CN1180065A - Antianaphylaxis, anti-asthma and anti-inflammatory new medicine - Google Patents

Abstract

After activity screening, separation and structural identification of more than 90 kinds of alpine medicinal plants, two new catechol derivatives were found, the structures of which are as follows:  is a single or double bond. Two new catechol derivatives have significant inhibitory activity against 5-lipoxygenase, and their IC ₅₀ are 43nM and 70nM, respectively. Therefore, these two active compounds may become drugs for treating diseases such as allergy, asthma and inflammation caused by leukotriene series substances produced by the action of 5-lipoxygenase in the process of human body lipid metabolism.

Description

A new anti-allergic, anti-asthma and anti-inflammatory drug

The invention belongs to a drug related to new catechol derivatives, their salts and 5-lipoxygenase activity inhibitors with them as active ingredients.

Due to the action of 5-lipoxygenase in the human body, arachidonic acid generates 5-hydroperoxyeicosatetraenoic acid, and then uses this compound as an intermediate to generate various leukotrienes. Among them, leukotriene B4 has a significant effect of promoting leukocyte swimming, which is the cause of inflammation. Leukotriene C4 and leukotriene D4 are allergy pathogenic factors.

Therefore, compounds that inhibit the activity of 5-lipoxygenase may be used to prevent and treat diseases caused by the metabolites of 5-lipoxygenase, such as bronchial asthma, nasal allergies, eye inflammation, and atopic reactions. Allergic diseases such as allergic dermatitis, edema, anemia, high blood pressure, anemia and brain disorders and other diseases of the circulatory system.

So far, the development of nearly 200 kinds of 5-lipoxygenase inhibitors, such as BW-755C developed by Wellcome, L-651896 developed by Merck, OND-LP-219 developed by Ono Pharmaceutical Co., Ltd. , absorbency and other issues and stopped.

The object of the present invention is to provide new catechol derivatives and their salts, and 5-lipoxygenase inhibitors containing them as active ingredients.

In order to solve the above problems, the present invention uses the inhibitory effect of 5-lipoxygenase activity as an indicator to separate and refine Chinese medicinal plants. The present invention has been accomplished by discovering a novel catechol derivative having a remarkable 5-lipoxygenase activity inhibitory effect.

According to the present invention, neocatechol derivatives represented by the following structural formulas can be provided.

(R represents a straight-chain or branched alkyl group containing 1 to 10 oxygen atoms or carbon atoms, preferably a lower alkyl group containing 1 to 4 carbon atoms such as methyl or ethyl; or a group containing 2 to 4 carbon atoms. Aliphatic and aromatic acyl groups with 10 carbon atoms, preferably lower aliphatic acyl groups such as acetyl and propionyl. represents a carbon-carbon single or double bond)

The compound of the present invention mainly exists in the wild plant Aletris glabra Bur.et Franch.(Liliaceae)] in Zhongtong and Sichuan Province. After drying and pulverizing the whole herb, extract it with 95% ethanol, remove the solvent, and separate and purify the obtained crude extract by appropriate separation methods such as column chromatography to obtain the compound represented by compound (I). The structure of the neocatechol derivative of the physiologically active substance represented by B(I) isolated by the above method was identified based on the analytical results of the spectroscopic method.

When the compound of the present invention is used as a medicine, the compound of the present invention is directly or added with the additives allowed in the medicine to make a pharmaceutical preparation for administration, or as a mixture of the two compounds, and the vermicelli containing them It is administered in the form of extract or extract (extract refers to the extract that removes the leaching solvent).

The pharmacologically acceptable salts in the present invention refer to: salts formed with inorganic bases, salts formed with organic bases, salts formed with amino acids, and the like. Inorganic base refers to: alkali metals, such as lithium, sodium, potassium; alkaline earth metals, such as magnesium, calcium and other inorganic bases formed, and other metals such as aluminum, lead, iron, etc. are also included in the present invention. Organic bases refer to amines, such as ammonia, primary amines, secondary amines, tertiary amines, nitrogen-containing heterocycles (including aliphatic and aromatic), etc. Specifically, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, propylamine, dipropylamine, isopropylamine, diisopropylamine, butylamine, dibutylamine, isobutylamine, tert-butylamine, Monoethanolamine, diethanolamine, triethanolamine, pyrrolidine, piperidine, morpholine, pyrrole, pyridine, etc.; amino acids refer to lysine, arginine, histidine, etc.

Since the present invention can inhibit the activity of 5-lipoxygenase, thereby inhibiting the generation of the metabolite leukotrienes of 5-lipoxygenase, it can treat and prevent mammals (such as people, cats, rabbits, guinea pigs, mice) and other allergic diseases such as bronchial asthma, nasal allergy, ophthalmia, atopic dermatitis, edema, anemia, hypertension, anemia and other circulatory system diseases caused by these metabolites.

The administration mode of the 5-lipoxygenase activity inhibitor in the present invention can be oral or parenteral. Dosage forms can be lozenges, tablets, capsules, powders, emulsifiers, suspensions, injections, etc. The above dosage forms are combined with their appropriate carriers, forming agents or other corresponding additives, and are produced by conventional methods.

The daily dosage of neocatechol derivatives in the present invention varies with the patient's condition, body weight, type of derivative, administration method and the like. For example, in the case of parenteral administration, subcutaneous, intravenous, intramuscular or rectal administration of 0.01-20 mg/kg or 0.1-10 mg/kg per day. In the case of oral administration, 0.1-1000 mg/kg or 1-10 mg/kg is administered daily.

The new catechol derivatives in the invention have inhibitory effect on 5-lipoxygenase activity and can be used as anti-allergic, anti-asthma and anti-inflammation drugs.

[Example]

The present invention is specifically described by the following examples, but the present invention is not limited to the following examples.

[instance 1]

1. The structure of 7-bis-(3,4-dihydroxyphenyl)-4,6-heptadiene-3-one is as follows:

The whole herb of Aletris glabra Bur.etFranch.(Liliaceae) collected in Sichuan Province, China, was dried, crushed, extracted with 95% ethanol at room temperature, and the extract was concentrated under reduced pressure below 60°C. 8.0 g of the obtained extract was subjected to centrifugal partition chromatography (CPC) (manufactured by Sanki Denki Co., Ltd.). The solvent system was a mixed liquid two-phase system of chloroform-methanol-isopropanol-water (7:6:2:5). Then, the silica gel 60F254 manufactured by Merck was used for separation, and the activities of the separated fractions were evaluated.

Active fraction obtained from CPC crude fraction: 164.7mg, Rf=0.3～1.0 (solvent: chloroform-methanol-isopropanol-water 4:3:1:2)

The active part obtained after crude fractionation of CPC is eluted with gradient chromatographic column chromatography of 20 grams of silica gel, and the solvent system is chloroform-methanol-water (100:10:1)→40:10:1→20:10:1→methanol . 26.0 mg of active fraction was obtained. Rf=0.4 (chloroform-methanol-water:70:10:1)

The above-mentioned active fraction was eluted by gradient chromatography using 16 g of silica gel, and the solvent was -chloroform-methanol-water 20:1:0→10:1:0→70:10:1. 16.6 mg of pure 5-lipase activity inhibitor was obtained.

The yield was 16.6mg. Rf=0.36 (chloroform:methanol:10:1)

With the said active pure product, according to the following spectral identification, its structure is the aforementioned novel catechol derivative compound. Proton NMR spectrum:

Using tetramethylsilane as an internal standard in deuterated chloroform, the proton nuclear magnetic resonance spectrum was measured at a frequency of 500 megacycles, and the data are as follows, unit: ppm.

δ2.78(m, 2H), 2.86(m, 2H), 6.22(d, 1H, J=15.6H ₂ ), 6.53(dd, 1H, J=8.1, 2.1Hz), 6.65(d, 1H, J =2.1Hz), 6.67(d, 1H, J=8.1Hz), 6.75(d, 1H, J=7.9Hz), 6.75(dd, 1H, J=15.6, 11.0Hz), 6.88(dd, 1H, J = 7.9, 2.1 Hz), 6.89 (d, 1H, J = 15.6 Hz), 6.99 (d, 1H, J = 2.1 Hz), 7.35 (dd, 1H, J = 15.6, 11.0 Hz). C-13 NMR spectrum:

C-13 NMR spectra were measured at a frequency of 125.8 megacycles in deuterochloroform with tetramethylsilane as an internal standard. The data are as follows, unit: ppm. δ31.2(t), 43.2(t), 114.7(d), 116.4(d), 116.6(d), 116.6(d), 120.7(d), 121.9(d), 125.0(d), 128.8(d ), 129.9(s), 134.2(s), 144.0(d), 144.6(s), 146.2(d), 146.2(s), 146.7(s), 148.5(s), 203.0(s).

[Example 2]

1,7-di-(3,4-dihydroxyphenyl)-1.4.6-heptatrien-3-one has the following structure:

Centrifugal distribution chromatographic crude fractionation obtained A～C, a～d 7 fractions, after activity evaluation of each fraction, 1.32 g of active fraction (part B) was obtained. Rf=0.3～1.0 (organic phase of chloroform-methanol-isopropanol-water 4:3:1:2).

The above-mentioned active part was eluted with column chromatography gradient elution with 75 g of silica gel, chloroform-methanol-water 100:10:1→60:10:1→40:10:1→20:10:1, and obtained A～O 15 parts, and activity evaluation was carried out. Wherein the yield of the active part (part F) is 78.2 mg, Rf=0.3 (chloroform-methanol-water, 70:10:1)

The aforementioned fractions were eluted with 16 g of silica gel, n-hexane-ethyl acetate 1:1→1:2→0:1→methanol for column chromatography to obtain 4 fractions A to D, and their activities were evaluated. The amount of active fraction (fraction D) was 43.1 mg, Rf = 0.35 (chloroform-methanol-water: 70:10:1).

The above-mentioned active fraction was purified by a medium-pressure column (type B), and the eluent was methanol-water 1:1→1:0, and the obtained fraction D was 11.9 mg of pure product of 5-lipoxygenase activity inhibitor. The yield of the active fraction (fraction D) was 11.9 mg, Rf = 0.38 (reverse phase, methanol-water 3:2).

The chemical structure of the above active ingredient was identified as a novel catechol derivative 1.7-bis-(3,4-dihydroxyphenyl)-1,4,6-heptatrien-3-one according to the following spectroscopic method. Mass Spectrum (EI-MS method) C ₁₉ H ₁₆ O ₅ : Calculated 324.0998, found 324.1024. Proton Magnetic Resonance Spectrum:

In deuterated chloroform, using tetramethylsilane as an internal standard, the proton nuclear magnetic resonance spectrum was measured at a frequency of 500 megacycles, and the data are as follows: unit ppm

δ6.64(d, 1H, J=15.1Hz), 6.76(d, 1H, J=8.4Hz), 6.80(d, 1H, J=8.3Hz), 6.86(dd, 1H, J=15.3, 10.7Hz ), 6.91(d, 1H, J=15.8Hz), 6.91(dd, 1H, J=8.4, 2.1Hz), 6.95(d, 1H, J=15.3Hz), 7.02(d, 1H, J=2.1Hz ), 7.04 (dd, 1H, J = 8.3, 2.1Hz), 7.12 (d, 1H, J = 2.1Hz), 7.52 (dd, 1H, J = 15.1, 10.1Hz), 7.59 (d, 1H, J = 15.8Hz). C-13 NMR spectrum:

In deuterated chloroform, tetramethylsilane was used as an internal standard, and the C-13 NMR spectrum was measured at a frequency of 125.8 megacycles. The data are as follows, in ppm.

δ114.8(d), 115.7(d), 116.7(d), 116.8(d), 122.0(d), 123.6(d), 123.7(d), 125.5(d), 128.5(s), 128.5(d) ), 130.1(s), 144.2(d), 145.7(d), 146.1(d), 146.9(s), 147.1(s), 148.6(s), 150.2(s), 191.9(s).

<Example 3>: 5-lipoxygenase activity inhibition experiment

The purpose of this experiment is to investigate the 5-lipoxygenase activity inhibitory effect of the compounds of the present invention.

1. Preparation of Enzyme Solution

Guinea pig basic leukemia cells (Rat Basophilic Leukemia Cell: RBL-1, ATCC CRL1378) were cultured in eagle medium containing 10% non-immobilized bovine fetal serum. , 0.1M HEPES, pH 7.0 solution in the proportion of 1 × 107/ml for dispersion. After the cells were crushed by ultrasonic waves, they were centrifuged at 13,000 rpm for 60 minutes, and the supernatant was the enzyme solution.

2. Determination of Enzyme Activity

Add 54ul of the above enzyme solution to a solution containing 1mM EDTA, 2mM calcium chloride, the substance to be tested (dissolved in DMSO), 0.1M HEPES, and a pH of 7.0, with a total volume of 120ul. After standing at 37°C for 10 minutes, add 6ul 0.4mg/ml arachidonic acid (methanol solution), react at 37°C for 60 minutes, add 120ul hexane to terminate the reaction, centrifuge at 13000 rpm for 10 minutes, and use high pressure The content of 5-hydroxyeicosatetraenoic acid contained in the supernatant was detected by liquid chromatography.

The results of the above assay are: the concentration at which 5-lipoxygenase activity is inhibited by 50% (IC ₅₀ ), 1.7-bis-(3,4-dihydroxyphenyl)-4,6-heptadiene-3- Ketone was 70 nM, 1.7-bis-(3,4-dihydroxyphenyl)-1,4,6-heptatrien-3-one was 43 nM.

Claims (4)

1. a kind of antiallergic, anti-asthma and anti-inflammation new medicine, it is characterized in that: active ingredient is new catechol derivatives and their pharmacologically allowed salt, structural formula is:

(R represents an oxygen atom or a linear or branched alkyl group with 1 to 10 carbon atoms, or an aliphatic and aromatic acyl group with 2 to 10 carbon atoms, Indicates that the carbon-carbon bond is a single bond or a double bond). 2. A new drug according to claim 1, characterized in that it is a drug composed of neocatechol derivatives and their pharmacologically acceptable salts. 3. A new drug according to claim 1, characterized in that: neocatechol derivatives and their pharmacologically acceptable salts are active ingredients of 5-lipoxygenase activity inhibitors. 4. A new drug according to claim 3, characterized in that it is used for treating allergies, anti-asthma or anti-inflammation.