DE10005275A1 - Novel glycoconjugates - Google Patents

Abstract

The invention concerns cytostatic agents which are more tumor-specific as a result of modification with carbohydrates. Urea or dicarboxylic acid units as appropriate spacers ensure serum stability and simultaneous intracellular effect. The compounds according to the invention have formula (I): K - Sp- L - AA1 - AA2 - C, wherein K represents an unsubstituted or regioselectively modified carbohydrate radical; Sp represents an optionally substituted arylene or alkylene; L represents a group of formula (a); AA1 represents a direct bond or an amino acid radical in the D or L configuration, optionally having protective groups or a second K-Sp-L grouping, in which K, Sp, L are independent from the other K-Sp-L grouping and may have the meaning cited above; AA2 represents a direct bond or an amino acid radical in the D or L configuration, optionally having protective groups or a second K-Sp-L grouping, in which K, Sp, L are independent from the other K-Sp-L grouping and may have the meaning cited above and C represents a cytostatic radical or the radical of a cytostatic agent or cytostatic agent derivative additionally having a hydroxy or amino group.

Description

The present invention relates to cytostatics which are modified by coal hydrates are tumor-specific. Urea or dicarboxylic acid units as suitable Spacers ensure serum stability and at the same time an intracellular effect.

Chemotherapy for tumor diseases is accompanied by mostly serious ones Side effects due to the toxic effects of chemotherapy drugs proliferating cells of tissue types other than tumor tissue can be attributed. Scientists have been dealing with the problem of verbs for many years improvement of the selectivity of active substances used. A widely followed approach is the synthesis of prodrugs that are more or less selective in target tissue for example by changing the pH (Tietze et al., e.g. DE-A-42 29 903), by enzymes (e.g. glucuronidases; Jacquesy et al., EP-A-0 511 917; Bosslet et al., EP-A-0 595 133) or by antibody-enzyme conjugates (Bagshawe et al., WO 88/07378; Senter et al., U.S. 4,975,278; Bosslet et al., EP-A-0 595 133) will. One problem with these approaches is the lack Stability of the conjugates in other tissues and organs, and in particular the ubiquitous drug distribution, which is based on the extracellular drug release in the Tumor tissue connects.

The pronounced lectin pattern on tumor cell surfaces (Gabius; Onkologie 12 , (1989), 175) opens up the possibility in principle of targeting tumor cells by linking appropriate carbohydrate building blocks to cytostatics. This perspective is limited by the fact that lectins with similar carbohydrate specificities (galactose, lactose, mannose, N-acetylglucosamine, fucose etc.) also occur in other tissues, especially in the liver (Ashwell et al., Annu. Rev. Biochem 46 (1982), 531; Stahl et al. Proc. Natl. Acad. Sci. USA 74 (1977), 1521; Hill et al., J. Biol. Chem. 262 (1986), 7433; Jansen et al. , J. Biol. Chem. 266 (1991), 3343). Accordingly, a significant accumulation of active ingredient-containing glycoconjugates in the liver and other lectin-rich organs must be expected if carbohydrates are used in this approach without any special modification that causes a selectivity towards tumor tissue.

The heterocyclic amine batracylin (1) has been shown in various Models have a good anti-tumor activity (US-4,757,072).

Peptide conjugates of (1) with good in vitro activity and more favorable solubility Properties (US-4 180 343) are less tolerable in animal experiments than free ones Batracylin. The fucose conjugates of batracycline described in EP-A-0 501 250 (1) disadvantageously accumulate very strongly in the liver.

The quinolone-a (2) 7 - [(3a-R, S, 4-R, S, 7a-S, R) -4-amino-1,3,3a, 4,7,7a-hexahydro- iso-indol-2-yl] -8-chloro-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinoline carbon In addition to its excellent antibacterial activity, acid also shows a very good one Efficacy against various tumor cell lines (EP-A-0 520 240, JP-4 253 973). However, there are considerable toxicological problems (e.g. Geno toxicity, bone marrow toxicity, high acute toxicity in vivo etc.).  

20 (S) -camptothecin is a pentacyclic alkaloid developed by Wall et al. was isolated (J. Am. Chem. Soc. 88, 3888 (1966)). It has a high anti-tumor Effective potential in numerous in vitro and in vivo tests. Unfortunately, however, the failed Realization of the promising potential in the clinical investigation phase 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 Find derivatives.

Salts from A-ring and B-ring were used to improve water solubility modified camptothecin derivatives and of 20-O-acyl derivatives with ionizable Groups described (Vishnuvajjala et al. US 4943579). The latter prodrug The concept was later transferred to modified camptothecin derivatives (Wani et al. WO 9602546). The 20-O-acyl prodrugs described, however, have in vivo a very short half-life and very quickly become the basic body columns.

Carbohydrate-modified cytostatics are described in WO 96/31532 through a novel combination of selectively modified carbohydrates with cytostatics (e.g. batracylin, quinolone-a, camptothecin) before pulled spacer and linker groups both serum stability and release of Cytostatic within the tumor cells as well as a specific enrichment of the Cytostatic in tumor tissue is achieved. In this document, however, are only coal hydrate-modified cytostatics described in which the linker unit a thio is urea or triazine group.

WO 98/15571 describes camptothecin derivatives which are a carbohydrate have free side chain at the C20 position, which includes a thio has urea group as a linker unit.

Carbohydrate-modified cytostatics are described in WO 98/51703 which the saccharide residue and the camptothecin ver over a unit Sp-LAA1-AA2 are linked, where L stands for a thiourea unit.

In WO 98/14459, WO 98/14468 and WO 98/15573 there are further carbons third-modified camptothecin derivatives described.

Tumor cells have numerous options for treatment with cytotoxic Means to survive, which is why there is still a need for new drugs Cancer treatment exists. Because of the numerous mechanisms involved in tumor cells can bypass the negative effects of cytotoxic agents still far are not clear, it is difficult to make any predictions about the extent to which  structural changes in known medicinal products with regard to their Effect on tumor cells.

It was the object of the present invention to further carbohydrate-modified To provide cytostatics for the treatment of cancer.

It has now surprisingly been found that the compounds of the present Er have a high effectiveness against different tumor cell lines.

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

K-Sp-L-AA1-AA2-C (I)

With
K = unsubstituted or regioselectively modified carbohydrate residue
Sp = optionally substituted arylene or alkylene

in which
m is an integer from 1 to 6
n means 0 or 1
where the link to Sp takes place via the NH group.

AA1 a direct bond or an amino acid residue in the D or L configuration, which optionally carries protective groups or a second grouping K-Sp-L-, in which K, Sp, L independently of the other grouping K-Sp- L- can have the meanings given above.
AA2 a direct bond or an amino acid residue in the D or L configuration, which optionally carries protective groups or a second grouping K-Sp-L-, in which K, Sp, L independently of the other grouping K-Sp- L- can have the meanings given above.
C is a cytotoxic residue or the residue of a cytostatic or cytostatic derivative which can additionally carry a hydroxyl or amino group and is bonded to AA2 via an ester or amide group,
and their isomers and salts.

According to a preferred embodiment, the present invention relates to compounds of the general formula (I),) in which
K carbohydrate residue with the general formula (II)

in which
A denotes methyl, hydroxymethyl, carboxy and esters and amides derived therefrom, alkoxymethyl, acyloxymethyl or carboxyalkyloxymethyl and esters and amides derived therefrom or CH ₂ -B,
in which
B in turn is a carbohydrate residue of the general formula (II) linked via the anomeric center.
R ₂ , R ₃ , R ₄ individually or together equal to H, hydroxy, alkyloxy, carboxy alkyloxy and esters and amides derived therefrom, hydroxyalkyloxy, aminoalkyloxy, acyloxy, carboxyalkylcarbonyloxy, sulfato, phosphato, halogen, or another modified and via the anomeric center linked carbohydrate radical (II), where R ₂ can also be amino or acylamino and / or two of the radicals R ₂ , R ₃ , R _{4 can} also together form an epoxy group,
and Sp, L, m, n, AA1, AA2 and C have the same meaning as described above.

According to a further preferred embodiment, the present invention relates to compounds of the formula (I) in which
Sp arylene, which is ortho-, meta- or para-modified with K and L and can also carry 1 to 4 further substituents which independently or equal to H, methyl, methoxy, hydroxy, carboxy, methyloxy carbonyl, cyano Can be nitro, halogen, sulfonyl or sulfonamide, or is a linear or branched alkylene radical,
and K, L, m, n, AA1, AA2 and C have the same meaning as described above.

According to a further preferred embodiment, the present invention relates to compounds of the formula (I) in which
C is a nucleoside, an endiin antibiotic or a cytotoxic peptide antibiotic, a quinolone or naphthyridonecarboxylic acid or batracylin, 5-fluorouracil, cytosine arabinoside, methotrexate, etoposide, camptothecin, daunomycin, doxorubicin, taxol, esinincininamincinincinincininamine, vinblastamine, vinblastamine, vinblastamine, vinblastamine, vinblastamine, vinblastamine, vinblastamine, vinblastamine, vinblastamine, vinblastine, vinblastine, vinblastine, vinblastine, vinblastine Quercetin, Suramin, Erbstatin, Cyclophospha mid, Mitomycin C, Melphalan, Cisplatin, Bleomycin, Staurosporin or another antineoplastic active substance, which can additionally carry a hydroxy or amino group and is bound to AA2 via an ester or amide group,
and K, Sp L, m, n, AA1 and AA2 have the same meaning as described above.

Compounds in which C is a campto are particularly preferred thecinrest or the rest of a camptothecin derivative.

According to a further preferred embodiment, the present Er relates Compounds of formula (I) in which n is 0. In particular are here compounds of the formula (I) are preferred, in which AA1 additionally Is direct binding.

According to the present invention under a carbohydrate residue K Polyhy droxyaldehyde (aldoses) or polyhydroxyketones (ketoses) as well as higher molecular weight Compounds which can be converted into such compounds by hydrolysis, be understood. In particular, monosaccharides, ie. H. monomeric polyhydroxy aldehydes or polyhydroxy ketones, or oligosaccharides, d. H. dimers to decamers (disaccharides, trisaccharides, etc.) polyhydroxy aldehydes or polyhydroxy ketones. Examples are pentoses and Hexoses such as B. ribose, xylose, arabinose, glucose, mannose, galactose, gulose, Called fructose, sorbose, fucose and rhamnose. As examples of oligosaccharides disaccharides such as sucrose, lactose and maltose may be mentioned. According to the invention the carbohydrates can be in the D or L form. Particularly preferred are L-fucose and D-galactose.

According to the invention, the carbohydrate residues K can be unsubstituted or modified regioselectively. With regioselective modification, the substitution of one or more hydroxyl groups of the carbohydrate residue by alkyloxy, carboxyalkyloxy and esters and amides derived therefrom, such as, for example, the corresponding C _1-6 -alkyl esters or C _1-6 -mono- or dialkylamides, hydroxyalkyloxy, aminoalkyloxy, Acyloxy, carboxyalkylcarbonyloxy, sulfato, phosphato, halogen or optionally amino or acylamino are understood. Furthermore, two of the radicals R ₂ , R ₃ , R _{4 can} also together form an epoxy group. In addition, one or more hydroxyl groups of the carbohydrate residue can be substituted by a further carbohydrate residue, likewise modified accordingly and linked via the anomeric center. However, one or more hydroxyl groups of the carbohydrate residue can also be replaced by hydrogen and in this way the carbohydrate residue can be converted into a so-called deoxy sugar residue.

The regioselective modification of the carbohydrate radical K by converting one or more hydroxy groups into a C _1-6 alkoxy radical such as methoxy, ethoxy, propoxy or butoxy, into a hydroxyalkoxy radical such as hydroxyethoxy, into a carboxyalkoxy radical or an ester is particularly preferred. or an amide derivative thereof such as, for example, carboxymethoxy, methylcarbonylmethoxy, aminocarbonylmethoxy, methylaminocarbonylmethoxy, ethylaminocarbonylmethoxy, propylaminocarbonylmethoxy, butylaminocarbonyl methoxy, in a carboxyalkyl radical or an ester or amide derivative thereof such as, for example, methylcarbonyloxymethyl derivative, in a carboxy group, for example, in a carboxy group, or in a carboxy group, such as a carboxy group, in a carboxy group a halogen radical such as Cl or Br or in a sulfate radical.

According to the invention, C ₁ -C ₆ -alkyl comprises methyl, ethyl, n- and i-propyl, n-, i-, sec- and tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl.

(C ₁ -C ₆ ) alkoxy represents a straight-chain or branched alkoxy radical having 1 to 6 carbon atoms. A straight-chain or branched alkoxy radical having 1 to 4 carbon atoms is preferred. Examples include: methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, n-pentoxy and n-hexoxy. A straight-chain or branched alkoxy radical having 1 to 3 carbon atoms is particularly preferred.

(C ₁ -C ₆ ) alkoxycarbonyl represents a straight-chain or branched alkoxy carbonyl radical having 1 to 6 carbon atoms. A straight-chain or branched alkoxycarbonyl radical having 1 to 4 carbon atoms is preferred. Examples include: methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, isopropoxy carbonyl and tert-butoxycarbonyl. A straight-chain or branched alkoxycarbonyl radical having 1 to 3 carbon atoms is particularly preferred.

In the context of the invention, (C ₁ -C ₆ ) acyl represents a straight-chain or branched acyl radical having 1 to 6 carbon atoms. Examples include: acetyl, ethylcarbonyl, propylcarbonyl, isopropylcarbonyl, butylcarbonyl, isobutyl carbonyl, pentylcarbonyl and hexylcarbonyl. A straight-chain or branched acyl radical having 1 to 4 carbon atoms is preferred. Acetyl and ethylcarbonyl are particularly preferred.

Are one or more hydroxyl groups of the carbohydrate residue K by kohlhy third-party residues, so these can be independent of the primary carbohydrate residue also regioselectively modified or unsubstituted as described above be. According to the invention, these further carbohydrate residues are always above it anomeric center linked to the primary carbohydrate residue.  

Through this regioselective modification, the connection can be specific to tumor cells are addressed and thus experience increased tissue selectivity and Effect.

The carbohydrate residue K is connected to the rest of the molecule via a spacer group Sp. According to the invention, Sp can be an optionally substituted alkylene (also referred to as alkanediyl) or arylene. A C ₂ -C ₈ -alkanediyl radical, ie a straight-chain or branched alkanediyl radical having 2 to 8 carbon atoms, is preferred according to the invention. A straight-chain or branched alkanediyl radical having 2 to 6 carbon atoms, particularly preferably having 2 to 4 carbon atoms, is particularly preferred. Examples include: ethylene, propylene, propane-1,2-diyl, propane-2,2-diyl, butane-1,3-diyl, butane-2,4-diyl, pentane-2,4-diyl, 2- Methyl pentane-2,4-diyl. According to the invention, arylene stands for an aromatic carbon hydrogen radical with 6 to 10 carbon atoms in the cyclic structure. Examples include 1,4-phenylene, 1,3-phenylene, 1,2-phenylene or naphthylene. The alkanediyl or arylene spacer can also be substituted one or more times, preferably up to four times, with one or more radicals from the group consisting of H, methyl, methoxy, hydroxy, carboxy, methyloxycarbonyl, cyano, nitro, halogen, Sulfonyl or sulfonamide.

According to the invention, the linker group L is a unit of the formula

in which
m is an integer from 1 to 6
n means 0 or 1
where the link to Sp takes place via the NH group.

According to the invention, preference is given to compounds in which n is 0 (i.e. L is a urine unit of matter), or where n is 1 and m is an integer from 1 to 4 be indicates (i.e. L is a malonic, succinic, glutaric or adipic acid unit).

The linker group L can either directly or via one or two amino acid groups AA1 and AA2 may be linked to the cytotoxic residue.

The amino acid residues AA1 and AA2 can independently in the D- or L configuration and optionally a second grouping Wear K-Sp-L- in which K, Sp, L regardless of the other grouping K-Sp-L- can have the meanings given above.

The amino acid residue AA1 can be given both via the α-amino group and if via side chain amino or hydroxy functions or also via both radio tions linked with L. If AA1 carries further functional groups, then can this unblocks or with protective groups known to those skilled in the art (i.e. organic Residues with which certain functional groups ent one of several active centers holding mol. are temporarily protected against attack by reagents protected) (see e.g. Kocienski, Protecting Groups, Stuttgart, Thieme 1994). 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-dimethoxybenzyloxy carbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 4-nitro benzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxy carbonyl, methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl (Boc), allyloxy carbonyl, vinyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, phthaloyl, 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-tri chloroacetyl, benzoyl, benzyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, Phthalimido, isovaleroyl or benzyloxymethylene, 4-nitrobenzyl, 2,4-dinitrobenzyl, 4-nitrophenyl or 2-nitrophenylsulfenyl. The Fmoc- Group and the Boc group.

The splitting off of protective groups in appropriate reaction steps can lead to Example by exposure to acid or base, hydrogenolytically or to others Way reductive.

The amino acid residue AA2 can be given both via the α-amino group and if via side chain amino or hydroxy functions or also via both radio be linked with AA1. If AA2 carries further functional groups, then so these can be unblocked or known to the person skilled in the art above AA1 described protective groups are protected.

According to the invention, AA1 and AA2 preferably represent those in nature occurring α-amino acids, but also includes their homologues, Isomers and derivatives. Enantiomers can be mentioned as examples of isomers will. Derivatives can, for example, amino acids provided with protective groups be.

AA1 and AA2 can occur in the L or D configuration or as Mixture of D and L forms. AA1 particularly preferably represents an amino acid with basic side chain. AA2 particularly preferably represents an amino acid sterically demanding or non-polar side chain.

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, respectively.

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.

Such a side chain K-Sp-L-AA1-AA2 described above is available from Ver Linking with a cytotoxic residue is able to select this into the tumor insert tissue where it can develop its cytotoxic effect. This be The principle of the invention described is broad on cytotoxic and cytostatic Connections applicable. The property of substances is called cytotoxicity understood to inactivate or kill other cells. According to the invention a group linked to the unit K-Sp-L- under a cytotoxic residue stood, which at least after the release from the molecule cytotoxic effect shows. These are known cytotoxic or released cytostatic compounds such as the rest of a nucleoside, one Endiin antibiotic or a cytotoxic peptide antibiotic e.g. B. from the Class of dolastatine or a quinolone or naphthyridonecarboxylic acid. C. For example, batracylin, 5-fluorouracil, cytosine arabinoside, methotrexate, Etoposide, camptothecin, daunomycin, doxorubicin, taxol, vinblastine, vincristine, Dynemicin, Calicheamycin, Esperamycin, Quercetin, Suramin, Erbstatin, Cyclo phosphamide, mitomycin C, melphalan, cisplatin, bleomycin, staurosporine or a other antineoplastic active ingredient. C Batracylin, metho is preferred trexat, quinolone-a, etoposide, melphalan, taxol, camptothecin, daunomycin or Doxorubicin or a quinolone or naphthyridonecarboxylic acid.

The cytostatic is linked to AA2 via amino or hydroxy functions.

The above-mentioned quinolone or naphthyridonecarboxylic acid building blocks C which are suitable according to the invention can be represented by the general structure of the formula (III)

TQ (III)

in which
Q a remainder of the formulas

means what
R ^a for alkyl with 1 to 4 carbon atoms which is monosubstituted or disubstituted by halogen or hydroxy, vinyl, optionally cycloalkyl with 3 to 6 carbon atoms which is substituted by 1 or 2 fluorine atoms, bicyclo [1.1.1] pent-1-yl, 1 , 1-dimethylpro pargyl, 3-oxetanyl, methoxy, amino, methylamino, dimethylamino, phenyl which is mono- or disubstituted by halogen, amino or hydroxy, or can also form a bridge together with R ^e ,
R ^b for hydroxy, alkoxy with 1 to 3 carbon atoms, nitromethyl,
R ^{c represents} hydrogen or methyl or can together with R ^{g form} a bridge described there,
R ^d for hydrogen, CH ₃ , CH ₂ F or = CH ₂ ,
X ¹ for hydrogen, halogen or nitro,
X ² for hydrogen, halogen, amino, hydroxy, methoxy, mercapto, methyl, halomethyl or vinyl,
Y is N or CR ^e , wherein
R ^{e represents} hydrogen, halogen, CF ₃ , OCH ₃ , OCHF ₂ , CH ₃ , CN, CH = CH ₂ or C∼CH or also together with R ^a a bridge of the structure - ^* O-CH ₂ -CH-CH ₃ , - ^* S-CH ₂ -CH ₂ -, - ^* S-CH ₂ -CH-CH ₃ , - ^* CH ₂ -CH ₂ -CH-CH ₃ or - ^* O-CH ₂ -NR ^f , whereby the atom marked with ^* is linked to the carbon atom of Y and in which
R ^{f represents} hydrogen, methyl or formyl,
can form, and
D represents N or CR ^g , wherein
R ^{g represents} hydrogen, halogen, CF ₃ , OCH ₃ , OCHF ₂ or CH ₃ or also together with R ^c a bridge of the structure - ^* O-CH ₂ -, - ^* NH-CH ₂ -, - ^* N (CH ₃ ) -CH ₂ -, - ^* N (C ₂ H ₅ ) -CH ₂ -, - ^* N (C ₃ H ₅ ) -CH ₂ - or - ^* S-CH ₂ -, which is marked with ^* Atom is linked to the carbon atom of D,
o 1, 2 or 3 and
T a residue of the formula

means what

stands where
R ^k for hydrogen or methyl and
R ^{i represents} hydrogen, C ₁ -C ₃ alkyl or cyclopropyl.

Compounds of the formula (III) in which
Q is a residue of the formula

means what
R ^a for alkyl having 2 to 4 carbon atoms optionally substituted by 1 fluorine atom, optionally cyclopropyl substituted by 1 fluorine atom, optionally phenyl mono- or disubstituted by fluorine,
R ^b for hydroxy, alkoxy with 1 to 2 carbon atoms,
R ^{c represents} hydrogen, methyl or together with R ^g can form a bridge described there,
X ¹ for fluorine,
X ² for hydrogen or amino,
Y is N or CR ^e , wherein
R ^{e represents} hydrogen, fluorine, chlorine, CF ₃ , OCH ₃ , OCHF ₂ , or C∼CH or together with R ^{a is} a bridge of the structure - ^* O-CH ₂ -CH-CH ₃ or - ^* O-CH ₂ -NR ^f ,
where the atom marked with ^* is linked to the carbon atom of Y and where
R ^f means methyl
can form
D represents N or CR ^g , wherein
R ^{g represents} hydrogen, fluorine, chlorine, CF ₃ , OCH ₃ or CH ₃ or also together with R ^c a bridge of the structure - ^* O-CH ₂ -, - ^* NH-CH ₂ -, - ^* N (CH ₃ ) Can form CH ₂ - or - * S-CH ₂ -,
where the atom marked with ^* is linked to the carbon atom of D and
T a residue of the formula

means what

stands in what
R ^{k represents} hydrogen or methyl,
R ^{i represents} hydrogen or methyl.

According to the invention, C is particularly preferably camptothecin or Camptothecin derivative such as 9-aminocamptothecin.

The compounds according to the invention can be in the form of their salts. In all in general, salts with organic or inorganic acids may be mentioned here. These salts can be reacted with the free compounds of the formula (I) organic or inorganic acids. As acids come here in accordance with the invention preferably hydrohalic acids, such as. B. the chlorine hydrochloric acid and hydrobromic acid, especially the hydrogen chloride acid, also phosphoric acid, nitric acid, sulfuric 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 as well as sulfone acids, such as B. p-toluenesulfonic acid, 1,5-naphthalenedisulfonic acid or camphor sulfonic acid in question.

The compounds of the invention can 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 also their mixtures.

The stereoisomer mixtures can, if necessary, in a known manner in the stereoisomerically uniform constituents are separated, for example by chro matography or by crystallization processes.

The present invention further relates to a process for the preparation of the compounds of the formula (I), comprising the reaction of a compound of the formula

K-Sp-NH ₂

in which
K and Sp have the meaning described above, with

• a) a chloroformate in the presence of a base in an organic solvent

or

• a) an alkanedicarboxylic acid or a dicarboxyl derivative thereof in the presence a base in an organic solvent

and the subsequent reaction with a compound of the formula

AA1-AA2-C

in which
AA1, AA2 and C have the meaning described above,
in the presence of a base in an organic solvent.

The preparation of compounds K-Sp-NH ₂ is described, for example, in WO 96/31532 (Examples 1 to 18), the content of which is hereby incorporated by reference.

The illustration of compounds AA1-AA2-C is also, for example, in the WO 96/31532 (Examples 1 to 18), the contents of which are hereby described is referenced.

Any esters such as, for example, methyl, Ethyl or p-nitrophenyl esters can be used. Is preferred according to the invention the chloroformic acid p-nitrophenyl ester.

The reaction of the compound of formula K-Sp-NH ₂ with the chloroformate can be carried out at various pressure and temperature ratios, for example 0.5 to 2 bar and preferably under normal pressure, or -30 to + 100 ° C. and preferably -10 to + 80 ° C, in suitable solvents such as dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane, chloroform, lower alcohols, acetonitrile, dioxane, water or in mixtures of the solvents mentioned. As a rule, reactions in dimethylformamide (DMF), dichloromethane, tetrahydrofuran (THF), dioxane / water or THF / dichloromethane at room temperature or under ice cooling and normal pressure are preferred. According to the invention, the reaction in tetrahydrofuran at room temperature and normal pressure is particularly preferred. The compounds are used in equimolar amounts or the chloroformic acid ester in a slight excess over the compound K-Sp-NH ₂ . The base is used either in equimolar amounts or in (for example a two-fold) excess. The reaction is usually complete after a few (approx. 5-45) minutes.

This reaction is carried out in the presence of a base. As examples for him bases suitable according to the invention are triethylamine, ethyldiisopropylamine, pyridine, N, N-dimethylaminopyridine or others conventionally used in such steps called turned bases.

Optionally, the compound of the formula K-Sp-NH _{2 can} also be reacted with an alkane dicarboxylic acid or a dicarboxyl derivative thereof in the presence of a base in an organic solvent. According to the invention, preference is given here to the use of the corresponding dicarboxylic anhydride. This reaction can be carried out under suitable pressure and temperature conditions, for example 0.5 to 2 bar and preferably under normal pressure, or -30 to + 100 ° C. and preferably -10 to + 80 ° C. in suitable solvents such as dimethylformamide (DMF). Tetra hydrofuran (THF), dichloromethane, chloroform, lower alcohols, acetonitrile, dioxane, water or in mixtures of the solvents mentioned. As a rule, reactions in dimethylformamide (DMF), dichloromethane, tetrahydrofuran (THF), dioxane / water or THF / dichloromethane at room temperature or under ice cooling and normal pressure are preferred. According to the invention, the reaction in DMF at room temperature and normal pressure is particularly preferred. The compounds are used in equimolar amounts or the chloroformate in a slight excess compared to the compound K-Sp-NH ₂ . The base is used either in equimolar amounts or in (for example a two-fold) excess. The reaction is usually complete after a few (approx. 5-45) minutes.

This reaction is also carried out in the presence of a base. As examples of bases suitable according to the invention are triethylamine, ethyldiisopropylamine, Pyridine, N, N-dimethylaminopyridine or others in such steps called commonly used bases.

The intermediate product obtained from one of these reactions is then reacted with the compound of the formula AA1-AA2-C in the presence of a base to give the compound of the formula (I). Examples of bases suitable according to the invention are triethylamine, ethyldiisopropylamine, pyridine, N, N-dimethylaminopyridine or other bases conventionally used in such steps. The compounds are used equimolar or the compound AA1-AA2-C in a slight excess compared to the compound K-Sp-NH ₂ . The base is used either in equimolar amounts or in (for example a two-fold) excess. This reaction can be carried out at various pressure and temperature conditions, for example 0.5 to 2 bar and preferably under normal pressure, or -30 to + 100 ° C. and preferably -10 to + 80 ° C, in suitable solvents such as dimethylformamide (DMF) , Tetrahydrofuran (THF), dichloromethane, chloroform, lower alcohols, acetonitrile, dioxane, water or in mixtures of the solvents mentioned. As a rule, reactions in dimethylformamide (DMF), dichloromethane, tetrahydrofuran (THF), dioxane / water or THF / dichloromethane at room temperature or under ice-cooling and normal pressure are preferred. According to the invention, the reaction in DMF or THF at room temperature and normal pressure is particularly preferred. If necessary, the reaction can be accelerated using ultrasound or can be carried out with better yields. The reaction can take between 30 minutes and several, for example 16 hours.

In the case of reacting the compound of formula AA1-AA2-C with a compound obtained from the reaction of K-Sp-NH ₂ with an alkanedicarboxylic acid or a derivative thereof, the free carboxyl function of the latter may be necessary Activate connection. For the activation of the carboxyl group come the coupling reagents known in peptide chemistry, such as z. B. in Jakubke / Jeschkeit: amino acids, peptides, proteins; Verlag Chemie 1982 or Tetrahedr. Lett. 34, 6705 (1993). For example, N-carboxylic acid anhydrides, acid chlorides or mixed anhydrides are preferred.

The formation of is also suitable for activating the carboxyl group 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 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-hyd roxybenzotriazole or N-hydroxysuccinimide esters.

The present invention will hereinafter be described by way of non-limiting example play and illustrated comparative examples.  

Examples A) Starting compounds

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

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

20 g (70 mmol) p-nitrophenyl-β-L-fucopyranoside (preparation see WO 96/31532) in 1 l absolute. Methanol is mixed with 26.13 g (105 mmol) of dibutyltin oxide and Heated under reflux for 23 h. The mixture is then concentrated and the residue is dried and then taken up in 1 liter of DMF. After adding 35 ml of methyl iodide Batch stirred at 70 ° C for 16 h. The solvent is removed in vacuo and the Residue taken up in dichloromethane. The suspension is filtered, the ver remaining solution again concentrated and a flash chromatography on silica gel (Dichloromethane / methanol 99: 1). After concentration, 17.4 g (83%) are obtained of the target product.

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

8.7 g (29 mmol) of p-nitrophenyl-3-O-methyl-β-L-fucopyranoside from Ex. Ia) are dissolved in 1.2 l of methanol and, after the addition of palladium on activated carbon, hydrogenated in a hydrogen atmosphere at a slight excess pressure . After filtering off the catalyst, precipitating with ether and washing the residue with methanol, the target product is taken up in water and lyophilized. 7.4 g (95%) are obtained. [TLC: dichloromethane / methanol 9: 1 R _f = 0.3].

II) 20 (S) -20-O-L-valyl camptothecin, trifluoroacetate II.a) 20 (S) -20-O- [N- (tert-Butoxycarbonyl) -L-valyl] camptothecin

A suspension of 40 g (115 mmol) 20 (S) -camptothecin in 2 l absolute dichloromethane is stirred with 56 g (230 mmol) N- (tert-butoxycarbonyl) -L-valine-N-carboxylic anhydride and 4 g 4 - (N, N-Dimethylamino) pyridine added. After 4 days of stirring under reflux in an argon atmosphere, camptothecin reacted. The solvent is evaporated and the residue is mixed with 500 ml of methyl tert-butyl ether. After stirring for 20 min, 750 ml of petroleum ether are added and filtered off. The filter residue is dried in a high vacuum. 61.5 g (97%) of the target compound are obtained. [TLC: acetonitrile / water 20: 1 R _f = 0.35].

II) 20 (S) -20-O-L-valyl camptothecin, trifluoroacetate

A solution of compound II.a (61 g, 111 mmol) in 1.7 l of dichloromethane is cooled to 5 ° C., mixed with 350 ml of anhydrous trifluoroacetic acid and then stirred for 1 h at room temperature. After concentration in vacuo to a small volume, the product is precipitated with 1 l of methyl tert-butyl ether and stirred for 10 min. The precipitate is filtered off. The crude product is dissolved again in 950 ml of methanol, filtered and the solution is then precipitated with 3.5 l of methyl tert-butyl ether. The precipitate is filtered off again and washed with methyl tert-butyl ether. The process may be repeated again. Yield: 38.6 g (60%) [TLC: aceto nitrile / water 20: 1 R _f = 0.38].

III) 20 (S) -20-O- {L-histidyl-L-valyl} camptothecin, trifluoroacetate IIIa) 20 (S) -20-O- [N- (tert-Butoxycarbonyl) -L-histidyl-L-valy] camptothecin

6.28 g (24.6 mmol) of N- (tert-butoxycarbonyl) -L-histidine and 5 g of 1-hydroxy-1H benzotriazole hydrate are dissolved in 500 ml of dimethylformamide and the solution is cooled to 0.degree. After adding 5.64 g of N-ethyl-N '- (dimethylaminopropyl) carbodiimide hydrochloride, the mixture is stirred at 0 ° C. for 30 minutes. Then compound II (11.5 g, 20.5 mmol) and 8.5 ml 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 taken up in 1 l of dichloromethane and extracted three times with 500 ml of water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is precipitated from dichloromethane with methyl tert-butyl ether and washed with methyl tert-butyl ether. 12.8 g (91%) of the target compound are obtained. [TLC: acetonitrile / water 10: 1 R _f = 0.33].

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

12.8 g (18.7 mmol) of compound III.a are taken up in 180 ml of dichloromethane and 90 ml of anhydrous trifluoroacetic acid are added at 5 ° C. The resulting solution is stirred for 30 min and allowed to warm to room temperature. The mixture is then stirred for a further hour at room temperature. After concentration to a small volume in vacuo, the product is precipitated by adding methyl tert-butyl ether and stirred for a further 30 min. The residue is filtered off and, after drying in vacuo, 12.9 g (85%) of the target compound are obtained [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.25].

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

22 g (47 mmol) N ^α - (tert-butoxycarbonyl) -N ^ε - (fluorenyl-9-methoxycarbonyl) -L-lysine and 9.5 g (70 mmol) 1-hydroxy-1H-benzotriazole hydrate are dissolved in 800 ml Dissolved methylformamide and cooled to 0 ° C. After adding 10.8 g (56 mmol) of N-ethyl-N '- (dimethylaminopropyl) carbodiimide hydrochloride, the mixture is stirred at 0 ° C. for 1 h. Then compound II (21.85 g, 39 mmol) and 24.3 ml of ethyl diisopropylamine are added and the mixture is stirred for a further 16 h at room temperature. After concentration in vacuo, 1.5 l of water are added to the residue and the mixture is stirred for 15 min. The resulting solid is filtered off, washed with water and then taken up in 800 ml of dichloromethane. After drying, the organic phase is concentrated to 150 ml and precipitated with 2 l of methyl tert-butyl ether. The residue is filtered off and dried. 28.4 g (81%) of the target product are obtained. [TLC: aceto nitrile R _f = 0.47].

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

The compound IVa (27.89 g, 31 mmol) is taken up in 600 ml of dichloromethane, cooled to 5 ° C. and mixed with 200 ml of anhydrous trifluoroacetic acid. The resulting solution is allowed to warm to room temperature and stirred for 2 hours. After concentration to a small volume in vacuo, the product is precipitated by adding methyl tert-butyl ether and dried. 26 g (92%) of the target compound are obtained [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.54].

B) working examples example 1

20 (S) -20-O- {N- [4- (3-O-Methyl-β-L-fucopyranosyloxy) phenylcarbamoyl] -L-histidyl-L-valyl} camptothecin, hydrochloride

1a) 20 (S) -20-O- {N ^α - (4- (3-O-Methyl-β-L-fucopyranosyloxy) -phenylcarbamoyl] -L-histidyl-L-valyl} -camptothecin

45 mg (0.22 mmol) p-nitrophenyl chloroformate and 48 mg ethyl diisopropylamine are dissolved in 10 ml of tetrahydrofuran and then with 50 mg (0.19 mmol) of compound I, which was previously dissolved in 10 ml of tetrahydrofuran, ver sets. After stirring for 5 min at room temperature, 130 mg (0.19 mmol) of compound III and a further 72 mg (0.56 mmol) of ethyl-diisopropylamine are added and the mixture is treated in an ultrasound bath at room temperature for 30 min. The solvent is evaporated in vacuo and the residue is purified by flash chromatography on silica gel with acetonitrile / water 10: 1. The corresponding fractions are concentrated and the residue is precipitated from dichloromethane / methanol with diethyl ether. After drying in a high vacuum, 52 mg (32.4%) of the target compound are obtained. [TLC: acetonitrile / water 10: 1 R _f = 0.15] [FAB-MS: m / z = 880 (M + H ⁺ )].

1) 20 (S) -20-O- {N ^α - [4- (3-O-Methyl-β-L-fucopyranosyloxy) -phenylcarbamoyl] -L-histidyl-L-valyl} -camptothecin, hydrochloride

48 mg (0.05 mmol) are dissolved in 10 ml of dioxane / water 1: 1 and mixed with 545 µl of a 0.1 N hydrochloric acid solution. The solution is then lyophilized. The residue is precipitated from dichloromethane / methanol with diethyl ether. After drying in a high vacuum, 52 mg (32.4%) of the target compound are obtained. [TLC: acetonitrile / water 10: 1 R _f = 0.15].

Example 2

20 (S) -20-O- {N ^α - [4- (3-O-Methyl-β-L-fucopyranosyloxy) phenylcarbamoyl] -L-valyl} camptothecin

90 mg (0.45 mmol) of p-nitrophenyl chloroformate and 77 mg of ethyl diisopropylamine are dissolved in 10 ml of tetrahydrofuran and then with 80 mg (0.3 mmol) of compound I, which was previously dissolved in 10 ml of tetrahydrofuran, ver sets. After stirring for 5 min at room temperature, 107 mg (0.19 mmol) of compound II and a further 77 mg (0.59 mmol) of ethyldiisopropylamine are added and the mixture is stirred overnight at room temperature. The solvent is evaporated off in vacuo and the residue is stirred with water and then filtered off. The residue is then precipitated twice more from dichloromethane / methanol with diethyl ether. After drying in a high vacuum, 104 mg (70.7%) of the target compound are obtained. [TLC: dichloromethane / methanol / ammonia 17% 15: 2: 0.2 R _f = 0.48] [FAB-MS: m / z = (M + H ⁺ )].

Example 3

20 (S) -20-O- {N- [4- (3-O-Methyl-β-L-fucopyranosyloxy) phenylcarbamoyl] -L-lysyl-L-valyl} camptothecin, hydrochloride

3a) 20 (S) -20-O- {N ^α - [4- (3-O-methyl-bL-fucopyranosyloxy) phenylcarbamoyl] - N ^ε - [fluorenyl-9-methoxycarbonyl) -] - L-lysyl- L-valyl} camptothecin

45 mg (0.22 mmol) of p-nitrophenyl chloroformate and 38 mg of ethyl diisopropylamine are dissolved in 8 ml of tetrahydrofuran and then with 40 mg (0.15 mmol) of compound I, which was previously dissolved in 10 ml of tetrahydrofuran, ver sets. After stirring for 5 min at room temperature, 114 mg (0.13 mmol) of compound IV and a further 48.5 mg (0.56 mmol) of ethyldiisopropylamine are added and the mixture is treated in an ultrasound bath at room temperature for 2 h. The solvent is evaporated in vacuo and the residue is purified by flash chromatography on silica gel with acetonitrile / water / glacial acetic acid 5: 1: 0.1. The corresponding fractions are concentrated and the residue is precipitated from dichloromethane / methanol with diethyl ether. After drying in a high vacuum, 54 mg (40%) of the target compound are obtained. [TLC :: dichloromethane / methanol / ammonia 17% 15: 2: 0.2 R _f = 0.42].

3b) 20 (S) -20-O- {N ^α - [4- (3-O-Methyl-β-L-fucopyranosyloxy) phenylcarbamoyl] -L-lysyl-L-valyl} -camptothecin

50 mg (0.05 mmol) of compound 3a) are dissolved in 5 ml of dimethylformamide, and 500 μl (0.15 mmol) of piperidine are then added. The mixture is stirred at room temperature for 30 minutes and concentrated. The residue is precipitated twice from dichloromethane / methanol with diethyl ether. After drying in a high vacuum, 33 mg (83%) of the target compound are obtained. [TLC: acetonitrile / water / glacial acetic acid 5: 1: 0.2 R _f = 0.17].

3) 20 (S) -20-O- {N ^α - [4- (3-O-Methyl-β-L-fucopyranosyloxy) phenylcarbamoyl] -L-lysyl-L-valyl} -camptothecin, hydrochloride

30 mg (0.04 mmol) are dissolved in 5 ml of dioxane / water 1: 1 and mixed with 320 ul of a 0.1 N hydrochloric acid solution. The solution is then lyophilized. After drying in a high vacuum, 31 mg (99%) of the target compound are obtained. [TLC: acetonitrile / water 10: 1 R _f = 0.15]. [ESI-MS: m / z = 870 = (M + H ⁺ )].

Example 4

20 (S) -20-O- {N- [3- (4- (3-O-Methyl-β-L-fucopyranosyloxy) phenylcarbamoyl) propionyl] -L-histidyl-L-valyl} -camptothecin, hydrochloride

4a) 3- (4- (3-O-Methyl-β-L-fucopyranosyloxy) phenylcarbamoyl] propionic acid

100 mg (0.37 mmol) of the starting material I are dissolved in 5 ml of dimethylformamide together with 52 mg of succinic anhydride and 10 mg of dimethylaminopyridine and stirred at room temperature for 30 min. The mixture is concentrated and the residue is precipitated from dichloromethane / methanol with diethyl ether and dried in a high vacuum. 95 mg (69%) of the target compound are obtained, which is used in the next step without further purification. [TLC: acetonitrile / water 10: 1 R _f = 0.22].

4b) 20 (S) -20-O- {N ^α - [3- (4- (3-O-Methyl-β-L-fucopyranosyloxy) -phenylcarbamoyl) - propionyl] -L-histidyl-L-valyl} - camptothecin

50 mg (0.12 mmol) of compound 4a) are dissolved in 15 ml DMF and at 0 ° C 25 mg (0.18 mmol) 1-hydroxy-1H-benzotriazole hydrate and 28 mg (0.15 mmol) N -Ethyl-N '- (dimethylaminopropyl) carbodiimide hydrochloride is added and the mixture is stirred for 30 min. Then compound III (100 mg, 0.14 mmol) and 48 mg of ethyl-diisopropylamine are added and the mixture is stirred for a further 16 h at room temperature. After concentration in vacuo, dichloromethane is added to the residue and the mixture is stirred for 15 min. It is then filtered off and the organic phase is concentrated. The residue is purified by flash chromatography on silica gel with dichloromethane / methanol / ammonia 17% 15: 2: 0.2 as the eluent. The corresponding fractions are combined, concentrated and the residue is then precipitated from dichloromethane / methanol with diethyl ether. The residue is filtered off and dried. 69 mg (60%) of the target product are obtained. [TLC: dichloromethane / methanol / ammonia 17% 15: 4: 0.5 R _f = 0.5].

4) 20 (S) -20-O- {N ^α - (3- (4- (3-O-Methyl-β-L-fucopyranosyloxy) -phenylcarbamoyl) - propionyl] -L-histidyl-L-valyl} - camptothecin, hydrochloride

59 mg (0.06 mmol) of compound 4b) are dissolved in 5 ml of dioxane / water 1: 1 and mixed with 630 μl of a 0.1 N hydrochloric acid solution. The solution is then lyophilized. After drying in a high vacuum, 60 mg (98%) of the target compound are obtained. [TLC: dichloromethane / methanol / ammonia 17% 15: 4: 0.5 R _f = 0.5].

Biological tests 1. Growth inhibition test to determine the cytotoxic properties

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 RPMI 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 ul cell suspension / well were placed in a 96 microwave plate and incubated for 1 day at 37 ° C in the CO ₂ incubator. Then another 100 µl RPMI medium and 1 µl DMSO with the test substances were added. 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 in a CO ₂ incubator at 37 ° C. for 5 hours. 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 595 nm was measured with a Multiplate Reader (Bio / Rad) 3550-UV.

The cytotoxic effect is given in Table 1 as the 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 Table 2 Inhibition of CSF-induced proliferation of mouse bone marrow stem cells

IC ₅₀ [ng / ml] example 1 10th Example 2 0.8 Example 3 100

3. HPLC method for determining the stability in the cell supernatant of HT-29

Equipment: Waters Alliance 2690; UV detection at 257 nM or 356 nM
Column: LiChrospher 100, RP-18 (5 µm) 250 x 4 mm; Merck 50833 with guard column
Conditions: Eluent A: 0.01 M KH ₂ PO ₄ in H ₂ O
Eluent B: 80% acetonitrile + 20% eluent A

Method of determining stability

HT-29 tumor cells (200,000 cells / ml) were in RPMI 1640 medium under Zu set of 10% FCS drawn 24 h. Aqueous solutions of the glycoconjugates in Concentrations of 10 µM are added and incubated for 24 hours. After that the supernatant is diluted with the same volume of methanol and then centrifuged. 50 ul of the resulting solution are injected into the HPLC and after analyzed above method. The measured area percentages of the glycoconjugate are compared with those of released active ingredient.

For example, the compound from Example 1 becomes 3% free after 24 h Camptothecin detected (0% at 0 h) while the starting glyco conjugate decreased from 91.5 to 87.6 area percent in the same period.

In contrast, the compound from Example 2 becomes very rapid in the cell supernatant split into camptothecin.  

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

For all in vivo experiments to investigate the inhibition of tumor growth athymic nude mice (NMRI nu / nu strain) were used. The selected ones Tumors were developed by serial passage in nude mice. The human The origin of the tumor was determined by isoenzymatic and immunohistochemical Methods proven.

Experimental setup

The tumor was subcutaneously in both flanks of 6 to 8 week old nu / nu naked implanted in mice. Treatment was based on doubling time starts as soon as the tumors have reached a diameter of 5-7 mm. The Mice were the treatment group and the control group (5 mice per group with 8-10 evaluable tumors) by randomization. The single ones Tumors in 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).

Relative tumor volume (RTV) values were for each individual tumor by dividing the tumor size on day X by the tumor size on day 0 (at Time of randomization). The mean 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 administered intravenously (IV), intraperitoneally (IV), oral (p. o.) or subcutaneous (s. c.).

Claims (8)

1. Compounds of the general formula (I)
K-Sp-L-AA1-AA2-C (I)
With
K = unsubstituted or regioselectively modified carbohydrate residue
Sp = optionally substituted arylene or alkylene
in which
m is an integer from 1 to 6
n means 0 or 1
where the link to Sp takes place via the NH group.
AA1 is a direct bond or an amino acid residue in the D or L configuration, which optionally carries protective groups or a second grouping K-Sp-L-, in the K, Sp, L independently of the other grouping K-Sp-L - Can have the meanings given above.
AA2 a direct bond or an amino acid residue in the D or L configuration, which optionally carries protective groups or a second grouping K-Sp-L-, in the K, Sp, L independently of the other grouping K-Sp-L - Can have the meanings given above.
C is a cytotoxic residue or the residue of a cytostatic or cytostatic derivative which can additionally carry a hydroxyl or amino group and is bonded to AA2 via an ester or amide group,
and their isomers and salts. 2. Compounds according to claim 1, wherein
K carbohydrate residue with the general formula (II)
in which
A denotes methyl, hydroxymethyl, carboxy and esters and amides derived therefrom, alkoxymethyl, acyloxymethyl or carboxyalkyloxymethyl and esters and amides derived therefrom or CH ₂ -B,
in which
B is in turn a carbohydrate radical of the general formula (II) linked via the anomeric center,
R ₂ , R ₃ , R ₄ individually or together equal to H, hydroxy, alkyloxy, carboxyalkyloxy and esters and amides derived therefrom, hydroxyalkyloxy, aminoalkyloxy, acyloxy, carboxyalkyl carbonyloxy, sulfato, phosphato, halogen, or another modified and via the anomeric center linked carbohydrate radical (II), where R ₂ can also be amino or acylamino and / or two of the radicals R ₂ , R ₃ , R ₄ can together form an epoxy group,
and Sp, L, m, n, AA1, AA2 and C have the same meaning as defined in claim 1. 3. Compounds according to claim 1, wherein
Sp arylene, which is ortho-, meta- or para-modified with K and L and in addition can carry 1 to 4 further substituents which are, independently or identical, H, methyl, methoxy, hydroxyl, carboxy, methyloxycarbonyl, cyano, nitro , Halogen, sulfonyl or sulfonamide, or is a linear or branched alkylene radical,
and K, L, m, n, AA1, AA2 and C have the same meaning as defined in claim 1. 4. Compounds according to claim 1, wherein
C is a nucleoside, an endiin antibiotic or a cytotoxic peptide antibiotic, a quinolone or naphthyridonecarboxylic acid or batracylin, 5-fluorouracil, cytosine arabinoside, methotrexate, etoposide, camptothecin, daunomycin, doxorubicin, taxol, esinincincinamine dynorcincinamine dyinolincinamine dynorcincinamine dynocycline, vinblinamine dynorcinol amine, Quercetin, Suramin, Erbstatin, Cyclophosphamide, Mitomycin C, Melphalan, Cisplatin, Bleomycin, Staurosporin or another antineoplastic active substance, which can additionally carry a hydroxy or amino group and is bound to AA2 via an ester or amide group,
and K, Sp L, m, n, AA1 and AA2 have the same meaning as defined in claim 1. 5. Compounds according to claim 4, wherein
C stands for camptothecin. 6. Compounds according to claim 1, wherein
n is 0
and K, Sp L, m, AA1, AA2 and C have the same meaning as defined in claim 1. 7. Compounds according to claim 1, wherein
n is 0
AA1 is a direct bond
and K, Sp, m, AA2 and C are as defined in claim 1. 8. A process for the preparation of compounds of formula (I) according to claim 1, comprising the implementation of a compound of formula