Preparation method and application of 7-(10-methyl undecyl) azacycloheptane-2-ketone

Abstract

The invention provides a preparation method and application of 7-(10-methyl undecyl) azacycloheptane-2-ketone, and the method comprises the following steps: fermenting and culturing wetland streptomyces 13-3 to obtain a fermentation product, extracting the fermentation product with ethyl acetate, and concentrating under reduced pressure to obtain a crude extract; the crude extract is subjected to rapid preparative chromatography, Sephadex LH-20 gel column chromatography and silica gel column chromatography purification, and 7-(10-methyl undecyl) azacycloheptane-2-one is obtained; according to the invention, the new compound 7-(10-methyl undecyl) azacycloheptane-2-ketone is obtained from wetland streptomyces for the first time, and the 7-(10-methyl undecyl) azacycloheptane-2-ketone has a relatively strong inhibition effect on alpha-glucosidase and can be used for preparing a medicine for treating diabetes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a preparation method of 7-(10-methylundecyl)azacycloheptan-2-one and application thereof. BACKGROUND

[0002] Natural products are an important source of new drug research and development, and their structural diversity and biological activity diversity provide a broad space for drug development. In recent years, the research on novel structure natural bioactive molecules has been the focus of attention. A large number of studies have shown that actinomycetes, especially Streptomyces, are the main microbial resources that produce a variety of active secondary metabolites. However, although wetland Streptomyces 13-3 has been confirmed to have strong biological activity, its specific active ingredients have not been systematically studied. There is a lack of efficient separation and purification method for the active ingredients of the strain in the prior art, which leads to the potential application value not being fully tapped. Therefore, in-depth research on the separation, identification and function of the active ingredients of wetland Streptomyces 13-3 will provide an important scientific basis for its further development and utilization. SUMMARY

[0003] The present application aims to at least solve one of the above technical problems in the art, i.e., the present application first obtains a new compound 7-(10-methylundecyl)azacycloheptan-2-one from wetland Streptomyces and verifies its inhibitory activity on alpha-glucosidase.

[0004] To this end, in a first aspect of the present application, a preparation method of 7-(10-methylundecyl)azacycloheptan-2-one is provided, comprising the following steps:

[0005] Strain culture: wetland Streptomyces 13-3 is activated to obtain a seed solution; the seed solution is inoculated on YMG solid culture medium and incubated at 28℃ for 7-8 days to obtain a fermentation product;

[0006] Crude extract extraction: the fermentation product is cut into pieces and extracted with ethyl acetate; the extraction solution is concentrated to dryness under reduced pressure, redissolved in methanol and filtered, and then concentrated to dryness under reduced pressure again to obtain a crude extract;

[0007] Fast preparation chromatography: the crude extract is dissolved in methanol and separated by fast preparation chromatography; methanol and water are used for gradient elution, each gradient elution is 2L, the flow rate is 50mL / min, the 100% methanol eluate is collected, and concentrated under reduced pressure at 35℃ to obtain component 1;

[0008] Gel column chromatography: component 1 was separated by Sephadex LH-20 gel column, methanol was used for elution, the flow rate was set to 10-12 seconds / drop, one tube was collected every 90 min, the target component was combined, and was concentrated under reduced pressure to obtain component 1-1; component 1-1 was separated again by Sephadex LH-20 gel column, dichloromethane:methanol = 2:1 solvent was used for elution, the flow rate was set to 10-12 seconds / drop, one tube was collected every 90 min, the target component was combined, and was concentrated under reduced pressure to obtain component 1-1-1;

[0009] Silica gel column chromatography: component 1-1-1 was separated by silica gel column chromatography, and petroleum ether and ethyl acetate were used for gradient elution, the eluent of petroleum ether: ethyl acetate = 30:1 to 6:1 was collected, and was concentrated to dryness under reduced pressure to obtain 7-(10-methylundecyl)azacycloheptan-2-one.

[0010] According to the preparation method of 7-(10-methylundecyl)azacycloheptan-2-one provided in the application, the fermentation product is obtained by fermenting and culturing Streptomyces humidus 13-3, the fermentation product is extracted with ethyl acetate, and is concentrated under reduced pressure to obtain a crude extract; the crude extract is purified by rapid preparation chromatography, Sephadex LH-20 gel column chromatography and silica gel column chromatography to obtain 7-(10-methylundecyl)azacycloheptan-2-one; the new compound 7-(10-methylundecyl)azacycloheptan-2-one is obtained from Streptomyces humidus for the first time, 7-(10-methylundecyl)azacycloheptan-2-one has a strong inhibitory effect on alpha-glucosidase, and can be used for preparing a drug for treating diabetes.

[0011] Optionally, when the strain is cultured, Streptomyces humidus 13-3 is inoculated into YMG liquid medium, and is cultured at 28℃ with 180 rpm oscillation for 3 days to obtain a seed liquid.

[0012] Optionally, when the crude extract is extracted, the fermentation product is added with an equal volume of ethyl acetate for extraction for 3 times, and the extraction solutions are combined.

[0013] Optionally, when the rapid preparation chromatography is performed, methanol and water are gradient eluted according to 0% methanol, 30% methanol, 50% methanol, 70% methanol and 100% methanol.

[0014] Optionally, the YMG solid medium is composed of yeast extract 4 g, malt extract 10 g, glucose 4 g, agar powder 15-20 g, and distilled water, and the volume is made up to 1000 mL, and the pH is 7.2.

[0015] In the second aspect of the application, 7-(10-methylundecyl)azacycloheptan-2-one prepared by the above preparation method is provided, and the structural formula of the 7-(10-methylundecyl)azacycloheptan-2-one is as follows:

[0016]

[0017] In a third aspect of the present application, there is provided the use of the above-mentioned 7-(10-methylundecyl)azepan-2-one in the manufacture of an alpha-glucosidase inhibitor.

[0018] In a fourth aspect of the present application, there is provided the use of the above-mentioned 7-(10-methylundecyl)azepan-2-one in the manufacture of a medicament for treating diabetes.

[0019] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and the attendant drawings or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 TLC analysis chart of the compound;

[0021] Figure 2 High resolution mass spectrum analysis chart of the compound;

[0022] Figure 3 Alpha-glucosidase inhibitory activity chart of the compound. DETAILED DESCRIPTION

[0023] The technical solutions of the present application are described below by specific examples. It should be understood that the one or more method steps mentioned in the present application do not exclude other method steps before and after the mentioned combination steps or other method steps inserted between the explicitly mentioned steps; it should also be understood that the examples are only used to illustrate the present application and not to limit the scope of the present application. Moreover, unless otherwise specified, the numbering of the method steps is only a convenient tool to identify the method steps and is not intended to limit the arrangement order of the method steps or to restrict the scope of the present application, and the change or adjustment of the relative relationship without substantial change of the technical content is also considered as the scope of the present application.

[0024] In order to better understand the above technical solutions, the exemplary embodiments of the present application are described in more detail below. Although exemplary embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough and complete understanding of the present application and to convey the full scope of the present application to those skilled in the art.

[0025] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market.

[0026] The application will be described below with reference to specific examples, it should be noted that these examples are merely descriptive, and do not limit the application in any way.

[0027] Example 1

[0028] Streptomyces garebi 13-3 was provided by the Plant Protection Institute of Fujian Academy of Agricultural Sciences, and was preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 22830.

[0029] A method for extracting 7-(10-methylundecyl)azacycloheptan-2-one from Streptomyces garebi 13-3, comprising the following steps:

[0030] (1) Strain culture: YMG liquid medium (yeast extract 4 g, malt extract 10 g, glucose 4 g, distilled water to 1000 mL, pH 7.2, high pressure sterilization) was prepared, Streptomyces garebi 13-3 was inoculated, and the seed liquid was obtained by culturing at 28℃ with 180 rpm shaking for 3 days.

[0031] YMG solid medium (yeast extract 4 g, malt extract 10 g, glucose 4 g, agar powder 15-20 g, distilled water to 1000 mL, pH 7.2, high pressure sterilization) was prepared; 500 μL of seed liquid was inoculated in YMG solid medium, and the fermentation product was obtained by culturing at 28℃ for 7-8 days.

[0032] (2) Crude extract extraction: the fermentation product was cut into small pieces, and equal volume of ethyl acetate was added for extraction 3 times, the extraction liquid was combined, and concentrated to dryness at 35℃ under reduced pressure, then dissolved in methanol and filtered, and concentrated to dryness again at 35℃ under reduced pressure to obtain the crude extract (13 g).

[0033] (3) Compound purification:

[0034] Flash preparation chromatography: the crude extract was dissolved in a small amount of methanol, separated by flash preparation chromatography, and gradient eluted with methanol and water (0% methanol, 30% methanol, 50% methanol, 70% methanol, 100% methanol), each gradient elution was 2 L, the flow rate was 50 mL / min, the 100% methanol eluate was collected, and concentrated at 35℃ under reduced pressure to remove the solvent to obtain component 1.

[0035] Gel column chromatography: Component 1 was separated by Sephadex LH-20 gel column, eluted with methanol, the flow rate was set at 10-12 seconds / drop, and one tube was collected every 90 min. The target component was combined and concentrated at 35°C under reduced pressure to remove the solvent to obtain component 1-1. Then component 1-1 was separated again by Sephadex LH-20 gel column, eluted with dichloromethane:methanol (2:1, v / v) solvent, the flow rate was set at 10-12 seconds / drop, and one tube was collected every 90 min. The target component was combined and concentrated at 35°C under reduced pressure to remove the solvent to obtain component 1-1-1.

[0036] Silica gel column chromatography: Component 1-1-1 was separated by silica gel column (10 mm x 200 mm) chromatography and eluted with petroleum ether and ethyl acetate in gradient (90:1, 70:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 6:1, v / v). The eluate of petroleum ether:ethyl acetate (30:1-6:1, v / v) was collected and concentrated to dryness at 35°C under reduced pressure to obtain the compound.

[0037] The compound was a white powder, and its TLC analysis was shown in Table 1, using high performance thin layer chromatography plate, developed in dichloromethane:methanol = 15:1 (v / v) developing agent, iodine coloration, and the result showed a single spot, indicating that the isolated compound was very pure. Figure 1

[0038] Example 2

[0039] The compound obtained in Example 1 was dissolved in deuterated chloroform (CDCI3), and NMR analysis of the compound was performed using a Bruker AV 500 nuclear magnetic resonance instrument, as shown in Table 1. The compound obtained in Example 1 was dissolved in mass spectrometry grade methanol, and mass spectrometry analysis was performed using a Waters Acquity UPLC shimadzu IT-TOF, ESI + mode, as shown in Table 1. Figure 2 Figure 2 The HRESIMS spectrum of the compound is shown in Figure 1, which gives the quasi-molecular ion peak m / z 282.2785 [M+H] + (molecular weight is 281.2797, C 18 H 35 NO). In combination with high resolution mass spectrometry and NMR spectroscopy data, it can be determined that the obtained compound is 7-(10-methylundecyl)azacycloheptan-2-one (C 18 H 35 NO), and its structural formula is as follows:

[0040]

[0041] ​​Table 1. NMR spectral data of the compounds

[0042]

[0043] Example 3

[0044] Analysis of the inhibitory activity of compound 7-(10-methylundecyl)azepan-2-one on a-glucosidase: 20 μL of compound (obtained in Example 1) sample of different concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mg / mL) was sequentially added in a 96-well plate, 112 μL of 0.05 mol / L phosphate buffer (pH 6.8) and 20 μL of 0.2 U / mL a-glucosidase solution were added, and incubated at 37°C for 15 min. Then 20 μL of 2.5 mmol / L substrate p-NPG was added, and incubated at 37°C for another 15 min. Finally, 80 μL of 0.2 mol / L Na2CO3 solution was added to terminate the reaction, and the absorbance of each well was measured at 405 nm wavelength using an enzyme marker. Acarbose was selected as a positive control, three parallel groups were set for each experiment, and the inhibition rate of each concentration sample was calculated according to the following formula:

[0045]

[0046] Wherein, A1 refers to the absorbance value of the determination system containing the sample, the substrate and a-glucosidase; A2 refers to the absorbance value of the determination system containing the sample and the substrate; A3 refers to the absorbance value of the determination system containing the blank solvent (methanol), the substrate and a-glucosidase; A4 refers to the absorbance value of the determination system containing the blank solvent (methanol) and the substrate.

[0047] The results are shown in Table 2. Figure 3 As shown in Table 2, within a certain concentration range (0-1.0 mg / mL), the compound 7-(10-methylundecyl)azepan-2-one showed a dose-dependent relationship between the concentration and the inhibition rate of a-glucosidase, and the inhibitory effect on a-glucosidase was significantly enhanced with the increase of the concentration of the compound. The inhibition curve of the compound on a-glucosidase was drawn with the concentration of the compound as the abscissa and the inhibition rate of a-glucosidase as the ordinate, linear fitting was performed, and the half effective inhibitory concentration (IC 50 ) was calculated. The results showed that the linear regression equation of the inhibition rate of the compound on a-glucosidase was y = 61.523x + 88.636 (R 2 = 0.9846), and the IC 50 value was 235 μg / mL, which was slightly higher than that of acarbose (173 μg / mL), indicating that the compound had a strong inhibitory effect on a-glucosidase.

[0048] In summary, according to the embodiment of the present application, the fermentation product is obtained by fermenting Streptomyces gabbatti 13-3, the fermentation product is extracted with ethyl acetate, concentrated under reduced pressure to obtain a crude extract, the crude extract is purified by fast preparation chromatography, Sephadex LH-20 gel column chromatography and silica gel column chromatography to obtain a compound, and the compound is further identified by TLC, nuclear magnetic resonance and high resolution mass spectrometry as 7-(10-methylundecyl)azacycloheptan-2-one. The present application obtains the new compound 7-(10-methylundecyl)azacycloheptan-2-one from Streptomyces gabbatti for the first time, and 7-(10-methylundecyl)azacycloheptan-2-one has a strong inhibitory effect on alpha-glucosidase and can be used for preparing a drug for treating diabetes.

[0049] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and integrate different embodiments or examples described in the present specification.

[0050] Although the embodiments of the present application have been shown and described above, it should be understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A method for preparing 7-(10-methylundecyl)azepan-2-one, characterized in that: The following steps are involved: Bacterial culture: Activate Streptomyces lycopersicum 13-3 to obtain seed liquid; inoculate the seed liquid into YMG solid medium and culture at 28°C for 7-8 days to obtain the fermentation product; Crude extract extraction: the fermentation product is cut into pieces, extracted with ethyl acetate, the extract is concentrated to dryness under reduced pressure, re-dissolved in methanol, filtered, and concentrated to dryness under reduced pressure again to obtain a crude extract; Flash preparative chromatography: The crude extract was dissolved in methanol and separated by flash preparative chromatography using a gradient elution of methanol and water (2 L per gradient elution) at a flow rate of 50 mL / min. The 100% methanol eluate was collected and concentrated under reduced pressure at 35°C to obtain fraction 1. Gel column chromatography: Fraction 1 was separated by passing it through a Sephadex LH-20 gel column, eluted with methanol at a flow rate of 10-12 seconds per drop, and collected in one tube every 90 minutes. The target fractions were combined and concentrated under reduced pressure to obtain fraction 1-1. Fraction 1-1 was separated again by passing it through a Sephadex LH-20 gel column, eluted with a solvent of 2:1 dichloromethane:methanol at a flow rate of 10-12 seconds per drop, and collected in one tube every 90 minutes. The target fractions were combined and concentrated under reduced pressure to obtain fraction 1-1-1. Silica gel column chromatography: Component 1-1-1 was separated by silica gel column chromatography and gradient eluted with petroleum ether and ethyl acetate. The eluate with a petroleum ether:ethyl acetate ratio of 30:1 to 6:1 was collected and concentrated to dryness under reduced pressure to obtain 7-(10-methylundecyl)azacycloheptane-2-one.

2. The preparation method according to claim 1, wherein During bacterial culture, Streptomyces palustris 13-3 was inoculated into YMG liquid culture medium and cultured at 28° C. and 180 rpm with shaking for 3 days to obtain seed solution.

3. The preparation method according to claim 1, wherein When extracting the crude extract, equal volume of ethyl acetate was added to the fermentation product for extraction three times, and the extracts were combined.

4. The preparation method according to claim 1, wherein During rapid preparative chromatography, methanol and water were used for gradient elution according to 0% methanol, 30% methanol, 50% methanol, 70% methanol, and 100% methanol.

5. The preparation method according to claim 1, wherein The YMG solid culture medium is composed of 4 g of yeast extract, 10 g of malt extract, 4 g of glucose, 15-20 g of agar powder, and distilled water to a volume of 1000 mL, with a pH of 7.

2.

6. 7-(10-methylundecyl)azepan-2-one prepared by the preparation method according to any one of claims 1 to 5, wherein the structural formula of the 7-(10-methylundecyl)azepan-2-one is as follows:

7. Use of 7-(10-methylundecyl)azepan-2-one as claimed in claim 6 in the preparation of α-glucosidase inhibitors.

8. Use of 7-(10-methylundecyl)azepan-2-one according to claim 6 in the preparation of a drug for treating diabetes.

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