A class of gabosine derivatives, their preparation methods and applications

Abstract

This divisional application discloses a class of gabosine derivatives, their preparation methods, and applications, belonging to the field of marine fungal active ingredient analysis technology. This invention utilizes the polarity difference of gabosine derivatives to extract and isolate a novel gabosine derivative from the fermentation culture of the marine fungus *Epicoccum sorghinum* GST-5. In vitro anti-β-glucuronidase assays show that the gabosine derivative provided by this invention has good β-glucuronidase inhibitory activity and low cytotoxicity, demonstrating promising development potential in the preparation of anti-β-glucuronidase drugs.

Description

[0001] This application is a divisional application of application number 202210955910.X, application date August 10, 2022, entitled "A class of polyhydroxycyclohexane derivatives and their preparation methods and applications". Technical Field

[0002] This invention relates to the field of marine fungal active ingredient analysis technology, specifically to a gabosine derivative extracted from Epicoccum sorghinum GST-5 and its application. Background Technology

[0003] Compared to terrestrial microorganisms, marine microorganisms are able to tolerate the extreme conditions unique to the ocean, such as high salinity, high pressure, low oxygen, and low light. This unique living environment leads to diversity in species, genetic composition, and ecological functions among marine microorganisms. The special characteristics of the marine environment, coupled with advancements in marine microbial resource acquisition technologies, have brought unprecedented opportunities for the research of natural medicinal compounds derived from marine microorganisms.

[0004] Marine fungi are a rich source of bioactive secondary metabolites. 70-80% of these metabolites possess biological activity, including small-molecule lactones; fungal toxins; novel substances with inhibitory activity against the central nervous system; 1-dodecyl alcohol, unsaturated hydrocarbons, acids, and esters; and lipopeptide antibiotics that can inhibit the synthesis of new target sites by fungal viruses acting on the fungal cell wall. Discovering natural products with specific structural types using marine fungi as raw materials is of great significance for the development of marine drugs.

[0005] β-glucuronidase (GUS) is an important hydrolytic enzyme produced by the gut microbiota. It catalyzes the hydrolysis of glucuronide conjugates to generate the corresponding aglycones. In the human body, GUS distributed in the gut can also catalyze the glucuronide hydrolysis of various drugs (such as indomethacin, diclofenac, and 7-ethyl-10-hydroxycamptothecin, the active product of irinotecan) and endogenous hormones. The released aglycones can be reabsorbed by the intestines, resulting in a bimodal pharmacokinetic phenomenon. Notably, this process can potentially trigger serious gastrointestinal adverse reactions. For example, GUS can catalyze the hydrolysis of 7-ethyl-10-hydroxycamptothecin-O-glucuronic acid (SN-38G) to generate the highly cytotoxic product SN-38. The latter's accumulation in the intestines can cause intestinal mucosal shedding and lead to severe delayed-type diarrhea, among other adverse reactions. Therefore, the development of highly effective and safe GUS inhibitors is of great significance for alleviating fatal diarrhea caused by drugs such as irinotecan and indomethacin (Weng Zimiao, Lü Shanshan, Ge Guangbo, Wang Ping, Hou Jie, Pharmaceutical Progress, Research Progress on Inhibitors of Enteric Bacteria β-glucuronidase, 2018, 42(03)).

[0006] There is currently limited research on the chemical composition of Epicoccum fungi. Hao Baocong et al. isolated (4R*, 5R*, 6S*)-4,5-dihydroxy-6-(6′-methylsalicylic acidoxy)-2-methoxymethyl-2-cyclohexen-1-one, (4R*, 5R*, 6S*)-4,5-dihydroxy-6-(6′-methylsalicylic acidoxy)-2-methyl-2-cyclohexen-1-one, (-)-gabosine E, theobroxide, 3-chlorogentiol, and 3-hydroxybenzyl alcohol from a dandelion endophytic fungus, *Epicoccum sorghinum*. (Hao Baocong, Zheng Yaoyao, Chen Xu, Chen Qiuxia, Ji Ruonan, Chen Min, *Acta Pharmaceutica Sinica*, An endophytic fungus of dandelion, *Epicoccum sorghinum*) Study of 1-2 secondary metabolites, 2022, 57(07)).

[0007] Yuan Chao et al. studied the chemical constituents of *Epicoccumnigrum 14one*, an endophytic fungus isolated from the lichen *Leptogium masiaticum*. They identified the following compounds as: one alkaloid, fusaricide; seven benzofuran compounds, epicoccone B, 4,6-dihydroxy-5-methoxy-7-methyl-1,3-dihydroisobenzofuran, 5-methyl-epicoccone B, 3,6,7-trihydroxy-5-methoxy-4-methylisobenzo furan-1(3H)-one, 3-methoxyepicoccone B, 2,3,4-trihydroxy-6-(hydroxymethyl)-5-methylbenzyl-alcohol, isoochracinic acid; and three epicolide compounds, epicocconigrones A, epicolide B, and epicocconigrones. B, among which alkaloids fusaricide and epicolide compounds have strong antibacterial activity. Among them, alkaloid fusaricide has good in vitro inhibitory activity against lung cancer cells M109 (Yuan Chao, Guo Yuhua, Zhang Yingbo, Hu Xuan, Wang Dan, Yu Fulai, Li Gang, Chinese Journal of Traditional Chinese Medicine, Study on secondary metabolites of a plant endophytic fungus Epicocum nigrum 14one, 2019, 44(18), 4021-4025).

[0008] However, no literature has been found to date reporting the isolation of chlorogentisyl alcohol and gabosine compounds that resist GUS enzymes from Acetococcus fungi. Summary of the Invention

[0009] The purpose of this invention is to extract natural active substances with medicinal value from marine Epicoccum fungi.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] This invention isolated 25 compounds from the fermentation products of the fungus *Epicoccum sorghinum* GST-5. Structural identification revealed that these 25 compounds are polyhydroxycyclohexane derivatives, belonging to the categories of chlorogentisyl alcohol derivatives (with a 2-chloro-6-(hydroxymethyl)benzene-1,4-diphenol structure), gabosine derivatives (with a 4,5,6-trihydroxy-2-(hydroxymethyl)cyclohexane-2-en-1-one structure), and derivatives with a hybrid skeleton of chlorogentisyl alcohol and gabosine. Specific structural formulas are shown below. Figure 1 As shown, they are numbered 1 to 25 respectively.

[0012] The fungus *Epicoccum sorghinum* GST-5 is a large marine plant isolated from hydrothermal vent sediments off Guishan Island, Taiwan Province, with accession number CGMCC 3.20238.

[0013] By consulting databases and literature, this invention identified 19 of the above 25 compounds as new compounds. Therefore, this invention provides a new class of gabosine derivatives with structural formulas as shown in any one of formulas (8), (20), (24)-(25), corresponding to compounds 8, 20, 21, 22, 24, and 25, respectively.

[0014]

[0015]

[0016] The present invention also provides a method for isolating and extracting the above-mentioned new compounds from the fermentation products of Epicoccum sorghinum GST-5, but the preparation method of the above-mentioned compounds in the present invention is not limited thereto.

[0017] A method for extracting the gabosine derivative from fermentation products includes the following steps:

[0018] (1) The activated fungus Epicoccum sorghinum GST-5 was inoculated into liquid culture medium and fermented.

[0019] (2) After the fermentation culture is completed, mycelium and fermentation broth are separated. Organic solvent is added to the mycelium for extraction, and the extract is separated. The extract is concentrated and suspended in distilled water to obtain an aqueous suspension. The aqueous suspension is then extracted with ethyl acetate or n-butanol to obtain extract A. The fermentation broth is stirred with diatomaceous earth and extracted by reflux with ethyl acetate or n-butanol to obtain extract B.

[0020] (3) After concentrating extract A or extract B, perform normal-phase silica gel column chromatography separation, and sequentially elute with dichloromethane / methanol mixtures with volume ratios of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, and 10:1. Collect the fraction eluted from the dichloromethane / methanol mixture with a volume ratio of 10:1, and then perform reverse-phase silica gel column chromatography and high-performance liquid chromatography separation to obtain the derivative.

[0021] In step (1), the fungus Epicoccum sorghinum GST-5 was fermented and cultured.

[0022] GST-5, a fungus belonging to the genus *Plasmodium*, can be fermented using conventional PDA liquid medium or malt extract medium. To increase yield and provide sufficient nutrients for microbial growth and metabolism, preferably, the liquid medium, per 1L volume, comprises the following ingredients: 1-5g starch, 10-20g wheat bran, 3-15g yeast extract, 1-8g KH₂PO₄, 0.1-0.8g MgSO₄·7H₂O, with the remainder being water.

[0023] Alternatively, the liquid culture medium, per 1L volume, comprises the following ingredients: 200-600g potato, 2-10g peptone, 1-5g yeast extract, 5-20g glucose, with the remainder being water; the initial pH of the culture medium is 6.0-7.0.

[0024] Alternatively, based on a volume of 1L, the liquid culture medium comprises the following ingredients: 10-40g sucrose, 5-20g corn flour, 1-4g NaNO3, 1-4g yeast extract, 0.2-0.8g KH2PO4, 0.2-1g MgSO4·7H2O, 0.2-1g KCl, 0.001-0.005g FeSO4, with the remainder being water;

[0025] Alternatively, the liquid culture medium, per 1L volume, comprises the following ingredients: 20-30g malt extract, 15-20g glucose, 1-2g casein peptone, with the remainder being water.

[0026] The fermentation culture conditions are static culture at 20-30℃ for 10-40 days. The static culture method refers to culture without shaking flasks.

[0027] Preferably, the fermentation culture temperature is 22-26°C. More preferably, it is cultured at 25°C for 20 days, under which the yield of the gabosine derivative is the highest.

[0028] In step (2), mycelium and fermentation broth are separated and obtained, and the gabosine derivative can be extracted and separated from both mycelium and fermentation broth.

[0029] In the process of obtaining gabosine derivatives using mycelium, the mycelium is soaked in an organic solvent for 7-14 days to fully disrupt the cell walls and effectively dissolve intracellular substances. The organic solvent is one or two of methanol, ethanol, ethyl acetate, and acetone.

[0030] In step (3), the separation and purification method is as follows: the extract is separated by normal-phase silica gel column chromatography, and the obtained fraction is then separated by recrystallization, reversed-phase silica gel column chromatography, and high-performance liquid chromatography. Through multi-step separation and purification, a high-purity gabosine derivative can be obtained.

[0031] Preferably, the fractions are subjected to reversed-phase silica gel column chromatography, with gradient elution using methanol / water mixtures at volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, each gradient eluted 5 times, and numbered sequentially from 1 to 45. Fractions numbered 1 to 3 are combined into subfraction 1, fractions 4 to 5 into subfraction 2, fractions 14 to 17 into subfraction 6, and fractions 38 to 39 into subfraction 12. Subsequently, high-performance liquid chromatography (HPLC) is used for separation. Subfraction 1, eluted with a 28% methanol / water mixture, has a peak retention time of 25.6 minutes, which corresponds to compound 20 with the structural formula shown in formula (20), R1 = Cl and R2 = CH3. 2. When eluted with a 35% (v / v) methanol / water mixture, the peaks with retention times of 35.6 min and 36.9 min were compounds 21 and 22, respectively, with structural formulas as shown in formula (20) R1 = Oxybutyl, R2 = H and formula (20) R1 = OH, R2 = Oxybutyl, respectively. When eluted with a 36% (v / v) methanol / water mixture, the peak with retention time of 30.6 min was compound 8, with structural formula as shown in formula (8). When eluted with a 25% (v / v) acetonitrile / water mixture, the peaks with retention times of 14.3 min and 18.3 min were compounds 24 and 25, respectively, with structural formulas as shown in formula (24) and formula (25), respectively.

[0032] This invention demonstrates that the gabosine derivative isolated from the fermentation culture of *Epicoccums orghinum* GST-5 using the above method exhibits good anti-β-glucuronidase (GUS) activity. Furthermore, this invention uses the inhibitory activity of the compounds on cells as its cytotoxicity data, and the results show that, except for compounds 12 and 14, the other compounds have low cytotoxicity to human cells. Therefore, this invention provides the application of the aforementioned gabosine derivative in the preparation of drugs against β-glucuronidase.

[0033] Furthermore, the anti-β-glucuronidase drug is an inhibitor of intestinal bacterial β-glucuronidase. Studies have shown that β-glucuronidase distributed in the intestine can cause gastrointestinal adverse reactions during the hydrolysis of drugs (such as indomethacin, irinotecan, etc.). The compound provided by this invention can alleviate drug-induced gastrointestinal adverse reactions by inhibiting GUS enzyme activity.

[0034] This invention provides the application of the aforementioned gabosine derivative in the preparation of drugs that alleviate gastrointestinal toxicity caused by irinotecan and indomethacin.

[0035] Furthermore, among the 25 compounds mentioned above, compounds with structural formulas as shown in (19) and (23) are known compounds, corresponding to compounds 19 and 23, respectively. This invention demonstrates that these compounds also possess anti-GUS enzyme activity. Therefore, this invention provides the application of compounds with structural formulas as shown in formulas (19) and (23) in the preparation of drugs against β-glucuronidase. Furthermore, the anti-β-glucuronidase drug is an inhibitor of intestinal bacterial β-glucuronidase.

[0036]

[0037] The drug is made with the gabosine derivative of the present invention as the main active ingredient, and with the addition of pharmaceutically acceptable excipients. It can be formulated according to pharmaceutical preparation methods. The formulation can be an injection, infusion, powder for injection, granules, tablets, granules, powders, oral liquids, sugar-coated tablets, film-coated tablets, enteric-coated tablets, lozenges, pills, ointments, boluses, sprays, drop pills, disintegrants, orally disintegrating tablets, microcapsules, etc.

[0038] The present invention also provides a method for isolating and extracting compounds 19 and 23 from the fermentation products of Epicoccum sorghinum GST-5, comprising the following steps: separating the above sub-fractions 1 and 2 by high performance liquid chromatography, specifically: the peak of sub-fraction 1 with a retention time of 20.8 minutes after elution with a methanol / water mixture of 28% (v / v) is compound 19; the peak of sub-fraction 2 with a retention time of 27.3 minutes after elution with a methanol / water mixture of 35% (v / v) is compound 23.

[0039] The beneficial effects of this invention are as follows:

[0040] (1) This invention utilizes the polarity difference of gabosine derivatives to extract and isolate a gabosine derivative with a novel structure from the fermentation culture of marine fungi. The method is simple to operate, has a high extraction yield and high product purity, and is suitable for large-scale production.

[0041] (2) In vitro anti-GUS enzyme assays showed that the gabosine derivative provided by this invention has good anti-GUS enzyme activity. Further cytotoxicity assays showed that the sugar compound provided by this invention has low cytotoxicity and has good development prospects in the preparation of drugs against GUS enzymes. Attached Figure Description

[0042] Figure 1 The structural formulas of the 25 chlorogentisyl alcohol and gabosine derivatives of this invention are shown below.

[0043] Figure 2 For compound 8 1 H NMR data (in DMSO-d6, 600MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data.

[0044] Figure 3 For compound 19 1 H NMR data (in DMSO-d6, 600MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data.

[0045] Figure 4 For compound 20 1 H NMR data (in DMSO-d6, 600MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data.

[0046] Figure 5 For compound 21 1 H NMR data (in CD3OD, 600MHz) and 13 C NMR data (in CD3OD, 150MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data.

[0047] Figure 6 For compound 22 1 H NMR data (in DMSO-d6, 600MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data.

[0048] Figure 7 For compound 23 1 H NMR data (in CD3OD, 600MHz) and 13 C NMR data (in CD3OD, 150MHz), where (A) is 1H NMR data, (B) is 13 C NMR data.

[0049] Figure 8 For compound 24 1 H NMR data (in DMSO-d6, 600MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data.

[0050] Figure 9 For compound 25 1 H NMR data (in DMSO-d6, 600MHz) and 13 C NMR data (in DMSO-d6, 150 MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data. Detailed Implementation

[0051] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0052] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0053] Example 1: Fungal Isolation

[0054] Large marine plants were collected from seabed sediments at the hydrothermal vent of Guishan Island, Taiwan Province. After the samples were brought back to the laboratory, they were first rinsed three times with sterile seawater to remove non-attached microorganisms. The plants were then placed in centrifuge tubes, and a small amount of seawater was added. The mixture was vortexed for 10 minutes, and the suspension after removing the algae was centrifuged for 20 minutes (5000 r / min). The supernatant was discarded. The precipitate was resuspended in a small amount of sterile seawater, and 0.1 mL was spread on Martin's medium (containing 8 U / L gentamicin) plates. After incubation at 20°C for 10 days, single colonies were picked, purified by streak plating, and then transferred to slant plates for storage at 4°C for later use.

[0055] Example 2: Identification of *Plasmodium* fungi

[0056] The isolated fungus was cultured on a PDA, and the 18S rDNA gene sequence of the strain was determined. The 18S rDNA sequence of the strain is shown in SEQ ID No. 1.

[0057] Based on the morphological characteristics and 18S rDNA sequence analysis, the strain was identified as belonging to the genus *Epicoccum*, and named *Epicoccum sorghinum* GST-5. It was deposited on July 12, 2021, at the China General Microbiological Culture Collection Center (CGMCC), and confirmed to be viable. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, accession number: CGMCC 3.20238.

[0058] Example 3: Fermentation culture of *Plasmodium* fungi

[0059] The activated fungus GST-5 was prepared into a spore suspension, which was then inoculated into the culture medium and statically fermented at 25°C for 20 days.

[0060] The culture medium formula is as follows: starch 3g, wheat bran 14g, yeast extract 6g, KH2PO4 5g, MgSO4·7H2O 0.4g, and water 1000mL.

[0061] Example 4: Fermentation culture of *Plasmodium* fungi

[0062] The activated Acetococcus fungus GST-5 was made into a spore suspension, which was then inoculated into the culture medium and statically fermented at 24°C for 20 days.

[0063] The culture medium formula is as follows: 400g potato, 6g peptone, 2g yeast extract, 10g glucose, and 1000mL water; the initial pH of the culture medium is 6.5.

[0064] Example 5: Fermentation culture of *Plasmodium* fungi

[0065] The activated fungus GST-5 was prepared into a spore suspension, which was then inoculated into the culture medium and statically fermented at 25°C for 20 days.

[0066] The culture medium formula is as follows: 25g sucrose, 10g corn flour, 2g NaNO3, 2g yeast extract, 0.5g KH2PO4, 0.5g MgSO4·7H2O, 0.5g KCl, 0.001g FeSO4, and 1000mL water.

[0067] Example 6: Fermentation culture of *Plasmodium* fungi

[0068] The activated fungus GST-5 was prepared into a spore suspension, which was then inoculated into the culture medium and statically fermented at 25°C for 20 days.

[0069] The culture medium formula is as follows: 25g malt extract powder, 15g glucose, 1.5g casein peptone, and 1000mL water.

[0070] Example 7: Preparation of chlorogentisyl alcohol and gabosine derivatives

[0071] After fermentation culture of *Plasmodium* fungi GST-5, 5 L of fermentation broth was collected, centrifuged, and the precipitate was used to obtain mycelium. The mycelium was soaked in methanol for one week, and the soaking solution was concentrated and then suspended in 1 L of distilled water. The aqueous suspensions were combined and extracted with 6 L of n-butanol. The n-butanol extract was concentrated to obtain 10 g of extract. The extract was mixed with silica gel (100 mesh, 100 g) and subjected to normal-phase silica gel column chromatography (200-300 mesh, 100 g; silica gel column size L 50 mm). The distillate was eluted sequentially with dichloromethane / methanol mixtures at volume ratios of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, and 10:1, using 300 mL of methanol each time. The fractions were analyzed by TLC. The fractions eluted with dichloromethane / methanol mixtures at volume ratios of 35:1 to 10:1 were collected and recrystallized from methanol.

[0072] Example 8: Preparation of chlorogentisyl alcohol and gabosine derivatives

[0073] After fermentation culture of *Plasmodium* fungi, 5 L of fermentation broth was taken, centrifuged, and the supernatant was collected to obtain the fermentation broth. The fermentation broth was concentrated, mixed with 10 g of diatomaceous earth, refluxed with 1 L of n-butanol, and subjected to normal-phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L50 mm). The dichloromethane / methanol mixtures with volume ratios of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, and 10:1 were used for gradient elution with methanol. 400 mL of each eluent was collected per elution, and each eluent was repeated 5 times. A total of 30 fractions were collected from the dichloromethane / methanol mixtures with volume ratios from 35:1 to 10:1, and numbered sequentially from 1 to 30. The fractions were analyzed by TLC, and similar components were combined: fractions 1-7 were combined into component 1, fractions 8-11 into component 2, fractions 12-15 into component 3, fractions 16-25 into component 4, and fractions 26-30 into component 5.

[0074] Fraction 5 was subjected to reversed-phase silica gel column chromatography using methanol / water (1:9-9:1) as the eluent. Gradient elution was performed sequentially with methanol / water mixtures at volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. For each eluent, 400 mL of fraction was collected per elution, and each eluent was repeated 5 times, resulting in 45 fractions collected from each eluent. These fractions were numbered sequentially from 1 to 45. The fractions were analyzed, and similar components were combined. Fractions 1-3 were combined into subfraction 5-1, fractions 4-5 into subfraction 5-2, fractions 6-8 into subfraction 5-3, and fractions 9-10 into subfraction 5-3. The fractions 5-4, 11-13 are combined to form sub-component 5-5, 14-17 are combined to form sub-component 5-6, 18-25 are combined to form sub-component 5-7, 26-28 are combined to form sub-component 5-8, 29-30 are combined to form sub-component 5-9, 31-33 are combined to form sub-component 5-10, 34-37 are combined to form sub-component 5-11, 38-39 are combined to form sub-component 5-12, 40-41 are combined to form sub-component 5-13, 42-43 are combined to form sub-component 5-14, and 44-45 are combined to form sub-component 5-15.

[0075] The compounds were then separated by high-performance liquid chromatography (HPLC) with methanol / water or acetonitrile / water as the mobile phase at a flow rate of 10 mL / min and a detection wavelength of 254 nm. Subfraction 5-1, with eluent of 28% (methanol-water), had retention times of 13.1 min, 15.1 min, 18.3 min, 20.8 min, and 25.6 min, respectively. Recrystallization from cyclohexane yielded compounds 1, 2, 3, 19, and 20. Subfraction 5-2, with eluent of 35% (methanol-water), had retention times of 29.3 min, 24.5 min, 35.6 min, 36.9 min, and 27.3 min, respectively. Recrystallization from cyclohexane yielded compounds 4, 5, 21, 22, and 23. Subfraction 5-6, with eluent of 36% (methanol-water), had retention times of 23.3 min, 25.6 min, 26.9 min, and 27.3 min, respectively. The peaks at 0.5 min, 30.6 min, and 33.3 min were recrystallized from cyclohexane to compounds 6, 7, 8, and 18; the peaks of subfractions 5-7 with retention times of 18.2 min, 24.4 min, 26.9 min, and 22.3 min, respectively, with eluent of 42% (methanol-water), were recrystallized from cyclohexane to compounds 9, 11, 12, and 10; the peaks of subfractions 5-8 with retention times of 15.2 min, 17.5 min, 19.6 min, 23.3 min, and 26.3 min, respectively, with eluent of 18% (acetonitrile-water), were recrystallized from cyclohexane to compounds 13, 14, 15, 16, and 17; and the peaks of subfractions 5-12 with retention times of 14.3 min and 18.3 min, respectively, with eluent of 25% (acetonitrile-water), were recrystallized from cyclohexane to compounds 24 and 25.

[0076] Example 9: Structural identification of chlorogentisyl alcohol and gabosine derivatives

[0077] The purity of the obtained compounds was determined by HPLC. Samples with a purity greater than 98% were structurally identified using mass spectrometry and nuclear magnetic resonance (NMR). NMR was performed using a Bruker AVANCE DRX-600 NMR spectrometer with TMS as an internal standard. High-resolution mass spectrometry (FTIRMS) was performed using a Bruker Apex Spectrometer. Electrospray ionization mass spectrometry (ESI-MS) was performed using a Bruker Esquire 3000. plus Spectrometer determination. The structural formulas of the above 25 compounds are as follows: Figure 1 As shown, the structural identification of the gabosine derivative is as follows:

[0078] Based on the one-dimensional NMR and mass spectrometry analysis results of compound 8 (see Table 1), the molecular formula of compound 8 is C8. 13 H 11 ClO5 has an unsaturation degree of 8. (Comparison of compounds...) 1 H NMR and 13 12C NMR data revealed that compound 8 is very similar to compound 5, both containing an α-pyranone structure. Two-dimensional NMR analysis confirmed that compound 8 is a new compound. The structure is shown below. Figure 1 As shown:

[0079] Table 1. NMR data of compound 8

[0080]

[0081]

[0082] Based on the one-dimensional NMR and mass spectrometry analysis results of compound 19 (see Table 2), the molecular formula of compound 19 is C7H9ClO4. Through database and literature review, compound 19 was identified as the known compound 5-dehydroxy-5S-chlorogabosine C, with the structure shown below. Figure 1 As shown:

[0083] Table 2. NMR data of compound 19

[0084]

[0085] Based on the one-dimensional NMR and mass spectrometry analysis results of compound 20 (see Table 3), the molecular formula of compound 20 is C8H. 11ClO4, with an unsaturation degree of 3. The NMR data of this compound are very similar to those of compound 19, except for the presence of an additional methoxy group. Two-dimensional NMR analysis identified compound 20 as a new compound. Its structure is as follows: Figure 1 As shown:

[0086] Table 3. NMR data of compound 20

[0087]

[0088] Based on the one-dimensional NMR and mass spectrometry analysis results of compound 21 (see Table 4), the molecular formula of compound 21 is C8H. 11 ClO4, with an unsaturation degree of 3. The NMR data of this compound are very similar to those of compound 19, with only the addition of a butyl signal. Two-dimensional NMR analysis identified compound 21 as a new compound. Its structure is as follows: Figure 1 As shown:

[0089] Table 4. NMR data of compound 21

[0090]

[0091] Based on the one-dimensional NMR and mass spectrometry analysis results of compound 22 (see Table 5), the molecular formula of compound 22 is C8H. 11 ClO4, with an unsaturation degree of 3. The NMR data of this compound are very similar to those of compound 21, with only slight changes in the C and H signals of the butyl substituent. Two-dimensional NMR analysis identified compound 22 as a new compound. Its structure is as follows: Figure 1 As shown:

[0092] Table 5. NMR data of compound 22

[0093]

[0094]

[0095] Based on the one-dimensional NMR and mass spectrometry analysis results of compound 23 (see Table 6), the molecular formula of the compound is C23. 15 H 16 O7. Through database and literature review, compound 23 was identified as the known compound 3,4-dihydroxy-5-(11-methylsalicyloxy)-7-hydroxymethyl-2-cyclohexenl-one. Its structure is as follows: Figure 1 As shown:

[0096] Table 6. NMR data of compound 23

[0097]

[0098] Based on the one-dimensional NMR and mass spectrometry analysis results of compound 24 (see Table 7), the molecular formula of compound 24 is C2. 13 H 14 O8, with an unsaturation degree of 7. The NMR data of this compound are very similar to those of compound 19, both belonging to the gabosine series. Two-dimensional NMR analysis identified compound 24 as a new compound. Its structure is as follows: Figure 1 As shown:

[0099] Table 7. NMR data of compound 24

[0100]

[0101] Based on the one-dimensional NMR and mass spectrometry analysis results of compound 25 (see Table 8), the molecular formula of compound 25 is C2. 19 H 18 O9, with an unsaturation degree of 11. The NMR data of this compound are very similar to those of compound 24, except for one additional α-pyranone signal. Two-dimensional NMR analysis identified compound 25 as a new compound. Its structure is as follows: Figure 1 As shown:

[0102] Table 8. NMR data of compound 25

[0103]

[0104]

[0105] Example 10: Analysis of the anti-GUS activity of gabosine derivatives

[0106] (1) Solution preparation

[0107] Sample preparation: Dissolve 1.0 mg of D-glucono-1,4-lactone (DSL) in DMSO to prepare a 10.0 mM sample and reference solution.

[0108] Preparation of PNPG substrate solution: Accurately weigh 3.0 mg of PNPG and dissolve it in PBS buffer (pH 7.4) to a concentration of 2.5 mM;

[0109] Preparation of 10% DMSO solution: Accurately measure 10.0 μL of DMSO and add 90.0 μL of PBS buffer (pH 7.4) to prepare a 10% DMSO solution;

[0110] Preparation of liquid sample stock solution: Accurately measure 100.0 μL of the above sample, add DMSO and mix to prepare a 10.0 mM sample solution (final concentration: 0.1 mM).

[0111] (2) Preliminary screening of GUS inhibitors

[0112] GUS inhibitors were screened using DMSO as a blank control and DSL as a positive control. The blank control consisted of a 100.0 μL mixture containing 10.0 μL GUS (final concentration: 2.0 μg / mL), 79.0 μL PBS buffer (pH 7.4), 1.0 μL 10% DMSO solution (final concentration: 100.0 μM), and 10.0 μL PNPG (final concentration: 250.0 μM).

[0113] Inhibitor and positive control: The total volume of the mixed system was 100.0 μL, containing 10.0 μL of Escherichia coli β-glucuronidase (final concentration: 2.0 μg / mL), 70.0 μL of PBS buffer (pH 7.4), 10.0 μL of inhibitor, DSL stock solution (final concentration: 100.0 μM), and 10.0 μL of PNPG (final concentration: 250.0 μM).

[0114] After sample addition, immediately measure the absorbance at 405 nm using a microplate reader. Incubate at 37℃ for 30 min, then repeat the measurement. Perform three parallel measurements and calculate the relative activity. Relative activity is the percentage of PNP content in the presence of inhibitors or DSL within 30 min compared to the blank control.

[0115] (3) GUS inhibitor IC 50 Value determination

[0116] The GUS inhibitors with strong inhibitory effects screened in (2) were subjected to IC50. 50 Value determination.

[0117] The reaction conditions were as follows: the total volume of the mixed system was 100.0 μL, containing 10.0 μL GUS (final concentration: 2.0 μg / mL), 70.0 μL PBS buffer (pH 7.4), 10.0 μL PNPG (final concentration: 250.0 μM), and 10.0 μL inhibitor solution (final concentration: 1.0 nM-100.0 μM).

[0118] After sample addition, the absorbance was immediately measured at 405 nm using a microplate reader. The sample was incubated at 37°C for 30 min, and the measurement was repeated in triplicate. The relative activities were calculated. Finally, Prism 6.0 (GraphPad Software, LaJolla, CA) was used to plot the concentration-response curve to calculate the IC50. 50 value.

[0119] (4) Inhibition kinetics of GUS inhibitors

[0120] The inhibition kinetics (competitive, non-competitive, and mixed) of the selected GUS inhibitors in (3) were investigated to explore the interaction between the substrate, inhibitor, and GUS. To determine the inhibition kinetics of the inhibitors, various concentrations of PNPG and various concentrations of inhibitors were used to determine the corresponding reaction rates, and the results were evaluated using the intersection points of Lineweaver-Burk plots.

[0121] (5) Experimental Results

[0122] Table 9. GUS inhibition rate and IC50 of the compounds 50 value

[0123] a ND indicates no detection

[0124] The initial screening data of the compounds in the table above show that compounds 19, 20, and 21 have an enzyme inhibition rate of 85% or higher at a concentration of 10 μM. Further screening of these compounds shows that their half-maximal inhibitory concentrations (IC50) range from 1.01 μM to 4.61 μM, which are significantly inhibitory compared to the positive control drug DSL. Their Ki inhibition constants range from 0.67 μM to 7.32 μM. In the enzyme-substrate binding type analysis, except for compound 20, which is a non-competitive inhibitor, the others are all mixed inhibitors.

[0125] Example 11: Analysis of the antitumor activity of gabosine derivatives

[0126] (1) Reagent preparation

[0127] 0.4% SRB solution: Weigh 0.8g SRB, dissolve in 200mL 1% acetic acid, and store at room temperature.

[0128] 50% TCA solution: Weigh 50g of TCA, add water to make up to 100mL, and store at 4℃.

[0129] 10mM Tris-base solution: Weigh 0.6057g Tris-base, add water to a final volume of 500mL, pH 10.5, and store at 4℃.

[0130] (2) Experimental apparatus

[0131] CO2 incubator (Thermo), mini shaker (Kylin-Bell Lab instruments), microplate reader (MD, M5 model).

[0132] (3) Method

[0133] Logarithmic growth phase cells were selected, and after trypsin digestion, the cell concentration was adjusted to 2 × 10⁴ cells / mL using RPMI 1640 medium containing 10% fetal bovine serum. 190 μl of cell suspension was seeded into each well of a 96-well plate and cultured at 37°C with 5% CO₂ for 24 h. For drug treatment wells, 10 μl of sample solution (final concentration: 5 μg / mL for the compound; 50 μg / mL for the mixture) was added. For positive control wells, 5-FU was added to a final concentration of 5 μg / mL. Control wells were added to medium containing an equal volume of solvent and cultured at 37°C with 5% CO₂ for 3 days. The medium was discarded, and 100 μl of 50% TCA-fixed cells (pre-chilled at 4°C) was gently added. The cells were incubated for 5 min, then transferred to 4°C for 1 h. The fixative was discarded, and the cells were washed 5 times with distilled water to remove TCA and air-dried for 1 h. 80 μl of 0.4% SRB solution was added to each well, and staining was performed at room temperature for 30 min. Discard the staining solution, wash five times with 1% acetic acid to thoroughly remove unbound SRB, and air dry. Dissolve in 150 μL of 10 mM Tris-base (pH 10.5), and shake on a microplate reader for 5 min. Measure the OD510 nm value using an M5 microplate reader.

[0134] (4) Calculation of results

[0135] Tumor cell survival rate (%) = 100% - (OD) 对照 -OD 药物 ) / (OD 对照 -OD 空白 )×100%

[0136] (5) Experimental Results

[0137] Table 10. Survival rates of the compounds in Namalwa and U266 cells.

[0138]

[0139] a ND indicates no detection

[0140] The cytotoxicity of compounds 19, 20, and 21 at a concentration of 20 μM was further determined using Namalwa and U266 tumor cell lines. The data showed that the compounds had low cytotoxicity to cells.

Claims

1. A gabosine derivative, characterized in that, The gabosine derivative has a structure as shown in any one of formula (8), (20), (24), (8)、 (20)、 (24) 。 2. The method for preparing the gabosine derivative as described in claim 1, characterized in that, The method comprises the following steps: (1) the fungi of the genus Claviporales with the preservation number CGMCC 3.20238 Epicoccum sorghinum ) GST-5 after activation is inoculated in a liquid culture medium for fermentation culture; (2) After the fermentation culture is completed, mycelia and a fermentation liquor are separated, the mycelia are extracted by adding an organic solvent, an extraction liquor is separated, the extraction liquor is concentrated, is suspended in distilled water to obtain a water suspension, and the water suspension is extracted by ethyl acetate or n-butanol to obtain an extraction liquor A; the fermentation liquor is stirred with diatomite, and is extracted by refluxing with ethyl acetate or n-butanol to obtain an extraction liquor B; (3) The extraction liquor A or the extraction liquor B is concentrated and then subjected to normal-phase silica gel column chromatography separation, gradient elution is performed with dichloromethane / methanol mixtures with volume ratios of 100:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1 and 10:1 in sequence, a fraction eluted by the dichloromethane / methanol mixture with a volume ratio of 10:1 is collected, and reverse-phase silica gel column chromatography and high-performance liquid chromatography separation are performed to obtain the derivative.

3. The production method according to claim 2, wherein In step (1), the liquid culture medium comprises the following raw materials in a volume of 1 L: starch 1-5 g, bran 10-20 g, yeast extract 3-15 g, KH2PO4 1-8 g, MgSO4·7H2O 0.1-0.8 g, and the rest is water; Alternatively, the liquid culture medium comprises the following raw materials in a volume of 1 L: potato 200-600 g, protein peptone 2-10 g, yeast extract 1-5 g, glucose 5-20 g, and the rest is water; and the initial pH value of the culture solution is 6.0-7.0; Alternatively, the liquid culture medium comprises the following raw materials in a volume of 1 L: sucrose 10-40 g, corn powder 5-20 g, NaNO3 1-4 g, yeast extract 1-4 g, KH2PO4 0.2-0.8 g, MgSO4·7H2O 0.2-1 g, KCl 0.2-1 g, FeSO4 0.001-0.005 g, and the rest is water; Alternatively, the liquid culture medium comprises the following raw materials in a volume of 1 L: malt extract powder 20-30 g, glucose 15-20 g, casein peptone 1-2 g, and the rest is water.

4. The production method according to claim 2, wherein In step (1), the fermentation culture is performed at 20-30 °C for 10-40 days.

5. The production method according to claim 2, wherein In step (3), the fraction is subjected to reverse phase silica gel column chromatography, gradient elution is carried out with methanol / water mixture in the volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, respectively, 5 times for each gradient elution, and numbered 1~45 in turn, the fractions numbered 1~3 are combined into sub-fraction 1, fractions 4~5 are combined into sub-fraction 2, fractions 14~17 are combined into sub-fraction 6, and fractions 38~39 are combined into sub-fraction 12; then separated by high performance liquid chromatography, the peak with retention time of 25.6 minutes in sub-fraction 1 eluted with methanol / water mixture in the volume ratio of 28% is compound 20 with structural formula as shown in formula (20) R1=Cl, R2=CH3; the peaks with retention time of 35.6 minutes and 36.9 minutes in sub-fraction 2 eluted with methanol / water mixture in the volume ratio of 35% are compounds 21 and 22 with structural formula as shown in formula (20) R1=oxybutyl, R2=H and formula (20) R1=OH, R2=oxybutyl, respectively; the peak with retention time of 30.6 minutes in sub-fraction 6 eluted with methanol / water mixture in the volume ratio of 36% is compound 8 with structural formula as shown in formula (8); the peak with retention time of 14.3 minutes in sub-fraction 12 eluted with acetonitrile / water mixture in the volume ratio of 25% is compound 24 with structural formula as shown in formula (24).

6. Use of the gabosine derivative of claim 1 in the preparation of a medicament for inhibiting β-glucuronidase.

7. Use according to claim 6, wherein The β-glucuronidase is intestinal bacterial β-glucuronidase.

8. Use of the gabosine derivative of claim 1 in the preparation of a medicament for relieving gastrointestinal toxicity caused by irinotecan and indomethacin.

9. Use of the compound with structural formula as shown in formula (19) and (23) in the preparation of a medicament for inhibiting β-glucuronidase, (19)、 (23)。

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