CA2028086A1 - Glycosyl-etoposide prodrugs, a process for the preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates - Google Patents
Glycosyl-etoposide prodrugs, a process for the preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugatesInfo
- Publication number
- CA2028086A1 CA2028086A1 CA002028086A CA2028086A CA2028086A1 CA 2028086 A1 CA2028086 A1 CA 2028086A1 CA 002028086 A CA002028086 A CA 002028086A CA 2028086 A CA2028086 A CA 2028086A CA 2028086 A1 CA2028086 A1 CA 2028086A1
- Authority
- CA
- Canada
- Prior art keywords
- group
- acyl
- tri
- hydrogen atom
- acetyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
- A61K47/6899—Antibody-Directed Enzyme Prodrug Therapy [ADEPT]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Nanotechnology (AREA)
- Biophysics (AREA)
- Epidemiology (AREA)
- General Engineering & Computer Science (AREA)
- Medical Informatics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Immunology (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
BEHRINGWERKE AKTIENGESELLSCHAFT HOE 89/B 039 - Ma 792 Dr. Ha/Sd Abstract of the disclosure Glycosyl-etoposide prodrugs, a process for the prepara-tion 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 tumor-specific enzyme conjugates for treating cancers, and specifically relates to 4'-O-glycosyl-etoposides as prodrugs which can be cleaved by the action of tumor-specific enzyme conjugates to give cytotoxic active sub-stances, the liberated active substance being suitable, by reason of its cytostatic activity, for treating cancers.
Description
202~ 3 BEHRINGWERKE A~TIENGESEL~SCHAFT 89/B 039 - Ma 792 Dr. Ha~Sd Glycosyl etoposide prodrugs, a process for the prepara-tion thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugate~
.. .... ...... __ _ __ The present invention relates to glycosyl etoposide prodrugs, a process ~or the pxepara~ion thereo~ and the use thereof in combination with functionalized tumor-specific enzyme con~ugates for treating cancers, andspecifically relates to 4~-O-g}ycosyl-etoposides as prodrugs which can be cleaved by the action of tumor-specific enzyme conjugates to give cytotoxic active sub-stances, the liberated active substance being suitable, by reason of its cytostatic activity, for treating cancers.
The combination of prodrug and tumor-specific antibody-enzyme conjugates for use as therapeutic agents is described in the specialist literature. Thiæ entailed antibodies which are directed against a particular tissue and are covalently bonded to a prodrug-clea~ing enzyme being in~ected into an animal which contains ~he trans-planted 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 con~ugate, which is anchored to the tissue, into the cytotoxin which exerts a cytotoxic effect on ~he trans-planted tissue.
A therapeutic system which contains ~wo componen~s and is composed of an antibody-enzyme component and of a prodrug component which can be activated by enzyme i8 described in WO 88/07378. In this case, the use of non-mammalian enzymes is described for the preparation of the antibody-enzyme conjugates, and that of endogenous enz~me~
is ruled out because of the non specific liberation of 2 2 ~
active compound. Since the exogenou~ enzymes are recog-nized by the body a~ foreign antigens, the use thereof is associated with the disPdvantage of an immune response to the nsn-end~genous 6ubstances, for which reason the S enzyme Lmmobilized on the antibody is inactivated and, where appropriate, the Pntire con~ugate i8 ~liminated. In addition, in this case p-bis-N-~2-chloroethyl)amino-benzylglutamic acid and derivatives thereof are used as prodrug, and their chemical half-life i~ only ~.3 to 16.5 hours. I~ is a disadvantage for a prodrug to be chemically unstable because of the side effect6 to be expected.
A therapeutic system which contains two component~ and in which the antibody-enzyme conjuga~e located on the tumor 13 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-.ummobilized alkaline phosphatases for liberating the etopo~ides, which is described therein inter alia, is disadvanta~eous because of the strong pre~ence of endogenous alkaline phospha-tases in the serum. As described in DE 38265662 Al, the etoposide 4'-phosphates are already used alone as thera-peutic antitumor agents, in which case the phosphatases present in the serum liberate the etoposide from the prodrug.
It has emergedr surprisingly, that the 6ynthetically prepared, hitherto unobtainable compound 4'-O-alpha-D-glucopyranosyl-etopo~ide can be cleaved in vitro into etoposide and D-glucose with the ~nz~me alpha-glucosidase as well as a tumor~pecific antibody-glucosida~e con~
jugate.
Based on this finding, and taking into account the dis-advantayes, described above, of combinations of prodrugs and antibody-enzyme conjugates, the objec~ of the present invention was to prepare synthetic, enzymatically 2 0 2 8 0 8 ~
.. .... ...... __ _ __ The present invention relates to glycosyl etoposide prodrugs, a process ~or the pxepara~ion thereo~ and the use thereof in combination with functionalized tumor-specific enzyme con~ugates for treating cancers, andspecifically relates to 4~-O-g}ycosyl-etoposides as prodrugs which can be cleaved by the action of tumor-specific enzyme conjugates to give cytotoxic active sub-stances, the liberated active substance being suitable, by reason of its cytostatic activity, for treating cancers.
The combination of prodrug and tumor-specific antibody-enzyme conjugates for use as therapeutic agents is described in the specialist literature. Thiæ entailed antibodies which are directed against a particular tissue and are covalently bonded to a prodrug-clea~ing enzyme being in~ected into an animal which contains ~he trans-planted 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 con~ugate, which is anchored to the tissue, into the cytotoxin which exerts a cytotoxic effect on ~he trans-planted tissue.
A therapeutic system which contains ~wo componen~s and is composed of an antibody-enzyme component and of a prodrug component which can be activated by enzyme i8 described in WO 88/07378. In this case, the use of non-mammalian enzymes is described for the preparation of the antibody-enzyme conjugates, and that of endogenous enz~me~
is ruled out because of the non specific liberation of 2 2 ~
active compound. Since the exogenou~ enzymes are recog-nized by the body a~ foreign antigens, the use thereof is associated with the disPdvantage of an immune response to the nsn-end~genous 6ubstances, for which reason the S enzyme Lmmobilized on the antibody is inactivated and, where appropriate, the Pntire con~ugate i8 ~liminated. In addition, in this case p-bis-N-~2-chloroethyl)amino-benzylglutamic acid and derivatives thereof are used as prodrug, and their chemical half-life i~ only ~.3 to 16.5 hours. I~ is a disadvantage for a prodrug to be chemically unstable because of the side effect6 to be expected.
A therapeutic system which contains two component~ and in which the antibody-enzyme conjuga~e located on the tumor 13 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-.ummobilized alkaline phosphatases for liberating the etopo~ides, which is described therein inter alia, is disadvanta~eous because of the strong pre~ence of endogenous alkaline phospha-tases in the serum. As described in DE 38265662 Al, the etoposide 4'-phosphates are already used alone as thera-peutic antitumor agents, in which case the phosphatases present in the serum liberate the etoposide from the prodrug.
It has emergedr surprisingly, that the 6ynthetically prepared, hitherto unobtainable compound 4'-O-alpha-D-glucopyranosyl-etopo~ide can be cleaved in vitro into etoposide and D-glucose with the ~nz~me alpha-glucosidase as well as a tumor~pecific antibody-glucosida~e con~
jugate.
Based on this finding, and taking into account the dis-advantayes, described above, of combinations of prodrugs and antibody-enzyme conjugates, the objec~ of the present invention was to prepare synthetic, enzymatically 2 0 2 8 0 8 ~
cleavable 4'-0-glycosyl-etoposide~ as well a~ functiona-lized tumor-specific ~nzymes, and to test the pharma-cological utility of the combination of the two compo-nents in suitable mammali~n 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 o~ cyto~tatic activity.
~ he invention relates ~o 4~-0-glycosyl-etoposides of the formula I
R' ~ o-~~
0~
R' < ~0 ~41~ I
R ~
~eO ~ o~e R'O- ~ ~ O
R6 R~
in which R1 is a methyl, benzyl or 2-thienyl ~roup, R2 i~ a hydrogen atom, an acyl or tri-C1-C4-alkylsilyl protective group, R3 is a hydroxyl group, an acyl or tri-Cl-C4-alkyl~ilyl protective qroup which is bonded via an oxYaen atcm, an amino, acetyl~,~no, benzYloxYcarbonYlam m o or dImethylam mo qroup, R4 is a hydrogen atom or a methyl group, R5 is a hydrogen atom, a hydroxyl group, an acyl or tri-Cl-C~-alkylsilyl protective group which i~ bonded via an o~ygen atom, or an amino, benzyloxycarbonyl-amino, azido or acetylamino group, R~ is a hydroxyl group, an acyl or tri Cl-C4~alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzylo~ycarbonylamino or azido group, R7 is a hydrogen a~om, an acyl ox tri-Cl-C4-alkylsilyl ~2~
protective group and R~ is a methyl or hydroxymethyl group or an acyl protective group which i~ 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 i~vention 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-associa~ed antigen, or is a biomolecule which accumulates in a tumor, such as E&F (epidermal growth factor), TGF-alpha (transforming growth factor alpha), PD~F
(platelet derived growth factor), IGF I+II (insulin like growth factor I+II) or a+b FGF (acidic + basic fibroblast growth factor) 20 E is a glycosidase which i8 not Lmmunogenic or is of low immunogenicity, preferably mammalian glycosidase, as alpha- or beta-glucosidase, alpha-galactosidase, alpha- or beta-mannosidase, alpha-fucosidase, N-acetyl-alpha-galactosaminidase, N-acetyl-beta-/N-acetyl-alpha-glucosaminidaseorbeta-glucuronidase, Sp (spacer) i6 ~ bifunctional sulfide- or disulfide-containing group of the formula III or IV
X(S)nY III ~(S)~ IV
or a polypeptide spacer, in which X or Y is -CO-R9-(N-succinLmido)- or -C(=Rl~-CH2-CH2-with R9 being -CH2-CH2 , 1,4-cyclohexylideneg 1,3- or 1,4-phenylene or methoxycarbonyl- or chloro-1,4-phenylene and Rl being O or NH, and furthermore 2~280~
Y is -C(=Rl)-CH2-CH2-, where R10 has the stated meaning, and n is 1 or 2.
The fusion gene composed o V~, C~l hinge and enzyme gene is cloned into an expression plasmid which i6 suitable for expression in eukaryotic cell~ and carries a selec-tion marker. The expression plasmid with the fu~ion 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 ~uitable antibiotic is followed by identification of transfectoma clones which contain the expression plas-mids. Suitable detection methods (BioDot, ELISA) sre used to identify those transfectoma clones which secrete the fusion protein of the formula II composed of antibody ~nd enzyme.
Preferred within the scope of the in~ention are compounds of the formula I in which the radicalæ
R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, an acetyl or chloroacetyl group or a tri-Cl-C4-alkylsilyl protecti~e group, R3 is a hydroxyl group, an acetyl, chloroacetyl or tri-C1-C4-alkylsilyl protective group which i8 bond~d via an oxygen atom, or an amino, acetylamino, benzyloxycarbonylamino or dimethylamino group, R4 is a hydrogen atom or a methyl group, Rs is a hydrogen atom, a hydroxyl group, or an acetyl, chloroacetyl or tri C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino, azido or acetylamino group, R~ is a hydroxyl group, an ace~yl, chloroacetyl or tri-Cl-C4-alkylsilyl protective ~roup which i~ bonded via an oxygen atom, or an amino, benzyloxycarbonyl-amino or azido group, R7 is a hydrogen atom, an acetyl, chloxoacetyl or tri-Cl-C4-alkylsilyl protective group and R3 is a methyl, hydroxymethyl, acetyloxy or chloro-acetyloxymethyl group or a benzyloxycarbonyl group, as well as a functionalized tumor-specific enzyme of the formula II in which A is an antibody or fra~ment thereof, which ha~e specificity for a tumor-associated antigen, or is a biomolecule which accumulates on or in the tumor, such as EGF (epidexmal growth factor), TGF-alpha (transforming growth factor alpha), PDGF (platelet derived growth factor), IGF I~II (in~ulin like growth factor I~II), a+b FGF (acidic ~ basic fibro-blast 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, alpha-galactosidase, alpha- or beta-mannosidase, alpha-fucosidase, N-acetyl-alpha-galactosaminidase, N-acetyl-beta-/N-acetyl-alpha-glucosaminidaseorbeta-glucuronidase, Sp is a bifunctional disulfide-containing group of the formula III or IV or a polypeptide spacer, in which X or Y is -C0-R9-(N-succinLmido)- or -C(=Rl)-CHz-CH2-with R9 being -CH2-CH2- or 1,4-phenylene and R10 being 0 or NH, Y is -C(=R1)-CH2-CH2-, where R10 has the stated meaning, and n is 1 or 2.
The proce~ according to the invention ~or preparing a compound of the formula I, which can be degraded by glycosidase, in which R~ is a methyl, benzyl or 2~thienyl gro~p, R2 is a hydrogen atom, R3 is a hydroxyl, amino or dLmethylamino ~roup, R4 is a hydrogen atom or a methyl group, R5 is a hydrogen atom, a hydroxyl group, an amino or acetylamino group, R5 is a hydroxyl group or an amino group, 20280~
R7 is a hydrogen atom, Ra 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 ~n a~yl protective group mean~ 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 ~olvent at -50C to 60C, an etoposide compound of the formula V
Rl_~R' <o~ V
~e~ ~ o~e in which Rl is a methyl, benzyl or 2-thienyl group, 5 R2 is a hydrogen atom, an acyl or a tri-Cl-C4-alkylsilyl protective group, R3 is a hydroxyl group, an acyl or tri-C,-C4-alkyl~ilyl protective group which is bonded via oxygen, or acetylamino, benzyloxycarbonylamino or dimethylamino group, and ~4 i~ a hydrogen a~om or a methyl group, with a carbohydrate component of the formula VI
R ~ .
R 0 ~ Z VI
in which 25 R5 is a hydrogen atom, a hydroxyl group, an acyl protective group which is bonded via an o~ygen atom, or benzylo~ycarbonylamino, azido vr acetylamino 2 ~
group, Rfi i5 an acyl protective group which i~ bonded via an oxygen atom, or a bsnzyloxycarbonylamino 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 a~om, preferably fluorine, chlorine or bromine, a hydroxyl group, a tri-Cl-C4-alkylsilyloxy group, or an acyl protective group which iB bonded ~ia an oxyqen atom, where the acyl protective group is an acetyl, monv-, di- or trihalogeno-acetyl group~ preferably with the halogen atom being fluorine or chlorine, to give a 4'-0-glyco~yl-etoposide derivative of the fonmula 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 hydroly is, 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 ~pecific procedure for this is as follows:
the glycosidation of etoposide derivatives of the formula V is carried out using ~unctionali~ed carbohydrate unit~
of the fo.rmula VI which are typically protected with acyl protective group~ on the 0-2, 0-3, 0-4 and, where appro-priate, 0-6 atoms. Preferred acyl protective group~ are acetyl, chloroacetyl or trifluoroacetyl groups. In the case of amino sugars, the amin~ group i 8 protected temporarily with a benzyloxycarbonyl group or permanently with an acetyl group. It is likewise possibl* to use ~zido sugars because they can be converted straight-forwardly into amino sugar~ by hydrogenolysis. The carbohydrate component must be suitably functionalized at the anomeric center. Used for thi6 purpose are ~lyco-syl halides, such as fluorides, chlorides or bromides, which can be prepared starting from l-0-acyl derivatives, 2~2~0~i g for example using HF, HCl, HBr or Ti~r4. The glyco~idation components which carry an O-acyl group or a hydroxyl group on the anomeric center are prepared by proce~se~
customary in carbohydrate chemistry.
The glycosidation of etoposides of the formula V with carbohydrate units of the formula VI i~ carried out in the presence of a promoter. ~he promoter used when glycosyl fluorides and the l-hydroxy or l-acetyloxy analogs thereof are employed is BF3 x ether or tri-Cl-C4-alkylsilyl trifluoromethanesulfonate. The promoters usedin the case of glycosyl chlorides or bromide~ are ~alts of silver or of mercury.
The glycosidation is carried out in an aprotic organic solvent such as acetonel 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 agent6 such as molecular ~ieves or magnesium sulfa~e are added. The reaction temperature is in the range from -50C to 0C
when glycosyl fluorides and the 1-hydroxy analog~ are employed and ~t 0C to 60C when glyco6yl chlorides or bromides are employed. ~he glycosyl etoposide~ produced in the reaction are deblocked by the following processe6:
the acyl protective groupR are removed by methanolysi~
catalyzed by zinc(II) salts or with alkaline ion ex-changers 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 earbon or palladium/
barium sulfate or, in the case of the azido group, con-verted into amino group. The compounds of the formula I
containing ~mino sugar~ can addi~ionally be con~erted into dLmethylamino derivative~ ~y reductive ~l~ylation with formaldehyde~sodium cyanoborohydride.
To prepare A~Sp-E, either the ~pacer (Sp) can be linked via an amino gxoup to an enzyme and to the antibody or lo 2~2,~$
the biomolecule via an HS group which has been introducad or generated by cleavage of the di6ulfide linkage, or nucleic a~id ~equences which code for the parts A, Sp and E are covalently linked with the aid of molecular bio-logical methods to result in a fusion gene, and A~Sp-E is prepared by genetic engineering proce~se~.
This can be carried out in a variety of ways:
A) A restriction cleavage ~ite A is introduced by ~pecific mutagene is a~ the 3~ end of the C~1 exon in the gene of the heavy chain of ~he immunoglobu-lin. The ~ame restriction cleavage site A is genera-ted at the 5' end of the oligonucleotide which codes for the oligopeptide which acts as ~pacer. ~oth restriction cleavage sites A are sited in such a way that the immunoglobulin gene can be linked to the oligonucleotide via the restriction cleavage ~ite A
without disturbing the reading frame.
A restriction cleavage si~e 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 se~uence coding for the mature protein starts. The enzyme gene is then linked via the restriction cleavage site ~ to the immunoglobulin gene-linked construct. ~he restriction cleavage sites B
are sited such that the reading frame i~ not di~turbed on linkage. The ~usion gene composed of the ~or the hsavy chains of the immunoglobulin V~ and C~1 linker enzyme iE
cloned into an expression plasmid which is suitable for expression in the eukaryotic cells and carries a sel c-tion marker.
The expression plasmid with the fusion gene i~ trans-fected together with an expression plasmid which carries the gene for the light chain belonging to the antibody into eukaryo~ic cells (for example myeloma cells~.
Selection wi~h suitable antibiotics is carried out to isolate cell clones which contain the plasmids with the 11- 2~2~
fusion gene and the gene for ~he light chains (transfectomas). Suitable detection methods (BioDot;
ELI5A) are used to identify those transfectomas 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 ~ is introduced ~ the 3' end of the hinge exon of the gene for the heavy chains of the immunoglobulin. ~he re~triction cleavage site ~ is introduced at ~he ~ite 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 V~, CB1 and hinge exons is linked via the restriction cleavage ~ite 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 V8, C~l hinge and enzyme gene is cloned into an expression pla~mid which is ~uitable for expression in eukaryotic cells and carries a ~elec-tion marker. The expression plasmid with the fusion gene is transfected together with an expression plasmid which contains the light-chain gene belonging to ~he antibody into eukaryotic expression cells. Selection with a suitable antibiotic iæ followed by identification of transfectoma clones which contain the expres~ion plasmids. Suitable detection methods (Bio~ot, ELISA) are used to identify those transfectoma clones which ~ecrete the fusion protein of the formula II composed of antibody and ~nzyme.
.
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. ~hose, I. Immunolog. Methods 59 (1983) 129-143; ToI~ Ghose et 3~
al. Methods in enzymology, Vol. 93 ~1983) 280-333~.
This entails ini~ial functionalization of the enzyme via its amino group using succinLmidyl N-maleLmido-alkylidene~, cycloalkylidene- or arylene-l-carboxylate, where the double bond of ~he maleimldo group enters into a reaction with the HS group of the functionalized antibody, fxagment thereof or the biomolecules, with the formation of a thioether func~ionality.
It is possible to use ~or the preparation of the antibody-enzyme con~ugates the monoclonal antibodies described in EP-A-0141079, preferably th~ antibodies 431J26, 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 immunohistochemi~try.
The nucleotide sequence of the V genes of these mono-clonal antibodies is described in German Patent Applica-tion DE-A-39Q9799.4.
To prepare the tumor-specific enzyme conjugates, it i8 possible for the enæymes which are mentioned hereinafter and from the identified source to be purified by the indicated literature procedureo - alpha-galactosidase from human liver, Dean, K.G. and Sweeley, C.C. (1979), J. Biol. Chem. 254, 994-1000 25 _ beta-glucuronida~e from human liver, ~o, 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 livert GrabDwski, G.A., Ikonne, J.U., Desnick, R.J. (19803 Enzyme 25, 13-2S
- beta-mannosidase from human placenta, Noeske, C., Mersmann, G. (1983) ~oppe Seylers Z Physiol. Chem.
364, 1645-1651 alpha-~lucosidase from human gastro~ntestinal mucosa, Asp, N.-G., Gudmand-Hoeyer, E., - 13 _ 2 ~2~r3~ ~
Christiansen, P~., Dahlquist, A. (1974) Scsnd. J.
Clin. Lab. Invest. 33, 239-245 - beta-glucosidase fr~m human liver, ~aniels, L.B., Coyle, P.J., Chiao, Y.-B., Glew, R.H. (1981) J.
S Biol. Chem. 256, 13004-13013 - beta-glucocerebrosidaæe from human placenta, Fu rbish, F.S., Blair, H.E., Shiloach, J.,Pentcheu, P~G., Brady, R.O. (1977) Pro~O Natl.
Acad. Sci. ~SA 74, 3560-3563 10 - alpha-N-acetylglucosaminidase from human placenta, Roehrborn, W., von Figura, R. (1~78) Hoppe Saylers Z Physiol. Chem. 359, 1353-1362 - beta-N-acetylglucos~minidase from human amniotic membrane, Orlacchio, A., Emiliani, C., Di Renzo, G.C., Cosmi, E.V. (1986~ Clin. Chim. Aota 159, 279-289 - alpha-N-acetylgalacto~minidase according to Salvayre, R., Negre, A., Maret, A., Douste-Blazy, L.
(1984) Pathol. Biol. (Paris) 32, 269-284.
The glycolytic activity of the functionalized tumor-specific enzymes wa~ determined in comparative investi-gations with p-nitrophenyl glycosides at the particular pH optimum.
The invention additionally relates to a pack contsining a glycosyletoposide according to the invention and a functionalized tumor-specific enzyme con~ugate in com-bination with functionalized tumor-6pecific enzyme con~ugates.
To test the efficacy of a combined ~equential 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 s~opped and regression occurred.
2 ~ 2 ~
~xample 1 Preparation of the glycosylation component Benzyl D-qlucuronate (compound l) Benzyl bromide (4.89 g, 28.59 mmol) was added to a solution of sodium D-glucuronate (5 g, 23.13 mmol) in DMF
(300 ml). The reaction mixture was stirred at 40C 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 l3C NMR
Benzvl 1,2,3,4-tetra-O-chloroacetvl-alpha and beta-D-lucuronate ~compound 2a and 2b) Benzyl D-glucuronate (3.80 g, 13.36 mmol) was suspended in dichloromethane (200 ml). Chloroacetyl chloxide (7.90 g, 69.94 mmol) was added and then the mixture was cooled to -30C, and pyridine (4.33 g, 54.74 mmol) dissolved in dichloromethane (50 ml) was added. The ~0 reaction mixture was stirred at -30C 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 ~odium 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 2,3,4-tri-O-chloroacetyl-alpha, beta-D-glucuronate besides the title compound, was used without further purification steps in the next stage.
Benzy~12,3,4-tri-O-chloroacetyl-alpha,beta-D-alucuronate (compound 3a, 3bl The crude product ~7.92 g) of compounds 2a/2b was dis-solved in 3:1 methanol/chloroform (320 ml), and aminated silica gel (11.09 g) was added. The reaction mixture wa~
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 petro-2~2~
leum ether/ethyl ac~tate.
Yield: 4.94 g (72% based on compounds 2a/2b).
Benzyl 1 deoxy-l-fluoro-alpha-D-qlucuronate (comPound 4) Sodium l-deoxy-l-fluoro-alpha~D-glucuronate (6.22 g, 28.51 mmol) was suspended in D~F ~350 ml), and benzyl bromide (4.89 g, 28.59 mmolJ was added. The reaction mixture was stirred at 60C ~or 24 h and then evaporated.
The residue wa~ dissolved in 3:1 chloroform/methanol, and magnesium sulfate (12 g) was added. ~he ~uspen~ion was ~tirred for 2 h and then filtered, and the filtrate was evaporated. The residue was purified by column chromato-graphy on 6ilica gel ~160 g) with 4:1 dichloromethane~
acetone.
Yield: 5.95 g (73~).
Benzyl 1-deoxy-l-fluoro-2,3,4-tri-O-alpha-D-qlucuronate ~compound 5) Compound 4 (5.0 g, 17.46 mmol) was suspended in dichloro-methane (120 ml) and, at 0C, benzyl bromide (9.85 g, 57.61 mmol) and silver oxide 111.2 g) were added. The reaction mixture was stirred at O~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 ~ilica gel (240 g) with 3:1 petrol~um ether/ethyl acetate.
Yield: 6.60 g ~68%).
Benzyl l-deoxy-l-fluoro-2,3,4-tri-O-chloroacetyl-alpha-D-alucuronate (compound 6) Compound 4 (5.0 g, 17.46 mmol) was ~uspended in dichloro-methane (260 ml) and, at o30QC, chloroacetyl chloride (g.86 g, 87.30 mmol) was added. After addition of lOsl dichloromethane/pyTidine (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 ~hen wi~h water. The organic phase was dried over ~odium sulfate and evaporated. The residue was purified by column ~28~
- ~6 -chromatography on silica gel ~260 g) with S:l petroleum ether/ethyl aceta~e.
Yield: 7.58 g (92~), 2,3,4,6-Tetra-0-chloroacetyl-alpha-D-~alactopyranosyl fluoride (compound_7) ~lpha-D-galactopyranosyl fluoride (2.30 g, 12.62 mmol) was dissolved in dry dichloromethane (lD0 ml~ and, at -25C, chloroacetyl chloride (9.0 g, 79.68 mmol) and lsl dichlorometha~e/triethylamine (55 ml) were added. The reaction mixture was stirred for 16 h and then chloro-acetyl chloride (9.0 g) and lsl dichloromethane/triethyl-amine (55 ml) were added. The reaction mixture was stirre~ for a further 24 h and then washed with sodium citrate buffer (pH 5.0, 5~ ml x 3) and then with water.
The organic phase was dried (sodium sulfate) and evapora-ted in vacuo. The residue was purified by column chroma-tography on silica gel l300 g) with 40:8:1 dichloro~
methane/petroleum ether/ethyl acetate.
Yield: 5.19 g (82%) [alpha]D +64.2 (c = 1, dichloromethane).
2,3,4,6-Tetra-0-benzyl-alpha-D-qalactopyranosyl fluoride (compound 8~
Alpha-D-~alactopyranosyl fluoride (2.30 g, 12.62 mmol) was dissolved in dry DMF (40 ml) and, at -20C, benzyl bromide (12.95 g, 75.72 mmol) and silver oxide (10 g) were added. The reaction mixture was stirred at ~20C for 5 h and then at room temperature for ~4 h. The ~alt~ 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%).
- 17 _ 2~2~
Example 2 Glycoside synthesis 4'-O-Demethyl-4-O ~2.3di-O-chloroacetyl-4,6,0-ethy-lidene-beta-D-~lucopyrano~yl~-4-epi-~odophyllotoxin (compound 9) 4'-O-Benzyloxycarbonyl~4-O-demethyl-4-O-(2,3-di-O-chloro-acetyl-4,6-0-ethylidene-beta-D-glucopyranosyl)-4-epi-podophyllotoxin (10 g, 11.42 mmol) wa~ hydrogenated in 2:1 ethyl acetate/methanol (200 ml) in the presence of 10% Pd/C (5.0 g) for 2 h. The reaction mixture was filtered, and the filtrate was evaporated in ~acuo. 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 201-203C; [alpha]D
-73.4 (c = 1, chloroform) Benzyl 4'-O-demethyl-4-O-(di-O-chloroacetyl-4~6-0-ethyli-dene-beta-D-qlucopyranosyl3-4-epi-4'-0-(2,3~4-tri-O-benzyl-beta-D-glucopyranosyl ! -uronate-podophyllotoxin (comPound 9~
Benzyl l-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 di-chlorome~hane ~220 ml), and 4 ~ molecular ~ieves (10 g) were added. BF3-ether (2.5 ml) was added at -40C to the reaction mixture, which was then stirred at -30C for 20 h. Triethylamine (7.0 ml) was added and then She mixture w~s filtered. The filtrate was washed with citrate buffe- (pH 5, 80 ml x 3) and water (120 ml x 3)r dried (sodium sulfate) and evaporated in vacuo. The residue was purified by column chromatography on ~ilica gel (360 g) with 5:5:1 dichloromethane/petroleum ether/
acetone.
Yield: Ç.49 g ~67%).
~2~
4'-0-Demethvl-4-O-(di-O-chloroacetyl-4,6-O-ekhylidene-beta-D-~lucopyranosyl)-4-epi-4'-0-(2,3,4,6-tetra-O-benzyl-alpha- and beta-D-~alactoPvranosyl)-pod lotoxin (compound lOa and lOb) 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 di~solved in dichloromethane (250 ml). 4 A molecular sieves (7.2 g) were added and then the reaction mixture was cooled to -40C, and 30~
strength BF3-ether (2.2 ml) was added dropwise. The mixture was stirred at -30C for 20 h and then, after addition of triethylamine (5.5 ml), filtered. The fil-~rate 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 chroma-tography on silica gel (370 g) with 5:5:1 petroleum ether~dichloromethane/acetone. Further separation by column chromatography provided the title compounds lOa (alpha-glycoside: 4.36 g (52~)) and lOb (beta-glycoside:
1.42 g (17%))-Example 3 Deblocking reaction for glycosyl etoposides Benzyl 4'-O demethyl-4-epi-4-O-t4,6-O-ethylidene-beta-D-qlucopyranosyl ! -4 ~ -o- ( 2,3,4-tri-O-benzyl-beta-D-gluco-pyranosyl)-uronate podophyllotoxin (compound 11 !
Glucuronide derivative (compound ~, 4.56 g, 3.57 mmol) was dissolved in 4:1 methanol/chloroform (120 ml), and DOWEX 1 x 8 (6.2 g) was added. The reaction mixture was stirred at room temperature for 3 h and then filtered.
Chloroform (lS0 ml) was added to the filtrate, which was then stirred with magnesium sulfate (10 g). ~he salt~
were filtered off and then the filtrate was evaporated in vacuo. The residue was purified by col~mn chromatography on 200 g of silica gel with -5:2:1 dichloromethane~
petroleum ether/acetone.
Yield: 3.29 g (B2%~.
2 0 2 8 0 ~ ~
4'-O-Vemethyl-4-epi-4-0-(4,6-O-ethylidene-beta-D-~luco-pyranosyl)-4'-O-(beta-D-alucopyranosyl)-uronic acid podophyllotoxin (compound 12) Deacylated glucuronidP derivative (compound 11, 3.62 g, 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 n vacuo. The residue was purified by column chromatography on RP-18 silica gel using methanol/hexane (gradient).
Yield: 1.82 g (74%).
4'-0-Demethyl-4-epi-4-0-(4.6-0-ethylidene-beta-D-~luco-pYranosyl)-4~-0-~2.3~4~6-tetra-O-benzyl-alpha-D-qalacto-pyranosyl)-podophyllotoxin (compound 13) Podophyllotoxin galactopyranoside (compound lOa, 3.0 g, 2.37 mmol) was dissolved in 3:1 methanol/chloroform, and Dowex 1 x 8 was added. The reaction mixture was stirred at room ~emperature for 3 h and filtered. The filtrate was evaporated in vacuo. The re~idue 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 wa5 purified by column chromatography on ~ilica gel (85 g) with 5:3:1 dichloromethane/petrole~m ether/acetone.
Yield: 2.27 g (86%).
4'-O-Demethyl-4-epi-4'-0-(alpha-D-qalactopyranosyl~-4-0-(4 ! 6-O-ethylidene-beta-D-alucopyranosyl~-podoph~llotoxin ~ comPound 14 !
Deacylated podophyllotoxin galactopyranoside (compound 13, 2.0 g, 1.80 ~mol) was dissol~ed in 3:1 methanol~ethyl acetate and hydrogenated in the prasence of 10% Pd/C
~2.5 g) for 24 h. The mixture was ~tirred 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%).
2~2~$~
Result:
Under the experLmental conditions it wa~ not po~sible to detect any enzyme activity for alpha-galactosidase and beta-~lucuronidase.
Example 5 Determination of ~he enzyme activity of beta-glucuroni-dase conjugates The beta-glucuronidase purified by the abovementioned procedure was coupled to ~he antibody/~he biomolecule, and the activity of the enzyme and of the conjugate was determined as follows:
500 ~1 of the enzyme ~olution to be determined were added to 500 ~1 of a 2.5 mM p-nitrophenyl beta-D-glucuronide solution in 100 mM HEPES ~N-2-hydroxyethyl-piperazine-N'-2-ethane-sulfonic acid), pH 5. The assay mixture was incubated at 37 and stopped after 6 min with 300 ~1 of a 0.4 M glycine solution, pH 10.8. The liberated p-nitrophenol was then determined by measuring the extinc-tion at 405 nm.
Result:
The conjugate showed an only inconsiderable raduc~ion in enzyme activity Example 9 In vivo antitumor effects of the glyco6yl-e~opo~ide prodrug system 30 NMRI nu/nu mice received on day 0 a ~ubcutaneous inoculation of pieces about 5 mm3 in size of CoCa 4 human tumor per anLmal. After the human tumor ti~sue had grown in the mice ~day 7-14), 5 animals in each of ~he group~
a,b,c xeceived 5 x 500 ~g of ~Ab BW 494/32-glucuxoni-dase conjugate, d received 5 x 500 ~g of ~Ab BW 494/32, e received S x 500 ~g of glucuronidase and f received 5 x 500 ~1 of PBS injected întravenollæly on S consecu~ive days.
2~2808~
On days 5, 6 and 7 after ~he end of the MAb BW 494~32-glucuronida~e, MAb B~ 494/32, glucuronidase or PBS
injection, the mice in groups a, d and e received one third of the maxLmum tolerable dose (MTD) of the glycosyl-etoposide in~ected 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 ~ignificantly from that in group f. Groups a, b and c exhibited distinct inhibition of tumor gr~wth, with the effects being most distinct in group a.
Comparable results were received with the MAbs 8W 431/26, BW 250/183 in ~he CoCa4 xenograft 6ystem and with the MAb BW 704 in the M21 xen~yraft system.
Example 10 a) Cleavaqe of 4'-0-alpha-D-qalactopyranosyl-etoPoside with alpha-galactosidase (from yreen coffee beans) 4'-0-~lpha-D galactopyranosyl-e~oposide was dis-solved in a concen~ration of 1 mg/ml in 20 mM sodium phosphate buffer, pH ~, and 0.3 U~ml of alpha-galactosidase (green coffee beans; Sigma Co.; 1 U z cleavage of 1 ~mol of p-nitrophenyl alpha-D-galacto-side per minute at pH 6.5 and 25 degrees) was added, and the mixture was incubated at 37 degrees. The breakdownof4'-0-alpha-D-galactopyranosyl-etoposide and the appearance of free etoposide ware investi-gated by HPLC. ~he half~ e was 15 minutes.
b) Cleavaqe of 4'-0-alpha-D-qalactopyranosyl-etopo~ide with alpha-galactosidase A_~from human pl2centa~
4'-0-Alpha-D-galactopyranosyl-etoposide w~s dis-solved in a concentration of 107 or 10.7 yg/ml in 20 mM sodium phosphate buffer, pH 5, and 0.36 U/ml of alpha-galactosidase A (i~olated from human 2 ~
placenta; 1 U = cleavage o~ 1 ~mol of 4-methumbelli-feryl alpha-D-galactoside per minute 3t pH 5 and 37 degrees) was added, and ~he mixture was incubated at 37 degrees. The breakdown of 4'-0-~lpha-D-galacto-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 etoposide6 General procedure:
Molecular sieves (1.4 g), ~ilver carbonate (0.857 g) and silver perchlorate were added at -20C to a solution of 2~',3''-di-0-chloroacetyl-etoposide (O.67 mm~l) and glycosyl halide (bromide or chloride, 1.2 mmol) in dichloromethane (50 ml)~ and the reaction mixture was 6tirred with exclusion of light for 60 h. Dichloromethane (50 ml) was added to the mixture, which was then fil-tered. The filtrate was washed with water and dried over magnesium sulfate> The residue wa~ chromatographed, resulting in the alpha- and ~-0-glycosidically linked compounds.
2'',3''-Di-0-chloroacetyl-4'-0-(2,3,4,6~tetra-0-ben~yl-alpha-D-galactopyranosyl)-etoposide The title compound was prepared starting from 2,3,4,6-tetra-0-benzyl-alpha-D-galactopyranosyl chloride (or bromide) and 2'',3''-di-0-chloroacetyl-etoposide by the abovementioned procedure.
Alpha-glycoside ~alpha]D +11.8 (c = 1, chloroform).
2'',3''-Di-0-chloroacetyl-4'-0-(~,3,4,6-~etra-0-benzyl-~-D-yalactopyranosyl)-etoposide The ti~le compound was prepared ~tartin~ from 2~3,4,6-tetra-0-benzyl-alpha-D-galactopyranosyl bromide and 2~,3''-di-0-chloroacetyl-etoposide by the abovementioned procedure.
~-Glyco~ide [alpha]D -39.8 (c - 1, chloroform3.
2a2~0~
2'~3''-Di-O-chloroacetyl-~'_0-~2~3~4l6-te~ra-o-ben alpha-D-glucopyranosyl~-etoposide The title compound was prepared starting from 2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl chloride (or S bromide~ and 2'',3''-di-O-chloroacetyl-etoposide by the abovementioned procedure.
Alpha-glycoside [alpha3D +16.2 (c = 1, chloroform).
2''~3''-Di-O-chloroacetyl-4'-O-~2~3,4~6-te~ra-0-benzyl-~-D-glucopyranosyl)-etopofiide The ti~le compound was prepared starting from 2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl bromide and 2'',3''-di-O-chloroacetyl-etoposide ~y the abovementioned proceduxe.
~-Glycoside ~alpha]D -44.5 (c = 1, chloroform).
lS 2'',3''-Di-O-chloroacetyl-4'-0-(2,3,4,6-tetra-O-benzyl-~-D-glucuronyl)-etoposide The title compound was prepared starting from benzyl 2,3,4-tri-O-benzyl-1-chloro (or -bromo)-1-deoxy-alpha-D-glucupyranuronateand2//,3''-di-O-chloroacety~-etoposide by the abovementioned procedure.
~-Glycoside lalpha] 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 glyco~yl-etoposide (O.75 mmol~ and Dowex 1 x 8 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 wa~hed with phosphate buffer (pH 6), dried ~odium sulfate) and evaporated in vacuo.
The residue was purified by column chromatography. The following compounds were prepared:
4'~0-(2,3,4,6-Tetra-O-benzyl-alpha-D-galactopyranosyl)-2 ~
etopo~ide alpha-Glycoside talpha]D f6.0 (C = 1, chloroform) 4'-0-(2,3,4,6-Tetra-O benzyl-~-D-gala~topyranosyl)-etoposide 4'-0-(2,3,4,6-Tetra-O-benzyl-alpha-D-glucopyrano~yl)-etoposide alpha-Glycoside [alpha~D +14.9 (c = 1, chloroform) 14~-0-(2,3,4,6-Tetra-O-benzyl-~-D-glucopyranosyl)-etoposide ~-Glycoside ~alpha~D -53~ (c = 1~ ~hloroform) 4'-O-(2,3,4,6-Tetra-O-benzyl-~-D-glucuronyl)-etoposide.
Elimination of benzyl protective groups in glycosyl-etoposides 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 cataly~t was filtered off and then the filtrate was evaporated in vacuo at about O~C. The residue was purified by column chromatography on silica gel, resulting in the ~ollowlng glycosyl-etoposides:
4'-O-(alpha-D-Galactopyrano6yl)-~toposide alpha-Glycoside [alpha]D ~6.2 (c = 1, chloroform) 4'-O-~-D GalactQpyrano~yl)-etoposide ~-Glyroside [alpha~D -73.1 (c = 1, chloroform) 4~-O-(alpha-D-Glucopyranosyl)-etopo~ide 4~-O-(~-D-Gtucopyranosyl~-e~oposide ~-Glycoside [alpha]D -76,6 (c = 0.9, chloroform) 4~-O-(~-D-Glucuronyl)-etoposide.
~ he invention relates ~o 4~-0-glycosyl-etoposides of the formula I
R' ~ o-~~
0~
R' < ~0 ~41~ I
R ~
~eO ~ o~e R'O- ~ ~ O
R6 R~
in which R1 is a methyl, benzyl or 2-thienyl ~roup, R2 i~ a hydrogen atom, an acyl or tri-C1-C4-alkylsilyl protective group, R3 is a hydroxyl group, an acyl or tri-Cl-C4-alkyl~ilyl protective qroup which is bonded via an oxYaen atcm, an amino, acetyl~,~no, benzYloxYcarbonYlam m o or dImethylam mo qroup, R4 is a hydrogen atom or a methyl group, R5 is a hydrogen atom, a hydroxyl group, an acyl or tri-Cl-C~-alkylsilyl protective group which i~ bonded via an o~ygen atom, or an amino, benzyloxycarbonyl-amino, azido or acetylamino group, R~ is a hydroxyl group, an acyl or tri Cl-C4~alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzylo~ycarbonylamino or azido group, R7 is a hydrogen a~om, an acyl ox tri-Cl-C4-alkylsilyl ~2~
protective group and R~ is a methyl or hydroxymethyl group or an acyl protective group which i~ 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 i~vention 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-associa~ed antigen, or is a biomolecule which accumulates in a tumor, such as E&F (epidermal growth factor), TGF-alpha (transforming growth factor alpha), PD~F
(platelet derived growth factor), IGF I+II (insulin like growth factor I+II) or a+b FGF (acidic + basic fibroblast growth factor) 20 E is a glycosidase which i8 not Lmmunogenic or is of low immunogenicity, preferably mammalian glycosidase, as alpha- or beta-glucosidase, alpha-galactosidase, alpha- or beta-mannosidase, alpha-fucosidase, N-acetyl-alpha-galactosaminidase, N-acetyl-beta-/N-acetyl-alpha-glucosaminidaseorbeta-glucuronidase, Sp (spacer) i6 ~ bifunctional sulfide- or disulfide-containing group of the formula III or IV
X(S)nY III ~(S)~ IV
or a polypeptide spacer, in which X or Y is -CO-R9-(N-succinLmido)- or -C(=Rl~-CH2-CH2-with R9 being -CH2-CH2 , 1,4-cyclohexylideneg 1,3- or 1,4-phenylene or methoxycarbonyl- or chloro-1,4-phenylene and Rl being O or NH, and furthermore 2~280~
Y is -C(=Rl)-CH2-CH2-, where R10 has the stated meaning, and n is 1 or 2.
The fusion gene composed o V~, C~l hinge and enzyme gene is cloned into an expression plasmid which i6 suitable for expression in eukaryotic cell~ and carries a selec-tion marker. The expression plasmid with the fu~ion 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 ~uitable antibiotic is followed by identification of transfectoma clones which contain the expression plas-mids. Suitable detection methods (BioDot, ELISA) sre used to identify those transfectoma clones which secrete the fusion protein of the formula II composed of antibody ~nd enzyme.
Preferred within the scope of the in~ention are compounds of the formula I in which the radicalæ
R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, an acetyl or chloroacetyl group or a tri-Cl-C4-alkylsilyl protecti~e group, R3 is a hydroxyl group, an acetyl, chloroacetyl or tri-C1-C4-alkylsilyl protective group which i8 bond~d via an oxygen atom, or an amino, acetylamino, benzyloxycarbonylamino or dimethylamino group, R4 is a hydrogen atom or a methyl group, Rs is a hydrogen atom, a hydroxyl group, or an acetyl, chloroacetyl or tri C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyloxycarbonylamino, azido or acetylamino group, R~ is a hydroxyl group, an ace~yl, chloroacetyl or tri-Cl-C4-alkylsilyl protective ~roup which i~ bonded via an oxygen atom, or an amino, benzyloxycarbonyl-amino or azido group, R7 is a hydrogen atom, an acetyl, chloxoacetyl or tri-Cl-C4-alkylsilyl protective group and R3 is a methyl, hydroxymethyl, acetyloxy or chloro-acetyloxymethyl group or a benzyloxycarbonyl group, as well as a functionalized tumor-specific enzyme of the formula II in which A is an antibody or fra~ment thereof, which ha~e specificity for a tumor-associated antigen, or is a biomolecule which accumulates on or in the tumor, such as EGF (epidexmal growth factor), TGF-alpha (transforming growth factor alpha), PDGF (platelet derived growth factor), IGF I~II (in~ulin like growth factor I~II), a+b FGF (acidic ~ basic fibro-blast 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, alpha-galactosidase, alpha- or beta-mannosidase, alpha-fucosidase, N-acetyl-alpha-galactosaminidase, N-acetyl-beta-/N-acetyl-alpha-glucosaminidaseorbeta-glucuronidase, Sp is a bifunctional disulfide-containing group of the formula III or IV or a polypeptide spacer, in which X or Y is -C0-R9-(N-succinLmido)- or -C(=Rl)-CHz-CH2-with R9 being -CH2-CH2- or 1,4-phenylene and R10 being 0 or NH, Y is -C(=R1)-CH2-CH2-, where R10 has the stated meaning, and n is 1 or 2.
The proce~ according to the invention ~or preparing a compound of the formula I, which can be degraded by glycosidase, in which R~ is a methyl, benzyl or 2~thienyl gro~p, R2 is a hydrogen atom, R3 is a hydroxyl, amino or dLmethylamino ~roup, R4 is a hydrogen atom or a methyl group, R5 is a hydrogen atom, a hydroxyl group, an amino or acetylamino group, R5 is a hydroxyl group or an amino group, 20280~
R7 is a hydrogen atom, Ra 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 ~n a~yl protective group mean~ 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 ~olvent at -50C to 60C, an etoposide compound of the formula V
Rl_~R' <o~ V
~e~ ~ o~e in which Rl is a methyl, benzyl or 2-thienyl group, 5 R2 is a hydrogen atom, an acyl or a tri-Cl-C4-alkylsilyl protective group, R3 is a hydroxyl group, an acyl or tri-C,-C4-alkyl~ilyl protective group which is bonded via oxygen, or acetylamino, benzyloxycarbonylamino or dimethylamino group, and ~4 i~ a hydrogen a~om or a methyl group, with a carbohydrate component of the formula VI
R ~ .
R 0 ~ Z VI
in which 25 R5 is a hydrogen atom, a hydroxyl group, an acyl protective group which is bonded via an o~ygen atom, or benzylo~ycarbonylamino, azido vr acetylamino 2 ~
group, Rfi i5 an acyl protective group which i~ bonded via an oxygen atom, or a bsnzyloxycarbonylamino 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 a~om, preferably fluorine, chlorine or bromine, a hydroxyl group, a tri-Cl-C4-alkylsilyloxy group, or an acyl protective group which iB bonded ~ia an oxyqen atom, where the acyl protective group is an acetyl, monv-, di- or trihalogeno-acetyl group~ preferably with the halogen atom being fluorine or chlorine, to give a 4'-0-glyco~yl-etoposide derivative of the fonmula 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 hydroly is, 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 ~pecific procedure for this is as follows:
the glycosidation of etoposide derivatives of the formula V is carried out using ~unctionali~ed carbohydrate unit~
of the fo.rmula VI which are typically protected with acyl protective group~ on the 0-2, 0-3, 0-4 and, where appro-priate, 0-6 atoms. Preferred acyl protective group~ are acetyl, chloroacetyl or trifluoroacetyl groups. In the case of amino sugars, the amin~ group i 8 protected temporarily with a benzyloxycarbonyl group or permanently with an acetyl group. It is likewise possibl* to use ~zido sugars because they can be converted straight-forwardly into amino sugar~ by hydrogenolysis. The carbohydrate component must be suitably functionalized at the anomeric center. Used for thi6 purpose are ~lyco-syl halides, such as fluorides, chlorides or bromides, which can be prepared starting from l-0-acyl derivatives, 2~2~0~i g for example using HF, HCl, HBr or Ti~r4. The glyco~idation components which carry an O-acyl group or a hydroxyl group on the anomeric center are prepared by proce~se~
customary in carbohydrate chemistry.
The glycosidation of etoposides of the formula V with carbohydrate units of the formula VI i~ carried out in the presence of a promoter. ~he promoter used when glycosyl fluorides and the l-hydroxy or l-acetyloxy analogs thereof are employed is BF3 x ether or tri-Cl-C4-alkylsilyl trifluoromethanesulfonate. The promoters usedin the case of glycosyl chlorides or bromide~ are ~alts of silver or of mercury.
The glycosidation is carried out in an aprotic organic solvent such as acetonel 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 agent6 such as molecular ~ieves or magnesium sulfa~e are added. The reaction temperature is in the range from -50C to 0C
when glycosyl fluorides and the 1-hydroxy analog~ are employed and ~t 0C to 60C when glyco6yl chlorides or bromides are employed. ~he glycosyl etoposide~ produced in the reaction are deblocked by the following processe6:
the acyl protective groupR are removed by methanolysi~
catalyzed by zinc(II) salts or with alkaline ion ex-changers 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 earbon or palladium/
barium sulfate or, in the case of the azido group, con-verted into amino group. The compounds of the formula I
containing ~mino sugar~ can addi~ionally be con~erted into dLmethylamino derivative~ ~y reductive ~l~ylation with formaldehyde~sodium cyanoborohydride.
To prepare A~Sp-E, either the ~pacer (Sp) can be linked via an amino gxoup to an enzyme and to the antibody or lo 2~2,~$
the biomolecule via an HS group which has been introducad or generated by cleavage of the di6ulfide linkage, or nucleic a~id ~equences which code for the parts A, Sp and E are covalently linked with the aid of molecular bio-logical methods to result in a fusion gene, and A~Sp-E is prepared by genetic engineering proce~se~.
This can be carried out in a variety of ways:
A) A restriction cleavage ~ite A is introduced by ~pecific mutagene is a~ the 3~ end of the C~1 exon in the gene of the heavy chain of ~he immunoglobu-lin. The ~ame restriction cleavage site A is genera-ted at the 5' end of the oligonucleotide which codes for the oligopeptide which acts as ~pacer. ~oth restriction cleavage sites A are sited in such a way that the immunoglobulin gene can be linked to the oligonucleotide via the restriction cleavage ~ite A
without disturbing the reading frame.
A restriction cleavage si~e 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 se~uence coding for the mature protein starts. The enzyme gene is then linked via the restriction cleavage site ~ to the immunoglobulin gene-linked construct. ~he restriction cleavage sites B
are sited such that the reading frame i~ not di~turbed on linkage. The ~usion gene composed of the ~or the hsavy chains of the immunoglobulin V~ and C~1 linker enzyme iE
cloned into an expression plasmid which is suitable for expression in the eukaryotic cells and carries a sel c-tion marker.
The expression plasmid with the fusion gene i~ trans-fected together with an expression plasmid which carries the gene for the light chain belonging to the antibody into eukaryo~ic cells (for example myeloma cells~.
Selection wi~h suitable antibiotics is carried out to isolate cell clones which contain the plasmids with the 11- 2~2~
fusion gene and the gene for ~he light chains (transfectomas). Suitable detection methods (BioDot;
ELI5A) are used to identify those transfectomas 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 ~ is introduced ~ the 3' end of the hinge exon of the gene for the heavy chains of the immunoglobulin. ~he re~triction cleavage site ~ is introduced at ~he ~ite 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 V~, CB1 and hinge exons is linked via the restriction cleavage ~ite 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 V8, C~l hinge and enzyme gene is cloned into an expression pla~mid which is ~uitable for expression in eukaryotic cells and carries a ~elec-tion marker. The expression plasmid with the fusion gene is transfected together with an expression plasmid which contains the light-chain gene belonging to ~he antibody into eukaryotic expression cells. Selection with a suitable antibiotic iæ followed by identification of transfectoma clones which contain the expres~ion plasmids. Suitable detection methods (Bio~ot, ELISA) are used to identify those transfectoma clones which ~ecrete the fusion protein of the formula II composed of antibody and ~nzyme.
.
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. ~hose, I. Immunolog. Methods 59 (1983) 129-143; ToI~ Ghose et 3~
al. Methods in enzymology, Vol. 93 ~1983) 280-333~.
This entails ini~ial functionalization of the enzyme via its amino group using succinLmidyl N-maleLmido-alkylidene~, cycloalkylidene- or arylene-l-carboxylate, where the double bond of ~he maleimldo group enters into a reaction with the HS group of the functionalized antibody, fxagment thereof or the biomolecules, with the formation of a thioether func~ionality.
It is possible to use ~or the preparation of the antibody-enzyme con~ugates the monoclonal antibodies described in EP-A-0141079, preferably th~ antibodies 431J26, 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 immunohistochemi~try.
The nucleotide sequence of the V genes of these mono-clonal antibodies is described in German Patent Applica-tion DE-A-39Q9799.4.
To prepare the tumor-specific enzyme conjugates, it i8 possible for the enæymes which are mentioned hereinafter and from the identified source to be purified by the indicated literature procedureo - alpha-galactosidase from human liver, Dean, K.G. and Sweeley, C.C. (1979), J. Biol. Chem. 254, 994-1000 25 _ beta-glucuronida~e from human liver, ~o, 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 livert GrabDwski, G.A., Ikonne, J.U., Desnick, R.J. (19803 Enzyme 25, 13-2S
- beta-mannosidase from human placenta, Noeske, C., Mersmann, G. (1983) ~oppe Seylers Z Physiol. Chem.
364, 1645-1651 alpha-~lucosidase from human gastro~ntestinal mucosa, Asp, N.-G., Gudmand-Hoeyer, E., - 13 _ 2 ~2~r3~ ~
Christiansen, P~., Dahlquist, A. (1974) Scsnd. J.
Clin. Lab. Invest. 33, 239-245 - beta-glucosidase fr~m human liver, ~aniels, L.B., Coyle, P.J., Chiao, Y.-B., Glew, R.H. (1981) J.
S Biol. Chem. 256, 13004-13013 - beta-glucocerebrosidaæe from human placenta, Fu rbish, F.S., Blair, H.E., Shiloach, J.,Pentcheu, P~G., Brady, R.O. (1977) Pro~O Natl.
Acad. Sci. ~SA 74, 3560-3563 10 - alpha-N-acetylglucosaminidase from human placenta, Roehrborn, W., von Figura, R. (1~78) Hoppe Saylers Z Physiol. Chem. 359, 1353-1362 - beta-N-acetylglucos~minidase from human amniotic membrane, Orlacchio, A., Emiliani, C., Di Renzo, G.C., Cosmi, E.V. (1986~ Clin. Chim. Aota 159, 279-289 - alpha-N-acetylgalacto~minidase according to Salvayre, R., Negre, A., Maret, A., Douste-Blazy, L.
(1984) Pathol. Biol. (Paris) 32, 269-284.
The glycolytic activity of the functionalized tumor-specific enzymes wa~ determined in comparative investi-gations with p-nitrophenyl glycosides at the particular pH optimum.
The invention additionally relates to a pack contsining a glycosyletoposide according to the invention and a functionalized tumor-specific enzyme con~ugate in com-bination with functionalized tumor-6pecific enzyme con~ugates.
To test the efficacy of a combined ~equential 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 s~opped and regression occurred.
2 ~ 2 ~
~xample 1 Preparation of the glycosylation component Benzyl D-qlucuronate (compound l) Benzyl bromide (4.89 g, 28.59 mmol) was added to a solution of sodium D-glucuronate (5 g, 23.13 mmol) in DMF
(300 ml). The reaction mixture was stirred at 40C 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 l3C NMR
Benzvl 1,2,3,4-tetra-O-chloroacetvl-alpha and beta-D-lucuronate ~compound 2a and 2b) Benzyl D-glucuronate (3.80 g, 13.36 mmol) was suspended in dichloromethane (200 ml). Chloroacetyl chloxide (7.90 g, 69.94 mmol) was added and then the mixture was cooled to -30C, and pyridine (4.33 g, 54.74 mmol) dissolved in dichloromethane (50 ml) was added. The ~0 reaction mixture was stirred at -30C 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 ~odium 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 2,3,4-tri-O-chloroacetyl-alpha, beta-D-glucuronate besides the title compound, was used without further purification steps in the next stage.
Benzy~12,3,4-tri-O-chloroacetyl-alpha,beta-D-alucuronate (compound 3a, 3bl The crude product ~7.92 g) of compounds 2a/2b was dis-solved in 3:1 methanol/chloroform (320 ml), and aminated silica gel (11.09 g) was added. The reaction mixture wa~
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 petro-2~2~
leum ether/ethyl ac~tate.
Yield: 4.94 g (72% based on compounds 2a/2b).
Benzyl 1 deoxy-l-fluoro-alpha-D-qlucuronate (comPound 4) Sodium l-deoxy-l-fluoro-alpha~D-glucuronate (6.22 g, 28.51 mmol) was suspended in D~F ~350 ml), and benzyl bromide (4.89 g, 28.59 mmolJ was added. The reaction mixture was stirred at 60C ~or 24 h and then evaporated.
The residue wa~ dissolved in 3:1 chloroform/methanol, and magnesium sulfate (12 g) was added. ~he ~uspen~ion was ~tirred for 2 h and then filtered, and the filtrate was evaporated. The residue was purified by column chromato-graphy on 6ilica gel ~160 g) with 4:1 dichloromethane~
acetone.
Yield: 5.95 g (73~).
Benzyl 1-deoxy-l-fluoro-2,3,4-tri-O-alpha-D-qlucuronate ~compound 5) Compound 4 (5.0 g, 17.46 mmol) was suspended in dichloro-methane (120 ml) and, at 0C, benzyl bromide (9.85 g, 57.61 mmol) and silver oxide 111.2 g) were added. The reaction mixture was stirred at O~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 ~ilica gel (240 g) with 3:1 petrol~um ether/ethyl acetate.
Yield: 6.60 g ~68%).
Benzyl l-deoxy-l-fluoro-2,3,4-tri-O-chloroacetyl-alpha-D-alucuronate (compound 6) Compound 4 (5.0 g, 17.46 mmol) was ~uspended in dichloro-methane (260 ml) and, at o30QC, chloroacetyl chloride (g.86 g, 87.30 mmol) was added. After addition of lOsl dichloromethane/pyTidine (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 ~hen wi~h water. The organic phase was dried over ~odium sulfate and evaporated. The residue was purified by column ~28~
- ~6 -chromatography on silica gel ~260 g) with S:l petroleum ether/ethyl aceta~e.
Yield: 7.58 g (92~), 2,3,4,6-Tetra-0-chloroacetyl-alpha-D-~alactopyranosyl fluoride (compound_7) ~lpha-D-galactopyranosyl fluoride (2.30 g, 12.62 mmol) was dissolved in dry dichloromethane (lD0 ml~ and, at -25C, chloroacetyl chloride (9.0 g, 79.68 mmol) and lsl dichlorometha~e/triethylamine (55 ml) were added. The reaction mixture was stirred for 16 h and then chloro-acetyl chloride (9.0 g) and lsl dichloromethane/triethyl-amine (55 ml) were added. The reaction mixture was stirre~ for a further 24 h and then washed with sodium citrate buffer (pH 5.0, 5~ ml x 3) and then with water.
The organic phase was dried (sodium sulfate) and evapora-ted in vacuo. The residue was purified by column chroma-tography on silica gel l300 g) with 40:8:1 dichloro~
methane/petroleum ether/ethyl acetate.
Yield: 5.19 g (82%) [alpha]D +64.2 (c = 1, dichloromethane).
2,3,4,6-Tetra-0-benzyl-alpha-D-qalactopyranosyl fluoride (compound 8~
Alpha-D-~alactopyranosyl fluoride (2.30 g, 12.62 mmol) was dissolved in dry DMF (40 ml) and, at -20C, benzyl bromide (12.95 g, 75.72 mmol) and silver oxide (10 g) were added. The reaction mixture was stirred at ~20C for 5 h and then at room temperature for ~4 h. The ~alt~ 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%).
- 17 _ 2~2~
Example 2 Glycoside synthesis 4'-O-Demethyl-4-O ~2.3di-O-chloroacetyl-4,6,0-ethy-lidene-beta-D-~lucopyrano~yl~-4-epi-~odophyllotoxin (compound 9) 4'-O-Benzyloxycarbonyl~4-O-demethyl-4-O-(2,3-di-O-chloro-acetyl-4,6-0-ethylidene-beta-D-glucopyranosyl)-4-epi-podophyllotoxin (10 g, 11.42 mmol) wa~ hydrogenated in 2:1 ethyl acetate/methanol (200 ml) in the presence of 10% Pd/C (5.0 g) for 2 h. The reaction mixture was filtered, and the filtrate was evaporated in ~acuo. 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 201-203C; [alpha]D
-73.4 (c = 1, chloroform) Benzyl 4'-O-demethyl-4-O-(di-O-chloroacetyl-4~6-0-ethyli-dene-beta-D-qlucopyranosyl3-4-epi-4'-0-(2,3~4-tri-O-benzyl-beta-D-glucopyranosyl ! -uronate-podophyllotoxin (comPound 9~
Benzyl l-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 di-chlorome~hane ~220 ml), and 4 ~ molecular ~ieves (10 g) were added. BF3-ether (2.5 ml) was added at -40C to the reaction mixture, which was then stirred at -30C for 20 h. Triethylamine (7.0 ml) was added and then She mixture w~s filtered. The filtrate was washed with citrate buffe- (pH 5, 80 ml x 3) and water (120 ml x 3)r dried (sodium sulfate) and evaporated in vacuo. The residue was purified by column chromatography on ~ilica gel (360 g) with 5:5:1 dichloromethane/petroleum ether/
acetone.
Yield: Ç.49 g ~67%).
~2~
4'-0-Demethvl-4-O-(di-O-chloroacetyl-4,6-O-ekhylidene-beta-D-~lucopyranosyl)-4-epi-4'-0-(2,3,4,6-tetra-O-benzyl-alpha- and beta-D-~alactoPvranosyl)-pod lotoxin (compound lOa and lOb) 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 di~solved in dichloromethane (250 ml). 4 A molecular sieves (7.2 g) were added and then the reaction mixture was cooled to -40C, and 30~
strength BF3-ether (2.2 ml) was added dropwise. The mixture was stirred at -30C for 20 h and then, after addition of triethylamine (5.5 ml), filtered. The fil-~rate 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 chroma-tography on silica gel (370 g) with 5:5:1 petroleum ether~dichloromethane/acetone. Further separation by column chromatography provided the title compounds lOa (alpha-glycoside: 4.36 g (52~)) and lOb (beta-glycoside:
1.42 g (17%))-Example 3 Deblocking reaction for glycosyl etoposides Benzyl 4'-O demethyl-4-epi-4-O-t4,6-O-ethylidene-beta-D-qlucopyranosyl ! -4 ~ -o- ( 2,3,4-tri-O-benzyl-beta-D-gluco-pyranosyl)-uronate podophyllotoxin (compound 11 !
Glucuronide derivative (compound ~, 4.56 g, 3.57 mmol) was dissolved in 4:1 methanol/chloroform (120 ml), and DOWEX 1 x 8 (6.2 g) was added. The reaction mixture was stirred at room temperature for 3 h and then filtered.
Chloroform (lS0 ml) was added to the filtrate, which was then stirred with magnesium sulfate (10 g). ~he salt~
were filtered off and then the filtrate was evaporated in vacuo. The residue was purified by col~mn chromatography on 200 g of silica gel with -5:2:1 dichloromethane~
petroleum ether/acetone.
Yield: 3.29 g (B2%~.
2 0 2 8 0 ~ ~
4'-O-Vemethyl-4-epi-4-0-(4,6-O-ethylidene-beta-D-~luco-pyranosyl)-4'-O-(beta-D-alucopyranosyl)-uronic acid podophyllotoxin (compound 12) Deacylated glucuronidP derivative (compound 11, 3.62 g, 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 n vacuo. The residue was purified by column chromatography on RP-18 silica gel using methanol/hexane (gradient).
Yield: 1.82 g (74%).
4'-0-Demethyl-4-epi-4-0-(4.6-0-ethylidene-beta-D-~luco-pYranosyl)-4~-0-~2.3~4~6-tetra-O-benzyl-alpha-D-qalacto-pyranosyl)-podophyllotoxin (compound 13) Podophyllotoxin galactopyranoside (compound lOa, 3.0 g, 2.37 mmol) was dissolved in 3:1 methanol/chloroform, and Dowex 1 x 8 was added. The reaction mixture was stirred at room ~emperature for 3 h and filtered. The filtrate was evaporated in vacuo. The re~idue 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 wa5 purified by column chromatography on ~ilica gel (85 g) with 5:3:1 dichloromethane/petrole~m ether/acetone.
Yield: 2.27 g (86%).
4'-O-Demethyl-4-epi-4'-0-(alpha-D-qalactopyranosyl~-4-0-(4 ! 6-O-ethylidene-beta-D-alucopyranosyl~-podoph~llotoxin ~ comPound 14 !
Deacylated podophyllotoxin galactopyranoside (compound 13, 2.0 g, 1.80 ~mol) was dissol~ed in 3:1 methanol~ethyl acetate and hydrogenated in the prasence of 10% Pd/C
~2.5 g) for 24 h. The mixture was ~tirred 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%).
2~2~$~
Result:
Under the experLmental conditions it wa~ not po~sible to detect any enzyme activity for alpha-galactosidase and beta-~lucuronidase.
Example 5 Determination of ~he enzyme activity of beta-glucuroni-dase conjugates The beta-glucuronidase purified by the abovementioned procedure was coupled to ~he antibody/~he biomolecule, and the activity of the enzyme and of the conjugate was determined as follows:
500 ~1 of the enzyme ~olution to be determined were added to 500 ~1 of a 2.5 mM p-nitrophenyl beta-D-glucuronide solution in 100 mM HEPES ~N-2-hydroxyethyl-piperazine-N'-2-ethane-sulfonic acid), pH 5. The assay mixture was incubated at 37 and stopped after 6 min with 300 ~1 of a 0.4 M glycine solution, pH 10.8. The liberated p-nitrophenol was then determined by measuring the extinc-tion at 405 nm.
Result:
The conjugate showed an only inconsiderable raduc~ion in enzyme activity Example 9 In vivo antitumor effects of the glyco6yl-e~opo~ide prodrug system 30 NMRI nu/nu mice received on day 0 a ~ubcutaneous inoculation of pieces about 5 mm3 in size of CoCa 4 human tumor per anLmal. After the human tumor ti~sue had grown in the mice ~day 7-14), 5 animals in each of ~he group~
a,b,c xeceived 5 x 500 ~g of ~Ab BW 494/32-glucuxoni-dase conjugate, d received 5 x 500 ~g of ~Ab BW 494/32, e received S x 500 ~g of glucuronidase and f received 5 x 500 ~1 of PBS injected întravenollæly on S consecu~ive days.
2~2808~
On days 5, 6 and 7 after ~he end of the MAb BW 494~32-glucuronida~e, MAb B~ 494/32, glucuronidase or PBS
injection, the mice in groups a, d and e received one third of the maxLmum tolerable dose (MTD) of the glycosyl-etoposide in~ected 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 ~ignificantly from that in group f. Groups a, b and c exhibited distinct inhibition of tumor gr~wth, with the effects being most distinct in group a.
Comparable results were received with the MAbs 8W 431/26, BW 250/183 in ~he CoCa4 xenograft 6ystem and with the MAb BW 704 in the M21 xen~yraft system.
Example 10 a) Cleavaqe of 4'-0-alpha-D-qalactopyranosyl-etoPoside with alpha-galactosidase (from yreen coffee beans) 4'-0-~lpha-D galactopyranosyl-e~oposide was dis-solved in a concen~ration of 1 mg/ml in 20 mM sodium phosphate buffer, pH ~, and 0.3 U~ml of alpha-galactosidase (green coffee beans; Sigma Co.; 1 U z cleavage of 1 ~mol of p-nitrophenyl alpha-D-galacto-side per minute at pH 6.5 and 25 degrees) was added, and the mixture was incubated at 37 degrees. The breakdownof4'-0-alpha-D-galactopyranosyl-etoposide and the appearance of free etoposide ware investi-gated by HPLC. ~he half~ e was 15 minutes.
b) Cleavaqe of 4'-0-alpha-D-qalactopyranosyl-etopo~ide with alpha-galactosidase A_~from human pl2centa~
4'-0-Alpha-D-galactopyranosyl-etoposide w~s dis-solved in a concentration of 107 or 10.7 yg/ml in 20 mM sodium phosphate buffer, pH 5, and 0.36 U/ml of alpha-galactosidase A (i~olated from human 2 ~
placenta; 1 U = cleavage o~ 1 ~mol of 4-methumbelli-feryl alpha-D-galactoside per minute 3t pH 5 and 37 degrees) was added, and ~he mixture was incubated at 37 degrees. The breakdown of 4'-0-~lpha-D-galacto-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 etoposide6 General procedure:
Molecular sieves (1.4 g), ~ilver carbonate (0.857 g) and silver perchlorate were added at -20C to a solution of 2~',3''-di-0-chloroacetyl-etoposide (O.67 mm~l) and glycosyl halide (bromide or chloride, 1.2 mmol) in dichloromethane (50 ml)~ and the reaction mixture was 6tirred with exclusion of light for 60 h. Dichloromethane (50 ml) was added to the mixture, which was then fil-tered. The filtrate was washed with water and dried over magnesium sulfate> The residue wa~ chromatographed, resulting in the alpha- and ~-0-glycosidically linked compounds.
2'',3''-Di-0-chloroacetyl-4'-0-(2,3,4,6~tetra-0-ben~yl-alpha-D-galactopyranosyl)-etoposide The title compound was prepared starting from 2,3,4,6-tetra-0-benzyl-alpha-D-galactopyranosyl chloride (or bromide) and 2'',3''-di-0-chloroacetyl-etoposide by the abovementioned procedure.
Alpha-glycoside ~alpha]D +11.8 (c = 1, chloroform).
2'',3''-Di-0-chloroacetyl-4'-0-(~,3,4,6-~etra-0-benzyl-~-D-yalactopyranosyl)-etoposide The ti~le compound was prepared ~tartin~ from 2~3,4,6-tetra-0-benzyl-alpha-D-galactopyranosyl bromide and 2~,3''-di-0-chloroacetyl-etoposide by the abovementioned procedure.
~-Glyco~ide [alpha]D -39.8 (c - 1, chloroform3.
2a2~0~
2'~3''-Di-O-chloroacetyl-~'_0-~2~3~4l6-te~ra-o-ben alpha-D-glucopyranosyl~-etoposide The title compound was prepared starting from 2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl chloride (or S bromide~ and 2'',3''-di-O-chloroacetyl-etoposide by the abovementioned procedure.
Alpha-glycoside [alpha3D +16.2 (c = 1, chloroform).
2''~3''-Di-O-chloroacetyl-4'-O-~2~3,4~6-te~ra-0-benzyl-~-D-glucopyranosyl)-etopofiide The ti~le compound was prepared starting from 2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl bromide and 2'',3''-di-O-chloroacetyl-etoposide ~y the abovementioned proceduxe.
~-Glycoside ~alpha]D -44.5 (c = 1, chloroform).
lS 2'',3''-Di-O-chloroacetyl-4'-0-(2,3,4,6-tetra-O-benzyl-~-D-glucuronyl)-etoposide The title compound was prepared starting from benzyl 2,3,4-tri-O-benzyl-1-chloro (or -bromo)-1-deoxy-alpha-D-glucupyranuronateand2//,3''-di-O-chloroacety~-etoposide by the abovementioned procedure.
~-Glycoside lalpha] 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 glyco~yl-etoposide (O.75 mmol~ and Dowex 1 x 8 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 wa~hed with phosphate buffer (pH 6), dried ~odium sulfate) and evaporated in vacuo.
The residue was purified by column chromatography. The following compounds were prepared:
4'~0-(2,3,4,6-Tetra-O-benzyl-alpha-D-galactopyranosyl)-2 ~
etopo~ide alpha-Glycoside talpha]D f6.0 (C = 1, chloroform) 4'-0-(2,3,4,6-Tetra-O benzyl-~-D-gala~topyranosyl)-etoposide 4'-0-(2,3,4,6-Tetra-O-benzyl-alpha-D-glucopyrano~yl)-etoposide alpha-Glycoside [alpha~D +14.9 (c = 1, chloroform) 14~-0-(2,3,4,6-Tetra-O-benzyl-~-D-glucopyranosyl)-etoposide ~-Glycoside ~alpha~D -53~ (c = 1~ ~hloroform) 4'-O-(2,3,4,6-Tetra-O-benzyl-~-D-glucuronyl)-etoposide.
Elimination of benzyl protective groups in glycosyl-etoposides 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 cataly~t was filtered off and then the filtrate was evaporated in vacuo at about O~C. The residue was purified by column chromatography on silica gel, resulting in the ~ollowlng glycosyl-etoposides:
4'-O-(alpha-D-Galactopyrano6yl)-~toposide alpha-Glycoside [alpha]D ~6.2 (c = 1, chloroform) 4'-O-~-D GalactQpyrano~yl)-etoposide ~-Glyroside [alpha~D -73.1 (c = 1, chloroform) 4~-O-(alpha-D-Glucopyranosyl)-etopo~ide 4~-O-(~-D-Gtucopyranosyl~-e~oposide ~-Glycoside [alpha]D -76,6 (c = 0.9, chloroform) 4~-O-(~-D-Glucuronyl)-etoposide.
Claims (8)
1. A process for the preparation of a compound of the formula I
I
in which R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, R3 is a hydroxyl, amino or dimethylamino group, R4 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 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 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
V
in which R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, an acyl or a tri-C1-C4-alkyl-silyl protective group, R3 is a hydroxyl group, an acyl or tri-C1-C4-alkylsilyl protective group which is bonded via oxygen, or acatylamino, benzyloxycarbonylamino or dimethylamino group, and R4 is a hydrogen atom or a methyl group, with a carbohydrate component of the formula VI
VI
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 acetyl amino group, R6 is an acyl protective group which is bonded via an oxygen atom, ox 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-C1-C4-alkyl-silyloxy 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 R1 to R8 retain their meaning as defined above, and eliminating the protective groups present in these compounds by hydrogenolysis or hydrolysis, and, where appro-priate, converting by means of reductive alkylation one of the resulting compounds containing amino groups into another compound of the formula containing dimethylamino groups.
I
in which R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, R3 is a hydroxyl, amino or dimethylamino group, R4 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 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 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
V
in which R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, an acyl or a tri-C1-C4-alkyl-silyl protective group, R3 is a hydroxyl group, an acyl or tri-C1-C4-alkylsilyl protective group which is bonded via oxygen, or acatylamino, benzyloxycarbonylamino or dimethylamino group, and R4 is a hydrogen atom or a methyl group, with a carbohydrate component of the formula VI
VI
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 acetyl amino group, R6 is an acyl protective group which is bonded via an oxygen atom, ox 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-C1-C4-alkyl-silyloxy 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 R1 to R8 retain their meaning as defined above, and eliminating the protective groups present in these compounds by hydrogenolysis or hydrolysis, and, where appro-priate, converting by means of reductive alkylation one of the resulting compounds containing amino groups into another compound of the formula 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 R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, an acyl or tri-C1-C4-alkyl-silyl protective group, R3 is a hydroxyl group, an acyl or tri-C1-C4-alkyl-silyl protective group which is bonded via an oxygen atom, an amino, acetylamino, benzyloxycarbonylamino or dimethylamino group, R4 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, benzyl-oxycarbonylamino, azido or acetylamino group, R6 is a hydroxyl group, an acyl or tri-C1-C4-alkyl-silyl 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-alkyl-silyl protective group and R8 is a methyl or hydroxymethyl group or an acyl protective group which is bonded via a methylene-oxy 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 R1 is a methyl, benzyl or 2-thienyl group, R2 is a hydrogen atom, an acetyl or chloroacetyl group or a tri-C1-C4-alkylsilyl protective group, R3 is a hydroxyl group, an acetyl, chloroacetyl or tri-C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, acetyl-amino, benzyloxycarbonylamino or dimethlamino group, R4 is a hydrogen atom or a methyl group, R5 is a hydrogen atom, a hydroxyl group, or an acetyl, chloroacetyl 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 acetyl, chloroacetyl or tri-C1-C4-alkylsilyl protective group which is bonded via an oxygen atom, or an amino, benzyl-oxycarbonylamino or azido group, R7 is a hydrogen atom, an acetyl, chloroacetyl or tri-C1-C4-alkylsilyl protective group and R8 is a methyl, hydroxymethyl, acetyloxy or chloro-acetyloxymethyl 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.
A-Sp-E II.
6. 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.
7. A functionalized tumor-specific enzyme or geneti-cally engineered product of the formula II in claim 5 or 6, containing a glycosidase.
8. The process as claimed in claim 1 and substantially as described herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3935016.9 | 1989-10-20 | ||
| DE3935016A DE3935016A1 (en) | 1989-10-20 | 1989-10-20 | GLYCOSYL ETOPOSIDE PRODRUGS, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE IN COMBINATION WITH FUNCTIONALIZED TUMOR-SPECIFIC ENZYME CONJUGATES |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2028086A1 true CA2028086A1 (en) | 1991-04-21 |
Family
ID=6391872
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002028086A Abandoned CA2028086A1 (en) | 1989-10-20 | 1990-10-19 | Glycosyl-etoposide prodrugs, a process for the preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0423747B1 (en) |
| JP (1) | JP3293086B2 (en) |
| KR (1) | KR910007948A (en) |
| AT (1) | ATE168695T1 (en) |
| AU (1) | AU638064B2 (en) |
| CA (1) | CA2028086A1 (en) |
| DE (2) | DE3935016A1 (en) |
| DK (1) | DK0423747T3 (en) |
| ES (1) | ES2119738T3 (en) |
| GR (1) | GR3027569T3 (en) |
| IE (1) | IE903764A1 (en) |
| PT (1) | PT95620B (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5851527A (en) * | 1988-04-18 | 1998-12-22 | Immunomedics, Inc. | Method for antibody targeting of therapeutic agents |
| US7241595B2 (en) * | 1989-10-20 | 2007-07-10 | Sanofi-Aventis Pharma Deutschland Gmbh | Glycosyl-etoposide prodrugs, a process for preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates |
| DE68928946T2 (en) * | 1989-12-11 | 1999-10-21 | Immunomedics Inc | METHOD FOR DETECTING DIAGNOSTIC OR THERAPEUTIC AGENTS BY ANTIBODIES |
| FR2676058B1 (en) * | 1991-04-30 | 1994-02-25 | Hoechst Lab | GLYCOSYLATED PRODUCTS, THEIR PREPARATION PROCESS AND THEIR USE IN THE TREATMENT OF CANCERS. |
| IT1250692B (en) * | 1991-07-23 | 1995-04-21 | PROCEDURE FOR THE PREPARATION OF DEMETYLEPIPODOPHYLOTOXY-BETA-D-GLUCOSIDES. | |
| DE4236237A1 (en) * | 1992-10-27 | 1994-04-28 | Behringwerke Ag | Prodrugs, their preparation and use as medicines |
| DE19702988A1 (en) * | 1997-01-28 | 1998-07-30 | Hoechst Ag | Isoxazole and crotonic acid amide derivatives and their use as pharmaceuticals and diagnostics |
| US6713454B1 (en) | 1999-09-13 | 2004-03-30 | Nobex Corporation | Prodrugs of etoposide and etoposide analogs |
| WO2001049693A1 (en) * | 2000-01-03 | 2001-07-12 | Korea Research Institute Of Chemical Technology | 4-O-[2-(N,N-DIALKYLAMINO)-2-DEOXY-4,6-O,O-beta-D-GLUCOSYL]-4'-O-DEMETHYL-EPI-PODOPHYLLOTOXIN, DERIVATIVES, AND AN ANTICANCER COMPOSITION CONTAINING SAME |
| AP2003002831A0 (en) * | 2001-10-26 | 2003-09-30 | Centre Nat Rech Scient | Derivatives of etoposide and analogs, and pharmaceutical compositions containing them |
| DE60237915D1 (en) | 2001-12-03 | 2010-11-18 | Universitaetsklinikum Charite | Podophyllotoxine als antiproliferative mittel |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3329184A1 (en) * | 1983-08-12 | 1985-02-21 | Behringwerke Ag, 3550 Marburg | MONOCLONAL ANTIBODIES WITH SPECIFICITY FOR MEMBRANE-ASSOCIATED ANTIGENS |
| GB8705477D0 (en) * | 1987-03-09 | 1987-04-15 | Carlton Med Prod | Drug delivery systems |
| NZ225599A (en) * | 1987-08-04 | 1991-09-25 | Bristol Myers Co | Antibody-enzyme conjugates and combinations with prodrugs for the treatment of tumour cells |
| DE4002888A1 (en) * | 1990-02-01 | 1991-08-08 | Behringwerke Ag | ANTHRACYCLIN-GLYCOSYL PRODRUGS, METHOD FOR THE PRODUCTION THEREOF AND THEIR USE IN COMBINATION WITH FUNCTIONALIZED TUMOR-SPECIFIC ENZYME CONJUGATES |
-
1989
- 1989-10-20 DE DE3935016A patent/DE3935016A1/en not_active Withdrawn
-
1990
- 1990-10-17 DK DK90119886T patent/DK0423747T3/en active
- 1990-10-17 DE DE59010838T patent/DE59010838D1/en not_active Expired - Lifetime
- 1990-10-17 EP EP90119886A patent/EP0423747B1/en not_active Expired - Lifetime
- 1990-10-17 ES ES90119886T patent/ES2119738T3/en not_active Expired - Lifetime
- 1990-10-17 AT AT90119886T patent/ATE168695T1/en not_active IP Right Cessation
- 1990-10-18 PT PT95620A patent/PT95620B/en not_active IP Right Cessation
- 1990-10-19 AU AU64778/90A patent/AU638064B2/en not_active Ceased
- 1990-10-19 CA CA002028086A patent/CA2028086A1/en not_active Abandoned
- 1990-10-19 IE IE376490A patent/IE903764A1/en not_active IP Right Cessation
- 1990-10-19 JP JP27953590A patent/JP3293086B2/en not_active Expired - Lifetime
- 1990-10-20 KR KR1019900016761A patent/KR910007948A/en not_active Application Discontinuation
-
1998
- 1998-08-04 GR GR980401753T patent/GR3027569T3/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| IE903764A1 (en) | 1991-04-24 |
| JPH03135993A (en) | 1991-06-10 |
| AU638064B2 (en) | 1993-06-17 |
| PT95620B (en) | 1997-08-29 |
| JP3293086B2 (en) | 2002-06-17 |
| ES2119738T3 (en) | 1998-10-16 |
| AU6477890A (en) | 1991-04-26 |
| EP0423747B1 (en) | 1998-07-22 |
| EP0423747A2 (en) | 1991-04-24 |
| DE59010838D1 (en) | 1998-08-27 |
| PT95620A (en) | 1991-09-13 |
| DK0423747T3 (en) | 1999-04-26 |
| GR3027569T3 (en) | 1998-11-30 |
| DE3935016A1 (en) | 1991-04-25 |
| ATE168695T1 (en) | 1998-08-15 |
| KR910007948A (en) | 1991-05-30 |
| EP0423747A3 (en) | 1991-09-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100254113B1 (en) | Glycosylated prodrugs, method of preparation and use | |
| US6218519B1 (en) | Compounds and methods for the selective treatment of cancer and bacterial infections | |
| US5773435A (en) | Prodrugs for β-lactamase and uses thereof | |
| US7728117B2 (en) | Heterobifunctional pan-selectin inhibitors | |
| US20090036386A1 (en) | Heterobifunctional compounds for selectin inhibition | |
| CA2030564A1 (en) | Immunoconjugates and prodrugs and their use in association for drug delivery | |
| JPH05501726A (en) | Novel linker for bioactive agents | |
| CA2028086A1 (en) | Glycosyl-etoposide prodrugs, a process for the preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates | |
| IE910334A1 (en) | Glycosyl prodrugs of anthracyclines, a process for the¹preparation thereof and the use thereof in combination with¹functionalized tumor-specific enzyme conjugates | |
| AU649275B2 (en) | Prodrugs for beta-lactamase and uses thereof | |
| EP0454783B1 (en) | Site specific in-vivo activation of therapeutic drugs | |
| Leenders et al. | β-Glucuronyl carbamate based pro-moieties designed for prodrugs in ADEPT | |
| KR100249576B1 (en) | Fusion protein for activating prodrug | |
| WO2018090045A1 (en) | Antibody-drug conjugates | |
| US6475486B1 (en) | Glycosyl-etoposide prodrugs, a process for preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates | |
| US6610299B1 (en) | Glycosyl-etoposide prodrugs, a process for preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates | |
| US7241595B2 (en) | Glycosyl-etoposide prodrugs, a process for preparation thereof and the use thereof in combination with functionalized tumor-specific enzyme conjugates | |
| MICHEL et al. | THERAPY (ADEPT) AND PRODRUG MONOTHERAPY | |
| Michel et al. | Prodrugs of natural anthracycleves suited for antibody directed enzyme prodrug therapy (ADEPT) and prodrug monotherapy (PMT) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |