Process for preparation of 3,24;16,22-di-0,O-isopropylideneprotoescigenin and crystalline forms thereof

Field of the invention

The present invention relates to preparation of diisopropylidene derivative of protoescigenin, 3,24; 16,22-di-O, O-isopropylideneprotoescigenin, and crystalline forms thereof. Protoescigenin is polyhydroxy triterpene aglycon obtained by hydrolysis of escin, isolated from Aesculum hippocastanum L.

3,24;16,22-Di-O, O-isopropylideneprotoescigenin, which systematic name and carbon atoms numbering according to IUPAC rules is: 3,24;16,22-di-O, O- isopropylidene-(3 β,4β,16α,21 p,22a)-olean- 12-en-3 , 16,21 ,22,23 ,28-hexol, represented by the structure (I),

can be used as the synthon to obtain natural saponin derivatives Background of the invention

The complex mixture of saponins known under the common name escin, is the major component of horse chestnut seed (Aesculus hippocastanum∑.). It is present in the three forms named a-escin, β-escin and cryptoescin. These saponins belong to polyhydroxy 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.

Until now 79 saponins have been isolated and characterized from the hydrolyzates of Aesculus hippocastanum L. extracts. Most of these compounds consist of a trisaccharide chain containing glucuronopyranosyl residue linked via a glycosidic bond to the C-3 atom of an aglycon, acyl groups at C-21, C-22 and C-28, and 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 represented by the formulae 2,

agfycone

glycone

by: Pharm. Crops, 201

wherein Ang - 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 Aesculus hippocastanum seeds extracts. The outcome of these works was 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.

PES - Protoescigenin

BAC - Barringtogenol C

Escin, due to its beneficial effects on venous tone, anti-inflammatory and anti- edema activity, is wildly used in medicine, mainly in the treatment of chronic venous insufficiency, but it found application 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 qualitative and quantitative determination of the natural compounds composition, impedes studies on pharmacokinetics 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 resulting in similar physico chemical properties, for example polarity, and in the end lack of chromophores, hinders detection of the analyzed molecules.

In general, saponins are isolated from the crude plant material by extraction with the mixture of water and alcohol, such as methanol or ethanol, followed by evaporation of the solvents under reduced pressure, reconstitution of the solid residue in a small amount of water and separation between n-butanol / water bi-phase system. For further purification, the column chromatography techniques or liquid-liquid chromatography separation methods are employed, but usually high-performance liquid chromatography (HPLC) 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 Aesculus 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 containing the pre-purified mixture of saponins furnished separation of escin la, lb, Ila, lib and Ilia. In 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. et 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 published procedure, n-butanolic extract of Aesculus seeds was condensed in vacuo furnishing a resin residue. This product was refluxed in ether resulting in precipitation of the solid of the crude mixture of saponins, which were 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 a mixture of four main compounds. The major sapogenin was purified by crystallization in methanol and precipitated as colorless needles, characterized by 300-307°C melting point. The physico chemical data, such as melting point, IR (KBr) spectra and TLC analysis were consistent with the structure of protoescigenin.

According to the aforementioned publications, cleavage of the glycosidic bond of deacylated escin occurs during 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, methods other than chromatography separation 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 chromatographic purification of either the mixtures or individual saponins. Following these multi-step purification processes, some of the pentacyclic triterpenes were successfully isolated and purified on laboratory scale. However, these elaborate and expensive methods cannot be implemented on industrial scale.

The main problem one must face while scaling-up the process, is the viability of the crude extract composition, the similar polarity, and the similarity of molecular weight of particular components of the saponin mixture. These physico chemical 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 ultrafiltration. Possibility of obtaining protoescigenin of high purity and at bulk quantities is crucial for the substrate to be used in synthetic modifications. In the molecule of protoescigenin 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 protoescigenin is contaminated with aglycons of the other saponins. In the aftermath of chemical reaction, complex mixtures of products of similar structures are formed, separation of which is impossible.

The results of experimental attempts to produce protoescigenin by hydrolysis of escin demonstrate that the reaction product is usually the mixture of sapogenins, containing from 40 to 60% of protoescigenin only, as determined by HPLC. Among the other main components of the mixture, barringtogenol C is also detected, which is accompanied by 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 protoescigenin isolation.

Efficient method of enrichment of sapogenin mixture, obtained by hydrolysis of escin under optimal conditions, has been described in the International Patent Application PCT/PL 2012/000102. Isolation of crystalline protoescigenin monohydrate of high chemical purity, essential for further chemical derivatizations, became possible following the disclosed procedure. Protoescigenin monohydrate can be used as the synthon in the regioselective synthesis of triterpene derivatives, selectively protecting hydroxyl groups, for example with isopropylidene moieties and subjecting the unprotected groups to further chemical transformations.

Preparation of mono- and di-O-isopropylidene derivatives of protoescigenin and barringtogenol C was disclosed inter alia, in Tetrahedron 1966, 22, 1899-1906 and Liebigs Ann. Chem. 729, 205, 212 (1969). Escin was hydrolyzed under acidic conditions, the obtained mixture of 28-tigloyl and/or 28-angeloyl protoescigenin esters was treated with acetone in presence of anhydrous copper sulfate to yield acetonide derivatives. The resulting mixture was subjected to chromatography to furnish 28- acylated mono- and di-isopropylidene protoescigenin derivatives, containing 3,24;16,22-di-0,O-isopropylideneprotoescigenin, which was separated by chromatography. The latter compound was hydrolysed under basic conditions. This method was not used for direct preparation of 3,24;16,22-di-0,O-isopropylidene- protoescigenin from protoescigenin. The attempts of preparative application of this procedure to obtain diisopropylidene-protoescigenin failed. The reaction of protoescigenien with acetone or 2,2-dimethoxypropane in presence of anhydrous copper sulfate proceeds very slowly, in addition the substrate is not entirely consumed and the inseparable mixture of protoescigenin acetonides is obtained. In Chem. Pharm. Bull. 18, 8, 1621-1630 formation of acetonide derivative of sapogenol extracted from Thea sinensis, upon acetone and dimethoxypropane treatment in presence of p-toluenesulfonic acid, has been disclosed. Similarly to the previously discussed protocols, following this methodology chromatographic separation and purification of the final product was necessary. To develop the laboratory scale synthesis of diverse protoescigenin derivatives, simplified bulky-scale method of 3,24;16,22-di-O, O-isopropylideneprotoescigenin preparation, avoiding chromatographic separation of this compound from the complex mixture of acetonides, was the challenging task.

The reaction of sapogenol acetonide formation, employing acetone, 2,2- dimethoxypropane and p-toluenesulfonic acid has some drawbacks such as, lack of selectivity and implementation of elaborate purification techniques to separate the expected products. Additional obstacle is low stability of the obtained acetonide derivative, which is attributed to presence of trace amounts of acidic impurities, like for example p-toluenesulfonic acid. 3,24;16,22-Di-O, O-isopropylideneprotoescigenin (I) is isolated as a solid, but when dissolved it easily undergoes disproportionation, yielding the mixture of acetonides and/or protoescigenin.

The present invention aims at development of technological process for preparation of the compound (I), furnishing the product of high purity and stability reproducibly, in high overall yield. Summary of the invention

The synthetic goal has been achieved owing to the finding, that acetonidation when carried out in acetone and/or 2,2-dimethoxypropane in presence of p- toluenesulfonic acid, results in delayed precipitation of the product identified as 3,24;16,22-di-0,0-isopropylideneprotoescigenin (I), from the crude mixture of acetonides.

This unexpected founding triggered the researchers to find optimal conditions of acetonidation such as, appropriate ratio of acetone and 2,2-dimethoxypropane in relation to amount of protoescigenin used, as well as reaction time, temperature and precautions to be taken while handling the unstable product.

Process for preparation of 3,24;16,22-di-O,O-isopropylidenoprotoescigenin (I) according to the present invention comprises:

1) acetonidation of protoescigenin monohydrate in the mixture consisting of 0 - 100% vol. of acetone and 100 - 0% vol. of 2,2- dimethoxypropane, in presence of catalytic amount of an acid, furnishing precipitation of 3,24;16,22-di-0,0- isopropylideneprotoescigenin from the reaction mixture, stirring the suspension of 3,24; 16,22-di-0,0- isopropylideneprotoescigenin and a base, which forms a salt with the acid used in step 1), said salt is soluble under reaction conditions,

3) isolation of the solid of 3,24;16,22-di-0,0- isopropylideneprotoescigenin from the reaction mixture,

4) purification of 3,24; 16,22-di-O, O-isopropylidenoprotoescigen.

Process according to the present invention can be realized in the following To the suspension of protoescigenin monohydate in the mixture consisting of 2,2-dimethoxypropane and acetone, which are the reagents and the solvents at the same time, catalytic amount of an acid is added to initiate the reaction and cause the dissolution of the solid. During the course of this process, the mixture of protoescigenin acetonides is formed. After some period of time, usually 15-30 min., 3,24;16,22-di-O,O- isopropylidenoprotoescigen is precipitating from the reaction mixture. To this suspension small amount of a base is added to neutralize the catalyst, continuing stirring for additional ca. 30 min. Then, the solid is isolated by filtration or decantation and washed with acetone to give crude 3,24;16,22-di-0,0-isopropylideneprotoescigenin. Preferably, acetonidation is carried out in the mixture consisting of 0 - 100% vol. of acetone and 100 - 0% vol. of 2,2-dimethoxypropane, calculated on total reaction volume. The reaction can be performed in either acetone or 2,2-dimethoxypropane itself, preferably the reaction is carried out in acetone with addition of some amount of 2,2- dimethoxypropane, for example, at 17: 1 volume ratio of acetone and 2,2- dimethoxypropane, which significantly shortens reaction time. Preferably, other volume ratios, 7:3, 3:2 or 1 :1 for instance, of acetone and 2,2-dimethoxypropane can be used in this process. The optimal volume ratio of acetone and 2,2-dimethoxypropane is 1 :1. When decreasing volume of acetone in favor of increased amount of 2,2-dimethoxypropane, slightly diminished reaction yield and improved purity of the final product are observed. The amount of precipitated 3,24;16,22-di-0,0-isopropylideneprotoescigenin depends on the volume of the solvents used in relation to the amount of initial protoescigenin monohydrate. The expected product precipitates in high yield when total solvents volume in relation to protoescigenin monohydrate does not exceed 50 mL/g. Minimum volume of solvents depends on the chemical equipment affordable for scaling down, usually is not smaller than 2,5 mL/g. Preferably, the minimum ratio accounts for 10 mL/g.

Reaction temperature does not essentially affect acetonidation course and can range from 10 to 50°C. As regards technological processes, it is convenient to carry out the reaction at 18-30°C temperature range. Acids used as catalysts in acetonidation may include inorganic acids such as, hydrochloric acid (30% aqueous solution), sulfuric acid (98%, about 1 : 1 solution in acetone), or organic acids such as, p-toluenesulfonic acid, trifluoroacetic acid or the other strong acids. Acetonidation carried out in presence of inorganic acids is accomplished from

30 min. to several hours, yielding 3,24;16,22-di-0, O-isopropylidenoprotoescigen as a high purity solid. The reaction progress is monitored by TLC. While using trifluoroacetic acid the process runs slower and the obtained product contains more impurities (TLC). Use of acetic acid results in formation of the mixture of products contaminated with the unconsumed substrate.

Most preferably, acetonidation is carried using p-toluenesulfonic acid.

To neutralize acid used as the catalyst, a base is added, especially aliphatic amines such as, diisopropylethylamine, triethylamine; aromatic amines such as, pyridine; and ammonia (25% aqueous solution) or other amines, which form, soluble in the reaction mixture, salts with acids used in the prior step. The base added also contributes toward stabilizing the obtained product.

Preferably, amine of choice is triethylamine.

Crude 3,24; 16,22-di-0,0-isopropylideneprotoescigenin, which is the solid product of acetonidation carried out according to the present invention, is characterized by 94-96% purity determined by HPL or UHPLC. This purity is satisfactory as far as synthetic application is concerned, but additional purification is appropriate prior further chemical transformations are undertaken.

There are two main groups of crude product impurities: by-products of higher polarity than 3,24;16,22-di-0,0-isopropylideneprotoescigenin (Rti _mpur ity _x < RTi, for the used HPLC method), characterized by RRT = 0.66; 0.79; 0.88 coefficient, total amount of these impurities content in the solid accounts for about 1.5-3%, the by-products of lower polarity than 3,24;16,22-di-0,0-isopropylideneprotoescigenin (Rtj _m p _U rity x < RTi, for the W

12

used HPLC method), characterized by RRT = 1.03; 1.07 coefficient, total amount of these impurities content in the solid accounts for about 1-4%.

The crude solid can be purified by suspending and stirring (so called 'maceration') 3,24;16,22-di-0,0-isopropylideneprotoescigenin in single selected solvent such as C _1-2 alcohol (methanol, ethanol), ketone (acetone), nitrile (acetonitrile), dichloromethane or in C _6-7 carbohydrate (n-hexane, cyclohexane, n-heptane), preferably in MTBE, n-heptane or ethanol. Maceration is carried out by heating the suspension of 3,24;16,22-di-0,0-isopropylideneprotoescigenin at given temperature for certain period of time in a selected solvent, filtrating the solid at elevated temperature or after cooling down the mixture and washing the obtained product with selected solvent.

Alternatively, the crude solid of 3,24;16,22-di-0,0- isopropylideneprotoescigenin is purified by crystallization in selected solvent such as, Ci- ₃ alcohol (propan-l-ol, propan-2-ol), ether (THF), chlorinated hydrocarbons (chloroform), amide (DMF), sulfonamide (DMSO), preferably propan-l-ol. Crystallization is carried out by suspending the solid in selected solvent, heating the suspension until the solid is entirely dissolved, optionally filtrating the solution, initiating precipitation of the solid by cooling down the solution, filtrating and washing the solid with selected solvent.

In another embodiment of the present invention, the crude solid of 3,24; 16,22- di-0,0-isopropylideneprotoescigenin is purified by crystallization in a mixture of two selected solvents, dissolving the solid in one solvent and initiate precipitation adding a second solvent. The first selected solvent is Ci _-3 alcohol (propan-l-ol, propan-2-ol), ether (THF), chlorinated hydrocarbons (chloroform), amide (DMF), sulfonamide (DMSO), the second selected solvent is C _1-2 alcohol (methanol, ethanol), ketone (acetone), nitrile (acetonitrile), dichloromethane or C _6-7 hydrocarbons (n-hexane, cyclohexane, n-heptane), preferably MTBE, n-heptane, ethanol, preferably THF-water and n-PrOH- water.

Crystallization in a two-solvent mixture is carried out by dissolving 3,24; 16,22- di-0,0-isopropylideneprotoescigenin in one selected solvent at ambient or elevated temperature, then adding second solvent to the resulting solution. Precipitation of the solid can be accelerated adding anti-solvent, decreasing reaction temperature or using both methods.

To avoid degradation of the final product caused by acidic impurities of the used solvents, a base, preferable amine (for example triethylamine, Et ₃ N), at experimentally adjusted amount, usually accounting up to 0,2% v/v is added.

On account of low solubility in majority of solvents, preferably, the crude solid of 3,24;16,22-di-0,0-isopropylideneprotoescigenin is purified by maceration in such solvents as acetone, n-heptane, tert-butyl methyl ether with addition of triethylamine. Most preferably, maceration is carried out in tert-butyl methyl ether.

It has been proved that 3,24;16,22-di-0,0-isopropylideneprotoescigenin could be isolated in one out of two different . crystalline forms, named form A and form B, depending on crystallization conditions.

Crystalline forms A and B are characterized by different X-ray powder diffraction patterns (XRPD), which were recorded on the diffractometer equipped with the copper anode of Kaj λ = 1,54056 A wave length. Infrared spectroscopy (IR) proved to be appropriate alternate diagnostic method to distinguish these two crystalline forms.

Crystallization conditions, which influence formation of 3,24;16,22-di-0,0- isopropylideneprotoescigenin crystalline form A and B are presented below in Table 1 : Table 1

3,24; 16,22-di-Q O-

Solvent XRPD IR

isopropylidene-protoescigenin

Acetone, 2,2-

Crude reaction product A A

DMP

Acetone, 2,2-

Crude reaction product A A

DMP

Crystallization DMF A A Maceration MTBE A A

Maceration MTBE A A

Maceration MTBE A A

Crystallization propan- l-ol A A

Crystallization THF A A

Maceration Acetone B B

Maceration Acetonitrile B B

Maceration Acetonitrile B B

Maceration Cyclohexane B B

Maceration Cyclohexane B B

Maceration n-Heptane B B

Maceration n-Heptane B B

3,24;16,22-di-0,0-isopropylideneprotoescigenin crystalline form A is characterized by X-ray powder diffraction pattern (XRPD), represented as relative intensities of diffraction peaks I/I ₀ , diffraction angles 2Θ and interplanar distances d, using scanning range from 3 to 40°, scanning rate 0,5°/min and step size 0,02°, which are collected in Table 2:

Table 2

2Θ, [°] d, [A] I I _m ax, [%]

4.46 19.818 5

7.25 12.189 15

10.34 8.544 47

12.19 7.255 29

13.23 6.689 100

13.52 6.546 78

13.98 6.328 24

15.12 5.856 45

16.86 5.253 48 18.37 4.825 15

18.67 4.748 18

18.99 4.670 1 1

19.55 4.536 9

20.70 4.288 27

22.74 3.907 9

23.03 3.859 11

24.73 3.598 7

27.26 3.269 9

28.74 3.104 10

34.21 2.619 8

3,24;16,22-di-0,0-isopropylideneprotoescigenin crystalline form B is characterized by X-ray powder diffraction pattern (XRPD), represented as relative intensities of diffraction peaks I/I ₀ , diffraction angles 2Θ and interplanar distances d, using scanning range from 3 to 40°, scanning rate 0,5°/min and step size 0,02°, which are collected in Table 3 :

Table 3

2Θ, [°] d, [A] I Imax, [%]

4.37 20.204 2

9.16 . 9.643 15

10.87 8.133 100

12.52 7.066 35

13.44 6.584 35

14.38 6.155 10

14.90 5.939 20

15.36 5.764 20

16.26 5.446 10

16.70 5.304 13

16.97 5.221 13 17.47 5.071 20

18.40 4.818 24

24.86 3.578 11

Exemplary X-ray powder diffractograms of 3,24;16,22-di-0,0- isopropylideneprotoescigenin crystalline form A and B are depicted on Fig. 1 and Fig. 2 respectively, comparative analysis of these two diffractograms is presented on Fig. 3.

Infra-red spectrum of 3,24;16,22-di-0,0-isopropylideneprotoescigenin crystalline form A and B embedded in KBr tablet are depicted on Fig. 4 and Fig. 5 respectively.

DSC profiles of 3,24;16,22-di-0,0-isopropylideneprotoescigenin crystalline form A and B, obtained using differential scanning calorimetry are depicted on Fig. 6 and Fig. 7 respectively.

DSC curve of form A is characterized by broad endothermic effect at temperature range from about 40 to about 170°C, which is attributed to solvents evaporation, and endothermic effect resulting from substance melting. Melting point of form A measured as onset accounts for 1245,60 °C. The compound when melting is decomposing, this phenomenon is observed as base line disturbances, which are shown after melting spike.

DSC curve of form B is characterized by broad endothermic effect at temperature range from about 40 to about 140°C, which is attributed to solvents evaporation, and endothermic effect resulting from substance melting. Melting point of form B measured as onset accounts for 196,26 °C. The compound when melting is decomposing, this effect is shown on base line sloping down after melting spike.

TG curves were obtained using theromogravimetric (TGA) analysis, heating under dynamic regime from 30 to 300°C at heating rate 10°C/min. of 3,24;16,22-di- 0,0-isopropylideneprotoescigenin crystalline form A and B are depicted on Fig. 8 and Fig. 9 respectively. On form A TGA curve mass loss of 5,70% is shown, when heating at temperature range from 30 to about 180°C. Additional mass loss occurs at temperature about 220°C or higher. Comparison of TGA and DSC results proves, the first effect results from solvents evaporation, the second one is caused by substance decomposition. On form B TGA curve mass loss of about 2,99% is shown, when heating at temperature range from 30°C to about 270°C. This phenomenon is attributed to evaporation of solvents.

Both 3,24;16,22-di-0,0-isopropylideneprotoescigenin crystalline forms are hydrates, containing from 1,54 to 3,64% of water - form A, and from 2,57 to 2,68% water - form B, respectively.

The present invention provides the method for preparation of 3,24;16,22-di-0,0- isopropylideneprotoescigenin in high total yield, which accounts for about 80%.

According to the present invention, 3,24;16,22-di-0,0- isopropylideneprotoescigenin is obtained as crystalline solid, characterized by > 99% purity determined by HPLC, which can be used as the synthon in further chemical transformations.

The present invention is illustrated by the following examples.

Examples

Analytical methods Infra-red (IR) spectra were performed in pressed KBr tablets, on Bruker Alpha spectrometer at measurement range from 4000 to 400 cm ^"1 and 4 cm ^"1 resolution.

Proton nuclear magnetic resonance spectra 1H NMR and carbon nuclear magnetic resonance spectra ⁱ³ C NMR were recorded on Varian VNMRS 600 MHz spectrometer.

X-Ray powder diffractograms were recorded on X-ray powder diffractometer type MiniFlex by Rigaku, with the following parameters:

• Radiation: ΟιΚα1, λ=1, 54056 A • Scanning range 2Θ: from 3 to 40°

• Step size: 0,02°

• Scanning rate Δω: 0,5°/min

• Temperature of measurement: ambient temperature

• Detector: scintillating counter.

Data collected from diffractometer were calculated using DHN_PDS software.

Differential scanning calorimetry (DSC) measurements were performed in the furnace sample chamber DSC822 ^e by Mettler Toledo, under following conditions:

• Melting pot: aluminum, 40 μΐ, capacity,

• Purge gas: N ₂ , flow rate 60 mL/min,

• Measurement conditions: the samples were heated under dynamic regime from 25 to 180 °C at heating rate 10 °C/min,

• Samples preparation: the crystalline substances weighting aboout 7 mg were placed in the melting pots without prior preparation. The melting pots were air-tight pressed and punctured before the measurement.

Thermogravimetry measurements (TGA) were performed in the furnace sample chamber TGA/SDTA851 ^e by Mettler Toledo, under following conditions:

• Melting pot: aluminum, 40 μΐ, capacity,

• Purge gas: N ₂ , flow rate 60 mL/min,

• Measurement conditions: the samples were heated under dynamic regime from 30 to 180 °C at heating rate 5 °C/min,

• Samples preparation: the crystalline substances weighting about 7 mg were placed in the melting pots without prior preparation. The melting pots were air-tight pressed and punctured before the measurement. The empty pot correction was included in the measurements. Water content measurement was determined by Karla Fischer volumetric titration following Ph. Eur. 2.5.12 procedures, on Methrom 701 KF Titrino, using Methrom 6.0338.100 electrode.

Purity of the compounds was determined by Ultra Performance Liquid Chromatography (UPLC) technique on Dionex Ultimate 3000 UHPLC chromatograph, equipped with PDA detector.

Parameters of UPLC analysis:

• UPLC column: Acquity BEH C 18 2, 1 x 50 mm, 1 ,7μηι

• Column temperature: 30°C

• Mobile phase flow rate of: 0,5 ml/min

• Injection: 2 μΐ

• Detection at wave length: 200 nm

• Sample concentration: 2 mg/ml

• Solvent: MeOH, UPLC purity

• Phase A: 10 mM ammonium acetate, pH 6,8, UPLC purity

Phase B: acetonitrile, UPLC purity

T [min] % A % B

0 80 20

4.7 0 100

8 0 100

8.1 20 80

9.5 20 80 Example 1

Protoescigenin monohydrate (PES H ₂ 0, 505 mg, 1.0 mmol, 93.17%) was suspended in the mixture of acetone (10 mL, purris. p.a.) and 2,2-dimethoxypropane (2,2-DMP, 6 mL, 98%) in ambient temperature giving dense, white suspension. To the vigorously stirred suspension catalytic amount of ;?-toluenesulfonic acid (p-TSA, 98.5%) was added and the solid was dissolved in no more than 30 s (giving clear, colorless solution). The vigorous stirring was continued at ambient temperature for few hours. The reaction was left overnight. The next day stirring was continued, and after few hours the solution became cloudy, the solid started to precipitate. The resulting suspension was left overnight. Next day triethylamine (few drops) was added and the mixture was stirred for 30 min. The solid was filtered off and dried (overnight, 40°C in a vacuum dryer).

3,24;16,22-di-0,0-isopropylideneprotoescigenin (I) was obtained as a solid (320 mg, 54.7% yield), 99.5% purity (HPLC).

Example 2

Protoescigenin monohydrate (PES H ₂ 0, 2.52 g, 4.97 mmol, 97.0%) was suspended in the mixture of acetone (50 mL, purity p.a.) and 2,2-dimethoxypropane (2,2-DMP, 30 mL, 98%) at ambient temperature (23-24°C), giving dense, white suspension. To the vigorously stirred suspension catalytic amount of / oluenesulfonic acid monohydrate (p-TSA, 65 mg, 0.34 mmol, 98.5%) was added, and the solid was dissolved within 30 s (giving clear, colorless solution). The vigorous stirring was continued at ambient temperature for two days (during this time color of the mixture can change to dark- orange). After two days a colorless solid precipitated. Triethylamine (5 drops, -0.08 mL) was added and the mixture was stirred for 1 h. The solid was filtered off then dried (overnight, 40°C in a vacuum dryer) to yield 3,24;16,22-O,O-diisopropylidene- protoescigenin (I) as a colorless solid (1.78 g, 61.6% yield, 98.2% purity (HPLC)). W 201

21

Example 3

Protoescigenin monohydrate (1.0 g, 1.97 mmol, 95.63%) was suspended in acetone (10 mL, purity p.a.) at ambient temperature (23-24°C) giving very dense, white suspension. Addition of acetone (7 mL) improved stirring of the mixture. To the vigorously stirred mixture catalytic amount of /?-toluenesulfonic acid monohydrate ( -TSA, 98.5%) was added. After 1 h solid was not dissolved, after addition of 2,2-dimethoxypropane (2,2-DMP, 1 mL, 98%) the solution became clear immediately. The reaction mixture was stirred at ambient temperature overnight, a colorless solid precipitated, the solution became orange. Triethylamine (3 drops, -0.05 mL) was added and the mixture was stirred for additional 30 min. The solid was filtered off and the flask was washed with cold acetone (1 mL). The solid was dried (overnight, 40°C in a vacuum dryer) to yield 3,24;16,22-di-0,0-isopropylideneprotoescigenin (I) as a colorless crystalline solid (1.10 g, 94.7% yield, 91.06% purity (HPLC)).

Example 4

The optimized synthesis

Protoescigenin monohydrate (PES ¾0, 20.0 g, 39.47 mmol, 97.59%) was suspended in the mixture of acetone (100 mL, purity p.a.) and 2,2-dimethoxypropane (2,2-DMP, 100 mL, 98%) at ambient temperature (23-24°C) giving dense, white suspension. To the vigorously stirred mixture catalytic amount of -toluenesulfonic acid monohydrate (p- TSA,130 mg, 0.68 mmol, 98.5%) was added, and the solid was immediately dissolved (within 30 s). Stirring was continued at ambient temperature. After 2 h the solution became turbid, and after 1 hour colorless solid started to precipitate. The formed suspension was stirred overnight. Triethylamine (5 drops, -0.08 mL) was added and the mixture was stirred for additional 30 min. The solid was filtered off, the flask was washed with cold acetone (5-10 mL). The solid was dried (overnight, 40° C in a vacuum dryer) to yield 3,24;16,22-di-0,0-isopropylideneprotoescigenin (I) as a colorless crystalline solid (20.18 g, 87.6% yield, 94.62% purity (HPLC)). TLC: hexanes - ethyl acetate (1 : 1), R _F = 0.28 - 0.33.

Example 5

Maceration in TBME Crude solid of 3,24; 16,22-di-0,0-isopropylideneprotoescigenin (I, 20.02 g, 94.62%) was suspended in tert-butyl methyl ether (TBME, 40 mL, 20 mL/g of 1) after addition of Et ₃ N (ca. 20 drops ~ 0.32 mL) dense, white suspension was obtained. The mixture was refluxed for 80 min. Heating was removed, and the mixture was allowed to cool down, the colorless solid of (1) was filtered off (18.41 g, 91.9% yield, 99.31% purity (HPLC)). The crystalline form was identified as polymorphic form A.

1H NMR (600 Hz, DMSO-d ₆ ), δ (ppm):

5.37 (1H, m, H12); 4.75 (1H, t, J = 4.8 Hz, C28-OH); 4.22 (1Η, m, C21-OH); 3.92 (1Η, d, 11.4 Hz, H24); 3.81 (1H, d, J = 9.0 Hz, H22); 3.66 (1H, dd, J = 4.8 and 9.0 Hz, H21); 3.41 (1H, m, H16); 3.37 (1H, dd, J = 4.8 and 9,0 Hz, H3); 3.31-3.14 (3H, m, H24 and 2 H28); 2.0-1.8 (4H, m, H2, Hl l, H15, H19); 1.63 (1H, m, H2); 1.53-1.47 (4H, m, HI, H6, H9, HI 5); 1.45 (1H, m, H7); 1.39 (1H, m, H7); 1.34 (3H, s, ½ x (CHj) ₂ C-); 1.33 (3H, s, ½ (CH^C-); 1.30 (1H, d, J = 5.4 Hz, H6); 1.25 (6H, s, (CH5) ₂ C-); 1.20 (1H, dd, J = 3.6 and 12.6 Hz, H19); 1.15 (3H, s, H27); 1.12 (3H, s, H23); 1.08 (3H, s, H25); 1.03 (1H, m, HI); 0.93 (3H, s, H29); 0.88 (1H, d, J = 12.0 Hz, H5); 0.78 (3H, s, H26); 0.77 (3H, s, H30);

¹³ C NMR (150 Hz, DMSO-d6), δ (ppm):

141.0 (C13); 121.7 (C12); 98.0 ((CH ₃ ) ₂ C-); 97.8 ((CH ₃ ) ₂ C-); 75.9 (C3); 75.7 (C21); 72.2 (C22); 68.3 (C16); 62.8 (C24); 62.3 (C28); 52.9 (C5); 46.8 (C9); 44.7 (C19); 43.1 (C17); 41.2 (C14); 40.3 (C18); 39.6 (C8); 36.9 (C4); 36.0 (CI); 35.8 (CIO); 35.3 (C20); 35.0 (CI 5); 32.2 (C7); 30.4 (C29); 30.4 (( H ₃ ) ₂ C-); 27.9 (C27); 25.7 (C23); 25.4 (( H ₃ ) ₂ C-); 24.4 (C2); 22.7 (Cl l); 18.3 (C30); 18.2 (C26); 17.5 (C6); 16.7 (C25);

Example 6

Addition of hydrochloric acid

Protoescigenin monohydrate (PES H ₂ 0, 2.0 g, 3.94 mmol) was suspended in the mixture of acetone (10 mL) and 2,2-dimethoxypropane (2,2-DMP, 10 mL, 98%) at ambient temperature (23-24°C) giving dense, white suspension. To the vigorously stirred suspension concentrated hydrochloric acid (HC1, 4 drops) was added, and the solid was dissolved within 30 s, (giving colorless, clear solution). Stirring was continued at ambient temperature. After c.a. 1 h the solution became turbid, and after another hour a colorless solid started precipitating. The formed suspension was stirred overnight at ambient temperature. After addition of triethylamine (5 drops, -0.08 riiL) the mixture was stirred for 1 h. The solid was filtered off, the flask was washed with cold acetone (2 x 5 mL) and dried (overnight, 40°C in a vacuum dryer) to yield crude (I) as a colorless crystalline solid (1.87 g).

Example 7 Addition of diisopropylethylamine (DIPEA) Protoescigenin monohydrate (PES H ₂ 0, 2.0 g, 3.95 mmol) was suspended in a mixture of acetone (10 mL) and 2,2-dimethoxypropane (2,2-DMP, 10 mL, 98%) at ambient temperature (23-24°C) giving dense, white suspension. To. the vigorously stirred suspension catalytic amount of ^-toluenesulfonic acid monohydrate (p-TSA,130 mg, 0.68 mmol, 98.5%) was added, the solid was dissolved within 30 s, resulting in a colorless, clear solution. Stirring was continued at ambient temperature. After 2 h the solution became turbid, and after another hour colorless solid started precipitating. The suspension was stirred overnight at ambient temperature. After addition of diisopropylethylamine (DIPEA, 0.2 mL) the mixture was stirred for 2 h. The solid was filtered off, the flask was washed with cold acetone (2 ^χ 10 mL). The solid was dried (overnight, 40°C in a vacuum dryer) yielding crude I as a colorless crystalline solid (2.07 g).

Example 8 Addition of ammonia

Protoescigenin monohydrate (PES H ₂ 0, 2.0 g, 3.95 mmol) was suspended in the mixture of acetone (10 mL) and 2,2-dimethoxypropane (2,2-DMP, 10 mL, 98%) at ambient temperature (23-24°C) giving dense, white suspension. To the vigorously stirred suspension catalytic amount of >-toluenesulfonic acid monohydrate (p-TSA,130 mg, 0.68 mmol, 98.5%) was added, the solid was dissolved within 30 s giving a clear colorless, solution. Stirring was continued at ambient temperature. After 2 h the solution became turbid, and after 1 hour a colorless solid started precipitating. The suspension was stirred overnight at ambient temperature. Aqueous solution of ammonia (25% N¾ aq, 0.2 mL) was added and the mixture was stirred for 2 h. The solid was filtered off, the flask was washed with cold acetone (2 x 10 mL). The solid was dried (overnight, 40°C in a vacuum dryer) yielding crude (I) as a colorless crystalline solid (1.91 g).

Example 9 Maceration in MeOH

Crude solid of 3,24;16,22-di-0,0-isopropylideneprotoescigenin (PES H ₂ 0, 500 mg, 95%) was suspended in methanol (25 mL, 50 mL/g of solid), after addition of triethylamine (Et ₃ N, ca 3 drops ~ 0.05 mL) dense, white suspension was obtained. The mixture was refluxed for 1 h. Heating was removed, the mixture was allowed to cool down, then filtered to yield a colorless solid of (I) (419 mg, 83.8%yield, 99.40% purity (HPLC)).

Example 10 Crystallization in i-PrOH

Crude solid of 3,24;16,22-di-0,0-isopropylideneprotoescigenin (1, 500 mg, 95%) was suspended in propan-2-ol (/-PrOH, 25 mL, 50 mL/g of solid) after addition of triethylamine (Et ₃ N, ca 3 drops ~ 0.05 mL) dense, white suspension was obtained. The mixture was refluxed until the solid was completely dissolved, then heating was removed and the solution was allowed to cool down. The solid started precipitating. The mixture was filtered to yield (I) as a colorless solid (351 mg, 70.2% yield, 99.64% purity (HPLC)).

Example 11 Crystallization in z ^' -PrOH-water

Crude solid of 3,24;16,22- 0, O-diisopropylidene-protoescigenin (I, 500 mg, 95%) was suspended in propan-2-ol (/ ^' -PrOH, 25 mL, 50 mL/g of I) afater addition of triethylamine (Et ₃ N, ca 3 drops ~ 0.05 mL) dense, white suspension was obtained. The mixture was refluxed until the solids was completely dissolved. After addition of water (1 mL, 2 mL/g of I) solid started precipitating. Heating was removed, and the suspension was allowed to cool down. The mixture was filtered to yield a colorless solid of (I) (432 mg, 86.4%yield, 99.49% purity (HPLC)).

Example 12 Crystallization in THF

Crude solid of 3,24;16,22-O, O-diisopropylidene-protoescigenin (I, 500 mg, 95%) was suspended in tetrahydrofurane (THF, 5 mL, 10 mL/g of I) after addition of triethylamine (Et ₃ N, 1 drop ~ 0.02 mL) dense, white suspension was obtained. The mixture was refluxed until the solids was completely dissolved, then heating was removed and the solution was allowed to cool down. The mixture was filtered to yield a colorless solid of (I) (31 1 mg, 62.2% yield, 99.87% purity (HPLC)).

Example 13

Crystallization in THF-water Crude solid of 3,24;16,22-di-0,0-isopropylideneprotoescigenin (I, 500 mg, 95%) was suspended in tetrahydrofurane (THF, 10 mL, 20 mL/g of I) after addition of triethylamine (Et ₃ N, 1 drop ~ 0.02 mL) dense, white suspension was obtained . The mixture was refluxed until the solid was comletely dissolved. After addition of water (1 mL, 2 mL/g of I) the solid started precipitating. Heating was removed and the suspension was allowed to cool down. The mixture was filtered to yield a colorless solid of (I) (458 mg, 91.6% yield, 99.58% purity (HPLC)).

Example 14 Maceration in TBME Crude solid of 3,24;16,22-di-0,0-isopropylideneprotoescigenin (I, 500 mg, 95%) was suspended in tert-butyl methyl ether (TBME, 10 mL, 20 mL/g of I) after addition of triethylamine (Et ₃ N, 1 drop ~ 0.02 mL) dense, white suspension was obtained. The mixture was refluxed for 2 h, then heating was removed. The mixture was allowed to cool down, the mixture was filtered to yield a colorless solid of (I) (472 mg, 94.4% yield, 98.28% purity (HPLC)).

Table 1. Purincation of 3,24;16,22-di-0,0-isopropylideneprotoescigenin (1) in a 0.5 g scale.

Ratio of solvent 1 [ml] to (1) [g] HPLC Purity, % of peak area

b ⁾ HPLC Purity, % of peak area The results of research on end product purification have proved, due to maceration in MTBE the polar and nonpolar impurities are effectively removed, furnishing the final product of high purity in high yield. Maceration in tert-butyl methyl ether (400 mL, 20 mL/1 g) was carried out on 20,0 g scale of 3,24;16,22-di-0,0- isopropylideneprotoescigemn under reflux for 1 h 20 min. Purified 3,24;16,22-di-0,0- isopropylideneprotoescigenin was obtained in 91.9 % yield and 99.3 % purity (HPLC).

Total yield of the process embracing preparation of 3,24;16,22-di-0,0- isopropylideneprotoescigenin from protoescigenin monohydrate and purification of the final product accounts for 79.40%.