CN113620914A - Andrographolide derivative and industrial chromatographic preparation method and application thereof - Google Patents
Andrographolide derivative and industrial chromatographic preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- BOJKULTULYSRAS-OTESTREVSA-N Andrographolide Chemical class C([C@H]1[C@]2(C)CC[C@@H](O)[C@]([C@H]2CCC1=C)(CO)C)\C=C1/[C@H](O)COC1=O BOJKULTULYSRAS-OTESTREVSA-N 0.000 title claims description 27
- 150000001875 compounds Chemical class 0.000 claims abstract description 17
- 206010035664 Pneumonia Diseases 0.000 claims abstract description 12
- 241000711573 Coronaviridae Species 0.000 claims abstract description 7
- 208000025721 COVID-19 Diseases 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 54
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 47
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 34
- 238000010828 elution Methods 0.000 claims description 34
- 239000012043 crude product Substances 0.000 claims description 28
- 239000003960 organic solvent Substances 0.000 claims description 28
- ASLUCFFROXVMFL-UHFFFAOYSA-N andrographolide Natural products CC1(CO)C(O)CCC2(C)C(CC=C3/C(O)OCC3=O)C(=C)CCC12 ASLUCFFROXVMFL-UHFFFAOYSA-N 0.000 claims description 20
- 239000003208 petroleum Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- 238000011068 loading method Methods 0.000 claims description 15
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 13
- 239000011347 resin Substances 0.000 claims description 13
- 229920005989 resin Polymers 0.000 claims description 13
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 239000012046 mixed solvent Substances 0.000 claims description 12
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 10
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 239000000741 silica gel Substances 0.000 claims description 8
- 229910002027 silica gel Inorganic materials 0.000 claims description 8
- 238000001179 sorption measurement Methods 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 239000003480 eluent Substances 0.000 claims description 7
- 239000000945 filler Substances 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- 238000002347 injection Methods 0.000 claims description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 6
- 208000027866 inflammatory disease Diseases 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 239000003463 adsorbent Substances 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000003826 tablet Substances 0.000 claims description 4
- 239000002775 capsule Substances 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 2
- 125000003158 alcohol group Chemical group 0.000 claims description 2
- 239000007919 dispersible tablet Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 4
- 239000008194 pharmaceutical composition Substances 0.000 claims 1
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 abstract description 20
- 239000002158 endotoxin Substances 0.000 abstract description 8
- 229920006008 lipopolysaccharide Polymers 0.000 abstract description 8
- 210000002540 macrophage Anatomy 0.000 abstract description 6
- 230000002401 inhibitory effect Effects 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000012404 In vitro experiment Methods 0.000 abstract 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 239000008213 purified water Substances 0.000 description 5
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- 230000006835 compression Effects 0.000 description 2
- 238000007906 compression Methods 0.000 description 2
- 230000003467 diminishing effect Effects 0.000 description 2
- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 241000207965 Acanthaceae Species 0.000 description 1
- 244000118350 Andrographis paniculata Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000010268 HPLC based assay Methods 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- -1 labdane diterpenoid compound Chemical class 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003186 pharmaceutical solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/60—Two oxygen atoms, e.g. succinic anhydride
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- 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]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
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- Communicable Diseases (AREA)
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- Engineering & Computer Science (AREA)
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Abstract
In-vitro experiments show that the compound has a remarkable inhibiting effect on the generation of Nitric Oxide (NO) in RAW264.7 macrophages induced by Lipopolysaccharide (LPS), and further can be used for treating novel coronavirus pneumonia (COVID-19); the preparation method of 10- (R) -17-hydro-7-dehydroandrographolide provided by the invention can be used for preparing the compound with high purity in a large scale by adopting an industrial chromatographic technology.
Description
Technical Field
The invention relates to an andrographolide derivative 10- (R) -17-hydro-7-dehydroandrographolide and a preparation method and application thereof.
Background
Andrographolide is a labdane diterpenoid compound extracted from the whole herb of Andrographis paniculata (Burm.F.) Nees of Acanthaceae, has the effects of resisting bacteria, diminishing inflammation, detoxifying and the like, and is an important active natural product. Andrographolide and its derivatives have been made into various dosage forms (tablet, dripping pill, capsule, etc.) widely used in clinic, such as Xiyanping injection, Lianbizhi injection, Yanhuning injection, andrographolide tablet, etc., all showing good heat-clearing and detoxicating, antibacterial and anti-inflammatory effects in clinic. Luvone et al believe that Nitric Oxide (NO) is associated with both the development of acute and chronic inflammation [ Luvone T, Carnuccio R, Di RM.modulation of grandioloma formation endogenous nitrile oxide [ J ]. Eur J Pharmacol,1994,265(1/2):89-92 ]. The andrographolide can obviously reduce the expression of inflammatory factors NO, tumor necrosis factor-alpha (TNF-alpha) and Interleukin (IL) -6 in mouse macrophage RAW264.7 induced by lipopolysaccharide, thereby inhibiting inflammatory reaction.
The active ingredient of the Xiyanping injection which is widely used clinically at present is andrographolide general sulfonate which is a derivative of andrographolide and has the functions of clearing away heat and toxic materials, resisting bacteria and diminishing inflammation, but the active ingredient of Xiyanping is a sulfonated product of andrographolide and is not of a single structure, so that a small molecular compound of the single structure is adopted to further define a mechanism, and the injection has important significance on the medicine effect and the toxicological research thereof; although andrographolide compounds have achieved some research results, there are no new andrographolide derivatives or analogues that are particularly effective in the clinical setting for a variety of diseases, particularly in the anti-inflammatory field. Therefore, the development of new andrographolide monomer drugs with anti-inflammatory activity has important social significance.
Disclosure of Invention
The invention provides a novel andrographolide derivative 10- (R) -17-hydro-7-dehydroandrographolide (formula I) with anti-inflammatory activity, a preparation method thereof and application thereof in preparing medicaments for treating inflammatory diseases.
In a first aspect of the present invention, there is provided a compound of formula (I) 10- (R) -17-hydro-7-dehydroandrographolide,
in a second aspect of the present invention, a method for preparing a compound of formula (i) 10- (R) -17-hydro-7-dehydroandrographolide, comprises the following steps:
(1) dissolving andrographolide sulfonate in water, loading onto macroporous adsorbent resin column, eluting with mixed solvent of organic solvent and water, mixing eluates containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating to obtain crude product 1;
(2) dissolving the crude product 1 with mixed organic solvent (A/B), loading onto silica gel column, eluting with mixed organic solvent (A/B), mixing eluates containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating to obtain crude product 2;
(3) dissolving the crude product 2 with a mixed solvent of an organic solvent and water, loading the solution on an HPLC preparative column, performing gradient elution with the mixed solvent of the organic solvent and the water, detecting the separation condition with an ultraviolet on-line detector, determining the start and stop time of eluent collection according to the peak appearance time and the chromatographic peak height, combining the eluates containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating.
Preferably, in the step (1), the macroporous adsorption resin is styrene type macroporous adsorption resin;
more preferably, the styrene type macroporous adsorbent resin is HPD-100S, D101S or LX-1180.
Preferably, in the step (1), the organic solvent is an alcohol, such as methanol or ethanol.
Preferably, the mixed solvent of the organic solvent and water is 0% to 70% (V/V) methanol-water solution (preferably 20% to 60% (V/V) methanol-water solution) or 0% to 70% (V/V) ethanol-water solution (preferably 20% to 60% (V/V) ethanol-water solution).
Preferably, in the step (1), the elution mode is gradient elution, such as elution sequentially with 20%, 40%, 60% ethanol-water solution by volume, or elution sequentially with 20%, 35%, 45%, 60% ethanol-water solution by volume, or elution sequentially with 20%, 35%, 50%, 60% methanol-water solution by volume.
Preferably, in the step (1), the concentration is a concentration under reduced pressure.
Preferably, in the step (1), the concentration temperature is 40-50 ℃.
Preferably, in the step (2), in the mixed organic solvent (a/B), a is one or more of petroleum ether or cyclohexane, and B is one or more of ethyl acetate, dichloromethane, chloroform or acetone;
more preferably, the mixed organic solvent (A/B) is preferably a petroleum ether/acetone solution;
more preferably, the mixed organic solvent (A/B) used in the dissolving process is a petroleum ether/acetone solution with the ratio of 5: 1-4: 1 (V/V); the mixed organic solvent (A/B) used in elution is a petroleum ether/acetone solution of 5:1 to 1:1(V/V) (preferably 4:1 to 1:1 (V/V)).
Preferably, in the step (2), the elution mode is gradient elution, such as elution with 4:1, 3:1, 2:1, 1:1(V/V) petroleum ether acetone solution in sequence.
Preferably, in the step (2), the concentration is preferably concentration under reduced pressure.
Preferably, in the step (2), the concentration temperature is preferably 40-50 ℃.
Preferably, in the step (3), the HPLC preparative column packing is reversed phaseC18The filler is preferably XAquA, ODS-A or ODS-AQ.
Preferably, in the step (3), the detection wavelength of the ultraviolet online detector is 200-300 nm.
Preferably, in the step (3), the organic solvent is methanol or acetonitrile;
more preferably, the mixed solvent of the organic solvent and water used in the dissolving process is 5-15% (V/V) methanol aqueous solution or 5-15% (V/V) acetonitrile aqueous solution; the mixed solvent of the organic solvent and water adopted during elution is 20-60% (V/V) methanol water solution or 20-60% (V/V) acetonitrile water solution.
Preferably, in the step (3), the concentration is a concentration under reduced pressure.
Preferably, in the step (3), the concentration temperature is 40-50 ℃.
In a third aspect of the present invention, a pharmaceutical preparation containing 10- (R) -17-hydro-7-dehydroandrographolide as the active ingredient is provided, wherein the preparation type includes but is not limited to injection, tablet, capsule or dispersible tablet.
In a fourth aspect of the present invention, there is provided the use of the above 10- (R) -17-hydro-7-dehydroandrographolide or the above pharmaceutical preparation in the preparation of a medicament for treating inflammatory diseases; the inflammatory disease is preferably pneumonia; the pneumonia is preferably novel coronavirus pneumonia; the novel coronavirus pneumonia is preferably COVID-19.
The invention has the beneficial technical effects
1. Provides a brand-new chiral 10- (R) -17-hydrogen-7-dehydroandrographolide compound shown in formula (I), which has proved to have better effect of treating inflammatory diseases; further, it can be used for treating pneumonia, such as novel coronavirus pneumonia (COVID-19).
2. The preparation method of 10- (R) -17-hydro-7-dehydroandrographolide has the advantages of convenient operation and high yield, and can be used for preparing a large amount of high-purity compound by adopting an industrial chromatographic technology and being used for industrial large-scale production.
Drawings
FIG. 1 is the NMR spectrum of 10- (R) -17-hydro-7-dehydroandrographolide of example 2;
FIG. 2 is the NMR carbon spectrum of 10- (R) -17-hydro-7-dehydroandrographolide of example 2;
FIG. 3 is an HPLC chromatogram of 10- (R) -17-hydro-7-dehydroandrographolide nuclei from example 2;
FIG. 4: the anti-inflammatory activity of 10- (R) -17-hydro-7-dehydroandrographolide of example 2 was tested.
Detailed Description
The chemical structural formula of 10- (R) -17-hydro-7-dehydroandrographolide indicated in the following examples (the Arabic numerals in the structure are the positions of carbon atoms in the chemical structure):
example 1: preparation of andrographolide general sulfonate
Taking 50L of absolute ethyl alcohol, placing the absolute ethyl alcohol in a reaction kettle, slowly adding 20L of concentrated sulfuric acid, stirring uniformly, adding 50.00kg of andrographolide, stirring, and standing at normal temperature for 72 hours. Controlling temperature, adding 50L 95% ethanol, stirring, adding 50% sodium hydroxide solution, adjusting pH to 7.0, adding ethanol until the alcohol content is 85%, standing for 24 hr, filtering, recovering ethanol from filtrate, concentrating into soft extract, and vacuum drying to obtain andrographolide total sulfonate.
Example 2: preparation of 10- (R) -17-hydro-7-dehydroandrographolide
Dissolving 200.01g andrographolide sulfonate in purified water, loading onto D101S macroporous adsorbent resin column, sequentially eluting with 20%, 40% and 60% ethanol-water solution, mixing eluates containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating at 50 deg.C under reduced pressure to obtain crude product 1;
dissolving the crude product 1 with a proper amount of 5:1(V/V) petroleum ether/acetone, loading the solution into a silica gel column, eluting the silica gel column with 4:1, 3:1, 2:1 and 1:1(V/V) petroleum ether/acetone solutions in sequence, collecting eluent, combining elution fractions containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating the elution fractions at 50 ℃ under reduced pressure to obtain a crude product 2;
dissolving the crude product 2 with A proper amount of 15% methanol-water solution, performing HPLC preparation (ODS-A, 10 μm filler), performing gradient elution with 20%, 35%, 45% and 60% methanol-water solution by volume ratio, receiving fractions according to absorption peak height and peak shape under 225nm wavelength detection condition, and concentrating under reduced pressure at 50 deg.C to obtain 3.21g of 10- (R) -17-hydro-7-dehydroandrographolide with purity of 95.05%.
The chemical structure of the compound 10- (R) -17-hydrogen-7-dehydroandrographolide is identified by modern spectral techniques such as NMR and ESI MS, the absolute configuration of the compound is determined by ECD calculation, and the physicochemical properties are as follows:
white powder with molecular formula of C20H30O5；
High resolution mass spectrum HRESIMS M/z373.1995[ M + Na ]]+(cald.for.C20H30O5Na,373.1991)。
Hydrogen spectrum of nuclear magnetic resonance1H-NMR(400MHZ) And nuclear magnetic resonance carbon spectrum13C-NMR(100MHZ) See fig. 1 and 2, and the data is shown in table 1. The nuclear HPLC assay is shown in fig. 3.
TABLE 110 Hydrogen and carbon Spectrum data (400/100MHz, DMSO) for (R) -17-hydro-7-dehydroandrographolide
Example 3: preparation of 10- (R) -17-hydro-7-dehydroandrographolide
Dissolving 199.97g andrographolide sulfonate in purified water, loading onto HPD100S macroporous adsorbent resin column, sequentially eluting with 20%, 35%, 50%, and 60% methanol-water solution, mixing eluates containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating at 40 deg.C under reduced pressure to obtain crude product 1;
dissolving the crude product 1 with a proper amount of 4:1(V/V) petroleum ether/acetone solution, loading the solution into a silica gel column, eluting with 4:1, 3:1, 2:1 and 1:1(V/V) petroleum ether/acetone solutions in sequence, collecting eluent, combining elution fractions containing 10- (R) -17-hydrogen-7-dehydro-andrographolide, and concentrating and drying at 40 ℃ under reduced pressure to obtain a crude product 2;
dissolving the crude product 2 with a proper amount of 5% methanol-water solution, performing HPLC preparation (ODS-AQ, 10 μm filler), performing gradient elution with 20%, 40%, 50% and 60% methanol-water solution by volume ratio, receiving fractions according to absorption peak height and peak shape under 225nm wavelength detection condition, and concentrating under reduced pressure at 40 deg.C to obtain 3.27g of 10- (R) -17-hydro-7-dehydroandrographolide with a purity of 94.87%.
Example 4: preparation of 10- (R) -17-hydro-7-dehydroandrographolide
Dissolving 200.15g of andrographolide total sulfonate in a proper amount of purified water, loading the dissolved andrographolide total sulfonate on an LX-1180 macroporous adsorption resin column, sequentially eluting with 20%, 35%, 45% and 60% ethanol-water solution by volume, combining elution fractions containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating the elution fractions under reduced pressure at 50 ℃ to obtain a crude product 1;
dissolving the crude product 1 with a proper amount of 5:1(V/V) petroleum ether-acetone, loading the solution into a silica gel column, eluting with 4:1, 3:1, 2:1 and 1:1(V/V) petroleum ether/acetone solutions in sequence, collecting eluent, combining elution fractions containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating and drying at 40 ℃ under reduced pressure to obtain a crude product 2;
dissolving the crude product 2 with A proper amount of 5% acetonitrile-water solution, performing HPLC preparation (ODS-A, 10 μm filler), performing gradient elution with 20%, 35%, 45% and 60% acetonitrile-water solution by volume ratio, receiving fractions according to absorption peak height and peak pattern under the detection condition of 225nm wavelength, and concentrating under reduced pressure at 50 ℃ to obtain 3.08g of 10- (R) -17-hydro-7-dehydroandrographolide with the purity of 94.93%.
Example 5: preparation of 10- (R) -17-hydro-7-dehydroandrographolide
Dissolving 199.25g of andrographolide total sulfonate in a proper amount of purified water, loading the dissolved andrographolide total sulfonate on an LX-1180 macroporous adsorption resin column, sequentially eluting with 20%, 35%, 45% and 60% ethanol-water solution by volume, combining elution fractions containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating the elution fractions under reduced pressure at 40 ℃ to obtain a crude product 1;
dissolving the crude product 1 with a proper amount of 4:1(V/V) petroleum ether/acetone solution, loading the solution into a silica gel column, eluting with 4:1, 3:1, 2:1 and 1:1(V/V) petroleum ether/acetone solutions in sequence, collecting eluent, combining elution fractions containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating and drying at 50 ℃ under reduced pressure to obtain a crude product 2;
dissolving the crude product 2 with a proper amount of 15% acetonitrile-water solution, performing HPLC preparation (XAqua, 10 mu m filler), performing gradient elution by using 20%, 30%, 40% and 60% acetonitrile-water solution in volume ratio, receiving fractions according to the absorption peak height and peak shape under the detection condition of 225nm wavelength, and performing reduced pressure concentration at 40 ℃ to obtain 3.15g of 10- (R) -17-hydro-7-dehydroandrographolide with the purity of 95.01%.
Example 6: preparation of 10- (R) -17-hydro-7-dehydroandrographolide
Taking 20.08kg of andrographolide total sulfonate, adding a proper amount of purified water to dissolve, loading the andrographolide total sulfonate on a D101S macroporous adsorption resin column, sequentially eluting with 20% by volume, 40% by volume and 60% ethanol-water solution, combining elution fractions containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating and drying at 50 ℃ under reduced pressure to obtain a crude product 1;
dissolving the crude product 1 with a proper amount of 5:1(V/V) petroleum ether/acetone, then purifying the solution by using an industrial chromatographic system (DAC-HB300 dynamic axial compression column and silica gel packing), eluting the solution by using 4:1, 3:1, 2:1 and 1:1(V/V) petroleum ether/acetone solutions in sequence, collecting eluent, combining elution fractions containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating and drying the elution fractions at 40 ℃ under reduced pressure to obtain a crude product 2;
dissolving the crude product 2 with A proper amount of 15% methanol-water solution, performing industrial chromatographic HPLC preparation (DAC-HB150 dynamic axial compression column ODS-A, 10 μm filler), performing gradient elution by using 20%, 40%, 50% and 60% methanol-water solution in volume ratio, receiving fractions according to the absorption peak height and peak shape under the detection condition of 225nm wavelength, and concentrating under reduced pressure at 40 ℃ to obtain 335.19g of 10- (R) -17-hydrogen-7-dehydroandrographolide with the purity of 95.13%.
Example 7: anti-inflammatory activity test of 10- (R) -17-hydro-7-dehydroandrographolide
The monomeric compound (10- (R) -17-hydro-7-dehydroandrographolide) acts on RAW264.7 mouse macrophage induced by Lipopolysaccharide (LPS), the level of NO in the culture supernatant is detected by a Griess reagent color development method, and the anti-inflammatory activity of the compound is evaluated by taking the capability of the tested drug for inhibiting NO release as a screening index.
1. Preparation of pharmaceutical solutions
The monomer compounds were dissolved in DMSO to prepare solutions of 1.564, 3.125, 6.250, 12.50, 25.00. mu.g/ml.
2. Test method
(1) Preparing single mouse macrophage suspension with 10% fetal calf serum culture solution, inoculating log-phase mouse macrophage RAW264.7 into 96-well plate, 10 per well5Cell number, 3 multiple wells were set.
(2) After 24 hours incubation, different concentrations of test drug were added to each well along with 1ug/mL LPS.
(3) After culturing at 37 ℃ for 24 hours, 100ul of Griess solution was added to each well and the culture was continued for 5 minutes, and the culture was stopped.
(4) The Optical Density (OD) value at a wavelength of 570m was measured by an enzyme-linked immunosorbent assay (ELISA) apparatus, and the NO inhibition rate was calculated.
3. Results of the experiment
The NO inhibition effect IC50 of the monomeric compound (10- (R) -17-hydro-7-dehydroandrographolide) on RAW264.7 cells is 6.237 mu g/ml. The results are shown in FIG. 4.
The result shows that the compound has obvious inhibition effect on the generation of mouse macrophage RAW264.7 NO induced by Lipopolysaccharide (LPS), and the compound has obvious anti-inflammatory activity, so that the compound can be used for preparing a novel anti-inflammatory active medicament. Further, the monomeric compound (10- (R) -17-hydro-7-dehydroandrographolide) can be used for treating novel coronavirus pneumonia (such as COVID-19).
Claims (7)
1.10- (R) -17-hydrogen-7-dehydroandrographolide compound (I),
the preparation method of the compound of formula (I) 10- (R) -17-hydro-7-dehydroandrographolide, which is characterized by comprising the following steps:
(1) dissolving andrographolide sulfonate in water, loading onto macroporous adsorbent resin column, eluting with mixed solvent of organic solvent and water, mixing eluates containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating to obtain crude product 1;
(2) dissolving the crude product 1 with mixed organic solvent (A/B), loading onto silica gel column, eluting with mixed organic solvent (A/B), mixing eluates containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating to obtain crude product 2;
(3) dissolving the crude product 2 with a mixed solvent of an organic solvent and water, loading the solution on an HPLC preparative column, performing gradient elution with the mixed solvent of the organic solvent and the water, detecting the separation condition with an ultraviolet on-line detector, determining the start and stop time of eluent collection according to the peak appearance time and the chromatographic peak height, combining the eluates containing 10- (R) -17-hydro-7-dehydroandrographolide, and concentrating.
3. The production method according to claim 2, wherein in the step (1),
the macroporous adsorption resin is styrene type macroporous adsorption resin; preferably, the styrene type macroporous adsorption resin is HPD-100S, D101S or LX-1180;
and/or, the organic solvent is an alcohol, such as methanol or ethanol;
and/or the mixed solvent of the organic solvent and the water is 0-70% (V/V) methanol-water solution or 0-70% (V/V) ethanol-water solution;
and/or, the elution mode is gradient elution;
and/or, concentrating to vacuum concentration;
and/or the concentration temperature is 40-50 ℃.
4. The production method according to claim 2, wherein in the step (2),
in the mixed organic solvent (A/B), A is one or more of petroleum ether or cyclohexane, B is one or more of ethyl acetate, dichloromethane, chloroform or acetone; preferably, the mixed organic solvent (A/B) is a petroleum ether/acetone solution; more preferably, the mixed organic solvent (A/B) used in the dissolving process is a petroleum ether/acetone solution with the ratio of 5: 1-4: 1 (V/V); the mixed organic solvent (A/B) adopted during elution is 5: 1-1: 1(V/V) petroleum ether/acetone solution;
and/or, the elution mode is gradient elution;
and/or, concentrating to vacuum concentration;
and/or the concentration temperature is 40-50 ℃.
5. The production method according to claim 2, wherein, in the step (3),
HPLC preparative column packing of reversed phase C18A filler, preferably XAquA, ODS-A or ODS-AQ;
and/or the detection wavelength of the ultraviolet online detector is 200-300 nm;
and/or the organic solvent is methanol or acetonitrile; preferably, the mixed solvent of the organic solvent and water used in the dissolving process is 5-15% (V/V) methanol aqueous solution or 5-15% (V/V) acetonitrile aqueous solution; the mixed solvent of organic solvent and water is 20-60% (V/V) methanol water solution or 20-60% (V/V) acetonitrile water solution;
and/or, concentrating to vacuum concentration;
and/or the concentration temperature is 40-50 ℃.
6. A pharmaceutical preparation containing 10- (R) -17-hydro-7-dehydroandrographolide as active ingredient is provided, and the preparation is in the form of injection, tablet, capsule or dispersible tablet.
7. Use of 10- (R) -17-hydro-7-dehydroandrographolide according to claim 1 or prepared by a method according to any one of claims 2 to 5 or a pharmaceutical formulation according to claim 6 for the manufacture of a medicament for the treatment of an inflammatory disease; the inflammatory disease is preferably pneumonia; the pneumonia is preferably novel coronavirus pneumonia; the novel coronavirus pneumonia is preferably COVID-19.
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| CN108148022A (en) * | 2016-12-02 | 2018-06-12 | 上海迪诺医药科技有限公司 | Andrographolide class compound, its pharmaceutical composition and application |
| CN108392478A (en) * | 2017-02-07 | 2018-08-14 | 江西青峰药业有限公司 | Application of the andrographolide class compound in terms of preparing for radioactive damage drug |
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| CN103145657A (en) * | 2012-04-12 | 2013-06-12 | 江西青峰药业有限公司 | 17-hydro-9-dehydroandrographolide compound and its preparation method and use in drug preparation |
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