WO2016135749A1 - Diosgenin acetate-isoxazole derivatives, process for preparation thereof and their antifungal activity - Google Patents

Diosgenin acetate-isoxazole derivatives, process for preparation thereof and their antifungal activity Download PDF

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WO2016135749A1
WO2016135749A1 PCT/IN2016/050062 IN2016050062W WO2016135749A1 WO 2016135749 A1 WO2016135749 A1 WO 2016135749A1 IN 2016050062 W IN2016050062 W IN 2016050062W WO 2016135749 A1 WO2016135749 A1 WO 2016135749A1
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diosgenin
dihydro
dehydro
formula
acetate
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Archana Moni DAS
Manash Protim HAZARIKA
Purnajyoti DEKA BHUYAN
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Council of Scientific and Industrial Research CSIR
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0036—Nitrogen-containing hetero ring
    • C07J71/0042—Nitrogen only
    • C07J71/0047—Nitrogen only at position 2(3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0036—Nitrogen-containing hetero ring
    • C07J71/0057—Nitrogen and oxygen
    • C07J71/0063—Nitrogen and oxygen at position 2(3)

Definitions

  • This invention relates to the synthesis of a novel class of Diosgenin acetate-isoxazole starting from Diosgenin and its relatives and their antifungal activities against plant pathogenic fungus Alternaria alternate.
  • the compound of formula A antifungal activities against plant pathogenic fungus Alternaria alternate prepared by the process of invention are found to have an overall yield 80% after purification.
  • the process of the present invention does not involve the use of any costly and environmentally toxic reagents and catalysts and has been developed using a non-toxic reagents under mild reaction conditions.
  • Isoxazoline derivatives of the steroid compounds solasodine and diosgenin [Prismetov, M. P.; Dzhiembaev, B. Zh.; Kiseleva, E. N.; Kharlamova, T. V., Seriya Khimicheskaya (2000), (3), Page No. 28-30.]
  • This article describes the method for the synthesis of isoxazolinosolasodine and diosgenin via reaction of steroid ketones with formamide in the presence of perchloric acid with structural formula as given below:
  • This document discloses antifungal activity of 22 derivatives of C-27 steroidal saponins and 6 steroidal sapogenins against Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, and Aspergillus fumigates with the following structures.
  • This could be useful as potent antifungal inhibitors shown in Fig.4.
  • the present invention provides a compound of formula A
  • R2 OAc or OH
  • R C 6 H 5 or CH 3 .
  • diosgenin acetate -Isoxazoles exhibits good antifungal activity.
  • diosgenin 1 i. optionally hydrogenating diosgenin 1 to obtain dihydrodiosgenin lb;
  • step (v) mixing the solution of C-26-oxime-5,6 dehydro-diosgenin acetate or C-26-oxime-5,6 dihydro-diosgenin acetate obtained in step (v) with phenyl acetylene or methyl acetylene, base and sodium hypochlorite in DCM (dichloromethane) at the temperature ranging between 30-50 °C under nitrogen atmosphere followed by the stirring at the temperature ranging between 30-50 °C for the period of time 25-48 hr;
  • DCM dichloromethane
  • step (vi) adding water to the reaction mixture obtained in step (vi) then extracted with water immiscible solvent followed by washing the combined organic layer with water and brine;
  • step (viii) drying the organic layer obtained in step (vii) followed by evaporating the solvent and purifying by column chromatography to get (22P,25R)-3P-acetoxyfurost-5,6,-dehydro-26-substituted isoxazole or (22P,25R)-3P-acetoxyfurost-5,6,-dihydro-26- substituted isoxazole a compound of formula A;
  • base is selected from the group consisting of pyridine, triethylamine.
  • protic solvent selected from methanol, ethanol, isoporyl alcohol.
  • water immiscible solvent selected from the group consisting of ethyl acetate, dichlorome thane.
  • composition comprising an antifungal compound of formula A or a pharmaceutically acceptable salt thereof along with pharmaceutically acceptable excipients.
  • compound of formula A is used for treating or preventing a fungal infection against plant pathogenic fungus Alternaria alternate.
  • Step C Oxidation of the hydroxyl group on C-26 of E-ring manipulation of diosgenin acetate of formula 4, starting from C-26-ol-5,6 dehydro-diosgenin acetate and C-26-ol- 5,6 dihydro-diosgenin acetate of formula 3 and their relatives under a mild reaction conditions as per literature procedures;
  • Step D Oxime formation on C-26 of E-ring manipulation of diosgenin acetate of formula 5, starting from C-26-aldehyde-5,6 dehydro-diosgenin acetate and C-26- aldehyde-5,6 dihydro-diosgenin acetate of formula 4 and their relatives under a mild reaction conditions ie.
  • Step E Isoxazole formation on C-26 of E-ring manipulation of diosgenin acetate of formula 7, starting from C-26-oxime-5,6 dehydro-diosgenin acetate and C-26-oxime- 5,6 dihydro-diosgenin acetate of formula 5a and 5b and of formula 6, under a mild reaction conditions ie.
  • Step D Formation of oxime [Step D] on C-26 of E-ring manipulation of diosgenin acetate of formula 5 starting from C-26-aldehyde-5,6 dehydro-diosgenin acetate and C-26-aldehyde-5,6 dihydro-diosgenin acetate of formula 4 and their relatives under a mild reaction conditions ie. reaction of C-26-aldehyde-5,6 dehydro-diosgenin acetate and C-26-aldehyde-5,6 dihydro-diosgenin acetate of formula 4a and 4b with acetylene in presence of hydroxylamine hydrochloric acid, pyridine and ethanol.
  • FIGURE 1 Reagents and conditions: (a) Pyridine/ Acetic anhydride/40-90 °C/ recrystallize [MeOH/EtOH/PrOH] (b) NaBH 3 CN [in CH 3 COOH/ C 3 H 7 COOH / C 4 H 9 COOH (2-5 mL)]/ Inert gas atmosphere/30-60 hrs/20-30 °C/CH 2 C1 2 / Base/ purification by column chromatography (c) PCC/CaC0 3 /Silica/ CH 2 Cl 2 /30-60 °C/ 4-12 hrs/ purification by column chromatography (d) Hydroxylamine hydrochloric acid/ Pyridine/Ethanol/ 60-100 °C/H 2 0/ extract with ethyl acetate (e) DCM/Acetylene/ Triethylamine/ sodium hypochlorite/0-40 °C/ 20-48 hrs/Nit
  • FIGURE 2 General formula according to the claim 1 is given below (Formula 1, 2, 3, 4, 5, 6 and 7):
  • FIGURE 3 Formula of final compounds
  • the present invention is to provide a compound of formula A
  • R2 OAc or OH
  • R C 6 H 5 or CH 3 .
  • the representative compounds are selected from;
  • Step C Oxidation of the hydroxyl group on C-26 of E-ring manipulation of diosgenin acetate of formula 4, starting from C-26-ol-5,6 dehydro-diosgenin acetate and C-26-ol- 5,6 dihydro-diosgenin acetate of formula 3 and their relatives under a mild reaction conditions as per literature procedures.
  • Step D Oxime formation on C-26 of F-ring manipulation of diosgenin acetate of formula 5, starting from C-26-aldehyde-5,6 dehydro-diosgenin acetate and C-26- aldehyde-5,6 dihydro-diosgenin acetate of formula 4 and their relatives under a mild reaction conditions ie. reaction of C-26-aldehyde-5,6 dehydro-diosgenin acetate and C- 26-aldehyde- 5,6 dihydro-diosgenin acetate of formula 4a and 4b with acetylene in presence of hydroxylamine hydrochloric acid, pyridine and 95% ethanol. The reaction mixture was refluxed, the progress of the reaction was monitored by TLC and the mixture was cooled with ice, washed with water and extracted with ethyl acetate. The solvent was removed under reduced pressure to get the desired products 5.
  • Step E Isoxazole formation on C-26 of E-ring manipulation of diosgenin acetate of formula 7, starting from C-26-oxime-5,6 dehydro-diosgenin acetate and C-26-oxime- 5,6 dihydro-diosgenin acetate of formula 5a and 5b and of formula 6, under a mild reaction conditions ie.
  • Antifungal activity test was performed using the plant pathogenic fungus Alternaria alternate with the inhibition percentage 85 as depicted in the Fig.4 and Table 1 as determined by the standard antifungal activity test, viz., Poison Food Technique.
  • R2 OAc or OH
  • Rl OH
  • acetylated the diosgenin viz. 5,6 dehydro-diosgenin and 5,6 dihydro-diosgenin [Step A] to acetylated diosgenin of the formula 2a and 2b.
  • the compound of formula 7a and 7b antifungal activities against plant pathogenic fungus Alternaria alternate as shown in Fig.4 and Table 1.
  • Example 1 is given by way of illustration of the working of the invention in actual practice and should not be construed to limit the scope of the present invention in any way.
  • Example 1 is given by way of illustration of the working of the invention in actual practice and should not be construed to limit the scope of the present invention in any way.
  • Step A Hydrogenation of Diosgenin : Diosgenin 1, two grams in 100 ml of ethanol was hydrogenated at 45 psi using 500 mg of 5% Pd/C for a period of 10 h. The reaction mixture was filtered and alcohol was distilled off under reduced pressure to furnish the crude hydrogenated product which was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as the eluent to furnish dihydro diosgenin compound lb in pure form of 95% yield.
  • Step B F-ring opening of Diosgenin acetate of formula 3: ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ - acetoxyfurost-26-ol : lg NaBH 3 CN was added to a 1 g solution of diosgenin acetate 2a in acetic acid 3 ml under argon atmosphere and stirred for 60 hrs at (30 °C). After that CH 2 CI 2 was added and then Na 2 C0 3 solution. The aqueous phase was extracted with CH 2 CI 2 and the combined organic layer dried over anhydrous Na 2 S0 4 and evaporated to get the product. Then the residue was purified by column chromatography (30% ethyl acetate/hexane) to give pure product 3a (white gummy liquid) with the yield of 0.716 g, 71%.
  • Step C Oxidization of the formula 3 to aldehyde of formula 4:((22 ⁇ ,2513 ⁇ 4)-3 ⁇ - acetoxyfurost-26-al:
  • Step E Isoxazole derivative of Diosgenin acetate of formula 7: ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ - acetoxyf urost-26- (5-pheny 1 (isoxazole (8a) :
  • Step A Hydrogenation of Diosgenin lb:
  • Diosgenin 1 two grams in 100 ml of ethanol was hydrogenated at 45 psi using 500 mg of 5% Pd/C for a period of 10 h.
  • the reaction mixture was filtered and alcohol was distilled off under reduced pressure to furnish the crude hydrogenated product which was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as the eluent to furnish dihydro diosgenin compound lb in pure form of 95% yield.
  • Step B F-ring opening of Diosgenin acetate of formula 3 : ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ - acetoxyfurost-26-ol) :
  • Step C Oxidization of the formula 3b to aldehyde of formula 4b: ((22 ,25R)-3 ⁇ - acetoxyfurost-26-al) :
  • Step D Oxime derivative of dihydrodiosgenin acetate of the formula 5: ((22 ,25R)-3 ⁇ -acetoxyfurost-26-oxime): A mixture of 4b, 0.196 g, hydroxylamine hydrochloric acid 0.14 g, pyridine 1 ml and 95% ethanol 20 ml at temperature 80 °C for 1 hr, the progress of the reaction was monitored by TLC. Then the mixture was cooled with ice, washed with water and extracted with ethyl acetate. The solvent was removed under reduced pressure. A white gummy liquid was obtained, 5b with the yield of O.lg (51%).
  • Step E Isoxazole derivative of dihydrodiosgenin acetate of formula 8b: ((22 P,25R)-3 ⁇ -dihydroacetoxyfurost-26-(5-phenyl)isoxazole 8b):
  • Step B F-ring opening of Diosgenin acetate of formula 2a : ((22 P,25R)-3 ⁇ - acetoxyfurost-26-ol) : lg NaBH 3 CN was added to a 0.31 g solution of diosgenin acetate 2a in butanoic acid 3 ml under helium atmosphere and stirred for 30 hrs at 30 °C(. After that CH 2 CI 2 was added and then Na 2 C0 3 solution. The aqueous phase was extracted with ethyl acetate and the combined organic layer dried over anhydrous Na 2 S0 4 and evaporated to get the product. Then the residue was purified by column chromatography (30% ethyl acetate/hexane) to give pure product 3a (white gummy liquid) with the yield of 0.13 g, 41%.
  • Step C Oxidization of the formula 3 to aldehyde of formula 4a: ((22 P,25R)-3 ⁇ - acetoxyfurost-26-al) :
  • Step D Oxime derivative of Diosgenin acetate of the formula 5a: ((22 p,25R)-3 ⁇ - acetoxyfurost-26-oxime) :
  • Step E Isoxazole derivative of Diosgenin acetate of formula 7 ((22 p,25R)-3 ⁇ - acetoxyfurost-26-isoxazole) :
  • Step A Hydrogenation of Diosgenin :
  • Diosgenin 1 two grams in 100 ml of ethanol was hydrogenated at 45 psi using 500 mg of 5% Pd/C for a period of 10 h.
  • the reaction mixture was filtered and alcohol was distilled off under reduced pressure to furnish the crude hydrogenated product which was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as the eluent to furnish dihydro diosgenin compound lb in pure form of 95% yield.
  • Step B F-ring opening of Diosgenin acetate of formula 3b: ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ - acetoxyfurost-26-ol :
  • PCC 0.273 g was added to a ice cold mixture of powdered CaC0 3 0.127 g, silica 1.0 g, and 3b, 0.70 g in CH 2 C1 2 of 25 mL at room temperature.
  • the reaction mixture was stirred for 12 hrs at 30 °C.
  • the reaction mixture was diluted with diethyl ether, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4b with white solid of 0.42 g (60%), mp.124 °C.
  • Step D Oxime derivative of Diosgenin acetate of the formula 5b: ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ - acetoxyf urost-26-oxime:
  • Step E Isoxazole derivative of Diosgenin acetate of formula 7b: ((22 ⁇ ,25 ⁇ )-3 ⁇ - acetoxyfurost-26-(5-methyl)isoxazole (7b):
  • Step B F-ring opening of Diosgenin of formula 3c : ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ -hydroxyfurost- 26-01) :
  • PCC 0.273 g was added to a ice cold mixture of powdered CaC0 3 0.127 g, silica 1.0 g, and 3c, 0.8 g in CH 2 C1 2 of 25 mL at 30 °C.
  • the reaction mixture was stirred for 7 hrs at temperature 40 °C.
  • the reaction mixture was diluted with diethyl ether, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4c with white solid of 0.6 g (75%), mp.124 °C.
  • Step D Oxime derivative of dehydrodiosgenin of the formula 5c: ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ - hydroxyfurost-26-oxime):
  • Step E Isoxazole derivative of dehydrodiosgenin of formula 8c: ((22 p,25R)-3 ⁇ - dehydrohydroxyfurost-26-(5-phenyl)isoxazole 8c):
  • Step A Hydrogenation of Diosgenin :
  • Diosgenin 1 two grams in 100 ml of ethanol was hydrogenated at 45 psi using 500 mg of 5% Pd/C for a period of 10 h.
  • the reaction mixture was filtered and alcohol was distilled off under reduced pressure to furnish the crude hydrogenated product which was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as the eluent to furnish dihydro diosgenin compound lb in pure form of 95% yield.
  • Step B F-ring opening of dihydrodiosgenin of formula 3d : ((22 P,25R)-3 ⁇ - hydroxyfurost-26-ol) :
  • Step C Oxidization of the formula 3d to aldehyde of formula 4d: ((22 p,25R)-3 ⁇ - hydroxyfurost-26-al) :
  • Step D Oxime derivative of dihydrodiosgenin of the formula 5d: ((22 P,25R)-3 ⁇ - acetoxyfurost-26-oxime) :
  • Step E Isoxazole derivative of dihydrodiosgenin of formula 8d ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ - hydroxyfurost-26-isoxazole) :
  • Step B F-ring opening of Diosgenin of formula 3c : ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ -hydroxyfurost- 26-01) :
  • Step C Oxidization of the formula 3c to aldehyde of formula 4c: ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ - hydroxyfurost-26-al) :
  • PCC 0.273 g was added to a ice cold mixture of powdered CaC0 3 0.127 g, silica 1.0 g, and 3c, 0.5 g in CH 2 C1 2 of 25 mL at 30 °C.
  • the reaction mixture was stirred for 7 hrs at temperature 40 °C.
  • the reaction mixture was diluted with diethyl ether, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4c with white solid of 0.32 g (64%), mp.124 °C.
  • Step D Oxime derivative of dehydrodiosgenin of the formula 5c: ((22 ⁇ ,2513 ⁇ 4)-3 ⁇ - hy droxyf urost-26-oxime) :
  • Step E Isoxazole derivative of dehydrodiosgenin of formula 7c: ((22 p,25R)-3 ⁇ - dehydrohydroxyfurost-26-(5-methyl)isoxazole 7c) :
  • Step A Hydrogenation of Diosgenin :
  • Diosgenin 1 two grams in 100 ml of ethanol was hydrogenated at 45 psi using 500 mg of 5% Pd/C for a period of 10 h.
  • the reaction mixture was filtered and alcohol was distilled off under reduced pressure to furnish the crude hydrogenated product which was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as the eluent to furnish dihydro diosgenin compound lb in pure form of 95% yield.
  • Step B F-ring opening of dihydrodiosgenin of formula 3d : ((22 P,25R)-3 ⁇ - hydroxyfurost-26-ol) :
  • Step C Oxidization of the formula 3d to aldehyde of formula 4d: ((22 p,25R)-3 ⁇ - hydroxyfurost-26-al) :
  • Step D Oxime derivative of dihydrodiosgenin of the formula 5d: ((22 P,25R)-3 ⁇ - hydroxyfurost-26-oxime) :
  • Step E Isoxazole derivative of dihydrodiosgenin of formula 7d ((22 P,25R)-3 ⁇ - hydroxyfurost-26-isoxazole) :

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Abstract

This invention also relates to novel class of diosgenin acetate-Isoxazole and the synthesis of diosgenin acetate-Isoxazole starting from Diosgenin and its relatives with five (5) synthetic routes, acetylation (A), F-ring opening of diosgenin acetate (B), Oxidation of the hydroxyl group on C-26 of E-ring manipulation of diosgenin acetate (C), Formation of oxime (D) and Isoxazole formation on C-26 of E-ring manipulation of diosgenin acetate (E) having potent Antifungal activity against Alternaria alternate fungus.

Description

DIOSGENIN ACETATE-ISOXAZOLE DERIVATIVES, PROCESS FOR PREPARATION THEREOF AND THEIR ANTIFUNGAL ACTIVITY
FIELD OF THE INVENTION
This invention relates to the synthesis of a novel class of Diosgenin acetate-isoxazole starting from Diosgenin and its relatives and their antifungal activities against plant pathogenic fungus Alternaria alternate.
This invention also relates to the synthesis of a novel class of Diosgenin acetate- isoxazole of the formula A ((22P,25R)-3P-acetoxyfurost-26-isoxazole) where R2= OAc from Diosgenin, viz. 5,6 dehydro-diosgenin and 5,6 dihydro-diosgenin of formula la and lb where Ri= OH and with acetylene derivatives of the formula 6 where R= C¾ or C6H5. The compound of formula A antifungal activities against plant pathogenic fungus Alternaria alternate prepared by the process of invention are found to have an overall yield 80% after purification. The process of the present invention does not involve the use of any costly and environmentally toxic reagents and catalysts and has been developed using a non-toxic reagents under mild reaction conditions.
BACKGROUND AND PRIOR ART OF THE INVENTION
It is pertinent to note that Diosgenin acetate-isoxazole is recently gaining importance owing to their potent biological activity. So far, no literature reported Doisgenin which has potent antifungal activity against plant pathogenic fungus Alternaria alternate.
Isoxazoline derivatives of the steroid compounds solasodine and diosgenin [Prismetov, M. P.; Dzhiembaev, B. Zh.; Kiseleva, E. N.; Kharlamova, T. V., Seriya Khimicheskaya (2000), (3), Page No. 28-30.] This article describes the method for the synthesis of isoxazolinosolasodine and diosgenin via reaction of steroid ketones with formamide in the presence of perchloric acid with structural formula as given below:
Figure imgf000003_0001
Synthesis of pyrazoles and isoxazoles of diosgenin [M.P. Irismetov, M. I, Goryaev, and G. Yu. Tsvetkova, Russian Journal of General Chemistry (1976), 46(6), Page No. 1407- 1409.]
This document discloses the synthesis of pyrazoles and isoxazole derivatives of Diosgenin with the following structures.
Figure imgf000003_0002
Modified steroids. VIII. Synthesis and study of heterocyclic compounds made of the methyl ester of A-3,4-5-ketonic acid from diosgenin [Irismetov, M. P.; Goryaev, M. I.; Mirzasaheva, N. A.; Rustembekova, G. B, Izvestiya Akademii Nauk Kazakhskoi SSR, Seriya Khimicheskaya (1982), (5), Page No. 55-59.] This document discloses the synthesis of heterocyclic compounds made of the methyl ester of A-3,4-5-ketonic acid from diosgenin with the following structures.
Figure imgf000004_0001
Antifungal Activity of C-27 Steroidal Saponins [Chong-Ren Yang,Ying Zhang,Melissa R. Jacob, Shabana I. Khan,Ying-Jun Zhang, and Xing-Cong Li, Antimicrobial Agents and Chemotherapy, (2006) 50(5), Page No. 1710-1714.]
This document discloses antifungal activity of 22 derivatives of C-27 steroidal saponins and 6 steroidal sapogenins against Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, and Aspergillus fumigates with the following structures.
1 Gal{4> t j6k:[(EM iKylj(2- 1 ¾e(-M H
Figure imgf000005_0001
2 Gal(4- 1 )Gte -l jGfc - 1 >Qfe(S- 1 )Rha
3 {3&{4-ljGte[{3-1 >X j(-M }<3Sc Gal^-I jGtet(3- 1 )Xyl](.£M (¾c(3- 1 )R a H M
4 Gal(4^ )Glc[{3- l )Xjilp-13<33c(3-1 )Xyi m H H OH
Figure imgf000005_0002
/« v/tro Antimicrobial Activity of Garhwal Himalaya Medicinal Plant Dioscorea deltoidea Tuber [Subhash Chandra, Sarla Saklani, Abhay P. Mishra, International Journal of Herbal Medicine, 2013, 1(4), Page No. 67-70.]
This document discloses the antimicrobial activity of the extract of Dioscorea deltoidea. Thus the Diosgenin acetate-isoxazole of formula 7 as described in the present invention are completely new and novel and have so far not been reported by any other workers.
OBJECTIVES OF THE INVENTION
The main objective of the present invention, therefore, is to provide a novel process to synthesize a novel class of Diosgenin acetate-isoxazole of formula A starting from 5,6 dehydro-diosgenin and 5,6 dihydro-diosgenin of formula la and lb where Ri= OH and with the reagent of formula 6 where R= CH3 or C6H5 shown in the drawing (Figure 1).
Another object of the present invention is to provide a general method of preparation of a novel class of Diosgenin acetate-isoxazole of formula A starting from 5,6 dehydro- diosgenin and 5,6 dihydro-diosgenin of formula la and lb and their relatives where Ri= OH with five(5) reactions steps and finely with the reagent of formula 6 where R= CH3 or C6H5 . This could be useful as potent antifungal inhibitors shown in Fig.4.
Yet another object of the present invention is to provide a novel process to synthesize a novel class of Diosgenin acetate-isoxazole of formula A where R2 = OAc and Ac representing acetyl group, starting from 5,6 dehydro-diosgenin and 5,6 dihydro- diosgenin of formula la and lb where Ri= OH and with the reagent of formula 6 where R= CH3 or C6H5 having potent antifungal activity against plant pathogenic fungus Alternaria alternate.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a compound of formula A
Figure imgf000007_0001
wherein R2= OAc or OH; R=C6H5 or CH3.
In an embodiment of the present invention, wherein the representative compounds comprising;
3a. (22p,25R)-3P -acetoxyfurost-5,6-dehydro-26-ol;
3b. (22p,25R)-3p -acetoxyfurost-5,6-dihydro-26-ol
4a. (22P,25R)-3P -acetoxyfurost-5,6-dehydro-26-al;
4b. (22p,25R)-3p -acetoxyfurost-5,6-dihydro-26-al
5a. (22p,25R)-3P -acetoxyfurost-5,6-dehydro-26-oxime;
5b. (22p,25R)-3P -acetoxyfurost-5,6-dihydro-26-oxime
7a. (22p,25R)-3P -acetoxyfurost-5,6-dehydro-26-(5-methyl)isoxazole
7b. (22p,25R)-3P -acetoxyfurost-5,6-dihydro-26-(5-methyl)isoxazole
7c. (22p,25R)-3P -hydroxyfurost-5,6-dehydro-26-(5-methyl)isoxazole
7d. (22p,25R)-3P -hydroxyfurost-5,6-dihydro-26-(5-methyl)isoxazole
8a. (22p,25R)-3P -acetoxyfurost-5,6-dehydro-26-(5-phenyl isoxazole)
8b. (22p,25R)-3P -acetoxyfurost-5,6-dihydro-26-(5-phenyl isoxazole)
8c. (22p,25R)-3P -hydroxyfurost-5,6-dehydro-26-(5-phenyl isoxazole) 8d. (22p,25R)-3P -hydroxyfurost-5,6-dihydro-26-(5-phenyl isoxazole)
In another embodiment of the present invention, wherein diosgenin acetate -Isoxazoles exhibits good antifungal activity.
In yet another embodiment of the present invention, wherein a process for preparation of diosgenin acetate -Isoxazoles of the formula A wherein R2= OAc or OH comprising the steps:
i. optionally hydrogenating diosgenin 1 to obtain dihydrodiosgenin lb;
ii. acetylating diosgenin 1 or dihydrodiosgenin lb using acetic anhydride in presence of a suitable base to obtain 3P-acetoxy dehydrodiosgenin (2a) or 3P-acetoxy dihydrodiosgenin (2b); iii. F-ring opening of 3P-acetoxy dehydrodiosgenin (2a) or 3β- acetoxy dihydrodiosgenin (2b) using sodium cyanoborohydride in acetic acid to obtain dehydro or dihydro (22β, 25R)-3P- acetoxyfurost-26-ol ;
iv. oxidizing dehydro or dihydro (22β, 25R)-3P-acetoxyfurost-26-ol using pyridinium chlorochromate (PCC) in dichloromethane to obtain C-26-aldehyde-5,6-dehydro-diosgenin acetate or C-26- aldehyde-5 ,6-dihydro-diosgenin acetate ;
v. refluxing the mixture of C-26-aldehyde-5,6-dehydro-diosgenin acetate or C-26-aldehyde-5,6-dihydro-diosgenin acetate, hydroxyl amine hydrochloric acid, base and protic solvent at a temperature ranging between 60-95 °C for a period of 1-3 hrs, followed by cooling, washing, extracting with a water immiscible solvent and removing of solvent under reduced pressure to get C-26-oxime-5,6 dehydro-diosgenin acetate C-26-oxime-5,6 dihydro-diosgenin acetate; vi. mixing the solution of C-26-oxime-5,6 dehydro-diosgenin acetate or C-26-oxime-5,6 dihydro-diosgenin acetate obtained in step (v) with phenyl acetylene or methyl acetylene, base and sodium hypochlorite in DCM (dichloromethane) at the temperature ranging between 30-50 °C under nitrogen atmosphere followed by the stirring at the temperature ranging between 30-50 °C for the period of time 25-48 hr;
vii. adding water to the reaction mixture obtained in step (vi) then extracted with water immiscible solvent followed by washing the combined organic layer with water and brine;
viii. drying the organic layer obtained in step (vii) followed by evaporating the solvent and purifying by column chromatography to get (22P,25R)-3P-acetoxyfurost-5,6,-dehydro-26-substituted isoxazole or (22P,25R)-3P-acetoxyfurost-5,6,-dihydro-26- substituted isoxazole a compound of formula A; and
ix. optionally hydrolyzing acetyl group of (22β,25Κ)-3β- acetoxyfurost-5,6,-dehydro-26-substituted isoxazole or (22P,25R)-3P-acetoxyfurost-5,6,-dihydro-26-substituted isoxazole with suitable base to obtain (22β,25Κ)-3β- hydroxyfurost-5,6,-dehydro-26-substituted isoxazole or (22P,25R)-3P-hydroxyfurost-5,6,-dihydro-26-substituted isoxazole a compounds of formula A.
In yet another embodiment of the present invention, wherein purification of the product by column chromatography with 10% ethyl acetate/hexane or 10% ethyl acetate /petroleum ether.
In yet another embodiment base is selected from the group consisting of pyridine, triethylamine. In yet another embodiment protic solvent selected from methanol, ethanol, isoporyl alcohol.
In yet another embodiment water immiscible solvent selected from the group consisting of ethyl acetate, dichlorome thane.
In yet another embodiment a composition comprising an antifungal compound of formula A or a pharmaceutically acceptable salt thereof along with pharmaceutically acceptable excipients.
In yet another embodiment compound of formula A is used for treating or preventing a fungal infection against plant pathogenic fungus Alternaria alternate.
In still another embodiment of the present invention, wherein a process for synthesizing or constructing novel class of Diosgenin acetate-isoxazole of formula A where R2 = OAc or OH and Ac representing acetyl group, with the reagent of formula 6 where R= CH3 or C6H5 shown in the drawing (Figure 1) accompanying this specification which comprises the following Steps:
Step A : Acetylation of diosgenin of formula 2 where R2= OAc or OH and Ac representing the acetyl group starting from 5,6 dehydro-diosgenin and 5,6 dihydro- diosgenin of formula la and lb and their relatives acetylated of diosgenin of formula 2 under a mild reaction conditions as per literature procedures;
Step B : F-ring opening of diosgenin acetate of formula 3 where R2= OAc or OH and Ac representing the acetyl group starting from 5,6 dehydro-diosgenin acetate and 5,6 dihydro-diosgenin acetate of formula 2a and 2b and their relatives under a mild reaction conditions as per literature procedures;
Step C : Oxidation of the hydroxyl group on C-26 of E-ring manipulation of diosgenin acetate of formula 4, starting from C-26-ol-5,6 dehydro-diosgenin acetate and C-26-ol- 5,6 dihydro-diosgenin acetate of formula 3 and their relatives under a mild reaction conditions as per literature procedures; Step D : Oxime formation on C-26 of E-ring manipulation of diosgenin acetate of formula 5, starting from C-26-aldehyde-5,6 dehydro-diosgenin acetate and C-26- aldehyde-5,6 dihydro-diosgenin acetate of formula 4 and their relatives under a mild reaction conditions ie. reaction of C-26-aldehyde-5,6 dehydro-diosgenin acetate and C- 26-aldehydride- 5,6 dihydro-diosgenin acetate of formula 4a and 4b with acetylene in presence of hydroxylamine hydrochloric acid, pyridine and 95% ethanol. The reaction mixture was refluxed, the progress of the reaction was monitored by TLC and the mixture was cooled with ice, washed with water and extracted with ethyl acetate. The solvent was removed under reduced pressure to get the desired products 5;
Step E : Isoxazole formation on C-26 of E-ring manipulation of diosgenin acetate of formula 7, starting from C-26-oxime-5,6 dehydro-diosgenin acetate and C-26-oxime- 5,6 dihydro-diosgenin acetate of formula 5a and 5b and of formula 6, under a mild reaction conditions ie. reaction of 5,6 dehydro-diosgenin acetate-C-26-oxime and 5,6 dihydro-diosgenin acetate - C-26-oxime of formula 5a and 5b with phenyl acetylene, triethyl amine and sodium hypochlorite at 0 °C under nitrogen atmosphere and stirred 30 °C room temperature for 48 h. The progress of the reaction was monitored by TLC analysis, added water in the reaction mixture and extracted with DCM. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2S04. Evaporation of the solvent in high vacuum followed by column chromatography to get the pure product 7.
In an another embodiment of the present invention, wherein process for the synthesis of a new and novel class of E-ring manupulated diosgenin acetate-Isoxazole of formula 7 starting from Diosgenin and its relatives of formula 1 can be effected by using pyridine, aniline, acetic anhydride at 70 °C for 2-7 hrs in stirring condition [Step A] to acetylated diosgenin of the formula 2. F-ring opening of diosgenin acetate of formula 3 in presence of NaBH3CN and acetic acid under argon/helium/nitrogen atmosphere in stirring condition for 50 hrs at 30 °C temperature [Step B]. Oxidation of the hydroxyl group on C-26 of E-ring manipulation of diosgenin acetate of formula 4 starting from C-26-ol-5,6 dehydro-diosgenin acetate and C-26-ol-5,6 dihydro-diosgenin acetate of formula 3 and their relatives [Step C] under PCC in ice cold mixture of powdered CaC03, silica and CH2CI2 at 35° temperature. Formation of oxime [Step D] on C-26 of E-ring manipulation of diosgenin acetate of formula 5 starting from C-26-aldehyde-5,6 dehydro-diosgenin acetate and C-26-aldehyde-5,6 dihydro-diosgenin acetate of formula 4 and their relatives under a mild reaction conditions ie. reaction of C-26-aldehyde-5,6 dehydro-diosgenin acetate and C-26-aldehyde-5,6 dihydro-diosgenin acetate of formula 4a and 4b with acetylene in presence of hydroxylamine hydrochloric acid, pyridine and ethanol. Isoxazoline formation on C-26 of E-ring manipulation of diosgenin acetate of formula A [Step E] starting from C-26-oxime-5,6 dehydro-diosgenin acetate and C-26- oxime-5,6 dihydro-diosgenin acetate of formula 5a and 5b and aryl/alkyl acetylene of formula 6 under a mild reaction conditions ie. reaction of 5,6 dehydro-diosgenin acetate-C-26-oxime and 5,6 dihydro-diosgenin acetate-C-26-oxime of formula 5a and 5b with aryl/alkyl acetylene of formula 6, triethyl amine and sodium hypochlorite at 0- 35 °C under nitrogen/argon/helium atmosphere and stirred at 30 °C temperature for 48 hrs. The progress of the reaction was monitored by TLC analysis, added water in the reaction mixture and extracted with Pet Ether/DCM/CHC13. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2S04 and purified by column chromatography to get pure product 7, white gummy product.
In yet another embodiment of the present invention, wherein these novel Diosgenin acetate-isoxazole of formula A starting from 5,6 dehydro-diosgenin and 5,6 dihydro- diosgenin of formula la and lb where Rl= OH and with the reagent of formula 6 where R= CH3 or C6H5 have been found to show potent antifungal activity against plant pathogenic fungus Alternaria alternate shown in Table 1.
In yet another embodiment of the present invention, wherein these noval Diosgenin acetate-isoxazole of formula A starting from 5,6 dehydro-diosgenin and 5,6 dihydro- diosgenin of formula la and lb where Rl= OH or H and with the reagent of formula 6 where R= CH3 or C6H5 also have been found to show with the inhibition percentage 85 as depicted in the Fig.4 and for the starting compound 10 which was determined by the standard antifungal activity test, viz., Poison Food Technique shown in Table 1.
BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1: Reagents and conditions: (a) Pyridine/ Acetic anhydride/40-90 °C/ recrystallize [MeOH/EtOH/PrOH] (b) NaBH3CN [in CH3COOH/ C3H7COOH / C4H9COOH (2-5 mL)]/ Inert gas atmosphere/30-60 hrs/20-30 °C/CH2C12/ Base/ purification by column chromatography (c) PCC/CaC03/Silica/ CH2Cl2/30-60 °C/ 4-12 hrs/ purification by column chromatography (d) Hydroxylamine hydrochloric acid/ Pyridine/Ethanol/ 60-100 °C/H20/ extract with ethyl acetate (e) DCM/Acetylene/ Triethylamine/ sodium hypochlorite/0-40 °C/ 20-48 hrs/Nitrogen atmosphere/argon atmosphere/ purification by column chromatography.
FIGURE 2: General formula according to the claim 1 is given below (Formula 1, 2, 3, 4, 5, 6 and 7):
FIGURE 3 : Formula of final compounds
FIGURE 4: Antifungal activity: C= Control, E & El = Experimental
for 7a & 7b, D= Diosgenin
Antifungal activity against plant pathogenic fungus Alternaria alternate.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is to provide a compound of formula A
Figure imgf000014_0001
wherein R2= OAc or OH; R=C6H5 or CH3.
The representative compounds are selected from;
3a. (22p,25R)-3P -acetoxyfurost-5,6-dehydro-26-ol;
3b. (22p,25R)-3p -acetoxyfurost-5,6-dihydro-26-ol
4a. (22P,25R)-3P -acetoxyfurost-5,6-dehydro-26-al;
4b. (22p,25R)-3p -acetoxyfurost-5,6-dihydro-26-al
5a. (22p,25R)-3P -acetoxyfurost-5,6-dehydro-26-oxime;
5b. (22p,25R)-3P -acetoxyfurost-5,6-dihydro-26-oxime
7a. (22p,25R)-3P -acetoxyfurost-5,6-dehydro-26-(5-methyl)isoxazole
7b. (22p,25R)-3P -acetoxyfurost-5,6-dihydro-26-(5-methyl)isoxazole
7c. (22p,25R)-3P -hydroxyfurost-5,6-dehydro-26-(5-methyl)isoxazole
7d. (22p,25R)-3P -hydroxyfurost-5,6-dihydro-26-(5-methyl)isoxazole
8a. (22p,25R)-3P -acetoxyfurost-5,6-dehydro-26-(5-phenyl isoxazole)
8b. (22p,25R)-3P -acetoxyfurost-5,6-dihydro-26-(5-phenyl isoxazole)
8c. (22p,25R)-3P -hydroxyfurost-5,6-dehydro-26-(5-phenyl isoxazole)
8d. (22p,25R)-3P -hydroxyfurost-5,6-dihydro-26-(5-phenyl isoxazole) Further a process for synthesizing or constructing a new class of Diosgenin acetate - isoxazole of formula A in an yield of 80% shown in the drawing (Figure 1) accompanying this specification which comprises the following Steps :
Step A : Acetylation of diosgenin of formula 2 where R2= OAc or OH and Ac representing the acetyl group starting from 5,6 dehydro-diosgenin and 5,6 dihydro- diosgenin of formula 1 and their relatives acetylated of diosgenin of formula 2 under a mild reaction conditions as per literature procedures.
Step B : F-ring opening of diosgenin acetate of formula 3 where R2= OAc or OH and Ac representing the acetyl group starting from 5,6 dehydro-diosgenin acetate and 5,6 dihydro-diosgenin acetate of formula 2 and their relatives under a mild reaction conditions as per literature procedures.
Step C : Oxidation of the hydroxyl group on C-26 of E-ring manipulation of diosgenin acetate of formula 4, starting from C-26-ol-5,6 dehydro-diosgenin acetate and C-26-ol- 5,6 dihydro-diosgenin acetate of formula 3 and their relatives under a mild reaction conditions as per literature procedures.
Step D : Oxime formation on C-26 of F-ring manipulation of diosgenin acetate of formula 5, starting from C-26-aldehyde-5,6 dehydro-diosgenin acetate and C-26- aldehyde-5,6 dihydro-diosgenin acetate of formula 4 and their relatives under a mild reaction conditions ie. reaction of C-26-aldehyde-5,6 dehydro-diosgenin acetate and C- 26-aldehyde- 5,6 dihydro-diosgenin acetate of formula 4a and 4b with acetylene in presence of hydroxylamine hydrochloric acid, pyridine and 95% ethanol. The reaction mixture was refluxed, the progress of the reaction was monitored by TLC and the mixture was cooled with ice, washed with water and extracted with ethyl acetate. The solvent was removed under reduced pressure to get the desired products 5.
Step E : Isoxazole formation on C-26 of E-ring manipulation of diosgenin acetate of formula 7, starting from C-26-oxime-5,6 dehydro-diosgenin acetate and C-26-oxime- 5,6 dihydro-diosgenin acetate of formula 5a and 5b and of formula 6, under a mild reaction conditions ie. reaction of 5,6 dehydro-diosgenin acetate-C-26-oxime and 5,6 dihydro-diosgenin acetate - C-26-oxime of formula 5a and 5b with phenyl acetylene, triethyl amine and sodium hypochlorite at 0 °C under nitrogen atmosphere and stirred 30 °C at room temperature for 48 hrs. The progress of the reaction was monitored by TLC analysis, added water in the reaction mixture and extracted with DCM. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2S04. Evaporation of the solvent in high vacuum followed by column chromatography to get pure product 7.
This is the first ever method where these new 5,6 dehydro-diosgenin acetate -isoxazole and 5,6 dihydro-diosgenin acetate -isoxazole of formula 7a and 7b where R2= OAc or OH and Ac representing the acetyl group, with the reagent of formula 6 where R= CH3 or C6H5 have been found to show potent antifungal activity against plant pathogenic fungus Alternaria alternate with the inhibition percentage 85 and for the starting compound ie. diosgenin 10 as depicted in the Fig.4 as determined by the standard antifungal activity test, viz., Poison Food Technique.
Antifungal activity test was performed using the plant pathogenic fungus Alternaria alternate with the inhibition percentage 85 as depicted in the Fig.4 and Table 1 as determined by the standard antifungal activity test, viz., Poison Food Technique.
The following specific examples are given by way of illustration of the invention in actual practice and therefore should not be construed to limit the scope of the present invention.
The invention particularly relates to the synthesis of a novel class of A and E-ring manipulated Diosgenin acetate-Isoxazole of formula A where R2= OAc or OH and related products shown in the diagram accompanying this specification from 5,6 dehydro-diosgenin and 5,6 dihydro-diosgenin of formula la and lb where Rl= OH . In the reaction route first acetylated the diosgenin, viz. 5,6 dehydro-diosgenin and 5,6 dihydro-diosgenin [Step A] to acetylated diosgenin of the formula 2a and 2b. Then F- ring opening of diosgenin acetate of formula 3 [Step B], Oxidation of the hydroxyl group on C-26 of E-ring manipulation of diosgenin acetate of formula 4 starting from C-26-ol-5,6 dehydro-diosgenin acetate and C-26-ol-5,6 dihydro-diosgenin acetate of formula 3 and their relatives [Step C], Formation of oxime [Step D] on C-26 of E-ring manipulation of diosgenin acetate of formula 5 starting from C-26-aldehyde-5,6 dehydro-diosgenin acetate and C-26-aldehyde-5,6 dihydro-diosgenin acetate of formula 4 and their relatives under a mild reaction conditions ie. reaction of C-26-aldehyde-5,6 dehydro-diosgenin acetate and C-26-aldehyde-5,6 dihydro-diosgenin acetate of formula 4a and 4b and Isoxazole formation on C-26 of E-ring manipulation of diosgenin acetate of formula 7a and 7b [Step E] starting from C-26-oxime-5,6 dehydro-diosgenin acetate and C-26-oxime-5,6 dihydro-diosgenin acetate of formula 5a and 5b and acetylene derivative of formula 6 where R= CH3 or C6H5.
Accordingly the present invention provides for the production of a novel class of Diosgenin acetate-Isoxazole of the formula 7a and 7b where R2= OAc or OH as shown in the drawing (Figure 1) accompanying this specification from Diosgenin, viz. 5,6 dehydro-diosgenin and 5,6 dihydro-diosgenin of formula la and lb where Rl=OH and with acetylene derivatives of the formula 6 where R= CH3 or C6H5. The compound of formula 7a and 7b antifungal activities against plant pathogenic fungus Alternaria alternate as shown in Fig.4 and Table 1.
EXAMPLES
The following examples are given by way of illustration of the working of the invention in actual practice and should not be construed to limit the scope of the present invention in any way. Example 1
Step A: Hydrogenation of Diosgenin : Diosgenin 1, two grams in 100 ml of ethanol was hydrogenated at 45 psi using 500 mg of 5% Pd/C for a period of 10 h. The reaction mixture was filtered and alcohol was distilled off under reduced pressure to furnish the crude hydrogenated product which was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as the eluent to furnish dihydro diosgenin compound lb in pure form of 95% yield. Acetylation of Diosgenin of formula 1: (synthesis of 3 β -acetoxy-diosgenin):
Diosgenin 1, 1.18 g and pyridine 1 ml were added to 25 ml acetic anhydride, and the mixture was heated and stirred at 90 °C for 2 hrs. The solution was poured into ice water and the precipitate was filtered. Filtrate was washed with water and dried under vacuum to yield a white solid, which was recrystallized with CH3OH to give compound 2a with the yield of 1.06 g, (89%), mp.194 °C as colourless needle crystals.
Step B: F-ring opening of Diosgenin acetate of formula 3: ((22 β,251¾)-3 β - acetoxyfurost-26-ol : lg NaBH3CN was added to a 1 g solution of diosgenin acetate 2a in acetic acid 3 ml under argon atmosphere and stirred for 60 hrs at (30 °C). After that CH2CI2 was added and then Na2C03 solution. The aqueous phase was extracted with CH2CI2 and the combined organic layer dried over anhydrous Na2S04 and evaporated to get the product. Then the residue was purified by column chromatography (30% ethyl acetate/hexane) to give pure product 3a (white gummy liquid) with the yield of 0.716 g, 71%.
Step C: Oxidization of the formula 3 to aldehyde of formula 4:((22 β,251¾)-3 β - acetoxyfurost-26-al:
PCC, 0.273 g was added to a ice cold mixture of powdered CaC03 0.127 g, silica 1.0 g, and 3a, 0.70 g in CH2CI2 of 25 mL at room temperature. The reaction mixture was stirred for 12 hrs at 30 °C. Then, the reaction mixture was diluted with diethyl ether, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4a with white solid of 0.492 g (70%), mp.124 °C. Step D: Oxime derivative of Diosgenin acetate of the formula 5: ((22 β,251¾)-3 β - acetoxyf urost-26-oxime :
A mixture of 4a, 0.2 g, hydroxylamine hydrochloric acid 0.14 g, pyridine 1 ml and 95% ethanol 20 ml was refluxed for 1 hr, the progress of the reaction was monitored by TLC. Then the mixture was cooled with ice, washed with water and extracted with ethyl acetate. The solvent was removed under reduced pressure. A white gummy liquid was obtained, 5a with the yield of 0.176g (88%).
Step E: Isoxazole derivative of Diosgenin acetate of formula 7: ((22 β,251¾)-3 β - acetoxyf urost-26- (5-pheny 1 (isoxazole (8a) :
To a solution of compound 5a, 0.2 g, in DCM, 20 mL, phenyl acetylene 6, 0.072g, triethyl amine, 0.83 g and sodium hypochlorite, 10 ml (12% in water) were added at 0 °C under nitrogen atmosphere and stirred at 30 °C for 48 hrs. The progress of the reaction was monitored by TLC analysis (15% EA/pet ether). Then, water (50 ml) was added and the reaction mixture was extracted with DCM. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2S04. Evaporation of the solvent in high vacuum followed by column chromatography (10% ethyl acetate/hexane) gives pure product 8a with white gummy product of yield 0.16 g (80%).
Example 2
Step A: Hydrogenation of Diosgenin lb:
Diosgenin 1, two grams in 100 ml of ethanol was hydrogenated at 45 psi using 500 mg of 5% Pd/C for a period of 10 h. The reaction mixture was filtered and alcohol was distilled off under reduced pressure to furnish the crude hydrogenated product which was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as the eluent to furnish dihydro diosgenin compound lb in pure form of 95% yield. Acetylation of dihydrodiosgenin of formula lb: (3 β -acetoxy- dihydrodiosgenin) : dihydrodiosgenin lb, 1.18 g and pyridine 1 ml were added to 25 ml acetic anhydride, and the mixture was heated and stirred at 75 °C for 3 hrs. The solution was poured into ice water and the precipitate was filtered. Filtrate was washed with water and dried under vacuum to yield a white solid, which was recrystalhzed with C2H5OH to give compound 2b with the yield of 0.62 g, (52%), mp.194 °C as colourless needle crystals.
Step B: F-ring opening of Diosgenin acetate of formula 3 : ((22 β,251¾)-3 β - acetoxyfurost-26-ol) :
lg NaBH3CN was added to a 1 g solution of dihydrodiosgenin acetate 2b in propionic acid 3 ml under argon atmosphere and stirred for 40 hrs at 30 °C. After that CH2CI2 was added and then Na2C03 solution. The aqueous phase was extracted with CH2CI2 and the combined organic layer dried over anhydrous Na2S04 and evaporated to get the product. Then the residue was purified by column chromatography (30% ethyl acetate/hexane) to give pure product 3b (white gummy liquid) with the yield of 0.491 g, 49%.
Step C: Oxidization of the formula 3b to aldehyde of formula 4b: ((22 ,25R)-3 β - acetoxyfurost-26-al) :
PCC, 0.273 g was added to a ice cold mixture of powdered CaC03 0.127 g, silica 1.0 g, and 3, 0.584 g in CH2CI2 of 25 mL at 30 °C. The reaction mixture was stirred for 9 hrs at temperature 40 °C. Then, the reaction mixture was diluted with diethyl ether, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4 b with white solid of 0.26 g (44%), mp.124 °C. Step D: Oxime derivative of dihydrodiosgenin acetate of the formula 5: ((22 ,25R)-3 β -acetoxyfurost-26-oxime): A mixture of 4b, 0.196 g, hydroxylamine hydrochloric acid 0.14 g, pyridine 1 ml and 95% ethanol 20 ml at temperature 80 °C for 1 hr, the progress of the reaction was monitored by TLC. Then the mixture was cooled with ice, washed with water and extracted with ethyl acetate. The solvent was removed under reduced pressure. A white gummy liquid was obtained, 5b with the yield of O.lg (51%).
Step E: Isoxazole derivative of dihydrodiosgenin acetate of formula 8b: ((22 P,25R)-3 β -dihydroacetoxyfurost-26-(5-phenyl)isoxazole 8b):
To a solution of compound 5b, 0.2 g, in DCM, 20 mL, phenyl acetylene 6, 0.072g, triethyl amine, 0.83 g and sodium hypochlorite, 10 ml (12% in water) were added at 0 °C under nitrogen atmosphere and stirred at temperature 35 °C for 30 hrs. The progress of the reaction was monitored by TLC analysis (15% EA/pet ether). Then, water (50 ml) was added and the reaction mixture was extracted with CHCI3. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2S04. Evaporation of the solvent in high vacuum followed by column chromatography (10% ethyl acetate/hexane) give pure product 8b with white gummy product of yield O.lg (50%).
Example 3
Acetylation of Diosgenin of formulal: (3p-acetoxy-diosgenin2a):
Diosgenin 1, 1.18 g and pyridine 1 ml were added to 25 ml acetic anhydride, and the mixture was heated and stirred at 50 °C for 6 hrs. The solution was poured into ice water and the precipitate was filtered. Filtrate was washed with water and dried under vacuum to yield a white solid, which was recrystallized with C3H-7OH to give compound 2a with the yield of 0.31 g, (26%), mp.194 °C as colourless needle crystals. Step B: F-ring opening of Diosgenin acetate of formula 2a : ((22 P,25R)-3 β - acetoxyfurost-26-ol) : lg NaBH3CN was added to a 0.31 g solution of diosgenin acetate 2a in butanoic acid 3 ml under helium atmosphere and stirred for 30 hrs at 30 °C(. After that CH2CI2 was added and then Na2C03 solution. The aqueous phase was extracted with ethyl acetate and the combined organic layer dried over anhydrous Na2S04 and evaporated to get the product. Then the residue was purified by column chromatography (30% ethyl acetate/hexane) to give pure product 3a (white gummy liquid) with the yield of 0.13 g, 41%.
Step C: Oxidization of the formula 3 to aldehyde of formula 4a: ((22 P,25R)-3 β - acetoxyfurost-26-al) :
PCC, 0.273 g was added to a ice cold mixture of powdered CaC03 0.127 g, silica 1.0 g, and 3a, 0.13 g in CH2CI2 of 25 mL at 30 °C. The reaction mixture was stirred for 7 hrs at temperature 50 °C. Then, the reaction mixture was diluted with dichloromethan, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4a with white solid of 0.05 g (38%), mp.124 °C.
Step D: Oxime derivative of Diosgenin acetate of the formula 5a: ((22 p,25R)-3 β - acetoxyfurost-26-oxime) :
A mixture of 4a, 0.05 g, hydroxylamine hydrochloric acid 0.14 g, pyridine 1 ml and 95% propanol 20 ml at temperature 70 °C for 2 hrs, the progress of the reaction was monitored by TLC. Then the mixture was cooled with ice, washed with water and extracted with chloroform. The solvent was removed under reduced pressure. A white gummy liquid was obtained, 5a with the yield of 0.02g (40%).
Step E: Isoxazole derivative of Diosgenin acetate of formula 7 ((22 p,25R)-3 β - acetoxyfurost-26-isoxazole) :
To a solution of compound 5a, 0.02 g, in acetone, 20 mL, methyl acetylene, 0.072g, triethyl amine, 0.83 g and sodium hypochlorite, 10 ml (12% in water) were added at 0 °C under nitrogen atmosphere and stirred at temperature 40 °C for 25 hrs. The progress of the reaction was monitored by TLC analysis (15% EA/pet ether). Then, water (50 ml) was added and the reaction mixture was extracted with CHCI3. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2SC>4. Evaporation of the solvent in high vacuum followed by column chromatography (10% ethyl acetate/hexane) give pure product 7a with white gummy product of yield 0.009g (45%).
Example 4
Step A: Hydrogenation of Diosgenin :
Diosgenin 1, two grams in 100 ml of ethanol was hydrogenated at 45 psi using 500 mg of 5% Pd/C for a period of 10 h. The reaction mixture was filtered and alcohol was distilled off under reduced pressure to furnish the crude hydrogenated product which was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as the eluent to furnish dihydro diosgenin compound lb in pure form of 95% yield.
Acetylation of dihydrodiosgenin of formula lb: (synthesis of 3 β -acetoxy- dihydrodiosgenin) :
Dihydrodiosgenin lb, lg and pyridine 1 ml were added to 25 ml acetic anhydride, and the mixture was heated and stirred at 90 °C for 2 hrs. The solution was poured into ice water and the precipitate was filtered. Filtrate was washed with water and dried under vacuum to yield a white solid, which was recrystalhzed with CH3OH to give compound 2b with the yield of 0.83 g, (83%), mp.194 °C as colourless needle crystals.
Step B: F-ring opening of Diosgenin acetate of formula 3b: ((22 β,251¾)-3 β - acetoxyfurost-26-ol :
lg NaBH3CN was added to a 1 g solution of dihydrodiosgenin acetate 2b in acetic acid 3 ml under argon atmosphere and stirred for 60 hrs at (30 °C). After that CH2C12 was added and then Na2C03 solution. The aqueous phase was extracted with CH2C12 and the combined organic layer dried over anhydrous Na2S04 and evaporated to get the product. Then the residue was purified by column chromatography (30% ethyl acetate/hexane) to give pure product 3b (white gummy liquid) with the yield of 0.70 g, 70%. Step C: Oxidization of the formula 3 to aldehyde of formula 4b:((22 ,25R)-3 β - acetoxyf urost-26-al :
PCC, 0.273 g was added to a ice cold mixture of powdered CaC03 0.127 g, silica 1.0 g, and 3b, 0.70 g in CH2C12 of 25 mL at room temperature. The reaction mixture was stirred for 12 hrs at 30 °C. Then, the reaction mixture was diluted with diethyl ether, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4b with white solid of 0.42 g (60%), mp.124 °C.
Step D: Oxime derivative of Diosgenin acetate of the formula 5b: ((22 β,251¾)-3 β - acetoxyf urost-26-oxime:
A mixture of 4b, 0.2 g, hydroxylamine hydrochloric acid 0.14 g, pyridine 1 ml and 95% ethanol 20 ml was refluxed for 1 hr, the progress of the reaction was monitored by TLC. Then the mixture was cooled with ice, washed with water and extracted with ethyl acetate. The solvent was removed under reduced pressure. A white gummy liquid was obtained, 5b with the yield of 0.17g (85%).
Step E: Isoxazole derivative of Diosgenin acetate of formula 7b: ((22 β,25ϋ)-3 β - acetoxyfurost-26-(5-methyl)isoxazole (7b):
To a solution of compound 5b, 0.2 g, in DCM, 20 mL, methyl acetylene 6, 0.072g, triethyl amine, 0.83 g and sodium hypochlorite, 10 ml (12% in water) were added at 0 °C under nitrogen atmosphere and stirred at 30 °C for 48 hrs. The progress of the reaction was monitored by TLC analysis (15% EA/pet ether). Then, water (50 ml) was added and the reaction mixture was extracted with DCM. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2S04. Evaporation of the solvent in high vacuum followed by column chromatography (10% ethyl acetate/hexane) gives pure product 8a with white gummy product of yield 0.15 g (75%).
Example 5
Step B: F-ring opening of Diosgenin of formula 3c : ((22 β,251¾)-3 β -hydroxyfurost- 26-01) :
lg NaBH3CN was added to a 1 g solution of diosgenin 1 in propionic acid 3 ml under argon atmosphere and stirred for 40 hrs at 30 °C. After that CH2C12 was added and then Na2C03 solution. The aqueous phase was extracted with CH2C12 and the combined organic layer dried over anhydrous Na2S04 and evaporated to get the product. Then the residue was purified by column chromatography (30% ethyl acetate/hexane) to give pure product 3c (white gummy liquid) with the yield of 0.78 g, 78%. Step C: Oxidization of the formula 3c to aldehyde of formula 4c: ((22 β,251¾)-3 β - hydroxyfurost-26-al) :
PCC, 0.273 g was added to a ice cold mixture of powdered CaC03 0.127 g, silica 1.0 g, and 3c, 0.8 g in CH2C12 of 25 mL at 30 °C. The reaction mixture was stirred for 7 hrs at temperature 40 °C. Then, the reaction mixture was diluted with diethyl ether, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4c with white solid of 0.6 g (75%), mp.124 °C.
Step D: Oxime derivative of dehydrodiosgenin of the formula 5c: ((22 β,251¾)-3 β - hydroxyfurost-26-oxime):
A mixture of 4c, 0.3 g, hydroxylamine hydrochloric acid 0.14 g, pyridine 1 ml and 95% ethanol 20 ml at temperature 80 °C for 2 hr, the progress of the reaction was monitored by TLC. Then the mixture was cooled with ice, washed with water and extracted with ethyl acetate. The solvent was removed under reduced pressure. A white gummy liquid was obtained, 5c with the yield of 0.23g (76%).
Step E: Isoxazole derivative of dehydrodiosgenin of formula 8c: ((22 p,25R)-3 β - dehydrohydroxyfurost-26-(5-phenyl)isoxazole 8c):
To a solution of compound 5c, 0.2 g, in DCM, 20 mL, phenyl acetylene 6, 0.072g, triethyl amine, 0.83 g and sodium hypochlorite, 10 ml (12% in water) were added at 0 °C under nitrogen atmosphere and stirred at temperature 30 °C for 20 hrs. The progress of the reaction was monitored by TLC analysis (15% EA/pet ether). Then, water (50 ml) was added and the reaction mixture was extracted with CHC13. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2S04. Evaporation of the solvent in high vacuum followed by column chromatography (10% ethyl acetate/hexane) give pure product 8c with white gummy product of yield 0.16g (80%).
Example 6
Step A: Hydrogenation of Diosgenin :
Diosgenin 1, two grams in 100 ml of ethanol was hydrogenated at 45 psi using 500 mg of 5% Pd/C for a period of 10 h. The reaction mixture was filtered and alcohol was distilled off under reduced pressure to furnish the crude hydrogenated product which was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as the eluent to furnish dihydro diosgenin compound lb in pure form of 95% yield.
Step B: F-ring opening of dihydrodiosgenin of formula 3d : ((22 P,25R)-3 β - hydroxyfurost-26-ol) :
lg NaBH3CN was added to a 1 g solution of dihydrodiosgenin lb in butanoic acid 3 ml under helium atmosphere and stirred for 25 hrs at 30 °C(. After that CH2C12 was added and then Na2C03 solution. The aqueous phase was extracted with ethyl acetate and the combined organic layer dried over anhydrous Na2S04 and evaporated to get the product. Then the residue was purified by column chromatography (30% ethyl acetate/hexane) to give pure product 3d (white gummy liquid) with the yield of 0.72 g, 72%.
Step C: Oxidization of the formula 3d to aldehyde of formula 4d: ((22 p,25R)-3 β - hydroxyfurost-26-al) :
PCC, 0.273 g was added to a ice cold mixture of powdered CaC03 0.127 g, silica 1.0 g, and 3b, 0.5 g in CH2C12 of 25 mL at 30 °C. The reaction mixture was stirred for 7 hrs at temperature 50 °C. Then, the reaction mixture was diluted with dichloromethan, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4d with white solid of 0.32 g (64%), mp.124 °C.
Step D: Oxime derivative of dihydrodiosgenin of the formula 5d: ((22 P,25R)-3 β - acetoxyfurost-26-oxime) :
A mixture of 4d, 0.4 g, hydroxylamine hydrochloric acid 0.14 g, pyridine 1 ml and 95% propanol 20 ml at temperature 60 °C for 2 hrs, the progress of the reaction was monitored by TLC. Then the mixture was cooled with ice, washed with water and extracted with chloroform. The solvent was removed under reduced pressure. A white gummy liquid was obtained, 5d with the yield of 0.27g (67%).
Step E: Isoxazole derivative of dihydrodiosgenin of formula 8d ((22 β,251¾)-3 β - hydroxyfurost-26-isoxazole) :
To a solution of compound 5d, 0.2 g, in acetone, 20 mL, phenyl acetylene, 0.072g, triethyl amine, 0.83 g and sodium hypochlorite, 10 ml (12% in water) were added at 0 °C under nitrogen atmosphere and stirred at temperature 40 °C for 25 hrs. The progress of the reaction was monitored by TLC analysis (15% EA/pet ether). Then, water (50 ml) was added and the reaction mixture was extracted with CHCI3. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2S04. Evaporation of the solvent in high vacuum followed by column chromatography (10% ethyl acetate/hexane) give pure product 8d with white gummy product of yield 0.14g (70%).Example 7
Step B: F-ring opening of Diosgenin of formula 3c : ((22 β,251¾)-3 β -hydroxyfurost- 26-01) :
lg NaBH3CN was added to a 1 g solution of diosgenin 1 in propionic acid 3 ml under argon atmosphere and stirred for 30 hrs at 30 °C. After that CH2C12 was added and then Na2C03 solution. The aqueous phase was extracted with CH2C12 and the combined organic layer dried over anhydrous Na2S04 and evaporated to get the product. Then the residue was purified by column chromatography (30% ethyl acetate/hexane) to give pure product 3c (white gummy liquid) with the yield of 0.73 g, 73%.
Step C: Oxidization of the formula 3c to aldehyde of formula 4c: ((22 β,251¾)-3 β - hydroxyfurost-26-al) :
PCC, 0.273 g was added to a ice cold mixture of powdered CaC03 0.127 g, silica 1.0 g, and 3c, 0.5 g in CH2C12 of 25 mL at 30 °C. The reaction mixture was stirred for 7 hrs at temperature 40 °C. Then, the reaction mixture was diluted with diethyl ether, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4c with white solid of 0.32 g (64%), mp.124 °C.
Step D: Oxime derivative of dehydrodiosgenin of the formula 5c: ((22 β,251¾)-3 β - hy droxyf urost-26-oxime) :
A mixture of 4c, 0.3 g, hydroxylamine hydrochloric acid 0.14 g, pyridine 1 ml and 95% ethanol 20 ml at temperature 80 °C for 2 hr, the progress of the reaction was monitored by TLC. Then the mixture was cooled with ice, washed with water and extracted with ethyl acetate. The solvent was removed under reduced pressure. A white gummy liquid was obtained, 5c with the yield of 0.20g (66%).
Step E: Isoxazole derivative of dehydrodiosgenin of formula 7c: ((22 p,25R)-3 β - dehydrohydroxyfurost-26-(5-methyl)isoxazole 7c) :
To a solution of compound 5c, 0.2 g, in DCM, 20 mL, methyl acetylene 6, 0.072g, triethyl amine, 0.83 g and sodium hypochlorite, 10 ml (12% in water) were added at 0 °C under nitrogen atmosphere and stirred at temperature 30 °C for 20 hrs. The progress of the reaction was monitored by TLC analysis (15% EA/pet ether). Then, water (50 ml) was added and the reaction mixture was extracted with CHCI3. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2SC>4. Evaporation of the solvent in high vacuum followed by column chromatography (10% ethyl acetate/hexane) give pure product 7c with white gummy product of yield 0.142g (71%).
Example 8
Step A: Hydrogenation of Diosgenin :
Diosgenin 1, two grams in 100 ml of ethanol was hydrogenated at 45 psi using 500 mg of 5% Pd/C for a period of 10 h. The reaction mixture was filtered and alcohol was distilled off under reduced pressure to furnish the crude hydrogenated product which was purified by column chromatography over silica gel using petroleum ether and ethyl acetate as the eluent to furnish dihydro diosgenin compound lb in pure form of 95% yield.
Step B: F-ring opening of dihydrodiosgenin of formula 3d : ((22 P,25R)-3 β - hydroxyfurost-26-ol) :
lg NaBH3CN was added to a 1 g solution of dihydrodiosgenin lb in butanoic acid 3 ml under helium atmosphere and stirred for 25 hrs at 30 °C(. After that CH2C12 was added and then Na2C03 solution. The aqueous phase was extracted with ethyl acetate and the combined organic layer dried over anhydrous Na2S04 and evaporated to get the product. Then the residue was purified by column chromatography (30% ethyl acetate/hexane) to give pure product 3d (white gummy liquid) with the yield of 0.69 g, 69%.
Step C: Oxidization of the formula 3d to aldehyde of formula 4d: ((22 p,25R)-3 β - hydroxyfurost-26-al) :
PCC, 0.273 g was added to a ice cold mixture of powdered CaC03 0.127 g, silica 1.0 g, and 3b, 0.6 g in CH2C12 of 25 mL at 30 °C. The reaction mixture was stirred for 7 hrs at temperature 50 °C. Then, the reaction mixture was diluted with dichloromethan, 50.0 mL and poured through a short column containing aluminum oxide (neutral). The solvent was removed under vacuum. Then the residue was purified by column chromatography (10% ethyl acetate/hexane) to give pure product 4d with white solid of 0.4 g (66%), mp.124 °C.
Step D: Oxime derivative of dihydrodiosgenin of the formula 5d: ((22 P,25R)-3 β - hydroxyfurost-26-oxime) :
A mixture of 4d, 0.4 g, hydroxylamine hydrochloric acid 0.14 g, pyridine 1 ml and 95% propanol 20 ml at temperature 60 °C for 2 hrs, the progress of the reaction was monitored by TLC. Then the mixture was cooled with ice, washed with water and extracted with chloroform. The solvent was removed under reduced pressure. A white gummy liquid was obtained, 5d with the yield of 0.24g (60%).
Step E: Isoxazole derivative of dihydrodiosgenin of formula 7d ((22 P,25R)-3 β - hydroxyfurost-26-isoxazole) :
To a solution of compound 5d, 0.2 g, in acetone, 20 mL, methyl acetylene, 0.072g, triethyl amine, 0.83 g and sodium hypochlorite, 10 ml (12% in water) were added at 0 °C under nitrogen atmosphere and stirred at temperature 40 °C for 25 hrs. The progress of the reaction was monitored by TLC analysis (15% EA/pet ether). Then, water (50 ml) was added and the reaction mixture was extracted with CHC13. The combined organic layer was washed with water followed by brine and dried over anhydrous Na2S04. Evaporation of the solvent in high vacuum followed by column chromatography (10% ethyl acetate/hexane) give pure product 7d with white gummy product of yield 0.134g (67%).
Example 9
Biological Activity of 5,6 dehydro-diosgenin acetate-Isoxazole and 5,6-dihydro-20 diosgenin acetate -Isoxazole of formula 7 :
In Vitro Antifungal Activity Determination:
In a preferred embodiment of the present invention : The new product E-ring manipulated 5,6 dehydro - diosgenin acetate-Isoxazole and 5,6-dihydro- diosgenin acetate-Isoxazole of the formula 7a and 7b where R2= OAc or OH have shown in good antifungal activity against plant pathogenic fungus Alternaria alternate as shown in Fig.4 and Table 1.
The inhibitory effects of the sample tested in vitro on mycelia growth of Alternaria alternata. Poisoned Food technique using to test the antifungal activity of the product of formula 7a and 7b in different concentrations of 100, 200, 300, 400 and 500 ppm. Petriplates (90 mm dia.) each containing 20 ml of potato dextrose agar (PDA) medium amended with the desired concentrations of samples inoculating with test fungus. A 5 mm diameter disc of the test fungus with a corkborer, cut from the periphery of an actively growing 8 -days old culture on PDA plates and placing at the center in each treated petriplate PDA plates without product of formula 7 served as control which consist of 100, 200, 300, 400 and 500 ppm of the solvent. The experiments conducting with three replications. Then plates were kept in incubator at temp. 25 ±1°C. After 72 hrs. of incubation radial growth of the colony measured and calculating the mycelia growth in percentage of inhibition from mean values of colony diameter in treated and control petridishes. Table 1 : The Inhibitory effects of the compounds on mycelial growth of Alternaria alternata 7days after inoculation (percent inhibition)
Figure imgf000032_0001
ADVANTAGES OF THE PRESENT INVENTION
1. Synthesis of a new and novel 5,6 dehydro- diosgenin acetate- Isoxazole and 5,6 dehydro- diosgenin acetate- Isoxazole.
2. Synthesis of a new and novel A and E-ring manipulated 5,6 dehydro- diosgenin acetate- Isoxazole and 5,6 dehydro- diosgenin acetate- Isoxazole.
3. Synthesized products have been found to exhibit potent antifungal activity against Alternaria alternate.
4. A new fungus using in the invention 'Alternaria alternate '.
5. The chemicals used are cheap and commercially available.
6. The process is reported for the first time.

Claims

WE CLAIM:
1. A compound of formula A
Figure imgf000034_0001
wherein R2= OAc or OH ; R=C6H5 or CH3.
2. A compound as claimed in claim 1 wherein the representative compounds are:
(22 P,25R)-3 β -acetoxyfurost-5,6-dehydro-26-(5-methyl isoxazole) (7a); (22 P,25R)-3 β -acetoxyfurost-5,6-dihydro-26-(5-methyl isooxazole) (7b); (22 P,25R)-3 β -hydroxyfurost-5,6-dehydro-26-(5-methyl isooxazole) (7c); (22 P,25R)-3 β -hydroxyfurost-5,6-dihydro-26-(5-methyl isooxazole) (7d); (22 P,25R)-3 β -acetoxyfurost-5,6-dehydro-26-(5-phenyl isoxazole) (8a); (22 P,25R)-3 β -acetoxyfurost-5,6-dihydro-26-(5-phenyl isooxazole) (8b);
(22 P,25R)-3 β -hydroxyfurost-5,6-dehydro-26-(5-phenyl isooxazole) (8c); (22 P,25R)-3 β -hydroxyfurost-5,6-dihydro-26-(5-phenyl isooxazole) (8d). A process for a process for preparation of diosgenin acetate -Isoxazoles of the formula A where in R2= OAc or OH ; R=C6H5 or CH3 comprising the steps:
i. optionally hydrogenating diosgenin 1 to obtain dihydrodiosgenin lb; ii. optionally acetylating diosgenin 1 or dihydrodiosgenin lb using acetic anhydride in presence of a suitable base to obtain 3P-acetoxy dehydrodiosgenin (2a) or 3P-acetoxy dihydrodiosgenin (2b) respectively;
iii. opening F-ring of 3P-acetoxy dehydrodiosgenin (2a) or 3P-acetoxy dihydrodiosgenin (2b) using sodium cyanoborohydride in acetic acid to obtain dehydro or dihydro (22β, 25R)-3P-acetoxyfurost-26-ol respectively;
iv. oxidizing dehydro or dihydro (22β, 25R)-3P-acetoxyfurost-26-ol as obtained in strep (iii) with pyridinium chlorochromate (PCC) in dichloromethane to obtain C-26-aldehyde-5,6-dehydro-diosgenin acetate or C-26-aldehyde-5,6-dihydro-diosgenin acetate respectively; v. refluxing the mixture of C-26-aldehyde-5,6-dehydro-diosgenin acetate or C-26-aldehyde-5,6-dihydro-diosgenin acetate, hydroxyl amine hydrochloric acid, base and protic solvent at a temperature ranging between 60-95 °C for a period of 1-3 hrs, followed by cooling, washing, extracting with a water immiscible solvent and removing of solvent under reduced pressure to obtain C-26-oxime- 5,6 dehydro-diosgenin acetate or C-26-oxime-5,6 dihydro-diosgenin acetate respectively;
vi. mixing the solution of C-26-oxime-5,6 dehydro-diosgenin acetate or C-26-oxime-5,6 dihydro-diosgenin acetate obtained in step (v) with phenyl acetylene or methyl acetylene, base and sodium hypochlorite in dichloromethane at the temperature ranging between 30-50 °C under nitrogen atmosphere followed by the stirring at the temperature ranging between 30-50 °C for the period of time 25-48 hrs;
vii. adding water to the reaction mixture obtained in step(ii) followed by extraction with water immiscible solvent followed by washing the combined organic layer with water and brine;
viii. drying the organic layer obtained in step (iii) followed by evaporating the solvent and purifying by column chromatography to obtain (22P,25R)-3P-acetoxyfurost-5,6,-dehydro-26-substituted isoxazole or (22P,25R)-3P-acetoxyfurost-5,6,-dihydro-26-substituted isoxazole a compound of formula A; and
ix. optionally hydrolyzing acetyl group of (22P,25R)-3P-acetoxyfurost- 5,6,-dehydro-26-substituted isoxazole or (22β,25Κ)-3β- acetoxyfurost-5,6,-dihydro-26-substituted isoxazole with suitable base to obtain (22P,25R)-3P-hydroxyfurost-5,6,-dehydro-26- substituted isoxazole or (22P,25R)-3P-hydroxyfurost-5,6,-dihydro- 26-substituted isoxazole a compounds of formula A.
The process as claimed in claim 3 wherein purification of the product by column chromatography with 10% ethyl acetate/hexane or 10% ethyl acetate /petroleum ether.
The process as claimed in claim 3, wherein the base is selected from the group consisting of pyridine, triethylamine.
The process as claimed in claim 3, wherein the protic solvent selected from methanol, ethanol, isoporyl alcohol.
The process as claimed claim 3, wherein water immiscible solvent selected from the group consisting of ethyl acetate, dichloromethane.
8. A compound obtained by the process as claimed in claim 3, where the compounds are
(22 P,25R)-3 β -acetoxyfurost-5,6-dehydro-26-ol (3a);
(22 P,25R)-3 β -acetoxyfurost-5,6-dihydro-26-ol (3b);
(22 P,25R)-3 β -acetoxyfurost-5,6-dehydro-26-al (4a);
(22 P,25R)-3 β -acetoxyfurost-5,6-dihydro-26-al (4b);
(22 P,25R)-3 β -acetoxyfurost-5,6-dehydro-26-oxime (5a);
(22 P,25R)-3 β -acetoxyfurost-5,6-dihydro-26-oxime(5b).
9. A composition comprising an antifungal compound of formula A.
10. The compound of formula A as claimed in claim 1 for use in treating or preventing a fungal infection against plant pathogenic fungus Alternaria alternate.
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