CN116262114B - Benzyl o-hydroxybenzoate and its pharmaceutical composition and use - Google Patents

Abstract

The present invention belongs to the field of biomedicine, and discloses a novel structural compound FN as shown in formula (I) obtained by separation from the natural drug Solidago asiatica and its pharmaceutically acceptable salt, a preparation method thereof, a pharmaceutical composition containing the compound, and the use of the compound in the preparation of drugs for inflammation, fibrosis-related diseases and acute lung injury. The active natural product FN or its pharmaceutically acceptable salt involved in the present invention has a simple preparation method and has obvious anti-inflammatory, anti-fibrosis and pharmacodynamic activity for the treatment of acute lung injury.

Description

Benzyl o-hydroxybenzoate and pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a preparation method of a novel structural compound FN or a pharmaceutically acceptable salt thereof, which is separated from a natural medicine solidago, a medicinal composition containing the compound or the pharmaceutically acceptable salt thereof, and application of the compound or the pharmaceutically acceptable salt thereof in inflammation, fibrosis and acute lung injury related diseases.

Background

Natural drugs are an important source of modern pharmaceutical compounds. Solidago (Solidago decurren) is a plant of the genus Solidago of the family Compositae, and is mainly produced in the east, south and west areas of China, and in the Shaanxi, taiwan and other areas. The goldenrod herb is used as a medicinal plant, has very wide application, has the effects of dispelling wind, clearing heat, detoxifying and relieving swelling, and is mainly applied to the treatment of wind-heat type common cold, headache, sore throat, lung-heat cough and other diseases. Modern pharmacological researches have shown that Solidago decumbens has antibacterial, diuretic, expectorant and antiasthmatic effects. So far, few researches on chemical components and pharmacological activities of solidago are reported, and the literature reports that main chemical components of solidago are flavone, saponin, benzyl benzoate, phenylpropionic acid and the like. FN (formula I) is a novel structural benzyl benzoate natural product found in the chemical composition study of solidago of the research group. Pharmacological experiment results show that FN has obvious effects of resisting inflammation, resisting fibrosis and treating acute lung injury, and is an active natural product with deep research value. The related literature search shows that the structure of the compound and the related pharmacological activity research are not reported so far.

Disclosure of Invention

The invention aims to solve the technical problem of providing o-hydroxybenzoic acid benzyl ester compounds, and a pharmaceutical composition and application thereof.

In order to solve the technical problems of the invention, the invention provides the following technical scheme:

According to a first aspect of the technical scheme, the invention provides a natural product FN shown as a formula (I) and pharmaceutically acceptable salts thereof.

The study firstly separates benzyl benzoate natural product FN with a new structure from a medicinal plant solidago, and determines the structural formula of the benzyl benzoate natural product FN as formula (I) through a spectrum analysis method.

The compound disclosed by the invention comprises a compound shown as a formula (I) and pharmaceutically acceptable salts thereof.

The second aspect of the technical scheme of the invention provides a pharmaceutical composition, which is characterized by comprising a compound shown in a formula (I) or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or auxiliary material.

The pharmaceutical composition is characterized by comprising a compound shown in a general formula (I), pharmaceutically acceptable salts thereof and a medicinal carrier and/or excipient.

Pharmaceutical compositions of the compounds of the present invention may be prepared according to methods well known in the art. For this purpose, the compounds of the invention may, if desired, be combined with one or more solid or liquid pharmaceutical excipients and/or auxiliaries to form suitable administration forms or dosage forms which can be used as medicaments.

The compound of the present invention or a pharmaceutical composition containing it may be administered in unit dosage form by the enteral or parenteral route such as oral, intravenous, subcutaneous, intraperitoneal, nasal, oral mucosa, ocular, pulmonary and respiratory tract, skin, vaginal, rectal, etc., preferably orally.

The dosage form may be a liquid, solid or semi-solid dosage form. The liquid dosage forms can be solutions (including true solution and colloid solution), emulsions (including o/w type, w/o type and multiple emulsion), suspensions, injections (including injection solutions, powder injections and infusion solutions), eye drops, nasal drops, lotions, liniments and the like. The solid dosage forms can be tablets (including common tablets, enteric coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets and orally disintegrating tablets), capsules (including hard capsules, soft capsules and enteric coated capsules), granules, powder, micropills, dripping pills, suppositories, films, patches, aerosol (powder) and sprays, and the semisolid dosage forms can be ointments, gels, pastes and the like.

The compound of the invention can be prepared into common preparations, and can also be sustained release preparations, controlled release preparations, targeted preparations and various microparticle administration systems.

For the purpose of shaping the unit dosage form into a tablet, various carriers known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc., wetting agents and binders such as water, gan Bo, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, dextrose solution, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc., disintegrants such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, methyl cellulose, ethyl cellulose, etc., disintegration inhibitors such as sucrose, glycerol tristearate, cocoa butter, hydrogenated oil, etc., absorption accelerators such as quaternary ammonium salt, sodium dodecyl sulfate, etc., lubricants such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets may be further formulated into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer and multilayer tablets.

For the preparation of the dosage unit into a pill, various carriers well known in the art can be widely used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oils, polyvinylpyrrolidone, gelucire, kaolin, talc, etc., binders such as acacia, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or batter, etc., disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.

For preparing the dosage unit into suppositories, various carriers well known in the art can be widely used. Examples of the carrier include polyethylene glycol, lecithin, cocoa butter, higher alcohols, enzymes of higher alcohols, gelatin, semisynthetic glycerases, and the like.

In order to capsule the dosage unit, the active ingredient is mixed with the various carriers described above, and the mixture thus obtained is placed in a hard gelatin capsule or a soft capsule. The active ingredients can also be made into microcapsule, suspension in aqueous medium to form suspension, or hard capsule or injection.

For example, the compositions of the invention are formulated for injection, such as solutions, suspension solutions, emulsions, freeze-dried powder injection solutions, which may be aqueous or non-aqueous, and may contain one or more pharmaceutically acceptable carriers, diluents, binders, lubricants, preservatives, surfactants or dispersants. For example, the diluent may be selected from water, ethanol, polyethylene glycol, 1, 3-propanediol, ethoxylated isostearyl alcohol, polyoxy isostearyl alcohol, polyoxyethylene sorbitol lipase, and the like. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and further, a conventional cosolvent, a buffer, a pH adjuster, and the like may be added. These adjuvants are commonly used in the art.

In addition, colorants, preservatives, flavors, flavoring agents, sweeteners, or other materials may also be added to the pharmaceutical formulation, if desired.

For the purpose of administration, the drug or the pharmaceutical composition of the present invention can be administered by any known administration method to enhance the therapeutic effect.

The dosage of the pharmaceutical composition of the present invention to be administered depends on many factors such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, character and individual response of the patient or animal, the administration route, the number of administrations and the like, and thus the therapeutic dosage of the present invention may vary widely. Generally, the dosages of the compounds of the present invention used are well known to those skilled in the art. The amount of the actual effective drug contained in the final preparation in the pharmaceutical composition of the present invention can be appropriately adjusted to meet the requirements of the therapeutically effective amount thereof, thereby achieving the purpose of preventing and treating inflammation, fibrosis-related diseases and acute lung injury of the present invention.

Typically, for patients weighing about 75 kg, the daily dose of the compound of the invention is 0.001mg/kg body weight to 400mg/kg body weight, preferably 1mg/kg body weight to 200mg/kg body weight. The above-mentioned dosages may be administered in a single dosage form or in divided dosage forms, for example, two, three or four dosage forms, which are limited by the clinical experience of the administering physician and the administration regimen. The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents.

In a third aspect, the present invention provides an application of the compound FN of the formula (I) and pharmaceutically acceptable salts thereof and pharmaceutical compositions thereof in preparing medicines for treating and/or preventing inflammation, fibrosis related diseases and acute lung injury.

The inflammatory diseases include arthritis, nephritis, tracheitis, rhinitis and pharyngitis. The fibrotic diseases include pulmonary fibrosis and hepatic fibrosis.

To achieve the object of the present invention, the present invention relates to the use of benzyl o-hydroxybenzoate FN and pharmaceutically acceptable salts thereof, pharmaceutically acceptable carriers and/or excipients, and pharmaceutical compositions thereof in inflammation, fibrotic diseases and acute lung injury.

Inflammation is a basic condition of inflammatory diseases such as arthritis, nephritis, tracheitis, rhinitis, pharyngitis and the like, and anti-inflammatory treatment is a necessary means for clinical treatment. FN has remarkable inhibition effect on croton oil induced otitis in mice, and the inhibition rates of the mice are 44.0%, 49.6%, 55.4% and 72.0% when the mice are dosed with body weight doses of 25, 50, 100 and 200mg/Kg, and the inhibition rate of the mice is obviously better than that of a positive control drug (26.0%) when the mice are dosed with equal doses (50 mg/Kg).

Anti-fibrosis can effectively slow down the disease progression of pulmonary fibrosis, hepatic fibrosis and the like. Hydroxyproline production in mouse lung fibroblasts (NIH 3T 3) induced by TGF-beta 1 is adopted, and when tissue fibrosis is simulated in vitro, collagen fibers in the tissue are increased to reflect the fibrosis degree. FN significantly reduced the rise in hydroxyproline production in TGF-beta 1-induced NIH3T3 cells at doses of both 10. Mu.M and 20. Mu.M, demonstrating significant anti-fibrosis effects.

Acute lung injury is injury to alveolar epithelial cells and capillary endothelial cells caused by direct or indirect factors, resulting in acute hypoxic respiratory insufficiency. The invention prepares a mouse Acute Lung Injury (ALI) model by LPS trachea instillation induction, FN can obviously reduce the count level of total white blood cells, neutrophils and lymphocytes in BALF of an ALI mouse under the dosage of 7.5, 15.0 and 30mg/kg, and can obviously improve the abdominal aortic blood pO ₂、 SaO₂ and the pH value level of the ALI mouse and reduce the blood pCO ₂ level under the dosage of 12.5, 25.0, 50 and 100 mg/kg. Mice showed no toxic or side effect after FN doses, but 4 mice died in the positive drug indomethacin (15.0 mg/kg) group, and the dexamethasone groups (12.5 and 15.0 mg/kg) showed a poor and listlessness and obvious toxic or side effect.

According to a fourth aspect of the technical scheme, the invention provides a preparation method of a natural product shown in a formula (I).

In order to achieve the aim of the invention, the invention prepares the natural product FN with a new structure from the solidago decumbens, the molecular formula of which is C ₁₆H₁₆O₅ and the name of which is 2, 6-dimethoxy phenyl methyl 2-hydroxybenzoate. The method is characterized by comprising the following separation preparation steps:

Step one, the whole herb of Solidago decumbens is crushed, soaked in hot water for 1 hour, filtered while the hot state is still hot, the filtrate is removed, the residual residues are continuously extracted with 75% ethanol under reflux for 2 times, each time for 2 to 3 hours, the extracting solutions are combined, and the ethanol extract is obtained by decompression concentration.

And step two, adding a proper amount of water into the ethanol extract to prepare a suspension, extracting for 3 times by using equal volume of dichloromethane, combining the extracting solutions, and concentrating under reduced pressure to obtain the dichloromethane extract.

Separating the dichloromethane extract by silica gel column chromatography, performing gradient elution on petroleum ether/ethyl acetate in a ratio of 4:1,2:1 and 1:2, collecting 4:1 eluent, and concentrating under reduced pressure to obtain a separation section rich in FN.

And step four, further performing silica gel column chromatography on the FN-enriched separation section, eluting with dichloromethane, collecting eluent, concentrating under reduced pressure, and drying to obtain a natural product FN sample (a compound with the structure shown in formula (I)).

Compared with the prior art, the invention has the beneficial technical effects that (1) the invention firstly separates and identifies benzyl o-hydroxybenzoate natural product FN with a new structure from a medicinal plant Solidago decumbens, (2) the invention firstly discovers and proves that the compound has stronger anti-inflammatory, anti-fibrosis and anti-acute lung injury activities and relatively smaller toxic and side effects, and (3) the compound has higher content in plants, easily obtained raw materials, simple and feasible extraction and separation methods and good economic value and application prospect.

Detailed Description

The following examples further illustrate the invention, but the invention is not limited to these examples. The test materials used in the examples below are commercially available, except for the compounds of the present invention which are prepared.

Example 1 isolation and Structure identification of Compound FN

5.0Kg of whole herb of Solidago decumbens, crushing, soaking for 1 hour in hot water, filtering while the whole herb is hot, removing filtrate, and extracting the residual residue with 75% ethanol under reflux for 2 times each for 2-3 hours. Mixing the extractive solutions, concentrating under reduced pressure to obtain ethanol extract, adding water into the ethanol extract, suspending, extracting with equal volume of dichloromethane for 3 times, mixing the extractive solutions, concentrating under reduced pressure to obtain dichloromethane extract, separating the dichloromethane extract by silica gel column chromatography, gradient eluting with petroleum ether/ethyl acetate (4:1, 2:1, 1:2), collecting 4:1 eluate, concentrating under reduced pressure, subjecting the obtained sample section to silica gel column chromatography, eluting with dichloromethane, concentrating the eluate under reduced pressure, and drying to obtain natural compound (FN) 1500mg.

2, 6-Dimethoxyphenylmethyl 2-hydroxybenzoate (FN) as white powder .¹H NMR(500MHz, acetone-d₆),δ10.90(1H,s),7.59(1H,d,J＝7.6Hz),7.40(1H,J＝8.4Hz),7.35(1H,t,J＝7.8 Hz),7.06(1H,d,J＝8.3Hz),7.02(1H,t,J＝7.5Hz),6.61(1H,d,J＝8.4Hz),6.57(1H,d,J＝ 8.3Hz),5.42(2H,s),3.89(3H,s),3.87(3H,s).¹³C NMR(500MHz,acetone-d₆),δ170.7,162.8, 161.5,157.9,135.5,130.0,129.3,125.0,121.2,111.3,110.3,105.7,103.5,63.1,56.5.55.9. HRESI m/z:289.1068[M+H]⁺,cald for C₁₆H₁₆O₅,289.1071[M+H]⁺.

Pharmacological experiments:

Experimental example 1 inhibition of croton oil induced otitis in mice by FN

The experimental method comprises the steps of taking 18-22 g of Kunming male mice, and randomly dividing the mice into a model group, an FN group (25, 50, 100, 200 mg/kg) group and a positive control group (50 mg/kg) of indomethacin, wherein each group comprises 10 mice. The intraperitoneal administration was performed, and an equal volume of physiological saline was administered to the model group. After 30min of administration, 50 μl of 2% croton oil was applied to both front and rear sides of left ear of the mice, the mice were euthanized after 4h, both ears were cut off along auricle baseline, round ear pieces were respectively put down at the same parts of both ears with a punch with a diameter of 8mm, and the ear swelling degree (ear swelling degree=left ear piece weight-right ear piece weight) and ear swelling inhibition rate [ ear swelling inhibition rate (%) = (model group average ear swelling degree-administration group ear swelling degree)/model group average ear swelling group×100% ].

Experimental results:

The experimental results are shown in table 1, the FN of each administration dose has remarkable inhibitory activity (P <0.05 or 0.001) on croton oil induced ear swelling of mice, the inhibition rate of 200mg/Kg dose can reach 72%, and the FN shows remarkable dose dependency. Compared with the positive control medicine with equal dosage, FN has stronger activity of inhibiting the mice otitis induced by the babysbreath oil.

Effects of fn on croton oil induced otitis in mice (mean±sd, n=10

Remarks p <0.05, p <0.001 compared to model control.

Experimental example 2 inhibition of TGF-beta 1 induced hydroxyproline production in mouse lung fibroblasts (NIH 3T 3) by FN

Hydroxyproline (HYP) is a unique amino acid in tissue collagen, and accounts for about 13% of the total amino acid content of collagen, and can be used as an important index of connective tissue collagen metabolism. When the tissue is fibrosed, the main component increased in the tissue is collagen fibers, and HYP is peculiar to collagen, so that the content of HYP in the tissue can reflect the degree of fibrosis. This experiment observes the effect of FN on the production of HYP in TGF-beta 1 induced mouse lung fibroblasts (NIH 3T 3) with its inhibitory effect on pulmonary fibrosis.

The experimental method comprises the following steps:

NIH3T3 cells in logarithmic growth phase were inoculated into 24-well plates at 1X 10 ⁵ cells/well in DMEM medium containing 10% FBS, cultured in an incubator at 37℃and 5% CO ₂ for 24 hours, and then further cultured for 6 hours by changing to DMEM medium without serum. TGF-beta 1 was added to each well to a final concentration of 10ng/ml except for the blank, and after incubation for 24 hours, the cell supernatants were collected and assayed according to the hydroxyproline assay kit instructions. After collection of the supernatant, MTT-containing medium was added to the well plate and incubated in a 37℃5% CO2 incubator for 4 hours, and the MTT method was used to determine cell proliferation.

Experimental results:

The experimental results are shown in table 2, and compared with the blank control group, the model group and the FN administration group have no significant effect on cell proliferation, which indicates that neither model stimulation nor FN has toxic inhibition effect on cells. Compared with the model group, FN can significantly reduce the rise of hydroxyproline production in the NIH3T3 cells induced by TGF-beta 1, which suggests that FN can inhibit fibrosis of the NIH3T3 cells to a certain extent.

Table 2. Effects of FN on TGF-. Beta.1 induced production of hydroxyproline in NIH3T3 cells (mean+ -SD, n=3)

Note that ^## p <0.01 compared to the blank control and p <0.001 compared to the model control.

Experimental example 3 Effect of FN on Acute Lung Injury (ALI)

(1) Effects of FN on LPS-induced pulmonary inflammation in mice ALI

The experimental method comprises the following steps:

18-22g male Kunming mice were randomly divided into a blank group, a model group, FN groups (7.5, 15, 30 mg/kg) and positive control groups (indomethacin 15mg/kg; dexamethasone, 15 mg/kg), 8 each. Each group of animals was given by intraperitoneal injection 1 time a day for 4 consecutive days, and the blank group and model group were given an equal volume of physiological saline. After 1h of administration on day 3, animals were anesthetized by intraperitoneal injection with 0.1ml/10g of 0.6% pentobarbital sodium, and 50 μl of LPS (2.4 mg/ml) solution was slowly instilled intratracheally, and an equal volume of physiological saline was administered to the blank group. Animals were sacrificed 1h after dosing on day 4, bronchoalveolar lavage fluid (BALF) was taken and leukocyte differential counts were performed on a five differential hematology analyzer.

Experimental results:

As shown in the results of table 3, total white blood cells, neutrophils, lymphocytes and monocytes/macrophages were all significantly elevated in BALF in ALI model mice compared to the blank, and FN, indomethacin and dexamethasone administration significantly reduced total white blood cell, neutrophil, lymphocyte count levels in BALF in model mice. The positive drug indomethacin group has the advantages that 4 mice die, and the dexamethasone group mice have weak body type and listlessness and show obvious toxic and side effects, compared with the positive drug indomethacin group, the FN group mice with each dose have good state and do not show obvious toxic and side effects.

TABLE 3 Effect of FN on differential count of leukocytes in ALI mouse BALF (mean+ -SD)

Remarks ^##p＜0.01,^### p <0.001 compared to the placebo group and p <0.01 compared to the model control, effect of FN <0.001 (2) on LPS-induced ALI mouse arterial blood gas

The experimental method comprises the following steps:

Male Kunming mice (18-22 g) were randomly divided into blank, model, FN (12.5, 25, 50, 100 mg/kg) and positive control (12.5 mg/kg dexamethasone) groups, each of which was 8. Each group of animals was given by intraperitoneal injection 1 time a day for 4 consecutive days, and the blank group and model group were given an equal volume of physiological saline. After 1h of administration on day 3, animals were anesthetized by intraperitoneal injection with 0.1ml/10g of 0.6% pentobarbital sodium, and 50 μl of LPS (2.4 mg/ml) solution was slowly instilled intratracheally, and an equal volume of physiological saline was administered to the blank group. Animals were anesthetized by intraperitoneal injection of 1.0% sodium pentobarbital (0.1 ml/10 g) 1h after administration on day 4, sampled from the abdominal aorta, and immediately subjected to blood gas analysis on a blood gas analyzer (pHOx, NOVA).

Experimental results:

As shown in the results of table 4, ALI model mice had significantly reduced arterial blood oxygen partial pressure (pO ₂), blood oxygen saturation (SaO ₂) and blood pH value (pH) and significantly increased blood carbon dioxide partial pressure (pCO ₂) compared to the blank group. The FN doses and dexamethasone can obviously raise the abdominal aortic blood pO ₂、SaO₂ and the pH value level of the model mice and lower the blood pCO ₂ level after being dosed. The FN mice in each dosage group are good in state, have no obvious toxic or side effect, and the dexamethasone mice in each dosage group are lean and listless in shape, and have obvious toxic or side effect.

Table 4. Effects of fn on ALI mice abdominal aortic blood gas (mean±sd, n=8)

Remarks are ^### p <0.001 compared to the blank and p <0.05, p <0.01, p <0.001 compared to the model group

In conclusion, the research results show that FN has remarkable anti-inflammatory activity, can remarkably inhibit TGF-beta 1 induced fibrosis, improve arterial blood gas injury of mice with acute lung injury, reduce lung inflammatory cell count of mice with acute lung injury, and has remarkable anti-inflammatory, anti-fibrosis and anti-acute lung injury pharmacodynamic activities. Moreover, compared with the positive medicine, FN has no toxic or side effect and has obvious low toxicity advantage. FN has very wide development and application prospect.

Claims (8)

1. A benzyl orthohydroxybenzoate compound FN of the formula (I) and pharmaceutically acceptable salts thereof:

2. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or adjuvant.

3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is selected from the group consisting of tablets, capsules, pills, and injections.

4. Pharmaceutical composition according to claim 2, characterized in that the pharmaceutical composition is selected from a slow release formulation and/or a controlled release formulation.

5. The pharmaceutical composition according to claim 2, wherein said pharmaceutical composition is selected from the group consisting of microparticle delivery systems.

6. Use of a compound according to claim 1 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment and/or prophylaxis of inflammatory, fibrotic related diseases and/or acute lung injury.

7. Use of a pharmaceutical composition according to claim 2 for the preparation of a medicament for the treatment and/or prevention of inflammation, fibrosis related diseases and/or acute lung injury.

8. The use according to any one of claims 6 or 7, wherein the inflammation-fibrosis related disorder includes arthritis, nephritis, tracheitis, pulmonary fibrosis, liver fibrosis.