CN117946006A - Efficient preparation method and application of benzoazepine alkaloid-containing compound - Google Patents
Efficient preparation method and application of benzoazepine alkaloid-containing compound Download PDFInfo
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 150000008038 benzoazepines Chemical class 0.000 title description 2
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- -1 alkaloid compound Chemical class 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 45
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
The invention discloses a high-efficiency preparation method and application of a benzoazepine-containing alkaloid compound. The invention prepares 3 compounds with neuraminidase inhibitory activity from Nodulisporium sp.SCSIO 41223, the structural formula is shown in formula (I), and the preservation number of the strain SCSIO 41223 is GDMCC No:64051. The invention realizes the efficient fermentation, preparation and directional separation of a novel alkaloid compound containing the benzazepine Zhuo Jituan by optimizing and screening the fermentation conditions of the strain. The compound has remarkable neuraminidase inhibition activity, provides an alternative compound for developing new anti-influenza medicines, and has important significance for developing marine medicinal microorganism resources in south China sea.
Description
Technical Field
The invention belongs to the technical field of natural medicines, and particularly relates to optimization of high-yield fermentation conditions of strains, and a novel directional separation of a alkaloid compound containing benzazepine Zhuo Jituan and application of the alkaloid compound in preparation of anti-influenza medicines.
Background
Influenza is a transmissible disease with high pathogenicity and higher mortality caused by influenza viruses, seasonal outbreaks occur between humans and animals worldwide every year, the occurrence of highly pathogenic avian influenza and swine influenza which are continuously exploded in recent years is a continuous threat to the life safety of humans, great economic loss is brought, and meanwhile, due to the continuous occurrence of influenza virus variants, the development of novel influenza treatment drugs is urgently needed. Neuraminidase plays an indispensable important role in assisting the release of mature influenza virions and infection of new host cells, and because of its highly conserved active site, is the most potential target for developing a therapeutic agent for influenza, the therapeutic effect is achieved by developing novel inhibitors of neuraminidase capable of blocking viral transmission pathways. Marine fungi are a kind of microorganism resource with important value, can produce secondary metabolic substances with abundant structures and remarkable biological activity, and are an important source for developing novel medicines.
Disclosure of Invention
It is a first object of the present invention to provide 3 benzazepine Zhuo Jituan alkaloid compounds nodulibenzones A, B, C or pharmaceutically acceptable salts thereof which have inhibitory activity against neuraminidase.
The structural formula of the 3 benzazepine Zhuo Jituan alkaloid compounds nodulibenzones A and B, C is shown as the formula (I),
The second object of the present invention is to provide a process for the preparation of compounds nodulibenzones A, B, C isolated from a fermentation culture of the northern bay marine black-edge tongue-tail beef co-producing fungus Nodulisporium sp.scsio 41223. The method comprises the following specific steps:
1) Preparing a fermentation culture of the fungus Nodulisporium sp.scsio 41223;
2) Adding ethyl acetate with the same volume into the fermentation culture, stirring, performing ultrasonic treatment, soaking overnight, concentrating ethyl acetate extract, and repeatedly soaking, concentrating and extracting with ethyl acetate for five times to obtain concentrated ethyl acetate phase extract; separating the crude extract by adopting a medium-pressure normal-phase silica gel column, and using petroleum ether: ethyl acetate: methanol is subjected to gradient elution according to the volume ratio of 100:0:0, 9:1:0, 4:1:0, 3:1:0, 2:1:0, 1:1:0, 20:20:1, 10:10:1, 5:5:1, 3:3:1, 2:2:1 and 0:0:100, four column volumes are required to be eluted for each gradient, and 11 fractions Fr 1-Fr 11 are obtained through detection and combination of thin layer chromatography; petroleum ether: ethyl acetate: fr.6 eluted with methanol in the volume ratio of 1:1:0 was purified by ODS medium pressure reverse phase column chromatography with methanol: carrying out gradient elution on water according to the volume ratio of 5:95-100:0, and separating to obtain 6 components Fr.6.1-Fr.6.6; methanol is added to the mixture: fr.6.6, obtained by eluting with water in a volume ratio of 9:1, was purified by semi-preparative HPLC, using acetonitrile: water: eluting with formic acid at a volume ratio of 40:60:0.01, and collecting subfractions Fr.6.6.4 with retention time of 15min to obtain a compound nodulibenzone B; the subfraction fr.6.6.5 with retention time of 23min was further purified by semi-preparative HPLC on methanol: water: eluting with formic acid at a volume ratio of 45:55:0.01, collecting the component with retention time of 12min to obtain compound nodulibenzone A, and collecting the component with retention time of 10min to obtain compound nodulibenzone C.
Preferably, the fermentation culture is prepared by the following method: inoculating Nodulisporium sp.SCSIO 41223 into seed culture medium, shake culturing to obtain seed culture solution, inoculating the seed culture solution into solid fermentation culture medium of rice, and culturing to obtain fermentation culture.
Preferably, the seed culture medium comprises the following formula: 15g of malt extract, 24g of sea salt, 1L of distilled water and pH of 7.4-7.8; the formula of the rice solid fermentation medium is as follows: 200g of rice, 6g of artificial sea salt and 250mL of distilled water.
A third object of the present invention is to provide the use of compound nodulibenzones A, B, C, or a pharmaceutically acceptable salt thereof, in the manufacture of an anti-influenza medicament.
Preferably, the anti-influenza drug is a neuraminidase inhibitor.
A fourth object of the present invention is to provide an anti-influenza drug comprising an effective amount of the compound nodulibenzones A, B, C or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.
A fifth object of the present invention is to provide the use of the fungus Nodulisporium sp.scsio 41223 for the preparation of compounds nodulibenzones A, B, C or pharmaceutically acceptable salts thereof.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides a novel method for realizing efficient fermentation, preparation and directional separation of a benzo-aza Zhuo Jituan alkaloid compound by optimizing and screening fermentation conditions of marine fungus Nodulisporium sp.SCSIO 41223. The compound has remarkable neuraminidase inhibition activity, provides an alternative compound for developing new anti-influenza medicines, and has important significance for developing marine medicinal microorganism resources in south China sea.
The fungus Nodulisporium sp.SCSIO 41223 was deposited at the microbiological bacterial collection center (GDMCC) of Guangdong province at 11/23/2023, under the address of building 5, 30/100, guangzhou city martyr, guangdong province, post code: 510070, accession number is: GDMCC No:64051.
Drawings
FIG. 1 is a key HMBC, COSY-related for Compounds 1 and 3.
Figure 2 shows the comparison of the measured CD with the theoretical calculated ECD spectra for compounds 1 and 2.
FIG. 3 shows a comparison of the measured CD of Compound 3 with the calculated ECD spectra.
FIG. 4 is the interaction of Compound 3 with neuraminidase (PDB code: 2HU 4).
Detailed Description
The invention is further illustrated in detail below in connection with specific examples which are provided solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1: preparation and structural identification of Compounds nodulibenzones A, B, C
1. Microbial fermentation conditions
The method comprises the steps of growing fungus Nodulisporium sp.SCSIO 41223 in a flat plate culture medium (15 g of malt extract powder, 32g of sea salt, 18g of agar, 1L of distilled water, pH of 7.4-7.8), stirring and mixing all components uniformly, adjusting pH, sterilizing and cooling to obtain the culture medium, inoculating the fungus to a seed culture medium (15 g of malt extract, 24g of sea salt, 1L of distilled water, pH of 7.4-7.8 after the fungus grows out of spores, preparing the culture medium, stirring and mixing all components uniformly, adjusting pH, sterilizing and cooling to obtain the culture medium), culturing at room temperature for 3 days (180 r/min) to obtain a seed culture solution, inoculating the seed culture solution to a rice solid fermentation culture medium (200 g of rice, 6g of artificial sea salt and 250mL of distilled water are respectively taken from each conical flask, stirring and mixing all components uniformly, sterilizing and cooling to obtain the culture medium), and culturing at 26 ℃ for 30 days.
2. Extraction and separation
Adding ethyl acetate with the same volume into the strain rice fermentation medium, stirring, performing ultrasonic treatment for 30min to extract secondary metabolite in the strain into an ethyl acetate layer, soaking overnight, repeating the extraction for five times, and concentrating to obtain 65.1g of ethyl acetate phase extract. Separating the crude extract by adopting a medium-pressure normal-phase silica gel column, mixing the crude extract with 300-400 meshes of silica gel, carrying out gradient elution by using a solvent system (V Petroleum ether ∶V acetic acid ethyl ester ∶V Methanol ＝100:0:0、9:1:0、4:1:0、3:1:0、2:1:0、1:1:0、20:20:1、10:10:1、5:5:1、3:3:1、2:2:1、0:0:100, with the flow rate of 100mL min -1), eluting 4 column volumes for each gradient, and then carrying out thin-layer chromatography detection and merging to obtain 11 fractions Fr 1-Fr 11. Separating 6 components Fr.6 (4.0 g) obtained by eluting with V Petroleum ether ∶V acetic acid ethyl ester ∶V Methanol =1:1:0 by ODS medium pressure reverse phase column chromatography (MeOH: H 2 O, volume ratio 5:95-100:0) to obtain 6 components Fr.6.1-Fr.6.6, purifying the component Fr.6.6 obtained by eluting with MeOH: H 2 O=9:1 by semi-preparative HPLC (ChromCore C 18,5μm,10*250mm;V Acetonitrile ：V Pure water ：V Formic acid =40:60:0.01; flow rate 3mL×min -1) to obtain subfractions Fr.6.6.1-Fr.6.6.5, wherein subfraction Fr.6.6.4 is compound nodulibenzone B (2.84 mg, t R =15 min); the subfractions fr.6.6.5 with retention time 23min were collected and further purified (ChromCore C 18,5μm,10*250mm;V Methanol ：V Pure water ：V Formic acid =45:55:0.01; flow rate 3ml×min -1) to give compound nodulibenzone A (5.39 mg, t R =12 min) and compound nodulibenzone C (3.1 mg, t R =10 min).
3. Structural identification of compounds
Nodulibenzone A: the yellow-green powder, high resolution mass spectrum (HRESIMS) showed an ion peak at m/z 349.1785[ M+H ] +, combined with 13 C NMR data, was assumed to have a chemical formula of C 18H24N2O5 and an unsaturation of 8. Hydrogen spectral data (table 1) shows that it contains 1 characteristic 1,2, 3-trisubstituted benzene ring [ delta H 6.00.00 (d, j=8.5 hz, h-9), 6.06 (d, j=8.0 hz, h-7), 7.21 (t, j=8.5 hz, h-8) ],3 double peaks methyl [ delta H 0.82.82 (d, j=6.0 hz, h-15), 0.85 (d, j=6.5 hz, h-16), 1.12 (d, j=6.5 hz, h-8) ] and 2 exchangeable protons [ delta H 12.58,8.13] signals. 13 C NMR (Table 1) in combination with HSQC data showed that it contained 18 carbon signals including 3 methyl groups, 3 methylene groups (1 methylene group attached to the nitrogen atom, delta C 52.9.9), 6 methines (3 ethylenic carbons, delta C 103.2,103.4,138.2,2, N methines, delta C 51.0,54.2) and 6 quaternary carbons (3 carbonyl groups, delta C 168.2,174.4,199.4 and 3 ethylenic carbons, delta C 105.5,150.1,162.3). By analyzing the two-dimensional nuclear magnetic data of 1H-1 H COSY and HSQC, the structure inference of the 3 fragments as described below was confirmed. HMBC spectra showed CH 3 -18 with C-2, C-3; h-3 and C-1, C-2, C-5, C-18; h-5 is associated with C-3, C-5a (FIG. 1), confirming the bicyclic moiety of the benzazepine ring. HMBC spectra show that the association of hydroxyl hydrogen (. Delta. H 12.58) with C-8, C-9a, C-1 confirms that the hydroxyl group is attached to C-9. HMBC spectra showed CH 3 -15 with C-13, C-14; h-12 is associated with C-13, C-14, C-17, indicating the presence of leucine units. HMBC spectra show that H-5,H-12 is associated with C-11, confirming that leucine units are linked to the bicyclic ring through ureido groups. The absolute configuration of compound 1, i.e. its absolute configuration 3s,12r, was further determined by comparing the literature with the ECD calculated data (fig. 2), and was designated nodulibenzone A.
Table 1: 1 H and 13 C NMR data for Compounds 1 and 2
Nodulibenzone B: a pale yellow powder, high resolution mass spectrum (HRESIMS) showed an ion peak at m/z 347.1612[ M-H ] -, indicating that it has the same molecular formula C 18H24N2O5 as compound 1. Compound 2 has similar NMR data as compound 1 (table 1), and by detailed analysis of two-dimensional NMR spectrum data of compound 2 (1H-1 H COSY and HMBC), it is shown that compound 2 has the same planar structure as compound 1. However, the CD data were different, and the absolute configuration of compound 2 was determined by performing an ECD calculation on compound 2 to determine the absolute configuration, and the result showed that the ECD spectrum obtained for the calculated configuration (3 s,12 s) -2 of compound 2 was identical to the experimental CD spectrum (fig. 2), and was designated nodulibenzone B.
Nodulibenzone C: pale yellow powder, and high resolution mass spectrum (hresis) showed its molecular formula as C 21H22N2O5. The one-dimensional nuclear magnetic data shows many similar structural features to compound 1 (table 2), except that the substitution of one single substituted benzene ring for (δH 7.14,d,J＝7.5Hz,H-16,H-18;δH 7.16,t,J＝7.5Hz,H-17;δH 7.22,m,H-15,H-19;δC 125.8,127.7,129.5,138.9), in compound 3 for two methyl groups and 1 methine group of compound 1 indicates that the leucine unit in compound 1 is represented as a phenylalanine structure in compound 3. The above conclusion is further confirmed by two-dimensional NMR spectrum (1H-1 H COSY, HMBC, HSQC) data of compound 3, HMBC spectra showing H-12 and C-11, C-13, C-14, C-20; h-13 is associated with C-12, C-14, C-15, C-19 (FIG. 1). The absolute configuration of compound 3 was determined by comparing the calculation with the experimental ECD profile of 3 (FIG. 3).
Table 2: 1 H and 13 C NMR data for Compound 3
From the above physicochemical data analysis, the specific structures of the compounds nodulibenzones A, B, C are shown in formula (I).
Example 2: detection of inhibitory Activity of Compounds nodulibenzones A, B, C on neuraminidase
1. Preparation for standard curve detection
(1) 70 Mu L of neuraminidase detection buffer solution is added into the 96-well fluorescent ELISA plate;
(2) Adding 0, 1, 2, 5, 7.5 and 10 mu L neuraminidase respectively;
(3) 0-20. Mu.L of Milli-Q water was added to make the total volume per well 90. Mu.L.
2. Preparation and detection of Compounds
(1) Preparing a compound to be tested into a concentration of 50 mug/mL by using DMSO;
(2) Adding 70 mu L of neuraminidase detection buffer, 10 mu L of neuraminidase and 10 mu L of compound sample to be detected in each hole into a 96-hole fluorescent ELISA plate respectively;
(3) Vibrating and mixing the ELISA plate for about 1min, incubating at 37 ℃ for 2min to enable the inhibitor to fully interact with neuraminidase, respectively adding 10 mu L of neuraminidase fluorogenic substrate, vibrating and mixing for about 1min, incubating at 37 ℃ for 30min, and performing fluorescence measurement;
(4) The excitation wavelength of the fluorescence measurement was 322nm, and the emission wavelength was 450nm. In order to avoid the influence of temperature, humidity and other conditions on experiments, the detection of the standard curve of neuraminidase and the detection of a sample to be detected are performed on the same 96-hole fluorescent ELISA plate, and three parallel detection is performed in each group of experiments so as to reduce experimental errors;
(5) The percent inhibition of neuraminidase by the sample can be calculated from the standard curve. For detection of an inhibitor found to be effective, IC 50 of the inhibitor can be determined by detecting the dose effect of the inhibitor.
3. Detection of neuraminidase inhibitory Activity of Compounds Nodulibenzones A and B, C
When the concentration of the compound is 50 mug/mL, the activity test result shows that the compounds nodulibenzones A and B, C have better inhibition activity on neuraminidase, the inhibition rate is more than 50%, the relevant standard curve is established in the research, the IC 50 measurement is carried out, the result shows that the inhibition activity of the compound nodulibenzone C on neuraminidase is most obvious, the IC 50 value is 4.82 mu M and is superior to a positive medicament, the inhibition activity of the compounds nodulibenzone A and B on neuraminidase is relatively weak, the IC 50 values are 120.67 and 160.75 mu M and Oseltamivir acid respectively as positive controls, and the IC 50 value is 20 mu M.
Molecular interaction between the compound nodulibenzone C and neuraminidase is further explored through molecular docking, and a research basis is provided for carrying out structural optimization and improving related activity in the future. The results in FIG. 4 show that compound nodulibenzone C is well positioned in the binding gap in a similar anchoring configuration, with a binding energy (S value) of 7.8kcal/mol. Compound nodulibenzone C interacts with neuraminidase protein (PDB code: 2HU 4), forming 3 hydrogen bonds with mainly amino acid residues TYR-347 and ARG-152 within the target protein, at the respective distancesAnd/>These intermolecular interactions allow for stable binding of compound nodulibenzone C to neuraminidase, and also provide a reasonable explanation for the interaction of compound nodulibenzone C with neuraminidase proteins, showing the potential of this compound for development into treatment of influenza.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (10)
1. Compounds nodulibenzones A and B, C shown in the structural formula (I) or pharmaceutically acceptable salts thereof;
2. A process for the preparation of a compound nodulibenzones A, B, C as defined in claim 1, characterized in that it is isolated from a fermentation culture of the fungus Nodulisporium sp.scsio 41223; the Nodulisporium sp.SCSIO 41223 has a deposit number GDMCC No:64051.
3. The preparation method according to claim 2, characterized by the specific steps of:
1) Preparing a fermentation culture of the fungus Nodulisporium sp.scsio 41223;
2) Adding ethyl acetate with the same volume into the fermentation culture, stirring, performing ultrasonic treatment, soaking overnight, concentrating ethyl acetate extract, and repeatedly soaking, concentrating and extracting with ethyl acetate for five times to obtain concentrated ethyl acetate phase extract; separating the crude extract by adopting a medium-pressure normal-phase silica gel column, and using petroleum ether: ethyl acetate: methanol is subjected to gradient elution according to the volume ratio of 100:0:0, 9:1:0, 4:1:0, 3:1:0, 2:1:0, 1:1:0, 20:20:1, 10:10:1, 5:5:1, 3:3:1, 2:2:1 and 0:0:100, four column volumes are required to be eluted for each gradient, and 11 fractions Fr 1-Fr 11 are obtained through detection and combination of thin layer chromatography; petroleum ether: ethyl acetate: fr.6 eluted with methanol in the volume ratio of 1:1:0 was purified by ODS medium pressure reverse phase column chromatography with methanol: carrying out gradient elution on water according to the volume ratio of 5:95-100:0, and separating to obtain 6 components Fr.6.1-Fr.6.6; methanol is added to the mixture: fr.6.6, obtained by eluting with water in a volume ratio of 9:1, was purified by semi-preparative HPLC, using acetonitrile: water: eluting with formic acid at a volume ratio of 40:60:0.01, and collecting subfractions Fr.6.6.4 with retention time of 15min to obtain a compound nodulibenzone B; the subfraction fr.6.6.5 with retention time of 23min was further purified by semi-preparative HPLC on methanol: water: eluting with formic acid at a volume ratio of 45:55:0.01, collecting the component with retention time of 12min to obtain compound nodulibenzone A, and collecting the component with retention time of 10min to obtain compound nodulibenzone C.
4. A process according to claim 3, wherein the fermentation culture is prepared by: inoculating Nodulisporium sp.SCSIO 41223 into seed culture medium, shake culturing to obtain seed culture solution, inoculating the seed culture solution into solid fermentation culture medium of rice, and culturing to obtain fermentation culture.
5. The method of claim 4, wherein the seed culture medium comprises the following formula: 15g of malt extract, 24g of sea salt, 1L of distilled water and pH of 7.4-7.8; the formula of the rice solid fermentation medium is as follows: 200g of rice, 6g of artificial sea salt and 250mL of distilled water.
6. Use of a compound nodulibenzones A, B, C, or a pharmaceutically acceptable salt thereof, as claimed in claim 1 in the manufacture of an anti-influenza medicament.
7. The use according to claim 6, wherein the anti-influenza drug is a neuraminidase inhibitor.
8. An anti-influenza agent comprising an effective amount of a compound nodulibenzones A, B, C, or a pharmaceutically acceptable salt thereof, as an active ingredient, according to claim 1, and a pharmaceutically acceptable carrier.
9. The anti-influenza drug of claim 8, wherein the anti-influenza drug is a neuraminidase inhibitor.
10. Use of the fungus Nodulisporium sp.scsio 41223 for the preparation of a compound nodulibenzones A, B, C as claimed in claim 1 or a pharmaceutically acceptable salt thereof; the Nodulisporium sp.SCSIO 41223 has a deposit number GDMCC No:64051.
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