Compounds with anti-inflammatory action in arnica roots and their preparation and use

Abstract

The application discloses alkaloid compounds extracted from anacyclus pyrethrum roots and an extraction method and application thereof, wherein the alkaloid compounds are obtained by adopting organic solvent extraction, extraction, and then separation through a combination of two, three or four of normal phase silica gel column chromatography, reverse phase silica gel column chromatography and semi-preparative high performance liquid chromatography from roots of anacyclus pyrethrum (Anacyclus pyrethrum (L.) DC.) (anacyclus pyrethrum roots). In addition, the compounds provided by the application can play an anti-inflammatory role by inhibiting cell nitric oxide (NO) and can be used for preparing anti-inflammatory drugs.

Description

[0001] This application is a divisional application of Chinese invention patent application filed on August 30, 2023, with application number 202311104612.0 and invention title "Compounds with anti-inflammatory effects in anachine root and their preparation and application". Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, specifically relating to compounds (compounds of formula 1, 2, 3 or 4) with anti-inflammatory effects from anachine root, and their preparation and application. Background Technology

[0003] Exploring lead compounds with significant pharmacological activity from traditional medicinal plants is a hot topic in drug research. Therefore, discovering novel anti-inflammatory compounds from medicinal plants is an effective way to develop new anti-inflammatory drugs.

[0004] Anacy root is the dried root of Roman pyrethrum (L.) DC., a perennial herb belonging to the genus Anacyclus in the tribe Anthemideae of the family Asteraceae. Other uses in traditional medicine include pharyngitis, tonsillitis, epilepsy, fever, and diabetes. It has strong stimulating properties and can be used as a saliva-inducing and nervous system tonic; root decoctions can be used to treat tonsillitis, toothache, sore throat, and tooth decay.

[0005] Modern pharmacological studies have shown that anachine possesses various biological activities, including anti-epileptic, immunomodulatory, male sexual function improvement, antimutagenic, anti-inflammatory, hepatoprotective, hypoglycemic, and antibacterial effects. Studies have reported the presence of N-alkylamide and piperidine alkaloids, but the correlation between these compounds and the pharmacological activities of anachine remains unclear, and the pharmacodynamic material basis of anachine has not yet been elucidated. Summary of the Invention

[0006] Through dedicated research, the inventors have isolated anti-inflammatory compounds (compounds of formula 1, 2, 3 or 4) from the root of Anachi for the first time. These compounds are novel skeletal alkaloids, and their structures have been identified. Furthermore, cell experiments have verified that they inhibit the release of nitric oxide (NO) from cells and can be used to prepare anti-inflammatory drugs.

[0007] Therefore, the present invention provides the following aspects:

[0008] Aspect 1. Selected from the following compounds or their isomers:

[0009] N-(1-(2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methyl-bridged cyclopentano[7,8]aza[4,5,6-ij]isoquinoline-2-yl)-2-methylpropyl-2-yl)acetamide (a compound of formula 1 or compound 1);

[0010] 8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyridino[3',2':3,4]cyclopentano[1,2-g]pyrrolo[3,2-b]indazine-10(1H)-one (a compound of formula 2 or compound 2);

[0011] 2,2,6,6,8,8-Hexamethyl-9a-(2-propionyl)-1,2,3,5,6,8,9,9a-octahydro-1H-pyrrolo[2,3-h]isoquinoline-1-one (a compound of formula 3 or compound 3); and

[0012] 2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrolo-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one (a compound of formula 4 or compound 4).

[0013] Aspect 2. The compound or isomer thereof according to aspect 1 above, wherein the isomer is an enantiomer.

[0014] Aspect 3. The compound or its isomers according to aspect 1 or 2 above, selected from compound (+)-1, compound (–)-1, compound (+)-2, compound (–)-2, compound (+)-3, compound (–)-3, compound (+)-4 and compound (–)-4, wherein:

[0015] Compound (+)-1 is: N-(1-((8R,9aS,10aS)-2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methyl-bridged cyclopentano[7,8]aza[4,5,6-ij]isoquinoline-2-yl)-2-methylpropyl-2-yl)acetamide;

[0016] Compound (–)-1 is: N-(1-((8S,9aR,10aR)-2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methyl-bridged cyclopentano[7,8]aza[4,5,6-ij]isoquinoline-2-yl)-2-methylpropyl-2-yl)acetamide;

[0017] The compound (+)-2 is: (5aR,8aR,11aR)-8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyridino[3',2':3,4]cyclopentano[1,2-g]pyrrolo[3,2-b]indazine-10(1H)-one;

[0018] The compound (–)-2 is: (5aS,8aS,11aS)-8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyridino[3',2':3,4]cyclopentano[1,2-g]pyrrolo[3,2-b]inazine-10(1H)-one;

[0019] The compound (+)-3 is: (S)-2,2,6,6,8,8-hexamethyl-9a-(2-propionyl)-1,2,3,5,6,8,9,9a-octahydro-1H-pyrrolo[2,3-h]isoquinoline-1-one;

[0020] The compound (–)-3 is: (R)-2,2,6,6,8,8-hexamethyl-9a-(2-propionyl)-1,2,3,5,6,8,9,9a-octahydro-1H-pyrrolo[2,3-h]isoquinoline-1-one;

[0021] Compound (+)-4 is: (R)-2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrolo-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one; and

[0022] The compound (–)-4 is: (S)-2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrolo-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one.

[0023] Aspect 4. The compound or its isomer according to any one of Aspects 1 to 3 above, having a structural formula selected from the following:

[0024] .

[0025] Aspect 5. A method for extracting the compound or its isomer according to any one of Aspects 1 to 4 above from the root of Anachi, comprising the following steps:

[0026] a. After drying and pulverizing the roots of Anachi, extract them using a 50-95% (v / v) aqueous solution of ethanol, methanol or chloroform as solvent (the ratio of medicinal material weight (Kg) to solvent (L) is 1:1.5-1:4). Extract by cold soaking, percolation, heating and reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain the extract.

[0027] b. After suspending the total extract from step a in water, disperse it with 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, then the pH is adjusted to approximately 10-12 with an alkali such as NaHCO3, Na2CO3, ammonia, or NaOH. It is then extracted again with an organic solvent such as dichloromethane, ethyl acetate, or n-butanol. The organic solvent is recovered by vacuum concentration to obtain the total alkaloids.

[0028] c. The total alkaloids from step b are separated by silica gel column chromatography, thin-layer chromatography, reversed-phase MCI column chromatography, dextran gel LH-20 column chromatography, high-performance liquid chromatography, or any combination thereof to obtain the compound or its isomers.

[0029] Aspect 6. The method according to aspect 5 above, wherein in step c, separation is performed using a combination of normal-phase silica gel column chromatography and reversed-phase silica gel column chromatography or reversed-phase MCI column chromatography or semi-preparative high-performance liquid chromatography, preferably, after gradient or isocratic elution using normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography or reversed-phase MCI column chromatography or semi-preparative high-performance liquid chromatography is used to obtain compounds of formula 1, formula 2, formula 3 or formula 4, wherein 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, or petroleum ether and ethyl acetate in a volume ratio of 500:1 to 0:1. The eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is an aqueous methanol solution with a volume ratio of 10-100% (v / v) or an aqueous acetonitrile solution with a volume ratio of 20-100% (v / v). The eluent used in the semi-preparative high-performance liquid chromatography for isocratic or gradient elution is an eluent with a volume ratio of 99-50% hexane / EtOH, a volume ratio of 99-50% hexane / 2-isopropanol, a volume ratio of 99:1:0.002 to 50:50:0.002 hexane / 2-isopropanol / diethylamine, or an aqueous methanol solution with a concentration of 20-100% (v / v).

[0030] Aspect 7. According to the method described in aspect 5 above, wherein in step c, separation is performed using a combination of normal-phase silica gel column chromatography, reversed-phase silica gel or reversed-phase MCI column chromatography, and semi-preparative high-performance liquid chromatography. Preferably, after gradient or isocratic elution using normal-phase silica gel column chromatography, gradient elution is performed using reversed-phase silica gel or reversed-phase MCI column chromatography, followed by semi-preparative high-performance liquid chromatography to obtain compounds of formula 1, formula 2, formula 3, or formula 4. More preferably, the eluent used in the normal-phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol, or trichloromethane in a volume ratio of 100:1 to 0:1. The eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution with a volume ratio of 10-100% (v / v) or an acetonitrile aqueous solution with a volume ratio of 20-100% (v / v), and the eluent used in the semi-preparative high-performance liquid chromatography for isocratic or gradient elution is a hexane / EtOH eluent with a volume ratio of 99-50%, a hexane / 2-isopropanol eluent with a volume ratio of 99-50%, a hexane / 2-isopropanol / diethylamine eluent with a volume ratio of 99:1:0.002 to 50:50:0.002, or a methanol aqueous solution with a concentration of 10-100% (v / v).

[0031] Aspect 8. According to the method described in Aspect 5 above, wherein in step c, separation is performed using a combination of normal-phase silica gel column chromatography, dextran gel LH-20 column chromatography, reversed-phase silica gel or reversed-phase MCI column chromatography, and semi-preparative high-performance liquid chromatography. Preferably, after gradient or isocratic elution using normal-phase silica gel column chromatography, the mixture is then subjected to dextran gel LH-20 column chromatography, followed by gradient elution with reversed-phase silica gel or reversed-phase MCI column chromatography, and finally semi-preparative high-performance liquid chromatography to obtain compounds of formula 1, formula 2, formula 3, or formula 4. 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 dextran gel LH-20 column chromatography employs methanol gradient or isocratic elution. The eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution with a volume ratio of 10-100% (v / v) or an acetonitrile aqueous solution with a volume ratio of 20-100% (v / v). The eluent used in the semi-preparative high-performance liquid chromatography for isocratic or gradient elution is n-hexane / EtOH with a volume ratio of 99-50% (v / v), n-hexane / 2-isopropanol with a volume ratio of 99-50% (v / v), n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002 to 50:50:0.002 (v / v), or a methanol aqueous solution with a concentration of 20-100% (v / v).

[0032] Aspect 9. The method according to any one of Aspects 5 to 8 above, wherein in step c, the silica gel column chromatography is atmospheric or pressurized column chromatography, and / or the packing material used is normal-phase silica gel or reversed-phase silica gel; and / or preferably, the method further includes the step of chirally resolving the obtained racemic mixture (i.e., the compound of formula 1, formula 2, formula 3 or formula 4) through a chiral chromatographic column to obtain the enantiomeric compound.

[0033] Aspect 10. Use of the compound or isomer thereof according to any one of Aspects 1 to 4 above in the preparation of an anti-inflammatory drug, preferably, the compound or isomer thereof exerts its anti-inflammatory effect by inhibiting the release of nitric oxide (NO) from cells.

[0034] In summary, this invention provides an alkaloid compound isolated from *Anacyclus pyrethrum* (L.) DC., along with its preparation method and uses. Using *Anacyclus pyrethrum* (L.) DC. as raw material, the method involves solvent extraction, acid-base treatment, and further solvent extraction. Separation is achieved through two, three, or four of the following methods: silica gel column chromatography, preparative thin-layer chromatography, dextran gel LH-20 column chromatography, or pHPLC. Analysis is performed using thin-layer chromatography or pHPLC to obtain four novel alkaloid compounds with different skeletons. The in vitro anti-inflammatory activity of these compounds was determined. Experimental results show that the novel alkaloid compounds isolated from *Anacyclus pyrethrum* possess certain anti-inflammatory activity and can be used to prepare anti-inflammatory drugs. Attached Figure Description

[0035] Figure 1 This is the X-ray single-crystal diffraction pattern of compound 1;

[0036] Figure 2 This is the X-ray single-crystal diffraction pattern of compound 3;

[0037] Figure 3 This is the X-ray single-crystal diffraction pattern of compound 4;

[0038] Figure 4 It is compound 1 1 H NMR spectrum;

[0039] Figure 5 It is compound 1 13 C NMR spectrum;

[0040] Figure 6 It is compound 2. 1 H NMR spectrum;

[0041] Figure 7 It is compound 2. 13 C NMR spectrum;

[0042] Figure 8 It is compound 3. 1 H NMR spectrum;

[0043] Figure 9 It is compound 3. 13 C NMR spectrum;

[0044] Figure 10 It is compound 4. 1 H NMR spectrum;

[0045] Figure 11 It is compound 4. 13 C NMR spectrum. Detailed Implementation

[0046] The purpose of this invention is to provide compounds with anti-inflammatory effects, their isolation and preparation methods, and their application value in the preparation of anti-inflammatory drugs.

[0047] According to a first aspect of the present invention, a compound having anti-inflammatory activity is provided, the structural formula of which (a compound of formula 1, formula 2, formula 3 or formula 4, respectively) is shown in the figure below;

[0048] .

[0049] Formula 1

[0050] .

[0051] Formula 2

[0052] .

[0053] Formula 3

[0054] .

[0055] Formula 4

[0056] in:

[0057] Compound (+)-1 is: N-(1-((8R,9aS,10aS)-2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methyl-bridged cyclopentano[7,8]aza[4,5,6-ij]isoquinoline-2-yl)-2-methylpropyl-2-yl)acetamide (a compound of formula 1);

[0058] Compound (–)-1 is: N-(1-((8S,9aR,10aR)-2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methyl-bridged cyclopentano[7,8]aza[4,5,6-ij]isoquinoline-2-yl)-2-methylpropyl-2-yl)acetamide (a compound of formula 1);

[0059] Compound (+)-2 is: (5aR,8aR,11aR)-8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyridino[3',2':3,4]cyclopentano[1,2-g]pyrrolo[3,2-b]indazine-10(1H)-one (a compound of formula 2);

[0060] Compound (–)-2 is: (5aS,8aS,11aS)-8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyridino[3',2':3,4]cyclopentano[1,2-g]pyrrolo[3,2-b]indazine-10(1H)-one (a compound of formula 2);

[0061] Compound (+)-3 is: (S)-2,2,6,6,8,8-hexamethyl-9a-(2-propionyl)-1,2,3,5,6,8,9,9a-octahydro-1H-pyrrolo[2,3-h]isoquinoline-1-one (a compound of formula 3);

[0062] Compound (–)-3 is: (R)-2,2,6,6,8,8-hexamethyl-9a-(2-propionyl)-1,2,3,5,6,8,9,9a-octahydro-1H-pyrrolo[2,3-h]isoquinoline-1-one (a compound of formula 3);

[0063] Compound (+)-4 is: (R)-2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrolo-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one (a compound of formula 4);

[0064] The compound (–)-4 is: (S)-2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrolo-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one (the compound of formula 4).

[0065] The extraction and separation method for the above-mentioned alkaloid compounds shall be carried out according to the following steps:

[0066] After drying and pulverizing the roots of Anachi, extract them using a 50-95% (v / v) aqueous solution of ethanol, methanol, or chloroform as solvents (the ratio of medicinal material weight (Kg) to solvent (L) is 1:1.5-1:4). Extract by cold soaking, percolation, heating and reflux, or ultrasonic extraction. The solvent is then recovered by vacuum concentration to obtain the extract.

[0067] b. After suspending the total extract from step a in water, disperse it with 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, ammonia or NaOH. Then, it is extracted with organic solvents such as dichloromethane, ethyl acetate or n-butanol, and the organic solvent is recovered by vacuum concentration to obtain the total alkaloids.

[0068] c. The total alkaloids in step b are separated by two, three or four of the following methods: silica gel column chromatography, thin layer chromatography, reversed phase MCI column chromatography, dextran gel LH-20 column chromatography, and high performance liquid chromatography.

[0069] Two separation methods are available:

[0070] The eluent used in normal-phase silica gel column chromatography was petroleum ether-ethyl acetate (v / v ratio) of 100:0-3:1, dichloromethane-methanol (v / v ratio) of 500:1-3:1, or petroleum ether / acetone (v / v ratio) for gradient elution. After elution, the compound was obtained by reverse-phase silica gel or MCI column chromatography or semi-preparative high-performance liquid chromatography.

[0071] Three separation methods:

[0072] The eluent used in normal-phase silica gel column chromatography was a gradient elution of petroleum ether-ethyl acetate, dichloromethane-methanol, or trichloromethane-methanol at a volume ratio of 100:1–0:1. Following this gradient elution, the mixture was subjected to reverse-phase silica gel or MCI column chromatography with a gradient elution of methanol-water solution or acetonitrile-water solution at a volume ratio of (v / v). Semi-preparative high-performance liquid chromatography was then employed, using hexane / EtOH at a volume ratio of 99–50% (v / v), hexane / 2-isopropanol at a volume ratio of 99–50% (v / v), hexane / 2-isopropanol at a volume ratio of 99:1:0.002–50:50:0.002 (v / v) hexane / 2-isopropanol / diethylamine as the eluent to obtain compounds of formula 1, 2, 3, or 4.

[0073] Four separation methods:

[0074] The eluent used in normal-phase silica gel column chromatography was a gradient elution of petroleum ether-ethyl acetate, dichloromethane-methanol, or trichloromethane-methanol at a volume ratio of 100:1–0:1 (v / v). After elution with a dextran gel LH-20 column using isocratic elution with methanol, the column was then subjected to reversed-phase silica gel or MCI column chromatography with a gradient elution of methanol-water solution or acetonitrile-water solution at a volume ratio of 20–100% (v / v). Semi-preparative high-performance liquid chromatography was then performed using hexane / EtOH, hexane / 2-isopropanol, or hexane / 2-isopropanol / diethylamine at a volume ratio of 99–50% (v / v), or hexane / 2-isopropanol / diethylamine at a volume ratio of 99:1:0.002–50:50:0.002 (v / v / v) to obtain compounds of formula 1, 2, 3, or 4.

[0075] The method for preparing alkaloids from Anachi root as described in step c is characterized in that the silica gel column chromatography used is atmospheric or pressurized column chromatography, the packing material used is normal silica gel or reversed silica gel, and the eluents are dichloromethane and methanol in a volume ratio of 500:1-3:1 (v / v); petroleum ether and ethyl acetate in a volume ratio of 1:0-3:1 (v / v); or methanol and water in a volume ratio of 1:9-1:0 (v / v), and isocratic or gradient elution is employed.

[0076] The method for preparing alkaloids from Anachi root described in step c is characterized in that the eluent for the dextran gel LH-20 column chromatography is methanol, and isocratic elution is used.

[0077] The method for preparing alkaloids from Anachi root as described in step c is characterized in that the eluent used in the high-performance liquid chromatography (HPLC) preparation is a methanol-water solution with a volume ratio of 10-100% (v / v), n-hexane / EtOH with a volume ratio of 99-50% (v / v), n-hexane / 2-isopropanol with a volume ratio of 99-50% (v / v), or n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002-50:50:0.002 (v / v / v) using isocratic or gradient elution.

[0078] According to another aspect of the present invention, a method for extracting and separating compounds with anti-inflammatory effects is provided. The root of *Anochias henryi* is dried and pulverized. The extract is then prepared using a solvent of 50-95% (v / v) ethanol-water solution, methanol, or chloroform (the ratio of medicinal material weight (kg) to solvent (L) is 1:1.5-1:4). Extraction is performed using cold soaking, percolation, reflux extraction, or ultrasonic extraction. The solvent is then recovered by vacuum concentration to obtain an extract. The total extract is suspended in water and then dispersed with an acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid. The resulting acidic aqueous layer is then... After removing non-alkaloids by dichloromethane extraction, the pH was adjusted to 10-12 using NaHCO3, Na2CO3, ammonia, or NaOH. Extraction was then performed again with organic solvents such as chloroform, ethyl acetate, or n-butanol. The organic solvent was recovered by vacuum concentration to obtain total alkaloids. The total alkaloids were separated by two, three, or four methods: silica gel column chromatography, thin-layer chromatography, dextran gel LH-20 column chromatography, or high-performance liquid chromatography (HPLC). Analysis using thin-layer chromatography or HPLC yielded eight new alkaloid skeletons. The silica gel column chromatography used was either atmospheric or pressurized, with normal or reversed-phase silica gel as the packing material. Eluents included dichloromethane and methanol (v / v), petroleum ether and ethyl acetate (v / v), or methanol and water (v / v), with isocratic or gradient elution. The preparative thin-layer chromatography method used was atmospheric pressure chromatography, with a developing system consisting of a mixture of dichloromethane and methanol, or a mixture of petroleum ether and acetone. The dextran gel LH-20 column chromatography method used chloroform-methanol or methanol at a volume ratio of 10:1, with isocratic elution. The preparative high-performance liquid chromatography method used hexane / EtOH at a volume ratio of 99-50% (v / v), hexane / 2-propanol at a volume ratio of 99-50% (v / v), hexane / 2-isopropanol / diethylamine at a volume ratio of 99:1:0.002 to 50:50:0.002 (v / v), or an aqueous methanol solution at a concentration of 20-100% (v / v), with isocratic or gradient elution.

[0079] According to another aspect of the invention, the application of the compound having anti-inflammatory activity in the preparation of anti-inflammatory drugs is provided.

[0080] The novel skeletal alkaloids of this invention were analyzed using a combination of various spectroscopic methods (high-resolution mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, and nuclear magnetic resonance spectroscopy) and X-ray single-crystal diffraction to determine the structures of compounds 1 to 4 prepared in the examples. The relative configurations of compounds 1, 3, and 4 were determined by X-ray single-crystal diffraction, as shown below. Figure 1 , Figure 2 and Figure 3 As shown.

[0081] Compound 1 (Anacyphrethine C): Yellow blocky crystals; optical rotation [α]25 D 128 (c 0.092, methanol, enantiomer compound (+)-1); [α]25 D -128 (c 0.092, methanol, enantiomer compound (-)-1); UV (methanol) λ max (log ε) 288 (3.92) nm, 417 (3.74) nm; infrared (KBr) max 3291, 2973, 2936, 2871, 1714, 1667, 1563 and 1178 cm⁻¹ -1 ECD (c 1.86×10 -3 M, methanol) λ max 215 (-1.17), 289 (2.22), 337 (-1.97), 435 (1.67), enantiomer (+)-1; ECD (c 1.86×10 -3 M, methanol) λ max (Δε) 219 (1.43), 285 (-2.15), 377 (2.17), 432 (-1.74) nm, enantiomer (-)-1; high-resolution mass spectrometry m / z 496.3532 [M + H] + (Calculated value C) 30 H 45 O3N3 + ,496.3534). 1 H and 13 The C NMR spectral data are shown in Table 1.

[0082] Table 1. Compound 1 1 H and 13 C NMR data (deuterated chloroform, 600 MHz)

[0083]

[0084] Compound 2 (Anacyphrethine D): Yellow powder; optical rotation [α]25 D 92 (c 0.08, methanol, enantiomer (+)-2); [α]25 D -92 (c 0.066, methanol, enantiomer (-)-2); UV (methanol) λ max(log ε) 268 (408) nm, 453 (4.14) nm; infrared (KBr) max 3342, 2970, 2928, 2870, 1713, 1571, 1365 and 1052 cm⁻¹ -1 ECD (c 1.83×10 -3 M, methanol) λ max (Δε)216 (1.32), 243 (0.75), 274 (-2.85), 306 (0.77), 381 (-1.10), 446 (1.12), enantiomer (+)-2; ECD (c 1.51×10 -3 M, methanol) λ max (Δε) 219 (-1.52), 243 (-1.44), 275 (5.01), 307 (-1.09), 383 (1.80), 446 (-1.71), enantiomer (-)-2; high-resolution mass spectrometry m / z 438.3114 [M + H] + (Calculated value C) 27 H 40 O2N3 + , 438.3115). 1 H and 13 CNMR spectral data are shown in Table 2.

[0085] Table 2. Compound 2 1 H and 13 C NMR data (deuterated chloroform, 600 MHz)

[0086]

[0087] Compound 3 (Anacyphrethine E): Colorless massive crystals; optical rotation [α]25 D -34 (c 0.2, methanol, enantiomer compound (-)-3); [α]25 D 34 (c 0.2, methanol, enantiomer compound (+)-3); UV (methanol)λ max (log ε) 260 (4.22) nm; infrared (KBr) max 2960, 2920, 1713, 1665, 1626, 1399, 1355 and 1162 cm⁻¹ -1 ECD (c 1.21×10 -3 M, methanol) λ max(Δε) 218 ​​(0.60), 257 (-4.05), 288 (4.78) nm, enantiomer (-)-3; ECD (c 1.21×10 -3 M, methanol) λ max (Δε) 216 (-0.54), 260 (4.39), 288 (-4.86) nm, enantiomer (+)-3; high-resolution mass spectrometry m / z 331.2376 [M + H] + (Calculated value C) 20 H 31 O2N2 + , 331.2380). 1 H and 13 The C NMR spectral data are shown in Table 3.

[0088] Compound 4 (Anacyphrethine F): Colorless massive crystals; optical rotation [α]25 D 66 (c 0.2, methanol, enantiomer (+)-4); [α]25 D -66 (c 0.2, methanol, enantiomer (-)-4); UV (methanol) λ max (log ε) 253 (3.86) nm; infrared (KBr) max 2970, 2927, 2866, 1697, 1662, 1618, 1466, 1448 and 1358 cm⁻¹ -1 ECD (c 6.62×10 -4 M, methanol) λ max (Δε) 216(22.06), 240 (-4.91), 267 (2.98), 336 (-1.81) nm, enantiomer (+)-4; ECD (c 6.62×10 -4 M, methanol) λ max (Δε) 216 (-22.20), 241 (4.41), 272 (-2.91), 337 (2.28) nm, enantiomer (-)-4; high-resolution mass spectrometry m / z 303.2063 [M + H] + (Calculated value C) 18 H 27 O2N2 + , 303.2067). 1 H and 13 The C NMR spectral data are shown in Table 3.

[0089] Table 3. Compounds 3 and 4 1 H and13 C NMR data (deuterated chloroform, 600 MHz)

[0090]

[0091] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages:

[0092] (1) Compounds 1 to 4 provided in this invention are novel skeletal compounds. Compound 1 is a pair of highly conjugated diamino 6 / 6 / 6 / 6 / 5 pentacyclic alkaloid enantiomers with a unique 6,13-diazapentacyclic [10.3.3] skeleton. 1,3 .1.0 2, 7 .0 8,12 The nonadecane ring system has a skeletal structure with three discontinuous chiral stereocenters. Compound 2 is a novel enantiomer of a pair of triamino 6 / 5 / 6 / 5 / 5 pentacyclic alkaloids, a unique 4,6,17-triazapentacyclic [10.7.0.0]. 4,11 .0 5,9 .0 13,18 The skeletal structure of the nonadecane ring system has three discontinuous chiral stereocenters. Compound 3 is a novel enantiomer of a pair of triamino 6 / 6 / 5 tricyclic alkaloids, a unique 5,12-diazatricyclic [7.4.0] 2,6 The nonadecane ring system has a skeletal structure with three discontinuous chiral stereocenters. Compound 4 is a pair of novel pyridone-pyrrole enantiomers with one chiral stereocenter.

[0093] (2) Compounds 1 to 4 provided in this invention have anti-inflammatory activity and are used to prepare anti-inflammatory drugs.

[0094] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but this does not limit the scope of the invention. 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 are within the scope of the invention.

[0095] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0096] The following are specific examples:

[0097] Example 1

[0098] Dried anacyclus pyrethrum (L.) DC., 15.0 kg, was pulverized and ultrasonically extracted with chloroform (30 L). The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The pH of the acid solution was adjusted to 10 with saturated NaHCO3 aqueous solution under ice-water bath with constant stirring to obtain an alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain the total alkaloids.

[0099] Example 2

[0100] Dried anacyclus pyrethrum (L.) DC., 15.0 kg, was pulverized and extracted with 95% ethanol by percolation. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The pH of the acid solution was adjusted to 10 with ammonia solution under ice-water bath with constant stirring to obtain an alkalized solution. The alkalized solution was fully extracted with ethyl acetate. The ethyl acetate extracts were combined and dried to obtain the total alkaloids.

[0101] Example 3

[0102] Dried anacyclus pyrethrum (L.) DC., 15.0 kg, was pulverized and extracted with 50% ethanol (40 L) by percolation. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 2% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The pH of the acid solution was adjusted to 10 with Na2CO3 aqueous solution under ice-water bath with constant stirring to obtain an alkalized solution. The alkalized solution was fully extracted with n-butanol, and the n-butanol extracts were combined and dried to obtain total alkaloids.

[0103] Example 4

[0104] Dried anacyclus pyrethrum (L.) DC., 15.0 kg, was pulverized and extracted with 75% ethanol (45 L) under reflux. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 1% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The pH of the acid solution was adjusted to 12 with NaOH aqueous solution under ice-water bath with constant stirring to obtain an alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain total alkaloids.

[0105] Example 5

[0106] Dried anacyclus pyrethrum (L.) DC., 15.0 kg, was pulverized and extracted with methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The pH of the acid solution was adjusted to 10 with saturated NaHCO3 aqueous solution under ice-water bath conditions with constant stirring to obtain an alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain total alkaloids.

[0107] Example 6

[0108] Dried anacyclus pyrethrum (L.) DC., 15.0 kg, was pulverized and extracted with 22.5 L of methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 1% sulfuric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The pH of the acid solution was adjusted to 10 with saturated NaHCO3 aqueous solution under ice-water bath with constant stirring to obtain an alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain total alkaloids.

[0109] Example 7

[0110] Dried anacyclus pyrethrum (L.) DC., 15.0 kg, was pulverized and extracted with 22.5 L of methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 2% sulfuric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The pH of the acid solution was adjusted to 10 with saturated NaHCO3 aqueous solution under ice-water bath with constant stirring to obtain an alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain the total alkaloids.

[0111] Example 8

[0112] Dried anacyclus pyrethrum (L.) DC., 15.0 kg, was pulverized and extracted with 22.5 L of methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% sulfuric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The pH of the acid solution was adjusted to 10 with saturated NaHCO3 aqueous solution under ice-water bath with constant stirring to obtain an alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain the total alkaloids.

[0113] Example 9

[0114] Any total alkaloids from Examples 1-8 were mixed with 100-200 mesh silica gel and subjected to silica gel column chromatography, followed by elution with a dichloromethane / methanol gradient (100:0~3:1, V / V). Identical fractions were combined to obtain six fractions of increasing polarity: Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, and Fr.F. Fraction 1, Fr.A (159.0 g), was mixed with 100-200 mesh silica gel and subjected to silica gel column chromatography, followed by elution with a petroleum ether / ethyl acetate gradient (100:0~3:1, V / V). Identical fractions were combined to obtain six fractions of increasing polarity: Fr.A1–Fr.A6. Fraction 2, Fr.A2 (76.1 g), was further eluted. g) After reversed-phase MCI column chromatography, elution was performed with an acetonitrile / water gradient (20:80~100:0, V / V). Identical components were combined to obtain seven subfractions Fr.A21–Fr.A27 with decreasing polarity. Subfraction Fr.A25 (10.0 g) was mixed with 200–300 mesh silica gel and subjected to silica gel column chromatography, followed by petroleum ether / ethyl acetate gradient elution (10:1~3:1, V / V). Identical components were combined to obtain three subfractions Fr.A251–Fr.A253 with increasing polarity. Subfraction Fr.A253 (9.0 g)… g) The sample was mixed with 200-300 mesh silica gel and subjected to silica gel column chromatography, followed by elution with a petroleum ether / ethyl acetate gradient (10:1~3:1, V / V). Identical fractions were combined to obtain six fractions Fr.A2531-Fr.A2533 with increasing polarity. Fractions Fr.A251 and Fr.A2531 (136.3 mg) were combined and separated by semi-preparative high-performance liquid chromatography (HPLC) on a C18 column with methanol / water (10:100-100:0, V / V) at a flow rate of 3 mL / min, yielding compound 4 (Anacyphrethines F, 45.0 mg, 0.000300%), with a retention time t0. R The reaction time was 20.0 min; the racemic mixture of compound 4 was chirally resolved by a chiral column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / EtOH = 97:3; column temperature: 25 ℃; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain the enantiomeric compound (+)-4 (13.0 mg, t R = 9.7 min) and enantiomer compound (-)-4 (13.3 mg, t) R= 11.2 min). Fraction Fr.A5 (22.9 g) was subjected to reversed-phase MCI column chromatography, eluted with a methanol / water gradient (10:90–100:0, V / V), and identical fractions were combined to obtain nine subfractions Fr.A51–Fr.A59 with decreasing polarity; fraction Fr.A58 (702.0 mg) was subjected to Sephadex LH-20 gel column chromatography, eluted with methanol, to obtain four subfractions Fr.A581–Fr.A584 with decreasing molecular weight; fraction Fr.A582 (120.0 mg) was subjected to semi-preparative high-performance liquid chromatography (HPLC) C1. 18 Column chromatography was performed with a mobile phase of methanol / water (70:30, V / V) at a flow rate of 3 mL / min to obtain compound 2 (Anacyphrethines D, 15.0 mg, 0.000300%), with a retention time t0. R The reaction time was 51.9 min; the racemic mixture of compound 2 was chirally resolved by a chiral column (DAICELCORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / EtOH = 95:5; column temperature: 25℃; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain the enantiomeric compound (+)-2 (6.5 mg, t R = 13.4 min) and enantiomer compound (-)-2 (6.3 mg, t) R = 18.4 min).

[0115] Fr.B (179.1 g) was subjected to reversed-phase MCI column chromatography, eluted with a methanol / water gradient (20:80–100:0, V / V), and identical fractions were combined to obtain two subfractions, Fr.B1–Fr.B2, with decreasing polarity. Fr.B1 (76.1 g) was subjected to silica gel column chromatography, then eluted with a petroleum ether / ethyl acetate gradient (1:0–3:1, V / V), and identical fractions were combined to obtain four subfractions, Fr.B1a–Fr.B1d, with increasing polarity. Fr.B1b (18.6 g) was subjected to reversed-phase MCI column chromatography, eluted with a methanol / water gradient (10:90–100:0, V / V), and identical fractions were combined to obtain six subfractions, Fr.B1b1–Fr.B1b6, with decreasing polarity. Fr.B1b4 (9.6 g)… g) was subjected to normal-phase silica gel column chromatography, eluted with dichloromethane / methanol (500:1~10:1, V / V), yielding three subfractions Fr.B1b41−Fr.B1b44 with increasing polarity; Fr.B1b42 (2.0 g) was subjected to Sephadex LH-20 gel column chromatography, eluted with methanol, yielding two subfractions Fr.B1b42a−Fr.B1b42b with decreasing molecular weight; Fr.B1b42b (1.9 g) was subjected to reverse-phase C18 silica gel column chromatography, eluted with acetonitrile / water gradient (20:80~100:0, V / V), and the same components were combined to yield eleven subfractions Fr.B1b42b1−Fr.B1b42b11 with decreasing polarity; Fr.B1b42b1 (552.0 g) was subjected to normal-phase silica gel column chromatography, eluted with dichloromethane / methanol (500:1~10:1, V / V), yielding three subfractions Fr.B1b4 ...2a−Fr.B1b42b11 with decreasing polarity; Fr.B1b42b1 (552.0 g) was subjected to normal-phase silica gel column chromatography, eluted with dichloromethane / methanol (500:1~10:1, V / V), yielding three subfractions Fr.B1b42b11 with decreasing polarity; Fr.B1b42b1 (552.0 g) was The sample (183.2 mg) was subjected to normal-phase silica gel column chromatography, eluted with dichloromethane / methanol (500:1~5:1, V / V), yielding five subfractions Fr.B1b42b1a−Fr.B1b42b1e with increasing polarity; the fraction Fr.B1b42b1c (183.2 mg) was subjected to Sephadex LH-20 gel column chromatography, eluted with methanol, yielding four subfractions Fr.B1b42b1c1−Fr.B1b42b1c4 with decreasing molecular weight; the fraction Fr.B1b42b1c2 (127.9 mg) was subjected to reverse-phase C24 chromatography. 18 Silica gel column chromatography was performed with a methanol / water gradient elution (10:90–100:0, V / V). Identical fractions were combined to obtain four subfractions, Fr.B1b42b1c2a–Fr.B1b42b1c2d, with decreasing polarity. Fraction Fr.B1b42b1c2d (15.5 mg) was further analyzed by semi-preparative high-performance liquid chromatography (HPLC). 18 Column chromatography was performed with methanol / water (36:64, V / V) as the mobile phase at a flow rate of 3 mL / min to obtain compound 3 (Anacyphrethines C, 17.7 mg, 0.000113%); retention time t RThe reaction time was 17.0 min; the racemic mixture of compound 3 was chirally resolved by a chiral column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / 2-propanol = 85:15; column temperature: 25 ℃; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain the enantiomeric compound (+)-3 (1.5 mg, t R = 17.6 min) and enantiomer compound (-)-3 (1.4 mg, t R = 12.6 min). Fraction Fr.B1b5 (2.6 g) was subjected to normal-phase silica gel column chromatography, eluted with dichloromethane / methanol (500:1~5:1, V / V), yielding three subfractions Fr.B1b51−Fr.B1b53 with increasing polarity; among them, fraction Fr.B1b52 (983.0 mg) was subjected to Sephadex LH-20 gel column chromatography, eluted with methanol, yielding two subfractions Fr.B1b52a−Fr.B1b52b with decreasing molecular weight; fraction Fr.B1b52b (736.9 mg)... Fr.B1b52b1–Fr.B1b52b6 was subjected to reverse-phase C18 silica gel column chromatography with a methanol / water gradient elution (20:80–100:0, V / V). Identical fractions were combined to obtain six subfractions, Fr.B1b52b1–Fr.B1b52b6, with decreasing polarity. Fr.B1b52b5 (175.0 mg) was subjected to normal-phase silica gel column chromatography with dichloromethane / methanol elution (500:1–5:1, V / V) to obtain three subfractions, Fr.B1b52b5a–Fr.B1b52b5c, with increasing polarity. Fr.B1b52b5b (170.0 mg) was further analyzed using Sephadex chromatography. LH-20 gel column chromatography, eluted with methanol, yielded three subfractions Fr.B1b52b5b1−Fr.B1b52b5b3 with decreasing molecular weight; subfraction Fr.B1b52b5b2 (123.0 mg) was subjected to normal-phase silica gel column chromatography, eluted with dichloromethane / methanol (300:1~5:1, V / V), yielding two subfractions Fr.B1b52b5b2a−Fr.B1b52b5b2b with increasing polarity; subfraction Fr. B1b52b5b2a (92.5 mg) was subjected to semi-preparative high-performance liquid chromatography (HPLC) C1. 18 Column chromatography was performed with acetonitrile / water (20:100–100:0, V / V) as the mobile phase at a flow rate of 3 mL / min to obtain compound 1 (Anacyphrethines C, 17.7 mg, 0.000113%); retention time t RThe reaction time was 27.3 min; the racemic mixture of compound 1 was chirally resolved by a chiral column (DAICELCORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / 2-propanol / Diethylamine = 98:2:0.002; column temperature: 25 ℃; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain the enantiomeric compound (+)-1 (6.2 mg, t R = 30.8 min) and enantiomer compound (-)-1 (6.0 mg, t R = 33.3 min).

[0116] Example 10

[0117] Any total alkaloids from Examples 1-8 were mixed with 100-200 mesh silica gel and subjected to silica gel column chromatography, followed by gradient elution with dichloromethane / methanol (100:0~3:1, V / V). Identical fractions were combined to obtain six fractions, Fr.A, Fr.B, Fr.C, Fr.D, Fr.E, and Fr.F, with increasing polarity. Fraction 1, Fr.A (159.0 g), underwent repeated silica gel column chromatography, using gradient elution with petroleum ether-ethyl acetate (100:0-3:1 v / v), dichloromethane-methanol (500:1-3:1 v / v), or petroleum ether / acetone (50:1-0:1 v / v), to obtain compound 4 (Anacyphrethines F, 45.0 mg, 0.000300%) and compound 2 (Anacyphrethines D, 15.0 g). mg, 0.000300%); the racemic mixture of compound 4 was chirally resolved by a chiral column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / EtOH = 99:1-50-50; column temperature: 25 ℃; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain the enantiomeric compound (+)-4 (13.0 mg, t R = 9.7 min) and enantiomer compound (-)-4 (13.3 mg, t) R = 11.2 min). The racemic mixture of compound 2 was chirally resolved by a chiral column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / EtOH = 99:1-50:50; column temperature: 25 ℃; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain the enantiomeric compound (+)-2 (6.5 mg, t R= 13.4 min) and enantiomer compound (-)-2 (6.3 mg, t) R = 18.4 min).

[0118] Fraction Fr.B (179.1 g) was subjected to repeated silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate (v / v), dichloromethane-methanol (v / v), or petroleum ether / acetone (v / v) to give compound 3 (Anacyphrethines C, 17.7 mg, 0.000113%) and compound 1 (Anacyphrethines C, 17.7 mg, 0.000113%). The racemic mixture of compound 3 was chirally resolved using a chiral column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / 2-isopropanol = 99:1-50:50; column temperature: 25 ℃; flow rate: 3 ml / min; detection wavelength: 254 nm) to give the enantiomeric compound (+)-3 (1.5 mg, t R =17.6 min) and enantiomer compound (-)-3 (1.4 mg, t R = 12.6 min); the racemic mixture of compound 1 was chirally resolved by a chiral column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / 2-isopropanol / diethylamine = 99:1:0.002-50:50:0.002; column temperature: 25 ℃; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain the enantiomeric compound (+)-1 (6.2 mg, t R = 30.8 min) and enantiomer compound (-)-1 (6.0 mg, t R = 33.3 min).

[0119] Example 11

[0120] The anti-inflammatory activity of the novel skeleton alkaloid compound isolated from Anachi root as described in this invention

[0121] 1. Cell culture:

[0122] Mouse macrophages Raw264.7 (purchased from BeNa Culture Collection, BNCC) were cultured in Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) (purchased from Giboco, USA), 1% penicillin and streptomycin, and high glucose medium (purchased from Hyclone, USA) at 37°C and 5% CO2.

[0123] 2. Testing the effect of the compounds described in this invention on cell viability:

[0124] The compound of the present invention was dissolved in dimethyl sulfoxide (DMSO). Raw264.7, which was in the logarithmic growth phase and in good growth condition, was dissolved in water at a concentration of 5 × 10⁻⁶ ppm. 3 Cells were inoculated into 96-well plates. Different concentrations of various compounds were added to the experimental groups, while dimethyl sulfoxide (DMSO) was added to the control group. After culturing for 24 hours, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt (CCK-8 reagent) was added to each well. The absorbance at 450 nm was measured using an ELISA reader, and the cell viability was calculated. The experimental results are shown in Table 4.

[0125] Table 4. Cell viability of compounds 1-4

[0126]

[0127] 3. Determination of nitric oxide (NO) content:

[0128] The intracellular NO release of Raw264.7 cells was tested using the Griess method (Arias-Negrete et al., Analytical Biochemistry 328.1(2004):14-21). After incubating the cells with the test monomer compound for 1 h, 1 μg / mL of lipopolysaccharide (LPS, Sigma, L4391) was added, and the cells were incubated for another 22 h. After incubation, the cell supernatant was collected, and the nitric oxide content in the cell supernatant was determined using the Griess method. Before measurement, GriessReagent I and II (Nitric Oxide Assay kit, Beyotine, S0021M) were removed and allowed to return to room temperature. The standards were diluted with complete culture medium (1-100 μM). The concentrations of the standards could be 0, 1, 2, 5, 10, 20, 40, 60, or 100 μM. At a concentration of 50 μM, the standard and the collected culture supernatant were added to a 96-well plate at 50 μL / well. Then, 50 μL of Griess Reagent I (restored to room temperature) and 50 μL of Griess Reagent II were added to each well sequentially. After shaking and mixing for 5 min, the absorbance was measured at 540 nm to create a standard curve. The NO content in the culture supernatant was calculated based on the standard curve. The initial screening concentration of the monomer compound was 40 μM, and the inhibition rate results are shown in Table 4.

[0129] Table 5. NO-inhibiting activities of compounds 1-4

[0130]

[0131] [a] Positive control drug: Andrograholide (AG, HY-N0191). [b] Each experiment was repeated 3 times.

[0132] Conclusion: Compounds 1 to 4 all exhibited certain inhibitory effects on NO release, with compounds (+)-1, (–)-1, (+)-2, (–)-2, (+)-4, and (–)-4 showing the highest IC50 values. 50 The values ​​were 56.21±2.14, 73.13±3.78, 44.14±1.47 μM, 41.10±3.15 μM, 65.82±1.77, and 84.13±2.37, respectively. Compounds (+)-2 and (–)-2 significantly inhibited the release of NO.

[0133] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Alkaloids extracted from the roots of Anachi: 8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyridino[3',2':3,4]cyclopentano[1,2-g]pyrrolo[3,2-b]indazine-10(1H)-one.

2. The alkaloid compound according to claim 1, wherein it is selected from compound (+)-2 or compound (–)-2, wherein: The compound (+)-2 is: (5aR,8aR,11aR)-8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyridino[3',2':3,4]cyclopentano[1,2-g]pyrrolo[3,2-b]indazine-10(1H)-one; Compound (–)-2 is: (5aS,8aS,11aS)-8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyridino[3',2':3,4]cyclopentano[1,2-g]pyrrolo[3,2-b]inzin-10(1H)-one. They each have the following structural formulas: or .

3. A method for extracting the alkaloid compound according to claim 1 or 2 from the root of *Anachia*, comprising the following steps: a. After drying and pulverizing the anatchi root, use 50-95% ethanol aqueous solution, methanol or chloroform as solvent, wherein the ratio of anatchi root by weight kg to solvent by volume L is 1:1.5-1:

4. Extract by cold soaking, percolation, heating reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain the extract. b. After suspending the extract from step a in water, disperse it with acid. Extract the resulting acidic aqueous layer with dichloromethane to remove non-alkaloids, adjust the pH to 10-12 with alkali, and then extract with organic solvents such as dichloromethane, ethyl acetate, or n-butanol. Concentrate under reduced pressure to recover the organic solvents, yielding the total alkaloids; and c. The total alkaloids from step b are eluted using normal-phase silica gel column chromatography with gradient or isocratic elution, followed by separation using a method selected from reversed-phase silica gel column chromatography, reversed-phase MCI column chromatography, and semi-preparative high-performance liquid chromatography to obtain the alkaloid compounds. The eluent used in the normal-phase silica gel column chromatography is petroleum ether and ethyl acetate (v / v), dichloromethane and methanol (v / v), petroleum ether and acetone (v / v), or methanol and water (v / v) at a ratio of 100:0 to 3:1, 500:1 to 3:1, 500:1 to 0:1, or 1:9 to 1:

0. The eluent used in phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution with a volume ratio of 10-100% or an acetonitrile aqueous solution with a volume ratio of 20-100%. The eluent used in the semi-preparative high-performance liquid chromatography for isocratic or gradient elution is a hexane / EtOH solution with a volume ratio of 99-50%, a hexane / 2-isopropanol solution with a volume ratio of 99-50%, a hexane / 2-isopropanol / diethylamine solution with a volume ratio of 99:1:0.002 to 50:50:0.002, or a methanol aqueous solution with a volume ratio of 20-100%.

4. A method for extracting the alkaloid compound according to claim 1 or 2 from the root of *Anachys*, comprising the following steps: a. After drying and pulverizing the anatchi root, use 50-95% ethanol aqueous solution, methanol or chloroform as solvent, wherein the ratio of anatchi root by weight kg to solvent by volume L is 1:1.5-1:

4. Extract by cold soaking, percolation, heating reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain the extract. b. After suspending the extract from step a in water, disperse it with acid. Extract the resulting acidic aqueous layer with dichloromethane to remove non-alkaloids, adjust the pH to 10-12 with alkali, and then extract with organic solvents such as dichloromethane, ethyl acetate, or n-butanol. Concentrate under reduced pressure to recover the organic solvents, yielding the total alkaloids; and c. The total alkaloids from step b are eluted using normal-phase silica gel column chromatography with gradient or isocratic elution, followed by gradient elution using reversed-phase silica gel column chromatography or reversed-phase MCI column chromatography, and then separated using semi-preparative high-performance liquid chromatography to obtain the alkaloid compounds. 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 reversed-phase silica gel or reversed-phase MCI column chromatography is... It is a methanol aqueous solution with a volume ratio of 10-100% or an acetonitrile aqueous solution with a volume ratio of 20-100%, and the eluent used in the semi-preparative high performance liquid chromatography method for isocratic or gradient elution is n-hexane / EtOH with a volume ratio of 99-50%, n-hexane / 2-isopropanol with a volume ratio of 99-50%, n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002 to 50:50:0.002, or a methanol aqueous solution with a volume ratio of 10-100%.

5. A method for extracting the alkaloid compound according to claim 1 or 2 from the root of *Anachia*, comprising the following steps: a. After drying and pulverizing the anatchi root, use 50-95% ethanol aqueous solution, methanol or chloroform as solvent, wherein the ratio of anatchi root by weight kg to solvent by volume L is 1:1.5-1:

4. Extract by cold soaking, percolation, heating reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain the extract. b. After suspending the extract from step a in water, disperse it with acid. Extract the resulting acidic aqueous layer with dichloromethane to remove non-alkaloids, adjust the pH to 10-12 with alkali, and then extract with organic solvents such as dichloromethane, ethyl acetate, or n-butanol. Concentrate under reduced pressure to recover the organic solvents, yielding the total alkaloids; and c. The total alkaloids from step b are eluted using normal-phase silica gel column chromatography with gradient or isocratic elution, followed by dextran gel LH-20 column chromatography, then gradient elution using reversed-phase silica gel column chromatography or reversed-phase MCI column chromatography, and finally separation using semi-preparative high-performance liquid chromatography to obtain the alkaloid compounds. 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 alcohol, wherein the dextran gel LH-20 column chromatography employs methanol gradient or isocratic elution, and the eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution with a volume ratio of 10-100% or an acetonitrile aqueous solution with a volume ratio of 20-100%, and the eluent used in the semi-preparative high-performance liquid chromatography for isocratic or gradient elution is n-hexane / EtOH with a volume ratio of 99-50%, n-hexane / 2-isopropanol with a volume ratio of 99-50%, n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002 to 50:50:0.002, or a methanol aqueous solution with a volume ratio of 20-100%.

6. The method according to any one of claims 3 to 5, wherein in step b, the acid used is hydrochloric acid with a concentration of 1-5% or sulfuric acid with a concentration of 1-5%.

7. The method according to any one of claims 3 to 5, wherein in step b, the base used is NaHCO3, Na2CO3, ammonia or NaOH.

8. The method according to any one of claims 3 to 5, wherein in step c, the silica gel column chromatography is atmospheric pressure or pressurized column chromatography, and the packing material used is normal-phase silica gel or reverse-phase silica gel.

9. The method according to any one of claims 3 to 5, wherein the method further comprises the step of chirally resolving the compound obtained as a racemic mixture through a chiral chromatographic column to obtain an enantiomer.

10. Use of the compound according to claim 1 or 2 in the preparation of an anti-inflammatory drug, wherein the compound exerts its anti-inflammatory effect by inhibiting the release of nitric oxide from cells.

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