NZ260430A - Intermediates useful in the preparation of epipodophyllotoxin glucoside phosphates - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £60430 26043 gncior tf-o 0: later. 22 -j:v Sp&cilicav.ori lei bGc.> u to 13 %£ Priority Dcto(o): .

Complete Spcc'.."ic3tion Fiiod: Jff". 01^9: .CCOFfPlUtfW Publication Date: ....? P.O. Journal, No: ..... i3sr„ *" ^»nnnw.n Initials NO DRAWINGS NEW ZEALAND PATENTS ACT, 1953 Divided out of No.: 242059 Date Filed: 2 May 1994 6 COMPLETE SPECIFICATION INTERMEDIATES IN PROCESS FOR THE PREPARATION OF 4'-DEMETHYLEPIPODOPHYLLOTOXIN GLUCOSIDE 4'-PHOSPHATES We, BRISTOL-MYERS SQUIBB COMPANY a Delaware corporation, USA of 345 Park Avenue, New York, New York 10154, United States of America, hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- 2 INTERMEDIATES IN PROCESS FOR THE PREPARATION OF 4'-DEMETHYLEPIPODOPHYLLOTOXIN GLUCOSIDE 4'-PHOSPHATES BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel process for the preparation of antitumor compounds, as well as to the novel intermediates produced in said process. More particularly, the novel process and intermediates are directed to the preparation of 4'-demethylepipodophyllotoxin glucoside 4'-phosphates. 2. Background Art Etoposide and teniposide, two 41-demethylepipodophyllotoxin glucoside derivatives, are presently widely used in clinical therapy of cancer and etoposide is currently approved in the United States for the treatment of small cell lung cancer and testicular cancer. However, these compounds have very low water solubility rendering them difficult to formulate into suitable pharmaceutical dosage forms.

US Patent 4,904,768 discloses water soluble prodrugs of 4'-demethylepipodophyllotoxin glucoside derivatives bearing a 4'-phosphate group, as ex-emplif ied by etoposide 4'-phosphate. Etoposide 4'- 3 phosphate was prepared by reacting etoposide with phosphorous oxychloride followed by hydrolysis, or by reacting etoposide with diphenyl chlorophosphate followed by hydrogenation to remove the phenyl groups.

Etoposide 4'-phosphate is also reported in Japanese Kokai 63/192,793 (published August 10, 1988) and it was prepared by reacting 2",3"-bis-0-(2, 2,2-trichloroethoxycarbonyl)etoposide with phosphorous oxychloride followed by hydrolysis, and then removal of the sugar hydroxy protecting groups by treatment with zinc.

The starting materials used in the above processes are obtained by condensing 4'-protected 4'-demethylepipodophyllotoxin with hydroxy-protected ethylidene-B-D-glucopyranose, and subsequently removing at least the 4'-protecting group.

Surprisingly and unexpectedly, the present inventors found that the 4 *-phenolic hydroxy group of 4'-demethylepipodophyllotoxin may be protected in the form of a phosphate triester which, following condensation with the glucopyranosyl moiety, may be cleaved to provide etoposide 4'-phosphate directly. The present process eliminates the necessity of separately protecting and deprotecting the 4'-phenolic hydroxy group of 4'-demethylepipodophyllotoxin, and therefore represents a more economical and efficient route over the other known art methods for the preparation of etoposide 4'-phosphate.

SUMMARY OF THE INVENTION One aspect of the present invention, which is claimed in our New Zealand Patent Specification 35 No. 242059, relates to a process for preparing a compound of formula (IV) 4 Rla0 rla0 ,0 ch opo3h2 (iv) wherein the two R1a groups together represent alkylidene, or a phannaceutically acceptable salt thereof, or a solvate thereof, which comprises: reacting a compound of formula (II) wherein R1 is a hydroxy protecting group, or the and R2 is a hydroxy protecting group, with a 30 compound of formula (III) two R1 groups together represent alkylidene, HO \ -OR' "OR" (III) wherein R3 is a phosphate protecting group, in the presence of a Lewis acid to form a compound of formula 20 (I) 0 \p--0R3 ||^0R3 0 (I) wherein R1, R2 and R3 are as defined above; removing the hydroxy protecting groups, and where R1 is hydroxy protecting group, reacting the resultant 6 product with a carbonyl compound having one to five carbon atoms or an acetal or a ketal equivalent thereof; and removing the phosphate protecting groups.

Another aspect of the present invention, also claimed in our New Zealand Patent Specification No 242059, provides a novel process for the preparation of a protected 4'-demethylepipodophyllotoxin glucoside 4'-phosphate intermediate of formula (I) which 10 comprises reacting a hydroxy protected glucopyranose of formula (II) and a phosphate protected 4'-demethylpodo-phyllotoxin 4'-phosphate (III), in the presence of a Lewis acid.

The present invention also contemplates the novel compounds of formula (III), described and claimed herein.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an improved method for preparing 41-demethylepipodophyllotoxin glucoside 4•-phosphates, pharmaceutically acceptable salts thereof, or solvates thereof; in particular, the process is suitable for the preparation of etoposide 25 4•-phosphate including its pharmaceutically acceptable salts and solvates. As used herein, "pharmaceutically acceptable salts" include mono- or di-alkali metal salts, and alkaline earth metal salts; preferably the pharmaceutically acceptable salt is the disodium salt. 30 "Solvates" are formed by crystallization or recrystallization from organic solvents such as ethanol or from water (hydrates). The term "alkylidene" includes straight and branched carbon chains, for example ethylidene, propylidene and 35 isopropylidene. The term "4'- demethylpodophyllotoxin," unless otherwise specified, will be understood to encompass 4'-demethylpodophyllotoxin and 4*- f*h r--> fa :: xi- ■■■. " f V> ■' J k ;? \ i £Jt % ? 3 v> xjr i % J} y demethylepipodophyllotoxin, individually and as a mixture thereof.

The process of the present invention comprises 5 reacting a phosphate-protected 41-demethylpodo- phyllotoxin of formula (III) with a hydroxy-protected glucopyranose of formula (II) in the presence of a Lewis acid to provide an intermediate of formula (I). The hydroxy and phosphate protecting groups on the 10 intermediate are then removed to give 4'- demethylepipodophyllotoxin glucoside 4'-phosphate of formula (IV) directly, where the two R1 groups together represent Cv5 alkylidene; or where R1 is hydroxy protecting group, the product is reacted with an 15 appropriate carbonyl compound or an acetal or ketal equivalent thereof to give compound of formula (IV).

In compounds of formulas (I) and (III), the phosphate protecting group may be any that is known in 20 the art, examples of which include, but are not limited to, 2,2,2-trichloroethyl, phenyl, substituted phenyl, benzyl and substituted benzyl; the substituent may be one or more groups selected from methoxy, nitro, and methyl. Preferably, the phosphate 25 protecting group is phenyl or benzyl; and most preferably it is benzyl.

In compounds of formulas (I) and (II) the hydroxy protecting group may be any that is conventionally 30 used and include, but is not limited to, ethers such as methyl, t-butyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl, trityl, methoxymethy1, methoxyethoxymethyl, ethoxyethyl, tetrahydropyranyl, tetrahydrothiopyranyl, and trialkylsilyl ethers such 35 as trimethylsilyl ether and t-butyldimethylsilyl ether; esters such as benzoyl, acetyl, phenylacetyl, 8 formyl, mono-, di-, and trihaloacetyl such as chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl; and carbonates such as methyl, ethyl, 2,2,2-trichloroethyl, benzyl, and p-nitrophenyl. More 5 preferably the hydroxy protecting group is one commonly used in podophyllotoxin chemistry; examples that may be mentioned include, but are not limited to, benzyl; esters such as C^alkanoyl and halogenated C2. 3alkanoyl groups, e.g. formyl, acetyl, mono-, di-, and 10 trihaloacetyl; and Chalky 1 or halogenated Chalky 1 carbonates such as ethyl, and mono-, di-, and trihaloethyl carbonates. Halogen includes fluorine, bromine, chlorine and iodine. Most preferably the hydroxy protecting group is 2,2,2-trichloroethyl 15 carbonate (i.e. the 2,2,2-trichloro-ethoxycarbonyl group).

Compounds of formula (III) may be obtained by reacting 41-demethylpodophyllotoxin with a 20 chlorophosphate CIP(O) (OR3)2 wherein R3 is as previously defined. The reaction is carried out in an inert organic solvent such as acetonitrile at ambient temperature and in the presence of an acid scanvenger, e.g. a tertiary amine base such as diisopropyl 25 ethylamine. Illustrative of this general process is the reaction of 4•-demethylepipodophyllotoxin with diphenyl chlorophosphate in the presence of diisopropylethylamine in acetonitrile at room temperature to provide 4'-demethylepipodophyllotoxin 30 4•-diphenylphosphate.

The stereochemical configuration at position 1 of the starting material of formula (III) is not critical as the condensation of a compound of formula (II) with 35 a compound of formula (III) under the present conditions generally results in a product having the p K'.-.y #■> 7 8 /' f\ J Lit 0 0 desired configuration at position 1, i.e. an epipodophyllotoxin. Thus the starting material may be phosphate-protected 4'-demethylepipodophyllotoxin, phosphate-protected 4'-demethylpodophyllotoxin, or a 5 mixture thereof.

Tetra-O-protected 6-D-glucopyranose derivatives of formula (II) and methods for their preparation are generally known in the art. Compounds of formula (II) 10 wherein the two R1 groups together represent CV5alkylidene may also be prepared by known methods, for example, by reacting glucose with an appropriate carbonyl compound, or an acetal or ketal equivalent thereof, in the presence of an acid catalyst to form 15 the cyclic acetal, and subsequently introducing the R2 hydroxy protecting groups. The carbonyl compound may be for example acetaldehyde, propionaldehyde, or acetone, and equivalents thereof such as paraldehyde, acetaldehyde dimethyl acetal, and acetone dimethyl 20 acetal. Thus, as an example, D-glucose is treated with paraldehyde in the presence of a mineral acid, e.g. sulfuric acid to provide 4,6-O-ethylidene-D-glucose which is treated with sodium hydroxide and then benzylchloroformate to convert the anomeric 25 hydroxy group to the benzyl carbonate. The compound formed is treated with 2,2,2-trichloroethyl chloroformate, and the resulting product is subject to catalytic hydrogenation to provide 4,6-O-ethylidene-2,3-bis-0-(2,2, 2-trichloroethoxycarbonyl)-B-D-30 glucopyranose. This compound is also disclosed in Japanese Kokai J58-225,096.

The coupling of the protected glucopyranose of formula (II) and the protected 4'-demethylpodophyllo-35 toxin 4'-phosphate of formula (III) to form a compound of formula (I) is carried out in an inert organic 9 R i] £ T f\ C \J y h? u i) solvent and in the presence of a Lewis acid, preferably boron trifluoride etherate. The solvent is preferably a halogenated hydrocarbon such as methylene chloride, dichloroethane or chloroform. The reaction 5 temperature is typically below 0°C and suitably about -15 to about -25°C.

The hydroxy and phosphate protecting groups of compounds of formula (I) are then removed to provide 10 the corresponding 4'-demethylepipodophyllotoxin glucoside 4'-phosphate derivatives. Deprotection may be effected by methods generally known in the art, and examples that may be mentioned include hydrolysis, hydrogenation, hydrogenolysis, reduction, and the 15 like. For example, the 2,2,2-trichloroethoxycarbonyl hydroxy protecting group may be removed with a zinc reagent such as zinc and acetic acid; the phenyl and benzyl phosphate protecting groups may be removed by catalytic hydrogenolysis, and the benzyl group may 20 also be removed by catalytic transfer hydrogenation using palladium on carbon and a reagent such as 1,4-cyclohexadiene and 1-methyl-l,4-cyclohexadiene.

Depending on the nature of the protecting groups the deprotection may be effected sequentially in which 25 case the order of removal is not particularly critical, or if suitable, the protecting groups are removed in one step.

Where R1 is hydroxy protecting group, the compound 30 produced after removing the hydroxy protecting group is reacted with an appropriate carbonyl compound, e.g. acetaldehyde, propionaldehyde, acetone, or an acetal or ketal equivalent thereof such as paraldehyde, acetaldehyde dimethyl acetal, and acetone dimethyl 35 acetal, in the presence of an acid catalyst to form the corresponding compound of formula (IV). 9 Pt n /; a (L \j %} ^ -<J \.i 11 In one specific embodiment illustrative of the present process, 4'-demethylepipodophyllotoxin 4'-diphenylphosphate is reacted with 4,6-0-ethylidene-2,3-bis-O- (2,2,2-trichloroethoxycarbonyl)-D-5 glucopyranose in dichloroethane at -20°C in the presence of boron trifluoride etherate to give 2,3-bis-O-(2,2,2-trichloroethoxycarbonyl)etoposide 41 -diphenylphosphate. Treatment with zinc and acetic acid to remove the 2,2,2-trichloroethoxycarbonyl 10 groups, followed by catalytic hydrogenolysis to remove the phenyl groups, provides etoposide 4'-phosphate. In another example, the benzyl group is used as phosphate protecting group, and is removed by catalytic transfer hydrogenation with e.g. palladium 1 5 on carbon and l-methyl-1,4-cyclohexadiene.

The 4'-demethylepipodophyllotoxin glucoside 4'-phosphate of formula (IV) may be converted to its pharmaceutically acceptable salt by contacting it with 20 a source of the appropriate cation; for example treating it with a base such as sodium carbonate results in the formation of the sodium salt thereof.

Solvates of the 4'-demethylepipodophyllotoxin glucoside 4'-phosphate of formula (IV) may also be 25 obtained by crystallizing or recrystallizing from organic solvents or water; thus, for example, the diethanolate of etoposide 4'-phosphate may be obtained from a saturated solution of etoposide 4'-phosphate in ethanol containing solvent system.

The present invention provides novel compounds of the formula (III) 12 HO \ 0 0CH3 -OR" -OR: £% tf\ 8 r>v,~ f* (] A \ I (III) wherein R3 is a phosphate protecting group; preferably R3 is selected from phenyl, substituted phenyl, benzyl, substituted benzyl, and 2,2,2-trichloroethyl; more preferably R3 is phenyl or benzyl; most preferably R3 is benzyl.

The following examples are offered in order to more fully illustrate the present invention and shall not be construed to limit the scope of the invention in any manner.

Example 1 41-Demethvlepipodophvllotoxin-4'-diphenylphosphate 30 (Compound 1) To a solution of 4'-demethylepipodophyllotoxin (1.44g), prepared from podophyllotoxin as described (Helv. Chim. Acta. 152. p. 944, 1969), in dry 35 acetonitrile (25 ml) was added diisopropylethylamine (941 fiL) and diphenyl chlorophosphate (821 /iL). The 26043 13 mixture was stirred at room temperature for 16 h and concentrated in vacuo. The residue was dissolved in CH2C12 and washed with H20 and brine. Purification was effected by medium pressure chromatography on silica 5 gel using 2% MeOH in CH2C12 followed by crystallization from EtOAc in hexane. The unoptimized yield of pure 1 was 700 mg (ca 30%).

'H-NMR (CDCIj) S 7.35-7.15 (m, 10H), 6.81(s,lH), 6.43(s,1H), 6.23 (s,2H), 5.94 (d,2H), 4.78 (t,lH), 4.54 (d,lH, J=5Hz), 4.35-4.20 (m,2H), 3.48 (s,6H), 3.24 (dd,1H,J=13.2 and 5Hz) , 2.73-2.63 (m, 1H) , 1.97 (d,1H,OH).

HRMS calcd. for C33HJ0OnP (M+H) : 633.1526 Found: 633.1532 Example 2 4 1 -Demethylepipodophyllotoxin-4' -dibenzvlohosphate (Compound 2) Dibenzyl chlorophosphate (6 equiv) was freshly 25 prepared from equimolar amounts of N-chlorosuccinimide and dibenzyl phosphite refluxing in CH2C12 (150 ml) for 20 min. This mixture was filtered and the filtrate was added to a solution of 4'-demethylepipodophyllotoxin (1.22 g), diisopropylethylamine (1.7ml), and 30 4-dimethyl-aminopyridine (37mg) in dry acetonitrile. The mixture was stirred for 16 h and then partitioned with CH2C12 and saturated NaHCOj. The organic extracts were washed with H20 and brine and dried (Na2S04) . Purification was effected by flash chromatography on 35 silica gel using 1% MeOH in CH2C12 followed by crystallization from EtOAc, 10% MeOH in CH2C12 followed 14 by crystallization from EtOAc, 10% MeOH in CH2C12, and hexane. The unoptiraized yield of pure 2 was 760 mg (ca 38%). 1H-NMR (CDC13) S 7.37-7.27 (m,10H), 6.83 (s,lH), 6.49 (s,1H), 6.27 (s,2H), 5.96 (d,2H), 5.25-5.17 (m,4H), 4.82 (d,1H,J=3.3Hz) , 4.60 (d,1H,J=5Hz), 4.40-4.30 (m,2H), 3.62 (s,6H), 3.28 (dd,lH,J=13 and 5Hz), 2.77-2.68 (m,lH).

Mass Spectrum, m/e=661 (M++H).

Example 3 2". 3"-bis-0-r 2.2.2-trichloroethoxv) carbonvl l -etoposide -4 ' -diphenylphosphate (Compound 3.) To a suspension of Compound 1. (50mg) and 4,6-0-ethylidene-2,3-bis-0-[(2,2,2-trichloroethoxy)-carbonyl]-6-D-glucopyranose (45mg) in dry 1,2-dichloroethane (0.5ml) at -20°C under argon was added boron trifluoride etherate (27 nl) dropwise over 5 min. After 2 h at -20°C, the mixture was quenched with pyridine (50 nl) and allowed to warm to room temperature. The mixture was diluted with CH2C12 (20 ml) and washed with cold 1 N HCl (2x), H20, and brine and dried (MgSOJ . Purification by preparative TLC using 1% MeOH in CH2C12 gave 74.5 mg (80.5%) of pure title compound, 3.

IR (KBr) 1778, 1600, 1488, 1284, 1260, 1232, 1190, 1162, 1132, 958, 822, 774 cm*1.

'H-NMR (CDClj) S 7.35-7.15 (m,10H), 6.72 (S,1H), 6.47 (S,1H), 6.15 (s,2H) , 5.98 (S,2H), 4.88 (d,lH), 4.82-4.45 (m,7H), 4.35 (m,lH), 4.25-4.18 (m,2H), 3.80-3.49 (m,4H), 3.48 (s,6H), 3.41-3.37 (m,1H), 3.13 (dd, 1H, J=14.1 and 5.2 Hz), 2.86-2.76 (m,lH), 1.31 (d,3H,J=5Hz).

Mass Spectrum, m/e=1171 (M++H).

Example 4 2".3"-bis-0-r2.2.2-trichloroethoxv)carbonvll-etoposide-4'-dibenzvlphosphate (Compound 4) The procedure described in Example 3 was repeated 15 using Compound 2 (145 mg) and 4,6-0-ethylidene-2,3-bis-0-[2,2,2-trichloroethoxy)carbonyl]-6-D-glucopyranose (135 mg) in dry 1,2-dichloroethane (1.5 ml) with boron trifluoride etherate (74 nL) to provide 240 mg of pure title compound, 4.

IR (KBr) 1768, 1599, 1505, 1485, 1458, 1420, 1381, 1338, 1260/ 1229, 938, 818 cm"1. 1H-NMR (CDClj) 7.35-7.25 (m,10H), 6.75 (S,1H), 25 6.51 (s,1H), 6.22 (s,2H), 5.98 (s,2H), 5.24-5.14 (m,4H), 4.91 (d,lH), 4.83-4.47 (m,7H), 4.37 (m,lH), 4.24-4.20 (m,2H), 3.80-3.45 (m,4H), 3.62 (s,6H), 3.44-3.35 (m,lH), 3.15 (dd,1H,J=14 and 5.2 Hz), 2.88-2.78 (m,1H), 1.31 (d, 3H,J=4.9Hz) .

Mass Spectrum, m/e=1199 (M*+H) . tf*\ r* 7 D7 i"r". ^ o 0 j u 16 Example 5 Etoposide 4'-diphenylphosphate.

To a solution of compound 3 (200 mg) in dry THF (8 ml) under N2 at room temperature was added zinc dust (468 mg) followed by acetic acid (4.8 ml). The reaction mixture was sonicated at 25-30°C for 30 min and then stirred for 3 h at room temperature. The 10 mixture was filtered through Celite and washed with CH2C12 (200 ml). The filtrate was washed with saturated NaHC03, H20 and brine, and dried (MgSOJ . Evaporation in vacuo followed by flash chromatography on silica gel using 0-3% MeOH in CH2C12 gave 116 mg 15 (83%) of pure title compound.

The conversion of the title compound to etoposide 4'-phosphate is described in U.S. Patent 4,904,768.

Example 6 Etoposide 4'-dibenzvlphosphate.

The procedure described in Example 5 was repeated using a solution of Compound 4 (152 mg) in dry dioxane (2ml), zinc dust (315 mg) and acetic acid (1.2 ml) to provide 126 mg (66%) of pure title compound.

Example 7 Etoposide 41-phosphate.

A solution of 4'-dibenzylphosphate etoposide (500 g, 0.59 mole) in methanol (2 1) is added to a tasry '*■ \ 0 > U 17 suspension of 10% Pd/C (50 g) in methanol (l 1), and the suspension is heated to about 37 °C. To this suspension is added slowly a solution of 1-methyl-l,4-cyclohexadiene (555 g, 660 ml, 5.89 mole, 10 eq) in 5 methanol (1 1), and the suspension is stirred at 40 -45°C until the reaction is complete as shown by TLC. The reaction mixture is filtered and the volume of the filtrate is adjusted to about 1 1 by concentrating or adding additional methanol, the resulting solution is 10 then added to absolute ethanol (4 1). The solution is seeded with etoposide 4'-phosphate reference standard and concentrated to about 2.5 1. Absolute ethanol (3.5 1) is added to the slurry and stirred at about 20°C for 18 - 72 hours. The solids are collected by 15 filtration, washed with absolute ethanol (2 x 250 ml) and dried under vacuum for 18 hours at about 20°C to provide 300 - 350 g of the title compound as the diethanolate (80 - 90% yield).

Melting point: 141 - 150°C (lose solvent); 160 - 172°C (melt).

Ethanol residue: 11.8 % (by NMR); 13.2 % (by thermogravimetric analysis). Calc. for C^jjO^P.2C2H60 25 12.1%.

Moisture content: 0.22% by Karl Fischer method A 18

Claims (3)

WHAT WE CLAIM IS:

1. A compound having the formula CH30 (111) wherein R3 is a phosphate protecting group.

2. A compound according to Claim 1 wherein R3 is selected from the group consisting of phenyl, substituted phenyl, benzyl, substituted benzyl, and 2,2,2-trichloroethyl.

3. A compound according to claim 1 wherein R3 is phenyl or benzyl. DATED THIS el DAY OFA?^ 19 A. J. PARK & SON PER AGENTS FOR THE APPLICANTS 35 N.Z. PATENT OFFICE - 2 MAY m r*M~ /■%<"- n / r~ f*\