Analgesic compound having nav1.2 inhibitory effect in radix anacycli pyrethri, and preparation and use thereof

Abstract

The present invention relates to a method for preparing a highly conjugated tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloid compound having a unique 8,14,18,24-tetraazaoctacyclic [21.2.21,4.1.02,21.03,18.05,17.09,16.011,15] nonacosane ring system, and the anti-inflammatory and analgesic use of the compound. With regard to the tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloid compound, the root of Anacyclus pyrethrum (L.) DC is extracted using an organic solvent, the resulting extract is extracted, separation is then performed by means of two to three methods of normal-phase silica-gel column chromatography, reversed-phase silica-gel column chromatography and semi-preparative high-performance liquid chromatography to obtain four tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloid monomer compounds having a new skeleton, and the structures of the compounds are identified by means of methods such as high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy. Further provided in the present invention are the Nav1.2 inhibitory effect and NO inhibitory effect of the compounds.

Description

Analgesic compound with Nav1.2 inhibitory effect from Anachia root and its preparation and application Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to analgesic / anti-inflammatory compounds (compounds of formula 1 and formula 2) in Anachia root that have Nav1.2 inhibitory effects and NO inhibitory effects, as well as their preparation and application. Background Art

[0002] Pain is the body's defense mechanism against disease and is considered the fifth vital sign. However, persistent and severe pain can impact patients' mental health, causing anxiety, distress, and weakened immunity, leading to a range of consequences, including, in severe cases, fatality or disability. However, traditional analgesics such as opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) have side effects such as tolerance, addiction, and gastrointestinal bleeding.

[0003] Ion channels regulate the passage of ions in and out of cells to adjust the voltage potential across the membrane. The flow of ions generates electrical pulses, which in turn prompt adjacent voltage-sensitive channels to open continuously, resulting in spontaneous electrical signals. By blocking ion channels and inhibiting the ongoing rhythmic potential, signal transduction can be prevented, thereby reducing pain responses. Currently, various types of ion channel inhibitors have been reported, including VGSCs, VGCCs, VGPCs, and TRPs. Sodium channels are responsible for the generation and propagation of action potentials and are the basic elements for all excitable cells (including nerve and muscle cells) to generate electrical signals. In the human body, there are nine subtypes of sodium channels, named Nav1.1-Nav1.9. For example, lidocaine reduces the response to pain by inhibiting Nav1.7 and Nav1.9. Therefore, it is very important to find new ion channel inhibitors with analgesic potential.

[0004] Exploring lead compounds with significant pharmacological activity from traditional medicinal plants is a hot topic in drug research. Therefore, discovering novel ion channel inhibitors and compounds with anti-inflammatory effects from medicinal plants is an effective way to develop new analgesic / anti-inflammatory drugs.

[0005] Anachy root is the dried root of the Roman pyrethrum (Anacyclus pyrethrum (L.) DC.) of the Asteraceae family. It is used to clear the brain (open the orifices), treat hemiplegia, vitiligo, epilepsy, headache, cough and phlegm, convulsions, etc.

[0006] Summary of the Invention

[0007] After intensive research, the inventors isolated for the first time analgesic / anti-inflammatory compounds (compounds of Formula 1 and Formula 2) with Nav1.2 inhibitory and NO inhibitory effects from the roots of Anachia. These compounds are novel skeleton compounds, which are highly conjugated tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloid compounds. Their structures were identified, and cell experiments verified that they have Nav1.2 inhibitory and NO inhibitory effects, and can be used to prepare analgesic and / or anti-inflammatory drugs.

[0008] Therefore, the present invention provides the following:

[0009] 1. A compound selected from the following compounds or their isomers:

[0010] 7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methanoaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepina[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione (compound of Formula 1); and

[0011] 7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepina[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione (compound of formula 2).

[0012] 2. The compound according to 1 above or an isomer thereof, wherein the isomer is an enantiomer.

[0013] 3. The compound according to 1 or 2 above or its isomer, which is selected from the group consisting of:

[0014] (4aR,7aR,11aS,14aR)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methanoaza[4,5,6-de]pyrrolo[3″',2″':4″,5″]cyclopenta[1″,2″:6′,7′]aza[4′,5′:4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione;

[0015] (4aS,7aS,11aR,14aS)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3″′,2″′:4″,5″]cyclopenta[1″,2″:6′,7′]azepina[4′,5′:4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione;

[0016] (4aR,7aR,11aR,14aS)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepino[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione; and

[0017] (4aS,7aS,11aS,14aR)-7a-Acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3″′,2″′:4″,5″]cyclopenta[1″,2″:6′,7′]azepina[4′,5′:4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione.

[0018] 4. The compound according to any one of 1 to 3 above or its isomer, which has a structural formula selected from the following:

[0019] 5. A method for extracting the compound according to any one of 1 to 4 above or its isomer from the root of Anachia, comprising the following steps:

[0020] a. Dried anacardia root was crushed and extracted with 50-95% (v / v) ethanol aqueous solution, methanol or chloroform as solvent (ratio of medicinal material weight (kg) to solvent (L) was 1:1.5-1:4) by cold soaking, percolation, heating under reflux or ultrasonic extraction, and the solvent was recovered by vacuum concentration to obtain an extract;

[0021] b. The total extract of step a is suspended in water and dispersed with an acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid. The obtained acidic aqueous layer is extracted with dichloromethane to remove non-alkaloids and then adjusted to pH 10-12 with a base such as NaHCO3, Na2CO3, ammonia water or NaOH. The extract is then extracted with an organic solvent such as dichloromethane, ethyl acetate or n-butanol, and the organic solvent is recovered by concentration under reduced pressure to obtain total alkaloids.

[0022] c. Separating the total alkaloids in step b by silica gel column chromatography, thin layer chromatography, reverse phase MCI column chromatography, Sephadex LH-20 column chromatography, high performance liquid chromatography, or any combination thereof to obtain the compound or its isomers.

[0023] 6. The method according to 5 above, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography and reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography. Preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography is used to obtain the compound of Formula 1 or Formula 2. More preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 100:0 to 3:1, dichloromethane and methanol in a volume ratio of 500:1 to 3:1, The volume ratio of petroleum ether and acetone is 50:1 to 0:1, or the volume ratio of methanol and water is 1:9-1:0. The eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution with a volume ratio of 20-100% (v / v) or a 20-100% (v / v) acetonitrile aqueous solution. The eluent for isocratic or gradient elution used in the semi-preparative high-performance liquid chromatography is n-hexane / EtOH with a volume ratio of 99-50% (v / v).

[0024] 7. The method according to 5 above, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography. Preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, gradient elution is performed using reverse phase silica gel or reverse phase MCI column chromatography, and then semi-preparative high performance liquid chromatography is used to obtain the compound of Formula 1 or Formula 2. More preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol, or chloroform and methanol in a volume ratio of 100:1 to 0:1, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is a methanol aqueous solution with a volume ratio of 20-100% (v / v) or a acetonitrile aqueous solution with a volume ratio of 20-100% (v / v), and the eluent for isocratic or gradient elution in the semi-preparative high performance liquid chromatography is n-hexane / EtOH with a volume ratio of 99-50% (v / v).

[0025] 8. The method according to 5 above, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, Sephadex LH-20 column chromatography, reverse phase silica gel or reverse phase MCI column chromatography, and semi-preparative high performance liquid chromatography. Preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, the mixture is subjected to Sephadex LH-20 column chromatography, gradient elution using reverse phase silica gel or reverse phase MCI column chromatography, and then semi-preparative high performance liquid chromatography to obtain the compound of Formula 1 or Formula 2. More preferably, the eluent used in the normal phase silica gel column chromatography is The eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution with a volume ratio of 20-100% (v / v) or a 20-100% (v / v) acetonitrile aqueous solution, and the eluent used in the semi-preparative high performance liquid chromatography for isocratic or gradient elution is n-hexane / EtOH with a volume ratio of 99-50% (v / v).

[0026] 9. The method according to any one of 5 to 8 above, wherein in step c, the silica gel column chromatography is normal pressure or pressurized column chromatography, and / or the filler used is normal silica gel or reverse phase silica gel.

[0027] 10. Use of the compound or its isomer according to any one of 1 to 4 above in the preparation of analgesic or anti-inflammatory drugs, preferably, the compound or its isomer exerts analgesic effect by inhibiting Nav1.2, and exerts anti-inflammatory effect by inhibiting NO.

[0028] Detailed Description of the Invention

[0029] The purpose of the present invention is to provide a compound having Nav1.2 inhibitory effect and / or anti-inflammatory effect, as well as a method for separating and preparing the compound and its application value in preparing analgesic drugs.

[0030] According to the present invention, in a first aspect, a compound having Nav1.2 inhibitory effect and / or anti-inflammatory effect is provided, and the structural formula of the compound is shown in the figure below;

[0031] in:

[0032] Compound (+)-1 is: (4aR,7aR,11aS,14aR)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepina[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione;

[0033] Compound (-)-1 is: (4aS,7aS,11aR,14aS)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepina[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione;

[0034] Compound (+)-2 is: (4aR,7aR,11aR,14aS)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepinazo[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione;

[0035] Compound (-)-2 is: (4aS,7aS,11aS,14aR)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepina[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione.

[0036] The extraction and separation method of the above-mentioned alkaloid compounds is carried out according to the following steps:

[0037] a. Dried anacardia root was crushed and extracted with 50-95% (v / v) ethanol aqueous solution, methanol or chloroform as solvent (ratio of medicinal material weight (kg) to solvent (L) was 1:1.5-1:4) by cold soaking, percolation, heating under reflux or ultrasonic extraction, and the solvent was recovered by vacuum concentration to obtain an extract;

[0038] b. The total extract of step a is suspended in water and dispersed with an acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid. The obtained acidic aqueous layer is extracted with dichloromethane to remove non-alkaloids and then adjusted to pH 10-12 with a base such as NaHCO3, Na2CO3, ammonia water or NaOH. The extract is then extracted with an organic solvent such as dichloromethane, ethyl acetate or n-butanol, and the organic solvent is recovered by concentration under reduced pressure to obtain total alkaloids.

[0039] c. Separating the total alkaloids in step b by silica gel column chromatography, thin layer chromatography, reverse phase MCI column chromatography, Sephadex LH-20 column chromatography, high performance liquid chromatography, or any combination thereof to obtain the compound or its isomers.

[0040] There are two separation methods:

[0041] The eluent used for normal phase silica gel column chromatography is petroleum ether-ethyl acetate in a volume ratio of 100:0-3:1 or dichloromethane-methanol in a volume ratio of 500:1-3:1 for gradient elution, followed by reverse phase silica gel or MCI column chromatography or semi-preparative high performance liquid chromatography to obtain a compound of formula 1 or formula 2;

[0042] Three separation methods:

[0043] The normal phase silica gel column chromatography eluent is petroleum ether-ethyl acetate, dichloromethane-methanol or chloroform-methanol with a volume ratio of 100:1-0:1 for gradient elution, followed by reverse phase silica gel or MCI column chromatography with a volume ratio of 20-100% (v / v) methanol aqueous solution or 20-100% (v / v) acetonitrile aqueous solution for gradient elution, and semi-preparative high performance liquid chromatography is performed with a volume ratio of 99-50% (v / v) n-hexane / EtOH as the eluent to obtain the compound of Formula 1 or Formula 2.

[0044] Four separation methods:

[0045] The normal phase silica gel column chromatography eluent is petroleum ether-ethyl acetate, dichloromethane-methanol or chloroform-methanol with a volume ratio of 100:1-0:1 for gradient elution, followed by isocratic elution with methanol on a Sephadex LH-20 column, followed by reverse phase silica gel or MCI column chromatography with a volume ratio of 20-100% (v / v) methanol aqueous solution or 20-100% (v / v) acetonitrile aqueous solution for gradient elution, and semi-preparative high performance liquid chromatography is performed with a volume ratio of 99-50% (v / v) n-hexane / EtOH as the eluent to obtain a compound of Formula 1 or Formula 2.

[0046] The preparation method of alkaloid compounds in the root of Anachia described in step c is characterized in that the silica gel column chromatography used is normal pressure or pressurized column chromatography, the filler used is normal silica gel or reversed phase silica gel, and the eluent is dichloromethane and methanol in a volume ratio of 500:1-3:1; petroleum ether / ethyl acetate in a volume ratio of 100:0-3:1; petroleum ether / acetone in a volume ratio of 5:2; or methanol and water in a volume ratio of 1:9-1:0 as the eluent, and isocratic or gradient elution is adopted.

[0047] The method for preparing alkaloid compounds from the root of Anachia described in step c is characterized in that the eluent of the Sephadex LH-20 column chromatography method is methanol, and isocratic elution is adopted.

[0048] The method for preparing alkaloid compounds from the root of Anacardiaceae described in step c is characterized in that the preparative high performance liquid chromatography uses n-hexane / EtOH with a volume ratio of 99-50% (v / v) as the eluent, and adopts isocratic or gradient elution.

[0049] According to another aspect of the present invention, there is provided use of the compound having Nav1.2 inhibitory effect for preparing analgesic drugs.

[0050] Combining a variety of spectral analysis methods (high resolution mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, and nuclear magnetic resonance spectroscopy), quantum chemical calculation method ( 13The structures of Compound 1 and Compound 2 prepared in the Examples were determined by comprehensive analysis using methods such as C-NMR DP4+ probability analysis and ECD) and X-ray single crystal diffraction. The relative configurations of Compound 1 and Compound 2 were determined by X-ray single crystal diffraction, as shown in Figures 1 and 4.

[0051] Compound 1 (Anacyphrethine A): Yellow needle-shaped crystals; optical rotation values ​​[α]25 D1584 (c 0.08, methanol, (+)-1); [α]25 D-1584 (c 0.1, methanol, (-)-1); UV (methanol) λ max (logε)427(4.15)nm,267(3.96)nm;Infrared (KBr)max 3297,2961,2924,1704,1623,and 1448cm -1 ;ECD(c 3.12×10 -4 M, methanol)λ max (Δε) 216 (-11.39), 253 (-2.36), 289 (-13.16), 417 (17.07) nm, enantiomer compound (+)-1; ECD (c 3.12 × 10 -4 M, methanol)λ max (Δε) 217 ​​(9.25), 253 (0.05), 290 (13.67), 416 (-21.25) nm, enantiomeric compound (-)-1; high-resolution mass spectrum m / z 641.4051 [M+H] + (Calculated value C 39 H 53 O4N4 + ,641.4066), 1 H and 13 C NMR spectrum data are shown in Tables 1 and 2.

[0052] Compound 2 (Anacyphrethine A): yellow needle-shaped crystals; optical rotation values ​​[α]25D-530 (c 0.03, methanol, (-)-2); [α]25D 530 (c 0.02, methanol, (+)-2); UV (methanol) λ max (logε)475(3.90)nm,281 (3.74)nm; IR (KBr)max 3316,2963,2926,2857,1699,1615,1445,and 1194cm -1 ;ECD(c 3.12×10 -4 M, methanol)λ max(Δε) 219 (-23.50), 265 (17.79), 300 (-35.20), 381 (25.45), 481 (-9.33), enantiomer compound (-)-2; ECD (c 3.12 × 10 -4 M, methanol)λ max (Δε) 220 (30.48), 264 (-25.61), 301 (46.37), 380 (-34.83), 480 (10.83), enantiomeric compound (+)-2; high-resolution mass spectrum m / z 641.4049 [M+H] + (Calculated value for C 39 H 53 O4N4 + ,641.4066), 1 H and 13 C NMR spectrum data are shown in Tables 1 and 2.

[0053] In general, the above technical solutions conceived by the present invention have the following technical advantages compared with the existing technology:

[0054] (1) Compounds 1-2 provided in the present invention are novel skeleton compounds. Compounds 1 and 2 are two unprecedented pairs of highly conjugated tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloid novel skeleton enantiomers with unique 8,14,18,24-tetraazaoctacyclic [21.2.2 1,4 .1.0 2,21 .0 3,18 .0 5,17 .0 9,16 .0 11,15 ]The skeleton structure of the nonacosane ring system has four discontinuous chiral stereo centers.

[0055] (2) Compounds 1-2 provided in the present invention have Nav1.2 inhibitory activity and NO inhibitory activity, and compound 2 has micromolar-level Nav1.2 inhibitory activity and has potential Nav1.2 inhibitory activity.

[0056] (3) The present invention uses computer molecular docking and combines the activity results of the compound with Nav1.2 to conclude that Gln332, Phe38, Asn361, Asp334, Tyr362, Asp949, Trp948, Pro921, Trp923, Tyr1429 and Met1425 residues in the Nav1.2 polypeptide chain are the active binding sites of the Nav1.2 polypeptide chain, providing a theoretical basis for the subsequent development of efficient Nav1.2 inhibitors.

[0057] The present invention will be further described in detail below with reference to the accompanying drawings and Examples, but the scope of the present invention is not limited thereby. Without departing from the spirit and substance of the present invention, modifications or replacements made to the method, steps, conditions, etc. of the present invention all fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIG1 is an X-ray single crystal diffraction pattern of compound 1;

[0059] FIG2 is an X-ray single crystal diffraction pattern of compound (+)-1;

[0060] FIG3 is an X-ray single crystal diffraction pattern of compound (-)-1;

[0061] FIG4 is an X-ray single crystal diffraction pattern of compound 2;

[0062] FIG5 is a diagram showing the molecular docking and molecular dynamics simulation results of compound (+)-2;

[0063] Figure 6 is a graph showing the 1 H NMR spectra;

[0064] Figure 7 is a graph showing the 13 C NMR spectrum;

[0065] Figure 8 is the 1 H NMR spectra;

[0066] Figure 9 is a graph showing the 13 C NMR spectrum. DETAILED DESCRIPTION

[0067] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only intended to illustrate the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0068] The following are specific embodiments:

[0069] Example 1

[0070] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) was pulverized and ultrasonically extracted with chloroform (30 L). The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was then placed in an ice-water bath with constant stirring and adjusted to pH 10 with a saturated aqueous NaHCO3 solution to obtain an alkaline solution. The alkaline solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain total alkaloids.

[0071] Example 2

[0072] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) was pulverized and extracted with 95% ethanol (40 L) by percolation. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% hydrochloric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was then placed in an ice-water bath with constant stirring and adjusted to pH 10 with aqueous ammonia to obtain an alkaline solution. The alkaline solution was thoroughly extracted with ethyl acetate, and the ethyl acetate extracts were combined and dried to obtain the total alkaloids.

[0073] Example 3

[0074] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) was pulverized and extracted with 50% ethanol (40 L) by percolation. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 2% hydrochloric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was then placed in an ice-water bath with constant stirring and adjusted to pH 10 with an aqueous Na2CO3 solution to obtain an alkaline solution. The alkaline solution was then fully extracted with n-butanol. The n-butanol extracts were combined and dried to obtain the total alkaloids.

[0075] Example 4

[0076] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) was ground and extracted with 75% ethanol (45 L) under reflux. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 1% hydrochloric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was then placed in an ice-water bath with constant stirring and adjusted to pH 12 with an aqueous NaOH solution to obtain an alkaline solution. The alkaline solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain the total alkaloids.

[0077] Example 5

[0078] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) was ground and extracted with 22.5 L of methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was then stirred in an ice-water bath and adjusted to pH 10 with a saturated aqueous NaHCO3 solution to obtain an alkaline solution. The alkaline solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain the total alkaloids.

[0079] Example 6

[0080] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) was ground and extracted with 22.5 L of methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 1% sulfuric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was then placed in an ice-water bath with constant stirring and adjusted to pH 10 with a saturated aqueous NaHCO3 solution to obtain an alkaline solution. The alkaline solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain the total alkaloids.

[0081] Example 7

[0082] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) was ground and extracted with 22.5 L of methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 2% sulfuric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was then placed in an ice-water bath with constant stirring and adjusted to pH 10 with a saturated aqueous NaHCO3 solution to obtain an alkaline solution. The alkaline solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain the total alkaloids.

[0083] Example 8

[0084] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) was ground and extracted with 22.5 L of methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% sulfuric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was then placed in an ice-water bath with constant stirring and adjusted to pH 10 with a saturated aqueous NaHCO3 solution to obtain an alkaline solution. The alkaline solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain the total alkaloids.

[0085] Example 9

[0086] The total alkaloids were mixed with 100-200 mesh silica gel, subjected to silica gel column chromatography, and then eluted with dichloromethane / methanol gradient (100:0-3:1, V / V). The same fractions were combined to obtain 6 fractions Fr.A-Fr.F with increasing polarity. Among them, the first fraction Fr.A (159.0 g) was mixed with 100-200 mesh silica gel, subjected to silica gel column chromatography, and then eluted with petroleum ether / ethyl acetate gradient (100:0-3:1, V / V). The same fractions were combined to obtain 6 fractions Fr.A1-Fr.A6 with increasing polarity. Among them, the second fraction Fr.A2 (76.1 g) was subjected to reverse phase MCI column chromatography and eluted with methanol / water gradient (20: 80~100:0, V / V), the same components were combined to obtain 7 sub-components Fr.A21-Fr.A27 with decreasing polarity; among them, component Fr.A25 (10.0g) was mixed with 200-300 mesh silica gel, subjected to silica gel column chromatography, and then eluted with petroleum ether / ethyl acetate gradient (10:1~3:1, V / V), and the same components were combined to obtain 3 components Fr.A251-Fr.A253 with decreasing polarity; component Fr.A253 (9.0g) was mixed with 200-300 mesh silica gel, subjected to silica gel column chromatography, and then eluted with petroleum ether / ethyl acetate gradient (10:1~3:1, V / V), and the same components were combined to obtain 6 components Fr.A2531-Fr.A2533 with decreasing polarity; among them, component Fr.A2533 (7.8g) was subjected to Sephadex LH-20 gel column chromatography, eluted with methanol, obtained three subfractions Fr.A25331-Fr.A25333 with decreasing molecular weight; component Fr.A25333 (6.1 g) was subjected to reverse phase C18 silica gel column chromatography, eluted with methanol / water gradient (20:80-100:0, V / V), and the same fractions were combined to obtain six subfractions Fr.A253331-Fr.A253336 with decreasing polarity; component Fr.A253336 (231.2 mg) was chromatographed on a normal phase silica gel column with dichloromethane / methanol (500:1 to 50:1, V / V) as eluent to obtain five subfractions Fr.A2533361 to Fr.A2533365 in increasing polarity. Component Fr.A2533363 (71.1 mg) was recrystallized from methanol to obtain compound 1 (i.e., the compound of formula 1) (Anacyphrethines A, 30.3 mg, 0.000202%). The racemate of compound 1 was chirally resolved on a chiral chromatography column (DAICEL CORPORATION Chiralpak ID 5μm 10×250mm; solvent: n-Hexane / EtOH=90:10; column temperature: 25°C; flow rate: 3 ml / min; detection wavelength: 360 nm) to obtain compound (+)-1 (14.0 mg, tR =12.3min) and compound (-)-1 (15.4mg, t R =18.0min). Fraction Fr.A253335 (214.3 mg) was chromatographed on a normal phase silica gel column using dichloromethane / methanol (500:1 to 50:1, V / V) as the eluent to obtain 6 subfractions Fr.A2533351 to Fr.A2533356 with decreasing polarity. Fraction Fr.A2533353 (61.3 mg) was chromatographed on a normal phase silica gel column using petroleum ether / acetone (5:2, V / V) as the eluent to obtain compound 2 (i.e., compound of formula 2) (Anacyphrethines B, 20.1 mg, 0.000134%). The racemate of compound 2 was purified by chiral chromatography on a DAICEL CORPORATION Chiralpak ID 5 μm 10×250mm; solvent: n-Hexane / EtOH=60:40; column temperature: 25℃; flow rate: 3ml / min; detection wavelength: 360nm) was used for chiral separation to obtain compound (+)-2 (7.5mg, t R =17.0min) and compound (-)-2 (7.3mg, t R =12.4min).

[0087] Example 10

[0088] The total alkaloids were mixed with 100-200 mesh silica gel, subjected to silica gel column chromatography, and then eluted with dichloromethane / methanol gradient (100:0-3:1, V / V), and the same components were combined to obtain 6 components Fr.A-Fr.F with increasing polarity; the first component Fr.A (159.0 g) was mixed with 100-200 mesh silica gel, subjected to silica gel column chromatography, and then eluted with petroleum ether / ethyl acetate gradient (100:0-3:1, V / V), and the same components were combined to obtain 6 components Fr.A1-Fr.A6 with increasing polarity; the second component Fr.A2 (7 The product (6.1 g) was subjected to reverse phase MCI column chromatography and eluted with a methanol / water gradient (20:80 to 100:0, V / V). The same fractions were combined to obtain 7 subfractions Fr.A21 to Fr.A27 with decreasing polarity. Among them, the component Fr.A25 (10.0 g) was mixed with 200-300 mesh silica gel and subjected to silica gel column chromatography. Then, the product was eluted with a petroleum ether / ethyl acetate gradient (10:1 to 3:1, V / V). The same fractions were combined to obtain 3 subfractions Fr.A251 to Fr.A253 with decreasing polarity. The component Fr.A253 (9.0 g) was mixed with 200-300 mesh silica gel and subjected to silica gel column chromatography. The sample was mixed with silica gel and subjected to silica gel column chromatography, followed by gradient elution with petroleum ether / ethyl acetate (10:1-3:1, V / V). The same fractions were combined to obtain 6 fractions Fr.A2531-Fr.A2533 with decreasing polarity. Among them, fraction Fr.A2533 (7.8 g) was subjected to MCI column chromatography and eluted with 20-100% acetonitrile water to obtain 3 subfractions Fr.A25331-Fr.A25333 with decreasing molecular weight. Fraction Fr.A25333 (6.1 g) was subjected to reverse phase C18 silica gel column chromatography and eluted with methanol / water gradient (20:80-10 0:0, V / V), and the same components were combined to obtain 6 subfractions Fr.A253331-Fr.A253336 with decreasing polarity; component Fr.A253336 (231.2 mg) was subjected to normal phase silica gel column chromatography and eluted with dichloromethane / methanol (500:1-50:1, V / V) to obtain 5 subfractions Fr.A2533361-Fr.A2533365 with decreasing polarity; component Fr.A2533363 (71.1 mg) was recrystallized from methanol to obtain compound 1 (i.e., the compound of formula 1) (Anacyphrethines A, 30.3 mg, 0.000202%); the racemate of compound 1 was subjected to chiral chromatography on a chiral column (DAICEL CORPORATION Chiralpak ID 5 μm 10×250 mm; solvent: n-Hexane / EtOH=90:10; column temperature: 25°C; flow rate: 3 ml / min; detection wavelength: 360 nm) to afford compound (+)-1 (14.0 mg, t R=12.3min) and compound (-)-1 (15.4mg, t R =18.0min). Fraction Fr.A253335 (214.3 mg) was chromatographed on a normal phase silica gel column using dichloromethane / methanol (500:1 to 50:1, V / V) as the eluent to obtain 6 subfractions Fr.A2533351 to Fr.A2533356 with decreasing polarity. Fraction Fr.A2533353 (61.3 mg) was chromatographed on a normal phase silica gel column using petroleum ether / acetone (5:2, V / V) as the eluent to obtain compound 2 (i.e., compound of formula 2) (Anacyphrethines B, 20.1 mg, 0.000134%). The racemate of compound 2 was purified by chiral chromatography on a DAICEL CORPORATION Chiralpak ID 5 μm 10×250mm; solvent: n-Hexane / EtOH=60:40; column temperature: 25℃; flow rate: 3ml / min; detection wavelength: 360nm) was used for chiral separation to obtain compound (+)-2 (7.5mg, t R =17.0min) and compound (-)-2 (7.3mg, t R =12.4min).

[0089] Example 11

[0090] The total alkaloids were mixed with 100-200 mesh silica gel and subjected to silica gel column chromatography. The mixture was then eluted with a dichloromethane / methanol gradient (100:0 to 3:1, V / V). Fractions of the same polarity were combined to obtain six fractions Fr.A to Fr.F, with increasing polarity. Fraction 1, Fr.A (159.0 g), was subjected to repeated silica gel column chromatography using a gradient elution of petroleum ether-ethyl acetate (volume ratio: 100:0-3:1), dichloromethane-methanol (volume ratio: 500:1-3:1), or petroleum ether / acetone (volume ratio: 50:1-0:1) to obtain compound 1 (i.e., compound of formula 1) (Anacyphrethines A, 30.3 mg, 0.000202%) and compound 2 (i.e., compound of formula 2) (Anacyphrethines B, 20.1 mg, 0.000134%). The racemate of compound 1 was subjected to chiral chromatography on a column (DAICEL CORPORATION Chiralpak ID 5μm 10×250mm; solvent: n-Hexane / EtOH=99:1-50-50; column temperature: 25℃; flow rate: 3ml / min; detection wavelength: 360nm) was used for chiral separation to obtain compound (+)-1 (14.0mg, t R =12.3min) and compound (-)-1 (15.4mg, t R=18.0min). The racemate of compound 2 was subjected to chiral separation on a chiral chromatographic column (DAICEL CORPORATION Chiralpak ID 5μm 10×250mm; solvent: n-Hexane / EtOH=99:1-50-50; column temperature: 25°C; flow rate: 3ml / min; detection wavelength: 360nm) to give compound (+)-2 (7.5mg, t R =17.0min) and compound (-)-2 (7.3mg, t R =12.4min).

[0091] Example 12

[0092] The Nav1.2 inhibitory activity of compounds 1 and 2 was evaluated by patch-clamp electrophysiological assays. HEK293T cells (ATCC Cell Bank) were cultured at 37°C in a 5% CO2 incubator in 90% DMEM / 10% fetal bovine serum (FBS). Cells were digested with 0.25% trypsin at a density of 80-90% and then subcultured or plated. Twenty-four hours after plating, cells were transfected using the Lipo2000 transfection kit (Thermo Fisher, Shanghai, China) with a 9:1 ratio of pcDNA3.1-SCN2A (Nav1.2 GenBank accession No. NM_001040142) plasmid (BGI, Beijing, China) to pcDNA3.1-EGFP plasmid (BGI, Beijing, China) (4000 ng total). Electrophysiological experiments were performed 18 hours after transfection.

[0093] Current-clamp recordings were performed using an Axon patch 700B amplifier (Axon Instruments, Molecular Devices, USA) and a Digidata 1440A digital-to-analog converter (Axon Instruments, Molecular Devices, USA). Signals were acquired using pClamp 10.0 software (Molecular Devices, USA), with a 2 kHz filter and a 10 kHz sampling rate. Patch-clamp electrodes were drawn using a P-97 horizontal electrode puller (Sutter Instruments, USA) using a multi-step procedure. The resistance of the electrodes was measured by filling the electrodes with solution to determine whether they were 3-5 MΩ. Recordings were performed at room temperature (23-25°C). The perfusion system was custom-made at a rate of approximately 2 mL / min, and the drug delivery system was a BPS-8 (ALA Scientific Instruments, USA). All electrophysiological data were processed using Clampfit 10.4 (Molecular Devices, USA) and analyzed using GraphPad Prism 5 (GraphPad Software, USA). The initial screening concentration of the monomer compound was 40 μM, and the inhibition rate results are shown in Table 3.

[0094] Table 3. Nav1.2 inhibitory activity of compounds 1-2

[0095] [a] Positive drug: TTX (tetrodotoxin, Jiangsu Kangte Bioengineering Co., Ltd.). [b] Each experiment was repeated 3 times.

[0096] Conclusion: Compound (+)-2 has a significant inhibitory effect on Nav1.2, and compound (+)-2 has a micromolar Nav1.2 inhibitory activity, and its IC 50 The value was 23.94±2.70μM.

[0097] Example 13

[0098] NO inhibitory activity of the compound of Formula 1 (Compound 1) and the compound of Formula 2 (Compound 2):

[0099] 1. Cell culture:

[0100] BV2 cells (purchased from BeNa Culture Collection, BNCC) were cultured in Dulbecco's modified eagle medium (DMEM) high-glucose medium (purchased from Hyclone, USA) containing 10% fetal bovine serum (FBS) (purchased from Giboco, USA) and 1% penicillin and streptomycin in an incubator at 37°C and 5% CO2.

[0101] 2. Test of the Effect of the Compounds of the Invention on Cell Viability:

[0102] The compound was dissolved in dimethyl sulfoxide (DMSO), and BV2 cells in the logarithmic growth phase and in good growth condition were taken and 5×10 3 Cells were plated in a 96-well plate. The experimental group was added with different concentrations of 12.5, 25, 50, and 100 μM of the compound of formula I, and the control group was added with dimethyl sulfoxide (DMSO). The cells were cultured for 24 hours, and then 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolium monosodium salt (CCK-8 reagent) was added to each well. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated. The experimental results are shown in Table 4.

[0103] Table 4. Cell viability of compounds 1-2

[0104] 3. Determination of nitric oxide (NO) content:

[0105] The Griess method (Arias-Negrete et al., Analytical Biochemistry 328.1 (2004): 14-21.) was used to test the release of NO in BV2 cells. Samples at different concentrations of 25, 50, and 100 μM were added and incubated for 2 h. Then, 1 μg / mL lipopolysaccharide (LPS, Sigma, L4391) was added and incubated for 22 h. After the incubation was completed, the cell supernatant was collected and the nitric oxide content in the cell supernatant was determined by the Griess method. Before the determination, Griess Reagent I and II (Nitric Oxide Assay) were removed. kit, Beyotine, S0021M) and return it to room temperature. Complete culture medium was used to dilute the standard (1-100 μM). The concentration of the standard could be 0, 1, 2, 5, 10, 20, 40, 60, or 100 μM. The standard and the collected culture supernatant were added to a 96-well plate at 50 μL / well. 50 μL of Griess Reagent I and 50 μL of Griess Reagent II, which had been returned to room temperature, were added to each well in sequence. After vortex mixing for 5 minutes, the absorbance was measured at 540 nm to create a standard curve. The NO content in the culture supernatant was calculated based on the standard curve. The initial screening concentration of the monomer compound was 40 μM, and the inhibition rate was shown in Table 5.

[0106] Table 5. NO inhibitory activity of compounds 1-2

[0107] [a] Positive drug: Andrographolide (AG, HY-N0191). [b] Each experiment was repeated 3 times.

[0108] Conclusion: Compounds 1-2 have a certain inhibitory effect on the release of NO, among which compounds ()-1 and ()-1 have an IC 50 The values ​​were 27.63±3.753 and 37.35±0.807 respectively.Compounds ()-1 and ()-1 had a significant inhibitory effect on the release of NO.

[0109] Example 14

[0110] In this example, Autodock 4.2.6 molecular docking software (The Scripps Research Institute, USA) was used to investigate the interaction mode of Compound 2 with Nav1.2 (pdb ID: 6J8E). Molecular dynamics simulations were performed to characterize the dynamic conformational changes and motion trajectories of the compound upon binding to the Nav1.2 protein. Nav1.2 is composed of a single polypeptide chain folded into four homologous repeats.

[0111] Conclusion: The docking results showed that (+)-2 bound to the top residues of the active pocket and occupied the pore of the Nav1.2 channel with a low affinity (-9.126 kcal / mol), which was consistent with the previous binding experiments. Detailed analysis of the interaction between the active site of the Nav1.2 channel and (+)-2 showed that the C-10 carbonyl group in (+)-2 acted as a hydrogen bond donor to form a hydrogen bond with the Nav1.2 channel residue Asn333. In addition, (+)-2 interacted with residues such as Gln332, Phe385, Asn361, Asp334, Tyr362, Asp949, Trp948, Pro921, Trp923, Tyr1429 and Met1425. These residues may be the action sites of Nav1.2, providing a theoretical basis for the subsequent development of efficient Nav1.2 inhibitors.

[0112] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound selected from the following or its isomers: 7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methanoaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepina[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione (compound of Formula 1); and 7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepina[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione (compound of formula 2).

2. The compound or isomer thereof according to claim 1, wherein the isomer is an enantiomer.

3. The compound according to claim 1 or 2 or its isomer, which is selected from the group consisting of: (4aR,7aR,11aS,14aR)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methanoaza[4,5,6-de]pyrrolo[3″',2″':4″,5″]cyclopenta[1″,2″:6′,7′]aza[4′,5′:4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione; (4aS,7aS,11aR,14aS)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepina[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione; (4aR,7aR,11aR,14aS)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepina[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione; and (4aS,7aS,11aS,14aR)-7a-acetyl-2,2,4a,6,6,9,9,11a,13,13,17,17-dodecamethyl-3,4a,5,6,7,7a,9,10,11a,12,13,14,16,17-tetradecahydro-11a,14a-methylaza[4,5,6-de]pyrrolo[3"',2"':4",5"]cyclopenta[1",2":6',7']azepina[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-trione.

4. The compound according to any one of claims 1 to 3 or an isomer thereof, which has a structural formula selected from the following:

5. A method for extracting the compound or isomer thereof according to any one of claims 1 to 4 from the root of Anabaena, comprising the following steps: a. Dry and crush the root of Anacardia, use 50-95% (v / v) ethanol aqueous solution, methanol or chloroform as solvent, wherein the ratio of Anacardia (kg) to solvent (L) is 1:1.5-1:4, extract by cold soaking, percolation, heating reflux or ultrasonic extraction, and recover the solvent by vacuum concentration to obtain an extract; b. After the total extract of step a is suspended in water, it is dispersed with an acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid. The obtained acid water layer is extracted with dichloromethane to remove non-alkaloids, and then the pH is adjusted to 10-12 with a base such as NaHCO3, Na2CO3, ammonia water or NaOH, and then extracted with an organic solvent such as dichloromethane, ethyl acetate or n-butanol, and concentrated under reduced pressure to recover the organic solvent to obtain total alkaloids; c. Separating the total alkaloids in step b by silica gel column chromatography, thin layer chromatography, reverse phase MCI column chromatography, Sephadex LH-20 column chromatography, high performance liquid chromatography or any combination thereof to obtain the compound or its isomers.

6. The method according to claim 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography and reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography, preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography is used to obtain the compound of formula 1 or formula 2, wherein more preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, volume ratio of 100:0 to 3:1, volume ratio of 1:1 to 1 ... and the compound of formula 1 or 2 is obtained by reverse phase silica gel column chromatography or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography. The invention relates to a method for preparing the semi-preparative high performance liquid chromatography method comprising: a dichloromethane and methanol in a volume ratio of 500:1 to 3:1, a petroleum ether and acetone in a volume ratio of 50:1 to 0:1, or a methanol and water in a volume ratio of 1:9-1:0; the eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution in a volume ratio of 20-100% (v / v) or an acetonitrile aqueous solution in a volume ratio of 20-100% (v / v); and the eluent for isocratic or gradient elution in the semi-preparative high performance liquid chromatography is n-hexane / EtOH in a volume ratio of 99-50% (v / v).

7. The method according to claim 5, wherein in step c, separation is performed by a combination of normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography. Preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, gradient elution is performed using reverse phase silica gel or reverse phase MCI column chromatography, and then semi-preparative high performance liquid chromatography is used to obtain a compound of Formula 1 or Formula 2. More preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol or chloroform and methanol in a volume ratio of 100:1 to 0:1, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is a methanol aqueous solution in a volume ratio of 20-100% (v / v) or an acetonitrile aqueous solution in a volume ratio of 20-100% (v / v), and the eluent for isocratic or gradient elution used in the semi-preparative high performance liquid chromatography is n-hexane / EtOH in a volume ratio of 99-50% (v / v).

8. The method according to claim 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, Sephadex LH-20 column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography, preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, Sephadex LH-20 column chromatography, gradient elution using reverse phase silica gel or reverse phase MCI column chromatography, and then semi-preparative high performance liquid chromatography to obtain the compound of formula 1 or formula 2, wherein more preferably, the elution used in the normal phase silica gel column chromatography is The eluent is petroleum ether and ethyl acetate, dichloromethane and methanol or chloroform and methanol in a volume ratio of 100:1 to 0:1, the Sephadex LH-20 column chromatography adopts methanol gradient or isocratic elution, the eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution in a volume ratio of 20-100% (v / v) or an acetonitrile aqueous solution in a volume ratio of 20-100% (v / v), and the eluent for isocratic or gradient elution in the semi-preparative high performance liquid chromatography is n-hexane / EtOH in a volume ratio of 99-50% (v / v).

9. The method according to any one of claims 5 to 8, wherein in step c, the silica gel column chromatography is normal pressure or pressurized column chromatography, and / or the filler used is normal silica gel or reversed phase silica gel.

10. Use of the compound or its isomer according to any one of claims 1 to 4 in the preparation of analgesic or anti-inflammatory drugs, preferably, the compound or its isomer exerts analgesic effect by inhibiting Nav1.2, and exerts anti-inflammatory effect by inhibiting NO.

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