IE903764A1 - Glycosyl-etoposide prodrugs, a process for the preparation¹thereof and the use thereof in combination with¹functionalized tumor-specific enzyme conjugates - Google Patents

Abstract

The present invention relates to glycosyl-etoposide prodrugs, process for their preparation and their use in combination with functionalised tumour-specific enzyme conjugates for the treatment of cancers, and it specifically relates to 4'-O-glycosyl-etoposides as prodrugs which can be cleaved to cytotoxic agents on exposure to tumour-specific enzyme conjugates, where the liberated agent is suitable, by reason of its cytostatic activity, for the treatment of cancers.

Description

-1IE 903764 BEHRINGWERKE AKTIENGESELLSCHAFT 89/B 039 - Ma 792 Dr. Ha/Sd Glycosyl-etoposide prodrugs, a process for the preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates The present invention relates to glycosyl-etoposide prodrugs, a process for the preparation thereof and the use thereof in combination with functionalized tumor10 specific enzyme conjugates for treating cancers, and specifically relates to 4'-O-glycosyl-etoposides as prodrugs which can be cleaved by the action of tumorspecific enzyme conjugates to give cytotoxic active substances, the liberated active substance being suitable, by reason of its cytostatic activity, for treating cancers.

The combination of prodrug and tumor-specific antibodyenzyme conjugates for use as therapeutic agents is described in the specialist literature. This entailed antibodies which are directed against a particular tissue and are covalently bonded to a prodrug-cleaving enzyme being injected into an animal which contains the transplanted tissue, and subsequently administering a prodrug compound which can be activated by the enzyme. The prodrug is converted by the action of the antibody-enzyme conjugate, which is anchored to the tissue, into the cytotoxin which exerts a cytotoxic effect on the transplanted tissue.

A therapeutic system which contains two components and is composed of an antibody-enzyme component and of a prodrug component which can be activated by enzyme is described in WO 88/07378. In this case, the use of nonmammalian enzymes is described for the preparation of the antibody-enzyme conjugates, and that of endogenous enzymes is ruled out because of the non-specific liberation of active compound. Since the exogenous enzymes are recognized by the body as foreign antigens, the use thereof is associated with the disadvantage of an immune response to the non-endogenous substances, for which reason the enzyme immobilized on the antibody is inactivated and, where appropriate, the entire conjugate is eliminated. In addition, in this case p-bis-N-(2-chloroethyl)aminobenzylglutamic acid and derivatives thereof are used as prodrug, and their chemical half-life is only 5.3 to 16.5 hours. It is a disadvantage for a prodrug to be chemically unstable because of the side effects to be expected.

A therapeutic system which contains two components and in which the antibody-enzyme conjugate located on the tumor tissue cleaves a prodrug compound to a cytotoxic active compound is likewise described in EPA 0302473 A2. The combined use of etoposide 4'-phosphate and derivatives thereof as prodrug and of antibody-immobilized alkaline phosphatases for liberating the etoposides, which is described therein inter alia, is disadvantageous because of the strong presence of endogenous alkaline phosphatases in the serum. As described in DE 38265662 Al, the etoposide 4'-phosphates are already used alone as therapeutic antitumor agents, in which case the phosphatases present in the serum liberate the etoposide from the prodrug.

It has emerged, surprisingly, that the synthetically prepared, hitherto unobtainable compound 4'-O-alpha-Dglucopyranosyl-etoposide can be cleaved in vitro into etoposide and D-glucose with the enzyme alpha-glucosidase as well as a tumor-specific antibody-glucosidase conjugate.

Based on this finding, and taking into account the disadvantages, described above, of combinations of prodrugs and antibody-enzyme conjugates, the object of the present invention was to prepare synthetic, enzymatically cleavable 4'-O-glycosyl-etoposides as well as functionalized tumor-specific enzymes, and to test the pharmacological utility of the combination of the two components in suitable mammalian test models. This object has been achieved by preparing compounds of the formula I and functionalized tumor-specific enzymes which, on combined use thereof, showed an effect in tests of cytostatic activity.

The invention relates to 4'-O-glycosyl-etoposides of the 10 formula I in which R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, an acyl or tri-Ci-C^-alkylsilyl protective group, R3 is a hydroxyl group, an acyl or tri-C1-C4-alkylsilyl protective group which is bonded via an oxvcen a tan, an amino acetylamino, benzyloxycarbonylamino or dimethylamino group, R* is a hydrogen atom or a methyl group, R5 is a hydrogen atom, a hydroxyl group, an acyl or tri-C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino, azido or acetylamino group, R6 is a hydroxyl group, an acyl or tri-Ci-Ct-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino or azido group, R7 is a hydrogen atom, an acyl or tri-C1-C4-alkylsilyl protective group and R is a methyl or hydroxymethyl group or an acyl protective group which is bonded via a methyleneoxy group, or a benzyloxycarbonyl group, where an acyl protective group means an acetyl, mono-, di- or trihalogenoacetyl group with halogen meaning fluorine or chlorine.

A functionalized tumor-specific enzyme means within the scope of the invention an enzyme of the formula II A-Sp-E II in which A is an antibody or one of the fragments thereof, which have specificity for a tumor-associated antigen, or is a biomolecule which accumulates in a tumor, such as EGF (epidermal growth factor), TGFalpha (transforming growth factor alpha), PDGF (platelet derived growth factor), IGF I+II (insulin like growth factor I+II) or a+b FGF (acidic + basic fibroblast growth factor) E is a glycosidase which is not immunogenic or is of low immunogenicity, preferably mammalian glycosidase, as alpha- or beta-glucosidase, alphagalactosidase, alpha- or beta-mannosidase, alphafucosidase, N-acetyl-alpha-galactosaminidase, Nacetyl-beta-/N-acetyl-alpha-glucosaminidase or betaglucuronidase , Sp (spacer) is a bifunctional sulfide- or disulfidecontaining group of the formula III or IV X(S)nY III X(S)n IV or a polypeptide spacer, in which X or Y is -CO-R0-(N-succinimido)- or -C (=R10)-CH2-CH2with R8 being -CH2-CH2-, 1,4-cyclohexylidene, 1,3- or 1,4-phenylene or methoxycarbonyl- or chloro-1,4phenylene and R10 being 0 or NH, and furthermore Y is -C(=R10)-CH2-CH2-, where R10 has the stated meaning, and n is 1 or 2.

The fusion gene composed of VH, CB1 hinge and enzyme gene 5 is cloned into an expression plasmid which is suitable for expression in eukaryotic cells and carries a selection marker. The expression plasmid with the fusion gene is transfected together with an expression plasmid which contains the light-chain gene belonging to the antibody into eukaryotic expression cells. Selection with a suitable antibiotic is followed by identification of transfectoma clones which contain the expression plasmids. Suitable detection methods (BioDot, ELISA) are used to identify those transfectoma clones which secrete the fusion protein of the formula II composed of antibody and enzyme.

Preferred within the scope of the invention are compounds of the formula I in which the radicals R1 is a 20 R2 is a or a R3 is a Ci-C^-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, acetylamino, benzyloxycarbonylamino or dimethylamino group, R* is a hydrogen atom or a methyl group, R5 is a hydrogen atom, a hydroxyl group, or an acetyl, chloroacetyl or tri-Cj-C^-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino, azido or acetylamino group, R6 is a hydroxyl group, an acetyl, chloroacetyl or triC1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino or azido group, R7 is a hydrogen atom, an acetyl, chloroacetyl or triCi-C^-alkylsilyl protective group and R8 is a methyl, hydroxymethyl, acetyloxy or chloroacetyloxymethyl group or a benzyloxycarbonyl group, as well as a functionalized tumor-specific enzyme of the formula II in which A is an antibody or fragment thereof, which have specificity for a tumor-associated antigen, or is a biomolecule which accumulates on or in the tumor, such as EGF (epidermal growth factor), TGF-alpha (transforming growth factor alpha), PDGF (platelet derived growth factor), IGF I+II (insulin like growth factor I+II), a+b FGF (acidic + basic fibroblast growth factor) E is a glycosidase which is not immunogenic or has low immunogenicity, preferably a mammalian glycosidase, for example an alpha- or beta-glucosidase, alphagalactosidase, alpha- or beta-mannosidase, alphafucosidase, N-acetyl-alpha-galactosaminidase, Nacetyl-beta-/N-acetyl-alpha-glucosaminidase or betaglucuronidase , Sp is a bifunctional disulfide-containing group of the formula III or IV or a polypeptide spacer, in which X or Y is -CO-R8-(N-succinimido)- or -C (=R10) -CH2-CH2with R9 being -CH2-CH2- or 1,4-phenylene and R10 being 0 or NH, Y is —C(=R10)-CH2-CH2-, where R10 has the stated meaning, and n is 1 or 2.

The process according to the invention for preparing a compound of the formula I, which can be degraded by glycosidase, in which R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, R3 is a hydroxyl, amino or dimethylamino group, R* is a hydrogen atom or a methyl group, R5 is a hydrogen atom, a hydroxyl group, an amino or acetylamino group, R6 is a hydroxyl group or an amino group, R7 is a hydrogen atom, R8 is a methyl or hydroxymethyl group or a carboxyl group or an acyl protective group which is bonded via a methyleneoxy group, or a benzyloxycarbonyl group, where an acyl protective group means an acetyl, mono-, di- or trihalogenoacetyl group with halogen meaning fluorine or chlorine, comprises reacting, in the presence of a promoter and, where appropriate, of an acid trap or drying agent in a solvent at -50*C to 60*C, an etoposide compound of the formula V in which R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, an acyl or a tri-Cj-C^-alkylsilyl protective group, R3 is a hydroxyl group, an acyl or tri-C1-C4-alkylsilyl protective group which is bonded via oxygen, or acetylamino, benzyloxycarbonylamino or dimethylamino group, and R* is a hydrogen atom or a methyl group, with a carbohydrate component of the formula VI r70 'Υ- νί in which R5 is a hydrogen atom, a hydroxyl group, an acyl protective group which is bonded via an oxygen atom, or benzyloxycarbonylamino, azido or acetylamino group, R6 is an acyl protective group which is bonded via an oxygen atom, or a benzyloxycarbonylamino or azido group, R7 is an acyl protective group, R8 is a methyl group, methyleneoxy-acyl protective group or a benzyloxycarbonyl group and Z is a halogen atom, preferably fluorine, chlorine or bromine, a hydroxyl group, a tri-C^-C^-alkylsilyloxy group, or an acyl protective group which is bonded via an oxygen atom, where the acyl protective group is an acetyl, mono-, di- or trihalogeno-acetyl group, preferably with the halogen atom being fluorine or chlorine, to give a 4'-O-glycosyl15 etoposide derivative of the formula I in which all the radicals R1 to R8 retain their meaning as defined above, and eliminating the protective groups present in these compounds by hydrogenolysis or hydrolysis, and, where appropriate, converting by means of reductive alkylation one of the resulting compounds containing amino groups into another compound of the formula I containing dimethylamino groups.

The specific procedure for this is as follows: the glycosidation of etoposide derivatives of the formula V is carried out using functionalized carbohydrate units of the formula VI which are typically protected with acyl protective groups on the 0-2, 0-3, 0-4 and, where appropriate, 0-6 atoms. Preferred acyl protective groups are acetyl, chloroacetyl or trifluoroacetyl groups. In the case of amino sugars, the amino group is protected temporarily with a benzyloxycarbonyl group or permanently with an acetyl group. It is likewise possible to use azido sugars because they can be converted straightforwardly into amino sugars by hydrogenolysis. The carbohydrate components must be suitably functionalized at the anomeric center. Used for this purpose are glycosyl halides, such as fluorides, chlorides or bromides, which can be prepared starting from 1-0-acyl derivatives, for example using HF, HC1, HBr or TiBr4. The glycosidation components which carry an O-acyl group or a hydroxyl group on the anomeric center are prepared by processes customary in carbohydrate chemistry.

The glycosidation of etoposides of the formula V with carbohydrate units of the formula VI is carried out in the presence of a promoter. The promoter used when glycosyl fluorides and the 1-hydroxy or 1-acetyloxy analogs thereof are employed is BF3 x ether or tri-Ci-C*10 alkylsilyl trifluoromethanesulfonate. The promoters used in the case of glycosyl chlorides or bromides are salts of silver or of mercury.

The glycosidation is carried out in an aprotic organic solvent such as acetone, ethyl acetate, ether, toluene, dichloromethane or dichloroethane or mixtures thereof. In order to trap the acid or water produced in the reaction, where appropriate, acid traps or drying agents such as molecular sieves or magnesium sulfate are added. The reaction temperature is in the range from -50 eC to 0*C when glycosyl fluorides and the 1-hydroxy analogs are employed and at 0eC to 60’C when glycosyl chlorides or bromides are employed. The glycosyl etoposides produced in the reaction are deblocked by the following processes: the acyl protective groups are removed by methanolysis catalyzed by zinc (II) salts or with alkaline ion exchangers in methanol, ethanol or mixtures thereof with chloroform, dichloromethane or ether. The benzyl or benzyloxycarbonyl groups or azido groups are eliminated by hydrogenolysis with palladium on carbon or palladium/ barium sulfate or, in the case of the azido group, converted into amino group. The compounds of the formula I containing amino sugars can additionally be converted into dimethylamino derivatives by reductive alkylation with formaldehyde/sodium cyanoborohydride.

To prepare A-Sp-E, either the spacer (Sp) can be linked via an amino group to an enzyme and to the antibody or the biomolecule via an HS group which has been introduced or generated by cleavage of the disulfide linkage, or nucleic acid sequences which code for the parts A, Sp and E are covalently linked with the aid of molecular bio5 logical methods to result in a fusion gene, and A-Sp-E is prepared by genetic engineering processes.

This can be carried out in a variety of ways: A) A restriction cleavage site A is introduced by specific mutagenesis at the 3' end of the CH1 exon in the gene of the heavy chain of the immunoglobulin. The same restriction cleavage site A is generated at the 5' end of the oligonucleotide which codes for the oligopeptide which acts as spacer. Both restriction cleavage sites A are sited in such a way that the immunoglobulin gene can be linked to the oligonucleotide via the restriction cleavage site A without disturbing the reading frame.

A restriction cleavage site B is generated at the 3' end of the oligonucleotide. This restriction cleavage site B is introduced at the site in the gene which codes for the enzyme at which the nucleic acid sequence coding for the mature protein starts. The enzyme gene is then linked via the restriction cleavage site B to the immunoglobulin gene-linked construct. The restriction cleavage sites B are sited such that the reading frame is not disturbed on linkage. The fusion gene composed of the for the heavy chains of the immunoglobulin VB and CH1 linker enzyme is cloned into an expression plasmid which is suitable for expression in the eukaryotic cells and carries a selec30 tion marker.

The expression plasmid with the fusion gene is transfected together with an expression plasmid which carries the gene for the light chain belonging to the antibody into eukaryotic cells (for example myeloma cells).

Selection with suitable antibiotics is carried out to isolate cell clones which contain the plasmids with the fusion gene and the gene for the light chains (transfectomas). Suitable detection methods (BioDot; ELISA) are used to identify those trans fee tomas which secrete the fusion protein of the formula A-Sp-E composed of the MAb Fab part, linker polypeptide and enzyme.

B) A restriction cleavage site A is introduced at the 3' end of the hinge exon of the gene for the heavy chains of the immunoglobulin. The restriction cleavage site A is introduced at the site in the enzyme gene at which the nucleotide sequence coding for the mature protein starts. The gene fragment of the heavy chains of the immunoglobulin with the VH, CH1 and hinge exons is linked via the restriction cleavage site A to the enzyme gene.

The restriction sites A are sited such that the reading frame is not disturbed on linkage. The hinge part of the antibody functions as the polypeptide spacer in this construction.

The fusion gene composed of VH, CH1 hinge and enzyme gene is cloned into an expression plasmid which is suitable for expression in eukaryotic cells and carries a selection marker. The expression plasmid with the fusion gene is transfected together with an expression plasmid which contains the light-chain gene belonging to the antibody into eukaryotic expression cells. Selection with a suitable antibiotic is followed by identification of transfectoma clones which contain the expression plasmids. Suitable detection methods (BioDot, ELISA) are used to identify those transfectoma clones which secrete the fusion protein of the formula II composed of antibody and enzyme.

The coupling between enzyme and antibody, fragment thereof or a biomolecule is carried out by processes described in the literature (A.H. Blair and T.I. Ghose, I. Immunolog. Methods 59 (1983) 129-143; T.I. Ghose et al. Methods in enzymology, Vol. 93 (1983) 280-333).

This entails initial functionalization of the enzyme via its amino group using succinimidyl N-maleimidoalkylidene-, cycloalkylidene- or arylene-1-carboxylate, where the double bond of the maleimido group enters into a reaction with the HS group of the functionalized antibody, fragment thereof or the biomolecules, with the formation of a thioether functionality.

It is possible to use for the preparation of the 10 antibody-enzyme conjugates the monoclonal antibodies described in EP-A-0141079, preferably the antibodies 431/26, 250/183, 704/152 and 494/32. The specificity of the antibodies for tumor-associated antigens has already been demonstrated on animals and humans by means of immunoscintigraphy and immunohistochemistry.

The nucleotide sequence of the V genes of these monoclonal antibodies is described in German Patent Application DE-A-3909799.4.

To prepare the tumor-specific enzyme conjugates, it is 20 possible for the enzymes which are mentioned hereinafter and from the identified source to be purified by the indicated literature procedure: alpha-galactosidase from human liver, Dean, K.G. and Sweeley, C.C. (1979), J. Biol. Chem. 254, 994-1000 - beta-glucuronidase from human liver, Ho, K.J. (1985) Biochim. Biophys. Acta 827, 197-206 alpha-L-fucosidase from human liver, Dawson, G., Tsay, G. (1977) Arch. Biochem. Biophys. 184, 12-23 alpha-mannosidase from human liver, Grabowski, G.A., Ikonne, J.U., Desnick, R.J. (1980) Enzyme 25, 13-25 beta-mannosidase from human placenta, Noeske, C., Mersmann, G. (1983) Hoppe Seylers Z Physiol. Chem. 364, 1645-1651 alpha-glucosidase from human gastrointestinal mucosa, Asp, N.-G.,. Gudmand-Hoeyer, E., Christiansen, P.M., Dahlquist, A. (1974) Scand. J. Clin. Lab. Invest. 33, 239-245 beta-glucosidase from human liver, Daniels, L.B., Coyle, P.J., Chiao, Y.-B., Glew, R.H. (1981) J.

Biol. Chem. 256, 13004-13013 beta-glucocerebrosidase from human placenta. Furbish, F.S., Blair, H.E., Shiloach, J.,Pentcheu, P.G., Brady, R.O. (1977) Proc. Natl. Acad. Sci. USA 74, 3560-3563 - alpha-N-acetylglucosaminidase from human placenta, Roehrborn, W., von Figure, K. (1978) Hoppe Seylers Z Physiol. Chem. 359, 1353-1362 beta-N-acetylglucosaminidase from human amniotic membrane, Orlacchio, A., Emiliani, C., Di Renzo, G.C., Cosmi, E.V. (1986) Clin. Chim. Acta 159, 279-289 alpha-N-acetylgalactosaminidase according to Salvayre, R.,Negre, A.,Maret, A., Douste-Blazy, L. (1984) Pathol. Biol. (Paris) 32, 269-284.

The glycolytic activity of the functionalized tumorspecific enzymes was determined in comparative investigations with p-nitrophenyl glycosides at the particular pH optimum.

The invention additionally relates to a pack containing a glycosyletoposide according to the invention and a functionalized tumor-specific enzyme conjugate in combination with functionalized tumor-specific enzyme conjugates.

To test the efficacy of a combined sequential use, transplanted mice were given the functionalized enzyme, then, after waiting until the plasma level of the enzyme had fallen virtually to zero, the glycosyletoposide was given and it was observed whether growth stopped and regression occurred.

Example 1 Preparation of the glycosylation component Benzyl D-qlucuronate (compound 1) Benzyl bromide (4.89 g, 28.59 mmol) was added to a 5 solution of sodium D-glucuronate (5 g, 23.13 mmol) in DMF (300 ml). The reaction mixture was stirred at 40’C for 2 h and then at 80°C for 16 h and evaporated in vacuo. The residue was purified by column chromatography on silica gel (130 g) with 80:20:1 chloroform/methanol/ water.

Yield: 4.87 g (74%). The title compound was characterized by 13C NMR.

Benzyl 1.2,3,4-tetra-O-chloroacetvl-alpha and beta-Dqlucuronate (compound 2a and 2b) Benzyl D-glucuronate (3.80 g, 13.36 mmol) was suspended in dichloromethane (200 ml). Chloroacetyl chloride (7.90 g, 69.94 mmol) was added and then the mixture was cooled to -30°C, and pyridine (4.33 g, 54.74 mmol) dissolved in dichloromethane (50 ml) was added. The reaction mixture was stirred at -30eC for 16 h and then chloroacetyl chloride (7.90 g) and pyridine (4.33 g) were added. The mixture was stirred for 16 h and then cold dichloromethane (150 ml) was added and the mixture was washed with 5% strength sodium citrate buffer (pH 5, 60 ml x 2) and ice-water (50 ml x 2). The resulting product (7.92 g), which contained about 30% benzyl 2,3,4tri-O-chloroacetyl-alpha, beta-D-glucuronate besides the title compound, was used without further purification steps in the next stage.

Benzyl 2,3,4-tri-O-chloroacetvl-alpha, beta-D-qlucuronate (compound 3a, 3b) The crude product (7.92 g) of compounds 2a/2b was dissolved in 3:1 methanol/chloroform (320 ml), and aminated silica gel (11.09 g) was added. The reaction mixture was stirred at room temperature for 6 h and filtered. The filtrate was evaporated and then the residue was purified by chromatography on silica gel (220 g) with 2:1 petroIE 903764 leum ether/ethyl acetate.

Yield: 4.94 g (72% based on compounds 2a/2b).

Benzyl 1-deoxv-l-fluoro-alpha-D-alucuronate (compound 4) Sodium 1-deoxy-l-fluoro-alpha-D-glucuronate (6.22 g, 5 28.51 mmol) was suspended in DMF (350 ml), and benzyl bromide (4.89 g, 28.59 mmol) was added. The reaction mixture was stirred at 60°C for 24 h and then evaporated.

The residue was dissolved in 3:1 chloroform/methanol, and magnesium sulfate (12 g) was added. The suspension was stirred for 2 h and then filtered, and the filtrate was evaporated. The residue was purified by column chromatography on silica gel (160 g) with 4:1 dichloromethane/ acetone.

Yield: 5.95 g (73%).

Benzyl 1-deoxv-l-fluoro-2,3,4-tri-O-alpha-D-qlucuronate (compound 5) Compound 4 (5.0 g, 17.46 mmol) was suspended in dichloromethane (120 ml) and, at 0’C, benzyl bromide (9.85 g, 57.61 mmol) and silver oxide (11.2 g) were added. The reaction mixture was stirred at 0*C for 5 h and then at room temperature for 28 h. The reaction mixture was filtered, and the filtrate was evaporated in vacuo. The residue was purified by chromatography on silica gel (240 g) with 3:1 petroleum ether/ethyl acetate.

Yield: 6.60 g (68%).

Benzyl 1-deoxy-l-fluoro-2,3.4-tri-O-chloroacetyl-alphaD-qlucuronate (compound 6) Compound 4 (5.0 g, 17.46 mmol) was suspended in dichloromethane (260 ml) and, at -30*C, chloroacetyl chloride (9.86 g, 87.30 mmol) was added. After addition of 10:1 dichloromethane/pyridine (100 ml), the reaction mixture was stirred at -30C for 18 h. Cold dichloromethane (80 ml) was added to the mixture, which was washed with sodium citrate buffer (pH 5.0, 80 ml x 3) and then with water. The organic phase was dried over sodium sulfate and evaporated. The residue was purified by column chromatography on silica gel (260 g) with 5:1 petroleum ether/ethyl acetate.

Yield: 7.58 g (92%). 2,3,4, 6-Tetra-O-chloroacetvl-alpha-D-qalactopvranosvl fluoride (compound 7) Alpha-D-galactopyranosyl fluoride (2.30 g, 12.62 mmol) was dissolved in dry dichloromethane (100 ml) and, at -25eC, chloroacetyl chloride (9.0 g, 79.68 mmol) and 1:1 dichloromethane/triethylamine (55 ml) were added. The reaction mixture was stirred for 16 h and then chloroacetyl chloride (9.0 g) and 1:1 dichloromethane/triethylamine (55 ml) were added. The reaction mixture was stirred for a further 24 h and then washed with sodium citrate buffer (pH 5.0, 50 ml x 3) and then with water.

The organic phase was dried (sodium sulfate) and evaporated in vacuo. The residue was purified by column chromatography on silica gel (300 g) with 40:8:1 dichloromethane/petroleum ether/ethyl acetate.

Yield: 5.19 g (82%) [alpha]D +64.2° (c « 1, dichloromethane). 2,3,4,6-Tetra-O-benzvl-alpha-D-galactopyranosyl fluoride (compound 8) Alpha-D-galactopyranosyl fluoride (2.30 g, 12.62 mmol) was dissolved in dry DMF (40 ml) and, at -20eC, benzyl bromide (12.95 g, 75.72 mmol) and silver oxide (10 g) were added. The reaction mixture was stirred at -20’C for 5 h and then at room temperature for 24 h. The salts were then filtered off and the filtrate was evaporated in vacuo. The residue was purified by column chromatography on silica gel (350 g) with 15:1 petroleum ether/ethyl acetate.

Yield: 5.20 g (76%).

Example 2 Glycoside synthesis ' -0-Demethyl-4-0-(2,3-di-0-chloroacetvl-4.6,0-ethvlidene-beta-D-qlucopyranosvl)-4-epi-podophvllotoxin (compound 9) ' -0-Benzyloxycarbonyl-4-0-demethyl-4-0- (2,3-di-O-chloroacetyl-4,6-O-ethylidene-beta-D-glucopyranosyl) -4-epipodophyllotoxin (10 g, 11.42 mmol) was hydrogenated in 2:1 ethyl acetate/methanol (200 ml) in the presence of % Pd/C (5.0 g) for 2 h. The reaction mixture was filtered, and the filtrate was evaporated in vacuo. The residue was filtered through a layer of silica gel (50 g) . The resulting product was crystallized with methanol/ethyl acetate.

Yield: 7.50 g (88.6%); melting point 201-203’C; [alpha]D -73.4° (c = 1, chloroform) Benzyl 4'-O-demethyi-4-O-(di-O-chloroacetyl-4,6-O-ethvlidene-beta-D-qlucopvranosvl)-4-epi-4'-0-(2,3.4-tri-Obenzvl-beta-D-qlucopvranosvl) -uronate-podophvllotoxin (compound 9) Benzyl 1-fluoroglucuronate (compound 5, 4.23 g, 7.60 mmol) and 2'', 3''-di-O-chloroacetyl-etoposide (compound 9, 5.63 g, 7.60 mmol) were dissolved in dichloromethane (220 ml), and 4 A molecular sieves (10 g) were added. BF3-ether (2.5 ml) was added at -40’C to the reaction mixture, which was then stirred at -30 *C for 20 h. Triethylamine (7.0 ml) was added and then the mixture was filtered. The filtrate was washed with citrate buffer (pH 5, 80 ml x 3) and water (120 ml x 3), dried (sodium sulfate) and evaporated in vacuo. The residue was purified by column chromatography on silica gel (360 g) with 5:5:1 dichloromethane/petroleum ether/ acetone.

Yield: 6.49 g (67%). 4'-O-Demethvl-4-0-fdi-O-chloroacetvl-4,6-O-ethvlidenebeta-D-glucopyranosvl1-4-epi-4·-0-(2,3.4,6-tetra-0benzvl-alpha- and beta-D-qalactopvranosvl)-podophvllotoxin (compound 10a and 10b) Tetra-O-benzyl-galactopyranosyl fluoride (compound 8, 3.60 g, 6.65 mmol) and etoposide derivative (compound 9, 4.93 g, 6.65 mmol) were dissolved in dichloromethane (250 ml). 4 A molecular sieves (7.2 g) were added and then the reaction mixture was cooled to -40*C, and 30% strength BF3-ether (2.2 ml) was added dropwise. The mixture was stirred at -30*C for 20 h and then, after addition of triethylamine (5.5 ml), filtered. The filtrate was washed with citrate buffer (75 ml x 3, pH 5) and water (70 ml x 2), dried over sodium sulfate and evaporated. The residue was prepurified by column chromatography on silica gel (370 g) with 5s5:1 petroleum ether/dichloromethane/acetone. Further separation by column chromatography provided the title compounds 10a (alpha-glycoside: 4.36 g (52%)) and 10b (beta-glycoside: 1.42 g (17%)) .

Example 3 Deblocking reaction for glycosyl etoposides Benzyl 4'-O-demethvl-4-epi-4-O-(4.6-O-ethvlidene-beta-Dqlucopyranosvl)-4'-0-(2,3,4-tri-O-benzvl-beta-D-qluco25 pyranosvl)-uronate podophyllotoxin (compound 11) Glucuronide derivative (compound 9, 4.56 g, 3.57 mmol) was dissolved in 4:1 methanol/chloroform (120 ml), and DOWEX 1x8 (6.2 g) was added. The reaction mixture was stirred at room temperature for 3 h and then filtered.

Chloroform (150 ml) was added to the filtrate, which was then stirred with magnesium sulfate (10 g). The salts were filtered off and then the filtrate was evaporated in vacuo. The residue was purified by column chromatography on 200 g of silica gel with 5:2:1 dichloromethane/ petroleum ether/acetone.

Yield: 3.29 g (82%).

' -O-Demethvl-4-epi-4-O- (4,6-O-ethvlidene-beta-D-qlucopyranosvl)-4 *-0-(beta-D-qlucopyranosvl)-uronic_acid podophyllotoxin (compound 12) Deacylated glucuronide derivative (compound 11, 3.62 g, 5 3.22 mmol) was dissolved in 4:1 methanol/ethyl acetate (180 ml) and hydrogenated in the presence of 10% Pd/C (2.76 g) for 20 h. The catalyst was filtered off and then the filtrate was evaporated in vacuo. The residue was purified by column chromatography on RP-18 silica gel using methanol/hexane (gradient).

Yield: 1.82 g (74%).

· -O-Demethvl-4-epi-4-O- ( 4.6-0-ethylidene-beta-D-qlucopyranosvl)-4·-0-(2.3,4.6-tetra-O-benzyl-alpha-D-qalactopyranosvl)-podophyllotoxin (compound 13) Podophyllotoxin galactopyranoside (compound 10a, 3.0 g, 2.37 mmol) was dissolved in 3:1 methanol/chloroform, and Dowex 1x8 was added. The reaction mixture was stirred at room temperature for 3 h and filtered. The filtrate was evaporated in vacuo. The residue was dissolved in chloroform and washed with phosphate buffer (pH 7, 30 ml x 3) and then with water. The organic phase was dried over sodium sulfate and evaporated. The residue was purified by column chromatography on silica gel (85 g) with 5:3:1 dichloromethane/petroleum ether/acetone.

Yield: 2.27 g (86%). 4·-O-Demethyl-4-epi-4·-O-(alpha-D-qalactopvranosvl)-4-0(4,6-0-ethylidene-beta-D-qlucopvranosvl)-podophyllotoxin (compound 14) Deacylated podophyllotoxin galactopyranoside (compound 13, 2.0 g, 1.80 mmol) was dissolved in 3:1 methanol/ethyl acetate and hydrogenated in the presence of 10% Pd/C (2.5 g) for 24 h. The mixture was stirred with magnesium sulfate and filtered. The filtrate was evaporated in vacuo. The residue was purified by column chromatography on RP-18 silica gel using methanol/ethyl acetate (gradient).

Yield: 1.37 g (72%).

Result: Under the experimental conditions it was not possible to detect any enzyme activity for alpha-galactosidase and beta-glucuronidase.

Example 5 Determination of the enzyme activity of beta-glucuronidase conjugates The beta-glucuronidase purified by the abovementioned procedure was coupled to the antibody/the biomolecule, and the activity of the enzyme and of the conjugate was determined as follows: 500 μΐ of the enzyme solution to be determined were added to 500 μΐ of a 2.5 mM p-nitrophenyl beta-D-glucuronide solution in 100 mM HEPES (N-2-hydroxyethyl-piperazine-N'15 2-ethane-sulfonic acid), pH 5. The assay mixture was incubated at 37’ and stopped after 6 min with 300 μΐ of a 0.4 M glycine solution, pH 10.8. The liberated pnitrophenol was then determined by measuring the extinction at 405 nm.

Result: The conjugate showed an only inconsiderable reduction in enzyme activity..

Example 9 In vivo antitumor effects of the glycosyl-etoposide prodrug system NMRI nu/nu mice received on day 0 a subcutaneous inoculation of pieces about 5 mm3 in size of CoCa 4 human tumor per animal. After the human tumor tissue had grown in the mice (day 7-14), 5 animals in each of the groups a,b,c received 5 x 500 μς of MAb BW 494/32-glucuronidase conjugate, d received 5 x 500 μς of MAb BW 494/32, e received 5 x 500 μς of glucuronidase and f received 5 x 500 μΐ of PBS injected intravenously on 5 consecutive days.

On days 5, 6 and 7 after the end of the MAb BW 494/32glucuronidase, MAb BW 494/32, glucuronidase or PBS injection, the mice in groups a, d and e received one third of the maximum tolerable dose (MTD) of the glycosyl-etoposide injected intravenously per animal and per day. The mice in group b each received 1/10 of the MTD, and those in group c received 1/20 of the MTD on the same days.

Result: Groups d and e exhibited a tumor growth which did not differ significantly from that in group f. Groups a, b and c exhibited distinct inhibition of tumor growth, with the effects being most distinct in group a.

Comparable results were received with the MAbs BW 431/26, BW 250/183 in the CoCa4 xenograft system and with the MAb BW 704 in the M21 xenograft system.

Example 10 a) Cleavage of 4'-O-alpha-D-qalactopvranosvl-etoposide with alpha-qalactosidase (from green coffee beans) 4'-O-Alpha-D-galactopyranosyl-etoposide was dissolved in a concentration of 1 mg/ml in 20 mM sodium phosphate buffer, pH 5, and 0.3 U/ml of alphagalactosidase (green coffee beans; Sigma Co.; 1 U = cleavage of 1 pmol of p-nitrophenyl alpha-D-galacto25 side per minute at pH 6.5 and 25 degrees) was added, and the mixture was incubated at 37 degrees. The breakdown of 4'-O-alpha-D-galactopyranosyl-etoposide and the appearance of free etoposide were investigated by HPLC. The half-life was 15 minutes. b) Cleavage of 4'-O-alpha-D-qalactopyranosvl-etoposide with alpha-qalactosidase A (from human placental 4'-O-Alpha-D-galactopyranosyl-etoposide was dissolved in a concentration of 107 or 10.7 /ig/ml in 20 mM sodium phosphate buffer, pH 5, and 0.36 U/ml of alpha-galactosidase A (isolated from human placenta; 1 U = cleavage of 1 μΐηοΐ of 4-methumbelliferyl alpha-D-galactoside per minute at pH 5 and 37 degrees) was added, and the mixture was incubated at 37 degrees. The breakdown of 4'-O-alpha-D-galacto5 pyranosyl-etoposide and the appearance of free etoposide were investigated by HPLC. The half-life was 4 or 7 hours respectively.

Example 11 A. Glycosylation of etoposides 10 General procedure: Molecular sieves (1.4 g), silver carbonate (0.857 g) and silver perchlorate were added at -20eC to a solution of 2'',3''-di-O-chloroacetyl-etoposide (0.67 mmol) and glycosyl halide (bromide or chloride, 1.2 mmol) in dichloromethane (50 ml), and the reaction mixture was stirred with exclusion of light for 60 h. Dichloromethane (50 ml) was added to the mixture, which was then filtered. The filtrate was washed with water and dried over magnesium sulfate. The residue was chromatographed, resulting in the alpha- and 0-O-glycosidically linked compounds. ' ', 3''-Di-O-chloroacetyl-4'-0-(2,3,4,6-tetra-0-benzylalpha-D-galactopyranosyl)-etoposide The title compound was prepared starting from 2,3,4,625 tetra-O-benzyl-alpha-D-galactopyranosyl chloride (or bromide) and 2,3''-di-O-chloroacetyl-etoposide by the abovementioned procedure.

Alpha-glycoside [alpha]D +11.8* (c = 1, chloroform). , 3 -Di-O-chloroacetyl-4 '-0- (2,3,4,6-tetra-O-benzyl30 ^-D-galactopyranosyl)-etoposide The title compound was prepared starting from 2,3,4,6tetra-O-benzyl-alpha-D-galactopyranosyl bromide and 2'', 3'' -di-O-chloroacetyl-etoposide by the abovementioned procedure.

^-Glycoside [alpha]D -39.8’ (c = 1, chloroform). 2'', 3 -Di-O-chloroacetyl-4'-0-(2,3,4,6-tetra-0-benzylalpha-D-glucopyranosyl)-etoposide The title compound was prepared starting from 2,3,4,6tetra-O-benzyl-alpha-D-glucopyranosyl chloride (or bromide) and 2'',3''-di-O-chloroacetyl-etoposide by the abovementioned procedure.

Alpha-glycoside [alpha]D +16.2’ (c = 1, chloroform). ' ', 3 ' ' -Di-O-chloroacetyl-4'-0-(2,3,4,6-tetra-0-benzyl0-D-glucopyranosyl)-etoposide The title compound was prepared starting from 2,3,4,6tetra-O-benzyl-alpha-D-glucopyranosyl bromide and 2'', 3' ' -di-O-chloroacetyl-etoposide by the abovementioned procedure. 0-Glycoside [alpha]D -44.5° (c = 1, chloroform). 2 ' ', 3 ' ' -Di-O-chloroacetyl-4'-0-(2,3,4,6-tetra-0-benzyl0-D-glucuronyl)-etoposide The title compound was prepared starting from benzyl 2,3,4-tri-0-benzyl-l-chloro (or -bromo)-1-deoxy-alpha-Dglucupyranuronate and 2'', 3'' -di-O-chloroacetyl-etoposide by the abovementioned procedure.

^-Glycoside [alpha]D -52.4 (c = 1, chloroform).

B. Deblocking of chloroacetyl protective group in glycosyl-etoposides General procedure: A mixture of a 2 '',3''-di-O-chloroacetylated glycosyletoposide (0.75 mmol) and Dowex 1x8 ion exchanger (3.0 g) in 3:2 methanol/dichloromethane (200 ml) was stirred at room temperature for 1 h. The resin was filtered off and then the filtrate was washed with phosphate buffer (pH 6), dried (sodium sulfate) and evaporated in vacuo.

The residue was purified by column chromatography. The following compounds were prepared: ' -0- (2,3,4,6-Tetra-O-benzyl-alpha-D-galactopyranosyl) IE 903764 etoposide alpha-Glycoside [alpha]D +6.0’ (c = 1, chloroform) 4'-0-(2,3,4,6-Tetra-O-benzyl-^-D-galactopyranosyl) etoposide 4 ' -0- (2,3,4,6-Tetra-O-benzyl-alpha-D-glucopyranosyl) etoposide alpha-Glycoside [alpha]D +14.9* (c = 1, chloroform) ' -0- ( 2,3,4,6-Tetra-0-benzyl-)9-D-glucopyranosyl) etoposide ^-Glycoside [alpha]D -53’ (c = 1, chloroform) 4'-0-(2,3,4,6-Tetra-O-benzyl-^-D-glucuronyl)-etoposide.

Elimination of benzyl protective groups in glycosyletoposides by hydrogenolysis General procedure: A mixture of a benzylated glycosyl-etoposide (0.64 mmol) in glacial acetic acid (10 ml) was hydrogenated in the presence of 10% Pd/C (1.0 g) for 24 h. The catalyst was filtered off and then the filtrate was evaporated in vacuo at about 0*C. The residue was purified by column chromatography on silica gel, resulting in the following glycosyl-etoposides: 4'-0-(alpha-D-Galactopyranosyl)-etoposide alpha-Glycoside [alpha]D +6.2* (c = 1, chloroform) 4'-0-(0-D-Galactopyranosyl)-etoposide ^-Glycoside [alpha]D -73.1’ (c = 1, chloroform) 4'-0-(alpha-D-Glucopyranosyl)-etoposide 4'-0-(0-D-Glucopyranosyl)-etoposide ^-Glycoside [alpha]D -76.6’ (c - 0.9, chloroform) 4'-0-(0-D-Glucuronyl)-etoposide.

HOE 89/B 039

Claims (10)

1. Patent claims 1. A process for the preparation of a compound of the formula I in which R 1 is a methyl, benzyl or 2-thienyl group, R 2 is a hydrogen atom, R 3 is a hydroxyl, amino or dimethylamino group, R 4 is a hydrogen atom or a methyl group, R 5 is a hydrogen atom, a hydroxyl group, an amino or acetylamino group, R 6 is a hydroxyl group or an amino group, R 7 is a hydrogen atom, R® is a methyl or hydroxymethyl group or a carboxyl group or an acyl protective group which is bonded via a methyleneoxy group, or a benzyloxycarbonyl group, where the acyl protective group is an acetyl, mono-, di- or trihalogenoacetyl group with halogen being fluorine or chlorine, which comprises reacting, in the presence of a promoter and, where appropriate, of an acid trap or drying agent in a solvent at -50 C to 60*C, an etoposide compound of the formula V in which R 1 is a methyl, benzyl or 2-thienyl group, R 2 is a hydrogen atom, an acyl or a tri-Ci-C^-alkylsilyl protective group, R 3 is a hydroxyl group, an acyl or tri-Cj-C*alkylsilyl protective group which is bonded via oxygen, or acetylamino, benzyloxycarbonylamino or dimethylamino group, and R 4 is a hydrogen atom or a methyl group, with a carbohydrate component of the formula VI in which R 5 is a hydrogen atom, a hydroxyl group, an acyl protective group which is bonded via an oxygen atom, or benzyloxycarbonylamino, azido or acetylamino group, R 6 is an acyl protective group which is bonded via an oxygen atom, or a benzyloxycarbonylamino or azido group, R 7 is an acyl protective group, R 8 is a methyl group, methyleneoxy-acyl protective group or a benzyloxycarbonyl group and Z is a halogen atom, preferably fluorine, chlorine or bromine, a hydroxyl group, a tri-Ci-C^-alkylsilyloxy group, or an acyl protective group which is bonded via an oxygen atom, where the acyl protective group is an acetyl, mono-, di- or trihalogeno-acetyl group, preferably with the halogen atom being fluorine or chlorine, to give a 4'-O-glycosyl-etoposide derivative of the formula I in which all the radicals R 1 to R 8 retain their meaning as defined above, and eliminating the protective groups present in these compounds by hydrogenolysis or hydrolysis, and, where appropriate, converting by means of reductive alkylation one of the resulting compounds containing amino groups into another compound of the formula I containing dimethylamino groups.

2. The process as claimed in claim 1, wherein the acyl protective group is an acetyl, chloroacetyl or trifluoroacetyl group.

3. A 4'-O-glycosyl-etoposide of the formula I, in which R 1 is a methyl, benzyl or 2-thienyl group, R 2 is a hydrogen atom, an acyl or tri-Cx-C^-alkylsilyl protective group, R 3 is a hydroxyl group, an acyl or tri-Cx-C^-alkylsilyl protective group which is bonded via an oxygen a ton, an amino, acetylamino, benzvloxvcaxbonvlamino or dimethylamino group, R* is a hydrogen atom or a methyl group, R 5 is a hydrogen atom, a hydroxyl group, an acyl or tri-Ci-C^-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino, azido or acetylamino group, R 6 is a hydroxyl group, an acyl or tri-Cj-C^-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino or azido group, R 7 is a hydrogen atom, an acyl or tri-Cj-C^-alkylsilyl protective group and R 8 is a methyl or hydroxymethyl group or an acyl protective group which is bonded via a methyleneoxy group, or a benzyloxycarbonyl group, where the acyl protective group is an acetyl, mono-, di- or trihalogenoacetyl group with halogen being fluorine or chlorine.

4. A compound as claimed in claim 3, in which R 1 is a methyl, benzyl or 2-thienyl group, R 2 is a hydrogen atom, an acetyl or chloroacetyl group or a tri-Ci-C^-alkylsilyl protective group, R 3 is a hydroxyl group, an acetyl, chloroacetyl or tri-Cx-C^-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, acetylamino, benzyloxycarbonylamino or dimethylamino group, R* is a hydrogen atom or a methyl group, R 5 * is a hydrogen atom, a hydroxyl group, or an acetyl, chloroacetyl or tri-Ci-C^-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino, azido or acetylamino group, R e is a hydroxyl group, an acetyl, chloroacetyl or tri-Ci-C^-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino or azido group, R 7 is a hydrogen atom, an acetyl, chloroacetyl or tri-Ci-C^-alkylsilyl protective group and R* is a methyl, hydroxymethyl, acetyloxy or chloroacetyl oxymethyl group or a benzyloxycarbonyl group.

5. A pharmaceutical containing a compound as claimed in claim 3 and a functionalized tumor-specific enzyme of the formula II A-Sp-E II -29in which A is an antibody or one of the fragments thereof, which have specificity for a tumor-associated antigen, or is a biomolecule which accumulates In a 15 tumor, such as EGP (epidermal growth factor), TGFalpha (transforming growth factor alpha), PDGF (platelet derived growth factor), IGF I+II (insulin like growth factor Σ+ΙΙ) or a+b FGF (acidic + basic fibroblast growth factor) 20 E is a glycosidase which is not immunogenic or is of low imraunogenicity, preferably mammalian glycosidase, as alpha- or beta-glucosidase, alphagalactosidase, alpha- or beta-mannosidase, alphafucosidase, N-acetyl-alpha-galactosarainidase, N25 acetyl-beta-/N-acetyl-alpha-glucosaminidaee or betaglucuronidase, Sp (spacer) is a bifunctional sulfide- or disulfidecontaining group of the formula III or IV X(S)„Y III X(S), IV 30 or a polypeptide spacer, in which X or Y is -CO-R*-(N-succinimido)- or -C(«R :o )-CH a -CH a with R* being -CH a -CH,-, 1,4-cyclohexylidene, 1,3- or 1,4-phenylene or methoxycarbonyl- or chloro-1,4phenylene and R ie being 0 or NH, and furthermore Y is —C( e R ie ) -CHj-CHj-, where R 10 has the stated meaning, and n is 1 or 2. -306. An agent as claimed in claim 5, wherein A-Sp-E is prepared by genetic engineering, where A and E have the meaning as in claim 5, and Sp is an oligo— or polypeptide.

6. 7. A functionalized tumor-specific enzyme or genetically engineered product of the formula II in claim 5 or 6, containing a glycosidase. θ· A process according to claim 1 for the preparation of a compound of the formula I given and defined therein, substantially as hereinbefore described and exemplified.

7. 9. A compound of the formula I given and defined in claim 1, whenever prepared by a process claimed in a preceding claim.

8. 10. A 4'-O-glycosyl-etoposide of the formula I defined in claim 3, substantially as hereinbefore described and exemplified.

9. 11. A pharmaceutical according to claim 5, substantially as hereinbefore described.

10. 12. A functionalized tumor-specific enzyme or genetically engineered product according to claim 7, substantially as hereinbefore described. Dated this the 19th day of October, 19' F. R. KELLY & CO.