A lycopodium alkaloid compound (-)-hupserrine A and a preparation method and application thereof

Abstract

The application discloses a lycopodium alkaloid compound (-) -Hupserrine A and a preparation method and application thereof, and an extraction and synthesis method thereof, and belongs to the technical field of medicines. The synthetic preparation method has many advantages, is novel in route, is excellent in efficiency, and can realize large-scale preparation, so that production demands can be fully met. The lycopodium alkaloid compound has a significant 5-hydroxytryptamine (5- HT3A) ligand gate ion channel inhibition effect, so as to regulate the ion channel function, and then regulate the release of neurotransmitters and signal transmission. The 5- HT3A receptor inhibitor is a core drug for treating nausea and vomiting caused by chemotherapy and radiotherapy, and is also widely applied in the fields of anxiety, depression, schizophrenia and neuralgia. The lycopodium alkaloid compound has an inhibition effect on the 5- HT3A receptor, so that it has potential application value in the treatment of central nervous system diseases (such as epilepsy, pain and sleep disorders).

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a lycophyte alkaloid compound (-)-Hupserrine A, its preparation method and application, as well as a pharmaceutical composition with (-)-Hupserrine A as the active ingredient. (-)-Hupserrine A can become a core drug for the treatment of central nervous system diseases, and can also be widely used in the treatment of anxiety, depression, schizophrenia and neuralgia. Background Technology

[0002] Lycopodium alkaloids possess complex and diverse chemical structures with a rich variety of skeleton types. Due to their unique structure and significant biological activity, pharmacological studies have revealed that lycopodium alkaloids, as a traditional herbal ingredient, have effects such as dispelling wind and dampness, relieving wind-cold paralysis, and treating traumatic injuries. Their activities in neuroprotection, cholinesterase inhibition, and cytotoxicity provide an important theoretical and practical foundation for modern medical research and drug development.

[0003] Advances in the research of the structure, function, and drug development of the 5-HT3A receptor: The 5-HT3A receptor belongs to the ligand-gated ion channel receptor subtype and is a member of the LGIC superfamily. It consists of five subunits, each containing an N-terminal extracellular domain and four transmembrane helices (M1-M4). The 5-HT3A subunits can form homopentamers independently, but usually require binding with other subunits (such as 5-HT3B) to form heteropentamers to achieve functional channel function. It has high sequence homology with the nicotinic acetylcholine receptor (nAChR), and the two may share similar three-dimensional structural features. Functional characteristics: The 5-HT3A receptor is widely distributed in the central and peripheral nervous systems, participating in the regulation of various physiological processes, including neurotransmitter release, pain signal transmission, and emotional and cognitive functions. In the central nervous system, it is mainly distributed in the dorsal horn of the spinal cord, brainstem, and cerebellum; in the peripheral nervous system, the 5-HT3A receptor is widely present in the gastrointestinal tract, regulating physiological functions such as intestinal motility, secretion, and the vomiting reflex. Furthermore, the 5-HT3A receptor is associated with a variety of diseases, such as depression, anxiety disorders, and irritable bowel syndrome. Due to its crucial role in multiple diseases, the 5-HT3A receptor has become an important target for drug development. Antiemetic drugs (such as ondansetron and palonosetron) alleviate nausea and vomiting induced by chemotherapy and radiotherapy by competitively inhibiting receptor activity. Competitive antagonists also include cloprothixone, lidocaine, morphine, and fentanyl, while non-competitive antagonists include vortioxetine, which, in addition to inhibiting the 5-HT3A receptor, also inhibits several other serotonin receptors (such as 5-HT1A, 5-HT1B, and 5-HT7), with a Ki value of 3.7 nM, indicating high selectivity for the 5-HT3A receptor. Antidepressants and anxiolytics may also exert their effects by modulating the function of the 5-HT3A receptor. In addition, isomeric modulators of the 5-HT3A receptor (such as positive and partial agonists) may provide new strategies for treating depression and other mental illnesses.

[0004] Recent research has yielded significant progress in the study of the 5-HT3A receptor. For example, using cryo-electron microscopy, researchers successfully resolved the structure of the 5-HT3A receptor in its resting state. This achievement not only provides an important foundation for understanding its function but also offers potential directions for developing novel therapeutic drugs. Antidepressants such as vortioxetine have been shown to improve mood disorders by inhibiting the 5-HT3A receptor, while cannabinoids, due to their allosteric inhibition of the 5-HT3A receptor, may have potential in treating anxiety and pain. The 5-HT3A receptor and its inhibitors play a crucial role in neuroscience and drug development. Future research should focus on developing more efficient and selective inhibitors and exploring their potential applications in various diseases.

[0005] To date, there have been no literature reports on the structure of (-)-Hupserrine A, a lycophyte alkaloid compound with 5-HA3A ligand-gated ion channel inhibitory activity as described in this invention, nor have there been any reports on its pharmacological effects. Summary of the Invention

[0006] This invention aims to develop a medicinal compound of lycopodium alkaloids with 5-HA3A ligand-gated ion channel inhibitory activity, and its extraction and synthetic preparation method. Inspired by biosynthetic pathways, a biomimetic synthetic strategy was adopted, combining key steps such as the Heck coupling reaction and the Mannich cyclization reaction. This invention successfully solved the problem of the scarcity of lycopodium alkaloids in plants, achieving their efficient and large-scale synthesis. Furthermore, the drug compounds developed based on the synthesized lycopodium alkaloids provide new options for the treatment of various diseases, demonstrating broad application prospects. This innovative achievement not only promotes the research and development of lycopodium alkaloids but also provides new ideas and methods for the synthesis and application of natural products. Moreover, lycopodium alkaloids are a multifunctional and important neurotransmitter receptor for the 5-HT3A receptor, playing a crucial role in physiological and pathological processes, making them valuable for the treatment of central nervous system diseases (such as epilepsy, pain, and sleep disorders) and the regulation of physiological functions such as intestinal motility, secretion, and vomiting reflexes.

[0007] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0008] The following structural formula shows the lycophyte alkaloid compound (-)-Hupserrine A.

[0009]

[0010] A pharmaceutical composition comprising a therapeutically effective amount of the lycopodium alkaloid compound (-)-Hupserrine A, as shown in the structural formula, and a pharmaceutically acceptable carrier.

[0011] The use of the lycophyte alkaloid compound (-)-Hupserrine A, as shown in the structural formula, or the pharmaceutical composition thereof, in the preparation of a medicament for treating epilepsy, pain, and sleep disorders.

[0012] The use of the lycophyte alkaloid compound (-)-Hupserrine A, as shown in the structural formula, or the pharmaceutical composition thereof, in the preparation of a medicament for regulating intestinal motility, secretion, and vomiting.

[0013] The use of the lycophyte alkaloid compound (-)-Hupserrine A, as shown in the structural formula, or the pharmaceutical composition thereof, in the preparation of a drug for treating anxiety and depression.

[0014] The use of the lycophyte alkaloid compound (-)-Hupserrine A, as shown in the structural formula, or the pharmaceutical composition thereof, in the preparation of 5-HA3A ligand-gated ion channel inhibitors.

[0015] A method for synthesizing (-)-Hupserrine A, an alkaloid compound derived from lycophytes, comprising the following steps:

[0016] (1) The compound shown in Formula 1 undergoes a retro-Aldol reaction under acidic conditions, and then undergoes an esterification reaction with dimethyl carbonate to generate the intermediate compound shown in Formula 2.

[0017] (2) The intermediate compound shown in Formula 2 undergoes a halogenation reaction with N-bromosuccinimide to generate the intermediate compound shown in Formula 3.

[0018] (3) The intermediate compound shown in Formula 3 undergoes a protective reaction with ethylene glycol to generate the intermediate compound shown in Formula 4;

[0019] (4) The intermediate compound shown in formula 4 undergoes a free radical elimination reaction in the photocatalyst to generate the intermediate compound shown in formula 5.

[0020] (5) The intermediate compound shown in Formula 6 undergoes a Michael addition reaction with the intermediate compound shown in Formula 5 under alkaline conditions, and then undergoes a deprotection reaction under acidic conditions to generate the intermediate compound shown in Formula 7.

[0021] (6) The intermediate compound shown in Formula 7 undergoes a Michael addition reaction with acrylonitrile to generate the intermediate compound shown in Formula 8;

[0022] (7) The intermediate compound shown in Formula 8 undergoes a decarboxylation reaction with lithium salt, and then a hydrolysis-cyclization reaction is carried out with the addition of palladium catalyst to generate the intermediate compound shown in Formula 9.

[0023] (8) The intermediate compound shown in Formula 9 undergoes a Heck reaction on a palladium catalyst to generate the intermediate compound shown in Formula 10;

[0024] (9) The intermediate compound shown in Formula 10 undergoes a protective reaction with benzyl bromide, followed by a demethylation reaction to generate the intermediate compound shown in Formula 11;

[0025] (10) The intermediate compound shown in Formula 11 undergoes a dearomatization reaction with samarium powder to generate the intermediate compound shown in Formula 12;

[0026] (11) The intermediate compound shown in Formula 12 undergoes a protective reaction with ditert-butyl dicarbonate to generate the intermediate compound shown in Formula 13.

[0027] (12) The intermediate compound shown in Formula 13 undergoes a deprotection reaction with the photocatalyst to generate the intermediate compound shown in Formula 14;

[0028] (13) The intermediate compound shown in Formula 14 undergoes a reduction reaction with a reducing agent, and then undergoes a dehydration reaction with an acid anhydride to generate the intermediate compound shown in Formula 15.

[0029] (14) The intermediate compound shown in Formula 15 undergoes a reduction reaction with a reducing agent to generate the intermediate compound shown in Formula 16;

[0030] (15) The intermediate compound shown in Formula 16 reacts with an acid in a Mannich reaction to produce the intermediate compound shown in Formula 17.

[0031] (16) The intermediate compound shown in Formula 17 undergoes an amidation reaction with an acid anhydride to generate the target product shown in Formula 18, lycopodium alkaloid compound (-)-Hupserrine A.

[0032] The reaction route is as follows:

[0033]

[0034] Boc represents tert-butyloxycarbonyl, and Bn represents benzyl.

[0035] In some embodiments, the raw material (+)-pulegone shown in Formula 1 in step (1) is added to water, cetylpyridinium chloride and concentrated sulfuric acid are added at 0°C, the temperature is raised to 120°C and reacted for 6 hours, then the oily compound is distilled off, saturated sodium chloride aqueous solution and dichloromethane are added for extraction, the compound is dissolved in tetrahydrofuran by vacuum distillation, sodium hydride and dimethyl carbonate are added at 0°C, and the mixture is refluxed for 6 hours. The molar ratio of the compound (+)-pulegone, cetylpyridinium chloride, concentrated sulfuric acid, sodium hydride and dimethyl carbonate shown in Formula 1 is 1:5%:2:1.5:1.2.

[0036] step (2) The intermediate compound 2 shown in Formula 2 is dissolved in dichloromethane, and azobisisobutyronitrile and N-bromosuccinimide are added at 0°C and reacted for 0.5 hours. The molar ratio of compound 2, azobisisobutyronitrile and N-bromosuccinimide is 1:0.2:2.5.

[0037] step (3) The intermediate compound 3 shown in Formula 3 is dissolved in ethylene glycol, and trimethylchlorosilane is added at room temperature. The mixture is heated to 40°C and reacted for 10 hours. The molar ratio of compound 3 shown in Formula 3 to trimethylchlorosilane is 1:2.5.

[0038] In step (4), intermediate compound 4 of formula 4 is dissolved in acetonitrile, and 2,4,5,6-tetrakis(9-carbazolyl)-isophthalonitrile is added at room temperature.

[0039] (OC-6-14)-[[2,3-butanedione 2,3-di(oxime-κN)](1-)][2,3-butanedione 2,3-di(oxime-κN)]cobalt dichloro, N,N-diisopropylethylamine, and potassium carbonate were reacted at room temperature under Blue LED irradiation for 16 hours. The molar ratio of compound 4 (as shown in Formula 4), 2,4,5,6-tetra(9-carbazolyl)-isophthalonitrile, (OC-6-14)-[[2,3-butanedione 2,3-di(oxime-κN)](1-)][2,3-butanedione 2,3-di(oxime-κN)]cobalt dichloro, N,N-diisopropylethylamine, and potassium carbonate was 1:2%:5%:20%:1.

[0040] In step (5), intermediate compound 6 of formula 6 is dissolved in tetrahydrofuran. Diisopropylaminolithium solution and hexamethylphosphoric triamine are added at -78°C and reacted for 10 minutes. Then, intermediate compound 5 of formula 5 is added dropwise to the reaction mixture in tetrahydrofuran solution and reacted for 2 hours. Then, 3M hydrochloric acid is added to adjust the pH to 2 and reacted for 2 hours. The molar ratio of intermediate compound 6 of formula 6, diisopropylaminolithium solution, hexamethylphosphoric triamine, intermediate compound 5 of formula 5, and 3M hydrochloric acid is 1:5:5:1:15.

[0041] step (6) The intermediate compound 7 shown in Formula 7 is dissolved in acetonitrile, and allyl nitrile and tetrabutylammonium hydroxide solution are added at 0°C and reacted for 6 hours. The molar ratio of compound 7, allyl nitrile and tetrabutylammonium hydroxide is 1:1.5:0.1.

[0042] In step (7), intermediate compound 8 of formula 8 is dissolved in an organic solvent, and anhydrous lithium chloride is added at room temperature. The mixture is then heated to 150°C and reacted for 3.5 hours. Then, palladium acetate, acetaldehyde oxime, and molecular sieve are added at room temperature, and the mixture is heated to 120°C and reacted for 10 hours. The molar ratio of compound 8 of formula 8, anhydrous lithium chloride, palladium acetate, and acetaldehyde oxime is 1:8:0.2:5.

[0043] In step (8), intermediate compound 9 of formula 9 is dissolved in toluene, and tris(dibenzylacetone)dipalladium, 1,2-bis(diphenylphosphine)benzene and cesium carbonate are added at room temperature. The mixture is heated to 140°C and reacted for 12 hours. The molar ratio of intermediate compound 9 of formula 9, tris(dibenzylacetone)dipalladium, 1,2-bis(diphenylphosphine)benzene and cesium carbonate is 1:0.3:0.3:2.

[0044] In step (9), intermediate compound 10 of formula 10 is dissolved in N,N-dimethylformamide, sodium hydrogen and benzyl bromide are added at room temperature and reacted for 3 hours, then sodium ethanethiol is added and the temperature is raised to 120°C and reacted for 6 hours. The molar ratio of intermediate compound 10 of formula 10, sodium hydrogen, benzyl bromide and sodium ethanethiol is 1:2:2:8.

[0045] In step (10), intermediate compound 11 of formula 11 is dissolved in tetrahydrofuran and 3M hydrochloric acid, and samarium powder is added in batches at 0°C and reacted for 20 minutes. The molar ratio of intermediate compound 11 of formula 11 to samarium powder is 1:16.

[0046] In step (11), intermediate compound 12 of formula 12 is dissolved in 1,4-dioxane, and 4-dimethylaminopyridine and ditert-butyl dicarbonate are added at 100°C and reacted for 10 minutes. The molar ratio of intermediate compound 12 of formula 12, 4-dimethylaminopyridine and ditert-butyl dicarbonate is 1:2:2.5.

[0047] In step (12), intermediate compound 13 of formula 13 is dissolved in N,N-dimethylformamide, and 3,5-di-tert-butyl-[1,1'-biphenyl]-4-phenol, 2,2,6,6-tetramethylpiperidine oxidol, and cesium carbonate are added at room temperature. The reaction is carried out under LED irradiation for 10 hours. The molar ratio of intermediate compound 13 of formula 13, 3,5-di-tert-butyl-[1,1'-biphenyl]-4-phenol, 2,2,6,6-tetramethylpiperidine oxidol, and cesium carbonate is 1:0.2:5:0.5.

[0048] In step (13), intermediate compound 14 of formula 14 is dissolved in tetrahydrofuran, and triethyl borohydride is added at -78°C and reacted for 10 minutes. Then, N,N-diisopropylethylamine, 4-dimethylaminopyridine, and trifluoroacetic anhydride are added, and the mixture is heated to room temperature and reacted for 10 minutes. The molar ratio of intermediate compound 14 of formula 14, triethyl borohydride, N,N-diisopropylethylamine, 4-dimethylaminopyridine, and trifluoroacetic anhydride is 1:1.25:3:0.2:2.8.

[0049] In step (14), intermediate compound 15 of formula 15 is dissolved in tetrahydrofuran, lithium aluminum hydride is added at room temperature, and the mixture is heated to reflux for 30 minutes. The molar ratio of intermediate compound 15 of formula 15 to lithium aluminum hydride is 1:3.

[0050] In step (15), intermediate compound 16 of formula 16 is dissolved in trifluoroethanol, trifluoroacetic acid is added at room temperature, and the temperature is raised to 120°C and reacted for 12 hours. The molar ratio of intermediate compound 16 of formula 16 to trifluoroacetic acid is 1:5.

[0051] In step (16), intermediate compound 17 of formula 17 is dissolved in tetrahydrofuran. Formic acid and acetic anhydride are dissolved in tetrahydrofuran at room temperature. The temperature is raised to 70°C and reacted for 1 hour. Then the temperature is lowered to 0°C and intermediate compound 17 of formula 17 is added dropwise to the reaction solution and reacted for 1 hour. The molar ratio of intermediate compound 17 of formula 17, formic acid and acetic acid is 1:15:5.

[0052] Understandably, the product of any corresponding step can be used as a starting material to directly prepare the above-mentioned intermediate compound of lycopodium alkaloid through a subsequent reaction. For example, intermediate compound 8 can be prepared by using the compound shown in Formula 1 as a starting material and performing steps (1)-(6) as described above, or intermediate compounds shown in Formulas 5 and 6 can be used as starting materials and performing steps (4)-(6) as described above. The product of any corresponding step can be used as a starting material to directly prepare the above-mentioned lycopodium alkaloid (-)-Hupserrine A through a subsequent reaction. For example, lycopodium alkaloid (-)-Hupserrine A can be prepared by using the intermediate compound shown in Formula 1 as a starting material and performing steps (1)-(16) as described above.

[0053] This invention also provides another method for preparing the aforementioned lycophyte alkaloid compound (-)-Hupserrine A, the method comprising the following steps:

[0054] The dried whole herb of *Huperzia serrata* was pulverized and extracted three times with methanol. After removing the methanol, an extract was obtained, acidified with 3% tartaric acid, extracted with ethyl acetate, and the pH was adjusted to 9 by saturating the aqueous layer with sodium carbonate. Chloroform extraction was then performed to obtain the total alkaloids. The total alkaloids were subjected to silica gel column chromatography, with eluents successively changing from 90:10:0.5–0:100:0 petroleum ether / acetone / diethylamine to 90:10–0:100 dichloromethane / methanol, yielding four fractions Fr.1–Fr.4. Fr.3 was further fractionated with chloroform / methanol at a ratio of 97:3–0:100 to obtain two fractions. Fr.3.1–Fr.3.2; Fr.3.1 was fragmented on a C18-CE column to obtain 6 fractions Fr.3.1.1–Fr.3.1.6; Fr.3.1.5 was fragmented on a silica gel column with petroleum ether / acetone / diethylamine in a ratio of 90:10:0.5–0:100:0.5 to obtain Fr.3.1.5.1 and Fr.3.1.5.2; Fr.3.1.5.1 was purified on a silica gel column with a ratio of chloroform / methanol in a ratio of 97:3–95:5 to obtain the lycopodium alkaloid compound (-)-Hupserrine A.

[0055] Specifically, the method includes the following steps:

[0056] Step (1) 17 kg of dried whole Huperzia serrata herb was crushed and extracted three times with methanol. The methanol was removed to obtain an extract, which was then acidified with 3% tartaric acid and extracted with ethyl acetate. The pH of the acidic aqueous layer was adjusted to 9 with saturated sodium carbonate, and chloroform extraction was performed to obtain 65 g of total alkaloids.

[0057] Step (2) The total alkali was subjected to silica gel column segmentation, with the eluent successively being petroleum ether / acetone / diethylamine (90:10:0.5–0:100:0.5) to dichloromethane / methanol (90:10–0:100), to obtain 4 components Fr.1–Fr.4;

[0058] Step (3): Fr.3 (8.8 g) was fragmented using chloroform / methanol (97:3–0:100) to obtain two fractions, Fr.3.1–Fr.3.2. Fr.3.1 was fragmented using a C18-CE column to obtain six fractions, Fr.3.1.1–Fr.3.1.6. Fr.3.1.5 was fragmented using a silica gel column (petroleum ether / acetone / diethylamine, 90:10:0.5–0:100:0.5) to obtain Fr.3.1.5.1 and Fr.3.1.5.2. Fr.3.1.5.1 was purified using a silica gel column (chloroform / methanol, 97:3–95:5) to obtain compound (-)-Hupserrine A (3 mg).

[0059] In addition, the present invention provides a 5-HA3A ligand-gated ion channel inhibitor comprising the lycophyte alkaloid compound (-)-Hupserrine A as shown in the structural formula.

[0060] The lycopodium alkaloid compound (-)-Hupserrine A of this invention can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90%, of the lycopodium alkaloid compound (-)-Hupserrine A of this invention, with the remainder being a pharmaceutically acceptable, non-toxic, and inert carrier for humans and animals. Various pharmaceutical excipients can be used to formulate solid dosage forms (tablets, capsules, granules, powders, etc.) or liquid dosage forms (injections, oral liquids, etc.).

[0061] The pharmaceutical composition of the present invention is preferably used in the form of a dose per unit body weight, and can be administered once or multiple times.

[0062] Compared with the prior art, the present invention has the following significant innovations and advantages:

[0063] The lycopodium alkaloids and their derivatives involved in this invention possess novel chemical structures, demonstrating strong innovation. This innovation is not only reflected in the diversity of their chemical structures but also in their potential pharmacological activities. This novel chemical structure fills a gap in the existing technology.

[0064] This invention proposes a novel synthetic method for preparing lycopodium alkaloids and their derivatives. This method not only solves the problem of insufficient lycopodium alkaloid content but also enables the rapid construction of a lycopodium alkaloid derivative library, providing a sufficient quantity of compounds for pharmacological activity screening. The raw materials used in this synthesis are readily available in large quantities and have low toxicity; the related reagents are inexpensive and readily available. Furthermore, the synthetic steps are simple and straightforward, and the operation is easy to perform. These characteristics make this invention extremely significant for promotion and application in the pharmaceutical field.

[0065] The inhibitory activity experiments of compound (-)-Hupserrine A and the positive control drug Quinine against 5-HA3A ligand-gated ion channels showed that both could inhibit the 5-HA3A receptor. Further studies indicated that both compound (-)-Hupserrine A and the positive control drug Quinine inhibited the 5-HA3A receptor in a concentration-dependent manner, with IC50 values ​​of 10.6 μM and 18.1 μM, respectively. Channel current-voltage curves plotted using whole-cell recordings more clearly reflected the inhibitory effect of compound (-)-Hupserrine A on the 5-HA3A receptor. Therefore, compound (-)-Hupserrine A, as an effective inhibitor of the 5-HA3A receptor, could serve as a lead compound for the treatment of central nervous system diseases (such as epilepsy, pain, and sleep disorders) and for regulating physiological functions such as intestinal motility, secretion, and vomiting reflexes.

[0066] The lycopodium alkaloid compound (-)-Hupserrine A of this invention has unique value in the field of drug development. This opens up a new and important avenue for drug development. This type of compound demonstrates core value in treating nausea and vomiting induced by chemotherapy and radiotherapy, and also shows broad application prospects in many other areas such as anxiety, depression, schizophrenia, and neuralgia. Furthermore, this invention also relates to the application of lycopodium alkaloid compounds as 5-HT3A receptor inhibitors. Lycopodium alkaloid compounds possess a rich structural skeleton, enabling them to specifically bind to multiple receptors, thereby exerting different pharmacological effects. In addition to their inhibitory effect on 5-HT3A receptors, lycopodium alkaloid compounds also exhibit good activity in cholinesterase inhibition, neuroprotection, and cytotoxicity. These compounds are mainly isolated from plants in the Lycopodiaceae, Humulaceae, and their close relatives. Research and Application Prospects: The discovery of lycopodium alkaloid compounds provides new ideas and directions for medical research and drug development. Attached Figure Description

[0067] Figure 1 This is a flowchart illustrating the preparation route of the lycophyte alkaloid compound (-)-Hupserrine A of this invention;

[0068] Figure 2 This is a representative current diagram of the channel current effect of the lycophyte alkaloid compound (-)-Hupserrine A of this invention;

[0069] Figure 3 IC50 diagram showing the channel current action of the lycophyte alkaloid compound (-)-Hupserrine A in this invention;

[0070] Figure 4 IC50 curve of channel current effect of the positive control drug Quinine;

[0071] Figure 5 This is a schematic diagram of the structure of the lycophyte alkaloid compound (-)-Hupserrine A of the present invention. Detailed Implementation

[0072] This invention discloses a series of key intermediate compounds for the synthesis of lycophyte alkaloids and their synthetic methods. Based on these intermediates, this invention also proposes a biomimetic synthetic strategy inspired by the biosynthetic pathway of lycophyte alkaloids for the synthesis of a series of lycophyte alkaloids and their derivatives. The implementation of this invention is diverse and not limited to the specific details described herein.

[0073] Those skilled in the art can make appropriate adjustments to the reaction conditions based on the content of this invention, such as changing the temperature, time, reagents, solvents, molar ratios, etc. It should be particularly noted that similar substitutions and fine-tuning of reaction conditions are common practices in the art and are all considered to be covered within the scope of this invention.

[0074] To further understand the content of this invention, the following detailed description is provided in conjunction with the accompanying drawings and embodiments, but this description is not intended to limit the scope of the invention.

[0075] Example 1:

[0076]

[0077] (R)-pulegone 1 (100 g, 656.8 mmol, commercially available), CPC (11.17 g, 32.84 mmol), and water (426 mL) were added to a 1 L round-bottom flask at room temperature. Concentrated sulfuric acid (73 mL, 1.31 mol) was added dropwise to the reaction solution at 0 °C. The reaction solution was then heated to 120 °C and reacted for 6 hours. The reaction solution was then distilled, and the distillate was extracted with saturated brine and dichloromethane. After drying with anhydrous sodium sulfate, the solution was directly distilled under reduced pressure to proceed to the next step without further purification.

[0078] The raw material, which did not require purification in the previous step, was dissolved in tetrahydrofuran (561 mL). Sodium hydroxide (60%, 33.7 g, 561.6 mmol) was added at 0 °C and reacted for 10 minutes. Then, dimethyl carbonate (60.7 mL, 673.9 mmol) was added dropwise to the reaction mixture. The reaction mixture was heated to 80 °C and reacted for 6 hours. The reaction was quenched by adding saturated ammonium chloride solution at 0 °C. Extraction was performed with dichloromethane, drying with anhydrous sodium sulfate, and vacuum distillation. The mixture was then subjected to column chromatography (petroleum ether:ethyl acetate = 50:1–25:1) to give compound 2, with an overall yield of 70%. The detection data for compound 2 are as follows: 1 H NMR (400MHz, CHCl3) δ12.09(s,1H),3.72(s,3H),2.40–2.22(m,2H),2.21–2.07(m,1H),1.89(dd,J=17. 9,9.9Hz,1H),1.71(dt,J=12.3,6.5Hz,2H),1.15(dtd,J=12.8,10.9,5.6Hz,1H),0.96(d,J=6.5Hz,3H); 13 C NMR (100MHz, CHCl3) δ173.1,171.9,97.2,51.5,37.3,30.7,28.4,22.2,21.4. = +89.28 (c = 8.33, CHCl3).

[0079] Example 2:

[0080]

[0081] Compound 2 was dissolved in dichloromethane (71 mL), and azobisisobutyronitrile (2.32 g, 14.1 mmol) and N-succinimide bromide (31.37 g, 14.1 mmol) were added at 0 °C. The reaction solution was reacted for 30 minutes at the same temperature. The reaction was quenched with saturated sodium bicarbonate solution, followed by extraction with dichloromethane. The extract was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (petroleum ether:acetone = 30:1-9:1) to give compound 3 in 91% yield. The detection data for compound 3 are as follows: 1 H NMR (400MHz, CHCl3) δ3.83(s,3H),2.87(dd,J=14.2,11.6Hz,1H),2.63(ddd,J=15.4,11.3,4.2Hz,1H),2.39(ddd,J=14.1,4.3,1.6Hz,1H),2.33(d 13C NMR(100MHz,CHCl3)δ200.5,168.2,65.7,53.9,44.8,37.6,34.1,29.7,21.7.HRMS(ESI+):m / zcalc'd for C9H 13 BrNaO3[M+Na] + :270.9946,found:270.9937.dr=3.6:1for 1 H NMR.

[0082] Example 3:

[0083]

[0084] Compound 3 (20.0 g, 80.29 mmol) was dissolved in ethylene glycol (160 mL), and trimethylchlorosilane (25.62 mL, 200.7 mmol) was added at room temperature. The mixture was heated to 40 °C and reacted for 10 hours. The reaction was quenched with water (200 mL), extracted with dichloromethane, dried over anhydrous sodium sulfate, purified by vacuum distillation, and purified by silica gel column chromatography (petroleum ether:acetone = 30:1-9:1) to give compound 4 in 76% yield. Compound 4 detection data: 1H NMR(400MHz,CHCl3)δ4.06(q,J=8.5,7.5Hz,1H),4.01–3.95(m,1H),3.95(m,1H),3.96–3.89(m,1H),3.76(s,3H),2.27(dd,J=12.3,3.7Hz,1H), 2.21(dt,J=15.2,3.7Hz,1H),1.88(dd,J=13.5,12.3Hz,1H),1.69(d,J= 1.9Hz,1H),1.64–1.55(m,2H),1.45–1.33(m,1H),0.92(d,J=6.5Hz,3H); 13 C NMR(100MHz,CHCl3)δ169.8,110.1,66.4,65.5,64.6,53.0,40.2,35.8,29.4,29.3,21.4.HRMS(ESI+):m / z calc'd for C 11 H 17 BrNaO4[M+Na] + :315.0208,found:315.0202.dr=3.6:1for 1 H NMR.

[0085] Example 4:

[0086]

[0087] Compound 4 (10.0 g, 34.0 mmol) was dissolved in acetonitrile (170 mL), and 2,4,5,6-tetrakis(9-carbazolyl)-isophthalonitrile (538.2 mg, 0.68 mmol), (OC-6-14)-[[2,3-butanedione 2,3-di(oxime-KN)](1-)][2,3-butanedione 2,3-di(oxime-KN)]cobalt dichloro(615 mg, 1.71 mmol), cesium carbonate (4.71 g, 34.1 mmol), and N,N-diisopropylethylamine (1.2 mL, 6.82 mmol) were added at room temperature. Argon gas was used for purging. The reaction solution was placed in a Blue LED and reacted for 16 hours. Saturated ammonium chloride aqueous solution was added for quenching. Acetonitrile was removed by vacuum distillation. Extraction was performed with water and dichloromethane. The mixture was dried over anhydrous sodium sulfate, purified by vacuum distillation, and then purified by silica gel column chromatography (petroleum ether:acetone = 20:1-10:1) to obtain compound 5, with a total yield of 92%. Detection data for compound 5 are as follows: 1H NMR(400MHz,CHCl3)δ7.08(dd,J=5.6,2.4Hz,1H),4.26–4.14(m,2H),4.00( td,J=6.3,5.7,3.4Hz,1H),3.96(d,J=6.5Hz,1H),3.69(s,3H),2.30(td,J= 19.2,z5.2,1.7Hz,1H),2.02–1.92(m,1H),1.89–1.83(m,1H),1.81(q,J=2. 3Hz,1H),1.79–1.72(m,1H),1.42(t,J=13.0Hz,1H),0.97(d,J=6.6Hz,3H); 13 C NMR(100MHz,CHCl3)δ165.6,145.5,131.2,106.5,66.0,65.2,51.4,43.6,34.3,27.0,21.2.HRMS(ESI+):m / z calc'd for C 11 H 17 O4[M+H] + :213.1127,found:213.1123. = -152.77 (c = 2.27, CHCl3).

[0088] Example 5:

[0089]

[0090] Compound 6 (26.9 mL, 194.35 mmol, commercially available) was dissolved in tetrahydrofuran. Diisopropylaminolithium (2.0 M solution in THF:Hexane, 323.9 mL, 647.8 mmol) and hexamethylphosphoric triamine (116 mL, 647.8 mmol) were added at -78 °C and reacted for 10 min. Then, a tetrahydrofuran solution of compound 5 (27.5 g, 129.57 mmol) was added dropwise to the reaction solution and reacted for 2 h. Hydrochloric acid was then added to adjust the pH of the reaction solution to 2, and the reaction was continued for 3 h. The reaction was quenched at 0 °C with 6 M sodium hydroxide aqueous solution. Tetrahydrofuran was removed by vacuum distillation. Extraction was performed with dichloromethane, followed by drying with anhydrous sodium sulfate. Purification was achieved by vacuum distillation and silica gel column chromatography (petroleum ether:ethyl acetate = 100:1-20:1) to obtain compound 7, with a total yield of 72%. Detection data for compound 7 are as follows: 1H NMR(400MHz,CHCl3)δ12.42(s,1H),7.62(d,J=8.6Hz,1H),6.47(d,J=8.6Hz,1H),3.89 (s,3H),3.71(s,3H),3.33–3.17(m,1H),3.06(dd,J=13.6,4.4Hz,1H),2.86(dd,J=10. 2,3.4Hz,1H),2.40(ddd,J=18.3,5.6,1.6Hz,1H),2.26–2.10(m,1H),1.92(dd,J=18.3 ,11.2Hz,1H),1.72–1.52(m,1H),1.14(td,J=12.9,5.3Hz,1H),0.97(d,J=6.5Hz,3H); 13 CNMR(100MHz,CHCl3)δ173.2,173.0,162.2,156.5,142.3,112.0,109.7,100.4,53.4,51.3,41.4,37.6,34.4,32.1,23.4,21.9.HRMS(ESI+):m / z calc'd forC 16 H 21 BrNO4[M+H] + :370.0654,found:370.0653. = +67.93 (c = 2.96, CHCl3).

[0091] Example 6:

[0092]

[0093] Compound 7 (25 g, 67.52 mmol) was dissolved in acetonitrile. Acrylonitrile (6.7 mL, 100.28 mmol) and a solution of tetrabutylammonium hydroxide·30 water (5.4 g, 6.75 mmol) in acetonitrile were added at 0 °C. The reaction was carried out at the same temperature for 6 hours. The reaction was quenched with saturated ammonium chloride aqueous solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, purified by vacuum distillation, and purified by silica gel column chromatography (petroleum ether:acetone = 20:1-9:1) to obtain compound 8 in 74% yield. The detection data for compound 8 are as follows: 1H NMR (400MHz, CHCl3) δ7.63(d,J=8.6Hz,1H),6.50(d,J=8.7Hz,1H),3.85(s,3H),3.78(s,3H),3.19(dd,J=15.0,10. 2Hz,1H),2.99–2.91(m,1H),2.94–2.86(m,1H),2.70(ddd,J=15.7,10.7,4.8Hz,2H),2.56(ddd,J=13.5,10.6,5.1Hz ,1H),2.45(ddd,J=15.9,10.7,5.1Hz,1H),2.39–2.28(m,1H),2.22(ddd,J=13.9,3.5,1.8Hz,1H),2.15(ddd,J=13. 5,10.7,4.7Hz,1H),2.03(ddd,J=14.0,12.2,4.9Hz,1H),1.49(dtd,J=14.1,3.6,1.8Hz,1H),0.86(d,J=7.1Hz,3H); 13 C NMR(100MHz,CHCl3)δ206.8,170.6,162.7,155.0,142.8,119.7,112.6,110.7,6 3.0,53.8,52.7,46.8,38.4,37.5,33.3,29.1,28.0,19.5,13.1.HRMS(ESI+):m / z calc'd for C 19 H 23 BrN₂NaO₄[M+Na] + :445.0739,found:445.0736. = -35.88 (c = 3.45, CHCl3).

[0094] Example 7:

[0095]

[0096] Compound 8 (5 g, 11.87 mmol) was dissolved in N,N-dimethylformamide, and lithium chloride (4 g, 94.95 mmol) was added at room temperature. The mixture was heated to 150 °C and reacted for 3.5 hours. Then, palladium acetate (265 mg, 1.18 mmol), acetaldehyde oxime (3.5 mL, 59.0 mmol), and [other compounds] were added at room temperature. Molecular sieve (2g) was reacted at 100℃ for 10 hours, and then quenched with saturated sodium bicarbonate aqueous solution. Extraction with ethyl acetate, drying with anhydrous sodium sulfate, purification by vacuum distillation, and silica gel column chromatography (petroleum ether:acetone = 9:1-4:1) yielded compound 9, with a yield of 86%. Detection data for compound 9 are as follows:1 H NMR(600MHz,CHCl3)δ7.62(d,J=8.6Hz,1H),7.08(s,1H),6.48(d,J=8.6Hz,1H),3.89(s,3H) ,2.98(dd,J=13.4,3.7Hz,1H),2.79(dd,J=13.4,10.5Hz,1H),2.69(dt,J=9.9,4.3Hz,1H),2 .52–2.49(m,1H),2.49–2.47(m,1H),2.48–2.40(m,1H),2.28–2.18(m,1H),2.12–2.05(m,1H ),2.00(dd,J=16.4,5.5Hz,1H),1.79–1.70(m,1H),1.60–1.53(m,1H),0.98(d,J=6.5Hz,3H); 13 C NMR(150MHz,CHCl3)δ171.6,162.5,156.5,142.6,129.4,113.1,112.4 110.1,53.7,40.0,37.7,35.3,34.6,31.3,24.5,24.3,21.9.HRMS(ESI+):m / z calc'd for C 17 H 22 BrN2O2[M+H] + :365.0865,found:365.0852. = +46.44 (c = 0.44, CHCl3).

[0097] Example 8:

[0098]

[0099] Compound 9 (2 g, 5.48 mmol) was dissolved in toluene (76 mL). Tris(dibenzylacetone)dipalladium (1.5 g, 1.64 mmol), 1,2-bis(diphenylphosphine)benzene (733.3 mg, 1.64 mmol), and cesium carbonate (3.57 g, 10.95 mmol) were added at room temperature. The mixture was heated to 140 °C and reacted for 12 hours. The reaction was quenched with saturated sodium bicarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, purified by vacuum distillation, and purified by silica gel column chromatography (petroleum ether:acetone = 4:1-9:1) to obtain compound 10 in 95% yield. The detection data for compound 10 are as follows: 1H NMR(400MHz,CHCl3)δ8.70(s,1H),7.45(d,J=8.3Hz,1H),6.49(d,J=8.3Hz,1H),5.14(d, J=5.0Hz,1H),3.91(s,3H),3.39(dd,J=16.6,6.6Hz,1H),2.51(s,1H),2.47(q,J=6.4,5. 9Hz,2H),2.36(dt,J=12.3,6.2Hz,1H),2.33–2.24(m,1H),1.90(dt,J=12.8,6.2Hz,1H), 1.83(q,J=7.6,6.6Hz,1H),1.58–1.48(m,1H),1.49–1.42(m,1H),1.05(d,J=7.0Hz,3H); 13 C NMR(100MHz,CHCl3)δ170.8,164.6,161.0,135.5,135.1,131.8,111.8,107.9 ,53.8,46.4,39.6,38.8,33.3,31.2,29.3,27.6,21.8.HRMS(ESI+):m / zcalc'd for C 17 H 21 N₂O₂[M+H] + :285.1603,found:285.1597. = +88.73 (c = 0.66, CHCl3).

[0100] Example 9:

[0101]

[0102] Compound 10 (2 g, 7.03 mmol) was dissolved in N,N-dimethylformamide (35 mL). Sodium hydroxide (60%, 562 mg, 14.07 mmol) and benzyl bromide (1.62 mL, 14.07 mmol) were added at room temperature, and the reaction was carried out for 3 hours. Then, sodium ethanethiol (4.4 g, 52.34 mmol) was added, and the reaction was heated to 120 °C for 6 hours. The reaction was quenched with 3 M hydrochloric acid, and N,N-dimethylformamide was removed by vacuum distillation. The mixture was extracted with water and dichloromethane, dried over anhydrous sodium sulfate, and then subjected to vacuum distillation and silica gel column chromatography (dichloromethane:methanol = 40:1-20:1) to obtain compound 11 in 65% yield. Monitoring data for compound 11 are as follows: 1H NMR(400MHz,CHCl3)δ7.36(t,J=7.2Hz,2H),7.29–7.25(m,3H),6.35(d,J=9.2Hz,1H), 5.49–5.34(m,1H),5.31(d,J=4.8Hz,1H),4.65(d,J=15.5Hz,1H),3.33(dd,J=17.3,7.4 Hz,1H),3.00–2.78(m,1H),2.72–2.54(m,3H),2.51–2.36(m,1H),2.13–1.99(m,2H),1 .66(ddd,J=15.8,9.1,4.0Hz,1H), 1.49(dt,J=13.5,4.1Hz,1H), 1.05(d,J=7.0Hz,3H); 13 C NMR(100MHz,CHCl3)δ169.2,166.3,149.4,139.3,139.1,137.5,128.6,127.2,127.0,12 3.0,116.8,114.5,48.9,47.5,41.6,35.0,32.6,32.1,29.9,27.5,21.4.HRMS(ESI+):m / z calc'd for C 23 H 25 N₂O₂[M+H] + :361.1916,found:361.1915. = +87.75 (c = 0.32, CHCl3).

[0103] Example 10:

[0104]

[0105] Compound 11 (500 mg, 1.39 mmol) was dissolved in 3 M hydrochloric acid and tetrahydrofuran (1:1, 80 mL). Samarium powder was added in portions at 0 °C and reacted for 20 minutes. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, purified by vacuum distillation, and purified by silica gel column chromatography (dichloromethane:methanol = 50:1) to obtain compound 12 in 62% yield. The detection data for compound 12 are as follows: 1H NMR (600 MHz, CHCl₃) δ 7.29 (t, J=7.6 Hz, 2H), 7.23–7.19 (m, 1H), 7.19–7.16 (m, 2H), 6.65 (s, 1H), 5.46 (d, J=15.6 Hz, 1H), 5.15 (d, J=2.4 Hz, 1H), 4.28 (d, J=15.6 Hz, 1H), 3.80 (ddt, J=9.6, 6.4, 3.2 Hz, 1H), 2.88–2.71 (m, 1H), 2.29–2.25 (m, 1H), 2.24 (dt, J=4.5, 2.8 Hz, 1H), 2.18–2.15 (m, 2H), 2.15–2.11 (m, 1H), 2.03 (q, J=6.3, 5.1 Hz, 2H), 1.92 (ddd, J=13.5, 11.6, 7.9 Hz, 1H), 1.80 (ddd, J=13.6, 7.2, 1.9 Hz, 1H), 1.65–1.59 (m, 1H), 1.55 (dt, J=13.3, 3.7 Hz, 1H), 1.42 (qd, J=13.3, 4.5 Hz, 1H), 1.29 (dtd, J=13.0, 5.1, 2.4 Hz, 1H), 1.19 (ddd, J=13.0, 10.1, 5.4 Hz, 1H), 0.89 (d, J=7.0 Hz, 3H); 13 C NMR (150 MHz, CHCl₃) δ 172.3, 168.6, 140.2, 137.6, 128.7, 127.1, 126.8, 116.6, 53.2, 48.9, 44.0, 43.5, 42.0, 38.3, 33.4, 33.3, 31.3, 29.9, 25.8, 21.9, 21.3. HRMS (ESI+): m / z calc'd for C 23 H 29 N₂O₂ [M+H] + : 365.2229, found: 365.2226. = +120.40 (c=0.20, CHCl₃).

[0106] Example 11:

[0107]

[0108] Compound 12 (312 mg, 0.85 mmol) was dissolved in 1,4-dioxane, and 4-dimethylaminopyridine (209 mg, 1.71 mmol) and di-tert-butyl dicarbonate (0.5 mL, 2.14 mmol) were added at 100 °C and reacted for 10 min. The reaction was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (petroleum ether:acetone = 9:1-4:1) to give compound 13 in 98% yield. Monitoring data for compound 13 are as follows: 1 H NMR(400MHz,CHCl3)δ7.23(t,J=7.4Hz,2H),7.16(d,J=7.4Hz,1H),7.15–7.08(m,2H),5.34(d,J=15.6Hz,1H),5 .10(d,J=2.3Hz,1H),4.28(dd,J=13.4,6.1Hz,2H),4.05(q,J=7.2Hz,1H),2.74–2.60(m,1H),2.35(ddd,J=17.6, 4.7,2.4Hz,1H),2.28–2.13(m,3H),2.07(tq,J=6.7,3.1Hz,1H),2.00(d,J=3.5Hz,1H),1.83(ttd,J=11.1,7.0,6 .3,3.6Hz,2H),1.46(s,9H),1.41–1.28(m,2H),1.19(d,J=7.1Hz,2H),1.16–1.10(m,1H),0.84(d,J=6.9Hz,3H); 13 C NMR(100MHz,CHCl3)δ170.6,168.4,153.0,139.8,137.4,128.5,127.0,126.7,116.5,83.2,60.4,57.0,4 8.4,43.3,42.5,41.9,36.1,34.2,33.3,33.0,29.8,28.0,25.7,21.8,21.0,14.2.HRMS(ESI+):m / zcalc'd for C 28 H 36 N₂NaO₄[M+Na] + :487.2573,found:487.2751. = +66.08 (c = 0.47, CHCl3)

[0109] Example 12:

[0110]

[0111] Compound 13 (390 mg, 0.84 mmol) was dissolved in N,N-dimethylformamide. 3,5-di-tert-butyl-[1,1'-biphenyl]-4-phenol (47.4 mg, 0.17 mmol), 2,2,6,6-tetramethylpiperidine oxidol (660 mg, 4.2 mmol), and cesium carbonate (136 mg, 0.42 mmol) were added at room temperature. The reaction was purged with argon and irradiated under a Blue LED lamp for 10 hours. The reaction was quenched with saturated sodium bicarbonate solution, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (petroleum ether:acetone = 4:1-3:2) to obtain compound 14 in 85% yield. Monitoring data for compound 14 are as follows: 1 H NMR(400MHz,CHCl3)δ8.53(s,1H),5.00(d,J=2.0Hz,1H),4.27(dt,J=12.3,6.3Hz,1H),2 .57(ddd,J=19.0,11.2,7.8Hz,1H),2.53–2.43(m,1H),2.43(d,J=3.7Hz,1H),2.34–2.25 (m,1H),2.26–2.13(m,2H),2.07–1.95(m,2H),1.80–1.72(m,2H),1.59–1.49(m,3H),1.4 6(s,9H),1.45–1.36(m,1H),1.11(ddd,J=13.1,11.2,6.2Hz,1H),0.96(d,J=6.9Hz,3H); 13 C NMR(100MHz,CHCl3)δ170.8,170.0,153.2,136.3,114.9,83.4,57.1,42.2,42. 0,42.0,36.0,34.5,34.2,32.8,29.5,28.2,25.1,21.9,21.4.HRMS(ESI+):m / z calc'd for C 21 H 30 N₂NaO₄[M+Na] + :397.2103,found:397.2103.

[0112] = +88.15 (c = 0.4, CHCl3).

[0113] Example 13:

[0114]

[0115] Compound 14 (270 mg, 0.72 mmol) was dissolved in tetrahydrofuran (35 mL), and triethylborohydride sodium (1.0 M solution in THF, 0.86 mL) was added at -78 °C and reacted for 10 min. Then, N,N-diisopropylethylamine (0.37 mL, 2.16 mmol), 4-dimethylaminopyridine (14.2 mg, 0.11 mmol), and trifluoroacetic anhydride (0.22 mL, 2.63 mmol) were added, and the reaction was allowed to return to room temperature for 10 min. The reaction was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, purified by vacuum distillation, and purified by silica gel column chromatography (petroleum ether:acetone = 50:1) to obtain compound 15 in 92% yield. Monitoring data for compound 15: 1 H NMR (400MHz, CHCl3) δ8.74 (d, J = 12.0Hz, 1H), 6.70 (dd, J = 8.5, 2.7Hz, 1H), 5.06 (d, J = 2. 1Hz,1H),4.70(ddd,J=8.5,6.0,2.3Hz,1H),4.33(dt,J=12.2,5.8Hz,1H),2.65–2.52(m ,1H),2.53–2.42(m,1H),2.03(dq,J=12.5,6.7Hz,4H),1.93–1.70(m,4H),1.49(d,J=5. 2Hz,9H),1.17(ddd,J=12.2,6.1,3.4Hz,1H),0.99(d,J=6.9Hz,3H),0.87–0.81(m,1H); 13 CNMR(100MHz,CHCl3)δ170.2,152.4,136.7,123.8,114.7,102.4,80.9,51.5,42.3, 41.6,37.5,34.0,33.8,32.2,29.5,28.3,25.1,21.7,20.8.HRMS(ESI+):m / zcalc'd for C 21 H 31 N₂O₃[M+H] + :359.2335,found:359.2326. = +126.19 (c = 0.21, CHCl3).

[0116] Example 14:

[0117]

[0118] Compound 15 (50 mg, 0.14 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and then LiAlH4 (16 mg, 0.42 mmol) was added at room temperature. The mixture was refluxed for 30 minutes. The solution was then purified by silica gel distillation under reduced pressure and silica gel column chromatography (ethyl acetate-dichloromethane:methanol = 4:1) to obtain compound 16. The starting material was recovered, and the yield was 81%. Monitoring data for compound 16 are as follows: 1 H NMR(400MHz,CHCl3)δ6.72(dd,J=8.5,2.7Hz,1H),4.73(d,J=8.3Hz,1H),4.33(dd,J=12.2,6.2Hz ,1H),4.17(dq,J=11.1,5.6,4.8Hz,1H),3.74–3.53(m,2H),3.32(q,J=9.5,8.5Hz,2H),2.55(dd, J=18.2,5.4Hz,2H),2.34–2.25(m,1H),2.17–2.10(m,1H),2.02–1.96(m,2H),1.87–1.81(m,1H), 1.77(dd,J=11.5,5.4Hz,2H),1.73–1.65(m,2H),1.48(s,9H),1.24(s,1H),0.93(d,J=6.5Hz,3H). 13 C NMR(100MHz,CHCl3)δ176.7,152.6,124.6,102.3,81.1,62.8,52.4,47.0,46. 6,45.4,42.9,34.0,35.7,30.3,28.5,25.9,21.9,21.7,19.3.HRMS(ESI+):m / z calc'd for C 21 H 33 N₂O₂[M+H] + :345.2542,found:345.2534. =-7.15(c=1.2,CHCl3).dr=1:1.64for 1H NMR.

[0119] Example 15:

[0120]

[0121] Compound 16 (17 mg, 0.047 mmol) was dissolved in trifluoroethanol (2 mL), and trifluoroacetic acid (16 μL, 0.24 mmol) was added at room temperature. The mixture was then heated to 120 °C and reacted for 12 hours. The reaction was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, purified by vacuum distillation, and purified by silica gel column chromatography (dichloromethane:methanol = 9:1-3:2) to obtain compound 17 in 75% yield. Monitoring data for compound 17 are as follows: 1 H NMR(600MHz,CHCl3)δ3.70(dtd,J=16.6,4.2,1.8Hz,1H),3.51(t,J=5.5Hz,1H),3.39(ddd,J=16.7,9.5,3.8Hz,1H ),3.09(t,J=6.3Hz,1H),2.91–2.76(m,1H),2.32–2.26(m,1H),2.27–2.21(m,1H),2.19(d,J=6.7Hz,1H),2.17(d, J=6.3Hz,1H),1.88–1.83(m,1H),1.85–1.79(m,1H),1.77(ddd,J=11.2,5.3,1.8Hz,1H),1.70–1.63(m,3H),1.62– 1.56(m,2H),1.53(ddd,J=13.8,6.1,2.6Hz,2H),1.49–1.43(m,1H),1.39(d,J=13.7Hz,1H),1.03(d,J=6.7Hz,3H). 13 C NMR(150MHz,CHCl3)δ177.7,59.8,56.7,51.0,50.4,49.7,49.6,49.2,42.8,37.4,36.2,30.8,23.6,23.3,20.8,17.2.HRMS(ESI+):m / z calc'd for C 16 H 25 N2[M+H] + :245.2018,found:245.2012. = +10.32 (c = 0.06, CHCl3).

[0122] Example 16:

[0123]

[0124] Formic acid (23 μL, 0.6 mmol) and acetic anhydride (21 μL, 0.2 mmol) were dissolved in tetrahydrofuran (1 mL), and the mixture was heated to 70 °C and reacted for 1 hour. The mixture was then cooled to 0 °C, and a tetrahydrofuran solution of compound 17 (10 mg, 0.04 mmol) was added dropwise to the reaction mixture and reacted for 1 hour. The reaction was quenched with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, purified by vacuum distillation, and purified by silica gel column chromatography (dichloromethane:methanol = 80:1-25:1) to obtain compound 18 (i.e., the target product "Hupserrine A" alkaloid compound), with a yield of 83%. Monitoring data for compound 18 are as follows: 1 H NMR (800MHz, Methanol-d4) δ8.11(s,1H),4.23(t,J=5.8Hz,1H),3.87(t,J=6.6Hz,1H),3.65(dt,J=16.9,4.0Hz,1H),3.40(ddd, J=16.7,9.9,4.2Hz,1H),2.37(dt,J=10.4,3.5Hz,1H),2.33–2.29(m,1H),2.27(dd,J=7.3,1.5Hz,1H),2.15(dd,J=6.3,4.6Hz,1 H),1.91–1.86(m,1H),1.81(dq,J=12.5,6.2Hz,1H),1.76(dd,J=8.3,4.2Hz,3H),1.73(ddd,J=9.8,4.8,3.2Hz,1H),1.69(ddd,J =11.6,4.4,2.2Hz,2H),1.67–1.64(m,1H),1.51(dddd,J=22.9,14.0,5.9,2.8Hz,2H),1.03(d,J=6.7Hz,3H),1.01–0.99(m,1H). 13 C NMR(200MHz,Methanol-d4)δ178.5,164.8,59.2,58.2,53.5,50.5,50.3,39.0,37.8,36.8,32.5,24.5,23.4,21.5,17.4.HRMS(ESI+):m / z calc'd for C 17 H 25 N₂O[M+H] + :273.1967,found:273.1964. = -47.62 (c = 0.79, MeOH).

[0125] Example 17:

[0126] (1) 17 kg of dried whole Huperzia serrata herb was crushed and extracted three times with methanol. After removing the methanol, the extract was obtained, acidified with 3% tartaric acid, and extracted with ethyl acetate. The pH of the acidic aqueous layer was adjusted to 9 with saturated sodium carbonate, and extracted with chloroform to obtain 65 g of total alkaloids.

[0127] (2) The total alkali was subjected to silica gel column segmentation, with the eluent successively being petroleum ether / acetone / diethylamine (90:10:0.5–0:100:0.5) to dichloromethane / methanol (90:10–0:100), to obtain 4 components Fr.1–Fr.4;

[0128] (3) Fr.3 (8.8 g) was fragmented using a chloroform / methanol column (97:3–0:100) to obtain two fractions, Fr.3.1–Fr.3.2. Fr.3.1 was fragmented using a C18-CE column to obtain six fractions, Fr.3.1.1–Fr.3.1.6. Among them, Fr.3.1.5 was fragmented using a silica gel column (petroleum ether / acetone / diethylamine, 90:10:0.5–0:100:0.5) to obtain Fr.3.1.5.1 and Fr.3.1.5.2. Fr.3.1.5.1 was purified using a silica gel column (chloroform / methanol, 97:3–95:5) to obtain compound (-)-Hupserrine A. Monitoring data for compound (-)-Hupserrine A: 1 H NMR (800MHz, Methanol-d4) δ8.11(s,1H),4.23(t,J=5.8Hz,1H),3.87(t,J=6.6Hz,1H),3.65(dt,J=16.9,4.0Hz,1H),3.40(ddd, J=16.7,9.9,4.2Hz,1H),2.37(dt,J=10.4,3.5Hz,1H),2.33–2.29(m,1H),2.27(dd,J=7.3,1.5Hz,1H),2.15(dd,J=6.3,4.6Hz,1 H),1.91–1.86(m,1H),1.81(dq,J=12.5,6.2Hz,1H),1.76(dd,J=8.3,4.2Hz,3H),1.73(ddd,J=9.8,4.8,3.2Hz,1H),1.69(ddd,J =11.6,4.4,2.2Hz,2H),1.67–1.64(m,1H),1.51(dddd,J=22.9,14.0,5.9,2.8Hz,2H),1.03(d,J=6.7Hz,3H),1.01–0.99(m,1H). 13C NMR(200MHz,Methanol-d4)δ178.5,164.8,59.2,58.2,53.5,50.5,50.3,39.0,37.8,36.8,32.5,24.5,23.4,21.5,17.4.HRMS(ESI+):m / zcalc'd for C 17 H 25 N₂O[M+H] + :273.1967,found:273.1964. = -47.62 (c = 0.79, MeOH).

[0129] Example 18:

[0130] Experimental study on the inhibitory activity of the 5-HA3A ligand-gated ion channel of the lycophyte alkaloid compound (-)-Hupserrine A in this invention:

[0131] (1) Cell culture and treatment;

[0132] HEK293 cells stably expressing 5-HT3A were cultured in 35 mm diameter cell culture dishes at 37°C in a 5% CO2 incubator. The culture medium consisted of 90% DMEM, 10% tetracycline-regulated fetal bovine serum (DF-FBS), 15 μg / mL blisterdin, and 50 μg / mL hygromycin B. When the cell density reached 80%-90%, 2 μg / mL doxycycline was added for induction, and the cells were used for patch-clamp assays the following day. On the day of assay, the cell culture medium was aspirated, the cells were rinsed once with extracellular fluid, and then 0.25% trypsin-EDTA solution was added for digestion at room temperature for 3-5 minutes. The digestion solution was aspirated, the cells were resuspended in extracellular fluid, and then transferred to experimental dishes for electrophysiological recording.

[0133] (2) Preparation of (-)-Hupserrine A and the positive control compound Quinine;

[0134] The alkaloid compound (-)-Hupserrine A was prepared into a 50 mM stock solution using DMSO. This stock solution was then serially diluted 3-fold with DMSO to obtain intermediate concentrations. These intermediate concentrations were then further diluted with extracellular fluid (containing 10 μM 5HT) to obtain the final concentration required for testing. The DMSO content in the final test concentration did not exceed 0.2%, as this concentration of DMSO had no effect on the 5-HT3A channel current.

[0135] (3) Electrophysiological recording process;

[0136] HEK293 cells stably expressing the 5-HT3A receptor channel were used to record whole-cell currents induced by 10 μM 5-HT stimulation at room temperature using whole-cell patch-clamp technique. The glass microelectrode was fabricated from a glass electrode blank (BF150 86-10, Sutter) using a stretching apparatus. The tip resistance after perfusion with electrode fluid was approximately 2-5 MΩ. The glass microelectrode was simply inserted into the amplifier probe to connect to the patch-clamp amplifier. Clamping voltage and data recording were controlled and recorded via computer using pClamp software, with a sampling frequency of 10 kHz and a filtering frequency of 2 kHz. After obtaining whole-cell recordings, cells were clamped at -60 mV. A rapid drug delivery system was used to administer 10 μM 5-HT by gravity for approximately 10 seconds to induce channel currents. Stimulation was repeated at least twice to observe the amplitude of the induced current, with a 1-minute interval between each stimulation. Once the induced current stabilized, the target lycopodium alkaloid compound (-)-Hupserrine A was administered for at least 1 minute, followed by stimulation with 10 μM 5-HT (containing the target lycopodium alkaloid compound (-)-Hupserrine A) to observe changes in current amplitude before and after administration. The lycopodium alkaloid compound (-)-Hupserrine A was administered continuously from low to high concentrations. At least 3 cells (n≥3) were tested for each concentration of the lycopodium alkaloid compound (-)-Hupserrine A.

[0137] (4) Data processing;

[0138] Data analysis and processing were performed using pClamp, GraphPad Prism 8, and Excel software. The inhibitory effect of different compound concentrations on channel current (whole-cell current amplitude induced by 10 μM 5-HT stimulation at -60 mV) was calculated using the following formula: Inhibition% = [1 – (I / Io)] × 100%, where Inhibition% represents the percentage of inhibition of the 5-HT3A channel current by the compound, and I and Io represent the current amplitude induced by 10 μM 5-HT stimulation before and after drug administration, respectively. The IC50 of the compound was calculated using GraphPad Prism 8 software by fitting the following equation: Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)), where X is the Log value of the detected concentration of the test sample, Y is the inhibition percentage at the corresponding concentration, and Bottom and Top are the minimum and maximum inhibition percentages, respectively.

[0139] (5) Quality control;

[0140] Whole-cell sealing impedance >1 GΩ, series resistance less than 10 MΩ, current amplitude change induced by two consecutive stimulations before drug administration not exceeding 10% and ≥400 pA.

[0141] (6) Test results (see Table 1 and Table 2).

[0142] Table 1 summarizes the data and IC50 values ​​of the effects of (-)-Hupserrine A and the positive control drug Quinine on the 5-HT3A channel current.

[0143]

[0144]

[0145] Table 2. Channel current (pA) of (-)-Hupserrine A and the positive control drug Quinine

[0146] and inhibition rate (inhibition%)

[0147] (-)-Hupserrine A(pA)

[0148]

[0149]

[0150] Quinine(pA)

[0151]

[0152] Formulation Example 1:

[0153] The alkaloid compound (-)-Hupserrine A from lycophyte was added with water for injection according to standard procedures, filtered, filled, and sterilized to prepare an injection solution. Example 2:

[0154] Take the alkaloid compound (-)-Hupserrine A from lycopodium, dissolve it in sterile water for injection, stir until dissolved, filter with a sterile suction funnel, then filter aseptically, dispense into ampoules, freeze-dry at low temperature, and then aseptically seal to obtain powder for injection.

[0155] Formulation Example 3:

[0156] The alkaloid compound (-)-Hupserrine A from lycopodium was added to the excipient in a weight ratio of 9:1 to obtain a powder.

[0157] Formulation Example 4:

[0158] Take the alkaloid compound (-)-Hupserrine A from lycopodium, add it to the excipient at a weight ratio of 1:5 to 1:10, and then granulate and compress the mixture into tablets.

[0159] Formulation Example 5:

[0160] The alkaloid compound (-)-Hupserrine A from lycopodium was prepared into an oral liquid using conventional oral liquid preparation methods.

[0161] Formulation Example 6:

[0162] Take the alkaloid compound (-)-Hupserrine A from lycopodium, add it to the excipient at a weight ratio of 5:1, and prepare it into capsules, granules, or powders.

[0163] Formulation Example 7:

[0164] Take the alkaloid compound (-)-Hupserrine A from lycopodium, add it to the excipient in a weight ratio of 3:1, and prepare it into capsules, granules, or powders.

[0165] The embodiments listed in this invention are intended to demonstrate possible implementations of the invention and are merely for reference only, not to limit the scope of the invention. Within the spirit and scope of this invention, those skilled in the art can make various adjustments, improvements, and optimizations to the specific details of the embodiments according to their own needs and actual conditions, thereby deriving a variety of different variations and modifications. These adjusted and improved solutions, as long as they do not exceed the core concept and technical scope of this invention, should be considered equivalent technical solutions to this invention and naturally fall within the protection scope of this invention. Therefore, the protection scope of this invention should not be limited to the specific embodiments described in the text, but should cover all equivalent technical solutions based on the core ideas of this invention.

Claims

1. The lycophyte alkaloid compound (-)-Hupserrine A, as shown in the following structural formula, 2. A pharmaceutical composition comprising a therapeutically effective amount of the lycopodium alkaloid compound (-)-Hupserrine A of the structural formula of claim 1, and a pharmaceutically acceptable carrier.

3. The use of the lycophyte alkaloid compound (-)-Hupserrine A as shown in the structural formula of claim 1, or the pharmaceutical composition of claim 2, in the preparation of a medicament for treating epilepsy, pain, and sleep disorders.

4. The use of the lycophyte alkaloid compound (-)-Hupserrine A as shown in the structural formula of claim 1, or the pharmaceutical composition of claim 2, in the preparation of a medicament for regulating intestinal motility, secretion, and vomiting.

5. The use of the lycophyte alkaloid compound (-)-Hupserrine A as shown in claim 1, or the pharmaceutical composition of claim 2, in the preparation of a medicament for treating anxiety and depression.

6. The use of the lycophyte alkaloid compound (-)-Hupserrine A as shown in claim 1, or the pharmaceutical composition of claim 2, in the preparation of 5-HA3A ligand-gated ion channel inhibitors.

7. The method for synthesizing and preparing the lycophyte alkaloid compound (-)-Hupserrine A as shown in claim 1, characterized in that, The method includes the following steps: Where Boc represents tert-butyloxycarbonyl and Bn represents benzyl. (1) The compound shown in Formula 1 undergoes a retro-Aldol reaction under acidic conditions, and then undergoes an esterification reaction with dimethyl carbonate to generate the intermediate compound shown in Formula 2. (2) The intermediate compound shown in Formula 2 undergoes a halogenation reaction with N-bromosuccinimide to generate the intermediate compound shown in Formula 3. (3) The intermediate compound shown in Formula 3 undergoes a protective reaction with ethylene glycol to generate the intermediate compound shown in Formula 4; (4) The intermediate compound shown in formula 4 undergoes a free radical elimination reaction in the photocatalyst to generate the intermediate compound shown in formula 5. (5) The intermediate compound shown in Formula 6 undergoes a Michael addition reaction with the intermediate compound shown in Formula 5 under alkaline conditions, and then undergoes a deprotection reaction under acidic conditions to generate the intermediate compound shown in Formula 7. (6) The intermediate compound shown in Formula 7 undergoes a Michael addition reaction with acrylonitrile to generate the intermediate compound shown in Formula 8; (7) The intermediate compound shown in Formula 8 undergoes a decarboxylation reaction with lithium salt, and then a hydrolysis-cyclization reaction is carried out with the addition of palladium catalyst to generate the intermediate compound shown in Formula 9. (8) The intermediate compound shown in Formula 9 undergoes a Heck reaction in a palladium catalyst to generate the intermediate compound shown in Formula 10; (9) The intermediate compound shown in Formula 10 undergoes a protective reaction with benzyl bromide, followed by a demethylation reaction to generate the intermediate compound shown in Formula 11. (10) The intermediate compound shown in Formula 11 undergoes a dearomatization reaction with samarium powder to generate the intermediate compound shown in Formula 12; (11) The intermediate compound shown in Formula 12 undergoes a protective reaction with ditert-butyl dicarbonate to generate the intermediate compound shown in Formula 13; (12) The intermediate compound shown in Formula 13 undergoes a deprotection reaction with the photocatalyst to generate the intermediate compound shown in Formula 14; (13) The intermediate compound shown in Formula 14 undergoes a reduction reaction with a reducing agent, and then undergoes a dehydration reaction with an acid anhydride to generate the intermediate compound shown in Formula 15. (14) The intermediate compound shown in Formula 15 undergoes a reduction reaction with a reducing agent to generate the intermediate compound shown in Formula 16; (15) The intermediate compound shown in Formula 16 reacts with an acid in a Mannich reaction to produce the intermediate compound shown in Formula 17. (16) The intermediate compound shown in Formula 17 undergoes an amidation reaction with an acid anhydride to generate the target product shown in Formula 18, lycopodium alkaloid compound (-)-Hupserrine A.

8. The method for synthesizing lycophyte alkaloids having (-)-Hupserrine A according to claim 7, characterized in that: In step (1), the raw material (+)-pulegone shown in Formula 1 is added to water, and cetylpyridinium chloride and concentrated sulfuric acid are added at 0°C. The mixture is heated to 120°C and reacted for 6 hours. Then, the oily compound is distilled off, and saturated sodium chloride aqueous solution and dichloromethane are added for extraction. The mixture is dissolved in tetrahydrofuran by vacuum distillation. Sodium hydride and dimethyl carbonate are added at 0°C, and the mixture is refluxed for 6 hours. The molar ratio of the compound (+)-pulegone, cetylpyridinium chloride, concentrated sulfuric acid, sodium hydride, and dimethyl carbonate shown in Formula 1 is 1:5%:2:1.5:1.

2. step (2) The intermediate compound 2 shown in Formula 2 is dissolved in dichloromethane, and azobisisobutyronitrile and N-bromosuccinimide are added at 0°C and reacted for 0.5 hours. The molar ratio of compound 2, azobisisobutyronitrile and N-bromosuccinimide is 1:0.2:2.

5. step (3) The intermediate compound 3 shown in Formula 3 is dissolved in ethylene glycol, and trimethylchlorosilane is added at room temperature. The mixture is heated to 40°C and reacted for 10 hours. The molar ratio of compound 3 shown in Formula 3 to trimethylchlorosilane is 1:2.

5. In step (4), intermediate compound 4 of formula 4 is dissolved in acetonitrile, and 2,4,5,6-tetrakis(9-carbazolyl)-isophthalonitrile is added at room temperature. (OC-6-14)-[[2,3-butanedione 2,3-di(oxime-κN)](1-)][2,3-butanedione 2,3-di(oxime-κN)]cobalt dichloro, N,N-diisopropylethylamine, and potassium carbonate were reacted at room temperature under Blue LED irradiation for 16 hours. The molar ratio of compound 4 (as shown in Formula 4), 2,4,5,6-tetra(9-carbazolyl)-isophthalonitrile, (OC-6-14)-[[2,3-butanedione 2,3-di(oxime-κN)](1-)][2,3-butanedione 2,3-di(oxime-κN)]cobalt dichloro, N,N-diisopropylethylamine, and potassium carbonate was 1:2%:5%:20%:

1. In step (5), intermediate compound 6 of formula 6 is dissolved in tetrahydrofuran. Diisopropylaminolithium solution and hexamethylphosphoric triamine are added at -78°C and reacted for 10 minutes. Then, intermediate compound 5 of formula 5 is added dropwise to the reaction mixture in tetrahydrofuran solution and reacted for 2 hours. Then, 3M hydrochloric acid is added to adjust the pH to 2 and reacted for 2 hours. The molar ratio of intermediate compound 6 of formula 6, diisopropylaminolithium solution, hexamethylphosphoric triamine, intermediate compound 5 of formula 5, and 3M hydrochloric acid is 1:5:5:1:

15. step (6) The intermediate compound 7 shown in Formula 7 is dissolved in acetonitrile, and allyl nitrile and tetrabutylammonium hydroxide solution are added at 0°C and reacted for 6 hours. The molar ratio of compound 7, allyl nitrile and tetrabutylammonium hydroxide is 1:1.5:0.

1. In step (7), intermediate compound 8 of formula 8 is dissolved in an organic solvent, and anhydrous lithium chloride is added at room temperature. The mixture is then heated to 150°C and reacted for 3.5 hours. Then, palladium acetate, acetaldehyde oxime, and molecular sieve are added at room temperature, and the mixture is heated to 120°C and reacted for 10 hours. The molar ratio of compound 8 of formula 8, anhydrous lithium chloride, palladium acetate, and acetaldehyde oxime is 1:8:0.2:

5. In step (8), intermediate compound 9 of formula 9 is dissolved in toluene, and tris(dibenzylacetone)dipalladium, 1,2-bis(diphenylphosphine)benzene and cesium carbonate are added at room temperature. The mixture is heated to 140°C and reacted for 12 hours. The molar ratio of intermediate compound 9 of formula 9, tris(dibenzylacetone)dipalladium, 1,2-bis(diphenylphosphine)benzene and cesium carbonate is 1:0.3:0.3:

2. In step (9), intermediate compound 10 of formula 10 is dissolved in N,N-dimethylformamide, sodium hydrogen and benzyl bromide are added at room temperature and reacted for 3 hours, then sodium ethanethiol is added and the temperature is raised to 120°C and reacted for 6 hours. The molar ratio of intermediate compound 10 of formula 10, sodium hydrogen, benzyl bromide and sodium ethanethiol is 1:2:2:

8. In step (10), intermediate compound 11 of formula 11 is dissolved in tetrahydrofuran and 3M hydrochloric acid, and samarium powder is added in batches at 0°C and reacted for 20 minutes. The molar ratio of intermediate compound 11 of formula 11 to samarium powder is 1:

16. In step (11), intermediate compound 12 of formula 12 is dissolved in 1,4-dioxane, and 4-dimethylaminopyridine and ditert-butyl dicarbonate are added at 100°C and reacted for 10 minutes. The molar ratio of intermediate compound 12 of formula 12, 4-dimethylaminopyridine and ditert-butyl dicarbonate is 1:2:2.

5. In step (12), intermediate compound 13 of formula 13 is dissolved in N,N-dimethylformamide, and 3,5-di-tert-butyl-[1,1'-biphenyl]-4-phenol, 2,2,6,6-tetramethylpiperidine oxidol, and cesium carbonate are added at room temperature. The reaction is carried out under LED irradiation for 10 hours. The molar ratio of intermediate compound 13 of formula 13, 3,5-di-tert-butyl-[1,1'-biphenyl]-4-phenol, 2,2,6,6-tetramethylpiperidine oxidol, and cesium carbonate is 1:0.2:5:0.

5. In step (13), intermediate compound 14 of formula 14 is dissolved in tetrahydrofuran, and triethyl borohydride is added at -78°C and reacted for 10 minutes. Then, N,N-diisopropylethylamine, 4-dimethylaminopyridine, and trifluoroacetic anhydride are added, and the mixture is heated to room temperature and reacted for 10 minutes. The molar ratio of intermediate compound 14 of formula 14, triethyl borohydride, N,N-diisopropylethylamine, 4-dimethylaminopyridine, and trifluoroacetic anhydride is 1:1.25:3:0.2:2.

8. In step (14), intermediate compound 15 of formula 15 is dissolved in tetrahydrofuran, lithium aluminum hydride is added at room temperature, and the mixture is heated to reflux for 30 minutes. The molar ratio of intermediate compound 15 of formula 15 to lithium aluminum hydride is 1:

3. In step (15), intermediate compound 16 of formula 16 is dissolved in trifluoroethanol, trifluoroacetic acid is added at room temperature, and the temperature is raised to 120°C and reacted for 12 hours. The molar ratio of intermediate compound 16 of formula 16 to trifluoroacetic acid is 1:

5. In step (16), intermediate compound 17 of formula 17 is dissolved in tetrahydrofuran. Formic acid and acetic anhydride are dissolved in tetrahydrofuran at room temperature. The temperature is raised to 70°C and reacted for 1 hour. Then the temperature is lowered to 0°C and intermediate compound 17 of formula 17 is added dropwise to the reaction solution and reacted for 1 hour. The molar ratio of intermediate compound 17 of formula 17, formic acid and acetic acid is 1:15:

5.

9. The method for preparing the lycophyte alkaloid compound (-)-Hupserrine A according to claim 1, characterized in that, The method includes the following steps: The dried whole herb of *Huperzia serrata* was pulverized and extracted three times with methanol. After removing the methanol, an extract was obtained, acidified with 3% tartaric acid, extracted with ethyl acetate, and the pH was adjusted to 9 by saturating the aqueous layer with sodium carbonate. Chloroform extraction was then performed to obtain the total alkaloids. The total alkaloids were subjected to silica gel column chromatography, with eluents successively changing from 90:10:0.5–0:100:0 petroleum ether / acetone / diethylamine to 90:10–0:100 dichloromethane / methanol, yielding four fractions Fr.1–Fr.

4. Fr.3 was further fractionated with chloroform / methanol at a ratio of 97:3–0:100 to obtain two fractions. Fr.3.1–Fr.3.2; Fr.3.1 was fragmented on a C18-CE column to obtain 6 fractions Fr.3.1.1–Fr.3.1.6; Fr.3.1.5 was fragmented on a silica gel column with petroleum ether / acetone / diethylamine in a ratio of 90:10:0.5–0:100:0.5 to obtain Fr.3.1.5.1 and Fr.3.1.5.2; Fr.3.1.5.1 was purified on a silica gel column with a ratio of chloroform / methanol in a ratio of 97:3–95:5 to obtain the lycopodium alkaloid compound (-)-Hupserrine A. 10.5-HA3A ligand-gated ion channel inhibitor, characterized in that... It contains the lycophyte alkaloid compound (-)-Hupserrine A as shown in the structural formula of claim 1.

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