JPH069462A - Aphidicolan derivative and its production - Google Patents

Abstract

PURPOSE:To provide the new 16beta-lower alkoxy-3alpha,17,18-trihydroxyaphidicolan having an antitumor action. CONSTITUTION:A compound of the formula (R is lower alkyl). The compound is obtained by reacting 3alpha,18-dihydroxy-16beta,17-epoxyaphidicolan with a lower alcohol (e.g. methanol) in the presence of an acidic substance (e.g. p- toluenesulfonic acid) as a catalyst at 0-100 deg.C. For example, 3alpha,17,18- trihydroxy-16beta-methoxyaphidicolan is produced as follows: introducing a 3alpha,18- isopropylidenedioxy group into 3alpha,16beta,18-trihydroxy-17-p- toluenesulfonyloxyaphidicolan, hydrolyzing the reaction product, introducing a trityloxy group at the 17-position of the hydrolyzed product, reacting the product with methyl iodide, converting the 16beta-position into a methoxy group, and subsequently hydrolyzing the 3alpha,18-isopropylidenedioxy group and the 17- trityloxy group.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an affidicorane derivative and a method for producing the same. More specifically, it relates to a novel affidicorane derivative having an antitumor effect and a method for producing the same.

[0002]

2. Description of the Related Art Aphidicolin, which is a typical Aphidicolin derivative, is a type of fungus, Verticillium.
It was discovered as an antiviral agent obtained from lecanii, is a poorly water-soluble substance having the structure of tetracyclic diterpene tetraol, and shows a high specific inhibitory action on eukaryotic cell-derived DNA polymerase α and δ types. It is used to discriminate the type of DNA, is ineffective for DNA polymerases derived from prokaryotic cells except for special viruses, and is mainly used for analysis of DNA replication and repair mechanism. Further, it is also known to have an antitumor action, an antiviral action, and a plant seed growth inhibitory action, and its use for such purposes has been attempted.

In recent years, attention has been paid to the antitumor activity of aphidicolin, and various derivatives have been synthesized (Japanese Patent Laid-Open Nos. 59-88438, 61-83138, and 62-155232). 62-155239, Nucleic Acid Research No. 1
Volume 7, pp. 6339-6348, 1989, J. Chem. Soc., Perkin T
rans. I 41-46, 1992). In particular, for aphidicolin-17-glycinate, which is a water-soluble ester derivative synthesized as a prodrug, clinical trials are in progress, producing aphidicolin in blood,
It is said to show antitumor activity (J. of Pharm. Sci.
Volume 8, No. 5, 399-401, 1989). However, it has been reported that the blood concentration of the produced aphidicolin is rapidly lowered (AACR Vol. 31, p. 180, 1990).

By the way, there have been reports of synthesizing derivatives obtained by substituting four hydroxyl groups contained in aphidicolin with various substituents and measuring their DNA polymerase α inhibitory activity (Chem. Pharm. Bull. 35). 1641 to 1644, 1987
), A compound in which only the 16β-position alcoholic hydrogen group is substituted with a methoxymethyl group as a protecting group is described therein, but it is stated that it does not exhibit an inhibitory effect on DNA polymerase α.

On the other hand, a compound in which only a stable alkyl group replaces the alcoholic hydrogen group at the 16β-position has not been reported yet, and therefore its biological activity is unknown.

[0006]

The object of the present invention is to provide 16β
-Lower alkoxy-3α, 17,18-trihydroxyaffidicolane, a synthetic intermediate thereof, and a method for producing the same.

[0007]

According to the present invention, the following 4
A class of affidichorane derivatives is provided.

[Chemical 1] 16β-lower alkoxy-3α, 17,18-trihydroxyaffidichorane [I] R = lower alkyl group

[Chemical 2] 16β-hydroxy-3α, 18-isopropylidenedioxy-17
-p-Toluenesulphonyloxyaffidicolane [II] R '= H, X = p-toluenesulphonyl group 16β-hydroxy-3α, 18-isopropylidenedioxy-17
-Trityloxyaffidichorane [III] R '= H, X = trityl group (triphenylmethyl group) 3α, 18-isopropylidenedioxy-16β-methoxy-17-
Trityloxy Affi di cholanic _{[IV] R'= CH 3,} X = trityl

Hereinafter, 3 of the compounds [I] will be described in the order of reaction steps.
The method for producing α, 17,18-trihydroxy-16β-methoxyaffidicolane will be described. (1) The compound [II] is obtained by reacting 3α, 16β, 18-trihydroxy-17-p-toluenesulfonyloxyaphidicorane with 2,2-dimethoxypropane in the presence of an acidic substance catalyst. The starting compound used here can be obtained by reacting aphidicolin with a p-toluenesulfonic acid halide in a pyridine solvent as described in JP-A-59-88438.

As the acidic substance catalyst, organic acids such as p-toluenesulfonic acid and inorganic acids such as hydrochloric acid are used. The reaction is usually carried out by suspending or dissolving the starting compound in a solvent such as acetone that does not interfere with the reaction, and adding about 2 to 100-fold molar amount of 2,2-dimethoxypropane and a catalytic amount of an acidic substance, It is carried out by reacting at -5 to 40 ° C, preferably about 0 to 20 ° C.

(2) Compound [II] thus obtained
Is dissolved in a water-miscible solvent inert to the reaction, such as dioxane, tetrahydrofuran, dimethylsulfoxide,
After reacting with a large excess amount of an alkali hydroxide aqueous solution at about 50 to 150 ° C, preferably about 80 to 120 ° C, 16β, 17-dihydroxy-
3α, 18-isopropylidenedioxyafidicolin
[V; compound in which R '= H and X = H in the above general formula] is obtained.

(3) The compound [V] is added to a pyridine solvent in an amount of 1.0 to
The compound [III] is obtained by reacting with a 1.5-fold molar amount of trityl halide at about 20 to 100 ° C, preferably about 50 to 80 ° C.

(4) The obtained compound [III] is treated with about 2% of a compound in aprotic polar solvent such as tetrahydrofuran, dimethylformamide and dimethylsulfoxide in the presence of a strong base such as n-butyllithium and sodium hydride. The compound [IV] is obtained by reacting with a methylating agent such as iodomethyl or dimethylsulfate in a molar amount of about 20 to 20 times at about 0 to 60 ° C, preferably about 20 to 50 ° C.

(5) The compound [IV] is treated with an acidic substance in the presence of water to hydrolyze both the isopropylidene group and the trityl group. In the hydrolysis reaction, compound [IV] is dissolved or suspended in water or a mixed solvent of a solvent inert to the reaction and water, and the reaction is performed in the presence of an acidic substance catalyst similar to the above.
This is carried out at 0 to 100 ° C. to obtain the final target compound [I].

3α, 17,18-trihydroxy-16β-methoxyaffidicolane is 3α, 18-dihydroxy-16β, 17-epoxyaffidichorane.

[Chemical 3] Can also be obtained by reacting with methanol, similarly, by reacting with other lower alcohol such as ethanol, n-propanol, isopropanol, n-butanol, n-pentanol, 16β-lower alkoxy-3α It is possible to obtain 17,17,18-trihydroxyaffidichorane.

The starting compound for this reaction, 3α, 18-dihydroxy-16β, 17-epoxyaphidicolane, is a known compound and is described in JP-A-59-88438. It can also be obtained by reacting -trihydroxy-17-p-toluenesulfonyloxyaphidicolane with a basic substance (see Japanese Patent Application No. 3-237489).

The reaction of this raw material compound with a lower alcohol is carried out by using a large excess of the lower alcohol which also serves as a solvent, and in the presence of a catalytic amount of an acidic substance, at about -5 to 100 ° C., preferably about 0.
It is carried out at a reaction temperature of ~ 30 ° C. At this time, the reaction can be performed by diluting with a solvent such as tetrahydrofuran or dioxane that does not react with the raw material compound under the reaction conditions.
Examples of the acidic substance used as a catalyst include organic acids such as p-toluenesulfonic acid and trifluoroacetic acid, inorganic acids such as hydrochloric acid and sulfuric acid, and π such as tetracyanoethylene.
Acid etc. are mentioned.

[0017]

INDUSTRIAL APPLICABILITY The present invention provides a novel affidichorane derivative having a lower alkoxy group at the 16β-position. Further, three kinds of affidichorane derivatives useful as synthetic intermediates thereof are provided.

The 16β-lower alkoxy derivative provided by the present invention is the first compound in which the 16β-position hydroxyl group is a stable alkyl ether type. At the cultured cell level, in addition to showing high antitumor activity, 16β-
Since it has a lower alkoxy group at the position, it may be metabolized differently from aphidicolin.

[0019]

EXAMPLES The present invention will now be described with reference to examples.

Example 1 8.04 g of 3α, 16β, 18-trihydroxy-17-p-toluenesulfonyloxyaphidicolin was suspended in 80 ml of acetone and ice-cooled. To this suspension, 4.0 ml of 2,2-dimethoxypropane and a catalytic amount of p-toluenesulfonic acid were added, and the mixture was stirred for 1 hour while maintaining the ice cooling temperature. The reaction mixture was poured into ice-cooled 2% aqueous sodium hydrogencarbonate solution (300 ml) and then extracted with ethyl acetate. The extract was washed with water, dried over anhydrous sodium sulfate, and concentrated to dryness. N-
It was treated with hexane to obtain 6.86 g of 16β-hydroxy-3α, 18-isopropylidenedioxy-17-p-toluenesulfonyloxyaphidicolin (melting point 142-145 ° C.) as a colorless powder. ¹ H NMR (CDCl ₃ , TMS) (270 MHz) δ: 0.71 (3H, s), 0.96 (3H,
s), 1.397 (3H, s), 1.40 (3H, s), 2.45 (3H, s), 3.22 (1H, d)
J = 12.2Hz), 3.62 (1H, d J = 12.2Hz), 3.62 (1H, b), 3.86
(2H, ABq J = 9.9Hz), 7.35 (2H, d J = 8.0Hz), 7.79 (2H, d J =
8.0Hz) ¹³ C NMR (CDCl ₃ , TMS) (68MHz) δ: 144.9, 132.7, 129.9,
128.0, 98.0, 75.4, 73.4, 73.4, 68.8, 49.0, 41.2, 4
0.0, 39.6, 35.2, 33.3, 32.3, 31.3, 29.5, 27.9, 26.
9, 26.6, 24.3, 24.1, 22.5, 21.7, 19.1, 17.3, 15.4

Example 2 3.27 g of 16β-hydroxy-3α, 18-isopropylidenedioxy-17-p-toluenesulfonyloxyafidicorane obtained in Example 1 was dissolved in 250 ml of dioxane and 250 ml of 1N potassium hydroxide aqueous solution. The mixture was added to the mixed solution and heated under reflux for 18 hours. After the reaction mixture was concentrated, ice water was added to the residue, and the precipitate was collected by filtration, washed with water and dried to give 16β, 17-dihydroxy-3.
2.21 g of α, 18-isopropylidenedioxyaphidicolane (melting point 75-85 ° C) was obtained as a colorless powder. ¹ H NMR (CDCl ₃ , TMS) (270 MHz) δ: 0.73 (3H, s), 0.99 (3H,
s), 1.41 (6H, s), 3.23 (1H, d J = 12.2Hz), 3.40 (2H, ABq J
= 11.2Hz), 3.63 (1H, b), 3.64 (1H, d J = 12.2Hz)

Example 3 2.05 g of 16β, 17-dihydroxy-3α, 18-isopropylidenedioxyafidichorane obtained in Example 2 and 3.03 g of trityl chloride were added to 3 ml of 20 ml of pyridine at 80 ° C.
Stir for hours. After cooling, pour the reaction mixture into ice water,
The precipitate was collected by filtration, washed with water and dried. Column chromatography of the dried product (Wako Gel C-300 75 g, eluent methanol
/ Chloroform = 1/199) and 16β-hydroxy-3
2.58 g of α, 18-isopropylidenedioxy-17-trityloxyafidicorane (melting point 241-244 ° C) was obtained as a colorless powder. ¹ H NMR (CDCl ₃ , TMS) (270 MHz) δ: 0.71 (3H, s), 0.97 (3H,
s), 1.40 (6H, s), 2.98 (2H, ABq J = 8.9Hz), 3.21 (1H, d J =
12.2Hz), 3.62 (1H, d J = 12.2Hz), 3.62 (1H, b), 7.20 to 7.
44 (15H, m) ¹³ C NMR (CDCl ₃ , TMS) (68MHz) δ: 143.9, 128.7, 127.8,
127.0, 98.0, 86.3, 74.2, 73.5, 68.8, 68.2, 49.1, 4
2.2, 40.1, 39.6, 35.2, 33.3, 32.7, 31.3, 29.5, 28.
9, 27.0, 26.6, 24.5, 24.3, 22.6, 19.1, 17.4, 15.4

Example 4 16β-Hydroxy-3α, 18-isopropylidenedioxy-17-trityloxyafidicorane obtained in Example 3
1.78 g are dissolved in 30 ml of tetrahydrofuran under a stream of nitrogen. To this solution was added 7.64 ml of an n-hexane solution having a concentration of 1.68 mol of n-butyllithium and stirred at room temperature for 1 hour, then 1 ml of methyl iodide was added and stirred at 50 ° C. for 3 hours. The obtained reaction solution further contains the above n-butyllithium solution
After adding 7.64 ml and stirring for 30 minutes, 2 ml of methyl iodide was added and stirred at 50 ° C. for 3 hours.

The reaction mixture was cooled to room temperature, poured into 100 ml of ice water, and extracted with ethyl acetate. The extract was washed with water, dried over anhydrous sodium sulfate, and concentrated to dryness. The residue was subjected to column chromatography (Wako Gel C-300 100
g, methanol / chloroform = 1/199), 3α, 18
1.52 g of -isopropylidene-16β-methoxy-17-trityloxyaphidicolin (melting point 196-199 ° C) was obtained as a colorless powder. ¹ H NMR (CDCl ₃ , TMS) (270 MHz) δ: 0.71 (3H, s), 0.96 (3H,
s), 1.40 (6H, s), 3.00 (2H, ABq J = 10.6Hz), 3.10 (3H,
s), 3.21 (1H, d J = 12.2Hz), 3.61 (1H, b), 3.62 (1H, d J = 1
2.2 Hz), 7.18 to 7.49 (15 H, m) ¹³ C NMR (CDCl ₃ , TMS) (68 MHz) δ: 144.1, 128.8, 127.7,
126.8, 98.0, 86.1, 78.7, 73.5, 68.8, 63.8, 49.4, 4
8.8, 40.3, 39.8, 39.6, 35.2, 33.3, 32.5, 31.5, 29.
6, 26.9, 26.6, 25.1, 24.5, 24.3, 22.6, 19.1, 17.
4, 15.4

Example 5 1.52 g of 3α, 18-isopropylidene-16β-methoxy-17-trityloxyaphidicolane obtained in Example 4 was added to 1
The mixture was stirred in 5 ml of 80% acetic acid aqueous solution at 80 ° C for 2 hours. The reaction mixture was concentrated to dryness, the residue was treated with 20 ml of ethyl acetate, and the precipitated powder was collected by filtration and subjected to column chromatography.
Purify with (Wakogel C-300 15g, eluent methanol / chloroform = 3/100) to give 3α, 17,18-trihydroxy-16
250 mg of β-methoxyaffidicorane (mp 182-186 ° C) was obtained as a colorless powder. ¹ H NMR (pyridine-d ₅ , TMS) (270
MHz) δ: 0.80 (3H, s), 1.02 (3H, s), 3.32 (3H, s), 3.58〜
3.85 (4H, m), 3.93 (1H, b), 5.69 (1H, b), 5.77 (1H, dd),
6.28 (1H, d J = 2.3Hz) ¹³ C NMR (pyridine-d ₅ , TMS) (68MHz) δ: 79.3, 76.3, 71.
9, 62.9, 49.3, 48.9, 41.0, 40.6, 40.1, 39.4, 34.
0, 33.0, 31.7, 27.4, 27.2, 25.4, 25.4, 23.5, 18.
1, 15.4

Example 6 1.025 g of 3α, 18-dihydroxy-16β, 17-epoxyaffidicolane was dissolved in 10 ml of methanol to obtain p-
1 mg of toluenesulfonic acid monohydrate was added, and the mixture was stirred at room temperature for 30 minutes. After pouring the reaction mixture into 200 ml of ice water,
The precipitate was collected by filtration and subjected to silica gel column chromatography.
(Wakogel C-300 150g, eluent: methanol / chloroform = 3/100), then recrystallize with ethyl acetate,
402 mg of 3α, 17,18-trihydroxy-16β-methoxyafidicolane was obtained as colorless prism crystals. The melting point, ¹ H NMR and ¹³ C NMR of this product were in perfect agreement with those obtained in Example 5 above.

Example 7 1 mg of p-toluenesulfonic acid monohydrate was added to a solution prepared by dissolving 658 mg of 3α, 18-dihydroxy-16β, 17-epoxyafidicolane in 8 ml of ethanol, and the mixture was allowed to stand at room temperature for 1 hour. It was stirred. The reaction mixture was poured into 100 ml of ice water and then extracted with 75 ml of ethyl acetate. 2 times the ethyl acetate layer with 100 ml of water
After washing twice, dry over anhydrous sodium sulfate, concentrate under reduced pressure,
756 mg of residue was obtained. This residue was flash chromatographed (20-40 μm particle size 15-40 μm Merck silica gel).
g, use, eluent: methanol / chloroform = 1/49) and further purify by HPLC (Inertosyl PREP-ODS, eluent: methanol / water = 4/1), then recrystallize with ethyl acetate, 240 mg of 16β-ethoxy-3α, 17,18-trihydroxyafidicolane (melting point 203-212 ° C) was obtained as colorless prism crystals. ¹ H NMR (pyridine-d ₅ , TMS) (270MHz) δ: 0.80 (3H, s), 1.0
3 (3H, s), 1.20 (3H, t J = 6.9Hz), 2.45 (1H, dd J = 3.6,13.2
Hz), 2.55 (1H, t J = 6.9Hz), 2.85 (1H, dd J = 3.0,12.2H
z), 3.59 (2H, q J = 6.9Hz), 3.64 ~ 3.86 (4H, m), 3.91 ~
3.96 (1H, b) ¹³ C NMR (pyridine-d ₅ , TMS) (68MHz) δ: 79.2, 76.3, 71.
9, 63.7, 56.2, 49.3, 41.0, 40.6, 40.1, 39.7, 34.
0, 33.1, 31.7, 27.4, 27.2, 25.7, 25.4, 23.5, 18.
1, 16.4, 15.4

[Tumor cell growth inhibition test] Mouse tumor cell YA
C-1 10 ⁶ cells / ml containing RPMI 1640 medium (10% FCS, 100 U /
100 μl of 100 ml of penicillin and 100 μg / ml streptomycin) was dispensed into each well of a 96-well microplate, and various concentrations of 3α, 17,18-trihydroxy-16β-methoxyaffidicolane (5% dimethyl sulfoxide solution) were added. Was diluted with the RPMI 1640 medium described above and adjusted to various concentrations), 100 μl was added to each well, and the mixture was cultured at 37 ° C. in a 5% CO ₂ incubator for 72 hours. The color change of the medium was compared with the control to determine the presence or absence of proliferation of the tumor cells.As a result, 3α, 17,18-trihydroxy-16β-methoxyafidicorane was found to have a tumor cell concentration of 0.39 μg / ml. Proliferation was completely suppressed. In addition, the corresponding 16β-ethoxy substitution completely suppressed the growth of tumor cells at a concentration of 0.78 μg / ml.

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Claims (10)

[Claims] 1. 16β-Lower alkoxy-3α, 17,18-trihydroxyaffidichorane. 2. 16β-Hydroxy-3α, 18-isopropylidenedioxy-17-p-toluenesulfonyloxyaphidicolin. 3. 16β-Hydroxy-3α, 18-isopropylidenedioxy-17-trityloxyaphidicolin. 4. 3α, 18-isopropylidenedioxy-16β
-Methoxy-17-trityloxyaphidicolane. 5. 16β-Hydroxy-3α, 18-isopropylidene diene characterized by reacting 3α, 16β, 18-trihydroxy-17-p-toluenesulfonyloxyaphidicorane and 2,2-dimethoxypropane Process for producing oxy-17-p-toluenesulphonyloxyaphidicolin. 6. 16β, 1 characterized by hydrolyzing 16β-hydroxy-3α, 18-isopropylidenedioxy-17-p-toluenesulfonyloxyafidicorane.
Process for producing 7-dihydroxy-3α, 18-isopropylidenedioxyafidicorane. 7. 16β-Hydroxy-3α, 18, characterized in that 16β, 17-dihydroxy-3α, 18-isopropylidenedioxyaphidicorane is reacted with trityl halide.
-A method for producing isopropylidenedioxy-17-trityloxyafidecorane. 8. 3α, 18-Isopropylidenedioxy-16β-methoxy-characterized in that 16β-hydroxy-3α, 18-isopropylidenedioxy-17-trityloxyafidicorane is reacted with a methylating agent. Method for producing 17-trityloxyaphidicolin. 9. 3α, 18-isopropylidenedioxy-16β
A process for producing 3α, 17,18-trihydroxy-16β-methoxyafidicolane, which comprises hydrolyzing -methoxy-17-trityloxyafidicorane. 10. A method for producing 3α, 17,18-trihydroxy-16β-lower alkoxyaphidicoranes, which comprises reacting 3α, 18-dihydroxy-16β, 17-epoxyaffidichorane with a lower alcohol.