CS252605B1 - 2a, 3a, 17β-trihydroxy-5α-androstan-6-one and a process for its preparation - Google Patents

Abstract

The solution relates to 2-alpha,3-alpha,17-beta-trihydroxy-5-alpha-androstan-6-one of formula I, i.e. a compound that affects plant growth in a similar manner to the plant hormone brassinolide, and a method for preparing this compound.

Description

The present invention relates to a novel 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one compound of the formula I having brassinolid activity by acting on plant growth and a process for its production .

Seven years ago [Grove M. D. et al .: Nature (London) 281, 216 (1979)], the plant hormone brassinolide, which causes cell division and elongation, has been isolated, thus affecting plant growth. The accessibility of brassinolide by isolation from natural material is quite difficult because it occurs at very low concentrations. E.g. in rape pollen [Grove M. D. et al., Nature (London) 281, 216 (1979). Its content is 0.1 ppm, in chestnuts [lkeda M. et al., Agr. Biol. Chem. 42, 655 (1983) .J 0.005 ppm, in Chinese cabbage [Morishita T. et al .: Phytochemistry 22, 1051 (1983J.J 0.000 003 ppm and the like).

The accessibility of brassinolide and its analogs by synthesis is also very difficult due to the complexity of the synthetic procedures and their low yields [see, for example: Abe et al., Agr.

Biol. Chem. 46, 2609 (1982); Fung, S. et al., J. Amer. Chem. Soc. 102, 6580 (1980); Ishiguro, M. et al., J. Chem. Soc., Chem. Commun. 1980, 962, Thompson M.J. et al., Steroids 38,

567 (1981), Thompson M. J. et al. (Chem. Abstr., 95, 127 425 (1981).), Sakakibara M. et al. : Heterocycles 17, 501 (1982), Sea K. et al., Tetrahedron 38, 2099 (1982) Sakakibara M. et al., Agr. Biol. Chem. 47, 663 (1983); Anastasia M. et al., J. Chem. Soc., Perkin Trans. I, 1983, 383, Takatsuto S. et al., J. Chem. Soc., Perkin Trans. I, 1984, 139, Japanese Patent 82-70900 (Chem. Abstr. 97, 163 330 (1982)) and Japanese Patent 82-163 400 (Chem. Abstr. 9ji, 198 606 (1983) J.

None of the compounds synthesized so far had at the same time two properties that are important for its practical utility: high potency and ease of preparation.

It is therefore desirable to prepare compounds having both of these properties.

During the study of the synthesis and efficacy of brassinolide analogues, it was unexpectedly found that 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one of formula I

shows unusually high potency in the second internode bean test [Mitchell J. W. et al .: Methods of Studying Plant Hormones and Growth Regulating Substances; 336, p. 26, US Government Printing Office, Washington D. C., 1968., a test that is used to determine brassinolide efficacy. The compound of the above formula (I) can be prepared in a very simple manner as described below.

The object of the present invention is 2-alpha, 3-alpha, 17-beta-trihydroxy-5alpha-androstan-6-one of the above formula I, characterized in that it affects the growth of plants.

A further object of the invention is a process for the preparation of a compound of the above formula I, characterized in that the compound of the formula II

(II)

Wherein R, R and R are independently of each other a suitable protecting group, preferably -123

CH 2 CO-, or at least one of R, R, and R is a suitable protecting group and the other is a hydrogen atom, in an alkaline or acidic environment (e.g., by treatment with an alkali metal hydroxide, preferably potassium or sodium hydroxide), of formula X.

The compound of general formula may be prepared, for example

3 of formula II, wherein R, R and R are as defined above, are as defined above

Wherein R and R are independently of each other a suitable protecting group as described above by treatment with a suitable peracid, for example trifluoroperacetic acid or m-chloroperbenzoic acid, preferably trifluoroperacetic acid, in a suitable inert solvent, preferably carbon tetrachloride, for two to 240 hours at room temperature.

3

A compound of formula II in which R, R and R are as defined above may also be prepared by reacting a compound of formula IV

(IV) wherein R is a suitable protecting group, preferably CH 2 CO-, hydroxylates, for example with osmium tetroxide or a mixture of osmium tetroxide and N-methylmorpholine-N-oxide, preferably in a second manner, to form a compound of formula II, in which í <

R 3 is both hydrogen and R is a suitable protecting group as described above.

The plant growth activity of the compound of the above formula I according to the invention was compared with that of epibrassinolide, a compound which is close to that of natural brassinolide. From Figure 1 (curve 1 represents the efficacy of epibrassinolide, curve 2 represents the efficacy of the compound of formula I) shows that the efficacy of the compound of formula I and epibrassinolide are very similar. Although the efficacy of Compound I is slightly lower than that of epibrassinolide, it is in favor of incomparably simpler synthetic accessibility.

The following examples are intended to illustrate the invention in more detail, but are not intended to limit the scope of the invention.

Example 1

Preparation of 2-alpha, 3-alpha, 17-beta-triacetate 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one

To a solution of trifluoroacetic anhydride (5.7 mL) in dichloromethane (32.1 mL), cooled to about 5 ° C, was carefully added hydrogen peroxide (1.07 mL, 50%). After about two minutes, a solution of 2-alpha, 3-alpha-dihydroxy-5-alpha-pregnane-6,20-dione 2,3-diacetate (3.21 g) in dichloromethane (32.1 ml) was added.

The mixture was allowed to stand at room temperature for two hours. Then the same solution of trifluoroacetic anhydride and hydrogen peroxide is added to the mixture as above. The mixture was allowed to stand for another 4 hours at the same temperature. Then the reaction mixture is poured into water, the product is extracted with dichloromethane, the organic phase is washed with water, potassium bicarbonate, water and dried. A mixture is obtained which contains three products: the starting compound, the more lipophilic product and the more polar product. This mixture was separated by silica gel column chromatography (300 g, eluting with 7: 3 benzene / ether).

Treatment of the fractions with the lipophilic product gave 1.0 g of 2,3,17-triacetate 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one, m.p. 203-205 ° 23 ° (c 1.2).

IC spectrum (chloroform): 1737, 1259 cm @ -1. Mass spectrum: m / Z 448 (M @ ⁺ ).

NMR Spectrum: 0.79 (s, 19-H), 0.83 (s, 18-H), 1.96, 2.02 and 2.06 (three singlets, three acetates), 4.42-5. O 2 (mt, 2-beta-H and 17-alpha-H), 5.37 (dd, J = 3 Hz, J 1 = 6 Hz, 3-beta-H). For ^C 25 ^H 36 ° 7 (448.7) calculated: 66.94 8 C, 8.09 8 H, found: 64.95 8 C,

8.08 8 H.

Example 2

Preparation of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one 17-acetate

To a solution of 17-beta-hydroxy-5-alpha-androst-2-en-6-one 17-acetate (600 mg) in acetone (30 mL) was added a solution of osmium tetroxide in tert-butanol (10%, 0.3 ml), t-butanol (0.7 ml), N-methylmorpholine-N-oxide (600 mg), water (1 ml) and the mixture was stirred under sodium at room temperature for 24 hours.

Excess osmium tetroxide is decomposed with 38 aqueous sodium thiosulphate solution. The product was extracted with chloroform. The organic phase was separated, washed with 58 hydrochloric acid, water and dried over sodium sulfate. Evaporation of the solvent yielded 600 mg of product which crystallized upon addition of ether. 459 mg of crystals were obtained, which were recrystallized from acetone-heptane to give 378 mg of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one 17-acetate, mp 205-207 ° C. For ^C 21 ^H 32 4 (348.5) calculated: 72.38 8 C, 9.26 8 H, found: 72.09 8 C, 9.10 8 H.

Example 3

Preparation of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one from the compound prepared according to example 1

To a solution of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one 2,3,17-triacetate (345 mg) prepared in Example 1 in methanol (17 ml) was added dropwise. potassium hydroxide (1 g). The mixture was refluxed for 30 minutes.

The reaction mixture was evaporated under reduced pressure to about 1/4 volume, poured into water and the product was extracted with chloroform. The chloroform phase is separated, washed with 58 hydrochloric acid, water, potassium bicarbonate, water, dried and the solvent is distilled off.

38 mg of colorless needles are obtained, mp 226-228 ° C. Evaporation of the mother liquors to dryness by crystallization of the residue from chloroform gave an additional 86 mg of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one, mp 220-225 ° C | appointing ² ® O ° (0.8).

IR (KBr): 3,640, 1,078, 1,049, 1,021 (hydroxyls), 1,710 (C = O) cm ^-1 . CD spectrum (dioxane) and? ₃₀₁ = -1.00, ^X H NMR (CDCl mixture and CDjOD): 0.74 and 0.77 (two singlets, 18-H and 19-H), 3.35 to

3.80 (mt, 2-beta-H, 3-beta-H and 17-alpha-H). For C ₁₉ H ₃₀ O ₄ (322.4) calculated: 70.77 8 C,. 9.38 8 H, found: 70.82 8 C, 9.21% H.

Example 4

Preparation of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one from the compound prepared according to example 2

To a solution of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one 17-acetate (60 mg) prepared according to Example 2 in methanol (10 mL) was added aqueous hydrogen carbonate solution. potassium (60 mg, 1 mL). The mixture was refluxed for two hours. After concentration to 1/3 volume by distilling off the solvent in vacuo, the mixture was poured into water. The product was extracted with chloroform.

The chloroform extract was washed with water, dried over sodium sulfate and the solvent was distilled off in vacuo. The evaporation residue (55 mg) from acetone-heptane gave 31 mg of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one, identical in all directions to the compound prepared according to Example 3.

Example 5

Preparation of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one from the compound prepared according to example 2

To a solution of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one 17-acetate (150 mg) prepared according to Example 2 in methanol (20 mL) was added concentrated hydrochloric acid (27%, 1 mL).

The mixture was allowed to stand overnight at room temperature. The reaction mixture was then evaporated to dryness in vacuo. The residue was dissolved in chloroform, washed with potassium bicarbonate, water, dried over sodium sulfate and the solvent was distilled off. The crystallization residue from acetone-heptane gave 85 mg of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one, identical in all respects to the compound prepared according to Example 3.

Example 6

Testing of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one on the second bean internode

Bean seeds (Phaseolus vulgaris, var. Pinto) are planted in 7.0 cm pots containing vermiculite and Hoagland's solution (half concentration, pH = 5.7). The plants are grown in a greenhouse at a temperature of 23-27 ° C with a light of 6000 lux for 16 hours a day.

Different amounts (in moles) of the test compound in 2 µl of lanolin are applied to groups of 8 plants 7 days old with a second internode 2 mm long. Lanolin alone is applied to control plants. The method of application is described in the literature (Mitchell J. W. et al .: Methods of Studying Plant Hormones and Growth Regulating Substances, Agriculture Handbook No. 336, p. 26, US Government Printing Office, Washington D. C., 1968.).

Evaluation is performed on the fifth day after application. The evaluation measures the length of the second internode of the test and control plants. The difference between these lengths (v in mm) is used as a measure of the potency of these compounds.

Test results of 2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one (compound of formula I, curve 2 in Figure 1) and its comparison with epibrassinolide £ (22R, 23R, 24R) - 2-alpha, 3-alpha, 22,23-tetrahydroxy-24-methyl-β-homo-7-oxa-5-alpha-cholestan-6-one (curve 1, Figure 1) are shown in Table I and Figure 1.

Table 1

Compound applied dose in moles of extension of the second internode of the bean at the given applied dose (in mm):

2-alpha, 3-alpha, 17-beta-trihydroxy-5-alpha-androstan-6-one 8.4 15.5 19.6 26.3 12.8 7.4 epibrassinolide - 20.4 30.8 32 , 3 18.6 12.9 7.2

Claims (2)

SUBJECT OF THE INVENTION 1. 2-alpha, 3-alpha, 17-beta-Trihydroxy-5-alpha-androstan-6-one of formula I A process for the preparation of a compound of formula (I), characterized in that the compound of formula (II): Wherein R, R and R independently of one another represent a suitable protecting group, preferably the group 12 3 CH 3 CO-, or at least one of R, R and R is a suitable protecting group and the other is a hydrogen atom, in an alkaline or acidic medium, is converted to a compound of formula I