Analgesic compounds with Nav1.2 inhibitory activity from anachi root and their preparation and use

Abstract

The present invention relates to a unique 8,14,18,24-tetraazaoctacyclo[21.2.2 1,4 .1.0 2,21 .0 3,18 .0 5,17 .0 9,16 .0 11,15 The present invention provides a method for preparing highly conjugated tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloid compounds with a nonacosane ring system, and their anti-inflammatory and analgesic uses. The tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloid compounds were extracted from the roots of Anacyclus pyrethrum (L.) DC. (anachi root) with an organic solvent and then separated by two or three methods: normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, and semi-preparative high-performance liquid chromatography. Four novel tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloid single compounds were obtained, and their structures were identified by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. The present invention also provides Nav1.2 inhibitory activity and NO inhibitory activity of these compounds.

Description

[Technical Field]

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to analgesic / anti-inflammatory compounds (compounds of formula 1 and formula 2) derived from Annatchi root that have Nav1.2 inhibitory activity and NO inhibitory activity, and their preparation and use. [Background technology]

[0002] Pain is the body's defense mechanism against illness and is the fifth vital sign. However, persistent and severe pain can affect patients' mental health, causing anxiety, sadness, and a weakened immune system, leading to a series of consequences that, in the most severe cases, can be fatal or disabling. However, conventional painkillers, such as opioids and nonsteroidal drugs, have side effects such as tolerance, addiction, and gastrointestinal bleeding.

[0003] Ion channels regulate the voltage potential across membranes by regulating the passage of ions into and out of cells. The flow of ions generates electrical pulses, which then stimulate the continuous opening of adjacent voltage-sensitive channels, resulting in the generation of spontaneous electrical signals. Inhibiting sustained rhythmic potentials by blocking ion channels prevents signal transduction and thereby reduces pain responses. Currently, various ion channel inhibitors have been reported, including VGSCs, VGCCs, VGPCs, and TRPs. Sodium channels are involved in the generation and propagation of action potentials and are the basic components of electrical signal generation in all excitable cells (including nerve and muscle cells). The human body has nine subtypes of sodium channels, designated Nav1.1–Nav1.9. For example, lidocaine reduces pain responses by inhibiting Nav1.7 and Nav1.9. Therefore, identifying new ion channel inhibitors with analgesic properties is crucial.

[0004] Searching for advanced compounds with significant pharmacodynamic activity from traditional medicinal plants is a hot topic in drug research, so discovering new ion channel inhibitors and compounds with anti-inflammatory activity from medicinal plants is an effective way to develop new analgesic / anti-inflammatory drugs.

[0005] Anaci root is the dried root of Anacyclus pyrethrum (L.) DC., a plant in the Asteraceae family, and its main functions are to clear the brain (revitalize), treat hemiplegia, vitiligo, epilepsy, headaches, relieve coughs and phlegm, and treat convulsions. DISCLOSURE OF THE INVENTION

[0006] As a result of intensive research, the present inventors have isolated for the first time from Anachi root analgesic / anti-inflammatory compounds (compounds of formula 1 and formula 2) with Nav1.2 inhibitory activity and NO inhibitory activity. These compounds have novel skeletons and are highly conjugated tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloid compounds. Their structures have been identified and cell experiments have verified that they have Nav1.2 inhibitory activity and NO inhibitory activity, and they can be used to prepare analgesic and / or anti-inflammatory drugs.

[0007] Thus, the present invention provides the following:

[0008] 1,7a-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']azepino[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-triketone (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-methanoaza[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)-triketone (compound of formula 2) A compound selected from the group consisting of:

[0009] 2. The compound or its isomer according to the above item 1, wherein the isomer is an enantiomer.

[0010] 3. (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']azepino[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-triketone, (4aS,7a S,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-methanoaza[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)-triketone, (4aR,7aR,11aR,14 aS)-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']azepino[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-triketone, and (4aS,7aS,11aS,14aR)-7a 3. The compound according to claim 1 or 2, selected from the group consisting of -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']azepino[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-triketone, or an isomer thereof.

[0011] 4. The compound according to any one of the above items 1 to 3, having a structural formula selected from the following, or an isomer thereof.

[0012] [ka]

[0013] 5. A method for extracting the compound or its isomer according to any one of the above items 1 to 4 from annatto root, comprising the steps of: a. Anachi root is dried and then crushed, and the extract is obtained by cold maceration, percolation, heating under reflux, or ultrasonic extraction using a solvent of 50-95% (v / v) ethanol, methanol, or chloroform (ratio of the weight of the drug (kg) to the solvent (L) is 1:1.5-1:4), followed by vacuum concentration to recover the solvent; b. The total extract from step a is suspended in water, and then dispersed in an acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid. The resulting acidic aqueous layer is extracted with dichloromethane to remove non-alkaloids, and then the pH is adjusted to 10-12 with an alkali such as NaHCO3, Na2CO3, aqueous ammonia, or NaOH. The extract is then further extracted with an organic solvent such as dichloromethane, ethyl acetate, or n-butanol, and concentrated under reduced pressure to recover the organic solvent, thereby obtaining the total alkaloids; c. The total alkaloids from step b are separated by silica gel column chromatography, thin layer chromatography, reversed-phase MCI column chromatography, Sephadex LH-20 column chromatography, high performance liquid chromatography, or any combination thereof to obtain the compound or its isomer.

[0014] 6. In step c, separation is performed by 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 by gradient or isocratic elution of normal phase silica gel column chromatography, followed by reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography to obtain the compound of formula 1 or formula 2, more preferably by using a normal phase silica gel column chromatography with a volume ratio of 100:0 to 3:1 petroleum ether as the eluent. 6. The method according to claim 5, wherein the eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a 20% to 100% (v / v) aqueous methanol solution or a 20% to 100% (v / v) aqueous acetonitrile solution, and the eluent for isocratic or gradient elution used in the semi-preparative high-performance liquid chromatography is a 99% to 50% (v / v) n-hexane / EtOH solution.

[0015] 7. The method of 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, the compound of Formula 1 or Formula 2 is obtained by gradient or isocratic elution using normal-phase silica gel column chromatography, gradient elution using reverse-phase silica gel or reverse-phase MCI column chromatography, and further semi-preparative high-performance liquid chromatography. More preferably, the eluent used in the normal-phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol, or trichloromethane 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 20 to 100% (v / v) aqueous methanol solution or a 20 to 100% (v / v) aqueous acetonitrile solution, and the eluent used for isocratic or gradient elution using the semi-preparative high-performance liquid chromatography is n-hexane / EtOH in a volume ratio of 99 to 50% (v / v).

[0016] 8. In step c, separation is performed by 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 by gradient or isocratic elution by normal phase silica gel column chromatography, followed by Sephadex LH-20 column chromatography, gradient elution by reverse phase silica gel or reverse phase MCI column chromatography, and further semi-preparative high performance liquid chromatography to obtain the compound of formula 1 or formula 2, more preferably by the normal phase silica gel column chromatography. 6. The method according to claim 5, wherein the eluent used in the above is petroleum ether and ethyl acetate, dichloromethane and methanol, or trichloromethane and methanol in a volume ratio of 100:1 to 0:1, the Sephadex LH-20 column chromatography is gradient or isocratic elution with methanol, the eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a volume ratio of 20 to 100% (v / v) aqueous methanol solution or 20 to 100% (v / v) aqueous acetonitrile solution, and the eluent for isocratic or gradient elution used in the semi-preparative high-performance liquid chromatography is a volume ratio of 99 to 50% (v / v) n-hexane / EtOH.

[0017] 9. The method according to any one of the above items 5 to 8, wherein in step c, the silica gel column chromatography is atmospheric pressure or pressurized column chromatography, and / or the filler used is normal phase silica gel or reverse phase silica gel.

[0018] 10. Use of the compound or its isomer according to any one of the above 1 to 4 in the preparation of an analgesic or anti-inflammatory drug, wherein the compound or its isomer preferably exerts an analgesic effect by inhibiting Nav1.2 and an anti-inflammatory effect by inhibiting NO.

[0019] Details of the invention The present invention aims to provide a compound having Nav1.2 inhibitory activity and / or anti-inflammatory activity, a method for isolating and preparing the compound, and its application value in the preparation of analgesic drugs. According to a first aspect of the present invention, there is provided a compound having Nav1.2 inhibitory activity and / or anti-inflammatory activity, the structural formula of which is shown below.

[0020] [ka]

[0021] however, 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-methanoaza[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)-triketone; 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-methanoaza[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)-triketone, 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-methanoaza[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)-triketone; 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-methanoaza[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)-triketone.

[0022] The method for extracting and separating the alkaloid compounds is carried out in the following steps. a. Anachi root is dried and then crushed, and the extract is obtained by cold maceration, percolation, heating under reflux, or ultrasonic extraction using a solvent of 50-95% (v / v) ethanol, methanol, or chloroform (ratio of the weight of the drug (kg) to the solvent (L) is 1:1.5-1:4), followed by vacuum concentration to recover the solvent; b. The total extract from step a is suspended in water, and then dispersed in an acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid. The resulting acidic aqueous layer is extracted with dichloromethane to remove non-alkaloids, and then the pH is adjusted to 10-12 with an alkali such as NaHCO3, Na2CO3, aqueous ammonia, or NaOH. The extract is then further extracted with an organic solvent such as dichloromethane, ethyl acetate, or n-butanol, and concentrated under reduced pressure to recover the organic solvent, thereby obtaining the total alkaloids; c. The total alkaloids from step b are separated by silica gel column chromatography, thin layer chromatography, reversed-phase MCI column chromatography, Sephadex LH-20 column chromatography, high performance liquid chromatography, or any combination thereof to obtain the compound or its isomer.

[0023] There are two types of separation methods: The compound of Formula 1 or Formula 2 is obtained by gradient elution using normal-phase silica gel column chromatography with an eluent of petroleum ether-ethyl acetate in a volume ratio of 100:0 to 3:1 or dichloromethane-methanol in a volume ratio of 500:1 to 3:1, followed by reverse-phase silica gel or MCI column chromatography or semi-preparative high-performance liquid chromatography.

[0024] Three separation methods: Gradient elution by normal-phase silica gel column chromatography using petroleum ether-ethyl acetate, dichloromethane-methanol, or trichloromethane-methanol in a volume ratio of 100:1 to 0:1 as the eluent is followed by gradient elution by reverse-phase silica gel or MCI column chromatography using a 20% to 100% (v / v) aqueous methanol solution or a 20% to 100% (v / v) aqueous acetonitrile solution as the eluent is followed by semi-preparative high-performance liquid chromatography using a 99% to 50% (v / v) volume ratio of n-hexane / EtOH as the eluent to obtain the compound of formula 1 or formula 2.

[0025] Four separation methods: Gradient elution by normal-phase silica gel column chromatography using petroleum ether-ethyl acetate, dichloromethane-methanol, or trichloromethane-methanol in a volume ratio of 100:1 to 0:1 as the eluent, followed by isocratic elution with methanol on a Sephadex LH-20 column, followed by gradient elution with 20-100% (v / v) aqueous methanol or 20-100% (v / v) aqueous acetonitrile on a reverse-phase silica gel or MCI column, followed by semi-preparative high-performance liquid chromatography using 99-50% (v / v) n-hexane / EtOH as the eluent, yields the compound of Formula 1 or Formula 2.

[0026] The method for preparing alkaloid compounds from annatto root according to step c is characterized in that the silica gel column chromatography used is atmospheric pressure or pressurized column chromatography, the filler used is normal phase silica gel or reverse phase silica gel, and elution is performed with dichloromethane and methanol in a volume ratio of 500:1 to 3:1, petroleum ether / ethyl acetate in a volume ratio of 100:0 to 3:1, petroleum ether / acetone in a volume ratio of 5:2, or methanol / water in a volume ratio of 1:9 to 1:0 as the eluent, using isocratic or gradient elution.

[0027] The method for preparing alkaloid compounds from Anchi root according to step c, wherein the eluent for the Sephadex LH-20 column chromatography is methanol and isocratic elution is used. The method for preparing alkaloid compounds from Anachi root according to step c, characterized in that the preparative high performance liquid chromatography uses n-hexane / EtOH in a volume ratio of 99 to 50% (v / v) as an eluent, and isocratic or gradient elution is used.

[0028] According to another aspect of the present invention, there is provided a use of the compound having Nav1.2 inhibitory activity in the preparation of an analgesic medicament.

[0029] Multiple spectral analysis methods (high-resolution mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance spectroscopy), quantum chemical calculation methods ( 13The structures of Compounds 1 and 2 prepared in the examples were identified through a comprehensive analysis using methods including C-NMR (DP4+ probability analysis and ECD) and X-ray single crystal diffraction. The relative configurations of Compounds 1 and 2 were determined by X-ray single crystal diffraction and are shown in Figures 1 and 4.

[0030] Compound 1 (Anacyphrethine A): Yellow needles; optical rotation values ​​[α]25 D1584 (c 0.08, methanol, (+)-1); [α]25 D1584 (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, enantiomeric 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 spectrometry m / z 641.4051 [M+H] + (Calculated value C 39 H 53 O4N4 + , 641.4066), that 1 H and 13 The C NMR spectral data are shown in Tables 1 and 2. Compound 2 (Anacyphrethine A): Yellow needle crystals; Optical rotation values ​​[α] D (c 0.03, methanol, (-)-2); [α] D (c 0.02, methanol, (+)-2); UV (methanol) λ max (logε) 475 (3.90)nm, 281 (3.74)nm; Infrared (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), enantiomeric 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 spectrometry m / z 641.4049 [M+H] + (Calculated value for C 39 H 53 O4N4 + , 641.4066), that 1 H and 13 C NMR spectral data are shown in Tables 1 and 2.

[0031] [Table 1-1] [Table 1-2]

[0032] [Table 2-1] [Table 2-2]

[0033] Overall, the above technical aspects of the present invention have the following main technical advantages over the prior art:

[0034] (1) Compounds 1 and 2 provided in this invention are novel scaffold compounds. Compounds 1 and 2 are two unprecedented pairs of highly conjugated tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloids with novel scaffold enantiomers, and possess unique 8,14,18,24-tetraazaoctacyclo[21.2.2 1,4 .1.02,21 .0 3,18 .0 5,17 .0 9,16 .0 11,15 ]It has a nonacosane ring system backbone structure and has four discontinuous chiral stereocenters.

[0035] (2) Compounds 1-2 provided by the present invention have Nav1.2 inhibitory activity and NO inhibitory activity, and compound 2 has Nav1.2 inhibitory activity at the micromolar level, and thus has potent Nav1.2 inhibitory activity.

[0036] (3) By performing molecular docking on a computer and combining the activity results of the compounds with Nav1.2, the present invention concludes that the 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.

[0037] The present invention will be described in more detail below with reference to the accompanying drawings and examples, but the scope of the present invention is not limited thereto. Any changes or substitutions made to the methods, steps, conditions, etc. of the present invention without departing from the spirit and essence of the present invention are all within the scope of the present invention. [Brief explanation of the drawings]

[0038] [Figure 1] FIG. 1 is an X-ray single crystal diffraction diagram of Compound 1. [Figure 2] FIG. 2 is the X-ray single crystal diffraction pattern of compound (+)-1. [Figure 3] FIG. 3 is the X-ray single crystal diffraction pattern of compound (-)-1. [Figure 4] FIG. 4 is an X-ray single crystal diffraction diagram of compound 2. [Figure 5] FIG. 5 shows the results of molecular docking and molecular dynamics simulation of compound (+)-2. [Figure 6] FIG. 6 is the 1H NMR diagram of compound 1. [Figure 7] FIG. 7 is the 13C NMR diagram of compound 1. [Figure 8] FIG. 8 is the 1H NMR diagram of compound 2. [Figure 9] FIG. 9 is the 13C NMR diagram of compound 2. DETAILED DESCRIPTION OF THE INVENTION

[0039] In order to clarify the objectives, technical aspects and advantages of the present invention, the present invention will be described in more detail below in combination with drawings and examples. It should be understood that the specific examples described herein are merely for the purpose of illustrating the present invention and do not limit the present invention. In addition, the technical features of each embodiment of the present invention described below can be combined with each other as long as they are not mutually inconsistent.

[0040] The following is a specific example. <Example> [Example]

[0041] Dried anachi root (Anacyclus pyrethrum (L.) DC., 15.0 kg) was crushed 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, oxidized with 5% hydrochloric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was stirred in an ice-water bath and the pH was adjusted to 10 with saturated aqueous NaHCO3 to obtain an alkalized solution. The alkalized solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain the total alkaloids. [Example]

[0042] Dried anachi root (Anacyclus pyrethrum (L.) DC., 15.0 kg) was crushed 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, oxidized with 5% hydrochloric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was stirred in an ice-water bath while adjusting the pH to 10 with aqueous ammonia to obtain an alkalized solution. The alkalized solution was thoroughly extracted with ethyl acetate, and the ethyl acetate extracts were combined and dried to obtain total alkaloids. [Example]

[0043] Dried anachi root (Anacyclus pyrethrum (L.) DC., 15.0 kg) was crushed 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, oxidized with 2% hydrochloric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was stirred in an ice-water bath and the pH was adjusted to 10 with aqueous Na2CO3 to obtain an alkalized solution. The alkalized solution was thoroughly extracted with n-butanol, and the n-butanol extracts were combined and dried to obtain the total alkaloids. [Example]

[0044] Dried anachi root (Anacyclus pyrethrum (L.) DC., 15.0 kg) was crushed and refluxed with 75% ethanol (45 L). The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, oxidized with 1% hydrochloric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was stirred in an ice-water bath and the pH was adjusted to 12 with aqueous NaOH to obtain an alkalized solution. The alkalized solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain total alkaloids. [Example]

[0045] Dried anachi root (Anacyclus pyrethrum (L.) DC., 15.0 kg) was crushed and extracted with 22.5 L of methanol at room temperature by cold immersion. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, oxidized with 5% hydrochloric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was stirred in an ice-water bath and the pH was adjusted to 10 with saturated aqueous NaHCO3 to obtain an alkalized solution. The alkalized solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain the total alkaloids. [Example]

[0046] Dried anachi root (Anacyclus pyrethrum (L.) DC., 15.0 kg) was crushed and extracted with 22.5 L of methanol at room temperature by cold immersion. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, oxidized with 1% sulfuric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was stirred in an ice-water bath and the pH was adjusted to 10 with saturated aqueous NaHCO3 to obtain an alkalized solution. The alkalized solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain total alkaloids. [Example]

[0047] Dried anachi root (Anacyclus pyrethrum (L.) DC., 15.0 kg) was crushed and extracted with 22.5 L of methanol at room temperature by cold immersion. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, oxidized with 2% sulfuric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was stirred in an ice-water bath and the pH was adjusted to 10 with saturated aqueous NaHCO3 to obtain an alkalized solution. The alkalized solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain total alkaloids. [Example]

[0048] Dried anachi root (Anacyclus pyrethrum (L.) DC., 15.0 kg) was crushed and extracted with 22.5 L of methanol at room temperature by cold immersion. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, oxidized with 5% sulfuric acid, and extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was stirred in an ice-water bath and the pH was adjusted to 10 with saturated aqueous NaHCO3 to obtain an alkalized solution. The alkalized solution was thoroughly extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain total alkaloids. [Example]

[0049] The total alkaloids were loaded onto 100-200 mesh silica gel, subjected to silica gel column chromatography, and gradient elution with dichloromethane / methanol (100:0 to 3:1, v / v). The same components were combined to obtain six fractions, Fr. A to Fr. F, ranging in polarity from least to most polar. The first fraction, Fr. A (159.0 g), was loaded onto 100-200 mesh silica gel, subjected to silica gel column chromatography, and gradient elution with petroleum ether / ethyl acetate (100:0 to 3:1, v / v). The same components were combined to obtain six fractions, Fr. A1 to Fr. A6, ranging in polarity from least to most polar. The second fraction, Fr. A2 (76.1 g), was purified by reverse-phase MCI column chromatography using a gradient elution with methanol / water (20:80 to 100:0, v / v). The same components were combined to yield seven subfractions, Fr. A21 to Fr. A27, ranging in polarity from most to least polar. Fraction Fr. A25 (10.0 g) was loaded onto 200-300 mesh silica gel for silica gel column chromatography. The same components were combined to yield three subfractions, Fr. A251 to Fr. A253, ranging in polarity from least to most polar. Fraction Fr. A253 (9.0 g) was loaded onto 200-300 mesh silica gel and subjected to silica gel column chromatography. Gradient elution with petroleum ether / ethyl acetate (10:1 to 3:1, v / v) yielded six fractions, Fr. A2531 to Fr. A2533, ranging in polarity from least to most polar. Fraction Fr. A2533 (7.8 g) was then purified by Sephadex LH-20 gel column chromatography and eluted with methanol to yield three subfractions, Fr. A25331 to Fr. A25333, ranging in molecular weight from most to least polar. Fraction Fr. A25333 (6.1 g) was purified by reverse-phase C18 silica gel column chromatography using a gradient elution with methanol / water (20:80 to 100:0, V / V). The same components were combined to obtain six subfractions, Fr. A253331 to Fr. A253336, ranging in polarity from most to least polar.Fraction Fr. A253336 (231.2 mg) was purified by normal-phase silica gel column chromatography using dichloromethane / methanol (500:1 to 50:1, V / V) to obtain five subfractions, Fr. A2533361 to Fr. A2533365, ranging in polarity from least to most polar. Fraction Fr. A2533363 (71.1 mg) was recrystallized in methanol to obtain Compound 1 (i.e., the compound of Formula 1) (Anacyphrethines A, 30.3 mg, 0.000202%). The racemic mixture of compound 1 was chirally separated using a chiral chromatography 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 obtain compound (+)-1 (14.0 mg, t R =12.3min) and compound (-)-1 (15.4mg, t R =18.0 min). Fraction Fr. A253335 (214.3 mg) was purified by normal-phase silica gel column chromatography and eluted with dichloromethane / methanol (500:1 to 50:1, V / V) to obtain six subfractions, Fr. A2533351 to Fr. A2533356, ranging in polarity from least to most polar. Fraction Fr. A2533353 (61.3 mg) was purified by normal-phase silica gel column chromatography and eluted with petroleum ether / acetone (5:2, V / V) to obtain compound 2 (i.e., the compound of formula 2) (Anacyphrethines B, 20.1 mg, 0.000134%). The racemic mixture of compound 2 was chirally separated using a chiral chromatography column (DAICEL CORPORATION Chiralpak ID 5 μm 10 × 250 mm; solvent: n-Hexane / EtOH = 60:40; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 360 nm) to obtain compound (+)-2 (7.5 mg, t R = 17.0 min) and compound (-)-2 (7.3 mg, t R =12.4 min). [Example]

[0050] The total alkaloids were loaded onto 100-200 mesh silica gel, subjected to silica gel column chromatography, and gradient elution with dichloromethane / methanol (100:0 to 3:1, v / v). The same components were combined to obtain six fractions, Fr. A-Fr. F, ranging in polarity from least to most polar. The first fraction, Fr. A (159.0 g), was loaded onto 100-200 mesh silica gel, subjected to silica gel column chromatography, and gradient elution with petroleum ether / ethyl acetate (100:0 to 3:1, v / v). The same components were combined to obtain six fractions, Fr. A1-Fr. A6, ranging in polarity from least to most polar. The second fraction, Fr. A2 (76.1 g), was purified by reverse-phase MCI column chromatography using a gradient elution with methanol / water (20:80 to 100:0, v / v). The same components were combined to yield seven subfractions, Fr. A21 to Fr. A27, ranging in polarity from most to least polar. Fraction Fr. A25 (10.0 g) was loaded onto 200-300 mesh silica gel for silica gel column chromatography. The same components were combined to yield three subfractions, Fr. A251 to Fr. A253, ranging in polarity from least to most polar. Fraction Fr. A253 (9.0 g) was loaded onto 200-300 mesh silica gel and subjected to silica gel column chromatography. Gradient elution with petroleum ether / ethyl acetate (10:1 to 3:1, v / v) yielded six fractions, Fr. A2531 to Fr. A2533, ranging in polarity from least to most polar. Fraction Fr. A2533 (7.8 g) was then purified by MCI column chromatography with 20-100% acetonitrile / water to yield three subfractions, Fr. A25331 to Fr. A25333, ranging in molecular weight from most to least polar. Fraction Fr. A25333 (6.1 g) was purified by reverse-phase C18 silica gel column chromatography using a gradient elution with methanol / water (20:80 to 100:0, V / V). The same components were combined to obtain six subfractions, Fr. A253331 to Fr. A253336, ranging in polarity from most to least polar.Fraction Fr. A253336 (231.2 mg) was purified by normal-phase silica gel column chromatography using dichloromethane / methanol (500:1 to 50:1, V / V) to obtain five subfractions, Fr. A2533361 to Fr. A2533365, ranging in polarity from least to most polar. Fraction Fr. A2533363 (71.1 mg) was recrystallized in methanol to obtain Compound 1 (i.e., the compound of Formula 1) (Anacyphrethines A, 30.3 mg, 0.000202%). The racemic mixture of compound 1 was chirally separated using a chiral chromatography 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 obtain compound (+)-1 (14.0 mg, t R =12.3min) and compound (-)-1 (15.4mg, t R =18.0 min). Fraction Fr. A253335 (214.3 mg) was purified by normal-phase silica gel column chromatography and eluted with dichloromethane / methanol (500:1 to 50:1, V / V) to obtain six subfractions, Fr. A2533351 to Fr. A2533356, ranging in polarity from least to most polar. Fraction Fr. A2533353 (61.3 mg) was purified by normal-phase silica gel column chromatography and eluted with petroleum ether / acetone (5:2, V / V) to obtain compound 2 (i.e., the compound of formula 2) (Anacyphrethines B, 20.1 mg, 0.000134%). The racemic mixture of compound 2 was chirally separated using a chiral chromatography column (DAICEL CORPORATION Chiralpak ID 5 μm 10 × 250 mm; solvent: n-Hexane / EtOH = 60:40; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 360 nm) to obtain compound (+)-2 (7.5 mg, t R = 17.0 min) and compound (-)-2 (7.3 mg, t R =12.4 min). [Example]

[0051] The total alkaloids were loaded onto 100-200 mesh silica gel and subjected to silica gel column chromatography. The resulting mixture was then gradient-eluted with dichloromethane / methanol (100:0 to 3:1, v / v). Six fractions, Fr. A to Fr. F, were obtained by combining the same components. The first fraction, Fr. A (159.0 g), was repeatedly loaded onto a silica gel column and eluted with petroleum ether-ethyl acetate (volume ratios: 100:0 to 3:1), dichloromethane-methanol (volume ratios: 500:1 to 3:1), or petroleum ether / acetone (volume ratios: 50:1 to 0:1). Compound 1 (i.e., the compound of formula 1) (Anacyphrethines A, 30.3 mg, 0.000202%) and compound 2 (i.e., the compound of formula 2) (Anacyphrethines B, 20.1 mg, 0.000134%) were obtained. The racemic mixture of compound 1 was chirally separated using a chiral chromatography column (DAICEL CORPORATION Chiralpak ID 5 μm 10 × 250 mm; solvent: n-Hexane / EtOH = 99:1 to 50:50; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 360 nm) to obtain compound (+)-1 (14.0 mg, t R = 12.3 min) and compound (-)-1 (15.4 mg, t R The racemic mixture of compound 2 was chirally separated using a chiral chromatography column (DAICEL CORPORATION Chiralpak ID 5 μm 10 × 250 mm; solvent: n-Hexane / EtOH = 99:1 to 50:50; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 360 nm) to obtain compound (+)-2 (7.5 mg, t R = 17.0 min) and compound (-)-2 (7.3 mg, t R =12.4 min). [Example]

[0052] The NaV1.2 inhibitory activity of compounds 1 and 2 was evaluated by patch-clamp electrophysiology. HEK293T cells (ATCC Cell Bank) were cultured at 37°C in a 5% CO2 incubator in 90% DMEM + 10% fetal bovine serum (FBS). When the cells reached a confluency of 80-90%, they were digested with 0.25% trypsin and then subcultured or plated. 24 h after plating, they were transfected using a Lipo2000 transfection kit (Thermo Fisher, Shanghai, China) with a 9:1 ratio (4000 ng total) of pcDNA3.1-SCN2A (NaV1.2 GenBank accession no. NM_001040142) plasmid (BGI Genomics, Beijing, China) and pcDNA3.1-EGFP plasmid (BGI Genomics, Beijing, China). Electrophysiological experiments were performed 18 h or later after transfection.

[0053] For current-clamp recording experiments, an Axon patch 700B patch-clamp amplifier (Axon Instruments, Molecular Devices, USA) was used, along with a Digidata 1440A digital-to-analog converter (Axon Instruments, Molecular Devices, USA). Signal acquisition was performed using pClamp 10.0 software (Molecular Devices, USA), with frequency filtering at 2 kHz and a sampling frequency of 10 kHz. Patch-clamp electrodes were prepared by multi-step traction using a horizontal traction electrode preparation system P-97 (Sutter Instrument, USA). The electrodes were filled with fluid and their electrical resistance was measured. They were ready for use when they were 3–5 MΩ. Recordings were performed at room temperature (23–25°C). The perfusion system was handmade, with a rate of approximately 2 mL / min. The administration system was a BPS-8 (ALA Scientific Instruments, USA). All electrophysiological data were processed with Clampfit 10.4 (Molecular Device, USA) and analyzed with GraphPad Prism 5 (GraphPad Software, USA). The initial screening concentration of single compounds was 40 μM, and the inhibition rate results are shown in Table 3.

[0054] [Table 3]

[0055] Conclusion: Compound (+)-2 has a significant inhibitory effect on Nav1.2, and among these, compound (+)-2 has micromolar-level Nav1.2 inhibitory activity, with an IC 50 The value is 23.94±2.70 μM. [Example]

[0056] NO Inhibitory Activity of Compound of Formula 1 (Compound 1) and Compound of Formula 2 (Compound 2)

[0057] 1.Cell culture 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.

[0058] 2. Testing the effect of the compounds of the present invention on cell viability The compounds were dissolved in dimethyl sulfoxide (DMSO), and 5 × 10 BV2 cells in the logarithmic growth phase were cultured. 3 Cells were seeded into a 96-well plate at 100 μM per well. The experimental group was treated with the compound of formula I at different concentrations of 12.5, 25, 50, and 100 μM. The control group was treated with dimethyl sulfoxide (DMSO). After 24 hours of incubation, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-benzenedisulfonic acid)-2H-tetrazole 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.

[0059] [Table 4]

[0060] 3. Measurement of nitric oxide (NO) content The amount of NO released in BV2 cells was measured using the Griess method (Arias-Negrete et al., Analytical Biochemistry 328.1(2004):14-21.). After incubation for 2 hours with different concentrations of 25, 50, and 100 μM samples, 1 μg / mL of lipopolysaccharide (LPS, Sigma, L4391) was added and the cells were co-cultured for 22 hours. After incubation, the cell supernatant was collected and the nitric oxide content in the cell supernatant was measured using the Griess method. Prior to measurement, Griess Reagent I and II (Nitric Oxide Assay kit, Beyotine, S0021M) were removed and warmed to room temperature. Standards were diluted (1–100 μM) with complete medium at concentrations of 0, 1, 2, 5, 10, 20, 40, 60, and 100 μM. 50 μL of the standards and collected culture supernatants were added to a 96-well plate. 50 μL of room-temperature Griess Reagent I and 50 μL of Griess Reagent II were added sequentially to each well. After shaking for 5 minutes, the absorbance at 540 nm was measured, and a calibration curve was constructed. The NO content in the culture supernatants was calculated according to the calibration curve. The initial screening concentration of the single compounds was 40 μM. The inhibition results are shown in Table 5.

[0061] [Table 5]

[0062] Conclusion: Compounds 1 and 2 both have a certain inhibitory effect on NO release, and the IC values ​​of compounds (+)-1 and (-)-1 were significantly higher than those of compounds (-)-1. 50 The values ​​are 27.63±3.753 and 37.35±0.807, respectively. Compounds (+)-1 and (-)-1 have a significant inhibitory effect on NO release. [Example]

[0063] In this example, we used Autodock 4.2.6 molecular docking software (The Scripps Research Institute, USA) to investigate the mechanism of action of compound 2 with Nav1.2 (pdb ID: 6J8E). Molecular dynamics simulations captured the dynamic structural changes and trajectories of the compound upon binding to the Nav1.2 protein. Nav1.2 consists of a single polypeptide chain that folds into four homologous repeat sequences. Conclusions: The docking results indicate that (+)-2 binds to the upper residues of the active pocket and occupies the Nav1.2 channel pore with low affinity (-9.126 kcal / mol), consistent with previous binding experiments. Detailed analysis of the interaction between the Nav1.2 channel active site and (+)-2 indicates that the C-10 carbonyl group of (+)-2 acts as a hydrogen bond donor and forms a hydrogen bond with Nav1.2 channel residue Asn333. Furthermore, (+)-2 interacts with residues such as Gln332, Phe385, Asn361, Asp334, Tyr362, Asp949, Trp948, Pro921, Trp923, Tyr1429, and Met1425, which may be the active site of Nav1.2, providing a rationale for the development of efficient Nav1.2 inhibitors.

[0064] Those skilled in the art can easily understand that the above description is only a preferred embodiment of the present invention, and does not limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. 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']azepino[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-triketone (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-methanoaza[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)-triketone (compound of formula 2) A compound selected from the group consisting of:

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

3. (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']azepino[4',5':4,5]pyrrolo[3,2,1-ij]quinoline-1,8,11(9H)-triketone, (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-methanoaza[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)-triketone, (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-methanoaza[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)-triketone, 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-methanoaza[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)-triketone 3. The compound according to claim 1 or 2, or an isomer thereof, selected from the group consisting of:

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

5. 10. A method for extracting the compound of any one of claims 1 to 4 or an isomer thereof from annatti root, comprising the steps of: a. Anathi root is dried and then crushed, and the extract is obtained by cold maceration, percolation, heating under reflux, or ultrasonic extraction using a solvent of 50-95% (v / v) ethanol aqueous solution, methanol, or chloroform at a volume fraction of 1:1.5-1:4, where the ratio of anathi root (kg) to solvent (L) is 1:1.5-1:4, followed by concentration under reduced pressure to recover the solvent; b. The entire extract from step a is suspended in water, and then dispersed in an acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid. The resulting acid aqueous layer is extracted with dichloromethane to remove non-alkaloids, followed by NaHCO ３ , Na ２ CO ３ The pH is adjusted to 10-12 with aqueous ammonia or an alkali such as 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 the total alkaloids; c) The total alkaloids from step b are separated by silica gel column chromatography, thin layer chromatography, reversed-phase MCI column chromatography, Sephadex LH-20 column chromatography, high performance liquid chromatography, or any combination thereof to obtain the compound or an isomer thereof.

6. In step c, separation is performed by 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 by gradient or isocratic elution of normal phase silica gel column chromatography, followed by reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography to obtain the compound of formula 1 or formula 2, more preferably by using an eluent of petroleum ether and acetic acid in a volume ratio of 100:0 to 3:1 in the normal phase silica gel column chromatography.

6. The method according to claim 5, wherein the eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a 20% to 100% (v / v) aqueous methanol solution or a 20% to 100% (v / v) aqueous acetonitrile solution, and the eluent for isocratic or gradient elution used in the semi-preparative high-performance liquid chromatography is a 99% to 50% (v / v) n-hexane / EtOH solution.

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

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

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

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

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