Analgesic compounds derived from anachan root that have Nav1.2 inhibitory activity, 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 anachan root that have Nav1.2 inhibitory activity and NO inhibitory activity, as well as 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 a patient's mental health, causing anxiety, sadness, and a weakened immune system, leading to a range of consequences, most severely potentially fatal or causing physical disability. However, conventional analgesics, such as opioids and nonsteroidal anti-inflammatory drugs, have side effects such as drug tolerance, addiction, and gastrointestinal bleeding.

[0003] Ion channels can regulate the voltage potential across the membrane by controlling the passage of ions inside and outside the cell. The flow of ions generates electrical pulses, which in turn prompt adjacent voltage-sensitive channels to open sequentially, generating spontaneous electrical signals. Blocking ion channels inhibits sustained rhythmic potentials, disrupting signal transduction and thereby reducing the pain response. Currently, various ion channel inhibitors, including VGSCs, VGCCs, VGPCs, and TRPs, have been reported. Sodium channels are involved in the generation and transmission of action potentials and are fundamental components of electrical signals generated by all excitatory cells (including nerve and muscle cells). The human body has nine subtypes of sodium channels, called Nav1.1 to Nav1.9. For example, lidocaine reduces the pain response by inhibiting Nav1.7 and Nav1.9. Therefore, finding new ion channel inhibitors with analgesic effects is extremely important.

[0004] Since the search for cutting-edge compounds with remarkable pharmacodynamic activity from conventional medicinal plants is a hot topic in drug research, discovering new ion channel inhibitors and anti-inflammatory compounds 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 belonging to the Asteraceae family. Its functions are primarily to refresh the brain (resuscitation), and to treat conditions such as hemiplegia, vitiligo, epilepsy, headaches, cough and phlegm relief, and convulsions. [Disclosure of the Invention]

[0006] As a result of diligent research, the inventors have isolated for the first time analgesic / anti-inflammatory compounds (compounds of formula 1 and formula 2) from the root of the Japanese honeysuckle (Anatina japonica) that exhibit Nav1.2 inhibitory activity and NO inhibitory activity. These compounds are novel skeletal compounds, highly conjugated tetraamino-6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloid compounds. Their structures have been identified, and cell experiments have verified that they possess Nav1.2 inhibitory activity and NO inhibitory activity, making them suitable for use in the preparation of analgesic and / or anti-inflammatory drugs.

[0007] Therefore, 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)-Tricetone (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)-Tricketone (compound of formula 2) A compound or its isomer selected from the group consisting of the above.

[0009] 2. The compound described in 1 above or its isomer, 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)-tricetone, (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)-tricetone, and (4aS,7aS,11aS,14aR)-7a A compound or isomer thereof 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)-tricetone.

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

[0012]

Chemical formula

[0013] 5. A method for extracting the compound or its isomer according to any one of the above items 1 to 4 from Anacyclus roots, comprising the following steps: a. After drying and pulverizing Anacyclus roots, using an aqueous ethanol solution, methanol or chloroform with a volume fraction of 50 - 95% (v / v) as a solvent (the ratio of the weight of the medicinal material (Kg) to the volume of the solvent (L) is 1:1.5 - 1:4), extract by cold soaking, percolation method, heating under reflux or ultrasonic wave, concentrate under reduced pressure to recover the solvent, and obtain an extract; b. Suspend the total extract of step a in water, then disperse it with an acid such as hydrochloric acid with a concentration of 1 - 5% or sulfuric acid with a concentration of 1 - 5%, extract the obtained acid aqueous layer with dichloromethane to remove non - alkaloids, then adjust the pH to 10 - 12 with an alkali such as NaHCO3, Na2CO3, aqueous ammonia or NaOH, and further extract with an organic solvent of dichloromethane, ethyl acetate or n - butanol, concentrate under reduced pressure to recover the organic solvent, and obtain total alkaloids; c. Separate the total alkaloids of step b by silica gel column chromatography, thin - layer chromatography, Reverse phase Ram chromatography, Sephadex Ska Ram chromatography, high - performance liquid chromatography or any combination thereof to obtain the compound or its isomer.

[0014] 6. In step c, separate by a combination of normal - phase silica gel column chromatography and Reverse phase Ram chromatography or semi - preparative high - performance liquid chromatography. Preferably, after gradient or isocratic elution by normal - phase silica gel column chromatography, Reverse phaseCompounds of formula 1 or formula 2 are obtained by ram chromatography or 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 in a volume ratio of 100:0 to 3:1, dichloromethane and methanol in a volume ratio of 500:1 to 3:1, petroleum ether and acetone in a volume ratio of 50:1 to 0:1, or methanol and water in a volume ratio of 1:9 to 1:0. Reverse phase The method according to 5, wherein the eluent used in RAM 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 used in the semi-preparative high-performance liquid chromatography for fixed composition or gradient elution is n-hexane / EtOH in a volume ratio of 99-50% (v / v).

[0015] 7. In step c, normal-phase silica gel column chromatography and Reverse phase Separation is performed by a combination of lamb chromatography and semi-preparative high-performance liquid chromatography, and preferably, gradient or fixed-composition elution is performed by normal-phase silica gel column chromatography. Reverse phase Gradient elution is performed by lamb chromatography, and further by 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 petroleum ether and ethyl acetate in a volume ratio of 100:1 to 0:1, dichloromethane and methanol, or trichloromethane and methanol. Reverse phase The method according to 5, wherein the eluent used in RAM 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 used in the semi-preparative high-performance liquid chromatography for fixed composition or gradient elution is n-hexane / EtOH in a volume ratio of 99-50% (v / v).

[0016] 8. In step c, normal-phase silica gel column chromatography and Sephadec Ska Lamb chromatography and Reverse phase Separation is performed using a combination of lamb chromatography and semi-preparative high-performance liquid chromatography, and preferably, gradient or fixed-composition elution is performed by normal-phase silica gel column chromatography, followed by Sephadec. Ska After lamb chromatography, Reverse phase The compounds are obtained by gradient elution by lamb chromatography, and further by semi-preparative high-performance liquid chromatography to obtain compounds 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 100:1 to 0:1, dichloromethane and methanol, or trichloromethane and methanol, and the Sephadec Ska In Rham chromatography, elution is performed using methanol gradient or fixed composition, as described above. Reverse phase The method according to 5, wherein the eluent used in RAM chromatography is a methanol aqueous solution with a volume ratio of 20-100% (v / v) or an acetonitrile aqueous solution with a volume ratio of 20-100% (v / v), and the eluent used in semi-preparative high-performance liquid chromatography for fixed composition or gradient elution is n-hexane / EtOH with a volume ratio of 99-50% (v / v).

[0017] 9. The method according to any one of items 5 to 8 above, 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 reversed-phase silica gel.

[0018] 10. The use of the compound or its isomer described in any one of items 1 to 4 above 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 object of the present invention is to provide compounds having Nav1.2 inhibitory activity and / or anti-inflammatory activity, methods for separating and preparing the same, and their applicability in the preparation of analgesic drugs. According to the present invention, in a first embodiment, a compound having Nav1.2 inhibitory activity and / or anti-inflammatory activity is provided, 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)-tricetone, 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)-tricetone, 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)-tricetone, 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)-tricetone.

[0022] The extraction and separation method for the aforementioned alkaloid compounds is carried out in the following steps. a. After drying the Anachini root, it is crushed and extracted using a 50-95% (v / v) volume fraction aqueous ethanol solution, methanol, or chloroform as a solvent (ratio of medicinal material weight (kg) to solvent (L) 1:1.5-1:4). The extract is obtained by cold maceration, percolation, heating under reflux, or sonication, and then concentrated under reduced pressure to recover the solvent; b. After suspending the entire extract from step a in water, disperse it with an acid such as hydrochloric acid or sulfuric acid at a concentration of 1-5%. Extract the resulting acidic aqueous layer with dichloromethane to remove non-alkaloids, then adjust the pH to 10-12 with an alkali such as NaHCO3, Na2CO3, aqueous ammonia, or NaOH. Further extract with an organic solvent such as dichloromethane, ethyl acetate, or n-butanol, concentrate under reduced pressure to recover the organic solvent, and obtain the total alkaloids. c. Total alkaloids in step b are analyzed by silica gel column chromatography, thin-layer chromatography, and reverse chromatography. Mutual Lamb chromatography, Sephadec Ska The compound or its isomers are separated by Lamb chromatography, high-performance liquid chromatography, or any combination thereof.

[0023] Among them, there are two types of separation methods: After gradient elution by normal-phase silica gel column chromatography using a petroleum ether-ethyl acetate or dichloromethane-methanol in a volume ratio of 100:0 to 3:1 as the eluent, Reverse phase Compounds of formula 1 or formula 2 are obtained by Lamb chromatography or semi-preparative high-performance liquid chromatography.

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

[0025] 4 types of separation methods: After gradient elution by normal-phase silica gel column chromatography with the eluent being petroleum ether-ethyl acetate, dichloromethane-methanol, or trichloromethane-methanol in a volume ratio of 100:1 to 0:1, Sephadec Ska After eluting with methanol by RAM chromatography, gradient elution is performed with a 20-100% (v / v) methanol aqueous solution or a 20-100% (v / v) acetonitrile aqueous solution by reverse-phase silica gel or MCI column chromatography. Finally, the compound of formula 1 or formula 2 is obtained by semi-preparative high-performance liquid chromatography using a 99-50% (v / v) n-hexane / EtOH eluent.

[0026] The method for preparing alkaloid compounds from Anachinan root according to step c, 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, and fixed composition or gradient elution is used.

[0027] The aforementioned Cepha Deck Ska The method for preparing alkaloid compounds from Anachinan root according to step c, characterized in that the eluent for lamb chromatography is methanol and fixed-composition elution is used. The method for preparing alkaloid compounds from Anachinan root according to step c, characterized in that the preparative high-performance liquid chromatography uses n-hexane / EtOH in a volume ratio of 99-50% (v / v) as the eluent, and either a fixed composition or gradient elution is used.

[0028] Another embodiment of the present invention provides for the use of a compound having the Nav1.2 inhibitory effect in the preparation of analgesic drugs.

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

[0030] Compound 1 (Anacyphrethine A): Yellow needle-shaped crystals; optical rotation values ​​[α]25 D1584 (c 0.08, methanol, (+)-1); [α]25 D1584 (c 0.1, methanol, (-)-1); ultraviolet (methanol) λ max(logε) 427 (4.15) nm, 267 (3.96) nm; infrared (KBr) max 3297, 2961, 2924, 1704, 1623, and 1448 cm -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), its 1 H and 13 C NMR spectral data are shown in Table 1 and Table 2. Compound 2 (Anacyphrethine A): yellow needle crystals; specific rotation value [α]25 D -530 (c 0.03, methanol, (-)-2); [α]25 D 530 (c 0.02, methanol, (+)-2); ultraviolet (methanol) λ max (logε) 475 (3.90) nm, 281 (3.74) nm; infrared (KBr) max 3316, 2963, 2926, 2857, 1699, 1615, 1445, and 1194 cm -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 The 13C 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-mentioned technical embodiments of the present invention have the following main technical advantages compared to the prior art.

[0034] (1) Compounds 1 and 2 provided in the present invention are novel skeletal compounds. Compounds 1 and 2 are novel skeletal enantiomeric isomers of two pairs of unprecedented highly conjugated tetraamino 6 / 6 / 6 / 5 / 7 / 5 octacyclic alkaloids, and are 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 It has a nonacosane ring system as its skeletal structure and possesses four discontinuous chiral stereocenters.

[0035] (2) Compounds 1 and 2 provided in the present invention have Nav1.2 inhibitory activity and NO inhibitory activity, and compound 2 has micromolar Nav1.2 inhibitory activity and potential Nav1.2 inhibitory activity.

[0036] (3) The present invention performs molecular docking using a computer and combines the activity results of the compound with those of Nav1.2 to conclude that the residues Gln332, Phe38, Asn361, Asp334, Tyr362, Asp949, Trp948, Pro921, Trp923, Tyr1429 and Met1425 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 attached drawings and examples, but the scope of the invention is not limited thereto. Any modifications or substitutions made to the methods, steps, conditions, etc. of the present invention, without departing from the spirit and essence of the invention, shall be included within the scope of the invention. [Brief explanation of the drawing]

[0038] [Figure 1] Figure 1 is the X-ray single-crystal diffraction pattern of compound 1. [Figure 2] Figure 2 shows the X-ray single crystal diffraction pattern of compound (+)-1. [Figure 3] Figure 3 is the X-ray single crystal diffraction pattern of compound (-)-1. [Figure 4] Figure 4 shows the X-ray single-crystal diffraction pattern of compound 2. [Figure 5] Figure 5 shows the molecular docking and molecular dynamics simulation results for compound (+)-2. [Figure 6] Figure 6 is the 1H NMR spectrum of compound 1. [Figure 7] Figure 7 is the 13C NMR map of compound 1. [Figure 8] Figure 8 shows the 1H NMR spectrum of compound 2. [Figure 9] Figure 9 is the 13C NMR spectrum of compound 2. [Modes for carrying out the invention]

[0039] To further clarify the object, technical modes, and advantages of the present invention, the invention will be described in more detail below in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not limiting. Furthermore, the technical features of each embodiment of the present invention described below can be combined with each other, insofar as they do not contradict each other.

[0040] The following are specific examples. <Examples> [Examples]

[0041] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) roots were crushed and ultrasonically extracted with chloroform (30 L). The extracts were concentrated under reduced pressure and combined to obtain the total extract. The total extract was suspended in water, oxidized with 5% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acidic aqueous solution was stirred in an ice bath and the pH was adjusted to 10 with saturated NaHCO3 aqueous solution 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. [Examples]

[0042] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) roots were crushed and extracted by percolation with 95% ethanol (40 L). The extracts were concentrated under reduced pressure and combined to obtain the total extract. The total extract was suspended in water, oxidized with 5% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acidic aqueous solution was stirred in an ice bath and the pH was adjusted 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 the total alkaloids. [Examples]

[0043] Dried Anacyclus pyrethrum (L.) DC. (15.0 kg) roots were crushed and extracted by percolation with 50% ethanol (40 L). The extracts were concentrated under reduced pressure and combined to obtain the total extract. The total extract was suspended in water, oxidized with 2% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acidic aqueous solution was stirred in an ice bath and the pH was adjusted to 10 with Na2CO3 aqueous solution 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. [Examples]

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

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

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

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

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

[0049] All of the above alkaloids were loaded onto 100-200 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with dichloromethane / methanol (100:0-3:1, V / V). The same components were combined to obtain six fractions Fr.A-Fr.F ranging from low to high polarity. The first fraction Fr.A (159.0g) was loaded onto 100-200 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with petroleum ether / ethyl acetate (100:0-3:1, V / V). The same components were combined to obtain six fractions Fr.A1-Fr.A6 ranging from low to high polarity. The second fraction, Fr.A2 (76.1g), was gradient eluted with methanol / water by reverse-phase MCI column chromatography (20:80~100:0, V / V), and the same components were combined to obtain seven sub-fractions Fr.A21~Fr.A27 ranging from highly polar to highly polar. Fraction Fr.A25 (10.0g) from these sub-fractions was loaded onto 200~300 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with petroleum ether / ethyl acetate (10:1~3:1, V / V). The same components were combined to obtain three fractions Fr.A251~Fr.A253 ranging from highly polar to highly polar. Fraction Fr.A253 (9.0g) was loaded onto 200-300 mesh silica gel, and silica gel column chromatography was performed. Gradient elution with petroleum ether / ethyl acetate (10:1-3:1, V / V) was then performed, and the same components were combined to obtain six fractions Fr.A2531-Fr.A2533 ranging from small to large polarity. Fraction Fr.A2533 (7.8g) was eluted with methanol by Sephadex LH-20 gel column chromatography to obtain three sub-fractions Fr.A25331-Fr.A25333 ranging from large to small molecular weight. Fraction Fr.A25333 (6.1g) was eluted with methanol / water gradient by reverse-phase C18 silica gel column chromatography (20:80~100:0, V / V), and the same components were combined to obtain six sub-fractions Fr.A253331~Fr.A253336 ranging from highly polar to less polar.Fraction Fr.A253336 (231.2 mg) was eluted with dichloromethane / methanol by normal-phase silica gel column chromatography (500:1~50:1, V / V) to obtain five sub-fractions Fr.A2533361~Fr.A2533365 ranging from low to high polarity. Fraction Fr.A2533363 (71.1 mg) was recrystallized in methanol to obtain compound 1 (i.e., compound of formula 1) (Anacyphrethines A, 30.3 mg, 0.000202%). The racemic mixture of compound 1 was chiral-resolved using a chiral chromatography column (DAICEL CORPORATION Chiralpak ID 5μm 10×250mm; solvent: n-Hexane / EtOH=90:10; column temperature: 25℃; flow rate: 3ml / min; detection wavelength: 360nm) to obtain compound (+)-1 (14.0mg, t). R =12.3min) and compound (-)-1 (15.4mg, t R A fraction of 18.0 min was obtained. Fraction Fr.A253335 (214.3 mg) was eluted with dichloromethane / methanol by normal-phase silica gel column chromatography (500:1~50:1, V / V) to obtain six sub-fractions Fr.A2533351~Fr.A2533356 ranging from low to high polarity. Fraction Fr.A2533353 (61.3 mg) was eluted with petroleum ether / acetone by normal-phase silica gel column chromatography (5:2, V / V) to obtain compound 2 (i.e., compound of formula 2) (Anacyphrethines B, 20.1 mg, 0.000134%). The racemic mixture of compound 2 was chiral-resolved using a chiral chromatography column (DAICEL CORPORATION Chiralpak ID 5μm 10×250mm; solvent: n-Hexane / EtOH=60:40; column temperature: 25℃; flow rate: 3ml / min; detection wavelength: 360nm) to obtain compound (+)-2 (7.5mg, t R =17.0min) and compound (-)-2 (7.3mg, t R We obtained (=12.4 min). [Examples]

[0050] All of the above alkaloids were loaded onto 100-200 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with dichloromethane / methanol (100:0-3:1, V / V). The same components were combined to obtain six fractions Fr.A-Fr.F ranging from low to high polarity. The first fraction Fr.A (159.0g) was loaded onto 100-200 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with petroleum ether / ethyl acetate (100:0-3:1, V / V). The same components were combined to obtain six fractions Fr.A1-Fr.A6 ranging from low to high polarity. The second fraction, Fr.A2 (76.1g), was subjected to reverse-phase MCI column chromatography, followed by gradient elution with methanol / water (20:80~100:0, V / V). The same components were then combined to obtain seven sub-fractions, Fr.A21 to Fr.A27, ranging from highly polar to highly polar. Fraction Fr.A25 (10.0g) was loaded onto 200~300 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with petroleum ether / ethyl acetate (10:1~3:1, V / V). The same components were then combined to obtain three fractions, Fr.A251 to Fr.A253, ranging from highly polar to highly polar. Fraction Fr.A253 (9.0g) was loaded onto 200-300 mesh silica gel, and silica gel column chromatography was performed. Gradient elution with petroleum ether / ethyl acetate (10:1-3:1, V / V) was then performed, and the same components were combined to obtain six fractions Fr.A2531-Fr.A2533 ranging from small to large polarity. Fraction Fr.A2533 (7.8g) was eluted with 20-100% acetonitrile water by MCI column chromatography to obtain three sub-fractions Fr.A25331-Fr.A25333 ranging from large to small molecular weight. Fraction Fr.A25333 (6.1g) was eluted with methanol / water gradient by reverse-phase C18 silica gel column chromatography (20:80~100:0, V / V), and the same components were combined to obtain six sub-fractions Fr.A253331~Fr.A253336 ranging from highly polar to less polar.Fraction Fr.A253336 (231.2 mg) was eluted with dichloromethane / methanol by normal-phase silica gel column chromatography (500:1~50:1, V / V) to obtain five sub-fractions Fr.A2533361~Fr.A2533365 ranging from low to high polarity. Fraction Fr.A2533363 (71.1 mg) was recrystallized in methanol to obtain compound 1 (i.e., compound of formula 1) (Anacyphrethines A, 30.3 mg, 0.000202%). The racemic mixture of compound 1 was chiral-resolved using a chiral chromatography column (DAICEL CORPORATION Chiralpak ID 5μm 10×250mm; solvent: n-Hexane / EtOH=90:10; column temperature: 25℃; flow rate: 3ml / min; detection wavelength: 360nm) to obtain compound (+)-1 (14.0mg, t). R =12.3min) and compound (-)-1 (15.4mg, t R A fraction of 18.0 min was obtained. Fraction Fr.A253335 (214.3 mg) was eluted with dichloromethane / methanol by normal-phase silica gel column chromatography (500:1~50:1, V / V) to obtain six sub-fractions Fr.A2533351~Fr.A2533356 ranging from low to high polarity. Fraction Fr.A2533353 (61.3 mg) was eluted with petroleum ether / acetone by normal-phase silica gel column chromatography (5:2, V / V) to obtain compound 2 (i.e., compound of formula 2) (Anacyphrethines B, 20.1 mg, 0.000134%). The racemic mixture of compound 2 was chiral-resolved using a chiral chromatography column (DAICEL CORPORATION Chiralpak ID 5μm 10×250mm; solvent: n-Hexane / EtOH=60:40; column temperature: 25℃; flow rate: 3ml / min; detection wavelength: 360nm) to obtain compound (+)-2 (7.5mg, t R =17.0min) and compound (-)-2 (7.3mg, t R We obtained (=12.4 min). [Examples]

[0051] All of the above alkaloids were loaded onto 100-200 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with dichloromethane / methanol (100:0-3:1, V / V). The same components were combined to obtain six fractions Fr.A-Fr.F ranging from low to high polarity. The first fraction Fr.A (159.0g) was repeatedly loaded onto silica gel column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate in volume ratios of 100:0-3:1, dichloromethane-methanol in volume ratios of 500:1-3:1, or petroleum ether / acetone in volume ratios of 50:1-0:1 as the eluent 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 racemic mixture of compound 1 was chiral-resolved using a chiral chromatography 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) to obtain compound (+)-1 (14.0mg, t R =12.3min) and compound (-)-1 (15.4mg, t R A racemic mixture of compound 2 was obtained (18.0 min). The racemic mixture of compound 2 was chiral-resolved using a chiral chromatography column (DAICEL CORPORATION Chiralpak ID 5 μm 10 × 250 mm; solvent: n-Hexane / EtOH = 99:1~50:50; column temperature: 25°C; flow rate: 3 ml / min; detection wavelength: 360 nm) to obtain compound (+)-2 (7.5 mg, t R =17.0min) and compound (-)-2 (7.3mg, t R We obtained (=12.4 min). [Examples]

[0052] The Nav1.2 inhibitory activity of compound 1 and compound 2 was evaluated by patch-clamp electrophysiological experiments. HEK293T cells (ATCC cell bank) were used in the experiment. Culture conditions were 37°C in a 5% CO2 incubator, with a medium containing 90% DMEM + 10% fetal bovine serum (FBS). When the cell density reached 80-90%, the cells were digested with 0.25% trypsin and then subcultured or plated. 24 hours after plating, transfection was performed using a Lipo2000 transfection kit (Thermo Fisher, Shanghai, China) with a ratio of pcDNA3.1-SCN2A (NaV1.2 GenBank accession No. NM_001040142) plasmid (BGI Genomics, Beijing, China) to pcDNA3.1-EGFP plasmid (BGI Genomics, Beijing, China) of 9:1 (total 4000 ng). Electrophysiological experiments were performed 18 hours after transfection.

[0053] For the current clamp recording experiment, an Axon patch 700B patch clamp amplifier (Axon Instruments, Molecular Devices, USA) was used, the digital-to-analog converter was a Digidata 1440A (Axon Instruments, Molecular Devices, USA), and the signal acquisition was performed using pClamp 10.0 software (Molecular Devices, USA). The frequency filtering was 2 kHz, and the sampling frequency was 10 kHz. Patch clamp electrodes were fabricated using a P-97 horizontal traction electrode fabricator (Sutter Instruments, USA) through a multi-step procedure. Electrodes were deemed usable if their electrical resistance, measured after filling them with fluid, was 3-5 MΩ. The recording process was performed under room temperature conditions (23-25°C). The perfusion system was handmade, with a flow rate of approximately 2 mL / min. The dosing 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 the individual compounds was 40 μM, and the inhibition rate results are shown in Table 3.

[0054] [Table 3]

[0055] Conclusion: Compound (+)-2 exhibits significant inhibitory activity against Nav1.2, and in particular, compound (+)-2 has micromolar-level Nav1.2 inhibitory activity, and its IC 50 The value is 23.94 ± 2.70 μM. [Examples]

[0056] NO inhibitory activity of compound 1 (compound 1) and compound 2 (compound 2)

[0057] 1.Cell culture BV2 cells (purchased from Kitano Biological BeNa Culture Collection, BNCC) were cultured in Dulbecco's modified eagle medium (DMEM) containing 1% fetal bovine serum (FBS) (purchased from Giboco, Inc., USA), 1% penicillin, and streptomycin in a high-glucose medium (purchased from Hyclone, Inc., USA) in an incubator at 37°C and 5% CO2.

[0058] 2. Testing the effect of the compound according to the present invention on cell viability. The compound was dissolved in dimethyl sulfoxide (DMSO), and BV2 cells in good growth condition during the logarithmic growth phase were selected in 5 × 10⁻⁶ units. 3 Cells were inoculated into 96-well plates at a rate of one cell / well. Different concentrations of formula I compound (12.5, 25, 50, and 100 μM) were added to the experimental group, and dimethyl sulfoxide (DMSO) was added to the control group. The cells were incubated for 24 hours, and then 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 cell viability was calculated. The experimental results are shown in Table 4.

[0059] [Table 4]

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

[0061] [Table 5]

[0062] Conclusion: Both compounds 1 and 2 exhibit a certain inhibitory effect on NO release, particularly on the IC of compounds (+)-1 and (-)-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. [Examples]

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

[0064] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and does not limit it, and that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should all be within the scope of protection 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)-Tricetone (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)-Tricketone (compound of formula 2) A compound selected from the group consisting of the following.

2. (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)-tricetone, (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)-tricetone, (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)-tricetone, 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)-tricetone A compound according to claim 1, selected from the group consisting of the following.

3. The compound according to claim 1 or 2, having a structural formula selected from the following. 【Chemistry 1】

4. A method for extracting the compound described in claim 1 from a rhododendron root, comprising the following steps: a. After drying the roots of the Anachi plant, they are ground and extracted using an aqueous solution of ethanol, methanol, or chloroform with a volume fraction of 50-95% (v / v), where the ratio of Anachi roots (kg) to solvent (L) is 1:1.5-1:

4. The extract is obtained by cold maceration, percolation, heating under reflux, or sonication, and the solvent is recovered by reducing the pressure and concentrating. b. After suspending the entire extract from step a in water, disperse it with an acid such as hydrochloric acid or sulfuric acid at a concentration of 1-5%. The resulting acidic aqueous layer is then extracted with dichloromethane to remove non-alkaloids, followed by NaHCO3. ３ Na ２ CO ３ The pH is adjusted to 10-12 with ammonia water or an alkali such as NaOH, and then extracted with an organic solvent such as dichloromethane, ethyl acetate, or n-butanol. The organic solvent is then recovered by reducing the pressure and concentration to obtain the total alkaloids. c. The total alkaloids from step b are separated by silica gel column chromatography, thin-layer chromatography, reverse-phase column chromatography, Sephadex column chromatography, high-performance liquid chromatography, or any combination thereof to obtain the compound.

5. The method according to claim 4, wherein in step c, separation is performed by a combination of normal-phase silica gel column chromatography and reverse-phase column chromatography or semi-preparative high-performance liquid chromatography.

6. The method according to claim 4, wherein in step c, separation is performed by normal-phase silica gel column chromatography and a combination of reverse-phase column chromatography and semi-preparative high-performance liquid chromatography.

7. The method according to claim 4, wherein in step c, separation is performed by a combination of normal-phase silica gel column chromatography, Sephadex column chromatography, reverse-phase column chromatography, and semi-preparative high-performance liquid chromatography.

8. The method according to any one of claims 4 to 7, 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 reversed-phase silica gel.

9. Use of the compound according to claim 1 in the preparation of analgesic or anti-inflammatory drugs.

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