CN113620914B - 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 31
- 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 29
- 206010035664 Pneumonia Diseases 0.000 claims abstract description 9
- 241000711573 Coronaviridae Species 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 52
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 43
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 30
- 239000012043 crude product Substances 0.000 claims description 28
- 238000010828 elution Methods 0.000 claims description 25
- 239000003960 organic solvent Substances 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 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 17
- 239000003208 petroleum Substances 0.000 claims description 17
- 238000011068 loading method Methods 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000003480 eluent Substances 0.000 claims description 13
- 239000011347 resin Substances 0.000 claims description 13
- 229920005989 resin Polymers 0.000 claims description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000012046 mixed solvent Substances 0.000 claims description 12
- 238000001179 sorption measurement Methods 0.000 claims description 10
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 claims description 10
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 9
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 8
- 238000004090 dissolution Methods 0.000 claims description 8
- 239000000945 filler Substances 0.000 claims description 8
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- 239000000741 silica gel Substances 0.000 claims description 7
- 229910002027 silica gel Inorganic materials 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 4
- 239000003826 tablet Substances 0.000 claims description 4
- 239000003463 adsorbent Substances 0.000 claims description 3
- 239000002775 capsule Substances 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 239000007919 dispersible tablet Substances 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 239000000825 pharmaceutical preparation Substances 0.000 claims 4
- 238000004519 manufacturing process Methods 0.000 claims 1
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 abstract description 22
- 150000001875 compounds Chemical class 0.000 abstract description 15
- 239000002158 endotoxin Substances 0.000 abstract description 8
- 229920006008 lipopolysaccharide Polymers 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 6
- 210000002540 macrophage Anatomy 0.000 abstract description 6
- 208000025721 COVID-19 Diseases 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 3
- 238000004587 chromatography analysis Methods 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 17
- 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
- 238000001035 drying Methods 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000008213 purified water Substances 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 208000027866 inflammatory disease Diseases 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 244000118350 Andrographis paniculata Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 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
- 210000004027 cell Anatomy 0.000 description 2
- 238000007906 compression Methods 0.000 description 2
- 230000006835 compression Effects 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
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 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
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000004432 carbon atom Chemical group C* 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
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram 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
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000033687 granuloma formation Effects 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 150000001761 labdane diterpenoid derivatives 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
- 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
- 238000000746 purification Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012306 spectroscopic technique Methods 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
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- 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
-
- 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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- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Virology (AREA)
- Pulmonology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The 10- (R) -17-hydrogen-7-dehydroandrographolide disclosed by the invention has an obvious inhibition effect on Nitric Oxide (NO) generation in RAW264.7 macrophages induced by Lipopolysaccharide (LPS), and can be further used for treating novel coronavirus pneumonia (COVID-19); the preparation method of the 10- (R) -17-hydrogen-7-dehydroandrographolide provided by the invention can be used for preparing a large amount of high-purity compounds by adopting an industrial chromatographic technique.
Description
Technical Field
The invention relates to andrographolide derivative 10- (R) -17-hydrogen-7-dehydroandrographolide, and a preparation method and application thereof.
Background
Andrographolide is a labdane diterpenoid extracted from whole plant of Andrographis paniculata Nees of Acanthaceae [ Andrographis paniculata (Burm. F.) ], has antibacterial, antiinflammatory, and toxic materials removing effects, and is an important active natural product. Andrographolide and its derivatives have been prepared into various dosage forms (such as tablet, dripping pill, capsule, etc.) for clinical application, such as Xikiping injection, lianbizhi injection, yanhuning injection, andrographolide tablet, etc., and have been shown to have good effects of clearing heat, detoxicating, resisting bacteria, and diminishing inflammation in clinic. Luvone et al recognized that Nitric Oxide (NO) is associated with the occurrence of both acute and chronic inflammation [ Luvone T, carnuccio R, di RM. Modulation of granuloma formation endogenous nitric oxide [ J ]. Eur J Pharmacol,1994,265 (1/2): 89-92 ]. Andrographolide can obviously down regulate the expression of inflammatory factors NO, tumor necrosis factor-alpha (TNF-alpha) and Interleukin (IL) -6 in lipopolysaccharide-induced mouse macrophage RAW264.7, thereby inhibiting inflammatory response.
The effective component of the existing camptothecine injection widely used clinically is andrographolide total sulfonate, is andrographolide derivative, has the functions of clearing heat and detoxicating, resisting bacteria and diminishing inflammation, and the like, but the active component of camptothecine is the sulfonated product of andrographolide and is not in a single structure, so that a small molecular compound with a single structure is adopted to further define a mechanism, and has important significance for the research on the efficacy and toxicology of the camptothecine; although andrographolide compounds have achieved some research results, there is still no new and particularly effective andrographolide derivative or analogue for clinical use in a variety of diseases, especially in the anti-inflammatory field. Therefore, the development of a novel andrographolide monomer medicament with anti-inflammatory activity has important social significance.
Disclosure of Invention
The invention provides a novel andrographolide derivative 10- (R) -17-hydrogen-7-dehydroandrographolide (formula I) with anti-inflammatory activity, a preparation method thereof and application thereof in preparing medicines for inflammatory diseases.
In a first aspect of the invention, there is provided a compound of formula (I) of 10- (R) -17-hydro-7-dehydroandrographolide,
in a second aspect, the present invention provides a method for preparing a compound of formula (i) 10- (R) -17-hydro-7-dehydroandrographolide, comprising the steps of:
(1) Dissolving andrographolide total sulfonate in water, loading onto macroporous adsorbent resin column, eluting with mixed solvent of organic solvent and water, mixing eluates containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating to obtain crude product 1;
(2) Dissolving the crude product 1 with a mixed organic solvent (A/B), loading the mixture on a silica gel column, eluting with the mixed organic solvent (A/B), combining eluates containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating to obtain a crude product 2;
(3) Dissolving crude product 2 with mixed solvent of organic solvent and water, loading onto HPLC preparation column, gradient eluting with mixed solvent of organic solvent and water, detecting separation condition with ultraviolet on-line detector, determining time for collecting eluate according to peak time and chromatographic peak height, mixing eluents containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating.
Preferably, in the step (1), the macroporous adsorption resin is styrene 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 is performed by gradient elution, for example, by sequentially using 20%, 40%, 60% ethanol-water solution, or sequentially using 20%, 35%, 45%, 60% ethanol-water solution, or sequentially using 20%, 35%, 50%, 60% methanol-water solution.
Preferably, in the step (1), the concentration is reduced pressure concentration.
Preferably, in the step (1), the concentration temperature is 40 to 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;
further preferably, the mixed organic solvent (A/B) used in the dissolution is a petroleum ether/acetone solution of 5:1-4:1 (V/V); the mixed organic solvent (A/B) used in the elution is a petroleum ether/acetone solution of 5:1-1:1 (V/V), preferably 4:1-1:1 (V/V).
Preferably, in the step (2), the elution is performed by gradient elution, such as elution with a petroleum ether acetone solution of 4:1, 3:1, 2:1, and 1:1 (V/V) in sequence.
Preferably, in the step (2), the concentration is preferably reduced pressure concentration.
Preferably, in the step (2), the concentration temperature is preferably 40 to 50 ℃.
Preferably, in the step (3), the HPLC preparation column packing is reversed phase C 18 The filler is more preferably Xaquse:Sup>A, ODS-A or ODS-AQ.
Preferably, in the step (3), the detection wavelength of the ultraviolet on-line detector is 200 to 300nm.
Preferably, in the step (3), the organic solvent is methanol or acetonitrile;
more preferably, the mixed solvent of the organic solvent and water adopted in the dissolution 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 reduced pressure concentration.
Preferably, in the step (3), the concentration temperature is 40 to 50 ℃.
In a third aspect of the present invention, there is provided a pharmaceutical formulation comprising 10- (R) -17-hydro-7-dehydroandrographolide as a pharmaceutically active ingredient, of the type including but not limited to injection, tablet, capsule or dispersible tablet.
In a fourth aspect, the present invention provides the use of the above-described 10- (R) -17-hydro-7-dehydroandrographolide or the above-described pharmaceutical formulation for the preparation of a medicament for the treatment of inflammatory diseases; the inflammatory disease is preferably pneumonia; the pneumonia is preferably novel coronavirus pneumonia; the novel coronavirus pneumonia is preferably covd-19.
The beneficial technical effects of the invention
1. Provides a novel chiral 10- (R) -17-hydrogen-7-dehydroandrographolide compound, which has better effect of treating inflammatory diseases through verification; further, it can be used for treating pneumonia such as novel coronavirus pneumonia (COVID-19).
2. The preparation method of the 10- (R) -17-hydrogen-7-dehydroandrographolide is convenient to operate and high in yield, and can be used for preparing a large amount of high-purity compounds by adopting an industrial chromatographic technique and is used for industrial large-scale production.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of 10- (R) -17-hydro-7-dehydroandrographolide according to example 2;
FIG. 2 is a nuclear magnetic resonance carbon spectrum of 10- (R) -17-hydro-7-dehydroandrographolide according to example 2;
FIG. 3 is a HPLC detection chart of 10- (R) -17-hydro-7-dehydroandrographolide nuclear in 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 (Arabic numerals in the structure are the index of carbon atoms in the chemical structure) referred to in the examples below:
example 1: preparation of andrographolide total sulfonate
Taking 50L of absolute ethyl alcohol, placing the absolute ethyl alcohol into a reaction kettle, slowly adding 20L of concentrated sulfuric acid, uniformly stirring, adding 50.00kg of andrographolide, stirring, and standing at normal temperature for 72 hours. Adding 50L of 95% ethanol at controlled temperature, stirring, adding 50% sodium hydroxide solution to adjust pH to 7.0, adding ethanol to ethanol content of 85%, standing for 24 hr, filtering, recovering ethanol from filtrate, concentrating into soft extract, and vacuum drying to obtain total sulfonate of andrographolide.
Example 2: preparation of 10- (R) -17-hydrogen-7-dehydroandrographolide
Taking 200.01g of total sulfonate of andrographolide, adding a proper amount of purified water for dissolution, loading the mixture on a D101S macroporous adsorption resin column, eluting the mixture by using ethanol-water solution with the volume ratio of 20%, 40% and 60%, merging eluting flow parts containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating the eluting flow parts 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, loading the solution on a sample silica gel column, eluting with 4:1, 3:1, 2:1 and 1:1 (V/V) petroleum ether/acetone solutions in sequence, collecting the eluent, combining the elution fractions containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating the eluent under reduced pressure at 50 ℃ to obtain a crude product 2;
dissolving the crude product 2 with 15% methanol-water solution, performing HPC preparation (ODS-A, 10 μm filler), gradient eluting with 20%, 35%, 45%, 60% methanol-water solution, and concentrating under reduced pressure at 50deg.C to obtain 10- (R) -17-hydrogen-7-dehydroandrographolide 3.21g with purity of 95.05%.
The chemical structure of the compound 10- (R) -17-hydrogen-7-dehydroandrographolide is identified by modern spectroscopic techniques such as NMR, ESI MS and the like, and the absolute configuration of the compound is determined by ECD calculation, and the physicochemical properties are as follows:
white powder with molecular formula of C 20 H 30 O 5 ；
High resolution mass spectrum HRESIMS M/z373.1995[ M+Na] + (cald.for.C 20 H 30 O 5 Na,373.1991)。
Nuclear magnetic resonance hydrogen spectrum 1 H-NMR(400MH Z ) Nuclear magnetic resonance carbon spectrum 13 C-NMR(100MH Z ) See fig. 1 and 2, and the data are shown in table 1. Core HThe PLC detection diagram is shown in FIG. 3.
TABLE 1 Hydrogen and carbon Spectrum data for 10- (R) -17-hydrogen-7-dehydroandrographolide (400/100 MHz, DMSO)
Example 3: preparation of 10- (R) -17-hydrogen-7-dehydroandrographolide
Taking 199.97g of total sulfonate of andrographolide, adding a proper amount of purified water for dissolution, loading the mixture on an HPD100S macroporous adsorption resin column, eluting with methanol-water solution with the volume ratio of 20%, 35%, 50% and 60%, combining the eluting flow containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating the combined flow at 40 ℃ under reduced pressure 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 on a sample silica gel column, eluting with 4:1, 3:1, 2:1 and 1:1 (V/V) petroleum ether/acetone solutions in sequence, collecting the eluent, combining the elution fractions containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating the eluent under reduced pressure at 40 ℃ 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 a volume ratio of 20%, 40%, 50% and 60% methanol-water solution, and concentrating under reduced pressure at 40deg.C to obtain 3.27g of 10- (R) -17-hydrogen-7-dehydroandrographolide with a purity of 94.87% according to absorption peak height and peak type receiving flow under 225nm wavelength detection condition.
Example 4: preparation of 10- (R) -17-hydrogen-7-dehydroandrographolide
Taking 200.15g of andrographolide total sulfonate, adding a proper amount of purified water for dissolution, loading the solution on an LX-1180 macroporous adsorption resin column, eluting with 20%, 35%, 45% and 60% ethanol-water solutions in sequence, combining elution fractions containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating and drying 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 mixture on a sample silica gel column, eluting with 4:1, 3:1, 2:1 and 1:1 (V/V) petroleum ether/acetone solutions in sequence, collecting the eluent, combining the elution fractions containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating the eluent at 40 ℃ under reduced pressure to obtain a crude product 2;
dissolving the crude product 2 with se:Sup>A proper amount of 5% acetonitrile-water solution, performing HPC preparation (ODS-A, 10 μm filler), performing gradient elution with se:Sup>A volume ratio of 20%, 35%, 45% and 60% acetonitrile-water solution, and concentrating and drying under reduced pressure at 50deg.C according to absorption peak height and peak type receiving flow under 225nm wavelength detection condition to obtain 3.08g of 10- (R) -17-hydrogen-7-dehydroandrographolide with purity of 94.93%.
Example 5: preparation of 10- (R) -17-hydrogen-7-dehydroandrographolide
Taking 199.25g of andrographolide total sulfonate, adding a proper amount of purified water for dissolution, loading the solution on an LX-1180 macroporous adsorption resin column, eluting with 20%, 35%, 45% and 60% ethanol-water solutions in sequence, combining elution fractions containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating and drying at 40 ℃ under reduced pressure 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 on a sample silica gel column, eluting with 4:1, 3:1, 2:1 and 1:1 (V/V) petroleum ether/acetone solutions in sequence, collecting the eluent, combining the elution fractions containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating the eluent under reduced pressure at 50 ℃ to obtain a crude product 2;
dissolving the crude product 2 with 15% acetonitrile-water solution, performing HPLC preparation (Xaqua, 10 μm filler), performing gradient elution with 20%, 30%, 40% acetonitrile-water solution by volume ratio, and concentrating under reduced pressure at 40deg.C to obtain 3.15g of 10- (R) -17-hydrogen-7-dehydroandrographolide with purity of 95.01% according to absorption peak height and peak type receiving flow under 225nm wavelength detection condition.
Example 6: preparation of 10- (R) -17-hydrogen-7-dehydroandrographolide
Taking 20.08kg of total sulfonate of andrographolide, adding a proper amount of purified water for dissolution, loading the solution on a D101S macroporous adsorption resin column, eluting with ethanol-water solution with the volume ratio of 20%, 40% and 60%, mixing the eluting flow containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating and drying under reduced pressure at 50 ℃ to obtain a crude product 1;
purifying the crude product 1 by using a proper amount of 5:1 (V/V) petroleum ether/acetone, loading the solution into an industrial chromatographic system for purification (DAC-HB 300 dynamic axial compression column and silica gel filler), eluting with 4:1, 3:1, 2:1 and 1:1 (V/V) petroleum ether/acetone solutions in sequence, collecting the eluent, combining the elution fractions containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating and drying at 40 ℃ under reduced pressure to obtain a crude product 2;
the crude product 2 is prepared by industrial chromatographic HPC after being dissolved by se:Sup>A proper amount of 15% methanol-water solution (DAC-HB 150 dynamic axial compression column ODS-A,10 μm filler), gradient elution is carried out by using methanol-water solution with volume ratio of 20%, 40%, 50% and 60%, the absorption peak height and peak type receiving flow under the detection condition of 225nm wavelength are carried out, and the pressure reduction concentration is carried out at 40 ℃ for drying, thus obtaining 335.19g of 10- (R) -17-hydrogen-7-dehydroandrographolide with purity of 95.13%.
Example 7: anti-inflammatory Activity assay of 10- (R) -17-hydro-7-dehydroandrographolide
The anti-inflammatory activity of the compounds was evaluated by applying a monomeric compound (10- (R) -17-hydro-7-dehydroandrographolide) to Lipopolysaccharide (LPS) -induced RAW264.7 mouse macrophages, detecting the NO level in the culture supernatant by a Griess reagent chromogenic method, and using the ability of the test drug to inhibit NO release as a screening index.
1. Preparation of pharmaceutical solutions
The monomer compounds were dissolved in DMSO to prepare 1.564, 3.125, 6.250, 12.50, 25.00. Mu.g/ml solutions.
2. Test method
(1) Single mouse macrophage suspension is prepared by using culture solution containing 10% of fetal bovine serum, taking log-phase mouse macrophage RAW264.7, inoculating into 96-well plate, 10% of each well 5 Cell number, 3 multiplex wells were set.
(2) After 24 hours of incubation, different concentrations of the test drug were added to each well with 1ug/mL LPS.
(3) After incubation at 37℃for 24 hours, 100ul of Griess solution was added to each well and incubation was continued for 5 minutes, and the incubation was stopped.
(4) Optical Density (OD) values at 570m wavelength were measured with an enzyme-linked immunosorbent assay and the NO inhibition was calculated.
3. Experimental results
The IC50 of the monomer compound (10- (R) -17-hydro-7-dehydroandrographolide) for inhibiting NO in RAW264.7 cells was 6.237 μg/ml. The results are shown in FIG. 4.
The results show that the compound has remarkable inhibition effect on the generation of Lipopolysaccharide (LPS) -induced mouse macrophage RAW264.7 NO, and the compound has remarkable anti-inflammatory activity, so that the compound can be used for preparing novel anti-inflammatory active medicaments. Furthermore, the monomeric compound (10- (R) -17-hydro-7-dehydroandrographolide) can be used for treating novel coronavirus pneumonia (such as COVID-19).
Claims (12)
1. An andrographolide derivative 10- (R) -17-hydrogen-7-dehydroandrographolide shown in formula (I):
2. a process for the preparation of andrographolide derivatives 10- (R) -17-hydro-7-dehydroandrographolide according to claim 1, characterized in that: the method comprises the following steps:
(1) Dissolving andrographolide total sulfonate in water, loading onto macroporous adsorbent resin column, eluting with mixed solvent of organic solvent and water, mixing eluates containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating to obtain crude product 1; the mixed solvent is 20-60% V/V methanol-water solution or 20-60% V/V ethanol-water solution;
(2) Dissolving the crude product 1 with a mixed organic solvent A/B, loading the solution on a silica gel column, eluting with the mixed organic solvent A/B, combining eluents containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating to obtain a crude product 2; the mixed organic solvent A/B adopted in the dissolution is a petroleum ether/acetone solution with the concentration of 5:1-4:1V/V; the mixed organic solvent A/B adopted during elution is a petroleum ether/acetone solution with the concentration of 5:1-1:1V/V;
(3) Dissolving the crude product 2 with a mixed solvent of an organic solvent and water, loading the solution on an HPLC (high performance liquid chromatography) preparation 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 time for collecting the eluent according to the peak time and the chromatographic peak height, merging the eluent containing 10- (R) -17-hydrogen-7-dehydroandrographolide, and concentrating; the organic solvent and water mixed solvent adopted in the dissolving process is 5-15% of V/V methanol aqueous solution or 5-15% of V/V acetonitrile aqueous solution; the mixed solvent of the organic solvent and water adopted during elution is 20-60% of methanol aqueous solution or 20-60% of acetonitrile aqueous solution.
3. The preparation method according to claim 2, characterized in that: in the step (1), the macroporous adsorption resin is styrene macroporous adsorption resin.
4. A method of preparation according to claim 3, characterized in that: the styrene type macroporous adsorption resin is HPD-100S, D101S or LX-1180.
5. The preparation method according to claim 2, characterized in that: in the step (1), the elution mode is gradient elution, and the concentration is reduced pressure concentration and the concentration temperature is 40-50 ℃.
6. The preparation method according to claim 2, characterized in that: in the step (2), the elution mode is gradient elution, the concentration is reduced pressure concentration and the concentration temperature is 40-50 ℃.
7. The preparation method according to claim 2, characterized in that: in the step (3), the HPLC preparation column packing is a reversed-phase C18 packing.
8. The method of manufacturing according to claim 7, wherein: the reversed phase C18 filler is Xaquse:Sup>A, ODS-A or ODS-AQ.
9. The preparation method according to claim 2, characterized in that: in the step (3), the detection wavelength of the ultraviolet on-line detector is 200-300 nm, and the concentration is reduced pressure concentration and the concentration temperature is 40-50 ℃.
10. A pharmaceutical preparation comprising the andrographolide derivative 10- (R) -17-hydro-7-dehydroandrographolide as a pharmaceutically active ingredient according to claim 1, characterized in that: the pharmaceutical preparation is an injection, a tablet or a capsule.
11. The pharmaceutical formulation according to claim 10, wherein: the pharmaceutical preparation is a dispersible tablet.
12. Use of an andrographolide derivative 10- (R) -17-hydro-7-dehydroandrographolide according to claim 1 or an andrographolide derivative 10- (R) -17-hydro-7-dehydroandrographolide prepared by the preparation method according to any one of claims 2 to 9, or a pharmaceutical preparation according to any one of claims 10 to 11, for the preparation of a medicament for the treatment of novel coronavirus pneumonia.
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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 |
| WO2016065264A1 (en) * | 2014-10-24 | 2016-04-28 | Biogen Ma Inc. | Diterpenoid derivatives and methods of use thereof |
| CN107973764A (en) * | 2016-10-24 | 2018-05-01 | 江西青峰药业有限公司 | Andrographolide class compound, its preparation method, pharmaceutical composition and application |
| 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 |
| WO2016065264A1 (en) * | 2014-10-24 | 2016-04-28 | Biogen Ma Inc. | Diterpenoid derivatives and methods of use thereof |
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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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