IE910334A1 - Glycosyl prodrugs of anthracyclines, a process for the¹preparation thereof and the use thereof in combination with¹functionalized tumor-specific enzyme conjugates - Google Patents

Glycosyl prodrugs of anthracyclines, a process for the¹preparation thereof and the use thereof in combination with¹functionalized tumor-specific enzyme conjugates

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IE910334A1
IE910334A1 IE033491A IE33491A IE910334A1 IE 910334 A1 IE910334 A1 IE 910334A1 IE 033491 A IE033491 A IE 033491A IE 33491 A IE33491 A IE 33491A IE 910334 A1 IE910334 A1 IE 910334A1
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Abstract

The present invention relates to glycosyl-anthracycline prodrugs, to processes for their preparation and to their use in combination with functionalised tumour-specific enzyme conjugates for the treatment of cancers, and it specifically relates to 14-O-glycosyl-anthracyclines as prodrugs which can be cleaved by the action of tumour-specific enzyme conjugates to cytotoxic agents, the liberated agent being suitable, by reason of its cytostatic activity, for the treatment of cancers. The invention relates to 14-O-glycosyl-anthracyclines of the formula I and to salts thereof with an inorganic or organic acid in which R<1>, R<2> and R<3> are, independently of one another, hydrogen, hydroxyl, methoxy, R<4>, R<5> and R<6> are, independently of one another, hydrogen, hydroxyl, halogen, aliphatic acyloxy (C1-C8), benzoyloxy or substituted benzoyloxy such as para-nitrobenzoyloxy, alkyloxy (C1-C8), allyloxy, benzyloxy or substituted benzyloxy, tetrahydropyranyloxy, amino, NH-acyl (C1-C8), NH-(9-fluorenylmethoxycarbonyl), morpholino or substituted morpholino, preferably 3-O-methylmorpholino or 2-cyanomorpholino, R<7> is a carbohydrate of the general formula II in which R<8> is methyl, hydroxymethyl, acyloxymethyl (C1-C8), alkyloxymethyl (C1-C8), benzyloxymethyl, allyloxymethyl, carboxyl, carboxymethyl or carboxyallyl, R<9>, R<10> and R<11> are, independently of one another, hydrogen, hydroxyl, acyloxy (C1-C8), benzoyloxy or substituted benzoyloxy such as para-nitrobenzoyloxy, alkyloxy (C1-C8), allyloxy, benzyloxy or substituted benzyloxy, amino, NH-acyl (C1-C8) or NH-(9-fluorenylmethoxycarbonyl). Excepted are compounds in which R<4> = R<5> = R<9> = R<10> = O-acetyl and R<6> = R<11> = H in the alpha-L-deoxyfucose conformation. A functionalised tumour-specific enzyme means within the scope of the invention an enzyme of the formula III A-Sp-E III in which A is an antibody or one of its fragments, which have specificity for a tumour-associated antigen, or a biomolecule which accumulates in a tumour, 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) or a+b FGF (acidic + basic fibroblast growth factor), E is a glycosidase which has little or no immunogenicity, preferably a mammalian glycosidase such as alpha - or beta -glucosidase, alpha -galactosidase, alpha - or beta -mannosidase, alpha -fucosidase, N-acetyl- alpha -galactosaminidase, N-acetyl- beta -N-acetyl- alpha -glucosaminidase or beta -glucuronidase, and Sp (spacer) is a bifunctional sulphide or disulphide containing group of the formula IV X(S)nY IV or (S)n in which X or Y is -CO-R<12>-(N-succinimido)- or -C(=R<13>)-CH2-CH2- where R<12> is -CH2-CH2-, 1,4-cyclohexylidene, 1,3- or 1,4-phenylene or methoxycarbonyl- or 1,4-chlorophenylene and R<14> is O or NH, Y is -C(=R<13>)-CH2-CH2- where R<13> has the stated meaning, and n is 1 or 2, or a polypeptide spacer.

Description

in which R8 is methyl, hydroxymethyl, acyloxymethyl (C1-C8), alkyloxymethyl (clC8), benzyloxymethyl, allyloxymethyl, carboxyl, carboxymethyl or carboxvallyl,R9, RIO and Rll are, independently of one another, hydrogen, hydroxyl, acvloxv (C iC8), benzoyloxy or substituted benzoyloxy such as para-nitrobenzoyloxy, alkyloxy (Cl-CS), allyloxy, benzyloxy or substituted benzyloxy, amino, ΝΉ-acyl (C1-C6) or NH-(9-fluorenylmethoxycarbonyl). Excepted are compounds in which R4 = R5 = R9 = RIO = O-acetyl and R6 = Rll = H in the alpha-L-deoxyfucose conformation, A functionalised tumour-specific enzyme means within the scope of the invention an enzyme of the formula III A-Sp-E III in which A is an antibody or one of its fragments, which have specificity for a tumour-associated antigen, or a biomolecule which accumulates in a tumour, such as EGF (epidermal growth factor), TGF-0 (transforming growth factor 0), 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 has little or no immunogenicity, preferably a mammalian glycosidase such as 0- or B-glucosidase, O-galactosidase, 0- or Bmannosidase, 0-fucosidase, N-acetyl-O-galactosaminidase, N-acetyl-B-N-acetyl-Oglucosaminidase or B-glucuronidase, and Sp(spacer) is a bifunctional sulphide or disulphide containing group of the formula IV X(S)nY IV or (S) n in which X or Y is -CO-R12-(N-succinimido)- or -C(=R13)-CH2-Ch2- where R12 is -CH2-Ch2-, 1,4cyclohexylidene, 1,3-or 1,4-phenylene or methoxycarbonyl- or 1,4-chlorophenylene and R14 is 0 or NH, Y is -C(=R13) -CH2-CH2- where R13 has the stated meaning, and n is 1 or 2, or a polypeptide spacer. 910334 IE i ABSTRACT Oifig Na bPaitinni The Patents Office s E IE 91334 PATENTS ACT, 1964 COMPLETE SPECIFICATION GLYCOSYL PRODRUGS OF ANTHRAACYCLINES, A PROCESS FOR THE PREPARATION THEREOF AND THE USE THEREOF IN COMBINATION WITH FUNCTIONALIZED TUMOR-SPECIFIC ENZYME CONJUGATES BEHRINGWERKE AKTIENGESELLSCHAFT, a Joint Stock Company organized and existing under the laws of the Federal Republic of germany, of D-3550 Marburg, Federal Republic of Germany.
BEHRINGWERKE AKTIENGESELLSCHAFT IE 91334 HOE 90/B 001 - Ma 816 Dr. Ha/Sd Description Glycosyl prodrugs of anthracyclines, a process for the 5 preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates The invention relates to glycosyl prodrugs of anthracyclines, a process for the preparation thereof and the use thereof in combination with functionalized tumor10 specific enzyme conjugates for treating cancers; it specifically relates to 14-0-glycosylanthracyclines as prodrugs which can be cleaved by the action of tumorspecific enzyme conjugates to cytotoxic active substances, where the liberated active substance is suitable because of its cytotoxic activity for the treatment of cancers.
A model in which combinations of prodrugs and tumorspecific antibody-enzyme conjugates can be tested as therapeutic agents is described in the specialist litera20 ture. This entails antibodies which are directed against particular tissue and to which a prodrug-cleaving enzyme is covalently bonded being injected into an animal to which this tissue has been transplanted. After the MAbenzyme conjugate has localized on the target tissue, a prodrug ccnpound which can be activated by the enzyme is administered. The action of the antibody-enzyme conjugate which is anchored to the tissue converts the prodrug into the cytotoxin which exerts a cytotoxic effect on the transplanted tissue.
WO 88/07378 describes 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 the enzyme. In this case, non-mammalian enzymes are used to prepare the antibody-enzyme conjugates, and the use of endogenous enzymes is ruled out because of possible nonIE 91334 specific release of active substance. Because the exogenous enzymes are recognized by the organism as foreign antigens, the use thereof is associated with the disadvantage of an immune response to these non-endogenous substances, which leads to inactivation of the enzyme immobilized on the antibody and, where appropriate, to elimination of the entire conjugate. In addition, in this case p-bis-N-(2-chloroethyl)aminobenzylglutamic acid and derivatives thereof are used as prodrug whose chemical half-life is relatively short and is preferably in the range from 5.0 to 17.0 hours. Chemical instability of a prodrug is a disadvantage because of the expected side effects.
EPA 0302473 A2 likewise describes a therapeutic system which contains two components and in which the antibodyenzyme conjugate localized on the tumor tissue cleaves a prodrug compound to a cytotoxic active substance.
The combined use, which is described herein inter alia, of an N-phenoxyacetyl derivative of adriamycin as prodrug and antibody-immobilized L6 penicillin V amidase for liberating adriamycin is disadvantageous because of the immunogenicity of the bacterial enzyme. Another disadvantage of such prodrugs is the increase, owing to the acylation of the amino group of adriamycin, in the lipophilicity of the prodrugs, which reduces the solubility in plasma, and non-specific adsorption of the prodrugs to cell membranes can take place.
The synthesis of 14-O-glycosylanthracyclines has to date been described only as undesired side reaction in the glycosidation of adriamycinone with 2-deoxy-L-fucose (Horton et al., Carbohydrate Research 94, 11-25, 1981).
It has emerged, surprisingly, that glycosidation of anthraoyclines in the 14-position is possible by employing particular patterns of protective groups on the anthracycline as well as on the glycosyl donors.
IE 91334 - 3 Based on this finding, and taking account of the disadvantages, described above, of previous combinations of prodrugs and antibody-enzyme conjugates, the object of the present invention was to prepare synthetic, enzymatically cleavable 14-0-glycosylanthracyclines as well as enzymes functionalized in a tumor-specific manner, that is to say enzymes which have been altered in such a way that they bind to tumor cells, which are suitable for treating cancers.
This object has been achieved by preparing compounds of the formula I as well as enzymes functionalized in a tumor-specific manner, which when used in combination showed an effect in the test for cytostatic activity.
The invention relates to 14-0-glycosylanthracyclines of the formula I and the salts thereof with an inorganic or organic acid R in which R1, R2 and R3 are, independently of one another, hydrogen, hydroxyl, methoxy, R4, R5 and R6 are, independently of one another, hydrogen, hydroxyl, halogen, aliphatic acyloxy (C1-C8), benzoyloxy or substituted benzoyloxy such as paranitrobenzoyloxy, alkyloxy (C1-C8), allyloxy, benzyloxy or substituted benzyloxy, tetrahydropyranyloxy, amino, NH-acyl (C1-C8), NH-(9-fluorenylmethoxycarbonyl), morpholino or substituted morpholino, IE 91334 _- 4 preferably 3-O-methylmorpholino or 2-cyanomorpholino, R7 is a carbohydrate of the formula II with R6 being methyl, hydroxymethyl, acyloxymethyl (C1-C8), alkyloxymethyl (C1-C8), benzyloxymethyl, allyloxymethyl, carboxyl, carboxymethyl or carboxyallyl, R9, R10 and R11 are, independently of one another, hydrogen, hydroxyl, acyloxy (C1-C8), benzoyloxy or substituted benzoyloxy such as para-nitrobenzoyloxy, alkyloxy (C1-C8), allyloxy, benzyloxy or substituted benzyloxy, amino, NH-acyl (C1-C8) or NH-(9fluorenylmethoxycarbonyl).
Compounds in which R4=R5=R9=R10=O-acetyl and R6=Rn=H in the alpha-L-deoxyfucose conformation are excepted.
An acyl group also means a mono-, di- or trihalogenoacetyl group, preferably with the halogen atoms fluorine or chlorine.
Substituted benzyloxy preferably means benzyloxy substituted by one or two methoxy groups.
A functionalized tumor-specific enzyme is intended to mean within the scope of the invention an enzyme of the formula III ; A-Sp-E III IE 91334 in which A is an antibody or one of its fragments which have specificity for a tumor-associated antigen, or a biomolecule which accumulates in a tumor, such as EGF (epidermal growth factor), TGF-o (transforming growth factor a), 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 of little or no immunogenic ity, 10 preferably a mammalian glycosidase such as a- or βglucosidase, ο-galactosidase, a- or £-mannosidase, α-fucosidase, N-acetyl-a-galactosaminidase, Nacetyl-0-/N-acetyl-a-glucosaminidase or ^-glucuronidase , and Sp (spacer) is a bifunctional sulfide or disulfidecontaining group of the formula IV X(S)nY IV or (S)n in which X or Y is -CO-R12-(N-succinimido)- or -C (=R13)-CH2-CH2- with 20 R12 being -CH2-CH2-, 1,4-cyclohexylidene, 1,3- or 1,4phenylene or methoxycarbonyl- or chloro-l,4-phenylene and R14 is 0 or NH, Y is —C (=R13) -CH2-CH2- where R13 has the stated meaning, and n is 1 or 2, or a polypeptide spacer.
For example, an enzyme functionalized in a tumor-specific manner, which represents a fusion gene composed of VH, CHI, hinge and enzyme gene, can be cloned into an expres30 sion 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 IE 91334 cells. After selection with a suitable antibiotic, trans fee toma clones which contain the expression plasmids are identified.
Suitable detection methods (BioDot, ELISA) are used to identify those transfectoma clones which secrete the fusion protein of the formula III composed of antibody and enzyme.
Compounds of the formula I preferred within the scope of the invention are those in which the radicals R1 is hydroxyl or methoxy, R2 and R3 are hydroxyl, R4 is hydrogen, hydroxyl, tetrahydropyranyloxy, amino or acyloxy (C1-C8), R5 is hydroxyl, acyloxy (C1-C8), amino, NH-acyl (C1-C8) or NH-(9-fluorenylmethoxycarbonyl), R6 is hydrogen, hydroxyl, halogen or acyloxy, R7 is a carbohydrate of the formula II with R8 being methyl, hydroxymethyl, acyloxymethyl (C1-C8), alkyloxymethyl, carboxyl, carboxymethyl, carboxy20 benzyl or carboxyallyl, and R9, R10 and R11 are, independently of one another, hydrogen, hydroxyl, acyloxy (C1-C8) or alkyloxy, excepting the compounds in which R4=R5=R9=R10=O-acetyl and R6=R11=H in the β-L-deoxyfucose conformation.
Also preferred is a functionalized tumor-specific enzyme of the formula III in which A is an antibody of fragment thereof which has specificity for a tumor-associated antigen, or a biomolecule which accumulates on or in the tumor, such as EGF (epidermal growth factor), TGF-o (transforming growth factor o), PDGF (platelet-derived growth factor), IGF I+II (insulin-like growth factor I+II) or a+b FGF (acidic + basic fibroblast growth factor) IE 91334 E is a glycosidase of little or no immunogenicity, preferably a mammalian glycosidase, especially an aor ^-glucosidase, α-galactosidase, o- or /9-mannosidase, α-fucosidase, N-acetyl-o-galaCtosaminidase, N-acetyl-0-/N-acetyl-o-glucosaminidase or βglucuronidase, Sp is a bifunctional disulfide-containing group of the formula IV X (S)n Y IV in which X or Y is -CO-R12-(N-succinimido)- or -C(=R13)-CH2-CH2 with R12 being -CH2-CH2- or 1,4-phenylene and R13 being 0 or NH, Y is —C(=R13)-CH2-CH2- where R13 has the stated meaning, and n is 1 or 2, or Sp is a suitable polypeptide spacer.
The invention also relates to a process for preparing a 20 compound of the formula I with the stated definitions, which comprises reacting an anthracycline of the formula V in which R1, R2, R3, R*, R5 and R6 have the meanings stated 25 above, IE 91334 — 8 — with a carbohydrate component of the formula VI 8 VI in which is methyl, acyloxymethyl (C1-C8), alkyloxymethyl (C1-C8), benzyloxymethyl, allyloxymethyl, carboxymethyl, carboxybenzyl or carboxyalkyl, R10 and Ru are, independently of one another, hydrogen, acyloxy (C1-C8), benzoyloxy or substituted benzoyloxy such as para-nitrobenzoyloxy, alkyloxy (C1-C8), allyloxy, benzyloxy, substituted benzyloxy, NH-acyl or NH-(9-fluorenylmethoxycarbonyl) and is a leaving group such as a halogen atom, a hydroxyl group, a tri-Cl-C4-alkylsilyloxy group, an acyl protective group bonded via an oxygen atom, or a trichloroacetimidate bonded via an oxygen atom. in the presence of a promoter and, where appropriate, of an acid trap and/or desiccant in a solvent at -50’ to +80’C to give a 14-O-glycosylanthracycline derivative of the formula I in which all the radicals R1 to R11 retain their meaning as defined above, and eliminating, by hydrolysis or with the aid of a suitable catalyst, the protective groups or some of the protective groups which are present in these compounds where appropriate.
The specific procedure for this is as follows: An anthracycline of the formula V must if it carries an IE 91334 amino group as one of the radicals R*, R5 or R6 initially be protected on this group, for example as NH-trifluoroacetate or NH-(9-fluorenylmethoxycarbonyl).
Employed for the glycosidation at the 14-position of 5 anthracycline are functionalized carbohydrate components of the formula VI which typically contain as radicals R9, R10 and R11 O-acyl, O-alkyl or O-allyl when they carry radicals bonded via oxygen. The radical R8 is likewise protected as acyloxymethyl, alkyloxymethyl or carboxy10 alkyl.
The carbohydrate derivatives must have suitable leaving groups Z at the anomeric center. Glycosyl halides such as fluorides, chlorides or bromides are prepared, for example, starting from 1-O-acyl derivatives using HF, HCI, HBr or TiBr*. Other compounds with leaving groups such as trichloroacetimidates as well as with various patterns of protective groups for R8 to R11 are prepared by the methods customary in carbohydrate chemistry.
A promoter is necessary for the glycosidation of the anthracycline which is capable of coupling with a glycosyl donor. When glycosyl fluorides, 1-hydroxyglycosides, 1-acyloxyglycosides or 1-trichloroacetimidates of carbohydrates are employed, Lewis acids such as BF3.ether or trimethylsilyl triflate are used, while silver or mercury salts are used in the case of glycosyl chlorides and bromides.
The glycosidation takes place in an aprotic organic solvent or solvent mixture such as acetone, ethyl acetate, ether, toluene, dichloromethane, dichloroethane, acetonitrile, nitromethane. The reaction is carried out at temperatures between -50’ and +80’C, depending on the reactivity of the glycosyl donor, where appropriate with the addition of an acid trap or desiccant, for example of a molecular sieve. The subsequent elimination of the protective groups is carried out by methods customary in IE 91334 carbohydrate chemistry.
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 can be linked via an HS group which has been introduced or generated by cleavage of a disulfide bridge, or nucleic acid sequences which code for parts A, Sp and E are covalently linked with the aid of molecular biology methods in such a way that the result is a fusion gene, and A-Sp-E is prepared by genetic engineering methods.
This can take place in a variety of ways: A) A restriction cleavage site A is introduced at the 3' end of the CHI exon in the gene of the heavy chain of the immunoglobulin by specific mutagenesis. The same restriction cleavage site A is generated at the 5' end of the oligonucleotide which codes for the oligopeptide which functions as spacer. Both restriction cleavage sites A are sited such 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 into the gene which codes for the enzyme at the point where the coding nucleic acid sequence for the mature protein starts. The enzyme gene is then linked via the restriction cleavage site B to the immunoglobulin gene-linker construct. The restriction cleavage sites B are sited so that the reading frame is not disturbed on linkage. The fusion gene composed of the for the heavy chains of the immunoglobulin VH and CHl-linker enzyme is cloned in an expression plasmid which is suitable for expression in eukaryotic cells and carries a selection marker.
The expression plasmid with the fusion gene is IE 91334 transfected together with another 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 (transfeetomas).
Suitable detection methods (BioDot; ELISA) are used to identify those transfectomas which secrete the fusion protein of the formula A-Sp-E composed of 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 point in the enzyme gene at which the coding nucleotide sequence for the mature protein starts. The gene fragment of the heavy chains of the immunoglobulin with the VH, CHI and hinge exons is linked via the restriction cleavage site A to the 0 enzyme gene.
The restriction sites A are sited so that the reading frame is not disturbed on linkage. The hinge part of the antibody acts as polypeptide spacer in this construction. The fusion gene composed of VH, CH125 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 another expression plasmid which contains the light chain gene belonging to the antibody into eukaryotic expression cells. After selection with a suitable antibiotic, transfectoma clones which contain the expression plasmids are identified. Suitable detection methods (BioDot, ELISA) are used to identify those transfectoma clones which secrete the fusion protein of the formula III composed of antibody and 91334 enzyme.
The coupling between enzyme and antibody, its fragments or a biomolecule is carried out by processes described in the literature (A.H. Blair and T.I. Ghose, J. Immunolog.
Methods 59 (1983) 129-143; T.I. Ghose et al. Methods in Enzymology Vol. 93 (1983) 280-333). This entails the enzyme initially being functionalized via its amino group using succinimidyl-N-maleimido-alkylidene-, 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, its fragment or the biomolecules with the formation of a thioether functionality. It is possible to use monoclonal antibodies, for example those described in EP-A-0 141 079, preferably the antibodies 431/26, 250/183, 704/152 and 494/32, for preparing the antibody-enzyme conjugates. The specificity of these antibodies for tumor-associated antigens has already been demonstrated on animals and humans by immunoscintigraphy and immunohistochemistry.
The nucleotide sequence of V genes of these monoclonal antibodies is described in German Patent Application DEA-39 09 799.4.
To prepare the tumor-specific enzyme conjugates, the enzymes specified hereinafter can be purified from the designated source by the method in the specified literature: - α-galactosidase A from human placenta: Bishop, D.F. (1981), J. Biol. Chem. 256, 1307-1316, or from transfected mammalian cells: Tsuji, S. (1987), Eur. J.
Biochem. 165, 275-280. ^-glucuronidase from human placenta: Brot, F.E. (1978) Biochemistry 17, 385-391 human ^-glucuronidase from transfected mammalian cells: Oshima, A. (1987) Proc. Natl. Acad. Sci. USA 84, 685-689 α-L-fucosidase from human liver: Dawson, G., Tsay, G.
IE 91334 (1977) Arch. Biochem. Biophys. 184, 12-23 - α-mannosidase from human liver: Grabowski, G.A., Ikonne, J.U., Desnick, R.J. (1980) Enzyme 25, 13-25 - l-mannosidase from human placenta: Noeske, C., Mersmann, G. (1983) Hoppe Seylers Z Physiol. Chem. 364, 1645-1651 - a-glucosidase from human gastric, intestinal mucosa: Asp, N.-G., Gudmand-Hoeyer, E., Christiansen, P.M., Dahlguist, A. (1974) Scand. J. Clin. Lab Invest. 33, , 239-245 - ^-glucosidase from human liver: Daniels, L.B., Coyle, P.J., Chiao, Y.-B., Glew, R.H. (1981) J. Biol. Chem. 256, 13004-13013 - 0-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 - α-Ν-acetylglucosaminidase from human placenta: Roehrborn, W., Von Figura, K. (1978) Hoppe Seylers Z.
Physiol. Chem. 359, 1353-1362 - l-N-acetylglucosaminidase from human amniotic membrane: Orlacchio, A., Emiliani, C., Di Renzo, G.C., Cosmi, E.V. (1986) Clin. Chim. Acta 159, 279-289 - α-Ν-acetylgalactosaminidase: by the method of 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 also relates to a pack containing a 14-Oglycosylanthracycline according to the invention and a functionalized tumor-specific enzyme conjugate.
To test the activity of a combined sequential use, 35; transplanted mice were given the functionalized enzyme and, after waiting until the plasma level of the enzyme IE 91334 had fallen virtually to zero, the 14-0-glycosylanthracycline was given and it was observed whether cessation of growth and regression of the transplanted tissue occurred.
Examples: The following examples explain the invention in more detail without restricting it: A Syntheses of the prodruqs: The structures of the prepared compounds were determined by *H- and 13C-NMR spectroscopy, including two-dimensional NMR methods and other multipulse NMR techniques, as well as MS, UV and IR spectroscopy. The progress of the reactions and the resulting compounds were examined by thin-layer chromatography or HPLC techniques.
Example 1: 2-O-Allyl-l, 3,4,6-tetra-O-acetyl-a-D-galacto-hexopyranose (1) To a solution of 1,3,4,6-tetra-O-acetyl-e-D-galactopyranose (3.48 g = 10 mmol) and allyl trichloroacetimidate (14 ml of a 30% strength solution in hexane) in dichloromethane (15 ml) was added trifluoromethanesulfonic acid (0.25 ml), and the mixture was stirred at 40°C for 4 h. It was filtered after cooling, and the filtrate was extracted by shaking with agueous sodium bicarbonate solution.
The organic phase was subjected to column chromatography (mobile phase: ethyl acetate/petroleum ether - 1:4). Yield: 1.52 g (3.9 mmol = 39%), mp 133“C, [«3d25 +-68.6 (c = 1.0 in dichloromethane). 3© XH NMR (300 MHz, CDC13) 6 6.40 (d, 1H, J1>2 = 3.6 Hz, H-l), 5.83 (dddd, 1H, allyl), 5.47 (dd, 1H, J3A = 3.3 Hz, IE 91334 J45 = 1.2 Hz, H-4), 5.32-5.17 (m, 3H, allyl, H-3), 4.30 (dt, JA>5 = 1.2 Hz, J5>6a = J56b = 6.6 Hz, H-5), 4.16-4.06 (m, 4H, H-6a, H-6b, allyl), 3.88 (dd, 1H, J1(2 = 3.6 Hz, J2>3 = 10.5 Hz, H-2), 2.17 (s, 3H, OAc), 2.15 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.03 (s, 3H, OAc). 13C NMR (50 MHz, CDC13) 6 117.4 (allyl), 93.5 (allyl), 89.7 (C-l), 71.8 (allyl), 71.7 (C-2), 69.0 (C-3), 68.2 (C-5), 67.4 (C-4), 61.0 (C-6), 20.6 (OAc), 20.4 (OAc), .3 (OAc), 20.1 (OAc).
Example 2: 2-O-Allyl-3,4,6-tri-O-acetyl-a-D-galacto-hexo-pyranosyl bromide (2) Compound 1 (940 mg = 2.42 mmol) was dissolved in dichloromethane (2 ml) and, at 0eC, HBr in glacial acetic acid (11 ml; 33% HBr solution) was added and the mixture was stirred at this temperature for two hours. The reaction mixture was poured into ice and extracted by shaking with dichloromethane. The organic phase was extracted by shaking with aqueous sodium bicarbonate solution until the reaction was slightly basic, dried over sodium sulfate and evaporated with toluene several times.
Yield: 930 mg (2.27 mmol = 94%), syrup.
*H NMR (200 MHz, CDC13) S 6.58 (d, IH, J1>2 = 3.8 Hz, H-l), .90 (dddd, IH, allyl), 5.49 (dd, IH, J3>4 = 3.4 Hz, J4>5 = 1.3 Hz, H-4), 5.38-5.20 (m, 3H, H-3, allyl), 4.48 (dt, IH, JA>5 = 1.3 Hz, J56e = J5,6b β 6.3 Hz, H-5), 4.23-4.04 (m, 4H, allyl, H-6a, H-6b), 3.77 (dd, IH, J12 = 3.8 Hz, J23 = 10.3 Hz, H-2), 2.17 (s, 3H, OAc), 2.08 (s, 3H, OAc), 2.03 (s, 3H, OAc). 13C NMR (50 MHz, CDC13) S 170.2 (OAc), 169.7 (OAc), 169.6 (OAc), 133.7 (allyl), 117.9 (allyl), 90.6 (C-l), 72.8 (C-2), 71.7 (allyl), 70.9 (C-5), 69.9 (C-3), 67.2 (C-4), 60.8 (C-6), 20.6 (OAc), 20.5 (OAc), 20.4 (OAc).
IE 91334 Example 3: N-(9-Fluorenylmethoxycarbonyl)-doxorubicin (3) Sodium methanolate solution (30% strength in methanol) was added to a suspension of doxorubicin hydrochloride (107 mg - 0.184 mmol) in absolute methanol (10 ml) until the reaction was slightly alkaline, whereupon the substance dissolved. Addition of sodium carbonate (anhydrous) (98 mg = 0.92 mmol) and 9-fluorenylmethoxycarbonyl chloride (142 mg - 0.552 mmol) was followed by stirring at room temperature for one hour. Filtration and concentration were followed by column chromatography (mobile phase: dichloromethane/methanol/formic acid 20:1:0.1).
Yield: 139 mg (0.181 mmol = 98%), mp 165 - 166’C, [o]D25 + 156° (c = 0.02 in chloroform).
*H NMR (200 MHz, CDC13) S 13.86 (s, 1H, OH-6), 13.09 (s, 1H, OH-11), 7.98-6.70 (m, UH, H-l, H-2, H-3, 8 Ftooc aromatic H), 5.39 (bs, 1H, H-l'), 5.14 (m, 2H, NH, H-7), 4.71 (s, 2H, H-14), 4.48 (s, 2H, Fmoc-CH), 4.25 (s, 1H, Fmoc-CH), 4.07 (q, 1H, H-5'), 3.99 (s, 3H, OMe), 3.80 (m, 2H, H-3', H-4'), 3.55 (bs, 1H, OH), 3.15 (d, 1H, J1Oe.iob = 18.7 Hz, H-lOa), 2.99 (bs, 1H, OH), 2.82 (d, 1H, = 18.7 Hz, H-lOb), 2.48-2.03 (m, 4H, H-8a, H-8b, H-2a', H2e'), 1.23 (d, 3H, J5>>6. = 6.5 Hz, CH3-6').
FAB-MS, m/z = 766 (M+H).
Example 4: 14-0-(2-O-Allyl-3,4,6-tri-O-acetyl-a-D-galacto-hexopyranosyl)-N-trifluoroacetyl-doxorubicin (4) Silver carbonate (90 mg - 0.33 mmol) and silver per30 chlorate (23 mg = 0.11 mmol) were added to a solution of N-trif luoroacetyl-doxorubicin (70 mg = 0.11 mmol) in a solvent mixture composed of absolute toluene and absolute nitromethane (7 ml; 1:1) with molecular sieve (4 A) and IE 91334 the mixture was stirred at room temperature for one hour. A solution of compound 2 (134 mg = 0.33 mmol) in absolute toluene (1 ml) was added to this mixture which was then stirred at room temperature for 22 hours. Filtration and washing with toluene and addition of dichloromethane were followed by shaking with sodium bicarbonate solution and then water, and the organic phase was dried over sodium sulfate. The product was obtained as crystals by column chromatography (mobile phase dichloromethane/methanol/ formic acid = 20:1:0.1).
Yield: 48 mg (0.05 mmol = 45%), mp 131-132’C, [o)D25 + 220 (c = 0.02 in chloroform).
NMR (300 MHz, CDC13) fi 14.01 (s, IH, OH-6), 13.23 (s, IH, OH-11), 8.04 (d, IH, J12 = 8.7 Hz, H-l), 7.79 (t, IH, J12 = J2>3 = 8.5 Hz, H-2), 7.40 (d, IH, J2>3 = 8.3 Hz, H3), 6.72 (d, IH, J3>ra = 8.2 Hz, NH'), 5.93 (dddd, IH, allyl), 5.54 (d, IH, J3..,v = 3.4 Hz, H-4), 5.50 (d, IH, Jr,2a· = 2.9 Hz, H-l'), 5.44-5.17 (m, 4H, H-7, H-3', 2 x allyl), 5.15 (d, IH, Jr.f2.. = 3.6 Hz, H-l), 5.00 (d, IH, Jua.ub = 19·5 Hz, H-14a), 4.86 (d, IH, JUa>14b - 19.5 Hz, H14b), 4.49 (t, IH, J5..,6a.. = J5.t6b.. = 6.5 Hz, H-5), 4.18 (m, 6H, 2 x allyl, H-3', H-5', H-6a, H-6b), 4.09 (s, 3H, OMe), 3.87 (dd, IH, Jx.>2.. = 3.5 Hz, J2..(3. = 10.5 Hz, H-2”), 3.67 (d, IH, J3.tA. = 3.7 Hz, H-4'), 3.27 (d, IH, J1Oa,iOb = 19.0 Hz, H-lOa), 2.98 (d, IH, J10a,10b = 19.0 Hz, H-lOb), 2.36-1.7 (m, 4H, H-8a, H-8b, H-2a’, H-2e'), 2.16 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.04 (s, 3H, OAc), 1.33 (d, 3H, J5. 6. = 6.5 Hz, CH3-6).
FAB-MS, m/z = 990 (M + Na).
Example 5: 14-0-(2-O-Allyl-3,4,6-tri-O-acetyl-e-D-galacto-hexopyranosyl)-N-(9-fluorenylmethoxycarbonyl)-doxorubicin (5) Compound 3 (93 mg = 0.122 mmol) was reacted under the conditions mentioned in Example 4 with silver carbonate (IH = 0.366 mmol), silver perchlorate (25 mg 18 IE 91334 0.122 mmol) and compound 2 (150 mg = 0.366 mmol) and was worked up.
Yields 65 mg (0.06 mmol = 49%), mp 148-149eC, [e]D20 * * * * 25 * * * * 30 + 170° (c = 0.02 in chloroform).
XH NMR (200 MHz, CDC13) 13.97 (s, 1H, OH-6), 13.20 (s, 1H, OH-11), 8.08-7.01 (m, 11H, H-l, H-2, H-3, 8 x Fmocaromatic H), 5.95 (dddd, 1H, allyl), 5.52 (d, 1H, J3.<4« = 3.0 Hz, H-4), 5.45 (d, 1H, Jr<2a. = 2.8 Hz, H-l'), 5.435.11 (m, 5H, 2 x allyl, H-7, H-l, H-3), 4.94 (m, 2H, H10 14a, H-14b), 4.57-4.03 (m, 13H, OMe, 2 x allyl, 3 x Fmoc, H-6a“, H-6b, H-3', H-5', H-5), 3.87 (dd, 1H, J1%2« = 3.3 Hz, J2.j3. = 10.4 Hz, 2-H), 3.67 (m, 1H, H-4'), 3.25 (d, 1H, J14a,14b = 19.0 Hz, H-14a), 2.93 (d, 1H, J14a>14b = 19.0 Hz, H-14b, 2.15 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.03 (s, 3H, OAc), 1.31 (d, 3H, J5, 6. = 6.3 Hz, CH3-6').
FAB-MS, m/z = 1116 (M + Na).
Example 6: 14-0-(3,4,6 -Tr i-O-acetyl-a-D-galacto-hexo-pyranosyl) -N(9-fluorenylmethoxycarbony1)-doxorubic in (6) A solution of l,5-cyclooctadiene-bis[methyldiphenylphosphine]iridium hexafluorophosphate (5 mg) in absolute tetrahydrofuran (5 ml) was hydrogenated under atmospheric pressure until decolorized, and a solution of compound 5 (44 mg = 0.04 mmol) in absolute tetrahydrofuran was added and the mixture was stirred for two hours. Then water (1 ml) and iodine were added and the mixture was stirred for a further 30 minutes. Dichloromethane was added to the reaction mixture which was extracted by shaking first with agueous sodium thiosulfate solution and then with agueous sodium bicarbonate solution. Drying with sodium sulfate and concentration were followed by purification by column chromatography (mobile phase: dichloromethane/ methanol/formic acid - 20:1:0.1). Yield: 15 mg (0.014 mmol = 35%). 91334 Example 7: 14-0- (3,4,6-Tri-0-acetyl-a-D-galacto-hexo-pyranosyl) doxorubicin (7) A solution of compound 6 (12 mg = 0.011 mmol) in morpho5 line (2 ml) was stirred at room temperature for three hours and then evaporated twice with toluene. Taking up with dichloromethane was followed by extraction by shaking with aqueous acetate buffer (pH 5).
Yield: 9 mg (0.0103 mmol - 94%).
XH NMR (200 MHz, CDC13) 5 8.03 (d, 1H, Jlf2 = 7.7 Hz, H-l), 7.79 (t, 1H, J1>2 = J2>3 = 8.0 Hz, H-2), 7.39 (d, 1H, J2>3 = 8.2 Hz, H-3), 5.52 (m, 1H, H-4), 5.46 (d, 1H, J1>>2a. = 3.3 Hz, H-l'), 5.34 (m, 1H, H-7), 5.26 (dd, 1H, J2-,3.. = .5 Hz, J3..v. = 3.2 Hz, H-3), 4.98 (d, 1H, Jr>2« = 3.8 Hz, H-l), 4.92 (m, 2H, H-14a, H-14b), 4.39 (t, 1H, J5..,6a.. = Js-.a- = 6-5 Hz, H-5), 4.14-3.97 (m, 6H, OMe, H-5', H6a, H-6b), 3.49 (s, 1H, H-4'), 3.27 (d, 1H, J10a,10b = 19.0 Hz, H-lOa), 2.99 (d, 1H, J1Oa.iOb = 19·θ «2/ H-10b), 2.15 (s, 3H, OAc), 2.07 (s, 6H, 2 x OAc), 1.36 (d, 3H, J5,t6. = 6.6 Hz, CH3-6).
Example 8: 14-0- (3,4,6-Tri-O-acetyl-e-D-galacto-hexo-pyranosyl) -Ntrifluoroacetyl-doxorubicin (8) Compound 4 was reacted under the conditions specified in Example 7.
*H NMR (200 MHz, CDC13) δ 14.05 (s, 1H, OH-6), 13.28 (s, 1H, OH-11), 8.06 (dd, 1H, J12 = 7.8 Hz, J1>3 = 0.8 Hz, H1), 7.80 (t, 1H, J1>2 = J2>3 = 8.0 Hz, H-2), 7.40 (dd, 1H, J23 = 8.7 Hz, J1>3 = 0.8 Hz, H-3), 6.67 (d, 1H, J3.>ira = 8.5 Hz, NH), 5.57 (d, 1H, J3..3- = 2.8 Hz, H-4), 5.45 (d, 1H, JVt2a. = 3.6 Hz, H-l'), 5.33 (m, 1H, H-7), 5.23 (dd, 1H, J2,.>3.. = 10.5 Hz, J3«4. = 2.9 Hz, H-3), 5.02 (d, 1H, Jrt2= 3.8 Hz, H-l), 4.87 (m, 2H, H-14a, H-14b), 4.37 (t, 1H, IE 91334 J5,6a = Js-.a·· = 6-5 Hz, H-5), 4.20 (q, IH, J5.(6. = 6.3 Hz, H-5'), 4.09 (s, 3H, OMe), 3.65 (m, IH, H-4'), 3.33 (d, IH, J10a>10b = 18.5 Hz, H-lOa), 2.98 (d, IH, J1Oa.iob = 18·5 Hz, H-lOb), 2.15 (s, 3H, OAc), 2.08 (s, 3H, OAc), 2.04 (s, 3H, OAc), 1.32 (d, 3H, J5.>6. = 6.5 Hz, CH3-6').
Example 9: 14-0- (2-O-Allyl-a-D-galacto-hexo-pyranosyl) -N-trifluoroacetyl-doxorubicin (9) A solution of compound 4 (15 mg = 0.015 mmol) in methanol Γ0 (2 ml) was mixed with a catalytic amount of sodium methanolate and stirred at room temperature for one hour.
The mixture was then neutralized with acidic ion exchanger e Dowex WX50), filtered and concentrated.
Yield 12 mg (0.014 mmol = 93%) JH NMR (400 MHz, CDC13 + CD3OD) S 14.02 (s, IH, OH-6), 13.28 (s, IH, OH-11), 8.03 (d, IH, Jli2 = 7.8 Hz, H-l), 7.80 (t, IH, J12 = J2>3 = 8.0 Hz, H-2), 7.40 (d, IH, J2>3 = 8.5 Hz, H-3), 6.01 (m, IH, allyl), 5.55 (m, IH, H-l'), 5.26 (m, IH, H-7), 5.17 (d, IH, J1%2.. = 3.5 Hz, H-l), 4.94 (d, IH, J14a,1Ab = 19.0 Hz, H-14a), 4.85 (d, IH, JUa.1Ab = 19.0 Hz, H-14b), 4.08 (s, 3H, OMe), 3.28 (d, IH, J1OajlOb = 18.5 Hz, H-lOa), 3.02 (d, IH, J10a>10b = 1θ·5 Hz, H-lOb), 1.32 (d, 3H, J5. 6. = 6.5 Hz, CH3-6').
Example 10: 14-O-a-D-Galactohexopyranosyl-doxorubicin (10) a) Compound 7 (4 mg - 0.005 mmol) was reacted under the conditions specified in Example 9.
Yield: 2.8 mg (0.004 mmol - 80%). b) A solution of compound 8 (11 mg s 0.012 mmol) in methanol (1 ml) and 0.5 N sodium hydroxide solution (0.5 ml) was stirred at room temperature for 90 minutes. Neutralization with dilute hydrochloric acid - 21 IE 91334 was followed by evaporation with toluene several times, then taken up with methanol, filtration through Celite and concentration. Purification was by HPLC (mobile phase: phosphate buffer/acetonitrile/tetra5 hydrofuran = 30:60:10).
XH NMR (200 MHz, CDC13 + CD3OD) 8 7.90 (d, 1H, J1>2 = 7.6 Hz, H-l), 7.73 (t, 1H, Jli2 = J2>3 = 8 Hz, H-2), 7.22 (d, 1H, J23 = 8.4 Hz, H-3), 5.37 (s, 1H, H-l'), 5.12 (s, 1H, H-7), 4.85 (s, 2H, H-14a, H-14b), 4.75 (m, 1H, H-l), 3.98 (s, 3H, OMe), 2.29 (d, 1H, J10,tl0b = 15·5 Hz, H-lOa), 2.06 (d, lH,J10e l0b = 15.5 Hz, H-lOb), 1.21 (d, 3H, J4.<5. = 6.6 Hz, CH3-6).
B Cleavage of the prodruqs bv enzymes: Example 11: Cleavage of 14-0-e-D-galacto-hexo-pyranosyl-doxorubicin (compound 10) by human placental α-galactosidase A 0.5 mg of the starting compound was dissolved in 0.5 ml of phosphate buffer, pH 5, containing 0.03 U/ml of egalactosidase A isolated from human placenta and was incubated at 37°C in the dark. Samples of the incubation mixture were taken after various incubation times and analyzed directly by high-pressure liquid chromatography (column: Nucleosil 100 (RP 18, 5 μία particle diameter, 125 x 4.6 mm; mobile phase: gradient of solution A and B: 0 min - 20% A, 10 min - 30% A, 15 min - 30% A, solution A - 100% acetonitrile, solution B = 0.02 M phosphate buffer pH 3.0; flow rate: 1 ml/min; detection: fluorescence, Ex 495 nm, Em 560 nm). The retention time of the starting compound under these chromatographic conditions was 9.0 min. The compound produced during the incubation had the same retention time as adriamycin at 12.6 min.
The half-life for the cleavage under the described conditions was about 54 h (extrapolated value). The IE 91334 compound remained stable over this period on incubation of the starting compound in enzyme-free buffer solution.
Example 12: Cleavage of 14-0-a-D-galacto-hexo-pyranosyl-doxorubicin 5 (compound 10) by coffee bean a-galactosidase The starting compound was incubated under the same conditions as described in Example 11 in the presence of 0.03 U/ml coffee bean α-galactosidase (Sigma). The halflife for the cleavage in the presence of the enzyme was about 20 min.
C Determination of the enzyme activity of enzyme conjugates: Example 13: Determination of the enzyme activity of ^-glucuronidase conjugates The ^-glucuronidase purified by the abovementioned procedure was coupled to the antibody or the biomolecule, and the activity of the enzyme and of the conjugate was determined as follows: 500 μΐ of the enzyme conjugate solution to be determined were added to 500 μΐ of a 2.5 mM p-nitrophenyl β-Ώglucuronide solution in 100 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 5. The assay mixture was incubated at 37” and, after 6 min, stopped with 500 μΐ of a 0.4 M glycine solution, pH 10.8. The liberated p-nitrophenol was then determined by measuring the extinction at 405 nm.
Result: The conjugate showed only a slight reduction in the enzyme activity.
IE 91334 - 23 Example 14: Determination of the enzyme activity of e-D-galactosidase A conjugates The α-galactosidase A purified by the abovementioned 5 procedure was coupled to the antibody or the biomolecule, and the activity of the enzyme and of the conjugate was determined as follows: 500 μΐ of the enzyme conjugate solution to be determined were added to 500 μΐ of a 2.5 mM p-nitrophenyl e-D10 galactopyranoside solution in 100 mM HEPES (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid), pH 5. The assay mixture was incubated at 37° and, after 6 min, stopped with 500 μΐ of a 0.4 M glycine solution, pH 10.8. The liberated p-nitrophenol was then determined by measuring the extinction at 405 nm.
Result: The conjugate showed only a slight reduction in the enzyme activity.
D Occurrence of the enzyme in human plasma: Example 15: Occurrence of 0-D-glucuronidase in human plasma 500 μΐ of human plasma were added to 500 μΐ of a 2.5 mM p-nitrophenyl 0-D-glucuronide solution in 100 mM HEPES (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid), pH 7.4. The assay mixture was incubated at 30° and, after 30 min, stopped with 500 μΐ of a 0.4 M glycine solution, pH 10.8. The liberated p-nitrophenol was then determined by measuring the extinction at 405 nm.
Result: No activity of ^-D-glucuronidase enzyme was detectable IE 91334 - 24 under the test conditions.
Example 16: Occurrence of α-D-galactosidase in human plasma 500 μΐ of human plasma were added to 500 μΐ of a 2.5 mM 5 p-nitrophenyl ο-D-galactopyranoside solution in 100 mM HEPES (N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid), pH 7.4. The assay mixture was incubated at 30° and, after 30 min, stopped with 500 μΐ of a 0.4 M glycine solution, pH 10.8. The liberated p»nitrophenol was then 1.0; determined by measuring the extinction at 405 nm.
Result: No activity of ο-D-galactosidase enzyme was detectable under the test conditions.
E In vivo anti-tumor effects: Example 17: In vivo anti-tumor effects of the 14-O-glycosylanthracycline prodrug system NMRI nu/nu mice received on day 0 a subcutaneous inoculation of pieces of CoCa 4 human tumor about 5 mm in size per animal. After the human tumor tissue had grown in the mice (day 7-14) 6 animals in each of groups a,b,c received 5 x 500 μς of MAb BW 494/32-galactosidase conjugate, d received 5 x 500 μ$ of MAb BW 494/32, e received 5 x 500 μ$ of galactosidase and 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 494/32, galactosidase, MAb BW 494/32, galactosidase or PBS injection the mice in groups a, d and e received per animal one third of the maximum tolerable dose (MTD) of the 14-O-glycosylanthracycline injected intravenously IE 91334 --------- 25 each 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: 5i The tumor growth found in groups d and e did not differ significantly from that in group f. Groups a, b and c showed a distinct inhibition of tumor growth, and the effects were most distinct in group a.
Comparable results were obtained with MAbs BW 431/26, BW 10 250/183 in the CoCa4 xenograft system and with MAb BW 704 in the M21 xenograft system.
IE 91334

Claims (6)

1. Patent Claims! HOE 90/B 001
1. Compound of the formula I and the salts thereof with an inorganic or organic acid R in which R 1 , R 2 and R 3 are, independently of one another, hydrogen, hydroxyl, methoxy, R*, R 5 and R 6 are, independently of one another, hydrogen, hydroxyl, halogen, aliphatic acyloxy (C1-C8), benzoyloxy or substituted benzoyloxy such * as para-nitrobenzoyloxy, alkyloxy (C1-C8), allyloxy, benzyloxy or substituted benzyloxy, tetrahydropyranyloxy, amino, NH-acyl (C1-C8), NH-(9fluorenylmethoxycarbonyl), morpholino or substituted morpholino, preferably 3-0-methylmorpholino or 2-cyanomorpholino, R 7 is a carbohydrate of the formula II O — Π with IE 91334 R 8 being methyl, hydroxymethyl, acyloxymethyl (ClC8), alkyloxymethyl (C1-C8), benzyloxymethyl, allyloxymethyl, carboxyl, carboxymethyl or carboxyallyl, 5 R 9 , R 10 and R 11 are, independently of one another, hydrogen, hydroxyl, acyloxy (C1-C8), benzoyloxy or substituted benzoyloxy such as para-nitrobenzoyloxy, alkyloxy (C1-C8), allyloxy, benzyloxy or substituted benzyloxy, amino, NH-acyl (C1-C8) 10 or NH-(9-fluorenylmethoxycarbonyl), where an acyl group is a mono-, di- or trihaloacetyl group, preferably with the halogen atoms fluorine or chlorine, excepting compounds in which R 4 =R 5 =R 8 =R 10 =Oacetyl and R 8 =R ll =H in the α-L-deoxyfucose conforma15 tion.
2. A compound as claimed in claim 1, wherein R 1 is methoxy, R 2 , R 3 are hydroxyl and the radicals R* to R 11 have the meaning stated in claim 1.
3. A compound as claimed in claim 1, wherein R 1 is 20 methoxy, R 2 and R 3 are hydroxyl and R* is hydroxyl or tetrahydropyranyloxy, R 5 is amino, R 6 is hydrogen and R 7 is a carbohydrate in the α-D-galactopyranosyl or 0-D-glucuronyl configuration with the meanings for R 8 to R 11 stated in claim 1. 25
4. A compound as claimed in claim 1, wherein R 1 is methoxy, R 2 , R 3 are hydroxyl and R* is hydroxyl or tetrahydropyranyloxy, R 5 is amino, R 8 is hydrogen, R 7 is α-D-galactopyranosyl with R 8 being hydroxymethyl and R 9 , R 10 and R 11 being hydroxyl. 30 5. A compound as claimed in claim 1, wherein R 1 is methoxy, R 2 , R 3 are hydroxyl and R* is hydroxyl or tetrahydropyranyloxy, R 5 is amino, R 8 is hydrogen, R 7 is 0-D-glucuronyl with R 8 being carboxyl and R 9 , R lp and R 11 being hydroxyl. IE 91334 - 28 6. A compound as claimed in claim 1, wherein R 1 is methoxy, R 2 , R 3 and R 4 are hydroxyl, R 5 is amino, R 6 is hydrogen, R 7 is β-D-galactopyranosyl with R® being hydroxymethyl and R 8 , R 10 and R 11 being hydroxyl.
5. 7. A compound as claimed in claim 1, wherein R l is methoxy, R 2 , R 3 and R 4 are hydroxyl, R 5 is amino, R® is hydrogen, R 7 is 0-D-glucuronyl with R® being carboxyl and R 8 , R 10 and R 11 being hydroxyl.
6. 8. A process for preparing a compound as claimed in 10 claim 1, which comprises reacting an anthracycline of the formula V
IE033491A 1990-02-01 1991-01-31 Glycosyl prodrugs of anthracyclines, a process for the¹preparation thereof and the use thereof in combination with¹functionalized tumor-specific enzyme conjugates IE910334A1 (en)

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AU633867B2 (en) * 1989-02-02 1993-02-11 Eli Lilly And Company Delivery of cytotoxic agents
DE4106389A1 (en) * 1991-02-28 1992-09-03 Behringwerke Ag FUSION PROTEINS FOR PRODRUG ACTIVATION, THEIR PRODUCTION AND USE
DE3935016A1 (en) * 1989-10-20 1991-04-25 Behringwerke Ag GLYCOSYL ETOPOSIDE PRODRUGS, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE IN COMBINATION WITH FUNCTIONALIZED TUMOR-SPECIFIC ENZYME CONJUGATES
US5776458A (en) * 1990-12-05 1998-07-07 Pharmacia & Upjohn S.P.A. Anthracycline-conjugates
GB9026491D0 (en) * 1990-12-05 1991-01-23 Erba Carlo Spa Anthracycline-conjugates
DE69233669T2 (en) * 1991-10-23 2007-10-25 Cancer Research Technology Ltd. BACTERIAL NITROREDUCTASE TO REDUCE CB 1954 AND ANALOGUE IT INTO A CYTOTOXIC FORM
DE4236237A1 (en) 1992-10-27 1994-04-28 Behringwerke Ag Prodrugs, their preparation and use as medicines
US5502037A (en) * 1993-07-09 1996-03-26 Neuromed Technologies, Inc. Pro-cytotoxic drug conjugates for anticancer therapy
EP0647450A1 (en) * 1993-09-09 1995-04-12 BEHRINGWERKE Aktiengesellschaft Improved prodrugs for enzyme mediated activation
ES2148259T3 (en) * 1993-09-22 2000-10-16 Hoechst Ag PRO-PROFARMACOS, ITS PRODUCTION AND USE.
DE69533615T2 (en) 1995-06-27 2005-10-13 Pharmachemie B.V. Anthracycline prodrugs, process for their preparation and their use in selective chemotherapy
RU2196604C1 (en) 2001-12-21 2003-01-20 Северин Евгений Сергеевич Polypeptide displaying analog of receptor-binding epidermal growth factor fragment from 21 to 31 amino acids, its conjugate with doxorubicin and pharmaceutical composition based on thereof
US6740509B2 (en) 2002-05-22 2004-05-25 Ikuko Ishii Karakasa Method for the production of mucin-type glycopeptide
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