KR102385511B1 - Synthesis method of coumarin derivatives from extract Angelica gigas Nakai - Google Patents

Abstract

DN (Decursin) and DA (Decursinol angelate) are known to have anticancer, anti-inflammatory and fatty liver effects, but studies on the pharmacological efficacy of single substances of DN or DA for alcohol-induced fatty liver have not been conducted. In the present invention, the present invention was completed by confirming the liver protection function of these DNs or DAs, and by confirming a method for synthesizing them simply through continuous research and development for DN and DA.
That is, the compound of Structural Formula (1) and the NN-dimethylamino pyridine compound of Structural Formula 4, which is a reaction accelerator, were dissolved in CH ₂ Cl ₂ and reacted at 0° C. for 24 hours in the presence of nitrogen (N ₂₎ to form the structural formula ( It relates to the invention of a method for preparing the compound of 2) and preparing the compound of the structural formula (3) by reacting the compounds of the structural formulas (1) and (5).

Description

Synthesis method of coumarin derivatives from extract Angelica gigas Nakai}

Recently, the development of anticancer drugs containing decursin as an active ingredient is being actively carried out. Deckercin is a natural substance extracted from Angelica decursiva Fr. et Sav. for the first time in Japan in 1966. Years (J. Pharm. Soc. Korea, 11, pp. 22-26, 1967 and 13, pp. 47-50, 1969) were found, and the other plants were the fruits of the oilseed vegetable (Peucedanum terevinthaceum Fisher et Turcz.). (Journal of the Korean Pharmacopoeia, 30(2), pp.73-78, 1986).

A number of coumarin-based compounds and polyacetylene-based compounds such as decursin, decursinol, and decursinol angelate are separated is being reported The present invention relates to a method for synthesizing a coumarin derivative useful for liver disease.

Due to modern eating habits and excessive drinking, the number of people suffering from liver disease is increasing, and it has been reported that about 60% of all deaths are caused by alcohol. Alcoholic fatty liver is caused by continuous alcohol intake, and the stage of liver damage progresses to simple fatty liver, steatohepatitis, cirrhosis, and liver cancer as shown in [Figure 1] below.

[Figure 1]

The stages of simple fatty liver and steatohepatitis, which are the early stages of liver damage, are reversible and can return to a normal liver if only treatment and management are performed. It is a stage in which it is impossible to return to a normal liver again. When alcohol is ingested, a small amount is absorbed in the stomach and the remainder is absorbed in the duodenum or small intestine. Alcohol absorbed in this way is decomposed into acetaldehyde through two methods in the liver. One is decomposed by alcohol dehydrogenase, and the other is decomposed by cellular redox. It is degraded to acetaldehyde by the protein [Cytochrome P450 2E1 (CYP2E1)]. Oxidation of alcohol induces expression of CYP2E1 in the liver, and CYP2E1 produces superoxide anion radical and hydrogen peroxide, which are strong oxidizing agents in the presence of an Fe catalyst. ) will cause damage.

Angelica gigas (Angelica gigas) is a perennial plant belonging to the Dicotyledonous family, Dicotyledonous family, Umbilicalaceae, and its leaves and stems are eaten raw or eaten raw, and the roots are called Angelica gigas.

Angelica gigas Nakai. There are coumarin, a fat-soluble substance, and polysaccharide, a water-soluble substance. In the coumarine family, decursin (hereinafter referred to as “DN”) and decursinol angelate (hereinafter referred to as “DA”). ”), nodakenin and decursinol (hereinafter defined as “DL”), etc. DN and DA, which are the main active ingredients of these index components, are structural isomers with different side chain structures from DL (Decursinol), but they are very difficult to separate due to their similar physical properties. Although some methods for synthesizing are known, there is a problem in that the synthesis step is long and the yield is low.

Looking at conventional techniques for extracting or synthesizing coumarin-based compounds from Angelica keis, Korean Patent No. 1101011 discloses (+)-decursinol, a structural isomer of (+)-decursinol,- 8-hydroxy-7,7-dimethyl-8,9-dihydropyrano [2,3-g] coumarin ((8s)-8-hydroxy-7,7-dimethyl-8,9-dihydropyrano [2, 3-g] coumarin) and (6s)-6-hydroxy-7,7-dimethyl-5,6-dihydropyrano[3,2-f]coumarin ((6s)-6-hydroxy-7,7 -dimethyl-5,6-dihydropyrano[3,2-f]coumarin) was prepared and synthesized, and the (+)-decurcinol-restructural isomer synthetic derivative compound of a novel structure using this as a precursor, and health functional food using the same Deckersinol, a precursor of the anticancer drug deckersin, is known in Korea Patent No. 0365650, which shows a strong lethal action on cancer cells, while showing a much lower lethal action on normal cells compared to the lethal action on cancer cells. Although a technique for a method for synthesizing in a high yield is known, this also goes through a multi-step manufacturing process to finally produce decursin, but the manufacturing process is complicated and the yield is low. Korean Patent No. 0715206 relates to a method for producing decursinol, which is a precursor of decursin derivative synthesis, comprising: a first step of pulverizing Angelica gigas and extracting it with a solvent; a second step of suspending the solvent extract with water and then solvent fractionation with ether; a third step of directly hydrolyzing the ether layer with a base; a fourth step of extraction with acid; A method of extracting deckercinol through a fifth-step manufacturing method in which an acid extract is treated with activated carbon, filtered and dried under reduced pressure to recrystallize with a solvent is disclosed. Disclosed is a method of manufacturing a candy for improving immunity for children using Korean Patent Application Laid-Open No. 2014-40413 discloses a method of extracting decurcinol angelate from Angelica basil leaf and an invention related to a composition comprising the extracted decursinol angelate. In addition, Korean Patent No. 1404168 discloses a method of increasing the content and antioxidant activity of decursin or decursinol angelate by irradiating far-infrared rays on Angelica keiskei.

And, although DN and DA are known to be effective in anticancer, anti-inflammatory and fatty liver, etc., studies on the pharmacological efficacy of a single substance of DN or DA for alcohol-induced fatty liver have not been conducted, so in the present invention, these DNs The present invention was completed by confirming the liver protective ability function of or DA and confirming a method for synthesizing them simply through continuous research and development for DN and DA.

Domestic Patent No. 1101011 Domestic Patent No. 0365650 Domestic Patent No. 0715206 Domestic Patent No. 1192280 Domestic Patent No. 1404168 Domestic Patent No. 1404168 Domestic Patent Publication No. 2014-40413

Reuben, A. (2008) Alcohol and the liver. Curr. Opin. Gastroenterol. 24: 328-338. Heit, C.; Dong, H.; Chen, Y.; Thompson, D. C.; Deitrich, R. A.; Vasilioua, V. (2013) The Role of CYP2E1 in alcohol metabolism and sensitivity in the central nervous system. Subcell Biochem. 67, 235-247. Kuthan, H.; Ullrich, V.; Estabrook, R, W. (1982) A quantitative test for superoxide radicals produced in biological systems. Biochem. J. 203, 551-558 Lee, J. H.; Bang, H. B.; Han, S. Y.; Jun, J.-G. A (2006) Convenient total synthesis of (+)-decursinol from resorcinol. Bull. Kor. Chem. Soc. 27, 2104-2106. Nemoto, T.; Ohshima, T.; Shibasaki, M. (2000) Enantioselective total synthesis of novel PKC activator (+)-decursin and its derivatives using catalytic asymmetric epoxidation of an enone. Tetrahedron Lett. 41, 9569-9574.

Methods for extracting DN (Decursin) and DA (Decursinol angelate) from Angelica were developed, and also DN (Decursin) of Structural Formula (2) and DA (Decursinol) of Structural Formula (3) from DL (Decursinol) of Structural Formula (1) angelate), various methods have been tried, but there were difficulties such as having to go through several intermediate steps, complicated separation process, and using expensive starting materials to synthesize it. An object of the present invention is to simply synthesize the compounds of the structural formulas (2) and (3) from the compounds of the structural formula (1).

Another object of the present invention is to use an intermediate that is easy to purchase and easily reacts in synthesizing the compound.

Another object of the present invention is to facilitate separation and purification of reactants when synthesizing the compound, and to improve the yield of a target material.

In order to solve the above problems, the present inventors have extracted DL (deckercinol) obtained by extracting When reacting with 3,3-dimethylacryloyl chloride (3,3-dimethylacryloyl chloride), it was confirmed that DN (decursin) can be obtained, and also, when reacting DL with angeloyl chloride , it was confirmed that DA (decurcinol angelate) can be obtained.

In addition, an object of the present invention is to obtain a high yield while simply synthesizing the compounds of Structural Formulas (2) and (3).

In the conventional synthesis of DN(2) and DA(3), there was a high production cost and difficulties in mass production, but in the present invention, 3,3-dimethyl as a reactant when synthesizing DN(2) and DA(3) DN (77%) and DA (82%) in high yield were synthesized by reaction using acryloyl chloride and angeloyl chloride.

In addition, when DN and DA are synthesized according to the method of the present invention, the synthesis method is simple, the purchase of reactants is easy, and DN(2) and DA(3) prepared by the synthesis method of the present invention are continuously produced. It was confirmed through blood test and protein expression (western blotting) that it was effective in alleviating inflammation, disease, and stress caused by liver ingestion due to alcohol ingestion.

Figure 1: ¹ H nuclear magnetic resonance analysis of deckercinol (1).
Figure 2: ¹³ C nuclear magnetic resonance analysis of deckercinol (1).
Figure 3: ¹ H nuclear magnetic resonance analysis of deckersine (2).
Figure 4: ¹³ C nuclear magnetic resonance analysis of deckersine (2).
Figure 5: ¹ H nuclear magnetic resonance analysis of deckercinol angelate (3).
Figure 6: ¹³ C nuclear magnetic resonance analysis of deckercinol angelate (3).
Figure 7: Liver histopathological analysis of the Angelica persimmon extract.
Figure 8: Hepatic immunohistopathological analysis using TNF-α distribution for Angelica persimmon extract.
Figure 9: Blood pathology analysis of the Angelica persimmon extract.
Figure 10: Analysis of CYP2E1 protein expression for Angelica persimmon extract.

As for the terms used in the present invention, general terms that are currently widely used are selected as possible, and in specific cases, there may be terms arbitrarily selected by the applicant, but this is not a simple term name, but the meaning described or used in the detailed description of the invention. It should be taken into account to understand its meaning.

<Isolation and purification of decursinol>

Deckercinol (8g, active ingredient 4.63 g, 14.1 mmol) obtained after primary purification of the extract obtained by extracting 5 kg of Angelica keiskei by column chromatography was dissolved in 100 mL of a solvent made by mixing methyl alcohol and water 1:1 with methyl alcohol. After dissolving, K ₂ CO ₃ (975 mg, 7.1 mmol) was added and refluxed at 100° C. for 24 hours. The reaction solution was cooled to room temperature, ethyl acetate was added, and the organic layer was separated. After removing moisture from the organic layer with magnesium sulfate, the obtained solid was purified by column chromatography to obtain DL (yield: 56%), and the structure was confirmed by a spectroscopic method ( FIGS. 1 and 2 ).

<Synthesis of Decursin>

DL(1) was reacted with 3,3-dimethylacryloyl chloride to obtain deckersine (2) in a yield of 77%, and the structure was confirmed by a spectroscopic method (Fig. 3). and Figure 4). First, in the ¹ H NMR spectrum of the starting material DL, it was confirmed that the peak due to OH at position 7 of δ 1.90 disappeared, δ 2.07 (d, J = 1.2 Hz) and δ 1.80 (d, J = 1.2 Hz) It can be seen that the peak at position 3 is due to the methyl group of . On the other hand, the proton bonded to the 2nd carbon of dimethylacryloyl appeared at δ 5.58. The characteristics of H ₆ observed in deckercinol are shown, H _A is ² J = 16.8 Hz, ³ J = 5.6 Hz, ⁴ J = 5.6 Hz at δ 3.12, and H _B is ² J = 16.8 Hz, ³ at d 2.79. J = 5.6 Hz, respectively. In particular, the shift to the left (down-field shift) compared to DL indicates that the esterification reaction has progressed.

〈Synthesis of Decusinol angelate〉

DL(1) was reacted with angeloyl chloride to obtain DA( 3 ) in a yield of 82%, and its structure was confirmed by spectroscopic methods (FIGS. 5 and 6). As in the previous DN(2), the peak of δ 1.90 due to the OH group of carbon 7 disappeared, and the resonance of H _3' of angeloyl appeared as a complex peak in the region of d 6.81-6.76, and the region of δ 1.77-1.74 The resonance of the two methyl groups was complexly shown in Fig. 1, which can be explained by ³ J by H _3' and 4-methyl and ⁴ J by H _3' and 2-methyl. All spectral data were in good agreement with the literature values.

〈 In-vivo experiment 〉

In order to evaluate the efficacy of alcohol-based fatty liver damage relief of the common Angelica index component, 4-week-old C57BL/6 line (Orient Bio, Korea) mice were purchased, and these mice were operated at room temperature 20± In an environment of 2°C, 50±5% relative humidity, and a light-dark cycle of 12 hours, individual cages were used and they were bred on a free diet.

In order to test the efficacy of alcohol-induced liver damage induction and the liver damage of chamomile index components, mice were divided into 6 groups (Table 1), and the experiment was conducted for a total of 12 weeks. These mice were acclimatized to the environment of the breeding room for at least one week before the start of the test, and were classified by the randomized complete block design to maintain the same conditions and body weight. All experimental groups were supplied with feed indefinitely, and in the control experimental group, which is a normal control group, distilled water was filled in a water bottle and fed indefinitely at the free will of the mice, as well as orally administered by 0.1 mL once a day. To the control group, the Alcohol test group, 25% (v/v) ethanol was filled in a bottle instead of distilled water, and 0.1 mL of 25% ethanol was orally administered once a day. In the DN, DA, DL, and MIX experimental groups, 25% ethanol was supplied instead of negative water as a group to which the Angelica basil index component was administered, and 0.1 mL of the Angelica basil index component was orally administered once a day at a concentration of 200 mg/kg. This experiment was conducted for a total of 12 weeks from the start date. The animal test conditions are shown in [Table 1] below.

Animal test conditions Group Number of mice drinking source Oral administration Control 8 Distilled Water 0.1 mL/day Alcohol 8 25% alcohol 0.1 mL/day dn 8 25% alcohol 0.1 mL/day +
DN 200 mg/Kg DA 8 25% alcohol 0.1 mL/day +
DA 200 mg/Kg DL 8 25% alcohol 0.1 mL/day +
DL 200 mg/Kg MIX 8 25% alcohol 0.1 mL/day +
MIX 200 mg/Kg

〈Effect of Quercus Angelica indices on alcoholic liver damage 〉

The results of H&E staining and immunostaining to confirm the liver histopathological change and fatty liver alleviation effect through the administration of alcohol and the administration of the Quercus parrotfish indicator component are shown in FIGS. 7 and 8 . First, in the normal control group that consumed only distilled water without any alcohol intake, no alcohol-induced liver damage was observed and only normal hepatocytes could be observed. ) was not observed, lipid accumulation was also not observed, and the shape of the nucleus was relatively clearly observed (Fig. 7A). Unlike the hepatocytes of the normal control group, damage to the liver tissue was clearly observed in the control group treated with alcohol during the experiment period (Fig. 7B). Balloon-shaped deformation of cells, mallysosomes, and destroyed nuclei were observed due to fat deposition in hepatocytes, and these types of cells and destroyed nuclei were used as indicators for judging fatty liver.

In order to find out the effect of the indicator component of Angelica larvae on alcoholic fatty liver, immunostaining was carried out using the pro-inflammatory enzyme TNF-α. didn't However, a strong positive reaction was observed centered on blood vessels in the group that consumed alcohol and the group that consumed DL (Fig. 8B, E), and a relatively weak positive reaction was observed in the group that consumed DA (Fig. 8B). , D).

〈Effects of Quercus Angelica indices on the biochemical composition of blood〉

As the liver is damaged by alcohol, there are significant hematologic changes. The results of the blood test confirming the changes in the biochemical components in the blood in the animal model ingested with the ingestion of the common Angelica index component and alcohol are shown in FIG. 9 . Changes in levels of ALT, AST, and triglycerides (TG), which are representative proteins related to liver damage, were measured in the blood serum of mice administered with alcohol and an index component of chamomile.

Blood ALT and AST are generally known as enzymes released into the blood when liver tissue is damaged and are widely used as indicators of liver damage. AL) treatment group showed significantly higher blood ALT and AST activity. In the mixture (hereinafter referred to as MIX) treatment group containing DN and DA, the activity of blood ALT and AST was significantly lower than in the alcohol treatment group, and in the DL treatment group, the activity of the mixture containing DN and DA was significantly lower than that of the normal control group. Significantly high ALT and AST activities were shown. In addition, as a result of comparing the levels of triglycerides (TG) in serum, it was confirmed that the DN and DA treatment groups had significantly lower triglyceride levels compared to the alcohol treatment group.

〈Effect of Quercus Angelica indices on the expression of CYP2E1〉

The cause of oxidative stress is an increase in free radicals caused by alcohol, which plays a key role in alcoholic liver damage. Accordingly, the living body shows a high level of expression of CYP2E1 to alleviate oxidative stress, which is a major indicator to measure the degree of liver damage. Therefore, in the present invention, protein expression (western blot) was performed to quantify the expression level of CYP2E1 in the liver. In the normal treatment group administered only with distilled water, the protein level of CYP2E1, the protein to be measured, was almost not expressed. It could be seen that the expression level of CYP2E1 was decreased in the group to which DN and DA were administered, and the (MIX) treatment group containing DN also showed a decreased expression level (FIG. 10). could

Claims (6)

Primary purification of the extract obtained by extracting Angelica keiskei by column chromatography;
Dissolving the first purified extract in a solvent made of methyl alcohol and water 1:1;
Putting K ₂ CO ₃ in the dissolved extract and refluxing at 100° C. for 24 hours to cool the reaction solution to room temperature;
adding ethyl acetate to the cooled extract and separating the organic layer;
obtaining decursinol of the structural formula (1) by secondary purification of the solid obtained after removing moisture by adding magnesium sulfate to the organic layer by column chromatography;
Dissolving deckercinol of the structural formula (1) and 3,3-dimethylacryloyl chloride (3,3-dimethylacryloyl chloride) of the structural formula (4) in CH ₂ Cl ₂ ; and
Reacting the dissolved compounds of Structural Formulas (1) and (4) at 0° C. for 24 hours in the presence of nitrogen (N ₂ ); manufacturing method.

delete delete delete Primary purification of the extract obtained by extracting Angelica keiskei by column chromatography;
Dissolving the first purified extract in a solvent made of methyl alcohol and water 1:1;
Putting K ₂ CO ₃ in the dissolved extract and refluxing at 100° C. for 24 hours to cool the reaction solution to room temperature;
adding ethyl acetate to the cooled extract and separating the organic layer;
obtaining decursinol of the structural formula (1) by secondary purification of the solid obtained after removing moisture by adding magnesium sulfate to the organic layer by column chromatography;
Dissolving deckercinol of the structural formula (1) and angeloyl chloride of the structural formula (5) in CH ₂ Cl ₂ ; and
Reacting the dissolved compounds of Structural Formulas (1) and (5) in the presence of nitrogen (N ₂ ) at 0° C. for 24 hours; decurcinol angelate, a compound of Structural Formula (3), including; : DA ) manufacturing method.

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