FIELD OF INVENTION

The invention relates to a method for preparation of purified galantamine hydrobromide, in particular to extraction of galantamine from plant raw material, its conversion into hydrobromide and its purification.

BACKGROUND OF THE INVENTION

Galantamine- (4aS,6R ,8aS)-4a,5,9, 10,11.12-hexahydro-3-methoxy-ll-methyl-6H-benzofu [3a,3,2 ef][2]benzazepin-6-ol; galantamine, lycoremine, with the following structural formula:

is a selective acetylcholinesterase inhibitor. The first records of its isolation date from 1952. The following are indicated as sources of galantamine: Amaryllidaceae, Caucasian snowdrops, Galanthus woronowii \Je\., Galanthus Nivalis, G. narcissus, G. leucojum, G. orinium, Narcissus iv. Salome, Narcissus pseudonarcissus, Narcissus leonensis, Narcissus nivalis, Galanthus elwesil. Hydrobromide of galantamine, independently or in a combination, is included in the composition of various medicines.

It is known that galantamine has been isolated from Amaryllidaceae trought the extraction of plant material pre-alkalized with ammonium using dichloromethane or another chlorinated hydrocarbon. The obtained extract is treated by diluted sulphuric acid, then it is precipitated with aqueous ammonia. The galantamine which has remained in the solution is extracted with diethyl acetate or with dichloromethane, and then it is purified (1).

An extraction with dichloroethane or with benzine from Narcissus Carlton pre-mixed with powdered sodium carbonate has been described. The obtained extract is evaporated to dryness; the residue is dissolved in dilute sulphuric acid; it is extracted with ether; the remaining water solution is alkalized to pH 9 and extracted with diethyl ether, it is concentrated to dryness, propanol is added until galantamine crystallization (2). The toxic solvents used in large volumes are a disadvantage of the above method.

A method has been described for isolation of galantamine from biological material (Amoryllidaceae as narcissi and crinum, in particular from narcissus pseudonarcissus Carlton and Crinum amabile) through extraction with non toxical solvent (diethyl ether or benzine with a special boiling point, which is preferred) and subsequent purification with liquid-liquid extraction which in the first stage is carried out at pH of about 4, and in the second stage at pH of about 9. Before the extraction the biological material is mixed with alkali powder (sodium carbonate). The obtained galantamine is pre-crystallized from a suitable solvent (isopropanol) to a purity of 99 % (3).

A semi-synthetic method for preparation of galantamine and its derivatives has been protected. Bulbs of yellow narcissus or of snowdrop are used as raw material, they are stirred with secondary amine, with a formula specified in the description, in water-phosphate buffer and ethanol. The obtained mixture is extracted with ethyl acetate and then concentrated (4).

A method for extraction of pure galantamine is suggested trough extraction from Amaryllidaceae with aqueous solution of inorganic or organic acids. The obtained extract is applied on adsorbent and rinsed with water, followed by elution with a aqueous-miscible solvent and concentration of the collected fractions. The obtained concentrate of alkaloids is adsorbed on cation-exchange polymer and is eluted with aqueous solution of inorganic base. This is followed again by concentration, re-elution but with a solvent that is non-miscible with water. The newly obtained concentrate is subjected to chromatographic purification on dialuminum trioxide using a solvent non-miscible with water as a mobile phase. The obtained galantamine crystallizes in a suitable solvent, and then it is subjected to pre-crystallization in methyl-isobutyl ketone or in tert-butyl methyl ether. Other options for purification are also provided - passing through a salt, etc. The stated purity of the obtained galantamine is above 90%, mostly 99% (5). However, the method is quite complicated by many operations, the need of specialized equipment, etc.

Use of centrifugal chromatography has been protected (liquid-liquid chromatography without solid carrier) for purification of galantamine. In example 1 is described obtaining of galantamine, from Leucojum aestivum leaves, which is moistened with ammonia (10%) in water; the obtained mixture is threated by ethyl acetate for 18 h; after leaching the ethyl acetate, the obtained solution is extracted repeatedly with water solution (3%) of sulphuric acid; after alkalization of the aqueous phase with ammonium hydroxide, several extractions with chloroform are carried out; the subsequent operations lead to preparation of common alkaloids which are then subjected to separation and purification using the suggested chromatographic method (in various options of implementation) - a number of operations are used which require long time, large quantities of solvents and special chromatographic equipment (6).

A method of purification of the hydrobromide of galantamine extracted from Amaryllidaceae : Galanthus , Narcissus, Crinum, Leucojum, has been protected.

The preparation of galantamine used in the protected method is illustrated in example 1 where milled bulbs of Narcissus Pseudonarcissus "Carlton" are mixed with 10% water solution of sodium carbonate and are extracted seven times with toluene at 65-70 °C; the collected extracts are concentrated and processed with 2% sulphuric acid; the obtained aqueous phase is alkalized with ammonium hydroxide up to pH 9 and is extracted four times with toluene; after evaporation to dryness in vacuum common alkaloids are obtained which contain 40% galantamine. Examples 2 and 3 illustrate the obtaining of hydrobromide with purity of 88% and 85% during interaction of alkaloids with 48% hydrobromic acid, in ethanol and isopropanol medium respectively. The obtained hydrobromide is suspended with water, diluted with 10% sodium carbonate and extracted five times, respectively seven times with tert-butyl methyl acetate. The method in the different stages uses large quantities of solvents and includes many repetitive operations (7).

A method is described for isolation of galantamine from biological material that includes acid extraction with subsequent elution with aqueous-miscible organic solvent of the absorbed organic compounds; washing with water and concentration of alkaloids. The biological material is Amaryllidaceae: Galanthus, Narcissus, Leucojum and/or Lycoris (8).

Protection is requested of an extract of hippeastrum papilio, rich in galantamine, which is obtained by acid extraction (2% sulphuric acid) of the chopped plant source, with subsequent alkalization with ammonium and extraction by using the chloroform. After several operations for purification, the mixture of alkaloids is obtained, the composition of which has been chromatogfaphically determined (9).

The specified above shows that the issue of extracting galantamine from plants and of preparation of its pharmacopoeial grade hydrobromide is still actual. Due to the wide and varied application of this alkaloid, a simple method is necessary, which is easy to apply and will provide good yields from a pharmacopoeial grade product.

TECHNICAL DESCRIPTION OF THE INVENTION

It has been unexpectedly established that pharmacopoeial grade galantamine hydrobromide can be obtained when the galantamine base extracted from Leucojum aestivum L or Narcissus Carton cv in aqueous medium or in low alcohol medium, alkalized with calcium hydroxide to pH 9 - 12, at temperature of 30 - 40°C. After filtration and concentration, the residue is extracted 2 to 4 times with methyl isobutyl ketone, ethyl acetate, butylacetate or n- butanol in a ratio of extract/extractant of 8:1 during conducting the extractions in aqueous medium, and respectively 2:1 during conducting the extractions in the presence of low alcohol. The collected organic extracts are concentrated from 1/20 to 1/30 of the initial volume, the solvent is replaced with ethanol and the obtained galantamine base was treated with hydrobromic acid to be converted into galantamine hydrobromide in a known way. The obtained galantamine hydrobromide was purified in aqueous medium with activated carbon, at temperature of 80 - 85°C. After filtration the obtained purified solution is cooled down to 20- 25°C and alkalized with ammonium hydroxide to pH 9-12. The obtained galantamine base is extracted 2-4 times with methyl isobutyl ketone, ethyl acetate or butyl acetate in a ratio of alkaline aqueous solution/organic solvent of 2:1 to 3:1 and after concentration at volume from 1/5 to 1/10 of the initial volume and cooling, the obtained galantamine base is trated with a selective reagent for N-desmethyl galantamine, in particular di-tert-butyl dicarbonate or diethyl pyrocarbonate in a molar overange of 1,12-1,2 to the content of N-desmethyl galantamine (determined by HPLC) in the alkaloid mixture under stirring during 7-10 hours, followed by extraction with mineral acid to pH 2-3 in aqueous medium, then is done then sequentially alkalization of the acid water extract of the salt of galantamine at pH 9-12, extraction of the released galantamine base with methyl isobutyl ketone, ethyl acetate or butyl acetate in a ratio of aqueous solution/organic solvent of 2:1 to 3:1, replacement of the solvent with ethanol and treatment with hydrobromic acid in a known way until galantamine hydrobromide of HPLC grade more than 99 % is obtained. An advantage of this method according to the invention is that Galantamine hydrobromide is obtained elegantly with pharmacopoeial grade of 99,9 % and high yield of about 90 - 92 % of the content of Galantamine hydrobromide in the technical product. The following examples illustrate the the method according to the invention without any limiting of it.

EXAMPLES:

Preparation of Galantamine hydrobromide (with purity of 80-85 %)

EXAMPLE 1

A) 500 kg of chopped bulbs of Narcissus Carton cv with Galantamine content of 0,09 % are extracted four times, each extraction with 1500 litres of aqueous solution of calcium hydroxide at a temperature of 30°C and pH 9. The obtained aqueous alkaline extracts after its filtration of the excess calcium hydroxide are extracted four times, each extraction with 200 litres of butyl acetate.

The obtained butyl acetate extracts containing the alkaloid are collected and concentrated under vacuum to a volume of 20 litres, then 50 I of 96 % ethanol is added. The obtained mixture is concentrated under atmospheric pressure to a volume of 6 I. The obtained alcohol solution of Galantamine base is cooled down at 5 - 10°C and treated at the same temperature with 48 % hydrobromic acid until pH value become constant between 2,0 - 3,0.

The obtained suspension of Galantamine hydrobromide is allowed to crystallize under continuous stirring at a temperature of 0-5°Cfor 12 hours, then filtered and dried it at a temperature of 40°C. 0,53 kg of Galantamine hydrobromide is obtained with HPLC grade of 83 %.

B) The process is similar to the method described in Example 1A), with the only difference that the obtained aqueous-alkaline extracts after filtration of the excess calcium hydroxide are extracted four times, each extraction with 200 litres of n-butanol, instead of butyl acetate. 0,56 kg of Galantamine hydrobromide is obtained with HPLC grade of 84 %.

C) The process is similar to the method described in Example 1A ), with the only difference that the obtained aqueous-alkaline extracts after filtration of the excess calcium hydroxide are extracted four times, each extraction with 200 litres of methyl isobutyl ketone, instead of butyl acetate. 0,54 kg of Galantamine hydrobromide is obtained with HPLC grade of 84 %. D) 500 kg of chopped bulbs of Narcissus Carton cv are extracted six times, each extraction ith 1500 litres of aqueous solution of calcium hydroxide at a temperature of 40°C and at pH 10. Then method follows the steps described in example 1A).

1,0 kg of Galantamine hydrobromide is obtained with HPLC grade of 80 % .

E) 500 kg of chopped bulbs of Narcissus Carton cv with Galantamine content of 0,09 % are extracted three times, each extraction is performed with 1500 litres of 75% ethanol alkalized with saturated solution of calcium hydroxide, at a temperature of 40°C and pH 10. The obtained ethanol extracts are collected, then the excess calcium hydroxide is filtered and concentrated under vacuum to a volume of 200 I. The obtained aqueous concentrate is extracted three times, each extraction with 100 I with butyl acetate. The obtained organic extracts containing the alkaloid mixture are mixed and concentrated under vacuum to a volume of 20 litres. 50 I of 96 % ethanol is added and concentrated the thus obtained mixture under atmospheric pressure to a volume of 6 I.

The resulting alcoholic solution of the galanthamine base is then treated as described in Example

1 (A).

0,56 kg of Galantamine hydrobromide is obtained with HPLC grade Of 86 % .

F) Follows the procedure described in Example IE) with the only difference that the combined ethanol extracts after filtration of the excess calcium hydroxide are extracted with n- butanol instead of butyl acetate.

0,53 kg of Galantamine hydrobromide is obtained with HPLC grade of 86 %.

G) Follows the procedure described in Example IE) with the only difference that the combined ethanol extracts after filtration of the excess calcium hydroxide are extracted with methyl isobutyl ketone instead of butyl acetate.

0,51 kg of Galantamine hydrobromide is obtained with HPLC grade of 86 %.

H) 500 kg of chopped bulbs of Narcissus Carlon cv with Galantamine content of 0,09 % are extracted four times, each extraction is performed with 1500 litres of 65 % ethanol alkalized with saturated calcium hydroxide solution, at a temperature of 40°C and at pH 9. Then follow the method described in Example IE). 0,46 kg of Galantamine hydrobromide is obtained with HPLC grade of 85 %.

I) 500 kg of chopped bulbs of Narcissus Carlon cv with Galantamine content of 0,09 % are extracted six times, each extraction is performed with 1500 litres of 70 % methanol alkalized with saturated calcium hydroxide solution, at a temperature of 40°C and at pH 9. The obtained methanol extracts are collected and the excess calcium hydroxide is filtered, then concentrated under vacuum to a volume of 200 I. The obtained aqueous concentrate is extracted three times, each extraction with 100 I of butyl acetate. The Obtained organic extracts containing the alkaloid mixture are collected and concentrated under vacuum to a volume of 20 litres. 50 I of 96 % ethanol is added, the obtained mixture is concentrated under atmospheric pressure to a volume of 6 1. Then the resulting alcohol solution of Galantamine base is treated as described in Example 1 A).

0,54 kg of Galantamine hydrobromide is obtained with HPLC grade Of 83 %.

J) The process is similar to the method described in Example 11) with the difference that the extraction is threefold, the collected methanol extracts after its filtration of calcium hydroxide and concentration are extracted with methyl isobutyl ketone instead of ethanol.

0,53 kg of Galantamine hydrobromide is obtained with HPLC grade of 83 %.

K) The process is similar to the method described in Example II) with the difference that the collected methanol extracts after its filtration of calcium hydroxide and concentration are extracted with h-butanol instead of ethanol.

0,54 kg of Galantamine hydrobromide is obtained with HPLC grade of 83 %.

EXAMPLE 2

A) 1080 kg of dried aerial part of Summer snowflake { Leucojum aestivuml ) with Galantamine content of 0,12 % are extracted three times, each extraction is performed with 3500 litres of 70 % ethanol alkalised with saturated calcium hydroxide solution, at a temperature of 30°C and pH 10. The obtained ethanol extracts after collecting, filtering and concentrating are extracted three times, each extraction with 100 I of butyl acetate. The organic extracts containing the alkaloid mixture are collected and concentrated under vacuum to a volume of 20 litres , then 50 I of 96 % ethanol is added and concentrated the thus obtained mixture under atmospheric pressure to a volume of 10 I. The obtained alcohol solution of Galantamine base is cooled down up to 5-10°C and treated at this temperature with 48 % hydrobromic acid until a constsnt pH value of the solution of 3,0 is reached. The obtained suspension of Galantamine hydrobromide is let to crystallize under continuous stirring and a temperature of 0-5°C for 12 hours, then is filterred and dried at a temperature of 40°C.

1,57 kg of Galantamine hydrobromide is obtained with HPLC grade of 85 %.

B) The process is similar to the method described in Example 2 A) with the only difference that the collected, filtered and concentrated ethanol extracts are extracted with n-butanol instead of butyl acetate.

1,55 kg of Galantamine hydrobromide is obtained with HPLC grade 80 %.

C) The process is similar to the method described in Exarnple 2 A) with the only difference that the collected, filtered and concentrated ethanol extracts are extracted with methyl isobutyl ketone instead of butyl acetate.

1,54 kg of Galantamine hydrobromide is obtained with HPLC grade of 80 % are obtained.

D) 1000 kg of dried powdered aerial part of Summer snowflake ( Leucojum aestivum L) with Galantamine content of 0,11 % are extraced four times, each extraction with 3000 litres of aqueous solution of calcium hydroxide at a temperature of 30°C and at pH 9. The obtained aqueous-alkaline extracts are filterred from the excess calcium hydroxide and extracted four times, each extraction with 200 litres of butyl acetate. The obtained butyl acetate extracts containing the alkaloid mixture are collected and concentrated under vacuum to a volume of 20 litres, then 50 I of 96 % ethanol is added and thus obtained mixture is concentrated under atmospheric pressure to a volume of 6 I. The resulting alcohol solution of Galantamine base is cooled at 5-10°C and treated at this temperature with 48 % hydrobromic acid until a pH value of the solution is reached 3,0. The obtained suspension of Galantamine hydrobromide is alowed to crystallize under continuous stirring at a temperature of 0-5°C for 12 hours, then filtered it and dried at a temperature of 40°C. 1,2 kg of Galantamine hydrobromide is obtained with HPLC grade of 81 %.

E) Follow the method described in Example 2 D) and the aqueous -alkaline extracts obtained after filtering the excess calcium hydroxide are extracted with n-butanol instead of butyl acetate. 1,2 kg of Galantamine hydrobromide is obtained with HPLC grade of 80 %.

F) Follow the method described in Example 2 D) and the aqueous-alkaline extracts obtained after filtering the excess calcium hydroxide are extracted with methyl isobutyl ketone instead of butyl acetate. 1,2 kg of Galantamine hydrobromide is obtained with HPLC grade of 80 % .

The resulting galantamine hydrobromide, regardless of the raw material or method of obtaining described above, is subjected to purification that includes treatment with selective reagent for N- desmethyl galantamine, illustrated below with the following non-limiting examples:

Example 3:

Preparation of Galantamine hydrobromide HPLC grade more than 99 %

A) By treatment with BOC anhydride (Di-tert-butyl dicarbonate)

Aa) by using methyl isobutyl ketone.

20 kg of Galantamine hydrobromide HPLC grade of 83 % are dissolved in 200 I of purified water at a temperature of 80 - 85°C and treated with 500 g of activated carbon for 20 min at this temperature, then filterred. The obtained aqueous solution of Galantamine hydrobromide is cooled down to 20 - 25°C, alkalized with 25 % ammonium hydroxide to pH 9 and extracted three times, each extraction is performed with 100 I of methyl isobutyl ketone. The collected organic extracts containing Galantamine base are concentrated to a volume of 80 I and cooled down to 20 - 25°C. The thus resulting solution of Galantamine base in methyl isobutyl ketone is treated with 1,49 kg of BOC anhydride (Di-tert-butyl dicarbonate) under continuous stirring for eight hours, then 100 I of purified water is add and the two-phase system is acidified with 36 % hydrochloric acid to 2.0 pH of the aqueous layer under continuous stirring. The resulting organic layer is removed and the rest acid water solution containing Galantamine hydrochloride is alkalized by 25 % ammonium hydroxide to pH 9, then tree times extraction is done, each extraction with 100 I of methyl isobutyl ketone. The collected alkaline organic extracts containing purified Galantamine base are concentrated under vacuum to dryness, then 80 I of 96 % ethanol is added, the resulting alcohol solution of Galantamine base is cooled down to 10 °C and treated at this temperature with 48 % hydrobromic acid until a constant pH value of the solution of 3,0 is reached. The suspension obtained from purified Galantamine hydrobromide is left to crystallize under continuous stirring and at a temperature of 5°C for 12 hours, then filterred and dried at a temperature of 40°C.

15,0 kg of Galantamine hydrobromide is obtained with HPLC grade of over 99 %.

Ab) by using ethyl acetate.

Following the method described in Example 3Aa) but methyl isobutyl ketone is replaced with ethyl acetate. 15,2 kg of Galantamine hydrobromide is obtained with HPLC grade of over 99 %.

Ac) by using butyl acetate.

Following the method described in Example 3Aa) but the methyl isobutyl ketone is replaced with butyl acetate. 15,0 kg of Galantamine hydrobromide is obtained with HPLC grade of over 99 %.

B) Preparation of Galantamine hydrobromide HPLC grade of over 99 % by treatment with Diethyl pyrocarbonate

Ba) by using methyl isobutyl ketone.

20 kg of Galantamine hydrobromide HPLC grade of 83 % is dissolved in 200 I of purified water at a temperature of 80 - 85 °C and treated with 500 g of activated carbon for 20 min at this temperature, then filterred. The resulted aqueous solution of Galantamine hydrobromide is cooled down to 20 - 25 °C, alkalized with 25 % ammonium hydroxide to pH 9 and extracted three times, each extraction with 100 I of methyl isobutyl ketone. The collected organic extracts containing Galantamine base are concentrated to a volume of 80 I and cooled down to 20-25°C. Thus resulted solution of Galantamine base in methyl isobutyl ketone is treated with 1,1 kg of diethyl pyrocarbonate under continuous stirring for eight hours. Then 100 I of purified water is added and the received two-phase system is acidified with 36 % hydrochloric acid to 2.0 pH of the aqueous layer under continuous stirring. The organic layer is removed and the resulted acid aqueous solution containing Galantamine hydrochloride is alkalized with 25 % ammonium hydroxide to pH 9, then extracted three times, each extraction with 100 I of methyl isobutyl ketone. The collected alkaline organic extracts containing purified Galantamine base is concentrated under vacuum to dryness, then 80 I of 96 % ethanol is added, cooled the obtained alcohol solution of Galantamine base to 10°C and treated at this temperature with 48 % hydrobromic acid until a constant pH value of the solution of 3,0 is reached. The resilted suspension of purified Galantamine hydrobromide is alOwed to crystallize under continuous stirring arid a temperature of 5°C for 12 hours, then interred and dried it at a temperature of 40°C.

15,4 kg of Galantamine hydrobromide is obtained with HPLC grade of more than 99 % . Bb) by using ethyl acetate.

Following the method described in Example 3Ba) but the methyl isobutyl ketone is replaced with ethyl acetate. 15,2 kg of Galantamine hydrobromide is obtained with HPLC grade of more than 99 % . Be) by using butyl acetate.

Following the method described in Example 3Ba) but the methyl isobutyl ketone is replaced with butyl acetate. 15,2 kg of Galantamine hydrobromide is obtained with HPLC grade of more than over 99 % .

Literature sources used:

1. DEI 193061

2. US5877172

3. EP0815112 B1

4. EP0853624 B1

5. W02006099635 A

6. EP1824857 B1

7. BG /EP1861105 T3

8. EP1858897 A

9. EP2999480 Al= BGl 11420 A