DESC:The following specification particularly describes the nature of the invention and the manner in which it is to be performed.

INTRODUCTION
Aspects of the invention include process for the preparation of Ingenol mebutate from Ingenol and intermediates thereof.
Ingenol mebutate is a protein kinase activator and an inducer of cell death. Ingenol mebutate is naturally occurring 3-angelate ester of Ingenol and can be isolated from various plant species such as Euphorbia, particularly from Euphorbia peplus and Euphorbia drummondii. Ingenol mebutate has a chemical name, (1aR,2S,5R,5aS,6S,8aS,9R,10aR)-5,5a-dihydroxy-4-(hydroxymethyl)-1,1,7,9-tetramethyl 11oxo-1a,2,5,5a,6,9,10,10a-octahydro-1H-2,8a-methanocyclopenta[a] cyclopropa[e] cyclodecen-6yl (2Z) 2 methylbut-2-enoate.” and its structure can be represented as given below.

Picato® has been approved by USFDA and EMEA as a colorless gel containing 150 mcg and 500mcg of Ingenol mebutate for the topical treatment of actinic keratosis.
Experimenta (1980), 36, 1206-1207 first discloses Ingenol mebutate and US2003195168A1 has first disclosed its isolation by extraction and purification by chromatography at example-19.
US7449492 also discloses similar process for the purification of diterpene esters from E-peplus wherein 17 kg of Euphorbia peplus plants yielded only 7 g of a thick crude oil containing various Ingenane esters and then the crude oil was subjected to chromatography to isolate PEP005 (Ingenol mebutate). It is evident that the yield of Ingenol mebutate by extraction from Euphorbia peplus and subsequent chromatography is extremely low.
Ingenol is also a natural product and may be readily extracted from seeds of Euphorbia lathyris. The process for extraction of Ingenol involves the hydrolysis of various esters of Ingenol and thus the amount of isolated Ingenol is increased making Ingenol more easily available than its angelate ester. Ingenol is also commercially available and Ingenol has previously been used as starting material for the preparation of many other Ingenol esters.
WO2012010172A1 discloses a process for the preparation of Ingenol mebutate from Ingenol comprising the steps of protecting 5 and / or 20 hydroxyl groups and esterifying the 3 hydroxyl group with angelic acid derivative such as angelic anhydride in the presence of alkali bis (trimethylsilyl) amide, followed by deprotection of 5 and / or 20 hydroxy groups to obtain Ingenol-3-angelate ester.
WO 2013110753 A1 discloses a process for the preparation of Ingenol mebutate comprising acylation of Ingenol optionally followed by de-acylation of a diacylated Ingenol derivative wherein at least one step is catalyzed by an enzyme.
WO 2014012836 A1 discloses a process for preparing Ingenol-3-angelate from 20-deoxy-ingenol derivatives.
However the processes disclosed in the prior art involves the use of costly and hazardous reagents such as bis(trimethylsilyl)amides or use of multistep synthesis and results in unwanted isomerization of angelate to tiglate with very low yields or lengthy reaction time cycles. Hence, there remains a need for an alternate process which is industrially viable, cost effective, non-hazardous to practice and avoids the formation of undesired products.

SUMMARY
Aspects of the invention include process for the preparation of Ingenol mebutate from Ingenol and intermediates thereof.
In an aspect, the application provides a process for the preparation of Ingenol mebutate, comprising the steps of:
a) protecting 5, 20 hydroxyl groups of Ingenol with a suitable protecting agent;

b) treating 5, 20 hydroxyl protected Ingenol with angelic acid or its activated derivative in the presence of a base, wherein the base is selected from the group consisting of metal hydrides such as sodium hydride; alkali metal alkoxides such as sodium methoxide, potassium tert. butoxide; organo metal bases such as n-butyl lithium, lithium tetramethyl piperidide and organo metal halides such as tert. butyl magnesium chloride, isopropyl magnesium bromide, cyclopropyl magnesium bromide.

c) deprotecting the 5, 20 hydroxyl protected Ingenol-3- angelate to obtain Ingenol-3 angelate of formula (I);

d) optionally, purifying Ingenol-3-angelate in a suitable solvent,
wherein R1& R2 are same or different selected from the group consisting of hydrogen, methyl, ethyl, isopropyl, isobutyl, tert. butyl, phenyl, benzyl or both R1& R2 together form a carbonyl group along with carbon to which they are linked.
In another aspect, the application provides a process for the purification of Ingenol-3 angelate, comprising the steps of:
a) dissolving Ingenol-3- angelate in a suitable solvent;
b) contacting the reaction mixture of step a) with an anti-solvent;
c) recovering Ingenol-3-angelate.
In another aspect, the present application provides the process for the preparation of amorphous form of Ingenol mebutate comprising the steps of
a) providing the solution of Ingenol mebutate in a suitable solvent
b) recovering amorphous Ingenol mebutate.
In another aspect, the application provides the process for the purification of Ingenol-3-angelate comprising the steps of
a) providing Ingenol-3-angelate
b) passing through a chromatography system.
c) recovering pure Ingenol-3-angelate.
In another aspect, the application provides the process for the crystalline Ingenol mebutate comprising the steps of
a) providing a solution of Ingenol mebutate in isopropyl acetate
b) evaporating the solvent to obtain crystalline Ingenol mebutate.
In yet another aspect, the present application provides pharmaceutical compositions comprising Ingenol mebutate obtained according to the processes of any of the aspects of present application and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION
Aspects of the invention include process for the preparation of Ingenol mebutate from Ingenol and intermediates thereof.
In an aspect, the application provides a process for the preparation of Ingenol mebutate, comprising the steps of:
a) protecting 5, 20 hydroxyl groups of Ingenol with a suitable protecting agent

b) treating 5, 20 hydroxyl protected Ingenol with angelic acid or its activated derivative in the presence of a base, wherein the base is selected from the group consisting of metal hydrides such as sodium hydride; alkali metal alkoxides such as sodium methoxide, potassium tert. butoxide; organo metal bases such as n-butyl lithium, lithium tetramethyl piperidide and organo metal halides such as tert. butyl magnesium chloride, isopropyl magnesium bromide, cyclopropyl magnesium bromide.

c) deprotecting the 5, 20 hydroxyl protected Ingenol-3- angelate to obtain Ingenol-3 angelate of formula (I)

d) optionally, purifying Ingenol-3-angelate in a suitable solvent,
wherein R1& R2 are same or different selected from the group consisting of hydrogen, methyl, ethyl, isopropyl, isobutyl, tert. butyl, phenyl, benzyl or both R1& R2 together form a carbonyl group along with carbon to which they are linked.

The individual steps of this aspect are described herein below.
Ingenol may be extracted / isolated from the natural source such as plant species according to the procedures known in art or may be prepared synthetically according to the methods known in art. Ingenol may be purified by suitable techniques known in art such as column chromatography, fractional distillation and the like methods know in the art.
In an embodiment, Ingenol may be extracted from Euphorbia species. Euphorbia species may include, but not limited to E. lathyris, E. acrurensis, E. antiquorum, E. biglandulosa, E. carta riensis, E. cooperi, E. cotini folia, E. deightonii, E. desmondi, E. drupifera, E. ebracteolata, E. esula, E. helioscopia, E. hermentiana, E. iberica, E. ingens, E. jolkini, E. kamerunica, E. kansui, E. leuconeura, E. matabelensis, E. megalantha, E. millii, E. myrsinites, E. nematocypha, E. nubica, E. palustris, E. peplus, E. petiolata, E. pilosa, E. quadrialata, E. quinquecostata, E. resin if era, E. royleana, E. seguieriana, E. serrata, E. sieboldiana, E. tirucalli, E. triangularis, E. trigone.
In preferred embodiment, Ingenol may be extracted from seeds of E. Lathryis according to any of the methods known in the art.
Step a) of the aspect may be carried out by protecting 5, 20 hydroxyl groups of Ingenol to obtain 5, 20 hydroxyl protected Ingenol.

wherein R1&R2 are same or different selected from the group consisting of hydrogen, methyl, ethyl, isopropyl, isobutyl, tert. butyl, phenyl, benzyl or both R1& R2 together form a carbonyl group along with carbon to which they are linked.
In embodiments, step a) may be carried out by reacting Ingenol with suitable protecting agent. Suitable protecting agent is a reagent which under suitable reaction conditions reacts with a hydroxyl group to form a hydroxyl protective group and suitable hydroxyl protective group may include, but not limited to an ether, acetal, ketal, silylether, or a sulfenate derived hydroxyl protective group, preferably a ketal protecting group.
In preferred embodiments, suitable protective group may be ketal such as isopropylidene ketal (acetonide), cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzophenone ketal, 1-tert- butylethylidene ketal or 1-phenylethylidene ketal, 3-pentylidene ketal, 2,4- dimethyl-3-pentylidene ketal, 2,6-dimethyl-4-heptylidene ketal, 3,3-dimethyl-2- butylidene ketal or the like.
In an embodiment, step a) of the present aspect may be carried out by reacting Ingenol with suitable ketone. Suitable ketone includes, but not limited to acetone, cyclopentanone, cyclohexanone, cycloheptanone, benzophenone, ethyl tert. butyl ketone, phenylethyl ketone, 3-pentanone, 2,4- dimethyl-3-pentanone, 2,6-dimethyl-4-heptanone, 3,3-dimethyl-2-butanone or the like. In preferred embodiments, Ingenol may be protected with isopropylidene ketal (acetonide) by reacting Ingenol with acetone or 2,2 - dimethoxy propane to obtain Ingenol-5,20-acetonide.
Step a) may be carried out optionally in the presence of a suitable inert solvent or may also be carried out in the presence of a protecting agent alone such as acetone or 2,2 - dimethoxy propane.
Preferably, step a) may be carried out under acidic conditions. In an embodiment, step a) may be carried out in the presence of an acid catalyst such as sulphonic acid derivatives or salts thereof. In preferred embodiments, the acid catalyst may include, but not limited to methane sulfonic acid, p-toluene sulphonic acid or their salts such as pyridinium sulphonic acid.
Step a) of the present aspect may be carried out at a suitable temperature in the range of about 0ºC to boiling point of the solvent and reaction may be carried out for sufficient time to complete the protection of hydroxyl groups.
The 5, 20 hydroxyl protected Ingenol obtained in step a) may be isolated by performing suitable work-up procedure known in art or methods described or exemplified in the present application.
Purification of the 5, 20 hydroxyl protected Ingenol obtained by the present step (a) may be effected, if desired, by any suitable procedures known in art. Alternatively, the resulted product may be used as such for the next reaction.
Step (b) of the present aspect may be carried out by treating 5, 20 hydroxyl protected Ingenol with angelic acid or its activated derivative in the presence of a base, wherein the base is selected from the group consisting of metal hydrides such as sodium hydride; alkali metal alkoxides such as sodium methoxide, potassium tert. butoxide; organo metal bases such as n-butyl lithium, lithium tetramethyl piperidide and organo metal halides such as tert. butyl magnesium chloride, isopropyl magnesium bromide, cyclopropyl magnesium bromide..

The commercially available Angelic acid may be used for the said reaction or may be prepared according to any of the processes known in the art. Activated derivatives such as its corresponding acid halides, angelate esters and anhydrides may be prepared according to the procedure known in the art or methods described or exemplified in the present application. Angelic acid or its activated derivatives may be purified by suitable methods before using in step b) of the present aspect.
Activated derivatives are the derivatives of angelic acid which under the chosen reaction conditions will react more readily than the corresponding acid with an Ingenol to form an angelate ester which include, but not limited to angeloyl halides such as angeloyl chloride, angelic anhydride, mixed anhydride such as angeloyl 2,4,6-trichlorobenzoyl anhydride or angeloyl 4- nitrobenzoyl anhydride.
Anhydrides of angelic acid such as angeloyl 2,4,6-trichlorobenzoyl anhydride or the like, that may be used in step b) of this aspect may be prepared according to the methods disclosed in the literature or procedures described in the instant application. In an embodiments, such anhydrides may be prepared by reacting angelic acid with suitable acid chlorides such 2,4,6-trichlorobenzoyl chloride in the presence of suitable catalyst like organic or inorganic base such as triethylamine, diisopropyl ethyl amine, diisopropyl amine, alkali metal hydroxides, alkali metal carbonate or bicarbonates under suitable reaction conditions.
In an embodiment, the reaction may be carried out by reacting 5, 20 hydroxyl protected Ingenol with anhydrides of angelic acid such as angelic anhydride, angeloyl 4- nitrobenzoyl anhydride or angeloyl 2,4,6-trichlorobenzoyl anhydride. Preferably, the reaction may be carried out by reacting 5, 20 hydroxyl protected Ingenol with angeloyl 2,4,6-trichlorobenzoyl anhydride.
In an embodiment, the reaction may be carried out either by preparing acid anhydride and then reacting it insitu with 5, 20 hydroxyl protected Ingenol without isolation or the acid anhydride formed may be isolated in its pure form and then reacting it with 5, 20 hydroxyl protected Ingenol. Preferably, the reaction may be carried out by reacting isolated acid anhydride in its pure form with 5, 20 hydroxyl protected Ingenol.
Acid anhydrides may be prepared according to any of the methods known in the art or according to the procedures described or exemplified in the present application.
Reaction between 5, 20 hydroxyl protected Ingenol and acid anhydride may be carried out in the presence of a suitable base. Suitable base may include, but not limited to metal hydrides such as sodium hydride; alkali metal alkoxides such as sodium methoxide, potassium tert. butoxide; organo metal bases such as n-butyl lithium, lithium tetramethyl piperidide; organo metal halides such as phenyl magnesium bromide, tert. butyl magnesium chloride, cyclopropyl magnesium bromide, isopropyl magnesium bromide; alkali metal carbonates such as sodium bicarbonate, cesium carbonate and the like.
Reaction between 5, 20 hydroxyl protected Ingenol and the acid anhydride may be carried out for sufficient time to complete the esterification of 3 hydroxyl group of Ingenol, preferably for atleast 30 minutes or more.
Reaction between the 5, 20 hydroxyl protected Ingenol and the acid anhydride may be carried out at a sutiable temperature for the esterification of 3 hydroxyl group of Ingenol, at about -40 °C to boiling temperature of the reaction solvent. In preferred embodiments the reaction may be carried out at about -10 to 10 °C.
In an embodiment, the reaction between 5, 20 hydroxyl protected Ingenol and acid anhydride may be carried out in the presence of a suitable inert solvent. Suitable inert solvent may be an ether solvent such as tetrahydrofuran; hydrocarbon solvent such as toluene; nitrile solvent such as acetonitrile; halogenated hydrocarbon such dichloromethane and the like.
The 5, 20 hydroxyl protected Ingenol -3 angelate obtained in step b) may be isolated by performing suitable work-up procedure known in the art or methods described or exemplified in the present application.
Purification of the 5, 20 hydroxyl protected Ingenol-3-angelate prepared by the process of step (b) may be effected, if desired, by any suitable procedure. Alternatively, the product may be used as such for the next reaction.
Step c) of the present aspect may be carried out by deprotecting the 5, 20 hydroxyl groups of its corresponding 3-angelate ester to obtain Ingenol-3 angelate of formula (I).

Deprotection of 5, 20 hydroxyl groups of Ingenol may be carried out by selective removal of the protective groups from 5 and 20 hydroxyl groups of Ingenol by methods known in the art or according to the procedures described or exemplified in the present application.
Deprotection may be carried out by treating 5, 20 hydroxyl protected Ingenol -3-angelate with suitable deprotecting agent. Suitable deprotecting agents may include but not limited to in the presence of a suitable acid such as hydrochloric acid, perchloric acid, acetic acid, trifluoroacetic acid, p-toluene sulfonic acid, methane sulfonic acid, phosphoric acid, formic acid or the like.
In an alternate method, deprotection may also be carried out by treating 5, 20 hydroxyl protected Ingenol -3-angelate with an enzyme, optionally in the presence of a suitable enzyme catalyst.
In an embodiment, Ingenol-3-angelate may be prepared by cleaving the ketal moiety such as isopropylidene ketal in the presence of a suitable acid under suitable reaction conditions.
Deprotection of 5, 20 hydroxyl protected Ingenol -3-angelate may be carried out in the presence of a sutiable solvent. Suitable solvent may include, but not limited to alcohol solvent such as methanol, isopropanol; ether solvents such tetrahydrofuran, dioxane, ethyl ether; nitrile solvent such as acetonitrile; water or mixtures thereof. Preferably, deprotection may be carried out in an alcohol solvent.
Deprotection of 5, 20 hydroxy protected Ingenol -3-angelate may be carried out by treating with suitable deprotecting agent for sufficient time to complete deprotection of 5, 20 hydroxyl groups, for example about 4 hours or more.
Deprotection of 5, 20 hydroxyl protected Ingenol-3-angelate may be carried out by treating with suitable deprotecting agent at suitable temperature for the deprotection of 5, 20 hydroxyl groups, preferably at about -25 °C to boiling point of the reaction solvent. Most preferably, deprotection may be carried out at -10 to 10 °C.
The Ingenol -3 angelate obtained in step c) may be isolated by performing suitable work-up procedure known in the art or methods described or exemplified in the present application.
Step d) of the present application may be carried out by re-crystallizing Ingenol-3-angelate prepared in step (c). Ingenol -3-angelate may be re-crystallized by any suitable methods known in the art or according to the procedure described or exemplified in the present application.
In an embodiment, Ingenol-3-angelate may be re-crystallized by any of the suitable the techniques which include but not limited to cooling the reaction mass, removal of solvent, combining with an anti-solvent, etc., or any combination of techniques thereof.
Re-crystallization by cooling crystallization which includes, but not limited to: crystallization by controlled cooling or crash cooling of the reaction mass and methods similar thereof.
Re-crystallization by solvent removal includes, but not limited to: solvent evaporation under atmospheric pressure or under reduced pressure / vacuum, spray drying, freeze drying and the like.
Re-crystallization by combining reaction mass with an anti-solvent wherein anti-solvent is a solvent in which Ingenol mebutate has low solubility. Anti-solvents include, but not limited to: C2-C6 aliphatic or cyclic ethers; C5-C8 aliphatic or aromatic hydrocarbons; water or mixtures thereof.
Ingenol-3-angelate obtained in step c) or step d) of this aspect may be in amorphous form or crystalline form.
In another aspect, the application provides a process for the purification of Ingenol-3 angelate, comprising the steps of:
a) dissolving Ingenol-3- angelate in a suitable solvent.
b) contacting the reaction mixture of step a) with an anti solvent
c) recovering Ingenol-3-angelate.
Ingenol-3 angelate of step (a) may be prepared according to any of the methods known in the literature or according to the procedures described or exemplified in the present application.
Ingenol-3 angelate of step (a) may be in crystalline or amorphous form or mixture thereof.
Step (a) of this aspect may be carried out by dissolving Ingenol-3-angelate in a suitable inert solvent.
Inert solvent may include, but not limited to alcohols such as methanol, 1-propanol, 1-butanol , 2 butanol, 1-pentanol, 2-pentanol, 3-pentanol; ketones such as diethyl ketone, methyl ethyl ketone; esters such as ethyl acetate, isopropyl acetate; ether such as tetrahydrofuran; halogenated hydrocarbons such as dichloromethane; amides such as dimethyl formamide; sulfoxides such as dimethyl sulfoxide; or mixture thereof. Preferably, suitable solvents may be 1-propanol, dimethyl formamide, Isopropyl acetate and dimethyl sulfoxide.
Ingenol-3-angelate of step (a) may be dissolved at suitable temperature of about 0 °C and above. Preferably, at about 25 °C and above.
Reaction mixture of step a) may be filtered to make particle free solution. Optionally, the obtained solution may be treated with a decolorizing agent, such as carbon, before making the reaction mixture as particle free.
Step b) of this aspect may be carried out by contacting with a suitable anti-solvent. Anti-solvent is a solvent in which Ingenol-3-angelate has minimum solubility.
Suitable anti-solvent includes, but not limited to water; hydrocarbons like n-pentane, n-hexane, cyclohexane, methyl cyclohexane; ethers like diethyl ether, di isopropyl ether; or mixtures thereof. Preferably, the suitable anti-solvent may be water.
Anti-solvent of step (b) may be contacted with the reaction mixture either by adding anti-solvent to the reaction mixture of step (a) or by adding the reaction mixture of step (a) to the anti-solvent.
Anti-solvent of step (b) may be contacted with reaction mixture of step (a) for sufficient time for the crystallization of Ingenol-3-angelate. In an embodiment, the anti-solvent of step (b) may be contacted with reaction mixture of step (a) either in one portion or multiple small portions or by drop wise addition.
Anti-solvent of step (b) may be contacted with reaction mixture at suitable temperature of about 0 °C and above. Preferably, at about 25 °C and above.
Anti-solvent may be contacted in sufficient quantity to crystallize Ingenol-3-angelate from the reaction mixture. In an embodiment, the ratio of quantity of anti-solvent to the quantity of solution containing Ingenol-3-angelate in a suitable solvent may be atleast about 1:1 to about 1: 50.
The reaction mixture of step (b) may be stirred for sufficient time to complete the crystallization of Ingenol-3-angelate at a suitable temperature. In an embodiment, the reaction mixture of step (b) may be stirred for about 0.5 hour or more. Preferably, for about 2 hours or more. Reaction mixture of step (b) may be stirred at suitable temperature of about 0 °C and above. Preferably, at about 25 °C and above.
In an embodiment, the reaction mixture of step (b) may be optionally cooled to temperatures which are relatively lower than initial temperature.
Alternatively, crystallization of Ingenol-3-angelate may be induced by cooling the reaction mixture of step a) to suitable temperatures to effect isolation of Ingenol-3-angelate. In an embodiment, reaction mixture of step a) may be cooled after the addition of seed crystals to the reaction mixture at suitable temperature.
Step c) of the instant aspect may be carried out by recovering Ingenol-3-angelate from the reaction mixture of step b). Ingenol-3-angelate may be recovered by the methods known in art such as filtration, evaporation, centrifugation, sublimation or any combination methods thereof or according to the procedures described or exemplified in the present application. Here the terms “recovering” or “isolating” are synonymous.
In an embodiment, Ingenol-3-angelate may be recovered by filtration of the reaction mixture of step (b) or step a). Filtration may be carried out at suitable temperature of about 0 °C and above.
In an embodiment, Ingenol-3-angelate may be recovered by evaporating the solvent from the reaction mixture of step b) or step a). Evaporation of the solvent may be effected at a suitable temperature, optionally under reduced pressure, spray drying, freez drying, flash drying, solvent evaporation in an agitated thin film drier or the like. Suitable temperature may be at about 0 °C to boiling point of the solvent used.
In an embodiment, Ingenol-3-angelate obtained in step (c) may be dried under suitable drying conditions. Drying may be carried out preferably at about 25 °C to boiling temperature of solvent. Drying may be carried out preferably until constant weight is obtained and for time sufficient to completely remove the traces of solvent. Drying may be carried out in Vacuum drier, Air drier, Freeze drier or any other drying equipment known in the art.
In an embodiment, Ingenol mebutate obtained by the process of the present aspect may be either in crystalline form or amorphous form.
In another aspect, the present application provides Ingenol-3-angelate in amorphous form or crystalline form. Amorphous form of Ingenol mebutate may be obtained by any suitable methods known in the art such as evaporative precipitation, anti-solvent induced precipitation, sublimative precipitation and the like or according to the procedures described or exemplified in the present application.
In another aspect, the present application provides the process for the preparation of Amorphous form of Ingenol mebutate comprising the steps of
a) providing the solution of Ingenol mebutate in a suitable solvent and
b) recovering amorphous Ingenol mebutate.
Step a) of this aspect may be carried out by combining Ingenol mebutate with an inert solvent optionally by heating the components at suitable temperature to form a clear solution.
Ingenol mebutate used in step a) of the present aspect may be either in crystalline or amorphous form, and may exist as an anhydrous state, a hydrate or a solvate.
In an embodiment, solution of Ingenol mebutate may be provided by directly taking the reaction mixture containing Ingenol mebutate without its isolation. Reaction mixture may be obtained from any of the synthetic procedures or natural extraction procedures known in art for Ingenol mebutate or the processes of any of the aspects of present application.
Inert solvents may include but not limited to alcohols such as methanol, ethanol, 2-propanol, 1-propanol, benzyl alcohol; C1-C6 ketones such as acetone, methyl ethyl ketone; esters such as ethyl acetate, isopropyl acetate; C1-C4 halogenated hydrocarbons such as dichloromethane; amides such as formamide, N, N-dimethyl formamide, N, N-dimethyl acetamide; sulphoxide such as dimethyl sulphoxide; C1-C6 ethers such as diethyl ether, di isopropyl ether; hydrocarbons such as hexane, heptane, benzene, toluene, Xylene or mixtures thereof.
Step a) of this aspect may be carried out at suitable temperature from about 0 °C to boiling point of the solvent used.
Step b) of this aspect may be carried out by suitable methods known in the art such as removal of solvent, cooling the reaction mass, combining with an anti-solvent, etc., or combination of techniques thereof or procedures described or exemplified in the present application.
In an embodiment, recovering amorphous form of Ingenol mebutate may be carried out by removal of solvent employing suitable techniques know in the art such as solvent evaporation in rotavapour under atmospheric pressure or reduced pressure, spray drying, freez drying, flash drying, solvent evaporation in an agitated thin film drier or the like. Here the terms “recovering” or “isolating” are synonymous.
In an embodiment, removal of solvent may be carried out at suitable temperature at about 0 °C to boiling point of the solvent used.
In preferred embodiments, the removal of solvent may be carried out under reduced pressure at a temperature of about less than 40 °C.
Ingenol mebutate obtained according to any of the aspects of present application may be further purified by any of the suitable chromatographic purification methods known in the art which include Gas chromatography, Liquid chromatography, Column chromatography, Planar chromatography, Affinity chromatography such as Supercritical fluid chromatography, Ion exchange chromatography, Size-exclusion chromatography, Expanded Bed Adsorption (EBA) Chromatographic Separation, Reversed-phase chromatography, Two-dimensional chromatography, Simulated moving-bed chromatography, Countercurrent chromatography or the like.

In another aspect, the application provides the process for the purification of Ingenol-3-angelate comprising the steps of
d) providing Ingenol-3-angelate
e) passing through a chromatography system.
f) recovering pure Ingenol-3-angelate.
In an embodiment, crude Ingenol-3-angelate of step a) may be obtained by any of the procedures described in any of aspects of instant application or by the methods known in art.
Ingenol-3-angelate may be taken directly from the synthetic reaction mass or may be isolated from the natural source before use. In an embodiment, Ingenol-3-angelate may be combined with a suitable solvent. In an embodiment, Ingenol-3-angelate may be dissolved in a suitable solvent. Suitable solvent may include but not limited to alcohols such as methanol, ethanol, 2-propanol, 1-propanol, benzyl alcohol; C1-C6 ketones such as acetone, methyl ethyl ketone; esters such as ethyl acetate, isopropyl acetate; amides such as formamide, N, N-dimethyl formamide, N, N-dimethyl acetamide; sulphoxide such as dimethyl sulphoxide; nitriles such as acetonitrile, propionitrile; C1-C6 ethers such as diethyl ether, di isopropyl ether; hydrocarbons such as hexane, heptane, benzene, toluene, Xylene, water or mixtures thereof.
Step b) involves passing Ingenol-3-angelate of step a) through a suitable chromatography system. Suitable chromatography system is the system which is designed to separate strongly bound impurities from Ingenol-3-angelate.
In an embodiment, the chromatography system may be a preparative chromatography system, flash chromatography system or a system with integrated flash and preparative LC capabilities.
In an embodiment, the chromatography system may be in flash mode. The maximum pressure of 200 psig may be used based on desirable flow rate with suitable gradient such as linear, isocratic, step or combination thereof.
In an embodiment, the chromatography system may be in compact preparative mode. The maximum pressure of 1700 psig may be used with suitable gradient such as linear, isocratic, step or combination thereof.
In preferred embodiment, the chromatography system may be in compact preparative chromatography system having E LSD, UV detection along with injector and fractionation operable in the 0-120 bar pressure range, flow range of 0 – 200 ml/min.
In an embodiment, a preparative chromatography system is packed or pre-packed with 5 to 30 um Silica, C-Aryl Silica, C-4 Silica, C-8 Silica and C-18 Silica columns that can be used are available with vendors such as Puritas, Diaso and Phenomenex
In an embodiment, a preparative chromatography system can be operated with 5, 10 um C18 silica with linear gradient program at 1.5 to 15 cm/min linear velocity and 1- 25 ml injector loop.
In an embodiment, a preparative chromatography system can be operated alternatively with following columns and linear velocities.
Column size Linear Velocity (cm/min.)
4.6 X 250, 4.6 X 150 4.21 to 12.04
10 X 250 4.00 to 7.6
21.2 X 250 1.98 to 5.5
50 X 250 2.3 to 2.80

In an embodiment, a mobile Phase for the chromatography system that can be employed may be water (A) and Acetonitrile (B).
In an embodiment, the chromatographic gradient may be represented as following
Time (minutes) %Mobile Phase A %Mobile Phase B
0 50 50
55 30 70
57 2 98
70 2 98

In an embodiment, a preparative chromatography system with the linear gradient program having end portion of 5:95 ratio of A: B may be used to separate strongly bound impurities from Ingenol-3-angelate.
In an embodiment, sample may be injected on the equilibrated column of chromatography system to achieve desired impurity separations.
Fractions collected may be analyzed for the fraction containing pure Ingenol-3-angelate. All the fractions containing product meeting desired purity may be pooled and the pure Ingenol-3-angelate can be isolated with suitable downstream operations known to person skilled in art or according to the procedures described in the present application.
Pure Ingenol-3-angelate can be isolated from the fractions according to methods known in art such as evaporation under atmospheric pressure or reduced pressure such as spray drying, agitated thin film drying, Buchi rotavapour evaporation; sublimation techniques such as freez drying; precipitation by cooling; anti-solvent addition or the like. Alternatively, pure Ingenol-3-angelate may be isolated according to procedures described in the instant application.
Ingenol-3-angelate obtained according to the process of present aspect may be having purity of greater than 99.9 % and single maximum impurity of less than 0.05 % as measured by HPLC.
Ingenol-3-angelate obtained according to the aspects of present application may be in either crystalline or amorphous state.
In another aspect, the application provides the process for the crystalline Ingenol mebutate comprising the steps of
a) providing a solution of Ingenol mebutate in isopropyl acetate.
b) evaporating the solvent to obtain crystalline Ingenol mebutate.
In an embodiment, Ingenol mebutate of step a) may be obtained by any of the procedures described in any of aspects of instant application or by the methods known in art. Ingenol mebutate of step a) may be in either crystalline or amorphous form and may exist as an anhydrous state, a hydrate or a solvate.
Step a) of this aspect may be carried out by combining Ingenol mebutate with isopropyl acetate at suitable temperature. In an embodiment, the mixture of Ingenol mebutate and isopropyl acetate may be heated to obtain the clear solution.
Step a) of this aspect may be carried out at suitable temperature from about 0 °C to boiling point of the isopropyl acetate.
In an embodiment, solution of Ingenol mebutate of step a) may be provided by directly taking the reaction mixture containing Ingenol mebutate in isopropyl acetate without its isolation. Reaction mixture may be obtained from any of the synthetic procedures or natural extraction procedures known in art or the processes of any of the aspects of present application.
In embodiments, the solution of step a) may be filtered to make particle free solution. Optionally, the obtained solution may be treated with a decolorizing agent, such as carbon, before making the reaction mixture as particle free.
Step b) of this aspect may be carried out by methods known in art such as evaporation under atmospheric pressure or reduced pressure such as spray drying, agitated thin film drying, Buchi rotavapour evaporation; freez drying, flash drying or any of the procedures described in the present application.
In an embodiment, removal of solvent may be carried out at suitable temperature at about 0 °C to boiling point of the isopropyl acetate.
In an embodiment, Ingenol-3-angelate obtained in step (b) may be dried under suitable drying conditions. Drying may be carried out preferably at about 25 °C to boiling temperature of solvent. Drying may be carried out preferably until constant weight is obtained and for time sufficient to completely remove the traces of solvent. Drying may be carried out in vacuum drier, air drier, freeze drier or any other suitable drying equipment known in the art.
Ingenol-3-angelate obtained according to the aspects of present application may be analyzed by any of the suitable analytical methods known in the art.
In a preferred embodiment, Ingenol-3-angelate may be analyzed by a HPLC method with chromatographic conditions as described below.
Column : X-Bridge- C 8 150x4.6 mm, 3.5µ.
Flow : 1.0 mL/min
Column oven temp : 15°C
Detection : UV 210nm
Load : 10µL
Diluent : ACN : Buffer (65:35)
Elution : Gradient
Runtime : 60 min
Buffer Preparation : 1.36 g of KH2PO4 buffer and 1.0ml of ortho phosphoric acid was taken in 1000ml of milli Q water. This was filtered through 0.45µ filter.

Mobile phase-A Preparation: Buffer: ACN (85:15) %V/V
Mobile phase-B Preparation: ACN: MeOH (85:15) %V/V
Gradient:
Time (Min) % Mobile phase-A % Mobile phase-B
0 60 40
25 35 65
38 20 80
53 15 85
54 60 40
60 60 40

In another aspect, the present application provides Ingenol-3-angelate and a pharmaceutical composition thereof with a purity of greater than about 99.5 %, preferably with purity greater than about 99.9 % as determined by using high performance liquid chromatography (HPLC).
In another aspect, the present application provides Ingenol-3-angelate and its pharmaceutical composition containing single maximum impurity less than about 0.1 %, preferably containing less than about 0.05 % as determined by using high performance liquid chromatography (HPLC).
In another aspect, the present application provides, Ingenol-3-angelate obtained according to the processes of the present application may be milled or micronized by any of the processes known in the art, such as ball milling, jet milling, wet milling and the like, to produce desired particle sizes and particle size distributions.
In another aspect, the present application provides pharmaceutical compositions containing a therapeutically effective amount of Ingenol-3-angelate obtained according to the processes of present application together with one or more pharmaceutically acceptable excipients.
Certain specific aspects and embodiments of the present application will be explained in greater detail with reference to the following examples, which are provided only for purposes of illustration and should not be construed as limiting the scope of the application in any manner. Variations of the described procedures, as will be apparent to those skilled in the art, are intended to be within the scope of the present application.
EXAMPLES
Example-1: Preparation of 5, 20 - acetonide Ingenol

To a mixture of Ingenol (5.0 g) and acetone (750 mL), Pyridinium p-toluene sulphonic acid (10 g) was added at 28 °C and stirred for 2 hours at the same temperature. Pyridinium p-toluene sulphonic acid (5 g) was added to the reaction mixture at 28 °C and stirred for another 2 hours at same temperature. 10% sodium bicarbonate solution (100 ml) was added to the reaction mixture and distilled the reaction mass at 35 °C. Ethyl acetate (50 mL) was added to the reaction mixture and separated the organic layer from aqueous layer and the aqueous layer was extracted with ethyl acetate (10 mL). Combined organic layer was washed with 10% sodium bicarbonate solution (50 ml) and 10% sodium chloride solution (50 ml). The organic layer was distilled at 30 °C to obtain crude compound. Ethyl acetate (20 mL) was added to the crude compound at 29 °C and stirred for 10 min at same temperature. n-hexane (40 mL) was added to the reaction mixture at 29 °C and stirred for 2 hours at the same temperature. Filtered the solid and washed with n-hexane (10 mL). Solid was dried under vacuum at 31 °C for 4 hours to obtain the title compound. Yield: 3.8 g (68.2%); Purity by HPLC: 99.11%
Example-2: Preparation of 5, 20 - acetonide Ingenol-3 angelate

A mixture of angelic acid (1.0 g) and dichloromethane (15 mL) was cooled to 2 °C and diisopropyl ethyl amine (1.54 g) was added at 2 °C in 5 minutes. 2,4,6-trichlorobenzoyl chloride was added to the reaction mixture at 5 °C in 15 minutes and stirred at the same temperature for 2 hours to complete the reaction. 10% sodium bicarbonate solution (15 mL) was added to the reaction mixture and separated the organic layer. Aqueous layer was extracted with dichloromethane (10 mL) and the combined organic layer was washed with 10% sodium chloride solution (15 mL). The organic layer was distilled at 32 °C to obtain crude compound. Crude compound was taken in tetrahydrofuran (24 mL) and 5, 20 - acetonide Ingenol (3 g) was added at 28 °C. Cooled the reaction mixture to 4 °C and sodium hydride (0.27 g) was added. The reaction mixture was stirred for 1.5 hours at the same temperature to complete the reaction. Water (3 mL) and dichloromethane (3 mL) was added at 5 °C. Raised the temperature of the reaction mixture to 28 °C and separated the organic layer. Aqueous layer was extracted with dichloromethane (6 mL). Combined organic layer was washed with 10% sodium chloride solution (15 mL) and distilled the organic layer at 35 °C to obtain crude compound. Crude compound was combined with methanol (18 mL) and cooled to 2 °C. The reaction mixture was stirred for 2 hours at the same temperature. Solid was filtered and washed with methanol (6 mL). Solid was dried under vacuum at 29 °C for 3 hours to obtain the title compound. Yield: 2.4 g (67.6%); Purity by HPLC: 99.72%
Example-3: Preparation of 5, 20 - acetonide Ingenol-3 angelate

To a mixture of 5, 20 - acetonide Ingenol (100 mg) and tetrahydrofuran (2 mL), 2,4,6-trichlorobenzoyl angeloyl anhydride (0.094 g) was added at 27 °C and cooled the reaction mixture to 4 °C. Potassium ter. butoxide (0.042 g) was added and stirred at same temperature for 1.5 hours to complete the reaction. 10% sodium bicarbonate solution (1 mL) and dichloromethane (1 mL) were added to the reaction mixture and separated the organic layer and extracted the aqueous layer with dichloromethane (1 mL). Combined organic layer was washed with 10% sodium chloride solution (1 mL) and distilled the organic layer at 37 °C to obtain title compound. Yield: 110 mg; Purity by HPLC: 92.30%
Example-4: Preparation of 5, 20 - acetonide Ingenol-3 angelate

To a mixture of 5, 20 - acetonide Ingenol (100 mg) and tetrahydrofuran (1 mL), 2,4,6-trichlorobenzoyl angeloyl anhydride (0.094 g) was added at 28 °C and cooled the reaction mixture to 4 °C. LiHMDS (0.38 mL) was added and stirred at same temperature for 2 hours. 10% sodium bicarbonate solution (1 mL) and dichloromethane (1 mL) were added to the reaction mixture and separated the organic layer and extracted the aqueous layer with dichloromethane (1 mL). Combined organic layer was washed with 10% sodium chloride solution (1 mL) and distilled the organic layer at 37 °C to obtain title compound. Yield: 100 mg; Purity by HPLC: 70.53%
Example-5: Preparation of 5, 20 - acetonide Ingenol-3 angelate

To a mixture of 5, 20 - acetonide Ingenol (100 mg) and tetrahydrofuran (1 mL), 2,4,6-trichlorobenzoyl angeloyl anhydride (0.094 g) was added at 28 °C and cooled the reaction mixture to 1 °C. n-butyl Li (0.4 mL) was added and stirred at same temperature for 2 hours. 10% sodium bicarbonate solution (1 mL) and dichloromethane (1 mL) were added to the reaction mixture and separated the organic layer and extracted the aqueous layer with dichloromethane (1 mL). Combined organic layer was washed with 10% sodium chloride solution (1 mL) and distilled the organic layer at 35 °C to obtain title compound. Yield: 110 mg; Purity by HPLC: 96.39%
Example-6: Preparation of 5, 20 - acetonide Ingenol-3 angelate

To a mixture of 5, 20 - acetonide Ingenol (100 mg) and tetrahydrofuran (1 mL), 2,4,6-trichlorobenzoyl angeloyl anhydride (0.094 g) was added at 28 °C and cooled the reaction mixture to 1 °C. Ter. butyl MgCl (3 mL) was added and stirred at same temperature for 2.5 hours. 10% sodium bicarbonate solution (1 mL) and dichloromethane (1 mL) were added to the reaction mixture and separated the organic layer and extracted the aqueous layer with dichloromethane (1 mL). Combined organic layer was washed with 10% sodium chloride solution (1 mL) and distilled the organic layer at 39 °C to obtain title compound. Yield: 110 mg; Purity by HPLC: 96.52%
Example-7: Preparation of 5, 20 - acetonide Ingenol-3 angelate

To a mixture of 5, 20 - acetonide Ingenol (100 mg) and tetrahydrofuran (1 mL), 2,4,6-trichlorobenzoyl angeloyl anhydride (0.094 g) was added at 28 °C and cooled the reaction mixture to 7 °C. Iso-propyl MgBr (3 mL) was added and stirred at same temperature for 3.5 hours. 10% sodium bicarbonate solution (1 mL) and dichloromethane (1 mL) were added to the reaction mixture and separated the organic layer and extracted the aqueous layer with dichloromethane (1 mL). Combined organic layer was washed with 10% sodium chloride solution (1 mL) and distilled the organic layer at 35 °C to obtain title compound. Yield: 100 mg; Purity by HPLC: 85.94%
Example-8: Preparation of 5, 20 - acetonide Ingenol-3 angelate

To a mixture of 5, 20 - acetonide Ingenol (100 mg) and tetrahydrofuran (1 mL), 2,4,6-trichlorobenzoyl angeloyl anhydride (0.094 g) was added at 28 °C and cooled the reaction mixture to 0 °C. Lithium tetramethyl piperidide (0.04 g) was added and stirred at same temperature for 3 hours. 10% sodium bicarbonate solution (1 mL) and dichloromethane (1 mL) were added to the reaction mixture and separated the organic layer and extracted the aqueous layer with dichloromethane (1 mL). Combined organic layer was washed with 10% sodium chloride solution (1 mL) and distilled the organic layer at 35 °C to obtain title compound. Yield: 110 mg; Purity by HPLC: 84.15%
Example-9: Preparation of Ingenol-3 angelate

A mixture of 5, 20 - acetonide Ingenol-3 angelate (300 mg) and 1% perchloric acid in methanol (24 mL) was cooled to 5 °C and stirred at the same temperature for 5 hours to complete the reaction. 10% sodium bicarbonate solution (1.5 mL) and diethyl ether (27 mL) were added to the reaction mixture. Separated the organic layer and extracted the aqueous layer with diethyl ether (15 mL). Combined organic layer was washed with 10% sodium chloride solution (1.5 mL) and distilled the organic layer under vacuum at 28 °C to obtain crude compound. Yield: 300 mg; Purity by HPLC: 95.18%
Example-10: Purification of Ingenol-3-angelate
Taken one of three parts of crude compound of example-9 and dissolved in n-propanol (0.5 mL) at 28 °C. Water (0.4 mL) was added to the reaction mixture at 28 °C and stirred for 1.5 hours at the same temperature. Solid was filtered and washed with water. Solid was dried under vacuum at 28 °C for 2 hours to obtain the title compound. Yield: 48 mg; Purity by HPLC: 95.8%
Example-11: Purification of Ingenol-3-angelate
Taken one of three parts of crude compound of example-9 and dissolved in dimethyl sulphoxide (0.5 mL) at 28 °C. Water (1 mL) was added to the reaction mixture at 28 °C and stirred for 1 hour at the same temperature. Solid was filtered and washed with water. Solid was dried under vacuum at 28 °C for 2 hours to obtain the title compound. Yield: 40 mg; Purity by HPLC: 95.3%.
Example-12: Purification of Ingenol-3-angelate
Taken one of three parts of crude compound of example-9 and dissolved in dimethyl formamide (0.2 mL) at 28 °C. Water (1 mL) was added to the reaction mixture at 28 °C and stirred for 2 hour at the same temperature. Solid was filtered and washed with water. Solid was dried under vacuum at 28 °C for 1 hour to obtain the title compound. Yield: 50 mg; Purity by HPLC: 96.6%
Example-13: Preparation of Ingenol-3 angelate

A mixture of 5, 20 - acetonide Ingenol-3 angelate (300 mg) and 1% perchloric acid in methanol (24 mL) was cooled to 0 °C and stirred at the same temperature for 5 hours to complete the reaction. 10% sodium bicarbonate solution (1.5 mL) was added at 0 °C and stirred for 15 min at same temperature and diethyl ether (27 mL) were added to the reaction mixture. Separated the organic layer and extracted the aqueous layer with diethyl ether (27 mL). Combined organic layer was washed with 10% sodium chloride solution (1.5 mL) and distilled the organic layer under vacuum at 25 °C to obtain crude compound. Crude compound was dissolved in dimethylformamide (0.6 mL) at 25 °C and water (1.2 mL) was added. Stirred the reaction mixture for 1.5 hours and solid was filtered and washed with water (0.3 mL). Solid was dried under vacuum at 25 °C for 5 hours to obtain title compound. Yield: 140 mg (51.8%); Purity by HPLC: 98.29%
Example-14: Preparation of crystalline Ingenol-3 angelate.
5, 20 - acetonide Ingenol-3 angelate (2 g) was added to 1.0% Perchloric acid methanol (160 ml) at 0 °C and stirred at the same temperature for 6 hours for the completion of reaction. Quenched the reaction mass with 10% sodium bicarbonate solution (10 mL) and extracted with diethyl ether (180 mL). Separated the aqueous layer and extracted with diethyl ether (90 mL). Combined organic layer was washed with 10% sodium chloride solution (10 mL) and dried over anhydrous sodium sulphate. Solvent was evaporated under vacuum to obtain crude compound, which was further purified by recrystallization to obtain the title compound. Yield: 1.4 g; Purity by HPLC: 98.22%
Example-15: Preparation of amorphous Ingenol-3 angelate.
Ingenol-3 angelate (1.3 g) was dissolved in dichloromethane (10 mL) at 28 °C and filtered the solution through 0.45 micron filter to obtain particle free solution. Solvent was evaporated under vacuum at 27 °C and dried for 2.5 hours at the same temperature under vacuum to obtain the title compound. Yield: 1.2 g
Example-16: Preparation of 5, 20 - acetonide Ingenol

A mixture of Ingenol (2.0 g) and acetone (100 mL) was cooled to 22°C and Pyridinium p-toluene sulphonic acid (0.72 g) was added. The reaction mixture was stirred for 6 hours at 22°C. Reaction mixture was quenched with 10% sodium bicarbonate solution (8 ml) and distilled the reaction mass at 32°C. Ethyl acetate (40 mL) and water (10 mL) was added to the reaction mixture and separated the organic layer from aqueous layer and the aqueous layer was extracted with ethyl acetate (10 mL). Combined organic layer was washed with 10% sodium chloride solution (10 ml). The organic layer was distilled at 31 °C to obtain crude compound. Ethyl acetate (6 mL) was added to the crude compound at 27°C and stirred for 10 min at same temperature. Reaction mixture was heated to 42°C and stirred for 15 min at same temperature. n-hexane (12 mL) was added to the reaction mixture at 27 °C and cooled to 5°C and stirred for 2 hours at the same temperature. Filtered the solid and washed with n-hexane (6 mL). Solid was dried under vacuum at 31 °C for 4.5 hours to obtain the title compound. Yield: 1.67 g (76%); Purity by HPLC: 99.15 %
Example-17: Preparation of angeloyl 2,4,6-trichlorobenzoyl anhydride.
To a mixture of angelic acid (5.0 g) and dichloromethane (50 mL) at 28°C, 2,4,6-trichlorobenzoyl chloride (13.4) was added and stirred at the same temperature for 5 minutes under nitrogen atmosphere. Reaction mixture was cooled to -8 °C and diisopropyl ethyl amine (14 mL) was added slowly at -8°C in 25 minutes. Reaction mixture was stirred at -5°C for 15 minutes and quenched with 10% citric acid (50 mL). Separated the organic layer and washed with 10% sodium bicarbonate solution (50 mL). Separated the organic layer and evaporated the solvent under vacuum at 38 °C to obtain crude compound. N-hexane (10 mL) was added to the crude compound and evaporated the solvent at 40°C. Again n-hexane (25 mL) was added and cooled the reaction mixture to 3°C. The reaction mass was stirred at 3°C for 1.5 hours and filtered. Wet compound was washed with pre-cooled n-hexane (5 mL) and dried the compound under vacuum at 28°C for 4 hours to obtain the title compound. Yield: 5.2 g; Purity by HPLC: 95.4%.
Example-18: Preparation of 5, 20 - acetonide Ingenol-3 angelate.

To a mixture of 5, 20 - acetonide Ingenol (12g) and ethyl acetate (480 mL), angeloyl 2,4,6-trichlorobenzoyl anhydride (11.4 g) was added at 28 °C. Cooled the reaction mixture to -9 °C and Potassium ter. butoxide (5.9 g) was added. The reaction mixture was stirred for 30 minutes at the same temperature. Extracted the reaction mixture with water (120 mL) at 28 °C and separated the organic layer. Washed the organic layer with 10% sodium bicarbonate solution (120 mL) and then with 10% sodium chloride solution (120 mL). The solvent was evaporated from the organic layer at 45 °C under vacuum to obtain crude compound. Crude compound was combined with methanol (24 mL) and again evaporated the solvent completely. The crude compound was dissolved in methanol (72 mL) at 50°C and cooled to 2°C slowly. Solid was filtered and washed with methanol (12 mL). Compound was dried under vacuum at 42 °C for 3 hours to obtain the title compound. Yield: 10.3 g (70.9 %); Purity by HPLC: 97.74%.
Example-19: Preparation of 5, 20 - acetonide Ingenol-3 angelate.

To a mixture of 5, 20 - acetonide Ingenol (50 mg) and tetrahydrofuran (5 mL), angelic anhydride (5 mg) was added at 28 °C. Cooled the reaction mixture to 10 °C and cyclopropyl magnesium bromide (1 mL) was added. The reaction mixture was stirred for 3.5 hours at 10°C and the reaction mass was quenched with saturated sodium bicarbonate solution. Extracted the product into dichloromethane and evaporated the solvent to obtain title compound. Purity by HPLC: 83.5%.
Example-20: Preparation of 5, 20 - acetonide Ingenol-3 angelate.
To a mixture of 5, 20 - acetonide Ingenol (50 mg) and dichloromethane (1 mL), angelic anhydride (50 mg) and sodium methoxide (40 mg) were added. The reaction mixture was stirred for 6 hours at 28°C and the reaction mass was quenched with water (2 mL). Separated the organic layer and evaporated the solvent to obtain title compound. Purity by HPLC: 80.5%.
Example-21: Preparation of Ingenol-3 angelate

A mixture of 1.5 % Perchloric acid in methanol (120 mL) was cooled to 0 °C and 5, 20 - acetonide Ingenol-3 angelate (2 g) was added at -1°C. The reaction mixture was stirred at same temperature for 4.5 hours. 10% sodium bicarbonate solution (10 mL) was added at 0 °C to quench the reaction and stirred for 15 min at same temperature. Water (30 mL) and dichloromethane (120 mL) were added to the reaction mixture and stirred. Separated the organic layer and extracted the aqueous layer with dichloromethane (60 mL). The combined organic layer was washed with water (10 mL) and then with10% sodium chloride solution (10 mL). Separated the organic layer and evaporate the solvent completely under vacuum at 28 °C. Solid was dried under vacuum at 28 °C to obtain title compound. Yield: 1.85 g; Purity by HPLC: 93.87%
Example-22: Preparation of crystalline Ingenol-3 angelate.
Ingenol-3 angelate (1.1 g) was dissolved in acetonitrile (5 mL) at 55°C and passed the solution through Reveleris® chromatography system at flow rate of 45 mL / min using water - formic acid mixture and acetonitrile - formic acid mixture as two mobile phases. Collected the fractions containing pure Ingenol-3-angelate and the combined fractions were evaporated under vacuum at 35°C to about 50% volume. Dichloromethane (215 mL) was added to the reaction mass (clear solution) and then water (215 mL) was added. Separated the organic layer and washed it with water (2 x 215 mL). Separated the organic layer and evaporated the solvent under vacuum at 33°C to obtain amorphous product. The amorphous product was dissolved in Isopropyl acetate (5.5 mL) at 75°C and evaporated the solvent under vacuum at same temperature. The solid was dried under vacuum at 79°C for 7 hours to obtain the title compound. Yield: 0.77 g; Purity by HPLC: 99.88%.
Example-23: Preparation of crystalline Ingenol-3 angelate.
Amorphous Ingenol-3 angelate (5 g) was dissolved in Isopropyl acetate (50 mL) at 28°C and evaporated the solvent at 55°C under vacuum and dried the compound under vacuum at 70°C for 12 hours to obtain the title compound. Yield: 5.0 g; Purity by HPLC: 99.97%
Example-24: Preparation of crystalline Ingenol-3 angelate.
Ingenol-3 angelate (3.3 g) was dissolved in acetonitrile (45 mL) at 55°C and passed the solution through Reveleris® chromatography system at flow rate of 45 mL / min using water - formic acid mixture and acetonitrile - formic acid mixture as two mobile phases. Collected the fractions containing pure Ingenol-3-angelate and the combined fractions were evaporated under vacuum at 35°C to about 50% volume. Dichloromethane (150 mL) was added to the reaction mass (clear solution) and then water (150 mL) was added. Separated the organic layer and again extracted the aqueous layer with Dichloromethane (150 mL). The combined organic layer was washed it with water (3 x 150 mL). Separated the organic layer and evaporated the solvent completely under vacuum at 33°C and Dichloromethane (150 mL) was added to it. Again the solvent was evaporated under vacuum to obtain amorphous product. The amorphous product was dissolved in Isopropyl acetate (30 mL) at 78°C and evaporated the solvent under vacuum at same temperature to obtain crystalline product. 0.4 g of crystalline product was combined with Dichloromethane (0.2 mL) and evaporated the solvent under vacuum at 75°C and again Dichloromethane (0.2 mL) was added to it and evaporated the solvent under vacuum at 76°C. The solid was dried under vacuum at 78°C for 2.5 hours to obtain 0.38 g of title compound. Purity by HPLC: 99.98%.
,CLAIMS:We Claim:

1. A process for the preparation of Ingenol mebutate, comprising the steps of:
a) protecting 5, 20 hydroxyl groups of Ingenol with a suitable protecting agent;

b) treating 5, 20 hydroxyl protected Ingenol with angelic acid or its activated derivative in the presence of a base, wherein the base is selected from the group consisting of metal hydrides; alkali metal alkoxides; organo metal bases and organo metal halides.

c) deprotecting the 5, 20 hydroxyl protected Ingenol-3- angelate to obtain Ingenol-3 angelate of formula (I);

d) optionally, purifying Ingenol-3-angelate in a suitable solvent,
wherein R1& R2 are same or different selected from the group consisting of hydrogen, methyl, ethyl, isopropyl, isobutyl, tert. butyl, phenyl, benzyl or both R1& R2 together form a carbonyl group along with carbon to which they are linked.
2. A process of claim 1, wherein metal hydride is sodium hydride; alkali metal alkoxide is selected from the group consisting of sodium methoxide and potassium tert. butoxide; organo metal base is selected from the group consisting of n-butyl lithium and lithium tetramethyl piperidide; organo metal halide is selected from the group consisting of tert. butyl magnesium chloride, isopropyl magnesium bromide and cyclopropyl magnesium bromide.
3. A process for the preparation of Ingenol mebutate, comprising the step of treating 5, 20 hydroxyl protected Ingenol with angelic acid or its activated derivative in the presence of a base, wherein the base is selected from the group consisting of metal hydrides; alkali metal alkoxides; organo metal bases and organo metal halides,

wherein R1& R2 are same or different selected from the group consisting of hydrogen, methyl, ethyl, isopropyl, isobutyl, tert. butyl, phenyl, benzyl or both R1& R2 together form a carbonyl group along with carbon to which they are linked.
4. A process of claim 3, wherein metal hydride is sodium hydride; alkali metal alkoxide is selected from the group consisting of sodium methoxide and potassium tert. butoxide; organo metal base is selected from the group consisting of n-butyl lithium and lithium tetramethyl piperidide; organo metal halide is selected from the group consisting of tert. butyl magnesium chloride, isopropyl magnesium bromide and cyclopropyl magnesium bromide.
5. A process for the purification of Ingenol-3 angelate, comprising the steps of:
a) dissolving Ingenol-3- angelate in a suitable solvent;
b) contacting the reaction mixture of step a) with an anti-solvent;
c) recovering Ingenol-3-angelate.
wherein the suitable solvent of step a) is selected from the group consisting of 1-propanol, dimethyl formamide, Isopropyl acetate, dimethyl sulfoxide or mixtures thereof and the anti-solvent of step b) is selected from the group consisting of water, n-pentane, n-hexane, cyclohexane, methyl cyclohexane, diethyl ether, di isopropyl ether; or mixtures thereof.
6. A process for the preparation of amorphous form of Ingenol mebutate, comprising the steps of
a) providing the solution of Ingenol mebutate in a suitable solvent
b) recovering amorphous Ingenol mebutate.
7. A process of claim 6, wherein recovering amorphous Ingenol mebutate at step b) is carried out by techniques selected from removal of solvent, cooling the reaction mass of step a), combining the reaction mass of step a) with an anti-solvent or combination thereof.
8. A process for the purification of Ingenol-3-angelate, comprising the steps of
a) providing Ingenol-3-angelate
b) passing through a chromatography system.
c) recovering pure Ingenol-3-angelate.
9. A process of claim 8, wherein chromatography system is system with integrated flash and preparative LC capabilities.
10. A process for the preparation of crystalline Ingenol mebutate comprising the steps of
a) providing a solution of Ingenol mebutate in isopropyl acetate
b) evaporating the solvent to obtain crystalline Ingenol mebutate.