GB2555866A

GB2555866A - A novel crystalline form of epocholeone, a process for its preparation and use the same

Info

Publication number: GB2555866A
Application number: GB1619315.3A
Authority: GB
Inventors: Timothy Bristow James
Original assignee: Rotam Agrochem International Co Ltd
Current assignee: Rotam Agrochem International Co Ltd
Priority date: 2016-11-15
Filing date: 2016-11-15
Publication date: 2018-05-16
Anticipated expiration: 2036-11-15
Also published as: CN108070017A; GB2555866B

Abstract

A crystalline form of epocholeone [(1R,3aS,3bS,6aS,8S,9R,10aR,10bS,12aS)-1-[(1S)-1-[(2R,3R)-3-[(1S)-1-ethyl-2-methylpropyl]-2-oxiranyl]ethyl]hexadecahydro-10a,12a-dimethyl-8,9-bis(1-oxopropoxy)-6H-benz[c]indeno[5,4-e]oxepin-6-one] of formula (I): may exhibit at least 3 of the reflexes 5.03, 7.02, 8.81, 10.47, 10.67, 10.83, 11.28, 11.52, 13.17, 13.63, 14.54, 16.25, 17.01, 17.36, 17.71, 17.92, 18.25, 18.46, 19.35, 20.15, 21.04, 21.68, 22.72, 23.23, 24.62, 25.37 and 28.22 as 2θ ±0.20 degrees in an X-ray powder diffractogram (X-RPD) recorded using Cu-Kα radiation at 25 ºC. The crystalline epocholeone may also exhibit an IR spectrum with vibration peaks at wavenumbers (cm-1, ±0.2 %) of one or more of about 2963, 2874 and 1724 cm-1. A crystalline modification of epocholeone may be prepared by dissolving epocholeone (e.g. amorphous epocholeone) in a solvent or solvent mixture; precipitating the doissolved compound as the crystalline modification and isolating it. Preferred crystallisation solvents include toluene and xylene. Various suspension concentrate and oil-bases suspension concentrate formulations are described using the crystalline propionyl brassinolide suitable as plant growth regulating compositions.

Description

(54) Title of the Invention: A novel crystalline form of epocholeone, a process for its preparation and use the same

Abstract Title: Crystalline epocholeone and its preparation (57) A crystalline form of epocholeone [(1R,3aS,3bS,6aS,8S,9R,10aR,10bS,12aS)-1-[(1S)-1-[(2R,3R)-3-[(1S)-1-ethyl-2methylpropyl]-2-oxiranyl]ethyl]hexadecahydro-10a,12a-dimethyl-8,9-bis(1-oxopropoxy)-6H-benz[c]indeno[5,4-e] oxepin-6-one] of formula (I):

may exhibit at least 3 of the reflexes 5.03, 7.02, 8.81, 10.47, 10.67, 10.83, 11.28, 11.52, 13.17, 13.63, 14.54, 16.25, 17.01, 17.36, 17.71, 17.92, 18.25, 18.46, 19.35, 20.15,21.04,21.68, 22.72,23.23, 24.62, 25.37 and 28.22 as 20 ±0.20 degrees in an X-ray powder diffractogram (X-RPD) recorded using Cu-Κα radiation at 25 °C. The crystalline epocholeone may also exhibit an IR spectrum with vibration peaks at wavenumbers (cm¹, ±0.2 %) of one or more of about 2963, 2874 and 1724 cm¹. A crystalline modification of epocholeone may be prepared by dissolving epocholeone (e.g. amorphous epocholeone) in a solvent or solvent mixture; precipitating the doissolved compound as the crystalline modification and isolating it. Preferred crystallisation solvents include toluene and xylene. Various suspension concentrate and oil-bases suspension concentrate formulations are described using the crystalline propionyl brassinolide suitable as plant growth regulating compositions.

1/3

CM

Lf) ostss

SSTZGZ

06’296S τύππγτττ^^πγίπΓτ**τί“

Πι»«ΓΎιΓΊΓΤιΓΤΊΓ^

Γ*»Τ|Γ»<>ηΓτ»^ΓΎ“Τ“ΤΎΗΠΤΎ“ΤΤ“1“ΤΤ“ί“Γ“Γ“ϊ“Γ“1“Γ ρτ-ττηΓΎ-ΎΠΓΤ-τ-ττητττ-ττ-τ-ττη-Ύ-τ-ττΟ CO CO 'srCNOOOCDM-CNQOQCDM-iNQOOCD’^iN ο σ> σ> σ> σ> σ> co co co οο co r- r- r- r- co co co co

4Q00 35Q0 3000 2500 2000 1500 1000

Wave numbers (em-1) eouepiLusueji %

2/3

386'SSj

Μ 002'OS,

M 618'8®i

U.89877 . I»] 808 2W o

LO

Sow.

12 17 w ®®3®®< [o] 86®'8F [o] 68®3®ί W 800' it

W 92 I/O® [o] 88688«

H 026' zc: [oj 68 8'28 ^:

Μ 82® 98, [ο] 2®6'58’^ί _r [o] 3L8'®8< 280 88 788

H 682 78--/.—/X _r, H 85248-p W 60®48—

W 625 83 [a] ggg 92--M -686'93*

W 5®8'S3.W 3Z8'S3— ' [_o] O39'®3* [°j ®83 r 3 ®1 l/Z/S [o] 22® ί3·Π-629

W 388'6 ojZLS

W 61

0	O	O
0	O	O
0	O	O
0	O	O
CO	CM	x—

o

340

CO

CM

CL

3/3

12 17

Figure 3

A NOVEL CRYSTALLINE FORM OF EPOCHOLEONE, A PROCESS FOR ITS PREPARATION AND USE THE SAME

Technical Field

The present invention relates to a crystalline form of epocholeone, also named as 5 propionyl brassinolide [(lR,3aS,3bS,6aS,8S,9R, 10aR,10bS,12aS)-l-[(lS)-l-[(2R,3R)-3[(lS)-l-ethyl-2-methylpropyl]-2-oxiranyl]ethyl] hexadecahydro-10a,12a-dimethyl-8,9bis(l-oxopropoxy)-6H-benz[c]indeno[5,4-e]oxepin-6-one], to its preparation processes and to its use in agrochemical preparations.

12 17

Background

Epocholeone, also named as propionyl brassinolide [(lR,3aS,3bS,6aS,8S,9R,10aR,10bS,12aS)-l-[(lS)-l-[(2R,3R)-3-[(lS)-l-ethyl-2methylpropyl]-2-oxiranyl]ethyl]hexadecahydro-10a,12a-dimethyl-8,9-bis(l-oxopropoxy)6H-benz[c]indeno[5,4-e]oxepin-6-one] is a plant growth regulator. It was discovered from rape pollen by M. D. Grove et al. in 1979 Nature, Vol. 281, pp. 216-217 (1979)], and ever since the plant growth promoting effects have been disclosed (Brassinosteroids. Chemistry, Bioactivity & Applications. ACS Symposium Series 474, American Chemical Society, Washington, D.C., 1991). It is extremely effective on root-crops, potatoes and taroes, cereals, com, cotton, fruity vegetables, pulses, leafy vegetables, fruit-trees, woody plants, flowering plants, and industrial crops.

Epocholeone has molecular formula of C Η O . Its chemical structure is:

56 7

H,C ' \ HX fe

£......Q Η

--------N'7\ H^Z N \Z ,A / CH_S fX''

V* (I)

12 17

The commercially available epocholeone, which is usually manufactured by the process described in U.S Pat. No. 5,763,366, is present in amorphous state. It has been found that amorphous epocholeone is not suitable for being prepared as formulation as it is very susceptible to hydrolysis. It will undergo significant hydrolysis when dissolved or dispersed in water. Hydrolysis can even occur during storage, particularly where the compound is exposed to moisture and heat. As a result, the plant growth regulator activity decreases, higher amounts of epocholeone have to be applied. Moreover, epocholeone has to be applied in shorter intervals. Therefore, there is a need to provide a novel form of epocholeone with anti-hydrolysis property.

Summary of the invention

In attempt to resolve some or all of the problems with existing amorphous form of epocholeone, a new and stable crystalline form of epocholeone has been prepared.

Aspects of the invention relate to a crystalline modification of [(lR,3aS,3bS,6aS,8S,9R,10aR,10bS,12aS)-l-[(lS)-l-[(2R,3R)-3-[(lS)-l-ethyl-2methylpropyl]-2-oxiranyl]ethyl]hexadecahydro-10a,12a-dimethyl-8,9-bis(l-oxopropoxy)6H-benz[c]indeno[5,4-e]oxepin-6-one] (epocholeone).

In a first aspect, the present invention provides a novel crystalline form of epocholeone [(lR,3aS,3bS,6aS,8S,9R,10aR,10bS,12aS)-l-[(lS)-l-[(2R,3R)-3-[(lS)-l20 ethyl-2-methylpropyl]-2-oxiranyl]ethyl]hexadecahydro-10a,12a-dimethyl-8,9-bis(loxopropoxy)-6H-benz[c]indeno[5,4-e]oxepin-6-one], termed “crystalline modification I”, exhibiting at least three of the following reflexes, in any combination, as 20+0.20 degree in an X-ray powder diffractogram (X-RPD) recorded using Cu—Ka radiation at 25°C:

26 = 5.03 + 0.2 = 7.02 + 0.2 = 8.81 + 0.2 (1) (2) (3)

2Θ = 10.47 ±0.2	(4)
2Θ = 10.67 ±0.2	(5)
2Θ = 10.83 ±0.2	(6)
2Θ = 11.28 ±0.2	(Ό
2Θ = 11.52 ± 0.2	(8)
2Θ = 13.17 ± 0.2	(9)
2Θ = 13.63 ±0.2	(1θ)
2Θ = 14.54 ±0.2	(11)
2Θ = 16.25 ±0.2	(12)
2Θ = 17.01 ±0.2	(13)
2Θ = 17.36± 0.2	(14)
2Θ = 17.71 ±0.2	(15)
2Θ = 17.92 ±0.2	(16)
2Θ = 18.25 ±0.2	(17)
2Θ = 18.46 ±0.2	(18)
2Θ = 19.35 ±0.2	(19)
20 = 20.15 ±0.2	(20)
20 = 21.04 ±0.2	(21)
20 = 21.68 ±0.2	(22)
20 = 22.72 ± 0.2	(23)
20 = 23.23 ±0.2	(24)
20 = 24.62 ± 0.2	(25)
20 = 25.37 ±0.2	(26)

2Θ = 28.22 ± 0.2 (27)

In an embodiment, the crystalline modification according to the first aspect of the invention, exhibits at least 3, 4, 5, 6, 7, 8 or all of the reflexes, in any combination, from the following:

= 5.03 + 0.2 (1) = 7.02 ± 0.2 (2)

20= 10.47 + 0.2 (4)

20= 10.68 + 0.2 (5)

20= 11.29 + 0.2 (7)

20= 11.52 + 0.2 (8)

20= 16.25 + 0.2 (12)

20=17.01 + 0.2 (13)

20= 17.36 + 0.2 (14)

20= 17.71 + 0.2 (15)

20= 17.92 + 0.2 (16)

20= 18.46 + 0.2 (18)

20= 19.35 + 0.2 (19) = 20.15 + 0.2 (20)

20 = 21.04 + 0.2 (21) = 22.72 ± 0.2 (23) = 23.23 + 0.2 (24)

12 17

In a second aspect, the present invention provides a crystalline modification I of epocholeone, optionally according to the first aspect of the invention, exhibiting an infrared (IR) spectrum with characteristic functional group vibration peaks at wavenumbers (cm-1, ± 0.2%) of one or more of about 2963, 2874 andl724 cm^-1.

In a third aspect, the present invention provides a crystalline modification I of epocholeone, optionally according to the first or second aspect of the invention, characterized by X-ray powder diffraction pattern as substantially shown in Figure 2, and/or characterized by an IR spectrum as substantially shown in Figure 1.

In a fourth aspect, the present invention provides a crystalline modification I of epocholeone, optionally according to optionally according to any one of the first to third aspects of the invention, obtainable by the process as substantially as described in Example 2 or 3.

In a fifth aspect, the present invention provides a crystalline modification I of epocholeone, optionally according to any one of the first to fourth aspects of the invention, obtainable by the process of the sixth aspect of the invention.

It has been found that the present crystalline modification I of epocholeone has a significant increase in its hydrolysis stability, which significantly decrease hydrolysis problem encountered in current commercially available formulations. In addition, it is found that the crystalline modification I of epocholeone is easier to to comminute or grind compared to amorphous epocholeone prepared in accordance with the disclosure of U.S. PATENT NO. 5,763,366 and that formulations prepared using this crystalline modification I is are stable after prolonged storage. This allows the preparation of commercial formulations such as suspension concentrates (SC) and oil-based suspension concentrates (OD). Hence, it is possible to prepare any formulations of epocholeone in crystalline modification I, which is disclosed hereinafter. By virtue of its high anti-hydrolysis property, the crystalline modification I of epocholeone is highly suitable for preparing compositions for promoting the plant growth.

In a sixth aspect, the present invention provides a process for preparing a crystalline modification I of epocholeone comprising steps of

12 17

i) dissolving epocholeone in a solvent, or mixture of solvents;

ii) precipitating the dissolved compound into crystalline modification I of epocholeone; and iii) isolating the precipitated crystalline modification I.

In an embodiment of the eighth aspect of the invention, the epocholeone in step i) is amorphous epocholeone.

Methods for preparing amorphous epocholeone are known in the art. Amorphous epocholeone is manufactured and available on a commercial scale. A particularly suitable method for preparing amorphous epocholeone is described in U.S. Pat. No. 5,763,366.

In an embodiment of the sixth aspect of the invention, the solvent is selected from the group consisting of halogenated hydrocarbons (for example, trifluoro methyl benzene, chlorobenzene, bromobenzene, dichlorobenzene, chlorotoluene and trichlorobenzene), ethers (for example, ethyl propyl ether, n-butyl ether, anisole, phenetole, cyclohexyl methyl ether, dimethyl ether, diethyl ether, dimethyl glycol, diphenyl ether, dipropyl ether, diisopropyl ether, di-n-butyl ether, diisobutyl ether, diisoamyl ether, ethylene glycol dimethyl ether, isopropyl ethyl ether, methyl tert-butyl ether, tetrahydrofuran, methyltetrahydrofuran, dioxane, dichlorodiethyl ether, methyl-tetrahydrofuran, polyethers of ethylene oxide and/or propylene oxide), nitrated hydrocarbons (for example, nitromethane, nitroethane, nitropropane, nitrobenzene, chloronitrobenzene and o20 nitrotoluene), aliphatic, cycloaliphatic or aromatic hydrocarbons (for example, pentane, nheptane, n-octane, nonane, ethyl benzene, mesitylene), cymene, petroleum fractions having a boiling range of from 70 °C to 190 °C, cyclohexane, methylcyclohexane, petroleum ether, ligroin, octane, toluene, xylene, esters (for example, malonates, acetic acid n-butyl ester (n-butyl acetate), methyl acetate, , isobutyl acetate, dimethyl carbonate, diethyl carbonate, dibutyl carbonate and ethylene carbonate), methyl ethyl ketone and mixtures thereof.

In an embodiment of the sixth aspect of the invention, the solvent is selected from the group consisting of toluene, xylene, chlorobenzene, dichlorobenzene, ethyl benzene,

12 17 trifluoro methyl benzene, mesitylene, nitrobenzene, methyl ethyl ketone, acetonitrile or mixture of THF-water.

In an embodiment of the sixth aspect of the present invention, the solvent is selected from the group consisting of xylene, toluene or a mixture thereof.

In an embodiment of the sixth aspect of the present invention amorphous epocholeone is dissolved in a solvent comprising xylene and/or toluene in step (i).

According to an embodiment of the sixth aspect of the present invention, step ii) is effected by concentration of the solvent and/or by cooling and/or by the addition of a solubility reducing solvent and/or by adding a seed crystal of the crystalline modification I of epocholeone.

According to an embodiment of the sixth aspect of the present invention, crystalline modification I of epocholeone is prepared by dissolving the amorphous epocholeone in a solvent or a solvent mixture as a concentrated solution by heating from ambient temperature to a temperature at or below the reflux temperature of the solvent or the solvent mixture. Optionally, the concentrated solutions can be prepared at the reflux temperature of the solvents. The concentration of the solution depends on the solubility of epocholeone in the corresponding solvent or solvent mixture.

In an embodiment of the sixth aspect of the present invention, the concentrated homogeneous solution thus prepared as in step (i) is then cooled to ambient temperature or cooled to about 0 to 20°C to crystallize the desired crystalline form from the solvent. The crystalline modification I of epocholeone can also be crystallized out by concentrating the homogeneous solution by removing the solvent or solvent mixture to a certain volume, with or without applying vacuum, and cooling to below the reflux temperature of the solvent or the solvent mixture.

In an embodiment of the sixth aspect of the present invention, crystallization of crystalline modification I of epocholeone can also be obtained by adding seed crystals of the desired crystalline form during crystallization into the solution prepared in step (i), which can promote or accelerate the crystallization.

12 17

The seed crystal amount added to the concentrated solution is typically in the range of 0.001% to 10% by weight, more particularly in the range of 0.005% to 0.5% by weight, based on the weight of epocholeone used for the preparation of concentrated solution in step (i). Optionally, the seed crystals are added to the concentrated solution at a temperature below the boiling point of the corresponding solvent or the solvent mixture.

In an embodiment of the sixth aspect of the invention, the precipitated crystalline modification I of epocholeone obtained from step (ii) is isolated by the usual solid component separating techniques from solutions, such as filtration, centrifugation or decantation. Then, the isolated solid will be washed with solvent one or more times.

Optionally, the solvent employed in the washing stage consists of one or more components of the solvent or solvent mixture employed for the preparation of concentrated solution in step (i), as described hereinbefore. The washing is usually carried out using the corresponding solvent or solvent mixture between room temperature and 0°C, depending on the solubility of the crystal in order to avoid the loss of crystal as far as possible in the corresponding washing solvent. In an embodiment of the sixth aspect of the invention, crystalline modification I of epocholeone is dissolved and recrystallized. The washings and/or the solvent of crystallization in any of the methods may be concentrated to obtain solid epocholeone which may be recycled.

In a seventh aspect, the present invention provides a crystalline modification I of epocholeone obtained according to the sixth aspect of the invention, having a crystalline epocholeone content of at least 98% by weight.

In an eighth aspect, the invention provides a composition comprising the crystalline modification I of epocholeone according to any one of the first to fifth and seventh aspects of the invention, and at least one auxiliary.

In a ninth aspect, the present invention provides a use of the crystalline modification I of epocholeone according to any one of the first to fifth and seventh aspects of the invention, or a composition according to the eighth aspect of the invention, for promoting plant growth.

12 17

In an embodiment of the eighth aspect of the invention, the amount of the crystalline modification I of epocholeone is less than 20% by weight of the composition, optionally less than 1% by weight of the composition, further optionally about 0.003% by weight of the composition.

The use of epocholeone as plant growth regulator is well known in the art and is used on a commercial scale. The crystalline modification I of epocholeone is also active in promoting the plant growth. As a result, the techniques of formulating and applying epocholeone known in the art with respect to amorphous epocholeone, for example as disclosed in the prior art documents discussed hereinbefore, can also be applied in an analogous manner to epocholeone in the crystalline modification I of the invention.. Accordingly, the present invention provides a plant growth regulator composition comprising epocholeone in the crystalline modification I as defined hereinbefore.

The invention additionally provides processes for preparing compositions for promoting plant growth using the crystalline modification I of epocholeone.

In an embodiment of the eighth aspect of the invention, the composition is in the form of a suspension concentrate (SC), an oil-based suspension concentrate (OD), a watersoluble granule (SG), a dispersible concentrate (DC), ab emulsifiable concentrate (EC), emulsion seed dressings, suspension seed dressings, a granule (GR), a microgranule (MG), a suspoemulsion (SE), a soluble liquid (SL) and a water-dispersible granule (WG).

Crystalline modification I of epocholeone can be included into these customary formulations in a known manner using suitable auxiliaries, carriers and solvents.

In an embodiment of the eighth aspect of the invention, the composition is in the form of a suspension concentrate (SC) and an oil-based suspension concentrate (OD).

In an embodiment of the eighth aspect of the invention, the crystalline modification

I of epocholeone may be present in a concentration of from about 0.003 to about 1% by weight of the total mixture, i.e., in amounts sufficient to achieve the required dosage. The formulations are prepared, for example, by extending the crystalline modification I of epocholeone with water, solvents and carriers, using, if appropriate, emulsifiers and/or dispersants, and/or other auxiliaries. These formulations are prepared in a known manner

12 17 by mixing the crystalline modification I of epocholeone with customary additives, for example, a carrier, an emulsifier, a dispersing agent, a wetting agent, an antifoam agent and any necessary adjuvants and other formulation ingredients.

Carriers include solid carriers and liquid carriers. Solid carriers include, but are not limited to, inorganic materials such as talc, bentonite, clay, kaolin, diatomaceous earth, white carbon, silicic anhydride, synthetic calcium carbonate, vermiculite, silica sand, mica, pumice, gypsum, calcium carbonate, dolomite, magnesium, calcium hydroxide, phosphorus lime, zeolite and ammonium sulfate; vegetable-based organic materials such as soybean powder, tobacco powder, walnut powder, wheat flour, wood flour, starch and crystalline cellulose; synthetic high polymeric substances and natural high polymeric substances such as cumarone resin, petroleum resin, alkyd resin, polyvinyl chloride, polyalkylene glycol, ketone resin, ester gum, copal gum and dammar gum; waxes such as carnauba wax and beeswax; and ureas. Liquid carriers include, but are not limited to paraffinic and naphthenic hydrocarbons such as kerosine, mineral oil, spindle oil and white oil; aromatic hydrocarbons such as toluene, xylene, ethylbenzene, cumene and methylnaphthalene; chlorohydrocarbons such as carbon tetrachloride, chloroform, trichloroethylene, monchloroethylene and o-chlorotoluene; ethers such as dioxane and tetrahydrofuran; ketones such as acetone, methyl ethyl ketone, diisobutyl ketone, cyclohexanone, acetophenone and isophorone; esters such as amyl acetate, ethylene glycol acetate, diethylene glycol acetate, dibutyl maleate and diethyl succinate; alcohols such as methanol, ethanol, isopropanol, butanol, n-hexanol, ethylene glycol, propylene glycol and diethylene glycol; ether-alcohols such as ethylene glycol phenyl ether and diethylene glycol butyl ether, and polar solvents such as dimethylformamide and dimethylsulfoxide; and water.

As the emulsifier and the dispersant, surface active agents are usually used, which include nonionic, cationic, anionic and ampholytic surface active agents. In general a nonionic surface active agent and/or an anionic surface active agent is preferably used. As specific examples of the nonionic surface active agent, there can be mentioned products prepared by polymerizing and adding ethylene oxide to higher alcohols such as lauryl io

12 17 alcohol, stearyl alcohol and oleyl alcohol; products prepared by polymerizing and adding ethylene oxide to alkylphenols such as isooctyl phenol and nonyl phenol; products prepared by polymerizing and adding ethylene oxide to alkylnaphthols such as butylnaphthol and octylnaphthol; products prepared by polymerizing and adding ethylene oxide to higher fatty acids such as palmitic acid, stearic acid and oleic acid; products prepared by polymerizing and adding ethylene oxide to monoalkyl or dialkyl phosphates such as stearyl phosphate and dilauryl phosphate; products prepared by polymerizing and adding ethylene oxide to amines such as dodecylamine and stearic amide; higher fatty acid esters of a polyhydric alcohol such as sorbitan, and products prepared by polymerizing and adding ethylene oxide to the higher fatty acid esters; products prepared by additionpolymerization of ethylene oxide with propylene oxide; and esters of a polyvalent fatty acid with an alcohol, such as dioctyl succinate. As specific examples of the anionic surface active agent, there can be mentioned alkylsulfate salts such as sodium laurylsulfate and oleylsulfate amines; alkyl-sulfonate salts such as dioctyl sodium sulfo succinate and 215 ethylhexyl sodium sulfonate; arylsulfonate salts such as sodium isopropylnaphthalenesulfonate, sodium methylenebisnaphthalenesulfonate, sodium ligninsulfonate and sodium dodecylbenzenesulfonate; and phosphate salts such as sodium tripolyphosphate. Sodium salt of naphthalene sulfonate condensate is particularly useful for the composition of the invention.

Wetting agents include, but are not limited to, alkyl sulfo succinates, laureates, alkyl sulfates, phosphate esters, acetylenic diols, ethoxyfluomated alcohols, ethoxylated silicones, alkyl phenol ethyoxylates, benzene sulfonates, alkyl-substituted benzene sulfonates, alkyl a-olefm sulfonates, naphthalene sulfonates, alkyl-substituted napthalene sulfonates, condensates of naphthalene sulfonates and alkyl-substituted naphthalene sulfonates with formaldehyde, and alcohol ethoxylates. Polyalkylene glycol ether is particularly useful for the composition of the invention.

Antifoam agents include all substances which can normally be used for this purpose in agrochemical compositions. Suitable anti-foam agents are known in the art and are available commercially. Particularly preferred antifoam agents include modified n

12 17 polydimethylsiloxane formulation, such as SAG 1529 from Momentive, modified polyether-polysiloxane, such as Breakthru® AF9902 from Evonik, mixtures of polydimethylsiloxanes and perfluroalkylphosphonic acids, such as the silicone antifoaming agents from GE or Compton.

Other formulation ingredients can also be used in the present invention, such as thickening agent, antifreeze agents, biocide, dyes, drying agents, and the like. These ingredients are known to one skilled in the art.

In an embodiment of the eighth aspect of the invention, the crystalline modification I of epocholeone according to the invention can be present in its commercially available formulations and in its use forms, prepared from these formulations, and as a mixture with other active compounds (such as insecticides, attractants, sterilizing agents, bactericides, acaricides, nematicides, fungicides, growth-regulating substances, herbicides, safeners, fertilizers and semiochemicals) or with agents for improving plant properties.

Preferred mixing partners of the “crystalline modification I” of epocholeone can be indol-3-ylacetic acid, gibberellic acid, jasmonic acid, anthranilic acid, sodium nitrophenolate, pyraclostrobin, prothioconazole, thiodicard, spinosad, abamectin, ftpronil, emamectin benzoate, diafenthiuron, pyriproxyfen, imidacloprid, thiamethoxam, azoxystrobin, cyproconazole, cymoxanil, difenoconazole, hexaconazole, metconazole, prochloraz, tebuconazole, tefluthrin.

Particular preferred mixing partners are spinosad, abamectin, ftpronil, emamectin benzoate, thiodicard, imidacloprid and their mixtures.

All plants and plant parts can be treated in accordance with the invention. In the present context, plants are to be understood as meaning all plants and plant populations such as desired and undesired wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants which can be obtained by conventional breeding and optimization methods, by biotechnological and genetic engineering methods, or by combinations of these methods, including the transgenic plants and the plant cultivars which can or cannot be protected by plant breeders' rights. Plant parts are to be understood as meaning all parts and organs of plants above and below the ground, such as shoot, leaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds, roots, tubers and rhizomes. Harvested materials, and vegetative and generative propagation materials, for example, cutting, tubers, meristem tissue, rhizomes, offsets, seeds, single and multiple plant cells and any other plant tissues, are also included.

Treatment according to the invention of the plants and plant parts with the compositions or formulations of the inventions is carried out directly or by allowing the compositions or formulations to act on their surroundings, habitat or storage space by the customary treatment methods. Examples of these customary treatment methods include dipping, spraying, vaporizing, fogging, broadcasting, painting on in the case of propagation material, and applying one or more coats particularly in the case of seed. Throughout the description and claims of this specification, the words “comprise” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to”, and do not exclude other moieties, additives, components, integers or steps. Moreover the singular encompasses the plural unless the context otherwise requires: in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Other features of the invention will become apparent from the following examples. Generally speaking the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims and drawings). Thus features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. Moreover unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

Where upper and lower limits are quoted for a property then a range of values defined by a combination of any of the upper limits with any of the lower limits may also be implied.

In this specification, references to properties are - unless stated otherwise - to properties measured under ambient conditions, i.e. at atmospheric pressure and at a temperature of about 20°C.

As used herein, the term “about,” when used in connection with a numerical 5 amount or range, means somewhat more or somewhat less than the stated numerical amount or range, to a deviation of ± 10% of the stated numerical amount or endpoint of the range.

The term “crystalline”, as used herein, refers to a solid state form wherein molecules are arranged to form a crystal lattice comprising distinguishable unit cells. In general, crystalline material may, for example, be identified by yielding diffraction peaks when subjected to X-ray radiation and/or exhibiting an endothermic melting peak profile with a characteristic sharp peak under differential scanning calorimetry (DSC).“Surrounding,” as used herein, refers to the place on which the plants are growing, the place on which the plant propagation materials of the plants are sown or the place on which the plant propagation materials of the plants will be sown.

Precipitation as used herein, refers to the sedimentation of a solid material (a precipitate), including the sedimentation of a crystalline material, from a liquid solution in which the solid material is present in amounts greater than its solubility in the amount of liquid solution.

All percentages are given in weight % unless otherwise indicated.

Embodiments of the present invention will now be described by way of the following examples which are provided for illustrative purposes only, and not intended to limit the scope of the disclosure.

Brief Description of Drawings

Various features and aspects of the embodiments of the invention disclosed herein can be more clearly understood by reference to the drawings, which are intended to exemplify and illustrate, but not to limit, the scope of the invention, and wherein:

12 17

FIG 1 is an infrared (IR) spectrum of the crystalline modification I of epocholeone.

FIG 2 is a X-ray powder diffractogram (X-RPD) of the crystalline modification I of 5 epocholeone.

FIG 3 is a X-ray powder diffractogram of amorphous epocholeone.

Detailed Description

Embodiments of the present invention will now be described by the following examples, in which the following measurement techniques have been employed, and which examples are provided for illustrative purposes only, and not intended to limit the scope of the disclosure.

All X-ray diffractograms were determined using powder diffractometer in reflection geometry at 25° C, using the following acquisition parameters:

X’Pert Pro MPD from PANalytical B.V.

Theta compensating slit and graphite monochromator Copper (K-alpha) radiation, 40 kV, 40 mA Step size: 0.03 degree 2-theta

Count time: 1.0 second

Maximum peak intensity: 1705 counts per second Scan range: 3-60 degrees 2-theta

The IR spectrum was measured with the resolution of 4 cm-1 and with the number of scans of 16 for the crystallized samples. The crystalline modification I of epocholeone can be identified by its characteristic functional group vibration peaks at wavenumbers (cm^-1, ± 0.2%) of one or more of 2962.20, 2873.55 and 1724.02 as shown in Figure 1.

All IR spectra were obtained using the following acquisition parameters:

FT-IR spectrometer	Nicolet™ iS 5
Diamond ATR unit	Thermo Scientific™ iD5 ATR
Wavelength range	550-4000 cm’¹
Resolution	4 cm^-1
Number of scans	16

12 17

Examples

Example 1: Preparation of amorphous epocholeone in accordance with the Example 1 in U.S. PATENT NO. 5,763,366

Step 1

Synthesis of 2a, 3a-dipropionyloxy-24S-ethyl-5a-cholest-22-ene-6-one (V)

To 20.5 g (49.92 mmol) of (22E, 24S)-24S-ethyl-5a-cholesta-2, 22-diene-6-one (III) [K. Mori, Agric. Biol. Chem., 44, 1211 (1980)], 7.3 g (54.42 mmol) of Nmethylmorpholine-N-oxide and 1000 ml of acetone were added and stirred. After purging the reaction container with nitrogen gas, a mixed solution of 22 ml of water and 32 ml of tbutyl alcohol containing 390 mg (1.5 mmol) of osmium tetroxide was added to react overnight at room temperature. Filtering the crystals (15.6 g) precipitating in the reaction solution, the filtrate was stirred for 1 hour by adding aqueous solution of sodium hydrogensulfite, and extracted by chloroform, washed in water, and dried on anhydrous magnesium sulfate, and the solvent was distilled away in vacuo, and crude product was obtained. This crude product was refined by silica gel column chromatography (toluene:ethyl acetate 30:1), and 3.77 g of 2a, 3a-dihydroxy-24S-ethyl-5a-cholest-22-ene6-one (IV) was obtained. Combining with the previously filtered crystals (19.37 g), 150 ml of pyridine, 76 ml of propionate anhydride, and 500 mg of 4-dimethylaminopyridine were added to react overnight at room temperature. Extracting with chloroform, washing in water, and drying on anhydrous magnesium sulfate, the solvent was distilled away in vacuo, and crude product was obtained.

This crude product was defined by silica gel column chromatography (benzene, ethyl acetate 30:1 to 15:1), and 21.06 g (75.7%) of 2a, 3a-dipropionyloxy-24S-ethyl-5acholest-22-ene-6-one (V) was obtained.

12 17

Step 2

Synthesis of (22R, 23R, 24S)-2a, 3a-dipropionyloxy-22, 23-epoxy-B-homo-7-oxa5a -stigmastan-6-one [compound (I)]

Dissolving 2.06 g (3.70 mmol) of 2a, 3a-dipropionyloxy-24S-ethyl-5a-cholest-22ene-6(V) obtained in step 1 in 100 ml of dichloromethane, 8.0 g (31.08 mmol) of m10 chloroperbenzoic acid was added, and the mixture was stirred for 15 days at room temperature. Afterwards, adding 8.0 g of powder calcium hydroxide, stirring for 1 hour and filtering, the solvent was distilled away in vacuo, and crude product was obtained. This crude product was refined by silica gel column chromatography (benzene:ethyl acetate 30:1 to 15:1), and 0.85 g (39.0%) of (22R, 23R, 24S)-2a, 3a-dipropionyloxy-22,

23-epoxy-B-homo-7-oxa-5a-stigmastan-6-one [compound (I)], that is, (lR,3aS,3bS,6aS,8S,9R, 10aR,10bS,12aS)-l-[(lS)-l-[(2R,3R)-3-[(lS)-l-ethyl-2methylpropyl]-2-oxiranyl]ethyl] hexadecahydro- 10a, 12a-dimethyl-8,9-bis(l-oxopropoxy)6H-benz[c]indeno[5,4-e]oxepin-6-one] was obtained.

Scheme 1. Synthesis of Epocholeone

12 17

As shown in Figure 3, the X-ray powder diffraction pattern of the resulting epocholeone product has no significant signals, which indicates the Epocholeone product prepared in accordance with the disclosure of 5,763,366U.S. PATENT NO. 5,763,366 is amorphous.

Example 2: Preparation of the crystalline modification I of epocholeone

Crystallization from xylene g of epocholeone sample prepared in Example 1 was taken in a 3 neck round 10 bottom flask along with 50 ml of xylene.The resulting slurry was heated to 65 °C to get a homogeneous solution. The insoluble particles, if any, were filtered and the solution was slowly cooled to 20-25°C. Upon cooling, fine crystals were formed and the resulting heterogeneous mixture was stirred at 20°C for 2h. Then, the slurry was filtered and washed with 3 ml of xylene at 20°C. The filtered crystals were dried under vacuum at 40 °C. The crystal product thus obtained was having a purity of >98% and the yield was found to be not less than 90%.

The crystals were analyzed by IR spectrometry, X-RPD and found out to bethe crystalline modification I of epocholeone as shown in Figure 1 and Figure 2. .

The IR spectrum of the crystalline modification I of epocholeone exhibited the 20 functional group characteristic vibrations peaks at wavenumbers of one or more of about at

2962.90, 2873.55 and 1724.02 cm¹ as shown in Figure 1.

The X-ray powder diffractogram of the crystals exhibited the reflexes in Figure 2 and the values are summarized in Table 1.

Table 1

Crystalline Modification I
2 6(°)	d(A)
5.025 ±0.2	17.585 ±0.05
7.019 ±0.2	12.594 ±0.05
8.814 ±0.2	10.034 ±0.05
10.470 ±0.2	8.450 ±0.05
10.675 ±0.2	8.288 ±0.05
10.832 ±0.2	8.168 ±0.05
11.285 ±0.2	7.841 ±0.05
11.517 ± 0.2	7.683 ±0.05
13.165 ±0.2	6.725± 0.05
13.625 ±0.2	6.499 ±0.05
14.543 ±0.2	6.091 ±0.05
16.253 ±0.2	5.454 ±0.05
17.011 ±0.2	5.213 ±0.05
17.356 ±0.2	5.110 ± 0.05
17.714 ±0.2	5.007 ±0.05
17.918 ±0.2	4.950 ±0.05
18.255 ±0.2	4.860 ±0.05

12 17

18.456 ±0.2	4.807 ±0.05
19.352 ±0.2	4.587 ±0.05
20.148 ±0.2	4.407 ±0.05
21.043 ±0.2	4.222 ± 0.05
21.679 ±0.2	4.100± 0.05
22.721 ±0.2	3.914 ±0.05
23.234 ±0.2	3.829 ±0.05
24.620 ± 0.2	3.616 ±0.05
25.372 ±0.2	3.511 ±0.05
28.224 ±0.2	3.162 ±0.05

Example 3: Preparation of the crystalline modification I of epocholeone

Crystallization from toluene

5 g of epocholeone sample prepared in Example 1 was taken in a 3 neck round bottom flask along with 30 ml of toluene. The resulting slurry was heated to 83 °C to get a homogeneous solution. The insoluble particles, if any, were filtered and the solution was slowly cooled to 20°C. Upon cooling, fine crystals were formed and the resulting heterogeneous mixture was stirred at 20°C for 2h. Then, the slurry was filtered, washed with 3 ml of toluene at 20°C and dried under vacuum at 45 °C. The crystal product thus obtained had a purity of >98% and the yield was found to be not less than 90%.

The crystals were characterized as being the crystalline modification I of epocholeone e using IR spectrometry and X-ray powder diffraction as described in Example 2.

Formulation examples

Example 4 - Preparation of suspension concentrate (SC) of amorphous Epocholeone

All the components list in Table 2 below were mixed uniformly and the resulting mixture was ground with a Dyno-Mill( manufactured by Willy A. Bachofen AG) to obtain a suspension concentrate.

12 17

Table 2

Content	Weight %	Function
Amorphous epocholeone, 98% (prepared in example 1)	0.0031	Active ingredient
Sodium alkylnaphthalenesulfonate,formaldehyde condensate(MORWET D-425® POWDER)	3.0	Dispersing agent
Polyalkylene glycol ether (ATLASTM G-5000)	2.0	Wetting agent
Xanthan gum(AG-RHO POL 23AV)	0.2	Thickening agent
Modified polydimethylsiloxane formulahon((SAG 1529)	0.5	Antifoaming agent
Propylene glycol	8.0	Antifreeze
l,2-Benzisothiazol-3(2H)-one (Proxel®)	0.2	biocide
Water	Balance to 100	Carrier

Example 5 - Preparation of Suspension Concentrate (SC) of epocholeone crystalline modification I

All the components list in Table 3 below were mixed uniformly and the resulting 5 mixture was ground with a Dyno-Mill( manufactured by Willy A. Bachofen AG) to obtain a suspension concentrate.

12 17

Table 3

Content	Weight %	Function
Epocholeone, crystalline modification I„ 98% (prepared in example 2)	0.0031	Active ingredient
Sodium alkylnaphthalenesulfonate,formaldehyde condensate(MORWET D-425® POWDER)	3.0	Dispersing agent
Polyalkylene glycol ether (ATLASTM G-5000)	2.0	Wetting agent
Xanthan gum(AG-RHO POL 23AV)	0.2	Thickening agent
Modified polydimethylsiloxane formulation((SAG 1529)	0.5	Antifoaming agent
Propylene glycol	8.0	Antifreeze
l,2-Benzisothiazol-3(2H)-one (Proxel®)	0.2	biocide
Water	Balance to 100	Carrier

Example 6- Preparation of oil based suspension concentrate (OD) formulation

All the components listed in Table 4 below were mixed uniformly and ground with a Dyno-Mill (manufactured by Willy A. Bachofen AG) to obtain an oil based suspension concentrate.

12 17

Table 4

Ingredients	Weights %	Function
Amorphous epocholeone, 98% (prepared in example 1)	0.0031	Active compound
Modified polyether-polysiloxane (Breakthru® AF9902 from Evonik)	0.5	Antifoaming agent
Ethoxylated castor oil (ALKAMULS OR/36)	15	Emulsifier
Sodium alkylnaphthalenesulfonate, formaldehyde condensate (MORWET D-425 POWDER)	5	Dispersing agent
Silica	2	Thickening agent
Com oil	Balance to 100%	Carrier

Example 7- Preparation of oil based suspension concentrate (OD) formulation

All the components listed in Table 5 below were mixed uniformly and ground with a 5 Dyno-Mill (manufactured by Willy A. Bachofen AG) to obtain an oil based suspension concentrate.

Table 5

Ingredients	Weights %	Function
Epocholeone, crystalline modification I, 98% (prepared in example 2)	0.0031	Active compound
Modified polyether-polysiloxane (Breakthru® AF9902 from Evonik)	0.5	Antifoaming agent
Ethoxy lated castor oil (ALKAMULS OR/36)	15	Emulsifier

Sodium alkylnaphthalenesulfonate, formaldehyde condensate (MORWET D-425 POWDER)	5	Dispersing agent
Silica	2	Thickening agent
Com oil	Balance to 100%	Carrier

12 17

Example 8: Anti-hydrolysis test

The anti-hydrolysis property of the epocholeone in these compositions was determined by aging samples in heated ovens having the same atmosphere therein, and then comparing the epocholeone content before and after aging to determine relative percentage of hydrolysis (RPH). RPH was calculated by the following equation:

(The final weiqht % of enocholenone — The initial weiqht % of enocholenone)

RPH = ------—--------, , -—-- x 100%

The initial weight % of epocholenone

Epocholeone content was determined by assaying the compositions with high10 pressure liquid chromatography (HPLC) using reverse phase columns and eluants.

Samples prepared in Examples 1, 2, 3, 4, 5, 6 and 7 were stored at 54 °C for 1 week. The procedures are followed according to CIPAC MT 46.3. The concentration of epocholeone was measured at the end of each storage time by HPLC. The results are listed in Table 6.

Table 6.

Sample	Compound	Weight (%) of epocholeone	RPH%
Example 1	Amorphous epocholeone	98	-50
Example 2	Epocholeone, crystalline	98	-2

	modification I,
Example 3	Epocholeone, crystalline modification I,	98	-3
Example 4	Amorphous epocholeone in SC	0.0031	-55
Example 5	Epocholeone, crystalline modification I in SC	0.0031	-3
Example 6	Amorphous epocholeone in OD	0.0031	-50
Example 7	Epocholeone, crystalline modification I in OD	0.0031	-3

12 17

Claims

05 12 17

5 1. A crystalline modification of epocholeone brassinolide [(lR,3aS,3bS,6aS,8S,9R, 10aR,10bS,12aS)-l-[(lS)-l-[(2R,3R)-3-[(lS)-l-ethyl-2-methylpropyl]-2-oxiranyl]ethyl] hexadecahydro-10a,12a-dimethyl-8,9-bis(l-oxopropoxy)-6H-benz[c]indeno[5,4-e]oxepin- 6-one] 10
2. A crystalline modification according to claim 1, exhibiting at least 3 of the following reflexes, in any combination, as 2Θ ± 0.20 degree in X-ray powder diffractogram (X-RPD) recorded using Cu—Ka radiation at 25°C: 20 = 5.03 + 0.2 (1) 20 = 7.02 + 0.2 (2) 20 = 8.81 + 0.2 (3) 15 20= 10.47 + 0.2 (4) 20= 10.67 + 0.2 (5) 20= 10.83 + 0.2 (6) 20= 11.28 + 0.2 (7) 20= 11.52 + 0.2 (8) 20 20= 13.17 + 0.2 (9) 20= 13.63 + 0.2 (10) 20= 14.54 + 0.2 (11) 20= 16.25 + 0.2 (12) 20= 17.01 + 0.2 (13)

05 12 17

26= 17.36± 0.2 (14) 26= 17.71 ±0.2 (15) 26= 17.92 ±0.2 (16) 26= 18.25 ±0.2 (17) 26= 18.46 ±0.2 (18) 26= 19.35 ±0.2 (19) 20 = 20.15 ±0.2 (20) 26 = 21.04 ±0.2 (21) 26 = 21.68 ±0.2 (22) 26 = 22.72 ± 0.2 (23) 26 = 23.23 ±0.2 (24) 26 = 24.62 ± 0.2 (25) 26 = 25.37 ±0.2 (26) 26 = 28.22 ± 0.2 (27)
3. The crystalline modification of epocholeone according to claim 1 or claim 2, exhibiting at least 3 of the following reflexes, in any combination, in an X-ray powder diffractogram recorded using Cu—Ka radiation at 25 °C:

26 = 5.03 ±0.2 (1)

2Θ = 7.02 ± 0.2 (2)

26= 10.47 ±0.2 (4)

26 = 10.68 ±0.2 (5)

26 = 11.29 ±0.2 (7)

26 = 11.52 ±0.2 (8)

05 12 17

26= 16.25 ±0.2 (12) 20= 17.01 ±0.2 (13) 26= 17.36 ±0.2 (14) 26= 17.71 ±0.2 (15) 26= 17.92 ±0.2 (16) 26= 18.46 ±0.2 (18) 20= 19.35 ±0.2 (19) 26 = 20.15 ±0.2 (20) 26 = 21.04 ±0.2 (21) 26 = 22.72 ± 0.2 (23) 26 = 23.23 ±0.2 (24)
4. A crystalline modification of epocholeone according to any preceding claim, exhibiting an IR spectrum with characteristic functional group vibrations peaks at

15 wavenumbers (cm-1, ± 0.2%) of one or more of about 2963, 2874 andl724 cm-1.
5. A crystalline modification of epocholeone according to any preceding claim, characterized by X-ray powder diffraction pattern as substantially shown in Figure 2, and/or characterized by an IR spectrum as substantially shown in Figure 1 and/or obtainable by the process as substantially as described in Example 2 or 3.

20
6. A crystalline modification of epocholeone, optionally according to any one of claims 1 to 5, obtainable by the process of any one of claims 7 to 12.
7. A process for the preparation of a crystalline modification of epocholeone according to any one of claims 1 to 5:

i) dissolving epocholeone in a solvent, or mixture of solvents;

25 ii) precipitating the dissolved compound into crystalline modification of

05 12 17 epocholeone; and iii) isolating the precipitated crystalline modification.
8. The process according to claim 7, where the epocholeone in step i) is amorphous epocholeone.

5
9. The process according to claim 7 or 8, wherein the solvent is selected from the group consisting of toluene, xylene, chlorobenzene, dichlorobenzene, ethyl benzene, trifluoro methyl benzene, mesitylene, nitrobenzene, methyl ethyl ketone, acetonitrile or mixture of THF-water.
10 10. The process according to any one of claims 7 to 9, wherein the solvent is xylene, toluene or a mixture thereof.
11. The process according any one of claims 7 to 10, wherein step ii) is effected by concentration of the solvent and/or by cooling and/or by the addition of a solubility reducing solvent and/or by adding a seed crystal of the crystalline modification of

15 epocholeone.
12. The process according to claim 11, wherein step ii) is effected by cooling to about 0 to 20°C.
13. A crystalline modification of epocholeone obtainable according to any one of claims 7 to 12 and having a crystalline epocholeone content of at least 98% by weight.

20
14. A composition comprising the crystalline modification of epocholeone according to any of claims 1 to 6 and 13 and at least one auxiliary.
15. The composition according to claim 14, wherein the auxiliary is selected from the group consisting of a carrier, an emulsifier, a dispersing agent, a wetting agent, an antifoam agent and any necessary adjuvants and other formulation ingredients.

25
16. The composition according to any one of claims 14 or 15, , which is in form of a suspension concentrate (SC), an oil-based suspension concentrate (OD), water-soluble granules (SG), a dispersible concentrate (DC), an emulsifiable concentrate (EC), an emulsion seed dressing, a suspension seed dressing, a granule (GR), a microgranule (MG), a suspoemulsion(SE), and a water-dispersible granule (WG).
17. The composition according to claim 16, which is in form of a suspension 5 concentrate (SC) or an oil-based suspension concentrate (OD).
18. The composition according to any of claims 14 to 17, which comprises crystalline modification of epocholeone in an amount of less than 20 % by weight.
19. The composition according to any of claims 14 to 18, which comprises crystalline modification of epocholeone in an amount of 0.003 % by weight.

10
20. Use of a crystalline modification of epocholeone according to any of claims

1 to 6 or a composition according to any of claims 14 to 19 for promoting plant growth.

05 12 17

Intellectual

Property

Office

Application No: GB1619315.3 Examiner: Dr Simon Grand