Field of invention

The invention relates to the process for preparation of protoescigenin from escin. Protoescigenin is a polyhydroxyl aglycone obtained during the hydrolysis of escin, the natural saponin present in Aesculum hippocastanum seeds. Protoescigenin may be a valuable substrate used in the synthetic modification and derivatization of the naturally occurring saponins. Background of the invention

The mixture of saponines known under the common name escin, is the major component of Aesculum hippocastanum seeds extracts. It is present in the three forms named a-escin, β-escin and cryptoescin. These saponins belong to polyhydroxyl triterpene glycosides, containing four different aglycones (sapogenins), such as escigenin, protoescigenin, barringtogenol C and barringtogenol D, which are characterized by different substituents at C-16/C-21 and C-24 hydroxyl groups. Up till now 79 saponins have been isolated from the hydrolysates of Aesculum hippocastanum seeds extracts and characterized. Most of these compounds consist of a trisaccharide chain containing glucuronopyranosyl residue linked via glycosidic bound to an atom C- 3 of an aglycone, acyl groups at C-21, C-22 and C-28, seldom at C-16 position. Acyl moieties embrace angeloyl, tigloyl, acetyl, 2-methylbutanoyl and 2-methylpropanoyl groups {Pharmaceutical Crops, 2010, 1, 24-51).

The extracts of Aesculus seeds usually vary in composition and the difference depends on a plant species as well as the origin of a plant growth. The chemical composition of saponins isolated from the horse chestnut Aesculus hippocastanum L. seeds, growing predominantly in Europe and North America was proposed by Tschesche in Liebig's Ann. 669, 171 (1963) and can be illustrated by the structure presented below: aglycone

glycone

by: Pharm. Crops, 2010 wherein Ang is angeloyl, Tig = tigloyl, Ac = acetyl, MP = 2-methylpropanoyl, MB = 2- methylbutanoyl, GlcA-p = β-D-glucuronopyranosyl acid, Glc-p = β-D-glucopyranosyl, Gal-p = galactopyranosyl, Xyl-p = β-D-xylopyranosyl.

The research of Yoshikawa and co-workers resulted in the isolation and identification of 12 saponins, which proved to be the main components of Aesculum hippocastanum seeds extracts. The outcome of these works were published inter alia in Chem. Pharm. Bull. 42(6), 1357-1359 (1994); Chem. Pharm. Bull. 44(8), 1454-1464 (1996); Chem. Pharm. Bull. 20(10), 1092-1095 (1997), Chem. Pharm. Bull. 46(1 1), 1764-1769 (1998). The identified compounds included escin la, lb, Ila, lib, Ilia, Illb, IV, V and VI and also isoescin la, lb, and V, the chemical structures of which are summarized in the table below. Name Aglycone Ri R ₂ R ₃ R4 Rs Re

Escin la PES H Tig Ac OH H Glc-p

Escin lb PES H Ang Ac OH H Glc-p

Escin Ila PES H Tig Ac OH H Xyl-p

Escin lib PES H Ang Ac OH H Xyl-p

Escin Ilia BAC H Tig Ac H H Gal-p

Escin Illb BAC H Ang Ac H H Gal-p

Escin IV PES H Ac Ac OH H Glc-p

Escin V PES H MP Ac OH H Glc-p

Escin VI PES H MB Ac OH H Glc-p

Isoescin la PES H Tig H OH Ac Glc-p

Isoescin lb PES H Ang H OH Ac Glc-p

Isoescin V PES H MP H OH Ac Glc-p

Protoescigenin PES H H H OH H -

Barringtogenol C BAC H H H H H -

PES - protoescigenin

BAC - barringtogenol C

Escin, due to its beneficial effects on venous tone, antiinflammatory and antiedema activity, is wildly used in medicine, mainly in the treatment of chronic venous insufficiency, but it found use in the cosmetics as well. Although the efficiency of escin has been proved in the traditional medicine as well as in the clinical treatment, the molecular basis of its activity has not been established yet. The complexity of the saponin mixture and the lack of validated analytical methods, necessary for a qualitative and a quantitative determination of the natural compounds composition, impedes studies of pharmacokinetic and biochemical mechanism.

Isolation of the individual saponins from the crude plant material, determination of their structure and their analysis require application of laborious and advanced analytical techniques. These difficulties result from the unique and complex chemical structures of saponins, the similarity of their structure and resulting similar physicochemical properties, for example polarity, and in the end lack of chromophores, which hinders detection of analyzed molecules. In general, saponins are isolated from the crude plant material by extraction with the mixture of water and an alcohol, such as methanol or ethanol, followed by evaporation of the solvents under reduced pressure, reconstitution of the residue in a small amount of water and separation between n-butanol / water diphase system. For further purifications, the column chromatography techniques or liquid-liquid chromatography separation method are employed, but usually high-performance liquid (HPLC) chromatography must be used. In most cases, obtaining the high purity saponins requires multiple chromatography, which involves the replacement of column filling and the change of eluting solvents.

For instance, according to the procedure published in Chem. Pharm. Bull. 44(8),

1454-1464 (1996), the crude methanolic extract of Aesculum hippocastanum L. seeds was chromatographed on Diaion HP-20 column, followed by another separation of methanolic fraction of saponins mixture in reversed phase, on the chromatography column filled with silica gel. Multiple HPLC chromatographies of 90% methanolic eluate containig the pre-purified mixture of saponins furnished separation of escin la, lb, Ila, lib and Ilia.

\n Justus Liebigs Annalen der Chemie 1963, 669, 183-188 Kuhn R. and Loew I. described the hydrolysis of escin in the solution of 4 N hydrochloric acid in ethanol, the separation of the intermediate, and its subsequent hydrolysis under basic conditions with potassium hydroxide in methanol. The hydrolysis products were separated by chromatography on silica gel, yielding protoescigenin and escygenin.

Yoshika I. and al. separated and determined the chemical structure of sapogenins isolated from Japanese Aesculus turbinata BLUME extract. They also assigned the configuration of carbon atoms bound to hydroxyl groups in ring E of protoescigenin. These findings were published in Chem. Pharm. Bull. 19(6), 1200-1213 (1971). According to their procedure, n-butanolic extract of Aesculus seeds was condensed under vacuo furnishing the resin residue. This product was refluxed in ether resulting in precipitation of the solid of the crude mixture of saponins, which was subsequently hydrolyzed in 4 N hydrochloric acid in ethanol at elevated temperature. The obtained mixture was diluted with water, condensed and diluted with water again to give a solid precipitate, which was hydrolyzed in the basic medium with 5% KOH methanolic solution. After water addition, the crude mixture of sapogenins precipitated out of the solution, the solid was purified by chromatography on the column filled with aluminum oxide and yielded the mixture of four main compounds. The major sapogenin was purified by the crystallization in methanol and precipitated in a colorless needles, characterized by 300-307°C melting point. The physicochemical data, such as the melting point, IR (KBr) spectra and TLC analyses proved the structure of protoescigenin.

According to the publications described above, cleavage of the glycosidic bond of deacylated escin occurs during the hydrolysis under acidic conditions, in this process protoescigenin (deacylated escin II methanolysis), and barringtogenol (deacylated escin III methanolysis) are formed. Under the basic conditions hydrolysis of saponin acyl groups takes place, liberating the molecules of acetic, tyglic and angelic acids.

Although different methods of hydrolysis are well known to those skilled in the art, other than chromatography separation methods of the products of saponin hydrolysis have not been found in the prior art. In all the procedures described in the publications mentioned above, hydrolysis is preceded by the chromatography purification of either the mixtures or individual saponins. Following these multi-step purification processes, some of the pentacyclic triterpenes were successfully isolated and purified at a laboratory scale. However, these elaborate and expensive methods cannot be implemented at a bigger scale.

The main problem one must face while scaling-up the process, is viability of the crude extract composition, the similar polarity and molecular weight of the particular saponin mixture components. These physicochemical properties of the crude mixture, as well as the lack of standardization methods designed for the plant raw materials and products, impede the separation of individual saponins either by crystallization or ulrafiltration. Possibility to obtain protoescygenin at high purity and at bulk quantities is crucial, if it is to be the substrate used in synthetic modifications. In the molecule of protoescygenin six hydroxyl groups are present, thus the number of possible products resulting from substitution of hydroxyl groups amounts for 63 (2 ⁶ -l). This number may dramatically increase, if protoescygenin is contaminated with aglycones of other saponins. In the aftermath of chemical reaction, complex mixture of products of similar structures is formed, separation of which is impossible.

The results of experimental attempts to obtain protoescygenin by the hydrolysis of escin demonstrated that the reaction product usually is the mixture of sapogenins, containing just only from 40 to 60% of protoescygenin, according to HPLC. Among the other main components of the mixture barringtogenol C was also detected, accompanied by the smaller amounts of other sapogenins, such as escigenin and barringtogenol D. Methods routinely used for the purification and isolation, for example the multiple crystallization, liquid - liquid or liquid - solid extractions, were not successful in the protoescygenin isolation.

It was the aim of the present invention to improve the process of hydrolysis of escin to make possible its implementation at technical scale, and to develop a method of isolation of protoescigenin from the mixture of sapogenins other than chromatography. It was also the aim of the present invention to affect preparation of high purity protoescigenin as the important synthon for further synthetic transformations.

These aims were accomplished due to the development of optimal conditions for hydrolysis of escin, effective process of enrichment of post-hydrolysis mixture of sapogenins with protoescigenin, and selective methods of isolation and purification of protoescigenin.

Brief description of the invention

The present invention provides the process for preparation of protoescigenin from escin, comprising: a) two-step hydrolysis of escin, consecutively under acidic and basic conditions, resulting in obtaining the crude mixture of sapogenins, b) process of enrichment of the crude mixture of sapogenins with protoescygenin, wherein the said process consists of the following steps: b-i) dissolving the hydrolysis product in a three-component mixture of solvents to obtain a clear mono- or bi-layer solution, b-ii) addition of water to the solution obtained in step b-i), until the precipitation of a solid of the pre-purified mixture of sapogenins containing protoescygenin as the major component occurs, c) isolation of the solid obtained in step b-ii), d) purification of the pre-purified mixture of sapogenins, e) isolation of protoescygenin.

Another embodiment of the present invention provides the process for isolation of protoescigenin of purity higher than 98% from the mixture of sapogenins, the said mixture comprises about 40-60% of protoescigenin, and barringtogenol C, escigenin and barringtogenol D as the main impurities, wherein the process comprises: i) dissolving the mixture of sapogenins in a three-component mixture of solvents until clear, mono- or bi-layer solution is formed, ii) addition of water to the solution obtained in step i), until the precipitation of a solid of the pre-purified mixture of sapogenins containing protoescygenin as the major component occurs, iii) isolation of the solid of the mixture of sapogenins obtained in step ii), containing approximately 70-90% of protoescigenin, iv) purification of the solid obtained in step iii) by crystallization, v) isolation of protoescigenin.

The other embodiment of the invention provides the process for purification of protoescigenin, comprising at least one crystallization in the mixture of solvents, consisting of an organic solvent and water, and at least one crystallization in the mixture of selected organic solvents with ethers, acetonitrile or saturated hydrocarbons. Another embodiment of the invention is a new compound identified as protoescigenin monohydrate, which has been isolated from the mixture of sapogenins, containing protoescigenin as the main component.

Protoescigenin monohydrate can be prepared by crystallization of protoescigenin in the mixture consisting of Ci-C ₃ alcohol and saturated hydrocarbon, especially cyclohexane.

Detailed description of the invention Process according to the present invention is depicted in Scheme 1 below.

EC-04

mixture of aglycones

Hydrolysis and separation of the crude mixture of spogenins

Step a) of the process according to the present invention comprises a two-step hydrolysis of escin, isolated from the horse chestnut plant (Aesculum hippocastanum), consecutively under acidic and basic conditions. The sequence of acidic and basic hydrolyses is optional, but it was proved experimentally that better separation of the crude mixture of sapogenins is achieved when the hydrolysis of escin in acidic medium is performed prior to the basic one.

Preferably, the hydrolysis of escin under acidic conditions is performed for several hours in alcohol, preferably methanol, under reflux, in the presence of inorganic acid such as sulfuric acid or hydrochloric acid. The product of hydrolysis precipitates after the addition of water, after the neutralization of the solution the solid is filtered, yielding a multi-component mixture of sapogenins and their esters. Temperature increase considerably accelerates the reaction progress and as a result shortens reaction time. At ambient temperature thehydrolysis proceeds very slowly (several days) leaving the substrate not entirely consumed.

The product of acidic hydrolysis is subsequently subject to hydrolysis under basic conditions in the presence of sodium or potassium hydroxide, in alcohols such as methanol, ethanol, propan-2-ol or the mixture thereof, optionally with the addition of water. The hydrolysis proceeds at a wide range of temperatures, the higher reaction temperature, the shorter hydrolysis time.

Preferably, a two-step hydrolysis is a 'one-pot reaction', without the separation of the intermediate obtained after acidic hydrolysis. There are several methods of separation of the hydrolysis products, yielding the crude mixtures of sapogenins of different compositions.

Typically, the solid of the crude mixture of sapogenins (EC-05.S) precipitates out of the reaction medium upon the addition of water, optionally, it is neutralized and washed with water. The product obtained this way is a wet solid in the form of a paste, containing a substantial amount of water (more than 50%, most often 80-90% ). It is dried on air or under reduced pressure or it is lyophilized, furnishing a dry solid of EC- 05. S, containing less than 10%, for example about 5-7%, of water.

The crude mixture of sapogenins EC-05.S, obtained in the process according to the invention contains protoescigenin and barringtogenol C as the main products, and minor amounts of other sapogenins with the structures as presented below. According to UPLC analysis, the solid of EC-05.S consists mainly of 40-60% of protoescigenin and 15-25% of barringtogenol C.

Barringtogenol C

Barringtogenol D

Preparation of the pre-purfied mixture of sapogenins enriched with protoescigenin In step b), the crude mixture of sapogenins EC-05.S obtained in step a) by the hydrolysis of escin is enriched with protoescigenin.

The process of enrichment with protoescigenin comprises dissolving the crude mixture of sapogenins EC-05.S in a three-component mixture of solvents, until a clear mono- or bi-layer solution is formed (primary solution), followed by the precipitation of the pre-purified mixture of sapogenins by the addition of water and finally separation of the solid enriched with high contents of protoescigenin (EC-05.T), that is more than 70%, most often 75-90%, according to UPLC.

The three-component mixture of solvents used to dissolve the crude mixture of sapogenins comprises: - Cj-C ₃ aliphatic alcohol, as the Component 1,

- water, as the Component 2, and

- a solvent selected from the group consisting of aliphatic and cyclic ethers, as the Component 3.

Preferably, the Component 1 is methanol, ethanol or propan-2-ol, more preferably methanol.

The Component 3 is selected from the group consisting of aliphatic ethers such as fert-butylmethyl ether, diisopropyl ether and cyclic ethers such as tetrahydrofuran or dioxane.

Preferably, the crude mixture of sapogenins EC-05.S is enriched with protoescigenin in the three-component mixture of solvents consisting of methanol, water, tert-butylmethyl ether. In another embodiment of the present invention, the crude mixture of sapogenins

EC-05.S is enriched with protoescigenin in the three-component mixture of solvents consisting of methanol, water, diisopropyl ether.

According to the present invention, the process of enrichment of the crude mixture of sapogenins EC-05.S with protoescigenin, can be performed directly after the second step of the hydrolysis. The mixture can be in the form of a solution or a suspension, in the form of a dry solid obtained by drying or lyophilization or as a wet paste.

Step b) of the present invention is accomplished by dissolving the crude mixture of sapogenins in the three-component mixture of solvents, at temperature range from ambient temperature to reflux, preferably at reflux.

To achieve the best effect, the crude mixture of sapogenins in the three- component mixture of solvents should form a clear mono- or bi-layer solution. It has been discovered, that to obtain the solid enriched with protoescigenin characterized by better physicochemical parameters, the solution must become clear before the nucleation and crystallization take place.

The formation of the mono- or bi-layer solution depends on the physicochemical properties of used solvents. Whenever the terms 'bi-layer solution' or 'two phase solution' are used throughout the description, they relate to the mixture of solvents, consisting of two separate liquid phases under atmospheric pressure and at a given temperature or at a temperature range from ambient temperature (about 20°C) to reflux. According to the above definition, a bi-layer solution is not formed by the miscible solvents.

In case when the liquid layers are not separated spontaneously, the addition of the appropriate volume of the Component 2 and/or the Component 3 to the mixture facilitates the formation of a bi-layer solution. Undissolved solid materials present at small quantities and/or inorganic solids are separated by filtration.

In step c), the pre-purified mixture of sapogenins EC-05.T enriched with protoescigenin is gradually precipitated from the primary solution, upon the addition of the Component 2 (ie. water). Optionally, the two layers of the primary solution containing the crude mixture of sapogenins EC-05.S are separated, the water layer is washed with selected ether, and the combined organic phases are used for the crystallization of the product. In this process, after the addition of water, the bi-layer solution is formed as well and the concomitant solid precipitation of the pre-purified mixture of sapogenins EC-05.T occurs. The solvents of the three-component mixture (primary solution), which is the reaction medium in the crystallization of the pre-purified mixture of sapogenins, are used at different volume ratios. The volumes of solvent components that are to be used, depend on the contents of sapogenin mixture. The quantitative ratio of sapogenins in their mixture results from the composition of escin used as the starting material in hydrolysis, hydrolysis conditions and the presence of some by-products, such as inorganic and organic salts. In the calculations of the water amount to be used in the three-component mixture that is necessary to dissolve EC-05.S, the water contents of the substrate solid should also be taken into account. As mentioned before, the crude mixture of sapogenins, when used in the form of a suspension or a paste, may contain up to 90% of water.

In general, the mixture comprising methanol - water - tert-butylmethyl ether solvent system, at amounts (given at volume percentage of the mixture) from 15 to 85% of methanol, from 10 to 50% of water and tert-butylmethyl ether up to 100%, provides good solubility of the crude mixture of sapogenins EC-05.S. In case when the crude EC-05.S subject to the crystallization is in the form of an aqueous paste, dried solid or lyophilized solid, the volume ratios of solvents are as follows, from 15 to50% of methanol, from 15 to 40% of water (water amount comprised in the solid was included) and from 20 to 65% of tert-butylmethyl ether, preferably from 15 to 40% of methanol, from 10 to 35% of water and from 30 to 65% tert-butylmethyl ether. The total volume of solvents, calculated in the relation to the amount of escin used in the hydrolysis, as the EC-05.S equivalent, ranges from 15 ml/g to 60 ml/g, preferably from 20 to 35 ml/g.

When EC-05.S is used in the enrichment process in the form of a solution or a suspension and the separation step of the crude sapogenin mixture from the post- hydrolysis mixture is omitted, the solvents volume ratio ranges from 20 to 70% of methanol, from 10 to 40% of water and from 10 to 50% of tert-butylmethyl ether, preferably from 30 to 70% of methanol, from 15% to 40% of water and from 20% to 50% of tert-butylmethyl ether. The total volume of solvents, calculated in relation to the amount of escin ranges from 40 ml/g to 75 ml/g, preferably about 55 ml/g.

In case when the enrichment process subsequently follows the hydrolysis of escin, EC-05.S is used in the form of a solution or a suspension, but inorganic salts formed during hydrolysis are removed by filtration, EC-05.S is not dissolved in the Component 3 (the initial amount of ether in the solution accounts for 0%). In such a case, ether is added later to form a three-component solution, in which the precipitation of EC-05.T can proceed according to the method described above. The adjustment of the solvents volume to form a bi-layer solution, thus enabling achievement of advantage effects of the present invention, remains within basic knowledge of those skilled in the art and are well acquainted with the physicochemical properties of solvents and multi-component mixtures thereof to follow the rules of required solvents volume selection. The water amount required for precipitation of the solid from the three- component solution in step b) results from the volume proportions of two other components, reached when the product crystallization occurs. Regardless of the chosen procedure, the final proportion of solvents amounts includes total volumes of solvents used in the whole process, for example, volumes of solvents used before and after the separation of layers are included in these calculations. In general, water volume required to precipitate the solid is adjusted experimentally. Usually, in the method without layers separation, a minimum 20% of water amount in relation to the primary solution total volume is required. In case when the procedure of two layers formation is followed, the precipitation may occur due to the addition of only 10% volume of water, calculated in relation to the total volume before the separation of phases.

The solid precipitation is performed at the temperature values ranging from ambient temperature to reflux. When the process is carried out without layers separation, the crystallization proceeds at elevated temperatures, at reflux or below. When the process is carried out with the separation of the primary solution liquid phases, the product precipitates at room temperature. The way of water addition to the reaction mixture does not affect the process of crystallization, however the slow addition of water is recommended, due to better morphology of the forming crystals and in order to avoid the unwanted formation of emulsion. The precipitation of the solid is favoured in case when the temperature of the reaction mixture is decreased after water addition as well as the nucleation of the solution with protoescigenin crystals of purity higher than 98%, and /or the addition of the additional amount of water (1/3 to 1/2 of the initial volume) after the precipitation.

The solid separated according to the invention is the pre-purified sapogenins mixture EC-05.T, which is enriched with protoescigenin in comparison with the crude sapogenins mixture EC-05.S. Protoescigenin contents in the mixture amounts for about from 70% to 90%.

Purification of the pre-purified mixture of sapogenins and isolation of crystalline protoescigenin

Regardless the enrichment of the pre-purfied mixture of sapogenins EC-05.T with protoescigenin in comparison with the crude post-hydrolysis mixture EC-05.S, the purity of EC-05.T is usually insufficient for use in the chemical synthesis. Apart from protoescigenin and barringtogenol C which is present at amount about 5 to 25%, the precipitate contains the substantial amounts of unidentified impurity characterized by RRT = 0,95 (up to 3%). In step d) according to the present invention, to remove plethora of different impurities the pre-purified mixture of sapogenins EC-05.T is subject to crystallization in the solvents of different physicochemical properties.

Preferably, the pre-purfied mixture of sapogenins EC-05.T is purified by at least one crystallization in a mixture of solvents consisting of an organic solvent selected from the group A combined with water, and at least one crystallization in a mixture of solvents selected from group B consisting of an organic solvent with admixture of ethers or saturated hydrocarbons.

Polar impurities characterized by RRT (RRT = RT _an aiysed peak / RTPES-OI) ⁼ 0.84, 0.87, 0.95 and 1.06, as well as 'ambient impurities' characterized by RRT = 1.09 ( there are at least two peaks of the impurity of RRT values ranging from 1.07 to 1.10; separation of these peaks is not successful, therefore they are treated as a total impurity, represented by 1.09 RRT value), especially impurities characterized by RRT = 0.95 and 1.09, are effectively removed if the pre-purfied mixture of sapogenins EC-05.T is dissolved in a solvent selected from the group A, comprising Ci-C ₃ alcohols such as methanol, ethanol, propan-l-ol, propan-2-ol and mixtures thereof with ethers; organic acids such as acetic or propionic acid; amides such as dimethylformamide, dimethylacetamide or N-methylpyrrolidone; and dimethylsulfoxide, at the temperature range from ambient temperature to reflux, preferably at elevated temperature, followed by the addition of water at ambient temperature or reflux. The solid precipitating during the addition of water or cooling the mixture down is filtered off, washed with water or the mixture of water and alcohol, and dried.

The impurities characterized by RRT = 1.09 and 1.14 (BAC-01) and RRT above 1.14, for example RRT = 1.47, 1.53, 2.17, are effectively removed by the crystallization in the mixture of solvents selected from the group B such as Ci-C ₃ alcohols, ie. methanol, ethanol, propan-l-ol, propan-2-ol, and the mixtures thereof, as well as their mixtures with acetonitrile, ethers or saturated hydrocarbons, for instance. EC-05.T is dissolved in the selected mixture of solvents at the temperature range from ambient temperature to reflux, preferably at elevated temperature, the resulting solution is cooled down or the same amount of alcohol is evaporated.

Another possibility of crystallization is achieved by dissolving the sapogenins mixture EC-05.T in an alcohol at reflux, followed by the addition of the co-solvent with concomitant temperature decrease. Preferable co-solvents are ethers such as tetrahydrofuran or diisopropyl ether, or saturated hydrocarbons such as hexane, hexane fraction, cyclohexane, heptane, and also acetonitrile. The isolated solid is washed with a selected solvent and dried. Purification can also be accomplished by the maceration of EC-05.T solid in an alcohol or acetone.

The method of purification depends on the purity of the starting sapogenins mixture EC-05.T, the use of at least two crystallization processes in different solvents, one - in a solvent selected from the group A with water, and another - in a solvent chosen from the group B, yields protoescigenin of purity higher than 98% (according to UPLC), containing less than 1% of barringtogenol C and less than 0,5% of unidentified impurities characterized by RRT = 0.95.

The number of crystallizations required to obtain protoescigenin of the purity higher than 98% can be chosen experimentally by those skilled in the art and it depends on the impurities contents in the pre-purfied mixture of sapogenins EC-05.T.

One crystallization in C1-C3 alcohol/water system and at least one crystallization in C]-C ₃ alcohol/ saturated hydrocarbon system are much more effective than multiple crystallizations of protoescigenin in methanol, reported in the state of art.

It has been discovered, that protoescigenin crystallizes at different polymorphic forms, which are distinguished by variable amount of crystalline water and characteristic X-Ray powder diffraction pattern (XRPD).

As was disclosed in Chem. Pharm. Bull. \9(6), 1200-1213 (1971), upon crystallization in methanol at least two polymorphic forms of protoescigenin were identified. For the purposes of the present invention these forms are denoted as forms II and VI. The water content in these forms is ranging from 1% to 2,8%, as determined by thermogravimetric analysis.

Preferably, the compound obtained in the process according to the present invention by the crystallization in the mixture of propan-2-ol and cyclohexane is isolated in the form of protoescigenin monohydrate, named Form III. Protoescigenin monohydrate is a new compound, having characteristic peaks in X-ray powder diffraction (XRPD) pattern recorded with CuKa, λ= 1,54056A, having the specific diffraction lines at diffraction angles 2Θ about 6,74; 8,45; 11,17; 13,61 and 14,57 ± 2°. Protoescigenin monohydrate has characteristic peaks in X-ray powder diffraction

(XRPD) pattern recorded with CuKa, λ= 1,54056A, presented by the reflection angles 2Θ [°], interplanar spacings d [A] and relative intensities in attitude to the most intensive diffraction peak, I/I ₀ [%], as set forth in Table 1 :

Representative X-ray powder diffractogram of proteoscygenin monhydrate is depicted in Fig.l .

Infrared spectrum of proteoscygenin monhydrate, performed in KBr pressed tablet, is depicted in Fig. 2. DSC profile of protoescygenin monohydrate recorded by differentiation scanning calorimetry is presented in Fig. 3, and is characterized by two specific endothermic effects. First effect, observed at 98.86°C is the melting point determined as Onset' (onset), with enthalpy -100.60J/g, and comes from the elimination of water of crystallization. Second effect, observed at 319.37°C (onset), with enthalpy -58.80J/g, comes from the melting of substance with accompanying decomposition.

Thermogravimetric curve (solid line in Fig. 2) shows a visible weight loss within the temperature range from 30 to 150°C. Another weight loss is from 320°C. The comparison of effects from TGA, SDTA (Single Differential Thermal Analysis; dashed line) and DSC curves proves that first effect results from a solvent evaporation and the second one is from the decomposition of substance.

In the TGA curve, measured with the scan speed of 5°C/min, weight loss 3.67 % calculated to 160°C was determined (Fig. 4). This mass loss is observed on the DSC curve as first endotherm. Percentage value of weight loss and amount of water determined by Karl Fischer method, correspond to stoichometric amount of water contained in proteoscygenin monohydrate crystals, which is 3.43%.

Protoescigenin monohydrate is stable under ambient conditions, up to 100°C. X-ray diffraction measurements of monohydrate were done at following temperature loop: 25°C→ 30 min at 1 10°C→ 25°C. It has been observed that form III after heating for 30 min at 1 10°C changes to a different form, called form V. According to TGA measurements, Form V contains from 1.35 to 1.65% of water that is consistent with the stoichiometric water content in the hemihydrate form (1.75%). Form V is characterized by X-ray powder diffractogram different from the ones obtained for forms II and VI and as well as form III of protoescigenin monohydrate, diffractograms of the mentioned forms are compared in Fig. 5. Forms II and VI contain from about 1 to 2.8% of water as well as from monohydrate form III. Diffractograms of the different protoescygenin crystalline forms are compared in Fig. 5.

The present invention enables the preparation of protoescigenin of purity higher than 98% by means of hydrolysis of naturally available escine, enrichment of the resulting crude mixture of sapogenins in protoescygenin and purification thereof by crystallization, without the need of laborious chromatography technique.

The present invention is further illustrated by the following preparative examples.

Examples

Analytical methods The IR spectrum was recorded on the Bruker Alpha spectrometer in the range of

4000 - 400 cm ^"1 , with spectral resolution of 4 cm ^"1 . Samples were measured in KBr pellets (-1.5 mg of substance / 200 mg KBr). The Ή and ¹³ C NMR spectra were acquired on the Varian VNMRS 600 spectrometer at 600 MHz transmitter frequency. S X-ray powder diffraction (XRPD) studies were performed by means of the

MiniFlex diffractometer (Rigaku Coporation, Tokyo Japan) using CuKa radiation (λ = 1.54056A) with following parameters:

• Scanning range 2Θ: from 3° to 40°

• Scanning speed: 0.5°/min

· Measurement step: 2Θ: 0.02 ⁰

• Measurement temperature: ambient temperature

• Detector: scintillator

Diffractograms were analyzed using the DHN_PDS software.

^■ S Differential scanning calorimetry (DSC) measurements were carried out by means of the DSC822 with IntraCooler (Mettler Toledo) with following parameters:

• Pan: aluminum 40 μΐ,

• Atmosphere: N ₂ , 60 mL/min

• Measurement: heating from 25 to 350°C at 10°C/min

· Sample preparation: accurately weighed samples (5-7 mg) were packed in the aluminum pan with the pierced lid.

•S Thermogravimetric analyses (TGA) were carried out by means of the TGA/SDTA851e (Mettler Toledo) with following parameters:

• Pan: aluminum 40 iL

· Atmosphere: N ₂ , 60 mL/min • Measurement: heating from 30 to 400°C at 10°C/min and heating from 30 to 180°C at 5°C/min. Measurements were blank curve corrected.

• Sample preparation: accurately weighed samples (5-7 mg) were packed in the aluminum pan with the pierced lid.

^• S Water content determination was done by Karl Fischer volumetric titration, according to Ph. Eur. 2.5.12, using the Methrom 701 KF Titrino apparatus and the Methrom 6.0338.100 electrode Specific rotation was calculated from an optical rotation measurement performed on the PEPvKIN ELMER 341 Polarimeter at the wavelength of 589 nm (sodium lamp), at 20 °C. A sample was dissolved in methanol (HPLC purity).

^• Related substances determination using Ultra Performance Liquid Chromatography (UPLC) was carried out employing UHPLC Dionex Ultimate 3000, equipped with PDA detector.

Method parameters:

UPLC Acquity BEH CI 8 2,1 x 50 mm, 1,7μιη column

Column temperature 30°C

Mobile chase flow: 0,5 ml/min

Injection Volume: 2 μΐ

Detection wavelength 200 nm

Sample concentration: 2 mg/ml

Diluent: MeOH, UPLC purity

Mobile chase A: 10 mM ammonium acetate, pH 6,8, UPLC purity

Mobile chase B: acetonitrile, UPLC purity

Mass spectrometry analysis was carried out using Applied Biosystems QTrap 3200, equipped with ESI ion Source. 0.1% solution of ammonium acetate in MeOH was used as a diluent. Sample concentration was 0.1 mg/ml. Example 1 β-Escin (commercialy available) (100 g) was dissolved in MeOH (350 ml) at RT (23 C). Concentrated H ₂ S0 ₄ (40 ml) in MeOH (60 ml) was added into the obtained solution. The resulting clear reaction mixture was refluxed for 1 h 30 min. The heating was removed and 30% NaOH solution (190 ml) was slowly added at 50 C. The temperature raised to 70°C and the reaction mixture was further stirred at this temperature for 20 min. Upon H ₂ 0 (600 ml) addition, a fine-crystalline solid precipitated. The solid was filtered off and washed with H ₂ 0 (4^200 ml). A creamy solid was obtained as a paste (water content 84%, by Karl-Fischer); yield 465 g, UPLC: 55.3% PES-01, 24.8% BAC- 01.

Example 2

β-Escin (commercialy available) (10 g) was dissolved in MeOH (40 ml) at RT (23 C). HCl _aq. (35%, 7 ml) in MeOH (6 ml) was dropped in to the obtained solution. The resulting clear reaction mixture was refluxed for 1 h 30 min. The heating was removed and 25% NH _{3 aq} . solution (190 ml) and 30% NaOH aq. solution (13 ml) were slowly added at 50 C. The temperature raised to 60°C and the reaction mixture was further stirred at this temperature for 20 min. Upon H ₂ 0 (150 ml) addition, fine-crystalline solid precipitated. The solid was filtered off and washed with H ₂ 0 (3x 100 ml). Creamy solid was obtained as a paste.

Example 3

The wet solid EC-05.S obtained in Example 1 (41.6 g) was dissolved in the mixture of MeOH (125 ml), tert-butylmethyl ether MTBE (125 ml) and H ₂ 0 (50 ml) at reflux temperature. The heating was removed and to the hot solution MTBE (100 ml) and H ₂ 0 (40 ml) were added. The clear, bi-layer solution was allowed to cool down to 30°C. The phases were separated, the organic phase was transferred to a flask and water (25 ml) was added at RT (23°C). A crystalline white solid EC-05.T precipitated from the reaction mixture. The mixture was stirred for some hours, then the solid was filtered off and washed with water. The solid was dried overnight in the laboratory drier at 45 °C. Yield 2,0 g, UPLC: 87.6 % PES-01 ; 5.1 % BAC-01. Example 4

The dried solid EC-05.S obtained in Example 1 (7.60 g; 16.3% of solid obtained from 100 g of escin) was dissolved in the mixture consisting of MeOH (125 ml), MTBE (125 ml) and H ₂ 0 (90 ml) at reflux. The heating was removed and to the hot solution MTBE (100 ml) and H ₂ 0 (50 ml) were added. The clear, bi-layer solution was allowed to cool down to 25°C. The phases were separated, the organic phase was extracted with MTBE (2 x 50 ml). The combined organic phases were transferred to a flask and water (25 ml) was added at RT (23 °C). After some minutes the crystalline white solid precipitated from the reaction mixture. The mixture was stirred for further 15 min, then the additional portion of water was added (25 ml) and the mixture was left overnight. The precipitated solid was filtered off and washed with water and acetonitrile. The product was dried in the laboratory drier overnight at 45°C. Yield 2.67 g, UPLC: 86.1 % PES-01 ; 6.27 % BAC-01.

Example 5

The dried solid EC-05.S with the water content 8,26%, UPLC: 46.1% PES-01 , 16.0% BAC-01, in the amount 7.60 g (18% of the solid obtained from 100 g of escin) was dissolved in the solvents mixture consisting of MeOH (125 ml), MTBE (125 ml) and H ₂ 0 (90 ml) at reflux. The heating was removed and to the hot solution MTBE (100 ml) and H ₂ 0 (30 ml) were added. The clear, bi-layer solution was allowed to cool down to 25 °C. The phases were separated, the organic phase was transferred to a flask and water (25 ml) was added at RT (23°C). The crystals of pure protoescygenine (UPLC: 98%) were added to the obtained suspension. After some minutes the crystalline white solid precipitated from the reaction mixture. The mixture was stirred for further 15 min, then a further portion of water was added (10 ml) and the mixture was left overnight. The solid was filtered off and washed with water and acetonitrile. The product was dried in the laboratory drier overnight at 45°C. Yield: 1.90 g, UPLC: 89.5 % PES-01 ; 4.7 % BAC-01. Example 6

The crystalline β-escin (200 g) was dissolved in 700 ml of MeOH at RT. The concentrated H ₂ S0 ₄ (80 ml) was added to the solution within ca. 20 min., and the addition funnel was rinsed with 100 ml MeOH. During the course of adding the acid the temperature of the solution raised from 24 to 45°C. The heating was turned on and the reaction mixture was refluxed for ca. 3 h, when the reaction was controlled by TLC (AcMe-H ₂ 0 8: 1). It was left overnight to cool down.

Water (3.0 L) and KOH (100 g) were placed in the flask, the mixture was heated to 45- 50°C. The post-reaction solution, after rinsing the flask with 100 ml MeOH, was put in the addition funnel and dropwise added to the flask within 10 min, while stirring vigorously. The pH of the reaction mixture was bring pH 7 by the use of 30% KOH _aq . The slurry was cooled down, the solid was filtered off and washed with water (3x200 ml). The obtained solid EC-04 (136 g) was dried in the vacuum drier over P ₂ 0 ₅ for 2 days, resulting in 1 10.5 g of the product EC-04. The part of the obtained solid EC-04 (55.3 g) was placed in the 1.5 L reactor equipped with a reflux condenser, a magnetic stirrer and a heater, and dissolved in methanol (250 ml). Then, the solution of KOH (24.3 g) in methanol (250 ml) was added and the reaction was heated under reflux for 4-5 h (TLC control, AcOEt-MeOH 9: 1 or CHC1 ₃ - AcOEt-MeOH 45:45:10). It was left overnight to cool down. The reaction mixture was added to the 4.5 L reactor equipped with a stirrer and the addition funnel, filled with 2 L of water, while stirring vigorously. A white solid precipitated. The suspension was neutralized to pH 7.5-8.0 by the addition of 3 M HCl solution. The reaction was stirred for 2 h, then it was transferred onto the Schott's funnel. The equipment and the solid were washed with water (1 x200 ml) under reduced pressure and the solid was additionally washed with water (2x200 ml). Then, the suspension was diluted with water and lyophilized overnight. The obtained product was dried in the vacuum drier (P ₂ 0 ₅ , 45°C, 1 day), to obtain 36.6 g of the lyophilized mixture of sapogenins EC-05.S with the water content 5.1%, UPLC purity: 48.6% PES- 01, 16.1% BAC-01. EC-05.S lyophilizate (7.10 g; 9.3% of the solid obtained from 100 g of escin) was dissolved in the mixture consisting of MeOH (130 ml), MTBE (110 ml) and H ₂ 0 (95 ml) under reflux. The heating was removed and a portion of 140 ml MTBE and 45 ml H ₂ 0 were added to the hot solution. The obtained clear, bi-layer solution was left overnight to cool down to 25°C. The layers were separated, the organic phase was transferred to the flask and water (50 ml) was added at RT. After some minutes the white solid precipitated from the reaction mixture. After 15 min stirring, the additional portion of water was added (25 ml) and the reaction was left overnight.

EC-05.T solid was filtered off, washed with water and acetonitrile and dried in the laboratory drier overnight at 45°C. Yield: 2.17 g, UPLC: 83.9 % PES-01 ; 3.4 % BAC- 01.

Example 7

The concentrated H ₂ S0 ₄ (8 ml) in MeOH (12 ml) was added to the solution of β-escin (20 g) in MeOH (60 ml) at RT. A strong exothermic effect was observed. The addition funnel was washed with MeOH (6 ml). The whole solution was stirred under reflux for ca. 2 h (TLC control, Si0 ₂ , CHCl ₃ -MeOH-water 16:8: 1). The solution was cooled down to ca. 40-50°C and the solution of NaOH (15.2 g) in water (35.5 ml) was added, while observing a strong exothermic effect. The resulting yellowish-orange suspension was stirred under reflux for ca. 2 h (TLC control, CHC1 ₃ - AcOEt-MeOH 45:45: 10). The suspension was cooled down and filtered, the solid was washed with MeOH (2x50 ml) and discarded. The resulting filtrate was transferred to the reactor and heated to ca. 50-55°C. MTBE (300 ml) was added and then water (500 ml) was slowly dropped in while keeping the temperature above 50°C, until the solid occurred at the phase boundary. The additional amount of 100 ml of water was dropped in. The suspension was left overnight to cool down. The solid was filtered off, washed with water (2x50 ml) and acetonitrile (2x30 ml). After drying, 3.41 g of the solid EC- 05.T was obtained, UPLC: 86.9 % PES-01 ; 2.77 % BAC-01.

Example 8

The wet solid EC-05.S (21 g; 5.8 % of the solid obtained from 100 g of β-escin, water content ca. 80%, UPLC: 51.2% PES-01, 19.8% BAC-01) was dissolved in the mixture consisting of MeOH (30 ml), tetrahydrofuran THF (40 ml) and H ₂ 0 (10 ml) under reflux. The heating was removed, a portion of 40 ml H ₂ 0 was added to the hot solution and the mixture was seeded with pure PES-01 (UPLC 98%). The additional amount of H ₂ 0 (25 ml) was added. After a white solid precipitated, the suspension was left overnight to cool down. The solid was filtered off, and washed with H ₂ 0 and acetonitrile. The solid was dried at 50-55°C for 16 h. Yield: 827 mg, UPLC: 70.12% PES-01, 24.14% BAC-01.

Example 9

The wet solid EC-05.S (42 g; 10.6% of the solid obtained from 100 g of β-escin, water content ca. 80%, UPLC 51.2% PES-01, 19.8% BAC-01) was dissolved in the mixture consisting of MeOH (90 ml), diisopropyl ether DIPE (50 ml) and H ₂ 0 (15 ml) under reflux. The additional amount of DIPE (100 ml) was added, forming two layers of the solution and the mixture was left to cool down. The layers were separated, H ₂ 0 (75 ml) was dropped to the ether layer at RT, while stirring vigorously. Two layers were formed and the white solid precipitated. The suspension was stirred for lh and the additional amount of water (25 ml) was added. The suspension was left overnight. The solid was filtered off, washed with H ₂ 0 and acetonitrile. The solid of EC-05.T was dried overnight in the vacuum drier at 40°C. Yield: 2.64 g, UPLC: 92.7% PES-01, 2.8% BAC-01.

Example 10

The wet solid of EC-05.S (19.5 g, obtained from β-escin according to the procedure in Example 1, UPLC 59.4% PES-01, 19.5% BAC-01) was dissolved in the mixture consisting of EtOH (100 ml), MTBE (100 ml) and H ₂ 0 (100 ml) under reflux. A portion of MTBE (75 ml) was added to the clear solution, resulting in two layers formation. The heating was removed and the solution was left to cool down to RT. The layers were separated, H ₂ 0 (40 ml) was added to the ether solution at RT, while stirring vigorously. Two layers were formed and a white solid precipitated. The suspension was stirred for lh and the additional amount of water (15 ml) was poured in. The suspension was left overnight. The solid was filtered off, washed with water and acetonitrile. The solid of EC-05.T was dried at 50-55°C for 16 h. Yield: 1.96 g, UPLC: 71.10% PES-01, 20.87% BAC-01. The solid was dried at 50-55°C for 16 h. Yield 0.57 g of EC-05.T, UPLC: 79.40% PES- 01, 4.52% BAC-01.

Example 11

The wet solid of EC-05.S (42 g, obtained from 10 g of β-escin according to the procedure in Example 1, UPLC: 48.3% PES-01, 19.5% BAC-01) was dissolved in the mixture consisting of i-PrOH (30 ml), MTBE (30 ml) and H ₂ 0 (100 ml) under reflux. A portion of 50 ml H ₂ 0 and 30 ml MTBE were further added to the clear solution, resulting in a two layers formation. The heating was removed, then the solution was left to cool down to RT and it was further stirred at the same temperature for 20 min. A white solid precipitated at the phase boundary. It was filtered off and washed with H ₂ 0 (50 ml). The solid of EC-05.T was dried at 50-55°C for 16 h. Yield: 0.57 g, UPLC: 79.40 % PES-01 , 4.52 % BAC-01.

Example 12

The concentrated H ₂ S0 ₄ (40 ml) in MeOH (300 ml) was added to the solution of β- escin (100 g) in MeOH (50 ml) at RT. The addition funnel was washed with MeOH (50 ml). The resulting solution was stirred under reflux for ca. 2 h (TLC control, Si0 ₂ , CHCl ₃ -MeOH-water 16:8: 1). The solution was cooled down to ca. 40-50°C and the solution of NaOH (76 g) in water (175 ml) was added. The resulting yellowish-orange suspension was stirred under reflux for ca. 2 h (TLC control, CHCl ₃ -AcOEt-MeOH 45:45:10). MeOH (850 ml), MTBE (1250 ml) and water (525 ml) were added to the heated suspension. A clear, bi-layer solution was formed. After the additional portion of water was added (650 ml), the heating was stopped and the solution was cooled down. After ca. 10 min a white solid occured. The additional amount of water (500 ml) was dropped in. The suspension was heated for ca. 1 h and was left overnight to cool down. The solid was filtered off, washed with some portions of H ₂ 0 (750 ml in total) to bring the pH of the filtrate below 8.0 and then the solid was washed with acetonitrile (1 x 150 ml). After drying, 22.1 g of the solid EC-05.T was obtained, UPLC: 87.2 % PES-01 ; 5.3% BAC-01. Example 13

The concentrated H ₂ S0 ₄ (6 ml) in MeOH (50 ml) was added to the solution of β-escin (20 g) in MeOH (15 ml) at RT. The addition funnel was washed with MeOH (15 ml). The resulting solution was stirred under reflux for ca. 2 h (TLC control, Si0 ₂ , CHCI3- MeOH-water 16:8: 1).

The solution was cooled down to ca. 40-50°C and the solution of NaOH (11.2 g) in water (35.5 ml) was added. The resulting yellowish-orange suspension was stirred under reflux for ca. 2 h (TLC control, CHCl ₃ -AcOEt-MeOH 45:45: 10). MeOH (210 ml), diisopropyl ether (250 ml) and water (300 ml) were added to the heated suspension. The additional portion of water was dropped in (300 ml). The heating was stopped and the solution was cooled down to RT. During the course of cooling, a white solid precipitated. The suspension was heated for ca. 1 h and was left overnight to cool down. The solid was filtered off, washed with H ₂ 0 (2x100 ml) to bring pH of the filtrate below 8.0 and then the solid was washed with acetonitrile (2x25 ml). After drying at 45°C, 5.61 g EC-05.T was obtained; UPLC: 70.11 % PES-01 ; 20.46 % BAC-01.

Example 14

The concentrated H ₂ S0 ₄ (8 ml) in MeOH (10 ml) was added to the solution of β-escin (20 g) in MeOH (60 ml) at RT. The addition funnel was washed with MeOH (10 ml). The resulting solution was stirred under reflux for ca. 2 h (TLC control, Si0 ₂ , CHC1 ₃ - MeOH-water 16:8: 1).

The solution was cooled down slightly to ca. 40-50°C and the solution of NaOH (15.2 g) in water (35,5 ml) was added. A strong exothermic effect was observed. The resulting yellowish-orange suspension was stirred under reflux for ca. 2 h (TLC control, CHCl ₃ -AcOEt-MeOH 45:45: 10).

MeOH (120 ml) and MTBE (220 ml) were added to the heated suspension. Then water was slowly dropped in. After dropping in 185 ml of water, a clear bi-layer solution was obtained. The solution was cooled down to 30°C. The layers were separated to give a water phase (1) and an organic phase (l).MTBE (100 ml) and MeOH (70 ml) were added to the water phase (1), and the mixture was stirred vigorously for 10 min. The phases were separated to give a water phase (2) and an organic phase (2).

The organic phase (1) was placed in the flask and water (30 ml) was added at RT (ca. 24C). The solid precipitated. After stirring, the additional amount of water (20 ml) was added. The reaction was stirred overnight. The solid was filtered off, washed with H ₂ 0- MeOH solution (7:3, 2x50 ml) and with acetonitrile (25 ml). After drying overnight at 45°C, a white solid EC-05.T, 2.99 g was obtained; UPLC: 89.6 % PES-01 ; 1.0% BAC- 01.

Comperative Example 15 Series 1.0 (Table 1)

EC-05.T was subject to some crystallization in methanol, according to the following description.

1.1 - 1. Mixture of sapogenins EC-05.T (6.1 g; UPLC: 73.83% PES-01 , 3.66% BAC-01 ,

1 1.65% of impurity of RRT 2, 17), was dissolved in MeOH (200 ml) and was allowed to cool down. The obtained solid was filtered off and washed with acetonitrile (3 x 12 ml). 3.65 g (59.9 %) of the solid 1.1 -1 was obtained; UPLC: 3.23% RRT(0.95), 78.51% PES-01 , 1.1 1% BAC-01.

1.2- 1. Solid 1.1 -1 (3.56 g) was dissolved in MeOH (131 ml) under reflux and was allowed to cool down to RT. The obtained solid was filtered off, washed with cold MeOH (3 10ml) and dried. The solid 1.2-1 , 1.97g (55.34 %) was obtained; UPLC: 4.28% RRT(0.95), 90.39% PES-01 , 0.39% BAC-01.

1.3- 1. Solid 1-2-1 was dissolved in MeOH (85 ml), then ca. 40 ml MeOH was distilled off. After a moment the solid started to precipitate. The suspension was cooled down to RT, solid was filtered off, washed with acetonitrile (3 x 10ml). The solid 1.3-1 , 1.43 g (75.26 %) was obtained; UPLC: 5.28% RRT(0.95), 92.65% PES- 01 , 0.21% BAC-01.

1.4- 1. Solid 1.3-1 (1 ,2 g) was dissolved in the boiling MeOH (40 ml) and allowed to cool down. The precipitated solid was filtered off, washed with cold MeOH (2x5 ml), and dried. The solid 1.4-1, 750 mg (62.42 %) was obtained; UPLC: 5.07% RRT(0.95), 93.20% PES-01, 0.07% BAC-01.

The results of product purity assays following the subsequent steps of crystallization are set forth in Table 1. Even a 4-fold crystallization in MeOH did not allow to purify EC- 05. T to the purity higher than 93%. The main impurity was the compound of RRT(0.95).

Example 15

Process for the purification of EC-05.T by crystallization in different solvent systems. Many attempts at crystallization have been performed in the aim of protoescygenin purification from the impurities present in EC-05.T mixture. The results are collected in Table 2 and Table 3, wherein RRT, ie. the relative retention time of the analyzed peak, was determined by the following formula:

RRT= RT_analvsed peak

RT PEs-oi , where RT is the recorded retention time.

There are two peaks within RRT = 1.07-1.10, for which no good resolution was achieved, so they are treated as the sum of impurities (1.09). The impurities of a minor content are not represented in the tables.

A. Removing of the impurities in the mixtures of solvents selected from among the Group B (,,ηοη-polar" impurities)

Series 2.0 (Table 2)

2.2-5. The mixture of sapogenins (UPLC: 94.12% PES-01, 1.77% BAC-01), 0.5 g, was suspended in acetone and heated to boiling temperature for 5 h, and after that the heating was stopped and the suspension was allowed to cool down overnight. The solid was filtered off, washed with acetone and dried overnight in the drier at 40°C. A white precipitate 2.2-5, 0.12 g (25.3%) was obtained, UPLC: 94.70% PES-01, 0.21% BAC-01. Series 5.0 (Table 2)

5-34. The mixture of sapogenins (UPLC: 87.16% PES-01, 5.29% BAC-01), 0,5 g, was dissolved under reflux in EtOH (3.8 ml). Then, maintaining the boiling temperature hexane fraction was dropped in (16 ml), the heating was turned off and the mixture was allowed to cool down. After some minutes a solid precipitated from the solution. The mixture was left overnight. The solid was filtered off, washed with hexane and dried. The white solid 5-34, 0.36 g (72.8%) was obtained; UPLC: 92.78% PES-01, 0.98% PES-01.

5-39. The mixture of sapogenins (UPLC: 87.16% PES-01, 5.29% BAC-01), 0,5 g, was dissolved under reflux in i-PrOH (5 ml). Then, maintaining the boiling temperature, cyclohexane was dropped in (37 ml), the heating was turned off and the mixture was allowed to cool down. After some minutes a solid precipitated from the solution. The mixture was left overnight. The solid was filtered off, washed with i-PrOH/cyclohexane (5:40) and dried. A white solid 5-39, 0.39 g (77.6%) was obtained; UPLC: 91.06% PES- 01, 1.17% BAC-01.

Conclusion: The impurities of RRT>1.47 are removed in Ci-C ₃ alcohols and the mixtures thereof, as well as in their mixtures with acetonitrile, ethers and hydrocarbons; their content after single crystallization decreases below the detection level. BAC-01 (RRT=1.14) is purified in all examined solvent mixtures, preferably in alcohol with the admixture of hydrocarbon or acetonitrile. The impurity of RRT=1.09 is preferably removed in the mixtures of alcohol and hydrocarbons or acetonitrile and by maceration with acetone. The impurity of R T=1.06 practically is not removable in the solvent systems tested herein (the assays of purity change are within the error of measurement). The impurity of RRT=0.95 is not removable in the systems tested herein.

B. Removing of impurities in the mixtures of solvents selected from among the Group A („polar" impurities)

In Table 3, the examples of removing the polar impurities, of RRT = 0.95 and 1.06, as well as intermediate of RRT = 1.09, are set forth.

Series 3.1 (Table 3) 3.1. The mixture of sapogenins EC-05.T (5.8 g; UPLC 75.8 % PES-01, 5.07% BAC-01, 10.98% impurities of RRT 2.17), was dissolved in the mixture consisting of MeOH (155 ml) and acetonitrile (100 ml) under reflux and was allowed to cool down to RT. The precipitated solid was filtered off, washed with acetonitrile (2x 12 ml), and dried in the vacuum drier at 40°C. The solid 3.1, 3.52 g (60.5%), UPLC: 3.09% RRT(0.95), 78.72% PES-01, 1.36% BAC 01, 8.50% RRT(2.17), was obtained.

3.2-6. The obtained solid 3.1 (0.50 g) was dissolved at 90-100°C in n-PrOH and water was added (10 ml). A solid precipitated. After cooling down to RT, the solid was filtered off, washed with n-PrOH/water and dried in the vacuum drier at 40°C. The solid 3.2-6, 0.30 g (60.5 %), UPLC: 96.89% PES-01, 1.85% BAC-01, was obtained.

Series 5.0 (Table 3)

5-15. The mixture of sapogenins EC-05.T, UPLC: RRT(0.95) - 3.03%, PES-01 - 87.16%, BAC-01 - 5.29%), 0,5 g, was dissolved in propionic acid (10 ml) at 90°C. Then water was dropped in (7.5 ml) and a solid started to precipitate. The additional amount of water was added (2.5 ml, 10 ml in total), the heating was turned off and the mixture was allowed to cool down. The solid was filtered off, washed with water and dried. The white solid 5-15, 0.32 g (64,2%), UPLC: 0.40% RRT(0.95), 90.64% PES-01, 7.26% BAC-01 , was obtained.

5-19. The mixture of sapogenins EC-05.T, UPLC: RRT(0.95) - 3.03%, PES-01 - 87.16%, BAC-01 - 5.29%), 0.5 g, was dissolved in N-methylpyrrolidone NMP (10 ml) at ca. 90°C. Water was added (6 ml), and the solution became turbid. The heating was turned off. After some minutes a precipitate occured. The suspension was allowed to cool down overnight. The solid was filtered off, washed with water and dried. The white solid 5-19, 0.34 g (67.8%), UPLC: 0.29% RRT(0.95), 91.36% PES-01, 6.87% BAC-01, was obtained.

5-28. The mixture of sapogenins EC-05.T, UPLC: RRT(0.95) - 3.03%, PES-01 - 87.16%, BAC-01 - 5.29%), 0.5 g, was dissolved in the mixture consisting of MeOH (10 ml) and MTBE (8 ml) under reflux. Water was added (4 ml) and a solid precipitated. The additional amount of water was added (1 ml). The heating was turned off and the mixture was left to cool down overnight. The solid was filtered off, washed with water and acetonitrile, and dried. The white solid 5-28, 0.30 g (60.8%), UPLC: 0.73% RRT(0.95), 89.79% PES-01, 7.76% BAC-01, was obtained.

Conclusion: The impurity of RRT=0.95 is removed well in all tested solvent systems. The impurity of RRT=1.06 is removed by crystallization in ethanol, propan-2-ol, propionic acid, higher amides and MeOH/ether solvent system, preferably in propan-2- ol/water. The impurity of RRT=1.09 is removed by crystallization in ethanol, propan-2- ol, acetic acid, N-methylpyrrolidone and methanol/ether solvent system, preferably in propan-2-ol/water. In the tested systems non-polar impurities of RRT>1.4 are remmoved, however BAC-01 (RRT=1.14) is not removed. In some samples after acid solvents treatment the small amounts of impurity of R T=1.19 are generated.

Example 16

Multi-step crystallization

Series 6.0 (Table 4) 6-1. The mixture of sapogenins EC-05.T (10.0 g; UPLC: 78.94% PES-01, 12.43% BAC-01), was dissolved in i-PrOH (340 ml) under reflux. The mixture was cooled down to 50°C and an undissolved brown solid was filtered off. The solid was washed with i-PrOH (55 ml), and heated to boiling. Water was dropped in (330 ml) under reflux, a solid started to precipitate. The mixture was left overnight to cool down to RT. The obtained solid was filtered off, washed with water, and dried overnight in the vacuum drier at 40°C. A white solid 6-1, 6.49 g, (64.90%), was obtained.

6-2. The solid 6-1 (6.39 g) was dissolved in i-PrOH (65 ml) and was heated to 60°C (ie. i-PrOH/cyklohexane aseotrope b.p.). Cyclohexane (CyH) (250 ml) was dropped in, a solid precipitated. The mixture was left overnight to cool down to RT. The precipitated solid was fitered off, washed with iPrOH/CyH (1 :3; 80 ml) and dried in the vacuum drier. A white solid 6-2, 2.458 g (69.5%), was obtained.

6-3. The solid 6-2 (4.50 g) was dissolved in i-PrOH (75 ml) and heated to 60°C. Cyclohexane (400 ml) was added. The heating was removed and the mixture was left to cool down overnight. A solid was filtered off, washed with i-PrOH - cyclohexane mixture (1 :4, 2x25 ml), and dried overnight in the laboratory drier at 40°C. A white solid 6-3, 3.78 g (83.9%), was obtained.

The purity of the products after subsequent steps of crystallization are set forth in Table 4. The end product obtained by the crystallization in the solvent system i-PrOH - cyclohexane was identified by XRPD, IR (KBr), DSC and TGA methods to be protoescygenine monohydrate (crystalline form III).

The chemical structure of the obtained compound was confirmed by Ή NMR and ¹³ C NMR.

1H NMR (DMSO-d ₆ ), δ (ppm): 5.19 (1H, m, H12), 4.96 (1H, d, J = 4.8 Hz, C3-OH), 4.43 (1H, dd, J = 4.4 and 5.8 Hz, C28-OH), 4.22 (1H, d, J = 4.4 Hz, C16-OH), 4.06 (1H, dd, J = 3.0 and 7.5 Hz, C24-OH), 4.03 (1H, m, H16), 3.97 (1H, d, J = 4.2 Hz, C21-OH), 3.82 (1H, m, H24), 3.81 (1H, d, J = 4.9 Hz, C22-OH), 3.79 (1H, dd, J = 4.3 and 9.6 Hz, H21), 3.60 (1H, dd, J = 5.0 and 9.6 Hz, H22), 3.27 (1H, dd, J = 7.6 and 1 1.0 Hz, H24), 3.18 (1H, m, H3), 3.15 (1H, dd, J = 6.0 and 10.2 Hz, H28), 2.99 (1H, dd, J = 4.3 and 10.2 Hz, H28), 2.35 (1H, m, H19), 2.27 (1H, dd, J = 4.2 and 14.2 Hz, H18), 1.80 (2H, m, Hl l), 1.62-1.59 (2H, m, H15 and H2), 1.56-1.50 (4H, m, H6, H2, HI and H9), 1.41 (1H, dd, J = 3.9 and 12.7 Hz, H7), 1,37 (1H, dd, J = 2.4 and 12.5 Hz, H6), 1.34 (3H, s, C14-CH ₃ (27)), 1.25 (1H, m, H7), 1.19 (1H, dd, J = 2.3 and 14.8 Hz, H15), 1.08 (3H, s, C4-CH ₃ (C23)), 0.94-0.90 (2H, m, H19 and HI), 0.87 (3H, s, C10-CH ₃ (C25)), 0.84 (3H, s, C20-CH ₃ (C29)), 0.81 (3H, s, C8-CH ₃ (C26)), 0.80 (3H, s, C20-CH ₃ (C30)), 0.75 (1H, dd, J = 1.8 and 12.0 Hz, H5).

^I3 C NMR (DMSO-d ₆ ), δ (ppm): 143.1 (C13), 121.8 (C12), 78.6 (C3), 76.8 (C21), 74.0 (C22), 66.6 (CI 6), 65.2 (C28), 63.0 (C24), 55.4 (C5), 47.2 (CI 9), 46.2 (C9), 46.0 (CI 7), 42.1 (C4), 40.9 (C14), 39.5 (C18), 39.2 (C8), 38.2 (CI), 36.3 (CIO), 35.3 (C20), 33.2 (C15), 32.8 (C7), 30.0 (CH ₃ 29), 27.2 (C2), 26.7 (CH ₃ 27), 23.2 (Cl l), 22.9 (CH ₃ 23), 18.8 (CH ₃ 30), 18.6 (C6), 16.3 (CH ₃ 26), 15.7 (CH ₃ 25).

Table 1. Crystallization in methanol (Comperative Example 15)

MeOH - methanol

Table 2. Crystallization - removal of the impurities in the solvents selected from group B ('non-polar") (Example 16)

* Amount added after solid precipitation; MeOH - methanol; EtOH - ethanol, iPrOH - propan-2-ol; nPrOH - propan-l-ol; iPr ₂ 0 - diisoprop ether; THF - tetrahydrofuran; CH ₃ CN - acetonitrile; Tol - toluene; CyH - cyclohexane; Hex - hexane; Hep - heptane

Table 3. Crystallization - removal of the impurities in the solvents selected from group A („polar" impurities) (Example 16)

MeOH - methanol; EtOH - ethanol, iPrOH - propan-2-ol; nPrOH - propan-l-ol; AcOH acetic acid; EtC0 ₂ H - propionic acid; DMF - N, dimethylformamid; DMAc - N,N-dimethylacetamid; NMP - N-methylpyrrolidone; DMSO dimethylsulfoxide; MTBE - tert-buthylmethyl eth

Table 4. Purification of the mixture of sapogenins EC-05.T in the multi-step crystallization (Example 17)

*) For impurities of RRT values ranging from 1.07 to 1.10 the chromatographic separation is not possible; they are treated as a total impuri represented by 1.09 RRT value.

4-

O