CN108997453B

CN108997453B - Diphenyl ethyl ketone glycoside compound and preparation method and application thereof

Info

Publication number: CN108997453B
Application number: CN201810806487.0A
Authority: CN
Inventors: 袁崇均; 陈帅; 罗森; 谭正怀; 张磊; 余梦瑶; 王笳
Original assignee: Sichuan Academy of Chinese Medicine Sciences SACMS
Current assignee: Sichuan Academy of Chinese Medicine Sciences SACMS
Priority date: 2018-07-20
Filing date: 2018-07-20
Publication date: 2021-01-01
Anticipated expiration: 2038-07-20
Also published as: CN108997453A

Abstract

The invention provides a diphenylethanone glycoside compound shown as a formula (I) or a pharmaceutically acceptable salt thereof, wherein Glc represents glucose; r₁、R₂、R₃Each independently selected from hydrogen and C₁～C₆Alkyl group of (1). In conclusion, the compound obtained by structurally modifying the tectoridin skeleton has better pharmacological activity than that of tectoridin and generates new drug effect.

Description

Diphenyl ethyl ketone glycoside compound and preparation method and application thereof

Technical Field

The invention relates to a diphenylethanone glycoside compound and a preparation method and application thereof.

Background

Tectoridin (tectoridin) is derived from various plants of Iris and Belamcanda of Iridaceae, such as Belamcanda chinensis and Belamcanda chinensis, and has chemical name of 5, 4' -dihydroxy-6-methoxy-benzopyran-4-one-7-O-glucose, and chemical structural formula as follows:

the tectoridin has wide pharmacological activity, such as antiinflammatory, antibacterial, antioxidant, antiviral, free radical scavenging, lipid peroxidation injury resisting, atherosclerosis resisting, blood lipid reducing, anti-tumor, estrogen-like effect, and myocardial protection effect on myocardial infarction mice.

The structural modification of effective drugs to discover new drugs and new therapeutic effects is a constant theme in the pharmaceutical field, but is limited to various conditions, and the modification of belamcandin mainly focuses on 4 ' -hydroxyl, 7-O-glucosyl, H at positions 8, 3 ', 5 ', and carbonyl at position 4, etc., without changing the basic skeleton of belamcandin isoflavone, namely A, B, C ring structure, so that the pharmacokinetics of the obtained compound is poor, and the compound cannot be upgraded to new drug research.

Disclosure of Invention

The invention aims to modify the structure of the skeleton of the belamcandin so as to obtain the belamcandin derivative with new drug effect or improved pharmacological activity.

The invention firstly provides a diphenylethanone glycoside compound shown as a formula (I) or pharmaceutically acceptable salt thereof:

wherein Glc represents glucose;

R₁、R₂、R₃each independently selected from hydrogen and C₁～C₆Alkyl group of (1).

Further, the compound is:

the present invention also provides a process for preparing the above compound, which comprises the steps of:

(1) taking tectoridin, reacting with an aqueous alkali solution in the presence of an organic solvent, and adjusting the pH value to 6-7 to obtain a reaction solution;

(2) desalting the reaction solution, and recrystallizing to obtain the compound.

Further, in the step (1), the organic solvent is 55-95% V/V ethanol, preferably 75% V/V ethanol; the alkali is selected from sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium carbonate and potassium bicarbonate.

Further, in the step (2), the desalting method comprises the following steps:

a: concentrating and drying the reaction liquid obtained in the step (1), dissolving and filtering the reaction liquid by using 95% V/V ethanol, and concentrating and drying the filtrate to obtain a desalted sample;

or

B: and (2) washing the reaction liquid obtained in the step (1) by using a macroporous resin column until an effluent liquid is free of chloride ion reaction, eluting by using 70% V/V ethanol, filtering, concentrating and drying to obtain a desalted sample.

Further, in the step (2), the crystallization method is as follows: recrystallizing the desalted sample with 70% V/V ethanol to obtain the compound.

The invention also provides application of the compound or the pharmaceutically acceptable salt thereof in preparing a medicine or a cosmetic for preventing skin photodamage.

The invention also provides the application of the compound or the pharmaceutically acceptable salt thereof in preparing medicines for treating degenerative nervous system diseases, tumors and viral diseases; wherein the nervous system diseases are senile dementia and vascular dementia.

Further, the tumor is colon cancer, breast cancer, lung cancer; the virus is Coxsackie virus or adenovirus.

The invention also provides a composition for preventing skin photodamage, treating degenerative nervous system diseases, resisting tumors and viruses, which is a preparation prepared by taking the compound or the pharmaceutically acceptable salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

The preparation is liquid injection, lyophilized powder for injection, tablet, powder, granule, capsule, pill, oral liquid, and external preparation. Preferably, the external preparation is sunscreen cream, ointment, gel, honey, lotion, oil, solution.

In conclusion, the compound obtained by structurally modifying the tectoridin skeleton has better pharmacological activity than that of tectoridin and generates new drug effect. Specifically, the compound is an effective ultraviolet absorbent, can shield the contact of ultraviolet rays to human skin by absorbing the ultraviolet rays, blocks the ultraviolet rays and achieves the aim of preventing the ultraviolet rays from damaging the skin, and can be used for preparing medicines or cosmetics for preventing the skin from photodamage. And, the compound of the present invention is p.H₂O₂The induced PC12 cell injury has obvious protective effect; shows certain activity on HCT116, MCF-7 and A549 cell strains, and the effect is obviously better than that of a belamcandin control group. Meanwhile, the compound of the invention resists coxsackie virus CVB₂Effect and anti-Adenoviral ADV₃The effect is better than that of belamcandin.

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

"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.

The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, e.g. prefix (C)_a～C_b) Alkyl means any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, (C)₁～C₆) The alkyl group means an alkyl group having 1 to 6 carbon atoms.

Said C is₁～C₆Alkyl is C₁、C₂、C₃、C₄、C₅、C₆The alkyl group of (1) is a straight or branched alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, and the like.

Pharmaceutically acceptable salts refer to acid and/or base salts of the compounds of the invention with inorganic and/or organic acids and 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. Or by mixing the compound with a certain amount of an acid or a base as appropriate (e.g., an equivalent amount). 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.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

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 is an infrared spectrum of a compound of the present invention.

FIG. 2 is a mass spectrum of a compound of the present invention.

FIG. 3 is a hydrogen spectrum of a compound of the present invention.

FIG. 4 is a partial magnified view of the hydrogen spectrum of the compound of the present invention.

FIG. 5 is a carbon spectrum of a compound of the present invention.

FIG. 6 is a graph showing an ultraviolet absorption spectrum of a compound of the present invention.

FIG. 7 is a graph showing the UV absorption spectrum of 2-hydroxy-4-methoxybenzophenone as a control compound.

Detailed Description

The raw materials and instruments used in the invention can be purchased from the market.

Example 1 preparation of a Compound of the invention

1. Extraction of belamcandin

Drying rhizoma Belamcandae crude drug at 60 deg.C, pulverizing into coarse powder, sieving with 20 mesh sieve, placing in an extraction container, extracting with 95% V/V ethanol under reflux for 3 times, each time for 1 hr, and the solvent amount is 4 times (V/w) of the amount of the crude drug. Filtering while hot, combining the filtrates, and recovering the solvent at 60 deg.C under reduced pressure to obtain syrup (brown yellow extract) with specific gravity of 1.2g/ml (measured at 60 deg.C), wherein the yield is 49-51% (w/w) of the medicinal material. Adding 95% V/V ethanol (1/5V/w) into the above extract, stirring, and vacuum filtering to obtain crude product of tectoridin. Taking crude tectoridin, adding 95% V/V ethanol, heating and refluxing for 1 hr (solvent amount is 2 times of crude tectoridin, V/w), standing overnight, precipitating to obtain light yellow precipitate, filtering, recrystallizing the precipitate with 95% V/V ethanol for 1 time as above to obtain refined tectoridin (colorless crystalline powder, yield is 4.5% of medicinal material amount, content is > 98%).

2. Preparation of the Compounds of the invention

2.1 chemical reaction

Adding tectoridin 2kg into 75% V/V ethanol 20 times in 100L enamel reaction tank, heating and stirring, boiling, adding 5% W/V NaOH or KOH or Na₂CO₃Reacting for 60min with 2L of aqueous solution, discharging reaction liquid, and adjusting the pH value to 6-7 with hydrochloric acid for later use.

2.2 desalination

The method comprises the following steps: concentrating the above reaction solution in 100L evaporation pan to obtain extract, and drying at 60 deg.C under reduced pressure to obtain brown yellow dry powder. Stirring with 10L 95% V/V ethanol for 5 times, filtering, mixing filtrates, recovering solvent, and drying at 60 deg.C under reduced pressure to obtain brown yellow dry powder.

The method 2 comprises the following steps: on the above reaction solution D₁₀₁Macroporous adsorbent resin column (column specification: diameter 10cm × length 65cm), washing with water until effluent liquid has no chloride ion reaction, eluting with 70% V/V ethanol, filtering, recovering solvent, and drying at 60 deg.C under reduced pressure to obtain brown yellow dry powder.

2.3 crystallization

Recrystallizing the sample obtained after desalting with 70% V/V ethanol to obtain the compound of the invention, which is light yellow crystalline powder with the yield of 54% (based on the feed amount of belamcandin). Melting point mp is 122-124 ℃, and UV lambda is_max285nm，IR、MS、¹HNRM、¹³The CNRM is shown in figures 1-5 and is identified as 2, 6, 4' -trihydroxy-3-methoxydiphenylethanone-4-O-glucoside, and the chemical structural formula is as follows:

example 2 measurement of ultraviolet Spectroscopy

1, material:

compound prepared in example 1, control compound: 2-hydroxy-4-methoxybenzophenone

2 measurement method and results

Taking 15mg of the compound or the control compound, placing the compound or the control compound in a 50ml volumetric flask, dissolving the compound or the control compound in 70% V/V ethanol, diluting the solution to a scale, shaking the solution uniformly, precisely sucking 1ml of the solution in the 50ml volumetric flask, diluting the solution with 70% V/V ethanol, fixing the volume to the scale, shaking the solution uniformly, scanning the solution in a wavelength range of 200-400 nm by using 70% ethanol as a blank according to an ultraviolet-visible spectrophotometry (four parts 0401 of Chinese pharmacopoeia 2015 edition), and recording a spectrogram. The map is shown in figures 6-7. The results of the value measurement are shown in Table 1

As can be seen from FIGS. 6 and 7, the compounds of the present invention and the control compound both have absorption maxima in the UVB region (280-320 nm) (λ max is 285nm, 289nm, respectively).

Values for the Compounds of Table 1

Name (R)	Molecular weight	Weighing (mg)	Concentration C (mg/ml)	λ_maxnm	A	ε
							Compounds of the invention	452	19.46	0.007784	285.00	0.260	1.51×10⁴
Control Compounds	228	12.27	0.004908	289.00	0.301	1.40×10⁴

Value calculation formula: (A/C). times.molecular weight

The value is the lambda possessed by the target compound_maxMolar absorption coefficient at wavelength, generally considered to be greater than 10⁴For strong absorption, 10³～10⁴Is medium absorption, less than 10³Is weakly absorbed. Value as anti-UV combinationPreferably, the standard for determination of the substance is more than 15000. As can be seen from the above table, the inventive compounds have values greater than 15000 and greater than the control compounds.

The advantageous effects of the present invention are described below by way of test examples.

Test example 1, safety study and pharmacodynamic test of sunscreen skin care product

(1) Preparation of skin care product

Prescription:

compounds of the invention or control Compounds	1g
		Glycerol	15g
Propylene glycol	10g
		P-hydroxybenzoic acid ethyl ester	0.1g
Sodium metabisulfite	0.1g
		Triethanolamine	0.4g
Glyceryl monostearate	5g
		Stearic acid	10g
Liquid paraffin	10g
		White vaseline	0.5g
Rose essential oil	0.2g
		Distilled water is added to	100g

The preparation method comprises the following steps: heating stearic acid, glyceryl monostearate, white vaseline, and liquid paraffin to melt, mixing, filtering with fine cloth, and keeping the temperature at about 80 deg.C; dissolving the compound or the reference compound, propylene glycol, triethanolamine, ethyl p-hydroxybenzoate and sodium pyrosulfite in hot distilled water, slowly adding into oil phase at about 80 deg.C, and stirring. Adding rose essential oil when the paste is cooled to below 50 ℃, continuing stirring uniformly, and naturally cooling to room temperature to obtain the sunscreen skin care product.

In addition, the basic drugs except the compound of the invention or the reference compound are prepared into 100g of blank paste (matrix) according to the same method in proportion for testing.

(2) Safety research of sunscreen skin care product

Taking 12 adult healthy rabbits with the weight of 2.5-3.0 kg, randomly and averagely dividing the rabbits into 2 groups of 6 rabbits each, and selecting 2 multiplied by 2cm on the backs of two ears²Shearing rabbit hair at the symmetrical epidermis part of the area, coating a sunscreen skin care product on one side, coating a blank matrix on the other side, and observing whether the skin surface has red swelling, pimple and the like. As a result: no irritation reaction is generated on the ear back epidermis of the tested rabbit, and the skin care product group and the matrix group have no obvious difference.

(3) Pharmacodynamic test of sun-screening skin care product

The 18 rats were divided into 3 groups of 6 rats each and the chest and abdomen were removedWool 4cm²Respectively applying 1g of 2 skin care products and blank matrix, covering the rest part, and applying 40 biological doses of ultraviolet radiation (irradiating for 20min at a distance of 50cm from the ultraviolet lamp) to the application part. After the irradiation, the skin care product is washed off, and the observation is continuously carried out for one week. The results are shown in Table 3.

TABLE 3 protective action of sunscreen skin care products on rat sunburn

As can be seen from Table 3, the skin care products made with the compounds of the present invention are superior to the control skin care products.

Test example 2 Compound of the present invention to H₂O₂Protective effect of inducing PC12 cell damage

Taking PC12 cells in logarithmic growth phase at 1X 10⁵one/L was inoculated into a 96-well plate, 100. mu.1 per well, at 37 ℃ with 5% CO₂Culturing in incubator for 24h, adding tectoridin with final concentration of 0 μ g/mL, 0.01 μ g/mL, 0.1 μ g/mL, 1.0 μ g/mL and the compound of the invention for pretreatment for 24h, respectively, except for blank control group, and adding positive medicinal water soluble V_EControl group) were added separately with H at a final concentration of 60. mu. mol/L₂O₂And (3) continuing to culture for 20h, adding 20 mu L of MTT (5mg/mL) into each hole, continuing to culture for 4h, absorbing and removing the upper layer liquid, adding 100 mu L of DMSO into each hole, culturing for 15min at 37 ℃, and detecting the light absorption value of each group of cells at 490nm by using an enzyme-labeling instrument after the purple crystals are completely dissolved. According to the method (formula:

) Cell viability was calculated and the results are shown in table 4.

TABLE 4 drug of the invention vs. H₂O₂Protective effect of inducing PC12 cell damage

Remarking: comparing the first step with the blank group,^ΔP<0.05，^ΔΔP<0.01; comparison with model group<0.05，**P<0.01

MTT experiment shows that H₂O₂The OD value of the group is obviously reduced, and the cell survival rate is obviously lower than that of a normal control group (P)<0.0 l); while the ratio of cell survival rate H of the drug of the invention at different concentrations₂O₂The damage groups are obviously improved, have obvious difference and are in dose correlation, and the cell survival rate of the compound of the invention is related to the positive drug (water-soluble V)_E) The control group and the blackberry lily glycoside control group have better effects. The experimental results show that the compound of the invention is paired with H₂O₂The induced PC12 cell injury has obvious protective effect.

Test example 3 Effect of the Compound of the present invention on Nuclear proliferation of HCT116, MCF-7 and A549

Respectively taking human colon cancer cell strain HCT116, human breast cancer cell strain MCF-7 and human lung cancer cell strain A549 in log phase, digesting with pancreatin, blowing, preparing cell suspension, counting microscopically, adjusting cell concentration, and processing according to 6 × 10³One/well was inoculated into a 96-well plate and incubated overnight at 37 ℃. Adding each compound diluted in gradient into the drug treatment group, setting blank control group and tectoridin positive drug control group at the same time, repeating each concentration for 3 times, adding 5% CO at 37 deg.C₂The culture was carried out in an incubator for 48 hours, 4 hours before the end of the test, the culture medium in a 96-well plate was aspirated, 100. mu.L of LPBS buffer and 10. mu.L of 5mg/ml MTT solution were added, and the mixture was incubated at 37 ℃ for 4 hours. Add 100. mu.L of 10% SDS solution. Incubate overnight at 37 ℃. Determination of OD₅₇₀Computing IC₅₀. The results are shown in Table 5.

TABLE 5 in vitro antitumor Activity of Compounds of the invention against various human cancer cells

The results show that the compound of the invention shows certain activity on HCT116, MCF-7 and A549 cell strains, and the effect is obviously better than that of a belamcandin control group.

Test example 4 in vitro antiviral action of the Compound of the present invention

1 Main Instrument and Equipment

Medicine preparation: 1# medicine of the invention, 2# belamcandin (content > 98%), 3# ribavirin (Sichuan Mei Da kang pharmaceutical Co., Ltd., 0.1 g/tablet, lot number: 170110), the sample was dissolved with DMSO, and the volume was supplemented with MEM holding solution with calf serum final concentration of 2%. Reagent: calf serum, MEM culture solution, Hepes solution, Tryple Select 1 Xdigestive enzyme, and DPBS.

MEM nutrient solution: 0.055g of sodium pyruvate (Sigma, P-2256) was added to MEM liquid medium (GIBCO, cat. No. 11095-080) to give a final concentration of 0.11g/L, and 5ml of Hepes buffer solution (HyClone, SH30237.01) was dispensed by filtration through a 0.22 μm disposable needle filter, and 90ml of the solution was dispensed into each bottle and stored in a refrigerator at 4 ℃.

Cell growth liquid: 10ml of calf serum is added into every 90ml of the prepared MEM nutrient solution until the final concentration is 10%.

Cell maintenance solution: 2ml of calf serum is added into each 98ml of the prepared MEM nutrient solution until the final concentration is 2%.

Cell lines: vero and Hep-2 cells were purchased from cell banks of the China academy of sciences type culture Collection, and were continuously passaged 3 times in MEM medium containing 10% calf serum for logarithmic phase for experiments, and the rest was preserved in liquid nitrogen for a long period.

Virus strain: coxsackie virus B₂Type (CVB)₂) Adenovirus type 3 (ADV)₃) Is provided by Sichuan province disease control center, and is passed through the animal experiment center P2 laboratory of the institute of traditional Chinese medicine of Sichuan province, and TCID is determined before experiment₅₀。

The main items are as follows: cell culture dish, 96-hole culture plate, 200 mul pipettor, 1ml pipettor, 300 mul eight-channel pipettor, 0.22 um disposable needle filter.

The main equipment is as follows: super clean bench, full-row type biosafety cabinet and CO₂Cell culture case, inverted microscope, full-automatic enzyme mark appearance, full-automatic cell count appearance, analytical balance.

2 method of experiment

2.1 use of CPE Process for CVB₂And ADV₃The virulence of (A) was determined and the TCID was calculated separately₅₀。

2.2 Virus 100TCID₅₀Acting cells, setting normal cell control, virus control and ribavirin positive control with final concentration of 156 μ g/ml of each compound as treatment concentration, and determining CVB of each compound under the concentration₂And ADV₃The inhibition ratio of (3).

3 results of the experiment

CVB₂And ADV₃TCID of virus₅₀Are respectively 10^-6.3And 10^-4The concentration of the test virus was 100TCID₅₀。

1-3 pairs of compounds CVB₂The inhibition ratios of (A) and (B) are respectively as follows: 59.7%, 32.1% and 61.3%.

1-3 pairs of ADV compounds₃The inhibition ratios of (A) and (B) are respectively as follows: 41.5%, 13.4% and 51.2%.

The compound of the invention can resist coxsackie virus CVB₂The effect is superior to that of belamcandin and is equivalent to that of ribavirin; the medicine of the invention is anti-adenovirus ADV₃The effect is better than that of belamcandin and slightly weaker than that of ribavirin.

Claims

1. A diphenylethanone glycoside compound or a pharmaceutically acceptable salt thereof, characterized in that: the compound is:

2. a process for preparing a compound of claim 1, wherein: it comprises the following steps:

3. The method of claim 2, wherein: in the step (1), the organic solvent is 55-95% V/V ethanol; the alkali is selected from sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium carbonate and potassium bicarbonate.

4. The method of claim 3, wherein: in the step (1), the organic solvent is 75% V/V ethanol.

5. A method according to claim 2 or 3, characterized in that: in the step (2), the desalting method comprises the following steps:

or

6. The method according to any one of claims 2 to 4, wherein: in the step (2), the crystallization method comprises the following steps: recrystallizing the desalted sample with 70% V/V ethanol to obtain the compound.

7. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament or cosmetic for preventing photodamage to skin.

8. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a degenerative nervous system disease, a tumor, a viral disease; wherein the nervous system diseases are senile dementia and vascular dementia.

9. Use according to claim 8, characterized in that: the tumor is colon cancer, breast cancer or lung cancer; the virus is Coxsackie virus or adenovirus.

10. A composition for preventing photodamage to skin, treating degenerative nervous system diseases, and resisting tumor and virus, which is characterized in that: the compound or the pharmaceutically acceptable salt thereof as the active ingredient is added with pharmaceutically acceptable auxiliary materials to prepare a preparation; the preparation is liquid injection, lyophilized powder for injection, tablet, powder, granule, capsule, pill, oral liquid, and external preparation.

11. The composition of claim 10, wherein: the external preparation is sunscreen cream, ointment, gel, honey, lotion, oil and solution.