Claims:
WE CLAIM:
1. A process for the preparation of 22R,23R-isomer of 28-Homocastasterone of formula (I), comprising:
reacting 2,22-unsaturated compound of formula (VI), for asymmetric hydroxylation, with potassium osmate, chiral ligand (DHQD)2-PHAL, potassium ferricyanide, potassium carbonate, methanesulphonamide, in t-butanol-water (1:1) to obtain
crude tetraol compound of formula (I), and
2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia); and
purifying the crude tetraol compound of formula (I) to obtain 22R,23R-isomer of 28-Homocastasterone of formula (I).
2. The process as claimed in claim 1, wherein the reaction is carried out at temperature ranging from -10°C to 50°C.
3. The process as claimed in claim 1, wherein the reaction is carried out at room temperature.
4. The process as claimed in claim 1, wherein the purification of the crude tetraol compound of formula (I) is achieved by carrying out column chromatography of the crude tetraol compound of formula (I) using a silica gel mesh and a solvent system with loading of silica gel 5 times to 18 times.
5. The process as claimed in claim 4, wherein the silica gel mesh used is selected from one of a 60-120 mesh, 100-200 mesh and 230-400 mesh.
6. The process as claimed in claim 4, wherein the solvent system is selected from one of a chloroform-methanol and hexane-ethyl acetate.
7. The process as claimed in claim 1, further comprises converting 2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia) to 22R,23R-isomer of 28-Homocastasterone of formula (I) by
reacting 2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia), for asymmetric hydroxylation, with potassium osmate, chiral ligand (DHQD)2-PHAL, potassium ferricyanide, potassium carbonate, methanesulphonamide, in t-butanol-water (1:1) to obtain crude tetraol compound of formula (I); and
purifying the crude tetraol compound of formula (I) to obtain 22R,23R-isomer of 28-Homocastasterone of formula (I).

Dated this the 18th day of December 2015
Signature

SANTOSH VIKRAM SINGH
Patent Agent
Agent of the Applicant
, Description:FORM 2

THE PATENTS ACT, 1970
[39 of 1970]
&
THE PATENTS RULES, 2003

COMPLETE SPECIFICATION
(Section 10; Rule 13)

A SYNTHETIC PROCESS FOR PREPARATION OF 22R,23R-ISOMER OF 28-HOMOCASTASTERONE

PHYTOTRON AGRO PRODUCTS (INDIA) PVT. LTD.
Thirumenahalli, Yelahanka, Bangalore - 560064,
Karnataka, India
[An Indian Company]

The following Specification particularly describes the invention and the manner in which it is to be performed

FIELD OF INVENTION
The present invention relates to synthetic production of plant hormones. More specifically, the present invention relates to a synthetic processes for the preparation of 22R,23R isomer of 28-Homocastasterone of formula (I) and 2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia), a major intermediate in the synthetic process thus improving the overall yield of 28-homocastasterone.
BACKGROUND
28-Homobrassinolide with the chemical name as 2a,3a,22R,23R-tetrahydroxy-24S-ethyl-B-homo-7-oxa-5a-cholestan-6-one, a group of polyhydroxy steroidal lactones, are now well-established as a class of plant growth regulators. 28-Homobrassinolide has been detected in seeds and sheaths of Brassica campestris var. perkinesis [Experientia.,39, 351 (1983)]. It is reported to be as active as brassinolide in the rice lamina inclination bioassay but less active in certain other assays, e.g. the bean first internode assay [Phytochemistry.,22, 2437 (1983)].
Synthesis of 28-Homobrassinolide has been reported [Chem. Pharm. Bull. (Japan) 30, 4181 (1982)]. The starting material was the readily available stigmasterol, which by well-known reactions, was converted to the target compound, but in low overall yield.
Till now there have been several attempts for increasing the overall yield of 28-Homobrassinolide, but not resulted in the desired result. Further, the low yield of the desired 28-Homobrassinolide results in extremely high cost of the same, making it almost inaccessible to users.
Wherefore, a synthetic process which could prepare 28-Homobrassinolide more efficiently at a higher yield is extremely desirable by the industry.

SUMMARY OF THE INVENTION
The present invention provides a synthetic process for preparation of 28-Homocastasterone of formula (I).
In an embodiment of the present invention, a process for the preparation of 22R,23R-isomer of 28-Homocastasterone of formula (I) is disclosed. The process includes two steps, first being reacting 2,22-unsaturated compound of formula (VI), for asymmetric hydroxylation, with potassium osmate, chiral ligand (DHQD)2-PHAL, potassium ferricyanide, potassium carbonate, methanesulphonamide, in t-butanol-water (1:1) to obtain crude tetraol compound of formula (I), and 2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia); and second being purifying the crude tetraol compound of formula (I) to obtain 22R,23R-isomer of 28-Homocastasterone of formula (I).
In a further embodiment of the present invention, a process for converting 2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia) to 22R,23R-isomer of 28-Homocastasterone of formula (I) is disclosed. The process includes following steps: reacting 2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia), for asymmetric hydroxylation, with potassium osmate, chiral ligand (DHQD)2-PHAL, potassium ferricyanide, potassium carbonate, methanesulphonamide, in t-butanol-water (1:1) to obtain crude tetraol compound of formula (I); and purifying the crude tetraol compound of formula (I) to obtain 22R,23R-isomer of 28-Homocastasterone of formula (I).
In another embodiment of the present invention, a process for synthesis of 28-Homocastasterone of formula (I) from stigmasterol of formula (II) is provided.

OBJECTIVES OF THE INVENTION
The main objective of the present invention is to provide a novel synthetic process for preparation of 28-Homocastasterone of formula (I) while overcoming drawbacks of the hitherto known processes.
Another objective of the present invention is to provide a novel synthetic process for conversion of dihydroxy compound of formula (Ia) to 28-Homocastasterone of formula (I).
DETAILED DESCRIPTION OF THE PRESENT INVENTION
The present invention discloses a synthetic process for preparation of 28-Homocastasterone of formula (I). The synthetic process demonstrates overall increase in the yield of 28-Homocastasterone of formula (I) while employing method of asymmetric hydroxylation reported to prepare 22R,23R-diol structure.
In the following detailed description of the embodiments of the invention, the embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that changes may be made without departing from the scope of the present invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims.
The specification may refer to “an”, “one” or “some” embodiment(s) in several locations. This does not necessarily imply that each such reference is to the same embodiment(s), or that the feature only applies to a single embodiment. Single features of different embodiments may also be combined to provide other embodiments.
As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless expressly stated otherwise. It will be further understood that the terms “includes”, “comprises”, “including” and/or “comprising” when used in this specification, specify the presence of stated features, and steps, but do not preclude the presence or addition of one or more other features and steps and/or groups thereof. As used herein, the term “and/or” includes any and all combinations and arrangements of one or more of the associated listed items.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
The present embodiments have been described with reference to specific example embodiments; it will be evident that various modifications and changes may be made to these embodiments without departing from the broader spirit and scope of the various embodiments.
Although the embodiments herein are described with various specific embodiments, it will be obvious for a person skilled in the art to practice the invention with modifications. However, all such modifications are deemed to be within the scope of the claims. It is also to be understood that the following claims are intended to cover all of the generic and specific features of the embodiments described herein and all the statements of the scope of the embodiments which as a matter of language might be said to fall there between.

In one of the embodiment of the present invention, a process for preparation of 22R,23R-isomer of 28-Homocastasterone of formula (I) is disclosed. The process starts with reacting 2,22-unsaturated compound of formula VI, for asymmetric hydroxylation, with potassium osmate, chiral ligand (DHQD)2-PHAL, potassium ferricyanide, potassium carbonate, methanesulphonamide, in t-butanol-water (1:1) to obtain crude tetraol compound of formula (I) (37% yield) as white solid, and 2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia) (45% yield) as white solid.

The 2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia) is a by-product of the present step.

The reaction is carried out at temperature ranging from -10°C to 50°C. However, the reaction is preferably carried out at room temperature.
Further, during next step the crude tetraol compound of formula (I) is purified to obtain the desired 22R,23R-isomer of 28-Homocastasterone of formula (I).
The purification of the crude tetraol compound of formula (I) is achieved by carrying out column chromatography of the crude tetraol compound of formula (I) using a silica gel 60-120 mesh or 100-200 mesh or 230-400. Out of these the silica gel 60-120 mesh is preferably used. The column chromatography is carried out using a solvent system chloroform-methanol or hexane-ethyl acetate, with loading of silica gel 5 times to 18 times.
In a further embodiment of the present invention, a process for conversion of 2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia) to 22R,23R-isomer of 28-Homocastasterone of formula (I) is provided. The process includes carrying out asymmetric hydroxylation of 2a,3a-dihydroxy-24S-ethyl-5a-chloest-22E-en-6-one of formula (Ia) by reacting it with potassium osmate, chiral ligand (DHQD)2-PHAL, potassium ferricyanide, potassium carbonate, methanesulphonamide, in t-butanol-water (1:1) to obtain crude tetraol compound of formula (I).
In the next step the crude tetraol compound of formula (I) is purified to obtain 22R,23R-isomer of 28-Homocastasterone of formula (I) (50% yield) as white solid. The purification the crude tetraol compound of formula (I) is carried out in the similar way as explained earlier.
In another embodiment of the present invention, a process for synthesis of 28-Homocastasterone of formula (I) from stigmasterol of formula (II) is provided. The 28-Homocastasterone is synthesized as shown in the following scheme:

In the given synthetic route, the starting stigmasterol (II) is a well-known steroidal compound [J.Am.Chem.Soc.,62, 2006 (1940)].
The step 1 to step 4 of the synthetic route are already known [J.Org.Chem.,28, 571 (1963), Phytochemistry.,Vol.36, No.3, 585 (1994) and J.Org.Chem.,58, 2338 (1993)].
In the step 5, the 2,22-unsaturated compound (VI) is oxidized to the 2,3,22,23-tetraol compound (I). The oxidation may be accomplished by catalytic osmylation [Tetrahedron Letters.,23, 1973 (1976)], preferably together with a tertiary amine N-oxide (e.g. N-methylmorpholine-N-oxide, pyridine N-oxide) in an inert solvent (e.g. aqueous tetrahydrofuran, aqueous dioxane). The performance of the oxidation in an inert gas atmosphere such as argon is usually favorable. In general, the amount of osmium tetroxide may be much smaller than the theoretical amount, e.g. about 1/20 mol to 1 mol of the double bond in the 2,22-unsaturated compound (VI). Thus, stigmasterol can be transformed in five steps to 28-homocastasterone in an overall yield of 48%.
28-Homocastasterone of formula (I) so produced is a bio-synthetic precursor to plant growth regulator like 28-Homobrassinolide. The 28-Homocastasterone of formula (I) is reacted with trifluoroperacetic acid in chlorinated solvent(s) to bring about oxidative ring expansion of 28-Homocastasterone, while maintaining reaction temperature between -10°C to 50°C, to obtain 28-Homobrassinolide (80% yield) of formula (VII) as white solid.

The present invention, therefore, provides a very efficient process for producing the 28-Homobrassinolide.

EXAMPLES
The present invention is explained further in the following specific examples which are only by way of illustration and are not to be construed as limiting the scope of the invention.
Example 1: Preparation of 2a,3a,22R,23R-tetrahydroxy-24S-ethyl-5a-cholestan-6-one (I)
A mixture of tert-butanol (675 ml), water (675 ml), potassium carbonate (45.43 g), potassium ferricyanide (108.23 g), potassium osmate (80 mg), (DHQD)2PHAL ligand (0.853 g) and methanesulfonamide (10.42 g) was cooled to 0°C, 24S-ethyl-5a-cholesta-2,22-dien-6-one (22.5 g) was added, the resultant mixture stirred at room temperature for 6 days. After 6 days solid sodium sulphite (41.36 g) was added and the mixture was stirred at room temperature for 2 hours. Tert-butanol was removed under reduced pressure and the residue was extracted with ethyl acetate. Combined ethyl acetate extracts were washed with water, 0.25M H2SO4 (3×112 ml) then with saturated sodium chloride solution, and dried with anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (60-120 mesh). Elution with Hexane-Ethyl acetate (5:5) provided less polar 2a,3a-dihydroxy-24S-ethyl-5a-cholest-22E-en-6-one (11.8 g, 45%). M.P. 233°-235°C. NMR: d (400 MHz, CDCl3) = 0.71 (3H, s, H-18), 0.89 (3H, s, H-19), 0.91-0.98 (12H, m, 4×Me), 3.11 (1H, dd), 3.58 (1H, d), 3.70-3.73 (2H, m), 4.03 (1H, br s).
Further elution with Hexane-Ethyl acetate (3:7) gave most polar 2a,3a,22R,23R-tetrahydroxy-24S-ethyl-5a-cholestan-6-one (9.7 g, 37%). M.P. 250°-252°C. NMR: d (400 MHz, CDCl3) = 0.67 (3H, s, H-18), 0.75 (3H, s, H-19), 0.78-0.86 (9H, m, 3×Me), 1.03 (3H, d, H-20), 2.28 (1H, dd), 2.69 (1H, dd), 3.75 (1H, m), 4.05 (1H, 1H, brs), 5.02 (1H, dd), 5.14 (1H, dd).
Example 2: Preparation of 2a,3a,22R,23R-tetrahydroxy-24S-ethyl-5a-cholestan-6-one (Ia)
A mixture of tert-butanol (300 ml), water (300 ml), potassium carbonate (9.3 g), potassium ferricyanide (22.2 g), potassium osmate (17 mg), (DHQD)2PHAL ligand (0.175 g) and methanesulfonamide (2.13 g) was cooled to 0°C, 2a,3a-dihydroxy-24S-ethyl-5a-cholesta-22E-en-6-one (10 g) was added, the resultant mixture stirred at room temperature for 6 days. After 6 days solid sodium sulphite (8.5 g) was added and the mixture was stirred at room temperature for 2 hours. Tert-butanol was removed under reduced pressure and the residue was extracted with ethyl acetate. Combined ethyl acetate extracts were washed with water, 0.25M H2SO4 (3×50 ml) then with saturated sodium chloride solution, and dried with anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (60-120 mesh). Elution with Hexane-Ethyl acetate (5:5) provided less polar 2a,3a-dihydroxy-24S-ethyl-5a-cholest-22E-en-6-one (4.5 g, 45%). M.P. 233°-235°C.
NMR: d (400 MHz, CDCl3) = 0.71 (3H, s, H-18), 0.89 (3H, s, H-19), 0.91-0.98 (12H, m, 4×Me), 3.11 (1H, dd), 3.58 (1H, d), 3.70-3.73 (2H, m), 4.03 (1H, br s).
Further elution with Hexane-Ethyl acetate (3:7) gave most polar 2a,3a,22R,23R-tetrahydroxy-24S-ethyl-5a-cholestan-6-one (4.8 g, 45%). M.P. 250°-252°C. NMR: d (400 MHz, CDCl3) = 0.67 (3H, s, H-18), 0.75 (3H, s, H-19), 0.78-0.86 (9H, m, 3×Me), 1.03 (3H, d, H-20), 2.28 (1H, dd), 2.69 (1H, dd), 3.75 (1H, m), 4.05 (1H, 1H, brs), 5.02 (1H, dd), 5.14 (1H, dd).
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention.