JPH0310640B2 - - Google Patents

Description

[Detailed description of the invention]

Cancer has become the number one cause of death in recent years, and this trend is expected to continue in the future. Therefore, cancer treatment, which used to be left to surgery and radiation therapy, is now increasingly being supported by chemotherapy. By the way, a considerable number of anticancer drugs are currently approved.
It is used clinically. Broadly speaking, they are: a) alkylating agents, b) metabolic inhibitors antimetabolites, c) anticancer antibiotics, and d) plant-derived substances.
products, e) Hormones and other hormones
and others. Also recent 10
In the year, 5-fluorouracil (5FU), ftoraful
(futraful, tegaful, FT-207), mitomycin C
(MMC), cytrabine (cytosine arabinoside,
Ara-C), Picibanil (OK-432), Krestin
With the advent of new drugs such as (PSK), chemotherapy alone is becoming more effective for childhood cancers and blood cancers. As described above, cancer treatment is making rapid progress, but creating new drugs to cure solid cancers is said to be a pressing issue. Of the anticancer drugs currently in clinical use, only 10 are effective against human solid tumors.
It is no more than a seed. In addition, drugs have organ specificity, and it is said that it is impossible to create a uniform anticancer drug for cancer. In order to meet these challenges, great efforts are still being made in research and development of new drugs. However, there are two main approaches to this, one of which is a synthetic method and the other one is derived from natural products. For example, the National Cancer Institute (NCI) in the United States
Every year, more than 20,000 specimens are submitted from all over the world, and more than half of them are synthetic substances, and the rest are derived from natural substances. Furthermore, when researching the development of anticancer drugs, one of the important points is the selection of the screening method. Of course, there are many excellent reviews on screening, but
It can be said that nothing has been established so far. Therefore, it is no exaggeration to say that we are simply investigating the activity of randomly separated or synthesized substances. The present inventor has been interested in effectively discovering highly useful antitumor active substances from plant resources. We have focused our efforts on research and development of particularly effective screening methods, and have now unexpectedly arrived at a process for producing a substance useful in the treatment of solid cancers using a relatively simple method. That is, the present invention relates to a method for producing a novel compound having therapeutic effects, particularly antitumor activity, from madder grass, which is a type of medicinal plant. Many attempts have been made to extract antitumor active ingredients from plants. Specifically, there are known methods for producing products containing alkaloids extracted from dog saffron, periwinkle, etc., a type of lignan from podophyllum, and anthamacrolide substances with a structure similar to rifamycin from plants in the Eustaceae family. It is being Furthermore, protein polysaccharide substances extracted and manufactured from lower plant mushrooms, for example, Coriolus versicolor, are also known and are already commercially available. However, there are still very few products that have been put to practical use as pharmaceuticals, and therefore, many studies are still being conducted to obtain substances that exhibit medicinal effects from plants as a single component, but in any case, none of them are satisfactory. There weren't many. On the other hand, many researchers have also conducted research on the artificial synthesis of this type of drug, but most of these efforts tend to be abandoned during the development stage. The main reason for this was the occurrence of side effects of the synthetic drug. Therefore, the present inventors focused on various medicinal herbs that have been used as folk medicine since ancient times and are still taken today as they have low toxicity and mild medicinal effects, and whose effects have recently been reconsidered. We have been conducting research and development. As a result, we discovered that a crude extract of madder grass from a nearly infinite variety of medicinal herbs has remarkable therapeutic effects.
By further purifying the crude extract, they succeeded in obtaining two types of single substances that also have strong antitumor effects, leading to the completion of the present invention. It is therefore an object of the present invention to provide a method for producing compounds with therapeutic effects, in particular antitumor activity, by extraction. The above objects of the present invention and its features will become clearer from the following description. That is, the present invention has the general formula: (However, in the general formula, R is hydrogen or a methyl group.) Hereinafter, for convenience, those in which R is hydrogen will be abbreviated as TPC-A, and those in which R is a methyl group will be abbreviated as TPC-B. The two compounds of the present invention are isolated from madder grass as described above. The raw material, madder grass, is a perennial vine that grows wild in the fields and mountains of Japan or China, and its name comes from the yellow-red color of its roots.It is used in Chinese medicine as a blood purifier, a medicinal agent, and a hemostatic agent, and is also used as a folk medicine. It is also said to be effective against colds, heart disease, and bruises. By the way, the ingredients of madder grass include anthraquinones, nepetalactones, triterpenoids,
Saponins, alkaloids, etc. are already known. However, I have never heard of any excellent medicinal ingredients being extracted and isolated. On the other hand, madder root has been used as a dye since ancient times, but due to the remarkable development of chemical dyes in recent years, it is almost no longer used. By the way, the compound obtained from madder grass of the present invention
TPC-A has a free hydroxyl group and has an excellent antitumor effect in itself, and is predicted to have numerous efficacy as other pharmaceuticals.It can also be useful by modifying the free hydroxyl group in various ways. Its usefulness as an intermediate for producing derivatives is immeasurable. For example, it has an effect as an emetic, an appetite suppressant, a psychotropic drug, a vasoconstrictor, a papaverine agonist, and an antiparkinsonian (compound TPC-A), and an emetic and appetite suppressant (compound TPC-B). This has already been suggested. The two therapeutically effective compounds of the present invention can be produced mainly by extraction as follows. First, compound TPC-A is obtained by extracting the raw material, dried madder grass, with a solvent such as methanol, ethanol, chloroform, or ethyl acetate, and then concentrating the extract to obtain a crude extract, which is then subjected to various chromatographs. It can be obtained by separation and purification using E. Compound TPC-B is obtained by separating and purifying it as a separate fraction during the above treatment, and is a new compound. The manufacturing method will be explained in more detail below. (i) Solvent extraction of madder grass Madder grass can be used either dried or undried. Similar to other plants, the roots of madder grass contain large amounts of active ingredients, but the leaves and stems also contain significant amounts, so the whole plant can be used, but in this case, Due to its volume, it is advantageous to use a dry product. Furthermore, it was found that cutting the madder grass into small pieces was more convenient for extracting the active ingredients than cutting it into small pieces. Alcohols such as methanol and ethanol, and solvents such as chloroform and ethyl acetate used as extraction solvents can be used alone or in combination, but even when a plurality of solvents are used, methanol is preferably used as one of the mixed solvents. In addition, when considering the rationality of extraction, it is possible to use a large amount of solvent and extract at once, but when considering the efficiency of subsequent processing and extraction amount, it is recommended to repeat the extraction several times using a small amount of solvent. was preferable. (ii) Chromatography of crude extract (separation/purification) Chromatography using silica gel is effective for separating and purifying the crude extract, but chromatography using activated carbon is also used to remove colored components. However, activated carbon chromatography can be installed before or after silica gel chromatography. Further, purification may be possible without using it in some cases. For separation and purification by chromatography, it is preferable to use a column since the amount of the active ingredient is small, but it goes without saying that batch separation can also be used. A mixed solvent of chloroform and methanol is suitable as the elution solvent, and this mixing ratio is, for example, 3:2 with less polarity (for example, 100:1).
The active ingredient could be obtained without damaging it by elution with a solvent of Hereinafter, the present invention will be explained in more detail with reference to Examples. Example: Approximately 500g of dry Akane grass raw material, approximately 1 part of methanol
was added and extracted by heating on a hot bath for about 3 hours, followed by filtration and separation into a filtrate and a residue. Approximately 1 part methanol was newly added to this residue and the same extraction was performed again. This operation was repeated several more times. The obtained extracts were combined and concentrated under reduced pressure to obtain about 27 g of crude extract. Approximately 25g of this crude extract, approx.
It was dissolved in 200 ml of methanol and adsorbed onto a column packed with about 250 g of activated carbon for chromatography (manufactured by Wako Co., Ltd.). After washing this column with methanol, the chloroform-methanol mixture and the fractions eluted with chloroform were collected and concentrated under reduced pressure to obtain about 10 g of a reddish-brown oil. Approximately 9 g of this oil was dissolved in a small amount of chloroform, adsorbed onto a column packed with approximately 400 g of silica gel, and washed sequentially with chloroform, a chloroform-ethyl acetate mixture, and ethyl acetate.
The fractions eluted with the methanol mixture were collected and concentrated under reduced pressure to obtain about 1.4 g of oil. Approximately 1.3 g of this oil was dissolved in a small amount of chloroform and adsorbed onto a column packed with approximately 150 g of silica gel. After washing with chloroform and a chloroform-methanol (100:1) mixture, the mixture was washed with a chloroform-methanol (100:3) mixture. The fractions eluted were collected and concentrated under reduced pressure to obtain about 250 mg of solid material. This solid was dissolved in a small amount of chloroform-methanol mixture, and chloroform-methanol (100:15) was added using a TLC plate Art 7544 (manufactured by Merck).
After developing with a mixed solvent, the dark blue bands around Rf 0.60 to 0.71 and around Rf 0.50 to 0.59 were packed into columns under 254 nm ultraviolet irradiation, and the fractions eluted with a chloroform-methanol mixture were collected. When collected and concentrated to dryness under reduced pressure, about 56 mg of a light brown solid was obtained from the low Rf value fraction and about 24 mg from the high Rf value fraction. 40 mg and 20 mg of the above solids, each dissolved in a 45% acetonitrile aqueous solvent, were prepared using a Waters Microbondapak C-18 column.
% acetonitrile aqueous solution as the developing solvent
Perform preparative HPLC using absorbance at 254 nm as an indicator, and concentrate the eluate separated from the solid obtained from the low Rf value area to dryness under reduced pressure to obtain approximately 24 mg of white powder.
was obtained (compound TPC-A). Further purification was possible by repeating preparative HPLC. The eluate separated from the solid fraction obtained from the high Rf value portion was concentrated to dryness under reduced pressure to obtain about 12 mg of white powder. This powder was recrystallized from methanol to obtain about 10 mg of colorless needle crystals (compound TPC-B). This substance was identified as compound TPC-B based on its NMR, IR, and other data. Table 1 shows the physical properties of the compounds TPC-A and TPC-B thus obtained. Further, charts of infrared absorption spectra are shown in Figures 1 and 2.

[Table] Animal test In this test, CDF1 mice were used to evaluate the effects of the compounds TPC-A and B of the present invention on transplanted tumors.
The in vivo antitumor effect was investigated. P-388 was injected intraperitoneally into six CDF1 mice each.
¹⁰⁶ cells/0.1ml and 105 L- ¹²¹⁰ cells/0.1ml were transplanted, and predetermined amounts of the compounds TPC-A and B of the present invention were intraperitoneally administered from 24 hours to 5 days after transplantation. did. On the other hand, P-388 and L-1210 were similarly transplanted to a control mouse group to which the compound of the present invention was not administered, and the difference in survival rate between them was observed. The results are shown in Table 2.

[Table] Toxicity test Compounds of the present invention (TPC-A and TPC-B)
Acute toxicity tests were conducted on Using 5 to 10 male 4-week-old CD-1 (ICR mice) per group, a predetermined amount of each compound of the present invention was suspended in a 0.5% CMC solution, and this was administered intraperitoneally or intravenously. did. Observe life and death and general symptoms for 7 days after administration, and determine the number of fatal individuals after 7 days.
LD ₅₀ values were calculated by the Litchfield-Wilcoxon method. The results are shown in Table 3.

[Table] From Table 3, it was found that both compounds TPC-A and B showed lower LD ₅₀ values when administered intraperitoneally than when administered intravenously, unlike common drugs. . Tests related to other drug effects In this study, drug effects other than antitumor effects were investigated. 1 to 20 mg/Kg of each compound of the present invention was intraperitoneally administered to three 4-week-old male CD-1 (ICR) mice (Japan Charles River KK) per group. Symptoms were observed for 180 minutes at 30-minute intervals after administration. The observation method was a neuropharmacological multidimensional observation method using a modified version of the Irwin method and the Smith method. By comparing the observed symptoms with the pharmacological action patterns of various existing drugs, it was found that each compound of the present invention has the following medicinal effects. Table 4 Compound Estimated medicinal efficacy TPC-A Emetic effect, appetite suppressant effect, psychotropic effect, vasoconstriction effect, papaverine effect, antiparkinsonian effect TPC-B Emetic effect, appetite suppressant effect

[Brief explanation of drawings]

The attached FIGS. 1 and 2 are charts showing infrared absorption spectra of the compounds TPC-A and TPC-B of the present invention, respectively.

Claims (1)

[Claims] 1. Extracting madder grass with a solvent, concentrating the extract,
General formula, characterized by subjecting the resulting crude extract to chromatography: (However, R′ represents hydrogen or a methyl group.) A method for producing a compound represented by: 2. The method according to claim 1, wherein the solvent is at least one of methanol, ethanol, chloroform, or ethyl acetate.