The crude extract is purified by vacuum silica gel column, Lobar LiChroprep RP-18 reverse phase silica gel column, 200-300 mesh silica gel column and Sephadex LH-20 column in turn.

[0021] Further, the specific operation of step (1) is as follows:

[0022] (11) A certain amount of Aspergillus tenuissimus 1022LEF is inoculated on PDA flat plate medium and cultured at 28℃ for 5 days;

[0023] (12) The mycelium on the surface of the PDA flat plate medium is transferred to rice medium for fermentation and cultured at 28℃ for 50 days;

[0024] The rice medium comprises rice and distilled water, and the mass / volume ratio of the two is 2:1 g / mL.

[0025] Further, the specific operation of step (3) is as follows:

[0026] (31) The crude extract is mixed with 100-200 mesh silica gel, and then subjected to component separation by vacuum silica gel column chromatography, eluted with petroleum ether-ethyl acetate as gradient eluent, and 4 components Fr.1-Fr.4 with decreasing polarity are obtained;

[0027] The volume ratio of petroleum ether to ethyl acetate in the gradient eluent is 10:1, 5:1, 2:1 and 1:1 in turn;

[0028] (32) The component Fr.4 is subjected to component separation by Lobar LiChroprep RP-18 reverse phase silica gel column, eluted with methanol-water as gradient eluent, and 5 sub-components Fr.4.1-Fr.4.5 with decreasing polarity are obtained;

[0029] The volume ratio of methanol to water in the gradient eluent is 10% to 100%;

[0030] (33) The sub-component Fr.4.2 is mixed with 100-200 mesh silica gel, and then subjected to 200-300 mesh silica gel column chromatography, and the eluent is dichloromethane and methanol with a volume ratio of 25:1, and the second column volume component is collected and concentrated under reduced pressure (vacuum degree-0.1 Mpa, concentrated to non-flow extract);

[0031] (34) Then, through Sephadex LH-20 column chromatography (with methanol as the eluent), the 2nd column volume component was collected and concentrated under reduced pressure (vacuum degree-0.1 Mpa, concentrated to non-flowing extract), and the compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol was purified.

[0032] Through the technical solutions described above, compared with the prior art, the present application has the following beneficial effects: (1) The fungal-derived benzo[d]thiazole compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol is a new natural product, which is searched by SciFinder database and compared with the currently reported benzo[d]thiazole compounds; (2) The preparation method provided by the present application can quickly locate and accurately separate the benzo[d]thiazole compound; (3) The benzo[d]thiazole compound of the present application has obvious insecticidal activity on the 2nd instar larvae of Spodoptera frugiperda (better than the positive drug sophoridine). BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0034] Figure 1 A molecular structure diagram of the new benzo[d]thiazole compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol in Example 2 of the present application;

[0035] Figure 2 A high-resolution mass spectrum diagram of the new benzo[d]thiazole compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol in Example 2 of the present application;

[0036] Figure 3 A hydrogen spectrum (500MHz, DMSO-d6) diagram of the new benzo[d]thiazole compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol in Example 2 of the present application;

[0037] Figure 4 A carbon spectrum (125MHz, DMSO-d6) diagram of the new benzo[d]thiazole compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol in Example 2 of the present application;

[0038] Figure 5 Single crystal diffraction pattern of the novel benzothiazole compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol in Example 2 of the present application. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0040] The required reagents in the present application are conventional experimental reagents, which are purchased from the market; and the experimental methods not mentioned are conventional experimental methods, which are not described here one by one.

[0041] Example 1

[0042] The specific process for preparing 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol in the present application is as follows:

[0043] (1) Under sterile conditions, a proper amount of Aspergillus tennesseensis 1022LEF strain preserved in agar-malt extract medium was inoculated on PDA plate medium, and placed in a 28℃ constant temperature incubator for culture for 5 days; a proper amount of 1022LEF strain on the surface of the above PDA plate medium was cut and transferred into a 1000mL triangular flask containing sterilized rice medium (containing 200g of rice and 100mL of distilled water).

[0044] (2) The above triangular flask was placed at 28℃ for fermentation culture for 50 days, and the rice medium containing Aspergillus tennesseensis 1022LEF strain after fermentation was subjected to ultrasonic extraction with 100mL of ethyl acetate, and vacuum concentration (vacuum degree-0.1Mpa, concentrated to non-flow extract) to obtain a crude extract extract.

[0045] (3) The above crude extract extract was added to 100-200 mesh silica gel for sample mixing, and then subjected to component separation by vacuum silica gel column chromatography under reduced pressure, and eluted with petroleum ether-ethyl acetate (volume ratio 10:1, 5:1, 2:1 and 1:1) as gradient eluent in order of increasing polarity of eluent, to obtain four components Fr.1-Fr.4 with polarity from small to large.

[0046] (4) Fraction Fr.4 (eluted by petroleum ether-ethyl acetate 1:1 fraction) was purified by Lobar LiChroprep RP-18 reversed-phase silica gel column (eluted by methanol-water, volume ratio 10% to 100%), to obtain five sub-fractions Fr.4.1 to Fr.4.5 in order of increasing polarity.

[0047] (5) Sub-fraction Fr.4.2 (eluted by methanol-water, volume ratio 40%) was mixed with 100-200 mesh silica gel and purified by normal-phase silica gel column chromatography (200-300 mesh silica gel, eluted by dichloromethane:methanol = 25:1), and the second column volume fraction was collected and concentrated under reduced pressure (vacuum degree -0.1 Mpa, concentrated to non-flowing extract);

[0048] (6) Then, the second column volume fraction was purified by Sephadex LH-20 column chromatography (eluted by methanol), and the second column volume fraction was collected and concentrated under reduced pressure (vacuum degree -0.1 Mpa, concentrated to non-flowing extract), to obtain the compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol.

[0049] Example 2

[0050] This example determines the molecular structure of the compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol by high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, and single crystal diffraction. Figure 1 ).

[0051] The physicochemical properties of the compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol are as follows:

[0052] Appearance: white amorphous powder.

[0053] Solubility: easily soluble in methanol and acetone, slightly soluble in dichloromethane, insoluble in petroleum ether and ethyl acetate.

[0054] Molecular formula: C9H9NO2S.

[0055] UV absorption spectrum (UV absorption spectrum) λ max (methanol, logε) 220 (2.31), 238 (4.25), 268 (3.36) nm.

[0056] High-resolution mass spectrometry (HRESI-MS): m / z 196.0443 [M+H] + (theoretical value C9H 10 NO2S, 196.0432) Figure 2 ).

[0057] Hydrogen spectrum data 1 HNMR, 500MHz, DMSO-d6): 9.11 (1H, s, H-2), 7.34 (1H, d, J = 1.3 Hz, H-5), 6.78 (1H, s, H-7), 2.76 (2H, dd, J = 7.0 Hz, H2-8), 3.62 (2H, dd, J = 12.1, 7.0, H2-9), 10.20 (1H, br s, 4-OH), 4.74 (1H, br s, 9-OH) Figure 3 ).

[0058] Carbon spectrum data 13 C NMR, 125MHz, DMSO-d6): 152.5 (CH, C-2), 141.8 (C, C-3a), 151.6 (C, C-4), 112.6 (CH, C-5), 139.1 (C, C-6), 112.9 (CH, C-7), 135.6 (C, C-7a), 39.6 (CH2, C-8), 62.6 (CH2, C-9) Figure 4 ).

[0059] The single crystal diffraction pattern is shown in Figure 5 .

[0060] Example 3

[0061] The present application uses a novel benzothiazole compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol for the insecticidal activity against Spodoptera exigua, and the specific process is as follows: the insecticidal activity is determined according to the Pesticide Indoor Biological Test Guideline - Artificial Feed Mixed Pesticide Method (NY / T 1154.10-2008) of the People's Republic of China.

[0062] Spodoptera exigua was provided by the Plant Protection Research Center of Tobacco Research Institute of Chinese Academy of Agricultural Sciences, and was bred indoors using artificial feed. The breeding conditions were as follows: the temperature was (25±1) °C, the relative humidity (RH) was (70±5) %, and the light cycle was L:D = (16:8) h. Adult insects were bred in an egg laying cage and fed with 10% honey water. To avoid the infection of bacteria on artificial feed and Spodoptera exigua, the insect breeding equipment was regularly disinfected.

[0063] A new benzothiazole compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol was prepared into a mother liquor with acetone, and then added into artificial feed to prepare toxic feed with final concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mg / mL. The feed was placed in a 24-well plate, and 2nd instar Spodoptera exigua larvae with consistent growth were selected, 1 larva was put into each well, 3 replicates were performed for each treatment, 20 larvae were used for each replicate, matrine treatment was used as a positive control, and acetone treatment was used as a blank control, and the final content of acetone in each treatment should not exceed 1%. The treated larvae were reared and observed under the conditions of temperature (25±1)℃, relative humidity (RH) (70±5)%, and photoperiod L:D=(16:8)h, and the number of dead larvae was counted after 48h. The dead larvae were gently touched with a fine brush, and those with no reaction or slight reaction were considered dead. The mortality rate and corrected mortality rate of Spodoptera exigua were calculated. The formula is as follows:

[0064] Mortality rate (%) = number of dead insects / total number of insects in treatment x 100%;

[0065] Corrected mortality rate (%) = (treatment group mortality rate-control group mortality rate) / (1-control group mortality rate) x 100%.

[0066] The results are shown in Table 1.

[0067] Table 1 Insecticidal activity of 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol on 2nd instar larvae of Spodoptera exigua

[0068]

[0069] Note: The data in the table are mean ± standard error. The same lowercase letters in the same column indicate significant differences (P<0.05) by Duncan's new range test.

[0070] It was found that the compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol had certain insecticidal activity on 2nd instar larvae of Spodoptera exigua, and the corrected mortality rate gradually increased with the increase of the concentration of the compound, as shown in Table 1. The half lethal concentration LC 50 of the compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol on 2nd instar larvae of Spodoptera exigua was 0.24 mg / mL, which was better than that of the positive drug matrine (LC 50 value of 0.33 mg / mL).

[0071] The various embodiments described in this specification are intended to be illustrative only and in no way limit the scope of the application. Changes and modifications can be made by those skilled in the art, which employ the principles of the application, without departing from the scope of the application. Accordingly, the application is not limited to the embodiments described herein, but instead has scope to encompass any choice whatsoever that is dependent on, or can be substituted in, the principal, new and inventive features that are described and claimed herein.

[0072] The above description of disclosed embodiments is intended to be illustrative only and not limiting of the application. Numerous modifications to these embodiments can be made by those skilled in the art without departing from the spirit or scope of the application. The scope of the application is not limited to the embodiments described herein, but rather extends to any that are dependent on, or can be substituted in, the principal, new and inventive features that are described and claimed herein.

Claims

1. A benzothiazole compound, characterized by, The compound is named as 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol, and the molecular formula is C9H9NO2S, and the molecular structure formula is as follows:

2. The use of the compound of claim 1 in the control of Spodoptera frugiperda.

3. A Spodoptera fugiperda insecticide, characterized by, The compound of claim 1.

4. A process for the preparation of a compound according to claim 1, characterized in that, The fermentation culture of Tennessee Aspergillus 1022LEF is taken, and the fermentation liquor is separated and purified to obtain the compound; The Tennessee Aspergillus 1022LEF has been preserved by the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 21469, the classification name is Aspergillus tennesseensis, the Latin name is Aspergillus tennesseensis, the preservation date is March 16, 2021, and the preservation place is No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard.

5. The production method according to claim 4, wherein The specific steps are as follows: (1) Tennessee Aspergillus 1022LEF is fermented in PDA plate culture medium and rice medium in sequence to obtain fermentation liquor; (2) The fermentation liquor is extracted with ethyl acetate and concentrated to obtain a crude extract; (3) The crude extract is purified by vacuum silica gel column, Lobar LiChroprep RP-18 reverse phase silica gel column, 200-300 mesh silica gel column and Sephadex LH-20 column in sequence to obtain the compound.

6. The production method according to claim 5, wherein The specific operation of step (1) is as follows: (11) A certain amount of Tennessee Aspergillus 1022LEF is inoculated on PDA plate culture medium, and cultured at 28°C for 5 days; (12) The mycelium on the surface of the PDA plate culture medium is cut and transferred to rice medium for fermentation, and cultured at 28°C for 50 days; The rice medium comprises rice and distilled water, and the mass / volume ratio of the two is 2:1 g / mL.

7. The production method according to claim 5, wherein The specific operation of step (3) is as follows: (31) The crude extract is added to 100-200 mesh silica gel for sample mixing, and then component separation is carried out by vacuum silica gel column chromatography, and elution is carried out with petroleum ether-ethyl acetate as gradient eluent, and 4 components Fr.1-Fr.4 with decreasing polarity are obtained; The volume ratio of petroleum ether to ethyl acetate in the gradient eluent is 10:1, 5:1, 2:1 and 1:1 in sequence; (32) Component Fr.4 is separated by Lobar LiChroprep RP-18 reverse phase silica gel column, and eluted with methanol-water as gradient eluent to obtain 5 sub-components Fr.4.1-Fr4.5 with decreasing polarity; The volume ratio of methanol to water in the gradient eluent is 10% to 100%; (33) Sub-component Fr.4.2 is added to 100-200 mesh silica gel for sample mixing, and then component separation is carried out by 200-300 mesh silica gel column chromatography, and elution is carried out with dichloromethane and methanol as eluent, and the volume ratio of the two is 25:1, and the second column volume component is collected and concentrated under reduced pressure; (34) The compound 6-(2-hydroxyethyl)benzo[d]thiazol-4-ol was then purified by Sephadex LH-20 column chromatography with methanol as eluent, the 2nd column volume fraction was collected and concentrated under reduced pressure.

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