CN115594707A

CN115594707A - Aconite alkaloid derivative and its use in preparing anti-inflammatory medicine

Info

Publication number: CN115594707A
Application number: CN202110770738.6A
Authority: CN
Inventors: 肖凯; 刘小宇; 李晓洁
Original assignee: Sichuan University; West China Hospital of Sichuan University
Current assignee: Sichuan University; West China Hospital of Sichuan University
Priority date: 2021-07-07
Filing date: 2021-07-07
Publication date: 2023-01-13

Abstract

The invention provides a aconite alkaloid derivative and application thereof in preparing an anti-inflammatory medicament, belonging to the field of pharmacy. The aconite alkaloid derivative has a structure shown in formula I, has excellent anti-inflammatory activity, and can effectively inhibit LPS induced RAW264.7 cells generate NO, and RAW264.7 cells activated by LPS are effectively inhibited from generating inflammatory factors IL-6 and TNF-alpha; it can inhibit the expression of iNOS and NLRP3 activated by LPS and the activation of MAPK/NF-kB signal channel to play the role of anti-inflammation. In addition, the aconite alkaloid derivative can obviously reduce ear swelling, reduce inflammatory cell infiltration, play an in-vivo anti-inflammatory role and effectively treat the ear swelling in vivo; can remarkably reduce the levels of inflammatory factors TNF-alpha and IL-6 in serum of a general sepsis model mouse, and can effectively treat sepsis in vivo; can also obviously improve the pathological injury of the colon of the mouse induced by the DSS. The aconite alkaloid derivative provided by the invention has excellent anti-inflammatory activity and low toxicity, and has wide application prospect in preparing anti-inflammatory drugs.

Description

Aconite alkaloid derivative and application thereof in preparing anti-inflammatory drugs

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a monkshood alkaloid derivative and application thereof in preparing an anti-inflammatory medicament.

Background

Inflammation is a complex and necessary physiological response to biological, chemical or physical stimuli. In the acute phase of the inflammatory response, cells of the immune system migrate to the site of injury according to a specific program under the influence of soluble mediators such as cytokines, chemokines, and acute phase proteins. Depending on the extent of the injury, the acute phase response may be sufficient to resolve the injury and initiate the healing process, protecting the body from the insult. However, long-term exposure to stimuli, or inappropriate responses to inflammatory mediators produced excessively by the body itself, may cause shock and multiple organ functional impairment, and inflammation to enter a chronic phase, during which tissue damage and fibrotic inflammatory responses, including continuous release of inflammatory mediators, recruitment of activated circulating leukocytes at the site of inflammation, may occur, leading to further mediator release. Chronic inflammation has a major impact on arthritis, asthma, autoimmune diseases, atherosclerosis, diabetes, cancer and other diseases, as well as aging.

Current clinical approaches to treating inflammation focus primarily on inhibiting the production of proinflammatory mediators and on inhibiting the signaling pathway activated by proinflammatory cytokines. The use of the commonly used anti-inflammatory drugs steroidal anti-inflammatory drugs and cyclooxygenase inhibitors may cause serious side effects such as glucocorticoid-induced osteoporosis, induction of exacerbation infection, cushing's syndrome, non-steroidal anti-inflammatory drugs-induced gastrointestinal reactions, kidney damage, liver damage, and the like.

Therefore, it is of great significance to develop an anti-inflammatory agent having low toxicity and superior anti-inflammatory effect.

Disclosure of Invention

The invention aims to provide a aconite alkaloid derivative and application thereof in preparing anti-inflammatory drugs.

The invention provides a compound shown in a formula I, a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a deuterated compound thereof:

wherein R is ₁ Selected from hydrogen, hydroxyl, = O, C _1～6 Alkyl radical, C _1～6 An alkoxy group; r is ₃ Selected from hydrogen, LR _a 、LOR _a L is selected from none, C _1～4 Alkylene, R _a Selected from hydrogen, protecting groups, C _1～6 An alkyl group; or, R ₁ 、R ₃ Connecting to form a ring;

R ₂ selected from hydrogen, hydroxy, C _1～6 Alkyl radical, C _1～6 Alkoxy, OCOR _b ，R _b Selected from unsubstituted or halogenated C _1～6 An alkyl group;

R ₄ selected from hydrogen, hydroxy, C _1～6 Alkyl radical, C _1～6 An alkoxy group;

R ₅ selected from hydrogen, hydroxy, C _1～6 Alkyl radical, C _1～6 Alkoxy, = O, = NR _c 、OR _d ，R _c Selected from hydrogen, hydroxy, C _1～6 Alkyl radical, R _d Selected from hydrogen, protecting groups;

R ₆ selected from hydrogen, hydroxy, C _1～6 Alkyl radical, C _1～6 Alkoxy radical, COR _e ；

R ₇ Selected from hydrogen, hydroxy, C _1～6 Alkyl radical, C _1～6 Alkoxy radical, COR _e ；

R ₈ Selected from hydrogen, hydroxy, C _1～6 Alkyl radical, C _1～6 Alkoxy radical, COR _e ；

R ₉ Selected from hydrogen, hydroxy, C _1～6 Alkyl radical, C _1～6 Alkoxy radical, COR _e ；

R _e Selected from hydroxy, C _1～6 An alkyl group.

Further, the structure of the compound is shown as formula II:

wherein R is ₁ Selected from hydrogen, hydroxyl, = O, C _1～4 Alkyl radical, C _1～4 An alkoxy group; r is ₃ Selected from hydrogen, LR _a 、LOR _a L is selected from the group consisting of _1～2 Alkylene radical, R _a Selected from hydrogen, hydroxy protecting groups, C _1～4 An alkyl group; or, R ₁ 、R ₃ Connecting to form a ring;

R ₂ selected from hydrogen, hydroxy, C _1～4 Alkyl radical, C _1～4 Alkoxy, OCOR _b ，R _b Selected from unsubstituted or halogenated C _1～4 An alkyl group;

R ₄ selected from hydrogen, hydroxy, C _1～4 Alkyl radical, C _1～4 An alkoxy group;

R ₅ selected from hydrogen, hydroxy, C _1～4 Alkyl radical, C _1～4 Alkoxy group, = O, = NR _c 、OR _d ，R _c Selected from hydrogen, hydroxy, C _1～4 Alkyl radical, R _d Selected from hydrogen, hydroxyl protecting groups;

R ₆ selected from hydrogen, hydroxy, C _1～4 Alkyl radical, C _1～4 Alkoxy radical, COR _e ，R _e Selected from hydroxy, C _1～4 An alkyl group;

the hydroxyl protecting group is preferably a TBS group.

Further, the structure of the compound is shown as formula III:

wherein R is ₂ Selected from hydrogen, hydroxy, C _1～4 Alkyl radical, C _1～4 Alkoxy, OCOR _b ，R _b Selected from unsubstituted or halogenated C _1～4 An alkyl group.

Further, the structure of the compound is selected from:

wherein Ac is acetyl and Piv is pivaloyl.

The invention also provides an anti-inflammatory drug which is a preparation prepared by taking the compound, the pharmaceutically acceptable salt thereof, the stereoisomer thereof or the deuterated compound thereof as an active ingredient and adding pharmaceutically common auxiliary materials.

The invention also provides the application of the compound, the pharmaceutically acceptable salt, the stereoisomer or the deuterated compound thereof, or the compound C-1, the pharmaceutically acceptable salt, the stereoisomer or the deuterated compound thereof, or the compound A-19, the pharmaceutically acceptable salt, the stereoisomer or the deuterated compound thereof in preparing anti-inflammatory drugs;

further, the anti-inflammatory drug is a drug for preventing and/or treating nonspecific inflammation.

Further, the anti-inflammatory drug is a drug for preventing and/or treating ear swelling, sepsis, colitis.

Further, the anti-inflammatory agent is capable of inhibiting the production of NO, TNF- α, IL-6; and/or, the anti-inflammatory drug can inhibit iNOS, NLRP3 expression; and/or, the anti-inflammatory drug is capable of inhibiting activation of the MAPK/NF- κ B signaling pathway.

The invention also provides the use of the compound, the pharmaceutically acceptable salt thereof, the stereoisomer thereof or the deuterated compound thereof, or the compound C-1, the pharmaceutically acceptable salt thereof, the stereoisomer thereof or the deuterated compound thereof, or the compound A-19, the pharmaceutically acceptable salt thereof, the stereoisomer thereof or the deuterated compound thereof in the preparation of an NO generation inhibitor;

it is well known to those skilled in the art that high levels of NO are also associated with chronic inflammation (e.g. asthma) and acute inflammation, which may cause vascular damage, skin rashes and skin flushing problems. And NO production inhibitors can solve these problems. Drugs classified as inhibitors of NO production may reduce NO concentration, helping to stabilize patients. Inhibitors of NO production may prevent tissue damage associated with shock and inflammation, and protect the internal organs from potentially serious complications. For patients with eczema and rosacea, the NO production inhibitor helps to reduce inflammation and swelling, make the skin look clean and healthy, and possibly prevent complications such as vascular injury.

Definitions of terms used in relation to the present invention: unless otherwise indicated, the initial definitions provided for by a group or term herein apply to that group or term throughout the specification; for terms not specifically defined herein, the meanings that would be afforded to them by a person skilled in the art, in light of the disclosure and context, should be given.

The minimum and maximum values of the content of carbon atoms in hydrocarbon groups are indicated by a prefix, e.g. prefix C _a～b Alkyl represents any alkyl group containing "a" to "b" carbon atoms. E.g. C _1～6 Alkyl means a straight or branched chain alkyl group containing 1to 6 carbon atoms。

"deuterated compound" refers to a compound wherein one or more hydrogen atoms in the compound have been replaced by deuterium.

By "pharmaceutically acceptable" is meant that the carrier, diluent, excipient, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.

"salts" are acid and/or base salts of a compound or a stereoisomer thereof with inorganic and/or organic acids and/or bases, and also include zwitterionic (inner) salts, as well as quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound, or a stereoisomer thereof, may be obtained by appropriately (e.g., equivalentlymixing) a certain amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization.

The pharmaceutically acceptable salt in the present invention may be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salt of the compound.

Halogen means fluorine, chlorine, bromine or iodine.

TBS group is tert-butyl dimethyl silicon base, which is a hydroxyl protecting group.

Ac group is acetyl.

The Piv group is pivaloyl.

Compound A-1 of the present invention may be represented by

Can also be expressed as

Other compounds are also similar.

Experimental results show that the aconite alkaloid derivative provided by the invention has excellent anti-inflammatory activity, can effectively inhibit LPS (LPS) from inducing RAW264.7 cells to generate NO, effectively inhibits RAW264.7 cells activated by LPS from generating inflammatory factors IL-6 and TNF-alpha, and can be used for preparing anti-inflammatory drugs and NO generation inhibitors. The aconite alkaloid derivative can play an anti-inflammatory role by inhibiting the expression of iNOS and NLRP3 activated by LPS and the activation of MAPK/NF-kB signal channels.

The experimental result also shows that the aconite alkaloid derivative provided by the invention can obviously reduce ear swelling, reduce inflammatory cell infiltration, play an in-vivo anti-inflammatory role and effectively treat ear swelling in vivo; the aconite alkaloid derivative provided by the invention can obviously reduce the levels of inflammatory factors TNF-alpha and IL-6 in serum of a general sepsis model mouse, and can effectively treat sepsis in vivo; the aconite alkaloid derivative provided by the invention can also obviously improve the pathological damage of mouse colon induced by DSS.

The aconite alkaloid derivative provided by the invention has excellent anti-inflammatory activity and low toxicity, and has wide application prospect in preparation of anti-inflammatory drugs and NO generation inhibitors.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the NO concentration (A) in the culture supernatant of LPS-induced RAW264.7 cell inflammation model cells and the NO concentration (B) after treatment with an iNOS inhibitor.

FIG. 2 shows the effect of the synthetic derivatives of diterpene alkaloids from monkshood on the LPS-induced production of NO by RAW264.7 cells.

FIG. 3 shows the effect of the derivatives of diterpene alkaloids from monkshood on the production of NO by RAW264.7 cells induced by LPS.

FIG. 4 shows the effect of the synthetic derivatives of diterpene alkaloids from monkshood on LPS-induced NO production by RAW264.7 cells.

FIG. 5 shows the effect of the derivatives of diterpene alkaloids from monkshood on the production of NO by RAW264.7 cells induced by LPS.

FIG. 6 shows the effect of the derivatives of diterpene alkaloids from monkshood on the production of NO by RAW264.7 cells induced by LPS.

FIG. 7 shows the effect of synthetic derivatives of aconite diterpene alkaloids on the production of IL-6, TNF- α by RAW264.7 activated by LPS; wherein, compared with the negative control group, ^# P<0.05; compared with model group<0.05。

FIG. 8 is the cytotoxicity of aconite diterpene alkaloid derivatives.

FIG. 9 shows the effect of A-8 on LPS-induced expression of iNOS in RAW 264.7: (A) LPS stimulation is carried out for 4h; (B) LPS stimulation is carried out for 6h; (C) LPS stimulation is carried out for 24h; (D) the relative expression of iNOS protein was semi-quantitative.

FIG. 10 shows the effect of A-8 on LPS-induced iNOS enzyme activity: (A) LPS stimulation for 4h; and (B) stimulating by LPS for 24h.

FIG. 11 shows the effect of A-8 on LPS-induced p-ERK1/2, p-p38, p-JNK: (A) detecting a result by Western blot; (B) semi-quantitative relative protein expression.

FIG. 12 is a graph showing the effect of A-8 on LPS-induced p-p 65: (A) Western blot detection result; (B) semi-quantitative relative protein expression.

FIG. 13 is a graph of the effect of A-8 on LPS-induced NLRP 3: (A) detecting a result by Western blot; (B) semi-quantitative relative protein expression.

FIG. 14 is a graph of the efficacy of A-8 on a model of TPA-induced ear swelling in mice; wherein, compared with the control group, ^# P<0.05, P compared to TPA model group<0.05。

Fig. 15 is a H & E staining picture of mouse ear tissue: (A) a normal control group; (B) TPA model group; (C) A-8 (0.1 mg/ear) + TPA group; (D) A-8 (0.3 mg/ear) + TPA group; (E) A-8 (1 mg/ear) + TPA group; (F) dexamethasone (10 mg/kg) + TPA.

FIG. 16 is TNF- α and IL-6 in LPS-induced serum of mouse sepsis model; it is provided withIn (e), compared with the normal control group, ^# P<0.05; p compared to LPS model group<0.05。

FIG. 17 shows the CREA and BUN in LPS-induced mouse sepsis model serum; wherein, compared with a normal control group, ^# P<0.05; p compared to LPS model group<0.05。

Fig. 18 is LPS-induced mouse sepsis model lung tissue H & E: (a) a normal control group; (b) LPS model group; (c) dexamethasone + LPS group; (d) a-8 high dose + LPS group; (e) A-8 Low dose + LPS group.

Fig. 19 is LPS-induced mouse sepsis model kidney tissue H & E: (a) a normal control group; (b) LPS model group; (c) dexamethasone + LPS group; (d) a-8 high dose + LPS group; (e) A-8 Low dose + LPS group.

Figure 20 shows body weight and percentage change in body weight for each group.

Figure 21 is the disease activity index score (DAI) for each group.

Figure 22 is the visual intestinal mucosal injury score (CMDI) for each group.

FIG. 23 shows the colon length/weight ratio for each group.

Figure 24 is DSS-induced colon injury H & E staining: a) A normal control group; b) A set of DSS models; c) Mesalazine group (200 mg/kg); d) A-8 Low dose group (80 mg/kg); e) A-8 high dose group (200 mg/kg).

Figure 25 is a DSS-induced colon injury pathology score; wherein, compared with a normal control group, ^# P<0.05; p compared to LPS model group<0.05。

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.

Example 1 preparation of Compound A-8 of the invention

A-7 (500mg, 1.18mmol) is dissolved in 20mL dry dichloromethane under the protection of argon, the temperature is reduced to-78 ℃ and Et is added successively ₃ N(0.57mL,4.13mmol) and TBSOTf (0.68mL, 2.95mmol). After 20 minutes of reaction, TLC checked that the reaction was complete, and saturated aqueous ammonium chloride (20 mL) was added to quench the reaction. Extraction was performed with dichloromethane (3X 10 mL), the combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. Purification by column chromatography (petroleum ether/acetone =4:1to 1) gave a-8 (pale yellow oily liquid, 399mg, yield 52%). ¹ H NMR(400MHz,CDCl ₃ )δ4.27(t,J＝7.2Hz,1H),3.88–3.85(m,1H),3.69(d,J＝6.8Hz,1H),3.47–3.41(m,2H),3.36(d,J＝7.6Hz,7H),3.32–3.25(m,2H),2.91(q,J＝6.8Hz,2H),2.48–2.39(m,2H),2.15(d,J＝6.8Hz,1H),2.06(dd,J＝12.0,8.8Hz,1H),1.88(s,1H),1.82(dd,J＝12.0,7.2Hz,1H),1.78–1.67(m,3H),1.55–1.48(m,2H),1.05(t,J＝6.8Hz,3H),0.99(s,3H),0.92(s,9H),0.90(s,9H),0.10(s,3H),0.09(s,3H),0.05(s,3H),0.04(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ87.3,85.1,83.8,81.6,71.6,70.0,67.6,66.8,58.3,57.3,57.2,56.5,50.5,50.1,43.6,41.7,39.6,35.5,29.1,28.5,27.2,26.2,25.9,18.2,18.1,15.7,–3.7,–5.5；ESIMS m/z 652[M+H] ⁺ .

Example 2 preparation of Compound A-1 of the invention

To a solution of A-19 (500mg, 0.94mmol) in THF (10 mL) at-78 deg.C was added NaHMDS (2.0M in THF) (0.75mL, 1.50mmol), after 1h of reaction, a pre-cooled solution of Davis reagent (1.4 g, 5.36mmol) in THF (28 mL) was added, after 2h of reaction continued, camphorsulfonic acid (350mg, 1.51mmol) was added, after stirring for 10min, the reaction was quenched by addition of saturated aqueous ammonium chloride, 50mL of water was added, and the solution was basified to pH with aqueous ammonia>9, dichloromethane extraction (50 mL. Times.3), anhydrous Na ₂ SO ₄ The residue obtained by drying and concentration under reduced pressure was eluted by silica gel column chromatography (cyclohexane acetone/6. ¹ H NMR(400MHz,CDCl ₃ ):4.32(1H,d,J＝10.4Hz),3.99(1H,t,J＝3.2Hz),3.40(1H,t,J＝10.0Hz),3.38,3.34(each 3H,s),1.11(3H,t,J＝7.2Hz),0.89(9H,s),0.83(3H,s),0.07,0.05(each 3H,s)； ¹³ C NMR(100MHz,CDCl ₃ ):δ216.9,210.7,82.7,82.2,80.1,72.4,70.8,70.1,67.4,64.7,57.4,56.7,53.1,52.8,49.0,44.5,42.5,42.1,39.3,

36.8,27.5,25.8,24.1,18.1,13.9,–5.6；ESIMS m/z 550[M+H] ⁺ .

To compound A-14 (200mg, 0.364mmol) in CH ₂ Cl ₂ DMAP (10mg, 0.088mmol), pyridine (50uL, 0.546mmol) and bromoacetyl bromide (92uL, 1.092mmol) were sequentially added to a solution (6 mL), and after stirring at room temperature for 2 hours, the reaction mixture was poured into ice water and basified with ammonia to pH>9, dichloromethane extraction (30 mL. Times.3), anhydrous Na ₂ SO ₄ The residue obtained by drying and concentration under reduced pressure was eluted by silica gel column chromatography (cyclohexane acetone/8, 1) to give compound a-1 (white amorphous powder, 215mg, 88%). ¹ H NMR(400MHz,CDCl ₃ ):δ5.66(1H,dd,J＝7.6,2.4Hz),3.99,3.96(each 1H,ABq,J＝12.8Hz),3.95(1H,overlapped),3.60(1H,s),3.60(1H,s),3.44,3.40(each 1H,ABq,J＝10.4Hz),3.37,3.34(each 3H,s),1.14(3H,t,J＝7.2Hz),0.91(3H,s),0.90(9H,s),0.08,0.06(each 3H,s)；ESIMS m/z 670[M+H] ⁺ .

EXAMPLE 3 preparation of Compound A-15 of the invention

To compound A-14 (50mg, 0.091mmol) in CH ₂ Cl ₂ DMAP (5mg, 0.044mmol), pyridine (12uL, 0.145mmol), chloroacetyl chloride (11uL, 0.138mmol) were added to the solution (1.5 mL) in this order, and after stirring at room temperature for 2 hours, the reaction mixture was poured into ice water and basified with ammonia to pH>9 dichloromethane extraction (30 mL. Times.3), anhydrous Na ₂ SO ₄ The residue obtained by drying and concentration under reduced pressure was eluted by silica gel column chromatography (cyclohexane acetone/8. ¹ H NMR(400MHz,CDCl ₃ ):δ5.67(1H,dd,J＝7.6,2.4Hz),4.23,4.15(each 1H,ABq,J＝15.2Hz),3.95(1H,t,J＝3.6Hz),3.60(1H,s,3.60(1H,s),3.43,3.39(each 1H,ABq,J＝10.0Hz),3.35,3.32(each 3H,s),1.12(3H,t,J＝7.2Hz),0.88(9H,s),0.87(3H,s),0.06,0.04(each 3H,s)； ¹³ C NMR(100MHz,CDCl ₃ ):δ210.6,208.7,166.8,82.7,82.2,80.1,73.7,72.4,70.2,67.4,65.4,57.5,56.8,52.9,52.7,49.0,44.5,42.6,40.7,39.2,38.7,36.8,27.6,26.9,25.8,24.5,18.2,13.9,-5.6.ESIMS m/z 648[M+Na] ⁺ .

Example 4 preparation of Compound A-17 of the invention

To a solution of Compound A-14 (20mg, 0.036mmol) in pyridine (0.2 mL) was added acetic anhydride (0.1 mL), and the mixture was stirred at room temperature for 3 hours, then the reaction mixture was poured into ice water, and basified with ammonia water to pH>9, extraction with dichloromethane (10 mL. Times.3), washing of the organic layer with saturated brine, anhydrous Na ₂ SO ₄ Drying, concentrating under reduced pressure, and vacuum drying to obtain compound A-17 (white amorphous powder, 21mg, 100%). ¹ H NMR(400MHz,CDCl ₃ ):δ5.66(1H,dd,J＝11.2,2.8Hz),3.97(1H,t,J＝3.6Hz),3.62(1H,s),3.61(1H,s),3.47(1H,t,J＝10.0Hz),3.37,3.34(each 3H,s),2.17(3H,s),1.14(3H,t,J＝7.2Hz),0.91(3H,s),0.90(9H,s),0.08,0.06(each 3H,s). ¹³ C NMR(100MHz,CDCl ₃ ):210.7,209.7,170.2,82.6,82.1,80.1,72.5,72.1,70.3,67.3,65.3,57.4,56.6,52.8,52.5,49.0,44.4,42.5,39.2,38.9,36.7,27.6,25.9,24.5,20.8,18.1,14.0,-5.6；ESIMS m/z 614[M+Na] ⁺ .

Example 5 preparation of Compound A-2 of the invention

Compound A-19 (200mg, 0.375mmol) was dissolved in EtOH-CH ₂ Cl ₂ The solvent (5). Stirring at 50 deg.C for 24h, adding water (30 mL), alkalifying with concentrated ammonia water to pH>9,CH ₂ Cl ₂ Extraction (30 mL. Times.3). The organic layers were combined and then dried over anhydrous Na ₂ SO ₄ Drying and concentrating under reduced pressure to obtain residual solid, and eluting with silica gel column chromatography (petroleum ether acetone/5, 1) to obtain A-2 (white amorphous powder, 148mg, 72%). ¹ H NMR(400MHz,CDCl ₃ ):δ4.58(1H,d,J＝2.0Hz),3.91(1H,t,J＝3.6Hz),3.50(1H,s),3.39,3.34(each 3H,s),1.03(3H,t,J＝8.0Hz),1.02(3H,s),0.88(9H,s),0.03,0.03(each 3H,s)； ¹³ C NMR(100MHz,CDCl ₃ ):δ214.9,166.0,84.1,82.4,80.9,73.1,66.5,65.9,58.3,57.4,56.9,56.6,53.2,46.9,42.2,41.8,38.9,37.1,35.9,29.7,29.5,25.8,25.2,18.1,13.9,–5.6；ESIMS m/z 549[M+H] ⁺ .

Example 6 preparation of Compound A-9 of the present invention

Compound A-13 (350mg, 0.84mmol) was placed in 6.5% HBr-AcOH (20 mL), stirred at 85 ℃ for 65 hours, the reaction solution was poured into ice water, basified to pH with aqueous ammonia>9 dichloromethane extraction (100 mL. Times.3), anhydrous Na ₂ SO ₄ The residue obtained by drying and concentration under reduced pressure was eluted by silica gel column chromatography (cyclohexane acetone/3. ¹ H NMR(400MHz,CDCl ₃ ):δ4.74(1H,s),2.13,2.08,2.01(each 3H,s),1.22(3H,s),1.01(3H,t,J＝7.2Hz). ¹³ C NMR(100MHz,CDCl ₃ ):δ207.9,176.2,170.5,170.0,169.9,88.9,88.9,74.6,73.2,71.9,71.5,66.5,49.0,48.2,48.2,41.7,40.3,39.9,38.3,29.5,26.8,23.9,22.6,20.9,20.9,19.9,12.8；ESIMS m/z 516[M+H] ⁺ .

Example 7 preparation of Compound A-19 of the invention

According to the literature (Qi-Feng Chen, feng-Peng Wang, xiao-Yu Liu, generating genetic similarity from the C ₁₉ Compound a-19 was prepared according to the method reported in (1) -a ring-deviation propaac, chem. Eur. J.2015,21, 8946-8950).

Example 8 preparation of Compound C-1 of the present invention

Compound C-1 was prepared according to the method described in the patent application (chinese patent application No. 201910183030.3).

The remaining compounds in Table 1 were prepared by the methods of examples 1-8.

TABLE 1 Structure and molecular weight of the Compounds

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1: anti-inflammatory action test of aconite alkaloid derivative of the invention

1. Experimental methods

(1) Cell culture

Culture of RAW264.7 cells DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin diabody was used. When the cells grow to about 80% -90% of the culture dish area, the cells are subjected to passage, and the passage ratio is 1. The cell freezing medium is DMEM medium containing 10% DMSO and 20% fetal bovine serum, and the cells are frozen in a programmed cooling freezing chamber and stored in a-80 deg.C refrigerator. When the cells are recovered, the frozen cells are taken out, rapidly thawed in a 37 ℃ water bath, centrifuged, the frozen solution is discarded, fresh culture medium is added, the concentration of CO is 5% ₂ And (4) culturing.

(2) Griess method for measuring cellular production of NO

NO produced by cells is easily oxidized into NO ₂ ^- The latter, theReacting with diazonium salt sulfanilamide under acidic condition to generate diazonium compound, further performing coupling reaction with naphthyl ethylene diamine to generate colored substance, comparing with NaNO ₂ The standard curve of the standard substance, the absorption value measured by a microplate reader at 540nm, can measure NO ₂ ^- To obtain the content of NO in the cell supernatant. The method comprises the following specific steps:

(2.1) cell inoculation culture

The cells in logarithmic growth phase were blown out to completely shed and counted, and the concentration of the cells was adjusted to 2X 10 with the medium ⁶ one/mL. The cell suspension was seeded at + 100. Mu.L/well in 96-well cell culture plates and cultured for 2h adherently. After cells were pretreated with different concentrations of alkaloids for 1h, lipopolysaccharide (LPS) was added to a final concentration of 20ng/mL, model groups were supplemented with LPS only, negative controls with complete medium only, positive controls with iNOS (indole nitrile oxide synthase) inhibitor S-Methylisothiourea Sulfate (SMT) and LPS. Incubate at 37 ℃ for 24h.

(2.2) measurement of NO in cell supernatant ₂ ^-

Griess Reagent I and II, stored at 4 deg.C, were removed and equilibrated to room temperature. The standard was diluted with DMEM medium at a concentration of 0,1,2,5,10,20,40,60,100. Mu.M. The standard or sample, griess Reagent I and Griess Reagent II were added sequentially to a 96-well plate at 50. Mu.L/well. The absorbance at 540nm was measured with a preheated microplate reader. Calculation of NO per well from Standard Curve ₂ ^- I.e. the amount of NO produced by the cells.

Inhibition rate of NO (%) = (C) of NO produced by RAW264.7 induced by LPS by drug to be tested _{Model (model)} -C _{Administration of drugs} )/(C _{Model (model)} -C _Control )×100％(C _{Model (model)} NO concentration, C, of cell supernatant stimulated with LPS alone _{Administration of drugs} Is administered LPS stimulated NO concentration, C _Control NO concentration without any treatment). The concentrations and the corresponding NO inhibition rates are fitted by software SPSS to obtain the half Effective Dose (ED) of the drug to be tested ₅₀ )。

(3) ELISA for measuring IL-6 and TNF-alpha in cell supernatant

Enzyme linked immunosorbent assay (ELISA) adopts the principle of double antibody sandwich method, anti-IL-6 monoclonal antibody is pre-coated on an ELISA plate, and IL-6 in a sample or a standard substance is combined with the monoclonal antibody and captured on the ELISA plate. Then adding biotinylated anti-IL-6 antibody and horse radish peroxidase-labeled avidin, and removing free unbound substrate in each step to form a compound fixed at the bottom of the ELISA plate. After the chromogenic substrate is added, the originally colorless chromogenic substrate turns blue by the horseradish peroxidase indirectly combined with the IL-6, the reaction system presents yellow by adding the stop solution, then the absorbance of the reactant is detected at 450nm, the IL-6 content in the cell supernatant can be calculated by comparing with a standard curve, and the TNF-alpha detection method is similar. The method comprises the following specific steps:

(3.1) preparation of solution

The kit was removed from 4 ℃ and returned to room temperature. Deionized water is used for preparing the working solution of the washing solution. And diluting and dissolving the freeze-dried standard substance by using corresponding diluent to prepare a standard curve. The 30 Xconcentrated biotinylated antibody and the 30 Xconcentrated enzyme conjugate were diluted to 1 Xworking solution with the corresponding dilutions, respectively, 20min prior to use.

(3.2) adding the sample or the prepared standard substance into the ELISA plate according to 100 mu L/hole. And adding standard substance and specimen universal diluent into blank holes, and incubating for 90min at 37 ℃. mu.L of washing solution was injected into each well, and the plate was washed 5 times at intervals of 30 s.

(3.3) adding biotinylated antibody working solution into each hole with the volume of 100 mu L, and adding biotinylated antibody diluent into each blank hole. Incubate at 37 ℃ for 60min. The plate was washed 5 times.

(3.4) adding 100 mu L/well of the working solution of the enzyme conjugate, adding the diluent of the enzyme conjugate into a blank well, and incubating for 30min at 37 ℃ in a dark place. The plate was washed 5 times.

(3.5) add chromogenic substrate 100 u L/hole. Incubate at 37 ℃ in the dark for 15min, add 100. Mu.L/well of stop buffer. After mixing, absorbance is detected at 450nm by an enzyme-linked immunosorbent assay within 3 min.

And (3.6) drawing a standard curve according to the standard substance, and substituting to obtain the concentration of the IL-6 in the sample to be detected (the same manner as the TNF-alpha detection method).

(4) CCK-8 method for determining cytotoxicity

CCK-8 (Cell Counting Kit-8) solution can be oxidized and reduced by dehydrogenase in cells to generate orange yellow formazan, and the content of the orange yellow formazan is measured at 450nm and is in direct proportion to the number of living cells. The method comprises the following specific steps: blowing the cells in logarithmic growth phase until the cells completely drop off, counting, and adjusting the concentration of the cells to be 2 x 10 by using a culture medium ⁶ one/mL. The cell suspension was seeded at 100. Mu.L/well in 96-well cell culture plates and cultured for 2h adherently. Adding different concentrations of alkaloid, and culturing at 37 deg.C for 24 hr. And (3) absorbing waste liquid, adding 100 mu L of working solution with the volume ratio of the culture medium to the CCK-8 solution being 10 into each hole, and detecting the absorbance at 450nm by using a microplate reader after 30-60 min.

(5) Statistical analysis

Data are presented as means ± standard deviation. The differences between the two groups were compared using one-way ANOVA (one-way ANOVA) and Least Significant Difference (LSD) methods using SPSS software analysis, and were considered statistically significant when P < 0.05.

2. Results of the experiment

(1) Inflammation model of LPS-induced RAW264.7 activation

After LPS treatment of RAW264.7 cells in different cell inoculation numbers and different concentrations, the NO concentration in the cell culture supernatant was measured using Griess reagent. When seeded with cells 2X 10 ⁶ Since NO concentration can be significantly increased in a 96-well plate at 100 μ L/well with LPS stimulus concentration of 1ng/mL to 1ug/mL (fig. 1A), NO production can be significantly inhibited when treatment with the iNOS inhibitor as a positive drug is performed at an intermediate concentration of 20ng/mL (fig. 1B).

The results show that the experiment successfully establishes a model for LPS to induce RAW264.7 to generate the inflammatory mediator NO.

(2) Aconite alkaloid derivative can inhibit LPS activation RAW264.7 to generate NO

TABLE 2 ED that each compound inhibits LPS-induced NO production by RAW264.7 cells ₅₀

In Table 2, "-" indicates NO inhibitory activity against the production of NO by RAW264.7 cells induced by LPS.

The RAW264.7 cells were pretreated with each test drug at different concentrations, and then activated cells were induced with LPS at a concentration of 20 ng/mL. The Griess method measures the concentration of NO produced after cell activation (fig. 2, fig. 3, fig. 4, fig. 5, fig. 6). The result shows that the aconitum carmichaeli alkaloid derivative provided by the invention can effectively inhibit LPS (lipopolysaccharide) to induce RAW264.7 cells to generate NO, particularly the compound A-1, A-8, A-2, A-9, C-1, E-1.

(3) Aconite alkaloid derivative can inhibit LPS activation RAW264.7 to produce cytokine

In order to further verify the influence of the aconite alkaloid derivative on the inflammatory factors, the effects of cytokines IL-6 and TNF-alpha generated by A-1, A-8, E-1, C-1 and A-9 after RAW264.7 cells are activated are respectively detected by an ELISA method. Compared with a negative control group, the IL-6 and TNF-alpha are obviously increased after cells are stimulated by LPS (20 ng/mL) (P < 0.05); compared with the model group, each aconite alkaloid derivative provided by the invention has obvious inhibition effect on the generation of IL-6 and TNF-alpha, and shows a certain dose dependence (figure 7).

(4) Cytotoxicity of aconite alkaloid derivatives

To determine whether the effect of the aconite diterpenoid alkaloid derivative on inhibiting NO generation is caused by reduction of cell number and function reduction due to toxicity on cells, the CCK-8 method is used for detecting the cytotoxicity of each alkaloid on RAW264.7, and the result shows that the aconite diterpenoid alkaloid derivative does not show obvious toxicity (figure 8).

The experimental results show that the aconite diterpenoid alkaloid derivative provided by the invention has excellent anti-inflammatory activity, can effectively inhibit LPS (lipid-soluble lipid) from inducing RAW264.7 cells to generate NO, can effectively inhibit LPS-activated RAW264.7 cells from generating inflammatory factors IL-6 and TNF-alpha, and can be used for preparing anti-inflammatory drugs and NO generation inhibitors.

Experimental example 2: the anti-inflammatory activity mechanism of the aconite alkaloid derivative A-8 in the in vitro cell inflammation model

1. Experimental method

1.1Western blot for intracellular iNOS protein assay

(1) Extraction of cellular proteins

A) Blowing RAW264.7 to completely shed cells in logarithmic growth phase, counting according to 2 × 10 ⁶ One cell/well was seeded in 6-well cell culture plates and cultured overnight adherent. Cells were pretreated with different concentrations of alkaloid A-8 for 1h, followed by LPS at a final concentration of 20 ng/mL. The negative control group is added with complete culture medium DMEM only, the model group is added with LPS only and is not added with drugs for treatment, and the culture is carried out for 4h,6h and 24h at 37 ℃.

B) The cell culture medium is aspirated, the cells are washed with precooled normal saline, the cells are blown and collected with normal saline into an EP tube, and the supernatant is removed after centrifugation at 250g for 5min. Per tube of cell pellet was added 200 μ L RIPA lysate containing the protease inhibitor Cocktail and the phosphatase inhibitor Cocktail (Cocktail: RIPA = 1. The mixture is placed on ice and shaken at a high speed on a decoloring shaking table for 15min. Taking out and centrifuging at 16000g for 10min in a centrifuge with precooling at 4 ℃, and collecting supernatant into a precooling centrifuge tube.

C) Protein content determination by BCA colorimetry

The principle is as follows: the protein can convert Cu under alkaline conditions ²⁺ Reduction to Cu ¹ Dicinchoninic acid (BCA) chelates with the latter, producing a dark purple product with strong linear absorption at 562nm, with absorbance increasing with increasing protein concentration. The protein concentration of the sample can be determined by comparison with a standard protein curve.

The method comprises the following specific steps: taking a bovine serum albumin standard (BSA) to dilute the BSA to a final concentration of 2000, 1500, 1000, 750, 500, 250, 125 and 25. Mu.g/mL with physiological saline, taking 25. Mu.L of each of a sample to be detected and a protein standard to add into a 96-well cell culture plate, adding 200. Mu.L of a working solution into each well, wherein the working solution comprises 50 parts of a BCA reagent A and a BCA reagent B, fully mixing the solution, incubating the mixture at 37 ℃ for 30min, and then measuring the absorbance value of the mixture at 562nm by using an enzyme-labeling instrument.

D) SDS-PAGE protein loading buffer (5X) is added to each sample according to the volume ratio of 4 to 1, and the samples are heated at 100 ℃ for 5min and stored at-20 ℃ for subsequent Western blot experiments.

(2) Western blot for protein immunoblotting experiment

A) Electrophoresis: separating gel is prepared according to the specification of the SDS-PAGE gel rapid preparation kit, carefully poured between glass plates (2-3 cm is reserved for pouring concentrated gel), and deionized water is added to fill and flatten the separating gel. The gel solidified at room temperature until a distinct boundary line appeared between the separating gel and water. Pouring out water, injecting concentrated glue between the glass plates, and inserting the glass plates into an electrophoresis comb. After the concentrated gel is solidified, the comb is carefully pulled out. And taking out the prepared protein sample, loading the protein sample, performing electrophoresis, wherein the electrophoresis condition of the concentrated gel part is 70V and 20min, the separation gel part is 140V, and performing electrophoresis until the bromophenol blue dye is about 1cm away from the bottom of the glass plate.

B) Film transferring: the PVDF membrane was cut to size and activated with methanol for 10s. The rotary membrane clamp comprises filter paper, separation glue, a PVDF membrane, filter paper and sponge in sequence from the negative electrode to the positive electrode. Preparing a film transfer liquid according to the specification, precooling at 4 ℃, and placing the film transfer liquid in an ice-water mixture for film transfer by a film transfer groove device. The membrane transfer condition was 400mA and 25min.

C) And (3) sealing: and (3) placing the PVDF membrane subjected to membrane conversion in 5% milk powder sealing solution, sealing for 1 hour at room temperature on a decoloring shaking table, and washing the membrane by TBST.

D) Incubation of primary antibody: primary antibody was added and incubated overnight at 4 ℃.

E) Incubation of secondary antibody: washing the membrane with TBST for three times, 5min each time, adding secondary antibody, decolorizing at room temperature and incubating for 1h with shaking table. Washing the membrane for three times by TBST, 5min for each time,

f) And (3) developing: preparing a developing solution according to the instruction, sucking the developing solution to soak the PVDF membrane, and carrying out chemiluminescence imaging analysis.

1.2iNOS enzyme Activity assay

(1) Cells RAW264.7 were cultured in 10cm dishes, and when about 50% of cells grew, 1. Mu.g/mL of LPS was added thereto, and the cells were cultured at 37 ℃ for 12 hours. For inducing the cells to express iNOS protein.

(2) Sucking out culture medium, washing cells with physiological saline, blowing to collect cells, and counting at 2 × 10 ⁶ One cell/well was seeded in 96-well cell culture plates,after 2h of adherence at 37 ℃ in the incubator, cells were treated with A-8 at different concentrations and medium was added to the negative control group. Culturing for 4h and 24h at 37 ℃.

(3) The NO content in the cell supernatant was determined as described above under 1.2.2.

1.3Western blot assay of intracellular inflammation pathway-associated proteins

The cells in logarithmic growth phase are blown with RAW264.7, and counted according to 2 × 10 ⁶ One/well was seeded in 6-well cell culture plates and cultured overnight adherent. Cells were pretreated with different concentrations of alkaloid A-8 for 1h, followed by LPS at a final concentration of 20 ng/mL. And adding complete culture medium DMEM only in the negative control group, adding LPS only in the model group without adding medicaments for treatment, respectively culturing for 1h at 37 ℃ for detecting NF-kB and MAPK pathway related proteins, and culturing for 24h for detecting NLRP3 proteins. Other steps are detailed in 2.2.1.

1.4 statistical analysis

Data are presented as means ± standard deviation. SPSS software is used for analysis, one-way ANOVA is adopted for multi-group comparison, the difference between two groups is compared by adopting a least significant difference method (LSD), and when P is less than 0.05, the difference is considered to have statistical significance.

2. Results of the experiment

2.1A-8 inhibition of LPS-induced iNOS expression in RAW264.7

The expression of iNOS is obviously increased after 4-24 h of LPS (20 ng/mL) stimulating RAW264.7 cells. Pretreatment with A-8 for 1h inhibited iNOS production at 4h and 6h early, and exhibited a dose-dependence (FIG. 9).

2.2A-8 does not affect iNOS enzymatic Activity

RAW264.7 cells were treated with LPS (20 ng/mL) for 16h, the LPS-containing medium was discarded and the cells were washed with physiological saline, at which time the content of iNOS in the cells did not change any more and the amount of NO produced by the cells indirectly reflected the activity of iNOS enzyme. Activated cells were treated with different concentrations of A-8 and the amount of NO production in the cell supernatant was measured after a certain period of time. The results showed that there was NO significant change in the amount of NO produced after treatment with A-8 for 4h (FIG. 10A) and 24h (FIG. 10B), respectively, indicating that A-8 did not affect the enzymatic activity of iNOS.

2.3A-8 inhibition of LPS-induced activation of the MAPK pathway

After 1h of LPS stimulation at 20ng/mL, the phosphorylated protein expression of MAPK pathway proteins p38, ERK1/2 and JNK was increased and the pathway was activated. After pretreatment of cells with A-8 and stimulation with LPS, A-8 inhibited p-ERK1/2, p-p38, p-JNK in cells, i.e., inhibited activation of MAPK pathway (FIG. 11).

2.4A-8 inhibition of LPS-induced NF- κ B pathway activation

After 1h of LPS stimulation at 20ng/mL, the intracellular p-p65 increased significantly, while A-8 treatment inhibited phosphorylation of the p65 protein, inhibiting LPS-induced NF- κ B pathway activation (FIG. 12).

2.5A-8 inhibition of LPS-induced increased NLRP3 expression

Intracellular NLRP3 increased significantly after 24h of LPS stimulation at 20ng/mL, whereas A-8 treatment inhibited the increase in NLRP3 (FIG. 13)

The experimental results show that the aconite alkaloid A-8 provided by the invention has a regulation effect on the LPS-induced RAW264.7 cell activation, can obviously reduce the expression of inflammatory mediators NO, TNF-alpha and IL-6, and is related to the inhibition of iNOS expression and the inhibition of NF-kappa B and MAPK signal pathway activation. The result shows that A-8 can play an anti-inflammatory role by inhibiting the expression of iNOS and NLRP3 activated by LPS and the activation of MAPK/NF-kB signal channels.

Experimental example 3: the aconite alkaloid derivative A-8 has the anti-inflammatory curative effect on an in-vivo model of TPA (terephthalic acid) induced ear swelling

1. Experimental methods

Experimental animals: male BALB/c mice, 6-8 weeks old and 20-22g in weight, were purchased from Beijing Wittiaxle animal technology Limited and housed in SPF-rated animal rooms (temperature 23 + -3 deg.C, relative humidity 40-70%, light/dark cycle 12h, free access to food and water). The environment was acclimatized for at least 3 days before the test, and healthy animals were selected as test animals. Animal experiment related operations are strictly regulated according to animal experiment ethics and related laws of Sichuan university.

1.1 preparation of solutions

(1) TPA acetone solution

1mg of 12-O-tetradecanoylphosphatide-13-acetate (TPA) was dissolved in 1mL of acetone to prepare a stock solution of 1mg/mL and stored at-20 ℃. The solution was diluted with acetone to 100. Mu.g/mL before use.

(2) A-8 solution

A-8 was dissolved in acetone to 0.05 mg/. Mu.L and diluted with acetone to 0.016 mg/. Mu.L and 0.005 mg/. Mu.L, respectively.

(3) Dexamethasone sodium phosphate solution

Dexamethasone sodium phosphate is prepared into 1mg/mL by using normal saline. Dexamethasone (Dexamethasone, DXMS for short).

1.2 TPA-induced mouse ear swelling model

(1) 30 BALB/c male mice were weighed and divided into 6 groups of 5 mice each by a randomized block method.

(2) The packet processing operation is as follows:

normal control group: the right ear was smeared with 20. Mu.L of acetone by pipette and was injected with 10mL/kg of saline intraperitoneally. Acetone was applied to the right ear at 20. Mu.L after 1h.

TPA model group: the right ear was smeared with 20. Mu.L of acetone and was injected intraperitoneally with 10mL/kg of physiological saline. After 1h, the right ear was smeared with TPA solution 100. Mu.g/mL, 20. Mu.L.

A-8 Low dose (0.1 mg/ear) + TPA group: the right ear was smeared with 0.005 mg/. Mu.L of A-8 solution 20. Mu.L, and was injected with normal saline at 10mL/kg i.p.. After 1h, the right ear was smeared with TPA solution at 100. Mu.g/mL, 20. Mu.L.

Dose A-8 (0.3 mg/ear) + TPA group: the right ear was smeared with 0.016 mg/. Mu.L of A-8 solution 20. Mu.L, and was injected intraperitoneally with 10mL/kg of physiological saline. After 1h, the right ear was smeared with TPA solution 100. Mu.g/mL, 20. Mu.L.

High dose of A-8 (1 mg/ear) + TPA group: the right ear was smeared with 0.05 mg/. Mu.L of A-8 solution 20. Mu.L, and was injected with normal saline at 10mL/kg i.p.. After 1h, the right ear was smeared with TPA solution at 100. Mu.g/mL, 20. Mu.L.

Dexamethasone + TPA group: the right ear was smeared with 20. Mu.L acetone and injected intraperitoneally with dexamethasone sodium phosphate solution at 10 mL/kg. After 1h, the right ear was smeared with TPA solution 100. Mu.g/mL, 20. Mu.L.

(3) Sampling of ear tissue

The mice were sacrificed 5h after molding, and tissues at the same positions of the left and right ears of the mice were taken with a metal punch having a diameter of 6mm and weighed. Calculation of ear swelling rate = (W) _{Right ear} -W _{Left ear} )/W _{Left ear} ×100％，W _{Right ear} Weight of right ear, W _{Left ear} Left ear weight. After weighing, the ear tissues were fixed in 10% formalin solution or frozen at-80 ℃ respectively. Fixed tissue is subjected to hematoxylin-eosin staining method (hematoxylin-eosin staining) to be used as H&And E, pathological section.

1.3 statistical analysis

2. Results of the experiment

2.1 ear swelling Rate determination

The left ear of the mouse was used as a control without any treatment, TPA was dissolved in acetone and applied to the auricle of the right ear of the mouse at a dose of 2. Mu.g/ear for molding, and the normal control group mice were treated with a solvent. After 5h of molding, the right ear of the molding group is observed to be obviously red and thickened by naked eyes. The same position of the left and right ears was uniformly sampled by a punch, and the untreated left ear was used as a control to evaluate the swelling rate of the modeled right ear. The results are shown in FIG. 14, and the results show that the A-8 can significantly reduce ear swelling caused by TPA (P < 0.05) by 1 mg/ear topical application or 1h pretreatment by intraperitoneal injection (10 mg/kg) of a positive dexamethasone sodium phosphate solution.

2.2 ear histopathology H & E Observation

The anti-inflammatory effect of A-8 was further evaluated by H & E staining. As shown in fig. 15, the ear tissue treated with TPA alone was much thicker than the normal control ear tissue with dense lymphocyte infiltration. Treatment with A-8 or dexamethasone significantly reduced edema and lymphocyte infiltration by TPA.

The mouse ear swelling model is a mature non-specific inflammation model. The experimental results show that the A-8 is locally coated on auricle of mice of an auricle swelling model for pretreatment, so that the auricle swelling can be obviously reduced, inflammatory cell infiltration is reduced, and an in-vivo anti-inflammatory effect is exerted, thereby showing that the aconite alkaloid derivative A-8 can effectively treat the auricle swelling in vivo.

Experimental example 4: the aconite alkaloid derivative A-8 has anti-inflammatory effect on an LPS (LPS) -induced mouse sepsis model

1. Experimental methods

Experimental animals: male BALB/c mice, 6-8 weeks old and 20-22g in body weight, were purchased from Beijing Wintolidian animal technology, inc. and were housed in SPF-rated animal rooms (temperature 23 + -3 deg.C, relative humidity 40-70%, light/dark cycle 12h, free access to food and water). The environment was acclimatized for at least 3 days before the test, and healthy animals were selected as test animals. Animal experiment-related procedures were strictly in accordance with ethical and related laws for animal experiments at the university of Sichuan.

1.1 solution preparation

(1) LPS solution

LPS is prepared into a solution with the concentration of 1mg/mL by using normal saline, and is administrated by intraperitoneal injection according to the administration volume of 10 mL/kg.

(2) A-8 solution

Dissolving the drug A-8 in pure DMSO to prepare a mother solution, then adding other solvents according to the following formula volume ratio, namely 5% of the mother solution (in DMSO) +40% of PEG400+5% of Tween80+50% of normal saline, fully dissolving the mother solution by ultrasound, respectively preparing solutions with final concentrations of 10mg/mL and 4mg/mL, and carrying out intragastric administration on mice in a high-dose administration group and a low-dose administration group according to the administration volume of 20 mL/kg.

(3) Dexamethasone sodium phosphate solution

Dexamethasone sodium phosphate is prepared into 1mg/mL by using normal saline, and is administrated by intraperitoneal injection according to the administration volume of 10 mL/kg.

1.2 LPS-induced mouse sepsis model

(1) Weighing body weight

30 mice were weighed individually and divided into 5 groups of 6 mice by a random method.

(2) Pretreatment for group administration

A-8 low dose group (80 mg/kg, p.o.), A-8 high dose group (200 mg/kg, p.o.), dexamethasone group (10 mg/kg, i.p.). The normal control group and LPS model group were intragastrically injected with a solvent (5% DMSO +40% PEG400+5% Tween80+50% saline) at an administration volume of 20mL/kg, each group was pretreated for 1h at 10 mL/kg.

(3) LPS (Low pressure polystyrene) molding

Except for the normal group, mice in other groups were subjected to intraperitoneal injection molding using LPS solution at a dose of 10mg/kg.

(4) Taking materials

After molding for 2h, taking blood in a separation gel accelerating tube by adopting a submaxillary venous blood sampling method, centrifuging at 5000r/min for 15min, collecting serum samples, preserving the serum samples at the temperature of minus 80 ℃ for detecting TNF-alpha in the serum by ELISA. The experimental endpoint was 5h later, and the mice were sacrificed to obtain material. Blood is collected from eyeballs after mice are anesthetized and is collected in a separation gel accelerating tube, the centrifugation is carried out for 15min at 5000r/min, serum is collected and is preserved at the temperature of minus 80 ℃, and the serum is used for detecting IL-6 in the serum by ELISA. The lung and kidney of the mouse are taken, the left and the right are respectively fixed by 10 percent formalin solution and quick frozen by liquid nitrogen and preserved at-80 ℃.

1.3 Biochemical detection of blood

The serum specimen is sent to the national New medicine safety evaluation center for blood biochemical detection. The detection indexes are as follows: urea Nitrogen (BUN); creatinine (creatinine).

1.4 statistical analysis

2. Results of the experiment

2.1A-8 inhibition of serum TNF-alpha and IL-6 levels

And B, performing intragastric administration pretreatment on the A-8 mice for 1h at the doses of 80mg/kg and 200mg/kg, performing the administration pretreatment for the same time by taking 10mg/kg of an intraperitoneal injection of dexamethasone sodium phosphate solution as a positive control, and performing 10mg/kg of intraperitoneal injection of LPS (fiber laser) for molding. As shown in FIG. 16, after 5h of molding, TNF-. Alpha.and IL-6 were significantly increased in the serum of mice in LPS model group (P < 0.05) compared to the normal control group; compared with a model group, the positive drug dexamethasone can effectively reduce the generation of inflammatory factors TNF-alpha and IL-6 in serum, and A-8 can also obviously reduce the generation of the two inflammatory factors.

2.2 Biochemical test results of blood

Mice were pretreated with A-8 and dexamethasone sodium phosphate for 1h, respectively, and then were injected intraperitoneally with LPS physiological saline solution for molding. The results show (fig. 17), after 4h, the Blood Urea Nitrogen (BUN) and Creatinine (CREA) of the mice in the LPS model group were significantly increased (P < 0.05) compared to the normal control group; dexamethasone reduced BUN concentrations in blood (P < 0.05) compared to the model group, but had no statistical significance for CREA effects. When the dosage of the oral gavage A-8 is 80mg/kg, the concentration of urea nitrogen in blood can be obviously reduced (P is less than 0.05), but no obvious effect on creatinine is generated; there was no significant effect on elevated BUN and CREA in serum when 200mg/kg A-8 was administered (P > 0.05).

2.3 pathological H & E observations

H & E staining of mouse lung (FIG. 18) and kidney (FIG. 19) tissues was performed, and no significant difference was observed between groups, and no pathological injury response was observed.

Sepsis is a systemic inflammatory response syndrome of life-threatening organ dysfunction caused by infection. The experimental results show that the application of A-8 can obviously reduce the levels of inflammatory factors TNF-alpha and IL-6 in the serum of a mouse model of systemic sepsis, and the aconite alkaloid derivative A-8 can effectively treat sepsis in vivo.

Experimental example 5: the aconite alkaloid derivative A-8 has anti-inflammatory effect on DSS mouse colitis model

1. Experimental method

Experimental animals: male C57BL/6 mice, 6-8 weeks old and 20-22g in body weight, were purchased from Beijing Wintonli animal technology, inc. and housed in SPF-rated animal rooms (temperature 23 + -3 deg.C, relative humidity 40-70%, light/dark cycle 12h, free access to food and water). The environment was acclimatized for at least 3 days before the test, and healthy animals were selected as test animals. Animal experiment-related procedures were strictly in accordance with ethical and related laws for animal experiments at the university of Sichuan.

1.1 solution preparation

(1) DSS solution

2.5% by volume DSS solution was prepared, 20g dextran sulfate sodium salt (DSS) powder (MW: 36000-50000) was dissolved thoroughly in 800mL autoclaved reverse osmosis deionized water. The solution can be stored at 4 ℃ for up to 4 weeks.

(2) Mesalazine enteric coated tablet suspension

Preparing 10mg/mL mesalazine enteric-coated tablet suspension, fully grinding mesalazine enteric-coated tablets (0.5 g), dissolving in 50mL of physiological saline to prepare suspension, and storing at 4 ℃. Mice were given intragastric administration at a dosing volume of 20 mL/kg.

(3) A-8 solution

Firstly, dissolving the drug A-8 in pure DMSO to prepare a mother solution, then adding other solvents of 5 percent of the mother solution (in DMSO) +40 percent of PEG400+5 percent of Tween80+50 percent of normal saline according to the following formula volume ratio, fully dissolving the solution by ultrasound, respectively preparing the solution with the final concentration of 10mg/mL and 4mg/mL, and carrying out intragastric administration on mice of low-dose administration group and high-dose administration group according to the administration volume of 20 mL/kg.

1.2A-8 pharmacodynamic experiment on DSS-induced acute colitis

(1) Molding and administering the drug

Animals were weighed and 50 male C57BL/6 mice were divided into 5 groups by weight in a randomized block method. 1) A normal control group; 2) A set of DSS models; 3) Mesalazine group (200 mg/kg); 4) A-8 Low dose group (80 mg/kg); 5) A-8 high dose group (200 mg/kg). The molding method comprises the following steps: 200mL of 2.5% DSS solution was added to the mice drinking water bottles, and fresh DSS solution was replaced every 3 days, allowing the mice to drink water freely. Normal control mice were fed reverse osmosis deionized water without DSS. The mesalazine group, the A-8 low dose group and the A-8 high dose group were subjected to intragastric administration 1 time every 3 days, and mice in the normal control group and the model group were subjected to intragastric administration with a solvent.

(2) Body weight and percent change in body weight

Recording the weight of the mouse every day from the beginning of molding to the end of the experiment, and calculating the weight change percentage according to the weight, wherein the calculation formula is as follows; percent weight change (%) = (BW-BW) ₀ )/BW ₀ X100%. BW is the body weight of the day ₀ The body weight was measured for the first day of molding.

(3) Disease Activity Index (DAI)

Mice were scored daily after molding according to DAI scoring criteria (table 3). DAI = (weight loss score + stool trait score + stool blood score)/3. Among them, occult blood in feces was detected using an occult blood kit (benzidine method), and the procedure was performed according to the kit instructions. And (3) evaluating the fecal character: normal feces: forming a particle-like stool; loosening feces: paste-like semi-formed feces not adhering to the anus are not broken; sparse feces: can be used for treating dilute water-like feces attached to anus.

TABLE 3DAI scoring criteria

(4) Visual intestinal mucosal injury score (CMDI)

On day 11 after molding and administration, colon tissue from mice was collected, intestinal contents were washed with physiological saline, colon length and colon weight were recorded, longitudinally sectioned, and gross lesions in the colon were visually observed and subjected to CMDI scoring against CMDI scoring criteria (table 4).

TABLE 4CMDI Scoring criteria Table

(5) Colon Length/colon weight ratio

After the animal is dissected, the colon is straightened but not stretched and placed on white paper, and the length is measured by using a ruler. And the colon wet weight is taken and the colon length/colon weight ratio is calculated.

(6) H & E staining pathology grading and morphological performance pathology scoring

After sacrifice, colon tissue was removed, intestinal contents were washed clean with PBS solution, and longitudinally split to make "swiss roll" sample fixed in 10% formalin solution. Pathological Scoring as per Table 5

TABLE 5H & E staining pathology scoring standard table

1.3 statistical analysis

2. Results of the experiment

2.1 weight and percent weight change

From the fifth day after the start of molding to the 11 th day at the end of the experiment, the mice in the other groups had a significant weight loss and no significant difference in weight between the groups compared to the normal water drinking group (fig. 20).

2.2 disease Activity index score (DAI)

From the start of the model building to the end of the experiment, the disease activity index was significantly increased in the other groups compared to the normal control group, but there was no major difference between the groups (fig. 21).

2.3 visual intestinal mucosal Damage score (CMDI)

And at the end point of the experiment, the colon mucosa of the mice is observed by naked eyes in an anatomical mode to be scored, and the colon tissue structure of the mice in a normal group is normal and has no pathological damage. The other groups had mild mucosal and submucosal inflammatory cell infiltration, edema, mucosal erosion but intact muscularis mucosae, and the model group had no statistical significance from the administration group (fig. 22).

2.4 Colon Length/Colon weight ratio

The length of colon was measured at the end of the experiment, and the colon was significantly thickened and shortened in the other groups compared to the normal group, but there was no statistical difference in the colon length/weight between the model group and the administered group (FIG. 23)

2.5 Colon H & E staining pathomorphism and pathological Scoring

As shown in FIG. 24, the tissue structure is normal, no obvious pathological damage is seen in the colon of the normal control group, large-area obvious inflammation and edema appear in the colon tissue of the model group, erosion enters submucosa, and a small amount of inflammatory cells enter the muscular layer. The mesalazine and a-8 high dose group improved pathological damage due to DSS, with statistical differences in pathology scores compared to the model group (fig. 25).

The experimental results show that the pathological injury of the colon of the mice induced by the DSS can be obviously improved by the high-dose administration of the A-8.

In conclusion, the invention provides a monkshood alkaloid derivative shown as a formula I and application thereof in preparing anti-inflammatory drugs. Experimental results show that the aconite alkaloid derivative provided by the invention has excellent anti-inflammatory activity, can effectively inhibit LPS (LPS) from inducing RAW264.7 cells to generate NO, effectively inhibits RAW264.7 cells activated by LPS from generating inflammatory factors IL-6 and TNF-alpha, and can be used for preparing anti-inflammatory drugs and NO generation inhibitors. The aconite alkaloid derivative can play an anti-inflammatory role by inhibiting the expression of iNOS and NLRP3 activated by LPS and the activation of MAPK/NF-kB signal channels. The experimental result also shows that the aconitum carmichaeli alkaloid derivative provided by the invention can obviously reduce ear swelling, reduce inflammatory cell infiltration, play an in vivo anti-inflammatory role and effectively treat ear swelling in vivo; the aconite alkaloid derivative provided by the invention can obviously reduce the levels of inflammatory factors TNF-alpha and IL-6 in serum of a general sepsis model mouse, and can effectively treat sepsis in vivo; the aconite alkaloid derivative provided by the invention can also obviously improve DSS-induced pathological injury of colon of mice. The aconite alkaloid derivative provided by the invention has excellent anti-inflammatory activity and low toxicity, and has wide application prospect in preparation of anti-inflammatory drugs and NO generation inhibitors.

Claims

1. A compound of formula I, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a deuterated compound thereof:

wherein R is ₁ Selected from hydrogen, hydroxyl, = O, C _1～6 Alkyl radical, C _1～6 An alkoxy group; r ₃ Selected from hydrogen, LR _a 、LOR _a L is selected from none, C _1～4 Alkylene radical，R _a Selected from hydrogen, protecting groups, C _1～6 An alkyl group; or, R ₁ 、R ₃ Connecting to form a ring;

R _e Selected from hydroxy, C _1～6 An alkyl group.

2. The compound, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a deuterated compound thereof, according to claim 1, wherein: the structure of the compound is shown as formula II:

wherein R is ₁ Selected from hydrogen, hydroxyl, = O, C _1～4 Alkyl radical, C _1～4 An alkoxy group; r is ₃ Selected from hydrogen, LR _a 、LOR _a L is selected from,C _1～2 Alkylene, R _a Selected from hydrogen, hydroxy protecting groups, C _1～4 An alkyl group; or, R ₁ 、R ₃ Connecting to form a ring;

the hydroxyl protecting group is preferably a TBS group.

3. The compound, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a deuterated compound thereof, according to claim 2, wherein: the structure of the compound is shown as the formula III:

4. The compound, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a deuterated compound thereof according to any one of claims 1-3, wherein: the structure of the compound is selected from:

wherein Ac is acetyl and Piv is pivaloyl.

5. An anti-inflammatory agent which is a preparation prepared by using the compound, the pharmaceutically acceptable salt, the stereoisomer or the deuterated compound thereof as an active ingredient, and adding pharmaceutically common auxiliary materials.

6. Use of the compound of any one of claims 1to 4, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a deuterated compound thereof, or compound C-1, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a deuterated compound thereof, or compound a-19, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a deuterated compound thereof, for the preparation of an anti-inflammatory drug;

7. use according to claim 6, characterized in that: the anti-inflammatory drug is a drug for preventing and/or treating nonspecific inflammation.

8. Use according to claim 7, characterized in that: the anti-inflammatory medicament is a medicament for preventing and/or treating ear swelling, sepsis and colitis.

9. Use according to any one of claims 6 to 8, characterized in that: the anti-inflammatory drug can inhibit NO, TNF-alpha and IL-6; and/or, the anti-inflammatory drug can inhibit iNOS, NLRP3 expression; and/or, the anti-inflammatory drug is capable of inhibiting activation of the MAPK/NF- κ B signaling pathway.

10. Use of the compound, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a deuterated compound thereof, or compound C-1, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a deuterated compound thereof, or compound a-19, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a deuterated compound thereof, according to any one of claims 1to 4, for the preparation of an NO generation inhibitor;