Chloro spiro-guaiacol derivatives, process for their preparation and use

Abstract

The present divisional application discloses a kind of chlorinated spirogenin derivatives, preparation method and application thereof, and belongs to the technical field of marine fungi active ingredient analysis.The chlorinated spirogenin derivative is the derivative with the hybrid skeleton of chlorogentisyl alcohol and gabosine, the present application utilizes the polarity difference of chlorogentisyl alcohol and gabosine derivative to obtain a kind of chlorinated spirogenin derivative with novel structure from marine fungi Epicoccum sorghinum A chlorinated spirogenin derivative with novel structure is isolated from the fermentation culture of GST-5, and through in vitro anti-beta-glucuronidase test, it is shown that the chlorinated spirogenin derivative provided by the present application has good beta-glucuronidase inhibitory activity and low cytotoxicity, and has development prospect in the preparation of anti-beta-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 the analysis of active ingredients from fungi of the genus *Plasmodium* (…). Epicoccum sorghinum Derivatives with a heterozygous backbone of gabosine and chlorogentisyl alcohol obtained from GST-5 and their applications. 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 in plants and humans. 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] Currently, regarding fungi of the genus *Plasmodium* ( Epicoccum There is limited research on the chemical composition of dandelion endophytes. Hao Baocong et al. obtained this information from an endophytic fungus in a dandelion. Epicoccum sorghinum (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, (4R,5R,6S)-4,5-dihydroxy-6-(6′-methylsalicylic acidoxy)-2-hydroxymethyl-2-cyclohexen-1-one, (-)-gabosine E, theobroxide, 3-chlorogentiol and 3-hydroxybenzyl alcohol were isolated from dandelion (Hao Baocong, Zheng Yaoyao, Chen Xu, Chen Qiuxia, Ji Ruonan, Chen Min, Acta Pharmaceutica Sinica, An Endophytic Fungus of Taraxacum mongolicum). Epicoccum sorghinum Study of 1-2 secondary metabolites, 2022, 57(07)).

[0007] Yuan Chao et al. isolated from lichen Leptogium masiaticum An endophytic fungus, *Pseudomonas nigricans* Epicoccum nigrumThe chemical composition of 14one was studied and identified as one alkaloid compound, 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-methylisobenzofuran-1(3H)-one, 3-methoxyepicoccone B, 2,3,4-trihydroxy-6-(hydroxymethyl)-5-methylbenzyl-alcohol, isoochracinic acid; and three epicocolide compounds, epicocconigrones A, epicocolide B, and epicocconigrones. B, among which alkaloids fusaricide and epicolide compounds have strong antibacterial activity, with fusaricide showing good in vitro inhibitory activity against lung cancer cells M109 (Yuan Chao, Guo Yuhua, Zhang Yingbo, Hu Xuan, Wang Dan, Yu Fulai, Li Gang, China Journal of Traditional Chinese Medicine, A plant endophytic fungus). Epicoccum nigrum Research on secondary metabolites of 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 obtain marine parasitic fungi (Plasmodium spp.) Epicoccum Natural active substances with medicinal value are extracted from them.

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

[0011] This invention is derived from fungi of the genus *Plasmodium* ( Epicoccum sorghinumTwenty-five compounds were isolated from the fermentation products of GST-5. Structural identification revealed that these 25 compounds were 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. Their specific structural formulas are shown below. Figure 1 As shown, they are numbered 1 to 25 respectively.

[0012] The fungi of the genus Acetococcus ( Epicoccum sorghinum GST-5 is a large marine plant isolated from hydrothermal vent sediments on the seabed of Guishan Island, Taiwan Province, China. Its accession number is 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 chlorospirocyclic gentiolic acid derivatives, namely derivatives with a chlorogentisyl alcohol and gabosine heterogeneous skeleton, with structural formulas as shown in any one of formulas (9), (16), and (17), corresponding to compounds 9, 10, 11, 12, 16, and 17, respectively.

[0014] (9) (16)

[0015] (17).

[0016] This invention also provides a method for obtaining fungi from the genus *Plasmodium* (… Epicoccum sorghinum The method for separating and extracting the above-mentioned new compounds from the fermentation products of GST-5 is described, but the preparation method of the above-mentioned compounds in this invention is not limited to this.

[0017] A method for extracting the derivative having a chlorogentisyl alcohol and gabosine heterozygous backbone from fermentation products includes the following steps:

[0018] (1) Fungi of the genus Acropora ( Epicoccum sorghinum After activation, GST-5 was inoculated into liquid culture medium for fermentation.

[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 fungi of the genus Acetococcus ( Epicoccum sorghinum GST-5 was used for fermentation culture.

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

[0025] Alternatively, the liquid culture medium, per 1L volume, comprises the following ingredients: 20-30 g malt extract, 15-20 g glucose, 1-2 g 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 chlorogentisyl alcohol and gabosine hybrid skeleton derivative is the highest.

[0028] In step (2), mycelium and fermentation broth are separated and obtained. The derivative with chlorogentisyl alcohol and gabosine hybrid skeleton can be extracted and separated from both mycelium and fermentation broth.

[0029] In obtaining derivatives with a heterozygous backbone of chlorogentisyl alcohol and gabosine from 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, high-purity derivatives with a heterogeneous skeleton of chlorogentisyl alcohol and gabosine can be obtained.

[0031] Preferably, the fractions are subjected to reversed-phase silica gel column chromatography, with gradient elution sequentially 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 being eluted 5 times. The fractions are numbered sequentially from 1 to 45. Fractions numbered 1 to 3 are combined into subfraction 1, and fractions 4 and 5 are combined into subfraction 2. Fractions 14-17 were combined into subfraction 6, fractions 18-25 into subfraction 7, fractions 26-28 into subfraction 8, and fractions 38-39 into subfraction 12. These fractions were then separated by high-performance liquid chromatography (HPLC). Subfraction 7, eluted with a 42% methanol / water mixture, showed peaks with retention times of 18.2 min, 24.4 min, 26.9 min, and 22.3 min, corresponding to compounds 9, 11, 12, and 10, as shown in formula (9). Subfraction 8, eluted with an 18% acetonitrile / water mixture, showed peaks with retention times of 23.3 min and 26.3 min, corresponding to compounds 16 and 17, with structural formulas as shown in formulas (16) and (17), respectively.

[0032] This invention demonstrates that the above method can be used to extract GST-5 (a fungus of the genus Acetococcus) Epicoccum sorghinumThe chlorogentisyl alcohol and gabosine hybrid backbone derivative isolated from GST-5 fermentation culture 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 compound 12, the other compounds have low cytotoxicity to human cells. Therefore, this invention provides the application of the aforementioned chlorogentisyl alcohol and gabosine hybrid backbone derivative in the preparation of anti-β-glucuronidase drugs.

[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 chlorogentisyl alcohol and gabosine heterozygous derivative in the preparation of drugs that alleviate gastrointestinal toxicity caused by irinotecan and indomethacin.

[0035] Furthermore, this invention has found that compound 12 has a significant inhibitory effect on Namalwa cell line and U266 cell line. Therefore, this invention provides the application of a compound with the structural formula shown in formula (12) in the preparation of antitumor drugs. Specifically, the tumor is myeloma or lymphoma.

[0036] (12).

[0037] The drug is formulated with the derivative of the present invention having a chlorogentisyl alcohol and gabosine heterozygous skeleton as the main active ingredient, and with the addition of pharmaceutically acceptable excipients. It can be formulated according to pharmaceutical preparation methods described herein. 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 beneficial effects of this invention are as follows:

[0039] (1) This invention utilizes the polarity difference between chlorogentisyl alcohol and gabosine heterozygous skeleton derivatives to extract and isolate a novel derivative with a chlorogentisyl alcohol and gabosine heterozygous skeleton 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.

[0040] (2) In vitro anti-GUS enzyme assays showed that the derivatives with a chlorogentisyl alcohol and gabosine heterozygous backbone provided by this invention have good anti-GUS enzyme activity, with compound 17 exhibiting the strongest activity at an IC50 value of 17. 50 The value is 0.24 M. Further cytotoxicity assays showed that the carbon sugar compounds provided by this invention have low cytotoxicity and show good development potential in the preparation of drugs against GUS enzymes.

[0041] (3) Through in vitro tumor cell experiments, the tumor cell lines used were Namalwa cells and U266 cells. The survival rate of compound 12 against Namalwa cells was -0.33%, which shows good development prospects in the preparation of anti-tumor drugs. 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 9 1 H NMR data (in DMSO- d6 (600 MHz) 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 9 1 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data.

[0045] Figure 4 For compound 101 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data.

[0046] Figure 5 For compound 11 1 H NMR data (in CD3OD, 600 MHz) 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 12 1 H NMR data (in CD3OD, 600 MHz) and 13 C NMR data (in CD3OD, 150MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data.

[0048] Figure 7 For compound 16 1 H NMR data (in DMSO- d6 (600 MHz) and 13 C NMR data (in DMSO- d6 , 150 MHz), where (A) is 1 H NMR data, (B) is 13 C NMR data.

[0049] Figure 8 For compound 17 1 H NMR data (in DMSO- d6 (600 MHz) 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

[0050] 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.

[0051] 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.

[0052] Example 1: Fungal Isolation

[0053] Large marine plants were collected from seabed sediments at the hydrothermal vent of Guishan Island, Taiwan Province, China. 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 min, and the suspension after removing the algae was centrifuged for 20 min (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.

[0054] Example 2: Identification of *Plasmodium* fungi

[0055] 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.

[0056] Based on the morphological characteristics and 18S rDNA sequence analysis, the strain was identified as belonging to the genus *Plasmodium*. Epicoccum sp.), and named it GST-5, a fungus of the genus Acetococcus. Epicoccum sorghinum GST-5 was deposited on July 12, 2021, at the China General Microbiological Culture Collection Center (CGMCC), identified as viable. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, accession number: CGMCC 3.20238.

[0057] Example 3: Fermentation culture of *Plasmodium* fungi

[0058] 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.

[0059] The culture medium formula is as follows: starch 3g, wheat bran 14g, yeast extract 6g, KH2PO4 5g, MgSO4·7H2O 0.4g, and water 1000mL.

[0060] Example 4: Fermentation culture of *Plasmodium* fungi

[0061] 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.

[0062] 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.

[0063] Example 5: Fermentation culture of *Plasmodium* fungi

[0064] 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.

[0065] 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.

[0066] Example 6: Fermentation culture of *Plasmodium* fungi

[0067] 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.

[0068] The culture medium formula is as follows: 25g malt extract powder, 15g glucose, 1.5g casein peptone, and 1000mL water.

[0069] Example 7: Preparation of chlorogentisyl alcohol and gabosine derivatives

[0070] After fermentation culture of *Plasmodium* fungi GST-5, 5 L of fermentation broth was taken, centrifuged, and the precipitate was collected to obtain mycelium. The mycelium was soaked in methanol for one week, and the soaking solution was concentrated and 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, Ø 12 mm). Gradient elution was performed with dichloromethane / methanol mixtures and methanol 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, 300 mL each time. mL; TLC detection of the fraction; collection of the fraction eluted from a dichloromethane / methanol mixture with a volume ratio of 35:1 to 10:1 and recrystallization with methanol.

[0071] Example 8: Preparation of chlorogentisyl alcohol and gabosine derivatives

[0072] After fermentation culture of *Plasmodium* fungi, 5 L of fermentation broth was collected, centrifuged, and the supernatant was obtained 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 L 50 mm, Ø 12). 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.

[0073] 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 into sub-component 5-5, 14-17 are combined into sub-component 5-6, 18-25 are combined into sub-component 5-7, 26-28 are combined into sub-component 5-8, 29-30 are combined into sub-component 5-9, 31-33 are combined into sub-component 5-10, 34-37 are combined into sub-component 5-11, 38-39 are combined into sub-component 5-12, 40-41 are combined into sub-component 5-13, 42-43 are combined into sub-component 5-14, and 44-45 are combined into sub-component 5-15.

[0074] 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. These peaks were recrystallized from cyclohexane to reconstitute 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. These peaks were recrystallized from cyclohexane to reconstitute 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.

[0075] Example 9: Structural identification of chlorogentisyl alcohol and gabosine derivatives

[0076] The purity of the synthesized compounds was determined by HPLC. Samples with a purity greater than 98% were subjected to structural identification 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 structures of derivatives with a chlorogentisyl alcohol and gabosine hybrid skeleton are identified as follows:

[0077] Based on the one-dimensional NMR and mass spectrometry analysis results of compound 9 (see Table 1), the molecular formula of compound 9 is C9. 14 H 12 Cl₂O₅ has an unsaturation degree of 8. (Comparison with other compounds...) 1 H NMR and 13 1 / 2C NMR data revealed that compound 9 is very similar to compound 1, both containing a 2-chloro-6-(hydroxymethyl)benzene-1,4-diol structure. Two-dimensional NMR analysis confirmed that compound 9 is a new compound. The structure is shown below. Figure 1 As shown:

[0078] Table 1. NMR data of compound 9

[0079]

[0080] Based on the one-dimensional NMR and mass spectrometry analysis results of compound 10 (see Table 2), the molecular formula of compound 10 is C10. 15 H 14 Cl₂O₅ has an unsaturation degree of 8. (Comparison with other compounds...) 1 H NMR and 13 C10 NMR data revealed that compound 10 is very similar to compound 9, with only one additional methoxy group signal. Two-dimensional NMR analysis confirmed that compound 10 is a new compound. Its structure is shown below. Figure 1 As shown:

[0081] Table 2. NMR data of compound 10

[0082]

[0083] Based on the results of one-dimensional NMR and mass spectrometry analysis of Compound 11 (see Table 3), it can be seen that the molecular formula of Compound 9 is C 14 H 12 Cl2O5, and the degree of unsaturation is 8. By comparing the 1 H NMR and 13 C NMR data of the compounds, it was found that Compound 11 is very similar to Compound 9, with only minor changes in the chiral carbon region. Through the results of two-dimensional NMR analysis, Compound 11 was identified as a new compound. The structure is as Figure 1 shown:

[0084] Table 3. NMR data of Compound 11

[0085]

[0086] Based on the results of one-dimensional NMR and mass spectrometry analysis of Compound 12 (see Table 4), it can be seen that the molecular formula of Compound 12 is C 14 H 12 Cl2O5, and the degree of unsaturation is 8. By comparing the 1 H NMR and 13 C NMR data of the compounds, it was found that Compound 12 is very similar to Compound 9, with only minor changes in the chiral carbon region. Through the results of two-dimensional NMR analysis, Compound 12 was identified as a new compound. The structure is as Figure 1 shown:

[0087] Table 4. NMR data of Compound 12

[0088]

[0089] Based on the results of one-dimensional NMR and mass spectrometry analysis of Compound 16 (see Table 5), it can be seen that the molecular formula of Compound 16 is C 21 H 24 Cl2O7, and the degree of unsaturation is 9. Combining the 1 H and 13 C-NMR data, it was found that this compound is similar to Compound 10, except that its methoxy group is replaced by an ampelomin group. Through the results of two-dimensional NMR analysis, Compound 16 was identified as a new compound. The structure is as Figure 1 shown:

[0090] Table 5. NMR data of Compound 16

[0091]

[0092] Based on the results of one-dimensional NMR and mass spectrometry analysis of Compound 17 (see Table 6), it can be seen that the molecular formula of Compound 17 is C21 H 24 Cl2O7 has an unsaturation of 9. Combining 1 H and 13 C-NMR data, it was found that this compound is similar to Compound 9, except that the chlorine substituent on its gabosine group is replaced by a 2-chloro-6-(hydroxymethyl)benzene-1,4-diol group. Through the results of two-dimensional NMR analysis, Compound 17 was identified as a new compound. The structure is as Figure 1 shown:

[0093] Table 6. NMR data of Compound 17

[0094]

[0095] Example 10: Analysis of the anti-GUS activities of chlorogentisyl alcohol and gabosine derivatives

[0096] (1) Preparation of solutions

[0097] Preparation of samples: 1.0 mg of the sample and the reference substance D-glucaric acid-1,4-lactone (DSL) were dissolved in DMSO to prepare 10.0 mM sample and reference substance solutions;

[0098] Preparation of the substrate PNPG solution: 3.0 mg of PNPG was accurately weighed and dissolved in PBS buffer (pH 7.4) to make its concentration 2.5 mM;

[0099] Preparation of 10% DMSO solution: 10.0 μL of DMSO was accurately measured and added to 90.0 μL of PBS buffer (pH 7.4) to prepare 10% DMSO solution;

[0100] Preparation of the liquid sample stock solution: 100.0 μL of the above sample was accurately measured, mixed with DMSO, and prepared into a 10.0 mM sample solution (final concentration: 0.1 mM).

[0101] (2) Preliminary screening of GUS inhibitors

[0102] Using DMSO as the blank control and DSL as the positive control, screening of GUS inhibitors was carried out. Among them, the blank control: the total volume of the mixed system was 100.0 μL, containing 10.0 μL of GUS (final concentration: 2.0 μg / mL), 79.0 μL of PBS buffer (pH 7.4), 1.0 μL of 10% DMSO solution (final concentration: 100.0 μM), and 10.0 μL of PNPG (final concentration: 250.0 μM).

[0103] 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).

[0104] After sample addition, immediately measure the absorbance at 405 nm using a microplate reader, incubate at 37 ℃ for 30 min, and repeat the measurement. Perform three parallel measurements and calculate the relative activity. Relative activity is the percentage of PNP content in the control group within 30 min with the addition of inhibitors or DSL.

[0105] (3) GUS inhibitor IC 50 Value determination

[0106] The GUS inhibitors with strong inhibitory effects selected in (2) were subjected to IC50. 50 Value determination.

[0107] 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).

[0108] 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, La Jolla, CA) was used to plot the concentration-response curve to calculate the IC50. 50 value.

[0109] (4) Inhibitory kinetics of GUS inhibitors

[0110] The inhibition kinetics (competitive, non-competitive, and mixed) of the selected GUS inhibitors in (3) were investigated to explore the interactions between the substrate, inhibitor, and GUS. To determine the inhibition kinetics of the inhibitors, various concentrations of PNPG and various concentrations of the inhibitor were used to determine the corresponding reaction rates, and the results were evaluated using the intersection points of Lineweaver-Burk plots.

[0111] (5) Experimental results

[0112] Table 7. GUS inhibition rate and IC50 of the compounds 50 value

[0113]

[0114] a ND indicates not detected

[0115] The initial screening data of the compounds in the table above show that compounds 9, 10, 12, and 17 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 0.24 μM to 2.83 μM, which are significantly inhibitory compared to the positive control drug DSL. Their Ki inhibition constants range from 1.09 μM to 4.43 μM, and all of them exhibit mixed inhibition in the enzyme-substrate binding type analysis.

[0116] Example 11: Analysis of the antitumor activity of chlorogentisyl alcohol and gabosine derivatives

[0117] (1) Reagent preparation

[0118] 0.4% SRB solution: Weigh 0.8g SRB, dissolve in 200mL 1% acetic acid, and store at room temperature.

[0119] 50% TCA solution: Weigh 50g of TCA, add water to make up to 100mL, and store at 4℃.

[0120] 10mM Tris-base solution: Weigh 0.6057g Tris-base, add water to a final volume of 500mL, pH 10.5, and store at 4℃.

[0121] (2) Experimental apparatus

[0122] CO2 incubator (Thermo), mini shaker (Kylin-Bell Lab instruments), microplate reader (MD, M5 model).

[0123] (3) Method

[0124] 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 (pre-chilled at 4°C) was gently added to fix the cells. The cells were incubated for 5 min, then transferred to 4°C and incubated 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 micro-shaker for 5 min. Measure the OD value at 510 nm using an M5 microplate reader.

[0125] (4) Calculation of results

[0126] Tumor cell survival rate (%) = 100% - (OD) 对照 -OD 药物 ) / (OD 对照 -OD 空白 ) × 100%

[0127] (5) Experimental Results

[0128] Table 8. Survival rates of the compounds in Namalwa and U266 cells

[0129]

[0130] a ND indicates not detected

[0131] The cytotoxicity of compounds 9, 10, 12, and 17 at a concentration of 20 µM was further determined using Namalwa and U266 tumor cell lines. Data showed that, except for compound 12, the other compounds exhibited low cytotoxicity. The survival rate of compound 12 against Namalwa cell lines was -0.33%, close to the 11.28% survival rate of the positive control drug 5-Fu against Namalwa cell lines, and the 2.59% survival rate of the positive control drug As2O3 against U266 cell lines. This indicates that compound 12 possesses dual antitumor efficacy while inhibiting GUS.

Claims

1. A chlorospirocyclic gentianol derivative, characterized in that, The chlorospirocyclic gentiolic acid derivative is a derivative having a chlorogentisyl alcohol and gabosine heterozygous skeleton, with the structural formula shown in any one of formulas (9), (16), and (17). (9)、 (16)、 (17)。 2. The method for preparing the chlorospirocyclic gentianol derivative as described in claim 1, characterized in that, Includes the following steps: (1) The fungus of the genus *Plasmodium* with accession number CGMCC 3.20238 ( Epicoccum sorghinum After activation, GST-5 was inoculated into liquid culture medium for fermentation. (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 then extracted by reflux with ethyl acetate or n-butanol to obtain extract B. (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.

3. The preparation method according to claim 2, characterized in that, In step (1), the liquid culture medium comprises the following raw materials in 1L volume: starch 1-5g, wheat bran 10-20g, yeast extract 3-15g, KH2PO4 1-8g, MgSO4•7H2O 0.1-0.8g, with the remainder being water; 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. Alternatively, the liquid culture medium, per 1L volume, comprises the following ingredients: 10-40 g sucrose, 5-20 g corn flour, 1-4 g NaNO3, 1-4 g yeast extract, 0.2-0.8 g KH2PO4, 0.2-1 g MgSO4•7H2O, 0.2-1 g KCl, 0.001-0.005 g FeSO4, with the remainder being water; Alternatively, the liquid culture medium, per 1L volume, comprises the following ingredients: 20-30 g malt extract, 15-20 g glucose, 1-2 g casein peptone, with the remainder being water.

4. The preparation method according to claim 2, characterized in that, In step (1), the fermentation culture conditions are static culture at 20-30℃ for 10-40 days.

5. The preparation method according to claim 2, characterized in that, In step (3), 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, eluting 5 times for each gradient, numbered sequentially from 1 to 45. Fractions 18 to 25 are combined into subfraction 7, and fractions 26 to 28 are combined into subfraction 8. Subfraction 7 is then separated by high-performance liquid chromatography (HPLC) at a volume ratio of 42. When eluted with a 18% methanol / water mixture, the peaks with retention times of 18.2 min, 24.4 min, 26.9 min, and 22.3 min were compounds 9, 11, 12, and 10, with structural formulas as shown in formula (9). When subcomponent 8 was eluted with an 18% acetonitrile / water mixture, the peaks with retention times of 23.3 min and 26.3 min were compounds 16 and 17, with structural formulas as shown in formulas (16) and (17), respectively.

6. The use of the chlorospirocyclic gentioyl alcohol derivative as described in claim 1 in the preparation of a drug for inhibiting β-glucuronidase.

7. The application as described in claim 6, characterized in that, The β-glucuronidase is an intestinal bacterial β-glucuronidase.

8. The use of the chlorospirocyclic gentiolate derivative as described in claim 1 in the preparation of a drug for alleviating gastrointestinal toxicity caused by irinotecan and indomethacin.

9. The use of a compound with the structural formula shown in formula (12) in the preparation of an antitumor drug, characterized in that, The tumor is a lymphoma. (12)。

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