IL37809A - A process for the selective separation of vinblastine,vinleurosine and vincristine or the salts thereof - Google Patents

Description

37&Q9/2. eoiaa i oi*n a*i ,?·»ΒΟ33»Ι n'a'ep o mian T» nn A process for the selective separation of vinblastine, vinleurosine and vincristine or the salts thereof Richter Gedeon Vegye*szeti Gy£r R.T.

This invention relates to process for the selective aloids of Vinca rosea L. form of the free bases c alkaloids are the generic name: vinleurosine (VLR, on: vinleurosine) and leu.rocristine (VCR, generic name: vincristine, further on: vincristine).

In the last few years pharmacological research workers all oyer .the world have made considerable efforts for the preparation of substances having cytostatic activity* These substances are intended to be used in the medical treatment of cancerous diseases. One of the most important results of these investigations was the discovery of the dimeric alkaloids of Vinca rosea L. and the introduction thereof into the therapy. In the field of oncology, for the last decade, these substances have proved to be the relatively most active agents in the clinical practice.

Vinblastine is an oncolytical agent known since 1959. Its clinical application results in a substantial decrease of the leukocyte-count in the blood. This substance proved to be effective in the treatment of Hodgkii£disease, acute leukaemia and resistant choriocarcinoma. (See e.g. Johnson I.S., Wright H.F., Svoboda G.H. and Vlantis J. : Cancer Res. 20^ 1016 / 9.60/ ) ' eu osine are less known but its Antitumoral effects of Vinca rosea alkaloids,. Proceeding first symposium of the ,G.E.A. Excerpta Medica Foundation, Amsterdam, 1966).

Vincristine was first isolated and described by Svoboda G.H. (Svoboda, G.H.: Alkaloids of Vinca rosea /Catharanthus roseus/ IX. Extraction and Characterization of Leurosidine and Leurocristine. Lloydia, 24, 173-178 /1961/). Research workers have soon discovered that, although the effects of this new alkaloid are similar to those of the already known vinblastine, vincristine is . the ic far more advantageous from/therapeutieal point of view.

In the course of in vitro investigations it has turned out that vincristine arrests the mitotic division of neoplastic cells in the mete-phase (see e.g. Cardinali, G., Aboul Enein, Π.Ι.Μ.: Studies on the Antimitotic Action of leurocristine Aincristine/. Blood, 21, 101 /1963/).

In clinical application, compared to other chemo-therapeutic agents used so far, vincristine proved to be very effective agent in the treatment of acute leukaemia in -fctee- childhood. While in the past this serious dis ase practically could not be treated or cured, the use of vincristine makes possible the total remission in about 50 per cent of the cases, i.e. the action of vincristine is life-saving. (See e.g. Haggard, M.E.: 1 Vincristine /NSC-67574/ therapy for acute -Leukaemia in children. Cane. Chemother. Rep. j>2, 477 /1968/.) I solid tumorous diseases of childhood in 65 cases of 94 a considerable regression could be achieved (Selavry, 0., Holland, J.F., Wolmsn, I.J.: Cane. Chemother. Rep. 2, 497 /T?68/).

The dimeric alkaloids of Vinca rosea applicable in therapy (vinblastine, vinleurosine and vincristine), are 'the minor alkaloids of the plant, i.e. from the more than 60 types of alkaloids just the ones that are present in a very low quantity. The quantity of vinblastine and vinleurpsine is about 1 to 2 % of the total alkaloid content of the plant, and the vincristine content of the plant on® as much is about ft/hundred ^¾e--_re-cK3-. It is obvious that the separation of these alkaloids from the others, their of purity isolation and preparation in a p.%«ii½y grade/ required for pharmaceutical purposes can be carried out by a far more complicated process than the usual methods for the separation of alkaloids.

There are several methods known for the isolation of vinblastine, vinleurosine and vincristine (Nobel, R.L. et al.: Ann. N.Y. Acad. Sci., £6, 882 /1958/j Brit. Pat. No. 870 , 723 ; U.S. Pat. Nos. 3.097.131 and 5.205* 220 j Svoboda, G.H. et al. : J.Am.Pharm. Assoc. Sci. ed. 48, 659 /1959Λ Hung. Pat. Nos. 143*200 and 1 .7 ; Belgian Pat. No. 624046). According to all of these known processes the plant dried is digested in acidic or alkaline medium or plant the dry -eLi«Ag is pre-treated with an acid or a base and it is subsequently extracted with a water-immiscible organic solvent; the alkaloids present in the aqueous phase are transferred into an organic solvent at one or two different pH-values, thereafter the mixture of alkaloids in the organic solvent is fractionated by chromatography.

As a result of the further development of these processes the three dimeric alkaloids of pharmaceutical plant use could be isolated from the with better and better quality and recovery, the multiple phase exchanges and, first of all, the complicated chromatographic separation, however, makes the industrial realization of the known processes still very difficult. The main source of the difficulties arising at the chromatographic separation of the alkaloids is the fact that the three dimeric alkaloids (vinblastine, vinleurosine and vincristine) present in an amount of about 1 to 2 % of the total alkaloid oontent, have to be enriched, isolated and converted into a pharmaceutically acceptable pure product from a mixture of more than 60 different alkaloids. The chromatographic separation of the total alkaloid content is a very complicated, time-consuming operation which should be carried out with a particular care, and requires very large chromatographic columns. Besides the dimensions of the column 1, the size of the chromatographic equipment can well be characterized by the quantity of λΐ^θ^ used for the separation. According to the process disclosed in Hung. Pat. No. 54.715, 240 kg of AlgO^ is required for the separation of 150 g vinblastine from 1 to» of drug.

According to the process of the present invention, the known processes based on multiple phase exchanges and complicated chromato raphical separation can be substantially simplified. When using the process according to the invention the disadvantages of the known processes can be overcome, the dimensions of the chromatographic equipments can be considerably reduced, and the desired three dimeric alkaloids can be isolated in a good purity grade and with yields superior to those of the know processes. This invention is based on the discovery that an alkaloid-salt mixture consisting mainly of vinblastine, vinleurosine and vincristine may be directly separated from the mixture of crude alkaloids plant isolated from the ebs¾g, and the desired components may be obtained by a relatively simple' process from this alkaloid-salt mixture.

We have found that the three dimeric alkaloids may be isolated in a way much simpler than that utilized in the known processes by enriching these alkaloids in the mixture before the chromatographic isolation, and subjecting the thus-obtained concentrate, containing the desired alkaloids in about 70 per cent, to chromatographic treatment. The enrichment can be carried out by extracting .plant the with neutral aqueous methanol Without subjecting plant the 4s¾g to acid or alkaline treatment either prior to or during the extraction. Thereby the three dimeric alkaloids are separated from the main part of the accompanying alkaloids in the form of acid addition salts, tive preferably sulphate salts, without exposing the sensifeie^ easily decomposing alkaloid mixture obtained from the extract to deleterious effects. According to the above method, the concentration of the three alkaloids becomes as high as to 40 times k¾gk»3?-~tefepa in the alkaloid mixtures used in the chromatographic process of the known that methods, so/ a much simpler and smaller chromatographic equipment is needed for the separation, since the dimeric alkaloids are to be separated only from a small amount of accompanying substances. The advantageous simplification of the chromatographic separation according to the method of the invention may be characterized by the quantity of AlpO-, needed for the chromatographic fractionation of an ·..- · plant alkaloid mixture obtained from 1 ton of -kntg-. This quantity decreases to about 20 kg. from the original value of about 240 kg.

This invention relates to a process for the selective separation of the three dimeric alkaloids (vinblastine, vinleurosine and vincristine) of Vinca rosea L. or the ; plant acid addition salts thereof by extracting the ¾i>*tg- with aqueous methanol, distilling off practically the total amount of methanol present in the extract , acidifying the concentrate, filtering the mixture, extracting the filtrate with a water-immiscible organic solvent, alkalinizing the aqueous phase, extracting the alkaloids into a water-immiscible organic solvent, isolating the alkaloids present in this solution by evaporatin the anhydrous solution to dryness and subjecting the alkaloid mixture to chromatographic separation on partially plant desactivated A^O^, characterized in that the ¾**¾-ΙΒ extracted with neutral methanol, the alkaloid mixture obtained by a method known per se is dissolved in ethanol or in ethanol containing at most 10 % quantity of an acid, the solution is acidified Af ethanol is used as solvent the solution is acidified, vinblastine, vinleurosine and vincristine are crystallized from the solution in the form of their acid addition salts, the enriched salt-mixture containing a small' amount of accompanying substances is filtered off, the mixture is subjected to chromatography on partially desactivated by water, either in the form of the acid addition salts or in the form of the free bases obtained by treating the salts with an alkali, the column is fractionally eluted, vinblastine, vinleurosine and vin-cristme or the acid addition salts thereof are isolated from the fractions, the substances are purified if necessary, and the individual dimeric alkaloids are optionally converted to their acid addition salts or the acid addition salts of the dimeric alkaloids are optionally converted to the free' bases.

A preferred embodiment of the process of the in-vention is the following: Tr plant Ground Vinca rosea et3*«g is extracted in several portions with methanol containing 5 to 20 %, preferably % water. The extracts are combined, filtered and evaporated under reduced pressure until practically the total amount of methanol is distilled off. The aqueous reaidue is acidified in order to separate the non-alkaloidal substances. The precipitate is removed by filtration, and the aqueous acidic filtrate is extracted several times with benzene* The aqueous phase is separated, rendered alkaline, and the alkaloids, contained in this alkaline solution are extracted into benzene.

The alkaline solution is extracted several times with beizene until a substantially alkaloidfree aqueous phase is obtained. The thus-obtained benzene solutions are conbined and evaporated to dryness under reduced pressure. As a loose, amorphous, brown coloured alkaloid' mixture is obtained. This alkaloid mixture is dissolved in dilute ethanolic sulphuric acid containing at most 10 % acid. During this procedure the pH of the solution is adjusted to 4.0 to 5.0 . The solution is. seeded with a crystal of vinblastine sulphate, and the crystallization is initiated by scraping the wall of the flask. The mixture is allowed to crystallize at room temperature for 2 days. The separated crystals are filtered off. The crystalline substance is a mixture of the sulphates of vinblastine, vinleurosine, vincristine and of the accompanying substances. The crystalline substance is dissolved in a 2:1 mixture of methylene, chloride and chloroform and is fractionated by chromato raphy on a column filled with AlgO^,partially deactivated by water. ! As eluent, a 2:1 mixture of methylene chloride and chloroform is used. Fractions 1 to 3 contain the main portion of the accompanying alkaloids still present in the mixture, while fractions 2 to 9 contain vinblastine and vinleurosine. Traces of vincristine first appear in fraction 9» and the elution of this component* is finished at fraction 18. The fractions con-taining vinblastine and vinleurosine are evaporated to dryness, and the residue is dissolved in ethanol. Vinleurosine separates from this solution in crystalline form. The crystals are removed by filtration, and the mother liquor is treated with dilute ethanolic sulphuric acid containing up to 10 % of acid. The separated solid vinblastine sulphate is collected by filtration and purified by crystallization.

The fractions containing vincristine are evaporated to dryness, the dry residue is dissolved in ethanol, and the solution is acidified with ethanolic sulphuric acid.

The separated vincristine sulphate is collected by filtration and purified by recrystallization.

The fractions containing vincristine may also be worked up as follows: The fractions are evaporated to o and vincristine base is' crystallized from the solution.

The obtained substance is purified as described above, i.e. it is dissolved in ethanol and the sulphate salt is separated by adding -dilute ethanolic sulphuric acid solution to the mixtu e; ···. ':, According to another method o the inventio , the alkaloid bases are subjected to cli oma o raphic separation. In this, case the crystalline mixture containing the sulphates of vinblastine, vinleurosine, vincristine and of the accompanying alkaloids is dissolved in water, the f . . ... ■ - ■ pH of the solution is adjusted to 8.0 to 9.0 with ammonia, and the alkaline solution is extracted several times with chloroform. The chloroform solutions containing the alkaloid bases are. combined, dried over anhydrous sodium sulphate, filtered and evaporated to dryness under reduced pressure. The residue, containing the three dimeric alkaloid bases as a major component, is dissolved in a 1:1 mixture of benzene and chloroform, and the solution is fractionated by chromatography on a column filled with A^O^ partially deeactivated by water. T e column is eluted with a 1:1 mixture of benzene and. chloroform.

Fractions 1 to 10 contain vinblastine and vinleurosine, while fractions 13 to 17 ^contain vincristine. (The alkaloids contained in the fractions are identified by . pape^chromatography or by tic ) The fractions containing vinblastine and vinleurosine or vincristine, respectively, are worked up as described above.

The invention is ..elucida ed in detail by the aid of the followin non-limitin Exam les.

-Example 1 100 kg. of dried and ground Vinca rosea L. (consisting mainly of the leaves) are extracted with 1x600 1. and 2x400 1. of 90 % aqueous methanol. The combined and filtered methanol solution is evaporated under reduced pressure to a final volume of 140 to 150 1, (this residue is practically methanol-free) . In order to separate the non-alkaloidal substances present in the solution, the aqueous residue is acidified to pH 2 with an equal volume of 5 % aqueous sulphuric acid. The separated dark precipitate is filtered off. The aqueous acidic filtrate is extracted wi h 3x500 1. of benzene, the aqueous phase containing the alkaloids is separated and alkalinized to pH 8.5 to 9.0 with concentrated aqueous ammonia. The aqueous alkaline solution is extracted alkaloid-free with 3 to 4 portions of benzene (600 ml. each). The benzene solutions are combined, dried over anhydrous sodium sulphate or potassium carbonate, filtered, and the filtrate is evaporated to dryness under reduced pressure. About 700 g. of a loose, brown-coloured alkaloid^mixture is obtained.

The alkaloid mixture is dissolved in ethanol containing up to 10 per cent sulphuric acid. The pH of the solution is to be maintained at a value of 4.0 to 5-0. The solution is seeded with a crystal of vinblastine sulphate, and the crystallization is initiated by scraping the wall of the vessel. The mixture is allowed to crystallize for 2 days at,.';room temperature^ thereafter the separated crystals are filtered on a sintered glass filter.

Yield: 20 to 100 g. of crystal mixture containing .the sulphates of vinblastine, vinleurosine, vincristine and of the accompanying alkaloids. The yield depends on the plant nature and quality of the 4¾s*g-» The obtained crude alkaloid sulphate crystal mixture is dissolved in 7 ml./g.. of a 2 : 1 mixture of methylene chloride and chloroform, and the solution is fractionated by chromatography on a column containing a hundredfold quantity of Αΐ,,Ο^ partially deeactivated by water.. The column is eluted with a 2 : 1 mixture of methylenechloride and chloroform.. The effluent is collected into fractions of J>0 to 50 ml. volume.

Fractions 1 to contain the major part of the accompanying alkaloids. Fractions 2 to 9 contain vinblastine and vinleurosine.. Traces of vincristine appear first in fraction 9> an the elution of this - substance is finished at fraction 18. Vinblastine, vinleurosine and vincristine present in the fractions are identified by paperchromato-graphy or tic, and the fractions are separately evaporated to dryness. Yield: 5 to 75 . of dry amorphous residue containing vinblastine and vinleurosine, and 0.6 to 3.0 g. of dry amorphous residue containing vincristine.

The residue containing vinblastine and vinleurosine is dissolved in a 15-fold amount of anhydrous ethanol, then the solution is allowed to stand at room temperature for 16. hours. Vinleurosine separates from the solution in crystalline form. The separated crystals are filtered off, washed and dried. Yield: 15 to 17 g. of vinleurosine, m.p.: 202-205 °C ( c<)^6 = +72° (in chlorofom) . The yield depends to a great extent on the nature of the starting plant The mother liquor, which contains only the vinblastine, is diluted with equal volume of ethanol, and the pH of the solution is adjusted to 4.0 with ethanolic sulphuric acid.

Vinblastine sulphate starts to crystallize aiaefl^y-during a the addition of the acid. The mixture is kept in/refrigerator for 16 hours, and the separated crystalline ijubstance is isolated. Yield: 15 to 70 g. of crude vinblastine sulphate, depending on the nature of the startin €ts»gplant.

The obtained crude vinblastine sulphate is dried and powdered, then it is dissolved in a 20 fold amount of anhydrous methanol at room temperature. The solution is 'filtered, and 4 fold "amount of anhydrous ethanol is added to the filtrate. Vinblastine sulphate starts to crystallize * The mixture is kept in refrigerator for 16 hours. This rocrystallxzation may be carried ou with a recovery of 9 %. Yield: 13 to 63 g. of pure vinblastine sulphate, dspending on the nature of the. starting etpwrgrplan . ( ).^6 =.-28° (in chloroform).

In order to identify the product, vinblastine sulphate is converted into the free base, and this substance is recrystallized from ether. The obtained vinblastine etherate melts at 201-211 °0 (<<) = +42° (in chloroform).

* · The 0.6 to 3.0 g of amorphous dry residue obtained from the vincristine-containing fractions is dissolved in a tenfold amount of anhydrous ethanol, and the pH of tlu. solution is adjusted to 4.0 with ethanolic sulphuric aci'L. Vincristine sulphate starts to crystallize. The mixtire is kept in ^refrigerator for 16 hours, thereafter the separated crystals of ^vincristine sulphate are filtered off* The obtained substance is about 60 to 70 % of the amorphous dry residue.

The thus-obtained crude vincristine sulphate is the dissolved in/minimal amount of ethanol, thereafter a fourfold quantity of anhydrous ethanol is added to the solution.

The solution is kept in/refrigerator for 16 hours, thereafter the separated crystalline substance is filtered off. 0.3 to ^5 g» of vincristine sulphate are obtained, m.p. : 218-220 0 C.

According to another method, pure vincristine is prepared as follows: 0.6 to 3*0 g. of the amorphous dry residue obtained from the vincristine-contaihing fractions is dissolved in a 5 to 10-fold quantity of anhydrous methanol, and vincristine base is crystallized from the solution. The obtained crude base is dissolved in anhydrous ethanol, thereafter the substance is precipitated in the form of its sulphate as described above. Pure vincristine sulphate is obtained.

Example 2 A crystal mixture containing the sulphates of vin-leurosine, vinblastine, vincristine and of the accompanying alkaloids is prepared as described in Example 1. 20 to 100 g. of the crystalline substance are obtained, de- plant pending on the nature of the starting dsug. This crystalline substance is dissolved in water, and the solution is alkalinized with ammonia to pH 8.0 to 9.0.

The alkaloids are removed from the aqueous-alkaline solution by extraction with 4x1 1. of chloroform. The chloroform solutions are combined, dried over anhydrous sodium sulphate, filtered and evaporated to dryness under reduced pressure. The obtained 16 to 80 g. of alkaloid* mixture is dissolved in a 3 to 4-fold amount of a 1:1 mixture of benzene and chloroform, and is subjected to ' chromatographic separation on a column filled with about a 40-fold amount of A^O^ partially deeactivated with water. The column is eluted with a 1:1 mixture of benzene and chloroform.

The first 2 to 10 1. of the effluent, containing the accompanying alkaloids in a minor amount, are separated, thereafter the effluent is collected intc 0.6 to 3.0 1. fractions. Fractions 1 to 10 contain vinblastine and vinleurosine, while fractions 11 and 12 are free of these alkaloids. Thereafter the column is eluted with 3 to 18 1. of a 1:1 mixture of benzene and chloroform (depending on the quantity of the dissolved crystalline substance), and the. effluent is collected into 0.6 to 3.0 1. fractions.

Fractions 13 to 17 contain vincristine.

The fractions containing vinblastine and vinleurosine, or vincristine, respectively, are worked up as described in Example 1. The alkaloids present in these fractions are identified by paperchromatography or by tic.

Claims (18)

— il —
1. What we claim is: „ - 1. A process for the selective separation of the three dimeric alkaloids (vinblastine, vinleurosine and vincristine) of Vinca rosea L, or the acid addition plant salts thereof by extracting the et¥¾tg with aqueous methanol, distilling off practically the total amount of methanol present, acidifying the concentrate, filtering the mixture, extracting the filtrate with a water-immiscible organic solvent, alkalinizing the aqueous phase, extracting the alkaloids into a water-immisci-ble organic solvent, isolating the alkaloids present in this solution by evaporating the anhydrous solutions to dryness and subjecting' the alkaloid mixture to chromatographic separation on partially desactivated A^O^., characterized in that the drug is extracted with neutral methanol, the . alkaloid mixture obtained by a method known per se is dissolved in ethanol or in ethanol containing at most 10 % the solution is acidified quantity of an acid, /if ethanol is used as solvent vinblastine, vinleurosine and vincristine are crystallized from the solution in the form of their acid addition salts, the enriched salt-mixture containing a small amount of accompanying substances is filtered off, the mixture is subjected to chromatography on AlgO^ partially desactivated by water, either in the form of the acid addition salts or in the form of the free bases obtained by treating the salts with an alkali, the. column is fractionally eluted, vinblastine, vinleurosine and vincristine or the acid addition salts thereof are isolated from the fractions, the substances are purified if necessary, and the individual dimeric alkaloids are optionally converted to their acid additio salts or the acid addition salts of the dimeric alkaloids are optionally converted to the free bases. '
2. 2. A process as claimed in claim 1, in which the enrichment of vinblastine, vinleurosine and vincristine is carried out by separating the alkaloids in the form of their' sulphates.
3. ( 3. A process as claimed in claims 1 or 2, in which the sulphates are separated by treating the alkaloids with ethanolic sulphuric acid.
4. 4. A process as claimed in any of claims 1 to 3» in which vinblastine, vinleurosine and vincristine are subjected to. chromatography in the form of their acid addition salts, preferably in the form of their sulphates.
5. 5. process as claimed in any of claims 1 to 3» in which vinblastine, vinleurosine and vincristine are subjected to chromatography in the form of. the free bases.
6. 6. A process as claimed in any of claims 1 to 4, in which the enriched mixture of the sulphates of vinblastine, vinleurosine and vincristine is dissolved ;in'. a 2:1 mixture of methylene chloride and chloroform, then the solution is subjected to chromatography.
7. 7. A process-as claimed in any of claims 1 to and 6, in which the elution is carried out with a 2:1 mixture of methylene chloride and chloroform,
8. 8. A process as claimed in claim 5i in which the enriched mixture of vinblastine, vinleurosine and vincristine is dissolved in a 1:1 mixture of benzene and chloroform, and the solution is subjected to. chromatography. - i? -
9. 9. A process as claimed in claims 5 to 8, in which the elution is carried out with 1:1 mixture of benzene and chloroform..
10. 10, A process as claimed in any of claims to 9, ir. which vinblastine and vinleurosine or their , salts are eluted together, and the two dimeric alkaloids are separated from each other in a subsequent step.
11. 11. A process as claimed in claim 10, in which vinleurosine is separated from the vinleurosine-vinblastine mixture obtained after the chromatographic separation by dissolving the mixture in anhydrous ethanol, and crystallizing the substance.
12. 12, A process as claimed in claim 11, in which vinblastine is separated from the mother liquor of vinleurosine in the form of its acid addition salt, preferably in the form of the sulphate,
13. .13. A process as claimed in claim 12, in which vinblastine is separated with ethanolic sulphuric acid, and the obtained substance is recrystallized, if necessary,
14. 14. A process as claimed in claim 11, in which vinblastine salt is converted to the free alkaloid base, a.ai the alkaloid base is purified if necessary.
15. 5. A process as claimed in any of claims 1 to 9» in which crude vincristine, obtained optionally by evei orating to dryness the vincristine-containing fractions, is dissolved in anhydrous ethanol, and vincristine sulphate is separated from the solution with ethai.olic hydrochloric acid.
16. !6, A process as claimed in claim 15» in which vincristine sulphate is purified by recrystallization.
17. 17. A process as claimed in claim 15» n which vi cristine sulphate is converted optionally to the free alkaloid baseband the free base is converted into an acid additio salt thereof, if desired.
18. 18* A process as claimed in any of the preceding claims j with special reference to the Examples. Por the Applicants Dr. Yitzhak Hess