Three-carbon-reduced ganoderma triterpenic acid compound, preparation method thereof and application of compound in prevention and treatment of influenza

Abstract

The invention belongs to the field of medicines, and discloses a three-carbon-reduced ganoderma triterpenic acid compound, a preparation method thereof and application of the compound in prevention and treatment of influenza. The invention relates to a three-carbon-reduced ganoderma triterpenoid acid compound 3, 7, 11-Trioxo-25, 26, 27-trinorlanosta-8-en-24-oic acid, which is shown as a formula I in the specification, and a preparation method of the three-carbon-reduced ganoderma triterpenoid acid compound. Experiments prove that the compound disclosed by the invention has relatively good influenza virus neuraminidase inhibitory activity, and the IC50 of the compound is 5.17 mu M. Researches show that the compound has an application prospect in further development of novel anti-influenza drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, specifically to a novel triterpenic acid compound 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid isolated from Ganoderma lucidum, its preparation method, and its use in anti-influenza virus therapy. Background Technology

[0002] Reishi mushroom was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), listed as a superior herb. It is sweet, neutral, and non-toxic, beneficial to essence and qi, and good for tendons and bones, improving complexion. Since 2000, reishi has been included as a traditional Chinese medicine in the *Pharmacopoeia of the People's Republic of China* (Part I), as the dried fruiting body of the fungus *Ganoderma lucidum* (Leyss. ex Fr.) Karst. or *Ganoderma inense* Zhao, Xu et Zhang, belonging to the Polyporaceae family. In 2023, reishi (the fruiting body of *Ganoderma lucidum* and *Ganoderma inense*) was also included in the list of substances traditionally used as both food and traditional Chinese medicine. The main chemical components of reishi include triterpenes, heteroterpenes, polysaccharides, steroids, alkaloids, and sesquiterpenes. Modern clinical and pharmacological studies suggest that reishi possesses various effects, including immunomodulation, sedation, tranquilization, anti-tumor, antiviral, anti-inflammatory, blood pressure lowering, and liver protection. Currently, the main type of artificially cultivated Ganoderma lucidum is Ganoderma lucidum (Leyss. ex Fr.) Karst.

[0003] Influenza viruses belong to the Orthomyxoviridae family and are enveloped, negative-sense RNA viruses. Based on antigenic characteristics, they can be classified into influenza A, B, C, and D viruses, with influenza A posing the greatest threat to human health. Influenza viruses possess two glycoproteins on their surface: hemagglutinin (HA) and neuraminidase (NA). NA helps release progeny viruses from host cells by cleaving sialic acid on the cell surface. Neuraminidase inhibitors exert their antiviral effect by inhibiting the activity of NA in influenza viruses. Currently, neuraminidase inhibitors approved by the FDA for the treatment of influenza include oseltamivir, zanamivir, and peramivir. The U.S. Food and Drug Administration (FDA) classifies drugs into five categories (A, B, C, D, and X) based on animal studies and clinical experience regarding their teratogenic effects on fetuses. Category C drugs are those for which animal studies have shown harm to the fetus (teratogenicity or embryonic death, etc.), or for which there are no controlled studies in pregnant women, or for which no studies have been conducted on pregnant women and animals. These drugs can only be used after weighing the benefits to the pregnant woman against the risks to the fetus. Currently, because there are no controlled trials in pregnant women, NA inhibitors are all classified as Category C according to the FDA. Ganoderma lucidum is considered a superior herb, non-toxic, suitable for long-term use, and has almost no toxic side effects.

[0004] The novel Ganoderma lucidum triterpene acid (3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid) involved in this study is a new compound, and its inhibitory activity against influenza virus neuraminidase is being discovered for the first time. To date, no patents or literature reports have been found to support this finding.

[0005] The purpose of this invention is to fully develop my country's traditional Chinese medicine resources, to find compounds with unique chemical structures, high activity, and low toxicity and side effects that inhibit influenza virus neuraminidase, in order to provide novel anti-influenza virus drugs for clinical research. Summary of the Invention

[0006] The technical problem solved by this invention is to provide 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid, its preparation method, and its use in anti-influenza virus therapy.

[0007] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0008] The first aspect of the present invention is to provide 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid, a triterpenic acid with a lower C3 content. Its characteristic is that the compound has the following structural formula:

[0009]

[0010] The second aspect of the present invention provides a method for preparing the 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid described in the first aspect:

[0011] 1) The dried fruiting bodies of Ganoderma lucidum were extracted three times by heating and refluxing with 90% ethanol-water. The extracts were combined and concentrated under reduced pressure to obtain an extract, which was then mixed with macroporous resin.

[0012] 2) Macroporous resin chromatography was performed with gradient elution using 30% ethanol-water, 60% ethanol-water, 80% ethanol-water, and 95% ethanol-water to obtain four fractions, namely DK-1-DK-4. DK-3 was further eluted by silica gel column chromatography with a gradient elution of a mixture of petroleum ether and ethyl acetate at volume ratios of 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50, followed by washing the column with ethyl acetate and methanol to obtain 25 fractions, namely Fr.1-Fr.25. Fr.17 was eluted by medium-pressure silica gel column chromatography with a gradient elution of petroleum ether:ethyl acetate (80:20-50:50, v / v), followed by washing the column with ethyl acetate to obtain 14 subfractions, namely Fr.17-1-Fr.17-14. Fr.17-7 was further eluted by C... 18 Reverse-phase column chromatography with gradient elution using methanol:water (50:50-75:25, v / v) as the eluent, followed by column washing with 100% methanol, yielded 25 subfractions, namely Fr.17-7-1-Fr.17-7-25. Fr.17-7-10 was separated by semi-preparative reversed-phase high-performance liquid chromatography with isocratic elution using 40% acetonitrile-water as the eluent to obtain ganoderic acid 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid (number LZ-GO-45).

[0013] LZ-GO-45 is a white amorphous powder. UV(MeOH)λ max (logε)212(3.91),252(3.79)nm. IRν max 1732, 1708, 1692, 1673cm -1The presence of 3 carbonyl groups and 1 carboxyl group is indicated, 1585cm. -1 This is a double bond absorption peak. HR-ESI-MS shows a quasi-molecular ion peak at m / z 441.2646 [MH]. – (calcd.forC 27 H 37 O5,441.2647), its molecule is determined to be C 27 H 38 O5 has an unsaturation degree of 9.

[0014] 1 The 1H-NMR spectrum (Table 1) shows 37 hydrogen signals. There are no hydrogen signals at the low field; all hydrogen signals are at the high field. Six methyl signals are observed in the high field region [δ]. H 1.28(3H,s,H-19), 1.22(3H,s,H-30), 1.13(3H,s,H-28), 1.11(3H,H-29), 0.91(3H,d,J=6.4Hz,H-21), 0.83(3H,s,H-18)], the remainder being the methylene or methine hydrogen signals of the compound.

[0015] 13 The C-NMR spectrum (Table 1) shows 27 carbon signals, including 3 carbonyl and 1 carboxyl carbon signals [δ]. C 215.8(C-3), 202.0(C-11), 201.4(C-7), 177.7(C-24)]; Two carbon signals from one double bond [δ C 151.6(C-8), 149.8(C-9)], 6 methyl carbon signals [δ C 27.5 (C-28), 25.9 (C-30), 20.4 (C-29), 18.1 (C-21), 18.0 (C-19), 17.0 (C-18)], plus 15 other aliphatic carbon signals [δ C 51.3(C-12),49.8(C-5),49.1(C-17),48.7(C-14),46.9(C-13),46.7(C-4),38.8(C-10),3 7.1(C-6),35.7(C-20),35.0(C-1),34.1(C-2),31.9(C-15),30.7(C-22,23),27.3(C-16)].

[0016] By HR-ESI-MS, 1 H-NMR and 13 C-NMR spectra suggest that LZ-GO-45 is a no-three-carbon lanostane-type triterpenoid.

[0017] Combined with HMBC spectrum, H3-28(δ) H 1.13), H3-29(δ) C 1.11) and C-3(δ) C 215.8) is related, indicating that the C-3 position is a carbonyl group; H-5 (δ H 2.26) and C-7(δ) C The correlation between 201.4 and H2 indicates that the C-7 position is a carbonyl group; H2-22 (δ H 1.35) and C-24 (δ) C The correlation of 177.7 indicates that the C-24 position of the side chain is a carbonyl group; H3-30 (δ H 1.22) and C-8(δ) C 151.6) related, H3-19 (δ) H 1.28) and C-9(δ) C (149.8) Related to this, indicating that C-8 and C-9 are double bonds.

[0018] Thus, the planar structure of the compound was established.

[0019] The planar structure of LZ-GO-45 is identical to that of the known synthesized euphorbia-type triterpenoid (5R,10S,13S,14S,17S,20S)-3,7,11-trioxo-25,26,27-trinortirucalla-8-en-24-oic acid (Studies on the constituents of Chinese drug "kanzui."8. KMnO4 oxidation of euphol,tirucalloland beta-euphorbol, Yakugaku Zasshi, 1967, 87:21–25). The difference lies in the reverse orientation of the three methyl groups (CH3-18 at C-13, CH3-30 at C-14, and CH3-21 at C-20) and H-17. H 0.83) / H3-19(δ H 1.28), H3-19 (δ) H 1.28) / H3-29(δ H 1.11), H-17(δ) H 1.69) / H3-21(δ H 0.91), H-17 (δ) H 1.69) / H3-30(δ H 1.22), H-5(δ) H 2.26) / H3-28(δ H1.13) It can be determined that the methyl groups at positions 18 and 19 are β-methyl, the methyl groups at positions 21 and 30 are α-methyl, and H-5 and H-17 are α-H. This configuration is consistent with the lanostane skeleton configuration. Finally, the absolute configuration of LZ-GO-45 was determined to be 5R,10S,13R,14R,17R,20R.

[0020] The structure of LZ-GO-45 was determined and named 3,7,11-trioxo-25,26,27-trinorlanos ta-8-en-24-oic acid. A Sci-finder search confirmed it as a novel compound.

[0021] Table 1. 3,7,11-Trioxo-25,26,27-trinorlanosta-8-en-24-oic acid 1 H NMR and 13 CNMR data

[0022]

[0023] 1 H NMR 400MHz and 13 C NMR 100MHz, in CDCl3.

[0024] A third aspect of the present invention is to provide a pharmaceutical composition, characterized in that: the pharmaceutical composition contains the 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid described in the first aspect, its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

[0025] The fourth aspect of the present invention is the use of the trioxo-25,26,27-trinorlanosta-8-en-24-oic acid described in the first aspect of the present invention in the preparation of a medicament for the prevention and / or treatment of influenza.

[0026] Beneficial technical effects

[0027] The inventors of this invention discovered 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid, a natural no-tricarbon Ganoderma lucidum triterpene acid, in natural products. Based on this, the inhibitory activity of the obtained compound against influenza virus neuraminidase was evaluated, and a novel structural compound (IC) exhibiting significant inhibitory activity against influenza virus neuraminidase was obtained. 50The concentration was 5.17 μM. The study revealed that 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid is a novel active compound with potential for further development as a new drug for treating influenza-related diseases. To date, no structural and activity studies of this compound have been reported in the literature. Existing literature and techniques do not report on 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid or its medically acceptable salts, nor on the use of this compound for treating influenza. Therefore, the present invention is significantly innovative. Attached Figure Description

[0028] LZ-GO-45

[0029] Figure 1 The structure of 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oicacid

[0030] Figure 2 High-resolution mass spectrometry of 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid Figure 3 UV spectrum of 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid Figure 4 Infrared spectrum of 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid Figure 5 :3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid 1 H NMR spectrum Figure 6 :3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid 13 C NMR spectrum Figure 7 HSQC spectrum of 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid Figure 8HMBC spectrum of 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid Figure 9 NOESY spectrum of 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid Detailed Implementation

[0031] Example 1: Isolation of 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid (a type of Ganoderma lucidum triterpenoid)

[0032] Molecular formula: C 27 H 38 O5

[0033] Nomenclature: 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid

[0034] Separation and preparation route:

[0035] The dried fruiting bodies of Ganoderma lucidum were extracted three times by heating and reflux with 90% ethanol-water. The extracts were combined and concentrated under reduced pressure to obtain a paste, which was then mixed with macroporous resin. Macroporous resin chromatography was performed, with gradient elution using 30% ethanol-water, 60% ethanol-water, 80% ethanol-water, and 95% ethanol-water to obtain four fractions, namely DK-1 to DK-4. DK-3 was further eluted by silica gel column chromatography with a gradient elution of a mixed solvent of petroleum ether and ethyl acetate at volume ratios of 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50. The column was then washed sequentially with ethyl acetate and methanol to obtain 25 fractions, namely Fr.1 to Fr.25. Fr.17 was eluted by gradient chromatography on a medium-pressure silica gel column with petroleum ether:ethyl acetate (80:20-50:50, v / v). The column was then washed with ethyl acetate to obtain 14 subfractions, namely Fr.17-1-Fr.17-14. Fr.17-7 was subjected to C... 18 Reverse-phase column chromatography with gradient elution using methanol:water (50:50-75:25, v / v) as the eluent, followed by column washing with 100% methanol, yielded 25 subfractions, namely Fr.17-7-1-Fr.17-7-25. Fr.17-7-10 was separated using a semi-preparative reversed-phase column YMC-Pack ODS-A (250×20 mm) with isocratic elution using 40% acetonitrile-water as the mobile phase at a flow rate of 12 mL / min. R=69 min yielded 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid (number LZ-GO-45).

[0036] Pharmacological experiments

[0037] Experimental Example 1:

[0038] 1.1.3,7,11-Trioxo-25,26,27-trinorlanosta-8-en-24-oic acid inhibitory effect on influenza virus neuraminidase

[0039] The influenza virus neuraminidase is located on the surface of the influenza virus and can cause the virus to be released from the host cell. The compound 4-methylumbelliferyl-N-acetyl-α-D-neuraminidase sodium hydrate (MUNANA) is a specific substrate of neuraminidase (NA). The metabolites produced under the action of neuraminidase can produce 450 nm fluorescence when excited by 360 nm irradiation. The change in fluorescence intensity can sensitively reflect the activity of neuraminidase.

[0040] Experimental methods:

[0041] In the enzyme reaction system, a certain concentration of the analyte compound and influenza virus neuraminidase were suspended in a reaction buffer (pH 6.5). The fluorescent substrate MUNANA was added to start the reaction, and the mixture was incubated at 37°C for 60 minutes. The reaction was then terminated by adding a stop solution. The fluorescence intensity was measured under the parameters of an excitation wavelength of 360 nm and an emission wavelength of 450 nm. The inhibition rate of the compound on neuraminidase activity was calculated based on the decrease in fluorescence intensity.

[0042] Experimental results:

[0043] IC50 of the inhibitory activity of 3,7,11-trioxo-25,26,27-trinorlanosta-8-en-24-oic acid against influenza neuraminidase 50 The IC50 value of the positive control drug Zanamivir was 5.17 μM. 50 It is 7.8×10 -4 μM.

Claims

1. A Ganoderma lucidum triterpenoid compound or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the compound is 2. The method for preparing the compound according to claim 1, characterized in that, 1) The dried fruiting bodies of Ganoderma lucidum were extracted three times by heating and refluxing with 90% ethanol-water. The extracts were combined and concentrated under reduced pressure to obtain an extract, which was then mixed with macroporous resin. 2) Macroporous resin chromatography was performed with gradient elution using 30% ethanol-water, 60% ethanol-water, 80% ethanol-water, and 95% ethanol-water to obtain four fractions, namely DK-1-DK-4. DK-3 was further eluted by silica gel column chromatography with a gradient elution of a mixture of petroleum ether and ethyl acetate at volume ratios of 85:15, 80:20, 75:25, 70:30, 60:40, and 50:50, followed by washing the column with ethyl acetate and methanol to obtain 25 fractions, namely Fr.1-Fr.

25. Fr.17 was eluted by medium-pressure silica gel column chromatography with a gradient elution of petroleum ether:ethyl acetate (80:20-50:50, v / v), followed by washing the column with ethyl acetate to obtain 14 subfractions, namely Fr.17-1-Fr.17-14. Fr.17-7 was further eluted by C... 18 Reverse-phase column chromatography with gradient elution using methanol:water (50:50-75:25, v / v) as the eluent, followed by washing the column with 100% methanol, yielded 25 subfractions, namely Fr.17-7-1-Fr.17-7-25. Fr.17-7-10 was separated by semi-preparative reversed-phase high-performance liquid chromatography with isocratic elution using 40% acetonitrile-water as the eluent to obtain the compound of claim 1.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of claim 1, its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier or excipient.

4. The use of the compound of claim 1 and its pharmaceutically acceptable salt in the preparation of medicaments for the prevention and / or treatment of influenza.

5. The application according to claim 4, characterized in that, The influenza mentioned includes influenza A and influenza B, among others.

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