CN117143100A - Analgesic compound with Nav1.2 inhibition effect in radix Arnafae, and preparation and application thereof - Google Patents
Analgesic compound with Nav1.2 inhibition effect in radix Arnafae, and preparation and application thereof Download PDFInfo
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- CN117143100A CN117143100A CN202311104618.8A CN202311104618A CN117143100A CN 117143100 A CN117143100 A CN 117143100A CN 202311104618 A CN202311104618 A CN 202311104618A CN 117143100 A CN117143100 A CN 117143100A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
The invention relates to a novel 8,14,18,24-tetraazaocta-ring [21.2.2 ] 1,4 .1.0 2,21 .0 3,18 .0 5,17 .0 9, 16 .0 11,15 ]A preparation method of a tetramino 6/6/6/5/7/5 octacyclic alkaloid compound with a highly conjugated icosahedan ring system and an anti-inflammatory and analgesic application thereof, wherein the tetramino 6/6/6/5/7/5 octacyclic alkaloid compound is prepared by extracting roots (ananape roots) of Roman pyrethrum (Anacyclus pyrethrum (L.) DC) with an organic solvent, separating by two to three methods of normal phase silica gel column chromatography, reverse phase silica gel column chromatography and semi-preparative high performance liquid chromatography to obtain 4 new-skeleton tetramino 6/6/6/5/7/5 octacyclic alkaloid monomer compounds by high resolution mass spectrometry, nuclear magnetic resonance spectrometry and the likeAnd carrying out structural identification on the strain. The invention also provides Nav1.2 inhibition and NO inhibition of these compounds.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to analgesic/anti-inflammatory compounds (a compound of formula 1 and a compound of formula 2) with Nav1.2 inhibition and NO inhibition in the root of Arna, and preparation and application thereof.
Background
Pain is a body's defense against disease and is the fifth largest vital sign. However, persistent and severe pain can have an impact on the mental health of the patient, causing anxiety, sadness, and reduced immunity, leading to a range of consequences, most severe leading to death or disability. However, traditional analgesics such as opioids and nonsteroidal drugs have side effects of drug resistance, addiction and gastrointestinal bleeding.
Ion channels can regulate the passage of intracellular and extracellular ions to regulate the voltage potential across the membrane. The ion current generates an electrical pulse which in turn causes adjacent voltage sensitive channels to open continuously, resulting in a spontaneous electrical signal. By blocking ion channels, inhibiting sustained rhythmic potentials, signal transduction can be prevented, thereby reducing pain response. Currently, various types of ion channel inhibitors have been reported, including VGSCs, VGCCs, VGPCs and TRPs. Sodium channels are responsible for the generation and propagation of action potentials and are the fundamental elements of all excitable cells (including nerve and muscle cells, etc.) that produce electrical signals. In humans, there are nine subtypes of sodium channels, designated Nav1.1-Nav1.9. For example, lidocaine reduces the response to pain by inhibiting nav1.7 and nav1.9. Therefore, it is very important to find new ion channel inhibitors with analgesic potential.
The search for lead compounds with significant pharmacodynamic activity from traditional medicinal plants is a hotspot in drug research, and therefore, the discovery of novel ion channel inhibitors and compounds with anti-inflammatory effects from medicinal plants is an effective way to develop novel analgesic/anti-inflammatory drugs.
The Arna root is dry root of Roman pyrethrum (Anacyclus pyrethrum (L.) DC) belonging to Compositae, and has effects in refreshing brain, inducing resuscitation, treating hemiplegia, vitiligo, cornu Naemorhedi, headache, relieving cough, eliminating phlegm, and exhausting air.
Disclosure of Invention
The inventor has conducted intensive studies to first separate analgesic/anti-inflammatory compounds (a compound of formula 1 and a compound of formula 2) having Nav1.2 inhibitory action and NO inhibitory action from the root of Arna, and these compounds are brand-new framework compounds, are highly conjugated tetra-amino 6/6/6/5/7/5 octacyclic alkaloids, and have been identified in their structures, and through cell experiments, they have been verified to have Nav1.2 inhibitory action and NO inhibitory action, and can be used for preparing analgesic and/or anti-inflammatory drugs.
Accordingly, the present invention provides the following:
1. a compound selected from the group consisting of:
7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methano-aza [4,5,6-de ] pyrrolo [3 ', 2': 4",5" ] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione (a compound of formula 1); and
7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2'; 4",5" ] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione (compound of formula 2).
2. The compound according to 1 above or an isomer thereof, wherein the isomer is an enantiomer.
3. A compound according to 1 or 2 above, or an isomer thereof, selected from the group consisting of:
(4 aR,7aR,11aS,14 aR) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methano-aza [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
(4 aS,7aS,11aR,14 aS) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
(4 aR,7aR,11aR,14 aS) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione; and
(4 aS,7aS,11aS,14 aR) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4',5' ] cyclopenteno [1 ', 2': 6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinolin-1,8,11 (9H) -trione.
4. A compound according to any one of claims 1 to 3 above, or an isomer thereof, having a structural formula selected from the group consisting of:
5. a method of extracting a compound according to any one of 1 to 4 above or an isomer thereof from the root of ananape, comprising the steps of:
a. drying and pulverizing radix Arnebiae, taking 50-95% (v/v) ethanol water solution, methanol or chloroform as solvent (weight ratio of medicinal material (Kg) to solvent (L) 1:1.5-1:4), extracting by cold soaking, percolating, heating and refluxing or ultrasonic extracting, concentrating under reduced pressure to recover solvent to obtain extract;
b. suspending the total extract in step a with water, dispersing with acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid to obtain acid water layer, extracting with dichloromethane to remove non-alkaloids, and extracting with alkali such as NaHCO 3 、Na 2 CO 3 Regulating pH to 10-12 with ammonia water or NaOH, extracting with organic solvent such as dichloromethane, ethyl acetate or n-butanol, concentrating under reduced pressure, and recovering organic solvent to obtain total alkaloids;
c. separating the total alkaloids from step b by silica gel column chromatography, thin layer chromatography, reversed phase MCI column chromatography, sephadex LH-20 column chromatography, high performance liquid chromatography or any combination thereof to obtain the compound or isomer thereof.
6. The method according to above 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography and reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography, preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, a compound of formula 1 or formula 2 is obtained by reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography, 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, petroleum ether and acetone in a volume ratio of 500:1 to 3:1, methanol 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, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is an aqueous methanol solution in a volume ratio of 20 to 100% (v/v) or an aqueous acetonitrile solution in a volume ratio of 20 to 100% (v/v), and the isocratic eluent used in the semi-preparative high performance liquid chromatography is gradient of normal phase of 99 to 99 v/v.
7. The method according to above 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography, preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, gradient elution is performed with reverse phase silica gel or reverse phase MCI column chromatography, and further, semi-preparative high performance liquid chromatography is performed to obtain the compound of formula 1 or formula 2, wherein more preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol or chloroform and methanol in a volume ratio of 100:1 to 0:1, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is an aqueous methanol solution of 20 to 100% (v/v) or an aqueous acetonitrile solution of 20 to 100% (v/v), and the isocratic or gradient elution eluent used in the semi-preparative high performance liquid chromatography is n-hexane/EtOH in a volume ratio of 99 to 50% (v/v).
8. The method according to above 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, sephadex LH-20 column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography, preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, sephadex LH-20 column chromatography, gradient elution with reverse phase silica gel or reverse phase MCI column chromatography, and further semi-preparative high performance liquid chromatography is performed to obtain a compound of formula 1 or formula 2, wherein more preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 100:1 to 0:1, dichloromethane and methanol or chloroform and methanol, the eluent used in the reverse phase silica gel LH-20 column chromatography is an aqueous methanol solution of 20 to 100% (v/v) or an aqueous acetonitrile solution of 20 to 100% (v/v), and the eluent used in the semi-preparative high performance liquid chromatography is a gradient of 99 to 99% (v/v) hexane.
9. The method according to any one of the preceding claims 5 to 8, wherein in step c the silica gel column chromatography is normal pressure or pressure column chromatography and/or the packing used is forward silica gel or reverse phase silica gel.
10. The use of a compound according to any one of 1 to 4 above or an isomer thereof for the preparation of an analgesic or anti-inflammatory drug, preferably, the compound or isomer thereof exerts an analgesic effect by inhibiting nav1.2 and an anti-inflammatory effect by inhibiting NO.
Detailed Description
The invention aims to provide a compound with Nav1.2 inhibition and/or anti-inflammatory effect, a separation preparation method thereof and application value in preparing analgesic drugs.
According to a first aspect of the present invention, there is provided a compound having nav1.2 inhibitory effect and/or having anti-inflammatory effect, the compound having the structural formula shown in the following figure;
wherein:
compound (+) -1 is: (4 aR,7aR,11aS,14 aR) -7 a-acetyl
-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2'; 4",5" ] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
compound (-) -1 is: (4 aS,7aS,11aR,14 aS) -7 a-acetyl
-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2'; 4",5" ] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
the compound (+) -2 is: (4 aR,7aR,11aR,14 aS) -7 a-acetyl
-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2'; 4",5" ] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
compound (-) -2 is: (4 aS,7aS,11aS,14 aR) -7 a-acetyl
-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2'; 4',5' ] cyclopenteno [1 ', 2': 6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinolin-1,8,11 (9H) -trione.
The extraction and separation method of the alkaloid compounds comprises the following steps:
a. drying and pulverizing radix Arnebiae, taking 50-95% (v/v) ethanol water solution, methanol or chloroform as solvent (weight ratio of medicinal material (Kg) to solvent (L) 1:1.5-1:4), extracting by cold soaking, percolating, heating and refluxing or ultrasonic extracting, concentrating under reduced pressure to recover solvent to obtain extract;
b. suspending the total extract in step a with water, and concentrating with acid such as 1-5% hydrochloric acid or concentrated solutionDispersing with 1-5% sulfuric acid to obtain acid water layer, extracting with dichloromethane to remove non-alkaloids, and extracting with alkali such as NaHCO 3 、Na 2 CO 3 Regulating pH to 10-12 with ammonia water or NaOH, extracting with organic solvent such as dichloromethane, ethyl acetate or n-butanol, concentrating under reduced pressure, and recovering organic solvent to obtain total alkaloids;
c. separating the total alkaloids from step b by silica gel column chromatography, thin layer chromatography, reversed phase MCI column chromatography, sephadex LH-20 column chromatography, high performance liquid chromatography or any combination thereof to obtain the compound or isomer thereof.
Two of the separation modes are as follows:
the volume ratio of the normal phase silica gel column chromatography eluent is 100:0-3:1 or 500:1-3:1 dichloromethane-methanol, and performing reversed phase silica gel or MCI column chromatography or semi-preparative high performance liquid chromatography to obtain compound of formula 1 or 2;
three separation modes:
the normal phase silica gel column chromatography eluent is petroleum ether-ethyl acetate, methylene dichloride-methanol or chloroform-methanol with the volume ratio of 100:1-0:1, reverse phase silica gel or MCI column chromatography is carried out, methanol aqueous solution with the volume ratio of 20-100% (v/v) or acetonitrile aqueous solution with the volume ratio of 20-100% (v/v) is adopted for gradient elution, semi-preparation high performance liquid chromatography is adopted, and normal hexane/EtOH with the volume ratio of 99-50% (v/v) is adopted as the eluent, so that the compound of the formula 1 or 2 is obtained.
Four separation modes:
the normal phase silica gel column chromatography eluent is prepared by gradient eluting petroleum ether-ethyl acetate, methylene dichloride-methanol or chloroform-methanol with the volume ratio of 100:1-0:1, subjecting to Sephadex LH-20 column chromatography, subjecting to isocratic eluting with methanol, subjecting to reverse phase silica gel or MCI column chromatography, subjecting to gradient eluting with methanol aqueous solution with the volume ratio of 20-100% (v/v) or acetonitrile aqueous solution with the volume ratio of 20-100% (v/v), subjecting to semi-preparative high performance liquid chromatography, and subjecting to normal hexane/EtOH with the volume ratio of 99-50% (v/v) as eluent to obtain the compound of formula 1 or 2.
The preparation method of the alkaloid compound in the root of the Arna is characterized in that the silica gel column chromatography is normal pressure or pressurized column chromatography, the filler is forward silica gel or reverse phase silica gel, and the volume ratio of dichloromethane and methanol is 500:1-3:1; the volume ratio is 100:0-3:1 petroleum ether/ethyl acetate; the volume ratio is 5:2 petroleum ether/acetone elution; or methanol water with the volume ratio of 1:9-1:0 is used as an eluent, and isocratic or gradient elution is adopted.
The preparation method of the alkaloid compound in the root of the argan in the step c is characterized in that the eluent of the sephadex LH-20 column chromatography is methanol and isocratic elution is adopted.
The preparation method of the alkaloid compound in the root of the Arna in the step c is characterized in that the preparation high performance liquid chromatography adopts normal hexane/EtOH with the volume ratio of 99-50% (v/v) as an eluent, and adopts isocratic or gradient elution.
According to another aspect of the present invention, there is provided the use of the compound having nav1.2 inhibiting effect for the preparation of an analgesic drug.
Combining various spectrum analysis methods (high resolution mass spectrum, ultraviolet spectrum, infrared spectrum and nuclear magnetic resonance spectrum) and quantum chemical calculation method 13 C-NMR DP4+ probability analysis and ECD) and X-ray single crystal diffraction, and the structures of the compound 1 and the compound 2 prepared in examples were determined. Wherein the relative configuration of compound 1 and compound 2 is determined by X-ray single crystal diffraction, as shown in fig. 1 and 4.
Compound 1 (anaryphthrine a): yellow needle-like crystals; optical rotation value [ alpha ]]25 D1584 (c 0.08, methanol, (+) -1); [ alpha ]]25 D-1584 (c 0.1, methanol, (-) -1); ultraviolet (methanol) lambda max (log ε) 427 (4.15) nm,267 (3.96) nm; infrared (KBr) max 3297,2961,2924,1704,1623,and 1448cm -1 ;ECD(c 3.12×10 -4 M, methanol) lambda max (Δε) 216 (-11.39), 253 (-2.36), 289 (-13.16), 417 (17.07) nm, enantiomer compound (+) -1; ECD (c3.12X10) -4 M, methanol) lambda max (Δε) 217 (9.25), 253 (0.05), 290 (13.67), 416 (-21.25) nm, enantiomer compound (-) -1; high resolution mass spectrum m/z 641.4051[ M+H ]] + (calculated value C) 39 H 53 O 4 N 4 + 641.4066), which 1 H and 13 the C NMR spectrum data are shown in Table 1 and Table 2.
Compound 2 (anaryphthrine a): yellow needle-like crystals; optical rotation value [ alpha ]]25 D-530 (c 0.03, methanol, (-) -2); [ alpha ]]25 D530 (c 0.02, methanol, (+) -2); ultraviolet (methanol) lambda max (log ε) 475 (3.90) nm,281 (3.74) nm; infrared (KBr) max 3316,2963,2926,2857,1699,1615,1445,and 1194cm -1;E CD(c 3.12×10 -4 M, methanol) lambda max (Δε) 219 (-23.50), 265 (17.79), 300 (-35.20), 381 (25.45), 481 (-9.33), enantiomer compound (-) -2; ECD (c3.12X10) -4 M, methanol) lambda max (Δε) 220 (30.48), 264 (-25.61), 301 (46.37), 380 (-34.83), 480 (10.83), enantiomer compound (+) -2; high resolution mass spectrum m/z 641.4049[ M+H ]] + (calculated value for C) 39 H 53 O 4 N 4 + 641.4066), which 1 H and 13 the CNMR spectrum data are shown in tables 1 and 2.
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In general, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
(1) The compounds 1-2 provided by the invention are brand new framework compounds. Compounds 1 and 2 are two unprecedented pairs of novel skeletal enantiomers of highly conjugated tetramino 6/6/6/5/7/5 octacyclic alkaloids with unique 8,14,18,24-tetraazaoctacyclic [21.2.2 ] 1,4 .1.0 2,21 .0 3,18 .0 5,17 .0 9,16 .0 11,15 ]The skeleton structure of the icosahedron ring system has 4 discontinuous chiral stereo centers.
(2) The compound 1-2 provided by the invention has Nav1.2 inhibitory activity and NO inhibitory activity, and the compound 2 has micromole-level Nav1.2 inhibitory activity and potential Nav1.2 inhibitory activity.
(3) According to the invention, through computer molecule docking, the active results of a compound and Nav1.2 are combined, residues of Gln332, phe38, asn361, asp334, tyr362, asp949, trp948, pro921, trp923, tyr1429 and Met1425 in the Nav1.2 polypeptide chain are summarized as active binding sites of the Nav1.2 polypeptide chain, and a theoretical basis is provided for subsequent development of efficient Nav1.2 inhibitors.
The invention will now be described in further detail with reference to the drawings and examples, which are not intended to limit the scope of the invention. Modifications and substitutions of the method, steps, conditions, etc. of the present invention without departing from the spirit and nature of the present invention are intended to be within the scope of the present invention.
Drawings
FIG. 1 is an X-ray single crystal diffraction pattern of Compound 1;
FIG. 2 is an X-ray single crystal diffraction pattern of compound (+) -1;
FIG. 3 is an X-ray single crystal diffraction pattern of compound (-) -1;
FIG. 4 is an X-ray single crystal diffraction pattern of compound 2;
FIG. 5 is a graph of molecular docking and molecular dynamics modeling results for compound (+) -2;
FIG. 6 is a diagram of Compound 1 1 H NMR chart;
FIG. 7 is a diagram of Compound 1 13 C NMR chart;
FIG. 8 is a diagram of Compound 2 1 H NMR chart;
FIG. 9 is a diagram of Compound 2 13 C NMR chart.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The following are specific examples:
example 1
Pulverizing dried radix Arnafiae (Anacyclus pyrethrum (L.) DC.,15.0 kg), ultrasonic extracting with chloroform (30L), concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract in water, acidifying with 5% hydrochloric acid, extracting with dichloromethane to remove non-alkaloid impurities, and stirring with saturated NaHCO under ice-water bath 3 The pH of the aqueous solution is adjusted to 10 to obtain an alkalized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 2
Pulverizing dried radix Arnafiae (Anacyclus pyrethrum (L.) DC.,15.0 kg), percolating with 95% ethanol (40L), concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract in water, acidifying with 5% hydrochloric acid, extracting with dichloromethane to remove non-alkaloid impurities, and regulating pH to 10 with aqueous ammonia solution under stirring to obtain alkalinized solution. The alkalization liquid is fully extracted by ethyl acetate, and the ethyl acetate extracts are combined and dried to obtain the total alkaloid.
Example 3
Pulverizing dried radix Arnafiae (Anacyclus pyrethrum (L.) DC, 15.0 kg), percolating with 50% ethanol (40L), concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract in water, acidifying with 2% hydrochloric acid, extracting with dichloromethane to remove non-alkaloid impurities, and adding aqueous acid solution ice waterThe bath is stirred continuously with Na 2 CO 3 The pH of the aqueous solution is adjusted to 10 to obtain an alkalized solution. And fully extracting the alkalization liquid with n-butanol, combining n-butanol extract and drying to obtain total alkaloids.
Example 4
Dried ananape root (Anacyclus pyrethrum (L.) dc.,15.0 kg) was crushed, extracted with 75% ethanol (45L) under reflux, concentrated under reduced pressure and combined to give a total extract, which was suspended in water, acidified with 1% hydrochloric acid, extracted with dichloromethane to remove non-alkaloid impurities, and the aqueous acid solution was pH adjusted to 12 with NaOH aqueous solution with constant stirring in an ice-water bath to give an alkalinized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 5
Pulverizing dried radix Arnafiae (Anacyclus pyrethrum (L.) DC.,15.0 kg), cold soaking in 22.5L methanol at room temperature, concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract in water, acidifying with 5% hydrochloric acid, extracting with dichloromethane to remove non-alkaloid impurities, and stirring with saturated NaHCO under ice-water bath 3 The pH of the aqueous solution is adjusted to 10 to obtain an alkalized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 6
Pulverizing dried radix Arnafiae (Anacyclus pyrethrum (L.) DC.,15.0 kg), cold soaking in 22.5L methanol at room temperature, concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract in water, acidifying with 1% sulfuric acid, extracting with dichloromethane to remove non-alkaloid impurities, and stirring with saturated NaHCO under ice-water bath 3 The pH of the aqueous solution is adjusted to 10 to obtain an alkalized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 7
Dried root (Anacyclus pyrethrum (L.) DC.,15.0 kg) of Arna, pulverizing, cold-soaking in 22.5L methanol at room temperature, and concentrating under reduced pressureMixing to obtain total extract, suspending the total extract with water, acidifying with 2% sulfuric acid, extracting with dichloromethane to remove non-alkaloid impurities, and stirring with saturated NaHCO under ice-water bath 3 The pH of the aqueous solution is adjusted to 10 to obtain an alkalized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 8
Pulverizing dried radix Arnafiae (Anacyclus pyrethrum (L.) DC.,15.0 kg), cold soaking in 22.5L methanol at room temperature, concentrating under reduced pressure, mixing the extractive solutions to obtain total extract, suspending the total extract in water, acidifying with 5% sulfuric acid, extracting with dichloromethane to remove non-alkaloid impurities, and stirring with saturated NaHCO under ice-water bath 3 The pH of the aqueous solution is adjusted to 10 to obtain an alkalized solution. The alkalization solution is fully extracted by methylene dichloride, and the methylene dichloride extract is combined and dried to obtain the total alkaloid.
Example 9
Mixing the total alkaloids with 100-200 mesh silica gel, performing silica gel column chromatography, then performing gradient elution with dichloromethane/methanol (100:0-3:1, V/V), and combining the same components to obtain 6 components Fr.A-Fr.F with polarity from small to large; wherein the 1 st component Fr.A (159.0 g) is mixed with 100-200 mesh silica gel, silica gel column chromatography is carried out, then petroleum ether/ethyl acetate gradient elution (100:0-3:1, V/V) is carried out, the same components are combined, and 6 components Fr.A1-Fr.A6 with the polarity from small to large are obtained; wherein the 2 nd component Fr.A2 (76.1 g) is subjected to reversed-phase MCI column chromatography, methanol/water gradient elution (20:80-100:0, V/V) is adopted, and the same components are combined to obtain 7 subfractions Fr.A21-Fr.A27 with the polarity from large to small; wherein the component Fr.A25 (10.0 g) is mixed with 200-300 mesh silica gel, silica gel column chromatography is carried out, then petroleum ether/ethyl acetate gradient elution (10:1-3:1, V/V) is carried out, the same components are combined, and 3 components Fr.A251-Fr.A253 with the polarity from small to large are obtained; the component Fr.A253 (9.0 g) was stirred with 200-300 mesh silica gel, subjected to silica gel column chromatography, then eluted with a petroleum ether/ethyl acetate gradient (10:1-3:1, V/V), and the same components were combined to obtain 6 components Fr.A2531-Fr with polarity from small to large.A2533; wherein the component Fr.A2533 (7.8 g) is subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain 3 subfractions Fr.A25331-Fr.A25333 with molecular weights from large to small; subjecting the component Fr.A25333 (6.1 g) to reverse phase C18 silica gel column chromatography, eluting with methanol/water gradient (20:80-100:0, V/V), and mixing the same components to obtain 6 subfractions Fr.A253331-Fr.A253336 with polarity from large to small; the fraction Fr.A253336 (231.2 mg) was subjected to normal phase silica gel column chromatography, eluting with methylene chloride/methanol (500:1-50:1, V/V) to give 5 subfractions Fr.A2533361-Fr.A2533365 with polarity from small to large; component fr.a2533363 (71.1 mg) was recrystallized from methanol to give compound 1 (i.e. compound of formula 1) (Anacyphrethines A,30.3mg, 0.000202%); chiral resolution of racemate of Compound 1 by chiral chromatography (DAICEL CORPORATION Chiralpak ID μm10×250mm; solvent: n-Hexane/EtOH=90:10; column temperature: 25 ℃ C.; flow rate: 3ml/min; detection wavelength: 360 nm) to give Compound (+) -1 (14.0 mg, t) R =12.3 min) and compound (-) -1 (15.4 mg, t R =18.0 min). The fraction Fr.A253335 (214.3 mg) was subjected to normal phase silica gel column chromatography, eluting with methylene chloride/methanol (500:1-50:1, V/V) to give 6 subfractions Fr.A2533351-Fr.A2533356 with polarity from small to large; component fr.a2533353 (61.3 mg) was subjected to normal phase silica gel column chromatography eluting with petroleum ether/acetone (5:2, V/V) to give compound of formula 2 (i.e. compound of formula 2) (Anacyphrethines B,20.1mg, 0.000134%); chiral resolution of racemate of Compound 2 by chiral chromatography (DAICEL CORPORATION Chiralpak ID μm10×250mm; solvent: n-Hexane/EtOH=60:40; column temperature: 25 ℃ C.; flow rate: 3ml/min; detection wavelength: 360 nm) to give Compound (+) -2 (7.5 mg, t) R =17.0 min) and compound (-) -2 (7.3 mg, t R =12.4min)。
Example 10
Mixing the total alkaloids with 100-200 mesh silica gel, performing silica gel column chromatography, then performing gradient elution with dichloromethane/methanol (100:0-3:1, V/V), and combining the same components to obtain 6 components Fr.A-Fr.F with polarity from small to large; wherein component 1, fr.A (159.0 g), is mixed with 100-200 mesh silica gel, subjected to silica gel column chromatography, and then eluted with petroleum ether/ethyl acetate gradient (100:0 to the extent)3:1, V/V), and combining the same components to obtain 6 components fr.a1-fr.a6 having a polarity ranging from small to large; wherein the 2 nd component Fr.A2 (76.1 g) is subjected to reversed-phase MCI column chromatography, methanol/water gradient elution (20:80-100:0, V/V) is adopted, and the same components are combined to obtain 7 subfractions Fr.A21-Fr.A27 with the polarity from large to small; wherein the component Fr.A25 (10.0 g) is mixed with 200-300 mesh silica gel, silica gel column chromatography is carried out, then petroleum ether/ethyl acetate gradient elution (10:1-3:1, V/V) is carried out, the same components are combined, and 3 components Fr.A251-Fr.A253 with the polarity from small to large are obtained; mixing the components Fr.A253 (9.0 g) with 200-300 mesh silica gel, performing silica gel column chromatography, then performing gradient elution with petroleum ether/ethyl acetate (10:1-3:1, V/V), and combining the same components to obtain 6 components Fr.A2531-Fr.A2533 with the polarity from small to large; wherein the component Fr.A2533 (7.8 g) is subjected to MCI column chromatography and eluted with 20-100% acetonitrile water to obtain 3 subfractions Fr.A25331-Fr.A25333 with molecular weights from large to small; subjecting the component Fr.A25333 (6.1 g) to reverse phase C18 silica gel column chromatography, eluting with methanol/water gradient (20:80-100:0, V/V), and mixing the same components to obtain 6 subfractions Fr.A253331-Fr.A253336 with polarity from large to small; the fraction Fr.A253336 (231.2 mg) was subjected to normal phase silica gel column chromatography, eluting with methylene chloride/methanol (500:1-50:1, V/V) to give 5 subfractions Fr.A2533361-Fr.A2533365 with polarity from small to large; component fr.a2533363 (71.1 mg) was recrystallized from methanol to give compound 1 (i.e. compound of formula 1) (Anacyphrethines A,30.3mg, 0.000202%); chiral resolution of racemate of Compound 1 by chiral chromatography (DAICEL CORPORATION Chiralpak ID μm10×250mm; solvent: n-Hexane/EtOH=90:10; column temperature: 25 ℃ C.; flow rate: 3ml/min; detection wavelength: 360 nm) to give Compound (+) -1 (14.0 mg, t) R =12.3 min) and compound (-) -1 (15.4 mg, t R =18.0 min). The fraction Fr.A253335 (214.3 mg) was subjected to normal phase silica gel column chromatography, eluting with methylene chloride/methanol (500:1-50:1, V/V) to give 6 subfractions Fr.A2533351-Fr.A2533356 with polarity from small to large; component fr.a2533353 (61.3 mg) was subjected to normal phase silica gel column chromatography eluting with petroleum ether/acetone (5:2, V/V) to give compound of formula 2 (i.e. compound of formula 2) (Anacyphrethines B,20.1mg, 0.000134%); racemate of Compound 2 is subjected toChiral chromatographic column (DAICEL CORPORATION Chiralpak ID μm10×250mm; solvent: n-Hexane/EtOH=60:40; column temperature: 25 ℃ and flow rate: 3ml/min; detection wavelength: 360 nm) gave compound (+) -2 (7.5 mg, t) R =17.0 min) and compound (-) -2 (7.3 mg, t R =12.4min)。
Example 11
Mixing the total alkaloids with 100-200 mesh silica gel, performing silica gel column chromatography, then performing gradient elution with dichloromethane/methanol (100:0-3:1, V/V), and combining the same components to obtain 6 components Fr.A-Fr.F with polarity from small to large; wherein component 1 fr.a (159.0 g) was subjected to repeated silica gel column chromatography using a volume ratio of 100:0-3:1, petroleum ether-ethyl acetate, 500:1-3:1 methylene chloride-methanol or petroleum ether/acetone with a volume ratio of 50:1-0:1 as eluent to obtain compound 1 (i.e. compound of formula 1) (Anacyphrethines A,30.3mg, 0.000202%) and compound 2 (i.e. compound of formula 2) (Anacyphrethines B,20.1mg, 0.000134%); chiral resolution of racemate of Compound 1 by chiral chromatography column (DAICEL CORPORATION Chiralpak ID μm10×250mm; solvent: n-Hexane/EtOH=99:1-50-50; column temperature: 25 ℃ C., flow rate: 3ml/min; detection wavelength: 360 nm) to give Compound (+) -1 (14.0 mg, t) R =12.3 min) and compound (-) -1 (15.4 mg, t R =18.0 min). Chiral resolution of racemate of Compound 2 by chiral chromatography column (DAICEL CORPORATION Chiralpak ID μm10×250mm; solvent: n-Hexane/EtOH=99:1-50-50; column temperature: 25 ℃ C., flow rate: 3ml/min; detection wavelength: 360 nm) to obtain compound (+) -2 (7.5 mg, t) R =17.0 min) and compound (-) -2 (7.3 mg, t R =12.4min)。
Example 12
The Nav1.2 inhibitory activity of compound 1 and compound 2 was evaluated by patch clamp electrophysiology experiments. Experiments were performed using HEK293T cells (ATCC cell bank) at 37℃with 5% CO 2 An incubator; the culture medium contains 90% DMEM+10% Fetal Bovine Serum (FBS), and after digestion with 0.25% pancreatin when the cell density reaches 80-90%, subculture or hole plating is performed. After 24h plating, transfection was performed with Lipo2000 transfection kit (Thermo Fisher, shanghai, china)The ratio of pcDNA3.1-SCN2A (NaV1.2 GenBank accession No. NM-001040142) plasmid (Huada Gene, beijing, china) to pcDNA3.1-EGFP plasmid (Huada Gene, beijing, china) was 9:1 (4000 ng in total), and electrophysiological experiments were performed 18h or more after transfection.
The current clamp recording experiments used an Axon patch 700B patch clamp amplifier (Axon Instruments, molecular Devices, usa), digital to analog converter Digidata 1440A (Axon Instruments, molecular Devices, usa), signal acquisition using pClamp 10.0 software (Molecular Devices, usa), filtering at 2kHz, sampling frequency at 10kHz. The patch clamp electrode is prepared by drawing a horizontal electrode drawing instrument P-97 (Sutter Instrument, U.S.) through a multi-step procedure, and the resistance of the filled electrode internal liquid is measured to be 3-5MΩ. The recording process is carried out at room temperature (23-25 ℃). The perfusion system is self-made at the speed of about 2mL/min; the drug delivery system was BPS-8 (ALA Scientific Instruments, USA). All electrophysiological data were processed using a Clampfit 10.4 (Molecular Device, usa) and then analyzed using GraphPad Prism 5 (GraphPad Software, usa). The initial concentration of the monomer compound was 40. Mu.M, and the inhibition ratio was shown in Table 3.
TABLE 3 Nav1.2 inhibitory Activity of Compounds 1-2
[a] Positive drug: TTX (tetrodotoxin, jiangsu Kangte bioengineering limited). [b] Each experiment was repeated 3 times.
Conclusion: the compound (+) -2 has remarkable inhibition effect on Nav1.2, wherein the compound (+) -2 has micromole-level Nav1.2 inhibition activity and IC thereof 50 The value was 23.94.+ -. 2.70. Mu.M.
Example 13
NO inhibitory activity of the compounds of formula 1 (compound 1) and 2 (compound 2):
1. cell culture:
BV2 cells (purchased from North Nami organism BeNa Culture Collection, BNCC) in 10% fetal bovine serum (FBS) (purchased from Giboco Inc. of the United states), du's broth (dulbecco's modified eagle medium, DMEM) high sugar medium (purchased from Hyclone Inc. of the United states) of 1% penicillin and streptomycin, at 37℃and 5% CO 2 Is cultured in an incubator;
2. test of the effect of the compounds of the invention on cell viability:
dissolving compound in dimethyl sulfoxide (DMSO), collecting BV2 cells in logarithmic growth phase and good growth state, and mixing with the obtained solution at a ratio of 5×10 3 The cells were inoculated into 96-well plates, the experimental groups were added with different concentrations of 12.5, 25, 50 and 100. Mu.M of the compound of formula I, the control group was added with dimethyl sulfoxide (DMSO), cultured for 24 hours, then 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, absorbance at 450nm was measured using a microplate reader, and cell viability was calculated, and the experimental results are shown in Table 4;
TABLE 4 cell viability of Compounds 1-2
3. Nitric Oxide (NO) content determination:
the intracellular NO release of BV2 was tested by the Griess method (Arias-Negrete et al, analytical biochemistry328.1 (2004): 14-21.), samples of different concentrations 25, 50 and 100. Mu.M were added for incubation for 2 hours, then 1. Mu.g/mL Lipopolysaccharide (LPS, sigma, L4391) was added for co-incubation for 22 hours, and after incubation, cell supernatants were collected and assayed for nitric oxide content by the Griess method; before measurement, firstly taking out Griess Reagent I and Griess Reagent II (Nitric Oxide Assay kit, beyotin, S0021M), allowing the Griess Reagent I and the Griess Reagent II to return to room temperature, diluting a standard substance (1-100 mu M) with a complete culture medium, wherein the concentration of the standard substance can be 0,1,2,5, 10, 20, 40, 60 and 100 mu M, adding the standard substance and a collected culture solution supernatant into a 96-well plate according to 50 mu L/hole, sequentially adding 50 mu L of Griess Reagent I and 50 mu LGriess Reagent II which return to room temperature into each hole, shaking and mixing for 5min, measuring absorbance at 540nm, making a standard curve, and calculating NO content in the culture solution supernatant according to the standard curve; the initial concentration of the monomer compound was 40. Mu.M, and the inhibition ratio was shown in Table 5.
TABLE 5 NO inhibitory Activity of Compounds 1-2
[a] Positive drug: andrographolide (AG, HY-N0191). [b] Each experiment was repeated 3 times.
Conclusion: compounds 1-2 have certain inhibition effect on NO release, wherein compounds (+) -1, (-) -1 IC 50 The values were 27.63.+ -. 3.753, 37.35.+ -. 0.807, respectively. The compounds (+) -1 and (-) -1 have remarkable inhibiting effect on the release of NO.
Example 13
This example investigated the mode of action of compound 2 with Nav1.2 (pdb ID:6J 8E) using Autodock 4.2.6 molecular docking software (The Scripps Research Institute, USA). Dynamic conformational changes and movement trajectories of the compound bound to the nav1.2 protein were reflected by molecular dynamics simulation. Nav1.2 consists of a polypeptide chain that is folded into four homologous repeats.
Conclusion: the docking results show that (+) -2 binds to the top residue of the active pocket and occupies the pores of the Nav1.2 channel with lower affinity (-9.126 kcal/mol), consistent with previous binding experiments. A detailed analysis of the interaction of the Nav1.2 channel active site with (+) -2 showed that the C-10 carbonyl group in (+) -2 forms a hydrogen bond with the Nav1.2 channel residue Asn333 as a hydrogen bond donor. In addition, (+) -2 interacts with residues Gln332, phe385, asn361, asp334, tyr362, asp949, trp948, pro921, trp923, tyr1429 and Met1425, which are possible sites of action for Nav1.2, providing a theoretical basis for subsequent development of potent Nav1.2 inhibitors.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A compound selected from the group consisting of:
7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methano-aza [4,5,6-de ] pyrrolo [3 ', 2': 4",5" ] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione (a compound of formula 1); and
7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl
-3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2'; 4",5" ] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione (compound of formula 2).
2. The compound of claim 1, or an isomer thereof, wherein the isomer is an enantiomer.
3. The compound according to claim 1 or 2, or an isomer thereof, selected from the group consisting of:
(4 aR,7aR,11aS,14 aR) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methano-aza [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
(4 aS,7aS,11aR,14 aS) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione;
(4 aR,7aR,11aR,14 aS) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4",5"] cyclopenteno [1",2":6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinoline-1,8,11 (9H) -trione; and
(4 aS,7aS,11aS,14 aR) -7 a-acetyl-2, 4a,6, 9,11a,13, 17-dodecamethyl- -3,4a,5,6, 7a,9,10,11a,12,13,14,16, 17-decatetrahydro-11 a,14 a-methylazao [4,5,6-de ] pyrrolo [3 ', 2' ". 4',5' ] cyclopenteno [1 ', 2': 6',7' ] azepino [4',5':4,5] pyrrolo [3,2,1-ij ] quinolin-1,8,11 (9H) -trione.
4. A compound according to any one of claims 1 to 3, or an isomer thereof, having a structural formula selected from:
5. a method of extracting a compound according to any one of claims 1 to 4, or an isomer thereof, from the root of ananape, comprising the steps of:
a. drying and pulverizing the root of Arna, taking ethanol water solution, methanol or chloroform with volume fraction of 50-95% (v/v) as solvent, wherein the ratio of the root of Arna (Kg) to the solvent (L) is 1:1.5-1:4, extracting by cold soaking, percolating, heating and refluxing or ultrasonic extracting, concentrating under reduced pressure, and recovering solvent to obtain extract;
b. suspending the total extract in step a with water, dispersing with acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid to obtain acid water layer, extracting with dichloromethane to remove non-alkaloids, and extracting with alkali such as NaHCO 3 、Na 2 CO 3 Regulating pH to 10-12 with ammonia water or NaOH, extracting with organic solvent such as dichloromethane, ethyl acetate or n-butanol, concentrating under reduced pressure, and recovering organic solvent to obtain total alkaloids;
c. separating the total alkaloids from step b by silica gel column chromatography, thin layer chromatography, reversed phase MCI column chromatography, sephadex LH-20 column chromatography, high performance liquid chromatography or any combination thereof to obtain the compound or isomer thereof.
6. The method according to claim 5, wherein in step c a combination of normal phase silica gel column chromatography and reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography is used for separation, preferably, after gradient or isocratic elution with normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography is used to obtain the compound of formula 1 or formula 2, wherein more preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 100:0 to 3:1, petroleum ether and acetone in a volume ratio of 500:1 to 3:1, methanol and acetone in a volume ratio of 50:1 to 0:1, or methanol and water in a volume ratio of 1:9-1:0, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is an aqueous methanol solution in a volume ratio of 20-100% (v/v) or an aqueous acetonitrile solution in a volume ratio of 20-100% (v/v), and the isocratic eluent used in the semi-preparative high performance liquid chromatography is gradient of 99-v/99% hexane.
7. The method according to claim 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography, preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, gradient elution is performed with reverse phase silica gel or reverse phase MCI column chromatography, and further, semi-preparative high performance liquid chromatography is performed to obtain the compound of formula 1 or formula 2, wherein more preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol or chloroform and methanol in a volume ratio of 100:1 to 0:1, the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is an aqueous methanol solution of 20-100% (v/v) or an aqueous acetonitrile solution of 20-100% (v/v), and the isocratic or gradient elution used in the semi-preparative high performance liquid chromatography is n-hexane/EtOH in a volume ratio of 99-50% (v/v).
8. The method according to claim 5, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography, sephadex LH-20 column chromatography, reverse phase silica gel or reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography, preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, sephadex LH-20 column chromatography is gradient-eluted with reverse phase silica gel or reverse phase MCI column chromatography, and semi-preparative high performance liquid chromatography is further adopted to obtain the compound of formula 1 or formula 2, wherein more preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 100:1 to 0:1, dichloromethane and methanol or chloroform and methanol, the eluent used in the reverse phase silica gel LH-20 column chromatography is methanol aqueous solution of 20-100% (v/v) or acetonitrile aqueous solution of 20-100% (v/v), and the eluent used in the semi-preparative high performance liquid chromatography is gradient of 99-99 v/v.
9. The process according to any one of claims 5 to 8, wherein in step c the silica gel column chromatography is atmospheric or pressurized column chromatography and/or the packing used is forward silica gel or reverse phase silica gel.
10. Use of a compound according to any one of claims 1 to 4 or an isomer thereof in the manufacture of an analgesic or anti-inflammatory medicament, preferably said compound or isomer thereof exerting an analgesic effect by inhibition of nav1.2 and an anti-inflammatory effect by inhibition of NO.
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