BACKGROUND OF THE INVENTION Etoposide is an antineoplastic agent having the following structure (1): Etoposide has been used effectively as an anti-tumor drug for a variety of conditions. For example, it has been employed in the treatment of acute monocytic leukemia (Schilling's leukemia), as well as medullary monocytic leukemia, and has proved effective for the treatment of recticulum cell sarcoma, tissue-cellular lymphoma, lymphasarcoma and Hodgkin's disease. Due to the well recognized activity of etoposide, a number of techniques have been developed directed to its synthesis.

One synthetic technique is that reported by Kuhn et al. in Swiss Patent No. 514,578, and related techniques disclosed in U. S. Patent Nos. 3,408,411 and 3,524,844. Kuhn et al. discloses the preparation of etoposide by the reaction of 4'-

demethyl-epipodophyllotoxin (2) with chloroformic acid benzyl ester (as a protecting group for the 4'-phenolic alcohol) to give 4'-carbobenzoxy-4'-demethyl- epipodophyllotoxin (3), followed by reaction of (3) with 2,3,4,6-tetra-O-acetyl-ß-D- glucose (4) in the presence of boron trifluoride diethyl etherate to give tetra-O-acetyl-4'- carbobenzoxy-4'-demethyl-epipodophyllotoxin-p-D-glucoside (5): The carbobenzoxy protecting group of compound (5) is removed to give tetra-O-acetyl-4'-demethyl-epipodophyllotoxin-ß-D-glucoside (6), which is then deacylated in the presence of zinc acetate to form 4'-demethyl-epipodophyllotoxin-ß-D- glucoside (7):

Conversion of 4'-demethyl-epipodophyllotoxin-p-D-glucoside (7) to etoposide is achieved by reacting with acetaldehyde-dimethylacetal and p-toluene sulphonic acid.

This synthetic method, however, in addition to requiring numerous reaction steps, is of low yield. That is, only about 18% etoposide based on 4'-demethyl-epipodophyllotoxin (2).

Another synthetic method is disclosed by Kurabayashi and Kalsuhiko et al. in Japanese Patent No. 84/98098. Unlike the method of Kuhn et al., 2,3-di-O- chloroacetyl- (4, 6-0-ethylidene)-p-D-glucopyranose (8)-as opposed to glucose (4) of Kuhn et al.-is reacted directly with a 4'- (protected)-4'demethyl-epipodophyllotoxin (9) in the presence of boron trifluoride etherate to give intermediate (10):

The resulting intermediate (10) is then converted to etoposide by reaction with zinc acetate. While this method represents an improvement of the technique of Kuhn et al., strict reaction conditions are required for controlling monoacylation of the 4'-phenolic hydroxyl group to generate compound (9) from 4'-demethyl-epipodophyllotoxin.

A further improvement to the synthesis of etoposide is disclosed in U. S.

Patent No. 5,206,350 by Wang et al. In that method, direct addition of 2, 3-di-O- chloroacetyl-(4, 6-O-ethylidene)-ß-D-glucopyranose(4, 6-O-ethylidene)-ß-D-glucopyranose (8) to 4'-demethyl- epipodophyllotoxin (2) is achieved in the presence of boron trifluoride etherate as catalyst without having to employ a protecting group on the 4'-phenolic hydroxyl group, giving 4'-demethylepipodophyllotoxin-4- (2, 3-di-O-chloroacetyl-4, 6-0-ethylidene)-p-D- glucopyranoside (11): After removal of the chloroacetyl protecting groups from compound (11) with zinc acetate in methanol, etoposide is obtained at a reported yield of 54% (based on 4'- demethyl-epipodophyllotoxin).

While eliminating the need to protect the 4'-phenolic hydroxyl group of compound (2), Wang et al. still suffers drawbacks with regard to yield, extended reaction times and isolation methodology. Accordingly, there exists a need in the art for improved synthetic methods for making etoposide which overcome these deficiencies.

The present invention fulfills these needs and provides further related advantages.

SUMMARY OF THE INVENTION This invention is directed to an improved synthetic procedure for the synthesis of etoposide. Due to the well know utility of etoposide, particularly in the context of cancer treatment, synthetic methods which result in higher yields of etoposide are of particular interest, especially with regard to commercial manufacture of the same. The present invention provides a relatively simple method of making etoposide at higher yields than existing techniques, and under more favorable reaction times and much simplified isolation procedures.

In one embodiment, a method for making etoposide is disclosed comprising the steps of : condensing 4'-demethyl-epipodophyllotoxin of formula (2) with a glucopyranose of formula (13) in an organic solvent at a temperature below-30°C and in the presence of trimethylsilyl triflate catalyst to give a compound of formula (14): where R, is-COCH3,-COCH2X,-COCHX or-COCX3, and each occurrence of X is independently selected from a halogen; and converting compound (14) to etoposide (1) having the following formula:

In more specific aspects of this embodiment, the compound of formula (13) is present in about 1.5 to about 2.0 equivalents based on the compound of formula (2), and trimethylsilyl triflate is present in about 1.5 to about 2.5 equivalents based on the compound of formula (2). Condensation of the compound of formula (13) and the compound of formula (2) is typically in the range from-40°C to-60°C, and may be performed in the presence of a drying agent such as dry molecular sieve or zeolite. The organic solvent is typically a halogenated or non-halogenated organic solvent, including (but not limited to) acetonitrile, acetone, diethylether, chloroform, dichloromethane, 1,2-dichloroethane, or mixtures thereof. Preferred R, groups of the glucopyranose of formula (13) include-COCHCI, and-COCH. CI.

In the condensing step, the trimethylsilyl triflate may be added to the mixture of the compound of formula (13) and the compound of formula (2) over a period of about 30 minutes, with the temperature of the mixture being maintained at about-50°C to about-40°C. The condensing step may be completed in about 1 to 2 hours.

In the converting step, such conversion may be accomplished by alcoholysis with, for example, a transesterification catalyst such as zinc acetate dihydrate. The zinc acetate dihydrate may be present in about 1.0 to about 2.0 equivalents based on the compound of formula (14). The converting step may be

performed in the presence of an organic solvent, including (but not limited to) a C, 4alkanol such as methanol. The compound of formula (14) and zinc acetate dihydrate may be heated to a temperature ranging from about 60°C to about 75°C for up to about 2 hours.

In a further embodiment, compound (14) may be eluted through a celite/basic alumina column, or a silica gel, prior to being converted to etoposide. In still a further embodiment, the resulting etoposide may be purified. Such purification may be accomplished by, for example, crystallization, extraction or column chromatography. Crystallization may be from a C, 4alkanol, a C, 4aliphatic ester, or a non-polar solvent, where the C, 4alkanol includes methanol and ethanol, the C, 4aliphatic ester includes ethyl acetate, and the non-polar solvent includes n-pentane or hexanes or petroleum ether. The temperature of such crystallization may be from-4°C to 0°C for 8 to 12 hours.

Preferably, the etoposide of this invention is at least 99% pure, is substantially free of a dimer of 4'demethyl-4-epipodophyllotoxin, and is substantially free of etoposide in the a-glucoside form. In this context, the term"substantially" means less than 0.5% by weight.

In another embodiment of this invention, a method for making etoposide is disclosed comprising the steps of : condensing 4'-demethyl-epipodophyllotoxin of formula (2) with 2,3-di- 0-dihaloacetyl- (4, 6-0-ethylidene)-p-D-glucopyranose of formula (13) in an organic solvent at a temperature below-20°C and in the presence of a Lewis acid catalyst to give 4'-demethylepipodophyllotoxin-4-(2, 3-di-O-dihaloacetyl-4, 6-O-ethyidene)-ß-D- glucopyranoside of formula (14):

H H O OH H O 1 Ri0 O CH3'\\p OR O/ O O/ OH Ru "",,, ! (13) OR p \"", Lewis Acid Catalyse I I CH10/+OCH3CH30"oCH3 OH (14) OH (2) where R, is-COCHX, and each occurrence of X is independently selected from a halogen; and converting the 4'-demethylepipodophyllotoxin-4- (2, 3-di-O-dihaloacetyl- 4,6-O-ethylidene)- (3-D-glucopyranoside of formula (14) to etoposide (1) having the following formula: In more specific aspects of this embodiment, the Lewis acid may be a tri (C, 4alkyl) silyltrifluoromethane sulfonate, such as trimethylsilyl triflate, or a boron trifluoride di-C, 4alkylether complex, such as boron trifluoride etherate. Further Lewis acids include (but are not limited to) ZnCI"DEAC, CF3SO3H or CF3SO3Ag. A

preferred R, for the 2,3-di-O-dihaloacetyl- (4, 6-O-ethylidene)-P-D-glucopyranose of formula (13) is-COCHCI,.

In still a further embodiment of this invention, a method for making etoposide is disclosed comprising the steps of : condensing 4'-demethyl-epipodophyllotoxin of formula (2) with a glucopyranose of formula (13) in an organic solvent at a temperature below-20°C and in the presence of a Lewis acid catalyst to give a compound of formula (14): H H po O OH 0 riz 0 RIO O/ O O/ R i O OH C I \O (13) ORi u 0 0 = o _ o Lewis Acid Catalyst I I CH30 CH30""VOCH3 OH (14) OH (2) where R, is-COCH3,-COCHX,-COCHX,, or-COCX3, and each occurrence of X is independently selected from a halogen; collecting compound (14) by elution through a celite/basic alumina column or silica gel; and converting the collected compound (14) to etoposide (1) having the following formula:

In more specific aspects of this embodiment, Lewis acid is a tri (C, 4alkyl) silyltrifluoromethane sulfonate, such as trimethylsilyl triflate, or a boron trifluoride di-C, 4alkylether complex, such as boron trifluoride etherate. Further Lewis acids include (but are not limited to) ZnCI,, DEAC, CF3SO3H or CF, S03Ag. A preferred R, for the glucopyranose of formula (13) is-COCHCI,.

These and other aspects of this invention will be evident upon reference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION As mentioned above, this invention is directed to a method for making etoposide at high yield and under simplified reaction conditions. The method involves the direct condensation of 4'-demethyl-epipodophyllotoxin (2) with 2,3-di-O- dichloroacetyl-(4, 6-O-ethylidene)-ß-D-glucopyranose (13)(4, 6-O-ethylidene)-ß-D-glucopyranose (13) in the presence of trimethylsilyl trifluoromethane sulfonate (TMSOTf) to yield 4'- demethylepipodophyllotoxin-4-(2, 3-di-O-dichloroacetyl-4, 6-O-ethylidene)-ß-D- glucopyranoside (14) as represented by the following Reaction Scheme 1:

Reaction Scheme 1 The above reaction is carried out at a temperature range of below-30°C and generally in the range of-40°C to-50°C, for a period of time ranging from 1 to 3 hours and typically from 1 to 2 hours. As with the prior technique of Wang et al., the condensation is performed without protecting the 4'-phenolic hydroxyl moiety of compound (2). However, unlike the prior technique, reaction product (14) may be collected by filtration through basic alumina, thereby avoiding the long isolation procedures reported by Wang et al. and Kuhn et al. For example, Kuhn et al. utilizes aqueous base treatment followed by repeated extractions with an organic solvent, and successive washings with hydrochloric acid solution, NaHCO3, water and drying over anhydrous sodium sulfate. Such a long work-up methodology generally leads to the formation of undesired side-products, which are avoided in the simplified isolation procedures of the present invention.

Regeneration of the alcoholic group at the 2-and 3-positions of the glycosidic moiety of compound (14) may be accomplished by alcoholysis using zinc acetate dihydrate. The resulting product of this transesterification reaction is etoposide (1).

In Reaction Scheme 1 above, 4'-demethyl-epipodophylloxin (2) may be obtained from podophyllotoxin by, for example, the techniques disclosed in U. S. Patent No. 3,524,844 to Kuhn et al. (incorporated herein by reference). Further, 2,3-di-O- dichloroacetyl-(4, 6-O-ethylidene)-ß-D-glucopyranose(4, 6-O-ethylidene)-ß-D-glucopyranose (13) may be prepared from 2,3-

di-O-dichloroacetyl-1-O-benzyloxycarbonyl- (4, 6-O-ethylidene)-P-D-glucopyranose (12) by hydrogenolysis using 10% palladium on activated carbon.

Thus, the overall reaction for synthesis of etoposide by the method of this invention may be represented by the following Reaction Scheme 2: Reaction Scheme 2 In an alternative embodiment of this invention, the R, groups of glucopyranose (13) may be-COCH3,-COCH2X,-COCHX2 or-COCX3, where each

occurrence of X is independently selected from a halogen. In a preferred embodiment, as represented by Reaction Schemes 1 and 2 above, R, is-COCHCI, However, in other embodiments suitable R, moieties include-COCH2CI and-COCH2Br, as well as the other R, moieties noted above.

A number of advantages are associated with the present invention. For example, this synthetic technique is a highly efficient process, wherein all reactions may be carried out within 1-2 hours, and providing much simplified isolation procedures.

Protection of the 4'-phenolic group of starting compound (2) is avoided, and no purification is involved in the individual steps, with only the final product being re- crystallized once. Further, all reactions are readily monitored by thin layer chromatography. Lastly, and perhaps most importantly, the overall yield is significantly higher that existing synthetic techniques.

Presently, the best synthetic method for making etoposide is the technique disclosed above by Wang et al. In that technique, the overall yield of etoposide-calculated based on compound (2)-was 54%. In contrast, utilizing trimethylsilyl triflate as the catalyst, the overall yield of etoposide according to the method of the present invention is about 68%-again, based on compound (2). This represents a 25% increase in yield over Wang et al. Furthermore, the present invention provides new isolation conditions for the intermediate (14), thus eliminating the formation of further side-products and any epimerized side-products.

It should be recognized that the technique of Wang et al. employs boron trifluoride etherate as the catalyst for formation of 4'-demethylepipodophyllotoxin-4- (2, 3-di-O-chloroacetyl-4, 6-O-ethylidene)-p-D-glucopyranoside (11) by the direct condensation 2, 3-di-O-chloroacetyl- (4, 6-O-ethylidene)-ß-D-glucopyranose (8) with 4'- demethyl-epipodophyllotoxin (2). While boron trifluoride etherate of Wang et al. and others, and trimethylsilyl trifluoromethane sulfonate of the present invention may both be classified as Lewis acids, it has been surprisingly found that trimethylsilyl trifluoromethane sulfonate works significantly better than boron trifluoride etherate, as well as significantly better than other Lewis acids tested, including DEAC, ZnCI,, CFSOH and CF3SO3Ag.

While not intending to be limited by the following, it is believed that trimethylsilyl trifluoromethane sulfonate may function by a different mechanism than that of boron trifluoride etherate. The latter is believed to generate a carbocation intermediate at the C4 position of the aglucone, which is then attacked by the free hydroxyl group of the glucopyranose. In contrast, trimethylsilyl triflate apparently reacts with the free hydroxyl group of the glucopyranose to form a good leaving group, which facilitates the later attachment of the lignan moiety to give compound (14) with retention of stereochemistry that is the same as that of the starting materials.

Furthermore, the use of trimethylsilyl triflate allows for a more efficient coupling, which results in 80% yield from lignan (2) to compound (14)--based on the lignan (2)-- which is achieved within 2 hours, as opposed to only about 60% yield by Wang et al.

Therefore, by use of trimethylsilyl triflate, significant advantages are achieved, including increased yield and reduced reaction times.

Furthermore, when the present invention employs boron trifluoride etherate as catalyst (the same catalyst disclosed by Wang et al.), improved yields are again obtained. that is, 60% yield compared to the 54% of Wang et al. This difference in yield is believed due to the use of a different glucopyranose (where R, is-COCHCI,, rather than-COCH, CI of Wang et al.), as well as being attributable to the simplified isolation procedures of this invention.

In addition to trimethylsilyl trifluoromethane sulfonate and boron trifluoride etherate, other Lewis acids may be employed, provide compound (14) is collected by elution through a celite/basic alumina column or a silica gel prior to converting the collected compound (14) to etoposide (1).

The following examples are presented by way of illustration, not limitation.

EXAMPLES Example 1 PREPARATION OF 2, 3-DI-0-DICHLOROACETYL-(4, 6-0-ETHYLIDENE)-ß-D- GLUCOPYRANOSE (HYDROGENOLYSIS) (13) An over-dried 100 mL three-necked round bottom flask fitted with a stir bar. low temperature thermometer, septa and H) inlet was charged with 2, 3-Di-O- dichloroacetyl-1-O-benzyloxycarbonyl-(4, 6-O-ethylidene)-ß-D-glucopyranose (1.(4, 6-O-ethylidene)-ß-D-glucopyranose (1. 8 mmol), in acetone (15-30% concentration) and 10% palladium on activated carbon powder (0.2 mmol). The solution was stirred until uniform and then cooled to-10°C to 0°C. After the reaction was over the catalyst was filtered over sintered glass containing a plug of celite under reduced pressure. The sintered glass is washed trice with one times the total reaction volume of anhydrous acetone and the filtrates are pooled and then concentrated to dryness under reduced pressure at a temperature close to 30°C.

The crude residue was dried under vacuum at ambient temperature and compound (13) was thus obtained as white foam in 98% yield with a melting point of 130-132°C (from acetone). DCI-MS: 428; IR: ,, CHCI3: 3650,1770,1390,1200 and 1100 cm-'.

Example 2 PREPARATION OF 4'-DEMETHYL-EP/-PODOPHYLLOTOXIN-4- (2, 3-DI-O-DICHLOROACETYL- 4,6-O-ETHYLIDENE)--D-GLUCOPYRANOSIDE (14) An oven-dried, three-neck 250 mL round bottom flask was fitted with a stir bar, low temperature thermometer, septa and argon inlet, was introduced with 4'-demethyl-epi-podophyllotoxin (2) (1 mmol), dry molecular sieve (1/16"pellets) and anhydrous dichloromethane (20-50% concentration). 2-3-Di-O-dichloroacetyl- (4, 6-O- ethylidene)-p-D-glucopyranose (13) (1.7 mmol) in dichloromethane (10-20% concentration) was added via double-ended needle. The suspension was stirred until homogenous and then cooled to-40°C to-60°C in an atmosphere of argon and in the absence of moisture. To the stirred suspension was added via a syringe, trimethylsilyl

trifluoromethane sulfonate (2 mmol) over 30 minutes. The reaction was held at between-50°C and-40°C for 30 minutes. The course of the coupling reaction was monitored by thin layer chromatography. The suspension was allowed to warm to about-30°C and filtered through a short celite/basic alumina column, eluting twice with one times the total reaction volume of dichloromethane. The pooled filtrate was evaporated under reduced pressure to yield the crude intermediate product (14) (80% based on the lignan (2)). This crude product is used directly in the next step without any purification. A sample was purified by the chromatraton for spectroscopic identification. The results are as follows: m. p: 242-243°C (from methanol); DCI-MS: 810.

Example 3 PREPAR : ATION OF 4-DEMETHYL-EP/-PODOPHYLLOTOXIN-4- (4, 6-O-ETHYLIDENE)--D- GLUCOPYRANOSE(ETOPOSIDE) To 0.8 mmol of 4-demethyl-epi-podophyllotoxin-4-(2, 3-di-O- dichloroacetyl-4,6-0-ethylidene)-p-D-glucopyranose (14) in 10-25% concentration in methanol is added 1.5 mmol of zinc acetate dihydrate. The reaction mixture is refluxed with stirring under heating for 90 minutes. After completion of the reaction, the mixture is cooled and the volume reduced to one third by rotary evaporation under reduced pressure. Working up is effected by diluting the reaction solution with 100 mL dichloromethane and 100 mL of water. The aqueous phase was washed with 50 mL of dichloromethane. The combined dichloromethane phases was washed twice with 50 mL water. 15 mL of methanol was added to the first wash to prevent precipitation of etoposide. The organic phase was dried over anhydrous sodium sulphate, filtered and concentrated by evaporation under vacuum to an amorphous solid. This solid was re- crystallized from methanol/n-pentane at-4°C to 0°C, thus obtaining colorless amorphous powder of Etoposide (68% yield based on the lignan (2), if the mother liquors are treated the yield will be higher). m. p: 256-258°C; DCI-MS: 588.

Example 4 PREPARATION OF ETOPOSIDE EMPLOYING 2, 3-DI-O-DICHLOROACETYL-(4, 6-0-ETHYLIDENE)-ß-D-GLUCOPYRANOSE AND BORON TRIFLUORIDE ETHERATE AS CATALYST 4'-demethyl-epi-podophyllotoxin (1 mmol) and 2, 3-di-O-dichloroacetyl- (4,6-0-ethylidene)-p-D-glucopyranose (2 mmol) were introduced into dry dichloromethane (20-50% concentration based on the lignan) under anhydrous condition. When the temperature was stabilized to-20°C to-30°C, boron trifluoride etherate (1.5 mmol) was added slowly with stirring. Reaction was continued at this temperature and monitored by thin layer chromatography. After the completion of the reaction as evidenced by TLC, the solution was washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford the crude intermediate product (14). This crude product was then converted to etoposide by following the procedure as outlined in Example 3. The yield of final product etoposide was about 60% based on the lignan.

It will be appreciated that, although specific embodiments of the invention have been described herein for purpose of illustration, various modifications may be made without departing from the spirit and scope of the invention.

Accordingly, the invention is not limited except as by the appended claims.