JPS5910548A - Pfaffic acid and derivative thereof - Google Patents

Abstract

NEW MATERIAL:Pfaffic acid and its derivative of the formula (R1 is H or acyl; R2 is H or alkyl). USE:An antitumor agent, capable of exhibiting improved inhibitory activity against melanoma cells, cervical cancer cells, cancer cells of the lung, etc. in a concentration as low as 4-6mug/ml, and administered in 10mg-1g, preferably about 100mg daily dose in the form of a powder, granule, capsule, solution, etc. PROCESS:The root of a dried Pfaffia paniculate Kuntze is pulverized and extracted with an alcohol, and sulfuric acid is then added to the resultant extract solution and refluxed under heating to distill away the solvent. The residue obtained by the distillation is then passed through a column packed with silica gel, and eluted with a mixture of chloroform with methanol at (50:1)-(20:1) ratio. The solvent is then distilled from the eluted fraction to recrystallize the residue from the alcohol and give the aimed pfaffic acid of the formula. The pfaffic acid of the formula is a crystalline substance having specific <=29C skeleton, and stable to acids and alkalis with 285-286 deg.C melting point.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel compound having antitumor activity represented by the following general formula (I): In the formula (1), R1 represents a hydrogen atom or an acyl group, and R2 represents a hydrogen atom or an alkyl group. 10 In formula (1), R
Compounds in which 1 and 2 are hydrogen, i.e. 3β-hydroxy-16,20-cyclo-30-fluorene-12-
EN-28-OITSUKU ASSOD is a Brazilian ginseng (
Pra [fiapaniculata kuntze
) is a compound with a completely novel structure isolated from puff acid (1'raffic ac
id).

Brazilian ginseng is a perennial plant of the Hull family that grows in South America, and has been used as a folk medicine since ancient times for its nourishing, tonic, and tonic properties.
For energizing, sedative purposes, or for diabetes, ulcers, leukemia,
It has been used to treat cancer, etc.

However, its active ingredients have not been revealed at all so far. As a result of many years of research to elucidate this active ingredient, the present inventors succeeded in isolating a substance that exhibits a remarkable inhibitory effect on the proliferation of cultured tumor cells, and as a result of structural analysis - the substance. was found to be the above puff acid.

Furthermore, the present inventors have discovered an alkyl puff acid obtained by esterifying puff acid (a compound that is a 1 (2-alkyl group) in formula (I)) and an acyl puff acid obtained by acylating it (a compound that is a 1 (2-alkyl group) in formula (■)). compound that is a group)
The present invention was completed based on the discovery that the same anti-tumor activity as puffed acid has excellent anti-tumor activity.

Puff acid can be extracted and isolated from Brazilian ginseng by the following method. That is, the dried Brazilian ginseng root is crushed and extracted with alcohol, and the resulting alcoholic extract is diluted with water and extracted with n-phthanol.

Next, the n-butanol extract is divided into a water-soluble part and a poorly soluble part. If the latter is purified by silica gel column chromatography, puff acid, which is present in free form in plants, can be obtained. On the other hand, the water-soluble part of the above n-butanol extract contains puff acid (saponin) bound to sugar, so this is hydrolyzed by an appropriate method, extracted with an organic solvent, and then silica gel is added. Puff acid, which is present in plants in the form of saponin, can be obtained by column chromatography.

As is clear from the above, puff acid itself is soluble in water and relatively low polar organic solvents, and the opposite is true for saponin, so water is used instead of alcohol as the extraction solvent. If an organic solvent such as acetone, ethyl acetate, chloroform, or ether is used, mainly free puff acid can be obtained.

Therefore, although it is possible to extract all the puff acid by extracting Brazilian ginseng alternately with water and the above-mentioned organic solvents, from a practical point of view it is preferred to extract with alcohol, especially methanol. In this case, it is also possible to directly hydrolyze the alcoholic extract containing all puffed acid, and this method is most preferred from an economic standpoint.

Normal acids or alkalis can be used to hydrolyze saponins, but if the sugar moiety is decomposed with periodate in advance, hydrolysis can be carried out under relatively mild conditions. However, as described below, puff acid is a fairly stable substance despite its complex structure, so the latter hydrolysis method is not particularly desirable. Note that this saponin can also be biochemically hydrolyzed by enzymes and bacteria such as soil bacteria and airborne bacteria.

The crude puff acid obtained by the above method can be purified by recrystallization from alcohol, column chromatography, preparative thin layer chromatography, or the like.

Alkyl puff acids and acyl puff acids can be derived from puff acids by conventional methods. That is, the former can be easily produced by alkylating with an alcohol and a dehydrating agent or diazomethane, and the latter can be easily produced by acylating with an acid anhydride or acyl halide.

As mentioned above, puff acid is a crystalline substance that is stable to acids and alkalis and has a unique bone core with 29 carbon atoms, which has been completely unknown until now, and its melting point is 285 to 286°C.

Puff acid and alkyl puff acid and T-acyl puff acid,
Mouse-derived melanoma cells (melanoma B) 6), mouse-derived lung cancer cells (3LL), and human-derived γ-miya!
4 to 6/for J region cancer cells (l1eLa S 3 )
' Low concentrations of K//mf inhibit its proliferation quite significantly.

On the other hand, as mentioned above, Brazilian carrots are 300
It is a plant that has been used by Indians for many years, so it is clear that the toxicity of this plant component is low. In fact, a hospital in Brazil reported that no side effects were observed when Brazilian ginseng powder was taken orally for 1 to 15 hours a day. Therefore, puffic acid, which is contained in Brazilian ginseng in free form or as a saponin glycoside at about 0.1 to 1%, is not toxic at least within the normal usage range (10 to 12 days). It is clear that it is not shown.

Those skilled in the art can readily infer that alkyl puff acids and acyl puff acids, which are simple derivatives of puff acid, also have low toxicity like puff acid.

In order to use the compound of formula (I) in actual treatment, it may be taken orally in the form of powder, granules, capsules, liquid, or the like. Alternatively, the compound of formula (■) can be suspended in water or dissolved in an oily solvent and used as an injection or external preparation. In addition, instead of puff acid, it is also possible to formulate a formulation using an extracted fraction, extract, etc. containing puff acid at a high concentration.

The dosage of the compound of formula (■) in humans can be about 10 mg to 1 y per day depending on the symptoms, but it is usually preferably around too+v/day.

The present invention will be explained in detail below with reference to Examples.

Example 1 Dried Brazilian ginseng roots are crushed and extracted by heating at 1,11600 z for 3 times of 18 Kywometa/-/l/200/te. The extract was concentrated under reduced pressure to obtain methanol extract 2 to 9. Of these, 8202 are water 500I+Ie
The suspension is suspended in water, extracted by shaking with 500 m of n-butanol at room temperature, and this operation is repeated 5 times. The butanol layer is collected and the solvent is distilled off under reduced pressure to obtain residue 1407.

Add 300 mff of water to this, stir, and after centrifugation,
”Excluding Erqing. Further, water 200IIIe is added to the precipitate and the same operation is repeated twice to obtain a water-insoluble precipitate 27y. Collect the supernatant separately.

This precipitate was dissolved in a 2:1 (7) mixture of chloroform and methanol, mixed with silica gel 407, and the solvent was completely evaporated while stirring on a water bath. Place the column on a silica gel column and sequentially elute with hexane, a hexane-ethyl acetate mixture, ethyl acetate, and an ethyl acetate-methanol mixture. From ethyl acetate to ethyl acetate JLI-
The fractions eluting in a 20:1 mixture of methanol are collected and the solvent is evaporated to give 15 g of a residue. This is 30y
The mixture is mixed with 5007 silica gel, placed on a 5007 silica gel column, and eluted with a chloroform-methanol mixture. Both fractions eluted with 50:1 to 20:1 of chloroform-methanol are collected, the solvent is distilled off, and the residue is recrystallized from methanol to obtain puff acid I FI Oq as colorless needle-like crystals.

mP 185~186”C,C (2 peaks, = 109.2°
(cm0.72 chloroform), insoluble in water, methanol, ethanol, pyridine, ethyl acetate, ether
iJ soluble, sparingly soluble in benzene, insoluble in hexane. Positive Liberman Birchyard reaction (reddish-purple color), positive tetranitromethane reaction (light yellow color). IR', KRr+11aX cm:3350.2950,'28'70.16
90,1460°1380.1035,995. M
S m/z: 440.

422.284,232,207,187,164° Elemental analysis (as C29”4403); WI calculated value:
C179,04; II, 10.06, experimental value: C,
79,32:11, l□, OB, NMR data are shown in Tables 1 and 2.

Example 2 Methanol-7/methanol extract 68o2 of real MAJ1
After adding Lz3.34-4 500-4N sulfuric acid and heating under reflux for 3 hours, methanol was distilled off under reduced pressure, and after cooling, the precipitate 1°60y was taken off the water. Dissolve this in a 2:1 mixture of chloroform and methanol, add 100 y of silica gel, mix, stir in a water bath to completely evaporate the solvent from the IS, and place on a column of 5007 siligel.
Operate in the same manner as in the case of water-insoluble precipitate in Example 1,
350 tq of puff acid is obtained.

Example 3 The supernatant obtained in Example 1 (water ijJ solution) was collected, passed through a layer of 350 g of activated carbon for column chromatography, and washed with 500 m of water.
After washing with e, elution is sequentially performed using ethanol, a mixture of ethanol and ethyl acetate, and ethyl acetate. Both eluted fractions ranging from ethanol-ethyl acetate to ethyl acetate are collected and the solvent is distilled off to obtain a pale brown crude saponin powder. 280 mg of this saponin is dissolved in 2N sulfur v50 mF, heated under reflux for 3 hours, allowed to cool, and the precipitate that precipitates is washed with water and recrystallized from methanol to obtain 28 my of puff acid.

Example 4 50 to 50% of the crude saponin obtained in Example 3 was mixed with 10 to 10% of methanol.
mp, add 100 mg of sodium periodate, stir at room temperature for 4 days, shake and extract with n-butanol for 3 mouths, wash the n-butanol layer with water, and remove the solvent under reduced pressure. do. 5' in the residue? I) Potassium hydroxide solution 5 m
Add e and heat under reflux in a nitrogen stream for 2 hours. The reaction mixture was neutralized with 10% phosphoric acid, extracted by shaking with n-butanol, and the 11-butanol layer was washed with water. The solvent was distilled off under reduced pressure, and the resulting residue was recrystallized from methanol.
Puff acid 12~ is obtained.

Example 5 40 ~ of puffic acid was dissolved in 5 ml of methanol, an excess of diazomethane ether solution was added, and after being left at room temperature overnight, the solvent was distilled off under reduced pressure. When the resulting residue was recrystallized from methanol, it became colorless. Acicular crystals of methylpuffic acid (formula (I) with R1=tl, R2=CI+3>38~ are obtained. mp 169~170''Co insoluble in water.

Soluble in methanol, ethanol, pyridine, ethyl acetate, ether, chloroform, benzene, hexane. 11
1ν,,,axtyn,3450,2950.28
70°1720.1455.13B0,995°MSm
/z :454.436,298,246,207,1
87,178° elemental analysis (as C30”4603);
a1 calculated value: C979, 25; o, 10. '20, Experimental value: C, 78,95; 11.10.40°N MR data are shown in Tables 1 and 2.

Example 6 Puff acid 35W was mixed with acetic anhydride-pyridine 1:1 mixture 6
,/, and after standing overnight at room temperature, the solvent was distilled off under reduced pressure and 5 me of water was added to the residue. When the precipitate is taken off the coast and recrystallized from water-containing methanol, colorless needle-like crystals of acetylpuffic acid (R1=c113Go, R
2,,11) 35■ is obtained. i hit 302-305
Insoluble in 0 water, soluble in methanol, ethanol, pyridine, ethyl acetate, benzene, soluble in hexane. IkVmax-, 2950, 2870°1730
．． 1690, 1455, 1375, 1240°1021
025o, /y, + 422,284,232,187
゜] 64゜ Elemental analysis (C31 [as I4604); Calculated value: C, 77, 14; It, '9.61, Experimental value: C, 76, 91; 11, 9.84゜ NMR data are shown in 1 and 2.

Example 7 30 rq of acetyl puffic acid was dissolved in 5 methanol of methanol, an excess of diazomethane ether solution was added, and the solution was left at room temperature overnight. After that, the solvent was distilled off under reduced pressure, and the residue was recrystallized from methanol to give a colorless plate. Crystals of ethyl methacetate, benzene, dissolved in hexane with O, IR, KRr, l: 29
50,2870.1?30,1455゜ax 1370.1270.10301030o/y,: 4
96° 436.298, 246, 187, 178° Elemental analysis (as C32"4804); J1 calculated value: C, 77
,38°11.9.74. Experimental value: C, 77, 15, I
I, 10.00° NMR data are shown in Tables 1 and 2.

Example 8 Effect of puff acid on black tumor cells Culture medium:) Synthetic medium of IAM, IIF-10 and Ri, -
A mixture of 15 and 3-1 was used by adding fetal bovine serum. The serum concentration was such that the number of cultured cells was 1/2 of the maximum value.

Procedure: Dissolve puff acid in the culture solution described in 4.5.
rd to culture 1 ((1) with a diameter of 60 mm, add 0.5 ml of suspension culture of mouse melanoma cells (melanoma 1316) to this.
me (containing 1X10' cells) and incubated for 4 days.
37° and 5% CO. After culturing in an incubator,
Remove the culture medium by aspirating, wash with saline, and add 0.12
5% trypsin-11ank's solution Q, 5ml! was added to detach the cells adhering to the culture vessel, and further 9.5 mF physiological saline was added to suspend the cells, and the number of cells was counted using a Coulter counter. The results are shown in Table 3. puff acid is 6
The proliferation of black tumor cells was significantly inhibited at the concentration of /'y/me.

Example 9 Effect of puff acid on cervical cancer cells The same procedure as in Example 8 was conducted using human cervical cancer cells (IIeL, aS 3 ). The results are shown in Table 4. Puff acid significantly inhibited the proliferation of human cervical cancer cells at a concentration of 4 py/me.

Example 10 The same procedure as in Example 8 was carried out using mouse lung cancer cells (3LL). The results are shown in Table 5. Puff acid is 4719/me
The proliferation of lung cancer cells was significantly inhibited at a concentration of .

Example 11 Tablets 20.0y of puffed acid, 49.6F of lactose, 49.6y of microcrystalline cellulose, 0.4f of cornstarch and 0.4g of macnesium stearate were mixed well, a slug was made using a tablet machine, and an oscillator was used. Coarsely crush, select, and form into granules. This granule (120.0y) is compressed according to a conventional method to produce 400 plain tablets. Puff acid 5 per tablet (300~)
Contains 0 Ing.

Example 12 Capsules Puff acid-containing dry extract (produced by concentrating the sulfuric acid hydrolyzate in Example 2, thoroughly washing the precipitate, and drying) 50.0 y (contains 5.0 y of puff acid) and lactose 50
．． The OF is mixed well and 500 capsules are manufactured using a capsule filling machine. Puff acid 10m2 per capsule
Contains.

Table 2 13C-NMRppm (C51,)5N
) 36-5 Shimonakasu, Nishisuka-cho, Tokushima City 0 Applicant: Nobuo Odajima 328 Kamishinmachi, Kanazawa, 1-numa, 14 (1) Showa 1) Philo-11122rl 'I diagonal'l IT I; Government 1 San L'-1jl'l's entry 11jt flll!') 71 Special Accommodation Application No.-11J3F
(7? No. 2 Kai, Ming's name Bano n So Otohi?'s intelligence guidance 1 book 3, it' f, l ro 11 things 1) Relationship with 1 i, l, M'l out ii+! '1 person (1 location Osaka ni Osaka Kakuno-ku Tatsumi Nishi 1NIJ'i Hanging No. 1 name n-
1. Seihin Co., Ltd.? 1 4, agent fl t'li〒h /I '1 n, tni if: 1st item of command; 1:1 intervention [i,
Supplement 11, Taku 1: Detailed explanation of the invention of Ming II Inyen, 1, 7,? One person on Mt. 11? ``Da details i'';'l D page i, l 1llll 1 B
ri/day 3 ti'(: lruIH+ 7 r'i
,'4 "2 RfX"('? l fJ J i
t'jV 11,・1゜1] 1

Claims (1)

[Scope of Claims] A compound represented by formula 1, in which R1 represents a hydrogen atom or an acyl group, and R2 represents a hydrogen atom or an alkyl group. 2. The compound according to item 1, wherein both R1 and R2 are hydrogen atoms and has the name puff acid. In the formula, R] represents a hydrogen atom or an acyl group, and R2 represents a hydrogen atom or an alkyl group. A method for extracting and isolating the compound represented by the formula [wherein k□ and R2 represent hydrogen atoms] from Brazilian ginseng, in which the dried Brazilian ginseng root is ground and extracted with alcohol, and the extract is added to the After adding sulfuric acid and heating to reflux, the solvent was distilled off, and the resulting residue was placed in a column packed with silica gel and mixed with chloroform/methanol (50: 1-20).
A method characterized by distilling off the solvent from both fractions eluted with : ) and recrystallizing the residue from alcohol.