JPS6311000B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the general formula The present invention relates to a method for producing triptolide (R=H) and tripdiolide (R=OH) represented by The substance is known as SMKupchan from the US.
In 1972, this compound was isolated from Kurozuru, a plant belonging to the family Irisaceae, and has strong antitumor activity. The present inventors previously discovered that the compound could be produced by tissue culturing Tripterygium wilfordii (Japanese name: Kurozuru) (Japanese Patent Application No. 1983).
−74926). Subsequently, as a result of research into a more advantageous production method for the compound, N-(2-substituted-4
It has been found that by adding an appropriate amount of (pyridyl) ureas or thioureas to the culture medium, the significant amount of the target product produced can be increased. The ureas have the general formula () [In the formula, R ₁ represents halogen, hydroxyl, lower alkoxy, lower alkylthio, lower alkoxycarbonyl, benzoylamino, lower alkoxycarbonylamino, cyano or trifluoromethyl, R ₂ represents hydrogen or lower alkyl, and R ₃ represents It refers to a compound [hereinafter referred to as compound ()], which represents oxygen or amber, and R ₄ represents an aromatic hydrocarbon unsubstituted or substituted with lower alkyl, lower alkoxy, hydroxyl, or halogen. In the definition of the above formula, halogen is a chlorine atom,
Includes bromine atom, iodine atom, and fluorine atom.
Lower alkoxy, lower alkylthio, lower alkoxycarbonyl, lower alkoxycarbonylamino, and lower alkyl mean alkyl or alkyl having 1 to 4 carbon atoms in the alkyl moiety;
Includes methyl, ethyl, propyl, i-butyl, n-butyl, t-butyl and the like. Aromatic hydrocarbons in R ₄ include phenyl,
An example is benzyl. Compound () is a known compound, and its manufacturing method is disclosed in JP-A-54-81275 and JP-A-55-62066. According to the present invention, tissue pieces from stems, leaves, buds, seeds, and other parts of plants belonging to the genus Tripterygium, or cell groups derived therefrom, so-called callus, are grown aerobically on a solid surface and in a liquid culture medium in the presence of a compound (). When the target compound is cultured in a tissue culture such as a proliferated callus, proliferated cells, partially or completely differentiated cells, and a culture medium, the target compound is collected from the tissue culture and/or the culture medium. can do. The plants used in the present invention belong to the genus Tripterygium, and any plant can be used as long as it has the ability to produce the target compound. Plants of the genus Tripterygium include Tripterygium wilfordii and Tripterygium wilfordii.
Examples include T. regelii and T. forrestii, and for Tripterygium willfolii, see Jizaburo Oi's "Japanese Flora" page 364.
For other information on Shibundo in 1965, please refer to R.Bruning.
et.al. Phytochemistry Volume 17, pages 1821-1858 (1978). More preferably, Tripterygium wilfoldii is used. For culturing, stems, leaves, roots, buds, fruits, and other tissue pieces or cell groups of plants belonging to the genus Tripterygium are used as materials, cut into desired sizes, the surface washed with deionized water, and then, for example, After sterilizing with sodium chlorite or ethyl alcohol, wash thoroughly with sterile water. The surface-sterilized pieces are then used, for example, in Murashige T. and Skoog F. “Physiol.
Plantarum” vol. 15, pages 473-497 (1962))
Sugar 3g/dl, kinetin 1mg/, 2,4-
Dichlorophenoxyacetic acid 1mg/and agar 0.8
After placing on an agar medium supplemented with g/dl, callus is induced by culturing at a constant temperature (15 to 35°C) for about two weeks. The callus induced as described above can be further cultured on a solid culture medium or in a liquid culture medium by applying operations similar to those for culturing normal microorganisms. For example, culture can be carried out by a shaking culture method or a tank culture method in which culture is carried out while aerating sterile air. Examples of the composition of these culture media include, but are not limited to, the above-mentioned media, such as those described by White, Gamborg, and Nitch, which are plant tissue culture media. Others (Plant Cell Tissue Culture, Practice, Applications, Prospects - Rigakusha, 1979, edited by Hiroshi Harada and Mu Komamine) can be used, and instead of sugar (sucrose), glucose, etc. can be used as the carbon source.
Fructose, mannose, molasses, starch and others can be used. Yeast extract, peptone, meat extract, casamino acids, etc. can also be added. Other cytokinins such as benzyladenine may be used instead of kinetin, and growth regulators such as indoleacetic acid and naphthaleneacetic acid may be used instead of 2,4-dichlorophenoxyacetic acid. Various inorganic trace components do not necessarily need to be added if natural products are used or tap water is used instead of deionized water. Compound () is added within an effective range depending on the type of compound, but is usually added in an amount of 0.01 to 5 g. A specific example of the compound used is N-(2
-chloro-4-pyridyl)-N'-(m-methyl-
phenyl)urea, N-(2-chloro-4-pyridyl)-N'-(o-chlorophenyl)urea, N-(2
-Fluoro-4-pyridyl)-N'-phenylurea, N-(2-methoxy-4-pyridyl)-N'-
Phenyl urea, N-(2-bromo-4-pyridyl)
-N'-phenylurea, N-(2-acetamido-
4-pyridyl)-N'-phenylurea, N-(2-
methoxy-4-pyridyl)-N'-phenylthiourea, N-(2-chloro-4-pyridyl)-N'-phenylurea, N-(2-chloro-4-pyridyl)-
Examples include N'-(o-methyl-phenylurea). Cultivation is carried out at a temperature of 25 to 35°C and a pH of 4.5 to 8.5, and is completed in 1 to 2 weeks. The cell group (callus) cultured and proliferated in this way and the differentiated plant cells are collected by sieving, the cells are destroyed using a homogenizer, etc., and the cells are collected from the supernatant or solution obtained by sieving or centrifuging, or from the culture. The target compound can be separated from the culture solution obtained by removing the plants and plant cells. These separation methods are similar to those of Kupchan et al. (Journal of American Chemical).
Society, Vol. 94, p. 7194, 1972), but preferably separation can be performed by the following method. The method for isolation from cultured cells is to freeze-dry the cells, add 95% ethyl alcohol, and extract using a Soxhlet extractor or heating in a beaker or flask. After condensing the extract under reduced pressure, water and n-hexane were added, and the mixture was shaken well using a separator to remove impurities by dissolving them in the n-hexane layer.
As another separation method, when cell extracts or culture fluids are used as materials, n-hexane is added to these fluids to transfer impurities into this layer. In any case, it is better to repeat the distribution operation using n-hexane two or three times to remove impurities soluble in n-hexane as much as possible. Next, ethyl acetate is added to the aqueous layer, partitioned using a separating funnel, and the ethyl acetate layer is collected. It is best to repeat this operation two or three times. The ethyl acetate layer thus obtained was concentrated under reduced pressure,
High performance liquid chromatography (column used,
Unisil Q C ₁₈ Gas Kuro Kogyo, solvent type 30%
Tripdiolide and triptolide can be separated using ethanol). The two diterpenoid triepoxides thus obtained were KB, L-1210, p-
It has been confirmed that it suppresses the growth of various tumor cells such as 388, and especially for KB cells, it can be used as a constant quantitative method for purification and quantification during culture. Examples will be described below. Example 1 Crozzle stems cut into lengths of about 1 cm are sterilized by soaking them in a sodium hypochlorite solution (3% available chlorine) for 10 minutes. This sterilized and washed Kurozuru stem is
Syuucrose 3 in Murashige-Skoog medium
g/dl, 2,4 dichlorophenoxyacetic acid 1 mg/
, one test tube was placed in 10 ml of medium (PH 6.3) containing 1 mg/dl of kinetin and 0.8 g/dl of agar,
Culture at 28℃ for about 1 month. The entire amount of callus that has been formed from the cut surface of the stem was added to 10 ml of a medium with the same composition as N-
Transplant into a medium supplemented with 0.1 mg/(2-chloro-4-pyridyl)-N'-phenylurea. Further 28℃
The callus that grew after culturing for one month was collected and freeze-dried. 95 for 20g of dried callus collected in this way.
Add 200 ml of % ethyl alcohol and perform extraction for 6 hours using a Soxhlet extractor. Perform this operation 3
The ethyl alcohol portion obtained by repeating the approx.
Concentrate 600ml to dryness under reduced pressure, then add 50ml of water.
Melt as much as possible. Add 50 ml of n-hexane to this and shake with a separating funnel to remove impurities. Repeat the same procedure twice more to collect the aqueous layer, add 50 ml of ethyl acetate, and transfer the target substance into the ethyl acetate layer using a separating funnel. This operation was repeated two more times, the ethyl acetate layer was collected, concentrated under reduced pressure, and then transferred to a UnisilQ-C ₁₈ column.
Perform high performance liquid chromatography using % ethanol. The peaks appearing at the same sites as the standard product (detected at UV 218 nm) were collected and concentrated to obtain 3.2 mg of tripdiolide and 0.7 mg of triptolide.
Incidentally, N-(2-chloro-4-pyridyl) was added to the culture medium.
-N'-Phenylurea-free medium,
The amount of tripdiolide accumulated when cultured is
The amount of triptolide was 0.6 mg, and the amount of triptolide was 0.1 mg. Example 2 The callus obtained in Example 1 (approximately 2 g as fresh weight) was added to Murashige-Skoog medium containing 0.1 mg of kinetin, 1 mg of naphthalene acetic acid, and 3 g of sucrose per dl. -Chlor-4-pyridyl)-N'-phenylurea 0.1 mg/
When planted in a 300 ml Erlenmeyer flask containing 100 ml of liquid medium supplemented with 100 ml of liquid medium and cultured at 180 rotations per minute at 28°C in the dark for 2 weeks, the growth rate was 15 mg per 1 ml of medium.
(dry weight). When the cells were collected by filtration and the target substances in the cells and culture medium were quantified, it was found that tripdiolide and triptolide were present in the cells at 1.18 μg and 0.09 μg, respectively (per 1 volume of the culture medium).
(converted per ml), tripdiolide and triptolide are 129 μg and 0.12 μg, respectively, in the culture solution.
(each calculated per ml of medium). Incidentally, when 1 mg of N-(2-chloro-4-pyridyl)-N'-phenylurea was added and cultured in the same manner, the amounts of tripdiolide and triptolide accumulated in the cells were the same, 1.02 μg and 0.05 μg (medium (converted per ml), 0.94 μg and 0.04 μg in the culture medium, respectively.
It was hot at g. In addition, when culturing was carried out in the same manner without adding N-(2-chloro-4-pyridyl)-N'-phenylurea, the amounts produced in the cells were 0.15 μg of tripdiolide and 0.02 μg of triptolide (each (converted per ml of culture medium), each in the culture solution
The amounts were 0.17 μg and 0.03 μg (each calculated per ml of medium). From 142 g (dry weight) of cells obtained from culture medium 10 containing 0.1 mg of N-(2-chloro-4-pyridyl)-N'-phenylurea, 8.4 mg of tripdiolide and 0.75 mg of triptolide were added. From the culture solution, white powders containing 8.9 mg of tripdiolide and 0.82 mg of triptolide were obtained. Example 3 Example 2 was repeated except that the compound shown in Table 1 was used in place of N-(2-chloro-4-pyridyl)-N'-phenylurea to obtain the results shown in Table 1.

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Claims (1)

[Scope of Claims] 1. Plants belonging to the genus Tripterygium and having the ability to produce triptolide and/or tripdiolide are defined by the general formula [In the formula, R ₁ represents halogen, hydroxyl, lower alkoxy, lower alkylthio, lower alkoxycarbonyl, benzoylamino, lower alkoxycarbonylamino, cyano or trifluoromethyl, R ₂ represents hydrogen or lower alkyl, and R ₃ represents oxygen or sulfur, and R ₄ is an aromatic hydrocarbon unsubstituted or substituted with lower alkyl, lower alkoxy, hydroxyl, or halogen], and the tissue is cultured in the presence of a compound represented by the following formula. A method for producing triptolide and/or tripdiolide, which comprises producing and accumulating a substance and collecting the accumulated substance.