POLYHYDROXYSPIROSTANNONES AS PLANT GROWTH REGULATORS

Abstract: This present invention is related with the biological chemical branch, and in particular with the preparation of new spirostanics analogues of brassinosteroids and with oxygenated functions in diferent positions of the A ring starting from sapogenins steroidals, through the performance of dihydroxylation reactions to a Δ 2 -steroid as principal reaction. The preparation of 6-oxo spirobrassinosteroids is described with oxygenated functions in positions 2 and 3 o 3 and 5, with the following general formula (2): where: Compounds R 1 R 2 R 3 2a CH 3 COO β CH 3 COO β H α 2b HO β CH 3 COO β H α 2c CH 3 COO β HO β H α 2d HO β HO β H α 2e HO β HO β H β 2f CH 3 COO β CH 3 COO β H β 2g CH 3 COO β CH 3 COO α H α 2h CH 3 COO β HO α H α 2i HO β HO α H α 2j HO β HO α H β 2k CH 3 COO β CH 3 COO α H β 2l CH 3 COO α CH 3 COO β H α 2m HO α HO β H α 2n HO α HO α H α 2ñ CH 3 COO α CH 3 COO α H α 2o H CH 3 COO β HO α 2p H HO β HO α 2q H HO β HO β 2r HO α HO α HO α 2s CH 3 COO α CH 3 COO α HO α These synthesized compounds present regulating activity of plant growth in very low concentrations, with wide perspectives for their use in Agriculture.

Description: This invention is related to the chemical-biological field and in particular to a procedure to obtain new spirostan analogs of brassinosteroids based on steroidal sapogenins, through partial synthesis. These spirostan analogs have a regulating effect on plant growth. Brassinosteroids are steroidal compounds, with a regulating activity on plant-growth and which are found in very small quantities in plants. For this reason extracting them from plants is economically prohibitive. Some of the natural compounds have been obtained sinthetically but with very low yields, thus the current tendency is to synthetisize analogs of these compounds. The majority of them possess (among other typical structural requirements), a 2α,3α- diol system in the A ring and a union trans of the A and B rings, of the steroidal system. Many scientists attribute a relevant importance to this peculiarity because of the biological activity that they are able to carry out. For that reason, some of the analogs of synthesized brassinosteroids present substituents in positions 2 and 3 of the A ring with alfha stereochemical, which exhibit activity as regulators of plant growth, (Kohout, L., Strnad, M., Collect. Czech. Chem. Commun. 57 , 1731, 1992 ; Cerny,V., et al Collect. Czech. Chem. Commun . 51 , 687, 1986 .) Also, the first similar spirostanics of brassinosteroids synthesized starting from steroidals sapogenin, possess α stereochemistry /orientation of the sustituents groups in the A ring in the positions 2,3 (Marquardt, V. et al, German Patent CO7J 63 / 00 273638A , 1989 ), but the synthesis of them involves the utilization of a very toxic reagent, osmium tetroxide. It is also interesting to study how variations in the positions of these hydroxylic groups in the A ring influence the biological activity that these compounds exert. The synthesis of compounds shown in this invention possess oxigenated function in the positions 3,5 with different stereochemistry, for example : the (25R) 3β-acetoxy-5-hydroxy-5α-spirostan-6-one 2a , (25R) 3β,5-dihydroxy-5α-spirostan-6-one 2b and (25R) 3β,5-dihydroxy-5β-spirostan-6-one 2c . These structural characteristics is unusual for this type of compounds with plant growth promoting activity. In 1975, the compound (25R) 3β,5-dihydroxy-5α-spirostan-6-one 2b was synthesized by Novosel'skaya, I.L. et al ( Chemistry of Natural Compounds, Vol 11 , No 2, 268-269, 1975 ) as intermediate for the purpose to obtain the ecdysones analogs from steroid sapogenins. The diosgenin was converted into the triol (25R) 5α-spirostane-3β,5,6β-triol through the method reported by Romo, J. et al ( J. Org. Chem ., 19 , 1509, 1954 ) with hydrogen peroxide/formic acid. On oxidation of the triol with N-bromosuccinimide in dioxane solution for seven days formed the compound 2b (Nawa, H. et al, Chem . Pharm. Bull. Jpn. 11, 139, 1963 ). The acetylation of the compound 2b gave the compound 2a. By the same way, in 1982, Dawidar, A.M. et al (Zeitschrift für Naturforschung , part B : Anorganische Chemie, Organische Chemie, vol 37B , No 7, 892-895, 1982 ) reported the synthesis of the compound 2b with in order to prepare 6-keto diosgenin derivatives which could be converted to the intermediate 6-methyl pregnane compounds. The reaction of diosgenin with hydrogen peroxide/formic acid has the inconvenient to produce 5α-pregnane-3β,5,6β,16β,20α-pentol (POL) as a by product. By the other hands, oxidation process with NBS required several days. In 1984 Schwantz, R. et al, ( Revue Roumanie of Chimie , 29 , 9-10, 755-759, 1984 ) published the synthesis of the (25R) 3β-acetoxy-5-hydroxy-5α-spirostan-6-one (2a) as an intermediary compound for the synthesis of 3β-acetoxy-B-nor-androst-5-en-3β-ol-17-one and 3β-acetoxy-B-nor-pregn-5-en-3β-ol-20-one, by means of acetilation reactions with acetic anhydride and pyridine, to obtain the acetate of diosgenin. Then an epoxidation of the double bond with sodium acetate, chloroform and peracetic acid at 14.5%, is performed to obtain the corresponding epoxide and subsequently it is oxidized utilizing acetone and chromic anhydride dissolved in water. In the present invention, with the purpose of obtaining the compound 2b in mass quantities, the previously described methods were repeated, until (25R)3β-acetoxy-5-hydroxy-5α-spirostan-6-one 2a , noting that in the epoxide oxidation with the above mentioned conditions, the reaction takes place with difficulty and low yields to produce the corresponding ketol. Also this process requires the acetylation of diosgenin with acetic anhydride and pyridine at room temperature for 24 hours, which, in addition to the time required involves the utilization of a pyridine reagent which is a toxic and relative expensive reagent. Bearing this in mind, it was decided to develop a procedure for the synthesis of spirostanones with oxygenating functions at 3,5 positions of the A ring with a different stereochemistry employing a feasible, continuous and higher yield procedures in their preparation. The method used to obtain new spirobrassinosteroids with the general formula is described below: Compounds R 1 R 2 R 3 2a H CH 3 COO β HO α 2b H HO β HO α 2c H HO β HO β The compounds with these general formulas present a regulating activity on plant growth. Diosgenin was direct acetilated by heating with (1-3) part in volume of acetic anhydride, the wet diosgenin acetate collected by filtration was suspended in (20-40) part in volume of acetone and treated with acetic anhydride/H 2 O 2 at 35 % at room temperature, followed by another oxidation with (1-3) parts in volume of the Jones reagent. These process can be realized in another solvents such as methylethylketone, isopropylmethylketone, isobutylmethylketone, dichloromethane, 1,2-dichloroethane, etc. The saponification of 2a utilizing (10-20) part in volume potassium hydroxide dissolved in 5% ethanol produces compound 2b. The C-5 isomerization of the compound 2b produces 2c . The synthetic scheme followed to obtain the products was: This invention made possible to obtain three new spirobrassinosteroids with oxygenating functions in 3,5 positions of the A ring with a different stereochemistry, that demonstrate activity as controllers of plant growth. The synthetic process utilizes common chemical reagents with low toxicity. The synthesized compounds facilitate the increased growth and development of the plants, as well as their agricultural yields. They are additionally use as an additive in the post-harvest conservation of the flowers. Example of the process. To a suspension containing 2g of diosgenin in 6 mL of anhydride acetic was heated under reflux for 1.5 h.. When the reaction was concluded, was cooled and the diosgenin acetate was filtered. The wet acetate was suspended in 10 mL of acetone and treated under mechanical stirring with 2mL of anhydride acetic, 0.6 g of sodium acetate, 1.4 mL of H 2 O 2 were added dropwise at 0-10 °C. When the reaction was concluded, the mixture washed with a saturated solution of sodium chloride and sodium hydrogen carbonate. The organic phase was heat under refluxing, 1.5 mL of Jones reagent were added dropwise. When the reaction was concluded, the usual work-up afforded the product 2a . Yield: 2.2 g (95%). The compounds referred in this invention can be utilized as the active principles of a formulation that presents properties as vegetal growth regulator and as an additive for the post-harvest conservation of flowers. As regulator of plant growth the compounds in this invention can be prepared in liquid form or in a water solution, mixing the active ingredient with one or various types of liquid agents including organic solvents such as ethanol (500-1000 mL) and dimethylformamide (20-60g), among others. If can be applied as well as a solid, or a liquid formulation, etc, mixed with fertilizers, pesticides, herbicides, etc. When preparing the formula an active surface agent such as tween-20 (0.05-0.1 %) can be added to improve its dispersability, when preparing a water solution for the concentrated liquid formula for its application to the plant. This formulation results stable to temperatures less than 35°C for periods until one year. The compounds referred in this invention can be applied in concentrations of 10 -4 to 10 ppm and in a dose within the range of 0.1 to 1000 mg/ha depending on the vegetative state of the plant and the edafoclimatics conditions, among other factors. The property as plant growth regulator of the synthesized products was determinated by means of bioassays : 1. Hypocotyl elongation and expansion of the radish colyledons 2. Retention of the degradation of the photosynthetic pigments in wheat leaves. In the first bioassay, solutions with a concentration of the active principle between 0.01-10 [ppm] were employed, to obtain an increase in the length of the hypocotyl between 1.1 ± 0.22 cm, as well as an increment in the weight of the cotyledons. Moreover, the bioactivity of the compounds was corroborated by means of the same field test up to 1 Ha with various cultivars, for example: CULTIVAR CONCENTRATION YIELD INCREASE (AVERAGE %) Onion 0.1-1 ppm 21-40 % Pepper 0.5-1 ppm 8-50 % Garlic 0.1-0.5 ppm 2-15 % Potato 0.1-10 ppm 18-25 % Corn 0.1-0.5 ppm 10-47 % Soy bean 0.1-1 ppm 9-34 % Sorghum 0.1-1 ppm Aproxim. 15 % Rice 0.1-1 ppm 12 % Tomato 0.1-1 ppm 6-11%

Claims: Spirostanones with oxygenating functions in the A ring, as regulator of growth and their preparation procedure, characterized by general formula (2): where: Compounds R 1 R 2 R 3 2a H CH 3 COO β HO α 2b H HO β HO α 2c H HO β HO β The procedure for the synthesis of spirobrassinosteroids derivatives according to claim No. 1 is characterized because such compounds are obtained by means of steroidal sapogenin through the following chemistry reactions:: a- The compound 2a is obtained through direct oxidation of diosgenin acetate utilizing between (20-40), (1-3) and (0.5-1) parts in volume of acetone, acetic anhydryde and hydrogen peroxide at 35% respectively and sodium acetate in molar proportion of (1-2) followed by a further oxidation with (1-3) parts in volume of Jones reagent. b- The saponification of 2a utilizing (10-20) part in volume of potassium hydroxyde disolution in 5% ethanol yield the compounds 2b . c- The C-5 isomerization of the compound 2b produces 2c . Spirostanones obtained according to claims 1 and 2 are characterized because their are used as regulator of vegetal growth. Spirostanics analogues of brassinosteroids according to claims from 1 to 3 are characterized because they are utilized as regulators of growth plant and as flowers preservative. Formulation that contains spirostanones or spirostanics analogues of brassinosteroids of claims (1 and 4), is characterized by being a liquid form that contains between 10 -4 - 100 ppm of the active principle and can be applied alone or mixed with herbicides, pesticides, fertilizers, etc, that are soluble in water. Use of spirostanones or spirostanics analogues of brassinosteroids of former claims as regulators of growth plant and like flowers preservative.

Inventors: ALONSO BECERRA ESTHER MARIA, CABRERA PEDROSO MARIA TERESA, COLL MANCHADO FRANCISCO, JOMARRON RODILES ISABEL MARIA, ROBAINA RODRIGUEZ CARIDAD MARC, ALONSO BECERRA, ESTHER MARIA, CABRERA PEDROSO, MARIA TERESA, COLL MANCHADO, FRANCISCO, JOMARRON RODILES, ISABEL MARIA, ROBAINA RODRIGUEZ, CARIDAD MARCELINA
Applicants: ALONSO BECERRA ESTHER MARIA, CABRERA PEDROSO MARIA TERESA, COLL MANCHADO FRANCISCO, JOMARRON RODILES ISABEL MARIA, ROBAINA RODRIGUEZ CARIDAD MARC, ALONSO BECERRA, ESTHER MARIA, CABRERA PEDROSO, MARIA TERESA, COLL MANCHADO, FRANCISCO, JOMARRON RODILES, ISABEL MARIA, ROBAINA RODRIGUEZ, CARIDAD MARCELINA
Application year: 1996
Main classification: C07J71
IPC codes: C07J71