DE19813137A1 - New camptothecin glyco-conjugate compounds - Google Patents

Abstract

Camptothecin (CT) glyco-conjugates of formula (I), containing a 3-O-methylated beta -L-fucose residue bonded to the 20-hydroxy group via a thiourea-modified peptide spacer, and their salts, stereoisomers and stereoisomer mixtures, are new. R1 = sterically hindered (i.e. beta - or psi -branched) non-polar side-chain of an aminoacid; R2 = basic-side-chain of an aminoacid; and Q = a group of formula (a)

Description

The present invention relates to glycoconjugates of 20 (S) -camptothecin in which a 3-O-methylated β-L-fucose building block via thiourea-modified peptide spacer is linked to the 20-hydroxyl group of a camptothecin derivative. The The invention further relates to processes for producing the ver according to the invention bonds and their use as medicines, especially in combination hang out with cancer.

20 (S) -camptothecin is a pentacyclic alkaloid developed by Wall et al. isolated (J. Am. Chem. Soc. 88, 3888 (1966)). It has a high anti-tumor effect potential in numerous in vitro and in vivo tests. Unfortunately, the Reali failed Development of the promising potential in the clinic of 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 balance with the ring-closed form. Clinical studies have not yet been conducted here either to success.

About 20 years later it was found that the biological activity on an enzyme inhibition of topoisomerase I is due. Since then, the research activities intensified again in order to make camptothecin To find derivatives.  

Salts from A-ring and B-ring modified camptothecin derivatives and of 20-O-acyl derivatives with ionized described groups (Vishnuvajjala et al. US 4943579). The latter prodrug The concept was later transferred to modified camptothecin derivatives (Wahi et al. WO 9602546). However, the 20-O-acyl prodrugs described have in vivo a very short half-life and are split very quickly into the basic body.

In WO 9631532 A1 sugar-modified cytostatics have been described in which the linkage of various cytotoxic or cytostatically active compounds with z. B. regioselectively modified carbohydrate building blocks via certain spacers led to an improvement in tumor selectivity. From the broadly described combinations of carbohydrate, spacer and active ingredient, we found it surprising that the linking of 3-position-modified β-L-fucose units via a thiourea-modified peptide spacer, consisting of a sterically demanding non-polar and a basic Amino acid to the 20-hydroxyl group of 20 (S) -camptothecin leads to very particularly preferred conjugates with the following properties:

• - By the ester-like connection of the carrier residue to the 20-hydroxyl The lactone ring in the camptothecin part, which is important for the effect, becomes the group stabilized.
• - Due to the special constellation of the dipeptide spacers, the conjugates in extracellular medium and in blood a compared to similar, previously in WO 9631532 conjugates with other spacers described again significantly improved stability. In particular, the invention Conjugates more stable than the 20-O-acyl prodrugs described in US 4943579 from Camptothecin.
• - The conjugates according to the invention are compared to similar conjugates from WO 9631532 more water soluble.  
• - The conjugates according to the invention show a high activity against in vitro about tumor cell lines and tumor xenografts.
• - The conjugates according to the invention show in vivo after i.v. application excellent therapeutic efficacy across multiple dose levels different tumors.
• - They show a significantly higher level than the underlying toxophore
• - Tolerability and tumor selectivity, especially with regard to bones market toxicity.

The invention relates to compounds of the formula (I)

wherein
R ^{1 stands} for a sterically demanding non-polar side chain of an amino acid and
R ^{2 represents} a basic side chain of an amino acid
and their salts, stereoisomers and stereoisomer mixtures.

Compounds of the formula (I) are preferred
wherein
R ^{1 represents} a branched alkyl radical having up to 4 carbon atoms and
R ^{2 represents} a radical of the formula - (CH ₂ ) _n -R ³ , where

means and
n stands for a number 1 to 4.

Compounds of the general formula (I) in which
R ¹ for a branched alkyl radical of the formulas

stands and
R ² for a remainder of the formulas

or

stands.

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

The amino acids can occur in the L or in the D configuration or else as a mixture of D and L forms.

The term "amino acids" denotes in particular those that occur in nature α-amino acids, but also includes their homologs, isomers and Derivatives. Enantiomers can be mentioned as examples of isomers. Derivatives can be, for example, amino acids provided with protective groups.

Amino acids with "sterically demanding" side chains are such Amino acids understood, the side chain of which has a branch in the β or γ position supply; examples include valine and isoleucine and leucine.

Typical examples of amino acids with non-polar side chains are:
Alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine.

Typical examples of amino acids with basic side chains are:
Lysine, arginine, histidine, ornithine, diaminobutyric acid.

The compounds according to the invention are preferably in the form of their salts. in the general salts with organic or inorganic acids may be mentioned here.  

The acids that can be added preferably include halogen water Substance acids, such as B. hydrochloric acid and hydrobromic acid, in particular especially hydrochloric acid, also phosphoric acid, nitric acid, Schwe rock acid, mono- and bifunctional carboxylic acids and hydroxycarboxylic acids, such as. B. Acetic acid, trifluoroacetic acid, maleic acid, malonic acid, oxalic acid, gluconic acid, Succinic acid, fumaric acid, tartaric acid, citric acid, salicylic acid, sorbic acid and Lactic acid and sulfonic acids, such as. B. p-toluenesulfonic acid, 1,5-naphthalenedi sulfonic acid or camphorsulfonic acid.

The glycoconjugates according to the invention can be produced, for example by linking 20 (S) -camptothecin with activated carboxyl components, which in turn are parts of protected amino acids, peptides or carbohydrate-modified Can represent peptides.

The glycoconjugate is preferably built up sequentially, starting with the Acylation of 20 (S) -camptothecin with an N-protected carboxyl-activated Building block of a non-polar sterically demanding amino acid in a suitable Solvents, if appropriate in the presence of a base, by customary methods. The amino protective group is then selectively selected using known methods cleaved. Then, if necessary, a suitably protected building block becomes one linked basic amino acid and then, if necessary, while maintaining the Side chain protecting group at the α-amino function unblocked. The key step is linked to the carbohydrate residue by transferring p-amino phenyl-3-O-methyl-β-L-fucopyranoside in the corresponding isothiocyanate and at closing linkage with the deblocked α-amino group of the peptidyl Camptothecins. If necessary, still existing side chain protection groups detached and the free amino group is optionally in a suitable ammo transferred sodium salt.

The invention thus further relates to a process for the preparation of the glyco conjugates of the formula (I)

wherein
R ^{1 stands} for a sterically demanding non-polar side chain of an amino acid and
R ^{2 represents} a basic side chain of an amino acid,
or of their salts, characterized in that the isothiocyanate of the formula (H)

with the peptidyl camptothecin of the formula (III) which may have a protective group in the side chain

wherein
R ^{1 has} the meaning given above and
R ^{2 'has} the meaning of the above-mentioned basic radical R ² , which can moreover carry a protective group customary in peptide chemistry on the basic group
to the glycoconjugate of formula (IV)

wherein
R ¹ and R ^{2 have} the meanings given above,
implements
the side chain amino protecting group which may be present is split off by customary methods and the compound obtained is optionally converted into the desired salt.

Another sequence of reaction steps in the construction of the target ver is also conceivable binding. Thus, according to a likewise preferred variant, the p-isothiocyanato phenyl-3-O-methyl-β-L-fucoside also first with the possibly suitable protected ten terminal basic amino acid are linked, and then this building block with the free amino group of the amino acid conjugate from 20 (S) -camptothecin and the non-polar, sterically demanding amino acid. Give If necessary, existing side chain protection groups are removed and the free amino group optionally converted into a suitable ammonium salt.

The invention therefore further relates to an alternative process for the preparation of compounds of the general formula (I) or of their salts, characterized in that the isothiocyanate of the formula (II)

with an optionally suitably protected terminal basic amino acid of the formula (V)

wherein R ^{2 stands} for a basic side chain of an amino acid whose basic group can be protected,
to an amino acid conjugate of the formula (VI)

in which R ^{2 has} the meaning given above,
this then with amino acid conjugates of the formula (VII)

where R ^{1 has} the meaning given above,
implements
the side chain protecting group is split off and the compounds are optionally converted into a suitable salt.

Especially after linking the first amino acid to camptothecin Mixtures of diastereomers are formed. Pure diastereomers of the invention Compounds can be, for example, according to the methods given above produce in which, after connecting the first amino acid building block to the Camptothecin and subsequent deprotection of the diastereomers in suitably separates. From a diastereomerically pure intermediate the target diastereomerically pure compound is prepared in the above-mentioned way become.

It can also the diastereomer mixture of the target compound in the usual way in the individual diastereomers are separated.

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, chloroform, lower alcohols, acetonitrile, dioxane, water or in mixtures of the above Solvents are carried out. Usually there are reactions in DMF, Dichloromethane or THF / dichloromethane at room temperature and normal pressure prefers.

Those known in peptide chemistry come for the activation of the carboxyl groups Coupling reagents such as. B. in Jakubke / Jeschkeit: amino acids, peptides, Proteins; Verlag Chemie 1982 or Tetrahedr. Lett. 34, 6705 (1993) are in question. For example, N-carboxylic anhydrides and acid chlorides are preferred 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 Car bonyl 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-isoxazolium-per chlorate, or acylamino compounds such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydro quinoline, or propanephosphonic anhydride, or isobutylchloroform, or benzo triazolyloxy tris (dimethylamino) phosphonium hexafluorophosphate, 1-hydroxy benzotriazole or N-hydroxysuccinimide esters. Furthermore, the amino acid com component can also be used in the form of a Leuchsian anhydride.

As bases, for example, triethylamine, ethyldiisopropylamine, pyridine, N, N- Dimethylaminopyridine or others can be used.

Protective groups for third-party functions of the amino acids can be those in the peptide Chemically known protective groups, for example urethane, alkyl, acyl, ester or amide type can be used.

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

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, phthaloyl, 2,2,2-trichloroethoxycarbonyl, 2,2,2-trichloro-tert-butoxy carbonyl, menthyloxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxy carbonyl (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-nitro  benzyl, 2,4-dinitrobenzyl, 4-nitrophenyl or 2-nitrophenylsulfenyl. Especially the Fmoc group and the Boc group are preferred.

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

The splitting off of protective groups in corresponding reaction steps can lead to Example by exposure to acid or base, hydrogenolytically or in another way done reductive.

The glycoconjugates according to the invention have both in vitro and in vivo surprisingly strong antitumor activity against various tumors, ins special lung, breast, pancreas, melanoma and colon tumors connected with a high selectivity towards non-malignant cells.

They are therefore suitable for the treatment of cancer, both in the Human as well as in veterinary medicine.

The present invention includes pharmaceutical preparations which, besides not toxic, inert pharmaceutically suitable excipients one or more inventions contain compounds according to the invention or the invention from one or more Active ingredients exist, as well as processes for the preparation of this preparation exercises.

The active ingredients can optionally be in one or more of the above Carriers are also available in microencapsulated form.

The therapeutically active compounds should be in the pharma listed above zeutischen 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.  

The pharmaceutical preparations listed above can apart from the Invention according to the compound also contain other active pharmaceutical ingredients.

In general, it has been found in both human and veterinary medicine proven advantageous, the active ingredient according to the invention in total amounts of about 0.5 to about 500, preferably 5 to 100 mg / kg body weight per 24 hours, if necessary in the form of several individual doses to achieve the desired results nisse to administer. A single dose contains the active ingredient (s) according to the invention substances preferably in amounts of about 1 to about 80, in particular 3 to 30 mg / kg Body weight.  

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 (CRL 6475) were grown in Roux dishes in RPMI 1640 medium with the addition of 10% FCS. It was then trypsinized and taken up in RPMJ plus 10% FCS to a cell number of 50,000 cells / ml for SW 480 and HT 29 and 20,000 cells for B16F10. 100 μl of cell suspension / well were placed in a 96 microwave plate and incubated for 1 day at 37 ° C. in a CO ₂ incubator. Then a further 100 µl RPMI medium and 1 µl DMSO were added with the test substances. The growth was checked after day 6. For this purpose, 25 μl of MTT solution (3- (4,5-dimethylthiazol-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 for 30 min with 100 μl H ₂ O, the absorbance at 595 nm was measured with a Multiplate Reader (Bio.) 3550-UV.

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 the glycoconjugate compared to underlying active ingredient material and methods

Bone marrow cells were rinsed from the mouse femur. 10 ⁵ cells were 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. Seven days later, the colonies (<50 cells) and clusters (17-50 cells) were counted.

Results

As shown in Table 2, the glycoconjugates examined show one against over the underlying active ingredient drastically reduced inhibition of Bone marrow stem cell proliferation.

Table 2

Inhibition of CSF-induced proliferation of mouse bone marrow stem cells

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) were used in growth. The selected LXFL 529 large cell lung carcinoma was identified by serial Passage developed in nude mice. The human origin of the tumor was demonstrated by isoenzymatic and immunohistochemical methods.

Experimental setup

The tumor was subcutaneously in both flanks of 6 to 8 week old nu / nu Implanted nude mice. Treatment became dependent on the doubling Development time started as soon as the tumors had a diameter of 5-7 mm had reached. The mice became the treatment group and the control group (5 mice per group with 8-10 evaluable tumors) Randomize assigned. The individual tumors in the control group grew all progressive.

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 with the tumor size of the Day 0 (at the time of randomization) calculated. The middle values of the RTV were then used for further evaluation.

Inhibition of tumor volume (relative tumor volume of the test group / control group × 100, T / C in%) was the final measured value.  

treatment

The compounds were applied, for example, on days 0, 1 and 2 after randomization intravenously (IV), the total dose per day over 2 Gifts were split.

Results

The therapeutic effectiveness of the glycoconjugates according to the invention Examples 1 and 2 are exemplary of the large cell human lung tumor xeno graft LXFL 529. The therapy leads to the maximum tolerable Dose (MTD) and at 1/2 MTD to complete to clear tumor remission. An excellent effect can also be demonstrated on other tumors be treated.  

Table 3

The long-lasting complete remission of the compound from Example 1 in the dose range of 16 to 8 mg / kg and the dose-dependence of the effect is shown in a further experiment with the therapy schedule day 1-3 iv in FIG. 1.

4. Hydrolytic stability

The compounds of Example 1, 2, 8 and 9 according to the invention are in water dissolved and after 24 h standing at room temperature in HPLC clearly below 1% Camptothecin release by area percent.

When 10 .mu.M of the compounds from Examples 1 and 2 were dissolved in RPMI medium plus 10% FCS and in 30% human whole blood in PBS buffer, after 24 hours standing there was only a camptothecin release below 5%.
Method: HPLC system Hewlett Packard HP 1050
Column: Nucleosil 120-5 C 18 250 mm × 4 mm (Macherey &Nagel; Germany)
Eluent:
A: 0.01 m KH ₂ PO ₄ in H ₂ O (H ₂ = Milli-Pore grade)
B: 80% acetohitrile / 20% eluent A
Flow: 1.2 ml
Gradient:
t ₀ : 20% B - t ₄₀ : 100% B
t ₄₅ : 100% B - t ₄₇ : 20% B
Detection: 240 nm or 370 nm

5. Lactone stabilization

The glycoconjugates according to the invention from Examples 1, 2, 8 and 9 are described in 80% water and 20% acetonitrile dissolved and with 2 equivalents of sodium hydroxide solution to pH 9 set. After standing at room temperature for 1 h, the lactone ring opening is located (Detection according to the above method) below 5%.

Examples Carbohydrate educt I) p-aminophenyl-3-O-methyl-β-L-fucopyranoside

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

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

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

3.81 g (12.73 mmol) of p-nitropheryl-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].

Peptidyl camptothecin starting materials II) 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -L-lysyl-L / D-leucyl} - camptothecin, trifluoroacetate II) L 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -L-lysyl-L-leucyl} - camptothecin, trifluoroacetate II) D 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -L-lysyl-D-leucyl} - camptothecin, trifluoroacetate II.a) 20 (S) -20-O- [N- (tert-Butoxycarbonyl) -L / D-leucyl] camptothecin

A suspension of 10 g (28.7 mmol) of 20 (S) -camptothecin in 250 ml of absolute dimethylformamide is stirred with 11.1 g (43 mmol) of N- (tert-butoxycarbonyl) - leucine-N-carboxylic anhydride and 1 g 4- (N, N-Dimethylamino) pyridine added. After 16 hours of treatment in an ultrasonic bath at room temperature, a further 3.7 g of N- (tert-butoxycarbonyl) leucine-N-carboxylic anhydride are added and the mixture is left for a further 2 hours at room temperature. The remaining undissolved camptothecin is then separated off and the filtrate is concentrated in vacuo. The residue is purified by flash chromatography [petroleum ether / ethyl acetate 1: 1 → 1: 4]. 13.55 g (84%) of the target compound IIa) are obtained. [TLC: acetonitrile R _f = 0.47].

II.b) 20 (S) -20-O-L / D-leucyl camptothecin, trifluoroacetate II.b) L 20 (S) -20-O-L-leucyl camptothecin, trifluoroacetate II.b) D 20 (S) -20-O-D-leucyl camptothecin, trifluoroacetate

A solution of compound II.a (13.55 g, 24.1 mmol) in a mixture of 100 ml of dichloromethane and 40 ml of anhydrous trifluoroacetic acid is for 30 min. stirred at room temperature. After concentration in vacuo to a small volume, the product is precipitated with diethyl ether and washed thoroughly with diethyl ether. In the thin layer chromatogram (acetonitrile / water 20: 1) a double spot with R _f values of 0.4 and 0.32 can be seen [yield: 9.5 g (68%)].

The mixture can be separated into two individual components II.b) L and II.b) D by repeated precipitation from dichloromethane / methanol with diethyl ether. Both forms prove to be diastereomers, which give slightly different ¹ H NMR spectra:
polar diastereomer:
400 MHz ¹ H NMR (CD ₂ Cl ₂ / CD ₃ OD 1: 1): δ
s CH (B ring) 8.63 ppm
s CH (D-ring) 7.4 ppm
non-polar diastereomer:
400 MHz ¹ H NMR (CD ₂ Cl ₂ / CD ₃ OD 1: 1): δ
s CH (B-ring) 8.60 ppm
s CH (D-ring) 7.32 ppm

In the further stages, both the mixture of the two forms and the purified diastereomerically pure forms can be used.

II.c) 20 (S) -20-O- [N ^α - (tert-butoxycarbonyl) -N ^ε - (fluorenyl-9-methoxy carbonyl) -L-lysyl-L / D-leucyl] -camptothecin II.c) L 20 (S) -20-O- [N ^α - (tert-butoxycarbonyl-N ^ε - (fluorenyl-9-methoxycarbonyl) -L-lysyl-L-leucyl] -camptothecin II.c) D 20 (S) -20-O- [N ^α - (tert-butoxycarbonyl) -N ^ε - (fluorenyl-9-methoxycarbonyl) -L-lysyl-D-leucyl] -camptothecin

25.6 g (54.6 mmol) N ^α - (tert-Butoxycarbony-N ^ε (fluorenyl-9-methoxycarbony-L-lysine) and 11.1 g (82 mmol) 1-hydroxy-1H-benzotriazole hydrate are dissolved in 500 ml of dimethylformamide was dissolved and 12.6 g (1.2 eq.) of N-ethyl-N '- (dimethylaminopropyl) carbodiimide hydrochloride were added and the mixture was stirred at room temperature for 1 h. Compound II.b ( 26.2 g, 0.83 eq. And 7.83 ml (1 eq.) Of ethyl-di-isopropylamine and the mixture is stirred for a further 16 h at room temperature, after concentration in vacuo and purification by flash chromatography [petroleum ether / ethyl acetate 1: 2 → ethyl acetate] gives the target compound (43.5 g, 87%) [TLC: aceto nitrile R _f = 0.44].

As in the other examples, the approach can be completely analogous with any of the purified diastereomerically pure forms can be carried out in II.b.

II) 20 (S) -20-O- {N ^ε- fluorenyl-9-methoxycarbonyl] -L-lysyl-L / D-leucyl} - camptothecin, trifluoroacetate II) L 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -L-lysyl-L-leucyl} - camptothecin, trifluoroacetate II) D 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -L-lysyl-D-leucyl} - camptothecin, trifluoroacetate

The compound II.c (43.3 g, 47.5 mmol) is taken up in 150 ml of dichloromethane and 50 ml of anhydrous trifluoroacetic acid are added. The resulting solution is stirred for 1 h at room temperature. After concentration to a small volume in vacuo, the product is precipitated by adding diethyl ether. 39.4 g (90%) of the target compound are obtained [TLC: acetonitrile / water 10: 1 R _f = 0.35].

III) 20 (S) -20-O- (L-histidyl-L / D-valyl) camptothecin, trifluoroacetate III.a) 20 (S) -20-O- [N- (tert-Butoxycarbonyl) -L / D-valyl] -camptothecin

A suspension of 10 g (28.7 mmol) of 20 (S) -camptothecin in 500 ml of absolute dimethylformamide is stirred with 21.5 g (3 equivalents) of N- (tert-butoxy carbonyl) -valine-N-carboxylic acid anhydride and 1 4- (N, N-Dimethylamino) pyridine was added. After 16 hours of treatment in an ultrasound bath at room temperature, the remaining undissolved camptothecin is separated off and the filtrate is concentrated in vacuo. The residue is purified by flash chromatography [petroleum ether / ethyl acetate 1: 3 → ethyl acetate]. 11.65 g (73%) of the target compound are obtained. [TLC: aceto nitrile R _f = 0.44]. If the same reaction is carried out in dichloromethane under reflux conditions instead of in dimethylformamide, the formation of the diastereomer with the L configuration of the valine building block is more favored.

III.b) 20 (S) -20-O-L / D-valyl camptothecin, trifluoroacetate III.b) L 20 (S) -20-O-L-valyl camptothecin, trifluoroacetate III.b) D 20 (S) -20-O-D-valyl camptothecin, trifluoroacetate

A solution of compound III.a (11.65 g, 21 mmol) in a mixture of 100 ml dichloromethane and 100 ml anhydrous trifluoroacetic acid is stirred for 1 h at room temperature. After concentration in vacuo to a small volume, the product is precipitated with diethyl ether and washed thoroughly with diethyl ether. The product is precipitated again from dichloromethane / methanol with diethyl ether and isolated as a mixture of diastereomeric forms. Yield: 10.9 g (92%) [TLC: aceto nitrile / water 20: 1 R _f = 0.31 and 0.39]. A higher order of the nonpolar diasteromer with the L configuration of the valine building block is possible at this stage by crystallization from methanol:
8 g of the product obtained with enriched non-polar diastereomer are dissolved in 80 ml of methanol, cooled to 0 ° C. and mixed with diethyl ether in 10 ml steps. After a total of 60 ml of diethyl ether has been added, the precipitated product is filtered off with suction and dried. 4.6 g (58%) of pure nonpolar diasteromer with the L configuration of the valine building block are obtained. Reprecipitation of the mother liquor with diethyl ether gives a further 730 mg (9%) of a product fraction with a diastereomer ratio of 1:18.

In the further reactions, both the diastereomer mixture and that purified non-polar or the polar diastereomer can be used. The order settings run completely analog.

III.c) 20 (S) -20-O- [N- (tert-butoxycarbonyl) -L-histidyl-L / D-valyl] - camptothecin III.c) L 20 (S) -20-O- [N- (tert-Butoxycarbonyl) -L-histidyl-L-valyl] -camptothecin III.c) D 20 (S) -20-O- [N- (tert-butoxycarbonyl) -L-histidyl-D-valyl] - camptothecin

5.95 g (23.3 mmol) of N- (tert-butoxycarbonyl) -L-histidine and 4.73 g of 1-hydroxy-1H benzotriazole hydrate are dissolved in 200 ml of dimethylformamide. After adding 5.38 g of N-ethyl-N '- (dimethylaminopropyl) carbodiimide hydrochloride, the mixture is stirred for 1 h at room temperature. Then compound III.b (10.9 g, 19.44 mmol) and 6.7 ml of ethyl-diisopropylamine are added and the mixture is stirred for a further 16 h at room temperature. After concentration in vacuo and purification by flash chromatography [acetonitrile / water 50: 1 → 20: 1], the target compound is obtained, [DC: acetonitrile / water 10: 1 R _f = 0.42], which is immediately reacted further.

III) 20 (S) -20-O- (L-histidyl-L / D-valyl) camptothecin, bis-trifluoroacetate III) L 20 (S) -20-O- (L-histidyl-L-valyl) camptothecin, bis-trifluoroacetate III) D 20 (S) -20-O- (L-histidyl-D-valyl) camptothecin, bis-trifluoroacetate

The compound III.c is taken up in 100 ml of dichloromethane and mixed with 50 ml of anhydrous trifluoroacetic acid. The resulting solution is stirred for 30 min at room temperature. After concentration to a small volume in vacuo, the product is precipitated by adding diethyl ether. 13.05 g (83%) of the target compound are obtained [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.2].

IV) 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -D-lysyl-L / D-leucyl} - camptothecin, trifluoroacetate IV) L 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -D-lysyl-L-leucyl} - camptothecin, trifluoroacetate IV) D 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -D-lysyl-D-leucyl} - camptothecin, trifluoroacetate

This compound is synthesized in complete analogy to the diastereomeric compound II. In stage II.c, the corresponding D-isomer is used instead of N ^α - (tert-butoxycarbonyl-N ^ε - (fluorenyl-9-methoxycarbonyl) -L-lysine used [TLC: acetonitrile / water 10: 1 R _f = 0.4].

V) 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -L-ornithyl-L / D-leucyl} - camptothecin, trifluoroacetate V) L 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -L-ornithyl-L-leucyl} - camptothecin, trifluoroacetate V) D 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -L-orhithyl-D-leucyl} - camptothecin, trifluoroacetate

This compound is synthesized in complete analogy to the corresponding lysine compound II. In step II.c, instead of N ^α - (tert-butoxycarbonyl) - N ^ε (fluorenyl-9-methoxycarbonyl) -L-lysine N ^α - (tert-Butoxycarbonyl) -N ^ε - (fluorenyl-9-methoxycarbonyl) -L-ornithine used [DC: acetonitrile / water 20: 1 R _f = 0.125].

VI) 20 (S) -20-O- (L-Arginyl-L / D-leucyl) camptothecin, tri-trifluoroacetate VI) L 20 (S) -20-O- {L-Arginyl-L-leucyl} camptothecin, tri-trifluoroacetate VI) D 20 (S) -20-O- {L-Arginyl-D-leucyl} camptothecin, tri-trifluoroacetate VI.a) 20 (S) -20-O- (tri-tert-butoxycarbonyl-L-arginyl-L / D-leucyl) camptothecin VI.a) L 20 (S) -20-O- {tri-tert-butoxycarbonyl-L-arginyl-L-leucyl} - camptothecin VI.a) D 20 (S) -20-O - {(Tri-tert-butoxycarbonyl-L-arginyl-D-leucyl} - camptothecin

200 mg (0.42 mmol) of tri-tert-butoxycarbonyl-L-arginine and 80 mg of 1-hydroxy-1H benzotriazole hydrate are dissolved in 20 ml of dimethylformamide. After adding 97 mg of N-ethyl-N '- (dimethylaminopropyl) carbodiimide hydrochloride, the mixture is stirred for 1 h at room temperature. Then compound II.b (200 mg, 0.35 mmol) and 217 μl of ethyl-diisopropylamine are added and the mixture is stirred for a further 3 d at room temperature. After concentrating in vacuo and treating with water, the target compound [DC: acetonitrile / water 5: 1: 0.2 R _f = 0.77] is obtained, which is immediately reacted further.

VI) 20 (S) -20-O- {L-Arginyl-L / D-leucyl} camptothecin, tri-trifluoroacetate VI) L 20 (S) -20-O- {L-Arginyl-L-leucyl} camptothecin, tri-trifluoroacetate VI) D 20 (S) -20-O- {L-Arginyl-D-leucyl} camptothecin, tri-trifluoroacetate

0.35 mmol of the compound from VI.a) are stirred in 10 ml of dichloromethane with 5 ml of anhydrous trifluoroacetic acid for 2 h at room temperature. The mixture is concentrated and the mixture is precipitated twice from dichloromethane / methanol with diethyl ether. 280 mg (82%) of the target compound are obtained [TLC: acetonitrile / water 10: 3: 1.5 R _f = 0.42].

VII) L 20 (S) -20-O- {N ^ε - [Fluorenyl-9-methoxycarbonyl] -L-lysyl-L-valyl} - camptothecin, trifluoroacetate VII.a) L 20 (S) -20-O- [N ^α - (tert-butoxycarbonyl) -N ^ε - (fluorenyl-9-methoxycarbonyl) -L-lysyl-L-valyl] -camptothecin

10.02 g (21.4 mmol) of N- (tert-butoxycarbonyl) -N- (fluorenyl-9-methoxycarbonyl) -L-lysine and 4.4 g (32.1 mmol) of 1-hydroxy-1H-benzotriazole hydrate are dissolved in 400 ml of dimethylformamide. After adding 4.92 g (1.2 eq.) Of N-ethyl-N '- (dimethyl aminopropyl) carbodiimide hydrochloride, the mixture is stirred at 0 ° C. for 30 min. Then 10 g (17.8 mmol) of the non-polar diastereomer III.b L of compound III.b and 9.2 ml (3 eq.) Of ethyl-diisopropylamine are added and the mixture is stirred for a further 16 h at room temperature. After concentration in vacuo, the residue is stirred for 30 min with 500 ml of water and suction filtered. The product is taken up in 400 ml of dichloromethane, the remaining water is removed, the solution is concentrated to 200 ml and then precipitated with 800 ml of diethyl ether. It is suctioned off and the residue is washed with diethyl ether. 14.712 g of the target compound (92%) are obtained [TLC: acetonitrile R _f = 0.6].

VII) L 20 (S) -20-O- {N- [fluorenyl-9-methoxycarbonyl] -L-lysyl-L-valyl} - camptothecin, trifluoroacetate

The compound VII.a) L (14.65 g, 16.3 mmol) is taken up in 300 ml dichloromethane and 50 ml of anhydrous trifluoroacetic acid are added at 0 ° C. The resulting solution was stirred for 2 hours under ice cooling. After concentration to a small volume in vacuo, the product is precipitated by adding diethyl ether. 13.8 g (93%) of the target compound are obtained [TLC: acetonitrile / water 10: 1 R _f = 0.35].

example 1 20 (S) -20-O- {N ^α - [O- (3-O-Methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -L-lysyl-L / D-leucyl} - camptothecin, hydrochloride

1.a) 20 (S) -20-O- {N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxyphenylamino-thiocarbonyl] -N ^ε - [fluorenyl-9- methoxycarbonyl] -L-lysyl-L / D-leucyl} -camptothecin

A solution of 9.82 g (36.5 mmol) of p-aminophenyl-3-O-methyl-β-L-fucopyranoside (Example I) in 500 ml of dioxane / water 1: 1 is stirred with 3.94 ml of thiophosgene (1.4 eq.). After 10 min. 4 equivalents of ethyldiisopropylamine are added, the mixture is then concentrated in vacuo and the residue is dried for 1 h in an oil pump vacuum. The isothiocyanate obtained is dissolved in 500 ml of absolute dimethylformamide and mixed with 30.4 g (32.8 mmol) of compound II and 22.6 ml of ethyl diisopropylamine. The mixture is stirred for 16 h at room temperature, then concentrated in vacuo and stirred with water. The residue is purified by flash chromatography [acetonitrile → acetonitrile / water 30: 1]. The product is reprecipitated from dichloromethane / methanol with diethyl ether and washed with diethyl ether. 28.7 g (78%) of the target product are obtained [TLC: acetonitrile / water 10: 1 R _f = 0.44].

1.b) 20-O- {N ^α - [O- (3-O-Methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino thiocarbonyl] -L-lysyl-L / D-leucyl} -camptothecin

28.6 g (25.5 mmol) of conjugate 1.a) are mixed with 200 ml dimethylformamide with 5 ml piperidine. After stirring for 2 h at room temperature, the mixture is concentrated and the residue is digested twice with dichloromethane and diethyl ether is added. Then is taken up in dimethylformamide / dichloromethane and precipitated with ether. This cleaning process is repeated twice. The product is filtered off and washed with ether. Yield: 19.5 g (85%) [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.3].

1) 20 (S) -20-O- {N ^α - [O- (3-O-Methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -Lysyl-L / D-leucyl} -camptothecin, hydrochloride

10 g (11.1 mmol) of the compound from Example 1.b are taken up in 100 ml of water, mixed with 11.1 ml (1 eq.) Of a 1N HCl solution and lyophilized. The product is then precipitated several times from dichloromethane / methanol with diethyl ether. Yield: 9.15 g (88%) [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.3].

Example 2 20 (S) -20-O- {N- [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino- thiocarbonyl] -L-histidyl-L / D-valyl} -camptothecin, hydrochloride

A solution of 7.14 g (26.5 mmol) of p-aminophenyl-3-O-methyl-β-L-fucopyranoside (carbohydrate starting material I) in 300 ml of dioxane / water 1: 1 is stirred with 2.86 ml of thiophosgene (1.4 eq.) added. After 10 min. 4 equivalents of ethyl diisopropylamine are added, the mixture is then concentrated in vacuo and the residue is dried for 1 h in an oil pump vacuum. The isothiocyanate obtained is dissolved in 500 ml of absolute dimethylformamide, and 17.45 g (22 mmol) of compound III and 22.7 ml of ethyldiisopropylamine are added. The mixture is stirred at room temperature for 16 h, then concentrated in vacuo and the residue is taken up in water. The pH is adjusted to 7.8 with 1 N aqueous sodium hydroxide solution and the product is filtered off with suction. After drying under high vacuum, the residue is reprecipitated twice from dichloromethane / methanol with diethyl ether and washed with diethyl ether. 17.97 g (91%) of the target product are obtained, which is then converted into the hydrochloride with 1 equivalent of 0.1 N aqueous hydrochloric acid [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0. 36] [FAB-MS: m / z = 896 (M + H⁺)].

Example 3 20 (S) -20-O- {N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -D-lysyl-D-leucyl} -camptothecin, Hydrochloride

The synthesis is carried out completely analogously to Example 1. The starting materials I and IV.D are used here, the polar diasteromer II.b) D being used from the stage of 20-O-leucyl camptothecin II.b. [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.31] [FAB-MS: m / z = 901 = M + H⁺]

Example 4 20 (S) -20-O- {N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -L-ornithyl-D-leucyl} -camptothecin, Hydrochloride

The synthesis is carried out completely analogously to Example 1. The starting materials I and V) D are used here, starting from the stage of 20-O-leucyl camptothecin II.b the polar diasteromer II.b) D with D-Leu configuration was used. [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.25].

Example 5 20 (S) -20-O- {N ^α - [O- (3-O-Methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -L-arginyl-D-leucyl} camptothecin

A solution of 73 mg (0.27 mmol) of p-aminophenyl-3-O-methyl-β-L-fucopyranoside (educt I) in 20 ml of dioxane / water 1: 1 is stirred with 30 µl of thiophosgene (1.4 Eq.). After 10 min. 4 equivalents of ethyldiisopropylamine 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 20 ml of absolute dimethylform amide, and 175 mg (0.18 mmol) of compound VI) D and 620 μl of ethyl diisopropylamine are added. The polar diastereomer II.b) D is used here for the synthesis of compound VI) D from the stage of 20-O-leucyl camptothecin II.b. The mixture is stirred at room temperature for 16 h, then concentrated in vacuo and stirred with dichloromethane. The residue is then reprecipitated from dichloromethane / methanol with diethyl ether and washed with diethyl ether. It is then lyophilized from dioxane / water and then crystallized again from dichloromethane / methanol with diethyl ether. 154 mg (90%) of the target product [DC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.39] [FAB-MS: m / z = 929 = M + H⁺].

Example 6 20 (S) -20-O- {N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -L-lysyl-D-leucyl} -camptothecin, Hydrochloride

The compound is prepared in analogy to Example 1, with the step of 20-O-leucyl camptothecins II.b the polar diastereomer II.b) D with D-leucine Configuration was used.

Alternatively, the compound from Example 1 z. B. also be separated into the individual isomeric forms by preparative HPLC.
Conditions:
Separation column: Macherey & Nagel 250 × 21 mm Nucleosil 100-7 C18 AB
Solvent A: H ₂ O + 0.1M KH ₂ PO ₄
Mobile solvent B: acetonitrile / water 4: 1 + 0.02M KH ₂ PO ₄
Flow: 10 ml / min
Inj. Volume: 1500 µl
Gradient:
0-30% B
4-30
20-90
22-90
24-30
Wavelength: 215 nm.

After HPLC separation, the corresponding fractions are lyophilized and the residue was then precipitated several times from dichloromethane / methanol with diethyl ether. Then it is adjusted to pH 8-9, filtered and the residue is transferred with 0.1 N hydrochloric acid in the hydrochloride.

In the ¹ H-NMR spectrum, the isomers in Examples 6 and 7 show different chemical shifts, in particular for the two singlets of the aromatic H atoms in the camptothecin B ring and D ring.
Diasteromer with D-leucine:
400 MHz ¹ H NMR (CD ₂ Cl ₂ / CD ₃ OD 1: 1): δ
s CH (B-ring) 8.52 ppm
s CH (D-ring) 7.42 ppm
[FAB-MS: m / z = 901 = M + H⁺]

Example 7 20 (S) -20-O- {N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -L-lysyl-L-leucyl} -camptothecin, Hydrochloride

The compound is prepared in analogy to Example 1, with the step of 20-O-Leucyl-Camptothecins II.b the non-polar diastereomer II.b) L with L-configuration ration of leucine was used.

Alternatively, the compound from Example 1 z. B. also by preparative HPLC in the individual isomeric forms are separated.

Conditions: as indicated in Example 6.

After HPLC separation, the corresponding fractions are lyophilized and the residue was then precipitated several times from dichloromethane / methanol with diethyl ether. Then it is adjusted to pH 8-9, filtered and the residue is transferred with 0.1 N hydrochloric acid in the hydrochloride.

In the ¹ H-NMR spectrum, the isomers in Examples 6 and 7 show different chemical shifts, in particular z. B. for the two singlets of the aromatic H atoms in the camptothecin B ring or D ring.
Diastereomer with L-leucine:
400 MHz ¹ H NMR (CD ₂ Cl ₂ / CD ₃ OD 1: 1): δ
s CH (B-ring) 8.6 ppm
s CH (D-ring) 7.35 ppm
[FAB-MS: m / z = 901 = M + H⁺]

Example 8 20 (S) -20-O - {(N ^α - [O- (3-O-Methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -L-lysyl-L-valyl} -camptothecin , Hydrochloride

The connection is made in the non-polar series starting from connection VII) L in Prepared analogy to Example 1.

The diastereomer ratio can be determined by analytical HPLC. If necessary, a further purification of the diastereomer with the L configuration of the valine can be achieved by crystallization from methanol (<20: 1).
HPLC conditions:
Separation column: Macherey & Nagel 250 × 4 mm Nucleosil 100-7 C18 AB
Solvent A: H ₂ O + 0.1M KH ₂ PO ₄
Mobile solvent B: acetonitrile / water 4: 1 + 0.02M KH ₂ PO ₄
Flow: 1 ml / min
Inj. Volume: 15 µl.

In the ¹ H-NMR spectrum, the pure diastereomer shows only one set of signals, e.g. B. for the two singlets of aromatic H atoms in the camptothecin B ring or D ring.
400 MHz ¹ H NMR (CD ₂ Cl ₂ / CD ₃ OD 1: 1): δ
CH (B ring) 8.55 ppm
CH (D-ring) 7.35 ppm
[FAB-MS: m / z = 887 = M + H⁺].

An alternative procedure for establishing the connection from example 8 is just if possible. The carbohydrate building block from Example I is first with a side chain protected lysine derivative linked:

8a) N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxyphenylamino-thiocarbonyl] -N ^ε - [fluorenyl-9-methoxycarbonyl] L-lysine

A solution of 6.8 g (25.3 mmol) of p-aminophenyl-3-O-methyl-β-L-fucopyranoside (Example I) in 600 ml of dioxane / water 1: 1 is stirred with 2.72 ml of thiophosgene (1.4 eq.). After 10 min. are mixed with 26 ml of ethyldiisopropylamine, stirred another 5 min at RT and then concentrated in vacuo to a volume of 150 ml on. 800 ml of dichloromethane are added and the phases are separated. The organic phase is washed twice with water, dried over sodium sulfate and concentrated. The residue is mixed with 200 ml of methyl tert-butyl ether and 200 ml of petroleum ether stirs and vacuumed. 7.26 g (92%) of the isothiocyanate are obtained.

2.92 g (9.4 mmol) of the isothiocyanate obtained are dissolved in 200 ml of dioxane / water 1: 1 and with 3.11 g (0.9 eq) of N ^ε - [fluorenyl-9-methoxycarbonyl] -lysine and 3.2 ml of ethyl diisopropylamine were added. The mixture is stirred for 16 h at room temperature, then concentrated in vacuo and the residue is taken up in water. By adjusting the pH to 2 with 1 N HCl, the product is precipitated and filtered off after 30 min. The residue is suspended in dichloromethane and the solvent is stripped off twice. After washing several times with ether and petroleum ether, 5.3 g (92%) of the target product [DC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.69].

8b) 20 (S) -20-O- {N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -N ^ε - [fluorenyl-9- methoxycarbonyl] -L-lysyl-L-valyl} -cam ptothecin

1.2 g (1.76 mmol) N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino thiocarbonyl] -N ^ε - [fluorenyl-9-methoxycarbonyl] -L -lysine (Example 8a) and 675 mg (1.2 mmol) of compound II.b) L are dissolved in 50 ml of dimethylformamide, the mixture is cooled to 0 ° C. and then with 346 mg (1.8 mmol) of N-ethyl -N'- (dimethylaminopropyl) carbodiimide hydrochloride added. The temperature is allowed to rise to RT overnight with stirring. The solvent is i. vac. evaporated and the residue stirred with water. It is purified by flash chromatography on silica gel, starting with acetonitrole as the eluent and later changing to aceto nitrile / water 50: 1. After concentration of the appropriate fractions, 1.06 g (76%) of the target product [DC: acetonitrile / water 20: 1 R _f = 0.34].

The deblocking and the subsequent conversion into the hydrochloride takes place in Analogy to example 1.

Example 9 20 (S) -20-O- {N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -L-histidyl-L-valyl} -camptothecin, Hydrochloride

The compound is derived from the non-polar diastereomer of 20-O-valyl Camptothecin, trifluoroacetate (III.b) L prepared in analogy to Example 2.

The diastereomer ratio can be determined by analytical HPLC. If necessary, a further purification of the nonpolar diastereomer with the L configuration of the valine can be achieved by crystallization from methanol (<20: 1).
HPLC conditions:
Separation column: Macherey & Nagel 250 × 4 mm Nucleosil 100-7 C18 AB
Solvent A: H ₂ O + 0.1M KH ₂ PO ₄
Mobile solvent B: acetonitrile / water 4: 1 + 0.02M KH ₂ PO ₄
Flow: 1 ml / min
Inj. Volume: 15 µl.

In the ¹ H-NMR spectrum, the pure diastereomer shows only one set of signals, e.g. B. for the two singlets of aromatic H atoms in the camptothecin B ring or D ring. Diastereomer with L-valine:
400 MHz ¹ H NMR (CD ₂ Cl ₂ / CD ₃ OD 1: 1): δ
s CH (B-ring) 8.58 ppm (superimposed by a CH aroma of histidine)
s CH (D-ring) 7.35 ppm
[TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.36] [FAB-MS: m / z = 896 (M + H⁺)].

Example 10 20 (S) -20-O- {N ^α - [O- (3-O-Methyl-β-L-fucopyranosyl) -4-hydroxy-phenylamino-thiocarbonyl] -L-arginyl-L-leucyl} camptothecin

The product is produced in analogy to Example 5. The non-polar diastereomer II.b) L is used at the level of 20-O-leucyl camptothecin II.b.
[TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.4] [FAB-MS: m / z = 929 = M + H⁺].
The diastereomers from Examples 5 and 10 can be distinguished using the HPLC conditions given under Examples 8 and 9.

Claims (9)

1. Camptothecin glycoconjugates of the formula (I)
wherein
R ^{1 stands} for a sterically demanding non-polar side chain of an amino acid and
R ^{2 represents} a basic side chain of an amino acid
and their salts, stereoisomers and stereoisomer mixtures. 2. Compounds of formula (I) according to claim 1, wherein
R ^{1 represents} a branched alkyl radical having up to 4 carbon atoms and
R ^{2 represents} a radical of the formula - (CH ₂ ) _n -R ³ , where
means and
n stands for a number 1 to 4,
and their salts, stereoisomers and mixtures of stereoisomers. 3. Compounds of formula (I) according to claim 1, wherein
R ¹ for a branched alkyl radical of the formulas
stands and
R ² for a remainder of the formulas
or
stands,
and their salts, stereoisomers and mixture of stereoisomers. 4. Process for the preparation of the glycoconjugates of the formula (I)
wherein
R ^{1 stands} for a sterically demanding non-polar side chain of an amino acid and
R ^{2 represents} a basic side chain of an amino acid,
or of their salts, characterized in that the isothiocyanate of the formula (II)
with the peptidyl camptothecin of the formula (III) which may have a protective group in the side chain
wherein
R ^{1 has} the meaning given above and
R ^{2 has} the meaning of the above-mentioned basic radical R ² , which can moreover carry a protective group customary in peptide chemistry on the basic group
to the glycoconjugate of formula (IV)
wherein
R ¹ and R ² have the meanings given above,
implements
the side chain amino protecting group which may be present is split off by customary methods and
if necessary, the compound obtained is converted into the desired salt. 5. A process for the preparation of compounds of the general formula (I) or salts thereof, characterized in that the isothiocyanate of the formula (II)
with an optionally suitably protected terminal basic amino acid of the formula (V)
wherein R ^{2 stands} for a basic side chain of an amino acid whose basic group can be protected,
to an amino acid conjugate of the formula (VI)
in which R ^{2 has} the meaning given above,
this then with amino acid conjugates of the formula (VII)
wherein R ^{1 has} the meaning given in claim 1,
implements
the side chain protecting group may be split off and the compounds may be converted into a suitable salt. 6. A compound according to claim 1, characterized in that it is 20 (S) -20-O- {N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenyl amino -thiocarbonyl] -L-lysyl-L-valyl} -camptothecin of the formula
and its salts. 7. A compound according to claim 1, characterized in that it is 20 (S) -20-O- {N ^α - [O- (3-O-methyl-β-L-fucopyranosyl) -4-hydroxy-phenyl amino -thiocarbonyl] -L-histidyl-L-valyl} -camptothecin of the formula
and its salts. 8. Use of compounds of formula (I) according to claim 1 for the Her provision of medicines. 9. Medicament containing compounds of formula (I) according to claim 1.