CN103739653B - A kind of 23-fall oleanane acid compound and preparation method thereof and the purposes in preparing glycosidase inhibitor - Google Patents

Abstract

The invention discloses compound 2 hydroxyl 3 carbonyl 23 and drop olive 1,4,12 triolefin 28 acid and preparation method thereof and the application in preparing glycosidase inhibitor.The present invention extracts from Three Akebia Decne Species and separates potent a glucosidase inhibitor, source of plant material is enriched, extraction preparation method is easily operated, and plant itself also can be made to be utilized for a long time without destruction when using fruit to extract, can increase economic efficiency, the most environmentally friendly, and this monomeric compound is stable, easy to store.Pharmacological evaluation shows, olive Isosorbide-5-Nitrae drops in compound 2 hydroxyl 3 carbonyl 23, and a glucosidase inhibitory active of 12 triolefin 28 acid is than strong about 16 times of a line diabetes medicament acarbose, it is expected to be further development of the medicine of clinical treatment type Ⅱdiabetes mellitus, promise well.

Description

A kind of 23-noroleananoic acid compound and its preparation method and in the preparation of glycosidase Uses in Inhibitor Drugs

Technical field:

The invention belongs to the field of natural medicinal chemistry, and in particular relates to a new 23-noroleanolic acid compound, namely 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28 - the acid, the preparation method of the compound, and the application of the compound or its pharmaceutically acceptable salt or its esterified derivative in the preparation of glycosidase inhibitor drugs.

Background technique:

Diabetes is a common clinical endocrine and metabolic disorder disease, which has an important correlation with the annual increase in cardiovascular diseases and cancers, and is a potentially important killer of human health. With the progress of society and the improvement of people's living standards, the incidence of diabetes is increasing worldwide. In my country, the prevalence of diabetes is more than 100 million, and it shows a trend of increasing year by year. Diabetes is causing more and more serious losses to our people's health and national economy.

Diabetes in western medicine is divided into type Ⅰ diabetes (or insulin-dependent, DM1) and type Ⅱ diabetes (or non-insulin-dependent, DM2). bigger. Competitive α-glucosidase inhibitors have the functions of delaying the digestion and absorption of carbohydrates, reducing the burden on the kidneys, controlling the sharp rise in blood sugar after meals, and reducing the fluctuation of blood sugar concentration within a day. Drugs can be developed for the treatment of type 2 diabetes. Important α-glucosidase inhibitors that have been developed and marketed and are currently being clinically tested as first-line drugs for the treatment of type 2 diabetes include acarbose (acarbose), voglibose, miglitol, and emigliate.

Diabetes is also known as diabetes in Chinese medicine. There are nearly 200 single Chinese medicines that can be used to treat diabetes in the Compendium of Materia Medica, which shows that the discovery of new α-glucosidase inhibitors from Chinese medicine or plant sources has important prospects. It has been reported in the literature that Akebia genus plants have pharmacological functions such as anti-inflammatory and diuretic, and are rich in compounds such as triterpenes and nortriterpenes. However, the research on pharmacological activities and components is generally not deep enough. Recently, we found that the plant tissue extracts of this genus have a certain activity of inhibiting α-glucosidase, so it has important potential in exploring new, efficient and safe α-glucosidase inhibitors.

Invention content:

The first object of the present invention is to provide a new 23-nortriterpenoid compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triterpenoid with α-glucosidase inhibitory activity En-28-acids.

The 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid of the present invention has a structural formula as shown in formula (I):

The second object of the present invention is to provide a preparation method of compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid, characterized in that compound 2- Hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid is derived from Akebia quinata (Thumb.) Decne., Akebiatrifolia (Thumb.) Koidz ), Akebia longeracemosa Matsumura, white Akebia (Akebiatrifolia(Thumb.) Koidz.Var.australis(Diels)Rehd) or Akebiatrifolia(Thumb.)Koidz..subsp.LongisepalaH.N. Qin) prepared from stems, leaves or fruits. The specific material can be dried or fresh, preferably dried fruit of the plant.

The specific steps are preferably:

a. Preparation of the total extract: crush the collected stems, leaves or fruit materials of Akebia trifoliata, Akebia trifoliata, Akebia trifoliata, Akebia trifoliata, or Akebia trifoliata, and then extract them with ethanol aqueous solution or acetone aqueous solution to extract Concentrate the liquid to remove the organic solvent to obtain the crude extract of the total extract, suspend the crude extract of the total extract in water, extract with petroleum ether or ethyl acetate, and obtain the total extract after the extract is concentrated;

b. Separation and purification: the total extract is subjected to normal phase silica gel column chromatography, using petroleum ether/acetone as the eluent, sequentially from the volume ratio of 100:0, 20:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 0:100 gradient elution, collect the fractions eluted with petroleum ether/acetone 5:1, and then go through normal phase silica gel column chromatography. Ratio 100:0, 10:1, 8:1, 6:1, 4:1, 2:1, 0:100 is the mobile phase gradient elution, collect petroleum ether/acetone 6:1 eluted fractions, and then Sephadex LH-20 gel column separation and purification was eluted with acetone, and the eluate was recrystallized to obtain the pure compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12 represented by formula (I) -triene-28-oic acid.

The ethanol aqueous solution or acetone aqueous solution is preferably an ethanol aqueous solution or acetone aqueous solution with a volume fraction greater than or equal to 70%.

The new compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid of the present invention has been confirmed by in vitro pharmacological experiments that it has a strong effect on α-glucosidase Inhibitory effect, its inhibitory activity (IC ₅₀ =24.8±0.28μM) was even stronger than the positive control acarbose (IC ₅₀ =408.78±5.67μM). Therefore, the new compound is a stronger alpha-glucosidase inhibitor than acarbose, can be developed for the preparation of drugs for preventing and treating type II diabetes, and has wide application potential.

The third object of the present invention is to provide 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid, its pharmaceutically acceptable salt or its esterified derivatives in Application in the preparation of α-glucosidase inhibitor drugs.

The fourth object of the present invention is to provide an α-glucosidase inhibitor drug, characterized in that it contains an effective amount of the compound 2-hydroxyl-3-carbonyl-23-norolean-1,4,12- Triene-28-acid or its pharmaceutically acceptable salt or its esterified derivatives, and commonly used pharmaceutical excipients or carriers.

The fifth object of the present invention is to provide the stems, leaves or fruits of Akebia clover, Akebia clover, Akebia changxu, Bai Akebia or Akebia longifolia in the preparation of compound 2-hydroxy-3-carbonyl-23-norqi Application of aconic-1,4,12-trien-28-oic acid.

The new compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid or its pharmaceutically acceptable salt or esterification derivative described in the present invention, its essential The active ingredients for inhibiting glycosidase are all compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid molecules. The pharmaceutically acceptable salt of the 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid, the essence of which inhibits α-glucosidase is in the human digestive tract in Under physiological conditions such as stomach acid, it can be converted into the active molecule 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid to function. The esterification derivative of the compound 2-hydroxyl-3-carbonyl-23-norolean-1,4,12-triene-28-acid refers to the compound 2-hydroxyl-3-carbonyl-23-norolean A derivative compound in which the 2-hydroxyl group in the 1,4,12-triene-28-acid molecule is esterified with an organic acid or the 28-position carboxyl group in the molecule is esterified with an alcohol compound, and the esterification derivative The compound can be a partial or full esterification of one or both of the 2-hydroxyl and 28-carboxyl functional groups, which are based on 2-hydroxy-3-carbonyl-23-norolean-1,4 , an esterified derivative of the molecular skeleton of 12-triene-28-acid, which can be easily converted into the active molecule 2-hydroxy-3-carbonyl-23-norolean in the human digestive tract under physiological conditions such as gastric acid or intestinal alkali Fruit-1,4,12-triene-28-acid, its essence is also the compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid from α-glucoside Enzyme inhibitory activity thus falls within the strict protection scope of the present invention. Wherein the organic acids and organic alcohols that are respectively esterified with the 2-position hydroxyl group and the 28-position carboxyl group can be in any form that can hydrolyze the relevant ester bonds under physiological acid-base conditions, preferably C1 to C4 that can enhance the water solubility of the entire molecule Small molecule organic acids and alcohols, as well as various small and medium molecular organic acids or organic alcohols from C6 to C10 containing benzene rings.

The new compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid of the present invention or its pharmaceutically acceptable salt or esterified derivative can be combined with pharmaceutically commonly used excipients Or combined with a drug carrier, prepared with 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid to inhibit the activity of α-glucosidase, which can be used to prevent and treat type II Medicine or pharmaceutical composition for diabetes. The medicine or pharmaceutical composition can adopt dosage forms such as wettable powder, tablet, granule, capsule, oral liquid, drop pill, injection, aerosol; nano formulations.

The present invention extracts and separates potent α-glucosidase inhibitors from widely distributed plants of the genus Akebia in my country. The source of the material is rich, the preparation process is simple and easy to operate, and the plant itself can be extracted without the use of plant fruits. It can be used for a long time after being destroyed, and it can also be environmentally friendly while achieving better economic benefits, and the monomer compound is stable and easy to store. The α-glucosidase inhibitory activity of the compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-oic acid is even higher than that of the clinical drug acarbose, and it is likely to be further developed It is an effective and safe new α-glucosidase inhibitor drug for the prevention and treatment of type Ⅱ diabetes, which has a good market prospect.

Description of drawings:

Fig. 1 is the ¹ HNMR spectrum of compound 2-hydroxyl-3-carbonyl-23-norolean-1,4,12-triene-28-acid;

Fig. 2 is the ¹³ CNMR spectrum of compound 2-hydroxyl-3-carbonyl-23-norolean-1,4,12-triene-28-acid;

Figure 3 is the HMBC spectrum of the compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-oic acid.

detailed description:

The following examples are further descriptions of the present invention, rather than limitations of the present invention, and simple improvements to the present invention according to the essence of the present invention all belong to the scope of protection of the present invention.

Example 1: Preparation of 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid in Akebia clover fruit

1.1 Instruments and reagents

The decompression concentration adopts N-1000 rotary evaporator of Tokyo Physical and Chemical Company, CCA-1110 circulating cooling box and SB-1000 electric heating constant temperature water bath; HPLC adopts Japan Shimadzu Company LC-20AT liquid chromatograph, SPD-M20A detection and Shim-PackPRC-ODS chromatographic column (particle size 5μm, pore size 12nm, 250mm×20mm); electrospray mass spectrometry (ESIMS) adopts MDSSCIEXAPI2000LC/MS/MS instrument from Applied Biosystems, USA, and uses methanol as solvent for direct sample determination; ¹ HNMR spectrum and ¹³ CNMR spectrum were measured by BrukerDRX-400 nuclear magnetic resonance instrument, and tetramethylsilane was used as internal standard. The color development method adopts 10% sulfuric acid ethanol solution or sulfuric acid vanillin treatment, and then heats the color or iodine vapor to develop the color.

1.2 Plant source and identification

The fruit samples of Akebiatrifolia (Thumb.) Koidz., the plant material used for extraction, were collected from Hunan Province in September 2009 and identified by Xing Fuwu, a researcher at South China Botanical Garden, Chinese Academy of Sciences.

1.3 Extraction and separation

The sample (Akebia trifoliate fruit, dry weight 2.0 kg) was crushed and extracted three times with 95% ethanol aqueous solution at room temperature, the combined filtrate was concentrated under reduced pressure to remove the organic solvent, and the crude extract of the total extract was obtained. Suspend the crude extract of the total extract in 500ml of water, then extract with an equal volume of petroleum ether, and concentrate the extract under reduced pressure to obtain the total extract of petroleum ether (32g). Dissolve the petroleum ether total extract with 1:1 chloroform/methanol (100mL), add normal phase silica gel (80-100 mesh) at a weight ratio of 1:1.5, mix the sample and evaporate to dryness, dry-pack the column (200-300 mesh , 800 g), dry loading, followed by petroleum ether / acetone = 100:0, 20:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 0 : 100v/v is mobile phase gradient elution. According to TLC detection, each fraction collects 9 components E1–E9 in sequence according to the difference in polarity; E5 (petroleum ether/acetone 5:1) The fractions that come down) are separated and purified by normal phase silica gel column chromatography (200-300 mesh, 50g grams), and the petroleum ether/acetone=100:0, 10:1, 8:1, 6:1, 4:1, 2:1, 0:100v/v is mobile phase gradient elution (each gradient elution is 300ml, every 15ml is collected as a component), according to the normal phase TLC plate detection, the eluents are collected and properly combined to obtain 7 Components E5-1-E5-7; E5-4 (petroleum ether/acetone 6:1 elution fraction) were separated and purified by SephadexLH-20 gel column (acetone), eluted with acetone, and collected according to TLC detection. Eluent, obtain 7 components E5-4-1-E5-4-7, component E5-4-7 (this component carries out normal phase TLC detection with chloroform/methanol 9:0.25 as developer, and with 10% sulfuric acid-ethanol was sprayed and heated to develop color, and the main component showed purple-red spots with Rf=0.8) and then recrystallized multiple times with methanol as a solvent to obtain a colorless (white) powder compound 1 (2-hydroxy-3- Carbonyl-23-norolean-1,4,12-trien-28-oic acid) (8 mg).

1.4 Structural identification of compound 1 (2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-oic acid)

The obtained compound 1 is a white amorphous powder with a molecular formula of C ₂₉ H ₄₀ O ₄ ; UV(MeOH)λ _max nm(logε): 203(4.13), 262(3.8); HRESIMS(pos.) m/z475.2816(calcd.forC ₂₉ H ₄₀ NaO ₄ ,475.2819); ESIMS(pos.)m /z475[M+Na]+, 491[M+K]+, (neg.)m/z451[MH]–, 487[M+Cl] ^– ; ¹ H-NMR and ¹³ C-NMR data are shown in Table 1 Shown:

Table 1. NMR data (inCD ₃ OD) of the obtained compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-oic acid

According to the comprehensive analysis of the above ultraviolet, mass spectrometry and one-dimensional and two-dimensional NMR data, the chemical structure of the new compound 1 is deduced as 2-hydroxy-3-carbonyl-23-norolean-1,4,12 -triene-28-acid, the structure of which is shown in formula (I).

Example 2: Preparation of 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid in the stems and leaves of Akebia trifoliata

2.1 Instruments and reagents: same as Example 1

2.2 Plant source and identification: same as Example 1

2.3 Extraction and separation

The sample (Akebia trifoliate stems and leaves, dry weight 2.0 kg) was crushed and extracted three times with 95% ethanol aqueous solution at room temperature, and the combined filtrate was concentrated under reduced pressure to remove the organic solvent to obtain the crude extract of the total extract. Suspend the crude extract of the total extract in 500ml of water, then extract with an equal volume of petroleum ether, and concentrate the extract under reduced pressure to obtain the total extract of petroleum ether (24g). Dissolve the total extract of petroleum ether with chloroform/methanol (100mL) with a volume ratio of 1:1, add normal phase silica gel (80-100 mesh) and mix the sample with a weight ratio of 1:1.5, evaporate to dryness, and dry-pack the column (200- 300 mesh, 800g), dry loading, sequentially use petroleum ether/acetone=100:0, 20:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1 , 0:100v/v is mobile phase gradient elution, according to TLC detection, each fraction collects 9 components F1-F9 according to the difference in polarity from small to large; F5 (petroleum ether/acetone 5:1 The fractions eluted) were separated and purified by normal phase silica gel column chromatography (200-300 mesh, 50g gram), with petroleum ether/acetone=100:0, 10:1, 8:1, 6:1, 4: 1, 2:1, 0:100v/v is the mobile phase gradient elution (each gradient elution is 300ml, and each 15ml is collected as a component), and the eluents are collected and properly combined according to the normal phase TLC detection to obtain Seven components F5-1-F5-7; F5-4 (petroleum ether/acetone 6:1 elution fraction) were separated and purified by Sephadex LH-20 gel column (acetone), eluted with acetone, and detected according to TLC , collect eluate, obtain 7 components F5-4-1-F5-4-7, component F5-4-7 (this component carries out normal phase TLC detection with chloroform/methanol 9:0.25 as developer, And spray heating with 10% sulfuric acid-ethanol to develop color, the main component presents a purple spot with Rf=0.8) and then use methanol as solvent to carry out multiple recrystallization to obtain a colorless (white) powder compound 2-hydroxy-3- Carbonyl-23-norolean-1,4,12-trien-28-oic acid (5 mg).

Example 3:

Take the stem, leaf or fruit of Akebia akebiae, Akebia changxu, Akebia basilicate, or Akebia clover as samples, and finally purify according to the extraction and separation methods described in Example 1 to obtain the pure compound of formula (I) 2,3 ,20-Trihydroxy-29-norolean-12-ene-28-oic acid.

Example 4: Detection of α-glucosidase inhibitory activity of 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-oic acid

4.1 Instruments and reagents

Experimental instrument: Genoismmicroplatereader (TecanGENios, Swizerland)

Reagents and compound samples: α-glucosidase was purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis, USA); 4-nitrophenol-α-D-glucopyranoside (PNPG) was purchased from Tokyo Chemical Industry Co., Ltd. (Japan); Acarbose, purchased from Tokyo Chemical Industry Co., Ltd. (Japan); 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-oic acid Prepared from the above experimental example

4.2 Test method:

a) Preparation of drug solution: 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-oic acid and acarbose were prepared from dimethyl sulfoxide (DMSO) respectively 10mg/ml solution, and prepare 67mmol of phosphate buffer solution (prepared with ultrapure water), PNPG substrate solution (5mM, prepared with phosphate buffer solution), and 0.2M NaCO ₃ solution (prepared with phosphate buffer solution).

b) Half of the compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-oic acid was tested against α-glucosidase in a 96-well cell culture plate using a colorimetric method The inhibitory concentration was determined. First, 20 μL of α-glucosidase (0.8 U) is added to the sample well, then the test sample solution is diluted in a certain proportion with phosphate buffer, and 120 μL of the sample solution is added to each well to make the test sample (2-hydroxyl-3- Carbonyl-23-norolean-1,4,12-trien-28-oic acid or acarbose) at final concentrations of: 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 μg/mL, 15.625 μg/mL, and finally add 20 μL (5 mM) of the reaction substrate 4-nitrophenol-α-D-glucopyranoside. After reacting in a water bath at 37°C for 15 minutes, 80 μL of NaCO ₃ (0.2M) was added to each sample well to terminate the reaction, and the colorimetric measurement was performed at a wavelength of 405 nm. The same volume of phosphate buffer was used instead of the enzyme solution. The inhibition rate of the compound is calculated from the OD value of the sample to the OD value of the blank and the control, and the calculation formula is as follows: Inhibition rate (%)=(OD _control –OD _neg )-(OD _test –OD _{test control} )/(OD _control –OD _neg )× 100%. The half inhibitory concentration (IC ₅₀ ) of the test compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid on α-glucosidase was obtained from the dose-effect curve.

4.3 See Table 2 for experimental data:

Table 2. α-glucosidase inhibitory activity of 2-hydroxy-3-carbonyl-23-norolean-1,4,12-trien-28-oic acid

4.4 Experimental conclusion:

α-glucosidase is an index test enzyme for drug screening of α-glucosidase inhibitors. Many drugs for treating diabetes are developed into hypoglycemic drugs based on the competitive inhibition of α-glucosidase. The results of this experiment show that the new compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid has a strong inhibitory effect on a-glucosidase. Its inhibitory activity is even about 16 times stronger than that of the positive control substance, that is, the first-line hypoglycemic drug acarbose, so it has strong development potential and is expected to be further developed into a new drug for the prevention and treatment of type Ⅱ diabetes. widely.

Claims (6)

1. The compound 2-hydroxyl-3-carbonyl-23-norolean-1,4,12-triene-28-acid or a pharmaceutically acceptable salt thereof, its structural formula is as shown in formula (I): 2. a method for preparing compound 2-hydroxyl-3-carbonyl-23-norolean-1,4,12-triene-28-acid according to claim 1, characterized in that, a. Preparation of the total extract: crush the collected stems, leaves or fruit materials of Akebia trifoliata, Akebia trifoliata, Akebia trifoliata, Akebia trifoliata, or Akebia trifoliata, and then extract them with ethanol aqueous solution or acetone aqueous solution to extract Concentrate the liquid to remove the organic solvent to obtain the crude extract of the total extract, suspend the crude extract of the total extract in water, extract with petroleum ether, and obtain the total extract after the extract is concentrated; b. Separation and purification: the total extract is subjected to normal phase silica gel column chromatography, using petroleum ether/acetone as the eluent, sequentially from the volume ratio of 100:0, 20:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 0:100 gradient elution, collect the fractions eluted with petroleum ether/acetone 5:1, and then go through normal phase silica gel column chromatography. Ratio 100:0, 10:1, 8:1, 6:1, 4:1, 2:1, 0:100 is the mobile phase gradient elution, collect petroleum ether/acetone 6:1 eluted fractions, and then Sephadex LH-20 gel column separation and purification was eluted with acetone, and the eluate was recrystallized to obtain the compound 2-hydroxy-3-carbonyl-23-norolean-1,4,12-triene-28-acid . 3. The preparation method according to claim 2, characterized in that, the aqueous ethanol solution or the aqueous acetone solution is an aqueous ethanol solution or an aqueous acetone solution with a volume fraction greater than or equal to 70%. 4. 2-hydroxyl-3-carbonyl-23-norolean-1,4,12-triene-28-acid or pharmaceutically acceptable salt thereof according to claim 1 is used in the preparation of α-glucosidase inhibitory application in pharmaceuticals. 5. An α-glucosidase inhibitor drug, characterized in that it contains an effective amount of the compound 2-hydroxyl-3-carbonyl-23-norolean-1,4,12-tri Alkene-28-acid or its pharmaceutically acceptable salt, and commonly used pharmaceutical excipients or carriers. 6. The stem, leaf or fruit of Akebia clover, Akebia clover, Akebia syringae, Akebia chinensis or Akebia syringae is preparing the compound 2-hydroxyl-3-carbonyl-23-norzidun according to claim 1 Application of fruit-1,4,12-trien-28-acid.