JPH01230599A - Production of compound having antitumor active compound from 'akane' - Google Patents

Abstract

NEW MATERIAL:A compound of the formula (R' is H, methyl). USE:Antitumor agent. PREPARATION:Rubia akane (madder) is extracted under heat with a solvent, preferably methanol, ethanol, chloroform or ethyl acetate on a water bath. The extract is concentrated under reduced pressure to give a crude extract. The crude extract is dissolved in a solvent and subjected to chromatography.

Description

[Detailed Description of the Invention] Cancer has become the leading 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 supported by chemotherapy.

By the way, a considerable number of anticancer drugs have been approved and are currently in clinical use. Broadly speaking, they are: a) alkylating agents, b) metabolic inhibitors antimetabolites, c) anti-cancer antibiotics, and d) plant products.

e) Hormones and ot
classified as hers. Also, in the recent decade, 5-fluorouracil (5F U),
ftoraful (futraful.

tegaful, FT-207), mitom
ycin C (MMC).

cytrabine (cytosine arabic)
noside, Ara-C).

Picibanil (OK-432), kre
With the advent of new drugs such as stin (PSK), pediatric cancers and blood cancers are becoming more successful with chemotherapy alone.

As described above, cancer treatment is making rapid progress, but the creation of new drugs that can cure solid cancers is said to be an urgent issue. Of the anticancer drugs currently used in clinical practice, only the IO type is effective against human solid tumors.

In addition, drugs have organ specificity, and it is said that there is no hope for 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 broad categories:
There are two directions, one is synthetic and the other is derived from natural products. For example, American National
To Cancer Institute (NCI),
More than 20,000 specimens are submitted from all over the world every year, 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 there is nothing established so far.

Therefore, if something is randomly separated or synthesized,
It is no exaggeration to say that we are simply investigating its activity.

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 Seigun, a type of medicinal plant.

Many attempts have been made to extract antitumor active ingredients from plants.

Specifically, there are methods for producing products containing alkaloids extracted from saffron, periwinkle, etc., a type of lignan from bodophyllum, and anthamacrolide substances with a structure similar to famycin from plants in the Eustaceae family. Are known.

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 (therefore, many studies are still being conducted to obtain substances that exhibit medicinal effects from plants as a single component, but in any case, the results are satisfactory). There were few.

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, Western crude extracts from a nearly limitless variety of medicinal herbs,
We discovered that it has a remarkable therapeutic effect, and by further purifying the crude extract, we succeeded in obtaining two single substances that also have strong antitumor effects, leading to the completion of the present invention. be.

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 relates to a method for producing a compound having the general formula: (However, in the general formula, R is hydrogen or a methyl group.) In addition, below, for convenience,
TPC-A is for those where R is hydrogen, and TPC is for those where R is a methyl group.
-Abbreviated as B.

The two compounds of the present invention are isolated from Seigun as described above. Seigun, the raw material, is a perennial vine that grows wild in the fields and mountains of Japan or China, and its roots are yellowish-red, hence its name. In Chinese medicine, it is used as a blood purifier, a medicine for blood purification, and a hemostatic agent.It is also said to be effective in treating colds, heart disease, bruises, etc. as a folk medicine.

By the way, anthraquinones, nebetalactones, triterpenoids, saponins, alkaloids, and the like are already known as the ingredients of Western medicine. However, I have never heard of any excellent medicinal ingredients being extracted and isolated.

On the other hand, Seigun 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 TPC-A obtained from Seigun of the present invention has a free hydroxyl group and has excellent antitumor effects in itself, and is predicted to have numerous efficacy as other pharmaceuticals. Its usefulness as an intermediate for producing useful derivatives by variously modifying the above-mentioned free hydroxyl groups is immeasurable.

For example, it has an effect as an emetic, an appetite suppressant, a psychotropic drug, a vasoconstrictor, a papaperine agonist, and an antiparkinsonian (Compound TPC-A), and an emetic and anorectic (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, the compound TPC-A is obtained by extracting the raw material, a dried product of Seigun, with a solvent such as methanol, ethanol, chloroform, or ethyl acetate, then concentrating the extract to obtain a crude extract, and then using various chromatographs. It can be obtained by separation and purification by graphography.

Compound TPC-B is obtained by separating and purifying the two components in the above treatment, and is a new compound.

The manufacturing method will be described in more detail below.

(i) Solvent extraction of Nishigun Nishigun can be used either dried or undried. Similar to other plants, Seigun contains a large amount of active ingredients mainly in the roots, but the leaves and stems also contain a considerable amount, so the whole plant can be used. In this case, it is advantageous to use a dried product due to its volume. In addition, the Western army found that cutting into small pieces was more convenient for extracting the active ingredients than it took.

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 them are used, it is preferable to use methanol as the - of the mixed solvent.

In addition, when considering the rationality of extraction, it is possible to use a large amount of solvent and perform extraction several times, but when considering the efficiency of subsequent processing and extraction amount, it is possible to perform extraction several times using a small amount of solvent. Things were good.

(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 can also be used in combination to remove colored components. However, activated carbon chromatography can be installed before or after silica gel chromatography.

In some cases, purification may be possible without using it.

In separation and purification by chromatography, it is preferable to use a column since the amount of active ingredient is small, but it is natural that batch separation can also be used.

A mixed solvent of chloroform and methanol is suitable as the elution solvent, and by eluating with a solvent with a lower polarity than 3:2 (for example, 100:1), it is possible to elute the active ingredient without damaging the active ingredient. I was able to obtain it.

Hereinafter, the present invention will be explained in more detail according to Examples.

Example: Approximately 11 g of methanol was added to 500 g of dry Western raw materials, and the mixture was extracted by heating on a hot bath for about 3 hours, followed by filtration and separation into a filtrate and a residue. To this residue, about I! ! of methanol was added 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. About 25 g of this crude extract was dissolved in about 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, both the chloroform-methanol mixture and the chloroform eluate were collected and concentrated under reduced pressure to obtain 10 g of a reddish brown oil.

Dissolve 9 g of this oil in a small amount of chloroform and adsorb it on a column packed with about 400 g of silica gel. After sequentially washing with chloroform, a chloroform-ethyl acetate mixture, and ethyl acetate, the fractions eluted with a chloroform-methanol mixture were collected. The mixture was concentrated under reduced pressure to obtain approximately 1.4 g of an oil.

1.3 g of this oil was dissolved in a small amount of chloroform and adsorbed on a column packed with about 150 g of silica gel, and chloroform, chloroform-methanol (100:1) was added.
After washing with a mixed solution, chloroform-methanol (100
: 3) Collect both fractions eluted in the mixture and concentrate under reduced pressure,
Approximately 250 mg of solid was obtained.

This solid was dissolved in a small amount of chloroform-methanol mixture and subjected to chloroform-methanol (100:15) using Art 7544 (manufactured by Merck & Co., Ltd.).
After developing with a mixed solvent, RfO is around 60-0.71 and Rfo is 50-0.5 under 254 nm ultraviolet irradiation.
When each band showing a dark blue color around 9 was packed into a column, the fractions eluted with a chloroform-methanol mixture were collected and concentrated to dryness under reduced pressure.
About 24 mg of a light brown solid was obtained from both the 6 mg and high Rf values.

each dissolved in 45% acetonyl aqueous solvent,
40μ and 20mg of the above solid were subjected to preparative HPLC using a Waters 11 Micro Bonderback C-18 column and a 45% acetonitrile aqueous solution as a developing solvent, using the absorbance at a wavelength of 254nm as an index, and the results showed low Rf values. The eluate separated from the solid material was concentrated to dryness under reduced pressure to obtain about 24 mg of white powder (compound TPC
-A). Further purification was possible by repeating preparative HPLC.

The eluate separated from the solid 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 form colorless needle-shaped crystals of approximately 10 m
I want to obtain compound TPC-B).

This substance was identified as compound TPC-B from its NMR, IR, etc. data.

The compounds thus obtained, TPC-A and TP
The physical properties of C-B are shown in Table 1. Further, charts of infrared absorption spectra are shown in FIGS. 1 and 2.

Table 1 Compound TPO-A Compound TPC-B Molecular weight 756 770 Properties Shape White powder Colorless needles Easy melting point Gradually turns brown from about 235°C 〉2
80°C (clear decomposition point (no clear decomposition point shown).
specific rotation, −220° (CHCls,
C=0.1) -224°(CH(L7z,
C=0.25) [α].

1, R, spectrum Figure 1
Fig. 2 282 ru++;
t = 3300 28
2 ns; g = 33000 molecular weight is FD-
Measured by MS.

of, R, were measured by the KBr method.

o N, M, R, are CDCj! as solvents. ! , using Si(CH3)a as internal standard, 100 MHz
Protons N, M, and R were measured.

Animal Test In this test, CDF 1 mice were used to examine the in vivo antitumor effects of the compounds TPC-A and B of the present invention against transplanted tumors.

Six CDF1 mice each had 106 P-388 cells (10.1mj) in their peritoneal cavities! and L-1210105 pieces 1
0.1 ml was 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. 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 2 TPC-8 10 17.8
± 0.58 187.47PC-841
6,2± 1.02 173.6 Control 9.
50±0.19 100TPC-8 10
9.67± 0.95 12
8.9TPC-8510,3± 0.21 137
．． 3 Control 7.50±OJ8 100Toxicity Test Acute toxicity tests were conducted on each compound of the present invention (TPC-A and TPC-B).

Using 5 to 10 male 4-week-old CD-1 mice (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. was administered. Observe life and death and general symptoms for 7 days after administration.
From the number of lethal individuals after 1 day, Litchf 1eld
- LD by the Wilcoxon method.

The value was calculated. The results are shown in Table 3.

Table 3 Intraperitoneal 16.0 (13,3-19,3) 1
0.0 (7,0-14,3) Intravenous 34.6 (2
8,1-41,2) 16.5(12,6-21,
5) From Table 3, both compounds TPC-A and B are
It was found that, unlike common drugs, the LD5° value was lower when administered intraperitoneally than when administered intravenously.

Tests on other drug effects In this study, drug effects other than antitumor effects were investigated.

Three 4-week-old male CD-1 (ICR) mice (Charles River K., Japan) were used per group, and 1 to 20 mg/kg of each compound of the present invention was intravenously administered to them. administered. Symptoms were observed for 180 minutes at 30 minute intervals after administration. The observation method was a neuropharmacological multidimensional observation method based on a modification 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.

TPC-8 Emetic action 1 Appetite suppressing action, psychotropic action, vasoconstriction action, papaverine action.

Antiparkinson effect TPC-B Emetic effect 9 Appetite suppressing effect

[Brief explanation of the drawing]

Attached Figures 1 and 2 show the compound TPC of the present invention, respectively.
It is a figure which shows the chart of the infrared absorption spectrum of -A and TPC-B.

Claims (2)

[Claims] (1) A general formula characterized by extracting madder grass with a solvent, concentrating the extract, and subjecting the resulting crude extract to chromatography: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, R' represents hydrogen or methyl group). (2) The method according to claim (1), wherein the solvent is at least one of methanol, ethanol, chloroform, or ethyl acetate.