阅读说明：本技术 一种穿心莲内酯衍生物及其工业色谱制备方法和应用 (Andrographolide derivative and industrial chromatographic preparation method and application thereof ) 是由 王章伟 郭正友 刘地发 张毅 刘尧奇 方礼 刘芳芳 周舟 王振 周鹏 谢标鹏 于 2020-05-08 设计创作，主要内容包括：本发明的10-(R)-17-氢-7-去氢穿心莲内酯,体外实验表明该化合物对脂多糖(LPS)诱导的RAW264.7巨噬细胞中一氧化氮(NO)的生成具有显著的抑制作用,进一步,可用于治疗新型冠状病毒肺炎(COVID-19)；本发明提供的10-(R)-17-氢-7-去氢穿心莲内酯制备方法,可采用工业色谱技术大量制备高纯度该化合物。(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.)

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.

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(400MH_Z) And nuclear magnetic resonance carbon spectrum¹³C-NMR(100MH_Z) 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).