DE19640969A1 - 20-0 linked glycoconjugates from camptothecin - Google Patents

Abstract

The invention concerns camptothecin glycoconjugates in which at least one carbohydrate component is suitably linked to camptothecin via an oligopeptide bridge. The invention further concerns processes for preparing the compounds according to the invention and their use as medicaments, in particular in connection with cancers.

Description

The present invention relates to glycoconjugates of camptothecin in which at least one carbohydrate component in a suitable manner via an oligo peptide bridge is linked to camptothecin.

The invention further relates to methods for producing the invention Compounds and their use as medicines, especially together association with cancer.

20 (S) -camptothecin is a pentacyclic alkaloid developed by Wall et al. iso (J. Am. Chem. Soc. 88, 3888 (1966)). It has a high anti-tumor potential in numerous in vitro and in vivo tests. Unfortunately, however, failed the realization of the promising potential in the clinic at toxicity and Solubility problems.

One opened by opening the E-ring lactone and formation of the sodium salt water-soluble compound obtained in a pH-dependent equilibrium with the ring-closed form. Clinical studies have also been carried out here so far not to success.

About 20 years later, it was found that the biological activity decreased to one Enzyme inhibition of topoisomerase I is due. Since then the For research activities intensified again in order to be more tolerable and effective in vivo Find camptothecin derivatives.

Salts from A-ring and B-ring were used to improve water solubility modified camptothecin derivatives and 20-O-acyl derivatives with ioni described groups (Vishnuvajjala et al. US 4,943,579). The latter The prodrug concept was later also applied to modified camptothecin derivatives wear (Wani et al. WO 9602546). The described 20-O-acyl prodrugs have  however, a very short half-life in vivo and very quickly become Split basic body.

Our pending patent application PCT / 96/01279 describes that the attachment of carbohydrate residues via amino acid or dipeptide Spacers on cytostatics, including the 20-hydroxyl group from Campto thecin to antineoplastic, highly effective and more tumor-selective Leads.

Surprisingly, it has now been found that an extension of the Peptide spacers with suitable amino acids the water solubility of the concerned Conjugate is improved dramatically. At the same time, these conjugates show a very good biological activity.

In particular, the camptothecin conjugates according to the invention have, for example, the following favorable properties:

• - By the ester-like connection of the carrier residues to the 20-hydroxy group is the lactone ring of the Camptothecin derivatives stabilized.
• - The conjugates obtained in this way show a high activity towards in vitro Tumor cell lines and tumor xenografts.
• - They clearly show a comparison to the underlying toxophores higher tolerance and tumor selectivity.
• - In vivo they show excellent therapeutic efficacy over several Dose levels.
• - They are much more stable than that in extracellular medium and in blood previously described 20-O-acyl prodrugs of camptothecin.

The invention relates to compounds of the general formula (I)

wherein
M stands for a peptide with 3 to 7 amino acid units which chains via the α-amino group and / or via amino and / or hydroxyl groups of the sides with 1 to n 'identical or different groups

-L-Sp-K

is linked, where further functional groups of the peptide may also carry protective groups and
(-L-Sp-K) _{n stands} for n identical or different groups -L-Sp-K,
in which
n denotes a number from 1 to n 'and n' corresponds to the maximum number of possible connecting points of M,
L for a group of formulas

stands
wherein
R ^{1 represents} chlorine or hydroxyalkylamino,
Sp stands for arylene with up to 10 carbon atoms or for alkylene with up to 8 carbon atoms, which are each optionally substituted.

K for a radical of the formula (II)

stands in what
A denotes methyl, hydroxymethyl, alkoxymethyl with up to 6 carbon atoms, acyloxymethyl with up to 6 carbon atoms or a radical of the formula -CH ₂ -B, in which
B represents a radical of the formula (II),
R ² , R ³ and R ⁴ independently of one another are hydrogen, hydroxy, optionally substituted by hydroxy-substituted alkoxy having up to 6 carbon atoms, optionally substituted by alkyl or acyl having up to 6 carbon atoms, amino, halogen, sulfate or a group of the formulas

mean what
R ⁵ and R ⁶ independently of one another represent hydroxyl or alkoxy having up to 6 carbon atoms or amino, which is optionally substituted by alkyl having up to 6 carbon atoms, and
s and t can independently assume the values 0, 1, 2, 3 or 4, in particular the values 1, 2, 3 or 4
or
R ² , R ³ and R ⁴ independently of one another represent a radical of the formula (II)
or
two of the radicals R ² , R ³ , R ⁴ together represent an epoxy group,
and their isomers, mixtures of isomers and salts.

The blocks L are usually with their -NH function, if available linked to the building block Sp.

Unless otherwise stated, the term "alkyl groups" is intended to be used in the sense of Invention straight-chain, branched, cyclic and cycloalkyl radicals containing Include alkyl radicals. This definition should also apply to all other alkyls groups containing groups apply, such as alkoxy, acyl, etc.

Preferred compounds of the general formula (I) are those in which M is a peptide stands, which is linked to the radicals L via amino functions, via an acyl function is linked to the camptothecin residue and its amino acid structure stones can possibly carry protective groups. Are particularly preferred Tri, tetra and pentapeptides, especially tripeptides.

The amino acid units are preferably selected from the group glycyl, Alanyl, valyl, leucyl, lysyl, seryl, glutamyl, threonyl, asparagyl, isoleucyl, Diaminopropionyl, diaminobutyryl, histidyl and / or ornithyl.

The amino acid building blocks glycyl, alanyl, valyl, leucyl, Lysyl, Seryl, Asparagyl, Histidyl and / or Glutamyl.

Compounds of the general formula are also preferred
wherein
L for a group of the formula

stands,
and their isomers, isomer mixtures and salts.

Also preferred are compounds of the general formula (I), in which Sp is Arylene with up to 10 carbon atoms, which may be one or multiple times by hydroxy, carboxy, carboxyalkyl with up to 4 carbon atoms, Nitro, cyano, halogen, alkyl with up to 4 carbon atoms, haloalkyl with up to 4 carbon atoms or by alkoxy with up to 4 carbon atoms is substituted, as well as their isomers, isomer mixtures and salts.

Particularly preferred substituents of Sp are halogen, nitro, alkyl having up to 4 carbon atoms, alkoxy having up to 2 carbon atoms, -OCF ₃ and / or - CF ₃ .

Preference is furthermore given to compounds of the general formula (I) in which K represents a radical of the formula (II)
in which
A denotes methyl, hydroxymethyl, methoxymethyl or acetoxymethyl,
R ^{2 is} hydrogen, hydroxyl, methoxy or a group of the formulas

means what
s and t can independently assume the values 1 or 2 and
R ⁵ and R ⁶ independently of one another represent hydroxyl or alkoxy having up to 4 carbon atoms,
or
R ^{2 represents} a radical of the formula (II),
R ^{3 is} hydrogen hydroxyl, halogen, alkoxy with up to 4 carbon atoms, sulfate or a group of the formulas

means what
s and t can independently assume the values 1 or 2 and
R ⁵ and R ⁶ independently of one another represent hydroxyl or alkoxy having up to 4 carbon atoms or amino, which is optionally substituted by alkyl having up to 4 carbon atoms,
R ^{4 is} hydroxyl, alkoxy with up to 4 carbon atoms, which is optionally substituted by hydroxy, amino, which is optionally substituted by alkyl or acyl with up to 4 carbon atoms, or a group of the formulas

means what
s and t can independently assume the values 1 or 2 and
R ⁵ and R ⁶ independently of one another represent hydroxyl or alkoxy having up to 4 carbon atoms,
or in what
R ² and R ³ together represent an epoxy group,
and their isomers, mixtures of isomers and salts.

According to a particularly preferred embodiment, the carbohydrate building blocks comprise K a maximum of two monosaccharide building blocks.  

The compounds of the invention can be in stereoisomeric forms for example as enantiomers or diastereomers, or as mixtures thereof for example as a racemate. The invention relates to both the pure Stereoisomers as well as their mixtures.

Stereoisomer mixtures can, if necessary, in a known manner in the ste reoisomerically uniform constituents are separated, for example by chro matography or by crystallization processes.

The stereochemistry at the anomeric center of the carbohydrate building blocks K can be α or be β. Due to the stereochemistry at the other centers, the gluco-, manno, galacto, gulo, rhamno or fuco configuration.

Like the carbohydrate building blocks, the amino acid building blocks can each be in in the D or L form.

The camptothecin building block Cp can be in the 20 (R) or in the 20 (S) configuration or as a mixture of these two steroisomeric forms. The is preferred 20 (S) configuration.

The compounds according to the invention can also be present in the form of their salts. In general, here are salts with organic or inorganic bases or Acids and internal salts called.

The acids that can be added preferably include halogen water Substance acids, such as B. the hydrochloric acid and hydrobromic acid, ins especially hydrochloric acid, also phosphoric acid, nitric acid, Schwe rock acid, mono- and bifunctional carboxylic acids and hydroxycarboxylic acids, such as e.g. B. acetic acid, trifluoroacetic acid, maleic acid, malonic acid, oxalic acid, glucon acid, succinic acid, fumaric acid, tartaric acid, citric acid, salicylic acid, sorbine acid and lactic acid and sulfonic acids, such as. B. p-toluenesulfonic acid, 1,5-naphthalenedisulfonic acid or camphorsulfonic acid.

Physiologically acceptable salts can also be metal or ammonium salts of the compounds of the invention, which be a free carboxyl group to sit. Z are particularly preferred. B. sodium, potassium, or magnesium Calcium salts, as well as ammonium salts, which are derived from ammonia or  organic amines such as ethylamine, di- or triethylamine, di- or triethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine, lysine, Ethylenediamine or phenethylamine.

The glycoconjugates according to the invention can be produced, for example by linking camptothecin with activated carboxyl components that in turn, for example, parts of protected amino acids, peptides or carbohydrate-modified peptides.

The invention therefore further relates to a process for the preparation of compounds of the general formula (I), characterized in that compounds of the formula (III)

with an activated carboxyl component Ma corresponding to the radical M defined above, which optionally carries protective groups, in a suitable solvent, if appropriate in the presence of a base, by customary methods, optionally selectively cleaving off one, more or all of the protective groups of M by known methods and the product with compounds of the general formula (IV)
K-Sp-La (IV),

wherein
K and Sp are as defined above and La represents a reactive precursor of group L, where appropriate the protective groups are split off selectively and other groups of the formula K-Sp-L- can gradually be introduced in a comparable manner,
or that, in a comparable manner, a first amino acid residue in the form of the corresponding optionally protective groups carrying the activated carboxyl component is introduced, optionally splitting off protective groups, furthermore optionally linking protective amino groups either successively or as a peptide building block, optionally splitting off protective groups, as indicated above introduces the formula K-Sp-L and splits off protective groups if necessary.

The reactions can take place at different pressure and temperature conditions, for example 0.5 to 2 bar, or -30 to + 100 ° C, in suitable solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane, chloro form, lower alcohols, acetonitrile, dioxane, water or in mixtures of mentioned solvents are carried out. Usually reactions are in DMF or THF / dichloromethane at normal pressure and at a temperature of 0 to 60 ° C, especially at room temperature, preferred.

For the activation of the carboxyl groups come those in peptide chemistry knew coupling reagents such as z. B. in Jakubke / Jeschkeit: amino acids, Peptides, proteins; Verlag Chemie 1982 or Tetrahedr. Lett. 34, 6705 (1993) be are in question. For example, acid chlorides, N-carbon are preferred acid anhydrides or mixed anhydrides.

The formation of is also suitable for activating the carboxyl groups Adducts with carbodiimides e.g. B. N, N'-diethyl, N, N'-diisopropyl, N, N'-dicyclo hexylcarbodiimide, N- (3-dimethylaminopropyl) -N'-ethyl-carbodiimide hydrochloride, N-cyclohexyl-N '- (2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate, or Carbonyl compounds such as carbonyldiimidazole, or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium-3-sulfate or 2-tert-butyl-5-methyl-isoxa zolium perchlorate, or acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl- 1,2-dihydroquinoline, or propanephosphonic anhydride, or isobutylchloroform, or benzotriazolyloxy-tris (dimethylamino) phosphonium hexafluorophosphate, 1-hydroxybenzotriazole or N-hydroxysuccinimide.

Triethylamine, ethyldiisopropylamine, pyridine, N, N-dimethylaminopyridine or others can be used.

As protective groups for possible third-party functions of the amino acids, the in Protective groups known in peptide chemistry, for example from urethane, alkyl, Acyl, ester or amide type can be used.  

Amino protecting groups in the context of the invention are the usual ones in peptide chemistry amino protecting groups used.

These preferably include: benzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxy benzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 2-nitro- 4,5-dimethoxybenzyloxycarbonyl, methoxycarbonyl, ethoxycarbonyl, tert-butoxy carbonyl (Boc), allyloxycarbonyl, vinyloxycarbonyl, 3,4,5-trimethoxybenzyloxy carbonyl, 2,2,2-trichloroethoxycarbonyl, 2,2,2-trichloro-tert-butoxycarbonyl, Menthyloxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), formyl, acetyl, propionyl, pivaloyl, 2-chloroacetyl, 2-bromoacetyl, 2,2,2-trifluoroacetyl, 2,2,2-trichloroacetyl, benzoyl, benzyl, 4-chlorobenzoyl, 4-bromo benzoyl, 4-nitrobenzoyl, phthalimido, isovaleroyl or benzyloxymethylene, 4-nitrobenzyl, 2,4-dinitrobenzyl, 4-nitrophenyl or 2-nitrophenylsulfenyl. The Fmoc group and the Boc group are particularly preferred.

Preferred carboxyl protecting groups are linear or branched C ₁ -C ₄ alkyl esters.

Modification of amino acid or peptide conjugates of camptothecin Derivatives with carbohydrate residues can be made using various methods and linkers groups take place. Preferably z. B. the transfer of p-aminophenylglycosi the in isothiocyanates and the linkage, for example, with amino groups. Furthermore, carboxyalkyl or aminoalkyl glycosides can also easily be used Amino or carboxyl groups are coupled. The Her is also conceivable provision of N- or O-glycopeptides.

Protective groups can be split off in appropriate reaction steps for example by exposure to acid or base, hydrogenolytically or on otherwise done reductively.  

Biological testing 1. Growth inhibition test to determine the cytotoxic properties create

The human colon cell lines SW 480 and HT 29 (ATCC No. CCL 228 and HBT-38) and the mouse melanoma cell line B16F10 were grown in Roux dishes in RPMI 1640 medium with the addition of 10% FCS. It was then trypsinized and taken up in RPMI plus 10% FCS to a cell number of 50,000 cells / ml. 100 µl cell suspension / Weil were placed in a 96 microwave plate and incubated for 1 day at 37 ° C in the CO ₂ incubator. Then a further 100 µl RPMI medium and 1 µl DMSO with the test substances were added. The growth was checked after day 3 and day 6. For this purpose, 40 μl of MTT solution (3- (4,5-dimethyithiazol-2-yl) -2,5-diphenyltetrazoline bromide) with an initial concentration of 5 mg / ml H ₂ O was added to each microwave. It was incubated for 5 hours in a CO ₂ incubator at 37 ° C. The medium was then suctioned off and 100 μl of i-propanol / well were added. After shaking with 100 μl H ₂ O for 30 min, the absorbance at 540 nm was measured with a Titertek Multiskan MCC / 340 (Flow).

The cytotoxic effect is given in Table 1 as an IC ₅₀ value for the SW 480 and HT 29 and B16F10 cell lines:

Table 1

2. Hematopoietic activity of glycoconjugates compared to underlying active ingredient material and methods

Bone marrow cells are washed out of the mouse femur. 10 ⁵ cells are in McCoy 5A medium (0.3% agar) together with recombinant murine GM-CSF (Genzyme; stem cell colonization) and the substances (10 ^-4 to 100 µg / ml) at 37 ° C and 7% CO ₂ incubated. 7 days later, the colonies (<50 rows) and clusters (17-50 cells) are counted.

Results

As shown in Table 2, the glycoconjugates examined show ge a drastically reduced compared to the underlying active ingredient Inhibition of bone marrow stem cell proliferation.

Inhibition of CSF-Induced Bone Marrow Stem Cell Proliferation mouse

Inhibition of CSF-Induced Bone Marrow Stem Cell Proliferation mouse

3. In vivo inhibition of tumor growth in the nude mouse model material

For all in vivo experiments to investigate tumor inhibition athymic nude mice (NMRI nu / nu strain) used. The selected large cell lung carcinoma LXFL 529 was developed by serial passage in nude mice. The human  Origin of the tumor was determined by isoenzymatic and immunohisto chemical methods documented.

Experimental setup

The tumor was subcutaneously in both flanks from 6 to 8 weeks old implanted nu / nu nude mice. Treatment became dependent on that Doubling time, started as soon as the tumors had a diameter of 5-7 mm had reached. The mice were the treatment group and the Control group (5 mice per group with 8-10 evaluable tumors) allocated by randomization. The individual tumors of the control group all grew progressively.

The size of the tumors was measured in two dimensions using a slide gauge. The tumor volume, which correlates well with the cell number, was then used for all evaluations. The volume was calculated according to the formula "length × width × width / 2" ([a × b ² ] / 2, a and b stand for two diameters arranged at right angles).

The relative tumor volume (RTV) values were for each individual Tumor by dividing the tumor size on day X by the tumor size of day 0 (at the time of randomization). The middle ones RTV values were then used for further evaluation.

Inhibition of the increase in tumor volume (tumor volume of the test group / control group, T / C, in percent) was the final measurement.

treatment

The compounds were applied on days 1, 2 and 3 according to Rando mization intravenously (IV).

Results

The therapeutic effectiveness is based on the compounds 1.1 and 1.2 the glycoconjugates according to the invention compared to the large-celled huma A lung tumor xenograph LXFL 529 is shown with IV application. The Therapy leads to the maximum tolerable dose (MTD) and 1/2 MTD on tumor remissions or significant inhibitions of tumor growth.  

Table 3

The compounds of the invention exhibit both in vitro and in vivo a surprisingly strong cytotoxic activity against different Tu mors, especially those of the lungs and colon connected to one great selectivity towards non-malignant cells.

They are therefore suitable for the treatment of cancer, in particular of those of the lungs and colon.

The present invention includes pharmaceutical preparations which, in addition to non-toxic, inert pharmaceutically suitable excipients one or more contain more compounds according to the invention or those of one or more Active substances according to the invention exist, and methods for producing them Preparations.  

The active ingredient (s) may optionally be present in one or more of the above specified carrier substances are also present in microencapsulated form.

The therapeutically active compounds are said to be in the phar pharmaceutical preparations preferably in a concentration of about 0.1 to 99.5, preferably from about 0.5 to 95% by weight of the total mixture is present be.

The pharmaceutical preparations listed above can in addition to the inventions Compounds according to the invention also contain other active pharmaceutical ingredients.

In general it has been in both the human and the veterinary medicine has proven to be advantageous in the active ingredient (s) according to the invention Total amounts from about 0.5 to about 500, preferably 5 to 100 mg / kg body weight each 24 hours, if necessary in the form of several individual doses, to educate to deliver the desired results. A single dose contains the or the active compounds according to the invention preferably in amounts of about 1 to about 80, especially 3 to 30 mg / kg body weight.  

Examples Carbohydrate educts Example I.1 p-aminophenyl-3-O-methyl-β-L-fucopyranoside

I.1.a) p-Nitrophenyl-3-O-methyl-β-L-fucopyranoside

6 g (21 mmol) p-nitrophenyl-β-L-fucopyranoside in 300 ml absolute. Methanol who added with 7.84 g (31.5 mmol) of dibutyltin oxide and under reflux for 2 h heated. It is then concentrated, the residue is dried and then in 300 ml of DMF added. After adding 15.7 ml of methyl iodide Batch stirred at 70 ° C for 40 h. The solvent is removed in vacuo and the residue was taken up in 300 ml of dichloromethane. The suspension becomes fil tried, the remaining solution concentrated again and a flash chromatography (Dichloromethane / methanol 99: 1). After concentration, 3.82 g are obtained (61%) of the target product.

I.1) p-aminophenyl-3-O-methyl-β-L-fucopyranoside

3.81 g (12.73 mmol) of p-nitrophenyl-3-O-methyl-β-L-fucopyranoside are dissolved in methanol and, after adding platinum dioxide, hydrogenated in a hydrogen atmosphere at a slight excess pressure. After filtering off the catalyst and precipitating with ether, 3 g (88%) of the target product are obtained. [TLC: dichloromethane / methanol 9: 1 R _f = 0.53].

Example I.2 p-aminophenyl-3-O-carboxymethyl-β-L-fucopyranoside

I.2.a) p-Nitrophenyl-3-O-methoxycarbonylmethyl-β-L-fucopyranoside

• 1 g (3.5 mmol) of p-nitrophenyl-β-L-fucopyranoside and 1.3 g (5.2 mmol) of dibutyl Tin oxide are refluxed in 50 ml of methanol for 2 hours. The solution will be concentrated, the residue taken up in 50 ml of dioxane, with 2 ml of bromoacetic acid acid methyl ester and 100 mg of tetrabutylammonium iodide are added and the mixture is kept for 16 h Reflux heated. The solvent is evaporated and the product through Flash chromatography (dichloromethane / methanol 99: 1) cleaned. After constriction the corresponding fractions and cases from methanol / ether give 455 mg (37%) of the target connection.

I.2) p-aminophenyl-3-O-carboxymethyl-β-L-fucopyranoside

282 mg (0.79 mmol) of p-nitrophenyl-3-methoxycarbonylmethyl-β-L-fucopyranoside are dissolved in 20 ml of methanol and mixed with 440 ul of an aqueous 2N lithium hydroxide solution. After stirring for 2 h at room temperature, the pH is adjusted to 3 using acidic SC108 ion exchanger and the mixture is filtered. 250 mg of palladium on activated carbon are added to the filtrate. The mixture is then hydrogenated with hydrogen at a slight excess pressure for 1.5 h, the catalyst is separated off and washed with methanol. Concentration, absorption in water and freeze-drying lead to the target product (212 mg) in 86% yield. [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.24].

The following derivatives, for example, can be used as further carbohydrate building blocks:
p-aminophenyl-β-L-fucopyranoside
p-aminophenyl-2-O-methyl-β-L-fucopyranoside
p-aminophenyl-2-O-hydroxyethyl-β-L-fucopyranoside
p-aminophenyl-4-O-methyl-β-L-fucopyranoside
p-aminophenyl-3-O-methyl-α-L-fucopyranoside
p-aminophenyl-3-on-propyl-β-L-fucopyranoside
p-aminophenyl-3-deoxy-β-L-fucopyranoside
p-aminophenyl-3,4-dideoxy-β-L-fucopyranoside
p-aminophenyl-3,4-epoxy-β-L-fucopyranoside
p-aminophenyl-4-deoxy-β-L-fucopyranoside
p-aminophenyl-3-O-methoxycarbonylmethyl-β-L-fucopyranoside
p-aminophenyl-3-O-hydroxyethyl-β-L-fucopyranoside
p-aminophenyl-2-O-carboxymethyl-β-L-fucopyranoside
p-aminophenyl-3-O-succinyl-β-L-fucopyranoside
p-aminophenyl-3,4-di-O-methyl-β-L-fucopyranoside
p-aminophenyl-3-O-carbamoylmethyl-β-L-fucopyranoside
p-aminophenyl-α-L-rhamnopyranoside
p-aminophenyl-β-D-galactopyranoside
p-aminophenyl-2-O-methyl-β-D-galactopyranoside
p-aminophenyl-3-O-methyl-β-D-galactopyranoside
p-aminophenyl-4-O-methyl-β-D-galactopyranoside
p-aminophenyl-6-O-methyl-β-D-galactopyranoside
p-aminophenyl-2,3-di-O-methyl-β-D-galactopyranoside
p-aminophenyl-2,4-di-O-methyl-β-D-galactopyranoside
p-aminophenyl-2,6-di-O-methyl-β-D-galactopyranoside
p-aminophenyl-3,4-di-O-methyl-β-D-galactopyranoside, acetate
p-aminophenyl-3,6-di-O-methyl-β-D-galactopyranoside
p-aminophenyl-4,6-di-O-methyl-β-D-galactopyranoside
p-aminophenyl-2,3,4-tri-O-methyl-β-D-galactopyranoside
p-Alninophenyl-2,3,6-tri-O-methyl-β-D-galactopyranoside
p-aminophenyl-2,4,6-tri-O-methyl-β-D-galactopyranoside
p-aminophenyl-3,4,6-tri-O-methyl-β-D-galactopyranoside
p-aminophenyl-3-deoxy-β-D-galactopyranoside
p-Alninophenyl-3,4-dideoxy-β-D-galactopyranoside
p-aminophenyl-6-O-acetyl-β-D-galactopyranoside
p-aminophenyl-3-O-methoxy-carbonylmethyl-β-D-galactopyranoside
p-aminophenyl-3-O-carboxymethyl-β-D-galactopyranoside, sodium salt
p-aminophenyl-3-O-carbamoylmethyl-β-D-galactopyranoside
p-aminophenyl-3-O- (N-methyl-carbamoylmethyl) -β-D-galactopyranoside
p-aminophenyl-α-D-mannopyranoside
p-aminophenyl-3-O-methyl-α-D-mannopyranoside
p-aminophenyl-2,3-di-O-methyl-α-D-mannopyranoside
p-aminophenyl-3-O-methoxycarbonylmethyl-α-D-mannopyranoside
p-aminophenyl-3-O-carboxymethyl-α-D-mannopyranoside
p-aminophenyl-3-O-carbamoylmethyl-α-D-mannopyranoside
p-aminophenyl-4-O- (β-D-galactopyranosyl) -β-D-glucopyranoside
p-Aminophenyl-4-O- (3'-sulfate-β-D-galactopyranosyl) -β-D-glucopyranoside, sodium salt
p-aminophenyl-4-O- (3'-O-methyl-β-D-galactopyranosyl) -β-D-glucopyranoside
p-aminophenyl-2-O-methyl-4-O- (3'-O-methyl-β-D-galactopyranosyl) -β-D-gluco pyranoside
p-aminophenyl-4-O- (3'-4'-di-O-methyl-β-D-galactopyranosyl) -β-D-glucopyranoside.

The synthesis of the following carbohydrate building blocks was already described in EP 501 250:
Carboxymethyl-β-L-fucopyranoside
5-carboxypentyl-β-L-fucopyranoside
These two building blocks can be linked to peptide conjugates in the manner described in EP 501 250.

Glycoconjugates Example 1.1 20 (S) -20-O- {N ^α , N ^ε -bis- [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -lysyl-lysyl-alanyl } -camptothecin, hydrochloride

1.1.a) 20 (S) -20-O- [N- (tert-Butoxycarbonyl) alanyl] camptothecin

A solution of 5 g (14.4 mmol) 20 (S) -camptothecin in 200 ml absolute di methylformamide is stirred with 7.27 g (36 mmol) of N- (tert-butoxycarb onyl) alanine-N-carboxylic anhydride and 500 mg (12 mmol) of 4- (N, N-dimethyl) amino) pyridine added. After 3 hours of stirring at room temperature, more are added 6.18 g (28.8 mmol) of N- (tert-butoxycarbonyl) alanine-N-carboxylic acid anhydride and 500 mg (12 mmol) of 4- (N, N-dimethylamino) pyridine and leaves the batch over React at night in an ultrasonic bath at room temperature. Then in Va concentrated in vacuo and the residue by flash chromatography [petrol ether / ethyl acetate 1: 1]. 4.7 g of 63% of the target product are obtained.

1.1.b) 20 (S) -20-O-alanyl camptothecin, trifluoroacetate

A solution of compound 1.1.a (4.41 g, 8.5 mmol) in a mixture of 290 ml of dichloromethane and 50 ml of anhydrous trifluoroacetic acid is for 30 min. stirred at room temperature. After concentration in vacuo, the product is precipitated from dichloromethane / methanol with diethyl ether and washed thoroughly with diethyl ether. Yield: 4.2 g (93%); [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.33].

1.1.c) 20 (S) -20-O- [N ^α - (tert-Butoxycarbonyl) -N ^ε - (fluorenyl-9-methoxy carbonyl) -lysyl-alanyl] -camptothecin

3.688 g (7.87 mmol) of N ^α - (tert-butoxycarbonyl) -N- ^ε (fluorenyl-9-methoxycarbonyl) - lysine and 1.595 g of 1-hydroxy-1H-benzotriazole hydrate are dissolved in 250 ml of dimethylformamide. After adding 1.81 g of N-ethyl-N '- (dimethylaminopropyl) carbodiimide hydrochloride, the mixture is stirred for 30 min. at room temperature. A solution of compound 1.1.b (3.5 g, 6.56 mmol) in 35 ml of dimethylformamide and 2.25 ml of ethyldiisopropylamine is then added and the mixture is stirred for a further 2 h at room temperature. After concentration in vacuo and purification by flash chromatography [petroleum ether / ethyl acetate 2: 1 → ethyl acetate], 5.212 g (91%) of the target compound are obtained [TLC: acetonitrile R _f = 0.65].

1.1.d) 20 (S) -20-O- [N ^ε - (fluorenyl-9-methoxycarbonyl) lysyl-alanyl] -campto thecin, trifluoroacetate

A suspension of the above compound (5.21 g, 5.99 mmol) in dichloromethane (300 ml) is mixed with anhydrous trifluoroacetic acid (50 ml) and the resulting solution for 30 min. stirred at room temperature. After concentrating in vacuo, the product is redistilled with methanol and then precipitated from dichloromethane by adding diethyl ether. The precipitate is filtered off. (4.8 g, 90%) [TLC: acetonitrile / water 20: 1 R _f = 0.1]

1.1.e) 20 (S) -20-O - {[N ^α , N ^ε -bis-tert-butoxycarbonyl-lysyl] - [N ^ε - (fluorenyl-9-methoxycarbonyl) -lysyl] -alanyl} -camptothecin

141 mg (0.41 mmol) of N- ^α , N ^ε -bis-tert-butoxycarbonyl-lysine and 83 mg of 1-hydroxy-1H-benzotriazole hydrate are dissolved in 20 ml of dimethylformamide. After adding 94 mg of N-ethyl-N '- (dimethylaminopropyl) carbodiimide hydrochloride, the mixture is stirred for 1 h at room temperature. Then a solution of compound 1.1.d (300 mg, 0.34 mmol) in 3 ml of dimethylformamide and 58 μl of ethyl-diiso propylamine is added and the mixture is stirred for a further 30 min. at room temperature. After concentration in vacuo, the residue is digested with water and suction filtered. The product is taken up in DMF and precipitated with diethyl ether. 338 mg (91%) of the target compound are obtained [TLC: acetonitrile / water 10: 1 R _f = 0.65].

1.1.t) 20 (S) -20-O- {lysyl- [N ^ε - (fluorenyl-9-methoxycarbonyl) -lysyl] alanyl] camptothecin, bis-trifluoroacetate

At 336 mg (0.306 mmol) of compound 1.1.e with 5 ml of trifluoroacetic acid acid in 30 ml dichloromethane in analogy to example 1.1.d the Boc protection groups removed. Yield: 338 mg (98%).

1.1.g) 20 (S) -20-O- {N ^α , N ^ε -bis- [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxyphenylamino-thiocarbonyl] -lysyl-N ^ε - (fluorenyl-9-methoxycarbonyl) lysyl alanyl} camptothecin

A solution of 104 mg (0.368 mmol) of p-aminophenyl-3-O-methyl-β-L-fucopyra noside (Example 1.1) in dioxane / water 2: 1 (15 ml) is stirred with thio phosgene (59 µl, 0.77 mmol) was added. After 10 min. 3 equivalents of ethyl-diisopropylamine are added, the mixture is then concentrated in vacuo and the residue is dried in an oil pump vacuum for 1 h. The isothiocyanate obtained is dissolved in 50 ml of solute dimethylformamide and mixed with 337 g (0.3 mmol) of compound 1.1.f and 205 μl of ethyl-diisopropylamine. The mixture is stirred for 16 h at room temperature, then concentrated in vacuo and purified by flash chromatography [aceto nitrile / water 50: 1]. 247 mg (54%) of the target product are obtained. [TLC: aceto nitrile / water 10: 1 R _f = 0.46].

1.1) 20 (S) -20-O- {N ^α , N ^ε -bis- [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxyphenylamino-thiocarbonyl] -lysyl-lysyl-alanyl } -camptothecin, hydrochloride 245 mg (0.161 mmol) of compound 1.1.g are reacted with 0.5 ml piperidine in 10 ml DMF for 30 min at room temperature. The mixture is concentrated, the residue is digested with dichloromethane and the solvent is distilled off again in vacuo. The residue is taken up again in dichloromethane / methanol and the product is precipitated with diethyl ether. 185 mg (88%) of the amino-blocked compound are obtained. [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.24]. This is suspended in water and converted into the hydrochloride with one equivalent of an O, IN HCl solution. The target compound is obtained from the aqueous solution by freeze-drying.

Example 1.2 20 (S) -20-O- {N ^α , N ^ε -bis- [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxyphenylamino-thiocarbonyl] -lysyl-lysyl-valinyl} - camptothecin, hydrochloride

The synthesis of this compound is completely analogous to the synthesis of compound 1.1 described above. Only in the first reaction step, instead of N- (tert-butoxycarbonyl) -alanine-N-carboxylic anhydride, N- (tert-butoxycarb onyl) -valine-N-carboxylic anhydride is reacted with 20- (S) -camptothecin. R _{f of} the target compound: 0.25 [acetonitrile / water / glacial acetic acid 5: 1: 0.2].

Claims (11)

1. Compounds of formula (I)
wherein
M stands for a peptide with 3 to 7 amino acid units, which has the same or different groups via 1 to n 'via the α-amino group and / or via amino and / or hydroxyl groups of the side chains
-L-Sp-K
is linked, where further functional groups of the peptide can optionally carry protective groups and
(-L-Sp-K) _{n stands} for n identical or different groups -L-Sp-K,
in which
n denotes a number from 1 to n 'and n' corresponds to the maximum number of possible connecting points of M,
L for a group of formulas
stands
wherein
R ^{1 represents} chlorine or hydroxyalkylamino,
Sp stands for arylene with up to 10 carbon atoms or for alkylene with up to 8 carbon atoms, which are each optionally substituted. K for a radical of the formula (II)
stands in what
A is methyl, hydroxymethyl, alkoxymethyl with up to 6 carbon atoms, acyloxymethyl with up to 6 carbon atoms or a radical of the formula -CH ₂ -B, in which
B represents a radical of the formula (II),
R ² , R ³ and R ⁴ independently of one another are hydrogen, hydroxy, optionally substituted by hydroxy-substituted alkoxy having up to 6 carbon atoms, optionally substituted by alkyl or acyl having up to 6 carbon atoms, amino, halogen, sulfate or a group of the formulas
mean what
R ⁵ and R ⁶ independently of one another represent hydroxyl or alkoxy having up to 6 carbon atoms or amino, which is optionally substituted by alkyl having up to 6 carbon atoms, and
s and t can independently assume the values 0, 1, 2, 3 or 4
or
R ² , R ³ and R ⁴ independently of one another represent a radical of the formula (II)
or
two of the radicals R ² , R ³ , R ⁴ together represent an epoxy group, and their isomers, isomer mixtures and salts. 2. Compounds of the general formula (I) according to Claim 1, in which M represents a peptide which is linked to the residues L via amino functions, is linked to the camptothecin residue via an acyl function and whose amino acid units can optionally carry protective groups,
and their isomers, isomer mixtures and salts. 3. Compounds according to claim 2, characterized in that it is Tri, tetra or pentapeptides. 4. Compounds according to claim 2 or 3, characterized in that the Amino acid building blocks of the peptide are selected from the group best starting from glycyl, alanyl, valyl, leucyl, lysyl, seryl, glutamyl, threonyl, Asparagyl, isoleucyl, diaminopropionyl, diaminobutyryl, histidyl and / or Ornithyl. 5. Compounds of the general formula (I) according to claim 1,
wherein
L for a group of the formula
stands,
and their isomers, isomer mixtures and salts. 6. Compounds of the general formula (I) according to claim 1,
wherein
Sp stands for arylene with up to 10 carbon atoms, optionally one or more times by hydroxy, carboxy, carboxy alkyl with up to 4 carbon atoms, nitro, cyano, halogen, alkyl with up to 4 carbon atoms, haloalkyl with up to 4 carbons atoms or substituted by alkoxy with up to 4 carbon atoms,
and their isomers, mixtures of isomers and salts. 7. Compounds of the general formula (I) according to Claim 1, in which K represents a radical of the formula (II),
in which
A denotes methyl, hydroxymethyl, methoxymethyl or acetoxymethyl,
R ^{2 is} hydrogen, hydroxyl, methoxy or a group of the formulas
means what
s and t can independently assume the values 1 or 2 and
R ⁵ and R ⁶ independently of one another represent hydroxyl or alkoxy having up to 4 carbon atoms,
or
R ^{2 represents} a radical of the formula (II),
R ^{3 is} hydrogen, hydroxyl, halogen, alkoxy with up to 4 carbon atoms, sulfate or a group of the formulas
means what
s and t can independently assume the values 1 or 2 and
R ⁵ and R ⁶ independently of one another represent hydroxyl or alkoxy having up to 4 carbon atoms or represent amino which is optionally substituted by alkyl having up to 4 carbon atoms,
R ^{4 is} hydroxyl, alkoxy with up to 4 carbon atoms, which is optionally substituted by hydroxy, amino, which is optionally substituted by alkyl or acyl with up to 4 carbon atoms, or a group of the formulas
means what
s and t can independently assume the values 1 or 2 and
R ⁵ and R ⁶ independently of one another represent hydroxyl or alkoxy having up to 4 carbon atoms,
or in what
R ² and R ³ together represent an epoxy group,
and their isomers, mixtures of isomers and salts. 8. A process for the preparation of compounds of the general formula (I) according to claim 1, characterized in that compounds of the formula (III)
with an activated carboxyl component Ma corresponding to the radical M defined above, which optionally carries protective groups, in a suitable solvent, optionally in the presence of a base, by customary methods, optionally selectively cleaving off one, more or all of the protective groups of M by known methods and the product with compounds of the general formula (IV)
K-Sp-La (IV),
wherein
K and Sp are as defined above and La represents a reactive precursor of group L, where appropriate the protective groups are split off selectively and other groups of the formula K-Sp-L- can gradually be introduced in a comparable manner,
or that, in a comparable manner, a first amino acid residue in the form of the corresponding activated carboxyl component, optionally containing protective groups, is introduced in a comparable manner, if necessary splitting off protective groups, furthermore optionally attaching amino acid residues bearing protective groups either successively or as a peptide building block, optionally splitting off protective groups, as indicated above of the formula K-Sp-L, and if necessary splits off protective groups,
and that the stereoisomers are optionally separated by customary methods and the compounds are optionally converted into their salts. 9. Use of compounds of formula (I) according to claim 1 for the preparation provision of medicines. 10. Medicament containing one or more compounds of the formula (I) according to claim 1.